4.7. Assay of COX-2 Enzymatic Activity

The in vitro inhibitory activity of histamine, FXF, osthole, and DuP-697 on purified COX-2 enzyme was determined using a colorimetric COX inhibitor screening assay kit according to the manufacturer’s instructions (Cayman Chemical Company, Michigan, USA). Briefly, a 160 μL assay buffer and 10 μL heme were added to the background wells, while a 150 μL assay buffer, 10 μL heme, and 10 μL COX-2 enzyme were added to the 100% initial activity wells. Tested substances in 10 μL at final concentrations were added to the sample wells and 10 μL of medium RPMI was added to the background wells. The plate was carefully shaken for a few seconds and incubated for five min at 25 °C. The colorimetric substrate solution (20 μL) followed by arachidonic acid (20 μL) were added to each well. The plate was again shaken carefully for a few seconds and incubated for 5 min at 25 °C. The absorbance at 590 nm was read using a microplate reader and the inhibition ratio of COX-2 enzymatic activity was calculated according to the manufacturer’s instructions.