Pharmacological Study

Antidiarrheal activity of Clitorea ternatea Linn. (Fabaceae)

ethanol leaf extract in rats
Ramdas Bhanudas Pandhare, Sangameswaran Balakrishnan1, Gaurav Dnyandeo Bangar2, Pramod Dnyandeo Dighe2, Vinayak Kashinath Deshmukh3
Associate Professor and Head Department of Pharmacology, 2Student, 3Principal and Professor, MES’s College of Pharmacy, Sonai, Newasa, Ahmednagar,
Savitribai Phule Pune University, Pune, Maharashtra, 1Principal and Professor, S.S.M. College of Pharmacy, Chinniampalayam, Jambai, Bhavani, Tamil Nadu, India

Abstract
Background: Clitorea ternatea Linn. (Fabaceae) is a plant which is traditionally used for the treatment of wide range of pharmacological
activities including antimicrobial, antipyretic, anti‑inflammatory, analgesic, diuretic, local anesthetic, antidiabetic, insecticidal, blood
platelet aggregation‑inhibiting and diarrhea in India. However, scientific evidence does not exist in any literature to corroborate the claim
of therapeutic success of the plant species in diarrhea. Aim of the Study: The core aim of the present study is to evaluate the antidiarrheal
activity of C. ternatea ethanol extract (CTE). Materials and Methods: The antidiarrheal activity was evaluated using castor oil and magnesium
sulfate (MgSO4)‑induced diarrhea method. The effects of CTE on gastrointestinal motility, intestinal transit and enteropooling were also
examined in rats. Results: CTE (100–400 mg/kg, p.o.) produced dose‑dependent and significant (P < 0.05–0.01) protection of rats against
castor oil and MgSO4‑induced diarrhea, inhibited intestinal transit and delayed gastric emptying. CTE dose dependently and significantly
delayed the onset of castor oil and MgSO4‑induced diarrhea, decreased the frequency of defecation and reduced the severity of diarrhea in
the rats compared with loperamide (10 mg/kg, p.o.). Conclusion: These findings confirm the ethno medicinal use of C. ternatea as a valuable
natural remedy for the treatment, management and/or control of diarrhea.

Keywords: Castor oil, Clitorea ternatea, diarrhea, loperamide, magnesium sulfate

Introduction diarrhea in developing countries, especially the discomfort and

inconvenience of frequent bowel movements, the World Health
Since ancient times, diarrhea has been recognized as one
Organization has introduced a program for diarrheal control
of the most important health problems afflicting humanity,
which involves the use of traditional herbal medicines. Several
particularly those populations in socioeconomically backward,
African medicinal plants have been reported to be useful in the
developing and third world countries. Globally, diarrhea has
treatment, management and/or control of diarrhea.[7‑10]
been estimated to kill about 2.2 million people annually, the
majority of whom are infants and children below the age of Clitorea ternatea L. (Family: Fabaceae) is a perennial twing
5 years.[1] herb, stems are terete, more or less pubescent. The roots have
a sharp bitter taste and are useful in severe bronchitis, asthma,
Diarrhea is a disorder characterized by discharge of semisolid
inflammation, diarrhea and fever.[11,12] The fatty acid content
or watery fecal matter from the bowel three or more times
of C. ternatea seeds includes palmitic, stearic, oleic, linoleic
in a day.[2,3] It involves an increase in the fluidity, volume
and linolenic acids.[13] The seeds also contain beta‑sitosterol.[14]
and frequency of bowel movements, increased frequency of
C. ternatea possesses sedative action.[15] It enhances memory[16]
bowel sound, wet stools and abdominal pain, accompanied by
and increases acetylcholine content in rats.[17] Its nootropic,
increased secretion and decreased absorption of fluid and thus
loss of water and electrolytes.[4,5]
Address for correspondence: Dr. Ramdas Bhanudas Pandhare,
In general, the treatment of diarrhea is nonspecific and is Department of Pharmacology, MES’s College of Pharmacy, Sonai Affiliated
to Savitribai Phule Pune University, Pune ‑ 414 105, Maharashtra, India.
usually aimed at reducing the discomfort and inconvenience
E‑mail: ramdaspandhare83@rediffmail.com
of frequent bowel movements.[6] To overcome the menace of

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DOI: How to cite this article: Pandhare RB, Balakrishnan S, Bangar GD,
10.4103/ayu.AYU_19_17 Dighe PD, Deshmukh VK. Antidiarrheal activity of Clitorea ternatea
Linn. (Fabaceae) ethanol leaf extract in rats. Ayu 2018;39:40-5.

40 © 2018 AYU (An International Quarterly Journal of Research in Ayurveda) |

Official publication of Institute for Post Graduate Teaching & Research in Ayurveda, Jamnagar | Published by Wolters Kluwer - Medknow
 Pandhare, et al.: Antidiarrheal activity of Clitorea ternatea
anxiolytic, antidepressant, anticonvulsant, [18] antipyretic,
anti‑inflammatory and analgesic activities,[19] antidiabetic,[20]
antiasthamatic[21] and hepatoprotective[22] properties are also
reported. Since a systematic study of its antidiarrheal activity
has not been undertaken, this study was done to evaluate the
antidiarrheal activity of C. ternatea ethanol extract (CTE) in
rats.
Materials and Methods
Ethical considerations
Experimental procedures and protocols used in this study were
approved by the Institutional animal ethics committee (MES/
COP/IAEC/01/2010‑11) and the care of the laboratory animals
was taken as per the current committee for the purpose of
control and supervision of experiments on animals regulations.
Plant materials
Fresh leaves of C. ternatea L. (Fabaceae) were collected in
September 2010 from the Mahatma Phule Krishi Vidyapeeth,
Rahuri, Maharashtra, India and were authenticated by
Dr. J. Jayanti, Botanist, Botanical Survey of India (BSI), Pune.
A voucher specimen (BSI/WRC/Tech./2011/581) has been kept
in herbarium, BSI for future reference.
Preparation of plant extract
Fresh leaves of C. ternatea L. were washed with distilled water,
shed dried and later powdered. This powder was then defatted
with petroleum ether which was further macerated with ethyl
alcohol for 72 h with occasional shaking. It was then filtered
and evaporated. The yield of CTE was 3.6%  w/w. Purified
aliquot portions of the crude plant extract were weighed and
dissolved in distilled water (at room temperature) for use on
each day of the experiments.
Animals
Healthy adult, wistar rats (Rattus norvegicus) of both
(male/female) sexes weighing 150–200  g were obtained
from the animal house. All the animals were housed in clean
polypropylene cages placed in well‑ventilated house conditions
of temperature and humidity with a 12 h light/dark cycle.
They were allowed free access to food (standard pellet diet)
and drinking tap water ad libitum, except when fasting was
required during the study. The research was conducted in
accordance with the internationally accepted principles for
laboratory animal use and care.
Acute toxicity testing
The present study was conducted according to the organization
for economic cooperation and development (OECD) revised
fixed‑dose procedure for acute toxicity testing (OECD
guideline 420, 2001). Two groups of five healthy female
albino wistar rats  (3  months old, 150–200  g b. wt. were
administered a limit dose of 2000 and 5000 mg/kg of the CTE
and animals were observed for mortality and clinical signs for
the 1st hour, then hourly for 3 h and finally periodically until
48 h. All of the experimental animals were maintained under
close observation for 14 days and the number of rats that died
AYU | Volume 39 | Issue 1 | January-March 2018
within the study period were noted. The lethal dose (LD50)
was predicted to be above 2000 or 5000 mg/kg if three or
more rats survived.[23]
Evaluation of antidiarrheal activity of Clitorea ternatea
ethanol extract
Castor oil‑induced diarrhea in rats
Wistar albino rats were divided into five groups of six animals
(n = 6) each. All rats were fasted for 18 h and received castor
oil at a dose of 1 ml/animal orally (p.o.) using orogastric tubes
for induction of diarrhea.[24] Thirty minutes after castor oil
administration, rats of group I (control) received 1 ml/100 g
of 0.9% NaCl in distilled water  (normal saline), group  II
(reference) received standard drug, loperamide (3 mg/kg p.o.)
and rats of groups III, IV and V received 100, 200 and
400 mg/kg CTE, p.o., respectively. The animals were placed
separately in metabolic cages over white clean Whatman filter
paper, which was changed every hour. The severity of diarrhea
was assessed each hour for 4 h. The total number of diarrhea
feces of the control group was considered 100%.
Magnesium sulfate‑induced diarrhea
Wistar albino rats were divided into five groups of six animals
(n = 6) each. All rats were fasted for 18 h and received
magnesium sulfate (MgSO4) at a dose of 2 g/kg orally (p.o.)
using orogastric tubes for induction of diarrhea.[25] Thirty
minutes after MgSO4 administration, rats of group I (control)
received 1 ml/100 g of 0.9% NaCl in distilled water (normal
saline), group II (reference) received standard drug, loperamide
(3 mg/kg p.o.) and rats of groups III, IV and V received 100,
200 and 400 mg/kg CTE, p.o., respectively. The animals
were placed separately in metabolic cages over white clean
Whatman filter paper, which was changed every hour. The
severity of diarrhea was assessed each hour for 4 h. The total
number of diarrheal feces of the control group was considered
as 100%.
% Inhibition of diarrhea = (total weight of feces of control
group − total weight of feces of test group) × 100/total weight
of feces of control group
Measurement of gastrointestinal transit time using
charcoal
Wistar albino rats were fasted for 18 h and divided into five
groups of six animals each. Castor oil (1 ml) was administered
orally to the animals. One hour later, group I (control)
was administered 1  ml/100  g of 0.9% NaCl in distilled
water (normal saline), group II (reference) received standard
drug, atropine sulfate (5 mg/kg p.o.) and rats of groups III, IV,
and V received 100, 200 and 400 mg/kg CTE p.o., respectively.
After 30 min of the administration, 1 ml of charcoal meal
(10% suspension in 5% gum acacia) as a marker diet was
given orally to rats in all groups and immediately rats were
sacrificed by ether (20% v/v) anesthesia and small intestine
was separated from mesentery avoiding being stretched. For
each animal, gastrointestinal (GI) transit was calculated as
percentage distance travelled by charcoal meal to the total
41
 Pandhare, et al.: Antidiarrheal activity of Clitorea ternatea
length of intestine. The inhibitory effect of CTE on GI transit
was calculated relative to atropine sulfate.[26]
Measurement of castor oil‑induced intestinal
enteropooling and fluid accumulation
Castor oil‑induced enteropooling was determined by the
method of Robert et al.[27] Wistar albino rats were fasted for
18 h and divided into five groups of six animals each. Castor
oil (1 ml) was administered orally to the animals. One hour
later, group I (control) was administered 1 ml/100 g of 0.9%
NaCl in distilled water (normal saline), group II (reference)
received standard drug, atropine sulfate (5 mg/kg p.o.) and
rats of groups III, IV and V received 100, 200 and 400 mg/kg
CTE, p.o., respectively. After 2 h of treatment, the rats were
sacrificed by ether anesthesia. The edges of the intestine from
pylorus to cecum were tied with thread and the intestine was
removed and weighed. Intestinal content was collected by
milking into a graduated tube and the volume was measured.
The intestine was reweighed and the differences between full
and empty intestines were calculated.
Statistical analysis
The results were expressed as mean ± standard error of
mean; the level of statistical significance for the observed
difference in the mean P < 0.05 was considered as statistically
significant. Statistical analysis was done using one‑way
analysis of variance followed by Dunnette’s multiple
comparison test.
Results
Preliminary phytochemical screening
The preliminary phytochemical study showed the presence of
steroids, terpenoids, alkaloids, tannins, phenolic compounds,
flavonoids, sugars and amino acids.
Acute toxicity study
Oral administration of CTE was found safe at a dose of
2000 mg/kg, p.o. and produced no signs of toxicity. However,
at 5 g/kg, CTE caused slow movement of animal, decreased
aggressiveness and altered touch and pain sensibility, but
did not caused any negative behavioral changes such as
excitement, respiratory distress, convulsions or coma. No
mortality was observed up to 14 days. Hence, the median LD50
of the CTE was then >2000 mg/kg body weight. Therefore,
doses 100, 200 and 400 mg/kg birth weight were selected for
all in vivo experiments.
Table 1: Effect of Clitorea ternatea leaf ethanol extract on castor oil‑induced diarrhea in rats
Treatment groups Dose mg/kg or ml/kg Total number of stools
Control ‑ 18.33±0.88
Loperamide 3 4.33±0.33**
CTE 100 15.00±0.57*
CTE 200 13.66±0.88*
CTE 400 6.33±0.33**
*P<0.05, **P<0.01 values are mean±SEM, n=6, when compared with control using one‑way ANOVA followed by Dunnette’s multiple
ANOVA: Analysis of variance, SEM: Standard error of mean, CTE: Clitorea ternatea ethanol extract
42
Castor oil‑induced diarrhea
The CTE was found to be effective against castor oil‑induced
diarrhea in rats at doses of 100, 200 and 400 mg/kg body
weight as compared to control reflected by slower onset of
diarrhea, reduced frequency of diarrhea and number of wet
stools. Further, at the end, there was a significant 79.25%,
20.87%, 41.75% and 70.87% reduction of diarrheal feces with
the loperamide and CTE (100, 200 and 400 mg/kg) respectively
in a dose‑dependent manner when compared with control
group [Table 1].
Magnesium sulfate‑induced diarrhea
The CTE was found to be effective against MgSO4 induced
diarrhea in rats at doses of 100, 200 and 400 mg/kg body weight
as compared to control reflected by slower onset of diarrhea,
reduced frequency of diarrhea and number of wet stools with
a significant 69.58%, 47.78%, 56.52% and 65.27% reduction
of diarrheal feces with the loperamide and CTE (100, 200 and
400 mg/kg), respectively, in a dose‑dependent manner when
compared with control group [Table 2].
Measurement of gastrointestinal transit using charcoal
meal
Administration of CTE shows significant reduction of GI
transit against castor oil‑induced diarrhea in rats with a
significant 43.52%, 12.04%, 18.05% and 26.86% reduction of
GI transit with atropine and CTE (100, 200 and 400 mg/kg),
respectively in a dose‑dependent manner when compared with
control [Table 3].
Measurement of castor oil‑induced intestinal enteropooling
and fluid accumulation
Administration of CTE was found to possess an antienteropooling
properties in castor oil‑induced experimental animals with a
significant inhibition of intestinal content by 84.42%, 43.71%,
59.38% and 71.85% from atropine and CTE (100, 200 and
400 mg/kg), respectively, in a dose‑dependent manner when
compared with control [Table 4].
Discussion
Diarrhea is the frequent passage of liquid feces and it involves
both an increase in the motility of the GI tract along with
increased secretion and decreased absorption of fluid and
thus a loss of electrolytes (particularly sodium) and water.[28]
Therefore, to restore personal comfort and convenience, many
patients require antidiarrheal therapy and treatment is carried
Number of wet stools Percentage protected
8.00±0.57 ‑
1.66±0.39** 79.25
6.33±0.33* 20.87
4.66±0.21* 41.75
2.33±0.57** 70.87
comparison test.
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 Pandhare, et al.: Antidiarrheal activity of Clitorea ternatea

Table 2: Effect of Clitorea ternatea leaf ethanol extract on magnesium sulfate‑induced diarrhea in rats
Treatment groups Dose mg/kg or ml/kg Total number of stools Number of wet stools Percentage protected
Control ‑ 12.00±0.57 7.66±0.33 ‑
Loperamide 3 2.33±0.88** 2.33±0.33** 69.58
CTE 100 6.00±0.57* 4.00±0.57* 47.78
CTE 200 4.33±0.33* 3.33±0.88* 56.52
CTE 400 3.00±0.57** 2.66±0.33** 65.27
*P<0.05, **P<0.01 values are mean±SEM, n=6, when compared with control using one‑way ANOVA followed by Dunnette’s multiple comparison test.
ANOVA: Analysis of variance, SEM: Standard error of mean, CTE: Clitorea ternatea ethanol extract

Table 3: Effect of Clitorea ternatea leaf ethanol extract on castor oil‑induced intestinal propulsion in rats (charcoal meal
study)
Treatment groups Dose mg/kg or ml/kg Intestinal length (cm) Distance traveled by CP Inhibition of motility (%)
Control ‑ 75.00±1.15 72.00±0.57 ‑
Atropine 1 74.33±0.88 40.66±0.88** 43.52
CTE 100 77.33±0.33 63.33±0.88* 12.04
CTE 200 81.00±0.57 59.00±0.57* 18.05
CTE 400 81.66±0.88 52.66±0.88** 26.86
*P<0.05, **P<0.01 values are mean±SEM, n=6, when compared with control using one‑way ANOVA followed by Dunnette’s multiple comparison test.
ANOVA: Analysis of variance, SEM: Standard error of mean, CTE: Clitorea ternatea ethanol extract, CP: Charcoal pool

the modulation of function of GI tract and stimulate motility

Table 4: Effect of Clitorea ternatea leaf ethanol extract
and secretion leading to diarrhea.[34] In this study, the results
on castor oil‑induced intestinal enteropooling and fluid
showed that CTE reduced castor oil‑induced diarrhea as well
accumulation in rats
as the number of diarrheal feces and total weight of feces in a
Treatment Dose mg/kg Intestinal Inhibition of intestinal dose‑dependent manner, which could be taken as antidiarrheal
groups or ml/kg fluid (ml) fluid volume (ml) (%) potential. Loperamide is one of the most efficacious and widely
Control ‑ 10.66±0.33 ‑ employed antidiarrheal drugs which effectively antagonized the
Atropine 1 1.66±0.33 84.42 diarrhea induced by castor oil[35] and prostaglandiner cholera
CTE 100 6.00±0.57 43.71 toxin.[36] The therapeutic effect of loperamide is believed to be
CTE 200 4.33±0.21 59.38
due to its antimotility and antisecretory activity.[37]
CTE 400 3.00±0.51 71.85
*P<0.05, **P<0.01 values are mean±SEM, n=6, when compared MgSO4 has been reported to induce diarrhea by increasing the
with control using one‑way ANOVA followed by Dunnette’s multiple volume of intestinal content through prevention of reabsorption
comparison test. ANOVA: Analysis of variance, SEM: Standard error of
of water. It has been demonstrated that it promotes the release
mean, CTE: Clitorea ternatea ethanol extract
of cholecystokinin from the duodenal mucosa, which increases
the secretion and motility of small intestine and thereby
out to achieve other objectives such as increased resistance
prevents the reabsorption of sodium, chloride and water.[38]
to flow  (segmental contraction, decreased propulsion and
The CTE was also found to reduce MgSO4‑induced diarrhea
peristalsis) and increased mucosal absorption or decreased
significantly which could be due to increased absorption of
secretion.[29,30] The present investigation involves evaluation of water and electrolytes.
the antidiarrheal potential of C. ternatea against castor oil and
MgSO4‑induced diarrhea. CTE effect was also investigated on The CTE exhibited a significant antidiarrheal effect on GI transit
GI transit using charcoal meal in castor oil‑induced diarrheal using charcoal meal in rats. Hypermotility characterizes forms
rats in comparison to concerning actions of drugs such as of diarrhea where the secreting component is not the causative
atropine sulfate in reducing GI transit. factor. The CTE suppressed the propulsive movement or GI
transit of charcoal meal, which clearly indicates that extract,
Castor oil, a very effective laxative is hydrolyzed in the upper may be capable of reducing the frequency of stools in diarrheal
small intestine to ricinoleic acid,[31] which can stimulate fluid conditions. Anticholinergic agents are known to inhibit GI
secretion, inhibit water and electrolyte absorption, reduce hyper‑motility. Castor oil‑induced GI hyper‑motility has been
active Na+ and K+ absorption and decrease Na+, K+‑ATPase suggested to be indirectly mediated by cholinergic system since
in the small intestine and colon.[24,32] Castor oil also increases it is inhibited by atropine sulfate, a known anticholinergic
the peristaltic activity and produces permeability changes in agent.[39] CTE was found to possess an antienteropooling
the intestinal mucosal membrane to electrolytes and water. in castor oil‑induced diarrheal rats by reducing both weight
Furthermore, ricinoleic acid can also lead to the release of and volume of intestinal content. These effects are direct
endogenous prostaglandins,[33] which play an important role in consequences of reduced water and electrolytes secretion in

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 Pandhare, et al.: Antidiarrheal activity of Clitorea ternatea
small intestine, suggesting that extract may enhance water and
electrolyte absorption from intestinal lumen. Phytochemical
screening revealed the presence of numerous constituents such
as steroids, terpenoids, alkaloids, tannins, phenolic compounds,
flavonoids, sugars and amino acids. Antidiarrheal properties of
medicinal plants were found to be due to tannins, flavonoids,
alkaloids, saponins, reducing sugar, sterol and terpenes.[40‑43]
Hence, tannins, reducing sugars and sterols may be responsible
for antidiarrheal potential of CTE.
Conclusion
C. ternatea ethanol extract (CTE) produced dose‑dependent
and significant (P < 0.05–0.01) protection of rats against castor
oil and MgSO4‑induced diarrhea, inhibited intestinal transit
and delayed gastric emptying and inhibited castor oil‑induced
enteropooling in rats when compared with standard drugs
such as loperamide and atropine sulfate, respectively. These
findings demonstrate that C. ternatea has the potential to treat
GI disorders such as diarrhea owing to its antidiarrheal effect.
Further studies are necessary to substantiate above claim and to
work out exact mechanism of action involved in antidiarrheal
activity of this plant.
Acknowledgment
The first author is sincerely thank to Dr.  V. K. Deshmukh
Principal, M.E.S’s College of Pharmacy, Prashant Patil
Gadakh President, Mula Education Society, Sonai, for
encouragement and availing of the laboratory facilities during
investigation.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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