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Drug and Chemical Toxicology
ISSN: 0148-0545 (Print) 1525-6014 (Online) Journal homepage: https://www.tandfonline.com/loi/idct20
Protective effect of the solvent extracts of
Portulacca oleracea against acidified ethanol
induced gastric ulcer in rabbits
Muhammad Shah Zeb Jan, Waqar Ahmad, Abdullah, Atif Kamil, Mir Azam
Khan, Maqsood Ur Rehman, Irfan ullah & Muhammad Saeed Jan
To cite this article: Muhammad Shah Zeb Jan, Waqar Ahmad, Abdullah, Atif Kamil, Mir Azam
Khan, Maqsood Ur Rehman, Irfan ullah & Muhammad Saeed Jan (2019): Protective effect of the
solvent extracts of Portulacca�oleracea against acidified ethanol induced gastric ulcer in rabbits,
Drug and Chemical Toxicology, DOI: 10.1080/01480545.2019.1691584
To link to this article: https://doi.org/10.1080/01480545.2019.1691584
Published online: 19 Nov 2019.
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RESEARCH ARTICLE
Protective effect of the solvent extracts of Portulacca oleracea against acidified
ethanol induced gastric ulcer in rabbits
Muhammad Shah Zeb Jan
a
, Waqar Ahmad
a
, Abdullah
a
, Atif Kamil
b
, Mir Azam Khan
a
, Maqsood Ur Rehman
a
,
Irfan ullah
a
and Muhammad Saeed Jan
a
a
Department of Pharmacy, University of Malakand, Chakdara, Pakistan;
b
Department of Biotechnology, Abdul Wali Khan University
Mardan, Pakistan
ABSTRACT
Portulacca oleracea L. has been used for treatment of different ailments. The aim of this study was to
investigate the effectiveness and possible mechanism of action involved in the anti gastric ulcerogenic
effect of Portulacca oleracea. Methanolic extract & subsequent fractions (100, 200 and 400 mg/kg) of
Portulacca oleracea (P. oleracea) were administered orally to experimental rabbits one hour before oral
administration of HCl/ethanol (40:60). Anti gastric ulcerogenic potential of P. oleracea was evaluated by
assessment of gastric pH, pepsin, free acidity, ulcer index, mucus content and total acidity. For the
investigation of possible mechanism of action malondialdehyde (MDA), histamine, and H þKþATPase
content were determined in the stomach homogenate. Histopathological study of stomach tissue was
carried out by H&E dye. Ethyl acetate fraction (EAF) of P. oleracea was the most potent fraction among
all fractions that exhibited efficient protection against acidified ethanol mediated gastric-ulcer. The
ethyl acetate fraction (EAF) significantly increased the pH of gastric juice, while pepsin and histamine
was observed to decrease significantly in comparison to acidified ethanol group (p0.001). The
EAF showed moderately H þKþATPase inhibitory activity. Moreover, it was also observed that EAF
decreased the malondialdehyde (MDA) level in the stomach tissue homogenate showing antioxidant
effect. Histopathological studies showed that among the tested fractions, EAF significantly prevented
acidified ethanol induced gastric mucosal damage. These results showed that mechanism of anti gas-
tric ulcerogenic potential of P. oleracea could be associated with the reduction in histamine level,
HþKþATPase inhibition and reduced MDA level.
ARTICLE HISTORY
Received 23 May 2019
Revised 31 October 2019
Accepted 2 November 2019
KEYWORDS
Portulacca oleracea; gastric
ulcer; antioxidant;
histamine; pepsin and
Hþ-K þATPase
Introduction
Ulcer is an injury or sore in the mucous membrane or outer
surface skin of the body. Ulcer in the lining of the stomach
or duodenum is a disease of digestive system that affect
many people around the world (S
anchez-Mendoza et al.
2011). It has been documented that fourteen million people
throughout the world are suffering from gastric ulcer with a
mortality rate of four million. Gastric ulcer occur as a result of
imbalance between aggressive (alcohol, pepsin and acid
secretion, poor diet, oxidative stress, NSAIDs and Helicobacter
pylori) and protective factors (mucosal blood flow, mucus
secretion, bicarbonate secretion and increased levels of anti-
oxidants etc.) in the stomach (Zakaria et al. 2016b). Gastric
mucosa is damaged when aggressive factors “overcome”
mucosal defensive mechanisms (Laine et al. 2008).
The ethanol-induced ulcer model is widely used for inves-
tigating the testing agents or studying the efficacy of poten-
tial drugs (Strasser et al. 2014). It is well known that ethanol
increase histamine secretion in the stomach which in turn
release hydrochloric acid from the parietal cells inside
mucosa of gastric tissue via proton pump (Kumar and Kumar
2009). A membrane-bound enzyme (H
þ
K
þ
ATPase), located in
the canalicular membrane of parietal cells, catalyzes an elec-
troneutral exchange of H
þ
versus K
þ
into the gastric lumen
with the expenditure of ATP (Reyes-Chilpa et al. 2006). It is
documented that gastric ulcer induced by ethanol causes
reactive oxygen species (ROS) production because ethanol
rapidly reaches the gastric mucosa and solubilize the protect-
ive mucous and finally releases free radicals and superoxide
anion. These free radicals react with lipid to form malondial-
dehyde (MDA), a marker of lipid peroxidation (Zakaria et al.
2011). The treatment of gastric ulcer has become a challenge
in the world. Although there are some approaches used for
treatment of gastric ulceration like acid neutralization,
improving antioxidant level in the stomach but the most
important approach is inhibition of gastric acid secretion
inside the stomach (Halim et al. 2017).
The current drugs available in the market for the treat-
ment of gastric ulcers are histamine inhibitors (H
2
-blockers
such as cimetidine, ranitidine etc.), proton pump inhibitors
(esomeprazole, omeprazole etc.) and mucosal protective
agents like sucralfate and bismuth compounds (Shu et al.
2013). But these drugs have many undesirable effects on the
human body including gynecomastia, arrhythmia and
CONTACT Waqar Ahmad drwaqarahmad@uom.edu.pk Department of Pharmacy, University of Malakand, Chakdara, Khyber Pakhtunkhwa, Pakistan
ß2019 Informa UK Limited, trading as Taylor & Francis Group
DRUG AND CHEMICAL TOXICOLOGY
https://doi.org/10.1080/01480545.2019.1691584
hematopoietic changes (Zakaria et al. 2016a). Therefore medi-
cinal plants are important alternative for the development of
new drugs to control gastric ulcer (Al-Wajeeh et al. 2016).
P. oleracea is an annual herb from family Portulacaceae. It
is widely distributed throughout the world (Okafor et al.
2014). This plant has been folklorically used for the treatment
of many diseases such as diarrhea, bacillary dysentery, diur-
etic, hemorrhoids, emollient, anthelmintic, antiscorbutic
(Guichard 2013) and as gastric sedative (Dalziel 1937). P. oler-
acea has been reported to have various pharmacological
activities like analgesic and anti-inflammatory (Chan et al.
2000), wound healing (Rashed et al. 2003), hepatoprotective
(Anusha et al. 2011), antitumor (Zhao et al. 2013), antioxidant,
hypoglycemic and pancreatic protective activity (Ramadan
et al. 2017). Different phytochemicals have been detected in
P. oleracea include flavonoids, fatty acids, alkaloids, polysac-
charides, terpenoids, vitamins, proteins, sterols and minerals
(Zhou et al. 2015). Many Flavonoids have been reported to
possess gastroprotective activity (Mota et al. 2009). Many
plants having saponins have been reported to possess gas-
troprotective activity (Ugwah et al. 2013). According to litera-
ture, ethanolic extract of P. oleracea has been reported to
have gastroprotective activity against HCl/ethanol induced
gastric ulcer in animal models (Gholamreza et al. 2004).
Rabbits are important animal models used for studying
gastroprotective activity (Okwuosa et al. 2011). Mammalian
stomachs possess histological similarities among species. On
the basis of the gastric wall, rabbits have a predominant
population of chief cells like humans stomach. Thus, rabbit
shares more structural similarities with human. The thickness
of human mucosa is 1 mm which is larger than rats and mice
mucosa. So rabbits are more suitable animal models for gas-
tric ulcer study (Maeng et al. 2013). Therefore on the basis of
folk use, saponins, flavonoids contents and reported in vivo
study, the current study was conducted to elucidate the anti
gastric ulcer potential of all fractions and the possible mech-
anism of P. oleracea.
Material and methods
Chemicals and drugs
Ranitidine (Cirin Pharmaceuticals Hattar Pakistan), Absolute
ethanol (Fisher Scientific, Loughborough, UK), HCl (Lab-Scan,
Analytical Sciences, Pathumwan, Bangkok), Bovine albumin,
Adenosine triphosphate (ATP), Thiobarbituric acid, Histamine
(Sigma-Aldrich, St. Louis, USA), O-phthal-aldehyde (JebChem,
Bangkok,Thailand), Trichloroacetic acid, Methyl orange, Folin
Ciocalteau reagent, Sodium carbonate, Magnesium chloride,
Potassium chloride, Sodium hydroxide and Ammonium
molybdate (Merck, Darmstadt, Germany), n-Butanol and
Phenolphthalein (BDH), Perchloric acid (Scharlu, Sentmenat,
Spain). Chloroform, n-hexane, ethyl acetate and methanol
were of commercial grade.
Plant material
Mature plant of P. oleracea was collected from Charsadda,
Khyber Pakhtunkhwa, Pakistan. Identification of this was
made by botanist (Dr. Nasrullah Khan) at University of
Malakand (Department of Botany) and deposited in herbar-
ium with voucher No. H.UOM.BG.174.
Extraction and fractionation
The plant was air dried in shady area for 60 days at room
temperature followed by crushing via cutter mill into powder
(5 kg) and subsequent soaking in 80% methanol for 3 weeks.
The plant extract was filtered with the help of muslin cloth
and evaporated by using the rotary evaporator to obtain the
methanolic extract of P. oleracea (MEE, 360 g). Methanolic
extract (300 g) was further fractionated with solvents like
chloroform, n-hexane, ethyl acetate and water (Zeb et al.
2014). The chloroform (CHF), ethyl acetate (EAF), n-hexane
(NHF), and aqueous fraction (AQF) obtained were, 18, 27, 43,
and 98 g respectively.
Animals
Adult rabbits (1 to 1.5 kg) of either sex were bought from
local market. Animals were kept in the animal house at
University of Malakand, with twelve hours dark and light
cycle having free access to water and standard diet. Before
the start of experiment, animals were fasted overnight while
water was withdrawn 2 h earlier. The experimental proce-
dures were approved by the Ethical Committee of the
Department of Pharmacy according to Animal Bye Laws 2008
of the University of Malakand (NO.Pharm/ECC/PO-142/12/17).
Acute oral toxicity
Rabbits were randomly allocated into 18 groups, each contain-
ing 6 animals. Crude extract was dissolved in distilled water
and was administered orally at various doses ranging from 5
to 2000mg/kg. After receiving the doses, the rabbits were
observed for next 72 h for mortality/minor abnormal behavior
as well as any other allergic symptoms (OECD 2000).
Induction of gastric ulcer using HCl/ethanol
Rabbits were grouped into 18 (n¼6). Group 1 was adminis-
tered distilled water (10 ml/kg p.o). Groups 2–4 were given
with 100, 200 and 400 mg/kg doses of liquid MEE orally.
Groups 5–7 were given with 100, 200 and 400 mg/kg doses
of liquid NHF orally.
Groups 8–10 were given with 100, 200 and 400 mg/kg doses
of liquid CHF orally.
Groups 11–13 were given 100, 200 and 400 mg/kg doses of
liquid EAF orally.
Groups 14–16 were given 100, 200 and 400 mg/kg doses of
liquid AQF orally.
Group 17 (Ranitidine, 50 mg/kg orally) and XVIII
(Omeprazole 20 mg/kg orally) were used as positive control
(for H
þ
K
þ
ATPase only). After 60 min each animal was given
1 ml/250 g of weight HCl/ethanol solution (150 mM HCl in
2 M. S. Z. JAN ET AL.
60% ethanol) orally. After two hours animals were euthanized
using diethyl ether and were sacrificed (Ateufack et al. 2015).
Measurement of ulcer index
The stomachs of rabbits were pierced from the site of greater
curvature and gastric contents were removed. The stomachs
were then washed with distilled water to examine ulcers in
the glandular portion of the stomach. The number of ulcers
per stomach was calculated with the aid of magnifying lens.
The scoring was given as 0 for normal stomach, 0.5 for red
coloration, 1 for spot ulcer, 1.5 for hemorrhagic streaks, 2 for
ulcer 35 mm and 3 for ulcer >5 mm. The ulcer index for
each animal was expressed as mean ulcer score (Chetan
et al. 2013).
Estimation of free acidity, total acidity and pH of
gastric juice
The gastric content obtained from dissected stomach was
centrifuged at 2500 rpm for 10 min to remove solid material.
The pH of the supernatant of gastric juice was measured
with help of pH meter. For the measurement of free acidity
of gastric juice, 2–3 drops of methyl orange reagent was
added to the gastric juice and titrated with 0.01 N NaOH till
the appearance of pale color. The volume of added NaOH
was noted. Then, 2–3 drops of phenolphthalein were added
to the same solution for measurement of total acidity. The
titration was carried out until pink color appeared. The total
volume of NaOH added to the solution was noted. Acidity
was measured with the help of the following formula (Katary
and Salahuddin 2017).
Acidity ¼Volume of NaOH Normality of NaOH
0:1100 meq=L
Estimation of gastric mucus production
The gastric mucosal layer of each rabbit was smoothly
scraped off with the aid of glass slide and the mucus
obtained was weighed via electronic balance and compared
with positive control (Sidahmed et al. 2013).
Determination of pepsin
Gastric juice (1 ml) was taken in test tube with 1 ml albumin
solution (0.5% BSA w/v in 0.06 N hydrochloric acid) and was
incubated at 37 C for 10 min. For the termination of the
reaction, 10% trichloroacetic acid (TCA) 2 ml was added and
centrifuged the sample at 1500 rpm for 20 min. Sodium car-
bonate 0.55 M (2.5 ml) and 1 ml of Folin’s reagent (FCR) were
added to the tubes containing supernatant and incubated at
room temperature for 30 min. The absorbance of each sam-
ple was determined via spectrophotometer at 660 nm. The
activity of pepsin was represented as mg of tyrosine/ml
(Moram et al. 2016).
Histopathological study
Stomach from each rabbit were removed and washed with
normal saline solution. The excised stomach of each group
was kept in 10% neutral buffered formalin. The specimen of
gastric tissue was placed in paraffin wax. The specimen was
then stained with hematoxylin and eosin dye and examin-
ation was carried out with the help of light microscope for
the assessment of histopathological changes (Laloo
et al. 2014).
Mechanistic activity of P. oleracea
Estimation of malondialdehyde (MDA) level
One gram of gastric tissue was homogenized in phosphate
buffer and incubated at 37 C. Homogenized gastric tissue
(1 ml) was mixed with 10% TCA (1 ml) and then centrifuged
for a period of 20 min at 2000 rpm. The supernatant was
mixed with 2 ml of 0.67% TBA and heated for 30 min at
100 C in a water-bath. After cooling, the suspension was
centrifuged at 2000 rpm for 10 min. The absorbance of pink
colored supernatant was read via spectrophotometer at
532 nm. The result of MDA content was expressed as nano
moles per gram of wet tissue (Ping et al. 2005).
Estimation of histamine content
One gram of stomach tissue was homogenized with the aid
of Teflon homogenizer in 0.4 N perchloric acid followed by
centrifugation at 3000 rpm for 10 min. The n-butanol was
used for extraction of histamine from supernatant and then
n-butanol was mixed with 2 ml of 0.1 N HCl and 15 ml of
n-heptane. After this, centrifugation at 2000 rpm for 10 min was
carried out. The heptane layer was removed and discarded
while the HCl layer was mixed with o-phthal-aldehyde (OPA) for
the formation of fluorescent product. Spectrofluorimeter
(450 nm emission & 360 nm activation) was used for the meas-
urement of histamine. Result was expressed as micro gram of
histamine released/g tissue (Laloo et al. 2013).
Determination of H
þ
K
þ
ATPase
Proton potassium ATPase was obtained by scrapings rabbit
gastric mucosa. One gram of mucosa was homogenized with
the help of homogenizer in 100 ml of Tris-HCl buffer and cen-
trifuged at 5000 rpm for 10 min. The resulting supernatant
was recentrifuged at 5000 rpm for 20 min. The protein con-
centration in the supernatant was determined using bovine
serum albumin as standard (Lowry et al. 1951). One ml reac-
tion mixture containing enzyme was mixed with 20 mM Tris-
HCl (0.2 ml) having pH 7.4, 2 mM MgCl
2
and 2 mM of KCl
(0.2 ml). Reaction was started by adding of 0.2 ml of 2 mM
ATP (adenosine triphosphate) and incubated at 37 C for
30 min. The reaction was terminated by adding one ml of
TCA and 0.5 ml ammonium molybdate. The sample was cen-
trifuged for 10 min at 2000 rpm. The absorbance was meas-
ured with spectrophotometer at 640 nm to determine the
release of inorganic phosphorus from ATP. The enzyme
DRUG AND CHEMICAL TOXICOLOGY 3
activity was expressed as nM of phosphate liberated/min/mg
protein (Selvamathy et al. 2010).
Statistical analysis
The results of all assays were expressed as mean ± standard
error mean (SEM). One Way Analysis of Variance (ANOVA)
was used for analysis of data followed by post hoc Dunnett’s
test for multiple comparisons of data. All the values were
considered significant when p<0.05.
Results
Acute oral toxicity
In the acute toxicity study, no mortalities were recorded in
animals treated with a single dose of 2000 mg/kg body
weight. There were no clinical signs in the eyes and mucus
membrane, skin and fur, respiratory rate, autonomic effects,
circulatory signs, and central nervous system at dose
2000 mg/kg body weight. Therefore, the approximate lethal
dose in the experimental rabbit was higher than 2000 mg/kg.
According to organization for economic cooperation and
development (OECD) guidelines for acute oral toxicity, an
LD50 dose of >300–2000 is categorized as category 4 and
hence the plant extract is found to be safe.
Macroscopic evaluation
The anti gastric ulcer activity of P. oleracea in HCl/ethanol-
induced gastric lesion model is shown in Figure 1. Ethanol/
HCL solution cause damaged to the gastric mucosa of rab-
bits. Different fractions of P. oleracea significantly reduced
the formation of the gastric lesion. Based on our result, EAF
was the most potent fraction and it reduced the gastric
mucosal ulcer that was comparable with raniti-
dine (standard).
Effect on gastric ulcer index
The effects of the crude methanolic extract and subsequent
fractions of P. oleracea in the acidified ethanol-induced gas-
tric ulcer models are shown in Figure 2. The results show
that the EAF (3.3 ± 0.54) significantly reduced the Ulcer index
at highest dose (400 mg/Kg), when compared to the negative
control group (30 ± 0.44). These results suggest that EAF
demonstrated better healing of the lesions induced by acidi-
fied ethanol than other groups.
Effects of P. oleracea on pH, free and total acidity of
gastric juice
The effects of crude methanolic extract and subsequent frac-
tions of P. oleracea on acidity of gastric juice of rabbit after
administration of acidified ethanol are shown in Table 1.
Among the fractions, the EAF significantly increased the pH
and decreased the free and total acidity of gastric juice of
rabbit stomach as compared to negative control group.
Effects of different fractions of P. oleracea on
mucus production
Rabbits pretreated with crude methanolic extract and subse-
quent fractions of P. oleracea increased the mucus secretion
as shown in Table 2. The negative control decreased the
mucus secretion as compared to standard drug. The EAF was
the most potent fraction and significantly increased the
mucus secretion closely related to positive control group.
Effects of P. oleracea on peptic activity
Negative control showed significant increase in the pepsin
content. The crude extract and subsequent fractions of
P. oleracea decreased the pepsin secretion after administra-
tion of acidified ethanol as shown in Table 3. The result
showed that the most potent fraction (EAF) significantly
inhibited the pepsin release inside the rabbit stomach closely
related to positive control group.
Effects of different fractions of P. oleracea on MDA
The negative ulcerated control showed an increased level of
MDA in the gastric tissue. Pretreatment with crude extract
and subsequent fractions of P. oleracea decreased the MDA
level as shown in Table 4. The results showed that the EAF
significantly decreased in the MDA level closely related to
standard drug (ranitidine).
Effects of different fractions of P. oleracea on
histamine level
The histamine level of rabbit gastric tissue of ulcer negative
control group was significantly increased as compared to
other groups. Pretreatment with crude extract and subse-
quent fractions of P. oleracea decreased the level of hista-
mine in gastric tissue as shown in Figure 3. The EAF
significantly decreased the level of histamine in gastric tissue
than other groups, closely related to positive control group.
Effects of different fractions of P. oleracea on H
1
K
1
ATPase enzyme
Rabbits treated with a standard drug (omeprazole) showing a
significant inhibitory effect on H
þ
K
þ
ATPase enzyme by
reducing the release of phosphate ions (Pi) as compared to
negative control group. The EAF of P. oleracea at high dose
(400 mg/kg) showed moderate inhibitory effect on H
þ
K
þ
ATPase enzyme as compared to standard drug. MEE and CHF
showed very moderately inhibitory effect on H
þ
K
þ
ATPase
enzyme as shown in Figure 4.
4 M. S. Z. JAN ET AL.
Histological evaluation
Histological observation using H&E staining confirm the abil-
ity of crude and subsequent fractions of P. oleracea to pre-
vent gastric damage of the gastric mucosa induced by
acidified ethanol (Figure 5). The negative control group
showed highly extensive gastric mucosal damage (black
arrow), edema (white arrow) and leucocytes infiltration (blue
arrow) as shown in (Figure 5(B)). Among different fractions
the EAF (Figure 5(C–E)) and Ranitidine (Figure 5(A)) confined
gastric tissue from the effect ethanol/HCL having very mild
mucosal disruption, no leucocytes infiltration and edema in
comparison to negative control group.
Figure 1. Effect of P. oleracea on gross appearance of the gastric mucosa in rabbits. (A1) Positive control group (ranitidine 50 mg/kg) protected gastric mucosa. (B1)
Gross appearance of group pretreated with distilled water (ulcer control). Extensive damage of the gastric mucosa was observed. (C1, D1 and E1) Rabbits pretreated
with EAF (100, 200 and 400 mg/kg) þAcidified ethanol reduced gastric lesion comparable to positive control.
DRUG AND CHEMICAL TOXICOLOGY 5
EAF
AQF
MEE
CHF
NHF
Acidfied ethanol
Raniditine
0
5
10
15
20
25
30
35
100 mg/ kg
200 mg/kg
400 mg/kg
5ml/kg
50 mg/Kg
***
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***
***
***
***
***
Ulcer Index
Figure 2. Effect of P. oleracea on ulcer index of rabbits. Values are represented as
mean and n¼6, p<0.001, p<0.01 and p<0.05 as compare to control.
Table 1. Effect of P. oleracea on gastric juice parameters.
Group Dose mg/kg pH
Free acidity
(mEq/L)
Total acidity
(mEq/L)
HCl/Ethanol –1.36 ± 0.10 121.33 ± 0.88 133 ± 1.26
Ranitidine 50 4.96 ± 0.18 15 ± 0.54 25 ± 0.54
100 3.06 ± 0.03 71.0 ± 1.14 84.4 ± 0.92
Methanolic 200 3.18 ± 0.16 66.0 ± 2.35 79.0 ± 0.63
400 3.86 ± 0.12 52.2 ± 1.16 64.2 ± 2.15
100 1.52 ± 0.14 85.4 ± 1.21 106 ± 1.58
n-hexane 200 1.88 ± 0.16 80.2 ± 1.02 97.0 ± 1.82
400 2.07 ± 0.01 74.8 ± 0.97 91.6 ± 1.29
100 2.40 ± 0.09 78.0 ± 0.95 94.2 ± 1.39
Chloroform 200 2.71± 0.03 69.2 ± 0.97 85.2 ± 1.24
400 2.98 ± 0.06 64.4 ± 1.66 79.6 ± 1.33
100 4.38 ± 0.07 42.0 ± 1.26 59.0 ± 1.87
Ethyl acetate 200 4.50 ± 0.05 39.6 ± 1.75 47.2 ± 1.11
400 4.64 ± 0.08 22.4 ± 0.98 30.0 ± 1.22
ns
100 4.11 ± 0.11 55.4 ± 0.51 67.4 ± 1.43
Aqueous 200 4.29 ± 0.05 46.2 ± 0.80 57 ± 1.52
400 4.56 ± 0.18 36.0 ± 1.30 49 ± 1.14
Values are represented as mean and n¼6, p<0.001, p<0.01 and
p<0.05. as compared to standard drug ranitidine.
Table 2. Effect of P. oleracea on mucus content.
Group Dose mg/kg Mucus content (gram)
HCl/Ethanol –1.45 ± 0.195
Ranitidine 50 4.60 ± 0.103
100 3.39 ± 0.092
Methanol 200 3.52 ± 0.135
400 3.79 ± 0.104
100 2.07 ± 0.217
n-Hexane 200 1.94 ± 0.145
400 1.74 ± 0.200
100 2.13 ± 0.164
Chloroform 200 2.77 ± 0.207
400 3.32 ± 0.155
100 3.92 ± 0.098
Ethyl acetate 200 4.29 ± 0.143
ns
400 4.47 ± 0.117
ns
100 3.56 ± 0.125
Aqueous 200 3.89 ± 0.200
400 4.21 ± 0.088
ns
Values are represented as mean and n¼6, p<0.001, p<0.01 and
p<0.05 as compared to standard drug ranitidine.
Table 3. Effect of P. oleracea on peptic activity.
Group Dose mg/kg
Pepsin activity
(mg Tyrosine released/ml)
HCl/Ethanol –28.32 ± 0.30
Ranitidine 50 5.30 ± 0.95
100 16.84 ± 0.24
Methanol 200 14.92 ± 0.22
400 13.20 ± 0.46
100 24.82 ± 0.93
n-Hexane 200 22.23 ± 0.57
400 22.34 ± 0.26
100 19.83 ± 0.73
Chloroform 200 18.75 ± 0.67
400 17.12 ± 0.39
100 10.70 ± 0.28
Ethyl acetate 200 9.80 ± 0.11
400 8.38 ± 0.40
100 15.63 ± 0.05
Aqueous 200 13.84 ± 0.64
400 12.16 ± 0.36
Values are represented as mean and n¼6, p<0.001, p<0.01 and
p<0.05 as compared to standard drug ranitidine.
Table 4. Effect of P. oleracea on MDA.
Group Dose mg/kg MDA nmol/g tissue
HCL/Ethanol –78.2 ± 0.30
Ranitidine 50 12.08 ± 0.49
100 46.76 ± 0.62
Methanol 200 34.28 ± 0.55
400 28.17 ± 0.47
100 76.96 ± 0.70
n-Hexane 200 70.48 ± 0.62
400 64.13 ± 0.73
100 57.06 ± 0.86
Chloroform 200 48.11 ± 0.87
400 38.80 ± 0.64
100 38.61 ± 0.49
Ethyl acetate 200 28.32 ± 0.30
400 17.16 ± 0.27
100 40.62 ± 0.28
Aqueous 200 30.15 ± 0.35
400 20.06 ± 0.42
Values are represented as mean and n¼6, p<0.001, p<0.01 and
p<0.05 as compared to standard drug ranitidine.
EAF
AQF
MEE
CHF
NHF
HCL/Ethanol
Raniditine
0
1
2
3
4
5
6
7
8
9
10
11 400 mg/kg
200 mg/kg
100 mg/kg
50 mg/kg
10mg/kg
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***
Histamine mcg/ml
Figure 3. Effect of P. oleracea on histamine content of stomach homogenate of
rabbits. Values are represented as mean and n¼6, p<0.001, p<0.01
and p<0.05 as compare to control.
6 M. S. Z. JAN ET AL.
Discussion
Gastric ulcer presents a major health problem having high
morbidity and mortality rate (Mejia and Kraft 2009). However,
the use of medicinal plants and their secondary metabolites
gain importance worldwide for the treatment of different dis-
eases. Literature reports revealed the gastro protective effect
of medicinal plants in different ulcer induced models in
rodents (Rahim et al. 2014).
The medicinal plants showed better antiulcer activity,
when compared to conventional agents like H
2
–blockers, pro-
ton pump inhibitors and cytoprotective agents (Onasanwo
et al. 2011).
In the present study, antiulcer potential of crude extract
and subsequent fractions of P. oleracea was investigated as
part of our aims to establish the pharmacological potential of
this plant scientifically. Various folk uses have been reported
for this plant as described earlier, which include the gastro
protective activity. In this study, the mechanistic activity of
P. oleracea was also determined in animal models using acidi-
fied ethanol as ulcerogenic agent (Carlos et al. 2010).
Through its direct action, acidified ethanol penetrates the
gastric mucosa rapidly causing damage to gastrointestinal
mucosa cells and membranes (Kumar et al. 2011). The pro-
cess of formation of gastric mucosal lesions start through
micro vascular injury, increase of the vascular permeability,
which affected mucus secretion, pepsin secretion, and the
loss of hydrogen ions and histamine into the lumen (Szabo
et al. 1985). In this study, the consequential lesions of differ-
ent sizes along the glandular part of the stomach were devel-
oped by acidified ethanol. Our results showed that among
the fractions, EAF decreased ulcer index of rabbit which was
comparable to ranitidine. Histamine released by acidified
ethanol increases the production of HCl in the stomach
which decreases gastric pH (Dorababu et al. 2006). In the
present study EAF at 400 mg/kg reduced the acidity and
increased the pH of the gastric juice.
On the other hand, gastric mucus provides protection to
the gastric tissue against gastric acid. Our results showed
that EAF at 400 mg/kg significantly increased the mucus con-
tent against acidified ethanol (Allen and Flemstr€
om 2005).
Pepsin also plays an important role in producing gastric
ulcer. It damages the junctional complex which make the
epithelial layer of stomach acid permeable, pepsinogen is
auto catalytically converted to pepsin at low pH. The pepsin
hydrolyze the collagen surface epithelium, especially type IV,
and damage mucosa. Therefore, inhibition of pepsin secre-
tion play important role in the management of gastric ulcer
(Selvamathy et al. 2010). Our results showed that EAF at
400 mg/kg significantly decreased pepsin content.
Moreover for the mechanistic study, three different path-
ways were determined. Histamine which is the main mediator
of gastric acid secretion, triggers a chain of events which
damage the mucosa (Mahmood et al. 2005). Histamine
increase gastric acid secretion via H
2
-receptors that are
coupled to G-protein system. Stimulation of adenyl cyclase
increases cAMP level, which is compulsory for activation of
protein kinases. The protein kinases cause the phosporylation
of certain phosphatases and activate the proton pump for
the production of HCl (Mesia-Vela et al. 2002). The oral
administration of P. oleracea extracts reduced the histamine
level which is the main pathway for blockade of gastric acid
secretion. Moreover, EAF at high dose (400 mg/kg, p.o.) inhib-
ited H
þ
K
þ
ATPase enzyme in comparison with positive control
group. The H
þ
K
þ
ATPase enzyme which is also known as pro-
ton pump, that exchange protons (H
þ
) and potassium (K
þ
)
ions across the plasma membrane of stomach and play vital
role in acid formation (Gumz et al. 2010). Similarly, acidified
ethanol can also augment the production of ROS i.e., super-
oxide anion and hydroxyl radicals (Bailey 2003). ROS react
with cellular lipids and form lipid peroxides. An effective indi-
cator of oxidative stress is MDA, the major metabolite of lipid
peroxidation (Rouhollahi et al. 2014). This study showed that
EAF at high dose (400 mg/kg, p.o.) prevented gastric ulcer-
ation by reducing MDA production against acidified ethanol.
Conclusion
This study explained that EAF of P. oleracea exhibit anti gas-
tric ulcerogenic effect through (i) decreasing histamine level
in tissue homogenate which is an important pathway of
decreasing gastric acidity, beside the solvent extracts also (ii)
decreased MDA levels in the tissue and (iii) moderately inhib-
ited H
þ
K
þ
ATPase. As the solvent extract inhibited gastric
ulcer via multiple pathways, so this study may provide an
important alternative, safe and effective remedy as compare
to available conventional treatment (PPIs and H
2
-blockers).
Isolation of active compounds from ethyl acetate fraction
responsible for anti gastric ulcer activity is our future plan.
EAF
AQF
MEE
CHF
NHF
Omeprazole
HCL/Ethanol
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
100 mg/kg
200 mg /kg
400 mg /kg
10 mg/kg
10 mg/kg
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
nMole of phosphat e/min/mg protien
Figure 4. Effect of P. oleracea on H
þ
K
þ
ATPase activity in rabbits. Values are
represented as mean and n¼6, p<0.001, p<0.01 and p<0.05 as
compare to control.
DRUG AND CHEMICAL TOXICOLOGY 7
Figure 5. Effect of P. oleracea on stomach tissue of rabbits (A) Micrograph of ranitidine þacidified ethanol (Positive control group) (B) Ulcer control group (C) EAF
(100 mg/kg þacidified ethanol (D) EAF 200 mg/kg þacidified ethanol (E) EAF 400 mg/kg) þacidified ethanol. ME: mucularis externa; M: Mucosa; SM: submucosa.
8 M. S. Z. JAN ET AL.
Disclosure statement
The authors declare that they have no competing interests.
References
Allen, A., and Flemstr€
om, G., 2005. Gastroduodenal mucus bicarbonate
barrier: protection against acid and pepsin. American Journal of
Physiology-Cell Physiology, 288(1), C1–C19.
Anusha, M., et al., 2011. Hepatoprotective activity of aqueous extract of
Portulacca oleracea in combination with lycopene in rats. Indian
Journal of Pharmacology, 43(5), 563.
Ateufack, G., et al., 2015. Gastroprotective and ulcer healing effects of
Piptadeniastrum africanum on experimentally induced gastric ulcers in
rats. BMC Complementary and Alternative Medicine, 15(1), 214.
Bailey, S.M., 2003. A review of the role of reactive oxygen and nitrogen
species in alcohol-induced mitochondrial dysfunction. Free Radical
Research, 37(6), 585–596.
Carlos, K.L., et al., 2010. Antiulcerogenic activity of extract, fractions, and
some compounds obtained from Polygala cyparissias St. Hillaire &
Moquin (Polygalaceae). Naunyn-Schmiedeberg’s Archives of
Pharmacology, 381(2), 121–126.
Chan, K., et al., 2000. The analgesic and anti-inflammatory effects of
Portulacca oleracea L. subsp. sativa (Haw.) Celak. Journal of
Ethnopharmacology, 73(3), 445–451.
Chetan, M., Prasad, K., and Nitin, M., 2013. Role of melatonin with lanso-
prazole in treating peptic ulcer in rats. Journal of Pharmaceutical
Sciences, 3(2), 47–51
Dalziel, J. M., 1937. The useful plants of west tropical Africa. London:
Crown Agents for the Colonies.
Dorababu, D., et al., 2006. Effect of aqueous extract of neem (Azadirachta
indica) leaves on offensive and defensive gastric mucosal factors in
rats. Indian Journal of Physiology and Pharmacology, 50(3), 241.
Gholamreza, K., Hosseinzadeh, H., and Ettehad, N., 2004. Evaluation of
the gastric antiulcerogenic effects of Portulacca oleracea L. extracts in
mice. Phytotherapy Research, 18(6), 484–487.
Guichard, R.H., 2013. Niebuhr in Egypt: European science in a Biblical
world. Eugene, OR: Wipf and Stock Publishers.
Gumz, M.L., et al., 2010. The renal Hþ-Kþ-ATPases: physiology, regula-
tion, and structure. American Journal of Physiology-Renal Physiology,
298(1), F12–F21.
Halim, S.Z., et al., 2017. Synergistic gastroprotective activity of methanolic
extract of a mixture of Melastoma malabathricum and Muntingia cala-
bura leaves in rats. BMC Complementary and Alternative Medicine,
17 (1), 488.
Katary, M.A., and Salahuddin, A., 2017. Gastroprotective effect of punica-
lagin against ethanol-induced gastric ulcer: the possible underlying
mechanisms. Biomarkers Journal, 03(01–08).
Kumar, A., Singh, V., and Chaudhary, A.K., 2011. Gastric antisecretory and
antiulcer activities of Cedrus deodara (Roxb.) Loud. in Wistar rats.
Journal of Ethnopharmacology, 134(2), 294–297.
Kumar, P., and Kumar, C. M., 2009. Clark’s clinical medicine. Edinburgh:
Saunders Elsevier.
Laine, L., Takeuchi, K., and Tarnawski, A., 2008. Gastric mucosal defense
and cytoprotection: bench to bedside. Gastroenterology, 135(1), 41–60.
Laloo, D., et al., 2013. Gastroprotective activity of ethanolic root extract
of Potentilla fulgens Wall. ex Hook. Journal of Ethnopharmacology,
146 (2), 505–514.
Laloo, D., et al., 2014. Gastroprotective activity of polyphenolic-rich
extract of Potentilla mooniana.Pharmaceutical Biology, 52(12),
1532–1542.
Lowry, O.H., et al., 1951. Protein measurement with the Folin phenol
reagent. Journal of Biological Chemistry, 193(1), 265–275.
Maeng, J.H., et al., 2013. Rabbit gastric ulcer models: comparison and
evaluation of acetic acid-induced ulcer and mucosectomy-induced
ulcer. Laboratory Animal Research, 29(2), 96–102.
Mahmood, A.A., Sidik, K., and Salmah, I., 2005. Anti-ulcer and gastro pro-
tective effects of honey in combination with Trigonella foenum grae-
cum seeds extract on experimental gastric ulcer in rats. International
Journal of Molecular Medicine and Advance Sciences, 1(3), 225–229.
Mejia, A., and Kraft, W.K., 2009. Acid peptic diseases: pharmacological
approach to treatment. Expert Review of Clinical Pharmacology, 2(3),
295–314.
Mesia-Vela, S., et al., 2002. Solanum paniculatum L.(Jurubeba): potent
inhibitor of gastric acid secretion in mice. Phytomedicine, 9(6), 508.
Moram, G.S., et al., 2016. Evaluation of antiulcer activity of red pepper
and garlic bulb against indomethacin-induced gastric ulcer in rats:
biochemical and histopathological study. European Journal of
Pharmaceutical and Medical Research, 3(2), 100–112.
Mota, L., et al., 2009. Flavonoids with gastroprotective activity. Molecules
(Basel, Switzerland), 14(3), 979–1012.
OECD. 2000. Guidelines for the testing of chemicals revised draft guide-
lines 423: acute oral toxicity. Paris: OECD.
Okafor, I.A., Ayalokunrin, M.B., and Orachu, L.A., 2014. A review on
Portulacca oleracea (Purslane) plant–Its nature and biomedical bene-
fits. International Journal of Biomedical Research, 5(2), 75–80.
Okwuosa, C.N., et al., 2011. Gastro-protective effect of crude hexane leaf
extract of Sesamum indicum in rabbits. Nigerian Journal of
Physiological Sciences, 26(1), 49–54.
Onasanwo, S. A., et al., 2011. Anti-ulcerogenic and proton pump (Hþ,
KþATPase) inhibitory activity of Kolaviron from Garcinia kola Heckel
in rodents. Indian Journal of Experimental Biology, 49(6), 461–468.
Ping, J., et al., 2005. Protective role of metallothionein in stress-induced
gastric ulcer in rats. World Journal of Gastroenterology, 11(18), 2739.
Rahim, N.A., et al., 2014. Gastroprotective effect of ethanolic extract of
Curcuma xanthorrhiza leaf against ethanol-induced gastric mucosal
lesions in Sprague–Dawley rats. BioMed Research International, 2014, 1.
Ramadan, B.K., Schaalan, M.F., and Tolba, A.M., 2017. Hypoglycemic and
pancreatic protective effects of Portulacca oleracea extract in alloxan
induced diabetic rats. BMC Complementary and Alternative Medicine,
17(1), 37.
Rashed, A.N., Afifi, F.U., and Disi, A.M., 2003. Simple evaluation of the
wound healing activity of a crude extract of Portulacca oleracea
L.(growing in Jordan) in Mus musculus JVI-1. Journal of
Ethnopharmacology, 88(2-3), 131–136.
Reyes-Chilpa, R., et al., 2006. Inhibition of gastric Hþ,Kþ-ATPase activity
by flavonoids, coumarins and xanthones isolated from Mexican medi-
cinal plants. Journal of Ethnopharmacology, 105(1), 167–172.
Rouhollahi, E., et al., 2014. Evaluation of acute toxicity and gastroprotec-
tive activity of Curcuma purpurascens BI. rhizome against ethanol-
induced gastric mucosal injury in rats. BMC Complementary and
Alternative Medicine, 14(1), 378.
S
anchez-Mendoza, M.E., et al., 2011. Bioassay-guided isolation of an anti-
ulcer compound, tagitinin C, from Tithonia diversifolia: role of nitric
oxide, prostaglandins and sulfhydryls. Molecules, 16(1), 665–674.
Selvamathy, S.M., Geetha, A., and Saranya, P., 2010. Gastroprotective
effect of Swertia chirayita –A study with ulcer induced rats.
Pharmacology Online, 1, 332–355.
Shu, M.H., et al., 2013. Anti-inflammatory, gastroprotective and anti-
ulcerogenic effects of red algae Gracilaria changii (Gracilariales,
Rhodophyta) extract. BMC Complementary and Alternative Medicine,
13(1), 61.
Sidahmed, H.M.A., et al., 2013. Gastroprotective effect of desmosdumotin
C isolated from Mitrella kentii against ethanol-induced gastric mucosal
hemorrhage in rats: possible involvement of glutathione, heat-shock
protein-70, sulfhydryl compounds, nitric oxide, and anti-Helicobacter
pylori activity. BMC Complementary and Alternative Medicine, 13(1),
183.
Strasser, M., et al., 2014. Antiulcerogenic potential activity of free and
nanoencapsulated Passiflora serratodigitata L. BioMed Research
International, 2014, 1–7.
Szabo, S., et al., 1985. Early vascular injury and increased vascular perme-
ability in gastric mucosal injury caused by ethanol in the rat.
Gastroenterology, 88(1), 228–236.
Ugwah, M.O., et al., 2013. Comparative studies of anti-ulcerogenic activ-
ities of three Nigerian medicinal plants: a preliminary evaluation.
Journal of Medicinal Plants Research, 7(9), 490–495.
Wajeeh, A., et al., 2016. The gastro protective effects of Cibotium baro-
metz hair on ethanol-induced gastric ulcer in Sprague–Dawley rats.
BMC Veterinary Research, 13(1), 27.
DRUG AND CHEMICAL TOXICOLOGY 9
Zakaria, Z.A., et al., 2016a. Gastroprotective activity of chloroform extract
of Muntingia calabura and Melastoma malabathricum leaves.
Pharmaceutical Biology, 54(5), 812–826.
Zakaria, Z.A., et al., 2011. In vitro antiproliferative and antioxidant activities
and total phenolic contents of the extracts of Melastoma malabathri-
cum leaves. Journal of Acupuncture and Meridian Studies, 4(4), 248–256.
Zakaria, Z.A., et al., 2016b. Mechanism (s) of action underlying the gastro-
protective effect of ethyl acetate fraction obtained from the crude
methanolic leaves extract of Muntingia calabura.BMC Complementary
and Alternative Medicine, 16(1), 78.
Zeb, A., et al., 2014. Investigations of anticholinestrase and antioxidant
potentials of methanolic extract, subsequent fractions, crude saponins
and flavonoids isolated from Isodon rugosus.Biological Research, 47(1),
1–10.
Zhao, R., et al., 2013. Antitumor activity of Portulacca oleracea L. polysac-
charides against cervical carcinoma in vitro and in vivo. Carbohydrate
Polymers, 96(2), 376–383.
Zhou, Y.X., et al., 2015. Portulaca oleracea L.: a review of phytochemis-
try and pharmacological effects. BioMed Research International,
2015, 1.
10 M. S. Z. JAN ET AL.
