Bis-ferulic Acid Inhibits Lipopolysaccharide-stimulated Cyclooxygenase-2 Expression in Macrophages

Objectives:Ferulic acid (FA) (4-hydroxy-3-methoxycinnamic acid), a eugenol-related compound, is a potent antioxidant. We previously found that bis-eugenol, a dimer of eugenol, inhibits LPS-induced cyclooxygenase (COX)-2 expression in macrophages. To explore compounds with potent anti-inflammatory activity, we synthesized bis-FA (4-cis,8-cis-bis(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-2,6-dione) and investigated its inhibitory effect on LPS- induced COX-2 expression in comparison with that of FA, iso-ferulic acid, eugenol, iso-eugenol or o-methoxy phenol. Methods:LPS-induced expression of COX-2 mRNA in RAW 264.7 cells was analyzed using Northern blotting. Escherichia coli LPS (100 ng/ml) was used as a stimulator. Radical-scavenging activity was defined as the amount of inhibitor necessary to reduce the initial DPPH radical concentration by 50% (ID50). Results: Bis-FA, iso-ferulic acid and o-methoxyphenol significantly inhibited LPS-induced COX-2 gene expression at concentrations of 10 microM, 250 microM, and 500 microM, respectively. The potent inhibitory effect of each compound was unaccompanied by cell damage. In contrast, eugenol, iso-eugenol and FA showed no inhibition within a concentration range of 1-500 mM. The potential to suppress COX-2 gene expression appeared to be correlated with antioxidant activity because LPS-induced gene expression was suppressed by reduction of oxidative stress. The ID50 (mM) for DPPH radicals declined in the order bis-FA (0.366) > FA (0.145) > eugenol (0.059) > iso-eugenol (0.053). Although eugenol, iso-eugenol and FA possess high anti-DPPH activity, they did not inhibit COX-2 expression. These compounds with a p-conjugated allyl group may have a quinone-like function due to high resonance stability during oxidation. In contrast, bis-FA, iso-ferulic acid and o-methoxyphenol may have a phenolic function. Conclusions:Bis-FA is a potent inhibitor of LPS-stimulated COX-2 expression, and may be useful for chemoprevention of oral inflammatory disease through inhibition of inflammatory responses.