GJRMI - Volume 2, Issue 8, August 2013 by Dr. Hari Venkatesh.K.Rajaraman

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Contact: Mobile: +919980952358, +919035087999 E- mail: raviamrita.kumar9@gmail.com

Consultant Physician: Dr. Ravi Kumar. M. (Specialized in different types of Keraliya Ayurvedic treatments especially in ENT & Eye diseases) Special Ayurvedic Treatments in the following diseases: Special keraliya treatments in Myopia, Astigmatism, Macular degeneration, Cataract, Glaucoma, Diabetic Retinopathy, Retinal detachment, Retinal pigmentation, Obesity, Filariasis, Asthma, Stress, Anxiety, Insomnia, Depression, Loss of Memory & Concentration

An International, Peer Reviewed, Open access, Monthly E-Journal

ISSN 2277 â&#x20AC;&#x201C; 4289 www.gjrmi.com Editor-in-chief Dr Hari Venkatesh K Rajaraman

Managing Editor Dr. Shwetha Hari

Administrator & Associate Editor Miss. Shyamala Rupavahini

Advisory Board Prof. Rabinarayan Acharya Dr. Dinesh Katoch Dr. S.N.Murthy Dr. Mathew Dan Mr. Tanay Bose Dr. Nagaraja T. M. Prof. Sanjaya. K. S. Dr. Narappa Reddy

Editorial board Dr. Kumaraswamy Dr. Madhu .K.P Dr. Sushrutha .C.K Dr. Ashok B.K. Dr. Janardhana.V.Hebbar Dr. Vidhya Priya Dharshini. K. R. Mr. R. Giridharan

Honorary Members - Editorial Board Dr Farhad Mirzaei Mr. Harshal Ashok Pawar

INDEX – GJRMI, Vol.2, Iss. 8, August 2013 MEDICINAL PLANTS RESEARCH Bio-Technology INHIBITORY EFFECT OF GYMNEMA SYLVESTRE, STEVIA REBAUDIANA, PHYLLANTHUS EMBLICA AND SYZYGIUM CUMINI ON PORCINE PANCREATIC AMYLASE Dayananda. K. S., Gopinath S. M., Murthy R. K.

554–561

Bio-Resource Technology STUDY ON SEED GERMINATION AND AGROTECHNIQUES OF CHEILOCOSTUS SPECIOSUS (J. KONIG) C. SPECHT IN MID-HILL OF WESTERN HIMALAYA

562–570

Gopichand, Singh. R. D., Meena. R. L.

Pharmacology WOUND HEALING ACTIVITY OF ETHANOLIC EXTRACT OF EUPHORBIA HIRTA LEAVES ON EXCISION WOUND MODEL IN RATS Rathnakumar. K., Verma Ranbir, Jaikumar. S., Sengottuvelu. S.

571–575

INDIGENOUS MEDICINE Ayurveda – Rasa Shastra & Bhaishajya Kalpana MICRO-MORPHOLOGICAL AND MICRO-METRIC EVALUATION OF MANGIFERA INDICA LINN. – LEAF Anupriya, Harisha.C.R., Patgiri. B. J., Prajapati. P. K.

576–581

Ayurveda – Dravya Guna DETAILED PHARMACOGNOSTICAL EVALUATION ON RHIZOME COADUNATA (Wall. Ex Hook & Grev.) C. Chr. - A FOLKLORE HERB Mori Hardik, Nishteswar. K, Patel. B. R., Harisha. C. R.

TECTARIA

582–588

Ayurveda – Manasa Roga STANDARDIZATION OF ASTANGA LAVANA - A HERBO-MINERAL AYURVEDIC COMPOUND Shetty Suhas. K., Bhat. N. P., Savitha. H. P., Sunil Kumar.K. N., Ravishankar. B

589–598

Ayurveda – Review article CLINICAL RESEARCH RELATED CAREER CHOICES FOR AYUSH GRADUATES IN INDIA Samal Janmejaya

599–604

Ayurveda – Review article SHARIRIKA PRAKRITI – AN ASTUTE OF HUMAN CONSTITUTION Amin Hetal, Vyas. M. K., Vyas H. A., Baghel. A. S., Dwivedi. R. R.

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – DUGDHIKA (EUPHORBIA HIRTA L.), OF THE FAMILY EUPHORBIACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA

605–612

Research article INHIBITORY EFFECT OF GYMNEMA SYLVESTRE, STEVIA REBAUDIANA, PHYLLANTHUS EMBLICA AND SYZYGIUM CUMINI ON PORCINE PANCREATIC AMYLASE Dayananda K S1*, Gopinath S M2, Murthy R K3 1,2,

Acharya Institute Of Technology Dept. of Biotechnology Engineering, Banglore-560090, India Jain University, Bangalore, Karnataka, India *Corresponding Author: Email: drksdayananda@gmail.com; Mobile: +919448355341 3

Received: 20/06/2013; Revised: 03/08/2013; Accepted: 06/08/2013

ABSTRACT Diabetes has become a common global health problem that affects >170 million people worldwide. It is one of the leading causes of death and disability. It is estimated that by 2030, the number will rise to 366 million (www.who.int). The majority of diabetes (~90%) is type 2 diabetes (T2D) caused by a combination of impaired insulin secretion from pancreatic beta cells and insulin resistance of the peripheral target tissues, especially muscle and liver. The ‘top’ three countries in terms of the number of T2D individuals with diabetes are India (31.7 million in 2000; 79.4 million in 2030), China (20.8 million in 2000; 42.3 million in 2030); and the US (17.7 million in 2000; 30.3 million in 2030). Clearly, T2D has become an epidemic in the 21st century where India leads the world with the largest number of diabetic subjects. Traditional Medicines obtained from medicinal plants are used by about 40-60% of the world’s population. Though there are many approaches to control diabetes and its secondary complications, herbal formulations are preferred due to lesser side effects and low cost. In this study,the inhibitory effect of Syzygium cumini, Gymnema sylvestre, Stevia rebaudiana and Phyllanthus emblica extract on porcine pancreatic amylase.The most significant inhibitory activity was obtained with the aqueous and ethanolic extracts of G. sylvestre and S. cumini. KEY WORDS: Amylase, Diabetes, Inhibition, Syzygium cumini, Gymnema sylvestre, Stevia rebaudiana, Phyllanthus emblica, Porcine Pancreatic amylase

Cite this article: Dayananda. K. S., Gopinath. S. M., Murthy. R. K., (2013), INHIBITORY EFFECT OF GYMNEMA SYLVESTRE, STEVIA REBAUDIANA, PHYLLANTHUS EMBLICA AND SYZYGIUM CUMINI ON PORCINE PANCREATIC AMYLASE, Global J Res. Med. Plants & Indigen. Med., Volume 2(8): 554–561

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INTRODUCTION Type 2 diabetes is a major public health issue in India and South Asians, when compared to Caucasians (IDF) and are more likely to develop Diabetes at younger ages (Banerji, 1999; McKeigue et al., 1991) and at lower body mass indices (McKeigue, 1991; Ramachandran et al., 2001). India has the highest number of diabetes cases worldwide (40 million) (Mohan V et al., 2006). Another 30 million Indians have pre-diabetes and are at high risk of developing T2DM.5 T2DM is an economically costly disease (Yusuf, et al., 2004) and a major cause of mortality and morbidity. Indians and other South Asians with diabetes have worse glycemic control, (chandelier, 2007; Ramachandran, et al., 2006) a higher prevalence of microalbuminuria, (Pan et al., 1997) hypertension, retinopathy, and cardiovascular disease, (Lindstrom et al., 2003) and a higher incidence and faster progression of renal disease than most other diabetic populations. (Ramachandran, et al., 2007) unexplained weight loss. The prevalence of T2DM risk factors, such as insulin resistance, increased fat mass, and central obesity are higher in South Asian populations. (Knowler, 2002; Eriksson, 1991; Narayan KM, et al., 2004).T2DM prevention is a priority for South Asian populations. Diabetes is a chronic disorder of carbohydrate, fat and protein metabolism characterized by increased fasting and postprandial blood sugar levels. The global prevalence of diabetes is estimated to increase by 5.4% by the year 2025. WHO has predicted that the major burden will occur in developing countries. Studies conducted in India in the last decade have highlighted that not only is the prevalence of diabetes high but also that it is increasing rapidly in the urban population (Alberti KG, et al., 2007). Type I diabetes (insulin dependent) is caused due to insulin insufficiency because of lack of functional beta cells. Though patho physiology of diabetes remains to be fully understood, experimental evidences suggest the involvement of free radicals in the pathogenesis of diabetes and

more importantly in the development of diabetic complications (Mather, 1998; Mukhopadhyay,2006; Snehalatha et al., 2003). Free radicals are capable of damaging cellular molecules, DNA, proteins and lipids leading to altered cellular functions. Many recent studies reveal that antioxidants capable of neutralizing free radicals are effective in preventing experimentally induced diabetes in animal models (WHO, Bjork, et al., 2003) as well as reducing the severity of diabetic complications (Mather, et al., 1998). For the development of diabetic complications, the abnormalities produced in lipids and proteins are the major etiologic factors. In diabetic patients, extracellular and long lived proteins, such as elastin, laminin, collagen are the major targets of free radicals. These proteins are modified to form glycoproteins due to hyperglycemia. (Dixon, et al., 2006). During diabetes, lipoproteins are oxidized by free radicals. There are also multiple abnormalities of lipoprotein metabolism in very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) in diabetes. Lipid peroxidation is enhanced due to increased oxidative stress in diabetic condition. Apart from this, advanced glycation end products (AGEs) are formed by non-enzymatic glycosylation of proteins. AGEs tend to accumulate on long-lived molecules in tissues and generate abnormalities in cell and tissue functions (Chandie, 2006; Retnakaran, et al., 2006). To manage post-prandial hyperglycaemia at digestive level, glucosidase inhibitors such as Acarbose, miglitol and voglibose are used. Although several therapies are in use for treatment, there are certain limitations due to high cost and side effects such as development of hypoglycemia, weight gain, gastrointestinal disturbances, liver toxicity etc (Raji, et al., 2001). Based on recent advances and the involvement of oxidative stress in complicating diabetes mellitus, efforts are on to find suitable antidiabetic and antioxidant therapy. To date, over 400 traditional plant treatments for diabetes have been reported, although only a small number of these have

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 8 | August 2013 | 554–561

received scientific and medical evaluation to assess their efficacy. The hypoglycemic effect of some herbal extracts has been confirmed in human and animal models of type 2 diabetes. The World Health Organization Expert Committee on diabetes has recommended that traditional medicinal herbs be further investigated. (Lucy Dey et al., 2002). Major hindrance in amalgamation of herbal medicine in modern medical practices is the lack of scientific and clinical data proving their efficacy and safety. There is a need for conducting clinical research in herbal drugs, developing simple bioassays for biological standardization, pharmacological and toxicological evaluation, and developing various animal models for toxicity and safety evaluation. It is also important to establish the active component/s from these plant extracts. It has been shown that the activity of the HPA (human pancreatic α-amylase) in the small intestine correlates to an increase in postprandial glucose levels, the control of which is therefore an important aspect in the treatment of type 2 diabetes. Herbal medicines are getting more importance in the treatment of diabetes as they are free from side effects and less expensive when compared to synthetic hypoglycemic agents. In India, indigenous herbal remedies such as Ayurveda and other Indian traditional medicine have, since ancient times used plants in treatment of diabetes Ethnobotanical studies of traditional herbal remedies used for diabetes have identified more than 1,200 species of plants with hypoglycemic activity. A number of medicinal plants and their formulations are used for treating diabetes in the traditional Indian Ayurvedic system as well as in ethnomedicinal practices. WHO (World Health Organization) (1980) has recommended the evaluation and mechanistic properties of the plants effective in such systems. Pharmacological properties α-glucosidase inhibitors such as acarbose that can also inhibit pancreatic α-amylase revealed that the complications of DM such as onset of renal, retinal, lens and neurological changes and the

development of ischaemic myocardial lesions are prevented or delayed. Long-term day-today management of diabetes, with acarbose is well tolerated and can improve glycaemic control as monotherapy, as well as in combination therapy Gymnema sylvestre, Stevia rebaudiana, Phyllanthus emblica and syzygium cumini are well known in Ayurveda to possess anti-diabetic properties. Structurally as well as mechanistically, PPA (Porcine pancreatic α-amylase) is closely related to the HPA (Human pancreatic α-amylase). Hence, sequential solvent extracts of the above mentioned plants were screened for the presence of PPA inhibitors, Pancreatic αamylase (E.C. 3.2.1.1), is a key enzyme in the digestive system and catalyses the initial step in hydrolysis of starch to maltose and finally to glucose. Degradation of this dietary starch proceeds rapidly and leads to elevated post prandial hyperglycemia (PPHG). It has been shown that activity of Human Pancreatic αamylase (HPA) in the small intestine correlates to an increase in post-prandial glucose levels, the control of which is therefore an important aspect of treatment of diabetes .Hence retardation of starch digestion by inhibition of enzymes such as α-amylase would play a key role in the control of diabetes. However, the discovery of specific high-affinity inhibitors of pancreatic α-amylase for the development of therapeutics has remained elusive. Inhibitors currently in clinical, as for example, acarbose, miglitol, and voglibose, are known to inhibit a wide range of glycosidases such as αglucosidase and α-amylase. Because of their nonspecificity in targeting different glycosidases, these hypoglycemic agents have their limitations and are known to produce serious side effects (Sebedio, et al., 1989). An effort has been made to study the inhibitory effect of the above said medicinal plants extract on PPA. MATERIALS AND METHODS Starch, porcine pancreatic α-amylase (PPA), methanol, isopropanol, acetone, methylbutyl-tertiary ether, cyclohexane, and dimethylsulfoxide (DMSO) were purchased

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from SRL Pvt. Ltd, Mumbai, India. 3,5dinitrosalicylicacid (DNSA) was obtained from HiMedia Laboratories, Mumbai, India. Human pancreatic α-amylase (HPA) and acarbose were purchased from Sigma Aldrich, USA. All other chemicals procured were of AR grade. Preparation of Plant Extracts The air-dried plant material (60–100 g) was crushed with liquid nitrogen, powdered, and successively extracted in polar to nonpolar solvent on an increasing degree of nonpolarity.The different extracts obtained sequentially were with cold water, hot water, methanol, isopropanol, acetone, methyl-butyltertiary ether, and cyclohexane. This kind of sequential extraction was performed taking into consideration the fact that traditional methods of preparing herbal formulations are mainly aqueous. Also, aqueous extracts contain peptides, proteins, or glycans, which would otherwise be denatured by organic solvents and high-temperature extraction. Distilled water was added to the crushed material in a ratio of 1 : 4 (w/v) and kept at 30°C (24 h) and 55°C (2 h) at 130 RPM for cold-and hot-water extracts, respectively. For each solvent, the extract was filtered, centrifuged, and the residue collected for subsequent solvent extraction. The organic solvents were added in a ratio of 1 :3 (w/v) and refluxed with the residue for 3 h at their respective boiling temperatures. Each extract was filtered and stored at −20°C

mL DNSA reagent, placed in boiling water bath for 5 min, cooled to room temperature, diluted, and the absorbance measured at 540 nm. The control reaction representing 100% enzyme activity did not contain any plant extract. To eliminate the absorbance produced by plant extract, appropriate extract controls were also included. One unit of enzyme activity is defined as the amount of enzyme required to release one micromole of maltose from starch per min under the assay conditions. For the determination of the inhibitor concentration at which 50% inhibition of enzyme activity occurs (IC50), the assay was performed as above except that the inhibitor/plant extract concentrations were varied from 0.1–150 μg. Acarbose was used as a positive control in a concentration range of 6.5 μg–32.8 μg. The IC50 values were determined from plots of percent inhibition versus log inhibitor concentration and calculated by logarithmic regression analysis from the mean inhibitory values. The IC50 values were defined as the concentration of the extract, containing the α-amylase inhibitor that inhibited 50% of the PPA. % Relative enzyme activity = (enzyme activity of test/enzyme activity of control) 100; % inhibition in the α-amylase activity = (100 − % relative enzyme activity). RESULTS

α-Amylase Inhibition PPA was used for preliminary screening of α-amylase inhibitors from the extracts. The inhibition assay was performed using the chromogenic DNSA method (28). The total assay mixture composed of 500 μL of 0.02 M sodium phosphate buffer (pH 6.9 containing 6 mM sodium chloride), 0.04 units of PPA solution, and extracts at concentration from 0.1–1.5 mg mL−1 (w/v) were incubated at 37°C for 10 min. After pre-incubation, 500 μL of 1% (v/v) starch solution in the above buffer was added to each tube and incubated at 37°C for 15 min. The reaction was terminated with 1.0

Screening of Plant Extracts for α-Amylase Inhibition The plants from the Indian subcontinent exhibiting potential hypoglycemic properties were sequentially extracted with polar to nonpolar solvents. It should be noted that while generally cold-and hot-water extracts are most commonly used in the traditional method of preparing medicines in Ayurveda, the chances of missing out on bioactive principles with better amylase inhibitory potential from lesspolar solvents are high. Hence the rationale for performing extractions from polar to non-polar

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solvents is not only to confirm and validate the inhibitory activity, if found, in any of the aqueous extractions performed in the traditional manner but also to search for newer and higher specific affinity inhibitors in less-polar solvents. PPA was used as a target enzyme for screening of inhibitory activity from the above mentioned plant extracts. The control reaction representing 100% enzyme activity was 0.20 U/mL for PPA. Extract samples dissolved in DMSO contained as a final yield of 1.5 mg mL−1 of the dried extract and the enzyme activity of PPA was not affected by DMSO at

the concentration used. The appropriately diluted plant extract was used for enzyme inhibition assay and the activity obtained with each extract was normalized to percent relative activity (figure-1) from which the percent inhibition was calculated.. The most significant inhibitory activity was obtained with the aqueous and ethanolic extracts of, Gymnema sylvestre and S. cumini the known PPA inhibitor, acarbose, taken as a positive control exhibited an IC50 value of 82.78 μgml (Table1).

Table-1: IC-50 OF ACARBOSE Standard drug

Concentrations (ug/ml)

% inhibition

Acarbose

100

59.53

48.56

39.63

28.34

22.11

19.10

Ic50 value

82.78

Figure-1: The percent relative enzyme activity (RA %) of hot water extracts on PPA

74 72 70 68

RA%

66 64 62 60 58 56 54 Stevia rebaudiana,

Gymnema sylvestre

syzimum cumini,

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Phyllanthus emblica

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DISCUSSION The aim of the present study was to investigate the PPA inhibitory activity of medicinal plants known in the Indian Ayurvedic system for their anti-diabetic properties. To date no reports of compounds responsible for PPA inhibition from these plants exist in the literature and we report here for the first time their inhibitory activity on PPA. These plants chosen in this study are used by the Indian population not only for food purposes but also form a part of the local pharmacopoeia for the treatment of diabetes. Our results indicate that retardation of starch hydrolysis by inhibition of PPA activity of some of these extracts leads to a reduction in glucose concentrations,Of the 4 plants and their extracts tested, Gymnema exhibited significant inhibition of PPA, suggesting that they contain compounds capable of PPA inhibition. Preliminary phytochemical analysis to indicate the kind of compounds present in these extracts suggests the occurrence of proteins/peptides and polyphenols in cold-and hot-water extracts while the tannins, alkaloids, flavonoids, and

saponins are found in non-polar extracts. Flavonoids and polyphenolics may be responsible for hypoglycemic activity. CONCLUSION Plants chosen in this study are used by the Indian population not only for food purposes but also form a part of the local pharmacopoeia for the treatment of diabetes. Our results indicate that retardation of starch hydrolysis by inhibition of PPA activity of some of these extracts leads to a reduction in glucose concentrations. Of the 4 plants and their extracts tested, Gymnema exhibited significant inhibition of PPA, suggesting that they contain compounds capable of PPA inhibition. Preliminary phytochemical analysis to indicate the kind of compounds present in these extracts suggests the occurrence of proteins/peptides and polyphenols in cold-and hot-water extracts while the tannins, alkaloids, flavonoids, and saponins are found in non-polar extracts. Flavonoids and polyphenolics may be responsible for hypoglycemic activity.

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AN, Raymond NT, Mughal S, (2006)Prevalence of microalbuminuria and hypertension inSouth Asians and white Europeans with type 2 diabetes: a report from the United Kingdom AsianDiabetes Study (UKADS). Diab Vasc Dis Res;3(1):22–25.

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Mather HM, Chaturvedi N, Kehely AM. Comparison of prevalence and risk factors for microalbuminuria in South Asians and Europeans with type 2 diabetes mellitus. Diabet Med. Aug1998;15(8):672–677. McKeigue PM, Shah B, Marmot MG. (1991) Relation of central obesity and insulin resistance with high diabetes prevalence and cardiovascular risk in South Asians. Lancet.; 337 (8738): 382–386. Mohan V, Deepa M, Deepa R, (2006) Secular trends in the prevalence of diabetes and impairedglucose tolerance in urban South India--the Chennai Urban Rural Epidemiology Study (CURES-17). Diabetologia.; 49 (6): 1175–1178. Mukhopadhyay B, Forouhi NG, Fisher BM, Kesson CM, Sattar N. (2006) A comparison of glycaemic andmetabolic control over time among South Asian and European patients with Type 2 diabetes:results from follow-up in a routine diabetes clinic. Diabetic Medicine. ;23(1):94–98. Narayan KM, Benjamin E, Gregg EW, Norris SL, Engelgau MM. (2004) Diabetes translation research: where are we and where do we want to be? Ann Intern Med.; 140 (11): 958–963. Pan XR, Li GW, Hu YH, (1997) Effects of diet and exercise in preventing NIDDM in people with impaired glucose tolerance. The Da Qing IGT and Diabetes Study. Diabetes Care.;20(4):537–544. Raji A, Seely EW, Arky RA, Simonson DC. (2001) Body fat distribution and insulin resistance in healthyAsian Indians and Caucasians. J Clin Endocrinol Metab. Nov ;86(11):5366–5371. Ramachandran A, Snehalatha C, Kapur A, Sep (2001) High prevalence of diabetes and impaired glucosetolerance in India: National Urban Diabetes Survey. Diabetologia.; 44 (9): 1094–1101.

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Ramachandran A, Snehalatha C, Mary S, Mukesh B, Bhaskar AD, Vijay V. (2006) The IndianDiabetes Prevention Program shows that lifestyle modification and metformin prevent type 2 diabetes in Asian Indian subjects with impaired glucose tolerance (IDPP1). Diabetologia; 49(2):289–297. Ramachandran A, Snehalatha C, Yamuna A, Mary S, Ping Z. (2007) Cost Effectiveness of the Interventions in the Primary Prevention of Diabetes among Asian Indians: Within trial results of the Indian Diabetes Prevention Program (IDPP). Diabetes Care.. Retnakaran R, Hanley AJ, Connelly PW, Sermer M, Zinman B. (2006) Ethnicity modifies the effect ofobesity on insulin resistance in pregnancy: a comparison of Asian, South Asian, and Caucasian women. J Clin Endocrinol Metab;91(1):93–97.

Source of Support: Nil

Sebedio JL, Grandgirard A (1989) Cyclic fatty acids: natural sources, formation during heat treatment, synthesis and biological properties. Progr Lipid Res, 28:303– 336 Snehalatha C, Viswanathan V, Ramachandran A. (2003) Cutoff values for normal anthropometric variables in asian Indian adults. Diabetes Care.;26(5):1380– 1384. The World Health Organization. Diabetes Facts and Figures: Level of Direct Worldwide, Countryand Regional Data. http://www.who.int/diabetes/en/. Yusuf S, Hawken S, Ounpuu S, (2004) Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet.; 364 (9438): 937–952.

Conflict of Interest: None Declared

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Research article STUDY ON SEED GERMINATION AND AGROTECHNIQUES OF CHEILOCOSTUS SPECIOSUS (J. KONIG) C. SPECHT IN MID-HILL OF WESTERN HIMALAYA

Gopichand1*, Singh R D2, Meena R L3 1, 2, 3

CSIR- Institute of Himalayan Bioresource Technology Palampur, Himachal Pradesh -176061, India *Corresponding Author: E-mail: gc57@rediffmail.com; Mobile: +919805537625

Received: 09/07/2013; Revised: 05/08/2013; Accepted: 08/08/2013

ABSTRACT Cheilocostus speciosus (J. Konig) C. Specht. (family: Zingiberaceae) is used in ayurvedic medicines. This plant is enlisted under endangered and threatened plant species in India. The seeds of C. specious were treated with growth hormones viz., indole acetic acid (IAA), indole butyric acid (IBA), naphthalene acetic acid (NAA), Salicylic acid and Tannic acid and were evaluated at 250, 500 and 750 mg/l concentration with distilled water as control treatment. Another field experiment was conducted to evaluate effect of plant spacing (25 × 50 cm2 and 50 × 50 cm2) and rate of farm-yard manure (FYM 0, 15, 30 and 45 t/ha) on crop growth and yield. The design of the experiment was Randomized Complete Block. The rate of seed germination was significantly influenced by the above mentioned chemical treatments. The highest seed germination (90.8 %) was recorded due to IBA 750 mg/l. IBA 500 mg/l, NAA 750 mg/l, Salicylic acid 750 mg/l, IAA 750 mg/l, and Tannic acid 750 mg were statistically comparable to IBA 750 mg/l in this regard. The crop-plant height and rhizome yield (15.33 t/ha dry rhizome) were significantly highest due to 45 t/ha FYM. The crop planted at 50 × 50 cm2 spacing, had significantly taller plants as compared to those planted at 25 × 50 cm2 spacing, however the crop yield was not affected by the plant spacing. KEYWORDS: Cheilocostus speciosus (J. Konig) C. Specht, growth hormones, agro techniques

Cite this article: Gopichand, Singh. R. D., Meena. R. L., (2013), STUDY ON SEED GERMINATION AND AGROTECHNIQUES OF CHEILOCOSTUS SPECIOSUS (J. KONIG) C. SPECHT IN MID-HILL OF WESTERN HIMALAYA, Global J Res. Med. Plants & Indigen. Med., Volume 2(8): 562–570

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 8 | August 2013 | 562–570

INTRODUCTION: Cheilocostus speciosus (J. Konig) C. Specht. (Family: Zingiberaceae) is commonly known as Kust or Keu-kand in Hindi and Kebuka in Sanskrit. Its rhizome is used in ayurvedic medicines. It is distributed throughout the country in moist tropical evergreen forests up to an altitude of 1200 m, and reported from Assam, Meghalaya, Bihar, Uttarakhand, Orissa, Madhya Pradesh, West Bengal, Himachal Pradesh, Punjab, Haryana, Rajasthan, and Gujarat (Anonymous, 2007; Gupta et al., 2008; Gupta, 2010; Srivastava et al., 2011). Pandey et al., (2011) have rationalized its agro-techniques and observed that its cultivation is possible up to 1500 m elevation. The botanical status of C. specious has been explained by Gamble (1987), Kirtikar and Basu (1987), and Sivarajan and Balachandran (1994). Cheilocostus speciosus is enlisted as an endangered and threatened plant species in India (Ayensu, 1986). Cheilocostus speciosus is an erect plant attaining about 2.5 to 3.0 m height. The stem is sub-woody at the base and root is tuberous. The leaves are large, 12–28 cm × 6.0–7.8 cm in average size, dorsally silky and sub-sessile, oblong and spirally arranged. Flowers are shell shaped, which open up during night (Moosad, 1983). The colour of flowers is white, clustered with dense spikes, short corolla tube and bracts are red in colour. A single stamen present is perfect lip large with incurved margins. The fruits of Cheilocostus are ovoid capsules with obovoid or sub-globose seeds (Khanna et al., 1977). Karthikeyan et al. (2012) have characterized bioactive compounds in C. specious. The roots and rhizomes of C. speciosus contain diosgenin, dioscin, sitosterol, prosapogenins A and B of diosceins, gracillin and quinines. Srivastava et al. (2011) have described its morphology, traditional uses, phyto-chemistry, and anti-diabetic, hypolipidemic, anti-bacterial, anti-fungal, anti-fertility, oestrogenic, anticarcinogenic, spasmolytic, anti-inflammatory and antipyretic properties. Verma and Khosa

(2012) have also studied its Pharmacognostic properties. In Ayurvedic system of medicine, rhizomes are considered bitter, astringent, acrid, cooling, purgative antihelmintic, aphrodisiac, depurative, febrifuge expectorant and tonic (Khanna et al., 1977). Cheilocostus has diversified uses in traditional medicines (Gupta, 2010; Bown Deni, 2008; Chopra et al., 2006; Khare, 2007; Nad Karni, 2009). It possesses anti-diabetic and hypolipidemic (Vishalakshi Devi, 2008, Bavarva et al., 2008. Eliza et al., 2009), anti-chilinesterase (Bhattacharya et al., 1972, Mishra et al., 2009), hepatoprotective (Mishra et al., 2009, Bhuyan and Zaman, 2008), antioxidant (Chakraborty, 2009), adaptogenic (Verma and Khosa, 2009) antibacterial (Malabadi, 2005), antifungal (Singh et al., 1992, Bandara, 1988), antifertility (Tiwari et al., 1973), oestrigenic (Singh et al., 1972, Tiwari et al., 1973, Rastogi et al., 2004), anticariogenic (Singh et al., 2008), spasmolytic (Banerji et al., 1982), anti-inflammatory and antipyretic (Binny et al., 2010) properties. Asolkar et al. (1992) have reported microbial activity in essential oil extracted from its rhizome. Its rhizomes are also used in curing dyspepsia, diabetes, edema, blood pressure (Chunekar, 1982 and Sharma, 1983). The rhizomes are also used for producing sexual hormones and contraceptives (Prakash and Mehrotra, 1996; Warrier et al., 1995). MATERIALS AND METHODS The studies were conducted at CSIRInstitute of Himalayan Bioresource Technology (IHBT), Palampur, Himachal Pradesh during 2006–2010. The soil of the experimental site was silty-clay loam in texture, acidic in reaction (pH 6.4), having high organic carbon (2.4 %), low available N (198 kg ha-1) medium in available P (24 kg ha-1), and high in available K (539 kg ha-1) content. The seeds of Cheilocostus specious were separated from fruits harvested from Biodiversity farm of CSIR-Institute of Himalayan Bioresource Technology (IHBT), Palampur in October 2006. After drying, the seeds were stored for about 4 months at room-

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temperature, before using for seed germination studies. Experiment of seed germination was conducted in the month of April 2007, at the experimental farm of CSIR-IHBT. Growth hormones viz., indole acetic acid (IAA), indole butyric acid (IBA), naphthalene acetic acid (NAA), Salicylic acid and Tannic acid were evaluated at 250, 500 and 750 mg/l concentration with distilled water as control treatment. Total 300 seeds were treated by each treatment, and replicated thrice. The seeds were dipped for 15 minutes in solution of each of the above mentioned treatments and then sown in field. The seed-beds were covered by agronet permitting 50 % light during initial 15 days after sowing (DAS). The rate of seed germination was recorded at weekly interval from 45 DAS (Table 1). Another field experiment was initiated on February 28, 2008 to evaluate effect of plant spacing (25 × 50 and 50 × 50 cm2) and rate of farm-yard manure (FYM 0, 15, 30 and 45 t/ha) on crop growth and yield. The design of the experiment was Randomized Complete Block. The unit plot size was 4 × 4 m2. The average weight of the rhizomes used for planting was 25 g, length 9.5 cm and diameter 38.2 mm. The crop plants started emerging in March 2008. The crop-plant height was recorded from 4 days after crop-emergence (DAE) to 60 DAE (Table 2). Observations on number of leaves per plant were also recorded at weekly interval from 18 to 60 DAE (Table 3). After 2 years from planting, the crop plants were uprooted and the rhizomes were separated from the plant to record fresh, and subsequently, dry weight of rhizomes for each treatment plot separately. RESULTS The findings of the above mentioned studies are as follows.

Seed germination: The rate of seed germination was significantly influenced by IAA, IBA, NAA, salicylic acid and Tannic acid in different concentrations (Table 1). It was observed that at 66, 73 and 80 DAS, the highest seed germination was recorded due to IBA 750 mg/l was 60.8, 82.0 and 90.8 %, respectively. At 80 DAT, IBA 500 mg/l, NAA 750 mg/l, Salicylic acid 750 mg/l, IAA 750 mg/l, and Tannic acid 750 mg were statistically comparable to IBA 750 mg/l in this regard (Table 1). Effect of FYM on crop growth: The plant height was significantly influenced by FYM application as evident from the data on all the dates of observation. It was significantly highest due to 45 t/ha FYM. The plant height in plots treated with 30 t/ha was at par to that due to 45 t/ha (Table 2). In terms of number of leaves per plant, 45 t/ha FYM was significantly superior than 15 t/ha FYM and untreated control (Table 3). FYM 30 t/ha was statistically comparable to FYM 45 t/ha in this regard on 53 and 60 DAE. Effect of plant spacing on crop growth: The crop planted at 50 × 50 cm2 had significantly taller plants as compared to those planted at 25 × 50 cm2 spacing, on all dates of observation (Table 2), however number of leaves per plant was not affected by the plant spacing (Table 3). Effect on crop yield: The yield of fresh as well as dry rhizomes showed significant increment with increase in the level of FYM from 0 to 45 t/ha (Fig. 1). FYM 45 t/ha had the highest rhizome yield (46.47 t/ha fresh and 15.33 t/ha dry rhizomes) followed by that due to FYM 30 t/ha. However, there was no significant effect of the plant spacing on the fresh and dry rhizome yield (Fig. 2).

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Table 1. Effect of growth hormones and chemicals on rate of germination of Cheilocostus seed Treatment IAA 250 mg/l IAA 500 mg/l IAA 750 mg/l IBA 250 mg/l IBA 500 mg/l IBA 750 mg/l NAA 250 mg/l NAA 500 mg/l NAA 750 mg/l Salicylic acid 250 mg/l Salicylic acid 500 mg/l Salicylic acid 750 mg/l Tannic acid 250 mg/l Tannic acid 500 mg/l Tannic acid 750 mg/l Control CD (P=0.05)

45 DAS 1.0 1.9 2.6 3.1 3.7 4.3 1.0 1.7 2.7 1.6 2.1 3.0 1.7 2.8 3.4 1.1 0.7

Germination (%) 59 DAS 66 DAS 27.9 43.6 29.4 45.3 35.0 58.9 35.6 55.8 40.7 59.6 45.6 60.8 29.3 45.2 31.1 48.7 34.9 57.2 24.8 42.0 30.1 45.2 36.6 54.4 24.6 35.8 25.8 40.3 36.2 42.7 22.8 34.7 1.2 1.8

52 DAS 5.6 6.4 7.1 6.8 8.3 9.7 6.2 7.6 8.7 5.0 7.4 9.0 6.3 6.7 8.4 3.9 0.7

73 DAS 67.0 69.4 74.7 68.8 74.0 82.0 68.4 71.0 79.3 71.2 76.3 77.8 68.2 70.6 74.7 66.8 2.8

80 DAS 83.3 86.1 87.2 83.1 87.2 90.8 81.6 87.0 90.1 83.7 85.8 89.4 81.3 86.9 87.4 74.7 3.7

Table 2. Effect of FYM level and plant spacing on plant height of Cheilocostus speciosus Treatment FYM (t/ha) 0 15 30 45 CD (P=0.05) Plant spacing (cm2) 25×50 50×50 CD (P=0.05)

46 DAE 74.01 84.72 89.22 93.67 5.56

53 DAE

60 DAE

29.07 36.18 46.19 51.56 9.00

Plant height (cm) 25 32 39 DAE DAE DAE 41.23 58.50 66.65 52.61 70.07 77.94 57.45 73.03 81.17 62.45 76.11 85.50 8.73 6.40 5.78

77.89 89.71 93.32 97.78 6.71

80.12 91.71 95.46 100.27 7.83

35.37 46.13 9.00

49.42 57.45 8.73

81.34 89.47 5.56

85.14 94.21 6.71

87.29 96.48 7.83

4 DAE 3.11 3.48 3.85 4.47 0.54

11 DAE 9.87 10.93 11.52 13.04 1.74

18 DAE

3.30 4.16 0.54

9.85 12.83 1.74

65.28 73.58 6.40

73.94 81.69 5.78

Table 3. Effect of FYM level and plant spacing on number of leaves per plant of C. speciosus Treatment FYM (t/ha) 0 15 30 45 CD (P=0.05) Plant spacing (cm2) 25 ×50 50 ×50 CD (P=0.05)

18 DAE 5.09 5.47 6.01 6.55 0.68

25 DAE 5.84 6.82 7.37 8.48 0.83

5.54 6.02 0.68

6.63 7.63 0.83

Leaves /plant (No.) 32 DAE 39 DAE 46 DAE 9.00 13.26 15.08 10.07 13.76 16.18 10.79 14.93 17.06 11.83 15.18 18.13 1.55 2.07 1.92 9.71 11.13 1.55

13.79 14.78 NS

16.29 16.94 NS

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53 DAE 17.04 17.79 18.37 19.45 1.68

60 DAE 17.04 17.94 18.41 19.53 1.55

17.73 18.60 NS

17.85 18.61 NS

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Fig. 1. Effect of farm yard manure (FYM) on yield of rhizome of Cheilocostus speciosus

(t/ha) Rhizome weight (t/ha)

Fresh rhizome weight

Dry rhizome weight

50 40.14 40

46.47

43.52

35.12

30 20

12.04

9.48

15.33

13.93

10 0 0

15 FYM (t/ha) 30

(t/ha) Rhizome weight (t/ha)

Fig. 2. Effect of plant spacing on yield of rhizome of Cheilocostus speciosus

50 45 40 35 30 25 20 15 10 5 0

40.34

Plant spacing 25 × 50 cm2 Plant spacing 50 × 50 cm2 42.29

12.39

Fresh rhizome

Dry rhizome

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DISCUSSION Observations on rate of seed germination reveal that the germination initiated at 45 DAS. The rate of seed germination showed an increasing trend with the concentration of the growth hormones and chemical used in this study. Maximum percentage of seed germination was recorded due to the treatment of IBA 750 mg/l (4.3 %) followed by that due to Tannic acid 750 mg/l. Similarly, at 80 DAS, significantly highest seed germination was observed due to IBA 750 mg/l (90.8 %), to which NAA 750 mg/l (90.1 %) and IBA 500 mg/l (87.2 %) were statistically comparable. The plant height, number of leaves per plant, fresh rhizome yield and dry rhizome yield showed significant increment with increase in the level of FYM application. However, plant spacing could influence only plant height. This could be attributed to better crop growth with increment in FYM level.

Yield of rhizomes of Cheilocostus was significantly higher (46.47 t/ha fresh and 15.33 t/ha dry rhizomes) with application of FYM 45 t/ha followed by that due to application of FYM 30 t/ha, which is in agreement with the findings of Pandey et al. (2011). Plant spacing could only influence the plant height and it had no effect on number of leaves per plant. Hence, there was no significant effect of plant spacing on rhizome yield. CONCLUSION The present study indicates that dipping Cheilocostus specious seeds in solution of IBA 750 or 500 mg/l or NAA 750 mg/l for 15 min enhances the rate of germination. For cultivation of C. specious, plant spacing of 50 × 50 cm2 and application of FYM 45 t/ha has been observed to enhance crop growth and yield of rhizome.

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Bhuyan B, Zaman K (2008). Evaluation of Hepatoprotactive activity of Rhizomes of Costus specious (J. Konig) Smith. Pharma cologyonline 3:119–126. Binny K, Kumar Sunil, Dennis Thomas G (2010). Antiinflammatory and Antipyretic properties of the rhizome of Costus Speciosus (Koen.) Sm. Journal of Basic and Clinical Pharmacy. 1(3): 177–181,

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Chakrabotry GS (2009). Free Radical Scavening Activity of Costus specious leaves. Indian journal of Pharmaceutical Education and Research 43(1): 96–98

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Karthikeyan J, Reka V and Raja V Giftson (2012). Characterisation of bioactive compounds in Costus specious (Keon). By reberse phase HPLC. International Journal of Pharmaceutical sciences and research. vol. 3(5): 1461–1465.

Chopra RN, Nayar SL, IC Chopra (2006). Glossary of Indian Medicinal Plants, National Institute of Science Communication and Information Resources. P. no. 79. Chunekar KC (1982). Bhavaprakashanighantu of Sri Bhavamishra. Commentary, Varanasi (in Hindi) CP Khare (2007). Indian medicinal plants: an illustrated Dictionary Published by, Springer-Verlaf; Berlin/Heidelberg. 181–182. Eliza J, Daisy P, Ignacimuthu S, Duraipandiyan V (2009). Antidiabetic and antilipidemic effect of eremanthin from Costus speciosus (Koen.)Sm., in STZinduced diabatic rats. ChemicoBiological Interactions, 182 (1) :67–72.

Khanna P, Sharma GL Rathore AL and Manot SK (1977). Effect of cholesterol on in vitro suspension tissue cultures of Costus specious(Keon.) Dioscorea floribunda, Solanum aviculare and solanum xanthcarpum. Ind. J. Exptl. Biol. 15:1025–1027. Kirtikar KR and Basu BD (1987). Indian Medicinal Plants. Book Distributors, Dehra Dun.p.2444–2449. Malabadi RB (2005). journal of Phytological Research, Antibacterial activity in the rhizome extracts of Costus speciosus (Koen.).18(1): 83-85. Mishra G, Sinha R, Verma N, Khosa RL, Garg VK, Singh P (2009). Hepatoprotective activity of alcoholic and aqueous extracts of Wedelia chinensis. Pharmacologyonline 1: 343–356.

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Moosad TCP (1983). AmarakosamCommentary. Vaidyasarathy Press, Kottyam. P 361. Nadkarni KM, Nadkarni AK (2007). Indian Materia Medica, Mumbai: Bombay Popular Prakashan, Pvt Ltd. Vol 1: 385–6. Okpuzorl J, Oloyede AM (2009). Antiinflammatory, Anti-pyretic and Antidiarrhoeal properties of an antihaemorrhoid tri-herbal pill. Nature and Science. 7(8): 89–94 Pandey Ankita, Gupta Somesh and Yadav K R (2011). Agro Techniques of Costus specious: An Important Endangered Medicinal Plant, National conference on Forest Biodiversity: Earth’s Living Treasure. 125–129. Prakash V, and Mehrotra BN (1996). Zingiberaceae of India’s Biological screening and ethnobotanical diversity: In proceeding of the Biological symposium on the family Zingiberaceae, South China Institute of Botany. 229–237. Rastogi RP, Mehrotra BN (2004). Compendium of Indian Medicinal plants, Central Drug Research Institute, Lucknow and National Institute of Science Communication and Information Resources, New Delhi. vol 2: 215.

Singh S, Sanyal AK, Bhattacharya SK, Pandey VB (1972). Estrogenic Activity of Saponins from Costus-Speciosus. The Indian Journal of Medical Research, 60(2): 287–90. Singh UP, Srivastava BP, Singh KP, Pandey VB (1992). Antifungal activity of steroid saonins and sapogenins from Avena sativa and Costrus specious.Naturalia (Sao Paulo). 17: 71– 77. Sivarajan VV and Balachandran I (1994). Ayurvedic drugs and their Plant Sources. Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi. P. 570. Srivastava Shruti, Singh Pardeep, Mishra Garima, Jain KK, Khosa RL (2011). Costus specious (Keukand): A review. DerPharmacia Sinica. 2 (1): 118–128. Tewari PV, Chaturvedi C, Pandey VB (1973). Experimental Study on Estrogenic Activity of Diosgenin Isolated from Costus-Speciosus. Indian Journal of Pharmacy 35: 35–6. Thomas Paul A, Devasagayam J (2007). Clin Biochem Nutr., Introduction to serial reviews: Recent advances in Indian herbal drug research. 40: 73.

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Verma N, RL Khosa (2009). Effect of Costus speciosus and Wedelia chinensis on brain neurotransmitlers and enzyme monoamine oxidase following cold immobilization stress Journal of Pharmaceutical Sciences and Research 1(2): 22–25.

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Vishalakshi Devi D, Urooj A (2008). Hypoglycaemic potential of iMorus indicaL and Costus igneus, Nak-A preliminary study.Indian journal of Experimental Biology. 46: 614â&#x20AC;&#x201C;616.

Source of Support: Nil

Warrier PK, Nambiar VPK and Ramankutty C eds (1993-95). Indian Medicinal Plants: A compendium of 500 species. Vols.15. Orient Longman, Madras-600 002.

Conflict of Interest: None Declared

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Research article WOUND HEALING ACTIVITY OF ETHANOLIC EXTRACT OF EUPHORBIA HIRTA LEAVES ON EXCISION WOUND MODEL IN RATS

Rathnakumar K1, Verma Ranbir 2, Jaikumar S3, Sengottuvelu S4* 1

Department of Ophthalmology, Sri Lakshminarayana Institute of Medical Sciences, Pondicherry, India Department of Pharmacology, Dreamz College of Pharmacy, Khilra, Sundernagar, Himachal Pradesh, India 3 Department of Pharmacology, Sri Lakshminarayana Institute of Medical Sciences, Puducherry, India 4 Department of Pharmacology, Nandha College of Pharmacy and Research Institute, Erode- 638052, Tamil Nadu, India *Corresponding Author: E-mail: sengt@rediffmail.com; Mobile: +919994426689 2

Received: 15/07/2013; Revised: 30/07/2013; Accepted: 05/08/2013

ABSTRACT The study was conducted to evaluate the wound healing effect of Euphorbia hirta leaves on excision wound model in Wistar albino rats. The ethanolic extract of Euphorbia hirta leaf was formulated as an ointment for topical application (5% and 10% W/W). The wound healing activity of the Euphorbia hirta leaf extract was studied by using excision wound, which was inflicted by cutting away 500 mm2 of the skin on the anterio-dorsal side of the rats under anesthesia. Povidone iodine ointment was used as reference standard for comparing the wound healing potential of Euphorbia hirta. The wound contraction was observed at different time intervals. Both the concentrations of Euphorbia hirta leaf extracts showed significant (P<0.001) wound contraction on excision wound model in rats. KEYWORDS: Euphorbia hirta, excision wound model and Wound healing.

Cite this article: Rathnakumar. K., Verma Ranbir, Jaikumar. S., Sengottuvelu. S., (2013), WOUND HEALING ACTIVITY OF ETHANOLIC EXTRACT OF EUPHORBIA HIRTA LEAVES ON EXCISION WOUND MODEL IN RATS, Global J Res. Med. Plants & Indigen. Med., Volume 2(8): 571–575

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 8 | August 2013 | 571–575

INTRODUCTION Euphorbia is a genus of plants belonging to the family Euphorbiaceae. Euphorbia hirta is a small annual, branched herb prostrate to ascending with branches reaching 70 cm in height, reddish or purplish in color, with abundant latex and is covered with short hairs. Its leaves are opposite, distichous, and simple; its obvious stipules are linear (Linfang Huang et al., 2012). The parts of Euphorbia hirta is traditionally used to treat various ailments like gastrointestinal disorders, asthma, bronchitis, coughs, colds, kidney stones, menstrual problems, ulcers, skin infections and venereal diseases. This plant is a very vital drug in Siddha & Ayurvedic system of medicine. It is termed as “Ammaan pacharisi” in tamil & “Dugdhika” in Sanskrit used in traditional medicines. The plant latex is used for curing corn in legs & it was used as a draught relief plant in olden tamil culture as it was cooked and eaten during draught or famines. In Ayurveda the plant is very well used in Asthmatic conditions. Ethno Medicinal Practices, Euphorbhia hirta has been used to induce the secretion of milk among the Binjhwar tribe of Chhattisgarh in India (Shukla Rajesh et al., 2013). Two flavonoids have been isolated from Euphorbia hirta, namely quercitrin and myricitrin (Johnson et al., 1999 & Chen., 1991). Sterols were isolated from Euphorbia hirta and chemically characterized as cycloarternol, 24methylene-cycloarternol, α-sitosterol, euphorbol hexacozonate, 1-hexacosanol, tinyaloxin, campesterol and stigmasterol (Atallah and Nicholas., 1972 & Galvez et al., 1993). Euphorbia hirta contains three hydrolysable tannins, namely, dimeric hydrolysable tannin, euphorbin E and the dimeric dehydroellagitannins, euphorbin A and euphorbin B (Yoshida et al., 1990). The triterpenes α -amyrin, taraxerone (EH-1), taxerol as well as α -amyrin acetate have been identified from Euphorbia hirta (Pinn., 2001 & Mukherjee et al., 2004). E. hirta has shown significant burn wound healing activity

(Jaiprakash et al., 2006). Anti cataract activity of Euphorbia hirta has been reported (Rathnakumar et al., 2013). The present study was conducted to evaluate wound healing effect of Euphorbia hirta on excision wound model in rats. MATERIALS & METHODS Chemicals and Reagents All chemicals and reagents used in the study were of analytical grade. Plant Material Aerial parts of Euphorbia hirta were collected from outskirts of Erode, Tamilnadu. Authentication was done by Prof. V. S. Kumar, Scientist (F) and Head of the Office, Tamilnadu Agriculture University, Coimbatore (Tamil Nadu, India). The voucher specimen (No.: BSI/ SRC/ 7/ 47/ 11- 12/ Tech. 221) was deposited in the herbarium for future references. Preparation of Extract (Vedha Vijaya et al., 2012) The aerial parts of E. hirta were washed with fresh water to remove adhering dirt and foreign particles. The plant was shade dried, crushed and grinded to get coarse powder. The coarse powder was then placed with 90% ethanolic solution in a round bottomed flask. 500 g of the coarse powder of the leaves of E. hirta in 1.0 liter of 90% ethanolic solution were macerated for 7 days. The mensturm was collected, concentrated by vacuum distillation and then air dried in an evaporating dish till constant weight was obtained. Animals Wistar albino rats of either sex weighing 150–200 g were used for this study. The animals were placed randomly and allocated to treatment groups in polypropylene cages with paddy husk as bedding. Animals were housed at a temperature of 24 ± 2oC and relative humidity of 30–70%. A12:12 light: day cycle

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was followed. All the animals were allowed free access to water and fed with standard commercial pelleted chaw (M/s.Hindustan Lever Ltd., Mumbai). All the experimental procedures and protocols used in this study were reviewed by (IAEC) Institutional Animal Ethics Committee (932/a/06/CPCSEA) of Sri Lakshminarayana Institute of Medical Sciences, Puducherry, India, and were in accordance with the guidelines of the IAEC. Excision wound model An excision wound was inflicted by cutting away approximately 500 mm2 full thickness of shaved skin at a predetermined area on the anterio-dorsal side of the alloxan induced diabetic rats under pentobarbitone (30 mg/kg., i.p) anesthesia. The entire wound was left open. Animals were closely observed for any infection and those which showed signs of infection were separated, excluded from the study and replaced. Experimental protocol (Udupa et al., 1994) Totally 30 animals were used in this study. The rats were divided into 4 groups of 6 animals each. Excision wound was inflicted in all the rats of 5 groups. Group I, animals were treated with simple ointment base. Group II and III, animals were treated with topical application of E. hirta extract (5% and 10% W/W) in simple ointment base respectively. Group IV, animals were treated with standard povidone iodine ointment. All the test drugs were applied topically on the wound, twice daily for 18 days. Wound contraction was monitored by metric measurement of the wound area once in 3 days upto 18th day. This was studied by tracing the raw wound area on a transparent polythene paper and the traced area was measured by using a graph paper. The wound contraction was measured as a percentage decrease of original wound size of 500 mm2 for each animal in all groups.

Statistical Analysis The values were expressed as mean ± SEM. The statistical analysis was carried out by one way analysis of variance (ANOVA) followed by Dunnet‟s „t‟ – test. P values < 0.05 were considered significant. RESULTS AND DISCUSSION The wound healing activity of Euphorbia hirta leaf extract was studied in excision wound model and the results are given in table 1. Wound contraction was observed for 18 days during the drug treatment. Topical application of E. hirta leaf extract (5% and 10%) promoted the contraction of wound in rats, when compared to normal control. Topical application of 10% Euphorbia hirta leaf extract showed significant (P < 0.05) wound th contraction on 4 day as compared to normal control animals. On 8th day observation both the concentrations (5% and 10%) of Euphorbia hirta leaf extracts and povidone iodine treated animals showed significant (P < 0.05) wound healing activity. On 12th day observation, 10% of E. hirta leaf extract and povidone iodine treated animals showed significant (P < 0.001) wound contraction but 5% of E. hirta leaf extract showed less significant (P < 0.01) wound contraction. On 16th and 18th day observations, both the concentrations of E. hirta leaf extracts and povidone iodine treated animals showed significant (P < 0.001) wound contraction as compared to control animals. Several medicinal plants are used traditionally for the treatment but there is the need for ascertain these claims scientifically. The current study was conducted to evaluate the wound healing activity of E. hirta leaf extract in excision wound model in rats. Plant extracts are used in treating wounds because of the effect of high levels of antioxidants (Houghton et al., 2005). The antioxidant and free radical scavenging activities of E. hirta had already reported (Sharma and Prasad., 2008). The free radical scavenging activity of the E. hirta on may be responsible for its wound healing potential in rats.

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Table 1: Effect of Euphorbia hirta leaf extract on excision wound model in albino rats.

0 day

4th day

Wound Contraction (mm2) 8th day 12th day 16th day

Normal Control

485.32 ± 21.1

446.33 ± 15.45

342.12 ± 11.24

196.54 ± 17.41

124.54 ± 11.44

98.66 ± 8.55

Euphorbia hirta (5%)

496.31 ± 15.44

432.58 ± 12.36

310.28 ± 8.65*

124.59 ± 5.66**

56.97 ± 3.57***

14.56 ± 166***

Euphorbia hirta (10%)

502.66 ± 19.65

426.58 ± 15.45*

296.32 ± 10.69*

98.25 ± 6.67***

25.84 ± 1.45***

6.97 ± 0.52***

Povidone Ointment

506.87 ± 17.45

448.36 ± 14.54

305.64 ± 11.32*

114.45 ± 7.42***

48.66 ± 1.55***

15.64 ± 1.12***

Drug Treatment

18th day

Values are presented as mean ± SEM (n = 6)*P < 0.05, **P < 0.01 and ***P < 0.001 Vs Normal Control

CONCLUSION From the results it was concluded that the topical application of ethanolic leaf extract of Euphorbia hirta showed significant wound healing activity on excision wound model in

rats. The wound healing activity of the E. hirta on excision wound model in rats may be due to its antioxidant property. Further phytochemical and pharmacological studies are required to isolate the active compounds responsible for wound healing activity.

REFERENCES Atallah AM, Nicholas HJ (1972). Triterpenoids and steroids of Euphorbia pilulifera. Phytochem. 2: 1860–1868. Chen L. Polyphenols from leaves of Euphorbia hirta (1991). Zhongguo Zhong Yao Za Zhi. 16(1): 38–39. Galvez J, Zarzuelo A, Crespo ME, Lorente MD, Ocete MA, Jimenez J (1993). Antidiarrhoeic activity of Euphorbia hirta extract and isolation of an active flavonoid constituent. Planta Medica. 59(4): 333–336. Houghton PJ, Hylands PJ, Mensah AY, Hensel A, Deters M (2005). In vitro tests and ethnopharmacological investigations: wound healing as an example. J. Ethnopharmacol. 100: 100–107.

Jaiprakash B, Chandramohan, Reddy DN (2006). Burn wound healing activity of Euphorbia hirta. Ancient Science of Life. 15 (3): 01–03. Johnson PB, Abdurahman EM, Tiam EA, Abdu-Aguye I, Hussaini IM (1999). Euphorbia hirta leaf extracts increase urine output and electrolytes in rats. J. Ethnopharmacol. 65: 63–69. Linfang Huang, Shilin Chen, Meihua Yang (2012). Euphorbia hirta (Feiyangcao): A Review on its Ethnopharmacology, Phytochemistry and Pharmacology. J. Med. Plants Res. 6(39): 5176–5185.

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Mukherjee KS, Mukhopadhyay B, Mondal S, Gorai D, Brahmachari G (2004). Triterpenoid Constituents of Borreria articularis. J. Chin. Chem. Soc. 51(1): 229–231.

Shukla Rajesh, Chakravarty Moyna, Goutam MP (2013). Ethno Medicinal Practices Among the Binjhwar Tribe of Chhattisgarh, India. Global J Res. Med. Plants & Indigen. Med. 2(7): 525–531.

Pinn G. Herbal therapy in respiratory diseases (2001). Aust. Fam. Phy. 30 (8): 775– 779.

Udupa SL, Udupa AL, Kulkarni DR (1994). Studies on the anti-inflammatory and wound healing properties of Moringa oleifera and Aegle marmelos. Fitoterap. 65: 119–123.

Rathnakumar K, Jaikumar S, Sengottuvelu S (2013). Effect of Euphorbia hirta in Napthalene Induced Cataract in Rats, Research J. Pharm. and Tech. 6(8): 908–911. Sharma NK, Prasad R (2008). Oxidative injury to protein and their protection by phenolic acid antioxidants from Euphorbia hirta leaves Abstracts. J. Biotech. 136: 717–742.

Source of Support: Nil

Vedha Vijaya T, Srinivasan D, Sengottuvelu S (2012). Wound healing potential of Melia azedarach L. Leaves in alloxan induced diabetic rats. Global J Res. Med. Plants & Indigen. Med. 1(7): 265– 271. Yoshida T, Namba O, Chen L, Okuda T (1990). Euphorbin E: A Hydrolysable tannin dimer of highly oxidized structure from Euphorbia hirta. Chem. Pharm. Bull. 38: 1113–1115.

Conflict of Interest: None Declared

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Research article MICRO-MORPHOLOGICAL AND MICRO-METRIC EVALUATION OF MANGIFERA INDICA LINN. – LEAF Anupriya1*, Harisha C R2, Patgiri B J3, Prajapati P K4 1

PG Scholar, Dept. of RS & BK, IPGT&RA, Gujarat Ayurveda University, Jamnagar. Gujarat, India. Head, Dept. of Pharmacognosy, IPGT&RA, GAU, Jamnagar.Gujarat, India. 3 Reader, Dept. of RS & BK, IPGT&RA, Gujarat Ayurveda University, Jamnagar.Gujarat, India. 4 Professor and HOD, Dept. of RS & BK, IPGT&RA, Gujarat Ayurveda University, Jamnagar. Gujarat, India. *Corresponding author:E-mail: dranupriya10@gmail.com, Mob:+919537505503. 2

Received: 15/04/2013; Revised: 15/06/2013; Accepted: 25/07/2013

ABSTRACT Leaves and fruits of Mangifera indica Linn. are widely used in traditional Ayurvedic preparations like Pushyanuga Churna, Amrapanaka, Khandabhrakam, Vridhagangadhara etc. Till date there are no pharmacognostical scientific work done on its leaf. The present study was planned to do the micro-morphological and micrometeric study on the leaf of Mangifera indica Linn. Histochemical tests were done by using thick sections to observe the presence of tannins, lignin, starch etc. Both upper and lower epidermis was used for the surface study through hand peeling method. Micrometric readings of both surface i.e. Stomatal length, stomatal index etc. were scientifically studied and the mean value was taken in to consideration. Leaf powder was used for micromorphological analysis. Histo-chemical test were done by using thick sections to observe the presence of tannins, lignin, starch etc. Results showed stone cells and starch grains in petiole, 2-3 layer of pallisade parenchyma, pitted vessels in leaf, stomatal index i.e. 26 in lower epidermis. Powder microscopy of shade dried powder showed prismatic crystals and oil globules. The information generated in this study will be helpful for the proper identification and authentication of leaf of this herb.

Cite this article: Anupriya, Harisha. C.R., Patgiri B.J,, Prajapati P.K., (2013), MICRO-MORPHOLOGICAL AND MICRO-METRIC EVALUATION OF MANGIFERA INDICA LINN. – LEAF, Global J Res. Med. Plants & Indigen. Med., Volume 2(8): 576–581

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KEY WORDS: Amra, histo-chemical test, micrometry, stomata, surface study. INTRODUCTION Mangifera indica belongs to the family Anacardiaceae. It is commonly known as mango or Amra. Its stem bark, leaf, fruit both ripened and unripened, seed etc. are widely used in traditional ayurvedic preparations e.g. Pushyanuga churna, Amrapanaka, Khandabhrakam, Vridhagangadhara churna, Jatiphala rasa, Jambavadi kvatha, Mehamihira taila, Ashokarishta, Madhukadyavaleha, Chandanadi churna (Sharma PC, 2001). Decoction of mango and Jamun leaves is used with honey to treat vomiting. In bleeding from nose, mango seed juice is given as Nasya (Bapa LG, 2007). It occurs wild or semi-wild nearly throughout India in tropical and sub-tropical hilly forests, particularly near nullahs and ravines. It is common in sub-tropical Himalayas, Chota Nagpur, Bihar, Orissa, West Bengal, Assam, hill forests of Eastern and Western Ghats, Deccan and Andaman Islands, cultivated throughout the tropics of both hemispheres. Native of Indo-Burma region (Saxena HO and Brahmam M, 1994). A large evergreen tree reaches up to 10–45 m high. Leaves are simple, linear - oblong or elliptic lanceolate, 10–30 cm long. Flowers are small, reddish white or yellowish green, in large terminal panicles. Fruits are variable in form and size, green, yellowish or red fleshy. Seeds solitary, ovoid - oblique, encased in a hard fibrous endocarp (Saxena HO and Brahmam M, 1994). Its root, bark, leaf, flower, fruit, seed kernel are considered medicinal. Despite its known numerous uses in traditional literature, it is not explored much in scientific world. No study has been found on microscopic study of its leaf. Hence, micro-morphological and micro-metric evaluation study of leaf is explored in this work. MATERIAL AND METHODS Pharmacognostical evaluation Fresh leaves of Amra (along with inflorescence) were collected in month of March from botanical garden of Institute of

Pharmaceutical Sciences, Jamnagar. Collected leaves were identified and authenticated in pharmacognosy lab, I.P.G.T & R.A. Pharmacognostical evaluation of fresh drug was carried out by taking free hand sections rest of the leaves were preserved for further studies Phm. 6048/12–13 (Wallis TE, 1985). Both upper and lower epidermis was used for the surface study through hand peeling method. Micrometric readings of both surface i.e. Stomatal length, stomatal index etc. were scientifically studied and mean value was taken in to consideration (Evans WC, 2009). Histochemical tests were done by using thick sections to observe the presence of tannins, lignin, starch etc. (Krishnamurty KV, 1988). Powder microscopy of shade dried powder was carried out with and without stain. Photomicrographs were taken using Carl Zeiss trinocular microscope attached with a camera. Botanical identification was carried out by using various floras (Gamble JS, 1967). RESULTS AND DISCUSSION Macroscopic Leaf is simple, linear-oblong or elliptic to lanceolate having dimension of 30  7.5 cm and exstipulate. Petiole is was pulvinated and slightly twisted with slightly wavy margin, acute tip. Young leaves are coppery in colour, coriacious, have reticulate venations with 20– 30 pairs of veins. Upper part of the leaf was dark green when compared to the lower which was parrot green in colour [Figure 1.1–1.3]. Microscopic Transverse section of petiole The transverse section of the petiole showed outer single layered compactly arranged barrel shaped epidermal cells covered with cuticle followed by 2–3 layers of hypodermis made up of collenchymas cells. Cortex was widely distributed, made up of parenchyma cells, large number of prismatic crystals of calcium oxalate, oil globules were

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present. A ring of stone cells in the outer area was followed by arch shaped pericyclic fibres and were lignified. Many lysogenous cavities were distributed all over the cortical region. Vascular bundles were circularly arranged all over the ground tissue. Vascular bundles consisted of one large vascular bundle in the midrib and numerous small vascular bundles in the mesophyll. Each bundle was conjoint, collateral and closed. This was surrounded by a

parenchymatous bundle sheath. Bundle sheath was externally covered with thick lignified parenchyma cells. Vascular bundles were radially arranged with metaxylem towards periphery and protoxylem towards pith. Xylem consisted of xylem parenchyma and its fibres. Phloem was present above the xylem with some sieve elements. Central large pith had parenchymatous cells and was heavily loaded by simple starch grains [Figure 1.4â&#x20AC;&#x201C;1.6].

Figure 1: Macro & Micro photographs of Mangifera indica Linn.

1.1 Natural habitat

1.4 TS of petioleUnstained

1.7 TS Through MidribUnstained

1.2 Branchlet with leaves

1.5 TS of petiole- stained

1.8 TS Through Midrib-Stained

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1.3 Individual leaf

1.6 Starch grains- Iodine stained

1.9 Mesophyll

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Figure 2: Microphotographs of Mangifera indica Linn.

2.1 Rosette & Prismatic crystals

2.2 Paracytic stomata

2.3 Oil globules

2.4 Prismatic crystals

2.5 Oil globules

2.6 Lignified fibres

2.7 Wavy parenchyma cells

2.8 Scleroids

2.9 Pitted vessels

Transverse section of leaf through mid rib Upper and lower epidermis was single layered and compactly arranged in barrel shaped cells. Upper epidermis lacked stomata, lower epidermis had stomata. Mesophyll was differentiated into palisade and spongy parenchyma. Palisade parenchyma was below the upper epidermis in one to two layers, the cells were compactly arranged in long and tubular shape. Chloroplasts were present. Spongy parenchyma formed rest of the tissue. Number of prismatic and rosette crystals of

calcium oxalate were found distributed all over the mesophyll tissue. The cells were small, varied in shapes and sizes, loosely arranged and enclosed small air spaces. A few air spaces lead to the stomatal openings which formed substomatal cavity. Vascular bundles consisted of large vascular bundle in the midrib and numerous small vascular bundles in the wings. Each bundle was conjoint, collateral and closed surrounded by parenchymatous bundle sheath. Bundle sheath surrounded by 3â&#x20AC;&#x201C;4 layers of thick lignified pericyclic fibres. Numbers of lysogenous cavities were present in the

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parenchymatous region. Larger vascular bundle had an extensive bundle sheath that extends both towards lower and upper epidermis. Metaxylem was situated towards the lower epidermis and protoxylem towards upper epidermis with xylem fibres and parenchyma. Phloem was situated below the xylem with some phloem fibres [Figure 1.7–1. 9].

Organolrptic characters showed leaf powder was dark green in colour, astringent in taste, aromatic in odour and coarse in touch. Diagnostic powder microscopic characters were oil globules, prismatic and rosette crystals of calcium oxalate, paracytic stomata. Epidermal cells, fragments of palisade cells, fragments of spiral and annular vessels, lignified fibres and lignified parenchyma cells [Figure 2.4–2.9].

Surface preparation Thin upper and lower surface were prepared by simple peeling method both the epidermis showed numerous stomata, stomata mainly of paracytic, epidermal cells, prismatic and rosette crystals of calcium oxalate and large quantities of oil distributed all over the surface [Figure 2.1–2.3].

Micrometric analysis Stomatal length, breadth, stomatal index and other constituents were scientifically measured (3 successive readings) the mean value was taken into consideration and results are shown in the Table 1.

Powder microscopy

Table 1: Micrometeric analysis of Mangifera indica Linn. leaf Sr. No. Characters

Lower epidermis Upper epidermis

Stomata length

10.25 µm

9.75 µm

Stomata breadth

7.5 µm

7.3 µm

Stamata radius

85 µm2

80 µm2

Stamatal Index

Pallisade ratio

2-4

Nil

Vein islets no.

18-20

---

Epidermal cells

205 µm2

Prismatic crystal

35 µm2

32 µm2

Starch grains (Petiole) 28 µm2

---

*μm- micrometer Table 2: Histochemical tests for Mangifera indica Linn. leaf Sr. No.

Reagents

Observation

Characteristics

Phloroglucinol+ Conc. HCl Red

Lignified cells

Iodine

Starch

Blue (Petiole)

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Phloroglucinol+ Conc. HCl Dissolved

Fecl3 solution

Dark blue to black Tannin cells

Sudan III

Red

Histo-chemical test of Mangifera indica Linn. Various Histochemical tests were conducted on sections of the Mangifera indica Linn. leaf. The results are depicted in Table 2.

Calcium oxalate crystals

Oil globules

CONCLUSION Pharmacognostical evaluation and the values extracted from micrometry of Mangifera indica Linn. were scientific and constant and may be considered for further research works.

REFERENCES Bapa LG. (2007), Nighantu Adarsha (Reprint edn), Chaukhamba Bharati Academy, Varanasi, Vol 1, pp 329–36.

Krishnamurty KV. (1988), Methods in the plant histochemistry, Vishwanandan Pvt. Limited, Madras, pp 1–7.

Evans WC. (2009), Cell differentiation and ergastic cell contents, Techniques in microscopy in Trease and Evans Pharmacognosy (16th edn), Saunders Elsevier, New York, pp 51–70.

Sharma PC, Yelne MB, Dennis TJ. (2001), Database on Medicinal plants used in Ayurveda (e book), CCRAS, Part 2.

Gamble JS. (1967), The flora of Presidency of Madras, Adlard and sons publishers, London, Vol 1, pp 1353.

Source of Support: Nil

Saxena HO, Brahmam M. (1994), The flora of Orissa, Orissa Forest Development Corporation Ltd., Bhubaneshwar, Vol 1, pp 362. Wallis

TE. (1985), Textbook of th pharmacognosy (15 edn), Churchill publications, London, pp 572–82.

Conflict of Interest: None Declared

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Research article DETAILED PHARMACOGNOSTICAL EVALUATION ON RHIZOME OF TECTARIA COADUNATA (Wall. Ex Hook & Grev.) C. Chr. - A FOLKLORE HERB Mori Hardik1*, Nishteswar K2, Patel B R3, Harisha C R4 1,

M.D. Scholar, Department of Dravyaguna, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India. 2 Professor & Head, Department of Dravyaguna, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India. 3 Assistant professor, Department of Dravyaguna, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India. 4 Head, Pharmacognosy, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India. *Corresponding Author: Email: Hardeik12gir@gmail.com

Received: 08/06/2013; Revised: 25/07/2013; Accepted: 30/07/2013

ABSTRACT Tectaria coadunata (Wall. Ex Hook & Grev.) C. Chr (Kukkutnakhi) is used by many Vaidyas in hyperlipidemic conditions and obesity. Also it is used in vegetable curry or salad. Till date, a detailed pharmacognostical study on its rhizome is not available. Hence, the present study was carried out to document, morphological, microscopical and histochemical features of the rhizome, following standard procedures. Macroscopic studies showed the rhizome of 10–20 cm in length and 3 cm in diameter, short creeping, up to 3 cm thick, densely scaly at the apex. Rhizome showed the presence of cuticle, parenchyma, meristele and vascular bundles. Powder microscopy of the dried rhizomes showed epidermal cells with tannin, starch grains, fragments of ramenta, and prismatic crystals. KEY WORDS: Tectaria coadunata, Kukkutnakhi, microscopy, starch grains.

Cite this article: Mori Hardik, Nishteswar K, Patel B R, Harisha C R (2013), DETAILED PHARMACOGNOSTICAL EVALUATION ON RHIZOME OF TECTARIA COADUNATA (Wall. Ex Hook & Grev.) C. Chr. - A FOLKLORE HERB, Global J Res. Med. Plants & Indigen. Med., Volume 2(8): 582–588

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INTRODUCTION Tribal as well as folk sources were utilized by Acharyas of Ayurveda while documenting Ayurvedic materia medica. Acharya Sushruta suggests, collecting the information about the drugs from cowhards, sages, hunters and other forest dwellers (Dalhanacharya, 2009). Several folklore claims are being subjected to scientific evaluation, The present drug Tectaria coadunata (Dryopteridaceae), commonly known as “Kukkutnakhi” is being used by certain folks of Maharashtra for the management of Aamvata (Rheumatic arthritis), Granthi (Cyst), Arbuda (Tumour) and other inflammatory diseases is being marketed under the name of Kukkutnakhiguggulu (Gokhale B V, 1989). It is a pteridophyte fern. Rhizome of T. coadunata was short creeping, densely scaly at the apex; apex was acuminate. Stipes were scattered. Lamina ovate or broadly ovate, about 30×40 cm; base cordate, bipinnate or bipinnatifid. Primary pinnae was opposite to subopposite or alternate, about 8 cm apart, slightly ascending; secondary pinnae was mostly adnate and decurrent, about five pairs, alternate about 2 cm apart. Multicellular hairs densely distributed all over the lamina except the abaxial side of the main rachis. Sori on the end of the veinlets arranged in two rows along the costules of the secondary pinnae or lobes of secondary pinnae, about 1.5 mm in diameter, compital; spores are reniform or planoconvex or spherical, pale brown with broad anasomosed winged perispore. (Manickam V S and Irudayaraj V, 1992). Young fronds are used as vegetable curry or as salad; the extract or decoction of fresh rhizomes is used for preventing diarrhea (Jain S K, 1991). The present study was hence undertaken to establish certain botanical standards for the identification of T. coadunata rhizome on which no much exploration on pharmacognostical grounds have been done. Synonyms: (Manickam V S and V Irudayaraj, 1992) - Aspidium cicutarium (L.) Sw - Sagenia macrodonta Fee - Aspidium coadunatum Hook. & Grew.

Sagenia coadunata J. Sm. Tectaria macrodonta (Fee) C. Chr. Tectaria viridifrons Ching Nephrodium cicutarium sensu Hook.

Preliminary research studies showed free radical scavenging activity of T. coadunata rhizome. Preliminary phytochemical screening showed the presence of phenols, tannins, flavonoids and sterols. Total phenolics and total tannin content was found to be 17.35% (w/w) and 7.98% (w/w) respectively. Methanolic extract of T. coadunata rhizome showed concentration dependent free radical scavenging activity comparable to that of the positive controls pyrogallol and ascorbic acid in the respective models (Ghoghari A M et al., 2006). MATERIALS AND METHODS Collection and preservation of the sample: Rhizomes of T. coadunata were collected personally from its natural habitat, Ambaghat, Kolhapur (Maharashtra), during December 2012 and were identified with the help of Taxonomists (Prof. S. R. Yadav & Sachin Patil, Dept of Botany, Shivaji University, Kolhapur) and texts Pteridophyte flora of Western GhatsSouth India (Manickam V S and Irudayaraj V, 1992). A sample specimen was deposited in Pharmacognosy lab, IPGT&RA, Jamnagar Specimen No- PHM 6067/2/01/2013 for future references. The rhizomes were washed, shade dried, powdered, sieved through 80 # and preserved in an air-tight glass vessel. For microscopical evaluation, fresh sample was preserved in a solution prepared from 70% ethyl alcohol:glacial acetic acid:formalin (AAF) in the ratio of 90:5:5 (Johnson Alexander Donald, 1940). Pharmacognostical evaluation: The morphological study was carried out with regards to the size, shape of the rhizome of T. coadunata.

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Organoleptic characters: (Trease and Evans, 1996)

Histochemical 1988)

Colour, taste, odour and nature of the powder were recorded by sensory characters.

Transverse sections of the bulbs treated with various reagents to detect the presence and absence of tannin, starch, calcium oxalate etc were also carried out.

Microscopical study (Wallis T E, 2002) Free hand transverse sections of rhizome were first observed with distilled water and then with phloroglucinol and concentrated HCl. Microphotographs were taken by using carlzeiss trinocular microscope attached with camera. Powder microscopy: (Gokhale S B, Kokate C K, 2009) Small quantity of powder studied first with distilled water and after staining with phloroglucinol and concentrated HCl, photographs were taken.

evaluation:

(Krishnamurty,

RESULTS AND DISCUSSION Morphological study: Macroscopic investigation of rhizome has showed following characters. Rhizome is short creeping, up to 3 cm thick, densely scaly at the apex. Scales were ovate-lanceolate, about 8 × 2 mm, pale brown to dark brown. Apex was acuminate, margin sparsely ciliated. Stipes were scattered, about 0.5 cm apart, 50 × 1 cm rounded abaxially, grooved adaxially, castaneous or greyish-brown, glabrous and glossy all over (Plate A 1–2).

Plate A: Morphology of Rhizome & Plant Tectaria coadunata (Wall. Ex Hook & Grev.)

1. Rhizome

2. Plant in Natural habitat Tectaria coadunata 

Plate B: Transverse Section of Rhizome  Tectaria coadunata (Wall. Ex Hook & Grev.)

1. Cut Surface of Rhizome

2. Epidermis, Outer Cortex-Inner Cortex

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3. Vascular Bundles

4. Xylem, Phloem, Tannin content material

5. Tannin, Starch Grains

6. Vascular Bundles, Cortex, Epidermis, Pitta

Plate C: Powder Microscopy of Tectaria coadunata (Wall. Ex Hook & Grev.)

1. Epidermal cell with Tannin

2. Fibers with wide Lumen

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3. Fragments of Ramenta

4. Oil globules &Aluerone Grains

5. Parenchyma cell

6. Prismatic Crystals

7. Simple Starch Grain with Prismatic Crystals

8. Stained Annular Vessels

9. Stained Sceleriform Vessels

10. Stained Starch Grains with Aluerone

Table 1: Histochemical test: (Plate D) Sl.no.

Reagent

Observations

Characteristics

Results

Phloroglucinol+Conc. HCl

Red

Lignified cells

Iodine

Blue

Starch grains

Fecl3 Solution

Dark blue

Tannin cells

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Plate D: Slide depicts the Histo-chemical Analysis of T. coadunata

Microscopical study: T.S of Rhizome of Tectaria coadunata (Wall. Ex Hook & Grev.) C. Chr Transverse section of the rhizome showed the outermost surface, a single-layered barrel shaped epidermal cells embedded with tannin content. The cortex could be differentiated into two zones, outer cortex consisting of a few layers of cells with lignified thick walls and inner cortex made up of several layers of parenchyma cells with thin cellulose walls. Some of the parenchyma cells consisting of prismatic crystals of calcium oxalate, oil globules, occasionally with oleoresin material and most of the cells were largely filled with starch grains. Large mass of ground tissue was made up of parenchyma cells and a vascular cylinder (vascular bundles strong circularly arranged). The vascular cylinder is a typical dictyostele, with a number of meristeles embedded in the ground tissue forming an irregular ring. Each meristele was either round or elliptic in outline. The general mass of ground tissue lying internal to meristelar ring was the pith, made up of simple parenchyma cells, while the tissue lying outside this ring constituted the cortex (Plate B 1–6). Powder microscopy Organoleptic characters showed the presence of brownish color with astringent aromatic in odour and taste. Diagnostic characters of powder microscopy showed the

epidermal cells with tannin, simple starch grains, prismatic crystals, aluerone grains, annular vessels, cork cells are filled with brownish content, sceleriforum vessels and parenchymal cells (Plate C 1–10). Histochemical evaluation: The histochemical tests performed, confirmed the presence of Lignified cells, starch cells & Tannin cells (Table 1 & Plate D). CONCLUSION Rhizome of Tectaria coadunata (Wall. Ex Hook & Grev.) C. Chr. (Dryopteridaceae) can be identified on the basis of key morphological characters like nail of peacock and ramenta. Microscopically, like tannin in the epidermal cells, parenchymatous cells contain starch grains, prismatic crystals of calcium oxalate, vascular bundles and meristele. These observed characters could be helpful to establish certain botanical standards for identification and standardization of T. coadunata rhizome. ACKNOWLEDGEMENT The authors are thankful to Director, IPGT & RA, Gujarat Ayurved University, Jamnagar and Department of AYUSH, for providing financial support and other facilities to carry out the research work. We acknowledge Dr. Ashok Wali and Dr. Harish Nangare for their help during the drug collection at Ambaghat, Kolhapur (Maharashtra).

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REFERENCES: Dalhanacharya, (2009), Susruta Samhita, edited by Jadavaji Trikamji Acharya, Sutrasthana, Chapter 36/10, reprinted edition 2009, Chaukhambha Orientalia, Varanasi, pp. 159. Ghoghari A M, Bagul M S, Anandjiwala S, Chauhan M G, Rajani M,(2006), Free Radical Scavengning Activity of Aspidium cicutarium Rhizome, Journal of Natural Remedies, Vol 6/2. Gokhale B V, (1989), ChikitsaPradeep, Vaidyamitra Prakashan, Pune. pp.74. Gokhale S B, Kokate C K, (2009), Practical Pharmacognosy. 13th ed., Nirali Prakashan; Delhi, pp.1–13.

Johnson Alexander Donald, (1940), Plant Micro technique. New York, London, Maccgrow Hill Book Company, pp. 105. Krishnamurty K V, (1988). Methods in the plant histochemistry. Vishwanandan Pvt Limited, Madras, pp.1–70. Manickam V S, Irudayaraj V,(1992). Pteridophyte Flora of the Western Ghats – South India, reprinted edition 2009, B. I. Publications Pvt Ltd. pp. 260–261. Trease and Evans, (1996).Pharmacognosy, 15th ed., W.B. Sunders Company Ltd. pp. 569–570. Wallis

Jain S K, (1991), Dictionary of Indian Folk Medicine and Ethnobotany, Deep Publications, New Delhi, pp. 176.

Source of Support: Nil

T E, (2002). Text book of Pharmacognosy, 5th ed., New Delhi: CBS Publishers & Distributors, pp. 123–132, 210–215.

Conflict of Interest: None Declared

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Research article STANDARDIZATION OF ASTANGA LAVANA - A HERBO-MINERAL AYURVEDIC COMPOUND Shetty Suhas Kumar1*, Bhat Narayana Prakash2, Savitha H P3, Sunil Kumar K N4, Ravishankar B5 1

Associate Professor, Department of Manasa Roga, SDM College of Ayurveda and Hospital, Hassan 573201, India 2 HOD, Department of Manasa Roga, SDM College of Ayurveda and Hospital, Hassan 573201, India 3 Associate Professor, Department of Manasa Roga, SDM College of Ayurveda and Hospital, Hassan 573201, India 4 Senior Research Officer, SDM Centre for Research in Ayurveda and Allied Sciences, Lakshminarayana Nagar, Kuthpady, Udupi 574118, India 5 Director, SDM Centre for Research in Ayurveda and Allied Sciences, Lakshminarayana Nagar, Kuthpady, Udupi 574118, India * Corresponding author: E-mail: drsuhasshetty@gmail.com Mob: +91 9449603234.

Received: 22/06/2013; Revised: 25/07/2013; Accepted: 01/08/2013

ABSTRACT Standardization of Ayurvedic compound is vital to assure therapeutic efficacy and safety. Astanga Lavana is a poly herbo mineral Ayurvedic formulation, consisting of eight drugs, used for Agni Deepana (Improving digestive fire), Sroto Shodhana (cleansing of micro channels of the body) and specifically used to treat Kaphaja Madatyaya (Kapha variety of Alcoholic disorders). This study was aimed at standardization of Astanga Lavana with respect to powder microscope study, thin layer chromatography (TLC), High performance thin layer chromatography (HPTLC) finger printing and densitogram profiling. Ingredients of sample formula were powdered separately and combined in prescribed proportion and subjected for detailed physico-chemical, TLC and HPTLC analyses as per standard procedures. The tests results obtained like HPTLC fingerprint profile would serve as diagnostic parameters for the identity of this poly herbo mineral formulation. The results obtained may be considered as tools for assistance to the regulatory authorities, scientific organizations and manufacturers for developing standard formulation of great efficacy. KEY WORDS: Astanga Lavana, Madatyaya, Powder microscopy, High Performance Thin Layer Chromatography, Herbo mineral compound

Cite this article: Shetty Suhas K, Bhat N P, Savitha H P, Sunil Kumar K N, Ravishankar B (2013), STANDARDIZATION OF ASTANGA LAVANA - A HERBO-MINERAL AYURVEDIC COMPOUND, Global J Res. Med. Plants & Indigen. Med., Volume 2(8): 589â&#x20AC;&#x201C;598

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INTRODUCTION Ayurveda, the Indian system of medicine is widely practiced in Indian sub continent since ancient times. With increasing demand for safer drugs, concern has been drawn to quality, safety, efficacy and standards of the Ayurvedic formulations (Humber 2002, Elamthuruthy 2005). Most of the tests for standardization described in ancient literature appear to be based on observations and seems to be subjective without valid scientific support. Hence there is need for standardization and development of reliable quality protocols of Ayurvedic formulations using modern techniques of analysis (Anantanarayana DB, 2002). Plant material when used in bulk quantity may vary in its chemical content and therefore, in its therapeutic effect according to different batches of collection e.g. collection in different season and/or collection from sites with

different environmental surrounding or geographical location. The increasing demand of the population and chronic shortage of authentic raw materials have made it incumbent, to maintain uniformity in the manufacture of Ayurvedic medicines so as to ensure quality control and quality assurance (WHO, 1992). Various formulations are described in Ayurvedic texts to treat Madatyaya (alcoholic disorders) (Shetty SK 2013). Astanga Lavana is one of them, comprising of eight drugs, being used for the treatment of alcoholic disorders. Ingredients, parts used with proportions of Astanga Lavana are summarized in Table 1 (Trikamji Yadavji, 2009). Considering therapeutic utility of Astanga Lavana, a thought was given to standardize the same for wide spectrum use. Development of a composite standardization protocol for Astanga Lavana in Churna (powder form) was aimed in the study.

Table 1: Ingredients of Astanga Lavana Sanskrit name

Botanical name

Part used

Quantity

Sauvarchala Lavana Ajaji (Sveta Jiraka) Vrkshamla Amlavetasa Twak Ela Maricha Sarkara

Unaqua sodium chloride Cuminum cyminum Linn Garcinia Indica Chois. Garcinia pedunculata Roxb. Cinnamomum zeylanicum Blume. Elettaria cardamomum Maton Piper nigrum Linn. Saccharam officinarum Linn

Black Salt Dry seed Fruit Fruit Stem Bark Seed Dried fruit Sugar

1 part 1 part 1 part 1 part ½ part ½ part ½ part 1 part

MATERIALS AND METHODS

Preparation of Astanga Lavana

Plant materials

As per the textual description (Parashurama, 2000) and guidelines in Ayurvedic formulary of India (AFI, 2003) the 8 drugs described above were powdered separately and mixed as per quantity mentioned above (Trikamji Yadavji, 2009). 5 kg of final output of powder was obtained after 15% of loss during processing. All the sample powder passed through 80 mesh size.

Required plant medicines were collected from authorized raw drugs suppliers of Sreedhareeyam Ayurvedic medicines Pvt Ltd, Koothattukulam, Kerala. The raw materials were first identified and authenticated by team of botanist at Sreedhareeyam Ayurvedic medicines Pvt Ltd, Koothattukulam, Kerala.

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The results of powder microscopy study of Astanga Lavana were as follows:

Instrumentation and techniques Powder microscope study and High Performance Thin Layer Chromatography (HPTLC) studies were done at SDM Centre for Research in Ayurveda and Allied Sciences, Kuthpady, Udupi, Karnataka, India as per standard procedure (I Stahl, 1969; PD Sethi, 1996; Khandelwal KR, 2005). Following techniques adopted for the study:

and

methods

were

a. Powder microscopy: About 2 g of the sample was washed thoroughly with water to remove salt and preserve sample particle using centrifuge. Washed Churna (powder) was warmed with a drop of chloral hydrate on a microscopic slide and mounted in glycerine. Diagnostic characters were observed and photographed using Zeiss AXIO trinocular microscope (Lala, 1993) b. Extract preparation: 5 g of the sample was dissolved in 50 ml water and then extracted with 25 ml of butanol. (Lala, 1993) c. High Performance Thin Layer Chromatography (HPTLC): For HPTLC, 10, 20 and 30 Âľl of the above extract was applied on a precoated silica gel F254 on aluminum plates to a band width of 8 mm using Linomat 5 TLC applicator. The plate was developed in Toluene : Ethyl acetate (5 : 2) and the developed plates were visualized and scanned under UV 254, 366, and after derivatisation in vanillinsulphuric acid spray reagent at 620 nm. Rf values, colour of the spots, densitometric scan and superimposability of densitogram were recorded. (Anonymous, 2003) RESULTS AND DISCUSSION As the main ingredient of Astanga Lavana was black salt and sugar, the preparation was predominant of Lavana rasa (Salt), Madhura Rasa (Sweet) and Katu rasa (Pungent), which are gadgets for assessment of taste as per principles of Ayurvedic pharmacology

Powder microscopy: Characters like thick-walled mesocarp parenchyma of the pulp (Vrikshamla) (Figure 1.1); polygonal strata of cork tissue in surface view and in obliquely cut view, isolates acicular crystal often in raphide bundles embedded in elongated parenchyma cells, thick-walled cortical parenchyma (Amlavetasa) (Figure 1.3, 1.9, 1.27, 1.39, 1.42); cortical parenchyma with tannin content; thick-walled fibres, mucilage cells, cork in surface view; phloem tissue, different types of stone cells, parenchyma of the medullary ray cells, forked tipped thick walled fibres, stone cells of the medullary rays (Tvak) (Figure 1.2, 1.7, 1.11, 1.18, 1.32, 1.34, 1.35, 1.38, 1.40, 1.41); uni- to multicellular thin-walled conical trichomes, long thin xylem tissue associated with elongated parenchyma cells forming the stalk of the fruit,thin-walled mesocarp cells with radial thickening, fragment of septate branched vittae with oil drops, pitted tracheidal fibre of the stalk of the fruit, polygonal epicarp in surface view (Sveta Jeeraka) (Figure 1.4, 1.6, 1.12, 1.17, 1.20, 1.25, 1.28, 1.31, 1.36); sclerenchyma of the testa in surface view, obliquely cut sclerenchyma of the testa, obliquely cut cells of cotyledon with aleurone grains and oil droplets; epidermis of the seed in surface view often attached to obliquely cut cotyledon or with overlying cells of cotyledon (Ela) (Figure 1.13, 1.14, 1.15, 1.19, 1.30, 1.37); polygonal epicarp cells in obliques, surface or transversely cut mode often attached with hypodermal highly pitted stone cells, plenty of stone cells, perisperm cells; spirally thickened xylem vessels attached to other xylem elements (Marica) (Figure 1.5, 1.8, 1.16, 1.21, 1.23, 1.24, 1.26, 1.29); cortical parenchyma with starch, xylem elements, parenchyma cells with starch grains present abundantly from the above ingredients of the churna. Characters of all the ingredients of Astanga Lavana Churna were identified from the microscopic mount of the powder.

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Figure 1. Powder microscopy of Astanga Lavana Churna

1.1. Thick-walled mesocarp parenchyma (V)

1.2. Cortical parenchyma (T)

1.3. Acicular crystals and raphide bundles (A)

1.4. Epicarp in surface view (S)

1.5. Perisperm cells (M)

1.6. Fragment of vittae (S)

1.7. Thick-walled fibres (T)

1.8. Transversely cut epidermis and hypodermis (M)

1.9. Cortical parenchyma (A)

1.10. Cortical parenchyma with starch (U)

1.11. Mucilage cells (T)

1.12. Pitted tracheidal fibre (S)

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1.13. Sclerenchyma of the testa in surface view (E)

1.14. Obliquely cut sclerenchyma of the testa (E)

1.15. Obliquely cut cells of cotyledon (E)

1.16. Epicarp in surface view (M)

1.17. Fragment of branched vittae (S)

1.18. Cork in surface view (T)

1.19. Epidermis of the seed in surface view (E)

1.20. Fragment of vittae with oil drops (S)

1.21. Perisperm cells (M)

1.22. Xylem elements (U)

1.23. Stone cells (M)

1.24. Perisperm cells (M)

1.25. Mesocarp cells with radial thickening (S)

1.26. Obliquely cut epicarp (M)

1.27. Raphide bundle (A)

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1.28. Thick walled unicellular trichome (S)

1.29. Xylem elements (M)

1.30. Epidermis of seed with obliquely cut cotyledon (E)

1.31. Tissue from the stalk of the fruit (S)

1.32. Phloem tissue (T)

1.33. Parenchyma cells with starch grains (U)

1.34. Stone cells (T)

1.35. Parenchyma forming the medullary ray cells (T)

1.36. Multicellular trichomes (S)

1.37. Epidermis of seed with overlying cotyledon cells (E)

1.38. Thick walled fibre (T)

1.39. Perisperm cell (M) and raphides (A)

1.40. Stone cells of the medullary ray (T)

1.41. Forked tipped thick walled fibre (T)

1.42. Cork in surface view and in obliquely cut (A)

A – Amlavetasa; E – Ela; M – Marica; S – Sveta Jiraka; T – Tvak; U – unidentified; V – Vriksamla

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Fig.2 TLC Photodocumentation of Astanga Lavana extract

At UV 254 nm

At UV 366 nm

After derivatisation with vanillin- Sulphuric acid

Track 1- Astanga Lavana extract 10 µl; Track 2- Astanga Lavana extract 20 µl; Track 3- Astanga Lavana extract 30 µl; Solvent system - Toluene : Ethyl acetate (5: 2)

Fig. 3 HPTLC Densitometric scan of Astangalavana extract 3a. Astanga Lavana extract 20µl at UV 254 nm

3b. Astanga Lavana extract 20µl at UV 366 nm

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3c. Astanga Lavana extract 20µl at 620 nm after derivatisation with vanillin sulphuric acid

HPTLC densitometric scan of chloroform and alcohol extract of Astanga Lavana. (3a): Chloroform extract using toluene:ethyl acetate (6.5:2.5); (3b): Alcohol extract using toluene:ethyl acetate:acetic acid:methanol (3:5.5:0.8:0.1).74 K.

Fig.4: 3D supermposibility of densitogram at various wavelengths

Table 2. Rf table of Astanga Lavana (AL) extract AL extract 20 µl At UV 254 nm − − 0.41 Green − − 0.53 Green 0.58 Green − 0.62 Green − 0.74 Green − 0.82 Green 0.90 Green 0.95 Green −

AL extract 20 µl At UV 366 nm 0.03 F.Pink − − 0.51 F.Light green 0.55 F.Green − − 0.60 F.Green − 0.72 F.Blue − − − − − −

AL extract 20 µl At UV 620 nm − 0.14 Blue − − − 0.53 Pink 0.58 Blue − − − − 0.77 Blue − − − 0.97 Dark blue

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Thin layer chromatography (TLC): TLC fingerprint profile is a systematic representation of all the constitution of samples resolved in the given chromatographic system. TLC photo documentation is presented in Figure 2. High Performance Thin Chromatography (HPTLC):

Layer

HPTLC fingerprint of butanol soluble portion of Astanga Lavana Churna has been developed. The purity of the band in the sample extracts was confirmed by comparing the absorption spectra recorded at start, middle, and end positions of the band. The video densitometric images of chromatoplate are depicted. HPTLC densitometric scan at UV 254, 366, 620 nm are presented in Figure 3a, 3b & 3c. The super imposability of tracks is presented in Figure 4. The Rf values are tabulated in Table 2. Rf values of the spots and their colour by TLC photo-documentation of Astanga Lavana extracts have been developed. Chloroform extract of Astanga Lavana at 254 nm showed 8 spots (0.41 Green, 0.53 Green, 0.58 Green, 0.62 Green, 0.74 Green, 0.82 Green, 0.90 Green, 0.95 Green) whereas under 366 nm it showed 5 spots (0.03 F. Pink, 0.51 F. Light green, 0.55 F. Green, 0.60 F. Green, 0.72

F. Blue) and 5 spots (0.14 Blue, 0.53 Pink, 0.58 Blue, 0.77 Blue, 0.97 Dark blue) after derivatisation using toluene:methyl acetate (6.5:2.5) as solvent system. CONCLUSION Despite the initiation of recent technology in standardization of multipart formulations, not many Ayurvedic poly herbal or herbo mineral medicines are standardized so far. Standardization of compound formulation has various challenges. This study was aimed at authentication of ingredients used and physico chemical characterization using latest technology. The physicochemical standards would serve as preliminary test for the standardization of the formulation. The unique Rf values, densitometric scan and densitogram obtained at different wavelengths pre- and postderivatisation can be used as fingerprint to identify Astanga Lavana. Standards for the poly herbal or herbo mineral formulation is important for the quality check of the formulation. The current investigation can be used as standardization test for the compound formulation. Further, detailed macro & microscopic examination of the raw drug individually and powder form would add to the standardization test of Astanga Lavana.

REFERENCES Anantanarayana DB (2002). Proceeding of International Congress on Ayurveda, 28–30th January. 2002; 67 Anonymous (2003). Quality standards of Indian medicinal plants, Vol-1, Indian council of medical research, New Delhi. Anonymous (2003). The Ayurvedic Formulary of India; Part-I, 2nd ed., Government of India, Ministry of Health and family welfare, New Delhi.

Elamthuruthy Shah CR, Khan TA, Tatke PA, Gabhe SY (2005). Standardization of marketed Kumaryasava-an Ayurvedic Aloe vera product. J Pharm Biomed Anal, 37, 937–41. Humber (2002). The role of complementary and alternative medicine: Accommodating pluralism. J Am Med Assoc, 288, 1655–6.

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I Stahl (1969). Thin Layer Chromatography, A Laboratory Hand Book. Berlin: Springer-Verlag. 52–86. Khandelwal KR (2005). Practical Pharmacognosy: Techniques & Experiments, Nirali Prakashan, Pune, 13th ed, 144–155 Lala

PB (1993). Lab manuals of th Pharmacognosy, 5 ed, CSI publishers & distributors, Calcutta

Parashurama Shastri (2000). Choorna Adhyaya, Madhyama Khandha, In: Sharngadhara Samhita with Deepika and Gudhartha Deepika of Adhamalla and KashiramaVaidya. (ed.) Chaukambha Orientalia; New Delhi: 178

Source of Support: Nil

PD Sethi, (1996). High Performance Thin Layer Chromatography, CBS Publishers and Distributors; 1st ed. New Delhi: 1–56 Shetty SK, Savitha HP, Narayana Prakash B (2013). Critical Review on role of Panchakarma in Madatyaya (Alcoholic Disorders), Journal of Ayurveda and Holistic Medicine, April 2013; 1 (1): 13–16 Trikamji Yadavji (2009). Madatyaya Chikitsa Adhyaya. In Charaka Samhita. Chaukhambha Sanskrit Sansthan. Varanasi: 590. WHO (1992) Organisation Mondiale De La Sante, Quality control methods for medicinal plant materials, (World Health Organisation, 559, rev.1, Original English, 1992), 159

Conflict of Interest: None Declared

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Review article CLINICAL RESEARCH RELATED CAREER CHOICES FOR AYUSH GRADUATES IN INDIA Samal Janmejaya1* 1

District Epidemiologist, Dist. Health Office, Gadchiroli, Maharashtra, India *Corresponding Author: Email: janmejaya_samal@yahoo.com; jaytheworld@gmail.com; Mob: +919438323843, +919901316384

Received: 11/06/2013; Revised: 20/07/2013; Accepted: 27/07/2013

ABSTRACT India is increasingly being recognized as a hub for global clinical trials owing to some of the prized attributes, such as large and diverse patients’ pool for conducting clinical trial; availability of medical (including AYUSH), pharmacy and science graduates; excellent infrastructure facilities; comparative cost advantage and change in patent law. India being a land known for Ayurveda, Unani, Siddha, and Homeopathy besides, allopath is growing as the preferred destination for global clinical trials. Earlier AYUSH graduates were limited to their own field of study with very few exceptions in allied areas. This was mainly due to lack of information access regarding career options and a sense of insecurity in a relatively newer field. But the scenario is changing day by day with information access, awareness about job opportunities and a great degree of personal and professional satisfaction in these domains which indirectly leads to an increased influx of these graduates in to these allied areas. Apart from their personal interest their background helps them in availing these options in many different ways. These could be their understanding about basic and applied clinical biomedicine which is not the case with life science and other graduates as they only know about the basic bioscience not the clinical medicine. In this context a review was carried out with an objective of exploring various career options available in clinical research and related areas in terms of scopes, opportunities and challenges for AYUSH graduates in India. This article also tries to kind of bring out issues related to the suitability of these graduates for clinical research related career choices. KEY WORDS: AYUSH Graduates, Career Choices, Clinical Research, India.

Cite this article: Samal Janmejaya (2013), CLINICAL RESEARCH RELATED CAREER CHOICES FOR AYUSH GRADUATES IN INDIA, Global J Res. Med. Plants & Indigen. Med., Volume 2(8): 599–604

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INTRODUCTION AYUSH is an acronym for Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homeopathy. These are the six Indian systems of medicine prevalent and practiced in India. All these six systems of medicine have got a formal education system in their respective disciplines in various states across the nation. The students in these courses are taught about the Non- clinical, Para-clinical and Clinical courses during their graduation like their Allopathic counterparts. As on 1-1-2010 there were 7, 85,185 registered AYUSH doctors in India. During 1980–2010, an average annual growth rate of 2.5% had been observed in number of AYUSH doctors with a maximum growth rate of 11.8% had been recorded in 2000 (Dept. of AYUSH, 2010). Until the very recent times, majority of AYUSH graduates were limited to their own field of study with few exceptions in to the field of clinical research and allied areas. The reason might be the lack of awareness, unavailability of suitable job opportunity or a sense of insecurity in a relatively offbeat domain of work. But these days have witnessed a paradigm shift; with increase in the information access, awareness about the job opportunities and a great degree of professional and personal satisfaction in other domain of work. One of those other domains include clinical research which is the branch of medical science that determines the safety and efficacy of medications, devices, diagnostic products and treatment regimen intended for human use (Wikipedia, 2013). India is increasingly being recognized as a hub for global clinical trials owing to some of the prized attributes, such as large and diverse patients’ pool for conducting clinical trial; availability of medical (including AYUSH), pharmacy and science graduates; excellent infrastructure facilities; comparative cost advantage and change in patent law (Institute of Clinical Research India, 2013). At the same time the Indian pharmaceutical industry is growing by leaps and bound with huge growth potential. With this background this article addresses the clinical research related career choices among AYUSH graduates in India with

a special focus on educational opportunities, job opportunities, suitability and challenges in availing and sustaining in clinical research industry. METHODOLOGY The study predominantly adopted a review based approach. Many published documents were reviewed to obtain information regarding the basic understanding of clinical research, Indian clinical research industry, career pathways and demand forecasting of clinical research professional in India and globally. An attempt was made to make a survey on the acceptance of AYUSH graduates in clinical research related career options by visiting various web portals of job sites and Clinical research organizations. Similarly internet based Google search engine was used to obtain the list of institutes catering to the need of clinical research training for AYUSH graduates in India. DISCUSSION: Understanding Clinical Research: As per the guidelines of Good Clinical practice (GCP) a clinical trial is any investigation in human subjects intended to discover or verify the clinical, pharmacological and/or the pharmaco-dynamic effects of an investigational product, and/or to identify any adverse reactions to an investigational product, and /or to study absorption, distribution, metabolism and excretion of an investigational product with the object of ascertaining its safety and efficacy. The term clinical trial and clinical research are synonymous (T K Pal et al., 2009). A clinical trial classically consists of four different phases known as Phase-1, Phase-2, Phase-3, and Phase-4. These phases do not necessarily come in a chronological manner. An investigational medicine is often evaluated in two or more phases simultaneously in different clinical trials. Also, some clinical trials may overlap two different phases (Spilker Bert, 1984).

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Clinical Research Industry - An Indian Context: India has become a signatory to TRIPS (Trade Related Aspects of Intellectual Property Rights). Consequently, a new patent law has come into effect from January 2005. The new keyword for the pharmaceutical industry is innovation and to introduce new drugs clinical trials are mandatory. They are to be conducted as per ICH GCP (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use- Good Clinical Practice) norms. The clinical trials that have been conducted in India till date have gone well (Institute of Clinical Research India, 2013). For majority of the studies, patient enrollment is a key advantage. According to McKinsey & Company report the Indian Pharmaceutical market will grow to USD 55 billion by 2020 driven by a steady increase in affordability and step jump in market access (McKinsey & Company, 2013). India is increasingly being recognized as a hub for global clinical trials owing to the following prized attributes, such as large and diverse patientsâ&#x20AC;&#x2122; pool for conducting clinical trial; availability of medical (including AYUSH), pharmacy and science graduates; excellent infrastructure facilities; comparative cost advantage and change in patent law. Again India is a home to one billion people, including 30 million with cardiovascular diseases, 25 million with type-2 DM, and 10 million with major psychiatric disorders. Large extended families still live in proximity to one another making them attractive to genetic linkage studies (Institute of Clinical Research India, 2013). In one of the surveys conducted by Cygnus Management consultancy a list of 69 clinical research companies have been recorded (Cygnus Research, 2013). According to industry estimations India is going to require 50,000 clinical research professional by 2013 whereas globally the requirement is forecasted to 2,50,000. India is the fourth largest pharmaceutical market in terms of volume and 13th in value terms. This indicates a huge

requirement of clinical research professional in India and globally (Clinical Research Education and Management Academy, 2013). Educational Opportunities Research and Allied Areas:

Clinical

Following (Table no. 1) are some of the educational institutes and courses available in India for AYUSH graduates in pharmaceutical and clinical research area. These institutes offer either an MSc clinical research or a PG Diploma in clinical research, regulatory affairs, medical writing, data management, Pharmaceutical Management etc. Career Pathways of Clinical Research Professionals: An entry in clinical and pharmaceutical industry sometimes but not always requires a formal training in clinical research. This may not be a case with a person who has substantial experience in any of the domain of clinical research. The same is also applicable to AYUSH graduates but the percentage of these professionals is very few. This is because these graduates do not get appropriate exposure to clinical research during their undergraduate level studies so a formal training in clinical research is highly recommended for those who wish to make a career in this field. Students after obtaining formal education in clinical research can work as Clinical Research Associate, Clinical Research Advisor, Project Manager, Group Project Manager, and Operation Director depending upon experience (Institute of Clinical Research India, 2013). The pharmaceutical management students can start a career either in the marketing and sales division or in the production division depending on educational background, training, and experience. These openings can be accessed from various professional job sites and the web portals of various clinical research organizations. Interested graduates should keep in touch with various web portal related to this domain as the same cannot be reproduced due to obvious reasons.

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Table No.1. List of some of the Institutes providing clinical research training in India Sl No

Name of the Institute

Courses Offered

Duration

Eligibility Status for AYUSH Graduates

MSc (Clinical Research),

Yes

MSc (Pharmacovigilance and Clinical Data management), PG Diploma in Clinical Research and Pahrmacovigilance) MPH (Clinical Research), MS (Clinical Trial), MSc (Epidemiology and Biostatistics) MS (Clinical Trial)

Regular Mode 1

Institute of Clinical Research India, http://www.icriindia.com/ Campuses at Delhi, Mumbai, Bangalore, Dehradun

SRM School of public health, http://www.srmuniv.ac.in/node/366

Birla Institute of Technology and Sciences, Mesra http://www.bitmesra.ac.in/ Maharashtra University of Health Sciences, Nashik http://www.muhs.ac.in/ Padmashree Institute of Clinical Research (Rajiv Gandhi University of Health Sciences), Bangalore http://www.padmashree.org/ Indian Institute of Public Health, New Delhi in Collaboration with Academy of Scientific and Innovative Research, New Delhi http://www.phfi.org/ Indian Institute of Public Health-Delhi http://www.phfi.org/ Indian Institute of Health Management Research - Jaipur http://www.jaipur.iihmr.org/ Indian Institute of Public Health, Hyderabad http://www.phfi.org/

4 5

7 8 9

10 11 12

Chennai,

International Institute of Population Sciences, Mumbai http://www.iipsindia.org/ Chettinad Academy of Research and Education, Tamilnadu http://www.chettinadhealthcity.com/university/index_care.htm Clinical Research Education and Management Academy http://www.cremaindia.org/clinical-research-courses-datamanagement-programs.html

1 2

Yes

MSc (Pharmaceutical Medicine) MSc (Clinical Research)

Yes

MSc, PhD (Clinical Trial)

5(2+3)

Yes

PGDCR PGDPM (Pharmaceutical Management) PG Diploma in Biostatistics and Data management MSc (Epidemiology and Biostatistics) MSc (Experimental Medicine) MSc (Clinical Research), PG Diploma and Diploma in Clinical Research

1 2

Yes Yes

Yes

Distance Mode 1

Sikkim Manipal University-Distance Education, Manipal http://smude.edu.in/

Punjab Technical University http://www.ptudep.org/

Indira Gandhi National Open University, New Delhi http://www.ignou.ac.in/ Global Open University, Nagaland http://www.tgouonline.com/

MSc (Clinical Research and Regulatory Affairs) PG Diploma in Clinical Research and Regulatory Affairs MSc (Clinical Research) PG Diploma in Clinical Reserach MSc (Clinical Trial) MSc (Clinical Research)

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Yes

1 2

Yes

1 2

Yes

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Career Choices and Suitability of AYUSH Graduates: Clinical research can be one of the preferred career choices for AYUSH graduates in India owing to several reasons, these could be; 1. Indian pharmaceutical industry is growing by leaps and bound from a market size of USD 12.6 Billion in 2009 to USD 55 Billion by 2020 with the potential to reach USD 70 Billion in an aggressive growth scenario (McKinsey & Company, 2013). 2. The demand for the clinical research professionals is very huge in the coming years with 2,50,000 vacancies worldwide and 50,000 in India (Clinical Research Education and Management Academy, 2013). 3. From Table No.1 it is evident that most of the institutes in India accept AYUSH graduates for admission in different types of clinical research training programmes. Interestingly all the institutes specifically mention the AYUSH degrees in the eligibility criteria as evidenced from the survey carried out for the purpose of this study using internet based Google search engine. 4. This otherwise indicates the suitability of AYUSH graduates for clinical research related professions felt by various industry segments. One keen observation from the author shows that many of Clinical Research Organizations (CRO) looks for AYUSH graduates for recruiting them in different clinical research related jobs. More over what matters in a clinical research industry is the in-depth understanding of complex human biology, biostatistics and IT skills which could be acquired with personal

interest and training irrespective of the background whether AYUSH or Allopath. 5. The greatest advantage of AYUSH graduates is their understanding of their own discipline, so CROs conducting research in their disciplines would prefer them in comparison to other graduates such as pharmacy or life science. Some of the biggest players such as Himalaya Drugs Company, Atrimed Pharmaceuticals and many of the modern pharmaceutical industries which come up with Ayurvedic and herbal products would prefer a trained Ayurveda graduates.

CONCLUSION AYUSH graduates can definitely find this field interesting and challenging as well. Apart from their own field of study if these graduates are involved in careers related to clinical research and allied areas, probably opportunities wonâ&#x20AC;&#x2122;t be a problem for the right one. From the discussion and related points mentioned above it can be concluded that clinical research related career choices could be a preferred career option for many AYUSH graduates based on their interest, basic understanding and the demand as well. However confusions may stem out regarding the acceptance and sustainability of AYUSH graduates in clinical research domain under the hegemony of MBBS graduates. The answer to this may be mixed i.e. there are some instances where MBBS graduates have better acceptance in comparison to AYUSH graduates but it all depends upon the potentiality of the candidate. The opinion and research shown in this article is only a guide for the AYUSH graduates in choosing a career option in clinical research related areas hence making a decision of career choice in this domain lies predominantly on the candidate itself.

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REFERENCES Clinical Research Education and Management Academy, Mumbai, India. http://www.cremaindia.org/ (Accessed on 30/05/2013) Cygnus: Business Consulting and Research (2005) Directory of Clinical Research Companies in India, Banjara Hills, Hyderabad -500034, India, http://firstclinical.com/journal/2006/060 5_India_Sample.pdf (Accessed on 30/05/2013) Department of AYUSH, Planning and Evaluation Cell, Ministry of health and Family welfare (2010), -AYUSH in India, Govt. of India, New Delhi.

Institute of Clinical Research, India, http://www.icriindia.com/ (Accessed on 28/05/2013) McKinsey & http://www.mckinsey.com/ on 30/05/2013)

Company, (Accessed

Spilker, Bert. (1984) Guide to Clinical Trials, Raven Press. Page XXii-Xxiii (http://www.virginia.edu/vpr/irb/HSR_d ocs/CLINICAL_TRIALS_Phases.pdf) (Accessed on 31/05/2013) T K Pal, Sangita Agarwal (2009) - Clinical Research: Practice and Prospects. CBS Publishers & Distributors, New Delhi. Wikipedia, http://www.wikipedia.org/, (Last viewed on 31/05/2013)

Source of Support: Nil

Conflict of Interest: None Declared

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Review article SHARIRIKA PRAKRITI – AN ASTUTE OF HUMAN CONSTITUTION Amin Hetal1, Vyas M K2, Vyas H A3, Baghel A S4, Dwivedi R R5 1

Ph.D Scholar, Dept. of Basic Principles, IPGT & RA, GAU, Jamnagar, Gujarat, India Associate Professor, Department of Basic principles, IPGT & RA, GAU, Jamnagar, Gujarat, India 3 Assistant Professor, Department of Basic principles, IPGT & RA, GAU, Jamnagar, Gujarat, India 4 Associate Professor, Department of Basic principles, IPGT & RA, GAU, Jamnagar, Gujarat, India 5 Head and Proff. Dr. R.R. Dwivedi, Department of Basic principles, IPGT & RA, GAU, Jamnagar, Gujarat, India *Corresponding Author: E-mail: dr.hetal1985@gmail.com; Mob: +919924194660 2

Received: 28/05/2013; Revised: 31/07/2013; Accepted: 04/08/2013

ABSTRACT The concept of Prakriti (Constituent) has been described in Ayurvedic classics since very beginning with its high thoughts. The need of present day is that it should be easier and more applicable. As per the theory of evolution Prakriti is the prime cause of universe. Prakriti is that which constitutes, distinguishes and characterizes person apart from others and consists of anatomical, physiological and psychological makeup of that person. Anatomical and physiological component of Prakriti can be considered as Deha or Dosha Prakriti (Body constituent) where as Psychological component can be considered as Manas Prakriti (Mental constituent). To get a detail idea regarding Prakriti or to expand conception regarding it is only possible only after a thorough understanding of the subject. Here an attempt has been made to understand Sharirika Prakriti (Body Constituent) through the concept of classics. KEY WORDS: Prakriti, Dosha, Deha

Cite this article: Amin Hetal, Vyas. M. K., Vyas H. A., Baghel. A. S., Dwivedi. R. R. (2013), SHARIRIKA PRAKRITI – AN ASTUTE OF HUMAN CONSTITUTION, Global J Res. Med. Plants & Indigen. Med., Volume 2(8): 605–612

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INTRODUCTION The word Prakriti means “Nature” a natural form of build and constitution of human body. “Pra” means the “beginning” “commencement” or “source of origin” and “kriti” means “to perform” or “to form”. Put together, Prakriti means “natural form” or “original form” or “original source”. According to Brahma Vaivarta Purana, Prakriti is formed from 3 letters viz, Pra, Kri and Ti. The letter Pra stands for the Prakrashta Guna i.e. superior quality- Satva, Kri for moderate one the Rajas and Ti for Tamas. Pra also suggests the superior or best and Kriti stands for creation. Therefore, the best of creation or the superior creation is Prakriti. Therefore it can be said that Prakriti is sum total of morphological, physiological and psychological traits in human beings. Prakriti of a man has genetic and acquired aspect, the genetic aspect depends upon Shukra and Shonita (Shastri Ambikadutta, 2003), while acquired constitution develops in relation to environmental factors like climate, season, time factor, age, race, familial inheritance (Trikamji Yadavaji, 2005). Meaning of Prakriti in current perspective: During the union of Sukra (Sperm) and Shonita (Ovum), Dosha that is dominant, forms Prakriti of an individual (Shastri Ambikadutta, 2003). Caraka has stated that Prakriti of a person is not only dependent on Shukra and Shonita, but also dependent on the Kala (Time), diet and regimen, behavior of mother, nature of Garbhashaya (Uterus) as well on the Mahabhuta (Trikamji Yadavaji, 2005). Vagbhatta has also favored the view of Caraka as he has explained that Prakriti is dependent not only on Shukra and Shonita but also on diet and behavior of the pregnant woman, nature of Garbhashaya and Kala (Atridev Gupt, 1997).

nourished by mother so identical type of Prakriti of human being is formed from embryonic life. These Prakriti are of three types having Vata, Pitta and Kapha as pillars (predominance) (Hemaraja Sarma, 1998). Rasavaiseshikakara explained that, in development of fetus, due to own reasons Dosha become intensified. This non-pathogenic intensified status of Dosha remains constant from birth till death and that is known as Prakriti (Bhadanta Nagarjuna, 1976). Therefore it can be said that, Prakriti is not output of those Dosha, which undergo constant variations. Dosha, which remain constant are responsible for Prakriti and they change only at the time of death (Shastri Ambikadutta, 2003). Aspects affecting fortitude of Prakriti: Ayurveda has explained many factors which take part in determination of Prakriti along with development of fetus. Caraka has described six Bhavas (Aspects)responsible for development of fetus, which can be considered as determinants in the development of human organism and its personality (Trikamji Yadavaji, 2005; Shastri Ambikadutta, 2003). Sadabhava: 1. Matrja(mother) 2. Pitrija (Father) 3. Atmaja (Soul) 4. Satmyaja (Wholesomeness) 5. Rasaja (Digestive food) 6. Satvaja (Mind). It has been mentioned in Astanga Samgraha and Caraka samhita that the following factors influences the Prakriti: 1. Jatiprasakta (racial peculiarities), 2. Kulaprasakta (familial predisposition), 3. Deshanupatini (Demographic), 4. Kalanupatini (Seasonal effect), 5. Vayonupatini (Natural changes according to age), 6. Pratyatmaniyata (Personal habits & individuality, idiosyncrasy) (Trikamji Yadavaji, 2005; Sharma Shivprasad, 2008). Deha Prakriti:

Prakriti is concluded during the time of union of Shukra and Shonita according to Dosha existing at the time of conception (Shastri Ambikadutta, 2003; Atridev Gupt, 1997). Kashyapa has explained that the fetus is

Even though so many meanings of Prakriti are enumerated, Ayurveda science principally refers to Prakriti of a human, as Dosha Prakriti or Deha Prakriti. Due to non partisan bond,

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qualities of Dosha are expressed on body. This is called Deha Prakriti (Trikamji Yadavaji, 2005). These expressions are by way of structure or morphology, by way of function or physiology, by way of mental reactions or psychology. Types of Deha Prakriti: Charaka Samhita in Sutrasthana mentions Prakriti for first time in Swasthya Chatushka that, people are borne with various proportions of Dosha. Their body constitution is referred accordingly. Those borne with equal proportion of three Dosha i.e. ratio of Vata, Pitta and Kapha equal to each other are Sama. These individuals are generally healthy people and remain healthy. Few of them show predominance of one Dosha. Those exhibiting predominance of Kapha are Shleshmala, with predominance of Pitta or Vata are called Pittala and Vatala respectively (Trikamji Yadavaji, 2005). Due to this predominance of Dosha proportion people show psychosomatic expressions called Deha Prakriti. Caraka, Sushruta, Vagbhatta and other Ayurvedic scholars have divided the human constitution into seven types on the basis of relative prevalence of three basic humors viz. Vatala, Pittala, Shleshmala, Vata-Pittala, VataShleshmala, Pitta-Shleshmala, Sama-Doshika. Subject regarding influence of Prakriti has been described by all authors. Both Vagbhatta say that individuals with Sama Prakriti are excellent while with mixed Prakriti are denounce; the person of Vata, Pitta and Kapha predominance are said to be inferior, mediocre and good respectively (Atridev Gupt, 1997). Individuals of Sama Prakriti are always healthy and of Vatika etc. always remain ill (Hemaraja Sarma, 1998). Bhela has given Sannipataj Prakriti (All three Dosha) in place of Samaprakriti (Girijadayalu Suklah, 1999).

Prakriti as Bhautika. Here Bhautika term stands for Pancha-Mahabhuta. Vayu (Air), Agni (Fire) and Jala (Water) Prakriti are just same as Vata, Pitta, and Kapha subtypes of Doshaja Prakriti. According to him body of a person of Parthivasharira is Sthira, Vipula and he is Sahanashila (Endurance power) (divine) in his temperament. The person of Nabhas Sharira is Pavitra, Dirghayu (Long Life) and the aperture of their nose ear etc. is larger in size (Shastri Ambikadutta, 2003). Eccentricity of different types of Deha Prakriti: Different Ayurvedic scholars like Caraka, Sushruta, Vagbhatta, Sharangadhara, Bhela, Harita, Yogaratnakara have described the characteristics of different types of Deha Prakriti (Trikamji Yadavaji,2005; Shastri Ambikadutta,2003; Sharma Shivprasad,2008; Tripathi Brahmanand,2010; Atridev Gupt,1997; Girijadayalu Suklah,1999; Ramavalamba Shastri,1985; Hemaraja Sarma,1998; Bhadanta Nagarjuna,1976; Laksmipati Sastri,1999). In Pauranic prose i.e. Garuda Purana and Agni Purana, characteristics of different types of Prakriti have been also mentioned. Caraka is more systematic than other Samhita. Caraka has put down great emphasis on the physical characteristic although psychological have been also mentioned. In Sushruta and Vagbhatta the nature of other animals and description of dreams is found in relation to Prakriti, which is not described in Caraka Samhita. Prakriti Pariksha (Examination of Constituent) and its objectives:

Based on Panchamahabhuta (Five major Attribute):

The importance of Prakriti of patients for a physician has been greatly emphasized in Ayurveda. Sushruta has given an opinion that patient should be treated by physician according to Prakriti of a person (Shastri Ambikadutta, 2003). Kasyapa has also given similar view (Hemaraja Sarma, 1998).

Sushruta while describing Dehaprakriti refers to the school of some scholar of Ayurveda, who regard the constitutional type of

Caraka has enumerated points which should be examined by physician through Pramana (Examination) of Pratyaksha

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(Inspection), Anumana (Imagination) and Upadesha for ascertaining the period of life left to the patient and he has included them in the examination of Prakriti (Trikamji Yadavaji, 2005). The Prakriti is determined by heredity, family, age, climate, season, periodic factor, and idiosyncrasy. These factors are responsible for individual variation among persons (Trikamji Yadavaji, 2005). A rash administration of medication without proper examination of Prakriti (Trikamji Yadavaji, 2005; Atridev Gupt, 1997) may be injurious to the life of patient. Caraka has given constitutional temperamental, psychological and emotional aspects of personality because Prakriti and Satva Pariksha have been included in Dashvidha-Aturapariksha (Tenfold examination of patient) (Trikamji Yadavaji, 2005). The clinical and therapeutic utility of the knowledge of Prakriti has been vividly described by Caraka, whereas the detailed regimen that is to be followed by different Doshaja Prakriti individuals has been dealt separately (Trikamji Yadavaji, 2005). Preventive aspects of Prakriti have been described by Caraka (Trikamji Yadavaji, 2005). Any kind of alteration in Prakriti is death indicator symptom for every individual so one have to careful if original nature of individuals is going to change (Shastri Ambikadutta, 2003). Prakriti has prime importance in both healthy and diseased persons i.e., importance of Prakriti in prescribing dietary regimen and life style management in healthy individuals and treatment point of view in diseased individuals. Types of Prakriti: According to Charaka, four factors which are responsible for influencing Prakriti. (Trikamji Yadavaji, 2005) They are: 1. Shukrashonita Prakriti (Sperm and Ovum), 2. Kalagarbhashaya Prakriti (Season and condition of uterus), 3. Maturaharvihara Prakriti (Food and regimen of mother) and 4. Mahabhutavikara Prakriti (Nature of mahabhutas comprising of fetus).

Sushruta has described the presence of seven types of Prakriti according to combinations of Dosha: three owing to single Dosha, i.e. Ekadoshaja; three owing to the combination of two Dosha i.e. Dvidoshaja and Samaprakriti owing to the combination of all the three Dosha. (Shastri Ambikadutta, 2003) Caraka has mentioned that Prakriti of an individual may be Ekadoshaja, Dvidoshaja or Samaprakriti owing to dominance of one Dosha, two Dosha or by equity of all three Dosha respectively (Trikamji Yadavaji, 2005). Vagbhatta view is very much similar to Caraka (Atridev Gupt, 1997). Caraka has described Ekdoshaja Prakriti as Vatala, Pittala and Shleshmala still to pay to dominance of Vata, Pitta and Kapha respectively. Theoretically, as these individuals are having dominance of Dosha, they should have been always sick; but practically it is not so. Sushruta has clarified this doubt by giving the following analogy: „The insect born in the poison does not die owing to its own poison and in the same way; Dosha that is dominant according to one‟s Prakriti does not harm the individual‟ (Shastri Ambikadutta, 2003). Dalhana has stated that there are two types of dominance of Dosha: (1) Prakrita (Normal) (2) Vaikrita (Abnormal) and Dosha that are dominant at the time of Prakriti formation are Prakrita in nature, so they do not harm the individual (Shastri Ambikadutta, 2003). Cakrapani has explained the concept of „dominance of Dosha’ stating that Utkattata of Dosha is of three types. (1) Hina (Lower) (2) Madhyama (Middle)(3) Uttama (High) and if Utkattata of Dosha at the time of Prakriti formation is „Hina,’ it will not harm the individual. Caraka has stated that Ekdoshaja Prakriti persons are rare and always sick while individuals those are having Samadoshaja Prakriti is always healthy (Trikamji Yadavaji, 2005). Entire science of Ayurvedic medicine has been developed in terms of three basic humors i.e Vata, Pitta and Kapha. They are the essential categories of biological constituents of human organism (Singh and Dubey, 1967). On the basis of this concept another classification

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of Prakriti is neurogenic, vasogenic and histogenic based on of relative preponderance of three principle neurotransmitters namely acetylcholine catecholamine and histamines respectively are very much correspond with Vatika, Paittika and Kaphja. (Udupa et al., 1975). Humoral constitution as concerned in Indian medicine is very much comparable with constitutional typology suggested by Sheldon et al., (1940). He has described Ectomorphy = cerbrotonia = Vattika, mesomorphy = somatotonia = Paittika; Endomorphy = viscerotonia = Kaphja Prakriti. Features of Prakriti: Caraka has described the features of Prakriti based on the Guna (attributes) of a Dosha. Sushruta has described the characteristics of Prakriti on the basis of morphological, behavioral and other aspects (Trikamji Yadavaji, 2005; Shastri Ambikadutta, 2003; Tripathi Brahmanand, 2010; Atridev Gupt, 1997). The body constitution of persons is named according to the predominance of Dosha. Some persons maintain equilibrium of Vata, Pitta and Kapha from the very time of conception; some are dominated by Vata, some by Pitta and some by Kapha. Those of the first category are not susceptible to diseases and the rest of them are always likely to suffer. (Trikamji Yadavaji, 2005) External constitution (phenotypic classification) broadly, gets into description criteria for defining anatomical features similar to body built, body frame, size and symmetry of body parts, physiology, physical endurance and aptitudes. Moreover, the questionnaire also detains information pertaining to ethnicity, family history of diseases etc. Each of the query has multiple options to choose from, and each of the options further refers to it being a property attributed to either V, P or K Individuals who had thin and narrow body frame, weakly developed body built, with irregular appetite, food and bowel habits, difficulty in gaining weight, quick at physical

activities, dry skin and hair, and less tolerance for cold temperature were considered as Vata Prakriti. Individuals with moderately developed built, high frequency of appetite and thirst, good digestive power, perspiration tendency higher than normal, tolerance for cold weather, moderately mobile with moderate physical strength were identified as Pitta Prakriti. Individuals who had broad body frames with well developed body built, tendency to gain weight, low appetite and digestion, preferred to be less mobile, less forgetful and with good healing power and cool temperament, were selected as Kapha Prakriti individuals. A Study concluded that, every individual from three most constitution exhibit striking difference with respect to biochemical and hematological parameters and at genome wide expression levels. (Bhavana Parasher et al., 2008) Consequence of awareness of Prakriti: The Ayurvedic system of diagnosing Prakriti offers a unique approach in understanding and assessing one‟s health. It is comprehensive in scope, spanning both physical and mental aspect. It is not merely a diagnostic tool but also a guide to action for good health. It provides detailed instructions to adopt one‟s food and behavior to suit one‟s Prakriti. The psychosomatic constitution of a person is considered to determine the following:(1) Bala (natural strength of body and mind) The Vata Prakriti persons are having less strength than Pittaja and Kaphaja Prakriti persons. (Trikamji Yadavaji, 2005) (2) Susceptibility to different diseases – The Kaphaja persons are more prone to Kaphaja Vikara like Agnimandhya, Pratishyaya etc. similarly Pitta and Vata Prakriti persons are more prone to Pittaja and Vataja Vikara respectively. (Trikamji Yadavaji, 2005) (3) Prakriti is also considered for deciding the line of management, eg. In Amaja Vyadhi

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like Jvara, for Kaphaja person one can adopt complete Apatarpanachikitsa whereas in case of Vataja person that cannot be adopted completely. (Atridev Gupt, 1997). (4) Drug Doses – Bheshaja Matra is decided according to Prakriti for example Vataja, Pittaja and Kaphaja persons, the Alpa, Madhya and Pravara Matra of Bheshaja are adopted respectively. (Trikamji Yadavaji, 2005) (5) Preventive measures – On the basis of Prakriti person can adopt Dinacarya and Ritucarya as per need of Prakriti to keep Dosha in equilibrium state and to maintain health, for example Kapha Prakriti persons need more exercise, Laghu Ahara to keep their body fit and they should avoid Divaswapna, whereas Vataja Prakriti persons are advised to take nutritious and heavy food (guruahara), less exercise and can enjoy Divaswapna. In core, if one understand the dominant element of own constitution and attributes, they understand how to achieve balance in lives by avoiding what do not need and increasing that which are lacking. DISCUSSION Prakriti is one‟s own constitution and attitude. It is an expression of body functions, in the form of morphology, physiology, behavior of an individual. Two intermingling sets of Dosha, namely Prakrita or Arambhaka Dosha and Vaikrita or Ahara-MalaSambhavaja Dosha, steer this expression by virtue of their properties of Guna. Prakrita are responsible for seven types of Dosha Prakriti. Constitution is not a general survey observed on mass scale. It is individual specific and idiosyncratic. The clinical and therapeutic utility of knowledge of Prakriti has been described by Caraka, where the detailed schedules that are to be followed by different Dosaja Prakriti have been dealt separately (Trikamji Yadavaji,

2005). Sushruta has advised that the patient should be treated by physician according to Prakriti of person. Charaka Samhita is the only compendium, which describes Prakriti characters in accordance to every attribute of predominant Dosha. Pathological change in any property of Dosha is indicated by change in characteristics dented to that property. This makes any physician to plan his line of treatment in precise fashion. In Ayurveda, Prakriti is outcome of two factors. One of them is Prakrita Dosha. This is constant factor. This is individual specific arrangement of union of three sets of Panchamahabhuta namely paternal, maternal and that of a soul. Prakrita Dosha are the constant factor and is chief decisive directive of future Prakriti. Environmental factors are intra uterine and extra uterine. They influence a flexible factor, Vaikrita Dosha, therefore any flexibility in characteristics of Prakriti are due always and always to Vaikrita Dosha. Characteristics of an individual, which is output of combined effects of Prakrita Dosha and Vaikrita Dosha are titled as Prakriti. Charaka Samhita in Sharirasthana dictates all intra uterine factors and effect is already learnt of these factors on Prakriti. Extra uterine factors are dictated in Indriyasthana of Charaka Samhita as well as in Sutrasthana. They represent internal and external environmental effects on Prakriti. While enumerating factors and types of Prakriti, importance and application of them must be emphasized. Today when refined techniques have made it possible to reach micro world, need has arisen to notice and discuss details of fundamentals offered by science of Ayurveda. It must be agreed that Dosha Prakriti is constitution or Prakriti of an individual, expressed by conjoint output of Prakrita and Vaikrita Dosha. This constitution is fixed with flexibility and is flexible within fixity. Hence togetherness of Prakrita and Vaikrita Dosha, not only balance each other but balance health of individual.

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CONCLUSION Charaka Samhita, as said earlier, describes Prakriti in Sutrasthana to indicate that it is important to consider Prakriti for meaning health with the help of appropriate diet and daily routine. This is explained in seventh chapter of this compendium, „Na Vegannadharaniya’. Tenfold examination of patient is done to assess vitiated Dosha, its degree of vitiation. With the help of original

body constitution and responses of body, a physician can identify vitiated Dosha, its degree of vitiation and decide line of treatment. This is very important for diagnostic purpose. It is strongly mentioned that once formed Prakriti is also influenced though not changed due to environmental factors. Change is so impossible that any real drastic change in original characters should be taken seriously by Ayurvedic physician.

REFERENCES Trikamji Yadavaji (2005), Charaka Samhita, Sanskrit Commentary Ayurveda-Dipika by Chakrapanidutta Edited by Vaidya Yadavaji Trikamji Acharya, Reprint, Chaukhamba Surabharti Prakshan, Varanasi; pg. 52, 277, 276, 277, 308, 328, 354. Shastri Ambikadutta (2003), Sushruta Samhita, Hindi Commentary AyurvedatatvaSandeepika by Ambikadutt Shastri 4th edition, Chowkhamba Sanskrit Sansthan, Varanasi; pg. 353, 358, 360, 361, 362, 363. Sharma Shivprasad (2008), AstangaSamgraha, with the Sanskrit Commentaries, Sashilekha of Indu, Edited by Dr.Shivprasad Sharma, 4th edition, Chaukhambha Sanskrit Series Office, Varanasi; pg. 345, 344. Tripathi Brahmanand (2010), Sharangdhar Samhita, Amatel with Dipika hindi commentary by Brahmanand tripathi, 2nd edition, Chaukhamba Surbharti Prakashan, Varanasi; pg.134, 135.

Atridev Gupt (1997), Ashtang Hriday with Vidyotini hindi commentary, twelth edition, 4th edition Chaukhamba Sanskrit Bhawan, Varanasi; pg. 546, 548. Girijadayalu Suklah (1999), Bhela Samhita, Chaukhambha Bharati Academy, Reprinted, Varanasi; pg.267, 268. Ramavalamba Shastri (1985), Harita Samhita with Asha Hindi commentary; First edition, Prachya Prakashan, Varanasi; pg. 98. Hemaraja Sarma (1998), Kasayapa Samhita by Vrddha Jivaka, revised by Vatsya; 6th Edition, Chaukhambha Sanskrit Sansthan, Varanasi; pg. 324. Bhadanta Nagarjuna (1976), Rasa Vaisesika Sutram, Edited by N. E. Muttuswamy; 5th edition Kerala State Government; pg. 124. Laksmipati Sastri (1999), Yogaratnakara; Seventh edition, Chaukhambha Sanskrit Sansthan, Varanasi; pg. 235.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

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