Collection of plant materials: Leaves of C. nurvala were collected from Chittagong University campus in April 2012 and identified by Dr. Sheikh Bokhtear Uddin, Associate Professor, Department of Botany, University of Chittagong, Bangladesh.
Animals: Swiss albino mice of both sex and weighing 32-37 g were collected from International Centre for Diarrheal Disease and Research, Bangladesh (ICDDR, B). The animals were housed in plastic cages having dimension of 28x22x13 cm under standard laboratory conditions (relative humidity 55-65%, room temperature 23.0±2.0°C and 12 h light: dark cycle) and acclimatized for 7 days and fed with standard diet and water. The ethical guidelines for the investigation of experimental animals were followed in all tests and the protocol was approved by the institutional committee.
Preparation of extract: The collected leaves were thoroughly washed with water, chopped, air dried for a week at 35-40°C and pulverized with an electric grinder. The powder obtained was extracted with methanol at room temperature for 7 days with occasional shaking and stirring. The filtrate so obtained was concentrated to dryness by evaporation of solvent using a rotary evaporator under reduced temperature and pressure.
Assay for sedative activity
Open field test: This experiment was carried out according to published method where the floor of an open field of half square meter was divided into a series of squares each alternatively colored black and white. The apparatus had a wall of 40 cm high. The number of squares visited by the mice was counted for 3 min, on 0, 30, 60, 90 and 120 min during the study period.
Hole cross test: This test was done for CNS depressant activity in mice. The animals were divided into three groups-negative control, positive control and test animals. The test groups received methanol extract of C. nurvala leaves at 400 mg kg-1 b. wt orally whereas the control group received vehicle (1% Tween 80 in water). A steel partition was made in the middle of a cage having a size of 30x20x14 cm. A hole of 3 cm diameter was made at a height of 7.5 cm in the center of the cage. The total number of passages of a mouse through the hole from one chamber to another was counted for a period of 3 min on 0, 30, 60, 90 and 120 min after the oral administration with test substances. In this test, diazepam (1 mg kg-1 b.wt.) was used as the positive control.
Elevated plus maze (EPM) test: This experiment was performed by the method, the experimental details of which could be found elsewhere. It utilizes an equipment consisted of two open arms (5x10 cm) and two closed arms (5x10xl5 cm) radiating from a platform (5x5 cm) to give the apparatus a plus sign in appearance. The apparatus was situated 40 cm above the floor in which the open arms edges were 0.5 cm in height to keep the mice from falling and the closed-arms edges were 15 cm in height. The maze floor and walls were made with dark opaque wood. Sixteen minutes after administration of the test agents, each animal was placed at the center of the maze facing one of the enclosed arms. During the five min test period, the number of open arms entries was recorded. The entry into an arm was defined as the point when the animal places all four paws onto the arm. This procedure was conducted in a sound free room and observations made from an adjacent corner.
Cytotoxic activity: The cytotoxicity assay was performed on brine shrimp nauplii by standard method using vincristine sulphate as standard. The test sample was prepared by dissolving in dimethyl sulfoxide solution, DMSO (not more than 50 μL in 5 ml solution) plus sea water (3.8% NaCl in water) to attain concentrations of 25, 50, 100, 200, 400 and 800 μg mL-1. A vial containing 50 μL DMSO diluted to 5 mL-1 with sea water was used as negative control. Then matured shrimps were applied to each of all experimental and control vials. After 24 h, the vials were inspected using a magnifying glass and the number of survived nauplii in each vial was counted. From the data, the percent (%) of mortality of the brine shrimp nauplii was calculated for each concentration using the following formula: Mortality )%) = N1 / N0 x 100, where, Nt is Number of killed nauplii after 24 h of incubation, N0 is Number of total nauplii transferred i.e., 20. The median lethal concentration, LC50 was then determined using Probit analysis.
Statistical analysis: All data were expressed as mean±STD and were analyzed by one way ANOVA followed by using Dunnett’s test. The difference was considered significant at p<0.05.