Effects of Naturally Occurring Flavonoids on Nitric Oxide Production in the Macrophage Cell Line RAW 264.7 and Their Structure-Activity Relationships

Flavonoids affect the inflammatory process of the mammalian system and possess antiinflammatory as well as immunomodulatory activities in vitro and in vivo. Since nitric oxide (NO)produced by inducible nitric oxide synthase (iNOS)is one of the inflammatory mediators, the effects of various naturally occurring flavonoids on NO production in LPS-activated RAW 264.7 cells were evaluated in vitro. Flavonoids such as apigenin, wogonin, luteolin, tectorigenin, and quercetin inhibited NO production, as measured by nitrite formation at 10-100 mM. The most active among 26 flavonoid derivatives tested were apigenin, wogonin, and luteolin, having IC50 values of 23, 17, and 27 mM, respectively, while AMT, a synthetic selective iNOS inhibitor, had an IC50 value of 0.09 mM. In contrast, flavanones, such as naringenin, and flavonoid glycosides, such as apiin, did not demonstrate significant inhibition up to 100 mM. These results clearly indicated that a C-2,3 double bond might be important, and that the potency of inhibition depended upon the substitution patterns of the flavonoid molecules. The inhibitory activity of flavonoids was not due to direct inhibition of iNOS enzyme activity because they did not reasonably inhibit iNOS activity, as measured by [3H]citrulline formation from [3H]arginine, up to 100 mM. In contrast, wogonin and luteolin concentration-dependently reduced iNOS enzyme expression, when measured by western blotting, at 10-100 mM. All these results clearly demonstrated that certain flavonoids inhibit NO production in ipopolysaccharide-activated RAW 264.7 cells, and their inhibitory activity might be due to reduction of iNOS enzyme expression.