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Evaluation of anti-diarrheal activity of Cleome viscosa L. extract in rats.

Summary

A study was undertaken to evaluate the effect of a methanol extract of the entire plant Cleome viscosa L. (CVME) (Family; Capparidaceae) for its anti-diarrheal potential against some of the experimental models of diarrhea in rats. CVME showed significant inhibitory activity against castor-oil-induced diarrhea and [PGE.sub.2], induced enteropooling in rats. The extract also showed a significant reduction in gastrointestinal motility in the charcoal meal test in rats. The results obtained establish the efficacy and substantiate the folklore claim as an anti- diarrheal agent.

Key words: Cleome viscosa, CVME, entire plant, methanol extract, anti-diarrheal

Introduction

The major cause of diarrhea among children in developing countries is malnutrition. To nullify the problem of diarrhea, which is the leading cause of mortality in developing countries, the World Health Organization (WHO 1964) has constituted a Diarrheal Disease Control program (CDD), which includes studies of traditional medicinal practices, together with the evaluation of health education and prevention approaches. Cleome viscosa L. (Capparidaceae) is a widely distributed sticky herb with yellow flowers, having strong penetrating odor. It is known as Hurhur (Hindi) Hurhuria (Bengali), Nayikkadugu (Tamil) in Indian traditional medicine, and is found throughout the greater part of India, often in waste places. The juice of the plant, diluted with water, is given internally in small quantity to treat fever (Anonymous, 1966).

The seeds are used as anthelmintic and the leaves are useful in healing external wounds and ulcers (Kirtikar and Basu, 1935). In the Ayurvedic system of medicine, this plant is used for fever, inflammations, liver diseases, bronchitis, infantile convulsions and diarrhea (Chatterjee and Pakrashi, 1991). On the basis of traditional use, we have chosen the plant to evaluate its anti-diarrheal potential in several animal models. Because methanol is the solvent which extracts most of the components present in any material, the present investigation was carried out using a methanol extract of the entire plant.

* Materials and Methods

Plant material

Flowering plants of Cleome viscosa L. were collected from Jhilimili, Bankura, West Bengal, India, during the months of July and August. Taxonomic identification of the plant was made by The Botanical Survey of India, Shibpur, Howrah, and West Bengal. The voucher specimen was preserved in our laboratory for future reference. The plants were collected fresh and dried under shade, pulverized in a mechanical grinder, passed through a 40-mesh sieve and stored in a closed vessel for further use.

Preparation of the extract

The powdered, entire plant material was extracted using 90% methanol in a Soxhiet apparation. The solvent was removed completely removed from the extract by distillation in vacuo and a greenish-brown colored, semisolid mass was obtained. (Yield 9% w/w, with respect to the dry powder). The extract was treated with several chemical reagents and showed positive results for tannins, steroids, and flavonoids. The presence of these constituents were confirmed by thin-layer chromatography. The methanol extract of Cleome viscosa L. (CVME) was stored in a desicator and a weighed quantity was dissolved in normal saline or 2% v/v aqueous Tween80 solution and used for the various experiments.

Animals used

Albino Wistar rats (either sex) weighing 180-200 g each were used for the study. The animals were divided into five groups of six each for every individual study and housed in standard metal cages with food and water ad libitum except in the castor-oil model, in which the animals were placed individually in separate cages. The animals were maintained under standard laboratory conditions for an acclimatization period of 7 days prior to performing the experiments. The animals were fasted for 18 h prior to the experiment.

Toxicity study

An acute toxicity study relating to the determination of [LD.sub.50] value was performed using different doses of the extract yccording the method described by Ghosh et al. (1984). From the toxicity study, it was observed that the plant extract is non-toxic and caused no death up to a dose of 3.2 g/kg body wt. (p.o.). It is safe and was used in different doses for further studies (Table 1).

Castor-oil-induced diarrhea in rats (Awouters et al. 1978) The doses of CVME were selected on a trial basis and administered orally (200, 400 and 600 mg/kg body wt.) by gavage to three groups of animals. The fourth group received Diphenoxylate (5 mg/kg body wt.) orally and the fifth group received neither drug nor extract but 2% v/v aqueous Tween 80 (1 ml) only and served as a control. After 60 min of drug treatment, each animal was administered 1 ml of castor oil (Sicco, Calcutta) orally by gavage and observed for defecation up to 4 h after castor oil administration. Characteristic diarrheal droppings were noted in the transparent plastic dishes placed beneath the individual perforated rat cages. The mean number of wet feces was calculated from the diarrheal droppings in the transparent plastic dishes (Mandal et al. 1997).

[PGE.sub.2]-induced enteropooling

For this evaluation, rats of the same stock as above were deprived of food and water for 18 h prior to the experiment. Five groups of six animals were used, which were placed in five perforated cages. The first three groups of rats were treated with CVME (200,400 and 600 mg/kg body wt., p.o) while the fourth and fifth groups received 1 ml of 5% v/v ethanol in normal saline (i.p.). The fourth group was then administered 1 ml of normal saline and used as control. Immediately afterwards, each rat was treated with [PGE.sub.2] (100 pg/kg body wt. in 5% v/v ethanol in normal saline; Astra-IDL Limited, India) administered orally. All the rats were sacrificed under mild anesthesia after 30 min. The entire length of the intestine from the pylorus to the caecum was dissected out, and its contents were collected and measured. (Subhash et al. 1997).

Gastrointestinal motility test

In this method rats were fasted for 18 h and placed in five metal cages, six in each. Each animal was given 1 ml of charcoal meal (3% deactivated charcoal in normal saline). The first three groups of animals were administered CVME orally (200, 400 and 600 mg/kg body wt.) immediately after the charcoal meal treatment. The fourth group received Atropine (0.1 mg/kg body wt., i.p.) as a standard drug for comparison. The fifth group was treated with normal saline as control. 30 min after administration of the charcoal meal, animals of each individual group were killed and the movement of charcoal from pylorus to caecum was measured. The charcoal movement in the intestine was expressed as a percentage (Sheba Rani et al. 1999).

Statistical analysis

For all the above experiments, results were expressed as mean [+ or -] SEM. Statistical significance tests were performed using the Student's t-test and p values were calculated by comparison with control groups (Woodson, 1987).

* Results

Toxicity study

From the toxicity study it was observed that the plant extract is non-toxic and caused no death up to a dose of 3.2 g/kg orally. It is safe and was used in different doses for further studies.

Inhibition of Castor-oil- induced diarrhea

The extract (CVME) inhibited the frequency of defecation significantly, like the standard drug (diphenoxylate) as compared to control (2% aqueous Tween 80 treated). The wetness of the fecal matter was also reduced greatly by both the standard drug and extract (CVME). The results are shown in Table 1.

Effects on gastrointestinal motility

The extract (CVME) decreased the propulsion of the charcoal meal through the gastrointestinal tract significantly with respect to the control group. The effect was comparable to the standard drug (Atropine 0.1 mg/kg body wt.). The results are shown in Table 1.

Anti-enteropooling activity

[PGE.sub.2] induced a significant increase in the fluid volume of the rat as compared to control animals receiving only ethanol in normal saline. The extract (CVME) of the plant material significantly inhibited [PGE.sub.2]-induced enteropooling in rats at almost all the doses used (see Table 3).

* Discussion

CVME extract at doses of 200, 400 and 600 mg/kg body wt. significantly inhibited frequency of defecation and wetness of fecal droppings, like the standard antidiarrheal agent (Diphenoxylate) as compared to untreated control rats (i.e., rats which were administered only castor oil; Table 2). The antimuscarinic drug atropine and extracts (CVME) at different dose levels (200, 400 and 600 mg/kg body wt.) decreased the intestinal propulsive movement in the charcoal-meal-treated model (Table 3). CVME (600 mg/kg body wt. showed activity similar to that of atropine. Similarly, the plant extract inhibited [PGE.sub.2]-induced enteropooling significantly (Table 4).

The above observation suggests that CVME in graded doses (200, 400 and 600 mg/kg body wt.) reduced diarrhea by inhibiting motility and [PGE.sub.2] enteropooling. The inhibitory effect of the extracts justifies the use of the plant in folk medicine, and its use as a nonspecific anti-diarrheal agent. Tannic acid and tannins are present in many plants and they denature proteins by formation of protein tannate, which makes the intestinal mucosa more resistant and reduces secretion (Tripathi, 1994). The tannin present in the plant extract (CVME) may be responsible for the antidiarrheal activity. We conclude that the present study supports claims by traditional medicine practitioners about the use of CVME in the treatment of diarrhea (Nadkarni, 1992).
Table 1.

Toxicity study.

Treatment Dose (mg/kg No. of No. of No. of [LD.sub.50] value
 body wt.) animals survival death

Control 2% tween 80 20 20 0 -
 Solution

CVME 100 20 20 0 -
 200 20 20 0 -
 400 20 20 0 -
 800 20 20 0 -
 1600 20 20 0 -
 3200 20 20 0 >3.2 g/kg body wt.

CVME -- Cleome viscosa L. Methanol Extract

Table 2

Inhibition of Castor-oil-induced diarrhea.

Oral pretreatment at 60 min Mean number of wet feces
(mg/kg body wt.) (mean [+ or -] SEM)

2% v/v aqueous Tween 80 (5) 28.0 [+ or -] 2.83
Diphenoxylate (5) 11.3 [+ or -] 1.33 (a)
CVME (200) 21.8 [+ or -] 1.72 (b)
CVME (400) 15.8 [+ or -] 1.17 (a)
CVME (600) 13.2 [+ or -] 1.2 (a)

(a) P <0.001

(b) P <0.01 as compared to control (n = 6)
CVME -- Cleome viscosa L. Methanol Extract

Table 3

Anti-enteropooling activity.

Treatment Volume of intestinal P-Value
 fluid ml
 (mean [+ or -] SEM)

Ethanol in saline 0.74 [+ or -] 0.12 -
[PGE.sub.2] in ethanol 2.90 [+ or -] 0.20 < 0.001 (a)
 (100 mg/kg body wt.)
CVME 1.79 [+ or -] 0.10 < 0.001 (b)
 (200 mg/kg body wt.)
CVME 1.14 [+ or -] 0.11 < 0.001 (b)
 (400 mg/kg body wt.)
CVME 1.07 [+ or -] 0.07 < 0.001 (b)
 (600 mg/kg body wt.)

(a) Significance -- with respect to ethanol in saline treatment

(b) With respect to [PGE.sub.2] treatment (n = 6)

CVME - Cleome viscosa L. Methanol Extract

Table 4

Effect on gastrointestinal motility.

Treatment Dose (mg/kg body wt.) Movement o charcoal
 meal (%)

Control 2% aqueous Tween 800 84.5 [+ or -] 2.8

Atropine 0.1 41.3 [+ or -] 2.3 (a)
CVME 200 80.3 [+ or -] 2.3
CVME 400 75.7 [+ or -] 2.4 (b)
CVME 600 60.7 [+ or -] 2.2 (a)

P-Value calculated with respect to control group (n = 6)

CVME - Cleome viscosa L. Methanol Extract

(a)P <0.001

(b) P <0.02


Acknowledgement

The authors are grateful to the All India Council of Technical Education, New Delhi for financial assistance to Dr. S. C. Mandal.

References

Anonymous (1966) The Wealth of India. Vol. I. Publication and Information Directorate, CSIR, New Delhi, 231

Awouters F, Nimegeers CJE, Lenaerts FM, Janssen PAJ (1978) Delay of castor oil diarrhoea in rats; a new way to evaluate inhibitors of prostaglandin biosynthesis. J of Pharmacy and pharmacology 30: 41-45

Chatterjee A, Pakrashi SC (1991) The Treatise on Indian Medicinal Plants Publication and Information Directorate. CSIR. New Delhi. I. 155

Ghosh MN (1984) Fundamental of Experimental pharmacology. 11 edn,Scientific Book agency, Calcutta 153

Kirtikar KR, Basu BD (1935) In: Blatter JF, Caius KS, Mhaskar (Eds) Indian Medicinal plants. Bishen Siagh and Mahendra Pal Siagh, Dehradun. 183-185

Mandal SC, Mukherjee PK, Saha K, Pal M, Saha BP (1997) Antidiarrhoeal evaluation of Ficus racemosa Linn. Leaf Extract. J Natural Product Sciences 3(2): 100-103

Nadkarni KM, Nadkami AK (1992) Indian Materia Medica. Vol I Popular Prakashan, Bombay 351

Rani S, Ahamed N, Rajaram S, Saluja R, Thenmozhi S, Murugesan T (1999) Anti-diarrhoeal evaluation of Clerodendrum phlomidis Linn leaf extract in rats. J of Ethnopharmacology 68: 315-319

Tripathi KD (1994) Essential of Medical pharmacology Jaypee brothers, Medical publishers (P) New Delhi 187

Woodson RF (1987) Statistical Methods for the analysis of Biomedicinal Data (Wiley Series in Probability and Mathematical Statistics) Wiley, New York 315-316

WHO Expert Committees (1964) In: WHO Technical Report Series of Enteric Infection 288

* Address

S. C. Mandal, Division of Pharmacognosy, Department of Pharmaceutical Technology, Faculty of Engineering & Technology, Jadavpur University, Calcutta 700 032, India

Tel.: ++91-33-4676316 (R); Fax: ++91-33-8381243; e-mail: subhashmandal@yahoo.com
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Author:Devi, B. Parimala; Boominathan, R.; Mandal, S.C.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:9INDI
Date:Dec 1, 2002
Words:2175
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