tetrahydrodaidzein and dihydrodaidzein

Topics: Actinobacteria; Equol; Gene Expression Regulation, Bacterial; Genes, Bacterial; Isoflavones; Multigene Family; Regulatory Elements, Transcriptional

Lactococcus strain 20-92 is a bacterium that produces equol directly from daidzein under anaerobic conditions. In this study, we reveal that the transcription of the gene encoding daidzein reductase in Lactococcus strain 20-92 (L-DZNR), which is responsible for the first stage of the biosynthesis of equol from daidzein, is regulated by the presence of daidzein. We analyzed the sequence surrounding the L-DZNR gene and found six novel genes, termed orf-US4, orf-US3, orf-US2, orf-US1, orf-DS1 and orf-DS2. These genes were expressed in Escherichia coli, and the resulting gene products were assayed for dihydrodaidzein reductase (DHDR) and tetrahydrodaidzein reductase (THDR) activity. The results showed that orf-US2 and orf-US3 encoded DHDR and THDR, respectively. DHDR in Lactococcus strain 20-92 (L-DHDR) was similar to the 3-oxoacyl-acyl-carrier-protein reductases of several bacteria and belonged to the short chain dehydrogenase/reductase family. THDR in Lactococcus strain 20-92 (L-THDR) was similar to several putative fumarate reductase/succinate dehydrogenase flavoprotein domain proteins. L-DHDR required NAD(P)H for its activity, whereas L-THDR required neither NADPH nor NADH. Thus, we succeeded in identifying two novel enzymes that are related to the second and third stages of the biosynthetic pathway that converts daidzein to equol.

Topics: Amino Acid Sequence; Biosynthetic Pathways; Cloning, Molecular; Coenzymes; Equol; Escherichia coli; Gene Expression; Genes, Bacterial; Isoflavones; Lactococcus; Molecular Sequence Data; NADP; Oxidation-Reduction; Oxidoreductases; Sequence Analysis, DNA; Sequence Homology, Amino Acid

Stereochemical course of isoflavanone dihydrodaidzein (DHD) reduction into the isoflavan (3S)-equol via tetrahydrodaidzein (THD) by the human intestinal anaerobic bacterium Eggerthella strain Julong 732 was studied. THD was synthesized by catalytic hydrogenation, and each stereoisomer was separated by chiral high-performance liquid chromatography. Circular dichroism spectroscopy was used to elucidate the absolute configurations of four synthetic THD stereoisomers. Rapid racemization of DHD catalyzed by Julong 732 prevented the substrate stereospecificity in the conversion of DHD into THD from being confirmed. The absolute configuration of THD, prepared by reduction of DHD in the cell-free incubation, was assigned as (3R,4S) via comparison of the retention time to that of the authentic THD by chiral chromatography. Dehydroequol (DE) was unable to produce the (3S)-equol both in the cell-free reaction and in the bacterial transformation, negating the possible intermediacy of DE. Finally, the intermediate (3R,4S)-THD was reduced into (3S)-equol by the whole cell, indicating the inversion of stereochemistry at C-3 during the reduction. A possible mechanism accounting for the racemization of DHD and the inversion of configuration of THD during reduction into (3S)-equol is proposed.

Topics: Actinobacteria; Biotransformation; Chromatography, High Pressure Liquid; Circular Dichroism; Equol; Humans; Isoflavones; Metabolic Networks and Pathways; Models, Biological; Stereoisomerism

Neointimal proliferation is a key element in atherosclerotic plaque formation and in arterial restenosis following angioplasty. Estrogen-like compounds, including naturally occurring plant phytoestrogens, are known to alter the extent of neointimal proliferation. This study investigates the anti-atherogenic/restenotic effect of several synthetic metabolites of isoflavone phytoestrogens (dihydrodaidzein, tetrahydrodaidzein and dehydroequol) (Novogen, Sydney, Australia). Acute neointimal proliferation was induced in the iliac artery of cholesterol-fed mice, by mechanically damaging the endothelium. Phytoestrogens were administered orally for 4 weeks and the damaged arteries harvested. Intimal area, as a percentage of the iliac artery wall area, was measured. Dihydrodaidzein significantly halved the intimal response (intima approximately 25% of wall area; p < 0.01) compared with placebo diet-fed mice (intima approximately 50% of wall area), while tetrahydrodaidzein and dehydroequol showed no inhibitory effects. Immunohistochemistry demonstrated that alpha-actin-positive vascular smooth muscle cells were the major cell type in the proliferating neointima. A single layer of endothelium covered the thickened intima by 4 weeks. Thus, a specific phytoestrogen isoflavone compound (dihydrodaidzein) can selectively inhibit neointimal proliferation, either by inhibition of vascular smooth muscle cell migration and proliferation, and/or by enhancing endothelial proliferation and function, and inhibition of endothelial apoptosis.

Topics: Angioplasty; Animals; Cell Proliferation; Coronary Restenosis; Iliac Artery; Isoflavones; Male; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phytoestrogens; Tunica Intima

Legume-derived isoflavones such as genistein, diadzein and equol have been associated with a reduction in risk of cardiovascular disease. In the current study, we explore the vascular activity of several isoflavone metabolites namely dihydrodaidzein, cis and trans-tetrahydrodaidzein and dehydroequol for potential cardioprotective properties. Rat isolated aortic rings were used. 17beta-oestradiol, equol, and all four of the metabolites studied significantly antagonized contractile responses to noradrenaline. The direct vasodilatory action of these compounds were examined and in contrast to 17beta-oestradiol, the vasodilatory effect of which was demonstrated to be endothelium independent, the dilatory action of all four compounds could be inhibited by endothelium denudation. Further, the dilatory action of both dihydrodaidzein and cis-tetrahydrodaidzein were inhibited by the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine (NOLA), by the soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and by 40 mM KCl. Dilatory responses to dehydroequol and trans-tetrahydrodaidzein, on the other hand, were inhibited by 40 mM KCL but not by NOLA nor ODQ. Finally, we examined the protective potential of these compounds in inhibiting endothelium damage by oxidized low density lipoprotein (ox-LDL). Trans-tetrahydrodaidzein was at least 10 fold more potent than 17beta-oestradiol in protecting against ox-LDL induced damage. We conclude that the isoflavone metabolites, dihydrodaidzein, cis- and trans-tetrahydrodaidzein and dehydroequol, may potentially represent a novel series of cardioprotective therapeutics.

Topics: Animals; Aorta; Endothelium, Vascular; Estradiol; In Vitro Techniques; Isoflavones; Lipoproteins, LDL; Male; Norepinephrine; Protective Agents; Rats; Rats, Sprague-Dawley; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents