Individual Isoflavones Research Articles

Analysis of isoflavone concentration and composition in soybean [Glycine max (L.)] seeds between the cropping year and storage for 3�years

The objective of this study was to examine the relation between storage period and isoflavone concentration and composition in soybean [Glycine max (L.) Merrill] seeds. Total isoflavones varied from 470.0 to 878.1 µg g−1 over the three cropping years and there were significant differences between the soybean cultivars. Generally, the malonyl conjugates were in higher concentrations than were the other compounds, and the individual isoflavones varied with each cropping year. The Myeongjunamul (1998: 936.7 µg g–1, 1999: 727.0 µg g–1, 2000: 807.8 µg g–1) cultivar had the highest concentration, on average. Regarding changes in isoflavones with storage period, the concentration decreased by 91.7 to 171.1 µg g–1. Malonyldaidzin and malonylgenistin concentrations also decreased, and the concentration of glucosides increased slightly over the 3 years. Acetyldaidzin, acetylglycitin and glycitein could not be detected or were only found in very small concentrations. Pureun (−66.2 µg g–1) and Myeongjunamul (−77.4 µg g–1) cultivars showed little variation in isoflavone levels, whereas the concentration in Muhan (–295.4 µg g–1) decreased markedly on average over the three storage years. There were also significant differences between the cultivars. Our study suggests that it might be feasible to improve the storage methods of soybeans to preserve them for longer periods, which would have benefits for both their import and their export.

Soy Isoflavones Affect Sterol Regulatory Element Binding Proteins (SREBPs) and SREBP-Regulated Genes in HepG2 Cells

Soy intake reduces cholesterol levels. However, both the identity of the soy component or components that contribute to this reduction and the cellular mechanism producing this reduction are unknown. Soy consists of protein, lipids, fiber, and phytochemicals including isoflavones. We propose that the isoflavone component of soy mediates this effect, at least in part, by affecting cellular sterol homeostasis. We investigated the effects of an isoflavone-containing soy extract and the individual isoflavones on the maturation of the sterol regulatory element binding proteins (SREBP) and the expression of SRE-regulated genes controlling lipid metabolism. We found a corresponding increase in the mature form of SREBP-2 in both soy extract- and isoflavone-treated HepG2 cells, whereas there was no significant change in the levels of SREBP-1. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase protein and HMG CoA synthase mRNA levels also increased. When HepG2 cells were transiently transfected with HMG CoA synthase and LDL receptor reporter plasmids there was an increase in expression in response to soy extract or isoflavone treatment from both of these promoters, but this induction was blunted in the presence of sterols (P < 0.05). The mechanism responsible for this effect may be via a statin-like inhibition of HMG CoA reductase enzyme activity or by enhanced SREBP processing via the SREBP cleavage activating protein. We hypothesize that maturation of SREBP and induction of SRE-regulated genes produce an increase in surface LDL receptor expression that increases the clearance of plasma cholesterol, thus decreasing plasma cholesterol levels.

Isoflavone content of soybean cultivars grown in eastern Canada

AbstractSoybean (Glycine max (L) Merr) seeds contain isoflavones that have positive impacts on human health. The objective of this study was to determine isoflavone concentrations of early maturing soybean cultivars grown in different environments and to determine their relation to other important seed characteristics. Twenty soybean cultivars were grown in replicated trials at two sites in Montreal, Canada in 2002/2003 and their total and individual isoflavone concentrations were determined by high‐performance liquid chromatography. Seed yield, 100‐seed weight and oil, crude protein and crude fiber concentrations were concurrently determined. Total and individual isoflavone concentrations were significantly affected by cultivar, site and year. Total isoflavone concentration ranged from 360 to 2241 µg g−1 and averaged 851 µg g−1 across environments and cultivars. Variation across environments ranged from 20 to 100% for specific cultivars. Total isoflavone concentration was, on average, 40% greater in 2003 than 2002, which was characterized by above average temperatures and severe drought. Despite significant cultivar × year × site interactions, cultivars with consistently high and low isoflavone concentrations across environments were identified. Weak but significant positive correlations were observed between total isoflavone concentration and seed yield, 100‐seed weight and crude fiber, indicating that isoflavone concentration is positively associated with other desirable seed characteristics. Copyright © 2004 Society of Chemical Industry

Effect of Red Clover Isoflavones on Cox-2 Activity in Murine and Human Monocyte/Macrophage Cells

: Long-term use of nonsteroidal anti-inflammatory drugs is associated with a reduction in the incidence of a range of cancers, the mechanism of which is thought to be cyclooxygenase (COX) inhibition. Because long-term ingestion of foods rich in isoflavones, such as legumes (beans, peas, lentils) has been associated with reduced cancer incidence, it was considered useful to examine the COX-inhibitory activities of individual isoflavones. Red clover dietary supplements also contain varying ratios of the 4 isoflavones commonly found in legume-based diets, namely, daidzein, genistein, formononetin, and biochanin. Using 2 separate cell assays, this study examined the ability of the isoflavones found in red clover to inhibit COX enzyme activity in both the murine macrophage cell line RAW 264.7 and human monocytes. Within the range of 1-40 μM in RAW 264.7 cells and 10-100 μM in human monocytes, isoflavones were able to reduce significantly the synthesis of prostaglandin E2 and/or thromboxane B2 (P < 0.001 to P < 0.05), indicating COX inhibition. Thus, it is possible that the lower rates of some cancers in populations with a high intake of dietary isoflavones is linked to their inhibition of COX activity.

Critical Issues in R&amp;D of Soy Isoflavone‐enriched Foods and Dietary Supplements

ABSTRACT: Increasing consumer awareness of soy as a healthy food ingredient has led to a tremendous growth in sales of soy isoflavone‐enriched foods and dietary supplements in the past 3 y. Because of their weak estrogenic activity, isoflavones are believed to have preventive effects for several hormone‐dependent diseases. This article discusses some critical issues to be considered in any R&amp;D program of novel soy and isoflavone products for the healthy‐food market, such as (1) Which isoflavone compounds should be in the product? (2) Which individual isoflavones are the most beneficial ones? (3) Can certain isoflavone groups be targeted to specific disease prevention goals? (4) Are there any safety concerns in isoflavone consumption? (5) How are isoflavones affected by thermal processing and storage? (6) How should isoflavones be analyzed? These questions may affect the choice of isoflavone source, processing conditions, quality control procedures, and marketing considerations.

Dietary exposure estimates of isoflavones from the 1998 UK Total Diet Study

The phyto-oestrogen contents of the 1998 UK Total Diet Study (TDS) food group composites were determined. Each TDS set consisted of composite samples, one for each of the 20 designated food groups. These composites then represented the average consumption of all the individual food elements in each group, processed into the form in which they were consumed. In the TDS, individual composites of the bread, processed meat and fish food groups contained >5 mg kg−1 of the individual isoflavones (daidzein, genistein and glycitein). Individual composites from the groups, miscellaneous cereals, other vegetables, fruit products and nuts contained >1 mg kg−1. After weighting for average consumption of food from each TDS food group, an estimated daily intake of 3 mg day−1 of combined isoflavone aglycones was obtained from the TDS sample collection model for the average (adult) consumer. The UK dietary intake of phyto-oestrogens is higher than previously estimated due in part to the use of soya in processed foods.

Enzyme-linked immunosorbent assay for the determination of isoflavones in alimentary important plants

The development of polyclonal antibody-based enzyme-linked immunosorbent assays (ELISA) for the determination of individual isoflavones, i.e. daidzein, genistein and biochanin and their homologues, is presented in this work. Isoflavone conjugates with bovine serum albumin were used as immunogens, coupled at the position C 7 and C 4’ via a carboxy methyl spacer. The developed ELISAs are highly specific, I&lt;sub&gt;50&lt;/sub&gt; values of the standard curves range between 0.3–1.2 ng/ml. The cross reactivities to other isoflavones are in acceptable range and the interference of non-isoflavonoid molecules is negligible. The immunoassays have been used for monitoring of changes in isoflavone levels in alimentary important plants, such as Medicago sativa, during germination; and during different vegetation stages of the Rutaceae family plants.

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.

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.

Variation in isoflavone of soybean cultivars with location and storage duration.

Fifteen soybean [Glycine max (L.) Merrill] cultivars were grown in Seoul, Suwon, and Kyongsan, Korea, in 1998, 1999, and 2000, and their isoflavone contents were assessed. After harvest, the beans were stored for 3 years at room temperature. Soybean isoflavones were analyzed using high-performance liquid chromatography (HPLC) within each crop year and after storage. Total isoflavone contents ranged from 188.4 to 685.6 mg 100 g(-1) in 1998, from 218.8 to 948.9 mg 100 g(-1) in 1999, and from 293.1 to 483.0 mg 100 g(-1) in 2000. The year x variety, and year x location x variety interactions were significantly different in 1998, the year x location, year x variety, and year x location x variety interactions were significantly different in 1999, and the year x variety interaction was significantly different in 2000 for total and individual isoflavone contents. Total isoflavone contents of soybeans stored for 1 year were only slightly higher than those of soybeans stored for 2 or 3 years. However, the concentrations of individual isoflavones, especially 6' '-O-malonyldaidzin and 6' '-O-malonylgenistin, decreased markedly in soybeans stored for 2 or 3 years. These data suggest that it may be feasible to improve soybean cultivars with higher antioxidative substances.

Isoflavones: their effects on cardiovascular risk and functions.

Isoflavones - plant polyphenols with oestrogenic activity - are becoming widely distributed in foods and sold as nutriceuticals. Their similarity to steroidal oestrogens has stimulated research into potential cardiovascular benefits. The two major areas, those of lipid lowering and of improved vascular function, are reviewed. A major plasma cholesterol lowering effect has not been sustained in recent publications. Relatively minor reductions in LDL-cholesterol or non-HDL-cholesterol have generally been reported, but studies in which no reduction was found have also been reported. Fewer publications have dealt with effects on vasculature, but these have been more consistent. Arterial compliance, a parameter of the elasticity or distensibility of large arteries, is improved by as much as has been reported for estrogens. Vasodilatory effects on the microcirculation with isoflavones or their metabolites may provide opportunities for pharmacological intervention. The metabolism of isoflavones is reviewed briefly in order to highlight its complexity. Although research has failed to demonstrate substantial cholesterol lowering with dietary isoflavones, consumption of legumes is generally to be encouraged as part of the overall strategy for lowering plasma lipids. The favourable effects of ingested or infused isoflavones on several parameters of arterial function suggest an alternative explanation for the apparent low prevalence of coronary disease in populations that eat legumes as a staple food. A better understanding of the pharmacokinetics and bioavailabilty of individual isoflavones is needed if definitive studies are to be designed.

Complementary and alternative medicine for menopausal symptoms: a review of randomized, controlled trials.

Women commonly use soy products, herbs, and other complementary and alternative medicine (CAM) therapies for menopausal symptoms. Randomized, controlled trials have evaluated the efficacy and short-term safety of these therapies. To review randomized, controlled trials of CAM therapies for menopausal symptoms in order to better inform practice and guide future research. Searches of MEDLINE for articles published from January 1966 through March 2002, of the Alternative and Complementary Database (AMED) of the British Library for articles published from January 1985 through December 2000, and of the authors' own extensive files. Search terms were hot flash/flush, menopause, and climacteric, combined with phytoestrogens, alternative medicine, herbal medicine, traditional medicine, Traditional Chinese Medicine (TCM ), Ayurveda, naturopathy, chiropractic, osteopathy, massage, yoga, relaxation therapy, homeopathy, aromatherapy, and therapeutic touch. 29 randomized, controlled clinical trials of CAM therapies for hot flashes and other menopausal symptoms were identified; of these, 12 dealt with soy or soy extracts, 10 with herbs, and 7 with other CAM therapies. Each author extracted information from half of the studies on the number of patients, study design, outcome measures, and results; the other author then checked these results. Soy seems to have modest benefit for hot flashes, but studies are not conclusive. Isoflavone preparations seem to be less effective than soy foods. Black cohosh may be effective for menopausal symptoms, especially hot flashes, but the lack of adequate long-term safety data (mainly on estrogenic stimulation of the breast or endometrium) precludes recommending long-term use. Single clinical trials have found that dong quai, evening primrose oil, a Chinese herb mixture, vitamin E, and acupuncture do not affect hot flashes; two trials have shown that red clover has no benefit for treating hot flashes. Black cohosh and foods that contain phytoestrogens show promise for the treatment of menopausal symptoms. Clinical trials do not support the use of other herbs or CAM therapies. Long-term safety data on individual isoflavones or isoflavone concentrates are not available.

Hydrolysis of Isoflavone Glycosides to Aglycones by β-Glycosidase Does Not Alter Plasma and Urine Isoflavone Pharmacokinetics in Postmenopausal Women

We investigated whether the bioavailability of isoflavones could be enhanced by enzymatic hydrolysis of glycosides to aglycones before consumption of a nonfermented soy food. Two drinks were formulated with an enriched isoflavone extract from soy germ (Fujiflavone P10), one of which was hydrolyzed enzymatically with beta-glucosidase to produce aglycones. In a randomized, double-blinded, cross-over study, six European, postmenopausal women consumed each soy drink at a 1-wk interval at a concentration of 1 mg total isoflavones/kg body. The plasma and urinary pharmacokinetics of daidzein, genistein and glycitein did not differ after consumption of the two beverages. Plasma total isoflavone concentrations reached 4-5 micro mol/L. The pharmacokinetics of glycitein were similar to those of daidzein. The isoflavone secondary metabolites detected were dihydrodaidzein in plasma and O-desmethylangolensin, equol, and dihydrogenistein in urine. The ratios of individual isoflavones to one another were not conserved from food to plasma to urine, indicating that the individual isoflavones do not have the same absorptions and body retentions. In conclusion, previous hydrolysis of glycosides to aglycones does not enhance the bioavailability of isoflavones in humans.

Differential interaction of Sophora isoflavonoids with lipid bilayers

The mechanisms of some biological effects exerted by flavonoids (e.g. activity against lipid oxidation, multidrug resistance modulation) may involve their interactions with lipid bilayers. Due to variety of substituents attached to the flavonoid nucleus individual isoflavones significantly differ in their properties; in particular they may differently interact with membranes. For this reason we have investigated the interactions of different isoflavones with lipid bilayers. The influence of four plant isoflavones on the phase transitions of dipalmitoylphosphatidylcholine (DPPC) and on liposome aggregation was studied, using microcalorimetry and absorption measurements, respectively. We found that isoflavones substituted with one or two prenyl groups less effectively induce liposome aggregation than more polar ones, possessing no prenyl groups. For aggregation-promoting compounds, rather small differences in the influence on phosphatidylcholine, phosphatidylserine and phosphatidylinositol liposomes were recorded. On the other hand, the alteration of DPPC phase transitions by prenyl-substituted isoflavones was more pronounced than changes induced by non-prenyl ones. On the basis of observed effects we conclude that prenyl-substituted isoflavones penetrate deeper into the lipid bilayer while more polar ones act closer to the membrane surface. Comparing our results with biological tests it seems that interactions with the hydrophobic core of membranes are responsible for the activity of the studied isoflavones.

Isoflavone aglycon and glucoconjugate content of high- and low-soy U.K. foods used in nutritional studies.

The isoflavone aglycon and glucoconjugate content of commercially prepared and "home-prepared" high- and low-soy foods selected for use in an on-going nutritional study were measured by LC-MS. The daidzin, daidzein, 6"-O-malonyldaidzin, 6"-O-acetyldaidzin, genistein, genistin, 6"-O-malonylgenistin, 6"-O-acetylgenistin, glycitin, glycitein, 6"-O-malonylglycitin, and 6"-O-acetylglycitin content are expressed in terms of individual isoflavones, total isoflavone equivalents, and milligrams of isoflavones per portion served. Soybeans (774 mg x kg(-1) total isoflavones) and soybean-containing foods had the highest isoflavone content of the foods examined. The low-soy foods all contained very low concentrations (<8 mg x kg(-1) total isoflavones) of the isoflavone aglycons and glucoconjugates. High- and low-soy 11 day rotating menus were constructed from the analyzed foods to deliver 100.0 and 0.5 mg of isoflavones per day, respectively.

Determination of Isoflavones in Soy and Selected Foods Containing Soy by Extraction, Saponification, and Liquid Chromatography: Collaborative Study

Isoflavones are biologically active compounds occurring naturally in a variety of plants, with relatively high levels found in soybeans. Twelve laboratories participated in a collaborative study to determine the aglycon isoflavone content of 8 test samples of soy and foods containing soy. The analytical method for the determination of isoflavones incorporates a mild saponification step that reduces the number of analytes measured and permits quantitation versus commercially available, stable reference standards. Test samples were extracted at 65 degrees C with methanol-water (80 + 20), saponified with dilute sodium hydroxide solution, and analyzed by reversed-phase liquid chromatography with UV detection at 260 nm. Isoflavone results were reported as microg/aglycon/g or microg aglycon equivalents/g. The 8 test samples included 2 blind duplicates and 4 single test samples with total isoflavone concentrations ranging from approximately 50 to 3000 microg/g. Test samples of soy ingredients and products made with soy were distributed to collaborators with appropriate reference standards. Collaborators were asked to analyze test samples in duplicate on 2 separate days. The data were analyzed for individual isoflavone components, subtotals of daidzin-daidzein, glycitin-glycitein, and genistin-genistein, and total isoflavones. The relative standard deviation (RSD) for repeatability was 1.8-7.1%, and the RSD for reproducibility was 3.2-16.1% for total isoflavone values of 47-3099 microg/g.

Optimization of acid hydrolysis conditions for total isoflavones analysis in soybean hypocotyls by using RSM

The optimum hydrolysis conditions of soybean hypocotyls with HCl for a quantitative HPLC determination of total isoflavones (TI) were studied by using response surface methodology (RSM). HCl concentration (HC), hydrolysis time (HT) and reaction temperature (RT) were assumed to be the most important factors affecting hydrolysis for the determination of TI. Optimum hydrolysis conditions for maximizing the determination of TI were: HC=3.42N, HT=205.5 min and RT=44.6°C. The model had a satisfactory coefficient of R2 (=0.967) and was verified experimentally. Furthermore, the recovery of individual isoflavones was 97–100% under the optimum hydrolysis conditions. The results suggested that the conditions were mild and useful for maximizing a quantitative HPLC determination of TI in soybean hypocotyls.

Influence of Genotype and Environment on Isoflavone Contents of Soybean

Isoflavones in soybean [Glycine max (L.) Merr.] may have positive impacts on human health. The objective of this study was to determine the role of the genotype, environment, and genotype × environment interactions on isoflavone contents of soybean. Nine isoflavones were measured in six cultivars grown at eight locations during 2 yr. The mean contents of total isoflavones and six of nine individual isoflavones were significantly higher in 1996 than 1995. There were significant differences among locations in one or more years for total and individual isoflavone contents. The year × location interactions were significant due to changes in rank and magnitude among the locations during the 2 yr. The genotype, genotype × year, genotype × location, and genotype × year × location interactions were significant for total and individual isoflavone contents. Despite the significant genotype × environment interactions, the differences between the cultivars with the highest and lowest total and individual isoflavone contents were relatively consistent among the 16 environments. It should be possible to breed for isoflavone content as a quantitative trait in a cultivar development program.

Dietary Intake and Sources of Isoflavones Among Japanese

We examined the dietary intake and sources of isoflavones (daidzein and genistein) among Japanese subjects based on dietary records (DRs). The subjects comprised two groups: 1,232 who completed one-day DRs (Group 1) and 88 men and women who kept four four-day (16-day) DRs. For quantitative data on the level of daidzein and genistein in soy foods, we extensively reviewed the literature, particularly for Japanese soy foods, and adopted the median value for each food. The median intake of daidzein was 12.1 and 9.5 mg/day among Groups 1 and 2, respectively, while the corresponding values for genistein were 19.6 and 14.9 mg/day. The top four foods (tofu, miso, natto, and fried tofu) covered about 90% of the population intake of daidzein and genistein. It did not seem feasible to estimate one's intake of isoflavones by using dietary recording/recall in epidemiological studies, since the day-to-day variation in intake was too large, the within-person coefficient of variation being 89.1% for daidzein and genistein. Therefore, we should use other methods, such as food-frequency questionnaires, focusing on the four major sources of isoflavones, to assess individual isoflavone intake.

Constitutive isoflavones as modulators of indole-3-acetic acid oxidase activity of acidic cell wall isoperoxidases from lupin hypocotyls

The effect of isoflavones on the indole-3-acetic acid (IAA) oxidase activity of acidic cell wall isoperoxidases from lupin hypocotyls was tested using as isoflavone source a mixture of isoflavones obtained from the whole hypocotyl, isoflavones isolated from five different growth zones, and individual isoflavones purified by thin-layer chromatography. The results showed that 2′-hydroxygenistein was the most potent inhibitor of the enzymatic activity, genistein and wighteone having similar, but lower, inhibitory effects. Since 2′-hydroxygenistein is accumulated in the cell walls of young tissues, it can be expected that the strong inhibitory effect shown by isoflavones isolated from the most apical zones is due mainly to 2′-hydroxygenistein. These results are discussed on the basis of the compartimentalization of auxin catabolism in both young epidermal cells and differentiating vascular tissues of lupin seedlings.