Shirisha Bongu / Vol 2 / Issue 2 / 2012 / 82-87.

e-ISSN 2249 - 7749 Print ISSN 2249 7757

International Journal of

Pharmacological Screening Methods

www.ijpsmjournal.com

ANIMAL MODELS IN EXPERIMENTAL GASTRIC ULCER SCREENING-A REVIEW

Shirisha Bongu1*, Subash Vijayakumar2
2

Department of Pharmacology, Vaagdevi College of Pharmacy, Hanamkonda, Warangal, Andhra Pradesh, India. Department of Pharmacy Practice, Vaagdevi College of Pharmacy, MGM Hospital, Warangal, Andhra Pradesh, India.

ABSTRACT Gastric ulcer is a very common global problem now days. Among various causes of gastric ulceration lesions caused by stress, alcohol consumption, H.pylori infection use of NSAIDs, Uneven food habits, Gastric irritants. Hyper secretion of gastric acid is a pathological condition, which occurs due to uncontrolled secretion of hydrochloric acid from the parietal cells of gastric mucosa through the proton pumping H+ K+ ATPase. The modern approach to control gastric ulcer is to inhibit gastric acid secretion, to promote gastro protection. key words: Gastric ulcer, Stress, NSAIDs, Gastric acid. INTRODUCTION Ulcers are the areas of degeneration and necrosis of gastro intestinal mucosa exposed to acid of the alimentary tract that is exposed to hydrochloric acid and pepsin they occur most commonly (98-99%) in either the duodenum or the stomach in the ratio 4:1 [1]. Ulcers can occur in the stomach, where they are called gastric ulcers or they can occur in the first portion of the intestine called as duodenal ulcers. "Peptic Ulcer" is the term used to describe either or both of these two types of ulcer [2]. Ulcers cause gnawing, burning pain in the upper abdomen. These symptoms frequently occur several hours following a meal, after the food leaves the stomach but while acid production is still high. Instead of pain, some patients experience intense hunger or bloating. Other patients have no pain but have black stools, indicating that the ulcer is bleeding. Bleeding is a very serious complication of ulcers [2]. Ulcers occurs due to imbalance between the offensive (gastric acid secretion ) and defensive (gastric mucosal integrity) factors. The aggressive and protective factors in the stomach are acid pepsin secretion, mucosal barrier, blood flow, cellular regeneration, prostaglandins and epidermal growth factors. Sometimes the gastric mucosa is continuously exposed to potentially injurious agents such as pepsin, bile acids, food ingredients, bacterial products (H.pylori) and drugs. Factors such as stress, smoking, nutritional deficiency and ingestion of NSAIDS all can increase the incidence of gastric ulcers. It is reported that prolonged anxiety, emotional stress, haemorrhagic surgical shock, burns and trauma are known to cause severe gastric irritations [3]. THE REGULATION OF ACID SECRETION BY PARIETAL CELLS The regulation of acid secretion by parietal cells is especially important in the pathogenesis of peptic ulcer, and constitutes a particular target for drug action. The secretion of the parietal cells is an isotonic solution of HCl (150 m mol/l) with a pH less than 1, the concentration of hydrogen ions being more than a million times higher than that of the plasma. The Cl- is actively transported into canaliculi in the cells that communicate with the lumen of the gastric glands and thus with the stomach itself. This Cl -

Corresponding Author:- Shirisha Bongu Email:- shirisha46@gmail.com

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secretion is accompanied by K+, which is then exchanged for H+ from within the cell by a K+/H+ ATPase (+ and bicarbonate ions. The later exchanges across the basal membrane of the parietal cell for Cl-. The principal stimuli acting on the parietal cells are: Gastrin (a stimulatory hormone) Acetylcholine (a stimulatory neurotransmitter) Histamine (a stimulatory local hormone) Prostaglandins E2 and I2 (local hormones that inhibit acid secretion). Gastrin Gastrin is a peptide hormone synthesised in endocrine cells of the mucosa of the gastric antrum and duodenum, and secreted into the portal blood. Its main action is stimulation of the secretion of acid by the parietal cells. Gastrin also indirectly increases pepsinogen secretion, stimulates blood flow and increases gastric motility. Release of this hormone is controlled both by neuronal transmitters and blood- borne mediators, as well as the chemistry of the stomach contents. Amino acids and small peptides directly stimulate the gastrin-secreting cells [4]. Acetylcholine Acetylcholine is released from (e.g.vagal) neurons and stimulates specific muscarinic receptors on the surface of the parietal cells and on the surface of histaminecontaining cells . Histamine Within the stomach, mast cells (or histaminecontaining cells similar to mast cells) lying close to the parietal cell release a steady basal release of histamine, which is further increased by gastrin and acetylcholine. The hormone acts on parietal cell H2 receptors, which are responsive to histamine concentrations that are below the threshold required for vascular H2 receptor activation. Prostaglandins Prostaglandins (mainly E2 and I2), synthesised in the gastric mucosa mainly by cyclo-oxygenase-1, stimulate mucus and bicarbonate secretion, decrease acid secretion and cause vasodilatation, all of which serve to protect the stomach against damage [5]. Drug therapy Different groups of drugs are used in the treatment of the ulcers those includes [6] Proton pump inhibitors (omeprazole, lansaprazole, pantaprazole, robeprazole) H2 receptor antagonists (cimetidine, ranitidine,famotidine,nizatidine) Prostaglandin analogue (mesoprostol) Acid neutralizers (antacids) magnesium trisillicate, aluminium hydroxide, alginates.

Ulcer protectants (sucralfate, bismuth chelate)

Animal models used in the screening of anti ulcer activity Various screening models are used for the screening of the anti ulcer activity .it helps to understanding the aetiology of the ulcer and screening of anti ulcer agents. Aspirin induced ulcers Ethanol induced ulcers Pylorus ligation induced ulcers Water immersion stress induced ulcers Indomethacin induced ulcers Histamine induced ulcers Reserpine induced ulcers Serotonin induced ulcers Acetic acid induced ulcers Hydrochloric acid induced ulcers Aspirin induced ulcers Principle Aspirin is a NSAID which inhibit the synthesis of prostaglandins. Prostaglandins protect the gastric mucosa by producing leukotrienes and bicarbonate ions. Aspirin also inhibit the gastric peroxidase and may increase mucosal hydrogen peroxide and hydroxyl ions level to cause oxidative mucosal damage [5]. Procedure Albino rats of either sex weighing between 150-200 gms are divided into five groups of six animals in group. The animals are fasted for 24 hours. The test drug in varying concentrations based on the design of the experiment is administered orally in 2% gum acacia solution 30 minute prior to aspirin at dose of 200 mg/kg. 4 hours later the rats are sacrificed by using anaesthetic ether and their stomachs dissected and they were opened along greater curvature for the determination of gastric lesions. Ulcer index calculated by noting the number of ulcers per animal and severity scored by observing the ulcers microscopically with the help of 10x lens and scoring is done as below [7] 0 - Normal stomach 0.5 - Red coloration 1 - Spot ulcers 1.5 - Haemorrhagic streaks 2 - Ulcer > 3 mm but 5 mm 3 - Ulcers > 5 mm Calculation of ulcer Index UI = UN + US + UP x 10-1 UI = Ulcer Index UN = Average of number of ulcer per animal US = Average of severity score UP = Percentage of animal with ulcer

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And percentage protection was observed by using the formula: %protection= Ethanol induced ulcers Principle Alcohol causes secretion of gastric juice and decrease mucosal resistance due to which protein content of gastric juice is significantly increased by ethanol. This could be leakage because of plasma protein in the gastric juice with weakening of mucosal resistance barrier of gastric mucosa, this leading to peptic ulcer [8]. Procedure Albino rats of either sex weighing between (150-200 gms) are divided into group. The animals are fasted for 24 hours with free access water. Animals are given test drugs or standard drug. 1 hour later 1ml/200gm of 99.80% alcohol is administered orally to each animal. The animals were anaesthetized 1 h latter with ether and stomach was incised along the greater curvature and ulceration was scored. The number of ulcers and the length of each ulcer were measured. Ulcer index was calculated using severity scores and avg no of ulcers per animal. Severity scores as below [9] 0 - Normal stomach 0.5 - Red coloration 1 - Spot ulcers 1.5 - Haemorrhagic streaks 2 - Ulcer > 3 mm but 5 mm 3 - ulcers > 5 mm Calculation of ulcer Index UI = UN + US + UP x 10-1 UI = Ulcer Index UN = Average of number of ulcer per animal US = Average of severity score UP- Percentage of animal with ulcer Percentage inhibition was calculated by the formula X 100 Histopathological studies were conducted by fixing stomach tissues in 10% formalin for 24 h. The formalin fixed specimens are embedded in paraffin and section (3-5m) and stained with haematoxylin and eosin dye. The histochemical sections are evaluated by light microscopy. Pylorus ligation induced ulcers Principle In Pylorus ligation pylorus part of the stomach was ligated it helpful to produce the ulcers in rats by stopping passage of gastric contents from stomach it creates the

acidic medium in stomach for longer time and produces the ulcer [10]. Procedure Albino Wistar rats of either sex weighing between (150-200 gms) are divided into groups of a animal. sIn this method albino rats are fasted in individual cages for 24 hours. Test drug or standard drug or control vehicle is administered 30 minute prior to pyloric ligation. Under light ether anaesthesia, the abdomen is opened and the pylorus was ligated. The abdomen is then sutured. At the end of 4 hours after ligation the animals are sacrificed with excess of anaesthetic ether , and the stomach is dissected out gastric juice is collected were drained into tubes and were centrifuged at 1000 rpm for 10 minutes and the volume is noted. The pH of gastric juice is recorded by pH meter. Then the contents are subjected to analysis for free and total acidity. The stomachs are then washed with running water to see for ulcers in the glandular portion of the stomach. The numbers of ulcers per stomach are noted and severity of the ulcers scored microscopically with the help of 10x lens. Histopathological studies were conducted by fixing stomach tissues in 10% formalin for 24 h. The formalin fixed specimens are embedded in paraffin and section (35m) and stained with haematoxylin and eosin dye. The histochemical sections are evaluated by light microscopy [11,12]. 0 = Normal stomach 0.5 = Red coloration 1 = Spot ulcers 1.5 = Haemorrhagic streaks 2 = Ulcer > 3 mm but > 5 mm 3 = ulcers > 5 mm Calculation of ulcer Index UI = UN + US + UP x 10-1 UI = Ulcer Index UN = Average of number of ulcer per animal US = Average of severity score UP = Percentage of animal with ulcer %protection= Determination of free acidity total acidity One ml of gastric juice is pipette into 100 ml conical flask, added 2 to 3 drops of topfers reagent and treated with 0.01 N sodium hydroxide until all traces of red colour disappears and the colour of the solution turns to yellowish orange. The volume of the alkali added was noted. This volume corresponds to free acidity. Then 2 to 3 drops of phenolphthalein solution is added and titration is continued until a definite red tinge reappears. Again the total volume of alkali added is noted. Acidity is calculated by using the formula

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Acidity = volume of NaOH X Normality of NaOHx100/0.1 m.eq/lit/100gms. Water immersion stress induced model Principle Stress can arise from prolonged anxiety, tension, and emotion, severe physical discomfort, haemorrhage and surgical shock, burns and trauma, thereby resulting in severe gastric ulceration. Procedure Stress induced ulcers were induced by force swimming in the glass cylinder (height 45cm diameter 35cm) containing water up to 35cm maintained at 35 oc for 3 hrs. Animals were fasted 24 hrs prior to the experiment .After the drug treatment (standard/test) animals were allowed to swim for 3hrs then animal were dissected stomachs were removed. All stomachs were opened along the greater curvature ulcer index and % inhibition was calculated and Histopathological studies conducted [13]. Indomethacin induced ulcers Principle Indomethacin is a non steroidal anti inflammatory drug that inhibits the synthesis of prostaglandins which are protective agents for gastric mucosa high doses of the NSAIDs causes ulceration [5]. Procedure All the animals were fasted 36 hours before administration of Indomethacin. The animals were divided into groups. Each rat was administered with the 20mg/kg Indomethacin orally.30 min prior to the administration of the Indomethacin standard/test drug was administered. The rats were anaesthetized with ether 1 hour latter the stomach was incised through the greater curvature and examined for the number of lesion under the dissecting microscope by titrating with 0.01N NaOH using phenolphthalein as an indicator. gastric juice estimated for pepsin. Ulcer index and % inhibition were calculated and histopathological studies conducted for the stomach tissues [14]. Histamine induced ulcers Principle Histamine causes gastric ulceration by enhancing the gastric acid secretion by directly acting on histamine receptors of parietal cells and it also having the vasoplastic activity [4]. Procedure Guinea pigs weighing (300-400 gm) were divided into groups of six each and the animals were fasted for 36 hours (water allowed) before experiment. One ml of histamine acid phosphate (50 mg base) was administered intraperitoneally. Promethazine hydrochloride 5 mg was injected intraperitoneally 15 min before and 15 min after

histamine administration. The standard/test drugs were administered by gavage 45 minutes before histamine. Four hours after administration of histamine, the guinea pigs were sacrificed by stunning. The anterior abdominal wall was opened and the stomach dissected out. Stomach was opened along the greater curvature ulcers were identified. Severity scores were calculated. Histopathological studies were conducted on the stomach tissues [15]. Reserpine induced ulcers Principle Reserpine-induced gastric ulceration has been attributed to the degranulation of gastric mast cells and consequent liberation of histamine which is believed to be a cholinergically mediated [16]. Procedure Adult albino rats weighing 150-180gms were fasted for 24 hr. Animals were divided into different groups following water ad libitum. Reserpine (5mg/kg) administered intramuscularly rats. 30 min after the administration of the standard or test drug or control vehicle (Distilled water) intraperitoneally. All the animals were sacrificed after 18 hr, their stomachs were removed, opened along the greater curvature and sum of lengths (mm) of all lesions for each rat was used as ulcer index and percentage protection of ulcers were calculated. Histopathological studies were performed on the stomachs tissues [17]. Serotonin induced ulcers Principle Serotonin-induced gastric ulceration is believed to arise from a disturbance of gastric mucosal microcirculation .The development of ulcers by serotonin usually takes about 18 hr10. Procedure Rats weighing 120-150gms were taken and they were randomly divided into groups. Animals kept fasting for 24hrs and water withdrawn 2hr before the experiment. Serotonin creatinine sulphate (20mg/kg) was administered subcutaneously to rats. The standard drug/test drug/ control vehicle (Distilled water) was administered intraperitoneally after 30 min prior to the serotonin injection. The animals were sacrificed after 18 hr, their stomachs were removed and opened along the greater curvature, and the ulcer index was determined. Percentage protection of the ulcers calculated. Histopathological studies were conducted [18,19]. Acetic acid induced ulcers Principle Acetic acid enhances the ulceration in stomach by increasing the acidity of stomach contents and acetic acid also causes gastric obstruction leads to the ulceration.

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Procedure Rats were anaesthetized with pentobarbitone (35 mg/kg, ip). The abdomen was opened and the stomach was visualized. A cylindrical glass tube (6 mm in diameter) was tightly placed upon the anterior serosal surface of the glandular portion of stomach 1 cm away from the pyloric end. 50% acetic acid (0.06 ml/animal) was instilled into the tube and allowed to remain for 60 sec on the gastric wall . After removal of acid solution, the abdomen was closed in two layers and animals were caged and fed normally. Standard and test drug administered orally 4 h after the application of acetic acid and continued up to 9 days after induction of ulcer. The animals were then sacrificed after 18 h of the last dose of drug on 10th day of experiment to assess the ulcer size and healing. Ulcer index was calculated based upon the product of length and width (mm2/ rat) of ulcers [20]. Hydrochloric acid induced ulcers Principle Hydrochloric acid induces the ulcer by enhancing the acidity of the stomach contents. Procedure

Rats weighing 150-180gms are taken and they were divided into groups. Thirty minutes after the test or reference drug or the control vehicle treatment, 0.6 M HCl was orally administered to each rat. After 1 h the rats were anaesthetised with excess of anaesthetic ether and stomach was cut open along the greater curvature, cleared of residual matter with saline and the inner surface was examined for ulceration. Ulcer index and % ulcer protection. Histopathological were conducted by Gastric tissue samples from each group were fixed in 10% formalin for 24 h. The formalin fixed specimens are embedded in paraffin and section (3-5m) and stained with haematoxylin and eosin dye. The histochemical sections are evaluated by light microscopy [21]. CONCLUSION Several methods are there to evaluate the anti ulcerogenic activity of the synthetic and natural products like aspirin induced ulcers, alcohol induced ulcers, pyloric ligation induced ulcers, indomethacin induced ulcers, histamine induced ulcer, reserpine induced ulcers, serotonin induced ulcer, acetic acid induced ulcers, Hydrochloric acid induced ulcers. Most of the researcher could evaluate the efficacy of various anti ulcer drugs by these methods.

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17. Jude E Okokon and Paul Nwafor. Anti ulcer and anti convulsant activity of Croton zambesicus. Pakistan Journal of pharmaceutical sciences, 22(4), 2009, 384-390. 18. Dabadi P, Koti BC,Vijay C,Manjuntha SK. Anti-ulcer activity of Mimsops elengi against serotonin induced ulcers in rats. International research journal of pharmacy, 2(8), 2011, 173-176. 19. Lucky L. Nwidu and Paul A. Nwafor: Gastroprotective effects of leaf extracts of Carpolobia lutea (polygalaceae) G. Don. in rats. Africal Journal of Biotechnology, 8(1), 2009, 12-19. 20. Aditi C, Bhavani G, Agarwal PK, Shalini G. Ulcer healing proprties of ethanolic extract of Euginia jambolana in diabetic rats:study on gastric mucosal dffensive factors. Indian Journal of physiology and pharmacology, 53(1), 2009, 16-24. 21. Deshpande SS, Shah GB, Parmar NS. Anti ulcer activity of Tephrosia purpurea in rats. Indian Journal of Pharmacology, 35, 2003, 168-172.

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