Plant materials: The fresh plant was collected from local area of Chittagong, Bangladesh and identified by Dr. Sheikh Bokhtear Uddin, Department of Botany, University of Chittagong.
Preparation of extract: The collected fresh plant leaves were washed thoroughly with water and then air dried for a week at 35-40°C and pulverized in electric grinder. The obtained powder was successively extracted in methanol and filtered by Whatman filter paper. The filtrate so obtained was then concentrated to dryness through the evaporation of solvent using rotary evaporator under reduced pressure.
Animals: The study was conducted on Swiss albino mice purchased from International Centre for Diarrheal Disease and Research, Bangladesh (ICDDR, B). They were five to six weeks of age, weighing about 30-35 g, which were housed in colony cages (six mice per cages) at an ambient temperature of 23±2°C and relative humidity 50-60% with 12 h light and dark cycles having proper ventilation in the room. The mice were fed normal diets purchased commercially from the vendors and water ad libitum. The animals were allowed to acclimatize to the laboratory environment for one week and then randomly divided into groups for experiments.
Brine shrimp lethality bioassay: This assay was performed on brine shrimp nauplii. In this experiment simple zoological organism (Artemia salina) was used as a convenient monitor for the screening. The eggs of the brine shrimp were collected from an aquarium shop of Chittagong, Bangladesh and hatched in artificial seawater (3.8% NaCl solution) for 48 h to mature shrimp called nauplii. The test sample of crude extract was prepared by dissolving them in DMSO (not more than 0.01% v/v) plus sea water (3.8% NaCl in water) to attain concentrations of 12.5, 25, 50, 100, 200 and 400 μg mL-1. A vial containing DMSO diluted in seawater was used as a control. Standard vincristine sulphate was used as positive control. Then matured shrimps were applied to each of all experimental vials and control vial. After 24 h, the vials were inspected using a magnifying glass and the number of survived nauplii in each vial was counted. From the obtained data, the percent (%) of mortality of the brine shrimp nauplii was calculated. The median lethal concentration, LC50 was then determined using Probit analysis.
Hole cross test: This test was performed for screening sedative activity in mice. The animals were divided into three groups-control, positive control and test. The test groups received methanolic extract of G. multiloculare leaves at the doses of 200 mg kg-1 body weight (b.wt.) orally whereas the control group received vehicle (1% Tween 80 in water) at dose of 10 mL kg-1 per oral (p.o). 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 other was counted for a period of 3 min on 0, 30, 60, 90 and 120 min after the oral treatment with test drugs. In this test diazepam was used in the positive control group as reference standard at the dose of 1 mg kg-1 intra peritoneal (i.p).
Open field test: The experiment was observed according to standard method. The dose for extract (200 mg kg-1 b.wt., p.o.) for vehicle (1% Tween 80 in water, 10 mL kg-1 p.o.) and for standard (Diazepam-1 mg kg-1, p.o.) was maintained throughout the experiment. The floor of an open field of half square meter was divided into a series of squares each alternatively colored black and white. The wall of this apparatus was 40 cm height. During the study period the total number of squares visited by the mice was counted for 3 min on 0, 30, 60, 90 and 120 min.
Elevated plus maze test: The instrument used here consists 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 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. Dark opaque wood was used to make maze floor and walls. Sixty minutes after administration of the test drugs; 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 entry and duration of staying into open arms was recorded. The entry into an arm was defined as the point when the animal places all four paws onto the arm. The sound free room and observations were made from an adjacent corner was conducted. The same dose and route of hole cross test was used for this test.
Thiopental sodium induced sleeping time test: Animals were randomly divided into three groups consisting of five mice each. The test groups were received methanolic extract of the leaves of G. multiloculare at dose 200 mg kg-1 (p.o.) body weight while the standard group was treated with diazepam (1 mg kg-1, p.o.) and control group with vehicle (1% Tween 80 in water, 10 mL kg-1 b. wt., p.o.). Twenty minutes later, thiopental sodium (40 mg kg-1, i.p.) were administered to each mouse to induce sleep. During the latent period (time between thiopental administrations to loss of righting reflex) and duration of sleep i.e., time between the loss and recovery of righting reflex the animals were observed.
Statistical analysis: All obtained data were expressed as mean±standard deviation (n = 5) and were analyzed by one way ANOVA followed by using Dunnett’s test. The differences were considered significant at p<0.05.