Cardamonin, a chalcone isolated from the fruits of a local plant Alpinia rafflesiana, has demonstrated anti-inflammatory activity in cellular models of inflammation. In this report, we evaluated the ability of cardamonin to suppress both NO and PGE2 synthesis, iNOS and COX-2 expression and enzymatic activity, and key molecules in the NF-kappaB pathway in order to determine its molecular target. Cardamonin suppressed the production of NO and PGE2 in interferon-gamma (IFN-gamma)- and lipopolysaccharide (LPS)-induced RAW 264.7 cells. This inhibition was demonstrated to be caused by a dose-dependent down-regulation of both inducible enzymes, iNOS and COX-2, without direct effect upon iNOS or COX-2 enzyme activity. Subsequently we determined that the inhibition of inducible enzyme expression was due to a dose-dependent inhibition of phosphorylation and degradation of I-kappaBalpha, which resulted in a reduction of p65NF-kappaB nuclear translocation. We conclude that cardamonin is a potential anti-inflammatory drug lead that targets the NF-kappaB pathway.

Summary Many plant-derived natural compounds have been reported previously to inhibit the production of important pro-inflammatory mediators such as nitric oxide, prostaglandin E 2 , TNF-a and reactive oxygen species by suppressing inducible enzyme expression via inhibition of the mitogen-activated protein kinase pathway and nuclear translocation of critical transcription factors. This study evaluates the effects of atrovirinone [2-(1-methoxycarbonyl-4,6-dihydroxyphenoxy)-3-methoxy-5,6-di-(3-methyl-2-butenyl)-1,4-benzoquinone)], a benzoquinone that we have previously isolated from Garcinia atroviridis, on two cellular systems that are repeatedly used in the analysis of anti-inflammatory bioactive compounds, namely, RAW 264.7 macrophage cells and whole blood. Atrovirinone inhibited the production of both nitric oxide and prostaglandin E 2 from LPS-induced and IFN-ginduced RAW 264.7 cells and whole blood, with inhibitory concentration (IC) 50 values of 4.62 ± 0.65 and 9.33 ± 1.47 mmol/L, respectively. Analysis of thromboxane B 2 (TXB 2 ) secretion from whole blood stimulated by either the cyclooxygenase (COX)-1 or the COX-2 pathway showed that atrovirinone inhibits the generation of TXB 2 by both pathways, with IC 50 values of 7.41 ± 0.92 and 2.10 ± 0.48 mmol/L, respectively. Analysis of IC 50 ratios showed that atrovirinone was more COX-2 selective in its inhibition of TXB 2 , with a ratio of 0.32. Atrovirinone also inhibited the generation of intracellular reactive oxygen species and the secretion of TNF-a from RAW 264.7 cells in a dose-responsive manner, with IC 50 values of 5.99 ± 0.62 and 11.56 ± 0.04 mmol/L, respectively. Lipoxygenase activity was also moderately inhibited by atrovirinone. Our results suggest that atrovirinone acts on important pro-inflammatory mediators possibly by the inhibition of the nuclear factor-kB pathway and also by the inhibition of the COX/lipoxygenase enzyme activity.