A newly isolated rod-shaped, gram-negative anaerobic bacterium from human feces, named Julong 732, was found to be capable of metabolizing the isoflavone dihydrodaidzein toS-equol under anaerobic conditions. The metabolite, equol, was identified by using electron impact ionization mass spectrometry,¹H and¹³C nuclear magnetic resonance spectroscopy, and UV spectral analyses. However, strain Julong 732 was not able to produce equol from daidzein, and tetrahydrodaidzein and dehydroequol, which are most likely intermediates in the anaerobic metabolism of dihydrodaidzein, were not detected in bacterial culture medium containing dihydrodaidzein. Chiral stationary-phase high-performance liquid chromatography eluted only one metabolite,S-equol, which was produced from a bacterial culture containing a racemic mixture of dihydrodaidzein. Strain Julong 732 did not show racemase activity to transformR-equol toS-equol and vice versa. Its full 16S rRNA gene sequence (1,429 bp) had 92.8% similarity to that ofEggerthella hongkongenisHKU10. This is the first report of a single bacterium capable of converting a racemic mixture of dihydrodaidzein to enantiomeric pureS-equol.

Equol, a microbial metabolite of the isoflavone daidzein, is currently receiving much attention because of its strong antiproliferative effect on hormone-related human breast cancer cells; however, in our previous study, we observed that racemic equol [(±)-equol] shows the highest antiproliferative effect on human hepatocellular carcinoma SMMC-7721 cells compared with other cells, including human breast cancer MCF-7 and MDA-MB-231 cell lines. In the present study, we use the SMMC-7721 cancer cell line to investigate the mechanisms of (±)-equol-induced, R-(+)-equol-induced, and S-(−)-equol-induced apoptosis. Our purpose was to provide some guidelines to introduce equol into a clinical situation. R-(+)-equol and S-(−)-equol were prepared from (±)-equol by chiral stationary phase high performance liquid chromatography. The antiproliferative effect of equol on SMMC-7721 cells was investigated by crystal violet staining. Equol-induced apoptosis was detected by acridine orange/ethidium bromide staining and by flow cytometry. Western blotting was performed to study the molecular mechanisms of equol-induced apoptosis. The results showed that (±)-equol, R-(+)-equol, and S-(−)-equol inhibited the proliferation of SMMC-7721 cells in a concentration-dependent manner. Exposure of SMMC-7721 cells to equol caused significant cell cycle arrest in the S-phase. In addition, equol was shown to induce endoplasmic reticulum stress-mediated apoptosis by activating caspase-12 and caspase-8, and by upregulating Chop and Bip. Mitochondrion-mediated apoptosis was caused by upregulation of Bax and downregulation of Bcl-2, followed by activation of caspase-9, caspase-3, and cleaved poly (ADP-ribose) polymerase, respectively. This is the first report that shows that R-(+)-equol, S-(−)-equol, and (±)-equol can induce apoptosis of human hepatocellular carcinoma SMMC-7721 cells through the intrinsic pathway and the endoplasmic reticulum stress pathway.