Limonene Suppresses Lipopolysaccharide-Induced Production of Nitric Oxide, Prostaglandin E2, and Pro-inflammatory Cytokines in RAW 264.7 Macrophages

The monoterpene D-limonene and its metabolites have been shown to exert chemopreventive and chemotherapeutic effects against different tumours in animal models and clinical trials. However, it is unknown whether these compounds modulate the inflammatory response in RAW 264.7 macrophage cells. The present study was therefore designed to elucidate the pharmacological and biological effects of D-limonene on the production of pro-inflammatory cytokines and inflammatory mediators in macrophages. The results indicate that D-limonene is an effective inhibitor of lipopolysaccharide (LPS)-induced NO and prostaglandin E(2) production in RAW 264.7 cells. These inhibitory effects of D-limonene included dose-dependent decreases in the expression of iNOS and COX-2 proteins. To evaluate the inhibitory effects of D-limonene on other cytokines, we also measured TNF-alpha, IL-1beta, and IL-6 levels in the cell supernatants of LPS-stimulated RAW 264.7 macrophages by enzyme-linked immunosorbent assay. In these assays, D-limonene decreased the expression of TNF-alpha, IL-1beta, and IL-6 in a dose-dependent manner. To assess the suitability of D-limonene for cosmetic applications, we also performed 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays on HaCaT keratinocytes. D-limonene did not display any cytotoxicity in these assays. From these results, we suggest that D-limonene may be considered a potential anti-inflammatory candidate.