Anti-inflammatory activities of ent-16alphaH,17-hydroxy-kauran-19-oic acid isolated from the roots of Siegesbeckia pubescens are due to the inhibition of iNOS and COX-2 expression in RAW 264.7 macrophages via NF-kappaB inactivation.

To isolate the anti-inflammatory components in Siegesbeckia pubescens root, we performed activity-guided fractionation using a carrageenan-induced edema rat model. Antinociceptive effects were followed using acetic acid-induced abdominal constriction and hot plate tests in mice. Chloroform extract was subjected to silica gel and octadesyl silane (ODS) column chromatography, and a diterpene was isolated which was identified as ent-16alphaH,17-hydroxy-kauran-19-oic acid (siegeskaurolic acid). Pretreatment with siegeskaurolic acid (20 or 30 mg/kg/day, p.o.) exhibited anti-inflammatory and antinociceptive effects in these animal models. To investigate the mechanisms underlying this anti-inflammatory action, we investigated the effect of siegeskaurolic acid on lipopolysaccharide (LPS)-induced responses in a murine macrophage cell line, RAW 264.7. Siegeskaurolic acid was found to significantly inhibit the productions of nitric oxide (NO), prostaglandin E(2) (PGE(2)), and tumor necrosis factor-alpha (TNF-alpha). Consistent with these findings, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, and iNOS, COX-2, and TNF-alpha mRNAs were found to be inhibited by siegeskaurolic acid. Furthermore, siegeskaurolic acid inhibited the nuclear factor-kappaB (NF-kappaB) activation induced by LPS, and this was associated with the prevention of inhibitor kappaB degradation (I kappaB), and subsequently with decreased nuclear p65 and p50 protein levels. Taken together, our data indicate that the anti-inflammatory and antinociceptive properties of siegeskaurolic acid may be due to iNOS, COX-2 and TNF-alpha inhibition via the down-regulation of NF-kappaB binding activity.