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REGISTRATION OF SUBJECT FOR SYNOPSIS DEPARTMENT OF PHARMACOLOGY VYDEHI INSTITUTE OF MEDICAL SCIENCES & RESEARCH CENTRE #82, EPIP Area, Nallurahalli, Whitefield, Bangalore - 560066 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE, KARNATAKA. 1 Name of the Candidate and Address 2 Name of the Institution 3 Course of Study and Subject 4 Date of Admission to Course 5 Title of the Topic Dr. ANAND. M. INGALE. DEPARTMENT OF PHARMACOLOGY, VYDEHI INSTITUTE OF MEDICAL SCIENCES AND RESEARCH CENTRE, #82, EPIP AREA, NALLURAHALLI, WHITEFIELD, BANGALORE-560066. Vydehi Institute of Medical Sciences & Research Centre, Bangalore. M.D. PHARMACOLOGY 22-04-2011 Experimental evaluation of anti-ulcer activity of Grape Seed Extract in Wistar Albino Rats. 1 6. BRIEF RESUME OF THE INTENDED WORK: 6.1 NEED FOR THE STUDY: Antioxidants are compounds that inhibit oxidation process and thereby eliminate the threat of pathological processes. During environmental stress, elevation of corticosteroids accelerates the generation of free radicals and suppresses immune function. Antioxidants are effective in sheerful conditions, as they readily give up their own electron to free radicals. It has been found that oxygen-derived free radicals are implicated in the mechanism of acute and chronic gastric mucosal ulceration, and scavenging these free radicals plays an appreciable role in healing ulcers. Phenolic compounds present in medicinal plants, have been reported to possess powerful antioxidant activity1. Herbal products are gaining a lot of demand these days because of their safety and efficacy. They are more compatible and economical. Grape seed is a waste product in winery with 6090% procyanidine, which is potent antioxidant. Grape Seed Extract (GSE) has been shown to possess various activities like antioxidant, antibacterial (Helicobacter Pylori, MethicillinResistant Staphylococcus Aureus), anticarcinogenic, antifungal, antihypolipidemic, antihepatogenic 1, 2. Since antiulcerogenic effect of GSE using various models simultaneously has not been done, this study is undertaken to establish the same. Establishment of its antiulcer activity will provide a newer and economically better modality of treatment for peptic ulcer, which is a more common present-day ailment. 6.2 REVIEW OF LITERATURE: Shi J et al of Korea, in 2003, have elaborately elicited the biochemistry and functions of grape seeds. Grape seed possess lipid, protein, carbohydrates, and polyphenols depending on the variety of grapes. Flavonols are major polyphenols of grapes. Flavanols have generated remarkable interest based on positive reports of their antioxidant properties and ability to serve as free radical scavengers. The beneficial effects of grape seed polyphenols is superior to other well known antioxidants, such as vitamin C, vitamin E and beta carotene. Antioxidant potency of proanthrocyanidins, a phenol, is 20 times greater than vitamin C and 50 times greater than vitamin E. The total extractable phenolics in grape are present at only about 10% or less in pulp, 28-35% in the skin and 60-70% in the seeds1. Maheswari U.M. of Coimbatore, in 2004, assessed the antihepatotoxic effect of grape seed oil in rats. It was found out that oral administration of Grape Seed Oil in rats, significantly decreased serum hepatic enzymes (malondialdehyde and hydroperoxide), which were elevated after carbon tetrachloride (CCl4) treatment. Grape Seed Oil had no effect in control rats. Hepatoprotective activity is attributed to the antioxidant mechanism which is brought about by protecting against free-radicals2. 2 Brown J. C. et al of America, in 2008, explored the alternate methods to treat Helicobacter Pylori [H. Pylori] by determining its activity using agar dilution, confocal laser scanning microscopy and cell proliferation assays following treatment with various grape extracts. The study was done in-vitro and their activity against H. Pylori was established. The study suggests that, higher phenolic levels do not necessarily determine overall anti-helicobacter pylori activity. Their study showed Muscadine grape skin to possess the strongest activity, followed by grape synergy (skin and seed) and seed3. Sreemantula S et al of Vishakapattanam, in 2005, assessed the adaptogenic and nootropic activities of grape seed. In their study, rats treated with grape seed, were subjected to forced swim test. 24 hour excretion of urinary vanillylmandelic acid (VMA) and ascorbic acid was assessed. Their levels were compared with that of normal rats. Comparison of their values established the anti-stress (adaptogenic) action of GSE. The cognition, as determined by the acquisition, retention and recovery, was found to be dose dependent. The conditioned avoidance response analysis, established its nootropic activity. The antioxidant activity was determined based on its ability to scavenge the hydroxyl radicals, as measured by studying the competition between deoxyribose and the extract for hydroxyl radicals4. Saito M et al of Japan, in 1998, assessed the gastroprotective activity of flavanols and procyanidines present in grape seed in rats. They used grape seed having lower flavanol content, higher flavanol content and procyanidins to assess their gastroprotective activity. The dose used by them was 200mg/kg body weight. Ulcer was induced by 60% Ethanol containing 150mM Hydrochloride. It was seen that the test compounds used strongly inhibited the stomach mucosal injury. The mechanism of flavanol was by protection against radical injury and of procyanidines by covering the gastric mucosal surface5. 3 6.3 OBJECTIVES OF THE STUDY: 1. To demonstrate the Anti-Ulcer Activity of GSE using the following Experimental Rat Ulcer Models: A. Aspirin (200mg/kg) and Pylorus ligation model with standard of Ranitidine (50mg/kg) -Anti-secretory mechanism B. Hydrochloric Acid [HCl] (0.3M) – Ethanol (60%) model with standard of Sucralfate (100mg/kg) -Cytoprotective mechanism C. Water-immersion Stress model with standard of Omeprazole (20mg/kg) - Proton Pump Inhibition mechanism 2. To compare the anti-ulcer efficacy of GSE with that of respective standard drug. 3. To evaluate the efficacy of treatment using two doses (100mg/kg body weight, 200mg/kg body weight) of GSE. 4. To determine the probable anti-ulcer mechanism of GSE. 7. MATERIALS AND METHODS: 7.1 Source of Data: Inbred Wistar Albino Rats of either sex, weighing 180 - 250 grams, from Institutional Animal House, housed under standard conditions, caged separately. 7.2 Methodology of Study: Design and Sample size: Three models, each model comprising of four groups, with each group having 6 rats, 72 rats in total. Groups: I. II. III. IV. Control(Gum Acacia) Standard(STD) drug GSE (100mg/kg body weight) GSE (200mg/kg body weight) o All drugs are administered orally. o Rats will be sacrificed by decapitation. 4 Methodology: o Model A: Rats are fasted (water allowed), 200mg/kg Aspirin is given once for 3 consecutive days. On the fourth day, test compounds are administered. After 30minutes, pylorus is ligated under anaesthesia. Four hours later, rats will be sacrificed6,7. o Model B: Rats are fasted (water allowed) for 1day. Next day, test compounds are given. After 30mins, 1ml HCl and Ethanol are administered. 1hour later, rats are sacrificed7. o Model C: Rats are fasted for 1day (no water). Next day, test compounds are given. Rats will be subjected to forced swimming in standard glass cylinder (height-45cms, diameter25cm) containing 35cms of water for 3 hours. Rats will be sacrificed. Analysis: Stomach is excised; Contents-drained into centrifuge tube; Stomach - histologically analysed. Statistical analysis employed: ANOVA and Student t test Outcome measures: Free and Total acidity Ulcer severity Ulcer index Does this study require any Investigations or Interventions to be conducted on patients or other humans or animals? If so, describe briefly Yes. Gross evaluation of ulcer number and ulcer size Biochemical evaluation of free and total acidity Has ethical clearance been obtained from your institution: Submitted to Institutional Animal Ethical Committee for approval 5 8. List of references: 1. Shi J, Yu J, Pohorly J.E., Kakuda Y. Polyphenolics in Grape Seeds- Biochemistry and Functionality. Journal of Medicinal Food. 2003; 6(4): 291-299. 2. Maheswari U.M., Rao P. G. M., Antihepatotoxic effect of grape seed oil in rat. Indian Journal of Pharmacology. 2004; 37(3): 179-182 3. Brown J.C., Huang G, Haley-Zitlin V, Jiang X. Antibacterial effects of grape extracts on Helicobacter pylori. Application of Environmental Microbiology. 2009; 75(3):84852. 4. Sreemantula S, Nammi S, Kolanukonda R, Koppula S, Boini K M. Adaptogenic and nootropic activities of aqueous extract of Vitis vinifera (grape seed): An experimental study in rat model. Biomed Central Complement of Alternate Medicine. 2005; 5:1. 5. Saito M, Hosoyama H, Ariga T, Kataoka S, Yamaji N. Antiulcer activity of Grape Seed Extract and Procyanidins. Journal of Agricultural Food Chemistry. 1998; 46(4): 1460-1464. 6. Shay H, Komarov S A, Fels S E, Meraze D, Gruenstein M, Siplet H. A simple method for the uniform production of gastric ulceration. Gastroenterology. 1945; 5 : 43–61. 7. Bickel M. Activity on gastrointestinal tract in Drug discovery and evaluation. Pharmacological assays. Editors: Vogel G H, Vogel W H, Scholkens B A, Sandow J, Muller G, Vogel W F. 2nd edition. Springer Publications. Germany. 2002: 867-869. 6 9 Signature of the candidate: 10 Remarks of the Guide: 11 11.1 Name & Designation Dr. (Mrs.) VIJAYA RAJENDRAN. M.D. of the Guide Professor , Department of Pharmacology, Vydehi Institute of Medical Sciences & Research Centre, Bangalore 560066. So far no study has been done on the antiulcer activity of grape seed extract in India, which is a most welcoming study in Bangalore. 11.2 Signature 11.3 Co-Guide (if any) 11.4 Signature 11.5 Head of Department Dr. B.N.SRINIVAS. M.D. Professor & Head of Department, Department of Pharmacology, Vydehi Institute of Medical Sciences & Research Centre, Bangalore 560066. 11.6 Signature 12. 12.1 Remarks of Chairman & Principal 12.2 Signature 7