The bark of the plant of Dillenia indica Linn was collected from the botanical garden of Pharmacy department, Jahangirnagar University, Bangladesh during January 2011. Mice was used as the test animal. The plant material was taxonomically identified by the National Herbarium of Bangladesh whose voucher specimen no. JU/32234 is maintained for future reference. The plant material was shade-dried with occasional shifting and then powdered with a mechanical grinder, passing through sieve #40, and stored in a tight container. The dried powder material (1.5 kg) was refluxed with MeOH for three hours. The total filtrate was concentrated to dryness, in vacuo at 400C to render the MeOH extract (490 g). Folin–Ciocalteu phenol reagent, were purchased from E. Merck (Germany). Galic acid and quercetin, were purchased from Sigma Chemical Co. Ltd, (St. Louis, MO, USA). All other chemicals and reagents were of analytical grade. The total phenolic content of extract was determined using Folin–Ciocalteu reagent. Extracts (100 μl) were mixed with the Folin–Ciocalteu reagent (500 μl) and 20% sodium carbonate (1.5 ml). The different concentration of extracts (0.5 ml) were separately mixed with 95% ethanol (1.5 ml), 10% aluminum chloride (0.1 ml), 1M potassium acetate (0.1 ml) and distilled water (2.8 ml). After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm. The amount of 10% aluminum chloride was substituted by the same amount of distilled water in blank. All the determinations were carried out in duplicates. These data were used to estimate the flavonoid contents using a standard curve obtained from various concentration of quercetin. Animals were divided into groups of five mice each. The test was performed using increasing doses of both test extracts, given orally, in a 10 ml/kg volume to different groups serving as test groups19. Another group of mice was administered saline (10 mL/kg, p.o.) as negative control. The mice were allowed food ad libitum during the 24 h test and kept under regular observation for mortality.
1. Castor oil-induced Diarrhea: The animals were all screened initially by giving 0.5 ml of castor oil. Only those showing diarrhea were selected for the final experiment. Group I received 1% CMC (10 ml/kg, p.o.), groups III-IV received orally MDIB extract (100 and 200 mg/kg), respectively. Group II was given Loperamide (3 mg/ kg, p.o.) in suspension. After 60 min, each animal was given 0.5 ml of castor oil, each animal was placed in an individual cage, the floor of which was lined with blotting paper which was changed every hour, observed for 4 h and the characteristic diarrhoeal droppings were recorded.
2. Magnesium sulphate-induced Diarrhea: Diarrhoea was induced by oral administration of magnesium sulphate at the dose of 2 g/kg to the animals 30 min after pre-treatment with vehicle (1% Tween 80 in water, 10 ml/kg, p.o.) to the control group, loperamide (3 mg/kg) to the positive control group, and the methanol extract (MDIB) at the doses of 100 and 200 mg/kg to the test groups21.
3. Effect on Gastrointestinal Motility: Animals were divided into four groups of five mice each and each animal was given orally 1 ml of charcoal meal (5% activated charcoal suspended in 1% CMC) 60 min after an oral dose of drugs or vehicle. Group I was administered 1% CMC (10 ml/kg) and animals in groups III-IV received extract of MDIB at the dose of 100 mg/kg and 200 mg/kg body weight, respectively. Group II received atropine sulfate (0.1 mg/kg,) as the standard drug.
After 30 min, animals were killed by light ether anaesthesia and the intestine was removed without stretching and placed lengthwise on moist filter paper. The intestinal transit was calculated as a percentage of the distance travelled by the charcoal meal compared to the length of the small intestine.
4. PGE2-induced Enteropooling: The method of Robert et al.23 was applied. Overnight fasted mice were divided into five groups of 5 animals each. Group I was given 2% gum acacia and kept as a control. Group III-IV received 100 and 200 mg/kg p.o. of MDIB extracts, respectively. Group II served as a vehicle control and received 2% gum acacia plus PGE2 (0.5 ml of 100μg/kg, i.p.).
Group V received loperamide and kept as a positive control. Immediately afterwards, diarrhea was induced by 0.5 ml of 100μg/kg, i.p., dose of PGE2 (Sigma Aldrich, USA). After 30 minutes, the animals were sacrificed, small intestine was removed, and intestinal contents were collected and measured in a syringe. The percentage inhibition in intestinal fluid was determined by comparing the values with vehicle control.
5. Antimicrobial activity: Sterile 6.0 mm diameter blank discs (BBL, Cocksville, USA) were impregnated with test substances of MDIB at the dose of 500μg/disc. This disc, along with standard discs (Ciprofloxacin, Oxoid Ltd, UK) and control discs were placed in petri dishes containing a suitable agar medium seeded with the test organisms using sterile transfer loop and kept at 4oC to facilitate maximum diffusion. The plates then kept in an incubator (370C) to allow the growth of the bacteria. The antibacterial activities of the test agents were determined by measuring the diameter of the zone of inhibition in terms of millimeter. Antimicrobial activity was tested against Staphylococcus aureus, Escherichia coli, Pseudomonus aeruginosa, Salmonella typhi, Shigella boydii, Shigella flexneri and Shigella dysenteriae were obtained from International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B).
All values were expressed as the mean ± standard error of the mean (SEM) of three replicate experiments and were analyzed using the GraphPad program (GraphPad, San Diego, CA, USA). The analysis was performed by using student’s t test. p<0.001 and p<0.05 were considered to be statistically significant.