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

Nutrients. 2020 Apr 15;12(4):1089. doi: 10.3390/nu12041089.


Red clover (Trifolium pratense) 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 (TNF)α, interleukin (IL), inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein (MCP)1, and cyclooxygenase (COX)2. LPS-stimulated intracellular reactive oxygen species (ROS) production also was prevented by both RC and RCA. NADPH oxidase 1 (NOX1) gene and phosphorylation of p47phox of NOX1 that were increased by LPS were inhibited in the cells treated with RCA. LPS-stimulated nuclear factor erythroid 2-related factor 2 (NRF2) 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.

Keywords: anthocyanins; anti-inflammation; antioxidation; red clover.

MeSH terms

  • Animals
  • Anthocyanins / isolation & purification
  • Anthocyanins / pharmacology*
  • Anti-Inflammatory Agents*
  • Antioxidants*
  • Gene Expression / drug effects*
  • Interleukin-1beta / genetics
  • Interleukin-1beta / metabolism
  • Lipopolysaccharides / adverse effects
  • Mice
  • NADPH Oxidase 1 / genetics
  • NADPH Oxidase 1 / metabolism
  • NF-E2-Related Factor 2 / genetics
  • NF-E2-Related Factor 2 / metabolism
  • NF-kappa B / genetics
  • NF-kappa B / metabolism
  • Nitric Oxide Synthase Type II / genetics
  • Nitric Oxide Synthase Type II / metabolism
  • Oxidative Stress / drug effects*
  • Oxidative Stress / genetics
  • Phosphorylation / drug effects
  • RAW 264.7 Cells
  • Reactive Oxygen Species / metabolism
  • Signal Transduction / drug effects
  • Trifolium / chemistry*
  • Tumor Necrosis Factor-alpha / genetics
  • Tumor Necrosis Factor-alpha / metabolism


  • Anthocyanins
  • Anti-Inflammatory Agents
  • Antioxidants
  • Interleukin-1beta
  • Lipopolysaccharides
  • NF-E2-Related Factor 2
  • NF-kappa B
  • Nfe2l2 protein, mouse
  • Reactive Oxygen Species
  • Tumor Necrosis Factor-alpha
  • Nitric Oxide Synthase Type II
  • Nos2 protein, mouse
  • NADPH Oxidase 1
  • NOX1 protein, mouse