Post on 08-May-2023
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.
Fitoterapia (2011), 82(4), 666-675
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.
Fitoterapia (2011), 82(4), 666-675
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).
Fitoterapia (2011), 82(4), 666-675
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
Fitoterapia (2011), 82(4), 666-675
(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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