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Volume 3 Issue 1 Jan-March, 2013 Editor-In-Chief JAYAPAL REDDY GANGADI M.Pharm, M.Phil.,FICCP, Ph.D

Associate Editors Mr.E.Venkateshwarlu Mr.K.Venu Mr.K.Naresh Mr.M.Satish

Department Of Pharmacology, India Department Of Pharmacology, India Department Of Pharmaceutical Chemistry, India Department Of Pharmaceutical Chemistry, India

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Research Article Biological Sciences SERUM PARAOXONASE-1 ACTIVITY, OXIDATIVE STRESS & LIPID PROFILE IN PATIENTS WITH CHRONIC LIVER DISEASE SUSHMA B JAGANNATHA*1, NAGARAJAPPA .K2 & MALLIKARJUNA. C.R3 1*, 2, 3

Department of Biochemistry, S.S. Institute of Medical Sciences and Research Centre, Davangere -577005, Karnataka, India. *Corresponding Author Email: Sushmabj1983@gmail.com

ABSTRACT Background/Aim: Chronic liver disease in the clinical context is a disease of the liver that involves a process of progressive destruction and regeneration of the liver parenchyma leading to fibrosis and cirrhosis. Oxidative stress influences the pathophysiological changes leading to chronic liver disease. Paraoxonase-1 [PON1] is an esterase, exclusively synthesized by the liver exerts a protective effect against oxidative stress. The present study has two objectives: to estimate and compare the standard liver function tests, lipid profile, serum basal PON1 activity & malondialdehyde [MDA] in chronic liver disease patients and healthy controls & to find the correlation between serum basal PON1 activity, MDA and standard LFTs. Materials and Methods: In this study we included 40 diagnosed cases of chronic liver disease and 40 healthy age and sex matched subjects from whom blood was drawn to measure paraoxonase-1 activity manually using spectrophotometer, malondialdehyde by thiobarbutric acid method. Liver function tests- bilirubin, total protein, albumin, alanine transaminase, alkaline phosphatase and lipid profile were measured using clinical chemistry auto analyzer. Results and observations: Serum paraoxonase-1 activity, total protein, albumin levels, high-density lipoproteins are decreased and malondialdehyde, bilirubin, alanine transaminase and alkaline phosphatase are increased in patients with chronic liver disease. Conclusion: Serum PON1 activity has decreased significantly & MDA levels were increased significantly in chronic liver disease. Determination of PON1 activity may serve as a useful marker to assess severity of chronic liver disease.

KEYWORDS Chronic liver disease, high-density lipoprotein, malondialdehyde, paraoxoanase-1

INTRODUCTION

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Chronic liver diseases are slow, progressive diseases characterized by advancing hepatocellular necrosis, inflammation and fibrosis. WHO estimates that about 3% of the worldâ&#x20AC;&#x2122;s population has been infected with Hepatitis C Virus and more than 170 million chronic carriers who are at risk of developing liver cirrhosis and hepatocellular carcinoma. Oxidative stress and inflammation plays a fundamental role in the onset and development of liver diseases. Oxygen free radicals cause lipid

peroxidation leading to destruction of PUFA producing toxic metabolites such as malondialdehyde (MDA) which is commonly used as a marker of lipid peroxidation.The ubiquitous presence of antioxidant enzymes may represent an important defence mechanism in diminishing the burden of the pro-oxidant stimuli. Paraoxonase-1 (PON1) is an enzyme synthesized in liver and has lactonase and esterase activities towards lipid peroxides and circulates in plasma bound to high-density lipoproteins (HDL)1.

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www.ijpbs.com (or) www.ijpbsonline.com MATERIAL AND METHODS The study was conducted in department of biochemistry, S.S Institute of Medical Sciences & Research Centre, Davangere between June 2010 to May 2011. Patients admitted to S.S Institute of Medical Sciences were enrolled in the study.

a. Subjects: The study consisted of a total of 80 subjects, 40 patients with chronic liver disease 40 healthy controls. Based on the etiology of the liver disease patients were divided into two groups [Table 1]. CLD was diagnosed based on clinical evidence, radiography, laboratory investigations.

Table 1: Division of patients in to 2 groups based on etiology of liver disease

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Groups Group I - Chronic viral hepatitis Group II - Cirrhosis Exclusion criteria: patients with diabetes, neoplasia, renal disease and cardiovascular disease were excluded from the study. b. Collection of blood samples After 12 hours of fasting about 5ml of blood was drawn under all aseptic precautions into plain vacutainers from the antecubital veins of healthy controls and patients. The blood was allowed to clot for about 30 minutes and then the serum was separated by centrifugation at 5000 rpm and stored at 40C until the analysis. c. Biochemical determinations Paraoxonase assay PON1 activity was estimated spectrophotometrically by the method using pnitrophenylacetate as a substrate. The increase in the absorbance at 412 nm due to formation of p-nitrophenol was measured. Briefly, the assay mixture consists of 3ml of 20mM/L Tris-HCl buffer, pH 8.0 containing 1 m Mol CaCl2, 15 mg of p-nitrophenylacetate dissolved in 0.5 ml of absolute ethanol & 50 Âľl of fresh serum. After mixing the contents kinetic measurements were taken immediately at every minute for 5 minutes at 412 nm at 250C. First absorbance reading is taken as zero minute reading and subsequent absorbance readings were obtained by subtracting one minute reading with zero minute reading, likewise the latter minute reading was subtracted from previous minute readings. The

No of patients 15 25 mean absorbance was calculated. Mean absorbance was used to determine PON1 activity. PON1 activity is expressed as Units/ml of serum i.e. 1 U= 1 nanomole of p-nitrophenol formed per minute2. Measurement of Serum MDA Concentration Serum MDA levels were measured according to a method described elsewhere. The principle of the method was based on the spectrophotometric measurement of the color obtained during the reaction of thiobarbituric acid with MDA. Concentration of thiobarbituric acid reactive substances (TBARS) was calculated by the absorbance coefficient of MDAthiobarbituric acid complex and expressed in nmol/ml3. Standard liver function and fasting lipid profile tests Serum total and direct bilirubin, alanine transaminase (ALT), alkaline phosphatase (ALP), total protein (TP), albumin, total cholesterol (TC), high density lipoprotein-cholesterol (HDLC-C), triglycerides (TAG) levels were determined using clinical chemistry analyzer (Erba 360). LDLcholesterol (LDL-C) values were derived from Friedewald formula4. d. Statistical analysis Statistical analysis was performed using statistical package for social sciences [SPSS-16]. The results were expressed as mean Âą standard

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RESULTS A total of 40 cases and 40 controls were studied. As shown in Table 2 & Table 3: The mean age (years) in controls was [42.23±11.67] and cases [40.87±15.10] in chronic hepatitis & [47.56±11.03] years in cirrhosis. Total bilirubin in controls was [0.75±0.21] as compared to cases [5.24±0.60 in chronic hepatitis 5.11±0.234 in cirrhosis]. Total protein in controls was [6.81±0.57] as compared to cases [5.71±0.48] in chronic hepatitis & [5.56±0.42] in cirrhosis. Albumin in controls was [3.95±0.41] as compared to cases [3.20±0.55] in chronic hepatitis & [2.84±0.59] in cirrhosis. ALT in

controls was [24.88±11.27] as compared to cases [123.8±12.94] in chronic hepatitis & [117.24±48.3] in cirrhosis. ALP in controls was [89.40±26.8] as compared to cases [177.8±22.18] in chronic hepatitis & [195.60±87.7] in cirrhosis. LDL cholesterol in controls was [137±2.3] as compared to cases [86 ±16.4] in chronic hepatitis & [80±12.3] in cirrhosis. HDL cholesterol in controls was [46±1.8] as compared to cases [40±1.5] in chronic hepatitis & [39±2.3] in cirrhosis. Total cholesterol in controls was [186±23.1] as compared to cases [138±13.5] in chronic hepatitis & [132±12.6] in cirrhosis. Table 4 & Figure 1: Serum MDA in controls was [2.17±0.56] as compared to cases [7.52±0.41] in chronic hepatitis & [8.67±1.83] in cirrhosis. Serum PON1 activity in controls was [175.80±24.30] as compared to cases [69.67±10.86] in chronic hepatitis & [59.14±11.9] in cirrhosis.

Table 2: Results of Standard liver function tests & Lipid profile of healthy controls and patients with CLD. Healthy controls

Chronic Hepatitis

Cirrhosis

p Value

Total bilirubin [mg/dl]

0.75±0.21

5.24±0.60

5.11±0.234

<0.001

Total protein [g/dl]

6.81±0.57

5.71±0.48

5.56±0.42

<0.001

Albumin [g/dl]

3.95±0.41

3.20±0.55

2.84±0.59

<0.001

ALT [U/L]

24.88±11.27

123.8±12.94

117.24±48.3

<0.001

ALP [U/L]

89.40±26.81

177.8±22.18

195.60±87.7

<0.001

LDL cholesterol

137 ± 2.3

86 ± 16.4

80 ±12.3

0.025

HDL cholesterol

46 ±1.8

40 ± 1.5

39 ± 2.3

0.042

Total cholesterol

186 ± 23.1

138 ±13.5

132 ± 12.6

0.030

Triglycerides

183 ± 13.2

83 ± 5.6

79 ± 6.8

0.012

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Variables

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Table-3: Demographic, PON 1 status, MDA in healthy controls and patients with CLD Variables

Healthy controls (n=40) 42.23±11.67 28/12 0.75±0.21 6.81±0.57 3.95±0.41 24.88±11.27 89.40±26.81 175.80±24.30 2.17±0.56

Age(years) Sex (M/F) Total bilirubin(mg/dl) Total protein (g/dl) Albumin (g/dl) ALT (U/L) ALP (U/L) PON1(U/L) MDA(µmol/L)

Chronic hepatitis (n=15) 40.87±15.10 11/4 5.24±0.60 5.71±0.48 3.20±0.55 123.8±12.94 77.8±22.18 69.67±10.86 7.52±0.41

Cirrhosis (n=35) 47.56±11.03 22/3 5.11±0.234 5.56±0.42 2.84±0.59 117.24±48.3 195.60±87.7 59.14±11.9 8.67±1.83

P value

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Table-4: PON1 status and MDA in chronic hepatitis and cirrhosis Variables

Chronic hepatitis

Cirrhosis

P value

PON1(U/L) MDA (µmol/L)

69.67±10.86 7.52±0.41

59.14±11.98 8.67±1.83

<0.008 <0.02

Figure 1: Graphical representation of PON1 activity and MDA levels in Healthy controls and patients with chronic liver disease.

200.00 150.00

100.00

PON1

50.00

MDA

0.00 healthy control

Chronic Hepatitis

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DISCUSSION Chronic liver diseases are slow, progressive diseases characterized by advancing hepatocellular necrosis, inflammation and fibrosis. WHO estimates that about 3% of the world’s population has been infected with HCV and more than 170 million chronic carriers who are at risk of developing liver cirrhosis and Hepatocellular Carcinoma. Oxidative stress and inflammation plays a fundamental role in the onset and development

Cirrhosis

of liver diseases. Oxygen free radicals cause lipid peroxidation leading to destruction of PUFA producing toxic metabolites such as malondialdehyde which is commonly used as a marker of lipid peroxidation. MDA can also initiate the formation of protein-aldehyde adducts. They are seen predominantly in the perivenous region and they coincide with signs of more advanced liver injury. It has been reported that patients with degenerative liver

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www.ijpbs.com (or) www.ijpbsonline.com disease had increased lipoperoxide levels in liver tissue and serum5. Recently serum PON1 has been studied extensively in relation to cardiovascular diseases, whereas in contrast, there is a paucity of data on hepatic enzyme.PON1 activity has been observed in rat and human hepatic microsomes. Some of this enzyme is secreted into the circulation bound to HDL where as another portion is stored in the liver 6, 7, 8. The physiologic role played by PON1 in the liver is unknown although preliminary observations suggest that this enzyme provides hepatic protection against oxidative stress. In the present study, the decrease in PON1 activity in serum of patients with chronic liver disease was related to the degree of liver damage. Previous authors have proposed two mechanisms to explain the decrease in activity in PON1 in liver disorder patients. First, as there is hepatic dysfunction, it is obvious that there is defective gene expression, which contributes to decreased PON1 in these patients. It has been reported that there was significant decrease in PON1 activity in Carbon tetra chloride induced liver cirrhosis secondary to increased free radicles9. Second, as a consequence of an altered synthesis and/or secretion of HDL-C, this may be due to impaired lecithin: cholesterol acyl transferase (LCAT) activity. We have observed positive correlation between PON1 activity and HDL-C levels in patients chronic liver disease. Several workers have proposed earlier that viral hepatitis is associated with oxidative stress. Further, PON1 activity associated with HDL-C in plasma is thought to protect LDL-C oxidation10. A previous study has stated that decrease in PON1 activity in patients with chronic liver diseases such as chronic hepatitis and cirrhosis, was related to degree of liver damage. Recently, Keskin et al. also have reported reduced baseline

and stimulated PON1 and arylesterase (ARE) activities in patients with CLD. Currently it is widely accepted that sensitivities of standard biochemical tests for liver function are low and insufficient for a reliable determination of the presence or absence of liver disease. Consequently a battery of tests to be performed to increase the sensitivity and specificity of the evaluation11. Results of our study demonstrate that serum PON1 activity measurement may add a significant contribution to liver function test. Its diagnostic accuracy is equivalent to that of ALT in patients with chronic hepatitis and far superior to that of other tests in patients with cirrhosis12.

CONCLUSION The results of our study suggests that there were higher oxygen free radicals production as evidenced by higher MDA and lower PON1 activity, supporting that there is increased oxidative stress in patients with CLD & decreased PON1 activity supports the decreased detoxification in CLD. Determination of PON1 activity may serve as useful additional test in assessing degree and severity of CLD.

STUDY LIMITATIONS Sample size in the present study was small. Large prospective studies in Indian population are needed to support the results of present study.

ACKNOWLEDGMENT We would like to thank all the staff, postgraduates and the technical staff of our department for their co-operation.I would specially thank my parentâ&#x20AC;&#x2122;s smt Aruna & Jagannatha for their support.

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REFERENCES 1.

Jordi Camps, Judit Marsillach, Jorge Joven. Measurement of serum paraoxonase-1 activity in the evaluation of liver function. World J Gastroenterol 2009; 15(16): 1929-33. Sarkar PD, Shivaprakash TM, Madhusudhan B. Association between Paraoxonase activity and lipid levels in patients with premature coronary artery disease. Clin Chim Acta. 2006; 373:77-1. Jain SK. Evidence for membrane lipid peroxidation during the in vivo aging of human erythrocytes. Biochem Biophys Acta 1988; 937:205-10. Friedwald WT, Levy RI, Fredrickson DS.Estimation of the concentration of low-density lipoprotein cholesterolin plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18:499-502. Hulya Aksoy, Mehmet Koruk, Fatih Akcay. The relationship between serum malondialdehyde and ceruloplasmin in chronic liver disease. Turk J Biochem 2003;28:32-4 Ruiz J, Blanche H, James RW, Blatter Garin MC, Vassie C. Gln-Arg192 polymorphism of paraoxonase and coronary heart disease in type 2 diabetes. Lancet 1995; 346:869-72. Gil F, Pla A, Gonzalvo MC, Hernandez AF, Villanueva E. Partial purification of Paraoxonase from rat liver. Chem Biol Interact 1993; 87:69-5. Gonzalvo MC, Gil F, Hernandez AF, Rodrigo L, Villanueva E, Pla A. Human liver Paraoxonase [PON1] :

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Subcellular distribution and characterization. J Biochem Mol Toxicol 1998; 12:61-9. Ferre N, Campus J, Cabre M, Paul A, Joven J. Hepatic Paraoxonase activity alterations and free radical production in rats with experimental cirrhosis. Metabolism 2001; 9:997-1000. Sabesh SM, Hawkins HL, Kuiken L, Ragland JB. Abnormal plasma lipoproteins and lecithin-cholesterol acyltransferase deficiency in alcoholic liver disease. Gastroenterology 1977; 72:510-18. Preisig R, Tygstrup N, Price C. Assessment of liver function. 3rd ed. London: WB Saunders. 1992:461-75. Natalia Ferre, Jordi Camps, Eduard Prats, Elisabet Vilella, Antoni Paul. Serum Paraoxonase activity: A new additional test for the improved evaluation of chronic liver damage. Clinical chemistry;48:261-68 Keslo GJ, Stuart WD, Richter RJ, Furlong CE, JordanStrack TC, Harmony JA. Apolipoprotein J is associated with Paraoxonase in human plasma. Biochemistry 1994; 33:832-39. Judit Marsillach, Gerard Aragones, Bharti Mackness, Anna Rull, Raul Beltran-Debon. Decreased Paraoxonase-1 activity is associated with alterations of high-density lipoprotein particles in chronic liver impairment. Lipids in health and disease 2010; 9:46. Mevlut Baskol, Gulden Baskol, Kemal Deniz, Omer Ozbakir. A new marker for lipid peroxidation: Serum Paraoxonase activity in non-alcoholic steatohepatitis. Turk J Gastroenterol 2005; 16:119-23.

*Corresponding Author:

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Dr.SUSHMA B JAGANNATHA* Department of Biochemistry,S.S Institute of Medical Sciences Davangere-577004, Karnataka, India.

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Research Article Biological Sciences SERUM FERRITIN - A POTENTIAL THREAT AND RISK FACTOR FOR ACUTE MYOCARDIAL INFARCTION Bharathi B.K.*1 and Shrikant chandrakar2 Department of Biochemistry, J. J. M. Medical College, Davangere -577004, Karnataka, India. *Corresponding Author Email: shri99xx@gmail.com

ABSTRACT Introduction: Elevated body iron stores have been suggested to be a risk factor for the atherosclerosis and ischemic heart disease but the results of epidemiological studies that relate iron stores to risk of coronary heart disease have been inconsistent. Objectives: (1) To study the relationship of serum ferritin with acute myocardial infarction(AMI) in univariate analysis. (2)To assess the relationship of increased serum ferritin with established risk factor for AMI. Materials and Methods: Hospital based study of 30 AMI cases and 30 age and sex matched controls without having AMI in the age group of 40-80 years. Results: Median serum ferritin levels were significantly associated higher in cases (325.5µg/L) as compared to controls (65.5µg/L) (P<0.001). In univariate analysis in addition to ferritin >200µg/L (Odds Ratio=11.67, 95%Confidence Interval=3.39-40.23,P<0.001), Diabetes, Hypertension, Smoking, Alcohol, BMI, Total Cholesterol, Triglyceride, HDL, LDL, CPK, SGOT, and LDH were found to be significantly associated with AMI. Conclusion: Increased ferritin level is strongly and independently associated with AMI.

KEYWORDS Acute myocardial infarction, Serum ferritin, Lipid profile, Cardiac markers

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INTRODUCTION The association of high iron stores and coronary heart disease was first suggested by Sullivan1 after that several observational and epidemiological studies have identified many new emerging potential risk factors like elevated blood levels of triglycerides, atherogenic lipoproteins fibrinogen, Homocysteine and number of genetic polymorphism are of particular interest. But apart from these there is strong evidence that oxidative free radicals have a role in the development of degenerative disease including coronary heart disease2, 3. Thus oxidized low density lipoprotein (LDL) exerts several potentially atherogenic effects 4, 5, 6 (1) is chemotectic for monocytes (but not neutrophils). (2) is cytotoxic to endothelial cells and (3) Rapidly taken up by macrophages

through the scavenger receptor on these cells reproducing in vitro the appearance of atherosclerotic plaque foam cells. Iron is a transition metal that can catalyze toxic redox reactions, and it has been suggested to be involved in many harmful biological processes and disease in the human body 7,8. Excessive iron has been proposed to be a potent risk factor for coronary heart disease, especially for acute myocardial infarction (AMI) 1,8-10. Supporting evidence comes from invitro lipid peroxidation and lipoprotein modification studies11-13, from cholesterol fed iron overloaded animal models14, 15 and from analysis of the composition of human atherosclerotic lesions 16,17. Since serum ferritin concentrations are directly proportional to intracellular ferritin concentration, it is considered to be the best clinical measure of

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body iron stores 18 , and the most feasible to use in epidemiologic studies19. The main objective of our study was to study the relationship of serum ferritin with acute myocardial infarction in univariate analysis and to assess the relationship of increased serum ferritin with established risk factor like smoking, alcohol intake, body mass index, hypertension, lipids, diabetes mellitus.

MATERIAL AND METHODS

All subjects were assessed by clinical examination ECG, serum- creatine kinase MB function. Height and weight were recorded. Body mass index was calculated by formula weight in kg/ height2 in meter. BMI > 25 was considered as risk factor for acute myocardial infarction. Cases and controls were investigated for conventional risk factors (lipid profile, BMI, Blood sugar).Venous blood was collected and centrifuged at 3000rpm for 10 minutes and serum was used immediately for the estimation of serum was estimated by using acculite ferritin CLIA microwells test and estimation of lipid profiles and cardiac marker by enzymatic method using Roche 400 autoanalyser.

Inclusion criteria: 30 consecutive cases of acute myocardial infarction admitted to the intensive cardiac care unit of Bapuji hospital. Diagnosis of acute myocardial infarction was based on fulfilling two of the following criteria: 1. Chest pain of <12 hrs duration. RESULTS 2. ST elevation >1mm in at least 2 consecutive leads. A total of 30 cases and 30 controls were studied. 3. Increased cardiac markers The mean age of controls and cases was similar 4. New onset bundle branch block. (56.5±8.8 years and 57.1±9.8 years) (age range Exclusion criteria: Cases with high ferritin levels 40-80 years). Males outnumbered females with a like hemochromatosis, liver disease, ratio of 1.5:1. Mean haemoglobin in cases and tuberculosis, chronic inflammatory disease, controls was similar (13.48 g% and 13.56 g %), those on iron therapy, past h/o of acute since they were matched for haemoglobin. The myocardial infarction or CHD. median serum ferritin values were significantly Controls: 30 age, gender and haemoglobin higher in cases (325.5 μg/L) as compared to matched control was recruited for each case controls (65.5 μg/L), (P < 0.001). Even median irrespective of presence of risk factors( serum CPK, SGOT, and LDH levels are hypertension, diabetes mellitus, smoking and significantly increased in cases as compared to alcohol intake) but without having acute controls. (Table 1 & Figure 1). myocardial infarction(in the past or present ) or any evidence of CHD assessed by symptoms, clinical examination and normal ECG. Table 1: Comparison of median serum ferritin and cardiac markers levels in cases and controls. Cases 325.5 483 252 408

Controls 65.5 139.5 29.2 211

P value <0.001 <0.001 <0.001 <0.001

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Variables Median serum Ferritin(µg/L) Median serum CPK (IU/L) Median serum SGOT (IU/L) Median serum LDH (IU/L)

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Figure 1: Bar graph showing increased median serum Ferritin, CPK, SGOT, and LDH in cases as compared to controls. Cases Controls

500 400 300 200 100 0 Median serum Ferritin(µg/L)

Median serum CPK (IU/L)

Median serum SGOT (IU/L)

Median serum LDH (IU/L)

The mean value of serum ferritin (μg/L) in controls and cases were found to be 96.3 ± 69.5 and 408.7± 252.8, respectively (P < 0.001). There was no significant difference of mean serum ferritin levels in males and females. High serum ferritin levels (> 200 μg/L) was significantly associated with AMI (OR = 11.67 (95% CI 5.37–74.5, P < 0.001) (Table 2). Table 2: Association of acute myocardial infarction with high serum ferritin and high serum cardiac markers. Variable Serum ferritin ≥200µg/L <200µg/L

Cases

Controls

Total

25 5

9 21

34 26

Serum CPK ≥195(IU/L) <195(IU/L)

22 8

5 25

27 33

Serum SGOT ≥37(IU/L) <37(IU/L)

25 5

11 19

36 24

Serum LDH ≥300(IU/L) <300(IU/L)

22 8

7 23

29 31

P value X2 -17.37 O.R -11.67 C. I -5.37-74.5 P <0.001 X2 -19.46 O.R -13.75 C. I -3.92-48.3 P <0.001 X2 -13.61 O.R -8.64 C. I -2.57-29.07 P <0.001 X2 -15.01 O.R -9.036 C. I -2.8-29.1 P <0.001

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In univariate analysis, alcohol intake, BMI, DM, hypertension, serum cholesterol, serum triglyceride, high-density lipoprotein (HDL) < 35 and smoking were found to be significantly associated with AMI (Table 3).

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TABLE-3: Comparison of conventional risk factors for myocardial infarction in cases and controls (Univariate Analysis) Cases

Controls

19 11

8 22

Hypertension Present Absent

18 12

6 24

Serum cholesterol ≥200mg/dl <200 mg/dl

17 13

5 25

Serum triglyceride ≥160 mg/dl <160 mg/dl

20 10

6 24

Serum LDL ≥150 mg/dl <150 mg/dl

18 12

5 25

Serum HDL <35 mg/dl ≥35 mg/dl

17 13

7 23

BMI ≥25Kg/m2 <25 Kg/m2

24 6

5 25

Alcohol intake Present Absent

15 15

5 25

Smoking Present Absent

24 6

7 23

P value X2 -8.148 O.R -4.75 C. I -1.58-14.25 P < 0.005 X2 -10 O.R -6 C. I -2.08-17.29 P < 0.005 X2 -10.33 O.R -6.54 C. I -1.97-21.74 P < 0.005 X2 -13.3 O.R -8 C. I -2.45-25.9 P < 0.001 X2 -11.6 O.R -7.5 C. I -2.24-25.1 P < 0.001 X2 -6.94 O.R -4.297 C. I -1.41- 13.1 P <0.01 X2 -24.1 O.R -20 C. I -5.38- 74.29 P < 0.001 X2 -10.33 O.R -6.54 C. I -1.97-21.74 P < 0.005 X2 - 19.29 O.R -13.14 C. I -3.84-45.01 P < 0.001

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Variable Diabetes mellitus Present Absent

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DISCUSSION Epidemiological studies have found a positive relationship between body iron stores and coronary artery diseases9, 20. Subsequently, evidence of an association of elevated serum ferritin and increased risk of AMI came from various authors 10, 21, which is similar to our findings. However results of some other studies did not show significant correlation between high ferritin and risk of AMI 22, 23. The main possibility that iron over load leads to increased lipid peroxidation and foam cell formation but apart from this the chemical properties of oxidized lipoproteins were found to be chemotactic to blood monocytes, facilitate the entry of lipoproteins by a cytotoxic endothelial injury, and give rise to smooth muscle cell proliferation 4,24,25. Native low density lipoprotein in contrasts lacks all these atherogenic potentials4, 25. One study yielded a strong relation between sonographically assessed carotid atherosclerosis and prominent iron stores in both genders particularly when associated with hypercholesterolemia 26. Lipid peroxidation therefore may constitute an initiating and crucial step in the development of fatty streaks and plaques. Blood donation has also been reported to be associated with decreased risk of cardiovascular events 27. High ferritin levels have been associated with established conventional risk factors like diabetes mellitus and hypertension by various authors 28,29. Reduced extraction of hepatic with increasing iron stores leading to peripheral hyper insulinemia was the proposed mechanism for diabetes mellitus30 and pronounced metabolic alteration is the proposed mechanism for high ferritin hypertensive 29. Significant association of LDL cholesterol and ferritin was also reported previously21,31. High ferritin levels have been observed in smokers. Cigarette smoke mediated iron mobilisation from ferritin and represents

specific pro-oxidant mechanism related to smoking32, 33.

CONCLUSION Our present findings suggest that there is strong and independent relationship of high serum ferritin with acute myocardial infarction and serum ferritin was significantly high in diabetics and smokers.

STUDY LIMITATIONS Sample size in the present study was small. Large prospective studies in Indian population are needed to support the results of present study. ACKNOWLEDGMENT We would like to thank all the staff, postgraduates and the technical staff of our department for their co-operation. I would specially thank Dr. Raginee Chandrakar & Chitralekha and A.R. Chandrakar for their support.

REFERENCES 1. 2. 3. 4.

Sullvian JL. Iron and sex difference in heart disease risk. Lancet 1981; 1:1293–4. Aviram M. Modified forms of low density lipoproteins and atherosclerosis. Atherosclerosis 1993; 98:1–9. Oliver MF. Antioxidant nutrients, atherosclerosis and coronary heart disease. Br Heart J 1995; 73:299–301. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low density lipoprotein that increase its atherogenicity. N Engl J Med. 1989; 320:915-924. Esterbauer H, Dieber-Rotheneder M, Waeg G, Striegl G, Jurgens G: Biochemical, structural, and functional properties of oxidized low-density lipoprotein. Chem Res Taxicol 1990; 3:77-92. Montgomery RR, Nathan CF, Cohn ZA: Effects of reagent and cell-generated hydrogen peroxide on the properties of low density lipoprotein. Pmc Natl Acad Sci USA 1986; 83:6631-6635. Gutteridge JM, Halliwell B. Iron toxicity and oxygen radicals. Baillieres Clin Haematol. 1989; 2:195–256.

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21. Delphine W, Silvia CR, Biswas S, Uthappa S, Shetty P. Ferritin—a potent threat for acute myocardial infarction. J Assoc Physicians (India) 2003; 51:947–50. 22. Reunanen A, Takkunen H, Knekt P, Seppanen R, Aromaa A. Body iron stores, dietary iron intake and coronary heart disease mortality. J Intern Med 1995; 238:223–30. 23. Gupta R, Rastogi S, Nagar R, Kastia S, Kaul V. Dietary and serum iron, body iron stores and coronary heart disease. J Assoc Physicians (India) 2000; 48:489–92. 24. Boulanger CM, Tanner FC, Bea ML, Hahn AWA, Werner A, Luscher TF. Oxidized low density lipoproteins induce mRNA expression and release of endothelin from human and porcine endothelium. Ore Res. 1992; 70:1191-1197. 25. Quinn MT, Parthasarathy S, Fong LG, Steinberg D. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/ macrophages during atherogenesis. Proc Natl Acad Sci USA. 1987; 84:2995-2998. 26. S Kiechl, F Aichner, et al. Body iron stores and the risk of carotid atherosclerosis. Prospective results from the Bruneck study. 27. Meyer DG, Strickland D, Maloeley PA, Seburg JJ, Wilson JE, McManus BF. Possible association of a reduction in cardiovascular events with blood donation. Heart 1997; 78:188 93. 28. Haidari M, Javadi E, Sanati A. Association of increased ferritin with premature coronary stenosis in men. Physiol Genomics 2003; 13:25–30. 29. Piperno A, Trombini P, Gelosa M. Increased serum ferritin is common in men with essential hypertension. J Hypertension 2002; 20:1513–8. 30. Niedery C, Berger M, Stremmet W. Hyperinsulinemia innon cirrhotic haemochromatosis impaired hepatic insulin degradation. Diabetologica 1984; 26:441–4. 31. Karml P, Potockova J, Koprivova H, et al. Ferritin oxidative stress and coronary atherosclerosis. Vintr Lek 2004; 50:183–5. 32. Lapenna D, De Givia S, Mezzette A. Cigarette smoke, ferritin, lipid peroxidation. Am J Respir Crit Care Med 1995; 151:431–519. 33. Grobusch KK, Koster JF, Grobbee DE. Serum ferritin and risk of myocardial infarction in the elderly; Rotterdam study. Am J Clin Nutr 1999; 69:1231–6.

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Halliwell B, Gutteridge JM. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol. 1990; 186:1– 85. Salonen JT, Nyyssonen K, Korpela H, Tuomilehito J, Seppanen R, Salonen R. High stored iron levels are associated with excess risk of myocardial infarction in Eastern Finnish men. Circulation 1992; 86:803-11. Salonen JT, Nyyssonen K, Salonen R. Body iron stored and the risk of coronary heart disease. NEJM 1994; 331:1159–60. Salonen JT, Yla-Herttuala S, Yamamoto R, et al. Autoantibody against oxidized LDL and progression of carotid atherosclerosis. Lancet 1992; 339:883-7. Balla G, Jacob HS, Eaton JW, Belcher JD, Vercellotti GM. Hemin: a possible physiological mediator of low density lipoprotein oxidation and endothelial injury. Arterioscler Thromb. 1991; 11:1700 –1711. Witztum JL. The oxidation hypothesis of atherosclerosis. Lancet. 1994; 344:793–795. Weiner MA, Paige SB, Bailey SR. Antioxidant therapy decreases atherosclerotic plaque burden in an iron loaded animal model. Eur Heart J. 1994; 425(suppl 15):2241. Abstract. Araujo JA, Romano EL, Brito BE, Parthe V, Romano M, Bracho M, Montano RF, Cardier J. Iron overload augments the development of atherosclerotic lesions in rabbits. Arterioscler Thromb Vasc Biol. 1995; 15:1172–1180. Smith C, Mitchinson MJ, Aruoma OI, Halliwell B. Stimulation of lipid peroxidation and hydroxyl-radical generation by the contents of human atherosclerotic lesions. Biochem J. 1992; 286:901–905. Swain J, Gutteridge JM. Prooxidant iron and copper, with ferroxidase and xanthine oxidase activities in human atherosclerotic material. FEBS Lett. 1995; 368:513–515. Cook JD, Lipschitz DA, Miles LEM, Finch CM. Serum ferritin as a measure of iron stores in normal subjects. Am J Clin Nutr 1974; 27:681-7. Beaton GH, Corey PN, Steele C. Conceptual and methodological issues regarding the epidemiology of iron deficiency and their implications for studies of the functional consequences of iron deficiency. Am J Clin Nutr 1989; 50:575-85. Solymoss BC, Marcil M, Gilfix BM. The place of ferritin among risk factors associated with coronary artery disease. Coron Artery Dis 1994; 5:231–5.

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Dr. Bharathi B.K.*1 & Dr. Shrikant chandrakar2 1- Professor, 2- Post graduate student, Department of Biochemistry,J.J.M. Medical College, Davangere-577004, Karnataka, India.

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Research Article Biological Sciences STUDY OF SERUM IRON, TIBC, TRANSFERRIN SATURATION AND FERRITIN IN IRON DEFICIENCY ANEMIA IN TERTIARY CARE HOSPITAL A. Veena 1a*, Amit D. Sonagra2b, Rekha M.3b, Jayaprakash Murthy D.S.4b a

Department of Biochemistry, S.S. Institute of Medical Science, Davangere, Karnataka (India). b Department of Biochemistry, JJM Medical College, Davangere-577004, Karnataka (India). *Corresponding Author Email: veena.karatagi@gmail.com

ABSTRACT Background: Amongst anemia, Iron deficiency anemia is the most common nutritional deficiency disorder in the world. Iron deficiency leads to giddiness, anorexia, decreased defense activity of the body, decreased alertness, cerebral ischaemia, menstrual irregularities leading to maternal morbidity and mortality. Objectives: To estimate serum iron, TIBC, transferrin saturation and ferritin in iron deficiency anemic patients. Materials & methods: 30 anemic patients were selected, 20 controls were taken and ferrokinetic study was done with commercially available kits. Statistical analysis was done by student â&#x20AC;&#x153;tâ&#x20AC;? test to study serum iron, transferrin and serum ferritin levels in iron deficiency subjects. Results: There was decreased serum iron, transferrin saturation and serum ferritin and increased total iron binding capacity in iron deficient patients. Conclusion: Estimation of serum iron, TIBC, transferrin saturation and ferritin gives accurate analysis of patients iron status and helps to take necessary interventions in planning, treating and thus prevents the risk of adverse events.

KEYWORDS Ferritin, Iron deficiency anemia, serum iron, total iron binding capacity, transferrin saturation %.

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INTRODUCTION Anemia constitutes a common problem in clinical practice and hematological Laboratories. It is neither a diagnosis in itself nor a specific entity but a manifestation of an underlying disease process which is often related to the severity of the disease process. [1] Iron deficiency is the most common nutritional deficiency disorder in the world, reported to affect 50-60% of young children and pregnant females & 20-30% of non-pregnant females in developing countries. It is the most common microcytic anemia. The consequences of iron deficiency are numerous as iron plays a central part in the transport of oxygen in the body and is also essential in many enzyme systems like cytochrome oxidase, xanthine oxidase. Iron

deficiency affects neurotransmitter systems in brain causing changes in behavior such as attention, memory and learning in infants & small children. It also negatively influences the normal defense system against infection. In pregnant women, iron deficiency contributes maternal morbidity & mortality & increases risk of fetal morbidity, mortality & low birth weight.[2] In children it is frequently caused by dietary deficiency, because milk has low iron content and in adults it is almost always the result of chronic blood loss or child bearing. Iron deficiency anemia develops when there is inadequate iron for hemoglobin synthesis and is the result of an imbalance between iron assimilation and iron loss. [3]

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www.ijpbs.com (or) www.ijpbsonline.com Iron absorbed from diet or released from stores circulates in the plasma bound to transferrin, the iron transport protein. The half-clearance time of transferrin-bound iron is very rapid  60-90 min. Most of iron transported by transferrin is delivered to the erythroid marrow. So, when erythropoesis is markedly stimulated, the pool of erythroid cells requiring iron increases and the clearance time of the iron from the circulation decreases. The half-clearance time of iron in the presence of iron deficiency is very short i.e. 10-15min. [5] The balance of iron in human is tightly controlled and conserve iron for reutilization. Iron is lost from the body by blood loss (via gastrointestional bleeding, menses or other forms of bleeding), loss of epithelial cells from the skin, gut and genito-urinary tract. Only route by which iron comes into the body via absorption from food or from medicinal iron. Iron also enters the body through red cell transfusion or injection of iron complexes. Amount of iron required from the diet to replace losses averages about 10% of body iron content a year in men & 15% in women of child bearing age. Iron bioavailability is affected by the nature of the food stuff, with heme iron (e.g. red meat) being most readily absorbed. Infants, children and adolescents require additional iron due to demands of body growth, lower dietary intake of iron. During the last two trimesters of pregnancy, daily iron requirement increases to 5-6mg. [4, 5] The progression to iron deficiency can be divided into three stages - (Fig 3): [6] 1) First stage: Negative Iron balance where demand of iron exceed the body’s ability to absorb iron from the diet. (Blood loss, pregnancy where the demands for red cell production by the fetus outstrip the mother’s ability to provide iron, rapid growth spurts in the adolescent, or inadequate dietary iron intake). During this

period- serum ferritin levels decrease. As long as iron stores are present and can be mobilized the serum iron, total iron binding capacity and red cell protoporphyrin levels remain within normal limits. Red cell morphology and indices are normal at this stage. 2) Second stage: (Iron deficient erythropoesis) when iron stores becomes depleted, when marrow iron stores are absent, when s.ferritin levels <15μg/L, the serum iron begins to fall. Gradualll, the TIBC increases. Once the transferrin saturation falls to 15-20%, hemoglobin synthesis becomes impaired. Evaluation of peripheral blood smear reveals the appearance of microcytic cells.[6] 3) Iron Deficiency Anemia (IDA): Hemoglobin & hematocrit begin to fall. Transferrin saturation is 10-15% in this stage.[5] In severe anemia (7-8g/dl), hypochromia and microcytosis is more prominent, target cells and poikilocytes appear in blood smear.[5] The objective of this study was to study and know the levels of serum iron , TIBC, transferrin saturation% (TSAT) and serum ferritin in relation with Hb and peripheral smear at Bapuji Hospital, Karnataka.

MATERIALS AND METHODS A prospective study was conducted in Iron deficiency anemia subjects from Bapuji Hospital and Chigateri General Hospital, Davangere (both attached teaching hospitals for J.J.M Medical college, Davangere). The controls were selected from surrounding community. Each participant gave an informed consent and this study was approved by the ethical and research committee of J.J.M. Medical College, Davangere to use human subjects in the research study. The patients and controls voluntarily participated in the study.

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www.ijpbs.com (or) www.ijpbsonline.com A) Selection of study subjects Based on inclusion and exclusion criteria a total number of 50 subjects (30 cases and 20 controls) were selected for the present study. Inclusion Criteria used to select the study subjects: Iron deficiency anemia was detected based on clinical history, Hb estimation and peripheral blood smear study. Patients having Hb levels equal to or less than 7 gm% percentage, microcytic hypochromic blood picture, and age group between 20-50 yrs were included in the study. Controls- It included 20 age matched healthy non-anemic people in age group of 20-50 yrs without any major illness and who are not on any medication. Exclusion Criteria: Patients with anemia of age group <20yrs and > 50yrs and anemia of chronic disease were excluded from the study. Based on the inclusion and exclusion criteria, age matched cases and controls were included in the present study after obtaining informed consent. A proforma was used to record relevant information and patient’s data. B) Collection of blood samples: About 5 ml of venous blood was drawn under aseptic precautions in a sterile bulb from selected subjects. Serum was separated by centrifugation and was used for analysis. Serum Iron, Total Iron Binding Capacity (TIBC), Serum Ferritin and TSAt% were estimated.

Serum Iron and TIBC were estimated by Iron and TIBC kit in semiautoanalyzer, (Erba Chem 5plus) which uses Ferrozine method.[7] Transferrin saturation was calculated as Serum iron × 100 / TIBC.[3] Serum ferritin was estimated by Chemiluminescence Immunoassay.[8] Values were calculated as mean ± SD and the statistical analysis was done using SPSS 17.0 software. Student’s unpaired t-test was used to study serum iron, TIBC transferring satutation% and serum ferritin levels in iron deficiency subjects. The p-value of less than 0.05 was considered as statistically significant.

RESULTS Table 2, 3 shows that serum iron, serum ferritin and transferrin saturation% was decreased significantly and TIBC levels were significantly increased as compared to controls. No significant difference was found in Hb percentage. Table 4 shows that maximum cases belong to age group of 31-40 yrs, followed by 41-50 yrs and least in 21-30 yrs group. Also Fig 4 shows that maximum IDA cases are of females i.e 18 cases out of total 30 cases. Table 5 shows that 60% of cases are severly anemic with ferritin levels <6ng/ml. Table 6 shows that maximum cases of IDA had Hb levels in the range of 6.1-7.0g%; followed by 5.1-6.0g%, 4.1-5.0g%, 3.1-4.0g% and 2.1-3.0g% respectively.

Table 2: Showing descriptive information of subjects. Hb%

Serum Iron(µg/dl)

TIBC (µg/dl)

Serum Ferritin(ng/ml)

Transferrin Sat%

Controls

12.3 ± 1.5

76.5 ± 32.5

322.4 ± 32.9

136.8 ± 34.0

24 ± 10.6

Cases

5.3 ± 1.2

28.6 ± 10.3

496.0 ± 99.5

5.1 ± 2.3

6.3 ± 2.7

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Groups

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Table 3: Hb %, serum iron, TIBC, serum ferritin and transferrin saturation in iron deficiency anemic patients. Groups

Hb%

Serum iron (µg/dl)

TIBC (µg/dl)

Serum ferritin (ng/ml)

Transferrin sat%

IDA

5.3 ± 1.2

28.6 ±10.3

496 ± 99.5

5.1 ± 2.3

6.3 ± 2.7

t-value

1.33

6.71

5.76

25.5

6.96

p-level

0.19, ns

<0.001

“t” = unpaired t-test, p <0.05=significant, p> 0.05= not significant, ns=non significant

Table 4: Age distribution in two groups Controls

IDA

Age (yrs) No

21-30

23.3

31-40

46.7

41-50

Total

100

Mean Age

36.8 years

36.6 years

Table 5: Classification of patients with IDA based on severity of ferritin levels. No

Percentage

Moderate (Serum Ferritin 6-10 ng/ml)

Severe (Serum Ferritin <6 ng/ml)

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Severity

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TABLE 6: Study of IDA at various ranges of hemoglobin percentage. IDA Hb percentage levels

2.1-3.0

3.2

3.1-4.0

12.9

4.1-5.0

22.6

5.1-6.0

25.8

6.1-7.0

35.5

Total

100.0

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Figure 1: Iron circulation [4]

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Figure 2: Iron transport in humans.[4]

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Fig 3: Stages of iron deficiency anemia

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DISCUSSION Iron deficiency anemia is one of the most common anemia worldwide accounting for about two billion people. In developed countries, it accounts for about 2-5% of adult men and post menpausal women and is a common cause of referral to gastroenterology clinic.[8] In India, it is a major public health problem especially for infants and pregnant women. Deficiency of iron during developmental stage of brain cause irreversible disturbances and damage to GABA neurotransmitters system thus causing poor learning capacity, varying degrees of impairment in cognitive performance etc.[9] Maximum number of patients in IDA group fall in the Hb range of 6.1 to 7.0 g/dl as compared to 39 patients of Hb range 4.6 to 6.5 g/dl according to Krishna Das [10]. Nutritional inadequacy is the most likely single aetiological factor which is augmented by other factors such as helminthic infestations, blood loss, malabsorption, failure to utilization of

nutrients, increased demand by the body, and dietary interactions which hinders iron absorption. Microcytic hypochromic anemia accounts for 76% and macrocytic anemia 7%. Maternal iron deficiency is reflected in the new born as reduction in the serum iron and lower levels of Hb.[10] Anemia is more common in females. Menstruating, pregnancy and lactation is a form of iron excretion. There is an average loss of 297mg/pregnancy in Indian women. As nearly 150mg of iron are conserved during pregnancy as a result of suppression of menstruation, the average net loss is about 150 mg/pregnancy. This is a recurring loss with each pregnancy and frequently occurs during the adolescent period when their own body need for iron is great due to growth in size of body.[11]. In our study 18 females had IDA out of total 30 cases. There were two peaks in iron deficiency anemia i.e 21-30 yrs and 41-50 yrs, suggesting that majority of patients with iron deficiency anemia were aged between 20-60yrs.

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www.ijpbs.com (or) www.ijpbsonline.com Capillary fragility is increased in iron deficiency. The role of iron in maintaining the integrity of vascular endothelium and mucosal cells is emphasized. In IDA, there is impairment of cell mediated immunity and is demonstrable by defective lymphocyte transformation. Iron deficiency also interferes with cellular immune mechenisms. Myeloperoxidase is reduced in Iron Defeciency. The cell mediated immune response and bactericidal activity of leucocytes are reduced when hemoglobin falls to 10 g/dl or less.[12] The typical Indian diet is based on cereals and pulses which contain more than 40% of total phosphorus as phytates and vegetables and plant food contain oxalates, which interfere with absorption of food iron inspite of high dietary intake.[3] Compared to controls, anemic patients have significantly (p value <0.001) increased levels of TIBC. This is in accordance with studies of Sharma.D.C[3] & Robert Hawkins.C[13]. Trnsferrin saturation % is decreased in IDA cases compared to controls which are in accordance with studies of Sharma. D.C [3], Neeta bahal[14] and Saul Nurko[15]. In IDA patients, there is increase in iron carrying protien transferrin and the amount of iron which is available to bind is reduced causing decrease in transferrin saturation. In circulation, tiny quantity of ferritin is found which in normal and iron depleted subjects, is in close direct co-relation with total body iron reserve[3]. In our study, 18 patients have ferritin levels <6ng/ml accounting for 60% showing that majority of IDA have ferritin levels less than 6ng/ml which is due to multiple factors including dietary deficiency , poor socioeconomic condition, worm infestation, multiple pregnancy , maternal bleeding etc in our region. The remaining 12 patients have moderate IDA with

ferritin levels in range of 6-10ng/ml, accounting for 40%. The lower the TSAT and serum ferritin , the higher the likelihood that a patient is iron deficient, and higher the TSAT, serum ferritin, the lower the likelihood that a patient is iron deficient. Estimation of ferritin is non invasive and an excellent alternative to bone marrow stainable iron and showed a significant positive correlation with it.[3] Complete exhaustion of iron stores is indicated by serum ferritin concentration <12 ng/ml and TSAT <15%.[8] Ferritin is an acute phase reactant and is increased in inflammatory diseases or cirrhosis. Serum ferritin concentration of <12Âľg/dl is diagnostic of iron deficiency.[16] In our study,12 patients belong to moderate IDA with ferritin levels 6-10ng/ml and 18 pateints belong to severe IDA with ferritin levels <6ng/ml which is in accordance with the study by Mohammed Idris[17]. Measurement of serum transferrin receptor is not routinely used in clinical practice as it is expensive and should only be carried out in patients in whom the quantitation of hypochromic red cells is not available or when the determination of serum ferritin , serum iron, transferrin and TSAT does not lead to an accurate classification of type of anemia.[18] Reticulocyte hemoglobin content (CHr) measures Hb entering reticulocytes during terminal differentiation and hence reflects the effectiveness of erythropoesis. It is highly accurate with lowest coefficient of variability, but is a least widely available assay.[8] Serum ferritin is to be used as an indicator of the iron stores rebuilt by i.v iron supplementation and to be determined after 2 weeks of last i.v iron dose.

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www.ijpbs.com (or) www.ijpbsonline.com Serum ferritin levels below 25 ng/ml were associated with lower hemoglobin concentration. Levels above 100ng/ml were not consistently associated with higher hemoglobin concentration.[19] Oral iron therapy is usually the first line therapy for patients with IDA. An increase in the Hb level of 1g/dl (10g/l) should occur every two or three wks on iron therapy and may take up to four months for the iron stores to return to normal after the hemoglobin has corrected. Laxatives, stool softeners and adequate intake of liquids can alleviate the constipating effects of oral iron therapy. Indications for use of intravenous iron include chronic uncorrectable bleeding, intestinal malabsorption, intolerance to oral iron, non adherence, or a hemoglobin level less than 6g/dl with signs of poor perfusion in patients who would otherwise receive transfusion (eg.those who have religious objections). Parenteral iron preparations like iron dextran, iron sucrose, sodium ferric gluconate can be used. Its side effects are anaphylactic reactions and delayed reactions like myalgias, headache, arthralgias which occur 24 to 48 hrs after infusion. NSAIDs are used to relieve these symptoms.[20,21]

ACKNOWLEDGEMENT We acknowledge all the study subjects for their participation in the study and their cooperation.

REFERENCES 1.

4. 5.

7. 8.

10.

11.

CONCLUSION

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13.

14.

15.

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The present study shows decreased serum iron, TSAT and ferritin levels with high levels of TIBC in IDA group whose iron levels may be improved by giving parenteral /oral iron therapy. They can be used as an alternative noninvasive technique to assess iron stores and response to treatment. So, iron level estimations should be done which helps in accurate analysis of patients iron status and thus helps in taking necessary interventions to prevent the risk for adverse events.

MM Eldibany, KF Tonochi: Usefulness of certain red blood cell indices in diagnosing & differentiating thalassemia trait from iron deficiency anemia: Am J Clin Path 111:676-682, (1999). Detels R. Iron deficiency. In: McEmen J, Beaglehole R, Tanakai H (edts). Oxford textbook of public health. Food & Nutrition. 4thed. 1999, pg 152- 153. Sharma D, Mathur R, Singh P. Iron metabolism: A Review. Indian Journal of Clinical Biochemistry, 8(2): 80-101, (1993). WHO. Techn. Rep. Ser. No. 405(1968). Kumar P. Hematological disease. In: Kumar P, Clark M (edts). Kumar & Clarkâ&#x20AC;&#x2122;s Clinical Medicine.7th Ed. Pg.392. Adamson J. Iron deficiency and other hypoproliferative anemias. In: Harrisonâ&#x20AC;&#x2122;s principles of internal medicine. 17th ed, pg 628. Seidel J et al. Clin Chem.30:975, (1984). Hussain A, Tayyab M, Tasneen T, Ahmed N, Chaudhary. serum Ferritin; An indicator of Bone marrow iron stores in hemodialysed patients. Kidney international:152-156,(1999). Goddard A, McIntyre A, Scott B. Guidelines for management of Iron deficiency anemia. Gut 46 (suppl IV): IV I-IV S,(2000). Batra J, Seth P. Effect of iron defeciency on developing rat brain. Indian Journal of Clinical Biochemistry 17(2) : 108-114, (2002). Krishna Das KV. Nutritional anemias in India. Review article. Jr.Asso.Phys.Ind 28:521-533, (1960). Meier P, Olson K, Berg R. Prevetion of iron deficiency in adolescent and adult pregnancies. Clinical Medicine and Research 1(1): 29-36,(2002). Besarab A, Horl W, Silverberg D. Iron metabolism, iron deficiency, thrombocytosis and the cardiorenal anemia syndrome. The Oncologist 14(suppl 1):22-33,(2009). Hawkins R. Total iron binding capacity or transferrin concentration alone outperforms iron and saturation indices in predicting iron deficiency. Clinica Chemica Acta 380: 203-207,(2007). Bahal N, Mara O. Anemia in patients with chronic kidney disease. In brief. Diabetes Spectrum 21(1), (2008).

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www.ijpbs.com (or) www.ijpbsonline.com 16. Nurko S. Anemia in chronic kidney disease:Causes, diagnosis, treatment. Cleveland Clinic Journal of Medicine 73(3): 289-297, (2006). 17. Idris M, Anis-ur-Rehman. Iron deficiency anemia in moderate to severely anemic pts. J Ayub Med Coll Abbottabad: 17(3),(2005). 18. Al-Sayesâ&#x20AC;&#x2122; F, Gari M, Qusti S, Bagation N, Abuzenadah A. Prevalence of Iron deficiency & iron deficiency

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|14-23 anemia among females at University stage. Journal of Medical Laboratory & Diagnosis 2(1) : 5-11,(2011). 19. Lutter C. Iron deficiency in young children in low income countries and new approaches for its prevention. J Nutr: 138: 2523-2528,(2008). 20. Kaltwasser J, Gottschalk R. Erythropoietin and Iron. Kidney International (55): suppl 69: s-49-s-56, (1999). 21. Killip S, Bennet J, Mara C. Iron deficiency anemia. Am Fam Physician: 75:671-8,(2007).

CONFLICT OF INTEREST: Nil FUNDS FOR STUDY: Nil

*Corresponding Author:

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Dr. A. Veena. * Assistant Professor, Department of Biochemistry, S.S. Institute of Medical Science, Davangere, Karnataka. Email id: veena.karatagi@gmail.com

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Review Article Pharmaceutical Sciences IN-VIVO AND IN-VITRO MODELS ON COLON CANCER Ajit B. Patil*1, Asha S. Jadhav2 1

Department of Pharmacology, Tatyasaheb Kore College of Pharmacy, Warananagar. Tal-Panhala, Dist- Kolhapur,416113, (M.S.), India. 2 Dr. D.Y.Patil IPSR College, Pimpri, Pune University, Pune *Corresponding Author Email: abpatil.tkcp@gmail.com

ABSTRACT In-Vivo and In-Vitro models are thought to be the best model’s for to obtain transferable results. The present review aims to give a short overview of the existing in-vivo and in-vitro models and their characteristics. Four different autochthonous tumor models; 1,2-dimethylhydrazine, N-methylnitrosourea, N-mthyl-NꞋ-nitro-Nnitrosoguanidine, N-nitroso-acetoxymethyl-methylamine, are the In-Vivo models and the In-vitro models; studies involving primary cell cultures, studies on cell line, Brine Shrimp Lethality Bioassay, MTT (Microculture Tetrazolium) assay, SRB (Sulphorhod Amine B) assay, Trypan Blue Exclusion and Crystal Violet Inclusion these are used worldwide for cell violability, cell growth studies of colon cancer cell line. The choice of the model for experimental investigation of colon cancer should be depends on the clinical equations.[5]

KEY WORDS In-Vivo and In-Vitro models-Experimental colorectal carcinoma.

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INTRODUCTION Colon cancer is one of the most common malignancies in many regions of the world is thought to arise from the accumulation of mutations in a single epithelial cell of the colon and rectum. The idea that this cancer might be a root cause for chemoprevention stems from epidemiological evidence that some factors in the diet may play important roles in its development, where others may reduce the risk. Experimental Colon carcinogenesis is a multistep process involving three distinct stages, initiation, that alters the molecular message of a normal cell, followed by promotion and progression that ultimately ends up with a phenotypically altered “transformed cell”. In animal studies, treated with a carcinogen, such as, 1,2 dimethylhydrazine (DMH), methylnitrosurea, Nmethyl-N’-nitro-N-nitrsoguanidine will induce colon tumors in experimental animals

particularly in rats and for invitro studies such as, MTT Assay, SRB Assay, Trypan Blue Exclusion Assay requires different cell line.

IN -VIVO TESTING Human cancer is autochthonic (Chemical induced) with the exception of choriocarcinoma and tumors transplanted with an organ from a cancer patients. It seems to that tumors in animals equal in their genesis to those in man are also comparable in biology chemosensitivity. It is accepted today that exogenous chemicals induce most of human cancer. Autochthonic means remaining at the site of formation. A blood clot that has not been carried in the bloodstream from its point of origin. Using this autochthonic model not only biology of cancer but also the etiology of these tumors can be studied. Today there is some evidence that autochthonic tumor models can give more

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www.ijpbs.com (or) www.ijpbsonline.com precise answer on questions concerning the induction, development and treatment of cancer than transplantation models on cancers. Criteria for autochthonic animal tumors as a model for chemotherapeutics studies [2].  Unilocular tumor occurrence.  Short, well defined collective tumor induction period,with little individual deviation.  Tumors diagnosable in time.  Tumor biology should parallel human counterparts.  Chemosensitivity should parallel human counterparts.

AUTOCHTHONIC MODELS

COLORECTAL

CANCER

between 180-240 days with a tumor rate of 90%. This includes all localizations. At a higher dosage of DMH the incidence of tumors beside the colon increases. The DMH model also leads to a high tumor spread (93%) in contrast to other autochthonous tumors. An important disadvantage is the high incidence of primary tumors in other organs. Because of tumor localization in the whole colon control of tumor size and growth is difficult. DMH-induced colon carcinoma in the rat is comparable to human colon cancer. Today it is the model most used to study colon cancer. B. Second autochthonous tumor model: Nmethylnitrosourea: N-methylnitrosourea (MNU) induces tumors by alkylation. It has been shown that methylation of the guanine base in DNA arises from AM, which in turn comes from DMH and MNU.Also alkylation of proteins and RNA has been reported. MNU is an exceptionally unstable substance with a very short biological half-life. Particularly following i.v. application tumors are found in many different organs (brain, bone, stomach, small bowel, colon, pancreas kidney, breast, and nervous system). Systematic investigations for the induction of colon tumors by intrarectal application resulted in a high incidence (up to 100%) of multiple colon tumors.Histopathologically these tumors are characterized as adenocarcinomas with a high grade of invasion and low spread. The disadvantage of this model is the time consuming application often necessary. [5]

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A. First autochthonic tumor model: 1,2 Dimethylhydrazine: 1, 2 Dimethylhydrazine(DMH) is very effective and selective carcinogen of the colon following subcutaneous application in BD rats. DMH is easily oxygenated to azomethane(AM)and azoxymethane (AOM) utilizing the same degradation pathway for all three substances. By oxidative dealkylation from AOM to methylazoxy methanol (MAM) it enters the degradation pathway of cycasin. The final alkylating carcinogen is produced by further metabolism of MAM and can be identified as a diazonium compound. [3] Organ manifestation and tumor distribution depend highly on the dose of the carcinogen. The optimal dosage for the selective induction of colonic tumors is 10 mg/kg for 25 weeks (total dose = 250 mg) [4]. The time of induction is

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Tumors in other locations were not reported. The disadvantage of this model is the long period of induction and latency.[6,7] D. Fourth autochthonous tumor model: Nnitroso-acetoxymethyl-methylamine: Like other nitrosamines N-nitrosoacetoxymethyl-methylamine (AMMN) is not per sea carcinogen but is degraded to the carcinogen diazohydroxide. Intrarectal instillation of 2 mg/kg every week for 10 weeks (total dose = 20 mg/kg) showed tumors in 97% of the treated animals. The latent time was up to 252 days.Tumor metastases were found in up to 28% of the animals.[8]

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C. Third autochthonous tumor model: Nmethyl-N'-nitro-N-nitrosoguanidine: Locally instilled N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) has been shown to have a direct carcinogenic effect. MNNG is degraded in alkaline milieu without enzymatic action to the carcinogen diazomethane. This rapid degradation to the carcinogen is the cause of the local cancer induction. After intrarectal instillation at a dose of 2 mg/kg three times a week (total dose = 150 mg/kg) tumors were observed at the instillation site between the 250th and 356th day of the trial. Tumors were found in 97% of the animals.

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www.ijpbs.com (or) www.ijpbsonline.com Drug 1,2-DMH MNU MNNG AMMN

Table 1: Comparison of the four autochthonous tumor models described Dose Application Time of Distribution in form induction(days) colon 10mg/kg/week 25 weeks S.C 180-240 Total 3x08mg/kg/week Intrarectal 175-245 Distal 3 x 2 mg/kg/week 25 weeks Intrarectal 230-350 Distal 2x10mg/kg/week Intrarectal 210-280 Distal

IN VITRO TESTING: Colorectal epithelial cells are constantly exposed to complex mixtures of compounds derived from the diet and the digestive process. These include genotoxic carcinogens and toxic or growth stimulating agents that can act as tumor promoters, but also protective compounds that counteract these effects. While demonstration of in vivo tumor induction is the only definite proof of carcinogenic or tumor-promoting activity, the experiments are expensive and time consuming. Both for the identification of candidate compounds worth testing in vivo and for the analysis of cell biological mechanisms in vitro models are of great importance. They have to satisfy two basic requirements: (1) Availability and easy handling for highthroughput testing. (2) Retention of tissue characteristics to support interpretation of results for the in vivo situation. Unfortunately tissue culture models for the target cellsâ&#x20AC;&#x201D;namely normal and premalignant colorectal epithelial cells (CEC)â&#x20AC;&#x201D;are very difficult to obtain.Therefore requirement of different cell cultures and different cell lines for in vitro studies which are given below [Carroll, 1981; Mahmoud et al., 2000].

STUDIES IN CELL LINES: While primary cultures have provided many insights into the growth control of CECs and the effects of tumor promoters on the CECs, they are not suitable for the analysis of large groups of compounds. Neither can they easily provide sufficient material for the investigation of protein or RNA expression. For these purposes cell lines have to be used. A multitude of different carcinoma cell lines have been successfully used for studies of regulatory mechanisms and for the identification of chemopreventive compounds that induce apoptosis in the tumor cells[Willson et al., 1987][11]. HT-29 Cell lines were obtained from the primary tumor of a Caucasian woman with colon adenocarcinoma[12].

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STUDIES USING PRIMARY CULTURES: Epithelial cells can be isolated from colorectal tissue specimen and put into primary culture.

Even cells isolated from normal mucosa that have very stringent culture requirements can be maintained for a few days. The advantage of primary cultures lies in the direct comparibility: they contain the same combination of cell populations that are also present in the tissue and not sufficient time has passed for selection mechanisms to have taken effect. For human CEC (colorectal Epithelial Cells) biopsy specimen can provide material for four to five cultures, larger surgical specimen can supply sufficient material for extended studies [Buset et al., 1986].

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Table 2: Examples of cell line studies of tumorogenic or protective compoundâ&#x20AC;&#x2122;s in colon Cell Line Test compound Effect Observed SW480 Carotene, retinoids Inhibitior of DG-induced growth. HT29, SW480 Anthraquinone Proliferation. HT29, SW480 Tyrphostines Induction of apoptosis HT29, SW480 H7, calphostine Induction of apoptosis HT29, SW480 Flavonoids Induction of apoptosis SW480, HT29, Caco2 Apigenin Cell-cycle arrest HT29 Dietary flavone Induction of apoptosis S/KS/FI Butyrate Induction of apoptosis HT29, S/KS NS398 Induction of apoptosis BRINE SHRIMP LETHALITY BIOASSAY: Many new natural compounds are isolated, characterized, and published without any biological testing whatsoever. Without accompanying biological data, the discovery of new medicinal plant constituents is nothing more than pure Phytochemistry. There is a real need for reliable, general bioassays which can detect a broad spectrum of pharmacologic activities in higher plants. Desiring a rapid, inexpensive, in-house, bioassay for screening and fractionation monitoring, of our physiologically active plant extracts, we have been using a tiny crustacean, brine shrimp, as the general bioassay tool. A general bioassay that appears capable of detecting a board spectrum of bioactivity present in crude extracts is the brine shrimp lethality bioassay (BSL). The technique is easily mastered, costs little, and utilizes small amount of test material. Brine shrimp have been previously utilized in various other bioassay systems, namely, analysis of pesticidal residues, mycotoxins, stream pollutants, anesthetics, dinoflagellate toxins, Morphine like compounds, toxicity of oil dispersants, di-n-alkyl pthalates and cocarcinogenic phorbol esters etc. The assay is also widely being used for evaluating the cytotoxicity of plant based compounds. Most of workers have made use of the hatched napulii, although

inhibition of hatching of eggs has also been studied. B.N. Mayer et al developed "Brine shrimp Lethality Bio-assay" in 1982. The basic principle behind this method is "the toxicology is simply pharmacology at higher doses. Thus if we could be able to isolate toxic compounds, a lower non toxic dose might elicit a useful, pharmacological, perturbation on physiological system, especially cytotoxic compounds for the treatment of cancer. The main advantage of this bioassay is simple, (do require few apparatus), rapid (overall screening is finished in 72 hrs), inexpensive (does not require any special apparatus & does not require aseptic condition) and is an in house bioassay. The eggs of brine shrimp, "Artemia salina leach", are readily available at low costs at any pet shop as a food for tropical fish, and they remain viable for years in the dry state. Upon being placed in a brine solution, the eggs hatch within 48 hrs providing large number of larvae (nauplii). The organism is now suggested as a convenient probe for pharmacologic activities in plant extracts which may be manifested as toxicity towards the newly hatched napulii. For the study, plant extracts, fractions, or pure compounds are tested at initial concentrations of 10, 100 and 1000 ppm (Îźg/ml) after being added to vials or test tubes containing brine and ten shrimps in each of three replicates. Survivors are counted after 24 hrs,

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www.ijpbs.com (or) www.ijpbsonline.com and the percentage of deaths at each dose are recorded. These data can then be analyzed with Finney computer program to determine LC50 values.[14-17] MTT-MICROCULTURE TETRAZOLIUM ASSAY: In the MTT assay, the tetrazolium salt, 3-(4, 5dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), is actively absorbed into cells and reduced in a mitochondrial dependent reaction to yield a formazan product. The product accumulates within the cell since it cannot pass through the cell membrane. Upon

addition of DMSO, isopropanol, or other suitable solvent, the product is solubilized and liberated, and is readily quantified colorimetrically. The ability of the cells to reduce MTT provides an indication of mitochondrial integrity and activity, which is interpreted as a measure of cell viability. The assay is suitable for a variety of cell lines displaying exponential growth in culture and a relatively high level of mitochondrial activity. It should be noted that some known compounds selectively affect mitochondria, resulting in an overestimation of toxicity.[20,21]

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Table 3: Other tetrazolium salts

SULPHORHODAMINE B (SRB) ASSAY: For the anticancer screen application, there were two especially troublesome problems encountered with the tetrazolium assays that eventually prompted the development of a third alternative microculture assay method. For either MTT or XTT, tetrazolium reduction was dependent on the cellular generation of NADH and NADPH. This raised concern about the influence of glucose concentration on the formation of the colored tetrazolium formazan, which was measured colorimetrically as an estimate of cellular growth or viability. Studies with MTT indicated that a progressive reduction in MTT specific activity (MTT formazan

formed/pg cell protein), which was observed during the course of a typical 7-d assay, was paralleled by a progressively decreasing glucose concentration. For XTT, there was a further problem resulting from the additional requirement of an electron transfer reagent, phenazine methylsulfate (PMS), to promote adequate cellular reduction of the tetrazolium. In an attempt to identify a suitable, nontetrazolium assay for use in the in vitro primary drug screen, a series of protein and biomass stains were investigated. These included anionic dyes that bound to the basic amino acid residues of proteins, as well as cationic dyes that bound to the negative, fixed charges of biological

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www.ijpbs.com (or) www.ijpbsonline.com macromolecules. Of all the reagents tested, sulforhodamine B (SRB) gave the best combination of stain intensity, signal-to-noise ratio, and linearity with cell number. SRB is a bright pink anionic dye that, in dilute acetic acid, binds electrostatically to the basic amino acids of TCA-fixed cells.[25] TRYPAN BLUE EXCLUSION: Measuring percentage Trypan Blue exclusion can rapidly assess cell viability and confirm whether cell culture conditions are ptimal. Cells that are viable exclude Trypan Blue, while cells that have died are stained by this dye. Trypan Blue exclusion measurement is also useful for determining whether cells in culture have escaped breakage or isruption and to assess apoptosis. Trypan Blue exclusion asurement therefore has utility in assessing viability and also in assessing cell mortality in response to environmental insult. The staining procedure with this dye is simple. Determination of percentage Trypan Blue exclusion requires only a light microscope. Typically, a small percentage of cells are harvested for trypan Blue exclusion measurement. Determination of Trypan Blue exclusion is performed at one or several predetermined time points.[24,25] CRYSTAL VIOLET INCLUSION: Crystal violet inclusion measurement detects cell lysis. This dye stains viable cells that adhere to their culture vessel. Lysed cells simply fall away from their vessel surface and are not stained by this dye. Crystal violet is used in a standard bioassay for TNF-α. In this bioassay, fibroblasts are grown as a monolayer in the presence of actinomycin D. To these cells, increasing concentrations of TNF-α are added. Cells that are lysed in the presence of TNF-α are subsequently stained by crystal violet, whereas intact cells are stained by this dye. Crystal violet-stained cells are then detected by spectrophotometric measurement. This method has the limitation of

detecting only adherent cells and only permits measurement at a single predetermined time point. In other words, no continuous monitoring can be performed by this method.[27]

DISCUSSION Comparison (Table 3) of these four autochthonous models shows differences in handling and biological behavior e.g., growth, invasion, and spread. In the DMH model induced tumors spread over the whole gut. Tumors also arose in other organs. This model allowed the hypotheses resulting from epidemiology or etiology regarding the whole bowel to be tested. In the three other models tumors were induced by rectal instillation. A high incidence of adenomas and carcinomas specifically in the large intestine were found. In addition, the site and time of tumor appearance was constant. Differences in the macroscopic appearance of the lesion - polypoid or ulcerative – allowes the choice of a specific model for different clinical questions. The advantage of these models is the constant localization of tumor appearance (distal colon), allowing control of tumor development by coloscopy. Also the cystostatic effectiveness of test substances in the colon can be evaluated within a short period by endoscopic observation of the tumor growth. For the form and rate of application and its results we prefer the AMMN model for our chemotherapy studies [5]. This tumor model which was developed by Schmfihl and coworkers is today one of the best techniques for inducing autochthonous colon cancers.Table 2 contains defferent cell lines with their comparative test compounds used for study and the purpose of study e.g. inhibition of growth, induction of apoptosis and cell cycle arrest. Such cell lines can be easily used for highthroughput screening. On the other hand, they have been established from carcinomas or even metastases that have already accumulated many

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www.ijpbs.com (or) www.ijpbsonline.com genetic defects and genetic instabilities [10] Table 3 shows the tetrazolium salts with their lambda max and solubility in water.

10. 11.

REFERENCES 1.

Habs M, Schm/ihl D, Wiessler M (1978) Carcinogenity of acetoxymethylmethyl-nitrosamine after subcutaneous intravenous and intrarectal application in rats. Z Krebsforsch 91:217-221 Amberger H (1986) Different autochthonous models of colorectal cancer in the rat. In: J Cancer Res Clin Oncol (1986) 111:157-159 Druckrey H (1970) Production of colonic carcinomas by 1, 2-dialkyMaydrazines and azoxyalkanes. In: Burdette WJ (ed) Carcinoma of the colon and antecedent epithelium. C.C. Thomas Springfield, O.: 267-269 Sych F, Habs M, Schmfihl D (1978) Chemotherapy studies in autochthonous tumors: Intestinal cancer. Z Krebsforsch 92:105-117 Schmfihl D (1976) In: Hellman K, Connors TA (eds) Utilisation of nitrosamine induced tumors as models for cancer chemotherapy. Chemotherapy Vol 7. Plenum Publishers, New York, pp 233-238 Amberger H (1984) Biological behavior of various autochthonous tumor models of colon cancer in the rat. In: Georgii A (ed) Aspects of clinical oncology. Gustav Fischer Verlag, Stuttgart, New York Amberger H (1986) Different autochthonous models of colorectal cancer in the rat. In: J Cancer Res Clin Oncol (1986) 111:157-159 Habs M, Schm/ihl D, Wiessler M (1978) Carcinogenity of acetoxymethylmethyl-nitrosamine after subcutaneous intravenous and intrarectal application in rats. Z Krebsforsch 91:217-221

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Marian B In vitro models for the identification and characterization of tumor-promoting and protective factors for colon carcinogenesis. Food and Chemical Toxicology 40 (2002) 1099–1104 Willson et al., 1987. Friedman, E., Isaksson, P., Rafter, J., Marian, B., Winawer, S., Newmark,H., 1989a. Fecal diglycerides as selective endogenous mitogens for premalignant and malignant human colonic epithelial cells. Cancer Research 49, 544–548. Friedman, E., Lipkin, M., Winawer, S., Buset, M., Newmark, H., 1989b. Heterogeneity in the response of familial polyposis epithelial cells and adenomas to increasing levels of calcium in vitro. Cancer 63, 2486– 2491. Mahmoud, N.N., Carothers, A.M., Grunberger, D., Bilinski, R.T., Churchill, M.R., Martucci, C., Newmark, H.L., Bertagnolli, M.M., 2000. Plant phenolics decrease intestinal tumors in an animal model of familial adenomatous polyposis. Carcinogenesis 21, 921–927. Brown JP. A review of the genetic effect of naturally occurring flavonoids, anthraquinones and related compounds. Mutat. Res. (1980) 75: 243-247. Muthiah Maridass, Evaluation of Brine Shrimp Lethality of Cinnamomum Species. Ethnobotanical Leaflets (2008) 12: 772-775. Jham, G.N., O.D., Dhingra, C.M. Jardin, and M.M. Valente, 2005. Identification of the major fungitoxic component of cinnamon bark oil. Fitopatol. Bras. 30: 404-408. Adoum, O. A., Determination of toxicity levels of some savannah plants using Brine shrimp test (BST). Bayero Journal of Pure and Applied Sciences, 2(1):135 – 138.

*Corresponding Author: Ajit B. Patil*

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Department of Pharmacology, Tatyasaheb Kore College of Pharmacy, Warananagar.Tal-Panhala, Dist- Kolhapur, 416113, (M.S.), India.

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Ajit B. Patil*et al www.ijpbs.com or www.ijpbsonline.com

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Research Article Biological Sciences SYNTHESIS AND SCREENING OF METHYL-2-(2-(ARYLIDENEAMINO) OXAZOLE-5-YLAMINO) BENZOXAZOLE-5-CARBOXYLATE DERIVATIVES AS CYCLOOXYGENASE INHIBITORS NAGESHWAR RAO CHILUMULA1*, DAYAKAR GADHE1 1

Department of Chemistry, Kakatiya University, Warangal, A.PIndia, 506009. *Corresponding Author Email: kirankrishna25@yahoo.com

ABSTRACT We have synthesized a series of methyl-2-(2-(arylideneamino) Oxcazole-5-ylamino)benzoxazole-5-carboxylate derivatives and investigated their ability to inhibit human cyclooxygenase-2 enzyme (COX-2). The active compounds were screened for cyclooxygenase-1 (COX-1) inhibition. Compound VIId is 368-fold and VIIh is more than 454 fold selective towards COX-2 compared to COX-1. Thus, this class of compounds may serve as excellent candidates for selective COX-2 inhibition.

KEYWORDS

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Benzaxozoles, synthesis, cyclooxygenase, evaluation, COX-2, COX-1

INTRODUCTION Cyclooxygenase (COX; prostaglandin endoperoxide synthase) metabolizes arachidonic acid to prostaglandin (PG) H2, which serves as the precursor for the biosynthesis of various PGs, thromboxanes, and prostacyclin [1]. COX activity originates from two distinct and independently regulated isozymes, COX-1 and COX-2 [2]. COX-1 is a constitutive enzyme, whereas COX-2 is inducible and short-lived. COX-2 is the product of an immediate-early gene, and its expression is stimulated by a host of growth factors, cytokines, and mitogens[3]. COX-1 appears responsible for the biosynthesis of PGs in the gastric mucosa and in the kidney, whereas COX-2 appears responsible for biosynthesis in inflammatory cells and the central nervous system[4]. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the two isoforms to different extents, and this feature accounts for their shared therapeutic properties and side effects [5]. The differential tissue distribution of the COX isozymes has provided a rationale for the

development of COX-2-selective inhibitors as nonulcerogenic, anti-inflammatory, and analgesic agents[6]. Most selective COX-2 inhibitors, including the recently approved drugs celecoxib[7] and rofecoxib[8] belong to the diarylheterocycle class of compounds[9-11]. Diarylheterocycles have been investigated extensively as COX-2 inhibitors since the description of the 2, 3-diaryloxazole, as a nonulcerogenic anti-inflammatory agent. In addition 2-Oxo-3H-benzoxazole derivatives exhibit a broad range of biological properties [13-16] including analgesic and antiinflammatory activity [17-22]. Among them, especially 3-substituted-2-oxo-3H-benzoxazoles are known to exhibit analgesic and antiinflammatory properties [23]. It has also been reported that mannich bases of 6-acyl-2-oxo-3Hbenzoxazoles resulted in compounds with potent analgesic activity [20]. Additional studies with some 3-aminoalkyl-2-oxo-3H-benzoxazole derivatives also demonstrated potent analgesic and anti-inflammatory activity, and showed that

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www.ijpbs.com (or) www.ijpbsonline.com these compounds exerted their in vivo activity by inhibiting the synthesis of prostaglandin E72. (2oxo-3H-benzoxazol-3-yl)propanamides also showed potent analgesic and anti inflammatory activity [24-27]. (6-acyl-2-oxo-3H-benzoxazol-3yl)alkanoic acids possessed potent analgesic and anti-inflammatory activity with reduced gastric toxicity [28]. In general, most of the research on this class of compounds included substitutions on positions 3 and 6 of the 2-oxo-3Hbenzoxazole nucleus. As a result, 2-oxo-3Hbenzoxazoles bearing N-alkyl, N-acyl, Ndiaminoalkyl and 6-acyl substituents were reported to have higher anti-inflammatory activity [29 - 30]. Berna et al described the synthesis of two novel 4-phenyl-and 4-(2chlorophenyl)-6-(5-chloro-2-oxo-3H-benzoxazol7-yl)-3(2H)-pyridazinone derivatives and showed potent anti-inflammatory activities without causing gastric lesions in the tested animals [31]. Hence these observations prompted us to synthesize a series of methyl-2-(2(arylideneamino) oxazole-5-ylamino) benzoxazole-5-carboxylate derivatives (VI 1VI15) and to evaluate their COX - 2 activity. The required starting material, methyl-3-amino-4hydroxybenzoate (II) was synthesized in good yield (85%) according to reported procedure [33].The starting material (II) on cyclization with cyanogen bromide on rapid stirring at room temperature gave the product, methyl-2aminobenzoxazole-5-carboxylate (III). The compound (III) on reaction with chloroacetyl chloride in dry benzene yields the compound, methyl-2-(2-chloroacetamido) benzoxazole-5carboxylate (IV). The compound (IV) on reaction with urea gave the compound methyl-2-(2aminooxazol-5-ylamino) benzoxazole-5carboxylate (V), finally which on reaction with various aromatic aldehydes conveniently converted into the targeted compounds methyl-

2-(2-(arylideneamino) oxazole-5-ylamino) benzoxazole-5-carboxylate derivatives (VI). The synthesized compounds were tested for their ability to inhibit human cyclooxgenase-2 (COX-2) enzyme and the more active compounds were tested for cyclooxgenase-1 (COX-1) inhibition in human whole blood assay [34]. Rofecoxib was used as active control in cyclooxygenase inhibition assay. The compound which shown IC50 less than 10 mM concentration were tested for COX-1 inhibition. Interestingly two compounds, namely VIId and VIIh have exhibited good activity with high selectivity towards COX-2 inhibition when compared to rest of the compounds. Compound VIId is 368 times more selective towards COX-2 when compared to COX-1 (COX-1 IC50=373mM; COX-2 IC50=lmM). Surprisingly compound VIIh is 454 times more selective towards COX-2 inhibition than COX-1 (COX-1 IC50=500; COX-2 IC50=1.04 mM), interestingly 100 fold more selective than compound VII d. However they are more selective and less potent than rofecoxib in human whole blood assay. Although compounds VI b, VIc, VI e, VI f and VI g possess good selectivity, they have shown moderate activity towards COX-2. In conclusion, these classes of compounds may serve as excellent candidates for selective COX-2 inhibition.

MATERIALS AND METHODS All melting points were taken in open capillaries on a veego VMP-1 apparatus and are uncorrected IR spectra were recorded as KBr pellets on a Perkin-Elmer FT IR 240-c spectrometer. The XH NMR spectra were recorded on Varian-Gemini 200 MHz spectrometer in DMSO-d6 using TMS as an internal standard and mass spectras were recorded on Schimadzu QP 5050A spectrometer.

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www.ijpbs.com (or) www.ijpbsonline.com The targeted compounds were synthesized as shown in Scheme-1. I.Synthesis of 4-carbomethoxy-2-nitrophenol (II) To a solution of aluminum nitrate (40g) in acetic acid- acetic anhydride [1:1] mixture (160ml), was added an appropriate phenol (I, 40g) in small portions, while cooling and shaking occasionally. The reaction mixture was left at room temperature for 1.5 h while shaking the contents intermittently to complete the nitration. The resulting brown solution was diluted to complete the nitration. The resulting brown solution was diluted with ice-cold water and acidified with concentrated nitric acid to get a bulky, yellow precipitate. It was filtered washed with small quantity of methanol and purified by recrystallization from alcohol to get a yellow crystalline solid (44g, 85%), m.p 73°C [33]. II.Synthesis of 4-carbomethoxy-2-aminophenol (III) 4-carbomethoxy-2-nitrophenol (II, 10 g) was dissolved in boiling alcohol (50%, 100ml) and sodium dithionite was added to this boiling alcohol solution until it becomes almost colourless. Then the alcohol was reduced to onethird of its volume by distillation and the residual liquid was triturated with crushed ice. The resulting colourless, shiny product was filtered, washed with cold water and dried in the air. Its purification was effected by recrystallization from benzene to get colourless, shiny scales (5.1 g; 60%) m.p 143°C. III. Synthesis of methyl-2-aminobenzoxazole-5carboxylate (IV) 1.3 mol of 4-carbomethoxy-2-aminophenol (III) was dissolved in Hit. Methyl alcohol and cooled the solution to 5°C by adding chopped ice. A cold suspension of 1.5 mol of cyanogenbromide in Hit of water was added over a period of 5min with rapid stirring. Continued the stirring for 0.75h at room temperature, 1.3 mol of solid sodium

bicarbonate in small portions over a period of 1.5 h was added to bring the pH 6.5 -7.0. Stirring was continued for another lh. The solid was separated by filtration, washed with cold water and on recrystallization from ethyl alcohol has resulted white solid, yield 70% m.p 238°C. IV. Synthesis of methyl-2-(2-chloroacetamido) benzoxazole-5-carboxylate (V) A mixture of methyl-2-aminobenzoxazole-5carboxylate (IV, 0.0lmol) and chloroacetyl chloride (0.0lmol) was taken in 20 ml of dry benzene and the reaction mixture was refluxed for 5h on a water bath. The solvent was evaporated and the residue was washed first with benzene and then with petroleum ether. The compound was recrystallized from suitable solvent(s). The compound was found to be containing yield 72% and m.p is 177°C. IV.Synthesis of methyl-2-(2-aminooxazol-5ylamino) benzoxazole-5-carboxylate (VI) Methyl-2-(2-chloroacetamido) benzoxazole-5carboxylate (VI, 0.0lmol) and urea (0.0lmol) were dissolved in 10ml of absolute alcohol in conical flask. The conical flask was hanged with a funnel and was subjected to microwave irradiation at 480 Watts for 5min in LG-Microwave oven. The reaction was monitored by TLC. After the completion of the reaction the contents were cooled and triturated with crushed ice the separated solid was filtered, washed with 1% NaHC03 solution and purified by recrystallization from ethanol and water mixture found to be containing yield 97% and m.p 199°C. V.Synthesis of methyl-2-(2-(arylideneamino) oxazol-5-ylamino) benzoxazole-5-carboxylate (VII) Methyl-2-(2-aminoxazol-5-ylamino) benzoxazole5-carboxylate (XII, 0.01mol) and appropriate aromatic aldehydes, Benzaldehyde, salicyladehyde, p-hydroxybenzaldehyde, anisaldehyde, p-dimethylaminobenzaldehyde, p-

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cooled and triturated with crushed ice; the separated solid was filtered, washed with 1% NaHC03 solution and purified by recrystallization from ethanol and water mixture. The compounds were characterized by spectral data. Physical data of all synthesized compounds given in Table 1.

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chlorobenzaldehyde, veratraldehyde, cinnam aldehyde and 3, 4, 5-trimethylbenaldehyde (0.015mol) were taken into a conical flask and were dissolved in 10ml of absolute alcohol. The conical flask was hanged with a funnel and was subjected to microwave irradiation at 480 Watts for 7min in LG-Microwave oven. The reaction was monitored by TLC. The reaction mixture was

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CompoundVIIa: Methyl-(2-(4-(dimethylamino) benzylideneamino) oxazol-5-ylamino) benzoxazole-5-carboxylate IR (KBr, cm-1): 3133(NH), 1693 (C=N), 1610 (C=C), 1582 (C=N), 1249 (C-O-C); 1 H-NMR (DMSO-d6) 8: 8.8 (s, 1H, CH), 8.6 (s, 1H, Ar-H), 8.1(d, 1H, Ar-H), 8.0(d, 1H, Ar-H), 7.6(s, 1H, Ar-H oxazole ring), 7.5(d, 2H, Ar-H), 6.8(d, 2H, Ar-H) 5.3(s, 1H, NH), 3.8(s, 3H, OCH3), 3.0(s, 6H, CH3); MS [m/z): M+: 406.1 Compound VIIb: Methyl-2-(2 (benzylideneamino) oxazol-5-ylamino) benzoxazole-5-carboxylate IR (KBr, cm-1): 3138(NH), 1696 (C=N), 1602 (C=C), 1576 (C=N), 1233 (C-O-C). 1 H-NMR (DMSO-d6) 8: 8.6 (s, 1H, CH), 8.5 (s, 1H, Ar-H), 8.1(d, 1H, Ar-H), 8.0(d, 1H, Ar-H), 7.8 (d, 2H, ArH), 7.7 (s, 1H, Ar-H oxazole ring), 7.5(t, 3H, Ar-H), 6.8(d, 2H, Ar-H) 5.3(s, 1H, NH), 3.8(s, 3H, OCH3); MS [m/z): M+: 363.1 Compound VIIc: methyl-2-(2-(2hydroxybenzylideneamino) oxazol-5-ylamino) benzoxazole-5-carboxylate IR [KBr, cm-1): 3137[NH), 1669 (C=N), 1620 (C=C), 1585 (C=N), 1241 [C-O-C). 1 H-NMR [DMSO-d6) 8: 11.2(s, 1H, OH), 8.8 [s, 1H, ArH), 8.3 [s, 1H, CH), 8.1[d, 1H, Ar-H), 8.0[d,

1H, Ar-H), 7.9 [s, 1H, ArH oxazole ring), 7.7 [d, 1H, Ar-H), 7.5[t, 1H, Ar-H), 7.1[t, 1H, Ar-H), 7.0[d, 1H, Ar-H), 5.5[s, 1H, NH), 3.9[s, 3H, OCH3); MS [m/z): M+: 379.1 Compound VIId: Methyl-2-(2-(4chlorobenzylideneamino) oxazol-5-ylamino) benzoxazole-5 carboxylate IR [KBr, cm-1): 3112[NH), 1685 [C=N), 1609 [C=C), 1564 (C=N), 1223 [C-O-C). 1 H-NMR [DMSO-d6) 8: 8.6[s, 1H, ArH), 8.3 [s, 1H, CH), 8.2[d, 1H, Ar-H), 8.0[d, 1H, Ar-H), 7.9 [s, 1H, ArH oxazole ring), 7.8 [d, 2H, Ar-H), 7.5[d, 2H, ArH), 5.1[s, 1H, NH), 3.6[s, 3H, OCH3); MS [m/z): M+: 397.1 Compound VIIe: Methyl-2-(2-(4methoxybenzylideneamino) oxazol-5-ylamino) benzoxazole-5-carboxylate IR [KBr, cm-1): 3114[NH), 1647 [C=N), 1615 [C=C), 1543 [C=N), 1212 [C-O-C). 1H-NMR [DMSO-d6) 8: 8.8[s, 1H, ArH), 8.5 [s, 1H, CH), 8.0[d, 1H, Ar-H), 7.8[d, 1H, Ar-H), 7.6 [s, 1H, ArH oxazole ring), 7.4 [d, 2H, Ar-H), 7.3[d, 2H, ArH), 5.4[s, 1H, NH), 3.9[s, 3H, OCH3), 3.6[s, 3H, OCH3); MS [m/z): M+: 393.1 Compound VIIf: Methyl-2-(2-(4hydroxybenzylideneamino) oxazol-5-ylamino) benzoxazole-5-carboxylate

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IR [KBr, cm-1): 3133[NH), 1690 [C=N), 1602 [C=C), 1592 [C=N), 1259 [C-O-C). 1 H-NMR [DMSO-d6) 8: 9.4[s, 1H, OH), 8.8[s, 1H, ArH), 8.6[s, 1H, CH), 8.1[d, 1H, Ar-H), 8.0[d, 1H, Ar-H), 7.9 [s, 1H, ArH oxazole ring), 7.8 [d, 2H, ArH), 6.8[d, 2H, Ar-H), 5.0[s, 1H, NH), 3.6 [s, 3H, OCH3); MS [m/z): M+: 379.1 Compound VIIg: methyl-2-(2-(2-hydroxy-4methoxybenzy-lideneamino) oxazol-5 ylamino) benzoxazole-5-carboxylate IR [KBr, cm-1): 3141[NH), 1659 [C=N), 1612 [C=C), 1568 [C=N), 1217 [C-O-C). 1 H-NMR [DMSO-d6) 8: 11.5[s, 1H, OH), 8.8[s, 1H, ArH), 8.7[s, 1H, CH), 8.4[d, 1H, Ar-H), 8.3[d, 1H, Ar-H), 7.7 [s, 1H, ArH oxazole ring), 7.6 [d, 1H, ArH), 6.6[d, 1H, Ar-H), 6.4[s, 1H, ArH), 5.2[s, 1H, NH), 3.9[s, 3H, OCH3), 3.3[s, 3H, OCH3); MS [m/z): M+: 409.0 CompoundVIIh: methyl-2-(2-(3cinnamylideneamino) oxazol-5-ylamino) benzoxazole-5-carboxylate IR [KBr, cm-1): 3103[NH), 1640 [C=N), 1600 [C=C), 1590 [C=N), 1219 [C-O-C). 1 H-NMR [DMSO-d6) 8: 8.6 [s, 1H, ArH), 8.1 [d, 1H, Ar-H), 8.0[d, 1H, Ar-H), 7.7[s, 1H, Ar-H oxazole ring), 7.6[d, 2H, Ar-H),7.5 [s, 1H, CH), 7.4[t, 1H, Ar-H), 7.3[t, 1H, Ar-H), 7.0 [s, 1H, CH), 5.3 [s, 1H, CH), 4.6 [s, H, NH), 3.8[s, 3H, OCH3); MS [m/z): M+: 389.0 CompoundVIIi: methyl-2-(2-(3, 4, 5trimethylbenzylideneamino) oxazol-5-ylamino) benzoxazole-5-carboxylate IR [KBr, cm-1): 3201[NH), 1670 [C=N), 1628 [C=C), 1588 [C=N), 1219 [C-O-C). 1 H-NMR [DMSO-d6) 8: 8.8 [s, 1H, ArH), 8.5 [d, 1H, CH), 8.1[d, 1H, Ar-H),8.0[s, 1H, ArH), 7.8[s, 1H, Ar-H oxazole ring), 7.3[s, 2H, Ar-H), 5.2 [s, 1H, CH), 5.3 [s, H, NH), 3.9[s, 3H, OCH3), 2.3[s, 6H, CH3), 2.13.9[s, 3H, CH3); MS [m/z): M+: 405.0

Biological Evaluation Cyclooxygenase Inhibitory Screening The compounds synthesized were tested for cyclooxygenase-1 and cyclooxygenase-2 inhibitory activity. The method of Copeland et al. [34] was followed to determine the IC50 values. The enzyme activity is measured using chromogenic assay based on oxidation of N,N,N',N'-tetramethyl-p phenylenediamine (TMPD) during the reduction of prostaglandin G2 to prostaglandin H2 by COX-1 and COX-2 enzymes. COX-1 enzyme is from Ram seminal vesicles (microsomal fraction) and COX-2 is Recombinant human enzyme purified from SF9 cells (microsomal fraction) were used in the assay. The compounds were dissolved in DMSO and stock solution is diluted to required assay concentration. The assay mixture consists of TrisHC1 buffer (pH 8.0, 100 mM), hematin (15 uM), EDTA (3 uM), enzyme (COX-1 or COX-2, l00mg) and test compound. The mixture was preincubated at 25째C for 15 min and then the reaction was initiated by the addition of arachidonic acid (l00uM) and TMPD (120uM) in total volume of 1.0 mL. The enzyme activity was measured by estimating the initial velocity of TMPD oxidation for the first 25 seconds of the reaction following the increase in absorbance at 603 nm. IC50 values are calculated from four parameter least squares non-linear regression analysis of the log dose vs. percentage inhibition plot.

RESULTS AND DISCUSSION The target compounds were synthesized as outlined in Scheme 1. The required starting material, Methyl-3-amino-4-hydroxybenzoate (III) was prepared in good yield (85%) according to reported procedure [33].The Methyl-3-amino4-hydroxybenzoate (III) on cyclization with cyanogen bromide on rapid stirring at room

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temperature gave the product, Methyl 2aminobenzoxazole-5-carboxylate (IV). The compound Methyl 2-aminobenzoxazole-5carboxylate (IV) on reaction with chloroacetyl chloride in dry benzene yields the compound, Methyl-2-(2-chloroacetamido) benzoxazole-5carboxylate (V). The compound Methyl-2-(2chloroacetamido) benzoxazole-5-carboxylate (V)

on reaction with urea gave the compound methyl-2-(2-aminooxazol-5-ylamino) benzoxazole-5-carboxylate (V), finally which on reaction with various aromatic aldehydes conveniently converted into the targeted compounds methyl-2-(2-(arylideneamino) oxazole-5-ylamino) benzoxazole-5-carboxylate derivatives (VII).

Table 2: Physical data of Methyl 2-(2-(arylideneamino) oxazol-5-ylamino)benzoxazole- 5-carboxylates (VII) O

H3CO

N NH

CHAr

O O

S.No

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1 2 3 4 5 6 7 8 9

Compound VIIa VIIb VIIc VIId VIIe VIIf VIIg VIIh VIIi

Chemical formula

COX2aIC50mM

p-Dimethylamino Salicylyl Phenyl Anisalyl Cinnamyl 3,4,5-Trimethylphenyl p-Hydroxy phenyl p-Chlorophenyl Veratralyl

C21H19N5O4 C19H14N4O5 C19H14N4O4 C20H16N4O5 C21H16N4O4 C22H20N4O4 C19H14N4O5 C19H13N4O4Cl C20H16N4O6

>10 >10 2.63 1.0 6.25 3.84 2.27 1.06 2.77

Whole blood as TXB2generation. IC50values were estimated from dose-response curve analysed by nonlinear regression using GraphPad software and values are average of three determinations, nt* samples those have IC50> 10 mM for COX-2 inhibition are not tested for COX-1 inhibition. In the case of compound VIIc having a simple phenyl group and compound VII g having hydroxyl group on 4-position of phenyl ring showed moderate activity towards COX-2

COXlbIC50m M nt* nt* >500 >368 >500 >500 >500 >373 >500

COXl/COX-2 â&#x20AC;&#x201D; >190 >368 >80 >130 >220 >454 >220

inhibition. Compound VII h chloro group at 4position of phenyl ring exhibited more inhibition (IC50=1.06 mM) when compare to compound VI c. In the case of compound VIIi, which bears veratryl group exhibited 2.5-fold less inhibition compared to compound VIIc. Compound VII d possessing methoxy group on the phenyl ring exhibited highest activity (IC50=lmM) among tested compounds. Remaining compounds are less active with an IC50 more than 10 mM.The compound which

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www.ijpbs.com (or) www.ijpbsonline.com shown IC50less than 10 mM concentration were tested for COX-1 inhibition. Interestingly two compounds, namely VII d and VIIh shown good activity with high selectivity towards COX-2 inhibition when compared to rest of the compounds. Compound VIId is 368 times more selective towards COX-2 when compared to COX1 (COX-1 IC50=3.84mM; COX-2 IC50=lmM). Surprisingly compound VIIh is 454 times more selective towards COX-2 inhibition than COX-1 (COX-1 IC50=>500; COX-2 IC50=1.06 mM), interestingly 100 fold more selective than compound VIId. However they are more selective and less potent than rofecoxib in human whole blood assay. Although compounds VIIc, VIIe, VIIg, VIIh and VIIi possess good selectivity, they have shown moderate activity towards COX-2 (results presented in Table-2). In conclusion, these classes of compounds may serve as excellent candidates for selective COX-2 inhibition.

CONCLUSION This study reports the successful synthesis of the title compounds in good yields and moderate to potent COX-2 of these derivatives containing benzoxazole moiety which is comparable with standard drug. It has been observed that the increased COX-2 inhibitory activity is attributed to the presence of pharmacologically active substituents like 2-(dialkylamino) acetamido group.

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REFERENCES [1] Hamberg M, Samuelsson B Proc Natl Acad Sci USA 70 (1973,/ 899-903. [2] Smith W L, Garavito R M, DeWitt D L JBiol Chem 271 (1996,/ 33157-33160. [3] Herschman H R Biochim Biophys Ada Lipids Lipid Metab 1299(1996,/125-140. [4] Needleman P, Isakson P C JRheumatol 24, Suppl 49 (1991) 6-8. [5] Vane J R, Bakhle Y S, Botting R M Annu Rev Pharmacol Toxicol 3 8 (1998,/ 97- 120.

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|32-40 [6] Seibert K, Zhang Y, Leahy K, Hauser S, Masferrer J, Isakson P Adv Exp Med Biol 400A (1997) 167-170. [7] Simon L S, Lanza F L, Lipsky P E, Hubbard R C, Talwalker S, Schwartz B D, Isakson P C, Geis G S Arthritis Rheum 41 (1998) 1591-1602. [8] Ehrich E W, Dallob A, De Lepeleire I, Van Hecken A, Riendeau D, Yuan W Y, Porras A, Wittreich J, Seibold J R, DeSchepp P, Clin Pharmacol Ther 65 (1999) 336-347. [9] Prasit P, Riendeau D, Annu Rep Med Chem 32 (1997) 211-220. [10] Talley J, J Prog Med Chem 36 (1999) 201-234. [11] Marnett L J, Kalgutkar A S, Curr Opin Chem Biol 2 (1998) 482-490. [12] Gans K R, Galbraith W, Roman R J, Haber S B, Kerr J S, Schmidt W K, Smith C, Hewes W E, Ackerman N R, J Pharmacol Exp Ther 254 (1990) 180-187. [13] S. Dalkara, U. Sunal, J. Pharm. Belg., 43 (1988) 372-378. [14] J. F. Delhomel, S. Yous, P. Depreux and D. Lessieur, J. Heterocyclic Chem., 36 (1999) 1241-1245. [15] D.D. Erol, M.D. Aytemir and N. Yulu, G Eur. J. Med. Chem., 30 (1995) 521-524. [16] D. Shi, T.D. Bradshaw, S. Wngley, CJ. McCall, P. Lelieveld, I. Fichtner, M.F.G. Stevens, J. Med. Chem. 39 (1996) 3375-3384. [17] J. Delarge and J.H Poupaert, J. Med. Chem., 41 (1998) 1138-1145. [18] E. Palaska, S. Unlu, H. Erdogan, C. Safak, B. Gumusel and R. Sunal, Eur. J. Med. Chem. 28 (1993) 963-967. [19] E. Palaska, S. Unlu, F. Ozkanl_, G Pilli, H. Erdogan, C. Safak, R. Demirdamar, B. Gumu_sel and S. Duru, Arzneim.-Forsch. Drug Res. 45, (1995) 693-696. [20] C.Safak, H.Erdogan, E.Palaska, R.Sunal and S.Duru, J.Med Chem.35(1992)1296-1299. [21] H Erdogan, S. Unlu and R. Sunal, Arch. Pharm. Pharm. Med. Chem. 322 (1989) 75-77. [22] H. Erdogan, M. Debaert and J. C. Cazin, Arzneim.Forsch. Drug Res. 41 (1991) 73-76. [23] D.S. Dogruer, S. Unlu, M.F.Sahin and E. Yesilada, Farmaco 53 (1998) 80-84. [24] J. Mercier, C. Lespagnol and M.R. Sestier, Bull. Soc. Lille., 35 (1953) 85-90. [25] D.S. Dogruer, S. Unlu, E. Yesilada and M.F.Sahin, Farmaco 52 (1997) 745-750. [26] D.S. Dogruer, S. Unl u, E. Yesilada and M.F.Sahin, Farmaco 53 (1998) 80-84. [27] T. Onkol, S. Ito, E. Yldrm and M.F.Sahin, Arch. Pharm. Pharm. Med. Chem. 334 (2001) 17-20. [28] T. Onkol, Y. Dundar, B.Srmagul, K. Erol and M.F.Sahin, J. Fac. Pharm. Gazi, 19 (2002)15-24. [29] S. Unlu, H. Erdogan, R. Sunal and B. Gumusel, J. Fac. Pharm. Gazi, 9 (1992) 75-80.

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www.ijpbs.com (or) www.ijpbsonline.com [30] G. Pilli, F. Ozkanl, C. Safak, H. Erdogan, S. Unlu, B. Gumusel and R. Demirdamar, Pharmazie, 49 (1994) 6364. [31] Berna Okcelik, Serdar Unl, Erden Banoglu, Esra Kupeli, Erdem Yesilada' M. Fethi Sahin, ArchivDer Pharmazie, 336 9 (2003) 406 - 412.

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|32-40 [32] B.Gopal Krishna, N. Raghunandan, J.V.Rao, S. Bari, B.Srinivas, A.Venkatesham and M. Sarangapani, Indian Drugs 42 (6) (2005) 182-187. [33] A. Finhorn and B.Ptyl, Ann. Chem., 311 (1900) 46-51. [34] Copeland R A, Williams J M, Giannars J, Nurnberg S, Covington M, Pinto D, Pick S & Trzaskos JM, Proc Natl Acad Sci USA, 91(1994) 11202.

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NAGESHWAR RAO CHILUMULA, Department of Chemistry, Kakatiya University, Warangal, A.P.,India, 506009.

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Research Article Pharmaceutical Sciences EVALUATION OF ANTIMICROBIAL ACTIVITY OF PLANT LEAF ARGEMONE MEXICANA Yashwant Bais1*, Sunil B.Chaudhari1, Sujit Belani1 and Arvind R.Umarkar2 1

Manoharbhai Patel Institute of Pharmacy Gondia M.S.India Shree.Sureshdada Jain Institute of Pharmaceutical education and Research Jamner Dist: Jalgaon *Corresponding Author Email: yashwantbais2011@gmail.com

ABSTRACT Increasing prevalence of multidrug resistance strains of micro-organism has initiated the exploration of alternate antimicrobial agent. Taking in account the medicinal important of Argemone mexicana (leaf) in this respect an attempt was made in the current study to investigate the antimicrobial potential of this plant. Authenticated fresh plant leaf were selected for determination of antimicrobial activity against eleven clinical isolates of Gram +ve (2), Gram –ve (4) and fungi (5).The methanol extracts of Argemone mexicana leaf were screened in vitro for antibacterial activity by well diffusion method it doesn’t show any antibacterial action against bacteria, but it quite sensitive to fungi. It shows remarkable action against Candida albicans, Aspergilus niger 24 mm and 22 mm respectively which was moderately to flucanazole. The MIC value 3.12mg/ml. Thus the current investigation leads to fresh source of new antimicrobial in future. The results suggest that argemone mexicana is a potential candidate plant for future exploitation in medical microbiology.

KEY WORDS Argemone Mexicana, Candida albicans, Aspergilus niger

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INTRODUCTION Argemone mexicana L (Papaveraceae) is an herb with branches, which has naturalized widely in many tropical and subtropical regions although it’s a native of tropical American1. It grows commonly in abandoned and cultivated fields of South-West, Nigeria where it is renowned for its high medicinal properties. A.Mexicana L. is known by many names in Nigeria, it is called “Kaju” in Yoruba, “Ahon ekun” in Ijebu land, “Kadinnia” among the Hausas It is an herb with bright yellow flowers and yellow juice. A. mexicana’s concoction from its ethnological survey in Nigeria is used in treatment of bacterial infection. It is widely believed that the latex from this plant cures cataract, reddening and itching in the eyes. Traditional healers in Mali use A. mexicana to treat Malaria2 Ayurveda reported

that the plant is purgative, diuretic and destroys worms. It cures skin-diseases, leprosy and inflammation bilious fevers. Roots are equally used to cure anthelmintic. Juice is used to cure opacity of cornea and ophthalmia. Seeds are purgative and sedative. In Mexico the seed is used as an antidote to snake poisoning and the fresh yellow milky seed extract contains proteindissolving substances, effective in the treatment of warts, coldsores, cutaneous infections, skin diseases, itches and also dropsy and jaundice 3. The present study was to screen the leaf of the plant, A. mexicana, against some selected bacteria and fungi often implicated in nosocomial and community infection 4, 5, 6. Even today plants are the almost exclusive source of drugs for the majority of the world population. People in developing countries

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www.ijpbs.com (or) www.ijpbsonline.com utilize traditional medicine for their primary health care needs7, 8. The potential of higher plants as a source for new drugs is thus still largely unexplored9. This is also true in India and only a small percentage of plants of this region been evaluated for antibacterial activity against human pathogens 10, 11. Thus considering the vast potentiality of plant as a source of new therapeutic agents, hence detail investigations were conducted to test the efficacy of some plant extract against important human pathogenic bacteria.

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MATERIALS AND METHODS Plant Materials: Authenticated Fresh plant materials Argemone mexicana (Papaveraceae) (Leaf) free from disease were collected from Vidarbha region of Maharastra, washed thoroughly 2-3 times with running tap water and once with sterile water, shade-dried, powdered and used for extraction. Preparation of Solvent Extract: Sample (250 gm) of the shade-dried powder of Argemone mexicana was extracted in a Soxhlet extractor successively with 1 L Petroleum ether, Chloroform, and Methanol until colourless extract was obtained on the top of the extractor. Each of the solvent extract was concentrated separately under reduced pressure. After complete solvent evaporation, each of these solvent extracts was weighed and subjected to antimicrobial activity assay. For only methanol extract, which recorded highest antibacterial activity, the minimal inhibitory concentration (MIC) was determined12, 13. Antimicrobial assays The methods of Hufford et al. (1975) were used with some modification. Agar-well diffusion assay was used to evaluate the antimicrobial activities of the leaf extract. Mueller-Hinton agar (Scharlau Chemie) was used for the culturing of bacteria while Sabouraud Dextrose agar (Difco)

was used for the fungi. Twenty milliliters of the specified molten agar (45°C) was aseptically mixed with 1 ml of bacterial suspension (3 × 108 CFU/ml) and poured into sterile Petri dishes. Once the agar has hardened, 6mm wells were bored using a sterile cork borer. From the various concentrations of 500, 1000 and 1500 μl/ml of the Leaf extracts, which was prepared using methanol as diluents, 0.1 ml of the extract was separately placed into each well. The plates were incubated for 24 h at 37°C for the bacteria and 24 - 72 h at room temperature for the moulds. Ampicillin (10 μg) serves as positive control for the bacteria species while Fluconazole serves as positive control for the Candida species. The antimicrobial activity was measured as the diameter (mm) of clear zone of growth inhibition. Methanol was included in every experiment as negative controls 14, 15. Antibacterial Activity Assay: Antibacterial activity was determined by cup diffusion method on MHA medium the sterile medium (20ml) was poured into a 9 cm petriplates. The medium was allowed to cool in a sterile condition and plates were then inoculated with cultures of test bacteria. Agar cup of 5 mm diameter were made in the plates with the help of sterile borers. The desired different concentrations of the extracts, fractions and pure compounds were prepared by first reconstituting in methanol then diluting in sterile distilled water. A 100μl volume of each dilution was introduced in triplicate wells into MHA plates already seeded with the standardized inoculums of the test bacterial cells. All test plates were incubated at 37°C for 24h. The least concentration of each extract showing a clear zone of inhibition was taken as the MIC. Negative controls were prepared using the same solvent employed to dissolve the extracts. Gentamicin and Streptomycin were used as

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www.ijpbs.com (or) www.ijpbsonline.com positive reference to determine the sensitivity of each bacterial species tested 16. Antifungal Activity Assay: To evaluate the antifungal activity, sterile agar plates were used according to the disc diffusion assay. Activated cultures of fungal strains in Sabouraud’s broth.100 μl of the inocullum was introduced to molten Sabouraud dextrose agar and poured in the sterile Petri plates. Sterile filter paper discs (7.0 mm diameter) were impregnated with 500 μg/disc, 250 μg/disc and 125 μg/disc of the plants extracts dissolved in 100% DMSO (dimethylsulphoxide) and dried. The discs were placed on fungal seeded plates incubated at 28°C for 48hrs. Disc impregnated with only 100% DMSO served as the negative control. As a positive control, Flucanazole.The (10 μg/disc) was used. Following an incubation period of 48hrs, plates were removed from the incubator and antifungal activity was evaluated by measuring zones of inhibition of fungal growth.

RESULT AND DISCUSSION

antibacterial activity against Gram positive such as Bacillus subtilis, Staphylococus aureus and negative bacteria such as E.coli, Pseudomonas aereaginosa, Salmonella typhi, Proteus vulgaris but it doesn’t shown inhibitory action. This study revealed that the extract of plant Argemone mexicana leaf has a poor antibacterial action. And then this extract evaluated by antifungal activity against human pathogenic yeast strain of Candida albicans, Candida tropicalis, Aspergilus niger, Aspergilus flavus, Aspergilus candidus. The antifungal activity of Methanolic extracts of the plant Argemone mexicana leaf against fungal strain was shown on Table No.01 from this table it is revealed that the methanol extracts of plant leaf Argemone mexicana having the more potent activity against Candida albicans as compared to other yeast strain but it is moderate to flucanazole.The. And the Minimum inhibitory concentrations of plant leaf extract were shown on the Table No.02. From this table it was found that the lowest MIC value 3.12mg/ml for methanol extract against the Candida albicans as compared to other fungal strain.

In the initial stages the Methanolic extracts of plant Argemone mexicana leaf was evaluated by Table No.1: Antifungal activity of Methanolic extracts against different fungal strains S.No Organisms Methanol extracts Fluconazole 01 Candida albicans 24 mm 26 mm 02 Candida tropicalis 20 mm 23 mm 03 Aspergilus niger 22 mm 24 mm 04 Aspergilus flavus 18 mm 24 mm 05 Aspergilus candidus 18 mm 24 mm Table No.2: Minimum inhibitory concentration of extracts against different fungal strain Solvent Candida Candida Aspergilus Aspergilus Aspergilus Extract albicans tropicalis niger flavus candidus 3.12mg/ml

6.25mg/ml

12.5mg/ml

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Methanol

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CONCLUSION In vitro evaluation of plants for antimicrobial property is the first step towards achieving the goal for developing eco-friendly management of infectious diseases of humans by search for new bio-molecules of plant origin. Considering these, plant Argemone mexicana, screened in vitro for antibacterial as well as antifungal activity against eleven human pathogenic bacteria and yeast strain known to cause diseases in humans. The plant was selected based on traditional medicine knowledge. On the basis of zone of inhibition, the result of the present investigation revealed that the leaf of the plant Argemone mexicana leaf has a potential source of antifungal action and its activity against various clinical isolates may be sufficient to perform further studies for isolation and identification for active principles.

9. 10.

11.

12.

13.

ACKNOWLEDGEMENTS The authors are thankful to head Manoharbhai Patel Institute of Pharmacy Gondia M.S.India for providing laboratory facilities.

REFERENCE 1.

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14.

Siddiqui IA; Shaukat SS, Khan GH, Zaki MJ (2002) Evaluation of Argemone mexicana for control of rootinfecting fungi in potato J. Phytopathol. 150: 321-329. Chopra RN, Nayer A, Chopra IC (1986). Glossary of Indian Medicinal plants, (including the supplement). Council of Scientific and Industrial Research, (CSIR), New Delhi. Hufford CD, Funderburk, JM, Morgan, JM, Robertson LW, Two antimicrobial alkaloids from heartwood of Liriodendron tulip feral. J.Pharm. Sci. 64, 789–792. A. Osho and T. Adetunji Department of Microbiology, Olabisi Onabanjo University, P. M. B. 2002, Ago-Iwoye, Ogun state, Nigeria. Antimicrobial activity of the essential oil of Argemone mexicana Linn. Journal of Medicinal Plants Research 4 January, 2010 ;Vol. 4(1): pp. 019-022, Harborne, J.B., Phytochemical methods: A guide to modern techniques of plant analysis. 1998; 3rd edition. Chapman & Hall Pub. London, UK D.C. Mohana, S. Satish and K.A. Raveesha Antibacterial Evaluation of Some Plant Extracts Against Some

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Human Pathogenic Bacteria Department of Studies in Botany University of Mysore, Manasagangotri, Mysore - 570 006, India Advances in Biological Research 2008; 2 (3-4): 49-55, R. Perumal Samy and P. Gopalkrishnakone; venom and toxin Research programme, Department of anatomy yong loo lin school of medicine, National University of Singapore, Singapore 117597 on “ Current status of herbal and their future perspectives;28 sept. 2007 Palombo, E.A. and S.J. Semple, Antibacterial activity of traditional medicinal plants. J. Ethnopharmacol. 2001; 77: 151-157. Cowan, M.M., Plant products as antimicrobial agents. Clinical Microbiol. Rev. 1999; 12: 564-582. Dubey, N.K., R. Kumar and P. Tripathi. Global promotion of herbal medicine: Indian’s opportunity Current Sci., 2004; 86: 37-41. Patwardhan, B., A.D.B. Vaidya and M. Chorghade. Ayurveda and natural products drug discovery Current Sci., 2004; 86(6): 789-799. Kumar, P. Valuation of medicinal plants for pharmaceutical uses. Current Sci., 2004; 86(7): 930937. Sanaa O. Yagoub, Shami El Haj Al Safi, Braaha Ahmed and Asha Z. El Magbol; Department of Microbioloy and Molecular Biology, Faculty of science and Technology, El Neelain University, Sudan, Medicinal and Aromatic Plants, Ministry and Technology, Khartoum, Sudan on Antimicrobial activity of some medicinal plants against some Gram positive, gram negative and Fungi, 2007. In vitro antifungal activity of methanol extracts of some Indian medicinal plant against pathogenic yeast and moulds; Jigna Parekh and Sumitra Chanda, Phytochemical, Pharmacological and Microbiological Lab, Department of Bioscience, Saurashtra University, Rajkot 360 005, Gujrat, India, African Journal of Biotechnology, 3 December, 2008;Vol. 7 (23), pp. 43494353. Bhusan Bhaskarwar, Prakash Itankar and Abhay Fulke; Department of Pharmaceutical science, R.T.M. Unioversity, Nagpur 440, Evaluation of antimicrobial activity of medicinal plant Jatropha podagrica (hook) Roumanian Biotechnology letters, 2008; vol.13, no.5, pp. 3873-3877. 1D.C. Mohana, 2S. Satish and 3K.A. Raveesha 1Medical Microbiology Laboratory, Department of Microbiology and Biotechnology, Bangalore University, Jnana Bharathi Campus, Bangalore - 560 056, India 2Department of Studies in Microbiology, 3Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore - 570 006, India Antibacterial Evaluation of Some Plant Extracts Against Some

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Human Pathogenic Bacteria, Advances in Biological Research, 2008;2 (3-4): 49-55

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Yashwant Bais [Pharmaceutical Biotechnology] Department of Pharmaceutics Manoharbhai Patel Institute of Pharmacy Gondia M.S.India Email: yashwantbais2011 @gmail.com Phone â&#x20AC;&#x201C; 09423618759,09096237171.

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Research Article Biological Sciences ZINGIBER OFFICINALE ETHANOLIC EXTRACT INHIBITS FORMATION OF PSEUDOMONAS AERUGINOSA BIOFILM M. F. Z. R. Yahya*, N. F. H. A. Saifuddin and U. M. A. Hamid School of Biology, Faculty of Applied Sciences, MARA Technology University, Shah Alam 40450 Shah Alam, Selangor, Malaysia *Corresponding Author Email: fakharulzaman@salam.uitm.edu.my

ABSTRACT This study was carried out to determine antibiofilm activity of Zingiber officinale ethanolic extract against Pseudomonas aeruginosa. Retention time of phytochemical compounds separated by gas chromatography mass spectrometry ranged from 17.00 to 39.00 and a major compound identified was 1, 3- Cyclohexadiene, 5–(1,5dimethyl-4-hexenyl)-2-methyl. Microbroth dilution showed minimum inhibitory concentration of Z. officinale ethanolic extract against P. aerugionsa was 12.5 mg/ml. In disc diffusion assay, Z. officinale ethanolic extract was found to exhibit inhibitory effect against P. aerugionsa under both aerobic (diameter of zone of inhibition ranging from 6.2±0.2mm to 22.0±1.6mm) and anaerobic (diameter of zone of inhibition ranging from 13.6±2.9mm to 22.4±2.8mm) conditions. In the context of antibiofilm activity which was determined using static microplate biofilm formation assay, Z. officinale ethanolic extract demonstrated lower optical biofilm density than that of control under both aerobic (optical biofilm density ranging from 0.059±0.002 to 0.080±0.008) and anaerobic (optical biofilm density ranging from 0.060±0.003 to 0.066±0.005) conditions at all test concentrations. Furthermore, the treatment of Z. officinale ethanolic extract caused a reduction in the amount of extracellular biofilm DNA compared to the negative control under aerobic (200 mg/ml- 0.6424±0.019; 50 mg/ml-0.6688±0.112) and anaerobic (50 mg/ml-0.6248±0.003 conditions. Taken together, Z. officinale ethanolic extract possesses a promising potential in combating P. aeruginosa biofilm.

KEY WORDS antibiofilm activity; biofilms; Pseudomonas aeruginosa; Zingiber officinale

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INTRODUCTION For decades, traditional folks frequently use spices as remedies for various human diseases because these plant species contain components of therapeutic values. The household spice such as ginger (Zingiber officinale) has been accepted as an alternative form of health care which has raised a great interest in exploring its potential in combating bacterial infection. Originating from South East Asia, the ginger which belongs to family Zingiberaceae is also used in Ayurvedic medicine [1]. It is widely known to have thick

tuberous rhizomes which possess important medicinal values such as anti-inflammatory, cholesterol-lowering, and antithrombotic properties [1,2]. According to [3], the rhizome is rich in the secondary metabolites such as curcumene, non-volatile hydroxyaryl compounds e.g. zingerone, gin- geroles and shogaoles (phenylalkanones), volatile ses- quiterpenes (e.g. zingiberene and bisabolene) and monoterpenoids (e.g. citral). Although the antibacterial activity of Z. officinale has been investigated [3-6], the potential of Z. officinale in

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www.ijpbs.com (or) www.ijpbsonline.com controlling biofilm growth remains to be elucidated. Nowadays there is a growing interest in studying the microbial biofilms in order to overcome various human diseases including cystic fibrosis which is commonly associated with Pseudomonas aeruginosa pathogen. This facultative anaerobic bacterium colonizes and subsequently forms a biofilm layer lining the lungs prior to adaption with environment of partial or total oxygen depletion [7] and development of resistance towards many antibiotics. Considering the previously reported antibacterial activity of Z. officinale and the severity of chronic lung infection by P. aeruginosa biofilm, the present study was carried out to determine inhibitory effect of Z. officinale against P. aeruginosa biofilm formation.

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In this study, the rhizome of Z. officinale was extracted using 100% ethanol solvent and subjected to gas chromatography mass spectrometry (GCMS) analysis and determination of minimum inhibitory concentration (MIC). The antibacterial activity was assessed using disc diffusion assay, while the antibiofilm activity was evaluated using a static microplate biofilm formation assay. Both disc diffusion and microplate biofilm formation assays were performed under aerobic and anaerobic conditions. Materials and methods a. Plant extract and test microorganism Fresh rhizomes were washed with tap water, ground and extracted using 100% ethanol solvent for three days at room temperature. The solvent was then filtered and removed using rotary evaporator. Pseudomonas aeruginosa strain ATCC 10145 was obtained from culture

collection of the School of Biology, Faculty of Applied Sciences, UiTM Shah Alam and maintained on nutrient agar media. A batch culture of P. aeruginosa strain ATCC 10145 was grown at 37 °C in nutrient broth and its purity was assessed regularly by Gram-staining and colony morphology. b. Gas chromatography mass spectrometry (GCMS) analysis The phytochemical profile of the plant extract was studied using gas chromatography mass spectrometry (GCMS) which was performed by Agilent 7809A GC/5975MSD system at the flow rate of 1ml/min. Initial temperature was set to 50°C and was held for 2 minutes. The first rate was set to 4°C/min until it reached 200°C. Then the temperature was increased by 10°C/min until it reached 300°C. The start and end m/z ratios were 40 and 350. Helium was used as the carrier gas. Identification of phytochemical compounds was achieved by comparing the mass spectra and GC retention times with data system library of the GC-MS equipment, NIST05. c. Determination of minimum inhibitory concentration (MIC) Minimum inhibitory concentration (MIC) value of plant extract was determined using microbroth dilution method in Mueller-Hinton broth (MHB). Inoculum was prepared at a density adjusted to 0.5 McFarland turbidity standard [(108colony forming units (CFU/ml)] and diluted 1:10. The plant extract concentration ranged from 3.125 to 100 mg/ml whilst the final volume in each well of microplate was 200 μl. A positive control (containing 20 μl of inoculum and 180 μl of MHB) and negative control (containing 20 μl of plant extract and 180 μl of MHB without inoculum) were included on the microplate which was then incubated at 37°C. The MIC value was defined as the lowest concentration of Z. officinale

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www.ijpbs.com (or) www.ijpbsonline.com ethanolic extract at which the test microorganism did not show visible growth or turbidity and it was recorded after 24 h of incubation.

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d. Disc diffusion assay Antibacterial activity of the plant extract was determined using disc diffusion assay with sterilized paper discs containing plant extract at various concentrations (12.5 mg/ml to 200 mg/ml). The discs were applied on the surface of nutrient agar plates, which were previously swabbed uniformly with the bacterial suspension of 108 cfu· ml-1 (turbidity = McFarland standard 0.5). Erythromycin and DMSO were used as a positive and negative control respectively. The inoculated plates were then incubated in an upright position at 37℃ for 24 hours. The diameter of inhibition zones (diameter of paper disc, 6 mm was included) was measured in mm and the results were recorded. The disc diffusion assay procedure was performed in five replicates under both aerobic and anaerobic conditions. The anaerobic condition was developed using candle jar and a strip of paper soaked in methylene blue dye. e. Determination of optical biofilm density Antibiofilm activity of plant extract was assessed using a static microplate biofilm formation assay. Inoculum was prepared at a density adjusted to 0.5 McFarland turbidity standard [(108colony forming units (CFU/ml)] and diluted 1:10. The plant extract concentration ranged from 12.5 to 200 mg/ml. The final volume in each well of microplate was 210 μl (containing 150 μl of fresh nutrient broth, 30 μl of inoculums and 30 μl of plant extract). Erythromycin and DMSO were used as a positive and negative control respectively. Following 24 hours incubation at 37℃, the media containing planktonic cells and Z. officinale extracts was discarded while

surface-attached biofilm cells were stained with crystal violet for five minutes. The excess stain was rinsed off with tap water and optical biofillm density was determined using microplate reader at a wavelength of 570 nm. The static microplate biofilm formation assay procedure was performed in five replicates under both aerobic and anaerobic conditions. The anaerobic condition was developed using candle jar and a strip of paper soaked in methylene blue dye. The same assay procedure was repeated for another microplate to determine extracellular biofilm DNA. e. Determination of extracellular biofilm DNA Extracellular biofilm DNA from the static microplate biofilm formation assay was measured using spectrophotometry. Following 24 hours biofilm incubation and removal of media containing planktonic cells and Z. officinale extract as described earlier, a volume of 200 μl of phosphate buffer saline (PBS) was added to each well of the microplate to solubilize the biofilm cells. The biofilm suspension was then incubated at room temperature for one hour and its extracellular DNA was measured using microplate reader at a wavelength of 260 nm. f. Statistical analysis All data from disc diffusion assay and biofilm formation assay were expressed as means±S.E.M. and ANOVA was performed to determine the degree of significance between the groups whereby p<0.05 was considered significant.

RESULTS AND DISCUSSIONS a.Phytochemical profile of Z. officinale ethanolic extract Phytochemical compounds are of key importance in explaining the biological activites

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of medicinal plants. In this study, the identified with acceptable quality value (>85) phytochemical profiling of Z. officinale ethanolic (Table 1). The major compound recorded from Z. extract was carried out using GCMS. Figure 1 officinale ethanolic extract was 1, 3 shows chromatogram of phytochemical Cyclohexadiene, 5 – (1, 5 – dimethyl – 4 compounds present in Z. officinale ethanolic hexenyl) – 2 - methyl (RT: 18.01; peak area: extract. The phytochemical compounds were 45.24%) which is commonly known as identified by mass spectral interpretation and Zingiberene. Our GCMS data has revealed the library searches. The retention time of the presence of phytochemical compounds in Z. chromatographically separated phytochemical officinale which are known to possess important compounds ranged from 17.00 to 39.00. medicinal value. However, there were only four compounds Figure 1 Chromatogram of Z. officinale ethanolic extract

Table 1: Identified phytochemical compounds with their retention time, composition and quality values Compounds Common name Retention Area Quality time % Benzene, 1-(1,5-dimethyl-4-hexenyl) -4-methyl Curcumene 17.70 12.9 99

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1, 3- Cyclohexadiene, 5 – (1,5-dimethyl-4-hexenyl)-2- Zingiberene methyl Cyclohexene, 1-methyl-4-(5-methyl-1-methylene-4- Bisabolene hexenyl) Cyclohexene, 3-(1,5-dimethyl-4-hexenyl)-6- β-esquiphellandrene methylene Recently, Bhargava et al. [6] performed a study on Z. officinale with respect to its phytochemical profile and antibacterial activity. In that study,

18.01

45.24

18.32

17.2

18.7

24.65

the rhizome of Z. officinale was extracted using 100% ethanol and methanol. Following the GCMS analysis, the Zingiberene compound was

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www.ijpbs.com (or) www.ijpbsonline.com found to be present in both plant extracts (20.57% and 15.32% respectively). Zingiberene was found to be the major compound in both plant extracts, which is agreement with the findings herein. b. Minimum inhibitory concentration (MIC) value and zone of inhibition Minimum inhibitory concentration (MIC) value is important to determine efficacy of antibacterial agent. Low MIC value may be an indication of high efficacy or that microorganism has no potential to develop resistance towards the bioactive compound. The MIC value against P. aeruginosa measured for Z. officinale ethanolic extract was 12.5 mg/ml. This MIC value was then being used for disc diffusion assay to determine antibacterial activity of Z. oficinale ethanolic extract.

Disc diffusion assay has been the most common method for antibacterial screening. Considering the pathogenicity of P. aeruginosa in both aerobic and anaerobic environments, the antibacterial potential of Z. officinale ethanolic extract was evaluated under both aerobic and anaerobic conditions. Based on Table 2, Z. officinale ethanolic extract exhibited inhibitory effect against P. aerugionsa under both aerobic (diameter of zone of inhibition ranging from 6.2±0.2mm to 22.0±1.6mm) and anaerobic (diameter of zone of inhibition ranging from 13.6±2.9mm to 22.4±2.8mm) conditions. At all test concentrations except 200 mg/ml, its antibacterial activity under anaerobic condition was significantly (p < 0.05) higher than that of aerobic condition. It was likely that Z. oficinale ethanolic extract has a greater antibacterial activity under anaerobic condition.

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Table 2: Antibacterial activity of Z. officinale ethanolic extract measured as diameter (mm) of zone of inhibition with =5 Concentration Aerobic Anaerobic Significant difference (p < 0.05) 200mg/ml 22.0±1.6mm 22.4±2.8mm No 100mg/ml

15.8±1.9mm

21.4±2.3mm

Yes

50mg/ml 25mg/ml 12.5mg/ml +ve control

14.4±2.2mm 10.4±1.7mm 6.2±0.2mm 18.0±9.0mm

19.6±2.8mm 17.8±3.1mm 13.6±2.9mm 15.2±0.6mm

Yes Yes Yes No

-ve control

0.0±0.0mm

Gao and Yang [5] reported the antibacterial activity of polysaccharide, flavonoid, aqueous and ethanolic extract from Z. officinale against various microorganisms including P. aeruginosa. However, that study did not include the antibacterial activity of Z. officinale ethanolic extract under anaerobic condition. Meanwhile, the antibacterial activity of vancomycin antibiotics has also been studied under aerobic and anerobic environments [8]. As shown by

flow cytometric data and CFU number, inhibitory effect of vancomycin antibiotics against Staphylococcus aereus under aerobic condition was greater than that of anaerobic condition despite its MIC value was similar in both environments. In the context of our study, the Z. officinale ethanolic extract has also the antibacterial activity against P. aeruginosa in the anaerobic environment thereby updating the

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www.ijpbs.com (or) www.ijpbsonline.com current information of medicinal value of Z. officinale ethanolic extract.

observed that the Z. officinale ethanolic extract inhibited P. aeruginosa biofilm formation under both aerobic (optical biofilm density ranging from 0.059±0.002 to 0.080±0.008) and anaerobic (optical biofilm density ranging from 0.060±0.003 to 0.066±0.005) environments at all test concentrations. At test concentration of 50 mg/ml, 100 mg/ml and 200 mg/ml, the antibiofilm activity of Z. officinale ethanolic extract under aerobic condition was not significantly (p > 0.05) different from that of anaerobic environment. This may suggest that antibiofilm activity of Z. oficinale ethanolic extract under aerobic condition was equivalent to that of anaerobic condition.

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c. Optical biofilm density Nowadays, microbial biofilm cells have been a great interest in development of antimicrobial agent due to its higher antibiotics resistance as compared to their planktonic counterparts. In the present study, inhibitory effect of Z. officinale ethanolic extract against formation of P. aeruginosa biofilm was determined using static microplate assay system. Table 3 illustrates optical biofilm density at wavelength of 570 nm. Inhibition of biofilm formation was indicated by optical biofilm density of the test concentrations lower than that of negative control. It was Table 3: Antibiofilm activity of Z. officinale ethanolic extract measured as optical biofilm density at a wavelength of 570 nm with n=5 Concentration Aerobic Anaerobic Significant difference (p < 0.05) 200mg/ml 0.064±0.005 0.060±0.003 No 100mg/ml 0.059±0.002 0.060±0.008 No 50mg/ml 0.063±0.004 0.065±0.007 No 25mg/ml 0.074±0.010 0.060±0.004 Yes 12.5mg/ml 0.080±0.008 0.066±0.005 Yes +ve control 0.076±0.002 0.078±0.002 No -ve control 0.082±0.011 0.109±0.028 Yes In the last few years, medicinal plant and marine bacterial products have widely been investigated for their antibiofilm potential. The natural resources has become a new interest in developing antimicrobial agents because there are many resistance problems reported to be associated with the commonly used antibiotics. In 2011, Vacheva et al. [9] reported the antibiofilm activity of 14 plant extracts against three clinical isolates of Escherichia coli. In that study, it was observed that the growth percentage of the biofilm treated with plant extracts ranged from 34% to 367%. Some plant

extracts were found to favor the E. coli biofilm growth which explained the growing resistance of E. coli isolates towards the selected plant extracts. We believe that the resistance of P. aeruginosa biofilm towards Z. officinale ethanolic extract was not observed in this study. A number of studies have attempted to explain the possible general antibiofilm mechanism using various experimental tools including electron microscopy. Recently, Chusry et al. [10] demonstrated that treatment of Quercus infectoria G. Olivier extract and tannic acid resulted in formation of clumps of

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staphylococcal cells with thickened and slightly rough cell wall. This observation was supported by another finding from the same study where those treatments increased staphylococcal cell surface hydrophobicity (CSH). They concluded that changes in hydrophobicity index mediated the antibiofilm activity of Quercus infectoria G. Olivier extract and tannic acid. Furthermore, understanding of antibiofilm mechanism has been greater when accounts for aspect of cellsurface interaction as reported by [11]. In that study, it was revealed that a bacterial exopolysaccharide (A101) inhibited the cellsurface interaction and multicellular aggregates formation happened on S. aureus and P. aeruginosa which was detected by phasecontrast microscopy with 600 magnifications. However, the A101 was only able to disrupt the multicellular aggregates of P. aeruginosa FRD1, but not that of S. aureus RN6390. They proposed that the antibiofilm activity of A101 may involve irreversible binding or adsorption in S. aureus RN6390, and reversible in P. aeruginosa FRD1. Considering the fact that a particular antibiofilm agent can inhibit biofilm either by causing formation of multicellular clumps or interfering with the microbial cell-surface interaction, it was possible that the Z. officinale ethanolic extract exhibited the same antibiofilm mechanism. d. Extracellular biofilm DNA Cell death and lysis associate with release of most intracellular contents into extracellular environment including deoxyribonucleic acid (DNA). Also, microbial biofilms release their DNA into extracellular environment in order to make them incorporated into extracellular polysaccharide (EPS) matrix [12] which protects them from antibiotics treatment. Considering these facts, one of the possible ways to investigate further the antibiofilm effect of the plant extract is determining amount of

extracellular DNA. In the present study, the extracellular biofilm DNA following treatment of Z. officinale ethanolic extract was measured spectrophotometrically. Table 4 depicts amount of extracellular biofilm DNA at a wavelength of 260nm. It was noted that the amount of extracellular biofilm DNA of several test concentrations were lower than that of negative control under aerobic (200 mg/ml-0.6424±0.019; 50 mg/ml-0.6688±0.112) and anaerobic (50 mg/ml-0.6248±0.003) environments. At all test concentrations except 50 mg/ml, the amount of extracellular biofilm DNA under aerobic condition was significantly (p < 0.05) different from that of anaerobic environment. A study of DNA release by microbial cells has been reported by [13] where the amount of extracellular DNA was measured using propidium iodide in order to determine effect of iron on DNA release. That experiment showed that a high level of iron (100 mM FeCl3) in the medium suppressed DNA release, structural biofilm development, and increased resistance of subpopulations towards antimicrobial compounds which might be due to suppression of pqs quorum-sensing systems. The extracellular DNA was suggested to be critical in the biofilm formation following treatment of DNAse I. However, in that study, planktonic cells were not separated from biofilm cells thereby the effect of iron was also accounting for the extracellular DNA of planktonic cells. According to [14] the extracellular DNA possess an antibacterial activity which contributed to cell lysis by chelating cations that stabilize lipopolysaccharide (LPS) and the outer membrane (OM). This DNA-mediated killing resulted in the release of cytoplasmic contents, including genomic DNA. Moreover, that study also demonstrated that the DNA release induced the PhoPQ- and PmrAB-regulated cationic

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www.ijpbs.com (or) www.ijpbsonline.com antimicrobial peptide resistance operon PA3552–PA3559 in P. aeruginosa which resulted in 640-fold increased resistance to aminoglycosides. They also concluded that the extracellular DNA caused development of cation gradients, genomic DNA release and antibiotic resistance. With regard to the cell lysis that associates with DNA release, Qin et al. [15] revealed that the release of genomic DNA into extracellular environment is mediated by autolysin. By using Staphylococcus epidermidis as a model of microbial biofilm, they suggested that the extracellular DNA is generated in bacterial populations through AtlE-mediated lysis of a subpopulation of the bacteria, and that the extracellular DNA promotes biofilm formation of the remaining population. The significance of extracellular DNA as a universal component of

the biofilm matrices has also been supported by [16] which proposed that the changes of the biofilm biomass in the presence of DNase I may also be observed in the biofilms of different unrelated gram-positive and gram-negative bacteria. Our result was in agreement with that suggestion where the negative control also released a certain amount of genomic DNA into extracellular environment under both aerobic and anaerobic conditions. Considering the possible antibiofilm mechanism of Z. officinale ethanolic extract with regards to inhibition of cell-surface interaction, we believe that the treatment of the plant extract should result in low extracellular biofilm DNA. Meanwhile, inconsistency of our data (Table 4) should be further validated using propidium iodide as described by [13].

Table 4: Antibiofilm activity of Z. officinale ethanolic extract measured as amount of extracellular biofilm DNA at a wavelength of 260 nm with n=5. Concentration Aerobic Anaerobic Significant difference (p < 0.05) 200mg/ml 0.6424±0.019 0.9514±0.062 Yes 100mg/ml 0.9168±0.067 0.7152±0.177 Yes 50mg/ml 0.6688±0.112 0.6248±0.003 No 25mg/ml 0.9222±0.636 0.825±0.2489 Yes 12.5mg/ml 0.9414±0.554 0.7118±0.1357 Yes +ve control 0.5842±0.0064 0.6078±0.0055 Yes -ve control 0.713±0.0310 0.685±0.0066 No

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CONCLUSION We have demonstrated that Z. officinale ethanolic extract has antibacterial activity against P. aeruginosa under both aerobic and anaerobic conditions. Further investigation showed that it is also able to inhibit P. aeruginosa biofilm formation under both aerobic and anerobic conditions. With respect to extracellular biofilm DNA, it seems that treatment of Z. officinale ethanolic extract has

also affected the DNA release by P. aeruginosa biofilm. We believe that the antibiofilm activity against P. aeruginosa observed in this study is due to the activity of zingiberene, the major compound in Z. officinale ethanolic extract.

ACKNOWLEDGEMENT This study was supported by Research Management Institute (RMI) UiTM under a

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REFERENCES [1]Tan B. K. H. and Vanitha J.. Immunomodulatory and Antimicrobial Effects of Some Traditional Chinese Medicinal Herbs: A Review. Current Medicinal Chemistry, Vol. 11, No. 11: 1423-1430. (2004). [2]Zaika L. L.Spices and Herbs: Their Antimicrobial Ac- tivity and Its Determination. Journal of Food Safety, Vol. 9, No. 21: 97-118. (1975). [3]Bensky D. and Gamble A. Chinese Herbal Medicine, Materica Medica. Eastland Press Inc, Seattle.(1993). [4]Kaushik P. and Goyal P..Evaluation of Various Crude Extracts of Zingiber officinale Rhizome for Potential Antibacterial Activity: A Study in Vitro. Advances in Microbiology, vol. 1: 7-12. (2011). [5]Gao D. and Zhang. Y. Comparative antibacterial activities of crude polysaccharides and flavonoids from Zingiber officinale and their extraction. Asian Journal of Traditional Medicines, vol.235-238. (2010). [6]Bhargava, S., Dhabhai, K. , Batra, A., Sharma A. and Malhotra B. Zingiber Officinale : Chemical and phytochemical screening and evaluation of its antimicrobial activities. Journal of Chemical and Pharmaceutical Research, vol. 4(1): 360-364. (2012). [7]Hassett. D. J. Anaerobic production of alginate by Pseudomonas aeruginosa: alginate restricts diffusion of oxygen. J. Bacteriol. vol. 178 (24): 7322–7325. (1996). [8]Suller M. T. and Lloyd D.The antibacterial activity of vancomycin towards Staphylococcus aureus under aerobic

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|46-54 and anaerobic conditions. J Appl Microbiol. 92(5):866-72. (2002). [9]Anna V., Byulent M., Jordanka S., Mariana M., Sonya K., Milka T.,Radka I., Stoyanka S.. Effects of extracts from medicinal plants on biofilm formation by Escherichia coli urinary tract isolates. Biotechnol. & Biotechnol. vol. 25(4):92-97. (2011). [10]Chusri S., Na P. P. and Voravuthikunchai S. P. Antibiofilm activity of Quercus infectoria G. Olivier against methicillin-resistant Staphylococcus aureus. Lett Appl Microbiol.;54 (6):511-517. (2012). [11]Jiang P, Li J, Han F, Duan G and Lu X. Antibiofilm Activity of an Exopolysaccharide from Marine Bacterium Vibrio sp. QY101.PLoS ONE 6(4): 1-12.(2011). [12]Whitchurch, C. B., Tolker-Nielsen, T., Ragas, P. C. and Mattick, J. S. Extracellular DNA required for bacterial biofilm formation. Science 295:1487. (2002). [13]Liang Y., Kim B. B, Mette E. S., Allan B. C., Michael G. and Tim T. N.. Effects of iron on DNA release and biofilm development by Pseudomonas aeruginosa.Microbiology. vol. 153:1318–1328. (2007). [14]Mulcahy H., Charron-Mazenod L. and Lewenza S. Extracellular DNA Chelates Cations and Induces Antibiotic Resistance in Pseudomonas aeruginosa Biofilms. PLoS Pathog 4(11): e1000213. [15]Qin Z, Ou Y, Yang L, Zhu Y, Tolker-Nielsen T, Molin S and Qu D. Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis. Microbiology. vol. 153:2083-2092. (2007). [16]George V. Tetz, Natalia K. Artemenko, and Victor V. Tetz. Effect of DNase and Antibiotics on Biofilm Characteristics. Antimicrobial Agents and Chemotherapy. vol. 53(3): 1204–1209. (2009).

*Corresponding Author:

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Mohd Fakharul Zaman Raja Yahya* School of Biology, Faculty of Applied Sciences MARA Technology University, Shah Alam 40450 Shah Alam, Selangor, Malaysia Email: fakharulzaman@salam.uitm.edu.my Tel: +60355444625, Fax: +60355444562

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Review Article Pharmaceutical Sciences OBSTRUCTING SHEDDING OF MIC: THE WAY FORWARD FOR CANCER TREATMENT D. O. ACHEAMPONG*, J. ZHANG and M. WANG Department of Molecular Biology, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China. *Corresponding Author Email: do.acheampong@yahoo.com

ABSTRACT One of the immunosurveillance mechanisms of the immune system is the expression of Major Histocompatibility Complex class I-related chain molecules A and B (MIC-A and B) on tumor cell surface. MIC-A and B are the ligands of an activating receptor, NKG2D expressed on the natural Killer cells (NK), therefore binding of NK cells to tumor cells through the interaction between NKG2D and MIC-A or MIC-B induces cytolysis of tumor cells. However, clinical observations of most of the human epithelial tumors are found to be MIC-positive rather than MICnegative, suggesting a functional compromise of the MIC ligand-NKG2D receptor system in cancer patients and therefore allow the growth of MIC positive tumor cells. This is made possible by the release of soluble forms of MIC-A/B from tumor cells which down regulates the NKG2D surface expression on effector cells. This review article therefore sought to discuss the mechanisms underlining the shedding of MIC from tumor cells and how they can be explored by researchers to design drugs for anti-cancer treatment. A literature search on the possible causes of MIC shedding was done. Endoplasmic reticulum protein 5 (ERp5) and A Disintegrin And Metalloproteinase (ADAM10 and ADAM17) have been implicated as responsible for the MIC shedding. Also the α3 ectodomain of the MIC has been identified as target site for these shedding agents. Anti-cancer drugs can possibly be designed using known inhibitors of ERp5, ADAM10 and ADAM17. We also believe, producing therapeutically effective amount of a purified antibody or a polypeptide comprising an antigen-binding fragment thereof that specifically binds to the α3 ectodomain of a MIC polypeptide is the way forward for cancer treatment.

KEY WORDS Major Histocompatibility Complex class I-related chain molecules A and B, Endoplasmic reticulum protein 5, A Disintegrin and Metalloproteinase, α3 ectodomain, NKG2D, immunosurveillance mechanisms

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INTRODUCTION The interaction between cancer and the immune system is basically characterized by three phases comprising; early immune-mediated tumor elimination, an equilibrium phase and the evasion of tumors from immunosurveillance [1, 2, 3]. As was stated by Waldhauer and his research group [1] scientists face major difficulty in explaining how the immune system is able to recognize malignant autologous cells and to mount an anti tumor immune response in spite of its self-telerant attitude. One of the

immunosurveillance mechanisms of the immune system is the expression of MHC class I-related chain molecules A and B (MIC-A and B) on tumor cell surface. MIC-A and B are the ligands of an activating receptor, NKG2D expressed on the natural Killer cells (NK) [4, 5]. Binding of NK cells to tumor cells through the interaction between NKG2D and MIC-A or MIC-B induces cytolysis of tumor cells [4, 5, and 6]. Nevertheless, clinical observations of most of the human epithelial tumors are found to be MIC-positive rather than MIC-negative [7-11]. This therefore suggests a

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www.ijpbs.com (or) www.ijpbsonline.com functional compromise of the MC ligand-NKG2D receptor system in cancer patients and therefore allows the growth of MIC positive tumor cells [11]. In vitro studies elsewhere have shown that engagement of soluble MIC-A/B to NKG2D results in significant reduction in surface NKG2D expression on NK and T cells [10, 11]. This leads to down regulation of NKG2D expression. Shedding of MIC-A/B from tumor cells surface accounts for most of the soluble MIC-A/B [12], therefore any therapeutic prevention of MIC-A/B shedding could lead to the discovery of new cancer treatments. This review article seeks to bring to light the relevance of MIC-A/B to cancer treatment and also some of the methods which have been used by other researchers elsewhere in obstructing the shedding of MIC-A/B as an option for cancer treatment.

human alleles [14, 15]. MIC-A/B is usually expressed minimally on normal cells but is frequently expressed on epithelial tumors and can also be induced by bacterial or viral infections, acting as stress proteins. Receptor for MIC-A/B, NKG2D, is an activating receptor expressed on NK cell, NKT cells, γδ T cells and CD8+ αβ T cells [14, 16]. The recognition of MICA/B by the receptor NKG2D results in the activation of cytolytic activity or cytokine production by the effector cells. This recognition is involved in tumor surveillance, viral infections, bacterial infections and autoimmune diseases. Studies elsewhere have shown that, the release of soluble forms of MIC-A/B from tumor cells down regulate the NKG2D surface expression on effector cells resulting in the impairment of the anti-tumor surveillance system [10, 14, and 16].

MATERIALS AND METHODS

NKG2D NKG2D is an activating receptor found on NK cells and CD8+ T cells (αβ and γδ). It was first identified in 1991 and encoded by the KLRK1 gene [17]. NKG2D is made up of two disulphidelinked type II transmembrane proteins with short intracellular proteins which are not capable of transducing signals. To overcome this difficulty, they use two adaptor proteins DAP10 and DAP12, which associate a homodimer to the NKG2D and therefore the entire receptor complex appears as hexamer [18]. NKG2D function as an activating receptor was first described in 1999 [19] and its ligands are stressed induced proteins which include MIC-A, MIC-B and ULBP1-6 for human NKG2D, and Rae1, Mult 1and H-60 for murine NKG2D [20]. These ligands are induced during cellular stress such as infections or genomic stress (cancer), which renders the cell susceptible to NK cell mediated lysis. The function of NKG2D on CD8 T cells is to send co-stimulatory signals to activate them [21]. As cancerous cells are "stressed", NKG2D

This review was done by compiling references from major databases like PubMed, Science Direct, Google scholar, Scopus, Online journals, Open J Gate, etc.

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MIC MIC-A/B (MHC class I chain-related gene A/B) are transmembrane glycoproteins that function as ligands for human NKG2D. MIC-A and MIC-B are closely related proteins in that, MIC-A shares 85% amino acid identity with MIC-B [4]. The two proteins are distantly related to the MHC class I proteins [4, 5]. They (MIC-A/B) are made up of three extracellular immunoglobulin-like domains but have no capacity to bind peptide or interact with β2-microglobulin [13]. The genes that encode MIC-A/B are found within the major histocompatibity complex (MHC) on human chromosome 6. The MIC-A locus is highly polymorphic with more than 50 recognized human alleles whereas MIC-B locus is polymorphic with a little over 15 recognized

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www.ijpbs.com (or) www.ijpbsonline.com ligands become upregulated, rendering the cell susceptible to NK cell-mediated lysis. It is therefore unsurprising that tumor cells have also developed strategies to evade NKG2D responses [18]. Two Protein Agents Implicated for MIC-A/B Shedding 1. ERp5 Endoplasmic reticulum protein 5 (ERp5) is a member of a group of proteins called Endoplasmic reticulum proteins (ERps) which are widely expressed proteins that are associated with Endoplasmic reticulum (ER) and may act as proteases, protein disulfide isomerases, thioldisulfide oxidases or phospholipases [22]. ERp5, also called PDIA6 (protein disulfide isomerase family A, member 6) or TXNDC7 is a 440 amino acid protein that contains two thioredoxin domains and belong to the protein disulfide isomerase family. It is localized to the melanosome as well as to the lumen of the ER [22]. ERp5 functions as a catalyst to the rearrangement of disulfide bonds in different proteins. Through its catalytic activity, ERp5 is able to reduce the disulfide bond that binds MIC-

A/B to tumor cells, thereby releasing MIC-A/B and reducing the rate of tumor expression and down regulating NKG2D receptor [22, 23]. ERp5 specifically binds to the amino acid sequence NGTYQT located in the Îą3 ectodomain of a MIC polypeptide therefore any therapeutic intervention should be targeted to this site to avoid ERp5 attachment which leads to the shedding of the MIC [24, 25]. 2. ADAM ADAM (A Disintegrin And Metalloproteinase) is a family of peptidase proteins [26, 27]. It is also referred to as the adamalysin family or MDC family (metalloproteinase-like, Disintegrin-like, Cysteine rich) [26, 28]. They are classified as sheddases because they are able to cut off extracellular portions of transmembrane proteins [29]. For example, according to Inja Waldhauer and his research group, ADAM10 and ADAM17 which are members of the ADAMâ&#x20AC;&#x2122;s family are associated with MIC-A shedding by tumor cells that promote tumor growth [30]. Therefore therapeutic ADAM inhibitors can potentially be anti-cancer therapy [30]. Fig. 1 illustrates the potential functions of the family members of ADAM.

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Fig. 1 Health and Disease Conditions Associated with ADAM members [31]

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www.ijpbs.com (or) www.ijpbsonline.com I. ADAM10 ADAM10 (A Disintegrin and metalloproteinase domain-containing protein10) is a protein encoded by the ADAM10 gene in human. It is known to involve in ectodomain shedding of various cell surface proteins such as growth factors, receptor and their ligands, cytokines and cell adhesion molecules [31]. As was reported by Inja Waldhauer and his group, it could play a role in the MIC-A shedding by tumor cells [30].

Although the exact mechanism of ADAM10 has not been thoroughly investigated, its active site is homologous to those of well studied zincproteases such as carboxypeptidase. Therefore it is believed that ADAM10 utilizes a similar mechanism as these enzymes. As shown in Fig. 2, in zinc proteases, the key catalytic elements have been identified as a glutamate residue and a Zn2+ ion coordinated to histidine residue [32].

Fig. 2 Diagram of an ectodomain shedding ADAM metalloprotease[33].

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II. ADAM17 ADAM metallopeptidase domain 17 (ADAM17), also called TACE (tumor necrosis factor- Îąconverting enzyme), is a 70-KDa enzyme [34]. It is known to be involved in the processing of tumor necrosis factor alpha at the surface of the cell. This process which is also known as shedding, involves the cleavage and release of a soluble ectodomain from membrane-bound proproteins [30, 34]. Share most of the features of ADAM10 described above [30].

Proposed Therapeutic interventions to the Shedding of MIC from Tumor cells As already mentioned, MIC shedding is considered a principal mechanism of tumor cells to escape from NKG2D mediated immunosurveillance in human. This does not only lead to reduction of MIC surface density but also generates soluble MIC, which eventually down regulate NKG2D and therefore results in immunosuppression [30, 35, 36]. This constituted the reason for the research by Inja Waldhauer and his group [30]. They came out with the following results after their research; (a) MICA cleavage occurs at the surface of tumor

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cells, (b) MICA is cleaved within the juxta membranous stalk, (c) MICA cleavage is dependent on the length but not on the sequence of the stalk, (d) MICA shedding is inhibited by broad-range metalloproteinase inhibitors but not by (Matrix metalloproteinase) MMP-specific Tissue inhibitor of metalloproteinase 2 (TIMP2), (e) MICA shedding is induced by protein kinase C (PKC), ( f ) constitutive and induced MICA shedding is variably affected by ADAM-specific inhibitors GI254023X and GW280264X, and (g ) shedding of MICA (and ULBP2) is suppressed on silencing of ADAM10 and/or ADAM17. They were however quick to state that, there is a possibility that other proteases additionally contribute to MIC-A shedding [30]. In another related research by Hermann C Altmeppen and his group on “Lack of a-disintegrin-and-metalloproteinase ADAM10 leads to intracellular accumulation and loss of shedding of the cellular prion protein in vivo”, their findings supported the fact that ADAM10 could play a role in the shedding of many proteins [37]. They used neuron-specific ADAM10 knockout mice to show that ADAM10 is the sheddase of PrPc and that its absence in vivo leads to increased amounts and accumulation of PrPc in the early secretory pathway by affecting its posttranslational processing [37]. All things being equal, application of appropriate ADAM inhibitors could obstruct the shedding of MIC from tumor cells. This could be explored further to develop treatment for cancer. Jennifer D. Wu worked on the topic “Methods of treating cancer by inhibiting MIC shedding” and subsequently received patent for her findings [38]. Her invention was based on the fact that expression of murine NKG2D ligands on tumor cells has been shown to be effective in activating NK-mediated tumor elimination experimentally [39]. Her invention describes methods for

treating cancer comprising administering a therapeutically effective amount of an agent that prevents MIC shedding mediated by the alpha-3 ecto domain of MIC of a tumor cell, thus rendering the tumor cell more sensitive to innate immune cell rejection [38]. One of the highlights of her invention was the proposed use of a therapeutically effective amount of a purified antibody or a polypeptide comprising an antigenbinding fragment thereof that specifically binds to the amino acid sequence NGTYQT (SEQ ID NO: 1) located in the α3 ectodomain of a MIC polypeptide; wherein the interaction of the MIC polypeptide and ERp5 is inhibited; and whereby the shedding of said MIC polypeptide is inhibited; whereby said MIC-positive cancer is treated [38]. This demonstrates clearly that ERp5 has something to do with the shedding of MIC from tumor cells and it does it by binding to α3 ectodomain of a MIC polypeptide. Therefore any therapeutic intervention that can bind to this specific site or prevent ERp5 from binding to this site as has been discovered by Jennifer D. Wu could prove effective for treating cancer. Another research by Jennifer D. Wu and her research team entitled “Obstructing Shedding of the Immunostimulatory MHC Class I ChainRelated Gene B Prevents Tumor Formation” actually identified the α3 ectodomain of MIC as the target site for shedding. After extensive research and study on the subject matter, they reported that partially replacing the α3 domain of MICB protects from tumor cell shedding. This was after they have generated mutant form MICB.A2 by replacing part of the α3 ectodomain of MICB (amino acids 215-274) with the corresponding residues from HLA-A2 and using ELISA assay to assess the degree of shedding, knowing very well that NKG2D only interacts with the α1 and α2 domain of MIC [11 ]. In another research entitled MHC Class I chainrelated protein A antibodies and shedding are

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www.ijpbs.com (or) www.ijpbsonline.com associated with the progression of multiple myeloma, Masahisa Jinushi and his research team made a very striking conclusion which implicated ERp5 as the cause of MIC shedding from the tumor cell they worked on [40 ]. They observed that, during full-blown multiple myeloma (MM), the up-regulation of ERp5 promotes efficient MICA shedding, which evokes NKG2D internalization and immune suppression. Thus, stage-specific alterations in MICA activity are associated with the conversion of immune equilibrium to immune escape [40]. In another development, Koji Yamanegi and his group’s quest to search for the remedy to the shedding of MIC from tumor cells led them to investigate the effect of sodium valproate (VPA), a histone deacetylase inhibitor, on the production of cell-surface and soluble MIC and NK cell-mediated cytotoxicity in four human osteosarcoma cells [6]. Their result showed that, VPA at 0.5 and 1.0 mM induced acetylation of histones bound to MICA and B gene promoters, increased cell-surface but not soluble MIC, and therefore augmented the susceptibility of osteosarcoma cells to NK cell-mediated cytotoxicity. They therefore concluded that VPA in combination with immunotherapy activating cytotoxic immune cells could be useful to treat osteosarcomas [6]. This should be investigated further since it could lead to the discovery of an anti-cancer treatment.

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CONCLUSION MIC shedding is considered a principal mechanism through which tumor cells escape from NKG2D-mediated immune surveillance in human [10, 11]. The shedding of MIC by tumor cells can therefore promote tumor growth. Considering the fact that MIC is a stress protein mostly expressed on stressed cells like tumor cells, it could have been easier to eliminate

tumor cell through NKG2D-mediated immunosurveillance but for its shedding tendencies. To overcome this difficulty, researchers have tried understanding the mechanism of MIC shedding through research and have implicated ERp5, ADAM10 and ADAM17 as the likely cause [22, 30]. Also the α3 ectodomain of the MIC has been identified as target site for the shedding agents [11]. Jennifer D. Wu has even gone further to identify the specific site the enzymes responsible for the shedding attaches [38]. We believe that armed with all this relevant information about MIC shedding, it is time researchers explored it to come out with efficient cancer treatment drugs, as it has been done by Jennifer D. Wu. Anticancer drugs can possibly be designed using known inhibitors of ERp5, ADAM10 and ADAM17. We also believe, producing therapeutically effective amount of a purified antibody or a polypeptide comprising an antigenbinding fragment thereof that specifically binds to the amino acid sequence NGTYQT (SEQ ID NO: 1) located in the α3 ectodomain of a MIC polypeptide as prescribed by Jennifer D. Wu in her patent report is the way forward for cancer treatment [38].

REFERENCE [1] Waldhauer I, et al: Tumor-Associated MICA is shed by ADAM Proteases. Cancer Res, 68(15): 6368-6376, (2008). [2] Dunn GP, Old LJ and Schreiber RD: The three Es of cancer immunoediting. Annu Rev Immunol, 22: 329-360, (2004). [3] Smyth MJ, Dunn GP and Schreiber RD: Cancer immunosurveillance and immunoediting: the roles of immunity in suppressing tumor development and shaping tumor immunogenicity. Adv Immunol , 90: 1-50, (2006). [4] Waldhauer I and Steinle A: NK cells and cancer immunosurveillance. Oncogene, 27: 5932-5958, (2008). [5] Nausch N. and Cerwenka A: NKG2D ligands in tumor immunity. Oncogene, 27: 5944-5958, (2008).

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www.ijpbs.com (or) www.ijpbsonline.com [6] Yamanegi K, et al: Sodium valproate, a histone deacetylase inhibitor, augments the expression of cellsurface NKG2D ligands, MICA/B, without increasing their soluble forms to enhance susceptibility of human osteosarcoma cells to NK cell-mediated cytotoxicity. Oncology Reports, 24: 1621-1627, (2010). [7] Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH and Spies T: Broad tumor-associated expression and recognition by tumor-derived gy Tcel ls of MICA and MICB. Proc Natl Ac ad Sci USA, 96:6879-6884, (1999). [8] Vetter CS, GrohV, thor Straten P, SpiesT, Brocker EB and Becker JC: Expression of stress-induced MHC class I related chain molecules on human melanoma. J Invest Dermatol, 118: 600-605, (20 02). [9] Jinushi M, Takehara T, Tatsumi T, et al: Expression and role of MICA and MICB in human hepatocellular carcinomas and their regulation by retinoic acid. Int J Cancer, 104:354-361, (2003). [10] Wu JD, Higgins LM, Steinle A, Cosman D, Haugk K, Plymate SR: Prevalent expression of the immunostimulatory MHC class I chain-related molecule is Counteracted by shedding in prostate cancer. J Clin Invest, 114:560-568, (2004). [11] Wu JD et al: Obstructing Shedding of the ImmunostimulatoryMHC Class I Chain-Related Gene B PreventsTumor Formation. Clin Cancer Res, 15(2): 632640 2009. [12] Marten A, von Lilienfeld-Toal M, Buchler MW, Schmidt J: Soluble MIC is elevated in the serum of patients with pancreatic carcinoma diminishing (γδ) T cellcytotoxicity. Int J Cancer, 119:2359-2365, (2006). [13] Trowsdale J, Lee J, Kelly A, et al: Isolation and sequencing of a cDNA clone for a human HLAABC antigen. Mol. Biol. Med, 2 (1): 53–61, (1984). [14] Marsh SG et al: Nomenclature for factors of the HLA System. Tissue antigens, 65: 301–369, (2005). [15] Brown MA, Crane AM and Wordsworth BP: Genetic aspects of susceptibility, severity, and clinical expression in ankylosing spondylitis. Curr Opin Rheumatol, 14(4): 354–60, (2002). [16] Carrington M, O'Brien SJ: The influence of HLA genotype on AIDS. Annu Rev Med, 54: 535–551, (2003). [17] Houchins J, et al: DNA sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on human natural killer cells. J. Exp. Med, 173: 1017– 1020, (1991). [18] Zafirova B et al: Regulation of immune cell function and differentiation by the NKG2D receptor. Cell Mol Life Sci, 68 (21): 3519–3529, (2011). [19] Bauer S et al.: Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science, 285 (5428): 727–729, (1999).

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|55-62 [20] Raulet DH: Roles of the NKG2D immunoreceptor and its ligands. Nature Reviews Immunology, 3: 781-790, (2003). [21] González S et al: NKG2D ligands: key targets of the immune response. Trends in Immunology, 29 (8), (2008). [22] Kaiser B.K., et al: Disulphide-isomerase-enabled shedding of tumour-associated NKG2D ligands. Nature, 447: 482-486, (2007). [23] Jordan PA, et al: A role for the thiol isomerase protein ERp5 in platelet function. Blood, 105: 1500-1507, (2005). [24] Wu JD, et al: Prevalent Expression of the Immunostimulatory MHC Class I Chain-related Molecule is Counteracted by Shedding in Prostate Cancer. J Clin Invest, 114: 560-568, (2004). [25] Salih, et al:Down-Regulation of MICA on Human Tumors by Proteolytic Shedding. J Immunol, 169: 4098102, (2002). [26] Edwards DR, Handsley MM and Pennington CJ: The ADAM etalloproteinases. Mol. Aspects Med, 29 (5): 258–289, (2008). [27] Brocker C, Vasiliou V and Nebert DW: Evolutionary divergence and functions of the ADAM and ADAMTS gene families. Human Genomics, 4 (1): 43-55, (2009). [28] Wolfsberg TG, Straight PD, Gerena RL, et al: ADAM, a widely distributed and developmentally regulated gene family encoding membrane proteins with a disintegrin and metalloprotease domain. Dev. Biol. 169 (1): 378– 383, (1995). [29] Liu PC, et al: Identification of ADAM10 as a major source of HER2 ectodomain sheddase activity in HER2 overexpressing breast cancer cells. Cancer Biology and Therapy 5 (6): 657–664, (2006). [30] Waldhauer I, et al: Tumor-Associated MICA Is Shed by ADAM Proteases. Cancer Res, 68(15): 6368-6376, (2008). [31] Reiss K and Saftig P. The A Disintegrin and Metalloprotease (ADAM) family of sheddases: Physiological and cellular functions. Seminar in Cell and Developmental Biology, 20(2):126-137, (2009). [32] Lolis E and Petsko GA:Transition-state analogues in protein crystallography: probes of the structural source of enzyme catalysis. Annual Review of Biochemistry, 59: 597–630, (1990). [33] Ectodomain shedding en.svg [34] Moss ML, Jin SL, Milla ME et al: Cloning of a disintegrin metalloproteinase that processes precursor tumournecrosis factor-alpha. Nature 385 (6618): 733–36, (1997). [35] Salih HR, Antropius H, Gieseke F, et al: Functional expression and release of ligands for the activating

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www.ijpbs.com (or) www.ijpbsonline.com immunoreceptor NKG2D in leukemia. Blood, 102: 13891396, 2003. [36] Groh V, Wu J, Yee C, and Spies T: Tumour-derived soluble MIC ligands impair expression of NKG2D and Tcell activation. Nature, 419:734â&#x20AC;&#x201C;738, (2002). [37] Altmeppen CH, et al: Molecular Neurodegeneration. Lack of a-disintegrin-and-metalloproteinase ADAM10 leads to intracellular accumulation and loss of shedding of the cellular prion protein in vivo. Molecular Neurodegeneration , 6(36) :1-12, (2011).

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|55-62 [38] Wu JD: Methods for treating cancer by inhibiting MIC shedding. United State Patent Wu. US 8,182,809 B1. May 22, (2012). [39] Cerwenka A, et al: Retinoic Acid Early Inducible Gene Define a Ligand Family for the Activating NKG2D Receptor in Mice. Immunity. 12: 721-727, (2000). [40] Jinushi M, et al: MHC class I chain-related protein A antibodies and shedding are associated with the progression of multiple myeloma. PNAS, 105(4):1285â&#x20AC;&#x201C; 1290, (2008).

*Corresponding Author:

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Desmond Omane Acheampong, Department of Molecular Biology, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China. Email: do.acheampong@yahoo.com

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Research Article Biological Sciences BIOCHEMICAL EVALUATION OF HYPOGLYCEMIC ANTIOXIDANT POTENTIAL OF HERBAL EXTRACT STUDIED IN (STZ) STREPTOZOTOCIN INDUCED DIABETIC RAT M.Syed Muzammila*, V.Dineshb, S.Rajeshc, S.Mahaboob Rahimand, V.Karthicke, J.Paul Rajf, S.Parsana1, F.Badriya 2 a,b,c,d,e,f

PG & Research Department of Biochemistry, Islamiah College (Autonomous) Vaniyambadi, Tamilnadu, India. 1 Department of Biochemistry, Marudhar Kesari Jain College for Women, Vaniyambadi, Tamilnadu, India. 2 PG & Research Department of Biochemistry, Muthayammal College of Arts & Science, Salem, Tamilnadu, India. *Corresponding Author Email: syed_bio2004@yahoo.co.in

ABSTRACT Diabetes Mellitus is a clinical condition characterized by increased blood glucose level due to the insufficient insulin. Streptozotocin is to induce hyperglycemic condition. Luffa acutancula and Gmelina arborea may be the good remedy for the treatment of diabetes mellitus. In this study, the oral administration of Luffa acutancula and Gmelina arborea showed the hypoglycemic activity and it could exert a beneficial action against biochemical alterations caused by the streptozotocin. Diabetes mellitus is a disease that affects more than 100 million people and may attain about five times more subjects in the next 10 years. In the search for new compounds, and within the exploratioration of natural resources, the hypoglycemic effect of plants which are reputed antidiabetic. The present study is carried out to identify new potential antidiabetic compounds of Luffa acutancula and Gmelina arborea. In the present study the antioxidative potential of Luffa acutancula and Gmelina arborea was assessed in streptozotocin induced diabetic rats. Lipid peroxide levels were also measured in normal, diabetic and treated animals. Malondialdehyde (MDA) levels were significantly higher and antioxidant activity was found low in diabetic groups as compared to the control groups, and significant alteration in both the MDA levels and antioxidant activity was also observed when the above herbal hypoglycemic agents were given to diabetic rats. On the basis of our results we conclude that Luffa acutancula and Gmelina arborea are not only useful in controlling the lipid peroxide levels but are also helpful in further strengthening the antioxidant potential. The probable mechanism by which Luffa acutancula and Gmelina arborea exerts its protective action against streptozotocininduced pancreatic metabolic alterations could be by the stimulation of pancreatic regeneration through an improved synthesis of protein or accelerated detoxification and exertion. Furthermore, comprehensive chemical and pharmacological research is required to reveal the mechanism of the anti-diabetic potential of Luffa acutancula and Gmelina arborea.

KEY WORDS Free radicals, Diabetes mellitus, Antioxidants, Luffa acutancula and Gmelina arborea, Streptozotocin (STZ).

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INTRODUCTION Our Country has immense wealth of huge medicinal plants. These plants have credited to the development of therapeutic agents for

various ailments and diseases. The medicinal plants documented in ‘Rig Veda’, (4500-1600 B.C) mentions 67 medicinal plants. ‘Yajur Veda’ 81 plants and ‘Atharvana Veda’, (4500-2500 B.C)

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www.ijpbs.com (or) www.ijpbsonline.com 290 species, which are still used in classified formulations in Ayurvedic system of medicine. Diabetes Mellitus is the first leading cause of death (after heart disease and cancer) in many developed countries. It affects about 2% to 3% of the general population complications of diabetes affects the eye, kidney and nervous system. Diabetes is a major cause of blindness, renal failure, amputation, heart attack and strokes. It is a clinical condition characterized by insulin is either not produced in sufficient quantity or insufficient in its action on the target tissues. Moreover, diabetes also induces changes in the tissue content and activity of the antioxidant enzymes Wohaieb S.A. et al., (1987), Asayama K. et al., (1989).Since the time of Charaka and Susruta many herbal medicines in different oral formulations have been recommended for Madhumeha and confident claims of cure are on record, Aslam M. et al., (1998). WHO Expert Committees, technical report series of Diabetes mellitus 1985,727. Diabetes mellitus remains a major health problem and prevention of diabetes still lies in the realm of future and until then tens of millions will continue to suffer from this disease. Oxidative stress is reported to be increased in patients with diabetes mellitus, Baynes J.W (1991). Role of oxidative stress in development of complications in diabetes. Diabetes 40, 405-412. Accumulating evidence suggests that oxidative cellular injury caused by free radicals contributes to the development of diabetes mellitus Bambolkar S et al., (1995). Reactive oxygen species generated in the cells are scavenged by antioxidant enzymes Genet S. et al., (2002). It is well known that the plants like Luffa acutancula and Gmelina arborea are not only possess hypoglycemic activity but some of them are hypotensive, hepatoprotective and also blood purifier Tiwari A.K et al., (2002), Grover J.K. et al., (2002).

In view of the above considerations the present study was designed to investigate the protective effect of Luffa acutancula and Gmelina arborea on plasma lipid peroxide levels and on antioxidant enzyme superoxide dismutase (SOD). Moreover, antioxidant molecules, uric acid and albumin content were also measured in streptozotocin induced diabetic rats. SCOPE OF OUR STUDY In this study the use of plants for medicinal purpose used locally in the treatment of various diseases and we examined for their antioxidant activity. The results of our studies conducted in animals have reported that useful in controlling the lipid peroxide levels but are also helpful in further strengthening the antioxidant potential. Therefore, the present investigation is part of continuing programme related to the biochemical screening of local plants used in Ancient Indian Medicine, Ayurveda, Siddha and Yunani. In several countries including India several plant species are administered orally to control the diabetes mellitus. Some of these plants have been pharmacologically provided to be of some value in diabetes mellitus. Luffa acutancula and Gmelina arborea may have the popular remedy for the treatment of diabetes mellitus.

MATERIALS AND METHODS CHEMICALS All the fine chemicals were purchased from Sigma chemical co., USA. All other chemicals used were of good quality and analytical grade. Luffa acutancula and Gmelina arborea as a gift from the Siddha Maruthuva Salai Vellore, Tamilnadu. All the plants were identified taxonomically by Dr.N.P.M.Mohammed Tariq (Botanist) Department of Biotechnology, Islamiah College (Autonomous) Vaniyambadi. PREPARATION OF PLANT EXTRACT

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www.ijpbs.com (or) www.ijpbsonline.com Luffa acutancula: Air dried plant leaves (100 g) were boiled in 200 ml of distilled water for 10minutes. After cooling to room temperature, the supernatant was filtered to obtain the decoction ready for animal treatment Satyanarayan K. et al.,(1978). Gmelina arborea: Air dried leaves powder was boiled in distilled water for 10minutes. After cooling to room temperature, the supernatant was filtered to obtain the decoction ready for animal treatment Luthy N. et al., (1964). ANIMALS Male albino rats weighing 150-200 g were used in the present study. All rats were kept at room temperature of 20째C in the animal room of our Department of Biochemistry, Islamiah College (Autonomous) Vaniyambadi. They were maintained on food pellets and water ad libitum. 18 rats, included for the study, were divided into 6 groups, each consisting of three animals. Out of 6 groups, seven were made diabetic with a single dose of streptozotocin (65 mg/kg b.w.) by intraperitoneal route Shibib B.A. et al., (1993). Diabetes was confirmed by the determination of fasting blood glucose concentration on the third day post administration of streptozotocin. Body weight and fasting blood glucose levels of all the rats were determined before the start of the experiment. Rats were divided into the following groups. SEGGREGATION OF EXPERIMENTAL GROUPS Group 1: Control given only saline (10 ml/kg/once a day, daily) Group 2: Streptozotocin induced diabetic given in saline (10 ml/kg/once a day, daily) Group 3: Diabetic rats treated with Luffa acutancula (10 ml/kg/ once in a day, daily) Group 4: Diabetic rats treated with Gmelina arborea (10 ml/kg/ once in a day, daily) Group 5: Diabetic rats treated with Insulin (5 units/ kg/ once a day, daily)

Group 6: Diabetic rats treated with Glibenclamide (500 mg/kg/ once a day, daily) After 30 days of treatment the body weight and fasting blood glucose of the animals were again determined. Blood was collected in heparinized vial and in plain vial for hemolysate preparation and for serum separation respectively. PREPARATION OF HEMOLYSATE The Collected blood was centrifuged for 10minutes at 3000 rpm. The plasma thus obtained was used for lipid peroxide estimation Ohkawa H. et al., (1979). Remaining packed RBCs were washed thrice with normal saline to remove the buffy coat. Hemolysis was performed by pipetting out 1 ml of washed red blood suspension in ice cold double distilled water. Erythrocyte ghosts were sedimented in a high speed refrigerated centrifuge at 12000 rpm for 40 minutes. The cell content was separated out carefully and used for superoxide dismutase estimation McCord J.M. et al., (1969). ISOLATION OF ERYTHROCYTES AND ERYTHROCYTE MEMBRANES Erythrocytes and their membranes were isolated from the control and experimental groups according to the method of Dodge et al., with slight modifications. Packed cells were washed with isotonic saline to remove buffy coat. Different aliquots of packed cells were thoroughly washed with Tris-buffer 0.31 M (pH7.4). These were used for the assay of various biochemical parameters. Then, another aliquot of packed cells were subjected to hemolysis by adding hypotonic Tris-buffer 0.015 M (pH7.2). After 4-6 hrs, the erythrocyte ghosts were sedimented by centrifugation at 12,000 rpm for 40-45 at 4o C, the supernatant (hemolysate) was used for the analysis of antioxidants. The erythrocyte membrane pellets were suspended in 0.02 M Tris-buffer (pH 7.2) and used for various other biochemical estimations.

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SEPARATION OF SERUM The blood collected in plain vial was kept for some time. Serum from blood after clotting separated out and collected in clean dry centrifuge tube and again centrifuged for 5minutes at 3000 rpm. The serum thus obtained was used for albumin and uric acid estimations Rodkey F.L. et al., (1965), Eichhorn E. et al., (1961). RESULTS AND DISCUSSIONS In streptozotocin induced diabetic rats there was a significant (p<0.001) increase in fasting blood glucose (133.34%) and a comparative decrease (p<0.001) in body weight and protein content. There was a slight increase in body weight and protein and a significant decrease in fasting blood glucose (Table 1) in diabetic rats treated with Luffa acutancula (55.13), Gmelina arborea (71.21%). These effects are quite similar to that obtained by insulin and glibenclamide. Table 2 shows a statistically significant increase in lipid peroxide levels (p<0.001) in streptozotocin induced diabetic rats with respect to normal controls and there was a significant decrease in

lipid peroxide levels in diabetic rats treated with herbal preparations of Luffa acutancula (p<0.001), Gmelina arborea (p<0.001). In contrast to this, the activity of the enzyme superoxide dismutase (SOD) was significantly decreased in diabetic rats as compared to that of normal control (Table 2). All the anti-diabetic agents including herbal preparations, insulin and glibenclamide used in the present study significantly elevated the activity of superoxide dismutase when compared with diabetic control. Another antioxidant, albumin was also found decreased in diabetic group as compared to normal control (Table 3). Following treatment with herbal preparations both showed some, significantly increased levels of albumin were found. Insulin and glibenclamide also showed significant (p<0.001) increase in albumin levels as compared to the control group (Table 3). Following treatment with both the herbal preparations increment (p<0.01) in the uric acid levels was observed, while insulin and glibenclamide exhibited non significant changes (Table 3).

Table 1: The effect of herbal hypoglycemic agent on body weight, blood glucose and protein in normal and diabetic rats. Values are expressed as mean + S.E. (n=6). Student’s t-test. S.no Groups Change in body Fasting Blood Glucose (mg/dl) Protein value weight (mg/ml) Pre-treated Post-treated 1 2 3 4 5 6

Control Diabetic control Diabetic rats + Luffa acutancula Diabetic rats + Gmelina arborea Diabetic rats + Insulin Diabetic rats + Glibenclamide

+35.0±5.12 -20.5±6.2*** +10.4±7.2*

84.83±6.75 192.60±6.91 192.28±7.77

86.06±8.32 195±10.78*** 109.25±10.58***

63.48±6.86 42.6±7.20*** 46.18±10.65*

+8.5±5.05*

197.93±10.62

78.45±6.48***

44.66±11.52*

+16.3±6.4**

197.46±6.79

81.42±8.03***

47.68±9.51**

+0.6±5.35**

198±6.77

109.92±10.15***

46.65±6.30**

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p-values: *p<0.1, **p<0.01, *** p<0.001.

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Table 2: Effect of herbal hypoglycemic agents on the levels of lipid peroxide and superoxide dismutase. Values are expressed as mean + S.E. (n=6). Student’s t-test. S.No. Groups MDA (nmol/ml) SOD (U/mg protein) 1 Normal Control 6.98±0.395 92.7±7.21 Groups 2 Diabetic control 10.81±0.586*** 65.5±6.45*** 3 Diabetic rats + 7.41±0.148*** 86.0±6.05*** Luffa acutancula 4 Diabetic rats + 7.38±0.059*** 77.5±5.31*** Gmelina arborea 5 Diabetic rats + 6.21±0.075*** 133.8±6.41*** Insulin 6 Diabetic rats + 6.83±0.261*** 107.3±9.36*** Glibenclamide *p<0.1, ***p<0.001 vs diabetic control group.

Table 3: Effect of herbal hypoglycemic agents on the levels of albumin and uric acid. Values are expressed as mean + S.E. (n=6). Student’s t-test. S.No. Groups Albumin (g/dl) Uric acid (mg/dl) 1 Normal Control Groups 6.74±0.173 0.685±0.020 2 Diabetic control 6.13±0.1030 0.572±0.059*** 3 Diabetic rats + Luffa 6.67±0.1545*** 0.493±0.0085** acutancula 4 Diabetic rats + Gmelina 6.29±0.1365** 0.667±0.0292** arborea 5 Diabetic rats +Insulin 6.74±0.1176*** 0.544±0.0642* 6 Diabetic rats + 6.67±0.0583*** 0.535±0.0070* Glibenclamide *p<0.1, **p<0.01 and ***p<0.001 Vs diabetic control group.

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CONCLUSION To conclude, our study suggested that the herbal plants posses the hypoglycemic effect or antidiabetic effect and antioxidant activities, which might be helpful in preventing or slowing the progress of diabetes. Further in vivo studies and investigations on the isolation and identification of active components in the plants may lead to chemical entities with potential for clinical use in the prevention and treatment of diabetes and various diseases and to evaluate the levels of metal ions such as copper, zinc,

magnesium, manganese and selenium, as altered metabolism of these metals have been reported to occur in both IDDM and NIDDM Walter R.M. et al., (1991). The results of our present study demonstrated that the elevated plasma lipid peroxide levels in streptozotocin-induced diabetic rats along with a significant decrease in the anti-oxidant enzyme, superoxide dismutase activity. Moreover, we also found reduced levels of serum albumin and uric acid in diabetic rats. Earlier there have been many reports documenting elevated serum lipid

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www.ijpbs.com (or) www.ijpbsonline.com peroxide levels and diminished antioxidant status in diabetic subjects Sato Y.et al.,(1979), Oberley L.W. et al.,(1988). They also significantly reduce the plasma lipid peroxide levels in diabetic rats. Moreover, following treatment the activity of the antioxidant enzyme superoxide dismutase and serum albumin content was also found increased. However, the serum uric acid content was not found significantly altered. Uric acid is one of the most abundant chain breaking antioxidants present in human serum and its levels are largely determined by genetic factors, purine intake and renal function Situnayake R.D. et al.,(1991). Moreover, excess accumulation of urate in serum and tissues induce gouty pathology, and is by no means beneficial from the medical point of view Asayama K. et al., (1993). SOD and albumin form the primary defense against reactive oxygen metabolites Mahdi A.A. (2002). Such metabolites have been implicated in the damage brought about by ionizing radiation, as well as in the effects of several cytostatic compounds Marklund S.L. et al., (1982). The decreased activity of antioxidant molecules along with elevated lipid peroxide levels in diabetic rats could probably be associated with oxidative stress and/or decreased antioxidant defense potential Mahdi A.A. et al., (1996).The reversal in their content following treatment may be due to decreased oxidative load. Elevated SOD activity and albumin levels in insulin treated group may probably be due to the anabolic role of this proteinous hormone. The herbal hypoglycemic agents may also act by either directly scavenging the reactive oxygen metabolites, due to the presence of various antioxidant compounds Gupta S.K. et al.,(2002) or by increasing the synthesis of anti-oxidant molecules.

ACKNOWLEDGEMENTS The authors are grateful to Dr.N.P.M.Mohammed Tariq and Dr.H.Abdul Jaffar Ali, Assistant Professors of the Department of Biotechnology, Islamiah College (Autonomous) Vaniyambadi for their immense help during the project. We acknowledge C. Khaiser Ahmed Sahib, General Secretary, Ghani Mohamed Jaweed Sahib, College Secretary and Dr. K.Prem Nazeer Sahib, Principal, Islamiah College, (Autonomous) Vaniyambadi for their great enthusiasm and wise advice.

REFERENCE o

o o o o

o o

Asayama, K., Kayashibe, H., Dobashi, K., Niitsu, T., Miyao, A. and Kato, K. (1989) Antioxidant enzyme status and lipid peroxidation in various tissues of diabetic and starved rats. Diabetes Res. 12, 85-91. Aslam, M., Jafri, M.A., Kalim, Javed and Surendra Singh (1998) Plant drug with hypoglycemic activity Glimpses in plant Research, Vol XII : 271-299. Today and Tomorrowâ&#x20AC;&#x2122;s Printers and Publishers, New Delhi â&#x20AC;&#x201C; 110005 (India). Asayama, K., Uchida, N., Nakane, T., hayashibe, H., Dobashi, K., Amemiya, S., Kato, K. and Nakazawa, S. (1993) Antioxidants in the serum of children with insulin dependent diabetes mellitus. Free Radic.Biol.Med. 15, 597-602. Andallu B, et al., (2000), Annapurna A, et al., (2001). Baynes, J.W. (1991) Role of oxidative stress in development of complications in diabetes. Diabetes 40, 405-412. Bopanna, K.N., Kannan, J., Sushma, G., Balaraman, R. and Rathod, S.P. (1997) Antidiabetic and antihyperlipaemic effects of neem seed kernel powder on alloxan diabetic rabbits. Ind.J.Pharmacol. 29, 162-167. Bambolkar, S. and Sainani, G. S. (1995). Evaluation of oxidative stress in diabetics with or without vascular complications. J. Asso. Phys. India, 43, 10-12. Bopanna KN, et al., (1997). Chandrasekar B, Mukherjee B and Mukherjee SK. Blood sugar lowering potentiality of selected Cucurbitaceae plants of Indian origin. Indian J. Med. Research 1989; 90: 300-305. Chandira M, Vankateswarlu BS, Gangwar RK, Sampathkumar KP, Bhowmik D, Jayakar B and Rao CV. Studies on anti stress and free radical scavenging

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activity of whole plant of Coccinia Indica Linn. Inter. J. Pharm Science 2010; 01: 0050. Chude MA, et al., (2001). Chaurasia AK, et al., (1994). Das AV, et al., (1996). Dubey GP, et al., (1994) Dhar, M.L., Dhar, M.M., Dhawan, B.N., Mehrotra, B.N. and Ray, C. (1968). Screening of Indian plants for biological activity (part-I). Ind.J.Exp.Biol. 6, 232-247. Deokate UA and Khadabadi SS. Pharmacology and phytochemistry of Coccinia indica. Journal of Pharmacognosy and Phytotherapy 2011; 3(11): 155159. Eichhorn, E., Zalmanwaki, S., Rotenburg, E.A. and Fanis, B. (1961) Uric acid estimation in serum and urine. J.Clin.Pathol. 14, 450-453. Genet, S., Kale, R. K. and Baquer, N.Z. (2002). Alterations in antioxidant enzymes and oxidative damage in experimental diabetic rat tissue; Effect of vanadate and fenugreek (Trigonella faenum graecum). Mol.Cell. Biochem. 236(1&2), 7-12. Grover, J.K., Yadav, S. and Vats, V. (2002). Medicinal plants of India with antidiabetic potential. J. Ethnopharmacol. 81, 81-100. Joy KL, et al., (1998). Khan AK, AKhtar S and Mahtab H. Treatment of diabetes mellitus with Coccinia indica. Brit. Med. J. 1980; 280(6220): 1044. Karunanayake, H., Eric, Jeevathayaparan S., Tennekoon, H. Kamani (1990). Effect of Momordica charantia fruit juice on streptozotocin induced diabetes in rats. J. Ethnopharmacol. 30, 199-204. Khosla, P., Sangeeta, B., Singh, J., Seth, S. and Srivastava, R.K. (2000). A study of hypoglycemic effects of A. indica (Neem) in normal and alloxan diabetic rabbits. Indian J. Physiol. Pharmacol. 44 (1), 69-74. McCord, J.M. and Fridovich, I. (1969) SOD enzyme function for erythrocuprein. J.Biol.Chem. 224, 60496055. Mahdi, A.A. (2002) Free radicals and other antioxidants. A text Book of Biochemistry by S.P. Singh, 3rd edn. CBS Publishers and Distributors, New Delhi. 545-555. Marklund, S.L., Westman, N.G., Lundgren, E. and Ross, G. (1982) Copper and zinc containing superoxide dismutase, manganese containing superoxide dismutase, catalase and glutathione peroxidase in normal and neoplastic human cell lines and normal human tissues. Cancer Res. 42, 1955-1961. Murugesan T, et al., (2000). Mahdi, A.A., Singh, R. and Singh, R.K. (1996) Role of reactive oxygen species and antioxidants in human

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disease: A overview Persp. Biol. (Rai, V., Naik, M.L. Manoharacharry, C. Eds.) School of Life Sciences, World Laser Graphics, Raipur, 55- 70. Nischino S, et al., (2000). Ohkawa, H., Ohishi, N. and Yagi, K. (1979) Assay for lipid peroxides in animal tissue by thio-barbituric acid reaction. Anal. Biochem. 95, 357358. Oberley, L.W. (1988) Free radicals and diabetes. Free Radical Biol. Med. 5, 113-124. Pari L, et al., (2001). Platel K, Srinivasan K. Plant foods in the management of diabetes mellitus: vegetables as potential hypoglycemic agents. Nahrung 1997;41(2): 68-74. Rodkey, F.L. (1965) Direct spectrophotometric determination of albumin in human serum. Clin.Chem. 11, 478-487. Reynolds JEF, et al., (1997). Satyanarayan, K., Murty, D., Narayana Rai D., Krishna Rao Gopalakrishna, and Murty LB. (1978). A preliminary study on hypoglycemic and antihyperglycemic effects of Azardirachta indica Ind.J.Pharmacol. 10(3), 247-250. Shibib, B.A., Khan, L.A. and Rahman, R. (1993). Hypoglycemic activity of Coccinia indica and Momordica charantia in diabetic rats: depression of the hepatic glyconeogenic enzymes glucose 6phosphatase and fructose 1,6 bisphosphatase and elevation of both liver and red cell count enzyme glucose-6-phosphate dehydrogenase. Biochem.J. 292, 267-270. Sheela, C.G. and Augusti, K.T. (1996). Antidiabetic effects of onion and garlic sulfoxide amino acids in rats. Planta Medica. 61(4), 356-7. Sato, Y., Hotta, N., Sakamoto, N., Matsuoka, S., Ohishi, N. and Yagi, K. (1979) Lipid peroxide level in plasma of diabetic patients. Biochem. Med. 21, 102-107. Situnayake, R.D., Thurnham, D.I., Kootathep, S., Chirico, S., Lunec, J., Davis, M. and McConkey, B. (1991) Chain breaking antioxidant status in rheumatoid arthritis: Clinical and laboratory correlates â&#x20AC;&#x201C; Ann. Rheum. Dis. 50, 81-86. Tiwari, A.K and Rao, M. (2002) Diabetes mellitus and multiple therapeutic approaches of phytochemicals: Present status and future prospects - Curr. Sci. 83, 3038. Upadhyay OP, et al., (1996). Vetrichelvan T, et al., (2002). Walter, R.M., Jr., Uri-Hare, J.Y., Olin, K.L., Oster, M.H., Anawalt, B.D., Critchfield, J.W. and Keen, C.L. (1991) Copper, zinc, magnesium status and complications of diabetes mellitus. Diabetes Care. 14, 1050-1056.

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WHO Expert Committees. In: WHO Technical report series of Diabetes mellitus 1985, 727. Wohaieb, S.A. and Godin, D.V. (1987) Alteration in free radical tissue defense mechanism in streptozotocininduced diabetes in rat. Diabetes 36, 1014-1018. Wealth of India. A dictionary of Indian raw materials and India products. Raw Material, New Delhi 1992; 4: 312.

Yanardag R, et al., (1998). Zacharis N.T., Sepastian K.L., Babu Philip and Augusti K.T. (1980). Hypoglycemic and hypolipidaemic effects of garlic in sucrose fed rabbits. Ind.J.Phys.Pharmacol. 24, 151-153.

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M. Syed Muzammil* PG & Research Department of Biochemistry, Islamiah College (Autonomous) Vaniyambadi-635 752, Tamilnadu State, INDIA.

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Research Article Biological Sciences DEPLETED ANTIOXIDANT VITAMINS AND ENHANCED OXIDATIVE STRESS IN UROLITHIASIS Bharathi B. K *1, H.B. Shivakumar2, Soumya N.S3 *1, 2,3

Department of Biochemistry, J. J. M. Medical College, Davangere -577004, Karnataka, India. *Corresponding Author Email: drsoumya034@gmail.com

ABSTRACT Background: Urolithiasis is the third most common urological disease with prevalence of 11% and recurrence rate of 50% and has a great impact on the economsically active population, representing significant health care cost burden. Crystal aggregation and retention are critical events for the formation of kidney stones. There is a close association between crystal development and free radical activity in vivo. Materials and Methods: In the present study 50 subjects presenting with urolithiasis were included. Serum levels of malondialdehyde, vitamin E and βcarotene were measured. These findings were compared with 50 age and sex matched control subjects. Unpaired‘t’ test was applied for statistical analysis. Results: There was a significant increase in MDA (p<0.001) and significant decrease in Vitamin E (p<0.01) and β-Carotene (p<0.001) levels in patients. There was significant negative correlation MDA and vitamin E and between MDA and β-carotene. Conclusion: It appears that a role of lipid peroxidation and oxidative function exists in the pathogenesis of urolithiasis.

KEY WORDS Oxidative stress, antioxidants, urolithiasis, vitamin E, β-carotene, malondialdehyde.

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INTRODUCTION Urolithiasis is calculus formation at any level in the urinary collecting system and is one of the most common diseases of the urinary tract which has tormented people throughout the ages[1]. It recurs at a very high rate without preventive measures. Most kidney stones are predominantly composed of calcium oxalate [2]. Despite enormous developments in nephrology and urology, we still do not know how to prevent them. Experiments performed on animals [3, 4], cultures [5] and human sera [6] have revealed that there is presence of enhanced oxidative stress in stone forming conditions. Oxalate is known to induce lipid peroxidation by unknown mechanism which causes disruption of the structural integrity of the membranes. Malondialdehyde is a major end product of free radical reaction on membrane fatty acids.

Although the cell is endowed with several antioxidant systems to limit the extent of lipid peroxidation, under certain conditions protective mechanism can be overwhelmed, leading to elevated tissue levels of peroxidation products [7]. Vitamin E and β-Carotene have proved to be efficient protectors to the membrane integrity. The present study was planned to quantitate the levels of serum malondialdehyde, Vitamin E and β-carotene and also to investigate their possible bearings in pathogenesis of urolithiasis.

MATERIALS AND METHODS The present study was carried out in the Department of Biochemistry, in collaboration of Department of Urology, J.J.M. Medical College attached to Bapuji and Chigateri hospitals, Davangere, Karnataka during the period April 2011 to April 2012.100 subjects were studied,

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www.ijpbs.com (or) www.ijpbsonline.com comprising of 50 healthy controls and 50 urolithiasis cases aged 15- 80 years that were diagnosed and confirmed by urologist. Patients with history of bowel disease, renal tubular acidosis, urinary tract anomalies, gout, diabetes mellitus and hypertension were excluded from the study. 50 healthy subjects, age and sex matched from public at large including medical undergraduate and post graduate students free from any history of smoking, alcoholism and co existence of any such disease which can also lead to similar changes in plasma levels of MDA, vitamin E and β- carotene levels were selected as controls. None of the subjects were on vitamin supplementation or used medications that could alter the study parameters. 6ml of venous blood was drawn under aseptic precautions into a plain bulb from selected subjects. Serum was separated by centrifugation at 3,000 rpm for 10 minutes and then Vitamin E was estimated by Baker and Frank method (8), serum β-carotene by Sobel and Snow method(9) and serum Malondialdehyde by Nadiger et al method (10) in all subjects within 24 hours of collection of the samples.

STATISTICAL ANALYSIS Results are expressed as mean ± SD and range values for all the variables. Unpaired‘t’ test is used to compare the mean values different biochemical parameters between cases and controls. Correlation analysis was performed to assess the relationship between different variables using Pearson’s correlation co-efficient. For all the tests p value of < 0.05 was considered as statistical significant.

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RESULTS The general description of the study subjects are given in table 1. Of the 50 controls, 40 were males and 10 were females with a mean age of 37.1 ± 13.4 years. Among 50 urolithiasis cases,

41 were males and 9 were females with a mean age of 39.8 ± 13.6 years. Table 2 shows comparative analysis of serum MDA, vitamin E and β-carotene levels between controls and urolithiasis cases. In this study, a significant increase in plasma MDA level (p<0.001) was observed in patients compared to controls (Table 2) that were similar to findings of other investigators[11, 12, 13]. Highly significant value (p<0.001) in the level of mean serum MDA is seen among the patients (5.48±0.81 nmol/ml) when compared to the controls (2.26±0.84 n mol/ml). Levels of mean serum vitamin E are found to be lower in urolithiasis patients (0.70±0.27 mg/dl) when compared to controls (1.39±0.24 mg/dl) and the difference was found to be statistically significant (p<0.001). Similarly levels of mean serum β-carotene was found to be significantly lower (p<0.001) in urolithiasis patients (137.1±20.5 μg/dl) when compared to controls (203.5±25.4 μg/dl) (Table 2). A negative correlationship was observed between MDA and vitamin E (r = -0.540, p< 0.01) and also between MDA with β-Carotene (r = 0.380, p<0.01) (Table 3). It means that decrease in the levels of these vitamins accelerate the lipid peroxidation thereby generating more MDA.

DISCUSSION Oxalate the major stone forming constituent has been reported to induce free radical generation, which results in peroxidative injury to renal epithelial cells. In this study, a significant increase in plasma MDA level (p<0.001) was observed in patients compared to controls (Table 2) that were similar to findings of other investigators [7, 11, 12, 13, 14]. Increase in MDA levels observed could be due to increased oxidative stress in kidney from various sources or decrease in antioxidant defense mechanism and vice-versa.

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www.ijpbs.com (or) www.ijpbsonline.com Lower levels of serum vitamin E (p<0.001) and serum β-carotene (p<0.001) were observed in urolithiasis cases when compared to controls (Table 2) which is similar to findings of others [6, 7, 11, 15, 16]. The decreased levels of serum Vitamin E, β-carotene in urolithiasis patients appears to be a consequence of increased oxidative stress due to oxalate induced free radical mediated lipid peroxidation[6]. Negative correlationship between MDA and antioxidants such as vitamin E and β- carotene (Table 2) suggest that the imbalance caused by the levels of these parameters may be the major factor leading to crystal adherence on the surface of

renal epithelial cells thereby leading to genesis of urolithiasis. Study was done with manual methods. Correlation between parameters and size, anatomical position of stone was not included. More studies may be required to substantiate the results and also to identify new antioxidant molecules which may well prove to be better preventive factors.

Table 1: Age and sex wise distribution of subjects

Number of subjects Age(years) Gender

Mean± S.D Range Male Female

Controls 50 37.1± 13.4 18 - 67 40 10

Cases 50 39.8±13.6 15 - 75 41 9

Table 2: Comparison of serum Malondihaldehyde, SOD activity, Vitamin E and β – Carotene in controls and urolithiasis cases MDA (nmol/ml)

β -carotene (μg/dl)

Vitamin E (mg/dl)

Range

Mean ± SD

Range

Mean± SD

Range

Mean± SD

Controls

0.51 ─4.77

2.26±0.84

0.69 ─ 1.69

1.39±0.24

105.8─248.1

203.5±25.4

Cases

2.20 ─ 7.11

5.48±0.81

0.27─1.58

0.70±0.27

102.6─218.0

137.1±20.5

Mean Diff.

3.22

0.69

66.4

t-value *

19.56

13.33

14.38

p-value

<0.001, HS

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*Unpaired t-test; HS: Highly Significant

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Table 3: Relationship between serum MDA, SOD activity, vitamin E and β-carotene among the urolithiasis cases

MDA and vitamin E MDA and β -carotene

Pearson’s correlation coefficient ‘r’ value ─ 0.54 ─ 0.38

Significance p – value <0.01, S <0.01, S

Graph 1: Relationship between serum antioxidants and serum MDA in patients.

CONCLUSION

REFERENCES

Oxidative stress is functional in urolithiasis as evident from increased lipid peroxidation and decreased antioxidants namely serum vitamin E and β-carotene in urolithiasis patients when compared to controls. From this study, it appears that a role of lipid peroxidation and nutritional antioxidants exists in the pathogenesis of urolithiasis as observed from the negative correlation between the serum MDA and other antioxidants. Further it emphasizes the importance of antioxidants in preventing stone formation.

ACKNOWLEDGMENT

We would like to thank all the staff, postgraduates and the technical staff of our department for their co-operation.

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Verkoelen CF. Crystal Retention in Renal Stone Disease: A

Crucial Role for the Glycosaminoglycan Hyaluronan? J Am Soc Nephrol 2006; 17:1673–1687. Yadav RD, Jain SK, Alok S, Mahor A, Bharti JP, Jaiswal M. Herbal plants used in the treatment of urolithiasis: a review. IJPSR 2011; 2(6):1412-1420. Selvam, R. and Kalaiselvi, P. (2001) Studies on calcium oxalate binding proteins: effect of lipid peroxidation.Nephron. 88, 163 – 167. Muthukumar, A. and Selvam, R. (1997) Renal injury mediated calcium oxalate nephrolithiasis: role of lipid peroxidation. Ren. Fail. 19(3), 401 – 408. Thamilselvan, S., Khan, S. R., and Menon, M. (2003) Oxalate and calcium oxalate mediated free radical toxicity in renal epithelial cells : effect of antioxidants. Urol. Res.31, 3 – 9. Singh, P. P. and Barjatia, M. K. (2002) Peroxidative stress and antioxidant status in relation to age in normal population and renal stone formers. Ind. J. Nephrol. 12(1), 10 – 15. Kato J, Ruram AA, Singh SS, Devi SB, Devi T, Singh WG. Lipid peroxidation and antioxidant vitamins in urolithasis. IJCB 2007; 22(1):128-130. Mc Murray W, Gowenlock AH. Vitamins. In: Gowenlock th edts. Varley’s Practical Clinical Biochemistry. 6 edn. London; Heinemann Medical Books 1988; p.902.

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Demetriou JA, Vitamins. In: Henry RJ, Cannon DC, Winkelman JW editors. Clinical chemistry. Principles and Technics. 2nd edn. New York: Harper and Row; 1974:p.1375-1381. 10. Nadiger HA, Marcus SR, Chandrakala MV, Kulkarni DD. Malondihaldehyde (MDA) level, in different organs of rats subjected to acute alcohol toxicity. Ind J Clin Biochem 1986; 1:133-136. 11. Bet VV, Deshpande KH, Suryakar AN, Ankush RD, Katkam RV. Depleted nitrite and enhanced oxidative stress in urolithiasis. IJCB 2006;21(2):177-180. 12. Mahmoud RH, Ewadh MJ, Al-Hamadani KJ. Roles of Malondialdehyde and Catalase in Serum of Urolithiasis. Medical Journal of Babylon 2009; 6(1):147-153.

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|71-75 13. Mahmoud RH, Ewadh MJ, Al-Hamadani KJ. Roles of Malondialdehyde, Glutathione Peroxidase and NAcetyl-Ă&#x;-D-glucosaminidase in Serum of Urolithiasis. Medical Journal of Babylon 2009; 6(1):138-146. 14. Mahmoud RH, Evadh MJ, Al-Hamadani KJ. Clinical Assessment of glutathione peroxidase and catalase to the status of malondialdehyde in urolithiasis. Pak J Med Sci 2009;25(5):738-743. 15. Kancha RK, Anasuya A. Contribution of vitamin A deficiency to calculogenic risk factors of urine: studies in children. Biochemical Medicine and Metabolic Biology 1992; 47(1):1-9. 16. Sakly R, Fekih M, Ben Amor A, Najjar MF, Mbazaa M Possible role of vitamin A and E deficiency in human idiopathic lithiasis. Ann Urol (Paris) 2003; 37(4):217-9.

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Dr. Bharathi B.K* Department of Biochemistry, J. J. M. Medical College, Davangere -577004, Karnataka, India.

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Research Article Biological Sciences PRELIMINARY PHYTOCHEMISTRY AND BACTERICIDAL ACTIVITY OF LEUCAS ASPERA S. Jaishree Jasutkar, Rupali Shirsat and Dipak Koche* Department of Botany, Shri Shivaji College, Akola (MS) India- 444001 *Corresponding Author Email: dipakkoche43@gmail.com

ABSTRACT For the study, the whole plant of Leucas aspera was first defatted and remaining material was successively extracted with water, ethyl acetate and methanol. All the extract were concentrated under vacuum to yield corresponding aqueous (AQ), ethyl acetate extract (EAE) and methanolic extract (ME). Extractive value was found to be 4.36% w/w, 5.28% w/w and 8.50% w/w, respectively. Preliminary phytochemical screening reveals the presence of alkaloids, glycosides, terpenoids and sterols in both the extracts. All the tree extracts were screened for its Bactericidal activity against two gram positive and three gram negative bacteria at different concentrations of 50, 100, 200, 300 and 400 Âľg/disc by agar diffusion method. The activities of both the extracts were compared with standard antibiotics, by measuring the dimension of the zone of microbial growth (zone of inhibition) around the disc. EAE and ME extracts exhibited a significant antibacterial activity against all the screened microorganisms.

KEY WORDS Leucas aspera, Bactericidal activity, Phytochemical screening.

INTRODUCTION Leucas aspera S. (Lamiaceae) is one of the common plant found in the wild and open agriculture waste lands. It has been reported to possess antipyretic, antiinflamatory, antiseptic and insecticidal Properties [1]. The leaf juice is used as an external application for psoriasis, chronic skin eruption, and painful swelling [2, 3]. Due to its wide ethnic medicinal potential, this plant was investigated for its bactericidal activity.

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MATERIAL AND METHODS The whole plant of Leucas aspera S. (Lamiaceae) was collected from Dr. P. D. K. V. Agricultural campus, Akola (MS) in February 2011. The sample was authenticated by Dr. D. G. Bhadange, Head. Department of Botany, Shri Shivaji College, Akola and a voucher specimen has been

preserved. Air dried and powdered, whole plant of Leucas aspera was defatted with hexane by maceration process. The defatted material was successively extracted with water, ethyl acetate and methanol. The extractive value was found to be 4.36%, 5.28% and 8.50% w/w, respectively. The preliminary phytochemical analysis was carried out using standard methods [4, 5]. Aqueous (AQ), Ethyl acetate extract (EAE) and methanolic extract (ME) was studied for its bactericidal activity using different clinically important strains at different concentrations of 50, 100, 200, 300 and 400 Âľg/disc by agar diffusion method [6] against two gram positive and three gram negative bacteria. The activities of extracts were compared with the standard antibiotics. The plates were incubated at 37oC for 48 hrs. The zone of inhibition was calculated by measuring the dimension of the zone of no

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microbial growth around the disc. For each value, averages of three determinations were recorded.

against all the tested microorganisms at the concentrations of 50, 100, 200, 300 and 400 Âľg/disc and comparable to the various antibiotics used for individual microorganism. The aqueous extracts revealed poor bactericidal activity. Present study also reveals that EAE was found to be highly active against Klebsiella pneumoniae, where as ME was highly active against Escherichia coli (Table 2). Our results indicate the potential usefulness of Leucas aspera, in the treatment of various bacterial infections. Further phytochemical studies are needed to identify the active principle responsible for the observed bactericidal activity.

RESULTS AND DISCUSSION The preliminary phytochemical analysis of Leucas aspera showed the presence of alkaloids, phenolics, tannins, terpenes, steroids, glycosides and fatty acids. Ethyl acetate is found to be the best extraction medium for the isolation of phytochemical compounds (Table 1). Except AQE other two EAE and ME of Leucas aspera exhibited moderate to significant and concentration dependent bactericidal activity

Table 1: Preliminary phytochemistry of Leucas aspera Phytochemicals Aqueous extract Ethyl acetate extract Methanolic extract (AQE) (EAE) (ME) Alkaloids ++ + Phenolics + + Tannins + +++ ++ Terpenes +++ ++ Steroids + +++ ++ Fatty acids + ++ + Glycosides ++ + Anthroquinones -

Microorganisms

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Bacillus subtilis (G+) Staphylococcus aureus (G+) Escherchia coli (G-) Klebsiella pneumoniae (G-) Proteus vulgaris (G-)

Table 2: Bactericidal activity of extracts of Leucas aspera AQE (Zone of inhibition at EAE (Zone of inhibition at ME(Zone of inhibition at mg/disc) mg/disc) mg/disc) 50 --

100 --

200 --

300 --

400 --

50 5.0

100 7.0

200 12.0

300 12.5

400 13.2

50 --

100 --

200 --

300 --

400 --

2.5

3.2

3.5

3.0

5.0

8.0

9.2

9.5

5.0

6.8

10.5

11.5

13.0

3. 5

3.8

5.2

6.5

7.0

8.0

9.8

12.0

13.2

15.0

12.5

14.0

16.2

17.5

19.5

3.2

3.5

10.3

12.5

15.2

16.5

18.5

8.7

10.2

12.6

14.5

15.5

7.5

8.2

8.0

9.5

Novobiocin (std) 30mg/disc, average zone of inhibition (31 mm) Chloramphenicol (Std) 30mg/disc, average zone of inhibition (26mm) Values are mean of triplicate analysis, Zone of inhibition is measured in mm, -: no inhibition.

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ACKNOWLEDGEMENT

The authors are thankful to Dr. D. G. Bhadange, Head Department of Botany for the taxonomic identification of plant. The authors also extend their thanks to the Principal, Shri Shivaji College, Akola for providing essential laboratory facility.

REFERENCES

1. 2.

Chopra RL, Nayar SL and Chopra IC. Glossary of Indian Medicinal Plants, NISC, New Delhi. 1996, p. 153. Anonymous, The Useful Plants of India, Publication & Information Directorate, CSIR, New Delhi, 1994, p. 326.

Koche DK, Syed Imran, Shirsat RP, Bhadange DG. Phytochemical screening of eight ethnomedicinal plants from Akola District (MS), International Journal of Pharma and Bioscience, 2010, 1(4): 256-0. Evans WC. Pharmacognosy, 15th Edn. Harcourt Publishers Limited 2002, p. 137. Harborne JB. Phytochemical Methods â&#x20AC;&#x201C; A Guide to rd Modern Techniques of Plant Aanlysis, 3 Edn., Springer (India) Private Limited, 2005, p. 91. Elizabeth KM. Antimicrobial activity of Terminalia belerica. Indian Journal of Clinical Biochemistry, 2005, 20(2): 150-153.

*Corresponding Author:

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Dr. D. K. Koche Assistant Professor, Department of Botany, Shri Shivaji College, Akola (MS) India

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Research Article Biological Sciences INCIDENCE OF METALLO-BETA-LACTAMASE PRODUCING PSEUDOMONAS, ACINETOBACTER & ENTEROBACTERIAL ISOLATES IN HOSPITALISED PATIENTS A. Hodiwala (Bhesania)*1, R. Dhoke2, A. D. Urhekar3 Department of Microbiology, MGM Medical College and Hospital, Sector-18, Kamothe, Navi Mumbai-410 209 *Corresponding Author Email: anahitapb@hotmail.com

ABSTRACT Introduction: The introduction of carbapenems like imipenem and meropenems into clinical practice represents a great advance especially for treatment of serious bacterial infections caused by beta lactam resistant bacteria. They are considered as a last resort most of the times for treatment of critically ill patients in intensive care units (ICUs) and high risk wards. Aims and Objectives: Hence the present study aims to find out the incidence of metallo-beta lactamase (MBL) production in Pseudomonas, Acinetobacter and Enterobacterial isolates from hospitalized patients. Materials and Methods: Isolates of Pseudomonas, Acinetobacter and members of Enterobacteriaceae family were identified by standard microbiological methods and antibiotic sensitivity was carried out by using Kirby Baur Disk Diffusion method as per CLSI guidelines. MBL production in imipenem resistant isolates was detected by using two methods i.e Imipenem-EDTA Double Disk Synergy Test (DDST) and ImipenemEDTA Combined Disk Diffusion Test (CDDT). Results: 289 isolates were included in the study, out of which 47 (16.2%) showed resistance to imipenem. Of these 47 strains 16 were P.aeruginosa, 16 were A.baumannii, 11 were E.coli, 2 were K.pneumoniae, and 1 was E. aerogenes and C.freundii each. 31(65.9%) of the imipenem resistant isolates tested positive for MBL production by both double disk synergy test (DDST) and combined disk diffusion test (CDDT). Conclusion: The detection of MBL producing isolates is of crucial importance and in the present scenario early detection will go a long way in making adjustments in empirical antimicrobial therapy and probably help a great deal in bringing down mortality rates for patients infected with MBL producing strains.

KEY WORDS Metallo-b-lactamase (MBL), Imipenem-EDTA double disc synergy test (DDST), Imipenem-EDTA combined disc test (CDDT), P. aeruginosa, A. baumanii

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INTRODUCTION The control of hospital-acquired infection caused by multi-drug resistant gram-negative bacilli has proved to be a particular problem over the last 20 years. Members of the family Enterobacteriaceae are among the most important bacterial human pathogens accounting for the majority of bacteria isolated from clinical samples.(1) That these Gram negative bacilli (GNBâ&#x20AC;&#x2122;s) are rapidly acquiring resistance to one or more antimicrobial agents

traditionally used for treatment is a matter of concern. Till now, extended spectrum betalactamase (ESBL) production by Gram negative bacilli was considered as the most important threat to clinical therapeutics. (2, 3) The introduction of carbapenems into clinical practice represented a great advance for the treatment of serious bacterial infections caused by beta-lactam resistant bacteria. The carbapenems available for use in India are imipenem and meropenem.(4) However, there

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www.ijpbs.com (or) www.ijpbsonline.com have been reports of resistance to carbapenems.(5,6) Carbapenem Resistance due to the production of metallo-beta-lactamases (MBL) in Gram-negative organisms is an increasing international public health problem.(7,8) The problem of MBL producing strains was originally confined to Pseudomonas and Acinetobacter. However, carbapenem resistance has been observed in members of Enterobacteriaceae family due to spread of MBL genes. The occurrence of an MBL positive isolate in a hospital environment poses not only a therapeutic problem, but is also a serious concern for infection control management.(9) Hence the present study aims at studying the incidence of MBL production in Pseudomonas, Acinetobacter & isolates of Enterobacteriaceae by two methods. 1) Imipenem-EDTA Double Disc Synergy Test (DDST) (10) & 2) Imipenem-EDTA Combined Disc Diffusion Test (CDDT). (13)

MATERIALS AND METHODS

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Various isolates of Pseudomonas, Acinetobacter and Enterobacteriaceae obtained from different clinical samples like blood, pus, sputum, endotracheal secretions, catheter tips, urine, cerebrospinal fluid and various body fluids (synovial, ascitic, pleural) and identified by standard microbiological procedures were included in the study. The isolates were tested for sensitivity to imipenem (10µg) using Kirby-Bauer method as recommended by National Committee for Clinical Laboratory Standards (NCCLS, now known as Clinical and Laboratory Standards Institute, CLSI).(9) Isolates of Pseudomonas, Acinetobacter and Enterobacteriaceae from various samples showing zone of inhibition less than 13mm were included in the study for MBL production by the following methods.

Imipenem-EDTA Double Disc Synergy Test (DDST): The Imipenem-EDTA Double Disk Synergy Test was performed as described by Lee et al (10). A 0.5 M EDTA solution was prepared by dissolving 186.1g of disodium EDTA. 2H20 in 1,000 ml of distilled water and adjusting it to pH 8.0 by using NaOH. The mixture was sterilised by autoclaving. (11)

Direct colony suspension of test organism adjusted to match 0.5 McFarland turbidity was prepared and inoculated onto Mueller-Hinton agar plate as recommended by the National Committee for Clinical Laboratory Standards.(12) An imipenem (10 µg) disc was placed 20 mm centre to centre from a blank disc containing 10 µl of 0.5 M EDTA (750µg). The inhibition zones of the imipenem and EDTA discs were compared after 16 to 18 hrs of incubation at 370C. Enhancement of the zone of inhibition in the area between imipenem and the EDTA disc in comparison with the zone of inhibition on the far side of the drug was interpreted as a positive result. (10) Imipenem-EDTA Combined Disc Diffusion Test (CDDT): The Imipenem-EDTA Combined Disc Diffusion Test was performed by Yong et al.(13) Test organisms were inoculated on to plates with Mueller Hinton agar as recommended by CLSI.(9) Two 10µg imipenem discs (Becton Dickinson) were placed on the plate, and appropriate amounts of 10µl of EDTA solution was added to one of them to obtain the desired concentration (750µg). The inhibition zones of the imipenem and imipenem-EDTA discs were compared after 16 to 18 hours of incubation at 370C. In the Combined Disc Test, the increase in inhibition zone with the Imipenem and EDTA disc was > 7 mm than the Imipenem disc alone, it was considered as MBL positive. (13)

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OBSERVATIONS AND RESULTS Out of the 289 isolates included in the study 76 were Pseudomonas aeruginosa, 68 Acinetobacter baumannii, 65 E.coli, 48 Klebsiella pneumonia, 24 Enterobacter aerogenes and 8 were Citrobacter freundii.

Of these 289 isolates, 47 (16.2%) showed resistance to imipenem and were therefore further tested for MBL production. 31 of these isolates showed positive results for Metallo beta lactamase production by both DDST and CDDT methods as shown in Table 1.

Table 1: Total number of organisms isolated from different clinical samples of IPD patients, number of Imipenem resistant and MBL Producers in them. Organism Total No. Imipenem MBL Positive MBL Positive Isolated R (%) by DDST (%) by CDDT (%) P.aeruginosa A.baumannii E.coli K.pneumoniae E.aerogenes C.freudii TOTAL

76 68 65 48 24 8 289

16 (21%) 16 (23%) 11 (16%) 2 (4%) 1 (4%) 1 (12%) 47

8 (50%) 9 (56%) 10 (91%) 2 (100%) 1 (100%) 1 (100%) 31

Photograph 1: Isolated imipenem resistant strains showing positive test for MBL by both the methods DDST & CDDT.

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DISCUSSION AND CONCLUSION MBLs have been identified from clinical isolates worldwide, with an increasing frequency over the past few years and strains producing these enzymes have been responsible for prolonged nosocomial outbreaks that were accompanied by serious infections, as reported by Senda K et al. (15)

In our study about 21% of P.aeruginosa strains showed resistance to Imipenem of which about 50% were detected as MBL producers which is much higher than that obtained in a study conducted by Navneeth et al who reported 12% of MBL mediated Imipenem resistance in P.aeruginosa.(16) However the incidence of P.aeruginosa has been reported to be 10-50%

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www.ijpbs.com (or) www.ijpbsonline.com from various clinical specimens across the country.(17) In a study carried out by R Hasan et al in a tertiary care hospital in India, MBL production in Imipenem resistant isolates of Acinetobacter was 96.6% which is very high as compared to our study which showed 56% MBL production in Imipenem resistant (18) Acinetobacter. Since there is no standard guideline for detection of MBL, different studies have reported the use of different methods. Our study included the use of Imipenem- EDTA DDST and Imipenem-EDTA CDDT for detection of such isolates. Both the methodologies showed positive MBL results for the same 47 (Imipenem resistant) strains. The above methods were simple to perform and the materials used were cheap, non-toxic and easily accessible, making it highly applicable to routine clinical laboratories. It was seen that both the methods ImipenemDDST and Imipenem-CDDT were equally effective for MBL screening. In our opinion, the detection of MBL producing isolates is of crucial importance not only in institutes with high prevalence of such isolates but also in those in which the phonotype of resistance have never been detected as this will go a long way in helping to make adjustments in empirical antimicrobial therapy and probably, the reduction of mortality rates for patients infected with MBL- producing strains.

10.

11.

12.

13.

REFERENCES 1.

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Eisenstein BI, Zaleznik DF. Enterobacteriaceae. In: Mandell GL, Bennett JE, Dolin R, editors. Principles and practice of Infectious diseases, 5th ed. Philadelphia, PA: Churchill Livingstone; 2000, p2294-2310. Livermore D. Metallo-beta-lactamase mediated resistance and opportunities for its control. J Antimicrob Chemotherapy 1998; 41: p25-41. Mathur P et al. Prevalence of extended spectrum beta lactamase producing gram negative bacteria in a tertiary care hospital. Indian J Med Res, 2002; 115; p153-7. Gupta E, Mohanty S, Sood S, Dhawan B, Das BK, Kapil A. Emerging resistance to carbapenems in a tertiary

14.

15.

care hospital in north India. Indian J Med Res 2006; 124; p95-8. Kurokawa H, Yagi T, Shibata N, Shibayana K, Arakawa Y, Worldwide proliferation of carbapenem resistant gram negative bacteria, Lancet 1999; 354; p955. Yano H et al. Plasmid coded metallo-b-lactamase (imp6) conferring resistance to carbapenems, especially meropenem. Antimicrobial agents chemother 2001; 45; p1343-8. Walsh TR et al. Metallo-beta-lactamses: The quiet before the storm? Clin Microbial Rev 2005; 18; p30625. Cornaglia G et al. Study group for antimicrobial surveillance (ESGARS). Metallo-beta-lactamases as emerging resistant determinants in gram negative pathogens: open issues. Int J Antimicro Agents 2007; 29; p380-8. National Committee for Clinical Laboratory Standards: Performance standards for antimicrobial disc th susceptibility test. 7 Edition. Villanova PA: NCCLS 2002; document M2-A7. Lee K et al. Evaluation of the Hodge test and the Imipenem-EDTA double disc synergy test for differentiation of metallo-beta-lactamases producing clinical isolates of Pseudomonas spp and Acinetobacter spp. J Clin Microbiol 2003; 41; p4623-9. Sambrook J et al. Imipenem â&#x20AC;&#x201C;EDTA disc method differentiation of Metallo-beta-lactamase producing clinical isolates of Pseudomonas spp and Acinetobacter spp, J Clinical Microbiol 2002 October; 40( 10); p37983801. National Committee for Clinical Laboratory Standards, 2001. Performance standards for antimicrobial susceptibility testing. Eleventh informational supplement. M100-S11. National Committee for Clinical Laboratory Standards, Wayne, Pa. Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem-EDTA disk method for differentiation of metallo-beta-lactamases producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2002; 40:3798-801. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility th testing, 16 informational supplements. CLSI Document M2-A9, Wayne, PA: 2006. Senda K, Y Arakawa, K Nakashima H. Ito, S Ichiyama, K Shimokata, N Kato, M Ohta. Multifocal outbreaks of metallo-Beta-lactamase producing Pseudomonas aeruginosa resistant to broad-spectrum beta-lactams, including carbapenems. Antimicrob. Agents Chemother. 1996; 40:349-53

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www.ijpbs.com (or) www.ijpbsonline.com 16. Navneeth BV, Sridaran D, Sahay D, Belwadi MR. A preliminary study on metallo-beta-lactamases producing Pseudomonas aeruginosa in hospitalized patients. Indian J Med Res 2002; 116:264-7. 17. Taneja N, Aharwal SM, Sharma M. Imipenem resistance in non-fermenters causing nosocomial urinary tract infection. Indian J Med Sci 2003; 57:294

*Corresponding Author:

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Dr Anahita V. Hodiwala (Bhesania), Everest Apt, 701, Edulji Road, Charai, Thane west-400601, Maharashtra, India Email ID: anahitapb@hotmail.com

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Research Article Biological Sciences STUDIES ON SEWAGE POLLUTION IN SOME RESIDENTIAL AREAS OF SRIKAKULAM, A.P A.V.L.N.S.H.HARIHARAN AND J.V.S.K.V.KALYANI Department of Chemistry, GIT, GITAM University. Visakhapatnam â&#x20AC;&#x201C; 530 045,India. *Corresponding Author Email: ahharan@gmail.com

ABSTRACT Sewage is water-carried wastes, in either solution or suspension that is intended to flow away from a community .This study comprises of analysis of domestic sewage from ten different residential sampling stations of Srikakulam town, Andhra Pradesh. This study includes the determination of physicochemical parameters of the water samples in those selected areas during the pre-and post monsoon periods .The values of BOD, NH4-N in certain cases and TDS (nearly approaching the upper limit) obtained as per the prescribed standards.

KEY WORDS Domestic Sewage -- Pollution- Srikakulam town

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INTRODUCTION Sewage is water-carried wastes, in either solution or suspension that is intended to flow away from a community. It is more than 99.9% pure water and is characterized by its volume or rate of flow, its physical condition, its chemical constituents, and the bacteriological organisms that it contains. Domestic or sanitary sewage is the spent water from residential areas -- body wastes, washing water, food preparation wastes, laundry wastes, and other waste products of normal living. Wastes that result from an industrial process or the production or manufacture of goods are classed as industrial wastes. Their flows and strengths are usually more varied, intense, and concentrated than those of sanitary sewage. These waste waters carry pathogenic organisms that can transmit disease to human beings and other animals; contain organic matter that can cause odor and nuisance problems of receiving water bodies; and can lead to ecotoxicity. Excessive deposition of chemical nutrients in

water bodies is called eutrophication. Degradation of water quality, reduction in the number of fish and increase in BOD, are the effects of eutrophication. Proper collection and safe, nuisance-free disposal of the liquid wastes of a community are legally recognized as a necessity in an urbanized, industrialized society [1]. Sewerage being the pipes, pumps and infrastructure through which sewage flows [2]. Power can also be obtained from sewage water. The technique uses Microbial fuel cells. Characterization of the sewage becomes essential for an effective and economical waste management program and to choose the treatment processed, deciding the extent of treatment methods and assessing the beneficial uses of the wastes. Some, which are present in lower concentration or below detection limits in supply water, tend to increase more than 98% in used water (i.e., domestic sewage). Reasons for the increment in heavy metals (in sewage) such as Zn, Pb are not only because of domestic uses, but also from other sources. For example, lead (Pb) may be entering into the sewage system

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through dust fall, soil erosion, leaching, urban body weight, the deficiency of which may cause waste discharges and runoff from streets and growth retardation. But excessive concentration other surfaces. This toxic metal may cause in the drinking water may cause undesirable anemia, kidney disease and nervous disorders aesthetic effects. Characteristics of drinking above the tolerance limits 0.05 mg/L. Similarly, water as well as discharge of effluents are shown zinc (Zn) is an essential element in human in Table 1. metabolism. A child requires 0.3 mg of Zn/kg of Table 1: Analysis of sewage waters collected in pre monsoon period (2012) Parameter S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Effluent Drinking discharge water std. std.(ISI) pH 7.39 7.92 7.65 7.63 8.02 7.92 7.59 7.90 7.46 7.50 5.5-9.0 6.5-8.5 Electrical 1.8 1.2 1.9 0.9 2.1 1.6 1.7 1.8 2.0 1.6 ------------Conductivity TDS 432.7 492.1 471.6 507.5 404.3 471.8 335.2 399.4 356.8 389.3 2100 500 TSS 76 68 73 89 62 91 69 72 83 61 100 NS Hardness 223 290 226 208 276 203 185 269 197 273 NS 300 Chloride 114 108.6 104.1 98.7 101.5 113.3 88.6 72.7 87.2 98.4 1000 250 DO BOD COD Iron Lead Zinc NH4 -N

5.1 5.3 4.8 5.2 4.9 4.9 5.0 4.7 4.8 5.2 55 61 72 58 77 68 82 65 78 64 102 114 98 87 103 71 82 116 123 139 0.016 0.072 0.032 0.003 0.048 0.016 0.001 0.025 0.048 0.051 0.002 ND ND 0.004 ND 0.002 0.003 ND 0.003 0.002 0.198 0.219 0.237 0.142 0.319 0.414 0.450 0.297 0.272 0.402 32

NS 30 250 3 0.1 5

NS NS NS 0.3 0.05 5

All the parameters expressed in mg/lit. Except pH and EC (mmhos) *All the data is based on average of five determinations. ND – non- detectable; NS- not specified

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EXPERIMENTAL The study has been carried out during the pre – monsoon and post –monsoon periods in 2012 in a residential area situated in Srikakulam town, A.P, India. The main source of water supply in this area is by deep bore wells and local municipality. The colony residents are having their individual overhead tanks. From this community, domestic or residential establishments contribute the main waste water portion. It is mainly the spent water from kitchens, bathrooms, lavatories etc. Domestic sewage water samples were collected from 10

different residential areas of the town S1Balaga/Krishna Park, S2 - Gujarathi peta, S3 – Purushottam Nagar Colony, S4 - Hayathi Nagar, S5 – Navabharat Colony, S6 – Visakha A colony S7-Illisupuram Colony, S8 - Peddapadu, S9 – Aadhivaram Peta and S10 – Seepannaidu peta. The samples were stored in plastic bottles. Parameters like pH, conductivity. TDS, chlorides, hardness were determined as per standard methods [3]. The concentrations of Fe, Pb, and Zn were determined with the help of atomic absorption spectrophotometer). The results obtained are compared against standards.

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RESULTS AND DISCUSSIONS The results obtained in the present investigation are provided in Tables 1&2 Table 2: Analysis of sewage waters collected in post monsoon period 2012 Parameter S1 S2 S3 S4 S5 S6 S7 S8 S9

pH Electrical Conductivity TDS TSS Hardness Chloride DO BOD COD Iron Lead Zinc

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NH4 -N

7.15 1.6

7.42 1.0

7.30 1.8

7.75 0.9

7.02 2.0

7.72 1.3

7.35 1.7

7.59 1.6

458.2 466.8 458.3 373.4 352.4 459.5 303.5 379.4 72 68 106 56 77 95 69 72 218 268 249 315 269 206 202 272 101.4 79.8 85.2 90.7 91.2 99.1 73.5 65.2 4.7 5.0 4.6 5.0 4.9 4.7 5.1 4.9 52 68 79 57 73 74 80 69 98 84 108 78 102 62 71 116 0.012 0.052 0.002 0.002 0.008 0.021 0.005 0.005 0.001 ND ND ND ND 0.002 0.003 ND 0.124 0.301 0.183 0.171 0.312 0.405 0.510 0.322 30

Colour and Odour: Domestic sewage has a slightly alkaline condition and earthy odor and a cloudy appearance. With lapse of time, due to microbial action, it darkened in colour and the smell of the sewage became more pronounced. pH and Conductivity: The pH of domestic sewage from different Indian cities has specified by WHO standards vary from 7.0 to 7.5. In the present investigation the pH of the fresh well water samples are within the limits (Table 1) [4]. The conductivity of the present water samples found varied between 0.9 â&#x20AC;&#x201C; 2.1mmhos. The reason of this is the contamination of the sewage effluents by ionic pollutants like NaCl etc. in some stations. TDS and SS: The total dissolved solids (TDS) in the domestic sewage are found in the range 321.9-507.5 and 61-106 mg/L. A comparison between the two results clearly indicates that

7.28 2.0

S10

7.23 1.5

365.7 503.9 83 66 174 253 92.7 95.8 4.8 5.0 62 59 121 92 0.008 0.031 0.002 ND 0.295 0.352 38

Effluent Drinking discharge water std. std.(ISI) 5.5-9.0 6.5-8.5 ------------2100 100 NS 1000 NS 30 250 3 0.1 5

500 NS 300 250 NS NS NS 0.3 0.05 5

the sewage effluents are contaminated with water insoluble solids more than water-soluble, solids.. Knowledge of the classification of these solids is important, as it constitutes lad on biological treatment processes. Chlorides, and Nitrates Chloride content of the water samples found in the range 65.2-108.6 mg/L after domestic use. The reason for the sharp increment is that the human excretions contain chlorides equal to the chlorides consumed (commonly NaCl as common salt) with food and water. This amount averages from 8gm of chloride/person/day. The nitrate nitrogen concentration in the water samples was found out to be in the range 21-62 mg/L after domestic use (Tables 1 & 2). This may be due to the presence of urea [CO (NH2)2] which is the major source of nitrogen in the domestic sewage. Generally, the nitrate pick-up in the

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www.ijpbs.com (or) www.ijpbsonline.com Indian domestic sewage has been reported as 20-40mg/L [5]. Hardness: The total hardness of the water samples was found in the range of 174 to 315mg/L. This may be due to addition of certain compounds (which may impart hardness) after domestic use of the water. This parameter also does not pose problems in the congenital water treatment process. It can be considered that the sampling was dined during pre-monsoon period, so the sewage was raw and highly concentrated. Biochemical oxygen demand (BOD): The BOD of the present studies are found in the range of 52 to 80 mg/L (present investigation), which is above reported value for different domestic sewage is 45-54 g/capita-day[6]. The probable reasons for this slightly higher value may be that the sampling was conducted in the pre monsoon period, so the sewage was raw and concentrated. A high BOD value may pose a great problem for the conventional water treatment processes, as it constitutes a high load. Chemical oxygen demand (COD): The COD of the domestic sewage comes found in the range of 71 to 139 mg/L in the present study. Generally the range of the COD for the Indian domestic sewage is about 1.6 to 1.9 times the value of BOD. In the present study, COD coming to be in the range of 1.4 to 1.7 times the value of BOD. If the ratio between COD to BOD is known, it becomes easier to assume the value of BOD of the sewage in a very short time. The ratio will vary from one waste to other and will change for the same waste as it is subjected to various treatment operations. Heavy Metals: A high iron (Fe) content of >2 mg/L imparts a taste to drinking water besides leaving stains on laundry and plumbing fixtures. In the present study, the Fe content in the drinking water

measured is ranged between 0.001 and 0.072 mg/L, which is not very high. In the domestic sewage the iron content is < 0.006mg/L [7]. The reason of such increment can be attributed due to the fact that water being stored in the overhead iron tanks before being supplied. The maximum permissible limit of Zn in the drinking water is 5 mg/L. In the present study, the Zn as well as Pb contents lie within the prescribed limits (Tables 1&2).

CONCLUSIONS The data presented in Tables 1 & 2 indicates that the sewage becomes polluted with ionic and organic pollutants. Organic pollutants like NH 4-N and BOD show slightly higher concentration which actually implies that these parameters are generally absent in drinking water and even a slight increment would result in higher value. The movement of these ionic and organic pollutants through the soils enhances the possibility of the contamination of the underground water resources also. Therefore, it is very much needed to take necessary measures to treat and dispose the sewage properly and safely to prevent pollution.

ACKNOWLEDGEMENTS Thanks are due to Principal, GIT and Management of GITAM University for providing necessary facilities

REFERENCES [1] Masters, M.Gilbert. Introduction to Environmental Engineering and Sciences, Prentice Hall of India (p) Ltd, New Delhi (1994). [2] Train, R.E, Quality Criteria for water, USEPA, Washington DC, (1979). [3] Standard Methods for the examination of water and waste water .APHA.AWWA, WEF,New York, 20th edition (1998). [4] Maiti, S.K. Handbook of methods in Environmental studies ABD Publishers, Jaipur, Vol 1 (2001).

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www.ijpbs.com (or) www.ijpbsonline.com [5] Arceivala S.J. Wastewater treatment for pollution control. TMH. New Delhi. (2nd edn.) (1998) [6] Siddiqui, H.R.Characterstics of domestic and municipal sewage in India, Indian J. Env. Hlth ; 55:85-88 (1975)

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A.V.L.N.S.H.HARIHARAN Department of Chemistry, GIT, GITAM University. Visakhapatnam â&#x20AC;&#x201C; 530 045, India.

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Research Article Biological Sciences STUDY OF CARDIAC TROPONIN-I AS A DIAGNOSTIC MARKER IN COMPARISION WITH CREATINE KINASE-MB IN MYOCARDIAL INFARCTION Mahalaxmi.S.Petimani1*, P.Suresh Babu2 Department of Biochemistry, JJM Medical College, Davangere – 577004, Karnataka (India). *Corresponding Author Email: mahalaxmi.petimani@gmail.com

ABSTRACT BACKGROUND: Myocardial infarction is a medical emergency that most often caused by an exclusive coronary thrombus which results in prolonged myocardial ischemia and irreversible cell death. Early diagnosis of myocardial infarction is crucial in planning the treatment modalities, which if instituted in time reduces the morbidity and mortality considerably. OBJECTIVES: To evaluate the usefulness of cardiac troponin-I in the early diagnosis of myocardial infarction and in assesing the severity of the infraction. The study also compares cardiac troponin-I with cardiac marker creatine Kinase-MB. METHODS: The present study comprises of 100 subjects which includes 50 healthy controls and 50 subjects with myocardial infarction, in whom the following parameters were analysed on admission and after 24hours. The parameters are cardiac troponin- I (Chemiluminesence immunoassay method), creatine- kinase –MB (optimized DGKC method by semi auto ananlyzer). RESULTS: Cardiac troponin –I is better than creatine kinase –MB in diagnosing myocardial infarction .On admission the increase in serum cardiac troponin –I is highly significant in subjects with MI (p<0.001)when comparied to creatine kinase-MB .In our study the cutoff value of cardiac troponin –I is 0.60ng/ml by taking the mean of healthy controls .cardiac troponin-I level greater than 0.60ng/nl is used as positive value .The sensitivity and specificity of troponin-I is 96% and 98% respectively which is higher than creatine kinase-MB(62%and 68%)respectively ,within the first 6 hours of myocardial infarction .The diagnostic efficiency of cardiac troponin-I is 97%,and creatine kinase –MB is 63% in diagonosis of acute MI. CONCLUSION: Serum cardiac troponin-I is highly sensitive and specific cardiac marker which can substantially improve the early diagnosis of acute mycocardial infartion so that adequate treatment modalities can be initiated early in these patients to minimise the risk of death.The estimation of serum cardiac troponin-I level can be routinely employed to confirm or rule out acute myocardial infarction as a part of laboratory evaluation of acute myocardial infarction .

KEY WORDS Cardiac troponin-I; Creatine kinase-MB; Myocardial infarction

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INTRODUCTION Cardiovascular diseases (CVD) comprise of a group of diseases of the heart and the vascular system which includes the major conditions like coronary heart disease (CHD), hypertension, congenital heart disease. Coronary heart disease is defined as “impairment of heart function due to inadequate blood flow to the heart compared

to its needs, caused by obstructive changed in the coronary circulation to the heart”. Coronary heart disease is now the leading cause of deaths in India accounting for 29% of all WHO has drawn attention to the fact that CHD is a mordern epidemic .CHD may manifest itself in many presentations such as myocardial infarction ,angina pectoris and sudden death ,of

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www.ijpbs.com (or) www.ijpbsonline.com which acute myocardial infarction is specific to CHD. [1] Acute myocardial infarction is the most dangerous manifestation of CHD.[2] Myocardial infarction (MI) results from reduced coronary flow such that oxygen demand of the myocardium is not met resulting in irreversible cell death and necrosis. [3] According to WHO criteria, diagnosis of MI is based on clinical symptoms ,ECG changes and characteristic changing pattern of serum cardiac markers .The complications after acute myocardial infarction are maximum in the first few hours .[4] The early detection and diagnosis of MI is vital for the institution of therapy to limit myocardial damage and preserve cardiac function.[5] There is an urgent need for a rapid, sensitive and specific cardiac marker that can help clinicians to make early diagnosis of MI.Now the commercial avaliability of rapid, sensitive and cardiac specific troponin tests have revolutionized the cardiac biomarker utility in the differentiation of MI from other causes of chest pain. [6] Troponin is a protien complex located on the thin filament of striated muscles having 3 subunits .Troponin –T(TnT),Troponin –I(TnI) and Troponin –C(TnC).cardiac troponin particularly troponin –I is the preferred marker for the diagnosis of MI.[6] The present study is an attempt to elucidate the usefulness of troponin-I in the early detection of myocadial injury enabling adequate interventions to be given towards those who are likely to be benefited.

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MATERIALS AND METHOD A study of serum cardia troponin-I, creatine kinase-MB activity in subjects of MI is carried out from May 2011 to May 2012. The study is carried out in subjects with myocardial infarction and healthy controls selected from Bapuji Hospital and Chigateri General Hospital, Davangere (both

these hospitalsd are attached to the teaching institute, J J M Medical College, Davangere). Each subject gave an informed consent and this study was approved by the ethical and research committee of JJM Medical College, Davangere to use human subjects in the research study. Based on the inclusion and exclusion criteria a total number of 100 subjects are selected for the present study which includes 50 cases with MI and 50 healthy controls.clinically proven cases of myocardial infarction in the age group of 3080yrs, who are admitted to the cardiac ICU are included in the study. The diagnosis of myocardial infarction was confirmed by ECG changes.and controls are healthy age and sex matched individuals without any major illness and not on any medications. And myocardial infarction patients with cardiac trauma (cardioversion, pacing), hepatic disease, renal disease, critically ill, history of MI in past 1 year, severe sepsis, hypothyroidism and patients with angina, pericarditis and pulmonary embolus were excluded from study. Under all aseptic precautions about 4ml of venous blood is collected in a sterile bulb after admission and also 24hours later. Serum is seperated after centrifugation and is used for the analysis. In the present study serum cardiac troponin-I and serum creatine kinase –MB were estimated.cardiac troponin-I is estimated using chemiluminesence immunoassay (CLIA) kit from Acculite in Lumax 4101 reader using principle of immunoenzymometric assay [7] with normal value in adults of < 1.3ng/ml, and creatine kinase-MB is analysed using kits from ERBA company in ERBA chem 5 semiautoanalyzer using immunoinhibition kinetic method.[8] with normal value of CK-MB < 25IU/L AT 37 0 C.

RESULTS The results obtained in this present study are from the total number of 100 subjects. Out of

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total 50 healthy controls, 36 were males and 14 females. And out of 50 cases 36 males and 14 females.serum troponin-I and creatine kinase –

MB is estimated in all the subjects. Their results are shown in the following tables.

Table 1: Age wise distribution of healthy controls and cases Controls Cases No of Subjects

Age (yrs) 31-40

41-50

51-60

>60

Mean ± SD

52.7± 11.0

55.7 ±10.0

Range

30-75yrs

38-75yrs

GRAPH 1: Age wise distribution of healthy controls and cases 25 20 15 Controls 10

Cases

5 0 Age (yrs)

31-40

41-50

51-60

>60

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Table 2: Serum levels of CK-MB and cTnI among healthy controls and MI cases on admission Groups n CK-MB (IU/L) CTnI(ng/ml) Controls

Mean± SD (Range)

Cases

Mean± SD (Range)

Mean diff. t-value* p-value

14.7±4.2 (7.5-22.4) 17.5±4.2 (9.2- 25.2) 2.8 3.30 <0.01, S

0.60±0.26 (0.20-1.35) 3.16±1.02 (1.30-5.95) 2.56 17.26 <0.001, HS

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www.ijpbs.com (or) www.ijpbsonline.com Table 2 shows levels (mean ± SD and range) of serum CK-MB and cardiac troponin-I in healthy controls and subjects with acute myocardial infarction at the time of admission. It is seen from the table that the estimated levels (mean ± SD) of serum CK-MB and Cardiac troponin-I in healthy controls were in the range of 14.7±4.2 IU/L and 0.60±0.26ng/ml respectively.In MI patients on admission, the mean value of serum CK-MB and cardiac

troponin-I were in the range of 17.5±4.2 IU/L and 3.16±1.02ng/ml respectively. The statistical analysis by unpaired t-test shows that cardiac troponin-I level is increased in patients with MI on admission when compared to healthy controls and it is statistically highly significant (p<0.001). The serum CK-MB level is slightly increased in patients with MI on admission and is statistically significant (p<0.01).

Table 3: Serum level of CK-MB and cTnI on admission and after 24 hrs amongs MI cases Parameters On admission After 24 hrs Difference t-value* p-level Mean ± SD Mean ± SD CK-MB (IU/L) 17.5±4.2 147.0±37.1 129.5 24.54 <0.001,HS CTnI(ng/ml) 3.16±1.02 16.50±3.38 13.34 31.65 <0.001,HS *Paired t-test

Table 3 shows the level (mean SD) of serum CKMB and Cardiac troponin-I levels on admission and after 24 hours in MI patients. On admission serum levels of CK-MB and Cardiac troponin-I levels were found to be 17.5±4.2 IU/L and 3.16 ±1.02ng/ml respectively. After 24hours the levels (mean ±SD) of serum CK-MB and cardiac troponin-I levels were in the range of 147.0±37.1 IU/L and 16.50±3.38 ng/ml respectively.

The statistical analysis by paired t-test shows that the difference between th e values of serum CK-MB and Cardiac troponin-I levels in subjects with MI on admission and after 24 hours is 129.5 and 13.34 respectively. It shows that CK-MB and Cardiac troponin-I levels are significantly increased after 24 hours which is statistically significant (p<0.001).

Table 4: Diagnostic value of cTnI and CK-MB in the diagnosis of AMI Markers CK-MB CtnI Cut-off value (>16.0 IU/L) (>1.3ng/ml) 62%

96%

Specificity

64%

98%

PPV

63%

98%

NPV

63%

96%

Diagnostic efficiency

63%

97%

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Sensitivity

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GRAPH 2: Diagnostic value of cTnI and CK-MB in the diagnosis of AMI 120% 100% 80% 60%

CK-MB (>16.0 IU/L)

40%

CtnI (>1.3ng/ml)

20% 0%

Sensitivity

Specificity

PPV

Table 4 and graph 2 shows the diagnostic utility of cardiac biomarkers (serum CK-MB and C ardiac troponin-I) in the diagnosis of MI in patients admitted to the emergency department within 3 to 6 hours after onset of chest pain. Median of the combined groups is used as the

NPV

Diagnostic efficiency

cutoff value for the serum CK-MB and Cardiac troponin-I for the diagnosis of MI.It is evident that cardiac troponin-I is the most sensitive and specific cardiac biomarker with highest diagnostic utility in the early diagnosis of myocardial infarction.

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Table 5: comparision of CK-MB and cTnI between MI cases on admission and the standard reference value Parameter Normal reference value On admission (mean ±SD) CK-MB(IU/L) 7.0-24.0 17.5±4.2 cTnI(ng/ml) 0.20-1.35 3.16±1.02

Table 5 shows the comparative analysis of serum levels of CK-MB and Cardiac troponin-I in patients with MI on admission are 17.5±4.2 and 3.16±1.02ng/ml respectively. The table shows the normal reference value of serum CK-MB and cardiac troponin-I as 7.024.0IU/L and 0.20-1.35ng/ml respectively. It is evident that cardiac troponin-I is elevated well above the normal reference value in patients with MI on admission to the emergency department. Serum CK-MB levels are within the normal reference range in patients with MI on admission.

DISCUSSION Myocardial Infarction is the most serious complication of coronary artery disease which occurs due to the perfusion imbalance between supply and demand within the coronary arteries as a result of an acute thrombotic process. [5],[9]. The diagnosis of myocardial infarction has been clasically based on a history of chest pain and echocardiographic documentation of new Q waves and evolving ST-T wave changes. It has been demonstrated that 4-8% of patients with MI may be missed, as some patients have atypical symptomsor suffer from “silent MI” .A

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www.ijpbs.com (or) www.ijpbsonline.com significant number of false positive and false negative results have been reported on ECG. [10] As a result, clinical laboratories have become increasingly relied upon either to establish or rule out the diagnosis of acute myocardial infarction.[11] Appropriate interventions like thrombolytic therapy and primary percutaneous angioplasty can limit the myocardial necrosis thus limiting myocardial damage which will improve the prognosis in patients with MI. There is a considerable difference in the outcum if these treatments can be administered early in the course of the illness.[6] According to the WHO, the diagnosis of MI requires the presence of atleast two of the following three criteria: (1) a history of ischemia type of chest pain (2) evolutionary changes on serial electrocardiograms (3) rise and fall in the serum cardiac markers. Today the cornerstone of these diagnostic criteria is the evaluation of serial cardiac markers.[12] The present study is conducted to determine the diagnostic performance of cardiac troponin-I for early diagnosis of acute MI in patients at the time of presentation to the emergency department. In this study the estimated levels (mean±SD) of serum creatine kinase-MB in healthy controls is and 14.7±4.2 IU/L in MI patients on admission it is 17.5± 4.2 IU/L respectively. In patients with MI, on admission the serum creatine kinase –MB level is 17.5±4.2 and after 24hours the level is 147.0±37.1 IU/L respectively. The statistical analysis by unpaired t-test shows that serum CK-MB level is slightly increased in patients with MI on admission is statistically significant (p< 0.01) The statistical analysis by paired test shows that the difference between the values of serum CKMB in subjects with MI on admission and after 24 hours is 129.5. It shows that serum CK-MB

level is significantly increased after 24 hours which is highly signiificant (<0.001). This is in accordance with Jesse E Admas et al [13],Wille Gerhaldt [14] , John Griffiths [15] , and John H Alexendaer et al [16]. Serum creatine-kinase activity starts to rise within 6-8 hours arter an acute attack of MI. It reaches a peak of two to ten folds in 24 hours and decline to the range within 3 to 4 days following the onset of chest pain.[2],[3],[17]. Serum CK-MB elevation following myocardial ischemia is indicative of myocardial necrosis. It is a reflection of enzymes release from the myocardial cells undergoing necrosis. The relationship between changes in the serum enzymes activity and infarct size appears to be quantitative. Thus analysis of changes in the serum CK-MB activity following MI helps in the accurate assessment of the extent of myocardial necrosis and its progression.[18],[19],[20]. Complements of CK-MB are found in skeletal muscle, brain and thyroid. So CK-MB is markedly increased in most patients with muscular dystrophy, inflammatory disease of muscle, alcohol intoxication and intramuscular injections because of the enzyme release from the muscle. The B-chain subunit of creatine kinase is predominantly present in the skeletal muscle during fetal and neonatal development. Expression of this B-chain is suppressed in adult skeletal muscle. After skeletal injury there is increased synthesis of this B-chain subunit by reexpression of previously suppressed B-subunit gene. it is also found increased in myxedema due to diminished catabolism of circulating enzyme.[19],[20],[21]. In this study the estimated levels (mean ±SD) of serum cardiac troponin-I in healthy controls is 0.60±0.26 ng/ml and in MI patients on admission is 3.16 ±1.02 ng/ml respectively. The statistical analysis by un-paired t-test shows that cardiac troponin –I level is increased in

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www.ijpbs.com (or) www.ijpbsonline.com patients with MI on admission when compared to healthy controls and is statistically highly significant (p<0.001). In patients with MI, on admission the serum cardiac troponin-I level is 3.16ﾂｱ 1.02ng/ml and after 24hrs the level is 16.50ﾂｱ3.38 ng/ml respectively. The statistical analysis by paired ttest shows that the difference between the values of serum cardiac troponin 窶的 levels in subjects with MI on admission and after 24 hours is 13.34. it shows that serum cardiac troponin -I levels is significantly increased after 24 hours whch is statistically highly significant ( p< 0.001). This is in accordance with the study of Kristin Newby et al [22], Daylily S et al [23], Jan Ravkilde et al [24]. The troponin complex (troponin-T, troponin-I, and troponin-C) along with tropomyosin is located on the actin filament and is essential for calcium for the calcium mediated regulation of skeletal and cardiac muscle contraction. Troponin 窶的 is a regulatory protein that binds to actin and inhibits calcium-mediated actin-myosin interaction. [2], [3], [25]. Cardiac troponin-I has unique amino acid sequence that is produced only in myocardium throughout development. It is a regulatory protein with a high specificity for cardiac injury. Cardiac troponin-T is expressed in the skeletal muscle during fetal development. Troponin-I is not found in skeletal muscle during neonatal develpoment or during adulthood. Thus cardiac troponin-I does not rise in skeletal muscle disease or injury where as cardiac troponin-T will be elevated in these conditions. [2],[3],[5].Cardiac troponin-I is tightly complexed to the contractile apparatus. It forms complexes with troponin-C and is released into the blood following MI predomintaly as a binary complex. Circulating levels are normally low , but they rise relatively rapidly after acute MI suggesting the pressure of troponins in the cytosol of the myocyte. The normal level of serum cTnI is 1.0-

1.3ng/ml due to yhe continuous microscopic loss of cardiomyocytes during normal life.[5] Troponin starts to rise within 4-6 hours of onset of myocardial necrosis because of rapid leak of the cytosolic pool (3% of troponin-I) of troponini. In any cardiac myocyte injury , this unbound pool of troponin-I os realeased first . thius is followed by slow release and degradation of the troponin bound to myofibrils (96% of the troponin-I). Troponin level peaks at 18-24 hours and declines over 10-14 days due to the continuous leashing of troponin-I from yhe neccrotic cell. The half life of troponin is 2 hours.[25] Studies have shown that patients with positive troponin-I marker at the time of admission have an increased risk of re-infarction and that recurrent events are associated with increased risk of death. The mortality risk appears to corelate with the level of troponin rise. These events may represent evidence of a refractorey pathophysiological process that progresses despite ongoing medical therapy. Our study is in accordance with the study of John F Tucker et al[26], Judd E Hollander[27] . To summarize an initial troponin-I determination drawn at the time of the patients presentation is a powerful diagnostic tool for a rapid diagnosis rather than serial CK-MB determination. This reflects the greater sensitivity and specificity of troponin-I for minor myocardial necrosis resulting from microembolization from active plaques, which predispose to subsequent major clinical events, but those activities may not be detectable by less sensitive markers like CK-MB until a large event occurs. [23]

CONCLUSION Acute myocardial infarction is the major cause of death and premature disability in the developing society .Serum cardiac biomarker testing is now the cornerstone in the diagnosis of MI.An ideal

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www.ijpbs.com (or) www.ijpbsonline.com Serum cardiac marker should have high sensitivity and high specificity so that the diagnosis of MI is not missed. The present study found a statistically highly significant increase in cardiac troponin-I levels in subjects with MI at the time of admission to the cardiac ICU when compared to CK-MB,.cardiac troponin-I showed a high diagnostic efficiency of 97%,with a very sensitivity(96%) and specificity(98%) and positive predictive value(96%).It is the earliest marker for confirmation and exclusion of acute MI which is detected as early as 3 hours after the infarction. The results of the study found that cardiac troponin-I has excellent sensitivity and specificity and it is superior to creatine kinase-MB as an indicator of myocardial ischemia. The routine use of cardiac troponin-I in the evaluation of patients with suspected MI, can eliminate the estimation of CK-MB in the diagnosis of MI. A single estimation of cardiac troponin-I can make the diagnosis of MI accurately rather than waiting for serial changes of serum cardiac markers. The estimation of serum cardiac troponin-I should be made as an essential part of the evaluation in all subjects with signs and symptoms of chest pain. The combined use of cardiac troponin-I along with ECG and clinical history of chest pain will help in early and accurate diagnosis of MI leading to early diagnosis, initiation of treatment modalities thus resulting in better prognosis of patients with chest pain.

3. 4.

10.

11.

12.

13.

ACKNOWLEDGEMENT We acknowledge all the study subjects for their participation in the study and their cooperation.

14.

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Hamshi CK, Woo KS. New diagnostics markers for myocardial infarction. JHK Cardiol, October,5:140145,(1997). Nigam PK. Biochemical markers of myocardial injury. Ind J Clin Biochem,22(1):10-17,(2007). Baheti R, Laddha P, Gehlot RS. Value of Troponin-T test in the diagnosiss of acute myocardial infarction. J Indian Academy of Clinical Medicine,3(1):55-8, (2002). Daubert MA, Jeremias A. The utility of troponin measurement to deyect myocardial infarction : review of the current findings. Vascular Health and Risk Management:691-699,(2010). Soreng K. Troponin I and Troponin T in myocardial infarction diagnosis and risk assessment. Bayer Health Care Diagnostics Division 2006;p:1-6. Apple FS, Jaffe AS. Cardiovascular disease. In: In : Burtis CA, Ashwoo ER, Burns DE, eds. Teitz Textbook of th fundamentals of clinical chemistry 6 edition. Philadelphia, Saunders; 2008:p:621-623.cTnI Acculite: Monobind Inc. Wille G, Johan W.Creatine kinase B subunit activity in serum after immnoinhibition of M-subunit activity. Clin Chem, 25:1274-1280, (1979). Warell DA, Cox TM, Firth JD. Clinical cardiology. In: th Oxford textbook of medicine 4 edition, Oxford University Press New York, 2003;vol 2:p388. Guzy PM. Creatine phosphokinase and the diagnosis of myocardial infarction. West J Med, 127:455-460, (1997). Winter RJ, Bholasingh R, Nieuwenhuijs AB, Koster RW, Peters RJG, Sanders GT et al. Ruling out acute myocardial infarction early with two serial creatine kinase- MB mass determinations. Eur Heart J ,20:967972, (1999). Alexander JH, Sparapani RA, Mahaffey KW, Deckers JM, Newby KL, Ohman ME et al. Association between minor elevations of creatine kinase-MB level and mortality in patients with acute coronary syndromes without ST-segment elevation. JAMA, 283:347-353, (2000). Adams JE, Schechtman KB, Yuonne L, Ladenson JH, Jaffe AS. Comparable detection of acute myocardial infarction by creatine kinase MB isoenzyme and cardiac troponin-I. Clin Chem,40:1291-1295, (1994). Griffiths J, Handschuh G. Creatine kinase isoenzyme MB in myocardial infarction: Methods compared. Clin Chem,23(3):567-570,( 1977). Henry R.T, Chiamori N, Goiub O.J. and Berkamn S, Am J Clin Path ,34(381), (1960). Apple FS, Pearce LA, Smith SW, Kaezmarker JM, Murakami MM. Role of Monitoring changes in sensitive cardiac troponin-I assay results for early

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of discordant creatine kinase- MB and troponin measurements in acute coronary syndromes. J Am Coll Cardiol, 47:312-318, (2006). Ooi SD, Isolato PA, Veinot JP. Correlation of antemortem serum creatine kinase, creatine kinaseMB, troponin-I and troponin-T with cardiac pathology. Clin Chem : 338-344, (2000). Ravkilde J, Nissen H, Horder M, Thygesen K. Independent prognostic vaue of serum creatine kinase isoenzyme MB mass, cardiac troponin T and myosin light chain levels in suspected acute myocardial infarction. J Am Coll Cardiol ,25:574-581, (1995). Lum G, Solarz De, Farney L. False positiv cardiac troponin results in patients without acute myocardial infarction. Labmedicine, 37(9):546-550, (2006). Tueker JF, Collins RA, Anderson AJ, Hauser J, Kalas J, Apple FS et al. Early diagnostic efficiency of cardiac troponin and Troponin-T for acute myocardial infarction. Acad Emerg Med 4:13-21, (1997). Hollander JE. Highly sensitive troponins. J Am Coll Cardiol, 54:1173-1175. (2009).

CONFLICT OF INTEREST: Nil FUNDS FOR STUDY: Nil

*Corresponding Author:

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Dr. Mahalaxmi.S.Petimani * Department of Biochemistry, JJM Medical College, Davangere, Karnataka. Email id:mahalaxmi.petimani@gmail.com

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Research Article Pharmaceutical Sciences EXPERIMENTAL EVALUATION OF DIURETIC ACTIVITY OF AEGLE MARMELOS IN RATS 1

Seema Singh*, 2Sandeep Kumar Singh, 3Shobit Srivastava, 3Pramod Singh, 4Mohit Trivedi, 3Pratap Shanker, 3R.K.Dixit, 5Romi Singh Rana 1

Department of Pharmacy, Northern India Engineering College, Lucknow, (U.P.) Department of Pharmaceutical Sciences, Dr. H. S. Gaur Central University, Sagar (M.P.) 3 Department of Pharmacology, King George Medical University, Lucknow (U.P) 4 Department of Pharmacology, E.L.M.C. Lucknow, (U.P.) 5 Department of Urology, King George Medical University, Lucknow (U.P.) *Corresponding Author Email: seema_b.pharm@rediffmail.com

ABSTRACT Purpose: The aim of the present study was to investigate the diuretic activity of ethanolic extracts and its fractions of Aegle marmelos fruit in experimental models. Ethanolic extracts and its fractions of Aegle marmelos ripe fruit were administered to experimental rats intraperitonealy at doses of 300, 400 and 500 mg/kg i. p. Control group received only normal saline (25ml/kg) through intraperitoneal route. Standard group received Furosemide (100mg/kg). The diuretic effect of the extracts was evaluated by measuring urine volume and sodium content in urine.

KEY WORDS Aegle marmelos, diuretic activity

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INTRODUCTION Diuretics are used in many clinical conditions including the edematous disorders and hypertension. Though all diuretics are used to increase renal excretion of sodium and water, they differ considerably in chemical derivation, efficacy, sites and mechanism of action. The choice of a diuretic clinically depend on is the objective of therapy and the pathophysiology of the patientâ&#x20AC;&#x2122;s disease. Patients with renal insufficiency require loop diuretics because they do not respond to other agents to a clinically relevant degree. Patients with cirrhosis are reported for have secondary hyperaldosteronism as a cause of sodium retention and diuretic treatment in such patients is initiated with an inhibitor of aldosterone, spironolactone. Effective use of diuretics requires knowledge of

the pharmacology of each diuretic agent coupled with an understanding of the pathophysiology of the patientâ&#x20AC;&#x2122;s disease. Other uses include the treatment of hypertension and the treatment of cerebral edema [1]. A global reliance on alternative system of medicine for chronic and acute ailments resulted in an intense area of research and discovery of a number of herbs with potential to curb diseases. Among them, ample number of herbs has been exploited for modulation of immune system from Ayurvedic formulations either alone or in combinations [2]. Traditionally, various parts of the plant, A. marmelos Corr. (Rutaceae) has been in use the treatment of a variety of disorders [3]. The plant is reported to have multiple therapeutic properties such as anti-inflammatory, antipyretic and analgesic [4] anti diabetic [5] anti diarrhoeal

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www.ijpbs.com (or) www.ijpbsonline.com [6] anti hyperlipidemic [7] antifungal [8] antimicrobial, antibacterial, anti parasitic [9] anti cancer [10] insecticidal activity [11] anti malaria [12] hepatoproctective [13] antigenotoxic activity [14] cytoprotective effect [15] immunomodulatory activity [16] anticonvulsant Activity [17] hypolipidemic activity [18] antifertility effect [19] and cardioprotective potentials [20]. However the diuretic activity of A. marmelos has more been reported so far. Therefore present study was planned to see the diuretic activity of A. Marmelos in animal in animal experimental model.

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MATERIALS AND METHODS Plant collection Ripe Fruits of Aegle marmelos (Bael) were collected in the month of March from local market of Lucknow (Uttar Pradesh). The fruits were authenticated as Aegle marmelos (Rutaceae) by pharmacognostic evaluation and a voucher specimen was deposited at Taxonomic Division of National Botanical Research Institute Lucknow, for future reference. (Voucher no. NBRI/CIF/148/2010). Preparation of ethanolic extract The plant materials were dried in the shade and size reduction of air-dried ripe fruit pulp of Aegle marmelos was done by a mechanical grinder. The powder of Aegle marmelos was initially defatted with petroleum benzene (60 - 80˚C) followed by 1000 ml of ethanol, by using a Soxhlet extractor for 72 hours at a temperature not exceeding the boiling point of the solvent. The extract was filtered using whattman filter paper (No.1) and then concentrated in a vacuum and dried at 45˚C for ethanol elimination and to obtain a dark-brown residue. The extracts were kept in a sterile bottle under refrigeration conditions of about 2-8˚C for further studies.

Experimental animals Male wistar rats weighing 100-200 gm were obtained from the animal house, faculty of pharmacy, NIEC, Lucknow (U.P.) the animals were housed in polypropylene cage with steel net and maintained under standard conditions of temperature 25±5ºc and 55±5 relative humidity with a regular 12 hours light and 12 hours dark cycles and allowed free access to standard laboratory food and water. All animals are treated humanely in accordance with guidelines from case of animals as set by IAEC. Diuretic activity The diuretic activity of ethanolic extracts of Aegle marmelos, petroleum ether fraction of Aegle marmelos, ethyl acetate fraction of Aegle marmelos, chloroform fraction of Aegle marmelos and Furosemide was carried out by using in-vivo, Lipschitz test method. The rats were divided into 6 groups each containing different no. of animal depending upon dose, at least 3 animals are required for each dosing and deprived of food and water for 18 hours. All the rats received priming dose of normal saline (25ml/kg) orally. Both the extracts and Furosemide (Standard) were dissolved in a normal saline.  Group I served as control in which only normal saline (25ml/kg) was administered through intraperitoneal route.  Group II served as standard received Furosemide (100mg/kg). Rest of the groups served as treated groups.  Group III received Ethanolic extract of a. marmelos at the dose levels of 300, 400 and 500 mg/ kg i.p., respectively.  Group IV received Petroleum ether extract of a. marmelos at the dose levels of 300 and 400 mg/ kg i.p., respectively.  Group V received Chloroform extract of a. marmelos at the dose levels of 300 and 400 mg/ kg i.p., respectively

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Group VI received Ethyl acetate extract of a. marmelos at the dose levels of 300 and 400 mg/kg i.p., respectively. Immediately after administration, the rats (one in each cage) were placed in metabolic cages specially designed to separate urine and faeces and kept at room temperature of 25±0.5ºC. The urine was collected in a measuring cylinder up to 24 hrs. During this period, no food or water was

made available to animals. The volume of urine collected was measured for all the groups. The parameters taken for each individual rat were body weight before and after test period, urine volume (concentrated for water intake during the test period), and concentration of Na+ in urine. The content of Na+ in the urine was estimated by flame photometry.

STATISTICAL ANALYSIS Statistical analysis by ANOVA followed by Dunnet’s multiple comparison tests. Results are expressed as mean ± standard error, n = 3 in each group. Significant difference compared to control group at p < 0.05. S.no.

Groups

Dose (mg/kg)

Urine vol. (ml/kg)

Conc. of excreted + Na ions (mEq/L)

Control (normal saline)(Cont.)

25 ml/kg

24.66 ± 1.452

78.63 ± 0.317 *

150 ± 1.154*

Standard (Furosemide)(Std.)

100 mg/kg

33.66 ± 1.855

Ethanolic Extract (E1) (E2) (E3) Pet. Ether Fraction(P1) (P2)

300 mg/kg 400 mg/kg 500 mg/kg 300 mg/kg 400 mg/kg

24 ± 0.577 27.33 ± 1.452 32.33 ± 1.452* 35 ± 1.732* 33 ± 1.527*

81.66 ± 0.881 83.33 ± 0.881 85 ± 1.154* 97 ± 1* 85.33 ± 2.027*

Ethylacet. Fraction (Et.1) (Et.2)

300 mg/kg 400 mg/kg

22 ± 1.154 22 ± 0.154

80.33 ± 0.881 80.33 ± 1.201

Chlorof. Fraction (C2)

300 mg/kg 400 mg/kg

20 ± 1.154 21 ± 0.577

85 ± 1.527* 81.66 ± 1.452

(C1)

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Graph no.4.1 showing concentration of excreted Na+ ions (mEq/L)

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Graph no. 4.2 showing amount of urine volume (ml/kg)

RESULTS The results of the preliminary phytochemical screening of ethanolic extracts and its fractions are given in Table No. 1. The ethanolic extract was found to produce significant increase in excretion of sodium at the higher dose tested (500 mg/kg i. p.). The order of activity of increase in urinary output and urinary electrolyte excretion was found to be as petroleum ether fraction > chloroform fraction > ethyl acetate fraction.

Results from the present study suggest that extract of A. marmelos possess diuretic activity. These results need further validation by doing experimental study in large scale. If the further studies support present study the effect can be further confirmed by clinical study. This plant may get place in near future in treatment of hypertension, renal failure, congestive heart failure and clinical conditions leading to edematous state of body.

REFERENCES 1.

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DISCUSSION The diuretic activity of ethanolic extracts as well as fractions (petroleum ether, ethyl acetate, chloroform) and Furosemide was carried out using Lipschitz test model. Statistical analysis was done using ANOVA followed by Dunnet’s multiple comparison tests. Results were expressed as mean ± standard error, n = 3 in each group. Among all test compounds used, the petroleum ether fraction was found to be most active which shows 35 ± 1.732* ml/kg (Urine vol.); 97 ± 1* mEq/L (Conc. of excreted Na+ ions) at 300 mg/kg dose and 33 ± 1.527* ml/kg (Urine vol.); 85.33 ± 2.027* mEq/L (Conc. of excreted Na+ ions) at 400 mg/kg dose which is significant from the control group, at 0.05 % Probability.

Holohan, P. D. General Principals and Renal Cellular Mechanisms of Drug Transport. In Principles of Pharmacology, Basic Concepts and Clinical Applications, 11th ed.; Manson, P. L., Ed.; Chapman and Hall, an International Thomson Publishing Company, New York, 1995; p 655. Patel, P.; Asdaq, S. M. B. Immunomodulatory activity of methanolic fruit extract of Aegle marmelos in experimental animals. Saudi Pharmaceutical Journal. 2010, 18, 161–165. Nadkarni, A. K., Dr. K. M. Nadkarni’s Indian Materia Medica, vol. 1. Popular Prakashan Company, Bombay (India), 1986, pp. 45–50. Arul, V., Miyazaki, S., Dhananjayan, R. Studies on the antiinflammatory, antipyretic and analgesic properties of the leaves of Aegle marmelos. J. Ethnopharmacol. 2005, 96, 159–163. Arumugam, S., Kavimani, S., Kadalmani, B., Ahmed, A. B. A., Akbarsha, M. A., Rao, M. V. Antidiabetic activity

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of leaf and callus extracts of Aegle marmelos in rabbit. Sci. Asia. 2008, 34, 317–321. Shoba, F.G., Thomas, M. Study of antidiarrhoeal activity of four medicinal plants in castor-oil induced diarrhea. J. Ethnopharmacol. 2001, 76, 73–76. Vijaya, C., Ramanathan, M., Suresh, B. Lipid lowering activity ethanolic extract of leaves Aegle marmelos in hyperlipidaemic models Wistar albino rats. Ind. J. Exp. Biol. 2001, 47, 182–185. Rana, B. K., Singh, U. P., Taneja, V. Antifungal activity and kinetics of inhibition by essential oil isolated from leaves Aegle marmelos. J. Ethnopharmacol. 1997, 57, 29–34. Ulahannan, R. K., Thomas, T., Sadasivan, C. Antibacterial action of leaves of Aegle marmelos. Int. J. (Sci.). 2008, 2, 134–138. Gangadevi, V., Muthumary, J. Taxol, an anticancer drug produced by an endophytic fungus Bartalinia robillardoides Tassi, isolated from a medicinal plant, Aegle marmelos Correa ex Roxb. World J. Microbiol. Biotechnol. 2008, 24, 717–724. Kumar, R.; Kumar, A.; Prasa, C. S.; Dubey, N. K.; Samant, R. Insecticidal activity aegle marmelos (l.) correa essential oil against four stored grain insect pests. Int. J. Food Safety. 2008, 10, 39-49. Elango, G., Abdul Rahuman, A., Bagavan, A., Kamaraj, C., Abduz Zahir, A., Venkatesan, C. Laboratory study on larvicidal activity of indigenous plant extracts against Anopheles subpictus and Culex tritaeniorhynchus. Parasitol. Res. 2009, 104, 1381– 1388. Singh, S.; Rao, H, R. Hepatoprotective effect of the pulp/seed of Aegle marmelos correa ex Roxb. against

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carbon tetrachloride induced liver damage in rats. Int. J. Green Pharmacy. 2008, 2 (4), 232-234. Kaur, P.; Walia, A.; Kumar, S.; Kaur. S. Antigenotoxic activity of polyphenolic rich extracts from Aegle marmelos (L.) correa in human blood lymphocytes and E.coli PQ 37. Rec. Nat. Prod. 2009, 3 (1), 68-75. Vinodhini, R.; Narayanan, M. Cytoprotective effect of Nelumbo nucifera and Aegle marmelos in common crap exposed to heavy metals. Int. J Integrative Biology. 2009, 7(1), 124-129. Patel, P.; Asdaq, S. M. B. Immunomodulatory activity of methanolic fruit extract of Aegle marmelos in experimental animals. Saudi Pharmaceutical Journal. 2010, 18, 161–165 Sankari, M.; Chitra, V.; Silambujanaki, P.; Raju, D. Anticonvulsant activity of ethanolic extract of Aegle marmelos (leaves) in mice. Int. J. Pharm. Tech. Res. 2010, 2 (1), 640-643. Devi, K.; Sivaraj, A. P.; Kumar, V.; Ahmed, K. S. Z.; Sathiyaraj, K.; Kumar, M. B. S.; David, E. Hypolipidemic effect of Aegle marmelos leaf extract in streptozotocin (stz) induced diabetic male albino rats Int. J. Pharm Tech Res. 2010, 2 (1), 259-265. Sathiyaraj, K.; Sivaraj, A.; Madhumitha, G.; Kumar, P. V.; Saral, A. M.; Devi, K.; Kumar, B. S. Antifertility effect of aqueous leaf extract of Aegle marmelos on male albino rats. Int. J. Current Pharm. Res. 2010, 2 (1), 26-29. Vimal, V., Devaki, T. Linear furanocoumarin protects rat myocardium against lipid peroxidation and membrane damage during experimental myocardial injury. Biomed. Pharmacother. 2004, 58, 393–400.

*Corresponding Author: Seema Singh

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Department of Pharmacy, Northern India Engineering College, Lucknow, (U.P.)

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Review Article Biological Sciences SUB-CLINICAL HYPOTHYROIDISM: COMPLICATIONS AND EFFECTS OF THYROXINE THERAPY S. P. JADKAR *1, J. V. GANU 1 and K. N. PUJARI1 1

Department of Biochemistry, Government Medical College, Miraj *Corresponding Author Email: drsangitajadkar@gmail.com

ABSTRACT Sub clinical hypothyroidism (SCH) is typically defined as elevated plasma TSH while free plasma T4 and T3 remain within normal range. SCH or mild thyroid failure is a common problem with a prevalence of 3% to 8% in the population. It increases with age and higher in women compared to men. There are various complications due to SCH such as, abnormal lipid metabolism, cardiac dysfunction, neurological, mental dysfunction, and neuromuscular dysfunction, psychiatric & cognitive dysfunction. The potential benefits and risks of therapy for SCH have been debated for two decades. The possible advantages of treating SCH generally fall in to three categories. First, progression to overt hypothyroidism, with its attendant morbidity, would be prevented by thyroxine therapy. Second, thyroxine therapy may improve the serum lipid profile & therapy potentially decreases the risk of death from cardiovascular causes. Finally, treatment may reverse the symptoms of mild hypothyroidism, including psychiatric and cognitive abnormalities. This review includes a discussion on diagnosis of SCH, its complications and effects of therapy.

KEY WORDS Sub-clinical hypothyroidism, Thyroxine therapy, Thyroid hormones.

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INTRODUCTION Sub clinical hypothyroidism (SCH) is typically defined as elevated plasma TSH while free plasma T4 and T3 remain within normal range [1]. With the advent of serum thyrotropin radioimmunoassay in the 1970s, the entity of mildly elevated TSH and normal thyroid hormones levels was recognized [2, 3]. Serum TSH has a log-linear relationship with circulating thyroid hormone levels. TSH is a sensitive and specific measure of thyroid function and serum TSH assay is the necessary test for diagnosis of mild thyroid failure when the peripheral thyroid hormone levels are within normal laboratory range [1]. The diagnosis of SCH is determined mainly by laboratory evaluations; because it has been observed that most patients exhibit few or no signs and symptoms of thyroid dysfunction

.Some studies suggest that few patients do indeed have clinical, biochemical or functional manifestations of mild thyroid failure such as abnormal lipid metabolism, cardiac dysfunction and neurological and mental dysfunction [4]. Prevalence: Sub clinical hypothyroidism or mild thyroid failure is a common problem with a prevalence of 3% to 8% in the population [5, 6], being higher in women (7-8%) as compared to men (2.84.4%). The age dependant increase in rates of SCH is higher in women than in men with near equal rates between genders around the mid 70s [5]. After the sixth decade of life, the prevalence in men approaches that of women, with a combined prevalence of 10% [5]. Antithyroid antibodies can be detected in 80% of patients

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www.ijpbs.com (or) www.ijpbsonline.com with SCH. Serum TSH of less than 10 mIU/L is observed in 80% of patients with SCH. Diagnosis: Other causes of raised TSH: Before diagnosis of SCH, other causes of an elevated TSH level, such as recovery from nonthyroidal illness, assay variability, presence of heterophile antibodies interfering with the TSH assay, and certain cases of central hypothyroidism with biologically inactive TSH and thyroid hormone resistance, should be excluded. However, the most common cause of elevated TSH is autoimmune thyroid disease [1]. Some drugs such as sulfonyureas, lithium, amiodarone, ethionamide, phenylbutazone, aminoglutethimide, and iodine can interfere with thyroid hormone production or release and secondarily result in a slight elevation of TSH. In addition, dopamine antagonist such as metaclopromide and domperidone may cause exaggerated TSH response to TRH stimulation by altering the inhibitory effect of dopamine on TSH secretion. Furosemide has also been shown to increase levels of TSH, especially in recovering critically ill patients. Other conditions that cause elevated TSH include thyroid hormone resistance, thyroid hormone secreting tumors (both should be associated with high free thyroxine level), psychiatric illness, adrenal insufficiency, renal failure, hyperprolactinemia and systemic illness [7]. Previous radioiodine therapy, thyroid surgery, and external radiation therapy can also result in mild thyroid failure. Transient SCH may occur after episodes of postpartum, silent, and granulomatous thyroiditis [1, 8]. Reference Range of Serum TSH: Lowering the upper limit of normal for the serum TSH level up to 3 to 2.5 mIU/L has been proposed. For this, the supporting argument was the higher level of antithyroid antibodies detected in persons with a serum TSH between

3.0 to 5.0 mIU/L and higher rate of progression to clinical thyroid disease [9]. But studies have shown that decreasing the reference range to 3.0 mIU/L will result in more than a 4 fold increase in diagnosis of hypothyroidism among patients without history of thyroid disease and no evidence supports a benefit for intervening at these levels of TSH [9]. Reference ranges for serum TSH was defined by an expert panel representing American Thyroid Asso, Am. Asso of Clin Endocrinologists as 0.45 to 4.5 mIU/L and SCH was defined as serum TSH more than 4.5 mIU/L with normal T3 and T4 values in the absence of symptoms [9]. The American thyroid association recommends screening for measurement of serum TSH beginning at the age of 35 yrs and every 5 yrs thereafter [10]. Because of potential implications of SCH for adverse outcome of pregnancy [11] and neuropsychiatric development of the fetus [12], aggressive case findings in pregnant women or in women anticipating pregnancy has been suggested [13]. Proposed Adverse Consequences of SCH: Although studies have pointed to some adverse effects of SCH, no consensus exists as to the clinical importance of the adverse effects and the benefits of levothyroxine therapy, particularly for the 80% of patients with SCH who have a TSH of less than 10miu/l, because of the different levels of TSH and degrees of thyroid dysfunction in the studies [14, 15]. The potential benefits and risks of therapy for SCH have been debated for two decades. The possible advantages of treating subclinical hypothyroidism generally fall into three categories. First, progression to overt hypothyroidism, with its attendant morbidity, would be prevented by thyroxine therapy. Second, thyroxine therapy may improve the serum lipid profile and thereby potentially decrease the risk of death from cardiovascular

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www.ijpbs.com (or) www.ijpbsonline.com causes. Finally, treatment may reverse the symptoms of mild hypothyroidism, including psychiatric and cognitive abnormalities [16]. A discussion of some of the proposed adverse effects of SCH and effect of levothyroxine therapy follows: Progression to Overt Hypothyroidism: Patients with SCH have a high rate of progression to clinically overt hypothyroidism, 2.6% each year if thyroperoxidase (TPO) antibodies are absent and 4.3% if they are present [17]. However, some persons do not show progression and some experience normalization. A TSH level greater than 10 mIU/L predicts a higher rate of progression, and a level of less than 6miu/l predicts a lower likelihood of progression. In a study in men and women older than 55 years with a mean follow-up of 32 months, the TSH level normalized in 52% of those with a serum TSH of less than 10 mIU/L [18]. Systemic Symptoms of Hypothyroidism: Several randomized studies of the effect of levothyroxine therapy in patients with SCH are available. One study limited to patients with serum TSH levels from 5 to 10 mIU/L did not show any benefit [19]. Some studies (range in TSH level, 3-32 mIU/L) showed improved symptom scores or improved memory in a quarter of patients. Many recent studies have not shown improvement in mood, anxiety, and cognition in older persons [20, 21]. In a previous scientific review in 2004 [13], available data were considered insufficient to support a benefit for levothyroxine therapy in patients with SCH, in particular for the group with TSH less than 10 mIU/L, and a similar conclusion can again be drawn in 2008 [22]. Lipid Abnormalities and Other Cardiac Risk Factors: The Colorado Health Fair study showed that the mean total cholesterol level was 216 mg/dL (to

convert to mmol/L, multiply by 0.0259) for euthyroid patient and 224 mg/dl for patients with SCH [23]. Several randomized studies have shown reduction of low-density lipoprotein cholesterol by levothyroxine therapy. However, most of the studies showing benefits are not categorized for serum TSH levels of 5.0 to 10.0 mIU/L. A meta-analysis of 13 studies concluded that lipid profile improved with therapy [24]. In a 2004 review, data were considered insufficient to show benefits of levothyroxine therapy on lipid levels [13]. In estimating, the possibility that lipid levels will improve with levothyroxine therapy is fair if the serum TSH level is greater than 10 mIU/L and questionable if the TSH level is less than 10 mIU/L. In a large population based study, emerging risk factors such as elevated C-reactive protein, homocysteines and lipoprotein (a) levels were not found to have a role in SCH [25]. Improvement of endothelial dysfunction and insulin resistance after levothyroxine therapy has been suggested [26, 27]. In the absence of large scale randomized trials, evidence remains inconclusive. The effects of sub clinical hypothyroidism on serum lipid levels remain controversial. Some [28], but not other [29], cross sectional studies have demonstrated that serum levels of total cholesterol and LDL cholesterol are higher in patients with SCH than in euthyroid controls. A recent meta-analysis of the effect of therapy for SCH on serum lipid levels demonstrated a mean reduction in the total cholesterol level of 7.9 mg/dl (0.2 mmol/L) & in the LDL cholesterol level of 10mg/dl (0.26 mmol/L) [30]. Changes in HDL cholesterol were heterogenous among the studies and were not statistically significant. Patients with higher cholesterol levels {â&#x2030;Ľ 240 mg/dl (6-21 mmol/L)} & patients with SCH as a result of inadequately treated overt hypothyroidism had greater reductions in

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www.ijpbs.com (or) www.ijpbsonline.com cholesterol levels. In patients with newly diagnosed SCH whose total cholesterol level was less than 240mg/dl, the mean reduction in total cholesterol was only 0.7 mg/dl (0.02mmol/L) which was not statistically significant. Small studies [31] have suggested that patients whose serum thyrotropin level is less than 10 mIU/L may have no reduction in cholesterol levels with thyroxine replacement, but the metaanalysis did not directly address this issue. In a cross-sectional cohort study of middle-aged Dutch women, those with SCH were approximately twice as likely as euthyroid control women to have “atherosclerosis” and a history of myocardial infarction, and the difference persisted after adjustment for body mass index, systolic and diastolic blood pressure, smoking status, and total and HDL cholesterol levels. “Nontraditional” coronary risk factors such as elevated lipoprotein (a) or homocysteine levels might explain the higher rate of atherosclerosis in SCH, but published data are few and conflicting [32]. Adverse Cardiac End Points: The cross-sectional Rotterdam Study showed an association of SCH with myocardial infarction and aortic calcification [33]. In contrast, the Wickham study [34] showed no increased cardiac mortality in a 20 year follow up. A more recent observational study did not show any association between unrecognized SCH and cardiovascular events or mortality [35]. However, several more recent meta-analyses of observational studies found an association between SCH and coronary artery disease [3638]. The risk is lower when higher quality studies are pooled [37]. A recent analysis of 7 cohort studies concluded that the relative risk of all cause mortality was increased compared with euthyroid controls, particularly in patients with comorbid conditions [39]. Another metaanalysis

of 15 studies showed an increased prevalence and incidence of cardiovascular mortality only in a relatively younger population [40]. Taken together, the findings of these recent metaanalysis suggest that a cardiovascular risk exists for persons younger than age 70 years with no effect for those aged 70 to 80 years and a possibly protective effect for those older than 80 years [41]. Thus, the cardiovascular risk issue remains controversial, and large scale, government sponsored, multicenter, randomized, placebo controlled studied are needed to assess the efficacy of levothyroxine therapy in risk reduction. Cardiac Dysfunction: Studies have shown slowed left ventricular relaxation time, increased vascular tone at rest, and left ventricular systolic dysfunction with exercise and impaired endothelial function [42]. Some studies have shown improvement of cardiac contractibility and systolic time interval with levothyroxine therapy [42]. No evidence exists to support an association between heart failure and a serum TSH level of less than 10.0 mIU/L. Again, most studies were not categorized for degrees of TSH elevation, and data remain insufficient for a TSH level less than 10miu/l but strongly suggestive for a TSH level greater than 10mIU/L. In some studies (not all) resting pre-ejection period (PEP), left ventricular ejection time (LVET) was increased in SCH and improved on T4 therapy. Isovolumetric relaxation time was increased in some studies. The time to ventricular filling rate was high and became normal with treatment. Thus some patients with SCH have subtle abnormalities in systolic time intervals, diastolic function and myocardial contractility that may improve during treatment [43].

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www.ijpbs.com (or) www.ijpbsonline.com Adverse fetal effects: A seminal study by Haddaw et al [12] showed a 7 point reduction in intelligence quotient in children aged 7 to 9 years whose mothers had SCH at pregnancy compared with the children of euthyroid mothers. Although this was a single study, it nevertheless points to the need for screening of pregnant women and therapy for mild thyroid failure in women who are pregnant or planning on becoming pregnant. Neuromuscular Dysfunction: It has been suggested that neuromuscular symptoms and dysfunctions are common in patients with SCH and can be reversed by levothyroxine treatment [44]. A definitive answer will require more studies with TSH levels stratified as less than or greater than 10 mIU/L. Psychiatric and Cognitive Dysfunction: Data related to aggravation of depression, bipolar disorder, and effects on cognitive function have been presented [45]. A recent study showed no association with anxiety, depression or cognitive dysfunction [20]. Nonetheless, it is still reasonable to have a low threshold for therapy for SCH in patients with depression, bipolar disorder and cognitive dysfunction. Effects on Symptoms, Mood and Cognition: The questions of whether persons with SCH have symptoms, and the extent to which the putative symptoms are reversible with thyroid hormone therapy, remain unanswered. Several studies have suggested that mild symptoms of hypothyroidism are more prevalent in patients with SCH than in age matched controls [29, 46, 47]. But not all studies have found this to be true [48]. Several studies have suggested that mild symptoms of hypothyroidism are more prevalent in patients with SCH than in age matched controls, but not all studies. There have been 3 published randomized, prospective, placebo-

controlled trials of therapy for SCH. Two reported significant improvements in the symptoms of hypothyroidism, whereas the third found no benefit of therapy. Overall, the percentage of patients whose condition improved ranged from 0 to 28 percent of those treated. In the trial that found no treatment benefit, however, the mean serum thyrotropin level (4.6m/L) remained in the high-normal range after therapy. On the basis of the two trials with positive findings, one would need to treat approximately four patients for one to benefit [49]. Patients with SCH have been reported to have higher scores on scales of anxiety or depression, although this finding has been inconsistent. In the studies in which cognitive function or memory was formally assessed before and after thyroxine reported small but statistically significant improvements. Limited data have suggested that therapy for SCH may decrease intraocular pressure, increase myocardial performance, and peripheral nerve function. In women with SCH and ovulatory dysfunction, thyroxine therapy may restore fertility. Although difficulty losing weight to decrease with thyroxine therapy [49]. Recommendations for SCH: Several randomized trials of thyroxine therapy for sub clinical hypothyroidism have been performed [50-53]. In one study, half the patients had fewer hypothyroid related symptoms with therapy [50]. In another study, thyroxine therapy improved psychometric test results. One study showed only improvement in memory scores. Some of these studies included patients with TSH levels as high as 39 mIU/L, and in some, fixed high doses of thyroxine were used [52]. Thus, the reported benefits are difficult to interpret. A recent randomized study in patients with serum TSH levels between 5 & 10 mIU/L

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www.ijpbs.com (or) www.ijpbsonline.com showed no clinical or metabolic benefit after 6 months of thyroxine therapy [53]. Thus, benefits of thyroxine therapy for patients with serum TSH levels between 5 & 10 mIU/L remain to be determined. The most convincing argument for therapy is the high rate of progression to overt hypothyroidism. The American college of physicians stated that the available evidence is insufficient to make recommendations for or against the treatment of SCH [54]. However, the issue is still controversial. At present, some practical recommendations can be given. For patients with serum TSH levels higher than 10mIU/L, no controversy exists, and treatment is recommended. This strategy is justified because the rate of progression to overt hypothyroidism is high, and low density lipoprotein is reduced 8% with thyroxine therapy [55]. Thyroxine therapy for patients with SCH may improve quality of life, muscle function, mood, fertility, and cardiac function. For patients with TSH levels between 5& 10 mIU/L, observation or treatment is recommended on an individual basis. Younger patients with a relatively large goiter and thyroid peroxidase antibody positivity are usually treated. Symptomatic patients and patient with fertility problems should receive therapy. Because of concern about possible aggravation of coronary disease symptoms, patients with SCH who have only mild elevation of serum TSH and normal serum thyroxine levels are usually followed up with a repeated TSH measurement & treated if there is progression of sub clinical thyroid disease. Children & aldolescents, pregnant women, & women contemplating pregnancy should receive treatment. Of note, in women with hypothyroidism who are receiving thyroxine therapy, the dose of thyroxine replacement may need to be increased 30% to 40% during pregnancy [56].

Observational data for possible adverse effects of SCH on the cardiovascular system are available 55. A cohort study from Rotterdam showed that SCH in women older than 55 years with serum TSH levels between 4 & 10 mIU/L is strong risk factors, independent of cholesterol level, for atherosclerotic diseases & myocardial infarction [57]. In patients with SCH, a daily dose of 0.025 to 0.075 mg of levothyroxine is usually adequate to normalize serum sensitive TSH. The required dose varies with the level of serum TSH, serum thyroid hormones, & the age & weight of the patient. If the patients are already taking thyroxine, only a small increase is necessary. Serum sensitive TSH levels should be determined after 6 to 8 weeks of treatment to adjust the dose if necessary. Some thyroidologists aim for a serum sensitive TSH need to increase the replacement thyroxine dose for patients who have sensitive TSH levels between 3 & 5 mIU/L is anecdotal [58]. Screening: Based on a computer model designed to assess cost effectiveness, Danse et al [59] recommended screening with serum sensitive TSH every 5 years in patients older than 35 years. Cost-effectiveness in women & patients older than 50 years is more substantial [59]. Recent recommendations from the American Thyroid Association are similar [60]. The American college of physicians recommends screening women who are older than 50 years but does not recommend screening younger women or asymptomatic men [54]. Consensus will be reached if more prospective randomized studies show benefits of therapy for SCH, the most common form of thyroid dysfunction. Recent evidence showed the adverse effects of SCH on neuropsychological development of the fetus, indicating the need for therapy for SCH in pregnant women [56]. These data point to

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Cooper DS. Subclinical hypothyroidism. N Engl J Med 2001; 345(4): 260-65. Klee GG,Hay ID.Biochemical testing of thyroid function.Endocrinol Metab Clin North Am 1997;26:76375. Hay ID, Klee GG. Thyroid dysfunction. Endocrinol Metab Clin North Am 1988; 17:473-09. Haddow JE, Palomaki GE, Allan WC,et al. Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. N Engl J Med 1999; 341:539-44. Hollowell JG, Staehling NW,Flanders WD, et al. Serum TSH,T(4), and thyroid antibodies in the united states population (1988 to 1944): National Health And Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 2002; 87(2):489-99. Karmisholt J, Andersen S, Laurberg P.Variation in thyroid function tests in patients with stable untreated subclinical hypothyroidism. Thyroid 2008; 18(3):30308. Arem R, Escalante D. Subclinical hypothyroidism: epidemiology, diagnosis, and significance. Advances in Internal Medicine 1996; 41:213-50. Fatourechi V. Subclinical hypothyroidism: when to treat, when to watch? Consultant 2004; 44(4):533-39. Wartofsky L, Dickey RA. The evidence for a narrower thyrotropin reference range is compelling. J Clin Endocrinol Metab 2005; 90(9):5483-88. Ladenson PW, Singer PA, Ain KB, et al. American Thyroid Association guidelines for detection of thyroid dysfunction. Arch Intern Med 2000; 160:1573-75. Hollowell JG, LaFranchi S, Smallridge RC, Spong CY, Haddow JE, Boyle CA. 2004 where do we go from here? Summary of working group discussions on thyroid function and gestational out comes. Thyroid 2005; 15(1):72-6. Haddow JE, Palomaki GE, Allan WC, et al. Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. N Engl J Med 1999; 341(8):549-55. Surks MI, Ortiz E, Daniels GH, et al. Subclinical thyroid disease: scientific review and guidelines for diagnosis and management. JAMA 2004; 291(2):228-38.

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|103-111 14) Chu JW, Crapo LM. The treatment of subclinical hypothyroidism is seldom necessary. J Clin Endocrinol Metab 2001; 86(10):4591-99. 15) McDermott MT, Ridgway EC. Subclinical hypothyroidism is mild thyroid failure and should be treated. J Clin Endocrinol Metab 2001; 86(10):4585-90. 16) Tunbridge WMG, Evered DC, Hall R, et al. The spectrum of thyroid disease in a community: The Whickham survey. Clin Endocrinol (Oxf) 1977; 7:48193. 17) Vanderpump MP, Tunbridge WM, French JM, et al. The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham Survey. Clin Endocrinol (Oxf) 1995; 43 (1):55-8. 18) DĂez JJ, Iglesias P. Spontaneous subclinical hypothyroidism in patients older than 55 years: an analysis of natural course and risk factors for the development of overt thyroid failure. J Clint Endocrinal Metal 2004; 89 (10): 4890-97. 19) Kong WM, Sheikh MH, Lumb PJ, et al. A 6-month randomized trial of thyroxine treatment in women with mild subclinical hypothyroidism. Am J Med. 2002; 112(5):348-54. 20) Roberts LM, Pattison H, Roalfe A, et al. Is subclinical thyroid dysfunction in the elderly associated with depression or cognitive dysfunction? Ann Intern Med 2006;145(8):573-81. 21) Gussekloo J, van Exel E, de Craen AJ, Meinders AE, FrĂślich M, Westendorp RG. Thyroid status, disability and cognitive function, and survival in old age. JAMA 2004; 292(21):2591-99. 22) Jorde R, Waterloo K, Storhaug H, Nyrnes A, Sundsfjord J, Jenssen TG. Neuropsychological function and symptoms in subjects with subclinical hypothyroidism and the effect of thyroxine treatment. J Clin Endocrinol Metab 2006 Jan; 91(1):145-53. 23) Canaris G, Manowitz NR, Mayor G, Ridgway EC. The Colorado thyroid disease prevalence study. Arch Intern Med 2000; 160(4):526-34. 24) Danese MD, Ladenson PW, Meinert CL, Powe NR. Effect of thyroxine therapy on serum lipoproteins in patients with mild thyroid failure: a quantitative review of the literature. J Clin Endocrinol Metab 2000; 85(9):2993-01. 25) Hueston WJ, King DE, Geesey ME. Serum biomarkers for cardiovascular inflammation in subclinical hypothyroidism. Clin Endocrinol. 2005; 63(5):582-87. 26) Duntas LH, Wartofsky L. Cardiovascular risk and subclinical hypothyroidism: focus on lipids and new emerging risk factors: what is the evidence? Thyroid. 2007; 17(11):1075-84.

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www.ijpbs.com (or) www.ijpbsonline.com 27) Razvi S, Ingoe L, Keeka G, Oates C, McMillan C, Weaver JU. The beneficial effect of L-thyroxine on cardiovascular risk factors, endothelial function, and quality of life in subclinical hypothyroidism: randomized, crossover trial. J Clin Endocrinol Metab. 2007 May; 92(5):1715-23. 28) Elder J, McClelland A, Oâ&#x20AC;&#x2122;Reilly DS, Packard CJ, Series JJ, Shepherd J. The relationship between serum cholesterol and serum thyrotropin, thyroxine and triiodothyronine concentrations in suspected hypothyroidism. Ann Clin Biochem 1990; 27:110-13. 29) Staub J-J, Althaus BU, Engler H, et al. Spectrum of subclinical and overt hypothyroidism: effect on thyrotropin, prolactin, and thyroid reserve, and metabolic impact on peripheral target tissues. Am J Med 1992; 92:631-42. 30) Danese MD, Ladenson PW, Meinert CL, Powe NR. Effect of thyroxine therapy on serum lipoproteins in patients with mild thyroid failure: a quantitative review of the literature. J Clin Endocrinol Metab 2000;85:2993-01. 31) Diekman T, Lansberg PJ, Kastelein JJ, Wiersinga WM. Prevalence and correction of hypothyroidism in a large cohort of patients referred for dyslipidemia. Arch Intern Med 1995; 155:1490-5. 32) Basak Cakal, Erman Cakal, Berrin Demirbas, etal. Homocysteine and fibrinogen changes with L-thyroxine in subclinical hypothyroid patients. J Koream Med Sci; 2007; 22:431-5. 33) Hak AE, Pols HAP, Visser TJ, Drexhage HA, Hofman A, Witteman JCM. Subclinical hypothyroidism is an independent risk factor for atherosclerosis and myocardial infarction in elderly women: the Rotterdam Study. Ann Intern Med. 2000; 132(4):270-78. 34) Vanderpump MP, Tunbridge WM, French JM, et al. The development of ischemic heart disease in relation to autoimmune thyroid disease in a 20-year follow-up study of an English community. Thyroid. 1996; 6(3):155-60. 35) Cappola AR, Fried LP, Arnold AM, et al. Thyroid status, cardiovascular risk, and mortality in older adults. JAMA. 2006; 295(9):1033-41. 36) Rodondi N, Aujesky D, Vittinghoff E, Cornuz J, Bauer DC. Subclinical hypothyroidism and the risk of coronary heart disease: a meta-analysis. Am J Med. 2006; 119(7):541-51. 37) Ochs N, Auer R, Bauer D, et al. Meta-analysis: subclinical thyroid dysfunction and the risk for coronary heart disease and mortality. Ann Intern Med. 2008 Jun 3; 148(11):832-45. 38) Singh S, Duggal J, Molnar J, Maldonado F, Barsano CP, Arora R. Impact of subclinical thyroid disorders on

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coronary heart disease, cardiovascular and all-cause mortality: a meta-analysis. Int J Cardiol. 2008 Mar 28; 125(1):41-8. Haentjens P, Van Meerhaeghe A, Poppe K, Velkeniers B. Subclinical thyroid dysfunction and mortality: an estimate of relative and absolute excess all-cause mortality based on time-to-event data from cohort studies. Eur J Endocrinol 2008 Sep; 159(3):329-41. Razvi S, Shakoor A, Vanderpump M, Weaver JU, Pearce SH. The influence of age on the relationship between subclinical hypothyroidism and ischemic heart disease: a metaanalysis. J Clin Endocrinol Metab. 2008 Aug; 93(8):2998-07. Biondi B, Cooper DS. The clinical significance of subclinical thyroid dysfunction. Endocr Rev. 2008 Feb; 29(1):76-131. Biondi B. Cardiovascular effects of mild hypothyroidism. Thyroid 2007; 17(7):625-30. Staub JJ, Althaus BU, Engler H, et al. Spectrum of subclinical and overt hypothyroidism: Effect on thyrotropin, prolactin, and thyroid reserve, and metabolic impact on peripheral target tissues. Am J Med 1992; 92:631-42. Christ-Crain M, Meier C, Huber PR, Staub J-J, Muller B. Effect of thyroxine replacement therapy on surrogate markers of skeletal and cardiac function in subclinical hypothyroidism. Endocrinologist. 2004; 14(3):161-66. Haggerty JJ Jr, Garbutt JC, Evans DL, et al. Subclinical hypothyroidism: a review of neuropsychiatric aspects. Int J Psychiatry Med. 1990; 20(2):193-08. Canaris GJ, Manowitz NR, Mayor GM, Ridgway EC. The Colorado thyroid disease prevalence study. Arch Intern Med 2000;160:526-34. Zulewski H, Muller B, Exer P, Miserez AR, Staub JJ. Estimation of tissue hypothyroidism by a new clinical score: evaluation of patients with various grades of hypothyroidism and controls. J Clin Endocrinol Metab1997; 82:771-6. Lindeman RD, Schade DS, LaRue A, et al. Subclinical hypothyroidism in a biethnic, urban community. J Am Geriatr Soc 1999;47:703-9. Kumana CR, Cheung BMY, Lauder IJ. Gauging the impact of statins using number needed to treat. JAMA 1999; 282:1899-01. Cooper DS, Halpern R, Wood LC, Levin AA, Ridgway EC. L- thyroxine therapy in subclinical hypothyroidism: a double-blind, placebo-controlled trial. Ann Intern Med 1984; 101:18-4. Jaeschke R, Guyatt G, Gerstein H, et al. Does treatment with L-thyroxine influence health status in middle-aged and older adults with subclinical hypothyroidism? J Gen Intern Med. 1996; 11:744-49.

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www.ijpbs.com (or) www.ijpbsonline.com 52) Nyström E, Caidahl K, Fager G, Wikkelsö C, Lundberg PA, Lindstedt G. A double-blind cross-over 12-month study of L-thyroxine treatment of women with ‘subclinical’ hypothyroidism. Clin Endocrinol (Oxf). 1988; 29:63-6. 53) Kong WM, Sheikh M, Lumb P, et al. A randomised controlled trial of thyroxine treatment in mild subclinical hypothyroidism [abstract]. Endocr Soc Annu Meet Program Abstr 2000; 82:597. 54) Helfand M, Redfern CC, American College of Physicians. Clinical guideline, part 2: screening for thyroid disease: an update [published correction appears in Ann Intern Med. 1999; 130:246]. Ann Intern Med 1998; 129:144-58. 55) Kahaly GJ. Cardiovascular and atherogenic aspects of subclinical hypothyroidism. Thyroid 2000; 10:665-79. 56) Glinoer D, Delange F. The potential repercussions of maternal, fetal, and neonatal hypothyroxinemia on the progeny. Thyroid 2000; 10:871-87.

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|103-111 57) Hak AE, Pols HAP, Visser TJ, Drexhage HA, Hofman A, Witteman JCM. Subclinical hypothyroidism is an independent risk factor for atherosclerosis and myocardial infarction in elderly women: the Rotterdam Study. Ann Intern Med. 2000;132:270-78. 58) Biondi B, Fazio S, Palmieri EA, et al. Left ventricular diastolic dysfunction in patients with subclinical hypothyroidism. J Clin Endocrinol Metab 1999; 84:2064-67. 59) Danese MD, Powe NR, Sawin CT, Ladenson PW. Screening for mild thyroid failure at the periodic health examination: a decision and cost-effectiveness analysis. JAMA 1996; 276:285-92. 60) Ladenson PW, Singer PA, Ain KB, et al. American Thyroid Association guidelines for detection of thyroid dysfunction. Arch Intern Med 2000; 160:1573-75.

*Corresponding Author:

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Sangita P. Jadkar Department of Biochemistry, Government medical College, Miraj-416410 (INDIA) Email: drsangitajadkar@gmail.com

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Research Article Biological Sciences ANTIOXIDANT ACTIVITY OF PROCESSED DRY FRUITS Neha Singh*1, Neeraj Mishra2, Ritu Gangwar3, Rajiv Rai4, Sonal Pandey5 1*, 2, 3, 4, 5

Department of Biotechnology, Saroj Institute of Technology & Management, Lucknow (U.P.), India *Corresponding Author Email: 19nehasingh86@gmail.com

ABSTRACT Various studies have been focussed on the protective and preventive effect of antioxidant activity on certain degenerative illnesses such as cardiovascular, cancer, neurological diseases, cataracts and oxidative stress dysfunction. The present work was conducted to evaluate the antioxidant activity of processed dry fruits. Total antioxidant activity was assessed by the reducing power and superoxide anion scavenging activity assays. The results of reducing power and scavenging ability revealed that the reducing power increases with concentration in two processing methods (roasting and boiling with milk) but decreases in other two processing methods (deep and shallow frying). The superoxide anion scavenging ability indicated that frying (deep and shallow) affects higher to the antioxidant activity while the other two processing (roasting and boiling with milk) had less effect on their antioxidant potential.

KEY WORDS Antioxidant Activity, Dry Fruits, Food Processing.

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INTRODUCTION Dried fruits are widely used in confectionaries, bakery products and sweet industries. Industrial food manufacturing units widely make use of dried fruits in soups, sauces, marinades, puddings, food formulas for children and infants, etc. As almost all dried fruits have healthy protective bioactive ingredients and essential nutrients they help to reduce the risk of chronic illnesses and also prevent them. As dried fruits have all the nutritive value like fresh fruits, they are now highly recommended by health care industries and are taken as dietary supplements. Numerous types of natural antioxidants have been identified but a lot of attention has recently been drawn to the addition of polyphenols to foods and biological systems, due to their known abilities to scavenge free radicals (Goli et al. 2005). Free radicals are highly reactive chemical molecules/ species such as superoxide radical,

hydroxyl radical and singlet oxygen that travel around the body and cause damage to the body cells. Diseases linked to oxygen radicals and reactive oxygen species (ROS) include cancer, atherosclerosis, heart disease, stroke, diabetes mellitus, rheumatoid arthritis, osteoporosis, ulcers, sunburn, cataracts and aging (Gulcin et al. 2003). Antioxidants enzymes (made in the body) and antioxidant nutrients (found in foods) can scavenge/deactivate this reactive free radicals turning them to harmless particles (Chu et al. 2002). The most likely and practical way to fight against degenerative diseases is to improve body antioxidant status, which could be achieved by higher consumption of vegetables and fruits (Oboh & Rocha 2007). Antioxidants protect by contributing an electron of their own. In so doing, they neutralize free radicals and help prevent cumulative damage to body cells and tissues (Alia et al. 2003). Foods

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www.ijpbs.com (or) www.ijpbsonline.com from plant origin usually contain natural antioxidants that can scavenge free radicals (Sun et al. 2002). Dried fruit is more commonly used in all food industries and is freely consumed by people as it helps to increase the fruit serving in the diet and delivers the same nutritive value, taste, flavour, etc. Dried fruits of any kind is the most healthiest and nutritious snack. Today Indian markets have a number of food materials (such as Kheer, Sewai, Mewa laddoos etc) containing dry fruits which are consumed widely, but these dry fruits are previously processed by any mean. These processing may influence the bio-activity of dry fruits. Earlier it have been reported that the different selected raw dry fruits almond (Prunus amygdales), walnut (Juglans regia), cashew nut (Anacardium occidentale L.), Raisins (Couma macrocarpa), chironji (Buchanania lanzan) were analyzed for their antioxidant capacity (Mishra et al. 2010). The present study aims to investigate the antioxidant activity of processed dry fruits.

MATERIALS AND METHODS

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Collection and Processing of Dry Fruits: All the selected dry fruits were obtained from local market. The various processing methods (deep frying, shallow frying, roasting and boiling with milk) of dry fruits during the food preparations were selected for the study. All the selected dry fruits (almonds, cashew, chironji, rasins and walnuts) were processed according to the regulations of food standards. The deep and shallow fried dry fruits were properly soaked to let out all the extra oil and the extra milk content of dry fruits boiled in milk were separated. Then each of the processed dry fruits was ground into mortar and pestle. Extract Preparation of Processed Dry Fruits: The ground dry fruits and ethanol were taken in the

ratio of 1:4 and kept on orbital shaker at 200 rpm for 24 hours. The dry fruits were then filtered out and the remaining alcohol content was evaporated in a rotary vaccum evaporator at 50oC. The prepared extract was taken as 100% concentration. The two concentrations (60% and 80%) of each extract were prepared in distilled water. Antioxidant Activity of Extracts Reducing Power: The reducing power of the extracts was determined as described by Oyaizu (1986). The suspension of prepared extracts (0.4–2.0 mg) in 1 ml of distilled water was mixed with 2.5 ml of 0.2M phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min. Subsequently, 2.5 ml of trichloroacetic acid was added and the mixture was then centrifuged at 3000 rpm for 10 minutes. A 2.5 ml aliquot of the upper layer was mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% FeCl3, and the absorbance of the mixture was taken at 700 nm. A higher absorbance indicates a higher reducing power. In the assay, the colour of test solutions changes to various shades of green and blue, depending on the reducing power of each extract. Superoxide Anion Scavenging Activity: The method of Markulund (1974) modified by Ekanayake et al. (2004) was used in this test. The method is based on the inhibition of the autoxidation of pyrogallol by phenolic compounds. To the assay mixture composed of a phosphate buffer solution (2.6 ml, 50 mM in water, pH 8.22 ± 0.03) with the analytical prepared extracts (0.3 ml) was added a freshly prepared solution of pyrogallol (0.1 mL of a 3 mM solution of pyrogallol in 0.010 M HCl (37.5 %).

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The autoxidation reaction rate of pyrogallol was determined at 400 nm by monitoring the absorbance every 30 seconds for a total period of 10 minutes, corresponding to the end of the reaction. The scavenging activity of the superoxide anion (O2 -) was calculated by the following formula (Sun et al. 2001): S = (K0-K1) /K0*100 Where K0 and K1 are autoxidation rates of the pyrogallol without and with the extract, respectively.

RESULTS The antioxidant activity may be due to different mechanism such as prevention of chain initiation, decomposition of peroxides and prevention of free radical scavenging reducing capacity and binding of transition metal ion catalysts (Mao et al. 2006). Thus various biochemical assays are performed to analyse the antioxidant characteristics depending on these mechanisms. The antioxidant properties of processed dry fruits were screened through Reducing Power Assay and Superoxide Anion Scavenging Activity Assay. The results of these assays are shown in Figure 1 and Figure 2.

1 0.9

Absornbance (700 nm)

0.8 0.7 0.6 0.5 0.4

Concentrations 60%

0.3

Concentrations 80%

0.2

Concentrations 100%

0.1

Deep Fry

Shallow Fry

Roast

Walnut

Raisin

Cashew

Chironji

Almond

Walnut

Raisin

Cashew

Chironji

Almond

Walnut

Raisin

Cashew

Chironji

Almond

Walnut

Raisin

Cashew

Chironji

Almond

Boilied with Milk

Name of Processings (Dry Fruits)

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Figure 1: Reducing Power of Different Processed Dry Fruits Extracts

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Figure 2: Superoxide Anion Scavenging Activity of Processed Dry Fruits. The reductive potential measures the ability of a sample to act as electron donor and therefore, reacts with free radicals converting them to more stable products and thereby terminated radical chain reactions. The reducing capacity of compound may serve as a significant indicator of its potential antioxidant activity (Ahmadi et al., 2007). The reducing power based on transformation of Fe3+ into Fe2+ in the presence of processed dry fruits extracts was assessed. Two processing (Roasting and Boiled with Milk) had demonstrated a positive correlation between the reducing power and concentrations of each dry fruits while the other two processing (deep fry and shallow fry) were found to be in indirect relationship. According to Hadafi et al. (1998), the reducing activity of the extracts is generally proportional to the concentration. Among all the prepared extracts, roasted almond and boiled milk walnut extracts had shown higher reducing activity. The deep fried chironji extracts had demonstrated lowest reduction (Figure 1).

Dioxygen form superoxide anions O2- by a single electron transfer during the pyrogallol autooxidation in basic solutions. The superoxide anions are scavenged by antioxidants and consequently, decrease the rate of pyrogallol autooxidation or even inhibit it. The ability of the phytochemicals from the ethanol extract to scavenge the superoxide anion was carried out using the pyrogallol autooxidation method. The results are reported in Figure 2. The higher scavenging activity was observed in the dry fruits extracts prepared after roasting (almond and cashew) and boiled with milk (chironji, raisin and walnut). The antioxidant activity assays indicated that among the selected food processing, frying (deep and shallow) affects higher to the antioxidant activity, while the other two processing (roasting and boiling with milk) had less effect on their antioxidant potential.

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DISCUSSION Some authors have earlier reported a direct correlation between antioxidant activity and total phenolic content (Velioglu et al. 1998; Barros et al. 2007). The antioxidant activity of phenolic constituents may be related to their redox properties, which allow them to act as reducing agents or hydrogen-atom donors, their ability to chelate metals, inhibit lipoxygenase and scavenge free radicals (Decker, 1997). The reduction power decreases inversely to the polarity of extraction solvent (Hadafi et al. 1998) and the phytochemicals, which contain non polar secondary metabolites, remain almost inactive (Tepe et al. 2011). But the heat treatment may affect antioxidant properties due to release of phenolic phytochemicals, hence contributing to the health promoting and disease preventing abilities (Oboh et al. 2010). The effect of frying on antioxidants of vegetables was reported by Kalogeropoulos et al. (2006). Besides water loss and oil absorption, shallow frying results in partial loss of all the antioxidants studied in frying oils. It was observed that oil interfered in the determination of antioxidant capacity of walnut (Arranz et al. 2009). Chandrasekara & Shahidi (2011) have earlier reported that the heat treatment (roasting) effectively enhances the antioxidant activity. Roasting had little effect on either free or total polyphenols in nuts. Raw and roasted walnuts had the highest total polyphenols (Vinson and Cai 2012). According to Garrido (2008), the roasted almond had higher antioxidant activity was than blanching + drying and the blanched (freeze-dried) almond samples and roasting is the most suitable type of industrial processing of almonds to obtain almond skin extracts with the greatest antioxidant capacity. Roasted cashew nut contains low amounts of hydroxy alkyl phenols (Trevisan et al. 2006); this suggests its lower antioxidant capacity.

According to Mishra et al. (2010), all the selected five dry fruits contain significant value of total phenols which contribute to their antioxidant ability. The results revealed that walnut and almond had higher antioxidant activity than other dry fruits. In comparison to their results of reducing power assay, the present study reveals that the food processing applied on dry fruits obstruct the antioxidant capacity of the dry fruits. This can be explained on account of the affect of food processing on total phenolic content of dry fruits. As far as we know, this is the first report which reveals the effect of four processing on antioxidant activity of dry fruits. The results showed that frying alters greatly the antioxidant capacity of dry fruits while roasting and boiling with milk affects lesser. Thus it may be concluded that frying of dry fruits can be avoided during food preparations because it diminishes the nutritive value of dry fruits.

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o o

Chu Y, Sun J, Wu X and Liu RH (2002) Antioxidant and antiproleferative activity of common vegetables. J Agric Food Chem 50(23): 6910-6916. Decker EA (1997) Phenolics: prooxidants or antioxidants? Nutr Rev 55: 396–407. Ekanayake P , Lee YD, Lee J (2004) Antioxidant activity of flesh and skin of Eptatretus burgeri (Hag Fish) and Enedrias nebulosus (White spotted Eel). Food Sci Tech Int 10 (3): 0171-0177. Garrido I, Monagas M, Gomez-Cordoves C, Bartolome B (2008) Polyphenols and antioxidant properties of almond skins: influence of industrial processing. J Food Sci 73(2):C106-C115. Goli AH, Barzegar M & Sahari MA (2005) Antioxidant activity & total phenolic compounds of pistachio (pistachio vera) hull extract. Food Chem 92(3): 521-525. Gulcin I, Oktay M, Kirecci E and Kufrevioglu O I (2003) Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts. Food Chem 83(3): 371382. Hadafi A, Ismaili Alaoui M, Chaouch A, Zrira S, Benjilli B (1998). Antioxidant activity of extracts of rosemary (Rosmarinus officinalis L.) and myrtle (Myrtus comminus). Effect of extraction solvent. 17th International Workshop Essential Oils Digne Les Bains. Kalogeropoulos N, Mylona A, Chiou A, Ioannou MS, Andrikopoulos NK (2007). Retention and distribution of natural antioxidants (α-tocopherol, polyphenols and terpenic acids) after shallowfrying of vegetables in virgin olive oil. Food Science and Technology 40 (6): 1008–1017. Markulund S, Markulund G (1974) Involvement of superoxide anion radical in auto-oxidation of pyrogallol and a convenient assay of superoxide dismutase. Eur J Biochem 47: 469-474. Mao LC, Pan X, Queand F, Fang XH (2006) Antioxidant properties of water and ethanol

extracts from hot air-dried and freeze dried daylily flowers. Eur Food Res Technol 222: 236–241. Mishra N, Dubey A, Mishra R, Barik N (2010) Study on antioxidant activity of common dry fruits. Food Chem Toxicol 48(12): 3316-3320. Oboh G, Adefegha SA, Ademosun AO, Unu D (2010) Effects of Hot Water Treatment on the Phenolic Phytochemicals and Antioxidant Activities of Lemon Grass (Cymbopogon citratus). EJEAFChe 9 (3): 503 - 513. Oyaizu M (1986). Studies on product of browning reaction prepared from glucose amine. Jpn J Nutr 44: 755-762. Sun T, Jia ZS, Chen WX, Jin YX, De Xu Z (2001) Active oxygen radical scavenging ability of WaterSoluble b-Alanine C60 adducts. Chin Chem Lett 12 (11): 997-1000. Sun J, Chu Y, Wu X, Liu R (2002) Antioxidant and antiproliferative activities of common fruits. J. Agric Food Chem 50: 7449-7454. Tepe B, Sarikurkcu C, Berk S, Alim N, Akpulat HA (2011). Chemical Composition, Radical Scavenging and Antimicrobial Activity of the Essential Oils of Thymus boveii and Thymus hyemalis Rec Nat Prod 5 (3): 208-220. Trevisan MT, Pfundstein B, Haubner R, Wurtele G, Spiegelhalder B, Bartsch H, Owen RW (2006) Characterization of alkyl phenols in cashew (Anacardium occidentale) products and assay of their antioxidant capacity. Food and Chem Toxico 44(2):188-197. Velioglu YS, Mazza G, Gao L, Oomah BD (1998) Antioxidant activity and total phenolics in selected fruits vegetables and grain products. J Agric Food Chem 46: 4113–4117. Vinson JA, Yuxing C (2012) Nuts, especially walnuts, have both antioxidant quantity and efficacy and exhibit significant potential health benefits. Food Funct 3: 134-140.

*Corresponding Author: Neha Singh*

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Assistant Professor Department of Biotechnology Saroj Institute of Technology & Management, Lucknow (U.P.), India

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Review Article Pharmaceutical Sciences NOVEL BIOABSORBABLE DRUG-ELUTING STENTS – A NEED IN INTERVENTIONAL CARDIOLOGY G.SIVA KRISHNA*, V.VENKATESULU, U.SHYAMALA, S.MOUNIKA Department of Pharmaceutics, Dr.K.V.Subba Reddy Institute of Pharmacy, Kurnool.-518218, Andhra Pradesh, INDIA. *Corresponding Author Email: shivapharmaco@gmail.com

ABSTRACT The development of stent has been a major advance in the treatment of obstructive coronary artery disease since the introduction of balloon angioplasty. However, neointimal hyperplasia occurring within the stent leading to instent restenosis is a main obstacle in the long-term success of percutaneous coronary intervention (PCI). The recent introduction of drug-eluting stents (DES) contributes a major breakthrough to interventional cardiology. Many large randomized clinical trials using DES have shown a remarkable reduction in angiographic restenosis and target vessel revascularization when compared with bare metal stents. The results of these trials also appear to be supported by evidence from everyday practice and noncontrolled clinical trials. However, the expanded applications of DES, especially in treating complex lesions such as left main trunk, bifurcation, saphenous vein graft lesions, or in-stent restenosis, are still under evaluation with on-going studies. With the availability of different types of DES in the market, the issue of cost should not be a deterrent and DES will eventually be an economically viable option for all patients. The adoption of DES in all percutaneous coronary intervention may become a reality in the near future. In this review article, we summarize the recent development and progress of DES as well as compare and update the results of clinical trials.This review focuses on describing next-generation drug-eluting stent systems based on the use of novel coatings and carrier systems developed to enhance DES safety.

KEY WORDS Drug-eluting stent, percutaneous transluminal coronary angioplasty, in-stent restenosis

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INTRODUCTION Background of drug-eluting stent development After the advent of cardiac catheterization in the late 1920s and the development of coronary angiographic technology in the late 1950s, balloon angioplasty (BA) was introduced in the mid 1960s. Balloon angioplasty was first applied to the revascularization of the femoral, popliteal, and renal arteries, and was adapted to the coronary arteries in the late 1970s (Forssmann 1929; Dotter and Judkins 1964; Hurst 1985, 1986). There were important limitations of coronary BA, including the risk of uncontrollable

plaque disruption and vascular recoil that may lead to periprocedural coronary occlusion and myocardial infarction, and a 20%–40% incidence of restenosis within 6–12 months after successful revascularization, which compromises the longterm prognosis (Miller et al 1999). Various atherectomy techniques such as rotational atherectomy (rotablation), Excimer Laser Coronary Angioplasty (ELCA), and Directional Coronary Atherectomy (DCA) were developed in late 1980s and early 1990s, but these devices did not significantly improve the long-term outcome due to a lack of an impact on restenosis rate (Mueller et al 1995; Karthikeyan

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www.ijpbs.com (or) www.ijpbsonline.com et al 2004). On the other hand, scaffolding metallic mesh, called stent, was developed during the same period to prevent restenosis after BA. The clinical efficacy of stent compared with conventional BA was studied in two landmark clinical trials. The North American Stent Restenosis Study (STRESS) showed a lower angiographic restenosis rate (31.6% vs 42.1%) and a lower target vessel revascularization (TVR) rate (10.2% vs 15.4%) in stent group than in BA group (Fischman et al 1994). The European comparison of balloon-expandable-stent implantation with BA in patients with coronary artery disease by the Benestent Study Group proved a similar, but more impressive, reduction of restenotic rate (22% vs 32%) (p = 0.02) and TVR rate (13.1% vs 22.9%) (p = 0.005) in stent group compared with BA group (Serruys et al 1994). Based on the result of these two studies, Palmaz-Schatz balloon-expandable stent (Cordis Corp; a Johnson and Johnson Company, Warren, NJ, USA) was approved as the first bare metal stent (BMS) for elective use by Food and Drug Administration (FDA) in 1994 after GianturcoRoubin coil stent (Cook Inc, Bloomington, IN, USA) was approved as the first BMS for acute closure in 1993 (Mueller et al 1995). However, the sudden occlusion of vessel due to subacute stent thrombosis (SAT) and late in-stent restenosis (ISR) are two major complications that were initially encountered with the widespread use of BMS. Although the SAT rate has been reduced to approximately 1% with adequate antiplatelet therapy (ie, aspirin and clopidogrel), the incidence of ISR is still a hindrance to the long-term success of the stenting procedure (Schomig et al 1996, 1997). When the use of BMS was expanded in the high-risk restenosis groups of patients such as those with small vessel, long and bifurcation lesions, and diabetes mellitus, ISR and TVR escalated to the range of 50%â&#x20AC;&#x201C;60% and 30%â&#x20AC;&#x201C;50%, respectively (Yokoi et al

1996). Extensive research was carried out in the late 1990s to seek a solution to the problem of ISR. Brachytherapy with insertion of radioactive devices in the coronary artery was initially developed to prevent ISR (Raizner et al 2000). Despite its moderate success, brachytherapy had limitations such as late thrombosis, geographic mismatch, relatively high cost, and requirement of radiation oncologists, which made it unsuitable for widespread and routine clinical practice (Raizner et al 2000). During the period when the brachytherapy was becoming the treatment of choice of in-stent restenosis, clinical trials of drug-eluting stents (DES) demonstrated a pristine outcome with a very high success rate and very low in-stent restenosis rate. DES has now become the mainstream therapy of coronary artery stenosis due to the expected very low rate of in-stent restenosis and brachytherapy has become a thing of the past. Drug-eluting stents (DES) were primarily conceived to reduce in-stent neointimal formation and therefore minimize the occurrence of restenosis, the major drawback of percutaneous coronary interventions with baremetal stents (BMS). The development of DES has been pioneered through a combination of the increased understanding of the biology of restenosis, the selection of drugs that target 1 or more pathways in the restenotic process, controlledrelease drug delivery strategies, and the use of the stent as a delivery platform. A drug-eluting stent (DES) is a peripheral or coronary stent (a scaffold) placed into narrowed, diseased peripheral or coronary arteries that slowly release a drug to block cell proliferation. This prevents fibrosis that, together with clots (thrombus), could otherwise block the stented artery, a process called restenosis. The stent is usually placed within the peripheral or coronary

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www.ijpbs.com (or) www.ijpbsonline.com artery by an Interventional cardiologist or Interventional Radiologist during an angioplasty procedure1. Drug-eluting stents in current clinical use were approved by the FDA after clinical trials showed they were statistically superior to bare-metal stents (BMS) for the treatment of native coronary artery narrowings, having lower rates of major adverse cardiac events (MACE) (usually defined as a composite clinical endpoint of death + myocardial infarction + repeat intervention because of restenosis). When blockages in the arteries of the heart (coronary arteries) develop, individuals may experience symptoms caused by inadequate blood supply to the heart muscle. This typically produces chest pain or pressure and/or shortness of breath. Treatment for this condition (coronary artery disease) will depend on the type of the blockage and its extent. Treatment options include medication, surgery (coronary artery bypass surgery), or catheter-based procedures, which are discussed below2. Patients should discuss these options with their physician to determine which may be best for them. Several types of catheter-based procedures are available. During balloon angioplasty, the physician passes a special balloon catheter into the narrowed segment of the artery and expands the balloon, which thus opens the artery and compresses the blockage against the wall of the artery. More than one third of patients who undergo balloon angioplasty may experience restenosis (renarrowing) of the diseased artery segment within 6 months of the procedure. Stents are very small metal tubes that can be inserted via a balloon catheter into the narrowed segment of the artery3. When the balloon is inflated, the stent expands and is embedded into the artery vessel wall, which thus opens the previously narrowed segment of artery. The balloon is then deflated and removed along with

the catheter, and the stent is left behind to serve as a metal framework for the artery. Although stented arteries have less chance of renarrowing than arteries opened with a balloon alone, instent restenosis can still occur in more than 1 in 5 patients after stent placement.

Figure-1. A, The stent is mounted on a balloon catheter and advanced to the diseased, narrowed portion of the heart artery. B, The balloon is inflated and the stent is expanded, which opens the narrowed section of the artery. C, The balloon is deflated and removed; the stent is embedded into the wall of the artery and stays in position. Medication coats drug-eluting stents and reduces the chance of renarrowing, or restenosis, of the blood vessel. Because restenosis within the stented region of a heart artery is caused by tissue growth, some stents (called drug-eluting stents) have medication on them to inhibit or prevent this tissue growth. Drug-eluting stents are placed in a fashion similar to other stents; however, their use markedly reduces the rate of renarrowing. In fact, about 1 in 10 patients develops renarrowing in the several years after drug-eluting stent implantation, a rate about half of that seen for stents without medication4. Because stents expose foreign material to the blood stream, a small risk exists that a blood clot may develop in the stent, a process called stent thrombosis. These blood clots can occur many months and even years after stent implantation and may lead to a heart attack or death. All stents can potentially be affected by stent thrombosis. For this reason, most patients with

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stents are instructed to take anticlotting medication, usually a combination of aspirin and clopidogrel or ticlopidine5. Each of these medications stops platelets (particles in the blood that help clots to form) from functioning to their full capacity. The precise duration of anticlotting medication depends on the type of stent placed by your doctor and your overall medical condition. If you have been prescribed anticlotting medications, you should not stop them (even for a few days) unless instructed to do so by your doctor. Concerns about the safety of drug-eluting stents have received much publicity, primarily related to a small increase in the number of blood clots that develop within drug-eluting stents late (more than 1 year) after implantation. In December 2006, the US Food and Drug Administration convened a panel of cardiovascular experts to review drug-eluting stent safety data. The panel concluded that for many patients, such as those with uncomplicated medical histories who undergo elective stenting of simple coronary blockages, drug-eluting stents remain a safe and appropriate therapy6. For others, such as those who have suffered an acute heart attack or those with multiple or complicated coronary blockages, current data are inadequate to determine whether drugeluting stents are better or worse than baremetal stents or coronary artery bypass surgery. Patients are advised to discuss with their physician which treatment option(s) may be most appropriate for them. In all cases, patients who receive drug-eluting stents are reminded to take their prescribed anticlotting medications without interruption for at least 1 year after stent implantation, unless otherwise instructed by their cardiologist.

Pathophysiology of ISR and mechanism of action of DES to prevent ISR Three distinct processes are involved in the pathogenesis of ISR as depicted in Figure 1. These include: (1) immediate vessel recoil after stretch injury, (2) negative arterial remodeling, and (3) neointimal hyperplasia (Hoffmann et al 1996; Mintz et al 1996; Liu et al 2002; Muhlestein et al 2002). Elastic recoil is the immediate shrinkage of vessel after percutaneous coronary intervention (PCI) due to the elastic properties of arterial wall, which usually occurs within 24 hours after procedure7, 8 . Negative remodeling is a process of local contraction of arterial wall and narrowing of the lumen at the injured vascular segment. It may be related to the healing process as well as the interaction between endothelial cells and nonlaminar blood flow (Liu et al 1989). Neointimal hyperplasia is the proliferation and migration of smooth muscle cells from the media, possibly circulating cells from bone marrow into the intima, and then encroaches on the vascular lumen (Liu et al 1989). Negative remodeling and neointimal proliferation usually occur weeks to months after PCI (Liu et al 1989). The first two pathological processes were the main causes of restenosis in BA, but were basically eliminated by use of stent. The third mechanism, neointimal hyperplasia, becomes the only major mechanism in the pathogenesis of ISR (Virmani and Farb 1999). Stent-based drug delivery system The main processes of ISR, smooth muscle cell activation and replication, occur locally at the site of injury. Therefore, one of the most logical approaches is a stent-based drug delivery system to locally deliver an appropriate concentration of an effective agent to stop this process without systemic toxicity. An effective system would consist of 3 components: (1) a metallic platform,

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www.ijpbs.com (or) www.ijpbsonline.com (2) a drug carrier vehicle that stores a therapeutic agent as well as allows the agent to diffuse into the vascular tissue in a controlled fashion, and (3) an effective therapeutic agent that reduces the neointimal growth induced by stent implantation9, 10. The cross-section of a stent strut with typical coating configuration can be seen in Figure 2. Therefore, an ideal DES to achieve the greatest clinical efficacy and safety is one that requires an optimization of these three essential parameters.

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Figure 2: Cross-section of a stent strut with a drug-loaded polymeric coating. Stent design in relation to even drug distribution to vessel wall The effect of different stent designs on the drug distribution pattern has been scrutinized in experimental studies and also tested in clinical trials (Hwang et al 2001; Takebayashi et al 2004). Recent experimental data suggest that the stent strut configuration directly determines the pattern and degree of drug delivery achieved by DES (Hwang et al 2001). The simple proximity of stent struts to vascular tissue does not ensure adequate drug delivery and distribution because most nonuniform distribution has been found in the layers of the artery closest to the stent (Hwang et al 2001). After deployment of even highly lipid-soluble and rapidly diffusing agents, homogeneous drug delivery throughout the vessel with uniform concentration at various depths of the vessel wall was not achieved in

their study11. In the same study, the uniformity of drug distribution was found to be increased with the strut number as well as significantly dependent on the strut pattern of distribution. Therefore, a symmetric expansion of stents with homogeneous distribution of struts is essential for the optimization of drug distribution.The importance of this concept was further verified by a recent clinical study using Sirolimus-eluting stent (SES) (Takebayashi et al 2004). In the latter study, a nonuniform stent strut distribution and a greater gap distance between struts after stent implantation resulted in more neointimal hyperplasia (Takebayashi et al 2004). Although a large number of stent designs have been developed to date, only the multicellular design is currently most commonly used; they can be categorized into “closed cell” and “open cell” configurations (Rogers 2002). A closed cell stent has a uniform cell expansion and constant cell spacing when deployed in a curved vascular segment, which gives more uniform drug distribution (Rogers 2002). An open cell stent has a greater variation in the surface coverage between the inner and outer curvatures in the curved segment, but gives better conformability to curved surface at the expense of less uniform drug distribution (Rogers 2002). The majority of current BMS use a closed cell design. In summary, the optimal stent design for drug delivery should have a large stent surface area, a small cell gap, and minimal strut deformation after deployment while maintaining conformability, radial support, and flexibility to reach the complex coronary lesions12.

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Figure 3: Uniform vsnonuniform drug distribution in closed cell vs open cell stents was shown in the longitudinal sections of the vessel wall after a deployment of a drug-eluting stent. Drug concentration was shown in the color intensity in the column. Upper red-brown... Coating matrix as a reservoir for drugs and controller of kinetic drug release Many methods of coating stents with drugs have been developed for DES (Figure 4). Some drugs can be bonded directly to a metal stent (eg, prostacyclin, paclitaxel), but most of the agents must be bonded to a matrix polymer, which acts as a drug reservoir to ensure drug retention during deployment and a uniform distribution on the stent (Sousa et al 2003a). The types, compositions, and designs of the polymers coated on the stent dictate the eluting kinetic of the sustain time release of the drug over a period of weeks or months following the implantation in situ13, 14. The coating materials can be categorized as organic vs inorganic, bioerodablevsnonbioerodable, and synthetic vs naturally occurring substances (Sousa et al 2003a). Generally, for long-term application, a nonbioerodable polymer is used in order to prevent triggering an inflammatory process. The most successfully tested DESs to date have been coated with synthetic polymers; poly-n-butyl methacrylate and polyethylene-vinyl acetate with sirolimus and a poly (lactide-co-Σ-

caprolactone) copolymer with paclitax-eleluting stents15. All naturally occurring organic materials are both bio- and hemo-compatible (Ratner 1993; De Scheerder et al 2000). Fibrin, cellulose, and albumin have been tested in animal models, but only phosphorylcholine is used for clinical purposes. Phosphorylcholine is a naturally occurring phospholipid polymer with less potential to elicit inflammation and to interfere with re-endothelialization of the stent surface (Lewis et al 2002). BiodivYsio® stents are phosphorylcholine-coated stents currently available (Galli et al 2000). Inorganic substances have also been tested for coating on the stent surface to improve electrochemical properties. One example is a stent coated with a nonporous 300 μm ceramic layer containing tacrolimusloaded nanocavities (Grube et al 2003).

Figure 4: Different types of stent-based drug delivery system: (A) Drug released by diffusion from polymer, (B) Drug released by diffusion through ratelimiting coating, (C) Drug released by swelling of coating, (D) Drug release directly from coating, (E) Drug loaded ... Therapeutic agents to inhibit neointimal growth Many agents with antiinflammatory or antiproliferative properties have been incorporated on the stent surface Tables 1 and .2). Many of the agents listed in the tables have more than one mechanism of action. The general mechanism of action for most of these drugs is

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www.ijpbs.com (or) www.ijpbsonline.com to stop cell cycle progression by inhibiting DNA synthesis16. Everolimus, sirolimus, tacrolimus (FK-506), ABT-578, interferon, dexamethasone, and cyclosporine all fall into this category. In this

group, sirolimus and its derivatives were shown to reduce intimal thickening (Sousa et al 2001; Sousa, Costa, et al 2003).

Table 1: Agents used in drug-eluting stent

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DIFFERENT TYPES OF STENTS BASED ON GENERATION 1. First-Generation Stents SES and PES revolutionized rates of restenosis after cardiac procedures. These stents were developed to prevent the proliferation of smooth-muscle cells and other cell types seen with restenosis. The FDA approved SES and PES for use in patients with newly diagnosed, previously untreated single lesions <28 mm to 30 mm in length and a vessel diameter between 2.5 mm and 3.75 mm17. Sirolimus is a macrocytic triene antibiotic that has immunosuppressive and antiproliferative properties and elutes slowly over 4 to 6 weeks.1 The efficacy of SES in preventing restenosis was demonstrated in the RAVEL, SIRIUS, and SCANDSTENT trials and the RESEARCH registry.28 RAVEL and SIRIUS, which compared SES with

BMS, included stented patients with stable or unstable angina who received DAPT for 6 to 9 months18. In the trials, there was a significant reduction of in-stent restenosis, late lumen loss, and target lesion revascularization (TLR) over 1 to 5 years. In all trials, there was no difference in rates of death or myocardial infarction (MI). Paclitaxel, an antineoplastic, works by disrupting the function of the microtubules responsible for proper chromosome segregation during cell division, and it is released bimodally over a 2week period.19 The efficacy of PES was demonstrated in the TAXUS II and TAXUS IV trials, which examined patients with low-risk lesions or previously untreated coronary stenosis who randomly received BMS or PES with either a slow or a moderate drug-release rate. All trials resulted in reduction of in-stent restenosis and TLR.10-12 Furthermore, TAXUS IV demonstrated

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that these benefits were maintained in subgroups, including patients with vessels <2.5 mm in diameter, those with lesions >20 mm in length, and those with renal insufficiency or diabetes.13 Pooled and long-term analysis also revealed a reduction in cardiovascular (CV) events.14,15 The newest member of the PES series is the TAXUS Liberté, which has thinner struts to improve deliverability and was shown to be noninferior to its prototype in the TAXUS ATLAS trial20. PES and SES were compared in several clinical trials, most of which concluded that SES were associated with lower rates of clinical restenosis and late lumen loss. The superiority of SES may be due to differences in mechanism of action, timing of drug delivery, and cellular inflammatory response for SES and PES at sites of overlapping stents. Second-Generation Stents The newer stents, EES and ZES, are thinner and more flexible and have a cobalt-chromium alloy platform, which makes them more deliverable than the first-generation stents. These stents may also be more biocompatible, thereby generating less inflammatory response and faster vessel endothelialization21. Everolimus, a sirolimus derivative, is a semisynthetic, lipophilic, highly absorbable macrolide immunosuppressant.25 Everolimus elutes over time, with 80% absorbed within the first month and the remainder eluting over a 4month period. EES demonstrated efficacy over BMS in the SPIRIT FIRST trial, with significantly lower in-stent late lumen loss at 6 months. EES’ superior performance relative to PES was further shown in a meta-analysis of four trials in which EES reduced the risk of stent thrombosis, MI, ischemic TLR, and death. In the SPIRIT II–IV trials, EES and PES were compared in patients with simple and complex coronary disease22. EES use

resulted in significantly lower rates of in-stent late loss and target-lesion failure (defined by cardiac death, target-vessel MI, ischemic TLR) up to 2 years later.28-33 Although no randomized trials have compared EES and SES, the X-SEARCH registry was used to evaluate the efficacy and safety of EES in higher-risk patients. EES were compared in three historical groups of patients who received BMS, SES, or PES. At 6 months, EES had a significantly lower rate of TLR, compared with BMS, and rates comparable to those with SES and PES23, 24. Zotarolimus, also a sirolimus derivative, is a lipophilic immunosuppressant. The polymer used in ZES mimics the cell membrane’s phospholipid phosphorylcholine. Ninety-five percent of zotarolimus elutes within the first 2 weeks. The efficacy of ZES has been examined in the ENDEAVOR trials. In ENDEAVOR I and II, which compared ZES with BMS, TLR was lower with ZES at up to 2 years.25,26 In ENDEAVOR III and IV, which compared ZES with SES and PES, respectively, angiographic in-segment late lumen loss—a surrogate for restenosis—was higher in the ZES group versus the SES group.38,39 Compared with PES, however, ZES was noninferior for the primary endpoint of targetvessel failure.38,39 The SORT-OUT III trial showed similar results in the primary composite endpoint of cardiac death, MI, and TLR, which occurred significantly more often with ZES than with SES.40 The ZEST trial, which compared ZES, SES, and PES, found no difference in the primary composite endpoint of death, MI, and TLR. In a recent trial assessing EES and ZES in complex clinical or lesion characteristics (renal insufficiency, low ejection fraction, recent acute MI, multiple or long bifurcations, bypass grafts, in-stent restenosis, unprotected left main artery, thrombus, or total occlusion), there was no difference in the primary endpoint of targetvessel failure.

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Antiplatelet Therapy with Stents Coronary rethrombosis and coronary restenosis are sequelae of stent placement. Coronary rethrombosis is defined as reocclusion of coronary vessels by thrombin formation, and coronary restenosis is reocclusion of coronary vessels and smooth-muscle endothelial 27 overgrowth. Thesesequelae can lead to devastating events such as MI and death. DES are associated with a reduced risk of restenosis but an increased risk of rethrombosis, specifically with early discontinuation of DAPT. Predictors of later DES thrombosis have been identified, including patient and angiographic characteristics. Patient characteristics consist of older age, diabetes mellitus, low cardiac ejection fraction, renal failure, and ACS. In addition, early discontinuation of antiplatelet medications has been identified as a risk factor for stent thrombosis. Angiographic characteristics such as long or overlapping stents, stent placement in small vessels, bifurcation lesions, and suboptimal stent results also increase the risk of DES thrombosis28. The American College of Clinical Cardiology Foundation/American Heart Association/Society for Cardiovascular Angiography and Intervention (ACCF/AHA/SCAI) guideline for PCI, updated in 2011, includes new recommendations for the prevention of stent thrombosis.29 The guideline stresses that PCI with coronary stenting (BMS or DES) should not be performed if the patient is unlikely to tolerate and comply with DAPT for the appropriate treatment duration based on the type of stent implanted. COMMERCIALLY AVAILABLE DES The sirolimus-eluting Cypher™ stent (Cordis Corp, a Johnson and Johnson Company, Miami, FL, USA) was approved by FDA in April 2003. It is coated with a layer of nonerodable polymer, of 5 μm–10 μm thickness, which is incorporated with

sirolimus (140 μgsirolimus/cm2 of stent surface area). An additional topcoat is placed on it as a diffusion barrier, which provides the vehicle for controlled release of the drug. It is designed as such that approximately 80% of the total dose of the agent is released in 4 weeks and the remainder over the course of the next 2 weeks (Wong and Chan 2004). The second commercially available DES is the Taxus™ stent (Boston Scientific, Natrick, MA, USA), which has a proprietary platform, the Express™ stent, and is coated with a proprietary polymer (Translute™) loaded with 1 μg of paclitaxel/mm2 of stent surface area. Although there are three drugrelease formulations (slow, moderate, and fast), only moderate- and slow-release formulations have been tested in clinical trials (Sahatjian 2003; Waugh and Wagstaff 2004). The moderate-release (MR) form of Taxus stent allows for an initial bolus release over the first 48 hours after stenting followed by a low-level release over at least the next 10 days. In the initial 10 days of drug release, the slow-release (SR) formulation of Taxus stent has a drug release concentration of 8–10 times lower than that of the MR formulation. Only SR formulation is used in FDA approved Taxus stents. (Sahatjian 2003; Waugh and Wagstaff 2004).

CURRENT ISSUES OF DES Expanded indications Although DES was proved to be a safe and effective method in the treatment of coronary artery stenosis by both randomized clinical trials and real world practice, its expanded indications in complex and high-risk lesions for restenosis such as totally occluded lesions, left main lesions, bifurcation lesions, ostial lesions, small and long lesions, saphenous vein graft lesion, ISR, and diabetes mellitus are still under evaluation with ongoing trials. The results of

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www.ijpbs.com (or) www.ijpbsonline.com clinical trials for some expanded indications are now available. Issue of stent-based delivery: incomplete stent apposition and uneven stent strut distribution Incomplete stent apposition (ISA), defined as one or more stent struts not in contact with vascular wall on IVUS at any point in time after stent implantation, was found in 21% of the SES arm in RAVEL vs 4% in the BMS arm at 6-month followup (Serruys et al 2002). It is possible that this is due to either an initial incomplete deployment of stent during implantation or positive remodeling of vessel wall but other mechanisms like plaque regression, cell necrosis, apoptosis, and allergic reaction to sirolimus have been postulated (Lemos et al 2003; Takebayashi et al 2004). Uneven stent strut distribution and incomplete wall apposition has been considered to be the causes of ISR after the DES implantation in two clinical studies (Lemos et al 2003; Takebayashi et al 2004). Economic burden One of the thorniest issues regarding DES is their cost and reimbursement. In the USA, a BMS costs approximately $900–$1200 each while a DES costs approximately $3065–$3195. However, in the cost-effective analysis of SIRIUS trial, the difference of cost between the 2 groups were only about US $300 at 1 year, despite an initial $3000 difference after hospitalization (Cohen et al 2003). The advent of more varieties of DES in near future will minimize the cost issue and make DES available to all patients. Role of the Pharmacist FDA advisories stress the importance of carrying out 12 months of DAPT after DES placement and advise educating the patient and health care providers about the hazards of premature discontinuation. The role of the pharmacist is to encourage patients to continue therapy through education and to promote the use of adherence programs provided through the pharmacy.

DES Systems with Bioabsorbable Polymers Since durable, thick polymers of first-generation DES seem to have a central role in perpetuating local vascular inflammatory reaction and potentially inducing the occurrence of late and very late stent thrombosis, the concept of a polymer that carries and controls the drug release during an proper period of time and after that erodes and vanishes from the vascular surface seems to be very attractive. Most of the systems presented in this section utilize poly-llactic acid (PLLA) and poly-d,l-lactide (PDLLA), which are progressively erode by shortened as ester bonds and ultimately will degrade into lactic acid30.

MAIN DES SYSTEMS WITH BIOABSORBABLE POLYMERS BioMatrix (Biosensors Inc) The BioMatrix (Biosensors Inc, Newport Beach, Calif) stent is a novel DES that incorporates the S-Stent platform, a thin, stainless steel, laser-cut, tubular stent with 16.3% to 18.4% metal surface area. The antiproliferative drug is biolimus A9, a highly lipophilic, semisynthetic sirolimus analogue with an alkoxy-alkyl group replacing hydrogen at position 42-O. At a cellular level, biolimus A9 forms a complex with intracellular FKBP-12, which binds to the mammalian target of rapamycin and reversibly inhibits cell-cycle transition of proliferating smooth muscle cells with a similar potency to sirolimus. On the basis of in vivo studies, the biodegradable polymer fully converted to lactic acid at 6 to 9 months (data on file at Biosensors). Cardiomind 0.014-Inch Sparrow (CardioMind, Inc) The Sparrow Coronary Stent System (CardioMind, Inc, Sunnyvale, Calif) is a 0.014-inch guide wire–based stent delivery platform combining a limus drug in a biodegradable

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www.ijpbs.com (or) www.ijpbsonline.com polymer matrix on the CardioMindnitinol stent platform with a novel release mechanism that uses a principle of electrochemical dissolution for stent release. ELIXIR-DES Program (Elixir Medical Corporation) Elixir Medical Corporation is currently working with 2 pharmaceutical agents, Novolimus, a metabolite of sirolimus, and Myolimus, a sirolimusanalog. Both drugs belong to the family of macrocyclic lactones with immunosuppressive and antiproliferative properties and have a similar mechanism of action to other macrocyclic lactones such as rapamycin. The Elixir Medical Drug-Eluting Coronary Stent Systems (Elixir Medical Corp, Sunnyvale, Calif) are designed to optimize safety and efficacy through the combination of a cobalt chromium stent platform, a low polymer loading with controlled release and a low pharmacological drug dose.

CONCLUSION After several years involved in the task of “bringing to life” novel devices to treat coronary disease, the recent introduction of DES in PCI is a major innovative advancement in interventional cardiology. DES dramatically reduces the ISR rate in all subgroups of patients in both randomized clinical trials and real-world practice. Continuing improvement in drug-delivery stent technologies and gradual reduction in cost would make DES an effective mainstay of therapy for coronary artery disease.

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G.SIVA KRISHNA D.NO-4/100, Kota Street, Yemmiganur-518360, Kurnool (dist), A.P. E-mail- shivapharmaco@gmail.com Contact No: +918985289221

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Research Article Biological Sciences PALM OIL SUPPLEMENTATION PREVENTS FURTHER TYROSINE DEPHOSPHORYLATION OF ERYTHROCYTES 30-KDA PROTEIN DURING PLASMODIUM BERGHEI INFECTION M. F. Z. R. Yahya1 and R. A. Kadir2 1 2

School of Biology, Faculty of Applied Science, UiTM Shah Alam, 40450 Shah Alam, Selangor, Malaysia School of Biosciences and Biotechnology, Faculty of Science and Technology, UKM Bangi, 43600 Bangi, Selangor, Malaysia *Corresponding Author Email: fakharulzaman@salam.uitm.edu.my

ABSTRACT This study was carried out to determine effect of palm oil (PO) supplementation on erythrocytes phosphotyrosine proteins in Plasmodium berghei (PB) infection. A number of 40 experimental mice were divided into observation (n=20) and sampling (n=20) groups which were then subdivided into four groups (group A: +PO+PB; group B: +POPB; group C: -PO+PB and group D: -PO-PB) with n=5 each. It was observed that parasitemia development of observation group was delayed in group A as compared to group C starting from day 7 post-infection onwards. Group A and C reached 40% parasitemia at day 13 and day 11 respectively. Immunoblotting of electrophoretically separated proteins revealed that there were four erythrocytes phosphotyrosine proteins present in all sampling groups namely 120-kDa, 60-kDa, 30-kDa, 17-kDa and 14-kDa. It was noted that PO supplementation did not affect all phosphotyrosine proteins in normal experimental mice apparently whilst PB infection affected 30-kDa phosphotyrosine protein significantly (p<0.05). Furthermore, PO supplementation was found to prevent further tyrosine dephosphorylation of 30-kDa protein by PB infection. We suggest that the delay of parasitemia development in infected experimental mice is due to prevention of tyosine dephosphorylation of 30-kDa protein.

KEY WORDS Palm oil; malaria; Plasmodium berghei and tyrosine phosphorylation.

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INTRODUCTION Plasmodium is known to modulate protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) as their strategy for invasion and multiplying in erythrocytes of host [1] which results in existence of many phosphoproteins during malarial development. This has been supported by [2] reporting that tyrosine phosphorylation - dephosphorylation mechanism is crucial in malarial infection. The abnormal protein phosphorylation in malaria may cause destruction of erythrocytes which lead to hemolysis and anemia [3]. Thus, the understanding on involvement of tyrosine

phosphorylation in malaria infection is crucial to improve the existing antimalaria treatment plan. Plasmodium berghei is one of the malaria parasite infecting rodents. Others include P. yoelli, chabaudi, P. knowlesi and P. vivax. This unicellular protozoan which serves as a good model for the human parasites has a complex life cycle comprising of sexual and asexual stages [4]. A report by [5] stated that P. berghei strain ANKA and strain NK65 were isolated from Anopheles dureni millecampsi but in different forest galleries, River Kasapa and River Kisanga respectively. Those forest galleries are located in Lubumbashi, Republic of Congo. Other isolated

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www.ijpbs.com (or) www.ijpbsonline.com P. berghei strains are LUKA, K173, KSP11 and SP11. For many years, P. berghei has been a good model to investigate cellular signaling in malaria infection. Palm oil (PO) is potent anti-oxidant rich oil which contains carotenoids, tocopherols, tocotrienols and lycopenes [4]. It is also high in palmitic (44 %) and oleic acid (40 %) [5]. The red palm oil is obtained from the fruit of the oil palm tree and is the most widely produced edible vegetable oil in the world. Its nutritional and health attributes have been well documented [6] while involvement of cellular signaling mechanism in PO-induced protection also has been demonstrated [7]. Therefore it is likely that various protective effects of PO are associated with complex molecular mechanism which remains to be elucidated. Because tyrosine kinase signaling is important in Plasmodium infection [8], this study was carried out to determine effect of PO supplementation on the profile of erythrocytes phosphotyrosine proteins during Plasmodium berghei infection. In this study, there were two scopes investigated with regard to the role of PO in malarial infection namely: i) monitoring of intraerythrocytic parasitemia development in experimental mice for 20 days and ii) protein analysis on both normal and infected experimental mice when the latter reach their 40% parasitemia. The intraerythrocytic parasitemia in the experimental mice was induced by intraperitoneal injection while its development was monitored through microscopic analysis of Giemsa-stained blood smears. The protein analysis was to determine level of phosphotyrosine protein in erythrocytes of the experimental mice, which involved a combination of SDS polyacrylamide gel electrophoresis, immunoblotting and densitometry. Our study provided an insight into how the intraerythrocytic parasitemia

development could possibly be controlled by PO supplementation.

MATERIALS AND METHODS Experimental design and malaria induction A total of 40 male HSD mice aged eight weeks were maintained in the animal house of Universiti Kebangsaan Malaysia, Bangi, Selangor. The experimental mice were divided into observation (n=20) and sampling (n=20) groups which were then subdivided into four groups (group A: +PO+PB; group B: +PO-PB; group C: PO+PB and group D: -PO-PB) with n=5 each. The mice from group A and B were supplemented with PO (0.3ml/mice/day) orally for four weeks prior to inoculation of PB parasites whereby a volume of 100 Âľl of infected blood (1.25 x 106 parasitized erythrocytes, 125 dilution with Alsever solution) was injected into the experimental mice intraperitoneally. To monitor parasitemia development in group A and C, blood films were prepared from mice tail, stained with Giemsa and examined using light microscopy within 20 days. Blood sampling for protein analysis Blood sampling was conducted on the experimental mice of sampling group. At 40% parasitemia, both normal and infected experimental were sacrificed in an air-tight container under chloroform anesthesia. The blood sample was then withdrawn from abdominal aorta of experimental mice using one ml syringe and was kept in Vacutainer tubes containing EDTA as anticoagulant. To obtain erythrocyte fraction for protein analysis, the blood samples were centrifuged at 4,000 rpm for 10 minutes. Protein fraction and protein analysis Erythrocytes protein fraction was prepared as reported by [9] whereby acetone solvent was used to precipitate cytosolic proteins following lysis of the blood sample (erythrocyte fraction)

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www.ijpbs.com (or) www.ijpbsonline.com and precipitation of haemoglobin using HEPES lysis buffer (pH 8.0, contained 1 mM EDTA, 1Mm EGTA, 5% PMSF and 1 mM Na3VO4) and a mixture of etanol:butanol (60:40) respectively. Protein concentration was then determined using Bradford assay at a wavelength of 595 nm [10] whereby bovine serum albumin was used as a standard. An amount of 30 μg of erythrocyte protein was resolved by electrophoresis in a 12% sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis [11]. Subsequently, the resolved proteins and molecular mass marker were transferred from SDS polyacrylamide gel onto a nitrocellulose membrane [12] which was then followed by incubation in 5% gelatin (two hours, room temperature), overnight incubation with primer antibody (polyclonal antiphosphotyrosine, 1000 dilution with 0.1% PBST, 40C) and two hours incubation with secondary antibody (HRP conjugated anti-IgG rabbit, 3000 dilution with 0.1% PBST, room temperature). Antibody binding was assessed by the enhanced chemiluminescence (ECL) method comprising of Reagent 1 (Enhanced luminal reagent) and Reagent 2 (Oxidizing reagent). Level of phosphotyrosine proteins were analyzed densitometrically at a wavelength range from 450 nm to 700 nm. The optical density (OD) values were expressed as the mean ± standard error measurement and T-test was performed to determine degree of difference between the groups where p<0.05 was considered significant. The protocol of protein analysis was performed in triplicates.

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RESULTS Intraerythrocytic parasitemia It is established that when the malaria parasite infect the erythrocytes, they induce intraerythrocytic parasitemia development. The successful invasion of the malaria parasites is well associated with changes in erythrocyte

morphology. Figure 1 depicts intraeryhrocytic parasitemia development in infected experimental mice while Figure 2 shows parasite morphologies detected inside the infected erythrocytes. The microscopic observation on Giemsa-stained blood films demonstrated that the intraerythrocytic parasitemia level in group A and C was low (<4%) for the first six days postinfection while the highest parasitemia level (90±0.98%) was achieved by group A at day 20. It was also found that the parasitemia development of group A (both observation and sampling) was delayed as compared to group C starting from day 7 post-infection onwards. On the other hand, merozoite morphology was noted to be dominant in the first seven days post-infection. Trophozoite morphology started to become dominant at day 8 onwards whilst schizont morphology was found to be dominant when the parasitemia level reached approximately 50%. Group A and C reached 40% parasitemia at day 13 and day 11 respectively. It was a strong likelihood that the delay of parasitemia development was due to the antioxidant contents in PO which scavenge the free radical abnormally generated during PB infection. Phosphotyrosine proteins Tyrosine phosphorylation is a post-translational modification process occurs on protein molecules by adding phosphate (PO43-) group to tyrosine amino residues. This process is catalyzed by protein tyrosine kinase (PTK) while the removal of that phosphate group is dependent on dephosphorylation by protein tyrosine phosphatase (PTP). A common method to study tyrosine phosphorylation dephosphorylation is a combination of SDS polyacrylamide gel electrophoresis and immunoblotting.Figure 3 shows phosphotyrosine proteins detected in erythrocytes of both normal

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www.ijpbs.com (or) www.ijpbsonline.com and infected experimental mice. It was revealed that there were four erythrocytes phosphotyrosine proteins present in all sampling groups namely 120-kDa, 60-kDa, 30-kDa, 17-kDa and 14-kDa. It was also noted that PO supplementation did not affect all phosphotyrosine proteins in normal experimental mice apparently whilst PB infection affected 30-kDa phosphotyrosine protein significantly (p<0.05). Moreover, PO supplementation was found to prevent further tyrosine dephosphorylation of 30-kDa protein by PB infection. Densitometric analysis demonstrated that the OD values of 30-kDa phosphotyrosine protein were as follows: 0.95±0.08 (group A), 1.07±0.15 (group B), 0.78±0.9 (group C) and 1.11±0.09 (group D). The level of 30-kDa phosphotyrosine proteins of group C was significantly lower (P<0.05) than that of group D. It seems that the PO was to retain the tyrosine phosphorylated state of 30kDa protein during PB infection.

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DISCUSSIONS PO supplementation as an important strategy to reduce severity of malaria For many years, PO has been consumed in several countries in order to control severity of malaria. Due to the fact that PO is rich in antioxidants and able to reduce oxidative stress [4], the PO supplementation has been an important agenda to improve protection against malaria in pre-school children [13]. In that study, the effect of PO supplementation on malaria was investigated in 207 children (aged 0-60 months) who presented with fever in August-October 1999 at several hospital clinics around Ile-Ife, Western Nigeria. The parameters measured were anthropometric data, body temperature, parasitaemia and plasma C-reactive protein (CRP), retinol, carotenoids and tocopherols. It was observed that median plasma

concentrations of both alpha-carotene (0.518 mumol/L) and beta-carotene (0.698 mumol/L) in the children were high. There were also some evidences that the PO supplementation associated with a lower severity of malaria. Therefore, it is in agreement with the in vivo data herein. Protective effect of PO and cellular signaling mechanism A number of studies have shown that PO exhibits effect on cellular signaling mechanism. According to [14] the PO supplementation improved cardiac function of animal and was mediated by cyclic nucleotide signaling. In particular, they demonstrated that the functional recovery in animal hearts was associated with an elevation in the cGMP level and decrease of cAMP level. Their finding suggested that dietary PO supplementation improved reperfusion function through mechanisms with respect to activation of the NO-cGMP and inhibition of the cAMP pathway. Furthermore, a study by [15] showed that the PO supplementation also modulated the phosphorylation mechanism. By using a combination of SDS polyacrylamide gel electrophoresis and immunoblotting, it was noted that p38 and PKB/Akt phosphorylation increased significantly during reperfusion when compared with control hearts. Besides, a significant decrease in JNK phosphorylation and attenuation of poly (ADP-ribose) polymerase cleavage were also observed in the POsupplemented group during reperfusion. It was suggested that the dietary PO supplementation caused differential phosphorylation of the MAPKs and PKB/Akt during ischemia/reperfusion-induced injury. The role of antioxidant in cellular signaling cascade was also evidenced by [16] in which it is useful to explain molecular mechanism of disease development and prevention. In the context of our study, prevention of further tyrosine

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dephosphorylation of 30-kDa protein of by PO may attribute to its role in controlling intraerythrocytic parasitemia development. Antioxidant and its influence on tyrosine phosphorylation The net cellular level of tyrosine phosphorylation is sustained by enzymes which catalyze the incorporation (protein tyrosine kinases) or removal (protein tyrosine phosphatases) of phosphate from tyrosine amino residues. With respect to modulation of tyrosine phosphorylation by phytochemical consitutents, there is a study that revealed the inhibition of tyrosine phosphorylation by an organic compound, kaempferol. According to [19], the kaempferol which is a flavonoid widely found in various natural sources such as onions and citrus fruits, exhibited cardioprotective effect via its antioxidant activity. In the primary cultured rat aortic vascular smooth muscle cells (VSMCs), it inhibited both c-fos mRNA expression and PDGF beta-receptor tyrosine phosphorylation which in

turn inhibited phosphorylation of the downstream signal transduction of PDGF such as ERK1/2, Akt and PLC-gamma 1. In addition, another study reported the role of antioxidant in inducing tyrosine phosphorylation. In 2010, Kaspar and Jaiswal [20] investigated effect of antioxidant treatment on the nuclear translocation of Bach 1, a transcription regulator protein which is dependent on phosphorylation of tyrosine 486. It was discovered that Bach1 levels inside the nucleus returned to normal at 4 h after antioxidant treatment in the absence but not in the presence of protein synthesis inhibitor cycloheximide. They concluded that antioxidantinduced tyrosine 486 phosphorylation leads to nuclear translocation of Bach1. In conjunction with these, it was possible that the antioxidant contents of PO prevent further tyrosine dephosphorylation of erythrocytes 30-kDa proteins by activating PTK and inhibiting PTP during PB infection.

Figure1: Intraerythrocytic parasitemia development in infected experimental mice. Data are represented by mean Âą standard error measurement (n=5). International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)

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Figure 2: Parasite morphologies detected inside infected erythrocytes. A, B and C indicates merozoite, trophozoite and schizont respectively.

OD value

Group A 0.95±0.08

Group B 1.07±0.15

Group C 0.78±0.31

Group D 1.11±0.09

Figure 3: Erythrocyte phosphotyrosine proteins in both normal and infected experimental mice. The phosphotyrosine protein bands were detected using primer antibody (polyclonal anti-phosphotyrosine) and secondary antibody (HRP conjugated anti-IgG rabbit). The intensity of phosphotyrosine protein bands were analyzed using densitometry method. Optical density values are represented by mean ± standard error measurement (n=5).

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CONCLUSION We have demonstrated that PO supplementation could delay the Intraerythrocytic parasitemia development which most probably due to its high antioxidant contents. Tyrosine dephosphorylation of 30-kDa proteins occurred following PB infection and it was substantially prevented by PO supplementation. It was proposed that the delay of intraerythrocytic parasitemia development in

the infected experimental mice is due to prevention of further tyosine dephosphorylation of 30-kDa protein.

ACKNOWLEDGEMENT The author would like to thank Animal House of Universiti Kebangsaan Malaysia, Bangi and Golden Jomalina Food Industries Sdn. Bhd., Teluk Panglima Garang for P. berghei PZZ1/00 stock and palm oil respectively.

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[12]Bradford , M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein - dye binding. Analytical Biochemistry 72 : 248 - 254 . (1976.) [13]Laemmli, U.K. Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature. 341: 152-154. (1970). [14]Towbin, H. , Staehelin , T. and Gordon , J.. Electrophoretic transfer of proteins from polyacrylamide to nitrocellulose sheets : Procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America. 76 : 4350 - 4354 . (1979). [15]Cooper KA, Adelekan DA, Esimai AO, Northrop-Clewes CA, Thurnham DI. Lack of influence of red palm oil on severity of malaria infection in pre-school Nigerian children. Trans R Soc Trop Med Hyg. 96(2):216-223. (2002). [16]Kruger, M., Engelbrecht, A. M., Esterhuyse, J., du Toit, E. .F, Rooyen, J. Dietary Red Palm Oil (RPO) reduces ischaemia/reperfusion injury in a hypercholesterolemic diet. British Journal of Nutrition. 97(4):653-60. (2007). [17]Engelbrecht, A. M., Esterhuyse, J., du Toit, E. F., Lochner, A. and Rooyen, J. p38-MAPK and PKB/Akt, possible role players in red palm oil-induced protection of the isolated perfused rat heart? Journal of Nutritional Biochemistry. 17: 265-271. (2006). [18]Palmer, H. J. and Paulson, K. E. Reactive oxygen species and antioxidants in signal transduction and gene expression. Nutrition Reviews 55(10) : 353-361. (1997). [19]Kim S. Y.,Jin Y. R., Lim, Y., Kim J. H., Cho M. R., Hong J. T., Yoo H. S. and Yun, Y. P. Inhibition of PDGF betareceptor tyrosine phosphorylation and ots downstream intracellular signal transduction in rat aortic vascular smooth muscle cells by kaempferol. 71 (7): 599-603. (2005). [20]Kaspar JW and Jaiswal AK. Antioxidant-induced phosphorylation of tyrosine 486 leads to rapid nuclear export of Bach1 that allows Nrf2 to bind to the antioxidant response element and activate defensive gene expression. J Biol Chem. 1;285(1):153-162. (2010).

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[1] Fischer, E.H. , Charbonneau , H. and Tonks , N.K.. Protein tyrosine phosphatase : A diverse family of intracellular and transmembranes enzyme. Science . 253 : 401-406. (1991). [2]Hasidah, M. S. , Chee, S.K. and Rohani, A. K.. Penglibatan protein fosfotirosin dalam infeksi Plasmodium berghei. Sains Malaysiana. 29 : 1-9. (2000). [3] Yuthavong , Y. and Limpaiboon , T.. The relationship of phosphorylation of membrane proteins with the osmotic fragility and filterability of Plasmodium bergheiinfected mouse erythrocytes. Biochimica et Biophysica Acta. 929 : 278-287 .( 1987). [4] V. Thomas. Parasitologi perubatan. First Edition. Dewan Bahasa Pustaka Kuala Lumpur. (1983). [5] R. Killick-kendrick. Parasitic protozoa of the blood of rodents : a revision of Plasmodium berghei. Parasitology. 69 : 225-237. (1974). [6]O' Holohan, D. R.. The story of A Major Edible Vegetable Oil and It's Role in Human Nutrition. Kuala Lumpur: Malaysian Palm Oil Promotion Council (MPOPC). (1997). [7]Nor Aini and Suria, M.S.A. Food uses of palm and palm kernel oils. In Advances in Palm Oil Research, pp. 9681035. (2000). [8]Chandrasekharan N., Sundram, K. and Basiron, Y.Changing nutritional and health perspectives on palm oil. Brunei International Medical Journal. 2: 417-427. (2000). [9]Rooyen, R. J., Esterhuyse, A. J., Engelbrecht, A. M. and Toit, E. F. D. Health benefits of a natural carotenoid rich oil: a proposed mechanism of protection against ischaemia/reperfusion injury. Asia Pacific Journal of Clinical Nutrition. 17: 316-319. (200.8) [10]Finlay, B. B. and Cossart, P. Exploitation of mammalian host cell function by bacterial pathogen. Science 276 : 718 - 725. (1997). [11]Bavil, A.A., Hayes, S., Goretzki, L., Kroger, M., Anders, J. and Hendriks, R. Convenient and versatile subcellular extraction procedure, that facilitates classical protein expression profiling and functional protein analysis. Proteomics 4: 1397-1405. (2004).

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Research Article Biological Sciences PROXIMATE ANALYSIS AND PRODUCTION OF PROTEIN HYDROLYSATE FROM KING FISH OF ARABIAN GULF COAST - SAUDI ARABIA *

Sheriff Sheik Abdulazeez 1, Baranitharan Ramamoorthy2 Ponmurugan Ponnusamy3

1* & 2

College of Applied Medical Sciences, Shaqra, Shaqra University, Kingdom of Saudi Arabia 3 K. S. Rangasamy College of Technology, Thiruchengode, Tamil Nadu, India *Corresponding Author Email: biosherif@yahoo.co.in

ABSTRACT Saudi Arabia documents continuous increase in the utilization of fish and fish based food products. However, the huge quantity of fishery wastes and by-products generated every year either doesnâ&#x20AC;&#x2122;t been utilized properly or simply dumped. The discarded wastes are rich sources of protein that can be made use in various commercial and industrial applications. The protein hydrolysates recovered from various fishes and fish parts were shown to have antioxidant, antihypertensive, immunomodulatory, neuroactive, and antimicrobial properties. The chemical methods used for the protein recovery does not produce the yields with required standards. Thus enzymatic recovery of protein hydrolysate is the preferred choice and this technique is gaining momentum now. The present study was designed to produce a protein hydrolysate from the skin of king fish (Scomberomorus commerson) by papain digestion. The results showed that the enzyme was capable of recovering significant quantity of protein from the skin of king fish. The protein hydrolysate produced was exhibiting good degree of hydrolysis to yield peptides in solution. Thus the protein hydrolysate is a interesting candidate to be explored for its bioactive properties.

KEY WORDS King fish, papain, protein hydrolysate, Saudi Arabia.

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INTRODUCTION The continuous increase in global fish resources results in 25% of wastage among total fish catch annually [1]. Fish and fish derived products are invariable diet of Saudi population. Fish consumption is an integral practice of Saudis and the rate of consumption is increasing continuously. Every year, huge quantity of solid waste in the form of fish head, vicera, skin, bones, frames, and some muscle tissue [2] are discarded from sea food processing plants. Either these marine wastes are underutilized to produce low market value products such as fish meal or fertilizer, or simply dumped. Complete utilization of fishery wastes for recovering high-

end products would be an ideal strategy for an economic gain. Fishery wastes and by-products are valuable sources of raw material for recovery of bioactive compounds. The fishery wastes converted by proteolytic hydrolysis into a more marketable and functional form is called as fish protein hydrolysate (FPH) [3]. Protein hydrolysates can be prepared by conventional chemical methods with strong chemicals and solvents. The usage of such chemicals makes the products unsuitable to be used in food industry. The protein hydrolysates produced were of low nutritional value with poor functional ability and could not be utilized for commercial applications. Enzyme mediated

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www.ijpbs.com (or) www.ijpbsonline.com protein recovery from fishery wastes have been a recent research arena. This method suits to produce fish protein hydrolysate with desired functional properties [4]. Digestion parameters such as choice of enzyme, time of incubation, temperature and pH are tightly controlled to produce fish protein hydrolysates with the desired functional and nutritional values. The Protein hydrolysates thus obtained are already gaining reputation and widely used in variety of food industries in various forms, such as milk replacers, stabilizers of beverages, flavor enhancers in confectionery products, protein supplements, animal food, and microbial media[5] etc. The development of fish protein hydrolysates as functional food ingredients have been gaining popularity due to an array of potential bioactive properties associated with them, including antioxidant, antihypertensive, immunomodulatory, neuroactive, antimicrobial, and mineral or hormone regulating abilities[6]. Production of protein hydrolysate from different fish species such as mackerel [7], herring [8], tuna cooking juice [9] by different enzyms have been reported earlier. The present study was designed to recover protein from the skin of king fish commonly known as Kanad in Arabic. King fish is one of the important commercial fish caught in Arabian Gulf coast of Saudi Arabia [10]. The proximate composition of crude sample and freeze dried hydrolysate were evaluated. The enzyme papain was selected for the study as it is a cheap, easily available, commercial enzyme.

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MATERIALS AND METHODS Collection of fish sample King fish, commonly known as kanad in Arabic, caught from the Arabian Gulf Coast of Saudi Arabia was purchased from Saudi Fisheries Company. The sample was immediately sealed in a plastic bag, placed in icebox and immediately transferred to the Shaqra University Research

Laboratory. In the University Laboratory, the skin portion was separated from the fishes, minced for uniformity and stored in plastic bags at -200C until used. The chemicals and reagents used were of analytical grade Preparation of Fish Protein Hydrolysate The proteolytic digestion of Scomberomorus commerson (Narrow-barred Spanish mackerel) was performed according to the method described by Je et al. (2007) [3] with minimal modification. To produce protein hyrolysate from fish skin, the enzymatic hydrolysis was carried out with the enzyme papain (phosphate buffer 0.1 M Na2HPO4- NaH2PO4; pH-6, temperature, 37 °C) at enzyme ⁄ substrate ratio (1 ⁄ 100 w⁄ w). The 300 g minced fraction of Scomberomorus commerson was homogenized with blender and then thoroughly mixed with 3g of enzyme. The mixture was incubated for 6 h with continuous stirring and then heated in a boiling water bath at 100°C for 10 min to inactivate enzyme activity. The content was then centrifuged at 10000 rpm for 15 minutes and supernatant obtained was the fish protein hydrolysate. The hydrolysate was lyophilized to get a powdered sample and was stored at -20 °C. Yield and Degree of Hydrolysis The extent of hydrolysis was determined by adapting the procedure described by Tang et al. (2009) [11]. Briefly, the sample was mixed with papain enzyme with different enzyme/substrate ratio (1/100, 2/100, 4/200 v/w) and the reaction was conducted at pH 8.0 and temperature 37 °C (optimal conditions) for 0.5, 1, 2, 3, 4, 5 and 6 h. The pH of the mixture was maintained constant during hydrolysis using 2 M NaOH. After hydrolysis, the pH of the broths was brought to 7.0, and the solutions were then heated at 100°C for 10 min to inactivate the Enzyme. The Hydrolysate was centrifuged at 10000g for 15 min, and the supernatant was lyophilized to get a powdered sample and was

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www.ijpbs.com (or) www.ijpbsonline.com stored at -20°C. The degree of hydrolysis (DH) is defined as the ratio between the number of broken peptide bonds (h) and the total number of peptide bonds per mass unit (htot) h DH% = ----- X100 htot The degree of hydrolysis (DH) of hydrolyzed protein was determined by measuing the amount of free α- amino groups based on the reaction between Sanger’s reagent of fluorodinitrobenzene (FDNB) and the amino groups in the amino acids which resulted a yellow complex of amino acids [12]. The absorbance was measured spectrophotometrically at 410 nm. The nitrogen recovery (NR) in the soluble fraction was calculated using the method of Benjakul and Morrissey (1997) [13] by the following formula.

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Total nitrogen in the soluble fraction NR = ------------------------------------------------ X 100 Total nitrogen in the substrate Proximate composition Moisture content was determined by placing approximately 2 g of sample into a pre-weighed aluminum dish. Samples were then dried in a forced-air convection oven at 105°C overnight or until a constant weight was reached [14]. The total crude protein (N X 6.25) content of samples was determined using the Kjeldahl method [14]. Total lipids in each sample were extracted with a mixture of chloroform and methanol as described by Bligh and Dyer [15]. The content of minerals (expressed as percent ash content) was determined by charring approximately 2 g of sample in a crucible over a Bunsen burner and then heating in a muffle furnace at 550°C until the ash had a white appearance [14]. Statistical Analysis

The statistical analysis of data was performed by using SPSS 16 for windows. The results were expressed as mean of triplicates ± SD.

RESULTS AND DISCUSSION Protein Recovery The degree of hydrolysis have been used as an indicator of the cleavage of peptide bond, whereas nitrogen recovery reflects the yield of proteins that can be recovered from the hydrolysis process. Enzymatic digestion of protein results in the release of Peptides during hydrolysis. Figure 1 describes the recovery of protein in terms of yield % with respect to incubation time. It’s common that yield of protein increased with increase in the time of hydrolysis. Maximum 63.3% of protein was recovered at 6 hours of incubation. The results suggest that papain digestion recovers a considerable amount of protein from the skin of Scomberomorus commerson. Degree of Hydrolysis DH estimates the change of peptide content in a hydrolytic reaction. It is generally used as a proteolysis monitoring parameter [16] and an important factor highly related with the hydrolytic process yield [17]. The results of DH are presented in Figure 2. The typical shape of curve obtained in the present study was reported earlier in many investigations by different investigators [8, 18-19]. As expected the DH increases with increase in incubation time. The DH was observed to be 22.2% for enzyme substrate ratio 1:100, 23.6% for 2:100 and 24.7% for 4:100 which was well within the range of earlier observations made from the skin of different fish species [20]. The varied pattern of DH was closely reliant upon applied enzyme concentration, namely the E/S ratio. With E/S ratio increasing (from 1:100, 2:100 and 4:100 w/ w), the rate of DH increase during initial phase (e.g., first hour) of hydrolytic process. However,

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www.ijpbs.com (or) www.ijpbsonline.com at the end of 6 hours the rate of degradation does not much influenced by the E/S ratio and all the three E/S ratios showing similar percentage of digestion. Proximate Analysis The composition of protein hydrolysates generally depends on choice of enzyme, pH, incubation time and analytical methods used for estimation. In the present study the papain enzyme with appropriate pH and temperature optima was used to get the protein hydrolysate. The proximate analysis in terms of protein, lipid,

moisture and ash contents of both the crude wet sample and freeze dried protein hydrolysate of king fish were measured. The results were expressed as a mean of triplicate ± SD and represented in Figure 3 and Figure 4 respectively. The minced skin sample of Scomberomorus commersoon showed higher moisture content (76.8%) and least lipid (0.43%) content. The lyophilized protein hydrolysate showed protein content of around 85.57% which was in par with earlier findings [21].

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Figure 1: Percentage protein recovery of hydrolysis from the skin of kingfish by papain. Values are expressed as mean of triplicates ±SD

Figure 2: DH and influence of E/S ratio during hydrolysis of skin sample from kingfish by papain enzyme. Values are expressed as mean of triplicates ±SD

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Figure 3: Proximate analysis of crude sample from the skin of king fish. Values are expressed as mean of triplicates ÂąSD

Figure 4: Proximate analysis of protein hydrolysate from the skin of king fish by papain enzyme. Values are expressed as mean of triplicates ÂąSD

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CONCLUSION The results of the present investigation clearly reveal that skin sample of Scomberomorus commerson could be utilized for the protein recovery by the proteolytic enzyme papain. The findings of the DH analysis clearly suggest that the sample is rapidly undergoing hydrolysis to produce small peptides during hydrolysis and not

much influenced by higher E/S ratio. Proximate analysis revealed higher protein content in the sample and was justified by higher protein recovery during hydrolysis. The protein yield was high enough for further characterization. From this study it can be concluded that skin protein hydrolysate of Scomberomorus commerson is an ideal choice of protein substrate for further

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FAO, La situation mondiale des peˆches et de l’aquaculture 2006. De´partement des peˆches et de l’aquaculture, Rome (2007) Awarenet (2004) Handbook for the prevention andminimization ofwaste and valorization of byproducts in European agro-food industries. Agro-food waste minimization and reduction network (AWARENET). Grow Programme, European Commission, pp 1–7 Je J.Y., Qian Z., Byun H.G., Kim S.K., Purification and characterization of an antioxidant peptide obtained from tuna backbone protein by enzymatic hydrolysis. Process Biochem, 42:849-846, (2007) Quaglia G.B., Orban E., Influence of enzymatic hydrolysis on structure and emulsifying properties of sardine (Sardina pilchardus) protein hydrolysate. Journal of Food Science 55: 1571–1573, (1990) Tanuja S., Viji P., Zynudheen A.A., Joshy C.G., Composition, functional properties and antioxidative activity of hydrolysates prepared from the frame meat of Striped catfish (Pangasianodon hypophthalmus). Egyptian Journal of Biology, 14: 27-35, (2012) Alasalvar, C., Shahidi, F., Quantick, P., Food and health applications of marine neutraceuticals. In: Alasalvar, C., Taylor, T., (Eds.), Seafoods- quality, technology and nutraceutical applications. Springer-Verlag, Berlin 2002, pp. 175-204, Wu H.C., Chen H.M., Shiau C.Y., Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus). Food Res Inter, 36: 949-957, (2003) Sathivel S., Bechtel P. J., Babbitt J., Smiley S., Crapo C., Reppond K.D., Prinyawawitkul W., Biochemical and functional properties of herring (Clupea harengus) byproduct hydrolysates. J Food Sci, 68: 2196–2200, (2003)

Jao C.L., Ko W.C., 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging by protein hydrolysates from tuna cooking juice. Fisheries Science, 68, 430–435, (2002) Younis E.M.,, Abdel-Warith A.A., Ali A., al-Asgah N.A., El-Shayia A. S., (2011) Chemical composition and mineral contents of six commercial fish species from the Arabian gulf coast of Saudi Arabia. Journal of Animal and vetenary advances, 10: 3053-3059, (2011) Tang C.H., Peng J., Zhen D.W., Chen Z., Physicochemical and antioxidant properties of buckwheat (Fagopyrum esculentum Moench) protein hydrolysates. Food Chem, 115:672–678, (2009) Goodwin J.F., The colorimetric estimation of plasma amino nitrogen with DNFB. Clin Chem, 14:1080-1090, (1968) Benjakul S., Morrissey M., 1997. Protein hydrolysates from Pacific whiting solid wastes. J Agric Food Chem, 45:3423–3430, (1997) AOAC, Official Methods of Analysis. Association of Official Analytical Chemists, Washington, DC, USA, (1995) Bligh E. G., Dyer W. J., (1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol, 37: 91 l-17, (1959) Guerard F., Guimas L., Binet A., Production of tuna waste hydrolysates by a commercial neutral protease preparation. J Mol Cat B; Enz, 19–20: 489–498 (2002). Adler-Nissen, J., Enzymatic Hydrolysis of Food Proteins. Elsevier, New York 1986. Hoyle N.T., Merritt J.H., Quality of fish protein hydrolysates from herring (Clupea harengus). J Food Sci, 59: 76–79, (1994). Liceaga-Gesualdo A.M., Li-Chan, E.C.Y., 1999. Functional properties of hydrolysates from herring (Clupea harengus). J Food Sci, 64: 1000–1004, (1999) Sathivel S., Huang J., Bechtel P.J., properties of pollock (Theragra chalcogramma) skin hydrolysates and effects on lipid oxidation of skinless pink salmon (Oncorhynchus gorbuscha) fillets during 4 months of frozen storage. J Food Biochem, 32: 247–263, (2008) Yin H., Pu J., Yutingwan B., Xiang, Bechtel P.J., Sathivel S., Rheological and functional properties of catfish skin protein hydrolysates. J Food Sci, 75:11-17, (2010)

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Assistant professor, Department of Biochemistry, College of applied medical sciences, Shaqra University, Post box.No.1383, Shaqra 11961. Kingdom of Saudi Arabia. Phone: +966 532949275 Email: biosherif@yahoo.co.in

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Research Article Biological Sciences ANTI ULCER ACTIVITY OF ACACIA FARNESIANA (L.) (AROMA) A LESSER KNOWN FOLK - MEDICINAL PLANT Dwarakanath V1*, B Dhanasree2, B Jayasimha Goud3, S Nizamuddin Basha4 1

Dept. of Biotechnology, University College of Science, Tumkur University, Tumkur, Karnataka. INDIA 2 Dept. Of Biochemistry, KVR Govt. Degree College, Kurnool, A.P. INDIA 3 Dept of Biochemisitry, SRN Adarsh College, Bangalore, Karnataka, INDIA 4Dept. Of Biochemistry, S.K. University, Anantapur, A.P. INDIA

ABSTRACT Aim of the study: To determine the pharmacological activity relevant to ulcer healing of Acacia farnesiana (aroma) leaf, a folk medicine used in the treatment of Ulcer. Materials and Methods: MEAF extract was done by using soxhelet apparatus and was further subjected to screening of the qualitative analysis of phyto chemicals. Consequently the Methanolic extract of leaf was tested against ulcer induced rats. Results: The methanol extract of leaf (200mg/Kg) has shown greater protection against ulcer damage to the tissue (58.35 compared to 61.12 % of RD) Conclusion: After performing of Preliminary Phytochemical screening of Acacia farnesiana (aroma), the Methanol extract of Acacia farnesiana (aroma) was chosen for further analysis, in other hand the chronic study was done in Ethanol induced ulcers of Male Wister albino rats and then the Ulcer Scoring as well as the Biochemical parameters were studied. Consequently we have evaluated the Anti Ulcer activity of MEAF. Hence, it establishes the fact that the Methanol extract of antiulcer activity of Acacia farnesiana (L.) (Aroma) is offering an ineffable support to the folk medicine, which is a traditional medicine for the treatment of ulcers in INDIA.

KEY WORDS Anti ulcer, Acacia farnesiana, Folk Medicine, Aroma

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1. INTRODUCTION Peptic ulcer is a conglomerate of heterogenous disorders which manifests itself as a break in the lining of the gastrointestinal mucosa bathed by acid and/or pepsin. NSAIDS ingestion is associated with erosions, petechiae type C gastritis, ulceration, interference with ulcer healing, Ulcer complications and injury to the small and large intestine16. Although a number of antiulcer drugs such as H2 receptor antagonists, proton pump inhibitors and cyto protectants are available for ulceration, but all these drugs have side effects and

limitations2. Herbal medicine considered safer because of the natural ingredients with no side effects4. In India, Acacia farnesiana (aroma) is known as Mulla tumma, Kampu tumma in local area and it is commonly known as Aroma and sweet acacia also. Grown throughout India, and often planted in gardens. If we see its yield, in India and other Eastern countries produce much for local use and Trees begin to flower from the third year, mainly from November to March5. The bark of this plant is used as astringent and demulcent. The leaves and roots are used for medicinal purposes. Woody branches used in

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www.ijpbs.com (or) www.ijpbsonline.com India as tooth brushes. The gummy roots also chewed for sore throat. The roots of this plant is also used for the antispasmodic, aphrodisiac, astringent, demulcent, diarrhea, febrifuge, rheumatism, and stimulant5.The flower infusion of this plant used as a stomachic9. It is also used for dyspepsia and neuroses. Mexicans sprinkle powdered dried leaves onto wounds. The flowers are added to ointment, rubbed on the forehead for headache. Green pods are decocted for dysentery and inflammations of the skin and raucous membranes. Colombians bathe in the bark decoction for typhoid. Costa Ricans decoct rhe gum from the trunk for diarrhea, using the pod infusion for diarrhea, leucorrhea, and uterorrhagia. Panamanians and Cubans used the pod to treat conjunctivitis. Cubans use the pod decoction for sore throat. For rheumatic pains, West Indians bind bark strips to the afflicted joint. The root decoction has been suggested as a folk remedy for tuberculosis. The decoction of the root, used in hot baths, is said to help stomach cancer. A plaster, made from the pulp, is said to alleviate tumors7. In preliminary phytochemical investigations it has found that the leaves contain lipids, carotenoids, alkaloids, flavonoids and reducing and non-reducing sugars and seven polyphenols (gallic acid, ellagic acid, m-digallic acid, methyl gallate, kaempferol, atomadendrin, and narigenin). Also found narigenin-7-glucoside and naringenin-7rhamnoglucoside (naringin), as well as naringenin, glucose, and gallic acid 6. Another Phytochemical compound Quercetin, of this plant is found to be shown antioxidant activity15. Some recent reports have indicated that many flavonoids possess antiulcerogenic activity. Oral treatment with the ether fraction of the flavonoid extract demonstrated a good level of gastric protection. Mucous content was increased and accompanied by proportionate increase in proteins and hexosamines1.

Quercetin, kaempferol, morin, myricetin and rutin when tested were found to inhibit the mucosal content of platelet activating factor (PAF) in a dose dependent manner suggesting that the protective role of these substances may be mediated by endogenous PAF10. Flavonoids exhibit several biological effects such as anti inflammatory, anti hepatotoxic and antiulcer actions3 In this present study we evaluated the antiulcer activity of MEAF leaf in ulcer induced experimental rats and It is further investigated and confirmed by using bio analytical studies of different antioxidant enzymes by taking the consideration of Phytochemical compounds of Acacia farnesiana(aroma).

2. MATERIALS AND METHODS: 2.1. Plant collection: The whole plant of Acacia farnesiana (Aroma) was collected from coastal area, near Chittoor district of A.P. INDIA, during the month of November. It is identified and authenticated. 2.2. Preparation of extract: The plant of Acacia farnesiana (Aroma) were shade dried and the leaves reduced to coarse powder in a mortar and pestle. The powdered material obtained was then subjected to successive extraction by hot percolation method using petroleum ether, chloroform and methanol solvents in a soxhelet apparatus. The different extracts obtained were evaporated at 450 C to get a semisolid mass. The extracts thus obtained were subjected to phyto-chemical analysis. The percentage yield alcoholic extract was found to be 55.5%w/w and the Methanolic extract was taken for further Phytochemical Screening Qualitative tests for the presence of plant secondary metabolites such as carbohydrates, alkaloids, tannins, flavonoids, proteins, saponins and glycosides were carried out on extracts using standard procedure studies.

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www.ijpbs.com (or) www.ijpbsonline.com 2.3. Animals used: Male albino Wister rats between 1 to 2 months of age and weighing 125-150g were procured and were maintained as per the guidelines of National Institute of Nutrition (NIN) Animal User’s Manual. Animals were acclimatized for 7 days to our animal house, maintained at temperature of 20-240 c. The light source in the animal room was regulated with 12 h light period followed by 12 h dark schedule. Two to three animals were housed per cage sized (41× 28 × 14 cm). Paddy husk was used for bedding and on every alternative day bedding was changed and washed thoroughly with water along with Domex, a disinfectant and a detergent. The rats were fed on a standard pellet diet purchased from Sai Durga Feeds and Foods Bangalore, and water ad libitem. 2.4. Experimental Design The animals were divided into four groups, each consisting of six rats. Group Ι represented the Normal control group, which received distilled water orally. Groups ΙΙ represented the Control group, which received Ethanol 1ml/100g.b.w13. Groups ΙΙI received Methanol extract of Acacia farnesiana (aroma) 200 mg/kg and, Ranitidine, in the dose of 20 mg/kg were administered orally for group ΙV as reference standard drug. The gastric ulcers were induced in rats by administrating absolute ethanol (95%) (1 ml/100 g b.w.) Orally13. After 45 min of Methanol extract and Ranitidine treatment .They were kept in specially constructed cages to prevent coprophagia during and after the experiment. The animals were anaesthetized 1hr latter with anesthetic ether and stomach was incised along the greater curvature and ulceration was scored.

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3. ULCER INDEX / SCORING OF ULCER 3.1. MEASUREMENT OF ULCER INDEX:

Stomach mucosa was flushed with saline and lesions in glandular portion were then exposed and examined under a 10x magnifying glass8. Ulcer index of each animal was calculated by adding the values and their mean values were determined by the following scoring system 8. For the macroscopic observations, the number, lengths and severity of ulcers were noted and scored. The ulcer index (U.I.) of each stomach was the sum of its scores. The ulcer index was reported as arithmetic means ± S.E. The significance of differences between means was evaluated by Student’s t - test for unpaired data. P< 0.05, versus control, was taken as significant. (i) Normal coloured stomach– 0, (ii) Red colouration ‐ 0.5, (iii) Spot ulceration – 1, (iv) Haemorrhagic streak – 1.5, (v) Ulcers (< 2mm) – 2, (vi) Ulcers (>2 < 4 mm) perforations – 3. (v) 3 Ulcers (< 4mm): -4 Percentage inhibition was calculated using the following formula: UI ulcer control - UI treated % Inhibition =________________ X 100 UI ulcer control 4. Sample collection and preparation for biochemical estimations and assays: 4.1. Measurement of gastric secretion and PH. 11 The stomach of aspirin induced ulcer rats was carefully excised keeping oesophagus closed and opened along greater curvature and luminal contents were removed. The gastric juice thus collected was centrifuged at 3000 rpm for 10 min and expressed in terms of ml/100 g of body weight. The pH of the supernatant was measured using digital pH meter 11. 4.2. Measurement of Free and total acidity12: Free and total acidity were determined by titrating with 0.01N NaOH using Topfer’s reagent

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www.ijpbs.com (or) www.ijpbsonline.com and phenolphthalein respectively as indicators and were expressed as meq/l per 100 g 12. 4.3. Measurement of gastric juice and pH11: The gastric juice was collected was centrifuged at 3000 rpm for 10 min and expressed in terms of ml/100 g of body weight. The pH of the supernatant was measured using digital pH meter. 4.4. Determination of free acidity and total acidity14: The gastric contents were centrifuged at 1000rpm for 10min. 1ml of supernant was diluted with 9ml of distilled water. A volume of 2ml diluted gastric juice was titrated with 0.1N Sodium hydroxide run from a micro burette

using 3-4 drops of Topfer's reagent as indicator until canary yellow colour was observed. Volume of NaOH required was noted. This corresponds to free acidity. Further 2-3 drops of phenolphthalein was added and titrated with NaOH until pink colour was restored. This gives total acidity. Free acidity and total acidity is expressed in terms of ml of 0.1N HCl per 100 gms of gastric contents. This is the same as mEq/lit. To obtain this figure multiply the burette reading obtained from titration by 10. Acidity was calculated by using the formula: Volume of NaOH X Normality of NaOH X 100 Acidity = ____________________________________ meq/L/100g 0.1

5. RELUTS ANTI ULCER EFFECT OF MEAF IN ETHANOL INDUCED MODEL TABLE A ANOVA TEST FOR ANTI ULCER ACTIVITY OF ETHANOL INDUCED MODEL S.NO

TREATMENT

ULCER INDEX

CONTROL

5.917 ± 0.2386

2. 3.

RANITIDINE MEAF

2.167 ± 0.3073 2.417 ± 0.3005

F, d f Value

54.569 (2/15)

P Value

P < 0.0001 * P < 0.01 when compared with Control

Figure A: Graphical Representation of Ulcer Index

ULCER INDEX

6 5 4

control

Ranitidine

MEAF

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0 control

Ranitidine

MEAF

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TABLE B ANOVA TEST FOR ANTI ULCER ACTIVITY OF PYLORUS LIGATED ULCER INDUCED MODEL: S.NO.

Treatment

Volume of Gastric juice(ml)

Free Acidity meq/l/100gm

Total Acidity meq/l/100gm

CONTROL

3.117 ± 0.1579

1.717 ± 0.04773

21.500 ± 0.9916

79.33 ± 1.333

RANITIDINE

1.217 ±0.04773

2.967 ± 0.1229

10.833 ± 0.6009

39.500 ± 1.147

MEAF

1.417 ± 0.06009

2.633 ± 0.1282

12.833 ± 0.6009

49.833 ± 1.078

106.05 (2/15)

37.151 (2/15)

56.547 (2/15)

301.22 (2/15)

P < 0.0001

8. F, ,d f Value P Value

* P < 0.01 when compared with Control 3.5

3.1

3 2.5 2 1.5

1.4

1.2

1 0.5 0 Control

Ranitidine

MEAF

Volume of Gastric juice(ml)

FIGURE B: ANOVA Test for Gastric Juice Volume

3.5

2.9

2.6

2.5

1.7

1.5 1 0.5 0 Control

MEAF

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Ranitidine

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21.5

20 15

12.8

10.8

10 5 0 Control

Ranitidine

MEAF

Free Acidity meq/100gm

FIGURE D: ANOVA Test for free acidity 100 80

79.3

49.8 39.5

40 20 0 Control

Ranitidine

MEAF

Total Acidity meq/100gm2

FIGURE E: ANOVA Test for Total acidity

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6. DISCUSSION Oral administration of methanol extract of Acacia farnesiana(aroma) leaves at a dose of 200 mg/kg exhibited dose dependent inhibition Percentage of 58.35 compared to the ulcer control, proving the antiâ&#x20AC;?ulcer activity of extract whereas ranitidine (20 mg/kg) produced 61.12 % inhibition of ulcer index against Ethanol induced ulcer. MEAF significantly protected gastric mucosa against the damage induced by ethanol and Curative ratio of the MEAF of 200 mg/kg was found to be 90.17%. Oral administration of MEAF 1 hr before the induction of stress reduced the

cold restrained stress induced ulcers. The MEAF exhibited a dose dependent inhibition percentage of 58.35 at doses of 200 mg/kg dose. The standard drug Ranitidine showed an inhibition percentage of 61.12. Ethanol administration (20mg/kg) resulted in the production of gastric mucosal damage. The ulcer index in control animals was (5.917). Methanol extract (2.417) significantly reduced the ulcer index (p<0.01) as compared to control. Ranitidine, a standard anti-ulcer drug showed ulcer index (2.167). The results are tabulated in Table A.

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www.ijpbs.com (or) www.ijpbsonline.com Pre treatment of rats with Acacia farnesiana (aroma) extracts produced a dose dependent protection in the ethanol induced ulceration model as compared to control group. However the protection was statistically significant reduced the severity of ulcer and caused a significant reduction of ulcer index in this model. Ranitidine produced significant gastric ulcer protection as compared to control group. Pre treatment with methanol extract of Acacia farnesiana(aroma) leaves produced significant anti‐ulcer effect which can observed by the effect of MEAF on gastric secretion in Ethanol induced ulcer. Gastric juice volume, total and free acidity significantly increased and pH decreased in ulcer control animal in comparison to normal animals. MEAF (200 mg/kg) produced dose dependent effect and decreased gastric juice volume, total and free acidity and increased pH significantly. Hence it establishes the fact that the Methanol extract of Acacia farnesiana(aroma) has shown the protection against the ulcers which was induced by the ethanol and it is further recommended to do the in vivo Bio analytical assays, such as enzymatic studies etc.,

5. 6.

7. 8.

10.

11.

12.

13.

14.

7. REFERENCES 1.

15. 16.

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Alarcon DLLC, Martin MJ, Lacasa C, Motilva V. Antiulcerogenic activity of flavonoids and gastric protection. J Ethnopharmacol 1994;42:161-70. Ariyoshi I, Toshiharu A, Sugimura F, Abe M, Mastsuo Y, Honda T. 1986. Recurrence during maintenance therapy with histamine H2 receptor antagonist in cases of gastric ulcer. Nihon Univ J Med 28: 69-74. Bors W, Heller W, Michel C, Saran M, Flavonoids as antioxidants:Determination of radical scavenging efficiencies. Methods Enzymol. 1990; 186:343-55.

Clouatre D Rosenbaum M 1994. The Diet and Health benefits of HCA (Hydroxy citric acid), Keats Publishing: New York, 31-32. Duke, J.A. 1981a. Handbook of legumes of world economic importance. Plenum Press. NewYork. El Sissi, H.I., El Ansari, M.A., and El Negoumy, S.I. 1973. Phenolics of Acacia farnesiana. Phytochemical reports. Phytochemistry 12:2303. Hartwell, J.L. 1967–1971. Plants used against cancer. A survey. Lloydia 30–34. Malairajan P, Gopalakrishnan G, Narasimhan S, Veni KJK, Kavimani S. Anti‐ulcer activity of crude alcoholic extract of Toona ciliate Roemer (heartwood). J Ethnopharmacol 2007; 110: 348‐51 Morton, J.F. 1981. Atlas of medicinal plants of Middle America. Bahamas to Yucatan. C.C. Thomas, Springfield, IL. Parmar NS, Shikha Parmar. Anti-ulcer potential of flavonoids. Indian J Physiology and Pharmacology 1998; 42:343-51. Patil KS, Kumar S, Bahuguna YM, Shinkar AS, Hugar DS. Antiulceractvity of leaves of Gossypium arboreum in aspirin induced rats and pylorus ligated rats. Indian Drugs 2008; 45: 325‐31. Rajkapoor B, Anandan R, Jayakar B. Anti‐ulcer effect of Nigella sativa Linn against gastric ulcers in rats. Current Science 2002; 83: 177‐9. Robert A, Nezamis JE, Lancaster C, Hanchar A. Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl, and thermal injury. Gastroenterology 1979; 77(03): 433-443. T. Gusdinar, R. Herowati, R.E. Kartasasmita and I.K. Adnyana, 2011. Anti-inflammatory and Antioxidant Activity of Quercetin-3, 3’, 4’-Triacetate. Journal of Pharmacology and Toxicology, 6: 182-188. Stanford T. Clinical diagnosis by laboratory methods. 14th ed.: Davidson and Henry; 1969. Wallace JL. 1992 Non – Steroidal anti-inflammatory drug gastropathy and cytoprotection – pathogenesis and mechanisms re-examined. Scand Journal of Gastroenterology 27: S192, 3-8.

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*Corresponding Author:

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Dwarakanath V Asst. Professor. Dept. Of Biotechnology, University College of Science Tumkur University, Tumkur, Karnataka, INIDA

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Research Article Pharmaceutical Sciences PREPARATION AND EVALUATION OF FAST DISSOLVING ORAL THIN FILM OF CAFFEINE FARHANA SULTANA*1, MOHAMMAD ARAFAT2, SAIFUL I. PATHAN3 1

State university of Bangladesh The University of Asia Pacific 3 State university of Bangladesh 2

*Corresponding Author Email: farhana_sultana_2005@yahoo.com

ABSTRACT The objective of this research was to prepare fast dissolving oral thin film (FDOTF) containing caffeine. Fast dissolving oral thin films of caffeine anhydrous were prepared using HPMC-2910 (15cps), sodium alginate and kollicoat速 IR white in various proportions. Total nine formulations were prepared and conducted various physicochemical evaluations including FTIR and in vitro dissolution studies .From the trinocular microscopic images 速 it appears that kollicoat IR white is more porous which may be due to the characteristic behavior of graft copolymer and was reflected in its lowest disintegration time (12sec.)and its cumulative percent release was 99.86% within 240seconds. Films in formulation F1 prepared with HPMC were very flexible, smooth and its in vitro disintegration time was 13 seconds. Its cumulative percent drug release was 100% within 120 seconds which is remarkable in comparison to other formulations.

KEY WORDS Cumulative percent drug release, FDOTF, HPMC, In vitro disintegration time, Kollicoat 速IR

INTRODUCTION

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153

Rapidly dissolving or quick dissolving dosage forms have acquired great importance in the pharmaceutical industry due [1][2][3] to their unique properties and advantages. They undergo disintegration in the salivary fluids of the oral cavity within a minute, where they release the active pharmaceutical ingredient. The major amount of the active pharmaceutical ingredient is swallowed orally with the saliva where subsequent absorption takes place in the gastro-intestinal tract.[4], [5] The rapidly dissolving dosage forms are referred by various names by researchers like quick disintegrating, orally disintegrating, mouth dissolve or melt in mouth dosage forms.[2][4][5]

Fast dissolving oral thin film drug delivery system is solid dosage form which dissolves in a short period of time when placed in the mouth without drinking water or chewing. These are also called fast dissolving oral thin films, Buccal films/ strips, Oral strips, Oral wafer. FDOTFs are the most advanced form of oral solid dosage form due to more flexibility and comfort. It improve the efficacy of APIs by dissolving within minute or seconds in oral cavity after the contact with saliva without chewing and no need of water for administration. It gives quick absorption and instant bioavailability of drugs due to high blood flow and permeability of oral mucosa is 4-1000 times greater than that of skin [6][7]. FDOFs are useful in patients such as pediatric, geriatrics, bedridden, emetic patients,

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diarrhoea, sudden episode of allergic attacks, or coughing for those who have an active life style. It is also useful whether local action desired such as local anesthetic for toothaches, oral ulcers, cold sores or teething. [8] The OTFs place as an alternative in the market due to the consumer’s preference for a fast-dissolving product over conventional tablets / capsules. The oral thin-film technology is still in the beginning stages and has bright future ahead because it fulfils all the need of patients. Eventually, film formulations having drug/s will be commercially launched using the OTF technology [9].

Bangladesh Ltd., HPMC was purchased from MERCK, India, Kollicoat® IR white was purchased from BASF, Germany, Sodium alginate& Sodium starch glycolate was purchased from Loba chemie, India. All other chemicals and reagents used were analytical grade. Distilled water was used in this study. Preparation of fast dissolving oral thin film of caffeine anhydrous The Table 1 shows the detailed composition of the oral thin film of caffeine anhydrous formulations which were used in the present study. Calculation of drug quantity: For 20cm x15cm glass rectangular plate, the total area of the plate is 20cmx15cm=300 cm2 Size of the individual film is 3x2 cm2 i.e. 6 cm2 So, theoretically total number of film will be 300/6=50 The total quantity of drug should be taken for 20x15 cm2 Plate will be (50x10)=500 mg.

MATERIALS AND METHODS

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MATERIALS Caffeine anhydrous was gift sample from Biopharma Ltd., Bangladesh. Sucralose was obtained from Drug International Ltd., Bangladesh as a gift sample, Glycerine was received as a gift sample from uniliver,

Table 1 Composition of various caffeine fast dissolving oral thin film formulations Ingredients Formulation Code F1 F2 F3 F4 F5 F6 F7 F8 Caffeine anhydrous (mg) 500 500 500 500 500 500 500 500

F9 500

HPMC 2910, 15cps (mg)

1500

2000

2500

Sodium alginate (mg) Sodium starch glycolate (mg) Kollicoat ® IR white(mg) Citric acid anhydrous (mg) Glycerin (ml)

400 2.5

750 750 400 2.5

750 1000 400 2.5

750 1250 400 2.5

1500 400 2.5

2000 400 2.5

2500 400 2.5

Sucralose (mg) Ethanol 96% (ml) Purified water (ml)

250 10 10

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www.ijpbs.com (or) www.ijpbsonline.com PREPARATION OF FAST DISSOLVING ORAL THIN FILM OF CAFFEINE [10] [11] Base film solution: Drug solution: Polymer was dispersed in a measured quantity of solvent (mixture of water and ethanol 96%). It involves the addition of all the ingredients except for base film solution. Initially the drug was added in a solvent. To this solution all other excipients were added with continuous stirring. At last plasticizer (glycerin) was added to this homogenous solution. To the base solution the drug solution was added slowly with a care to eliminate air entrapment. The final film solution was casted on glass plate. The casted films were dried in oven at 60°C for three hours or until dryness. The duration of drying depended on the properties of each polymer. Individual films

were prepared by cutting the films into strips of regular dimension of 2cm x 3cm with a stainless steel cutter. The samples were packed in a high density polyethylene sheet, sealed and stored in desiccators at room temperature.

EVALUATION OF FAST DISSOLVING ORAL THIN FILM OF CAFFEINE Variation of film mass [12] The mass of the films was determined by using analytical balance. When manufacturing the oral films,the film solutions were cast into sheets and then cut into smaller strips of 6 cm2 (3cm×2 cm). Oral films were cut from different sheets and the variability between the respective polymers as well as the variability between the polymers were investigated.The results are shown in Table 2.

Table 2: Evaluation of the caffeine fast dissolving oral thin films Code

#Thickness (mm)

*Mass uniformity (mg)

# Content uniformity (mg)

#D.T.(Secs)

0.048

52.10±1.20

10.06±0.206

F2 F3 F4 F5

0.111 0.188 0.92 0.96

82.8±1.23 110.6±1.43 86.6±0.88 91.52±1.24

9.9±0.56 9.87±0.25 9.75±0.12 9.80±0.025

20 45 33 25

F6 F7 F8 F9

0.101 0.06 0.076 0.087

95.91±1.76 60.4±0.74 86.55±1.28 106.25±2.09

9.83±0.049 9.98±0.35 9.99±0.044 10±0.13

22 12 16 27

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*All the values are expressed as mean± SD, n=10. #All the values are expressed as mean ± SD, n=3.D.T.= Disintegration time in seconds.

Thickness [13] The thickness of film was measured by micrometer screw gauge at different strategic locations. Each film was measured at 5 positions (center and four corners) and the mean thickness was calculated. This is essential to determine uniformity in the thickness of the film as this is directly related to the accuracy of

dose in the film. The results are shown in Table 2. Folding endurance [14] Folding endurance is determined by- repeated folding of the film at the same place till the film breaks. The number of times the film is folded without breaking is computed as the folding

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endurance value. The value is given in results and discussion. pH value [12] The pH value was determined by dissolving one oral film in 2 ml distilled water and measuring the pH of the obtained solution. The pH was measured by using pH paper. Differences were expected because various polymers were used as well as the addition of API. Disintegration testing [16] In vitro disintegration time was determined visually in a glass beaker of 25 ml distilled water with swirling every 10 seconds. The disintegration time is the time when the film starts to break or disintegrates. The results are shown in Table 2. Assay [15] The assay was performed to ensure the drug loading onto each film. This test was performed by dissolving a 6 cm2 area of film in 50 ml of pH 6.8 phosphate buffer with stirring. The resultant solution was filtered using a whatman filter paper, and the filtrate was diluted to 100 ml with the same buffer in a volumetric flask. Then 1 ml of the filtrate was further diluted to 10ml with buffer. This solution was analyzed using a spectrophotometer at 272 nm. Content uniformity The content uniformity test was used to ensure that every film contains the intended amount of drug substance with little variation among films

within a patch. Three pieces, each 6 cm2 (3 ×2 cm), were cut from the whole patch, and assayed for drug content. Same procedure was repeated for all the nine batches. The results are shown in Table 2. In vitro dissolution [12] The in vitro drug release study of film was carried out using a USP 23 type 2 rotating paddle dissolution test apparatus. 250ml of phosphate buffer (pH 6.8) was used, and maintained at 37±5°C while the basket was set at 50 rpm. A film sample of 6 cm2 (3 cm×2 cm) was fixed onto the specially designed SS disk with the help of cyanoacrylate adhesive. The disk was put at the bottom of the dissolution vessel so that the patch remained on the upper side of the disk. Five milliliters of samples were taken at a interval of 60 sec., and the same amount was replaced with fresh buffer. The withdrawn samples were filtered through whatman filter paper and then 1ml of the filtered sample was further diluted to 10ml of the same medium and analyzed using a spectrophotometer at a wavelength of 272 nm. The cumulative percentage release for different formulations was calculated. The relationship between time and percentage release were plotted. The results of invitro dissolution studies of all formulations were shown in Figure 1.

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In vitro release profile of caffeine anhydrous film using HPMC 2910,15cps,sodium alginate and kollicoat IR white

120 cumulative % release

100

150

200

Time(Seconds)

250

300

350

Figure 1: In vitro drug release profile of fast dissolving oral thin film of caffeine

FT-IR solid sample preparation Sample preparation for FTIR, Shimadzu, Japan 13mm KBr discs were prepared by grinding different sample (2mg) each with KBr (200mg) and then compressing the whole into discs using hydraulic press.Finally, the disk was inserted into the IR sample holder. Then run the spectrum. Sample run for FTIR, Nicolet iS5, USA For solid sample, 2mg of the sample was entered into the sampling accessories and got the spectrum.

RESULTS AND DISCUSSION

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In the present study, fast dissolving oral thin films of caffeine anhydrous were prepared successfully by using different polymer such as hydroxy propyl methyl cellulose(HPMC) 2910 15cps, kollicoat® IR white, and sodium alginate as film former using solvent casting method. Total nine formulations were prepared. Hydroxy propyl methyl cellulose 2910 (15cps) was used as film former in formulation F1-F3, whereas sodium alginate in the formulation F4-F6,and kollicoat® IR white was used in formulation F7F9, in various proportion. Mixture of water and

ethanol 96% (1:1) was used as solvent .The drying time was 3 hour. The films were evaluated for various properties including thickness, mass uniformity, pH, folding endurance, drug content uniformity, drug content, cumulative percent release as well as drug –excipients interaction. The mass of the films of all batches were between 50mg to 120mg. A very low standard deviation (1.5 to 2.5%) value indicates that the method used was reproducible and consistent. The folding endurance of the all the batches were F1(230), F2(250), F3(300), F4(280), F5(250), F6(230), F7(220), F8(240),F9(280).Batch with higher amount of HPMC (F3) scores higher folding endurance than the batch with lower HPMC (F1).Drug content of all the films were between 9.75mg to 10.06mg. The results are shown in Table 2.The results of weight and drug content uniformity test showed that the film was homogenous and drug was also uniformly distributed in the films. Films were having thickness adequate for handling and use. The disintegration times of the films were evaluated using phosphate buffer (pH6.8).

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The appearance of the film formulations F1 to F3 by visual inspection was totally homogenous, flexible and both side smooth surface,F4 to F6 was homogenous, flexible and both side smooth surface,F7-F9 was homogenous, flexible and one side smooth surface. FT-IR study: It appears from the FTIR Spectrum that the combination in formulation F1-F3 (caffeine anhydrous + HPMC) and formulation F7-F9 (caffeine anhydrous +kollicoat IR white ) showed better results when compared to the characteristic peak values of the pure drug (caffeine anhydrous ) while combination in formulation F4-F6 (caffeine anhydrous + sodium alginate + sodium starch glycolate) showed significant changes in the peak

Trinocular microscopic images Images of the caffeine anhydrous films were taken and evaluated. The surface of the film and distribution of polymer and drug within the films were examined. From the images below, it appears that more porous images were found from the kollicoat 速IR white containing film(F7) which may be due to the characteristic behavior of graft copolymer and reflected in its lowest disintegration time (12sec.). On the other hand, hydroxy propyl methyl cellulose containing film (F1) is also porous and showed less disintegration time (13sec) and smooth surface as well.

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Figure 2: FT-IR spectrum (FTIR , Shimadzu, Japan) of caffeine anhydrous , caffeine anhydrous +HPMC(F1-F3), caffeine anhydrous+ sodium alginate+ sodium starch glycolate(F4-F6).

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Figure 3: FT-IR spectrum (FTIR, ID5ATR, Nicolet iS5, USA) of caffeine anhydrous + kollicoat 速 IR white (F7-F9)

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HPMC containing caffeine film

Sodium alginate containing caffeine film

Kollicoat 速IR (white) containing caffeine film Figure 4: Trinocular microscopic images (Magnification:40) of HPMC containing caffeine anhydrous film, sodium 速 alginate containing caffeine anhydrous film, kollicoat IR containing caffeine anhydrous film.

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CONCLUSION Fast dissolving oral thin films are intended for the application in the oral cavity and they are an innovative and promising dosage form especially for use in pediatrics and geriatrics. The in vitro studies have shown that this is a potential drug delivery system for caffeine anhydrous with a considerably good physicochemical characteristics and release profile. Based on the above evaluations it is quite evident that the prepared fast dissolving oral thin film of caffeine anhydrous containing film forming agent hydroxyl propyl methyl cellulose (F1) is the best choice which provides faster release of drug within short time. Future studies are warranted to confirm these results including stability and DSC and other in vivo studies.

The authors are sincerely thankful to State university of Bangladesh, department of pharmacy, Dhaka, Bangladesh for providing us infrastructure facilities and moral support to carry out this research work.

10.

12.

13.

REFERENCES

11.

ACKNOWLEDGEMENT

Renuka Mishra and Avani Amin*,Formulation and Characterization of Rapidly Dissolving Films of Cetirizine hydrochloride using Pullulan as a Film Forming Agent, Ind J Pharm Edu Res, Jan-Mar, 2011/ Vol 45/ Issue 1, Liang AC, Chen LH. Fast Dissolving Intraoral Drug Delivery Systems. Exp. Opin. Ther. Patents 2001;11(6):981–6 Borsadia S, O'Halloran D, Osborne JL. Quick Dissolving Films-A Novel Approach to Drug Delivery. Drug Delivery Technology 2003; 3(3):63-66.

14. 15. 16.

Klancke J. Dissolution Testing of Orally Disintegrating Tablets. Dissolution Technologies 2003;10(2):6–8. Parakh SR, Gothoskar AV. Review of Mouth Dissolving Tablet Technologies. Pharm Tech 2003; 27(11):92–100 M.D. Nehal Siddiqui, Garima Garg and Pramod Kumar Sharma,A Short Review on “A Novel Approach in Oral Fast Dissolving Drug Delivery System and Their Patents”, Advances in Biological Research 5 (6): 291303, 2011 Galey, W.R., H.K. Lonsdale and S. Nacht, 1976. The in vitro permeability of skin and buccal mucosa to selected drugs and tritiated water. J. Investigative. M.D. Nehal Siddiqui, Garima Garg and Pramod Kumar Sharma,A Short Review on “A Novel Approach in Oral Fast Dissolving Drug Delivery System and Their Patents”, Advances in Biological Research 5 (6): 291303, 2011 Oral Thin Films,” in Orally Disintegrating Tablet and FilmTechnologies, 4th ed. (Technology Catalysts International, Falls Church, VA, 2006), pp: 18-31.. Sumitha Ch, Karuna Sree N, Divya B, Madhavi K, Vimal Kumar Varma M, Charbe NN. Int J Chem Research 2009; 1(2): 24-27. Kiran Kumar S*, Senthil Kumar S, Sundaramoorthy K, Shanmugam S and Vetrichelvan T, Research Journal of Pharmaceutical, Biological and Chemical Sciences ,Formulation and In-vitro evaluation of rizatriptan benzoate rapimelt tablets and oral thin films – A novel approach, 2011 ,Volume 2 Issue 2 ,108 Preparation and evaluation of fast dissolving oral thin film for pediatric use, heinrich heine universitate dusseldorf, M. Pharm dissertation (modified NA Nafee; NA Boraie; FA Ismail; LM Mortada. Acta Pharm 2003, 53, 199-212. VK Devi, S Saisivam, GR Maria, PU Deepti. Drug Dev. Ind. Pharm, 2003, 29, 495–503. R Patel, N Shardul, J Patel, A Baria. Arch Pharm Sci & Res, 2009, 1(2), 212 – 217. Shivani Singh*, Satyam Gangwar Garima Garg, Vipin Garg, P. K Sharma,Formulation and evaluation of rapidlydisintegrating film of Levocetrizin

*Corresponding Author:

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Farhana Sultana* State university of Bangladesh Email:farhana_sultana_2005@yahoo.com, Mobile no: +08801726020159 International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)

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Review Article Biological Sciences CLINICO-PATHOLOGICAL STUDY OF BENIGN & MALIGNANT LESIONS OF PROSTATE *1

Chandanwale Shirish, 2P. S. Jadhav,3S. C. Anwekar,4 H. Kumar,5A. C. Buch ,6U. S. Chaudhari

*1, 2, 3, 4, 5, 6

Department of Pathology, Padm. Dr. D. Y. Patil Medical College, Pimpri, Pune 411018 India. *Corresponding Author Email: shirishchandanwale@gmail.com

ABSTRACT Aims: Benign prostatic hyperplasia and adenocarcinoma are common diseases that account for considerable morbidity and mortality of ageing population. In cancer related deaths in men, the prostatic cancer is the second most common to lung cancer. Purpose of this study is to analyze various clinicopathological features in benign and malignant prostatic lesions, to correlate of benign and malignant prostatic lesions with serum prostate specific antigen (PSA) and to analyze the utility of Alcian Blue (AB) and Elastin Von Gieson (EVG) stain in evaluation of prostatic adenocarcinoma. Methods: All the prostatic specimens received in the histopathology department of our institute, over the period of 2 years, from June 2010 to July 2012 were analyzed. Results: 83%(n=83) were BPH , 17% (n=17) were adenocarcinoma. 2 cases showed changes of prostatic intraepithelial neoplasia(PIN) which were associated with adenocarcinoma. Out of 22 specimens in which PSA was available, 10 cases were adenocarcinoma. PSA was raised in all 10 cases. In our study wispy blue material was seen in 17.6% cases, while A.B. stain demonstrated mucin in 35.2% of cases. Conclusion: Benign prostatic hyperplasia was the commonest lesion. PSA level of >10ng/ml has high positive predictive value. EVG stain clearly highlighted the neoplastic acini and was useful in upgrading Gleason score in one case. Alcian Blue stain confirms the acidic mucinous nature of luminal secretions which are diagnostic of neoplastic acini, as against the neutral mucin seen in the non neoplastic acini.

KEY WORDS Prostate, Hyperplasia, Adenocarcinoma, PSA, Gleason score.

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INTRODUCTION The prostate gland is the largest accessory reproductive organ in male. The prostate is an exocrine gland and forms a significant component of seminal fluid. Benign prostatic hyperplasia and adenocarcinoma are common diseases that account for considerable morbidity and mortality of ageing population. In cancer related deaths in men, the prostatic cancer is the second most common to lung cancer.1 Prostate cancer is responsible for 3% of all deaths in men over age of 55 years.2 Incidence of prostatic cancer increases rapidly with age than any other

cancer. Thus, the numbers of prostate cancer cases are expected to increase, as average age of men is increasing.3 The prostatic biopsies, total prostatectomy specimens and prostatic chips obtained by transurethral resection of prostate (TURP) forms a significant volume of surgical pathology material received in histopathology department of our institute, accounting for 1.97% of all surgical specimens. Due to work done by various authors on prostate in last two decades, histological spectrum of benign prostatic hyperplasia has broadened and it has considerably expanded our knowledge

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www.ijpbs.com (or) www.ijpbsonline.com about pathology and biology of prostatic adenocarcinoma, special histological variants and most importantly about possible precursor lesions and prognostic factors. High-grade prostatic intraepithelial neoplasia (PIN) is considered as premalignant condition of prostatic adenocarcinoma. But some authors consider it as a separate entity. Establishing, or ruling out, the diagnosis of carcinoma of prostate has been a well known challenge for pathologists for many years and has become an even greater problem in recent times because of increased number of biopsy specimens and often limited amount of carcinoma, or questionable carcinoma, in such samples. There are many pitfalls associated with evaluation of prostatic adenocarcinoma, as there are many benign lesions, which mimic prostatic adenocarcinoma and requires considerable experience for correct diagnosis.Many investigators have studied various histomorphological features and tried to assess their usefulness in diagnosing or excluding prostatic adenocarcinoma. As most of the patients of prostatic adenocarcinoma present at old age, it is necessary to weigh the benefits of aggressive treatment against possible morbidity, while treating the patients of prostatic adenocarcinoma. For this reason accurate staging and grading of prostatic carcinoma is mandatory. Many investigators have proposed various methods for grading of prostatic adenocarcinoma. But TNM staging and Gleasonâ&#x20AC;&#x2122;s grading system.is accepted worldwide.4 Considering the magnitude of the problem and limited literature on prostatic lesions in india, the purpose of this study is to analyze various clinicopathological features in benign and malignant prostatic lesions, to correlate of benign and malignant prostatic lesions with serum prostate specific antigen (PSA) and to analyze the utility of Alcian blue

(AB) and Elastin Von Gieson (EVG) stain in evaluation of prostatic adenocarcinoma.

MATERIALS AND METHODS This is a prospective study of all the prostatic specimens received in the histopathology department of our institute, which is a tertiary public health care centre, during the period of 2 years, from June 2010 to July 2012.The prostatic material included prostatic biopsies, transurethral resection of prostate [TURP] chips and prostatectomy specimen. Our study included 100 prostatic specimens, which comprised of 22 prostatic biopsies, 77 TURP chips, and 1 prostatectomy specimens In cases of prostatic biopsies, all the tissue received was fixed and processed. In cases of TURP chips 3 to 4 cassettes were prepared in each case, which accommodated approximately 50% of total tissue, and weighed approximately 9 to 12gms. Specimens weighing <=12 grams were submitted entirely. In general, random chips were submitted; however, if some chips were firmer or had a yellow or orange-yellow appearance, they were preferentially submitted. If a carcinoma was detected in a TURP specimen that was not entirely submitted then all the remaining tissue was processed entirely irrespective of the amount. In case of prostatectomy specimens, multiple sections were made at the distance of 3 to 5mm. The slice in which tumor appears closest to the resection margin, is submitted entirely after dividing into adequate number of sections. All the tissues were fixed in 10% buffered formalin and paraffin processed. 3 to 5 micron sections were cut and stained with with routine Hematoxylin and Eosin (HE) stain. Alcian Blue (AB) and Elastin Von Geison (EVG) stains were done in cases of adenocarcinoma of prostate. All the slides were thoroughly evaluated for histological features. All prostatic lesions were

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www.ijpbs.com (or) www.ijpbsonline.com categorized into benign and malignant. In cases of benign enlargement of prostate, number of bits predominantly comprised of stromal component was counted and accordingly cases of benign prostatic hyperplasia were categorized as: Stromal predominant: >60% of the bits comprising of predominantly stromal component with presence of stromal nodules. Epithelial predominant: >60% of the bits comprising of predominantly epithelial component. Mixed glandular stromal: >40% but <60% bits comprising of predominantly stromal component. Inclusion criteria: Any prostatic tissue received in department of pathology for histopathological examination. Exclusion criteria: Non prostatic tissue.

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OBSERVATIONS AND RESULTS Out of 100 specimens of prostatic lesions received for histopathological examination, 83%(n=83) were BPH , 17% (n=17) were adenoccarcinoma. 2 cases showed changes of PIN which were associated with adenocarcinoma. Out of 100 cases, maximum specimens received were TURP chips 77 %, followed by prostatic biopsy material 22 % and prostatectomy specimens were only 1%.Most of the patients(94%) in our study presented with obstructive urinary tract symptoms viz. acute and chronic urinary retention, hesitancy, weak stream, terminal dribbling while 45% had urgency, increased frequency, dysuria and nocturia. Only 03 patients came with history of fever and 01 patient had the complaint of bone pain. Majority of the cases of both BPH and adenocarcinoma were in the age group of 61-70 years, accounting for 46.98% and 47.0% respectively. Approximately 6% cases of both BPH and carcinoma occurred in the fourth decade. Only 01 case of BPH was found in third

decade, while there was no case of carcinoma below the age of 40years. [Table 1] Majority of the cases of both BPH and adenocarcinoma were in the age group of 61-70 years, accounting for 46.98% and 47.0% respectively. Approximately 6% cases of both BPH and carcinoma occurred in the fourth decade. Only 01 case of BPH was found in third decade, while there was no case of carcinoma below the age of 40years. Around half of the cases (50.64 %) showed stromal predominance type of hyperplasia, followed by mixed pattern of hyperplasia (33.76%).Prostatic hyperplasia with epithelial predominance was seen in only 15.60% cases.Cystically dilated glands and Basal cell hyperplasia were the prominent features noted in our study. Basal cell hyperplasia was seen in 23 cases (27.71). Squamous metaplasia was seen in 08 cases (09.63%).Corpora amylacea was seen in 50 cases (60.24%).Infarcts were found in 4.81% cases [Table2] Chronic inflammatory cells of varying degree were found in 87.9% (n=77) cases in our study. More than half of the cases (n=44) showed only mild chronic inflammation, while moderate to severe chronic inflammation was seen in 34% (n=29) cases. Acute inflammation was found in 4.81%(n=4) cases, while only 02 cases of granulomatous prostatitis were found, out of which one case was xanthogranulomatous prostatitis, and one case was post TURP granuloma. No case of tuberculous inflammation was found. Only 03 cases of adenosis were found out of which 02 in TURP chips of BPH, while 01 was seen in adenocarcinoma. Adenosis was not detected in prostatic biopsies.Only 2 cases of PIN were noticed which were associated with adenocarcinoma. Both the cases of PIN showed tufted pattern were in high grade category. Out of 22 biopsy specimens in which PSA was available, 10 cases were denocarcinoma. Out of

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these 10 cases PSA was raised in all 10 cases. Out of 48 cases of TURP/ prostatectomy, 41 were BPH and 07 were carcinoma cases. Out of 07 carcinomas PSA was raised >10 ng/ml in 06 cases.Out of 41 BPH cases PSA was raised >10ng/ml in 06 cases and normal in 15 cases. [Table 3] Out of 22 prostatic biopsies, 10 cases had shown carcinoma i.e. 45.4%. In 12 cases i.e. 54.6%, biopsies did not show any evidence of malignancy. But in these cases benign etiology i.e; glandular proliferation associated with inflammation was noted and on follow up, TURP was done and these cases turned out to be BPH so they were considered as BPH as our final diagnosis and were included in our 83 cases of BPH out of 100 cases. Out of 17 cases, 10 cases (58.82%). of carcinoma were diagnosed on prostatic needle biopsies. In 06 cases, carcinoma was diagnosed on TURP chips, 02 of which represented incidental carcinoma in our study. Incidence of incidental carcinoma in our study is 02 out of total 77 TURP chips i.e.; 2.5%.

Age group (Years) 31-40

In our study pattern 3 was most common primary pattern (64.7%), while pattern 4 was most common secondary pattern. Tertiary pattern was not identified. We did not have pattern 1 and 2 in our study.[Table 4] 52.94% of prostatic carcinomas were of Gleason score 7, which fall under moderately poor category. In score 7, 07 cases were 3+4, and only 02 cases were 4+3 tumors. [Table5] In our study, 76.4 % cases showed infiltrating pattern. About half the cases (52.9) showed hyperchromasia, and 07 cases had prominent nucleolus. Amphophilic cytoplasm was seen in 29.4 % cases, while pale to clear cytoplasm was noted in (35.2%) cases. Wispy blue tinged mucinous secretions were noted in 17.6% carcinomas, perineural invasion was noted in 11.7% carcinomas. Mitoses were seen in 2 cases.[Table 6] In our study wispy blue material was seen in 17.6% cases, while A.B. stain demonstrated mucin in more number of cases i.e. 35.2%. No case of mucinous adenocarcinoma was detected in our study[Table7]

TABLE 1: show correlation of Age and Type of Lesion Benign Prostatic Hyperplasia Adenocarcinoma (BPH) No. Of Cases % No. Of Cases 01 1.22 0

% 0

7.22

5.7

51-60

27.72

17.7

61-70

46.98

47.0

71-80

13.25

17.3

81-90

3.61

11.7

91-100

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41-50

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TABLE 2: show secondary Changes in Benign Prostatic Hyperplasia (BPH) Features

No Of Cases (%)

A) Epithelial features Basal cell 23 (27.71) hyperplasia Squamous 08 (09.63) metaplasia Cystically dilated 29 (34.43) glands B) Intraluminal features

PSA (ng/ml) 0 to 4 4 to 10

Corpora amylacea

50 (60.24)

Crystalloids

Blue tinged mucin

C) Infarct

04 (4.81)

TABLE 03: Correlation of PSA Biopsy (n=22) TURP/Prostatectomy (n=48) Carcinoma(n=10) No e/o Carcinoma BPH (n=41) Carcinoma (12) (n=07). 0 02 0 15 02 06 01 20

>10

TABLE-4: Gleason Pattern Primary

Secondary

Tertiary Pattern

Pattern (N=17)

Pattern (n=17)

(n=00)

No. Of Cases (%)

00 (0)

00(0)

0(0)

00 (0)

00(0)

0(0)

11 (64.7)

05 (29.41)

0(0)

06 (35.3)

09 (53)

0(0)

03 (17.65)

0(0)

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Gleason Pattern

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TABLE- 5: Gleason Score No. Of Cases (n=17) %

Gleason Score

Differentiation

17.64

MODERATE

7* 8

09 03

52.94 17.64

MODERATELY POOR POOR

11.76

* In score 7, 07 cases were 3+4, and only 02 cases were 4+3 tumors.

TABLE 6: Ancillary Features Seen In Carcinoma: On H&E Staining Ancillary Features Seen InCarcinoma

% Of Cases

A) Infiltrating pattern

(76.4)

B) Nuclear features Nucleomegaly

13 (76.4)

Irregular nuclear membrane

07 (41.1)

Hyperchromasia

09 (52.9)

Prominent nucleoli

07 (41.1)

Marginated nucleoli

05 (29.4)

Mitotic figures

02 (11.7)

C) Cytoplasmic features Amphophilic cytoplasm

05 (29.4)

Clear cytoplasm

06 (35.2)

D) Intraluminal secretions Blue tinged mucin

03(17.6%)

Eosinophilic amorphous material

09 (52.9)

Eosinophilic crystalloids

02 (11.7)

Collagenous micronodules

E) Perineural invasion F) Retraction clefting

02 (11.7) 01(8.9) 02 (11.7)

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G) Associated PIN

0 5

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TABLE 7: Mucinous Differentiation of Adenocarcinoma of Prostate Type Of Mucinous H&E-Stain (n=03) After AB â&#x20AC;&#x201C; Pool Stain (n=06) No. Of Cases % No. Of Cases Intraluminal Mucin Extraluminal Mucin Pool

03 00

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DISCUSSION Benign prostatic hyperplasia is extremely common disorder in men over age 50.5 The prevalence of this disease is believed to be highly significant in most communities. In cancer related deaths in men, the prostatic cancer is the second only to lung cancer.1 Prostate cancer is responsible for 3% of all deaths in men over age of 55 years.2 In this prospective study, 100 prostatic specimens received over the period of two years in a tertiary public health care centre were analyzed. In our study BPH (83%) was the commonest lesion in specimens obtained at surgical pathology, followed by adenocarcinoma (17%). Out of 17 cases of adenocarcinoma two were associated with PIN. Our findings are in concordance with the study of Brawn et al.6 in which out of 2842 prostatic specimens 14% cases were of adenocarcinoma where as BPH was diagnosed in 79% of cases. Our findings are similar to his study. In about l/3rd of the cases, in which hard nodular prostate was palpated on digital rectal examination needle biopsy was performed. Transurethral resection of prostate (TURP) was done in patients having enlarged firm prostate with evidence of prostatic enlargement on sonography. Prostatectomy was done only in 01 patient. As compared to western literature, prostatectomy is done less frequently in India, and formed only 1% of total 100 prostatic specimens received in our institute. The possible explanation would be the fact that open prostatectomy is the treatment of choice for

17.64 00

06 00

% 35.29 00

early prostate cancer, and in India, because of lack of public awareness and proper screening methods; prostate cancer is often diagnosed at a late stage. TURP chips formed bulk of the specimens in our study, accounting for 77% of total specimens. (Table 2) This can be explained by the fact that TURP is the treatment of choice of BPH, as it is a simple procedure with fewer complications as compared to open prostatectomy. Also, BPH is much more common prostatic lesion than adenocarcinoma, and our study included 83 (83%) cases of BPH. In study done by Brawn et al.6, 2842 prostate specimens were included. Out of these, TURP chips formed 83.7% of total cases. Our findings were in accordance with his study. Most of the patients came with obstructive urinary tract symptoms, while irritative lower urinary tract symptoms were the next common mode of presentation.[Table 3] Obstructive symptoms included hesitancy, weak stream, terminal dribbling and acute or chronic retention of urine. Irritative symptoms included urgency, increased frequency, dysuria and nocturia. 94% of patients presented with obstructive symptoms in our study. In a study by Gaudin et al.7 in 1998; the most common presentation was retention of urine that is obstructive symptom. In a study of 50 cases by Herawi et al.8 in 2006; the major presenting signs and symptoms were urinary obstructive symptoms in more than 50% of patients. In another study by Wade et al.9 in 2001; obstructive symptoms were the most

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www.ijpbs.com (or) www.ijpbsonline.com common presentation. In a study done by Khan et al.10, in 2005, a total of 345 BPH patients were included in the study. Of these 270 (78.3%) patients presented with urinary retention. Our findings were in accordance to the above studies. In a study by Rijal et al,in 201111 the most common symptom associated with BPH with prostatitis was dysuria, however, there was a lot of overlap of the symptoms. In our study only 3 patients presented with fever. One of the patients came with bone pain. None of the patient came with the complaint of painful ejaculation. Adenocarcinoma and BPH most commonly presented in the age group 61-70 years (7th decade). The mean age of presentation for BPH and adenocarcinoma was 63.8 years and 66.07years respectively. [Table 4] Youngest patient of BPH was 38 years, and the oldest was 86 years. In adenocarcinoma the youngest patient was 48 years, and the oldest was 83 years. According to study done by Brawn et al, the average age of presentation for BPH and adenocarcinoma were 69 and 67 years respectively.12 In study done by Quian et al.13, mean age for carcinoma was 64.4 years (44 to 77years). According to study done by Di Silverio et al.14, mean age for BPH was 68.9years. In a study done by Kyungeun et al.15 mean age was 64.4 yrs (42-78yrs) in 148 cases. Our findings are similar to the above studies. There were 83 cases of BPH, which were diagnosed on 71 TURP chips and 12 on biopsy specimens (Table 5). According to study done by Vigilone et al.16 with the exception of stromal nodules, glandular proliferation and inflammation, histological findings on biopsy are not specific for either clinical or pathological BPH. In a study done by Shakya et al.17 all (106) specimens included in the series having BPH showed glandulostromal proliferation of which maximum cases showed predominantly stromal

pattern. These findings were similar with our study. In our study, out of the 77 cases, 39 (50.64%) cases showed predominantly stromal hyperplasia with presence of stromal nodules, 26 (33.76%) cases showed mixed glandular-stromal pattern of hyperplasia, while only 12 (15.6%) cases showed predominantly epithelial hyperplasia. (Table 5) Most of the cases showing stromal predominant hyperplasia came with obstructive urinary symptoms such as acute or chronic retention of urine. In one study, Shapiro et al.18 noted that stromal predominant nodules are more symptomatic than those of the other types. Our findings were in accordance with this study. TURP is usually done in the patients, which are clinically symptomatic. Thus it is obvious to find predominantly stromal hyperplasia in TURP specimens. BPH is classified in various different methods. The classification based on relative proportion of stromal and epithelial component is the simplest one, and was used in our study by applying the following criteria.4,18 1. Stromal predominant: >60% bits comprising of predominantly stromal component with presence of stromal nodules. 2. Epithelial predominant: >60% bits comprising of predominantly epithelial component. 3. Mixed glandular-stromal: >40% but <60% bits comprising of predominantly stromal component. In stromal nodules, capillary like blood vessels are surrounded by small, bland spindle cells with tapered nuclei and little cytoplasm along with marked edema with mild mononuclear infiltrate, giving them pale nodular appearance on scanner view. Predominant epithelial hyperplasia usually occurs within the transition zone. The glands are usually medium to large, sometimes cystic and may show architectural complexity and papillary infoldings The epithelium usually has a distinct

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www.ijpbs.com (or) www.ijpbsonline.com double layer of secretory and basal cells, but the basal cells are not always conspicuous. The cytoplasm is abundant and clear. Nuclei are uniform and nucleoli are inconspicuous. Mitosis is rare.19 Secondary changes associated with BPH were in the form of cystic dilation of glands, squamous metaplasia, basal cell hyperplasia, intraluminal secretions, inflammation, (Table 6) Similar changes were found in many studies.17,20-23 Adenosis and PIN are considered preneoplastic lesions of prostate although the evidence linking adenosis and adenocarcinoma is considerably weaker than the relating PIN and cancer. 5 Adenosis is usually seen in transition zone and PIN is usually seen in peripheral zone5,24 In our study, out of 100 specimens, adenosis was diagnosed in 3 cases out of which 2 were diagnosed in TURP specimens in association with BPH and one in prostatectomy specimen which was associated with carcinoma. Therefore the incidence of adenosis in carcinoma in our study was 1%. We did not encounter any case of adenosis in biopsy material. In two major studies done by Bostwick et al.25 and Brawn et al.[6] adenosis has been identified in 1.5 and 6.1% of transurethral resectates respectively. It is uncommon in needle biopsy specimens. Our findings are in accordance with these studies. Prostatic Intraepithelial Neoplasia (PIN): PIN is characterized by intraluminal proliferation of secretory epithelium that displays a spectrum of cytologic changes culminating in those that are indistinguishable from carcinoma. In our study, following prominent histological features helped us in diagnosing and grading PIN.4 1). Increased cellularity. 2] Pseudostratification 3] Intraluminal papillary formation 4] Bridging of lumen 5] Cribriform formation. PIN can be graded into a] Low grade PIN which is characterized by slight increase in cellularity, some variation in nuclear size,

focal hyperchromasia and appearance of small nucleoli. b] High graded PIN is characterized by definitive increase in cellularity, nuclear pseudostratification , hyperchromasia and presence of large nucleoli. Various normal structures, benign, metaplastic, reactive and neoplastic conditions can be confused with PIN. As PIN was considered preneoplastic lesion, various investigators studied incidence and tried to evaluate its preneoplastic role. In autopsy study, Sakr et al.26found that incidence of PIN precedes that of carcinoma by 10 years. The three major studies of PIN were done by McNeal and Bostwick.27, Kovi et al.28 and Troncoso et al.29 All these studies documented a greater incidence of PIN of some grade in carcinomatous glands than in benign glands. Also noted was a correlation between the quantity of PIN and multifocality of concurrent carcinoma. Like carcinoma, PIN was mainly identified in the peripheral zone.5,25 Davidson et al.30 found adenocarcinoma in 35% of subsequent biopsies from patients with previous diagnosis of PIN, as compared with 13% in control group without PIN. Incidence of PIN: Qian et al.13 encountered PIN in upto 16.5% of contemporary needle biopsies. He also noted PIN in 86% of prostatectomy specimens with diagnosed adenocarcinoma. Pacelli et al.31 found PIN in 2.4% of TURP chips. In our study, there were 2 cases of PIN which were found in carcinoma giving the incidence of 2%. In our study the finding was similar with Picelliet al.29 and Shakya et al.17 In our study incidence of PIN in carcinoma was low, as compared with the literature. This can be attributed to the fact that cases of carcinoma in our study were predominantly needle biopsies providing limited material. The other main reason could be the single core biopsies done in our institute as against multiple core biopsies in other studies.

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www.ijpbs.com (or) www.ijpbsonline.com Bostwick et al.32 studied various architectural patterns of PIN. Four common patterns of PIN were identified, usually with multiple patterns in each case: tufting (87%), micropapillary (85%), cribriform (32%), and flat (28%). In our study we found only tufted pattern. Both the cases of PIN were high grade. Prostate specific antigen (PSA): PSA is the best marker for prostatic carcinoma. Serum PSA is usually advised to the patients who come with obstructive urinary symptoms and in whom hard nodule is palpable on digital rectal examination. Normal serum PSA is 0-4ng/ml. There are few theoretical limitations to the use of this serum marker. A normal PSA level does not exclude diagnosis of carcinoma. About 33% of cancers were detected in men who had PSA levels within normal limits. Moreover false positive results are also common; since PSA levels are often elevated in men with common benign conditions such as BPH or acute prostatitis 2, 30 In our study, serum PSA was available in 70 cases. Out of these cases, 22 cases were biopsy specimens, 47 cases were TURP specimens, while 1 case was prostatectomy specimen. Out of these 22 biopsy specimens with available PSA levels, 10 were carcinoma, while 12 cases were negative for malignancy. PSA was raised in all 10 (100%) cases. 10 biopsies in clinically suspected cases with high PSA were negative for malignancy. Reasons may be that in our institute usually single core biopsies are done, so the chances of missing focus of malignancy are high. Out of 48 TURP/prostatectomy specimens with available PSA levels, 7 cases were carcinoma while 41 were BPH. Out of these 7 cases, PSA was raised in all 7 cases. Out of 41 BPH cases, 26 cases had high PSA. The reasons for this false positivity can be attributed to acute prostatitis, severe chronic inflammation, increasing age. In our study positive predictive

value and negative predictive value of PSA were calculated as follows: True positive (TP): Serum PSA >4ng/ml or the respective range with histological evidence of carcinoma. True negative (TN): Serum PSA within normal range (0-4ng/ml) and no histological evidence of carcinoma. False positive (FP): serum PSA >4ng/ml or the respective range but no histological evidence of carcinoma. False negative (FN): Serum PSA within normal range (0-4ng/ml) with histological evidence of carcinoma. Positive predictive value=TP/TP+FP x 100 Negative predictive value= TN/TN+FN x 100The positive predictive value for PSA level >10ng/ml was 58.33% in our study. The positive predictive value for PSA levels 4-10ng/ml was 10.3% in our study. According to studies done by Brawer et al. positive predictive value for serum PSA >10ng/ml was 60-70%, while it was 20-30% for serum PSA 4-l0ng/ml.33 Our findings were in accordance with Brawer et al.33 certain variations are may be due to difference in sample size in studies. We did not calcuated the negative predictive value because out of 100 cases in all the 17 cases diagnosed as carcinoma, the PSA value was not in the normal range. It was raised in all cases. Total number of biopsies out of 100 cases were 22. The indication of prostatic biopsy almost always is to rule out prostate carcinoma. In our study adenocarcinoma was detected in 10 cases (45.4%) in prostatic biopsies, which is considerably expected. 12 biopsies (54.6%) were negative for adenocarcinoma. Reasons could be single core biopsies done in our institute. So the chances of missing focus of malignancy were high. But in these cases benign etiology i.e; glandular proliferation associated with inflammation was

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www.ijpbs.com (or) www.ijpbsonline.com noted and on follow up, TURP was done and these cases turned out to be BPH so they were considered as BPH as our final diagnosis and were included in our 83 cases of BPH out of 100 cases. In a study done by Cheville et al.34 out of 1000 consecutive needle biopsies, almost 5% were reported as ‘Suspicious of carcinoma’. Most common factors responsible include dense inflammation obscuring morphology of glands and relatively scanty amount of tissue with few atypical glands. In our study no case was reported as ‘Suspicious of carcinoma’ may be due to difference in sample size. Establishing, or ruling out, the diagnosis of carcinoma of prostate has been a well known challenge for pathologists for many years and has become an even greater problem in recent times because of increased number of biopsy specimens and often limited amount of carcinoma, or questionable carcinoma, in such samples. In our study we have tried to analyze various histomorphological features such as various types of intraluminal secretions, morphological features of nucleoli, and tried to assess their usefulness in diagnosing or excluding prostatic adenocarcinoma. In our study there were 17 cases of adenocarcinoma, which accounts for (17 out of 100) 17% of total cases. Out of these 17 cases, 10 (58.82%) cases were detected on needle biopsies, 6 (35.29%) cases were detected on TURP chips and 1 (5.88%) cases were detected on prostatectomy. When prostatic tissue removed for clinically benign hyperplasia of the prostate and histological examinations reveals carcinoma, it is called incidental prostatic carcinoma. According to study by Mai et al35, there was a decrease in incidence of incidental carcinoma over the period of last 10 years. Specifically, there was a significant decrease in T1b carcinoma over time, while the incidence of Tla carcinomas remained unchanged with the

introduction of PSA screening. Furthermore, incidental carcinomas from the period 1997-1999 were associated with a higher proportion of cases of low-grade carcinoma. In our study, 06 out of 77 TURP chips showed presence of carcinoma out of which 2 cases (2.5%), represents incidental prostate cancer. Gleason grading: In our study we used Gleason grading system 36 which is most popular worldwide. Important features of various patterns can be summarized as follows: Pattern-1 Closely packed, single, separate, round, uniform glands with well defined margins. Pattern-2 Similar to pattern 1, but the glands are less uniform and less well defined margins. Pattern -3 The size of glands is variable. Both small and large glands and a papillary or cribriform pattern appear. Margins are poorly defined. Pattern-4 small fused glands; the glands may have papillary, Cribriform or solid pattern. Pattern-5 Few discernible glands; a comedo pattern is usually present. Tumor cells infiltrate the stroma as single cells or as ill defined cords. The presence of necrosis in any pattern automatically upgrades it to pattern 5. The Gleason score: It is the sum of the primary (most predominant) Gleason grade and the secondary (second most predominant) Gleason grade. Where no secondary Gleason grade exists, the primary Gleason grade is doubled to arrive at a Gleason score. Tertiary pattern is least common, which show 3 different Gleason patterns[36]. According to recent literature37,38 tertiary pattern should be reported in diagnosis only if it is Gleason pattern 4 or 5. It should be reported even if it is less than 5%, because presence of even small amount of high grade tumor affects the prognosis. The primary and secondary grades should be reported in parenthesis after the Gleason score,

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www.ijpbs.com (or) www.ijpbsonline.com i.e. Gleason score 7(3+4) or 7(4+3). When multiple needle biopsy specimens are submitted and they have differing Gleason scores, an overall (composite) Gleason score for the case should be clearly reported in a note.36,-38 Differentiation score: 39 a) Well-differentiated 2-4 b) Moderately differentiated 5-6 c) Moderately differentiated / poorly differentiated 7 d) Poorly differentiated.8-10 In pattern 7, 3+4 tumor has been found to have better prognosis than 4+3 tumor. This grading system has been used in classifying carcinoma in this study. The rarest pattern is pattern 1 and consists of tightly packed collection of small to medium sized acini, with relatively little variation in size and shape. Pattern 2 carcinoma shows greater separation of acini with limited infiltration of adjacent prostatic tissue.5 Distinction between Gleason grade 1 and 2 is often difficult but of no practical significance. Most of these tumors arise in the transition zone and are typically encountered in the transurethral resectates or in the prostatectomy specimens. Gleason pattern 2 and 1 were not encountered in our study, which could be explained as follows.In our study carcinoma was detected predominantly in needle biopsy specimens. According to Young et al.5 and Epstein et al.40 Pattern 2 and 1 are almost never diagnosed on needle biopsy. This is because the caliber of needle core does not generally enable all the edges of nodule to be seen. In addition lowgrade cancers are predominantly located anteriorly in the prostate within transition zone and tend to be small. There is poor reproducibility in its diagnosis even among urologic pathologic experts. Recently in studies by Rajal B. Shah41, current perspectives on the Gleason Grading were discussed. The

carcinomas, which constitute Gleason grade 3, show greater degree of gland separation, greater variation in the size and shape. An intermingling of neoplastic acini with the nonneoplastic ones is an important feature of Gleason grade 3. Infiltration is usually readily evident in grade 3 carcinoma in transurethral resectates and prostatectomy specimens. In some of our problematic or suspicious cases of adenocarcinoma, EVG stain highlighted the single layer of malignant cells and the clefting of neoplastic acini. The neoplastic acini have appreciable cytoplasm on high power evaluation with single layer cells, a feature much more in keeping with grade 3 adenocarcinoma. In Gleason scheme, grade 3C consists of generally rounded, smooth, circumscribed masses of glands with a cribriform or papillary architecture. There could be focal grade 4 carcinoma due to fusion of cribriform glands. If necrosis is present, grade becomes 5A. Gleason grade 4 carcinoma is characterized by growth as fused glands or chains of acini with little or no stroma within the aggregate. It is very important that Gleason grade 4 tumors should be recognized, since they are associated with a significant deterioration in prognosis compared to tumors that are Gleason grade 3 or lower. Strict criteria should be applied in recognizing the gland fusion that is definitional for Gleason grade 4 neoplasms. Most of these tumors are composed of cells with relatively scant eosinophilic or amphophilic cytoplasm, but some have abundant clear cytoplasm. The latter pattern has been referred to as hypernephroid , which is Gleason grade 4B. It has been linked to renal cell carcinoma, but the resemblance is only superficial. A cribriform pattern is common in grade 4 neoplasms and should not lead to confusion with the cribriform pattern that represents grade 3 neoplasms.

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3.68% ), and 9 (26 cases 10.65% ). In a study conducted in 2002 by Egevad L. et al.43 out of 305 cancers, 22% had a Gleason score of 4–5, 29% of 6, 18% of 7 and 32% of 8–10. The overall Gleason score in the study done by Falzarano et al44 was 6 in 21 (34%), 7 in 34 (55%) and 9 in 4 (6%) cases. In our study the Gleason score was 6 in 17.64% cases, 7 in 52.94% of cases, 8 in 17.64 % of cases and 9 in 11.76 % of cases. Our findings are almost similar with the studies of Babaian et al.42 and Falzarano et al.44. Reproducibility of Gleason score can be defined as the percentage of grades that remained unchanged on second grading and are a measure of intraobserver variability.According to study done by Harada et al.45 reproducibility of the same examiner for the same slides on two different examinations was 38%. Gleason’s own reproducibility was 80%.46 Cintra and Billis 47 had regraded 139 radical and transurethral resection prostatectomy specimens on 2 occasions and compared different grading systems. These authors found an intraobserver reproducibility of 63%.Ozdamar et al.44 had regarded 96 prostatic carcinomas and found an intraobserver reproducibility of 78%. In our study reproducibility of Gleason score by the same examiner on two different occasions was 94.11%. There was upgradation of Gleason score in 1 (5.57%) case. The upgradation was from pattern 3 to pattern 4. This was attributed to appreciation of Gleason patterns and associated ancillary features with the aid of special stain EVG. The frequency of various ancillary features that help in establishing diagnosis of adenocarcinoma of prostate in needle biopsy specimens as seen in studies done by Epstein et al.40, Thorson et al.48 and Varma et al.49 old are tabulated below

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Solid growth without specific features represents Gleason grade 5B. Sheets, cords and irregular aggregates of tumor cells may show focal lumens or cytoplasmic vacuoles. These formations are haphazardly arranged than in Gleason grade 4 carcinoma, and individual infiltrating tumor cells sometimes arranged in thin cords are also common. At times it is difficult to appreciate the tumor as adenocarcinoma except for focal luminal differentiation or the accompanying lower grade patterns that may be present. Some grade 5 carcinomas grow in large trabeculae, which may be difficult to distinguish from transitional cell carcinoma or as solid nests, which rarely have rosette-!ike structures. In Gleason grade 5 tumors, the nuclear morphology is highly variable. Small, dark and irregular nuclei with relatively inconspicuous nucleoli may be seen but in most cases, atypical nuclei with enlarged nucleoli are conspicuous. In general the nuclear pleomorphism of prostatic carcinoma is less striking than in most other carinomas.36-38,41 Gleason grade 3 was the most common in 11 cases (64.7%) primary pattern in our study. The most common secondary pattern was Gleason grade 4 in 9 cases (53%), while tertiary pattern was not identified. In our study, most common Gleason score was 7 in 9 (52.94%) cases. Out of these 9 cases, 7 (77.77%) cases had Gleason score 3+4. Only 2 (22.22) cases showed 4+3 pattern. Moderately differentiated tumors (Gleason score6) were 17.64 %, while 29.4% tumors were poorly differentiated (Gleason score8-10). In a study done by Babaian RJ42 et al. the overall Gleason scores in 244 cases were 4 (one case 0.4% ), 5 (63 cases 25.81% ), 6 (114 cases 46.72% ), 7 (151 cases 61.88%), 8 (9 cases

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FEATURES

STUDIES

A) Infiltrating pattern

Epstein et al -

Thorson et al 88%

Varma et al -

Our Study 76.4%

76.4% 52.9% 52.952.9 % 41.1%

B) Nuclear features 77% 96%

Nucleomegaly Hyperchromasia

30%

Prominent nucleoli

76%

64%

78%

Mitotic figures

11%

11.7%

29.4%

52%

17.6%

78%

86.7%

52.9% 11.7%

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C) Cytoplasmic features Amphophilic cytoplasm 39% 36% D) Intraluminal secretions Blue tinged mucin 34% 53%

Eosinophilic amorphous Material Crystalloids

25%

22%

40.6%

Collagenous micronodules

E) Perineural invasion

22%

11.7%

F) Retraction clefting

38.6%

8.9%

G) Associated PIN

13%

40%

In our study we have done a special stain EVG in all cases of carcinoma to evaluate its utility in diagnosis of carcinoma. In EVG stain, collagen is stained red; nuclei are stained blue/black, while rests of the tissues are stained yellow/green. We found that certain features were highlighted on EVG which is discussed in cases given below: Case-1 - In one case, few atypical acini were seen with abundant pale cytoplasm, irregular nuclei, and smudging artifact. Few of them showed few fibroblasts close to acini, which were mistaken for basal cells. Sometimes it is difficult to discern them on light microscopy. The EVG stain easily highlighted single layered glands with surrounding retraction clefting. Case-2- In another case of carcinoma diagnosed on needle biopsy, one focus showed 2-3 acini with abundant pale cytoplasm were seen

11.7%

adjacent to large benign gland, which appeared suspicious for carcinoma. However, lack of prominent nucleoli and other nuclear abnormality precluded the diagnosis of carcinoma. The EVG stain highlighted the two layers of acini and similar nuclear features as that of adjacent benign gland. Case-3 -In one of our cases Gleason pattern 3 tumor was identified. As no other grade was identified, Gleason grade given was 6. On EVG stain of the same slide, another bit showed few tumor cells with pale cytoplasm and dark nucleus infiltrating the stroma in cord like pattern i.e. pattern 5 tumor. When H&E stained slides were reviewed, featureless growth of poorly differentiated malignant cells (pattern 5) was noticed. On review Gleason score given was 3+5=8.The EVG stain in our study upgraded

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CONCLUSION Thus our study concluded that benign prostatic hyperplasia was the commonest lesion and most common pattern of inflammation associated with BPH was chronic inflammation. The commonest age group of presentation for both carcinoma and BPH was seventh decade and obstructive urinary symptoms were the most common mode of presentation. Incidence of PIN associated with carcinoma was less in our study. Both the cases with high grade PIN were associated with carcinoma. Strong correlation of PSA levels with adenocarcinoma was seen in our study. The range of 4-10ng/ml has a low positive predictive value. PSA level of >10ng/ml has high positive predictive value. The percentage of positivity of biopsy material was satisfactory. We concluded that in diagnosing prostatic adenocarcinoma, evaluating a constellation of architectural, cytoplasmic and nuclear features along with ancillary features is essential. Gleason grade 3 was the commonest pattern seen. Majority of cases were of Gleason score 7 i.e. of moderately poor differentiation.EVG stain clearly highlighted the neoplastic acini and was useful in upgrading Gleason score in one case. Alcian Blue stain confirms the acidic mucinous nature of luminal secretions which are diagnostic of neoplastic acini, as against the neutral mucin seen in the non neoplastic acini.

10.

11.

12. 13.

14.

15.

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www.ijpbs.com (or) www.ijpbsonline.com 46. Murphy GP, Whitmore WF. A report of the workshops on the current status of the histologic grading of prostate cancer. Cancer. 1979;44(4):1490-1494. 47. Cintra ML, Billis A. Histologic grading of prostatic adenocarcinoma: intraobserver reproducibility of the Mostofi, Gleason and Bocking grading systems. Int Urol Nephrol. 1991; 23(5):449â&#x20AC;&#x201C;454. 48. Thorson P, Vollmer Rt, Arcangeli C, Kectch DW, Humphrey PA. Minimal carcinoma in prostate needle

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|162-178 biopsy specimens: diagnostic features and radical prostatectomy follow up. Mod Pathol. 1998;11(6):543551. 49. Varma M, Amin MB. Morphologic criteria for the diagnosis of prostatic adenocarcinoma in needle biopsy specimens. A study of 250 consecutive cases in routine surgical pathology practice. Arch Pathol Lab Med. 2002;126(5):554-561.

*Corresponding Author:

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*Dr. Chandanwale Shirish S , Professor MD Department of Pathology, Padm. Dr. D. Y. Patil Medical College, Pimpri, Pune India. Email- shirishchandanwale@gmail.com, Mobile no. 09890144517, Fax 020- 27420439

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Research Article Pharmaceutical Sciences A NEW RP – HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF NAPROXEN AND SUMATRIPTAN IN TABLET DOSAGE FORM Y.SWAPNA*1, G.NAGARJUN REDDY1, CHANDRA K SEKHAR2 *1 Department of Pharmaceutical Analysis, KLR College of Pharmacy, Kakatiya University, Khammam, India 2 Quality Control, Bio-Leo Analytical Labs India Pvt Ltd, Prasanthinagar, Kukatpally, Hyderabad, India. *Corresponding Author Email: swapna.yampati@gmail.com

ABSTRACT A new simple fast accurate and economical reverse phase high performance liquid chromatographic method was developed for the determination of Naproxen Sodium [NPS] and Sumatriptan succinate [STS] in bulk and tablet dosage form. The separation was eluted on a Hypersil BDS C18 column (100 mm x 4.6 mm; 5µ) using a mobile phase mixture of phosphate buffer 6.5 and acetonitrile in a ratio of 60:40 v/v at a flow rate of 1.0ml/min. The detection was made at 235 nm. The retention times were 2.28min for [STS] and 3.14min for [NPS]. Calibration curve was linear over the concentration range of 125-750 µg/ml for [STS] and 20 to 120 µg/ml for [NPS]. The propose method was validated as per the ICH guidelines parameters like Linearity, specificity, precision, accuracy, robustness and ruggedness. The method was accurate, precise, specific and rapid found to be suitable for the quantitative analysis of the drug and dosage form.

KEY WORDS

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179

Method development and validation, Naproxen, Tablets, C18 column, RP-HPLC.

1. INTORDUCTION

2. MATERIAL AND METHODS

Naproxen sodium is 2-Napthaleneacetic acid, 6methoxy-α-methyl-, sodium salt and Sumatriptan succinate 1H-Indole-5methanesulfonamide,3-[2(dimethylamino)ethyl]N-methyl-, butanedioate (1:1)1. NPS and STS are official in U.S. Pharmacopoeia 1.but there is no official method for the combination. Both drugs in combination of tablet dosage form in the ratio of 85:500 mg STS: NPS. As per literature survey many methods have been reported the estimation of STS and NPS individually or in combination with some other drugs2-13. With this present proposed method both STS and NPS estimates simple and economical in tablet formulation.

2.1 Chromatographic Conditions Waters e 2695 separation module with high pressure liquid chromatographic instrument provided with a Hypersil BDS C18 column (100 mm x 4.6 mm ; 5µ) and 2489 UV-Visible detector, auto injector, auto sampler with Empower 2 software from Waters corporation, Milford USA was employed in the study. HPLC grade acetonitrile, water were purchased from Ranbaxy, India, and Potassium dihydrogen phosphate, ortho phosphoric acid AR grade were purchased from SD Fine Chem Mumbai, India were used in the study. 2.2 Drug Samples The reference samples were obtained from M/s. Bio-Leo Analytical Labs India Pvt Ltd, Hyderabad,

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www.ijpbs.com (or) www.ijpbsonline.com India, the formulation samples were purchased from local market. 2.3 Mobile phase A mixture of phosphate buffer pH 6.5 and acetonitrile in the ratio 60:40 v/v was filtered through 0.22µ membrane filter and was degassed. Mobile phase was used as diluent for preparing the working solution of the drug. The mobile phase was filtered and sonicated by using Bio-Technics india, Mumbai before use. The flow rate of the mobile phase was maintained at 1ml/min. The column temperature was maintained at 30ºC and the detection of the drug was carried out at 235nm. 2.4 Preparation of stock and working standard solution of Sumatriptan and Naproxen About 8.5mg of Sumatriptan succinate and 50 mg of Naproxen Sodium was weighed accurately on Sartorius semi micro balance model-CPA225D and transfers in to 50ml volumetric flask the solution was sonicated and the resulting solution was diluted with the mobile phase to get a working standard solution of 17 µg/ml STS AND 100 µg/ml NPS.

2.5 Sample Preparation Weighed accurately previously weighed and crushed 20 tablets powder equivalent to 8.5mgof STS and 50 mg of NPS transferred to 50ml volumetric flask make up to the mark with mobile phase sonicated and filtered through whatman filter paper. Further dilute 10 ml to 100 ml with mobile phase. 2.6 Linearity and Construction of Calibration Curve Linearity of the peak area response was determined by taking measurement at Six concentration prints (6 replicates at each point) working standard dilution of STS and NPS in the range of 4.25-25.5µg/ml and 25 to 150 µg/ml respectively. 20µl quantity of the dilution was injected each time in to the column. The drug elutes was monitored at 235 nm and the corresponding chromatograms were obtained. Form these chromatograms the mean peak areas were calculated and a plot of concentration over the peak area was constructed. This regression equation was later used to estimate the amount of STS and NPS in pharmaceutical dosage form. A representative chromatogram for the separation of STS and NPS presented in Figure 1.

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Figure 1: Chromatogram of STS (17mcg/ml) & NPS (100mcg/ml)

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www.ijpbs.com (or) www.ijpbsonline.com 2.7 System Suitability Testing The system suitability parameters such as Theoretical plates, tailing Factor and resolution

were performed to verify the system is adequate for the analysis to be performed. The results are performed in Table 1.

Table 1: System suitability parameters Parameters Sumatriptan Naproxen Tailing Factor 1.11 1.16 Theoritical plates 3197 4948 Resolution -5.13 LOD(µg/ml) 0.5407 2.9543 LOQ(µg/ml) 1.6387 8.9526

RESULTS AND DISCUSSION The present study was aimed at developing a simple economical precise and accurate HPLC method for the analysis of STS and NPS in bulk drug and in pharmaceutical dosage form. In order to achieve optimum separation of the component peaks, mixture of acetonitrile with water in different combinations were tested as mobile phase on a C18 stationary phase. A mixture of Phosphate buffer pH 6.5: acetonitrile in a proportion of 60:40 v/v was selected as the chromatographic peaks were well defined and resolved with no tailing. The retention time obtained for STS was 2.28±0.1 min and for NPS was 3.15±0.1min. Each of the samples was

injected Six times and the Sample retention times were observed in all cases. The peak areas of STS and NPS were reproducible as indicated by low coefficient of variation. A good linear relationship (r2 = 0.999) was observed for STS and (r2=0.999) was observed for NPS. The regression concentration and areas are given in Table 2. And the regression characters are given in Figure 2 & 3. When test solutions were analysed by the proposed method for finding out intra and interday variation, low co-efficient of variation was observed. The absence of additional peaks indicated non-interference of common excipients used in the tablets.

Table 2: Calibration data of the proposed method Sumatriptan

Naproxen

Mean Area

Conc (mcg/ml)

Mean Area

4.25

180282

2725561

8.5

356084

5454925

12.75

524727

8093281

701523

100

10742052

21.25

874029

125

13278587

25.5

1013140

150

15642564

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Conc (mcg/ml)

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Figure 2: Linearity of Sumatriptan

Linearity of Sumatriptan

y = 41025x + 3547. R² = 0.999

1000000 800000 600000 400000 200000 0 0

Figure 3: Linearity of Naproxen y = 10474x + 13518 R² = 0.999

Linearity of Naproxen 18000000 16000000 14000000 12000000 10000000 8000000 6000000 4000000 2000000 0 0

100

150

200

Table 3: Accuracy data (Triplicate values at 50,100 &150 percent levels) Amount added (µg)

Amount found (µg)

Percent Recovery

Percentage of mean recovery

8.5

8.487

99.85

17.0 25.5

16.95 25.31

99.72 99.27

49.85

99.69

100 150

99.69 149.88

99.69 99.92

Sumatriptan

Naproxen

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*Each value is a mean of three readings

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www.ijpbs.com (or) www.ijpbsonline.com High recovery values obtained from the different dosage form by the proposed method indicates the method is accurate. The drug content in tablets was quantified using the proposed analytical method are given in Table 3. The deliberate changes in the method have not much affected the peak tailing, Theoretical plates and the percent assay. This indicated the robustness of the method. The robustness study results are presented in Table 4. The lowest value of LOD and LOQ as obtained by the

183 Page

proposed method by calculated using 3.3xstdev/slope for LOD and 10xstdev/slpoe for LOQ. The standard solution of the drug was stable up to 24 hrs as the difference in percent assay during the above period is within limit system suitability parameters were studied with six replicates standard solution of the drug and the calculated parameters are within the acceptance criteria. The tailing factor and the number theoretical plate are in the acceptable limits.

Table 4: Robustness Study Chromatographic parameters Retention Area Height Theoretical plates time

Drug name

Variations

Sumatriptan

Buffer change ± 5% 55% v/v 60%v/v 65% v/v Change in flow rate at ±0.1ml/min 1.flow rate at 0.90ml/min 2.flow rate at 1.0ml/min 3.flow rate at 1.10ml/min Buffer change ± 5% 55% v/v 60%v/v 65% v/v

Naproxen

Change in flow rate at ±0.10ml/min 1.flow rate at 0.90ml/min 2.flow rate at 1.0ml/min 3.flow rate at 1.10ml/min

Asymmetry

2.021 2.284 2.588

689708 698156 697898

114346 114687 114598

3121 3189 3132

1.10 1.11 1.13

2.485 2.262 2.050

697890 696548 695650

114646 114536 114502

3176 3178 3156

1.12 1.11 1.10

2.845 2.275 3.487

10603245 10616250 10632446

1554657 1555163 1556324

4932 4948 4978

1.14 1.16 1.17

3.478 2.284 2.769

10632560 10615467 10605454

1556032 1555098 1555002

4933 4965 4945

1.18 1.16 1.15

The system precision was established by six replicate injections of the standard solution containing analytes of interest. The values of relative standard deviation were found within the limit, indicating the injection repeatability of the method. The method precision was established by carrying out the analyte six times using the proposed method. The relative

standard deviation was found within the limit, indicating the injection repeatability of the method. The results were presented in Table 5 & 6. The diluted preparations of marketed tablets were injected in duplicate and the results were calculated and presented in Table 7.

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Table 5: Precision Study Sumatriptan

Naproxen

S.No.

Area

2.283

684394

3.147

10631207

2.283

698154

3.147

10616250

2.283

692607

3.143

10621465

2.285

684783

3.145

10640231

2.284

698216

3.144

10617065

2.281

698547

3.146

10620674

Avg

2.283167

692783.5

3.145333

10624482

Stdev

0.001329

6723.022

0.001633

9376.966

%RSD

0.06 0.97 0.05 Table 6: Method Precision study Sumatriptan

184

Naproxen

S.No.

Area

2.282

697205

3.144

10596700

2.282

698542

3.143

10607200

2.282

698316

3.141

10578197

2.284

696982

3.142

10589457

2.283

698782

3.145

10611543

2.282

698128

3.141

10565387

avg

2.2825

697992.5

3.142667

10591414

stdev

0.000837

733.422

0.001633

17539.9

%RSD

0.04

0.11

0.05

0.17

Drug Sumatriptan Naproxen

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Table 7: Assay Results Amount present/tablet 84.58 mg 499.75 mg

The specificity of the HPLC method was determined by the complete separation of STS and NPS. When it was subjected to forced degradation as per ICH guidelines which was carried out with 0.1N HCL, 0.1N NaOH and Heat degradation. The method does not permit detection of degradation product for STS and NPS. Hence it can be concluded that the proposed HPLC method is evident very fast and economical compared to the literature available.

% of Assay 99.51 99.95

ACKNOWLEDGEMENT The authors are thankful to M/s Bio-Leo Analytical Labs India (P) Ltd, Hyderabad for providing standards of Sumatriptan and Naproxen and facilities. The authors are also thankful to Department of pharmaceutical Analysis, KLR College of pharmacy, Kakatiya University, Khammam, India for encouragement.

REFERENCES 1.

United States of Pharmacopoeia-35/National Formulary-30. 2012; Page No. 3997 & 4733

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Rajesh kumar nayak, sunil kumar swain, susanta kumar panda, kanchu charana sahu, debananda mishra, sanjay kumar, Method development and validation of Sumatriptan in bulk and pharmaceutical Dosage forms by UV spectrophotometric method, IJPBA, 2011, 2(4) : 1100-1105. R.P.Gondalia, AP Dharamsi, Spectrophotometric simultaneous estimation of naproxen sodium and Sumatriptan succinate in tablet dosage forms, Int J Pharm Biomed Sci, 2010, 1(2) : 24-26. Trinath.M, Saurabh K.Banerjee, Hari Hara Teja.D, C.G.Bonde, Development and validation of spectrophotometric method for simultaneous estimation of Sumatriptan and Naproxen sodium in tablet dosage form, Der Pharmacia Sinica, 2010, 1(1) : 36-41. Pakhuri Mehta, Chandra Shekhar Sharma, Deepak Nikam, M.S.Ranawat, Development and validation of related substances method by HPLC for analysis of naproxen in naproxen tablet formulations, IJPSDR, 2012,4(1) : 63-69. Akiful haque, S.Hasan Amrohi, Mahesh Nasare, Prashanth kumar.K, Pradeep kumar.T, Nivedita.G, Prakash V Diwan, Analytical method development and validation for the estimation of Naproxen using RPHPLC, IOSR Journal of Pharmacy, 2012, 2(4) : 19-24. Minakshi Pandey, Pooja Chawla, Shubhini A. saraf, Sumultaneous Estimation of Sumatriptan succinate, Naproxen and domperidone by reverse phase HPLC, Asian Journal of Pharmaceutical and Clinical Research, 2012, 5(3) : 176-178.

10.

11.

12.

13.

Sagar D.Solanki, Dr.Paresh U Patel, Development and validation of Reserved-phase HPLC method for simultaneous estimation of sumatriptan succinate and naproxen sodium in pharmaceutical dosage form, IJPPS, 2012, 4(1) : 276-278. Krishna babu A, Kiranmai K, Rojy George, Meenanaduri K, R.Ravinder reddy, pani kumar AD, Simultaneous estimation of Naproxen sodium and sumatriptan succinate in tablet dosage forms by RP-HPLC method, Journal of pharmacy research, 2011, 4(9) : 3021-3023. Rajesh Nuni, M.Sumithra, B.V.V.Sandeepkumar, Panjagala Satyanarayana, D.Narisimha Rao, Analytical method development and validation of sumatriptan and naproxen by RP-HPLC, IJAPR, 2011, 2(7) : 363-379. Kamepalli sujana, D.Gowri sankar, konda abbulu, simultaneous estimation of sumatriptan succinate and naproxen sodium by RP-HPLC in bulk and pharmaceutical dosage form, IJPSR, 2012, 3(9) : 34333437. Riddhi Gondalia, abhay dharamsi, HPTLC method for simultaneous determination of naproxen sodium and sumatriptan succinate in pharmaceutical dosage form, IJPSR, 2011, 2(1): 116-120. Palavai sripal reddy, shakil sait, gururaj vasudevmurthy, mathivanam natarajan, vure Prasad and s.jayapal reddy, impurities profiling method and degradation studies for sumatriptan succinate in sumatriptan succinate and naproxen tablets, JOCPR, 2012, 4(6) : 3263-3274.

*Corresponding Author:

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Y.SWAPNA Department of Pharmaceutical Analysis, Kakatiya University, Khammam, Andhra Pradesh, India, Email: swapna.yampati@gmail.com

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Research Article Biological Sciences BACTERIAL CONTAMINATION OF STETHOSCOPES USED BY HEALTH CARE WORKERS IN A TERTIARY CARE HOSPITAL IN NAVI MUMBAI Gurjeet Singh1*, A.D. Urhekar2, Anahita V. Hodiwala3, Neha Singh4, Bhaskar Das5 1*, 2,3,4,5

Department of Microbiology, MGM Medical College, Kamothe, Sector-18, Navi Mumbai-410209, Maharashtra, India. *Corresponding Author Email: gurjeetsingh360@gmail.com

ABSTRACT Background/Aim: Stethoscopes are widely used by doctors and medical students for clinical examination of patients. However they can act as vehicle for transfer of bacteria from one patient to another and contribute to hospital acquired infections. Hence, this study was undertaken with an to study the presence of bacteria and their species types on the stethoscopes of doctors and other HCW. Materials and Methods: Swabs were collected from stethoscopes by rubbing sterile, moist swabs on the entire surface of the diaphragm. In the second part of the study, the same stethoscope diaphragm was cleaned with alcohol rubs, allowed to act for 3-5 minutes and again second swab was collected from the same stethoscope. The swabs were directly inoculated on blood agar, MacConkey agar plates. Standard methods were followed for isolation and identification. All Staphylococcal strains were tested for MRSA. Antibiotic sensitivity was performed by Kirby-Bauer disc diffusion method as per CLSI guidelines to study antibiotic susceptibility pattern. Results: 100 swabs each were obtained before and after applying alcohol rub. 90 (90%) of stethoscopes showed bacterial contamination (Ď&#x2021; 2 = 163.64, d.f. =1, P<0.05) showed statistically significant different, isolates were Staphylococcus aureus (56%) followed Bacillus species (42%), Micrococci (24%), CoNS (04%), Pseudomonas aeruginosa (04%), Diptheroids (04%), Enterobacter species (2%) and Candida species (02%). Out of 56 isolates of the Staphylococcus aureus tested for MRSA, 20 (35.72%) were MRSA and 36 (64.28%) were MSSA (Ď&#x2021;2 = 6.36, d.f. =1, P<0.05) showed statistically significant different. Swabs collected after alcohol rub did not show any bacterial growth in any samples. Conclusion: Our study highlights the need to disinfect the stethoscopes diaphragms by simply applying the alcohol rubs to prevent any spread of bacteria from patient to patients.

KEY WORDS Hospital acquired infection, stethoscope, transmission, bacteria, health care workers.

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INTRODUCTION Infection transmission in the hospital environment (nosocomial infection) remains a significant hazard for hospitalized patients, and health-care workers are potential sources of these infections. Many pathogens can be transmitted through hands [1], which is a major reason that all health-care workers must wash their hands before and after examining each

patient [2]. Transmission of infections on contaminated medical devices is also possible and outbreaks of hospital-acquired infections have been linked to devices such as electronic thermometers, blood pressure cuffs, stethoscopes, latex gloves, masks, neckties, pens, badges and white coats [1,3-6]. Stethoscopes are commonly used to assess the health of patients and have been reported to be potential

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www.ijpbs.com (or) www.ijpbsonline.com vectors for nosocomial infections in various parts of the world [3,7-10]. Following contact with infected skin, pathogens can attach and establish themselves on the diaphragms of stethoscopes and subsequently be transferred to other patients if the stethoscope is not disinfected. [1113]

There are increasing reports of the risk of transmitting antibiotic resistant microorganisms from one patient to another on stethoscopes. [3, 14, 15, 16]

Swiping stethoscopes with alcohol pads is currently the gold standard for cleaning these instruments, but physicians do not consistently use alcohol pads for this purpose, as this requires an extra step of purchasing alcohol pads and their disposal. [17] There are increasing reports of the risk of transmitting antibiotic resistant microorganisms from one patient to another on stethoscopes. [18, 19, 20] These antibiotic-resistant organisms are capable of initiating severe infections in a hospital environment and could require contact isolation and aggressive treatment to prevent the spread of the organisms. [21] Examples of such antibiotic-resistant organisms are ceftazidime-resistant Klebsiella pneumoniae, vancomycin-resistant enterococci, methicillinresistant staphylococci, ciprofloxin-resistant Pseudomonas aeruginosa, gentamicin-resistant P. aeruginosa, and penicillin-resistant pneumococci. [22-26]

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MATERIALS AND METHODS The study was conducted in June 2012 to December 2012 at Department of Microbiology, MGM Medical College, Navi Mumbai. Samples that were taken from health care worker and tests were done at the Microbiology laboratory.

Laboratory testing was conducted within one hour of sample collection. Two swabs were taken-one before cleaning and second after cleaning with alcohol rub. The swabs taken from the stethoscope of health workers were inoculated directly onto blood agar and MacConkey agar and incubated at 37ÂşC for 24 hours. The colony morphology was recorded. Bacterial isolates were identified by standard methods. Antibiotic sensitivity test of isolated bacteria was done by Kirby-Bauer method as per CLSI guidelines [27]. Gram-positive isolates were tested for susceptibility against ampicillin/sulbactam, cefotaxime, linezolid, gentamicin, cloxacillin, roxithromycin, tetracycline, and ciprofloxacin and the Gramnegative isolates were tested for susceptibility against amikacin, gentamicin, ciprofloxacin, lomefloxacin, cefoperazone, ceftazidime, and csefuroxime.

RESULTS AND DISCUSSION This project was undertaken to study the presence of bacteria and their species types on the diaphragm of the stethoscopes of different health care workers groups-doctors working in the medicine and surgery department, MBBS internship students and nursing staff. Out of 100 stethoscopes studied, 90 (90%) of the total stethoscopes showed growth of bacteria. However 10 (10%) showed no growth on culture plate. The frequency of bacteria isolates was 100% for medicine, 88.89% for surgery, 85.71% MBBS internship students and 80% for nursing staff. Chi-square (Ď&#x2021;2) = 6.60, d.f. =3, P value >0.05, not significant (means there is no statistically significant difference in the number of stethoscopes showing bacterial growth from different groups) [Table No.1].

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Table 1: shows ward wise distribution. Ward Sample tested No growth Bacterial growth (%) Medicine 34 34 (100) Surgery 18 02 16 (88.89) MBBS Intern 28 04 24(85.71) Nursing 20 04 16(80) Total 100 10 90

In a study conducted by Chigozie J. et al (2010), of the 107 stethoscopes surveyed, 84 (79%) were contaminated with bacteria; 59 (81%) of the contaminated stethoscopes belonged to physicians and 25 (74%) were from other health workers. [16] In another study by Uneke CJ et al (2008), on stethoscopes of medical students, bacterial

contamination was found on 80% stethoscopes. [18]

In our study the maximum number of bacteria isolated were Staphylococcus aureus 56% followed by Bacillus species 42%, Micrococcus 24%, Coagulase Negative Staphylococci 4%, Pseudomonas aeruginosa 4%, Diphtheroids 4%, Enterobacter species 2% and Candida species 2% [Table no.2].

Table 2: shows all bacterial isolates from stethoscopes Total No. Sr. No. Isolated Bacteria % 56 1. Staphylococcus aureus (56%) 42 2. Bacillus species (42%) 24 3. Micrococcus (24%) 04 4. Coagulase Negative Staphylococci (4%) 04 5. Pseudomonas aeruginosa (2%) 04 6. Diptheroids (4%) 02 7. Enterobacter species (2%) 02 8. Candida species (2%)

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Total

In a study conducted by Chigozie J. et al (2010), of the 107 stethoscopes surveyed, isolates included Staphylococcus aureus (54%),

138

Pseudomonas aeruginosa (19%), Enterococcus faecalis (14%), and Escherichia coli (13%). [16] In another study conducted by Uneke CJ et al (2008), on the stethoscopes of medical students

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www.ijpbs.com (or) www.ijpbsonline.com in Nigeria, Staphylococcus aureus and Pseudomonas aeruginosa were major isolates. [18]

In our study the stethoscope without cleaning with alcohol rub showed 90% growth of bacteria and after cleaning with alcohol rub did not show

any type of growth. Chi-square (χ2) = 163.64, d.f. =1, P value <0.05, significant (means there is statistically significant difference with results of bacterial cultures before and after alcohol rub) [Table no.3].

Table 3: shows effectiveness of alcohol rub on the diaphragm of stethoscopes. Parameter Growth No growth Total Before applying alcohol rub 90 10 100 After applying alcohol rub 0 100 100 Total 90 110 200

In a study conducted by Chigozie J. et al (2010), contamination was significantly higher on stethoscopes cleaned with only water (100%) compared to those cleaned with alcohol (49%) (χ2 = 30.17, P<.05). Significantly fewer (9%) stethoscopes from health workers who washed their hands after seeing each patient were contaminated when compared with the instruments (86%) of those who did not practice hand washing (χ2 = 23.79, P < .05). [16] Another study conducted by Uneke CJ et al (2008), stethoscopes from students who cleaned them after use on each patient and from those who practised handwashing after contact with each patient had significantly lower

bacterial contamination (chi2 = 26.9; p < .05 and chi2=31.9, p < 0.05, respectively). [18] Ward wise distribution of bacterial isolates was from medicine ward Staphylococcus aureus (48%), Bacillus (16%), Micrococcus (16%), CoNS (8%), P. aeruginosa (8%) and Candida (4%). From surgery ward Bacillus (63.64%), Staphylococcus aureus (18.18%) and Micrococcus (18.18%). From MBBS internship students Staphylococcus aureus (45.45%), Bacillus species (22.73%), Micrococcus (18.18%), Diptheroids (9.09%) and Enterobacter species (4.55%). From nursing staff Staphylococcus aureus (36.36%), Bacillus species (45.45%), Micrococcus (18.18%) [Table no. 4].

Table 4: shows number and bacterial isolates.

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Bacteria isolated from Stethoscope

Doctors stethoscopes

No. (%) Nurses stethoscopes No. (%)

Total No.

20 (45.45) 10 (22.73) 08 (18.18)

08 (36.36) 10 (45.45) 04 (18.18)

56 42 24

04 (8) 0 0 02(4)

0 0 0 0

0 04(9.09) 02(4.55) 0

0 0 0 0

04 04 02 02

50 (100)

22 (100)

44 (100)

22 (100)

138

Medicine Ward

Surgery ward

MBBS Interns

Staph aureus Bacillus spp. Micrococcus

24 (48) 08 (16) 08 (16)

04 (18.18) 14 (63.64) 04 (18.18)

CoNS

04 (8)

P. aeruginosa Diptheroids Enterobacter spp. Candida spp. Total

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In this study (35.71%) the MRSA were isolated whereas MSSA were (64.29%). The MRSA isolated from medicine ward 4/20 (20%), surgery ward 4/20 (20%), MBBS intern 4/20 (20%) and nurses staff 8/20 (40%). Chi-square (Ď&#x2021;2) = 6.36,

d.f. =1, P<0.05, significant (means there is statistically significant difference of MRSA between doctors and nurses). Stethoscopes of doctors showed higher number of MRSA [Table no. 5].

Table 5: shows differentiation between MSSA and MRSA from total Staphylococci. Doctors stethoscopes Bacteria isolated from Stethoscope MRSA (out of 56 Staphylococcus aureus) MSSA (out of 56 Staphylococcus aureus) Staph aureus (Total)

Medicine Ward

Surgery ward

04 (16.67) 20 (83.33) 24 (100)

04 (100)

No. (%) MBBS Interns 04 (25) 12 (75) 16 (100)

0 04 (100)

Youngster I et al (2008), studied 43 stethoscopes belonging to senior physicians, residents, interns and medical students at the paediatric ward. Bacterial cultures and antibiotic sensitivity testing were carried out. All but six bacterial cultures were positive (85.7%). Staphylococcal species were the most common contaminants (47.5%). One case of methicillin-resistant Staphylococcus aureus was encountered. Gramnegative organisms were isolated in nine

Nurses stethoscopes No. (%) 08 (66.67) 04 (33.33) 12 (100)

Total No. (%)

20/56 (35.71) 36/56 (64.29) 56 (100)

different samples (21%) including one case of Acinetobacter baumannii in the neonatal intensive care unit. [26] In our study of antibiotic susceptibility testing of isolated bacteria, Gram positive cocci were 100% resistant to roxithromycin and cefotaxime, whereas linezolid and ciprofloxacin and vancomycin showed 100% sensitivity [Table No. 6].

Table 6: shows antimicrobial susceptibility of Gram positive bacteria from stethoscopes S. aureus Antibiotics Concentration n=56 (%) 30 mcg

56 (100)

Tetracycline

30 mcg

38 (67.86)

Gentamicin

10 mcg

45 (80.36)

Ciprofloxacin

5 mcg

56 (100)

Roxithromycin

30 mcg

Ampicillin/Sulbactam

20 mcg

36 (64.29)

Cefotaxime

30 mcg

Cloxacillin

1 mcg

36 (64.29)

Vancomycin

30 mcg

56 (100)

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Linezolid

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ceftazidime. However amikacin, pefloxacin was 100% sensitivity [Table No. 7].

Table 7: shows antimicrobial susceptibility of Gram negative bacteria from stethoscopes Antibiotics Concentration P. aeruginosa n=4 Enterobacter spp. n=2 (%) (%) Amikacin

30 mcg

04 100 01 (25)

02 (100) 02 (100)

Gentamicin

10 mcg

Cefuroxime

30 mcg

Lomefloxacin

30 mcg

Ofloxacin

5 mcg

Ciprofloxacin

5 mcg

Pefloxacin

5 mcg

2 (50) 04 (100)

02 (100) 02 (100) 02 (100)

Ceftazidime

30 mcg

Another study conducted by Uneke CJ et al (2008), isolates of Staphylococcus aureus showed the highest susceptibility to antibiotics, while the most effective antibiotics were Ciprofloxacin and Erythromycin. [18]



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CONCLUSION Our study revealed following findings Incidence of bacterial contamination of stethoscopes ranged from 80 – 100%.  Physician stethoscopes showed 100% bacterial contamination.  Most commonly organisms isolated S. aureus, Bacillus species, Micrococcus, CoNS, P. aeruginosa, Diptheroids, Enterobacter species and Candida species.  MRSA was isolated in doctors stethoscope higher than nurses stethoscope. (χ2 = 6.36, d.f. =1, P<0.05) statistically significant difference between doctors and nurses stethoscopes.



01 (50)

Antibiotic susceptibility testing of Gram positive cocci showed 100% resistance roxithromycin and cefotaxime, whereas linezolid and ciprofloxacin and vancomycin showed 100% sensitivity. Antibiotic susceptibility testing of Gram negative bacilli showed 100% resistance to cefuroxime, lomefloxacin, ofloxacin and ceftazidime. However amikacin, pefloxacin was 100% sensitivity. Antibiotic sensitivity patterns of bacterial isolates from different study groups was similar, indicates similar strain in the environment. Our study highlights the need to disinfect the stethoscopes diaphragms by simply applying the alcohol rubs to prevent any spread of bacteria from patient to patients. While it is impossible to destroy all bacteria or eliminate all infections in environment, many infections can be prevented with this

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www.ijpbs.com (or) www.ijpbsonline.com simple procedure of cleaning the stethoscopes. Alcohol rub is already in use for stethoscopes cleaning procedure which requires no added cost and no additional time.

ACKNOLEDGEMENTS Author wants to thank the Dean (MGM Medical College, Navi Mumbai), Medical Superintendent (MGM Hospital Kamothe / Kalamboli) for their permission, Head of Department of Medicine, Surgery and Paediatrics for their support and also thanks to all staff of Microbiology Department. LIMITATION OF THE STUDY  Other contaminating organisms like fungi, viruses, anaerobic bacteria were not studied.  Non alcohol based products – not studied.  It is not known whether the alcohol rub will damage stethoscope diaphragms. SCOPE FOR FURTHER RESEARCH Further research is necessary to determine whether the reduction of bacterial growth also corresponds to a reduction in clinically related diseases. The results of this study provide evidence that alcohol rub, decontaminated the stethoscopes. The research can also be made on comparing the use of stethoscope before and after hand wash.

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10.

11. 12.

13.

REFERENCES 1.

World Health Organization (2009) ‘WHO Guidelines for Hand Hygiene in Health Care’ *First Global Patient Safety Challenge Clean Care is Safer Care. Geneva: WHO 270p]. World Health Organization (2009) ‘Save lives clean your hands-Guide to Implementation’ *A Guide to the Implementation of the WHO Multimodal Hand Hygiene Improvement Strategy WHO/IER/PSP/2009.02. Geneva: WHO 48p].

14.

15.

Uneke CJ, Ogbonna A, Oyibo PG, Ekuma U (2008) ‘Bacteriological assessment of stethoscopes used by medical students in Nigeria: implications for nosocomial infection control’ *World Health Popul 10: 53-61]. Steinlechner C, Wilding G, Cumberland N (2002) ‘Microbes on ties: do they correlate with wound infection’ *Ann R Coll Surg Engl 84: 307-309]. Kotsans D, Scott C, Gillespie EE, Korman TM (2008) ‘What’s hanging around your neck? Pathogenic bacteria on identity badges and lanyards’ *Med J Aust 188: 5–8]. Treakle AM, Thom KA, Furuno JP, Strauss SM, Harris AD, Perencevich EN (2009) ‘Bacterial contamination of health care workers' white coats’ *Am J Infect Contr 37: 101-105]. Youngster I, Berkovitch M, Heyman E, Lazarovitch Z, Goldman M (2008) ‘The stethoscope as a vector of infectious diseases in the paediatric division’ *Acta Paediatr 97: 1253-1255]. Zuliani-Maluf ME, Maldonado AF, Bercial ME, Pedroso SA (2002) ‘Stethoscope: a friend or an enemy?’ *Sao Paulo Med J 120: 13-15]. Schroeder A, Schroeder MA, D'Amico F (2009) ‘What's growing on your stethoscope? (And what you can do about it)’ *J Fam Pract 58: 404-409]. Saloojee H, Steenhoff A (2001) ‘The health professional's role in preventing nosocomial infections’ *Postgrad Med J 77: 16-19]. Sanders S (2003) ‘The stethoscope and crossinfection’ *British J Gen Pract 53: 971-972]. Madar R, Novakova E, Baska T (2005) ‘The role of non-critical health-care tools in the transmission of nosocomial infections’ *Bratisl Lek Listy, 106: 348-350]. Whittington AM, Whitlow G, Hewson D, Thomas C, Brett SJ (2009) ‘Bacterial contamination of stethoscopes on the intensive care unit’ [Anaesthesia 64: 620-624]. Fenelon L, Holcroft L, Waters N (2009) ‘Contamination of stethoscopes with MRSA and current disinfection practices’ *J Hosp Infect 71: 376-378]. Merlin MA, Wong ML, Pryor PW, Rynn K, Marques-Baptista A, Perritt R, Stanescu CG, Fallon T (2009) ‘Prevalence of methicillinresistant Staphylococcus aureus on the

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16.

17.

18.

19.

20.

21.

stethoscopes of emergency medical services providers’ *Prehosp Emerg Care 13: 71-74]. Chigozie J. Uneke1, Annayo Ogbonna2, Patrick G. Oyibo3, Christian M. Onu4 ‘Bacterial contamination of stethoscopes used by health workers: public health implications’ *J Infect Dev Ctries 2010; 4(7):436-441]. Maryellen A. Schroeder, Frank D’Amico ‘Alcoholbased foam can do double duty, cleansing hands and stethoscope heads with a single scrub’ *ORIGINAL RESEARCH August 2009 • Vol. 58, No. 8: 404-409]. Uneke CJ, Ogbonna A, Oyibo PG, Ekuma U (2008) ‘Bacteriological assessment of stethoscopes used by medical students in Nigeria: implications for nosocomial infection control’ *World Health Popul 10: 53-61]. Fenelon L, Holcroft L, Waters N (2009) ‘Contamination of stethoscopes with MRSA and current disinfection practices’ *J Hosp Infect 71: 376-378]. Merlin MA, Wong ML, Pryor PW, Rynn K, Marques-Baptista A, Perritt R, Stanescu CG, Fallon T (2009) ‘Prevalence of methicillinresistant Staphylococcus aureus on the stethoscopes of emergency medical services providers’ *Prehosp Emerg Care 13: 71-74]. Gupta A, Della-Latta P, Todd B, San Gabriel P, Haas J, Wu F, Rubenstein D, Saiman L (2004). ‘Outbreak of extended-spectrum beta-lactamaseproducing Klebsiella pneumoniae in a neonatal intensive care unit linked to artificial nails’ *Infect Contr Hosp Epidemiol 25: 210-215].

22. Gastmeier P, Groneberg K, Weist K, Rüden H (2003) ‘A cluster of nosocomial Klebsiella pneumoniae bloodstream infections in a neonatal intensive care department: Identification of transmission and intervention’ [Am J Infect Contr 3: 424-430]. 23. Kerr JR, Martin H, Chadwick MV, Edwards A, Hodson ME, Geddes DM (2002) ‘Evidence against transmission of Pseudomonas aeruginosa by hands and stethoscopes in a cystic fibrosis unit’ *J Hosp Infect 50: 324-326]. 24. Lange CG, Morrissey AB, Donskey CJ (2000) ‘Point-prevalence of contamination of healthcare workers' stethoscopes with vancomycin-resistant enterococci at two teaching hospitals in Cleveland, Ohio’ *Infect Contr Hosp Epidemiol 21: 756]. 25. Parmar RC, Valvi CC, Sira P, Kamat JR (2004) ‘A prospective, randomised, double-blind study of comparative efficacy of immediate versus daily cleaning of stethoscope using 66% ethyl alcohol’ [Indian J Med Sci 58: 423-430]. 26. Youngster I, Berkovitch M, Heyman E, Lazarovitch Z, Goldman M (2008) ‘The stethoscope as a vector of infectious diseases in the paediatric division’ *Acta Paediatr 97: 1253-1255]. 27. CLSI. Performance Standards for Antimicrobial Susceptibility Testing; ‘Twenty-First Informational Supplement. CLSI document M100-S21’ *Wayne, PA: Clinical and Laboratory Standards Institute; 2011].

*Corresponding Author:

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193

Gurjeet Singh Department of Microbiology, MGM Medical College, Kamothe, Sector-18, Navi Mumbai-410209, Maharashtra, India.

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Research Article Pharmaceutical Sciences ROLE OF HYDROPHILIC POLYMER ON ETODOLAC FLOATING TABLETS G.Anusha*, Kiran.Ch1, Ajaykumar.Bâ&#x20AC; , Venkatesham.A1, P.Gayathri1, Pradeep.B1 S.V.S School of Pharmacy, Bheemaram, Hanmakonda, Warangal-506 001, Andhra Pradesh, India â&#x20AC; Project Coordinator, Richer Pharmaceuticals, Hyderabad. *Corresponding Author Email: anushareddy.gurrala1711@gmail.com

ABSTRACT The purpose of this research was to prepare floating matrix drug delivery system of Etodolac. Floating matrix tablets of Etodolac were developed to prolong gastric residence time. Rapid gastrointestinal transit could result in incomplete drug release from the drug delivery system above the absorption zone leading to diminished efficacy of the administered dose. Floating matrix tablets containing 100 mg Etodolac were developed using different hydrophillic combinations. The tablets were prepared by direct compression method, using polymers such as hydroxypropylmethylcellulose (HPMC K15, k4m), Carbomer 971p and other standard excipients. Sodium bicarbonate was incorporated as a gas-generating agent. The effects of sodium bicarbonate on drug release profile and floating properties were investigated. The formulation was optimized on the basis of acceptable tablet properties, floating lag time and total duration of floating and in vitro drug release. The resulting formulation produced tablets with optimum hardness, uniform thickness, consistent weight uniformity and low friability. The results of dissolution studies, floating lag time indicated that formulations F9 exhibited good and controlled drug release up to 18hrs as the polymer concentration increases the dissolution rate was decreased. Applying the linear regression analysis and model fitting showed the selected formulation F9 showed diffusion drug release mechanism followed first order kinetics.

KEY WORDS Etodolac; Floating tablets; HPMC, carbopol; Sustained release

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INTRODUCTION Etodolac belongs to a class of nonsteroidal antiinflammatory drugs (NSAIDs). Other members of this class include aspirin, ibuprofen (Motrin, Advil, Nuprin, etc.), naproxen (Aleve,Naprosyn), indomethacin (Indocin), nabumetone (Relafen) and numerous others. These drugs are used for the management of mild to moderate pain, fever, and inflammation. They work by reducing the levels of prostaglandins, which are chemicals that are responsible for pain and the fever and tenderness that occur with inflammation. Hydrophilic polymer matrix systems are widely used in oral controlled drug delivery because of

their flexibility to obtain a desirable drug release profile, cost effectiveness, and broad regulatory acceptance. The purpose of controlled release systems is to maintain drug concentration in the blood or in target tissues at a desired value as long as possible. In other words, they are able to exert a control on the drug release rate and duration. In recent years, considerable attention has been focused on hydrophilic polymers in the design of oral controlled drug delivery system because of their flexibility to obtain a desirable drug release profile, cost effectiveness and broad regulatory acceptance1.

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www.ijpbs.com (or) www.ijpbsonline.com The need for Gastroretantive dosage forms (GRDFs) has led to extensive efforts in both academic and industry towards the development of such drug delivery systems. Prolonging the gastric residence of a dosage form may be of therapeutic value. Amongst the methods available to achieve this, floating dosage forms show considerable promise2. Floating tablet of Etodolac will have the following advantages than other conventional dosage forms; 1. Improved drug absorption, because of increased GRT and more time spent by the dosage form at its absorption site 2. Delivery of drugs for local action in the stomach 3. Minimizing mucosal irritation by drugs, by drug releasing slowly at a controlled rate 4. Treatment of gastrointestinal disorders such as gastro-esophageal reflux 5. Ease of administration and better patient compliance Hence the present work has been proposed. The aim of this proposed work is to role of HPMC on floating tablets of Etodolac.

MATERIALS AND METHODS

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195

Etodolac gift sample from Bioleo analytical labs, prashanthinagar. HPMC K15, HPMC K4M, Carbopol940, lactose, NaHCO3, Magnesium sterate was obtained from Drugs India, Hyderabad. All reagents and solvents used were of analytical grade satisfying pharmacopoeial standards. The Compositions of formulations with different polymers are given in the following tables (1.6). Accurately weighed quantities of hydrophilic polymers, Lactose were taken in a mortar and mixed geometrically. To this mixture required quantity of Etodolac was added and mixed

slightly with pestle. This mixture was passed through 32# and later collected in a plastic bag and blended for 5 min. Later sufficient quantity of Magnesium Stearate was added and the final blend was again passed through 32#. Then they obtained blend was mixed thoroughly for 10 min and compressed into tablets with 8.7 mm round concave Punches and corresponding dies at a hardness of 5.5 kg/cm twelve station tablet punching machine. Here each tablet weight is kept constant for 250mg3.

EVALUATION PARAMETERS The prepared floating tablets were evaluated for Uniformity of weight using 20 tablets, Hardness, Friability, In Vitro buoyancy, Swelling behavior (Water uptake studies) and In Vitro dissolution studies. (1) Hardness: The hardness of ten tablets was measured using Hardness tester. Mean and standard deviation were computed and reported. It is expressed in kilopascal (kp) 5. (2) Friability: The friability of the tablets was determined using thermionic friabilator. It is expressed in percentage (percentage). 10 tablets were initially weighed and transferred into the friabilator. The friabilator was operated at 25 rpm for four minutes. After four minutes the tablets were weighed again. The % friability was then calculated using the formula5. %Friability = initial weight â&#x20AC;&#x201C; final weight x 100 Initial weight (3) Weight variation: Twenty tablets were selected at random and the average weight of the tablets was determined. The weight of individual tablets was compared with the average weight6. (4) Drug content uniformity:

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www.ijpbs.com (or) www.ijpbsonline.com Prepared tablets were accurately weight and finely powdered by pestle in a mortar. A weighed portion of each powder equivalent to 1 mg/ml of prepared tablet was transferred in to a volumetric flask and the drug was extracted with methanol as the solvent. The contents of the flask were sonicated for 10 min and diluted with 0.1 N HCl as the solvent. The samples were analyzed spectrophotometrically at 274 nm6. (5) In Vitro Buoyancy Studies The in vitro buoyancy was determined by floating lag time, per the method described by the tablets were placed in a 100-mL beaker containing 0.1N HCl. The time required for the tablet to rise to the surface and float was determined as floating lag time7. (6) Swelling study Swelling of hydrophilic polymer such as Hydroxy Propyl Methyl Cellulose greatly depends upon the contents of the stomach and the osmolarity of the medium. This eventually influences the release, slowing action and the residence time. For each formulation, one tablet was weighed and placed in a beaker containing 200 ml of 0.1 N Hcl. After each hour the tablet was removed from beaker and weighed again up to 8 hours. The percentage weight gain by the tablet was calculated by using the formula7. Swelling index (S.I) = {(Wt‐Wo)/WO} x 100 Where, S.I. = swelling index Wt = Weight of tablet at time t WO = Weight of tablet before immersion. (7) In Vitro Dissolution Studies The release rate of Etodolac from floating tablets (n = 3) was determined using United States Pharmacopeia (USP) 23. Dissolution Testing Apparatus 2 (paddle method). The dissolution test was performed using 900 mL of 0.1N HCl, at 37 ± 0.5°C and 50 rpm. A sample (5ml) of the solution was withdrawn from the dissolution apparatus hourly for 18 hours, and the samples were replaced with fresh dissolution medium.

The samples were filtered through a 0.45-μ membrane filter and diluted to a suitable concentration with 0.1N HCl. Absorbance of these solutions was measured at 274 nm using a Shimadzu UV-1601 UV/Vis double-beam spectrophotometer. Cumulative percentage drug release was calculated using an equation obtained from a standard curve. (8) Stability Study The selected batch (F9) was kept at 40° C with 50% RH and the samples were withdrawn at 30, 60 and 90 days for physical and in vitro evaluation of drug release. (9) Drug release Kinetics: Zero order release rate kinetics To study the zero order release kinetics the release rate data are fitted to the following equation F= K0t Here, F is the fraction of drug release K0 is the rate constant T is the release time First order model This model has also been used to describe absorption and /or elimination of some drug, the release of the drug which followed first order kinetic can be expressed by the equation LogC = logC0-Kt/2.303 Where, C0 is the initial concentration of drug K is the first order rate constant t is the time Higuchi release model To study the Higuchi release kinetics, the release rate data was fitted to the following equation F = KH.t 1/2 Where, F is the amount of drug release KH is the release rate constant t is the release time Korsmeyer and peppas model The release rate data were fitted to the following equation, Mt/M8 =KM.tn

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Where, Mt/M8 is the fraction of drug release KM is the release constant t is the release time

before proceeding to direct-compression. The physical parameters are recorded in Table 1.1. a Angle of repose: 26 to 30 indicating good, 3035 indicating passable. b Compressibility index: 19 to 23 indicating passable and 25-28 indicating poor. c Hausner ratio: 1.24 to 1.34 indicating passable and 1.35 to 1.39 indicating poor.

RESULTS AND DISCUSSION Flow Properties: Etodolac along with other excipients were evaluated for bulk density, tap density, angle of repose, compressibility and Hausner ratio,

Formulation F1 F2 F3 F4 F5 F6 F7 F8 F9

Table No 1.1 Preformulation parameters of Etodolac Bulk Density Tapped Carr’s Index Hausner’s Ratio 2 (gm/cm ) Density(gm/cm2) 0.390 0.500 22 1.28 0.378 0.510 25.88 1.35 0.387 0.496 22 1.28 0.364 0.506 28.06 1.39 0.393 0.510 22 1.32 0.416 0.516 19.37 1.24 0.374 0.514 27.23 1.37 0.388 0.526 26.23 1.36 0.396 0.511 25.50 1.29

Angle of Repose 28.1 30.9 27.2 31.3 32.6 34.6 28.3 26.7 29.4

0.1 mm. The hardness varied from 5.5  1.0 kg\cm2 found satisfactory. The friability test was passed. The percent content uniformity was 100  2 and therefore was satisfactory.

Evaluation Studies The important parameters in the production of tablets were evaluated and reported in Table 1.2. The weight variation of the tablets was within the range. The thickness varied from 4.1

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Table No 1.2 Evaluation studies of Etodolac Formulation

Average weight of tablets (mg)

Hardness (Kg/cm2)

Friability (%)

% Drug content

Buoyancy lag Time (sec)

Total floating time (hrs)

250

5.5

0.19

100.01

113

>20

F2 F3 F4 F5 F6 F7 F8 F9

249 248 248 250 252 251 250 250

5.2 5.5 5 5.2 5 5 5.2 5.5

0.14 0.21 0.15 0.12 0.21 0.40 0.13 0.20

100.31 101.31 101.20 99.77 100.52 98.21 99.58 100.97

129 174 150 165 140 96 109 105

>20 >20 >20 >20 >20 >20 >20 >22

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Swelling index The swelling studies were conducted on matrix tablets of Etodolac on the basis of weight. The

weight was taken after 12 hours. The data are recorded in Table 1.3. Swelling was uniform for all formulation.

Table No 1.3 Swelling index Formulation

Swelling index (%) Time (hr) 1

12.06

22.46

63.21

87.74

14.04

29.94

70.38

98.56

18.29

40.34

81.92

109.95

13.31

34.52

72.77

99.39

20.38

45.74

79.8

115.18

26.62

55.75

91.07

122.50

22.04

44.08

80.67

105.3

27.45

54.89

90.65

119.76

32.86

63.63

99.80

131.6

Kinetics Data The Higuchi equation suggests that the drug release by diffusion. Table No 1.4 Dissolution table of formulations in 0.1N HCL 1hr

2hr

4hr

6hr

8hr

10hr

12hr

14hr

16hr

32.36 26 23.3 33.51 30.05 25.62 36.6 24.07 21.57

42.57 38.14 33.52 45.46 42.95 37.75 50.08 35.63 34.29

55.86 51.62 47.2 59.91 55.09 49.9 67.42 52.78 43.53

68.58 65.5 54.7 80.9 72.23 64.73 89 66.07 53.75

78.21 75.21 61.64 96.7 87.84 78.98 96.31 80.9 60.1

84.95 78.8 76.5

95.9 87.07 77.25

94 85.72

96.51

97.86 88.61

98.28

90.73 66.65

95.93 74.74

82.83

91.88

18hr

98.44

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Time in hrs F1 F2 F3 F4 F5 F6 F7 F8 F9

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STABILITY STUDIES Table No.1.5: Stability Data of Formulation 9 at 30±2oc/65 ± 5%RH and 40  20C / 75  5% RH Hardness (kg/cm²)

Drug content (%)

Floating lag time ( s )

5.67 ± 0.14

99.76 ± 0.81

36.10 ± 0.36

At 30 ± 2°C 65 ± 5%RH

5.57 ± 0.10

99.06 ± 0.50

37.50 ± 0.2

At 40 ± 2°C 75 ± 5%RH

5.57 ± 0.10

98.80 ± 0.70

37.7 ± 0.3

At 30 ± 2°C 65 ± 5%RH

5.50 ± 0.05

98.14 ± 0.30

38.7 ± 0.3

At 40 ± 2°C 75 ± 5%RH

5.50 ± 0.05

97.21 ± 0.45

39.2 ± 0.4

Time

(Days) 0

Table No.1.6 Formulation table of Etodolac Ingredients Etodolac Lactose monohydrate HPMC k4m Carbomer 971p HPMC k15 NaHCO3 MS Talc Tablet weight

F1 100

F2 100

F3 100

F4 100

F5 100

F6 100

F7 100

F8 100

F9 100

102.5

82.5

77.5

102.5

82.5

77.5

102.5

82.5

77.5

25 --12.5 7.5 2.5 250

37.5 --12.5 7.5 2.5 250

50 --12.5 7.5 2.5 250

-25 -12.5 7.5 2.5 250

-37.5 -12.5 7.5 2.5 250

-50 -12.5 7.5 2.5 250

--25 12.5 7.5 2.5 250

--37.5 12.5 7.5 2.5 250

--50 12.5 7.5 2.5 250

FIGURES 120 % drug release

100

80 60 40 20 0

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Time(hrs)

Fig 1.1: Graphs of Dissolution Studies

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Zero order

R² = 0.944

120

% CDR

100 80 60 40 20 0 0

time in hours Fig No.1.2: Zero order kinetic graph

Log % CDR remaining

First order

R² = 0.488

2.5 2 1.5 1 0.5 0 0

time in hours

Fig No.1.3: First order kinetic graph

% CDR

Higuchi equation 120 100 80 60 40 20 0

R² = 0.991

SQRT

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Fig No.1.4.Higuchi equation graph

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Peppas Log % CDR

2.5

R² = 0.607

2 1.5 1 0.5 0 0

0.5

1.5

Log time Fig No.1.5:Peppas-korsemeyer equation graph 2.

CONCLUSION Drug release was delayed efficiently by hydrophilic polymers SR as a matrix forming excipient and was found to be governed by Higuchi model. For estimating food effects on application of floating oral drug delivery effects it is of great importance to study floating performance not only regarding floating duration but floating strength as well. All formulations remained float for a time interval of 18 h and showed an increased floating strength for samples with a higher polymer/drug ratio. As the polymer concentration increases lag floating time and release rate was retarded.

REFERENCES Gothi GD, Porish BN, Patel TD, Prajapati ST, Patel DM, Patel CN. Study of design and development of sustained release tablets of Etodolac. Journal Global Pharma Technol 2010; 2(2): 69-74.

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Rao BP, Kottan NA, Snehith VS, Ramesh C. Development of Gastro retentive drug delivery system of cephalexin by using factorial design. ARS Pharmaceutical 2009; 50:8-24. Rahman Z, Mushir A, Khar RK. Design and evaluation of bilayer floating tablets of captopril. Act a Pharma 2006; 56:49-57. Prashant Khemariya, Sanjay J. Ingale, Akhilesh K. Singhai, Mohit Bhargava, Gaurav Saraogi, Sanjay K. Singhai and Mayank Nagar, formulation and characterization of sustained Release gastroretentive drug delivery system of Ofloxacin, International Journal of Biopharmaceutics. 2010; 1(2): 85-91. Thakkar VT, Shah PA, Soni TG, and Parmar MY, Gohel MC, Gandhi TR. Fabrication and evaluation of levofloxacin hemihydrate floating tablets. Res Pharm Sci 2008; 3:1-8. Dave BS, Amin AF, Patel MM. Gastroretentive Drug Delivery System of Ranitidine Hydrochloride: Formulation and In Vitro Evaluation. AAPS Pharm Sci Tech 2004; 5: Article 34. J. Padmavathy,D. Saravanan, D. Rajesh, “Formulation And Evaluation Of Ofloxacin Floating Tablets Using Hpmc”, Int J Pharm Pharm Sci, Vol 3, Issue1, 170173.

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G.Anusha* S.V.S School of Pharmacy, Bheemaram, Hanmakonda.

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Review Article Pharmaceutical Sciences Clitoria ternatea (APARAJITA): A REVIEW OF THE ANTIOXIDANT, ANTIDIABETIC AND HEPATOPROTECTIVE POTENTIALS Manju Lata Zingare1, *, Prasanna Lata Zingare1, Ashish Ku Dubey2, Md. Aslam Ansari3 1

Department of Biotechnology, Govt. Digvijay Autonomous P.G. College, Rajnandgaon, C.G., 491441, India. 2 Department of Botany, Govt. Shaheed Bhagat Singh P.G. College, Jaora, Ratlam. M.P., 486001, India. 3 Department of Zoology, Govt. L.C.S. College, Ambagarh Chowki, Rajnandgaon, C.G., 491665, India. *Corresponding Author Email: mzingare@yahoo.co.in

ABSTRACT Clitoria ternatea commonly known as Butterfly pea is a medicinal plant belonging to the family Fabaceae. The plant is reported to be used in insect bites, skin diseases, asthma, burning sensation, ascites, inflammation, leucoderma, leprosy, hemicrania, amentia and pulmonary tuberculosis. It is commonly called “Shankpushpi” in the Sanskrit language where it is reported to be a good “Medhya” (brain tonic). The major phytoconstituents found in Clitoria ternatea are the pentacyclic triterpenoids such as taraxerol and taraxerone, ternatins, alkaloids, flavonoids, saponins, tannins, carbohydrates, proteins, resins and starch. Clitoria ternatea has been evaluated for its medicinal properties and shows promising effects as having antioxidant, antidiabetic and hepatoprotective activities.

KEY WORDS Antidiabetic, Antioxidant, Hepatoprotective, Clitoria ternatea, Ethanol extract, Flavonoids, Methanol extract, Phenolic compounds.

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INTRODUCTION Clitoria ternatea commonly known as Butterfly pea belonging to the family Fabaceae and subfamily Papilionaceae is a perennial leguminous twiner. Clitoria Linn. comprises 60 species distributed mostly within the tropical belt with a few species found in temperate areas. The mostly frequently reported species is Clitoria ternatea. The plant is mainly used as a forage as it is highly palatable for live-stock and it is well adapted to various climates [12]. Native to the island of Ternate in the Molluca archipelago, this species is now widely grown as ornamental, fodder or medicinal plant [17]. The plant originated from tropical Asia and later was distributed widely in South and Central America,

East and West Indies, China and India, where it has become naturalized [4]. Clitoria ternatea is commonly also called Clitoria, blue-pea, kordofan pea (Sudan), cunha (Brazil or pokindong (Philippines). This plant is known as Aparajit (Hindi), Aparajita (Bengali), and Kokkattan (Tamil) in Indian traditional medicine [43] . It has several synonyms in Ayurvedic scriptures like: Sanskrit names: Aparajita, Girikarnu, Asphota and Vishnukranta. English names: Butter-fly pea, Mazerion and Winged leaved Clitoria. Local names: Aparajita (Hin), Aparajita (Beng), Gorani (Guj), Gokarna (Mar) and Buzrula (Arabic).

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www.ijpbs.com (or) www.ijpbsonline.com The juice of flowers is reported to be used in insect bites and skin diseases [1]. The roots are useful in asthma, burning sensation, ascites, inflammation, leucoderma, leprosy, hemicrania, amentia, pulmonary tuberculosis, ophthalmology and reported as bitter, refrigerant, ophthalmic, laxative, diuretic, cathartic, aphrodisiac, tonic [36]. Consequently they are used in the treatment of a number of ailments including body-aches, infections, urinogenital disorders and as antihelmintic and antidote to animal stings. Seeds are cathartic and useful in visceralgia. They are considered safe for colic, dropsy and enlargement of abdominal viscera [32]. The root, stem and flower are recommended for the

Fig 1: A Clitoria ternatea twiner

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HISTORY From ancient times “Shankhpushpi” is known as reputed drug of Ayurveda and reported as a brain tonic, nervine tonic and laxative. It is considered as a “Medhya- Rasayana” in Ayurvedic texts. It comprises of entire herb with following botanicals viz. Convolvulus pluricaulis (Convolvulaceae), Evolvulus alsinoides (Convolvulaceae), Clitoria ternatea (Papilionaceae) and Conscora decusata (Gentianaceae). It is an Ayurvedic drug used for its action on the CNS (Central Nervous System), especially for boosting memory and improving intellect [52]. The flowers of the plant Clitoria

treatment of snakebite and scorpion sting in India [26].

PLANT DESCRIPTION Clitoria ternatea has twining fine stems, 0.5-3 m long. The leaves are pinnate, with 5-7 elliptic to lanceolate leaflets, 3-5 cm long and shortly pubescent underneath (Fig. 1). Flowers are solitary, deep blue to blue mauve; very short pedicellate and 4-5 cm long (Fig. 2). Pods are flat, linear, beaked, 6-12 cm long, 0.7-1.2 mm wide and slightly pubescent with upto 10 seeds. The seeds are olive, brown or black in colour, often mottled, 4.5-7 mm long and 3-4 mm wide [14].

Fig 2: A Clitoria ternatea flower

ternatea resemble a conch shell; therefore it is commonly called “Shankpushpi” in the Sanskrit language where it is reported to be a good “Medhya” (brain tonic) drug and, therefore, used in the treatment of “Masasika Roga” (mental illness) [10]. Extracts of this plant have been used as an ingredient in Medhya- Rasayana, are juvenating recipe used for treatment of neurological disorders [28]. PHYTOCONSTITUENTS Roots, seeds and leaves are the reported plant part used from ancient times. The major phytoconstituents found in Clitoria ternatea are

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the pentacyclic triterpenoids such as taraxerol and taraxerone [5, 6]. Phytochemical screening of the roots shows the presence of ternatins, alkaloids, flavonoids, saponins, tannins, carbohydrates, proteins, resins, starch, taraxerol and taraxerone [61]. A new simple, sensitive, selective and precise High Performance Thin Layer Chromatography method has been developed for the determination of taraxerol in Clitoria ternatea Linn. which was being performed on Thin Layer Chromatography aluminium plates [28]. A wide range of secondary metabolites including triterpenoids, flavonol glycosides, anthocyanins and steroids has been isolated from Clitoria ternatea Linn. [35]. Four kaempferol glycosides I II, III and IV were isolated from the leaves of Clitoria ternatea Linn. Kaempferol-3- glucoside (I), kaempferol-3 rutinoside (II) and kaempferol-3neohesperidoside (III) were identified by Ultra Violet, Protein Magnetic Resonance and Mass Spectrometry. (IV), C33H40O19, mp: 198, was characterized as Kaempferol-3-orhamnosyl glucoside from spectral data and was named Clitorin [33]. The seeds contain nucleoprotein with its aminoacid sequence similar to insulin, delphinidin3,3,5-triglucoside, essential amino-acids, pentosan, watersoluble mucilage, adenosine, an anthoxanthin glucoside, greenish yellow fixed oil,[21] a phenol glycoside, 3,5,7,4-tetrahydroxyflavone-3-rhamoglycoside, an alkaloid , ethyl Dgalactopyranoside, p-hydroxycinnamic acid polypeptide, a highly basic protein-finotin, a bitter acid resin, tannic acid, 6% ash and a toxic alkaloid [45]. According to Yoganarasimhan seeds contain g-sitosterol, Ă&#x;-sitosterol, and [57] hexacosanol and anthocyanin glucoside . It also contains anti-fungal proteins and has been shown to be homologous to plant defensins [42]. Another study demonstrated that minor

delphinidin glycosides, eight anthocyanins (ternatins C1, C2, C3, C4, C5 and D3 and preternatins A3 and C4 were isolated from the young Clitoria ternatea flowers [60]. Recent study showed that malonylated flavonol glycosides were isolated from the petals of Clitoria ternatea with different petal colors [26]. It was also reported that five new anthocyanins, ternatins A3, B3, B4, B2 and D2 were isolated from Clitoria ternatea flowers [60]. ANTIOXIDANT POTENTIAL OF Clitoria ternatea Antioxidants act as radical scavengers, inhibit lipid peroxidation and other free radicalmediated processes, and therefore they protect the human body from several diseases attributed to the reactions of radicals. Various phenolic antioxidants such as flavonoids, tannins, coumarins, xanthenes and, more recently, procyanidins have been shown to scavenge radicals in a dose-dependent manner and therefore are viewed as promising therapeutic drugs for free radical pathologies [49]. Phenolic compounds are a large and diverse group of phytochemicals, which includes many different families of aromatic secondary metabolites in plants [15]. They are known to exert various physiological effects in humans, such as inhibiting platelet aggregation [11], reducing the risk of coronary heart disease and cancer and preventing oxidative damage of lipid and lowdensity lipoprotein [34, 53, 56]. Phenolic compounds have strong in vitro and in vivo antioxidant activities associated with their ability to scavenge free radicals, break radical chain reactions and chelate metals [54]. Flavonoids and other classes of phenolic Compounds are important phytochemicals [20]. Flavonoids are very effective antioxidants [65] that constitute a large group of naturally occurring plant phenolic compounds including flavones,

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www.ijpbs.com (or) www.ijpbsonline.com flavonols, isoflavones, flavonones and chalcones. Flavonoids contain a characteristic C6–C3–C6 structure, with free hydroxyl groups attached to aromatic rings, and they inhibit lipid oxidation by scavenging free radicals or by other mechanisms such as singlet oxygen quenching, metal chelation, and lipoxygenase inhibition [65]. Many plant phenolics exhibiting antioxidant properties have been studied and proposed for protection against oxidation [41, 62]. Natural antioxidants occur in all parts of the plant (wood, bark, stems, pods, leaves, fruit, roots, flowers, pollen, and seeds) [46]. Flower is an important part of plant which contains a great variety of natural antioxidants, such as phenolic acids, flavonoids, anthocyanin and many other phenolic compounds [24, 68].

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Medicinal plants are considered as potential sources of antioxidant compounds. There is an increasing interest in the investigation of naturally occurring antioxidants from plants [39]. One of the plants that deserve attention is Clitoria ternatea. The ethanolic extract of Clitoria ternatea Linn. was evaluated for its in vitro antioxidant activities [43] by DPPH free radical method. DPPH (Diphenyl picryl hydrazine) is a free radical at room temperature which produces violet colour in ethanol. It is reduced in the presence of an antioxidant molecule, giving rise to uncoloured solution. Ascorbic acid was used as the standard drug for the determination of the antioxidant activity and the EC50 value of ascorbic acid was found to be 6.1 μg/ml. An increased EC50 value was observed (36.5μg/ml) for the plant extract when compared with standard drug ascorbic acid (6.1μg/ml). The extract exhibited potent antioxidant activity with an EC50 of 36.5μg/ml. The antioxidant properties of Clitoria ternatea has also being assayed by using the free radical

scavengers Feric reducing power assay (FRAP), super oxide dismutase (SOD), Di phenyl picryl hydrazyl (DPPH) and total poly phenols [29]. The study showed that methanolic extract showed good antioxidant activity than hexane and chloroform extracts. The antioxidant activities of the ethanol extract of Clitoria ternatea on acetaminophen (APAP) induced toxicity in rats suggest that the ethanol extract of Clitoria ternatea can prevent renal damage from APAP (Acetaminophen) induced nephrotoxicity in rats and it is likely to be mediated through active phytoconstituents and its antioxidant activities [50]. Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safely employed for a wide range of treatments [66]. Phytoconstituents like 1 Cycloprop[e]azulene,1a,2,3,5,6,7,7a,7boctahydro-1,1,4,7-tetramethyl-,[1aR(1aa,7a,7aa,7ba)] [Synonyms: Varidiflorene], Pterocarpin, 6H-Benzofuro[3,2-c][1]benzopyran, 6a,11a-dihydro-3,9-dimethoxy-, (6aR-cis)[Synonyms: Homopterocarpin], Isoparvifuran, Hexadecanoic acid, ethyl ester, Myo-Inositol, Propane, 1,1-diethoxy- were identified from ethanol extract of Clitoria ternatea by using a gas chromatograph-mass spectrograph (GC MS). The antioxidant studies revealed that the levels of renal SOD (superoxide dismutase), CAT (catalase), GSH (reduced glutathione) and GPx (glutathione peroxidase) in the APAP treated animals increased significantly along with a reduced MDA (malondialdehyde) content in ethanol extract of Clitoria ternatea treated groups [50]. The white flowered leaves had higher content of all the enzymic antioxidants analyzed than the blue flower [22]. The enzymatic antioxidant activity of Clitoria ternatea was analyzed by using goat liver slices, in both blue flowered leaf

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www.ijpbs.com (or) www.ijpbsonline.com and white flowered leaf of Clitoria ternatea and H2O2 was used as oxidant. The total phenolic compounds (TPC) and 1, 1diphenyl-2-picrylhydrazyl (DPPH) scavenging activity in the flowers and leaves of Clitoria ternatea has been analysed [47] and the presence of antioxidant activity in both leaves and flowers showed that Clitoria ternatea have the potential to be an alternative source of natural antioxidants. It is concluded that scavenging activity expressed by Clitoria ternatea flower is affected by the amount of total phenolic compound.

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Phytochemical analysis has revealed that the stem contains phytosterols, phenolic compound, flavonoids and carbohydrates [18]. Various in vitro models were applied to evaluate anti oxidant property of these extracts. In vitro studies included Free Radical Scavenging Capacity (RSC) on DPPH Radicals, Scavenging capacity for hydroxyl radicals, (by measuring the degradation of 2 - deoxyribose with OH radicals generated in Fenton reaction), scavenging capacity for super oxide radicals (NBT reduction assay, Nitro blue Tetrazolium assay) and Antioxidant using Î˛ Carotene linoleate model system (Î˛-CLAMS). The phytoconstituents responsible for antioxidant activity were isolated by preparative TLC method. The methanolic extract showed the maximum free radical scavenging capacity as compared to acetone extract. Comparative evaluation of in vitro antioxidant activity of root of blue and white flowered varieties of Clitoria ternatea [44] showed that methanol extracts of blue and white flowered varieties of Clitoria ternatea showed a very powerful antioxidant activity in DPPH radicalscavenging assay. Methanol extracts of Clitoria ternatea also showed significant reductive ability

as well as hydroxyl radical scavenging activity. Methanol extract of white flowered variety of Clitoria ternatea showed more significant antioxidant activity as compared to blue flowered variety of Clitoria ternatea. The phenolic compounds and antioxidant capacities of free and bound phenolics from 12 available Thai edible flowers which have long been consumed as vegetable and used as ingredients in cooking, has been investigated [23], Clitoria ternatea was one of them. Major phenolic acids identified in these analyses were gallic acid, ferulic acid and sinapic acid, while predominant flavonoids were quercetin and rutin. The soluble as well as bound fractions of edible flowers are rich sources of phenolic compounds with antioxidant, DPPH radicalscavenging activity and reducing power. DPPH radical scavenging capacity of bound phenolic fraction was found to be 17.6% in Clitoria ternatea, this suggests that screening edible flowers as potential sources of bioactive components with high antioxidant properties may be of interest to consumers and public health workers. A polyherbal formulation (Rheumatone) made using five medicinal plants namely Clitoria ternatea, Sida cordifolia, Cleodendron serratum, Bacopa monnieri, Cardiospermum Halicacabum, does not exhibit any side effects and it has the enzymatic antioxidant activity [55]. There was a significant reduction in the levels of Super oxide dismutase (SOD), Catalase, Peroxidase and Glutathione peroxidase (GPx) in the liver and kidney of adjuvant induced arthritic rats and there was an elevated level of Super oxide dismutase, Catalase, Peroxidase noted in the Liver and Kidney of rats that were treated with the polyherbal formulation Rheumatone compared with the toxic rats. There was a

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www.ijpbs.com (or) www.ijpbsonline.com signiciant increase of GPx in Liver and Kidney of rats treated with Rheumatone compared with Group II rats treated with Freundâ&#x20AC;&#x2122;s Complete Adjuvant (FCA).

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ANTIDIABETIC POTENTIAL OF Clitoria ternatea Diabetes mellitus is a syndrome characterized by chronic hyperglycemia and disturbances of carbohydrate, fat and protein metabolism associated with absolute or relative deficiency in insulin secretion or insulin action [19]. Diabetes mellitus is also associated with an increased risk for developing premature atherosclerosis due to independent risk factors such as [51] hypertriglyceridemia and hypertension . Insulin therapy and oral hypoglycemic agents offer effective glycemic control; yet, their shortcomings limit their usage [2]. The world health organization has also recommended the evaluation of the effectiveness of plants in conditions where we lack safe modern drugs [64]. Phytochemicals isolated from plant sources are used for the prevention and treatment of cancer, heart disease, diabetes mellitus and high blood pressure [63]. Plants are reputed in the indigenous systems of medicine for the treatment of various diseases [3], the available literature shows that there are more than 800 plant species showing hypoglycemic activity [30] and Clitoria ternatea is one of them. Chronic administration of plant extracts (100mg/kg) for 14 days reduces the blood glucose level of the diabetes induced animals (Wistar Albino rats) as compared to diabetic control group [13]. There was significant decrease in the blood glucose level in the 7th and 14th days of the diabetes induction, showing antidiabetic effect. The effect was comparable to that of standard antidiabetic drug Glibenclamide. Hyperglycemia was induced by intra peritoneal injection of freshly prepared aqueous solution of

alloxan monohydrate. Extensive damage to the islets of langerhans and reduced dimensions of islets were found in control animals. Restoration of normal cellular populatation and size of islets with hyperplasia were seen in extract treated groups. The partial restoration of normal cellular population and enlarged size of Î˛-cells with hyperplasia were indicative of the antidiabetic potential of the plant. Aqueous extracts of Clitoria ternatea plant showed antihyperglycemic activity in streptozotocin treated rats and this effect is because of increase in glucose uptake and glycogen deposition in isolated rat hemi diaphragm. Clitoria ternatea leaf and flower extracts exhibit antihyperglycaemic effect in rats with alloxaninduced diabetes mellitus [10]. The effect of orally administered aqueous extracts (400 mg/kg body weight) of Clitoria ternatea leaves and flowers on serum glucose, glycosylated hemoglobin, and insulin were examined in control and extracttreated diabetic rats. The aqueous extracts of Clitoria ternatea leaves and flowers significantly reduced serum glucose, glycosylated hemoglobin and the activities of gluconeogenic enzyme, glucose-6- phosphatase, but increased serum insulin, liver and skeletal muscle glycogen and the activity of the glycolytic enzyme, glucokinase. For all the biochemical tests performed, the leaf extract-treated rat showed essentially the same profile as those treated with the flower extract. The alcoholic root extract of Clitoria ternatea has shown significant gross impact in preventing the possible complications related to brain hippocampal area CA3 and pancreatic tissue in juvenile diabetic rat experimental models [31]. These benefits could be due to interference of number of chemical compounds present in this extract. Encephalopathy is a major complication in juvenile diabetes mellitus which cripples the

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www.ijpbs.com (or) www.ijpbsonline.com potential physiomorphological growth and development in early childhood. It is very essential to diagnose and initiate the treatment at the earliest to prevent the possible complications. The ancient medical science Ayurveda mentions number of remedies to treat cognitive dysfunctions, the herbal root of Clitoria ternatea plant is one among them.

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Clitoria ternatea leaf extract shows the synergetic effect along with Trichosanthes dioica leaf extract on the Streptozotocin-induced diabetic rats [25]. The ethanolic extracts of Trichosanthes dioica leaf and Clitoria ternatea leaf exhibited higher degree of antihyperglycaemic activity. With regard to the mechanisms, it cannot be excluded that Trichosanthes dioica leaf extract and Clitoria ternatea leaf extract may contain some biomolecules that may synthesize the insulin receptor to insulin or stimulate the beta cells of islets of Langerhans to release insulin which may finally lead to improvement of carbohydrate metabolizing enzymes towards the establishment of normal glucose levels. Significant and higher degree of antihyperglycaemic efficacy was achieved with combination (200 mg/kg of Trichosanthes dioica leaf + 200 mg/kg of Clitoria ternatea leaf) when compared to the extent of efficacy that was obtained with 400 mg/kg dose of individual plant extracts of Trichosanthes dioica leaf and Clitoria ternatea leaf. HEPATOPROTECTIVE POTENTIAL OF Clitoria ternatea Despite remarkable advances in modern medicine, hepatic disease remains a worldwide health problem, thus the search for new medicines is still ongoing. Hepatic cells participate in a variety of metabolic activities; therefore the development of liver protective

agents is of paramount importance in the protection from liver damage. The literature has constantly shown that hepatoprotective effects are associated with plant extracts rich in antioxidants [16, 38, 7]. Many compounds and extracts from plants have thus been evaluated for hepatoprotective and antioxidant effects against chemically-induced liver damage [8, 67, 9]. Many studies have been done on the hepatoprotective activity of Clitoria ternatea. Ethanolic extract of leaves of Clitoria ternatea (EECT, 200 and 400 mg/kg) was evaluated for prophylactic and therapeutic hepatoprotective activity against carbon tetrachloride induced hepatic damage [58]. Silymarin (100 mg/kg) was used as standard drug. Hepatoprotective effect of EECT was evident in prophylactic and therapeutic groups at doses of 200 and 400 mg/kg. Histopathology of liver ascertained the effect of EECT and carbon tetrachloride on cytoarchitecture of the liver. The liver section of normal control animals indicated the presence of normal hepatic parenchyma, whereas administration of carbon tetrachloride in animals showed severe centrilobular necrosis, fatty changes, vacuolization and ballooning degeneration indicating severe damage of liver cytoarchitecture. The EECT 200 mg/kg in both prophylactic and therapeutic studies showed recovery and protection from hepatocyte degeneration, centrilobular necrosis, fatty infiltration, whereas EECT 400 mg/kg showed mild to normal cytoarchitecture that indicated the dose dependent hepatoprotection of EECT. The silymarin treated animals showed slightly altered hepatic parenchyma and uniform spread sheets of hepatocytes which indicated functional liver, on account of regenerative activity. The possible prophylactic and therapeutic hepatoprotective effect of Clitoria ternatea leaves was attributed due to the presence of

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www.ijpbs.com (or) www.ijpbsonline.com flavonoids which contributed to its antioxidant property. Methanolic extracts of blue and white flowered varieties of Clitoria ternatea have potent hepatoprotective action against carbon tetrachloride induced hepatic damage in rats [44]. Methanol extract of white flowered variety (MEWFV) effectively control SGOT (serum glutamate oxaloacetate transaminase), SGPT (serum glutamate pyruvate transaminase) and ALP (serum alkaline phosphatase) as compared to methanol extract of blue flowered variety (MEBFV). MEWFV of Clitoria ternatea showed more significant hepatoprotective activity as compared to MEBFV of Clitoria ternatea . The possible mechanism of this activity was due to free radical-scavenging and antioxidant activity, which may be due to the presence of phenolic compounds in the extracts.

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The hepatoprotective effect against paracetamol-induced liver toxicity in mice of ME (Methanol Extract) of Clitoria ternatea leaf was studied [4] by monitoring the levels of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and billirubin along with histopathological analysis. The mice treated with the ME of Clitoria ternatea leaf (200 mg/kg) showed a significant decrease in ALT, AST, and bilirubin levels, which were all elevated in the paracetamol group; this confirmed the hepatoprotective effect of Clitoria ternatea leaf extract against the model hepatotoxicant paracetamol. The hepatoprotective action was likely related to its potent antioxidative activity. A polyherbal formulation named “Ayush-Liv.04” consisting of Clitoria ternatea leaves 20% as one of its constituents was evaluated for its hepatoprotective activity against ethanol and CCl4 induced liver damage in rats [37]. The

activities of liver marker enzymes in serum namely AST (Aspartate aminotransferase), ALT (Alanine aminotransferase), ALP (Serum alkaline Phosphatase) , ACP( Serum acid Phosphatase) and serum bilirubin level (total) were increased in toxic group animals. But the activities of these enzymes were significantly lowered in posttreated group of rats. This suggests antihepatotoxicity of “Ayush-Liv.04”.

CONCLUSION The scientific research on Clitoria ternatea suggests a huge antioxidant, antidiabetic and hepatoprotective potential of this plant. The plant is a rich source of phytochemicals, with high levels of phenolic compounds and antioxidant activities. The study also indicates that the leaf and flower extracts of Clitoria ternatea have a hypoglycaemic effect. The extracts were effective in regulating the biochemical indices associated with diabetes mellitus. Clitoria ternatea possesses strong hepatoprotective potential. The hepatoprotective activity of Clitoria ternatea leaf may be due to its free radical-scavenging and antioxidant activity, resulting from the presence of some phenolic compounds in the extracts. Further studies are in progress to better understand the mechanism of action of Clitoria ternatea responsible for the observed hepatoprotective and antioxidant activity. The organic and aqueous extracts of Clitoria ternatea could be further exploited in the future as a source of useful phytochemicals compounds for the pharmaceutical industry and the antioxidant mechanisms and the anti- Proliferative properties of the extracts should be further studied to gain more application for use as natural antioxidants.

REFERENCES

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www.ijpbs.com (or) www.ijpbsonline.com [1] Agrawal, P., Deshmukh, S., Al,i A., Patil, S., Magdum, C.,S., Mohite, S., K. and Nandgude, T., D., Wild Flowers as Medicines, International Journal of Green Pharmacy, 1(1): 12, (2007). [2] Anuradha, K., Hota, D., Pandhi, P., Investigation of central mechanism of insulin-induced hypoglycemic convulsions in mice. Indian J. Exp. Biol. 42: 368-372, (2004). [3] Arise, R., O., Malomo, S., O., Adebayo, J., O. , Igunnu, A., Effects of aqueous extract of Eucalyptus globules on lipid peroxidation and selected enzymes of rat liver. J. Med. Plant Res. 077-081, (2009). [4]Barik, D., P., Naik, S., K., Mudgal, A., Chand, P.,K., Rapid plant regeneration through in vitro axillary shoot proliferation of butter-fly pea (Clitoria ternatea L.) – a twinning legume, In Vitro Cell.Dev.Biol.-Plant, 43 : 144148, (2007). [5] Banerjee, S., K., Chakravarti, R., N., Taraxerol from Clitoria ternatea, Bull Calcutta School Trop Med, 11: 106-107, (1963). [6] Banerjee, S., K. , Chakravarti, R., N., Taraxerone from Clitoria ternatea, Bull Calcutta School Trop Med, 12: 23, (1964). [7] Bhaskar, V., H.; Balakrishnan, N., Protective effects of Pergularia daemia roots against paracetamol and carbon tetrachloride-induced hepatotoxicity in rats. Pharm. Biol. 48: 1265-1272, (2010). [8]Bhathal, P., S.; Rose, N., R.; Mackay, I., R., Whittingham, S. Strain differences in mice in carbon tetrachlorideinduced liver injury. Br. J. Exp. Pathol. 64: 524-533, (1983). [9]Choi, J.,H.; Choi, C.,Y.; Lee, K.,J.; Hwang, Y.,P.; Chung, Y.,C.; Jeong, H.,G., Hepatoprotective effects of an anthocyanin fraction from purple-fleshed sweet potato against acetaminophen-induced liver damage in mice. J. Med. Food. 12: 320-326, (2009). [10]Daisy, P. and Rajathi, M., Hypoglycemic Effects of Clitoria ternatea Linn. (Fabaceae) in Alloxan-induced Diabetes in Rats. Tropical Journal of Pharmaceutical Research, 8 (5): 393-398, (2009). [11]Daniel, O., Meier, M. S., Schlatter, J., and Frischknecht, P., Selected phenolic compounds in cultivated plants: ecologic functions, health implications, and modulation by pesticides. Environmental Health Perspectives, 107: 109–114, (1999). [12]Gomez, S., M., Kalamani, A., Butter-fly Pea (Clitoria ternatea): A Nutritive Multipurpose Forage Legume for the Tropics- An Overview, Pakistan Journal of Nutrition, 2 (6): 374-379, (2003). [13]Gunjam M., Ravindran, M., Sengamalam, R., Goutam, K., J., Jha, A., K., Pharmacognostic and antidiabetic study

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|203-213 of Clitoria ternatea. International Journal of Phytomedicine 2: 373-378, (2010). [14]Hall, T., J., Adaptation and Agronomy of Clitoria ternatea L. in Northern Australia, Tropical Grasslands, 19(4): 156-163, (1985). [15]Harborne, J. B., and Williams, C. A., Advances in flavonoid research since 1992. Phytochemistry, 55: 481504, (2000). [16]Huang, B.; Ban, X.; He, J.; Tong, J.; Tian, J.; Wang, Y., Hepatoprotective and antioxidant activity of ethanolic extracts of edible lotus (Nelumbo nucifera Gaertn.) leaves. Food Chem. 120: 873-878, (2010). [17] Jain, N., N., Ohal, C., C., Shroff, S., K., Bhutada, R., H., Somani, R., S., Kasture, V., S., Kasture, S., B., Clitoria ternatea and the CNS, Pharmacology, Biochemistry and Behaviour, 75, 529-536, (2003). [18] Jain R., A., Shukla S., H., and. Saluja A., K., Invitro evaluation of Clitoria ternatea stem extract for antioxidant property. IJPSR, Vol. 1 (12): 88-94, (2010). [19]Jayakar, B., Suresh, B., Antihyperglycemic and hypoglycemic effect of Aporosa lindleyana in alloxaninduced diabetic rats. J. Ethnopharmacol. 84: 247-249, (2003). [20] Johnson, I., T., Antioxidants and antitumour properties. In J. Pokorny, N. Yanishlieva, and M. H. Gordon (Eds.), Antioxidants in food: Practical applications (pp. 100– 123). Cambridge: Woodhead Publishing Ltd.. (2001). [12] Joshi, S., S., Shrivastava, R.,K., Shrivastava, D., K., Chemical examination of Clitoria ternatea Seeds, Journal of American Oil Chemical Society, 58(6): 714-715, (1981). [22]Jayachitra, A., and Padma, P., R., Antioxidant potential of Clitoria ternatea leaf extracts in vitro. Int J Pharm Bio Sci; 3(4): 753 – 763, (2012). [23] Kaisoon, O., Siriamornpun, S., Weerapreeyakul, N., and Meeso, N., Phenolic compounds and antioxidant activities of edible flowers from Thailand. Journal of functional foods 3: 88-99, (2011). [24] Kaur, G., Alamb, M. S., Jabbar, Z., Javed, K., and Athar, M., Evaluation of antioxidant activity of Cassia siamea flowers. Journal of Ethnopharmacology, 108, 340–348, (2006). [25]Kavitha, R., and Premalakshmi, V., Studies on the Synergetic Effect of Trichosanthes dioica and Clitoria ternatea Leaf Extract on the Streptozotocin-Induced Diabetic Rats. International Journal of Research in Pharmaceutical and Biomedical Sciences, 3 (3): 10561064, (2012). [26]Kazuma, K., Noda, N., Suzuki, M., Malonylated flavonol glycosides from the petals of Clitoria ternatea, Phytochemistry, 62 :229-237, (2003).

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www.ijpbs.com (or) www.ijpbsonline.com [27]Kirtikar, K., R., Basu, B., D., Indian Medicinal Plants, Vol. III, Basu LM, Allahabad, 802, (1935). [28] Kumar, V., Mukherjee, K. , Kumar, S., Mal, M., Mukherjee, P., K., Validation of HPTLC Method for the Analysis of Taraxerol in Clitoria ternatea, Phytochemical Analysis, 19: 244-250, (2008). [29] Madhavarao, B., Sabithadevi, K., Vinnakoti, In -vitro antimicrobial and free radical scavenger assay of two medicinal plants Clitoria ternatea and Cardiospermum halicacabum, International Journal of Chemical and Analytical Science. 2(11): 1253-1255, (2011). [30]Marles, R., J. and Farnsworth, N., R., Antidiabetic plants and their active constituents. Phytomedicine 2: 137-189, (1995). [31]Mathada, R., V., Jevoor, P., R., Ravishankar, R., Effect of Clitoria ternatea Linn. Root extract on the hippocampal area Ca3 and pancreas of juvenile diabetic rats- A preliminary investigation. Spatula DD, 2(1): 9-16, (2012). [32]Morris, J., B., Legume genetic resources with novel value added industrial and pharmaceutical use. In: Janick, J. (Ed.), Perspectives on Newcrops and New Uses. ASHS Press, Alexandria, VA, USA, 196-201, (1999). [33]Morita, N., Arisawa, M., Nagase, M., Hsu, H.,Y., Chen, Y.,P., Studies on the Constituents of Foramosan Leguminosae. L.The Constituents in the Leaves of Clitoria ternatea L., Pharmaceutical Society of Japan, 97: 649-653, (1977). [34]Morton, L. W., Abu-Amsha, C., Puddey, I. B., & Croft, K. D., Chemistry and biological effects of dietary phenolic compounds: Relevance to cardiovascular diseases. Clinical and Experimental Pharmacology and Physiology, 27, 152–159, (2000). [35]Mukherjee, P., K, Kuma,r V., Kumar, N., S., Heinrich, M., The Ayurvedic medicine Clitoria ternatea- From traditional use to scientific assessment, Journal of Ethnopharmacology, 120 :291-301, (2008). [36]Nadkarni, K., M., Indian Materia Medica, Popular Publications, Bombay, 14 : 354-355, (1976). [37] Narayanasamy, K. and V. Selvi, V., Hepatoprotective effect of a poly herbal formulation (Ayush – Liv.04) against ethanol and CCl4 induced liver damage in rats. Ancient Science of Life, 15 (1) : 28-33, , (2005). [38] Nayak, S., S.; Jain, R.; Sahoo, A., K., Hepatoprotective activity of Glycosmis pentaphylla against paracetamolinduced hepatotoxicity in Swiss albino mice. Pharm. Biol. 49, 111-117, (2011). [39]Nickavar, B., Kamalinelad, I., Yahya, M., H., and Shalagh,i B., Comparison of the free radical scavenging activity of six Iranian Achillea species, PharmBiol,4:208212, (2006).

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|203-213 [40]Nithianantham, K., Shyamala, M., Chen, Y., Yoga Latha, L. Subramanion, L., J. and Sreenivasan , S., Hepatoprotective Potential of Clitoria ternatea Leaf Extract Against Paracetamol Induced Damage in Mice. Molecules, 16: 10134-10145, (2011). [41]Oktay, M., Guloin, I., and Kufrevioglu, O. I., Determination of in vitro antioxidant activity of fennel (Foeniculum vulgare) seed extracts. LWT – Food Science and Technology, 36, 263–271, (2003).s [42]Osborn, R.,W., Samblanx, G., W., De, Thevissen, K., Goderis, . I, Torrekens, S., Van, Leuve,n F., et al. Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae, FEBS Letters, 368: 257-262, (1955). [43]Parimaladevi, B., Boominathan, R., Mandal, S., C., Antiinflammatory, analgesic and anti-pyretic properties of Clitoria ternatea root, Fitoterapia 74: 345-349, (2003). [44] Patil, A., P., and Patil, V., R., Comparative Evaluation of in vitro Antioxidant Activity of Root of Blue and White Flowered Varieties of Clitoria ternatea Linn. International Journal of Pharmacology, 7: 485-491, (2011). [45]Potsangbam, L., Ningombam, S., Laitonjam, W., S., Natural dye yielding plants and indigenous knowledge of dyeing in Manipur, Northeast India, Indian Journal of Traditional Knowledge, 7(1): 141-147, (2008). [46] Pratt, D., E., Natural antioxidants from plant material. In I. M. T. Huang, C. T. Ho, and C. Y. Lee (Eds.), Phenolic compounds in food and their effects on health. New York: American Chemical Society : 54–72, (1992). [47] Rabeta, M. S. and An Nabil, Z., Total phenolic compounds and scavenging activity in Clitoria ternatea and Vitex negundo linn. International Food Research Journal 20(1): 495-500, (2013). [48] Ramaswami, V., Varghese, N., Simon, A., An investigation on cytotoxic and antioxidant properties of Clitoria ternatea L. International Journal of Drug Discovery. 3(1) : 74-77, (2011). [49] Repetto, M.G and Llesuy, S.F, Antioxidant properties of natural compounds used in popular medicine for gastric ulcers, Brazilian Journal of Medical and Biological Research, 35: 523-534, (2002). [50] Sarumathy, K., Dhana Rajan M. S., Vijay,T., Jayakanthi J., Evaluation of phytoconstituents, nephro-protective and antioxidant activities of Clitoria ternatea. Journal of Applied Pharmaceutical Science 01 (05): 164-172, (2011). [51] Schwartz, C., J., Valente, A., J. , Sprague, E., A., A modern view of atherogenesis. American J. Cardiol. 71: 9-4, (1993). [52] Sethiya, N., K., Nahata, A., Mishra, H., Dixit, V., K., An update on Shankhpushpi, a cognition- boosting

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www.ijpbs.com (or) www.ijpbsonline.com Ayurvedic medicine, Journal of Chinese Integrative Medicine, 7(11): 1001-1022, (2009). [53]Shahidi, F., Antioxidants in food and food antioxidants. Nahrung, 44, 158–163, (2000). [54]Shahidi, F., and Naczk, M., Phenolics in food and nutraceuticals. Boca Raton, FL: CRC Press., . (2004). [55]Shamina, S., Effect of polyherbal formulation Rheumatone on enzymatic antioxidant levels in liver and kidney of Freud’s complete adjuvant (FCA) induced arthritic. World Journal of Pharmacy and Pharmaceutical Sciences, 1( 3): 1147-1154, (2012). [56]Shui, G., and Leong, L. P., Residue from star fruit as valuable source for functional food ingredients and antioxidant nutraceuticals. Food Chemistry, 97, 277– 284, (2006). [57] Sinha, A., Studies on the unsaponifiable matter of the seeds of Clitoria ternatea Linn. And isolation of sitosterol, Proceedings of the National Academy of Sciences, 29 : 23-26, (1960). [58]Somania, R., Vadnala, P., Deshmukhb, S., Patwardhana, S., Hepatoprotective activity of Clitoria ternatea L. leaves against carbon tetrachloride induced hepatic damage in rats. Journal of Pharmacy Research, 4(10): 3540-3544, (2011). [59]Terahara, N., Eight new anthocyanins, Ternatins C1-C5 and D3 and Preternatins A3 and C4 from young Clitoria ternatea flowers, Journal of Natural Products, 61(11): 1361-1367, (1998). [60]Terahara, N., Five new anthocyanins, ternatins A3, B4, B3, B2 and D2 from Clitoria ternatea Flowers, Journal of Natural Products, 59(2): 139-144, (1996). [61]Uma, B., Prabhaka, K., Rajendran, S., Phytochemical Analysis and AntiMicrobial Activity of Clitoria ternatea Linn against Extended Spectrum Beta Lactamase

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|203-213 Producing Enteric and Urinary Pathogens, Asian Journal of Pharmaceutical and Clinical Research, 2(4): 94- 96, (2009). [62]Van der Sluis, A., A., Dekker, M., Skrede, G., and Jongen, W,. M., F., Activity and concentration of polyphenolic antioxidants in apple juice. I. Effect of existing production methods. Journal of Agricultural and Food Chemistry, 50, 7211–7219, (2002). [63]Waltner-Law, M., E., Wang, X., L., Law, B., K., Epigallocatechin gallate, a constituent of green tea, represses hepatic glucose production. J. Biol. Chem. 277: 34933-34940, (2002). [64]World Health Organisation, Second report of the WHO expert committee on diabetes mellitus. Technical Report Series 646: 66, (1980). [65]Yanishlieva-Maslarova, N., V., Inhibiting oxidation. In J. Pokorny, N. Yanishlieva, & M. H. Gordon (Eds.), Antioxidants in food: Practical applications ( 22–70). Cambridge: Woodhead Publishing Limited, (2001). [66]Yapar K, Kart A, Karapehlivan M, Atakisi O, Tunca R, Erginsoy S and Citil M., Hepatoprotective effect of lcarnitine against acute acetaminophen toxicity in mice. Exp. And Toxicolo Pathology 59: 121-128, (2007). [67]Yousef, M., I.; Omar, S., A.; El-Guendi, M., I.; Abdelmegid, L., A., Potential protective effects of quercetin and curcumin on paracetamol-induced histological changes, oxidative stress, impaired liver and kidney functions and haematotoxicity in rat. Food Chem. Toxicol. 48: 3246-3261, (2010). [68]Youwei, Z., Jinlian, Z., and Yonghong, P., A comparative study on the free radical scavenging activities of some fresh flowers in southern China. LWT – Food Science and Technology, 41, 1586–1591, (2008).

*Corresponding Author: Manju Lata Zingare*

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Address- Qtr. No. 6/A, Street No. 31, Sector No. 8 Bhilai Nagar, Distt. Durg, C.G, 490009, India. (Department of Biotechnology, Govt. Digvijay Autonomous P.G. College, Rajnandgaon, C.G., 491441, India.) Email; mzingare@yahoo.co.in

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Review Article Pharmaceutical Sciences MICROSPONGE: COMPREHENSIVE REVIEW OF APPLICATION Swati Kale Valmik*1 Shalini R1. Kanchan M2.Ashwini C3, Eknath P4 1*, 1, 2, 3

Indira college of pharmacy,Vishnupuri, Nanded 4 Worked at Ciplapvt.ltd. Goa.

*Corresponding Author Email: kswati1234@yahoo.com

ABSTRACT Microsponge technology has been introduced in topical drug products to facilitate the controlled release of active drug into the skin in order to reduce systemic exposure and minimize local cutaneous reactions to active drugs. When applied to the skin, the Microsponge releases its active ingredient on a time mode and also in response to other stimuli (rubbing, temperature, pH, etc) Microsponge consists of macroporous beads, typically 5 - 300μm microns in diameter, loaded with active agent. That are used mostly for topical and recently for diagnosis of diseases like Heart diseases, HIV, cancer. Now a days used in RNA silencing, Microsponge delivery system (MDS) can provide increased efficacy for topically active agents with enhanced safety, extended product stability, enhanced formulation flexibility, reduced side effects and improved aesthetic properties in an efficient and novel manner. In addition these are non-irritating, non-mutagenic, non-allergenic, and non-toxic. This technique reaching the goal of controlled and site specific drug delivery system. The present review introduces Microsponge technology along with its Advantage over the other Dosage form and release mechanism of MDS.

KEY WORDS Microsponge, PBNC, RNA silencing.

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INTRODUCTION The Microsponge technology was developed by Won in 1987 [1]. Microsponges are patented delivery systems composed of porous microspheres. They are tiny sponge like spherical particles that consist of a myriad of interconnecting voids within anon-collapsible structure with a large porous surface. The size of the microsponges ranges from 5 - 300μm (Figure 1) in diameter and a typical 25μmsphere can have up to 250000 pores(Figure 2) and an internal pore structure equivalent to 10ft in length, providing a total pore volume of about 1ml/g. These microsponges have the capacity to entrap a wide range of active ingredients such as emollients, fragrances, essential oils, sunscreens and anti-infective, etc. are used as a topical

carrier system [2]. Further, these porous microspheres with active ingredients can be incorporated in to formulations such as creams, lotions and powders. Release of drug into the skin is initiated by a variety of triggers, including rubbing and higher than ambient skin temperature [3]. Their high degree of crosslinking results in particles that are insoluble, inert and of sufficient strength to stand up to the high shear commonly used in manufacturing of creams, lotions, and powders. Their characteristic feature is the capacity to adsorb or “load” a high degree of active materials into the particle and on to its surface. Its large capacity for entrapment of actives, up to three times its weight, differentiates microsponge products from other types of dermatological delivery

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www.ijpbs.com (or) www.ijpbsonline.com systems. The active payload is protected in the formulation by the microsponge particle; it is delivered to skin through controlled diffusion. This sustained release of actives to skin over time is an extremely valuable tool to extend the efficacy and lessen the irritation. Several probable and reliable systems were developed

for systemic drugs under the heading of transdermal delivery system (TDS) using the skin as portal of entry. It has improved the efficacy and safety of many drugs that may be better administered through skin. But TDS is not practical for delivery of materials whose final target is skin itself [4, 5].

Figure 1: Microspong

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Figure 2: Highly porous nature of Microspong Characteristics Of Materials That is Entrapped in Microsponges. Microsponges can absorb oil up to 6 times its weight without drying. It provides continuous action up to 12 hours i.e. extended release. Liquids can be converted in to powders improving material processing. It has flexibility to develop novel product forms.MDS can improve bioavailability of the drugs. Microsponge formulations are stable over range of pH1 to 11 and at the temperature up to 130 oC.

Microsponge formulations are compatible with most vehicles and ingredients and they are self sterilizing as their average pore size is 0.25Îźm where bacteria cannot penetrate into them, they can be cost effective [6]. Recent application of Microsponges derived from seaweed may help to diagnose heart disease, cancers, HIV and other diseases quickly and at far lower cost than current clinical

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www.ijpbs.com (or) www.ijpbsonline.com methods. The biomarkers are sequestered in tiny sponges set into an array of inverted pyramidshaped funnels in the microprocessor heart of the credit card-sized PBNC [7]. Most liquid or soluble ingredients can be entrapped in the particles. Actives that can be entrapped in microsponges must meet following requirements,  It should be either fully miscible in monomer or capable of being made miscible by addition of small amount of a water immiscible solvent.  It should be water immiscible or at most only slightly soluble.  It should be inert to monomers.  The solubility of actives in the vehicle must be limited to avoid cosmetic problems; not more than 10 to 12% w/w microsponges must be incorporated into the vehicle. Otherwise the vehicle will deplete the microsponges before the application.  The spherical structure of microsponges should not collapse.  Polymer design and payload of the microsponges for the active must be optimized for required release rate for given time period. It should be stable in contact with polymerization catalyst and conditions of polymerization. Advantages over Conventional Formulations Conventional formulations of topical drugs are intended to work on outer layers of the skin. Such products release their active ingredients upon application, producing a higher concentrated layer of active ingredient that is rapidly absorbed. When compared to the microsponge system can prevent excessive accumulation of ingredients within the epidermis and the dermis. Potentially, the microsponge system can reduce significantly the irritation of effective drugs without reducing their efficacy. For example, by delivering the active ingredient

gradually to the skin like MDS. Benzoyl peroxide formulations have excellent efficacy with minimal irritation. Advantages over Microencapsulation And Liposomes The MDS has advantages over other technologies like microencapsulation and liposomes. Microencapsules cannot usually control the release rates of actives. Once the wall is ruptured the actives contained within microcapsules will be released. Liposomes suffer from lower payload, difficult formulation, limited chemical stability and microbial instability. While microsponge system in contrast to the above systems are stable over range of pH 1 to 11, temperature upto 130°C;compatible with most vehicles and ingredients; self-sterilizing as average pore size is 0.25μm where bacteria cannot penetrate; higher payload (50 to 60%), still free flowing and can be cost effective. Advantages Over Ointments Ointments are often aesthetically unappealing, greasiness, stickiness etc. That often results into lack of patient compliance. These vehicles require high concentrations of active agents for effective therapy because of their low efficiency of delivery system, resulting in irritation and allergic reactions in significant users. Other drawbacks of topical formulations are uncontrolled. Evaporation of active ingredient, unpleasant odour and potential incompatibility of drugs with the vehicles, when microsponge system maximize amount of time that an active ingredient is present either on skin surface or within the epidermis, while minimizing its transdermal penetration in to the body [8-11]. Advantages of Microsponge Delivery System:  Microsponges can absorb oil up to 6 times its weight without drying.

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www.ijpbs.com (or) www.ijpbsonline.com         

  

It provides continuous action up to 12 hours i.e. extended release. Improved product elegancy. Lessen the irritation and better tolerance leads to improved patient compliance. It can also improve efficacy in treatment. They have better thermal, physical and chemical stability. These are non-irritating, non-mutagenic, non-allergenic and non-toxic. MDS allows the incorporation of immiscible products. They have superior formulation flexibility. In contrast to other technologies like microencapsulation and liposomes, MDS has wide range of chemical stability, higher payload and are easy to formulate. Liquids can be converted in to powders improving material processing. It has flexibility to develop novel product forms. MDS can improve bioavailability of the drugs [12].

compared with mineral oil containing microcapsules, mineral oil containing microsponge showed much more softening effect. The duration of emolliency was also much more for the Microsponge systems. 2. Temperature change: Some entrapped actives can be too viscous at room temperature to flow spontaneously from Microsponges onto the skin. Increased in skin temperature can result in an increased flow rate and hence release. 3. Solubility: Microsponges loaded with watersoluble ingredients like anti-prespirants and antiseptics will release the ingredient in the presence of water. The release can also be activated by diffusion taking into consideration the partition coefficient of the ingredient between the microsponges and the outside system. 4. pH triggered systems :Triggering the pH-based release of the active can be achieved by modifying the coating on the microsponge. This has many applications in drug delivery [13]. APPLICATION Microsponges are used mostly for topical, oral administration as well as biopharmaceutical delivery. It offers the formulator a range of alternatives to develop drug and cosmetic products. These are developed to deliver an active ingredient efficiently at the low dose and also to enhance stability, reduce side effects and modify drug release. Microsponge drug delivery system unique, novel and versatile and extremely attractive in cosmetic world. Recent applications of microspong from sea weed were to detect the diseases andalso microspong drugdelivery in RNA silencing. Some applications of MDS are describe in Table 1 [14].

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Release Mechanisms: By proper manipulation of the aforementioned programmable parameters, microsponges can be designed to release given amount of active ingredients over time in response to one or more external triggers. 1. Pressure: Rubbing/ pressure applied can release active ingredient from microsponges onto skin. The amount released depends upon various characteristics of the sponge. By varying the type of material and different process variables, the Microsponge best suited for a given application may be optimized. When

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Applications Sunscreens: Long lasting product efficacy, with improved protection against sunburns and sun related injuries even at elevated concentration and with reduced irritancy and sensitization. Anti-acne: E.g. Benzoyl peroxide Maintained efficacy with decreased skin irritation and sensitization. Anti-inflammatory: E.g. hydrocortisone Long lasting activity with reduction of skin allergic response and dermatoses. Anti-fungals: Sustained release of actives Ingredient. Anti-dandruffs: E.g. zinc pyrithione, selenium sulfide. Reduced unpleasant odour with lowered irritation with extended safety and efficacy Antipruritics: Extended and improved activity. Skin depigmenting: E.g.hydroquinone. Improved stabilization against oxidation with improved efficacy and aesthetic agents appeal. Rubefacients: Prolonged activity with reduced irritancy greasiness and odour. Table 1: Application of Microsponge

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TOPICAL DRUG DELIVERY MICROSPONGE TECHNOLOGY

USING

Conventional formulations of topical drugs are in-tended to work on the outer layers of the skin. Typically, such products release their active ingredients upon application, producing a highly concentrated layer of active ingredient that is rapidly absorbed. The Microsponge system can prevent excessive accumulation of ingredients within the epidermis and the dermis. Potentially, the Microsponge system can reduce significantly the irritation of effective drugs without reducing their efficacy. Further these porous microspheres with active ingredients can be incorporated in to formulations such as creams, lotions and powders. Microsponges consisting of non-collapsible structures with porous surface through active ingredients are released in a controlled manner. Depending upon the size the

total pore length may range up to 10 ft and pore volume up to 1 ml/g [15]. Products under development or in the market place utilize the Topical Microsponge systems in three primary ways: 1. As reservoirs releasing active ingredients over an extended period of time. 2. As receptacles for absorbing undesirable substances, such as excess skin oils. 3. As closed containers holding ingredients away from the skin for superficial action. Dicyclomine, itâ&#x20AC;&#x2122;s an anticholinergic drug, has direct smooth muscle relaxant action, and it shows antispasmodic action. Dicyclomine causes gastrointestinal (GI) side effects like other antispasmodic drugs. Its plasma half life is 4 - 6 h. The present study was designed to formulate a drug which reduces the (GI) side effects. Dicyclomine loaded, Eudragit based microsponges were prepared using a quasi

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www.ijpbs.com (or) www.ijpbsonline.com emulsion solvent diffusion method. Cumulative release for the microsponges over 8 hours ranged from 59 - 86 %. Flurbiprofen, Microsponge system containing flurbiprofen was formulated for the colonic delivery of the drug for targeted action. Benzylperoxide, Benzoyl peroxide (BPO) is commonly used in topical formulations for the treatment of acne and athletesâ&#x20AC;&#x2122; foot. Normal ointments produce Skin irritation itâ&#x20AC;&#x2122;s a common side effect. Therefore, the ethylcellulose Microsponge system was formulated containing BPO which were able to control the release of BPO to the skin. And it has been shown that controlled release of BPO from a delivery system to the skin could reduce the side effect while reducing percutaneous absorption. Fluocinolone acetonide, (FA) is a corticosteroid primarily used in dermatology to reduce skin inflammation and relieve itching. FA entrapped microporous microparticles (microsponges) were formulated to control the release of drug to the skin, which reduces the skin irritation and produce controlled release of active medicament. Tretinoin photo-damage treatment, icrosponge product used for photo damage, premature aging of skin and skin cancer [16]. Hydroquinone (HQ) Disorders of hyper pigmentation such as melisma and post inflammatory hyper pigmentation (PIH) are common, particularly among people with darker skin types. Bleaching creams are considered the gold standard for treating hyper pigmentation. Recently, a new formulation of HQ 4% with retinol 0.15% entrapped in microsponge reservoirs was developed for the treatment of melasma and PIH. Microsponges were used to release HQ gradually to prolong exposure to treatment and to minimize skin irritation [17].Hydroquinone prevents the overproduction

of melanin, while lightening the brown spots on the skin [18, 19]. Aceclofenac is a NSAID having excellent antiinflammatory and analgesic activity but NSAID produces GIT ulceration, liver and kidney trouble especially in case of oral administration. In view, of adverse drug reaction associated with oral formulations, aceclofenac is increasingly administered by topical route. Aceclofenac loaded microsponge are prepared by using quasi-emulsion solvent diffusion method. It is incorporated in gel base and various parameters are studied [20]. Microsponge Technology in Cosmetics An interesting application of the microsponge technology could be in oral cosmetics, such as tosustain the release of volatile ingredients, thus increasing the duration of the 'fresh feel'.Microsponges of such volatile ingredients may be easily incorporated in tooth pastes or mouthwashes. Colours entrapped in microsponges may be used in a variety of coloured cosmetic products such asrouge or lipsticks to make them long lasting. As stated above, microsponges help in uniform spreading and improving covering power. Thus, coloured cosmetics formulated with microsponges would be highly elegant [21]. Marketed formulation using the MDS includes Dermatological products which can absorb large amounts of excess of skin oil, while retaining an elegant feel on the skin's surface. Among these productsare skin cleansers, conditioners, oil control lotions, moisturizers, deodorants, razors, lipstick, makeup, powders, and eye shadows; which offers several advantages, including improved physical and chemical stability, greater available concentrations, controlled release of the active ingredients, reduced skin irritation and sensitization, and unique tactile qualities [22].Retinol is a highly pure form ofvitamin A which has demonstrated a remarkable ability for

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maintaining the skinâ&#x20AC;&#x2122;s youthful appearance. However, it becomes unstable when mixed with other ingredients. Stabilized retinol in a formulation which is cosmetically elegent and which has a low potential for skin irritation is successfully developed and marketed [23]. Oral Drug Delivery System Using Microsponge Technology Microsponge system offers the potential for active ingredients to remain within a protected environment and provide controlled delivery of oral medication to the lower gastrointestinal (GI) tract, where it will be released upon exposure to specific enzymes in the colon. If this approach is successful then it should open up entirely new opportunities for MDS. It has been shown that microsponge system enhances the solubilization of drugs which are poorly soluble by entrapping these drugs in their pores. As these pores are very small, the drug is in effect reduced to microscopic particles and drastically increased surface area consequently, increases the rate of solubilization. Additionally, the time it takes the microsponge system to pass through the small and large intestine is considerably increased as a result maximizing the amount of drug that is absorbed. Controlled oral delivery of ibuprofen microsponges is achieved with an acrylic polymer, Eudragit RS, by changing their intraparticle density. The release of ketoprofen incorporated into modified release ketoprofen microsponge 200 mg tablets and Profenid Retard 200 mg was studied in vitro and in vivo. The formulation containing ketoprofen microsponges yielded good modified release tablets. An in vivo study was designed to evaluate the pharmacokinetic parameters and to compare them with the commercially available ketoprofen retard tablets containing the same amount of the active drug.

Commercial ketoprofen retard tablets showed a more rapid absorption rate than modified release tablets and peak levels were reached within almost 3.6 h after administration. However, the new modified release tablets showed a slower absorption rate and peak levels were reached 8 h after administration [24]. Paracetamol loaded Eudragit based microsponges were prepared using quasiemulsion solvent diffusion method, then the colon specific tablets were prepared by compressing the microsponges followed by coating with pectin: hydroxyl propyl methyl cellulose (HPMC) mixture [25]. Reconstruction Of Vascular Wall Using Microsponge Technology The tissue-engineered patch was fabricated by compounding a collagen microsponge with a biodegradable polymeric scaffold composed of polyglycolic acid knitted mesh, reinforced on the outside with woven polylactic acid. Tissueengineered patches without precellularization were grafted into the porcine descending aorta (n = 5), the porcine pulmonary arterial trunk (n = 8), or the canine right ventricular outflow tract (as the large graft model; n = 4). Histological and biochemical assessments were performed 1, 2, and 6 months after the implantation.There wasno thrombus formation in any animal. Two months after grafting, all the grafts showed good in situ cellularization by hematoxylin/eosin and immune staining [26].The quantification of the cell population by polymerase chain reaction showed a large number of endothelial and smooth muscle cells 2 months after implantation. In the large graft model, the architecture of the patch was similar to that of native tissue 6 months after implantation and this patch can be used as a novel surgical material for the repair of the cardiovascular system [27].

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CardiovascularEngineering Using Microsponge Technology A biodegradable material with autologous cellseeding requires a complicated and invasive procedure that carries the risk of infection. To avoid these problems, a biodegradable graft material containing collagen microsponge that would permit the regeneration of autologous vessel tissue has developed. The ability of this material to accelerate in situ cellularization with autologous endothelial and smooth muscle cells was tested with and without precellularization. Poly (lactic-co-glycolic acid) as a biodegradable scaffold was compounded with collagen microsponge to form a vascular patch material. These poly (lacticco-glycolic acid)-collagen patches with (n =10) or without (n = 10) autologous vessel cellularization were used to patch the canine pulmonary artery trunk. Histologicand biochemical assessments were performed 2 and 6 months after the implantation. There was no thrombus formation in either group, and the poly (lactic-co-glycolic acid) scaffold was almost completely absorbed in both groups. Histologic results showed the formation of an endothelial cell monolayer, a parallel alignment of smooth muscle cells, and reconstructed vessel wall with elastin and collagen fibers. The cellular and extracellular components in the patch hadincreased to levels similar to those innative tissues at 6 months. This patch shows promise as a bioengineered material for promoting in situ cellularization and the regeneration of autologous tissue in cardiovascular surgery [28]. Microsponge Based Delivery System For Bone Substitute Compounds were obtained by mixing prepolymerized powders of poly methyl methacrylate and liquid methyl methacrylate

monomer with two aqueous dispersions of atricalcium phosphate (a-TCP) grains and calcium deficient hydroxyapatite (CDHA) powders. The finalcomposites appeared to be porous. Osteo conductivity and osteo inductivity of the final composites were tested in vivo by implantation in rabbits. Formation of new trabecular bone was observed inside the pores where the inorganic powders had been placed. The material produced shows a good level of biocompatibility, good osteo integration rate and osteogenetic properties [29]. Microsponges for Biopharmaceuticals Delivery The microsponge delivery system (MDS) is employed for both in the delivery of biopharmaceuticals as well as in tissue engineering. Dai 2010et al. developed 3D scaffolds hybrid structures that have advantages of natural type I collagen and synthetic PLGA knitted mesh. The collagen microsponges facilitated cell seeding and tissue formation and mechanically. Strong PLGA mesh served as a skeleton. The scaffolds were divided into three groups: a) Thin: collagen microsponge formed in interstices of PLGA mesh; b) Semi: collagen microsponge formed on one side of PLGA mesh c) Sandwich: collagen sponge formed on both sides of PLGA mesh. In the scaffolds Bovine chondrocytes were cultured and transplanted subcutaneously into nude mice for 2, 4, and 8 weeks. All transplants showed natural chondrocyte morphology, homogeneous cell distribution, and abundant cartilaginous ECM deposition. Production of GAGs per DNA and the expression of type II collagen and aggre can mRNA were much higher in the Semi and Sandwich groups than in the Thin group. Young's modulus showed 54.8, 49.3%mechanical strength of the engineered

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www.ijpbs.com (or) www.ijpbsonline.com cartilage and in stiffness 68.8, 62.7%, respectively, in Semi and Sandwich when compared to native articular cartilage. These scaffolds could be used for the tissue engineering of articular cartilage with adjustable thickness. Developed a biodegradable graft material containing collagen microsponge that would allow the regeneration of autologous vessel tissue in order to avoid these problems. Poly (lactic-co-glycolic acid) has been used as a biodegradable scaffold which was compounded with collagen microsponge to form a vascular patch material. The poly (lactic-co-glycolic acid) collagen patches with or without autologous vessel cellularization were used to patch the canine pulmonary artery trunk. Biochemical and histologic assessments were performed 2nd and 6th months after the implantation. Resulting, there was no thrombus formation in either group but the poly (lactic-co-glycolic acid) scaffold was approximately completely absorbed in both groups. Histologic results showed the formation of an endothelial cell monolayer, a parallel alignment of smooth musclecells, and reconstructed vessel wall with elastin and collagen fibers. The cellular and extra-cellular components in the patch had enlarged to levels analogous to those in native tissue at 6 months. This patch also shows promise as a bioengineered material for promoting in-situ cellularization and the regeneration of autologous tissue in cardiovascular surgery [30]. Tateishi et al., has also been studied developed biodegradable porous scaffolds for tissue engineering. 3D biodegradable porous scaffolds play a vital role in tissue engineering. A novel method were used for preparing porous scaffolds which consists of synthetic biodegradable polymers and developed by combining porogen leaching and freeze-drying techniques utilizing pre-prepared ice particulates as the porogen material. Biodegradable hybrid

porous sponges of synthetic polymer and collagen have been prepared by hybridizing synthetic polymer sponges with collagen microsponges. The collagen microsponges were produced in the pores of synthetic polymer sponges. Hybrid sponges of synthetic polymer, collagen and inorganic hydroxyapatite were prepared by depositing hydroxyapatite particulates on the surfaces of the collagen microsponges in the synthetic polymer-collagen sponges. The synthetic polymer sponge were used as a mechanical skeleton to aid the formation of these hybrid sponges into desired shapesand contributed good mechanical strength and handling whereas the collagen and hydroxyapatite are used to promote cell interaction and facilitate cell seeding[31]. Microspong From Seaweed Use In Diagnose The Diseases. Microsponges derived from seaweed may help diagnose heart disease, cancers, HIV and other diseases quickly and at far lower cost than current clinical methods. The microsponges are an essential component of Rice University's Programmable Bio-Nano-Chip (PBNC) (Figure 3) and the focus of a new paper in the journal Small PBNCs capture biomarkers molecules that offer information about a person's health found in blood, saliva and other bodyfluids. The biomarkers are sequestered in tiny sponges set into an array of inverted pyramid-shaped funnels in the microprocessor heart of the credit card-sized PBNC. When a fluid sample is put into the disposable device, micro fluidic channels direct it to the sponges, which are infused with antibodies that detect and capture specific biomarkers. Once captured, they can be analyzed within minutes with a sophisticated microscope and computer built into a portable, toaster-sized reader.

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www.ijpbs.com (or) www.ijpbsonline.com The biomarker capture process is the subject of the Small paper. The microsponges are 280micrometer beads of agarose, a cheap, common, lab-friendly material derived from seaweed and often used as a matrix for growing live cells or capturing proteins. The beauty of agarose is its ability to capture a wide range of targets from relatively huge protein biomarkers to tiny drug metabolites. In the lab, agarose starts as a powder, like Jell-O. When mixed with hot water, it can be formed into gels or solids of any size. The size of the pores and channels in agarose can be tuned down to the nano scale. The team found that agarose beads with a diameter of about 280 micrometers are ideal for real-world applications and can be mass-produced in a costeffective way. These agarose beads retain their

efficiency at capturing biomarkers, are easy to handle and don't require specialized optics to see. The agarose beads serve the dual role of analyte capture and signal generation and reagent capture is observed both on the bead exterior and interior [32]. Employing 3D â&#x20AC;&#x153;nanonetsâ&#x20AC;? composed of agarose strands supported within beads and a fluorescent signal output from nanoparticles (nano), the PBNC immobilizes and quantitates medically relevant species (bio) from complex samples within an enclosed miniature flow chamber (chip). This chemical processing unit uses an etched silicon or stainless-steel chip populated with sensitized beads to quantify proteins, oligonucleotides, small molecules, and ions [33, 34, 35, 36].

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Figure 3: ProgramableBio-nano Cheap. Various gold-standard systems, such as enzymelinked immune sorbent assay (ELISA), the PBNC has assay times measured in minutes rather than hours, limits of detection two or more orders of magnitude lower, and a multiplex capacity of 6 or more concurrent analytes with internal controls. Like ELISA, the PBNC utilizes a sandwich immunoassay; however, the immune complexes are present throughout the 3D bead matrix, rather than deposited on a 2D flat surface. These initial observations provide some information about the nature of molecular transport within

the beads. However, they also catalyze the emergence of additional questions related to the exact mode of transport and the influence the agarose density/pore size has on the time course of reagent capture [37]. Self-Assembling RNAi Microsponges Nature Materials describe Microsponges that act as both carrier and cargo for the delivery of gene-silencing RNA (siRNA) into cells. The work reports that, compared with conventional siRNA delivery vehicles, one thousand times lower

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www.ijpbs.com (or) www.ijpbsonline.com concentration of the microsponges achieve the same degree of gene-silencing effect in tumourcarrying mice. siRNA delivery has so far been hampered by carriers that inefficiently encapsulate RNA, and by its degradation prior to cellular uptake. Using RNA-amplification techniques, Paula Hammond and colleagues made very long chains of connected hairpin RNA strands from circular DNA templates. They observed that the chains self-assembled into sponge-like microspheres of pleated crystalline sheets. Because hairpin RNA is cleaved to form siRNA only inside the cell, the hairpin-RNA microsponges function both as a stable cargo and a carrier. To enhance cellular uptake, the authors made the microsponges ten times smaller by coating them with a highly charged polymer. They show that each polymer-coated microsponge delivers over half a million copies of siRNAper cell [38]. Once we generated the microsponges, we did several tests to ensure that we had all RNA sequences. We confirmed that the sponges degrade completely in the presence of RNAse, but not with DNAse, and confirmed the secondary structure of the RNA. We essentially did a basic material analysis of the system, looking at the size distribution and net charge these are very highly charged because of the RNA sequences. Then we wrapped up the microsponge with one adsorbed layer of a polycation. This allowed us to compact the microsponge into a nanostructure. Once we did that, we looked at internalization of the microsponge in vitro and saw that cell uptake was quite high in these systems. We actually have hundreds to thousands of copies of the siRNA in each chain that is generated. These chains are extremely long, and they form what appear to be polymeric crystals. The way polymers crystallize is that the repeat sequences begin to fold back on themselves and form a

sheet-like structure that can radiate outward from a spherical core, which is what we see with the siRNAs. We believe that we've created a siRNA-polymeric species of a high molecular weight, which is generated in high enough concentrations to form superstructures that are crystalline versions of the polymer. They are selforganized and mono disperses, so all these microsponges are the same size. The construction technique first involved preparing long linear single-stranded DNA encoding complementary sequences of both the antisense and sense sequences of anti-luciferase siRNA. This long strand was then hybridized with short DNA strands containing the T7 promoter sequence, to form circular DNA. The circle was closed using T4 DNA ligase, and the circular DNA then used to produce RNA transcripts by RCT, which encode both antisense and sense sequences of the anti-luciferase siRNA. This effectively generates nanoscale pleated sheets of hairpin RNA that self-assemble into sponge-like microspheres. Critically, once internalized in cells, the RNA is split up into the 21 nucleotidelong RNA component units by activity of the enzyme Dicer, and then converted to active siRNAs by theRNA-induced silencing complex. The authors believe their RNAi-microsponge could represent a more cost-effective and efficient means of delivering high concentrations of siRNAs to target cells for therapeutic applications. The crystalline form of the polymeric RNA protects the RNA itself from degradation during delivery, while the polymerization approach can easily be modified, which means multiple RNA species could be included in the constructs for combination therapy. “The RNAi-microsponge presents a novel materials system in general owing to its unique morphology and nanoscale structure within the polymer particle,” the team concludes, “and provides a promising self-

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www.ijpbs.com (or) www.ijpbsonline.com assembling material that spontaneously generates a dense siRNA carrier for broad clinical applications of RNAi delivery using the intrinsic biology of the cell.”[39].

CONCLUSION The microsponge drug delivery technology is widely applicable to the dermatological drug delivery products. The microsponge delivery technology of controlled release system in which active pharmaceutical ingredients are loaded in the microporous beads and initiates reduction in side effects with improved therapeutic efficacy. The microsponge drug delivery system has properties like improved stability and enhanced flexibility in formulation. MDS is originally developed for topical delivery of drugs like antiacne, anti-inflammatory, anti-fungal, antidandruffs, antipruritics, rubefacientsetc. But MDS also expands its application in oral drug delivery, bone and tissue engineering, in detecting the diseases and inRNAi silencing Hence, the microsponge drug delivery system focus as an important tool for future inventions in controlled drug delivery system.

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Won R., Method for delivering an active ingredient by controlled time release utilizing a novel delivery vehicle which can be prepared by a process utilizing the active ingredients as a Porogen Patent US 4690825, 1987. Vyas S., Khar R., Targeted and controlled drug delivery Novel carrier system. CBS publication, New Delhi, 1st edition, 453, (2002). Delattre L., Delneuville I., Biopharmaceutical aspects of the formulation of dermatological vehicles. J Eur Acad Dermatol Venereol (5): 70-71, (1995) . Kydonieus A., Berner B., Transdermal Delivery of Drugs. CRC Press, Boca Raton, (1987) Delattre L., Delneuville I., Biopharmaceutical aspects of the formulation of dermatological vehicles. Journal of the European Academy of Dermatology and Venereology (5):70, (1995) Aritomi H., Yamasaki Y., Yamada K., Honda H., Koshi M.,Development of sustained release formulation of

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chlorpheniramine maleate using powder coated microsponges prepared by dry impact blending method. Journal of Pharmaceutical Sciences and Technology, 56(1): 49-56, (1996). Microsponges from seaweed may save lives Rice University scientists refine process at heart of diagnostic Bio-Nano-Chip. Kawashima Y., Niwa T., Takeuchi H., Hino T., Ito Y., Control of Prolonged Drug Release and Compression Properties of Ibuprofen Microsponges with Acrylic Polymer, Eudragit RS, by Changing Their Intraparticle Porosity.Chemical & pharmaceutical bulletin, 40(1):196-201, (1992). D’ souza J., Masvekar R., Pattekari P., Pudi S., More H., Microspongic Delivery Of Fluconazole For Topical Application, 1st Indo- Japanese International Conference On Advances In Pharmaceutical Research And Technology, Mumbai, India, 25-29, (2005). Wester R., Patel R., Nacht S., Leydan J., Malendres J., Maibch H.,Controlled release of benzoyl peroxide from a porous microsphere polymeric system can reduce topical irritancy. J. Am. Acad. Dermatol, 24:720-726, (1991). Tansel C., Preparation and in vitro evaluation of modified release ketoprofen microsponge II Farmaco, 58:101-106, (2003) Guoping C., Sato T., Ohgushi H., Takashi U., Tetsuya T., Junzo T., Culturing of skin fibroblasts in a thin PLGA– collagen hybridmesh, Biomaterials, 26: 2559–2566, (2005). Embil K., Nacht S., The Microsponge Delivery System (MDS) - a topical delivery system with reduced irritancy incorporating multiple triggering mechanisms for the release of actives. J Microencapsul ,13(5): 575-88, (1996) Khopade A., Jain S., Jain N., “The Microsponge”. Eastern Pharmacist, 49-53, (1996). Patel G., Patel J., Use of a Microsponge in Drug Delivery Systems, Pharmaceutical processing, 158, (2008). Jain V., Singh R., Dicyclomine-loaded eudragit based microsponge with potential for colonic delivery Preparation and characterization. Tropical Journalof Pharmaceutical Research, 9(1): 67-72, (2010). Shelke O., Sable K., Gadhave M., Gaikawad D., Microsponge Drug Delivery System: An Emerging Tool for Topical Drug Delivery System. The GlobalJournal of Pharmaceutical Research, 1(4): 805-818, (2012). Kawashima Y., Niwa T., Takeuchi H., Hino T., Itoh Y., Furuyama S., Characterization of polymorphs of tranilast anhydrate and tranilastmonohydrate when crystallizedby two solvent change spherical

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material for reconstitution of vascular wall. Ann Thorac Surg, 80(5): 1821-1827, (2005). D’souza J., Harinath M., The Microsponge Drug Delivery System: For Delivering an Active Ingredient by Controlled Time Release.Pharmaceutical Reviews 6(3), (2008). Shaheen S., BollaK., Vasu K., Antimicrobial activity of the fruit extracts of Coccini indica. African journal of Biotechnology. 8(24): 7073-7076, (2009). Dean J., Robert C., Frederick H., Richard A., Philip G., Runstadler J., Weighted collagen microsponge for immobilizing bioactive materials, US Patent 4863856,1989. Talisuna A., Bloland P., Alessandro U., History, Dynamics&Public Health importance of Malaria parasite resistance.Clinical MicrobiologyReview 17: 235-254, (2004). Goodey A., Lavigne J., Savoy S., Rodriguez M., Curey T., Tsao A., Simmons G., Wright J., Yoo S., Sohn Y., Anslyn E., Shear J., .Neikirk D., McDevitt J., J Am Chem Soc. 123: 2559–2570, (2001). Goodey A., McDevitt J., J Am Chem Soc. 125:2870– 2871 (2003). Jokerst J., Raamanathan A., Christodoulides N., Floriano P., Pollard A., Simmons G., Wong J., Gage C., Furmaga W., Redding S., McDevitt J. Biosens Bioelectron. 24: 3622–3629, (2009). Ali M., Kirby R., Goodey A., Rodriguez M., Ellington A., Neikirk D., McDevitt J., Anal Chem.75: 4732– 4739, (2003). Jokerst J., McDevitt J,. Nanomedicine.5:143–155, (2010). Thompson J., Bau H., J Chrom B., 878:228–236, (2010). Nature MaterialsFebruary 27, (2012). MIT's Paula Hammond on siRNA Delivery Via AllRNA Microsponges March 01, (2012).

*Corresponding Author:

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Kale Swati Valmik Indira College of Pharmacy, Nanded, Vishnupuri- 431605.

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Research Article Biological Sciences STUDIES ON PHYTOPLANKTON DIVERSITY IN VADUVUR LAKE AT THIRUVARUR DISTRICT, TAMILNADU, INDIA Karthi N*, Vachanth M.C, and Sridharan G P.G and Research Department of Zoology, Rajah Serfoji Government College (Autonomous), Thanjavur – 613 005 Tamilnadu, India. *Corresponding Author Email: nkarthiphdzoology@gmail.com

ABSTRACT Diversity of phytoplankton was analyzed in Vaduvur lake (10.4°N; 79.19°E) water situated in Thiruvarur District, during the period of October 2010 to September 2011. Qualitative and Quantitative estimation of phytoplankton from study site was carried out with help of Sedgwick Rafter counting cell and identified using standard literature. The study period totals of 32 genera were observed in phytoplankton. Those genera, were present in different divisions were Bacillariophyceae (15 species), Cholorophyceae (10 species) and Cyanophyceae (7 species). Total number of phytoplankton 8325 Nos /lit were observed in throughout the year. The maximum phytoplankton population was found during the month of February 2011 and minimum phytoplankton population was found during the various month of December 2010, April 2011 and August 2011, (Chlorophyceae, Bacillaiophyceae and Cyanophyceae).

KEYWORDS Phytoplankton, Vaduvur Lake, Monthly variations.

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INTRODUCTION The Vaduvur Lake is oldest and largest Lake of Tamil Nadu. This is very significant lake in South India. This lake is important ecosystem for fishes and birds. The lake being used for multipurpose utility such as irrigation, migratory birds, fish catching washing and bathing. Biodiversity means the variability among the living organisms from all source including terrestrial, lake, marine and other aquatic ecosystem and ecological complex of which they are part (Ali, 1999). Phytoplankton is a predominant type of a plant found in most lake water. The quality and quantity of phyto plankton is a good indicator of water quality. The high relative abundance of chlorophyta is a indicator of productive water (Boyd, 1981).

Phytoplankton forms the vital source of energy as primary producers and serves as a direct source of food to the other aquatic plants and animals (Saha et al., 2000). Systematic and ecological studies on chlorophyceae of North India and their relationship with water quality were made (Dwivedi et al., 2005). In these systems phytoplankton is of great importance as a major source of organic carbon located at these bases (Gaikwad, et al., 2004). Phyto plankton is small organisms that play a crucial role in the food chain. While increased amounts of phytoplankton provide more food for organisms at higher tropic levels, too much phyto plankton or toxin producing phyto plankton can harm the over health of the Bay (Jana, 1973: Garcia and Lopez, 1989).

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MATERIALS AND METHODS The present study was carried out on Vaduvur Lake which is situated (10.4째N; 79.19째E) 21 km East of Thanjavur and 21 km west of Mannargudi, Thiruvarur District. This study was conducted during October 2010 to September 2011. Plankton sample were collected from the lake water on monthly basis. The collection of plankton was made by plankton net. Plankton samples were collected by filtering about 200 liters of the surface through the net. Immediately after collection of plankton samples were preserved in 10% formalin 10cc formalin diluted to 10cc of distilled water. Qualitative and quantitative estimation of phytoplankton from study site was carried out with the help of Sedgwick Rafter counting cell and identified using standard literature (Edmonson, 1959; Anand, 1998).

RESULTS AND DISCUSSION In the present study on the qualitative and quantitative analysis of phyto plankton of Vaduvur lake water taken monthly pattern and the density of phyto planktons identified. They belongs to the family of Bacillario phyceae (15 species), chlorophyceae (10 species), Cyanophycea (7 species). The phyto plankton analysed from the lake water samples were identified and listed (Table. 1).

The population density trend showed gradual increase during post monsoon period and monsoon season (Sukunan, 1980), Chlorophyceae, Bacillario phyceae and Cyanophyceae were recorded in large numbers during the study period and the Bacillariophyceae was dominant. There are several reports available on the distribution, density, species diversity and ecology of plankton in different water bodies (Fritsch, 1961; Rawson, 1956). Hence based on the diversity of phyto plankton population highly abundance in the month of December (Monsoon). The phyto plankton density due to the presence of high photo synthetic activity in the lake waters. Many reports are available on the plankton diversity of Indian lakes (Zafaar, 1986; Mani, 1992; Eswari, 2002; Rajasekar, et. al., 2005; Tiwari and Chauthan, 2006).

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The maximum number of chlorophyceae was 350 nos/lit was found in the month of February 2011 and the minimum was found 100 nos/lit was found in during the month of December 2010. The totals number of chlorophyceae of 2350 nos/lit were analyzed from the lake water during October 2011 to September 2011 (Table. 2).

The maximum number of Bacillario phyceae was 300 non/lit found in the month of December 2010. The minimum 100 nos/lit was found in the month of April 2011. The total number of Bacillario phyceae 2450 nos/lit were analysed from the lake water during the period of October 2010 to September 2011 (Table. 2). The maximum number of Cyanophyceae was 375 nos/lit found in the month of December 2010 and minimum number of cyanophyceae 200 nos/lit was found in the month of August 2011. The total number of cyanophyceae 3525 nos/lit were found in the period of October 2010 to September 2011 (Table. 2).

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Table 1: Showing the major groups of phytoplankton in Vaduvur lake (October 2010 to September 2011) PHYTO PLANKTONS S. No Bacillario phyceae 1 Fragillaria Crotonesis 2 Fragillaria Capunia 3 Synedra acus 4 Synedra vaucheriae 5 Aequalis sp., 6 Nitxshia diosipata 7 Nitzhiapalea 8 Navicula anglica 9 Navicla gracilis 10 Navicula gastrum 11 Pinnularia undulate 12 Navicula cuspidate 13 Gomphonema consrictum 14 Cymbella tumida 15 Syndera ulna

Chloro phyceae Pediastrum boryanum Polycedriopsis Selenastrum Pleurosigma Pediastrum duples Clostecidium tumdum Spirogyra Euglena Volvox Pandorina sp.

Cyano phyceae Anabea Oscillatoria putrida Merismopedia Spirulina Nostoc Oscillatora putrida Oscillatoria chlorine

Table 2: Monthly Variation of Phytoplankton (nos /lit) in Vaduvur lake During October 2010 to September 2011 Major Taxonomic Groups S. No

Month and year

1 2 3 4 5 6 7 8 9 10 11 12

October 2010 November 2010 December 2010 January 2011 February 2011 March 2011 April 2011 May 2011 June 2011 July 2011 August 2011 September 2011 Total number of phytoplankton (nos/lit)

Bacillario phyceae 250 200 300 170 150 250 100 150 250 150 200 280

Cyano phyceae 350 350 375 300 350 250 300 250 350 250 200 250

2350

2450

3525

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Chloro phyceae 130 120 100 150 350 250 200 150 150 200 250 300

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REFERENCES o o o o

o o

Ali, S.S., 1999, Fresh Water Fisheries Biology I ed. Naseem Book, Depot. Hyderabad. Anand, N., 1998. Freshwater Micro alagae. Bishen Singh Mahendra Pal Singh, Dehradun, India. 94. Boyd, C.E., 1981. Water quality in warm water fish ponds craft master printers. Inc. Opelika, Alabama. Dwivedi, S., Misra, P.K., Tripathi, R.D., Rai, U.N., Dwived, C.P., Baghal, V.S., Suseela, M.R., and Srivastava, M.N., 2005. Systematic and ecological studies on chlorophyceae of North India and their relationship with water quality. J. Environ. Biol. 26: 495–505. Edmendson, W.J., 1959. Fresh water biology, 2nd Edn., John Wiley and Sons, New York, 1248. Eswari,Y.N.K., and Ramani Bai, 2002. Distribution and abundance of phytoplankton is estuarine waters of Chennai, South East coast of India. J. Mar. Biol. Ass. India. 44: 205 – 211. Fritsch, F.E., 1961. The structure and reproduction of Algae. Vol. 1. Cambridge at University Press. Cambridge. Gaiwad, S.R., Tarot, S.R., and Chavan, T.P., 2004. Diversity of plankton and Zooplankton with respect to population status of river Tapi in North Maharastra region. J. Curr. Sci., 5: 749 – 754. Garcia, J.R., and Lopez, J.M., 1989. Seasonal patterns of phyto plankton productivity, zooplankton

o o

abundance and hydrological condition in laguna, Jogyunda, Puerto rico, 2(3): 625 – 631. Jana, B.B., 1973. Seasonal periodicity of plankton in a fresh water pond, West Bengal, India, Intl. Rev. Ges. Hydrological. 58: 127 – 143. Kumar, S., and Dutta, S.P.S., 1991. Studies on Phytoplankton population dynamics in Kunjawani pond, Jammu, J. hydrobiol., 7(1): 55 – 89. Mani, P. and Krishnamoorthy, K., 1989. Ecology of phytoplankton blooms in the velar estuary. East coast of India. Int. J. Mar. Sci. 15: 24 – 28. Rajasekar, K.J., Perumal, P., Santhanam, P., 2005. Phytoplankton diversity in the coleroon. Estuary, South East coast of India. J. Mar. Sci., Biol. Assoc. India. 47: 127 – 132. Rawson, D.S., 1956. Algal indicators of Tropic lake type. Limnology and oceano graphy. 1: 18 – 25. Saha, S.B., Bhattacharya, S.B., and Choudhary, A., 2000. Diversity of phytoplankton of a sewage pollution brackish water tidal ecosystem. J. Environ Biol., 21(1): 9 – 14. Sukunan, V.V., 1980. Seasonal fluctuations of plankton of Nagarajuna Sagar reservoir, Andhra Pradesh, India. J. Inland Fish. Soc. India. 12 : 79 – 91. Tiwari, A., and Chauthan, S.V.S., 2006. Seasonal phytoplankton diversity of Kitham Lake. Agra J. Environ. Biol. 27: 35 – 38. Zafaar, A.R., 1986, Seasonality in phytoplankton in some South Indian lakes Hydrobiol. 138: 117 – 187.

*Corresponding Author: Karthi N*

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P.G and Research Department of Zoology, Rajah Serfoji Government College (Autonomous), Thanjavur – 613 005 Tamilnadu, India. * Email: nkarthiphdzoology@gmail.com

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Research Article Pharmaceutical Sciences NEPHROTOXICITY INDUCED BY PAN MASALA IN SWISS MICE AND ITS PROTECTION BY ELETTARIA CARDAMOMUM (L.) MATON Sweety Kumari 1*, Abhijit Dutta1, Saabiya Farooqui1, Musarrat Naaz1, Swati Soren1, Sunita Dutta2, Abha Prasad2, Neeta Lal3, Anjali Smita4 1

Department of Zoology, Ranchi University, Ranchi, India Department of Zoology, Womenâ&#x20AC;&#x2122;s College, Ranchi, India 3 Department of Zoology, RLSY College, Ranchi, India 4 Department of Zoology, Nirmala College, Ranchi, India

*Corresponding Author Email: sweety25j@gmail.com

ABSTRACT This study was designed to investigate the protective effect of cardamom on renal tissue damage caused by pan masala. Experimental animals were divided into 3 groups, control, pan masala treated and cardamom along with pan masala treated. They were exposed till 12 months to observe changes histologically and ultrastructurally. Male Swiss mice were given orally pan masala at a dose of 2% of the feed which caused acute tubular necrosis along with dilation and atrophied glomerulus seen in its light microscopic structures, whereas ultrastructural changes showed pyknotic nucleus, swollen mitochondria and loss of membrane integrity. When cardamom was given at a dose of 0.2% along with pan masala or alone, damages were less showing normal glomerulus with less inflammation, and normal nucleus. It is, therefore, concluded that cardamom may be beneficial in preventing pan masala induced renal tissue damage and shows potential for clinical use.

KEY WORDS Cardamom, kidney, necrosis, pan masala

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INTRODUCTION The mammalian kidney is a target organ for a wide variety of toxic agents due to its prime function as a blood filter during the excretory process and is also sensitive to drug induced injury1. In this experiment, kidney is studied to assess the nephrotoxicity of pan masala and the protective properties of cardamom against it. Pan masala is a dry mixture of tobacco, areca nuts, slaked lime, catechu, and flavouring agents such as menthol, camphor, sugar, rosewater, aniseed, mint, or other spices are sometimes added in different regions. These have been found to promote excessive and harmful use and also lead to dependence2. It is sold in small

pouches in India, Pakistan, Srilanka, Bangladesh and in migrant populations from these regions in other countries3. It has become a very serious health hazard and its increasing trend has given rise to a number of human health problems like cancer, reproductive and developmental defects, cardiovascular problem etc. Various organs have been studied but the data on kidney is scarce. Thus, the present study was designed to evaluate the damages imposed by pan masala on kidney and its protection by cardamom.

MATERIALS AND METHODS The experiment was cleared by the Ethical committee, Ranchi University, Ranchi, for

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www.ijpbs.com (or) www.ijpbsonline.com conducting research on Swiss mice. Thirty male Swiss albino mice weighing 22Âą 5g acquired from B. N. Ghosh and Company, CIT Road, Kolkata, were housed in the laboratory under natural condition and allowed water ad-lib. Animals were randomly divided into three groups: control (fed with formulated feed), PMT (fed with formulated diet along with pan masala; 2% of the feed) and PMCT (formulated diet mixed with fixed combination of pan masala and cardamom, 2% and 0.2% of the feed) and were kept for 9 months4. Afterwards, mice from the group PMT and PMCT were continued with only cardamom along with the feed for another 3 months to check its ameliorating property. At the end of the experiment, mice from all groups were sacrificed by cervical dislocation under anaesthesia, and kidney was excised. The fragments from harvested tissue were fixed in

Bouinâ&#x20AC;&#x2122;s fixative, embedded in paraffin, stained and observed under light microscope. Another portion was fixed with a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer and processed to observe its ultrastructure by Philips CM-10 transmission electron microscope (Netherland) at AIIMS, New Delhi.

RESULTS Under the light microscope, the kidney of male mice in the control and sham control group showed a typical cortex represented by vascular glomerulus and convoluted tubules, which are lined by cylindrical epithelial cells (figure not shown). However, vascular changes with interstitial edema, severe inflammation and shrunken glomerulus were visible in PMT mice along with tubular dilation (Fig.1).

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Figure 1: Photomicrograph of PMT mice showing severe inflammation (*), also shown in the insat (40x). Dilation of tubules (arrow) and shrunken glomeruli (bounded by square). (9 months; H&E, 10x)

Cortical renal tubules show various degenerative changes with focal tubular necrosis, atrophied and fragmented glomerulus (Fig. 2). After 12 months including last 3 months of cardamom treatment, PMT still showed dilated tubule with slogged of epithelium and also tubules undergoing necrosis and atrophied glomerulus (Fig.3). Fig.4 represents the section of PMCT after 9 months of treatment of pan

masala along with cardamom. Normal glomerulus was visible, inflammation was less. Intact exfoliated tubular cells in tubular lumen were seen in the section showing ischemic injury. Signs of amelioration were clearer in PMCT, after cardamom treatment excluding pan masala for last 3 months, with normal glomerulus and tubules in renal cortex. (Fig. 5)

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Figure 2: Photomicrograph of PMT mice showing necrosis (*) and atrophied glomerulus (large arrow) alongwith fragmented glomerulus (small arrow). (9 months; Malloryâ&#x20AC;&#x2122;s triple stain, 40x)

Figure 3: Photomicrograph of PMT mice showing dilated Proximal Convoluted Tubule (T) and atrophied glomerulus (G). (12 months; Malloryâ&#x20AC;&#x2122;s triple stain, 40x)

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Figure 4: Photomicrograph of PMCT mice showing normal glomerulus (G) along with ischemic injury (*). (9 months; H&E, 40x)

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Figure 5: Photomicrograph of PMCT mice showing normal glomerulus (arrow) and necrotic tubules (*) in one part of the section showed. (12 months; Crossman stain, 10x)

Electron microscopy of injured tubules from PMT mice revealed swollen brush border microvilli and thinning of basal membrane in the proximal tubules while the control group showed array of

vertically oriented mitochondria and extensive infolding of the basolateral plasma membrane (Fig. 6 and Fig.7)

Figure 6: An electron micrograph of control kidney’s proximal convoluted tubule. mitochondria (M), nucleus (N), basal membrane (BL). (9 months; 1100x, scale bar-2µm).

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Figure 7: An electron micrograph of PMT kidney. There was a increase in the space between the interdigitations (arrow) and the basal membrane. Brush borders were swollen (**). (9 months; 1100x, scale bar-2µm).

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www.ijpbs.com (or) www.ijpbsonline.com Tubular necrosis was visible with the presence of pyknotic nucleus, swollen mitochondria, alterations in lysosomes and loss of other cytoplasmic organelles (Fig.8). Degenerative changes in mitochondria, including swelling and

loss of cristae are often visible (Fig.9). PMCT mice showed signs of amelioration with the presence of almost normal brush border, normal nucleus, and mitochondria in the proximal convoluted tubule (Fig.10).

Figure 8: An electron micrograph of PMT kidney. Note pyknotic nucleus (arrow), lysosomal vacuoles (*), and swollen mitochondria (M). (9 months; 1100x, scale bar-2µm).

Figure 9: An electron micrograph of PMT mice showing reduced invisible cristae (**) and, in some cases, exhibit lost membrane integrity (*). (9 months; 1100x, scale bar-2µm).

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Figure 10: An electron micrograph of PMCT mice showing amelioration. Basal membrane (BL), nucleus (N), mitochondria (M) (9 months; 1100x, scale bar-2µm).

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DISCUSSION The results of this work indicate that pan masala is directly nephrotoxic to mouse glomeruli and tubular kidney cells in vivo. Human victims can also develop severe renal damages with clinical signs and symptoms such as acute renal failure, usually due to acute tubular necrosis by consuming pan masala. The habit of using it is increasing because of the cheapness, bright pouches, easy availability, sweet taste and forceful misleading advertisements. Chewing tobacco could result in significantly greater deleterious cardiovascular effects due to a larger overall exposure owing to prolonged absorption 5 . Enhanced toxicity in proximal tubular cells in PMT occurs due to the extensive cellular uptake of pan masala by endocytosis and other transport pathways6, 7, 8. It also occurs via basolateral delivery of endogenous and exogenous organic ions (anions and cations) by peritubular capillaries6, 7. Transport of toxicants into cells, followed by movement through the intracellular space via various regulated carrier proteins, and subsequent exit from the cells via apical transport proteins enhances toxicity in proximal tubular cells. Loss of function mutations in and competition for apical secretory transporters9, which reduces toxin efflux from cell into urine, may promote accumulation of toxic substances within proximal tubular cell and cause cellular injury via apoptosis or necrosis. This extensive trafficking increases renal tubular exposure and risk for elevated concentration of pan masala. Another cause is the tissue inflammation due to pan masala exposure as observed in the PMT group which changes cell immune function, increased plasma concentration of cytokines, ACTH, cortisol, adrenaline, nor adrenaline and glucagon. Plasma free cortisol flows through glomeruli cause

increase in serum creatinine level and different degree of renal impairment10,11,12 . There are several toxic metals in pan masala such as lead and cadmium. They carry serious health risks as they can accumulate in the body and in the food chain and also pass through kidney during filteration process13. The kidney also possesses CYP450 enzymes that participate in drug metabolism14,15. Mutation of this gene by toxic pan masala could increase nephrotoxic risk as well6. Significant renal exposure to potential nephrotoxins, such as pan masala, occurs due to the high rate of this toxin delivery to the kidney, a result of the high blood flow to the kidney, which approaches 25% of cardiac output. Many renal cells, particularly those in the loop of Henle, exist in a relatively hypoxic environment due to the high metabolic rates required to actively transport many solutes via Na+窶適+ ATPase driven transport. This excess cellular workload and hypoxic environment promotes increased sensitivity to injury when exposed to pan masala, a potential nephrotoxic substance16, 17 . Many herbs are proved to decrease high blood pressure and improve kidney functions, including cardamom18. It prevents DNA damage, improves liver function, kidney functions, regulates pH balance and acts as a detoxifying agent. Cardamom diet showed also pronounced improvement of renal function19. Cardamom is a highly aromatic and distinctive spice possessing 8% essential oils. Constituents in cardamom include 1, 8 cineole, alpha-terpinyl acetate, limonene, and myrcene along with many other volatile terpenoids 20. 1, 8 cineole (eucalyptol) is a terpenoid compound that has been shown to possess anti-inflammatory capacity in the rat paw edema model of inflammation21.Thus, it can be concluded that the properties of cardamom should be utilized in the medical fields to protect

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CONFLICT OF INTEREST There is no conflict of interest in the research. 11.

ACKNOWLEDGEMENT The Authors like to thank AIIMS, New Delhi for providing TEM facility.

12.

REFERENCES 1.

13. 14.

15.

16.

17.

18. 19.

20.

21.

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Singh, N.P., Ganguli, A., Prakash, A., Drug-induced Kidney Diseases. J. Assoc. Physicians India, 51:970-979, (2003). Huang, Z., Xiao, B., Wang, X. et al., Betel nut indulgence as a cause of epilepsy. Seizure, 12: 406-408, (2003). Winstock, A.R., Trivedy, C.R. et al., A dependency syndrome related to areca nut use: Some medical and psychological aspects among areca nut users in the UK. Addiction Biol., 5: 173-179, (2000). Kumari S, Dutta A, Histochemical studies on the induced toxicity of Pan masala on various organs of Swiss mice and the protective effect of Elettaria cardamomum (L.) Maton. Annals of Biological Research, 3 (4):1919-1922, (2012). Gupta, B.K., Kaushik, A., Panwar, R.B. et al., Cardiovascular risk factors in tobacco-chewers: a controlled study. J. Assoc. Physicians India. 55: 27-31, (2007). Ciarimboli, G., Koepsell, H., Iordanova, M. et al., Individual PKC-phosphorylation sites in or-ganic cation transporter 1 determine substrate selectivity and transport regulation. J. Am. Soc. Nephrol., 16: 1562– 1570, (2005). Enomoto, A., Endou, H., Roles of organic anion transporters (OATS) and urate transporter (URAT1) in the pathophysiology of human disease. Clin. Exp. Nephrol., 9: 195– 205, (2005). Orbach, H., Tishler, M., Shoenfeld, Y., Intravenous immuno-globulin and the kidney—A two-edged sword. Semin. Arthritis Rheum., 34: 593–601, (2004).

Lang, F., Regulating renal drug elimination. J. Am. Soc. Nephrol., 16: 1535–1536, (2005). Allen, C. K.C., Lit, L.C.W.,et al., Diminished urinary free cortisol excretion in patients with moderate and severe renal impairment. Clin. Chem., 50(4): 757-759, (2004). Icapino, A.M., Cutler, C.W., Pathophysiological relationships between periodontitis and systemic disease: Recent concepts involving serum lipids. J. Periodontol., 71: 1375-1384, (2000). Fentoglu, O., Koroglu, B.K., et al., Pro-inflammatory cytokine levels in association between periodontal disease and hyperlipidaemia. J. Clin. Periodontol., 38 (1): 8-16, (2011). Rx Pharma ., Gutka (Asian Pan Masala) its use & side effects, (2011). Harty, L., Johnson, K.,Power, A., Race and ethnicity in the era of emerging pharmacogenomics. J. Clin. Pharmacol., 46:405–407, (2006). Ulrich, C.M., Bigler, J., Potter, J.D., Non-steroidal antiinflammatory drugs for cancer prevention: promise, perils and pharmacogenetics. Nature Review, 6: 130– 140, (2006). Cummings, B.S., Schnellmann, R.G., in: Schrier, R.W. (Ed.), Pathophysiology of nephrotoxic cell injury, Diseases of the Kidney and Uro-genital Tract. Lippincott Williams & Wilkinson, Philadelphia PA 2001, pp. 1071–1136, Kaloyanides, G.J., Bosmans, J.L., DeBroe, M.E., in: Schrier, R.W. (Ed.), Antibiotic and Immunosuppressionrelated renal failure. Diseases of the Kidney and Urogenital Tract. Lippincott Williams & Wilkinson, Philadelphia PA 2001, pp 1137–1174. www.secondary-nephropathy.com/hypertensivecommon-sense/264.html, (2012). El-Yamani, M.A.S., Cinnamon, cardamom and ginger impacts as evaluated on hyperglycemic rats. Research Journal Specific Education., 20: 665-678, (2011). Marongiu, B., A. Piras, S. Porcedda, Comparative analysis of the oil and supercritical C02 extract of Elettaria cardamomum (L.) Maton. J. Agric. Food Chem., 52(20): 6278-6282, (2004). Santos, F.A., Rao, V.S.N., Antiinflammatory and antinociceptive effects of 1,8-cineole a terpenoid oxide present in many plant essential oils. Phytother. Res., 14(4): 240-244, (2000).

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Department of Zoology, Ranchi University, Ranchi-834008, India

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Research Article Biological Sciences ATR-FTIR ANALYSIS OF TOPICAL CREAMS FORMULATED WITH CHROMOLAENA ODORATA ETHANOLIC EXTRACT AND HONEY: A WOUND HEALING STUDY M. F. Z. R. Yahya* and N. Ramlee School of Biology, Faculty of Applied Sciences, MARA Technology University, Shah Alam, 40450 Shah Alam, Selangor, Malaysia *Corresponding Author Email: fakharulzaman@salam.uitm.edu.my

ABSTRACT This study was carried out to evaluate wound healing effect of topical creams formulated with Chromolaena odorata ethanolic extract and honey. The topical creams were formulated as follows: cream A (95% base + 5% C. odorata ethanolic extract), cream B (90% cream base + 5% C.odorata ethanolic extract + 5% honey) and cream C (95% cream base + 5% honey). The experimental animals were segregated into five groups (each group consisting of five rats) as follows: group 1 (treated with cream A), group 2 (treated with cream B), group 3 (treated with cream C), group 4 (treated with positive control - Aloe vera cream) and group 5 (treated with negative control cream base). Epithelization period less than 14 days was observed in group 1 and group 4 while the other animal groups showed the epithelization period more than 14 days. Cream A demonstrated the highest wound healing rate, followed by cream C and cream B. Attenuated total reflectance fourier transform infrared spectroscopy (ATRFTIR) analysis demonstrated that there were five new absorption peaks [wavenumbers: 1159.2 cm-1 (amines), 1259.4 cm-1 (amines), 1456.7 cm-1 (alkanes), 1508.8 cm-1 (aromatics) and 2921.9 cm-1 (carboxylic acids)] present in spectrum of cream B as compared with those of other formulated topical creams and cream base. We proposed that aromatic diamines were produced during formulation of cream B and caused the delay of wound healing process in the experimental animals.

KEY WORDS Topical cream; wound healing; Chromolaena odorata; honey

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INTRODUCTION Wound is a type of tissue damage associated with disruption of its anatomic continuity [1]. It could be caused by physical, chemical, thermal, microbial or immunological insult to the tissue. Restoration of wounded tissue basically consists of integrated cellular and biochemical processes which lead to re-establishment of physical, structural and functional integrities of the wounded tissue. In particular, the wound healing process which begins just after surface lesions and or harmful exposures, involves various body responses such as coagulation, inflammation,

formation of granulation tissue and tissue remodeling [2]. Currently, there has been a great interest in exploring natural resources for wound healing potential. Chromolaena odorata, a perennial belonging to the plant family Asteraceae, is widely distributed in Southern Asia and Western Africa. It is a weed of 13 crops in 23 countries and has been described as the worldâ&#x20AC;&#x2122;s worst weed [3]. In addition to its antibacterial activity [4], C. odorata leaf extract has also been reported to exhibit promising wound healing effect.

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www.ijpbs.com (or) www.ijpbsonline.com According to [5], C. odorata ethanolic extract demonstrated protective effect on fibroblasts and keratinocytes against oxidative stress, which was due to its antioxidant content. By using liquid chromatography coupled with UV spectroscopy and mass spectrometry, the antioxidant contents of C. odorata ethanolic extract were found to be phenolic acids (protocatechuic, p-hydroxybenzoic, p-coumaric, ferulic and vanillic acids) and lipophilic flavonoid aglycones (flavanones, flavonols, flavones and chalcones). They concluded that a mixture of powerful antioxidant compounds contained in C. odorata ethanolic extract might be one of potential mechanism contributing to enhanced wound healing. Another in vitro study on wound healing activity of C. odorata ethanolic extract was conducted by [6]. It was observed that for fibroblasts, toxicity of hydrogen peroxide or hypoxanthine xanthine oxidase on cells was dose-dependent. At 400 and 800 microg/ml, C. odorata ethanolic extract showed maximum and consistent protective cellular effect on oxidant toxicity at low or high doses of oxidants while at 50 microg/ml concentration, it also had significant and slightly protective effects on fibroblasts against hydrogen peroxide and hypoxanthine-xanthine oxidase induced damage. Meanwhile, for keratinocytes, a dose-dependent relationship of oxidant toxicity was only seen with hydrogen peroxide but the protective action of C. odorata ethanolic extract correlated with oxidant dosage. C. odorata ethanolic extract at 400 and 800 microg/ml showed dosedependent effects with both low and high concentration of oxidants whilst at 50 microg/ml, it had no effect on keratinocytes. In that study, it was suggested that protection of cells against destruction by inflammatory mediators may be one of the ways in which C. odorata ethanolic extract contributes to wound healing. Taken together, C. odorata ethanolic

extract possesses a high wound healing potential. However, its synergistic wound healing effect in combination with honey which is well known to have wound healing activity remains uncertain. Therefore, this study was carried out to evaluate wound healing effect of topical creams formulated with C. odorata ethanolic extract and honey using excision wound model and attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR).

MATERIALS AND METHODS Plant extract Leaves of C. odorata and pure honey were collected from Kuala Rompin, town in the Rompin district of the State of Pahang, Malaysia. The leaves were washed, dried and ground into powder form. One hundred grams of the powder was then soaked in one liter of 100% ethanol. The solvent was then filtered using a Whatman No. 1 paper and removed using a rotary evaporator. Topical creams Topical creams were formulated using a combination of fermented rice as a cream base, C. odorata ethanolic extract and pure honey as follows: cream A (95% base + 5% C. odorata ethanolic extract), cream B (90% cream base + 5% C.odorata ethanolic extract + 5% honey), cream C (95% cream base + 5% honey). Phytochemical screening Chemical test for saponins, tannins and flavonoids were performed on the formulated topical creams using standard protocols. Experimental animals and wound healing activity A total of 25 male Sprague Dawley rats of approximately the same age, weighing about 150-250 g were used for the study. They were

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www.ijpbs.com (or) www.ijpbsonline.com fed with standard diet and water ad libitum. They were housed in polypropylene cages and maintained under standard conditions (12/12 hr light/dark cycle; 250C-300C). They were segregated into five groups (each group consisting of five rats) as follows: group 1 (treated with cream A), group 2 (treated with cream B), group 3 (treated with cream C), group 4 (treated with positive control - Aloe vera cream) and group 5 (treated with negative control - cream base). The experimental animals were subjected to ether anesthesia and their dorsal skin was shaved. Excision wound (four cm length) was created on the dorsal back of experimental animals using a forceps, surgical blade and pointed scissor. The formulated creams were then applied topically on the wound site on daily basis. Within 14 days of wound healing period, wound length and epithelization period were monitored. The procedures used in this animal study were in accordance with the ethical standard from the UiTM Research Committee on the Ethical Use in Research (UiTM Care).

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Attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) The samples (cream base and formulated topical creams) were placed in direct contact with horizontal attenuated total reflectance (ZnSe crystal) at controlled ambient temperature. The infra red spectra were recorded in the frequency range of 4000 cm-1 to 400 cm-1. Comparison of infra red spectra was made between the cream base and formulated creams. Statistical analysis Wound length (cm) was expressed as mean ± standard error measurement. The significant difference (p<0.05) between the animal groups was determined using one-way ANOVA.

RESULTS AND DISCUSSIONS Solvent extraction has been a common method to extract phytochemical constituents from medicinal plants. In this study, ethanol solvent was used because it is known to be effective in extracting the important bioactive compounds that exhibit wound healing potential. The leaves of C. odorata were extracted using 100 % ethanol solvent in four different duration of soaking times namely 24, 48, 72 and 96 hours. Table 1 shows effect of soaking time on C. odorata plant extract yield. It was found that 72 hour soaking time produced the highest percentage (13.12±0.29%) of C. odorata plant extract yield whilst 24 hour soaking time produced the lowest percentage (11.11±0.09%) of C. odorata plant extract yield. It seems that the soaking time did not apparently affect the percentage of C. odorata plant extract yield. Phytochemical constituents play key role in determining biological activities of medicinal plants. In this study, topical creams were prepared by formulating cream base (fermented rice) with C. odorata ethanolic extract and pure honey. With respect to wound healing activity of C. odorata ethanolic extract, the phytochemical screening was conducted on the formulated topical creams in order to determine the presence of saponins, tannins and flavonoids compounds. Table 2 shows the presence (indicated by +) or absence (indicated by -) of those chemical compounds in base (fermented rice) and formulated topical creams. It was observed that saponin compounds were absent in cream base and cream A, and present in cream B and C. Meanwhile, tannin and flavonoid compounds were present in all test samples except cream base. This result was slightly different from a previous study [7] reporting the presence of saponins in the ethanolic extract of C. odorata, which could be attributed to

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www.ijpbs.com (or) www.ijpbsonline.com different geographical locations where the plants were collected. The leaves of C. odorata in that

Soaking Time (Hours) 24 48 72 96

study were collected from the University of the Philippines Diliman, Quezon City, Philippines.

Table 1: Soaking time and percentage of plant extract yield Percentage of plant extract yield (%) 1 2 3 Average 13.11 13.22 13.00 11.11 11.89 12.83 12.41 12.38 13.44 13.18 12.74 13.12 12.82 12.32 13.27 12.95

S.E.M 0.09 0.38 0.29 0.39

Creams

Table 2: Phytochemical screening of formulated creams Bioactive components Saponins Tannins

Flavonoids

Cream base Cream A Cream B

C.odorata ethanolic extract C.odorata ethanolic extract + honey

+ +

Cream C

honey

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Table 3 Effect of topical application of formulated creams on epithelization period Animal groups Creams Epithelization period (days) Group 1 Cream A < 14 Group 2 Cream B > 14 Group 3 Cream C > 14 Group 4 +ve Control < 14 Group 5 -ve Control > 14 Epithelization period is a common method used in the study of wound healing activity of the medicinal plants. In brief, epithelization means growth of epithelium at the centre of wound site after injury. In this study, the epithelization period was determined within 14 days following topical application of the formulated creams on dorsal back of experimental animals. Table 3 shows the epithelization period recorded for all animal groups. The epithelization period less than 14 days was observed in group 1 (Cream A â&#x20AC;&#x201C; cream base + C. odorata ethanolic extract) and group 4 (positive control â&#x20AC;&#x201C; Aloe vera cream) while the other animal groups showed the epithelization period more than 14 days. It was

suggested that the wound healing activity of cream A was comparable with that of positive control. In addition to the epithelization period, wound length also has been one of the common parameters to evaluate wound healing activity of medicinal plant. It is normally measured on daily basis after wound excision has been made on dorsal back of animals. In this study, the wound length was measured using analytical scale within 14 days following topical application of the formulated creams. Table 4 and Figure 1 show length and healing of wound of different animal groups. To evaluate the wound healing effect of the formulated topical creams clearly, day 2, 8 and 14 post surgical wound were

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highlighted. It was observed that, the wound length apparently reduced from day 2 onwards in all animal groups except group 5. At day 8, it was also found that the wound healing activity of cream A (1.36±0.15 cm) was comparable with that of positive control (1.32 ± 0.43 cm). Meanwhile at day 14, all animal groups exhibited the wound length less than one cm except group 5 (2.20±0.45 cm). Taken together, cream A demonstrated the highest wound healing rate, followed by cream C and cream B. Because the cream B was formulated with C. odorata ethanolic extract and pure honey, it was possible that the formulation suppressed its wound healing effect. In vivo wound healing effect of C. odorata leaf extract and pure honey has also

been investigated by [8]. In that study, C. odorata aqueous extract and pure honey were topically applied on the posterior neck area of Sprague Dawley rats. In particular, group 1, 2, 3, and 4 were treated with normal saline (negative control), honey, a combination of honey and C. odorata aqueous extract, and solcoceryl jelly (positive control) respectively. It was found that the wounds treated with solcoceryl jelly and honey in combination with C. odorata aqueous extract healed significantly earlier than honey alone which indicated that the synergistic effect resulting from the combination was comparable with the positive control. However, our finding was not in agreement with [8] which might be due to different plant extraction method.

Table 4 Effect of topical application of formulated creams on wound length (cm). Values are expressed as mean ± standard error measurement with n=5.

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Animal groups

Creams

Wound length (cm) Day 2

Day 4

Day 6

Day 8

Day 10

Day 12

Day 14

Group 1

Cream A

3.06±0.17

2.44±0.09

1.94±0.24

1.36±0.15

1.00±0.23

0.48±0.20

0.10±0.14

Group 2

Cream B

3.06±0.23

2.62±0.22

2.36±0.38

2.18±0.38

1.92±0.50

1.30±0.41

0.92±0.37

Group 3

Cream C

3.12±0.19

2.94±0.05

2.40±0.26

2.22±0.26

1.74±0.27

1.04±0.29

0.36±0.18

Group 4

+ve Control

3.12±0.41

2.68±0.32

2.12±0.40

1.32±0.43

0.68±0.26

0.12±0.16

0.00±0.00

Group 5

-ve Control

3.54±0.11

3.22±0.11

2.92±0.23

2.76±0.34

2.62±0.37

2.40±0.44

2.20±0.45

In the context of medicinal agent, synergistic effect is an increased intensity of healing or protective effect due to a combination of two or more bioactive substances. C. odorata ethanolic extract was mixed with pure honey of known wound healing agent in order to investigate the possible synergistic wound healing effect of the formulated topical cream. However, based on Table 4 and Figure 1, the cream B (cream base + C.odorata ethanolic extract + honey) was found

to exhibit wound healing effect less than cream A and C. Therefore, to understand this, ATR-FTIR analysis was carried out to determine chemical composition of all the formulated topical creams. Figure 2 displays ATR-FTIR spectra of the formulated topical creams and cream base. It was demonstrated that there were five new absorption peaks present in spectrum of cream B as compared with those of other formulated topical creams and cream base. The

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www.ijpbs.com (or) www.ijpbsonline.com wavenumber of those new absorption peaks were as follows: 1159.2 cm-1 (amines), 1259.4 cm-1 (amines), 1456.7 cm-1 (alkanes), 1508.8 cm-1 (aromatics) and 2921.9 cm-1 (carboxylic acids). This indicated that aromatic diamines were possibly formed when the cream base formulated with C. odorata ethanolic extract and pure honey. In 2005, Shin et al. [9] studied suppressive effect of novel aromatic diamines on nuclear factor-kappa B (NF-KB), an inflammatory factor. It was noted that the aromatic diamine compound (N1-benzyl-4-methylbenzene-1, 2diamine, BMD) suppressed activation of NF-KB which in turn reduced the inflammation. It was also shown that the BMD compound had therapeutic potential in nitric oxide (NO)associated inflammatory diseases. Furthermore, dermal absorption of the aromatic diamines has been investigated by [10]. They found that the aromatic diamines (2, 5-toluylenediamine, 2, 5TDA) which is a contaminant in hair dye

formulation, was rapidly absorbed dermally based on GCMS method. After a distribution phase of 12 hours, 2, 5-TDA was excreted with a half-time of 8 hours. Excretion was 90% complete within 24 hours after application. The doses of 2, 5-TDA excreted within 48 hours were 700 Îźg for application of a brown-reddish hair dye cream and 1.5 mg for the application of a brown-black hair dye cream. In relation to these, the aromatic diamines observed in cream B might play a role in the wound healing effect. Considering the fact that the inflammation is one of the important stages in acceleration of wound healing [2], it was proposed that the aromatic diamines partially suppressed the inflammation which in turn delayed the wound healing process in the experimental animals topically treated with cream B. However, it remains poorly understood how the aromatic diamines could be produced during formulation of cream B.

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Figure 1 Photographs of rats showing various phases of wound healing. A: cream A (cream base + C.odorata ethanolic extract); B: cream B (cream base + C.odorata ethanolic extract + honey); C: cream C (cream base + honey); D: positive Control (Aloe Vera cream).

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Figure 2: ATR-FTIR spectra of cream base and formulated topical creams. Arrows indicate the new absorption peaks [wavenumbers: 1159.2 cm-1 (amines), 1259.4 cm-1 (amines), 1456.7 cm-1 (alkanes), 1508.8 cm-1 (aromatics) -1 and 2921.9 cm (carboxylic acids)] in comparison of all spectra.

Base

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Cream A (95% base + 5% C. odorata ethanolic extract)

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Cream B (90% cream base + 5% C.odorata ethanolic extract + 5% honey)

Cream C (95% cream base + 5% honey)

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CONCLUSION We have demonstrated that cream A (cream base + C. odorata ethanolic extract) exhibited the highest wound healing rate, followed by

cream C (cream base + honey) and cream B (cream base + C. odorata ethanolic extract + honey). The ATR-FTIR spectrum of cream B was found to contain five new absorption peaks

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www.ijpbs.com (or) www.ijpbsonline.com which correspond to amines, alkanes, aromatics and carboxylic acids, when compared with spectra of other formulated topical creams and cream base. It was likely that the aromatic diamines were produced during formulation of cream B and caused the delay of wound healing process in the experimental animals.

REFERENCES [1] [2]

[3]

[4]

[5]

Bennet, R.G. Fundamentas of cutaneous surgery. St. Louis : C. V. Mosby, 778. (1988). Degim, Z., Celebi, N., Sayan, H., Babul, A., Erdogan, D. and Take, G. An investigation on skin wound healing in mice with a taurine-chitosan gel formulation. Amino Acids, 22, 187-198. (2002). Holme G.L.R, Plucknet D.L., Pancho J.V. and Herberger J.P. Chromolaena odorata, The Worldâ&#x20AC;&#x2122;s Worst Weeds: Distribution and Biology: The University Press of Hawaii (1977). Sukanya, S. L., Sudisha J., Hariprasad P., Niranjana S. R.,. Prakash H. S and Fathima S. K. Antimicrobial activity of leaf extracts of Indian medicinal plants against clinical and phytopathogenic bacteria. African Journal of Biotechnology Vol. 8 (23), pp. 6677-6682. (2009). Phan, T., Wang, L., See, P., Grayer, R. J., Chan, S.Y. and Lee, S.T. Phenolic compounds of Chromolaena odorata protect cultured skin cells from oxidative

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|239-247 damage: implication for cutaneous wound healing. Biological & Pharmaceutical Bulletin, 24 (12), 13731379. (2001). [6] Thang, P.T., Patrick, S., Teik, L.S. and Yung, C.S. Antioxidant effects of the extracts from the leaves of Chromolaena odorata on human dermal fibroblasts and epidermal keratinocytes against hydrogen peroxide and hypoxanthineâ&#x20AC;&#x201C;xanthine oxidase induced damage. Burns 27 (4), 319-327. (2001). [7] Pierangeli G.V. and Windell L.R. Antimicrobial activity and cytotoxicity of Chromolaena odorata (L. f.) King and Robinson and Uncaria perrottetii (A. Rich) Merr. extracts. Journal of Medicinal Plants Research Vol. 3(7), pp. 511-518 (2009). [8] Mahmood, A.A., Sidik, K., Suzainur, K.A.R., Indran, M. and Salmah, I. Evaluation of In-vivo wound healing activity of Chromolaena odorata leaf extract on excision wound model in rats. Journal of food technology, 3 (2), 126-129. (2005). [9] Shin H.M., Byung H. K., Eun Y.C., Jung S.H., Yeong S. K., Kyung R. M. and Kim Y. Suppressive effect of novel aromatic diamine compound on nuclear factorkappaB-dependent expression of inducible nitric oxide synthase in macrophages. Eur J Pharmacol. 521(1-3):1-8 (2005). [10] Schettgen T., Heinrich K., Kraus T. and Gube M. Determination of 2,5-toluylenediamine (2,5-TDA) and aromatic amines in urine after personal application of hair dyes: kinetics and doses. Arch Toxicol. 85(2):127133 (2011).

*Corresponding Author:

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Review Article Pharmaceutical Sciences TABLET COATING INDUSTRY POINT VIEW- A COMPREHENSIVE REVIEW *

B.VENKATESWARA REDDY, K. NAVANEETHA, B.RASHMITHA REDDY

Department of Pharmaceutics, St.Paul's College of Pharmacy, Turakayamjal village, Hayath Nagar Mandal, Ranga Reddy Dist-501510, A.P. *Corresponding Author Email:

ABSTRACT Tablet Coating is a process by which an essentially dry, outer layer of coating material is applied to the surface of a dosage form in order to confer specific benefits over uncoated variety. The advantages of tablet coating are taste masking, odor masking, physical and chemical protection, protection of the drug from the gastric environment etc. The important reason to coat a pharmaceutical dosage form is to control the release profile and bioavailability of the active ingredient. The various techniques of coating such as sugar coating, film coating, enteric coating. The amount of coating solution applied and the thickness of the coating layer determines the release of the drug from the delivery system. Tablets are usually coated in conventional coating pans by spraying the coating solution on the free surface of the tablet bed and subsequent drying of the solution. The main focus of this review is, to study various key factors associated with coating, , latest techniques of coating such as dip coating, laminated coating, electrostatic, vacuum film coating and problem encountered during the coating process.

KEY WORDS Tablet coating, laminated coating, Pharma industry, gastric environment, bioavailability.

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INTRODUCTION TABLET: A tablet is a pharmaceutical dosage form. It comprises a mixture of active substances and excipients, usually in powder form, pressed or compacted into a solid. Tablets Dosage form is one of a most preferred dosage form all over the world. Almost all drug molecules can be formulated in a tablet and process of manufacturing of tablets is very simple, and is very flexible. One can administered 0.01 mg of a drug dose to 1 gm of a drug dose by oral route of administration, by formulating as a tablet. TABLET COATING: Coating is a process by which an essentially dry, outer layer of coating material is applied to the surface of a dosage form in order to confer specific benefits over uncoated variety.

Coating may be applied to a wide range of oral solid dosage form, such as particles, powders, granules, crystals, pellets and tablets. When coating composition is applied to a batch of tablets in a coating pan, the tablet surfaces become covered with a tacky polymeric film. Before the tablet surface dries, the applied coating changes from a sticky liquid to tacky semisolid, and eventually to a nonsticky dry Surface pans. The entire coating process is conducted in a series of mechanically operated acorn-shaped coating pans of galvanized iron stainless steel or copper. The smaller pans are used for experimental, developmental, and pilot plant operations, the larger pans for industrial production.

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Objectives to coat a tablet THERAPY • To avoid irritation of oesophagus and stomach • To avoid inactivation of drug in the stomach • To modify the drug release • To improve patient compliance • To mask the bitter taste TECHNOLOGY • To decrease the influence of moisture and atmosphere. • Reduces the risk of interaction between incompatible material • Improve the drug stability • To prolong the shelf life of the drug MARKETING • To avoid bitter taste • To improve product identity • To improve the appearance and acceptability • In improving product robustness Disadvantages of tablet coating • Disadvantages of sugar coating such as relatively high cost, long coating time and high bulk have led to the use of other coating materials. • However the process of coating is tedious and time-consuming and it requires the expertise of highly skilled technician. KEY FACTORS 1. TABLET PROPERTIES • Shape • Tolerance • Surface area 2. COATING PROCESS • Equipments • Parameters • Facility and ancillary equipment • Automation 3. COATING COMPOSITION • Polymers • Solvents • Plasticizers • Colorants

• Tablets that are to be coated must possess the proper physical characteristics. In the coating process, the tablets roll in a coating pan or cascade in the air stream of an air suspension coating as the coating composition is applied. • To tolerate the intense attrition of tablets striking other tablets or walls of the coating equipment, the tablets must be resistant to abrasion and chipping. • Tablet surfaces that are brittle, that soften in the presence of heat, or that are affected by the coating composition tend to become rough in the early phase of the coating process and are unacceptable for film coating. • The ideal tablet shape for coating is a, sphere, which allows the tablets to roll freely in a coating pan, with minimum tablet to tablet contact. The more convex the surface is, the fewer difficulties will be encountered with tablets agglomeration. • The surface properties of the tablet depend on the chemical nature of the ingredients utilized in the formulation. 2. COATING PROCESS Three types of equipments A. Conventional Pan Systems a) Pellegrini system b) Immersion-sword system c) Immersion –tube system B. Perforated Pans Systems a) Accela-coata b) Hi-coater systems c) Driacoater d) Glatt coater C. Fluid Bed Systems

A. Conventional pan systems: The standard coating pan system consists of a circular metal pan mounted somewhat angularly on a stand. The pan is 8-60 inches

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diameter and is rotated on its horizontal axis by a motor. Heated air is directed into the pan and onto the tablet bed surface, and is exhausted by means of ducts positioned through the front of the pan.

Pellegrini pan: • Has a baffled pan and diffuser for uniform distribution of drying air. • It is enclosed and automated.

B. Perforated pan system: It consists of a Perforated or partially perforated drum that rotates on its horizontal axis in an enclosed housing. a) Accela-Coata and Hi-coater system: Drying air is directed in to drum, is passed through bed, and is exhausted through perforations in to drum.

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b) Immersion-sword system: • Drying air is introduced through a perforated metal sword immersed in the tablet bed. • The drying air flows upward through bed. • Coating solutions are applied by an atomized spray system directed onto the tablet bed surface.

c) Immersion-tube system: • The tube immersed delivers heated air and • Coating solution is applied through spray nozzle built in the tip of tube • The drying air flows upward through the tablet bed and is exhausted by a conventional duct.

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Hi-coater system

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b) Driacoater: • Introduces drying air through hollow perforated ribs located inside periphery of the drum. • As the coating pan rotates, ribs dip into tablet bed • Drying air passes up through and fluidizes the tablet bed • Exhaust is from the back of the pan.

c) Glatt coater : • Drying air is directed from inside the drum through the tablet bed and out an exhaust duct • It consists of an optional splitchambered plenum, drying air can be directed in the reverse manner up through the drum perforations for partial fluidization of the tablet bed. • Several air flow configurations are possible.

C. Fluidized bed (Air suspension system): • These are highly efficient drying systems • Fluidization of tablet bed is achieved in a columnar chamber by the upward flow of drying air. • The airflow is controlled so that more air enters the center of the column, causing the tablets to rise in the center. • The movement of tablets is upward through the center of the chamber. They then fall towards the chamber wall and move downward to re-enter the air stream at the bottom of the chamber. • Coating solutions are continuously applied from a spray nozzle located at the bottom of the chamber or are sprayed onto the top of the cascading tablet bed by nozzles located in the upper region of the chamber.

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Spray application System Two basic types of spray system differ in manner in which atomization of liquid is achieved 1. High-pressure, air less system - Liquid pumped at high pressure (250-3000 psig) through small orifice, fine spray. Degree of atomization depends on Fluid pressure, Orifice size,Viscosity of liquid

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2. Low-pressure, Air atomized - Liquid pumped through large orifice at low pressure (5-50 psig). Air contacts liquid stream at tip of atomizer and fine spray is produced. Controlling variables-Fluid pressure, Fluid cap orifice, Viscosity of liquid, Air pressure, Air cap design Choice depends on coating solution formula and on the process developed for a particular product.

Parameters In a continuous coating operation, the coating operation is maintained essentially at equilibrium, where the rate of application of the coating composition equals the rate of

evaporation of volatile solvent. Deviation from these results serious coating problems. Mathematical modeling for automated aqueous coating process. Inlet A (T 1, H 1) +C 1 (S) +pSA 1 A (T 2, H 2) +C 2 +pSA 2 + Exhaust Where, A (T, H) = Air capacity, C(S) = Coating Composition, pSA = Tablet surface area, E = Equipment efficiency. • Air Capacity • Coating Composition • Tablet Surface Area • Equipment efficiency Air Capacity: It represents the quantity of water or solvent that can be removed during coating process. This depends on: • Quantity of air flow through tablet bed, • Temperature of air, • Water content of inlet air. Coating Composition: The coating contains the ingredients that are to be applied on the tablet surface and the solvents, which acts as a carrier for the ingredients. • The inlet air provides the heat to evaporate the water/ solvent. • The exhaust air becomes cooler and contains more water, owing to the evaporation of the solvent from the coating composition. • If the tablet surfaces are permeable to the applied coating solution, it can cause coating difficulties. • Most of the coating composition is solvent, so rapid removal is necessary to prevent adverse effects on tablet integrity. • Thin, rapidly drying formulations dry quickly on the tablet surface, allowing constant application by efficient atomization of coating solution Tablet surface area: • The size of the tablet and presence of debossed features affects the coating conditions.

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www.ijpbs.com (or) www.ijpbsonline.com • The total surface area per unit weight decreases significantly from smaller to larger tablets. • For same thickness of film, smaller tablets requires more coating composition as compared with larger tablets • Size of atomized coating droplets must be smaller and better controlled as the features to be coated become smaller. Equipment efficiency, E:

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Net increase in coated tablet weight Coating Efficiency, E = --------------------------------------------Non volatile coating weight applied to tablets

• Ideally, 90 to 95% of the applied film coating should be on the tablet surface. • Coating efficiency for conventional sugar coating is much less and 60% would be acceptable. • This significant difference in coating efficiency between sugar coating and film coating relates to the quantity of coating material that accumulates on the pan walls. • With an efficient film coating process, little coating material accumulates on the wall, but with sugar coating, the pan walls become thickly covered with coating. • A common cause of low film coating efficiency is that the application rate is too slow for the coating conditions (large tablet surface area, high airflow, and high temperature). • This results in drying part of the coating composition before it reaches the tablet surfaces, so that it is exhausted as dust. Facility and Ancillary Equipment  The facility required for any coating operation should be designed to meet the requirements of cGMP.  Adequate space is needed for the coating equipment, solution preparation and inprocess storage.

 The safety requirements depending on nature of solvent, where explosive or toxic concentrations of organic solvents could occur, during either solution preparation or the coating operation, electrical explosionproofing, specialized ventilation are required.  Exhaust air treatment may be done to recover solvent or to prevent entry in to atmosphere.  Federal EPA defines limits of organic solvents and particulate allowed in atmosphere.  Other Equipments needed to support the coating operation are Tanks, filters, mixers, mills, jacketed tanks, portable pressure tanks or pumping systems. Automation  It involves the development of a process in which all the important variables are predetermined.  Through a series of sensors and regulating devices for temperature, airflow, spray rate and pan speed, a feed back control of the process is maintained. TYPES OF COATING PROCESSES Three main types of coatings used in the pharmaceutical industry are • Sugar coating • Compression coating • Film coating 1. SUGAR COATING: • It involves successive application of sucrose based coating formulations to tablet core, in suitable coating equipment. • Water evaporates from the syrup leaving a thick sugar layer around each tablet. • Sugar coats are often shiny and highly colored. • Typically, tablets are sugar coated by panning technique, using traditional rotating

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•

sugar-coating pan with a supply of drying air (thermostatically controlled). The pan is automatically rotated, allowing tablets to tumble over each other while making contact with the coating solutions which are gently poured or sprayed, portion wise onto the tablets with warm air blown to hasten drying. Each coat is applied only after the previous coat is dried.

WHY? • In order to round off the tablet edge. Much of this material build-up occurs during this stage and is achieved by adding a bulking agent such as Calcium carbonate, to the sucrose solution. • Antiadherents e.g. Talc may be added after partial drying to prevent sticking of the tablets together. 3. Smoothing / syrup coating • To cover and fill in the imperfections in tablet surface caused by subcoating. • To impart desired color • The first syrup coat contains some suspended powders and are called “grossing syrups” • Dilute colorants can be added to provide tinted base that facilitates uniform coating in later steps. • Syrup solutions containing the dye are applied until final size and color are achieved. 4. Finishing • Final syrup coating step • Few clear coats of syrup may be applied. 5. Polishing • Desired luster is obtained in this final step • Clean standard coating pan, canvas-lined coating pans • Application of powdered wax or warm solution of waxes in suitable volatile solvent

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STEPS IN SUGAR COATING: • Seal coating • Sub coating • Syrup coating/Smoothing • Color coating • Polishing 1. Sealing (Waterproofing) • This involved the application of one or more coats of a water proofing substance in the form of alcoholic spray, such as pharmaceutical Shellac (traditionally) or synthetic polymers, such as CAP. • (WHY Sealing?) • Sugar-coatings are aqueous formulations which allow water to penetrate directly into the tablet core and thus potentially affecting product stability and possibly causing premature tablet disintegration. 2. Subcoating • Large quantities of sugar-coatings are usually applied to the tablet core ,typically increasing the tablet weight by 50- 100%

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2. COMPRESSION COATING • Although less popular, it gained increased interest in recent years for creating modifiedreleased products involves the compaction of granular materials around preformed tablet core using specially designed tableting equipment. • Compression coating is a dry process. • After tablet / core manufacture, it is transferred ( centrally positioned) to a slightly larger die that is partially filled with a coating powder. More coating powder is filled on the top of the core and compressed again resulting in tablet with in tablet. • Mechanically, it a complex process, as a tablet may be tilted when transferred to a secondary die cavity.

Need for compression coating • Traditionally, to separate incompatible material (one in core and other in coat). There is an interface between two layers and thus compromise product stability. It is possible to apply an inert placebo coating

layer first, to separate core from the final coat. • Used to create modified release product. 3. Film Coating Film coating and sugar coating shares the same equipments and process parameters. Two methods, 1) Pan-Pour method: • Same as that of pan-pour sugar coating • Method is relatively slow and relies heavily on skill and technique of operator • Aqueous based film coating is not suitable due to localized over-wetting. 2) Pan-Spray method: • Use of automated spraying system Types of film coating • Immediate release • Modified release Film coating formulation (Composition of the coating liquid) 1. POLYMER / FILM FORMER: A film former capable of producing smooth thin films reproducible under the prescribed coating conditions. Classified as 1. Non enteric materials E.g. HPMC, MHEC, EC, HPC, Povidone, SCMC, PEG, Acrylate Polymers

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2. Enteric materials E.g. CAP, Acrylate Polymers, HPMCP, PVAP. POLYMERS FOR FILM COATING Immediate release coating polymers

Modified release coating polymers

1.Cellulose derivatives: The mostly widely used of cellulosic polymers is HPMC (WHY?)

Extended release They are dissolved in Organic solvent or dispersed in aqueous medium (why for enteric coating?) Cellulosic derivatives: Highly substituted cellulosic ether, thus rendering the polymer water-insoluble. E.g. Ethyl cellulose

It is readily soluble in aqueous media

Forms film with good mechanical properties ( strength, flexibility and adhesion to the tablet core)

- Easy application of the coat Other examples are MC & HPC 2. Vinyl derivatives: PVP, it has a limited use in film coating because of its inherent tackiness. A copolymer of PVP and vinyl acetate forms better films.

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ENTERIC MATERIALS:  Reasons for enteric coating  Protect acid labile drugs from gastric fluid e.g. enzymes  Prevent gastric distress or nausea due to irritation from drug, e.g. Sodium Salicylate  To deliver drugs for local action in intestines, e.g. Intestinal antiseptic (Kanamycin)  For drugs optimally absorbed in small intestine  Provide delayed release component for repeat-action tablets Material should have following properties:  Resistance to gastric fluids  Ready susceptibility or permeability to intestinal fluids  Stability with coating composition and drug substrate

Enteric coating 1.Methacrylic acid copolymers: The presence of carboxylic acid groups renders this class to be insoluble in water at low PH (stomach) but gradually becomes soluble as the PH rises. E.g. Ethyl cellulose towards neutrality (upperpart of the small intestine). 2. Phthalate esters: e.g. cellulose acetate phthalate

   

Stability alone and in coating solution Formation of continuous film Nontoxic, Low cost Ease of application without special equipments  Ability to be printed or to allow film to be applied to debossed tablets 2. PLASTICIZER: These afford flexibility and elasticity to the coat and thus provide durability. They are of two types: • Internal plasticizers: Chemical modification of the polymer that alters the physical properties. o Degree of substitution o Type of substitution o Chain length. • External plasticizers : They are non-volatile or the other polymer, which when include with primary polymeric film former, changes the o Flexibility

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o Tensile strength, or o Adhesion properties of the resulting film. Concentration of Plasticizer Expressed as the amount of polymer being plasticized. Recommended Level of Plasticizer: 1 to 50% by weight of the film former. EXAMPLES • Castor oil; propylene glycol of 200 and 400 series; and surfactants eg; Tweens; Spans; and organic acid esters. • Water- soluble plasticizer: PEG, propylene glycol. • Organic- soluble plasticizer : castor-oil and Spans 3. COLORANTS • Colorants may be soluble in the solvent system or suspended as insoluble powders. • Used to provide distinctive color and elegance to a dosage form. • To achieve proper distribution of suspended colorants in the coating solutions requires the use of finepowdered colorants (< 10 microns ). • Most of colorants are synthetic DYES or LAKES OF DYES approved by the FD&C and D&C. LAKES: derived from dyes by precipitating with carriers. Eg; alumina or talc. • Lakes contains 10 to 30 % of the pure dye content. • For very light shade, concentration: less than 0.01 %. • For dark shade, concentration : more than 2.0 % • Examples – Inorganic materials: iron oxides – Natural coloring materials: Anthocyanins, caramel, carotenoids, chlorophyll, indigo, flavones, turmeric, and carminic acid.

4. OPAQUANT-EXTENDERS • These are very fine inorganic powders used in the coating solution formulation to provide more pastel colors and increase film coverage. • Opaquant provides a white coating or mask the color of the tablet core, and thus the less amount of the colorants are required. • Examples: Silicates (talc, aluminium silicate); Carbonates (magnesium carbonate); Sulfates (calcium sulfate); Oxides (Mg oxides) 5. SOLVENTS Volatile organic solvents may be used to allow good spreadability of the coat components over the tablet and allowing rapid evaporation, but they are expensive and show environmental hazards. Aqueous vehicles are safer, but they show slower evaporation and may affect drug stability. Ideal characters of coating material  Solubility in the coating solution.  Capacity to produce elegant looking product.  Stability in presence of water, heat, moisture, air, and substrate being coated and no change in properties with aging.  Essentially no odor or taste.  Compatibility with common coating solution additives.  Nontoxic and ease of application.  Resistance to cracking and should act as barrier.  No bridging or filling of the debossed tablet surfaces by the film former.  Ease of printing procedure on highspeed equipment.  Low cost & Ease of application without specialized equipment. Advances in tablet coating Specialized coating techniques include

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1. Dip coating 2. Electrostatic coating 3. Laminated coating 4. Vacuum film coating 5. Dry coating 1. DIP COATING:  In this, cores to be coated are a held in a suitable device eg: baskets  Dipped into coating solution and then dried taking care to prevent adherence to one another.  For obtaining more perfect or heavier coats the dipping and drying steps may be repeated several times one after another.  Several dipping arrangements are obtainable, amongst them the

sophisticated devices comprise tiny suction tubes, which hold the individual tablets apart until drying is accomplished.  Before proceeding to coat additional tablets or begin recoating cycles. 2. ELECTROSTATIC-COATING  Electrostatic coating is an efficient method of applying coating to conductive substances.  In this, an ionic charge is imparted to the core and an opposite charge to the coating material. This technology ensures thin, continuous and electronically perfected film to the surface.

3. LAMINATED-COATING  Laminated coating provides multiple layers for incorporation of medicament.  For example  Repeat-action tablet, here a portion of the drug is kept in outer lamella or coating.  Enteric tablet, here one drug could be made available for gastric absorption while another for release in intestine.  Buccal-swallow tablet, this could first be administered sublingually, and upon a signal, such as release of flavour from

the inner core, the same may be swallowed as a normal peroral tablet. 4. VACCUM FILM-COATING  This employs a specially designed baffled pan, which is water-jacketed and could be sealed to achieve vacuum.  Tablets are placed in the sealed pan, the vacuum is applied and the coating material is introduced through airless hydraulic spray system. Since the pan is completely sealed.

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Organic solvents could be effectively used with minimal environmental or safety concern.

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5. DRYCOATING  Dry coating avoids the use of water or, at least, allows it to be reduced to very small amounts with respect to the coating material, thus overcoming the need for time- and energy-consuming drying phases, as well as possible drug stability issues.  In this technology, powdered coating materials are directly coated onto solid dosage forms without using any solvent, and then heated and cured to form a coat. Problems encountered in Coating 1. Sticking & Picking 2. Roughness 3. Orange peel effect 4. Bridging 5. Filling 6. Blistering 7. Hazing/Dull film 8. Color variation 9. Cracking 1. STICKING & PICKING • Over wetting or excessive film thickness causes tablets to stick each other or to the coating pan. • On drying at the point of contact, a piece of film may remain adhere to pan or tablet, giving “picked” appearances to the tablet surface. Resulting in a small exposed area of the core. Remedies • Reduction in liquid application rate. • Increase in drying air temperature and air volume. 2. ROUGHNESS

•

A rough or gritty surface observed when the coating is applied by spray. Some of the droplets may dry too rapidly before reaching the tablet bed and spray- dried particles deposits on tablet surface instead of finely divided droplets of coating solution. Surface roughness also increases with pigment concentration and polymer concentration in the coating solution. Remedies • Moving the nozzle closer to the tablet bed or • Reducing the degree of atomization can decrease the roughness due to spray drying. 3. ORANGE PEEL EFFECT • Inadequate spreading of coating solution before drying causes a bumpy or Orange –peel effects on the coating. This Indicates that spreading is impeded by too rapid rate of drying or by high solution viscosity. Remedies • Thinning of coating solution with additional solvents may correct this problem. • Adjustment of speed of coating pan 4. BRIDGING • During drying film may shrink and pull away from the sharp corners of bisect, resulting in a “Bridging” of surface depression. This defect can be so severe that the monogram or bisect is completely obscured. Remedies • Increase in plasticizer contents or • Change in plasticizer 5. FILLING • Applying too much solution, resulting in a thick film that fills and narrows the monogram or bisects. In addition, if solution applied too fast, over wetting

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www.ijpbs.com (or) www.ijpbsonline.com may cause the liquid to quickly fill and be retained in the monogram. Remedies • Judicious monitoring of the fluid application rate. • Thorough mixing of tablets in the pan prevent filling. 6. BLISTERING • Evaporation of solvents from the core in the oven. And effect of high temperature on the strength, elasticity and adhesion of the film may results in blistering. Remedies • Controlled milder drying conditions. 7. HAZING/DULL FILM • Also called as bloom. • It can occur when too high a processing temperature is used for a particular formulation. Dulling is particularly evident when cellulosic polymers are applied out of aqueous media at high processing temperature. Also occur if the coated tablets are exposed to high humidity conditions and partial solvation of film results. 8. COLOR VARIATION • Improper mixing, uneven spray pattern and insufficient coating may results in color variation. The migration of soluble dyes, plasticizer and other additives give the coating a mottled or spotted appearance. Remedies • Use of lake dyes eliminates dye migration. • Reformulation with different plasticizer and additives is the best way to solve film instability. 9. CRACKING • Cracking occurs if internal stresses in the film exceed the tensile strength of the film. The tensile strength of the film can

be increased by using higher molecular weight polymers or polymer blends. Remedies • Adjusting the plasticizer type and concentration can minimize internal stresses. • Adjusting the pigment types and concentration can minimize internal stresses.

CONCLUSION In recent decades, coating of pharmaceutical dosage forms has been subject of remarkable developmental efforts aiming to ensure and enhance end product quality. Improvements regarding particle movement, heat and energy transfer, film distribution, drying efficiency and continuous processing have contributed to significantly develop this technology. In future there is enormous developments has to be done in the area of tablet coating

REFERENCES 1.

Remington J. Remington: The Science and Practice of Pharmacy; Nineteenth Edition: Volume II, pg.no 16151641. Lachman L., Liberman H., and Kanig J. The Theory and Practice of Industrial Pharmacy; Third Edition, pg.no 346-373. Lachman L., Lieberman, H. A., Joseph L. K. The Theory and Practice of Industrial Pharmacy; Third Edition; pg.no 297-321. Aulton M. Pharmaceutics: The Science of Dosage Form Design; International Student Edition: pg. no 304-321, 347-668. Ansel H., Allen L., Jr. Popovich N. Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems; Eighth Edition: pg.no 227-259. Neelam D. Kamble*, Prafulla S.Chaudhari1, Dr. Rajesh J. Oswal, Sandip S. Kshirsagar, Rishikesh V Antre, Innovations in tablet coating technology: A review , International Journal of Applied Biology and Pharmaceutical Technology, Volume: 2, Issue-1: JanMar -2011; 214-218. Gupta Ankit,A.et al tablet coating techniques: concepts and recent trends, www.irjponline.com.

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www.ijpbs.com (or) www.ijpbsonline.com 8.

Tech tips, Aqueous coating 101, techceuticals solutions for pharma and neutral manufactures since 1989 (cited 2012 Aug 2005.) Available from http:// www.techceuticals.com/techtips/show_news.phs?sub action=showfull&id=1079300125&archive=&star Gohel.M.Tabletcoating, 2009 (cited 2012 feb 23), Available from http://www.pharmainfo.net/tabletsruling-dosage-form-years/tablets-coating.

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|248-261 10. Behzadi S. innovations in coating technology, recent patents on drug delivery & formulations, 2008; 209230. 11. Pawar A.et al Advances in Pharmaceutical coating, International journal of chemtech Research, 2010, 733737

*Corresponding Author: B.Venkateswara Reddy

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Department of Pharmaceutics, St.Paul's College of Pharmacy, Turakayamjal village, Hayath Nagar Mandal, Ranga Reddy Dist-501510, A.P.

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Research Article Pharmaceutical Sciences EFFECT OF OG-1 CARDIOACTIVE PRINCIPLE FROM CHLOROFORM EXTRACT OF Ocimum gratissimum (Linn.) LEAVES ON ISCHAEMIA AND REPERFUSION-INDUCED MYOCARDIAL INJURY Yogita Dobhal1,2* , Versha Parcha3 and D.C.Dhasmana4 1

Department of Pharmaceutical sciences, Sardar Bhagwan Singh PG Institute of biomedical Sciences & Research, Balawala, Dehradun, Uttarakhand, India. 2 Department of Pharmaceutical Sciences, Jodhpur National University, Jodhpur, Rajasthan, India. 3 Department of Chemistry, Sardar Bhagwan Singh PG Institute of biomedical Sciences & Research, Balawala, Dehradun, Uttarakhand, India. 4 Departments of Pharmacology, H.I.M.S.Doiwala, Dehradun, Uttarakhand, India. *Corresponding Author Email: yogita_sharma05@rediffmail.com

ABSTRACT Objective:The present study has been designed to investigate efficacy Of OG-1 cardioactive principle from chloroform extract of Ocimum gratissimum (Linn.) Leaves on ischaemia and reperfusion-Induced myocardial injury. Methods: Various extracts of O. gratissimum (Linn.) leaves were prepared viz. Pet.ether, chloroform, acetone and methanol. Among all the extracts chloroform extract reduce myocardial injury. The chloroform extract further purified using column chromatographic technique that results four major fractions viz F1, F2, F3 and F4. Among these fractions F4 was found to be hot fraction. OG1 (active principle) was isolated from the F4 using column chromatography technique and evaluated for cardioprotective effect. Results: The present study demonstrated that Chloroform extract of O.gratissimum (Linn.) leaves and further fraction F4 of chloroform extract significantly prevented myocardial infarct size as compared with that of standard ramipril. OG1 (active principle) was isolated from fraction F4 of chloroform extract showing cardioprotective effect at dose level of 50mg/kg body weight as compared with that of standard ramipril (1mg/kg body weight). Conclusion:These results suggest that OG1 is active cardioprotective principle of O. gratissimum (Linn.) chloroform leaves extract.

KEY WORDS Ischaemia, Reperfusion, Ocimum gratissimum, Ramipril, Chromatography.

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1. INTRODUCTION As per Ayurveda, Indian medicinal plants are rich sources of substances that have several therapeutic properties including cardio protection. About 75-85% of the worldâ&#x20AC;&#x2122;s population, plant derived products is still play an essential role in primary health care, mainly in the developing countries. The use of herbal preparation is increasing in the treatment of cardiovascular disease because of various

possible mechanism involved in the cardio protection. Therefore, herbal extracts that are traditionally used, evaluated against in limiting the deleterious effects of ischaemia and reperfusioninduced myocardial injury. Furthermore, the results are statistically analysed and validated for prophylactic approaches and as an adjunct to standard treatment of ischaemia and reperfusion-induced myocardial injury [4] [5]. The word ischaemia is

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www.ijpbs.com (or) www.ijpbsonline.com derived from “ischo” meaning to hold back and “hamia” meaning blood. Thus, myocardial ischaemia means decrease in coronary blood flow which is unable to meet the oxygen demand of myocardium. The persistent myocardial ischaemia leads to death of cardiomyocytes leading to myocardial infarction [1]. Reperfusion is prerequisite to salvage ischaemic myocardium. The restoration of blood flow after transient ischaemia has been noted to produce myocardial hibernation, myocardial stunning, reperfusion induced arrhythmia, endothelial dysfunction leading to no-reflow state and extension of infarct size. Reperfusion after exacerbate myocyte cell death and increase in infarct size, a phenomenon termed as ‘reperfusion injury’ *2+ [3]. Genus ocimum is widespread over Asia, Africa and Central and southern America. Tulsi was probably first put to cultivation in India. Among the plants known for medicinal value, the plants of genus ocimum belonging to family lamiaceae are very important for their therapeutic potentials. Ocimum sanctum L. (tulsi), ocimum gratissimum (ram tulsi), ocimum canum (dulal tulsi), ocimum basilicum (ban tulsi), ocimum kilimandscharicum, ocimum ammericanum, ocimum camphora and ocimum micranthum are grown in different parts of the world and are known to have medicinal properties[6] [7]. Ocimum gratissimum found throughout India as a common weed in road sides and in waste places.The most important aroma components are euginol, 1, 8-cineole, α-pinene, β-pinene, (Z)ocimene, α-terpineole, (E)-β-caryophyllne, αhumulene, γ-muurolene, β-selinene, α-selinene. The composition of the essential oil is known to depend on climate, soil, genetic strain and season [8]. In order to establish the therapeutic uses of genus ocimum in modern medicine, in last few decades several Indian scientists and researchers have studied the pharmacological

effects of essential oils and extract of tulsi on immune system, central nervous system, gastric system, reproductary system, blood biochemistry and described the therapeutic significance in management of various ailments[9] [10].

2. MATERIALS AND METHODS 2.1 Drugs and chemicals: Ramipril is taken as a gift sample from USV Baddi, Himanchal pradesh, Himanchal, India. All the reagents used in this study were of analytical grade and were always freshly prepared before use. 2.2 Plant material: Leaves of Ocimum gratissimum (Linn.) was collected from Indian Institute of Integrative Medicine, Jammu (Formerly Regional Research Laoratiory), CSIR, India. The reference number is [RRL (J) OG-14]. 2.3 Animals: Adult Wister rats of either sex, weighing 250 to 300g were used in the study. The experimental protocol was reviewed and approved by the Institutional Animal Ethics Committee (Reg.no.273/CPCSEA). Animals were obtained from IVRI Bareilly, India and were maintained under standard laboratory conditions in the departmental animal house of SBSPGI, Dehradun, Uttarakhand, India. 2.4 Preparation of extracts: The fresh leaves of O. gratissimum (Linn.) were dried in shade and room temperature for 2days followed by drying [40-50oC] for 3-4hrs and powdered to obtained coarse powder. 1.5kg of powder of O. gratissimum (Linn.) leaves were extracted with pet ether, chloroform, acetone and methanol to get four extracts. The solvent was removed by evaporation under reduced pressure to obtain a semisolid mass. The resultant extracts were kept in a desicator

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www.ijpbs.com (or) www.ijpbsonline.com followed by weighing to give percentage yield of each extract. 2.5 Isolation and purification of principle constituent from active fraction: The chloroform extract showing good cardioprotective effect was subjected to column chromatography using silica gel mesh size 200400Âľ and Pet ether: ethyl acetate as mobile phase in different ratio leadind to the isolation of four fractions, F1, F2, F3 and F4. The cardioprotective activity was evaluated for all four fractions in which fraction F4 of chloroform extract was found significantly effective. Further purification of fraction F4 as above said process resulted in isolation of OG1, OG2 and OG3. Among all these the compound OG1 further evaluated for cardioprotective activity and it was more significant than other compounds. 2.6 Acute toxicity study: Albino mice of 10 animals per group and weighing 20-25g were administered graded dose (100-2000 mg/kg body weight, orally) of the chloroform extract of O.gratissimum (Linn.). After administration of extract mice were observed for toxic effects after 48hr of treatment. The toxicological effects were observed in terms of mortality expressed as LD50. The number of animals dying during the period was noted. The LD50 of the extract was determined by Litchfield and Wilcoxon, 1949 method [11]. No mortality was observed therefore the extract is safe to use even at the doses of 2000mg/kg of body weight orally. 2.7 Isolated rat heart preparation: Rats were heparinised (500 IU/L, i.p.) and sacrificed after 20min by cervical dislocation. The heart was rapidly excised and immediately mounted on Langendorffâ&#x20AC;&#x2122;s apparatus *12+. The temperature was maintained at 37oC by circulating hot water. The preparation was perfused with krebs Henseleit (K-H) buffer (NaCl 118 Mm; KCl 4.7 Mm; CaCl2 2.5 Mm;

MgSO4.7H2O 1.2 mM; KH2PO4 1.2mM; C6H12O6 11 mM), pH 7.4 and bubbled with 95% O2 and 5% CO2. The coronary flow rate was maintained 6-9 ml/min and perfusion pressure was kept constant at 70 mmHg. Global ischemia was produced for 30min by completely closing the inflow of physiological solution and followed by 120min of reperfusion. The coronary effluent was collected before ischaemia, immediately, 5min, 30min and 120min after reperfusion for estimation of LDH and CK-MB. 2.8 Assessment of myocardial injury: The myocardial infarct size was measured using the triphenyltetrazolium chloride (TTC) staining method. The level of LDH and CK-MB (Siemens Medical Solution Diagnostic Ltd.,Baroda, India)in coronary effluents was estimated using commercially available kits. Values of LDH and CK-MB were expressed in international units per litre (IU/L). 2.9 Assessment of myocardial infarct size: Heart was removed from the Langendorff,s apparatus. Both the auricals and the root of aorta were excised, and ventricles were kept overnight at temperature of -4oC. Frozen ventricles were sliced into uniform sections of 12mm thickness. The slices were incubated in 1% w/v TTC solution in 0.2M Tris-chloride buffer,pH 7.8 for 20min at 37oC. The normal myocardium was stained brick red while the infracted portion remained unstained. Infarct size was measured by macroscopic volume method [13]. 3. Experimental protocol: In all groups, isolated rat heart was perfused with K-H solution and allowed to stabilize for 10min. Group 1: (Sham control; n=5) After stabilization isolated rat heart was perfused continuously with K-H buffer for 160min. without subjecting it to global ischaemia. Group 2: (Vehicle control; n=5) Rats were administered 1% Tween 80 orally for 7days;

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www.ijpbs.com (or) www.ijpbsonline.com thereafter, on the 7th day ,isolated rat heart after stabilization, was subjected to 30min of global ischaemia followed by reperfusion for 120min. Group 3: (Standard; n=5) Ramipril (1mg/kg) was dissolved in 1% Tween 80 and administered orally once daily to rats for 7days; thereafter, on the 7th day ,isolated rat heart after stabilization, was subjected to 30min. of global ischaemia followed by reperfusion for 120min. Group 4: (Chloroform extract; n=5) Chloroform extract (100mg/kg) was dissolved in 1% Tween 80 and administered orally once daily to rats for 7days; thereafter, on the 7th day ,isolated rat heart after stabilization, was subjected to 30min of global ischaemia followed by reperfusion for 120min.

Group 5: (Fraction 4 of Chloroform extract; n=5) Fraction 4 of chloroform extract (100mg/kg) was dissolved in 1% Tween 80 and administered orally once daily to rats for 7days; thereafter, on the 7th day, isolated rat heart after stabilization, was subjected to 30min of global ischaemia followed by reperfusion for 120min. Group 6: (Active principle OG1of chloroform extract; n=5) Active principle OG1 of chloroform extract (50mg/kg) was dissolved in 1% Tween 80 and administered orally once daily to rats for 7days; thereafter,on the 7th day ,isolated rat heart after stabilization, was subjected to 30min of global ischaemia followed by reperfusion for 120min.

400

a c

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c c c

200

a c b

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V/V Sham Control Control

Standard Chloroform

20% fraction OG1

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Standard

20% fraction

Control

Chloroform

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Figure 2: Effect of Ischaemia and Reperfusion on LDH release. LDH was estimated in coronary effluent after stabilization (Basal), Immediately (Imm’Rep.) and 30min. after reperfusion (30’ Rep.). Values are expressed as mean ±SEM .a= P˂0.05 vs. Sham control; b= P˂0.05 vs. Control; c= P˂0.05 vs. Standard. ANOVA followed by Tukey’s multiple comparison test.

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Figure 1: Assessment of Myocardial Infarct Size. Infarct size was measured by volume method. Values are expressed as mean ±SEM .a= P˂0.05 vs. Sham control; b= P˂0.05 vs. Control; c= P˂0.05 vs. Standard. ANOVA followed by Tukey’s multiple comparison test

30 m in .

l sa ba

0m in .

0 0

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CK-MB(IU/L)

200

150

c c

100

ba sa l

in .

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Figure 3: Effect of Ischaemia and Reperfusion on CK-MB release. CK-MB was estimated in coronary effluent after stabilization (Basal) and 5min. after reperfusion (5’ Rep.). Values are expressed as mean ±SEM .a= P˂0.05 vs. Sham control; b= P˂0.05 vs. Control; c= P˂0.05 vs. Standard. ANOVA followed by Tukey’s multiple comparison tests.

4. Statistical Analysis: All values for enzymatic data (LDH and CK-MB) and infarct size were expressed as mean ±SEM. Statistical analysis was performed using Graph Pad Prism Software.The values were statistically analysed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Value of P˂0.05 was considered to be statistically significant.

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5. RESULTS 5.1 Effect on Myocardial Infarct Size: Various extracts of O.gratissimum (Linn.) leaves viz. petroleum ether, chloroform, acetone and methanol were evaluated on ischaemia and reperfusion induced increase in myocardial infarct size,respectively. Among all the extracts chloroform extract of O.gratissimum (Linn.) leaves found to be active. Further purification of active extract was carried out using column chromatography which resulted isolation of four fraction viz. F1, F2, F3 and F4. Which were again

evaluated for above said effect and among all the fractions fraction F4 significantly attenuated ischaemia and reperfusion induced increase in myocardial infarct size. Further purification of fraction F4 resulted in isolation of OG1, OG2 and OG3. OG1 further evaluated for cardioprotective activity and it was more significant than other compounds. However, treatment with standard (ramipril,1mg/kg) was significantly more effective to reduce myocardial infarct size as compared to compound OG1, measured by macroscopic volume method (Figure 1). 5.2 Effect on Ischaemia and Reperfusion Induced release of LDH Various extracts of O.gratissimum (Linn.) leaves viz. Petroleum ether, chloroform, acetone and methanol were evaluated on ischaemia and reperfusion induced increase in release of LDH in coronary effluent measured immediately and 30 min. after reperfusion, respectively. Similarily, among all the extracts chloroform extract of O.gratissimum (Linn.) leaves and the isolated

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www.ijpbs.com (or) www.ijpbsonline.com fraction F4 from chloroform extract significantly reduced release of LDH in coronary effluent. Further the active principle OG1 isolated from fraction F4 of chloroform extract significantly attenuated release of LDH in coronary effluent measured immediately and 30 min. after reperfusion. Moreover, treatment with standard (ramipril,1mg/kg) markedly reduced release of LDH in coronary effluent as compared to active compound OG1, measured immediately and 30 min. after reperfusion (Figure 2). 5.3 Effect on Ischaemia and Reperfusion Induced release of CK-MB Various extracts of O.gratissimum (Linn.) leaves viz. Petroleum ether, chloroform,acetone and methanol were evaluated on ischaemia and reperfusion induced increase in release of CK-MB measured in coronary effluent collected after 5min. of reperfusion, respectively. Similarily, among all the extracts chloroform extract of O.gratissimum (Linn.) leaves and the isolated fraction F4 from chloroform extract significantly reduced release of CK-MB in coronary effluent. Further the active principle OG1 isolated from fraction F4 of chloroform extract significantly attenuated ischaemia and reperfusion induced increase in release of CK-MB in coronary effluent collected after 5 min. of reperfusion. Moreover, treatment with standard (ramipril,1mg/kg) markedly reduced release of CK-MB in coronary effluent as compared to the active compound OG1, collected 5 min. of reperfusion (Figure 3).

study because it permits the use of pharmacological interventions without any interference due to change in systemic circulation. Various extracts of O.gratissimum (Linn.) leaves viz. petroleum eather, acetone, chloroform and methanol at a dose level of 100mg/kg were evaluated for ischaemia and reperfusion induced myocardial injury. Further purification of active extract was carried out using column chromatography which resulted isolation of four fraction viz. F1, F2, F3 and F4. These fractions were again evaluated for above said effect and among all the fractions F4 significantly attenuated ischaemia and reperfusion induced increase in myocardial infarct size. Further purification of fraction F4 resulted in isolation of OG1, OG2 and OG3. All the compounds were screened for above said activity.Compound OG1 significantly decreased the infarct size, release of LDH and CK-MB in coronary effluent during perfusion period compared to control group. The present findings suggests that compound OG1 from fraction F4 of chloroform extract of O.gratissimum (Linn.) leaves significantly effective to ameliorate myocardial ischaemic injury as compared to ischaemia and reperfusion induced control group. Moreover some extensive work in this direction could also lead to explore the possible mechanism of O.gratissimum (Linn.) against ischaemia and reperfusion induced myocardial injury. Further, chemical structure elucidation of active principle OG1 is in progress.

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6. DISCUSSION Inspite of the disadvantage of high mortality, high heart rate and high rate of drug metabolism, albino rats are used in the present study because they are small in size having low cost and readily available. Moreover histological sectioning and quantification is easy in rat hearts due to small size. Isolated perfused rat heart preparation has been employed in the present

ACKNOWLEDGEMENT The authors gratefully acknowledge to Director and Management of SBSPGI, Balawala, Dehradun, India for providing the necessary facilities during this experimental study.

REFERENCES

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www.ijpbs.com (or) www.ijpbsonline.com [1] American Heart Association Heart and Stroke Statistics, 2011 http://www.americanheart.org/statistics/index.htmlAcc essed January 15, 2011 [2] Yang X., Cohen M V., Downey J M. Mechanism of cardioprotection by early ischemic preconditioning. Cardiovasc Drugs Ther, 2010; 24: 225234. [3] Gerczuk P Z, Kloner R A. A review of the latest adjunctive therapies to limit myocardial infarction size in clinical trials. J Am Coll Cardiol. 2012; 59(11): 969978. [4] Heusch G, Boengler K, Schulz R. Cardioprotection: Nitric Oxide, Protein Kinases, and Mitochondria. Circulation. 2008; 118: 1961-1970. [5] Jain J A T and Devasagayam T P A. Cardioprotective and other beneficial effects of some indian medicinal plants. J.C.B.N. 2006; 38: 19-18. [6] Ramesh B and Satakopan V N . In vitro antioxidant activities of ocimum species: Ocimum basilicum and Ocimum sanctum. J. Cell and Tissue Res. 2010; 10: 21452150. [7] Balaji R , Prakash G , Suganya Devi P , Aravinthan K M . Antioxidant activity of methanol extract of Ocimum tenuiflorum (dried leaf and stem). IJPRD, 2011 ; 3: 20-27

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|262-268 [8] Nayive Pino Benitez1, Erika M. Mel茅ndez Le贸n1, and Elena E. Stashenko2. Eugenol and Methyl Eugenol Chemotypes ofEssential Oil of Species Ocimum gratissimum L.and Ocimum campechianum Mill. from Colombia. J. Chromatographic Sci .2009; Vol. 47:800803. [9] Prabhu K S, Lobo R, Shirwaikar A A and Shirwaikar A. Ocimum gratissimum: Areview of its chemical, pharmacological and ethnomedical properties. Open Complementry Medicine Journal, 2009; 1: 1-15. [10] Abdurahman F I, Tijjani M A and Osuji U O. Proximate content and chemical composition of Ocimum viridis leaf and Ocimum gratissimum leaf. IRJP, 2012; 3(4): 153-156. [11] Litchfield, J.T. and Wilcoxon, F., J. Pharmacol. Exp. Ther. 1949; 96: 99-113. [12] Sill B., Hammer P E., Cowan D B. Optical Mapping of Langendorff-perfused Rat Hearts. J. Vis. Exp. 2009; (30): e1138, DOI: 10.3791/1138. [13] Ludman A J., Yellon D M., Hausenloy D J. Cardiac preconditioning for ischaemia: lost in translation. Dis Model Mech, 2010; 3: 35-38.

*Corresponding Author: Yogita Dobhal* 1

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Review Article Pharmaceutical Sciences INTRODUCTION TO BIO-FUEL AND ITS PRODUCTION FROM ALGAE: AN OVERVIEW Aman Kothari1, Sarbjeet Singh Gujral*2 1

Northern India Engineering College (Aff. To G.G.S. Indraprastha University) Maharaja Surajmal Institute of Pharmacy, New Delhi (Aff. To G.G.S. Indraprastha University) *Corresponding Author Email: sarbjeet_singh18@yahoo.in

ABSTRACT In today's scenario where there is too much dependence on fuel for meeting the requirements of the common masses and industries, and due to failure in the replenishment of non-renewable natural sources, arises an urge to supplement the requirements from renewable natural resources. Biofuel or in simple language fuel obtained from biological sources like crop wastes, exhausted mineral oils or fats etc. is a cogent alternative but none has proven to be a complete surrogate for petroleum and coal due to various reasons. In this context, a path breaking development of mass production of biofuel form algae comes into picture. In this following article the production of biofuel and methods of obtaining the same has been discussed.

KEY WORDS Bio-fuel, Algae.

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INTRODUCTION Now-a-days the common notion around the world is to reduce the consumption of fossil fuels and bring down effects of global warming. It will be an ideal state to live in a pollution free world without cutting on power requirements, however for this kind of environment we urgently need to reduce our dependency on fossil fuels. Thinking tanks find it difficult to come up with such an alternative fuels which could replace conventional fuels. No energy source has still been proven perfect. Like some fuels are environment friendly but not effective for mass production due to high initial prices etc. environmental friendly fuel derived from natural source is termed as biofuel.[1]. Bio-fuel has become attention-grabbing topic; this is attributed to the higher demands of fuel, power, depletion of fossil fuels, green house effect, rise in crude oil prices. Thus this is being worked out

as an alternate source of energy to the present day liquid fuels. So what is a biofuel? A biofuel is a type of fuel whose energy is derived from biological carbon fixation. Biofuels include fuels derived from biomass conversion, as well as solid biomass, liquid fuels and various biogases. Classification of biofuel: biofuel can be classified as:

biofuel first generation biofuel second generation biofuel.

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www.ijpbs.com (or) www.ijpbsonline.com FIRST GENERATION BIOFUEL Bioalcohol Alcohol is produced by fermentation, generally from carbohydrates from starch rich plants like sugarcane, molasses to name a few and Bioethanol is a type of alcohol. Ethanol is also being produced from cellulose biomass which is being derived from non-food sources such as trees and grasses. Use of ethanol as a fuel is not new to the industry as earlier also it has been used as an additive up to 15% with gasoline fuel to increase the octane number of it, in turn increasing efficiency. Biobutanol (also called biogasoline) is often claimed to provide a direct replacement for gasoline, because it can be used directly in a gasoline engine. Methanol is currently produced from natural gas, a nonrenewable fossil fuel. It can also be produced from biomass as biomethanol. Methanol could be an alternative to the hydrogen economy, compared to today's hydrogen production from natural gas. Butanol is considered of higher value as compared to other bio-fuels as it can be directly used with the current gasoline engines (without modification to the engine or car),[2] and they would produce more energy. ABE

fermentation [acetone, ethanol] yields butanol. (Without modification to the engine or car), [2] and is less corrosive and less water soluble than ethanol. Biodiesel The most common biofuel in Europe is Biodiesel. Trans-esterification from oils or fats yields a liquid similar in composition to the crude oil byproduct diesel (see chemical reaction for the synthesis of biofuel from transesterification of oils below). Chemically, it consists mostly of fatty acid methyl (or ethyl) esters. Animal fats, vegetable oils, jatropha, mahua, mustard, flax, sunflower, palm oil, hemp, field pennycress, pongamia pinnata and algae are amentionable few of biodiesel feedstocks. Pure biodiesel (B100) is the lowest emission diesel fuel. In a number of countries, 5% biodiesel blend is widely used and is available at thousands of gas stations.[3][4] Biodiesel is an oxygenated fuel, meaning that it contains a reduced carbon content and higher hydrogen and oxygen content than conventional diesel. This improves the combustion of biodiesel and reduces the particulate emissions from un-burnt carbon.

Production of biodiesel

CoA Ligase ATP

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Glycerol-3-phosphate

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AcylCoA

1,2-diacylglycerol-3-phosphare

1-Acylglycerol-3-phosphate

Hydrolysis, fattyCoA

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Fig:2 Chemical reactions involving production of biodiesel from exhausted fats/oils

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The estimated world consumption of biodiesel is as follows:[15]

Green diesel Green diesel result from hydrocracking of biological oil feedstocks which include vegetable oils and animal fats.[5][6] Long chain hydrocarbons aree broken down into smaller chains in hydrocracking which is a method that

uses elevated temperatures and pressure in the presence of a catalyst to break down the long chains.[7] Vegetable oil Lower quality oil can and has been used for this purpose. Used vegetable oil is increasingly being processed into biodiesel, or (more rarely) cleaned of water and particulates and used as a fuel. Vegetable oil fuel must be heated to reduce its viscosity to that of diesel, either by electric coils or heat exchangers. This is easier in warm or temperate climates. Oils and fats can be hydrogenated to give a diesel substitute. The resulting product is a straight chain hydrocarbon with a high cetane number, low in aromatics and sulfur and does not contain oxygen. Hydrogenated oils can be blended with diesel in all proportions.[8] Biogas

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Biogas is methane(CH4) produced by anaerobic digestion of organic material by anaerobic microbes.[9] It can be produced either from biodegradable waste materials, and the use of energy crops fed into anaerobic digesters to supplement gas yields. Farmers produce biogas from manure from their cows and other farm wastes by using an anaerobic digester (AD).[10] Syngas Syngas is a mixture of hydrogen, carbon monoxide and other hydrocarbons. Partial combustion of biomass, the process proceeds with combustion with an amount of oxygen that is not sufficient to convert the biomass completely to carbon dioxide and water.[8] Before partial combustion the biomass is dried, and sometimes pyrolysed. • Can be used directly in internal combustion engines, turbines.[11] • Can be used to produce methanol, hydrogen, to produce a diesel substitute, or a mixture of alcohols that can be blended into gasoline. Gasification normally relies on temperatures >700°C. Solid biofuels Solid bio-fuels include wood, sawdust, grass trimmings, domestic refuse, charcoal, agricultural waste, non-food energy crops, and dried manure. One of the disadvantages of using the oldest solid bio-fuel wood is the amount smoke it creates. Advantage of solid biomass fuel is that it is often a by-product, a residue or waste-product of other processes, such as farming, animal husbandry and forestry, this way we do not invest separately to get the fuel.[12] In theory this means there is no competition between fuel and food production, although this is not always the case.[30] The other disadvantages of raw biomass are that it emits considerable amounts of pollutants such as particulates and PAHs (polycyclic aromatic

hydrocarbons). A derivative of solid biofuel is biochar, which is produced by biomass pyrolysis. Bio-char made from agricultural waste can substitute for wood charcoal. As wood stock becomes inadequate this alternative is gaining ground. For example, in eastern Democratic Republic of Congo, biomass briquettes are marketed as alternatives to charcoal to protect Virunga National Park from deforestation related with charcoal production.[13] Second generation biofuels (advanced biofuels) These are also known as advanced biofuels. Many of the biofuel in this class has been derived from algae and more recently from microalgae which are genetically modified version of algae of common use. Co-cultures of Shewanella and Synechococcus which have been genetically modified has is now commercially utilized for biofuel production but many of such biofuels are under development, examples of these include Cellulosic ethanol, Algae fuel [14]. BioDME, Fischer-Tropsch diesel, DMF, bio-hydrogen, biohydrogen diesel. Why biofuel from algae is more preferred to biofuel from plant courses?

Even though many crops like corn can be utilized for the production of biofuel, but none of the methods can be utilized effectively when yield is a major concern. Biofuel from plant source gives very low yield per hectare of land. Fuel obtained from Algae yields considerably more energy than other bio-fuels (per unit area). Algae can be grown on land that has become unsuitable for agriculture, and arable lands as well. This advantage is also commercially exploited for

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mass production of biofuels. Companies include Solazyme, Sapphire Energy and OPXBIO. Further the yield can be increased by using photo-bio reactors which can drastically change the yield of the biofuels from few kgs. to gallons. [16]

Fig: a photo bio-reactor Algae-based biofuels has both its pros and cons which includes: Pros • Reduction in the use of Crude Oil based fuel • Reduction in carbon dioxide (CO2) levels • Increased energy output as compared to other bio-fuel substituent • Reduction in degradation of land due to oil mining • Renewable source of energy can be grown anywhere provided temperature conditions are met Cons • High initial cost as in case of every other renewable source of energy • Needs some rare elements in extraction of fuel • Requirement of proper temperature conditions and sizeable amount of land for growth Methods of extraction of biofuel from algae: Lipid Extraction from algae Lipid extraction from algae is one of the methods for the production of biofuel .Lipid extraction includes the subsequent approaches: • Solvent-based extraction depending on microwaves

•

sonication for cell interruption for example using solvents to extract algal cells without disrupting cellular functions • Extraction bypass schemes that try to obtain algal systems that exude products directly into the growth medium. Mechanical Disruption (i.e., Cell Rupture) For the biofuels dependent on the accretion of intra-cell lipids to be successful, • extracting solvent should infiltrate through the lipid enclosing matrix • Physically contact the lipid material • Solvate the lipid Use elevated temperatures and pressures which force the solvent into the required biopolymer contact and be compensated for by effective mechanical disruption. Mechanical disruption can include bead mills (or bead-beating), cell homogenizers, autoclaving [17], ultrasounds. Non-mechanical techniques include processes such as freezing, acid, base, and enzyme reactions [17], use of organic solvents, and osmotic shock to name a few. The use of microwaves to disturb cells and augment efficiency of vegetable lipid and oil extraction is a noteworthy advancement [18], even if applications outside methodical labs are blurred. Treatment of sewage sludge with pulsed sonication has enhanced methane gas production and reduction in biosolids in sludge digestion [19]. Organic Co-solvent Mixtures In the co-solvent extraction procedure [20], after the reaction is accomplished, water is added to the co-solvent (chloroform) mixture until a twophase system develops where water and chloroform break up into two immiscible layers. Lipids can be recovered for analysis as they separate to the chloroform layer. Subsequently, other combinations of co-solvents projected for the extraction of lipids: hexane/isopropanol for tissue [21]; hexane/ethanol for microalgae [22];

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www.ijpbs.com (or) www.ijpbsonline.com and hexane/isopropanol for microalgae [23]. The hexane system is supported because hexane and alcohol will readily separate into two separate phases when water is added, thereby improving downstream separations. Likewise, less volatile and hazardous alcohols have been suggested in place of methanol. One example is the hexane/ethanol extraction co-solvent system [24]. In other cases, single alcohol (e.g., 1butanol and ethanol) solvents have been tested [23]. In these applications, the alcohol is first added as the extracting solvent. Separation is then achieved by adding both hexane and water in proportions that create a two phase system (hexane and an aqueous hydroalcoholic) that partition the extracted lipids into the nonpolar hexane [25]. The results suggest that the most vital condition when selecting a co-solvent system to extract lipids is: • The capability of a more polar co-solvent to disturb the cell membrane and thus make it adequately porous and • The ability of a second less polar co-solvent to better match the polarity of the lipids being extracted. Application of Organic Two-Solvent Systems for Lipid Extraction from Microalgae: The sequence of solvent addition also affects extraction [26]. Starting from freeze dried biomass, it has been verified that, the extraction of lipids was notably more efficient when solvents were added in order of increasing polarity (i.e. chloroform, methanol, and then water)[26]. The results can be justified as, in terms of initial contact of the biomass with nonpolar solvents deteriorating the connection between the lipids and cell structure, preceding their dissolution in the monophasic system of water, chloroform, and methanol. These results have a key influence on liquid phase extraction systems applied to moist biomass as, it is

suggested that the water will form a solvent covering around the lipids, making it difficult for less polar solvents such as chloroform to connect, solubilise, and extract the lipids. Direct Transesterification of Lipids into Fatty Acid Methyl Esters (FAMES) Lepage and Roy (1984) offered the direct transesterification of human milk without prior extraction or refining for improved recovery of fatty acids. In general, the approach proposed a one-step reaction that added the alcohol and acid catalyst directly to the biomass sample and followed with heating at 100˚C for an hour under sealed conditions would increase fatty acid concentrations measured, and provide relatively high recoveries of volatile medium chain triglycerides, and eliminate the need to use antioxidants to protect unsaturated lipids. Efforts in this direction have shown that when employing direct transesterification with an acid catalyst (i.e., acetyl chloride), the efficiency of the reaction improved when a second “less polar” solvent for example diethyl ether or toluene were mixed with the methanol to alter the polarity of the reaction medium [27]. These results suggest that the usefulness of the second co-solvent depends upon its ability to solubilise the target lipids along with its miscibility with methanol. However the earlier co-solvent systems stay largely bench-scale methods which are difficult to scale up to industrial processes due to solvent toxicity and the low carrying capacity of the solvents. As a result, single solvent systems at elevated temperature and pressure have the following advantages: • High temperature and pressure increase the rate of mass transfer and degree of solvent access • Increased pressures reduce the dielectric constant of otherwise immiscible solvent [28]. Accelerated Solvent Extraction

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www.ijpbs.com (or) www.ijpbsonline.com Accelerated solvent extraction was proposed in the mid 1990s [29], using the technique on 1 - 30 g samples of dried biomass. It uses organic solvents at high pressure and temperatures usually above their boiling point. The process involves, enclosing a solid sample in a sample cartridge which is prior filled with an extraction fluid and used to statically extract the sample under elevated temperature (50 - 200Ë&#x161;C) and pressure (500 - 3000 psi) conditions for short time periods (5 - 10 min). Compressed gas is used to remove the sample extract from the cell into a collection vessel. This method is applicable to solid and semi-solid samples that can be retained in the cell during the extraction phase. Apart from improving yields and reducing extraction time, Accelerated Solvent Extraction can also be applied to efface co-extractable material from various processes, to selectively extract polar compounds from lipid-rich samples. Accelerated Solvent Extraction process is more effective if extracting solvent, sample-solvent ratio, extraction temperature, and time have been optimized [30]. The performance of Accelerated Solvent Extraction was compared to that of traditional Folch method for microalgae grown on dairy manure effluent [31]. The Accelerated Solvent Extraction method, depending on the solvent, extracted 85 - 95% of the fatty acid substance in the reaped microalgae compared to 44 - 55% of the fatty acids by the Folch method in the first solvent extraction cycle. Subcritical Water Extraction This method is based on the use of water, at temperatures below the critical temperature, and pressure high enough to keep it in the liquid state [32]. The technique, was initially applied to biomass hemicellulose as a pre-treatment to its use as a fermentation substrate [33]. However, now it has been tested for the selective extraction of essential oils from plant matter

[34], functional ingredients from microalgae [35], and saponins from oil-seeds [36]. In technique the water, under condition consideration, becomes less polar and organic compounds tend to be more soluble than at room temperature. In addition, products can easily be separated as the water is cooled back down to room temperature, and products miscible at the high temperature and pressure become immiscible at lower temperatures. A major constraint, however, is the difficulty with proposing a large scale system and the high-energy load required to heat the system up to subcritical temperatures. Additional energy challenges are created as a large-scale design would require a considerable cooling system to cool down the product to room temperature to avoid product degradation. Heterotrophic Production One of the many other methods for extraction and fractionation is the production of oils using heterotrophic algae. In this approach nonphotosynthetic algae are developed using sugars as energy source and using traditional industrial fermentation equipment [37]. Several companies have contrived algae that secrete oil into the fermentation media which can be recovered and later refined into a biofuel; this methodology drastically reduces capital and operating cost for the extraction procedure. The benefits of this approach range from the use of conventional fermentation systems ease of scale-up, averting expensive extraction schemes, ability to maintain the integrity of the fermentation catalyst to the use of sugar-based feedstock. Direct Production of Biofuels from Algae Heterotrophic growth has certain benefits in terms of process cost as it can eliminate many process steps and also permits for maintaining controlled conditions. Such a system can engender very high biomass and a high proportion of that biomass as lipid. There are several biofuels that can be produced directly

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from algae, including alcohols, alkanes, and Alcohols [38]

hydrogen.

Fig: scheme for production of ethanol in algae Production of alcohols like ethanols is headspace of the reactor, purified, and stored.) accomplished through the creation and storage This expertise is estimated to yield 4,000 - 6,000 of starch during photosynthesis inside the algae, gallons per acre per year, with prospective or by feeding sugar to the algae directly, and increaseing up to 10,000 gallons per acre per subsequent anaerobic fermentation of these year within a few years with noteworthy carbon sources to produce ethanol under dark research. It is theoretically estimated that one conditions. Algae, such as Chlorella vulgaris and ton of CO2 is converted into approximately 60 Chlamydomonas perigranulata, are competent of 70 gallons of ethanol with this technology. yielding ethanol and other alcohols during Alkanes heterotrophic fermentation of starch [39]. If In addition to alcohols, alkanes may be produced alcohols can be mined directly from the algal directly by heterotrophic metabolic pathways culture media, the process can be considerably using algae. Rather than growing algae in ponds less energy demanding than competitive algal or enclosed in plastic tubes that utilize sunlight biofuel processes. This process usually consists and photosynthesis, algae can be grown inside of closed photo-bioreactors employing sea-water closed reactors without sunlight. The algae are with metabolically superior cyanobacteria [40]. fed sugars, the cheap availability of which is a (One key aspect of the system is that a source of key consideration for cost-effective production cheap carbon, such as a power plant, is typically of biofuels; these sugars are themselves used to supply CO2 to the bioreactors to available from renewable feedstock such as accelerate the algae growth. An example of this lignocellulosic biomass, in a pressure and heatprocess technology links sugar production to controlled environment. algal photosynthesis. There are claims that this Hydrogen process may consume more than 90% of the Biological production of hydrogen technologies systemâ&#x20AC;&#x2122;s CO2 through photosynthesis, wherein a present wide range of methods to generate portion of the carbon in these sugars is hydrogen which include indirect biophotolysis, converted into ethanol. The ethanol is secreted direct biophotolysis, photo-fermentation, and into the culture media and is collected in the dark-fermentation. Yet several challenges wait to International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)

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CONVERSION OF ALGAL EXTRACTS Chemical Transesterification This process is engaged to change triacylglycerols extracted from algae to fatty acid methyl esters (FAMe), which is simply a process of replacement of an alcohol group from an ester by another alcohol [41]. It can be achieved via catalytic or non-catalytic reaction systems using different heating systems required to initiate the reaction. This technology is relatively mature and has been verified to be a standard in the conversion of vegetable oils into biodiesel [42]. Vaporizing under high vacuum, biodiesel is separated from the ether. Acid catalyzed transestrification is another way of converting algal extracts. This involves soluble bases being replaced liquid acid catalyst some of which are H2SO4, HCl [43]. Acid catalysts have advantages, yet they at present are not chosen due to their lower activity than the conventional transesterification alkaline catalysts. Recently, it has been verified that HPA-catalyzed transesterification of vegetable oil accomplishes higher reaction pace than standard mineral acids due to their higher acid strength [44]. The apparent higher activity of certain HPAs with respect to polyoxometallates of higher strength resulted in lower pre-treatment temperatures. Biochemical Conversion (Enzymatic) The advantage of chemical processes giving high conversions of triscylglycerols to esters is

dwarfed by the shortcomings such as the process being energy demanding, intricacy in separating the glycerol, and require elimination of alkaline catalyst from the product and treatment of alkaline wastewater. A solution to these limitations is the use of biocatalysts (lipases) in transesterification of triacylglycerols for biodiesel manufacturing [45]. One vital point that needs to be addressed is the solvent and temperature limitations of the enzymes in order to facilitate efficient biocatalytic processing. Although enzymatic approaches have become more attractive, the process stands unproven at a large scale mainly due to the rather high price of lipase and its ephemeral life caused by the effects of excessive methanol and co-product glycerol. The presence of solvents is occasionally necessary to augment the solubility of the triacylglycerols during the extraction process, and the enzymes used in the downstream change must be able to fulfil their purpose in the presence of these solvents to varying degrees to enable cost-effective biofuel production [46]. Solvent engeering method can also be used to enhance the lipase lipase-catalyzed methanolysis of triacylglycerols for biodiesel production [47][48]. Much research is still necessary in discovery, engineering, and optimization of enzymatic processes, which are competent of producing the reactions in a range of environments and on different types of oil feedstocks [49]. Bioprospecting for the enzymes in extreme environments may produce enzymes with convincing characteristics that are more suitable for industrial applications [50]. Enzyme immobilization would play a vital role in developing an economic method of biocatalytic transesterification [51]. Catalytic Cracking Although catalytic transestrification is a very effective and relatively economical, these catalysts require purification and removal from

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www.ijpbs.com (or) www.ijpbsonline.com the product stream, which increases the overall cost of execution. One likely solution to this is the improvement of immobilized heterogeneous and/or homogeneous catalysts that are very efficient and inexpensive [52]. Acid and basic catalysts are usually classified as Brönsted or Lewis catalysts. Lewis acid catalysts, AlCl3, ZnCl2 to name a few; have been verified as a feasible means of converting triacylglycerols into fatty acid methyl esters. One of the many other methods include, catalysts derived from titanium compounds having the general formula ATixMO; where A is a hydrogen atom/an alkaline metal atom, M a niobium atom or a tantalum atom, and x is number less than 7; is employed in vegetable oil transesterification. The catalysts achieved are steady and give high glycerol yield. A typical FAME yield of 91% and glycerol yield of 91% were obtained in a fixed-bed reactor at 200˚C and 35 bar, using HTiNbO3 as the catalyst. Vanadate metal compounds are stable, active catalysts during transesterification, with TiVO4 being the most active [53]. This catalyst is also more active than HTiNbO3, producing the same yields with lower residence times. (no language to edit in blue) Double-metal cyanide Fe-Zn proved to be promising catalysts resulting in active transesterification of oil. These catalysts are Lewis acids, hydrophobic (at reaction temperatures of about 170˚C), and insoluble. Moreover, they can be used even with oils containing significant amounts of free fatty acids and water, probably due to the hydrophobicity of their surface. The catalysts are active in the esterification reaction, reducing the concentration of free fatty acids in non-refined oil or in used oil. Other catalyst examples include MgO, CaO, and Al2O3. At these temperatures about (220-240 C), the process pressure is high (40 - 60 bar), which interprets to high construction requirements and high plant design capital. The catalysts used must

most importantly resist poisoning and the leaching of active components. One particular concern is the consistency and the life of the catalysts in the reaction environment.

RECENT APPLICATIONS OF BIO-FUEL 150mpg Algae-Powered Toyota Prius One of the first successful test was an algae fuel powered vehicle which was a modified Toyota Prius known by the name Algaeus which gave a mileage of about 63 kilometers on a litre of green fuel. This car was launched in SanFrancisco. It derived power from green fuel, which was developed by Sapphire Energy. The only drawback was its cost. Engineers Tap Algae Cells for Electricity Light energy is converted to chemical energy and stored in bonds of sugar by plants during the photosynthesis process. This is later utilized as their source of energy. The process of conversion takes place inside a chloroplast, which is believed to be the source of power. Chloroplasts are responsible for the green shade of the plants and algae. The actual process that takes place during photosynthesis is that the water is broken down to oxygen, the negative charged electrons and the positive charged protons. The electrons are stored in the chloroplast, this reaches excited levels when leaves are exposed to sunlight. The excited electrons pass through a number of layers of proteins where they are absorbed. After this the energy from the electrons is used to make sugar until the entire energy is utilized. The New Role of Microbes in Bio-Fuel Production Plants and microbes both are used to refine and synthesize to produce bio-fuel. This helps conserve fossil fuels and are considered to be conservational and renewable sources of energy. Recent studies indicate that microbes can generate fuel from proteins, as a substitute to

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CONCLUSION Summing up, bio-fuels based on algae are being seen as an alternative source of energy. It is still in the embryonic stages as all results are being analyzed before starting the substitution primary source of energy, Crude Oil. The main factors that have to be looked upon the use of these fuels are their production, the by-products of their use, and the cost of production. The source of energy from algae seems to be promising, provided the above mentioned factors are taken care of.

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REFERENCE [1]Demirbas, A.(2009). "Political, economic and environmental impacts of biofuels: A review". Applied Energy86:S108S117.doi:10.1016/j.apenergy.2009.04.03 6. edit [2] "ButylFuel, LLC Main Page". Butanol.com. 2005-08-15. Retrieved 2010-07-14. [3] "ADM Biodiesel: Hamburg, Leer, Mainz". Biodiesel.de. Retrieved 2010-07-14. [4] RRI Limited for Biodiesel Filling Stations. "Welcome to Biodiesel Filling Stations". Biodieselfillingstations.co.uk. Retrieved 2010-07-14. [5] Brown, Robert; Jennifer Holmgren. "Fast Pyrolysis and Bio-Oil Upgrading". Retrieved 15 March 2012. [6] "Alternative & Advanced Fuels". US Department of Energy. Retrieved 7 March 2012. [7] Knothe, Gerhard (2010), Biodiesel and renewable diesel: A comparison, Progress in Energy and Combustion Science [8] Evans, G. "Liquid Transport Biofuels - Technology Status Report", National Non-Food Crops Centre, 2008-04-14. Retrieved on 2009-05-11. [9] Redman, G., The Andersons Centre. "Assessment of onfarm AD in the UK", National Non-Food Crops Centre, 2008-06-09. Retrieved on 2009-05-11. [10] "BIOGAS: No bull, manure can power your farm." Farmers Guardian (25 September 2009): 12. General OneFile. Gale. [11] Electricity from wood through the combination of gasification and solid oxide fuel cells, Ph.D. Thesis by Florian Nagel, Swiss Federal Institute of Technology Zurich, 2008

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|269-280 [12] Frauke Urban and Tom Mitchell 2011. Climate change, disasters and electricity generation. London: Overseas Development Institute and Institute of Development Studies [13] "Threat to Great Apes Highlighted at Virunga Meeting". America.gov. Retrieved 2010-07-14. [14] Scott SA, Davey MP, Dennis JS, Horst I, Howe CJ, LeaSmith DJ, Smith AG. 2010. Biodiesel from algae: challenges and prospects. Current Opinion in Biotechnology. 21(3):277-86 [15]http://domesticfuel.com/wpcontent/uploads/2008/09/ worldbiodiesel.gif [16]http://www.massey.ac.nz/massey/fms/Massey%20New s/2009/04/magazine-images/bio-reactor.jpg [17] Mata et al., 2010 [18] Cravotto et al., 2008; Virot et al., 2008 [19] Rittman et al., 2008 [20] Bligh and Dyer, 1959 [21] Hara & Radin, 1978 [22] Cartens et al., 1996 [23] Nagle & Lemke, 1990 [24] Grima et al., 1994 [25] Fajardo et al., 2007 [26] Lewis et al., 2000 [27] Carvalho and Malcata, 2005 [28] Herrero et al., 2006 [29] Richter et al., 1996 [30] Denery et al., 2004 [31] Mulbry et al., 2009 [32] Soto and Luque de Castro, 2001 [33] Mok et. al., 1992 [34] Eikani et al., 2007 [35] Herrero et al., 2006 [36] GueĂ§lue-UestuendaÄ&#x; et al., 2007 [37] Barclay et al., 1994 [38]http://media.treehugger.com/assets/images/2011/10/ Algenol-algae-image1.jpg [39] Hon-Nami, 2006; Hirayama et al., 1998 [40] Hirano et al., 1997 [41] Demirbas, 2009 [42] Hossain et al., 2008 [43] Ataya et al., 2007 [44] L. Xu et al., 2008 [45] Svensson and Adlercreutz, 2008 [46] Fang et al., 2006 [47] Su and Wei, 2008 [48] Liao et al., 2003 [49] Lopez-Hernandez et al., 2005 [50] Guncheva et al., 2008 [51] Yamane et al., 1998 [52] McNeff et al., 2008 [53] Cozzolino et al., 2006

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www.ijpbs.com (or) www.ijpbsonline.com [54] Sheehan, John; et al. (July 1998). "A Look Back at the U. S. Department of Energy's Aquatic Species Program:

*Corresponding Author: Sarbjeet Singh Gujral* Maharaja Surajmal Institute of Pharmacy (Aff. To GGS Indraprastha University) C-4, Janakpuri, New Delhi-110058, India Phone: 9871334787

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E-mail ID: sarbjeet_singh18@yahoo.in

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Review Article Pharmaceutical Sciences Caralluma fimbriata: AN OVERVIEW S.S. Naingade*1, A. S. Jadhav2, S. B. Surve3 1

Tatyasaheb Kore College of Pharmacy, Warananagar, Kolhapur, (M.S.), India. 2 D.Y. Patil, IPSR, College, Pune University, Pune, (M.S.), India. 3 Rajarambapu College of Pharmacy, Kasegaon, (M.S.), India. *Corresponding Author Email: snehalnaingade1989@gmail.com

ABSTRACT The claimed effect is â&#x20AC;&#x153;helps to reduce body weightâ&#x20AC;?. Pregnane glycosides are plant steroids, which are common to all the Caralluma genus, and various congeners are found in all Caralluma species. The species of Caralluma found in India are edible and form part of the traditional medicine system of the country. An investigation was carried out to find out the effect of Caralluma fimbriata extract on appetite, food intake, Diabetic and anthrometry in adult Indian men and women. Phytocomponents including phenolics, flavonoids, tanninsproanthocyanidins and various plants or herbal extracts have been reported to be Radical Scavengers and inhibitors of Lipid Peroxidation.

KEY WORDS Caralluma Fimbriata, Obesity, Safety, Weight loss.

INTRODUCTION

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Diabetes mellitus is a global health problem throughout the world [1]. Which is characterized by derangement in carbohydrate, fat and protein metabolism, affecting nearly 10% of the population [2]. In the year 2000, 150 million people world-wide had diabetes, and this is expected to double by 2010 This global pandemic principally involves type 2 diabetes, and is associated with several contributory factors such as increased longevity, obesity, unsatisfactory diet, sedentary lifestyle [3]. Globally, type 1 DM affects considerable percentage of population and it leads to morbidity and mortality of the diabetic patients. The plasma lipids usually raised in Diabetic which causes risk factor for Coronary Heart disease [4].

The treatment of Diabetic mellitus is based on oral hypoglycaemic agents and insulin [5]. Lack of insulin affects the metabolism of carbohydrate, protein and fat, and causes a significant disturbance of water and electrolyte homeostasis, the actions of insulin are also impaired by insensitivity of target tissues [3]. The oral hypoglycaemic agents currently used in clinical practice have characteristic some serious side effects. In both type 1 and type 2 diabetes, the actions of insulin are also impaired by insensitivity of target tissues. While this is a fundamental defect in type 2 diabetes, hyperglycaemia can also reduce insulin secretion by the effect of glucose toxicity on beta cell function. The pathophysiological processes in type 1 and 2 diabetes are shown in Fig: [3]

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Hence, there is a need to search for newer antidiabetic agents that retain therapeutic efficacy and reduce the side effects and also risk factor like hyperlipidemia, hypertension and so on [6]. there is an increased demand by patients to use natural products with antidiabetic activity. Hence, today there is need for finding the alternatives which will minimize the side effects and the cost of drug. Therefore it become necessary to make use of vast reserves of plant origins for medical purposes which will help to search effective as well as safer drug remedy for diabetes mellitus. Caralluma fimbriata plant having synuonym caralluma ascendens which belongs to family Asclepiadaceae. It is wisdly found in Africa, the Canary Islands, India, Arabia, southern Europe, Afaganisthan. Caralluma fimbriata is a dry herb growing in the dry parts of the india. Caralluma fimbriata, a traditional Indian â&#x20AC;&#x153;famine foodâ&#x20AC;? with no history of adverse effects[7].The key phytochemical constituents of the herb are

pregnane glycosides, flavone glycosides, megastigmane glycosides, and saponins. Some of the active components present in this plant are CaratubersideA, CaratubersideB, Bouceroside IX, Tomenkogenin, Sitosterol etc [14]. The plant is found to have anti-oxidant activity hence it can be used in the treatment of diabetes mellitus. Fresh leaf extract of Caralluma fimbriata has been reported to reduce blood glucose in normal and alloxan diabetic rabbits. In this study the prolonged effect (up to 14 day) of the ethanolic extracts of whole plant of Caralluma fimbriata in fasting blood glucose (FBG) and biochemical parameters such as serum total cholesterol (TC), LDL, HDL, creatinine, urea, and alkaline phosphatase were studied in alloxan induced diabetic rats. This study was designed to examine the hypoglycemic and antilipidemic effects of Caralluma fimbriata s on alloxaninduced Diabetes Mellitus based on the local uses of the plant for the treatment of diabetes mellitus.

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MECHANISM OF ACTION “Caralluma fimbriata addresses three key underlying issues related to weight gain: an inability to burn fat, constant hunger and poor muscle tone.Caralluma fimbriata contains pregnane glycosides, a phytochemical that blocks the enzyme citrate lyase. When this enzyme’s activity is stopped, your body cannot produce fat [8]. Further,Carallurna fimbriata also blocks another enzyme called Malonyl Coenzyme A. By blocking this enzyme, fat formation is further Blocked and the body is forced to burn its fat reserves. Thus, the rate of fat is losing by the body. Proposed mechanism of action: Kreb’s cycle or Citric acid cycle: Mitochondria are knowm as body’s powerhouse as they generate energy to the body from the

food we eat. Kreb’s Cycle or the Citric Acid Cycle, takes place in the mitochondria, which are found in the cells of most living beings. The body obtains energy when glucose molecules are broken down through a process called catabolism. This process is coupled with the synthesis of a high-energy molecule called ATP (adenosine tri-phosphate) which provide chemical energy to the body. Ghcose is broken down into a compound called pyruvic acid these enters the mitochondria. then after Pyruvic acid is broken down to acetic acid and ultimately, it is converted to acetyl co-enzyme A and citric acid, hence the name Citric Acid Cycle. During this cycle, ATP is formed. The Krebs Cycle is a critical pathway for the body because it generates ATP, which in turn generates the energy the body needs for its day-to-day activities. [9]

Glucose molecule Breakdown into Pyruvic acid Then it entres Mitochondria

Pyruvic acid breakdown into

Acetic acid Converted into Acetyl co-enzyme A and Citric acid

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HOW FAT IS FORMED? The Kreb’s Cycle described above, generates energy from glucose. When glucose rises, fat is formed in adipose (fat) tissue and in the liver

through a process called gluconeogenesis. Fat is formed after the Kreb’s Cycle and is synthesized in the form of fatty acids. The

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www.ijpbs.com (or) www.ijpbsonline.com basic building block of fatty acids is Acetyl coenzyme A, which is mentioned above. Now, for the formation of Acetyl Co A, a vital enzyme called citrate lyase is required. If this enzyme is blocked, then fat cannot be made by the body. The liver produces glucose by gluconeogenesis and glycogen breakdown. The main substrates for gluconeogenesis are shown

in Fig: Insulin suppresses gluconeogenesis and promotes glycogen synthesis and storage. It promotes the peripheral uptake of glucose, particularly in skeletal muscle, and encourages storage (as muscle glycogen) and protein synthesis. It also promotes lipogenesis and suppresses lipolysis. These processes are reversed in the absence of insulin.

Fig: Major gluconeogenic substrates and their tissues of origin

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the principles present in the widely consumed Indian food plant C. fimbriata extract (CFE) suppress appetite, and provide antiobesogenic and metabolic benefits. The data substantiate CFEâ&#x20AC;&#x2122;s reported anorexigenic effects. CFE treatment also gives protection against atherogenesis.CFE also possesses antiobesogenic and antiatherosclerotic properties [10]. Safety: Entire history of Caralluma fimbriata shows to be a little or no adverse effect.It is commonly used as vegetable in several parts of India. It also finds use today as an appetite suppressant and famine food during times of famine, in the semi-arid regions of India(15) There were a mild gastrointestinal upset, which may have

been caused by the gelatin capsules, rather than the extract itself as some subjects in the placebo group complained of identical symptoms. the Department of Pharmacology, St John's Medical College in Bangalore, India testing the safety of Caralluma fimbriata. Doses of 2g/kg body weight and 5g/kg body weight were given to both male and female.after the observation of 14 days all animals were survive at very high dose.thus,Caralluma fimbriata was not toxic [15]. Clinical trials of Caralluma fimbriata extract: 1. USE OF Caralluma Fimbriata EXTRACT TO REDUCE WEIGHT [11] Health and Clinical Research

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A double-blind, placebo-controlled, randomized clinical trial on Caralluma fimbriata extract was done on 50 human subjects.following are the observayions made in the trials Caralluma fimbriata extract was well tolerated.  Caralluma fimbriata extract shows minimal side effect  Caralluma fimbriata extract shows significant weight loss. 2. USE OF CARALLUMA fIMBRIATA EXTRACT FOR OBESITY. [12] This study was conducted 26 overweight patients. 19 on active compound and 7 on placebo compound followed for 4 weeks. Following are the observaions made in the trials Caralluma fimbriata Extract was well tolerated  Caralluma fimbriata Extract has shown long safety with little adverse effect.  Caralluma fimbriata shows significant reduction in body weight. Caralluma fimbriata-Safety pharmacology the acute oral toxicity of Caralluma fimbriata Extract by oral administration in male and female Wistar rats is carried out by dose 2g/kg body weight and 5g/kg body weight.observation is done for 14days. The symptoms subsided within a week in all subjects [13] All animals was well survive. thus, Caralluma fimbriata is safe as it does not show any toxic effect.Caralluma fimbriata extract did not produce signs of toxicity at very high doses of 5 g/kg and it could be classified in the classification of : LD50 more than 5 g/Kg. Clinical tests: The following parameters were estimated Fat analysis: Determines the amount of fat in body

 Blood chemistry and systemic function test: otal cholesterol, LDL, HDL, triglyceride Renal function tests comprsing serum creatinine and blood urea Liver function tests comprising total protein.these tests were done to ensure any adverse effect of Caralluma fimbriata extract on systemic function of body.  Cardiovascular function: Blood pressure ,ECG  Appetite suppressant: Hunger, Urge of eat, fullness.

CONCLUSION: From the clinical trials and clinical test it is concluded that extrat of caralluma fimbriata demonstrate the reduction of weight circumstance, reduction in body weight, reduction in fat.Extract of Caralluma fimbriata also shows appetite suppressant action. Extract of Caralluma fimbriata is capable of decreasing appetite, prevent deposition of fats and reduce obesity.This is because,Caralluma fimbriata blocks the formation of 2 enzymes i.e Acetyl Co-Enzyme A and Malonyl Co-Enzyme A, which are the building blocks of fat synthesis. Caralluma inhibits fat synthesis also increase burning of fats (15). Caralluma fimbriata is believed to act directly on the appetite control center of your brain, specifically the hypothalamus.When you are hungry, your hypothalamus sends your brain a message that you need to eat. Once you do eat and your belly is full, the hypothalamus then tells your brain you’ve had enough and to stop eating (15).

ACKNOWLEDGEMENT: We are thankful to Tatyasaheb Kore college of Pharmacy, Warananagar.

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REFRENCES: 1.

3. 4.

A.H. Morkadad, E.S. Ford, B.A. Bowman, D. E. Nelson, M.M. Engelgau, F. Vincor and J. S. Marks.(2000), Diabetes trends in the U.S: 1990-1998. Diabetes Care 23, 1278-1283.] Kirithikar, K.R., Basu, B.D., An, I.C.S., 1995. Indian Medicinal Plants. Vol.1, International book distributors, Dehradun, India, P. 371 – 372. DEVIDSONS PRINCIPLES AND PRACTICE OF MEDICINE 19 TH EDITION.PG NO.641. [B.-K. Yang, S.-C. Jeong, H.-J. Lee, D.-H. Sohn, and C.-H. Song,“Antidiabetic and hypolipidemic effects of Collybia confluens mycelia produced by submerged culture in streptozotocin-diabetic rats,” Archives of Pharmacal Research, vol. 29, no. 1,pp. 73–79, 2006..] (Chattopadhyay et al. , 1993; Ponnachan and Panikkhar, 1993; Subramonium et al. 1996) . The oral hypoglycaemic agents currently used in clinical practice have characteristic profiles of serious side effects ( Prout, 1974; Holm an and Tuener, 1991). Kumar et al. 249 Prout TE Malaisse WJ, Pirart J (1974) . Proceedings VIII Congress of International Diabetes Federation, Excerpta Medica, Amsterdam, pp. 162. Rajash Kumar G, Achycet Narayan K, Geeta W, Murthy PS, ramesh C, Kapil M and Vibha T (2005) Bansi Saboo, Dia care,Hemant Zaveri, Recent update in management of obesity and Overweight Patients: Standardized extract of Caralluma Fimbriata safe and effective Therapy. International Journal of Clinical Cases and Investigations 2011. Volume 2 (Issue 1), 5:9, 6th February 2011 Lawrence, RM, and Choudhary, S. Caralluma Fimbriata in the Treatment of Obesity. 12th Annual World Congress of Anti-Aging Medicine, December 2004, Las Vegas, USA.

10.

11.

12.

13.

14.

Ronold M.Lawrance and Suneeta choudhary Caralluma Fimbriata in the treatment of Obesity, Western Ceriatric research Institute, Los Angeles Californin,USA. Soundararajan Kamalakkannan, Ramaswamy Rajendran, Ramasamy V. Venkatesh,Paul Clayton, and Mohammad A. Akbarsha Antiobesogenic and Antiatherosclerotic Properties of Caralluma fimbriata Extract. Journal of Nutrition and Metabolism; 2010: 285301. Prof Anura V Kurpad, MD, PhD, Dean, Institute of Population. Use of Caralluma fimbriata extract to reduce weight, Conducted at: Division of Nutrition, St John’s Medical College and Hospital, sajapur Road, Bangalore 560 034 INDIA. Ronold M.Lawrance and Suneeta choudhary Caralluma Fimbriata in the treatment of Obesity, Western Ceriatric research Institute, Los Angeles Californin,USA. Kuriyan R, Tony R, Srinivas SK, Vaz M, Rajendran R and Kurpad AV. Effect of Caralluma Fimbriata extract on appetite, food intake and anthropometry in adult Indian men and women. Appetite May 2007; 48(3): 338-348. Scientific Opinion on the substantiation of a health claim related to ethanol-water extract of Caralluma fimbriata (Slimaluma and helps to reduce body weight pursuant to Article 13(5) of Regulation (EC) No 1924/20061 EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA)

15. Kuriyan. R., et al. Effect of Caralluma Fimbriata Extract on appetite, food intake and anthropometry in adult Indian men and women. Appetite, (2007).

*Corresponding Author:

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S.S. Naingade* Tatyasaheb Kore College of Pharmacy, Warananagar, Kolhapur, (M.S.), India.

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Research Article Biological Sciences ERYTHROCYTE ABNORMALITIES IN A FRESHWATER FISH, LABEO ROHITA EXPOSED TO TANNERY INDUSTRY EFFLUENT G. K WALIA*, D. HANDA, H. KAUR and R. KALOTRA Department Of Zoology and Environmental Sciences, Punjabi University, Patiala-147002 (Punjab) India. *Corresponding Author Email: gurinderkaur_walia@yahoo.co.in

ABSTRACT The erythrocyte abnormality test was analyzed in kidney cells of freshwater fish, Labeo rohita exposed to tannery industry effluent. The 96h LC50 value of tannery effluent against fish was calculated by Finney method and it comes out to be 7.07%. Three sublethal concentrations on basis of 96h LC50 value (3.53%, 1.76% and 0.88%) were selected. Fishes were exposed to these concentrations for 24h, 48h, 72h and 96h. Erythrocyte abnormalities (Nuclear and cellular) other than micronuclei have drawn major attention in recent years. Eleven types of abnormalities (5 nuclear and 6 cellular) were observed. Nuclear abnormalities included Nuclear Extrusion (NE), Blebbed (B), Binucleated (BN), Lobed (L) and Notched (N) nuclei, whereas cellular abnormalities included Enucleated (EC), Vacuolated (VC), Deformed (DC), Echinocytic (EC), Spindle shaped (SC) and Apoptotic (AC) cells. The importance of these erythrocyte abnormalities as biomarkers of exposures has not been previously exploited. In present study effort has been made to compare all erythrocytes abnormalities in fishes exposed to the sublethal concentrations of tannery industry effluent. 3.53% and 1.76% concentration of tannery industry effluent proved to be more toxic concentrations and induced abnormalities which damage erythrocytes completely. However, at 0.88% concentration erythrocyte abnormalities were seen but decreased at 96h as compared to other two concentrations. This concentration can act as safe concentration for disposal of effluent in aquatic ecosystem. The increase in erythrocyte abnormalities indicated that tannery industry effluent increased the clastogenic effects on peripheral erythrocytes of Labeo rohita and may have similar effects on the human population located around the river and consume fishes.

KEY WORDS Erythrocyte abnormality test, aquatic pollution, tannery industry effluent, Labeo rohita.

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1. INTRODUCTION The aquatic environment plays a vital role in functioning of ecosystem. However, industrialization, urbanization and nonsustainable developments are posing serious threat to the aquatic system. Water bodies like rivers, lakes and ponds are polluted due to the discharge of industrial effluents, agricultural runoff and domestic wastes into them. In Punjab (India) due to industrialization the condition is more severe as River Satluj which is the largest

and lifeline of the state is continuously being subjected to effluents from industries. Tannery industries located at Jalandhar contributes its major share of effluents in river Satluj. Further, tannery effluent is very toxic having foul smell and is damaging the aquatic flora and fauna. Fishes are excellent subject for the study of genotoxicity because they are in direct contact with the toxicants present in the water. They can also metabolize, concentrate and store water borne pollutants [1]. Fish erythrocytes are distinct because they possess a nucleus and their

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www.ijpbs.com (or) www.ijpbsonline.com interpretation in form of morphological changes became an important bioindicator of pollution. Erythrocyte abnormality test (change in nucleus and cell structure of erythrocytes) is one of the best diagnostic tools to judge the genotoxicity caused by the pollutants present in the aquatic ecosystem. Over recent years changes in erythrocyte nuclei have been increasingly used to evaluate genotoxic effects of various industrial effluents from textile mill, paper mill, peteroleum refinery and polycyclic aromatic. [2]. The formation of erythrocyte alterations especially nuclear alterations was first described by Carrasco et al. [3] and various other abnormalities like blebbed, Notched, Binucleate and Lobed nuclei have also been used as possible indicators of genotoxicity [4-6]. In the present study, genotoxic effect of tannery industry effluent on Labeo rohita has been done. Genotoxic evaluation in terms of erythrocyte abnormalities have not been previously studied in Punjab. Tannery industry is included in the list of red industries [7], and their effluents are highly toxic to the aquatic environment. There are about 30 tannery industries in Jalandhar which dump their effluents in Kala Sanghia drain which flows into chitti bein and then joins river Satluj. People living nearby have various skin diseases and are continuously exposed to this effluent. Family Cyprinidae is one of the largest families of freshwater fishes, having 2400 species belonging to 220 genera. These fishes are abundant in rivers of Punjab. Thus, they are ideal model to monitor genotoxic effect. Labeo rohita is selected because of its abundance, easy availability and high consumption by the people of Punjab. The main aims of the present study are 1) To determine LC50 for the preparation of three sublethal concentrations 2) To detect erythrocyte abnormalities (nuclear as well as cellular).

2. MATERIAL AND METHODS

2.1 Collection of specimen Freshwater fish, Labeo rohita of about 6-8 cm in length and 32â&#x20AC;&#x201C;58 gms in weight were collected from government fish seed farm Patiala in wide mouthed plastic bags containing freshwater and oxygen. They were acclimatized in laboratory for 20 days and were treated with 0.1% of KMnO4 solution for 30 minutes to remove any external infections. They were fed with pelted feed and feeding was stopped 24h prior to commencement of genotoxicity tests. They were not fed during experimental periods. 2.2 Effluent from tannery industry Effluent of tannery industry was taken directly from the waste outlet of an industry unit based at Jalandhar. Tannery industry effluent contains several complex organic and inorganic components like sulphides (sodium sulphide, sodium hydrosulphite and calcium hydrosulphide), sodium chloride, cyanides, dimethyl amines, chromium sulphate salts, oil, grease, alum salts and suspended solids [8]. 2.3 Determination of LC50 and selection of concentrations The 96h LC50 of tannery industry effluent was calculated by following the method given by Finney [9]. Three sublethal concentrations 3.53%, 1.76% and 0.88% (1/2, 1/4, 1/8 of 96h of LC50 value) were selected. Apparently healthy, uninjured and uninfected fish specimens were used. 2.4 Experimental design Fishes were released in tubs containing water (control) and three sublethal concentrations (3.53%, 1.76% and 0.88%) for 24h, 48h, 72h and 96h by using the method given by Ayllon et al. [4]. A of total 50 fishes were used for the experiment. 2.5 Measurement of erythrocytes abnormalities Kidney Blood was used to make smear on a clean slide. Erythrocytes abnormalities were classified according to Claxton et al. [10]. Five fishes were

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www.ijpbs.com (or) www.ijpbsonline.com used for each concentration. From each fish, four slides were prepared. For each hour 4000 cells (1000 cells from each slide) of control and treated groups were observed and photomicrographed. 2.6 Statistical analysis Data of erythrocyte abnormalities were subjected to ANOVA and Tukey test. Statistical analysis was done by using computer software ‘Graph pad prism’. p<0.05% was considered to be the level of significance. Statistical significance of erythrocyte abnormalities for control and treated groups of each concentrations and durations was also evaluated. Frequencies of erythrocytes abnormalities were expressed as Mean (%) ± S.E.

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3. RESULTS The 96h LC50 value of tannery industry effluent against Labeo rohita came out to be 7.07%. In Labeo rohita, a normal erythrocyte is elliptical in shape with centrally placed nucleus in the clear cytoplasm (Fig. 1). Such normal erythrocytes were observed in control fishes. Erythrocyte abnormalities induced by effluent is summarized in the Table. Nuclear abnormalities are considered as precursors of micronuclei whereas cellular abnormalities results into cell death. Five types of nuclear abnormalities viz., Nuclear Extrusion (NE, Fig. 2), Blebbed (B, Fig. 3), Binucleated (BN, Fig. 4), Lobed (L, Fig. 5) and Notched (N, Fig. 6) nuclei were observed. In 3.53% and 1.76% concentrations, nuclear abnormalities increased from 24h to 96h whereas in 0.88% concentration, these abnormalities increased from 24h to 48h then decreased upto 96h except in Lobed nuclei which decreased from 24h to 96h and Notched nuclei which increased from 24h to 72h then decreased sharply at 96h. Six types of cellular Abnormalities viz., Enucleated (EC, Fig. 7), Vacuolated (VC, Fig. 8), Deformed (DC, Fig. 9),

Echinocytic (EC, Fig. 10), Spindle shaped (SC, Fig. 11) and Apoptotic (AC, Fig. 12) cells were observed. In 3.53% and 1.76% concentrations, abnormalities increased from 24h to 96h whereas in 0.88% Vacuolated, Deformed, Echinocytic and Apoptotic cells increased from 24h to 48h then decreased up to 96h, while Enucleated and Spindle shaped cells decreased from 24h to 96h. Mean percentage of erythrocyte abnormalities increased with increase in concentration and duration of tannery industry effluent. At 72h of all the three concentrations, frequency of erythrocyte abnormalities rose steadily from 47.52 ± 0.66c (0.88%) to 58.22 ± 1.15c (1.76%) and 74.27 ± 0.88c (3.53%). However, in extreme exposure (96h), the initial frequency of erythrocyte abnormalities increased from 41.82 ± 2.03d to a maximum of 71.05 ± 0.66d and 80.55 ± 2.52d in 0.88%, 1.76% and 3.53% respectively as shown in Histogram. Thus, as the concentration increased, the cell alteration registered a quantitative increase with the increase of exposure time. Overall percent frequencies of cells with erythrocyte abnormalities in each concentration as well as control are shown in Pie Charts (a-d).

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Table. Frequencies of erythrocyte abnormalities in kidney cells of Labeo rohita after treatment with tannery industry effluent. Experimental groups

Control

Duration of exposure (h)

24 48 72 96

4000 4000 4000 4000

0 0 0 11 11

0 12 0 12 24

12 11 0 0 23

0 0 11 19 30

24 48 72 96

4000 4000 4000 4000

70 137 67 116 390

85 115 90 99 389

15 35 25 28 103

225 155 26 141 547

4000

48 72 96

4000 4000 4000

24 48 72 96

4000 4000 4000 4000

124 136 232 578 148 159 162 188 657

84 96 103 357 85 110 146 162 503

17 21 37 86 127 172 188 196 683

153 183 242 638 94 115 129 142 480

24 37 56 125 92 154 166 178 590

Total

0.88% Total

1.76% Total

3.53%

Nuclear abnormalities

Number of abnormal cells Cellular abnormalities

Total

Mean (%) ±S.E

EnC

0 0 0 0 0 12 0 0 0 12 Treated 10 24 54 15 63 18 44 7 171 64

0 0 0 0 0

12 13 18 15 58

0 0 0 0 0

15 16 15 13 59

13 15 0 13 41

52 67 56 83 258

1.30±0.33 1.67±0.88 1.40±0.66 2.07±0.88

34 134 48 114 330

796 507 651 425 2379

90 138 110 122 460

667 423 542 323 1955

195 273 261 254 983

2271 2086 1901 1673 7931

56.77±1.45 a 51.15±1.76 b 47.52±0.66 c 41.82±2.03 d

347

606

1372

34.30±0.66

30 61 99 200 353 461 471 479 1764

114 127 154 413 243 288 272 302 1105

487 533 553 1920 560 610 680 711 2561

87 96 171 433 125 139 198 255 717

780 864 955 3205 343 391 412 450 1596

112 155 240 580 93 135 147 159 534

2012 2329 2842 8555 2263 2734 2971 3222 11190

50.30±0.33 b 58.22±1.15 c d 71.05±0.66

56.67±1.20 a b 68.35±1.20 74.27±0.88 c 80.55±2.52 d

a, b, c and d: significant differences at 24h, 48h, 72h and 96h respectively from the control at p<0.05, T= total number of cells, t= total number of abnormal cells. NE= nuclear

extrusion, B= blebbed, L= lobed, N= notched, EnC= enucleated cell, VC= vacuolated cell, DC= deformed cell, EC= echinocytic cell, SC= spindle shaped cell, AC= apoptotic cell.

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Fig. 1: Normal erythrocyte (Control), Erythrocyte abnormalities (Treated): Nuclear abnormalities: Fig. 2: Nuclear extrusion, Fig. 3: Blebbed nuclei Fig. 4: Binucleate, Fig. 5: Lobed nuclei, Fig. 6: Notched nuclei Cellular abnormalities: Fig. 7: Enucleated cells, Fig. 8: Vacuolated cell

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Fig. 9: Deformed cells, Fig. 10: Echinocytic cells, Fig. 11: Spindle shaped cells, Fig. 12: Apoptotic cells

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Percent frequency of erythrocyte abnormalities in Labeo rohita after treatment with tannery industrial effluent.

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Percent frequency of erythrocyte abnormalities in Labeo rohita after treatment with tannery industrial effluent.

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4. DISCUSSION The impact of industrial effluent on aquatic ecosystem is a growing problem and studies on industrial effluents indicated that genotoxicity tests have a viable role in environmental quality monitoring and control. Although there are large number of genotoxicity assays, only a relative number have been used for the evaluation of industrial effluents [10]. In Punjab there are no or minimal use of treatment plants for treating tannery industry effluents. Thus, untreated effluent is discharged into rivers which makes water turbid, emits foul smell, make oily layer and toxic. This toxic water from tannery industries of Jalander is carried by a rivulet (chittin bein) to River Satluj which ultimately damage aquatic flora and fauna. Genotoxic effect of other industrial effluents have been reported by authors [5, 6, 11-14]. Cavas and Ergene-Gozukara [6] determined the increase in number of cells with nuclear abnormalities in fishes exposed to petroleum

refinery and chromium processing factory effluent. Matsumoto et al. [15] found frequency of erythrocyte abnormalities to be more in Oreochromis niloticus exposed to water receiving Tannery industry effluent. Eleven types of abnormalities were found in the present study. Increase in frequency of erythrocyte abnormalities exhibited time and concentration dependent response. Tannery industry effluent mainly contains chromium and heavy metals. Chromium undergoes intercellular reduction from chromium (VI) to chromium (III) and oxygen generates as highly reactive free radical which can react with DNA. Similar work has been reported by other authors and suggested that nuclear budding in interphase cause Blebbed and Lobed nuclei. The entire process represents the mechanism of elimination of amplified genes from the nuclei [16, 17]. Further, Von Sonntag and Steenken [18, 19] hypothesized that these abnormalities arise due to damage caused to the genetic material by

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www.ijpbs.com (or) www.ijpbsonline.com free radical produced under oxidative stress due to the toxicants. Aneuploidy is another abnormality that resulted due to tubulin failure and mitotic fuses caused by aneugenic actions of toxicants and resulted in formation of Binucleated cells and Notched Nuclei [20, 21]. Ateeq et al. [22] elaborated the sequence of cellular degradation under the impact of toxicants and also suggested that toxicants cause hypoxic conditions which result in depression of ATP that lead to abnormal shape of erythrocytes. Further, toxicants interrupted the lipid solubility of membranes of erythrocytes resulting in Vacuolated and Echinocytic cells and ultimately leading to apoptosis. Thus, presence of chromium and heavy metals induced such changes in fishes which are not reversed and cause cytotoxic damage resulting in death of fishes. Fishes are major protein source and reflect water quality. Their stock depletion reflects the water quality of major rivers.

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5. CONCLUSION The results obtained from this study Indicated that the tannery industry effluent being discharged into the river have genotoxic potential and is capable of causing significant ecological disruption in the receiving environment. The increase in the formation erythrocytes abnormalities (nuclear and cellular) in erythrocytes of Labeo rohita were identified as good genotoxic biomarkers for monitoring impact of industrial effluents in the environment. Since the assessement of cytotoxicity markers is quite helpful in terms of erythrocytes abnormalities. Examination of the biomarkers should be included into routine genotoxic survey to determine cytotoxicity at subcellular damage caused by environmental pollution. It is, therefore suggested that tannery industry effluent should be passed through treatment

plants before being discharged into the aquatic ecosystem.

REFERENCES [1] Al-Sabti, K., Handbook of genotoxic effects and fish chromosomes. Kligerman, A. D. (Eds.) Jozef Stefan Institute, Yugoslavia, Jozef Stefan Institute Press. pp. 221, (1991) [2] Gravato, C and Santos, M.A., Juvenile sea bass liver P 450, EROD induction and erythrocytic genotoxic responses to PAH and PAH-like compounds. Ecotoxicol. Environ. Saf. 51, 115-127, (2002) [3] Carrasco, K.R., Tilbury, K.L and Meyers, M.S., Assessment of the piscine micronucleus test as an insitu biological indicator of chemical contaminant effects. Can. J Fish. Aquat. Sci. 47, 2123-2136, (1990) [4] Ayllon, F., Suciu, R., Gephard, S., Juanes, F. and GarciaVazquez, E., Conventional armament wastes induce micronuclei in wild brown trout Salmo trutta. Mutat. Res., 470, 169-176, (2000) [5] Cavas, T. and Ergene-Gozukara, S., Micronuclei, nuclear lesion and interphase silver-stained nucleolar-organizer (AgNORs) as cyto-genotoxicity induicators in Oreochromis niloticus exposed to textile mill effluent. Mutat. Res., 538: 81-91, (2003) [6] Cavas, T. and Ergene-Gozukara, S., Micronucleus test in fish cell: A bioassay for in situ monitoring of genotoxic pollution in the marine environment. Environ. Mol. Mutagen., 46:64-70, (2005a) [7] Punjab Pollution Control Board., List of red category of industries, Punjab, India, (1999) [8] EPR., Environment (protection) third (amendment) rules. Ministry of Environment and Forests Notification, India, (2010) [9] Finney, D. J., Probit analysis. Cambridge University Press, London, (1971) [10] Claxton L.D, Houk V.S and Hugles T.J., Genotoxicity of the industrial wastes and effluents. Mutat. Res., 410: 237-243, (1998) [11] Da Silva Souza, T. and Fonntanetti, C.S, Micronucleus test and observation of nuclear alteration in erythrocytes of Nile tilapia exposed to waters affected by refinery effluent. Mutat. Res. 605, 87-93, (2006) [12] Hafez, A. M., Mugil cephalus Genome: A sensitive monitor for genotoxicity and cytotoxicity in aquatic environment. Aus. J. Bas. Appl. Sci., 3(3), 2176-2187, (2009) [13] Kirschbaum, A. A., Seriani, R., Pereira, C. D. S., Assuncao, A., Abessa, D. M. S., Rotundo, M. M. and Ranzani-Paiva, M. J. T., Cytotoxicity biomarkers in fat snook, Centropomus parallelus from Cananeia and Sao

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www.ijpbs.com (or) www.ijpbsonline.com Vicente estuaries, Brazil. Genet. Mol. Biol., 32(1), 151154, (2009) [14] Saleh, K. A. J., The intensity of pollutant genotoxicity in Lake Uluabat. Int. J. Biotechnol. Biochem., 6(4), 625-631, (2010) [15] Matsumoto, S. T., Mantovani, M. S., Malaguttii., M. I. A., Dias, A. L., Fonseca, I. C. and Marin-Morales, M. A., Genotoxicity and mutagenicity of water contaminated with tannery effluents, as evaluated by the micronucleus test and comet assay using the fish, Oreochromis niloticus and chromosome aberrations in onion root-tips. Genet. Mol. Biol., 29(1), 148-158, (2006) [16] Shimizu, N., Itoh, N., Utiyama, H. and Wahl, G. M., Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase. J. Cell Biol., 140, 1307-1320, (1998) [17] Crott, J. W., Mashiyama, S. D., Ames, B. C. and Fenech, M., The effect of folic acid deficiency and MTHFR C677T polymorphism on chromosome damage in human lymphocytes in vitro. Cancer Epidemiol. Biomark. Prevent., 10, 1089-1096, (2001)

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|287-295 [18] Von Sonntag, C., The Chemical Basis of Radiation Biology. New York: Taylor and Francis. p. 516, (1987) [19] Steenken, S., Purine basis, nucleosides and nucleotides: aqueous solution redox chemistry and transformation reactions of their radical cations and e and OH adducts. Chem. Rev., 89, 503-520, (1989) [20] Ventura, B. C., Angelis, D. F. and Marin-Molares, M. A., Mutagenic and genotoxic effects of the atrazine herbicide in Oreochromis nilotics (Perciformes, Cichlidae) detected by the micronuclei test and the comet assay. Pest. Biochem. Physiol., 90, 42-51, (2008) [21] Fernandes, T. C. C., Mazzeo, D. E. C. and MarinMolares, M. A., Mechanism of micronuclei formation in polyploidizated cells of Allium cepa exposed to trifluralin herbicide. Pest. Biochem. Physiol., 88, 252-259, (2007) [22] Ateeq, B., Farah, M. A., Ali, M. N. and Ahmed, W., Induction of micronuclei and erythrocyte alterations in the catfish, Clarias batrachus by 2,4dichlorophenoxyacetic acid and butachlor. Mutat. Res., 518, 135-144, (2002)

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J-1/2B, Gobind Colony, Rajpura-140401, Patiala, Punjab, India. Telephone number: 01762-223720 (Res.), 09815788644 (Mob.) E-mail address- gurinderkaur_walia@yahoo.co.in

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Letter To Editor Biological Sciences DRUG – PROTEIN BINDING (Some care must be taken to determine the value of KB correctly in the special case of r=1)

Manjunath.R #16/1, 8th Main Road, Shivanagar, Rajajinagar, Bangalore560010, Karnataka, India *Corresponding Author Email: manjunath5496@gmail.com

The binding of a drug to its target protein is the event that leads to medicinal activity. Therefore, the amount of drug - protein complex formed by a drug ultimately determines the medicinal effects displayed by that compound. This is described by a simple equilibrium reaction. D + P ⇌ DP With dissociation constant K = [D] [P]/ [DP] (1) In this equation [D] = [D] T – [DP] and [P] = [P] T – [DP] Where [D] T and [P] T are the initial total concentrations of the drug and protein, respectively. Using the equilibrium relationship K [DP] = [D] [P] and Substituting, [P] = [P] T – [DP] Gives: K [DP] = [D] {[P] T – [DP]} From which, bound fraction, FBP, of the protein is given by: FBP = [DP] / [P] T = [D] / K+ [D] (2) On the other hand, Using the equilibrium relationship K [DP] = [D] [P] and Substituting, [D] = [D] T – [DP] Gives: K [DP] = [P] {[D] T – [DP]} from which, bound fraction, FBD, of the drug is given by: FBD = [DP] / [D] T = [P] / K+ [P] (3)

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Dividing Eq. (2) by (3) we thus get FBP/ FBD = [D] T / [P] T = [D] {K + [P]} / [P] {K+ [D]} Setting [D] T = [P] T =[X] T, along with [D] = [P] =[X], then above expression turns into [X] T / [X] T = [X] {K + [X]} / [X] {K+ [X]} which is just a tautology, 1=1. We think this makes sense intuitively: if the starting (total) concentrations of drug and protein are equal, then it is easy for the free concentrations to be equal. However, if the starting concentrations are unequal, then it is hard for the free concentrations to be equal. If we set now [D]/ [P] = r Then we get FBP/ FBD = r {K + [P]} / {K+ [D]} International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)

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FBP {K+ [D]} = r FBD {K + [P]} FBPK+ FBP [D] = r FBDK + [D] FBD

On rearrangement this leads to K {FBP − r FBD} = [D] {FBD − FBP} Or

K / [D] = {FBD − FBP} / {FBP − r FBD}

(4)

Right after this expression, we reach the conclusion that, when r=1, it follows that K = 0. This implies a situation in which all the protein is completely bound up in drug-protein complexes. It is a limiting case in which the binding constant KB = 1/K is infinite. From Eq. (1) KB follows as KB= [DP] / [D] [P] If we set now [P] = [D] / r Then the above expression turns into KB = r [DP] / [D] 2 When r =1, it follows that KB = [DP] / [D] 2 However, in special case of r=1, KB does not need to be infinite, because we just showed above that, for r=1, KB= [DP] / [D] 2. Therefore, it is not a given that KB will go infinity; indeed some care must be taken to determine the value of KB correctly in the special case of r=1.

REFERENCES 1. Shargel, Leon (2005). Applied Biopharmaceutics & Pharmacokinetics. New York: McGraw-Hill, Medical Pub. Division. 2. Binding D B by Michael K. Gilson, pharmacy.ucsd.edu/labs/Gilson/BindingDB-Intro.pdf (2009)

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(Email: manjunath5496@gmail.com) #16/1, 8th Main Road, Shivanagar, Rajajinagar, Bangalore560010, Karnataka, India

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Research Article Pharmaceutical Sciences IN VITRO ANTIBACTERIAL ACTIVITY OF SEVEN SPICES AGAINST CLINICAL ISOLATES OF ENTEROCOCCI *SHARMA REVATI, CHAPAGAIN BIPIN, PAI (BHAT) CHITRA Department of Microbiology, MGM Medical College and Hospital, Kamothe, Navi Mumbai â&#x20AC;&#x201C; 410219 *Corresponding Author Email: revupattani@gmail.com

ABSTRACT Aim: To explore the in vitro antibacterial activity of seven ethanolic extracts of spices against the clinical isolates of enterococci. Methods: 215 enterococcal strains were isolated from clinical samples. Ethanolic extracts of Cumin (Cuminum cyminum), Cinnamon (Cinnamomum zeylanicum), Ginger (Zingiber officinale), Fenugreek (Trigonella foenum graecum) , Cloves (Syzygium aromaticum), Cardamom (Elettaria cardamomum Maton) and Black pepper (Piper nigrum) were prepared using Soxhlet Apparatus. The antibacterial effect of the extracts was studied using well diffusion method. Statistical analysis was carried out by Chi-square test using SPSS 17 software. Results: Only Cinnamon and Ginger were found to have activity against all the isolates whereas Cumin and Cloves had varied effect on the strains. Fenugreek, Black pepper and Cardamom did not show any effect on the isolates. The zone diameter of inhibition obtained for Cinnamon, Ginger, Cloves and Cumin ranged from 31-34mm, 27-30mm, 2526mm and 19-20mm respectively. Conclusion: C.zeylanicum and Z.officinale showed the maximum antibacterial activity against the enterococcal isolates followed by S.aromaticum and C.cyminum. The findings of the study shows that spices used in the study can contribute in the development of potential antimicrobial agents for inclusion in the anti enterococcal treatment regime.

KEY WORDS Enterococci, antimicrobial sensitivity testing, ethanolic extracts of spices.

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INTRODUCTION Enterococci classified as Group D streptococci are the normal intestinal flora in humans. They are second only to Escherichia coli as agent of nosocomial UTI and third behind Staphylococcus aureus and Coagulase negative staphylococci as agents of nosocomial bacteremia.1,2 Enterococcus faecalis is the species which dominate among the enterococcal human infections while majority of the remainder are caused by Enterococcus faecium. A major contributing factor to pathogenesis of enterococci is their resistance to a wide variety of antibiotics. Enterococci have intrinsic

resistance as well as can acquire resistance against a wide variety of antibiotics. Antibiotic resistance has become a serious threat around the globe. This has forced mankind to search for newer safe and effective alternative from natural resources available on the earth and one such alternative is plant sources. Herbal extracts are gaining popularity in terms of natural antimicrobial preservatives or additives. 3 People of India as well as other parts of the world are using spices to enhance flavor and aroma of the foods. They have a long standing practice of using them in the treatment of ailments.158 Antibacterial activities of extracts of different plants against various microorganisms

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www.ijpbs.com (or) www.ijpbsonline.com have been reported by many scientists.4,5 Some medicinal herbs have also been assessed. Some spices were specifically tested for anti-microbial activities.6However there are scanty reports of the antimicrobial activity of the various Indian kitchen spices specifically against multi drug resistant clinical isolates of enterococci possessing diverse genetic machinery of virulent factors. Many studies have only dealt with standard strains of enterococci but not the clinical isolates.7,8 The present study aims at evaluating the effect of seven spices which are commonly used in Indian cuisine on different species of enterococci obtained from divergent clinical manifestations. If proved of value they can be systematically screened for any novel active compounds.

MATERIALS AND METHODS

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A) Enterococcal isolates: A total of 215 enterococci strains were isolated from different clinical samples received in the Department of Microbiology of a tertiary care center. They were speciated in accordance to a biochemical key as given by Facklam and Collins.9 B) Spices: Seven types of typical Indian spices and herbs namely Piper nigrum (black pepper , Cuminumcyminum (cumin), Trigonella foenum graecum (fenugreek), Cinnamomum Zeylanicum (cinnamon), Elettaria cardamomum Maton (cardamom), Syzygium aromaticum (cloves) and Zingiber officinale (ginger) in the form of whole fruit, seed and rhizomes barks, and buds were purchased from a local retail market. All the spices were washed thoroughly with distilled water to make them completely free from any contaminated particulate matter. The spices were then air dried and used for further procedure.

1) Extraction of spices: The extraction of spices was carried out according to a procedure given by Manoj Kumar Singh et al which is as follows.10 a) Preparation of the powdered spices: The air dried materials were powdered using a mixer grinder. b) Soxhlet extraction: 150g of the powdered plant material was subjected to Soxhlet extraction using ethanol (500mL) as an extracting solvent. The extraction temperature was 780C. The time of extraction was 3-6 hrs or till the color solvent appeared in the siphon. The crude extract was kept at room temperature for ten days to remove any solvent remaining in the extract. The extracts were stored in dark colored bottles, labeled and stored at 40C until use. 2) Preparation of the working solution: The crude extract was dissolve in dimethyl sulphoxide (DMSO) (10-40%) by dissolving 1gm extract in 10ml of it. The concentration of the stock solution becomes 100mg/ml. The solutions were stored in refrigerator. 3) Inoculum preparation: Three or four colonies of bacteria were transferred to the test tube containing 5ml of sterile nutrient broth. It was incubated at 37ÂşC for 3 or 4 hr. The tubes were compared with McFarland Nephelometer Standard 0.5 (turbidity standard). A blank nutrient broth was used as a control. 4) Evaluation of the antibacterial activity of the spices extract: The crude extracts of spices were screened for its antimicrobial activity against the organisms by agar well diffusion method given by Dingle et al.11 Sterile cotton swab was dipped in to the prepared inoculums and seeded all over the MHA plate by rotating through an angle of 60Â° .After each swabbing finally. the swab was passed round the edges of the agar surface and

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www.ijpbs.com (or) www.ijpbsonline.com left to dry for few minutes at room temperature with lid closed. Then with the help of sterile cork borer (6mm), wells were made in the inoculated plate and labeled. 50μl of the working suspensions of the spices extract were dispensed in the respective wells with the help of the micropipette. The solvent DMSO itself was tested for its activity as a control as the same time. The plates were left for half an hour with the lid closed. Then the plates were incubated at 37°C for 24 hr then observed for the zone of inhibition which is suggested by the clear area around the well.

Statistical analysis The experiments were done at least twice and their mean values were represented. All statistical analyses including Chi square and Fischer’s exact test were done in SPSS Version 17.0. Differences were considered significant when p< 0.05.

RESULTS The 215 isolates of enterococci were subjected to a series of biochemical tests as per the key given by Facklam and Collins the result of which is as depicted in the following table.

Table 1 – Percentage distribution of various species of enterococci isolated from clinical samples. Enterococci species isolated n = 215 Number of isolates (%) E. faecalis 113 (58%) E. faecium 70 (33%) E. avium 11 (5%) E. raffinosus 8 (4%) E. casseliflavus 5 (2%) E. durans 6 (3%) E. gallinarum 2 (1%) The spice extracts were obtained using Soxhlet apparatus and the physical characteristics of the same studied which is presented in the table given below.

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Table 2: Physical characteristics of the spice extract Extract Color Texture Solubility Black pepper Black Oily DMSO Cardamom Green Oily DMSO Cinnamon Red Oily DMSO Fenugreek Yellow Oily DMSO Cloves Brown Gummy DMSO Ginger Yellowish brown Gummy DMSO Cumin Brown Watery DMSO

Smell Spicy Spicy Spicy Spicy Spicy Spicy Spicy

The antimicrobial activity of the above seven spices was tested against the multi drug enterococcus by using agar well diffusion assay. The comparative bioactivity of the spices is as shown in the Fig 1

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Fig 2: Antimicrobial activity of the seven spices used in the study. Antimicrobial activity of spices (%) 120 Percentage

100

94 100

100

80 60 40 20

Sensitive

Resistant

Spices

The crude ethanolic extract of cinnamon, cloves, ginger, cardamom and cumin showed significant antibacterial activity against all the clinical isolates of enterococci. The spices differed in their antimicrobial effect as shown in the above figure. (Fig 2) Maximum antibacterial effect was shown by cinnamom and ginger with all 215 isolates

inhibited when tested with it. The maximum diameter of zone of inhibition against the isolates achieved was by cinnamon (34 mm) followed by cloves (26 mm), ginger (20 mm) cardamom 18 mm and the least was shown by cumin (14mm). Fenugreek and black pepper did not have any antibacterial effect on enterococcal isolates.

Fig 3: Zone diameter of the seven spices for the enterococcal isolates. Zone diameter of the seven spices for enterococci (mm) 140 120

100 80

0 12

61 5 0 17

60 40

30 23

1 12 21

15-16

17-18

19-20

cumin cloves

39 40

12 21-22

23-24

0 25

25-26

27-30

Ginger Cinnamon

0 25 31-34

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13-14

0 19

cardamom

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Table 2: Antimicrobial sensitivity pattern of different spices for E.faecalis and E.faecium. Organism E.faecalis n=113 E.faecium n=70

Cinnamon

Ginger

Cloves

Cumin

Cardamom

113(100%)

106(94%)

1(1%)

8 (7%)

70 (100%)

68 (97%)

1(1%)

3 (4%)

Fenugreek

Pepper

Table 3: Antimicrobial sensitivity pattern of different spices on unusual enterococcal species. Organism Cinnamon Ginger Cloves Cumin Cardamom E.avium 11 (100%) 11(100%) 9 (82%) 0 0 n=11 E.casseliflavus 5 (100%) 5 (100%) 5(100%) 0 0 n=5 E.gallinarum 2 (100%) 2 (100%) 2(100%) 0 1 (50%) n=2 E.durans 6 (100%) 6 (100%) 6(100%) 0 0 n=6 E.raffinosus 8 (100%) 8 (100%) 8(100%) 0 0 n=8 The above table shows that all the different species of enterococcus was maximally inhibited by cinnamon and ginger. Cumin had an inhibitory effect only on one isolates of E.faecalis eight (7%) and E.faecium 3 (4%) each, whereas cardamom showed an antibacterial activity against E.faecalis, E.faecium and one (50%) E.gallinarum. Statistical analysis: The Ď&#x2021;2-test was employed to compare the antibacterial activities of all the spices, The significant differences in activity were found between cinnamon and clove (p=0.03), as well as between Cinnamon and cardamom (p=0.020). Similarly difference in the bioactivity was found for ginger and cloves as well as ginger and cardamom.

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DISCUSSION It is becoming more and more common to find many bacteria developing resistance to most common antibiotics. As the resistant strains

develop ways around our traditional methods, it may be important to look into alternative methods to fight back. The alternatives may be as simple as looking in your spice cabinet. With this aim we tested antimicrobial activity of seven spices against the multi drug clinical isolates of enterococci. From the early stages of synthetic antibiotic use, bacteria were already evading our efforts. Bacteria become resistant by actually destroying the drug, some staphylococci create penicillinase, which destroys penicillin. Some bacterial sites where the antibiotic attaches are altered making them less susceptible to the drug.12 The overuse of antibiotics and the rate that bacteria replicate make the antibiotics less effective, which in turn creates more resistant strains. Because of this bacterial behavior, it has been important for scientists to conduct research that will aid them in changing antibiotics to increase their effectiveness. The most promising spices that displayed antibacterial properties according

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www.ijpbs.com (or) www.ijpbsonline.com to this study are cinnamon and ginger which showed antimicrobial effect on all the isolates while cloves cardamom and cumin showed activity against some isolates. It could also be said that the concentration makes a difference. The C. zeylanicum bark is rich in cinnamaldehyde (50.5%), which has been proven to be active against many pathogenic gram-positive and gram-negative bacteria.13 Ali et al. reported cinnamaldehyde as the active agent to inhibit the growth of both antibiotic-sensitive and resistant strains of Helicobacter pylori.14 It has been reported that S. aromaticum oil contains high (75%) eugenol, and the antibacterial activity of S. aromaticum is attributed to this compound.13 Another important antimicrobial compound is tannin, in S. aromaticum, which also aids the process of antimicrobial action. The antibacterial activity of C. cyminum essential oil is perhaps attributable to the high levels of cumin aldehyde (16.1%), the other main component includes a-pinene (11.4%).15, 16. Here, we have not studied the antibacterial activity of the components within the extracts; however, the compounds mentioned above may be responsible for the anti-enterococcal activity of the extracts, as has been investigated in our study. Many studies have reported the antibacterial activity of different spices against a broad range of Gram positive and Gram negative strains. In all these studies a standard strain (E.faecalis ATCC 15753) of enterococci along with other microorganisms were tested for its susceptibility and resistance against the spices.8,10 Several studies have reported that cinnamon have shown a significant antibacterial activity against the standard strain which is similar to our study with enterococcal isolates.17,18,19 Some studies have also reported equal or high broad spectrum antimicrobial activity of the spices when compared with standard antibiotics tested.10

Our study only analysed the zone of inhibition as a parameter for the antibacterial activity of spices against the isolates. However for accurate description of the antimicrobial property of the spices we suggest MIC and Kill kinetics (rate and extent of bacterial killing) to be done. Studies have shown it displays a better sensitivity trends to physicians than disc diffusion methods. 13 Time kill experiments should also be considered as tool to determine whether the activity of the spices is dosage and time dependent so that it would imply a more rational basis for determining the optimal dosage of antimicrobial treatment regimens in order to combat the spread of antimicrobial resistance.13

CONCLUSION The degree of antibacterial activity of the spices tested can be represented in the following order: Cinnamon = Ginger > Cloves > Cardamom > Cumin. All the five spices were found to be excellent bactericidal agents. Thus, the above five spices could be selected for use as potential anti-enterococcal agents, which will also be of great benefit in combating antibiotic resistance of enterococcus causing serious human infections as found in the current study. However, further studies are required to determine the toxicity and proper dose selection, but before that; these agents can easily be utilized topically. The potential of these spices can be further investigated into the field of pharmacology, phyto-chemistry or food chemistry for better drug discovery. References: 1.

Das RN, Chandrashekhar TS, Joshi HS. Frequency and susceptibility profile of pathogens causing urinary tract infections at a tertiary care hospital in western Nepal. Singapore Med J. 2006;47(4):281-285. Tenover FC, Tokars J, Swenson J. Ability of clinical laboratories to detect antimicrobial agent resistant enterococci. J Clin Microbiol. 1993;31(7):1695-1699.

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www.ijpbs.com (or) www.ijpbsonline.com Cox S, Abu-Ghannam N, Gupta, S. An assessment of the antioxidant and antimicrobial activity of six species of edible Irish seaweeds. International Food Research Journal 2010;17: 205-220. 4. Nair R, Chanda S. Activity of some medicinal plants against certain pathogenic bacterial strains. Indian Journal of Pharmacology 2006;38: 142-144. 5. Shan B, Cai Y, Brooks J. D and Corke, H. The in vitro antibacterial activity of dietary spice and medicinal herb extracts. International Journal of Food Microbiology 2007;117(1): 112-119. 6. Sagdic O, Karahan, A. G, Ozcan M, Ozkan, G. Effect of some spice extracts on bacterial inhibition. Food Science and Technology International 2003;9(5): 353358. 7. Kumar A, Sharma V.K. Antibacterial activity of allicin from Allium sativum against antibiotic resistant uropathogens. The internet journal of Infectious disease 2012;8(1) 8. Meenal NG, Nitin DG. Comparison of Antimicrobial Efficacy of Ginger Extract and 2% Sodium Hypochlorite against Enterococcus faecalis using Agar Diffusion Method. Journal of the Indian Dental Association.2010;4(10):347-349. 9. Facklam RR, Collins MD. Identification of enterococcus species isolated from human infection by a conventional test scheme. J Clin Microbiol 1989; 27:731-734. 10. Singh MK and Singh N.Comparison of antimicrobial activity of herbs and spices and their phytochemical determination.International Journal of Green Pharmacy 2011; 5(3):229-235 11. Dingle J, Reed WW Solomons GL. The enzymatic degradation of pectin and other polysaccharides II. Application of the cup assay method to the estimation

12.

13.

14.

15.

16.

17.

18.

19.

of enzyme. J. Science. Food and Agriculture. 1953; 4:149-153. Hughes T. (n.d.). Word Constructions. Retrieved on 12.17.2012 from http://www.wordconstructions.com/articles/health/a ntibioticresist.html Mandal S, Debmandal M, Saha K, Pal NK. In Vitro Antibacterial Activity of three Indian Spices Against Methicillin-Resistant Staphylococcus aureus.Oman Med J 2011 Sept;26(5):319-323. Ali SM, Khan AA, Ahmed I, Musaddiq M, Ahmed KS, Polasa H, et al. Antimicrobial activities of Eugenol and Cinnamaldehyde against the human gastric pathogen Helicobacter pylori. Ann Clin Microbiol Antimicrob 2005;20(4):4-20. Helander IM, Alakomi HL, Latva-Kala K, MattilaSandholm T, Pol I, Smid EJ, et al. Characterization of the action of selected essential oil components on gram-negative bacteria. J Agric Food Chem 1998; 46:3590-3595. Iacobellis NS, Lo Cantore P, Capasso F, Senatore F. Antibacterial activity of Cuminum cyminum L. and Carum carvi L. essential oils. J Agric Food Chem 2005. Jan;53(1):57-61. Dilek Keskin. Studies on antimicrobial activities of solvent extracts of different spices. Journal of Environmental Biology 2011;32: 251-256. Syed Abdul Rahman M, Thangaraj S, Mohamed Salique S, et al. Antimicrobial and Biochemical Analysis of Some Spices Extract against Food Spoilage Pathogens. Internet Journal of Food Safety 2010; 12:71-75. Suree Nanasombat et al. (2005). Antibacterial activity of crude ethanolic extracts and essential oils of spices against salmonellae and other enterobacteria. KMITL Sci. Tech. J. 2005; 5(3):527-538.

*Corresponding Author: Revati Sharma*

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404, Angel ark Sector 8, Kalamboli, Navi Mumbai 410218 Email:revupattani@gmail.com

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Review Article Biological Sciences FLORA OF THE INDIAN EPIC PERIOD: ONCE LOST, THE DIVERSITY OF GENE POOLS CANNOT BE RESTORED - OUR EVOLUTIONARY RESPONSIBILITY Ashish Dubey1, Manju Lata Zingare *, Md. Aslam Ansari, Prasanna Lata Zingare2 1

Department of Botany, Govt. Shaheed Bhagat Singh P.G. College, Jaora, Ratlam, M.P., 457226, India Department of Biotechnology, Govt. Digvijay Autonomous P.G. College, Rajnandgaon, C.G., 491441, India *Corresponding Author Email: mzingare@yahoo.co.in

ABSTRACT The Indian epic flora are a fundamental part of the Indian culture and apart from having medicinal importance they also have religious value. Their present diversity of species is the result of a very long and slow process of genetic change and adaptation. The time necessary for the emergence of new species, and even for the accumulation of genetic variants at individual gene loci within species, greatly exceeds the time since the emergence of Homo sapiens. New techniques of molecular biology combined with recent theories in population genetics allow us to assess the time dimension of genetic change; these suggest that some genetic polymorphisms may have originated over a million generations ago. In other words, once lost, any particular genetic adaptation cannot be regained in any realistic time interval. These plants provide many services that we take for granted. However due to growing population, increasing anthropogenic activities, rapidly eroding natural ecosystem, etc. the natural habitat for a great number of herbs and trees are dwindling. Biotechnological approaches can prove beneficial for the conservation of these important plants. These plants have maintained their existence to date since the epic period and if once lost, they cannot be regained in any realistic time interval. So it is our evolutionary responsibility to conserve these plants for the future generations.

KEY WORDS Conservation, Flora, Indian epic period

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305

INTRODUCTION India was one of the foremost developed countries in ancient times. Learned persons of vedic culture were aware regarding unimaginable obligation of plants for the sustenance of life. There are a number of verses in ancient literature depicting this generosity of plant kingdom. No wonder that many such plants species have been revered as God [8]. One of the oldest treaties in the world is Rigveda (4500 BC-1000 BC) where healing properties of some herbs are mentioned in the form of sonnets, which were often recited in religious rituals. Later on a special faculty was

developed known as Ayurveda, mostly dealing with human philosophy of health including utilization of medicinal plants [8]. There are records in ancient scripts regarding periodic conferences, seminars and also workshops in selected areas where exchange of knowledge was often manifested. Even it was mentioned that women scholars like Maitrai, Gargi contributed some knowledge about medicinal plants and their maintenance. We produce here the names of few plants and trees of epic period, in Sanskrit language, their

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botanical names, local Hindi or English names and Sargas (Chapters) in which these species have been mentioned (Table 1, Fig. 1).

(http://en.wikipedia.org/wiki/Flora_of_the_Indian _epic_period)

Table 1. The names of flora of Indian epic period, in Sanskrit language, their botanical names, local Hindi or English names and Sargas (Chapters) in which these species have been mentioned.

S.no

Sanskrit name of plant (Devanagari) Agnimukha (अग्ननमुख)

Botanical name

Semecarpus anacardium

Arjuna 2.

(अर्ुन)

Asoka (अशोक)

Terminalia arjuna

Saraca asoca

Indian names

Indian epic

Parvaa

Shloka

Location in epics

Bhilawa, Bhela, Bhallaataka

Ramayana

Aranya Kanda Sarga 73

3.73.5

Matanga hermitage

Ramayana

Kishkindha kanda Sarga 1

4.1.81

Pampa Lake

Ramayana

Aranya kanda Sarga 11

3.11.74

Agastya’s hermitage

Anusasna parva

XIII.54.4

King Kusika country

Arjuna, Arjunasaadaddaa, Sanmadat,

Ashoka Mahabharata

Ashvakarna (अश्वकर्ु)

Vateria indica

Dhupa, Ralla

Ramayana

Bala Kanda Sarga 24

1.24.15

Badari (बदरी)

Zizyphus mauritiana

Ber, Bora

Ramayana

Bala Kanda Sarga 24

1.24.16

Bansha (ब ांस)

Dendrocalamus strictus

Bamboo, bansalochana

Ramayana

Aranya kanda Sarga 15

3.15.21

Panchvati

Dillenia indica

Bhava, Bhavya, Bhavishya, Bhavan, Vaktrashodhan, Pichchilbeeja

Anusasana Parva

XIII.54.5

King Kusika country

Bhavya (भव्य)

Mahabharata

Ramayana

Bilva (बबल्व )

Aegle marmelos

Pampa Lake Kishkinda kanda Sarga 1 Van Parva

Bel Mahabharata

306

Page

Malada and Karusha provinces Malada and Karusha provinces

10.

Champaka

4.1.78

III.174.23

Dvaita forest kurukshetra Saraswati river

(चम्ऩक)

Michelia champaca

Champa

Mahabharata

Anusasana parva

XIII.54.5

King kusika country

Chandana (चांदन)

Santalum album

Chandana

Ramayana

Kishkindha kanda Sarga 1

4.1.82

Pampa lake

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Deodar trees

Ramayana

Dhavda, Bakli , Dhau , Dhawa, Dhawra, Dhaora

Ramayana

12.

Dhava (धव)

13.

Hintala (हहन्त ऱ)

Cycus circinalis

Jangli madan mast ka phool

Ramayana

14.

Inguda (इङ्गुद)

Balanites roxburghii

Hinganbet, Ingudi, Hingoli, Hingun

Mahabharata

15.

Jambu (र्ांब)ू

Syzygium cumini

Jamun, Jambul,

Ramayana

16.

Kadamba (कदां ब)

Anthocepalus Cadamba

Kadamba

17.

Karavira (करवीर)

Nerium indicum

Kannhera

18.

Karira (करीर)

Capparis deciduas

kerda, kair, karir, kirir, karril Amaltas

Karnikara

Ramayana

Mahabharata Ramayana

Kishkindha kanda Sarga 43 Kishkindha kanda Sarga 1 Kishkinda kanda Sarga 1

4-43-13

Himalayas

4.1.81

Pampa lake

4.1.83

Pampa lake

IX.36.58

Sarasvati River

3.73.3

Matanga hermitage

3.73.4

Matanga hermitage

3.73.4

Matanga hermitage

Shalya Parva

IX.36.58

Sarasvati River

Kishkinda kanda Sarga 1

4.1.73

Shalya Parva Aranya kanda Sarga 73 Aranya kandaSarga 73 Aranya kandaSarga 73

Pampa Lake

19.

(कर्र्ुक र)

Cassia fistula

20.

Kasha (क श)

Saccharum spontaneum

Kans grass

Ramayana

Aranya kanda Sarga 15

3.15.22

Panchvati

(क श्मयु)

Berberis vulgaris

Kashmal

Mahabharata

Shalya Parva

IX.36.58

Sarasvati River

22.

Ketaka (केतक)

Pandanus tectorius

Kewada, Ketaki, Keura, Gagandhul

Mahabharata

XIII.54.4

King Kusika country

23.

Khadira (खहदर)

Acacia catechu

Khair, Khadira

Ramayana

3.15.18

Panchvati

Kichaka Venu

Bambusa arundinacea

Kaantaa baans

4.43.37

Where River Sailoda flows

(ककां शक ु )

Butea monosperma

Palas, Dhak, Khakara, Kakracha

Ramayana

4.1.75

Pampa lake

Kharjura (खर्रूु )

Phoenix dactylifera

Pindakhajur

Ramayana

Anusasana parva Aranya kanda Sarga 15 Kishkindha kanda Sarga 43 Kishkindha Kanda Sarga 1 Aranya kanda Sarga 15

3.15.18

Panchvati

Kovidara

Bauhinia variegata

Kachanar

Mahabharata

Drorna parva

VII.153.24

Kurukshetra war

Barleria prionitis

Vajradanti, Koraantipiwali

Ramayana

Kishkinda kanda Sarga 1

4.1.80

Pampa lake

24.

25.

26.

27.

307

(दे वद रु)

Cedrus deodara

Anogeissus latifolia

21.

Page

Devadaru

28.

Kashmarya

(कीचक वेर्)ू Kimshuka

(कोववद र) Kurantaka (कुरण्टक)

Mahabharata

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30.

Kurvaka (कुरवक) Kuṭasalmali

Lawsonia inermis

Ramayana

Ceiba pentandra

Kapok tree

Madhavi

Gaertnera racemosa

Vasanti, Madhumalati, Haladvel, Madahavilataa

Ramayana

(मधक ू )

Madhuka indica

Mahuwa, Mahuli

Ramayana

33.

Malati (म ऱती)

Jasminum sambac

Bel/Beli, Mogra, Mallika, Kampupot, Melati

Ramayana

34.

Malati (म ऱती)

Jasminum grandiflorum

Chameli, Jati

Ramayana

35.

Naga (न ग)

Messua ferrea

Nagachampa, Naagakeshara, Naagachaafaa,

Ramayana

Pongamia pinnata

Karanja, kiramal, Kidamar

Ramayana

32.

36.

37.

(म धवी) Madhuka

Naktamala (नक्तम ऱ) Narikela (न ररकेऱ)

Cocos nucifera

Nila (नीऱ) 38.

Ficus bengalensis Nyagrodha (न्यग्रोध)

308

Mehandi, Mendee English:Henna, Hina,

(कूटश ल्मऱी)

31.

Page

Coconut Palm

Plaksha, Bengal fig, Indian fig, East Indian fig, Indian Banyan or simply Banyan, also borh, nyagrodha and wad or Vad/Vat Hindi:Kamal, English:Indian lotus, sacred lotus, bean of India, or simply lotus

Ramayana

Mahabharata

Ramayana

39.

Padma (ऩद्म)

Nelumbo nucifera

40.

Padmaka (ऩद्मक)

Prunus cerasoides

Himalayan wild cherry

Ramayana

41.

Panasa (ऩनस)

Artocarpus heterophyllus

Kat-hal

Mahabharata

42.

Parnasa (ऩर् ुस)

Ocimum sanctum

Tulasi

Erythrina indica

Pangara, Dadap, Mandar, Ferrud, Panara

43.

Paribhadraka (ऩररभद्रक)

Ramayana

Kishkindha kanda Sarga 1 Kishkindha kanda Sarga 40 Kishkindha Kanda Sarga 1 Aranya kanda Sarga 11 Kishkindha kanda Sarga 1 Kishkindha Kanda Sarga 1 Kishkinda kanda Sarga 1 Kishkinda kanda Sarga 1 Kishkinda kanda Sarga 42

4.1.82

Pampa lake

4.40.39

Eastern side of the Jambudvipa

4.1.77

Pampa Lake

3.11.74

Agastya hermitage

4.1.76

Pampa lake

4.1.76

Pampa lake

4.1.78

Pampa lake

4.1.82

Pampa lake

4.42.11

Cities Murachi, Jatapura, Avanti and Angalepa Pampa Lake

Kishkinda kanda Sarga 1

4.1.79

Drorna parva

VII.153.24

Kurukshetra war

Kishkinda Sarga 1

4.1.76

Pampa lake

Kishkindha kanda Sarga 43

4.43.13

Himalayas

Shalya parva

IX.36.58

Sarasvati river

3.15.18

Panchvati

3.73.5

Matanga hermitage

Aranya kanda Sarga 15 Aranya kanda Sarga 73

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45.

(ऩ ररर् त)

Nyctanthus arbortristis

Paarijaat, Praajakt, Harsinghar, Seoli, Khurasli

Pilu (ऩीऱ)ु

Salvadora oleoides

Jaal, Pilu

Parijata

Mahabharata

Mahabharata Mahabharata

46.

47.

48.

49.

50.

51.

52.

53.

Plaksha (प्ऱऺ)

Ficus religiosa

Pipal, Pimpalla, Bodhi

Ramayana

Priyala (विय ऱ)

Buchanania lanzan

Chironji, Chanhar, Piyal, Achar

Ramayana

Punnaga (ऩुन्न ग)

Calophyllum inophyllum

Undi, Undala, Unang, Surangi, Surpunka, Sultan champa,

Ramayana

Rakta (रक्त )

Rubia cordifolia

Indian Madder

Ramayana

Rohitaka

Tecomella undulata

Rohida, Desert teak

Sahakaras

Mangifera indica

Mango, Indian: Aamba, Aamra, Aam, Amb

Sanjivani

Sellaginella byropteris

Sanjivani

Bombax ceiba

Semal, Shaalmali, laala-saanwar, Deokapaas, Shimal, Savari, Shembal

(रौहीतक)

(सांर्ीवनी) Shalmali (श ल्मऱी)

Shailya parva

IX.36.60

Sarasvati river

Shailya parva

IX.36.59

Sarasvati river

IX.36.58

Sarasvati river

3.73.3

Matanga Hermitage

Shailya parva Aranya Kanda sarga 73 Aranya kanda Sarga 73

3.73.3

Matanga hermitage

Aranya Kanda Sarga 15

3.15.16

Panchvati

Kishkindha kanda Sarga 1

4.1.82

Pampa Lake

Vana parva

III.174.23, III. 241.67)

Dvaita forest Kurukshetra Sarasvati river

Anusasana Parva

XIII.54.4

King Kusikas’s Country

Ramayana

Yuddha kanda Sarga 89

6.89.16

Mt. Dronagiri Himalayas

Ramayana

Kishkindha kanda Sarga 1

4.1.82

Pampa lake

Sarga 15

3.15.22

Sabha parva

II.47.4

Kamboja country

Van Parva

III.174.23

Dvaita Forest, Kurukshetra, Sarasvati River

Anusasana Parva

XIII.54.6

King Kusika Country

Kishkindha Kanda Sarga 1

4.1.81

Pampa Lake

Mahabharata

Ramayana

Page

309

54.

Shami (शमी)

Prosopis cineraria

55.

Shirisha (शशरीष)

Albizzia lebbeck

56.

Shyama (शय म)

Salvadora persica

57.

Simsupa (शशांशुऩ)

Dalbergia latifolia

Khejdi, sami, Jant/Janti, Sangri

Siras, Shirisha, Kalasiris, Chichola, Chichwa Khaankann, mirajollee, khakhin, miraj, jhak, pilva, kharjal, rhakhan, thorapilu Simsipa, Sinsipa, Krishnasara, Gurusara, Krishnasimsapa

Mahabharata

Ramayana

Panchvati

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59.

(शसन्धव ु र)

Vitex negundo

Nirgundi, Nirguddi, Sambhalu, Shivari, Nisida, Nigudi

Surakta (सुरक्त)

Pterocarpus santalinus

Rakta chandana, Laal chandan

Sindhuvara

Aparajita, saukarnika, ardrakarni, girikarnika, Sankhupushpam Tari (Hindi), Tal (Bengali), Nungu (Tamil), Thaati/Munjalu (Telugu), Munjal (Urdu)

Ramayana

60.

Supuṣpi (सुऩुष्ऩी)

Clitoria ternatea

61.

Tala (त ऱ)

Borassus flabellifer

62.

Tilaka (ततऱक)

Cinnamomum iners

Daalachini, Tejpat, Tamaal saala

Ramayana

63.

Tinduka (ततन्दक ु )

Diospyros melanoxylon

Tendu

Ramayana

64.

Tinisa (तततनश)

Lagerstroemia speciosa

Taaman, Jarul, Motabondara

Ramayana

(उद्द ऱक)

Cordia myxa

Lasora, Bhokara

Ramayana

Vakula (वकुऱ)

Mimusops elengi

Vakula, Bakulla, Maulsari, Ovalli

Ramayana

65.

66.

Uddalaka

Varanapushpa (व रर्ऩुष्ऩ)

Calophyllum inoplyllum

68.

Vasanti (व सन्ती)

Hiptage benghalensis

69.

Vetas (वेतस)

Calamus rotang

Vibhitaka

Terminalia bellirica

67.

(ववभीतक)

4.1.81

Pampa Lake

3.73.5

Matanga hermitage

Kishkinda Sarga 1

4.1.77

Pampa lake

Aranya Kanda Sarga 15

3.15.16

Panchvati

Kishkindha Kanda Sarga 1 Bala Kanda Sarga 24 Aranya Kanda Sarga 15 Kishkindha Kanda Sarga 1 Kishkindha Kanda Sarga 1

4.1.78

Pampa Lake

1.24.15

Malada and Karusha

3.15.16

Panchvati

4.1.81

Pampa Lake

4.1.78

Pampa Lake

Mahabharata

Anusasana parva

XIII.54.6

King Kusika country

Ramayana

Kishkindha Kanda Sarga 1

4.1.77

Pampa Lake

Rattan Palm

Mahabharata

Van Parva

III.174.23

Dvaita Forest, Kurukshetra, Sarasvati River

Baheraa, Behaddaa, Bibheeta

Mahabharata

Shalya Parva

IX.36.58

Sarasvati river

Page

310

70.

Sanskrit- Punnaga Indian:Undi, Undala, Unang, Surangi, Surpunka, Sultan champa Vasanti, Madhumalati, Haladvel, Madahavilataai

Ramayana

Kishkindha Kanda Sarga 1 Aranya kanda Sarga 73

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Figure 1. Some of the flora of Indian epic period

Kashmarya (Kashmal)

Ketaka (Kewada)

Kimshuka (Palas)

Kovidara (Kachanar]

Berberis vulgaris

Pandanus tectorius

Butea monosperma

Bauhinia variegata

[Berberidaceae]

[Fabaceae]

Kurvaka (Mehendi)

Kutasalmali (Kapok tree)

Naga (Naagakeshara)

Barleria prionitis

Lawsonia inermis

Ceiba pentandra

Messua ferrea

[Acanthaceae]

[Lythraceae]

[Malvaceae]

[Calophyllaceae]

Nila (Indian fig)

Padma (Kamal)

Paribhadraka (Pangara)

Parijata ( Harsinghar)

Ficus bengalensis

Nelumbo nucifera

Erythrina indica

Nyctanthus arbortristis

[Moraceae]

[Nelumbonaceae]

[Fabaceae]

[Oleaceae]

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Kurantaka (vajradanti)

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Rohitaka (Rohida)

Sanjivani (Sanjivani)

Shalmali (Semal)

Shami (khejdi)

Tecomella undulate

Selaginella bryopteris

Bombax ceiba

Prosopis cineraria

[Bignoniaceae]

[Selaginellaceae]

[Malvaceae]

[Fabaceae]

Sindhuvara (Nirgundi)

Supuspi (Aparajita)

Tinisa (Jarul)

Vakula (Maulsari)

Vitex negundo

Clitoria ternatea

Lagerstroemia speciosa

Mimusops elengi

[Lamiaceae]

[Fabaceae]

[Lythraceae]

[Sapotaceae]

NEED FOR CONSERVATION

312

Homo sapiens arose and became the dominant species on earth in the last 1 / 20,000 of the time elapsed since the origin of life. In this relatively short time, humans have altered both the physical and the biological worlds in profound ways. The changes that we are making in our environment are detrimental to biological diversity and ultimately to ourselves. The biodiversity of plants that surrounds us provides us with food, fiber, medicine and energy [12].

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The accumulation of this biodiversity has been a very slow process when measured in human timescales. Biodiversity is the product of a vast history of evolutionary change - about 3.5 billion

years. The colonization of the terrestrial environment by life forms began approximately 500 to 600 million years ago, and during this most recent 10% of evolutionary history all of the diverse forms of terrestrial life that comprise our environment appeared. We cannot repopulate our world with species that have been lost, nor can we expect to regain the use of lost genetic variants within the timescale of human existence [12]. To gain perspective on our biological resources and to formulate wise strategies for managing our world, we must consider the following questions: What do we know about the processes that have produced the biological diversity of our world? And how have we attempted to place a value on

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www.ijpbs.com (or) www.ijpbsonline.com biological diversity through our conservation activities. a. Timescales and diversity How long does it take to acquire the unique genetic attributes that mark distinct species? The temporal thread that binds generations is the transmission of the hereditary information encoded in DNA (deoxyribonucleic acid). The preservation of form and function depends on a highly efficient system for the replication of DNA, so that the information transfer from one generation to the next is nearly error free. Paradoxically, some errors are essential to provide evolutionary flexibility. The ultimate source of biological diversity derives from mutational change in DNA molecules [12].

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Owing to the powerful tools of molecular biology, our understanding of the genetic dimension of evolutionary change has advanced enormously over the past decade. These tools have provided us with a direct means of studying the pattern of mutational changes in DNA molecules among diverse life forms. Based on comparative studies, we now know that the error rate for DNA replication is very low (approximately 5 x 10-9 base substitutions per nucleotide per year) [13]. We have also learned that a number of mechanisms cause mutational change, including the insertion and deletion of DNA sequences and the transposition of DNA sequences (e.g., with respect to the chloroplast genome [2]. If we can determine the number of mutations that separate different species and if the mutation rate is constant, we can calculate the time it took to accumulate the observed level of mutational divergence. This notion of a molecular clock has been widely employed in evolutionary biology. To cite but one example of a molecular clock argument, it is estimated from the accumulation

of mutational change in molecules that the monocotyledonous class of flowering plants (e.g., grasses, palms, orchids) separated from within the dicotyledonous class (eg, cotton, sunflowers, apple trees and so on) approximately 200 million years ago [17] . Let us move from these ancient events in terrestrial evolution to the accumulation of genetic diversity within species. A commonly accepted definition of species is a group of individuals that are able to breed with each other [9] . As a consequence, the members of a species share a common gene pool. As the populations that compose a species diverge from one another through time, evolution barriers to reproduction begin to emerge. These include chromosomal rearrangements, behavioral divergence and changes in flowering time. New daughter species are born. The essential characteristic of a species is that the members share a common evolutionary future [12]. How extensive is the genetic diversity contained within species' gene pools? What factors control diversity levels and how long does it take to reach a given degree of diversity within a species' gene pool? According to biochemical assays of genetic diversity conducted over the past 25 years, most of the 470-plus tested plant species have extensive levels of genetic diversity [5] and plant breeders exploit genetic diversity to improve domesticated species. Similarly, natural selection depends absolutely on genetic diversity to produce adaptive responses to environmental changes [12]. Levels of genetic diversity within species are controlled by mutation rates, the size of the breeding population (effective population size), and the pattern and strength of natural selection. (Effective population size is calculated as the

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harmonic mean of population sizes taken over time.) While mutation rates are reasonably constant across most life forms, patterns of effective population size and selection are highly specific and depend on the unique history of the species in question. For example, species that have expanded from glacial refugia may have much larger current numbers but their effective population size is still dominated by the bottleneck imposed by the glacial era. (Refugia are areas of relatively unaltered climate inhabited by plants and animals during a period of continental climatic change.) Hence the time it took to achieve a given degree of genetic diversity depends on the species. The age of genetic variants within a species can be estimated by coalescence theory [6]. Coalescence theory is a recent development in population genetics that relates mutational diversity for a particular gene to past episodes of selection and to the effective population size can, in turn, be related to the age of genetic variants. To apply coalescence theory, researchers obtain DNA from a sample of individuals. For each individual, DNA sequence data are determined for a specific gene. According to the theory, the present-day sequences all trace back to a common ancestral sequence called the coalescent. The age of the coalescent depends on mutation rate and effective population size. Gene genealogies can also be used to detect natural selection. b. Space needed for evolution Species are composed of systems of populations called metapopulations [7] that are spread across an environment or landscape. A given environment is spatially heterogeneous [15], that is, local environments differ from each other. In each local environment, particular genetic variants of a species are more likely to survive and reproduce successfully, and natural selection favors those variants. Over time a given population adapts to its local environment. While genetically different,

these locally adapted populations remain part of the same species because genetic migration among populations maintains a common evolutionary trajectory for the species as a whole. Species cannot exist as dynamic evolutionary entities without sufficient habitat. One of the most fundamental generalizations of ecology is the relation between the size of a habitat and the number of species that can live there [11]. Reducing the size of a habitat means reducing the number of species that live there. In addition, habitat loss can ultimately reduce the ecosystem services required to sustain human activities. The enormous expansion of the global human population has engendered an unavoidable conflict between biological diversity and the activities necessary to accommodate population growth. The battle field is habitat. To expand our agricultural, urban, industrial and other needs, we must convert habitat that supported a variety of biological activities into space for human use. How do we manage the environment to sustain human life in the long term while still meeting the needs of present populations? The obvious answer is that we adopt societal rules to conserve habitat and thereby to conserve the biological heritage upon which we depend. c. Saving our ultimate resources The loss of species and valuable gene pools is proceeding at an accelerating pace. Once gone, this lost genetic diversity will not be regained for a long time - vastly longer than the total history of human existence. Man depends on the biological world for survival. Other species are the ultimate source of the energy, food, fiber and many of the medicines that we consume. While we have managed to convert the biological and physical resources of the earth to human use with increasing efficiency, we have simultaneously degraded the resource base for future human

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www.ijpbs.com (or) www.ijpbsonline.com generations. This, coupled with a vastly expanding human population, threatens our ability to sustain our current standard of living into the future [12]. BIOTECHNOLOGICAL METHODS FOR CONSERVATION OF INDIAN EPIC FLORA Most of the medicinal plants either do not produce seeds or seeds are too small and do not germinate in soils. Thus mass multiplication of disease free planting material is a general problem. In this regard biotechnology is a boon for conservation of these important plants.

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1. Micropropagation (Invitro regeneration) Micropropagation is the technique of in vitro multiplication of large number of plants from its part, whether it is leaves, seeds, nodes and tubers etc. In the recent years, tissue culture has emerged as a promising technique to obtain genetically pure elite populations under in vitro conditions. It a fast and dependable method for production of a large number of uniform plantlets in a short time. Moreover, the plant multiplication can continue throughout the year irrespective of season and the stocks of germplasm can be maintained for many years [9]. 2. Mycorrhization Plant production by micro propagation technology is limited by the acclimatization stage, one of the most critical stages of this process. A high percentage of micro propagated plantlets are lost or damaged during transfer from test tube conditions to in vivo environment. It would be useful to acclimatize plantlets during the in vitro period to reduce the stress during transfer ex vitro. For this reason, mycorrhizal technology can be applied. Inoculation of arbuscular mycorrhizal fungi (AMF) into the roots of micropropagated plantlets plays a advantageous role [16, 1]. As a result, the mycorrhizal technology can be applied for the conservation of rare and endangered

medicinal plants, by inoculation of growthpromoting fungi. 3. Genetic Transformation Genetic transformations improve yield and quality of medicinal plants, which involve the alteration or introduction of genes which improve the secondary metabolite synthesis in plant, which are mainly responsible for their medicinal properties. Genome manipulation is the general aim of the genetic transformation with medicinal plants by developing techniques for desired gene transfer into the plant genome in order to improve the biosynthetic rate of the compounds of interest. An essential strategy in this regard is the choice of the correct marker genes for genetic transformation, as it assists to analyze the transformed cell. Many researchers are mainly focusing on the mechanism of transfer and integration of the marker and reporter genes. Agrobacterium tumefaciens and A. rhizogenes are virulent for plants. They contain a large megaplasmid (more than 200 kb), which plays a key role in tumor induction. During infection the T-DNA, a mobile segment of Ti or Ri plasmid, is transferred to the plant cell nucleus and integrated into the plant chromosome and transcribed. Genetic transformation facilitates the growth of medicinal plants with multiple durable resistances to pests and diseases. There are more than 120 species belonging to 35 families in which transformation has been carried out successfully by using Agrobacterium and other transformations techniques [3]. 4. Establishment of DNA banks The establishment of DNA banks is one of the ex situ conservation method which is planned activity. The extraction of genetic material, and storage should be made readily available for molecular applications. DNA resources can be maintained at -20ยบC for short- and midterm storage (i.e. up to 2 years), and at -70ยบC or in

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www.ijpbs.com (or) www.ijpbsonline.com liquid nitrogen for longer periods. Other objectives of the creation of DNA banks may be related to training or distribution to scientists with an interest in different areas of biology. DNA banks assembled as a means to replace traditional methods of conserving genetic resources. For many species that are difficult to conserve by conventional means (either as seeds or vegetatively) or that are highly threatened in the wild, DNA storage may provide the ultimate way to conserve the genetic diversity of these genetic diversity of these species and their populations in the short term, until effective methods can be developed [4].

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5. Cryopreservation Cryopreservation is an important technique for long term storage of tissues/plants. This requires liquid nitrogen (-196ÎżC). Some important DNA banks are as below: (i) The Royal Botanic Garden, Kew, UK, which contains PGR DNA specimens, and presently the worldâ&#x20AC;&#x2122;s largest and the most comprehensive PGR DNA bank, consisting of over 20,000 DNA specimens representative of all plant families. (ii) The US Missouri Botanical Garden has collection of more than 20,000 plant tissue samples, and provide raw material for the extraction of DNA for its subsequent use in conservation research. (iii) The Australian Plant DNA Bank of Southern Cross University, which was established in June 2002. It contains representative genetic information from the entire Australian flora. (iv) DNA bank of Leslie Hill Molecular Systematics Laboratory of the National Botanical Institute (NBI) in Kirstenbosch, South Africa, in collaboration with the Royal Botanic Garden, Kew, which preserves genetic material of the South African flora [14]. CONCLUSION The Indian epic flora are a fundamental part of the Indian culture. In view of the tremendously

growing world population, increasing anthropogenic activities, rapidly eroding natural ecosystem, etc the natural habitat for a great number of herbs and trees are dwindling. Many of them are facing extinction. To cope up with alarming situation, the recent exciting developments in biotechnology are proving to be beneficial. India has a long history of conservation policy aimed at preserving useful genetic variants for agriculture, animals as well as ecosystems that are crucial to the quality of human life. Today, population pressures are intensifying the conflict between the need to preserve biological resources and the need of an expanding population to use land and raw materials. A major challenge will be to develop approaches to conservation that meet our obligations to both present and future generations. Certainly, a concerted effort involving collaborations of field biologists familiar with the status of threatened and endangered species with reproductive scientists, geneticists, and others with expertise and resource banking should be undertaken to match conservation and technological opportunities. Identification of the taxa at risk and the systematic collection of samples as opportunities arise, consistent with the conservation management of threatened and endangered species, offer increased opportunities for preventing extinction and for the preservation of gene pools. The plants of the Indian epic period are a part of our cultural heritage and religious beliefs, these plants have maintained their existence to date since the epic period and if once lost, they cannot be regained in any realistic time interval. So it is our evolutionary responsibility to conserve these plants for the future generations. REFERENCES [1]. Chandra, S., Bandopadhyay, R., Kumar, V., Chandra, R., O. : Acclimatization of tissue cultured plantlets: from laboratory to land. Biotechnol Lett (DOI: 10.1007/s10529-010-0290-0), (2010).

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www.ijpbs.com (or) www.ijpbsonline.com [2]. Clegg, M., T., Gaut, B,.S., Learn, G.,H. and Morton, B., R. : Rates and patterns of chloroplast DNA evolution. Proc. Natl. Acad. Sci. USA, (1994). [3]. Cucu, N., Gabriela, G., L. : Genetically modified medicinal plants. II. Transfer and expression of a marker kanamycine resistance gene in Atropa belladonna plants. Roumanian Biotech Lett (7) : 869-874, (2002). [4]. Dulloo, E., Nagamura, Y., Ryder, O. : DNA storage as a complementary conservation strategy. In: Vicente MC de, Andersson MS (eds.). DNA banks-providing novel options for gene banks? Topical reviews in agricultural biodiversity. International Plant Genetic Resources Institute, Rome, Italy, (2006). [5]. Hamrick, J., L. and Godt, M., J. : Allozyme diversity in plant species. In Plant Population Genetics, Breeding and Genetic Resources. A. H.D. Brown, M., T., Clegg, A., L., Kahler and B., S., Weir, (eds.). Sinauer Associates, Sunderland, MA. , (1990). [6]. Hudson, R., R. : Gene geneologies and the coalescent process. Oxford Surveys in Evol. Biol. 7: 1-44, (1990). [7]. Levins, R. : Extinction. In Some Mathematical Questions in Biology, Vol II M, Gerstenhaber, ed. American Mathematical Society, Providence, RI, (1970). [8]. Joshi, S., G. : Medicinal Plants, Oxford and IBH Publishing Co. Pvt. Ltd. New Delhi, ISBN 81-204-1414-4, p.xi, (2004). [9]. Malik, C., P., : Applications of biotechnology innovations in pharmaceutics and nutraceutics in multitherapeutic medicinal and special plants Vol ii. ed Karan singh, M., L., jahdon and D singh. Aavishkar publishers, Jaipur pp; 243265, (2007).

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|305-321 [10]. Mayr, E. :Animal Species andEvolution. Belknap Press of Harvard University Press. Cambridge, MA. (1963) [11]. Mc Arthur R., H., and Wilson, E., O. : The Theory of Island Biography. Princeton University Press, Princeton, N.J. (1967). [12]. Michael, T. C. : Millions of generations old. . .Once lost, diversity of gene pools cannot be restored. California agriculture : 49(6): 34-39, (1995). [13]. Nei, M. Molecular Evolutionary Genetics. Columbia University Press, New York, (1987). [14]. Rice, N., Henry, R., Rossetto, M. : DNA banks: a primary resource for conservation research. In: Vicente MC de, Andersson MS (eds.). DNA banks-providing novel options for gene banks? Topical reviews in agricultural biodiversity. International Plant Genetic Resources Institute, Rome, Italy, (2006). [15]. Risser, P. G. 1987. Landscape ecology: state of the art. In Landscape Heterogeneity and Disturbance. M. G. Turner, ed. Springer-Verlag, New York. [16]. Sylvia, D., M., Alagely, A., K., Kane, M., E., Philman, N., L. : Compatible host/mycorrhizal fungus combinations for micropropagated sea oats. I. Field sampling and greenhouse evaluations. Mycorrhiza 13 (4): 177-183, (2003). [17]. Wolfe, K., H., M., Gouy, Y., W., Yang, P., M., Sharp and W.J. Li. Date of the monocotdicot divergence estimated from chloroplast DNA sequence data. Proc. Natl. Acad. Sci. USA 86: 6201-05, (1989).

*Corresponding Author:

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Manju Lata Zingare* Email: mzingare@yahoo.co.in

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Research Article Biological Sciences CONGENITAL HEART DISEASE IN INFANTS IN GULBARGA REGION OF KARNATAKA Sandeep V.Harshangi, Laxmi Nagaraj Itagi, Venkatesh Patil, Vijayanath.V 1

Assistant Professor, Department of Pediatrics, M.R.Medical College, Gulbarga,Karnataka,Indi Assistant Professor, Department of obstetrics & Gynecology, M.R.Medical College, Gulbarga,Karnataka,India 3 Associate Professor, Department of Pharmacology, Navodaya Medical College, Raichur,Karnataka, India 4 Associate Professor, Department of Forensic Medicine & Toxicology, VMKV Medical College Salem, Tamil Nadu, India 2

ABSTRACT Congenital heart diseases are primarily disease of neonates, infants and children. The burden of CHD in India is likely to be enormous due to very high birth rate. The reported incidence is 8-10/ 1000 live births. To know the pattern of clinical presentation of various congenital heart diseases in 0-1 year age group. 50 cases of CHD proven by 2D echocardiography were studied for 2 years period. Present study included all infants in birth â&#x20AC;&#x201C; one year age group and excluded all preterm babies and patient with persistent pulmonary hypertension. Out of the 50 cases of congenital heart disease. Growth retardation and CCF were the commonest complication. The mortality rate was 18% with VSD as the leading cause of death among the CHD. The commonest cause of death among these patients was refractory CCF. A cardiac evaluation with echocardiography is also necessary in all cases of LRTI and FTT. Early diagnosis, close monitoring and timely intervention in cases of CHD will go a long way in reducing the morbidity and mortality to a large extent.

KEY WORDS Congenital heart disease; 2D echocardiography

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INTRODUCTION Congenital Heart Diseases (CHD) are primarily seen in neonates, infants and children, although in our country it is not uncommon to see adults with uncorrected CHD. The burden of congenital heart disease in India is likely to be enormous due to a very high birth rate. This heavy burden emphasizes the importance of this group of heart diseases. The reported incidence of CHD are 8-10/1000 live births according to various studies from different parts of the world.1 It is believed that this incidence has remained constant worldwide.2 Nearly one-third of these CHD are critical requiring intervention in the first year of life itself.3 Rapid advances have taken place in the

diagnosis and treatment of CHD over the last six decades. There are diagnostic tools available today by which an accurate diagnosis of CHD can be made even before birth. With currently available treatment modalities, over 75% of infants born with congenital heart disease can survive beyond the first year of life and many can lead normal lives thereafter. However, this privilege of early diagnosis and timely management is restricted to children in developed countries only. Unfortunately majority of children born in developing countries with CHD do not get the necessary care, leading to high morbidity and mortality. There is no community based data for the incidence of congenital heart disease at birth in

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www.ijpbs.com (or) www.ijpbsonline.com India. Since a large number of births in our country take place at home, mostly unsupervised by a qualified doctor, hospital statistics are unlikely be truly representative. The congenital heart disease has been the subject of innumerable studies both regarding prevalence and clinical features in western countries. The few studies carried out in India were either community or school based and they did not indicate the clinical profile of CHD. It was therefore decided to carry out hospital based study of clinical profile of congenital heart disease in infancy at Basaveshwar Teaching & General Hospital and Sangameshwar Teaching Hospital attached to M.R.Medical College, Gulbarga.

OBJECTIVES To know the pattern of clinical presentation of various congenital heart disease in 0-1 year age group.

METHODOLOGY Methods of collection of data: Infants presenting with features suggestive of congenital heart disease and proven by 2D echocardiography were studied over a period of 2 years from October 2005 to September 2007 admitted in Sangameshwar Hospital, Gulbarga and Basaveshwar Teaching & General Hospital, Gulbarga. Inclusion Criteria: All patients presenting with clinical features like breathlessness, recurrent LRTI, failure to thrive,

cyanotic spells, congestive cardiac failure, murmur in the age group of term neonates to first birthday. Exclusion Criteria:  Preterm neonates.  More than one year.  Patients with persistent pulmonary hypertension.

RESULTS In this clinical study, compromising of age 0-1 years, 50 cases of proven CHD were studied in the Department of Paediatrics of M.R.Medical College, Gulbarga at Basaveshwar Teaching & General Hospital and Sangameshwar Hospital, Gulbarga from October 2005 to September 2007. Following observations were made in the present study. Table-1: Age specific incidence of CHD Age No. of cases Percent Birth – 1 week 15 30.00 1 week – 1 month 6 12.00 1 month – 1 year 29 58.00 Total 50 100.00 Majority of patients in this study i.e., 58% presented in the 1 month to 1 years period, whereas 42% presented in the neonatal life, of which 30% were in the early neonatal period and 12% in late neonatal period.

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Table-2: Sex of Children Sex

No. of cases

Percent

Male

52.00

Female

48.00

Total

100.00

52% of the children were male, while 48% were female with a ratio of 1.08:1. International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)

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Table-3: Residence Residence No. of cases Percent Urban 37 74.00 Rural 13 26.00 Total 50 100.00 Majority of the patients i.e., 74% of cases were from urban area and 26% were from rural area.

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Table-4: Consanguinity Consanguinity No. of cases Percent First degree --Second degree 7 14.00 Third degree 5 10.00 Non-consanguinity 38 76.00 Total 50 100.00 In the present study, 76% of the cases reported a non-consanguineous marriage and 24% has history of some degree of consanguinity with 14% showing a second degree and 10% having third degree consanguinity marriage. Table-5: Birth order Birth order No. of cases Percent First 10 20.00 Second 16 32.00 More than two 24 48.00 Total 50 100.00 In 48% of the cases, birth order was more than 2 and in 32% birth order 2 and only 20% had birth order of one. Table-6: Immunization Status of Patient Immunization Status No. of cases Percent Completely immunized 20 40.00 Partial 9 18.00 Unimmunized 21 42.00 Total 50 100.00 In this study 42% of patients were unimmunized and 58% had some immunization with 40% complete and 18% had partial immunization. Table-7: Birth History Birth history No. of cases Percent Uneventful 40 80.00 Birth asphyxia 5 10.00 MAS 5 10.00 Total 50 100.00

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Majority of children had an uneventful delivery, while 20% had significant birth history in the form of birth asphyxia and meconium aspiration syndrome. Table-8: Age and Sex Incidence of Specific CHD Defects Acyanotic VSD ASD PDA AVSD Dextro cardia HOCM COA Cyanotic TOF TGV TAPVC Ebstein DORV Dextrocar dia with pulmonar y atresia with PDA Dextrocar dia with tricuspid atresia with ASD Total

Birth – 1 week Male Female

Total

One week – one month Male Female Total

1 month – 1 year Male Female

Total

1 1 --1

1 1 1 -1

2 2 1 -2

-1 1 ---

1 --1 --

1 1 1 1 --

5 3 2 ---

7 2 1 1 --

12 5 3 1 --

---

1 --

-1

---

-1

-2 1 1 -1

1 -1 ---

1 2 2 1 -1

-------

-1 -----

3 --1 ---

2 ---1 --

5 --1 1 --

1 1 16 6 3 2 2 1 1

Total 34 15 8 5 2 2

In present study VSD (30%) was the commonest CHD followed by ASD (16%) in the acyanotic group and among cyanotic CHD, TOF (12%) was the commonest followed by TGV (6%).

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DISCUSSION Congenital heart disease (CHD) occurs in 0.5 – 0.8% of live born children with a higher percentage in those aborted spontaneously or still born. CHD has become an important cause of morbidity and mortality in infancy and accounts for two third of all major birth defect along with neural tube defect. 2 – Dimensional

echocardiography with colour Doppler has revolutionized the diagnosis and management of cardiac malformation. It is a non invasive investigation that can precisely diagnose most congenital heart disease as well as offer treatment options, whether medical or surgical. In view of this we undertook the present study to evaluate the clinical profile of CHD in infancy

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at Basaveshwar teaching and general hospital and Sangmeshwar hospital of M R Medical

College Gulbarga over a period of two years from October 2005 to September 2007.

Profile of CHD and Comparison of our findings with other studies Present study (n=50)

Tank S et al (2004) (n=147)

Kasturi et al (1999) (n=108)

Vashisth a et al (1993) (n=44)

Rao VS et al (1974) (n=400)

Jain KK et al (1971) (n=55)

Bidwai et al (1971) (n=378)

VSD

15 (30.00)

45 (40.90)

25 (45.40)

91 (24.00)

8 (16.00)

5 (11.40)

26 (23.60)

2 (3.60)

20 (21.96)

PDA AVSD Dextro cardia HOCM COA Cyanotic

5 (10.00) 2 (4.00)

29 (27.00) 26 (24.00) 7 (6.00) --

18 (40.90)

ASD

54 (36.73) 18 (12.25) 7 (4.76) 3 (2.04)

2 (2.40) --

39 (35.40) --

3 (5.40) --

39 (10.30) --

2 (4.00)

1 (0.68)

1 (2.00) 1 (2.00)

1 (0.68) 1 (0.68)

---

-1 (1.80)

---

TOF

6 (12.00)

10 (9.00)

6 (13.60)

53 (28.60)

10 (18.60)

TGV TAPVC Ebstein DORV Dextrocardia with pulmonary atresia with PDA Dextrocardia with tricuspid atresia with ASD

3 (6.00) 2 (4.00) 2 (4.00) 1 (2.00)

26 (17.68) 8 (5.42) 4 (2.72) 5 (3.40) 2 (1.36)

3 (3.00) 1 (1.00) 1 (1.00) --

1 (2.30) ----

25 (13.5) 4 (2.16) 4 (2.16) --

1 (1.80) ----

-----

1 (2.00)

Type of defect

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Acyanotic

Present study had 34 cases of acyanotic CHD (68%) and 16 cases of cyanotic CHD (32%). The preponderance of acyanotic CHD is in concordance with the results of other Indian3 and western studies.4,5 Similar incidence was reported by Tank S et al9 2004 (5.4%). In our study there were two cases each of Ebsteins anomaly (4%), TAPVC (4%), AVCD (4%),

Dextrocardia (4%). We found one case each of cardiomyopathy (2%), CoA (2%), DORV (2%) and tricuspid atresia (2%). This observation is slightly higher than other Indian studies5 as shown in the above table. Sex Distribution: Following studies show male preponderance, Samanek M et al6 1990 (1.09:1), Rao VS et al7

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www.ijpbs.com (or) www.ijpbsonline.com 1974(1.25:1), Kinare SG et al8 1981(1.23:1), Tank S et al7 2004 (1.88:1) In the present study, 26 patients (52%) are male and 24 patients (48%) were female with a male to female ratio of 1.08:1. It shows slight male preponderance for CHD. Present study is in corcordance with Samnek M et al6 (1990). There are gender differences in the occurrence of specific heart lesions. Age Specific Incidence When considering the age of presentation, maximum cases were in infancy (58%). There were only 42 % cases in neonatal period, as was also seen with other Indian studies. In west, however there is higher number of patients in neonatal period which could be due to the fact that fetal echocardiography forms part of their routine antenatal examination. Locality In the present study nearly 3/4th cases were from urban areas (74%) and 1/4th were from rural area (26%). This distribution may not be a reflection of true incidence and this discrepancy can be attributed to the availability of specialized care, specialist doctors and advanced facilities in urban areas. Etiological Factors According to Nora10 study, the most vulnerable time for damage due to exposure to teratogenic agents is between 18-60 days. In the present study, no significant antenatal history of any drug intake or infection or exposure to radiation could be elicited. Sibling Affected Nadas and Fyler11 have mentioned that 2% of sibling may suffer from similar complaints and Nora has observed that 0.3% of sibling can have similar cardiac malformation. The present study reported occurrence of 2 cases which had complaints among siblings with congenital diseases. One had tracheo-oesophageal fistula and 1 patient with TOF had a sib who died at

early age because of a cyanotic CHD which could not be diagnosed. The findings in the present study correlates with observation of Nadas and Fyler.11 Consanguinity A study by Susan MB et al12 1979 reported that first degree consanguinity was significantly associated with CHD. Shafi T et al13 2003 reported that there was a significant association between children born of consanguineous marriage and risk of CHD. In this present study, it was found that 12 patients (24%) were products of consanguineous marriage, 7 patients (14%) were 2nd degree and 5 patients (10%) were products of 3rd degree consanguineous marriage. Rest 38 patients (76%) had no history of consanguinity. Hence it is clear that consanguinity is a risk factor for development of CHD. Birth Order Tay JS et al14 (1982), studied association of birth order and CHD in 100 chinese children and 100 controls and he reported that higher incidence of CHD was present in children with higher birth order. Zhan SY et al15 (1991) reported that higher incidence of CHD was present in higher order babies. Incidence of CHD in 1st born babies was 20%, 2nd born was 32% and more than 2 was 48% in our study. The present study is in concordance with the above studies. Immunisation Tank S et al9 (2004) reported 15.46% of unimmunised cases, 55% cases completely immunized while 29.25% were partially immunized. Immunization was given according to UIP schedule in the study group. In the present study 20 patients (40%) were completely immunized till age, while 9 patients (18%) were partially immunized, 21 patients (42%) were unimmunized however these patients were all neonates. Present study reported slightly lower incidence when compared to this study. The

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www.ijpbs.com (or) www.ijpbsonline.com common reason stated for non immunization or partial immunization were recurrent LRTIâ&#x20AC;&#x2122;s, FTT, ignorance and illiteracy about immunization. Anthropometric Status In this study, anthropometric status was assessed by ICMR charts. Length and weight were markedly affected in cases of CHD, with 82% patients of less than 50th percentile, out of which 52% patients were less than 10th percentile, and 30% were between 10 to 50th percentile. 9 patients (18%) had normal anthropometric status. Present study reported the similar observation as reported by other Indian3 and western studies.4,5

CONCLUSION Out of the 50 proven cases of congenital heart disease studied from October 2005 to September 2007, the highest incidence was noted in 1 month to 1 year age group. Out of the 50 cases of congenital heart disease. Breathlessness, LRTI, FTT, fever, tachypnea, tachycardia, cyanosis were the common clinical presentation of majority of cases. Male preponderance was noted in both cyanotic and acyanotic CHD. These children were deprived of basic medical care in the form of immunization. In our study 40% reported complete immunization while 18% were partial immunized and 42% were unimmunised. Malnutrition was seen in 82% having less than 50th percentile anthropometric data.Musculoskeletal anomalies were commonest extra cardiac anomaly with Down syndrome the next most common.

REFERENCES 1.

2. 3. 4.

7. 8.

10.

11. 12.

13.

14.

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Fyler DC, Buckley LP, Hellenbrand WE, Cohn HE. Report of the New England Regional Infant Care program. Pediatrics. 1980; 65: Suppl: 375-461. Abdullah R. What is the prevalence of congenital heart disease? Ped Cardio. 1997; 18: 268. Hoffman JI, Kaplan S. The incidence of congenital heart disease. J Am Cardiol. 2002; 39: 1980-1900. Jackson M, Poskitt EM. The effects of high energy feeding on energy balance and growth in infants with congenital heart disease. Br J of Nutrition 1991; 65: 131-43. Mitchell IM, Logan RW, Polloc JC, Jamieson MP. Nutritional status of children with congenital heart disease. Br Heart J 1995; 73: 277-283. Samanek M, Slavik Z, Zbarilova B, Hrobonava V, Variskova M and Skovranek J. Prevalence anaqlysis of 91823 live born children. Pediatr Cardiol. 1989 Fall: 10(4): 205-11. Rao VS, Reddi YR. Profile of heart in children. Indian J Pediatr. 1974; 41: 244-248. Kinare SG, Sharma S. Congenital heart disease in first year of life (an autopsy study of 270 cases). Indian J Pediatr. 1981; 48: 745-754. Tank S, Sushma M, Surekha J. Epidemiology of congenital heart disease among hospitalized patients. www.bhj.org/ journal/ 2004 4602 april.html. epidemiol. Nora JJ and Nora AJ. The evolution of specific genetic and environmental counseling in congenital heart disease. Circulation 1978; 57: 205. Nadas AS (1963). Pediatric Cardiology 790, Saunders, Philadelphia. Susan M, Zoair AH, Corozon M, Richard MP. Consanguinity and congenital heart disease in Saudi Arabia. Am J Med Genetics. 1999; 99(1): 8-13. Shafi T, Khan MK, Atiq M. Congenital heart disease and associated malformation in children with cleft lip and palate in Pakistan. Br J Plast Surg. 2003 Mar; 56(2): 106-9. Tacy JS, Yip WC and Joseph R. Parental age and birth order in Chiese children with congenital heart disease. Journal of Med Genetics. 1982; 19: 441-443. Zhan SR, Lian ZH, Zheng DZ, Gao L. Effect of fathersâ&#x20AC;&#x2122; age and birth order on occurrence of congenital heart disease. J Epidemiol Community Health. 1991 Dec; 45(4): 299-301.

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*Corresponding Author: Dr.Sandeep Harshangi*

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Assistant Professor,Department of Pediatrics M.R.Medical College, Gulbarga,Karnataka,India

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Research Article Biological Sciences BIODECOLORIZATION AND DEGRADATION OF TEXTILE DIAZO DYE REACTIVE BLUE 171 BY MARINOBACTER SP. NB-6 â&#x20AC;&#x201C; A BIOREMEDIAL ASPECT *

R.S. Shertate and P.R. Thorat

P.G. Department of Microbiology and Research Center, Shri Shivaji Mahavidyalaya, Barshi â&#x20AC;&#x201C; 413411, Dist. - Solapur, MS, India. *Corresponding Author Email: rubinamicro13@gmail.com

ABSTRACT The discharge of highly coloured effluents containing dyes can be damaging to the receiving marine water bodies and can result in serious environmental pollution problems. Hence, considerable attention has been given in determining the ability of marine microorganism in decolorization and degradation of textile dyes. Decolorization and Degradation of Reactive Blue 171 was carried out using the acclimatized Marinobacter sp. NB-6 (Accession No. HF568873) isolated from soil. The decolorization of dye Reactive Blue 171 in 24 hours was up to 95.00 % in nutrient broth having 8.0% NaCl and also it showed 93.11% decolorization in half strength nutrient broth having the same NaCl concentration. The percent decolorization of the dye was also studied by cell-free extract and was observed that the isolate can decolorize the dye 90.00 % in 24 hours. The percent decolorization of the dye was determined spectrophotometrically at 590nm. The percent COD reduction of the dye by the isolate was 86.00%. The degradation products formed after degradation were analyzed by GC-MS technique and it was found that this culture degraded Reactive Blue 171 to the products having molecular weights 98, 99, 149, 150, 223, 70, 86, 125, 154, 155, 149, 150, 223, 149, 150, 223, 57, 113, 149, 167 and 279. The microbial toxicity study revealed the degradation of Reactive Blue 171 into non-toxic products by Marinobacter sp. NB-6. From the study performed, we conclude that, this acclimatized species can prove better option for bioremediation of textile dye in wastes containing high salts and in marine environment.

KEY WORDS COD Reduction, Decolorization, Degradation, GCMS, Marine Bacteria,Reactive Blue 171

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INTRODUCTION Synthetic dyes and pigments are extensively used in the textile industries. So, synthetic dyes are release in textile effluent. Dye effluents are among the major pollutants discharged into the environment. Coloured wastewater from textile industry is rated as the most polluted in all industrial sectors. The colour of synthetic dyes in effluent is one of the most obvious indicators of water pollution. The discharge of highly coloured effluents containing dyes can be damaging to the receiving bodies [1] and can result in serious

environmental pollution problems. These effluents have common characteristics as their high coloration since a small amount of residual dye (of the order of mg l/1) can be sufficient to cause a significant visual effect and affects the aesthetic merit, water transparency and gas solubility in lakes, rivers and other water bodies. Different methods are available for the remediation of dye wastewaters. These include physicochemical methods, like adsorption, chemical oxidation, precipitation, coagulation, filtration, electrolysis, photo degradation, and

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www.ijpbs.com (or) www.ijpbsonline.com biological, and microbiological methods. Adsorption on activated carbon is an effective method for the removal of colour, but it is too expensive [2]. The major disadvantage of physicochemical methods has been largely due to the high cost, low efficiency, limited versatility, interference by other wastewater constituents, and the handling of the 252 Water Air Soil Pollut (2010) waste generated [3,4]. Copper complexed direct dyes were found to be highly toxic to fishes like Daphnia magna than unmetallized new dyes [5].Traditional wastewater treatment technologies have proven to be markedly ineffective for handling wastewater of synthetic textile dyes because of the chemical stability of these pollutants [6]. The development of efficient and environmentally friendly technologies to decrease dye content in wastewater to acceptable levels at affordable cost is of utmost importance [7]. Biological methods are generally considered environmentally friendly as they can lead to complete mineralization of organic pollutants at low cost [8]. Bioremediation may be the most effective method of treating industrial dyes wastewater [9]. Large number of textile industries are located on the coastal areas due to ease of transport to the various places in world and help in building nations economy, but on the contrary the effluents released from these industries are proving a great problem for the marine life. Therefore, industrial effluents containing dyes must be treated before their safe discharge into the environment. Reactive dyes have been identified as the most problematic compounds in textile dye effluents [10,11]. In the present study, a bacterium was isolated from marine environment capable of decolorizing and degrading Reactive blue 171 textile dye. This species was studied for decolorization of the dye Reactive blue 171 in

various different conditions like in complete nutrient medium, in half strength nutrient medium, cell-free extract and in presence of different co-substrates. The decolorization of the dye was monitored spectrophotometrically (Systronics-106) at its specific absorbance maxima (λmax) 590nm. Percent COD reduction of the dye was calculated.

MATERIALS AND METHODS 

Samples – Soil samples were collected from salterns (Saltpan), areas nearby waste disposal sites of the textile industry, sewage, sludge, effluent treatment plants and compost as the source of microorganisms.  Dye– Reactive Blue 171 (λmax-590nm) Preparation of the dye solutions and isolation of microorganisms Dye solutions of 1% are prepared in distilled water, which are stored as stock solutions and used for further study. After acclimatization process of the dye for 2 months isolation was carried out by adding the different NaCl concentrations and dye solution in soil collected from salterns (Saltpan), areas nearby waste disposal sites of the textile industry, sewage, sludge, effluent treatment plants and compost in increasing concentration. Isolation of microorganisms was done from the acclimatized soil. For isolation of microorganisms, the soil was mixed with nutrient broth having different NaCl concentrations and incubated for 24 hours at ambient temperatures for the growth of microorganisms. This nutrient broth was then streaked on the nutrient agar plates containing the dye and different NaCl concentrations. After incubation the colonies showing decolorization of the dye were selected for further study. Acclimatization of microorganismsSoil samples from salterns (Saltpan), area nearby waste disposal site of textile industries, sewage,

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sludge, ETP along with compost were collected and homogenized properly. The micro-flora from the samples were acclimatized in the dye Reactive Blue 171 (1%) and 8.0% NaCl concentrations for the period of one month. One gram of acclimatized soil was inoculated in the nutrient broth with 8.0% NaCl concentrations, after incubation isolation was carried-out on nutrient agar incorporated with same NaCl and dye concentration. The colonies showing decolorization were selected for the further studies.

Determination of Biodegradation Assay Acclimatized, 24 hours old culture of the isolate NB-6 was inoculated in nutrient medium containing dye Reactive Blue 171 (Figure 1) and 8.0% NaCl concentration at a concentration 10ml of 1% dye from stock and incubated at 37oC. An aliquot of 5 ml was removed after different time intervals. The aliquot was centrifuged at 10,000 rpm for 20 min to remove the cell mass. The supernatant was then used to investigate the decolorization of the dye by observing the change in the absorbance at maximum absorption wavelength (λ max) 590nm nm on spectrophotometer (Systronics-106 model).

Figure 1- Structure and properties of Reactive Blue 171 Structure SO3Na HN

NH2

SO3Na

N N

NaO3S

SO3Na

N N

NH N

N N

SO3Na

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Properties: Molecular Formula = C40H23Cl2N15Na6O19S6 Molecular weight =1418.93 Composition =C (33.86%) H (1.63%) Cl(5.00%) N (14.81%) Na (9.72%) O (21.42%) S (13.56%)

Decolorization of Dye in Nutrient Broth The selected microorganism was inoculated in 20 ml of nutrient broth containing 8.0% NaCl and 1000µg/ml concentration of dye. The tube was then incubated at ambient temperature for 24 hrs and observed for decolorization of the dye. Decolorization of dye in Half Strength Nutrient Broth The selected culture was then inoculated in 20 ml of half strength nutrient broth containing 8.0% NaCl and 1000µg/ml concentration of dye.

The tube was then incubated at ambient temperature for 24 hrs and decolorization pattern was studied. Cell-free extract studies on Decolorization of Dye The cells grown in nutrient broth with 8.0% NaCl concentrations were lysed by using Sonicator (Vibra Cell System) and centrifuged in cooling centrifuge (BIO-LABS 165-R) at 10000 rpm for 15 min. The supernatant was added with 1000µg/ml concentration of dye solution in nutrient broth

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www.ijpbs.com (or) www.ijpbsonline.com with same NaCl concentrations and incubated at ambient temperature. The percent decolorization studies were done by using spectrophotometer (Systronics – 106 models). Decolorization of dye in presence of different Co-substrates The promising isolate was inoculated in 20ml nutrient broth containing 8.0% NaCl concentrations, 1000µg/ml concentration of dye and 1% Glucose. Tube was then incubated at ambient temperature for 24 hours and observed for decolorization of the dye. Additionally, nutrient medium containing 1% Starch and 1% Yeast Extract with the same dye and 8.0% NaCl concentrations were also used to test the ability of Marinobacter sp. NB-6 to decolorize the dye Reactive Blue 171. Determination of Chemical Oxygen Demand (COD) Percent COD reduction value of the dye decolorized in nutrient medium having 8.0% NaCl were calculated by using strong oxidizing agent potassium dichromate (K2Cr2O7). GCMS analysis To study the products formed after degradation of dye Reactive Blue 171, decolorized samples were analysed by GCMS. The isolate was inoculated in 100mL of sterile nutrient broth containing 1000 μg/mL of dye Reactive Blue 171 and 8.0% NaCl. The broth was then incubated at ambient temperature for 24 hrs in separate flasks. The decolorized broth was then centrifuged at 10,000 rpm for 15 minutes in cooling centrifuge. Centrifugate was mixed with equal amount of dichloromethane in separating funnel. Samples were shaken vigorously for 15 minutes and kept for 10-15 minutes to separate solvent and aqueous phases. After separation, aqueous phase was discarded and solvent phase allowed for partial evaporation. Partially evaporated samples were analysed by GCMS technique.

Microbial Toxicity of Dyes and their Biodegradation Products Testing It is very important to know whether biodegradation of a dye leads to detoxification of the dye or not. Agar well bioassay is the most common technique used to evaluate the microbial toxicity. This can be achieved by microbial toxicity tests with the original dyes and their biodegradation products. The microbial toxicity was tested on three test organisms viz. Azotobacter sp., Pseudomonas sp. Rhizobium sp. The 24 hours old culture of the test organisms were used for the toxicity testing. After the confirmation of dye degradation, the degraded solution (decolorized broth) was poured in the wells prepared in nutrient agar previously spreaded with the test organisms. These plates were incubated at ambient temperatures for 24 hours. The zone of inhibition around the wells proved the toxicity of the dye and their degraded products. Identification of the isolate and sequencing of 16S rRNA gene The 16S rRNA was determined in National Center for Cell Sciences, University of Pune Campus, Pune. Genomic DNA isolation of isolate was carried using Qiagen DNA isolation kit as per manufacturer’s instruction. Its presence was checked by running in agarose gel (0.8%) stained with ethidium bromide. The sequence was deposited to European Bioinformatics Institute (EBI). Sequence was analyzed at the Ribosomal Database Project (RDP-II) (http://rdp.cme.msu.edu/) for closed homology. The sequences downloaded from the RDP II database were aligned by using CLUSTAL X2 multiple sequence alignment tools. The Phylogenetic tree was constructed by the neighbor joining method using Kimura-2parameter distances in MEGA 4.0 [12].

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RESULTS Isolation and Identification The organism was isolated from the soil on nutrient agar containing 8.0% NaCl and was identified by using biochemical observations and 16s rRNA analysis technique. From the analysis the isolate was identified as Marinobacter sp. NB-6. Biochemical results showed that the isolate was unable to hydrolyze Starch and

Gelatin, able to reduce nitrate, whereas fermentation of glucose, sucrose, mannose, maltose, lactose showed acid and gas production. The isolate was Oxidase and Catalase positive whereas Urease negative. Biochemical results are given in (Table 1). The phylogenetic tree was developed by using Neighbor joining method by Kimura-2-parameter with 1000 replicates in MEGA 4.0. [13] (Figure 2).

Table 1 Biochemicals of the isolate Characteristics of isolate NB-6 Gram nature and Motility

Marinobacter sp. NB-6 Gram negative motile rods

Optimal growth temperature ( C)

370C

Optimal NaCl Concentration (%)

8.0%

Utilization of D-Glucose

Sucrose

Mannose

Maltose

Lactose

Hydrolysis of Starch

Gelatin

Casein

Enzyme activity +

Oxidase

Catalase

Urease

Nitrate reduction

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Amylase

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54 35

EF028328 Marinobacter salsugin AY517632 Marinobacter flavimar

AJ609271 Marinobacter bryozoor 16

EF486354 Marinobacter salicamp AJ609270 20

EU293412 Marinobacter mobilis DQ414419 Marinobacter gudaonen

EF157832 Marinobacter segnicre EU047505 Marinobacter lacisals

AF288157 Marinobacter lutaoens 58

DQ325514 Marinobacter koreensi 99 EU496088 Marinobacter santorin

EU164778 Marinobacter szutsaon AJ704395 Marinobacter maritimu

99 30

DQ060402 Marinobacter psychrop EF660754 Marinobacter goseonge

NB-6

38 51

AY147906 Marinobacter lipolyti

0.005

Figure 2: Phylogenic tree of Marinobacter sp. NB-6. Phylogenetic analysis of 16s rRNA gene sequence of Marinobacter sp. NB-6. The percent numbers at the nodes indicate the levels of bootstrap support based on neighbor-joining analyses of 1,000 replicates. The scale bar (0.005) indicates the genetic distance.

Percent Decolorization of the Isolate Marinobacter sp. NB-6 was studied for its percent decolorization capacity in nutrient broth with 8.0% NaCl concentration and Dye

1000µg/ml concentration. The results of percent decolorization of dye Reactive Blue 171 in nutrient broth is given in (Table 2 and Figure 3).

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Table 2: Percent Decolorization in Nutrient Broth, ½ Strength Nutrient Broth and Cell-Free Extract in 24 hrs at λmax-590nm and percent COD reduction value Culture Code Identified As % Decolorization in COD Reduction Value Nutrient Broth ½ strength Cell-Free Nutrient Broth NB-6 Marinobacter 95.00 93.11 90.00 86.00 NB-6

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Figure 3: Percent Decolorization of Reactive Blue 171 in Nutrient medium having different NaCl concentrations 0 at 37 C in 24 hours

Percent Decolorization in Half (½) Strength Nutrient Broth Marinobacter sp. NB-6 was studied for its ability to decolorize the dye in half (½) strength nutrient broth with 8.0% NaCl concentration and the dye in same concentration 1000µg/ml as in complete nutrient broth. The results of percent decolorization of dye Reactive Blue 171 in half

strength nutrient broth is given in (Table 2 and Figure 4). Percent Decolorization in Cell-Free Extract Marinobacter sp. NB-6 was studied for its ability to decolorize the dye in Cell-Free extract. The results of percent decolorization of dye Reactive Blue 171 in cell-free extract is given in (Table 2 and Figure 4).

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Figure 4: Percent Decolorization of Reactive Blue 171 by Marinobacter sp. NB-6 in different nutrient medium having 8.0% NaCl concentration at 370C in 24 hours

Percent COD reduction The Percent COD reduction of the dye after decolorization of the dye by the Marinobacter sp. NB-6 is given in (Table 2).

Percent Decolorization of Dye in presence of different Co-substrates Marinobacter sp. NB-6 was further studied for its ability to decolorize the dye Reactive Blue 171 in nutrient medium containing 8.0% NaCl

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www.ijpbs.com (or) www.ijpbsonline.com concentration and 1% Glucose, 1% Starch and 1% Yeast extract as Co-substrates and 1000渭g/ml concentration of dye. The results of

percent decolorization of dye Reactive Blue 171 in presence of different Co-substrates is given in (Table 3 and Figure 5).

Table 3 Percent Decolorization in presence of Different Co-Substrates viz 1% Glucose, 1% Yeast Extract and 1% Starch in 24 hrs at 位 max-590nm Culture Code Identified As % Decolorization in 1% Glucose 1% Yeast Extract 1% Starch NB-6 Marinobacter 95.22 96.00 95.11 NB-6

Figure 5: Percent Decolorization of Reactive Blue 171 by Marinobacter sp. NB-6 in nutrient medium having 1% Co-substrates and 8.0% NaCl concentration at 370C in 24 hours GC-MS analysis The GC-MS analysis reports of the dye are shown in Figure 6, 7, and 8 respectively. The reports showed that the dye was degraded by the isolate having different molecular weights (Table 4). The results showed that the isolate from the acclimatized soil have good decolorization and

degradation of the dye Reactive Blue 171. Confirmation of the biodegradation of the dye Reactive Blue 171 was done by analysing the samples with GC-MS. The degradation products of the dye were of much lower mass than the original compounds.

Table 4 Molecular weights of the degraded products Identified As

NB-6

Marinobacter sp. NB-6

Molecular weights of the degraded products 98, 99, 149, 150, 223, 70, 86, 125, 154, 155, 149, 150, 223, 149, 150, 223, 57, 113, 149, 167 and 279 respectively

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Culture Code

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Figure 6: GCMS analysis report of degraded products of Reactive Blue 171 dye by Marinobacter sp. NB-6.

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Figure 7: GCMS analysis report of degraded products of Reactive Blue 171 dye by arinobacter sp. NB-6.

Molecular weights of the degraded products of dye The GC-MS analysis report showed that the dye Reactive Blue 171 was degraded and not only

decolorized. The molecular weights of the degraded products are given in (Table 4) Microbial Toxicity Studies Microbial toxicity of the dye Reactive Blue 171 was studied on microorganisms viz. Azotobacter

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www.ijpbs.com (or) www.ijpbsonline.com sp., Pseudomonas sp. and Rhizobium sp. The toxicity of the dye and its degradation products was studied by the agar well assay. The results showed that the wells which were poured with decolorized broth had no zone of inhibition and

wells with original dye solution had zone of inhibition. This confirmed that the original dye solution 1000 Âľg/ml was toxic to the bacteria but its degradation products were non toxic to the bacteria (Figure 9)

Figure 8: GCMS analysis report of degraded products of Reactive Blue 171 dye

Figure 9: Microbial toxicity testing against Azotobacter sp., Pseudomonas sp. and Rhizobium sp.

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DISCUSSION In present study, the decolorization of Reactive Blue 171took place in nutrient medium containing 8.0% NaCl, this suggests that the presence and availability of a co-substrate is necessary, because it acts as an electron donor for the azo dye reduction (Nigam et al., 2006). Effect of co-substrates viz. Glucose, Starch and Yeast Extract was studied which showed slight increase in the percent decolorization of the dye was observed in 24 hours. From the percent COD reduction values it can be concluded that dye was degraded by the isolate. The isolated marine organism carried out biotransformation of Reactive Blue 171, which was confirmed by GC-

MS analysis. These results were similar to Khalid, et al., (2008b). Khalid, et al., (2008b) reported enhanced decolorization of azo dyes by Shewanella putrefaciens strain AS96 in presence of yeast extract as co-substrate under hypersaline condition. Rania, M.A., (2008) used Glucose, Sucrose, Starch and Sodium citrate as carbon sources among which Starch was best for decolorization of Crystal violet up to 96 % . Gondaliya and Parikh, (2012) reported the highest percentage decolourization 97.04% of Reactive Orangeâ&#x20AC;&#x201C;16 was obtained by Serratia marcescens when additional supplement of glucose (1 g/l) was added in Nutrient broth. These study shows that additional supplement of

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www.ijpbs.com (or) www.ijpbsonline.com carbon source give rise in percentage of decolourization but among them addition of yeast extract give highest percentage decolourization in Reactive Blue 171. In the present study, the halophilic Marinobacter sp. NB-6 isolated from acclimatized soil decolorized the azo dye Reactive Blue 171 to an extent of 85.20% in nutrient medium, 84.25% in half strength nutrient medium. This observation was found to be in accordance with the previous studies of Shertate and Thorat, (2012) who reported the decolorization of azo dye Mordant Orange-1 by Marinobacter algicola MO-17 at 15% salinity. This result was also very much similar to the decolorization of azo dye Reactive Blue 171by Marinobacter sp. NB-6. This rate of decolorization may be due to the high metabolic diversity being observed in the halophiles due to their extremophilic nature (Oren et al., 1992; Ventosa et al., 1998). These results were similar to Jeremy Martin et al., (2011) who reported three marine-derived fungal strains, Phialophora sp. (MF 6), Penicillium sp. (MF 49) and Cladosporium sp. (EMF 14) which showed complete decolorization of 0.01% Congo red and up to 91 % decolorization of 0.01% crystal violet at 33g/L marine salts. Similar result was obtained by Bumpus and Brock (1998) in their experiment on the degradation of crystal violet by P. chyrosporium. The results of microbial toxicity indicate that the azo dye degradation products formed after biodegradation by Marinobacter sp. NB-6 were less toxic compounds compared to the original azo dye. These results are in agreement with result of Kalyani et al., (2009) and Mane et al., (2008) who found that the metabolites products after biodegradation of Reactive Red 2 and Reactive Blue 59 were less toxic compared to the original dye. The use of such marine microorganism able to degrade azo dye in presence of salt could help

prevent costly dilution to lower the salinity, or the removal of salt by physico-chemical methods before biological treatment and can be used for the treatment of effluents containing high salt content.

ACKNOWLEDGMENT We would like to offer our sincere thanks to Staff of Indian Institute of Technology Powai, Mumbai for GCMS analysis and National Center for Cell Sciences, University of Pune Campus for the 16s rRNA sequencing of the isolate. Our sincere thanks to Principal, Shri Shivaji Mahavidyalaya, Barshi and Head, Department of Microbiology for providing the Laboratory facilities.

REFERENCES 1.

7. 8.

Nigam P., Banat I.M., Singh D., and Marchant R., Microbial Process for the Decolorization of the Textile Effluent containing Azo Diazo and Reactive Dyes. Process Biochem, 31:435 – 442, (2006) Fu Y. and Viraraghavan T., Fungal decolorization of dye wastewaters: a review. Bioresour Technol, 79:251–262, (2001) Van der Zee F. P. and Villaverde S., Combined anaerobic-aerobic treatment of azo dyes—A short review of bioreactor studies. Water Research, 39:1425–1440, (2005) Kaushik P. and Malik A., Fungal dye decolourization: Recent advances and future potential. Environment International, 35:127–141, (2009) Bae J.S. and Freeman H.S., Aquatic toxicity evaluation of copper-complexed direct dyes to the Daphnia magna. Dyes Pigments 73:126–132, (2005) Forgacs E., Cserhati T. and Oros G., Removal Of Synthetic Dyes From Wastewaters: A Review. Environ Int ,30: 953-71, ( 2004) Couto S. R., Dye Removal By Immobilized Fungi. Biotechnology Advances, 27: 227–235, ( 2009) Pandey A., Singh P, and Iyengar L., Bacterial decolorization and degradation of azo dyes. International Biodeterioration & Biodegradation, 59:73-84,( (2007) Nozaki K., Beh C. H., Mizuno M., Isobe T., Shiroishi M. and Kanda T., Screening And Investigation Of Dye Decolorization Activities Of Basidiomycetes. Journal of Bioscience and Bioengineering, 105: 69–72,( 2008)

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www.ijpbs.com (or) www.ijpbsonline.com 10. ECD guidelines for testing of chemicals.1996: vol. 2 Test 302B 11. El-Dein A.M., Libra J.A. and Wiesmann U., Kinetics of decolorization and mineralization of the azo dye Reactive Black 5 by hydrogen peroxide and UV light. Water Sci. Technol, 44:295-301, ( (2001) 12. Tamura K., Dudley J., Nei M. and Kumar S., MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol, 24:1596–1599, (2007) 13. Saitou N. and Nei M., The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4:406-425, (1987): Available from http://mbe.oxfordjournals.org/content/4/4/406.full.pd f+html 14. Khalid A., Arshad M. and Crowley D.E., Decolorization of azo dyes by Shewanella spp. under saline conditions. Appl. Microbiol Biotechnol, 79:1053-1059. DOI: 10.1007/s00253-008-1498-y, (2008b) 15. Rania M.A., Decolorization and Biodegradation of Cryastal Violet an Malachite Green by Fusarium solani (Martius) Saccardo. A Comparative Study on Biosorption of Dyes by Dead Fungal Biomass. American-Eurasian Journal of Botany, 1(2):17 – 31, (2008) 16. Gondaliya Nidhi and Parikh Samir., Decolorization of Reactive Orange-16 by Serratia marcescens. Life Sciences Leaflet, 11:86-96, (2012) 17. Shertate R.S. and Thorat P.R. Decolorization of Mordant Orange-1 by Marinobacter algicola MO-17.

18.

19.

20.

21.

22.

23.

On Line Journal of Biological Sciences, 12 (1):1-5, (2012) DOI: 10.3844/ojbsci.2012.1.5 http://thescipub.com/issue-ojbs/12/1 Oren A., Gurevich P., Azachi M. and Henis, Y. M., Microbial degradation of pollutants at high salt concentrations. Biodegradation, 3:387-398, (1992): DOI: 10.1007/BF00129095 Ventosa A., Nieto J.J. and Oren, A., Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Rev., 62:504-544, (1998) Jeremy Martin O.T., Christine V.C., Llewelyn S.M., Ana Patricia A. G., and Thomas Edison E.C., Dye Decolorization Activities of Marine-Derived Fungi Isolated from Manila Bay and Calatagan Bay, Philippines. Philippine Journal of Science, 140 (2):133143, (2011). Retrieved from www.researchgate.net Bumpus J.A. and Brock B.J., Biodegradation of crystal violet by the white rot fungus Phanerochate chrysosporium. Appl Environ Microbiol, 54:1143–1150, (1998). Kalyani DC., Telke AA., Dhanve RS. and Jadhav JP., Ecofriendly biodegradation and detoxification of Reactive Red 2 textile dye by newly isolated Pseudomonas sp. Journal of Hazardous Materials,163: 735-742, (2009) Mane UV., Gurav PN., Deshmukh AM., and Govindwar SP., Degradation of textile dye reactive navy-blue Rx ( Reactive blue-59) by an isolated Actinomycete Streptomyces Krainskii SUK-5. Malaysian Journal of Microbiology, 4: 1-5, (2008)

*Corresponding Author: *

R.S. Shertate and P.R. Thorat

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P.G. Department of Microbiology and Research Center, Shri Shivaji Mahavidyalaya, Barshi – 413411, Dist. - Solapur, MS, India. E-mail ID: rubinamicro13@gmail.com

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Research Article Pharmaceutical Sciences DEVELOPMENT AND VALIDATION OF UV SPECTROPHOTOMETRIC METHOD FOR THE SIMULTANEOUS ESTIMATION OF CILNIDIPINE AND TELMISARTAN IN TABLET DOSAGE FORM UTILISING SIMULTANEOUS EQUATION AND ABSORBANCE RATIO METHOD *M.Haripriya1, Neethu Antony1, P. Jayasekhar 2 *1

College of Pharmaceutical Sciences, Government Medical College, Trivandrum, Kerala-695011 2 Oman Medical College, Azaiba, Post box 620, Postal code.130 Muscat , Sultanate of Oman *Corresponding Author Email: riyasiva@yahoo.co.in

ABSTRACT Two validated uv spectrophotometric methods for the simultaneous estimation of Cilnidipine and Telmisartan in pure powder and in two component dosage forms have been developed, utilising simultaneous equation and absorbance ratio method. The method is based on the measurement of absorbance of Cilnidipine and Telmisartan at their respective wavelengths of 240 nm and 297nm and at the iso absorptive wavelength of 270 nm in methanol. Cilnidipine and Telmisartan at their respective λmax240 nm and 297nm obeyed Beer’s law in the concentration range 4-10µg/ml and 6-18µg/ml respectively with correlation coefficient 0.9998 and 0.9992 for Cilnidipine and 0.9998 and 0.9991 for Telmisartan. The results have been validated statistically as per ICH guidelines.

KEY WORDS Cilnidipine; Telmisartan; Simultaneous estimation; absorbance ratio, Validation.

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INTRODUCTION Cilnidipine (CIL) O3-(2-methoxyethyl) O5-[(E)-3phenylprop-2-enyl] 2, 6-dimethyl-4-(3nitrophenyl)-1,4-dihydropyridine3,5dicarboxylate is a novel and unique dihydropyridine calcium channel blocker that possesses a slow-onset, long-lasting vasodilating effect1. Telmisartan (TEL), 4-((2-n-propyl-4-methyl-6-(1methylbenzimidazol – 2 – yl) – benzimidazol – 1 yl) methyl ) biphenyl-2- carboxylic acid, is an angiotensin II receptor antagonist that shows high affinity for the angiotensin II receptor type 1 (AT1), with a binding affinity 3000 times greater for AT1 than AT22. Literature review revealed only one method for the simultaneous estimation of Cilnidipine and Telmisartan in two component dosage forms,

which is an HPTLC method3.Hence it was proposed to develop economical, rapid and simple uv spectrophotometric methods for the simultaneous estimation of these drugs in dosage forms.

MATERIALS All the chemicals and reagents used were of analytical grade. Cilnidipine and Telmisartan hydrochloride were obtained as gift samples from J B Chemicals and Pharmaceuticals,Daman and Akums Drugs and Pharmaceuticals limited, Hardwar. The combined dosage form was purchased from local market.Methanol HPLC was procured from SD Fine-Chem limited, Mumbai.

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www.ijpbs.com (or) www.ijpbsonline.com EQUIPMENT

DERIVATION OF EQUATIONS

The Jasco double beam uvvis spectrophotometer with spectral band width 2.0 nm wavelength accuracy 0.5nm and matched quartz cells of 1 cm path length were used for all spectral and absorbance measurements. Class A volumetric glass wares were used.

METHOD I Simultaneous equation method From the absorptivity values determined for CIL and TEL, simultaneous equations are derived for determination of these two drugs in combination in their pharmaceutical formulations. A1=0.0850Cx+0.0605Cy …… at 240nm (1) A2=0.0146Cx+0.0494Cy ……..at 297 nm (2) The absorbance and the absorptivity values at the particular wavelength were calculated and substituted in the following equation, to obtain the concentration. Cx = (A1ax2 – A2ax1) / (ax2ay1 – ax1ay2). Cy = (A2ay1 – A1ay2) / (ax2ay1 – ax1ay2). Where, Cx = Concentration of CIL Cy = Concentration of TEL A 1&A2 absorbance of sample at 240 nm and 297 nm respectively ax1&ax2 absorptivity of CIL at 240 nm and 297 nm respectively ay1&ay2 absorptivity of TEL at 240 nm and 297 nm respectively. METHOD II Absorbance ratio/Q value method In Q analysis method, from the overlay spectra of CIL and TEL absorbances were measured at selected wavelength i.e.270 nm (isoabsorptive point) and at 297 nm (λ max of TEL).The absorptivity coefficient of each drug at both the wavelengths were determined. The concentration of each drug in the tablet formulation were determined by substituting the absorbances and absorptivity coefficients in the equation Cx = (Qm - Qy)/(Qx - Qy)×A1/ax1 Cy =(Qm – Qx)/(Qy - Qx)×A1/ay1 Where, Qm = Absorbance of sample at 297 nm / Absorbance of the sample at 270 nm

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EXPERIMENTAL METHODS

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Standard solutions and Calibration curves Stock solutions for spectrophotometric measurements were prepared by dissolving CIL and TEL in methanol to obtain concentration of 1mg/ml for each compound. For calibration, series of above solutions were prepared containing CIL4.0, 5.0,6.0,7.0,8.0.9.0,10.0 µg/ml and TEL 6.0,8.0,10.0,12.0,14.0,16.0,18.0 µg/ml by diluting the stock standard solution with methanol in standard volumetric flasks(10ml).The solutions were scanned in the range of 220-350nm. Selection of wavelengths Method I. Simultaneous equation method Overlain spectra for both the drugs are shown in Fig.1.Two wavelengths selected for the use of simultaneous equation were 240 and 297nm. The absorbance was recorded at the selected wavelengths and the absorptivity values were determined for Cilnidipine and Telmisartan. Statistical parameters like slope, intercept, coefficient of correlation and SD were determined. (Table 1). Method II. Absorbance ratio/Q value method From the overlain spectra of the two drugs, the isoabsorptive wavelength of 270nm and the λ max of telmisartan at 297 nm were selected for this method. The absorptivity values were calculated at 270nm and 297nm. Statistical parameters like slope, intercept, coefficient of correlation and SD were determined. (Table 1)

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www.ijpbs.com (or) www.ijpbsonline.com Qx = Absorptivity of CIL at 297 nm / Absorptivity of CIL at 270 nm Qy = Absorptivity of TEL at 297 nm / Absorptivity of TEL at 270 nm A1 = Absorbance of the sample of sample at 270nm (isoabsorptive point) ax1= Absorptivity of CIL at 270 nm ay1 = Absorptivity of TEL at 270 nm

ANALYSIS OF FORMULATION Twenty tablets of brand Cilacar T (J B Chemicals and Pharmaceuticals) containing 10 mg of CIL and 40 mg of TEL were weighed, average weight determined and finely powdered. Appropriate quantity of powder equivalent to 10 mg of CIL and 40 mg TEL was accurately weighed, transferred to a 100 ml volumetric flask and volume was made up to 100 ml with methanol and shaken vigorously for 15 minutes. The solution was then sonicated for 5 minutes and filtered through the Whatman filter paper no.41. Necessary dilutions of filtrate were made with methanol to get final concentration 10 μg/ml of CIL and 40 μg/ml of TEL. Absorbance of this solution was measured at 240 nm (λ max of CIL) 297nm ((λ max of TEL), and 270 nm (Isoabsorptive Point), The values obtained were substituted in the respective formulae of Method 1 & 2 to obtain concentrations of CIL and TEL. The results are shown in Table 3.

curves were plotted over the seven different concentrations in the range 4-10 μg/mL and 6-18 μg/mL for CIL and TEL, respectively (Fig.2 and Fig.3). The optical parameters and statistical parameters are depicted in Table 1. Accuracy (% Recovery) The accuracy of the method was determined by calculating recoveries of CIL and TEL by the standard addition method. Known amount of standard of CIL and TEL (80%, 100%, and 120%) were added to the sample solutions of tablet dosage forms. The amounts of CIL and TEL were estimated by equations in method I and II. The results are shown in Table 2. The values prove that the method is accurate. 5.3. Method Precision (Repeatability) The precision of the instruments was checked by repeatedly scanning (n = 5) standard solutions of CIL and TEL (10 μg/ml). The RSD values were found to be below 2% which indicate that the proposed methods are repeatable (Table 2). Intermediate Precision (Reproducibility) The intermediate precision for the proposed method was determined by estimating standard solution of CIL and TEL for three different concentrations for three times on the same day(intraday) and on three different days(interday). The results are reported in terms of relative standard deviation (RSD). The RSD values were found to be below 2% which indicate that the proposed methods are reproducible (Table 2). Robustness Solutions of both the drugs in methanol were studied for their stability at ambient temperature for 24 h. Absorbance variation was found to be less than 1%.

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Method Validation4 Calibration curve (linearity of the method) Calibration curves were constructed by plotting absorbance vs. concentrations of CIL and TEL,at their respective λmax and the regression equations were calculated. The calibration

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Fig. 1: Overlain Absorption spectra of Cilnidipine and Telmisartan

Fig.2 Calibration plot of fundamental spectra of CIL

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Fig.3. Calibration plot of fundamental spectra of TEL

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Table 1: Optical characteristics Data Parameters

TEL

CIL

TEL

Working λmax

240nm

297 nm

270 nm

Beer’s law limit

4-10µg/ml

6-18µg/ml

4-10µg/ml

6-18µ/ml

Correlation coefficient

0.999892

0.999179

0.999876

0.999071

Intercept

-0.03717

-0.03736

0.057311

-0.04226

Slope Regression equation

0.090775 Y=0.090775x -0.03717

0.0528680 Y=0.052868x -0.03736

0.029225 Y=.029225x+ 0.057311

0.040509 Y=0.040509x -0.04226

Accuracy % Precision (RSD, %) Repeatability(n=5) Intraday(n=3) Interday(n=3) Robustness

Brand name

347

Method II Q Absorbance ratio method

CIL

Parameter

Page

Method I Simultaneous equation method

Cilacar T

Table 2: Summary of validation parameters for the proposed methods CIL TEL Method I Method II Method I 99.72 98.94 100.10

Company

0.19 0.46-0.56 0.88-1.24 Robust

0.2 0.21-0.39 0.98-1.10 Robust

0.25 0.22-0.84 1.02-1.06 Robust

0.16 0.35-0.78 0.85-0.93 Robust

Table 3.Compilation of results of commercial formulation Formulation Label Claim Amount found

J B Chemicals Pharmaceuticals

Tablet

CIL 10mg TEL 40mg

Method II 98.95

(mg)

Method I

Method II

9.89±0.10 39.91±0.45

10.11±0.12 39.87±0.38

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www.ijpbs.com (or) www.ijpbsonline.com CONCLUSION The methods discussed in the present work provide a convenient and accurate way for simultaneous estimation of CIL and TEL. In simultaneous equation method, wavelengths selected for analysis were 240 nm (λmax of CIL) and 297 nm (λmax of TEL). In Q-analysis method wavelengths selected were 270 nm (isoabsorptiv e point) and 297 nm (λmax of (TEL). In both the methods linearity for detector response was observed in the concentration range of 4-10 μg/ml (for CIL) and 6-18 μg/ml (for TEL). Absorptivity coefficient were calculated for both the drugs at selected wavelengths and substituted in equations for determining concentration of CIL and TEL in its tablet dosage form. Percent label claim for CIL and TEL in tablets was found by simultaneous equation method and by Q-analysis method. Accuracy of proposed methods was ascertained

by recovery studies. Hence the proposed methods can be employed for routine quality control of Cilnidipine and Telmisartan in its combined dose formulations.

REFERENCES 1.

Yoshimoto R, Dohmoto H, Yamada K, Goto A. Prolonged inhibition of vascular contraction and calcium influx by the novel 1,4-dihydropyridine calcium antagonist cilnidipine (FRC-8653) Jpn J Pharmacol. 1991; 56:225–229. Benson, S. C.; Pershadsingh, H.; Ho, C.; Chittiboyina, A.; Desai, P.; Pravenec, M.; Qi, N.; Wang, J. et al. (2004). "Identification of Telmisartan as a Unique Angiotensin II Receptor Antagonist with Selective PPAR Modulating Activity". Hypertension 43 (5): 993. Pawar Prajakta, et al. “HPTLC Determination of Cilnidipine and Telmisartan in combined tablet dosage forms” . International Research Journal of Pharmacy. 2012:3(6) 219-222 ICH Harmonized Tripartite Guidelines, Validation of analytical procedures: Text and Methodology, Q2 (R1), 2005, Geneva.

*Corresponding Author:

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M. Haripriya* College of Pharmaceutical Sciences, Government Medical College, Trivandrum, Kerala-695011

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Research Article Pharmaceutical Sciences QUALITY CONTROL STUDIES ON CETIRIZINE HYDROCHLORIDE TABLETS AVAILABLE IN BANGLADESHI DRUG MARKET Asif Hasan, Syed Masudur Rahman Dewan*, Sayed Koushik Ahamed, Md. Masud Kaisar Department of Pharmacy, Noakhali Science and Technology University, Sonapur, Noakhali- 3814, Bangladesh *Corresponding Author Email: pharmasud@operamail.com

ABSTRACT Drugs are those Things which are directly related to the human health; therefore, quality of a drug is essential to save the human being from a severe health hazard. Various quality control parameters of pharmaceutical products i.e., weight variation, friability, content uniformity, disintegration time, and in vitro dissolution profiles can ensure their quality as well as bioavailability and optimum therapeutic activity. The present study was carried out aiming to ensure the quality and therapeutic activity of cetirizine hydrochloride tablets of different brands available in Bangladeshi pharma market. To demonstrate the differences between the commercial brands, difference (f1) and similarity (f2) data were analyzed. The results showed that all the selected brands met the specification given by pharmacopeia (USP-NF).

KEY WORDS Cetirizine Hydrochloride, Disintegration, Dissolution, Quality Control, Pharmacopeia

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INTRODUCTION Quality control is a process that is carried out to ensure a desired level of quality in a product or service. It might include whatever actions a business deems necessary to provide for the control and verification of certain characteristics of a product or service. Most often, it involves thoroughly examining and testing the quality of products or the results of services. ISO 84021986 standard defines that quality is the totality of features and characteristics of a product or service that bears its ability to satisfy stated or implicated needs 1. Pharmaceutically, we can say quality is checking and directing the degree and grade of experience of process and products 2. This process is carried out to validate the product quality, to produce medication of superior efficacy, safety, and to provide assurance to physician, pharmacists and patients

as well that given product performs satisfactorily and uniformly. When a number of different formulations are available for the same drug, it becomes essential to ensure the equivalency (relationship followed by bioavailability, therapeutic response, or a set of established standards of one drug product to another 3) of the products pharmaceutically. Cetirizine is a long acting antihistamine and unlike conventional or first generation antihistamines it causes less sedation and psychomotor impairment and as well causes no behavioral changes. It is a major metabolite of hydroxyzine, and a racemic selective H1 receptor antagonist used in the treatment of allergies, hay fever, angioedema, and urticaria. The basic goal of our study was to ensure that the products (cetirizine tablets) that are saturated in Bangladeshi drug market meet specific requirements and characteristics, such

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MATERIALS AND METHODS MATERIALS Cetirizine (standard) was donated by Globe Pharmaceuticals Ltd., Bangladesh. In our study, nine commercial Bangladeshi brands (selected randomly) containing 10 mg of Cetirizine Hydrochloride were purchased from nearby retail pharmacy shop and here denoted by CTZ 1, CTZ 2, CTZ 3, CTZ 4, CTZ 5, CTZ 6, CTZ 7, CTZ 8, and CTZ 9. In the study we used 2.9 mL/L phosphoric acid in water to prepare buffer solution. Analytical Method Accurately weighed cetirizine was dissolved in the buffer solution in a 100 ml volumetric flask. From the mother solution, different dilutions were prepared to generate a calibration curve by measuring absorbance using UV spectrophotometer (UV1800, UV-VIS spectrophotometer, Shimadzu, Japan) at 230 nm. The concentration of cetirizine was calculated using the linear regression equation of the calibration curve. Weight Variation For each brand, ten tablets were randomly selected and weighed individually using an analytical balance (ELB 3000, Shimadzu, Japan). The average weights were determined and the percentage deviations from mean values were calculated. Then the standard deviation, and percentage of related standard deviation (RSD) were determined. Hardness Test The hardness of randomly selected ten tablets was determined for all the brands using ‘Monsanto’ type hardness tester (Intech, Korea). The mean crushing strengths were determined.

Friability Test Ten tablets from each commercial brand were weighed individually, and each set of tablets was put into the friabilator (EF-2, Electrolab, India). Then the tablets were rotated at 100 rpm for 1 minute. Then the tablets were removed and weighed again. The friability percentage was calculated for each batch. Test for Content Uniformity The amount of cetirizine in each brand was determined according to USP. A standard solution and sample solutions were prepared of 20 tablets for each brand. The absorbance of each prepared solution was determined at 230 nm by UV spectrophotometer. The amount of cetirizine in each brand was calculated using the equation of the calibration curve. Disintegration Time Test USP-30 4 disintegration apparatus (Electrolab, India) containing 6 glass tubes that are 3 inches long, open at the top and held against a 10-mesh screen at the bottom end of the basket rack assembly was used in the study. To test for disintegration time, one tablet was placed in each tube and the basket rack is positioned in a 1L beaker containing buffer solution at 370C, such that the tablets remain 2.5 cm below the surface of the media on their upward movement and descent not closer than 2.5 cm from the bottom of the beaker. A standard motor driven device is settled to move the basket assembly containing tablets up and down through a distance of 5.3 to 5.7 cm at a frequency of 29 t o 32 cycles per min. The disintegration time of each tablet was determined and the average disintegration time was calculated. In vitro Dissolution Rate Studies The dissolution studies were carried out according to the USP paddle method 5. The stirring rate was 50 rpm at 37±0.50 C. the dissolution medium was 900 ml buffer solution. The samples were withdrawn at ten minute

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f1 = {[St = 1n (Rt – Tt)] / [St = 1n. Rt]}. 100 f2 = 50. Log {[1 + (1/n). St = 1n (Rt – Tt) 2]-0.5. 100} Where, n is the number of dissolution sample times, and Rt and Tt are the mean percent dissolved at each time point t for the reference and test dissolution profiles respectively.

intervals up to 30 minutes and assayed spectrophotometrically at 230 nm. The percentage of cumulative drug release of each tablet was determined using the linear regression equation of the calibration curve. Comparison of Dissolution Profiles As model-independent approaches, here, two fit factors (f1 and f2) that compare the dissolution profile of a pair of drug products were applied to the dissolution data. Values of f1 between 0 and 15, and values of f2 between 50 and 100 are used to define equivalence of two dissolution profiles 6 .

RESULTS AND DISCUSSION Calibration Curve The calibration curve (Figure 1) of cetirizine HCl was obtained by using the method which was explained in ‘analytical method’ section.

0.4 0.364 0.344

0.35

Absorbance

0.3

y = 36.18x + 0.010 R² = 0.998

0.301 0.263 0.235

0.25 0.2

0.19 0.156 0.118 0.079 0.049

0.15 0.1 0.05

Abs Linear (Abs)

0 0

0.002 0.004 0.006 0.008

0.01

0.012

Concentration

Figure 1: Calibration curve for cetirizine HCl test. If the tablet is too hard, it may not disintegrate in the required period of time to meet the dissolution specification. Conversely, hardness should not be so low that tablets are soft and friable. For a satisfactory tablet quality, hardness should be between 4 and 8 kg 7. Results of the brand products (Table 2) for hardness test were satisfactory.

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Weight Variation According to USP, the weight variation range for each cetirizine tablet is ±5% (w/w), and the weights from different brand products showed in Table 1, were within the acceptance limit. Hardness Test Tablet hardness is defined as the force required for breaking a tablet in a diametric compression

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Table 1: Weight variation Measurement (n = 10) % Relative Brand products Average weight Standard Standard deviation of Cetirizine (mg) Deviation (SD) (RSD) CTZ 1 195.39 0.016 0.003 CTZ 2 129.40 0.004 0.003 CTZ 3 178.47 0.009 0.005 CTZ 4 195.29 0.004 0.002 CTZ 5 135.59 0.014 0.010 CTZ 6 103.49 0.015 0.014 CTZ 7 194.38 0.005 0.003 CTZ 8 200.10 0.011 0.005 CTZ 9 83.96 0.003 0.004

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Table 2: Results of hardness, friability, content uniformity, disintegration time, and dissolution tests Average Drug Hardness Average Brand products Content release (%) (kg-ft) Friability (%) Disintegration of Cetirizine Uniformity (%) after 30 (mean ± SD) time (min) minutes CTZ 1 7.25 ± 0.39 0.56 95 4.86 102.39 CTZ 2 4.09 ± 1.14 0.78 97 0.38 101.29 CTZ 3 4.77 ± 1.32 1.00 102 3.54 91.95 CTZ 4 4.65 ± 1.44 0.78 99 1.77 106.63 CTZ 5 4.20 ± 1.99 0.51 103 10.96 80.88 CTZ 6 4.88 ± 1.35 0.58 99 8.39 90.70 CTZ 7 7.82 ± 0.35 0.89 95 2.88 90.56 CTZ 8 4.85 ± 1.29 0.95 109 19.04 80.70 CTZ 9 6.41 ± 1.09 0.71 105 8.65 93.66

Friability Test For satisfactory tablet, the friability value must be less than 0.5 – 1 % 8. From Table 2, it is shown that all brands were within the friability limit. Content Uniformity According to USP 36 5, contents of cetirizine tablets must be not lower than 90.0 % and not more than 110.0 % of the labeled amount of active drug. The results (Table 2) show that, all the brand products meet the criteria. Disintegration time (DT) test It has been established that no correlation can be established between disintegration and dissolution. Disintegration is used as a guide to the formulator in the preparation of a

satisfactory tablet formula and as an in-process control test. So, to ensure lot-to-lot product uniformity, DT test is very important. According to USP, it is said that the disintegration time must not more than (NMT) 30 minutes. The results, shown in Table 2 for the brand products, meet the criteria. Dissolution Rate Studies Oral bioavailability of a drug fully depends on the dissolution rate of the drug. So, it is very important to evaluate the dissolution data and comparison of dissolution profiles for different available market products. Table 2 shows the average percentage of drug release after an hour. Difference (f1) and similarity (f2) tests were

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applied to the release rate, and are shown in Table 3. Table 3: f1 (Difference) and f2 (similarity) factors for reference (CTZ 4) vs. test products (CTZ 1, 2, 3, 5, 6, 7, 8 and 9) Factors f1 f2

CTZ 1 13.64 89.19

CTZ 2 14.23 87.90

CTZ 3 35.14 47.94

CTZ 5 37.59 39.74

According to USP, the dissolution rate for cetirizine tablets should not be less than 80 % within 30 minutes, and the brand products meet the requirement (Table 2). Table 2 shows that CTZ 4 released the maximum amount of drug (106.63 %), whereas CTZ 8 released the lowest amount of drug after 30 min. So, CTZ 4 was considered as the reference for maximum drug release. Table 3 indicates that the dissolution profile of CTZ 1, CTZ 2 and CTZ 9 are similar to the profile of reference (CTZ 4), whereas the profiles of CTZ 3, 5, 6, 7 and 8 are not similar to that of the reference.

CTZ 6 42.79 47.79

353 Page

The authors are thankful to the Globe Pharmaceuticals Ltd., Bangladesh for giving the

CTZ 9 14.98 87.94

REFERENCES 1.

ACKNOWLEDGEMENT

CTZ 8 35.23 38.91

standard sample of cetirizine. The authors are also thankful to the Department of Pharmacy, Noakhali Science and Technology University for providing laboratory facilities.

CONCLUSION The investigation shows that the marketed cetirizine HCl tablets manufactured by the Bangladeshi pharmaceutical companies are of satisfactory quality. They met the USP standards in all aspects. Though, the results indicate the differences for release profiles, all the brand products released 80% of drug labeled amount according to USP, so they can satisfy patient needs. In fine, further investigation is suggested to establish in vivo-in vitro correlation to reveal the accurate pattern of drug release in vivo environment from marketed cetirizine formulations.

CTZ 7 44.54 49.19

Luthra, V. Quality definition with objectives and manufacturing retrieved from http://www.businessdictionary.com/definition/quality. html. 01-02, (2007). Levi, L., Walker, G. Quality Controls of pharmaceuticals. The Canadian Medical Association, Le journal de L association medicale canadienne. 91 (15), (2010). Shargel, L., Wu-Pong, S., Yu, A. B. C. Bioavailability and Bioequivalence. In: Applied Biopharmaceutics and Pharmacokinetics, 5th ed. McGraw-Hill: New York, pp 453- 499, (2004). The United States Pharmacopeia and National Formulary USP30- NF25; the United States Pharmacopeial Convention, Inc., (2007). The United States Pharmacopeia and National Formulary USP36- NF31; the United States Pharmacopeial Convention, Inc., (2012). http://www.usp.org/usp-nf/official-text/revisionbulletins/cetirizine-hydrochloride-tablets. Prior, A., Frutos, P., Correa, C. P. Comparison of dissolution profiles: current guidelines. Decaccia. 507509. Parrot, E. L., Saski, W. Solid Pharmaceuticals. In: rd Experimental Pharmaceutical Technology, 3 ed.; Burgess Publishing Co.: Minneapolis, MN, pp 58- 106, (1971). Moore, J. W.; Flanner, H. H. Mathematical comparison of dissolution profiles. Pharm Tech. 20 (6): 64- 70, (1996).

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Department of Pharmacy Noakhali Science and Technology University Sonapur, Noakhali- 3814 Bangladesh E-mail: pharmasud@operamail.com Contact number: +8801716026148

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Research Article Biological Sciences INFLUENCE OF STRESS ON OBESITY Pallavi Chitnis*, Neelam Rane, Vinu Vij, Vijay kumar Gupta Department of Physiology, Padmashree Dr D Y Patil Medical College and Hospital, Nerul, Navi Mumbai, India. *Corresponding Author Email: pallavichtns@gmail.com

ABSTRACT Background - Obesity is fast assuming epidemic proportions throughout the world in both children and adults. This study has been done to analyze the correlation between stress and obesity. Stress is one of the contributing factors for obesity, as stressful conditions often lead to irregular diet, irregular sleep, addiction and lack of exercise. Aim â&#x20AC;&#x201C; The present study was done to study the prevalence of stress in obese subjects. Materials and Methods: A total of st 171 female students of 1 year medical and paramedical sciences, age group of 17-21 years were included in the study. According to BMI, the study population was divided into four groups - underweight, normal, overweight and obese. Perception of stress level was measured by the Perceived Stress Scale (PSS) which is the most widely used scale. Result: Perceived Stress Scale score was higher in the obese subjects as compared to the normal subjects, which was found to be statistically significant. Perceived Stress Scale score was lower in the underweight subjects as compared to the normal subjects which was statistically insignificant. Conclusion: Stress is one of the contributing factors for obesity. Effective counseling for management of stress helps to reduce obesity and its related complications. Longitudinal studies are needed, however, to support inclusion of stress management or mindfulness techniques in obesity prevention efforts.

KEY WORDS BMI, Overweight, Obese, Perceived stress scale (PSS), Stress

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INTRODUCTION The high prevalence of obesity is a major public health problem because of the association of obesity with chronic health conditions such as coronary heart disease, type 2 diabetes, and some cancers [1]. Stress occurs when environmental demands tax or exceed the adaptive capacity of an organism; the demands result in physiological or psychological processes that put the organism at risk for disease [2]. Adults experience numerous types of stress (eg, work, finances, family), and each type contributes to overall stress. Perceived stress is associated with direct changes to both physiological (eg, hormonal response) and psychological processes. Chronically elevated

levels of perceived stress affect cortisol levels, which have been associated with increased risk for central obesity.[3] Chronic stress is assumed to have role in the development of obesity by interacting with mechanisms underlying energy intake and expenditure, and stimulating visceral fat accumulation in favour of abdominal obesity.[4] Stress is one of the contributing factors for obesity, as stressful conditions often lead to irregular diet, irregular sleep, addiction and lack of exercise. One important variable is how individuals use food to cope with stress and emotions. Eating has been recognized as a coping mechanism for alleviating and dealing with stress and emotions [5-8] by either undereating or over-eating [9].

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www.ijpbs.com (or) www.ijpbsonline.com Medical education is stressful throughout the whole course of training. The amount of material to be absorbed, social isolation, pressure of examination, discrepancies between expectation and reality all can be anticipated to bring psychological stress. [10] It is observed that people cope up with negative emotions generated by stressful events by engaging in emotional eating [11]. Emotional eating involves eating without hunger and usually without any planning. Thus, one is liable to eat unhealthy foods. Adolescents report that stress is associated with a shift towards unhealthy eating practices [12].

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MATERIAL AND METHODS A cross-sectional study was conducted on a total of 171 female students of 1st year medical and paramedical sciences, belonging to age group of 17-21 years. A signed consent was obtained from all the students. Stress was taken as an independent variable and overweight /obesity as the dependent variable. Perceived Stress Scale (5 point) accepted worldwide for assessment of Stress was given in the form of Clinical Questionnaire to the subjects. The subjects filled the questionnaire. The 10-item scale assesses feelings and thoughts during the last month. The PSS measures the degree to which situations in one’s life are perceived as stressful. It is more strongly related to life event impact scores as opposed to the number of stressful events, thereby representing one’s appraisal of the events as being stressful. The subjects were asked to rate their feelings on a scale of 0–4, with 0 indicating never and 4 indicating very often. The PSS scores range from 0 to 40, with higher scores indicating higher levels of stress. [13] BMI was categorized into four types: Underweight (<18.5), Normal (18.5- 24.9), overweight (25-29.9) and Obese (>30). [14, 15]

Statistical Analysis The results were expressed as mean ± SD of each variable. The comparison between the mean values was performed by unpaired t test using graph pad software online. P-value of 0.05 or less was interpreted as significant for the analysis.

RESULTS Out of 171 students who filled the perceived stress test questionnaire, 12.87% were underweight, 54.39% were normal weight, 23.4% were overweight and 9.36% were obese. Table-I Distribution of Subjects: BMI No. of Subjects <18.5 22 (Underweight) 18.5-24.9 93 (Normal) 25-29.9 40 (Overweight) >30 16 (Obese) The mean ± SD PSS scores of underweight, normal, overweight and obese subjects were 15.045 ± 5.65, 16.989 ± 5.5, 18.02±6.01 and 17.75 ±7.41 respectively. The maximum PSS scores were higher for overweight and obese subjects (34 and 36 respectively) as compared to the normal weight subjects i.e. 29. Comparison of PSS scores of obese subjects with normal weight subjects showed a p-value <0.001, which was statistically significant. In underweight subjects, the mean ± SD PSS score was lower as compared to the normal weight subjects, but it was statistically insignificant.

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www.ijpbs.com (or) www.ijpbsonline.com BMI Underweight (<18.5) Normal (18.5 â&#x20AC;&#x201C; 24.9) Overweight (25 â&#x20AC;&#x201C; 29.9) Obese (> 30)

Mean 15.045

Table-II Correlation of PSS with BMI SD SEM Minimum 5.65 1.205 2

Maximum 22

p-Value >0.1

16.989

5.5

0.57

>0.1

18.02

6.01

0.949

>0.1

17.75

7.41

1.852

<0.001*

*p-value <o.o5 significant

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DISCUSSION In the present study, we have evaluated the stress level using PSS scores in 171 female students of 1st year medical and paramedical sciences. This study reveals high prevalence of stress in obese subjects as compared to the normal weight subjects. People characterized as stress- or emotionaleaters tend to choose calorically dense foods to blunt their stress response or reduce negative emotions [3]. The relationship between restrained eating and stress is more complex; restrained eaters tend to eat less during normal conditions and overeat when stressed. [3] It has been widely reported that psychological stress and food consumption are related. [16, 17, 18] Emotional eating has often been examined as a possible risk factor for obesity. [18, 19] In some people stress can induce overeating and in others, overeating can induce stress. The results of studies on stress and eating are mixed [20]. The consumption of comfort foods may lead to a more positive dispositional state for several reasons including sensory pleasure, reduction of hunger, and the diminution of aversive physiological symptoms. [21] Stress may contribute to HPA axis dysregulation, tendency to overeat, which in turn contributes to a cascade starting with obesity and ending with type 2 diabetes and CVD. [22]

Eating in response to emotions has been found to be a predictive variable for long-term change [23]. Blair, Lewis, and Booth [24] found that individuals who decreased their emotional eating lost substantially more weight than those who did not decrease their emotional eating. In addition, individuals who are overweight exhibit more compromised coping skills with greater severity of binge eating [25-27]. Geliebter and Aversa found that overweight [28] individuals have substantially greater eating ratings, indicating a greater urge to eat, in response to negative emotions and negative situations than normal weight individuals. Prolonged sleep deprivation increases both food intake and energy consumption, as also a hormone imbalance is observed, leading to weight gain. [29] Bjornto rp and Rosmond describe the neuroendocrine abnormalities associated with visceral obesity and report decreased cortisol variability in relation to increased abdominal obesity. [30, 31] A key component of obesity is intra-abdominal accumulation of fat, which is responsible for a great portion of the increased CVD risk associated with obesity. [32]

CONCLUSION The present study shows that the stress levels are higher in obese subjects as compared to the

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www.ijpbs.com (or) www.ijpbsonline.com normal weight subjects. As obesity grows as a public health problem the challenge for health researchers and professionals is to develop more effective and innovative strategies for managing psychological stress which reduce stress induced eating. Effective stress management has been identified as an important factor in successful weight loss maintenance through increasing coping capacity [33, 34, 35]. The management of stress can be one of the modifiable risk factors among obesity and related disorders. Early identification of stress and aggressive management strategies are necessary to delay the onset and progression of obesity related complications.

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REFERENCES [1] NIH, NHLBI Obesity Education Initiative. Clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults. Accessed December 14, 2011. [2] Cohen S, Janicki-Deverts D, Miller GE. Psychological stress and disease. JAMA 2007;298(14):1685-7. [3] Adam TC, Epel ES. Stress, eating and the reward system. Physiol Behav 2007;91(4):449-58. [4] De Vriendt T, Moreno LA, De Henauw S: Chronic stress and obesity in adolescents: scientific evidence and methodological issues for epidemiological research. Nutr Metab Cardiovasc Dis 2009, 19: 511-519. [5] National Institutes of Health. National Heart, Lung, and Blood Institute North American Association for the Study of Obesity. The practical guide: Identification, evaluation & evaluation and treatment of overweight and obesity in adults. NIH Publication Number 004084; 2000 [6] Popkess-Vawter S, Wendel S, Schmoll S, O’Connell K. Overeating, reversal theory and weight cycling. West J Nurs Res. 1998;20:67-83. [7] Solomon MR. Eating as both coping and stressor in overweight control. J Adv Nurs. 2001;36:563-573. [8] Timmerman G, Acton GJ. The relationship between basic need satisfaction and emotional eating. Issues Ment Health Nurs. 2001; 22:691-701. [9] Geliebter A, Aversa A. Emotional eating in overweight, normal weight and underweight individuals. Eat Behav. 2003; 3:341-347. [10]Cohen S, Williamson GM. Perceived stress in a probability sample of the United States. In: Spacapan S,

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|355-359 Oskamp S, eds. The social psychology of health. Beverly Hills, CA: Sage Publications, 1998:31–67. [11] M., Simons G. Emotions and eating in everyday life. Appetite.2000; 35(1):65-71. [12] Cartwright M, Wardle J, Steggles N, Simon CE, Croker H. Stress and dietary practices in adolescents. Health Psycol. 2003; 22(4):362-369. [13] Cohen S, Kamarck T, Mermelstein R. A global measure of perceived stress. J Health Soc Behav 1983;24(4):385–96. [14]Wellen KE, Hotamisligil GS. Obesity-induced inflammatory changes in adipose tissue. The Journal of clinical investigation 2003; 112(12):1785- 8. [15]Clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults: Executive summary. Expert panel on the identification, evaluation, and treatment of overweight in adults. Am J Clin Nutr 1998; 68(4):899–917. [16]Lattimore PJ. Stress Induced Eating: An alternative methods for inducing ego threatening stress. Appetite: 2001; 36(2):187-188. [17] Oliver G., Wardle .J. Perceived Effects Of stress on food choice.PhysiolBehav.1999;66(3):511-515. [18] Greeno CG, Wing RR.Stress induced eating.Pyschol Bull.1994; 115(3):444-464. [19]Faith MS, Allision DB, Geliebter A. Emotional eating and obesity. Theoretical consideration and practical recommendation. In: Datton S, editor. Overweight and weight management. The Health Professional Guide to Understanding and Practice. Gaitherburg Ma. Aspen Publishers, 1997; 739-765. [20] Stone, A.A. & Brownell, K. (1994). The stress-eating paradox: multiple daily measurement in adult males and females. Psychological Health, 9, 425-436. [21]Gibson EL. Emotional influences on food choice: Sensory, physiological and psychological pathways.Physiol Behav.2006;89(1);53-61. [22] McEwen BS. Protective and damaging effects of stress mediators. N Engl J Med 998;338(3):171–9. [23]Lavery MA, Lowey JW. Identifying predictive variables for long-term weight change after participation in a weight loss program. J Am Diet Assoc. 1993;93;10171024. [24] Blair AJ, Lewis VJ, Booth DA. Does emotional eating interfere with success in attempts at weight control? J Clin Psychol. 1990;15:151- 157. [25] Crowther JH, Sanftner J, Bonifazi DZ, Shepherd KL. The role of daily hassles in binge eating. Int J Eat Disord. 2001; 29:449-454. [26] Agras WS, Telch CF. the effects of caloric deprivation and negative affect on binge-eating in obese being-

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www.ijpbs.com (or) www.ijpbsonline.com eating disordered women. Behav Ther. 1998; 2:491503. [27] Brownell KD, Wadden TA. Etiology and treatment of obesity: Understanding a serious, prevalent and refractory disorder. J Clin Psychol. 1992; 60:505-517. [28] Geliebter A, Aversa A. Emotional eating in overweight, normal weight and underweight individuals. Eat Behav. 2003;3:341-347. [29] Taheri S, Lin L, Austin D, Young T, Mignot E. Short sleep duration is associated with reduced leptin, elevated ghrelin and increased body mass index. 2004; LoSMed1 (3):e62.doi.10.1371/journal.pmed.0010062.Assesed on 1 September 2009 [30] Dalan MF, la Fleur SE, Pecoraro NC, Gomez F, Houshyar H, Akana SF. Minireview: Glucocorticoids—Food intake, abdominal obesity, Global Journal of Medical Research 46 © 2011 Global Journals Inc. (US) Clinical Significance of Perceived Occupational Stress Influencing Body Mass and Osteopenia: A Pilot Study and wealthy nations in 2004. Endocrinologlmy 2004; 145(6):2633–8

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|355-359 [31] Brunner EJ, Chandola T, Marmot MG. Prospective effect of job strain on general and central obesity in the Whitehall II Study. Am J Epidemiol 2007;165(7):828–37. [32] Janssen I, Heymsfield SB, Allison DB, Kotler DP, Ross R. Body mass index and waist circumference independently contribute to the prediction of nonabdominal, abdominal subcutaneous and visceral fat. Am J Clin Nutr 2002;75(4):683–8. [33] Elfhag, K., & Rossner, S. (2005). Who succeeds in maintaining weight loss? A conceptual review of factors associated with weight loss maintenance and weight regain. Obesity Reviews, 6, 67–85. [34] Foreyt, J. P., & Poston, W. S., 2nd. (1998). The role of the behavioral counselor in obesity treatment. Journal of the American Dietetic Association, 98(10 Suppl 2), S27–S30. [35] Senekal, M., Albertse, E. C., Momberg, D. J., Groenewald, C. J., & Visser, E. M. (1999). A multidimensional weight-management program for women. Journal of the American Dietetic Association, 99, 1257–1264.

*Corresponding Author: Pallavi Chitnis*

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Department of Physiology, Pad. Dr D Y Patil Medical College and Hospital, Nerul, Navi Mumbai, India. Email id – pallavichtns@gmail.com Telephone No. 9869480450.

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Research Article Biological Sciences ANTI-MALARIA AND HEMATOLOGICAL ANALYSES OF ETHANOL LEAF EXTRACT OF MORINGA OLEIFERA ON MALARIA INFECTED MICE 1

UGWU,Okechukwu P.C.*; 1NWODO, Okwesili F.C; 1JOSHUA, Parker E. , 1ODO, Christian E., 2 BAWA, Abubakar , 1OSSAI Emmanuel C. and 3ADONU Cyril C. 1

Department of Biochemistry, University of Nigeria,Nsukka, Enugu State,Nigeria. Department of Preliminary and Remedial Studies, Federal Polytechnic Mubi, Adamawa State, Nigeria. 3 Department of Pharmaceutics, University of Nigeria,Nsukka, Enugu State, Nigeria. * Corresponding Author Email and Contact: oky9992000@yahoo.com and +2348030799187

ABSTRACT Percentage parasitaemia and haematological parameters of ethanol leaf extract of Moringa oleifera were evaluated in a twenty four mice consisting of six groups. Groups 1 (positive control) and 6 (negative control) were treated with 5mg/kg body weight of distilled water, group 5 (standard control) was treated with 5mg/kg body weight of artesunate while groups 2, 3 and 4 were treated with 45, 90 and 180 mg/kg body weight of Moringa oleifera ethanol leaf extract. The results showed that percentage parasitaemia of the mice treated with ethanol leaf extract of moringa oleifera increased significantly (p<0.05) in group 1 (positive control) when compared to group 6 (negative control) and other groups. Group 4 (180 mg/kg body weight of the extract), group 5 (5 mg/kg body weight of Artesunate) showed no significant difference (p>0.05) in percentage parasitaemia compared to group 6 (negative control). The haematological parameters of packed cell volume (PCV), haemoglobin concentration of the cell (Hb) and total red blood cell counts (TRBC) increased significantly (p<0.05) in groups 4 (180 mg/kg body weight of the extract), group 5 (5 mg/kg body weight of Artesunate) and group 6 (negative control) compared to group 1 (positive control) while the haematological parameter of total white blood cell (TWBC) increased significantly (p<0.05) in groups 3 (90 mg/kg body weight of the extract) and group 6 (negative control) compared to group 1 (positive control).

KEY WORDS Moringa oleifera, percentage parasitaemia ,haematological parameters.

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INTRODUCTION Malaria is a mosquito-borne disease of humans caused by eukaryotic protists of the genus Plasmodium. It is transmitted from one human to another by a bite of an infected female anopheles mosquito. It is widespread in tropical and sub-tropical regions, including much of SubSahara Africa, Asia and the Americas (Clark and Cowden, 2003). Plasmodium species are generally host specific and vector specific in that each species will only infect a limited range of

hosts and vectors. Four species of plasmodium can infect and be transmitted by humans. They are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. Malaria caused by Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae is generally milder and rarely fatal. The fifth species, Plasmodium knowlesi is a zoonosis that causes malaria in Macaques but can also infect humans.Severe disease results largely from Plasmodium falciparum.

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www.ijpbs.com (or) www.ijpbsonline.com In humans, the parasites called sporozoites travel to the liver, where they mature and release another form, the merozoites. These enter the bloodstream and infect the red blood cells. The parasites multiply inside the red blood cells, which then ruptures after 48 to 78 hours, infecting more red blood cells (Trampuz et al., 2003). The first symptoms usually occur 10 days to 4 weeks after infection, though they can appear as early as 8 days or as long as a year after infection. The symptoms occur in cycles of 48 to 72 hours. The majority of symptoms are caused by the massive release of merozoites into the bloodstream, the anaemia resulting from the destruction of the red blood cells and the problems caused by large amount of free hemoglobin released into circulation after red blood cells rupture. Malaria can also be transmitted from a mother to her unborn baby (congenitally) and through blood transmission (Clark and Cowden, 2003). Malaria is transmitted by mosquitoes in temperate climates, but the parasites disappear over the winter. The disease is a major health problem in most of the tropics and sub-tropics (Clark and Cowden, 2003). WHO (2005) estimates that there are three hundred to five hundred million cases of malaria each year and more than one million people die. It presents a major health hazard for travelers to warm climates. In some areas of the world, mosquitoes that transmit malaria have developed resistance to insecticides. In addition, the parasites have developed resistance to some antibiotics. This has led to difficulties in controlling both the rate of infection and the spread of the disease. Symptoms of malaria include flu-like illness with fever, chills, muscle aches and headache. Some patients develop nausea, vomiting, cough and diarrhoea. Cycles of chills, fever and sweating that repeat every one, two or three days are

typical. There can be sometimes vomiting, diarrhoea, coughing and yellowing (jaundice) of the skin and whitening of the eyes due to destruction of red blood and liver cells (Mueller et al., 2007). People with severe Plasmodium falciparum malaria can develop bleeding problems, shocks, liver and kidney failure, central nervous system problems and they can die from infection or its complications. Celebral malaria (coma, altered mental status or seizures) can occur with severe Plasmodium falciparum infection. It can be lethal if not treated quickly. Even with treatment, about 15 -20% die (Adams et al., 2002). The classic symptoms of malaria is cyclical occurrence of sudden coldness followed by rigour, then fever and sweating lasting four to six hours, occurring every two days in Plasmodium vivax and Plasmodium ovale infections, while every three days for Plasmodium malariae. Malaria due to Plasmodium falciparum can give recurrent fever every 36 â&#x20AC;&#x201C; 48 hours or a less pronounced and almost continous fever. For reasons that are poorly understood, but that may be related to high intracranial pressure, children with malaria frequently exhibit abnormal posturing, a sign indicating severe brain damage (Idro et al., 2005). Malaria has been also found to cause cognitive impairments, especially in children. It causes widespread anaemia during a period of rapid brain development and also direct brain damage. The neurologic damage results from celebral malaria in which children are more vulnerable (Trampuz et al., 2003). Celebral Malaria is associated with retinal whitening, which may be a useful clinical sign in distinguishing between malaria and other causes of fever (Trampuz, et al., 2003). Severe malaria is almost exclusively caused by Plasmodium falciparum infection and usually arises 6 â&#x20AC;&#x201C; 14 days after infection.

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www.ijpbs.com (or) www.ijpbsonline.com Consequences of severe malaria include coma and death if untreated. Young children and pregnant women are more vulnerable. Splenomegaly (enlarged spleen), severe headache, celebral ischemia, hepatomegally (enlarged liver), hypoglycemia and hemoglobinuria with renal failure may occur. Renal failure is a feature of blackwater fever, where hemoglobin from lysed red blood cells leak into the urine. Severe malaria can progress extremely rapidly and cause death within hours or days (Makintosh et al., 2004). In most severe cases of the disease, fatality rate can exceed 20% even with intensive care and treatment (Makintosh et al., 2004). In endemic areas, treatment is often less satisfactory and the overall fatality rate for all cases of malaria can be as high as one in ten (Trampuz et al., 2003).The long term developmental impairments have been documented in children who have suffered episodes of severe malaria. Malaria has been and is still the cause of major human morbidity and mortality (Clark and Cowden, 2003). It is the most important parasitic disease worldwide with an incidence of almost three hundred million clinical cases and over one million deaths yearly (WHO, 2000). Malaria is directly responsible for one in five childhood deaths in Africa and indirectly contributes to illnesses and deaths from other diseases (WHO, 1999). Pregnant women and children under five years of age are the most vulnerable. In the absence of an effective vaccine, the fight against malaria depends on chemotherapy, the reduction and prevention of anopheles mosquito contacts with human (Winstainley, 2000). The loss in effectiveness of chemotherapy due to the emergence of resistant strains constitutes the greatest threat to the control of malaria. Therefore, to overcome malaria, new knowledge, products, and tools especially new drugs are urgently needed (Omulokoli et al.,

1997). Traditional methods of treatment and control of malaria could be a promising source of potential anti-malaria drugs. (Wright and Phillipson, 1990; Venkat et al., 2012; and Sumalata and Sreedevi, 2012) Moringa oleifera was massively grown and promoted by the local media in Uganda in the 1980s as a plant which is capable of curing a number of diseases ,including malaria, and relieving some symptoms of HIV/AIDS. Moringa oleifera is referred to as a MIRACLE TREE (Fuglie, 2001). This is due to its socio-economic, nutritional, pharmacological and industrial benefits (Makkar and Becker, 2007). As a result of the impact of malaria on the human race and claimed effectiveness of Moringa oleifera in curing diseases such as diabetes,typhoid and high blood pressure, it was considered necessary to investigate the anti-malarial effect of Moringa oleifera. This study was designed to determine the percentage parasitaemia and haematological parameters of ethanol leaf extract of Moringa oleifera.

MATERIALS AND METHODS Plant material Fresh leaves of Moringa oleifera were obtained from Ovoko, Igbo-Eze South L.G.A of Enugu State, Nigeria.The leaves were identified by Mr. O. Chijioke of the Hebarium unit of the Department of Botany, University of Nigeria, Nsukka. Animals The experimental animals used for this study were white albino mice of either sex weighing 20-34g.The mice were between 3-4 months old and were obtained from the animal unit of Faculty of Veterinary Medicine, University of Nigeria, Nsukka.

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www.ijpbs.com (or) www.ijpbsonline.com Chemicals/Reagents All chemicals used in this study were of analytical grade and products of May and Baker, England; BDH, England and Merck, Darmstand, Germany. Reagents used for the assays were products of Radox commercial kits. Extraction Procedure The fresh leaves of Moringa oleifera plant were plucked and dried under room temperature at (29ᵒC-35ᵒC) for three weeks, after which the leaves were pulverized into coarse form with a crestor high speed milling machine. The coarse form (130g) was then macerated in absolute ethanol. This was left to stand for 48 hours. After that the extract was filtered through muslin cloth on a plug of glass wool in a glass column.The resulting ethanol extract was concentrated and evaporated to dryness using rotary evaporator at an optimum temperature of between 40 and 45ᵒC to avoid denaturation of the active ingredients. The concentrated extract was stored in the refrigerator.

EXPERIMENTAL DESIGN Twenty-four white albino mice of either sex weighing 20 – 34kg were housed in separate cages, acclimatized for one week and then divided into six groups of four mice each. The route of administration (treatment) was via oral with the aid of an oral intubation tube. Group 1 was the (positive control) inoculated with malaria parasite (Mp+) and treated with 5mg/kg body weight of distilled water.

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Percentage (%) yield =

Group II was inoculated with malaria parasite and treated with 45mg/kg body weight of Moringa oleifera ethanol leaf extract. Group III was also inoculated with malaria parasite and treated with 90mg/kg body weight of Moringa oleifera ethanol leaf extract. Group IV was inoculated with malaria parasite and treated with 180mg/kg body weight of Moringa oleifera ethanol leaf extract. Group V which was also inoculated with malaria parasite (standard control) and was treated with 5mg/kg body weight of artesunate (standard drug). Group VI was the negative control which was not inoculated with malaria parasite and was finally treated with 5mg/kg body weight of distilled water. Before the treatments, the mice in Groups I – V were inoculated with malaria parasite and 3 days after that analyses were carried out to determine the baseline parameter in all the groups, then, two days later, treatment began. The treatment lasted for 5 days during which analyses were done on day 3, day 5 of treatment and 28 days post treatment Determination of percentage yield of extract The percentage yield of the extract was determined by weighing the coarse Moringa oleifera leaf before extraction and the Moringa oleifera ethanol leaf extract after concentration and then calculated using the formula.

Weight (g) of concentrated extract 100 Weight (g) of ground Moringa leaves

Determination of percentage parasiteamia The determination of malaria parasitemia (Mp+) was carried out according to the Method of Dacie and Lewis (2000).

Determination of haematological parameters The determination of haematological parameters of total red blood cell count, total white blood cell count, packed cell volume and haemoglobin

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concentration were carried out according to the method of Dacie and Lewis (2000).

RESULTS Percentage yield of the extract Table 1: The percentage yield of the ethanol leaf extract of Moringa oleifera Initial weight of ground extract (g) Final weight of Extract (g) Percentage (%) yield of extract 130

23.20

17.85

From the result in Table 1 the (%) yield of the ethanol leaf extract of Moringa oleifera was found to be 17.85%.

Effect of Ethanol Leaf Extract of Moringa oleifera on Percentage Parasitaemia 9

Mean Parasitaemia (%)

3 Days of Inoculation Day 3 of Treatment Day 5 Treatment Day 28 of Post-Treatment

0 Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Group Fig. 1: Effect of ethanol leaf extract Moringa oleifera on percentage parasitaemia in mice

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Group 1= Positive Control Group 2= 45mg/kg b.w of Moringa oleifera Group 3=90mg/kg b.w of Moringa oleifera

Group 4=180mg/kg b.w of Moringa oleifera Group 5=5mg/kg b.w of Artesunate Group 6=Negative Control

Fig 1:shows that 3 days of inoculation mean values for Percentage Parasitaemia of mice in groups 4 and 5 significantly decreased (p<0.05) compared to the values of mice in groups 1 (positive control), 2 and 3. On day 3 of treatment the mean values for Percentage Parasitaemia in all the groups significantly decreased (p<0.05) compared to the mean percentage parasitaemia of mice in groups 1 (positive control) and 2. Also, on day 5 of treatment the mean percentage parasitaemia in all the groups significantly decreased (p<0.05) compared to the values for the percentage parasitaemia of mice in group 1 (positive control). Hence,on day 28 of post treatment the mean values of the percentage parasitaemia significantly decreased in all the groups compared to the mean percentage parasitaemia of group 1 (positive control). Finally on day 28 of post treatment also showed significant (p<0.05) clearance of the parasitaemia in groups 4 and 5 compared to the mean values of the percentage parasitaemia in groups 1 (positive control), 2 and 3 animals.

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Effect of Ethanol Leaf Extract of Moringa oleifera on Haemoglobin Concentration 16 Day 3 After Inoculation Day 5 of Treatment Day 28 of Post-Treatment

Mean Hb Conc. (g/dl)

0 Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Group Fig. 2: Effect of ethanol leaf extract of Moringa oleifera on haemoglobin concentration concentration in mice

Group 1= Positive Control Group 2= 45mg/kg b.w of Moringa oleifera Group 3=90mg/kg b.w of Moringa oleifera

Group 4=180mg/kg b.w of Moringa oleifera Group 5=5mg/kg b.w of Artesunate Group 6=Negative Control

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Fig. 2: shows that 3 days after inoculation the mean values for heamoglobin in all the groups were essentially similar, while the value obtained for group 4 was significantly (p<0.05) lower than for mice in group 1 (positive control) . On day 5 of treatment the mean values for heamoglobin in groups 4, 5 and 6 significantly increased (p<0.05) compared to group 1 (positive control). Finally, on day 28 of post treatment the mean values for heamoglobin in groups 4, 5 and 6 (negative control) significantly increased (p<0.05) compared to group 1 (positive control).

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Effect of Ethanol Leaf Extract of Moringa oleifera on Total White Blood Cell Count in Mice 25

Day 3 After Inoculation Day 5 of Treatment

Mean Total WBC (109/L)

Day 28 of Post-Treatment

0 Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Group Fig. 3: Effect of ethanol leaf extract of Moringa oleifera on total white blood cell count in mice

Group 1= Positive Control Group 2= 45mg/kg b.w of Moringa oleifera Group 3=90mg/kg b.w of Moringa oleifera

Group 4=180mg/kg b.w of Moringa oleifera Group 5=5mg/kg b.w of Artesunate Group 6=Negative Control

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Fig.3: Shows the effect of ethanol leaf extract of Moringa oleifera on total white blood cell count. The TWBC (baseline) count obtained 3 days after inoculation for mice in groups 1, 2 ,3, 5 and 6 were essentially similar ,while the value obtained for mice in group 4 was significantly lower than that for mice in group 1. On day 5 after commencement of treatment, mean value for group 2 mice was significantly (p<0.05) lower than that of group 1 mice,while mean values for mice in groups 3 and 5 were significantly (p<0.05) higher than that of group 1 mice.There was no significant difference (p>0.05) between the mean values for mice in groups 4 and 6 when compared with that for mice in group 1. TWBC count on day 28 of treatment in group 3 mice was essentially similar to the value of TWBC in group 6 (negative control) mice,while the values obtained for mice in groups 2,3,4,5 and 6 were significantly (p<0.05) higher than that for group 1 (positive control) mice.

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Effect of Ethanol Leaf Extract of Moringa oleifera on Packed Cell Volume in Mice 50

Day 3 After Inoculation 45

Day 5 of Treatment Day 28 of Post-Treatment

Mean PCV (%)

0 Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Group Fig. 4: Effect of ethanol leaf extract of Moringa oleifera on packed cell volume in mice

Group 1= Positive Control Group 2= 45mg/kg b.w of Moringa oleifera Group 3=90mg/kg b.w of Moringa oleifera

Group 4=180mg/kg b.w of Moringa oleifera Group 5=5mg/kg b.w of Artesunate Group 6=Negative Control

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Fig.4: Shows that 3 days after inoculation mean values for PCV of mice in groups 2 ,3,5, and 6 were not significantly (p>0.05) different from the value obtained for mice in group 1 (positive control) ; but the value obtained for mice in group 4 was significantly (p<0.05) lower than that for mice in group 1 (positive control).On day 5 of treatment , mean values for PCV for groups 2 and 3 were essentially similar to that of animals in group 1. On the other hand,values obtained for mice in groups 4, 5 and 6 showed significant (p<0.05) increases above the value for animals in the group 1 (positive control). For day 28 post treatment,whereas the mean PCV values for groups 4 , 5 and 6 animals were significantly (p<0.05) higher than that of group 1 mice ,mean values for those in groups 2 and 3 showed no significant (p>0.05) difference when compared with the value for group 1 mice.Also , mean PCV values for mice in groups 4 ,5 and 6 were similar.

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Effect of Ethanol Leaf Extract of Moringa oleifera on Red Blood Cell Count in Mice 20

Mean RBC Count (x106)

Day 3 After Inoculation Day 5 of Treatment

Day 28 of Post-Treatment

0 Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Group Fig. 5: Effect of ethanol leaf extract of Moringa oleifera on red blood cell count in mice

Group 1= Positive Control Group 2= 45mg/kg b.w of Moringa oleifera Group 3=90mg/kg b.w of Moringa oleifera

Group 4=180mg/kg b.w of Moringa oleifera Group 5=5mg/kg b.w of Artesunate Group 6=Negative Control

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Fig. 5: Shows that mean RBC baseline obtained 3 days after inoculation for mice in groups 2 , 3 ,4, 5 and 6 (negative control ) mice were not significantly (p>0.05) different compared to the value in group 1 (positive control ) mice. On day 5 of treatment showed significant increase (p<0.05) in RBC count of groups 2, 3 ,4, 5 and 6 (negative control) mice compared to the mean value for RBC count of mice in group 1 (positive control). Also day 28 of post treatment, showed significant increase (p<0.05) in the mean RBC count of mice in groups 2,3,4,5 and 6 (negative control) mice when compared to the mean RBC count of group 1 (positive control) mice .But group 4 mice was essentially similar to group 6 (negative control) mice.

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DISCUSSION Malaria is a major public health problem and cause of much suffering and premature death in the poorer areas of the Tropical Africa, Asia and Latin America. Human beings are exposed to malaria through the bite of an infected female anopheles mosquito, blood transfusion and congenitally from mother to her child (Bruce, 1981). In many endemic areas, it is becoming difficult to control, because of the parasite resistance to antimalarial drugs and the failure of vector control measures. Due to resistance to some of the conventional drugs used for the treatment of malaria and the impact of malaria to world health, it is therefore necessary to search for new, cheap and easily available drug that will be used for the treatment of malaria (Dondorp, 2007). The medicinal uses of many plants like Moringa oleifera cannot be overemphasised. The choice of this plant for the research work was based on its numerous ethnomedicinal properties. The observation on the effect of ethanol leaf extract of Moringa oleifera on percentage parasitaemia in mice showing a significant (p<0.05) clearance of parasitaemia in group 4 (180mg/kg body weight of the extract) and group 5 (5mg/kg body weight of artesunate) when compared to group 1 (positive control) is consistent with the findings of Monzon (1995) in Phillipines, who administered Moringa oleifera leaf extract in mice that were infected with malaria and other parasitic diseases. The result showed that the extract might be effective against the parasites. The result also showed a significant increase (p<0.05) in parasitaemia in group 1 (positive control) treated with 5mg/kg distilled water which could lead to the destruction of the liver, blood cells, kidney and other vital organs in the mice (Trampuz et al., 2003). This could be as a result of the infection of the liver by the sporozoites and the resultant

multiplication of the merozoites in the blood cells. The result of the effect of ethanol leaf extract of Moringa oleifera on the haematological parameter of packed cell volume showed a non significant difference in packed cell volume (p>0.05) in group 4 (180 mg/kg body weight of the extract) and group 5 (5 mg/kg body weight of the artesunate) compared to group 6 (negative control). But, a significant reduction (p<0.05) in packed cell volume was observed in group 1 (positive control) when compared to group 6 (negative control). This showed that Moringa oleifera ethanol leaf extract has ameliorated the effect of malaria parasitaemia on the packed cell volume. This agrees with the work of Ambi et al., (2006) who showed that Moringa oleifera leaf extract boosted heamatological parameters of packed cell volume in rats. Packed cell volume is used to asses anaemia, erythrocytosis, haemodilution and haemoconcentration.A decrease in packed cell volume indicates anaemia (Dacie and Lewis, 2000). The result of the effect of ethanol leaf extract of Moringa oleifera on red blood cell count showed a non significant difference (p>0.05) in group 4 (180 mg/kg body weight of the extract) compared to group 6 (negative control).This, also corroborates with the work of Ambi et al. ,(2006) showing that Moringa oleifera leaf extract boost red blood cell counts in rats.. There was a significant increase (p<0.05) in red blood cell count in group 4 (180mg/kg body weight of the extract) when compared to group 1 (positive control) . A decrease in red blood cell could be as a result of anaemia (Dacie and Lewis,2000). Moringa oliefera ethanol leaf extract has probably repaired the damages caused by merozoites to the red blood cell in mice that were infected with malaria. The effect of ethanol leaf extract of Moringa oleifera on haemoglobin concentration in mice

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www.ijpbs.com (or) www.ijpbsonline.com showed a significant increase (p<0.05) in haemoglobin in group 4 (180 mg/kg body weight of the extract), group 5 (5mg/kg body weight of the artesunate) and group 6 (negative control) when compared to group 1 (positive control). But, group 4 (180mg/kg body weight of the extract) and group 5 (5 mg/kg body weight of the artesunate) showed no significant difference (p>0.05) in haemoglobin concentration when compared to group 6 (negative control). This corroborated with the work of Ambi et al., (2006) who showed that Moringa oleifera leaf extract boosted haemoglobin concentration in rat. A complete blood count is used to asses symptoms such as weakness, fatigue, anaemia, infection and other disorders. Haemoglobin molecule fills up the red blood cells. It transports oxygen and gives the blood cell its red colour. The higher the haemoglobin concentration, the higher its ability to transport oxygen throughout the body. The effect of ethanol leaf extract of Moringa oleifera on total white blood cell count showed a significant increase (p<0.05) in total white blood cell count in other groups when compared to group 1 (positive control) .The Moringa oleifera ethanol leaf extract increased the total white blood cell in group 2 (45 mg/kg body weight of the extract), group 3 (90 mg/kg body weight of the extract) and group 4 (180 mg/kg body weight of the extract) when compared to group 1 (positive control). This is also consistent with the work of Ambi et al., (2006) that showed the potency of Moringa oleifera leaf extract in increasing white blood cell counts in rat. This could be the reason for reduced parasitaemia in groups 2 (45mg/kg body weight of the extract) and group 3 (90 mg/kg body weight of the extract) and total clearance of the parasitaemia in group 4 (180 mg/kg body weight of the extract) and group 5 (5 mg/kg body weight of the artesunate).

CONCLUSION The results above have shown why ethanol leaf extract of Moringa oleifera have been used in numerous ethnomedicinal practises to combat malaria.

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Adams, S., Brown, H. and Turner, G. (2002). Breaking down the blood-brain barrier. Signaling a path to cerebral malaria. Trends in Parasistol, 18: 6-360. Ambi, A.A., Abdurahman, E.M. Ibrahim, N.D., Pateh, U.U. and Sule, I. (2006). Metal constituents and effect of Moringa oleifera leaf extract on some hematological parameters in rat. Journal of Pharmacy and Bioresources, 3: 102. Bruce, C.L. (1981). Alphonse Laveran’s discovery 100 years ago and today’s global fight against Malaria. J.R. Soc Med, 74: 531-600. Clark, I.A. and Cowden, W. B. (2003). The Pathophysiology of Falciparum malaria. Pharmacology and Therapeutics, 99: 221-260. Dacie, J.V. and Lewis, S.M. (2000). Practical Haematology. 9th Edition Churchill Livingstone. Dondorp, A.M (2007). The treatment of severe Malaria. Trans R Soc Trop Med Hyg, 101: 633-634. Fuglie, L. (2001). The miracle Tree. The multiple attributes of Moringa, Dakar. PP 25-30. Idro, R., Jenkins, N.E. and Newton, C.R.J.C. (2005). Pathogenesis, clinical features and neurological outcome of cerebral malaria. The Lancet Neurology, 7: 827-840. Mackintosh, C.L. Beeson, J.G. and Marsh, K. (2004). Clinical features and pathogenesis of severe malaria, Trends in Parasitology, 20: 597-603. Makkar, H. and Becker, K. (2007). Nutrients and antiquality factors in different morphological parts of the Moringa oleifera tree. Journal of Agricultural Science, 123: 311-322. Monzon, R.B. (1995). Traditional medicine in the treatment of Parasitic Disease in Philippines. South East Asian Journal of Tropical Medicine, 7: 4-6. Mueller, I., Zimmerman, P.A. and Reeder, J.C. (2007). Plasmodium malariae and Plasmodium ovale. The bashful malaria parasites. Trends in parasistol, 6: 278290. Omulokoli, E., Khan, B. and Chhabra, S.C. (1997). Antiplasmodial activity of four Kenyan medicinal plants. Journal of Ethnopharmacology, 65: 133-137.

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*Corresponding Author:

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Ugwu Okechukwu P.C, Dept. of Biochemistry University of Nigeria Nsukka)

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Review Article Biological Sciences CYSTATIN C: A BETTER INDICATOR THAN CREATININE TO ASSESS THE RENAL FUNCTIONS Shilpasree A S*, Satya Prakash, M. Itagappa Dept. of Biochemistry, Santosh Medical College, Ghaziabad *Corresponding Author Email: kirancapricorn@yahoo.com

ABSTRACT Plasma or Serum creatinine is most widely used to measure Glomerular filtration rate (GFR) in clinical practice. Although most widely used, creatinine cannot be considered as an ideal marker due to its various preanalytical and analytical limitations. The major disadvantage of creatinine being it is unable to detect mild reduction in GFR (60-80ml/min/1.73m2). To overcome its limitations, various creatinine based formulas have been introduced to estimate the GFR from creatinine concentration with the correction for age, muscle mass and sex. The Cockcroft and Gault (C&G) formula and modification of diet in renal disease (MDRD) formula are most commonly used in adults and Schwartz formula is used in children. However use of these formulas to estimate the GFR does not bypass inherent limitations of creatinine. Cystatin C is a low molecular weight protein used to estimate GFR. Cystatin C is considered superior to creatinine due to its various properties like constant production independent of age, sex, muscle mass and not being secreted or reabsorbed by the renal tubules. Major advantage of Cystatin C is 2 its ability to detect mild reduction in GFR (60-80ml/min/1.73m ). Many studies have demonstrated that cystatin C is a better marker than creatinine but few other studies have concluded that cystatin C is equivalent to creatinine but provides no advantage. So we have extensively reviewed the literature to compare the usefulness of Cystatin C over Creatinine in normal subjects as well as in individuals with Acute Renal Failure, Chronic Renal Failure, Diabetes, pediatric population and in elderly subjects.

KEY WORDS

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Creatinine, GFR- Glomerular filtration rate, Cystatin C

Glomerular filtration rate (GFR) is considered as a reliable measure of functional capacity of the kidneys. It is defined as the volume of plasma that can be completely cleared of a particular substance by the kidneys in a unit of time. Clearence of a variety of exogenous and endogenous markers have been used to estimate the GFR. The â&#x20AC;&#x153;gold standardâ&#x20AC;? for determining GFR is to measure the clearance of exogenous substances such as inulin, iohexol, 51Cr labeled ethylenediaminetetra-acetic acid (EDTA), 99mTc-labeled diethylenetriamine pentaacetic acid (DTPA), or 125I-labeled iothalamate. These techniques, however, are

time-consuming, labor-intensive, expensive, and require administration of substances that make them incompatible with routine monitoring. Thus, the measurement of endogenous blood substances to estimate GFR is a common practice. Properties of an ideal endogenous blood substance to estimate GFR should include release into the blood stream at a constant rate, free filtration by the glomerulus, no reabsorption or secretion by the renal tubules, and exclusive elimination via the kidneys.1 Most widely used endogenous marker for GFR is creatinine expressed either as its plasma concentration or renal clearance. Creatinine

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www.ijpbs.com (or) www.ijpbsonline.com fulfills most, but not all of the requirements for a perfect filtration marker. It is a low molecular weight substance, not protein bound; freely filtered, not metabolized by the kidney and it is physiologically inert.2 Nevertheless, serum creatinine remains a crude marker of GFR because its concentration is affected by various factors like age, gender, muscle mass, exercise, nutritional status etc.3 Serum creatinine is also insensitive for detecting small decreases in GFR (60-80 ml/min/1.73m2) because of the nonlinear relationship between its plasma concentration and GFR. 2 A small but significant proportion of creatinine is excreted in the urine is derived from tubular secretion. This leads to overestimation of the GFR. Further, the proportion of total renal creatinine excretion due to tubular secretion increases with decreasing renal function. This amplifies the overestimation of GFR. Tubular secretion of creatinine is not constant and varies, not only within an individual, but between individuals.4,5 Several substances can interfere with laboratory measurements of creatinine. Glucose, uric acid, ketone bodies, plasma proteins, and cephalosporins may lead to falsely high creatinine values when the Jaffe colorimetric method is used 6. Creatinine clearance determinations involving timed urine collections may provide greater accuracy but are difficult for patients to perform, time-consuming, and impractical for routine use. Inaccuracies may still arise if the specimens represent “under-” or “over”collections. Many creatinine based formulas have been introduced to estimate the GFR from creatinine concentration with the correction for age, muscle mass and sex. The Cockcroft and Gault (C&G) formula and MDRD formula are most commonly used in adults and schwartz formula is used in children. 1 However these formulas developed for estimating creatinine clearance in healthy men, may not be

appropriate for estimating creatinine clearance in women or in patients with renal disease and the use of formulas to estimate creatinine clearance does not bypass inherent limitations of creatinine as a filtration marker.2 Eventhough creatinine is most commonly used to assess the renal functions, due to its various limitations the search for ideal marker continues. Cystatin C is a small 13kDa protein that fulfils all the basic requirements for an endogenous filtration marker.1 Cystatin C is produced by all nucleated cells at a constant rate, regulated by a so-called housekeeping’ gene. The production rate of cystatin C is remarkably constant over the entire lifetime and elimination from the circulation is almost completely via glomerular filtration. In the absence of significant tubular damage, cystatin C is reabsorbed and metabolised by the proximal tubular epithelial cells and is not returned to the circulation.The cystatin C plasma concentration is independent of the muscle mass. Thus, the strong association with sex age and Muscle mass seen with creatinine is not observed for cystatin C. The increase of cystatin C with ageing (>50 years of age) reflects the natural decrease of renal function in advanced age. Only a few circumstances have been identified that have an impact on cystatin C plasma concentration: Highdose glucocorticoid therapy and Thyroid dysfunction. Many studies have confirmed the high sensitivity and specificity of Cystatin C for glomerular filtration rate (GFR) estimation; in most studies cystatin C was clearly superior to creatinine with regard to renal function assessment.7 So the purpose of this review is to compare the efficiency of cystatin C with the creatinine to assess the renal functions in different population groups including normal subjects and patients with Acute Renal Failure, Chronic Renal Failure, Diabetic nephropathy, Pediatric patients and in elderly individuals.

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www.ijpbs.com (or) www.ijpbsonline.com Various studies have shown the usefulness of cystatin C to assess the GFR. Frans J. Hoek et al compared the diagnostic accuracy of cystatin C with creatinine and creatinine based Cockcroft and Gault (C&G) formulafor estimation of GFR. The area under the curve (AUC) of the receiver operating curves (ROCs), a measure of diagnostic accuracy, for cys C (0.931) and C&G (0.938) were equal (P 0.815) and both better than the creatinine AUC (0.848; P 0.006). The day-to-day variation (biological and analytical) for cys C was small in diabetic patients. In the follow-up study in diabetic patients, cys C was the parameter which had the best correlation (r 0.66) with changes in GFR. Cys C gave a good estimate of GFR, more accurate and precise than C&G formula.8 In a study conducted by Stefan hergetrosenthal,et al among 85 patients at high risk to develop ARF,44 patients developed ARF and 41 served as controls. ARF was defined according to the Risk of renal dysfunction, Injury to the kidney, Failure of kidney function, Loss of kidney function, and ESRD (RIFLE) criteria. The increase of cystatin C significantly preceded that of creatinine. Specifically, Serum cystatin C increased already by ≥50% 1.5 ± 0.6 days earlier compared to creatinine. Serum cystatin C demonstrated a high diagnostic value to detect ARF as indicated by area under the curve of the ROC analysis of 0.82 and 0.97 on the two days before the criteria was fulfilled by creatinine. Cystatin C detected ARF with a sensitivity of 55% and 82% on these days respectively. Since there is absence of effective, specific therapies for ARF, the early and accurate detection of ARF by using cystatin C is crucial to prevent its progression, and thereby, to potentially improve its outcome.9 Ahlström A et al showed that among 202 patients admitted to intensive care unit 54(27%) patients developed ARF. Serum Cystatin C

showed excellent positive predictive value for ARF in critical illness by ROC analysis. Abnormal values of serum Cystatin C and plasma Creatinine appeared equally quickly (median 3 days). Serum Cystatin C was as good as plasma Creatinine but neither marker was clinically useful in predicting mortality 10 In a study conducted by Tarif N et al in 73 patients with ARF and 300 healthy individuals, Cystatin C correlated significantly with serum Creatinine and estimated GFR and this correlation was much greater in patients with deteriorating renal functions than in patients with improving renal functions. Hence cystatin c is a good marker of renal functions in ARF patients with worsening renal functions 11 Many studies have been conducted to study the usefulness of cystatin c in progression and staging of chronic kidney disease (CKD). Katharina-Susanne Spanaus et al did follow up of 177 patients with non diabetic chronic kidney disease for 7 years. 65 patients had a progressive CKD These patients were older and had a lower GFR and higher serum Creatinine, and Cystatin C values at baseline (all P <0.001) compared with the patients who did not progress. Cox proportional hazard regression analysis revealed that both clearance markers were equally strong predictors of CKD progression, even after adjustment for age, sex, GFR, and proteinuria.12 In a study conducted by Shani Shastri, MD et al Cystatin C level was a risk factor for incident CKD stage 3 and added information beyond that provided by baseline GFR estimated from serum Creatinine .This was attributed to two reasons first one is Cystatin C is a better estimator of measured GFR in those with eGFR >60 mL/min/1.73 m 2, it is the range in which the Creatinine based equations are less accurate. Alternatively, Cystatin C level reflects other factors independent of measured GFR that are associated with kidney disease progression 13

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www.ijpbs.com (or) www.ijpbsonline.com Lesley A. Stevens, et al estimated GFR using Creatinine alone and using Cystatin C with Creatinine in 3408 patients with CKD. GFR was estimated using the 4 new equations based on serum Cystatin C alone, serum Cystatin C, serum Creatinine, or both with age, sex, and race. Serum Cystatin C level alone provided GFR estimates that are nearly as accurate as serum Creatinine level adjusted for age, sex, and race, thus providing an alternative GFR estimate that is not linked to muscle mass.14 Danilo Fliser, MD et al showed that Serum cystatin C concentration is a better marker of renal dysfunction than plasma creatinine concentration, at least in elderly subjects with plasma creatinine concentrations within the normal range. They studied the con of cystatin C, serum creatinine and GFR in young normotensive patients and elderly normotensive and hypertensive subjects. GFR was measured using insulin clearance. The correlation between serum cystatin C concentration and Cln (r = 0.65; P<0.001) was considerably better than between plasma creatinine concentration and Cln (r = -0.30; P<0.02).15 Stefan Herget-Rosenthal, et al studied the efficiency of cystatin c as a screening test to detect reduced GFR in 226 patients with different nephropathies including glomerular and tubular damage. Cystatin C detected reduced GFR with higher sensitivity (97 vs. 83%), and higher negative predictive value (96 vs. 87%) compared to creatinine. In parallel, sensitivity of cystatin C as derived from receiver-operating characteristic plot was significantly higher (p < 0.05). In the subgroups with glomerular or tubular impairment, cystatin C and creatinine did not significantly differ with regard to efficacy.16 Diabetes mellitus due to its various microvascular and metabolic complecations has multiple effects on renal functions and creatinine metabolism. Studies in patients with type 1 and

type 2 DM have demonstrated 25 to 50% increase in GFR compared to normal subjects. Long-standing diabetes mellitus is associated with the development of renal failure in 30-50% of type 1DM patients and in 5% of type 2 DM patients. Detecting the early decrease in renal function in diabetic nephropathy by means of the serum creatinine concentration is difficult because of initially increased GFR and creatinine clearance and the lack of precision of measurements of serum creatinine within the normal range. As in other forms of chronic renal disease, the serum creatinine concentration is insensitive for detecting decreased GFR in Diabetic nephropathy.2 Michele mussap et al compared diagnostic accuracy of serum cystatin C and creatinine for estimating GFR in 52 patients with type 2 DM. The overall reciprocal relationship between cystatin C and GFR was significantly stronger (r 0.84) than those between serum creatinine and GFR (r - 0.65) and between Cockcroft and Gault estimated GFR and GFR (r - 0.70). Diagnostic accuracy of serum cystatin C (90%) was significantly better than those of serum creatinine (77%) and Cockcroft Gault estimated GFR (85%) in discriminating between type 2 diabetic patients with normal GFR (>80 ml/min per 1.73 m2) and those with reduced GFR (<80 mL/min/1.73 m2). So serum cystatin C may be considered as more accurate marker in discriminating type 2 diabetic patients with reduced GFR from those with normal GFR.17 CysC has advantage over SCr in pediatric populations because of the low muscle mass in children, which leads to very low SCr values, where increased assay imprecision is present. Therefore, it is difficult to accurately detect small changes in GFR with SCr in children. On the other hand, the plasma concentration of CysC appears to be constant in children >1 year of age and similar to that of adults .18, 19

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www.ijpbs.com (or) www.ijpbsonline.com Guido Filler et al assessed the diagnostic accuracy of Beta trace protein and compared with cystatin C, Beta 2 microglobulin and creatinie in 225 children with various renal pathologies. The reciprocal correlation of GFR with BTP, Cystatin C, and the Schwartz GFR estimate were significantly higher (r =0.653, 0.765, and 0.706, respectively; P <0.05) than with the reciprocal of creatinine or β2-MG (r =0.500 and 0.557, respectively). ROC analysis showed a significantly higher diagnostic accuracy of BTP, Cystatin C, and Schwartz GFR estimate for the detection of impaired GFR than serum creatinine (P <0.05). 20

REFERENCES 1.

4. 5.

CONCLUSION

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From the extensive review of various publications, cystatin C is considered as a better indecator of renal functions than creatinine both in healthy subjects and in patients with impaired renal functions. This is due to the unique properties of cystatin C like constant production independent of age, sex, and muscle mass and not being secreted or reabsorbed by the renal tubules. The major advantage of cystatin C over creatinine is its ability to detect mild reduction in GFR to which creatinine is insensitive. Since there are no specific therapies, early detection of impaired renal functions is crucial to prevent the progression of renal disease and to improve the patient outcome. The main disadvantage of cystatin C being high cost of its immunoassay. Although all the studies reviewed here have demonstrated the distinct advantage of cystatin C over creatinine it is important to document the advantage of cystatin C to improve the patient outcome. Replacement of creatinine which is most widely used from a new marker cystatin C ultimately depends on the results of patient outcome studies.

10.

11.

12.

13.

Delany MP, Price CP and Lamb EJ. Kidney function and disease. In: Brutis CA Ashwood ER and Bruns DE eds. Teitz fundamentals of clinical chemistry, 6th edn. Philadelphia : WR Saunders Co; 2008: p631-654 Perrone RD, Madias NE and Levey AS. Serum Creatinine as an Index of Renal Function: New Insights into Old Concepts. Clin Chem 1992;38(10); 1933-1953 James GD, Sealey JE, Alderman M, Ljungman S, Mueller FB, Pecker MS, Laragh JH. A longitudinal study of urinary creatinine and creatinine clearance in normal subjects: race, sex, and age differences. Am J Hypertens 1988;1:124–131 Swan SK. The Search Continues—An Ideal Marker of GFR. Clin Chem 1997;43(6); 192-194 Levey AS, Berg RL, Gassman JJ, Hall PM, and Walker WG. Creatinine filtration, secretion and excretion during progressive renal disease. Modification of Diet in Renal Disease (MDRD) Study Group. Kidney Int Suppl. 1989; 27:S73-80. Gerard SK, Khayam-Bashi H. Characterization of creatinine error in ketotic patients: a prospective comparison of alkaline picrate methods with an enzymatic method. Am J Clin Pathol 1985; 84:659–64. Carola Wagner. Cystatin C, Renal Function and Cardiovascular Risk. European Nephrology, 2010;4:49– 54 Hoek FJ, Kemperman FAW and Kridiet RT. A comparison between cystatin C, plasma creatinine and the Cockcroft and Gault formula for the estimation of glomerular filtration rate. Nephrol Dial Transplant (2003) 18: 2024–2031 Rosenthal SH, Marggraf G, Hosing J, Goring F and Pietruck F et al. Early detection of acute renal failure by serum cystatin C. Kidney International 2004; 66; 1115–1122 Ahlström A, Tallgren M, Peltonen S, and Pettilä V. Evolution and predictive power of serum cystatin C in acute renal failure. Clinical Nephrology 2004;62 (5);344-350 Tarif N, Alwakeel JS, Mitwalli AH, Durdana H and Memon NA et al. Serum Cystatin C as a marker of renal function in patients with Acute Renal Failure. Saudi J Kidney Dis Transplant 2008;19(6);918-923 Spanaus KS, Kollerits B, Ritz E, Hersberger M and Kronenberg F et al. Serum Creatinine, Cystatin C, and Beta Trace Protein in Diagnostic Staging and Predicting Progression of Primary Nondiabetic Chronic Kidney Disease. Clinical Chemistry 2010; 56(5) 740–749 Shastri S, Katz R, Shilpak MG, Kestenbaum B and Peralta CA et al. Cystatin C and Albuminuria as Risk

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14.

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Factors for Development of CKD Stage 3. Am J Kidney Dis. 2011;57(6):832-840. Stevens LA, Coresh J, Schmid CH, Feldman HI and Froissart M et al Estimating GFR Using Serum Cystatin C Alone and in Combination With Serum Creatinine: A Pooled Analysis of 3,418 Individuals With CKD. American Journal of Kidney Diseases 2008; 51(3);395406 Fliser D and Ritz E. Serum cystatin C concentration as a marker of renal dysfunction in the elderly. Am J Kidney Dis. 2001 ;37(1):79-83. Herget-Rosenthal s, Trabold s , Pietruck F, Holtmann M, Philipp T et al Cystatin C: Efficacy as Screening Test for Reduced Glomerular Filtration Rate. Am J Nephrol 2000;20:97-102 Mussap M, Vestra M, Fioretto P, Saller A,and Varagnolo M et al. Cystatin C is a more sensitive

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|372-377 marker than creatinine for the estimation of GFR in type 2 diabetic patients. Kidney International 2002; 61; 1453–1461 18. Randers E, Krue S, Erlandsen EJ, Danielsen H, Hansen LG. Reference interval for serum cystatin C in children. Clin Chem1999; 45:1856–8. 19. Bo¨kenkamp A, Domanetzki M, Zinck R, Schumann G, Byrd D, Brodehl J. Cystatin C—a new marker of glomerular filtration rate in children independent of age and height. Pediatrics 1998;101: 875–81. 20. Filler G, Priem F, Lepage N,Sinha P and Vollmer I et al. Beta Trace Protein, Cystatin C, Beta 2-Microglobulin, and Creatinine Compared for Detecting Impaired Glomerular Filtration Rates in Children. Clinical Chemistry 2002; 48(5)729–736

*Corresponding Author: Dr. Shilpasree A S

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Assistant Professor Dept. of Biochemistry Santosh Medical College, Santosh Nagar, Pratap Vihar, Ghaziabad (U.P) Pin- 201009

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Review Article Pharmaceutical Sciences CHRONOTHERAPY- A NEW VISTA IN NOVEL DRUG DELIVERY: An Overview Shweta B.Surve1*, Akshay D.Sapakal1, Snehal S. Naingade2 1

Rajarambapu College of Pharmacy, Kasegaon (Maharashtra) India. Tatyasaheb Kore college of pharmacy, Warananagar(Maharashtra)India. *Corresponding Author Email: surve.shweta638@gmail.com

ABSTRACT Chronotherapy is useful in the treatment of disease, in which drug availability is timed to match rhythms of disease, in order to optimize therapeutic effect and minimize side effects. The specific time that patients take their medication is very important as it has significant impact on success of treatment. If symptoms of a disease display circadian variation, drug release should also vary over time. Drug pharmacokinetics can also be time dependent; therefore, variations both in a disease state and in drug plasma concentration need to be taken into consideration in developing drug delivery systems intended for the treatment of disease with adequate dose at appropriate time.

KEY WORDS Cicardian rhythm, Chronotherapeutics, Chronopharmacology, chronotherapeutic drug delivery system.

INTRODUCTION

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In order to increase the effectiveness of drug there are many approaches have been applied, here one of the technique is described which chronotherapeutic drug delivery system is. Many functions of the human body vary considerably in a day. These variations cause changes both in disease state and in plasma drug concentrations. Human circadian rhythm is based on sleep activity cycle, is influenced by our genetic makeup and hence, affects the body’s functions day and night (24-hour period).[1] The dependence of bodily functions in certain disease states on circadian rhythm is well known. A number of hormones are released by the brain in the morning, while others are released during sleep. Blood pressure and heart rate are highest during the hours of 6.00 a.m. to 12.00 noon. [2] To introduce the concept of chronopharmaceutics, it is important to define the concepts of Chronobiology and pharmaceutics. Chronobiology is the study of

biological rhythms and their mechanisms. The term ‘‘circadian’’ was coined by Franz Halberg from the Latin circa, meaning about, and dies, meaning day. Oscillations of shorter duration are termed ‘‘ultradian’’ (more than one cycle per24 h). Oscillations that are longer than 24 h are ‘‘infradian’’ (less than one cycle per 24 h)rhythms. Ultradian, circadian, and infradian rhythms coexist at all levels of biologic organization.[3] Pharmaceutics is an area of biomedical and pharmaceutical sciences that deals with the design and evaluation of pharmaceutical dosage forms (or drug delivery systems) to assure their safety, effectiveness, quality and reliability . Chronotherapeutics The first chronotherapy to be widely applied in clinical practice was introduced in the 1960s — the alternate-day morning schedule of conventional tablet corticosteroid medication . Other chronotherapies have since been widely used in clinical medicine in the U S, Europe , and

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www.ijpbs.com (or) www.ijpbsonline.com Asia ; these include special evening theophylline systems for chronic obstructive pulmonary disease , conventional evening H2-receptor antagonists for peptic ulcer disease , and conventional evening cholesterol medications for hyperlipidemia . Chronopharmacology and chronotherapeutics are the two scientific domains that study specifically when drugs produce their best effectiveness and least side effects.

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The term "chrono" basically refers to the observation that every metabolic event undergoes rhythmic changes in time. Researchers have concluded that all living organisms are composites of rhythms with varying frequencies that may range from seconds to seasons. Perhaps the best known and studied chronobiologic frequency is the circadian rhythm which approximates the earth's 24-hour rotation around the sun. [4] Researchers have recently concluded that both disease states and

drug therapy are affected by a multitude of rhythmic changes that occur within the human body[5] Chronotherapeutics refers to a treatment method in which in vivo drug availability is timed to match rhythms of disease in order to optimize therapeutic outcomes and minimize side effects. It is based on the observation that there is an interdependent relationship between the peak to trough rhythmic activity in disease symptoms and risk factors, pharmacologic sensitivity, and pharmacokinetics of many drugs.[6] The tradition of prescribing medication at evenly spaced time intervals throughout the day, is an attempt to maintain constant drug levels throughout a 24-hour period, may be changing as researchers' report that some medications may work better if their administration is coordinated with day-night patterns and biological rhythms.[7]

Fig. 1 Circadian time structure (The term “circadian” derives from the Latin phrase “circa diem,” which means “about a day.”) International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)

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The biological rhythm studies help in defining the temporal organization of human beings. One means of illustrating the human circadian time structure is to depict the peak time of 24-h rhythms on a clock Shown is the approximate peak time of circadian (24-h) rhythms of selected biological variables in persons adhering to a normal routine of daytime activity (6–7 a.m. to 10–11 p.m.) alternating with nighttime sleep. For example ,the Circadian rhythms in the blood level of adrenocortical tropic hormone (ACTH), follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, cortisol, catecholamines, renin activity, aldosterone, and angiotensin peak near the end of nighttime sleep or start of daytime activity. The morning peak of the rhythm in vaso-active entities contributes to the morning peak time of the circadian rhythms in heart rate, blood pressure, arterial compliance, and vascular resistance in normotensive and uncomplicated essential hypertension persons, and the morning peak of the circadian rhythm in blood catecholamines gives rise to the morning peak of the circadian rhythm in platelet aggregation.The activity in light and sleep in darkness in a daily routine determines the phasing of all circadian rhythms. Together, the phasing (peak time) of these and numerous other 24-h rhythms in biological processes and functions make up the circadian time structure of human beings, giving rise to day–night patterns in disease activity, with the potential for varying-in-time requirements for pharmacotherapy, as well as administration-time differences in the kinetics and dynamics of medications. Molecular Mechanisms of biological timekeeping Daylight with a high blue component has an activating effect. It stimulates the receptors in the eye and therefore the control center in the

brain to a much greater extent than light with a high red component. Light acts via the photoreceptors in the eye on the control center in the brain. These photoreceptors are very evenly distributed over the retina. The signal to the brain and therefore the biological effect is greatest when as many photoreceptors as possible are stimulated simultaneously. Indirect lighting in which light is reflected from a large bright surface therefore has a stronger impact than the concentrated light from a spotlight. The key breakthrough in understanding the circadian system was discovery of the molecular mechanisms of the circadian clock (“molecular clock”). Molecular mechanisms underlying circadian rhythms are conceived as a series of interlocking molecular loops, involving rhythmic transcription of specific “clock genes”, and interactions of the proteins they encode. In a simplified model, these clock genes comprise “positive elements” such as clock and Bmail1, whose protein products dimerize, enter the nucleus, and stimulate transcription of negative elements period 1, 2, and 3 (Per 1-3) and cryptochrome (Cry 1-2). The protein products of these genes (per 1-3, cry 1-2) in turn oligomerize, enter the nucleus, and suppress the activity of the clock/Bmail1 complex.[8] .These circadian rhythms are controlled by an inherited master clock network composed of the paired supra chiasmatic nuclei (SCN) which are situated in the ventro-rostral part of the hypothalamus function as the master pacemaker of an endogenous circadian timekeeping system.[9]. In mammals, the paired suprachiasmatic nuclei (SCN) located in The SCN receive photic input from the retina via direct and indirect pathways, thus forming the prime relay between external and internal times. The SCN synchronises the periphery via humoral and neuronal pathways and via the regulation of activity, nutrient uptake and body temperature by means of transcriptional control

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www.ijpbs.com (or) www.ijpbsonline.com of ccgs peripheral oscillators translate time information into physiologically relevant signals. Perturbations of the circadian system have a profound impact on health and wellbeing. The physiological and psychological disturbances following trans-meridian travel (jet lag) are rooted in a transient state of internal desynchronisation while the body's clocks struggle to adapt to an abruptly shifted external light/dark cycle. In recent years, multiple molecular feedback loops (molecular clock machinery) were identified, which further fine-tune generation of intracellular circadian rhythms. A number of important nuclear receptors such as REV-ERB, ROR or PPAR, interact closely with circadian feedback loops. The regulation of clock genes, by nuclear receptors, renders the clock responsive to numerous circulating hormones (e.g. cortisol,

estrogen), nutrients signals (e.g. fatty acids derivatives) and cellular redox status (NADH/NAD+ratio). Nuclear receptors are now recognized as key intermediaries between the molecular clock machinery and a wide array of physiological processes [8]. Biological timekeeping is an evolutional adaptation to an environment that is organized in time, displaying discrete and important cyclic phenomena. Thus, the temporal organization of biological processes and functions during the 24-h period ensures peak functioning of the diurnal human species during daytime activity and restoration and repair during nocturnal rest; during the menstrual cycle, it ensures fertility and perpetration of the species; and during the year it ensures a priori biological adjustment to predictable-in-time changes and challenges associated with the different seasons of the year.

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Fig. 2 supra chiasmatic nuclei (SCN) controlling circadian rhythm.

BIOLOGICAL MARKERS The classic phase markers for measuring the timing of a mammal's circadian rhythm are:

1. Core body temperatureFor temperature studies, subjects must remain awake but calm and semi-reclined in near

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declining phase of the melatonin levels was more reliable and stable than the termination of melatonin synthesis.[10] One method used for measuring melatonin offset is to analyze a sequence of urine samples throughout the morning for the presence of the melatonin metabolite 6-sulphatoxymelatonin (aMT6s). Laberge in Quebec in 1997 used this method in a study that confirmed the frequently found delayed circadian phase in healthy adolescents. [12] The regulator for our internal clock Biorhythms dictate when we wake up, when we become tired and when we fall; they even have an effect on our body temperature and much more. This internal clock is influenced to a large extent by light. Although our genetic makeup determines our circadian rhythm, this rhythm has to be resynchronized by daylight each and every day. If light, the most important zeitgeber, is lacking then our internal clock soon goes out of sync. As a result we may suffer from sleep disorders, chronic fatigue and in the worst case clinical depression. ď&#x192;&#x2DC; Circadian rhythm hormone secretion The hormones responsible for the circadian rhythm in humans are melatonin, which is released in response to increasing levels of darkness and which promotes sleep and cortisol which is the biological opposite of melatonin and an indicator of the level of human activeness.

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darkness while their rectal temperatures are taken continuously. The average human adult's temperature reaches its minimum at about 05:00 (5 a.m.), about two hours before habitual wake time, though variation is great among normal chronotypes[10] 2. Plasma level of cortisol-A third marker of the human pacemaker is the timing of the maximum plasma cortisol level. Klerman in 2002 compared cortisol and temperature data to eight different analysis methods of plasma melatonin data, and found that "methods using plasma melatonin data may be considered more reliable than methods using CBT or cortisol data as an indicator of circadian phase in humans. [11] 3. Melatonin secretion by the pineal gland: Melatonin is absent from the system or undetectably low during daytime. Its onset in dim light, dim-light melatonin onset (DLMO), at about 21:00 (9 p.m.) can be measured in the blood or the saliva. Its major metabolite can also be measured in morning urine. Both DLMO and the midpoint (in time) of the presence of the hormone in the blood or saliva have been used as circadian markers. However, newer research indicates that the melatonin offset may be the more reliable marker. Benloucif et al. in Chicago in 2005 found that melatonin phase markers were more stable and more highly correlated with the timing of sleep than the core temperature minimum. They found that both sleep offset and melatonin offset were more strongly correlated with the various phase markers than sleep onset. In addition, the

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Fig. 3 Effect of light on secretion of harmones. Molecular Genetics of Circadian Rhythms As discussed previously, the properties of circadian clocks suggested cyclic changes in the expression of certain genes as a possible mechanism underlying the internal pacemaker. This hypothesis was supported by the demonstration in a number of species that the expression of genes and the production of proteins encoded by those genes were required for normal clock function. Nevertheless, a completely different experimental approach ultimately led to the identification of molecular circadian clock components. Researchers used chemical agents to introduce numerous, random mutations into the DNAs of the fruit fly, Drosophila melanogaster,[13] and of the filamentous fungus Neurospora. The resulting mutant organisms then were screened for rhythm abnormalities. This mutagenesis approach led to the identification of the first circadian clock mutants, which were called period (per) and frequency (frq, pronounced â&#x20AC;&#x153;freakâ&#x20AC;?). The genes that carried the mutations in these organisms were cloned in the 1980s [14]. However, considerable frustration ensued as researchers sought to isolate the equivalent genes in mammals (i.e., mammalian homologs). Finally, in the early 1990s, researchers began a

similar mutagenesis screening approach in the mouse and described the first mouse circadian mutation, called Clock, in 1994 . In 1997 the gene affected by this mutation became the first mammalian circadian clock gene to be cloned [14]. Like the mutants of the Per and Frq genes, the altered Clock gene both affected the freerunning rhythm period (i.e., lengthened the period) and caused a loss of persistence of circadian rhythms under constant environmental conditions. Both the Clock mutant in mice and the Per mutant in flies were the first animals of their respective species identified using such a mutagenesis approach in which the mutation manifested as altered behavior rather than an altered physiological process. Since the discovery of the Clock gene in mice, the list of circadian clock genes identified in mammals has grown in a remarkably short period of time. For example, researchers have identified not one, but three mammalian genes that correspond to the per gene in both their structure (i.e., nucleotide sequence) and their function [14]. Some of the pro-posed circadian clock genes have been identified solely based on their similarity in sequence to Drosophila clock genes and have not been confirmed to have clock function based on an examination of the

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www.ijpbs.com (or) www.ijpbsonline.com behavior of the corresponding mutants. Nevertheless, the findings to date clearly indicate the outline of a pacemaker that is based on a feedback cycle of gene expression. Basic Types of chronotherapy Bright Light Therapy The administration of correctly timed, properly dosed, high-intensity fluorescent light to treat various forms of depression. [15] Wake Therapy (Sleep Restriction) The use of prolonged periods of wakefulness, with intervening periods of recovery sleep, to induce rapid improvement in depressive symptoms.[16][17] Sleep Phase Advance Moves the time of sleep forward to the early evening to potentiate the action of other antidepressant interventions.[18] Triple Chronotherapy The combined use of Wake and bright light therapies along with sleep phase advance to generate a fast and sustained antidepressant response. [15-18] Dawn Simulation Use of a progressive illumination signal, administered during the end of sleep, to treat Seasonal Affective Disorder. [19] Chronobiotics Circadian rhythm-modifying compounds such as melatonin and certain psychiatric medications that treat depression and sleep disorders. [20, 21] Interpersonal Social Rhythm Therapy A problem-solving therapy that increases the routine of one's everyday activities to improve mood stability in bipolar disorder.[22,23]

minimizing the negative impact of side effects associated with therapy. In this respect, chronopharmaceutics will be a clinically relevant and reliable discipline if pharmaceutical scientists could delineate(describe) a formal and systematic approach to design and evaluate drug delivery system that matches the biological requirement. The key component for the success of ChrDDS design for the treatment of diseases is the elucidation of control-relevant models for drug delivery. A control-relevant model is the one that has: (i) Predictive capability in terms of the process input â&#x20AC;&#x201C; output behavior. (ii) Utility in performing on-line calculations for control or optimization purposes. Because of the complexity of identified biological oscillators, two physical descriptors have been discussed to illustrate the mathematical description of such system: the linear mass -spring oscillator and the non-linear electrical oscillator described by vanderPol . The latter provides a simple example of an oscillator in which the variation of one parameter alters the system from being relatively insensitive to noise to one that is very sensitive. (iii) A general introduction to the mathematics of biological oscillators can be found in the monograph by Pavlidis . A number of modeling approaches are available in the broad area of hemodynamic variable regulation, cancer chemotherapy, and glucose concentration control.

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CONCLUSION Theoretical and formal approaches to chronopharmaceutics When treating human diseases, the overall goal is to cure or manage the disease while

Advances in chronobiology and chronopharmocology has demonstrated the importance of biological rhythms in treatment of disease and this has led to a new approach to

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www.ijpbs.com (or) www.ijpbsonline.com the development of novel drug delivery systemChrDDS (Chronotherapeutical Drug Delivery System). The overall success of chronopharmaceutics will depend on the successful integration of knowledge from future advances in development timing, system biology and nanotechnology. The selection of the appropriate chronopharmaceutical technology should take into considerations the application range (e.g. targeted drugs of different physico-chemical properties), the ease of manufacturing, the costeffectiveness, and the flexibility in the pharmacokinetic profile. By selecting optimal time to achieve the desired effect, treatment opportunities may arise and undesired side effects can be minimized. As timing of drug administration in disease therapy has significant impact upon treatment success, optimal clinical outcome cannot be achieved if drug plasma concentrations are constant. If symptoms of a disease display circadian variation, drug release should also vary over time. ChrDDS in future is certainly going to gain popularity.

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REFERENCE [1] Evans R. M and Marain C.,Taking Your Medication: A Question of Timing.Chicago, IL: American Medical Association; 3-8(1996) [2] Michael P. L. Chronobiology and Chronotherapeutics Possible Strategy for Hypertension and Ischemic Heart Disease (Cited 2009 May 28) *3+ Smolensky M.H., D’Alonzo G.E. Biologic rhythms and medicine, Am. J. Med; 85 :34– 46 (1988) [4] Lamberg L. American Pharmacy , NS31(11): 20-23, (1991) [5] Ura J., Shirachi D., Ferrill M. Paper name please California Pharmacist, 23(9): 46-53, (1992) [6] Traynor K, Newton DW, Hrushesky JM, Reiter RJ. American Pharmacy. , NS32(3): 261-269, (1992)

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|378-386 [7] Evans R. M. and Marain C.,Taking Your Medication: A Question of Timing.Chicago, IL: American Medical Association; 3-8(1996) [8]Cermakian N, Boivin DB. The regulation of central and peripheral circadian clocks in humans. Obes Rev 2009; 10(Suppl 2): 25-36 [9] Ohdo S. ,Chronotherapeutic strategy: rhythm monitoring, manipulation and disruption. Adv Drug Deliv Rev.,62:859-875(2010). [10] Benloucif, S.; Guico, M.J.; Reid, K.J.; Wolfe, L.F.; L'hermite-Balériaux, M.; Zee, P.C. ,"Stability of melatonin and temperature as circadian phase markers and their relation to sleep times in humans". Journal of Biological Rhythms ,20 (2): 178–88, (April 2005). [11]klerman, Elizabeth B.; Hayley B. Gershengorn, Jeanne F. Duffy, Richard E. Kronauer. "Comparisons of the Variability of Three Markers of the Human Circadian Pacemaker". Journal of Biological Rhythms , 17 (2): 181–193. (April 2002) Retrieved 2010-10-09. "In summary, because they have a lower variance, methods using plasma melatonin data may be considered more reliable than methods using CBT [core body temperature] or cortisol data collected during CRs [constant routines] as an indicator of circadian phase in humans." [12]Laberge, L.; Lesperance, P.; Tremblay, R.; Lambert, C.; Montplaisir, J. (1997). "Phase delay of 6sulphatoxymelatonin in normal adolescents". Sleep Research (Québec, Canada: Centre d'etude du Sommeil, Hôpital du Sacré-Coeur, Département de Psychologie, Département de Pharmacologie, Departement de Psychiatrie, Université de Montréal) 26: 727. http://www.websciences.org/cftemplate/NAPS/archiv es/indiv.cfm?issn=19979287 Retrieved 2007-12-18. [13]Hall, J.C. Genetics of biological rhythms in drosophila. Adv. Genet. 38, 135–184 (1998). [14]King, D.P. & Takahashi, J.S. Molecular genetics of circadian rhythms in mammals., Annu. Rev. Neurosci. 23, 713–742 (2000). [15] Smolensky M.H., D’Alonzo G.E. Biologic rhythms and medicine, Am. J. Med; 85 :34– 46 (1988) [16] Loving R.T., Bright light augments antidepressant effects of medication and wake therapy. Depression & Anxiety, 16(1): 1-3, (2002) [17] Wu J.C., Rapid and Sustained Antidepressant Response with Sleep Deprivation and Chronotherapy in Bipolar Disorder. Biological Psychiatry, 66(3): 298-301, (2009) [18] Voderholzer U., Is the antidepressive effect of sleep deprivation stabilized by a three day phase advance of the sleep period? A pilot study. European Archives of

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www.ijpbs.com (or) www.ijpbsonline.com Psychiatry & Clinical Neuroscience, 253(2): 68-72, (2003) [19] Avery D. H., Dawn simulation and bright light in the treatment of SAD: a controlled study. Biological Psychiatry, 50(3): 205-16, (2001) [20] Lewy A. J., The circadian basis of winter depression. Proceedings of the National Academy of Sciences of the United States of America,103(19): 7414-9, (2006) [22] Kasper S., Efficacy of the novel antidepressant agomelatine on the circadian rest-activity cycle and depressive and anxiety symptoms in patients with

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|378-386 major depressive disorder: a randomized, double-blind comparison with sertraline. Journal of Clinical Psychiatry, 71(2): 109-0, (2010) [23] Frank E., The Role of Interpersonal and Social Rhythm Therapy in Improving Occupational Functioning in Patients With Bipolar I Disorder. American Journal of Psychiatry; 165(12): 1559-1565, (2008) [24]Frank E., Two-Year Outcomes for Interpersonal and Social Rhythm Therapy in Individuals With Bipolar I Disorder. Archives of General Psychiatry, 62(9): 9961004, (2005)

*Corresponding Author:

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Shweta B. Surve, Rajarambapu College of Pharmacy, Kasegaon (Maharashtra) India

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Research Article Pharmaceutical Sciences HERBAL DRUG AWARENESS AND RELATIVE POPULARITY IN JAMNER AREA R.E. Mutha1*, R.D. Shimpi2, P.S. Gayakwad2, A.J. Gavit2 1

H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dist. Dhule (M.S.) Shree Sureshdada Jain Institute of Pharmaceutical Education and Research, Jamner, Dist. Jalgaon (M.S.) *Corresponding Author Email: rakeshmutha123@yahoo.co.in

ABSTRACT The aim of this study was to find out the awareness, misconceptions and myths about the herbal drugs in rural areas. A sample of 450 persons was assessed by a proper set of questionnaire called proforma by door to door visit. Present study indicates that very little population (36%) is aware about the herbal drugs against a huge population (64%). So the appropriate steps are required to be taken to spread the awareness among the society related to herbal drugs.

KEY WORDS Awareness, Herbal drugs, Population, Questionnaire, Rural area

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INTRODUCTION Ayurveda is a medical system primarily practiced in India that has been known for nearly 5000 years. It includes diet and herbal remedies, while emphasizing the body, mind and spirit in disease prevention and treatment [1]. The herbal plants are used as medicines in folk and traditional medical practice based on the use of plants and plant extracts. The World Health Organization (WHO) has recently defined traditional medicine (including herbal drugs) as comprising therapeutic practices that have been in existence, often for hundreds of years, before the development and spread of modern medicine and are still in use today [2]. Herbal drug constitutes only those traditional medicines which primarily use medicinal plant preparations for therapy [2]. Herbal medicines may be made from the whole plants or a part of the plant, including the bark, stem, root, or seed and can be purchased fresh, dried, in pills, capsules, and in tinctures that are preserved in alcohol, glycerin, or another liquid. Herbal medicines are

also in great demand in the developed world for primary health care because of their efficacy, safety and lesser side effects [3]. India is a vast repository of medicinal plants that are used in traditional medical treatments. Certain herbs have become popular over the last some years, but herbal medicines are still poorly understood by the public, medical practitioners and the media [4]. The various indigenous systems such as Siddha, Ayurveda, Unani and Allopathy use several plant species to treat different ailments [5]. Because of the toxicity and side effects of allopathic medicines, the use of herbal medicine becoming popular. This led to sudden increase in the number of herbal drug manufactures [6]. The present study was conducted to find out the awareness about the herbal drugs in rural areas as the number of herbal drug manufacturers has been increased. The study also assess certain misconceptions, myths into the minds of general public regarding role of Herbal drugs in the treatment of diseases thereby promoting the use of these drugs.

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MATERIAL AND METHODS Study Design This was a questionnaire based study involving general public. The study was conducted, over a period of 4 months from July 2011 to October 2011. It was conducted in Jamner Tahsil, Dist Jalgaon (M.S.) India. Four hundred fifty persons (both male and female) in the age group of 18-70 years consisting different educational level responded to the questionnaire by door to door visit. Material used A set of questionnaire called proforma was drafted keeping above said objectives, in mind. For each question two choices were given except question no. 1 (Yes or No). The language was kept very simple so as to present no problem in understanding the spirit of question. The objectives were duly explained to each and every respondent and doubts, if any, were clarified. Subjects The study included 450 people residing in Jamner Tahsil, Dist Jalgaon (M.S.). Study setting This population categorized into two sections based upon a basic question that â&#x20AC;&#x153;Which system of medicine do you prefer in disease conditionâ&#x20AC;? as A] Residents Using Ayurvedic Drugs: This population helps to know their depth of knowledge about the herbal drugs. B] Residents Not Using Ayurvedic Drugs: with the help of such population, efforts were made to find out the reasons for what they are not using herbal drugs. Following set of questionnaire was prepared in order to evaluate the use of Herbal drugs in rural areas. 1. Which system of medicine do you prefer in disease condition? 2. Do you think whether herbal products are costly?

3. Do you use herbal medicine? 4. Do you think that herbal products are prepared according to rules and regulation? 5. Do you use herbal cosmetic? 6. Do you think herbal product (Drug/cosmetic) have purity, safety and efficacy? 7. Do you use herbal drugs only for acute conditions? 8. Do you use herbal drugs only for chronic conditions? 9. Do you use herbal drugs for both Acute as well as chronic conditions? 10. Which herbal dosage form do you prefer? Based upon the information gathered reasons for not preferring the herbal drugs were evaluated. Questions and their responses are being presented.

RESULTS During the door to door visit, the first basic question was asked that enabled to categorize the population according to different systems of medicine they are using in disease condition. As shown in figure 1, more than half population (51%) uses Allopathy, 36% population uses Ayurvedic system of medicine & 13% population uses Homeopathic system of medicine. Interesting fact is that nobody knows about the use of Unani drugs. It indicates that 36% of population believes to use Ayurvedic drugs and 64% do not believe in use of ayurvedic drugs. A] Residents using Ayurvedic drugs (36%) B] Residents not using Ayurvedic drugs (64%) Residents using Ayurvedic drugs (36%): This population was analyzed by using Q2 to Q9 as mentioned in experimental method, in order to recognize their depth of awareness about the herbal drugs. In response to this, population members responded differently to each question as shown in figure 2.

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Lastly on asking about dosage form mostly preferred by them it also gives different views as shown in figure 3.

Residents not using Ayurvedic drugs (64%):- This population was analyzed by using Q2, Q4 and Q6 as shown in figure 4 and efforts were made to find out reason for not using herbal drugs.

Figure 1: System of medicine mostly used

0% Allopathy

13%

Ayurvedic

51% 36%

Homeopathy Unani

Figure 2: Residents using ayurvedic drugs

120.00% 100.00% 80.00%

33.33%

21.59%

8.82%

18.18%

26.26% 47.06% 61.77%

60.00% 40.00%

91.18% 66.66%

79.41%

91.17%

81.81%

Yes

73.73% 52.94% 38.23%

20.00%

8.82%

0.00%

Q3 Q2

Q4 Q3

Q5 Q4

Q6 Q5

Q7 Q6

Q8 Q7

Q9 Q8

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Q1Q2

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Figure 3: Herbal dosage forms mostly used

Ointment 2% Cream 23%

Facewash 4%

Liquid 7%

Pastes 17% Powder 11%

Tablet 14% Shampoo 11%

Gel 11%

Figure 4: Residents not using ayurvedic drugs

100% 90%

16.67% 28.78%

80%

50.77%

70% 60% No

50% 40%

83.33%

Yes

71.22%

30%

49.23%

20% 10% 0%

Q4 Q3

Q6 Q5

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Q2 Q1

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www.ijpbs.com (or) www.ijpbsonline.com DISCUSSION AND CONCLUSION Present study indicates that very little population (36%) is aware about the herbal drugs against a huge population (64%). By using a particular set of questionnaire we tried to find out the reasons for not using the herbal drugs. According to this population (not using herbal drugs) herbal drugs are 1. Costly: 83% of population do not use herbal product because of their higher cost. 2. Safety: 29% of populations do not think herbal products are safe. 3. Rules and regulation: 50% of population thinks that herbal drugs are not prepared according to rules and regulations. So the appropriate steps are required to be taken to spread the awareness about importance of the herbal drugs in the society. This can be made possible by various ways like involvement of ayurvedic practitioners, govt. awareness programmes, medical camps by pharmaceutical

companies, conduction of seminars in educational institutions to create awareness among the young generation. Such studies are also required to be carried out in each corner of country to find out the depth of awareness and measures to be taken.

REFERENCES 1.

2. 3. 4.

Morgan C., Medicine of the Gods: Basic Principles of Ayurvedic Medicine, Mandrake of Oxford, 2002. [http://www.compulink.co.uk/â&#x2C6;źmandrake/ayurveda.h tm] Fahn A., Plant Anatomy, Pergamon Press, Oxford, 3rd Edn, 1982. Kamboj V.P., Herbal medicine. Curr Sci, 78 (1): 35, (2000). Chopra RN, Nayar SL Chopra IC: In; Glossary of Indian medicinal plants, Council of Scientific and Industrial Research, New Delhi: 197, (1956). Rabe T., Staden J.V., Antibacterial activity of South African plants used for medicinal purposes. J. Ethnopharmacol, 56: 81-87, (1997). Agarwal A., Critical issues in Quality Control of Herbal Products, Pharma Times, 37 (6): 9-11, (2005).

*Corresponding Author: Rakesh Eshwarlal Mutha

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Assistant Professor, H. R. Patel Institute of Pharmaceutical Education and Research, Karwand Naka, Shirpur, Dist. Dhule (M.S.) 425405 Contact No. : +91-9423189359, Fax No. : 02563-257599 E.mail ID: rakeshmutha123@yahoo.co.in

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Research Article Biological Sciences ANTICANCER ACTIVITY OF EXCOECARIA AGALLOCHA LEAF EXTRACT IN CELL LINE MODEL Ahmed John Syed Batsa and Kumar Periyasamy* P. G & Research department of botany, Jamal Mohamed College, Tiruchirappalli Tamil Nadu, India-620020 *Corresponding Author Email: Kumarbiotechnology.periyasamy@gmail.com

ABSTRACT Objective: To identify the anti-cancer activity of Excoecaria agallocha (Euphorbiaceae) is a Mangrove Plant used in folklore medicine for the treatment of several diseases. The present study was aimed at using leaf crude extract as anti cancer agent. The methanol extract was treated with under various concentrations and similarly treated with chloroform extract. The results are clearly indicated the anti cancer cell line activity of E. agallocha. Method: To analyze the in vitro studies using the crude extract. The leaf extract of E. agallocha was used for this study. Result: The cell viability was maximum at the lower concentration (µg/ml) when compare to higher concentration. At 3.906 (µg/ml) concentrations, the cell viability of 85.32 and 81.96 were found to occur at 1:128 dilution of Methanol and Chloroform extracts respectively. At the same concentration of 31.25 and at the dilution of 1.16, cell viability was observed 65.55 in methanol and 45.55 in chloroform extracts. At the higher concentration the cell viability of 22.35 and 8.12 was recorded in the Methanol and Chloroform extracts.

KEYWORDS Mangrove; Exchocaeria; Anticancer; Cell line

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1. INTRODUCTION The Mangrove forest is distributed in two sub divisions like tropical and subtropical coast of the world. In the world, mangrove forests spreads over 30 countries and covers a total area of 99,300 sq Km. The world’s largest mangrove forest exist in Indonesia and decreasingly exits in Brazil, Australia, India and Nigeria. The developing country loses acres of Mangrove forest in the last three decades. The mangrove forest exists in Indian coastal region and in Islands of India. As per the data collected during the year 1987, mangrove forest extended to 6740 sq Km .The largest mangrove forest occurs in West Bengal spreading over in 4200 sq Km and followed by Andaman and Nicobar Islands and in small levels mangrove forest exists in Gujarat, Maharashtra, Andhra Pradesh , Goa ,Orissa, and Tamil Nadu. In

Tamil Nadu, the mangrove forest got well developed in Pitchavaram area, Muthupet. In the mangrove plants of Pitchavaram area more than 45 species are available. To contain the degradation of mangrove forest, research foundation of M. S. Swaminathan, a non government organization made trenches in some areas to enhance flushing of tidal water. During early period of the December 2004, Tsunami level of exploitation of Pitchavaram areas mangrove far exceeded in sustainable level [1,7]. In ancient times, Natural product of plant has been used for several years to treat various diseases in countries like Egypt, China, India and Greece. They practiced and used to prepare medicine from plants in the ancient time and an impressive number of modern drug have been developed in the developing country. About 120

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2. MATERIALS AND METHOD  Monolayer culture bottle of Hep 2 cells  5ml, 10ml serological pipette  Minimal essential media (MEM) with 10%, 2% foetal calf serum  TPVG (Trypsin, PBS, versene, glucose)  Discarding jar, inverted microscope, dessicator  Gloves, spirit, cotton, label pad, marker pen 2.1. Maintenance of cell line Maintenance of cells involves the following operations: 1. Dispersion and Sub culturing (seeding) of cells. 2. Preservation of cells in repository. 3. Revival of cells from repository. 2.3. Sub culturing and maintenance of cell line 1. Bring the medium and TPVG to room temperature for thawing. 2. Observe the tissue culture bottles for growth, cell degeneration, pH and turbidity by seeing in inverted microscope. 3. If the cells become 80% confluent it goes for sub culturing process 4. Wipe the mouth of the bottle with cotton soaked in spirit to remove the adhering particles. 5. Discard the growth medium in a discarding jar keep distance between the jar and the flask. 6. Then add 4 – 5 ml of MEM without FCS and gently rinsed with tilting. The dead cells and excess FCS are washed out and then discard the medium. 7. TPVG was added over the cells. And incubate at 37º C for 5 minutes for dis aggregation. The cells become individual and it’s present as suspension.

8. Add 5ml of 10% MEM with FCS by using serological pipette. 9. Gently give passaging by using serological pipette. If any clumps are present then repeat the process. 10. After passaging split the cells into 1:2 and 1:3 ratios for cytotoxic studies for plating method. 2.4. Seeding of cells After homogenize take one ml of suspension and pour in to 24 well plates. In each well add 1ml of the suspension and kept in a desiccators in 5% CO2 atmosphere. After 2 days incubation observes the cells in inverted microscope, if the cells became 80% confluent [3]. 2.5. Performance of drug cytotoxic assay Cytotoxic is the toxicity or damage caused to the cells on addition of drug. After the addition of the drug, cell viability is estimated by staining techniques, where by cells are treated with Trypan blue. Trypan blue is excluded by live cells, but stains dead cells blue. The results are confirmed by additional metabolic intervention experiments such as MTT assays. 2.6. Materials Required 1. Monolayer cultures in log phase. 2. Drug extract (different concentrations) 3. MEM without FCS 4. 0.45µ filter 5. 5mL sterile storage vial 6. Tissue paper, Marker pen, Spirit, Cotton and Gloves 7. 1 mL, 2 mL pipettes, Micropipette and tips 8. Discarding jar with 1 % Hypochlorite solution 2.7. Stock drug concentration 10 mg of drug is dissolved in 10 ml of serum free MEM giving a concentration of 1mg / 1 ml. The stock is prepared fresh and filtered through 0.45  filter before each assay. Working concentrations of drug ranging from 1mg/ml to 7.8125 mg/ml are prepared as follow

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www.ijpbs.com (or) www.ijpbsonline.com 2.8. Preparation of working stock of 1 mg /ml To 4.5 ml MEM add 0.5 ml of stock to give a working concentration of 1mg/mL. Drug concentration can be prepared from the working stock in MEM without FCS. Prepare required volume of test sample for each concentration. 1. 48hr monolayer culture of Hep2 cells at a concentration of one lakh /well seeded in 24 well titer plates. 2. The plates were microscopically examined for confluent monolayer, turbidity and toxicity if the cells become confluent. 3. The growth medium (MEM) was removed using micropipette. Care was taken so that the tip of the pipette did not touch the cell sheet. 4. The monolayer of cells was washed twice with MEM without FCS to remove the dead cells and excess FCS. 5. To the washed cell sheet, add 1ml of medium (without FCS) containing defined concentration of the drug in respective wells. 6. Each dilution of the drug ranges from 1:1 to 1:64 and they were added to the respective wells of the 24 well titer plates. 7. To the cell control wells add 1ml MEM (w/o) FCS. 8. The plates were incubated at 37ºc in 5% CO2 environment and observed for cytotoxicity using inverted microscope. 2.9. MTT assay MTT assay is called as (3-(4, 5-dimethyl thiazol2yl)-2, 5-diphenyl tetrazolium bromide. MTT assay was first proposed by Mossman in 1982. [8] (Graphs) Procedure  After incubation, remove the medium from the wells carefully for MTT assay.  In each well wash with MEM (w/o) FCS for 2 – 3 times. And add 200µl of MTT conc of (5mg/ml).

 And incubate for 6-7hrs in 5% CO2 incubator for Cytotoxicity.  After incubation add 1ml of DMSO in each well and mix by pipette and leave for 45sec  If any viable cells present formazan crystals after adding solublizing reagent (DMSO) it shows the purple color formation.  The suspension is transferred in to the cuvette of spectrophotometer and an O.D values is read at 595nm by taking DMSO as a blank.  Cell viability (%) = × 100

3. RESULT AND DISCUSSION In the present investigation, the anti-cancer activity of Exchocaria agollocha was screened from 3.906 to 1000 µg/ml of concentrations with the dilution leads to 1:1 to 1:128 using Methanol and Chloroform extracts. The anti-cancer activity of Exchocaria agollocha , the cell viability was maximum at the lower concentration (µg/ml) when compare to higher concentration.. At 3.906 (µg/ml) concentrations, the cell viability of 85.32 and 81.96 were found to occur at 1:128 dilution of Methanol and Chloroform extracts respectively. At the same concentration of 31.25 and at the dilution of 1.16, cell viability was observed 65.55 in methanol and 45.55 in chloroform extracts. At the higher concentration the cell viability of 22.35 and 8.12 was recorded in the Methanol and Chloroform extracts. In general, the cell viability was more in Methanol when compare to Chloroform extracts at higher concentration in particular.

ACKNOWLEDGEMENTS The author hart full thanks to the management, Jamal Mohammad College, Research deportment of botany Tiruchirapplai for having provided the facilities to carry out this study.

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Tabe-1 Cytotoxicity effect of Excoecaria agallocha (Methanol Extract) S. no.

Concentration (µg/ml)

Dilutions

Cell viability

1000

Neat

22.35

2 3

500 250

1:1 1:2

29.63 48.56

125

1:4

54.32

62.5

1:8

61.05

31.25

1:16

65.55

15.625

1:32

75.23

7.8125

1:64

78.90

3.906

1:128

85.32

Cell control

100

Table- 2 Cytotoxicity effect of Excoecaria agallocha (Chloroform Extract) Concentration (µg/ml)

Dilutions

Cell viability

1000

Neat

8.12

500

1:1

14.22

250

1:2

19.63

125

1:4

21.85

62.5

1:8

29.63

31.25

1:16

45.55

15.625

1:32

53.62

7.8125

1:64

75.28

3.906

1:128

81.96

Cell control

100

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S. no.

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Figure: 1 Cytotoxicity effect of Excoecaria agallocha (Methanol Extract)

Toxicity 1 (1000µg/ml)

Toxicity 2 (62.5µg/ml)

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(31.25µg/ml)

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Figure: 2 Cytotoxicity effect of Excoecaria agallocha (Chloroform Extract)

Toxicity 1 (1000µg/ml)

Toxicity 2 (500µg/ml)

Toxicity 2 (62.5µg/ml) Sample (Methanol Extract)

MTT ASSAY

R² = 0.964

100 90 80

Cell viability

IC 50

60 50

40 30 20

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10 0 1000

500

250

125 62.5 31.25 Concentration (µg/ml)

15.625

7.8125

3.906

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Chloroform extract

MTT ASSAY

R² = 0.943

Cell viability

70 60

IC 50

40 30 20 10 0 1000

500

250

125 62.5 31.25 Concentration (µg/ml)

4. REFERENCES [1]Planning commission, Government of India, Report visit to pitchavaram in Tamil Nadu, a wetland included under national wetland conservation and mangrove programme of The Ministry of environmental programme of the Ministry of environmental forests – OCT-2008. [2] V. Selvam, P. Eganathan, V.M Karunagaran, T. Ravishankar and R. Ramasubramanian, Mangrove plants in Tamil Nadu., M.S.Swaminathan research foundation ChennaiOCT- 2004. [3] Y L N Murthy, Mallika Devarapalli, Damodar reddy.G, Phytochemical investigation of the bark Excoecaria agallocha Linn. Journal of Pharmacy research 2010, 3 (1),1-5. [4] G. Agramoorthy ,M. Chandrasekaran ,V. Venkaresalu ,M. J. Hsu., Antibacterial and antifungal activity of fatty acid methyl esters of the blind –your-eye mangrove from India .,Brazilian journal of Microbiology (2007) 38:739742.

15.625 7.8125

3.906

[5] R.C. Patil, Sonal M.Manohar, Anti Reverse Transcriptase and Anticancer activity of stem ethanol extracts of Excoecaria agallocha., Ceylon Journal of Science, 40 (2): 147 – 155, 2011. [6] Vundru Anil KUMAR, Kandru AMMANI, Busi Siddhardha, In vitro anti microbial activity of leaf extract of certain mangrove plants collected from Godavari estuarine of Konaseemma Delta, India.., Int. J.Med. Aroma Plants, 132 – 136, Sep 2011. [7] Takao KONOSHIMA,*,a Tenji KONISHI,a Midori TAKASAKI,a Kiyonori YAMAZOE,a and Harukuni TOKUDA, Anti-tumor-Promoting Activity of the Diterpene from Excoecaria agallocha. Biol. Pharm. Bull. 24(12) 1440—1442 (2001) [8] J.Margaret Beula ,M.Gnanadesigan,P.Banerjee Rajkumar,S.Ravikumar,M.Anand ,Antiviral ,antioxidant and toxicological evalution of mangrove plant from south east coast of india.Asian Pacific journal of Tropical biomedicine(2012)S352-S357.

*Corresponding Author: Kumar Periyasamy*

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P. G & Research department of botany, Jamal Mohamed College, Tiruchirappalli Tamil Nadu, India-620020 E-Mail:Kumarbiotechnology.periyasamy@gmail.com

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Research Article Biological Sciences IDENTIFICATION OF POTENTIAL ANTI-TUMORIGENIC TARGETS FOR ROSEMARY COMPONENTS USING DUAL REVERSE SCREENING APPROACHES SAVITA DESHMUKH*1, SHIVAKUMAR B MADAGI2, VIPRA SAVADATTI3 1*, 2, 3

Department of Bioinformatics, Karnataka State Womenâ&#x20AC;&#x2122;s University, Bijapur-586108, Karnataka, INDIA. *Corresponding Author Email: savitajayarao@gmail.com

ABSTRACT Introduction: Pioneering research on the role of phytochemicals for the treatment of various ailments in the traditional medicines has highlighted rosemary as one of the promising plant for cancer prevention and treatment. The anti-neoplastic properties of rosemary can be attributed to its major bioactive components like Carnosol, Carnosic acid (CA), Ursolic acid (UA), Rosmarinic acid (RA), Rosmanol and other diterpenes. However, only few targets for these bioactive components have identified. Objectives: (1) To identify the molecular targets for carnosol, carnosic acid, rosmarinic acid and ursolic acid.(2)To classify the targets based on their action as antiinflammatory, anti-apoptotic, signal transduction modulators, antioxidants, antimutagenic, etc. (3) To find the experimental proofs for the each bioactive component and the target. Material and Methods: The putative target identification was done by dual virtual reverse screening approach with the help of two servers namely, PharmMapper and idTatget. The target proteins with anti-neoplastic acivity ranked by fit score and binding energy were obtained and were classified based on their action. The targets obtained were validated from experimental studies. Results: The potential ani-neoplastic targets identified from both the servers were experimentally verified. The study also identified targets for rosemary components which are anti-neoplastic in nature but the experimental proof of their interaction with these components was not studied invivo. Conclusion: The present work using dual inverse screening strategy has revealed anti-tumorigenic targets for 4 bioactive components of rosemary. It also provides tractable set of anti-cancer targets for these components which can be further validated by invivo and invitro study.

KEY WORDS Anti-tumorigenic targets, molecular targets, reverse screening, rosemary.

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INTRODUCTION Of the drugs prescribed for cancer treatment majority of them are chemopreventive phytochemicals derived from traditional herbs1. The Southeast Asian countries are less prone to different types of cancers due to their dietary habits involving curcumin, onion, garlic, ginger, cruciferous vegetables, tomatoes, chilies etc. that suppress the transformative, hyper proliferative and inflammatory processes that initiate carcinogenesis2. The anti-cancer activities

of these plants are due to the presence of active components like Curcumin (diferuloylmethane) in Turmeric, Zerumbone in Ginger, isothiocyanates, indole-3-carbinol, isoflavones, protease inhibitors, saponins, phytosterols, inositol hexaphosphate, vitamin C, d-limonene, lutein, folic acid, Î˛-carotene, lycopene, selenium from different types of fruits and vegetables3. Rosemary (Rosmarinus officinalis L.) a member of Lamiaceae has been studied in the last decade

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www.ijpbs.com (or) www.ijpbsonline.com for many of its therapeutic effects on cenral nervous system (CNS), circulation, hepatoprotective effects, anti-tumorigenic effects etc., its chemo preventive effect is most widely studied4. Many researchers have shown its chemopreventive properties on neoplastic cells and experimental evidence indicating towards the anti-carcinogenic and antitumor activities both in invitro and invivo platforms 5-10, 17-20 . Rosemaryâ&#x20AC;&#x2122;s bioactive components include phenolic diterpenes or triterpenes, flavonoids and phenolic acids8 such as carnosic acid (CA), carnosol, ursolic acid (UA), caffeic acid, betulinic acid, rosmaridiphenol, rosmanol and rosmarinic acid (RA). The antitumor activities of the rosemary constituents are reported for carnosol, CA, UA and RA9, which have shown to inhibit tumor formation and promotion stages of tumorigenesis5, 10, 11-13. The antioxidant and antiinflammatory properties of the rosemary help the cells of the body to protect from damage. A study conducted on rosemary as a preclinical perspective has revealed that effects of rosemary are not species specific as its effects are consistent both in human cell lines and rodent models9. Rosemary has shown to suppress or block pro-inflammatory pathways in different cancer cells3. Its extracts have been studied for its antitumor or antineoplastic activities on different types of cancer cell lines/ rat or mice models and only one on human9. The probable mechanism for rosemary polyphenols in cancer prevention or progression has been revealed as arresting cell cycle at multiple phases, inhibition of tumor promoting pathways or by inducing cancer preventive markers. Many invivo studies have reported some of the molecular targets for rosemary components for their antioxidant, anti-inflammatory, antiapoptotic targets, signal transduction modulators, antimutagenic and epigenetic activity3-5, ,9,11-14, 21-22. Here in this study, we have

characterized major pharmacological therapeutic targets to which bioactive components of rosemary bind. In the present work, we have used comparative study of two reverse screening servers namely PharmMapper and the idTarget to identify the potential anti-tumorigenic targets for rosemary components. Both are open-source online servers which fishes putative therapeutic targets for the given molecule. PharmMapper uses reverse pharmacophore mapping strategy where as idTarget perform divide and conquer approach to predict possible binding targets. Thus dual reverse screening strategy was used to identify the potential anticancerous and antitumorigenic targets for the bioactive active components of the rosemary. Reverse screening approach searches a protein database against a given molecule. It involves reverse docking and Pharmacophore mapping which is gaining as quick and computationally rigorous alternative to fish molecular targets.The components of the rosemary bind to many therapeutic targets when searched through Reverse Pharmacology approach using PharmMapper and idTargets. In this study, we have identified and also classified the targets as antimutagenic, anti-inflammatory, antioxidant and signal transducing modulator for four of the rosemary components namely, carnosic acid, rosmarinic acid, carnosol and ursolic acid. The purpose of this work was to identify the potential targets for the bioactive components of rosemary that would help to predict the mechanism of their anti-neoplastic activity invivo. Recently several inverse docking tools have been reported such as INVDOCK15, Tarfisdock16, PharmaMapper17, idTarget18, Inverse Screening @tome server19. One such approach was used to identify the potential antineoplastic targets of tea polyphenols such as epigallocatechin gallate (EGCG), epigallocatechin

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www.ijpbs.com (or) www.ijpbsonline.com (EGC), epicatechin gallate (ECG) and epicatechin (EC) 20. In this work we have taken the targets from PharmaMapper and idTargets to analyse the anti-cancerous activity of rosemary bioactive components. The present work has identified anti-tumorigenic targets for 4 bioactive components of rosemary and also provides tractable set of anti-cancer targets for these components which can be further validated by invivo and invitro study. MATERIALS AND METHODS Retrieval of 3D Structures of Rosemary Polyphenols The three dimensional structures of four components of rosemary namely carnosol, carnosic acid, RA and UA were retrieved from PubChemâ&#x20AC;&#x2122;s database with pubChem IDs CID 442009, CID 65126, CID 5281792 and CID 64945 respectively. The .sdf file formats were then converted to .mol2 using MarvinSketch. This step was essential for both PharmMapper and idTarget servers to predict the molecular targets

for active components prior to submission. The.mol2 files were then submitted to both PharmMapper and idTarget for the identification of possible therapeutic targets for each of these components. Potential Therapeutic Target Identification Using PharmMapper and idTarget The PharmMapper is a web server (http://59.78.96.61/pharmmapper) that identifies potential target candidates for the given small molecule. During submission a maximum of 300 conformation generations were chosen and only human target set were considered to perform Genetic Algorithm (GA). The pharmocophore targets were then analysed based on the fit score. The idTarget, is also a web server (http://idtarget.rcas.sinica.edu.tw) which predicts possible binding of targets of a small chemical molecule via a divide and conquer docking approach. The idTarget reported an average of 7000 molecular targets for each of these components.

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RESULTS AND DISCUSSION

Figure 1. Structure of rosemary components Carnosol, CA, Rosmanol, UA and RA.

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The bioactive components studied for anticancer targets in this study include carnosol, carnosic acid (CA), ursolic acid (UA) and rosmarinic acid (RA) as shown in Figure 1. PharmMapper listed top 300 targtes based on the fit score for each of the component of rosemary. The idTarget server predicted 7527 targets for carnosol, 7535 for CA, 7526 targets for RA and 7278 for UA. Since idTarget server docks given molecule with whole set of PDB database only targets that are predicted by both PharmMapper and idTarget were considered for further analysis. Potential Anti-tumorigenic Targets for Carnosol Carnosol is a ortho-diphenolic diterpene which constitutes approximately 5% of the dry weight of the rosemary. Carnosol has been widely studied for its anti-cancer activities 6, 7, 11-14.The potential therapeutic targets for carnosol identified by reverse screening procedures were collected from PharmMapper that gives pharmacologically important targtes. The server predicted majority of targets that are proven experimentally for carnosol. Among the target screened, many were involved in anti-tumor activities which can explain the anti-cancer property of carnosol. The targets so obtained were classified based on their functions as antimutagenic, antioxidant, anti-inflammatory, signal transducing modulators, epigenetic and others. The targets were then confirmed by screening the results of targets predicted by idTarget server. Only those targets which are predicted by both the servers were considered since the approaches used by both the servers are different. There were total 80 targets under these categories predicted by both the servers. 25 targets were of Anti-inflammatory; 41 signal transduction modulators; 02 of anti-mutagenic; 01 epigenetic; 05 anti-oxidant atrgtes and 06 other receptors and enzymes involved in cancer.

Potential targets for carnosol identified by reverse screening procedures and experimentally compared data are listed in Table 1. Computed binding free energies and experimental references for several carnosolprotein complexes are also included. Carnosol binds to proteins of MAPK pathway which are the important targets of inflammation. Of the 80 targets that are predicted by both the serversâ&#x20AC;&#x2122; majority of them were signal transduction modulators which are involved in inflammation, antioxidant and antiapoptotic activities. Experimentally proven targets for carnosol are listed in the Table 1. Carnosol inhibits the early inflammation during cancer progress by binding to Mitogen-activated protein kinase 14 a stress related protein kinase , Cell division protein kinase 2 (CDK2) that controls cell division and Mitogen-activated protein kinase 10 involved in cell proliferation21,22. It binds to Glucocorticoid receptor which is the potent antiinflammatory target23. The anti-mutagenic experiment have proved the interaction of carnosol with Glutathione S-transferase A124. A study has shown that carnosol acts as an antagonist by binding to ligand binding site of androgen receptor25. Carnosol binds to PPARgamma which inhibits the transcriptional activation of COX-2 a protein produced during transformed cells and human malignancies26. This target was predicted by PharmMapper and not by idTarget. Carnosol also binds to Leukocyte elastase , Alcohol dehydrogenase, Glutathione-Stransferase-P, NAD(P)H dehydrogenase [quinone], Catheaspin S , Caspase 3, etc which are also therapeutic targets for cancer are not been been predicted by idTarget. Carnosol interaction with these targets are also not been studied till date. The list of anti-tumor targets to which the carnosol binds is shown along with the

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references of targets as anti-tumors in the Table 2. Table 1: Carnosol possible targets found by screening procedures compared with experiment. Target Name PDB IDs Predicted by Implicated by Energy Reference or Server Experiment Score Related (kcal/mol) Disease Mitogen-activated 1OUY PharmMapper/ Yes -8.08 [22] protein kinase 14 idTarget 2ZB0 -8.9 1ZZL -7.38 1BL7 -8.47 3FC1 -7.45 1ZYJ -8.11 1OZ1 -8.73 Cell division protein 1PYE PharmMapper/ Yes -7.97 [22] kinase 2 (CDK2) idTarget 3EZR -6.92 1R78 -7.58 2VTP -7.2 2BHE -8.32 1OIQ -7.95 Androgen receptor 3B67 PharmMapper/ Yes -9.21 [25] idTarget 1GS4 -9.28 1Z95 -9.01 2AX8 -8.67 Glucocorticoid receptor 3CLD PharmMapper/ No -9.23 Inflammation idTarget [23] 1NHZ -8.42 1M2Z -9.49 Mitogen-activated 1PMN PharmMapper/ Yes -8.02 [22] protein kinase 10 idTarget Glutathione 1GSE PharmMapper/ Yes -8.06 [24] S-transferase A1 idTarget 1GUH -8.45

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Table 2: Other Experimentally proven anti-tumor targets predicted only by ParmMapper to which Carnosol binds Target Name PDB IDs Rank Reference PPAR gamma 1RDT 146 [26] 2I4P 210 Glutathione-S- Transferase-P (GST-P) Alcohol dehydrogenase class-3 NAD(P)H dehydrogenase [quinone ]-1 Methionine Aminopeptidase Leucocyte elastase Cathespin S Mineralocorticoid Receptor

11GS 1MA0 1DXO 1BOA 1H18 1NQC 2AA5

83 95 142 8 27 140 177

[24] [27] [28] [29] [21] [30] [31]

Caspase-3

1RHR

184

[32]

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HSP90 Proteooncogene synthase tyrosine ptotein kinase ABL1 Tyrosine kinase ITK/TSK Baculoviral IAP repeat-containing protein 4 (XIAP) Histone-N-Methyltransferase Vascular endothelial growth factor Proto-ncogene tyrosine protein kinase src

1UYH 2HZI

69 263

[33] [34]

1SNU 1TFQ

269 290

[35] [36]

1JQE 1YWN 1O4A

100 113 221

[37] [38] [39]

Histone deacetylase-8 3-phosphoinositide-dependent kinase 1 Cyclin-A2

1T64 2PE1

261 21

[40] [41]

2IW6

156

[42]

protein

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Potential Anti-tumorigenic Targets for CA, RA and UA The anti-cancer properties of other components of the rosemary have also implicated in number

of experiments3-5,8-10. Therefore the anti-tumor targets for these components were also studied. The potential anti-tumor targets to which CA, RA and UA are listed In the Table 3.

Table 3: Potential anti-tumor targets for Carnosic acid (CA), Rosmarinic acid (RA) and Ursolic acid (UA) Ligand Target Name PDB ID Energy Score kcal/mol Name CA Glucocorticoid receptor 1M2Z -9.02 cAMP-specific 3,5-cyclic phosphodiesterase 4B 1XOS -8.9 Mitogen-activated protein kinase 14 1OVE -8.3 Macrophage metalloelastase 1ROS -8.74 Mineralocorticoid receptor 2AAX -9.24 Proto-oncogene tyrosine-protein kinase LCK 3BYU -8.43 Cell division protein kinase 2 1OIR -8.43 Caspase-3 1NMS -7.83 cAMP-specific 3,5-cyclic phosphodiesterase 4D 1Y2K -8.92 Glutathione S-transferase P 4PGT -7.79 Serine/threonine-protein kinase Chk1 2E9U -7.13 Vascular endothelial growth factor receptor 2 2P2I -8.72 Interleukin-2 1PY2 -8.34 Estrogen receptor 1YIN -8.91 Androgen receptor 1GS4 -8.8 3-phosphoinositide-dependent protein kinase 1 1UU9 -8.26 Tyrosine-protein phosphatase non-receptor type 1 1Q1M -8.15 RA Cathepsin K 1TU6 -8.47 Mitogen-activated protein kinase 14 3D83 -8.84 cAMP-specific 3,5-cyclic phosphodiesterase 4B 1Y2H -8.33 Mitogen-activated protein kinase 12 1CM8 -8.99 Matrix metalloproteinase-9 1GKC -10.67 International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)

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Ras-related protein Rap-2a Caspase-3 3-phosphoinositide-dependent protein kinase 1 Cell division protein kinase 2 Cell division protein kinase 6 ADAM 33 Glutathione S-transferase P Glutathione S-transferase Mu 1 Glutathione S-transferase A1 Alcohol dehydrogenase class-3 Mast/stem cell growth factor receptor Serine/threonine-protein kinase 6 Trifunctional purine biosynthetic protein adenosine-3 Cellular retinoic acid-binding protein 2 Serine/threonine-protein kinase Chk1 Proto-oncogene tyrosine-protein kinase Src Hypoxanthine-guanine phosphoribosyltransferase Tyrosine-protein phosphatase non-receptor type 1 Neprilysin Glutathione S-transferase P Dipeptidyl peptidase 4 Mitogen-activated protein kinase 10 Mitogen-activated protein kinase 14 Glucocorticoid receptor Macrophage metalloelastase cAMP-specific 3,5-cyclic phosphodiesterase 4B Interleukin-2 Cell division protein kinase 2 Calmodulin 3-phosphoinositide-dependent protein kinase 1 Serine/threonine-protein kinase Chk1 cAMP-specific 3,5-cyclic phosphodiesterase 4D Vascular endothelial growth factor receptor 2 Proto-oncogene serine/threonine-protein kinase Pim-1 Epidermal growth factor receptor Caspase-3 Proto-oncogene tyrosine-protein kinase LCK Glutathione S-transferase A1 Estrogen receptor Retinoic acid receptor RXR-alpha E3 ubiquitin-protein ligase Mdm2 Tyrosine-protein phosphatase non-receptor type 1

The PharmMapper gives the recurring targets with different PDB id. Therefore the above data

1KAO 1NMS 2PE2 2C6I 1XO2 1R55 4PGT 1XW6 1GUH 1MC5 1PKG 1MQ4 1MEN 1CBS 2HOG 1O47 1HMP 1ECV 1R1J 4PGT 2IIV 1PMN 1OVE 3CLD 1ROS 1XMU 1QVN 1OIR 1CTR 2PE1 2AYP 1Y2K 1Y6B 2BIK 3BEL 1NMS 1QPE 1GUH 1XP9 2P1U 1T4E 1Q6S

-10.53 -9.52 -8.19 -8.27 -9.7 -8.96 -7.42 -8.15 -8.1 -9 -9.93 -9.53 -8.86 -8.87 -6.31 -7.61 -7.87 -8.85 -9.73 -9.02 -9.12 -8.95 -9.32 -9.85 -9.05 -9.11 -8.3 -9.38 -8.3 -8.91 -7.94 -9.23 -8.32 -9.15 -9.11 -8.72 -8.49 -8.8 -7.19 -8.24 -9.34 -9.39

has been prepared based on the energy score given by idTarget. Only those targets among

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www.ijpbs.com (or) www.ijpbsonline.com them with highest binding energy were considered. The above data are support the anticancer properties of the rosemary components. Many of the targets that are listed are have been experimentally proven for the particular rosemary components. In this study, dual reverse screening approach was used to identify the potential targets for the bioactive components of Rosemary such as carnosol, CA, RA and UA. The results reveal that the reverse screening using PharmMapper and idTarget has characterized those targets for bioactive components of rosemary many of which are experimentally validated as their antineoplastic targets. Firstly the result revealed that targets like MAPK-14, CDK2, AR, PPAR gamma, are the experimentally proven for cancer. Screening also identified targets which are clinical targets with anti-cancer effects or enzymes that are involved in antitumor drug design. This work would help to enlighten on the anti-tumorigenic abilities of the bioactive components of the rosemary. The binding potential of rosemary ingredients to their novel set of potential targets can be further validated by invivo and invitro bioassays. This new reverse screening approach can be used as an alternative computational strategy to for quick identification of potential therapeutic targets in phytochemicals and medicinal plants. ACKNOWLEDGEMENT The authors would like to acknowledge Department of Bioinformatics, Karnataka State Women’s University, Bijapur for the continuous support during the research work.

10.

11.

12.

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Maria Russo, Carmela Spagnuolo, Idolo Tedesco and Gian Luigi Russo. Phytochemicals in Cancer Prevention and Therapy: Truth or Dare? Toxins. 2010; 2:517-551.

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Block G, Patterson B, Subar A. Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr Cancer. 1992; 18:1–29. Bharat B Aggarwal, Haruyo Ichikawa, Prachi Garodia, Priya Weerasinghe, Gautam Sethi, Indra D Bhatt, Manoj K Pandey, Shishir Shishodia and Muraleedharan G Nair. From traditional Ayurvedic medicine to modern medicine: identification of therapeutic targets for suppression of inflammation and cancer. Expert Opin. Ther. Targets 2006; 10:87-118. Al-Sereiti MR, Abu-Amer KM and Sen P. Pharmacology of rosemary (Rosmarinus officinalis Linn.) and its therapeutic potentials. Indian J Exp Biol. 1999; 37:12430. Ozlem Yesil-Celiktas, Canan Seviml, Erdal Bedir and Fazilet Vardar-Sukan. Inhibitory Effects of Rosemary Extracts, Carnosic Acid and Rosmarinic Acid on the Growth of Various Human Cancer Cell Lines. Plant Foods Hum Nutr. 2010; 65:158–163. Julia Bauer, Susanne Kuehnl, Judith M. Rollinger, Olga Scherer, Hinnak Northoff, Hermann Stuppner, Oliver Werz, and Andreas Koeberle. Carnosol and carnosic acids from Salvia officinalis inhibit microsomal prostaglandin E2 synthase-1. JPET Fast Forward. 2012; 1-35 Jeremy J. Johnson. Carnosol: a promising anti-cancer and anti-inflammatory agent. Cancer Lett 2011; 305:17. Ho C T, Wang M, Wei GJ, Huang TC and Huang MT. Chemistry and antioxidative factors in rosemary and sage. Biofactors. 2000; 13:161-6 Ngo SN, Williams DB and Head RJ. Rosemary and cancer prevention: preclinical perspectives. Crit Rev Food Sci Nutr. 2011; 51:946-54. Einbond LS, Wu HA, Kashiwazaki R, He K, Roller M, Su T, Wang X and Goldsberry S. Carnosic acid inhibits the growth of ER-negative human breast cancer cells and synergizes with curcumin. Fitoterapia. 2012; 83:11608. Valdés A, García Cañas V, Rocamora Reverte L, Gómez Martínez A, Ferragut JA and Cifuentes A. Effect of rosemary polyphenols on human colon cancer cells: transcriptomic profiling and functional enrichment analysis. Genes Nutr. 2013; 8:43-60. Tai J, Cheung S, Wu M and Hasman D. Antiproliferation effect of Rosemary (Rosmarinus officinalis) on human ovarian cancer cells in vitro. Phytomedicine. 2012; 19:436-43. Barni MV, Carlini MJ, Cafferata EG, Puricelli L and Moreno S. Carnosic acid inhibits the proliferation and migration capacity of human colorectal cancer cells. Oncol Rep. 2012; 27:1041-8.

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www.ijpbs.com (or) www.ijpbsonline.com 14. López Jiménez A, García Caballero M, Medina MA, Quesada AR. Anti-angiogenic properties of carnosol and carnosic acid, two major dietary compounds from rosemary. Eur J Nutr. 2011; 52:85-95. 15. Chen YZ and Ung CY. Prediction of potential toxicity and side effect protein targets of a small molecule by a ligand-protein inverse docking approach. J. Mol. Graph. Model. 2001; 20:199–218 16. Li H, Gao Z, Kang L, Zhang H, Yang K, Yu K, Luo X, Zhu W, Chen K and Shen J. TarFisDock: A web server for identifying drug targets with docking approach. Nucleic Acids Res. 2006; 34:W219–W224. 17. Xiaofeng Liu, Sisheng Ouyang, Biao Yu, Kai Huang, Yabo Liu, Jiayu Gong, Sisuan Zheng, Zhihua Li, Honglin Li and Hualiang Jiang. PharmMapper Server: a web server for potential drug target identification via pharmacophore mapping approach. Nucl. Acids Res. 2010; 38:W609W614. 18. Jui Chih Wang, Pei Ying Chu, Chung Ming Chen and Jung Hsin Lin. idTarget: a web server for identifying protein targets of small chemical molecules with robust scoring functions and a divide-and-conquer docking approach. Nucl. Acids Res. 2012; 40:W393W399. 19. Jean Luc Pons and Gilles Labesse. @TOME-2: a new pipeline for comparative modeling of protein–ligand complexes.Nucl. Acids Res. 2009; 37:W485-W491. 20. Rong Zheng, Tuan sheng Chen and Tun Lu. A Comparative Reverse Docking Strategy to Identify Potential Antineoplastic Targets of Tea Functional Components and Binding Mode; Int. J. Mol. Sci. 2011; 12: 5200-5212. 21. Poeckel D, Greiner C, Verhoff M, Rau O, Tausch L, Hörnig C, Steinhilber D, Schubert-Zsilavecz M and Werz O. Carnosic acid and carnosol potently inhibit human 5-lipoxygenase and suppress pro-inflammatory responses of stimulated human polymorphonuclear leukocytes. Biochem Pharmacol. 2008; 76:91-7. 22. Johnson JJ, Syed DN, Heren CR, Suh Y, Adhami VM and Mukhtar H. Carnosol, a dietary diterpene, displays growth inhibitory effects in human prostate cancer PC3 cells leading to G2-phase cell cycle arrest and targets the 5'-AMP-activated protein kinase (AMPK) pathway. Pharm Res. 2008; 25:2125-34 23. Smoak KA and Cidlowski JA. Mechanisms of glucocorticoid receptor signaling during inflammation. Mech Ageing Dev. 2004; 125:697-706. 24. Hayes JD and Pulford DJ, The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol. 1995; 30:445-600.

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|399-408 25. Johnson JJ, Syed DN, Suh Y, Heren CR, Saleem M, Siddiqui IA and Mukhtar H. Disruption of androgen and estrogen receptor activity in prostate cancer by a novel dietary diterpene carnosol: implications for chemoprevention. Cancer Prev Res. 2010;.3:1112-23. 26. Kotha Subbaramaiah, Philip A Cole and Andrew J. Dannenberg. Retinoids and Carnosol Suppress Cyclooxygenase-2 Transcription by CREB-binding Protein/p300-dependent and independent Mechanisms. Cancer Res. 2002; 62:2522. 27. Reisdorph R and Lindahl R. Constitutive and 3methylcholanthrene-induced rat ALDH3A1 expression is mediated by multiple xenobiotic response elements. Drug Metab Dispos. 2007; 35:386-93. 28. Li R, Bianchet MA, Talalay P and Amzel LM. The threedimensional structure of NAD (P) H: quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction. PNAS. 1995; 92: 88468850. 29. Eric C Griffith, Zhuang Su, Benjamin E Turk, Shaoping Chen, Yie Hwa Chang, Zhuchun Wu, Klaus Biemann and Jun O Liu. Methionine aminopeptidase (type 2) is the common target for angiogenesis inhibitors AGM-1470 and ovalicin. Chemistry & Biology. 1997; 4:461–471. 30. Nomura T and Katunuma N. "Involvement of cathepsins in the invasion, metastasis and proliferation of cancer cells". J. Med. Invest. 2005; 52:1–9. 31. Chantong B, Kratschmar DV, Nashev LG, Balazs Z and Odermatt A.Mineralocorticoid and glucocorticoid receptors differentially regulate NF-kappaB activity and pro-inflammatory cytokine production in murine BV-2 microglial cells. J Neuroinflammation. 2012; 28:260. 32. Qian Huang, Fang Li, Xinjian Liu, Wenrong Li, Wei Shi, Fei-Fei Liu, Brian O'Sullivan, Zhimin He, Yuanlin Peng, Aik-Choon Tan, Ling Zhou, Jingping Shen, Gangwen Han, Xiao-Jing Wang, Jackie Thorburn, Andrew Thorburn, Antonio Jimeno, David Raben, Joel S Bedford and Chuan Yuan Li. Caspase 3–mediated stimulation of tumor cell repopulation during cancer radiotherapy. Nature Medicine. 2011; 17:860–866. 33. Jane Trepe., Mehdi Mollapou., Giuseppe Giaccone and Len Neckers. Targeting the dynamic HSP90 complex in cancer. Nature Reviews Cancer. 2010; 10: 537-549. 34. Goldberg Z, Vogt Sionov Ronit, Berger Michael, Zwang Yaara, Perets Ruth, Van Etten Richard A, Oren Moshe, Taya Yoichi and Haupt Ygal. "Tyrosine phosphorylation of Mdm2 by c-Abl: implications for p53 regulation". EMBO J. 2002; 21:3715–27. 35. Bagga V, Silakari O, Ghorela VS, Bahia MS, Rambabu G and Sarma J. A three-dimensional pharmacophore modelling of ITK inhibitors and virtual screening for

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novel inhibitors. SAR QSAR Environ Res. 2011; 22:17190. Qiang Song Tong, Li Duan Zheng, Liang Wang, Fu Qing Zeng, Fang Min Chen, Ji-Hua Dong and GongCheng Lu.Downregulation of XIAP expression induces apoptosis and enhances chemotherapeutic sensitivity in human gastric cancer cells. Cancer Gene Therapy. 2005; 12:509–514. Albert M., Helin K. Histone methyltransferases in cancer. Semin Cell Dev Biol 2010; 21:209-20. Duncan TJ, Al Attar A, Rolland P, Scott IV, Deen S, Liu DT, Spendlove I and Durrant LG. Vascular endothelial growth factor expression in ovarian cancer: a model for targeted use of novel therapies? Clin Cancer Res. 2008; 14:3030-5. Ottenhoff-Kalff AE, Rijksen G, Van Beurden EACM, Hennipman A, Michels A. A and Staal GEJ.

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|399-408 Characterization of Protein Tyrosine Kinases from Human Breast Cancer:Involvement of the c-src Oncogene Product1. Cancer Res. 1992; 52:4773-4778. 40. Hsiang Yu Lin, Chang Shi Chen, Shuan Pei Lin, Jing Ru Weng and Ching Shih Chen. Targeting histone deacetylase in cancer therapy. Medicinal Research Reviews. 2006; 26:397–413. 41. Kulp SK, Yang YT, Hung CC, Chen KF, Lai JP, Tseng PH, Fowble JW, Ward PJ, Chen CS. 3-phosphoinositidedependent protein kinase-1/Akt signaling represents a major cyclooxygenase-2-independent target for celecoxib in prostate cancer cells. Cancer Res. 2004; 64:1444-51. 42. Yam CH, Fung TK and Poon RY. Cyclin A in cell cycle control and cancer. Cell Mol Life Sci. 2002; 59:1317-26.

*Corresponding Author: SAVITA DESHMUKH

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Department of Bioinformatics, Karnataka State Women’s University, Bijapur-586108, Karnataka, INDIA.

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Research Article Biological Sciences EFFECT OF CADMIUM ON ANTIOXIDANT METABOLIC MODULATIONS IN HEART AND MUSCLE OF FEMALE RABBITS K. Subba Rao, J.A.Pradeepkiran, V.Praveen chakravarthi, M.Bhaskar* Division of Animal Biotechnology, Department of Zoology, Sri Venkateswara University, Tirupati - 517502, AP, India. *Corresponding Author Email: matchabhaskar2010@gmail.com

ABSTRACT Disturbance in pro-oxidant/anti-oxidant systems and oxidative challenges of cadmium induced altering the antioxidant system in both heart and muscle tissue of female rabbits were studied. The cadmium induced oxidative stress leads to the tissue destruction by the elevated free radicals by suppression of antioxidant scavenger system. The imbalance between the free radicals and scavenger antioxidants leads to the tissue damage caused by the free radicals and peroxidants in rabbits. The present findings suggest that cadmium induction decreased Superoxide dismutase (SOD) activity is observed in both tissues like myocardial heart and muscle of rabbits over control rabbits. The decreased SOD activity on induction of cadmium envisages reduced neutralization of superoxide anions which might leads to cause an increase in superoxide radicals. The Glutathione-S-transferase (GST) activity was significantly inhibited in both heart and muscle on induction of cadmium. The rate of inhibition was maximum in heart myocardial tissue than in muscle when compared to control. Suppressed Reactive oxygen species (ROS) formation in both heart and muscle tissues by inhibiting Xanthine oxidase (XOD) activity on induction of cadmium. Decreased XOD can be envisaged that the XOD probably inter converting into xanthine dehydrogenase enzyme either by reversible sulphydryl oxidation or by irreversible photolytic modification. We concluded that the cadmium toxicity could have induced oxidative damage in both heart and muscle by enhancing peroxidation of membrane lipids by inducing inhibition of the antioxidant enzymes.

KEY WORDS Cadmium, antioxidants, Reactive oxygen species (ROS)

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INTRODUCTION The antioxidant system plays an important role in the prevention of oxidative damage caused by free radicals. Antioxidants are also widely used to preventing diseases such as cancer, coronary heart disease and neurological diseases [1]. Cadmium causes hypertension and arteriosclerosis without renal dysfunction in rats [2, 3, 4]. Recent investigation have established that free radicals may be important contributors to cardiac dysfunction and myocardial damage [5, 6] and free radical mediated cellular damage

and reactive oxygen species (ROS) had been the raised as contributing to metal mechanism of toxicity [7], cadmium plays an important role on antioxidant system in different animals [8, 9], it is essential to understand the impact of cadmium on antioxidant cascade. The superoxide dismutase is a primary antioxidant enzyme and is essential to the organism to fight against oxidative effects of free radicals. It is well established that catalase and glutathione peroxidase also plays a vital role in reducing the risk of oxidative stress [10, 11]. The highly

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www.ijpbs.com (or) www.ijpbsonline.com reactive free hydroxyl radicals (OH-) by the Fenton reaction, which is widely believed to be mainly responsible for oxidative damage [12,13]. Glutathione peroxidase converts H2O2 and/or other lipid peroxides to H2O and hydroxyl lipids and in the process glutathione is converted to oxidized glutathione (GSSG). The GST catalyses the conjugation of glutathione (GSH) with wide variety of several other organic compounds, including hydroperoxides thereby shares peroxidase activity [14]. The inhibition in the GST activity indicates that the breakdown of toxic compounds such as glutathione and hydroperoxide could have been reduced. In this present study we conducted an experiment to understand the role of cadmium toxicity on antioxidant system which includes the enzymes superoxide dismutase, catalase, glutathione-S-transferase, xanthine oxidase and lipid peroxidation status was tested in heart myocardial tissue and muscle tissues of rabbits.

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MATERIALS AND METHODS Twelve healthy female New Zealand white breed rabbits (Oryctolagus cuniculus) with an age group of 2 Months, (weight: 1.0 Âą 0.2 kg) were obtained from Veterinary College, Tirupati, India. And divided into two groups, each group consists of six individuals, group one is kept as control and another as experimental group. After procurement they were thoroughly examined and acclimatized to lab conditions prior to start the experiment. The rabbits were maintained at laboratory conditions (26 Âą 2oC; 12 h light & 12 h dark cycle) throughout the study. The animals had free access to standard laboratory chow food and clean water adlibitum. All care and management procedures for rearing of rabbits were in accordance with the National Institutes of Health Guidelines on the Care and Use of Laboratory Animals, and the approval of the Ethical Committee of the S.V. University was

obtained prior to the study. Rabbits in group 1(control) were administered single dose of cadmium chloride (25mg/kg body weight) every day orally for a period of two weeks. Rabbits in group 2 receive only normal water was given orally. The cadmium in form of CdCl 2 dissolved in normal saline was administered to the rats at a dose of 25mg/kg body weight two weeks to the termination of the experiment. At the end of 2 weeks experimental study period animals were sacrificed by cervical dislocation. The biochemical estimations and myocardial tissue damage was excised after two weeks period of exposure.

BIOCHEMICAL ESTIMATIONS The scavenger enzymes which includes the Superoxide dismutase, Catalase, Glutathione-Stransferase, Xanthine oxidase all these enzymes activity studied in the present investigation, and the assays were standardized in both experimental and control tissues by conducting preliminary tests to determine the optimal PH, temperature, enzyme and substrate concentrations and these optimal conditions were subsequently followed for each enzyme assay. Superoxide dismutase activity was measured as the inhibition of photo reduction of nitro blue tetrazolium (NBT) by the enzyme as per the method of [15]. The reaction samples were placed under fluorescence light for 30 minutes and the resulting color was read at 560 nm against the reagent blank and kept in dark place. The activity of the enzyme was expressed as units/ mg protein/ min. Catalase activity was determined by a slightly modified version of [16]. In this method decreased optical density of Catalase was measured at 240 nm for 60 seconds. The molar extinction coefficient of 43.6 M cm-1 was used to determine Catalase activity. Glutathione-s-transferase activity was measured with its conventional substrate 1-chloro 2, 4-

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www.ijpbs.com (or) www.ijpbsonline.com dinitrobenzene (CDNB) at 340 nm as per the method of [17]. In this method the reaction mixture glutathione and enzyme source was initiated by the addition of glutathione and the absorbance was read at 340 nm against reagent blank. Xanthine oxidase activity was estimated by the dye reduction method of [18]. The reaction formazan formed was estimated extracted over night in to 5 ml of toluene and intensity of the color was read at 495 nm in spectrophotometer against toluene blank.

ESIMATION OF LIPID PEROXIDATION Lipid peroxidation can be determined by estimating the levels of thiobarbituric reactive substances (TBARS) levels following the method of Miller and Aust (1989). Tissue homogenate (1 mg protein) in 0.15 M KCl, 0.25 M Tris-HCl buffer, 2 m mole ADP and 10 μ moles FeSO4 were incubated at 37 °C for 5 min. The reaction (final volume of 1.0 ml) was initiated by adding 0.1 mM ascorbic acid. The reaction was terminated after 30 min by adding 2 ml of thio-barbituric acid reagent (0.375% TBA, o.2 N HCL, 15% TCA) and aldehydes formed were determined.

STATISTICAL ANALYSIS

was made with the help of Independent –Sample t-Test and the P<0.01 was considered as significant.

RESULTS Decreased antioxidant system which includes superoxide dismutase (SOD) after two weeks dosing with Cd 25mg/kg body weight significantly (P>0.01) The level of experimental rabbits was lowered in heart (-34.207) and muscle (-26.984) over control animals. (Table1).followed by of catalase level of experimental rabbits was reduced in heart (-36.269) and muscle (-28.482) than control were observed and the percent change of glutathione-stransferase level of experimental rabbits was decreased in heart (-39.429) and muscle (43.951) over control animals. And finally of xanthine oxidase level of experimental rabbits was considerably reduced in heart (-59.561) and muscle (-20.00) over control. Which includes evidenced that lipid peroxidation increased in the level of experimental rabbits was considerably in heart (+76.923) and muscle (+93.061) over control. The oxidative stress may be due to imbalance of antioxidants and peroxidants which might be induced due to several factors and heart and muscle of myocardial tissue destruction observed by the continues exposure of free radicals induced by cadmium. The antioxidant system consists of glutathione, superoxide dismutase (SOD), catalase and xanthine oxidase (XOD).The data is presented in (Table 1). The analysis of various enzymes was studied at the sub cellular level in heart and muscle of both control and experimental female rabbits.

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Statistical analysis (ANOVA) analysis of variance used to test the differences between treatment effects and the control to analyze various enzymes was studied at the sub cellular level in heart and muscle of both control and experimental female rabbits was tested and performed by using statistical package, SSPS 16.0.1 to determine means and standard deviations were calculated. The percent change over control was calculated and presented for each parameter. Comparison between means

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Table-1: Antioxidant Enzyme levels heart and muscle tissues. Parameters

Tissue

Control

Experimental

Superoxide dismutase (SOD)( Superoxide anion reduced /mg protein/min)

Heart

7.63+0.241

5.02 + 0.259

Muscle

8.82+0.280

6.44+0.420

-26.984 P<0.001

Heart

1.93+0.082

1.23+0.092

-36.269 P<0.001

Muscle

1.12+0.029

0.801+0.022

-28.482 P<0.001

Heart

3.83+0.35

2.32+0.117

-39.429 <0.001

Muscle

2.48+0.124

1.39+0.083

-43.951 P<0.001

Heart

0.319+0.026

0.129+0.008

-59.561 P<0.001

Muscle

0.165+0.012

0.132+0.010

-20.00 P<0.001

Heart

1.95+0.036

3.42+0.053

+76.923 P<0.001

Muscle

2.45+0.023

4.73+0.083

+93.061 P<0.001

Catalase (µ moles of H2O2 reduced /mg protein/min) Glutathione -STransferase (GST) (µ moles of NADPH oxidized / mg protein/ min) Xanthine oxidase µ moles of formazan formed ( XOD) (Units/mg protein/min) Lipid peroxidation (µ moles of TBARS cleaved/g wet/of tissue/h

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DISCUSSION Cadmium could not generate free radicals by its own even though the byproducts of superoxide radical, hydroxyl radical and nitric oxide radicals could be generated indirectly through competitive binding kinetics with antioxidant defense enzymes. It does not participate in Fenton-type reactions [19] but can indirectly favour the production of different ROS, such as hydrogen peroxide (H2O2), superoxide (O2 ·2), and hydroxyl radical (·OH), by unknown mechanisms, giving rise to an oxidative burst [20,21,22].The enzymes superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX) are involved in the detoxification of O2 ·2 (SOD) and H2O2 (CAT, GST), thereby preventing the formation of ·OH

%Change -34.207 P<0.001

radicals.The present findings suggest that cadmium induction decreased antioxidant system which includes the primary antioxidant enzyme SOD decreased activity is observed in both the tissues like heart myocardial and muscle of rabbits over control rabbits. The primary reason could be attributed that this enzyme is naturally present in every living organism and its presence always regulates the formation of peroxidative free radicals and each in turn prevents the cells from damage caused by imbalance between oxidant and antioxidant systems. Hence the decreased SOD activity could have been accelerated the oxidative stress which might be induced by the cadmium treatment. Usually the superoxide anions are formed by addition of one electron with a negative electric

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www.ijpbs.com (or) www.ijpbsonline.com charge on molecular oxygen. This mechanism could have been obstructed in both heart and muscle tissues due to the inhibition of SOD activity [23] suggested acute cadmium exposure caused increased oxidative stress by producing superoxide anions. All the above reports provided ample evidence and support the result exhibits the decreased SOD activity due to increased ROS production in both heart myocardial tissue and muscle tissues [24]. The decreased SOD in heart and muscle might result in reduced conversion of free radicals. The decreased SOD activity might depend on the imbalanced expression of the antioxidative enzymes SOD, and elevated ROS leads to the cellular destruction by the continuous exposure of free radicals. These results initiated to study another important peroxidation enzyme, catalase to understand the impact of cadmium on free radical metabolism in heart and muscle. Another scavenger enzyme Catalase is present in the peroxisomes of nearly all aerobic cells and preserves to protect the cell from the toxic effects of H2O2 by catalyzing its decomposition into molecular oxygen and water without the production of free radicals. Catalase is one of the most important antioxidant enzymes which cleave the toxic H2O2 substrate into water. One molecule of catalase can convert millions molecules of H2O2 into water and oxygen per second [25]. Catalase also involves in the removal of toxic H2O2 from the cell. The inhibition of catalase activity could have altered the protection of cells from the toxic injury caused by free radicals in both heart and muscle. Catalase activity was inhibited maximum in heart tissue than muscle on induction of cadmium in the present study which indicates that the conversion of H2O2 is reduced more in both the tissues (Table-1). The cytosolic enzyme catalase activity was inhibited in both heart and muscle tissues on treatment with cadmium indicates

that the cadmium induction might cause damage to the peroxisomes, which in turn might lead to the loss of detoxificatory mechanism. Numerous reports depicted that cadmium intoxication significantly increased the malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) Free radicals generated by cadmium were scavenged by GSH directly or via the GSH peroxidase/GSH system. Acute intoxication of animals with cadmium has shown increased activity of antioxidant defense enzymes like copper-zinc containing superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase. Glutathione-stransferase (GST) is another important detoxification enzyme which regulates the formation of glutathione compounds in the cells. Especially the GST participates in the detoxification of reactive electrophilic compounds by catalyzing their conjugation to glutathione. The GST activity was significantly inhibited in both heart and muscle on induction of cadmium. The rate of inhibition was maximum in heart myocardial tissue than in muscle when compared to control (Table 1). It indicates that the cadmium induction caused maximum loss of detoxificatory mechanism in heart myocardial tissue than in muscle. The GST catalyses the conjugation of glutathione (GSH) with wide variety of several other organic compounds, including hydroperoxides thereby shares peroxidase activity [14]. It indicates that GST not only participates independently in the detoxificatory mechanism by cleaving the glutathione compound but also associates peroxidases to convert certain species of hydroperoxides. The inhibition in the GST activity in the present study indicates that the breakdown of toxic compounds such as glutathione and hydroperoxide could have been reduced. Xanthine oxidase enzyme activity (XOD) which is another important enzyme which

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www.ijpbs.com (or) www.ijpbsonline.com involving in the cascade of ROS generation, it is well known that the XOD enzyme catalyzed the oxidation of hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric acid. The XOD activity was significantly decreased in both heart and muscle tissues in cadmium treated animals when compared to their respective controls. This inhibition of these antioxidant enzymes will make such tissues prone to damage by free radicals and in part would have contributed to the observed increase in MDA levels of heart and muscle.

CONCLUSION: In conclusion that myocardial tissue injury refers to irreversible cellular injury (necrosis) induced by detrimental scavenger system due to elevated ROS with continuous exposure of free radicals to the cell leads to necrosis. Despite being essential for the ultimate survival of the myocardial cell, the sudden reintroduction of molecular oxygen can be unfavourable to the ischaemic myocyte. The cadmium toxicity could have induced oxidative damage in both heart and muscle by enhancing peroxidation of membrane lipids by inducing inhibition of the antioxidant enzymes.There is a complex interrelationship among the metal induced toxicity that contribute to free radical exposure leads to tissue injury due to imbalanced antioxidant system leads to ROS are produced by the injured endothelium, and the ischaemic myocardium itself Free radical scavengers, including SOD, catalase and SOD mimetics, decrease the contribution of superoxide (O2) and hydroxyl radicals (OH2-) leads to myocardial cell injury.

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IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|409-415 [2] Perry, H.N. and Erlanger, M.E. Metal – induced hypertension following chronic feeding of low doses of cadmium and mercury. J Lab, Clin. Med 83:541. (1974). [3] Revis NW, Zinsmeister AR, Bull R. Atherosclerosis and hypertension induction by lead and cadmium ions: an effect prevented by calcium ion. Proc Natl Acad Sci USA. 78:6494–6498. (1981). [4] Subramanyam, G., Bhaskar, M. and Govindappa, S. The role of cadmium in induction of Atherosclerosis in Rabbits. Heart Journal Vol.44, No.3, PP.177-180. (1992). [5] Mary Keith, RD; Amir Geranmayegan, Michael J Sole, Regina Kurian, Andrew Robinson, Ahmad S Omran, Khursheed N Jeejeebhoy. Increased Oxidative Stress in Patients With Congestive Heart Failure 1. J Am Coll Cardiol. 31(6):1352-1356. (1998). [6] Pichardo J, Palace V, Farahmand F and Singal PK. Myocardial oxidative stress changes during compensated right heart failure in rats. Mol Cell Biochem; 196:51–57, (1999). [7] Stohs, S.J., Bagchi,D., Hassoun, E. and Bagchi, M.M. Oxidative Mechanisms in the Toxicity of Chromium and Cadmium Ions. J.Environ.Pathol.Toxical.Oncol.19. pp. 201-213. (2000). [8] Ebru Beytut, Mesut Aksakal. The effect of long-term supplemental dietary cadmium on lipid peroxidation and the antioxidant system in the liver and kidneys of Rabbits. Turk j vet anim Sci 26: 1055-1060. (2002). [9] Yalin S, Comelekoglu U, Bagis S, Sahin NO, Ogenler O, Hatungil R. Acute effect of single-dose cadmium treatment on lipid peroxidation and antioxidant enzymes in ovariectomized rats. Ecotoxicol Environ Saf; 65:140–4. (2006). [10] Mc Cord J M and Fridovich I. Superoxide dismutase an enzymatic function for erythrocuprein (haemocuprein); J. Biol. Chem. 244 6049-6055. (1969). [11] Nissen, HP. & Kreysel, HWA. Superoxide dismutase in human semen. Klin Wochenschr, 61 (1): 63-65, ISSN 0023-2173. (1983). [12] Fridovich I. The biology of oxygen radicals. Science, 201, 875–880. (1978). [13] Halliwell, B. H. & Gutteridge, J. M. C. Iron and free radical reactions: Two aspects of antioxidant protection. Trends biochem. Sci. 11, 372–375. (1986). [14] Habig, W.H., Pabst, M.J., and Jakpby, W.B. GlutathioneS-transferases. J.Boil. Chem. 249:7130-7139. (1974). [15] Beauchamp, C. and Fridovich, I. Anal. Biochem., 44, 276. (1971). [16] Aebi, H. Catalase in Vitro. Methods Enzymol.105: 121126. (1984). [17] Srikanthan, T.N., Krishnamurthy, C.R., J Sci Indust Res, 14, 206-207. (1955).

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www.ijpbs.com (or) www.ijpbsonline.com [18] Stohs, S.J., Bagchi,D., Hassoun, E. and Bagchi, M.M. Oxidative Mechanisms in the Toxicity of Chromium and Cadmium Ions.J.Environ.Pathol. Toxical.Oncol.19. pp. 201-213. (2000). [19] Olmos E, Martinez-Solano JR, Piqueras A, Hellin E .Early steps in the oxidative burst induced by cadmium in cultured tobacco cells (BY-2 line). J Exp Bot 54: 291– 301. (2003). [20] Romero-Puertas MC, Rodríguez-Serrano M, Corpas FJ, Gómez M, del Rio LA, Sandalio LM .Cadmium-induced subcellular accumulation of O2− and H2O2 in pea leaves. Plant Cell Environ 27: 1122–1134. (2004) [21] Garnier L, Simon-Plas F, Thuleau P, Agnel JP, Blein JP, Ranjeva R, Montillet JL .Cadmium affects tobacco cells by a series of three waves of reactive oxygen species

[22]

[23]

[24] [25]

that contribute to cytotoxicity. Plant Cell Environ 29:1956–1969. (2006). Koizymi, T. and Li, Z.G. Role of oxidative stress in singledose cadmium induced testicular cancer. J. Toxicol. Environ. Health 37, 25-36. (1992). Casalino, E., Calzaretti, G., Sblano,C. and Landriscina, C. Molecular inhibitory mechanism of Antioxidant enzymes in rat liver and kidney by cadmium. Toxicol. 179, 3750. (2002). Fita I, Rossmann MG. The active center of catalase. J. Mol. Biol. 185, 21-37. (1985). Habig, W. H. and Jakoby, W. B. Glutathione Stransferase (rat and human). Meth. Enzymol. 77, 218– 231. (1981).

*Corresponding Author: M.Bhaskar

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Division of Animal Biotechnology, Department of Zoology, Sri Venkateswara University, Tirupati - 517502, AP, India. *Corresponding Author Email: matchabhaskar2010@gmail.com

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Research Article Biological Sciences DISTRIBUTION OF VARIOUS CASTES IN DIFFERENT PARTS OF THE MOUND OF THE TERMITES ODONTOTERMES OBESUS RAMBUR (ISOPTERA-TERMITIDAE) A.Nageswara Rao*1, E.Narayana2, Ch. Sravanthy3 and Ch.Sammaiah4 1, 2, 3, 4

Department of Zoology Kakatiya University, Warangal-506009 *Corresponding Author Email: drapkanagesh@gmail.com

ABSTRACT Population density(per 100 gm unit of fungus garden) of workers, soldiers and nymphs of the termite Odontotermes obesus from different parts of the viz., Peripheral fungus garden, Ground level fungus garden, the royal chamber were investigated. The percentage of major workers in peripheral fungus garden was higher compared to the fungus garden around the royal chambers 70.37%, 61.45%, respectively. However, percentage of soldiers and nymphs were found high in the fungus around royal chambers than the peripheral fungus garden workers, soldiers and nymphs 16.35%, 9.75% and 9.66% respectively. It is evident from the above results that various castes of this species in different parts of the mound ate distributed according to their functional behavior. It is apparent that the proportion of workers in a colony varies with the season on which also depend the foraging activities of the subterranean termites. It has further been observed that soldier population is invariably associated with an increase in population of nymphs.

KEY WORDS Termites, Fungus garden, Density, Foraging, and Royal chamber.

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INTRODUCTION Population of various castes and their relative percentage in different species of termites has been studied by Holdaway et al (1935)1. Coptotermes lactens by Mukharjee and Mitra (1949)2. Odontotermes redemanni by Gupta (1953)3, O. assumuthi and O. wallonensis by Agarwal (1976)4, some observations on the nesting behavior of O. obesus and O. wallonesis from North India have been studied Mathur (1960)5, Roonwal (1970)6. O. obesus and O. micro dentatus Blum (1977)7, Veeranna and Basalingappa (1981)8 have studied the total population and relative percentage of foraging forms of O. wallonensis from the covered runway raised on the eucalyptus trees. The size of relationship in nest population and mound parameters in the termite Macrotermes

michaelseni in Kenya (Darlingtonand dransdfield, 1987)9. Population in nests of the termite (Darlignton, 1990)10. Abundance, distribution and colony size estimates Reticuletermes sp. (Howard et al., 1982)11. The total populations, proportion of various castes and seasonal fluctuation have been studied in detail especially in the family Termitidae (Darlington, 1990; Aktar and Rashid, 2001)12.Since there are more reports regarding the distribution of various castes in different parts of the mounds in this semi â&#x20AC;&#x201C; arid zone of peninsular India population density of various castes in different parts of the mound of the termites Odonototermes wallonensis studied by Vasantkumar and Vijaykumar (2010)13. Solider are sometimes larger than workers, with darker heads. Nesting pattern of the termites Odontotermes studied by Farzaua et al.,

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www.ijpbs.com (or) www.ijpbsonline.com (2010)14.Since there are more reports regarding the distribution of various castes in different parts of the mound in this semi-arid zone of peninsular India. Hence the present investigation was under taken to study the population density of Ondototermes obesus

OBJECTIVES

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The present investigation was under taken to study the diversity and population density of various castes in different parts of the termite mound with following objectives 1) To know the diversity and population pattern of various castes. 2) To know the distribution pattern of soldiers, workers, nymphs in different parts of the mound that helps to assess the activity of these members during the foraging etc., activity that inter help the to know the infestation of these members over the different plant species during different seasons. 3) To know the structural pattern of the mound

MATERIALS AND METHODS The Odontotermes obesus mounds were cut opened by digging the mound soil sampling was carry out at different ranging from Above (peripheral) ground level fungus garden, ground level fungus garden, Below ground fungus garden.Freshly collected royal chamber, fungus gardens (from Above ground level, Ground level, Below ground fungus gardens) from the mound nests in the field, the workers, soldiers and nymphs from covered. Population density of major and minor workers, soldiers and nymphsfrom the royal chamber was determined by “whole count” method. Population size of various castes from different parts of the fungus garden was estimated according to the “hand sorting” method and flotation technique. For the comparison of various castes in different parts of the mound, the P values were less than 0.05 which was statistically significant.

RESULTS AND DISCUSSION Population density of workers, soldiers and nymphs of the termite, Odonototermes obesus from foraging covered runways and from different parts of the mound, namely, above ground level fungus garden, ground level fungus garden, below ground fungus gardens itself is given in Table 1.

Table-1 Number of workers, soldiers and nymphs from fungus combs distributed in different parts of mound O. obesus (Rambur) in Bhadrachalam forest region Population Count Weight of Different parts of the Average fungus Mound total Workers Soldiers Nymphs Combs Above ground level 349.2 ± 8.12 42.8 ± 2.03 40.4±1.32 432 103 fungus gardens Ground level 173.4 ± 7.15 45.4 ± 3.69 63.01 ± 4.59 fungus gardens 281 100 Below ground level 122.0 ± 4.30 82.0 ± 3.39 1107.6±23.97 1311 106 fungus gardens

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www.ijpbs.com (or) www.ijpbsonline.com The number of workers, soldiers and nymphs from fungus combs distributed in different part of the mound of O. obesus in Bhadrachalam forest region. The number of different casts in fungus garden at above ground level of the mound (peripheral) recorded 349.2 ± 8.12, 42.8 ± 2.03, and 40.4 ±1.32 workers, soldiers and nymphs respectively. The fungus garden weight was 103 grams recorded in the peripheral region of the mound. The average total number of workers, soldiers, nymphs was recorded 432. The number of workers, soldiers and nymphs at ground level fungus garden 173.4 ± 7.15, 45.4 ± 3.69, and 63.01 ± 4.59 were recorded respectively. The weight of ground level fungus garden was 100g. The total populations of workers, soldiers, nymphs were recorded 281. At below ground level fungus garden 122.0 ± 4.30, 82.0 ± 3.39 and 1107 ± 23.97 workers, soldiers and nymphs respectively. The weight fungus comb was 106g was recorded and total number (1311) of workers, soldiers, nymphs were recorded

REFERENCES 1.

CONCLUSION 1)

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In the present investigation the population density of O. Obesus the percentage of minor workers was high in the royal chamber when compared to other parts of the mound. The high percentage of minor workers might be for the purpose of feeding the royal couple and young ones and for transporting eggs from the royal chamber to fungus garden. High percentage of soldiers in the royal chamber was presumably for guarding the royal couple and to get food from minor workers. Though the royal pair all the time was found well protected in. The royal chamber, the presence of high percentage of soldiers might be for facing the

rare invasion (s) of predatory ants as found in odontotermes obesus. High percentage of nymphs population in the fungus garden around the royal chamber is reasonable because thousands of eggs laid per day by the large physogastric queen were to be transported from the royal chamber and stocked in masses for incubation. Thus it is an evident in the present investigation the various castes of this species in different parts of the mound are distributed according to their functional behavior, that helps the understand activity in the different seasons of this species. During different activities like foraging, defensive, mound repair work etc.,

5. 6.

7. 8.

Holdaway F.G., Gay, F.J. and Greaves, G.The termite population of the mound colony of Eutermes exitiosus. J. Counc. Sci. Ind. Res. Melbourne, (8): 42– 48, New Delhi, UNESCO.(1935). Mukharjee and Mitra Mukerjee, D. and Mitra, P.K. (1949).Ecology of mound building termite.Odontotermes redemanni. Proc. Nat. Inst. Sci. India, 35(5): 361 – 367.(1949). Gupta S.D. Ecological Studies of termites. Part I: Population of the mound building termite Odontotermes obesus Proc. Nat. Inst. Sci. India, 19 (5): 697 – 704.(1953) Agarwal, V.B. Seasonal fluctuations of proportions of different castes as found in the fungus combs of Odontotermes obesus (Rambur) and O. microdentatus (Roonwal and sen-sarma). Proc. Soil Biol. Ecol., University of Agricultural Science, Bangalore,(22): 192 – 196.(1976). Mathur R.N. Indian J. Sci., 86 623 – 624 (1960) Roonwal, M.L. Termites of the Oriental region in K. Krishna and F.M. Weesner, Eds, Biology of termites.Academic press, New York, PP. 320 – 391. (1970). Blum Inspection for dry wood termites. 346 So. Sci 2nd 356.(1977) Veeranna and BasalingappaForaging behavior of the termite odonototermes wollanensis wasmann. India Zoologist 5: 5-9.(1981) Darlington, J.P.E.C and Dransfield, R.D Size relationships in nest population and mound parameters in the

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www.ijpbs.com (or) www.ijpbsonline.com termite macrotermes michealseni in Kenya. Insects Sociaux, 34: 165 – 180.(1987). 10. Darlignton, Population in nests of the termite Macrotermes Subhyalinus in Kenya. Insects Sociaux. 37: 158 – 168.(1990). 11. Howard, R.W., Jones, S.C., Mauldin, J.K. and Beal, R.H. Abundance, distribution and colony size estimates for Reticuitermes spp. (Isoptera: Rhinotermitide) in Southern Mississippi. Environ. Entomol. 11: 1290 – 1293).(1982). 12. Akthar, M.S.and Rashid, I.M. Studies on population density and diversity of termites of district

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|416-422 Bahawalnagar. Pakistan Journal of Zoology, 12(2): 95 – 104.(2001). 13. VasantKumar .B. K. Vijay Kumar Population density of various castes in different parts of the mound of the termite Odototermes wallonensis wasmann (Isopteran: Termitidae) in wet lands, biodiversity and climate change 1-4.(2010). 14. Farzana, J. Sangamma, I., Rajashekhar, M., Vijaykumar, K., Burli, P. and Chimkod, V.N. Nesting Pattern of the termites Odontotermes brunneus and Odontotermes wallonensis (Isoptera: Termitidae)(2010)

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Eggs and nymphs were recorded on fungus comb

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Early stage of fungus garden surrounding of royal chamber below ground level.

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Queen of O.obesus in broken royal chamber at bottom of the mound

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The mound of O.obesus above ground level distribution of fungus comb

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The mound of O.obesus surrounding royal chamber distribution of fungus combs.

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Different castes of O.obesus near royal chamber at bottom

The mound of O. obesus ground level distribution of fungus combs

*Corresponding Author:

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E-mail:drapkanagesh@gmail.com Telephone: 91+9849043610

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Research Article Biological Sciences GENOTOXIC EFFECTS OF DYEING INDUSTRY EFFLUENT ON A FRESHWATER FISH, CIRRHINUS MRIGALA BY CHROMOSOMAL ABERRATION TEST H. KAUR, R. KALOTRA, G. K. WALIA* and D. HANDA DEPARTMENT OF ZOOLOGY AND ENVIRONMENTAL SCIENCES, PUNJABI UNIVERSITY, PATIALA-147002 (PUNJAB) INDIA. *Corresponding Author Email: gurinderkaur_walia@yahoo.co.in

ABSTRACT Pollution of water resources is a serious and growing problem, despite the existence of relevant legislations. Genotoxic studies on industrial pollutants are very important as they tend to accumulate in aquatic animals. Thus use of aquatic organisms to detect the genotoxicity is very useful in environment monitoring. For this chromosomal aberration test was employed to study genotoxicity caused by dyeing industry effluent on a freshwater fish, Cirrhinus mrigala. Kidney tissue was used for the present study. The 96h LC50 was calculated and three sublethal concentrations 24.48%, 12.24% and 6.12% were prepared. Chromosomal preparations of control and treated fishes were made after 24h, 48h, 72h and 96h of exposure period. Somatic metaphase plate was prepared from control fishes to study normal chromosome complement and compared with aberrated somatic metaphase plates of treated fishes. Chromosomal aberrations included Chromosomal fragmentations (Cf), Ring chromosomes (Rc), Terminal chromatid deletions (Tcd), Minutes (M), Centromeric gaps (Cg), Stickiness (Stk), Clumping (C), Pycnosis (Py), Stretching (Stch) and Pulverization (P). Results revealed that dyeing industry effluent to be potentially genotoxic (p<0.05). Centromeric gaps, Clumping and Ring chromosomes were predominant aberrations observed in all concentrations. Higher concentration (24.48%) and maximum exposure period (96h) induces more chromosomal aberrations. 6.12% and lower than this concentration however can act as safe disposal concentration of effluent to be dumped in rivers. This type of study could be used as criteria for determining genotoxicity caused by industrial effluents, which can be used to avoid its toxic effects on aquatic environment.

KEY WORDS

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Chromosomal aberration test, aquatic pollution, dyeing industry effluent, Cirrhinus mrigala.

1. INTRODUCTION Pollution of the aquatic ecosystem is recognized as a potential threat to all living organisms. It is produced by man himself; therefore pollution and its effects are considered manâ&#x20AC;&#x2122;s greatest crimes against himself. Water quality is being unswervingly affected by man activities towards development. Maximum aquatic pollutants come from industrial effluents and agricultural run-off.

The river Satluj and its tributaries form the largest river system in Punjab. At Ludhiana, effluents from industries dealing with dyeing, hosiery, machine parts, paint, chemical, tannery and electroplating are added via a tributary, the Buddha Nallah while at Harike, industrial wastes from Jalandhar and Kapurthala enter Satluj via a tributary, the East-Bain. Industrial wastes are known to contain heavy metals and other poisonous substances. These pollutants may be

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www.ijpbs.com (or) www.ijpbsonline.com genotoxic and may lead to several human afflictions like cancer, atherosclerosis, cardiovascular diseases and premature ageing. The increasing degradation of the aquatic environment by anthropogenic contaminants has been the motivation behind increasing need and intensive efforts being made to determine critical concentrations of toxicants in rivers and other water bodies and to evaluate effects of pollutants in biological systems. Bioassays of pollutants can be performed in vitro and in vivo to assess their effects. Measurement of chromosomal aberrations offers an acceptable parameter for monitoring mutagenic substances in water. Changes in DNA or chromosomal functions may be a step in the pathway to carcinogenesis. Chromosomal aberrations in animals thus could serve as useful indicators of the presence of clastogenic chemicals. Chromosomal aberration test (CAT) is a promising tool for assessing the genotoxicity in animals. Several reports are available on chromosomal aberrations in fishes exposed to polluted aquatic environment [1-5]. In river Satluj, several species of fishes belonging to the family Cyprinidae are found. The people of Punjab consume a large amount of fish. In Punjab, there are 268 dyeing industries in Ludhiana alone which discharge their wastes into Buddha Nallah that joins the river Satluj at Gorsian, Kedarbaksh in the north-western corner of Ludhiana district. Punjab Pollution Control Board [6] has identified 85 industries as red industries whose wastes are highly toxic to aquatic environment and dyeing industry is one of these red industries. The river Satluj is highly contaminated by dyeing industry effluent. Most fishes sold in market are captured from local water areas of the river Satluj. In the present study, Cirrhinus mrigala a popular edible fish of Punjab has been selected to observe genotoxic effects of dyeing industry effluent. The present

study is aimed to investigate 1) To calculate 96h LC50 of the dyeing industry effluent 2) To determine three sublethal concentrations to observe chromosomal aberrations 3) To compare chromosomal aberrations with normal chromosomal complement 4) To determine the relative toxicity of three sublethal concentrations of the effluent.

2. MATERIALS AND METHODS 2.1 Collection of specimen Specimens of Cirrhinus mrigala measuring 6-8 cm in length and 30 â&#x20AC;&#x201C; 55gms in weight, were collected from government fish seed farm, Patiala. Fishes were treated with 0.1% KMnO4 solution for 30 minutes to remove any external infections and were acclimatized in laboratory for 20 days. Fishes were fed with pelted feed. Feeding was stopped 24h prior to commencement of genotoxicity tests and fishes were not fed during experimental periods. 2.2 Collection of dyeing industry effluent Effluent of dyeing industry was taken directly from the waste outlet of an industrial unit based in Ludhiana to conduct genotoxicity test against the fish. The dyeing industry effluent contain mercury, chromium, copper, zinc, nickel, lead, manganese, cadmium, chlorides, sulphates, phenolic compounds, oil and grease [7]. 2.3 Determination of 96h LC50 and selection of sublethal concentrations 96h LC50 was determined by the method suggested by Finney [8]. Three sublethal concentrations 24.48%, 12.24% and 6.12% (1/2, 1/4, 1/8 of 96h LC50 value) were selected. Experimental design Fishes were released in tubs containing water (control) and three sublethal concentrations (24.48%, 12.24% and 6.12%) for 24h, 48h, 72h and 96h by using the method given by Manna and Sadhukhan [9]. Three sets of experiments

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www.ijpbs.com (or) www.ijpbsonline.com were performed for each concentration. A total of 72 fishes were used for the experiment. 2.4 Measurement of chromosomal aberrations Kidney tissue was used to make metaphasic plates. Slides were stained by the method given by Tijo and Whang [10]. Twenty four fishes (6 per hour) were used for each concentration. For each duration, thirty slides were prepared. For each hour 300 plates (100 plates from ten slides) of control and treated groups were observed and photomicrographed. 2.5 Statistical analysis Data of chromosomal aberrations were subjected to ANOVA and Tukey test. Statistical analysis was done by using computer software ‘Graph pad prism’. p<0.05% was considered to be the level of significance. Statistical significance of chromosomal aberrations for control and treated groups of each concentrations and durations was also evaluated. Frequencies of chromosomal aberrations were expressed as Mean (%) ± S.E.

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3. RESULTS The 96h LC50 value of dyeing industry effluent against Cirrhinus mrigala using Finney method (1971) came out to be 48.97%. Somatic metaphase plates obtained from kidney tissue revealed diploid number of 50 chromosomes (Fig. 1). Observations pertaining to aberrations induced in chromosomes of fishes were recorded after 24h, 48h, 72h and 96h of exposure is summarized in Table. Control fishes showed almost negligible chromosomal aberrations when compared to those exposed to dyeing industry effluent. Ten types of aberrations were determined from three sublethal concentrations and these were Chromosomal fragmentations (Cf, Fig.3), Ring chromosomes (Rc, Fig.4), Terminal chromatid deletions (Tcd, Fig.5), Minutes (M, Fig.6), Centromeric gaps (Cg, Fig.7), Stickiness (Stk,

Fig.8), Clumping (C, Fig.9), Pycnosis (Py, Fig.10), Stretching (Stch, Fig.11) and Pulverization (P, Fig.12). At 24.48% concentration, Chromosomal fragments, Ring chromosomes, Terminal chromatid deletions, Minutes, Centromeric gaps and Pycnosis increased from 24h to 96h. Clumping and Stretching increased up to 72h then decreased and Stickiness and Pulverization decreased from 24h to 96h. Centromeric gaps were predominant and Stretching was lowest. At 12.24% concentration there was increase in chromosomal aberrations from 24 h to 96 h duration of time. Centromeric gaps and Ring chromosomes showed progressive increase with increase in duration of time. Clumping was the highest and Stickiness was the lowest among various aberrations. At 6.12% concentration, there was increase in chromosomal aberrations with increase in duration of exposure up to 72h but afterwards, there was a decrease. Centromeric gaps were observed to be the highest and Stickiness to be the lowest. Mean percentage of chromosomal aberrations increased with increase in concentration and exposure duration of dyeing industry effluent. At extreme exposure 96h, of all the three concentrations, frequency of chromosomal aberration rose steadily from 48.66±2.96d (6.12%) to 59.33±2.33d (12.24%) and 83.00±1.73d (24.48%) as shown in Histogram. Thus, as the concentration increased, the chromosomal aberrations registered a quantitative increase with the increase of exposure time. Overall percent frequencies of each chromosomal aberration in each concentration as well as control are shown in Pie Charts (a-d).

4. DISCUSSION In view of ever increasing levels of pollution caused by a wide variety of toxic substances in various water bodies, testing for potential

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www.ijpbs.com (or) www.ijpbsonline.com genotoxic effect on aquatic organisms has assumed a considerable significance. Chromosomal aberration test is one of the best tests for assessing genotoxicity of a pollutant in fishes with relative large chromosome number. It is not widely done due to its laborious work of studying metaphasic plates. Cirrhinus mrigala a popular edible fish found abundantly in rivers of Punjab was selected. There was a quantitative relationship between the occurrence of chromosomal aberrations and dyeing industrial effluent. This toxic effluent can alter the genetic material of fish following bioaccumulation in fish organs. Ten types of chromosomal aberrations were found in the present study. Increase in frequency of chromosome aberrations exhibited time and concentration dependent response. Dyeing industry effluent mainly contains heavy metals which affect replication, translation and repair of genetic materials. Similar work has been reported by other authors and suggested heavy metals disrupt DNA duplications during S phase, interfere with nucleotide synthesis and misreplicate damaged DNA leading to malformation of DNA molecules [11-13]. It has further been suggested that toxins may have strong oxidative effect on membrane phospholipid proteins and nucleic acids [14]. Heavy metals also generate reactive free radicals [15]. According to Natarajan and Obe [16], OH- and O2 - radicals are most relevant oxygen species which react with DNA causing DNA breaks that ultimately lead to chromosomal aberrations. Earlier workers also examined the effect of polluted water from industrial, agricultural and sewage runoff on fishes. Al-Sabti and Kurelec [1] recorded chromosomal aberrations (breaks and fragments) in Mytilus galloprovincialis collected from ten locations of Rovinj area. Kumari and Ramkumaran [2] observed that 65% of Channa punctatus collected from polluted Hussainsagar

Lake showed chromosomal aberrations. Hafez [3] observed higher frequency of Stickiness, fragments, gaps and deletions in Mugil cephalus at the most polluted site of Abu-qir bay. Mahmoud et al. [4] observed more Chromatid gaps, Chromatid deletions and Chromosome fragments in two fishes, Oreochromis niloticus and Tilapia zillii at highly polluted sites receiving sewage and other discharges. Similar observations were also recorded by Rose et al. [5] in freshwater fish, Hypophthalmus molitrix present in polluted sites of the river Coovum. Direct effect of dyeing industrial effluent was not studied before on fishes in Punjab. The present study revealed chromosomal aberrations increased with increase in concentration and time. Concentration 24.48% was more toxic than 12.24%. 6.12% and lower than this concentration, however can act as safe disposal concentration of effluent to be dumped in rivers.

5. CONCLUSION Industrial effluents contain large amounts of heavy metals which are genotoxic. These when present in water get incorporated in fish through the food chain may enter the human body and affect the health. Thus, it is very important and urgent to study the genotoxic effects of dyeing industrial effluent on fish. From present study it is clear that dyeing industry effluent is highly genotoxic for fishes and should be passed through effluent treatment plant before being discharged into the rivers. Legal actions should be taken to avoid any further damage to the fish fauna.

REFERENCES [1] Al-Sabti, K. and Kurelec, B., Induction of chromosomal aberrations in the mussel, Mytilus galloprovincialis. Bull. Environ. Contam. Toxicol., 35, 660-665, (1985) [2] Kumari, S.A., and Ramkumaran, S., Chromosomal aberrations in Channa punctatus from Hussainsagar

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[11] Evans, H.J., Molecular mechanisms in the induction of chromosome aberrations. D. Scott and B. A. Bridges (Eds.), Sobier/North Holland, Amsterdam, pp. 57-74, (1977) [12] Landolt, M.L., Kocan, R.M., Fish cell cytogenetics: A measure of genotoxic effects of environmental pollutants. In: Aquat. Toxicol, J. O. Nriagu (Ed.), John wiley and Sons Ins, pp. 335-352, (1983) [13] Matter, E.E., Elserafy, S.S., Zowail, M.E.M., and Awwad, M.H., Genotoxic effect of carbamyl insecticide on the grass carp, Ctenopharygodan idella. Egypt. J. Histol., 15(1), 9-17, (1992) [14] Chorvatovicova, D., Kovacikov, Z., Sandula, J. and Navarova, J., Protective effect of sulphoethylglucan against hexavalent chromium. Mutat Res. 302, 207211, (1992) [15] Tsalev, D.L., Zaprianov, Z.K., Atomic absorption spectrophotometry in occupational and environmental health practice. Vol. 1, CRC, Boca Raton, FL, USA, pp. 224-246, (1983) [16] Natarajan, A.T. and Obe, G., Molecular mechanisms involved in the protection of chromosomal aberrations. Mutat. Res., 52, 137-149, (1978)

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Lake, Hyderabad (A. P). Ind. J. fish., 53(3), 359-362, (2006) [3] Hafez, A.M., Mugil cephalus Genome: A sensitive monitor for genotoxicity and cytotoxicity in aquatic environment. Austral. J. Basic Appl. Sci., 3(3), 21762187, (2009) [4] Mahmoud, A., Mohamed, Z., Yossif, G., and Sharafeldin, K., Cytogenetical studies on some River Nile species from polluted and nonpolluted aquatic habitats. Egypt. Acad. J. Biol. Sci., 2(1), 1-8. (2010) [5] Rose, M.H., Sudhakar, K., Sudha, P.N., Effect of water pollutants on the freshwater fish, Hypophthalmicthys molitrix. The Ecoscan, 4(1), 31-35, (2010) [6] Punjab Pollution Control Board, List of red category of industries, Punjab, India, (1999) [7] Environment protection Rules, Ministry of Environment and Forests Notification, India, (2010) [8] Finney, D.J., Probit analysis. Cambridge University Press, London, pp. 333, (1971) [9] Manna, G.K., Sadhukhan, A., Use of cells of gills and kidney of Tilapia fish in micronucleus test. Curr. Sci., 55, 498-501, (1986) [10] Tijo, J.H., Whang, J., In: Human Chromosome Methodology, New York, Academic press, pp. 51-56, (1965)

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Table: Frequencies of chromosomal aberrations in kidney cells of Cirrhinus mrigala after treatment with dyeing industry effluent Experimental groups

Control

Chromosomal aberrations (Number of plates)

Duration of exposure (h)

24 48 72 96

Tcd

300 300 300 300

2 0 1 0 3

3 0 1 2 6

0 1 0 0 1

24 48 72 96

300 300 300 300

24 48 72 96

300 300 300 300

24 48 72 96

300 300 300 300

3 28 23 10 64 1 18 19 9 47 11 17 22 34 84

26 24 35 22 107 16 19 24 40 99 10 17 19 48 94

8 30 35 17 90 2 12 29 21 64 3 8 26 33 70

Total

6.12% Total

12.24% Total

24.48% Total

0 0 0 0 0 0 0 0 0 0 Treated 19 20 19 34 17 50 10 23 65 127 13 13 24 18 23 24 8 41 68 96 12 22 15 17 23 29 22 56 72 124

Mean(%)±S.E

0 0 0 0 0

7 3 3 2

2.33±0.66 1.00±0.57 1.00±0.57 0.66±0.33

11 6 4 2 23 10 3 6 3 22 17 6 5 3 31

114 186 199 146

38.00±2.89a 62.00±1.15b 66.33±1.86c 48.66±2.96d

84 153 162 178

28.00±0.33a 51.00±1.86b 54.00±1.86c 59.33±2.33d

117 125 187 249

39.00±1.00a 41.66±1.33b 62.00±2.19c 83.00±1.73d

Stk

Stch

0 1 0 0 1

1 1 0 0 2

0 0 1 0 1

1 0 0 0 1

0 2 7 12 16 0 4 4 3 11 17 3 3 15 38

19 34 17 34 104 20 30 21 29 100 17 23 45 16 101

4 3 4 6 17 7 21 5 20 53 2 16 7 21 46

4 6 7 10 27 2 4 7 4 17 6 3 8 1 18

a, b, c and d: significant differences at 24 h, 48 h, 72 h and 96 h respectively from the control at p<0.05. T= total number of metaphase plates, t= total number of metaphase plates with chromosomal aberrations. Cf= chromosome fragmentation, Rc= ring chromosome, Tcd= terminal chromatid deletion, M= minutes, Cg= centromeric gaps, Stk= stickiness, C= clumping, Py= pycnosis, Stch= stretching, P= pulverization.

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Fig.1 Normal metaphase complement

Figs. (2-9): Chromosomal aberrations: Cf= Chromosome fragmentation (Fig. 2), Rc= Ring chromosome (Fig. 3), Tcd= Terminal chromatid deletion (Fig. 4), M= Minutes (Fig. 5), Cg= Centromeric gaps (Fig. 6), Stk= Stickiness (Fig. 7), C= Clumping (Fig. 8), Py= Pycnosis (Fig. 9)

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Figs. (10-11): Chromosomal aberrations: Stch= Stretching (Fig. 10), P= Pulverization (Fig. 11).

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Frequency of chromosomal aberrations in kidney cells of Cirrhinus mrigala after treatment with dyeing industry effluent.

Percent frequency of chromosomal aberrations in kidney cells of Cirrhinus mrigala after treatment with dyeing industry effluent.

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J-1/2B, Gobind Colony, Rajpura-140401, Patiala, Punjab, India. Telephone number: 01762-223720 (Res.), 09815788644 (Mob.) E-mail address- gurinderkaur_walia@yahoo.co.in

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Research Article Biological Sciences FUSINUS NICOBARICUS â&#x20AC;&#x201C; AN EXCELLENT NEOGASTROPOD FOR PREPARATION OF GOOD RECIPES 1 1&3

P. Subavathy, 2*V. Chandramathi, 3R.D. Thilaga and 4M. Tamilselvi

P.G. & Research Department of Zoology, St. Mary's College (Autonomous), Thoothukudi P.G. & Research Department of Zoology, V.O. Chidambaram College, Thoothukudi. 4 V.V. Vannia perumal College for women, Virudhunagar

ABSTRACT Sea food is an excellent source of protein of high biological value. Like other aquatic organisms, the gastropods meat are also consumed as food, due to its high nutritional and medicinal value. The gastropod meat is not only tasty but also nutritious and free of cholesterol. In the present study, the precooked meat of foot and mantle of a gastropod Fusinus nicobaricus is used for the preparation of good and tasty recipes such as soup, curry and cutlets. It can be taken as fresh or in the frozen form.

KEY WORDS Fusinus nicobaricus, recipes, sea food

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INTRODUCTION Seafood is an excellent source of protein of high biological value and is one of the thrust sectors for augmenting countryâ&#x20AC;&#x2122;s export earnings. Fish, shell fish and other aquatic organisms suitable for food and feed are of worldwide importance. They are excellent sources of high quality proteins, superior to those in meat and poultry. Approximately 14 % of the animal protein consumed by human beings comes from marine fisheries (Pigott and Tucker, 1990). As the world population is growing, the per capita consumption of seafood is also increasing rapidly. Shell fishes are suitable for food and are of worldwide importance. The demand for these types of fishery products becomes increasing day by day among the non-vegetarian population throughout the world. The gastropod meat is not only tasty but also a nourishing food. The meat of gastropod Strombus canarium is used

for the preparation of good recipes such as soup, noodles, curry, cutlet and chilli (Arularasan et al., 2010). Some of the earlier works on the preparations of recipes in gastropods include those of Chicoreus ramosus and Faciolaria trapezium (Ragunathan et al., 1992; Ramesh and Ayyakannu, 1992; Hylleberg, 1992; Patterson Edward and Ayyakannu, 1992; Ayyakannu, 1994; Patterson et al., 1994; Gopakumar, 1996). Generally meat of molluscs considered to be highly nutritious, owing to its content of essential aminoacid, proteins, rich vitamins and minerals (Thanonkaew et al., 2006). Giese (1969) reported that the protein is the dominant organic constituent in molluscs than any other biochemical constituents. The biochemical composition of marine gastropod is a nutritional assurance of millions of malnourished people. The gastropod meat is free of cholesterol but contains high nutritive substances. It is used for the preparation of good recipes such as stews,

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www.ijpbs.com (or) www.ijpbsonline.com soups, salads, appetizers and hotpot mixed with other seafood in high end restaurants. Fusinus nicobaricus is one of the commercially important sources of sea food, their utilization is not popular like other sea food due to lack of perception combined with the conservative food habits of the people in India. Hence, the gastropod Fusinus nicobaricus meat was used for the preparation of good recipes such as soup, curry and cutlet. They have the potential to become a regular food item in the household diet, with consumer acceptance.

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MATERIALS AND METHOD Pre-cooking process: Fusinus nicobaricus were collected from Gulf of Mannar Coastal region with the help of divers. The animals were brought to the laboratory, cleaned and boiled in water for 30 minutes and the soft parts were removed from the shell. The edible portions such as foot and mantle were cut into small pieces according to each dish. After thorough washing the pieces of the meat were pressure-cooked until the meat becomes soft. The pre-cooked meat was used for preparing all dishes. The following dishes were prepared from the meat of Fusinus nicobaricus. The ingredients used and methods of preparations are given below: Soup Ingredients: Foot meat - 500g Large onion (Finely - 200g chopped) Ghee - 50g Garlic Paste - 25g Ginger paste - small piece Cumin seeds - 25g Spices - 2g 4 slices of dry bread toasted ¾ cup of tomato sauce

¼ cup of sweet chilli sauce ¼ cup of soya sauce 1 ½ teaspoons of salt All spices -required amount Method: The meat was boiled and the juice was extracted. The spices and ajinomotto were added at required levels. The tomato, chilli and soya sauce were added in required quantities. The cooked soup was filtered well and at last the toasted dry bread was added and served hot. Cutlet Ingredients: Potato - 2 Carrot - 2 Beans - 5 Onions - 2 (finely chopped) Coriander leaf - half bunch Ginger garlic paste - 1 spoon Chilli powder - 1 ½ spoon Garam masala - 1 ½ spoon Green chillies - 2 Meat - 1 cup Salt - required amount Method: Boiled potatoes were smashed well. After frying the meat was added. Finely chopped and boiled carrot, beans and onion were added to the smashed potatoes. Ginger garlic paste, garam masala, shopped green chillies, pepper powder and chilli powder were added to the smashed potatoes. Meat was added and smashed well. Then the mixture was made into cutlet shape and fried in oil. The cooked cutlets were served along with chatni. Curry Ingredients: Meat - 5 nos. Onion - 2 (chopped) Chillies - 2 (chopped) Ginger garlic paste - 1 cup Curd - 1 cup

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www.ijpbs.com (or) www.ijpbsonline.com All spices - 1 teaspoon Chilli powder - 1 teaspoon Turmeric powder - 1 teaspoon Salt- required amount Coconut paste - 1 cup Coriander and mint leaf- required amount Method: Oil was taken in a pan. Spices, chopped onion, chilli and tomatoes, ginger, garlic paste and curd were added one by one till it turned brown. Then the meat was added to it by adding chilli powder, turmeric powder and salt at the required amount, water for boiling if necessary was added. Coconut paste was added to the

curry as per the required taste. It was boiled well and coriander leaves were added. At last garnishing was done using mint and coriander leaves. The sensory evaluations of the dishes were carried out by serving the sample to the investigators. The organoleptic scores of dishes include color, appearance, flavor, texture, taste and overall acceptance was determined by using hedonic scale of 1 to 9 (Amerine et al., 1965). The evaluated average organoleptic scores of all the recipes for the study animal Fusinus nicobaricus were shown in Table 1. The prepared dishes were displayed in plates 1– 3.

Table 1. Average organoleptic scores of the dishes prepared from Fusinus nicobaricus

S. No. 1. 2. 3.

Dishes Soup Curry Cutlet

Color 4 4 4

Appearance 5 5 4

Flavor 5 5 5

Texture 4 4 5

Taste 4 4 4

Excellent -5; Good-4; Fair-3

Recipes of Fusinus nicobaricus

Plate 1. Soup

Plate 2. Curry

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RESULTS AND DISCUSSION Next to fishes, molluscs form a good source of animal protein and have become highly esteemed delicious seafood. Seafood and its recognized important lipid components are attaining unpredicted popularity as important contribution to man’s diet. Although seafood in the diets has long been considered to provide

Plate 3. Cutlet

health benefits, only in the past decades has emphasis moved from “low fat, low calories and high protein” to other positive effects such as prevention from blood cells less rigid. In the present study, Fusinus nicobaricus is an important food source to supply daily nutritional needs. This is motivated us to prepare the tastiest recipes as soup, curry and cutlet. The

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www.ijpbs.com (or) www.ijpbsonline.com preparation of recipes in gastropods includes Chicoreus ramosus and Fasciolaria trapezium by Ragunathan et al., (1992). Gopakumar (1996) prepared different molluscan products. Arularasan et al., (2010) prepared various dishes from the meat of Strombus canarium and it may also be marketed as fresh, frozen, cooked (completely) or as pickled meat. The pickled gastropod meat is also marketed in canned form. The sensory evaluation of the soup, curry and cutlet was carried out by serving the sample to the evaluators. All the evaluators accepted the taste, consistency, appearance, color, texture, flavor are excellent, good and fair. Dishes with average organoleptic scores are recorded. Food security is an essential feature of country’s independence and sustenance. The food availability in a country has to commensurate with population size and nutritional requirements of its people. The first appraisal of food is by sight and the colour. The shape, size, and surface all register impressions. Part of the acceptance of a food depends on how it looked. Thus the appearance of food plays an important factor in its evaluation. The colour must be neither too pale nor too intense. It should have a uniform natural colour. It is concluded that the present study reveals that the preparation of soup, curry and cutlet from the foot and mantle of Fusinus nicobricus provides a way for the use of marine resources. To meet the protein requirements and malnutrition of the ever increasing population, the non-conventional sources like gastropods can be used.

REFERENCES Amerine, M.A., R.M. Pangborn and E.B. Roessler, 1965. Principles of sensory evaluation of foods, Academic Press, New York, pp: 349.

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Arularasan, S., P.S. Lyla, K. Kesavan and S. Ajmal Khan, 2010. Recipes for the mesogastropod – Strombus canarium. Adv. J. of food science and Technology., 2(1):31-35. Ayyakannu, K., 1994. Hand book on a delicacy in sea food Chicoreus recipe series – 1. Tropical Marine Mollusc Programme, A DANIDA sponsored Programme, Published by CAS in Marine Biology, Annamalai University, India. Giese, A.C., 1969. A new approach to the biochemical composition of the mollusc body. Oceanography marine Biology Annu. Rev., 7:115-129. Gopakumar, K., 1996. Post harvest handling, processing and quality control of molluscan products. Proceedings of the sixth workshop of the TMMP at CAS in Marine Biology, Annamalai University, India. Phuket Mar. Biolo. Cent. Spec., 16:17-22. Hylleberg, J., 1992. The Thai way of cooking KING ABLONE, alias Chicoreus ramosus, with a note on cooking in India. Phuket Mar. Biol. Cent. Spec., 10:1113. Patterson Edward, J.K., and K. Ayyakannu, 1992. Economic importance of the gastropod Fasciolaria trapezium, an important sea food resource occurring along the Southeast Coast of India. Phuket Mar. Biol. Cent., Spec, 10:17-19. Patterson, J.K., M.X. Ramesh and K. Ayyakannu, 1994. Recipes for the gastropod, Chicoreus ramosus. Proceedings of the fourth workshop of TMMP at Prince of Songkla University, Thailand. Phuket Mar. Biol. Cent. Spec., 13:17-28. Pigott, G.M. and B.W. Tucker., 1990. Sea food: Effects of Technology on Nutrition, Marcel Dekker. Inc., New York and Basel, pp:362 Ragunathan, C., J.K. Patterson Edward and K. Ayyakannu, 1992. Utilization of the non-edible meat of the gastropods Chicoreus ramosus and Fasciolaria trapezium as a supplementary diet for penacid prawn Penaeus indicus. Phuket Mar. Biol.Cent.Spec., 11:9-15. Ramesh, M.X. and K. Ayyakannu, 1992. The effect of long term cooking on the nutritive value of the edible portions of Chicoreus ramosus. Phuket Mar. Biol. Cent. Spec., 11:23-26. Thanonkaew, A. Benjakul, S., Visessanguan, W., 2006. Chemical composition and thermal property of cuttle fish (Sepiapharaonis) muscle, Journal of Food Composition and Analysis, 19:127-133.

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P.G. & Research Department of Zoology, V.O. Chidambaram College, Thoothukudi.

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Research Article Pharmaceutical Sciences EFFECTS OF CERTAIN ADRAVYABHUTA CHIKITSA IN RESTORING COGNITION IN CHILDREN Deepmala Yadav*1, Banshidhar Behera2, Abhimanyu Kumar3 1, 2

Research Fellow, National institute of Ayurveda, Jaipur-302002 Professor and Head, Dept. Of BalRoga, National Institute of Ayurveda, Jaipur-302002 *Corresponding Author Email: drdeeyashri2011@rediffmail.com

ABSTRACT Cognition involves various mental actions such as memory, attention, language ability, reasoning, problem solving, decision making etc. Out of these memory is the fundamental component of learning and it affects other mental actions immensely. Hence in current study, memory, attention and language ability were used as assessment parameter to verify the effects of certain Adravyabhuta chikitsa (Non-drug therapy) of Ayurveda in children. This study comprises chanting of “Om” mantra and shirodhara (pouring of milk on forehead) as Adravyabhuta chikitsa. Weschler’s Intelligence scale in children (WISC), Draw-a-man test and Vernier chronoscope were the assessment criteria adopted for the study. Both the therapies showed statistically significant result in almost all the spheres of memory and attention.

KEY WORDS

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Cognition, Memory, WISC, Om-mantra chanting

INTRODUCTION Cognition is a group of mental performances which involves memory, attention, producing and understanding language, reasoning, problem Solving and decision making1. Resoration of Cognition needs the appropriate functioning of these components. Out of these memory plays a key role and affects other mental processes. It is the ability to memorize the information which is previously acquired and stored. In Ayurveda, memory can be termed as Smriti which is an ability of an individual to recall the things that are directly perceived, heared and experienced2. In perspective of cognition there are certain treatment procedures in Ayurveda which are undoubtedly helpful in the enhancement and restorative effect of memory, attention and various other processes. Out of which, certain adravyabhuta chikitsa ( Non-drug therapy) like

chanting of OM mantra and panchakarma procedures like shirodhara have proved highly effective in making equilibrium among tridoshas, trigunas and manovaha strotas and consequently effective in improving memory with other cognitive processes. Therefore in current study we aimed to verify and provide an update of the practical implication of Adrvyabhuta chikitsa in the management of restoration of cognition in children. AIMS AND OBJECTIVES OF THE STUDY  To enhance the mental performance.  To get relief from the problem associated with memory.  To improve school performance by achieving positive impact on memory status of child.  To verify the efficacy of non-drug therapy in restoring cognition w.s.r. to different Ayurvedic Classics.

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Selection of Cases  Source -Children for the present study were screened out from OPD/IPD of Balroga, Department of National Institute of Ayurveda, Jaipur and from various schools, situated in Jaipur by survey method.  Age group -Children between 6 to 16 years were considered for the study.  Numbers of cases- Total 52 children were registered out of which 12 children discontinued the treatment.  Grouping of patients-Selected children were randomly divided into following two groups keeping in mind that both the groups had children from various grades (classes), schools and socio-economics status. Group A - 20 cases treated by meditation through "Chanting of OM" mantra. Group B - This group of 20 cases were treated by Shirodhara therapy (Ayurvedic Panchkarma Technique) DIAGNOSTIC CRITERIA Inclusion Criteria  Children aged 6-16 years of either sex satisfying, criteria.  Children with average /normal I.Q. Exclusion Criteria  Children with physical disability.  Children with psychiatric illness.  Children with gross brain damage causing mental retardation  Children with any genetic disorder.  Children able to recall digit span of more than 9 digits. Discontinuation Criteria  Any acute or severe illness.  Parents not willing to continue the therapy.

Assessment criteria  Intelligence scale Wechsler intelligence scale for children (WISC) was adopted for its following subtest. i) Working Memory index (WMI) which includes  Digit span – to testify auditory short term memory and focused attention.  Letter number sequencing-Auditory STM, focused attention, sequential memory, visualization ability and language ability. ii) Processing speed index (PSI) which includes  Coding- Visual S.T.M.  Symbol search -short term memory, visual motor skill, speed and motivation.  Draw-a-man test for IQ -assessment.  Memory scale - P.G. I memory scale for Long Term Memory test.  Reaction time - Vernier chronoscope (Electronic) for assessment of reaction time and attention span.

METHOD OF ADMINISTRATION Shirodhara3 (ksheerodhara) This specific technique of shirodhara includes the pouring of simple cow milk over the forehead of patient in the form of regular stream from a specific height of 8 cm in a fixed form of oscillatory movements, i.e. to and fro movement up to 30-45 minutes daily for 2 weeks. “Om" mantra meditation4 The meditation group children were first taught the procedure of "OM" mantra meditation and then they were undergone through the 10 min session daily regular for15 day’s meditation schedule. This method was planned to verify the efficacy of non-drug therapy (Adravyabhuta Chikitsa) on memory status with special reference to different Ayurvedic Samhitas.

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Table no.1: Pattern of clinical improvement in tests applied in Digit Span Groups

Mean

Dif.

% of Change

SE ()

Remark

20 17.30 16.15 1.15

6.65

3.98

0.89

1.29

>0.10

Insigni.

20 15.10 12.15 2.95

19.54

3.59

0.80

3.67

<0.01

Signi.

As evident in above table, group A which is meditation group shows statistically insignificant result (P>0.10) with gain percent 6.65% while group B which is a shirodhara group shows statistically significant result (P<0.01) with gain percent 19.54%. According to our assumption, group A should show significant result but if it’s not so an extensive study should be performed with large sample and for longer duration. Table no.2: Showing pattern of clinical improvement in coding Groups

Mean

% of Change

Dif.

SE ()

Remark

63.15

53.30

9.85

15.60

6.57 1.47

6.70 <0.001

Highly signi.

38.15

30.15

8.00

20.97

7.50 1.68

4.77 <0.001

Highly signi.

Above table shows, group A and B both having statistically highly significant result (P<0.001) with gain percent 15.60% and 20.97% resp. This shows that Adravyabhuta chikitsa is effective in visual short term memory and attention span. Table no.3 showing pattern of clinical improvement in Symbol search Groups

A B

Mean

Dif.

% of Change

SE ()

Remark

32.75

27.30

5.45

16.64

6.44

1.44

3.78

<0.01

Signi.

25.20

23.35

1.85

7.34

4.28

0.96

1.93

<0.10

Insigni.

As per table no.3 group A shows statistically significant result (P<0.01) and group B shows insignificant result (P<0.10) which reflects chanting ‘Om’ mantra is effective in short term memory, visual motor skill, speed and motivation.

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Table no.4: Showing pattern of clinical improvement in Letter-Number sequencing. Mean

Groups

18.25

16.20

% of Change

SE ()

Remark

15.55 2.70

14.79

4.07

0.91

2.97

<0.01

Signi.

13.30 2.90

17.90

3.71

0.83

3.49

<0.01

Signi.

Dif.

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Table no.4 reflects group A and group B, both showing statistically significant effect (P<0.01) with gain percent 14.79 and 17.90%.This may indicate both the procedures are useful in working memory problems. Table no.5: Showing pattern of clinical improvement in Long term memory test Mean

Groups

12.45

10.95

% of Change

SE ()

Remark

11.35 1.10

8.84

2.38

0.53

2.07

<0.10

Insigni.

9.24

11.42

2.31

0.52

2.42

<0.05

Signi.

Dif.

1.25

Group B shows statistically mild significant effect (P<0.05) with gain percent 11.42% while group A shows insignificant effect (P<0.10).This may suggest the therapies should also prolonged for few more days for longer action potential of neuronal pathway & stronger consolidation of information. Table 6: Showing pattern of clinical improvement in IQ test Mean

97.00

89.15 7.85

8.09

94.55

94.05 0.50

0.53

Dif.

% of Change

Groups

SE ()

Remark

13.20 2.95

2.66

<0.02

Signi.

4.52

0.49

>0.10

Insigni

1.01

Above table shows group A has statistically significant effects (P<0.02) in IQ test with very less gain percent 8.09% and group B shows insignificant effect (P>0.10).This may suggests that both procedures should prolonged for few more days. Table 7: Showing pattern of clinical improvement in Reaction time test Groups

A B

Mean

Dif.

% of Change

0.51

0.21

0.31

60.16

0.61

0.40

0.21

34.07

SE ()

Remark

0.16 0.04

8.56

<0.001

Highly signi.

0.16 0.03

5.95

<0.001

Highly signi.

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Above table shows both the procedures are statistically highly significant (P<0.001) in reaction time which shows that Adryavyabhuta Chikitsa are very effective in enhancing attention span of an individual.

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Graph showing pattern of clinical improvement in tests applied in Group A

% of improvement

60%

60.16%

40%

15.60% 16.64% 14.79%

20%

8.84%8.09%

6.65% 0% Digit Span

Coding Symbol LN Seq Search

LTM

Tests

Reac Time

Graph showing pattern of clinical improvement in tests applied in Group B

% of improvement

40% 34.07% 30%

20.97% 19.54%

17.90%

20%

11.42% 7.34%

10%

0.53% 0%

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Digit Span

Coding

Symbol Search

Discussion regarding probable mode of action Shirodhara The probable mode of action of shirodhara can be explained considering the following facts-

LN Seq

LTM

Tests

Reac Time

When a constant stream of any liquid is poured over the forehead from a fixed height it results in pressure on the skin over the forehead. This pressure stimulates the pacinion or mechanoreceptors present on the skin, which in

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www.ijpbs.com (or) www.ijpbsonline.com turn lead to mechanical deformation of the receptors, which result in the change in the membrane potential of the receptor and a receptor potential is generated.5 The receptor potential then leads to generation of action potential, which is then passed to the cerebral cortex via brain stem or the RAS. In this way the information from outside of reaches finally to the cerebral cortex which comprises areas of various cognitive functions5. The pressure input from the skin over the head region is conveyed by the ophthalmic branch of trigeminal nerve to the reticulospinal neurons via a disynaptic pathway6. When sensory information reaches the cerebral cortex, only a small fraction of that information causes an immediate motor response. Much of the remainder is stored for future control of motor activities and for use in higher order cognitive functions such as conceptualization of thoughts, reasoning, decision making etc.5 Repeated stimulus input leads to consolidation of the information, which needs 5 to 10 minutes for minimal consolidation and 1 hour or more for stronger consolidation5. This fact is consistent with duration of shirodhara of 30-45 minutes practiced daily. To achieve permanent effect of shirodhara, there must be change in response characteristics of different neuronal pathways and to attain this, activation of the second messenger pathway is needed which might be attained through the process of shirodhara . The process or the mode of action here is similar to that of neuro-feedback or the EEG biofeedback technique, in which individuals are provided with real time feedback about their brainwave activity and taught to use that information to modulate certain aspects of their mind. In an animal study, it was found that, the responses evoked by stimulation of either the head or the tail were three or four times larger

than those elicited by mid body stimulation7. Thus it may be interpreted that same response is true with the man. This point explains why dhara on forehead is more effective for mental disorders. Finally, it can be concluded that shirodhara may simply be a specific way of changing the excitabilities in dysfunctional circuits of the brain so that to make the individual to perform normally. Probable mode of action of Om mantra meditation Om mantra meditation causes various changes in brain having neurophysiological8, neurochemical and neuro-electrical effects. Brain waves are associated with different mental processes e.g. Alpha patterns of brain waves are associated with relaxation, visualization and creativity, theta patterns are associated with attention, intuition and memory while beta patterns are associated with alertness, cognition and concentration9. The primary findings of several meditation studies have implicated increase in theta, alpha and delta band power and decreases overall frequency10, 11, 12, 13

These studies help to conclude the fact that the brain wave activity is affected by â&#x20AC;&#x153;Omâ&#x20AC;? mantra meditation which may alter the long term neuroelectric profile and improve the human cognition.

CONCLUSION Multimodal approach of certain Ayurvedic procedures including Shirodhara, and Om mantra meditation (Adravyabhuta chikitsa) can be effectively used in management of restoration of memory. Shirodhara and Om meditation, both have shown efficacy in all areas like working memory, processing memory, visualization, and visualmotor skill except IQ and LTM respectively which suggest the administration of procedures should be prolonged for few more days. Both the therapies showed highly significant improvement

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www.ijpbs.com (or) www.ijpbsonline.com in Reaction Time which directly reflects the efficacy of therapies on attention span. Further extensive study is needed to authenticate the results of the current study, with larger samples and more precise assessment criteria.

REFERENCES 1. 2.

Sternberg R. J. & Sternberg K,Cognitive psychology (6th Ed.) Belmont, CA; Wadsworth, Cenage learning. (2009) Charak Samhita Sutrastan,chapter 1/149. Charaka Samhita with “Vidyotini” Hindi commentary by Pt. Kashinath Shastry and Dr. Gorakhnath Chaturvedi, Part 1 and 2, Chaukhambha Bharati Academy, (1996). Ashtanga Hridaya sutrasthan chapter 2/27. Ashtanga Hridaya with commentaries “Sarwanga Sundara” of Arunadutta and “Ayurveda Rasayana” of Hemadri, Chaukhambha Orientalia, Varanasi (2002). Charak Samhita Sutrastan,chapter 11/54. Charaka Samhita with “Vidyotini” Hindi commentary by Pt. Kashinath Shastry and Dr. Gorakhnath Chaturvedi, Part 1 and 2, Chaukhambha Bharati Academy, (1996). Textbook of Medical physiology (11th Ed.) by Arthur C. Guytan & John E. Hall; pg.572- 584. Elsvier Saunders publication.( 2006) Gonzalo Viana Di Prisco, Yoshihiro Ohta, Fulvia Bongianni, Sten Grillner, Réjean Dubuc,Trigeminal

9. 10.

11.

12.

13.

inputs to reticulospinal neurones in lampreys are mediated by excitatory and inhibitory amino acids,Brain Research 1016:0006-8993(1995) Gonzalo Viana Di Prisco, Edouard Pearlstein, Didier Le Ray, Richard Robitaille,and Re jean Dubuc, A Cellular Mechanism for the Transformation of a Sensory Input into a Motor Command,The Journal of Neuroscience, November 1 20(21):8169–8176.(2000) Wallance RK, Benson H & Wilson AF, A wakeful hypometabolic physical state. American Journal of Physiology, 229:795-799.( 1971) www.synthesislearning.com, Brainwave frequency changes states of consciousness. Anderson J, Meditation meets behavioural medicine: The study of experimental research on meditation. Journal of consciousness studies, 7:17-73.( 2002) Banquet JP, Spectral analysis of the EEG in meditation Electroencephalography and clinical Neurophysiolgy, 35:143-157.( 1973) Kasamatsu A. & Hirai T, An electroencephalography study on Zen Meditation. Folia Psychiatrica at neurologica japonica, 20: 315-336.( 1966) Deepak KK, Manchanda SK, & Maheshwari, MOLECULE, Meditation improves clinic electro encephalographic measures in drug resistant epileptics. Biofeedback & self-regulation, 19, 40.( 1994)

*Corresponding Author: Dr. Deepmala Yadav,

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Department of BalRoga, Gaur Brahman Ayurdedic College, Rohtak, Haryana – 124001, E. Mail- drdeeyashri2011@rediffmail.com; Phone: 9414893921, 09414458895

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FUNGAL POLYSACCHARIDE Available Online through www.ijpbs.com (or) www.ijpbsonline.com

Research Article Biological Sciences FUNGAL POLYSACCHARIDE PROTECTS HUMAN LYMPHOCYTES FROM RADIATION INDUCED DAMAGE Thulasi G. Pillaia, Dharmendra Kumar Mauryab, Veena P. Salvib, K.K.Janardhananc and Cherupally Krishnan Krishnan Naird a

Division of Forest Ecology and Biodiversity Conservation, Kerala Forest Research Institute, Peechi, Thrissur-690653, Kerala, India. b Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai, India c Amala Cancer Research Centre, Thrissur, Kerala, India Pushpagiri Institute of Medical Sciences and Research Centre, Thiruvalla, Kerala. *Corresponding Author Email: thulasigpilla@yahoo.co.in

ABSTRACT The radioprotective properties of polysaccharides isolated from the macro fungi Ganoderma lucidum was assessed by Single cell gel electrophoresis (Comet assay).Human lymphocytes were exposed to 0Gy, 2 Gy and 4 Gy gamma radiation in the presence and absence of polysaccharides. The comet parameters - % DNA, Tail length, Tail moment and olive tail moment were reduced by the presence of polysaccharides. The results indicate that the polysaccharides of G. lucidum possessed significant radioprotective activity. The findings suggest the potential use of this mushroom for the prevention of radiation induced cellular damages.

KEY WORDS Medicinal mushroom, Ganoderma.lucidum, Polysaccharides, Comet assay, Radioprotection.

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INTRODUCTION Ganoderma, commonly known as Reishi has been considered as a panacea for all types of diseases. Reishi has attracted significant attention in recent years due to its large number of pharmacological properties [1]. The fruiting bodies of this mushroom contain a variety of chemical substances. Recent investigations carried out in our laboratory have shown that aqueous extract of G.lucidum possessed significant radio protective properties [2]. Radiation protection has significant importance in radiotherapy of cancer, nuclear accidents and even in nuclear warfare. Radiation induced cell damage results from either damage to cell membrane or DNA [3-4]. Lesions in DNA can be induced either by direct ionization of DNA or

indirectly through the reaction of aqueous free radicals leading to base damage, intra or inter strand cross-linking and single or double strand breaks [3,5]. Protection of normal tissues against this cellular damage is important in radiotherapy. The major problem associated with cancer radiotherapy is the severe side effects and damage to normal tissue. Ionizing radiation is one of the well-established and widely used therapeutic modalities either for curative or palliative treatment of tumors in man. In radiotherapy of cancer, normal tissues need to be protected while cancers are exposed to high doses of radiation. A large number of compounds natural and synthetic have been evaluated for this purpose [6]. However most of them were not successful clinically because of toxicity and side

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www.ijpbs.com (or) www.ijpbsonline.com effects. Hence search for an ideal radioprotector without side effects is a compelling urgency. In the present study we examined the radioprotective effect of the polysaccharides from G.lucidum.

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MATERIALS AND METHODS Chemicals Tris base, high melting agarose, low melting point agarose, Na2-EDTA, TritonX-100, sodium sarcosinate, DMSO and propidium iodide were obtained from Sigma Chemicals (St.Louis, Missouri). All other chemicals used were of analytical grade and procured locally. Collection of human blood: Human blood samples were collected from three healthy nonsmoking volunteers, having a mean age of 252 years. Irradiation 60 Co-gamma rays in a Gamma Cell 220 (AECL, Canada) at a dose rate of 5.3Gy/ min and Junoir Theratron unit (AECL, Ottawa, Canada) with a dose rate of approximately 0.35Gy/min at 38 cm was used for irradiation purpose. Isolation of polysaccharides The fruiting bodies of G.lucidum were collected from the outskirts of Thrissur district, Kerala. The type specimen was deposited in the herbarium of Centre for Advanced Studies in Botany, University of Madras, Chennai, India (HERB. MUBL. 3175). Sporocarps were cut into small pieces, dried at 40-50°C for 48 hours and powdered. Polysaccharides were isolated by method of Mizuno [7] with slight modification The powdered sporocarps were defatted with petroleum ether and extracted with double distilled water at 800C for 8-10 hours in several batches. The extract were combined, filtered, and concentrated to about one third of the original volume and chilled ethanol about 5 times the original volume was added and kept at 40C for 48 hrs. The precipitate was collected after centrifugation, redissolved in

distilled water and treated with Sevags reagent [8] several times to remove protein and then dialyzed against deionised water for 48 hrs at 40C. The dissolved precipitate were again precipitated with ethanol and the precipitate thus obtained was lyophilized to obtain the polysaccharides. Confirmation of the polysaccharides was done by Anthrone [9] and Phenol sulphuric acid tests [10]. Comet assay Alkaline single-cell gel electrophoresis: Alkaline single-cell gel electrophoresis was performed using the method of Singh (2000) [11] with minor modifications [12-13]. In order to estimate DNA damage in blood leukocytes, 10μl heparinised whole blood was mixed with 200μl of low melting point agarose at 37°C and layered on frosted slides pre-coated with 200μl high melting point agarose. After solidification of agarose, the cover slips were removed and the slides were kept in pre-chilled lysing solution containing 2.5M NaCl, 100mM Na2-EDTA: pH10.0, 10mM Tris HCI, 1% sodium sarcosinate with freshly added 1% Triton X-100 and 1% DMSO at 4°C for 1 hour. The slides were removed from the lysis solution and placed on a horizontal electrophoresis tank filled with the alkaline buffer (300mM NaOH, 1mM Na2EDTA, 0.2%DMSO, pH >13.0). The slides were equilibrated in the same buffer 20 minutes. Electrophoresis was carried out for 20 minutes at 25 V, 300mA using a compact power supply. After electrophoresis, the slides were stained by layering on the top with 50μL of propidium iodide (20μg/ml) and visualized using a Carl Ziess Axioskop microscope with bright field, phase contrast and epi-fluorescence facility (HBO 50 high pressure mercury lamp), 40X camera adaptor lens. The integral frame grabber used in his system (Cvfb01p) was a PC based card made in the Electronics Division of Bhabha Atomic Research Centre, and it accepted color composite video output of the camera.

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The quantitation of the DNA strand breaks of the stored images was done using the imaging software CASP by which the percentage DNA in tail, tail length, tail moment, and Olive tail moment could be obtained directly [13]. The tail length of comet indicated the extent of damage because the smaller molecules moves faster on the agarose gel. Thus, the longer tails of the comets indicated that the strand breaks were more frequent and the DNA was fragmented into several small molecules. The tail moment was a commonly accepted unit of DNA damage that normalizes the difference in the size of the nucleus studied (e.g., blood leukocytes) [12-13]. It is the product of the percent DNA in the tail of the comet and tail length. For olive tail moment, distance of center of gravity of DNA is considered instead of usual tail length.

RESULTS An exposure of human peripheral blood leukocytes to 0 Gy and 4Gy -radiation ex vivo resulted in increase of comet parameters such as % DNA in tail, tail length, tail moment and olive tail moment and the presence of polysaccharides at 500 µg/ml during irradiation reduced these parameters. (Figure.1 and Table.1). These results thus suggest a protection of cellular DNA by G.lucidum polysaccharides from radiation damage. The Olive tail moment at 2 Gy was reduced by polysaccharide from 2.67240.0881 to 1.83140.2041. Similarly at 4 Gy the Olive tail moment was reduced from 9.1036  0.9719 to 7.1745  0.1434.

Figure 1: Protection of human blood leucocytes against gamma radiation-induced strand breaks by polysaccharides isolated from Ganoderma lucidum (500g/ml)

Control Total poly. 7

3.0 2.5

Olive tail moment

Percent DNA in tail

6 5

2.0

1.5

1.0

0.5

1 --

0.0

2.5

2.0

Tail moment

Tail length ()

15 10 5

1.5 1.0 0.5

0.0

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Table 1: Comet parameters in the presence and absence of polysaccharides at different doses of gamma irradiation. Treatment 0 Gy 4 Gy % DNA Control 1.9718±0.21096 6.6679±0.3743 1.5941±0.1947 3.1772±0.4270 500 g/ml Control 500 g/ml Control 500 g/ml Control 500 g/ml

Tail length 9.7137±0.3456 8.2476±0.6154

21.7296±0.6288 16.5423±1.1118

Tail moment 0.3051±.0.0463 0.2324±0.0501

2.0148±0.1665 0.6887±0.1243

Olive tail moment 0.7326±0.0637 0.3617±0.0691

2.7278±0.14507 1.1745±0.1434

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DISCUSSION DNA constitutes the primary vital target for cellular inactivation of living systems by ionizing radiation. Ionizing radiation damage to cellular DNA are mainly strand breaks, elimination of bases and sugar damage. Exposure of human leucocytes to 4Gy radiation cause severe damage as is reflected from the comet assay. The polysaccharides from the mushroom, G. lucidum causes decrease in the comet attributes. Administration of G. lucidum extract has been reported to provide significant relief from the side effects of chemotherapy [14]. . The damages by ionizing radiation to DNA can cause the loss of viability of the cells exposed to radiation. The alkaline comet assay is an elegant and effective technique to monitor the extent of the DNA damage and its protection. One of the deleterious consequences of DNA damage from exposure to ionizing radiation is the induction of cancer. Protecting cellular DNA from radiation damage might result in the prevention of the cancers induced by the radiation. Fungal polysaccharides of comparable structure and function as those found in Ganoderma have undergone rigorous clinical trials and based on such indirect experimental evidence, it is

hypothesized that this medicinal mushroom polysaccharide might render significant relief from the side effects of both chemotherapy and radiotherapy. Polysaccharides from G.lucidum also possess DNA repairing ability in human lymphocytes [15]. The result of present investigation reveals the potentials of G.lucidum in radiation protection not only in radiotherapy but also in accidental radiation exposure. The findings also suggest the possibility of using this medicinal mushroom polysaccharides as adjunct therapy in cancer radiotherapy.

REFERENCES 1.

Tim L, Yihuai G., Shufeng Z. Global marketing of medicinal Ling Zhi mushroom G.lucidum products and safety concerns, Int. J. Med. Mushr, 6:189 – 194,(2004). Thulasi G.P, Salvi V. P., Maurya D.K., Nair. C.K.K. and Janardhanan K.K. Prevention of radiation induced damages by aqueous extract of Ganoderma lucidum occurring in Southern parts of India. Current Science, 91: 341-344, (2006). Haimovitz-Friedman, A. Radiation-induced signal transduction response, Radiat Res, 150:102 – 108, (1998). Jonathan, E.C., Bernhard, E. J. and Mckenna, W. G. How does radiation kill cells? Curr Opin Chem Biol, 3: 77 – 83, (1999).

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www.ijpbs.com (or) www.ijpbsonline.com 5.

Ross, G.M., Induction of cell death by radiotherapy, Endocr Relat Cancer, 6:41 â&#x20AC;&#x201C; 44, (1999). 6. Nair,C.K.K., Parida,D.K., Nomura,T. Radiation protectors in radiotherapy J.Radiat.Res., 42: 21-37(2001). 7. Mizuno, T. Development of an antitumor biological response modifier from Phellinus linteus Teng. (Review). Int.J. Med.Mushrooms. 21-33, (2000). 8. Staub, A.M. Removal of protein, Sevag method. Methods in carbohydrate Chemistry, 5: 5-6, (1965). 9. Yemn, E.W., Wills, A.J. The estimation of carbohydrate in plant extract by anthrone. Biochem. J. 57: 508-514, (1954). 10. Dubois, S.M.,Gilles, G.A., Hamilton, J.K. Colorimetric estimation of Carbohydrates by Phenol Sulphuric acid method, Anal.Chem, 28: 350-356, (1956) 11. Singh,N.P. Microgel for estimation of DNA strand breaks, DNA protein crosslinks and apoptosis. Mutat Res. 455:111-127, (2000).

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|444-448 12. Maurya, D.K., Salvi, V.P, Nair,C.K.K.,. Radioprotection of Normal Tissues in Tumour-bearing Mice by Troxerutin. J. Radiat. Res. 45: 221-228, (2004). 13. Konca, K., Lankoff, A., Banasik. A., Lisowska, H., Kuszewski, T., Gozdz, S., Koza, Z., Wojcik, A.,. A cross platform public domain PC image analysis program for the comet assay. Mutat. Res. 534: 15-20, (2003). 14. Shi, J.H. PSP for the protection of the tumor patients during chemotherapy. In PSP Intl Symposium, Yang Q Y and Kwok C Y (eds), Fudan U, Press, Shanghai, 271-272, 1993. 15. Thulasi G.P., Nair C.K.K., Janardhanan K.K. Enhancement of repair of radiation induced DNA strand breaks in human cells by Ganoderma mushroom polysaccharides, Fd. Chem. 119:1040-1043, (2009).

*Corresponding Author: Thulasi G. Pillai*

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Division of Forest Ecology and Biodiversity Conservation, Kerala Forest Research Institute, Peechi, Thrissur-690653, Kerala, India. E-Mail:thulasigpilla@yahoo.co.in Tel : 91-487-2690100, Fax : 91-487-2690100

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Research Article Pharmaceutical Sciences CHEMICAL STABILITY OF BORTEZOMIB SOLUTIONS IN ORIGINAL MANUFACTURER VIAL AT ROOM TEMPERATURE AND IN SYRINGE AT 40C J. Berruezo Garcíaa, M. Espinosa Boschb, F. Sánchez Rojasa, C. Bosch Ojedaa a

Department of Analytical Chemistry, Faculty of Sciences, University of Málaga, Campus Teatinos s/n, 29071 Málaga, Spain b Department of Pharmacy, General Hospital, University Hospital “Virgen del Rocío”, Manuel Siurot s/n, 41013, Sevilla, Spain 1Corresponding

author: E-mail address: fsanchezr@uma.es

ABSTRACT Bortezomib is a drug used in the treatment of myeloma multiple. The experiments carried out demonstrate that bortezomib is cytotoxic for different types of neoplastic cells and reduces the tumor-like growth “in vivo” in many preclinical models of tumor, including myeloma multiple. In a recent study, was evaluated the effectiveness and security of the subcutaneous administration of bortezomib as opposed to the conventional intravenous administration. It is necessary to emphasize that the concentration of the solution used for subcutaneous administration is 2.5 mg mL -1, unlike the dissolution for intravenous injection is prepared to 1 mg mL-1. The objective of this work is to evaluate the stability of the bortezomib dissolution reconstituted with NaCl 0.9 % at 2.5 mg mL-1 concentration in original manufacturer vial at room temperature and in syringe at 40C.

KEYWORDS Bortezomib, stability study, room temperature, syringe, HPLC, subcutaneous administration.

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INTRODUCTION Bortezomib is a medicine that uses in the treatment of the multiple myeloma. His mechanism of action defines like inhibitor of the proteosome. According to the note technical of the product, has designed specifically to inhibit the activity of the 26S proteosome in cells of mammal. The 26S proteosome is a protein complex of big size that degrades certain proteins. The inhibition of the 26S proteosome affects to multiple waterfalls of intracellular signaling, what originates in the last resort the death of the neoplastic cell. The experiments realized show that the bortezomib is cytotoxic for distinct types of neoplastic cells and that reduces the growth of tumor “in vivo” in many models preclinical of tumor, included the multiple myeloma.

The multiple myeloma constitutes, by frequency, the second hematological tumor after the lymphoma. The first clinical assays with bortezomib effected in patients with refractory multiple myeloma or in relapse [1, 2]. In them the tax of answer was of 35% (10% of complete answer) using the bortezomib in dose of 1,3 mg m-2 by intravenous via the days 1, 4, 8 and 11 each 21 days, being able to associate with dexamethasone in case of incomplete answer. These data were confirmed in an essay in phase III [3] with 669 patients with refractory multiple myeloma, in those who the bortezomib was more effective that the conventional treatment with dexamethasone in high doses, so much regarding the tax of answer (a 43 in front of 18%) like the time until the progression (6, 2 in front of 3, 4 months) and the

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www.ijpbs.com (or) www.ijpbsonline.com global survival to the year (a 80 in front of 67%). Later they have carried out other clinical essays in combination with other cytotoxic drugs, as well as in different profiles of patients with multiple myeloma. At present, the indications approved according to technical note by the European Medicine Agency are: - in combination with melphalan and prednisone in the treatment of patients with multiple myeloma that have not been previously treaties and that are not candidates to receive treatment with high dose of previous chemotherapy to a transplantation of bone marrow. - as monotherapy in the treatment of the multiple myeloma in progression in patients that have received previously at least a treatment and that have been subjected or are not candidates to a transplantation of bone marrow. In a recent study carried out by Moreau et al. [4], was evaluated the efficiency and security of the administration subcutaneous of bortezomib. It treats of a clinical essay phase III of no inferiority in front of the conventional intravenous administration, both to a dose of 1, 3 mg m-2 the days 1, 4, 8 and 11 each 21 days in patients with multiple myeloma in relapse. The study was realized with 222 patient to receive subcutaneous bortezomib (n=148) or intravenous (n=74). The tax of global answer after four cycles was of 42% in both groups, showing the no inferiority. Neither had they found significant differences in the time until progression neither in the global survival to a year between both groups. The incidence of hematological adverse effects was similar in both groups. However it observed a decrease in the incidence of neuropathy peripheral in the patients to which administered by subcutaneous. The results of this study show that the subcutaneous administration is a valid alternative to the intravenous, as it presents the same profile of efficiency and better profile of

security. It is appropriate to underline that the concentration of the solution for subcutaneous administration is 2.5 mg mL-1, unlike the intravenous that prepares to 1 mg mL-1, without that this affect of significant manner to the pharmacokinetic parameters of the bortezomib. The subcutaneous administration offers some advantages on the intravenous, like the absence of need of catheter intravenous and the possibility of domiciliary administration, which can signify in important reductions of sanitary costs as well as improvements in the comfort of the patients. Each vial of velcade(R) contains 3.5 mg of bortezomib in dust for solution injectable. The recommendations for the reconstitution that appear in the technical note are the destined to the administration by direct intravenous route to a final concentration of 1 mg mL-1. According to this, the chemical and physical stability of the reconstituted solution is of 8 hours to 250C conserved in the vial original and/or a syringe before the administration. The information about the real stability of the drugs after the reconstitution or dilution in different vehicles is crucial for the preparation units. This is even more important in the case of the antineoplastic drugs, since in his majority prepare to individual dose by patient, and treats of drugs with high toxicity and narrow therapeutic margin. Unfortunately, these data no always are available since the laboratories manufacturers frequently limit his results of stability to a maximum of 8, 12 or 24 hours, by reasons of microbiological stability or because the studies carry out only in these short periods of time, the only demanded to obtain the register by part of the regulatory agencies. In the majority of the countries, the antineoplastic drugs prepare in units centralized inside the pharmacy hospital, under strict conditions of asepsis, by what, elimination the microbial pollution, the important stability is the

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www.ijpbs.com (or) www.ijpbsonline.com physical-chemistry that has to take into account the distinct concentrations, vehicles, primary conditionings or other environmental conditions that can affect to this stability in the practical usual clinic. In this sense, has published recently a guide [5] for the design of practical studies of stability of antineoplastic drugs by means of European consensus. At present we have of diverse studies of stability of the solution of bortezomib 1mg/ml for intravenous administration [6-10]. In a previous work, we investigated the stability of bortezomib solution (2.5 mg mL-1) in the manufacturer vial stored at 40C in the dark for up to 30 days following reconstitution [11]. The aim of this investigation was to assess the stability of bortezomib solution (2.5 mg mL-1) in the manufacturer vial stored at room temperature and in syringe at 40C, both in the dark.

solution of bortezomib and stored in vial at room temperature and in syringe at 40C. The product information states that reconstituted -1 bortezomib at 1.0 mg mL is stable for 8 hours when stored at <25oC and protected from light, and for 3 hours in a syringe. No information was available at this moment about the stability of bortezomib solution at 2.5 mg mL-1 concentration stored in vial at room temperature protected from light and at the same concentration stored in syringe protected from light and stored at 40C. Stability study in vial at room temperature On study day 0, two vials of bortezomib were each one reconstituted with 1.4 mL of 0.9% NaCl to prepare solutions of concentration 2.5 mg mL1 . One of the vials was stored into vial at room temperature for stability study and the other was stored in the freezer for daily preparation of standards, both protected from light.

EXPERIMENTAL INSTRUMENTATION HPLC analysis was performed at room temperature (~250C) using an Agilent Technologies, model LC 1220 Infinity including isocratic pump (maximum pressure 600 bar), manual injector valve 20μL and detector with variable wavelength (working wavelength at 270 nm). The signal from the detector was recorder and integrated with a PC HP Pro 3010 Desktop VN934EA; a Zorbax Eclipse XDB-C18, 4.6x250 mm, (5 µm) column was employed. The mobile phase consisted of acetonitrile: water (40:60, v/v).

Bortezomib is commercialized by Millennium Pharmaceuticals (Mass, USA) in the US and Janssen-Cilag in Europe under the trade name Velcade. The vials are reconstituted with 1.4 mL of sterile NaCl 0.9% to obtain 2.5 mg mL-1

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MATERIALS

Stability study in syringe at 40C On study day 0, two vials of bortezomib were each one reconstituted with 1.4 mL of 0.9% NaCl to prepare solutions of concentration 2.5 mg mL1 . One of them was stored into syringe in the refrigerator at 4oC for stability study and the other was stored in the freezer for daily preparation of standards; both (vial and syringe) were protected from light. Physical stability at room temperature Vial and syringe On all days studied, samples were drawn for analysis of concentration and were inspected visually for changes in colour and presence of particulate matter (vial and syringe). Bortezomib analysis Vial On each study day (0, 1, 5, 8, 12, 19, 26, 33), a 100 μL aliquot of thawed solution was used to

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www.ijpbs.com (or) www.ijpbsonline.com prepare standards with final concentration of 50, 125 and 250 μg mL-1 into NaCl 0.9%. These standards combined with a blank solution allowed the construction of a calibration curve. On the other hand, a quality control sample with bortezomib concentration of 125 μg mL-1 was prepared from the solution stored at room temperature in the dark. Triplicate 20 μL quantities of each prepared sample, quality control sample, standards and blank were injected manually in the column. The area under the bortezomib peak at 270 nm was subjected to least squares linear regression, and the bortezomib concentration in each sample was determined by interpolation from the calibration curve. The bortezomib was eluted at 3.3 minutes with a flow rate of 1.5 mL min-1. Syringe On each study day (0, 2, 7, 16, 23), analysis of bortezomib stored in syringe at 40C in the dark was carried out at the same manner described into before paragraph. Accelerated degradation analysis Five different studies were carried out for this purpose over bortezomib solution stored at room temperature: acid, base, heat, hydrogen peroxide and sodium hypochlorite.

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RESULTS AND DISCUSSIONS Bortezomib stability study Physical stability All solutions, as reconstituted in the original manufacturer’s glass vials and syringe, were initially clear and colourless and remained so for the duration of the study. Also, no visible particles were observed in any solution throughout the study period. Bortezomib analysis During the study period, the concentration in all study sample retained at least 92 % of the initial concentration of bortezomib both in original manufacturer’s vial and syringe. Table 1 provides

stability data of bortezomib (2.5 mg mL-1) stored at room temperature in the dark over 33 days, tested at a diluted concentration of 125 µg mL1 .Table 2 shows the same study realized over the sample contained in syringe at 40C in the dark. Accelerated degradation analysis The subsequent studies were made over bortezomib solution stored at room temperature. pH study The ultraviolet spectrum of bortezomib (200-365 nm) shows no variation in acid, neutral and basic medium with a maximum wavelength at 270 nm in all cases. On the other hand, an aliquot of 300 µL of 125 ppm of sample were added different amounts of HCl 0.1 M (50, 100, 150, 200 and 300 µL); the concentration of bortezomib in these samples were (107.14, 93.75, 83.33, 75.0 and 62.5 ppm), the chromatograms of these samples let to obtain a calibration graph with a slope similar to the chromatograms obtained when were added different amounts of NaOH (between 0.01-0.1 M) for to obtain the same concentration of bortezomib in basic medium. Both slopes were of the same order with respect to slope obtained in neutral medium. The higher difference observed in these chromatograms were the displacement of the retention time as consequence of diverse peaks corresponding to a degradation products, principally in basic medium as can be seen in Figure 1. On the other hand, in Figure 2 was represented the variation of the signal with respect to time for different additions of HCl and NaOH solutions, measured each one at retention time appears in Figure 1. Heat study A sample of 125 ppm of bortezomib was heat at different temperatures (400, 600, 700, 1000C) during different times. No significant change was observed when the sample is heated at 400C or 600C during different time periods still 180 min.

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www.ijpbs.com (or) www.ijpbsonline.com In the case of 700C and 1000C the chromatograms of bortezomib obtained after heat the sample during different time periods appear clearly the degradation products at retention time between 1.3-3.9 min. The results obtained were shown in Figure 3 for 1000C. Influence of hydrogen peroxide Degradation of bortezomib with hydrogen peroxide occurs quickly. At ambient temperature, 300 µL of 125 µg mL-1 of bortezomib solution was degraded when 20, 50, 100 and 200 µL of hydrogen peroxide solution 0.03% were added and the degradation products appear to 2.2 and 4.9 min. Figure 4 shows the evolution of the signal for retention time 3.3 and 4.9 with different amounts of H2O2 added.

Influence of sodium hypochlorite Degradation of bortezomib with sodium hypochlorite also occurs quickly (Figure 5). At ambient temperature, to 300 µL of 125 µg mL-1 of bortezomib solution were added 20, 50 and 100 µL of sodium hypochlorite solution 0.02 M, and the evolution of peak area were studied with the time (Figure 5a) at Rt=3.3 min and Figure 5b) at Rt=4.9 min). As can be seen from this Figure bortezomib was degraded completely when 50 µL of sodium hypochlorite solution 0.02 M was added and degradation product appears at 4.9 min when the sample is chromatographied immediately. After 10 min, this degradation product was newly degraded in other products (Rt= 2, 2.2, 8.5 min).

Day 1

124.9 ± 0.6

99.2

Day 5

122.4 ± 0.7

97.9

Day 8

120.7 ± 2.6

96.6

Day 12

121.9 ± 2.1

97.5

Day 19

115.5 ± 2.0

92.4

Day 26

113.8 ± 1.4

92.0

Day 33

121.0 ± 1.0

96.8

Table 2. Stability of bortezomib at 40C stored in syringe in the dark Study day Concentration of bortezomib Percent of bortezomib (mean ± SD, µg mL-1) remaining Day 0 126.0 ± 2.0 100.8 Day 2 124.4 ± 1.0 99.5 Day 7 127.2 ± 1.0 101.7 Day 16 123.7 ± 2.6 99.0 Day 23 124.6 ± 0.6 99.7

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Table 1. Stability of bortezomib at room temperature in the dark Study day [bortezomib] (mean ± SD, µg mL-1) Percent of bortezomib remaining Day 0 124.5 ± 0.6 99.6

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Figure 1. Variation of retention time as function of NaOH solution added at different concentrations. 3,5

Retention time

3 2,5

2 1,5

1 0,5

0 0 µL

100 µL 0.01M

20 µL 0.05M

50 µL 0.05M

100 µL 0.05M

50 µL 0.1M

100 µL 0.1M

NaOH

Figure 2. Variation of the bortezomib signal with the time for different additions of HCl 0.1 M (a) and NaOH(b) a)

25000000

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HCl 0.1 M

0 200 µL

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35000000 30000000 25000000 0

20000000 Peak area 15000000

10 20 30

10000000 5000000

30 20 10

0 100 µL 20 µL 0.01M 0.05M 50 µL 100 µL 0.05M 0.05M 50 µL 0.1M NaOH

Time (min)

100 µL 0.1M

Figure 3. Evolution of different degradation products with respect the heat time at 1000C

35000000 30000000 25000000 3.3 2.2

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10000000 55

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5000000 35

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1.6

3.7

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Figure 4. Influence of different amounts of H2O2 0.03% solution over peak areas at time 0 min

25000000 20000000 15000000 Peak area

3.3

10000000

4.9

5000000 0 20 µL 50 µL

4.9 Rt (min) 3.3 100 µL

200 µL

H2O2 0.03 %

Figure 5. Variation of peak area over bortezomib signal as function of the time when different amounts of NaClO 0.02 M solution were added: a) At Rt=3.3 min; b) At Rt=4.9 min a)

40000000 0

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35000000

30000000

25000000 Peak area

180 120 60 30 Time (min) 20 10

20000000 15000000 10000000 5000000 0 0

20 30 60 120 180

0 20 µL

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NaClO 0.02 M

CONCLUSIONS -1

Reconstituted bortezomib 2.5 mg mL for subcutaneous administration was physically and chemically stable at least for 33 days in the original manufacturer vial at room temperature and at 4oC at least for 23 days in syringe, both in the dark which could make an important contribution towards reducing drug waste and, consequently, improving cost efficiency.

REFERENCES

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[1] Richardson PG NEJM 2003; 348:2609-2617. [2] Richardson PG Cancer 2006; 106:1316-1319. [3] Richardson PG NEJM 2005; 352:2487-2498. [4] Moreau P, Pylypenko H, Grosocki S, et al. Lancet Oncol 2011; 12:431-440.

[5] Bardin C, Astier A, Vulto A, et al. Annales Pharmaceutiques Francaises 2011; 69:221-231. [6] Andre P, Cisternino S, Chiadmi F, et al. Annal of Pharmacotherapy 2005; 39:1462-1466. [7] Walker SE, Milliken D, Law S, Can J Hosp Pharm 2008; 61:14-20. [8] Vanderloo JP, Pomplun ML, Vermeulen LC, Kolesar JM, J Oncol Pharm Pract 2010; 17:400-402. [9] Bolognese A, Esposito A, Manfra M, Catalano L, Petruzziello F, Martorelli MC, Pagliuca R, Mazzarelli V, Ottiero M, Scalfaro M, Rotoli B, Advances in Hematology; Volume 2009 (2009), Article ID 704928, 5 pages; doi:10.1155/2009/704928. [10] Perissutti M, Vigneron J, Zenier H, May I, Eur J Hosp Pharm 2012; 19:175. doi:10.1136/ejhpharm-2012000074.238. [11] Espinosa Bosch M, Sánchez Rojas F, Bosch Ojeda C, Inter J Pharm Biomed Sci 2012; 2:344-350.

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*Corresponding Author: F. Sรกnchez Rojas*

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Department of Analytical Chemistry, Faculty of Sciences, University of Mรกlaga, 29071, Mรกlaga, Spain

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Research Article Pharmaceutical Sciences MEDICATION KNOWLEDGE AND ADHERENCE IN NEPHROLOGY PATIENTS F Islahudin*, S H Tan Faculty of Pharmacy, University Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia. *Corresponding Author Email: faridaislahudin@yahoo.com

ABSTRACT Medication adherence is vital in patients with chronic disorders. The number of medications taken and its complexity has proved to be a challenge. Therefore, it is critical to ensure that patients have adequate knowledge about their medications in order to improve adherence. This study was performed to investigate the extent of medication knowledge and adherence in nephrology patients in a local tertiary hospital. This work focuses on ensuring that patients understand and adhere to their medications. The medication was grouped into: anti-anaemic, anti-diabetic, anti-hypertension, diuretic and lipid lowering drugs. The study, in which 70 patients were included, was performed prospectively. From the results it was found that, patients had a lower knowledge score of both anti-anaemic and lipid lowering agents compared to the other drug groups tested (F=12.84, p=0.0001). Interestingly, adherence was also low for both these groups of drugs compared to other medication groups such as the anti-diabetic, antihypertensive and diuretics (F=3.65, 0=0.0001). Patient factors that were found to associate with differences in both medication knowledge and adherence comprised of the levels of education (F=6.38, p=0.0001 and F=10.54, p=0.0001 respectively). Therefore, evidence from this work supports the fact that the level of knowledge could possibly influence the levels of adherence to medications in nephrology patients. More importantly, this work demonstrates that pharmacists can play a more active role in ensuring proper information is disseminated based on the education levels of the patients. Further research could be performed to understand the differences in information required for different groups of patients.

KEY WORDS Medication knowledge, adherence, nephrology, renal

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INTRODUCTION Medication regimens prescribed to patients have become more complex and complicated. If a patient is prescribed more than one medication, complex and complicated medication regimens may affect their adherence. Those afflicted with chronic illness such as nephrology patients have been demonstrated to take less than the amount of medications prescribed to them [1]. The number of drugs taken by this group of patient is notably high. Adherence is vital in ensuring effectiveness of treatment. However, poor

adherence with medications is a major problem as the benefits get undermined [2, 3]. It has also been identified as a major concern amongst the public as it imposes a considerable burden to health care costs [4]. Non adherence relates to many factors such as forgetting to take medication, adverse drug effects, number of medications received, lack of symptoms, high cost and efficacy [5, 6, 7]. Nonetheless, limited knowledge and understanding of medications remains a major

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www.ijpbs.com (or) www.ijpbsonline.com contributor [8, 9, 10] to the issue. A basic knowledge of a patient’s medication such as the name, indication, dosage, frequency and side effects are considered vital information [8]. An understanding of medications taken is usually approximated based on the extent of the patient’s ability to recall all of these basic information. Nonetheless, approximately twothirds of the information provided by a health care provider to a patient has been shown to be forgotten immediately [11]. Complexity of drug regimens and lack of communication are often contributors towards the lack of understanding towards treatment. This is especially true in elderly patients that are unable to follow complicated instructions [3,4,11]. This is a particular concern as better knowledge of medications may improve the patient’s adherence. Therefore, this study was performed to investigate the extent of medication knowledge and adherence in nephrology patients and identify the groups of medication in which patients were more likely to not comply.

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METHODS Study design and procedure It was a cross sectional, prospective study performed in a local tertiary hospital. Approval was obtained from the Committee of Medical Research and Ethics. Patients admitted into the nephrology ward within the six months of the study period were eligible for the study. Patients were interviewed with informed consent, for knowledge and adherence of their medications prior to admission. Demographic data such as gender, race, age, duration of illness, and past medication history were obtained from patient medical records. Patients included in the study comprised of those above 18 years of age who were already on medication before admission to the hospital. Patients, who refused to be interviewed, were unable to communicate or

dependent on a caregiver were excluded. Patients were also excluded if they were readmitted within the duration of the study. Medication knowledge and adherence Patients were interviewed on admission based on a set of questions to assess for knowledge of medications. The questions were based on a Patient Medication Knowledge Assessment Form [12]. Medication knowledge was assessed for name, dose, frequency and indication of medication. There was a four-tiered grading scale that graded the patients as 0, 1, 2 and 3 according to their response. The expected responses were: 3=answering the questions correctly, 2=answering the questions partially, 1=answering when prompted and 0=not answering the questions correctly. A total scale score of 12 was given if the patient answered all components in the medication knowledge form, correctly. Adherence was assessed by interviewing patients based on a set of questions using the Medication Adherence Report Scale (MARS) [13, 14]. An instance of the questions is mentioned here: “Some people forget to take their medicines. How often does this happen to you?” The response scale was reported based on a five-point scale where 5=never, 4=rarely, 3=sometimes, 2=often and 1=very often. The scores were then summed up to a scale range of 4-25 in which the higher scores reported adherence. Data analysis Data was analyzed using the Statistical Package for Social Sciences (SPSS) version 15.0 for MS Windows and was tested with the appropriate inferential and descriptive statistics. Parametric tests such as t-tests and ANOVA were also used, whereas categorical data were determined by non-parametric tests such as the Whitney U test and Kruskal Wallis. Probability values of less than

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www.ijpbs.com (or) www.ijpbsonline.com 0.05 (p < 0.05) were accepted as statistically significant.

RESULTS AND DISCUSSION Patient demographics A total of 70 patients were included in this study. There were a higher number of females (n=44, 63%) in the study population compared to males (n=26, 37%). The mean age of patients was 51.71 ± 15.74 years. A total of 56 (80%) patients were between 18–65 years of age, whereas the remaining 14(20%) patients were above 65 years

in age. In terms of racial distribution, a majority of the patients were Malay (n=40, 57%), followed by the Chinese (n=27, 39%), the Indians (n=2, 3%) and others (n=1, 1%). Most of the subjects were educated till the secondary levels (n=32, 46%). The average duration of disease was 3.74 ± 5.74 years. Patients were on medications which were divided into the anti-diabetic, anti-hypertensive, anti-anaemic, diuretics and lipid modifying agents. It was demonstrated that 17(24%) patients admitted to taking alternative medications in addition to those prescribed.

Table 1: Demographic characteristic such as gender, age, race, level of education and duration of disease of patients included in the study.

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Parameter Gender (n, %) Male Female Age (mean ± SD) 18-35 years old 36-65 years old >65 years old Race Malay Chinese Indian Others Educational level Primary Secondary Tertiary Illiterate Duration of disease (mean ± SD)

Medication knowledge score Table 2 summarizes the average medication score for different type of medications taken. When comparing the group of medications taken, patients scored highest in their knowledge of antihypertension agents compared to the antianaemia and lipid lowering agents (F=12.84, p=0.0001). Interestingly, in this present study,

N=70 26 (37%) 44 (63%) 51.71 ± 15.74 17 ± 5.4 39 ± 8.6 69 ± 2.1 40 (57%) 27 (39%) 2 (3%) 1 (1%) 19 (27%) 32 (46%) 12 (17%) 7 (10%) 3.74 ± 5.74

most patients found it a challenge to remember the names and indications of the anti-anaemia drugs that they were taking when compared to the frequencies and dosages of these agents (F=44.91, p=0.0001). This was similarly reflected in previous studies that demonstrated that patients had trouble remembering drug names [15]. Alibhai et al. [16] also stated that less than

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half (43%) of their patients were able to recall the names of all their medications. The anti-anaemic agent consists of oral ferrous fumarate, vitamin B complex and folic acid. Patients found it difficult to remember the names of these medications in view of the generic packaging provided by the pharmacy. Although appropriate labelling was

provided on each package, patients were found to forget names of anti-anaemic drugs more often than any other group of drug. This was compared to the boxed original packaging of lipid lowering agents and anti-diabetic drugs which were easily identified by the patients.

Table 2: Medication knowledge between different groups of medication (N=70). Medication taken by patients were divided into 5 main groups; anti-anaemia, anti-diabetic, anti-hypertensive, diuretics and lipid-modifying agents. Results are calculated as knowledge score and represented as mean ± SD (N=70). Medication AntiAntiAnti-hypertensive Diuretics Lipid modifying agent anaemia diabetic Name 1.28 ± 1.36 2.82 ± 1.48 2.82 ± 1.36 2.81 ± 1.41 2.79 ± 1.35 Frequency 2.77 ± 0.63 2.89 ± 0.57 2.79 ± 0.72 2.77 ± 0.76 2.78 ± 0.79 Indication 1.72 ± 1.22 2.64 ± 1.19 2.78 ± 1.24 2.76 ± 1.39 1.41 ± 1.20 Dose 2.93 ± 0.53 2.83 ± 0.64 2.81 ± 0.71 2.80 ± 0.54 2.85 ± 0.65 Total Score 8.70 ± 2.18 11.18 ± 2.80 11.20 ± 2.80 11.14 ± 2.79 9.83 ± 2.51 Table 3: Adherence score of patients taking medication amongst nephrology patients. Results are calculated as knowledge score and represented as mean ± SD (N=70). Adherence AntiAntiAntiDiuretics Lipid anaemic diabetic hypertensive modifying agent I forgot to take my medicines 4.82 ± 0.41 4.81 ± 0.67 4.87 ± 0.55 4.87 ± 1.05 4.02 ±0.34 4.72 ± 0.93

4.93 ± 0.93

4.83 ± 0.52

4.87 ± 0.96

4.83 ±0.42

4.01 ± 0.12

4.81 ± 0.43

4.83 ±0.93

4.83 ± 0.79

4.83 ±0.33

4.98 ± 0.52

4.96 ± 0.77

4.82 ±0.83

4.83 ± 1.33

4.83 ±0.33

4.93 ±0.93

4.82 ± 0.23

4.87 ±0.76

4.92 ± 0.83

4.81 ±0.44

23.46±3.11

24.33 ± 2.12

24.12 ±1.31

24.32 ± 1.11 23.32 ±2.33

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I alter the dose for my medicines. I stop taking my medicines for a while. I decide to miss out a dose for my medicines. I take less medicine than instructed. Total Score

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Table 4: Patient factors associated to medication knowledge and adherence in nephrology patients. Results are calculated as knowledge score and represented as mean ± SD (N=70). Factor Medication p value Medication p value knowledge Adherence score Report Scale (mean ± SD) (mean ± SD) Gender Male 2.22 ± 0.51 p>0.05 4.98 ± 0.43 p>0.05 Female 2.42 ± 0.11 4.87 ± 0.34 Age 18–35 years old 2.33 ± 0.73 p = 0.0001 a 4.89 ± 0.23 p>0.05 36–65 years old 2.01 ± 1.03 F = 8.74 4.92 ± 0.55 >65 years old 1.12 ± 0.29 4.85 ± 0.46 Race Malay 2.11 ± 0.88 p>0.05 4.83 ± 0.54 p>0.05 Chinese 2.13 ± 0.49 4.87 ± 0.85 Indian 1.99 ± 1.07 4.78 ± 0.83 Others 1.91 ± 0.00 4.91 ± 0.33 Educational level Primary 1.92 ± 0.92 p = 0.0001a 4.74 ± 0.31 p = 0.0001a Secondary 2.01 ± 0.51 F = 6.38 4.87 ± 0.45 F=10.54 Tertiary 2.81 ± 0.59 4.92 ± 0.33 Illiterate 1.44 ± 1.08 4.02 ± 0.21 a ANOVA test with level of significance p<0.05 Overall, patients were able to remember the dosages and frequencies of all the medications taken. However, a significant number of patients found it difficult to remember the indications for lipid lowering agents compared to the name, frequency and dose (F=31.75, p=0.0001). Interestingly, previous work among nephrology patients has shown that knowledge on medication doses was the lowest compared to factors like name, indication and frequency [10]. However, in this present work, it was identified that although patients were able to name the drug, most were unaware that they were taking lipid lowering agents. Patients were more likely to relate obesity to lipid lowering agents and therefore, did not understand the need for these drugs. The lack of symptoms therefore led

patients to believe that the lipid-lowering agents were not necessary. Adherence score Based on the Medication Adherence Report Scale, scores of 20 and above were categorized as high adherence [14]. This cut-off point represented 80% of the total adherence score. Overall, the nephrology patients in this study had a high level of adherence to their medication (n=69, 98.57%). There have been mixed results when it comes to adherence to drugs in patients with chronic diseases. A high non-adherence rate of 20–50% has been reported amongst patients with kidney diseases [1, 17, 18, 19]. However, there have also been reports of approximately 80% adherence rates in patients, which are comparable to results

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Table 3 summarizes the adherence score for each adherence item. Based on the total score, patients were observed to show a lower adherence level in anti-anaemic and lipid modifying agents compared to the anti-diabetic, anti-hypertensive and diuretic agents (F=3.65, 0=0.0001). The lower adherence to the antianaemic and lipid lowering agents was comparable to the lower knowledge scores of both drugs mentioned above (Table 2). A few patients displayed lower adherence to antianaemic agents by stopping the medication in view of its adverse effects (F=24.93, p=0.0001). These patients reported concerns of gastrointestinal disturbances such as constipation caused by the anti-anaemic drugs. Patients were also shown to occasionally forget to take lipid modifying medications, which lowered their adherence to the medications (F=64.53, p=0.0001). Having to take the lipid lowering medications before bedtime was found to be the main cause. Most patients found that as most other drugs were taken with respect to the food intake, it was a challenge to remember to take the lipid lowering drugs later on during the night. Association between medication knowledge, adherence and patient factor Table 4 shows the relationship between medication knowledge and adherence with patient factors. It was demonstrated that age and education levels influenced the levels of medication knowledge amongst nephrology patients. Patients above the age of 65 years were shown to have lower medication knowledge (F=8.74, p=0.0001). Elderly patients have been

demonstrated to have a lower understanding of their medications due to their dependence on a caregiver [20], which is further supported in this current work. Furthermore, lower adherence in elderly patients has been attributed to poor memory [21]. A higher educational background also determined a better knowledge of medication amongst nephrology patients (F=6.38, p=0.0001). Educational levels of the patients were also significantly associated with the adherence scores (F=10.54, p=0.0001). It was demonstrated that with a higher level of education, level of knowledge and adherence significantly improved. Indeed, the levels of education have similarly been determinants of medication knowledge in previous work [10]. Interestingly, this was also demonstrated amongst nephrology patients [1], which substantiated the findings from this current study.

CONCLUSION Patients with chronic diseases receive a high number of medications. This proves a challenge for both the patients and the prescribers as adherence to medication is vital in this group of patients. The nephrology patients in this study demonstrated an overall high level of medication knowledge and adherence to their medications. Knowledge of medications has previously been shown to be an important determinant in affixing adherence [22]. Indeed, this was similarly demonstrated in this work. Interestingly, the slightly lower level in the understanding of antianaemic and lipid lowering agent in these patients was portrayed in terms of the lower adherence levels in the same group of drugs. The association of the level of education with both knowledge and adherence level provides additional insight into the cause for nonadherence amongst nephrology patients. To that

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www.ijpbs.com (or) www.ijpbsonline.com end, this provides a great opportunity for pharmacists and medical providers to target different groups of patients. Medication information could be tailored based on the levels of education in order to ensure a full understanding of the drugs taken. This could possibly lead to better adherence to medications amongst chronic disease patients.

REFERENCES

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[1] Ronita J. Bland, Randall R. Cottrell, Liliana R. Guyler. Medication compliance of haemodialysis patients and factors contributing to non-compliance. Dial Transplant. 37 (5): 174–178, (2008) [2] Loghman-Adham M. Medication Noncompliance in Patients with Chronic Disease: Issues in Dialysis and Renal Transplantation. The American Journal of Managed Care. 9 (2):155–171, (2003) [3] Roberta BC, Edith O, Paulette S. Compliance and rehabilitation in ESRD patients. Semin Dial. 10 (1):52–54, (1997) [4] Morris LS, Schulz RM. Patient compliance: an overview. Journal of Clinical Pharmacy and Therapeutics. 17: 183– 195, (1992) [5] Dolce JJ, Crisp C, Manzella B, Richards JM, Hardin JM, Bailey WC. Medication adherence patterns in chronic obstructive pulmonary disease. Chest. 99 (4):837–841, (1991) [6] Becky AB, Jerry HG, Stephen BS. Patients at-risk for costrelated medication non-adherence: a review of literature. J Gen Intern Med. 22 (6): 864–871, (2007) [7] Chambers SA Raine R, Rahman A, Isenberg D. Why do patients with systemic lupus erythematosus take or fail to take their prescribed medications? A qualitative study in a UK cohort. Rheumatology. 48:266–271, (2009) [8] Hope CJ, Wu J, Tu W, Young J and Murray MD. Association of medication adherence, knowledge, and skills with emergency department visits by adults 50 years or older with congestive heart failure. Amer J of Health Sys Pharm. 61 (19): 2043–2049, (2004) [9] Morrow DG, Weiner M, Young J, Steinley D, Deer M, Murray MD. Improving medication knowledge among older adults with heart failure: a patient- centered

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Research Article Biological Sciences PHYTOCHEMICAL ANALYSIS AND ANTIBACTERIAL ACTIVITY OF ENDOPHYTES OF EMBELIA TSJERIAM COTTAM LINN Chandrappa C.P.*1, Anitha R.1, Jyothi P.1, Rajalakshmi K.1, Seema Mahammadi H.1, Govindappa M1 & Sharanappa P.2 1

Shridevi Institute of Engineering and Technology, Department of Biotechnology, Sira Road, Tumkur 572 106, Karnataka, India. Ph: +91-9686114872, Fax: +91-816-2212629. 2 University of Mysore, Department of Studies in Biosciences, Hemagangothri, Hassan 573201, Karnataka, India. Ph: +91-08172-240578 *Corresponding Author Email: chandrappacp@gmail.com

ABSTRACT Four different fungal endophytes (Cladosporium cladosporiodes, Penicillium sp., Aspergillus niger and Alternaria sp.) were isolated from buds and leaf of Embelia tsjeriam cottam. Three different solvents were used to analyze the phytochemicals in all the endophytes. The Cladosporium cladosporiodes yielded flavonoids (in ethyl acetate), phenols, tannins and cardiac glycosides (in petroleum ether and hexane). Saponins and tannins (in ethyl acetate), phenols (in hexane) and cardiac glycosides present in Penicillium sp. Aspergillus niger showed the presence of saponins (in ethyl acetate), phenols and cardiac glycosides (in all solvents). Alternaria sp yielded the flavonoids (in petroleum ether), phenols (in hexane) and cardiac glycosides (in hexane and ethyl acetate). In antibacterial activity, the Aspergillus niger(ethyl acetae extract) inhibited the Pseudomonas aeuroginosa, Bacillus subtilis and Shigella flexneri strongly whereas hexane also inhibited the growth of Klebseilla pneumoniae and Bacillus subtilis. Ethyl acetate extract of Alternaria sp controlled the growth of Klebseilla pneumoniae and Bacillus subtilis and ethyl acetate extract of Penicillium sp inhibited the growth of Klebseilla pneumoniae and Bacillus subtilis. The activity may be due to the presence of phytochemicals (phenols, flavonoids, tannins or cardiac glycosides). Cladosporium cladosporiodes have not evaluated antibacterial activity against tested bacteria.

KEY WORDS

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Embelia tsjeriam cottam,endophytes , phytochemicals, antibacterial activity

Bioactive natural compounds produced by endophytes have been promising potential usefulness in safety and human health concerns, although there is still a significant demand of drug industry for synthetic products due to economic and time-consuming reasons (Strobel et al., 2004). Medicinal plants are reported to harbour endophytes, which inturn provide protection to their host from infectious agents and also provide adaptability to survive in adverse environmental conditions. It is therefore important to determine the entophyte diversity of medicinal plants (Strobel et al., 2002). Embelia tsjeriam cottam

Linn. (Myrsinaceae) commonly known as Vidanga, Ambati (common name) is a climbing shrub distributed in the mountains of the Western Ghats of Karnataka, Kerala and Malabar. It has considerable reputation as a potent medicament in the treatment of various ailments such as antifertility, antioestrogenic, and anthelminitic. One of its components, embelin, is reported to possess anti-inflammatory activity (Vite et al., 2011). In the present investigation was aimed to identify and isolate different fungal endophytes from different parts of Embelia tsjeriam cottam, their

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MATERIALS AND METHODS Collection of plant material The Plant material was collected from the surroundings of Alur, near Shakaleshapura, Hassan district of Karnataka, India during the month of January 2012. The plant was identified by their vernacular names and later it was compared with the herbarium of the Department of Studies in Botany, Manasa Gangothri, University of Mysore, Mysore and Government Ayurvedic College, Mysore, India. Isolation of endophytes The method used for isolation of other endophytes was followed (Rungjindamai et al., 2008; Oses et al., 2008; Theantana et al., 2009) for isolation of endophytes from Embelia tsjeriam cottam with slight modifications. The plant tissues were washed in running tap water for one hour. Twenty five segments of leaves from plant were cut into 5 mm pieces. Endophytic fungi were isolated from the leaf of the plant (25 segments) and 10 segments (2mm diameter) of the buds. The total 35 segments of plant material were treated by triple surface sterilization techniques (Bussaban et al., 2001). Each piece was then placed on malt extract agar (malt extract 20 gm. L¯), Rose Bengal (0.033 gm. L¯), chloramphenicol (50 mg. L¯; agar 15 gm. L¯). All plates were incubated at 26±2°C until mycelium grew out; hyphal tips were cut and transferred to Potato Dextrose Agar (PDA). Half strength PDA was used for subculture and stock culture. Identification was based on colony, hyphal morphology of the fungal cultures and characteristics of the spores (Sadananda et al., 2011). Mass culturing of endophytes

The isolated endophytes were mass cultured on a Potato Dextrose Broth (PDB) and incubated at 26± 2°C for 1 week for the development of fungal mycelia mats and then the cultures were taken out and filtered through sterile cheesecloth to remove the mycelia mats and grinded. The fungal metabolites from different endophytic mycelia mats were extracted by using solvents such as hexane, ethyl acetate and petroleum ether and the sample was filtered using Whatman filter paper No 2. Equal volume of the filtrate and solvents were taken in a separating funnel and was shaken vigorously for 10 min. The solution was then allowed to stand, the cell mass got separated and the solvent so obtained, was collected. All solvents were evaporated and the resultant was dried in vacuum evaporator using MgSO4 to yield the crude extract (Sadananda et al., 2011). Phytochemical screening Phytochemical analysis is intended to serve as a major resource for information on analytical and instrumental methodology in the plant sciences. This determines the presence of bioactive compounds in extracts of endophytes. Phytochemical analysis was carried out according to Bandoni et al (Sadananda et al., 2011) with slight modifications. Test for saponins 1 ml aliquots of the various endophytic extracts were combined with 5 ml water which is at 60°C, then, shaken for 2 min, as saponins are known to possess frothing activity, the volume of froth produced in this experiment was observed and recorded every 10 min for a period of 30 min (Sadananda et al., 2011). Test for phenolic compounds 1 ml of test solution was treated with 10% ethanolic ferric chloride. Phenolic compounds were considered present when a colour changes to blue green (Sadananda et al., 2011). Test for anthraquinones

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The Borntrager test was used for the detection of anthraquinones. 2 ml of the test sample was shaken with 4 ml of hexane. The upper lypophilic layer was separated and treated with 4 ml of dilute ammonia. If the lower layer changed to violet pink, it indicates the presence of anthraquinones (Sadananda et al., 2011). Test for steroids 1 ml of the respective endophytic extract was treated with three drops of acetic anhydride and one drop of concentrated sulphuric acid. A colour change from deep green, turning to brown indicated the presence of sterols (Sadananda et al., 2011). Cardiac glycosides 1 ml of sample solution was mixed with 1 ml of glacial acetic acid then treated with one drop of 5% ethanolic ferric chloride solution. 1ml of concentrated sulphuric acid was carefully poured down the sides of test tube. The appearance of a brownish ring between the two layers with lower acidic layer turning blue green upon standing indicates the presence of cardiac glycosides (Sadananda et al., 2011) Antibacterial activity The antibacterial activity of fungal (endophytic) extracts was done by using agar well diffusion method (Ganjewala et al., 2010). Growth and maintenance of test microorganism for antibacterial activity Bacterial cultures of Pseudomonos aeuroginosa (MTCC1034), Klebseilla pneumoniae (MTCC39), Shigella flexneri (MTCC1457) and Bacillus subtilis (MTCC441)were obtained authenticated cultures from Institute of Microbial Technology (IMTECH) Chandigarh. The bacteria were maintained on nutrient broth (NB) at 370C (Mahesh et al., 2008). Agar well diffusion method

The strains that had been incubated for 24 h were used for the assay. A sterile cotton swab was dipped into the bacterial suspension and then evenly streaked over the entire surface of a sterile Mueller Hinton agar plate to obtain a uniform inoculum. Wells were made on the seeded plates using a 1000 µl sterile micro tip (8 mm) and the plates and allowed to dry for 5 min. The endophytes extracts (20 µl) were dispensed into each well using a sterile micropipette. Dimethyl sulfoxide was used as a negative control and ampicillin (10 µl) was used as a positive control. The plates were incubated overnight at 37°C and the antibacterial activity was determined by measuring the diameter of zone of inhibition (mm) with slight modification (Mahesh et al., 2008).

RESULTS Isolation of endophytes The buds and leaf of the Embelia tsjeriam cottam were subjected to isolate the endophytes and the results are presented in Table 1. Phytochemical analysis We have used three different solvents to analyze the phytochemicals from different fungal endophytes. The entophytes were subjected to phytochemical analysis to check the presence of phytochemicals and the results are presented in Table 2. Antibacterial activity Size of zone of inhibition against bacterial strains Klebsiella pneumonia, Pseudomonas aeuroginosa, Bacillus subtilis and Shigella flexneri was determined by measuring the clear zone surrounding the well made on the nutrient agar and results are presented in Table 3 and Table 4.

Table 1. Incidence of endophytes in Embelia tsjeriam cottam Parts Endophytes Bud Leaf Cladosporium cladosporioides + -

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Phytochemicals

Alkaloids Flavonoids Saponins Phenols Tannins Anthraquinones Cardiac glycosides Phlobatannins Terpenoids Cardenolides

Penicillium sp + Aspergillus niger + Alternaria sp + *+ = Present *- = Absent, Repeated the each experiment thrice. Table 2. Phytochemical analysis of Embelia tsjerium cottam Endophytes Cladosporium Penicillium sp Aspergillus niger cladosporioides PE HE EA PE HE EA PE HE EA ++ + + + + ++ ++ + + + + + + + + + ++ ++ + + ++ ++ -

Alternaria sp PE + -

HE + + -

EA ++ -

*PE= Petroleum Ether, HE= Hexane, EA= Ethyl Acetate *++ = More presence, + = Moderate presence, - = Not present, Repeated the each experiment thrice.

Table 3. Antibacterial activity of ETC leaf (Aspergillus niger) Bacterial strains Inhibition zone (mm) Klebsiella Pseudomonas Bacillus subtilis pneumoniae aeuroginosa Dimethyl sulfoxide (DMSO) Ampicillin 16 20 Ethyl acetate extract 11 10 Hexane extract 10 13 Petroleum ether extract -

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Repeated the each experiment thrice. Table 4. Antibacterial activity of ETC leaf (Alternaria sp) Bacterial strains â&#x2020;&#x2019; Inhibition zone (mm) Klebsiella Pseudomonas Bacillus subtilis pneumoniae aeuroginosa Dimethyl sulfoxide (DMSO) Ampicillin 12 Ethyl acetate extract 8 8 Hexane extract -

Shigella flexneri -

Petroleum ether extract

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Bacterial strains â&#x2020;&#x2019;

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Shigella flexneri

Repeated the each experiment thrice. Table 5: Antibacterial activity of ETC bud (Pencillium sp) Inhibition zone (mm) Klebsilla Pseudomonas Bacillus subtilis pneumoniae aeuroginosa

Shigella flexneri

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DISCUSSION

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Totally four different fungal endophytes were isolated from buds and leaf of Embelia tsjeriam cottam. The buds have shown the presence of Cladosporium cladosporoides and Penicillium sp. whereas the leaf part has yielded Aspergillus niger and Alternaria sp. The presence of phytochemicals in endophytes is an indicator that they can be potential source of precursors in the development of synthetic drugs (Sadananda et al., 2011). Cardiac glycoside, flavonoids and phenols are present in high concentrations, tannins, terpenoids, cardenolids and saponins are present whereas alkaloids, anthraquinones and phlobatannins are totally absent. Petroleum ether, hexane and ethyl acetate extracts of Cladosporium cladosporioides isolated from buds of Embelia tsjeriam cottam have revealed the presence of phenols and tanins. Petroleum ether and hexane extracts of Cladosporium cladosporioides isolated from buds also have shown the presence of cardiac glycosides and presence of flavonoids in ethyl acetate extract. Alkaloids, saponins, anthroquinones, phlobatannins, terpenoids and cardenolides were not present in petroleum ether, hexane and ethyl acetate extracts of Cladosporium cladosporioides. Cardiac glycosides were present in petroleum ether, hexane and ethyl acetate extracts of Penicillium sp isolated from buds of Embelia tsjeriam cottam. Ethyl acetate extract of Penicillium sp have shown the presence of saponins. Phenols were also present in the hexane extract of Penicillium sp and petroleum ether, hexane and ethyl acetate have not revealed the presence of alkaloids, flavonoids,

12 8 -

tannins, anthraquinones, phlobatannins, terpenoids and cardenolides in Penicillium sp isolated from buds of Embelia tsjeriam cottam. Petroleum ether, hexane and ethyl acetate extracts of Aspergillus niger isolated from leaf of Embelia tsjeriam cottam has been revealed the presence of cardiac glycosides and phenols. Flavonoids and tanins were also present in the extracts of ethyl acetate. Extracts of petroleum ether, hexane and ethyl acetate have not shown the presence of alkaloids, saponins, anthraquinones, phlobatannins, Terpenoids and cardenolides in Aspergillus niger. Cardiac glycosides were present in hexane and ethyl acetate extract and also present flavonoids, cardenolides and phenols in the extracts of petroleum ether and hexane respectively of Alternaria sp. Alkaloids, saponins, tannins, anthraquinones, phlobatannins and terpenoids were not present in petroleum ether, hexane and ethyl acetate extracts of Alternaria sp isolated from Embelia tsjeriam cottam. Ethyl acetate extract of Aspergillus niger have shown maximum inhibition zone against Shigella flexneri followed by Pseudomonas aeuroginosa and Bacillus subtilis whereas no inhibition zone has been showed against Klebseilla pneumonia. Hexane extract of Aspergillus niger isolated from leaf of Embelia tsjeriam cottam have shown maximum inhibition zone against Bacillus subtilis followed by Klebseilla pneumonia whereas no activity against Pseudomonas aeuroginosa and Shigella flexneri. Petroleum extracts of Aspergillus niger have not shown inhibition zone against all the bacterial starins. It was found that the ethyl acetate extract of Alternaria sp isolated from leaf of Embelia tsjeriam cottam have shown

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www.ijpbs.com (or) www.ijpbsonline.com maximum inhibition zone against Klebsiella pneumonia and Bacillus subtilis whereas other extracts of Alternaria sp have not shown any inhibition zone against Pseudomonas aeuroginosa and Shigella flexneri. The size of zone of inhibition of extracts of Pencillium sp against bacterial strains Klebsiella pneumonia, Pseudomonas aeuroginosa, Bacillus subtilis and Shigella flexneri are presented in table 5. The ethyl acetate extract of Pencillium sp have shown least inhibition zone against Klebsiella pneumonia and Bacillus subtilis whereas petroleum ether, hexane extracts have not any inhibition zone against all the bacterial strains mentioned. Endophytic microorganisms are excellent sources of bioactive natural products that can be used to satisfy demand of Pharmaceutical and Medical Industries (Baby et al., 2011). A large number of antimicrobial compounds isolated from endophytes, belonging to several structural classes like alkaloids, peptides, sterols, terpenoids, phenols, quinines and flavonoids (Cushnie et al., 2005). The four different fungal endophytes have exhibited the presence of flavonoids, phenols, tannins and cardiac glycosides at different concentration. The antibacterial activity is due to potent activity present phytochemicals in endophytes. Our results are confirmed with the finding of Cushine and Lamb (2005), Saravanakumar et al. (2009) (Saravanakumar et al., 2009, Doss et al., 2009 and Maneemegalai et al., 2010). This is the preliminary and first attempt was taken up to add more work in plant. Further work needs to fullfil the gaps in the plants and endophytes and these endophytes can be exploiting to use phytochemicals for treatment of various diseases.

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endophytes exhibited antibacterial activity against four bacteria and therefore these can be considered as a source of antibacterial agents and for further analysis in future.

ACKNOWLEDGMENT Authors are thankful to Dr. M.R.Hulinaykar, Managing Trustee, Sri Shridevi Charitable Trust (R.) and Dr. K.Sukumaran, Principal, Shridevi Institute of Engineering and Technology, Tumkur for providing Laboratory facilities.

REFERENCE o

o o

CONCLUSION

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The antibacterial activity of the endophytes isolated from Embelia tsjeriam cottam can be due to the presence of various phytochemicals. The

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BABY, J. - MINI, P. R. 2011. Bioactive compounds from endophytes and their potential in Pharmaceutical Effect. American Journal of Biochemistry and Molecular Biology, vol.1, 2011, no. 3, p. 291-309. CUSHNIE, T. P. - LAMB, A.J. 2005. Antimicrobial activity of flavonoids. International Journal of Antimicrobial Agents, vol. 26, 2005, no. 5, p.34356. SARAVANAKUMAR, A. - VENKATESHWARAN, K. VANITHA, J. - GANESH, M. - VASUDEVAN, M. SIVAKUMAR, T. 2009. Evaluation of antibacterial activity, phenol and flavonoid contents of

Thespesia populnea flower extracts. Pakistan Journal of Pharma Science, vol. 22, 2009, no. 3, p. 282-286. DOSS A, H. - MUBARACK, M. - DHANABALAN, R. 2009. Antibacterial activity of tannins from the leaves of Solanum trilobatum Linn. Indian Journal of Science and Technology, vol. 2, 2009, no. 2, p. 41-43. MANEEMEGALAI, S. - NAVEEN, T. 2010. Evaluation of antibacterial activity of flower extracts of Cassia auriculata. Ethnobotanical Leaflets, vol.14, 2010, p. 8-20.

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