Metabolism of the soy isoflavones daidzein, genistein and glycitein in human subjects. Identification of new metabolites having an intact isoflavonoid skeleton

Epidemiological studies have associated high soy intake with a lowered risk for certain hormone-dependent diseases. Soy and soy foods are rich sources of isoflavones, which have been shown to possess several biological activities. In this study, the metabolism of soy isoflavones daidzein, genistein and glycitein was investigated in human subjects. The aim was to find and identify urinary phase I metabolites of isoflavones, which have an intact isoflavonoid skeleton, and which might possess some bioactivity. Six volunteers included three soy bars per day into their normal western diet for a 2-week period. Daily urine samples were collected before, and after the supplementation period. Urine samples were hydrolyzed with Helix pomatia, extracted with diethyl ether, purified with Sephadex LH-20 chromatography, and analyzed as trimethylsilyl derivatives using gas chromatography–mass spectrometry (GC–MS). The structures of the isoflavone metabolites were identified using authentic reference compounds. The metabolites, for which authentic reference compounds were not available, were identified by the interpretation of mass spectra. Several new isoflavone metabolites were identified, and the presence of previously reported metabolites confirmed. The metabolic pathways of daidzein, genistein and glycitein are presented on the basis of the identification of the metabolites in human urine after soy supplementation.     

Pharmacokinetics of isoflavones,daidzein and genistein,after ingestion of soy beverage compared with soy extract capsules in postmenopausal Thai women

Background  

Isoflavones from soybeans may provide some beneficial impacts on postmenopausal health. The purpose of this study was to compare the pharmacokinetics and bioavailability of plasma isoflavones (daidzein and genistein) after a single dose of orally administered soy beverage and soy extract capsules in postmenopausal Thai women.     

Effects of isoflavone supplements vs. soy foods on blood concentrations of genistein and daidzein in adults

The objective of this investigation was to examine the pharmacokinetics of isoflavone concentrations over a 24-h period among healthy adults consuming either soy foods or soy isoflavone tablets at different doses. This randomized, cross-over trial was conducted with 12 generally healthy adults. The three phases of the intervention included isoflavone tablets at (1) 144 mg/day or (2) 288 mg/day and (3) soy foods designed to provide a calculated 96 mg isoflavones/day (doses in aglycone equivalents). Doses were spread out over three meals per day. After 6 days on each study phase, plasma isoflavone concentrations were determined on the seventh day at 0, 4, 8, 10, 12 and 24 h. Average levels of total isoflavone concentrations at 8, 10 and 12 h were >4 micromol/L for the soy food phase and for the higher dose tablet phase. Genistein concentrations were higher overall in the soy food vs. both the lower and the higher dose supplement phases of the study (P<.05). When comparing plasma concentrations for the two doses of tablets, saturation appeared more evident for genistein than for daidzein at the higher dose level. In conclusion, we observed important differences in the pharmacokinetics of genistein and daidzein contrasting the sources and doses of isoflavones when administered three times daily, including a possible advantage for increasing serum concentrations of isoflavones from consuming soy foods relative to isoflavone supplements.     

Biotransformation and recovery of the isoflavones genistein and daidzein from industrial antibiotic fermentations

The objective of this study was to follow the metabolic fate of isoflavone glucosides from the soybean meal in a model industrial fermentation to determine if commercially useful isoflavones could be harvested as coproducts from the spent broth at the end of the fermentation. The isoflavone aglycones, genistein, and daidzein together make up 0.1–0.2 % of the soybean meal by weight but serve no known function in the manufacturing process. After feeding genistein to washed cells of the erythromycin-producing organism, Saccharopolyspora erythraea, the first biotransformation product (Gbp1) was determined by X-ray crystallography to be genistein-7-O-α-rhamnoside (rhamnosylgenistein). Subsequent feeding of rhamnosylgenistein to growing cells of Saccharopolyspora erythraea led to the production of a second biotransformation product, Gbp2. Chromatographic evidence suggested that Gbp2 accumulated in the spent broth of the erythromycin fermentation. When the spent broth was hydrolyzed with acid or industrial enzyme preparations, the isoflavone biotransformation products were returned back to their parental forms, genistein and daidzein, which were then recovered as coproducts. Desirable features of this method are that it does not require modification of the erythromycin manufacturing process or genetic engineering of the producing organism to be put into practice. A preliminary investigation of five additional antibiotic fermentations of industrial importance also found isoflavone coproduct potential.     

Formation of polyhydroxylated isoflavones from the soybean seed isoflavones daidzein and glycitein by bacteria isolated from tempe

Five tempe-derived bacterial strains identified as Micrococcus or Arthrobacter species were shown to transform the soybean isoflavones daidzein and glycitein to polyhydroxylated isoflavones by different hydroxylation reactions. All strains converted glycitein and daidzein to 6,7,4′-trihydroxyisoflavone (factor 2) and the latter substrate also to 7,8,4′-trihydroxyisoflavone. Three strains transformed daidzein to 7,8,3′,4′-tetrahydroxyisoflavone and 6,7,3′,4′-tetrahydroxyisoflavone. In addition, two strains formed 6,7,8,4′-tetrahydroxyisoflavone from daidzein. Conversion of glycitein by these two strains led to the formation of factor 2 and 6,7,3′,4′-tetrahydroxyisoflavone. The structures of these transformation products were elucidated by spectroscopic techniques and chemical degradation. Revision received: 9 September 1995 / Accepted: 21 September 1995     

Solvent extraction selection in the determination of isoflavones in soy foods

Acetonitrile is superior to acetone, ethanol and methanol in extracting the 12 phytoestrogenic soy isoflavone forms found in foods. At 53% organic solvent in water, raw soy flour, tofu, tempeh, textured vegetable protein and soy germ were evaluated for isoflavone extraction efficiency. The efficiency of acetonitrile extraction was demonstrated in mass balance evaluations of toasting of soy flour and soymilk heating.     

Chemoprotective activity of the isoflavones, genistein and daidzein on mutagenicity induced by direct and indirect mutagens in cultured HTC cells

Isoflavones are phenolic compounds widely distributed in plants and found in a high percentage in soybeans. They have important biological properties and are regarded as potential chemopreventive agents. The aim of this study was to verify the preventive effect of two soy isoflavones (genistein and daidzein) by a micronucleus assay, analysis of GST activity, and real-time RT-PCR analysis of GSTa2 gene expression. Mutagens of direct (doxorubicin) and indirect (2-aminoanthracene) DNA damage were used. Hepatoma cells (HTC) were treated with genistein or daidzein for 26 h at noncytotoxic concentrations; 10 μM when alone, and 0.1, 1.0 and 10 μM when combined with genotoxic agents. The micronucleus test demonstrated that both isoflavones alone had no genotoxic effect. Genistein showed antimutagenic effects at 10 μM with both direct and indirect DNA damage agents. On phase II enzyme regulation, the current study indicated an increase in total cytoplasmic GST activity in response to genistein and daidzein at 10 μM supplementation. However, the mRNA levels of GSTa2 isozymes were not differentially modulated by genistein or daidzein. The results point to an in vitro antimutagenic activity of genistein against direct and indirect DNA damage-induced mutagenicity.     

Survey of aflatoxin-producing fungi in certain fermented foods and beverages in Thailand

Aflatoxin-producing fungi were found in fermented foods and beverages: fermented rice (kaomak), soybean sauce (taotjo), peanut butter, soy sauce (shoyu), Thai red and white wine, and rice sugar wine. These foods were extracted directly and tested for aflatoxins by thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC).Four strains of aflatoxin-producing fungi were isolated from peanut butter, taotjo, and shoyu. Direct extracts of 10% of the peanut butters tested and 5% of the kaomak tested contained large amounts of aflatoxins. The HPLC procedure used in this experiment utilized chloroform-ethyl acetate (31)     

Soy protein with or without isoflavones, soy germ and soy germ extract, and daidzein lessen plasma cholesterol levels in golden Syrian hamsters

Dietary isolated soy protein (ISP, containing approximately equal amounts of daidzein and genistein), ethanol-extracted ISP (ISP (-)), soygerm or soygerm extract (containing large amounts of daidzein and glycitein and little genistein) and the isoflavone, daidzein, were hypothesized to lessen plasma cholesterol in comparison with casein. Sixty male and 60 female golden Syrian hamsters (6-8 weeks of age) were randomly assigned to six treatments fed for 10 weeks. Four of the experimental diets (ISP, daidzein, soygerm, and soygerm extract) contained 1.3 mmol total isoflavones/kg. The ISP (-) diet contained 0.013 mmol isoflavone/kg, whereas the casein diet contained no isoflavones. Hamsters fed ISP, ISP (-), daidzein, soygerm, and soygerm extract had significantly less plasma total cholesterol (by 16%-28%), less non-HDL cholesterol (by 15%-50%) and less non-HDL/HDL cholesterol ratios compared with hamsters fed casein (P < 0.01). For male hamsters, there were no differences among treatments in plasma HDL concentrations. Female hamsters fed ISP (-) had significantly greater HDL levels (P < 0.01) than females fed casein or daidzein. Triglyceride concentration was significantly less in hamsters fed ISP (-) compared with the casein-fed females. Because soy protein with or without isoflavones, soygerm and soygerm extract, and daidzein lessened plasma cholesterol to an approximately equal extent, soy protein alone, varying mixtures of isoflavones, and other extractable components of soy are responsible for cholesterol-lessening effects of soy foods, mainly due to their effects to lessen LDL cholesterol.     

Metabolism of daidzein by fecal bacteria in rats

Daidzein (4',7-dihydroxyisoflavone), a soy phytoestrogen, is a weakly estrogenic compound that may have potential health benefits. Biotransformation of daidzein by the human gut microflora after ingestion converts it to either the highly estrogenic metabolite equol or to nonestrogenic metabolites. We investigated the metabolism of daidzein by colonic microflora of rats. Fecal samples, obtained before and after rats were exposed to daidzein at 250 or 1000 parts per million, were incubated in brain-heart infusion (BHI) broth with daidzein under anaerobic conditions. Samples were removed from the cultures daily and analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry. The fecal bacteria of all rats, regardless of prior daidzein exposure, metabolized the added daidzein to dihydrodaidzein. Both compounds disappeared rapidly from BHI cultures incubated for more than 24 h, but no other daidzein metabolites were detected. Only daidzein and dihydrodaidzein were found in a direct analysis of the feces of rats that had consumed daidzein in their diets. Unlike the fecal bacteria of humans and monkeys, the rat flora rapidly metabolized daidzein to aliphatic compounds that could not be detected by HPLC or mass spectral analysis.     

Bioconversion of soy isoflavones daidzin and daidzein by <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains

Twenty-two strains of Bifidobacterium, representative of eight major species of human origin, were screened for their ability to transform the isoflavones daidzin and daidzein. Most of the strains released the aglycone from daidzin and 12 gave yields higher than 90%. The kinetics of growth, daidzin consumption, and daidzein production indicated that the hydrolytic activity occurred during the growth. The supernatant of the majority of the strains did not release the aglycone from daidzin, suggesting that cell-associated β-glucosidases (β-Glu) are mainly responsible for the metabolism of soybean glyco-conjugates. Cell-associated β-Glu was mainly intracellular and significantly varied among the species and the strains. The lack of β-Glu was correlated with the inability to hydrolyze daidzin. Although S-equol production by anaerobic intestinal bacteria has been established, information on S-equol-producing bifidobacteria is contradictory. In this study, 22 bifidobacteria failed to transform daidzein into reduced metabolites under all the experimental conditions, excluding any role in the reductive pathway of daidzein toward the production of S-equol. These results suggest that selected probiotic strains of Bifidobacterium can be used to speed up the release of daidzein, improving its bioavailability for absorption by colonic mucosa and/or biotransformation to S-equol by other intestinal microorganisms.     

Role of soy isoflavones in the hypotriglyceridemic effect of soy protein in the rat

The present study was undertaken to determine whether isoflavones present in soy protein isolate contribute to the triglyceride-lowering effect of the protein relative to casein. Plasma triglyceride concentrations, their secretion rate into blood circulation, and post-heparin plasma lipoprotein lipase activity (a major determinant of intravascular catabolism of triglycerides) were measured in the fasted state in male Sprague-Dawley rats fed for 21 days one of three experimental diets varying in protein source (20% weight/weight): soy protein isolate, casein or casein to which 1.82 mg/g isoflavones (genistein and daidzein) were added to match the isoflavone content of soy protein isolate. Body weight gain was slightly lower in soy protein fed rats than in casein fed rats, but this effect was not statistically significant (P = 0.22). Casein plus isoflavones diet induced intermediary weight gain. A decrease in plasma total triglycerides was observed in rats fed soy protein and casein plus isoflavones compared with casein (P < 0.05), and there was a tendency to a positive correlation between weight gain and plasma triglyceride concentrations (r = 0.35, P = 0.06). However, no significant effect was observed on hepatic triglyceride concentrations, triglyceride secretion rate by the liver and post-heparin plasma lipoprotein lipase activity. These results show that soy protein isolate, in comparison with casein, has a hypotriglyceridemic effect in the rat and suggest that isoflavones may be responsible, at least in part, for this effect. The lowering effect of soy protein isolate and isoflavones on plasma triglyceride concentrations may be mediated by an alteration in energy balance, and possibly by the hepatic production of lipoproteins more susceptible to intravascular hydrolysis. Subtle but sustained changes in triglyceride secretion and post-heparin plasma lipoprotein lipase activity may also be implicated.     

传统大豆发酵食品在心脑血管疾病防治上的研究现状

人类生活水平的提高以及生活压力的增加,使心脑血管疾病的发生频率显著增加,并日趋成为威胁人类健康的重要原因。传统大豆发酵食品中的各种生理活性物质通过降血压和调节体内脂质、糖类代谢等多种作用来有效地防治心脑血管疾病;与传统药物相比,有效果显著与无毒副作用等优点,因此传统大豆发酵食品有广阔的发展应用前景。     

Increase of isoflavones in soybean callus by Agrobacterium-mediated transformation

Plant secondary metabolites have always been a focus of study due to their important roles in human medicine and nutrition. We transferred the isoflavone synthase (IFS) gene into soybean [Glycine max (L.) Merr.] using the Agrobacterium-mediated transformation method in an attempt to produce transformed soybean plants which produced increased levels of the secondary metabolite, isoflavone. Although the trial to produce transgenic plant failed due to unestablished hygromycin selection, transformed callus cell lines were obtained. The induction rate and degree of callus were similar among the three cultivars tested, but light illumination positively influenced the frequency of callus formation, resulting in a callus induction rate of 74% for Kwangan, 67% for Sojin, and 73% for Duyou. Following seven to eight subcultures on selection media, the isoflavone content of the transformed callus lines were analyzed by high-performance liquid chromatography. The total amount of isoflavone in the transformed callus cell lines was three- to sixfold higher than that in control callus or seeds. Given the many positive effects of isoflavone on human health, it may be possible to adapt our transformed callus lines for industrialization through an alternative cell culture system to produce high concentrations of isoflavones.     

The phytoestrogens daidzein and genistein enhance the insulin-stimulated sulfate uptake in articular chondrocytes

Clinical observations have suggested a relationship between osteoarthritis and a changed estrogen metabolism in menopausal women. Phytoestrogens have been shown to ameliorate various menopausal symptoms. Proteoglycans (PG) consisting of low and high sulfated glycosaminoglycans (GAG) are the main components of articular cartilage matrix, and their synthesis is increased by insulin in growth plate cartilage. We have investigated whether GAG synthesis and sodium [35S]sulfate incorporation in female bovine articular chondrocytes are affected by daidzein, genistein, and/or insulin. For comparative purposes, estradiol incubations were performed. Articular chondrocytes were cultured in monolayers at 5% O2 and 5% CO2 in medium containing serum for 7 days followed by the addition of 10(-11) M-10(-4) M daidzein, genistein, 17beta-estradiol, or 5 microg/ml insulin in a serum-free culture phase of 2 days. Photometrically analyzed GAG synthesis was significantly suppressed by high doses (10(-5) M-10(-4) M) of daidzein, genistein, and 17beta-estradiol. Although insulin raised the sodium [35S]sulfate uptake significantly, different concentrations of daidzein, genistein, or 17beta-estradiol showed no significant effects. However, the stimulating effect of insulin on sulfate incorporation was enhanced significantly after preincubation of cells with 10(-11) M-10(-5) M daidzein or 10(-9) M-10(-5) M genistein but not by 17beta-estradiol. In view of the risks of long-term estrogen replacement therapy, further experiments should clarify the potential benefit of phytoestrogens and insulin in articular cartilage metabolism.     

Enhanced bioavailability of soy isoflavones by complexation with beta-cyclodextrin in rats

In order to improve the solubility and bioavailability of a soy isoflavone extract (IFE), inclusion complexes (IFE-beta-CD) of the isoflavone extract with beta-cyclodextrin (beta-CD) were prepared and studied for their solubility and bioavailability. The aqueous solubility of the complexes of IFE with beta-CD (2.0 mg/ml) was about 26 times that of IFE itself (0.076 mg/ml). The same dosages of IFE and IFE-beta-CD were orally administered to SD rats (Sprague-Dawley) on an isoflavone glycoside (IFG) basis (daidzin, genistin and glycitin), and the plasma concentrations of daidzein, genistein and glycitein were measured over time to estimate the average AUC (area under the plasma concentration versus time curve) of the isoflavones. After the oral administration, the AUC values for daidzein, genistein and glycitein were 340, 11 and 28 microg x min/ml, respectively. In contrast, the respective AUC values after the administration of IFE-beta-CD were 430, 20 and 48 microg x min/ml. The bioavailability of daidzein in IFE-beta-CD was increased to 126% by the formation of inclusion complexes with beta-CD, compared with that in IFE. Furthermore, the bioavailability of genistein and glycitein in IFE-beta-CD formulation was significantly higher by up to 180% and 170%, respectively, compared with that of IFE p=0.008 and p=0.028, respectively). These results show that the absorption of IFE could be improved by the complexation of IFE with beta-CD (IFE-beta-CD).     

Inactivation of thyroid peroxidase by soy isoflavones,in vitro and in vivo

Soy-containing foods and dietary supplements are widely consumed for putative health benefits (e.g. cancer chemoprevention, beneficial effects on serum lipids associated with cardiovascular health, reduction of osteoporosis, relief of menopausal symptoms). However, studies of soy isoflavones in experimental animals suggest possible adverse effects as well (e.g. enhancement of reproductive organ cancer, modulation of endocrine function, anti-thyroid effects). This paper reviews the evidence in humans and animals for anti-thyroid effects of soy and its principal isoflavones, genistein and daidzein.