4.5. Cyclooxygenases (COX 1 and 2) Assays

The Cyclooxygenase enzymes (COX-1 and 2) inhibitory potential of our test compounds was determined according to the previously reported procedure [64]. Initially, different concentrations of the test compounds, ranging from 62.50 to 1000 μg/mL, were prepared. The enzyme solutions with a concentration of 0.7–0.8 μg/10 μL (COX-1) and 300 U/mL (COX-2) were prepared. To start the assay, for enzyme activation, 10 µL enzyme solution (kept in a cool temperature of 4 °C for 5 min) and cofactor containing hematin (50 µL, 1 mM), glutathione (0.9 mM) and TMPD in a Tris-buffer (0.1 M) with a pH 8.0 was added. After that, 60 µL of the enzyme solution was added to 20 µL of the test samples of various concentrations and incubated at room temperature for 5 to 10 min. Then, 20 µL of arachidonic acid (30 mM) was added to start the reaction. The mixture was then again incubated for 15 min at 37 °C, and absorbances were measured at 570 nm with UV-visible spectrophotometer. In this assay, the standard drug was aspirin and celecoxib for COX-1 and COX-2 enzymes, respectively. The % enzyme inhibitions were calculated by using the previously reported formula.