2.1 Collection and Proper Identification of Plant The leaves of S. pulcherrima were collected from hill tract area of Banshkhali, (22.04: latitude and 91.95: longitude), Chittagong district, Bangladesh. Collected leaves were identified and authenticated by Dr. Shaikh Bokhtear Uddin, Associate Professor, Department of Botany, University of Chittagong.
2.2 Preparation of Extract The leaves were dried for a period of 2 weeks under shade at room temperature and made fine powder by grinding them in a suitable grinder. Then the powdered leaves (250 gm) were soaked in sufficient amount of methanol (1:3) for one week with proper shaking and then filtered through a cotton plug followed by Whitman filter paper number 1. The solvent was evaporated at 45ºC temperature in water bath to yield methanolic semisolid crude extract that was preserved in a refrigerator at 2-4ºC for further use. About 25 to 30 gm of extract was found with the yield value of 10-12%.
2.3 Experimental Animals Swiss Albino mice weighing 25-30 gm of both gender were collected from International Center for Diarrheal Diseases Research, Bangladesh (ICDDRB) and housed in polypropylene cages under controlled conditions. The animals were exposed to alternative 12:12 hours light and dark cycle at an ambient temperature of 26±2ºC. Animals were allowed free access to drinking water and pellet diet. Mice were acclimatized for 7 days in the laboratory environment prior to the study.
2.4 Hole Cross Test The hole cross test was performed according to the method, described by Takagi (1971), for screening CNS depressant activity in mice. The animals were divided into three groups as control, positive control and test. The test groups received methanolic extract of S. pulcherrima at the doses of 200 and 400 mg/kg, p.o. separately whereas the control group received vehicle (1% Tween 80 in water) with the dose of 10 ml/kg, p.o. In the test, a wood- made cage having a size of 30 × 20 × 14 (cm) with a fixed partition in the middle of the cage having a hole of 3 cm in diameter at the lower side of partition. In the determination of in-vivo sedative effect the number of passage of a mouse from one chamber to another was counted for a period of 3 minutes at 0, 30, 60, 90 and 120 min. after oral administration of extract solution. Standard diazepam (1 mg/kg, i.p.) was used as positive control. The results were represented by graphical representation plotting the number passage against the respective groups of mice.
2.5 Open Field Test The open field test was performed according to the method, described by Gupta (1971), where the animals were divided into control, positive control and test groups. The test groups received S. pulcherrima methanolic leaf extracts at the doses of 200 and 400 mg/kg body weight orally whereas the control group received vehicle (1% Tween 80 in water) with the dose of 10 ml/kg, p.o. The floor of an open field having a size of 16×16 square inch. was divided into 16 square areas with black and white colored areas alternatively. The apparatus had a 40 cm height wall. To determine in-vivo sedative effect in this test the number of square areas, passed by a mouse, were counted for a period of 3 minutes at 0, 30, 60, 90 and 120 min. after oral administration of extract solution or administration of reference standard drug. In current study diazepam (1 mg/kg, i.p.) was used as reference standard drug. The results were displayed by graphical representation by plotting the number of square areas, passed by the mice, against the respective group of mice.
2.6 Thiopental Sodium Induced Hypnosis Test Thiopental sodium induced hypnosis was performed according to the method, described by Ferrini, where the animals were randomly divided into three groups as control, standard and test groups consisting of 3 mice each. The test groups received methanol extract solution of the leaves of S. pulcherrima at the dose of 400 mg/kg (p.o.) and 200 mg/kg (p.o.) while the standard group was treated with diazepam (1 mg/kg, i.p.) and control group with vehicle (1% Tween 80 in water) at the dose 10 ml/kg, (p.o.). Twenty minutes later, thiopental sodium (40 mg/kg, i.p.) were administered to each mice to induce sleep. The animals were observed for about 3 hours while the latent period (time between thiopental administrations to loss of righting reflex) and duration of sleep (time between the loss and recovery of righting reflex). The results were expressed by graphical representation by plotting the time of onset of sleep versus respective group of mice the duration of sleep versus respective group of mice.
2.7 Elevated Plus-Maze (EPM Test) The elevated plus maze (EPM) is a rodent model of anxiety that is used as a screening test for putative anxiolytic or anxiogenic compounds and as a general research tool in neurobiological anxiety research. The EPM apparatus consists of two open arms (5×10 cm) and two closed arms (5×10×l5 cm) radiating from a platform (5×5 cm) to form a plus sign figure. The apparatus was situated 40 cm above the floor. 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. 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 5-min test period, the number of open and enclosed arms entries, plus the time spent in open and enclosed arms, was recorded. Entry into an arm was defined as the point when the animal places all four paws onto the arm. The procedure was conducted in a sound-attenuated room; observations made from an adjacent corner.
2.8 Statistical Analysis The data was expressed as mean ± standard error of mean (S.E.M.). Statistical comparisons were performed using one-way ANOVA followed by Dunnett’s multiple comparison test. The values obtained were compared with the vehicle control group and were considered statistically significant when P<0.05.