Fitoterapia (2011), 82(4), 666-675

Anti-ulcer constituents of Annona squamosa Twigs

Dinesh K. Yadav

a,1, Neetu Singh

b,1, Rolee Sharma

c, Mahendra Sahai

d, Gautam Palit

b, Rakesh

Mauryaa,*

aDivision of Medicinal and Process Chemistry, Central Drug Research Institute, CSIR,

Lucknow - 226001, India. bDivision of Pharmacology, Central Drug Research Institute, CSIR, Lucknow - 226001,

India. cDepartment of Biotechnology, Integral University, Lucknow - 226026, India.

dDepartment of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu

University, Varanasi - 221005, India. *Correspondence: Rakesh Maurya

Telephone Number: +91-(522)-2612411-18 Ext.4235

Fax Number: 091-(522)-2623405/2623938/2629504

E-mail: mauryarakesh@rediffmail.com 1

These authors contributed equally to this work

Abstract Phytochemical investigation of Annona squamosa twigs, resulted in isolation and

identification of 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 from natural 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 these compounds (+)-O-methylarmepavine (2), N-

methylcorydaldine (3), isocorydine (6) showed promising anti-secretory activity. Activity of

these compounds, comparable to standard drug omeprazole is novel to our finding. Moreover,

there is no information accessible regarding the pharmacological effect of Annona squamosa

on the gastrointestinal system. This study is the first of its kind to show significant anti-ulcer

effect of Annona squamosa. Present study aimed to evaluate the gastroprotective effect of

Annona 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 model and 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, HA model and

displayed 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 in

plasma gastrin level. Cytoprotection of AS was apparent with protection in AL, ASP models

and enhanced mucin level in PL.

Key words: Phytochemical, anti-ulcer, omeprazole, antisecretory, proton pump, aporphine.

1. Introduction

Annona squamosa, belongs to the family Annonaceae and commonly known as sugar apple.

It is a fruit tree native to Central America and is now cultivated throughout tropics mainly for

its edible fruit, taste of fruit pulp is sweet because of high sugar content (58% of dry mass), it

is clear that fruit pulp is having high calorie value [1]. This plant is reputed to possess several

medicinal properties [2]. Folkloric record reports its use as an insecticide and an anti-tumor

agent [3], anti-diabetic [4], anti-oxidant and anti-lipidimic activity [5], anti-inflammatory

Fitoterapia (2011), 82(4), 666-675

activities due to presence of cyclic peptides [6]. In addition, the crushed leaves are sniffed to

overcome hysteria and fainting spells, they are also applied on ulcers and wounds, and a leaf

decoction is taken in case of dysentery [6].

The previous phytochemical investigation of this plant has proved to have variety of

compounds like acetogenins are responsible for antifeedant, antimalarial, cytotoxic and

immunosuppressive activities [7, 8]. Diterpenes isolated from the title plant have anti-HIV

principle and anti-platelet aggregation activity [9, 10]. The partially purified flavonoids

reported from the same source are responsible for antimicrobial and pesticidal activities [11].

Some lignans and hydroxyl ketones are also found in this plant [12, 13]. The number of

alkaloids reported from this plant belongs to different groups such as aporphine [14, 15],

benzoquinazoline [7]. The above provided evidences suggest that the plant is known for its

various medicinal values, but to the best of our knowledge this plant is not yet, known for its

anti-ulcer activity.

Peptic ulcer disease (encircling gastric ulcer and duodenal ulcers) affects a large population

of the world. It is now generally agreed that gastric lesions develop when the delicate 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 pathological condition, which occurs due to uncontrolled

secretion of hydrochloric acid from the parietal cells of the gastric mucosa through the proton

pumping H+

K+-ATPase. Modern approach to this includes proton pump inhibitors, histamine

receptor blockers, drugs affecting the mucosal barrier and prostaglandin analog, but there are

reports of development of tolerance, incidence of relapses and side effects on clinical

evaluation make their efficacy arguable. This has been the basis for the development of new

anti-ulcer drugs, which includes herbal drugs.

As a part of our ongoing studies aimed to phytochemically and pharmacologically

characterize the title plant, we found that EtOH extract of twigs of A. squamosa showed

significant protection against cold restraint induced ulcer model in rats. Therefore, we

decided to carry out a detailed study aimed to investigate the chemical composition of A.

squamosa. Chloroform fraction yielded twelve compounds 1-tritriacontanol (1) [17], (+)-O-

methylarmepavine (2) [15], N-methylcorydaldine (3) [18], lanuginosine (4) [19, 20], (+)-

anomuricine (5) [21], isocorydine (6) [22], N-methyl-6,7-dimethoxyisoquinolone (7) [23],

6,7-dimethoxy-2- methylisoquinolinium (8) [24, 25], β-sitosterol (9) and β-sitosterol-3-O-β-

D-glucopyranoside (10) [26], 1-(4-β-D-glucopyranosyloxyphenyl)-2-(β-D-

glucopyranosyloxy)-ethane (11) [26] and Rutin (12) [28] (Figure 1).

N

H3CO

H3CO

H3CO

R2

N

H3CO

H3CO

HO

H3CO

CH3

N

H3CO

H3CO CH3

N

O

O

O

OCH3

N

H3CO

H3CO

O

CH3

6

4 32: R1= H, R2= CH3

5: R1= OH, R2= H

8

N

H3CO

H3CO CH3

O

7

11

R1

O

O

OH

O

OH

HO

HOOH

OH

OHHO

O

Figure 1. Structure of chemical constituents isolated from Annona squamosa.

Fitoterapia (2011), 82(4), 666-675

Among these, compounds 1, 3, 5, 7, 8 and 11 have been isolated first time from the title plant.

The known compounds were identified by using spectroscopic methods including, mass, 1D

and 2D NMR analysis and also by comparison data already reported in the literature. Thus,

the present study aimed to investigate the anti-ulcer activity of ethanolic extract of Annona

squamosa twigs against different experimental gastric and duodenal ulcer models and to

identify the active constituents through bioassay-guided fractionation.

2. Materials and Methods

2.1. General procedures

Optical rotations were measured on a Perkin-Elmer model 241 digital polarimeter. UV

spectra were obtained on a Perkin-Elmer λ-15 UV spectrophotometer. IR spectra were

recorded on a Perkin-Elmer RX-1 spectrophotometer using KBr pellets. 1H and

13C NMR

spectra were recorded on a Bruker DRX 300 MHz NMR spectrometer. ESMS on an

Advantage Max LCQ Thermo-Finnigan mass spectrometer and FABMS were carried out on

a JEOL SX 102/DA-6000 mass spectrometer. CC was performed using silica gel (230-400

mesh). TLC was carried out on precoated silica gel plates 60 F254 or RP-18 F254 plates

(Merck). Spots were visualized by UV light or by spraying with H2SO4–MeOH or

dragondorffs reagent.

2.2 Plant material

The twigs of Annona squamosa was collected from Lucknow, India in the month of march

2009.The identified plant (CDRI plant No. 4738) has been preserved in the investigator

laboratory.

2.3 Phytochemical Screening

Liquid-liquid partition of the ethanolic extract of A. squamosa yielded four fractions hexane,

chloroform, butanol and water. The fractions were evaluated in cold restraint induced gastric

ulcer model in rats. Among these chloroform and hexane soluble fraction were found to be

active and thus tested in other acute gastric ulcer models and further in vitro assay of H+ K

+-

ATPase activity. Chloroform fraction showed potent anti-ulcer activity, whereas low order of

activity was seen in hexane soluble fraction. Butanol and aqueous fractions were found to be

less active. So we purified active chloroform soluble fraction by repeated column

chromatography over silica gel yielded twelve pure compounds. The known compounds (1-

10, 12) were identified comparing their spectroscopic data with those previously reported in

literature.

The chloroform fraction (80.0 g) was subjected to column chromatography over silica gel

(60-120 mesh) and eluted with a gradient of hexane-ethyl acetate (95:05) to ethyl acetate-

methanol (95:05), fifty five fractions were collected (500 ml each) and their composition was

monitored by TLC, those fractions showing similar TLC profile grouped into nine major

fractions (F1-F9). Successive flash chromatography of fraction F1 using hexane-EtOAc as

binary mixture of increasing polarity yielded compound 1 (200 mg), as light brown powder.

Flash column chromatography over neutral alumina of fraction 2 using hexane-EtOAc as

binary mixture of increasing polarity yielded compound 2 (21 mg). Again CC of fraction F3

using hexane-EtOAc as binary mixture yielded compound 3 (6.0 mg). Compound 4 (180 mg)

was obtained as yellow amorphous powder from flash chromatography of fraction F4 using

hexane-EtOAc eluate. Flash chromatography of fraction F5 by elution with hexane-EtOAc as

binary mixture of increasing polarity yielded compound 5 (19 mg), which was again purified

by loading over neutral alumina using eluent as chloroform, hexane (90:10). Successive flash

Fitoterapia (2011), 82(4), 666-675

chromatography of fraction F6 using hexane-EtOAc as binary mixture of increasing polarity

yielded compound 6 (80 mg). Again flash chromatography of fraction F7 using hexane,

EtOAc as binary mixture of increasing polarity, yielded mixture of two compounds so these

were again purified on neutral alumina using chloroform as eluent, yielded compound 7 (23

mg) and Chloroform, Methanol (95:5) yielded compound 8 (24mg). Compound 9 (200 mg)

was crystallized in ethanol, from the residue of fraction F7. Fraction F8 was subjected to

dianion HP20 resin then eluted with methanol-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 then reverse chromatographed using gradient of water: methanol (8:2) resulted

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 prevent coprophagy and were kept in environmentally controlled rooms (25 +

2°C, 12 hours light and dark cycle). Animals were fed with standard laboratory food pellets

and water was provided ad libitum. Guinea pigs of either sex, weighing 300–350 g were used

for histamine-induced ulcer model, which were also housed under standard conditions as

described above. All experimental protocols were approved by our Institutional Ethical

Committee following the guidelines of CPCSEA (Committee for the Purpose of Control and

Supervision of Experiments on Animals) which complies with International norms of INSA

(Indian National Science Academy).

2.5 Materials

All chemicals used were purchased from Sigma Chemical Co. (St. Louis, MO, USA) except

otherwise stated. Sucralfate was obtained from Meranani Pharmaceuticals, India.

2.6 Treatment schedule

Ethanolic extract of A. squamosa twigs (EtOH extract), its chloroform (CF), hexane (HF),

butanolic (BuF) and aqueous fractions (AF), standard drugs like omeprazole (Omz) (10

mg/kg) and sucralfate (SUC) (500 mg/kg) were prepared in 1% carboxymethyl cellulose

(CMC) as suspension and administered orally 45 min prior to exposure of ulcerogens to the

animals at a volume of 1ml/200g of body weight. All animals were deprived of food for 16 h

before ulcerogens exposure 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

(20mg/kg, p.o.), HF (20 mg/kg, p.o.), BuF (20 mg/kg, p.o.), AF (20 mg/kg, p.o.) were tested

against Cold restraint ulcer (CRU) model to identify the effective dose and selected for

further studies in other ulcer models.

3. 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 induced duodenal 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) [29]

The rats were subjected to cold-stress paradigm after 45mins 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 40C in an environmental chamber for 2 hours and

then sacrificed. The stomach was cut along the lesser curvature and ulcers were scored with

the help of magnascope.

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3.2 Aspirin induced gastric ulcer model (ASP) [30]

Aspirin at a dose of 150 mg/kg was administered to induce ulcer after 45mins of treatment 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.). The animals were sacrificed 5 hr after aspirin treatment, the stomach was dissected out,

incised along the lesser curvature, and the lesion was scored.

3.3 Alcohol induced gastric ulcer in rats (AL) [31]

Gastric ulcer was induced in rats by administering absolute alcohol at the dose of 1 ml/200g,

body weight. EtOH extract (50mg/kg, p.o.), CF (20 mg/kg, p.o.), HF (20 mg/kg, p.o.) and

sucralfate (500 mg/kg, p.o.) were administered 45mins before alcohol treatment. The animals

were sacrificed after one hour and stomach was cut along the greater curvature to observe the

gastric lesions, which appear as hemorrhagic bands along the mucosal ridges of the stomach.

The lesions were analyzed through trinocular stereo-zoom microscope and the lengths of the

lesions were measured using biovis image analyzer software and summated to give a total

lesion score.

3.4. Pyloric ligation induced ulcer model (PL) [32]

This method was done by ligating the pyloric end of rat stomach under chloral hydrate

anesthesia (300 mg/kg, i.p.). After 45min 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.) administration, the abdomen was opened

below the xiphoid process. The pyloric end of the stomach was ligated avoiding any damage

to the adjacent blood vessels. Stomach was replaced carefully and the abdomen was stitched.

After 4 hr the animals were sacrificed and the stomach was dissected out. Lesions were

scored and gastric fluid was collected and centrifuged at 2000 rpm for 10 min. The collected

supernatant 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 with 0.01N NaOH, using Topfer’s

reagent and phenolphthalein as indicators respectively, and were expressed in terms of

µeq./ml [33]. Peptic activity was determined by measuring the amount of liberated tyrosine

by the action of pepsin on hemoglobin as substrate and expressed in terms of U/ml [34].

Mucin level in gastric juice was quantified with a fluorometric assay and expressed as µg of

mucin/ml of gastric juice [35].

3.6. Histamine induced duodenal ulcer in guineapigs (HA) [36]

Duodenal ulcers were induced in guinea pigs by intramuscular application of histamine acid

phosphate at a dose of 0.25 mg/kg at every 30 mins interval for 4 hr and the animals were

sacrificed after 30 mins of the last dose. Animals were treated with EtOH extract (50 mg/kg,

p.o.), CF (20 mg/kg, p.o.), HF (20mg/kg, p.o.) and Omz (10 mg/kg, p.o.) 45 min prior to

histamine administration. Stomach was cut along the lesser curvature down to the duodenum

to observe the formation of ulcer on the anterior and posterior wall of duodenum.

3.7. Measurement of ulcer index [37]

Ulcers were scored with the help of magnascope under 5X magnification using the ulcer

scoring criteria. The following scoring system was used to grade the incidence and severity of

the lesions: (i) shedding of epithelium = 10; (ii) petechial and frank hemorrhages = 20; (iii)

one or two ulcers = 30; (iv) more than two ulcers = 40; (v) Perforated ulcers = 50. Length of

hemorrhagic band is measured in AL model using Biovis Image Analysis Software.

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Percentage protection is calculated as follows:

% protection = (Uc − Ut) × 100 / Uc

Where Uc = ulcer index in control group; Ut = ulcer index in treated group.

4. In vitro assay of H+, K

+-ATPase activity [38]

The H+, K

+-ATPase containing gastric microsomes were isolated from non-stimulated rat

stomach [39]. Gastric microsomes, incubated with or without different concentrations of

as well as standard drug (omeprazole) for 10min at 37°C, were added to an assay buffer

containing 150 mM KCl, 10 mM PIPES, 1 mM MgSO4, 5 mM Mg-ATP, 1 mM EGTA and

0.1 mM ouabain, at pH 7.2 and 10 µg/ml valinomycin, 2.5 µg/ml oligomycin. The reaction,

carried out at 37°C for 20min was stopped by adding 10% ice-cold trichloroacetic acid. After

centrifugation (2000 g for 1 min), inorganic phosphate release was determined from the

resulting supernatant spectrophotometrically at 310nm wavelength

and expressed as

µM/hr/mg protein.

5. Gastrin measurement

In order to determine the gastrin levels in plasma, blood was collected by cardiac puncture,

centrifuged, and the plasma was analyzed for gastrin levels using rat gastrin I enzyme

immunoassay kit (assay designs, Hines Drive Ann Arbor, U.S.A) following the

manufacturer’s instructions. The results were expressed as pg/ml.

6. PGE2 estimation

For measurement of COX activity, PGE2 was determined in mucosal tissue samples obtained

from sham, control and treatment groups. Briefly, mucosa was scrapped and rapidly rinsed

with ice-cold saline. The tissue was weighed and homogenized in 10 volumes of phosphate

buffer (0.1M, pH-7.4) containing 1mM EDTA and 10 µM indomethacin. The homogenate

was centrifuged (10 000 rpm, 10 min, 4oC), and the supernatant was processed for PGE2

estimation using the Biotrak enzyme immunosorbent assay kit (Amersham Biosciences,

Piscataway, NJ), following the manufacturer’s instructions. Results were expressed as pg

PGE2/mg protein.

7. Statistical analysis

All values shown in the figures and tables represent the means ± S.E.M. IC50 values with 95%

confidence limits were estimated using Maximum Likelihood Iterative Procedure [40].

Statistical analysis was performed with Prism version 3.0 software using one-way analysis of

variance (ANOVA) followed by Dunnett’s multiple comparison test. P<0.05 was considered

to be statistically significant.

8. Results

8.1 Anti-ulcer effect of EtOH extract of AS against cold restraint induced ulcer in rats

The preliminary biological evaluation in CRU ulcer model, EtOH extract of AS, using doses

of 25, 50, 100 mg/kg body weight showed percentage protection of 50% (P<0.05), 87.50%

(P<0.01), 81.20 (P<0.01) respectively, whereas omeprazole (10mg/kg, p.o.) showed a

protection of 77.40% (P<0.01) with reference to the control group. Based on these outcomes,

50 mg/kg dose was chosen as the lowest effective one for further studies (Figure 2).

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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 ANOVA

followed by Dunnett's Multiple Comparison Test. *Statistically significant at P < 0.05 and **P < 0.01, in comparison to control (n = 6 in each group).

8.2 Anti-ulcer effect of EtOH extract of AS against other acute gastric ulcer models

In ASP and PL induced gastric ulcer model, EtOH extract of AS (50 mg/kg, p.o.) showed

percentage protection of 50% (P<0.05) and 66.63% (P<0.01) respectively, whereas

omeprazole (10 mg/kg, p.o.) exhibited 37.70% (P<0.05) and 69.42% (P<0.01) protection

respectively. Pre-treatment of rats with EtOH extract of AS (50 mg/kg, p.o.) produced

76.74% (P<0.01) protection against gastric mucosal damage, induced by absolute alcohol.

Sucralfate (500 mg/kg, p.o.), the standard drug, exhibited 64.50% (P<0.05) protection under

the same condition, when both values were compared with the control group. In HA model,

72.70% (P<0.01) protection was observed with EtOH extract (50 mg/kg, p.o.) as compared to

control. Omz (10mg/kg, p.o.) showed percentage protection of 70.60% (P<0.01) in HA

model. The results are graphically represented in Figure 3.

Fig. 3. Effect of EtOH extract of AS and standard drug (Omz and SUC) on percentage protection of ulcer against pyloric ligation, aspirin, alcohol induced ulcer model in rats and duodenal ulcer in guinea pigs. Data expressed as

mean % protection ± S.E.M. Statistical analysis was done by One Way ANOVA followed by Dunnett's Multiple Comparison Test. *Statistically significant at P < 0.05 and **P < 0.01, in comparison to control (n = 6 in each

group).

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

Fractionation studies of EtOH extract of AS twigs yielded CF (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

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(10 mg/kg, p.o.) exerted 81.20% (P<0.01), 62.40% (P<0.05), 43.60%, 38.62% and 77.40%

(P<0.01) protection respectively in CRU model as compared to control. These results are

shown in Figure 4.

Fig. 4. Effect of CF, HF, BuF, AF 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 ANOVA

followed by Dunnett's Multiple Comparison Test. *Statistically significant at P < 0.05 and **P < 0.01, in comparison to control (n = 6 in each group).

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% (P<0.01), 44.43% (P<0.05) and 86.3% (P<0.01) respectively, whereas

omeprazole (10 mg/kg, p.o.) exhibited 69.42% (P<0.01), 37.70% (P<0.05) and 70.60%

(P<0.01) protection respectively. Pre-treatment of rats with CF (20 mg/kg, p.o.) also exerted

65.89% (P<0.01) protection against gastric mucosal damage induced by AL compared with

sucralfate (500 mg/kg, p.o.), the standard drug, which exhibited 64.50% (P<0.01) with

reference to the control group. The results are graphically represented in Figure 5.

Fig. 5. Effect of CF of AS and standard drug (Omz and SUC) on percentage protection of ulcer against pyloric ligation, aspirin, alcohol induced ulcer model in rats and duodenal ulcer in guinea pigs. Data expressed as mean

Fitoterapia (2011), 82(4), 666-675

% protection ± S.E.M. Statistical analysis was done by One Way ANOVA followed by Dunnett's Multiple Comparison Test. *Statistically significant at P < 0.05 and **P < 0.01, in comparison to control (n = 6 in each

group).

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 ESIMS molecular ion peak [M+Na] +

observed at m/z 485, and its 1H and

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 a para-substituted phenyl ring, a triplet at δH 4.67 due to the methylene protons (-

O-CH2-) with the multiplet signal of the other benzylic methylene protons at δH 3.27. Two

anomeric protons 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).

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

Position δH (J in Hz) δC

1 - 158.2

2 7.07 d (8.6) 118.2

3 7.18 d (8.5) 130.9

4 - 131.8

5 7.18 d (8.5) 130.9

6 7.07 d (8.6) 118.2

C-α 4.11-3.06 m 33.7

C-β 4.67 t (7.23) 77.6

1 4.32 d (7.23) 105.4

2′ 4.11-3.06 m 75.0

3′ 4.11-3.06 m 77.9

4′ 4.11-3.06 m 71.5

5′ 4.11-3.06 m 77.7

6′ 4.11-3.06 m 69.86

1 4.86 d (7.4) 102.3

2 4.11-3.06 m 75.0

3 4.11-3.06 m 77.7

4 4.11-3.06 m 71.3

5 4.11-3.06 m 77.5

6 4.11-3.06 m 69.9

The coupling constants of the signal resulting from the anomeric proton of the

glucopyranoside indicated the glucosidic linkage to have β-configuration. The complexity of

the upfield signals was resolved by 1H-

1H COSY spectrum.

13C NMR analysis confirmed

presence of twenty carbon atoms in the molecule, eight of which belongs to aglycon part of

the molecule. Acid hydrolysis afforded p-hydroxyphenylethanol and D-glucose. The sugar

was identified by co TLC with standard sample. The optical rotation [α]D25

+9.8° (c 0.12,

H2O) of the hydrolyzed sugar indicated that it is the D-glucose. After detailed study of DEPT

and 13

C NMR it was concluded that molecule had four methylines, fourteen methines and two

quaternary carbons. HSQC (Heteronuclear Single Quantum Correlation) experiment,

correlated all proton resonances with those of each corresponding carbon. The HMBC

(Heteronuclear Multiple Bond Correlation) experiment which showed long-range correlations

Fitoterapia (2011), 82(4), 666-675

between C-β (δ 77.6) of the aglycon and H-1 (δ 4.32), C-α (δ 33.7) and H-3, 5 (δ 7.18), C-1

(158.2) and H-1 (δ 4.86), allowed to determine the position of each sugar residue at C-β and

C-1 of the aglycone moiety. Hence compound 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% (P<0.01), 63.87% (P<0.01) and 72.60% (P<0.01) respectively, whereas

omeprazole (10 mg/kg, p.o.) exhibited 69.42% (P<0.01), 37.70% (P<0.05) and 70.60%

(P<0.01) protection respectively. Pre-treatment of rats with HF (20mg/kg, p.o.) also exerted

69.82% (P<0.01) protection against gastric mucosal damage induced by AL compared with

sucralfate (500 mg/kg, p.o.), the standard drug, which exhibited 64.50% (P<0.01) with

reference to the control group. The results are graphically represented in Figure 6.

Fig. 6. Effect of HF of AS and standard drug (Omz and SUC) on percentage protection of ulcer against pyloric ligation, aspirin, alcohol induced ulcer model in rats and duodenal ulcer in guinea pigs. Data expressed as mean

% protection ± S.E.M. Statistical analysis was done by One Way ANOVA followed by Dunnett's Multiple Comparison Test. *Statistically significant at P < 0.05 and **P < 0.01, in comparison to control (n = 6 in each

group).

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

The effects of EtOH extract, CF and HF on different factors such as free acidity, total acidity,

peptic activity and defensive factors, mucin, that play a crucial role in the pathogenesis of

gastric ulcers, were studied by the analysis of the gastric juice from PL model. EtOH extract

(50mg/kg, p.o.) reduced free acidity, total acidity and peptic activity by 76.24% (P<0.01),

65.62% (P<0.01) and 34.52% (P<0.05) respectively. CF (20mg/kg, p.o.) reduced free acidity,

total acidity and peptic activity by 33.47% (P<0.05), 48.43% (P<0.05) and 33.56% (P<0.05).

HF (20 mg/kg, p.o.) reduced free acidity, total acidity and peptic activity by 59.39%

(P<0.01), 64.25% (P<0.01) and 40.98% (P<0.05). Omeprazole (10mg/kg, p.o.) significantly

reduced free acidity, total acidity and peptic activity by 49.60% (P<0.05), 75.03% (P<0.01)

and 76.74% (P<0.01) respectively, compared with the control group. The same dose of EtOH

extract, CF and HF and omeprazole (10mg/kg, p.o.) increased the mucin secretion by 56.53%

(P<0.01) and 48.62% (P<0.01), 50.71% (P<0.01) and 43.12% (P<0.05) respectively,

compared with the control. These results are represented in Table 2.

Fitoterapia (2011), 82(4), 666-675

Table 2. Effect of EtOH extract (50mg/kg, p.o.), chloroform fraction (20mg/kg, p.o.), hexane

fraction (20mg/kg, p.o.) of Annona squamosa and omeprazole (10mg/kg, p.o.) on free

acidity, total acidity, pepsin and mucin contents in pyloric ligation model (n= 6 in each

group). *Statistically significant at P<0.05 and ** P< 0.01, in comparison to control. n = 6 in

each group.

8.8 Effect of EtOH extract, CF, HF and Omeprazole on H+ K

+-ATPase activity

The antisecretory mechanism of action of EtOH extract (10-50µg/ml), CF (10-100µg/ml) and

HF (10-100µg/ml) has been confirmed through the inhibition of gastric H+

K+-ATPase

activity in comparison with control with an IC50 value of 31.43, 55.98 and 65.24 µg/ml

respectively. Omeprazole (10-50 µg/ml) used as positive control reduced the enzyme activity

with an IC50 value of 30.24 µg/ml (Table 3).

8.9 Effect of isolated pure compounds from CF and Omeprazole on H+ K

+-ATPase activity

Compound (+)-O-Methylarmepavine (2) (10-100µg/ml) N-methylcorydaldine (3) (10-

100µg/ml), and Isocorydine (6) (10-100 µg/ml) inhibited the gastric H+ K

+-ATPase activity

in comparison to control with an IC50 value of 111.83 µg/ml, 60.98 and 88.42 µg/ml

respectively. Omeprazole (10-50 µg/ml) used as positive control reduced the enzyme activity

with an IC50 value of 30.24 µg/ml (Table 3).

Table 3. Effect of EtOH extract (10-50µg/ml), chloroform fraction (10-100µg/ml), hexane

fraction (10-100µg/ml) of Annona squamosa, its active compounds (+)-O-methylarmepavine

(2), N-methylcorydaldine (3), isocorydine (6) (10-100µg/ml) each and standard drug

omeprazole (10-50µg/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).

Groups and Dose

mg/kg, p.o

Free acidity

(eq/ml)

Total acidity

(eq/ml)

Pepsin

(units/ml) Mucin (g/ml)

Control 46.3 ± 3.22 73.3 ± 4.37 97.6 ± 3.52 687.5 ± 17.92

Ethanol extract

(50)

11 ± 3.98** 25.2 ± 2.798* 63.9 ± 3.33* 1581.8 ± 29.74**

Chloroform

fraction (20)

30.8 ± 8.84* 37.8 ± 9.52* 64.8 ± 3.45* 1338.01 ±

29.97**

Hexane fraction

(20)

18.8 ± 4.35** 26.2 ± 3.99** 57.6 ± 6.48* 1395 ± 79.36*

Omeprazole (10) 23.3 ± 1.28* 18.3 ± 1.31** 22.7 ± 1.15** 1208.6± 19.92*

Treatment (g/ml) Percentage

inhibition

H+K

+

ATPase

inhibition

IC50 (g/ml)

95% Confidence of IC50 limit

Lower limit Upper limit

Control

Ethanolic extract 76.19 31.43 28.94 34.58

Chloroform fraction 44.86 55.98 52.19 59.13

Hexane 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 67.06

Isocorydine (6) 35.46 88.42 85.23 91.3

Omeprazole 80.07 30.24 27.52 33.25

Fitoterapia (2011), 82(4), 666-675

9 Effect of O-Methylarmepavine, N-methylcorydaldine, Isocorydine on gastrin hormone

concentration

The concentration of gastrin hormone in the plasma of ulcer control group (ethanol treated)

was 127.5±3.7 pg/ml. Pretreatment with O-Methylarmepavine (20mg/kg) significantly

(P<0.05) reduced the plasma gastrin level (102.8±6.6 pg/ml) in comparison to control.

Pretreatment with N-methylcorydaldine (20mg/kg) and Isocorydine (20mg/kg) reduced the

plasma gastrin level 96.8±8.9 pg/ml (P<0.05) and 112.0±10.2 pg/ml respectively.

Lansoprazole (30mg/kg) used as reference drug gives 39.6±8.6 pg/ml (P<0.01) plasma

gastrin level (Fig.7).

Fig. 7. Effect of O-methylarmepavine (2), N-methylcorydaldine (3), isocorydine (6) and omeprazole on plasma

gastrin hormone concentration in ethanol induced ulcer model. Results are expressed as mean ± S.E.M. (n = 6). *Statistically significant at P < 0.05, in comparison to control (n = 6 in each group).

9.1 Effect of compound O-Methylarmepavine, N-methylcorydaldine, Isocorydine and

Omeprazole on gastric PGE2 level

The PGE2 generation in the ulcer control group was 2589.329±208.0 pg/mg tissue protein.

The PGE2 value of O-Methylarmepavine, N-methylcorydaldine, Isocorydine and Omeprazole

treated group was found to be 3119.012±328.6, 2908.318±439.2, 2878.219±114.4,

4253.415±404.7 (P<0.05) respectively (Table 4).

Table 4 Effect 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±208.0

O-Methylarmepavine 3119.012±328.6

N-methylcorydaldine 2908.318±439.2

Isocorydine 2878.219±114.4

Omeprazole 4253.415±404.7*

Data expressed as mean ± S.E.M. Statistical analysis was done by One Way ANOVA

followed by Dunnett's Multiple Comparison Test. *Statistically significant at P<0.05, in

comparison to control (n=6 in each group).

10. Discussion

Research on natural products often is guided by ethnopharmacological knowledge, and has

brought substantial contributions to drug innovation by providing novel chemical structures

Fitoterapia (2011), 82(4), 666-675

and/or mechanisms of action [41]. In India; a large number of herbal extracts are used in folk

medicine to treat various types of disorders. Annona squamosa (AS) is renowned for its

various medicinal properties, but no scientific information is available regarding its anti-ulcer

activity. The present study has, therefore been conducted to evaluate the anti-ulcer activity of

ethanolic extract of Annona squamosa twigs and its fractions using different in vivo ulcer

models in rats and guineapigs and further confirmed through the in vitro assay of H+ K

+-

ATPase inhibitory activity, followed by its phytochemical investigation. Cold restraint stress-

induced ulcer has been suggested as the model for rapid massive screening of peptic ulcer, so

we performed anti-ulcer study of ethanolic 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 gastric ulcers, in which peripheral sympathetic activation and

increased acid secretion play important roles [42]. Ethanolic extract of AS showed significant

protection in a dose dependent manner in the CRU model, with utmost protection observed at

50 mg/kg, p.o. and thus selected for further studies in other models. Chloroform and hexane

fraction also showed significant protection against this model. The ethanolic extract and its

chloroform and hexane fraction also exert a protective effect against ethanol-induced gastric

lesions in contrast to standard drug, sucralfate. Since ethanol damages the superficial

epithelial layers and inhibit the release of mucosal prostaglandins [43] and depresses the

gastric defensive mechanisms, these agents appear to enhance the gastric mucosal defense

[44] indicating the cytoprotective potentials of AS and its chloroform and hexane fractions.

In pyloric-ligation model, gastric acid is an important factor for the genesis of ulceration. In

this model, auto-digestion of mucosa by gastric acid and pepsin results in the development of

ulcers [45] Ethanolic extract of AS and its chloroform and hexane fractions significantly

reduced free, total acidity and pepsin level in this model, which suggests its anti-secretory

potency.

Duodenal ulcer is caused mainly by an increase in acid and pepsin load and gastric

metaplasia in the duodenal cap [46]. Protective effect of ethanolic extract of AS and its

chloroform and hexane fraction against histamine induced duodenal ulcer in guinea pigs

signifies its role in control of injury mediated by gastric acid secretion suggesting its

antisecretory activity.

In order to clarify the mode of action of ethanolic extract of AS and its chloroform and

hexane fraction, through the anti-secretory pathway, its influence on gastric secretion was

studied using inhibition of H+

K+-ATPase (Proton pump). This proton pump is the common

and final pathway of all stimulation of acid production. Hence, its inhibitor will be a potent

anti-secretory agent. The results obtained with gastric microsomes isolated from rat stomach

showed that ethanolic extract of AS and its chloroform and hexane fraction, potently inhibited

the H+

K+-ATPase activity comparable to the positive control omeprazole, thus suggesting

that ethanolic extract of AS and its chloroform and hexane fraction might be imparting anti-

ulcer activity through decrease in acid secretion via proton pump inhibition.

Further, to substantiate the antisecretory potential of Methylarmepavine, N-

methylcorydaldine, Isocorydine, their effect on plasma concentration of gastrin hormone in

ulcerated rats was determined. Gastrin hormone is a known modulator of gastric acid

secretion [47] which stimulates the parietal cell to hypersecrete acid, resulting in the

development of gastric ulcer. O-Methylarmepavine, N-methylcorydaldine, Isocorydine

decreases the gastrin secretion in ethanol induced ulcer model, which further confirmed its

antisecretory potential. Thus, the antisecretory activity of O-Methylarmepavine, N-

methylcorydaldine, Isocorydine appears to be mainly related to the inhibition of H+K

+ATPase

activity and suppression of gastrin release.

Fitoterapia (2011), 82(4), 666-675

Cytoprotective ability of ethanol extract of AS and its chloroform and hexane fraction can be

corroborated with increased mucin content of gastric juice in comparison to omeprazole. To

further validate its cytoprotective effect we have evaluated the efficacy of AS and its

chloroform and hexane fraction against aspirin induced ulcer model. Aspirin/ NSAIDs

induces ulcers due to their effect on cyclooxygenase enzyme leading to reduced prostaglandin

production and increase in acid secretion [48]. Ethanolic extract of AS and its chloroform and

hexane fraction significantly reduced the ulcer, which further supports their cytoprotective

potential, which may be mediated by prostaglandins.

Further exploration of the active fractions for its chemical constituents demonstrated the

presence of (+)-O-methylarmepavine (2), N-methylcorydaldine (3), lanuginosine (4), (+)-

anomuricine (5), isocorydine (6), N-Methyl-6,7-dimethoxyisoquinolone (7), 6,7-dimethoxy-

2- methylisoquinolinium (8), 1-(4-β-D- glucopyranosyloxyphenyl)-2-(β-D-

glucopyranosyloxy)-ethane (11). Out of which (+)-O-methylarmepavine (2), N-

methylcorydaldine (3), isocorydine (6) inhibited the in vitro assay of H+ K

+-ATPase activity

and compared with proton pump inhibitor, omeprazole. Thus, the antisecretory activity of AS

might be due to presence of these compounds.

Conclusively, the present study demonstrated that ethanol extract of Annona squamosa and

its chloroform and hexane fraction imparts gastroprotection via inhibition of H+ K

+-ATPase

(proton pump) activity and simultaneous strengthening of mucosal defense mechanism. The

compounds 2, 3 and 6 are the active principles of the plant. These compounds may serve as

the starting point for design of novel semi-synthetic and synthetic compounds as anti-ulcer

agents in the future.

Acknowledgements Dinesh K. Yadav and Neetu Singh are thankful to Council of Scientific and Industrial

Research, New Delhi and Indian Council of Medical Research, New Delhi for award of

Senior Research Fellowship. The authors are thankful to SAIF Division, Central Drug

Research Institute for recording spectral data.

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