Pharmacology, Biochemistry and Behavior 75 (2003) 529 – 536
www.elsevier.com/locate/pharmbiochembeh
Clitoria ternatea and the CNS
Neeti N. Jain, C.C. Ohal, S.K. Shroff, R.H. Bhutada, R.S. Somani, V.S. Kasture, S.B. Kasture*
Natural Products Laboratory, M.V.P. Samaj’s College of Pharmacy, Nashik 422 002, India
Received 6 December 2002; received in revised form 2 April 2003; accepted 10 April 2003
Abstract
The present investigation was aimed at determining the spectrum of activity of the methanolic extract of Clitoria ternatea (CT) on the CNS.
The CT was studied for its effect on cognitive behavior, anxiety, depression, stress and convulsions induced by pentylenetetrazol (PTZ) and
maximum electroshock (MES). To explain these effects, the effect of CT was also studied on behavior mediated by dopamine (DA),
noradrenaline, serotonin and acetylcholine. The extract decreased time required to occupy the central platform (transfer latency, TL) in the
elevated plus maze (EPM) and increased discrimination index in the object recognition test, indicating nootropic activity. The extract was more
active in the object recognition test than in the EPM. The extract increased occupancy in the open arm of EPM by 160% and in the lit box of the
light/dark exploration test by 157%, indicating its anxiolytic activity. It decreased the duration of immobility in tail suspension test (suggesting
its antidepressant activity), reduced stress-induced ulcers and reduced the convulsing action of PTZ and MES. The extract exhibited tendency
to reduce the intensity of behavior mediated via serotonin and acetylcholine. The effect on DA- and noradrenaline-mediated behavior was not
significant. In conclusion, the extract was found to possess nootropic, anxiolytic, antidepressant, anticonvulsant and antistress activity.
Further studies are necessary to isolate the active principle responsible for the activities and to understand its mode of action.
D 2003 Elsevier Science Inc. All rights reserved.
Keywords: Clitoria ternatea; Cognitive behavior; Anxiety; Depression; Stress; Convulsions
1. Introduction
Clitoria ternatea Linn (Family Fabaceae) is commonly
known as ‘‘Butterfly pea.’’ The plant is a twining evergreen
herb, which will grow up to 3 m (9 ft) high, climbing over
any available prop. The stems are pubescent and spindly. The
compound leaves are made of three to nine oval or elliptical
leaflets. The flowers are 2– 4 cm long and in various shades
of blue with a yellow throat or pure white with a big standard
petal. The fruits are pods, resembling thin peas. Native to the
island of Ternate in the Molluca archipelago, this species is
now widely grown as ornamental, fodder or medicinal plant.
The roots and seeds have powerful laxative effects, the
flowers are used to make collyrium and the leaves are used
in Madagascar to relieve joint pain. The plant may start
flowering 4 months after sowing. Roots, seeds and leaves of
C. ternatea are commonly used in the Ayurvedic system of
medicine. The roots are bitter, refrigerant, laxative, intellect
promoting, diuretic, anthelmintic and tonic and are useful in
dementia, hemicrania, burning sensation, leprosy, inflam-
* Corresponding author. Tel.: +91-253-346266; fax: +91-253-580250.
E-mail address: Kasture_sb@hotmail.com (S.B. Kasture).
mation, leucoderma, bronchitis, asthma, pulmonary tuberculosis, ascites and fever. The leaves are useful in otalgia and
hepatopathy, whereas seeds are cathartic (Anonymous,
1995). C. ternatea contains antifungal proteins and has been
shown to be homologous to plant defensins (Osborn et al.,
1995). Rai et al. (2001), using passive avoidance test and
spatial learning T-maze, have shown that the aqueous root
extract of C. ternatea enhances memory in rats. Taranalli and
Cheeramkuczhi (2000) reported that the alcoholic extracts of
aerial and root parts of C. ternatea at 300 and 500 mg/kg po
doses in rats attenuated electroshock-induced amnesia. The
extract at 300 mg/kg dose produced significant memory
retention, and the root parts were found to be more
effective. The authors suggested that C. ternatea extract
increased rat brain acetylcholine content and acetylcholinesterase activity in a similar fashion to the standard
cerebroprotective drug pyritinol. Because the other activities
of C. ternatea have not been studied, we investigated the
nootropic, anxiolytic, antistress and anticonvulsant activities
using conventional animal models and also the effects on
behavior mediated via dopamine (DA) (haloperidol-induced
catalepsy), noradrenaline (clonidine-induced hypothermia),
serotonin (lithium-induced head twitches) and acetylcholine
(sodium nitrite-induced respiratory arrest).
0091-3057/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0091-3057(03)00130-8
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N.N. Jain et al. / Pharmacology, Biochemistry and Behavior 75 (2003) 529–536
2. Materials and methods
2.1. Extraction
The aerial parts of C. ternatea were collected from
garden. Dr. D.R. Mahajan, a botanist at the KTHM College,
Nashik, identified the plant material, and the specimen was
deposited at the Botanical Survey of India, Pune (Voucher
Specimen BSI 163826). The plant material was shade dried.
One kilogram of the plant material was defatted with
petroleum ether (60 – 80 C) and then extracted with methanol. The methanolic extract of C. ternatea (CT, 6.36% w/w)
was concentrated under reduced pressure. The dried extract
was dissolved in distilled water and administered orally. The
volume of injection in mouse was 0.1 ml and rat was 0.5 ml.
2.2. Animals
Male albino mice (Swiss, 22– 25 g) and rats (Wistar,
125 –150 g) were housed in groups of five under standard
laboratory conditions of temperature, humidity and lighting.
Animals had free access to food and water, except during
experiment. They were deprived of food but not water 6 h
before the drug administration. Each group consisted of five
animals. All experiments were carried out during the light
period (1100 – 1300 h). The studies were carried out in
accordance with the guidelines given by the Indian Council
for Medical Research and the Committee for the Purpose of
Control and Supervision of Experiments on Animals
(CPCSEA), New Delhi (India) and the Institutional Animal
Ethical Committee approved the study.
2.3. Drugs
Piracetam (Uni-UCB, India), clonidine (German Remedies, India), diazepam injections (Sigma, India), haloperidol (Searle, India), pentylenetetrazol (PTZ, Sigma, USA)
and lithium sulfate (Glenmark Laboratories, India) were
used in this study. Solvents used in this study were of
analytical grade. All drugs were dissolved in distilled water.
2.4. Assessment of nootropic activity
The nootropic activity of CT was screened by using
elevated plus maze (EPM) and object recognition test.
2.4.1. EPM test
The EPM consisting of two open arms (35 6 cm) and
two enclosed arms (35 6 15 cm) was elevated to the
height of 25 cm. Mice were placed individually at the end of
an open arm facing away from the central platform, and the
time it took to move from the end to either of the closed
arms (transfer latency, TL) was noted (Itoh et al., 1990). On
the first day, mice (n = 5) were allowed to explore the maze
for 5 min after the measurement of TL. On the following
day, mice received vehicle, piracetam (50 mg/kg ip) 30 min
before or CT (100 mg/kg po) 60 min before the test, and the
TL was noted for each animal. The TL was also measured
on the ninth day. The TL was expressed as inflexion ratio
(IR) using the formula described earlier by Jaiswal and
Bhattacharya (1992):
IR ¼ ðL1 L0 Þ=L0
where L0 = TL after 24 h or on the ninth day and L1 = initial
TL (s).
2.4.2. Object recognition test
The apparatus consisted of white colored plywood
(70 60 30 cm) with a grid floor that could be easily
cleaned with hydrogen peroxide after each trial. The apparatus was illuminated by a 60 W lamp suspended 50 cm
above the box. The object to be discriminated was also
made of plywood in two different shapes of 8 cm height and
colored black.
On the day before test, mice (n = 5) were allowed to
explore the box (without any object) for 2 min. On the day
of test in the first trial (T1), two identical objects were
presented in two opposite corners of the box, and the
amount of the time taken by each mouse to complete 20 s
of object exploration was recorded. Exploration was considered as directing the nose at a distance less than 2 cm to
the object and/or touching with nose. During the second trial
(T2, 90 min after T1), a new object replaced one of the
objects presented in T1, and mice were left individually in
the box for 5 min. The time spent for exploring the familiar
( F) and the new object (N) was recorded separately, and
discrimination index (D) was calculated as (N F)/(N + F).
Care was taken to avoid place preference and the influence
of olfactory stimuli by randomly changing the role (familiar
or new object) and the position of the two objects during T2
and cleaning the apparatus with hydrogen peroxide (Bartolini et al., 1996). The animals received vehicle or CT (100
mg/kg po) 60 min prior to the first trial, whereas piracetam
(50 mg/kg ip) was given 30 min before the first trial.
2.5. Assessment of anxiolytic activity
Anxiolytic activity was assessed by using EPM and the
light/dark exploration test.
2.5.1. Elevated plus maze
The EPM as described earlier by Lister (1987) was used
in this study. Mice were placed individually in the center of
the EPM facing an enclosed arm. The time spent by the
mouse during the next 5 min on the open and closed arms
was recorded. Mice (n = 5) were treated with vehicle, CT (30,
100, 200 and 400 mg/kg po) 60 min before and diazepam (1
mg/kg ip) 30 min before their placement on the maze.
2.5.2. Light/dark exploration test
The apparatus consisted of two boxes (25 25 25 cm)
joined together. One box was made dark by covering its top
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with plywood and a 10 W lamp illuminated the other box.
The light source was placed 25 cm above the open box. The
mice were placed individually in the center of the lit box and
observed for the next 5 min for the time spent in lit and dark
boxes (Crawley and Goodwin, 1981). Mice (n = 5) were
treated with vehicle, CT (30, 100, 200 and 400 mg/kg po)
60 min before and diazepam (1 mg/kg ip) 30 min before
their placement in the lit box.
2.6. Assessment of anticonvulsant activity
The anticonvulsant activity was assessed using PTZ- and
maximum electroshock (MES)-induced seizures in mice.
Mice were treated with CT 100 mg/kg, the dose that
exhibited significant anxiolytic activity in the EPM test
and light/dark exploration test.
2.6.1. PTZ-induced convulsions
Male mice were divided in groups of five each. The
animals were pretreated with vehicle, CT (100 mg/kg po) 60
min before or diazepam (1 mg/kg ip) 30 min before the
ED95 dose of PTZ (80 mg/kg sc). The latency to seizures
and the number of animals surviving after 24 h were noted
for each animal (Swinyard and Woodhead, 1982).
2.6.2. MES-induced seizures
Mice were divided in groups of five each. They were
treated as described for the PTZ-induced seizures and an
electric shock (42 mA, 0.2 s) was applied through the
corneal electrode. The duration of hindlimb extension was
recorded for each animal (Swinyard and Woodhead, 1982).
2.7. Assessment of antidepressant activity
2.7.1. Tail suspension test
The method described by Steru et al. (1985) was used.
Mice were divided in groups of five each. They were
suspended by tying a thread to their tail from a height of
50 cm above the table top. Duration of immobility was
recorded for 6 min (after discarding activity in the first 2
min because animals try to escape during this period). Mice
were considered immobile only when they hung passively
and remain motionless. Mice were treated with vehicle, CT
(100 and 400 mg/kg po) 60 min before the test and
fluoxetine (10 mg/kg ip) 30 min before the test.
2.8. Assessment of antistress activity
2.8.1. Cold restraint stress (CRS)-induced ulcers:
The rats were divided into groups of five each and fasted
for 18 h. Rats were treated with vehicle, CT (100, 200 and
400 mg/kg po) 60 min before and diazepam (1 mg/kg ip) 30
min before the test. Immediately after vehicle or drug
administration, each rat was subjected to CRS by strapping
the rats on a wooden plank and keeping them for 2 h at 4– 6
C. The stomach of each animal was cut longitudinally
along the grater curvature and the severity of gastric ulcers
was assessed in terms of mean ulcer index as described
earlier by Alphine and Word (1969).
2.9. Lithium-induced head twitches
The rats were divided into groups of five each. Piracetam
(50 mg/kg ip) was administered 30 min before and CT (100
mg/kg po) was administered 60 min before the injection of
lithium sulfate (3 mEq/kg ip). The number of head twitches
was observed for 60 min after the administration of lithium
sulfate as described earlier by Wielosz and Kleinrok (1979).
2.10. Clonidine-induced hypothermia
Clonidine (0.1 mg/kg ip) was administered 60 min after
vehicle or CT (100 mg/kg po) and 30 min after piracetam
(50 mg/kg ip) to groups of five mice and rectal temperature
was recorded at 0, 30, 60, 90 and 120 min as described
earlier by Drew et al. (1977).
2.11. Sodium nitrite-induced respiratory arrest
Sodium nitrite (250 mg/kg ip) was used to induce
chemical hypoxia. Sodium nitrite reduces the oxygen-carrying capacity of blood by converting hemoglobin to
methemoglobin. This dose produces death due to respiratory
arrest in vehicle-treated mice (Hock, 1993). Drugs increasing cholinergic transmission delay or prevent the onset of
respiratory arrest. CT (100 mg/kg po) was given 60 min
Table 1
Effect of piracetam and CT on TL and IR in EPM
Treatment dose (mg/kg)
Vehicle (5)
Piracetam (50)
CT (100)
TL (mean ± S.E.M.) (s)
IR
Day 1
Day 2
Day 9
Day 2
Day 9
41.8 ± 1.98
77.66 ± 2.38*
47.9 ± 2.02
20.16 ± 1.35
12.33 ± 1.78*
14.25 ± 1.60*
13.16 ± 1.16
9.50 ± 1.23*
7.33 ± 1.42*
1.07 ± 0.96
5.29 ± 0.33**
2.36 ± 0.116
2.17 ± 0.70
7.17 ± 0.93**
5.5 ± 0.44***
n = 5 mice.
* P < .05, compared with the vehicle-treated group (one-way ANOVA followed by Dunnett’s test).
** P=.003, compared with vehicle-treated group (Student’s t test).
*** P=.004, compared with vehicle-treated group (Student’s t test).
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Table 2
Effect of piracetam and CT on T1 and T2 sessions and discrimination index
in object recognition test
Treatment
(mg/kg)
T1 (mean ±
S.E.M.)
Vehicle (5)
24.5 ± 2.34
Piracetam (50) 10.0 ± 3.5
CT (100)
18.0 ± 2.98
T2 (mean ± S.E.M.)
N
F
Table 4
Effect of CT on time spent in lit/dark box in light/dark exploration test
Treatment (mg/kg)
D=(N F)/
(N + F)
17.28 ± 1.79 10.88 ± 1.69 0.24 ± 0.03
15.25 ± 1.29
7.0 ± 1.20# 0.37 ± 0.03**
13.25 ± 1.90 3.75 ± 1.80# 0.55 ± 0.03*
n = 5 mice.
* P < .0001.
* * P=.015, compared with the vehicle-treated group (Student’s t test).
#
P=.02 compared with the new object (Student’s t test).
before and piracetam (50 mg/kg ip) was administered 30
min before sodium nitrite. The time between injection of
sodium nitrite and death was recorded. Each group consisted of five mice.
2.12. Haloperidol-induced catalepsy
Rats divided into groups of five each received vehicle or
CT (100 mg/kg po) and haloperidol (1 mg/kg ip). Catalepsy
was scored using ‘‘Bar test’’ at 0, 5, 15, 30, 45, 60, 90, 120
and 150 min after haloperidol as described by Ferre et al.
(1990).
2.13. Statistical analysis
The observations are given as means ± S.E.M. The parametric data were assessed by Student’s t test or one-way
analysis of variance (ANOVA) followed by Dunnett’s test.
For nonparametric data, Kruskal –Wallis ANOVA was followed by Dunnett’s test. P < .05 was considered significant.
3. Results
Vehicle (5)
CT (30)
CT (100)
CT (200)
CT (400)
Diazepam (1)
Time spent (mean ± S.E.M.) (s)
Lit box
Dark box
69.75 ± 9.43
89.25 ± 18.32
110.5 ± 6.00 *
131.75 ± 10.00 *
143.50 ± 10.10 *
176.00 ± 6.62 *
229.50 ± 9.34
207.50 ± 17.59
189.00 ± 6.01 *
166.00 ± 10.10 *
154.75 ± 9.78 *
123.00 ± 6.64 *
n = 5 mice.
* P < .0001 (ANOVA followed by Dunnett’s test).
piracetam (50 mg/kg) required least time on both second
and ninth days ( F2,12 = 431.18, P < .0001) to enter the
central platform. The IR increased after piracetam on the
second and ninth days significantly ( P=.003), whereas CT
increased the IR significantly on ninth day only ( P=.004).
The observations are given in Table 1.
3.1.2. Object recognition test
The mice in the first trial required 10 ± 3.5 to 24.5 ± 2.34
s to explore the objects. In the second trial, when a new
object replaced one of the objects, CT- and piracetamtreated mice required significantly less time to explore the
familiar object as compared with the new object. Both CT
and piracetam significantly reduced the discrimination
index. The observations are given in Table 2.
3.2. Assessment of anxiolytic activity
3.2.1. Elevated plus maze
Vehicle-treated mice spent 55.5 ± 3.37 s in the open arm.
The oral administration of CT (100 – 400 mg/kg) dosedependently increased the time spent in the open arm
3.1. Assessment of nootropic activity
3.1.1. Elevated plus maze
On the first day, the mice entered the central platform
(TL) 41.8 ± 1.98 to 77.66 ± 2.38 s after their placement in
the EPM. On the second and ninth days, the TL was
significantly reduced in all groups. The animals treated with
Table 3
Effect of CT on time spent in open/enclosed arm in EPM
Treatment (mg/kg)
Vehicle (5)
CT (30)
CT (100)
CT (200)
CT (400)
Diazepam (1)
Time spent (mean ± S.E.M.) (s)
Open arm
Enclosed arm
55.5 ± 3.37
61.5 ± 2.62
88.5 ± 4.87 *
121.75 ± 6.18 *
132.75 ± 5.92 *
157.50 ± 6.27 *
235.50 ± 5.42
230.33 ± 6.17
204.00 ± 6.17 *
168.00 ± 6.89 *
165.50 ± 5.39 *
114.25 ± 6.78 *
n = 5 mice.
* P < .0001 (ANOVA followed by Dunnett’s test).
Fig. 1. Effect of CT on PTZ-induced convulsions in mice (n = 5). Values are
means ± S.E.M. * P < .05, compared with vehicle-treated group (ANOVA
followed by Dunnett’s test).
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Table 5
Effect of CT on time of immobility in tail suspension test
Treatment (mg/kg)
Time of immobility
(mean ± S.E.M.) (s)
Vehicle
CT (100)
CT (400)
Fluoxetine (10)
211.00 ± 8.35
165.60 ± 8.35 *
123.70 ± 8.80 *
151.66 ± 8.90 *
n = 5 mice.
The duration of immobility was observed for 6 min after discarding activity
during first 2 min.
* P < .001, compared with the vehicle-treated group (one-way ANOVA
followed by Dunnett’s test).
( F5,24 = 65.21, P < .0001). A dose of 30 mg/kg was without
any significant effect. The observations are given in Table 3.
3.2.2. Light/dark exploration test
The vehicle-treated mice spent 69.75 ± 9.43 s in the lit
box and 229.5 ± 9.34 s in the dark box. CT (30 mg/kg) was
without any significant effect on the time spent in lit or dark
box, whereas the higher doses (100, 200 and 400 mg/kg)
increased the time spent in the lit box ( F5,24 = 12.55,
P < .0001). The duration of time spent in the dark box
decreased in a dose-dependent manner. The observations
are given in Table 4.
3.3. Assessment of anticonvulsant activity
3.3.1. PTZ-induced seizures
The subcutaneous injection of PTZ produced convulsions after 123.0 ± 6.63 s and the CT and diazepam significantly delayed the onset of convulsions ( F2,12 = 47.48,
P < .0001). The animals that received vehicle died immediately after seizures. Three out of five animals died in the
group that received CT, whereas only one animal died in
diazepam-treated group (Fig. 1).
3.3.2. MES-induced seizures
In the vehicle-treated mice, the duration of tonic hindlimb extension was 34.7 ± 1.25 s. The animals treated with
CT (100 mg/kg po) and diazepam (1 mg/kg ip) exhibited
tonic hindlimb extension for 16.5 ± 2.62 and 9.28 ± 2.18 s
( P < .0001), respectively.
Fig. 2. Effect of CT on number of head twitches induced by lithium sulfate
in rats (n = 5). * P < .05, compared with vehicle-treated group (one-way
ANOVA followed by Dunnett’s test). The values are means ± S.E.M. The
doses of piracetam and CT were 50 and 100 mg/kg, respectively. The head
twitches were counted for 60 min after lithium sulfate.
3.4. Assessment of antidepressant activity
3.4.1. Tail suspension test
The total duration of immobility in vehicle-treated mice
was 211.0 ± 8.35 s. Oral administration of CT (100 and
400 mg/kg) significantly reduced the duration of immobility ( P < .001). Fluoxetine (10 mg/kg ip) also reduced the
duration of immobility. The observations are given in
Table 5.
3.5. Assessment of antistress activity
After the CRS, the ulcer index in the vehicle-treated
group was 267.5 ± 6.29. The treatment with CT (400 mg/kg)
and diazepam significantly ( P < .05) reduced the ulcer
index. The effect produced by the lower doses of CT (100
and 200 mg/kg) was also significant. The observations are
given in Table 6.
3.6. Lithium head twitches
Lithium induced 36.5 ± 6.0 head twitches in 1 h. Piracetam (50 mg/kg) and CT (100 mg/kg) reduced the number of
Table 7
Effect of CT on clonidine-induced hypothermia in mice
Time
(min)
Table 6
Effect of CT on CRS-induced ulcers in rats
Treatment (mg/kg)
Ulcer index
Vehicle
CT (100)
CT (200)
CT (400)
Diazepam (1)
267.5 ± 6.29
207.5 ± 8.53 *
132.5 ± 10.30 *
27.5 ± 6.29 *
20.0 ± 4.08 *
n = 5, H = 17.65, P=.001.
* P < .05, compared with vehicle-treated group (Kruskal – Wallis
ANOVA followed by Dunnett’s test).
0
30
60
90
120
Change in temperature (C)
Clonidine
(0.1 mg/kg)
Piracetam
(50 mg/kg) +
clonidine
(0.1 mg/kg)
CT
(100 mg/kg)
CT (100 mg/kg) +
clonidine
(0.1 mg/kg)
37.63 ± 0.33
35.19 ± 0.19
34.56 ± 0.16
35.72 ± 0.0.29
36.22 ± 0.05
37.42 ± 0.18
35.38 ± 0.35
34.55 ± 0.35
36.22 ± 0.35
36.38 ± 0.23
37.45 ± 0.25
36.50 ± 0.27
36.88 ± 0.20
36.44 ± 0.20
36.22 ± 0.26
37.55 ± 0.26
35.66 ± 0.21
35.11 ± 0.30
36.22 ± 0.21
36.55 ± 0.21
n = 5, H = 2.498, P=.648. The differences were not significant (Kruskal –
Wallis ANOVA).
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Table 8
Effect of CT on duration of haloperidol-induced catalepsy in rats
Time
Duration of catalepsy (mean ln ± S.E.M.)
Treatment (mg/kg)
0
15
30
45
60
90
120
150
Vehicle
CT
1.38 ± 1.32
2.15 ± 1.04
3.03 ± 0.95
3.19 ± 0.77
3.6 ± 0.56
3.2 ± 0.56
3.06 ± 1.09
2.97 ± 0.68
0.53 ± 0.0
3.00 ± 0.71
3.51 ± 0.09
3.66 ± 1.13
3.15 ± 0.92
3.15 ± 0.92
3.05 ± 0.67
2.99 ± 0.05
n = 5. The differences were not statistically significant.
head twitches to 19.2 ± 2.7 and 18.5 ± 2.37, respectively.
The differences were significant at P=.037 ( F2,12 = 4.4)
(Fig. 2).
3.7. Clonidine-induced hypothermia
Clonidine induced hypothermia in vehicle-treated mice.
The peak effect was observed 60 min after clonidine. The
CT per se was without any effect on the rectal temperature. Piracetam and CT did not significantly altered clonidine-induced hypothermia. The observations are given in
Table 7.
3.8. Sodium nitrite-induced respiratory arrest
The mice treated with sodium nitrite died after 19.75 ±
0.85 min of its administration, whereas death occurred after
15.3 ± 0.41 and 16.71 ± 2.51 min in the CT- and piracetamtreated mice. The effects of CT and piracetam were not significant ( P=.158).
3.9. Haloperidol-induced catalepsy
The haloperidol-induced catalepsy was measured until
150 min after haloperidol. The maximum catalepsy was
noted 60 min after haloperidol. The intensity of catalepsy
was more prominent in the group that received CT before
haloperidol. In CT-treated group, maximum catalepsy was
noted 45 min after haloperidol. The CT potentiated haloperidol-induced catalepsy only unto 45 min. However, after
normalization of data (converting to ln) as suggested by
Ferre et al. (1990), the difference between the effects
produced by vehicle and CT were not significantly different.
CT alone was without any cataleptic activity. The observations are given in Table 8.
4. Discussion
In the Ayurvedic system of medicine, the roots, seeds and
leaves of C. ternatea have long been in clinical use. In many
Ayurvedic formulations, the C. ternatea is used as a
substitute for Evolvulus alsinoids (Anonymous, 1995).
Recently, Taranalli and Cheeramkuczhi (2000) reported
memory-enhancing activity of C. ternatea. The results of
the present investigation suggest that C. ternatea possess a
wide spectrum of CNS activity. The CT, though weak,
exhibited nootropic, anxiolytic, antistress, antidepressant
and anticonvulsant activities. The extract reduced lithiuminduced head twitches, showed tendency (though statistically insignificant) to inhibit clonidine-induced hypothermia and reduce sodium nitrite-induced respiratory arrest.
The effect on haloperidol-induced catalepsy was not significant.
The nootropic drugs facilitate intellectual performance,
learning and memory (Giurgea, 1973). However, the neurological basis of such action is not known. Although
involvement of cholinergic system is well established, the
role of other neurotransmitters cannot be ignored (Hollander
et al., 1986). We have reported decrease in brain contents of
g-aminobutyric acid (GABA) and DA after piracetam
(Chintawar et al., 2002). It has been reported earlier that
increase in the serotonergic transmission can interfere with
learning acquisition and memory consolidation (Ogren,
1982). The role of DA is, however, controversial. Davis
(1989) has shown that learning and memory can proceed
normally despite depletion of brain DA. Piracetam, an
established nootropic drug, is known to augment dopaminergic activity (Nyback et al., 1979). However, in another
study, Bhattacharya et al. (1989) could not notice any
significant effect of piracetam on DA levels. Brain DA
level was found to decrease after administration of oil of
Celastrus paniculatus, which possesses nootropic activity
(Nalini et al., 1995). The increase in the IR and discrimination index by CT per se has proved that the plant possesses
nootropic activity. The CT met a major criterion for nootropic activity, namely, improvement of memory in absence
of cognitive deficit (Poschel, 1988). The decrease in TL by
CT in the EPM is in accordance with the hypothesis of Itoh
et al. (1990) that nootropic drugs decrease TL. The EPM test
is useful in screening effect on long-term memory, whereas
the object recognition test is useful to study the short-term
memory. The improvement in IR by CT on the ninth day
indicated its weak effect on long-term memory.
CT exhibited a weak anxiolytic activity in both animal
models of anxiety, the EPM and light/dark exploration test.
Increase in occupancy of the animals in the open arm or
decrease in the time spent in the enclosed arm indicates the
anxiolytic activity of drug (Pellow et al., 1985). Many
researchers have shown an inverted U-shaped dose –
response curve with anxiolytics (Weiss et al., 1981; Insel
et al., 1984; Nutt and Glue, 1991), but this was not observed
with CT. CT dose-dependently increased the time spent in
the open arm. The light/dark exploration test measures
natural aversion of mice and rats to brightly lit places.
Several researchers have used this model for evaluation of
anxiolytic agents (Imaizumi et al., 1996; Sanchez, 1995;
N.N. Jain et al. / Pharmacology, Biochemistry and Behavior 75 (2003) 529–536
Bilkiei-Gorz et al., 1998). The observation that CT
increased time spent in the lit box is in congruence with
these studies.
An imbalance between excitatory and inhibitory neurotransmitters is responsible for seizures. Many drugs that
increased the brain content of GABA have exhibited anticonvulsant activity against seizures induced by MES and
PTZ. The MES is probably the best validated method for
assessment of antiepileptic drugs in the generalized tonicclonic seizures (Fisher, 1989; Loscher et al., 1991). The
PTZ-induced seizures are similar to the symptoms observed
in the absence seizures, and the drugs useful in the treatment
of absence seizures suppress PTZ-induced seizures (McNamara, 1996). CT significantly delayed the onset of convulsions in PTZ-induced convulsions and also delayed the
duration of tonic hindlimb extension in MES-induced convulsions. These observations suggest possibility of usefulness of CT in treatment of seizures.
Existence of cognitive problem in depressive illness is
well known (Allain et al., 1990). Amitriptyline and imipramine have anticholinergic effects and this may attribute to
the most adverse effects on memory. Because of increasing
incidence of Alzheimer’s disease and depression, there is a
need for developing an antidepressant that could be useful in
Alzheimer’s disease and depression. A disturbed receptor
balance could be contributing to the symptomatology of
depression and cognitive impairment (Berendsen, 1995;
Meneses and Hong, 1997). Fluoxetine, a selective serotonin
reuptake inhibitor, reduces total duration of immobility.
Both clinical and animal studies have strongly implicated
a critical role of 5-HT1A, 5-HT1B and 5-HT2A receptors in
antidepressant response (Barnes and Sharp, 1999). Total
duration of immobility was decreased by CT. In addition,
CT did not produce sedation and behavioral toxicity but
rather improved cognitive abilities.
Stress is known to alter the physiological homeostasis of
the organism. Stress elicits various endocrinal and visceral
changes including gastric mucosal integrity (Ander, 1984).
Stress increases brain serotonin level (Bhattacharya and
Bhattacharya, 1982) and leaves a lasting imprint on cognitive behavior (Jaiswal and Bhattacharya, 1993). Ray et al.
(1991) have shown that peripheral and central injections of
diazepam attenuate several stress responses like gastric
ulcerogenesis. CT decreased ulcer index dose-dependently
and showed antistress activity.
The increase in serotonergic transmission can interfere
with learning acquisition and memory consolidation (Ichihara et al., 1993; Arnsten et al., 1997; Barnes et al., 1990).
Lithium-induced head twitches are due to increased formation of serotonin in the CNS (Wielosz and Kleinrok, 1979).
Both piracetam and CT reduced the head twitches significantly, and CT at the same dose exhibited increased IR in
EPM, suggesting a link between cognitive improvement and
decreased serotonergic transmission by CT.
It is well known that amphetamine, which markedly
augments central noradrenergic activity, leads to mental
535
confusion and retards memory consolidation. The amnesic
effect of electroconvulsive shock, which is attenuated by
piracetam, is known to produce marked increase in the
turnover of rat brain NA (Bhattacharya et al., 1989).
Clonidine, a presynaptic a-adrenoceptor agonist, induces
hypothermia by reducing noradrenergic release (Drew et al.,
1977). Piracetam and CT failed to reverse clonidine-induced
hypothermia, indicating that noradrenergic mechanism was
not involved in the central effects of CT.
Sodium nitrite is known to convert hemoglobin into
methemoglobin, thereby reducing oxygen-carrying capacity
and cholinergic transmission and ultimately leading to
death (Hock, 1993). Piracetam and CT failed to decrease
the effect of sodium nitrite. This indicated that CT did not
increase the cholinergic transmission in the CNS. Anticholinergics are useful as initial drugs in Parkinson’s disease,
but they deteriorate cognitive behavior in Alzheimer’s
disease. This is the most important observation of the
present study because the modern medicine does not have
any drug that would be useful in treatment of Alzheimer’s
disease and Parkinson’s disease simultaneously. This
strongly suggests that there can be a category of drugs
useful in Alzheimer’s disease and Parkinson’s disease.
Thus, plants can provide a drug that would be useful in
these diseases.
The striatum, the ventrorostral region and the nucleus
accumbens septi have been implicated as the major brain
structures involved in the antipsychotic-induced catalepsy
(Duvoisin, 1967). Antipsychotic-induced catalepsy appears
to be due to blockade of DA neurotransmission (Janssen,
1965; Carlesson, 1990; Seeman, 1980). In the present study,
CT did not significantly increase haloperidol-induced catalepsy. Brain DA level was found to decrease after administration of oil of C. paniculatus (Nalini et al., 1995). This
suggests that CT does not significantly alter DA levels in the
substantia nigra. Thus, in conclusion, the extract was found
to possess nootropic, anxiolytic, antistress and anticonvulsant activities. The present study is based on behavioral
effects, and specific binding studies and measurement of
neurotransmitter levels in freely moving animals are necessary to understand the mode of action and suitability of the
extract for human use.
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