2.8. Cyclooxygenase-2 (COX-2) Inhibition Assay

The COX inhibition screening assay directly measures PGF2α by stannous chloride reduction of COX-derived PGH2 produced in the COX reaction [24]. The reaction system consists of reaction buffer, haem, enzyme, and plant extract preincubated at 37°C for 20 min with background and enzyme controls. The reaction was initiated with the addition of arachidonic acid and incubated for 2 min at 37°C. The reaction was stopped with the addition of saturated stannous chloride solution for 5 min at room temperature. The prostaglandins were quantified by enzyme immunoassay technique (EIA). An aliquot of these reactions was added to the precoated plates in triplicate together with acetylcholinesterase (AChE) tracer and antiserum and incubated for 18 hours at room temperature on an orbital shaker. The plate was then finally developed with Ellman's reagent and kept on an orbital shaker in the dark at room temperature for 60 minutes. The absorbance was read at 420 nm. The data were plotted as %B/B0 (standard bound/maximum bound) versus log concentration using a 4-parameter logistic curve fit. The concentration of each sample was determined from a standard curve with appropriate dilutions and used to calculate the percent inhibition as per the following formula:

The results were expressed as IC₅₀, i.e., concentration of the extracts and controls that resulted in 50% COX-2 inhibition plotted on a graph.