Collection of plant materials: The leaves of Clerodendrum indicum Linn. were collected from Baldha Garden, Dhaka, Bangladesh on 2009 and its aerial part was identified taxonomically by the experts of Bangladesh National Herbarium, Mirpur, Dhaka (Accession No. DACB 31184).
Preparation of plant extracts: The leaves were dried by shed drying for about one week and then grounded into a coarse powder (1.0 kg) with the help of a suitable blender. Two hundred and fifty grams of powered material was extracted with 850 mL of 80% ethanol for 17 days with occasional shaking and stirring upto 2 inch height above the sample surface as it could sufficiently cover the sample surface. Then, it was filtered through whatman filter paper. After filtration the remaining portion of the plant extract was given for re-extraction for 7 days with another 250 mL of ethanol. The liquid extract was then concentrated on a water bath to give a greenish black type residue of 3.2 g (yield 2.13%).
Experimental animals: The experiments were carried out on 20 swiss albino mice aged 4 to 5 weeks and weighing 25 to 30 g of both sexes collected from the Animal Research Branch of the International Centre for Diarrhoeal Disease and Research, Bangladesh (ICDDRB). They were housed under room temperature, relative-humidity and light/dark cycle and were fed ICDDRB formulated rodent food and water ad libitum. Before starting of the experiment, all the mice were subjected to acclimatize for one week under standard experimental conditions (relative humidity 55 to 65% room temperature 25±2.0°C and 12 h light/dark cycle).
Chemicals and standard drug: Acetic acid and ethanol were collected from LOBA Chemicals Pvt. Ltd., India. Diclofenac sodium was obtained from Incepta Pharmaceuticals Ltd., Bangladesh. All the chemicals used in the entire study were of analytical grade.
Preliminary phytochemical screening: Preliminary phytochemical analysis of the ethanolic extract of Clerodendrum indicum was carried out based on the standard methods to identify the presence of alkaloid, steroid, flavonoid, tannin, reducing sugar, saponin and gum.
Experimental model: The antinociceptive activity of Clerodendrum indicum leaves extract was measured by acetic acid induced writhing method as previously described with minor modifications. In the writhing model, 1% acetic acid was used to induce pain through intraperitoneal administration. For experiments with Clerodendrum indicum Linn. leaves extract, mice were randomly divided into four groups of five mice each where Group-1 served as control and was administered 1% Tween solution in water (10 mg kg-1 body weight). On the other hand, Diclofenac sodium (25 mg kg-1 body weight) was administered to Group-2. In addition to these, Groups 3 and 4 received ethanolic extract of Clerodendrum indicum leaves at doses of 250 and 500 mg kg-1 body weight, respectively before intraperitoneal administration of acetic acid. A period of 5 min was given to each mouse to make sure the bioavailability of acetic acid and the number of abdominal contractions (writhings) was observed for 5 min after stimulation for a period of 10 mins.
Statistical analysis: The percentage inhibition of writhing was obtained using the following formula:
The results of the experiment were expressed as Means±Standard Error of Mean (SEM). Student’s t-test (GraphPad Software) was used to determine a significant difference between the control and experimental groups where p values of less than 5% (p<0.05) was chosen as the level of significance.