Drugs and reagents
Diazepam, ciprofloxacin, imipramine were obtained from Sigma-Aldrich Corporation (USA). Ethyl acetate (Merck, Germany), and other chemicals necessary for experiment were analytical graded which were taken from the laboratory of Dept. of Pharmacy, NSTU, Bangladesh.
Plant parts
Fruits of D. malabarica were collected from Bahaddar hat, Chittagong, Bangladesh on november, 2016. After collection, fruits were washed thoroughly, and later authenticated by Naimur Rahman (Taxonomist, Bangladesh National Harberium, Dhaka, Bangladesh); and the specimen was kept there for future correspondence.
Preparation of D. malabarica extract
The seeds of D. malabarica after peeling the fruits were collected. Seeds were allowed to air-dry for 15 days period with shaded condition, which was followed by grinding into course powder. Crude powder materials were macerated into 2000 ml ethyl acetate (> 99% pure) for 18 days at room temperature in a sterilized beaker which was wrapped by aluminum foil to avoid direct exposure of sunlight (cold extraction). After the incubation period, solution was filtered through filter cloth and later by Whatman filter paper. Filtrate was allowed to evaporate by the rotary vacuum evaporator under the reduced pressure to get concentrated semi-solid filtrate. After few days of drying in room temperature, we found the brownie granular sticky substance which was designated as crude ethyl acetate extract of D. malabarica [10].
Experimental animals
Swiss albino mice (25 ± 5 g) were procured from the animal house of Jahangirnagar University, Bangladesh. These were kept in metal cages (condition: 20 ± 5 °C and light/dark cycle for 12 h), and provided to feed on rodent foods and water ad-libitum from seven days before the commencement to finish the experiment.
Test microorganisms
We have used Staphylococcus aureus ATCC 25923, Salmonella typhi ATCC 14028, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 for studying antibacterial activities; these strains were collected from the laboratory of the Department of Microbiology, Noakhali Science and Technology University (NSTU), Bangladesh.
Behavioral studies of anxiolytic activities
Open field test (OFT)
The OFT apparatus was built using white plywood (72 cm × 72 cm × 36 cm). Mice can be seen from the outside wall (made by glass) of the apparatus. 16 squares were drawn on the floor for observing the movement of mice [11]. Mice were divided into four groups (each group consists of 5 mice): control (distilled water: 0.1 ml/mice, oral), standard (diazepam: 1 mg/kg b.w. of mice, i.p.), D. malabarica (200 mg/kg b.w. of mice, oral), and D. malabarica (400 mg/kg b.w. of mice, oral). After administration of the respective doses, every mouse was observed for 3 min periods at the time of (0, 30, 60, 90, and 120 min) to count the number of squares crossed. After finishing a trail, the OFT apparatus was wiped by (10%) ethanol for cleaning.
Hole cross test (HCT)
We have followed the process described by Hossain et al. for HCT [12]. A wood partition was set in center of the cage (30 cm × 20 cm × 14 cm). There was a hole (D- 3 cm) at 7.5 cm above from the ground in center of every partition. Animals were divided into four groups (each group consists of 5 mice): control (distilled water: 0.1 ml/mice, oral), standard (diazepam: 1 mg/kg b.w. of mice, i.p.), D. malabarica (200 mg/kg b.w. of mice, oral), and D. malabarica (400 mg/kg b.w. of mice, oral). Number of transit of mice among the chambers through the hole was recorded for 3 min spell at 0, 30, 60, 90 and 120 min after administering the samples and drug.
Elevated plus-maze (EPM) test
EPM test is a widely accepting and authentic research study for finding the new drug with potential anxiolytic effects. The details methodology of this study was described in our previous paper, Rashid et al. [13]. The apparatus consists of two open arms (35 cm × 5 cm × 35 cm) crossed by two closed arms of similar size which are interconnected by a central square of (5 cm × 5 cm). The experimental room was dimly illuminated, and EPM apparatus was kept on approx. 40 cm higher from the ground level. Experimental mice were grouped (each group consists of 5 mice) as control (distilled water: 0.1 ml/mice, oral), standard (diazepam: 1 mg/kg b.w. of mice, i.p.), D. malabarica (200 mg/kg b.w. of mice, oral), and D. malabarica (400 mg/kg b.w. of mice, oral). 1 h after treatment, animal was taken individually on the apparatus, and the number of entries in every arm was registered for 5 min spells. After finishing each session, EPM apparatus was cleaned by ethanol (70%) and allow drying for few minutes.
Hole board test (HBT)
In HBT, head-dipping is generally considered as a measure of exploitation which is somehow distinct from the motor activity. An increase count of head dips compared to control is considered as anxiolytic-like effect. HBT was performed in a box (40 × 40 × 25 cm) made by wood with 16 equidistant holes (D- 3 cm) was used in this experiment. Apparatus was kept at 35 cm above from the ground [14]. Mice were selected and grouped into 4 groups randomly (each group consists of 5 mice) and administered different samples accordingly. Mouse was kept on board, and its movement and head dipping in the hole was counted for 5 min duration. A single head-dip was counted when a mouse put into the hole at least up to the eye level; repeated dips into same hole were not consider as countable head dips if they can’t be separated by locomotion.
Light--dark transition (LDT) test
LDT test is used for assessing anxiety-like behavior of animal. Natural aversion to bright illumination and exploration in mild stressors of the mice are the basis of this test. We have followed the process described by Hascoet and Bourin for this study [15]. LDT test apparatus consists of a box (42 cm × 21 cm × 25 cm), in where there are separated dark chamber and brightly illuminated chamber. Mice were taken in light compartment and allowed to move freely through a (3 cm × 4 cm) opening. Animal were select randomly and provide the respective dosages accordingly. Then, the residual time in each chamber for every mouse were recorded.
Antidepressant activity test
Forced swimming test (FST)
FCT is another common rodent behavioral model for the exploration of new antidepressant compounds. We have followed the FST method described by Con et al. with minor modification [11, 16]. Cylindrical tanks (30 cm × 20 cm) made of glass were used in where water level was kept almost 15 cm from the bottom of tank. We had recorded the immobile time of each mouse (time to keep the head above the water) for 360 s (6 min). The last 240 s data from this recorded period were considered for analysis. Data was taken for each mouse of every group after treating with respective dosages. We used a stopwatch (can measure milliseconds) for counting time.
Tail suspension test (TST)
TST (a well-known behavioral test of mice) method was adopted from Steru et al. [17]. TST box is made of plastic (Dimension: 55 cm × 60 cm × 11.5 cm). Mouse was suspended from middle of the compartment, so that it can’t attach with any wall. Mice were selected and grouped into 4 groups randomly (each group consists of 5 mice) and administered different samples accordingly. Mouse was hung by its tail which was attached on a string (75 cm above the surface) by the help of adhesive tape. Immobile time was counted when mouse hung motionlessly. Observation was done for 6 min period; and the immobile time during this period was recorded.
Antimicrobial activity test
Antibacterial efficacy of the ethyl acetate extract of D. malabarica seeds were assessed through (Kirby-Bauer’s disc diffusion method) by following Rashid et al. [18]. Samples were prepared by dissolving them in relevant solvents. Sterilized paper discs (D- 6 mm) of the samples were impregnated into the swab plates (Muller-Hinton agar media) containing microorganisms [2 × 106 colony forming units (CFU/mL)]. Aliquot of 50 μL crude extract (concentration: 500 mg/mL) was added in each disc. These plates were stand at 4 °C for 2 h which was followed by incubation at 37 °C for 24 h. Zone of inhibition (in millimeter) on the plates were measured for assessing the antibacterial efficacy of crude extract. Ciprofloxacin (5 μg/disc) and blank (solvents) discs served as positive and negative control respectively.
Statistical analysis
Data found in the experiment was analyzed statistically using one-way ANOVA followed by Dunnet’s t-test. *p ≤ 0.05 was considered significant, whereas **p ≤ 0.001 was highly significant value. Origin Pro (ver. 8.5, Origin Lab. Corp., USA) was used for preparing graphical presentations.