Inhibition of inducible nitric oxide synthase by sumaflavone isolated from Selaginella tamariscina.

We previously reported that amentoflavone isolated from Selaginella tamariscina inhibits the production of nitric oxide (NO) in macrophages via nuclear factor-kappaB (NF-kappaB) inactivation. Here, we isolated two other bi-flavonoid compounds (sumaflavone and robustaflavone) from Selaginella tamariscina and studied their effects on inducible nitric oxide synthase (iNOS) gene expression. Sumaflavone inhibited NO production in a concentration-dependent manner and blocked the lipopolysaccharide (LPS)-induced expression of iNOS. In contrast, robustaflavone only marginally affected iNOS gene expression and NO production. To identify the transcriptional factors affected by sumaflavone, we investigated NF-kappaB and AP-1 activation. Reporter gene analysis using AP-1-specific luciferase reporter and gel shift analysis showed that enhanced AP-1 activity by LPS was significantly diminished in macrophages pretreated with sumaflavone. However, sumaflavone did not inhibit LPS-inducible NF-kappaB reporter activity. Western blot analyses also confirmed that the extent of I-kappaBalpha phosphorylation and the nuclear level of p65 were unaffected by sumaflavone. These results suggest that the inhibitory effect of sumaflavone on AP-1 activation may be associated with the potent NO blocking and anti-inflammatory effects of Selaginella tamariscina extract.