Collection of the plant sample Leaves of Ziziphus mauritiana were collected from Gazipur district, Dhaka, Bangladesh in February, 2013. This plant was identified by botanists of the Botany Department of Dhaka University. The reference sample for the plant was DUSH, Accession Number 4257 and calls no 01. Swiss albino mice of either sex, aged 4-5 weeks were the experimental animal and were obtained from the Animal Resource Branch of the International Centre for Diarrheal Diseases and Research, Bangladesh (ICDDR, B). Institution of Animal Ethical Committee which was maintained by Faculty of Biological Science, University of Dhaka gave approval for this project to collect and utilize Swiss albino mice as experimental animal. The approval reference number was Ref-DU/BD/IACE-A143. They were kept in standard environmental condition and fed ICDDR, B formulated rodent food and water. Experimental animals were collected, handled and kept by following standard protocol based on the ethical committee of our university.
Preparation of the plant material After collection of leaves it was washed, sun dried and made coarse powder. Around 1 kg of powdered material was soaked in 2.5 litter of methanol for several days with occasional stirring. It was then extracted at room temperature and concentrated by evaporator. About 5 gm of dried extract was partitioned into pet ether, chloroform, carbon tetrachloride and aqueous soluble fractions by standard protocol by modified kupkan partitioning method. The practical yield values of pet ether were 1.85 gm, chloroform 0.85 gm, carbon tetrachloride 1.07 gm and aqueous soluble fraction 0.65 gm respectively.
Evaluation of neuropharmacological activity Methanolic crude extract of Z. mauritiana leaves was subjected for evaluation of neuropharmacological activity by phenobarbitone induced sleeping time test. The study was carried out using Swiss albino mice (25-30 g) of either sex. Fifteen experimental animals were randomly selected and divided into three groups denoted as group-I (control group), group-II (A) and group-II (B) as experimental group consisting of 5 mice in each. The experimental groups were administered with test samples prepared with normal saline water and tween-80 at doses of 200 and 400 mg/kg body weight, while the control group was administered normal saline water containing 1% tween-80 solution. Thirty minutes later, phenobarbitone sodium (25 mg/kg body weight) was administered intraperitoneally to all the groups to induce sleep. The onset of sleep and total sleeping time were recorded for both control and experimental groups.
Preparation of the test samples In order to administer the crude extract at doses of 200 and 400 mg/kg body weight of mice, 50 and 100 mg of the dried extract were measured respectively and were triturated unidirectional way by the addition of small amount of suspending agents Tween. After proper mixing of extract and suspending agent, normal saline was slowly added and made 2.5 ml. Water for injection was added with morphine to dilute it so that 0.3 ml of the diluted solution will have 10 mg /kg body weight of morphine.
Evaluation of anti-diarrheal activity The anti-diarrheal activity of the methanolic crude extract of Z. mauritiana leaves (at a dose of 200 and 400 mg/kg body wt) was evaluated by castor oil induced diarrhea in mice . According to this model each mice was fed 1 ml of highly pure analytical grade castor oil to induce diarrhea. Twenty mice were taken and divided into four groups (Group I, Group II, Group III and Group IV). Group I was used as negative control, while Group II served as the positive control or standard group treated with Loperamide. Group III and Group IV were the test groups. Each mouse was fed with the test samples. Then thirty minutes later they were given 1 ml of castor oil to induce diarrhea. The mice were kept under observation for the next four hours. For each mouse the number of times it defecated was recorded. The observation of the experimental groups was compared with the positive control to evaluate the anti-diarrheal activity of the samples. % Reduction of number of defecation = [(NDC - NDT)/NDC] x 100 Where, NDC= Mean number of defecation of control group and NDT = Mean number of defecation of experimental group.
Evaluation of antimicrobial activity Methanolic crude extract of Z. mauritiana leaves and its different fractions were subjected for the evaluation of antimicrobial activity against 5 gram positive, 7 gram negative and 3 fungi by following standard disc diffusion method. In this classical method, antibiotics diffuse from a confined source through the nutrient agar media and create a concentration gradient. Dried and sterilized filter paper discs (6 mm diameter) containing the test samples at a dose of 400µg/disc were placed on nutrient agar medium uniformly seeded with the test microorganisms. Standard antibiotic (ciprofloxacin at a dose of 30 µg/disc) discs and blank discs are used as positive and negative control. These plates are kept at low temperature (4°C) for 24 hours to allow maximum diffusion of the test materials to the surrounding media10. The plates are then inverted and incubated at 37°C for 24 hours for optimum growth of the organisms. The test materials having antimicrobial property inhibit microbial growth in the media surrounding the discs and thereby yield a clear distinct area defined as zone of inhibition. The antimicrobial activity of the test agent is then determined by measuring the diameter of zone of inhibition expressed in millimeter.