In Vivo Evaluation of Antidiarrhoeal Activity of the Seed of Swietenia macrophylla King ( Meliaceae )

Purpose: The seeds of Swietenia macrophylla are used in traditional medicine for the treatment of diarrhoea. Thus the petroleum ether extract of Swietenia macrophylla (Meliaceae) seeds was investigated for its anti-diarrhoeal property in Wister albino rats to substantiate folklore claim. Methods: Petroleum ether extract of the seeds of this plant, at graded doses (25, 50 & 100mg/kg body weight) was investigated for anti-diarrhoeal activity in term of reduction in the rate of defecation and consistency of faeces in castor oil induced diarrhoea. To understand the mechanism of its antidiarrhoeal activity, its effect was further evaluated on intestinal transit and castor oil induced intestinal fluid accumulation (enteropooling). Results: At various doses (25, 50 & 100mg/kg body weight) the extract showed a remarkable antidiarrhoeal activity evidenced by the reduction in the rate of defecation and consistency of faeces. Results are comparable to that of standard drug diphenoxylate (50mg/kg body weight). A single oral dose of Swietenia macrophylla extract of 100mg/kg body weight produced a significant decrease in the severity of diarrhoea. Extract produced profound decrease in intestinal transit (4.45 34.60%) also significantly inhibited castor oil induced enteropooling comparable to that of intraperitoneal injection of standard drug atropine sulphate at doses of 0.1 mg/kg body weight and 3 mg/kg body weight respectively. Conclusions: Experimental findings showed that petroleum ether extract of seeds of Swietenia macrophylla possess significant anti-diarrhoeal activity and may be a potent source of anti-diarrhoeal


INTRODUCTION
Diarrhoea is characterized by increased frequency of bowel movement, wet stool and abdominal pain 1 .It is a leading cause of malnutrition and death among children in the developing countries of the world today 2 .Many governments and international organizations are trying to control this disease but the rate of incidence is still high, about 7.1 million per year 3 .Many synthetic chemicals like diphenoxylate, loperamide and antibiotics are available for the treatment of diarrhoea but they have some side effects.The natural drugs are used as antidiarrhoeal drugs, which are not always free from adverse effects 4 .Therefore, the search for safe and more effective agents has continued to be an important area of active research.Since ancient times, diarrhoea has been treated orally with several medicinal plants or their extracts based on folklore medicine.The plant Swietenia macrophylla (Family: Meliaceae) is a beautiful, lofty, evergreen large tree native to tropical America, Mexico and South America usually 30-40m in height and 3-4m in girth 5 .The seed of Swietenia macrophylla has been reported for its anti-inflammatory, antimutagenecity and antitumor activity 6 .Swietenine, swietenolide 7 , swietemahonin, khayasin, andirobin, augustineolide, 7deacetoxy-7-oxogedunin, 6-deoxy swietenine, proceranolide, 6-O-acetyl swietonolide, 2hydroxy swietenine have been isolated from the seeds of this plant 8 .Seed of Swietenia macrophylla is traditionally used by the local healers of East Midnapore, West-Bengal, India for curing diarrhoea.The present study was undertaken to evaluate the antidiarrhoeal potential of petroleum ether extract of seeds of Swietenia macrophylla in normal and castor oil induced diarrhoeal rats.

Plant material
Seeds of Swietenia macrophylla King.(Family: Meliaceae) were collected in the months of December and January, from the villages of Midnapore (E), West Bengal, India.The plant was authenticated by the Botanical Survey of India.A voucher specimen number entitled CNH/1-1(64) was deposited at our institute for future reference.

Preparation of extract
The powdered seeds of Swietenia macrophylla (600 g) were extracted with petroleum ether using Soxhlet apparatus.The resulting extract was evaporated in vacuum (Yield = 12.42%) and stored in desiccators for future use.The crude extract was dissolved 2% Tween 80 prior to the experiment and used.Animals Swiss albino rats (150 -180 g) of either sex were selected for the experiments.Animals were allowed to be acclimatise for a period of 2 weeks in our laboratory environment prior to the study.Animals were housed in polypropylene cages (4 animals per cage), maintained under standard laboratory conditions (i.e.12:12 hour light and dark sequence; at an ambient temperature of 25±2ºC; 35-60% humidity); the animals were fed with standard rat pellet diet (Hindustan Liver Ltd.Mumbai) and water ad libitum.The principles of Laboratory Animal Care (NIH, 1985) were followed and instructions given by our institutional animal ethical committee were maintained throughout the experiment.

Castor oil-induced diarrhoea
Rats were fasted for 18 h and divided into five groups of six animals per group.Castor oil at a dose of 1 ml/animal orally, was given to all groups of animals for the induction of diarrhoea 9 .Thirty minutes after castor oil administration, the first group (control group) received vehicle (0.5% v/v Tween 80 in distilled water), while the second, third and fourth groups were given petroleum ether extract at doses of 25, 50 and 100 mg/kg body weight respectively by oral route.The fifth group received the reference drug, diphenoxylate (50 mg/kg body weight) 10 .Animals of all groups were placed separately in individual cages lined with filter paper.The filter papers were changed every hour and the severity of diarrhoea was assessed hourly for six hours.The total number of faeces excreted and the total weight of faeces were recorded within a period of six hour and compared with the control group.The total number of diarrhoeal faeces of the control group was considered 100%.The results were expressed as percentage of inhibition of diarrhoea 11 .

Gastrointestinal motility test
This experiment was done by using charcoal meal as a diet marker 12 .The rats were divided into five groups of six animals each and fasted for eighteen hours before the experiment.The first group (the control group) was orally administered the vehicle (0.5% Tween 80 in distilled water).The second, third and fourth groups orally received petroleum ether extract at doses of 25, 50 and 100 mg/kg body weight respectively.The fifth group received the standard drug, atropine sulphate (0.1 mg/kg body weight intraperitoneal).Thirty minutes later each animal was given 1 ml of charcoal meal (10% activated charcoal in 5% gum acacia) orally.Each animal was sacrificed thirty minutes after administration of charcoal meal.The distance covered by the charcoal meal in the intestine was expressed as a percentage of the total distance traveled from the pylorus to the caecum 13 .

Castor oil-induced enteropooling
Intraluminal fluid accumulation was determined by the method of Boominathan et al. 2005.Over night fasted rats were divided into five groups of six animals each.Group 1 which received normal saline (2 ml/kg intraperitoneal) served as the control group.Group 2 received atropine (3 mg/kg intraperitoneal) and groups 3, 4 and 5 received extract of 25, 50 and 100mg/kg intraperitonealy, respectively, one hour before the oral administration of castor oil (1 ml).Two hours later, the rats were sacrificed; the small intestine was removed after tying the ends with threads and weighed.The intestinal content was collected by milking into a graduated cylinder and their volume was measured.The intestine was reweighed and the difference between the full and empty was calculated.

Statistical analysis
Data were analyzed by one-way ANOVA followed by Dunnett's t-test using computerized GraphPad InStat version 3.05 (Graph Pad software, U.S.A.).

RESULTS
The petroleum ether extract was found to be effective against castor oil induced diarrhoea on experimental rats at various doses of 25, 50 & 100 mg/kg body weight has been shown in Table 1.A single oral dose of Swietenia macrophylla extract of 100 mg/kg body weight produced a significant decrease in the severity of diarrhoea in terms of reduction in the rate of defecation and consistency of faeces in Wistar albino rats.The percentage inhibition for the number of wet faeces as well as wet mass indicates the presence of antidiarrhoeal activity in extract as compared with that of control group.Experimental results reflect the activity is more pronounced at the dose of 100mg/kg body weight.The percentage of inhibition of number of wet faeces as well as wet mass found 67.23% and 69.46% respectively at the dose of 100 mg/kg body weight very much comparable to that of standard drug diphenoxylate (50mg/kg body weight).The petroleum ether extract produced profound decrease in intestinal transit of 4.45-34.60%at the dose range of 25-100 mg/kg body weight and significantly inhibited castor oil induced enteropooling in terms of volume and weight of intestinal content comparable to that of intraperitoneal injection of standard drug atropine sulphate at doses of 0.1 mg/kg body weight and 3mg/kg body weight respectively as indicated in Tables 2 and 3.

DISCUSSION
Diarrhoea results from an imbalance between the absorptive and secretory mechanisms in the intestinal tract accompanied by hurry resulting in an excess loss of fluid in the faeces.In some diarrhoea the secretory component predominates while other diarrhoea is characterized by hypermotility 14 .Castor oil causes diarrhoea due to its active metabolite, ricinoleic acid 15,16 , which stimulates peristaltic activity in the small intestine, leading to changes in the electrolyte permeability of the intestinal mucosa.Its action also stimulates the release of endogenous prostaglandin 17 .Castor oil reported to induce diarrhoea by increasing the volume of intestinal contents by preventing the re-absorption of water.The liberation of ricinoleic acid results in irritation and inflammation of intestinal mucosa leading to release of prostaglandin 18 .In this study, the petroleum   19,20 .Hence, steroid may be responsible for the antidiarrhoeal activity.

CONCLUSION
In conclusion, the results of this investigation revealed that petroleum ether extract contains pharmacologically active substance(s) with antidiarrhoeal properties.This provides the rationale for the use of the seed extract of Swietenia macrophylla as an anti-diarrhoeal drug by traditional healers.Further research is to be carried out to fractionate and purify the extract, in order to find out the molecule responsible for the anti-diarrhoeal activity observed.

Table 1 :
Effect of the petroleum ether extract of S. macrophylla seeds at different dose levels on castor oil-v induced diarrhoea.

Table 2 :
Effect of the petroleum ether extract of S. macrophylla seeds at different dose levels on charcoal-induced gut transit changes.

Table 3 :
Effect of the petroleum ether extract of S. macrophylla seeds at different dose levels on castor oil enteropooling.