2.1. Plant collection, identification, and preparation of methanol extract (MEHC) The mature leaves of H. caustica were collected, with permission, in the month of October 2016, from Kaptai National Park (22°30′08″N 92°12′04″E), Rangamati district, Chittagong division, Bangladesh. After collection, the sample was identified and authenticated by Dr. Shaikh Bokhtear Uddin, Professor, Department of Botany, University of Chittagong, Bangladesh, with a voucher specimen (accession no: SBU 1622) deposited in the Herbarium of the University of Chittagong (CTGUH). After thorough cleaning, the collected leaves were cut and shade dried for a week by maintaining a suitable temperature (55–60 °C), and pulverized into a coarse powder using an automatic grinder. Thereafter, the fine powder (370 g) was soaked in 800 ml methanol for 15 days at room temperature, with temporal shaking and stirring on a Rotary Shaker, model VTRS-1 (Nunes Instruments, Tamil Nadu, India). Afterwards, the solvent extract was filtered through a sterilized cotton plug followed by Whatman filter paper No. 1. The solvent was evaporated using a rotary evaporator at room temperature to yield a semisolid extract (MEHC; 15 g), which was placed in a refrigerator until further analysis.
2.2. Experimental animals and ethical statements Adult Swiss albino mice (20–25 g) of both sexes were obtained from Jahangir Nagar University, Savar, Bangladesh. The animals were sheltered in 120 × 30 × 30 cm polypropylene cages and maintained under standard laboratory conditions (room temperature 25 ± 2 °C; relative humidity 55–60%, 12 h light/dark cycle) along with pellets (mice food) and clear water ad libitum during the adaptation period. Animals were acclimatized for 14 days and fasted overnight prior to starting all experiments. The experimental animals were managed according to the Ethical Principles and Guidelines for Scientific Experiments on Animals (1995) directed by “The Swiss Academy of Medical Sciences and the Swiss Academy of Sciences.” All tests were performed in a remote and noiseless ambiance between 9.00 a.m. and 5.00 p.m. The P&D Committee the of Department of Pharmacy, International Islamic University Chittagong, Bangladesh granted consent to all study protocols (Pharm-P&D-61/08′16–123).
2.3. Drugs and chemicals The following chemicals were used in our experiments: diclofenac sodium (Novartis Bangladesh Ltd.), morphine sulfate (Gonoshasthaya Pharmaceuticals Ltd., Bangladesh), naltrexone hydrochloride (Navana Pharmaceuticals Ltd., Bangladesh), acetic acid (Merck, Germany), methanol (Merck, Germany), formalin (Merck, Germany), methylene blue (Merck, Germany) and glibenclamide (Square Pharmaceuticals Ltd., Bangladesh). 2.4. Preliminary phytochemical screening of MEHC Qualitative phytochemical analysis of the methanol extract of H. caustica leaves was carried out following standard procedures.
2.5. Gas chromatography-mass spectroscopy (GC-MS) analysis of MEHC extract GC-MS analysis of the MEHC extract was explored using an Agilent (7890A) Technologies capillary gas chromatograph along with a mass spectrometer. The column utilized was a fused silica capillary column of 95% dimethyl-poly-siloxane and 5% phenyl (HP-5MSI; length: 90 m, diameter: 0.250 mm and film: 0.25 µm). Parameters for GC-MS detection were an injector temperature of 250 °C, initial oven temperature of 90 °C gradually raised to 200 °C at a speed of 3°C/min for 2 min and a final increase to 280 °C at 15 °C/min for 2 min. Total GC-MS run time was 36 min, with Helium 99.999% as carrier gas, used at a column flow rate of 1 ml/min. The GC to MS interface temperature was fixed at 280 °C, and an electron ionization system was set on the MS in scan mode. The mass range investigated ranged 50–550 m/z where MS quad and source temperatures were maintained at 150 °C and 230 °C respectively. The “NIST-MS Library 2009” was used to search and identify each component. Measurement of the relative percentage amounts of each compound was determined using peak area expression of the “TIC” (total ionic chromatogram), with calculations being done automatically.
2.6. Acute oral toxicity of MEHC Using standard laboratory conditions for acute toxicity testing under OECD guidelines (No, 2001), the allocated animals (n = 6) of each group (control and test) were administered a single oral dose (5, 50, 300 or 2000 mg/kg body weight) of the test extract (MEHC). Before administration of the extract, mice were kept fasting overnight, and food was also delayed for between 3 and 4 h after administration. All experimental animals were observed individually, with particular monitoring for possible unusual responses including behavioral changes, allergic syndromes (itching, swelling, skin rash), and mortality over the next 72 h.
2.7. Anti-nociceptive activity and analysis of the possible mechanism of action of MEHC
2.8. Anti-inflammatory activity of MEHC in carrageenan-induced paw oedema The anti-inflammatory response of MEHC was determined by the introduction of carrageenan into the sub-plantar surface of the right hind paws of test animals according to the method of Lanhers et al. (1991). For this experiment, experimental animals were separated into four groups (each n = 6) where Group I (control) received 1% Tween 80 in distilled water. The positive control (Group II) was treated with the standard drug (diclofenac sodium, 10 mg/kg body weight). MEHC at oral doses of 200 and 400 mg/kg body weight respectively constituted groups III and IV. Thirty minutes after treatment of all four groups, acute inflammation was induced by sub-plantar injection of a newly made carrageenan suspension (0.05 ml) with 1% Tween 80 into the right hind paw. Afterward, micrometer slide callipers were used to determine the paw volume at intervals of 1, 2, 3 and 4 h. The percentage inhibition by a test dose was calculated using the following formula: percentage inhibition of inflammation [(Vc-Vt)/Vc] × 100, where Vc refers to mean degree of inflammation with the control group and Vt the average degree of inflammation among the test groups.
2.9. Evaluation of locomotor activity of MEHC by open field test The spontaneous locomotor performance of mice was evaluated using the open field test (Archer, 1973) to verify possible nonspecific muscle relaxant or sedative effects of MEHC; the apparatus used in this test was 40 × 60 × 50 cm in size, with the floor of the apparatus divided into 25 equal squares. Mice (n = 6 per group) were administered vehicle or MEHC (200 and 400 mg/kg) 55 min before the experiment, then each mouse was placed into the centre of the test chamber, allowing free ambulation, and locomotor performance was monitored for 5 min.
2.10. Statistical analysis Data were analyzed by using SPSS 20.0 statistical software. Results were presented as mean ± SEM (standard error of mean), and one-way ANOVA followed by post hoc testing (Dunnett's and Bonferroni’s) was applied. A p-value less than 0.05 were considered as significant.