Analgesic and Neuropharmacological Activities of Methanolic Leaf Extract of Clitoria ternatea Linn.

Abstract

The present study was carried out on methanolic extract of Clitoria ternatea Linn. for pharmacological investigations. In this study, analgesic activities of the methanolic extract of Clitoria ternatea Linn. leaves were examined at the doses of 200 mg/kg and 400 mg/kg of body weight on mice. The analgesic activities were investigated using acetic acid induced writhing test. The plant extract’s Central Nervous System (CNS) depressant activity was evaluated by using hole cross and open field tests. Acetic acid induced writhing test revealed that the extract at the lower dose inhibited 82.67% and at the higher dose produced a maximum of 87.87% inhibition of writhing that is comparable to the reference drug Diclofenac Sodium. The results of CNS depressant activity showed that the extract decreased the dose dependent motor activity and exploratory behavior of mice in hole cross and open field test. The number of field crossed in open field test and hole crossed in hole cross test decreased as time approached.

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Journal of Pharmacognosy and Phytochemistry 2014; 2 (5): 110-114
ISSN 2278-4136
JPP 2014; 2 (5): 110-114
Received: 28-11-2013
Accepted: 25-12-2013
Shammy Sarwar
Laboratory of Pharmacognosy &
Pharmacology,
Department of Pharmacy, Stamford
University Bangladesh, Dhaka-1217,
Bangladesh.
Md. Rashidur Rahman
Department of Pharmacy,
Jessore University of Science &
Technology, Jessore-7408,
Bangladesh.
Kamrun Nahar
Department of Pharmacy, Stamford
University Bangladesh, Dhaka-1217,
Bangladesh.
Muhammad Ashikur Rahman
Department of Pharmacy,
Jahangirnagar University, Savar,
Dhaka-1342, Bangladesh.
Correspondence
Md. Rashidur Rahman
Department of Pharmacy,
Jessore University of Science &
Technology, Jessore-7408,
Bangladesh.
E-mail: sumon_pharmacy@yah oo.co.uk;
Tel: +8801727510926
Analgesic and Neuropharmacological Activities of
Methanolic Leaf Extract of Clitoria ternatea Linn.
Shammy Sarwar, Md. Rashidur Rahman, Kamrun Nahar, Muhammad Ashikur Rahman
Abstract
The present study was carried out on methanolic extract of Clitoria ternatea Linn. for
pharmacological investigations. In this study, analgesic activities of the methanolic extract of Clitoria
ternatea Linn. leaves were examined at the doses of 200 mg/kg and 400 mg/kg of body weight on
mice. The analgesic activities were investigated using acetic acid induced writhing test. The plant
extract’s Central Nervous System (CNS) depressant activity was evaluated by using hole cross and
open field tests. Acetic acid induced writhing test revealed that the extract at the lower dose inhibited
82.67% and at the higher dose produced a maximum of 87.87% inhibition of writhing that is
comparable to the reference drug Diclofenac Sodium. The results of CNS depressant activity showed
that the extract decreased the dose dependent motor activity and exploratory behavior of mice in hole
cross and open field test. The number of field crossed in open field test and hole crossed in hole cross
test decreased as time approached.
Keywords: Clitoria ternatea Linn., Open Field Test, Hole Cross Test, Analgesic and Neuropharmacological
Activities.
1. Introduction
Clitoria ternatea Linn. is a strangling and climbing herb with a strong base, ovate-oblong leaves
and beautiful blue flowers, has been grown as an ornamental plant in the garden. This plant
belongs to the family Papilionaceae and is commonly known as Butterfly pea plant in English
and locally known as Aparajita, Nila in Bengali [1]. Clitoria ternatea Linn. is a vine native to
tropical and equatorial Asia, but has been introduced to Africa, Australia and the rest.
Availability in India is either as wild or cultivated plant. The rest worlds are Egypt, Syria, Iraq,
Iran, Afghanistan etc. It grows well in moist neutral soil and requires little care. It is grown as a
revegetation species. It fixes nitrogen and is therefore also used to improve soil quality. It
requires summer rainfall of 500 mm over 3 months but grows best between 700-1,500 mm
AAR. This plant is drought tolerant and will survive in years which have only 400 mm rainfall
and a dry season of 5-6 months or longer even if heavily grazed. The useful parts are leaf, root,
bark, seeds and flowers. The plant used in colic gonorrhea and skin disease. Root is used as
laxative and demulcent, aperients. Roasted and powdered seeds are used in the treatment of
ascites, enlargement of abdominal viscera, weakness of sight, sore throat, tumors, and dropsy
and skin diseases [2]. It has been claimed to have antihyperglycaemic effect in rats with alloxan-
induced diabetes mellitus [3]. This plant has also been studied to find out antibacterial activities
[4]. An experiment showed that Clitoria ternatea Linn (Fabaceae) root possesses significant anti-
diarrheal activity [5]. Anti-oxidant activities has been proven by Clitoria ternatea Linn. extract in
in-vitro method [6]. Plant have been used for the treatment of diseases all over the world before
the advent of modern clinical drugs and are known to contain substances that can be used for
therapeutic purposes or as precursors for the synthesis of useful drugs [7]. Thus over 50% of
these modern drugs are of natural products origin and as such these natural products play an
important role in the drug development in the pharmaceutical industry [8]. Therefore, we have
been done this investigation to know about analgesic and Neuropharmacological activities of
Clitoris ternatea Linn. extract in animal behavioral model.

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2. Materials and Methods
2.1 Collection and Identification of plants
The leaves of Clitoria ternatea Linn. were collected from Boldha
Garden, Dhaka, Bangladesh and were identified by the expert at
the Bangladesh National Herbarium, Mirpur, Dhaka where the
Voucher specimen number 35202 has been deposited. The
collected plant parts were separated and dried for one week.
2.2 Preparation of Methanolic Extract
The dried leaves of Clitoria ternatea Linn. were coarsely powered
by a milling machine and extracted with a mixture of methanol:
water (7/3, v/v) by Soxhlet apparatus at 50 oC for 72 hours. When
the powders became exhausted of its chemical constituents as
evident from cycles of colorless liquid siphoning in the Soxhlet
apparatus, extraction was considered to be complete. After
completion of the extraction from leaves of Clitoria ternatea
Linn., the extracts were filtered using a sterilized cotton filter.
Then solvent was completely removed and obtained dried crude
extract which were used for investigations.
2.3 Animals
Swiss-Albino mice of either sex (20-25 gm body weight) were
collected from animal resources branch of the International
Center for Diarrheal Disease Research, Bangladesh (ICDDR, B)
and used for the experiments. The animal were kept in the
standard polypropylene cages and provided with standard diets
(ICDDR, B formulated). The animals were acclimatized in animal
house (Department of Pharmacy, Stamford University
Bangladesh) under standard laboratory conditions (relative
humidity 55-60%, room temperature 25±2 °C and 12 hours light:
dark cycle) for period of 14 days prior to performing the
experiments [9]. All experimental protocols were approved by the
Institutional Ethics committee of the Stamford University
Bangladesh.
2.4 Chemicals and drugs
All chemicals and drugs were obtained commercially and were of
analytical grade. Diclofenac Sodium and Diazepam was collected
from Square Pharmaceuticals Ltd., Bangladesh. Acetic acid was
purchased from Merck, Germany.
2.5 Analgesic activity test of the extract of Clitoria ternatea
Linn
The study of analgesic activity of the extract of Clitoria ternatea
Linn. was performed in animal models. For the screening of
analgesic activity against peripheral mechanism of pain, acetic
acid induced writhing test was considered.
2.5.1 Acetic acid induced Writhing test
The antinociceptive activity of the samples was studied using
acetic acid-induced writhing model in mice [9, 10]. The animals
were divided into control, positive control and test groups with
five mice in each group. The animals of test groups received test
samples at the doses of 200 and 400 mg/kg body weight. Positive
control group received standard drug Diclofenac Sodium at the
dose of 10 mg/kg body weight and vehicle control group was
treated with 1% Tween 80 in water at the dose of 10 ml/kg body
weight. Test samples and vehicle were administered orally 30 min
before intraperitoneal administration of 0.7% acetic acid but
Diclofenac Sodium was administered 15 min before injection of
acetic acid. After an interval of 5 min, the mice were observed for
specific contraction of body referred to as ‘writhing’ for the next
10 min [11]. After inducing the plant extract and control every
mice of all groups was observed carefully to count the number of
writhing which made within 10 minutes.
2.6 Neuropharmacological activity test of Clitoria ternatea
Linn.
The effects of drugs on the CNS with reference to the
neurotransmitters for specific circuits, attenuation should be
developed to general organizational principles of neurons. The
concept is that synapses represent drug-modifiable control points
within neuronal networks. It requires explicit delineation of the
sites at which given neurotransmitters may operate and the degree
of specificity with which such site that may be affected [12]. Open
field and hole cross tests were performed to determine the
Neuropharmacological activity of Clitoria ternatea Linn. extracts.
2.6.1 Open Field Test
The Open Field Test is clearly the most frequently used of all
behavioural tests in pharmacology and neuroscience. Despite the
simplicity of the apparatus, however, open field behaviour is
complex. Consequently, it has been used to study a variety of
behavioural traits, including general motor function, exploratory
activity and anxiety-related behaviours. Open field behavioral
assays are commonly used to test both locomotor activity and
emotionality in rodents.
2.6.1.1 Experimental Design
In open field test, the animals were divided into control, positive
control, and test groups containing five mice each. The test
groups received methanolic extract at the doses of 200 and 400
mg/kg body weight orally whereas the control received vehicle
(1% Tween 80 in water). Animals in positive control group
received diazepam (1 mg/kg body weight). The floor of an open
field of half square meter was divided into a series of squares
each alternatively colored black and white. The apparatus had a
wall of 40 cm height. The number of squares visited by the
animals was counted for 3 min at 0, 30, 60, 90, and 120 min after
oral administration of the test drugs and the standard [13].
2.6.2 Hole Cross Test
The method was carried out as described by Takagi et al. (1971)
[14]. A steel partition was fixed in the middle of a cage having a
size of (30 × 20 × 14) cm. A hole of 3 cm diameter was made at a
height of 7.5 cm in the centre of the cage. The animals were
divided into control, positive control, and test groups containing
five mice each. The test groups received extract of 200 and 400
mg/kg body weight orally whereas the control and positive
control groups received vehicle (1% Tween 80 in water) and the
standard drug diazepam (1 mg/kg body weight) respectively. The
number of passage of a mouse through the hole from one chamber
to other was counted for a period of 3 min at 0, 30, 60, 90 and 120
min after oral administration of the test drugs and the standard.
3. Results and Discussion
3.1 Test for Analgesic Activity of Clitoria ternatea Linn.
3.1.1 Acetic Acid-Induced Writhing Test
The analgesic effect of the methanolic extract on acetic acid-
induced writhing in mice was determined. The extract
significantly inhibited writhing response induced by acetic acid in
a dose dependent manner. The result was comparable to the
reference drug Diclofenac Sodium.

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Table 1: Results of Acetic acid induced writhing test
Administered Substance SEM Mean ± SEM % of Inhibition
Control 2.12 40.4±2.12 0
Positive control 2.00 9±2.00 77.72
Group-1 1.60 7±1.60 82.67
Group-2 1.13 4.9±1.13 87.87
Values are expressed as Mean ± SEM (n=5)
Here, Control = 1% Tween 80 in Water (0.2 ml/Mouse), Positive Control = Diclofenac Sodium (10 mg/Kg), Group 1 and Group 2 (200 mg /Kg and 400 mg/Kg body weight
respectively)
Fig 1: Graphical Representation of effect of leaves parts of Clitoria ternatea Linn. on acetic acid induced writhing test in mice
3.2 Tests for Neuropharmacological activity
In both open field and hole cross tests, the extract significantly
decreased the locomotor activity of the mice (Table 2 and 3). The
locomotor activity lowering effect (depressant effect) was evident
at the 2nd observation time (30 min) and continued up to the 5th
observation period (120 min) at the doses of 200 & 400 mg/kg
body weight and the results were dose dependent, the maximum
being at the dose of 400 mg/kg body weight. In the open field
test, the number of squares travelled by the mice at all doses of
the extract (200 and 400 mg/kg body weight) was reduced
significantly from the initial score. The results were comparable
to those of the reference drug, Diazepam. The maximum
reduction was exhibited at 90 and 120 min after administration of
the drug.
Table 2: Effect of Clitoria ternatea Linn. extract on Hole Cross Test
Group Route of
Administratio
n
Observation
0 min 30 min 60 min 90 min 120 min
Positive
control Oral 15.4±0.91 6±0.94 2±0.79 1.6±1.04 1.2±0.42
Control Oral 17±1.97 17.8±1.92 17.6±2.28 17.2±1.56 18.4±0.91
Group-1 Oral 12.4±1.44 5.2±1.14 4±0.78 2.6±0.76 0.8±0.55
Group-2 Oral 8.6±1.26 5.4±0.76 2.2±0.65 0.8±0.65 0.4±0.27
Values are expressed as Mean ±SEM (n=5)
Here, Control = 1% Tween 80 in Water (0.2 ml/Mouse), Positive control = Diazepam (1 mg/Kg), Group 1 and Group 2 (200 mg /Kg and 400 mg/Kg
body weight respectively)

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Fig 2: Graphical Representation of effect of Clitoria ternatea Linn. on Hole cross test in mice
Table 3: Effect of Clitoria ternatea Linn. extract on Open Field Test
Group Route of
Administrati
on
Observation
0 min 30 min 60 min 90 min 120 min
Positive
control Oral 118.4±7.04 67.8±5.63 41.6±3.65 19.6±2.61 10.8±2.04
Control Oral 121.6±5.35 117.8±3.94 116.6±2.61 110.8±7.53 117.2±4.62
Group-1 Oral 48.2±10.3 23.8±7.9 19.0±7.6 13.4±5.3 7.2±2.9
Group-2 Oral 64.4±8.3 18.8±6.9 9.8±2.2 11±1.6 2±1.2
Values are expressed as Mean ±SEM (n=5)
Here, Control= 1% Tween 80 in Water (0.4 ml/Mouse), Positive control = Diazepam (1 mg/Kg), Group 1 and Group 2 (200 mg /Kg and 400 mg/Kg body
weight respectively)
Fig 3: Graphical Representation of effect of Clitoria ternatea Linn. on Open field test in mice
The present study has established analgesic potential of Clitoria
ternatea Linn. using acetic acid-induced writhing test for visceral
pain. Acetic acid-induced writhing in mice is a model of visceral
pain which is highly sensitive and useful for screening
peripherally acting analgesic drugs. Clitoria ternatea Linn. plant
caused dose-dependent antinociception against chemical induced
pain in mice. Methanolic extract of the leaves parts of Clitoria
ternatea Linn. were treated in test animals at a dose of 200 & 400
mg/kg body weights. The leaves parts of Clitoria ternatea Linn.
extracts at the dose of 400 & 200 mg/kg body weight
respectively, was found to exhibit the highest (87.87%) &
(82.67%) writhing response inhibitory effect respectively, where
the reference drug Diclofenac Sodium shown about 77.72%
writhing inhibitory response at a doses of 10 mg/kg. Acetic acid-
induced writhing method is not only simple and reliable but also
affords rapid evaluation of peripheral type of analgesic action.
This model represents pain sensation by triggering localized
inflammatory response. Such pain stimulus leads to the release of

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free arachidonic acid from tissue phospholipids [15]. So, the
observed analgesic activity may be attributed to these compounds.
An important step in evaluating drug acting on CNS is to observe
its effect on locomotor activity of the animal. The activity is a
measure of the level of excitability of the CNS and this decrease
may be closely related to sedation resulting from depression of
the central nervous system [16]. The extracts significantly
decreased the locomotor activity as shown by the results of the
open field and hole cross tests. The locomotor activity lowering
effect was evident at the 2nd observation (30 min) and continued
up to 5th observation period (120 min). Both hole cross and open
field tests showed that the depressing acting of the extracts was
evident from the 2nd observation period in the test animals at the
doses of 200 & 400 mg/kg body weight. Maximum depressant
effect was observed from 3rd (60 min) to 5th (120 min)
observation period. From the result this is observed that, Clitoria
ternatea Linn. has CNS depressant activity by using both open
field & hole cross tests, which is comparable to the reference drug
Diazepam at doses of 1 mg/kg.
4. Conclusion
Based on the results of the present study, it can be concluded that
the leaves parts of crude methanolic extract of Clitoria ternatea
Linn. possesses remarkable CNS depressant and analgesic
potential in animal behavioral model. Hence, further studies are
suggested to be undertaken to pinpoint the exact compounds and
to better understand the mechanism of such actions.
5. Acknowledgement
The authors are thankful to Chairman, Department of Pharmacy,
Stamford University Bangladesh, Dhaka for providing laboratory
facilities to carry out analgesic and Neuropharmacological studies
with necessary reagents and animals.
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