Forms Of Isoflavones Research Articles

Protective effects of soy-isoflavones in cardiovascular disease

Epidemiological studies indicate that the consumption of soy-containing food may prevent or slow-down the development of cardiovascular disease. In endothelial cells application of a soy extract or a combination of the most abundant soy isoflavones genistein and daidzein both inhibited apoptosis, a driving force in atherosclerosis development, when applied in combination with oxidized LDL or homocysteine. Proteome analysis revealed that the stressor-induced alteration of protein expression profile was reversed by the soy extract or the genistein/daidzein mixture. Only few protein entities that could be functionally linked to mitochondrial dysfunction were regulated in common by both application forms of isoflavones. A dietary intervention with isoflavone-enriched soy extract in postmenopausal women, who generally show strongly increased cardiovascular risk due to diminished estrogen production, led to significant alterations in the steady state levels of proteins from mononuclear blood cells. The proteins identified by proteome analysis revealed that soy isoflavones may increase the anti-inflammatory response in blood mononuclear cells thereby contributing to the atherosclerosis-preventive activities of a soy-rich diet. By proteome analysis protein targets were identified in vitro in endothelial cells that respond to soy isoflavones and that may decipher molecular mechanisms through which soy products exert their protective effects in the vasculature.

Characterization of yeast cell surface displayed Aspergillus oryzae β-glucosidase 1 high hydrolytic activity for soybean isoflavone

The yeast Saccharomyces cerevisiae GRI-117-UK was transformed to either display or secrete β-glucosidase 1 (BGL1) from the koji mold, Aspergillus oryzae. The β-glucosidase activity of BGL1-displaying yeast strains reached 405.9 U/g dry cell mass after 72 h of cultivation in YPD medium. The optimal pH and temperature for BGL1 displayed on the cell surfaces of the yeast were 5.0 and 55 °C, while the optima for BGL1 secreted by the yeast were 4.0 and 55 °C. The displayed BGL1 was stable at higher pH compared with the secreted BGL1. In addition, the thermostability of BGL1 was improved by displaying the enzyme on the yeast cell surfaces. In addition, the displayed and secreted forms of BGL1 had similar substrate specificity. β-Glucosidase hydrolyzes daidzin and genistin, which are the glycoside forms of soybean isoflavones, to the aglycones. Isoflavone aglycones were efficiently produced by BGL1-displaying yeast from an isoflavone mixture; at optimal temperature and pH the rate of aglycone production was at least 15.8 g/(l h). After 144 h of reaction, almost isoflavones were converted to its aglycone by BGL1-displaying yeast. The results of the present study demonstrate that BGL1-displaying yeast strains are effective whole cell biocatalysts of isoflavone aglycone production.

Influence of inulin on plasma isoflavone concentrations in healthy postmenopausal women

Background: Bacterial intestinal glucosidases exert an important role in isoflavone absorption. Insoluble dietary fibers such as inulin may stimulate the growth of these bacteria in the colon and, hence, stimulate the absorption of these substances in subjects who may need isoflavone supplementation.Objective: The objective was to assess the influence of inulin on plasma isoflavone concentrations after intake of soybean isoflavones in healthy postmenopausal women.Design: Twelve healthy postmenopausal women participated in a randomized, double-blind, crossover study. They consumed 40 mg of a conjugated form of soybean isoflavones (6 mg daidzein and 18 mg genistein as free form) with or without 3.66 g inulin twice daily in two 21-d experimental phases. Blood samples were collected 0, 1, 2, 3, 4, 6, 10, 12, and 24 h after intake of isoflavones with breakfast and dinner at the end of each 21-d experimental phase. Plasma concentrations of isoflavones were assessed by HPLC with an electrochemical detector.Results: Plasma 24-h areas under the curve indicated that the intake of soybean isoflavones with inulin for 21 d was followed by higher plasma concentrations of daidzein and genistein (38% and 91%, respectively) compared with the formulation without inulin. Furthermore, the time for the maximum concentration of daidzein and genistein appeared to be lower after the 21-d intake of soybean isoflavones, with or without inulin. However, the time for the maximum concentration of daidzein and genistein after supplementation with the inulin-containing formulation on day 21 was not significantly different from that after supplementation with the formulation without inulin.Conclusions: Inulin may increase the apparent plasma concentrations of the soybean isoflavones daidzein and genistein in postmenopausal women. The higher plasma concentrations of the 2 isoflavones suggests that the absorption of each was facilitated by the presence of inulin.

Bioavailability of soy isoflavones in rats Part I: Application of accurate methodology for studying the effects of gender and source of isoflavones

There are limited and controversial reports about the effects of gender and source of isoflavones on their bioavailability. Moreover, several previous studies have not used appropriate methodology to determine the bioavailability of soy isoflavones, which requires comparing the area under the plasma concentration-time curve after both oral and intravenous injection (IV) administration. Therefore, the present study was conducted to determine the bioavailability of isoflavones from different sources following both oral and IV administration in male and female rats. Three sources of isoflavones; Novasoy (a commercial supplement), a mixture of synthetic aglycones (daidzein, genistein and glycitein) and a mixture of synthetic glucosides (daidzin, genistin and glycitin) were tested. Following administration, blood samples were collected at several time points (0, 10, 30 min and 1, 2, 8, 24, 48 h post oral gavage and 0, 10, 30, 45 min and 1, 2, 3, 4, 8 h post-IV dosing) and plasma isoflavones were measured by LC/MS. Bioavailability values for daidzein, genistein and glycitein were significantly (p <0.05) higher (up to sevenfold) in Novasoy and the glucoside forms of isoflavones compared with those of the aglycone forms. Moreover, significant (p <0.05) gender differences in the bioavailability of 7-hydroxyl-3-(4'-hydroxyphenyl)-chroman (a metabolite of daidzein), glycitein and daidzein were observed for Novasoy, with higher values in male rats. In summary, the source of isoflavones and the sex of rats had significant effects on isoflavone bioavailability.

Urinary isoflavone excretion in Korean adults: comparisons of fermented soybean paste and unfermented soy flour

AbstractBACKGROUND: This study compared the 24 h urinary excretion of isoflavone algycones after the consumption of fermented soybean paste or unfermented soy flour in a population of healthy Koreans (four males and five females). An equivalent amount (20 mg) of total isoflavone was consumed as either 52 g of soybean paste or 12.2 g of soy flour in a randomised, crossover trial consisting of two single‐time‐point feedings and having 3 day run‐in and washout periods between feedings.RESULTS: Analysis of starting materials indicated that daidzein (1.4%) and genistein (1.4%) constituted a smaller proportion of the total isoflavones in soy flour as compared with soybean paste (55.3% daidzein and 45.7% genistein). Free and glycosidic forms of isoflavones differed significantly between the two soy products. Levels of β‐glycosides as well as total glycosides (β‐glycosides, malonyl and acetyl forms) were two‐fold higher in soy flour as compared with soybean paste. Malonyl forms predominated in soy flour.CONCLUSIONS: The consumption of soybean paste resulted in significantly higher urinary excretion of both daidzein and genistein as compared with soy flour (P &lt; 0.05 and P &lt; 0.01 respectively). Copyright © 2007 Society of Chemical Industry

How isoflavone levels in common rodent diets can interfere with the value of animal models and with experimental results

Phytoestrogens are a diverse group of plant-derived compounds structurally or functionally mimicking mammalian oestrogens. A variety of effects, in vitro, in animal models and in humans, have been attributed to these compounds. It is generally not appreciated that many commercial rodent diets are formulated with soy as a source of protein and thus large daily doses of phytoestrogens in the form of isoflavones are delivered to the animals. The content of isoflavones in laboratory diets depends on a number of factors and, therefore, varies considerably, both between different formulations, and also from batch to batch of the same formulation. Dietary isoflavones can influence a number of different endpoints, rendering the dietary levels of such compounds an important experimental factor to consider. The levels of isoflavones in common laboratory diets are reviewed in relation to the dietary levels influencing different endpoints.

Isoflavone content and β-glucosidase activity in soybean cultivars of different maturity groups

Soy isoflavones are phenolic compounds comprised of aglucones, β-glucosides and β-glucosides conjugated with malonyl and acetyl groups. The β-glucosidases are a heterogeneous group of enzymes which hydrolyse β-glucosidic links of oligosaccharides and other glucosides conjugates releasing glucose. Thus the objective of this study was to determine the isoflavone forms content, β-glucosidase activity and their correlation in 18 soybean cultivars of different maturity groups under the same cultivation conditions. The predominant isoflavone forms were malonyldaidzin and malonylgenistin in all cultivars, corresponding to 67.0%, while β-glucoside and aglucone forms corresponded to 31.0% and 2.0%, respectively, of the total isoflavones; the acetylated form was not detected. This proportion among different forms was similar to all soybean cultivars studied. Regression analysis between number of days of maturity and all isoflavone forms were not significant. The relationship between maturity days and β-glucosides and malonyl-glucosides showed no correlation. However, a correlation was observed between maturity days and aglucones ( P<0.08). Regression analysis and correlation between β-glucosidase activity and cultivars of different number of days of maturity were not significant, and β-glucosidase activity did not correlate with isoflavone forms contents ( P<0.05).

Inheritance of isoflavone contents in soybean seeds

We studied the genetic basis of isoflavone content inheritance in soybean seeds. The progenitors BARC-8 (low isoflavone content), IAC-100 (high isoflavone content), the F1 and F2 populations derived from reciprocal crosses, and backcross populations were analyzed for isoflavone content and composition. Six isoflavones were detected: daidzin (DZ), genistin (GT), glycitin (GC), malonyldaidzin (MDZ), malonylgenistin (MGT) and malonylglycitin (MGC). DZ, GT, MDZ and MGT contents were influenced by the cytoplasm and the nuclear genes of the maternal parent. For this reason, a genetic model was considered that included the cytoplasmic effect and epistasis between nuclear and cytoplasmic genes. Except for GT, the additive effect was the most important one. For GT content the cytoplasmic effect was the most important. Except for MDZ, the epistatic effects were significant for all the isoflavone forms. Our data indicate that genetic improvement for these traits should explore the additive genetic variances in superior lines or the cytoplasmic effect and the epistatic interactions between cytoplasmic and nuclear genes to obtain the largest selection gains.

INFLUENCE OF HEAT TREATMENT AND GRAIN GERMINATION ON THE ISOFLAVONE PROFILE OF SOY MILK

Soy foods have attracted much attention for their possible effects on human health because of their phytochemical content, mainly isoflavones. Twelve forms of isoflavones have been identified in soybeans and soy products. Technological processes like heat treatment or fermentation can change the isoflavone profile of soy products. In this study, the effects of heat treatment and grain germination on isoflavone profile of soy milk prepared from BRS 213 soybean variety were evaluated. Isoflavone forms were separated and quantified by reversed-phase high-performance liquid chromatography. Heat treatment by autoclaving for 5 min did not affect the total isoflavone content, but led to a 90% decrease in malonyl forms, whereas β-glycosides increased by 70%. Autoclaving for 15 min led to a 20% loss of total isoflavones. Germination for 3 days did not change the total isoflavone content, but changed the distribution profile (15% increase of malonyl forms and 30% decrease of β-glycosides). Germination for 7 days increased the bioavailability of aglycone isoflavones in soy milk by seven times.

Distribution and molecular forms of soy isoflavones in organs/tissues of rats and pigs

In order to assess the potential risks and benefits of soy isoflavones and mechanisms by which health effects occur, it is essential to have a complete understanding of their concentrations and molecular forms in target tissues. Sprague-Dawley rats (n=6) and piglets (n=3) were fed soy protein isolates. Thirteen or twenty organs/tissues were collected from rats or pigs for isoflavone analysis. Rats were perfused and pig tissues were rinsed with 0.09% saline to remove residue blood. A new extraction method enabled us to measure genistein, daidzein, O-DMA, and equol, including aglycone and conjugated forms. Total isoflavone contents varied markedly in different organs of rats (genistein or daidzein, 0.01–14 nmol/g; equol, 0.06–47 nmol/g). The lowest contents were found in brain, spleen, and fat; and the highest in intestines and colon. Approximately 22–45% of total isoflavones were free aglycones, with 21–66% being glucuronides and reminders (14%–42%) being sulfates in rat organs. Total isoflavone contents in pig organs/tissues ranged from 0 to 5.3 nmol/g and 0.4 to 14 nmol/g for genistein and daidzein, respectively (lowest in lung, highest in intestines). The organs of rats and pigs differed in contents of isoflavones as well as proportion of aglycones and conjugates. All rat organs contained equol as the major metabolite of daidzein, whereas pig tissues contained very low level of equol (0–0.2 nmol/g). This study delineated isoflavone profiles in tissues and identified new target tissues of isoflavones, which may help to understand their mechanisms of action in vivo. (USDA CRIS 6251-51000-004-06S)

Phytoestrogens as Cardioprotective Agents

Epidemiologic studies have shown that women with a higher dietary intake of phytoestrogens, plant-derived compounds with partial estrogen agonist properties, have a lower incidence of cardiovascular disease and breast and uterine cancer than women with a lower dietary intake of these substances. The most-studied category of phytoestrogens is the isoflavones found in soybeans, chick peas, and other legumes. Genistein and daidzein are the 2 major forms of isoflavones and have demonstrated lipid-lowering properties, especially in hypercholesterolemic subjects. The mechanism for the hypocholesterolemic effects is unclear, but may involve altered hepatic metabolism, antioxidant properties, tyrosine kinase inhibition, and improved vascular reactivity. Other phytoestrogens, including coumestans and lignans, have shown unfavorable results with regard to their role in the prevention of coronary artery disease. Food and food supplements containing phytoestrogens are often advocated as an alternative to hormone replacement therapy (HRT) in women with contraindications to the use of conventional estrogen replacement or those wanting a natural alternative. In light of the recent trial results with HRT (estrogen plus progesterone), it would be prudent not to recommend phytoestrogens as cardioprotective substances until adequate safety and efficacy studies are completed.

A simplified HPLC method for total isoflavones in soy products

Soybeans and soy products are the main source of isoflavones, a well-studied group of phytoestrogens with numerous biological effects. Several methods have been published to quantify soy isoflavones in food samples. We have earlier applied enzyme hydrolysis followed by acid hydrolysis to measure isoflavones as total aglycone equivalents. Alternatively, other published methods have mainly been developed to measure all 12 forms of isoflavones. In order to simplify the analyses, a new extraction method for determination of total amount of isoflavones was developed and validated. Acetyl and malonyl glucosides were hydrolysed to 7- O-glucosides with 80% EtOH containing 1 M HCl to remove the acetyl and malonyl groups. Aglycones and 7- O-glucosides were analysed with HPLC using coulometric electrode array detection.

Soybean Isoflavones: Effects of Processing and Health Benefits

Isoflavones belong to a group of compounds known as flavanoids that share a basic structure consisting of two benzene rings linked through a heterocyclic pyrane C ring. Soybean provides the most abundant source of isoflavones, which have been linked to the prevention of several diseases, including cancer (prostate, colon, and breast cancer), hypercholesterolemia, cardiovascular disease, atherosclerosis, osteoporosis, and relief of menopausal symptoms in certain women. Currently, isoflavone use is targeted as a nutraceutical. In this article, we review the structural aspects and the effect of processing on the different forms of isoflavones in soy protein. We also discuss the impact of these changes on the health benefits of eating foods containing isoflavones. Isoflavones, as a class of phytoestrogens, are gaining a lot of interest in clinical nutrition.

Genetic parameters relating isoflavone and protein content in soybean seeds

Isoflavones are a class of compounds present in high amounts in soybean seeds, which can be used for prevention and treatment of several chronic diseases. Proteins present in soybean seeds are the basis for the high nutritional value and versatility of this leguminous species in animal and human feeding. The main goals of this work were to estimate heritabilities for isoflavone contents in soybean seeds and the correlation between isoflavone and protein contents. Commercial variety IAC-100 (high isoflavone and normal protein contents) and the line BARC-8 (low isoflavone and high protein contents) were crossed, and one single F1 plant derived 97 F2 seeds, which were used to obtain F3 seeds. A sample of F3 seeds from each F2 plant was used for isoflavone determination by HPLC and protein by the Kjeldahl method. Six isoflavone forms were detected: daidzin, genistin, glycitin, malonyldaidzin, malonylgenistin and malonylglycitin. Total isoflavone contents ranged from 427.92 to 965.89 μg per gram of dry seed and the protein content ranged from 45.17 to 34.95% in BARC-8 and IAC-100, respectively. Our results indicate that it is possible to select for high isoflavone content in early breeding generations because the broad sense heritabilities for the contents of the various isoflavone forms were higher than 90%. In addition, high correlation values among the contents of the individual isoflavone forms were observed (between 0.80 and 0.98). However, negative correlation values were obtained between isoflavone and protein contents, ranging from −0.51 to −0.37 for the different isoflavone forms. The correlation value of −0.47 between total isoflavone and protein contents confirmed the negative correlation between these two parameters, as reported by other authors.

Soy-based formulas and phyto-oestrogens: a safety profile.

Phyto-oestrogens are non-steroidal plant-derived compounds that possess oestrogenic activity and act as selective oestrogen receptor modulators (SERMs). Among the dietary oestrogens, the isoflavone class enjoy a wide-spread distribution in most of the members of the Leguminosae family, including such prominent high-content representatives as soybean. Phyto-oestrogen research has grown rapidly in recent years owing to epidemiological studies suggesting that diets rich in soy may be associated with potential health benefits. There is a paucity of data on endocrine effects of soy phytochemicals during infancy, the most sensitive period of life for the induction of toxicity. The safety of isoflavones in infant formulas has been questioned recently owing to reports of possible hormonal effects. Infants fed soy formula receive high levels of phyto-oestrogens in the form of isoflavones (genistein, daidzein and their glycosides). To date, no adverse effects of short- or long-term use of soy proteins have been observed in humans and exposure to soy-based infant formulas does not appear to lead to different reproductive outcomes than exposure to cow milk formulas. Soy formula seems to be a safe feeding option for most infants. Nevertheless, much closer studies in experimental animals and human populations exposed to phyto-oestrogen-containing products, and particularly soy-based infant formulas, are necessary.

Soy-based formulas and phyto-oestrogens: a safety profile

Phyto-oestrogens are non-steroidal plant-derived compounds that possess oestrogenic activity and act as selective oestrogen receptor modulators (SERMs). Among the dietary oestrogens, the isoflavone class enjoy a wide-spread distribution in most of the members of the Leguminosae family, including such prominent high-content representatives as soybean. Phyto-oestrogen research has grown rapidly in recent years owing to epidemiological studies suggesting that diets rich in soy may be associated with potential health benefits. There is a paucity of data on endocrine effects of soy phytochemicals during infancy, the most sensitive period of life for the induction of toxicity. The safety of isoflavones in infant formulas has been questioned recently owing to reports of possible hormonal effects. Infants fed soy formula receive high levels of phyto-oestrogens in the form of isoflavones (genistein, daidzein and their glycosides). To date, no adverse effects of short- or long-term use of soy proteins have been observed in humans and exposure to soy-based infant formulas does not appear to lead to different reproductive outcomes than exposure to cow milk formulas. Soy formula seems to be a safe feeding option for most infants. Nevertheless, much closer studies in experimental animals and human populations exposed to phyto-oestrogen-containing products, and particularly soy-based infant formulas, are necessary.

Bioavailability of soybean isoflavones from aglycone and glucoside forms in American women

Test results on the bioavailability of isoflavones in the aglycone or glucoside form in Eastern and Western human subjects are contradictory. The objective was to investigate the bioavailability of the soy isoflavones daidzein and genistein in American women with typical American dietary habits after ingestion of the aglycone or glucoside form of isoflavones. Fifteen American women aged 46 +/- 6 y participated in a randomized, double-blind study. Blood samples were collected 0, 1, 2, 4, 8, 12, 24, and 48 h after consumption of aglycone or glucoside tablets with breakfast. The plasma curves for daidzein, genistein, and equol were constructed and the postprandial maximum concentration (C(max)), time to the maximum concentration (t(max)), and area under the curve (AUC) were determined. Isoflavone concentrations peaked early (1-2 h) in plasma and peaked again at 4-8 h. Mean C(max), t(max), and AUC values for genistein were not significantly different after ingestion of aglycone or glucoside. However, C(max) and AUC values, but not t(max), were significantly higher for daidzein after aglycone ingestion, which was partly due to its higher content in the aglycone tablets. Equol appeared after 4 h and remained elevated after 48 h. Despite a higher content of daidzein in the aglycone tablets, the AUC for equol was significantly higher after ingestion of the glucoside tablets, probably because of the metabolic action of intestinal bacteria during the long intestinal transit time of glucoside. The apparent bioavailability of genistein and daidzein is not different when consumed as either aglycone or glucoside by American women.

Variations in isoflavone levels in soy foods and soy protein isolates and issues related to isoflavone databases and food labeling.

The reliability of databases on the isoflavone composition of foods designed to estimate dietary intakes is contingent on the assumption that soy foods are consistent in their isoflavone content. To validate this, total and individual isoflavone compositions were determined by HPLC for two different soy protein isolates used in the commercial manufacture of soy foods over a 3-year period (n = 30/isolate) and 85 samples of 40 different brands of soy milks. Total isoflavone concentrations differed markedly between the soy protein isolates, varying by 200-300% over 3 years, whereas the protein content varied by only 3%. Total isoflavone content varied by up to 5-fold among different commercial soy milks and was not consistent between repeat purchases. Whole soybean milks had significantly higher isoflavone levels than those made from soy protein isolates (mean +/- SD, 63.6 +/- 21.9 mg/L, n = 43, vs 30.2 +/- 5.8 mg/L, n = 38, respectively, p < 0.0001), although some isolated soy protein-based milks were similar in content to "whole bean" varieties. The ratio of genistein to daidzein isoflavone forms was higher in isolated soy protein-based versus "whole bean" soy milks (2.72 +/- 0.24 vs 1.62 +/- 0.47, respectively, p < 0.0001), and the greatest variability in isoflavone content was observed among brands of whole bean soy milks. These studies illustrate large variability in the isoflavone content of isolated soy proteins used in food manufacture and in commercial soy milks and reinforce the need to accurately determine the isoflavone content of foods used in dietary intervention studies while exposing the limitations of food databases for estimating daily isoflavone intakes.

Exposure to soy-based formula in infancy and endocrinological and reproductive outcomes in young adulthood.

A large body of evidence documents the role of phytoestrogens in influencing hormone-dependent states. Infants fed soy formula receive high levels of phytoestrogens, in the form of soy isoflavones, during a stage of development at which permanent effects are theoretically possible. However, a paucity of data exists on the long-term effects of infant soy formulas. To examine the association between infant exposure to soy formula and health in young adulthood, with an emphasis on reproductive health. Retrospective cohort study conducted from March to August 1999 among adults aged 20 to 34 years who, as infants, participated during 1965-1978 in controlled feeding studies conducted at the University of Iowa, Iowa City (248 were fed soy formula and 563 were fed cow milk formula during infancy). Self-reported pubertal maturation, menstrual and reproductive history, height and usual weight, and current health, compared based on type of formula exposure during infancy. No statistically significant differences were observed between groups in either women or men for more than 30 outcomes. However, women who had been fed soy formula reported slightly longer duration of menstrual bleeding (adjusted mean difference, 0.37 days; 95% confidence interval [CI], 0.06-0.68), with no difference in severity of menstrual flow. They also reported greater discomfort with menstruation (unadjusted relative risk for extreme discomfort vs no or mild pain, 1.77; 95% CI, 1.04-3.00). Exposure to soy formula does not appear to lead to different general health or reproductive outcomes than exposure to cow milk formula. Although the few positive findings should be explored in future studies, our findings are reassuring about the safety of infant soy formula.

Improved methods for the extraction and analysis of isoflavones from soy-containing foods and nutritional supplements by reversed-phase high-performance liquid chromatography and liquid chromatography–mass spectrometry

An improved method for extraction and analysis of isoflavones from soy protein, soy foods and nutritional supplements is presented. The method uses acetonitrile extraction without acidification, with apigenin as internal standard. Samples extracted in acetonitrile–water are diluted to 50% acetonitrile and directly injected for gradient HPLC separation on a C 18 reversed-phase column. This method saves significant time during sample preparation and improves accuracy and precision. Conventional and rapid HPLC analysis methods compatible with the extraction scheme were developed. During development of the methods, unexpected minor forms of malonyl and acetyl isoflavones were discovered in extracts of soy proteins and in pure isoflavone standard preparations. By LC–triple MS, these peaks have identical composition to the respective 6″- O-malonyl- and 6″- O-acetyl-isoflavones from which they form. These minor forms are believed to be malonyl and acetyl isoflavones where the site of attachment is a hydroxyl other than the 6′-OH of the glucose. These compounds can represent significant minor isoflavone components of foods, which contain high concentrations of malonyl or acetyl isoflavones.