The intake of weakly estrogenic isoflavonoids (phytoestrogens) is high in countries with a low incidence of estrogenrelated cancers, such as breast and prostate cancers. 1) Two ERs (a and b ) have been identified to date and the physiological responses of estrogens are known to be mediated within specific tissues by at least these two receptors.2,3) The ERs are a 3A member of the nuclear hormone receptor family and act as a ligand-activated nuclear transcription factor. 4)In a previous paper, we reported the results of systematic examination of the estrogenic activities of soy isoflavones (e.g. daidzin, genistin and glycitin) and their metabolites (e.g. daidzein, genistein, glycitein, equol, dihydrogenistein and dihydroglycitein) by enteric bacteria, 5) and the activities of several other isoflavone derivatives isolated from Pueralia lobata and P. thomsonii. 6)Usually, when soy foods are consumed, the soy isoflavones are metabolized into their aglycones and the related compounds by enteric bacteria. We have proved in in vitro experiments that the soy isoflavone glycosides are less estrogenic than the metabolites, i.e., they are activated by enteric bacteria. The metabolites produced by enteric bacteria are absorbed through the intestinal membranes and transported to the liver where they undergo re-metabolization by hepatic enzymes. To evaluate the bioactive compounds in soy products, investigation of the compounds which are actually absorbed within the body is necessary. 7,8) Herein, we describe the results of an investigation on the estrogenic activities of soy isoflavone metabolites isolated from human urine samples. Their effects on the estrogen dependent growth of MCF-7 cells, 9,10) on human ERs (hERs a and b )-dependent b-galactosidase induction, and their binding behavior to hERs were studied. We also examined the estrogenic activities of some synthetic isoflavone sulfates for the purpose of comparison. [2,4,6, H(N)]-17b-Estradiol (72 Ci/mmol) was purchased from Dai-Ichi Pure Chemicals Co. Ltd. RPMI 1640 medium (with or without phenol red), fetal bovine serum (FBS), trypsin/EDTA, Dulbecco's phosphate-bufferd saline (PBS), and kanamycin sulfate were purchased from Lifetech (Rockville, MD, U.S.A.). MATERIALS AND METHODS ChemicalsIsoflavone Metabolites Isoflavone metabolites examined in this work are shown in Fig. 1. M-1-9 were isolated from human urine samples collected for 12 h after feeding soy bean curds.11) Dihydrodaidzein and O-desmethylangolensin (O-DMA) were synthesized following the method described by Wahara et al.12) Genistein 4Ј-O-sulfate (S-1), 4Ј,7-di-Osulfate (S-2), daidzein 4Ј-O-sulfate (S-3), and 4Ј,7-di-O-sulfate (S-4) were synthesized following the method described by Peterson et al. 13)Cells MCF-7 cells were supplied by the Cell Resource Center for the Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University.Growth of MCF-7 Cells MCF-7 cells were cultured in phenol red-free RPMI 1640 medium supplemented with 10% (v/v) FBS and kanamycin sulfate (100 mg/ml) in 25 cm...

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The flowers from Pueraria, which are called Puerariae Flos, have been used since ancient times for recovery from alcohol intoxication. We elucidated the microbial transformation of the main isoflavones (1, 1a and 2) by using 29 commercially available human intestinal bacterial strains together with the bioactivation of the hepatoprotective activity of their metabolites. Tectoridin (1a), which contains only one glucosyl moiety, was metabolized to its aglycone 1b by various bacterial strains. On the other hand, the metabolism of 1 and 2, which both have disaccharide groups, was limited to specific bacterial strains. The metabolites 1c and 2c obtained from the Peptostreptococcus productus strain were completely different from those produced by the other strains. These metabolites were identified as 6-hydroxygenistein and 6-hydroxybiochanin A, respectively. The glycosides 1, 1a and 2 did not show any hepatoprotective activity, whereas aglycones 1b and 2b showed moderate activity. Furthermore, the hepatoprotective activity of the demethylated metabolites 1c and 2c was extremely potent. Although not all people have P. productus in their gastrointestinal tract, the O-demethylated compounds might become one of the bioactivated metabolites when Puerariae Flos is administered orally.