Formalin induced paw licking test

The procedure was similar to that described by Hunskaar and Hole[18] and Okokon and Nwafor.[19] Adult albino rats (23 – 27 g) of both sex randomised into five groups of 3 rats each were used for the experiment. The animals were fasted for 24 h before administration (p.o) but allowed access to water. The animals in Group 1 (negative control) received 10 ml/kg of normal saline, Group 2 received 100 mg/kg of diclofenac Na (positive control) while Groups 3-5 received 450, 900 and 1350 mg/kg doses of the seed extract, 30 min before pain was initiated with buffered formalin administration. A 20 μl of 2.5% formalin solution (0.9% formaldehyde) made up in phosphate buffered saline (PBS) of pH 7.2 (PBS concentration: NaCl, 100 mM, and phosphate buffer, 25 mM) was injected subcutaneously under the surface of the right hind paw of each animal. The number of times spent licking the injected paw was noted down and considered as indication of pain. The first of the nociceptive response normally peaks 5 minutes after injection and the second phase 15-30 min after formalin injection, representing the neurogenic and inflammatory pain responses, respectively. The responses were measured for 5 min after formalin injection (neurogenic response) and 15–45 min after formalin injection (inflammatory response). The experiment was conducted under conditions of no disturbance that may affect animals’ response. Percentage inhibition was determined by comparing the results of extract with control group using the following formula;

Percentage inhibition = (A − T)/A × 100

Where A is the average number of paw licking of control and T is the average number of paw licking of test group.