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.     

Oxidative metabolism and genotoxic potential of major isoflavone phytoestrogens

The soy isoflavones daidzein, genistein and glycitein are extensively metabolized by rat liver microsomes to a variety of catechol metabolites. Hydroxylated metabolites of daidzein and genistein have also been demonstrated in incubations with human hepatic microsomes and in the urine of humans after ingestion of soy food. Although the microsomal metabolism of formononetin and biochanin A is dominated by demethylation to daidzein and genistein, respectively, catechols of the parent isoflavones and of the demethylation products are also formed. Thus, oxidative metabolism appears to be common among isoflavones and may have implications for their biological activities. As genistein but not daidzein exhibits clastogenic activity in cultured mammalian cells, the role of oxidative metabolism for the genotoxicity of isoflavones is of particular interest.     

Metabolism of isoflavones in human subjects

Isoflavones form a group of plant compounds that occur mainly in legumes, soy being the most important source in human diet. The high levels of isoflavones in the diet have been associated with a lowered risk for hormone-dependent diseases, including breast and prostate cancers, osteoporosis and cardiovascular disease. The metabolism of isoflavones in humans has been studied to a certain extent, but detailed studies are lacking. This paper reviews the current knowledge on metabolism of isoflavones and presents some preliminary results of a comprehensive soy feeding study, in which the phase I metabolites of soy isoflavones, daidzein, genistein and glycitein, were identified by GC-MS. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genistein and daidzein induce cell proliferation and their metabolites cause oxidative DNA damage in relation to isoflavone-induced cancer of estrogen-sensitive organs

The soy isoflavones, genistein (5,7,4'-trihydroxyisoflavone) and daidzein (7,4'-dihydroxyisoflavone), are representative phytoestrogens that function as chemopreventive agents against cancers, cardiovascular disease, and osteoporosis. However, recent studies indicated that genistein and/or daidzein induced cancers of reproductive organs in rodents, such as the uterus and vulva. To clarify the molecular mechanisms underlying the induction of carcinogenesis by soy isoflavones, we examined the ability of genistein, daidzein, and their metabolites, 5,7,3',4'-tetrahydroxyisoflavone (orobol), 7,3',4'-trihydroxyisoflavone (7,3',4'-OH-IF), and 6,7,4'-trihydroxyisoflavone (6,7,4'-OH-IF), to cause DNA damage and cell proliferation. An E-screen assay revealed that genistein and daidzein enhanced proliferation of estrogen-sensitive breast cancer MCF-7 cells, while their metabolites had little or no effect. A surface plasmon resonance sensor showed that binding of isoflavone-liganded estrogen receptors (ER) to estrogen response elements (ERE) was largely consistent with cell proliferative activity of isoflavones. Orobol and 7,3',4'-OH-IF significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in human mammary epithelial MCF-10A cells, while genistein, daidzein, and 6,7,4'-OH-IF did not. Experiments using isolated DNA revealed a metal-dependent mechanism of oxidative DNA damage induced by orobol and 7,3',4'-OH-IF. DNA damage was enhanced by the addition of endogenous reductant NADH, formed via the redox cycle. These findings suggest that oxidative DNA damage by isoflavone metabolites plays a role in tumor initiation and that cell proliferation by isoflavones via ER-ERE binding induces tumor promotion and/or progression, resulting in cancer of estrogen-sensitive organs.     

Metabolism of the soy isoflavones daidzein and genistein by fungi used in the preparation of various fermented soybean foods

The ability of fungi used in the preparation of fermented soybean foods to metabolize the soy isoflavones daidzein and genistein was investigated. A total of 21 fungal strains from dou-chi, miso, sake, soy sauce, and sufu were screened. The genera of the tested fungi included Actinomucor, Aspergillus, Candida, Debaryomyces, Monascus, Mucor, Rhizopus, Saccharomyces, and Zygosaccharomyces. The results were that all tested Aspergillus strains from these soybean foods, including five A. oryzae strains, one A. sojae strain, and one A. tamarii strain, metabolized both daidzein and genistein. In contrast, no other tested fungi from the fermented soybean foods metabolized either daidzein or genistein. The metabolites of daidzein and genistein by Aspergillus strains were identified as 8-hydroxydaidzein and 8-hydroxygenistein, respectively, based on their mass, (1)H-, and (13)C-NMR spectra.     

Conversion of Daidzein and Genistein by an Anaerobic Bacterium Newly Isolated from the Mouse Intestine

The metabolism of isoflavones by gut bacteria plays a key role in the availability and bioactivation of these compounds in the intestine. Daidzein and genistein are the most common dietary soy isoflavones. While daidzein conversion yielding equol has been known for some time, the corresponding formation of 5-hydroxy-equol from genistein has not been reported previously. We isolated a strictly anaerobic bacterium (Mt1B8) from the mouse intestine which converted daidzein via dihydrodaidzein to equol as well as genistein via dihydrogenistein to 5-hydroxy-equol. Strain Mt1B8 was a gram-positive, rod-shaped bacterium identified as a member of the Coriobacteriaceae. Strain Mt1B8 also transformed dihydrodaidzein and dihydrogenistein to equol and 5-hydroxy-equol, respectively. The conversion of daidzein, genistein, dihydrodaidzein, and dihydrogenistein in the stationary growth phase depended on preincubation with the corresponding isoflavonoid, indicating enzyme induction. Moreover, dihydrogenistein was transformed even more rapidly in the stationary phase when strain Mt1B8 was grown on either genistein or daidzein. Growing the cells on daidzein also enabled conversion of genistein. This suggests that the same enzymes are involved in the conversion of the two isoflavones.     

Comparison of hormonal activity (estrogen,androgen and progestin) of standardized plant extracts for large scale use in hormone replacement therapy

Extracts from red clover (Trifolium pratense), soybean (Glycine max.) and black cohosh (Cimicifuga racemosa) are frequently used as alternative compounds for hormone replacement therapy (HRT) to treat menopausal disorders. Fifteen commercially available products made either from red clover, soybean or black cohosh were tested in in vitro assays in this study. The main polycyclic phenolic compounds of soy and red clover products were biochanin A, genistein, daidzein, formononetin, and glycitein. In red clover products glycitein was not abundant. All the compounds showed clear estrogenic activity through estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) and affinity to progesterone receptor (PR) and androgen receptor (AR), whereas the compounds from black cohosh did not. This was corroborated by synthetic isoflavones such as biochanin A, daidzein, genistein and formononetin. They exerted affinity to PR and AR in the range of 0.39-110 mM. Statistical analysis applying principal component analysis (PCA) revealed that all red clover and soy products are grouped in different clusters. Red clover products showed a higher affinity to AR and PR than soy products, which is explained by the higher amount of isoflavones present. In vitro assays and chemical analysis showed that theoretical estrogenic activity expressed as equivalent E2 concentration is in the same range as recommended for synthetic estrogens. Broader spectrum of action and hypothesized lower side effects by action through ERbeta make them suitable for alternative hormone replacement therapy.     

Neutrophil myeloperoxidase chlorinates and nitrates soy isoflavones and enhances their antioxidant properties

Soy isoflavones and other polyphenolics have a number of potentially important beneficial effects on the pro-oxidant aspects of chronic inflammation. The impact of inflammatory cell-specific metabolism of polyphenolics, which can include halogenation and nitration, on the properties of these compounds has not been examined. Using either human neutrophils or differentiated human leukemia cells (HL-60) stimulated with phorbol ester to elicit a respiratory burst, the hypothesis that local generation of reactive oxygen and nitrogen species may metabolize and modify the biological properties of the soy isoflavones was examined. Coincubation of the stimulated cells with genistein or daidzein had no effect on the respiratory burst. Medium from stimulated cells in the presence of the isoflavones and NO(2)(-) increased the inhibition of copper-induced LDL oxidation. Mass spectrometry analysis of this medium revealed that monochlorinated, dichlorinated, and nitrated isoflavones, formed through a myeloperoxidase-dependent mechanism, were present. The consumption of genistein in the presence of cells was both extensive and rapid with > 95% of the genistein converted to either the chlorinated or nitrated metabolites within 30 min. Chemically synthesized 3'-chlorogenistein and 3'-chlorodaidzein increased the inhibition of LDL oxidation by approximately 4-fold and 2-fold over genistein and daidzein, respectively. These results lead to the hypothesis that inflammatory cell-specific metabolism of polyphenolics can modify the properties of these compounds at the local site of inflammation.     

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.     

Effect of genistein and daidzein obtained by acid hydrolysis of their glycosides on phospholipid peroxidation

A mixture of isoflavones was obtained by acid hydrolysis of isoflavone glycosides isolated from the products of soybean processing by a successive extraction with aqueous acetone and methanol. Homogeneous isoflavones genistein and daidzein were isolated from the aglycone mixture by adsorption chromatography and identified by spectral and chromatographic methods. The effect of both isoflavones on lipid peroxidation of soy phospholipids in multilamellar vesicles was studied at various concentrations. These aglycones were found to inhibit the formation of lipid hydroperoxides and malonic dialdehyde at the concentrations as low as 1 mM.     

Isoflavones Daidzein and Genistein: Preparation by Acid Hydrolysis of Their Glycosides and the Effect on Phospholipid Peroxidation

A mixture of isoflavones was obtained by acid hydrolysis of isoflavone glycosides isolated from the products of soybean processing by successive extraction with aqueous acetone and methanol. The homogeneous isoflavones daidzein and genistein were isolated from the aglycone mixture by adsorption chromatography and identified by spectral and chromatographic methods. The effect of both isoflavones on lipid peroxidation of soy phospholipids in multilamellar vesicles was studied at various concentrations. These aglycones were found to inhibit the formation of lipid hydroperoxides and malonic dialdehyde at concentrations as low as 1 mM.     

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.     

Soybean isoflavonoids and their metabolic products inhibit in vitro lipoprotein oxidation in serum

Isoflavonoids are compounds present in many legumes, but are derived in the human diet mainly from soybeans and various soybean-based food products. The major isoflavonoids occurring in soy are the glycosides of genistein and daidzein. The metabolic products of genistein metabolism in humans have not been clearly shown. The two main products of daidzein metabolism in humans appear to be equol and O-desmethylangolensin. Increasing evidence suggests that oxidative modification to low-density lipoprotein is involved in atherogenesis, and that natural antioxidants that prevent or inhibit oxidative damage to low-density lipoprotein may beneficially influence atherogenesis. In the present experiments, the effects of genistein and daidzein, and the daidzein metabolites equol and O-desmethylangolensin on Cu²⁺-induced oxidation of lipoproteins in serum were examined. Three concentrations of each compound (0.1 μM, 1 μM, 10 μM) were tested for antioxidant activity in six individual serum samples. All compounds tested inhibited lipoprotein oxidation. The minimum concentration for significant inhibition was 1 μM for genistein and daidzein (P < 0.05), and 0.1 μM equol and O-desmethylangolensin (P < 0.05). Equol and O-desmethylangolensin were more potent inhibitors of in vitro lipoprotein oxidation in serum than the two major dietary isoflavonoids. This study has demonstrated that soybean isoflavonoids and metabolic products of daidzein metabolism inhibit lipoprotein oxidation in vitro. Human intervention studies are needed to determine if these compounds can influence oxidation in vivo.     

Absorption of isoflavones in humans: effects of food matrix and processing

If soy isoflavones are to be effective in preventing or treating a range of diseases, they must be bioavailable, and thus understanding factors which may alter their bioavailability needs to be elucidated. However, to date there is little information on whether the pharmacokinetic profile following ingestion of a defined dose is influenced by the food matrix in which the isoflavone is given or by the processing method used. Three different foods (cookies, chocolate bars and juice) were prepared, and their isoflavone contents were determined. We compared the urinary and serum concentrations of daidzein, genistein and equol following the consumption of three different foods, each of which contained 50 mg of isoflavones. After the technological processing of the different test foods, differences in aglycone levels were observed. The plasma levels of the isoflavone precursor daidzein were not altered by food matrix. Urinary daidzein recovery was similar for all three foods ingested with total urinary output of 33-34% of ingested dose. Peak genistein concentrations were attained in serum earlier following consumption of a liquid matrix rather than a solid matrix, although there was a lower total urinary recovery of genistein following ingestion of juice than that of the two other foods.     

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.     

兔肠道大豆异黄酮还原菌株的分离鉴定及其转化特性

【目的】从兔新鲜粪样中分离对大豆异黄酮黄豆苷原和染料木素具有转化作用的特定细菌菌株。【方法】在厌氧工作站内对獭兔新鲜粪样进行梯度稀释后涂板,挑取单菌落与底物黄豆苷原和染料木素分别厌氧混合培养,用高效液相色谱检测底物被转化情况。【结果】分离得到一株对大豆异黄酮黄豆苷原和染料木素均具有转化作用的革兰氏阳性严格厌氧细菌菌株AUH-JLR41(KJ188150)。根据产物的高效液相保留时间、紫外吸收图谱和质谱分析结果,将菌株AUH-JLR41代谢底物黄豆苷原和染料木素生成的产物分别鉴定为二氢黄豆苷原和二氢染料木素。经手性高效液相系统检测,产物二氢黄豆苷原和二氢染料木素均呈现两个等面积物质峰,表明这两个产物的对映体过量率均为0。通过转化动态研究发现,菌株AUH-JLR41分别在底物黄豆苷原和染料木素加入48 h和72 h后将底物全部转化为产物,该菌株能转化底物黄豆苷原和染料木素的最大浓度均为0.6 mmol/L。经BLAST比对,菌株AUH-JLR41的16S r RNA基因序列与斯奈克氏菌属菌株Slackia equolifaciens DZE(EU377663)的相似性高达99.6%。【结论】兔肠道分离的斯奈克氏菌属菌株Slackia sp.AUH-JLR41在厌氧条件下能将大豆异黄酮黄豆苷原和染料木素分别还原为二氢黄豆苷原和二氢染料木素。     

大豆异黄酮对大鼠乳腺癌细胞内cAMP/PKA信号途径的影响

本实验研究了大豆异黄酮对SHZ-88大鼠乳腺癌细胞内cAMP／PKA信号途径的影响。实验设3组：空白对照组、50μg／ml大豆黄酮及15μg／ml染料木素组。采用放射免疫测定法（RIA）检测了胞内cAMP的浓度、腺苷酸环化酶（adenylate cyclase,AC）和磷酸二酯酶（phosphodiesterase,PDE）的活性,用（γ-^32P）ATP掺入法测定cAMP依赖性PKA的活性,半定量RT-PCR法分析cAMP反应元件结合蛋白（cAMP response element binding protein,CREB）mRNA表达的变化。结果表明：在处理后5min,大豆黄酮组和染料木素组细胞的cAMP浓度分别比对照组升高了9．5％和11．0％（P〈0．05）：10min时,分别比对照组升高31．0％和40.3％（P〈0．01）。3组细胞的AC活性在处理时间内没有明显变化。但在处理后5min,大豆黄酮组和染料木素组细胞的PDE活性分别降至对照组的71．8％和71．6％（P〈0．05）。处理后20min,大豆黄酮组和染料木素组细胞PKA活性分别上升到对照组的125．8％和122．3％（P〈0．05）;到40min时仍维持在高水平。大豆黄酮组和染料木素组细胞CREB mRNA的表达量在处理后3h分别比对照组增加31．6％和51．1％（P〈0．05）;6h后开始下降。这些结果提示,大豆异黄酮能够激活大鼠乳腺癌细胞内cAMP／PKA信号途径;而且是通过抑制磷酸二酯酶的活性,导致胞内cAMP浓度升高而实现的。     

Daidzein and genistein content of fruits and nuts

Dietary phytoestrogens such as the isoflavones daidzein and genistein are thought to protect against chronic diseases that are common in Western societies, such as cancer, osteoporosis, and ischemic heart disease. In addition, there are concerns regarding the deleterious effects of hormone-like compounds, especially with respect to the development of infants. However, there is little information regarding the phytoestrogen content of foods, and therefore epidemiologic investigations of phytoestrogens are limited. As part of a study quantifying the consumption of phytoestrogens, the objective of this work was to assess the daidzein and genistein content of fruits and nuts commonly eaten in Europe. Eighty different fruits and nuts were sampled, prepared for eating, and freeze-dried. Daidzein and genistein were extracted from the dried foods, and the two isoflavones were quantified after hydrolytic removal of any conjugated carbohydrate. Completeness of extraction and any procedural losses of the isoflavones were accounted for using synthetic daidzin (7-O-glucosyl-4'-hydroxyisoflavone) and genistin (7-O-glucosyl-4'5-dihydroxyisoflavone) as internal standards. Of the 80 foods assayed, 43 contained no detectable daidzein or genistein, at a limit of quantification of 1 microg/kg dry weight of food. Nine foods contained more than 100 microg of the two isoflavones combined per kilogram wet weight, and 28 contained less than this amount. Currants and raisins were the richest sources of the isoflavones, containing 2,250 microg and 1,840 microg of the two isoflavones combined per kilogram of wet weight of food. Although fruits and nuts are not as rich in isoflavone phytoestrogens as are soy and other legumes, this is the first documentation of levels of daidzein and genistein occurring in these foods.     

Isolation of human intestinal bacteria metabolizing the natural isoflavone glycosides daidzin and genistin

Fecal bacteria from a healthy individual were screened for the specific bacteria involved in the metabolism of dietary isoflavonoids. Two strains of bacteria capable of producing primary and secondary metabolites from the natural isoflavone glycosides daidzin and genistin were detected. The metabolites were identified by comparison of their HPLC/mass, 1H NMR and UV spectra with those of standard and synthetic compounds. Both Escherichia coli HGH21 and the gram-positive strain HGH6 converted daidzin and genistin to the their respective aglycones daidzein and genistein. Under anoxic conditions, strain HGH6 further metabolized the isoflavones daidzein and genistein to dihydrodaidzein and dihydrogenistein, respectively. The reduction of a double bond between C-2 and C-3 to a single bond was isoflavonoid-specific by strain HGH6, which did not reduce a similar bond in the flavonoids apigenin and chrysin. Strain HGH6 did not further metabolize dihydrodaidzein and dihydrogenistein. This is the first study in which specific colonic bacteria that are involved in the metabolism of daidzin and genistin have been detected.