# Introduction zadirachta indica (AI) has been advocated for the treatment of disorders like cough, nausea, vomiting, fever, jaundice, gonorrhea, intestinal warm infestation and leprosy in indigenous system of medicine 1 and reported to have antiulcerogenic property. [2][3] The biological, medicinal and industrial uses of various parts of AI and the compounds isolated from it have been reviewed. [4][5][6] AI barks contained condensed tannins to the extent of 15% along with other nonisoprenoid constituents like flavonoids and phenolics. 7 Peptic ulcer, one of the most common gastrointestinal disease, is caused by multiple factors including stress, smoking, nutritional deficiencies, noxious agents such as alcohol, NSAID and Helicobacter pylori infection, among others. [8][9] Plant extracts are some of the most attractive sources of new drugs and have been shown to produce promising results in the treatment of gastric ulcers. 10 This is an important reason to investigate the antiulcer effect of AI bark extracts that have been used traditionally against gastric diseases. As to pharmacological effects, different extracts of leaves, seeds and stem barks of AI showed antimicrobial [11][12] , antioxidant 13 and antiulcer activities. [14][15] In our previous study, antioxidant effect of hydro alcoholic root bark extract was tested. 16 Given the association between biological constituents present and antiulcerogenic effects of the AI root bark, the present study was carried out by two approaches. First, we performed phytochemical screening of root bark successive solvent extracts. Second, we selected root bark methanol extract to assess its antiulcerogenic activity in ethanol induced gastric ulcer in mice. # II. # Materials and Methods # a) Chemicals and reagents All reagents and chemicals used were of analytical grade. Folin-ciocalteu reagent (Merck Pvt. Ltd. India), Sodium carbonate (Merck Pvt. Ltd. India), standard omeprazole was the kind gift from Aurobindo Pharma Ltd., Hyderabad. # b) Plant material The root bark of AI was collected from agriculture land of Deshmukhi village of Andhrapradesh, India and the authentication of plant material was done by a botanist at Osmania University, Hyderabad and the voucher no was 0125. # c) Preparation of root bark extracts Root barks were shade dried and powdered mechanically after cutting into small pieces. The powdered plant material was extracted in a soxhlet extractor by successive soxhlet extraction method based on polarity order of solvents. Solvents employed were pet ether, chloroform, ethyl acetate and methanol. The extracts were cooled at room temperature, filtered and evaporated to dryness under reduced pressure in a rotary evaporator 17 . Resultant successive extracts of root barks were subjected to qualitative chemical analysis for the presence of biologically active constituents. 18 Thin layer chromatography was performed for all the extracts by taking Quercetin as biomarker. Mobile phase employed was ethyl acetate: formic acid: glacial acetic acid: water (100: 11: 11: 26). 19 e) Determination of total phenolics content The Folin-Ciocalteu reagent (FCR) or Folin's phenol reagent is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric assay of phenolics and polyphenolic antioxidants. It works by measuring the amount of the substance being tested needed to inhibit the oxidation of the reagent. 20,21 Total phenol contents in the extracts were determined by the modified Folin-Ciocalteu method. An aliquot of the extract was mixed with 5 ml Folin-Ciocalteu reagent (previously diluted with water 1:10 v/v) and 4 ml (75 g/l) of sodium carbonate. The tubes were vortexed for 15 sec and allowed to stand for 30 min at 40 o C for color development. Absorbance was then measured at 765 nm using the Shimadzu UV-1800 spectrophotometer. Samples of extract were evaluated at a final concentration of 0.1 mg/ml. Total phenolics content were expressed as mg/g tannic acid equivalent using the following equation based on the calibration curve: y = 0.1216x, R 2 = 0.9365, where x was the absorbance and y was the tannic acid equivalent (mg/g). 22 f) Animals Swiss albino mice (24-30 g) of either sex maintained under standard husbandry conditions (temp 23±2 o C, relative humidity 55±10% and 12 hours light dark cycle) were used for the screening. Animals were fed with standard laboratory food and ad libitum during the study period. The experimental protocol has been approved by Institutional Animal Ethics Committee (IAEC NO.1330/AC/10/CPCSEA). g) Ethanol induced gastric ulcer 23,24 The methanol extract of root bark of AI was selected as it is having significant amount of biologically active constituents (from the results of phytochemical analysis) to evaluate anti ulcer activity by ethanol induced gastric ulcer in albino mice. After 12 hour of fasting Swiss albino mice weighing 24-30 g of either sex were divided into 5 groups, each group consists of 6 animals. Group 1 served as a control received 1.0 ml/kg p.o 80% Tween 80. Group 2 served as standard control received 30 mg/kg, p.o Omeprazole. After 1h all the animals were treated with 0.2 ml of ethanol p.o to induce gastric ulcer. Animals were sacrificed by cervical dislocation one hour after administration of ethanol. The stomach was excised and lesion index was determined by measuring each lesion in mm along its greater length. # h) Determination of gastric parameters Collection of gastric juice: After post operative period, animals were sacrificed by cervical dislocation and the stomach was dissected out as a whole by passing a ligature at the esophageal end. Gastric content was evacuated into graduated tube by cutting along the greater curvature of the stomach, and was centrifuged at 3000 rpm for 10min. Volume of gastric juice: The volume of the centrifuged sample was expressed as ml/ 100 g body weight. pH of gastric juice: pH of gastric juice was measured with the help of pH meter. Free and total acidity: Gastric juice (1ml) was pipette into a 100ml conical flask and diluted with 9ml distilled water. Two or three drops of Topfer's reagent was then added and titrated with 0.01 N sodium hydroxide until all traces of red colour disappeared and the colour of the solution was yellowish-orange. The volume of alkali added was noted. This volume corresponds to free acidity. Two or three drops of phenolphthalein were then added and the titration was continued until a definite red ring appeared; the volume of alkali added was noted. The volume corresponds to total acidity. The sum of the two titrations was total acidity. Acidity was expressed in terms of mEq/L. Acidity was expressed as: Acidity = 0.1 Estimation of gastric ulcerative index changes: The stomach was opened along the greater curvature and it was washed with running tap water. Then the ulcerative area was counted by placing it on a flat wooden plate. The following arbitrary scoring system was used to grade the incidence and severity of lesion.0 = Normal, 1 = Red coloration, 2 = Spot ulcers, 3 = Hemorrhagic streaks, 4 = Ulcers > 3 but < 5 and 5 = Ulcers > 5. Ulcer index and % protection were calculated by following formulas. # Volume of # Results and Discussion Qualitative chemical analysis results (table-2) were exhibiting the presence of alkaloids, glycosides, flavonoids, tannins, saponins and terpenoids. TLC results (table-1) were qualitatively confirming the presence of flavonoids in successive extracts of root bark by using quercetin as biomarker. Total phenolic contents were quantified by standard procedures and results were given in table 1. Results depicts that phenolic content was significantly found in ethylacetate extract followed by methanol extract. The anti-ulcer activity of root bark of AI was evaluated by employing ethanol induced gastric ulcer in mice. Ethanol induced gastric injury is associated with significant production of oxygen free radicals leading to increased lipid peroxidation, which causes damage to cell and cell membrane. 26 Pretreatment of mice with root bark extracts produced a dose dependent protection in the ethanol induced ulceration model as compared to control group. However the protection was statistically significant reduced the severity of ulcer and caused a significant reduction of ulcer index in this model. Omeprazole produced significant gastric ulcer protection as compared to control group (Table 3). Ethanol damages the plasma membrane and leads to intracellular accumulation of sodium and water by increasing the membrane permeability. These changes ultimately cause cell death and gastric mucosal exfoliation. 27 Ethanol is also known to release the endogenous ulcerogenic mediators. These could precipitate mucosal injury either by causing vascular changes like mucosal edema and increased mucosal permeability or by nonvascular effects like mucus depletion and enzyme release in the stomach. 28 The decrease in volume of gastric juice may also attributed to its anti secretory ear 2012 Y potential of the drug. The anti secretory potential may also relate towards gastric juice and interference of gastric blood circulation are responsible for the induction of ulceration. 29 AI root bark methanol extract significantly decreased the gastric juice volume as compared to control. Methanol extract significantly increased p H as compared to control and nearer to standard. The excessive secretion of hydrochloric acid in the stomach was considered to be an important factor in the formation of peptic ulcer. Hydrochloric acid is known to produce ulceration and digestion of the stomach tissues as well as to reduce the neutralizing capability of the stomach mucus secretions. [30][31][32] As a measurement of free hydrogen ion, pH indirectly represents the hydrochloric acid concentration in the stomach. Increase in pH is usually affected by either the reduction of the acid secreted in the stomach or the increase in the volume of alkaline and neutral fluids (mucus). The variation in the pH level among the groups shows tendency of protective effects of them towards gastric ulceration. The decrease in acidity was at its maximum level for the reference standard group followed by extract treatment group. The least decrease in acidity was shown by methanol extract treated group at its 500mg/kg dose. Macrocsopic examination of ethanol induced gastric ulcer in mice was shown in figure 1. suggest that flavonoid quercetin promotes a decrease in ulcerative lesions due to its antioxidant effect. In addition, a review of antiulcer drugs of plant origin shows that triterpenes, because of their ability to strengthen defensive factors such as stimulation of mucus synthesis or maintenance of the prostaglandin contents of gastric mucosa at high levels, are compounds with potential antiulcerogenic activity (Lewis and Hanson, 1991). Dose dependent ulcer index results were given in figure 2. Comparison of ulcer protection of root bark methanol extract with that of standard control was shown in figure 3. Based on this data, it is suggested that the gastro protection observed in this study could be related to the presence of phenolics and flavonoids in the methanol extract of root bark of AI extract. (a) (b) (c) IV. # Conclusion In conclusion, the results show that the methanol extract of root bark of Azadirachta indica present antiulcer activity, as evidenced by ethanol induced gastric ulcer model in albino mice. Results suggest that the effectiveness of the extract as anti ulcerogenic agent may be due to presence of flavonoids and phenolics compounds. The results of this study showed that the root bark of Azadirachta indica contains appreciable amount of phenolic contents along with other biologically active constituents. ![Group 3 received 100 mg/kg, p.o methanol extract of AI. Group 4 received 200 mg/kg, p.o methanol extract of AI. Group 5 received 500 mg/kg, p.o methanol extract of AI.](image-2.png "") ![GlobalJournal of Medical Research Volume XII Issue VI Version I © 2012 Global Journals Inc. (US) Evaluation of Total Phenolic Contents and Antiulcerogenic Activity of Root Bark of Azadirachta Indica Name of the chemical constituent PE CHCl 3 EtOAc MeOH HA](image-3.png "25") 1![Figure 1 : Macroscopic examination of ethanol induced gastric ulcer in mice a=control, b= standard control, c=treated control (conc.500mg/kg)](image-4.png "Figure 1 :") 2![Figure 2 : Dose dependent changes in ulcer index of treated and control group of animals](image-5.png "Figure 2 :") d) Phytochemical evaluation Ulcer index= Arithmetic mean of intensity in group+ Number of ulcer positive animals Total number of animals×2% Protection =Control mean index-Test mean index Control mean index× 100III.ear 2012YVolume XII Issue VI Version IMedical ResearchNaOH×Normality×100mEq/L/100gGlobal Journal ofUlcer Index 25 1ExtractPetetherChloroformEthylacetateMethanol% 80 Ethanol% Yield w/w2.503.801.724.701.29R f Value0.870.890.920.890.89Total phenolics content,98.19±1.6619.73±0.41821.54±2.70740.10±0.13380.75±2.78µg/mlTotal phenolic contents were expressed in Mean±SEM. 2 3TreatmentDoseVol. ofpHFree AcidityTotal AcidityUlcer index %Protection(mg/kg)gastric(mEq/L)(mEq/L)juice(ml)Control-1.65±0.812.9±0.2031±0.8974.5±1.875.25±0.480Std.control301.48±0.073.9±0.139.3±0.8123.3±2.751.02±0.2 ***75Treated1001.53±0.052.77±0.0828.8±0.9861.3±1.863.16±0.214212501.53±0.083.22±0.1116±3.5742.3±5.682.33±1.7041.755001.23±0.23.70±0.1011.21±1.2329.5±6.101.58±0.47 ***60.50Values are expressed in mean±SEM Statistical comparison was performed by using ANOVA coupled with student's'test. *** P<0.001 were consider statistically significant when compared to control group. © 2012 Global Journals Inc. (US) © 2012 Global Journals Inc. (US) © 2012 Global Journals Inc. (US) * A review of work on Indian medicinal plants including indigenous drugs and poisonous plants. Indian council of medical research, Special research series RNChopra ICChopra 1995 27 30 * Antigastric activity of nimbidin Indian NRPillai DSuganthan CSeshadri Santha J Med Res 169 68 1978 * VBalakrishnan MNarendranathan ASSubair EKRaji NRPillai Santha kumara G Nimbidin in duodenal ulcer Trop gastroenterol 6 1985 * In the neem tree edited by Schmutterer H Weinheim federal republic of Germany: VCH 1995: 1 HSchmutterer * Medicinal properties of neem leaves: a review RSubapriya SNagini Curr Med Chem-Anticancer agents 5 2005 * Biological activities and medicinal properties of neem KausikBiswas IshitaChattopadhyay KRanajit UdayBanerjee Bandyopadhyay Azadirachta indica) Curr Scien 11 2002 * Neem: A great boon to mankind www.shamskm MWasimAktar DwaipayanSengupta 2008 * PCDandiya SKKulkarni Pharmacology Vallabh prakashan New Delhi 247 2005 * Observation survey of NSAID related upper gastrointestinal adverse events in Belgium JBelaiche ABurette MDevos ELouis MHuybrechts MDettenre Acta Gastroenterol Belgium 65 2002 * Exploring Indian medicinal plants for antiulcer activity PDharmani GPallt Ind J Pharmacol 38 2 2006 * Nair BN Preliminary study of antibacterial substances from Melia azadirachta Indian ICChopra KCGupta J Med Res 1952 * Ansory R Activity of East African medicinal plants against Helicobactor pylori Chemotherapy WFebry POkema 1996 315 42 * Antioxidant activity of Saimese neem tree SPogtip SRoongtawan GWandee J Ethanopharmacol 1 2005 * UBandyopadhyay RChatterjee RBandyopadhyay Us Patent5 1998 730 986 Corresponding to Indian patent: 1100/Del/95 * Ogle CW The gastric antiulcer effects of the leaves of the neem tree GPGarg SKNigma Planta Med 215 59 1993 * Free radical scavenging activity of neem tree root bark extract MKiranmai MDIbrahim MahendraKumar Asia J Pharma Clin Res 2011 4 * Ashokkumar P, Rajkumar, Kanimozhi M, Phytochemical screening and antimicrobial activity from five Indian medicinal plants against human pathogens KNAsha RChowdhury MHChoudhury MARashid Mid Eas J Scie Res 54 2004. 2010 Acta pharma * Thin-layer chromatographic analysis of flavonoids, phenolic acids, and amino acids in some Croatian Hypericum taxa HMale J. Planar Chromatogr 17 2004 * Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. 299 VernonLSingleton Rudolf;Orthofer Lamuela-Raventos MRosa 1999 152 * Dried fruits: excellent in vitro and in vivo antioxidants JVinson LZubik PBose NSamman JProch J Am Coll Nutr 24 1 1 February 2005 * KWolfe XWu RHLiu Antioxidant J Agric Food Chem 51 2003 * Veni K Evalution of anti-ulcer activity of Polyalthia longifolia (Sonn.) Thwaites in experimental animals PMalairajan GGeetha SNarasimhan JessiKala Indian J Pharmacol 40 3 2008 * Activity of Wild Punica granatum Ethanolic Seed Extract NSGill SDhawan AJain RArora MBali Anti-UlcerogenicAntioxidant Res J Med Plant 6 2012 * Lipid peroxidation in biological systems in DNA and free radicals Kh Cheesman B Haliwell and OI Aruoma 1993 Ellis Horwood 12 London * Pathogenesis of peptic ulcer disease and current trends in therapy JKDesai RKGoyal NSParmar Ind J Pharmacol 41 1997 * Duodenal ulcer induced by MPTP (1-methyl 4-methyl 1,2,3,6,-tetrahydropyridine) SSzabo Brown HPinam Dli Newmeyer Proc Soc Exp Bio Med 180 1985 * Dual effects of zinc sulphate on ethanolinduced gastric injury in rats: Possibly mediated by an action on mucosal blood flow CHCho BWChen YKPoon MMNg WMHai Lam J Pharm Pharmacol 41 1989 * Antiulcer activity and the mechanism of action of magaldrate in gastric ulceration models of rats Av DDPatel RKSantani Goel Ind J Physiol. Pharmacol 44 2000 * The experimental production of gastric and duodenal ulcers in laboratory animals by the intramuscular injection of histamine in beeswax LHay RVarco CCode Surg Gynecol Obstet 75 1942 * Production of gastric and duodenal ulcer in the cat by intramuscular implantation of histamine SWalpole RVarco CCode Proc Soc Exp Bio Med 44 1940 * Evaluation of antiulcer activity of the main phenolic acids found in Brazilian Green Propolis Lr PDragstedt ; M MBarros ELLemos MFMaistro JP BLeite JKSousa SFBastos Andrade Contributions to the physiology of the stomach. XXXVII 1917. 2008 68 JAMA * The antioxidative and antihistaminic properties of quercetin in ethanolinduced gastric lesions AKahraman NFErkasap TKoken MSerteser FAktepe SErkasap Toxicology 183 2003 * Anti-ulcer drugs of plant origin DALewis PJHanson Progress Medicinal Chemistry GPEllis GBWest Elsevier Science Publishers 1991. 2012 28