Fitoterapia 82 (2011) 666–675

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Fitoterapia

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Anti-ulcer constituents of Annona squamosa twigs☆

Dinesh K. Yadav a,1, Neetu Singh b,1, Kapil Dev a, Rolee Sharma c, Mahendra Sahai d,Gautam Palit b, Rakesh Maurya a,⁎a Division of Medicinal and Process Chemistry, Central Drug Research Institute, CSIR, Lucknow, 226001, Indiab Division of Pharmacology, Central Drug Research Institute, CSIR, Lucknow, 226001, Indiac Department of Biotechnology, Integral University, Lucknow, 226026, Indiad Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India

a r t i c l e i n f o

☆ CDRI communication No. 8029.⁎ Corresponding author. Tel.: +91 522 2612411 18

2623405/2623938/2629504.E-mail address: mauryarakesh@rediffmail.com (R.

1 These authors contributed equally to this work.

0367-326X/$ – see front matter © 2011 Elsevier B.V.doi:10.1016/j.fitote.2011.02.005

a b s t r a c t

Article history:Received 22 November 2010Accepted in revised form 9 February 2011Available online 20 February 2011

Phytochemical investigation of Annona squamosa twigs, resulted in isolation and identificationof twelve known (1–12) compounds among them one 1-(4-β-D-glucopyranosyloxyphenyl)-2-(β-D-glucopyranosyloxy)-ethane (11) is synthetically known but first time isolated fromnatural sources. Their structures were elucidated using 1D and 2D NMR spectroscopic analysis.The isolated compounds (2–8, 11) were evaluated for H+ K+-ATPase activity. Three of thesecompounds (+)-O-methylarmepavine (2), N-methylcorydaldine (3), isocorydine (6) showedpromising anti-secretory activity. Activity of these compounds, comparable to the standarddrug omeprazole is novel to our finding. Moreover, there is no information accessible regardingthe pharmacological effect of A. squamosa on the gastrointestinal system. This study is the firstof its kind to show the significant anti-ulcer effect of A. squamosa. The present study aimed toevaluate the gastroprotective effect of A. squamosa (AS) and to identify its active constituents.Anti-ulcer activity was evaluated against cold restraint (CRU), pyloric ligation (PL), aspirin(ASP), alcohol (AL) induced gastric ulcer and histamine (HA) induced duodenal ulcer modeland further confirmed through in vitro assay of H+ K+-ATPase activity and plasma gastrin level.AS and its chloroform and hexane fraction attenuated ulcer formation in CRU, PL, HAmodel anddisplayed anti-secretory activity in vivo through reduced free, total acidity and pepsin in PL,confirmed by in vitro inhibition of H+ K+-ATPase activity with corresponding decrease inplasma gastrin level. Cytoprotection of AS was apparent with protection in AL, ASP models andenhanced mucin level in PL.

© 2011 Elsevier B.V. All rights reserved.

Keywords:PhytochemicalAnti-ulcerOmeprazoleAntisecretoryProton pumpAporphine

1. Introduction

Annona squamosa, belongs to the family Annonaceae and iscommonly known as sugar apple. It is a fruit tree native toCentral America and is now cultivated throughout tropicsmainly for its edible fruit. The taste of the fruit pulp is sweetbecause of its high sugar content (58%of drymass), and it is clearthat the fruit pulp contains a high calorie value [1]. This plant is

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Maurya).

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reputed to possess several medicinal properties [2]. Folkloricrecord reports its use as an insecticide and an anti-tumor agent[3], anti-diabetic [4], anti-oxidant, anti-lipidimic [5], and anti-inflammatory activities due to the presence of cyclic peptides[6]. In addition, the crushed leaves are sniffed to overcomehysteria and fainting spells, and they are also applied on ulcersand wounds. A leaf decoction is taken in case of dysentery [6].

The previous phytochemical investigation of this plant hasproved that it has a variety of compounds like acetogeninswhich are responsible for antifeedant, antimalarial, cytotoxicand immunosuppressive activities [7,8]. Diterpenes isolatedfrom the title plant have anti-HIV principle and anti-plateletaggregation activity [9,10]. The partially purified flavonoidsreported from the same source are responsible for antimicrobial

667D.K. Yadav et al. / Fitoterapia 82 (2011) 666–675

and pesticidal activities [11]. Some lignans and hydroxyl ke-tones are also found in this plant [12,13]. The number ofalkaloids reported from this plant belongs to different groupssuch as aporphine [14,15], and benzoquinazoline [7]. The aboveprovided evidences suggest that the plant is known for itsvarious medicinal values, but to the best of our knowledge thisplant is not yet, known for its anti-ulcer activity.

Peptic ulcer disease (encircling gastric ulcer and duodenalulcers) affects a large population of the world. It is nowgenerally agreed that gastric lesions develop when thedelicate balance between some gastroprotective (mucin,prostaglandin, bicarbonate, nitric oxide and growth factors)and aggressive factors (acid, pepsin, and Helicobacter pylori)is lost [16]. Hypersecretion of gastric acid is a pathologicalcondition, which occurs due to uncontrolled secretion ofhydrochloric acid from the parietal cells of the gastric mucosathrough the proton pumping H+ K+-ATPase. Modern ap-proach to this includes proton pump inhibitors, histaminereceptor blockers, and drugs that affect the mucosal barrierand prostaglandin analog, but there are reports of develop-ment of tolerance, incidence of relapses and side effects onclinical evaluation that make their efficacy arguable. This hasbeen the basis for the development of new anti-ulcer drugs,which includes herbal drugs.

As a part of our ongoing studies aimed to phytochemicallyand pharmacologically characterize the title plant, we foundthat EtOH extract of twigs of A. squamosa showed significantprotection against cold restraint induced ulcer model in rats.Therefore, we decided to carry out a detailed study aimed toinvestigate the chemical composition of A. squamosa. Chloro-form fraction yielded twelve compounds 1-tritriacontanol (1)

N

H3CO

H3CO

H3CO

R2

N

H3CO

H3CO

H3CO

HO

H3CO

H3COO

CH3

6

32: R1= H, R2= CH35: R1= OH, R2= H

H3CO

H3CO

7

11

R1

OO

OH

HOHO

OH

Fig. 1. Structure of chemical constituent

[17], (+)-O-methylarmepavine (2) [15], N-methylcorydaldine(3) [18], lanuginosine (4) [19,20], (+)-anomuricine (5) [21],isocorydine (6) [15], N-methyl-6,7-dimethoxyisoquinolone(7) [22], 6,7-dimethoxy-2- methylisoquinolinium (8) [23,24],β-sitosterol (9) and β-sitosterol-3-O-β-D-glucopyranoside(10) [25], 1-(4-β-D-glucopyranosyloxyphenyl)-2-(β-D-glucopyranosyloxy)-ethane (11) [26] and rutin (12) [27](Fig. 1). Among these, compounds 1, 3, 5, 7, 8 and 11 havebeen isolated for the first time from the title plant. The knowncompounds were identified by using spectroscopic methodsincluding, mass, 1D and 2D NMR analysis and also by com-parison data already reported in the literature. Thus, the presentstudy aimed to investigate the anti-ulcer activity of ethanolicextract of A. squamosa twigs against different experimentalgastric and duodenal ulcer models and to identify the activeconstituents through bioassay-guided fractionation.

2. Materials and methods

2.1. General procedures

Optical rotations were measured on a Perkin-Elmer model241 digital polarimeter. UV spectra were obtained on aPerkin-Elmer λ-15 UV spectrophotometer. IR spectra wererecorded on a Perkin-Elmer RX-1 spectrophotometer usingKBr pellets. 1H and 13C NMR spectra were recorded on aBruker DRX 300MHz NMR spectrometer. ESMS on anAdvantage Max LCQ Thermo-Finnigan mass spectrometerand FABMS were carried out on a JEOL SX 102/DA-6000 massspectrometer. CC was performed using silica gel (230–400mesh). TLC was carried out on precoated silica gel plates 60

N

N

O

O

O

OCH3

N

H3CO

H3CO

CH3

CH3CH3

4

8

N

O

O

OH

OHOH

HOO

s isolated from Annona squamosa.

668 D.K. Yadav et al. / Fitoterapia 82 (2011) 666–675

F254 or RP-18 F254 plates (Merck). Spots were visualized byUV light or by spraying with H2SO4–MeOH or dragondorff'sreagent.

2.2. Plant material

The twigs of A. squamosawas collected from Lucknow, Indiain themonthofMarch2009.The identifiedplant (CDRI plant no.4738) has been preserved in the investigator's laboratory.

2.3. Phytochemical screening

Liquid–liquidpartitionof theethanolic extract ofA. squamosayielded four fractions of hexane, chloroform, butanol andwater.The fractions were evaluated in cold restraint induced gastriculcermodel in rats. Among these chloroformandhexane solublefraction were found to be active and thus tested in other acutegastric ulcer models and further in vitro assay of H+ K+-ATPaseactivity. Chloroform fraction showed potent anti-ulcer activity,whereas low order of activity was seen in hexane solublefraction. Butanol and aqueous fractions were found to be lessactive. So we purified active chloroform soluble fraction byrepeated column chromatography over silica gel and yieldedtwelve pure compounds. The known compounds (1–10, 12)were identified comparing their spectroscopic data with thosepreviously reported in literature.

The chloroform fraction (80.0 g) was subjected to columnchromatography over silica gel (60–120 mesh) and elutedwith a gradient of hexane-ethyl acetate (95:05) to ethylacetate–methanol (95:05), fifty five fractions were collected(500 ml each) and their composition was monitored by TLC,those fractions showing similar TLC profiles were groupedinto nine major fractions (F1–F9). Successive flash chroma-tography of fraction F1 using hexane-EtOAc as binary mixtureof increasing polarity yielded compound 1 (200 mg), as lightbrown powder. Flash column chromatography over neutralalumina of fraction 2 using hexane-EtOAc as binary mixtureof increasing polarity yielded compound 2 (21 mg). Again CCof fraction F3 using hexane-EtOAc as binary mixture, yieldedcompound 3 (6.0 mg). Compound 4 (180 mg) was obtainedas yellow amorphous powder from flash chromatography offraction F4 using hexane-EtOAc eluate. Flash chromatographyof fraction F5 by elutionwith hexane-EtOAc as binarymixtureof increasing polarity yielded compound 5 (19 mg), whichwas again purified by loading over neutral alumina usingchloroform, and hexane (90:10) as eluent. Successive flashchromatography of fraction F6 using hexane-EtOAc as binarymixture of increasing polarity yielded compound 6 (80 mg).Again flash chromatography of fraction F7 using hexane,EtOAc as binary mixture of increasing polarity, yieldedmixture of two compounds so these were again purified onneutral alumina using chloroform as eluent, and this yieldedcompound 7 (23 mg) and chloroform, and methanol (95:5)as eluent yielded compound 8 (24 mg). Compound 9(200 mg) was crystallized in ethanol, from the residue offraction F7. Fraction F8 was subjected to dianion HP20 resinthen eluted withmethanol–water (40:60) to methanol:water(70:30) yielded two compounds 10 (73 mg) and 11(152 mg). Fraction 9 was purified over HP-20 resin thenreverse chromatographed using gradient of water:methanol(8:2) resulted in compound 12 (2.0 g).

2.4. Experimental animals

Adult Sprague Dawley rats of either sex, weighing 180–200 g were housed in raised bottom mesh cages to preventcoprophagy and were kept in environmentally controlledrooms (25±2 °C, 12 h light and dark cycle). Animals were fedwith standard laboratory food pellets andwater was providedad libitum. Guinea pigs of either sex, weighing 300–350 gwere used for histamine-induced ulcer model, which werealso housed under standard conditions as described above. Allexperimental protocols were approved by our InstitutionalEthical Committee following the guidelines of CPCSEA(Committee for the Purpose of Control and Supervision ofExperiments on Animals) which complies with Internationalnorms of INSA (Indian National Science Academy).

2.5. Materials

All chemicals used were purchased from Sigma ChemicalCo. (St. Louis, MO, USA) except otherwise stated. Sucralfatewas obtained from Meranani Pharmaceuticals, India.

2.6. Treatment schedule

Ethanolic extract of A. squamosa twigs (EtOH extract), itschloroform (CF), hexane (HF), butanolic (BuF) and aqueousfractions (AF), standard drugs like omeprazole (Omz)(10 mg/kg) and sucralfate (SUC) (500 mg/kg) were preparedin 1% carboxymethyl cellulose (CMC) as suspension andadministered orally 45 min prior to exposure of ulcerogens tothe animals at a volume of 1 ml/200 g of body weight. Allanimals were deprived of food for 16 h before exposure toulcerogens and were divided into three groups, (n=6).

1. Control group of animals were treated with vehicle 1%CMC.

2. Ethanolic extract of A. squamosa twigs (25, 50 and 100 mg/kg, p.o.) and its fractions CF (20 mg/kg, p.o.), HF (20 mg/kg, p.o.), BuF (20 mg/kg, p.o.), AF (20 mg/kg, p.o.) weretested against cold restraint ulcer (CRU) model to identifythe effective dose and selected for further studies in otherulcer models.

3. The experimental group was treated with standard anti-ulcer drugs such as Omz (10 mg/kg, p.o.) in (CRU), aspirin(ASP), pyloric ligation (PL), and histamine inducedduodenal ulcer (HA) and SUC (500 mg/kg, p.o.) in alcohol(AL) induced ulcer model.

3. Anti-ulcer studies

3.1. Cold restraint induced gastric ulcer (CRU) [28]

The rats were subjected to cold-stress paradigm after45 min of treatment of EtOH extract (25, 50, 100 mg/kg, p.o.)and its fractions CF (20 mg/kg, p.o.), HF (20 mg/kg, p.o.),BuF (20 mg/kg, p.o.), AF (20 mg/kg, p.o.) and Omz (10 mg/kg,p.o.). All the animals were immobilized in a restraint cages,kept at 4 °C in an environmental chamber for 2 h and thensacrificed. The stomach was cut along the lesser curvatureand ulcers were scored with the help of a magnascope.

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3.2. Aspirin induced gastric ulcer model (ASP) [29]

Aspirin at a dose of 150 mg/kg was administered to induceulcer after 45 minof treatmentof EtOHextract (50 mg/kg, p.o.),CF (20 mg/kg, p.o.), HF (20 mg/kg, p.o.) and Omz (10 mg/kg,p.o.). The animals were sacrificed 5 h after aspirin treatment,the stomach was dissected out, incised along the lessercurvature, and the lesion was scored.

3.3. Alcohol induced gastric ulcer in rats (AL) [30]

Gastric ulcerwas induced in rats by administering absolutealcohol at the dose of 1 ml/200 g, body weight. EtOH extract(50 mg/kg, p.o.), CF (20 mg/kg, p.o.), HF (20 mg/kg, p.o.) andsucralfate (500 mg/kg, p.o.) were administered 45 min beforealcohol treatment. The animals were sacrificed after 1 hourand the stomach was cut along the greater curvature toobserve the gastric lesions, which appear as hemorrhagicbands along the mucosal ridges of the stomach. The lesionswere analyzed through trinocular stereo-zoom microscopeand the lengths of the lesions were measured using Biovisimage analyzer software and summated to give a total lesionscore.

3.4. Pyloric ligation induced ulcer model (PL) [31]

This method was done by ligating the pyloric end of ratstomach under chloral hydrate anesthesia (300 mg/kg, i.p.).After 45 min of EtOH extract (50 mg/kg, p.o.), CF (20 mg/kg,p.o.), HF (20 mg/kg, p.o.) and Omz (10 mg/kg, p.o.) admin-istration, the abdomen was opened below the xiphoidprocess. The pyloric end of the stomach was ligated avoidingany damage to the adjacent blood vessels. The stomach wasreplaced carefully and the abdomen was stitched. After 4 hthe animals were sacrificed and the stomach was dissectedout. Lesions were scored and gastric fluid was collected andcentrifuged at 2000 rpm for 10 min. The collected superna-tant was used for the estimation of gastric secretion studies,mucin estimation and peptic activity.

3.5. Gastric secretion study

Free and total acids in the gastric juice were titrated with0.01 N NaOH, using Topfer's reagent and phenolphthalein asindicators respectively, and were expressed in terms of μeq./ml [32]. Peptic activity was determined by measuring theamount of liberated tyrosine by the action of pepsin onhemoglobin as substrate and expressed in terms of U/ml [33].Mucin level in gastric juice was quantifiedwith a fluorometricassay and expressed as μg of mucin/ml of gastric juice [34].

3.6. Histamine induced duodenal ulcer in guinea pigs (HA) [35]

Duodenal ulcers were induced in guinea pigs by intra-muscular application of histamine acid phosphate at a dose of0.25 mg/kg at every 30 min interval for 4 h and the animalswere sacrificed after 30 min of the last dose. Animalswere treated with EtOH extract (50 mg/kg, p.o.), CF (20 mg/kg, p.o.), HF (20 mg/kg, p.o.) and Omz (10 mg/kg, p.o.) 45 minprior to histamine administration. Stomach was cut along thelesser curvature down to the duodenum to observe the

formation of ulcer on the anterior and posterior wall ofduodenum.

3.7. Measurement of ulcer index [36]

Ulcers were scored with the help of magnascope under 5×magnification using the ulcer scoring criteria. The followingscoring system was used to grade the incidence and severityof the lesions: (i) shedding of epithelium=10; (ii) petechialand frank hemorrhages=20; (iii) one or two ulcers=30;(iv) more than two ulcers=40; and (v) perforatedulcers=50. Length of hemorrhagic band is measured in ALmodel using Biovis Image Analysis Software.

Percentage protection is calculated as follows:% protec-tion=(Uc−Ut)×100/Ucwhere Uc=ulcer index in controlgroup; Ut=ulcer index in treated group.

4. In vitro assay of H+, K+-ATPase activity [37]

The H+, K+-ATPase containing gastric microsomes wasisolated from non-stimulated rat stomach [38]. Gastricmicrosomes, incubated with or without different concentra-tions of as well as standard drug (omeprazole) for 10 min at37 °C, were added to an assay buffer containing 150 mM KCl,10 mM PIPES, 1 mM MgSO4, 5 mM Mg-ATP, 1 mM EGTA and0.1 mM ouabain, at pH 7.2 and 10 μg/ml valinomycin, 2.5 μg/ml oligomycin. The reaction, carried out at 37 °C for 20 minwas stopped by adding 10% ice-cold trichloroacetic acid. Aftercentrifugation (2000 g for 1 min), inorganic phosphaterelease was determined from the resulting supernatantspectrophotometrically at 310 nmwavelength and expressedas μM/h/mg protein.

5. Gastrin measurement

In order to determine the gastrin levels in plasma, bloodwas collected by cardiac puncture, centrifuged, and theplasma was analyzed for gastrin levels using rat gastrin Ienzyme immunoassay kit (assay designs, Hines Drive AnnArbor, U.S.A) following the manufacturer's instructions. Theresults were expressed as pg/ml.

6. PGE2 estimation

For measurement of COX activity, PGE2 was determined inmucosal tissue samples obtained from sham, control andtreatment groups. Briefly, mucosa was scrapped and rapidlyrinsed with ice-cold saline. The tissue was weighed andhomogenized in 10 volumes of phosphate buffer (0.1 M, pH-7.4) containing 1 mM EDTA and 10 μM indomethacin. Thehomogenate was centrifuged (10000 rpm, 10 min, 4 °C), andthe supernatant was processed for PGE2 estimation using theBiotrak enzyme immunosorbent assay kit (Amersham Bios-ciences, Piscataway, NJ), following the manufacturer'sinstructions. Results were expressed as pg PGE2/mg protein.

7. Statistical analysis

All values shown in the figures and tables represent themeans±S.E.M. IC50 values with 95% confidence limits wereestimated usingMaximum Likelihood Iterative Procedure [39].

670 D.K. Yadav et al. / Fitoterapia 82 (2011) 666–675

Statistical analysis was performed with Prism version 3.0software using one-way analysis of variance (ANOVA) followedby Dunnett's multiple comparison test. Pb0.05 was consideredto be statistically significant.

Fig. 3. Effect of EtOH extract of AS and standard drug (Omz and SUC) onpercentage protection of ulcer against pyloric ligation, aspirin, alcoholinduced ulcer model in rats and duodenal ulcer in guinea pigs. Dataexpressed as mean % protection±S.E.M. Statistical analysis was done by OneWay ANOVA followed by Dunnett's Multiple Comparison Test. *Statisticallysignificant at Pb0.05 and **Pb0.01, in comparison to control (n=6 in each

8. Results

8.1. Anti-ulcer effect of EtOH extract of AS against cold restraintinduced ulcer in rats

The preliminary biological evaluation in CRU ulcer model,EtOH extract of AS, using doses of 25, 50, 100 mg/kg bodyweight showed percentage protection of 50% (Pb0.05),87.50% (Pb0.01), 81.20 (Pb0.01) respectively, whereasomeprazole (10 mg/kg, p.o.) showed a protection of 77.40%(Pb0.01) with reference to the control group. Based on theseoutcomes, 50 mg/kg dose was chosen as the lowest effectiveone for further studies (Fig. 2).

group).

8.2. Anti-ulcer effect of EtOH extract of AS against other acutegastric ulcer models

In ASP and PL induced gastric ulcer model, EtOH extract ofAS (50 mg/kg, p.o.) showed percentage protection of 50%(Pb0.05) and 66.63% (Pb0.01) respectively, whereas ome-prazole (10 mg/kg, p.o.) exhibited 37.70% (Pb0.05) and69.42% (Pb0.01) protection respectively. Pre-treatment ofrats with EtOH extract of AS (50 mg/kg, p.o.) produced 76.74%(Pb0.01) protection against gastric mucosal damage, inducedby absolute alcohol. Sucralfate (500 mg/kg, p.o.), the standarddrug, exhibited 64.50% (Pb0.05) protection under the samecondition, when both values were compared with the controlgroup. In HA model, 72.70% (Pb0.01) protection wasobserved with EtOH extract (50 mg/kg, p.o.) as compared tocontrol. Omz (10 mg/kg, p.o.) showed percentage protectionof 70.60% (Pb0.01) in HA model. The results are graphicallyrepresented in Fig. 3.

Fig. 2. Effect of EtOH extract of AS and standard drug omeprazole (Omz)against cold restraint induced ulcer model in rats. Data expressed as mean %protection±S.E.M. Statistical analysis was done by One Way ANOVAfollowed by Dunnett's Multiple Comparison Test. *Statistically significantat Pb0.05 and **Pb0.01, in comparison to control (n=6 in each group).

8.3. Anti-ulcer effect of CF, HF, BuF and AF against cold restraintinduced ulcer in rats

Fractionation studies of EtOH extract of AS twigs yieldedCF (95 g) (20 mg/kg, p.o.), HF (100 g) (20 mg/kg, p.o.), BuF(136 g) (20 mg/kg, p.o.), AF (168 g) (20 mg/kg, p.o.) and Omz(10 mg/kg, p.o.) exerted 81.20% (Pb0.01), 62.40% (Pb0.05),43.60%, 38.62% and 77.40% (Pb0.01) protection respectivelyin CRUmodel as compared to control. These results are shownin Fig. 4.

8.4. Effect of CF against other acute gastric ulcer models

In PL, ASP and HA induced gastric ulcer model, CF (20 mg/kg, p.o.) showed percentage protection of 67.0% (Pb0.01),44.43% (Pb0.05) and 86.3% (Pb0.01) respectively, whereasomeprazole (10 mg/kg, p.o.) exhibited 69.42% (Pb0.01),

Fig. 4. Effect of CF, HF, BuF, AF and standard drug omeprazole (Omz) againstcold restraint induced ulcer model in rats. Data expressed as mean %protection±S.E.M. Statistical analysis was done by One Way ANOVAfollowed by Dunnett's Multiple Comparison Test. *Statistically significantat Pb0.05 and **Pb0.01, in comparison to control (n=6 in each group).

Table 1NMR data of compound 2-(4-β-D-glucopyranosyloxy phenyl)-ethanol (11).

Position δH (J in Hz) δC

1 – 158.22 7.07 d (8.6) 118.23 7.18 d (8.5) 130.94 – 131.8

671D.K. Yadav et al. / Fitoterapia 82 (2011) 666–675

37.70% (Pb0.05) and 70.60% (Pb0.01) protection respective-ly. Pre-treatment of rats with CF (20 mg/kg, p.o.) also exerted65.89% (Pb0.01) protection against gastric mucosal damageinduced by AL compared with sucralfate (500 mg/kg, p.o.),the standard drug, which exhibited 64.50% (Pb0.01) withreference to the control group. The results are graphicallyrepresented in Fig. 5.

5 7.18 d (8.5) 130.96 7.07 d (8.6) 118.2C-α 4.11–3.06 m 33.7C-β 4.67 t (7.23) 77.61′ 4.32 d (7.23) 105.42′ 4.11–3.06 m 75.03′ 4.11–3.06 m 77.94′ 4.11–3.06 m 71.55′ 4.11–3.06 m 77.76′ 4.11–3.06 m 69.861″ 4.86 d (7.4) 102.32″ 4.11–3.06 m 75.03″ 4.11–3.06 m 77.74″ 4.11–3.06 m 71.35″ 4.11–3.06 m 77.56″ 4.11–3.06 m 69.9

8.5. Characterization of 1-(4-β-D-glucopyranosyloxyphenyl)-2-(β-D-glucopyranosyloxy)-ethanol (11)

Compound (11), purified as off-white needle shape crystals,had a molecular formula of C20H30O12 according to its ESIMSmolecular ion peak [M+Na]+ observed at m/z 485, and its 1Hand 13C NMR spectroscopic data. The 1H NMR spectra (Table 1)exhibited two signals in the aromatic region at δH 7.18 (d,J=8.5 Hz, H-3, 5) and 7.07 (d, J=8.6 Hz, H-2, 6) indicating apara-substituted phenyl ring, a triplet at δH 4.67 due to themethylene protons (−O-CH2-) with themultiplet signal of theother benzylic methylene protons at δH 3.27. Two anomericprotons were appeared at δH 4.86 (d, J=7.4 Hz; δC 102.3, C-1″)and δH 4.32 (d, J=7.2 Hz; δC 105.4, C-1′). The couplingconstants of the signal resulting from the anomeric proton ofthe glucopyranoside indicated the glucosidic linkage to have β-configuration. The complexity of the upfield signals wasresolved by 1H-1H COSY spectrum. 13C NMRanalysis confirmedthe presence of twenty carbon atoms in the molecule, eight ofwhich belongs to the aglycone part of the molecule. Acidhydrolysis afforded p-hydroxyphenylethanol and D-glucose.The sugar was identified by co TLC with standard sample. Theoptical rotation [α]D25+9.8° (c 0.12, H2O) of the hydrolyzedsugar indicated that it is the D-glucose. After a detailed study ofDEPT and 13C NMR it was concluded that molecule had fourmethylines, fourteen methines and two quaternary carbons.HSQC (Heteronuclear Single Quantum Correlation) experi-ment, correlated all proton resonances with those of eachcorresponding carbon. The HMBC (Heteronuclear MultipleBond Correlation) experiment which showed long-range

Fig. 5. Effect of CF of AS and standard drug (Omz and SUC) on percentageprotection of ulcer against pyloric ligation, aspirin, alcohol induced ulcermodel in rats and duodenal ulcer in guinea pigs. Data expressed as mean %protection±S.E.M. Statistical analysis was done by One Way ANOVAfollowed by Dunnett's Multiple Comparison Test. *Statistically significantat Pb0.05 and **Pb0.01, in comparison to control (n=6 in each group).

correlations between C-β (δ 77.6) of the aglycone and H-1′ (δ4.32), C-α (δ 33.7) and H-3, 5 (δ 7.18), C-1 (158.2) and H-1″ (δ4.86), allowed one to determine the position of each sugarresidue at C-β and C-1 of the aglycone moiety. Hencecompound 11 is 2-(4-β-D-glucopyranosyloxy phenyl)-ethanol,a new naturally occurring compound.

8.6. Effect of HF against other acute gastric ulcer models

In PL, ASP and HA induced gastric ulcer model, HF (20 mg/kg, p.o.) showed percentage protection of 58.50% (Pb0.01),63.87% (Pb0.01) and 72.60% (Pb0.01) respectively, whereasomeprazole (10 mg/kg, p.o.) exhibited 69.42% (Pb0.01),37.70% (Pb0.05) and 70.60% (Pb0.01) protection respective-ly. Pre-treatment of rats with HF (20 mg/kg, p.o.) also exerted69.82% (Pb0.01) protection against gastric mucosal damageinduced by AL compared with sucralfate (500 mg/kg, p.o.),the standard drug, which exhibited 64.50% (Pb0.01) withreference to the control group. The results are graphicallyrepresented in Fig. 6.

8.7. Effect of EtOH extract, CF and HF on gastric secretion

The effects of EtOH extract, CF and HF on different factorssuch as free acidity, total acidity, peptic activity and defensivefactors, mucin, that play a crucial role in the pathogenesis ofgastric ulcers, were studied by the analysis of the gastric juicefrom PL model. EtOH extract (50 mg/kg, p.o.) reduced freeacidity, total acidity and peptic activity by 76.24% (Pb0.01),65.62% (Pb0.01) and 34.52% (Pb0.05) respectively. CF (20 mg/kg, p.o.) reduced free acidity, total acidity and peptic activity by33.47% (Pb0.05), 48.43% (Pb0.05) and 33.56% (Pb0.05). HF(20 mg/kg, p.o.) reduced free acidity, total acidity and pepticactivity by 59.39% (Pb0.01), 64.25% (Pb0.01) and 40.98%(Pb0.05). Omeprazole (10 mg/kg, p.o.) significantly reducedfree acidity, total acidity and peptic activity by49.60% (Pb0.05),75.03% (Pb0.01) and 76.74% (Pb0.01) respectively, comparedwith the control group. The same dose of EtOH extract, CF andHF and omeprazole (10 mg/kg, p.o.) increased the mucin

Fig. 6. Effect of HF of AS and standard drug (Omz and SUC) on percentageprotection of ulcer against pyloric ligation, aspirin, alcohol induced ulcermodel in rats and duodenal ulcer in guinea pigs. Data expressed as mean %protection±S.E.M. Statistical analysis was done by One Way ANOVAfollowed by Dunnett's Multiple Comparison Test. *Statistically significantat Pb0.05 and **Pb0.01, in comparison to control (n=6 in each group).

Table 3Effect of EtOH extract (10–50 μg/ml), chloroform fraction (10–100 μg/ml),hexane fraction (10–100 μg/ml) of Annona squamosa, its active compounds(+)-O-methylarmepavine (2), N-methylcorydaldine (3), isocorydine (6) (10–100 μg/ml) each and standard drug omeprazole (10–50 μg/ml) on H+ K+-ATPase isolated from rat gastric microsomes. Data expressed as mean±S.E.M.of experiments performed in triplicates (n=3 in each group).

Treatment (μg/ml) Percentageinhibition

H+ K+-ATPaseinhibitionIC50 (μg/ml)

95% confidenceof IC50 limit

Lowerlimit

Upperlimit

ControlEthanolic extract 76.19 31.43 28.94 34.58Chloroform fraction 44.86 55.98 52.19 59.13Hexane fraction 53.96 65.24 62.1 70.92(+)-O-methylarmepavine (2)

53.84 111.83 99.58 125.59

N-methylcorydaldine (3) 71.43 60.9 55.46 67.06Isocorydine (6) 35.46 88.42 85.23 91.3Omeprazole 80.07 30.24 27.52 33.25

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secretion by 56.53% (Pb0.01) and 48.62% (Pb0.01), 50.71%(Pb0.01) and43.12% (Pb0.05) respectively, comparedwith thecontrol. These results are represented in Table 2.

8.8. Effect of EtOH extract, CF, HF and omeprazole onH+ K+-ATPase activity

The antisecretory mechanism of action of EtOH extract(10–50 μg/ml), CF (10–100 μg/ml) and HF (10–100 μg/ml)has been confirmed through the inhibition of gastric H+ K+-ATPase activity in comparison with control with an IC50 valueof 31.43, 55.98 and 65.24 μg/ml respectively. Omeprazole(10–50 μg/ml) used as positive control reduced the enzymeactivity with an IC50 value of 30.24 μg/ml (Table 3).

8.9. Effect of isolated pure compounds from CF and omeprazoleon H+ K+-ATPase activity

Compound (+)-O-methylarmepavine (2) (10–100 μg/ml)N-methylcorydaldine (3) (10–100 μg/ml), and isocorydine(6) (10–100 μg/ml) inhibited the gastric H+ K+-ATPaseactivity in comparison to control with an IC50 value of111.83 μg/ml, 60.98 and 88.42 μg/ml respectively. Omepra-zole (10–50 μg/ml) used as positive control reduced theenzyme activity with an IC50 value of 30.24 μg/ml (Table 3).

Table 2Effect of EtOH extract (50 mg/kg, p.o.), chloroform fraction (20 mg/kg, p.o.), hexane fon free acidity, total acidity, pepsin and mucin contents in pyloric ligation modelcomparison to control. n=6 in each group.

Groups and dose mg/kg, p.o Free acidity (μeq/ml) Total ac

Control 46.3±3.22 73.3±4Ethanol extract (50) 11±3.98** 25.2±2Chloroform fraction (20) 30.8±8.84* 37.8±9Hexane fraction (20) 18.8±4.35** 26.2±3Omeprazole (10) 23.3±1.28* 18.3±1

8.10. Effect of O-methylarmepavine, N-methylcorydaldine,isocorydine on gastrin hormone concentration

The concentration of gastrin hormone in the plasma of ulcercontrol group (ethanol treated) was 127.5±3.7 pg/ml. Pre-treatment with O-methylarmepavine (20 mg/kg) significantly(Pb0.05) reduced the plasma gastrin level (102.8±6.6 pg/ml)in comparison to control. Pretreatment with N-methylcory-daldine (20 mg/kg) and isocorydine (20 mg/kg) reduced theplasma gastrin level 96.8±8.9 pg/ml (Pb0.05) and 112.0±10.2 pg/ml respectively. Lansoprazole (30 mg/kg) used as areference drug gives 39.6±8.6 pg/ml (Pb0.01) plasma gastrinlevel (Fig.7).

8.11. Effect of compound O-methylarmepavine,N-methylcorydaldine, isocorydine and omeprazole ongastric PGE2 level

The PGE2 generation in the ulcer control group was2589.329±208.0 pg/mg tissue protein. The PGE2 value of O-methylarmepavine, N-methylcorydaldine, isocorydine andomeprazole treated group was found to be 3119.012±328.6,2908.318±439.2, 2878.219±114.4, 4253.415±404.7(Pb0.05) respectively (Table 4).

9. Discussion

Research on natural products often is guided by ethno-pharmacological knowledge, and has brought substantial

raction (20 mg/kg, p.o.) of Annona squamosa and omeprazole (10 mg/kg, p.o.)(n=6 in each group). *Statistically significant at Pb0.05 and **Pb0.01, in

idity (μeq/ml) Pepsin (units/ml) Mucin (μg/ml)

.37 97.6±3.52 687.5±17.92

.798* 63.9±3.33* 1581.8±29.74**

.52* 64.8±3.45* 1338.01±29.97**

.99** 57.6±6.48* 1395±79.36*

.31** 22.7±1.15** 1208.6±19.92*

Fig. 7. Effect of O-methylarmepavine (2), N-methylcorydaldine (3),isocorydine (6) and omeprazole on plasma gastrin hormone concentrationin ethanol induced ulcer model. Results are expressed as mean±S.E.M.(n=6). *Statistically significant at Pb0.05, in comparison to control (n=6 ineach group).

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contributions to drug innovation by providing novel chemicalstructures and/or mechanisms of action [40]. In India; a largenumber of herbal extracts are used in folk medicine to treatvarious types of disorders. A. squamosa (AS) is renowned forits various medicinal properties, but no scientific informationis available regarding its anti-ulcer activity. The present studyhas, therefore been conducted to evaluate the anti-ulceractivity of ethanolic extract of A. squamosa twigs and itsfractions using different in vivo ulcer models in rats andguinea pigs and further confirmed through the in vitro assayof H+ K+-ATPase inhibitory activity, followed by its phyto-chemical investigation. Cold restraint stress-induced ulcerhas been suggested as the model for rapid massive screeningof peptic ulcer, so we performed an anti-ulcer study ofethanolic extract of AS and its chloroform (CF), hexane (HF),aqueous (AF) and butanolic (BuF) fractions in CRU model.CRU is a well-accepted model for the induction of gastriculcers, in which peripheral sympathetic activation andincreased acid secretion play important roles [41]. Ethanolicextract of AS showed significant protection in a dosedependent manner in the CRUmodel, with utmost protectionobserved at 50 mg/kg, p.o. and thus selected for furtherstudies in other models. Chloroform and hexane fraction alsoshowed significant protection against this model. Theethanolic extract and its chloroform and hexane fractionalso exert a protective effect against ethanol-induced gastriclesions in contrast to standard drug, sucralfate. Since ethanol

Table 4Effect of O-methylarmepavine (2), N-methylcorydaldine (3), isocorydine (6)of Annona squamosa and standard drug omeprazole on gastric PGE2 level.

Groups Prostaglandin PGE2 (pg/mg protein)

Ulcer control group 2589.329±208.0O-methylarmepavine 3119.012±328.6N-methylcorydaldine 2908.318±439.2Isocorydine 2878.219±114.4Omeprazole 4253.415±404.7 ⁎

Data expressed as mean±S.E.M. Statistical analysis was done by One WayANOVA followed by Dunnett's Multiple Comparison Test.⁎ Statistically significant at Pb0.05, in comparison to control (n=6 in each

group).

damages the superficial epithelial layers and inhibit the re-lease of mucosal prostaglandins [42] and depresses the gas-tric defensive mechanisms, these agents appear to enhancethe gastric mucosal defense [43] indicating the cytoprotectivepotentials of AS and its chloroform and hexane fractions.

In pyloric-ligation model, gastric acid is an important factorfor the genesis of ulceration. In this model, auto-digestion ofmucosabygastric acid and pepsin results in the development ofulcers [44] Ethanolic extract of AS and its chloroform andhexane fractions significantly reduced free, total acidity andpepsin level in this model, which suggests its anti-secretorypotency.

Duodenal ulcer is causedmainly by an increase in acid andpepsin load and gastric metaplasia in the duodenal cap [45].Protective effect of ethanolic extract of AS and its chloroformand hexane fraction against histamine induced duodenalulcer in guinea pigs signifies its role in control of injurymediated by gastric acid secretion suggesting its antisecre-tory activity.

In order to clarify themode of action of ethanolic extract ofAS and its chloroform and hexane fraction, through the anti-secretory pathway, its influence on gastric secretion wasstudied using inhibition of H+ K+-ATPase (Proton pump).This proton pump is the common and final pathway of allstimulation of acid production. Hence, its inhibitor will be apotent anti-secretory agent. The results obtained with gastricmicrosomes isolated from rat stomach showed that ethanolicextract of AS and its chloroform and hexane fraction, potentlyinhibited the H+ K+-ATPase activity comparable to thepositive control omeprazole, thus suggesting that ethanolicextract of AS and its chloroform and hexane fraction might beimparting anti-ulcer activity through decrease in acid se-cretion via proton pump inhibition.

Further, to substantiate the antisecretory potential ofmethylarmepavine, N-methylcorydaldine, and isocorydine,their effect on plasma concentration of gastrin hormone inulcerated rats was determined. Gastrin hormone is a knownmodulator of gastric acid secretion [46] which stimulates theparietal cell to hypersecrete acid, resulting in the develop-ment of gastric ulcer. O-methylarmepavine, N-methylcory-daldine, and isocorydine decreases the gastrin secretion inethanol induced ulcer model, which further confirmed itsantisecretory potential. Thus, the antisecretory activity of O-methylarmepavine, N-methylcorydaldine, and isocorydineappears to be mainly related to the inhibition of H+ K+-ATPase activity and suppression of gastrin release.

Cytoprotective ability of ethanol extract of AS and itschloroform and hexane fraction can be corroborated withincreased mucin content of gastric juice in comparison toomeprazole. To further validate its cytoprotective effect wehave evaluated the efficacy of AS and its chloroform andhexane fraction against aspirin induced ulcer model. Aspirin/NSAIDs induces ulcers due to their effect on cyclooxygenaseenzyme leading to reduced prostaglandin production andincrease in acid secretion [47]. Ethanolic extract of AS and itschloroform and hexane fraction significantly reduced theulcer, which further supports their cytoprotective potential,which may be mediated by prostaglandins.

Further exploration of the active fractions for its chemicalconstituents demonstrated the presence of (+)-O-methylarme-pavine (2), N-methylcorydaldine (3), lanuginosine (4), (+)-

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anomuricine (5), isocorydine (6), N-methyl-6,7-dimethoxyiso-quinolone (7), 6,7-dimethoxy-2-methylisoquinolinium (8), and1-(4-β-D-glucopyranosyloxyphenyl)-2-(β-D-glucopyranosy-loxy)-ethane (11), out of which (+)-O-methylarmepavine (2),N-methylcorydaldine (3), isocorydine (6) inhibited the in vitroassay of H+ K+-ATPase activity and compared with protonpump inhibitor, omeprazole. Thus, the antisecretory activity ofAS might be due to the presence of these compounds.

Conclusively, the present study demonstrated that etha-nol extract of A. squamosa and its chloroform and hexanefraction imparts gastroprotection via inhibition of H+ K+-ATPase (proton pump) activity and simultaneous strength-ening of mucosal defense mechanism. The compounds 2, 3and 6 are the active principles of the plant. These compoundsmay serve as the starting point for the design of novel semi-synthetic and synthetic compounds as anti-ulcer agents in thefuture.

Acknowledgements

Dinesh K. Yadav and Neetu Singh are thankful to theCouncil of Scientific and Industrial Research, New Delhi andthe Indian Council of Medical Research, New Delhi for awardof Senior Research Fellowship. The authors are thankful toSAIF Division, Central Drug Research Institute for recordingspectral data.

References

[1] Andrade EHAA, Zoghbi MDGB, Maia JGS, Fabricius H, Marx F. Chemicalcharacterization of the fruit of Annona squamosa L. occurring in theAmazon. J Food Comps Anal 2001;14:227–32.

[2] Asolkar LV, Kakkar KK, Chakre OJ. In: glossary of Indian medicinal plantswith active principles. New Delhi: Publications and InformationDirectorate; 1992. p. 72–3.

[3] Cheema PS, Dixit RS, Koshi T, Perti SL. Insecticidal properties of the seedoil of Annona squamosa L. J Sci Ind Res 1985;17:132–6.

[4] Shirwaikar A, Rajendran K, Kumar CD, Bodla R. In vitro antioxidantstudies of Annona squamosa Linn. leaves. Indian J Exp Biol 2004;42:803–7.

[5] Gupta RK, Kesari AN, Diwakar S, Tyagi A, Tandona V, Chandra R,Watal G.In vivo evaluation of anti-oxidant and anti-lipidimic potential of Annonasquamosa aqueous extract in Type 2 diabetic models. J Ethnopharma-cology 2008;118:21–5.

[6] Yang YL, Hua KF, Chuang PH,Wu SH,Wu K, Chang FR,Wu YC. New cyclicpeptides from the seeds of Annona squamosa L. and their anti-inflammatory activities. J Agric Food Chem 2008;56:386–92.

[7] Morita H, Sato Y, Chan KL, Choo CY, Itokawa H, Takeya K, Kobayashi J,Samoquasine A. A benzoquinazoline alkaloid from the seeds of Annonasquamosa. J Nat Prod 2000;63:1707–8.

[8] Fujimoto Y, Eguchi T, Kakinuma K, Ikekawa N, Sahai M, Gupta YK.Squamosin, a new cytotoxic bis-tetrahydrofuran containing acetogeninfrom Annona squamosa. Chem Pharm Bull 1988;36:4802–6.

[9] Wu YC, Hung YC, Chang FR, Cosentino M, Wang HK, Lee KH.Identification of ent-16β, 17-dihydroxykauran-19-oic acid as an anti-HIV principle and isolation of the new diterpenoids annosquamosins Aand B from Annona squamosa. J Nat Prod 1996;59:635–7.

[10] Yang YL, Chang FR, Wu CC, Wang WY, Wu YC. New ent-kauranediterpenoids with anti-platelet aggregation activity from Annonasquamosa. J Nat Prod 2002;65:1462–7.

[11] Kotkar HM, Mendki PS, Sadan SVGS, Jha SR, Upasani SM, MaheshwariVL. Antimicrobial and pesticidal activity of partially purified flavonoidsof Annona squamosa. Pest Management. Sciences 2002;58:33–7.

[12] Shanker KS, Kanjilal S, Rao BVSK, Kishore KH, Misra S, Prasad RBN.Isolation and antimicrobial evaluation of isomeric hydroxyl ketones inleaf cuticular waxes of Annona squamosa. Phytochem Anal 2007;18:7–12.

[13] Yang YL, Chang FR, Wu YC. Squadinorlignlside: a novel 7,9-dinorlignanfrom the stem of Annona squamosa. Helv Chim Acta 2005;88:2731–7.

[14] Bhaumik PK, Mukherjee B, Juneau JP, Bhacca NS, Mukherjee R. Alkaloidsfrom leaves of Annona squamosa. Phytochemistry 1979;18:1584–6.

[15] Bhakuni DS, Tewari S, Dhar MM. Aporphine alkaloids of Annonasquamosa. Phytochemistry 1972;11:1819–22.

[16] Hoogerwerf WA, Pasricha PJ. Pharmacotherapy of gastric acidity, pepticulcers, and gastroesophageal reflux disease. In: Brunton LL, Lazo JS,Parker KL, editors. The pharmacological basis of therapeutics.New York: Mc Graw Hill Press Inc.; 2006. p. 967–81.

[17] Mishra PK, Singh N, Ahmad G, Dube A, Maurya R. Glycolipids and otherconstituents from Desmodium gangeticum with antileishmania andimmunomodulatory activities. Bioorg Med Chem Lett 2005;15:4543–6.

[18] Venkov AP, Statkova-Abeghe SM. Synthesis of 3,4-dihydroisoquino-lines, 2-alkyl (Acyl)-1(2 H)-3,4-dihydroisoquinolinones, 2-alkyl-1(2 H)-isoquinolinones and 1-alkyl-2(2 H)-quinolinones by oxidationwith potassium permanganate. Tetrahedron 1996;52:1451–60.

[19] Wijeratne EMK, Hatanaka Y, Kikuchi T, Tezuka Y, Gunatilaka AAL. Adioxoaporphine and other alkaloids of two annonaceous plants of SriLanka. Phytochemistry 1996;42:1703–6.

[20] Zang Z, Elsholy HN, Jocob MR, Pasco DS, Walker LA, Clark AM. Newsesquiterpenoids from the roots of Guatteria multivenia. J Nat Prod2002;65:856–9.

[21] Nishyama Y, Moriyasu M, Ichimaru M, Iwasa K, Kato A, Mathenge SG,Chalo Mutiso PB, Juma FD. Secondary and tertiary isoquinoline alkaloidsfrom Xylopia parviflora. Phytochemistry 2006;67:2671–5.

[22] Wu WN, Beal JL, Doskotch RW. Isolation and characterization ofalkaloids from the root of Thalictrum alpinum. J Nat Prod 1980;43:373–81.

[23] Janssen Richard HAM, Lousberg Robert JJC, Wijkens P, Kruk C, TheunsHG. Assignment of 1H and 13C NMR resonances of some isoquinolinealkaloids. Phytochemistry 1989;28:2833–9.

[24] Nishiyama Y, Moriyasu M, Ichimaru M, Iwasa K, Kato A, Mathenge SG,Chalo Mutiso PB, Juma FD. Quaternary isoquinoline alkaloids fromXylopia parviflora. Phytochemistry 2004;65:939–44.

[25] Moghaddam FM, Farimani MM, Salahvarzi S, Amin G. Chemicalconstituents of dichloromethane extract of cultivated Satureja khuzis-tanica. eCAM 2006:1–4.

[26] Zofia K, Witold W, Boguslaw B. 2-Phenyl etanol glycosides 1. synthesisof 1-(4- β- D- glucopyranosyloxy phenyl), 2-(β-D-glucopyranosyloxy)ethane and 2-(4-β-D-glucopyranosyloxy phenyl) ethanol. Acta PolPharm 1972;29:137–44.

[27] Lee HJ, Ku CH, Beak NI, Kim SH, Park HW, Kim DK. Phytochemicalconstituents from Diodia teres. Arch Pharm Res 2004;27:40–3.

[28] Suleyman H, Demirezer LO, Buyukokuroglu ME, Akcay MF, GepdiremenA, Banoglu ZN, Gocer F. Antiulcerogenic effect of Hippophae rhamnoidesL. Phytother Res 2001;15:625–7.

[29] Goel RK, Das DG, Sanyal AK. Effect of vegetable banana powder onchanges induced by ulcerogenic agents in dissolved mucosubstances ofgastric juice. Indian J Gastroenterol 1985;4:249–51.

[30] Suleyman H, Demirezer LO, Kuruuzum-Uz A. Effects of Rumex patientiaroot extract on indomethacine and ethanol induced gastric damage inrats. Pharmazie 2004;59:147–9.

[31] Shay M, Kamarov SA, Fels D, Meraaze D, Grueinstein H, Siplet H. Asimple method for the uniform production of gastric ulceration in therat. Gastroenterology 1945;5:43–61.

[32] Anoop A, Jegadeesan M. Biochemical studies on the anti-ulcerogenicpotential of Hemidesmus indicus R.Br. var. indicus. J Ethnopharmacol2003;84:149–56.

[33] Debnath PK, Gode KD, Das D, Sanyal AK. Effects of propanolol on gastricsecretion in albino rats. Br J Pharmacol 1974;51:213–6.

[34] Crowther RS, Wetmore RF. Anal. fluorometric assay of o-linkedglycoprotein by reaction with 2 cyanoacetamide. Biochemistry1987;163:170–4.

[35] Parmar NS, Desai JK. A review of the current methodology for theevaluation of gastric and duodenal anti-ulcer agents. Indian J Pharmacol1993;25:120–35.

[36] Srivastava SK, Nath C, Gupta MB, Vrat S, Sinha NJ, Dhawan NK, Gupta GP.Protection against gastric ulcer by verapamil. Pharmacol Res 1991;23:81–6.

[37] Sanui H. Measurement of inorganic orthophosphate in biologicalmaterials: extraction properties of butyl acetate. Analyt Biochem1974;60:489–504.

[38] Berglindh T. Gastric glands and cells: preparation and in vitro methods.Method enzymol 1990;192:93–107.

[39] Finney DJ. Probit analysis; a statistical treatment of the sigmoidalresponse curve. 2nd ed. New York London: Cambridge Univ. Press;1952. pp 318.

[40] Rates SMK. Plants as source of drugs. Toxicon 2001;39:603–13.[41] Djahanguiri B, Taubin HL, Landsburg L. Increased sympathetic activity in

the pathogenesis of restraint ulcer in rats. J Pharmacol Exp Ther1973;184:163–8.

[42] Miller TA, Henagan JM. Indomethacin decreases resistance of gastricbarrier to disruption by alcohol. Dig Dis Sci 1984;29:141–9.

675D.K. Yadav et al. / Fitoterapia 82 (2011) 666–675

[43] Kinoshita M, Tsunehisa N, Tamaki H. Effect of a combination of ecabetsodium and cimetidine on experimentally induced gastric-lesions andgastric-mucosal resistance to ulcerogenic agents in rats. Biol Pharm Bull1995;18:223–6.

[44] Goel RK, Bhattacharya SK. Gastroduodenal mucosal defence andmucosal protective agents. Indian J Exp Biol 1991;29:701–14.

[45] Dore MP, Graham DY. Pathogenesis of duodenal ulcer disease: therest of the story. Baillières Best Pract Res Clin Gastroenterol 2000;14:97–107.

[46] Walsh JH, editor. New York: Gastrin Raven Press; 1993. p. 273.[47] Bhargava KP, GuptaMB, Tangri KK. Mechanism of ulcerogenic activity of

indomethacin and oxyphenbutazone. Eur J Pharmacol 1973;22:191–5.