Red clover extract: a source for substances that activate peroxisome proliferator-activated receptor alpha and ameliorate the cytokine secretion profile of lipopolysaccharide-stimulated macrophages

Abstract

Objective: Inflammation and hyperlipidemia or dyslipidemia contribute to an increased risk of atherosclerosis and cardiovascular disease. Cardiovascular disease represents one of the major causes of premature death worldwide. The activation of peroxisome proliferator-activated receptor (PPAR) alpha, a drug target for hyperlipidemia and dyslipidemia, leads to an improved blood lipid profile. In this study, we determined the putative anti-inflammatory and PPARalpha stimulatory activities of red clover, an alternative to the classic hormone therapy used currently to treat menopausal symptoms.

Methods: Lipopolysaccharide-induced macrophages were used as a model for anti-inflammatory activity, and a chimeric GAL4-PPARalpha system was used as a model for putative hypolipidemic activity.

Results: Red clover extract and the isoflavones genistein and biochanin A were moderate PPARalpha activators. Daidzein only slightly activated PPARalpha, but its metabolite 6-hydroxydaidzein exerted a much higher PPARalpha activity. Similarly, the metabolite 3'-hydroxygenistein achieved higher activation efficiency than its precursor, genistein, did. In lipopolysaccharide-stimulated macrophages, red clover extract and its compounds reduced the secretion of proinflammatory cytokines, interleukin-6 and tumor necrosis factor alpha, increased the secretion of the anti-inflammatory interleukin-10, and/or reduced the expression of nuclear factor-kappaB, inducible nitric oxide synthase, and/or cyclooxygenase 2. Tumor necrosis factor alpha production was most efficiently reduced by biochanin A and genistein. Interleukin-6 levels were most efficiently reduced by genistein and equol.

Conclusions: Owing to its PPARalpha activation and modulation of the secreted cytokine profile, red clover extract is a putative candidate for preventing atherosclerosis and, thus, cardiovascular disease.