Screening of ubiquitous plant constituents for COX-2 inhibition with a scintillation proximity based assay

A rapid semi-homogeneous cyclooxygenase-2 (COX-2) enzymatic assay using scintillation proximity assay (SPA) technology was developed, and 49 ubiquitous plant secondary metabolites were screened for inhibition of COX-2-catalyzed prostaglandin E-2 (PGE(2)) biosynthesis. Assay conditions were optimized with respect to reaction time, amount of antibody, radiolabeled PGE(2), and SPA beads, and the kinetic parameter, K-m, was estimated. The assay was validated with two natural triterpenoids, ursolic and oleanolic acid, known to inhibit COX-2, as well as with four synthetic COX inhibitors, NS-398, rofecoxib, indomethacin, and aspirin. Plant metabolites of different biosynthetic origin representing several substance classes, including alkaloids, anthraquinones, flavonoids, phenylpropanes, steroids, and terpenes, were screened for inhibition of COX-2-catalyzed PGE(2) production. Of these 49 plant metabolites, eugenol, pyrogallol, and cinnamaldehyde (with IC50 values of 129, 144, and 245 muM, respectively) were found to inhibit COX-2. This study showed that a COX-2-catalyzed PGE(2) assay using SPA is suitable for screening natural compounds with respect to COX-2 inhibition.