Soy Isoflavone Aglycones Research Articles

Enhanced Cholesterol-Lowering and Antioxidant Activities of Soymilk by Fermentation with Lactiplantibacillus plantarum KML06.

This study aimed to evaluate the cholesterol-lowering and antioxidant activities of soymilk fermented with probiotic Lactobacillaceae strains and to investigate the production of related bioactive compounds. Lactiplantibacillus plantarum KML06 (KML06) was selected for the fermentation of soymilk because it has the highest antioxidant, cholesterol-lowering, and β-glucosidase activities among the 10 Lactobacillaceae strains isolated from kimchi. The genomic information of strain KML06 was analyzed. Moreover, soymilk fermented with KML06 was evaluated for growth kinetics, metabolism, and functional characteristics during the fermentation period. The number of viable cells, which was similar to the results of radical scavenging activities and cholesterol assimilation, as well as the amount of soy isoflavone aglycones, daidzein, and genistein, was the highest at 12 h of fermentation. These results indicate that soymilk fermented with KML06 can prevent oxidative stress and cholesterol-related problems through the production of soy isoflavone aglycones.

Effects of soybean isoflavone aglycone on osteoporosis in ovariectomized rats.

Postmenopausal osteoporosis is one of the most common metabolic diseases in old women, and supplementing estrogen through bioactive substances is one of the important ways to improve menopausal syndrome. Some studies have confirmed that soybean isoflavone has estrogenic activity, and the main active component of soybean isoflavones is isoflavone aglycones. However, few studies have investigated the improvement effect of high-purity soy isoflavone aglycones on postmenopausal osteoporosis. Thus, the effect of different doses of high-purity soybeans isoflavone aglycone on the ovariectomized female osteoporosis rat model was evaluated by oral gavage. The rats were divided into seven experimental groups including SHAM, OVX, EE, SIHP, AFDP-L, AFDP-M, and AFDP-H, which was administered for 60 days from 30 days after ovariectomy. We collected blood from the abdominal aorta of rats on the 30th, 60th, and 90th days respectively, analyzed its serum biochemistry, and took out the femur for micro-CT imaging and bone microstructure parameter analysis. Results showed that the intervention effect of AFDP-H group on osteoporosis rats at 60 and 90 days was similar to that of EE group, and superior to the OVX group, SIHP group, AFDP-L group, AFDP-M group. The AFDP-H group inhibited the decrease in serum bone markers, bone density, trabeculae quantity, trabeculae thickness, and bone volume fraction, and increased the trabecular separation caused by ovariectomy, thereby significantly improving bone microstructure. It also prevented continuous weight gain and increased cholesterol levels in female rats. This study provided theoretical to application of soybean isoflavone aglycone in the intervention of osteoporosis. and confirmed that could replace chemical synthetic estrogen drugs.

Optimization of Fermentation Process for Improving Soy Isoflavones Aglycone Content in Bean Dregs by Lactobacillus plantarum PL70a

Aglycone-type soy isoflavones have higher biological activity than glycoside-type soy isoflavones. Bean dregs are rich in glycoside-type soy isoflavones. In order to improve the biological activity of soy isoflavones in bean dregs, the Lactobacillus plantarum PL70a was screened and the fermentation process of converting glycoside-type soy isoflavones into aglycone-type in bean dregs was optimized by single-factor experiments and the response surface methodology. The optimal fermentation process was as follows: (NH4)2SO4 was added in an amount of 0.17%, glucoamylase was added in an amount of 0.87%, inoculation amount was 17%, sucrose was added in an amount of 1.3%, and fermentation time was 3 days. Under this process, the content of aglycone-type soy isoflavones in bean dregs significantly increased. Trypsin inhibitors and antigen proteins were almost removed. The fermentation process provides a good reference for the low-cost processing of high-quality soy isoflavone aglycone food.

Anti-aging effect of Lactobacillus plantarum HFY09-fermented soymilk on D-galactose-induced oxidative aging in mice through modulation of the Nrf2 signaling pathway

Abstract After soymilk fermented by L. plantarum HFY09, the bound soybean isoflavones content was reduced, but free soybean isoflavones and peptides increased. In vivo results show that L. plantarum HFY09-fermented soymilk increased the T-SOD, MDA, GSH-Px, GSH, CAT, and NO in the serum, liver, and brain tissues of mice. The aforementioned soymilk also increased the Col I and hyaluronic acid, and reduced the contents of Col III, H2O2, and AGEs in the skin. L. plantarum HFY09-fermented soymilk upregulated Nrf2, HO-1, NOS1, NOS3, SOD1, SOD2, CAT, Nqol, Gclm expression in the liver and the spleen, reduced the NOS2 expression, and upregulated SOD1 SOD2, CAT, GSH-Px, TIMP1, and TIMP2 expression in skin, downregulated MMP3, and MMP9 expression. These results show that L. plantarum HFY09-fermented soymilk could effectively alleviate aging in D-galactose-induced aging mice, the mechanism may be related to the activation of Nrf2 signaling pathway by soy isoflavone aglycone and small molecule peptides.

Isoflavone Aglycones Attenuate Cigarette Smoke-Induced Emphysema via Suppression of Neutrophilic Inflammation in a COPD Murine Model.

Chronic obstructive pulmonary disease (COPD), a lung disease caused by chronic exposure to cigarette smoke, increases the number of inflammatory cells such as macrophages and neutrophils and emphysema. Isoflavone is a polyphenolic compound that exists in soybeans. Daidzein and genistein, two types of isoflavones, have been reported to have anti-inflammatory effects in various organs. We hypothesized that the daidzein-rich soy isoflavone aglycones (DRIAs) attenuate cigarette smoke-induced emphysema in mice. Mice were divided into four groups: the (i) control group, (ii) isoflavone group, (iii) smoking group, and (iv) isoflavone + smoking group. The number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) and the airspace enlargement using the mean linear intercept (MLI) were determined 12 weeks after smoking exposure. Expressions of neutrophilic inflammatory cytokines and chemokines were also examined. In the isoflavone + smoking group, the number of neutrophils in BALF and MLI was significantly less than that in the smoking group. Furthermore, the gene-expressions of TNF-α and CXCL2 (MIP-2) in the isoflavone + smoking group were significantly less than those in the smoking group. Supplementation of the COPD murine model with DRIAs significantly attenuates pathological changes of COPD via suppression of neutrophilic inflammation.

Biofunctional Attributes and Storage Study of Soy Milk Fermented by Lactobacillus rhamnosus and Lactobacillus helveticus.

SUMMARYDifferent soybean products are considered as traditional functional food among the Mongolian population in Northeast India. In the present study, the effect of different flavours (mango, orange, vanilla and white rose), inoculation rates of Lactobacillus rhamnosus K4E (KX950834) and Lactobacillus helveticus K14 (KU644578), and mass fractions of skimmed milk and sugar on the acceptability of soy yoghurts was studied. Physicochemical (pH, titratable acidity) and microbial analyses (total bacterial, total coliform, yeast and mould count) were conducted, and organoleptic (aroma, taste, colour, mouthfeel, texture and overall acceptability) and biofunctional properties (angiotensin-converting enzyme (ACE) inhibitory activity, antioxidant and antimicrobial activities and biotransformation of isoflavones) were evaluated during storage up to 10 days under refrigeration conditions (6–8 °C). Panellists preferred white rose soy yoghurt more than other flavours. The pH was from 5.65 to 4.20, the titratable acidity (expressed as mass fraction of lactic acid) was from 0.33 to 0.51% and total Lactobacillus count ranged from 6.81 to 8.69 log CFU/mL during storage. The ACE inhibitory activity increased from 21.17% on day 0 to 81.03% on day 5, followed by a decrease of the activity after 10 days (38.85%). The antioxidant activity was the highest on day 5 (87%). White rose soy yoghurt had the highest antimicrobial activity against Listeria monocytogenes, followed by Bacillus subtilis, Staphylococcus aureus, Salmonella typhi and Escherichia coli. RP-HPLC analysis showed that after 18 h, the production of soy isoflavone aglycones genistein and daidzein in yoghurt was 87.3 and 58.4%, respectively.

Genistein decreases basal hepatic cytochrome P450 1A1 protein expression and activity in Swiss Webster mice

Soy consumption has been associated with risk reduction for chronic diseases such as cancer. One proposed mechanism for cancer prevention by soy is through decreasing cytochrome P450 1A1 (Cyp1a1) activity. However, it is not known with certainty which soy components modulate Cyp1a1, or the characteristics or mechanisms involved in the responses after short-term (<20 days) dietary treatment without concomitant carcinogen-mediated induction. Therefore, the objective was to test the hypothesis that physiologic concentrations of dietary genistein and/or daidzein will decrease basal hepatic Cyp1a1 protein expression and activity in male and female Swiss Webster mice via inhibiting the bindings of aryl hydrocarbon receptor (AhR)–AhR nuclear translocator (ARNT) and estrogen receptor-α to the Cyp1a1 promoter region xenobiotic response element. The mice were fed the AIN-93G diet supplemented with 1500 mg/kg of genistein or daidzein for up to 1 week. Genistein, but not daidzein, significantly decreased basal hepatic microsomal Cyp1a1 protein expression and activity. AhR protein expression was not altered. Molecular mechanisms were investigated in Hepa-1c1c7 cells treated with 5 μmol/L purified aglycones genistein, daidzein, or equol. Cells treated with genistein exhibited inhibitions in ARNT and estrogen receptor-α bindings to the Cyp1a1 promoter region. This study demonstrated that genistein consumption reduced constitutive hepatic Cyp1a1 protein expression and activity, thereby contributing to the understanding of how soy isoflavone aglycones modulate cytochrome P450 biotransformation enzymes.

The genome of Bifidobacterium pseudocatenulatum IPLA 36007, a human intestinal strain with isoflavone-activation activity.

BackgroundBifidobacterium species, including Bifidobacterium pseudocatenulatum, are among the dominant microbial populations of the human gastrointestinal tract. They are also major components of many commercial probiotic products. Resident and transient bifidobacteria are thought to have several beneficial health effects. However, our knowledge of how these bacteria interact and communicate with host cells remains poor. This knowledge is essential for scientific support of their purported health benefits and their rational inclusion in functional foods.ResultsThis work describes the draft genome sequence of Bifidobacterium pseudocatenulatum IPLA 36007, a strain isolated as dominant from the feces of a healthy human. Besides several properties of probiosis, IPLA 36007 exhibited the capability of releasing aglycones from soy isoflavone glycosides. The genome contains 1,851 predicted genes, including 54 genes for tRNAs and fie copies of unique 16S, 23S and 5S rRNA genes. As key attributes of the IPLA 36007 genome we can mention the presence of a lysogenic phage, a cluster encoding type IV fimbriae, and a locus encoding a clustered, regularly interspaced, short, palindromic repeat (CRISPR)-Cas system. Four open reading frames (orfs) encoding β-glucosidases belonging to the glycosyl hydrolase family 3, which may act on isoflavone glycosides, were encountered. Additionally, one gene was found to code for a glycosyl hydrolase of family 1 that might also have β-glucosidase activity.ConclusionThe availability of the B. pseudocatenulatum IPLA 36007 genome should allow the enzyme system involved in the release of soy isoflavone aglycones from isoflavone glycosides, and the molecular mechanisms underlying the strain’s probiotic properties, to be more easily understood.

Therapeutic effects of isoflavones on impaired salivary secretion.

Dry mouth, which is characterized by decreased salivation, has a number of causes; the involvement of estrogen has been suggested as symptoms typically develop in middle-aged females. However, there is a lack of consensus regarding the treatment of this condition. Soy isoflavones, a subgroup of flavonoids, are abundantly found in the soy germ. They are thought to exert a number of effects by specifically binding to estrogen receptors due to their structural similarity to estrogen. Recently, soy isoflavones have been found to exert antioxidant effects, ameliorating disorders caused by reactive oxygen/free radicals. Based on these observations, the effects of soybean isoflavones on impaired salivary secretion were studied in patients with dry mouth. Soy isoflavone aglycones were administered at 25 mg per day to 15 subjects with an average age of 67.9 ± 8.0 years for 2 months, and salivary secretion was analyzed. The results showed a significant improvement based on the saliva flow rate and self-completed questionnaire, thus suggesting the usefulness of isoflavones in improving the symptoms of salivary gland hypofunction.

Conversion of major soy isoflavone glucosides and aglycones in in vitro intestinal models

This study compares conversion of three major soy isoflavone glucosides and their aglycones in a series of in vitro intestinal models. In an in vitro human digestion model isoflavone glucosides were not deconjugated, whereas studies in a Caco-2 transwell model confirmed that deconjugation is essential to facilitate transport across the intestinal barrier. Deconjugation was shown upon incubation of the isoflavone glucosides with rat as well as human intestinal S9. In incubations with rat intestinal S9 lactase phlorizin hydrolase, glucocerebrosidase, and cytosolic broad-specific β-glucosidase all contribute significantly to deconjugation, whereas in incubations with human intestinal S9 deconjugation appeared to occur mainly through the activity of broad-specific β-glucosidase. Species differences in glucuronidation and sulfation were limited and generally within an order of magnitude with 7-O-glucuronides being the major metabolites for all three isoflavone aglycones and the glucuronidation during first pass metabolism being more efficient in rats than in humans. Comparison of the catalytic efficiencies reveals that deconjugation is less efficient than conjugation confirming that aglycones are unlikely to enter the systemic circulation. Altogether, the data point at possible differences in the characteristics for intestinal conversion of the major soy isoflavones between rat and human, especially with respect to their deconjugation.

Inhibitory Effects of Isoflavones on Tumor Growth and Cachexia in Newly Established Cachectic Mouse Models Carrying Human Stomach Cancers

Cachexia, a negative prognostic factor, worsens a patient's quality of life. We established 2 novel cachexia models with the human stomach cancer cell line MKN-45, which was subcloned to produce potent cachexia-inducing cells by repeating the xenografts in immune-deficient mice. After subsequent xenografts, we isolated potent cachexia-inducing cells (MKN45cl85 and 85As2mLuc). Xenografts of MKN45cl85 cells in mice led to substantial weight loss and reduced adipose tissue and musculature volumes, whereas xenografts of 85As2mLuc cells resulted in highly metastatic and cachectic mice. Surgical removal of tumor tissues helped the mice regain body-weight in both mouse models. In vitro studies using these cells showed that isoflavones reduced their proliferation, implying that the isoflavones possess antiproliferative effects of these cancer cell lines. Isoflavone treatment on the models induced tumor cytostasis, attenuation of cachexia, and prolonged survival whereas discontinuation of the treatment resulted in progressive tumor growth and weight loss. The inhibitory effects of tumor growth and weight loss by isoflavones were graded as soy isoflavone aglycone AglyMax > daidzein > genistein. These results demonstrated that the 2 novel cachectic mouse models appear useful for analyzing the mechanism of cancer cachexia and monitoring the efficacy of anticachectic agents.

The Effect of Genistein on the Content and Activity of α‐ and β‐Secretase and Protein Kinase C in Aβ‐Injured Hippocampal Neurons

Genistein (Gen), a derivative of soy isoflavone aglycone, has been shown to exert significant protective effect on Aβ-induced neurotoxicity and neuroinjury. However, its underlying mechanism remains elusive. The objective was to investigate the inhibitory effect of Gen on Aβ-induced neurotoxicity and to elucidate the underlying mechanism. Primary rat hippocampal neurons were pre-treated with Gen for 2 hr followed by incubation with Aβ 25-35 for an additional 24 hr. The cell viability was assessed by MTT assay. The content and activity of α-, β-secretase and protein kinase C (PKC) were measured, and the antagonistic effect of PKC inhibitor Myr was also analysed to clarify the molecular mechanism of Gen inhibition of Aβ-induced toxicity to hippocampal neurons. The results showed that pre-treatment with Gen significantly increased the cell viability and presented the best effect at the final concentration of 0.375 µg/mL. Gen increases the activity of α-secretase but down-regulates the β-secretase activity. It also enhances the expression and activity of PKC. Myr, a PKC inhibitor, partially blocks the activation effect of Gen. Gen exerts protective effect on Aβ-induced neurotoxicity via activating the PKC signalling pathway, which further regulates the activities of α- and β-secretase and thereby inhibits the formation and toxicity of Aβ.

ANTIOXIDANT PROPERTIES OF ETHANOLIC AND METHANOLIC EXTRACTS FROMMONASCUS-FERMENTED SOYBEANS

ABSTRACT Soybeans are very common foods in oriental countries used as a meat substitute. Monascusspecies possess functional components and have been used on traditional fermented food. Solid-state fermentation of the soybean substrate by Monascus species is a new area of investigation. In the present study, methanolic extracts from three samples exhibited better antioxidant activity in the conjugated diene method, reducing power, scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals and chelating ability on ferrous ions. Monascus species inoculated onto the soybean substrate contributed higher reducing power, scavenging and chelating abilities and higher amounts of phenol components than uninoculated soybean product. Overall, Monascus-fermented soybeans showed better antioxidant properties and can be developed as a new dietary supplement and functional food. PRACTICAL APPLICATION Soybeans and soy products are rich in isoflavones and are very common foods used as a meat substitute in oriental countries. Fungus Monascus has been used as a traditional fermented food and its metabolic products are also utilized as food pigments or biological agents in oriental countries for centuries. Many reporters showed that soy isoflavone aglycones and Monascus spp. metabolite exhibited physiological activities. In addition, previous studies showed a fermentation process using microorganisms to inoculate onto a nonsoluble material that can produce chemicals and enzymes. Therefore, the ethanolic and methanolic extracts from Monascus-fermented soybeans were evaluated for antioxidant properties, and the information obtained would be more valuable than that from soybeans to develop new functional foods.

Long-term consumption of isoflavone-enriched foods does not affect bone mineral density, bone metabolism, or hormonal status in early postmenopausal women: a randomized, double-blind, placebo controlled study

Background: Osteoporosis is a major health problem. It was hypothesized that isoflavone-containing products may be a potential alternative to hormone replacement therapy for preventing bone loss during the menopausal transition.Objective: The objective was to investigate whether the consumption of isoflavone-enriched foods for 1 y affects bone mineral density, bone metabolism, and hormonal status in early postmenopausal women.Design: This was a randomized, double-blind, placebo-controlled, parallel, multicenter trial. Two hundred thirty-seven healthy early postmenopausal women [mean (±SD) age of 53 ± 3 y and time since last menses of 33 ± 15 mo] consumed isoflavone-enriched foods providing a mean daily intake of 110 mg isoflavone aglycones or control products for 1 y while continuing their habitual diet and lifestyle. Outcome measures included bone mineral density of the lumbar spine and total body, markers of bone formation and bone resorption, hormones, isoflavones in plasma and urine, safety variables, and adverse events.Results: Consumption of isoflavone-enriched products did not alter bone mineral density of the lumbar spine and total body or markers of bone formation and bone resorption. Hormone concentrations did not differ between the isoflavone and control groups. Consumption of isoflavone-enriched products resulted in increased isoflavone concentrations in plasma and urine, whereas control products did not. This finding indicated good compliance with treatment. Subgroup analysis did not support an effect of equol phenotype on bone density. The intervention had no effect on a range of safety variables and reported adverse events.Conclusion: Consumption of foods containing 110 mg/d of soy isoflavone aglycone equivalents for 1 y did not prevent postmenopausal bone loss and did not affect bone turnover in apparently healthy early postmenopausal white women. This trial was registered at clinicaltrials.gov as NCT00301353.

Soy Isoflavone Aglycone Modulates Expression of Cell Surface Antigensin Vitroandin Vivo

Soy isoflavone aglycones (IFAs) have a wide range of biological actions. We investigated in this study the effect of IFAs on myeloid cells. The cell surface expression of both CD80 and CD86 was up-regulated by treating myeloid cells with IFAs in vitro and in vivo. The findings suggest that IFAs could modulate the myeloid cell function.

Oral Intake of Soy Isoflavone Aglycone Improves the Aged Skin of Adult Women

A double-blind, placebo-controlled trial was conducted to clinically evaluate the effect of soy isoflavone aglycone on the aged skin of middle-aged women. Twenty-six women volunteers in their late 30s and early 40s were randomly assigned to receive either a test food (13 women, the test food group) or a placebo food (13 women, the control group). These groups were given the test food (40 mg of soy isoflavone aglycone per day) and placebo, respectively, for 12 wk. The extent of linear and fine wrinkles at the lateral angle of the eyes was selected as the major evaluation criterion to assess the effects of foods, and the wrinkles' area ratio was used as the evaluation parameter. The extent of skin microrelief at the lateral angle of eyes and that of malar skin elasticity were used as secondary evaluation criteria, and the skin microrelief's area ratio and recovery of skin elasticity were used as the respective evaluation parameters. These parameters were assessed 4, 8, and 12 wk after the start of the test food or placebo intake. The test food group showed a statistically significant improvement of fine wrinkles at week 12 (p<0.05) and of malar skin elasticity at week 8 (p<0.05), compared with the control group. Although there was no significant difference between the test food group and the control group regarding effects on skin microrelief at lateral angle of the eyes, a significant intragroup improvement was observed at week 8 in the test food group (p<0.05). As for the test food safety, none of the subjects presented adverse symptoms during the study period or discontinued the intake of the test food. These data indicate that the oral intake of 40 mg soy isoflavone aglycones per day improves the aged skin of middle-aged women.

Soy Isoflavone Aglycone Modulates A Hematopoietic Response in Combination with Soluble .BETA.-Glucan: SCG

Soy isoflavone aglycones (IFAs) have a wide range of biological actions that suggest they may be of use in cancer prevention. On the other hand, a branched beta-glucan from Sparassis crispa (SCG) is a major 6-branched 1,3-beta-D-glucan in an edible/medicinal mushroom: Sparassis crispa showing antitumor activity. We have previously reported that both oral and intraperitoneal administration of SCG enhanced the hematopoietic response in cyclophosphamide (CY)-induced leukopenic mice. In this study, we investigated the hematopoietic response due to IFA in combination with SCG in CY-induced leukopenic mice. The oral administration of IFA in combination with SCG synergistically enhanced the number of white blood cells, and increased spleen weight. Analyzing the leukocyte population by flow cytometry, the combination of IFA and SCG increased the number of monocytes and granulocytes in the spleen. Taken together, the combination of IFA and SCG synergistically provides the hematopoietic responses that are enhanced over IFA or SCG alone.

Soy isoflavones suppress invasiveness of breast cancer cells by the inhibition of NF-κB/AP-1-dependent and -independent pathways

High consumption of soy products in some Asian countries has been linked to the low incidence of breast cancer in women. The chemopreventive effect of the soy isoflavone, genistein, has been observed through the suppression of cell proliferation, inhibition of angiogenesis and stimulation of apoptosis in breast carcinoma cells. Cancer metastasis consists of interdependent processes, including cell adhesion, migration and invasion. In the present study, we compare the effect of soy isoflavones in the form of aglycones (genistein, daidzein and glycitein) and glucosides (genistin, daidzin and glycitin) on the behavior of highly invasive breast cancer cells. Here we demonstrate that genistein suppresses cell adhesion and migration by inhibiting the constitutively active transcription factors NF-kappaB and AP-1, resulting in the suppression of secretion of urokinase-type plasminogen activator (uPA) in breast cancer cells MDA-MB-231. In addition, we show that all tested soy isoflavone aglycones (genistein, daidzein, glycitein) and glucosides (genistin, daidzin, glycitin) markedly reduced motility of MDA-MB-231 cells. However, only genistein and daidzein inhibited constitutively active NF-kappaB and AP-1 and suppressed secretion of uPA from breast cancer cells. Taken together, our results suggest that dietary soy isoflavones inhibit adhesion and motility of highly invasive breast cancer cells by distinct signaling pathways.

LC/UV/ESI-MS analysis of isoflavones in Edamame and Tofu soybeans.

High-performance liquid chromatography coupled with ultraviolet and electrospray ionization mass spectrometry (HPLC/UV/ESI-MSD) was applied to the study of isoflavones in both Edamame and Tofu soy varieties, from which the immature fresh soybeans or the mature soybean seeds are consumed, respectively. Positive atmospheric pressure interface (API) MS and MS/MS were used to provide molecular mass information and led to the identification of a total 16 isoflavones, including three aglycones, three glycosides, two glycoside acetates, and eight glycoside malonates. The major isoflavones in soybean seeds were daidzein and genistein glycoside and their malonate conjugates. Trace levels of daidzein and genistein acetyl glycosides were found only in the mature dry soybean seeds. To facilitate quantitative analysis, acid hydrolysis during extraction of soy samples was selected to convert the various phytoestrogen conjugates into their respective isoflavone aglycones, allowing accurate quantitation of total phytoestrogens as aglycones. On the basis of HPLC combined with UV and MS detection, all three targeted soy isoflavone aglycones, daidzein, genistein and glycitein in hydrolyzed extracts were successfully quantified within 25 min with formononetin used as the internal standard. The standard curves of UV detection were fitted in the range of 14.16-29000 ng/mL for daidzein, 15.38-31500 ng/mL for genistein, and 11.72-24000 ng/mL for glycitein. For MS detection, the standard curves were established in the range of 3.54-1812.5 ng/mL for daidzein, 3.85-1968.75 ng/mL for genistein, and 2.93-1500 ng/mL for glycitein. Good linearities (r(2) > 0.999 for UV and r(2) > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and precision (RSD) were within 10% for UV detection and 15% for MS detection (n = 10). Using this method, the phytoestrogen levels of total isoflavone aglycones from 30 soybean seed varieties were then evaluated for confirmation of the technique. Total isoflavones ranged across the varieties from 0.02 to 0.12% in the Edamame varieties, which are harvested while the seeds are still immature, and from 0.16 to 0.25% in Tofu varieties, harvested when the seeds are physiologically mature. While the literature has focused on the isoflavone content of soy products and processing soy, this report provides a reliable analytical technique for screening of authenticated fresh immature Edamame soybeans and Tofu soybeans.

Effects of Dietary Isoflavone Aglycones on the Reproductive Tract of Male and Female Mice

We assessed the effects of dietary consumption of soy isoflavone aglycones on the reproductive tract of sexually mature male and female mice. Isoflavone concentrates with a ratio of 10:1, 2:1 or 1:10 genistein:daidzein (G:D) were added to provide 120 mg total isoflavones/1800 Calories (approximately 40 mg/kg body weight) to diets having either casein/lactalbumin or soy protein isolate as the source of protein. After 16 weeks, mice were necropsied and gross and histopathologic assessments of uterus, vagina, testes and accessory sex glands were completed. Effects of the 10G:1D isoflavone concentrates were absent or minimal in females but in males included atrophy of accessory sex glands. In contrast, the 2G:1D and 1G:10D concentrates caused dramatic estrogenic effects in both male and female mice. Effects in females included endometritis and effects typical of estrogenic stimulation (i.e., uterine enlargement, keratinization of vaginal epithelium, increased height of endometrial surface epithelial cells, and uterine squamous metaplasia). Effects in males included reduced plasma testosterone concentrations, atrophy of seminiferous epithelium, atrophy of accessory sex glands, and squamous metaplasia of seminal vesicles. Some effects varied with protein source. We conclude that a diet containing approximately 40 mg/kg soy isoflavone aglycones with a genistein:daidzein ratio of 2:1 or less has marked estrogenic effects on the reproductive system of male and female mice.