3.1. In Vitro Cyclooxygenase Inhibitor Assay

The cyclooxygenase inhibitory potential of compounds A1–13 was assessed using the COX inhibitor Screening Assay Kit (Catalog No. 560131, Cayman Chemical, Ann Arbor, MI, USA). The kit utilizes an enzyme immunoassay (EIA) in order to quantify the prostanoid product resulted from COX catalyzed reaction. The inhibitory potential of our molecules was tested against the ovine COX-1 and human recombinant COX-2 enzymes. The COX inhibition reaction was performed by a 10 min incubation at 37 °C in the presence of reaction buffer, heme, COX-1 or COX-2 enzymes, and the tested inhibitor. The reaction was initiated by adding arachidonic acid and incubating at 37 °C for 2 min. Enzymatic catalysis was stopped by adding HCl. Stannous chloride was subsequently added to perform the reduction of COX-derived prostaglandin H2 (PGH2) produced in the reaction of COX leading to prostaglandin F2α (PGF2α). PGF2α was then quantified using the EIA kit. Prostanoid containing solutions obtained from the COX reaction were then diluted and transferred to plates that were pre-coated with monoclonal anti-rabbit IgG antibodies produced in mouse. They were incubated overnight in the presence of PG-acetylcholinesterase (AchE) conjugate and specific PG antiserum. The reaction mixture was then removed from the wells, the plates were washed to remove all unbound reagents and Ellman’s reagent (which contains the AchE substrate) was then added for plate development. After a 1 h incubation period, the yellow product of the AchE reaction was measured spectrophotometrically at 412 nm. The intensity of the color is inversely proportional to the amount of PG found in the sample. The PG quantity was determined using a PG standard curve generated on the same plate. All determinations were performed according to the manufacturer’s instructions and similar with other literature reports [30,31,32,33]. The IC₅₀ values were calculated using a sigmoidal concentration-inhibition response curve (duplicate determinations). Every compound was assayed in the COX reaction on a range of concentrations from 0.03 μM to 300 µM in two distinct determinations. Each COX reaction sample was then EIA assayed at two dilutions and each dilution was tested in duplicate. Meloxicam, celecoxib and indomethacin provided by Cayman Chemical were used as reference compounds. Using IC₅₀ values, the selectivity index (SI) was calculated as the ratio IC₅₀COX-1/IC₅₀COX-2.