Red clover isoflavone metabolite bioavailability is decreased after fructooligosaccharide supplementation.

BACKGROUND: Red clover is an important source of isoflavones; which has been made commercially available as dietary supplements for the treatment of menopausal symptoms. Bioavailability and metabolism of these red clover isoflavones (RCI) have not been studied in detail. Fructooligosaccharides (FOS) stimulate the growth of intestinal bacteria and play an important role in the formation of certain isoflavone metabolites, such as equol and O-desmethylangolensin. OBJECTIVE: To determine the bioavailability of RCI metabolites and analyse whether FOS supplementation could influence their bioavailability. METHODS: Seventeen healthy adults were enrolled in the study carried out in two periods. In the first, compound bioavailability was determined after consumption of 80 mg of RCI (MF11RCE). In the second, a 6-day supplementation of 2×3000 mg/day of FOS was administered before isoflavone consumption. RESULTS: Biochanin A and formononetin were rapidly absorbed and both reached maximum concentrations at an average of 5-7h. Demethylation was a major reaction in the metabolic pathway. Daidzein serum level peaked after about 12.6h. Supplementation with FOS led to a significant decrease in the bioavailability of daidzein, dihydroformononetin, dihydrogenistein and dihydrodaidzein. An increase in equol production was also observed which did not reach statistical significance (p>0.05). CONCLUSION: This study is the first to provide detailed data on RCI bioavailability in humans and determine no influence of FOS yet a trend toward increased equol production. More research is warranted involving a greater sample size.

Receptor binding and transactivation activities of red clover isoflavones and their metabolites.

Red clover extracts contain a variety of isoflavones, which have affinity toward estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), androgen receptor (AR), and progesterone receptor (PR). Upon ingestion, they undergo various metabolic transformations. For a complete evaluation of red clover extracts and possible health benefits, the resulting metabolites should also be investigated. Biochanin A, formononetin, genistein, daidzein, dihydrobiochanin A, dihydroformononetin, dihydrogenistein, dihydrodaidzein, 3'-hydroxygenistein, 6-hydroxydaidzein, 6-hydroxydesmethylangolensin, equol, O-desmethylangolensin, angolensin, and p-ethylphenol were tested for their transactivation potential toward ERalpha, AR, and PR in yeast. Competitive binding assays with radiolabeled 17beta-estradiol, 17alpha-methyltrienolone or progesterone assessed binding to the respective ERalpha and ERbeta, AR, and PR. The compounds showed only weak binding affinity to AR and PR, with IC(50) values being greater (i.e., lesser affinity) than 10(-5)M for the respective receptor. So far, beneficial health effects have been attributed to the production of equol. We propose that other metabolites can also contribute to these effects. However, more detailed information for the formation of these metabolites in humans and for bioavailability data are required to confirm our assumptions.

GFP-reporter for a high throughput assay to monitor estrogenic compounds.

In vitro test systems using yeast cells are a useful tool for the determination of the estrogenic activity of estrogens, phyto- and xeno-estrogens and can be used for monitoring large sample numbers in a routine analysis procedure. Our conventional transactivation assay functions with an expression plasmid expressing estrogen receptor alpha (ERalpha) under the control of a copper-inducible CUP1 promoter and a reporter plasmid expressing beta-galactosidase under the control of the vitellogenin estrogen response element (ERE). In the novel yeast screen system the lacZ gene in the reporter plasmid was substituted by a gene for green fluorescent protein (GFP). Incubation of yeast with various concentrations of estrogenically active substances led to expression of the reporter gene product GFP in a dose dependent manner. The yeast transactivation assay was further down-scaled to be performed in a microplate scale, which is an important step to facilitate handling of large sample numbers. The sensitivity and reproducibility of the novel test system could be confirmed by analysis of the potencies of various estrogenically active substances. Thus, the newly developed yeast estrogen screen using GFP as a reporter can substitute the assay that has been used for a period of several years.