Anti-Inflammatory and Antioxidant Effects of Anthocyanins of Trifolium pratense (Red Clover) in Lipopolysaccharide-Stimulated RAW-267.4 Macrophages

Abstract

Red clover (<i>Trifolium pratense</i>) possesses various dietary compounds that improve human health. However, the functions of anthocyanins in red clover remain unclear. Here we examined anti-inflammatory and antioxidant effects of red clover extract (RC) and red clover anthocyanins fraction (RCA) using lipopolysaccharide (LPS)-treated RAW 264.7 macrophages and identified dietary compounds. RC and RCA suppressed LPS-induced expression of genes such as tumor necrosis factor (<i>TNF</i>)<i>α</i>, interleukin (<i>IL</i>)<i>1β</i>, inducible nitric oxide synthase (<i>iNOS</i>), monocyte chemoattractant protein (<i>MCP</i>)1, and cyclooxygenase (<i>COX</i>)<i>2</i>. LPS-stimulated intracellular reactive oxygen species (ROS) production also was prevented by both RC and RCA. NADPH oxidase 1 (NOX1) gene and phosphorylation of p47^phox of NOX1 that were increased by LPS were inhibited in the cells treated with RCA. LPS-stimulated nuclear factor erythroid 2-related factor 2 (<i>NRF2</i>) gene expression and nuclear translocation of nuclear factor kappa B (NF-kB) subunit p65 were suppressed together with reduced iNOS and COX2 proteins by RCA. Additionally, 27 polyphenols and 7 anthocyanins from RC were identified and quantified. In conclusion, RC, especially RCA, exerted anti-inflammatory and anti-oxidative activities in vitro by regulating NF-κB and NRF2 signaling pathways, suggesting that anthocyanins in red clover are the potential candidates to reduce inflammation and oxidative stress.