| dc.description.abstract | Cyclooxygenase (COX) is a key enzyme required for the conversion of arachidonic acid (AA) to various prostaglandins (PGs), thromboxanes (TXs), and hydroxyeicosatetraenoic acids (HETEs), by which AA exert numerous biological actions in the body such as inflammation and platelet aggregation. Therefore, the regulation of the levels of these autacoids is crucial for normal physiological functions. Experiments were performed to investigate the hypothesis that some of the bioflavonoids are naturally-occurring, physiological co-substrates and activators for COX I and II in the body, and to determine the mechanisms by which bioflavonoids modulate the catalytic activity of COXs. To investigate the effect of bioflavonoids on COX-mediated AA metabolism, a total of 20 naturally-occurring bioflavonoids were first tested for their ability to modulate the catalytic activity of COXs in vitro. Some of these bioflavonoids, such as quercetin, myricetin, fisetin and morin, were found to be powerful direct stimulators of the catalytic activity of COXs in vitro assays, increasing the formation of PG products by up to 11-fold over the controls. Additional studies using intact cells in culture showed that some of these dietary compounds also stimulated the formation of PGE2 (a representative PG). Two representative dietary bioflavonoids, quercetin and myricetin, were further studied to determine if they could modulate the plasma and tissue levels of PG products in normal Sprague-Dawley rats. Both compounds strongly stimulated the formation of several representative PG products in vivo, in time- and dose-dependent manners. Computational modeling studies further revealed that bioflavonoids could bind to the peroxidase active site of COXs and directly interact with the hematin component of COXs and facilitate the electron transfer from the bioflavonoids to hematin. Biochemical analysis and site-directed mutagenesis experiments were conducted to verify these computational findings. Biochemical analysis revealed that when the cyclooxygenase activity of COXs was selectively inhibited by chemical inhibitors, myricetin could still stimulate the conversion of prostaglandin G2 to PGE2, catalyzed by the peroxidase activity. Using the site-directed mutagenesis assay, it was found that Q189 at the peroxidase site of COX II was essential for the binding of bioflavonoids. Additional computational molecular modeling and structure-activity relationship studies revealed that the hydroxyl groups in the B-ring of various bioflavonoids played a crucial role in stimulating the COX's catalytic activity. Galangin, a representative bioflavonoid without a hydroxyl group in its B-ring, could function as an inhibitor of the catalytic activity of COXs both in vitro and in vivo when the enzymes were stimulated by quercetin. In conclusion, some of the bioflavonoids, such as myricetin, quercetin, fisetin and morin, were found to have a powerful direct stimulatory effect on the COX I and II activity at physiologically-relevant doses. Based on the results obtained from these studies, it was suggested that one of the important biological functions of bioflavonoids in the human body might be to serve as the naturally-occurring co-substrates for the COX enzymes through binding tightly into the peroxidase active site, and interacting directly with the hematin component of the COX enzymes to facilitate the electron transfer from bioflavonoids to hematin. Besides, some of the dietary compounds with no hydroxyl group on their B-rings, such as galangin, can function as inhibitors of COXs. These studies provide a platform for the future development of novel modulators (stimulators or inhibitors) of the human COX I and II activity. | |
