2.7. Anti-Inflammatory Activity Test

The test extract was given as suspension, in 0.5% of tween 80 solutions [16].

For this, 2 mL of formaldehyde was poured into a 100 mL volumetric flask and diluted up to exact 100 mL by using distilled water to prepare a 2% v/v fresh formalin solution.

Anti-inflammatory activity was evaluated by adopting the previously established formalin-induced paw edema method [7, 16–18]. Swiss albino mice were divided into 15 groups (5 animals in each group). Animals of all groups were injected with 0.2 mL of 2% v/v formalin, in the right hind paw, prepared by using distilled water.

  Group I animals (normal control) received 500 μL of 0.5% tween 80 only.

  Group II animals (formalin control) received distilled water p.o., 30 min prior to formalin injection.

  Group III, the standard reference group, was given p.o. an aqueous solution of indomethacin (5 mg/kg), 30 min prior to formalin injection.

  Groups IV and V received p.o. ethanolic extract of A. vulgaris of the tropical region, 200 mg/kg and 400 mg/kg of 0.5% tween 80, respectively.

  Groups VI and VII received ethyl acetate extract of A. vulgaris of the tropical region, 200 mg/kg and 400 mg/kg of 0.5% tween 80, respectively.

  Groups VIII and IX received p.o. ethanolic extract of A. vulgaris of the subtropical region, 200 mg/kg and 400 mg/kg of 0.5% tween 80, respectively.

  Groups X and XI received p.o. ethyl acetate extract of A. vulgaris of the subtropical region, 200 mg/kg and 400 mg/kg of 0.5% tween 80, respectively.

  Groups XII and XIII received p.o. ethanolic extract of A. vulgaris of the temperate region, 200 mg/kg and 400 mg/kg of 0.5% tween 80, respectively.

  Groups XIV and XV received p.o. ethyl acetate extract of A. vulgaris of the temperate region, 200 mg/kg and 400 mg/kg of 0.5% tween 80, respectively.

All the extracts were administered 30 min prior to formalin injection. The treatment was continued for three consecutive days at a fixed time but formalin was injected only on the first day. The paw volume of the mice was measured by using a vernier caliper and observed before and 1 h, 2 h, 3 h, 24 h, 48 h, and 72 h after formalin injection. The percentage inhibition of edema was calculated as follows: