Yasa Swetha*, Sunanda,
Rajanikar, Ashwini K. Reddy, Srivally, Md. Masoom.
Dept
of Pharmacology, Malla Reddy Institute of Pharmaceutical Sciences (MRIPS),
JNTU, Hyderabad 500054.
ABSTRACT:
Study was aimed to
evaluate the possible antiulcer activity of methanolic extract of leaves of Gymnema sylvestre for its ability to inhibit
gastric secretion and to protect gastro duodenal mucosa. G. sylvestre leaves were successively extracted with methanol and is
subjected to phytochemical screening to identify different phytoconstituents. Ld50
studies were conducted up to the dose level of 2g/kg by following OECD guidelines.
No mortality was observed with the extract up to the maximum dose level of 2g/kg.
Anti-Ulcer activity was evaluated in various animal models like pylorus ligation,
indomethacin an non steroidal anti inflammatory drug (NSAID) and forced swim stress
induced gastric ulcers in rats. Preliminary phytochemical studies revealed the presence of
saponins, sterols, glycosides, alkaloids, resins, carbohydrates, flavonoids, tannins,
proteins, triterpenoids, phenolic compounds in the extract of G. sylvestre. Further the Methanolic Extract
of G. sylvestre (MEGS) at 400mg/kg p.o.
significantly (P < 0.01) reduced ulcer index when compared with the methanolic
extracts at 200mg/kg and 100mg/kg p.o in rats. Ranitidine (30mg/kg p.o.) was used
as the reference drug. The present study revealed that the antiulcer activity of
MEGS may be due to the presence of phytochemical
constituents such as saponins, flavanoids, tannins, sterols, glycosides,
alkaloids, resins, carbohydrates, proteins, triterpenoids as these phytochemical
constituents were already reported for the above mentioned effects.
KEYWORDS: Gymnema
sylvestre, Anti-ulcer, Pylorus
ligation, NSAIDs, Ulcer index.
INTRODUCTION:
Peptic ulcer is a gastrointestinal disorder that requires a well targeted
therapeutic strategy. A number of
drugs including proton pump inhibitors and H2 receptor antagonists
are available for the treatment of peptic ulcer, but clinical evaluation of
these drugs has shown incidence of relapses, side effects and drug interactions1.
That is the rationale for the development of new anti-ulcer drugs, and the
search for novel molecules has been extended to herbal drugs that would offer
better protection and decreased relapse. Drugs of plant origin are increasing
in popularity and are being investigated for a number of disorders, including
peptic ulcer2. The plant Gymnema
sylvestre (family Asclepiadaceae), popularly known as ‘Gurmarbooti’,
a
large woody, much branched, climber with pubescent young parts, found
throughout India in a dry forest up to 600m, growing wild in the hilly regions of central India and
western Ghats and also in tropical Africa. The leaves have pleasant and
aromatic odour. The leaves contain pentriacontane, hentriacontane, phytin,
d-quercitol, gymnemic acids (antisweet compound), saponins, flavanoids,
alkaloids, resins, glycosides. According to the Ethanomedical information of Gymnema
sylvestre, it is being used
as antidiabetic, stomachic, stimulant, laxative and diuretic.
It was also found to be useful in hepatosplenomegaly, dyspepsia, constipation,
cardiopathy, jaundice, helminthiasis and amenorrhea3. It is also
said to be useful in biliousness, parageusia, furunculosis and as an antidote
against snake bites.
The herb is
recommended in Ayurvedic medicines for the control of diabetes mellitus. The
leaves of G.sylvestre are reported to lower
blood sugar, stimulate the heart, uterus, circulatory system and exhibit
antisweet and hepatoprotective activities4. It is also used
as Antibiotic, in stomach pains, as a blood purifier and in rheumatism5.
As far as our literature survey could ascertain, there is no in vivo study
reported for anti-ulcer activity of leaf extract Gymnema sylvestre
Therefore, the aim of the study was in vivo evaluation of anti-ulcer
activity of Gymnema sylvestre leaf extract.
MATERIALS AND METHODS:
Plant material and extraction: Fresh, matured leaves of G. sylvestre were
collected locally and were identified by an experienced botanist of the Osmania
University, India. Air-dried powder of leaves of G.sylvestre(1kg) (screened
through sieve no.60) was extracted by cold maceration in a closed conical flask for 24h at room
temperature with 100ml of 85%(v/v) methanol.
The suspension after maceration was centrifuged and the supernatant
evaporated under reduced pressure6.
Animals: Adult albino Wister rats of both sex (Sprague Dawley strain from
National Institute of Nutrition, Hyderabad, India), weighing 180-200g, were
used in this study. Animals were housed in groups of eight per cage in an
air-conditioned room with a constant temperature of 23°C and a 12h light/12h darkness photo period and allowed free access to water.
Toxicity studies7: Male albino mice of either sex weighing
18-26g were used for acute toxicity studies. All animals were maintained in the
animal house at ambient temperature with food and water available ad libitum.
Study protocol was approved by Institutional Animal Ethics Committee (IAEC).
Procedure:
Toxicity and gross behavioural studies of methanolic extract were
carried out in oral doses of 100 to 2000mg/kg weight using albino mice. After
the extract administration, the animals were kept under observation for 2hr to
note the behavioural changes.
Evaluation of Anti-ulcer activity
Pylorus ligated (PL) rat:
Rats were fasted
for 48hr and pylorus ligation was performed under light ether anesthesia. Animals were divided into six groups each
containing six animals. Immediately after pylorus ligation Group I treated as
vehicle control (Normal saline 10ml/kg), group II treated as pylorus ligation
control, group III received Standard drug (Ranitidine-30mg/kg p.o.), group IV,
V, VI received methanolic extracts 100mg/kg p.o., 200mg/kg p.o., and 400mg/kg
p.o. respectively. Nineteen hr after pylorus ligation animals were sacrificed,
stomachs were isolated and the contents were collected, measured and
centrifuged. The free and total acidity were estimated. The stomach was cut
open along the greater curvature and the surface was examined for ulceration.
The pH of the solution was noted using pH strips. The ulcer index was
calculated by using the below equation8
Scoring of Ulcer9
0 = Normal coloured stomach
0.5 = Red colouration
1 = Spot ulcer
1.5 = Haemorrhagic streaks
2 = Ulcers ≥ 3 but ≤ 5
3 = Ulcers >5
Calculation of
Ulcer Index10
U1 = UN + US + UP x 10-1
U1 = Ulcer Index
US = Average of severity score
UN = Average of number of ulcer per animal
UP = Percentage of
animal with ulcer
Determination of acidity11:
Acidity = Vol. of NaOH × N × 100
mEq/L
0.1
Determination of %protection11:
% Inhibition of ulcer =
(Ulcer index Control-Ulcer index Test) × 100
Ulcer index Control
Indomethacin
(IND) induced ulcer model
Thirty six rats
of either sex were randomly allotted to six groups each consists of six
animals. Group I served as the vehicle control (Normal saline 10ml/kg), Group II served as the Indomethacin control
(25mg/kg b.w); Group III served as standard drug (Ranitidine–30 mg/kg); Group
IV, V and VI received methanolic
extracts at the dose levels of 100mg/kg b.w, 200mg/kg b.w and 400mg/kg b.w
respectively were administered orally by gavage. The animals were fasted for
24hr before the test substance administration but had free access to water. The
test drugs were administered orally in normal saline 10min prior to oral
indomethacin in a dose of 25mg/kg. Six hours later, the rats were sacrificed by
cervical dislocation and their stomachs
removed. Stomach was cut along the greater curvature and observed for ulcers
and its content drained and centrifuged at 3000rpm for 10min. The volume of the
gastric content was measured. The pH of
the solution was noted using pH strips. Ulcer Index is calculated as described
above12.
Forced swim stress induced ulcer model (FSSU)
Thirty six rats
of either sex were randomly allotted to six groups each consists of six
animals. Group I served as the vehicle
control (Normal saline 10ml/kg) Group II served as the Forced swim stress
control (3h). Group III received
standard drug (Ranitidine–30mg/kg); Group IV, V and VI received
methanolic extracts at the dose levels of 100mg/kg b.w, 200mg/kg b.w and
400mg/kg b.w respectively were administered orally by gavage. The animals were
fasted for 24hr before the test substance administration but had free access to
water.
Table I. Effect of methanolic leaf
extract of Gymnema sylvestre on Pylorus ligation – induced ulcer in rats
|
Groups |
Treatment |
Mean pH |
Mean Total acidity |
Mean Ulcer Index |
% Inhibition of Ulcer |
|
I II III IV V VI |
Vehicle control(10
ml/kg) Pylorus
ligation(PL) control PL+Ranitidine(30mg/kg) PL+MEGS(100mg/kg) PL+MEGS(200mg/kg) PL+MEGS(400mg/kg) |
2 2.500±0.223 4.833±0.307*** 3.667±0.333* 3.667±0.6146* 4.500±0.341*** |
0 69.17±7.350 33.50±1.996*** 54.17±1.537* 48.83±0.872** 42.50±1.87*** |
3.050±0.2754 6.833±0.2789 1.417±0.200*** 2.710±0.1005 2.767±0.4883 1.750±0.214*** |
- - 79.4% 55.4% 60.2% 74.2% |
Values are
expressed as mean ± S.E.M. n = 6. Significant values were compared with* P <0.05,
** P <0.01 and *** P <0.001 Control Vs treated groups using one way
ANOVA followed by Dunnett’s test.
Table II. Effect of methanolic leaf
extract of Gymnema sylvestre on NSAIDs – induced ulcer in rats
|
Groups |
Treatment |
Mean pH |
Mean Ulcer Index |
% Inhibition of Ulcer |
|
I II III IV V VI |
Vehicle control (10
ml/kg) Indomethacin
control Indomethacin+Ranitidine(30mg/kg) Indomethacin+MEGS(100mg/kg) Indomethacin+MEGS(200mg/kg) Indomethacin+MEGS(400mg/kg) |
2.333±0.2108 2.667±0.4216 4.500±0.4282*** 3.333±0.6146 3.667±0.3333 4.167±0.3073* |
1.217±0.2949 3.500±0.4830 1.167±0.1054*** 2.627±0.1551 1.977±0.1744 1.750±0.2094* |
- - 69.5% 25.7% 45.7% 51.4% |
Values are
expressed as mean ± S.E.M. n = 6. Significant values were compared with* P <0.05,
** P <0.01 and *** P <0.001 Control Vs treated
groups using one way ANOVA followed by Dunnett’s test.
NORMAL
CONTROL PYLORUS CONTROL
STANDARD (Ranitidine) MEGS(400mg/kg)
MEGS(200mg/kg) MEGS(100mg/kg)
Figure 1: Effect
of MEGS on pylorus ligation induced ulcer model in rats
Table III. Effect
of methanolic leaf extract of Gymnema sylvestre on Forced swim stress – induced
ulcer in rats
|
Groups |
Treatment |
Mean pH |
Mean Ulcer Index |
% inhibition of Ulcer |
|
I II III IV V VI |
Vehicle control (10
ml/kg) Forced swim
stress(FSS) control FSS+Ranitidine(30mg/kg) FSS+MEGS(100mg/kg) FSS+MEGS(200mg/kg) FSS+MEGS(400mg/kg) |
2.000±0.0 2.500±0.223 4.833±0.3073*** 3.167±0.4014* 3.500±0.2236** 4.333±0.2108** |
3.050±0.2754 4.500±0.4830 0.9167±0.1537*** 2.710±0.1005 1.833±0.2108* 1.417±0.1537** |
- - 79.7% 40% 60% 68.8% |
Values are expressed
as mean ± S.E.M. n = 6. Significant values were compared with* P <0.05,
** P <0.01 and *** P <0.001 Control Vs treated groups using one way ANOVA
followed by Dunnett’s test.
Stress ulcers
were induced by forced swimming in the glass cylinder (height 45cm, diameter
25cm) containing water to the height of 35cm maintained at 25°C for 3hr. After
the drug treatment animals were allowed to swim in water for 3hr. The animals
were sacrificed by cervical dislocation, the stomachs were removed and its content
drained and centrifuged at 3000rpm for 10min. The stomach of each animal was
removed, incised along the greater curvature. The pH was determined using pH
strips and the extent of gastric damage was assessed. Ulcer Index is calculated as described above13.
RESULTS:
In the present
study, the phytochemical screening of methanolic
extract of Gymnema sylvestre revealed the
presence of saponins, flavonoids, tannins,
sterols, proteins, alkaloids, resins, carbohydrates, phenols,
glycosides, triterpenoids. The pharmacological studies evaluated for its
anti-ulcer activity against pylorus ligation, NSAIDs and forced swim stress
induced gastric ulcer model. The results of study are tabulated in Table-I
Table–II and Table–III.
In pylorus
ligation induced gastric ulcer three doses of G.sylvestre leaf extract showed significant reduction in ulcer
index, free acidity, total acidity and gastric volume but raised pH of gastric
Juice as compared to the control groups (figure 1). It showed significant
gastro protective activity of 55.2%, 64.2% and 74.2% at a dose of 100, 200 and
400mg/kg respectively when compared with standard drug Ranitidine showed
79.4%(Table-I). In NSAIDs induced ulcer model the plant extract at a dose of
100, 200 and 400mg/kg showed significant gastro protective activity of 25.7%,
45.7% and 51.4% when compared with standard drug Ranitidine showed
69.5%(Table-II). In forced swim stress induced ulcer model the plant extract at
a dose of 100, 200 and 400mg/kg showed significant gastro protective activity
of 40%, 60% and 68.8% respectively when compared with standard drug Ranitidine
showed 79.7% (Table-III).
DISCUSSION:
Peptic ulcers are caused when the natural imbalances
between the aggressive factors of acid, pepsin, defensive mechanisms of mucus,
bicarbonate, mucosal turnover and blood supply (mucosal barrier) are disturbed14.
The etiology of peptic ulcer is unknown in most of the cases, yet it is
generally accepted that it results from an imbalance between aggressive factors
and the maintenance of mucosal integrity through the endogenous defence
mechanisms. Generally various non-specific methods are used to restore these
imbalances including regular food intake, adequate rest and avoidance of
ulcerogenic agents (e.g. tobacco, alcohol and coffee). Their aims are to
attenuate and possibly block the gastric acid secretion or to enhance the
mucosal defense mechanisms15. The latter can be achieved through
increasing mucus production, stabilizing the surface epithelial cells, or
interfering with the prostaglandin synthesis. In addition, there are also
drugs, such as pump inhibitors, histamine (H2)-antagonists, anticholinergics
and antacids, used in the treatment of ulcer16. Despite the
availability of many pharmaceutical products for the treatment of gastric
ulcers, their successes were limited by presence of several adverse effects
(e.g. anaphylaxis reactions, gynecomastia, thrombocytopenia, nephrotoxicity and hepatotoxicity) 15,17,18.
Due to the reported side effects of available antiulcer drugs, focus have
been shifted towards natural products as the new sources of antiulcer agents.
With the increasingly growing interest in natural medicine, various plants have
been studied based on the traditional knowledge of their pharmacological
properties and confirmed to be useful in treating and managing ulcer16.
G.sylvestre has been reported to exert several
pharmacological properties such as anti-diabetic, anti-hypolipidemic,
antiobesity, anticancer, antioxidant,
antiviral, antiallergic, antimicrobial, anti-inflammatory, hepatoprotective activity19. A great variety and
diver-sity of compounds isolated from plants have showed anti-ulcer activities,
which include saponins, flavonoids, tannins, pro-teins, carbohydrates, phenols,
alkaloids and others11,20. Despite claim of these
compounds potential in the treatment of gastric ulcer, this plant has been
screened for its anti-ulcer activity using pylorus ligation, indomethacin
induced ulcers and forced swim stress induced ulcers in rats. Gastric ulcers
have multiple etiopathogenesis. Ulcers caused by PL are due to increased
presence of acid and pepsin in the stomach and damage by indomethacin are due
to decrease in Prostaglandin synthesis which are essential for the integrity of
mucosa. Stress ulcers are due to both physiological and psychological factors,
which is crucial for gastrointestinal defense and increased accumulation of acid and pepsin leading to autodigestion of the gastric
mucosa21 or may be due to exposure of the unprotected lumen
of the stomach to the accumulating acid22.
MEGS showed significant dose
dependent ulcer protective effect against PL, IND and FSSU induced gastric
ulcers.
The preliminary phytochemical screening of G.sylvestre
revealed
the presence of saponins, alkaloids,
resins, carbohydrates, flavonoids,
tannins, proteins, phenols, triterpenoids and
glycosides. Previous studies proved that
several plants containing high amounts of
saponins have been shown to possess significant
antioxidant, anti-ulcer23 and ulcer healing activity in
several experimental ulcer models. The protective activities of all these
active saponins are not due to inhibition of gastric acid secretion but
probably due to activation of mucous membrane protective factors24. Additionally, flavanoids, tannins, alkaloids, phenols and resins of some plants are also known to possess antiulcer activity25,26.
Though, we have not studied the active principles
responsible for the anti-ulcer activity of MEGS
it is likely that presence of saponin content
and other bioactive compounds in this plant
may be involved in the ulcer preventing action.
In conclusion, the oral administration of MEGS
display a significant antiulcer activity without any apparent toxicological
effects, which supports the use of Gymnema sylvestre
in herbal medicine of India for ulcer therapy. Further experiments and
detailed phytochemical analysis are underway to determine the
phytoconstituent(s) responsible for anti-ulcer mechanisms involved.
ACKNOWLEDGEMENT:
The authors are thankful to the principal and
management of Malla Reddy Institute of Pharmaceutical Sciences, Maisammaguda,
Hyderabad AP, India for providing necessary facilities to carrying out the
present research work.
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Received on 09.09.2012
Modified on 01.10.2012
Accepted on 09.10.2012
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Research J. Pharmacology and
Pharmacodynamics. 4(6): November
–December 2012, 368-372