Pharmaceutical Biology 1388-0209/00/3801-0051$15.
00
2000, Vol. 38, No. 1, pp. 51–56
INFLUENCE OF CLITORIA TERNATEA EXTRACTS ON MEMORY AND
CENTRAL CHOLINERGIC ACTIVITY IN RATS
A.D. Taranalli* and T.C. Cheeramkuzhy
Department of Pharmacology, K.L.E.S. College of Pharmacy, J.N. Medical College Campus, Nehru Nagar,
Belgaum-590 010, Karnataka, India
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ABSTRACT
Clitoria ternatea, commonly known as Shankpushpi, is
widely used in the traditional Indian system of medi-
cine as a brain tonic and is believed to promote mem-
ory and intelligence. We examined the effectiveness of
alcoholic extracts of aerial and root parts of C. ter-
natea at 300 and 500 mg/kg doses orally in rats in
attenuating electroshock-induced amnesia. Extracts at
For personal use only.
300 mg/kg dose produced significant memory reten-
tion, and the root parts were found to be more effec-
tive. In order to delineate the possible mechanism
through which C. ternatea elicits the anti-amnesic
effects, we studied its influence on central cholinergic
activity by estimating the acetylcholine content of the
whole brain and acetylcholinesterase activity at dif-
ferent regions of the rat brain, viz., cerebral cortex,
midbrain, medulla oblongata and cerebellum. Our
results suggest that C. ternatea extracts increase rat
brain acetylcholine content and acetyl cholinesterase
activity in a similar fashion to the standard cerebro
protective drug Pyritinol.
INTRODUCTION
Clitoria ternatea Linn. (Papilionaceae) is a common
garden plant known as Shankpushpi. It is a pretty
perennial twiner with conspicuous white or blue flow-
ers, and seeded pods. In the present study, only the
white flowered variety was used for extraction. Shank-
pushpi has been used in hectic fever, gonorrhea, irrita-
Keywords: Acetylcholine, acetylcholinesterase, anti-amnesic,
central cholinergic activity, clitoria ternatea, memory.
Address correspondence to: A.D. Taranalli, Dept. of Pharma-
cology, K. L. E. S’s College of Pharmacy, J. N. Medical Coll-
ege Campus, Nehru Nagar, Belgaum–590 010, Karnataka,
India.
© Swets & Zeitlinger
tion of bladder and urethra, in tuberculous glands,
asthma and chronic bronchitis. It is also used for its
analgesic, anthelmintic, gastro-protective and aphro-
disiac effects (Nadakarni, 1976). Clitoria ternatea is
widely grown in southern India. Though studies have
been carried out to demonstrate its local anaesthetic,
tranquillising and hypothermic effects (Kulkarni et al.,
1988) the anti-amnesic effects of this plant have not
been investigated.
Cognitive dysfunction and memory loss are associ-
ated with various psychiatric and neurodegenerative
disorders such as Alzheimer’s disease, epilepsy, Pick’s
disease, Down’s Syndrome, and AIDS (Reddy, 1997).
Many drugs in current therapy like anticholinergics,
benzodiazepines, narcotics and neuroleptics produce
cognitive dysfunction as a side-effect (Meador, 1998).
A variety of pharmacological agents like piracetam
and selegiline, have been used to ameliorate memory
deficits. However, they have not proved clinically use-
ful (Kuruvilla et al., 1994).
There is extensive evidence linking the central
cholinergic system to memory (Ghelardini et al., 1998;
Peng et al., 1997; Olney, 1990). Cognitive dysfunction
has been shown to be associated with impairment of
cholinergic function and the facilitation of central
cholinergic activity improves memory and learning
(Bhattacharya et al., 1993). Selective loss of choliner-
gic neurons and decrease in cholineacetyltransferase
activity was a characteristic feature in senile dementia
of the Alzheimer’s type (Agnoli et al., 1993).
Subjecting an animal to electroshock treatment soon
after the acquisition of a passive avoidance response
causes its disruption with concomitant reduction in brain
acetylcholine levels (Poschel et al., 1983; Bhattacharya
et al., 1993). In this study, we employed a passive avoid-
ance response paradigm for evaluating the memory in
rats. The reversal or attenuation of passive avoidance
 52 A.D. TARANALLI AND T.C. CHEERAMKUZHY
after electroshock treatment was tested. Cholinergic
activities, acetylcholine (Ach) content and acetyl-
cholinesterase (AchE) in the brains were determined.
Pyritinol (pyrithioxine), an enhancer of cerebral
cholinergic transmission (K.D., Tripati, 1999) that is
marketed as a nootropic agent, was used as a standard
for comparison.
MATERIALS AND METHODS
Plant Material
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Alcoholic extracts of aerial parts containing leaves,
stem, flowers and root parts of Clitoria ternatea (white
flower var.) from identified plants collected from its
natural habitats in Central Kerala were supplied by the
Dept. of Research and Development, Nagarjuna Herbal
Concentrates Ltd. Thodupuzha, Kerala. The dry roots
and aerial parts were extracted by using ethyl alcohol
(95%) at 65°C with herb extract ratio of 6.4:1 for root
extract and 11:1 for aerial parts. Further taxonomic
identification was conducted by Prof. T.R. Rajan, Head
of Dept. of Pharmacognosy, R. L. S. College, Belgaum.
For personal use only.
Chemicals
Pyritinol (Encephabol® suspension) (E. Merck India
Ltd.), acetylcholine chloride (Sigma), gallamine tri-
ethiodide (Sigma), eserine sulphate (Sigma), 5,5-dithio
-bis 2–nitro benzoic acid, (Ellman’s reagent; (Sigma),
acetylthiocholine iodide (Sigma), trichloroacetic acid
(S. D. Fine Chemicals, Bombay), and ingredients of
frog ringer solution were obtained from Nice Chemical
Co. Cochin.
Animals
Adult albino Wistar rats of both sex, weighing between
160–190 g were used for the study. Rats were experi-
mentally naive and were 90–110 days old at the start of
the experiment. Swiss Albino mice used for toxicity
study were obtained from the experimental animal
house, Dept. of Livestock Production and Manage-
ment, Govt. Veterinary College Hebbal, Bangalore.
They were housed in clean acrylic cages with standard
pellet chow and water ad libitum. Frogs (Rana tigrina)
whose rectus abdominis muscle was used for bioassay
of acetylcholine (Perry 1970), were obtained from cen-
tral animal facility, J. N. Medical College, Belgaum.
Toxicity Study
Both the extracts were administered orally to different
groups of mice in doses ranging from 200–3000 mg/kg.
There was no lethality in any of the groups. Mice which
received extracts in doses above 2000 mg/kg exhibited
ptosis (dropping of upper eyelids) and were found
lethargic. A characteristic observation was the cathar-
tic effect indicated by profuse watery stools in all the
root extract treated mice. 1/10th to 1/6th of the maxi-
mum dose of the extract used for testing LD50 were
chosen for the present study, i.e., 300 and 500 mg/kg.
Grouping of Rats for the Experiment
Seven groups of rats, each consisting of 12 rats, were as
follows:
Group 1–Normal rats, which received a sham electro-
shock and no drug.
Group 2–Electroshocked rats (control) received dis-
tilled water (vehicle).
Group 3–Standard drug treated rats (pyritinol 54
mg/kg)
Group 4–C. ternatea aerial part extract 300 mg/kg.
Group 5–C. ternatea aerial part extract 500 mg/kg.
Group 6–C. ternatea root extract 300 mg/kg
Group 7–C. ternatea root extract 500 mg/kg.
Experimental Schedule
The rats of all groups were dosed once daily with the
respective drugs for seven days. The extracts were dis-
solved in water and administered through the oral
route. On the seventh day, 1 h after the last dosing, the
animals were conditioned in the experimental cham-
ber for passive avoidance. Once they acquire the pas-
sive avoidance response, they were subjected to
electroshock treatment, and 25 min later tested again
for retention of passive avoidance response. Of 12 rats
per group, 6 were used for the acetylcholine estima-
tion and 6 for determination of acetylcholinesterase
activity.
Screening Test for Memory (Conditional Avoidance
Response Paradigm)
Naive rats were placed on a wooden platform (8  8 
1 cm) fixed on the corner of an open chamber (30  30
 50 cm), having an electrifiable grid floor. When the
rats stepped off the platform, they received a continuos
foot shock of 10 V (8 mA) from the grid floor. The nor-
mal reaction of the rats was to jump back to the wooden
platform. Whenever the rats stepped down from the
platform, they received this aversive foot shock. After
about 4–5 trials, the rats acquired the passive avoidance
response and they refrained from stepping down. The
criterion was reached when the animals remained on
the platform for at least 60 sec.
 INFLUENCE OF CLITORIA TERNATEA EXTRACTS ON MEMORY 53

Five minutes later the control and test rats received
an electroshock (30 mA for 0.3 sec) through a pair of
silver ear electrodes from an Electroconvulsiometer
and the group one rats received a sham electroshock,
being subjected to the application of electrodes, with-
out receiving a shock. After 25 min, each rat was
placed back on the wooden platform and tested again
for their step down latency, keeping the time period of
60 sec as the criterion. The percentage of animals
matching the above criterion were determined in each
group (Bhattacharya et al., 1993).
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homogenized in a tissue homogenizer using 20 mg/ml
of phosphate buffer, pH 8.0. Reaction mixtures con-
tained 0.4 ml of homogenate, 2.6 ml of phosphate
buffer, 100 l of Ellman’s reagent, and substrate
acetylthiocholine iodide 20 l was added. The change
in optical absorbance was measured every minute at
412 nm in a Jasco 530 UV VIS spectrophotometer to
provide a measure of enzyme activity (Ellman et al.,
1961).
Statistical Analysis
Values of rat brain acetylcholine content and acetyl-

Estimation of Rat Brain Acetylcholine cholinesterase activity of different groups were

Rats were decapitated 30 min after the electroshock
treatment. The brain was removed and placed on ice.
Acetylcholine was extracted into 10% ice cold
trichloroacetic acid according to the modified proce-
dure of the McIntosh and Perry (1951). The concentra-
tion of the acetylcholine in the extract was determined
by bioassay on frog Rectus abdominis muscle (Perry,
1970). Eserinised (10–6 M) Frog ringer was used as the
physiological solution. The contraction of the rectus
(skeletal) muscle was confirmed to be due to acetyl-
For personal use only.
analyzed using one-way analysis of variance (ANOVA)
followed by Dunnet’s test for individual comparison of
groups with control.
RESULTS
Dosage
The aerial and root parts of Clitoria ternatea were eval-
uated for their anti-amnesic activity along with their

choline from the extract, by demonstrating its compet-
itive reversible antagonism with gallamine. The
concentration of the acetylcholine in the extract was
calculated from the bioassay tracing by the method of
equivalence, equating the height of contraction pro-
duced by a known concentration of standard acetyl-
choline with the height produced by a known volume of
the extract.
Determination of Acetylcholinesterase Activity in
Different Parts of Rat Brain
The cerebral cortex, midbrain, medulla oblongata and
cerebellum were dissected on ice as described by
Glowinsky and Iversen (1966), suspended in phosphate
buffer and weighed. The different parts of brain were
influence on rat brain acetylcholine content and acetyl-
cholinesterase activity in various parts of the brain. C.
ternatea aerial and root extracts, up to 3000 mg/kg by
the oral route, failed to produce any lethality in mice.
However, animals showed signs of central nervous sys-
tem depression indicated by ptosis and decreased loco-
motor activity at doses 1500 mg/kg and above. All the
mice which received the extracts in the dose 2900
mg/kg and above through the intraperitoneal route died
in 6 h, due to severe CNS depression. On the basis of
the above data, the extracts of C. ternatea aerial parts
and root parts were tested for anti-amnesic activity in
the dose of 300 and 500 mg/kg, which are 1/10th and
1/6th of the maximum dose tested for toxicity. All the
drugs were administered through the oral route.

Table 1. Effect of C. ternatea on memory, Ach content and AchE activity.

Treatment Dose % of rats Acetylcholine content

retaining memory in g/g of brain AchE activity in nmol/min/g of tissue (n6) mean  S.D.
n  12 (n6) mean  S.D.
Cortex Midbrain Medulla Cerebellum

Control (ve)  0 1.70  0.09 2.17  0.06 3.00  0.15 3.03  0.30 1.16  0.04
Normal  100 2.07  0.02* 2.96  0.29 3.96  0.05* 3.33  0.22* 3.33  0.22
Pyritinol 54 mg/kg 91.33 1.89  0.05* 2.43  0.04 5.18  1.03* 3.04  0.78 1.48  0.11
C. ternatea aerial 300 mg/kg 66.66 1.79  0.15* 2.89  0.22* 3.21  0.54* 2.61  0.30 1.21  0.13
C. ternatea aerial 500 mg/kg 50 1.76  0.09 2.05  0.07 2.77  0.01 2.44  0.44 1.10  0.17
C. ternateaa root 300 mg/kg 83.33 1.81  0.02 * 1.99  0.23 2.13  0.13 2.17  0.01 1.03  0.21
C. ternatea root 500 mg/kg 83.33 1.80  0.07* 2.84  0.16* 2.28  0.36 1.98  0.36* 1.36  0.29

* Dunnets test P  0.05

 54 A.D. TARANALLI AND T.C. CHEERAMKUZHY
Step Down Latency, Brain Ach Content and AchE
Activity of Normal Rats
All of the rats (100%) acquired the passive avoidance
response as per the criterion in the paradigm, after
training. The sham electroshocked normal rats of group
one showed step down latency more than 60 sec, which
indicated the retention of passive avoidance response or
memory. The Ach content of normal rats was 2.07 
0.02 g/g of brain. AchE activity in the cortex, mid-
brain, medulla oblongata and cerebellum were, respec-
tively, 2.13  0.28, 3.96  0.05, 3.33  0.22, and 1.22
 0.09 nmol/min/g.
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Effects of Electroshock on Step Down Latency,
Brain Ach Content and AchE Activity of Rats
The group 2 (control) rats were subjected to elec-
troshock of 30 mÅ for 0.3 sec, five min after training.
This induced clonic-tonic convulsions lasting approxi-
mately for one minute, which totally disrupted the
acquired passive avoidance response, with 100% of the
trained rats stepping down from the platform within 60
sec, when tested 25 min later. This indicated the loss of
short term memory with electroshock treatment.
For personal use only.
The acetylcholine content of the normal
sham–electroshocked rats were significantly more
(2.07  0.02) as compared to electroshock treated
control group. The AchE activity of normal rats was
significantly increased in mid brain and medulla
oblongata (3.96  0.05 and 3.33  0.22) as compared
electroshock treated control group. However, there
was no change in the AchE activity of cerebral cortex
and cerebellum.
Effect of Aerial Parts of C. ternatea on Step Down
Latency, Brain Ach Content AchE Activity of
Electroshocked Rats
The C. ternatea aerial parts at the dose of 300 mg/kg
induced an inhibition of electroshock induced disrup-
tion of passive avoidance, with 66.66% of the animals
retaining their memory as compared to 0% in the con-
trol. The Ach content also was significantly increased
(1.79  0.15 g/g of brain).
The AchE activity was increased in the cortex and
midbrain (2.89  0.22 and 3.21  0.5 nmol/min/g,
respectively) but there was no significant change in
medulla oblongata and cerebellum.
Aerial parts of 500 mg/kg produced memory reten-
tion in only 50% of the tested animals which was less
compared to 300 mg/kg dose. The change in brain Ach
content and AchE activity was not significant.
Effect of Root Parts of C. Ternatea on Step Down
Latency, Brain Ach Content and AchE Activity of
Electroshocked Rats
The root parts at 300 mg/kg produced retention of
memory in 83.33% of the animals as compared to 0%
in electroshocked control. The Ach content was also
significantly increased (1.813  0.02 g/g of brain).
The AchE activity in midbrain and medulla oblongata
was decreased but the decrease was not significant.
There was no change in cerebral cortex and cerebellum
AchE activities. Roots parts at 500 mg/kg produced
retention of memory in 83.33% of the tested animals
which was equal to 300 mg/kg. The Ach content of the
brain was significantly increased: 1.80  0.07 g/g of
brain. The AchE activity was significantly increased in
the cerebral cortex (2.843  0.16 namol/min/g), sig-
nificantly decreased in medulla oblongata (1.98  0.10
nmol/min/g) and slightly decreased in midbrain, with
no change in cerebellum.
Effect of Pyritinol (Standard Drug) on Step Down
Latency, Ach Content of Brain and AchE Activity
of Electroshocked Rats
Pyritinol, in a dose 54 mg/kg, produced memory reten-
tion in 91.66% of the animals; Ach content of the
brains were increased significantly compared to control
(1.89 g/g of brain). AchE activity did not change sig-
nificantly in cerebral cortex, medulla oblongata and
cerebellum. However, there was a highly significant
increase in AchE activity in the midbrain (5.18  1.03
nmol/min/g).
DISCUSSION
Cholinergic modulation of memory is now well
accepted, based on extensive experimental and clinical
data. Disruption of the passive avoidance response by
electroshock with a concomitant reduction in brain
acetylcholine levels, is in keeping with this hypothesis.
In the present study, the anti-amnesic effect of C. ter-
natea in relation to its central cholinergic activity was
studied. The electroshock induced amnesia model was
used in the present study, which produces retrograde
amnesia, interfering with memory consolidation, with
concomitant reduction in brain acetycholine levels.
In the present study, the electroshock treatment sig-
nificantly decreased step down latency and the Ach con-
tent of brains in control rats. Our result supports the
earlier reports that electroshock treatment disrupts
 INFLUENCE OF CLITORIA TERNATEA EXTRACTS ON MEMORY
memory and decreases brain acetylcholine levels
(Poschel et al., 1983; Bhattacharya et al., 1993). AchE
activity was significantly decreased in midbrain and
medulla oblongata suggesting that the Ach decrease cor-
relates well with AchE activity decrease, especially in
midbrain and medulla oblongata, where the cholinergic
activity is preponderantlly existing (Rang & Dale, 1996).
The aerial parts produced significant retention of
memory, and also increased the Ach content of brain.
This confirms the beneficial effect of the aerial parts
300 mg/kg of C. ternatea in amnesia, and also its rela-
tionship with cholinergic activity. Again, the AchE
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activity was also increased parallel to increased acetyl-
choline content.
Surprisingly, at 500 mg/kg, the aerial parts produced
memory retention only in 50% of animals, compared to
66.66%, 300 mg/kg, and there was no change in Ach
content, and AchE activity in general, which again sug-
gests a link between amnesia and cholinergic activity.
The root parts of Clitoria ternatea at 300 mg/kg pro-
duced memory retention in 83.3% of animals,
increased Ach content of brain, but did not affect the
AchE activity in general. However, the root parts at the
For personal use only.
higher dose of 500 mg/kg produced memory retention
equal to the 300 mg/kg dose. The Ach content was also
increased, whereas the AchE activity was increased in
cortex significantly.
From this study, we observe that there was a good
correlation of loss of memory and loss of cholinergic
activity which includes both Ach content and AchE
activity. We also observe that retention of memory was
associated in general with increased Ach content and
AchE activity. However, there are exceptions in our
results to the above statement where increase in mem-
ory is not associated with the increase in AchE activity.
The relationship between Ach content and AchE activ-
ity is controversial. Madepalli et al. (1994) reported
that Ach content and AchE activity have an inverse
relationship, whereas Agnolli et al. (1993) reported that
Ach content and AchE activity were decreased in senile
dementia, suggesting a direct relationship between
them. In the present study, we have measured the Ach
content of the whole brain and AchE activity of differ-
ent parts of brain, and this may be the reason for some
exceptions observed in our study.
The standard drug pyritinol produced memory reten-
tion in 91.6% of animals, increased the whole brain Ach
content, AchE activity of mid brain very significantly.
The standard drug pyritinol’s, effect on Ach content
and AchE activity, clearly established beyond doubt,
55
the relationship between memory and cholinergic activ-
ity of the brain.
To conclude, root parts of C. ternatea were found to
be more effective in attenuating memory deficits as
compared to aerial parts, and the mechanism by which
C. ternatea produced memory retention appears to be
similar to the standard drug pyritinol, since aerial parts,
root parts and pyritinol have similar influence on
cholinergic activity of the brain.
ACKNOWLEDGEMENT
We thank Dr. F.V. Manvi Principal KLE’s College of Phar-
macy for providing necessary facilities and the management
of Nagarjuna Herbal Concentrates Ltd. Thodpuzha, Kerala
for providing the extracts and financial help.
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Accepted: July 27, 1999