Pharmacologyonline 2: 678-686 (2009) Newsletter Roslida and Noor

EVALUATION OF GASTROPROTECTIVE EFFECTS OF

THE ETHANOLIC EXTRACT OF PEPEROMIA
PELLUCIDA (L) KUNTH

Roslida AH1* and Noor Aini Z1

1
Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, University
Putra Malaysia , 43400 Serdang, Selangor, Malaysia.

Summary

This work was carried out to investigate the anti-

ulcerogenic activity of Peperomia pellucida (L.) Kunth in
necrotizing agent ie (ethanol , sodium chloride, sodium
hydroxide and hydrochloric acid) and indomethacin-
induced models in rats. The 70% of ethanolic extract of
aerial part of Peperomia pellucida (PPE) was prepared.
Four doses ie 10, 30, 100 and 300 mg/kg were selected for
further study. Ulcer effects were determined by counting
the total surface area of lesion in mm2. Results showed that
PPE provided significant protection in various
experimental models used. Pretreatment with the PPE at
all doses (10,30,100 and 300 mg/kg) has produced
significant inhibition of gastric mucosal damage induced by
80% EtOH, 25% NaCl, 0.6 M HCl, 0.2 M NaOH and 30
mg/kg indomethacin. The result suggests that PPE
possesses anti-ulcer properties.
.

Key words: Peperomia pellucida, anti-ulcer, Ethanol-induced ulcer, Indomethacin –

induced ulcer, necrotizing agents

Corresponding author: Dr Roslida AH,

Department of Biomedical Sciences,
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
E-mail: [email protected]

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Introduction

Global expansion of consumption of alcohol and non-steroidal anti-inflammatory drugs

(NSAID) and inappropriate diets have contributed to growing ulcer etiopathology (1). In
this way, the peptic ulcer is considered a disease of modern times, related to the
addictions that are increasingly frequent in the society and to its stressful lifestyle.
Treatment with natural products presents promise of a cure. Plants have been used as
sources of raw material for the synthesis of many drugs and they remain an important
source of new therapeutic agents (2). Borrelli and Izzo (3) presented a review that
demonstrated the enormous variety of chemical substances isolated from plants that
present antiulcerogenic activity, indicating their great potential in the discovery of new
therapies for ulcers.

Peperomia pellucida is an annual herb that is widely distributed in many South

American and Asian countries (4). The plant grows to a height of 15 to 45 cm, and its
shiny light-green leaves are succulent, well spaced, and heart shaped. The species
develops during rainy periods (often in the spring) and thrives in loose, humid soils under
the shade of trees. (4). The plant can be utilized as a vegetable and in salads, but the shiny
bush has also medicinal applications. In Malaysia, the plant is known as ‘ketumpangan
air” and it has been used for treating various ailments such as abdominal pain,
indigestion, abscess, acne, boils, colic, fatigue, gout, headache, renal disorders, and
rheumatic pain, and to treat breast cancer, impotence, measles, mental disorders, and
smallpox (5).
.
Previous chemical investigation on this plant indicated the existence of flavonoids (6-7) ,
phytosterols, apiols, substituted styrenes, and a dimeric ArC2 compound (4). Carotol
(13.41%) was the major hydroxylated sesquiterpene in a chemical analysis of Peperomia
pellucida. Other compounds, like the peperomins, have cytotoxic or anticancer activity in
vitro (8). Isolated flavonoids include acacetin, apigenin, isovitexin, and pellucidatin.
Phytosterols such as campesterol and stigmasterol have also been isolated (9). Xu et al
(8) isolated secolignans, lignans and highly methoxylated dihydronaphthalenone from the
whole plant.

The plant has been reported to possess analgesic, antipyretic, anti-inflammatory, anti-
bacterial, anti-fungal and CNS depressant effect (10-16), in either leaves or aerial part of
the plant. However to the author’s knowledge, none has reported on its anti-ulcer activity
yet. Therefore, the present study was undertaken with the aim to assess the anti-
ulcerogenic properties claimed by the ethnobotanic information and also the traditional
system of medicine

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Methods

Preparation of Plant Extract

The whole plants were collected from Serdang, Selangor, Malaysia . The botanical
identification and authentication of the plant was done by Dr Richard Chung, the Curator
of the Herbarium of Forest Research Institute in Kepong, Selangor, Malaysia where a
voucher specimen FRI 65401 (KEP) was deposited for future reference.

The aerial parts of Peperomia pellucida were dried in an oven at 40 to 42ºC for 3 days.
Then, they were weighed as dried weight. Later, they were grounded into powdered form
and were weighed once again The grounded form of Peperomia pellucida was macerated
in 70% ethanol for two days. The extracted solvent was then filtered and was
concentrated by using rotary evaporator until the solvent was completely removed and
crude extract was obtained. The crude extract of Peperomia pellucida was then dissolved
with 10% Tween 80 and was prepared into desired dose concentrations for
pharmacological testing.

Animals and Experimental Design

Healthy Sprague dawley rats of either sex weighing between 170-250 g were obtained
from Animal Unit of Faculty of Medicine & Health Sciences, Universiti Putra Malaysia .
The animals were kept in metal cages at room temperature under standard environmental
condition and were fasted 24 hours before the experiment but were allowed free access of
water. All the procedures were conducted in accordance with the guide line for Animal
Ethic Committee.

Gastric ulcers induced by necrotizing agents (cytoprotective studies)

Cytoprotective studies were carried out according to the method of Robert et al (17) with
some modifications. 1 ml of necrotizing agent viz 80% (v/v) aqueous ethanol, 25%
NaCl, 0.6 M HCl and 0.2 M NaOH was administered orally to induce the ulcer. SD rats
of either sex weighing between 170-200 g were divided into 5 groups of 6 animals each
and fasted for 24 hours with water ad libitum prior to experiment. The animal of group 1
were pretreated with vehicle (1% Tween 80) and the animals of group 2, 3, 4 and 5 were
pretreated with 70% ethanolic extract at 10, 30, 100 and 300 mg/kg respectively. 1 ml of
necrotizing agent (80%, (v/v) aqueous ethanol, 25% NaCl, 0.6 M HCl and 0.2 M NaOH)
was administered to all the animals of group 1 -5, 60 minutes after the respective
treatments. The animals were sacrificed by cervical dislocation after 8 hours of
necrotizing agent administration and stomach was incised along the greater curvature to
determine the lesion damage. The percentage protection was calculated based on the total
surface area of lesion in treated group compared with the lesions in control group.

NSAIDs (Indomethacin)-induced ulcer

The experiments were performed according to the method of Hayden et al (18) with some
modifications. SD rats of either sex weighing between 170-200 g were divided into 5
groups of 6 animals each and fasted for 24 hours with water ad libitum prior to
experiment. The animal of group 1 were pretreated with vehicle (1% Tween 80) and the
animals of group 2 and 3 were treated with standard ie cimetidine 50 mg/kg and 150

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mg/kg respectively. Similarly, the animals of group 4, 5, 6 and 7 were pretreated with
70% ethanolic extract at 10, 30, 100 and 300 mg/kg respectively. Indomethacin (30
mg/kg, po) was administered to all the animals of group 1 -7, 60 minutes after the
respective treatments. The animals were sacrificed by cervical dislocation after 8 hours of
indomethacin administration and stomach was incised along the greater curvature to
determine the lesion damage. The percentage protection was calculated based on the total
surface area of lesion in treated group compared with the lesions in control group

Statistical analysis
Data was expressed as mean ± S.E.M. The results of of the experiments were expressed
as changes of percentage from control values. Data was analyzed by two-way analysis of
variance (ANOVA), followed by Duncan’s multiple comparison test for post-hoc
comparison of group means. Student’s t-test was used to compare between two groups
For all tests, effects with probability of p<0.05 were considered significant.

Results

Gastric ulcers induced by necrotizing agents (cytoprotective studies)

The anti-ulcer effect of PPE induced by various necrotizing agents are summarized in
Table 1. The inhibitory action exerted by PPE on the ulcers induced by 80% ethanol,
25% NaCl, 0.6 M HCl and 0.2 M NaOH t was dose dependent and highly significant.
The inhibition of ulceration being in the range of 63-98% .

.
Indomethacin – induced ulcer
NSAIDs ie indomethacin used in the present study resulted in the production of gastric
ulcers, mainly in the glandular segment of the stomachs. As shown in Table 1, in
indomethacin-induced gastric ulceration model, pretreatment with PPE significantly
inhibited gastric ulceration at all doses administered but not in dose dependent manner.
Percentage inhibition of gastric erosions was in the range of 71-85%. At lower dose, ie 10
mg/kg, PPE exhibited maximum inhibition of 84.8% and comparable to 50 mg/kg
cimetidine.

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Table 1 : Effects of 70% ethanolic extract of Peperomia pellucida (PPE) on different models of acute gastric lesion induced in rats
Gastric lesion models Treatment (p.o) Dose (mg/kg) Number of animals Total area of lesions Inhibition (%)
(mm2)
80% EtOH Vehicle - 6 322.67 ± 22.00 -
PPE 10 6 84.00 ± 15.53** 73.97
30 6 46.60 ± 28.14** 85.86
100 6 44.00 ± 11.33** 86.36
300 6 10.17 ± 3.37** 96.85
25% NaCl Vehicle - 6 296.50 ± 23.64 -
PPE 10 6 110.60 ± 37.82* 62.70
30 6 29.00 ± 4.02** 90.22
100 6 1.60 ± 0.40** 99.46
300 6 4.60 ± 0.75** 98.45
0.6 M HCl Vehicle - 6 351.33 ± 12.12 -
PPE 10 6 87.75 ± 25.11** 75.02
30 6 5.20 ± 1.77** 98.52
100 6 2.33 ± 0.84** 99.34
300 6 4.83 ± 1.76** 98.63
0.2 M NaOH Vehicle - 6 270.50 ± 48.04 -
PPE 10 6 43.80 ± 15.83* 83.80
30 6 8.00 ± 1.53** 97.04
100 6 5.50 ± 2.09** 97.97
300 6 2.80 ± 0.80** 98.96
Indomethacin 30 mg/kg Vehicle - 6 152.67 ± 24.46 -
Cimetidine 50 6 23.56 ± 6.21** 84.57
150 6 1.00 ± 1.00** 99.34
PPE 10 6 23.20 ± 4.09** 84.80
30 6 43.83 ± 15.11* 71.29
100 6 34.40 ± 7.71** 77.47
300 6 39.17 ± 11.77 74.34
Data presented as means± S.E.M. Asterisks indicate significant differences from controls (*P<0.05; **P<0.001 ;Dunnett’s test)

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Discussion

PPE is effective orally against gastric damage in various experimental models. The result
of this study showed that PPE inhibits the formation of gastric ulcer induced by different
ulcerogenic drugs by cytodestructive agents. PPE exerts a dose dependent inhibitory
action on gastric mucosal lesions caused by various necrotizing agents. A key feature of
(gastric) cytoprotection is that a variety of gastric mucosal damage produced by different
nectrotizing agents (e. g. 0.6 M HCl, 0.2 M NaOH, 96 % ethanol, 25 % NaCl or thermal
stress) without affecting gastric acid secretion (17). Such cytoprotection has commonly
been referred to as the ability of prostaglandins to reduce the severe of injury of the
gastrointestinal tract induced by an array of noxious agents. Cytoprotection may occur as
result of the capacity that some compounds have to induce prostaglandin production,
fundamental for mucus protection because they stimulate mucus and bicarbonate
synthesis (19). Therefore we can postulate that PPE may have cytoprotective factors
based due to the reduction of total lesion area when induced with necrotizing agents
.
Ethanol-induced damages are induced by disturbance of mucosal microcirculation,
ischaemia and appearance of free radicals, endotelin release, degranulation of mast cells,
and inhibition of prostaglandins and decrease of mucus production (20). Ethanol-induced
gastric lesion formation may be due to stress in gastric blood flow that contributes to the
development of the hemorrhage and necrotic aspects of tissue injury (21). This chemical
agent also increases Na+ and K+ flux into the lumen and increases pepsin secretion along
with histamine release.

Furthermore, it is well known that ethanol-induced ulcers are not inhibited by the anti-
secretary agents, but are inhibited by agents that enhance mucosal defensive factor such
as prostaglandin (22) The incidence of ethanol-induced ulcer which is predominant in the
glandular part of the stomach has also been reported to stimulate the formation of
leukotriene C4 (LTC4) resulting in the damage of gastric mucosa (23-24). 70% PPE
significantly protected the gastric mucosa against ethanol challenge as shown by reduced
total surface area of lesion as compared to control group, suggesting its potent
gastroprotective effect. Similarly, NSAID’s like indomethacin inhibits COX-2 thereby
inhibits the prostaglandin synthesis.

Consequently lipoxygenase pathway is enhanced liberating leukotrienes and these

leukotrienes are reported to have a role in ulcerogenesis. In addition there is some
evidence that NSAIDs may induce ulcer by causing the back diffusion of H+ ion in to
mucosal cells (25). Therefore the gastroprotective effect of PPE may be due to its ability
to inhibit prostaglandin/leukotrienes synthesis.

On the other hand, ethanol-induced gastric damage may be due to stasis in gastric blood
flow, which contributes to the development of the hemorrhage and necrotic aspects of
tissue injury (21). This action is direct on the gastric epithelium also causing perturbation
of mast cells and release of a vasoactive mediator such as histamine (26).

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Some reports show that changes in gastric circulation after ethanol administration
remains unknown, but it has been reported that microcirculation damage can be
prevented by prostaglandin administration (21). On the other hand, ethanol-induced
gastric lesions are thought to arise as a result of direct damage of gastric mucosal cells,
resulting in the development of free radicals (27) and hyperoxidation of lipids (28). These
data suggest that antioxidant compounds could be active in this experimental model,
producing antiulcerogenic effects.

Numerous studies have demonstrated that the effect of absolute ethanol on the gastric
mucosa is related to the production of free radicals, the increase of lipoperoxidation and
the decrease of the levels of nonprotein SH compounds in the gastric mucosa (29).
Exposition to ethanol increases, in a dose dependent way, the generation of superoxide
anions and the extension of cellular damage (30)

The gastrointestinal irritant properties of nonsteroidal anti-inflammatory drugs (NSAIDs)

are the major impediment to their use as anti-inflammatory drugs (31). Non-steroidal
anti-inflammatory drugs like indomethacin are known to induce gastric damage,
particularly due to inhibition of biosynthesis of prostaglandins by inhibition of the
cyclooxygenase pathway of arachidonic acid metabolism (32). The present work
demonstrated that PPE significantly inhibited ulcer lesion area induced by indomethacin
but not in dose dependant manner. The gastrotoxicity of the NSAIDs including
indomethacin in animals can be attributed to their ability to induce the reactive oxygen
metabolites which may in turn promote lipid peroxidation (33).

Ulceration due to NSAIDs is also believed to occur because of non-selective inhibition of

cyclooxygenases that hampers the release of mucus due to reduction in prostaglandin
synthesis (34-35). However instead of indomethacin can cause gastric damage by
inhibiting cytoprotective prostaglandin, it can also affect enzymatic and non-enzymatic
oxidant and anti-oxidant mechanism such as GSH, CAT, SOD, MPO, and MDA (36).
Therefore, further works ie biochemical analyses to determine the enzymes activity
should be done to confirm its definite mechanism.

In conclusion, ethanolic extract of Peperomia pellucida prevented necrotizing agent and

indomethacin induced ulcer in rats. The findings has justified the traditional use of the
plant to treat abdominal pain. The phytochemical constituent might be contributory to the
anti-ulcer effect of Peperomia pellucida, and as the plant also exhibited anti-
inflammatory and analgesic activities, it is suggested that the constituents of the plant
extract might possess COX-2 inhibitory properties as well. However, further
investigations are required to identify the active constituents as much as to determine the
exact mechanism responsible for the anti-ulcer activity of Peperomia pellucida

Acknowledgement

The authors wish to thank the Faculty of Medicine and Health Sciences, Universiti Putra
Malaysia for the facilities and funding provided for the study.

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