DEVELOPMENT OF A RADIOCHEMICAL CYCLOOXYGENASE-1 AND CYCLOOXYGENASE-2 IN-VITRO ASSAY FOR IDENTIFICATION OF NATURAL-PRODUCTS AS INHIBITORS OFPROSTAGLANDIN BIOSYNTHESIS

A radiochemical enzyme assay for studying cyclooxygenase (COX)-catalyz ed prostaglandin biosynthesis in vitro was optimized with respect to b oth COX-1 and COX-2 activity. The assay can be used to assess the rela tive selectivity of plant-derived inhibitors on COX-1 and COX-2. Assay conditions were optimized for both enzymes with respect to concentrat ion of cofactors (l-epinephrine, reduced glutathione, and hematin), ac tivation time (enzyme and cofactors), reaction time, and pH. Moreover, the kinetic parameters, K-m and K-cat, of both enzymes were estimated . Five COX inhibitors were used to validate the assay, indomethacin, a spirin, naproxen, ibuprofen, and the arylsulfonamide NS-398, all with different COX selectivity and time dependency. Time-dependent inhibiti on was determined by comparing the inhibition, with and without preinc ubation of enzyme and inhibitor. Two flavonoids, (+)-catechin and quer citrin, were examined with respect to inhibition of COX-catalyzed pros taglandin biosynthesis. (+)-Catechin showed equal inhibitory effects o n the two enzymes. Quercitrin was found to be inactive toward both COX -1- and COX-2-catalyzed prostaglandin biosynthesis. The optimization p rocedure resulted in a considerable reduction of the amount of enzyme required for adequate prostglandin biosynthesis and a reliable method suited to evaluate natural products on inhibition of COX-2-catalyzed p rostaglandin biosynthesis, as well as on COX-1.