3.4. COX-1 and COX-2 Inhibition Assays

Inhibitory potency of the compounds on COX-1 and COX-2 enzymes was determined by using fluorimetric COX-1 and COX-2 inhibition screening kits from BioVision (Milpitas, CA, USA). The experimental protocol as described in the guides provided by the supplier was followed [51,52]. All of the pipetting were performed using a robotized system to increase speed, accuracy and precision. Fluorescence intensities of the samples were kinetically measured at 25 °C for 5–10 min by monitoring emissions at 587 nm after excitation at 535 nm. Two appropriate points (T1 and T2) in the linear range of the plot were chosen and the corresponding fluorescence values (RFU1 and RFU2) were obtained. The slope for all samples including Enzyme Control (EC) was calculated by dividing the net ΔRFU (RFU2 − RFU1) values by the time ΔT (T2 − T1). Percentage of relative inhibition was calculated by using the following equation:

where S is the term representing the tested compound [51,52]. The initial in vitro assays were performed at 10⁻³ M and 10⁻⁴ M concentrations for all of the test compounds; the ones which showed inhibition above 50% were assayed by the same protocol within a wider range of concentrations (10⁻⁵ M and 10⁻⁹ M) to determine their IC₅₀ against COX-1 and COX-2 enzymes. The IC₅₀ values were calculated using the plots of the enzyme activity against concentrations by applying regression analyses.