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Gastroprotective effect
of Cissus quadrangularis
extract in
rats with experimentally
induced ulcer
Background & objectives:
Most of the
non-steroidal
anti-inflammatory drugs
(NSAIDs) including
aspirin cause gastric
ulcer. In order to study
the gastroprotective
effect of Cissus
quadrangularis
extract (CQE), this
study was undertaken on
aspirin-induced
ulcerogenesis in pyloric
ligated
(ASP-PL) model in rats.
Methods: To assess the
possible antiulcer
effect of CQE, lesion
index, gastric
secretions
glycoprotein levels,
non-protein sulphydryls
(NPSH) and adherent
mucus content were
determined in ASP-PL
induced gastric mucosal
injury in rats.
Results: Pretreatment
with CQE significantly
prevented the gastric
mucosal lesion
development
and decreased the
gastric toxicity
produced by ulcerogen.
In addition, ulcerated
rats showed
depletion of gastric
wall mucus,
glycoproteins and NPSH
levels whereas treatment
with CQE
reverted this decline in
ASP-PL induced rats.
Histological studies
confirmed the results.
Interpretation &
conclusion: The present
finding suggests that
CQE promotes ulcer
protection
by the decrease in ulcer
index, gastric
secretions and increase
in the glycoprotein
level, gastric
mucin content and NPSH
concentration. CQE may
protect the gastric
mucosa against
ulceration
by its antisecretory and
cytoprotective property.
Key words Antiulcer
activity - aspirin -
Cissus quadrangularis
extract - histopathology
- mucus content
Non-steroidal
anti-inflammatory drugs
(NSAIDs) associated
gastric ulceration
occurs in
30 per cent of users
that led to
hospitalization and
is also associated with
high mortality1. Aspirin
(ASP)
one of the most widely
used NSAIDs, damages
gastrointestinal mucosa
by irritant action,
causing
alterations in mucosal
permeability and/or
suppression of
prostaglandin
synthesis2.
Plant extracts are
attractive sources of
new
drugs and have been
shown to produce
promising results in the
treatment of gastric
ulcers3. Cissus
quadrangularis Linn. (Vitaceae)
commonly known
as ‘bone setter’, is
frequently used as a
common
food item in India4. The
stout fleshy
quandrangular
stem of C.
quadrangularis, is an
edible plant found
throughout the hotter
parts of India, Malaya,
West
Africa and Ceylon5. The
stem is used for the
treatment of eye and ear
diseases, irregular
menstruation, asthma,
piles, tumours,
fractures of
bones, wounds and
scurvy6. Previous
studies
revealed that this plant
possesses analgesic7,
antioxidant8 and
fracture healing
property9. The
analysis showed that the
stem of C.
quadrangularis
contains 398 mg of
vitamin C, 267 mg of
b-carotene
and 0.73 per cent of
calcium10. The stem has
been
used in the treatment of
stomach ulcer and
dyspepsia
in traditional systems
of medicine in India11,
which
make it a potential
antiulcer drug for
evaluation,
Preliminary studies have
shown that
C. quadrangularis
extract (CQE) is well
known
to stimulate cell
proliferation, gastric
mucus synthesis
and secretion in
indomethacin-induced
gastric ulcer
model12.
Experimental studies to
determine the role of
CQE in gastric ulcer are
very limited. Apart from
the anitulcerogenic
activity of CQE, it is
also
important to monitor
acid output and status
of the
mucosal barrier in
gastric ulcer. The
present study
was therefore undertaken
to describe the effect
of CQE on acid-pepsin
secretion, mucus content
and sulphydryl groups in
aspirin-induced
ulcerogenesis in pyloric
ligated (ASP-PL) rat
models, which might be
useful for the treatment
of gastric ulcer.
Material & Methods
Drugs and chemicals:
Haemoglobin, alcian
blue,
Tris-EDTA,
5,5’-dinitrobenzoic acid
(DTNB),
sodium acetate, sucrose,
trichloro acetic acid
were
all purchased from the
Sigma Chemical Company,
USA. Aspirin (ASP) was
obtained from SRL,
India.
All other reagents used
for the experiment were
of analytical grade.
Preparation of C.
quadrangularis extract:
The
stem of C.
quadrangularis was
purchased from
Native Care and Cure
Center, Chennai, India,
and
authenticated with the
standard sample
preserved
in Pharmacognosy
Department, Captain
Srinivasa
Murthy Drug Research
Institute for Ayurveda,
Chennai. Dried parts
were coarsely powdered
and
1 kg of this powdered
plant material was
soaked
in 2 l methanol for 48
h, and the extract was
filtered
and distilled on a water
bath. The last traces of
the solvent were removed
under vaccum drier and
the solid brown mass
obtained was stored
at -4oC until further
use. The yield of the
extract
was 5.2 per cent w/w of
powdered methanolic
extract. For
administration, the
extract was
suspended in distilled
water.
Animals: Male albino
rats weighing 175-200g
purchased from Tamil
Nadu University of
veterinary
and Animal Sciences,
Chennai, were housed at
27
± 2oC, 55 per cent
humidity, and a 12:12 h
light -
dark cycle. They were
fed with standard
laboratory
chow (Hindustan Lever
Foods, Bangalore, India)
and provided with water
ad libitum. Experimental
protocols were approved
by the institutional
ethical
committee for animal
experimentation.
Toxicity studies: For
acute oral toxicity
studies,
rats were divided into 5
groups of six animals
each.
Group I served as
control received only
distilled
water while groups II,
III, IV, and V were
orally
fed with CQE at the
doses 0.5, 1.5, 3.0 and
5.0 g/kg b. w/day
respectively for 14
days. On
day 14, the animals were
sacrificed, blood was
collected and analyzed
for red blood cell count
(RBC), white blood cell
count(WBC), haemoglobin
(Hb), haematocrit (HCT),
mean corpuscular volume
(MCV), blood sugar,
cholesterol and protein
with
the use of autoanalyzer
(Hitachi 911, Germany).
Treatment protocol for
antiulcer activity:
Animals
were divided into 4
groups of six animals
each.
Group 1— received
distilled water orally
for 7
consecutive days.
Group 2— received
distilled water orally
for 7
consecutive days. On the
last day rats were given
aspirin (ASP) (200 mg/kg
body weight) suspended
in 1 per cent
carboxymethyl cellulose
orally13.
Group 3— received
pretreatment with CQE
(500 mg/kg body weight)
orally for 7 consecutive
days. On the last day 30
min after the extract
treatment, rats received
ASP (200mg/kg body
weight).
Group 4— Received CQE
(500 mg/kg body
weight) alone orally for
7 consecutive days. The
dosage and duration of
treatment for the
above mentioned groups
have been fixed based
on the previous
results14. At the end of
the
experimental period, the
animals were fasted for
24 h and care was taken
to avoid coprophagy.
Animals were
anaesthetized using
pentobarbitone
(35 mg/kg body weight, i
p), the abdomen was
opened
and pyloric end of the
stomach was ligated
without
causing any damage to
its blood supply. The
stomach was replaced
carefully and the
abdomen
wall was closed in two
layers with interrupted
sutures. The animals
were recovered fully
from
the anaesthetic agent
and deprived of water
during
post-operative period.
Aspirin was administered
orally to the animals of
groups 2 and 3 after
pyloric
ligation. After 4 h of
aspirin induction,
animals were
sacrificed and stomach
was dissected out after
tying the oesophageal
end. The stomach was cut
open along the greater
curvature and the
contents
were collected into
tubes, centrifuged at
1000 rpm
for 10 min and the
sediment/supernatent
used for
the estimation of
various biochemical
parameters.
The stomach was then
inflated with normal
saline
and the inner surface is
examined for ulceration
as described by Szabo et
al15. Erosion of at
least
1mm in diameter were
considered and the sum
of
the length of all
lesions per stomach was
calculated
for each group of rats
(in mm).
Total acid output of
gastric juice was
determined
by titrating with 0.01N
NaOH, using
phenolphthalein
as indicator and was
expressed as mEq/4h.
Peptic
activity was determined
using haemoglobin as
substrate and was
expressed as mmol of
tyrosine/4h16. Dissolved
mucosubstances were
estimated in 90 per cent
alcoholic precipitate
of the gastric juice.
The precipitate thus
obtained
was either dissolved in
1 ml of 0.1NaOH or 1 ml
H2SO4. The former was
used for the estimation
of protein17, total
hexoses18, hexosamine19
and
fucose20, while the
latter was used for the
estimation
of sialic acid21.
Assessment of adherent
gastric mucus content:
Alcian blue binding to
gastric wall mucus was
determined by the method
of Corn et al22. Animals
from all groups were
sacrificed, the gastric
mucosal
tissues were scrapped,
weighed and incubated in
tubes containing 1 per
cent alcian blue
solution
(0.16M sucrose in 0.05M
sodium acetate, pH 5.8)
for 2 h. The alcian blue
binding extract was
centrifuged at 3000rpm
for 10 min and the
absorbance of
supernatent was measured
at 489nm.
Estimation of
non-protein sulphydryl
(NPSH)
groups: Gastric mucosal
NPSH was determined
by the method of Sedlak
and Lindsay23. 200mg
gastric mucosal tissues
were homogenized in
2.0ml
of 20 mM EDTA at 4oC in
a homogenizer. To
measure NPSH content,
2.0 ml of water was
added
to 1.0 ml of homotenate
which was then treated
with 1.0 ml of 10 per
cent TCA and
centrifuged.
From this, 2.0 ml of
supernatent was taken
and
treated with 4.0ml of
Tris-EDTA (pH 8.0) and
0.1ml of DTNB in
methanol. The contents
were
mixed well and
absorbance read at 412
nm.
Histological studies:
Gastric tissue samples
from
each group were fixed in
10 per cent formalin for
24 h. The formalin fixed
specimens were embedded
in paraffin, sectioned
(3.5mm) and stained with
haematoxylin and eosin.
The histochemical
sections
were evaluated by light
microscopy.
Statistical analysis:
The data were analysed
using
Dunnett’s T3 multiple
comparison test and
ANOVA
(one-way analysis of
variance) using SPSS
package
version 7.0.
Results
ASP-PL induced animals
showed extensive
gastric lesions that
were confined to the
glandular
portion of the stomach
as compared to control
rats
(P<0.001). In contrast,
oral administration of
CQE
at 500 mg/kg body wt.
for 7 days before ulcer
induction lowered the
lesion index values
significantly (P<0.001)
in group 3 rats. No
gastric
mucosal injury was seen
in either the control
animals
or in those given CQE
alone orally (Table I).
non-significant changes
in these parameters when
compared with control
(Table, II).
A significant decrease
(P<0.001) in adherent
gastric mucosal content
was seen in ASP-PL
induced
rats when compared to
control rats.
Pretreatment
with CQE significantly
attenuated these changes
and showed a protective
effect on gastric mucosa
in ASP-PL induced rats.
CQE per se in group 4
rats showed significant
(P<0.005) elevation in
adherent gastric mucosal
content as compared to
control group (Fig.1).
ASP-PL was found to
significantly (P<0.001)
decrease NPSH
concentration in the
gastric mucosa of group
2 rats
as compared to control
group. Administration of
CQE (500 mg/kg body
weight) brought about a
significant increase
(P<0.001) in NPSH
concentration in ASP-PL
induced rats (group 3)
as compared to group 2
animals (Fig.1).
In the microscopic
observation of control
rats
showed normal apperance
of gastric mucosa
(Fig.2a). ASP-PL induced
rats (Fig.2b) showed
ulcer
Pretreatment with CQE
showed significant
antiulcer effect (P
<0.001) by reducing the
volume
of gastric juice, acid
output, pepsin output,
protein
level and a significant
increase (P<0.001) in pH
and (P<0.001)
glycoprotein levels such
as total
hexose, hexosamine,
fucose, sialic acid and
total
carbohydrate: protein
ratio when compared to
group
2 animals. CQE per se in
group 4 rats showed
crater with distorted
gastric glands, damaged
mucosal epithelium,
inflammatory exudates
and
cellular debris were
found in ulcerated wall
of the
stomach. Protection
against these
histopathological
changes was observed by
apparent
epithelializations,
glandular organization,
maintenance of mucularis
mucosa and reduced size
of ulcer crater in CQE
pretreated rats (Fig.
2c). The histological
picture
of CQE alone treated
rats showed normal
cytoarchitecture of
gastric mucosa with no
pathological changes
(Fig. 2d).
Discussion
The present results
suggested that
pretreatment
with CQE markedly
ameliorated the ulcer
index,
histological and
biochemical changes of
ASP-PL
induced gastric
ulceration in rats.
Administration
of aspirin produces
severe gastric
haemorrhagic
erosions and has not
been found to increase
the
aggressive factors (acid
and pepsin) but
significantly
decreased the gastric
output because of so
called
back diffusion of HCl
through the broken
barrier, inhibition of
mucosal blood flow and
acute
inflammation3,26 that is
consistent with the
present
report. CQE demonstrated
significant antiulcer
effect by decreasing the
ulcer lesions, volume of
gastric juice, acid
output and pepsin output
and an
increase in pH. The CQE
not only reduced the
volume of gastric
secretion, but it also
decreased
total acid output and
pepsin concentration
indicating
its antisecretory
effect.
Mucus secretion is a
crucial factor in the
protection of gastric
mucosa from the gastric
lesions
and has been regarded as
an important defensive
factor in the gastric
mucus barrier. A
decrease in
the synthesis of
sulphated mucus
glycoprotein has
been implicated in the
aetiology of gastric
ulcer24.
The increase in total
carbohydrate :protein
(TC:P)
ratio is the direct
reflection of mucin
activity, which
is indicated by the
enhanced level of
individual
mucopolysaccharides like
hexose, hexosamine,
fucose and sialic
acid25. Decrease in
protein content
in the gastric juice
also signifies decrease
in leakage
from the mucosal cells
indicating mucosal
resistantce. The wide
distribution of adherent
mucus
content in the
gastrointestinal tract
plays a pivotal
role in cytoprotection
and repair of the
gastric
mucosa26. The results
showed increased levels
of
adherent mucus content
of gastric tissue
pretreated
Fig.2. Histological
examinations of gastric
mucosal tissue sections
of control and
experimental rats
(hematoxylin and eosin,
20×). Control rat (2a);
ASP-PL-induced rats
(2b); CQE pretreated +
ASP-PL
induced rats (2c); CQE
alone treated rats (2d).
JAINU et al: ANTIULCER
ACTIVITY OF CISSUS
QUADRANGULARIS 805
with CQE indicating its
cytoprotective action on
experimentally induced
gastric ulcer.
In the present study,
the ulcerated region had
less mucosal NPSH
content than the intact
region
in normal rats. An
increase in NPSH content
limits
the production of oxygen
free radicals and could
be related with gastric
protection in ASP-PL
model.
In the present study,
rats pretreated with CQE
showed a significant
increase in NPSH
concentration, which
might attribute to its
direct
cyroprotection and
antioxidant activities.
In histological study,
pretreatment with CQE
was found to preserve
the functional
cytoarchitecture of the
entire gastric mucosa.
CQE
treatment showed not
only the maintenance but
also the regeneration of
gastric mucosa in the
damaged regions. These
findings confirm the
cytoprotective nature of
CQE12.
The significant
reduction in basal
gastric
secretion and ulcers
formation by CQE after
pylorus
ligation suggests that
the cytoprotective
mechanism
on the extract on
gastric mucosa may
involve direct
reduction of gastric
secretion. C.
quadrangularis
significantly reduced
gastric secration and
acidity,
which might be due to
the enhancement of mucus
and HCO3 secretion by
CQE27. We have earlier
reported that CQE
prevented
indomethacin-induced
ulcer lesions by its
cytoprotective
property12.
The antiulcer activity
of CQE might be
attributed
to the presence of
biological compounds
such as
triterpenoids,
glycosides, saponins,
tannins,
b-sitosterol and
aminoacids28 in the
extract.
Triterpenoids and
glycosides have been
shown
to inhibit gastric acid
secretion and
enhancement
in gastric mucus content
against several
experimental ulcer
models29,30. These plant
constitutents present in
CQE might have the
ability toprotect
against ulceration
induced by
ASP-PL.
In conclusion, CQE
showed significant
antiulcer
activity in
experimentally induced
ulcer in rat model
by decreasing the
gastric secretions and
by
enhancing glycoprotein
levels. Further studies
are
needed to isolate and
purify the active
principle
involved in the
antiulcer activity of
this plant.
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