Abstract
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)1β, 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.
Highlights
Trifolium pratense, which belongs to the bean family Fabaceae, is a medicinal plant that improves various health conditions such as asthma, whooping cough, cancer, and gout [1]
LPS increased the expression of TNFα, IL1β, inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein (MCP)1, and COX2 genes
No additional reduction was observed in the cells that were treated with 20 μg/mL red clover extract (RC)
Summary
Trifolium pratense (red clover), which belongs to the bean family Fabaceae (or Leguminosae), is a medicinal plant that improves various health conditions such as asthma, whooping cough, cancer, and gout [1]. Phytoestrogenic isoflavones of red clover such as daidzein, genistein, biochain A, and formononetin improved menopausal symptoms and vaginal cytology on menopausal women [2,3,4,5]. Red clover is a rich source of isoflavones, it is expected to have anthocyanins due to its color. Anthocyanins of red clover are not identified. Anthocyanins are the plants’ pigments ranging from orange and red to purple and blue, which is classified as a subgroup of flavonoids with various health benefits such as antioxidants and anti-inflammation [6,7,8]. Inflammation is a response against infection, illness, and injury by producing cytokines such as tumor necrosis factor (TNF)α, interleukin (IL)1β, and IL6, and further by generating reactive oxygen species (ROS) [9]
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