PHYTOTHERAPY RESEARCH

Phytother. Res. 16, 455–460 (2002)

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.971

Anxiolytic and Anticonvulsive Activity of

Sesbania grandiflora Leaves in Experimental
Animals

Veena S. Kasture,1* V. K. Deshmukh2 and C. T. Chopde2

1
College of Pharmacy, Nashik 422 002, India
2
Department of Pharmaceutical Sciences, Nagpur University, Nagpur 400 010, India

Various parts of Sesbania grandiflora have been used in the Indian system of medicine, in particular, the
leaves of S. grandiflora are used in Ayurveda for the treatment of epileptic fits. In the present study we
have evaluated the anticonvulsive activity of S. grandiflora leaves using a variety of animal models of con-
vulsions. Bioassay guided separation was also carried out to identify the fraction possessing anticonvul-
sant activity. The benzene:ethyl acetate fraction (BE) of the acetone soluble part of a petroleum ether
extract significantly delayed the onset of convulsions in pentylenetetrazol (PTZ) and strychnine (STR)-
induced seizures in mice and reduced the duration of tonic hindleg extension in the maximum electrocon-
vulsive shock (MES) induced seizures in mice. The BE contained a triterpene as a major component. In
addition, the BE also inhibited electrically induced kindled seizures in mice and lithium-pilocarpine-
induced status epilepticus in rats. It prolonged the duration of sleep induced by pentobarbital and antag-
onized the effect of D-amphetamine. Mice treated with BE preferred to remain in the open arm of the
elevated plus maze indicating anxiolytic activity. The BE raised the brain contents of gamma-aminobuty-
ric acid and serotonin. Thus the triterpene containing fraction of S. grandiflora exhibits a wide spectrum
of anticonvulsant profile and anxiolytic activity. Copyright # 2002 John Wiley & Sons, Ltd.
Keywords: Sesbania grandiflora; anticonvulsant; CNS activity; serotonin; gamma-aminobutyric acid (GABA).

INTRODUCTION vulsant activity. The fraction exhibiting anticonvulsant

In the Ayurvedic system of medicine, the leaves of
Sesbania grandiflora, Linn., (Family:Leguminosae) have
long been in clinical use. The juice of the leaves and
flowers is a popular remedy for nasal catarrh and
headache when taken as snuff. In epileptic fits various
preparations made from leaves are used. The external
application is useful in leprous eruptions and a poultice of
the leaves is a popular remedy for bruises. For congenital
bronchitis or cold in babies, leaf juice mixed with honey
is recommended in infants (Nadkarni, 1982). The plant
also possesses antifertility activity (Shrivastav and Jain,
1993).
During preliminary studies on the petroleum ether
extract of leaves of S. grandiflora, we noted a general
depressant activity on spontaneous motor activity in
mice. The preliminary study indicated that the acetone
soluble part of the petroleum ether extract of S. grand-
iflora protected animals from maximum electroshock
(MES) and pentylenetetrazol (PTZ)-induced seizures. In
the present study, the acetone soluble part was succes-
sively fractionated using benzene, ethyl acetate and
methanol and the fractions were screened for anticon-
* Correspondence to: Dr V. S. Kasture, College of Pharmacy, Nashik 422
002, India.
Email: kasture_sb@hotmail.com
activity was further studied against convulsions induced
by strychnine, electrical stimulus and lithium-pilocar-
pine. We further studied the acute toxicity, behavioural
effects, amphetamine antagonism and the effect on
pentobarbital induced sleep. Since gamma-aminobutyric
acid (GABA) and serotonin (5-HT) are known to be
major endogenous anticonvulsants (Pasini et al., 1992;
Hattori et al., 1985), the effect on the brain contents of
GABA and 5-HT was also investigated.
MATERIALS AND METHODS
Plant material. The plant material was collected in May
1999 and identified by Dr V. K. Deshmukh. Voucher
specimen No. AHMA 17390 was deposited at the
Agharkar Research Institute, Pune.
Animals. Male Sprague-Dawley rats weighing 200–
225 g and male mice (NIN strain) weighing 25–30 g were
housed in groups of 5–8, in standard laboratory
conditions of temperature (23 °  1 °C), lighting from 8
a.m. to 8 p.m. with food and water freely available. They
were transferred to the laboratory at least 1 h before the
start of each experiment. Most of the experiments were
performed during the light portion of the day (8 a.m.–
4 p.m.).

Received 18 December 1999

Copyright # 2002 John Wiley & Sons, Ltd. Accepted 5 July 2000
456 V. S. KASTURE ET AL.
Preparation of extract. 1 kg of dried leaves of S.
grandiflora were powdered and extracted with petroleum
ether (60 °–80 °C) using a Soxhlet extractor. The solvent
was evaporated under vacuum and dried in air, yielding
8.0 g of crude extract. This was suspended in 0.5% w/w
gum acacia immediately before the test and administered
orally to assess its anticonvulsive property using PTZ and
MES- induced seizures (Swinyard and Woodhead, 1982).
Isolation of active fraction. The dried extract was
fractionated into acetone- soluble and insoluble parts.
Both were screened for anticonvulsive activity. The
acetone soluble part (6.4 g), which exhibited anticon-
vulsant activity, was partitioned into benzene (3.8 g),
equal volumes of benzene and ethyl acetate (0.8 g), ethyl
acetate (0.7 g) and methanol (1.0 g) by column chromato-
graphy using neutral alumina as a stationary phase. The
fractions were dried and suspended in 0.5% acacia and
screened for anticonvulsant activity 60 min after oral
administration in a dose of 100 mg/kg using the method
of Swinyard and Woodhead (1982). The benzene:ethyl
acetate fraction that exhibited anticonvulsant activity was
named BE. The chemical nature of BE was examined
(Harborne, 1984) and it was found to be a triterpene
fraction.
Anticonvulsant activity. The preliminary study was
carried out using eight mice per group against MES and
PTZ-induced seizures. In the case of the MES test, the
petroleum ether extract, the acetone soluble and insoluble
parts (25–200 mg/kg) and the fractions of the acetone
soluble part (200 mg/kg p.o.) were administered 60 min
before application of an electric shock (42 mA for 0.2 s)
using corneal electrodes and the duration of tonic hindleg
extension and the incidence of seizures were noted. A
decrease in the duration of tonic hindleg extension
indicated anticonvulsant activity. Phenytoin (25 mg/kg
i.p.) was used as the reference standard.
Similarly, the benzene fraction (BE) was suspended in
acacia (0.5% w/v) and administered orally (10–
100 mg/kg) 60 min before the MES test. The dose
protecting 50% of the animals from seizures (PD50)
was determined using the graphical method of Miller and
Tainter (1944). This PD50 was used in further study,
except where stated otherwise.
For the PTZ-induced seizure, groups of mice (n = 8),
were treated in a similar way. They received the
petroleum ether extract (25–125 mg/kg) or its acetone
soluble /insoluble part (50–100 mg/kg) or fractions of the
acetone soluble part (200 mg/kg) orally 60 min before
PTZ (80 mg/kg s.c.). The incidence and onset of
convulsions were noted. Either delay or abolition of
clonic convulsions was taken as the criterion of anti-
convulsant activity.
Strychnine-induced seizures in mice. Eight mice were
used in each group. The animals received strychnine
(1.2 mg/kg) i.p. 60 min after oral administration of
45 mg/kg of BE or vehicle or diazepam (2 mg/kg i.p.).
The onset of spasms and time to death were noted in each
group. The animals were observed for 30 min after
strychnine.
Lithium-pilocarpine-induced seizures. The effect of
BE was studied on seizures induced by lithium-
pilocarpine. The albino rats received lithium sulphate
Copyright # 2002 John Wiley & Sons, Ltd.
(3 meq/kg i.p.), 24 h before pilocarpine (30 mg/kg i.p.).
The animals received BE (PD50 = 45 mg/kg), diazepam
(2 mg/kg i.p.) or vehicle, 30 min before pilocarpine. The
severity of convulsions was assessed, until the maximum
effect was produced, using the following scoring system
used by Patel et al. (1988): no response, 0; fictive
scratching, 1; tremors, 2; head nodding, 3; forelimb
clonus, 4; rearing, falling and clonus, 5.
Electrically induced seizures. Five rats were used in
each group. The animals received two subconvulsive
electric shocks per day (21 mA, 0.1 s duration, 3 h apart)
as previously described by Palmer et al. (1991) until all
animals exhibited full blown clonic convulsions. Ten
shocks were required to induce convulsions in each
animal. Each animal, after one shock-free day, received
fraction BE by oral route, 60 min before electrical
stimulation. The severity of seizures was graded as
described earlier by McNamara et al. (1993) according to
the following criteria: grade I, facial movements; grade
II, prominent head nodding; grade III, raising of forelimb
and mild forelimb clonus; grade IV, marked rearing with
oral movements and forelimb clonus; grade V, repeated
falling on back and clonic seizures.
Behavioural studies
Acute toxicity and behavioural assessment. Varying
doses (50, 100, 150, 200, 400, 800 or 1000 mg/kg) were
administered orally and the percent mortality was
observed after 24 h. To investigate the central actions
of the compound (50 and 100 mg/kg, p.o.), the method
described by Irwin et al. (1968) was employed. The
procedure involved an initial phase of undisturbed
observations and later a manipulative phase during which
animals were subjected to the least provoking stimuli. In
the initial phase, the animals were observed for body
position, locomotion, rearing, respiration, tremors, gait,
and in the later phase, the effect on passivity, grip
strength, passivity, pain response, righting reflex and
lacrimation was noted. The animals were observed for 2 h
after the treatment.
Effect on locomotion and rearing. Five mice were used
in each group and the effect of BE on rearing and
locomotion was studied in an open field as described
earlier by Sakina and Dandiya (1990). The apparatus
consisted of a box, 60  60  25 cm, with 16 squares
drawn on the floor. The number of rearings and the
number of squares traversed by the animal were recorded
for 5 min, before drug and 60 min after the oral
administration of the PD50 of BE. Chlorpromazine
(10 mg/kg i.p.) was used as a standard drug for
comparison.
Effect on motor coordination. The effect on motor
coordination was assessed using a rotarod apparatus as
described earlier (Dunham and Miya, 1957). In brief,
mice were trained to remain for 5 min on the rod rotating
at a speed of 25 rpm. On the next day diazepam (1 mg/kg
i.p.) or BE (45 mg/kg) was administered orally and the
ability of the animal to remain on the rotating rod was
assessed before and 30 min after the administration. The
fall-off from the rod was noted for each animal and the
test was terminated after 3 min.
Phytother. Res. 16, 455–460 (2002)
 ANTICONVULSIVE ACTIVITY OF SESBANIA GRANDIFLORA 457

Table 1. The effect of petroleum ether extract of S. grandiflora and its acetone soluble and insoluble parts on MES-induced
convulsions in mice

Duration of tonic hindleg

Treatment Dose extension (s) Incidence of convulsions in Animals survived
n=8 (mg/kg) (mean  SEM) (n) mice (%)

Vehicle ± 13.6  2.4 8 0

Petroleum ether extract 25 9.4  1.7 8 0
50 6.1  0.4a 6 25
100 3.4  0.4a 4c 50
125 2.3  0.6b 2c 75
150 nil 0 100
Acetone soluble part
25 10.1  2.2 7 12.5
100 4.2  1.1a 4c 50
200 nil 0 100
Acetone insoluble part
25 13.7  2.4 8 0
50 13.2  3.1 8 0
100 12.9  2.4 8 0
200 13.1  2.9 8 0
Phenytoin 25 i.p. nil 0 100
a
p < 0.05.
b
p < 0.01 (Student's t-test).
c
p < 0.01 different from control (Fisher exact test).

Pentobarbital-induced sleep. Mice were pretreated with
45 mg/kg (PD50 MES) of the fraction BE, (30 min before
pentobarbital) or clonidine (0.1 mg/kg i.p.), 15 min before
pentobarbital (40 mg/kg i.p.). The sleeping time was
measured as the period in which the mice lost the righting
reflex after receiving pentobarbital (Turner, 1965).
Amphetamine antagonism. The mice were pretreated
with BE (45 mg/kg) or chlorpromazine (10 mg/kg i.p.),
30 min before amphetamine (1 mg/kg i.p.). The animals
were observed for latency to grooming and biting as
described earlier by Kandi et al. (1998).
Elevated plus maze. The plus maze consisted of two
opposite open arms, 50  10 cm, crossed with two arms
of the same dimensions with a wall 40 cm high. The arms
were connected with a central square, 10  10 cm, to give
the apparatus a plus sign appearance. The maze was
elevated 70 cm above the floor in a dimly lit room. Mice
(n = 5) were treated with BE or diazepam or vehicle
30 min before placing them individually in the central
square facing an enclosed arm. The time spent by the
mice, during the next 5 min, on the open and closed arms
was recorded (Pellow et al., 1985).
Estimation of brain gamma-aminobutyric acid and
serotonin levels. Mice (n = 5) treated with the PD50 of
BE were killed 60 min after the treatment by cervical
dislocation and the brains were isolated and weighted
immediately. The brain GABA and 5-HT contents were
estimated by the method of Maynert and Klingmann
(1962) and Curzon and Green (1968), respectively.
Statistical analysis. The data obtained were analysed
using one-way analysis of variance (ANOVA) followed
by Dunnett’s test or Student’s t-test. The Kruskal–Wallis
test was used for non-parametric data and the complete
effect or lack of effect was analysed using Fisher’s exact
Copyright # 2002 John Wiley & Sons, Ltd.
test. Differences were considered significant at the 5%
level.
RESULTS
Anticonvulsant activity
The petroleum ether extract and its acetone soluble part
protected mice against seizures induced by MES. There
was a significant decrease in the duration of tonic hindleg
extension (p < 0.05 or 0.01). The acetone soluble part
was ineffective in doses up to 200 mg/kg p.o. (Table 1),
whereas the fraction (BE) protected all animals against
MES induced seizures but the other fractions were not
effective. The dose of BE that protected 50% animals was
determined by the method of Miller and Tainter (1944)
and was 43.6  7.4 mg/kg p.o. It was as effective as
phenytoin administered i.p. calculated as 45.2 
4.2 mg/kg (Table 2).
The petroleum ether extract, its acetone soluble part
and BE exhibited anticonvulsive activity against PTZ-
induced seizures. The other fractions were ineffective
(Table 3) except for the total acetone soluble fraction
(100 mg/kg) which displayed a small effect. The BE
delayed the latency to seizures from 264.3  12.4 s to
356.6  23.1 s (p < 0.05) and all the animals receiving
BE survived. Lithium-pilocarpine induced status epilep-
ticus in rats. BE significantly delayed the onset of
seizures and also reduced the severity of the seizures
from 4.25  1.5 to 1.25  0.3 (p < 0.05) and diazepam
completely inhibited the seizures (Fig. 1). BE reduced the
peak severity of electrically induced kindling from
3.86  0.2 to 0.6  0.1 (p < 0.05) and diazepam
(2 mg/kg i.p.) inhibited seizures completely.
Phytother. Res. 16, 455–460 (2002)
458 V. S. KASTURE ET AL.

Table 2. The effect of various doses of benzene:ethyl acetate (BE) fractions of acetone soluble part of petroleum ether extract
of S. grandiflora on MES-induced convulsions in mice

Dose Duration of tonic hindleg Incidence of convulsions

Treatment n = 8 (mg/kg p.o.) extension (s) (mean  SEM) in (n) mice PD50

Vehicle ± 14.3  1.4 8

BE 10 13.7  1.7 7
25 10.4  2.5 6
50 8.7  1.6a 4c 43.6  7.5
75 4.0b 1c
100 nil 0
Phenytoin 10 14.1  1.8 8
25 11.2  0.9 6
50 7.5  1.4a 4c 45.2  4.2
75 2.9  1.1b 2c
100 nil 0
a
p < 0.05.
b
p < 0.01 (Student's t-test).
c
p < 0.01 different from control (Fisher exact test).

Acute toxicity and behavioural assessment (1.0 mg/kg i.p.) treated mice remained on the rotating rod
for 23.2  5.6 s only.
No mortality was observed up to a dose of 1 g/kg. BE (50
and 100 mg/kg p.o.) showed some signs of central
nervous system depression such as reduced palpebral
Pentobarbital-induced sleep
opening, muscle weakness, reduced spontaneous motor
activity, abdominal and body tone. Grooming, pain
The mice receiving pentobarbital (40 mg/kg) slept for
response and righting reflex were normal but a staggering
116.6  16.5 min, whereas the animals treated with BE
gait and passivity were present (Table 4).
and clonidine prolonged the sleeping time to
296.6  28.4 min and 321.2  35.4 min, respectively
(p < 0.05).
Locomotion and rearing

Both locomotion and rearings were significantly reduced

by BE (p < 0.05). Rearings were completely inhibited.
Chlorpromazine reduced locomotion as well as rearing
significantly (Table 5).
Effect on motor coordination
Mice treated with vehicle or BE remained on the rotating
rod for the complete 180 s period, whereas diazepam
Amphetamine antagonism
In vehicle treated mice amphetamine induced grooming
and sniffing was observed after 3.0  0.3 min and
5.0  0.4 min, respectively. The latency to grooming
and sniffing was postponed by BE to 16.5  2.5 min and
32.5  3.9 min, respectively (p < 0.05). Both the signs of
stereotyped behaviour of amphetamine were blocked by
chlorpromazine.

Table 3. Effect of petroleum ether extract of S. grandiflora and its acetone soluble and insoluble parts on pentylenetetrazol
(80 mg/kg s.c.)-induced seizures in mice

Onset of clonic convulsions (s)

Treatment n = 8 Dose (mg/kg) (mean  SEM) Protection (%)

Vehicle ± 88.5  4.7 0

Petroleum ether extract 25 227.5  16.2a 25
50 1020.0  64.8a 50b
100 1500.0  71.4a 62.5b
125 1672.0 87.5b
Acetone soluble part
50 98.5  6.4 0
100 108.0a 87.5b
Acetone insoluble part
50 80.2  5.9 0
100 77.3  4.6 0
Diazepam 2 nil 100b

Except diazepam all other treatments were given orally. Diazepam was given i.p.
a
p < 0.05 (Student's t-test).
b
p < 0.01 (Fisher exact test).

Copyright # 2002 John Wiley & Sons, Ltd. Phytother. Res. 16, 455–460 (2002)
 ANTICONVULSIVE ACTIVITY OF SESBANIA GRANDIFLORA 459

Table 5. Effect of benzene:ethyl acetate fraction (BE) of the

acetone soluble part of the petroleum ether extract of
S. grandiflora on locomotion and rearing in mice

Treatment Locomotion and rearing at

(mg/kg)
0 30 0 30 min

Vehicle 20.66  3.5 18.3  5.7 7.33  2.3 6.2  1.0

BE (45) 23.5  3.9 6.6  2.8a 6.5  2.1 0.0a
CPZ (10) 25.4  6.3 8.4  3.6a 7.4  1.4 3.2  1.3a

n = 5, CPZ, chlorpromazine. BE was given orally, whereas

CPZ was given i.p.
a
p < 0.05 compared with vehicle treated group.

DISCUSSION

The BE fraction of Sesbania grandiflora was found to be

Figure 1. Effect of benzene:ethyl acetate (BE) fraction of
acetone soluble part of petroleum ether extract of Sesbania
grandi¯ora on the severity of lithium-pilocarpine induced
status epilepticus in rats. * p < 0.05 (Mann-Whitney U test)
Elevated plus maze
Vehicle treated mice remained for 190.5  12.5 s in the
closed arm, whereas BE treated mice remained in the
closed arm for 280.5  6.0 min (p < 0.05). The fraction
produced a significant decrease in the time spent in the
open arm (Fig. 2).
triterpenoid and it inhibited seizures induced by MES,
PTZ, strychnine, lithium-pilocarpine and electrical kind-
ling. The basic mechanism involved in PTZ-induced
seizures is not yet known but since PTZ acts via a specific
interaction with a GABA-coupled chloride ionophore
(Corda et al., 1991), the role of GABAergic neurons is a
possibility. Kindling is an accepted model of chronic
epilepsy (Lothman and Williamson, 1994). The BE
fraction inhibited seizures due to electrical kindling also.
Lithium alone does not have a proconvulsant effect in rats
(Clifford et al., 1985; Ormandy et al., 1991), however,
rats pretreated with lithium have limbic seizures,
following subconvulsant doses of pilocarpine. The
combined treatment with lithium-pilocarpine results in

Estimation of brain gamma-aminobutyric acid and

serotonin levels

The PD50 of BE increased the brain content of GABA

from 51.72  4.7 mg/g to 77.59  3.5 mg/g. The brain
content of serotonin was raised from 0.9  0.04 mg/g to
1.8  0.1 mg/g of tissue (p < 0.05).

Table 4. Behavioural assessment of the benzene:ethyl

acetate fraction of the acetone soluble part of
petroleum ether extract of leaves of Sesbania
grandiflora

Dose (mg/kg)
Behavioural sign 50 100

Twitches ± ±
Depression 5/8 8/8
Palpebral closure 3/8 6/8
Lacrimation ± ±
Muscle weakness 4/8 7/8
Staggering gait 3/8 8/8
Passivity 2/8 6/8
Loss of righting re¯ex ± ±

Mice were pretreated orally with the fraction and behavioural Figure 2. Effect of benzene:ethyl acetate (BE) fraction of
signs were observed for 2 h. The data showed the number of Sesbania grandi¯ora on time spent in closed and open arm.
mice having abnormal signs compared with the control mice. * p < 0.05 (Mann±Whitney U test)

Copyright # 2002 John Wiley & Sons, Ltd. Phytother. Res. 16, 455–460 (2002)
460 V. S. KASTURE ET AL.
an accumulation of inositol monophosphate and a
reduction in cortical inositol about 10 times greater than
the effects obtained with either drug alone (Sherman et
al., 1985). Status epilepticus induced by co-administra-
tion of lithium and pilocarpine is associated with a
massive increase in the concentration of acetylcholine
and choline in the cerebral cortex and the hippocampus of
rat, which may play a role in seizure maintenance and
lethality associated with status epilepticus (Jope and Gu,
1991). Both BE and diazepam prevented the occurrence
of lithium-pilocarpine induced seizures (Sherman, 1991).
Since epilepsy is linked to low GABA levels in the brain
(Meldrum, 1995), the effect of the BE on the brain
content of GABA was investigated. An increase in the
GABA content of the brain was observed and this could
be the likely mechanism of anticonvulsant action of BE,
as drugs increasing GABAergic transmission inhibit
seizures (Gupta and Malhotra, 1997; Rang et al., 1999).
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