Received date: 13 January 2014; Revised Date: 26 Feburary 2014; Accepted date: 15 March 2014
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This present study was undertaken to investigate antidiarrhoeal activity of ethanolic extract of unripe fruit of Aegle marmelos (EEAM) in castor oil, prostaglandin E2 (PGE2), 5-Hydroxytryptamine (5-HT), and carbachol induced diarrhoea in mice. The higher dose of EEAM (800mg/kg) completely prevented the diarrhoea whereas the lower doses (200 and 400mg/kg) significantly increased the time of induction of diarrhea, decreased the number and weight of wet faeces in castor oil induced diarrhoea. The higher doses of EEAM (400 and 800 mg/kg) significantly increased time of induction of diarrhoea, decreased the number and weight of wet faeces in PGE2, 5-HT and carbachol induced diarrhoea, whereas lower doses had no effect on these parameters.
Aegle marmelos, Castor oil, Carbachol, 5-HT, PGE2, Antidiarrhoeal activity
Aegle marmelos commonly known as Bael/Bilva belonging to the family Rutaceae has been widely used in indigenous system of Indian medicine due to various medicinal properties  Extensive studies have been reported on biological activities of various extracts of Aegle marmelos including antidiabetic , antiulcer , anticancer , antihyperlipidaemic , antispermetogenesis , Antidiarrhoeal activity of Aegle marmelos fruit has been reported in castor oil induced diarrhea , Secretory diarrhoea is a common form of acute diarrhoea manifested as dehydration due to excessive fluid and electrolyte loss. Various toxins induced diarrhoea is mediated through release of varieties of endogenous secretagogues including 5-HT, PGE2 and cholinergic pathway .Antidiarrhoeal activity of Aegle marmelos against various secretagogues induced diarrhoea is poorly reported. So, the present study was designed to evaluate antidiarrhoeal activity of ethanolic extract of Aegle marmelos in various secretagogues induced diarrhoea in mice.
Castor oil IP ( Oom Laboratories, Shimoga, Karnataka), Carbachol (Merck Mumbai), Prostaglandin E2 and 5-Hydroxytryptamine (Cayman chemical company, Michigan, USA).
The unripe fruits of Aegle marmelos were collected from local areas of Bellary district, Karnataka, during July-September. The plant material was taxonomically identified and authenticated by Dr. Govindraj, HOD, Department of Botany, Smt. A.S.M. College for Women, Bellary, Karnataka, India.
Preparation of crude extract
Freshly collected unripe fruits of Aegle marmelos were thoroughly washed under running water to remove adherent impurities. Fruits were chopped and the pulp along with pericarp and seeds were subjected to shade drying at room temperature and coarsely powdered (#40).The powdered drug (100g) was macerated with 16 parts of ethanol (90%) for a week and filtered. The obtained extract was concentrated in a rotary vacuum evaporator under reduced pressure to obtain a reddish brown semi-solid mass. The percentage yield of the extract was12.64 % w/w with respect to air dried plant material. The extract was stored at low temperature (4 to 80 C) for evaluation of phytochemical, toxicological and pharmacological studies.
In order to determine the presence of phytoconstituents, a preliminary phytochemical study of the extract was performed using specific reagents .
Swiss albino mice of either sex weighing 25-30 gwere procured from M/S Venkateshwara enterprises, Bangalore.They were housed in polypropylene cages and maintained under standard laboratory conditions (12:12 h light and dark cycles; temperature 25±20C and relative humidity 55±10%). Animals were fed with standarddiet and water ad libitum. Each experimental group consisted of eight animals housed in separate cages. Before the experimental study the animals were fasted overnight with free access to water.
The study protocolwas approved by Institutional Animal Ethics Committee and experiments were performed in accordance with the current guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) .
Acute toxicity (LD50) study
Acute toxicity study of the extract was performed in overnight fasted albino mice by following fixed dose method as per OECD guidelines No.423. Mortality & toxic symptoms in the treated animals were observed continuously for the first 3 h after dosing, periodically during the first 24 h and then daily observation for a total period of 14 days .
Castor oil induced diarrhea
The method reported by Awouters et.al  with modifications has been used in the present study. Swiss albino mice of either sex (24-30 g) were screened initially by giving 0.3ml of castor oil orally and only those showing diarrhoea were selected for the final experiment. Mice were deprived of food overnight before the experiment but had free access to water. The animals were divided into control and test groups containing eight mice in each group.
Each animal was placed in an individual cage having mesh grid with blotting paper placed on the floor of the cage. Diarrhoea was induced by oral administration of castor oil (0.3ml p.o). The different groups of animals received either vehicle or ethanolic extract of Aegle marmelos(200, 400 and 800 mg/kg) 30 min prior to castor oil administration. During observation period of 4hours, the onset of diarrhoea, the number and weight of wet stools excreted by the animals were recorded and used for assessing the antidiarrhoeal activity.
PGE2, 5-HT, and carbachol induced diarrhea
Different groups of mice were administered with PGE2 (0.2mg/kg i.p.), 5-HT (1mg/kg i.p) or carbachol (0.5 mg/kg i.p.) to induce diarrhoea [13,14,15].Diarrhoea was observed at around 7, 8, or 14 minutes after dosing PGE2, 5-HT or carbachol respectively. The EEAM (200,400,800mg/kg) or vehicle was given orally 1h before the dosing of each diarrhoeogenic agent. To assess antidiarrhoeal activity, the time of induction of diarrhea, the total number and weight of wet faeces were recorded for a period 2 h.
The result are expressed as mean ± SEM (n=8). Statistical difference between control and experimental values were analyzed by one-way analysis of variance (ANOVA), followed by Dunnet’s t-test(Graph Pad software).P values< 0.05 were considered significant.
Preliminary phytochemical analysis of the ethanolic extract of Aegle marmelos revealed the presence of tannins, steroidal glycosides, flavonoids, alkaloids, coumarins and terpenoids. Acute toxicity studies were carried out to evaluate toxicity and to determine the minimum lethal dose of the test extract using Swiss albino mice. Fixed dose method of OECD Guideline No.423 was adopted for toxicity studies. It was found that no mortality and changes in the behavior were observed up to dose 2000 mg/kg body wt. Therefore, 200, 400 and 800 mg/kg p.o extract doses were selected for screening of antidiarrhoeal activity.
The EEAM at dose 800mg/kg produced a marked antidiarrhoeal effect with no incidence of diarrhoea in any of the animals in the group (results not shown).The other two doses of extract (200mg and 400mg/kg) significantly prolonged the time of induction of diarrhoea along with reduced number and total weight of wet faeces during 4 hours of observation period (Table 1)
The higher test doses (400 and 800mg/kg) of EEAM significantly prolonged the time of induction of diarrhoea along with decreased number and total weight of faeces during the 2 h of observation period in all three secretagogues induced diarrhoea, whereas lower dose (200mg/kg) did not alter any of the parameters in all three secretagogues induced diarrhoea (Table 2). Secretory diarrhoea occurs as a result of increased intestinal secretion or decreased intestinal absorption of fluid and electrolytes or a combination of thesemechanisms . It is well accepted that various enterotoxins induced diarrhoea and inflammatory diarrhoea generally believed to be mediated through altering the intestinal absorption as well as increased intestinal secretion. It is well established that castor oil releases ricinoleic acid on oral administration which induces changes in mucosal fluid and electrolyte transport resulting in hypersecretory response and diarrhea [17,18].Further, ricinoleic acid induced water and electrolyte secretion in the small intestine is mediated through release of PGE2. In the present study EEAM not only prevented castor oil induced diarrhoea, it also reduced the severity of PGE2 induced diarrhoea. (Table 3). Therefore it can be speculated that Aegle marmelosmay alter PGE2 induced secretory process and thus prevent the diarrhoea.
In the present study EEAM prevented severity of 5-HT induced diarrhoea by prolonging the time of induction of diarrhoea along with decreasing the number and weight of wet faeces induced by 5-HT. (Table 4). Similar effects of EEAM in the present study were also observed in carbachol induced diarrhoea. It is reported that 5-HT has acetylcholine like effects on enterocytes  and also stimulates white cells in lamina propria to release prostaglandins . A number of intracellular mediators like cyclic AMP, cGMP, Ca2+contribute in regulation of water and electrolyte movement across intestinal luminal membrane. Secretory /antiabsorptive stimuli of 5-HT,PGs and Acetylcholine in intestinal tract is mediated through increase in intracellular Ca2+ and activation of PKC.The results of the present study indicate that the extract may exert its effect by modulating Ca2+ activated secretory/antiabsorptive stimuli. However, further studies required to establish mechanism involving antidiarrhoeal effect.
The flavanoids and terpenoid derivatives are known for inhibiting release of autocoids and prostaglandins and thereby inhibit motility and secretion induced by castor oil . The presence of flavonoids and terpenoids in the EEAM may contribute to antidiarrhoeal effect observed in the present study, however further studies involving isolation of active principles of EEAM and evaluation of their antidiarrhoeal activity may support these findings.
In conclusion our results demonstrate that EEAM is effective against various secretagogues induced diarrhoea and provide rationale for its traditional use as an antidiarrhoeal agent.