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ANTIULCER EFFECT OF LIVINA, A HERBAL FORMULATION AGAINST ETHANOL INDUCED ACUTE GASTRIC ULCER IN MICE Dr. Soumendra Darbar1*, Shyamaprasad Chattopadhyay1
1

Dr. S. Darbar

Research and Development Division, Deys Medical Stores (Mfg.) Ltd., 62, Bondel Road, Kolkata-700019, West Bengal, India Email:darbar_soumen@yahoo.co.in

ABSTRACT Peptic ulcer is the most common GIT disorder in the present day life of the industrialized and civilized world. The prevention or cure of peptic ulcers is one of the most important challenges confronting medicine nowadays, as it is certainly a major illness affecting 8 to 10% of the global population and of these 5% suffer from gastric ulcer. This study was designed to evaluate the gastro protective effect of Livina, a polyherbal formulation on ethanol (50%) induced gastric ulcers in mice. Forty young white male Swiss albino mice were divided to five groups (8 mice/group). Three case groups received Livina (50, 100, 200 mg/kg) and control negative and positive groups received distilled water and ranitidine respectively. Animals were killed and their stomachs were removed and macroscopic and microscopic ulcer index were determined. Data were subjected to one-way ANOVA followed by Dennetts t-test. The results indicated that polyherbal formulation, Livina (50,100,150 mg/kg) significantly decreased the ulcer index (p<0.05) and these doses of formulation exerted macroscopic curative ratios of 67.63%, 75.11% and 81.09% respectively. However, Livina at doses of 100 and 200 mg/kg significantly (P< 0.05) showed an antiulcer effect characterized by reduction of acid volume (AV), free acidity (FA), total acidity (TA), and increasing rate of pH, when compared to the control group. The present findings demonstrate that, Livina has gastroprotective effect on ethanolinduced gastric ulcer in mice model. Key Words: Gastric ulcer, Livina, Free acidity, mucosal damage. INTRODUCTION Peptic ulcer is the most common GIT disorder in the present day life of the industrialized and civilized world. It is a chronic inflammatory disease characterized by ulceration in the regions of upper gastrointestinal tract where parietal cells are

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found and where they secrete hydrochloric acid (HCl) and pepsin. The anatomic sites where ulcer occurs commonly are stomach and duodenum, causing gastric and duodenal ulcer, respectively[1]. Pathophysiology of ulcer is due to an imbalance between aggressive factors (acid, pepsin, h. pylori and non-steroidal antiinflammatory agents) and local mucosal defensive factors (mucus bicarbonate, blood flow and prostaglandins). Integrity of gastroduodenal mucosa is maintained through a homeostatic balance between these aggressive and defensive factors[2]. Infection of the stomach mucosa with helicobacter pylori - a Gram-negative spiral-shaped bacterium is now generally considered to be a major cause of gastro-duodenal ulcer [1]. Several drugs are widely used to prevent or treat gastro-duodenal ulcers; these include H2-receptor antagonists (cimetidine), proton pump inhibitors (omeprazole, lansoprazole) and cytoprotectives (misoprostol) [2]. Antacids, e.g. aluminium hydroxide and magnesium hydroxide, are used often to neutralize excess gastric acidity in the stomach. Due to problems associated with recurrence after treatment, there is therefore the need to seek alternative drug sources against GI ulcers [1]. Ethanol is a well-known cause of gastric mucosal damage, but its pathogenetic mechanisms are not well understood. The oxygen-derived free radicals generated by ethanol may be responsible for the induction of gastric damage [4,5]. Free radicals are extremely reactive products leading to oxidative damage through lipid peroxidation [6,7]. Herbal prescriptions and natural remedies are commonly employed in developing countries for the treatment of various diseases, this practice being an alternative in orthodox pharmacotherapy. This herbal medicines derived from various plant extracts are being increasingly utilized to treat a wide variety of clinical diseases [8]. Livina, a

polyherbal formulation is very useful as an natural hepatoprotective medicine, which compose of a number of Indian medicinal plants [9,10] . Our recent work established that Livina protect gastric mucosal damage and maintain mucosal lipid profile [11]. The aim of this study is to determine the protection afforded by Livina, a polyherbal formulation against ethanol-induced gastric mucosal damage in mice. MATERIALS AND METHODS Drugs and Chemicals Livina capsule was obtained from Deys Medical Stores (Mfg.) Ltd., 62, Bondel Road, Kolkta-700019, India. Ethanol, HCl, NaCl and NaOH were obtained from Merck, Germany All other chemicals and solvents were of analytical grade commercially available. Animals Male Swiss albino mice (30-35 g) were used in this experiment. The animals were maintained in cages with raised floors of wide wire mesh to prevent coprophagy and were housed in an ambient temperature of 22 1C in a 12 h lightdark cycle. They were fed a standard balanced diet and given free access to water ad libitum. All animals were fasted for 48h before use to ensure an empty stomach [12] During the fasting period rats received a nutritive solution of 8% sucrose in 0.2% NaCl to avoid excessive dehydration[13]. The experiment was carried out in accordance with CPCSEA guidelines and the study approved by Institutional animal Ethics Committee (IAEC) of Deys Medical Stores (Mfg.) Ltd. (Approval No. Ayur/Deys/01/2007). Composition of Livina Each Livina capsule contains:

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1. Solanum nigrum, 20 mg; 2. Holarrhena antidysenterica 10 mg; 3. Tephrosia purpurea 40 mg; 4. Andrographis paniculata 10 mg; 5. Phyllanthus niruri 20 mg; 6. Tinospora cordifolia 10 mg; 7. Terminalia chebula 10 mg; 8. Asteracantha longifolia 20 mg; 9. Alstonia scholaris 20 mg; 10. Berberis aristata 40 mg; 11. Cichorium intybus 10 mg; 12. Picrorhiza kurroa 20 mg. Preparation of test solution 10 Livina capsules dissolved in 5% methanol solution with a continuous mechanical staring. Stay the solution over night and filtered it. The test solution was stored in normal refrigerator. 50, 100, 200 mg/kg sample applied on animal during experiment. Experiment Design Three treatment groups received 50, 100, 200 mg/kg methanol extract Livina powder orally via a stainless steel intubation needle. Two doses were given at 08:00 and 16:00 and a third dose was given on the second day 1.5 h before induction of gastric ulceration [14]. As the same time as case groups: a negative control group was given distill-ed water (10 mL/kg) and a positive control group was given Ranitidine at 50 mg/kg [15]. All animals were given ethanol (Merck) 50 % (v/v) (in distilled water) at 1ml/kg orally to induce gastric ulceration [14]. One hour after ethanol administration, all rats were killed by an overdose of chloroform and the stomachs were rapidly removed, opened along their greater curvature and gently rinsed under running tap water and spread on a paraffin plate. Lesions in the glandular part of the stomach were measured with a graticules (Heerbrugg Switzerland wild) under a stereomicroscope (Lei-ca zoom 2000). Long lesions were counted and measured along their greater length. Petechial lesions were counted. Each five petechial lesions were taken as 1mm of ulcer [14]. The sum of the total length long ulcers and petechial

lesions in each group of rats was divided by its number to calculate the ulcer index (mm). The macroscopic curative ratio was determined by the formula:
(Control ulcer index) (Test ulcer index)

Curative ratio = (Control ulcer index)

X 100

Determination of free acidity and total acidity The gastric contents were centrifuged at 1000 rpm for 10 min. 1 ml of supernatant was diluted gastric juice was titrated with 0.1N NaOH using 3-4 drops of Topfers reagent as an indicator until canary yellow colour was noted. This corresponds to free acidity. Further 2-3 drops of phenolphthalein was added and titrated with 0.1 N NaOH until pink colour was restored. This gives total acidity. Free acidity and total acidity is expressed in terms of (mEq/L) of gastric contents [16]. Histological evaluation of gastric lesions Immediately after macroscopic evaluation the stomachs were fixed in neutral buffered formalin (10%) then glandular parts were divided to four segments and routine histologic processing was carried out. 5-6m sections were stained by H&E method and were evaluated microscopically (Olympus CH30). Microscopic ulcer index was obtained using published methods [17] by two pathologists, separately and a mean index was calculated. Normal tissue = 0; Local damage to gastric pits cells = 1; Local damage to gastric glands = 2; Deep damage to gastric glands = 3 Microscopic ulcer index = (number of lesion 1) + (number of lesion 2) 2+ (number of lesion3) 3 Statistical Analysis

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The data were expressed as mean SEM and tested for significance by one way ANOVA and Dunnett t-test and results were regarded as significant when p < 0.05. RESULTS Morphological investigation Ethanol (50%) induced gastric damage showed marked gross mucosal lesion, including long hemorrhage bands and petechial lesion. On gross examination these hemorrhagic bands were characterized by different sizes along the longitudinal axis of the glandular part of stomach (Fig 1A). Animals pretreated with Livina showed very mild lesions with interstitial hemorrhage and sometimes no lesion at all (Fig 1B). Investigation of gastric lesions Doses of Livina significantly increased macroscopic curative ratio compared with control groups (Table 1). Morphometric evaluation was also carried out to evaluate the extent of ulcer. The ulcer index was significantly (P<0.01) reduced in animals pretreated with Livina compared to distilled water and ranitidine treated rats (Table 1). Biochemical investigation Moreover, Livina (200 mg/kg) significantly reduced the gastric ulcer in ethanol induced gastric ulcer model by confirmation of significant decreases (P<0.05) in acid volume and increases (P<0.05) in pH when compared with (P<0.05) (Table-2) ethanol treated group.

mucosal epithelial loss was seen in pre-treated rats. DISCUSSION Gastric ulcer is known as damage of the mucosal integrity of the stomach, and duodenum defect produced due to active inflammation [18]. Some noxious agents like (acid, pepsin, bile acids, pancreatic enzymes, drugs and bacteria) attacking on the gastro duodenal mucosa by a host of integrity is maintained by an intricate system that provides mucosal defense and repair. Mucus bicarbonate layer formed an intricate biologic system, surface epithelial cells and a rich sub mucosal micro-circulatory bed which provides bicarbonate ions which neutralize the acid generated by parietal cell section (HCl), during removing toxic metabolic, the adequate supply of micronutrients and oxygen is supplied by microcirculatory bed [19]. The finding of present study demonstrated that hydroalcolic extract of F.vulgaris significantly protected against mucosal damage induced by ethanol (50%) and curative ratios of plant extracts 50, 100 and 150 mg/kg were 67.63%, 75.11% and 81.09% respectively. It is remarkable that these doses produced a greater protection than ranitidine (50 mg/kg) against the ethanol. Narcotizing agents such as ethanol, when given intragastrically to rats produce severe gastric hemorrhagic erosions. Ethanol induced both long ulcers and petechial lesions with-in a short time, which makes this technique suitable for screening experiments for investigation of antiulcer drugs. The genesis of ethanol-induced gastric lesion is of multifactorial origin with the decrease in gastric mucus amount also it is associated with significant production of free radicals leading to increased lipid peroxidation which inturn causes damage to cell and cell

Histopathological investigation On microscopic examination, ethanol treated rats showed mucosal hemorrhage (Fig 2A), segmental mucosal necrosis of gastric epithelium, oedema and ample infiltration of leukocytes in submucosa (Fig 2B). Only patchy

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membranes [20]. There was decrease in gastric volume and reduction in free and total acidity in the animals treated with Livina and was found to be devoid of ulcerogenic potential. The above discussion shows that Livina, the herbal formulation is said to produce beneficial anti ulcer activity. In conclusion, to our knowledge, this study provides for the first time evidence that showed gastroprotective effect of Livina, a polyherbal formulation against ethanol induced ulcer. ACKNOWLEDGEMENT The authors are thankful to Prof. (Dr.) T.K.Pal, Department of Pharmaceutical Technology, Jadavpur University, Kolkata700032 or his valuable suggestions and Mr. Gautam Dey, M.D., Mr. Ranajit Dey, Jt. M.D., Mr. Subharthee Dey, Whole time Director, Mr. S.K. Dasgupta, Advisor, H.R., and Mr. Ambarish Mukherjee, GM, Works for facilities and encouragement during this investigation. They are also thankful to Mr. Swapan Kumar Mitra, Manager, MIS, Mr. S.P. Basuchoudhury, Deputy Manager Purchase and Mr. utpal Dutta, Asst. manager for their ungrudging co-operation and help in carrying out this study. REFERENCES: 1. 1. Rang HP, Dale MM, Ritter M, Moore PK. (eds.). Pharmacology, 5th edition. Churchill, Livingstones, Edinburgh, 2003; pp. 797. 2. Raskin JB, White RH, Jackson JE, Weaver AL, Tindall EA, Lies RB, Stanton DS. Misoprostol dosage in the prevention of non-steroidal anti-inflammatory druginduced gastric and duodenal ulcers. A comparison of three regimens. Ann Int Med 1995; 123: 344-350. 3. Narayan S, Devi RS, Jainu M, Sabitha KE, Shyamala Devi CS. Protective effect of a Polyherbal Drug Ambrex in ethanol induced

gastric mucosal lesions in experimental rats. Indian J Pharmacol 2004; 36: 34-37. 4. Pihan G, Regillo C, Szabo S. Free radicals and lipid peroxidation in ethanol-and aspirin-induced gastric mucosal injury. Dig Dis Sci 1987; 32:1395-1401. 5. Szelenyi I, Brune K. Possible role of oxygen free radicals in ethanol-induced gastric mucosal damage in rats. Dig Dis Sci 1988; 33:865-871. 6. Yoshikawa T, Ueda S, Naito Y. Role of oxygen-derived free radicals in gastric mucosal injury induced by ischemia or ischemia reperfusion in rats. Free Radical Research Communication 1989; 7:285-291. 7. Bagchi D, Carryl O, Tran M. Stress, diet and alcoholinduced oxidative gastrointestinal mucosal injury in rats and protection by bismuth subsalicylate. J Applied toxicol 1998; 18 [Suppl 1]:3-13. 8. Hemnani T, Parihar MS. Reactive oxygen species and oxidative DNA damage. Indian J Physiol Pharmacol 1998; 42:440-452. 9. Darbar S, Ghosh B, Chattopadhyay SP, Ghosh, B. Antioxidant and hepatoprotective activity of Livina, a polyherbal liquid formulation. Asian J Chem 2009; 21(2):1495-1499. 10. Darbar S, Chakraborty MR, Chattopadhyay SP, Ghosh, B. Protective effect of Livina, a polyherbal liquid formulation against ethanol induced liver damage in rats. Ant sci life 2009; 28(3):14-17. 11. Darbar S, Chattopadhyay SP, Ghosh B, Chakraborty, MR. Effect of a polyherbal liquid formulation on aceclofenac induced gastric mucosal damage in albino wistar rats. J Pharm Res 2008; 7(2):62-65. 12. Garg GP, Nigam SK, Ogle CW. The gastric antiulcer effects of leaves of the neem tree. Planta Medica. 1993;59:215-217. 13. Glavin GB, Mikhail AA. Stress and ulcer etiology in the rat. Physiol Behav 1976;16:135-139.

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14. Alkofahi A, Atta AH. Pharmacological screening of anti-ulcerogenic effects of some Jordanian medicinal plants in rats. J Ethnopharmacol. 1999;67:341-345. 15. Alvares A, Pomar F, Sevilla MA, Montero MJ. Gastric antise-cretory and antiulcer activities of an ethanolic extract of Bidens pilosa L. var. radiate Schult. J Ethnopharmacol. 1999;67:333-340. 16. Godkar PB, Clinical biochemistry principles & practice. 7th ed. Bhalani Publishing House, Bombay, India 2000; p.168-170. 17. Pandit S, Sur TK, Jana U, Bhattacharyya D, Debnath PK. Anti-ulcer effect of Shankha bhasma in rats: a preliminary study. In-dian J Pharmacol. 2000;32:378-380. 18. Chaturvedi A, Kumar MM, Bhawani G, Chaturvedi H, Kumar M, Goel RK. Effect of ethanolic extract of Eugenia jambolana

seeds on gastric ulceration and secretion in rats. Indian J Physiol Pharmacol. 2007; 51: 131-140. 19. Gregory M, Vithalrao KP, Franklin G, Kalaichelavan V. Anti-Ulcer (UlcerPreventive) Activity of Ficus arnottiana Miq. (Moraceae) Leaf Methanolic Extract. American Journal of Pharmacology and Toxicology 2009; 4: 89-93. 20. Reshma, S, Vijay Kumar K, Naidu MUR, Ratnacar KS. Effects of Gingko biloba extract on ethanol-induced gastric mucosal lesions in rats. Indian J Pharmacol. 2000;32:313-7.

TABLES AND FIGURES: Table1. Effect of Livina on ethanol-induced gastric ulcer Groups Distilled water (10ml/kg) Ranitidine (50 mg/kg) Livina (50 mg/kg) Livina (100 mg/kg) Livina (200 mg/kg) Ulcer index SEM (Macroscopically) 12.34 2.09 5.24 1.06 4.19 0.75* 2.98 0.62* 2.31 0.48* Ulcer index SEM (Microscopically) 13.65 1.98 7.66 1.25 6.54 0.77* 4.33 0.81* 2.87 0.62* Macroscopic Curative ratio (%)

57.52 67.63 75.11 81.09

All values are mean S.D., n=8 mice in each group. *P<0.01 as compared with the negative control animal.

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Table 2. Effect of Livina on acid volume, pH, free acidity and total acidity against ethanol-induced gastric ulcer Groups Distilled water (10ml/kg) Ranitidine (50 mg/kg) Livina (50 mg/kg) Livina (100 mg/kg) Livina (200 mg/kg) Acid volume(ml) 5.81 0.21 4.03 0.11 2.98 0.19* 2.11 0.17* 1.96 0.12** pH 2.65 0.26 4.58 0.34 4.94 0.29* 5.76 0.41* 6.29 0.37** Free acidity (mEq/L) 71.64 3.25 56.78 2.15 49.22 2.66* 38.75 3.02* 32.65 1.93** Total acidity (mEq/L) 161.23 4.06 134.76 3.75 122.07 3.14* 115.43 2.99* 99.87 3.51**

All values are mean S.D., n=8 mice in each group. *P<0.01, **P<0.05 as compared with the negative control animal.

Fig. 1A

Fig. 1B

Fig. 1 Antiulcer activity of Livina, a polyherbal formulation, (A) stomach of mice showing mucosa with hemorrhagic erosion when treated with ethanol (50% v/v); (B) stomach of mice ethanol (50% v/v) + 200mg Livina treated group showing normal mucosa.

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Fig. 2A

Fig. 2B

Fig. 2 Histological section of the gastric mucosa in a mice, (A) animals treated only distilled water (negative control). There is severe disruption of the surface epithelium, deep penetration of necrotic lesions into mucasa and edema of the submucosa layer with leukocyte infiltration of ulcerative tissues; (B) mice pre-treated with Livina (200 mg/kg), compared with negative control, the disruption of the surface epithelium is very mild and there is no submucosal edema and no leucocytes infiltration (H&E stain, 40x).

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