2.6. In Vitro COX-2 Inhibitory Assays

COX-2 inhibition assay in vitro was performed using COX-2 (human) inhibitor screening assay kits according to the manufacturer’s instructions to evaluate the COX-2 inhibition activity of compounds and verify the results of UF-LC-MS/MS [26,27]. Firstly, samples were dissolved in DMSO, and prepared into a series of solutions with different concentrations. COX-2 cofactor working solution, COX-2 working solution, COX-2 probe, and COX-2 substrate were prepared according to manufacturer’s instructions, and then diluted 10 times with COX-2 assay buffer, respectively. Secondly, 150 µL Tris-HCl (pH = 7.8), 10 µL COX-2 cofactor working solution, 10 µL COX-2 working solution, and 10 µL sample solution were sequentially added in the 96-well black plates, mixed and incubated at 37 °C for 10 min. The COX-2 working solution of the blank control group was replaced with an equal volume of COX-2 assay buffer, and the sample was replaced with an equal volume of DMSO; the sample of the 100% enzyme activity control group was replaced with an equal volume of DMSO. Thirdly, 10 µL COX-2 probe was added into each well. Finally, 10 µL of COX-2 substrate was quickly added into each well, and incubated at 37 °C in the darkness for 5 min, and followed by the fluorescence measurement. The excitation and emission wavelengths were 560 nm and 590 nm, respectively. Indomethacin was set as a positive control. The experiments were performed in triplicate. The COX-2 inhibitory activity was expressed as IC₅₀. The inhibition of COX-2 was calculated according to the following formula:

where COX-2 inhibitory activity (%) was plotted against the concentration of samples to acquire the IC₅₀. RFU_100%Enzyme, relative fluorescence unit of 100% enzyme control group; RFU_Sample, relative fluorescence unit of sample; RFU_Blank, relative fluorescence unit of blank group.