IJPCBS 2015, 5(3), 748-758 Lakshmana Rao et al. ISSN: 2249-9504

INTERNATIONAL JOURNAL OF PHARMACEUTICAL, CHEMICAL AND BIOLOGICAL SCIENCES

Available online at www.ijpcbs.com Research Article

GASTROPROTECTIVE ACTIVITY OF MICHELIA NILAGIRICA IN RATS: POSSIBLE INVOLVEMENT OF H+-K+-ATPASE INHIBITION SK. Aminabee1, A. Lakshmana Rao1* and M. Chinna Eswaraiah2 1Department of Pharmacology, V. V. Institute of Pharmaceutical Sciences, Gudlavalleru, Andhra Pradesh, . 2Department of Pharmacognosy, Anurag College of Pharmacy, Kodad, Nalgonda, Telangana, India.

ABSTRACT Objective: Different fractions isolated from ethanolic extract of whole of Michelia nilagirica is investigated for protective effect and mechanism as gastroprotective agent. Methods: Adult male Wistar rats weighing 120-180 gm were used for the experiment. All the animals were fasted for 24 hrs prior to the experiments. Groups were treated with different fractions 30 min before Aspirin at a dose of 500 mg/kg was administered. After 3 hrs, of Aspirin treatment, rats were anesthetized under mild ether and 748sacrificed via cervical decapitation the animals were sacrificed by cervical dislocation and the stomach was incised along the greater curvature and measure ulcer index. Gastroprotective activity is also found out by pylorus ligation induced ulceration. Results: The present study revealed that fraction B (150 mg/kg) shown maximum percentage of protection against aspirin induced ulcer and pylorus ligation induced ulceration. Conclusion: Protection may be due to presence of valuable terpinoids that was found out in phytochemical study.

Keywords: Michelia nilagirica, ulceration, pyloric ligation, aspirin.

INTRODUCTION more sensitive to the ulcer causing effects of Peptic ulcer disease (PUD), also known as a NSAIDs. The diagnosis is typically suspected due peptic ulcer or stomach ulcer, is a break in the to the presenting symptoms with confirmation lining of the stomach, first part of the small by either endoscopy or barium swallow. H. intestine, or occasionally the lower esophagus1. pylori can be diagnosed by testing the blood for An ulcer in the stomach is known as a gastric antibodies, a urea breath test, testing the stool ulcer while that in the first part of the intestines for signs of the bacteria, or a biopsy of the is known as a duodenal ulcer2. The most stomach. Other conditions that produce similar common symptoms are waking at night with symptoms include stomach cancer, coronary upper abdominal pain or upper abdominal pain heart disease, and inflammation of the stomach that improves with eating. The pain is often lining or gallbladder1. Diet does not play an described as a burning or dull ache. Other important role in either causing or preventing symptoms include belching, vomiting, weight ulcers5. Treatment includes stopping smoking, loss, or poor appetite. About a third of older stopping NSAIDs, stopping alcohol, and people have no symptoms1. Complications may medications to decrease stomach acid. The include bleeding, perforation, and blockage of medication used to decrease acid is usually the stomach. Bleeding occurs in as many as 15% either a proton pump inhibitor (PPI) or an H2 of people3. Common causes include the bacteria, blocker with four weeks of treatment initially Helicobacter pylori and non-steroidal anti- recommended1. Ulcers due to H. pylori are inflammatory drugs (NSAIDs)1. Other less treated with a combination of medications such common causes include tobacco smoking, stress as amoxicillin, clarithromycin, and a PPI. due to serious illness, Behcet disease, Zollinger- Antibiotic resistance is increasing and thus Ellison syndrome, Crohn disease and liver treatment may not always be effective6. cirrhosis, among others[4]. Older people are Bleeding ulcers may be treated by endoscopy,

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with open surgery typically only used in cases in protected from direct sunlight and heat until which it is not successful3. Peptic ulcers are required for extraction. present in around 4% of the population1. About 10% of people develop a peptic ulcer at some Preparation of extracts point in their life7. They resulted in 301,000 The powdered materials of Michelia nilagirica deaths in 2013 down from 327,000 deaths in (whole plant) were extracted successively each 19908. for 18 hrs with petroleum ether, ethyl acetate, Michelia nilagirica, belonging to the chloroform, ethanol and distilled water in () is a native to tropical soxhlet apparatus. The extracts were and subtropical South and , concentrated to dryness in rota evaporator till including southern . It is widely used in free from the solvents. both Ayurveda and Homeopathic medicine. buds of Michelia champaca Linn. is Isolation of fractions commonly used by many traditional healers in Thin-layer chromatography method was carried most of the herbal preparations for diabetes9 out using silica gel aluminum plate 60F-254, 0.5 and kidney diseases10. Traditionally, it is being mm (TLC plates, Merck). The spots were used in fever, colic, leprosy, post partum visualized in UV light and 10% of H2SO4 in protection11 and in eye disorders12. It has been methanol. The ethanolic extract was subjected reported to possess antipyretic, antiulcer, anti- to column chromatography (silica gel # 60-100) inflammatory13, insecticidal14, antioxidant, for further purification. The equilibration of antimicrobial11 and leishmanicidal15 activities. column was carried for one hour with The active constituents reported in this plant petroleum ether at flow rate 5ml/min. The are alkaloids, tannins, saponins, sterols, sample was (2gm dissolve in acetone) loaded on flavonoids and triterpenoids11. Herbal medicines to the column, 8 fractions were collected using have recently attracted much attention as petroleum ether:ethyl acetate (4:1), petroleum alternative medicines16 useful for treating or ether:ethyl acetate (1:1), petroleum ether:ethyl preventing life style related disorders and acetate (2:3), ethyl acetate (100%), relatively very little knowledge is available chloroform:methanol (9:1), about their mode of action. There has been a chloroform:methanol (1:1) and growing interest in the analysis of plant chloroform:methanol (2:8). products which has stimulated intense research Above yielded product were pooled into five on their potential health benefits. In Ayurveda, fractions based on TLC. The yield and traditional usages of are most commonly appearance of the five fractions was fraction A in their aqueous extracts form only. 50 mg/gm & yellow, fraction B 300 mg/gm & Concurrently, some of the papers searched focus black, fraction C 150 mg/gm & green, fraction D for testing these plants in their ethanolic or 200 mg/gm & darkish brown and fraction E 150 aqueous extracts and some have also reported mg/gm & saffron. activity in petroleum ether, benzene and chloroform extracts17-19. Keeping these facts in Phytochemical analysis view, the present study was undertaken to Phytochemical analysis20 of fractions was create a scientific base for the use of the extract carried out for the presence of alkaloids, of Michelia nilagirica as a gastroprotective tannins, saponins, glycosides, terpenoids, agent. carbohydrates, flavonoids, proteins, amino acids, fixed oils, steroids & sterols by different MATERIALS AND METHODS methods. Plant material The whole plant of Michelia nilagirica was Animals collected from the deciduous forest of Tirumala Albino rats of wistar strain weighing 150-200 Hills in Andhra Pradesh State, India. Samples gm were purchased from National Institute of were authenticated by Dr. K. Nutrition, Hyderabad. The rats were kept in Madhava Chetty, Plant Taxonomist, Department polypropylene cages (3 in each cage) at an of , Sri Venkateswara University, ambient temperature of 25±2°C and relative Tirupati, India. The whole plant of Michelia humidity of 55–65%. A 12 hrs light and dark nilagirica were sorted, cleaned and air-dried at schedule was maintained in the air conditioned room temperature for one week. By using the animal house. All the rats were fed with laboratory hammer mill these were ground to common diets for 1 week after arrival and then powder. Powdered samples were collected and divided into groups with free access to food and stored in air- and water-proof containers water.

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Acute toxicity studies Spot ulcer : 1.0 Acute toxicity studies were performed according Hemorrhagic stress: 1.5 to organization for economic co-operation and Deep ulcer : 2.0 development (OECD) guidelines21. Animals were Perforations : 3.0 divided in groups (n=5). The animals were Deep perforations : 4.0 fasted for 4 hrs with free access to water only. The extracts were administered orally in doses Mean ulcer score for each animal will be of 2500 and 5000 mg/kg to different groups of expressed as ulcer index24. The percentage of mice and observed over 14 days for mortality ulcer protection was determined as follows: and physical/behavioral changes. Ulcer index (UI) was measured by using following formula: ASSESSMENT OF ANTI-ULCER ACTIVITY UI = UN + US + UP Χ 10−1 Pyloric ligation induced gastric ulceration Where, Albino rats of either sex were divided into eight UI= Ulcer Index; groups of six animals each. Animals were fasted UN = Average number of ulcers per animal; for 24 h before the study, but had free access to US = Average number of severity score; UP = water. Percentage of animals with ulcers Group I : Received only distilled water Percentage inhibition of ulceration was equivalent to the volume of calculated as below: plant fraction Group II : Pyloric ligation (Ulcer % Inhibition of Ulceration = Control) (Ulcer indexcontrol - Ulcer indextest) × 100 Group III : Omeprazole (50 mg/kg) Ulcer indexcontrol Group IV : Fraction A (150 mg/kg) Group V : Fraction B (150 mg/kg) Determination of pH Group VI : Fraction C (150 mg/kg) An aliquot of 1ml gastric juice was diluted with Group VII : Fraction D (150 mg/kg) 1ml of distilled water and pH of the solution was Group VIII : Fraction E (150 mg/kg) measured using pH meter.

After 1hr of treatment, they were anaesthetized Determination of total acidity with the help of anesthetic ether; the abdomen An aliquot of 1ml gastric juice diluted with 1ml was opened by a small midline incision below of distilled water was taken into a 50 ml conical the xiphoid process. Pyloric22 portion of the flask and two drops of phenolphthalein stomach was slightly lifted out and ligated indicator was added to it and titrated with according to method of Shay et al.23 avoiding 0.01N NaOH until a permanent pink colour was traction to the pylorus or damage to its blood observed. The volume of 0.01N NaOH consumed supply. The stomach was replaced carefully and was noted. The total acidity is expressed as the abdominal wall was closed by interrupted mEq/L by the following formula sutures. Rats were sacrificed by an over dose of anesthetic ether after four hours of pyloric Acidity = Vol. of NaOH × N × 100 mEq/L ligation. The abdomen was opened, cardiac end 0.1 of the stomach was dissected out and the contents were drained into a glass tube. The Determination of free acidity volume of the gastric juice was measured and Instead of phenolphthalein indicator, the centrifuged at 2000 rpm for 10 min. From the Topfer's reagent was used. Aliquot of gastric supernatant, aliquots (1 ml of each) were taken juice was titrated with 0.01N NaOH until canary for the determination of pH, total and free yellow colour was observed. The volume of acidity. Each stomach was examined for lesions 0.01N NaOH consumed was noted. The free in the fore stomach portion and indexed acidity was calculated by the same formula for according to severity. the determination of total acidity.

Macroscopic evaluation of stomach Aspirin induced ulcer model The stomachs were opened along the greater The ulcer was induced by administering ethanol. curvature, rinsed with saline to remove gastric All the animals were fasted for 36 hours before contents and blood clots and examined by a 10Χ administration of ethanol. The Albino rats of magnifier lens to assess the formation of ulcers. either sex were divided into five groups, each The numbers of ulcers were counted. consisting of six rats. Normal coloration : 0 Group I : Received only distilled water Red coloration : 0.5 equivalent to the volume of plant fraction

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Group II : Aspirin (500 mg/kg) Acute toxicity studies Group III : Omeprazole (50 mg/kg) Acute toxicity studies were carried by up-down Group IV : Fraction A (150 mg/kg) regulation method. It was found that the extract Group V : Fraction B (150 mg/kg) at a limit dose from 1500 to 3000 mg/kg is safe Group VI : Fraction C (150 mg/kg) and does not show any mortality. Group VII : Fraction D (150 mg/kg) Group VIII : Fraction E (150 mg/kg) Pyloric ligation induced gastric ulceration Effect of ethanolic extract of Michelia nilagirica All the animals were fasted for 24 hrs prior to on pyloric ligation induced gastric ulceration is the experiments. Groups were treated with shown in the table 2, 3 and figure 1, 2. The different fractions 30 min before aspirin at a pyloric ligation has caused the accumulation of dose of 500 mg/kg was administered. After 3 gastric secretions of 9.28±0.21, pH to 1.56±0.03, hrs of aspirin25 treatment, rats were free acidity to 34.16±0.28, total acidity to anesthetized under mild ether and sacrificed via 63.21±0.25 and ulcer index to 10.8. cervical decapitation the animals were sacrificed Pretreatment with omeprazole significantly by cervical dislocation and the stomach was reduced the gastric volume to 3.58±0.11, pH to incised along the greater curvature and measure 4.23±0.01, free acidity to 10.45±0.24, total ulcer index. acidity to 21.76±0.21 and ulcer index to 2.3. The ulcer inhibition is 78.70%. Further it was Assay of H+-K++ATPase activity observed that pretreatment with fraction B (150 Proton potassium ATPase26 was prepared from mg/kg) significantly reduced the gastric volume mucosal scrapings of fresh sheep stomach to 4.01±0.08, pH to 3.98±0.02, free acidity to obtained from slaughter house. Immediately 12.36±0.16, total acidity to 24.13±0.18 and ulcer after opening the stomach, the mucosa was index to 3.3. The ulcer inhibition is 69.44%. In scrapped to obtain random sample of addition there is no significant change in the approximately 100 mg of mucosa. The mucosa gastric volume, pH, free acidity, total acidity, was then homogenized in 20 mM Tris–HCl, pH ulcer index and % ulcer inhibition with fraction 7.4. The contents were centrifuged for 10 min at A, C, D & E. The gastroprotection offered by the 2000 gm and the resulting supernatant was fraction B is compared with the standard subsequently centrifuged for 5000 gm for 20 omeprazole (50 mg/kg). min. The parietal cell extract thus obtained was used to determine H+ K+ -ATPase activity. The Histopathological examination assay was done by the method of Reyes-Chilpa The control group shows a normal histological et al. The enzyme source of 0.1 ml was added structure of rat gastric mucosa. Pyloric ligated with various concentrations of extracts (50–250 group resulted in severe congestion of blood g/ml) in water and pre-incubated at 370C for 60 vessels, hemorrhage, tissue infiltrations, tissue min. After incubation, the reaction mixture was necrosis, edema and several ulcers. Omeprazole added with 0.2 ml of Tris–HCl (20 mM, pH 7.4); showed better protection of gastric mucosa as 0.2 ml of MgCl2 (2 mM); 0.2 ml of KCl (2 mM); seen by mild blood vessels congestion and mild 0.2 ml of ATP (2 mM) and incubated at 370C tissue infiltrations. Ethanolic extract of Michelia for 30 min. The reaction was terminated by the nilagirica fraction B showed mild blood vessels addition of 1 ml of 10% TCA followed by congestion, mild tissue infiltrations, no necrosis centrifugation at 2000g for 10 min. The amount & no ulcers and fraction D showed partial loss of of inorganic phosphorus liberated from ATP was mucosal surface, sub mucosal congestion, determined pectrophotometrically at 640 nm. interruption of serosal layer and no necrosis The assay was performed in triplicates and the (Figure 3). results were averaged. Omeprazole was used as reference compound. The amount of protein Aspirin induced ulcer model present was estimated using bovine serum Aspirin at a dose of 500 mg/kg showed deep albumin as standard by Bradford method. ulcers and perforations in control rats. However animals pretreated with ethanolic extract of RESULTS Michelia nilagirica fraction B and D showed Preliminary phytochemical screening significant reduction in the number of ulcers Phytochemical screening revealed the presence and ulcer index (Table 4). They showed 66.66% of flavonoids & amino acids in fraction A, and 54.16% ulcer inhibition respectively where terpenoids & proteins in fraction B, terpenoids as omeprazole showed 74.27% ulcer inhibition. in fraction D and alkaloids, tannins, Anti-ulcerogenic effect of Michelia nilagirica in carbohydrates & flavonoids in fraction E (Table aspirin induced ulcers was comparable to that of 1). omeprazole, 50 mg/kg.

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Assay of H+-K+-ATPase activity related to the ability of these agents to suppress In-vitro H+-K+-ATPase inhibition activity of prostaglandin synthesis29-31. In the stomach, ethanolic extract of Michelia nilagirica was prostaglandins play a vital protective role, evaluated in sheep parietal cells where stimulating the secretion of bicarbonate and omeprazole was used as positive control. mucus, maintaining mucosal blood flow and Compared to fractions A, C and E fraction B and regulating mucosal cell turnover and repair. D showed maximum inhibition of 63.1% and Thus, the suppression of prostaglandin 57.5% respectively at 320 micro gm/ml (Table synthesis by NSAIDs results in increased 5). The maximum inhibition values of the susceptibility to mucosal injury and fractions are compared with the standard gastroduodenal ulceration32. One of the omeprazole. mechanisms by which aspirin damages the gastric mucosa is the increased production of % of inhibition of H+-K+-ATPase activity = NO due to the over expression of Inos33. NO is a Absorbance of Control – Absorbance of Test X 100 mediator not only of gastrointestinal mucosal Absorbance of Control 34 35 defense , but also of its damage . It has been DISCUSSION shown that different concentrations of NO have 36 The etiology of peptic ulcer is unknown in most completely opposite effects on the same tissue . of the cases, yet it is generally accepted that it In general, the mucosal and endothelial NOS results from an imbalance between aggressive isoforms produce low amounts of NO. However, factors and the maintenance of mucosal the high quantity of NO produced by iNOS 37 integrity through the endogenous defense damages the epithelium . The excessive release mechanisms. To regain the balance, different of NO from gastric epithelial cells induced by therapeutic agents including plant extracts may aspirin has been reported to exert detrimental 38 be used27. Ethanolic extract of Michelia nilagirica effects . is one such herbal drug used in the present The preliminary phytochemical analysis of study primarily to evaluate the anti-ulcerogenic Michelia nilagirica showed the presence of in pylorus ligation and aspirin induced ulcers in alkaloids, annins, terpinoids, carbohydrates, rats. The causes of gastric ulcer pyloric ligation flavanoids, proteins and aminoacids. The are believed to be due to stress induced increase antiulcer property of Michelia nilagirica in in gastric hydrochloric acid secretion and/or pylorus ligation model is evident from its stasis of acid and the volume of secretion is also significant reduction in free acidity, total acidity, an important factor in the formation of ulcer due number of ulcers and ulcer index. Michelia to exposure of the unprotected lumen of the nilagirica treated animals significantly inhibited stomach to the accumulating acid27. Pylorus the formation of ulcers in the pylorus ligated ligation induced ulcers are due to auto digestion rats and also decreased both the concentration of the gastric mucosa and breakdown of the and increased the pH, it is suggested that gastric mucosal barrier. These factors are Michelia nilagirica can suppress gastric damage associated with the development of upper induced by aggressive factors. Aspirin induced gastrointestinal damage including lesions, ulcers ulcer model also significantly decreased the and life threatening perforation and formation of ulcers. Fraction B showed hemorrhage. Aspirin, phenylbutazone, significant reduction in ulcer index i.e., 63.42% indomethacin and some non-steroidal anti- and 63.08 in pylorus ligation and aspirin inflammatory drugs are also known to cause induced ulcer model respectively. The phenolic duodenal and gastric ulceration. Prostaglandin antioxidants terpenoids are reported as potent H+-K+-ATPase blockers. The present study E2 and I2 are predominantly synthesized by the gastric mucosa and are known to inhibit the reported protective phytochemical terpenoids secretion of gastric acid and stimulate the present in fraction B contribute towards the + + 26 secretion of mucus and bicarbonate. inhibition of H -K -ATPase blockers activity, Hydrophobic surfactant-like phospholipids which is believed to play a tremendous role in secretion in the gastric epithelial cells is also reducing an acidic condition in gastric lumen. stimulated by the prostaglandin. It is also showed development of gastric ulcers in pyloric CONCLUSION ligation model. Volume of gastric secretion is an Terpenoids are a class of secondary plant important factor in the production of ulcer due phenolics found in fruits, vegetables and foods, to exposure of unprotected lumen of the which act as pharmacological active compound stomach to the accumulating acid28. in many medicinal plants. The phytoconstituents Nonsteroidal anti-inflammatory drugs (NSAIDs) terpenoids, have been reported in several anti- such as aspirin have the ability to cause ulcer literatures as possible gastroprotective gastroduodenal ulceration and this effect is agents. It is suggested that these compounds

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will be able to stimulate mucus, bicarbonate and B contains valuable terpenoids that shown prostaglandin secretion and counteract with the gastroprotective activity in both pylorus ligation deteriorating effects of reactive oxidants in and aspirin induced ulceration. gastrointestinal lumen39. Present study fraction

Table 1: Preliminary phytochemical screening of ethanolic extract of Michelia nilagirica S. No. Phytochemicals Fraction A Fraction B Fraction C Fraction D Fraction E 1 Alkaloids ------+ 2 Tanins ------+ 3 Saponins ------4 Glycosides ------5 Terpinoids ---- + ---- + ---- 6 Carbohydrates ------+ 7 Flavonoids + ------+ 8 Proteins ---- + ------9 Aminoacids + ------10 Fixed oils ------11 Steroids & Sterols ------

Table 2: Effect of ethanolic extract of Michelia nilagirica on gastric volume pH, free acidity and total acidity on pylorus ligation induced gastric ulceration Gastric Free Acidity Total Acidity Group Treatment Volume pH (mEq/l) (mEq/l) (ml) I Control 6.52±0.12 2.14±0.02 11.91±0.31 25.06±0.30 Pyloric ligation II 9.28±0.21 1.56±0.03 34.16±0.28 63.21±0.25 (Ulcer control) Omeprazole III 3.58±0.11 4.23±0.01 10.45±0.24 21.76±0.21 (50 mg/kg) Fraction A IV 6.25±0.09 2.78±0.04 21.89±0.18 54.36±0.16 (150 mg/kg) Fraction B V 4.01±0.08 3.98±0.02 12.36±0.16 24.13±0.18 (150 mg/kg) Fraction C VI 5.99±0.20 2.11±0.05 19.64±0.20 45.28±0.25 (150 mg/kg) Fraction D VII 6.23±0.16 1.98±0.01 18.51±0.22 51.01±0.24 (150 mg/kg) Fraction E VIII 5.63±0.31 2.16±0.02 21.31±0.17 55.19±0.22 (150 mg/kg)

Table 3: Effect of ethanolic extract of Michelia nilagirica on ulcer index and % ulcer inhibition on pylorus ligation induced gastric ulceration Ulcer Index % Ulcer Group Treatment US UN UP (UI) inhibition I Control 0 0 0 0 --- Pyloric ligation II 4 4 100 10.8 --- (Ulcer control) III Omeprazole (50 mg/kg) 0.5 1 25.0 2.65 75.46 IV Fraction A (150 mg/kg) 1 3 75.0 7.8 27.77 V Fraction B (150 mg/kg) 0.5 105 37.5 3.95 63.42 VI Fraction C (150 mg/kg) 1.5 3 75 7.95 26.38 VII Fraction D (150 mg/kg) 0.5 2 50.0 5.25 51.38 VIII Fraction E (150 mg/kg) 1 2.5 62.50 6.6 38.88

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Table 4: Effect of ethanolic extract of Michelia nilagirica on ulcer index and % ulcer inhibition on aspirin induced gastric ulceration % Ulcer Group Treatment US UN UP Ulcer Index (UI) inhibition I Control 0 0 0 0 --- II Aspirin (500mg/kg) 3 4 100 10.7 --- Omeprazole (50 mg/kg) + III 0.5 1 25.0 2.65 75.23 Aspirin (500 mg/kg) Fraction A (150 mg/kg) + IV 2 3 75.0 8.0 25.23 Aspirin (500 mg/kg) Fraction B (150 mg/kg) + V 0.5 1.5 37.5 3.95 63.08 Aspirin (500 mg/kg) Fraction C (150 mg/kg) + VI 3 2.5 62.5 6.8 36.44 Aspirin (500 mg/kg) Fraction D (150 mg/kg) + VII 2 1.75 43.75 4.75 55.60 Aspirin (500 mg/kg) Fraction E (150 mg/kg) + VIII 2 2.5 62.5 6.7 37.38 Aspirin (500 mg/kg)

Table 5: Assay of H+-K+-ATPase activity Absorbance (660 nm) Con. (micro S. No. Phosphate gm/ml) Omeprazole A B C D E buffer 0.145 0.279 0.169 0.256 0.185 0.249 1 10 0.313 (53.67) (10.86) (46.0) (18.21) (40.89) (20.44) 0.147 0.298 0.218 0.276 0.195 0.279 2 20 0.354 (58.47) (15.81) (50.84) (22.03) (44.91) (21.18) 0.165 0.357 0.197 0.324 0.220 0.331 3 40 0.438 (62.32) (18.49) (55.02) (26.02) (49.77) (24.42) 0.169 0.411 0.223 0.350 0.249 0.375 4 80 0.531 (68.17) (22.59) (58.0) (34.08) (53.10) (29.37) 0.216 0.631 0.336 0.521 0.381 0.581 5 160 0.858 (74.82) (26.45) (60.83) (39.27) (55.59) (32.28) 0.258 0.856 0.462 0.721 0.53 0.799 6 320 1.254 (79.42) (31.73) (63.1) (42.5) (57.7) (36.28)

Fig. 1: Effect of ethanolic extract of Michelia nilagirica on gastric volume pH, free acidity and total acidity on pylorus ligation induced gastric ulceration

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Fig. 2: Effect of ethanolic extract of Michelia nilagirica on ulcer index and % ulcer inhibition on pylorus ligation induced gastric ulceration

Fig. 3: Histopathological examination of stomach sections on pylorus ligation induced gastric ulceration I) Control, II) Pyloric ligation (ulcer control), III) Omeprazole (50 mg/kg), IV) Fraction A (150 mg/kg), V) Fraction B (150 mg/kg), VI) Fraction C (150 mg/kg), VII) Fraction D (150 mg/kg), VIII) Fraction A (150 mg/kg)

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Fig. 4: Effect of ethanolic extract of Michelia nilagirica on ulcer index and % ulcer inhibition on aspirin induced gastric ulceration

Fig. 5: Histopathological examination of stomach sections on aspirin induced gastric ulceration I) Control, II) Aspirin (500 mg/kg), III) Omeprazole (50 mg/kg) + Aspirin (500 mg/kg), IV) Fraction A (150 mg/kg) + Aspirin (500 mg/kg), V) Fraction B (150 mg/kg) + Aspirin (500 mg/kg), VI) Fraction C (150 mg/kg) + Aspirin (500 mg/kg), VII) Fraction D (150 mg/kg) + Aspirin (500 mg/kg), VIII) Fraction A (150 mg/kg) + Aspirin (500 mg/kg).

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