Cyclooxygenase (COX-1 and COX-2) inhibition assay

A stock solution of F. solani fermentation extract was prepared in methanol as 1 mg/mL concentration. Test concentrations of 25, 50 and 100 µg/mL were prepared by serial dilution. The extract was tested in triplicates using a commercial COX (Ovine) colourimetric inhibitory screening assay kit following the instructions recommended by the manufacturer (Cayman test kit-760111, Cayman Chemical Company, Ann Arbor, MI) (George et al. 2014). The COX (ovine) inhibitor screening assay measures the peroxidase activity of COX which is assayed colourimetrically by monitoring the appearance of N,N,N′,N′-tetramethyl-p-phenylenediamine. Diclofenac (MW = 296.14) was used as the positive control for inhibition of COX-1 and COX-2. A mixture of 10 μL of test extract in 0.1 M Tris–HCl pH 8.0 (assay buffer), 150 µL assay buffer, 10 µL Heme and 10 µL of enzyme (COX-1or COX-2) in the inhibitor wells were pre-incubated with the enzyme at 25 °C for 10 min prior to the addition of arachidonic acid (AA). The reaction was initiated by addition of 20 μL of 10 mM AA after the addition of 20 µL of colourimetric substrate and the plate was incubated at 25 °C for another 2 min. Plate was read at 590 nm in ELISA plate reader. The reading was repeated at intervals of 2–5 min for over 30 min to determine the reaction rate and the apparent IC₅₀ value.