Bioassay Development for Identification of Cyclooxygenase-2 Inhibitors of Natural Origin

Abstract: To examine cyclooxygenase-2 (COX-2) inhibitory effects of natural compounds and structurally related compounds, a radiochemical assay for measuring inhibition of COX-2 and COX-1 catalysed prostaglandin biosynthesis was optimised. That process resulted in the development of a reliable assay for finding COX-2 inhibitors in plants, and for investigating plant constituents pertaining to the inhibition of COX-2 and COX-1. By means of bioassay-guided isolation of the plant Plantago major L., several inhibitors of COX-2 were found: ursolic, oleanolic, a-linolenic and linoleic acid. Subsequently, structural derivatives of these triterpenoids and fatty acids were investigated. The polyunsaturated and the thioether containing fatty acids were the most potent COX-2 and COX-1 inhibitors, with a-linolenic and all-(Z)-5-thia-8,11,14,17-eicosatetraenoic acid expressing selectivity toward COX-2. For rapid evaluation of plant constituents, a COX-2 assay using scintillation proximity assay-technology was used to evaluate 49 ubiquitous plant metabolites of different biosynthetic origin for inhibition of COX-2. Eugenol, cinnamaldehyde, and pyrogallol expressed inhibition. Later, in an attempt to find COX-2 inhibitors in plants, but without prior purification, several approaches to the surface plasmon resonance technique were examined, with the measurement of prostaglandin E2 being most successful.In mouse macrophage cells, two fatty acids were analysed for effects on COX-2 and iNOS, mRNA, protein, prostaglandin E2, and nitrite levels. 5-Thia-8,11,14,17-eicosatetraenoic acid decreased COX-2 protein expression.This thesis contributes to our growing knowledge of methods for measuring COX-2 inhibition, and also knowledge of COX-2 inhibitory effects of commonly occurring compounds in plants, herbal drugs and functional food. Thus the experimental results can facilitate future searches for new COX-2 inhibitors of natural origin.