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EVALUATION OF ANTI-DIABETIC ACTIVITY OF ETHANOLIC EXTRACT OF HOLARRHENA ANTIDYSENTRICA LEAVES. MASTER OF PHARMACY DISSERTATION PROTOCOL SUBMITTED TO THE RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE BY JAISAL .K .K Under The Guidance of MR. MOSES SAMUEL RAJAN M.PHARM DEPARTMENT OF PHARMACOLOGY, SRINIVAS COLLEGE OF PHARMACY MANGALORE – 574 143 2012 – 2013 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE, KARNATAKA. ANNEXURE-II PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION 1.0 NAME AND ADDRESS OF THE CANDIDATE 2.0 NAME OF THE INSTITUTION 3.0 COURSE OF STUDY AND SUBJECT 4.0 SRINIVAS COLLEGE OF PHARMACY VALACHIL, MANGALORE- 574 143 MASTER OF PHARMACY IN PHARMACOLOGY DATE OF ADMISSION TO COURSE 5.0 JAISAL . K .K KARIKULAKKATTIL HOUSE CHERIYAMUNDAM (P.O) MACHINGAPPARA MALAPPURAM- 676 106 KERALA TITLE OF THE TOPIC 26-06-2012 “EVALUATION OF ANTI-DIABETIC ACTIVITY OF ETHANOLIC EXTRACT OF HOLARRHENA ANTIDYSENTRICA LEAVES”. 2 6.0 BRIEF RESUME OF THE INTENDED WORK : 6.1 NEED FOR THE STUDY Diabetes mellitus has been known to medical sciences longer than any other hereditary metabolic diseases. It is a chronic metabolic disorder characterized by hyperglycemia caused by insulin deficiency often combined with insulin resistance. Two main types of diabetes mellitus: type I and type II. In type I diabetes, there is an absolute deficiency of insulin resulting from autoimmune destruction of β cells. Without insulin treatment, such patients will ultimately die with diabetic ketoacidosis. Type II diabetes is accompanied by both insulin resistance and by impaired insulin secretion. Such patients are often obese and usually present in adult life, the incidence rising progressively with age as β cell function declines. In conventional therapy, type I diabetes is treated with exogenous insulin and type II with oral hypoglycemic agents 1. India is the diabetic capital of the world, predicted to have 57.2 million diabetic populations by the year 2025 2. Diabetes mellitus is a disease in which homeostasis of carbohydrate, protein and lipid metabolism is improperly regulated by hormone insulin resulting in elevation of fasting and postprandial blood glucose levels 3. The major chronic complications associated with diabetes include retinopathy, neuropathy, nephropathy, atherosclerotic coronary artery disease and peripheral atherosclerotic vascular diseases. Besides hyperglycemia, several other factors like hyperlipidemia and enhanced oxidative stress play a major role in diabetic pathogenesis. Despite the great strides that have been made in the understanding and management of this disease, the graph of diabetes-related mortality is rising unabated. Although a number of synthetic drugs are available in the market, diabetes and its related complications still remain uncontrolled 4. The existing methods of treatment of diabetes are not completely satisfactory owing to low efficacy, associated adverse effects and compliance issues. Among the therapies non pharmacologic therapy (e.g. diet, exercise and weight loss) remains to be critical 3 component in diabetes treatment. Dietary management includes the use of traditional medicines mainly derived from plants. Traditional medicinal plants have been used successfully since ancient times by physicians to treat diabetes and its related complications, presenting a stirring prospect for the expansion of an alternative way of treatment of diabetes. The multiple activities of plant based medicinal preparations offer an enormous scope for not only to control the hyperglycemia but also its late complications. Moreover, WHO study groups have emphasized strongly the optimal rational uses of traditional medicines because the theory of polyherbal formulations having synergistic, potentiative, agonistic, antagonistic pharmacological actions within themselves work together in a dynamic way 5. Glibenclamide is an anti-diabetic drug coming under the class 2nd generation sulfonylurea. As of 2007, it is one of only 2 oral anti-diabetic drugs in the World Health Organization Model list of Essential Medicines. It is one of the major drugs used in diabetic therapy extensively due to its abundant availability and low cost. It acts by inhibiting ATP sensitive K+ channel in pancreatic β cell which finally leads to stimulation of insulin release. It is 150 times more potent than 1st generation sulfonylurea like tolbutamide, hence may cause severe hypoglycemia and may lead to even coma state 6. The drug is poorly soluble or practically insoluble in water. Advanced studies show that it is associated with a marginal decrease in circulating lipids. Also cause rare side effects like weight gain due to fluid retention and edema, photosensitization reaction, cholestatic jaundice 7. Holarrehena antidysentrica: belonging .to family Apocynaceae, commonly known as kutaja, or kurchi. Distributed in Asia, tropical area of Africa. Madagascar, India. . Growing up to an altitude of 1300 m in the Himalayas, often gregariously in deciduous forests. Open waste land and abundant in sub Himalaya tract. The plant has been employed for long time in folkfore therapy. Kurchi bark is a important traditional drug used in various ailments. The drug is astringent, anthelmentic, stomachic, antipyretic and also anti diabetic. Kurchi is given either alone or with other astringent drugs. A hot decoction of the drug is used as a gargle in toothache. It consist certain 4 biochemical constituent namely alkaloid, glycoside, phenolic compounds and tannins. the drug is collected from the wild source and varies in constituent and efficacy due to the geographical diversity. Holarrhena antidysentrica is an important plant employed in various indigenous system of medicine against several disease, and almost every part of plant has diverse medicinal properties, the results of the study could be useful in setting some diagnostic indices for the identification and preparation of a monograph of the plant8. 6.2 REVIEW OF LITERATURE: Holarrhena antidysentrica: Description: . H. antidysenterica is a deciduous laticiferous shrub or small tree, up to 13 m high and 1.1 m in girth with clear bole of 3-7 m. Leaves 15-30 x 4-12 cm, base obtuse, rounded or acute, nerves 10-14 pairs, opposite, sessile, elliptic or ovate, oblong, membranous, strong, arched; petiole up to 1.5 cm; cymes 3-6 cm diameter. Corymbose sessile terminal; bracts small, ciliate; pedicels slender. Flowers inodorous white, in terminal corymbose cyme. Calyxlobe 2.5-3 mm long, oblong-lanceolate, acute, ciliate.Corolla puberulous outside; tube 8-13 mm long . A large shrub or small tree grows up to 13 meters in height. Leaves simple, opposite, ovate, acute-acuminate, membranous; flowers white in axillary or terminal corymbose cymes; fruits long are pair of long follicles seeds long, brown, with silky hairs, aided in dispersion., Is an important plant used in indigenous system of medicine as remedy for bronchitis, hematuria spermatorrhoea, epilepsy, asthma, piles, leprosy eczema, diarrhea, fever and jaundice. Various part of holarrhena antidysentrica have been reported to possess antibacterial activity. The bark has been reported to possess astringent and anti diarrheal properties. leaves of the plant are used to cure scabies. 5 However, a major constituent which has hindered the acceptance of alternative medicine is the lack of documentation and stringent quality control with this backdrop, it is of the prime important to make an effort towards standardization of the plant material. The process of standardization is achieved pharmacognostic by step wise studies which in turn help to identify and authenticate plant material9. Chemical constituent: Conessydjene, connessimine, conkurchine, holadiene, holarrhejne, holarrhimine, kurchine, holarrhine, kuchisyne. Kurchi phylammine. Synonyms: Kuda (Hindi) Kurchi (English) Kodipla (Telungu) Kodagapaia (Malayalam) Veppajaj (Tamil) Part used : Seed, Leaf, Stem bark, Reported activities: Blood purifier, Anti-pyretic, Skin diseases, Abdominal disorders, Anti-diabetic activity 6.3 OBJECTIVES OF THE STUDY: The main objectives of the present study are as follows:1. To prepare the ethanolic extract of the leaves of kurchi (Holarrhena antidysentrica) 2. To study the anti-diabetic activity of Kurchi by using following animal models 6 I. Alloxan Induced Diabetes In Rats The antidiabetic activity will be evaluated by using following parameters a) Serum glucose level will be checked for every alternate day b) Body weight will be measured weekly once. c) Water and s intake will be measured once in a week. Biochemical Estimation: After the completion of treatment, blood will be withdrawn from retro orbital sinus and estimated for following parameters i) Serum glutamic pyruvate transaminase (SGPT) ii) Serum glutamic oxaloacetate transaminase (SGOT) II. Streptozotocin Induced Diabetes In Rats The antidiabetic activity will be evaluated by using following parameters a) Serum glucose level will be checked for every alternate day b) Body weight will be monitored weekly once. c) Water and food intake will be measured once in a week. Biochemical Estimation: After the completion of treatment, blood will be withdrawn from retro orbital sinus and estimated for following parameters i) Serum glutamic pyruvate transaminase (SGPT) ii) Serum glutamic oxaloacetate transaminase (SGOT) III Oral Glucose Tolerance Test in rats The blood glucose concentrations will be determined at 30, 60, 120 and 180 minutes after dosing 7.0 MATERIALS AND METHODS: 7.1 SOURCE OF DATA: Experiment will be performed as described in the standard bibliography, literatures and text books. The reputed journals and publications are obtained from college library and through web search. 7 7.2 ANIMALS Wistar albino rats (180-200 g) of either sex will be used for the study. They will be maintained under standard conditions (temperature 22 ± 2oC, relative humidity 50±5% and 12 h light/dark cycle) and have free access to standard pellet diet and water ad libitum. All experimental protocols will be reviewed and approved by the Institutional Animal Ethical Committee (IAEC) prior to the initiation of the experiment and the care of the laboratory animals will be taken as per the CPCSEA regulations. 7.3 CHEMICALS Alloxan monohydrate, Streptozotocin will be procured from Loba chemie, Mumbai, India. Glibenclamide will be purchased from Cipla ltd ,mumbai, India. Diagnostic kits to estimate serum parameters will be obtained from Mannheim diagnostic kits India Ltd. All other chemicals used in the experiment will be of analytical grade obtained from local suppliers. 7.4 COLLECTION OF THE DRUGS AND PREPARATION OF THE EXTRACTS: The leaves of holarrhena antidysentrica is procured either from commercial herbal stores or from natural source. Fresh Leaf of holarrhena antidysentrica will be cleaned, cut in to small pieces and shade dried or, in an incubator for 2-3 days at 400 C. Crushed it in an electric grinder and then powdered. Make extracts of powder by using Ethanol. Extracts will be prepared by over night maceration technique. The concentration of this kurchi preparation was considered to have 300mg/ml on the basis of the weight of the starting material according to the formula. 8 7.5 EXPERIMENTAL METHODS : 1. I. ALLOXAN INDUCED DIABETES IN RATS 9 Diabetes will be induced in overnight fasted animals by single intraperitoneal injection of alloxan monohydrate prepared in normal saline (100mg/kg body weight). After a fortnight, diabetes will be confirmed by hyperglycemia. The animals showing fasting blood glucose level more than 250mg/dl will be considered to be diabetic and included for the study. EXPERIMENTAL DESIGN : Animals will be randomly divided into 4 groups of 5 each. The different groups will be assigned as follows Group I: Vehicle control (normal saline). Group II: Diabetic control (Alloxan100mg/Kg). Group III: Diabetic + Glibenclamide (1mg/Kg) Group IV: Diabetic + Kurchi(300mg/kg13) EVALUATION: Starting from the first day of treatment, blood will be collected from all animals and blood glucose level will be estimated every alternate day. Daily body weight, food and water intake will be monitored for 30 days. On 30th day post treatment blood will be collected and serum will be separated by centrifugation and evaluated for biochemical parameters. II. STREPTOZOTOCIN INDUCED DIABETES IN RATS 10 Diabetes will be induced in overnight fasted animals by single intraperitoneal injection of Streptozotocin (65 mg/kg) prepared in normal saline. After 48 hours of STZ injection, blood samples will be taken and glucose estimation is done. Animals having fasting blood glucose level more than 250mg/dl will be chosen for studies. In spite of diabetes developed after 48h of STZ injection, the animal will be stabilized up to 6 days. The treatment will be started from 7th day of STZ injection once daily post orally for next 30 days. 9 EXPERIMENTAL DESIGN : Animals will be randomly divided into 4 groups of 5 each and assigned as below. Group I: Vehicle control (normal saline). Group II: Diabetic control (Streptozotocin 65mg/Kg). Group III: Diabetic + Glibenclamide (1mg/Kg) Group IV: Diabetic + Kurchi(300mg/kg13) EVALUATION: Starting from the first day of treatment, blood will be collected from all animals and blood glucose level will be estimated. Daily body weight, food and water intake will be monitored for 30 days. On 30th day post treatment blood will be collected and serum will be separated by centrifugation and evaluated for biochemical parameters. 2. III. ORAL GLUCOSE TOLERANCE TEST (OGTT) IN RATS 11 After overnight fasting (16h) the blood glucose level of rats will be determined. The animals will be treated with test samples after administrating oral glucose of 2g/Kg body weight in 0.2ml water. EXPERIMENTAL DESIGN : The animals will be randomly divided into 3 groups of 5 each and assigned as below Group I: Glucose control. Group II: Glucose load + Glibenclamide (1mg/Kg). Group III: Glucose load+ Kurchi (300mg/kg13) EVALUATION: The blood glucose concentration will be determined at 30, 60, 120 and 180 min after dosing. 10 7.5 STATISTICAL ANALYSIS: The data will be expressed as Mean value + SEM and will be analyzed by the one-way ANOVA. 7.6 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY. Yes. Study requires investigation on Wistar rats. 7.7 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION? Yes. Ethical clearance has been obtained. (Copy enclosed) 11 8.0 REFERENCES: 1) Rang HP, Dale MM, Ritter JM, Flower RJ. Rang and Dale’s pharmacology. 6th ed. Churchill Livingstone Elsevier Ltd; 2007. p. 402-4. 2) Pradeepa R, Deepa R, Mohan V. Epidemiology of diabetes in India- current perspective and future projections. J Indian Med Assoc;100: 144-8. 3) Tiwari AK, Rao JM. Diabetes mellitus and multiple therapeutic approaches of phytochemicals: Present status and future prospects. Curr Sci 2002;83: 30-8 4) Kaczmar T. Herbal support for diabetes management. Clin Nutr Insights 1998;6: 1-4 5) Sundaram EN, Reddy PUM, Singh KP. Effect of alcoholic extracts of Indian medicinal plants on the altered enzymatic activities of diabetic rats. Indian J Pharm Sci 2009;71(5): 594-8 6) Sharma HL, Sharma KK. Principles of pharmacology. Paras Medical publisher; 2007. p. 652-3 7) David EG, Armen H, Tashijan N, Ehrin JA, April WA. Principles of pharmacology. 2nd ed. Lippincott’s, William & Wilkins; 2008. p. 540 8) Parwaiz Akhtar, Mohd ali, M P Sharma, Humaira Farooqi, Showkat R. Mirand Hmid Nawaz Khan. Development of quality standard of Holarrena antidysentrica ( linn) Bark. RRST 2011; 3(1):73-80 9) P.S. Sujan ganapathy,Y.L Ramachandran, H.v sudeep. Pavan kumar bellamokondi. pharmacognostic and phytochemicalevaluation of Holarrhena antidysentrica wall. AAJPSB 2009; 3(1): 47-50 10) Sharma N, Garg V. Anti-hyperglycemic and anti-oxidative potential of hydro alcoholic extract of Butea monosperma Lam flowers in alloxan-induced diabetic mice. Indian J Exp Biol 2009;47: 571-6 11) Bajaj S, Srinivasan BP. Investigations into the anti-diabetic activity of Azadirachta indica. Indian J Pharmacol 1999;31: 138-41 12 12) Bhavana S, Santosh K, Satapathy N. Hypoglycemic and hypolipidemic effect of Aegle marmelos leaf extract on STZ induced diabetic mice. Int J Pharmacol 2007;3(6): 444-52. 13) Keshri umarshankar pd, satish Chandra, janardan Sharma. Antidiabetic activityof ethanolic extract of holarrhena antidysentrica seeds in streptoztocine-induced diabetic rats and its influence in certain biochemical parameters. JDDT 2012;2(4):159-162. 13 9. SIGNATURE OF THE CANDIDATE 10. REMARKS OF THE GUIDE 11. NAME AND DESIGNATION Recommended and Forwarded MR. MOSES SAMUEL RAJAN ASSO. PROFESSOR DEPARTMENT OF PHARMACOLOGY SRINIVAS COLLEGE OF PHARMACY 11.1 GUIDE 11.2 SIGNATURE OF GUIDE 11.3 CO - GUIDE (IF ANY) Not Applicable 11.4 SIGNATURE - 11.5 HEAD OF THE DEPARTMENT MR. MOSES SAMUEL RAJAN ASSO. PROFESSOR DEPARTMENT OF PHARMACOLOGY SRINIVAS COLLEGE OF PHARMACY MANGALORE- 574 143. 11.6 SIGNATURE OF HEAD OF DEPARTMENT 12 12.1 REMARKS OF THE PRINCIPAL 12.2 NAME & SIGNATURE OF PRINCIPAL DR. A. R. SHABARAYA PRINCIPAL, SRINIVAS COLLEGE OF PHARMACY MANGALORE- 574 143. 14