Jreim jan march, 2012 vol xviii (1) 1 64 e version

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THE JOURNAL OF RESEARCH AND EDUCATION IN INDIAN MEDICINE An International Quarterly

Volume XVIII : 1

e-Version

Jan. - March, 2012

Prokinetic Effect of Herbomineral Unani Formulation ( Dolabi ) in Diabetic Rats Rahul Somani, Abu Shaikh, Dilpesh Jain and Rajkumar Shete Energy Dispersive X-Ray Spectroscopy in Quality Control of Powdered Herbal Formulation - Avipattikar Churna K. Jayaram Kumar and Mouli Nandi Clinical Trial of Garbhpal Ras in Pregnancy Outcome Deepa Mishra, Mukta Sinha and Vikas Kumar Inhibitory Effect of the Root of Sida acuta Burm. F. on Calcium Oxalate Crystal Growth T. Vimala and S. Gopalakrishnan Spectroscopic Investigations of Palakarai (Cowrie shell) Parpam S. Joseph Vedhagiri, K. Ganesan and P.C. Jobe Parabakar Hypoglycaemic Activity of Indian Medicinal Plants in Streptozotocin Diabetic Rats E.N. Sundaram, K.P. Singh and P. Umamaheswara Reddy Pharmacological Screening of Cassine albens (Retz.) Kosterm (Celastraceae) for Antidepressant and Anxiolytic Activity in Rodents P.H. Patil, M.B. Gagarani, K.R. Patil and S.J. Surana Screening of Ximenia americana L. for it’s Anti-inflammatory Activity M. Siddaiah, K.N. Jaya Veera, P. Mallikarjuna Rao, K. Yogananda Reddy and C. Madhusudhana Chetty In-vitro Antioxidant Capacity of Graded Doses of Methanolic Extract from Luffa cylindrica (L) Seeds K. Nagarajan, Satyajit Dutta, Sumit Das, Surabhi Singhal, Pallavi Saxena, Avijit Mazumder and L.K. Ghosh Antibacterial Activity of Alcoholic Extract of Aloe vera L. by Disc Diffusion Method G.S. Niture, M.K. Patil, A.G. Karpe and A.V. Bhonsle

e-Version J. Res. Educ. Indian Med., Vol. XVIII (1) : 2012 Conten EDITOR -

IN

- CHIEF

Prof. Em. R.H. SINGH

CONTENTS ABMS, Ph.D, D.Sc.

Professor Emeritus Banaras Hindu University Formerly Vice Chancellor, Rajasthan Ayurveda University; Dean, Faculty of Ayurveda, IMS, BHU, Varanasi - 221005 UP (India)

FOUNDING Editor Prof. (Dr.) Suresh Kumar M.D.(Ay.), Ph.D. Kayachikitsa (BHU) Formerly Dean, Faculty of Ayurveda, Himachal Pradesh University, Shimla Director, Indian Institute of Panchakarma, CCRAS (AYUSH,MoH&FW,GoI) (Kerala)

CONSULTANT SUBJECT EDITORS Prof. Ranjana Patnaik Ph.D. (BHU) Professor, School of Bio Medical Engg, Institute of Technology, BHU, Varanasi - 221005 (India)

Dr. Sanjeev Rastogi

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ADVISORY-EDITORS (INTERNATIONAL) M.Sc., Ph.D.

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ASSISTANT EDITORS Dr. Dr. Dr. Dr.

Energy Dispersive X-Ray Spectroscopy in Quality Control of Powdered Herbal Formulation - Avipattikar Churna K. Jayaram Kumar and Mouli Nandi ... 7-11 Clinical Trial of Garbhpal Ras in Pregnancy Outcome Deepa Mishra, Mukta Sinha and Vikas Kumar

... 13-19

Inhibitory Effect of the Root of Sida acuta Burm. F. on Calcium Oxalate Crystal Growth T. Vimala and S. Gopalakrishnan ...

21-26

Spectroscopic Investigations of Palakarai (Cowrie shell) Parpam S. Joseph Vedhagiri, K. Ganesan and P.C. Jobe Parabakar ... 27-32

M.S.(Ay.) (BHU)

Assistant Professor-Shalakya Tantra Institute of Medical Sciences, BHU, Varanasi - 221005 (India)

Dr. Jagat Kanwar

Prokinetic Effect of Herbomineral Unani Formulation ( Dolabi ) in Diabetic Rats Rahul Somani, Abu Shaikh, Dilpesh Jain and Rajkumar Shete ... 1-6

M.D.(Ay.) (BHU)

Head, Dept. of Pancha Karma State Ayurvedic College & Hospital Lucknow - 226004 (India)

Dr. Manoj Kumar

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JREIM ADMIN. OFFICE: Dr. (Mrs.) Laxmi Bhargava

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Executive Editor

Hypoglycaemic Activity of Indian Medicinal Plants in Streptozotocin Diabetic Rats E.N. Sundaram, K.P. Singh and P. Umamaheswara Reddy ...

33-43

Pharmacological Screening of Cassine albens (Retz.) Kosterm (Celastraceae) for Antidepressant and Anxiolytic Activity in Rodents P. H. Patil, M.B. Gagarani, K.R. Patil and S.J. Surana ... 45-50 Screening of Ximenia americana L. for it’s Anti-inflammatory Activity M. Siddaiah, K.N. Jaya Veera, P. Mallikarjuna Rao, K. Yogananda Reddy and C. Madhusudhana Chetty ... 51-54 In-vitro Antioxidant Capacity of Graded Doses of Methanolic Extract from Luffa cylindrica (L) Seeds K. Nagarajan, Satyajit Dutta, Sumit Das, Surabhi Singhal, Pallavi Saxena, Avijit Mazumder and L.K. Ghosh Antibacterial Activity of Alcoholic Extract of Aloe vera L. by Disc Diffusion Method G.S. Niture, M. K. Patil, A.G. Karpe and A.V. Bhonsle

... 55-59

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THE JOURNAL OF RESEARCH & EDUCATION IN INDIAN MEDICINE Journal of Research and Education in Ayurveda, Yoga, Naturopathy, Unani, Siddha, Homeopathy, Complementary and Alternative Medicine, Integrative Medicine, Medicinal and Aromatic Plants, Pharmaceutical Sciences …… An International Quarterly

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J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 1-6

ISSN 0970-7700

PROKINETIC EFFECT OF HERBOMINERAL UNANI FORMULATION (DOLABI) IN DIABETIC RATS RAHUL SOMANI,1* ABU SHAIKH,1 DILPESH JAIN1 AND RAJKUMAR SHETE2 Sinhgad College of Pharmacy,1 Pune - 411041 Maharashtra (India) RD’s College of Pharmacy,2 Bhor - 412206 Maharashtra (India) Abstract: This study was undertaken to investigate the prokinetic activity of Unani herbomineral formulation (Dolabi) in streptozotocin induced diabetic rats and its in vitro antioxidant activity. Rat model of diabetes was established by intraperitoneal injection of streptozotocin (55 mg/kg, i.p).

Rats were divided into three groups: Normal control, diabetic control and treatment groups. After two weeks of treatment, rats were administered with phenol red meal followed by last dose of Dolabi and they were screened for gastric emptying (GE), intestinal transit (IT) and in vitro study of distal colonic smooth muscle. In vitro antioxidant activity of Dolabi was assessed on the basis of radical scavenging activity of the stable Diphenyl-2-picryl-hydrazyl (DPPH) free radical.

Percentage of GE and IT was significantly (P < 0.05) decreased in diabetic rat as compared to normal control groups. In streptozotocin induced diabetic rats, Dolabi significantly (P < 0.05) accelerated both GE and IT as compared to diabetic control rats. Significant (P<0.01) increase in EC50 of ACh in rat distal colon was observed in diabetic rats as compared to normal rats. Where as diabetic rats treated with Dolabi showed significant (P<0.01) decrease in EC50 of ACh in distal colon as compared to diabetic control group. In in vitro study, Dolabi showed potent radical scavenging activity to stable DPPH-free radical with IC50 value of 231.09. Dolabi may exert its prokinetic effect by reducing oxidative stress and therefore can be used as drug for treating diabetic patients with gastrointestinal impairments.

Keywords: Dolabi, Unani medicine, Herbal medicine, Diabetes, Oxidative stress, Medicinal plants, Diabetic neuropathy.

Introduction Oxidative stress play an important role in the pathogenesis of chronic complications of diabetes mellitus (Ziegler and Gries, 1997) including gastroparesis (James et al., 2008). The conditions that lead to the over production of the precursors of ROS and/or reduce the efficiency of scavenging system are shown to be responsible for the development of oxidative stress.

Hyperglycemia play an important role in generation of reactive oxygen species (ROS) and that lead to chronic diabetic complications including diabetic autonomic neuropathy.

Gastroparesis is the most common symptom of diabetic autonomic neuropathy (Kong et al., 1999) which is reported to cause considerable morbidity in patients. Gastroparesis leads to abnormal gastric motility, characterized by delayed gastric emptying (GE) and intestinal 1* Corresponding Author

transit (IT) (Shamaila et al., 2009). The abnormal gastrointestinal motility among diabetic patients seemes to be a clinical manifestation of diabetic autonomic neuropathy (Punkkinen et al., 2008) and some reports concluded that these gastrointestinal disturbances may be due to the damage of peripheral cholinergic neurons as a result of oxidative stress (Bijender et al., 2003; De Winter et al., 2005).

Herbomineral formulation (Dolabi) is used for treatment of diabetes and its complications in an Unani system of medicine. It contains Gymnema sylvestre, Eugenia jambolana, Bambusa arundinacea, Rumex vescarricus, Acacia arabica, oxide of egg shell, oxide of iron rust, zinc oxide (Table 1). Some of these ingredients have been reported to possess both anti-diabetic as well as antioxidant activity such

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Somani et al.

IAEC/Approval/2008-09/05) by the Institutional Animal Ethics Committee. Animals were housed under standard laboratory conditions at controlled temperature 25 Âą 1ÂşC with 50-60% relative humidity in a normal 12 h light and dark cycle with free access to water and standard laboratory feed ad libitum.

as E. jambolana (Sagrawat et al., 2006), A. arabica (Wadood et al., 1989; Sundaram and Mitra, 2007), zinc oxide (Robert and Silvestro, 2000) where as Gymnema sylvestre known for its antidiabetic effect (Gholap and Kar, 2003). So far there is no scientific evidence about efficacy of this formulation in preclinical models of impaired gastrointestinal motility and in vitro antioxidant activity. The present investigation was under taken to evaluate the effect of Dolabi on impaired gastrointestinal motility, colonic smooth muscle response to exogenous acetylcholine (ACh) in streptozotocin (STZ) induced diabetic rats and in vitro antioxidant activity.

Overnight fasted rats were injected with streptozotocin (55 mg/kg, i.p.) dissolve in 0.1 M cold citrate buffer (pH 4.45). The blood was withdrawn 48 h later by retro orbital method, serum was separated and fasting serum glucose level was determined using the glucose oxidase -peroxidase method (Miskiewicz et al., 1973).

Rats with a serum glucose level > 250 mg/dl were considered as diabetic and used for further study. Age matched five non-diabetic rats were used as normal control group and received 0.5ml of 0.1 M cold citrate buffer (pH 4.45).

Materials and Methods Drug and Chemicals Dolabi (Hamdard) a herbomineral formulation, purchased from local market.

Streptozotocin, phenol red and DPPH were purchased from Sigma (U.S.A.).

Study design for gastrointestinal transit and in vitro study on rat distal colon After persistent hyperglycemia for two weeks, diabetic rats were divided into two groups.

Group one was diabetic control, received distilled water (10 ml/kg) and second group served as treatment, received suspension of Dolabi in distilled water (140 mg/kg, p.o) for next two

Rat model of diabetes Wistar rats of either sex weighing 180230g were purchased from Haffkine Bio-Pharma Corporation Ltd., Mumbai (India). All the experimental procedures and protocols used in this study were reviewed and approved (SCOP/

Table 1. Contents of Unani herbomineral formulation (Dolabi) Sr. No

Name of content Unani Name

1

Aqaqiya

2

Banslochen

3

Tukhm Hammaz

4

Gurmar Booti

5

Maghz Jamun Labba Buz

6

Kushta Baiz Murgh

7 8 9 10

Kushata Khabsul Hadeed Kushta Jast Gond Safaid Labba Buz

Botanical Name (Family)

Acacia arabica (Fabaceae) Bambusa arundinaceae (Bambusaceae) Rumex vesicarius (Polygonaceae) Gymnema sylvestre (Asclepiadaceae) Syzygium cumini (Myrtaceae) ------

Quantity

English Name

Gum Arabic Tree

166.6 mg

Thorny bamboo

132.0 mg

Rosy Dock, Dock Sorrel, Bladder Dock Gymnema

83.3 mg 27.7 mg

Black Plum, Java Plum

27.7 mg

Egg gallius domestics (oxide of egg shell) Iron (oxide of iron rust) Zinc oxide ---

13.8 mg 13.8 mg 41.6 mg 41.6 mg 200.0 mg

Prokinetic Effect of Herbomineral Formulation in Diabetic Rats

weeks. Age matched five non-diabetic rats were used as normal control group and received distilled water (10 ml/kg). Gastrointestinal transit and in vitro study on rat distal colon were performed.

Gastric emptying and intestinal transit After administration of last dose of Dolabi to the overnight fasted rats, 1.5 ml of a phenol red meal, consisting of phenol red (0.05%, w/w) in 1.5% methylcellulose, was given through gavage feeding. Twenty minutes later, the rats were sacrificed by cervical dislocation. Their stomachs were clamped with a string above the lower oesophageal sphincter and a string beneath the pylorus to prevent leakage of phenol red. Gastric emptying was determined spectrophotometrically.

The stomach of each rat resected just above the lower oesophageal sphincter and pyloric sphincter. Phenol red remained partly in the lumen of the stomach. The stomach and its contents were put into 5 ml of 0.1 mol/l NaOH. The stomach was minced. The samples containing the total amount of phenol red present in the stomach were further diluted to 25 ml with 0.1 mol/l NaOH and left at room temperature for 1 h. The supernatant (5 ml) was then centrifuged at 800 g for 20 min.

The absorbance was read at a wavelength of 546 nm on a spectrophotometer (Shimadzu, Japan) and the phenol red content in the stomach was calculated. Percentage of gastric emptying of the phenol red was calculated as [(infusion amount-remains) / infusion amount] × 100.

The intestinal transit (IT) of phenol red meal was determined by modified Janseen method (Janseen and Jagenerous, 1957). The small intestine was removed from the pyloric sphincter to the ileocecal junction and the distance travelled by the phenol red meal was noted and expressed as percentage of intestinal transit calculated as [distance traveled by phenol red meal/ total length of small intestine] × 100 In vitro study on rat distal colon Immediately after cleaning and measuring the length of large intestine, 2 cm distal colon

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was cut and used for in vitro study. The distal colon was dissected out and mounted under resting tension of 0.5 g in an organ bath containing continuously aerated tyrode’s solution.

Dose response curves were obtained with ascending doses of ACh (100μg/ml). EC50 values were calculated from graph plotted using percent responses against log dose.

In vitro antioxidant activity In vitro antioxidant activity of Dolabi was assessed on the basis of radical scavenging effect of the stable DPPH-free radical. Free radical scavenging activity of different concentration of Dolabi and ascorbic acid were measured using DPPH, employing method of Blois (Blois, 1958).

Solutions of different concentration (50, 100, 150, 200, 250 μg/ml) of Dolabi and ascorbic acid were added to 0.01mM, solution of DPPH in methanol. After 30 min, absorbance was measured at 517 nm, using spectrophotometer (Shimadzu, Japan). 0.01mM solution of DPPH in methanol was used as control. All tests were performed in triplicate. IC50 value was calculated. The DPPH radical scavenging activity was calculated according to the following equation, DPPH radical scavenging activity (%) = A0 – A1/ A0 x 100 Where, A0 is the absorbance of DPPH, A1 is the absorbance of DPPH solution in presence of the extract.

Statistical analysis Statistical analysis of data was conducted using one-way ANOVA followed by Dunnett’s test. Data were expressed as mean ± SEM, P<0.05 was considered statistically significant.

Results Effect of Dolabi on delayed gastric emptying in diabetic rats Gastric emptying (GE) was significantly (P < 0.01) decreased in diabetic rats as compared to normal rats (46.24 ± 3.64 vs 57.57 ± 1.96 %,

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Somani et al.

Figure 1). In STZ induced diabetic rats, Dolabi significantly (P<0.05) accelerated gastric emptying (GE) as compared to diabetic control rats (59.05 ± 2.09 vs 46.24 ± 3.64 %, Figure 1).

Effect of Dolabi on delayed small intestinal transit in diabetic rats Intestinal transit (IT) was significantly (P < 0.05) decreased in diabetic rats compared to normal rats (45.43 ± 1.94 vs 60.93 ± 5.00 %, Figure 2). In STZ induced diabetic rats, Dolabi significantly (P<0.05) accelerated intestinal transit (IT) as compared to diabetic control rats (64.32 ± 4.16 vs 45.43 ± 1.942 %, Figure 2).

as compared to normal rats (41.52±3.94 vs 20.89±3.25 μg, Figure 3).

In STZ induced diabetic rats, Dolabi show significant (P<0.01) decrease in EC50 value of ACh in distal colon as compared to diabetic control rats (14.23±2.59 vs 41.52±3.94 μg, Figure 3).

Effect of Dolabi on in vitro antioxidant activity Dolabi exhibited in vitro antioxidant activity with IC 50 value of 232.11 μg/ml (Figure 4).

Effect of Dolabi on EC50 of ACh in distal colon of diabetic rats EC50 value of Ach in rat distal colon was significantly (P<0.01) increased in diabetic rats

Discussion Propulsive motility is termed as peristalsis and it is subserved by a complex pattern of neural reflexes that aim to relax intestinal muscle downstream (descending inhibitory reflex) and

Figure 1. Effect of two weeks repeated dose treatment of Dolabi on % of gastric emptying (GE %) in STZ induced diabetic rats

Figure 3. Effect of two weeks repeated dose treatment of Dolabi on EC50 of ACh in rat distal colon in STZ induced diabetic rats

Data represented as mean ± SEM. ## P< 0.01, compared to normal control group; **P< 0.05, compared to diabetic control group (ANOVA followed by Dunnett’ test)

Data represented as mean ± SEM. ## P< 0.01, compared to normal control group; **P< 0.01, compared to diabetic control group (significance by one way ANOVA followed by Dunnett’ test)

Figure 2. Effect of two weeks repeated dose treatment of Dolabi on % of intestinal transit (IT %) in STZ induced diabetic rats

Figure 4. Effect of Dolabi on DPPH free radical scavenging activity

Data represented as mean ± SEM # P< 0.05, compared to normal control group; * P< 0.05, compared to diabetic control group (significance by one way ANOVA followed by Dunnett’ test)

Data expressed as mean ± SEM from three observations

Prokinetic Effect of Herbomineral Formulation in Diabetic Rats

contract the muscle upstream (ascending excitatory reflex) of the intestinal bolus. Intestinal transit is controlled by both neural and myogenic mechanisms (Huizinga et al., 1998). An increase of the contractile activity of the smooth muscle layers is in general responsible for acceleration of intestinal propulsion. Several mediators and neurotransmitters are responsible for these motor patterns. Acetylcholine is the main excitatory neurotransmitter in the enteric nervous system, whereas NO is the major transmitter of the inhibitory motor neurons (Waterman and Costa, 1994). Disorder of autonomic functions (Punkkinen et al., 2008 ) and extrinsic nerve supply to the gut are known to be responsible for the disturbed gastric motility associated with diabetes.

In the present study, STZ induced diabetic rats had mild or moderate gastroparesis which is characterized by slow gastric emptying and intestinal transit as compared with normal controls. Similar delayed gastric emptying, intestinal transit were seen in the STZ diabetic rats which is in agreement with previous studies (Bijender et al., 2003; Young et al., 2006; ElSalhy, 2002a; Anjaneyulu and Ramarao, 2002; El-Salhy, 2002b). Thus, rat with STZ induced diabeties could be used as an animal model of diabetic gastroparesis. Disturbed motility of gastrointestinal tract was also reported in the human with diabetes mellitus (Russo et al., 1997).

The exact cause of slow gastrointestinal transit in diabetic patients is not known, but several mechanisms have been proposed. Most important among them, is damage of peripheral cholinergic nerve as a result of oxidative stress (Bijender et al., 2003). A significant reduction in the contractile response of distal colonic smooth muscle to exogenous Ach was reported in STZ induced diabetic rats.

Treatment with antioxidant vitamin E, significantly increase contractile response of distal colonic smooth muscle to exogenous Ach as well as accelerate small intestinal transit in diabetic rats which confirm role of oxidative stress in damage of peripheral cholinergic neuron

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associated with diabetic autonomic neuropathy (Bijender et al., 2003).

Acute change in blood glucose concentration has also major effect on gastrointestinal motor function in healthy subjects (Russo et al., 1997). In particular, acute hyperglycemia inhibits both the gastrointestinal and ascending components of peristaltic reflex.

Poor glycemic control has the potential to cause delayed gastrointestinal transit in diabetic patients (Jung et al., 2003). Therefore drug which control both blood glucose as well as having antioxidant activity will be the best one, for treating diabetic associated gastrointestinal trouble.

To the best of our knowledge, this is the first report on the effect of herbomineral formulation (Dolabi) on the gastrointestinal dysmotility in streptozotocin induced diabetic rats and its in vitro antioxidant activity using DPPH. Dolabi significantly accelerate gastric emptying, intestinal transit and increase contractile response of distal colonic smooth muscle to exogenous ACh in STZ induced diabetic rats. It also exhibit in vitro antioxidant activity to DPPH. Mechanism underlying the action of Dolabi on impaired gastric motility may be neuronal dependent and its antioxidant activity may play an important role. Our data strongly reveals that Dolabi exert a prokinetic action on gastric emptying and intestinal transit in diabetic rats.

The present study also suggests that antioxidant property of Dolabi may be responsible for halting progressive changes of chronic diabetes leading to gastric impairment. However, further detailed studies like estimation of endogenous antioxidant enzyme levels are needed.

Acknowledgments The authors wish to thank Prof. M.N. Navale, Founder President, STES and Dr. K.S. Jain, Principal, SCOP for providing facilities to carryout this work.

1.

References Anjaneyulu, M. and Ramarao, P: Studies on gastrointestinal tract functional changes in diabetic

6

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

Somani et al. animals. Methods Find. Exp. Clin. Pharmacol.

24: 71-75 (2002).

Bijender, S., Harish, D., Rishi, S. and Patil B.M: Effect of vitamin E on the impaired gastrointestinal activity of streptozotocin induced diabetic rats.

Ind. J. Pharmacol. 35: 186-187 (2003).

Blois, M.S: Antioxidant determination by the use of stable free radical. Nature. 29: 1199-1200 (1958).

De Winter, B.Y., Van Nassauw, L., De Man, J.G., De Jonge, F., Bredenoord, A.J., Seerden, T.C., Herman, A.G., Timmermans J.P. and Pelckmans P.A: Role of oxidative stress in the pathogenesis of septic ileus in mice.

Neurogastroenterol. Motility. 17(2): 251-61(2005).

El-Salhy, M: Gastrointestinal transit in an animal model of human diabetes type 2: relationship to gut neuroendocrine peptide contents. Ups. J. Med. Sci. 107: 101-110 (2002a).

El-Salhy, M: Gastrointestinal transit in relation to gut endocrine cells in animal models of human diabetes. Ups. J. Med. Sci. 107: 23-33 (2002b).

Gholap, S. and Kar A: Effect of Inula racemosa root and Gymnema sylvestre leaf extract in the regulation of corticosteroid induced diabetes mellitus. involvement of thyroid hormones.

Pharmazie. 58: 413-415 (2003).

Huizinga, J.D., Ambrous, K. and Der-Silaphet, T: Co-operation between neural and myogenic mechanisms in the control of distension-induced peristalsis in the mouse small intestine. J. Physiol.

506: 843–856 (1998).

James, L.E., Andrea, M.V., Hsinlin, T.C. and Eva, L.F: Diabetic neuropathy: Mechanisms to management.

Pharmacol. Therapeut. 120: 1–34 (2008).

Janseen, P.A. and Jagenerous, A.H: New series of potent analysis. J. Pharm. Pharmacol. 6: 38-40 (1957).

Jung, H.K., Kim, D.Y., Moon, I.H. and Hong, Y.S: Colonic transit time in diabetic patients comparison with healthy subjects and the effect of autonomic neuropathy. Yonsei. Med. J. 44: 265-272 (2003).

Kong, M.F., Horowitz, M., Jones, K.L., Wishart, J.M. and Harding, P.E: Natural history of diabetic gastroparesis. Diab. Care. 22(3): 503-507 (1999).

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

Miskiewicz, S.J., Arnett, B.B., and Simon, G.E: Evaluation of a glucose oxidase-peroxidase method adapted to the single channel autoanalyzer and SMA 12-60. Clin. Chem. 19: 253-257 (1973).

Punkkinen, J., Farkkila, M., Matzke, S., Korppi-Tommola, T., Sane, T., Piirila, P. and Koskenpato: Upper abdominal symptoms in patients with Type 1 diabetes: unrelated to impairment in gastric emptying caused by autonomic neuropathy. Diab. Med. 25(5): 570-577 (2008).

Robert, A. and Silvestro D: Zinc in relation to diabetes and oxidative disease. J. Nutr. 130: 1509S-1511S (2000).

Russo, A., Sun, W.M., Sattawatthamrong, Y., Fraser, R., Horowitz, M., Andrews, J.M. and Read, N.W: Acute hyperglycaemia affects anorectal motor and sensory function in normal subjects. Gut. 41: 494-499 (1997).

Sagrawat, H., Mann, A.S. and Kharya, M.D: Pharmacological potential of Eugenia jambolana: A review. Pharmacog. Mag. 2(6): 96-105 (2006).

Shamaila, W., Baharak, M., and Peter V.D: Gastroparesis: Current diagnostic challenges and management considerations. World. J. Gastroenterol. 15(1): 25-37 (2009).

Sundaram, R. and Mitra S.K: Antioxidant activity of ethyl acetate soluble fraction of Acacia arabica bark in rats. Ind. J. Pharmacol. 39: 33-38 (2007).

Wadood, A., Wadood, N. and Shah, S: Effect of Acacia arabica and Caralluma edulis blood glucose levels of normal and alloxan diabetic rabbits. J. Pak. Med. Assoc. 39: 208-212 (1989).

Waterman, S.A. and Costa, M: The role of enteric inhibitory motor neurons in peristalsis in the isolated guinea-pig small intestine. J. Physiol.

477: 459–468 (1994).

Young, C., Shou-Dong, L., Guey-Hwa Y. and Paulus S.W: Influence of blood glucose levels on rat liquid gastric emptying. Dige. Dise. Sci. 41(3): 528-532 (2006).

Ziegler, D., and Gries F.A: Alpha-lipoic acid in the treatment of diabetic peripheral and cardiac autonomic neuropathy. Diabetes. 46(2): 62-66 (1997).

Address for correspondence: Prof. Rahul Somani, Sinhgad College of Pharmacy, Pune - 411041 Maharashtra (India). E-mail: rahulsomani2001@yahoo.com 011_2010

J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 7-11

ISSN 0970-7700

ENERGY DISPERSIVE X-RAY SPECTROSCOPY IN QUALITY CONTROL OF POWDERED HERBAL FORMULATION - AVIPATTIKAR CHURNA K.JAYARAM KUMAR* AND MOULI NANDI Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi - 835215 Bihar (India) Abstract: Energy dispersive X-ray spectroscopy (EDX) is an analytical technique used for the elemental analysis and chemical characterization of a sample. Many of the Ayurvedic formulations contain salt and trace elements, which help to optimize the therapeutic efficacy of the main drugs.

The elements generally present are sodium, potassium, chlorine, mercury, sulphur, silver etc. EDX not only helps to quantify the minerals or salts added as one of the ingredients but also helps to quantify the presence of the elements of a formulation as well. It can be used as an important measure for quality control of Ayurvedic formulation. Thus, it can be used for their quantification even in minor amounts. In the present study quantification of different trace elements of two different marketed formulations and in house formulation and a formulation without salt of Avipattikar churna has been done by flame photometry and EDX. The result shows the presence of sodium, potassium, chlorine, calcium, copper and zinc. Quantification of sodium, potassium has been done by flame photometry but the results obtained by EDX showed better sensitivity, precision and accuracy.

Keywords: Energy dispersive X-ray spectroscopy, Flame photometry, Trace elements, Quality control, Avipattikar churna, Ayurvedic drugs.

Introduction According to an estimate of World Health Organization (W.H.O.) nearly 80% of population of developing countries relies on traditional medicines, mostly on plant drugs for their primary health care needs. Most of the traditional systems of medicines are effective but the need is just to validate them. Validation of herbal medicines is one of the toughest challenges for the scientist. There is a need to develop standards to bring this system of medicine in the main stream of health sciences (Mukherjee, 2003). In India Ayurveda, Unani and Siddha have been used for many centuries. Central Council for Research in Ayurveda and Siddha (C.C.R.A.S) has given preliminary guidelines for standardization of these formulations but for the uniformity of batches in production of Ayurvedic formulations. It is necessary to develop methods using phytochemical markers (Sane, 2005).

However, the analysis and the structure of the * Reader in Pharmacognosy

finished product, safety and side effects of the formulations, correct doses and duration of the treatment, mode of action remains unanswered (Prakash, 1999). Energy dispersive x-ray spectroscopy (EDX) is a technique used for the elemental analysis and chemical characterization of a sample. Different sample types (solid materials, liquids, powders, metals, minerals, etc.) can be analyzed with simple sample preparation over a wide range of concentrations (from traces to main components) is possible without dilution (Juillet, 1996). It is a new technique used to identify and quantify the trace elements present in a sample. Many of the Ayurvedic formulations contain salt and trace elements, which help to optimize the therapeutic efficacy of the main drugs. So EDX can be used as an important tool in standardization of Ayurvedic formulation.

In classical Ayurvedic books, Avipattikar churna is mentioned to be used in digestive and irritable bowel disorders. It is composed of twelve

8

Kumar and Nandi Table 1. Sodium and potassium content by flame photometer.

Sample

Sodium content (% weight)

Potassium content (% weight)

A

0.2508

0.322

B

0.3812

0.417

In house

0.325

0.473

In house without salt

0.1142

0.3554

chart_ back_ bw_final.pdf]. accessed on date 13.11.2010.

Vida lavana contains a small amount of natural amounts of trace elements such as iron and magnesium. It is carminative and tonic to the digestive system. It replenishes salt lost in exercise and adding to trace elements essential for normal health and fitness. Potassium, sodium, chloride helps in regulation of Na/K/CA ATPase, glucose transport, transport of some amino acids including alanine, proline, tryptophan and tyrosine.

Table 2. Trace element obtained by EDX (in % weight).

Sample

Sodium

Potassium

Calcium

Copper

Zinc

Chloride

A

0.50

0.46

0.55

1.39

1.24

0.72

B

0.58

0.82

0.46

3.15

2.71

1.04

In house

-

0.60

1.42

2.58

2.24

0.65

In house without salt

-

0.34

0.40

1.94

1.89

-

Table 3. Comparison of sodium and potassium by flame photometry and EDX (in % weight).

Flame photometry

EDX

Table 4. Constituents of herbal formulation Avipattikar churna.

Sodium

Potassium

Sodium

Potassium

Sl. No.

A

0.2508

0.322

0.50

0.46

B

0.3812

0.417

0.58

0.82

In house

0.325

0.473

-

0.60

Sample

In house without salt

0.1142

0.3554

-

0.34

different herbs and spices along with sugar and salt (Table 4).

Herbs include Emblic myrobalan, Ginger, Belliric myrobalan, Nutgrass, Myrobalan, Long pepper, Black pepper, Vidang, Cardamom, Indian cinnamon, Clove, Trivrt (Indian jalap), sugar and Vida lavana (black salt). Though Indian jalap being the main active ingredient for therapeutic efficacy but the presence of sugar and salt is also important as sugar reduces pitta. People with a predominantly pitta constitution are thought to be susceptible to hypertension, heart disease, infectious diseases, and digestive conditions[http://www.amritaveda. c o m / l e a r n i n g / a r t i c l e s / Ta s t e

Sanskrit / Hindi Name

Botanical Name

1

Shunthi

(Zingiber officinale)

2

Maricha

(Piper nigrum)

3

Pippali

(Piper longum)

4

Amalaki

(Embelica officinalis)

5

Vibhitaki

(Terminalia bellirica)

6

Haritaki

(Terminalia chebula)

7

Vidang

(Embelia ribes)

8

Nagarmotha

(Cyperus rotundus)

9

Ela

(Elettaria cardamomum)

10

Tejapatara

(Cinnamomum zeylanicum)

11

Launga

(Syzygium aromaticum)

12

Nishotha

(Operculina turpethum)

13

Candy sugar

Potassium ions are involved in a number of essential physiological processes, such as the maintenance of intracellular acid-base balance and tonicity. Copper aid enzymes, specifically in oxidation of iron. Zinc is essential in reactions involving synthesis such as carbohydrates, lipids, proteins and nucleic acids. [http://web.uct.ac.za/ depts/git/ibd/vits.htm# Minerals2] accessed on date 13.11.2010.

EDX in Quality Control of Herbal Formulations

9

Figure 2

Figure 1

Figure 4

Figure 3

Figure 6

Figure 5

Figure 1,3,5,7 shows the surface electron image of formulation A, B, in house and in house without salt respectively Figure 2,4,6,8 shows the graph showing the quantity of the present trace element of formulation A,B, in house and in house without salt respectively.

10

Kumar and Nandi

Figure 8

Figure 7

Materials and Methods Instruments and Chemicals Systronics flame photometer 128 was used for flame photometry and energy dispersive X-ray spectroscopy was done in JEOL-JSM 6390LVâ&#x20AC;&#x201C;SEM analyzer. Millipore water was used for flame photometry.

Methods for Flame Photometry 1000 ppm standard solution of analytical grade sodium chloride and potassium chloride was prepared. From that stock solution 20,40,60,80,100 ppm solution of sodium chloride and potassium chloride was prepared.

The sample was prepared by taking 1 g of four different formulations in 100 ml of water.

It is then filtered and 10 ml of the filtrate was taken and volume was made up to 100 ml. Their sodium content and potassium content was measured by Systronics flame photometer 128 (McNaught and Wilkinson, 1997).

They were converted to percentage weight for the convenience of comparison as the results obtained from EDX were in percentage weight.

Methods for Energy Dispersive X-ray Spectroscopy EDX analysis was done in JEOL- JSM 6390LV â&#x20AC;&#x201C;SEM analyzer. At first the atmospheric air was poured inside the chamber and vacuum was released. It is called Vent mode. Then the chamber was opened and the sample was placed.

Again vacuum was regenerated with pumps. After

creation of vacuum, the sample was moved towards the beam. Accelerating voltage was 20 kv, working distance was 10 m.m. and spot size was 40-70. Few milligrams of the samples were used in for the analysis. Comparison of results obtained from flame photometry and EDX was done (Seiichi and Hiroyuki, 1986; Kazuo et al., 1996).

The results obtained from flame photometry and EDX are given in Table 1 and Table 2. Comparison of the amount of sodium and potassium by flame photometry and EDX are shown in Table 3.

Results In house formulation without salt also shows the presence of sodium, potassium, chlorine and other trace elements. The results of flame photometry show presence of sodium ranges from 0.1142% to 0.3812% weight and potassium ranges from 0.322% to 0.473%. In EDX the presence of sodium ranges from 0.50% to 0.58% weight and potassium ranges from 0.34% to 0.82% weight. In EDX presence of other trace elements was also detected and quantified.

Discussion The results obtained in EDX and flame photometry shows quite a difference. However, in EDX presence of other trace elements was

EDX in Quality Control of Herbal Formulations

found like chlorine, copper, zinc calcium, etc. In house formulation without salt also shows the presence of sodium, potassium, chlorine and other trace elements. It indicates the presence of trace elements in the ingredients other than the salt. Thus EDX gives a better comprehension of trace elements.

Acknowledgements Financial help rendered by AICTE to carry out this research work is gratefully acknowledged. We are thankful to the Head, Department of Pharmaceutical Sciences, BIT, Mesra, Ranchi (India) for permission to carry out this experimental work.

1.

2.

3.

4.

References McNaught AD and Wilkinson A: IUPAC. Compendium of Chemical Terminology. 2nd edition (the Gold Book). Blackwell Scientific Publications, Oxford (1997).

Ayurvedaâ&#x20AC;&#x2122;s Six Tastes and their Effects on the Doshas http://www.amritaveda.com/learning/ articles Taste_chart_back_bw_final.pdf(13.11.10) Backgrounder Ayurvedic Medicine: An Introduction http://nccam.nih.gov/health/ayurveda/ D287_BKG.pdf(13.11.10) The Ayurvedic Formulary: Government of India Ministry of Health and Family Planning, Department of Health, Part-1, 1 st edition.

5.

6.

7.

8.

9.

10.

11.

12.

Government of India Press, Faridabad, Controller of Publication, New Delhi, India. pp 87 (1978).

Juillet HM: Spectroscopy for the determination of trace and main elements. SPECTRO Analytical Instruments, Kleve. Journal de physique IV. Colloque, supplkment au Journal de Physique III,C4 619-625 (1996).

Kazuo F, Mitsuaki O, Mitsuhiro A and Tetsuya S: Practical performance of energy dispersive xray spectroscopy with a high- voltage TEM up to 1,000 kv. Journal of Electron Microscopy. 45(4): 285-290 (1996).

Lee. R and Kligler B: Interactive Medicine: Principles for Practice. McGraw-Hill Companies (2004).

Mukherjee PK: Exploring botanicals in Indian system of medicine-regulatory perspectives.

Clinical Research and Regulatory Affairs. 20: 249â&#x20AC;&#x201C; 264 (2003).

Prakash VB: In Standardisation of Ayurvedic products including GMP of Ayurvedic industry, Kerala. Ayurvedic Medicine Manufacturer Association, Palakkad, Kerala, India (1999).

Sane VC: Past, present and future of Ayurvedic medicines in India. The Pharma Review. 3: 29-33 (2005).

Seiichi S and Hiroyuki K: Energy dispersive X-ray spectroscopy in medium voltage electron microscope. Journal of Electron Microscopy. 35(4): 335-342 (1986).

Vitamins, Minerals and Trace Elements http:// web.uct.ac.za/depts/git/ibd/vits.htm# Minerals2(13.11.10)

Address for correspondence: K. Jayaram Kumar, Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi - 835215 Bihar (India). E-mail: jayaram_res@yahoo.com 036_2009

11

J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 13-19

ISSN 0970-7700

CLINICAL TRIAL OF GARBHPAL RAS IN PREGNANCY OUTCOME DEEPA MISHRA,1* MUKTA SINHA2 AND VIKAS KUMAR3 Department of Prasuti Tantra,1,2 Institute of Medical Sciences, Pharmacology Laboratory, Department of Pharmaceutics,3 Institute of Technology, Banaras Hindu University, Varanasi – 221005 (India) Abstract: Introduction: Ayurvedic sages suggest that a wholesome diet along with sheetvirya, balya, rasayan and madhur drugs during the period of pregnancy is must. This ultimately results in fetal growth, maternal health and post delivery lactation. Garbhpal ras, a drug mentioned in the text of 17th century namely ‘Ras Chandanshu’ has such properties and is being used since then.

It was claimed that Garbhpal ras is a panacea for feto-maternal well being. The aim of present study was to assess the efficacy of Garbhpal ras in pregnancy outcome. Method: In present study, 170 pregnant women were registered. Haematological and biochemical parameters were observed.

Neonatal parameters like gestational age, weight, height, head circumference, chest circumference, mid arm circumference and Apgar score were assessed after delivery. Result and conclusion: Normal haematological picture, maintained renal and liver function tests show non-toxic nature of Garbhpal ras. Thus, it can be recommended safely in pregnancy at therapeutic doses.

Keywords: Garbhpal ras, Rasayan, Balya, Garbhsrava, Anupan, Antioxidant, Immunomodulatory, Pregnancy, Prevention of abortion, Ayurvedic medicine, Lactation, Meternal health.

Introduction Ayurveda, the oldest existing medical system, recognized by World Health Organization, is widely practiced. Obstetric care in ayurveda is an unique feature. Ayurveda ensures a safe and natural delivery of a healthy baby, which is achieved by a regulated diet and regimen during pregnancy and administration of herbal preparations needed in each month. A special unusual glow, beauty, serenity is seen on a pregnant woman’s face. This glow is the indicator of health of baby inside her body.

Garbhpal ras, a drug mentioned in the text of 17th century namely ‘Ras Chandanshu’ has occupied a respectable place in ayurveda, for preventing miscarriage and ensures better nourishment to fetus (Rasyog sagar, 1907-10; Chhangadi, 1999; Goyal and Mahajan, 1988; Indian Medical Science Series, 1994).

Garbhpal ras contains minerals like Hingula (Cinnabar/Hgs), Nag (lead/Pb), Vang (Tin/Sn)

and Loh bhasma (Iron/Fe). Herbal contents of Garbhpal ras include Dalchini (Cinnamomum zeylanicum), Ela (Elettaria cardamomum), Tejpatra (Cinnamomum tamala), Shunthi (Zingiber officinale), Marich (Piper nigrum), Dhanyak (Coriandrum sativum), Chavya (Piper retrofractum), Krishna jeerak (Carum bulbocastanum), Draksha (Vitis vinifera) and Devdaru (Cedrus deodara). All ingredients in same quantity (1 karsh each) except Loh bhasma (1/2 karsh) were triturated in extract of Vishnukranta (Clitoria ternatea) for seven days.

(1 karsh = 12 grams) Prior to this clinical study of Garbhpal ras in pregnant women, this drug has been evaluated for acute, sub-chronic and chronic toxicity. It was also evaluated for teratogenic effects, if any, in dams and was found safe at therapeutic doses (Mishra et al., 2008; Mishra et al., 2009a; Mishra et al., 2009b; Mishra et al., 2009c).

Garbhpal ras was also found helpful to reduce symptoms like nausea, vomiting, leg cramps, heart

*1. Incharge and Lecturer, Department of Prasuti Tantra, A & U Tibbia College and Hospital, New Delhi (India).

2. Professor and Head of Department, Prasuti Tantra, IMS, BHU, Varanasi (India).

3. Associate Professor, Department of Pharmaceutics, IT, BHU, Varanasi (India).

14

Mishra, Sinha and Kumar

burn, low back pain, abdominal pain, oedema, constipation and flatulence during pregnancy (Mishra et al., 2009d; Mishra et al., 2009e).

The aim of this study was to assess the efficacy of Garbhpal ras in pregnancy outcome, if any.

Materials and Methods Inclusion criteria Women belonging to age group, 18 to 38 years, having amenorrhoea due to pregnancy (as early as pregnancy was detected and up to five month of pregnancy), history of abortion, history of intra uterine death/still birth, history of preterm delivery or any other obstetric problem during previous pregnancies.

Exclusion criteria Women having essential hypertension, severe aneamia, diabetes mellitus, hypo/ hyperthyroidism and severe degree of pregnancy induced hypertension. Patients having tuberculosis (except women already on ATT for suspected genital tuberculosis), liver or renal disorder were also excluded.

Initially 178 pregnant women were randomly selected, out of which 170 cases registered from out patient department (during period of Aug. 2006 to Sept. 2007), Prasuti Tantra, Sir Sunderlal Hospital, BHU, Varanasi (India). At the time of registration written consent for becoming a volunteer of this study was taken from each pregnant woman.

All the registered cases have been categorized in 4 groups as follows: Group 1. Patients registered from first trimester having normal course of pregnancy with no specific obstetric or medical history.

Group 2. Patients registered from first trimester with high risk factor like history of abortion, pregnancy induced hypertension in previous pregnancy, preterm delivery and previous caesarean section within two years, elderly primigravida, TORCH infection, on anti tubercular treatment.

Group 3. Patients registered from second trimester having normal course of pregnancy with no specific obstetric or medical history.

Group 4. Patients registered from second trimester with high risk factor like history of abortion, pregnancy induced hypertension in previous pregnancy, preterm delivery and previous caesarean section within two years, elderly primigravida, TORCH infection, on anti-tubercular treatment.

Each group was further subdivided into control and trial: Control group (C1 ,C2,C3 ,C4): Patients received iron (Livogen), calcium (Calcimax forte) after the first trimester, and folic acid from the very beginning till last month. Natural progesterone 200 Îźg once or twice a day advised in cases having history of spontaneous abortion.

Trial group (T 1 ,T 2 ,T 3 ,T 4 ): Patients additionally received Garbhpal ras (120 mg bid with milk and munakka or draksha). As the women registered for the study, they were advised Garbhpal ras to be taken with anupan after meals.

Maximum duration for drug administration was six months.

In all the groups folic acid was advised as soon as pregnancy was detected till delivery and iron (Livogen), calcium (Calcimax forte) were advised after the first trimester. Blood samples were taken before initiation of treatment and in the last month of gestation for analysis. Pregnant women were reviewed for routine antenatal checkup ( Fortnightly up to 12 weeks, monthly till 32 weeks, fortnightly up to 36 weeks and weekly afterwards).

Parameters studied 1. Neonatal parameters (Anthropometry like weight, height, head circumference, chest circumference, mid arm circumference, gestational age and Apgar score at the time of delivery).

2. Outcome in cases of previous history of spontaneous abortion(s) (Table 1).

3. Haematological and bio-chemical blood parameters (Table 2 and 3).

Statistical analysis All data were expressed as MeanÂąSD. Treated and control groups were compared with

Clinical Trial of Garbhpal ras in Pregnancy Outcome Table 1. History of spontaneous abortion(s).

15

independent sample ‘t’ test, intragroup comparison was done with paired ‘t’ test. A 95% confidence level was used to determine statistically significant differences between Garbhpal ras treated and control groups.

Effect of Garbhpal ras on heamatological parameters Haemoglobin was improved overall, but statistically significant difference was observed in pregnant women registered in first trimester treated with Garbhpal ras in comparison to control group. It shows that Garbhpal ras causes no interference/ impairment in absorption of iron rather it might have synergistic effect.

It was observed that average mean value of total leukocytes count was increased significantly in last month, though it was within normal limits. It shows that Garbhpal ras does not produce any haemopoitic toxicity (Table 2).

Results and Discussion Results were assessed on the basis of differences in neonatal parameters (like gestational age at the time of delivery, weight, height, head circumference, chest circumference, mid arm circumference and Apgar score), successful outcome in cases of history of spontaneous abortion(s) and untoward effect on haematological and bio-chemical blood parameters, if any.

Effect of Garbhpal ras on biochemical parameters of blood Level of blood sugar was found within normal range in all groups. No incidence of hypo/ hyperglycemia was observed in any of the case.

Liver function (serum bilirubin, SGOT, SGPT and alkaline phosphatase) and renal function (blood urea, serum creatinine) are maintained during antenatal period in trial group as well as

Group

Number of cases

Percentage (%)

Cases having 1, 2 or 1

2

3

C2(n=19)

11

57.89

10

1

0

T2(n=25)

15

60.00

9

5

1

C4(n=14)

08

57.14

6

2

0

T4(n=20)

13

65.00

9

3

1

3 no. of abortion(s)

Table 2. Effect of Garbhpal ras on haematological and biochemical parameters.

Group

Haemoglobin (gm%)

TLC (mm3)

FBS (mg/dl)

Bl Urea (mgdl)

Serum Creat (mg/dl)

BT

AT

BT

AT

BT

AT

BT

AT

BT

AT

T1 (n=30)

10.25 ±1.22

12.14 ±1.10††

8347.22 ±1103.37

11244.44 ±1162.87††

72.04 ±12.98

73.25 ±10.51*

16.29 ±3.08

16.76 ±2.12

0.65 ±0.08

0.70 ±0.08

C1 (n=20)

10.23 ±1.23

11.58 ±1.10††

8652.15 ±1192.18

11808.71 ±1240.20††

78.76 ±11.98

79.42 ±10.58

17.20 ±3.20

17.9 ±2.32†

0.66 ±0.13

0.67 ±0.09

T2 (n=25)

10.32 ±1.63

12.20 ±0.92††*

9373.33 ±1038.03

11295.65 ±1157.38††

76.06 ±11.97

76.29 ±9.38

16.36 ±2.25

16.33 ±1.90

0.68 ±0.12

0.72 ±0.09

C2 (n=19)

10.50 ±1.39

11.81 ±0.88†

9121.08 ±1003.10

11989.47 ±1191.12††

75.05 ±11.29

77.93 ±10.64

17.98 ±3.32

16.77 ±3.50

0.69 ±0.13

0.71 ±0.10

T3 (n=25)

9.77 ±1.04

11.85 ±0.73††

9733.33 ±829.52

12013.33 ±1046.00††

71.64 ±10.71

73.74 ±10.82

17.20 ±4.99

16.95 ±3.82

0.65 ±0.05

0.66 ±0.06

C3 (n=17)

10.32 ±1.44

11.88 ±1.00††

9494.12 ±872.12

12276.47 ±1056.84††

77.54 ±10.62

76.65 ±10.66

17.34 ±4.19

17.91 ±3.69

0.65 ±0.09

0.68 ±0.09

T4 (n=20)

10.16 ±1.57

11.85 ±1.22††

9552.00 ±1037.87

12468.75 ±1189.8††

73.43 ±13.06

71.57 ±6.32*

15.08 ±0.93*

15.04 ±0.83

0.68 ±0.07

0.70 ±0.08

C4 (n=14)

10.24 ±1.26

11.78 ±1.15††

9186.67 ±1010.56

12320 ±1166.96††

75.09 ±11.01

80.36 ±12.44†

17.49 ±4.10

15.85 ±2.62†

0.67 ±0.09

0.69 ±0.08

BT= Before treatment ; AT= At last month of pregnancy ; * p<0.01, compared to control ; † p<0.01, compared to BT ; †† p<0.001, compared to BT.

16

Mishra, Sinha and Kumar

Table 3. Effect of Garbhpal ras on biochemical parameters.

Group

Serum Bilirubin (mg/dl)

SGOT (U/ml) AT

SGPT (U/ml)

BT

AT

BT

BT

T1 (N=30)

0.64 ±0.12

0.60 ±0.11

28.03 ±4.67

27.56 ±4.40

35.12 ±4.78

C1 (N=20)

0.68 ±0.08

0.56 ±0.11

29.01 ±5.88

29.10 ±4.94

T2 (N=25)

0.62 ±0.13

0.57 ±0.12

27.78 ±6.28

C2 (N=19)

0.63 ±0.12

0.60 ±0.13

T3 (N=25)

0.62 ±0.11

C3 (N=17)

Alk Phosp (U)

AT

Serum Protien (gm/dl)

Serum Alb (gm/dl)

BT

AT

BT

AT

AT

BT

35.32 ±4.65

142.77 ±24.34

149.80 ±23.50†

6.48 ±0.58

6.44 ±0.50

3.59 ±0.40

3.56 ±0.41

36.05 ±6.39

33.90 ±5.06

154.45 ±25.03

158.90 ±25.68

6.67 ±0.60

6.62 ±0.58

3.51 ±0.43

3.60 ±0.40

27.53 ±4.19

35.40 ±4.37

35.23 ±4.16

142.44 ±37.12

147.22 ±32.28

6.47 ±0.49

6.58 ±0.43

3.36 ±0.41

3.40 ±0.40

27.32 ±6.57

27.21 ±4.21†

34.69 ±6.65

35.79 ±5.80

144.21 ±31.16

140.63 ±34.16

6.38 ±0.49

6.60± 0.36

3.44 ±0.37

3.55 ±0.36

0.57 ±0.10

24.91 ±5.55

26.02 ±4.17

32.26 ±6.15

31.7 ±6.01

147.64 ±29.14

157.21 ±30.41†

6.66 ±0.64

6.70 ±0.50

3.47 ±0.56

3.66 ±0.41

0.63 ±0.11

0.61 ±0.11

27.52 ±6.89

28.28 ±4.63

35.61 ±4.55

36.38 ±5.50

143.29 ±27.85

156.29 ±27.43†

6.25 ±0.52

6.50 ±0.41

3.45 ±0.46

3.56 ±0.41

T4 (N=20)

0.59 ±0.12

0.55 ±0.11

26.66 ±6.28

27.95 ±6.39

36.16 ±4.06

37.29 ±4.30

137.21 ±42.33

140.50 ±46.09

6.24

6.41

3.28

±0.31*

±0.26*

±0.32*

3.37 ±0.36

C4 (N=14)

0.59 ±0.10

0.58 ±0.09†

25.14 ±7.35

26.07 ±4.92

32.21 ±8.28

32.64 ±8.12

163.29 ±24.36

167.86 ±41.65

6.99 ±0.67

6.98 ±0.45

3.74 ±0.40

3.76 ±0.40

* p<0.01, compared to control ; † p<0.01, compared to BT

Table 4. Effect of Garbhpal ras on neonatal parameter.

Gestational age (weeks)

Weight (gm)

Height (cm)

CC (cm)

HC (cm)

MAC (cm)

APGAR score (1stmin)

APGAR score (5thmin)

APGAR score (10thmin)

C1 (n=20)

38.40 ±1.60

2.66 ±0.31

48.13 ±1.70

31.89 ±1.25

33.50 ±1.14

9.23 ±0.75

6.42 ±0.61

7.52 ±0.51

8.63 ±0.50

T1 (n=29)

39.17 ±0.93

2.99 ±0.33**

49.74 ±1.27**

32.24 ±1.24

34.10 ±0.91

9.61 ±0.51*

6.69 ±0.89

7.79 ±0.68

8.79 ±0.68

C2 (n=15)

38.40 ±1.12

2.63 ±0.32

48.00 ±1.80

32.50 ±1.10

33.68 ±1.12

9.04 ±0.70

6.50 ±0.76

7.82 ±0.50

8.78 ±0.43

T2 (n=22)

38.91 ±1.15

2.94 ±0.29**

49.45 ±1.41*

32.75 ±1.32

33.81 ±0.72

9.69 ±0.81*

6.68 ±0.65

7.64 ±0.63

8.82 ±0.40

C3 (n=14)

38.43 ±1.02

2.65 ±0.31

48.23 ±1.79

32.92 ±1.71

33.42 ±1.32

9.19 ±0.50

6.61 ±0.65

7.75 ±0.45

8.77 ±0.44

T3 (n=16)

39 ±1.21

2.92 ±0.40

49.10 ±1.12

32.34 ±1.29

33.97 ±0.85

9.78 ±1.10

6.81 ±0.54

7.88 ±0.50

8.88 ±0.50

C4 (n=13)

38.23 ±1.24

2.54 ±0.46

47.75 ±1.91

31.71 ±1.71

32.96 ±1.57

9.25 ±0.69

6.42 ±1.00

7.41 ±1.00

8.42 ±1.00

T4 (n=12)

38.67 ±0.94

2.76 ±0.36

48.83 ±1.47

31.46 ±0.87

33.50 ±0.80

9.33 ±0.89

6.62 ±0.52

7.61 ±0.51

8.69 ±0.48

Group

* p<0.05, compared to control ; ** p<0.01, compared to control.

Clinical Trial of Garbhpal ras in Pregnancy Outcome

control group (Table 2 and 3). Normal levels of bio-chemical parameters till the end of treatment, show non-toxic nature of Garbhpal ras.

Effect of Garbhpal ras on outcome of pregnancy We are able to ascertain here the outcome of 141 pregnancies. There were 143 live births including 2 twins in Garbhpal ras group. No stillbirth, spontaneous abortion or major malformation was observed in any group during study. However there were 3 cases of intrauterine growth restriction, one case of intrauterine death (due to pregnancy induced hypertension) and 2 preterm deliveries (of gestational age 34-35 weeks) came across during study in control group (Table 6). Two patients of preterm delivery (of gestational age 35-36 weeks) were also observed in Garbhpal ras group (T2), though they were positive for TB IgM and VDRL respectively. Total 62.5% and 57.52% women out of total trial and total control group respectively had history of spontaneous abortion(s) in their previous pregnancies (Table 1). Women belong to control group, having previous history of spontaneous abortion advised progesterone therapy during present study, while women of trial group did not received such therapy. It showed that Garbhpal ras might have some role in successful outcome of pregnancy. But its efficacy and mode

17

of action yet to be determined on larger population. Ayurvedic sages have suggested several precautions to minimize the risk of abortions. If the uterus has vitiated doshas, then it expels the fetus, termed garbhsrava / garbhpata (S.S.Ni.8/10; M.Ni.64/2; B.P.Chi.70/72; Y.R.Stri Garbh Roga Ni.). Such uterus does not cause proper upbringing of fetus and sometimes baby may die soon after birth. Amongst all doshas, vata is predominant. By its dynamic and randomness nature, it expels the fetus. Garbhpal ras contains balya and vata-shamak herbs. They Table 5. Incidence of spontaneous vaginal delivery/ caesarean section.

Group

SVD

LSCS

C1 (n=20)

15

5

T1 (n=29)

18 (1LPV)

11

C2 (n=15)

7

8

T2 (n=22)

12 (1-Br, 1-LPV, 2-PT)

10

C3 (n=14)

9 (1-IUGR)

5

T3 (n=16)

11

5

C4 (n=13)

3 (1-IUD, 2PT)

10

T4 (n=12)

5

7

Control (n=62)

34 (54.84%)

Trial (n=79)

46 (58.23%)

28 (45.16%) 33 (41.77%)

Table 6. Indication for caesarean section.

FD

IUGR

PIH

Post Dated

FPOL + LPV

Abn. Presen.

CPD

Twins

Previous

C1 (n=20)

1

-

-

-

-

1

2

-

1

T1 (n=29)

1

-

-

-

1

1

4

-

4

C2 (n=15)

2

-

1

-

2

-

-

-

3

T2 (n=22)

1

-

-

-

-

1

3

1

4

C3 (n=14)

1

1

1

-

-

-

-

-

2

T3 (n=16)

1

-

-

1

-

-

1

-

2

C4 (n=13)

3

-

-

-

1

1

2

-

3

T4 (n=12)

1

-

-

1

-

1

-

1

3

Group

(SVD= Spontaneous vaginal delivery; LSCS=Lower segment caesarean section; PIH=Pregnancy induced hypertension; FPOL=Failed progress of labour; Br=Breech presentation; PT=preterm delivery ( 36 weeks gestational age);

FD=Fetal distress; LPV= Leaking per vaginum;

IUGR=Intra uterine growth restriction; CPD=Cephalo-pelvic disproportion; IUD=Intrauterine death of fetus)

CS

18

Mishra, Sinha and Kumar

may probably help in strengthening the ligaments which hold the uterus in place, supporting the weight of the fetus and prevent garbhsrava or garbhpata.

The incidence of spontaneous vaginal delivery was observed little more in Garbhpal ras treated group (58.23%) in comparison to control (54.84%) (Table 5).

Effect of Garbhpal ras on neonatal parameters Average mean of anthropometry of neonate (weight, height, head circumference, chest circumference, mid arm circumference) were found improved in Garbhpal ras group in comparison to control group. It was also observed that pregnant women registered during first trimester of pregnancy, treated with Garbhpal ras have statistically significant changes in weight of baby at birth (2.99 kg in T1 and 2.94 kg in T2) with respective control (2.66 kg in C1 and 2.63 kg in C2). Length (crown heel length) and mid arm circumference in T1 and T2 groups had also shown statistically significant changes with respective control (Table 4). Apgar score and gestational age at the time of delivery had not shown significant difference in comparison to control (Table 4). It means Garbhpal ras has not any effect on these parameters.

Health status of women along with good nutrition is prime key to the programming of the proper fetal growth in the uterus (C.S.Sh.2/6).

Good maternal body composition due to good nutrition ensures good immune adaptations and it also provides plenty of antioxidants and micronutrients for benefit of fetal growth. Ginger (Asnani and Verma, 2007), Cinnamon (Jayaprakash et al., 2006), Black pepper (Gulcin, 2005), Long pepper (Singh et al., 2007) and Munakka (Koga et al., 1999) have antioxidant activity. In a state of oxidative stress i.e. pregnancy, antioxidants help to protect the body from the formation of free radicals which impair the immune system (Palep, 2007).

Piperine has immunomodulatory and cytoprotective activity, thus pregnant women * Devdaru (Devdar) = Cedrus deodara

taking Garbhpal ras have better immunity to overcome several diseases (Pathak and Khandelwal, 2007; Choi et al., 2007). Herbs like ginger, black pepper, long pepper and coriander helps in digestion, thus nourish mother as well as fetus.

Most of the contents of the Garbhpal ras (Cinnamon, long pepper, cardamom coriander, ginger, Devdaru*) have madhur vipak, hence being anabolic, it improves weight of mother as well as fetus. Small doses of mercury to human beings cause an increase in the number of red blood corpuscles, while body weight and general nutrition are also improved (Nadkarni, 1982).

In such condition, fetus gets better nutrition.

Ginger also plays a role in increased growth of fetus (Wilkinson, 2000). Anupan of Garbhpal ras i.e. milk and draksha are madhur in ras, add glucose in diet and consequently improve general nutrition of fetus. These may be the possible reasons of increase in weight of fetus of trial group in comparison to control group. No gross congenital anomaly was observed in any of the neonates in all the groups.

Conclusion Normal haematological picture, maintained renal and liver function tests show non-toxic nature of drug. Improvement in hemoglobin percentage and normal bio-chemical parameters verify safe use of Garbhpal ras. Successful outcome in cases of previous spontaneous abortion with improved neonatal parameters justify the efficacy of Garbhpal ras. It gave a crystal clear impression that Garbhpal ras can be recommended safely at therapeutic dose in pregnancy.

Acknowledgements Authors are thankful to Dabur India Ltd., Sahibabad, U.P. (India) for providing gift sample of Garbhpal ras.

1.

References Asnani, V. and Verma, R.J: Antioxidative effect of rhizome of Zinziber officinale on paraben induced lipid peroxidation: an in vitro study. Acta Pol Pharm. 64(1): 35-7 (2007).

Clinical Trial of Garbhpal ras in Pregnancy Outcome 2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

Bhav Prakash: Vidyotini Hindi commentary and notes by Pt. Brahm Shankar Mishra. Chaukhambha Sanskrit Sansthan, Varanasi. Ed. VIII. Vikram samvat (2050).

Charak Samhita: Hindi Commentry by Kashinath Shastri and Dr. Gorakh Nath. Chaukhambha Sanskrit Sansthan, Varanasi. Ed. XVII (1991).

Chhangadi, G.S: In Ras Tantra Sar Va Siddhi Prayog Sangrah. Krishna Gopal Ayurveda Bhavan, Ajmer. Ed. IX. Vol. I. pp 554-55 (1999).

Choi, B.M., Kim, S.M., Park, T.K., Li, G., Hong, S.J., Park, R., Chung, H.T. and Kim, B.R.: Piperine protects cisplatin-induced apoptosis via heme oxygenase-1 induction in auditory cells. J Nutr Biochem. 18(9): 615-22 (2007).

Goyal, R.K. and Mahajan, R: In Adhyatan Ras Shastra. Chaukhambha Surbharati Prakashan, Varanasi. Ed. I. pp 309 (1988).

Gulcin, I: The antioxidant and radical scavenging activities of black pepper (Piper nigrum) seeds. Int J Food Sci Nutr. 56(7): 491-9 (2005).

Indian Medical Science Series: No. 3. Clinical Application of Ayurvedic Research and a list of Ayurvedic preparations by a panel of Vaidyas. Sri Satguru Publication, Delhi. Ed. III. (ISBN 81-7030101-07) pp160-61 (1994).

Jayaprakasha, G.K., Ohnishi-Kameyama, M., Ono, H., Yoshida, M. and Jaganmohan Rao, L: Phenolic constituents in the fruits of Cinnamomum zeylanicum and their antioxidant activity. J Agric Food Chem. 8; 54(5): 1672-9 (2006).

Koga, T., Moro, K., Nakamori, K., Yamakoshi, J., Hosoyama, H., Kataoka, S. and Arigo, T: Increase of antioxidative potential of rat plasma by oral administration of proanthocyanidin-rich extract from grape seeds. J Agric Food Chem.

47(5): 1892-1897 (1999).

Madhav Nidan: Vidyotini Hindi commentary and notes by Sri Sudarshan Shastri. Chaukhambha Sanskrit Sansthan, Varanasi. Ed. XVIII (1989).

Mishra, D., Sinha, M., Singh, P.N. and Kumar, V: Acute and sub-chronic toxicity study of Garbhpal ras. Electronic J Pharmacol Therapy.

1: 31-34 (2008).

Mishra, D., Sinha, M., Kumar, M. and Kumar, V: Chronic toxicity study of ‘Garbhpal ras’, An Ayurvedic medicine. J Herb Med Toxicol.

3(1): 13-17 (2009a).

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

Mishra, D. , Sinha, M. and Kumar, V: Teratological study of ‘Garbhpal ras’, An Ayurvedic formulation. J Herb Med Toxicol.

3(1): 37-40 (2009b).

Mishra, D. , Sinha, M. and Kumar, V: Safety evaluation of Garbhpal ras, an Ayurvedic formulation. In National Scientific Seminar on Reproductive Health of Women through Ayurveda.

Rashtriya Ayurveda Vidyapeeth, Research papers, New Delhi. pp 251-261 (2009c).

Mishra, D., Sinha, M. and Kumar,V: Effect of ‘Garbhpal ras’ on pregnancy induced nausea and vomiting (NVP). Ayurvedic Rennaince. (2009d).

Accepted for publication.

Mishra, D., Sinha, M. and Kumar, V: Clinical evaluation of ‘Garbhpal ras’ in pregnancy related gastrointestinal disorders. Ayurvedic Rennaince.

7(2): 13-16 (2009e).

Nadkarni, A.K: Indian Materia Medica. Vol 2.

Bombay Popular Prakashan. pp 72-73 (1982).

Palep, H.S. Beyond safe motherhood to programming for a super baby II (2007). available on www.

Pharmabiz.com/article/ detnews.asp?Arch=&articleid=11331&sectioned=46.

Pathak, N. and Khandelwal, S: Cytoprotective and immunomodulating properties of piperine on murine splenocytes: an in-vitro study. Eur J Pharmacol (2007). In press.

Rasyog Sagar: By Hari Prapanna Sharma. Krishna Das Academy, Varanasi. Garbhpal ras II 1907-10/ Ras Chandanshu. Vol. I. pp 374-75 (1983).

Singh, S.K., Shanmugavel, M., Kampasi, H., Singh, R., Mondhe, D.M., Rao, J.M., Adwankar, M.K., Saxena, A.K. and Qazi, G.N: Chemically standardized isolates from Cedrus deodara stem wood having anticancer activity. Planta Med. 73(6): 519-26 (2007).

Sushrut Samhita: Translation of Ayurveda Tatva Sandeepika by Kaviraj Ambika Dutta Shastri.

Chaukhambha Sanskrit Series, Varanasi. Ed. VII (1990).

Wilkinson, J. M: Effect of Ginger tea on the fetal development of Sprague- Dawley rats. Reprod Toxicol. 507-512 (2000).

Yog Ratnakar: Vidyotini Hindi commentary by Vaidya Sri Lakshmi Pati Shastri edited by Sri Brahma Shankar Shastri. Chaukhambha Sanskrit Sansthan, Varanasi. Ed. II (1973).

Address for correspondence: Dr. Deepa Mishra, Incharge and Lecturer, Department of Prasuti Tantra, A & U Tibbia College and Hospital, New Delhi - 110005 (India). E-mail: deepamishra219@gmail.com 009_2009

19

J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 21-26

ISSN 0970-7700

INHIBITORY EFFECT OF THE ROOT OF SIDA ACUTA BURM. F. ON CALCIUM OXALATE CRYSTAL GROWTH T. VIMALA AND S. GOPALAKRISHNAN* Department of Chemistry, Manonmaniam Sundaranar University, Abishekappatti, Tirunelveli - 627012 Tamil Nadu (India) Abstract: The root of Sida acuta Burm. F. is useful in nervous and urinary diseases, disorders of blood and bile and in chronic bowel complaints. Calcium oxalate is the most important constituent of urinary stone crystals and several research workers have been attracted to carry out in-vitro studies on urinary stone crystals. However, the inhibitory effect of the root of S. acuta on calcium oxalate crystal growth in-vitro has not been carried out. In the present work an attempt has been made to grow crystals of calcium oxalate and to study the inhibitory effect of the methanolic and aqueous extracts of the root of S. aucta on calcium oxalate crystal growth invitro. Calcium oxalate crystals were grown in silica gel media in Haneâ&#x20AC;&#x2122;s tubes by single diffusion method. The crystal growth after the addition of the methanolic and aqueous extracts in 20 mg/ 5 ml and 10 mg/5 ml doses was studied. In both the extracts there were reductions in sizes of calcium oxalate crystal columns and also the size of the individual calcium oxalate crystals when compared to that of the controls. The results of in-vitro experimental models, IR and SEM together attribute an inhibitory capacity with respect to calcium oxalate crystals for the methanolic and aqueous extracts of Sida acuta.

Keywords: Urinary disorders, Calcium oxalate, Crystal growth, Sida acuta.

Introduction Sida acuta Burm.F. (syn. S. carpinifolia Mast; family â&#x20AC;&#x201C; Malvaceae) is an erect, perennial undershrub or shrub, distributed throughout the hotter parts of India and Nepal. It is commonly known as Vattatiruppi in Tamil and Bala in Sanskrit. This species is not only important as a medicine, but also yields a good fibre. The root is commonly used for nervous and urinary disorders and also for curing various ailments (Anonymous, 1952). The major alkaloid of S. acuta was cryptolepine and this exhibited antimicrobial activity against Proteus vulgaris (Leslie et al., 1980). The aqueous extract of the whole plant showed significant hepato protective activity against carbontetrachloride, paracetamol and rifampicin induced hepato toxicities in experimental albino rats (Rao, 1998).

Chemical analysis of the whole plant has led to the isolation of many compounds (Saraswathy et al., 1998; Prakash et al., 1981). Ethanolic * Professor and Head, Pharmaceutical Chemistry

extract of the whole plant of S. acuta showed partial neutralization effect against venom lethal effect (Otero et al., 2000).

Deposition of calcium oxalate microcrystal in human body can be a significant problem and it is recognised that 70 - 80 % of kidney stones contain calcium oxalate (Seftel and Resnick, 1990). Barring D- penicillamine and allopurinol for cystine and uric acid stones, no effective drug therapy is available in Allopathy for the treatment or prevention of other types of stones.

However, a large number of indigenous drugs have been used for this purpose in our country since ancient times (Chopra et al., 1956).

Cystone is claimed to have properties of crushing stones in-situ and expelling them without surgery (Dandia et al., 1976). Saxifraga liqulata and Tribulus terrestris are two common herbs of this herbo - mineral formulation (Handa and Kapoor, 2000). The chances of another stone formation can be minimised and dissolution of existing

22

Vimala and Gopalakrishnan

stones enhanced if the composition of urine is modified. The rationale behind the use of herbal remedies is not well established except for a few plants. Studies on the growth of urinary crystals in the presence of some substances which act as inhibitors or promoters of crystal growth may help to pick out potential substances for use by recurrent stone formers (Natarajan et al., 1997).

In the present work an attempt has been made to study the inhibitory effect of the methanolic and aqueous extracts of the root of Sida acuta on calcium oxalate crystal growth in-vitro.

Materials and Methods The roots of S. acuta were collected from Tirunelveli, Tirunelveli District, Tamil Nadu, India during the month of September. The botanical identity was confirmed by comparing the sample with the Herbarium specimens preserved in the Department of Botany, by Dr. V. Chelladurai, Govt. Siddha Medical College, Palayamkottai, Tirunelveli District, Tamil Nadu, India. A voucher specimen of the plant had been deposited at the Herbarium, Department of Chemistry, Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India [Voucher specimen number MSU 054]. The plant material was thoroughly washed with water, cut into small pieces, dried under shade and powdered.

The powdered root was successively extracted with petroleum ether (40º- 60ºC), benzene, chloroform, methanol and water. The methanol and aqueous extracts were condensed and evaporated to dryness under vacuum.

There are previous reports on the growth of calcium oxalate crystals from aqueous solution and gel methods (Sindhu et al., 1992; Srinivasan and Natarajan, 1996; Aravindakshan and Jayanthi Bai, 1996). In the present study calcium oxalate crystals were grown in -vitro in silica gel media in Hane’s tubes by single diffusion method. The gel was prepared by treating sodium metasilicate solution of density 1.03 g/ml with 3M acetic acid and the pH was adjusted to 6.3. One of the reactants,

calcium chloride (1M) was incorporated inside the gel. After gellation, 1M oxalic acid was slowly added over the gel along with ethanol/water as the supernatant solution for control group and dried methanolic extract dissolved in ethanol / aqueous extract of the test plant for other groups.

The crystals appeared as cloudy precipitate and in due course, the depth of the column was found to increase. The thickness (length) of crystal column was measured in centimeter on days 1, 3, 7, 10, 15, 20, 25 and 30. The sizes of the crystals formed were noted in microns (μm) on days 1, 3, 7, 14, 21 and 28 using an optical microscope and the values thus obtained were compared with each other. The whole experiment was carried out at room temperature (27o + 3oC). The inhibitory effect of methanolic and aqueous extracts were studied at 20 mg/5 ml and 10 mg/5 ml doses. The experiments were repeated six times and the average value was taken. All the results were expressed as mean + SE. The test of significance was statistically analyzed using Student’s t-test (Fisher, 1950). After the crystal growth, the crystals were cleared off the gel by repeated washing with distilled water, filtered and air dried. The purity of the crystals thus obtained was assessed by infra-red spectroscopy using JASCO FT-IR-410

by KBr pellet method. For SEM studies a JEOL JSM 35 - C scanning electron microscope was used.

Results and Discussion The inhibitory effect of the methanolic and aqueous extracts of the root of S. acuta on calcium oxalate crystal growth have been studied in-vitro at 20 mg/5 ml and 10 mg/5 ml doses and the results are presented in Tables 1 and 2.

Concentration dependent inhibitory effect was observed in methanolic extract of S. acuta where the degree of inhibition was more with 20 mg/ 5 ml concentration. In the case of aqueous extract, the size of the crystal columns were almost equal for higher and lower concentrations but slightly less than the size of crystal columns in the control set up. Reductions in the lengths

Effect of Sida acuta on Calcium Oxalate Crystal Growth

of crystal columns were observed even on the first day of the experiment for methanolic extract and the reduction in growth rate was more significant from fifth day onwards. Aqueous extract of S. acuta did not produce significant reductions in the lengths of crystal columns.

The mean lengths of the crystal columns in the control set up were 3.48 cm and 3.97 cm respectively on the 30th day when ethanol and distilled water were used. The mean lengths of crystal columns in the extract set up were 2.88 cm (20 mg/5 ml) and 2.93 cm (10 mg/5 ml) for methanolic extract and 3.83 cm (20 mg/5 ml) and 3.83 cm (10 mg/5 ml) for aqueous extract.

Calcium oxalate crystals from the extract treated tubes when viewed under microscope showed considerable reduction in size when compared to that of the control. The mean lengths of the grown crystals on 28th day of the experiment in the control set up were 180 μm and 294 μm

23

respectively when ethanol and distilled water were used. The mean lengths of the crystals in the extract set up were 96 μm (20 mg/5 ml) and 162 μm (10 mg/5 ml) for methanolic extracts and 246 μm (20 mg/5 ml) and 262 μm (10 mg/ 5 ml) for aqueous extracts on 28th day. The results are presented in Fig. 1 and Fig. 2.

The drug treated crystals are found to exhibit IR spectra similar to that of the control suggesting no possibility of any type of chemical bonding between the drug and the oxalate crystals. These IR spectra are shown in Fig. 3-6.

The calcium oxalate crystals were also subjected to SEM analysis for understanding the morphological changes if any. SEM of in-vitro grown calcium oxalate crystals by single diffusion method in silica gel media are presented in

Table 1. Effect of methanolic extract of Sida acuta root on the lengths of calcium oxalate crystal columns in cm.

Days

1 3 7 10 15 20 25 30

Control

Sida acuta

1.783 + 0.08 2.200 + 0.05 2.716 + 0.01 3.016 + 0.03 3.266 + 0.03 3.483 + 0.04 3.533 + 0.04 3.483 + 0.04

20 mg/ 5 ml

10 mg/ 5ml

1.700 + 0.00 2.150 + 0.00 2.383 + 0.01 ** 2.533 + 0.03 ** 2.783 + 0.01 ** 2.883 + 0.01 ** 2.883 + 0.01 ** 2.883 + 0.01 **

1.633 + 0.03 2.133 + 0.03 2.383 + 0.03 * 2.550 + 0.05 * 2.783 + 0.08 * 2.933 + 0.08 * 2.933 + 0.08 * 2.933 + 0.08 *

All values are mean + SEM of six experiments in each set * P< 0.05; ** P< 0.005

Table 2. Effect of aqueous extract of Sida acuta root on the lengths of calcium oxalate crystal columns in cm.

Days

1 3 7 10 15 20 25 30

Control

1.733 + 2.566 + 3.133 + 3.616 + 4.016 + 4.683 + 3.966 + 3.966 +

Sida acuta

0.02 0.02 0.02 0.02 0.02 0.04 0.07 0.07

All values are mean + SEM of six experiments in each set * P< 0.05; ** P< 0.005

20 mg/5 ml

10 mg/5 ml

1.700 + 2.533 + 3.083 + 3.566 + 3.950 + 4.566 + 3.833 + 3.833 +

1.750 +0.03 2.583 +0.03 3.166 +0.03 3.650 +0.03 4.050 +0.04 4.566 +0.04 3.766 +0.03 3.833 +0.04

0.02 0.03 0.04 0.03 0.03 0.02 0.02 0.03

24

Vimala and Gopalakrishnan

Fig. 7-10. The crystals from control experiments contained individual prismatic crystals, twinned crystals and some aggregates.

Crystals from methanolic extract set up contained some deformed and smaller crystal aggregates in addition to single and twinned crystals. Addition of the extract resulted in partial dissolution of the crystals with cracks and crevices and a tendency for chipping away of the parts as compared to control. Similar results were reported by Aravindakshan and Jayanthi Bai (1996). Aqueous extract showed a marked reduction in the tendency to form crystal aggregates as is evident from Fig. 10. Aqueous extract when included in the crystal growth set up also resulted in corrosion, crevices in the crystals which could indicate a dissolution effect for drug. Agglomeration of calcium oxalate crystals plays a major role in urinary stone formation and has the potential to produce very large crystals in a short period of time with no

Fig. 3. IR spectra of calcium oxalate crystals treated with ethanol.

Fig. 4. IR spectra of calcium oxalate crystals treated with water

Fig. 1. Effect of methanolic extract of S. acuta on the size of the crystals.

Fig. 2. Effect of aqueous extract of S. acuta on the size of the crystals.

Fig. 5. IR spectra of calcium oxalate crystals treated with methanolic extract of S. acuta

Fig. 6. IR spectra of calcium oxalate crystals treated with aqueous extract of S. acuta

Effect of Sida acuta on Calcium Oxalate Crystal Growth

7a

7b

Fig. 7. Scanning electron micrographs of calcium oxalate crystals (a) On adding ethanol (b) On adding ethanol showing sharp edges.

8a

8b

Fig. 8. Scanning electron micrographs of calcium oxalate crystals (a) On adding methanolic extract of S. acuta (b) On adding methanolic extract of S. acuta showing corrosion.

dissolution of the crystals. Natarajan et al.

(1997) have reported that no nucleation, reduction in the number and size of the crystals, reduction in total mass of the crystals formed, the change in the morphology of crystals and change in the crystalline quality may be considered as inhibitory effects in crystal growth experiments.

In the present investigation the morphology of the crystals have changed with uneven surfaces and less defined edges. Platy crystals were obtained (Fig. 8b) when methanolic extract was used. Change in the lengths of crystal columns, shape, size, transparency were also observed indicating the inhibitory effect of the two extracts of Sida acuta.

Acknowledgements One of the authors (T. Vimala) wishes to thank the University Grants Commission, New Delhi and the Principal and Management of St. Judeâ&#x20AC;&#x2122;s College, Thoothoor, Kanyakumari District (India) for selecting her under FIP Programme.

The authors also thank Dr. Y.M. Fazil Marickar, Govt. Medical College, Trivandrum for technical assistance and Dr. Peter Koshy, R. R. L. Trivandrum (India) for SEM analysis.

1.

9a

9b

2.

Fig. 9. Scanning electron micrographs of calcium oxalate crystals (a) On adding water showing twinning (b) On adding water showing aggregation.

3.

4.

10a

10b

Fig. 10. Scanning electron micrographs of calcium oxalate crystals (a) On adding aqueous extract of S. acuta showing minimal aggregation. (b) On adding aqueous extract of S. acuta showing less defined edges.

reduction of supersaturation (Hounslow et al., 1988). The crystals also showed less defined edges with dots of corrosion which could facilitate

25

5.

6.

7.

References Anonymous: Wealth of India, Raw Materials.

CSIR, New Delhi. pp322-323 (1952).

Aravindakshan, C. and Jayanthi Bai, N: Effect of Premna latifolia Roxb and Imperata arundinacea Cyril on in-vitro oxalate crystal growth. Ind. J. Clinical. Biochem. 11: 42-45 (1996).

Chopra, R.N., Nayar, S.L. and Chopra, I.C: Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi (1956).

Dandia, D.S., Bhatia, S., Narula, I.M.S. and Pendse, A.K: Role of cystone in prevention of urolithiasis : An experimental study in dogs.

Ind. J. Surg. 3: 122 (1976).

Fisher, R.A: Statistical Methods for Research Workers. Oliver and Boyd, Edinburgh (1950).

Handa, S.S. and Kapoor, V.V: Pharmacognosy.

2nd ed. Vallabh Prakashan, Delhi. pp215 (2000).

Hounslow, M.J., Ryall, R.L. and Marshall, V.R: A discretized population balance for nucleation, growth and aggregation. AICHE Journal.

34: 1821-32 (1988).

26 8.

9.

10.

11.

Vimala and Gopalakrishnan Leslie Gunatilaka, A.A., Sotheeswaran, S., Balasubramaniam, S., Chandrasekara, A.I. and Badra Sriyani, H.T: Studies on medicinal plants of Sri Lanka III : Pharmacologically important alkaloids of some Sida species.

Planta Med. 39: 66-72 (1980).

Natarajan, S., Ramachandran, E. and Blisin Suja, D: Growth of some urinary crystals and studies on inhibitors and promoters. II. X–ray studies and inhibitory or promotery role of some substances.

Cryst. Res. Technol. 32: 553-559 (1997).

Otero, R., Nunez, V., Jimenez, S.L., Fonnegra, R., Osorio, R.G., Garcia, M.E. and Diaz, A: Neutralization of lethal and enzymatic effects of Bothropsatrox venom. J. Ethnopharmacol.

71: 505-511 (2000).

Prakash Anand, Varma, R.K. and Ghosal, S: Chemical constituents of Malvaceae Part III. Alkaloidal constituents of Sida acuta, S. humilis, S. rhombifolia and S. spinosa. Planta Med. 43: 384-388 (1981).

12.

13.

14.

15.

16.

Rao, K.S. and Mishra, S.H: Anti–inflammatory and hepato protective activities of Sida acuta.

Indian Drugs. 35: 92-97 (1998).

Saraswathy, A., Susan, T., Gnana Ravi, R., Govindarajan, S. and Kundu, A.B: Chemical investigation of Sida acuta Burm. F. Bulletin of Medico – Ethnobotanical Research.

19: 176-180 (1998).

Seftel, A. and Resnick, M.I: Metabolic evaluation of urolithiasis. Urol. Clin. North Am. 17: 159-69 (1990).

Sindhu, S., Krishnamoorthy, H., Thomas, N.E., Roshni, S.V., Vathsala, K.R., Aravindakshan, C. and Marickar, Y.M.F: Diffusion controlled growth of calcium oxalate.

Indian J. Pure Appl. Phys. 30: 82-83 (1992).

Srinivasan, N. and Natarajan, S: Growth of some urinary crystals and studies on inhibitors and promoters. I standardisation of parameters for crystal growth and characterization of crystals.

Indian J. Phys. 70A: 563-568 (1996).

Address for correspondence: S. Gopalakrishnan, Department of Chemistry, Manonmaniam Sundaranar University, Abishekappatti, Tirunelveli - 627012 Tamil Nadu (India). E-mail: sgkmsu@yahoo.com 020_2009

J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 27-32

ISSN 0970-7700

SPECTROSCOPIC INVESTIGATIONS OF PALAKARAI (COWRIE SHELL) PARPAM S. JOSEPH VEDHAGIRI,* K.GANESAN AND P.C. JOBE PRABAKAR Research and PG Department of Physics, T.B.M.L. College, Porayar - 609307 Nagai District, Tamil Nadu (India) Abstract: Parpam (Bhasma) is one form of internal medicine widely and effectively used in Siddha system of medicine. Shells of Cypraea moneta Linn. is commonly known as cowrie or cowry. In Siddha, it is known as Palakarai and in Ayurveda it is used as Varatika bhasma. The ingredients that are being used for the preparation of a parpam have to be subjected to several stages of pudam, known as calcination process. The three intermediate stages during the preparation of Palakarai parpam were taken for the study. All the three stages were subjected to particle size analysis, atomic and weight percentage of elemental variations using Scanning Electron Microscopy and Energy Dispersive Spectroscopy (SEM/EDS). Also the vibrational frequencies and their related functional groups were assigned using Fourier Transform Infrared Spectroscopy (FTIR) and finally the crystallographic phase changes by X-ray diffraction (XRD) were analyzed. The results of SEM/ EDS, FTIR Spectra and XRD are reported and discussed.

Keywords: Cypraea moneta shells, Palakarai parpam, SEM – EDS analysis, FTIR and XRD analysis, Siddha medicine, Varatika bhasma.

Introduction Shells of Cypraea moneta Linn. commonly known as cowries are called chozhi (or) palakarai in Tamil. It belongs to the phylum mollusca and the class gastropoda. Similar to the shells of oyster, palakarai also is the shell for protecting the sea mollusc Cypraea moneta. The shell, palakarai is obtained abundantly along the Indian coast. In ancient days palakarai were valued as currency equal to gold, and used as ornamental materials. In traditional system of medicine the shells of Cypraea moneta, have been used as medicine to cure various ailments mainly related with stomach. Palakarai parpam was found to be effective in anti-pyretic and anti-inflammatory, in experimentally induced albino rats (Devanathan et al., 2002). Also used in treating stomach poisoning and all toxaemic states, colic, retention of urine, gonorrhoea and inflammation of urino-genital tract (Anonymous, 1993).

Materials and Methods The samples of initial, intermediate and final stages during the preparation of palakarai parpam * Associate Professor (Physics)

were collected and labeled as JVP1, JVP2, and JVP3. Scanning electron microscope (SEM-Model: JEOL JSM 5610 Series) was used to examine the morphology and the particle size of the three stages and SEM images of four hundred magnifications of three stages are considered. Probable elements present in atomic and weight percentage at each stage are analyzed using EDS. The fundamental vibration band frequencies are also assigned for all the three stages by recording FTIR spectra over the region 400-4000 cm-1 at 4 cm-1 resolution using KBr wafer on a Nicolet Avatar-360 Series.

The XRD technique is employed to assess the phase purity and crystallographic changes during calcinations process of parpam. The XRD patterns of initial and final stages of parpam were recorded with high-resolution powder X-ray diffractometer, PANalytical–Philip–Netherlands / Model XPert Pro, with Cu-Kα 1 radiation (λ = 1.54056 A°) at 40 kev and 30 mA at the scanning rate of 2° per minute and 2θ was varied from 5 to 60°.

28

Vedhagiri, Ganesan and Prabakar

Results and Discussions SEM Particle size Analysis Figure 1a is representing SEM images of 400 magnifications of the three stages. It is observed that the morphology of the particles is of granular amorphous nature, varies from a minimum to maximum size. In comparing all the three stages most significant changes of particle size have been observed due to calcinations process. The range of particle size is varied from stage one to stage two from 41.67-16.66 μm to 33.67-8.34 μm due to first time calcinations.

The maximum size of the particle at the second stage is further reduced to 12.82 μm from 33.97 μm and the minimum size has been retained to 8.34 μm at the final stage which clearly indicates the particle size reducing due to the repeated calcinations. The size of the particles is decreasing in the order of JVP1 > JVP2 > JVP3.

The FTIR spectra of the samples could reveal the significant vibrational bands (Petsch et al., 1989).

The presence of hydroxyl O-H stretching and bending bands are observed around ~3660 to 3800 cm-1 and ~1650cm-1 (Ana et al., 1996) in all the stages and are pronounced less in the final stage which could be attributed to the reduction in the hydrogen bonded structure due to repeated high temperature treatment of the sample.

The presence of calcium carbonate is confirmed in all the three stages due to strong vibrational band frequencies prevailed around ~1420 to 1500 cm-1 and 860 to 875 cm -1 (Prabakaran et al., 2005).

The vibrational band observation around ~1080 cm-1 is attributed to the presence of calcium carbonate in the form of aragonite. At the final stage due to calcinations this band gets diminished, which clearly indicates that the form of calcium carbonate is not completely getting changed, from aragonite to calcite. It is noticeable that calcite has no vibrational band assignment at ~1080 cm-1 (Narasimhulu and Lakshmanan, 2000).

SEM-EDS Analysis The Electron Density Spectrum of 400 magnifications of the three stages is given in Figure 1b. The elemental compositions of palakarai parpam found in the three stages are given in Table 1.The weight percentage of calcium for all the stages are found to be prominent. First stage JVP1 is found to contain 75.07 wt% of calcium and rose to 93.56 wt% at the second stage after calcinations. Finally the weight percentage of calcium is maintained to 80.02% at the last stage. Magnesium, zinc, strontium and barium were the other elements present as listed in Table 1. After three repeated calcinations, palakarai parpam is found to contain calcium as the major constituent to the value of 80.02 wt%. It is concluded that palakarai parpam is consisting mainly of calcium and magnesium, barium and a trace amount of strontium as reported.

The presence of calcium carbonate in all the three stages is further confirmed by the existence of vibrational frequencies at ~875 cm-1 and ~712 cm-1 (Konwar et al., 2004). In the final stage, calcium carbonate has been further confirmed by the strong vibrational band frequencies exhibited at ~1420, ~874 and ~712 cm-1 (Gosh, 1978). In comparing the FTIR spectra of JVP1 and JVP2, it is observed that the vibrational band frequencies due to methyl amide and hydroxyl compounds related C-H, NH and O-H stretching have been shifted. At the final stage all these bands are found weak indicating that due to calcinations, the organic substances have been progressively reduced.

FTIR Analyses The recorded FTIR spectrum of the three stages is given in Figure 2. Each compound has its own unique absorption pattern in the finger print region ~1500 to 650 cm-1. (Kalsi, 1995).

XRD Analyses The XRD patterns are analyzed and the peaks are indexed. XRD patterns of JVP1 and JVP3 are presented in Figure 3. Powdered XRD patterns of initial stage showed the characteristic

Spectroscopic Investigations of Palakarai (Cowrie Shell) Parpam

Figure 1a: Stage I

Figure 1b: Stage I

Figure 1a: Stage II

Figure 1b: Stage II

29

Figure 1b: Stage III Figure 1a: Stage III Figure 1a: Three stages of SEM images of Palakarai Parpam (400 magnification)

Figure 1b: Three stages of EDS of Palakarai Parpam (400 magnification)

Vedhagiri, Ganesan and Prabakar

%T

30

%T

Wave numbers (cm-1)

Wave numbers (cm-1)

%T

Figure 3. XRD Palakarai Parpam

Wave numbers (cm-1) Figure 2. FTIR – Spectrum of Palakarai Parpam

peaks of poorly crystalline calcium carbonate. All the peaks are indexed from their observed d-values. The structure is found to be orthorhombic with lattice parameters a =3.8218 A°, b = 4.9827 A°, c = 24.2633 A° and α = β = γ = 90° with unit cell volume 462.04 (A°). 3 The lattice parameters of JVP3 (a =3.8502 A°, b = 10.8675 A°, c = 23.3284 A° and α = β = γ = 90° with unit cell volume 976.19 A°)3 are slightly changed and crystal system prevails the same as orthorhombic. It is a known fact that calcite form of calcium carbonate is always of hexagonal structure. If it changes from aragonite to calcite, the crystal system would be of hexagonal.

Spectroscopic Investigations of Palakarai (Cowrie Shell) Parpam

31

Table 1. Elemental composition of Palakarai Parpam.

Sl. No.

Element and Symbol

Weight %

Atomic %

JVP1

JVP2

JVP3

JVP1

JVP2

JVP3

1

Calcium, C

75.07

93.56

80.02

83.03

96.68

78.38

2

Magnesium, Mg

-

00.20

11.70

-

00.34

18.90

3

Iron, Fe

11.50

00.10

-

9.13

00.07

-

4

Zinc, Zn

06.06

-

-

04.11

-

-

5

Strontium, Sr

07.36

06.14

02.19

03.73

02.90

00.98

6

Barium, Ba

-

-

06.08

-

-

01.74

No such change of crystal structure from initial to final stage is observed, except the variations in sides and volume of unit cell. Moreover the peak around 2T of 29째 at the initial stage has not been shifted and remains almost the same in the final stage, confirming no phase transition of crystal system.

The increase in the intensity of the peaks is indicating the improvement of crystallinity due to calcinations process. The increase in unit cell volume is attributed to the increase of surface area which is a vital factor in drug interaction. The peaks around 2T of 29.2째 to 29.6째 are attributed to the presence of calcium carbonate (Davis et al., 1997). The sharp peaks observed at JVP3 clearly imply that the repeated calcinations promote crystallinity which is accountable for bioavailability and dissolution rate.

in aragonite form is observed. The XRD study reveals that the crystallinty has been improved due to calcinations. The orthorhombic crystal structure has not been changed and remains as orthogonal up to the final stage.

Acknowledgements Authors are grateful to Kaviraj Pharmaceuticals, Erode, Tamil Nadu (India) for providing the samples. Research grant received under a minor research project of UGC by one of the authors (SJV) is gratefully acknowledged.

1.

2.

3.

Conclusion The particle size of parpam is reduced due to repeated heat treatment. SEM-EDS analysis reveals that palakarai parpam is having calcium as prominent element and magnesium, barium and strontium as trace elements. The organic substances are progressively reduced from initial to the final stage. The presence of calcium carbonate

4.

5.

References Ana H Delgado, Ralph M, Paroli and James J Beaudock: Applied Spectroscopy. 50: 970 (1996).

Anonymous: Formulary of Siddha Medicines.

IVth ed. IMPCOPS, Chennai. pp 228 (1993).

Davis EE, Fisher AT and Firth JV Procee: Ocean Drilling Program - Initial Reports. Vol. 168: 35-45 (1997).

Devanathan Deva R, Prema S and Saraswathy Jeyaendra: Proce. of Fifth International Congress on Traditional Asian Medicine. pp 10 (2002).

Gosh SN: Jour. Mat. Sci. Vol. 13: 1877-1886 (1978).

32 6.

7.

8.

Vedhagiri, Ganesan and Prabakar Konwar R, Changmai R and Baruah GD: Indian Jour. of Pure and Applied Phy. Vol. 42: 812-815 (2004).

Kalsi PS: Spectroscopy of Organic Compounds.

IInd ed. New Age International Ltd., New Delhi. pp 74 (1995).

Narasimhalu, KV and Lakshmana RJ: Spectrochim. Acta- A. Vol. 56: 1345-1353 (2000).

9.

10.

Petsch, Clerc, Seibel and Simon: Tables of Spectral Data for Structure Determination of Organic Compounds, IInd ed. Springer â&#x20AC;&#x201C; Verlag, Hong Kong (1989).

Prabakaran K, Balamurugan A and Rajeswari S: Bull. Mater. Sci. Vol. 28(2): 115-119 (2005).

Address for correspondence: S. Joseph Vedhagiri, Research & PG Department of Physics, T.B.M.L. College, Porayar - 609307 Nagai District, Tamil Nadu (India). E-mail: jvedha@rediffmail.com 008_2009

J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 33-43

ISSN 0970-7700

HYPOGLYCAEMIC ACTIVITY OF INDIAN MEDICINAL PLANTS IN STREPTOZOTOCIN DIABETIC RATS E. N. SUNDARAM,1 K. P. SINGH2 AND P. UMAMAHESWARA REDDY3 Drug Standardisation Unit,1,2 Department of Zoology,3 Osmania University, Hyderabad – 500007 Andhra Pradesh (India) Abstract: In the present study, the hypoglycaemic effect of alcoholic extracts of M. charantia, A. marmelos and E. jambolana was studied in streptozotocin (STZ) induced diabetes. Rats were made diabetic by intraperitoneal injection of STZ (30 mg/kg) in citrate buffer. On confirmation of diabetes after 48 hrs of injection, alcoholic extract of medicinal plant (250 or 500 mg/kg) or glibenclamide (300 μg/kg) administered orally to rats for 30 days. These three plants produced dose and duration dependent hypoglycaemia very similar to that of glibenclamide. At the end of one month, serum glucose levels of STZ diabetic rats with daily doses of 500 mg/kg of any one of the alcoholic extract were ‘more or less’ comparable to that of normal rats. The anti-diabetic effect of these plants might be due to enhanced insulin secretion from the viable β-cells of islets of Langerhans as evidenced by presence of more viable β-cells and less necrotic changes in the pancreas of diabetic rats as compared to that of control diabetic rats. Thus, these plants appear to be better alternative for the diabetic patients who are prone to develop side effects with the regular use of synthetic hypoglycaemic drugs as these are also plants devoid of any untoward/toxic effects.

Keywords: STZ diabetic rats, Hypoglycaemic effect, Histopathology, Momordica charantia, Aegle marmelos, Eugenia jambolana.

Introduction In the present era, diabetes mellitus is a global problem. There are an estimated 143 million people worldwide, suffering from diabetes.1 This number may probably double by the year 2030.2 The diabetes enters in man’s life unnoticed as its symptoms remain silent for many years and is considered to be the mother of many diseases. Reports from the World Health Organization (WHO) indicate that diabetes mellitus is one of the major killers of our time, with people in South - East Asia and Western Pacific being most at risk. 3 Prolong and continuous use of currently available oral hypoglycaemic drugs in modern medicine not only associated with side effects like anorexia, nausea, vomiting, flatulence, abdominal discomfort but their use is also contraindicated in patients suffering from renal, hepatic, cardiorespiratory insufficiency, alcoholism and in advanced age. Likewise, repeated injections of

insulin may lead complications like insulin allergy, insulin resistance and localized lipoatrophy at the site of injections.4, 5 On the contrary, long before the use of insulin, medicinal plants have been used since ancient times by the physicians and laymen to treat not only the diabetes but also a great variety of human diseases such as heart diseases, cancer etc.6, 7 The text of Charaka, Sushruta, Vagbhatta and Ayurveda also elaborated in details the clinical features of diabetes mellitus, its complications and treatment with medicinal plants. The therapeutic approach of traditional medicines is more holistic because multifactorial pathogenicity of diabetes demands multi - model therapeutic approach.8 Considering the economic resource constraints, easy availability and cheapness and day to day use of M. charantia, A. marmelos and E. jambolana in diabetes mellitus by laymen since ancient time. The present study was undertaken to elucidate the effectiveness of these three plants as

1. Research Officer (Endocrinology) 2. Research Officer (Pharmacology)

3. Professor

34

Sundaram, Singh and Reddy

anti-diabetic drugs in streptozotocin (STZ) diabetic rats in a comparative way as compared to that of glibenclamide, a standard oral hypoglycaemic drug used in clinical practice in order to provide a scientific basis for their rationale use.

Materials and Methods Animals Albino rats of both sexes, weighing between 175 - 200 g, were procured from National Centre for Laboratory Animal Sciences, National Institute for Nutrition, Hyderabad and housed (12/ 12 hrs, light/dark cycles, room temp. 22 Âą 1.0oC.) in polypropylene cages (47 x 34 x 20 cm) lined with husk. The rats were acclimatized to the laboratory conditions for approximately 10 days before use and had free access to nutritious balanced diet and water through out the study.

Prepration of Alcoholic Extracts Mature and unripe fruits of Momordica charantia, mature and half ripe fruits of Aegle marmelos and fully ripe fruits of Eugenia jambolana were collected locally (Figures 1-8).

The whole fruit including seeds of M. charantia, and seeds of E. jambolana were cut into small pieces and pulp of the fruits of A. marmelos were all air dried and made into course powder in an electric grinder. The powder was filled in Soxhlet apparatus and extracted with 95% alcohol in a ratio of 1:4 until the alcohol extract coming out of the Soxhlet becomes completely colourless. Later on, alcohol extract was concentrated by evaporating the alcohol in a distillation apparatus and lyophilized under vacuum. Lyophilized material was weighed and made into suspension (50 mg/ml) with distilled water before use.

Drugs and Chemicals Streptozotocin (Sigma, USA), glibenclamide (Aventis Pharma, Mumbai, India), readymade kits/ reagents (M/S. Excel Diagnostic Pvt. Ltd., Hyderabad, India), haematoxylin-eosin and aldehyde-fuchsin stains and all other chemicals used in this study were of analytical grade.

1

3

2

Figure 1. Momordica charantia Linn.

Figure 2. M. chrarantia (fruits) Figure 3. M. chrarantia (Twig with fruit)

5

4 Figure 4. Aegle marmelos (L.) Corr.

Figure 5. A. marmelos (Twigs with fruits)

6 6

7

Figure 6. Eugenia jambolana Lam. Figure 7. E. jambolana (Twigs with fruits) Figure 8. E. jambolana (fruits)

8

Hypoglycaemic Activity of Medicinal Plants

Collection of Blood Samples The blood samples were collected from overnight fasted rats by retro-orbital puncture through heparinized coated microcapillaries into nonheparinized tubes before and after 48 hrs of STZ injection, on 11th day, 21st day and on 31st day of administration of alcoholic extract of medicinal plants or glibenclamide. The serum was separated from clotted blood by decanting.

Estimation of Serum Glucose The concentration of glucose in serum was determined by enzymatic method as described

by Trinder.9 In brief, 0.01 ml of serum sample was added into test tube containing one ml of glucose reagent. A parallel standard sample and a blank were also run simultaneously. The contents were mixed well and incubated at 37oC for 10 min. The absorbance of the serum samples and the standard sample were measured on spectrophotometer (Systronic) at 505 nm after calibrating it against blank and calculated as:

Glucose in serum (mg/100m l) =

Absorbance of test sample x conc. of standard sample Absorbance of standard sample

Histopathological Studies On last day of the experiment, rats were sacrificed. Pancreas, liver and kidneys were dissected out, examined macroscopically and fixed in 10% formalin. After processing the tissues for paraffin embedding,10 sections of the tissues (5μ thicknesses) were cut with a rotatory microtome and stained with haematoxylin-eosin and aldehyde-fuchsin stains.11 The sections were examined under binocular microscope for histopathological changes.

Experimental Protocol The experimental protocol was approved by the Institutional Animals Ethic Committee.

Measurement of Body Weights The body weights of the rats were taken on the day before injection of STZ and thereafter on 11th, 21st and 31st day of administration of alcoholic extract of medicinal plants or glibenclamide.

35

Induction of Diabetes Mellitus In overnight fasted rats, diabetes was induced by a single intra-peritoneal injection (30 mg/kg) of freshly prepared solution of STZ in citrate buffer of pH 4.5 at a rate of 1 ml/kg.12 Forty eight hrs after STZ administration, serum glucose concentration of each rat was estimated.

Rats having serum glucose concentrations between 250 -300 mg/100 ml, were considered diabetic and included in the study.

Experimental Design A total of 54 rats (six nondiabetics and forty eight STZ diabetics) were used. The animals were divided into following nine groups of six each: i) Nondiabetic (normal) rats without any treatment, ii) STZ diabetic rats administered vehicle (normal saline), iii & iv) STZ diabetic rat administered orally 250 mg and 500 mg/kg/ day respectively alcoholic extract of M. charantia fruit, v & vi) STZ diabetic rat administered orally 250 mg and 500 mg/kg/day respectively alcoholic extract of A. marmelos fruit, vii & viii) STZ diabetic rat administered orally 250 mg and 500 mg/kg/day respectively alcoholic extract of E. jambolana seeds and ix) STZ diabetic rat administered orally 300 μg/kg/day glibenclamide (in distilled water) for 30 days.13 Statistical Analysis The mean value and the standard error of the mean of each group were calculated. The level of significance of difference between the groups were analysed by student’s-‘t’ test.14 Results 1. Effect of Alcoholic Extracts of M. charantia, A. marmelos and E. jambolana on the Body Weight of STZ Diabetic Rats The average initial body weight of normal rats was 186.7 ± 3.57 g. There was a progressive increase in the body weights of normal rats.

After one month period, the average body weight of normal rats was 201.2±4.6g (7.8%) (Table 1).

On the other hand, STZ diabetic control rats showed a progressive decrease in their body

36

Sundaram, Singh and Reddy

Table 1. Effect of alcoholic extracts of M. charantia, A. marmelos and E. jambolana on body weight of streptozotocindiabetic rats Treatment

Body weightMean ± S.E.M (g) Before STZ injection

On days after initiation of the treatment On 11th Day

On 21st Day

On 31st Day

Percent change(+/-) @

Non diabetic (Control)

186.7 ± 3.57

190.8 ± 3.75NS

196.7 ± 4.94NS

201.2 ± 4.69a

7.8 (+)

STZ diabetic (Control)

184.2 ± 4.36

179.2 ± 4.36NS

167.5 ± 4.23a

165.0 ± 4.47a

10.4 (-)

M.charantia 250mg 500mg

187.5 ± 3.82

188.3 ± 3.80 NS 183.3 ± 2.47 NS

196.7 ± 5.72 NS 198.3 ± 4.01b

201.7 ± 3.33 a 205.8 ± 3.96 c

7.6 (+) 13.3 (+)

A.marmelos 250mg 500mg

185.8 ± 3.96

185.8 ± 3.96 NS 183.3 ± 1.67 NS

187.5 ± 4.95 NS 195.0 ± 4.65 a

195.8 ± 4.72 NS 195.8 ± 3.51b

5.4 (+) 7.3 (+)

E.jambolana 250mg 500mg

185.0 ± 4.83

185.8 ± 4.36 NS 185.0 ± 2.89 NS

189.2 ± 3.00 NS 190.8 ± 1.54 NS

190.8 ± 3.27 NS 193.3 ± 1.67a

3.1 (+) 5.5 (+)

Glibenclamide 300 ȝg

184.2 ± 3.75

185.8 ± 3.52 NS

190.0 ± 2.89 NS

196.7 ± 3.57 a

6.8 (+)

181.7 ± 3.33

182.5 ± 2.14

183.3 ± 3.33

Values NS - not significant (p> 0.05) and significant at p - value a < 0.05, b < 0.01 and c < 0.001 from the initial body weight taken before STZ injection. Values represent the Mean ± S.E.M. of six animals.@ Percent change (+ increase/- decrease) in the body weight relative to the initial body weight before STZ injection in the same group of rats Treatment was given 48 hrs after STZ injection and was considered as day 1 of the treatment.

weights. The body weights of STZ diabetic control rats decreased by 10.4% (165.0 ± 4.47 g) during one month period from their initial body weights of 184.7 ± 4.36 g taken before STZ injection (Table 1).

Alcoholic extracts of M. charantia, A. marmelos and E. jambolana showed a progressive dose and duration dependent increase in body weights of STZ diabetic rats which was apparent only after 20 days of the treatment. On 31st day, M. charantia, A. marmelos and E. jambolana in the daily doses of 250 mg/kg increased the body weights of STZ diabetic rats by 7.6% (201.7 ± 3.33 g), 5.4% (195.8 ± 4.72 g) and 3.1% (190.8 ± 3.27 g) respectively while in daily doses of 500 mg/kg, the respective increase in the body weights of STZ diabetic rats was of the order of 13.3% (205.8 ± 3.96 g), 7.3% (195.8 ± 3.51 g) and 5.5% (193.3 ± 1.67 g) when compared with that

of their respective initial body weights taken before STZ injections (Table 1).

The increase in the body weights of STZ diabetic rats at the end of one month period with three alcoholic extracts (500 mg/kg daily) and glibenclamide (300μg/kg daily) was statistically significant and was comparable to each other.

Of the three, M. charantia was comparatively most effective (Table 1).

2. Hypoglycaemic Activity of Alcoholic Extracts of M. charantia, A. marmelos and E. jambolana in STZ Diabetic Rats The average serum glucose concentrations of normal rats varied between 85.5 ± 3.67 mg/ 100 ml to 93.0 ± 2.77 mg/100 ml on different days of the experiments during one month period (Table 2).

Intraperitoneal injection of STZ (30 mg/kg) in 12 hrs fasted rats increased serum glucose

Hypoglycaemic Activity of Medicinal Plants

37

Table 2. Antidiabetic activity of alcoholic extracts of M. charantia, A. marmelos and E.jambolana in streptozotocin diabetic rats.

Serum glucose concentration Mean ± S.E.M ( mg/100ml) After 48 hrs. of STZ injection

On days after initiation of the treatment

Non diabetic (Control)

Treatment

th

On 11 day

On 21 day

On 31 day

85.5 ± 3.67

87.7

± 3.21

93.0 ± 2.77

91.5 ±

STZ diabetic (Control)

273.5 ± 8.10*

282.8 ± 9.51*

322.0 ± 18.06*

326.0 ± 10.58*

M.charantia 250mg 500mg

284.2 ± 7.51

252.0 ± 7.43 a

289.7 ± 9.31

236.7 ± 9.09 c

206.3 ± 10.53 c 173.8 ± 12.59 c

142.5 ± 8.48 c 100.0 ± 7.30 c

290.3 ± 7.59

265.7 ± 8.91 NS

287.7 ± 8.79

251.5 ± 9.80 a

215.7 ± 6.28 c 198.0 ± 9.53 c

161.3 ± 9.45 c 106.8 ± 9.45 c

283.8 ± 7.55

280.3 ±

9.66 NS

282.5 ± 7.13

230.0 ±

15.62a

235.8 ± 17.86 b 201.7 ± 9.45 c

180.7 ± 10.18 c 110.5 ± 8.42 c

279.7 ± 6.96

211.2 ±

22.44 a

117.3 ± 10.56 c

103.8 ± 7.03 c

A.marmelos 250mg 500mg

E.jambolana 250mg 500mg

Glibenclamide 300μg

st

st

Percent change (+/-)@

2.90 19.0 (+) 49.7 (-) 65.5 (-)

44.4 (-) 62.9 (-)

36.3 (-) 60.9 (-) 62.9 (-)

Values significant at p-value: - * < 0.001 from nondiabetic control, and a < 0.05, b < 0.01, c < 0.001and NS- Not significant (> 0.05) from STZ diabetic control of the same day Values represent the Mean ± S.E.M. of six animals. @ Percent change in the serum glucose conc. from its initial value observed after 48hrs of STZ injection in the same group.

Treatment was given 48 hrs after STZ injection and was considered as day 1 of the treatment.

concentrations to a great extent. In diabetic rats serum glucose concentrations raised to 273.5 ± 8.10 mg/100 ml (219.5%) to 290.3 ± 7.59 mg/ 100 ml (239.5%) from its initial normal values in different groups when measured after 48 hrs of STZ injection.

During one month of study, glucose concentrations in serum further increased by 19% (i.e. 326.0 ± 10.58 mg/100 ml) in diabetic control rats (Table 2).

Alcoholic extracts of M. charantia, A. marmelos and E. jambolana showed a progressive dose and duration dependent decrease in serum glucose concentrations in STZ diabetic rats. The decrease in serum glucose concentrations observed on last day of the experiments (i.e. on 31st day) was of the order of 49.7% (142.5 ± 8.48 mg/100 ml) with a daily oral doses of 250 mg/kg of M. charantia while in the daily doses of 500 mg/kg, it decreased

the serum glucose concentrations by 65.48% (100.0 ± 7.30 mg/100 ml) from the initial values observed after 48 hrs of STZ injection (Table 2).

Likewise, A. marmelos and E. jambolana also decreased serum glucose concentrations of STZ diabetic rats by 44.4% (161.3 ± 9.45 mg/100 ml) and 36.3% (180.7 ± 10.18 mg/100 ml) respectively with the daily doses of 250 mg/kg and 62.9% (106.8 ± 9.45 mg/100 ml) and 60.9% (110.5 ± 8.42 mg/100 ml) with the daily doses of 500 mg/kg respectively. The hypoglycaemic effect of the three alcoholic extracts was statistically significant and was comparable with that of glibenclamide (300 μg/kg daily) which also decreased serum glucose concentrations by 62.9% (103.8 ± 7.03 mg/100 ml) on 31 st day of experiment. Of the three, M. charantia was comparatively more effective followed by A. marmelos and E. jambolana (Table 2).

38

Sundaram, Singh and Reddy

3. Effect of Alcoholic Extracts of M. charantia, A. marmelos and E. jambolana on β - Cells of islets of Langerhans in Pancreas and on Liver and Kidney Tissues in STZ Diabetic Rats Pancreas Histology of the pancreas of nondiabetic rats showed dark stained granules of densely packed viable β- cells of the islets of Langerhans.

On the contrary, diffused necrotic changes were seen in the pancreas of STZ diabetic rats.

Occasional viable β- cells of islets of Langerhans could be seen in few rats only.

Histopathological studies of pancreas also revealed that daily oral administration of alcoholic extracts of M. charantia, A. marmelos and E. jambolana in doses of 250 and 500 mg/kg and glibenclamide in doses of 300 μg/kg for a period of 30 days significantly improved the histological architecture of the pancreas as evidenced by the presence of more viable β - cells of islets of Langerhans with less necrotic changes as compared to that of untreated STZ diabetic controls. The degree of improvement in the architecture of β - cells of islets of Langerhans of STZ damaged pancreas was more with 500 mg/kg dose than that of 250 mg/kg dose. The necrotic changes observed in pancreas of M. charantia group were less followed by A. marmelos, glibenclamide and E. jambolana.

However, the presence of viable β - cells of islets of Langerhans in pancreas of treated STZ diabetic rats was still less than that of normal rats (Figure 1).

Liver Histology of the liver of nondiabetic rat showed normal architecture of parenchymal cells.

Hepatic lobules with portal tracts and central veins with hepatic sinusoids radially arranged the central veins were visible. The hepatic veins were normal in size and portal tracts showed the collection of lymphocytes. On the other hand, histology of the liver of STZ diabetic rats showed hypertrophy and the distortion in the arrangement of the hepatic

cells around central veins, but such changes were very less in the liver of STZ rats treated with any one of the alcoholic extract of the plants and glibenclamide for 30 days (Figure 2).

Kidney The histology of the kidney of nondiabetic rat showed normal architecture of Bowman’s capsule. The basement membrane of glomerular capsule was thin. There was sufficient space between basement membrane and the tuft of the glomerulus (Figure 3). On the contrary, in STZ diabetic rats, there was a slight hypertrophy of the glomeruli and thickening of the basement membrane of glomerular capsule, thereby, decreasing the free space between the two. The walls of the renal tubules were also thickened due to hyalinization, thereby, decreasing the lumen of the renal tubules during 30 days period. Such changes in the architecture of the kidney of diabetic rats administered with three alcoholic extracts (M. charantia, A. marmelos and E. jambolana) in doses of 500 mg/kg or glibenclamide in doses of 300 μg/kg for 30 days were very less as compared to STZ diabetic control rats (Figure 3).

Discussion Diabetes mellitus or ‘Madhumeham’ known for centuries as a disease related to sweetness is a serious metabolic disorder with micro and macro-vascular complications that results in significant morbidity and mortality. The incidence of its occurrence in the population is increasing day by day at an alarming rate due to urbanization and modernization of civilization. The use of oral hypoglycaemic drugs and insulin which controls the blood glucose concentrations within normal range, do little in alleviating late clinical manifestations15 of the disease. Hence, it becomes necessary to look for new and if possible more efficacious drug; therefore, the research for such a hypoglycaemic drug has to be an area of active research.16,17 More than 400 medicinal plants are available globally for the medication of the diabetes. Their therapeutic effectiveness varied

Hypoglycaemic Activity of Medicinal Plants

39

Figure 1(a) Pancreas of healthy rat showing densely packed β -cells of islets of Langerhans (x 360), Figure 1(b) Pancreas of STZ diabetic rat showing necrosis of β - cells of islets of Langerhans (x 360), Figure 1 (c, d, e, f ) Diabetic pancreas showing presence of β - cells of islets of Langerhans of varying degree with less necrotic changes (x 360) after treatment with M. charantia, A. marmelos, E. jambolana alcoholic extracts (500 mg) and glibenclamide (300 μg) respectively

40

Sundaram, Singh and Reddy

Figure 2(a) Liver of healthy rat showing radially arranged normal parenchymal cells (x 360) around central vein.

Figure 2(b) Liver of STZ diabetic rat showing distortion of usual arrangement of parenchymal cells (x 360).

Figure 2(c, d, e & f) Showing protective effect on histology of liver by M. charantia, A. marmelos, E. jambolana alcoholic extracts (500 mg) and glibenclamide (300 Îźg) respectively (x 360).

Hypoglycaemic Activity of Medicinal Plants

41

Figure 3(a) Kidney of healthy rat showing normal structure of Bowman’s capsule and renal tubules (x 360).

Figure 3(b) Kidney of STZ diabetic rat showing expanded glomerulus of Bowman’s capsule and hyalinization of the walls of renal tubules (x 360).

Figure 3(c, d, e & f) Kidney of STZ diabetic rat showing protective effect on histology of kidney by M. charantia, A. marmelos, E. jambolana alcoholic extracts (500 mg) and glibenclamide (300 μg) respectively (x 360).

42

Sundaram, Singh and Reddy

from one study to other depending upon the methodologies and the experimental models used.18,19 We have chosen M. charantia, A. marmelos and E. jambolana plants because they are extensively grown and their fruits not only been eaten either as a raw fruit (Bael, Jamun) or as a vegetable (Karela) by the public but have also been used by the laymen for the treatment of the diabetes in the ancient time.

There are reports that the body weights of STZ diabetic rats decreased as they consumed less food.20 In present study also, body weights of STZ diabetic rats decreased by 10.4% at the end of one month from the initial body weights taken before STZ injection. On the other hand rats administered alcoholic extracts of all the three plants showed dose and duration dependent increase in the body weights which was apparent only after 20 days of the treatment with 500 mg/ kg dose. The restoration of the body weights in diabetic rats indicate that these rats properly utilized the glucose for their energy production which might be due to enhanced insulin secretion. In addition, M. charantia being a bitter might also acted as an appetizer hence more gain in the body weights when compared to that of normal rats.

The present results indicate that alcoholic extracts of M. charantia, A. marmelos and E. jambolana produced dose and duration dependent decrease (p<0.001) in serum glucose concentrations of STZ diabetic rats. At the end of one month study, the serum glucose concentrations observed in STZ diabetic rats with all three alcoholic extracts (in daily doses of 500 mg/kg) and that of glibenclamide (300 μg/kg) were ‘more or less’ comparable with that of serum glucose concentrations of normal rats.

The present findings are in conformity with the findings of earlier workers for their hypoglycaemic effects. 17,21,22 The order of effectiveness was M. charantia > A. marmelos = Glibenclamide > E. jambolana.

Present results showed that oral administration of alcoholic extracts of all the three plants and glibenclamide for one month had protective effect against the damage caused by

STZ as evidenced by the presence of more number of viable β - cells of islets of Langerhans in pancreas and practically normal architecture of liver and kidney tissues on histological examination. The present findings are in agreement with those of earlier workers for their protective effect on the pancreas by M. charantia,23 A. marmelos 2 4 and E. jambolana 2 5 respectively. The appearance of more number of dark stained granules may be due to activation of survival/ dormant β - cells which were not functional in normal pancreas. Some workers, however, also pointed out the regeneration of the β - cells of islets of Langerhans with medicinal plants.24,26 In brief, present result showed that the antidiabetic effect observed with all three medicinal plants is due to enhanced insulin secretion as evidenced by the presence of more viable β - cells of islets of Langerhans in STZ damaged pancreas of treated rats which was dose dependent.

Acknowledgements Author (ENS) is grateful to Prof. (Dr) C. Nayak, Director General, Central Council for Research in Homoeopathy, New Delhi for granting permission to take up this study.

1.

2.

3.

4.

5.

References Kingh, H., Aubert, R. and Herman, W.H: Global burden of diabetes 1995-2025. Diabetes Care. 21, pp. 1414-1431 (1998).

Harris, M.I., Flegal, K.M., Cowie, C.C., Eberhardt, M.S., Goldstein, D.E., Little, R.R., Wiedmeyer, H.M. and Bryd-Holt, D.D: Prevalence of diabetes impaired fasting glucose, and glucose tolerance in U.S. adults. Diabetes Care. 21, pp. 518-524 (1998).

Firshein, R: Eliminating diabetes in the 21st Century. Bio Med. 8, pp. 67-69 (2001).

Frier, B.M. and Fisher, B.M: Davidson’s Principles and Practice of Medicine. In: Haslett, C., Chilvers, E.R., Boon, N.A., Colledge, N. R. and Hunter, J.A.A. Churchill Livingstone, London. pp. 641-682 (2002).

Masharani, U: Current Medical Diagnosis and Treatment. In: Tierney, L.M.J.R., Mc Phee, S.J. and Papadakis, M.A. Lange Medical Books /

Hypoglycaemic Activity of Medicinal Plants

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

McGraw â&#x20AC;&#x201C; Hill, New York, New Delhi. pp. 1157-1201 (2005).

Havsteen, B: Flavonoids - a class of natural products of high pharmacology potency. Biochem Pharmacol. 32, pp. 1141-1148 (1983).

Middleton, E.J.R., Kandswami, C. and Theoharides, T.C: The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev. 52, pp. 671-751 (2000).

Tiwari, A.K. and Madhusudana Rao, J: Diabetic mellitus and multiple therapeutic approaches of phytochemicals: present status and future prospects. Curr Sci. 83, pp. 30-38 (2002).

Trinder, P: Determination of glucose using glucose oxidase with an alternative oxygen acceptor. Annals of Clinical Biochemistry. 6, pp. 24-27 (1969).

Gordon, K. and Bradbury, P: Theory and Practice of Histological Techniques. In: Gordon, K. and Bradbury, P. Churchill Livington, New York. pp. 61-80 (1990).

Gomori, G: Aldehyde-Fuchsin: a new stain for elastic tissue. Am J Clin Path. 20, pp. 665-666 (1950).

Siddique, O., Sun, Y., Lin, J.C. and Chain, Y.W: Facilitated transdermal transport of insulin.

J Pharm Sci. 76, pp. 341-345 (1987).

Kamalakkannan, N. and Stanely Mainzen Prince, P: Anti-diabetic and antioxidant activity of Aegle marmelos extract in streptozotocin diabetic rats. Indian J Exp Biol. 41, pp. 1285-1288 (2003).

Gupta, B.N: Statistics - Theory and Practice. In: Gupta, B.N. Sahitya Bhawan, Agra. pp. 186-584 (1989).

Vats, V., Yadav, S.P. and Grover, J.K: Ethanolic extract of Ocimum sanctum leaves partially attenuates streptozotocin induced alterations in glycogen content and carbohydrate metabolism in rats. J Ethnopharmacol. 90, pp. 155 -160 (2004).

Pari, L. and Uma Maheswari, J: Hypoglycemic effect of Musa sapientum L. in alloxan induced diabetic rats. J Ethnopharmacol. 68, pp. 321-325 (1999).

Stanely Mainzen Prince, P., Kamalakkannan, N. and Menon, V.P: Antidiabetic and antihyperlipidaemic effect of alcoholic Syzium

18.

19.

20.

21.

22.

23.

24.

25.

26.

43

cumini seeds in alloxan induced diabetic albino rats.

J Ethnopharmacol. 91, pp. 209-213 (2004).

Chakravarthy, B.K., Gupta, S., Gambheer, S.S. and Gode, K.D: Pancreatic beta cell regeneration -a novel antidiabetic mechanism of Pterocarpus marsupium (Roxb.). Indian J Pharmaco. 12, pp. 123-127 (1980).

Bopanna, K.N., Kannan, J., Sushma, G., Balaraman, R. and Rathod, S.P: Antidiabetic and antihyperlipaemic effects of neem seed kernel powder on alloxan diabetic rabbits. Indian J Pharmacol. 29, pp. 62-167 (1997).

Sivajothi, V., Dey, A., Jayakar, B. and Rajkapoor, B: Antihyperglycaemic property of Tragia cannabina in streptozotocin induced diabetic rats. J Medicinal Food. 10, pp. 361-365 (2007).

Upadhya, S., Shanbhag, K., Suneetha, G., Naidu, M.B. and Upadhya, S.A: Study of hypoglycaemic and antioxidant activity of Aegle marmelos in alloxan induced diabetic rats. Indian J Physiol Pharmacol. 48, pp. 476-480 (2004).

Viridi, J., Sivakami, S., Shahani, S., Suthar, A.C., Banavalikar, M.M. and Biyani, M.K: Antihyperglycemic effect of three extracts from Momordica charantia. J Ethnopharmacol. 88, pp. 107-111 (2003).

Ahmed, I., Adeghate, E., Sharma, A.K., Pallot, D.J. and Singh, J: Effects of Momordica charantia fruit juice on islet morphology in the pancreas of the streptozotocin-diabetic rats.

Diabetes Res Clin Pract. 40, pp. 145-151 (1998).

Kamalakkannan, N. and Prince, P.S: The effect of Aegle marmelos fruit extract in streptozotocin diabetes: a histopathological study.

J Herb Pharmacother. 5, pp. 87-96 (2005).

Grover, J.K., Vats, V., Rathi, S.S. and Dawar, R: Traditional Indian antidiabetic plants attenuate progression of renal damage in streptozotocin induced diabetic mice. J Ethnopharmacol. 76, pp. 233-238 (2001).

Chakravarthy, B. K., Gupta, S. and Gode, K. D: Functional beta cell regeneration in the islets of pancreas in alloxan induced diabetic rats by epicatechin. Life Sci. 31, pp. 2693-2697 (1982).

Address for correspondence: Dr. E. N. Sundaram, Research Officer (Endocrinology), Drug Standardisation Unit, Osmania Unversity Building No. 32, Street No. 4, Vikrampuri, Habsiguda, Hyderabad â&#x20AC;&#x201C; 500007 A.P. (India).

E-mail: sundaram_ccrh@yahoo.in 030_2009

J. Res. Educ. Indian Med., Jan.-March, 2012 Vol. XVIII (1) : 45-50

ISSN 0970-7700

PHARMACOLOGICAL SCREENING OF CASSINE ALBENS (RETZ.) KOSTERM (CELASTRACEAE) FOR ANTIDEPRESSANT AND ANXIOLYTIC ACTIVITY IN RODENTS P. H. PATIL,* M. B. GAGARANI, K. R. PATIL AND S. J. SURANA Department of Pharmacology, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur – 425405 Dhule, Maharashtra (India) Abstract: Cassine albens (R.) Kosterm belongs to the family celastraceae which is the rich source of triterpenes and alkaloids. In present study hydroalcoholic extract of stem bark of Cassine albens was investigated for antidepressant and anxiolytic activity in rodents. The dose dependant study was performed at the doses of 100, 200, 400 and 800 mg/kg, p.o. Cassine albens showed significant antidepressant activity in forced swim and tail suspension test. We observed significant anxiolytic effect at the dose of 400 and 800 mg/kg, p.o. in elevated plus maze and marble burying test in rodents. The present study substantiates folkloric use of Cassine albens in the treatment of psychosomatic disorders.

Keywords: Cassine albens, Neurotransmitter, Antianxiety, Antidepressant.

Introduction Cassine albens (R.) Kosterm (Celastraceae) (synonym: Elaeodendron glaucum or Cassine glauca) is found throughout India and Sri Lanka (Patil D.A., 2003). This plant is popularly known as ‘Bhutakes’ at Toranmal, a hill station located in North Maharashtra, which is used in the treatment of CNS related disorders (Patil and Bhaskar, 2006). In folkloric system of medicine, it is used for the treatment of hysteria, syncope and normal headache (Kirtikar and Basu, 2005).

Fresh root bark of this plant is used for swelling and cold water extract is used as emetic which may be fatal in overdose. In Sonbhadra district of Uttar Pradesh (India) this plant is used in cholera, menstrual disorders and to promote sterility in women (Singh et al., 2002). A paste prepared from root of this plant is used with cow’s milk in snake bite (Parinitha et al., 2005).

Celastraceae family is rich source of sesquiterpenoids (Aleksandra et al., 2007).

Lupeol, octacosanol, friedelin, betulonic acid, 23hydroxybetulin, β- sitosterol and its glucosides are isolated from its leaves and stem bark. The cardiac glycosides isolated from this plant are named as elaeodendroside A ,B, C, D, E, H, I, J * Head, Department of Pharmacology

(Rastogi and Malhotra 2001; Anjaneyulu and Narayanrao, 1980; Shimada et al., 1985).

Infusion of Cassine albens was used in headache and in psychosomatic disorders by local tribes residing in Nandurbar district of North Maharashtra (Patil and Bhaskar, 2006). Extensive review of literature revealed that the plant was not investigated systemically for its central nervous system activity.

The Cassine albens is used in psychosomatic illness by certain tribes residing in the Satpuda hill region of Maharashtra. Therefore Cassine albens seem to affect neuronal pathway involved in anxiety and depression. In the present study, we have evaluated the effect of hydroalcoholic extract of Cassine albens for its antidepressant and anxiolytic activity in various animal models.

Material and Methods Plant material and extract preparations Stem bark of Cassine albens was collected from the Satpuda hill region (Village: Legapani; Taluka: Dhadgaon; Dist: Nandurbar) of Maharashtra. The plant material was authenticated

by Dr. D. A. Patil, Department of Botany, SSVPS Science College, Dhule (Maharashtra). The stem bark of Cassine albens was shade dried for

46

Patil, Gagarani and Surana

15 days. The dried material was pulverized to obtain coarse to fine powder. The powder was subjected to extraction with ethanol and water (1:1) by cold maceration for three days. The material was filtered and the filtrate was concentrated. The concentrated material was air dried to obtain hydroalcoholic extract of Cassine albens. This extract was used for pharmacological studies in the form of suspension made in 1% carboxy methyl cellulose (CMC).

Drugs Fluoxetine and Diazepam were obtained as a gift sample from Cadila Pharmaceuticals Ltd., Ahmedabad and SVIZERA Healthcare Ltd., Mumbai (India) respectively.

Animals and Treatment All the experimental procedures were approved by Institutional Animal Ethical Committee. Swiss albino mice (20-25 g) and Albino wistar rats (220-230 g) of either sex were purchased from central animal house of RCPIPER, Shirpur, India. All animal were maintained under control condition of temperature (22 ± 2º C) and 12 hour light–dark cycle with free access of food (rodent lab standard diet) and water. All experiments were conducted in between 10.00 AM to 5.00 PM in a noise free room. Animals were divided into various groups, containing six animals in each group, the animals received different treatments like vehicle (1% CMC), Fluoxetine (20 mg/kg p.o.) or Diazepam (1 mg/ kg, i.p.) and hydroalcoholic extract of Cassine albens at the doses of 100, 200, 400 and 800 mg/ kg, p.o. of body weight daily for 7 days.

Neurobehavioral profile screening Neuropharmacological screening of hydroalcoholic extract of Cassine albens (HCA) was studied using mice. Different neuropharmacological test batteries were used during study as reported by Moscardo et al.

(2007). The scoring of test was done as per Irwin scale (Turner, 1996).

Spontaneous locomotor activity One hour after the oral administration each mouse was placed individually in digital activity cage (Besto Instrument) for about 10 minutes. At

the end of observation period mouse was removed and reading was recorded.

Forced swim test (FST) FST is the most widely used model for antidepressant studies (Herrera et al., 2006).

All animals were dosed for 7 days, on the 6th day all the animals were individually placed in a tank made up of PVC (20 × 40 cm and 25 cm deep) containing clean water at 25ºC for 15 minutes (pretest session). 24 hour later, on the 7th day, one hour after the oral administration of drug, each animal was subjected to 5 minutes swim test. During the test session the total immobility period was recorded. A rat was considered to be immobile when it remained floating in the water without struggling. After the end of observation period animal was taken out of cylinder then dried and returned to their respective home cages.

Tail suspension test In this test mouse was suspended on the edge of wire 50 cm above the floor by adhesive tape placed approximately 1 cm from the tip of tail. On the 7th day of drug treatment the total duration of immobility induced by tail suspension was measured during 6 minute test session.

Animal was considered to be immobile when it did not show any movement of body and hanged passively.

Elevated plus maze The EPM evokes the conflict between the need to explore the novel area and need to avoid more vulnerable (aversive) areas of elevated plus maze (Wijeweera et al., 2006). Apparatus consist of two open arms (50× 10 × 40 cm) with an open roof, arranged such that two open arms were opposite to each other; the apparatus was elevated to 50 cm from the floor. The apparatus was carefully cleaned after each test session to remove any stress of odor. On 7th day one hour after the oral administration of different doses of HCA, and 30 minutes after Diazepam (1 mg/kg, i.p.) administration, each rat was placed in Central Square of the maze facing towards the open arm. During 5 minute test period, the number of open and closed arm entries were recorded.

Pharmacological Screening of Cassine albens

Marble burying test in mice The 5 cm layer of sawdust was placed in polyethylene cage and 20 glass marbles were evenly distributed on the sawdust in the cages.

One hour after oral administration of drugs, animal was placed individually in cage. During the 15 minutes observation period, the total number of marbles buried was counted. Only those marbles to be taken into account which were buried twothird of its size (Matsushita et al., 2005; Laurent et al., 2006).

Statistical analysis The statistical analysis was done by using PRISM Graph pad (4.0) program. All values were expressed as Mean ± SEM. Data were analyzed by one way ANOVA followed by Dunnet’s ‘t’ test for group comparison.

Results A) Neurobehavioral profile Neurobehavioral profile of HCA tested at the dose of 1000 mg/kg and 2000 mg/kg did not show any significant alterations in animal behavior.

47

B) Spontaneous locomotor activity The HCA at all the four doses studied was unable to produce significant change in motor activity. This suggests that antidepressant and anxiolytic activity is devoid of motor activity (Kulkarni and Sharma, 1991).

C) Effect of HCA on immobility time in Forced Swim Test (FST) HCA at 200 mg/kg, 400 mg/kg and 800 mg/kg doses significantly (p< 0.01) decreased immobility period as compared to the control group. Fluoxetine (20 mg/kg) treated group showed more reduction in immobility time as compared with control group (Table 1).

D) Effect of HCA on immobility time in Tail Suspension Test (TST) HCA (400 mg/kg) significantly (p<0.01) decreased the immobility time as compared to control group. However HCA (100 mg/kg, 200 mg/kg and 800 mg/kg) did not show significant decline in immobility time. Fluoxetine treated mice showed greater decline in immobility time and

Table 1. Effect of hydroalcoholic extract of Cassine albens on immobility period in Forced Swim Test in rats.

Group

Treatment

Dose

Immobility Period in FST(Sec)

I

Control

10 ml/kg

240 ± 1.3

II

Fluoxetine

20 mg/kg

170 ± 2.4**

III

HCA

100 mg/kg

230 ± 3.3*

IV

HCA

200 mg/kg

220 ± 5.7**

V

HCA

400 mg/kg

180 ± 1.7**

VI

HCA

800 mg/kg

200 ± 3.3**

Data presented as mean ± SEM. Statistical analysis: One way ANOVA followed by Dunnet’s ‘t’ test. *p < 0.05; **p< 0.01 as compared with control group.

Table 2. Effect of hydroalcoholic extract of Cassine albens on immobility period in Tail Suspension Test in mice.

Group

Treatment

Dose

Immobility period in TST (Sec)

I

Control

10 ml/kg

136 ± 6.0

II

Fluoxetine

20 mg/kg

72 ± 1.6**

III

HCA

100 mg/kg

139 ± 4.2

IV

HCA

200 mg/kg

130 ± 5.4

V

HCA

400 mg/kg

113 ± 2.5**

VI

HCA

800 mg/kg

122 ± 3.4

Data presented as mean ± SEM. Statistical analysis: One way ANOVA followed by Dunnet’s ‘t’ test. **p< 0.01 as compared with control group

Patil, Gagarani and Surana

48

showed significant (p<0.01) reduction in immobility time with compared to control group (Table 2).

E) Effect of HCA in Elevated Plus Maze (EPM) Test In the present study HCA at the doses of 400 mg/kg, 800 mg/kg and Diazepam (1 mg/kg) showed significant increase in open arm entries as compared to control group. HCA at 100 mg/ kg and 200 mg/kg dose was failed to show anxiolytic activity. The increase in open arm entry as preference was observed at the oral dose of 800 mg/kg (Table 3).

F) Effect in Marble Burying Test In this test, HCA (400 mg/kg and 800 mg/ kg) significantly (p<0.05) reduced the number of marbles buried as compared to control group.

The Fluoxetine treated animals showed significantly (p<0.01) reduction in marble burying behavior as compared to control animals (Table 4).

Discussion Celastraceae family is a rich source of sesquiterpenoids of remarkably diverse structures ranging from small alkaloids and oligopeptide to very complex macrocyclic arrays (Alexandra et al., 2007). In the present study different doses of hydroalcoholic extract of Cassine albens showed antidepressant and anxiolytic activity in stress-evoked models. The major cause of mental disorders is the imbalance between neurotransmitter within the brain. Most of antidepressant and anxiolytic compounds targets neurochemical processes that occur during these disorders. Antidepressant compound acts through preventing the reuptake of noradrenalin and/or 5- hydroxytryptamine at synaptic nerve ending and increases the availability of neurotransmitter in brain. MAOI inhibits enzyme monoamine oxidase and increase the availability of noradrenalin in brain (Goodman and Gilman, 2001; Kasper et al., 2001). Currently, the treatment of anxiety and depression is restricted

Table 3. Effect of hydroalcoholic extract of Cassine albens on number of entries in an open arm in Elevated Plus Maze test in rats.

Group

Treatment

Dose

Number of entries in open arm

I

Control

10 ml/kg

0.71 ± 0.29

II

Diazepam

01 mg/kg

3.0 ± 0.38**

III

HCA

100 mg/kg

0.57 ± 0.20

IV

HCA

200 mg/kg

0.43 ± 0.20

V

HCA

400 mg/kg

2.1 ± 0.26**

VI

HCA

800 mg/kg

1.9 ± 0.26*

Data presented as mean ± SEM. Statistical analysis: One way ANOVA followed by Dunnet’s ‘t’ test. *p < 0.05, **p< 0.01 as compared with control group

Table 4. Effect of hydro alcoholic extract of Cassine albens on number of Marbles Buried by mice.

Group

Treatment

Dose

Number of marbles buried

I

Control

10 ml/kg

6.3 ± 0.6

II

Fluoxetine

20 mg/kg

1.4 ± 0.3**

III

HCA

100 mg/kg

IV

HCA

200 mg/kg

4.6 ± 0.3

V

HCA

400 mg/kg

4.0 ± 0.4*

VI

HCA

800 mg/kg

4.1 ± 0.51*

6.0 ± 0.8

Data presented as mean ± SEM. Statistical analysis: One way ANOVA followed by Dunnet’s ‘t’ test. *p < 0.05, **p< 0.01 as compared with control group

Pharmacological Screening of Cassine albens

to some typical classes such as Benzodiazepines, SSRI, TCA, MAOI, etc. Some atypical drugs like Buspirone came into existence but they are not therapeutically as effective as conventional drugs. In the treatment of anxiety Benzodiazepines are now slowly replaced by antidepressants which are not only efficacious in depression but also in acute and long term treatment of anxiety. When Cassine albens was studied at the doses of 100, 200, 400 and 800 mg/kg for effect on locomotor activity, we donâ&#x20AC;&#x2122;t observe any significant change in locomotor activity of animal. This suggests that the antidepressant activity of Cassine albens may be devoid of locomotor activity (Dhingra and Kumar, 2007). These results confirmed the assumption that antidepressant like effect of extract is specific. In forced swim test we recorded the immobility period of animal when forced to swim. Among the doses studied Cassine albens at the doses of 200 mg and 800 mg/kg showed little activity. Whereas we observed significant activity at the oral dose of 400 mg/kg as compared with control group. Fluoxetine, a serotonin reuptake inhibitor was used as a reference drug in this test. The effect of hydroalcoholic extract of Cassine albens on immobility time in FST was comparable with Fluoxetine. Hence, it can be proposed that antidepressant effect shown by Cassine albens may involve serotoninergic pathway. Tail suspension test induces a state of immobility known as behavior despair in animals and claimed to reproduce a condition similar to human depression (Dhingra and Sharma, 2006). This test is based on the observation that animals following initial escape oriented movements develop an immobile posture. The tail suspension test is less stressful and has greater sensitivity (Bhattamisara et al., 2008). In the present study Cassine albens at a dose of 400 mg/kg showed significant activity as compared to control group. The elevated plus maze is considered to be etiologically valid animal model, because it uses natural stimuli that is the fear of novel open space and fear of balancing on a relatively narrow, raised platform that can induce

49

anxiety in humans (Grundmann et al., 2007).

The validity of the EPM test for evaluation of anxiolytic or anxiogenic effects of drugs has been well documented (Kulkarni and Sharma, 1991). Diazepam (1 mg/kg) was used as reference standard in EPM test which significantly increased the number of entries in open arm. It was also demonstrated that the behavioral anxiety in animal on elevated plus maze was indirect correlation with decrease the number of GABA and Benzodiazepines receptor in cerebral cortex in radioligand studies (Kulkarni and Sharma, 1991).

In present study Cassine albens at 400 mg/kg and 800 mg/kg showed increase in number of entries in open arm. Hence it may postulate that the anxiolytic activity of hydroalcoholic extract may involve GABAergic pathway. However detailed investigations are needed to confirm this mechanism. In marble burying test Cassine albens at the doses of 400 mg/kg and 800 mg/kg showed decrease in number of marbles buried by animal.

The marble burying test is a valid model for obsessive compulsive disorders which is considered as one of the type of anxiety. Anxiolytic agents such as diazepam have been shown to reduce burying, behavior indeed antidepressants; particularly SSRIs are effective in the treatment of obsessive compulsive disorders.

Conclusion In present study, hydroalcoholic extract of Cassine albens shown significant activity in stress evoked animal models of depression and anxiety in rodents. The results of present study substantiate the folk use of Cassine albens in the treatment of central nervous system related ailments. Further detailed investigations are needed to elucidate the exact mechanism of Cassine albens behind its antidepressant and antianxiety effects.

1.

References Aleksandra, S., Cuperly, D., White, A. J. and Anthony, G. M: Total synthesis of the tricyclic skeleton of the natural Celastraceae sesquiterpenoids and related synthetic analogs.

Tetrahedron. 63(26): 590-5917 (2007).

50 2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

Patil, Gagarani and Surana Anjaneyulu, A. and Narayanrao, M: Elaeodendrol and elaeodendradiol new nortriterpene from Elaeodendron glaucum, Phytochemistry. 19(6): 1163-1169 (1980).

Bhattamisra, S. B., Khanna, V. K., Agrawal, A.K., Singh, P.N. and Singh, S. K: Antidepressant activity of standardized extract of Marsilea minnuta Linn. Journal of Ethnopharmacology. 117: 51-57 (2008).

Dhingra, D. and Kumar, V: Pharmacological evaluation for antidepressant –like activity of Asparagus racemosus wild in mice.

Pharmacologyonline. 3: 133-152 (2007).

Dhingra, D. and Sharma, A: Antidepressant – like activity of Glycyrriza glabra L. in mouse model of immobility test. Progress in Neuropsychopharmacology and Biological Psychiatry.

30: 449-454 (2006).

Goodman, S. L. and Gilman, A: The Pharmacological Basis of Therapeutics. 10th edition. Mcgrew Hill Publisher, Medical Division.

304-312 (2001).

Grundmann, O., Nakajima, J., Seo, S. and Butterweck, V: Anti-anxiety effect of Apocynum venetum L. in the elevated plus maze. J of Ethnopharmacology. 110: 406-411 (2007).

Herrera-ruiz, M., Garcia-beltran, Y., Mora, S., Diaz-veliz, G., Viana, S. B., Tortoriello, J. and Ramirez, G: Antidepressant and anxiolytic effect of hydroalcoholic extract from Salvia elegans. J of Ethnopharmacology. 107: 53-58 (2006).

Kasper, Braundwald, Fauci, Houser and Longo: Harrison’s Principle of Internal Medicine. 16th edition. Mcgrew Hill Medical Publishing Division.

New York (2001).

Kirtikar, K. R. and Basu, B. D: Indian Medicinal Plant. International Book Publisher, Deharadun.

Vol. I: 580-581 (2005).

Kulkarni, S. K. and Sharma, A. C: Elevated Plus- Maze: A novel paychobehavioural tool to measure anxiety in rodents. Meth Find Exp Clin Pharmacol. 13(8): 573-577 (1991).

Laurent, B. N., Yeter K. and Eric, P. M: A combined marble burying – locomotor activity in mice : A practical screening test with sensitivity to

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

different classes of anxiolytics and antidepressant.

European Journal of Pharmacology. 547: 106-115 (2006).

Matsushita, M., Egashira, N., Harada, S. and Okano, R: Perospiron: a novel antipsychotic drug inhibit marble- burying behavior via 5 HT1A receptor in mice : implication for obsessive compulsive disorders. J Pharmacol Sci. 99: 154-159 (2005).

Moscardo, E., Maurin, A., Dorigatti, R., Champeroux, P. and Richard, S: An optimized methodology for the neurobehavioral assessment in rodent. Journal of Pharmacological and Toxicological Methods. 56(2): 239-255 (2007).

Parinitha, M., Srinivasa, B.H. and Shivanna, M. B: Medicinal plants wealth of local communities in some villages in Shimoga District of Karnataka, India. J Ethnopharmacol. 98: 307-312 (2005).

Patil, D. A: Flora and Fauna of Dhule and Nandurbar districts (Maharashtra). Bishen Singh Mahendrapal Singh Publisher, Deharadun. 139-140 (2003).

Patil, H. M. and Bhaskar, V. V: Medicinal uses of plant by tribal men of Nandurbar district in Maharashtra. Natural Product Radiance. 5(2): 125-130 (2006).

Rastogi, R. M. and Malhotra, B. N: Compendium of Indian Medicinal Plants, 1980-1984. Central Drug Research Institute, Lucknow. 3: 146-147 (2001).

Shimada, K., Kyuno, T., Uchida, I. and Nmbara, T: Structure of elaeodendrosides B, C, F, G, K and L: A series of cardiac glycoside isolated from Elaeodendron glaucum. Phytochemistry. 24(6): 1345-1350 (1985).

Singh, A. K., Raghubanshi, A. S. and Singh, J. S: Medical ethnobotany of the tribals of Sonaghati of Sonbhadra district, Uttar Pradesh, India. Journal of Ethnopharmacology. 81: 31-41 (2002).

Turner, R. A: Organization of Screening, Methods in Pharmacology. Academic Press, Elsevier. Vol. 1: 22-41 (1996).

Wijeweera, P., Arnason, J. T., Koszycki, D. and Merali, Z: Evaluation of anxiolytic properties of Gotukola- (Centella asiatica) extract and asiaticoside in rat behavioral models. Phytomedicine. 13: 668-676 (2006).

Address for correspondence: Prof. P. H. Patil, Head, Department of Pharmacology, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur - 425405 Dhule, M.S. (India). E-mail: phpatil@rediffmail.com 042_2008

J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 51-54

ISSN 0970-7700

SCREENING OF XIMENIA AMERICANA L. FOR IT’S ANTI-IFLAMMATORY ACTIVITY M. SIDDAIAH,1 K.N. JAYA VEERA,2 P. MALLIKARJUNA RAO,3 K. YOGANANDA REDDY4 AND C. MADHUSUDHANA CHETTY1* Annamacharya College of Pharmacy,1 Rajampet, Kadapa, Andhra Pradesh (India) Department of Chemistry,2 Jawaharlal Nehru Technological University, College of Engineering, Ananthapur, Andhra Pradesh (India) International Medical University,3 Sesama Center, Plaza Komenwel, Kulalmpur – 57000 (Malysia) International Science Tech. Research Institute,4 Anathapur, Andhra Pradesh (India) Abstract: The anti-inflammatory activity of petroleum ether ethyl acetate and methanolic extracts of Ximenia americana L. was investigated in chemical models - using carrageenan induced paw oedema. Ximenia americana is the most common plant widely grown in tropic and sub tropical climatic conditions and is used in rheumatism and dropsy.1 The preliminary phytochemical screening of the plant shows the presence of sterols, terpenoids, alkaloids, tannin, saponin and phenolic compounds 3 which are responsible for various pharmacological activities. The leaf extract of the Ximinia americana were used to evaluate anti-inflammatory activity. The result proved that petroleum ether and methanolic extract of the plant has anti-inflammatory activity.

Keywords: Ximenia americana, Ximenia spinosa salisb, Anti-inflammatory, Plethysmograph, Carrageenan, Formalin, Paw oedema.

Introduction For many centuries medical treatment has relied to a large extent on the use of plants.

Natural products in current use possess nearly every conceivable type of biological activity. The usage of herbal drugs in various ailments is increasing in the modern civilization. W.H.O. estimates that four billion people from all over the world are using herbal medicines. The discovery of a vegetable, the active principle and it’s subsequent chemical characterization will help in synthesis of analogues relating the essential structural features for improved therapeutic activity.

For the present study Ximenia americana was used. It is also called Ximenia spinosa salisb.

The various plant parts like leaf is used as analgesic, anti-inflammatory in dropsy etc.1 in traditional systems of medicine. The plant is known to posses antitumer, antimalarial, antileprotic activity.1,2 But so far no report is available in the literature regarding the antiinflammatory activity. Hence the following study 1. Research Scholar

2. Director

3. Senior Lecturer

was carried out to evaluate scientifically the antiinflammatory activity by using carrageenan induced paw oedema method in albino rats and compared with the standard drug Diclofenac sodium.

Materials and Methods Plant Material Fresh leaves of Ximenia americana was collected form the surrounding villages of Chintala Dhornala Mandal Prakasham District A.P. (India) in the month of October 2006 and was taxonomically identified by an eminent botanist, S.V. University and a Specimen Voucher No. 1295 is kept for future reference at S.V. University. The plant leaves were washed thoroughly and shade dried and powdered in mechanical grinder and stored in air tight containers for further studies.

Preparation of Extracts The shade dried powder of Ximenia americana was subjected to successive extraction 4. Research Associate

1* Principal

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Siddaiah et al.

using solvents of increasing polarity like petroleum ether, ethyl acetate and methanol using soxhlet apparatus. The solvent was removed after the completion of extraction under reduced pressure.

Animals used Wister albino rats (150-200g) were used for the present study. The animals were kept in healthy constant temperature (22±2°C), humidity (55%) and light-dark conditions (12/12 light/dark) and provided with standard pellet diet (Hindustan Lever) and water ad libitum. The experiments were performed under the guidance of animal’s Ethical committee of Annamacharya College of Pharmacy, Rajampet, Kadapa, Andhra Pradesh, India (Registration No: 122/ a/ CPCSE/ACP).

Chemicals and Drugs Shade dried leaf powdered Ximenia americana petroleum ether AR, ethyl acetate methanolic AR and methanol (S.D. fine chemicals), soxlet apparatus, carrageenan, Diclofenac sodium and plethysmograph were used as received.

Phytochemical Screening Preliminary phytochemical screening of petroleum ether, ethyl acetate and methanol extracts were tested for the presence of carbohydrates steroids flavonoids, tannins, alkaloids glycosides, phenolic compounds, terpinoids, saponins resins, proteins and amino acids by using standard procedure as described

by Kandalwal (2003).4 Statistical Analysis The Experimental results were expressed as the mean +SEM. N= 6 animals in each group.

Statistical significant test for comparision was done by using unpaired ‘t’ test.

Acute toxicity study Wister albino rats weighing 200-250gm were choosen for the present study. They were divided into 6 groups of six animals each and then they were fasted for 24 hours prior to

treatment with all the extracts of Ximenia americana leaf. There after the test extract was administered orally as suspension in tween 80 (3ml of 1% solution) to different groups in increasing dose level of 10,100, 1000,4000, 6000 mg/kg body weight.

The animals were then observed continuously for three hours for general behavioral nemological, anatomical profiles, atoxia, diarrhea or diuresis and for every 30 minutes for next three hours and finally death after 24 hours.

Anti-inflammatory activity Carrageenan induced paw oedema Anti-inflammatory activity of petroleum ether ethyl acetate and methanolic extracts was determined by carrageenan induced paw oedema using plethysmograph.5 Albino rats of either sex weighing 150-200gm, were divided into eleven groups and each group consists six animals.

Group I served as control and received 3ml of tween 80 in normal saline. Group XI served as standard and received 10mg/kg of body wt. of Diclofenac sodium orally. Group III, IV, V VI, VII, VIII, IX and X, served as test and received 200, 400 and 600 mg/kg of body wt. of pet ether ethyl acetate and methanolic extracts respectively. Tween 80 in normal saline, Diclofenac sodium, extracts were administered one hour before the carrageenan administration.6 Carrageenan 1% w/v, in the normal saline was injected into sub-plantar region of the left hind paw of all groups of animals and right hind paw served as control. The volume of hind oedema was measured by plethysmograph at after mean increase in paw volume and % inhibition of inflammation were calculated. The percent inhibition of inflammation produced by the extracts of Ximenia americana was compared with the standard. Percentage inhibition was calculated by using the following formula.

Percentage of inhibiton = 100 (1-Vt/Vo) Where Vt=volume of the extract treated animals: Vo= volume of the control (saline).

Anti-inflammatory Activity of Ximenia americana

Results Phytochemical screening Preliminary phytochemical screening of petroleum ether and methanol extracts indicates the presence of alkaloids, carbohydrates, flavonoids, glycosides, terpinoids, steroids, tannins. Amino acids and resins were absent.

The results were presented in the Table 1.

Acute toxicity study In acute toxicity study no mortality was observed during the 24 hours period at the doses Table 1. Results of qualitative tests for phytoconstituents of Ximenia americana leaf.

Sl. No

1

Tests

Methanol

-ve

-ve

+ve

-ve -ve -ve

-ve -ve -ve

ve -ve +ve

+ve +ve +ve +ve

+ve +ve +ve +ve

++ve ++ve ++ve ++ve

Carbohydrates a) Anthrone test b) Benedict’s test c) Fehling’s test d) Molisch’s test

3

Ethyl Acetate

Alkaloids a) Dragendorff’s test b) Hager’s test c) Wagner’s test d) Mayer’s test

2

Pet. Ether

Flavanoids a) Shinoda’s test

+ve

++ve

-ve

+ve

++ve

a) Liebermann – Burchard test

+ve

+ve

++ve

6

Resins

-ve

-ve

7

Saponins

-ve

+ve

8

Steroids

4

Glycosides a) Molisch test

5

Triterpenoids

a) Liebermann Burchard’s test b) Salkowski reaction

+ve

9

Tannins

+ve

10

Proteins

11

tested and the animals showed no stereotypical symptoms associated with toxicity, such as convulsions, ataxia, diarrhea or increased diuresis and the results were shown in Table 2.

Carrageenan induced paw oedema In carrageenan induced paw oedema method,5 the oral administration of petroleum ether ethyl acetate and methanolic extract of Ximenia americana at dose of 600 mg/kg produced significant reduction in paw volume when compared with control. The maximum effect was seen in the oral dose of 600 mg/kg b.w which showed significant. The antiinflammatory activity in this dose of the test drug was comparable to standard, Diclofenac sodium (10mg/kg). The maximum antiinflammatory effect was observed at 4th hour in all the doses of test drug.

The yield value was maximum with ethyl acetate extract and methanolic and less with pet.

Table 2. Toxicity study of pet. ether, ethyl acetate and methanolic extract of Ximenia americana leaf.

Sl. No.

1 +ve

+ve

+ve

-ve

++ve

+ve +ve

++ve

Biuret test

+ve

+ve

++ve

Amino acids

-ve

-ve

-ve

++ve = Present , + = Traces, -ve = absent

53

Treatment

Control

Dose mg/kg body

Number of Animals

%

LD50 Value

weight

*

**

***

1%

6

6

0

0

---

Tween 80

2

MEXMA

10

6

6

0

0

---

3

MEXMA

100

6

6

0

0

---

4

MEXMA

1000

6

6

0

0

---

5

MEXMA

4000

6

6

0

0

---

6

MEXMA

6000

6

6

0

0

7

EEXMA

10

6

6

0

0

---

8

EEXMA

100

6

6

0

0

---

9

EEXMA

1000

6

6

0

0

---

10

EEXMA

4000

6

6

0

0

---

11

EEXMA

6000

6

6

0

0

12

PEXMA

10

6

6

0

0

---

13

PEXMA

100

6

6

0

0

---

14

PEXMA

1000

6

6

0

0

---

15

PEXMA

4000

6

6

0

0

---

16

PEXMA

6000

6

6

0

0

>6.0 gm/kg B.W

* = No of Animals; ** = No.of Survived; *** = No.of Death; % = % of Mortality

54

Siddaiah et al.

Table 3. Anti-inflammatory activity of the petroleum ether, ethyl acetate, methanolic extract Ximenia americana in carrageenan induced paw oedema method.

Group

Treatment Design

Dose (mg/kg /b.w/p.o.)

Oedema volume(ml)

Inhibition (%)

I

Control (Tween 80, 1%)

……

0.15 ± 0.010

……

II

PEEXMA

200

0.12 ± 0.007 **

20.00

III

......

400

0.09 ± 0.003*

40.00

IV

......

600

0.07 ± 0.012 *

53.33

V

EAEXMA

200

0.12 ± 0.004**

13.34

VI

......

400

0.09 ± 0.014*

40.75

VII

......

600

0.06 ± 0.005 *

60.00

VIII

MEEXMA

200

0.12 ± 0.010**

20.00

IX

......

400

0.08 ± 0.006*

46.66

X

......

600

0.06 ± 0.011 *

60.00

XI

Diclofinac sodium

100

0.05 ± 0.010*

66.67

It was concluded that the Ximenia americana possess considerable anti-inflammatory activity in all extracts. Our pharmacological studies explains the use of Ximenia americana leaf extract as an anti-inflammatory agent. This gives valuable information regarding the treatment of antiinflammatory condition with lesser side effects and toxicities, which are encountered in convential pain killer drugs. The common adverse effects of conventional pain killer are ulcer, hepatotoxicity and nephrotoxicity on prolonged use in condition like arthritis etc. These adverse effects can be minimized by using the extracts of Ximenia americana. Inhibition of carrageenan induced paw oedema in rats is one of the most suitable test to screen anti arthritic and anti-inflammatory agents as it closely resembles human arthritis.

1.

2.

PEEXMA= Petroleum Eather Extract Ximenia americana; EAEXMA = Ethyl Acetate Extract Ximenia americana; MEEXMA = Methanolic Extract Ximenia americana; * p < 0.001 compared to control; ** p < 0.01 compared to control.

Values are mean ± SEM of six animals in each group. Data was analysed by unpaired ‘t’ test.

ether extract. Pharmacological studies of Ximenia americana revealed that the % inhibition of inflammation for both methanolic and ethyl acetate extracts were remarkable i.e. 60% and 60% at 4th hour respectively. The comparison between petroleum ether, ethyl acetate and methanolic extracts with the Diclofenac sodium are represented in Table 3.

3.

4.

5.

6.

7.

8.

9.

10.

Discussion After the observations of the results obtained on carrageenan induced rat paw oedema.

11.

References Ogunleye DS and Ibitoye SF: Trop. J. Pharm.

Res. 2(2): 239-241 December (2003).

Madhava Chetty K, Sivaji K and Tulasi Rao K: Flowering Plants of Chittoor District A.P. India.

1st edition. Student Offset Printers, Tirupathi. pp 85 (2008).

VSSC. E. YME, Berger MR. PMID: 16005923 (Pub. Med-indexed for MEDLINE) Khandelwal KR: Practical Pharmacognsoy.

10th edition. Nirali Prakashan, Pune, India (2003).

William M Carey, Jeevan Manibabu D, Venkata Raju N and Krishna Mohan G: J. of Pharmacy and Chemistry. Vol. 2(3): 133-138 (2003).

Shetty SC, Bhagat VC, Kore KJ and Shete RV: Indian Drugs. 45(3) March (2008).

The Wealth of India: Vol. XI X-2 CSIR, pp 6 – 8 (1976).

Malaya Gupta, Upal Kanti Mazumder, Ramanathan, Sampath Kumar and Thangavel Siva Kumar: Iranian J. of Pharmacology and Therapeutics (IJPT). Vol. 2(2) (2003).

Paradesi Goldee, Gadgoli Charya, Vaidya Madhava D, et al.: Pharmacologyonline.

1: 111-116 (2008).

Goverdan P and Bobbale Diwakar: Ethno.

Botanical Leaflets. 13: 65-72 (2009).

Yasodha Krishna Janapati, Jayaveera K N, et al. : J. Pharm. and Chemistry. Vol. 2: 156–160 July (2008).

Address for correspondence: Dr. M. Siddaiah, Annamacharya College of Pharmacy, Rajampet, Kadapa, Andhra Pradesh (India). E-mail: siddaiah.m@rediffmail.com 038_2009

J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 55-59

ISSN 0970-7700

IN-VITRO ANTIOXIDANT CAPACITY OF GRADED DOSES OF METHANOLIC EXTRACT FROM LUFFA CYLINDRICA (L) SEEDS K. NAGARAJAN,1 SATYAJIT DUTTA,2 SUMIT DAS,2 SURABHI SINGHAL,3 PALLAVI SAXENA,4 AVIJIT MAZUMDER5 AND L. K.GHOSH6 Division of Bio-Medicinal Chemistry, R&D Laboratory, Department of Pharmacy,1,2,4 Department of Microbiology,3 IIMT College of Medical Sciences, ‘O’ Pocket, Ganga Nagar, Mawana Road, Meerut - 250001 U.P. (India) School of Pharmacy,5 Technology and Management, NMIMS University, Mumbai - 400053 M.S. (India) Department of Pharmaceutical Technology,6 Jadavpur University, Kolkata - 700032 W.B. (India) Abstract: Luffa cylindrica (L) (Family: Cucurbitaceae) commonly called sponge gourd plant have antibacterial, antifungal, anti-HIV and antiasthmatic activity. The seed powder was extracted with methanol under 60-80°C by continuous hot percolation process using Soxhlet apparatus and the solvent was removed by distillation under reduced pressure and the residue was stored in desiccator. The total antioxidant activity of the extract was determined with phosphomolybdenum method using α-tocopherol as the standard. Among the graded doses (1 μg/ml, 2 μg/ml, 3 μg/ml and 4 μg/ml) selected for the study, the maximum total antioxidant potency was observed with higher dose of seed extract of Luffa cylindrica (L) at a concentration of 3 μg/ml which is slightly greater than the standard α-tocopherol. The antioxidant activity was statistically significant at 1% and 5% level of significance, which clearly indicates the validation for the biological work accomplished.

Keywords: Luffa cylindrica, Antioxidant, α-tocopherol, Seed extracts.

Introduction There is an increased evidence for the participation of free radicals in the etiology of various diseases like cancer, diabetes, cardiovascular diseases, autoimmune disorders, neurodegenerative diseases, aging, etc.

(Bandopadhyay et al., 1999). An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules which scavenge the free radicals and prevent the damage caused by them. They can greatly reduce the damage due to oxidants by neutralizing the free radicals before they can attack the cells and prevent damage to lipids, proteins, enzymes, carbohydrates and DNA (Fang et al., 2002). Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells (Umamaheswari and Chatterjee, 2008). Antioxidants terminate these 1. Assistant Professor

2. Lecturer

chain reactions by removing free radical intermediates and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols, ascorbic acid or polyphenols.

Natural plants have been used in the treatment of various diseases from the time of immemorial the use of plants as a source of medicine lies deep in the history of mankind. A wide range of antioxidants from both natural and synthetic origin has been proposed for use in the treatment of various human diseases (Cuzzocrea et al., 2001). Loofa is derived from the cucumber and marrow family and originates from America (Mazali and Alves, 2005). Luffa [Luffa cylindrica (L) Rome syn L. aegyptiaca Mill] (Family: Cucurbitaceae) commonly called sponge gourd, loofa, vegetable sponge, bath sponge or dish cloth gourd, plant have antibacterial, antifungal, anti-HIV, antiasthmatic, immunomodulatory and antitumor

3. Assistant Professor and Head

4. Research Scholar

5 & 6.. Professor

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Nagarajan et al.

activitiy (Ng et al., 1992). In oriental medicine, L. cylindrica has effect on the treatment of fever, enteritis and swell etc. The extracts from vines alive are used as an ingredient in cosmetics and medicine (Lee and Yoo, 2006). Immature fruit is used as vegetables, which is good for diabetes (Bal et al., 2004). When extracted and analysed, the seed of L. cylindrica contained 40% oil which is used as oil sources and oil cake obtained is used as fertilizer and feed (Lee and Yoo, 2006). It has been suggested as an immobilization matrix for plant, algal, bacterial and yeast cells (Iqbal and Zafar, 1993 a, b; Iqbal and Zafar, 1994).

The fruits of Luffa cylindrica (L) has been already tested for their antioxidant activity with the radical scavenging effect on the 1,1-diphenyl2-picrylhydrazyl radical (Du et al., 2006). Thus, this study was designed to evaluate the in-vitro total antioxidant activity of methanolic extract of Luffa cylindrica (L) seeds.

Materials and Methods Collection of plant material Luffa cylindrica (L) was collected from the road sides of Meerut, Uttar Pradesh, India, in the month of January, 2009. The seeds were dried under shade with occasional shifting and then powdered with a mechanical grinder and stored in an airtight container.

Identification of the plant The plant was identified and authenticated by the botanist and a voucher specimen was deposited to Dr. Surabhi Singhal, Department of Microbiology, IIMT College of Medical Sciences, Meerut, Uttar Pradesh, India (voucher specimen no. IIMT/BD/01/04/2009/02/Tech.2005).

Preliminary phytochemical screening The powdered material were subjected to preliminary phytochemical qualitative screening by using different organic solvents for the presence or absence of various primary or secondary metabolites like alkaloids, glycosides, steroids, terpenoids, flavanoids, carbohydrates

and saponins following standard procedures (Kokate, 2005; Harborne, 1984).

Phytochemical extraction About 120 g of seed powder of Luffa cylindrica was extracted with 500 mL. methanol under 60-80°C by continuous hot percolation process using Soxhlet apparatus until the solvent became colorless. After completion of the extraction, the extracts were filtered and evaporated to dryness by Rotary Vacuum evaporator (Buchi Rota vapour, Switzerland) and the residue was stored in desiccator (Keiichi et al., 1990). The extract was purified by preparative TLC by using 60% ethyl acetate and 40% hexane as mobile phase.

Chemicals and solvents used The chemicals and solvents used for the experimental work were procured from E. Merck, India. Silica gel G (30–70 mesh) used for chromatography (TLC) was obtained from E. Merck. Standard α-tocopherol was procured from Cadila Pharmaceuticals, Ahmedabad, India, as a gift sample. All other chemicals and solvents used in the study were obtained commercially and were of analytical grade.

Total antioxidant efficacy The total antioxidant activity of the extract was determined by phosphomolybdenum method using α-tocopherol as the standard (Guddadarangavvanahally et al., 2004; AsokKumar et al., 2009; Jayaprakasha et al., 2002).

The assay is based on the reduction of Mo(VI)-Mo(V) by the extract and a subsequent formation of a green phosphate / Mo(V) complex at acid PH. Different graded doses were prepared with the extracts (1 μg/ml, 2 μg/ml, 3 μg/ml and 4 μg/ml) solution and each was combined with 1.0 ml of reagent (0.6 M H2SO4, 218 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in a boiling water bath at 95°C for 90 minutes. After the samples had cooled to room temperature, the

Antioxidant Efficacy of Luffa cylindrica Seeds

maximal absorption was measured at 695 nm against the blank solution in Photoelectric Colorimeter (Systronics-113, India) (Hecker and Ullrich, 1989).

57

methanolic seed extracts give highly active result for Rochan test whereas for steroids, it gives moderately active result for Salkovaski test. None other organic extracts have shown positive result for terpenoids and steroids. Terpenoids present in the seed extract was further confirmed with TLC analysis having an hRf value of 84 with 60% ethyl acetate : n-hexane and iodine as visualizing reagent.

Results Phytochemical investigation The results of the preliminary phytochemical screening of Luffa cylindrica seeds are expressed in Table 1. From the results of Table 1, it was concluded that the methanolic extracts having more active constituents like terpenoids and steroids than the other active constituents. For the terpenoids,

Total antioxident capacity The results of the various graded doses of Luffa cylindrica seed extracts and its total antioxidant capacity are expressed in Table 2.

Table 1. Preliminary Phytochemical screening of Luffa cylindrica Seeds.

Alkaloids

Glycosides (Borntrager’s test)

(Salkovaski test)

Steroids

Terpenoids

Flavonoids (Shinoda test)

Carbohydrates (Benedict’s test)

Saponins

-

-

-

-

-

-

-

Chloroform

++

-

-

-

-

++

-

Benzene

++

-

-

-

-

-

-

n-Butanol

-

-

-

-

-

+

-

Ethanol

-

-

-

-

-

+

-

Ethyl acetate

+

-

-

-

-

-

-

Methanol

-

-

+

++

-

-

-

Water

-

-

-

-

-

-

++

Solvents

(Hager’s test)

Petroleum ether

(Rochan test)

(Foam test)

++ Highly active; + Moderately active; - Not present

Table 2. Total Antioxidant Capacity of Methanolic extracts of Luffa cylindrica Seeds.

Sl. No.

Sample Accession Number

Sample Concentration Used

Absorbance at 695 nm

Standard Accession Number & Concentration Used for Standard

Absorbance at 695 nm

Total Antioxidant Capacity (Pg Vitamin E equivalent/mg)

1

LCSE1

1 Pg/ml

0.98

DT1 (0.6 Pg/ml)

0.30

0.0009 IU

2

LCSE2

2 Pg/ml

1.01

DT2 (1.2 Pg/ml)

0.34

0.0018 IU

3

LCSE3

3 Pg/ml

1.13

DT3 (1.8 Pg/ml)

0.70

0.0027 IU

4

LCSE4

4 Pg/ml

1.10

DT4 (2.4 Pg/ml)

0.24

0.0036 IU

LCSE1: Luffa cylindrica Seed Extract (1 Pg/ml); LCSE2: Luffa cylindrica Seed Extract (2 Pg/ml); LCSE3: Luffa cylindrica Seed Extract (3 Pg/ml); LCSE4: Luffa cylindrica Seed Extract (4 Pg/ml); IU: International Unit, 1mg of Tocopherol= 1.5 IU

DT1: D-tocopherol (standard) (0.6Pg/ml); DT2: D-tocopherol (standard) (1.2Pg/ml); DT3: D-tocopherol (standard) (1.8Pg/ml); DT4: D-tocopherol (standard) (2.4Pg/ml);

58

Nagarajan et al.

The maximum total antioxidant potency was observed with higher dose of seed extract of Luffa cylindrica (L) at a concentration of 3 μg/ml which is slightly greater than the standard α-tocopherol.

The antioxidant capacity of phosphomolybdate method is expressed as the number of equivalents of α-tocopherol among the extracts tested the dose at which the concentration contains 0.0009 IU, 0.0018 IU, 0.0027 IU, 0.0036 IU Vitamin E equivalent/mg.

Hence the seed extract (3 μg/ml) was found to have more pronounced antioxidant potency invitro, than the standard drug α-tocopherol used.

The antioxidant activity were statistically significant at 1% and 5% level of significance (calculated ‘t’=29.567; calculated ‘F’=20.5104

by ANOVA), which clearly indicates the validation for the biological work accomplished.

Discussion The total antioxidant capacity assessed in the methanolic seed extract of Luffa cylindrica was proved to be significant therapeutically invitro than the standard α-tocopherol with the above mentioned results of Table 2. This is mainly due to the presence of terpenoid as predominant bio-active constituent in the plant part with the findings from qualitative chemical analysis and TLC. To be discussed as health promoting or biofunctional, the significant impact of a tested extract was either on human metabolism or on well-defined and appropriate biomarkers (Wagner and Elmadfa, 2003). Three main ways of antioxidant action of carotenoid type terpenoids have been detected until now (i.e., quenching of singlet oxygen, hydrogen transfer, or electron transfer). Low-density lipoprotein oxidation is believed to play an important role in the development of atherosclerosis disorders (Grassmann, 2005). Several secondary plant metabolites have been tested for and therefore a high resistance of LDL against oxidation may prevent atherogenesis and accompanying their ability to prevent oxidation of LDL and many phenolic terpenoids as well as carotenoids have

been shown to enhance LDL oxidation resistance (Milde et al., 2007). Hence the probable mechanism of terpenoid bio constituent in the seeds of Luffa cylindrica may be due to their ability to prevent the oxidation of LDL in peripheral tissues, which has to further extensively investigated for cardio-protective effects only with clinical in-vivo studies in near future.

Acknowledgements The authors are very much thankful to Rtn. Yogesh Mohanji Gupta, Chairman, IIMT Group of Colleges, Meerut and Rtn. Abhinav Agarwaal, Secretary General, IIMT Group of Colleges, Meerut U.P. (India) for their constant encouragement and continuous support throughout the project work.

1.

2.

3.

4.

5.

6.

7.

8.

References AsokKumar, K., UmaMaheswari, M., Sivashanmugam, A. T., SubhadraDevi, V. N. and Subhashiniand Ravi, T. K: Free radical scavenging and antioxidant activities of Glinus oppositifolius (carpet weed) using different in vitro assay systems. Pharmaceutical Biology. Vol. 47(6): 474-482 (2009).

Bal, K. E., Bal, Y. and Lallam, A: Gross morphology and absorption capacity of cell-fibers from the fibrous vascular system of Loofah (Luffa cylindrica). Textile Res. J. Vol. 74: 241- 247 (2004).

Bandyopadhyay, U., Das, A. and Bannerjee, R. K: Reactive oxygen species: Oxygen damage and pathogenesis. Curr. Sci. Vol. 77(5): 658-666 (1999).

Cuzzocrea, S., Riley, D.P., Caputi, A.P. and Salvemini, D: Antioxidant therapy: A new pharmacological approach in shock, inflammation and ischemia or reperfusion injury. Pharmacol.

Rev. Vol. 53: 135-159 (2001).

Du, Q., Xu, Y., Li, L., Zhao, Y., Jerz, G. and Winterhalter, P: Antioxidant constituents in the fruits of Luffa cylindrica (L.) Roem. J. Agric.

Food Chem. Vol. 54(12): 4186-4190 (2006).

Fang, Y., Yang, S. and Wu, G: Free radicals, antioxidants and nutrition. Nutrition. Vol. 18: 872-879 (2002).

Grassmann, J: Terpenoids as plant antioxidants.

Vitam Horm. Vol. 72: 505-535 (2005).

Guddadarangavvanahally, K., Jayaprakasha, Rao, L. J. and Kunnumpurath, K. S: Antioxidant

Antioxidant Efficacy of Luffa cylindrica Seeds

9.

10.

11.

12.

13.

14.

15.

activities of flavidin in different in vitro model systems.

Bioorganic and Medicinal Chemistry. Vol. 12(19): 5141-5146 (2004).

Harborne, J.B: Phytochemical Methods. Chapman and Hall, London, New York (1984).

Hecker, M. and Ullrich, V: On the mechanism of prostacyclin and thromboxane A2 biosynthesis. J. Biol. Chem. Vol. 264(1): 141-150 (1989).

Iqbal, M. and Zafar, S. I: The use of fibrous network of matured dried fruit of Luffa aegyptiaca as immobilizing agent. Biotechnol. Tech. Vol. 7: 15-18 (1993a).

Iqbal, M. and Zafar, S. I: Vegetable sponge: A new immobilizing medium for plant cells. Biotechnol. Tech.

Vol. 7: 323-324 (1993b).

Iqbal, M. and Zafar, S. I: Vegetable sponge as a matrix to immobilize microbes: a trial study for hyphal fungi, yeast and bacteria. Lett. Appl. Microbiol. Vol. 18: 214-217 (1994).

Jayaprakasha, G. K., Jena, B. S. and Negi, P. S: Antioxidant activities of polyphenol containing extracts from citrus. J. Naturforsch. Vol. 57C: 828-835 (2002).

Keiichi, W., Yuji, M. and Gunki, F: Isolation and partial characterization of three protein synthesis inhibitory proteins from the seeds of Luffa of three protein synthesis inhibitory proteins from the seeds of Luffa cylindrica. Agric. Biol. Chem. Vol. 54(8): 2085-2092 (1990).

16.

17.

18.

19.

20.

21.

22.

59

Kokate, C.K: Practical Pharmacognosy.

Vallabh Prakashan, New Delhi (2005).

Lee, S. and Yoo, J. G: (WO/2006/019205) Method for preparing transformed Luffa cylindrica Roem (World Intellectual property organization) http:// w w w . w i p o . i n t / p c t d b / e n / wo.jsp?IA=KR2004002745&DISPLAY=STATUS. (2006).

Mazali, I. O. and Alves, O. L: Morphosynthesis: high fidelity inorganic replica of the fibrous network of loofa sponge (Luffa cylindrica). An. Acad. Bras. Cien. Vol. 77(1): 25-31 (2005).

Milde, J., Elstner, E. F. and Grassmann, J: Synergistic effects of phenolics and carotenoids on human low-density lipoprotein oxidation. Mol. Nutr. Food Res. Vol. 51(8): 956-961 (2007).

Ng, T. B. and Chan, W. Y: Proteins with abortifacient, ribosome inactivating, immunomodulatory, antitumor and anti-AIDS avtivities from Cucurbitaceae plants. General Pharmacology. Vol. 23(4): 575-590 (1992).

Umamaheswari, M. and Chatterjee, T. K: In-vitro antioxidant activities of the fractions of Cocciniagrandis L. leaf extract. African Journal of Traditional, Complementary and Alternative Medicines. Vol. 5(1): 61 – 73 (2008).

Wagner, Karl-Heinz and Elmadfa, I: Biological relevance of terpenoids overview focusing on Mono-, Di- and Tetraterpenes. Annals of Nutrition and Metabolism. Vol. 47: 95-106 (2003).

Address for correspondence: Dr. K. Nagarajan, Assistant Professor, Division of Bio-Medicinal Chemistry, R&D Laboratory, Department of Pharmacy, IIMT College of Medical Sciences, ‘O’ Pocket, Ganga Nagar, Mawana Road, Meerut - 250001 U.P. (India). E-mail: nagarajan_mph@yahoo.co.in, sanku6@gmail.com 035_2009

J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 61-63

ISSN 0970-7700

ANTIBACTERIAL ACTIVITY OF ALCOHOLIC EXTRACT OF ALOE VERA L. BY DISC DIFFUSION METHOD G.. S. NITURE,1 M. K. PATIL,2 A. G. KARPE3 AND A.V. BHONSLE4 Department of Microbiology,1,4 Department of Pharmacology,2 College of Veterinary and Animal Sciences, Udgir - 413517 District Latur, Maharashtra (India) Abstract: The study was carried out to assess antibacterial activity of 50% alcoholic cold extract of Aloe vera against pathogenic strains of E.coli, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa using extract impregnated disc by disc diffusion method. The present study revealed that 50% alcoholic cold extract of Aloe vera L. (37.52 + 0.77 mg/ disc) was effective against all the bacteria by disc diffusion method. The study concluded that 50% alcoholic cold extract of Aloe vera L. had significant antibacterial activity but lesser than reference antibiotic ciprofloxacin.

Keywords: Aloe vera L., Antibacterial activity, Medicinal Plants.

Introduction Aloe vera (Family â&#x20AC;&#x201C; Lilliaceae) commonly known as Grithkumari, Korphad is found in semi wild dry parts of India. Aloe vera possess antibacterial, anthelmentic, purgative, stomachic, cooling, bitter, carminative and digestive properties. It is useful in the treatment of eye diseases, tumours, enlargement and inflammation of spleen, liver complaints, vomiting, bronchitis, fever, skin diseases, jaundice, chronic ulcers, muscular pain and constipation (Anjaria, 2002; Rajpal, 2002). The study on antibacterial activity is meager, therefore the present investigation is undertaken to screen the antibacterial property of Aloe vera extract.

Materials and Methods 1. Preparation of extract Aloe vera L. leaves was used. The gel was subjected to drying and the powder was subjected for preparation of 50% alcoholic cold extract as per method described by Rosenthaler (1930).

2. Preparation of extract impregnated disc The sterile blank disc was obtained from M/ s. HiMedia Laboratory Ltd. Mumbai, India. Extract

impregnated discs were prepared using dissolved extracts in the 50% alcoholic solvent and impregnated onto the disc, until the discs get fully Table 1. Amount of alcoholic extract of Aloe vera impregnated on to the discs.

Extract

Weight (mg) Blank discs*

Extract impregnated discs*

Extract in each disc

Cold

65.00 65.00 65.00

327.00 330.00 326.00

37.43 37.86 37.28

Mean + SE

65.00 +0.00

327.67 +1.20

37.52 +0.77

* Weight of 7 discs at each time

saturated and was air dried. The extract impregnated discs were collectively weighed before and after impregnation of the extract. The amounts of the extract actually absorbed onto the disc were recorded as shown in Table 1.

3. Test organism The typed pathogenic bacterial culture of Escherichia coli (MTCC 723), Staphylococcus aureus (MTCC 96), Streptococcus pyogenes (MTCC 442) and Pseudomonas aeruginosa

1. M. V. Sc. Scholar, Veterinary Microbiology 2. Assistant Professor, Veterinary Pharmacology 3. Associate Dean, KNP College of Veterinary Sciences, Shirwal - 412801 District Satara, Maharashtra (India) 4. Assistant Professor, Veterinary Microbiology

62

Niture et al.

Table 2. Zone of inhibition by 50% alcoholic cold extract of Aloe vera L. against test bacteria.

Sensitivity pattern

Sr. No.

Extract discs

1

50 % Alcohol

2

50 % alcohol Aloe vera extract discs

3

Ciprofloxacin

+: Less sensitive (Zone < 10 mm) +++: Moderately sensitive (Zone between 15 â&#x20AC;&#x201C; 20 mm)

Staphylococcus aureus

Stereptococcus pyogenes

E.coli

Pseudomonas aeruginosa

No zone

No zone

No zone

No zone

++

++

++

++

++++

++++

++++

++++

++: Sensitive (Zone between 10 â&#x20AC;&#x201C; 15 mm) ++++: Highly sensitive (Zone > 20 mm)

(MTCC 741) were obtained from Microbial Typed Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh (India). The pathogenic bacterial cultures were sub cultured and maintained on nutrient agar (MM 012) and in nutrient broth (M 088).

4. Determination of antibacterial activity The antibacterial effectiveness of 50% alcoholic cold extract of Aloe vera L. was determined by disc diffusion method (Bauer et al., 1966).

Disc Diffusion Test Each of the four bacterial culture viz., Escherichia coli Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa was grown in nutrient broth at 37oC for overnight incubation. The bacterial culture was diluted 10-3, 10-4, 10-5, 10-6 using sterile nutrient broth.

Each dilution of four bacteria was plated on Petri- plates aseptically. The extract impregnated discs Aloe vera L. were placed in Petri-plates containing diluted four bacterial cultures on nutrient agar with the help of sterile forceps and incubated at 37oC. After 24 hours of incubation the Petri-plates were examined for zone of inhibition, measured in mm scale as shown in Table 2. The data of this investigation were statistically analyzed by Student-t-test (Snecdor and Cochran, 1968).

Results and Discussion The weight of the extract adsorbed on each disc is depicted in Table 1. The adsorption

of 50% alcoholic cold extract was 37.52 + 0.77.

Table 2 depicts zone of inhibition by 50% alcoholic cold extract of Aloe vera against test bacteria. It is evident that 50% alcohol discs did not have any antibacterial activities which were used as control.

50% alcohol Aloe vera extract discs were effective on the test bacteria. Ciprofloxacin discs which were used as control positive revealed significant antibacterial activity on all the test bacteria.

Conclusion Fifty percent alcoholic Aloe vera dry gel extract exhibited antibacterial activity by disc diffusion method against all test bacteria, whereas 50% alcohol control base disc (which was used to check the residual effect of alcohol) was found ineffective against all test bacteria. Fifty percent alcoholic Aloe vera dry gel extract showed Comparatively lesser antibacterial activity than Ciprofloxacin. After further study, dose of Ciprofloxacin may be reduced to half in the treatment of diseases caused by S. aureus, S. pyogenes, E. coli and P. aeruginosa by using Aloe vera extract preparation, thereby promoting use of herbal drugs and reducing side effects of chemical drug on animal body.

Acknowledgements The authors are thankful to Associate Dean, College of Veterinary and Animal Sciences, Udgir, M.S. (India) for providing necessary facilities to undertake the work.

Antibacterial Activity of Aloe vera

1.

2.

3.

References Anjaria, J: Inventory of Traditional Medicinal Practices in India. Ministry of Agriculture, Govt. of India. pp. 194 (2002).

Bauer, A.W., Kirby, M.M., Sherris, T.C. and Truck, M: Antibiotic susceptibility testing by standardized single disc method. Am J. Clin.

Pathol. 45: 493-496 (1966).

Nadkarni, K.M. and Nadkarni, A.K: Medicinal Plants Indian Materia Medica. Vol. 1. Popular

4.

5.

6.

Prakashan Private Limited, Bombay, India. pp. 28 (1976).

Rajpal, V: Standardization of Botanical Testing and Extraction Method of Medicinal Herbs.

Eastern Publisher, New Delhi. pp. 11-20 (2002).

Rosenthaler, L: The Chemical Investigation of Plants. 1st Edn. Bell and Sons, London. pp. 36 (1930).

Snedecor, G.W. and Cochran, W.G: Statistical Methods. Oxford and IBH Publication Co., Calcutta. pp. 534 (1968).

Address for correspondence: Dr. G. S. Niture, Niture Niwas, Near H.S. College, Dam Road, Udgir - 413517 Dist. Latur, Maharashtra (India). E-mail: ganeshniture@gmail.com 031_2009

63

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