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

Tested compounds were dissolved in DMSO. Each compound was tested in triplicates using a COX inhibitory screening assay kit according to the manufacturer (Cayman test kit-560131, Cayman Chemical, Ann Arbor, MI). The COX inhibitor screening assay depends on direct measurement of the amount of PG2α produced in the COX reaction. Celecoxib, rofecoxib, and indomethacin were used as the positive control for inhibition of COX-1 and COX-2. An aliquot of 20 µl of each test compound or solvent (100% initial activity) was added to 950 µl of Reaction Buffer (0.1 M Tris–HCl, pH 8.0, containing 5 mM EDTA and 2 mM phenol), 10 µl of haem, and 10 µl of COX-1 or COX-2, then incubated with the enzymes at 37 °C for 10 min. The reaction was initiated by addition of 10 µl AA to all the test tubes and incubation at 37 °C for an additional 2 min. Enzyme catalysis was terminated by addition of 50 µl 1 M HCl and 100 µl of the saturated stannous chloride solution. The PGs are quantified by enzyme immunoassay (EIA) at 410 nm.