Experimental animals Swiss albino mice (20-25 g) of both sexes were obtained from International Centre for Diarrhoeal Disease Research and Jahangirnagar University, Bangladesh. Animals were housed in groups of six in cages (40 cm x 30 cm x 17 cm; made up of polypropylene base and stainless-steel net) under a standard 12 h light: 12 h dark cycle (light phase 7 a.m. – 7 p.m.) in a room maintained at 23-25ºC and at approximately 50-55% relative humidity. Food and water were allowed ad libitum during the study period. In all experiments, mice were divided into four groups and each group consisted of six mice. All animal procedures and experimental protocols were approved by the Departmental Research Ethics Committee of the institution.
Plant materials The leaves of A. calcarata were collected from botanical garden, Dhaka, Bangladesh and were authenticated by National Herbarium, Dhaka, Bangladesh. The leaves were sorted, cleaned, dried at room temperature and finally pulverized. About 400 g powdered plant material was taken separately in a clean, flat bottomed glass container and soaked in 1500 ml of 80% methanol at room temperature for fifteen days with occasional shaking and stirring. Then the solution was filtered using filter cloth and Whatman filter paper No. 1 and concentrated with a rotary evaporator (RE-EV311- V, LabTeck S.R.L, Italy). It rendered a gummy concentrate of greenish black color. The gummy concentrate was designated as a crude methanolic extract.
Chemicals and drugs Methanol (Merck, Germany) was used as a solvent during extraction. 0.6% acetic acid (98% v/v) (Loba Chemie, India) aqueous solution was prepared and was used to induce writhing on mice. Formaldehyde, 37% (Loba Chemie, India) was used for the preparation of 2.5% formalin. Aspirin (dose used: 100 mg/kg.bw; Albion Laboratories Limited, Bangladesh) was used as a positive control or standard during the study (acetic acid induced writhing and formalin-induced paw licking test); diazepam (1 mg/kg.bw) for neuropharmacological tests was also collected from the same source. Distilled water was used as control or vehicle. Distilled water, aspirin or diazepam and plant extracts were administered orally.
Open field test (OFT) Open field test was performed in a box (72 cm x 72 cm x 36 cm) made of plywood and clear Plexiglas. The floor of the box was divided and marked into sixteen (18 cm x 18 cm) squares. Controls and extracts were administered orally 30 min prior to the test. Each mouse was then put into the apparatus and the number of square blocks visited by each mouse was calculated for 5 min on 0, 30, 60, 90 and 120 min intervals (Consolini et al., 2006).
Elevated plus maze test (EPM) The apparatus used for elevated plus maze test was comprised of two open arms (35 cm x 5 cm) across from each other and perpendicular to two closed arms (35 cm x 5 cm x 15 cm) with a center platform (5 cm x 5 cm) with minor variations in the related literature (Hritcu et al., 2011). The open arms were exposed having no wall whereas the closed arms were enclosed by walls 15 cm high. Each mouse was placed in the center area of the maze with its head directed toward a closed arm after 1 h of treatment with samples and allowed to move freely about the maze for 5 min. After each trial, all arms and the center area were cleaned with 10% ethanol.
Light-Dark test (LDT) The apparatus used for the light/dark transition test consisted of a cage (21 cm x 42 cm x 25 cm) divided into two sections by a partition with door (7 cm x 7 cm); the light compartment is 2/3 of the box, is brightly lit and open, the dark compartment is 1/3 of the total box and is covered and dark. Experimental animals were kept for an hour in a dark testing room to adapt with new environment before subjected to light-dark test. Then, each mouse was placed at the center of the light compartment with its back to the dark compartment, and then transition behavior over 5 min was observed with the following parameters- time spent in light compartment time and number of transitions. Typically, mouse was expected to move around the periphery of the compartment until they find the door. All four paws must be placed into the opposite chamber to be considered an entry.
Statistical analysis Data were presented as mean ± SEM values. One-way ANOVA with Dunnett’s test was performed using GraphPad Prism (version 8.3). A probability level of 0.05 (adjusted P value according to GraphPad Prism) or less was accepted as significant; a p < 0.05, b p < 0.01, c p < 0.001 vs. vehicle; α p < 0.05, β p < 0.01, γ p < 0.001 vs. either aspirin or diazepam.