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Some
Neuropharmacological
Effects of the
Methanolic Root Extract
of Cissus
Quadrangularis in Mice
ABSTRACT
In this study, some
neuropharmacological
effects of methnolic
root extract
of Cissus quadrangularis
Linn. (CQ) belongs to
family Vitaceae were
studied in mice using
various models. The CQ
root extract
significantly
inhibited acetic
acid-induced writhings
and increased tail flick
withdrawal
response in mice. The
effects of CQ on CNS
were studied by using,
spontaneous motor
activity, exploratory
behaviour, rota-rod
performance
and potentiation of
pentobarbitone sleeping
time in mice.
Preliminary
phytochemical evaluation
and acute toxicity
values were also carried
out
and LD50 was found to be
1000 mg/kg by i p route.
The extract (50,100
and 200 mg/kg i. p.)
produced reduction in
spontaneous motor
activity,
exploratory behaviour
and motor coordination
and prolonged
pentobarbitone sleeping
time. Preliminary
qualitative chemical
studies
indicated the presence
of triterpenoids,
flovonols, sapononis,
and alkaloids
in the extract. The
results suggest that the
methanolic root extract
of CQ
contains some active
principles, which may be
sedative in nature.
(Afr. J.
Biomed. Res. 9:69 – 75,
May 2006)
Keywords: Cissus
quadrangularis;
Sedation; Spontaneous
motor activity,
Exploratory
Behaviour, Motor
coordination,
Pentobarbital sleeping
time
INTRODUCTION
Cissus quadrangularis
Linn (CQ) is a medicinal
herb
reputed to be of
beneficial effect in the
traditional
system of medicine. The
CQ is commonly called as
Hajoda (Fam. Vitaceae)
is one of the most
widely used
ingredients in
alternative medicine (Ayurveda)
for the
treatment of piles,
anorexia, indigestion,
chronic
ulcers, asthma,
otorrhoea, wounds and in
augmenting
fracture healing process
(Agarwal 1997 and Rajpal,
2002). The alcoholic
extract of this plant
has evaluated
by Udupa et al reported
to facilitate healing of
fractured bones in
albino rats (Udupa,
1965) by
intramuscular
administration.
Phytochemical studies
reveal the presence of
known flavonols such as
quercetin and kaempferol
along with resveratrol,
piceatannol, pallidol,
ascorbic acid,
ketosteroid and
carotene (Saburi, 1999
and Sen, 1966).
Flavonoids are
some of the widest
spread phenolic
compounds in the
plant world and having a
wide range of
pharmacological effects.
Best of our knowledge,
studies on the CQ on
central nervous system
is not
reported. The pilot
studies indicated that
root extracts
CQ have role on CNS. On
this view/basis, we
investigated the
activity of the
methanolic extract of
the CQ on analgesic
activity, motor
coordination,
spontaneous motor
activity; pentobarbital
induced
sleeping time and
exploratory behavior in
mice.
Preparation of plant
extract
The root of the herb
cissus quadrangularis
was
collected in the month
of September and
authentication
of the plant was done by
Dr .Ganesh. R. Hegde,
Karnataka Univercity,
Dharwad, India. Root
extracts
were prepared by using
methanol in a sohxlet
apparatus according to
the previously published
method. The resultant
extract was stored in a
desiccator prior to use
and it gave a mean yield
of
0.76% w/w.
Phytochemical screening
The Preliminary
phytochemical studies
showes the
presence of
triterpenoides,
flovonols, sapononis,
and
alkaloids in the extract
and were tested using
standard
procedures.
Animals
The pharmacological
experiments were
conducted
using Swiss Female
albino mice weighing
20-25g.
Animals were maintained
under standard
nutritional
and environmental
conditions of 50 ± 10 %
RH and 12
h light and 12 h dark
cycle throughout the
experiment.
The animals were used
after an acclimatization
period
of at least 5 days to
the laboratory
environment and
provided with standard
food pellets and water
ad
libitum.. The animals
were deprived of food
24h
before experimentation.
The animal ethical
committee
clearance was obtained
from the institution for
the
present study.
Acute toxicity test
Mice were divided into
groups of ten each and
CQ
was injected i.p. in
doses from 50 to 2000
mg/kg.
Death within 24 h was
recorded. The LD50 was
estimated from the graph
of percent mortality
against
log-dose of the extracts
using the Miller and
Tainter
(1944) method.
Analgesic activity
Analgesic activity was
measured against acetic
acid
induced writhing and
tail flick painful
stimuli method.
The mice were divided
into five groups of six
each.
Group first received
normal saline (0.1 ml/10
g i. p.).
Group second, third, and
fourth received extract
of
CQ at doses of 50, 100
and 200 mg/kg, by i. p
respectively. Group five
received aspirin (200
mg/kg p
o.). Treatment was given
30 minutes before to
writhing were produced
by injecting 1ml/100gm
of
1% solution of acetic
acid i. p to all groups.
The
writhing response was
observed by the method
of
Turner (1965). The time
of writhing and number
of
writhing in 15 min were
noted. A reduction in
the
writhing numbers as
compared to the group
first was
considered evidence for
analgesia.
% inhibition =
WC – WT X 100
WC
Where,
WC = Mean number of
writhes in control group
WT =Number of writhes in
test group)
Tail flick test
To evaluate the central
analgesic effects of
methanolic
root extract. Tail flick
test was performed by
time
taken for mouse to
withdraw the tail when
immersed
in water maintained at
55±0.5° C was measured
(Turner, 1965). The
animals were divided
into five
groups of six mice each.
Group one received
normal saline (0.1
ml/10gm) and groups
second, third and
fourth received CQ
extract 50, 100 and 200
mg/kg i.
p. respectively. Group
five treated with
pentazocine
(10 mg/kg, i. p.).
Spontaneous motor
activity (SMA)
Spontaneous motor
activity was performed
using
Actophotometer (Techno
LE3806, India). Mice
were
grouped of six each and
treated with saline or
the CQ
extract (50,100 and 200
mg/Kg i.p.) or received
diazepam 1mg/kg i.p.
Activity was
automatically
recorded 30 min after
treatment and at every
10 min.
The experiments were
repeated at an interval
of 30
min, for a total of 120
min. Results of the
treated
groups were compared
with those of control
group at
each time interval (Amos
et al, 2001). SMA
measurements started 30
min after the
administration
of the extract and the
results were compared
with
those of control.
Exploratory behavioral
pattern
The study was carried
out using wooded board
measuring 40x40cm with
16 evenly spaced holes
(Perez et al., 1998).
Mice were grouped (n=6)
and
treated with saline or
extract (dose 50,100 and
200
mg/kg) or received
diazepam 1 mg/kg i. p.
Thirty
minutes after treatment
the mice were placed
singly on
an board with 16 evenly
spaced holes and the
number
of times the mice dipped
their heads into the
holes
during 5 min trial was
counted. Results were
expressed as means for
the various treatment
groups
at different time
intervals.
Motor coordination
Rota-rod (Techno, India)
biological research
apparatus
was used for the test.
The instrument (a
horizontal
rotation device) was set
at a rate of 16
revolutions per
minute (Fujimori and
Perez, 1998). Mice were
placed
on the rod and those
that were able to remain
on the
rod longer than 3 min
were selected for the
study.
Group 1 was treated with
saline, while group 2, 3
and
4 received the extract
(50,100 and 200 mg/kg
i.p.).The
group 5 received
diazepam 1 mg/kg i.p.
Mouse unable
to remain on the rod at
least for three min was
considered as a positive
test and the time of its
fall was
recorded.
Pentobarbital-sleeping
time
Albino mice were grouped
of six each. They were
treated as follows;
Group 1 received normal
saline,
groups 2, 3 and 4
received the extract
(dose 50,100
and 200 mg/kg). Animals
were administered with
sodium pentobarbitone
(40 mg/kg i.p.) 30 min
later
and index of hypnotic
effect recorded. The
effects
were recorded as
follows: Time elapsed
between the
administrations of
pentobarbital until loss
of righting
reflex was recorded of
as the onset of sleep,
while the
time from the loss to
its recovery was
considered as
the duration of sleep
(Ming-Chin Lu, 1998).
Statistical analysis
All the data obtained
were expressed as mean ±
standard error.
Differences in means
were estimated
by means of ANOVA
followed by Dunnet's
post hoc
test. Results were
considered significant
at P < 0.05.
RESULTS
Acute toxicity and
general behavioral
studies
The LD50 of the extract
by i.p. route in mice
was 1000
mg/kg. While conducting
the toxicity studies
animals
were observed
continuously for any
general behavioral
changes and significant
reduction of spontaneous
locomotor motility,
drowsiness and
remarkably quiet
were observed.
Analgesic activity
Analgesic activity was
investigated by the
acetic acidinduced
writhing test and tail
flick test in mice.
Results
of writhing studies in
mice are presented in
Fig.1. The
maximum writhes were
produced by saline
treated
mice. Extract of CQ
(50,100, and 200 mg/kg,
i.p.)
showed a significant
dose-dependent reduction
in the
number of writhing with
approximately 44%, 61%
and
84% of inhibition
respectively. The
maximum
inhibition was observed
at the dose of 200
mg/kg,
which was statistically
similar to the standard
drug
aspirin (100mg/kg). The
difference in tail flick
latency (sec) before and
after treatment, in
saline
treated group was
3.82±0.70 (Table 1). CQ
pretreatment induced
dose dependent related
changes
in tail-withdrawal
latencies when compared
to control
group. The maximum
analgesic effect reached
at 60
min after
administration. number
of writhing while Fig 1b
shows the percentage
inhibition compared with
the control values are
mean ±
SME; n=6 in each group.
**Significantly
different at P<0.01.
Spontaneous motor
activity
CQ produced significant
decrease in the
spontaneous
motor activity in mice.
This effect was dose
dependent
and the effect was
observed within 30 min
of drug
administration and
persisted for 120 min
(Table 2).
Exploratory behavior
pattern
On head dip test in mice
treated with different
doses of
CQ (50,100 and 200
mg/kg), there was a
significant
reduction in head dip
responses when compared
with
control (Table 3).
Motor coordination
Results of motor
coordination test are
presented in
Table 4. It was found
that, the CQ exhibited a
marked
reduction in motor
coordination in mice and
mice were
unable to hold on the
rotating rod. This
effect was
dose-dependent and
varied with time in
mice.
Pentobarbitone induced
sleeping time
Prior administration of
CQ significantly
potentiated
pentobarbitone–induced
sleeping time in mice.
Various
sleep time of mice
treated with
penotobarbitone with
or without extract are
shown in (Table 5). The
normal sleeping time was
found to be 39 min in
mice treated with
pentobarbitone alone.
Prior
administration of CQ
significantly
potentiated onset of
action and duration of
action of pentobarbitone
–
induced sleeping time in
mice. The maximum
duration
of sleeping was observed
at a dose of 200 mg/kg
of
CQ and was approximately
87%.
DISCUSSIONS
The present study
reports some
neuropharmacological
activities of methanolic
root extract of Cissus
quadrangularis in mice.
Results indicated that
the CQ
significantly reduced
acetic acid induced
writhings in
mice and increased in
tail flick withdrawal
response.
The dose-dependent
inhibition of acetic
acid induced
writhing indicated a
peripheral effect, which
was more
potent than aspirin.
Tail flick analgesic
testing is
usually considered
suitable for centrally
acting
analgesic though clear
cut dose response
relationship
was observed. The
efficacy of the most
herbal
remedies is attributed
to various active
principles in
combination. The extract
was found to produced
alteration in general
behavior pattern,
significant
reduction of spontaneous
motor motility,
exploratory
behavior pattern, motor
coordination and
potentiation
of pentobarbitone
induced sleeping time in
a dosedependent
fashion. The present
findings suggest that
CQ possesses
CNS-depressant action.
The extract
significantly reduced
spontaneous motor
activity. The
activity is a measure of
the level of
excitability of the
CNS (Mansur, et al,
1971) and this decrease
may be
closely related to
sedation resulting from
depression of
the central nervous
system (Ozturk, et al,
1996). The
CQ root extract
possessed central
nervous system
depressant activity as
indicated by the
decrease in
exploratory behavior
[Adzu, 2002] in mice as
demonstrated by the
reduction of the
head-dip test. It
also showed a marked
sedative effect as
indicated by
the reduction in gross
behavior and
potentiation of
pentobarbitone induced
sleeping time. Earlier
studies
have related
prolongation of barbital
hypnosis to
pentobarbital metabolic
inhibition or action on
the CNS
involved in the
regulation of sleep
(Kaul and Kulkarni,
1978).
It is generally accepted
that the sedative effect
of
drugs can be evaluated
by measurement of
spontaneous motor
activity and
pentobarbitone induced
sleeping time in
laboratory animal model
(Ming-chin,
1998). These results
corroborate those of
(Fujimori
1995) who proposed that
the enhancement of
barbital
hypnosis is a good index
of CNS depressant
activity.
Results of the
exploratory behavior
test (table) further
support the
neurosedative activity
and its possible
application in anxiety
condition (Amos 2001).
Present
findings of analgesic
activity are similar to
those
reported for pentozocin
and aspirin (Distasi et
al,
1988). It has been
reported that the
saponins show a potent
sedative activity when
tested in similar models
and also inhibit
spontaneous motor
activity in mice
(Dubois, et al, 1986).
Therefore, the saponin
content
of this extract might be
contributing in part to
the
experimental
pharmacological effects.
Further studies
are planned to establish
mechanism of
CNS-depressant
action of CQ by using
various agonists and
antagonists.
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