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.     

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.     

Identification of equol producers in a Japanese population by high-performance liquid chromatography with coulometric array for determining serum isoflavones.

Using a method of high-performance liquid chromatography (HPLC) with coulometric array, we measured isoflavone levels in sera from seven volunteers before and after three days of ingesting Soyaflavone E (an isoflavones powder) and from 129 female farmers (Japanese Multiple Environmental Toxicants Study; JMETS). Results showed that the serum isoflavone concentrations rose dramatically after three days of ingesting Soyaflavone E in all subjects except for the serum equol concentrations in two subjects. The geometric mean concentrations of daidzein, genistein, and equol in the serum of 129 Japanese women were 25.0 ng/ml of daidzein, 94.1 ng/ml of genistein, and 9.6 ng/ml of equol. Interestingly, there existed two dominant groups in terms of serum equol concentrations in an independent manner of soy-derived product intake among the study participants.     

Equol producer status, salivary estradiol profile and urinary excretion of isoflavones in Irish Caucasian women, following ingestion of soymilk

Equol production, isoflavone excretion, and the salivary estradiol profile among 36 females, native Irish Caucasian volunteers following ingestion of 200mL soymilk is reported. The soymilk contained daidzein (73+/-6.7mg) and genistein (86+/-10.2mg). Volunteers provided personal and family medical history. Dietary analysis revealed that all volunteers regularly consumed soy-based or soy-supplemented food products. The mean age, mean age at menarche, and body mass index of volunteers were 46.6+/-12.3 years, 13.1 years and 26.1, respectively. The average number of children per volunteer was 2.13. Twelve (34%) of the volunteers were found to be first-degree relatives of breast cancer patients. Following consumption of the soymilk, equol was detected in the urine of 18 (51%) of the volunteers. Mean urinary daidzein and genistein concentrations during the hours following soymilk ingestion were 13.5 and 16.7microg/mg creatinine, respectively, however, some volunteers excreted little (less than 4.0microg/mg) or no isoflavone. Salivary estradiol in most (24) volunteers had decreased from 51.5+/-28.67pmol/L pre-ingestion to 29.75+/-16.13pmol/L 5h after drinking the soymilk. However, the salivary estradiol in 12 subjects (34%) increased from 33.76+/-13.4pmol/L to 137.4+/-65.64pmol/L over the same period. Individuals whose salivary estradiol increased had significantly less children (1.58 (P<0.05)), were more likely to (a) return urine samples with low isoflavone content (50.3% compared to 25%), (b) to be equol producers (67% compared to 41.7%), and (c) to be first-degree relatives of breast cancer patients (41.7% compared to 25%). Volunteers who reported a first-degree link to breast cancer were more likely to have a higher body mass index (29.0 compared to 26.1 (P<0.05)), to be equol producers (75% compared to 51%), and to excrete isoflavones in low quantities only (60% compared to 50%). First-degree relatives also had fewer children (1.75 (P<0.05)). The results indicate a significant, distinctive variation in equol production, isoflavone excretion and salivary estradiol profile among individual volunteers following ingestion of soymilk.     

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.     

Anti-obesity activities of fermented soygerm isoflavones by Bifidobacterium breve

Soygerm isoflavones were subjected to fermentation by Bifidobacterium breve. Most of isoflavone glycosides (daidzin, glycitin and genistin) in soygerms were deglycosylated to their corresponding isoflavone aglycones (daidzein, glycitein and genistein) within 24 h fermentation. Fermented isoflavones significantly inhibited pancreatic lipase activity in fermentation-time and dosage dependant manner. When fermented isoflavones were orally administered with olive oil to SD rats, the triglyceride (TG) level in plasma after 2 h of ingestion was significantly lower than the control of only olive oil administered group whereas no such significant decrease in plasma TG was observed in unfermented isoflavone administered group. This result indicates that oral administration of fermented isoflavones effectively suppressed absorption of excessive lipid into a body. Addition of either unfermented or fermented soygerm isoflavones effectively inhibited adipocyte differentiation from 3T3-L1 in a dose dependent manner. In conclusion, B. breve successfully converted soygerm isoflavones into their aglycones, and these aglycones were more effective in suppressing lipid absorption as well as adipocytes differentiation than their glycosides.     

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.     

Incorporation of esterified soybean isoflavones with antioxidant activity into low density lipoprotein.

We have recently reported that dietary intake of soybean isoflavone phytoestrogens resulted in increased oxidation resistance of isolated low density lipoprotein (LDL). In order to explore the underlying mechanisms we designed two types of in vitro experiments. First, we prepared several different isoflavone fatty acid esters to increase their lipid solubility and studied their incorporation into LDL. Second, the oxidation resistance of the isoflavone-containing LDLs was investigated with Esterbauer's 'conjugated diene' method using Cu2+ as prooxidant. Unesterified daidzein and genistein as well as genistein stearic acid esters were incorporated into LDL to a relatively small extent (0.33 molecules per LDL particle, or less) and they did not significantly influence oxidation resistance. The oleic acid esters of isoflavones were incorporated more effectively, reaching a level of 2.19 molecules per LDL particle or more, and the 4',7-O-dioleates of daidzein and genistein exhibited prolongations of lag times by 46% (P<0.05) and 202% (P<0.01), respectively. A smaller but significant increase in lag time (20.5%, P<0.01) was caused by daidzein 7-mono-oleate. In summary, esterification of soybean isoflavones daidzein and genistein with fatty acids at different hydroxyl groups provided lipophilicity needed for incorporation into LDL. Some isoflavone oleic acid esters increased oxidation resistance of LDL following their incorporation.     

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.     

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.     

Antiproliferative efficacy of lipophilic soy isoflavone phytoestrogens delivered by low density lipoprotein particles into cultured U937 cells

Some fat-soluble bioactive substances incorporated into low density lipoprotein (LDL) may be delivered into cells via LDL receptor pathway influencing cellular functions. In this study, we synthesized a number of fat-soluble isoflavone esters and investigated their incorporation into LDL as well as their delivery into U937 cells. Using an artificial transfer system (Celite dispersion), genistein and daidzein oleates and daidzein dilinoleate were efficiently incorporated into LDL with concentrations ranging between 2.7 to 16.9 isoflavone molecules/LDL particle, while much smaller amounts of unesterified isoflavones and genistein stearates were transferred into LDL. LDL containing 7-oleates or 4',7-dioleates of genistein and daidzein significantly reduced U937 cell proliferation by 36-43%. The strongest inhibitory effect was shown by daidzein 4',7-dilinoleate with 93% reduction of cell proliferation. Neither of the 4'-oleates of genistein and daidzein contained in LDLs exhibited antiproliferative effects although they were incorporated into LDL. In summary, we demonstrated that isoflavones made fat-soluble by esterification can be incorporated into LDL in vitro and delivered into cultured U937 cells via the LDL-receptor pathway, reducing the cell proliferation.     

Glucuronidation of the soyabean isoflavones genistein and daidzein by human liver is related to levels of UGT1A1 and UGT1A9 activity and alters isoflavone response in the MCF-7 human breast cancer cell line

The soyabean isoflavones genistein and daidzein, which may protect against some cancers, cardiovascular disease and bone mineral loss, undergo substantial Phase 2 metabolism, predominantly glucuronidation. We observed a correlation between rates of metabolism of marker substrates of specific UGTs and rates of glucuronidation of genistein and daidzein in vitro by a panel of human liver microsomes, demonstrating that UGT1A1 and UGT1A9, but not UGT1A4, make a major contribution to the metabolism of these isoflavones by human liver. These findings were substantiated by observations that recombinant human UGT1A1 and UGT1A9, but not UGT1A4, catalysed the production of the major glucuronides of both genistein and daidzein in vitro. Recombinant human UGT1A8 also metabolised both genistein and daidzein, whereas UGT1A6 was specific to genistein and UGTs 2B7 and 2B15 were inactive, or only marginally active, with either isoflavone as substrate. The intestinal isoform UGT1A10 metabolised either both isoflavones or genistein only, depending on the commercial supplier of the recombinant enzyme, possibly as a result of a difference in amino acid sequence, which we were unable to confirm. Daidzein (16 microM) increased cell death in the MCF-7 human breast cancer cell line and this effect was reversed by glucuronidation. In view of a well-characterised functional polymorphism in UGT1A1, these observations may have implications for inter-individual variability in the potential health-beneficial effects of isoflavone consumption.     

Developmental and nutritional regulation of isoflavone secretion from soybean roots

Isoflavones play important roles in plant–microbe interactions in rhizospheres. Soybean roots secrete daidzein and genistein to attract rhizobia. Despite the importance of isoflavones in plant–microbe interactions, little is known about the developmental and nutritional regulation of isoflavone secretion from soybean roots. In this study, soybeans were grown in hydroponic culture, and isoflavone contents in tissues, isoflavone secretion from the roots, and the expression of isoflavone conjugates hydrolyzing beta-glucosidase (ICHG) were investigated. Isoflavone contents did not show strong growth-dependent changes, while secretion of daidzein from the roots dramatically changed, with higher secretion during vegetative stages. Coordinately, the expression of ICHG also peaked at vegetative stages. Nitrogen deficiency resulted in 8- and 15-fold increases in secretion of daidzein and genistein, respectively, with no induction of ICHG. Taken together, these results suggest that large amounts of isoflavones were secreted during vegetative stages via the hydrolysis of (malonyl)glucosides with ICHG.     

丹贝发酵过程中大豆异黄酮组分与含量的变化

用HPLC方法检测大豆[Glycine max(Linn.)Merr.]发酵食品－丹贝的发酵过程中异黄酮各组分含量变化。在发酵的最初24h内,大部分异黄酮由糖甙转化成甙元,随着发酵时间的延长转化率逐渐降低,发酵终产物中异黄酮主要以大豆甙元和染料木素形式存在,发酵64h的丹贝中总异黄酮摩尔含量比未发酵的大豆高51．25％。     

The characterisation of structural and antioxidant properties of isoflavone metal chelates

Isoflavone metal chelates are of interest as isoflavones act as oestrogen mimics. Metal interactions may enhance isoflavones biological properties so understanding isoflavone metal chelation is important for the commercial application of isoflavones. This work aimed to determine if isoflavones, daidzein (4',7-dihydroxyisoflavone) and genistein (4',5,7-trihydroxyisoflavone) could chelate with metals as isoflavone chelates. Biochanin A (4'-methoxy-5,7-dihydroxyisoflavone) was also examined for it's ability to chelate with Cu(II) and Fe(III). This study found daidzein does not chelate with Cu(II) and Fe(III) but genistein and biochanin A chelate with a 1:2 M/L stoichiometry. The copper and iron chelates were synthesised and characterised by elemental analysis, FTIR, thermogravimetric analysis (TGA) and electrospray ionisation mass spectrometry (ESI-MS). These studies indicated a 1:2 M/L stoichiometry and suggested the isoflavones bind with the metals at the 4-keto and the 5-OH site. 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition assays showed that copper isoflavone chelates have higher antioxidant activity than free isoflavones while the iron isoflavone chelates showed pro-oxidant activity compared to the free isoflavone. Synergistic DPPH studies with 0.02 mM ascorbic acid revealed copper chelates exhibit reduced antioxidant activity versus free isoflavones whereas the iron chelates showed lower pro-oxidant activity except at 1.0 mM.     

Quantitation of soy-derived phytoestrogens in human breast tissue and biological fluids by high-performance liquid chromatography

A new and reliable HPLC method for the quantitation of daidzein, equol, and genistein in human breast tissue has been developed. The method was applied to biopsies from women undergoing breast reductions, who, prior to surgery, had ingested either a soy isoflavone preparation or a placebo tablet. The results were compared with data collected for urine and serum of the same subjects using standard methods. The limits of detection in the breast tissue homogenate were 24.7 nmol/l for daidzein, 148.0 nmol/l for equol, and 28.4 nmol/l for genistein (S/N of 3). The chromatographic limits of quantitation were 62.5 nmol/l for daidzein and genistein, and 125.0 nmol/l for equol, for which the accuracies were 86.0%, 83.6%, and 81.8%, respectively. The coefficients of variation of these measurements were all below 20% (11.1% for daidzein, 16.4% for genistein, and 13.2% for equol). The sample preparation comprised a concentration step and the absolute limits of quantitation were, therefore, 4.7 nmol/l, 18.8 nmol/l, and 0.94 nmol/l for daidzein and genistein, and 9.4 nmol/l, 37.5 nmol/l, and 1.9 nmol/l for equol in urine, serum, and breast tissue homogenate, respectively. Recoveries were between 70% (+/-5.6%) in breast tissue homogenate and 100% (+/-14.1%) in urine and serum for all three compounds. Equol (less than 1 micromol/l homogenate) was found to be the predominant phytoestrogen in breast tissue and its concentrations exceeded those in serum. The concentrations of phytoestrogens were at least 100-fold higher in urine than in serum and breast tissue.     

Quantitative analysis of urinary daidzein and equol by gas chromatography after solid-phase extraction and high-performance liquid chromatography

Daidzein and its main metabolite equol are isoflavone phytoestrogens. Several studies have suggested that intake of an isoflavone-rich diet may prevent hormone-related cancer and estrogen-related disorders (cardiovascular disease, osteoporosis and menopausal symptoms). To better understand the role of isoflavones in preventing such severe disease, several methods have been developed to measure these compounds in biological fluids. However, the analytical procedures to measure isoflavones are often time-consuming and require highly skilled technicians. In this paper we describe a method for urinary daidzein and equol measurement that combines solid phase extraction and HPLC purification before gas chromatographic determination. The specificity of the method was confirmed by the gas chromatography-mass spectrometry technique. The mean recovery of daidzein and equol was 94.6% and 97.0%, respectively. The repeatability of the method was in the range of 2.0-7.4% for daidzein and 1.3-4.9% for equol. A linear relationship between observed and expected values was found in the dilution (r2=0.9983 for daidzein; r2=0.9982 for equol) and addition (r2=0.9984 for daidzein; r2=0.9989 for equol) assays. The method is suitable to measure changes in the urinary excretion of isoflavones and to investigate urinary isoflavonoids as biomarkers of isoflavone exposure.