A COMPARATIVE EVALUATION OF THE ANTI-ULCER ACTIVITY OF THE EXTRACTS OF SEED AND SKIN OF VITIS VINIFERA (GRAPE) IN WISTAR ALBINO RATS

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1 Pharmacology International Journal of Clinical And Diagnostic Research Volume 2, Issue 2, Mar-Apr GloriginLifesciences Private Limited. Original Research A COMPARATIVE EVALUATION OF THE ANTI-ULCER ACTIVITY OF THE EXTRACTS OF SEED AND SKIN OF VITIS VINIFERA (GRAPE) IN WISTAR ALBINO RATS Ingale Anand M. 1, Rajendran Vijaya 2, Venkata BharatKumar Pinnelli 3 Abstract Background: Peptic ulcer disease is a most common disease, whose incidence is increasing presently on account of demanding stressful life. Objectives: To evaluate and compare the antiulcer activity of the extracts of seed and skin of Vitis Vinifera (Grape) using two different doses (100 & 200 mg/kg). Methods: The extracts of seed and skin of Vitis Vinifera were investigated for their anti-ulcer activity using two doses (100 & 200 mg/kg) against aspirin plus pylorus ligation induced gastric ulcer in rats. Wistar albino rats ( grams) of either sex were divided into 6 groups, with 6 rats per group. All drugs were given orally. All received 200mg/kg Aspirin for 3 days along with Distilled water / Ranitidine / Test compound (water, food ad libitum). On the 4th day, pylorus was ligated under light ether anaesthesia (after 36 hours fasting). 4 hours later, rats were sacrificed by decapitation. The stomach was opened. Contents were collected and titrated against 0.01N NaOH(Sodium hydroxide) for free acidity and total acidity using indicators Topfer s reagent and Phenolphthalein. Ulcers were examined under magnification. Results: A significant reduction (p <0.001) was observed in ulcers, gastric volume, free and total acidity in all groups as compared to the control group. Grape seed 200 mg/kg showed maximum effect in all parameters. Conclusion: Our study indicates that the extracts of seed and skin of Vitis Vinifera has potential anti-ulcer activity in aspirin plus pylorus ligation model, with grape seed 200mg/kg having the best activity. Author Affiliations: 1 Post Graduate Student, Department of Pharmacology, 2 Professor, Department of Pharmacology, 3 Associate Professor, Department of Biochemistry. Vydehi Institute of Medical Sciences and Research Centre, #82, EPIP Area, Nallurhalli, Whitefield, Bangalore , Karnataka Keywords: Vitis vinifera, Anti-ulcer, Aspirin, Pylorus ligation, Free acidity, Total acidity * Corresponding Author: Dr. Venkata BharatKumar Pinnelli, PhD, Associate Professor, Department of Biochemistry, Vydehi Institute of Medical Sciences and Research Centre, #82, EPIP Area, White Field, Bangalore Contact # , ID: pvbharatkumar@yahoo.co.in.

2 1. INTRODUCTION Peptic ulcer disease (PUD) remains one of the most important health conditions of the alimentary tract affecting approximately 5 to 10 % of the population during their lifetime [1] with the estimated lifetime prevalence being 12 % for men and 9% for females. [2] Its prevalence is quite high in South India, where rice is the staple food. [3] The current pharmacological treatment of peptic ulcer is generally based on triple therapy regimen (Anti - Helicobacter Pylori drugs), inhibition of gastric acid secretion by histamine H 2 - antagonists, proton pump inhibitors, mucosal protective therapy provided by sucralfate and bismuth. [4] Although a number of anti-ulcer drugs are available, all these drugs have side effects and limitations. A large number of plant products, including Indian medical plants [5], are being evaluated for their anti-ulcer effects to achieve a favorable outcome. [6] Grapes [Vitis vinifera (V. vinifera)] are deciduous plants characterized by woody, climbing vines that are generally twelve to twenty meters long, mostly found in Europe and parts of Asia. Bulk of grapes is utilized winery, of which the Grape seeds are the waste products. Grape seed extract (GSE) is composed of nearly 60-90% of procyanidine, which is potent antioxidant and scavenge free radicals. [7, 8, 9,10,11,12] GSE possesses various activities. Grape skin extract (GKE) possesses 10-15% of proanthocyanidin and is proven of many activities. [13,14,15] The present study was undertaken to evaluate / investigate the anti-ulcer activity of the extracts of grape seed and skin extract against Aspirin plus Pylorus ligation induced gastric ulcers in rats (to evaluate H 2 blocker mechanism) by determining and comparing the ulcer number, ulcer inhibition; free and total acidity; ulcer severity and ulcer index. 2. MATERIALS AND METHODS: The study protocol is carried out only after being approved by our Institutional Animal Ethical Committee. The Guidelines of Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA) have been followed throughout the study. 2.1 Plant extract: The ethanolic extract of Grape Seed was procured from Olive life sciences, Bangalore and the ethanolic extract of Grape Skin was procured from Vidya Herbs, Bangalore. 2.2 Animals:

3 Inbred Wistar albino rats (Ratus Norvegicus) of either sex weighing 150 to 250 grams, acclimatized to the laboratory conditions for two weeks, were used for the study. They were housed in clean polypropylene cages, in a well-ventilated room at controlled temperature ( C) with a 12 hour light and dark cycle. All experiments were performed during the same time of the day to avoid diurnal variations of gastric functions. 2.3 Drugs and chemicals: All the chemicals used in this evaluation were of high grade and purity. Ranitidine powder [Saraca Laboratories Limited] Aspirin powder [Medopharm] Topfers reagent [Merck & Co.] Phenolphthalein indicator, Sodium hydroxide (0.01N) [S. D. Fine Chem. Ltd] 2.4 Aspirin plus pylorus ligation induced gastric ulcer in rats: The procedure was followed according to Malairajan et al. [16] The rats were divided into 6 groups (n=6). All the animals received 200 mg/kg of aspirin (suspended in 1% carboxy methyl cellulose) once daily for three days. Group I (Control) received 1ml distilled water. Group II (Standard) treated with 50 mg/kg Ranitidine. Groups III and IV treated with 100 mg/kg GSE and GKE respectively. Groups V and VI treated with 200mg/kg of GSE and GKE respectively. On the fourth day, pylorus part was ligated following 36 hour fasting under light ether anaesthesia and replaced carefully. Four hours after the pyloric ligation the animals were sacrificed by decapitation. The following parameters were analyzed: Gastric volume (in ml): The stomach content was centrifuged at the speed of 1000 revolutions per minute for 20 minutes and its volume was estimated. [17] Free and total acidity: 1ml of supernatant liquid is pipetted and diluted it to 10ml with distilled water. The solution was titrated against 0.01N NaOH using Topfer s reagent (Dimethyl-amino-azobenzene with phenolphthalein) as indicator. The end point was noted when the solution turns to orange color; this corresponds to the free acidity. Titration was continued further till the solution regained pink color to evaluate total acidity. Acidity was calculated by using the formula: Acidity = Volume of NaOH x Normality x 100 / 0.1mEq/lt/100g. [18] Ulcer number: The gastric mucosa was observed macroscopically under 10x magnifications for the number of superficial ulcers, deep

4 ulcers and perforations. The total number of ulcers in each rat was recorded. [17] Ulcer severity: The ulcers in each rat were graded [19] as: 0 - no ulcers; 1 - superficial ulcers; 2 - deep ulcers; 3 perforations Average severity score of each group was calculated Ulcer index: Ulcer index in each group was calculated by the formula [19] : UI = UN + US + UP*10-1 Where, UI = Ulcer Index; UN = Average of number of ulcer per animal; US = Average of severity score and UP = Percentage of animal with ulcer. 2.5 Statistical analysis: All data were expressed as Mean ± SEM. The statistical analysis of all the results was carried out using One-way Analysis Of Variance followed by Dunnet s multiple comparisons using graph pad in stat 3 and all the results obtained in the study were compared with the vehicle control group. Values were considered statistically significant at p < RESULTS: In Aspirin plus Pylorus ligation induced gastric ulcer model, pretreatment with the extract of the grape seed of 100mg/kg and 200mg/kg showed significant (p<0.05) reduction in number of ulcers, free and total acidity as compared to the control group. Pretreatment with Standard [Ranitidine 50mg/kg/p.o.] significantly reduced the gastric volume, free and total acidity and the number of ulcers. The average severity score was 1.5 and the ulcer index was Pretreatment with 100 mg/kg/p.o. of the grape seed extract caused significant reduction in the gastric volume, free and total acidity and number of ulcers. The average severity score was 3 and the ulcer index was Pretreatment with 100 mg/kg/p.o. of the grape skin extract caused significant reduction in the gastric volume, free and total acidity and number of ulcers. The average severity score was 3 and the ulcer index was Pretreatment with 200 mg/kg/p.o. of the grape seed extract caused significant reduction in the gastric volume and number of ulcers. The average severity score was 2 and the ulcer index was Pretreatment with 200 mg/kg/p.o. of the grape skin extract caused significant reduction in the gastric volume, total acidity and number of ulcers. The average severity score was 2.67 and the ulcer index was

5 TABLE 1: Effect of Extracts of Seed and Skin of V. Vinifera on Aspirin plus Pylorus Ligation Induced Gastric Ulcer in Rats Gastric volume (in ml) CONTROL STANDARD GSE-1 GKE-1 GSE-2 GKE ± ± 0.07 * 2.38 ±0.06 * 2.4 ± 0.06 * 2.16 ± 0.19 * 2.3 ± 0.1 * Free Acidity (in meq/l/100g) 60.5 ± ± 1.26 * ±2.77 * ± 4.41 * ± ± 1.67 Total Acidity (in meq/l/100g) 89.5 ± ± 0.84 * ± 2.6 * ± 6.02 * 70 ± ± 2.23 * Values are expressed as Mean ± SEM (n=6). *p < 0.05 as compared to control; TABLE 2: Effect of Extracts of Seed and Skin of V. Vinifera on Aspirin plus Pylorus Ligation Induced Gastric Ulcer in Rats Number of ulcers CONTROL STANDARD GSE-1 GKE-1 GSE-2 GKE ± ± 1.08 * ± 1.52 * ± 2.22 * ± 1.66 * ± 1.4 * Values are expressed as Mean ± SEM (n=6). *p < 0.05 as compared to control TABLE 3: Effect of Extracts of Seed and Skin of V. Vinifera on Aspirin plus Pylorus Ligation Induced Gastric Ulcer in Rats Ulcer inhibition (in %) CONTROL STANDARD GSE-1 GKE-1 GSE-2 GKE Ulcer severity score Ulcer index

6 4. DISCUSSION In the model tested, the extracts of seed and skin using doses of 100 and 200mg/kg body weight were used for the experiment. [20] They caused improvement in the parameters tested in our study indicating their antiulcer activity. The extract of the seed and skin of grapes reduced the gastric volume, free and total acidity, number of ulcers and ulcer index showing the antisecretory mechanism involved in the extracts for their antiulcer activity. Ulcer index parameter was used for the antiulcer evaluation since ulcer formation is directly related to factors such as gastric volume, free and total acidity. Here, the extract of seed with higher dose (200mg/kg) showed maximum activity and efficacy as an antiulcer drug. Ligation of the pylorus causes stasis of the acidic gastric contents in the stomach, which further causes auto digestion of the gastric mucosa precipitating an imbalance between the aggressive and defensive factors operating in the gastric lumen giving rise to ulcers in the rat stomach. There is also breakdown of the gastric mucosal barrier. [17] Aspirin, which is a NSAID, causes irreversible inhibition of the cyclo-oxygenase enzyme that synthesizes prostaglandins, which have a protective role in the gastric mucosa. Inhibition of prostaglandins diminishes the defensive ability of the stomach giving rise to ulcer formation. Aspirin increases acid secretion and back diffusion of H + ions into the mucosa [21] and increase the stress in the gastric lumen. They increase the generation of free radicals that precipitates the oxidative stress in the gastric lumen leading to ulcer formation. Synergistic effect of pylorus ligation and aspirin precipitates ulcer formation and enhances the efficacy of screening anti-ulcer agents. [19] The antiulcer activity of the extracts could be correlated to their proanthocyanidine (flavonoid) content. Flavonoids are proven antioxidants and are also demonstrated to have antiulcer activity. [22] The concentration of the flavonoids is rich in the seed and less in the skin. [23] Hence the antiulcer activity is more with the seed extract than the skin extract and the higher dose has higher activity than the lower dose used. CONCLUSION: The study shows that the ethanolic extract of grape seed has potential antiulcer activity than that of grape skin extract. The

7 extract with the dose of 200mg/kg shows maximum anti-ulcer activity. Establishment of the antiulcer activity of GSE may provide a newer and economically better modality of treatment for peptic ulcer which may have a better safety and efficacy. ACKNOWLEDGEMENTS: The authors gratefully acknowledge the Director of Vydehi Institute of Medical Sciences and Research Centre, White Field, Bangalore, for their kind support throughout our study. We extend our thanks to Olive Life Sciences, Bangalore for providing the Grape Seed Extract and Vidya Herbs, Bangalore for providing the Grape Skin Extract. Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. REFERENCES 1. Muchhandi AA, Chandrashekhar VM. Antiulcer and gastroprotective potential of streptospermum suaveolens in wistar rats. JPharmacol. 2011;2(2): Kalra P, Sharma S, Suman, Kumar S. Antiulcer effect of the methanolic extract of Tamarindus indica seeds indifferent experimental models. Journal of pharmacy and bioallied sciences.2011;3(2): Sharma MP, Ahuja V. Peptic ulcer disease and non ulcer dyspepsia. In: Tandon BN. Tropical Hepatogastroenterology. New Delhi: Elsevier Publishers; 2008.p McPee SI, Papadakis MA. Current Medical Diagnosis And Treatment. New Delhi: McGraw Hill Lange Publications; Umashanker M, Shruthi S. Traditional Indian Herbal Medicine used as Antipyretic, Antiulcer, Antidiabetic and Anticancer. International Journal of Research in pharmacy and Chemistry. 2001;1(4): Wandre RA, Bhagwat GB, Solunke RS, Yadav MB, Shaik AM. A Review On Medicinal Plants With Anti-Ulcer Activity. Journal of Pharmacognosy and phytochemistry. 2013; 2(1): Ahn HS, Jeon TI, Lee JY, Hwang SG, Lim Y, Park DK. Antioxidative activity of persimmon and grape seed extract: in vitro and in vivo. Nutritional research. 2002; 22: Brown JC, Huang G, Haley-Zitlin V, Jiang X. Antibacterial effects of grape extracts on Helicobacter pylori. Appl.

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