The Association between Plasma Concentration of Phytoestrogens and Hypertension within the Korean Multicenter Cancer Cohort.

In order to examine the association between plasma phytoestrogen concentration (genistein, daidzein, equol and enterolactone) and hypertension, we conducted a nested case-control study for 229 hypertension cases including 112 prehypertension and 159 healthy controls derived from the Korean Multi-center Cancer Cohort (KMCC). The concentration of plasma phytoestrogens was measured using time-resolved fluoroimmunoassay. We assessed the association between plasma phytoestrogens and hypertension using logistic regression models using odds ratio (OR) and 95% confidence interval (95%CI). The highest tertile of plasma equol and enterolactone concentration exhibited a significantly decreased risk of hypertension (equol, OR = 0.34, 95%CI 0.20-0.57; enterolactone, OR = 0.32, 95%CI 0.18-0.57), compared with the lowest tertile. Equol and enterolactone showed reduced ORs for prehypertension (the highest tertile relative to the lowest tertile, OR = 0.50, 95%CI 0.26-0.96; OR = 0.38, 95%CI 0.19-0.75, respectively) and hypertension (OR = 0.42, 95%CI 0.22-0.81; OR = 0.28, 95%CI 0.14-0.54, respectively). There was a stronger association in hypertension (the highest tertile relative to the lowest tertile in obesity vs. non-obesity; equol, OR = 0.06 vs. 0.63; enterolactone, OR = 0.07 vs. 0.46; both p-heterogeneity < 0.01). This study suggests that equol and enterolactone may contribute to prevent primarily prehypertension and hypertension, and control cardiovascular disease (CVD) based on the continuum of hypertension and CVD. Further study to assess hypertension risk based on useful biomarkers, including phytoestrogens, may contribute to primary prevention of hypertension.

Dietary daidzein induces accumulation of S-equol in enterohepatic circulation to far higher levels than that of daidzein in female rats with and without ovariectomy.

We previously found that daidzein decreased food intake in female rats. To understand the mechanism of anorectic action of dietary daidzein, it is necessary to determine distributions of daidzein and S-equol, a metabolite of intestinal bacterial conversion from daidzein, in the body. In the present study, we measured the concentrations of daidzein and S-equol in serum and bile in sham-operated and ovariectomized female rats fed a diet containing 150 mg/kg daidzein for 7 days. Dietary daidzein increased serum and bile concentrations of S-equol to far higher levels than those of daidzein. S-equol concentration was more than several hundred fold-higher in bile than in serum, regardless of ovariectomy. Moreover, to investigate whether accumulation of S-equol is facilitated by efficient enterohepatic circulation during continuous intake of daidzein and S-equol, female rats were fed diet containing daidzein or S-equol (both 150 mg/kg), or control diet for 1, 2, 3, or 5 days. Dietary daidzein significantly increased serum and bile concentrations of S-equol in a time-dependent manner, but not those of daidzein. These results indicated that substantial proportion of dietary daidzein was converted to S-equol, which underwent efficient enterohepatic circulation and predominantly accumulated there.

Circulating isoflavone and lignan concentrations and prostate cancer risk: a meta-analysis of individual participant data from seven prospective studies including 2,828 cases and 5,593 controls.

Phytoestrogens may influence prostate cancer development. This study aimed to examine the association between prediagnostic circulating concentrations of isoflavones (genistein, daidzein, equol) and lignans (enterolactone and enterodiol) and the risk of prostate cancer. Individual participant data were available from seven prospective studies (two studies from Japan with 241 cases and 503 controls and five studies from Europe with 2,828 cases and 5,593 controls). Because of the large difference in circulating isoflavone concentrations between Japan and Europe, analyses of the associations of isoflavone concentrations and prostate cancer risk were evaluated separately. Prostate cancer risk by study-specific fourths of circulating concentrations of each phytoestrogen was estimated using multivariable-adjusted conditional logistic regression. In men from Japan, those with high compared to low circulating equol concentrations had a lower risk of prostate cancer (multivariable-adjusted OR for upper quartile [Q4] vs. Q1 = 0.61, 95% confidence interval [CI] = 0.39-0.97), although there was no significant trend (OR per 75 percentile increase = 0.69, 95 CI = 0.46-1.05, ptrend = 0.085); Genistein and daidzein concentrations were not significantly associated with risk (ORs for Q4 vs. Q1 = 0.70, 0.45-1.10 and 0.71, 0.45-1.12, respectively). In men from Europe, circulating concentrations of genistein, daidzein and equol were not associated with risk. Circulating lignan concentrations were not associated with the risk of prostate cancer, overall or by disease aggressiveness or time to diagnosis. There was no strong evidence that prediagnostic circulating concentrations of isoflavones or lignans are associated with prostate cancer risk, although further research is warranted in populations where isoflavone intakes are high.

Pharmacokinetics and safety profile of single-dose administration of an estrogen receptor ß-selective phytoestrogenic (phytoSERM) formulation in perimenopausal and postmenopausal women.

OBJECTIVE: Selected estrogen receptor ß-selective phytoestrogen (phytoSERM), a preparation of genistein, daidzein, and S-equol, has an 83-fold selective affinity for estrogen receptor (ER) ß, and may promote neuronal survival and estrogenic mechanisms in the brain without exerting feminizing activity in the periphery. The aim of this study was to assess the safety, tolerability, and single-dose pharmacokinetics of the phytoSERM formulation in perimenopausal and postmenopausal women. METHODS: Eighteen women aged 45 to 60 years from a 12-week clinical trial evaluating cognitive performance and vasomotor symptoms were randomly assigned to placebo, 50 mg, or 100 mg phytoSERM treatment groups. Plasma levels of the three parent phytoestrogens and their metabolites were measured before and at 2, 4, 6, 8, and 24 hours after ingestion by isotope dilution high-performance liquid chromatography (HPLC) electrospray ionization tandem mass spectrometry. RESULTS: Plasma concentrations of genistein, daidzein, and S-equol peaked at 9, 6, and 4 hours, respectively, for the 50-mg dose, and at 6, 6, and 5 hours, respectively, for the 100-mg dose. The maximum concentration (Cmax) and area under the curve (AUC) for the three parent compounds were greater in the 100-mg dose group, indicating a dose-dependent change in concentration with the phytoSERM treatment. No adverse events were elicited. CONCLUSIONS: A single-dose oral administration of the phytoSERM formulation was well-tolerated and did not elicit any adverse events. It was rapidly absorbed, reached high plasma concentrations, and showed a linear dose-concentration response in its pharmacokinetics. These findings are consistent with previously reported parameters for each parent compound (Clinicaltrials.gov NCT01723917).

Significant inverse association of equol-producer status with coronary artery calcification but not dietary isoflavones in healthy Japanese men.

Equol, a metabolite of the dietary isoflavone daidzein, is produced by the action of gut bacteria in some individuals who are termed as equol-producers. It is proposed to have stronger atheroprotective properties than dietary isoflavones. We examined a cross-sectional association of dietary isoflavones and equol-producer status with coronary artery calcification (CAC), a biomarker of coronary atherosclerosis, among men in Japan. A population-based sample of 272 Japanese men aged 40-49 years recruited from 2004 to 2007 was examined for serum isoflavones, serum equol, CAC and other factors. Equol-producers were classified as individuals having a serum level of equol >83 nm. The presence of CAC was defined as a coronary Ca score ≥10 Agatston units. The associations of dietary isoflavones and equol-producers with CAC were analysed using multiple logistic regression. The median of dietary isoflavones, equol and CAC were 512·7 (interquartile range (IQR) 194·1, 1170·0), 9·1 (IQR 0·10, 33·1) and 0·0 (IQR 0·0, 1·0) nm, respectively. Prevalence of CAC and equol-producers was 9·6 and 16·0 %, respectively. Dietary isoflavones were not significantly associated with CAC. After multivariable adjustment, the OR for the presence of CAC in equol-producers compared with equol non-producers was 0·10 (95 % CI 0·01, 0·90, P<0·04). Equol-producers had significantly lower CAC than equol non-producers, but there was no significant association between dietary isoflavones and CAC, suggesting that equol may be a key factor for atheroprotective properties of isoflavones in Japanese men. This finding must be confirmed in larger studies or clinical trials of equol that is now available as a dietary supplement.

Kanamycin inhibits daidzein metabolism and abilities of the metabolites to prevent bone loss in ovariectomized mice.

BACKGROUND: Daidzein is an isoflavone derived from soybeans that exerts preventive effects on bone loss in ovariectomized (OVX) animals. These effects have been correlated with increasing serum equol levels. In the present study, we investigated the effects of antibiotic intake on equol metabolism from daidzein, and the corresponding levels of bone loss in OVX mice. METHODS: Eight-week-old female ddY mice (n = 42) were either ovariectomized (OVX) or subjected to a sham operation (sham). OVX mice were then divided into six dietary subgroups: control diet (control), 0.3 % kanamycin diet (KN), 0.1 % daidzein diet (Dz), 0.1 % daidzein and 0.0375 % kanamycin diet (Dz+KN3.75), 0.1 % daidzein and 0.075 % kanamycin diet (Dz+KN7.5), and 0.1 % daidzein and 0.3 % kanamycin diet (Dz+KN30). The mice were fed their respective diets for 4 weeks. RESULTS: Uterine weight and femoral bone mineral density (BMD) were significantly lower in the OVX mice compared in the sham mice. No significant differences in uterine weight were observed among all OVX dietary subgroups. The Dz subgroup was found to exhibit higher plasma equol and O-desmethylangolensin (O-DMA) concentrations, as well as greater femoral BMD, compared to all other OVX subgroups. Furthermore, when compared to the Dz group, kanamycin intake decreased plasma equol and O-DMA concentrations, as well as femoral BMD in the OVX mice. CONCLUSIONS: These results suggest that kanamycin intake inhibited the conversion of daidzein to equol and O-DMA, blocking the preventive effects of daidzein on bone loss in OVX mice. Therefore, the bone-protective effects of daidzein intake may be predominantly associated with increased plasma concentrations of either equol or O-DMA.

Plasma isoflavones in Malaysian men according to vegetarianism and by age.

Epidemiological studies indicate lower prevalences of breast and prostate cancers and cardiovascular disease in Southeast Asia where vegetarianism is popular and diets are traditionally high in phytoestrogens. This study assessed plasma isoflavones in vegetarian and non-vegetarian Malaysian men according to age. Daidzein, genistein, equol (a daidzein metabolite), formononetin, biochanin A, estrone, estradiol and testosterone were measured by validated liquid chromatography tandem mass spectrometry (LCMSMS). Plasma isoflavone and sex hormone concentrations were measured in 225 subjects according to age (18-34, 35-44 and 45-67 years old). In all age groups, vegetarians had a higher concentration of circulating isoflavones compared with non-vegetarians especially in the 45-67 year age group where all isoflavones except equol, were significantly higher in vegetarians compared with omnivores. By contrast, the 18-34 year group had a significantly higher concentration of daidzein in vegetarians and significantly higher testosterone and estrone concentrations compared with non-vegetarians. In this age group there were weak correlations between estrone, estradiol and testosterone with some of the isoflavones. This human study provides the first Malaysian data for the phytoestrogen status of vegetarian and nonvegetarian men.

Acute benefits of the microbial-derived isoflavone metabolite equol on arterial stiffness in men prospectively recruited according to equol producer phenotype: a double-blind randomized controlled trial.

BACKGROUND: There is much speculation with regard to the potential cardioprotective benefits of equol, a microbial-derived metabolite of the isoflavone daidzein, which is produced in the large intestine after soy intake in 30% of Western populations. Although cross-sectional and retrospective data support favorable associations between the equol producer (EP) phenotype and cardiometabolic health, few studies have prospectively recruited EPs to confirm this association. OBJECTIVE: The aim was to determine whether the acute vascular benefits of isoflavones differ according to EP phenotype and subsequently investigate the effect of providing commercially produced S-(-)equol to non-EPs. DESIGN: We prospectively recruited male EPs and non-EPs (n = 14/group) at moderate cardiovascular risk into a double-blind, placebo-controlled crossover study to examine the acute effects of soy isoflavones (80-mg aglycone equivalents) on arterial stiffness [carotid-femoral pulse-wave velocity (cfPWV)], blood pressure, endothelial function (measured by using the EndoPAT 2000; Itamar Medical), and nitric oxide at baseline (0 h) and 6 and 24 h after intake. In a separate assessment, non-EPs consumed 40 mg S-(-)equol with identical vascular measurements performed 2 h after intake. RESULTS: After soy intake, cfPWV significantly improved in EPs at 24 h (cfPWV change from 0 h: isoflavone, -0.2 ± 0.2 m/s; placebo, 0.6 ± 0.2 m/s; P < 0.01), which was significantly associated with plasma equol concentrations (R = -0.36, P = 0.01). No vascular effects were observed in EPs at 6 h or in non-EPs at any time point. Similarly, no benefit of commercially produced S-(-)equol was observed in non-EPs despite mean plasma equol concentrations reaching 3.2 µmol/L. CONCLUSIONS: Acute soy intake improved cfPWV in EPs, equating to an 11-12% reduced risk of cardiovascular disease if sustained. However, a single dose of commercially produced equol had no cardiovascular benefits in non-EPs. These data suggest that the EP phenotype is critical in unlocking the vascular benefits of equol in men, and long-term trials should focus on confirming the implications of EP phenotype on cardiovascular health. This trial was registered at clinicaltrials.gov as NCT01530893.

Evaluation of Levothyroxine Bioavailability after Oral Administration of a Fixed Combination of Soy Isoflavones in Post-menopausal Female Volunteers.

BACKGROUND: Post-menopausal women under treatment with levothyroxine for their medical conditions may take concomitantly dietary supplements containing soy isoflavones in combination to treat their post-menopausal symptoms. The aim of this study was to investigate the effect of a fixed combination of soy isoflavones on the oral bioavailability of levothyroxine in post-menopausal female volunteers. METHODS: 12 healthy post-menopausal female, who were on stable oral levothyroxine as replacement/supplementation therapy for hypothyroidism, received a single recommended oral dose of a food supplement containing 60 mg of soy isoflavones (>19% genistin and daidzin) concomitantly with (test) and 6 h later (reference) the administration of levothyroxine in a randomized, open label, crossover fashion. Plasma concentrations of levothyroxine and soy isoflavones (daidzin, daidzein, genistin, genistein, S-equol) were determined by LC-MS/MS. Pharmacokinetic (PK) parameters were determined by non-compartmental analysis. No effect of soy isoflavones was assumed if the 90% confidence intervals (CIs) for the estimated ratio test/reference was included in the acceptance limits 0.80-1.25 for PK parameters Cmax and AUCt. RESULTS: The test/reference ratios Cmax and AUCt of levothyroxine were very close to unity (1.02 and 0.99, respectively) and the corresponding 90% CIs (0.99-1.04 and 0.88-1.12, respectively) fell entirely within the acceptance bioequivalence limits. CONCLUSION: The combination of soy isoflavones used in the present investigation does not affect the rate and extent of levothyroxine absorption when administered concomitantly in post-menopausal women.

Red clover isoflavone metabolite bioavailability is decreased after fructooligosaccharide supplementation.

BACKGROUND: Red clover is an important source of isoflavones; which has been made commercially available as dietary supplements for the treatment of menopausal symptoms. Bioavailability and metabolism of these red clover isoflavones (RCI) have not been studied in detail. Fructooligosaccharides (FOS) stimulate the growth of intestinal bacteria and play an important role in the formation of certain isoflavone metabolites, such as equol and O-desmethylangolensin. OBJECTIVE: To determine the bioavailability of RCI metabolites and analyse whether FOS supplementation could influence their bioavailability. METHODS: Seventeen healthy adults were enrolled in the study carried out in two periods. In the first, compound bioavailability was determined after consumption of 80 mg of RCI (MF11RCE). In the second, a 6-day supplementation of 2×3000 mg/day of FOS was administered before isoflavone consumption. RESULTS: Biochanin A and formononetin were rapidly absorbed and both reached maximum concentrations at an average of 5-7h. Demethylation was a major reaction in the metabolic pathway. Daidzein serum level peaked after about 12.6h. Supplementation with FOS led to a significant decrease in the bioavailability of daidzein, dihydroformononetin, dihydrogenistein and dihydrodaidzein. An increase in equol production was also observed which did not reach statistical significance (p>0.05). CONCLUSION: This study is the first to provide detailed data on RCI bioavailability in humans and determine no influence of FOS yet a trend toward increased equol production. More research is warranted involving a greater sample size.

Enhanced Estrogenic Activity of Soybean Isoflavones by Coadministration of Liuwei Dihuang Pills in Ovariectomized Rats.

Soybean isoflavones are beneficial for treating hormone-related diseases. Simultaneous consumption of soybean isoflavones and Liuwei Dihuang pills (LWPs) is effective for treating perimenopausal period syndrome. However, why the combination of isoflavones and LWPs is more effective than ingestion of each component alone remains unknown. Here, we show that enhanced estrogenic activities would appear when the ovariectomized rats were fed with a soybean diet in combination of LWPs treatment. Our further studies explored enhancements of Lactobacillus (19-fold) and Bifidobacterium (12-fold) contents in the intestine of rat and 1.84-fold higher intestinal ß-glucosidase activity in LWPs treatment group compared with the control group. As a result, steady-state concentrations of genistein (1.20-fold), daidzein (1.36-fold), and equol (1.43-fold) in serum were significantly elevated in the combination group compared with the soybean alone group. The results present the first evidence of the mechanism of enhanced estrogenic activity of dietary soybean isoflavones in combination with LWPs. Our study indicates that alterations of gut bacteria after LWPs treatment play a key role in the enhanced estrogenic effect of dietary soybean, suggesting a direct relationship between dietary soybean, LWPs, and gut flora.

Dietary soy isoflavones increase metastasis to lungs in an experimental model of breast cancer with bone micro-tumors.

Bone is one of the most common sites for metastasis in breast cancer (BC). Micro-metastasis in bone marrow was detected in 30% of patients with stage I, II, or III BC at primary surgery and is a strong indicator of poor prognosis. The role dietary soy isoflavones play in BC with bone micro-metastasis is unclear. In this study, we examined the effects of genistein, daidzein, (-)-equol or a mixture of soy isoflavones on BC with bone micro-metastasis using an experimental model of murine mammary cancer 4T1 cells engineered with luciferase. A small number (1000) of 4T1 cells were injected into the tibia of female Balb/c mice to establish micro-tumors in bone. Soy isoflavones were supplemented in the AIN-93G diet at 750 mg/kg and were provided to mice from 3 weeks before to 3 weeks after cell injection. Bioluminescent imaging was conducted on day 2 (D2), D6, D8, D16 and D20 post cell injection and the results indicated dietary soy isoflavones enhanced the growth of bone micro-tumors on D8. Furthermore, dietary soy isoflavones stimulated metastatic tumor formation in lungs and increased Ki-67 protein expression in these metastasized tumors. In vitro, soy isoflavones (<10 µM) had limited effects on the growth, motility or invasion of 4T1 cells. Thus, the in vivo stimulatory effect could be likely due to systemic effects between the host, 4T1 tumors and soy isoflavones. In conclusion, soy isoflavones stimulate BC with bone micro-metastasis in mice and further investigations are needed regarding their consumption by BC survivors.

Gene polymorphisms in the ornithine decarboxylase-polyamine pathway modify gastric cancer risk by interaction with isoflavone concentrations.

BACKGROUND: The study aimed to examine the association between genes encoding molecules in the ornithine decarboxylase (ODC)-polyamine pathway (ODC1, AMD1, NQO1, NOS2A, and OAZ2) and gastric cancer risk and whether the gene-phytoestrogen interaction modifies gastric cancer risk. METHODS: Among 76 gastric cancer cases and their 1:4 matched controls within the Korean Multi-center Cancer Cohort, a total of 30 SNPs in five genes involved in the ODC pathway were primarily analyzed. The second-stage genotyping in 388 matched case-control sets was conducted to reevaluate the significant SNPs interacting with phytoestrogens during the primary analysis. The summary odds ratios (ORs) [95 % confidence intervals (CIs)] for gastric cancer were estimated. Interaction effects between the SNPs and plasma concentrations of phytoestrogens (genistein, daidzein, equol, and enterolactone) were evaluated. RESULTS: In the pooled analysis, NQO1 rs1800566 showed significant genetic effects on gastric cancer without heterogeneity [OR 0.83 (95 % CI 0.70-0.995)] and a greater decreased risk at high genistein/daidzein levels [OR 0.36 (95 % CI 0.15-0.90) and OR 0.26 (95 % CI 0.10-0.64), respectively; p interaction < 0.05]. Risk alleles of AMD1 rs1279599, AMD1 rs7768897, and OAZ2 rs7403751 had a significant gene-phytoestrogen (genistein and daidzein) interaction effect to modify the development of gastric cancer. They had an increased gastric cancer risk at low isoflavone levels, but a decreased risk at high isoflavone levels (p interaction < 0.01). CONCLUSIONS: Our findings suggest that common variants in the genes involved in the ODC pathway may contribute to the risk of gastric cancer possibly by modulating ODC polyamine biosynthesis or by interaction between isoflavones and NQO1, OAZ2, and AMD1.

Effect of soy isoflavone supplementation on endothelial dysfunction and oxidative stress in equol-producing postmenopausal women.

Dietary isoflavones are considered to be cardioprotective because of their structural similarity to estrogen. Equol is a metabolite of daidzein that is more potent than daidzein itself. The effect of equol on endothelial function and oxidative stress remains unclear. The objective of this study was to determine the effect of 100 mg/day soy isoflavone supplementation on the following markers: vascular cell adhesion molecule-1 (VCAM-1) and nitric oxide (NO) as markers of vascular endothelial function, and malonyldialdehyde (MDA) as oxidative stress marker in equol-producing postmenopausal women. A stratified randomized double blind controlled trial was conducted among 190 postmenopausal Indonesian women aged 47 to 60 years. They were stratified into equol producers and equol nonproducers. The random allocation of intervention was carried out separately in each stratum. The intervention group received tablets consisting of 100 mg soy isoflavones and calcium carbonate 500 mg, and the control group received 500 mg calcium carbonate. The concentrations of VCAM-1, NO and MDA were measured at baseline and post-supplementation at 6 months. After 6 months of supplementation the MDA concentrations were significantly lower in the soy-isoflavone equol producers compared with equol nonproducers (p=0.021). Similar results were also found for VCAM-1 and NO concentrations, but these were statistically not significant (p = 0.413 and p= 0.724, respectively). This study demonstrates that isoflavone supplementation in postmenopausal women with equol-producer status had a more beneficial effect by decreasing the MDA concentration, but did not improve VCAM-1 and NO concentrations.

Large inter-individual variation in isoflavone plasma concentration limits use of isoflavone intake data for risk assessment.

BACKGROUND/OBJECTIVES: Isoflavones are present in soy foods and soy-based supplements. Despite low plasma isoflavone concentrations in the general Western population, concentrations in supplement users exceed those suggested to be beneficial for health in Asian populations, raising concerns for adverse effects. To aid risk assessment, quantification of the relation between isoflavone intake and plasma concentrations is essential. SUBJECTS/METHODS: Plasma samples were collected from postmenopausal women in three placebo-controlled crossover studies with 8-week periods for supplements (two studies, ~100 mg isoflavones/day, n=88) or 4-week periods for soy foods (one study, ~48 mg isoflavones/day, n=15). Plasma isoflavone concentrations (daidzein, equol, genistein and glycitein) were quantified using high-performance liquid chromatography and electrochemical detection. The association between plasma concentrations and isoflavone intake, equol producer status, intake-producer interaction and background dietary intake was assessed based on the assumption of a log-linear relation. RESULTS: Median plasma total isoflavone concentrations after the soy food and supplement interventions were respectively 2.16 and 3.47 µmol/l for equol producers and 1.30 and 2.39 µmol/l for non-producers. Regression analysis showed that doubling isoflavone intake increased plasma concentrations by 55-62% (±s.e. 1-2%, R(2)>0.87) for daidzein, genistein, equol (only for producers) and total isoflavones; for glycitein the association was weaker (15±1%, R(2)=0.48). Adjustments for energy, carbohydrate and fat intake did not affect these estimates. Inter-individual variation, estimated based on repeated measures in one of the studies, was 30-96%. CONCLUSIONS: Although the relation between isoflavone intake and plasma concentrations was adequately quantified, the use of isoflavone intake data for risk assessment needs caution due to large inter-individual variation in plasma concentrations.

The effects of dietary treatment with S-equol on learning and memory processes in middle-aged ovariectomized rats.

The use of over-the-counter botanical estrogens containing isolated soy isoflavones, including genistein and daidzein, has become a popular alternative to traditional hormone therapies. Menopausal women use these products as an aide in healthy aging, including for the maintenance of cognitive function. The safety and efficacy of many of these commercial preparations remain unknown. Previous research in our lab found that treatment of ovariectomized (OVX) female Long-Evans rats with genistein impaired working memory in an operant delayed spatial alternation (DSA) task and response learning in a plus-maze, but enhanced place learning assessed in the plus-maze. The present study further examined the effects of isolated isoflavones on working memory and place learning by treating middle-aged (12-13 month old) OVX female Long-Evans rats with S-equol, the exclusive enantiomer produced by metabolism of daidzein in the mammalian gut. S-equol binds selectively to ERß with an affinity similar to that of genistein but has low transcriptional potency. For DSA testing, S-equol at 1.94, 0.97 mg, or 0mg (sucrose control) was orally administered to animals daily, 30 min before behavioral testing, and again both 4 and 8 hours after the first treatment. Rats were tested on the DSA task following the first, morning dose. For place learning, rats received 0.97 mg S-equol every 4 hours during the light portion of the cycle beginning 48 hours prior to behavioral testing (total exposure 8.7 mg S-equol). S-equol treatment was largely without effect on the DSA and place learning tasks. This is the first study to test the behavioral effects of isolated S-equol in OVX rodents, and shows that, unlike genistein or estradiol, repeated daily treatment with this isoflavone metabolite does not alter learning and memory processes in middle-aged OVX rats.

Effects of short-term fructooligosaccharide intake on equol production in Japanese postmenopausal women consuming soy isoflavone supplements: a pilot study.

BACKGROUND: Recent studies suggest that some of the clinical effectiveness of soy or daidzein, which is a type of isoflavone, may be attributed to a person's ability to produce equol from daidzein. Equol, which is a metabolite of one of the major soybean isoflavones called daidzein, is produced in the gastrointestinal tract by certain intestinal microbiota where present. Habitual dietary patterns may alter the intestinal bacterial profile, and influence the metabolism of isoflavones and the production of equol. Fructooligosaccharides (FOS) have a prebiotic activity as well as being a dietary fibre. The purpose of the present study was to determine whether FOS supplementation increases equol production in equol producers and stimulates equol production in equol non-producers in Japanese postmenopausal women. METHODS: A soy challenge was used to assess equol-producer status prior to the start of the study in healthy postmenopausal Japanese women. The study involved 4 separate groups in randomised crossover design. First, subjects were classified as equol producers (n = 25) or non-producers (n = 18), and then they were randomly assigned to the FOS or control group. All subjects received a daily dose of 37 mg isoflavone conjugates in the capsule (21 mg aglycone form) and either FOS (5 g/day) or sucrose as control, in a randomised crossover study design. Equol -production was assessed by testing the serum and urine before and after the 2-week supplementation period. RESULTS: The analyses were conducted on 34 subjects completed the study, 21 (61.8%) were classified as equol producers, and 13 (38.2%) as non-producers. Significant differences were observed in the interaction effect of time × equol state after 1 week of intervention (p = 0.006). However there were no effects after 2 weeks of intervention (p = 0.516). Finally, in both equol producers and non-producers, FOS supplementation did not affect the serum equol concentration or the urinary equol to daidzein concentration ratios. CONCLUSIONS: We have reported that FOS intervention (5 g/day for 2 weeks) does not significantly modulate the capacity of intestinal microbiota to produce equol in postmenopausal Japanese women, in either equol producers or non-producers in this pilot study. Further larger investigations that explore the roles of specific intestinal microbiota in equol production will enable the establishment of dietary conditions that are required to enhance equol production.

The inhibitory effect of black soybean on hepatic cholesterol accumulation in high cholesterol and high fat diet-induced non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is defined as excess of fat in the liver. We investigated the effects of black soybean on the cholesterol metabolism and insulin resistance of mice fed high cholesterol/fat diets. Mice were randomly allocated into four groups that were fed different diets: the normal cholesterol/fat diet; high cholesterol/fat diets (HCD); and HCD with 1%, and 4% black soybean powder (1B-HCD, and 4B-HCD). Liver total cholesterol and triglyceride concentrations were significantly lower in the black soybean-supplemented groups than that in the HCD group. PCR revealed significantly lower hepatic SREBP2 and HMG-CoA reductase mRNA levels of black soybean-supplemented mice. Real-time PCR revealed significantly higher hepatic ABCA1 mRNA level of black soybean-supplemented mice, which may increase cholesterol efflux. Liver bile acids concentration was significantly high in the 4B-HCD group. Black soybean stimulated secretion of adiponectin, activation of pAMPK, and eliminated free fatty acids in the liver. Black soybean supplementation decreased MDA and nitrate level. The activities of SOD, catalase, and GPx were restored by black soybean supplementation. Our data strongly indicate that black soybean influences the balance between oxidative and antioxidative stress. We suggest that black soybean improves cholesterol metabolism, insulin resistance, and alleviates oxidative damage in NAFLD.

A six-month randomized controlled trial of whole soy and isoflavones daidzein on body composition in equol-producing postmenopausal women with prehypertension.

OBJECTIVES: This paper reported the effects of commonly used whole soy foods (soy flour) and purified daidzein (one of the major isoflavones and the precursor of equol) on changes in anthropometric measurements and body composition in a 6-month double-blind, randomized, placebo-controlled trial among prehypertensive postmenopausal women who are also equol producers. METHODS: 270 eligible women were randomized to either one of the three treatments: 40 g soy flour (whole soy group), 40 g low-fat milk powder + 63 mg daidzein (daidzein group), or 40 g low-fat milk powder (placebo group) daily each for 6 months. Anthropometric indicators and body composition were measured before and after intervention. RESULTS: 253 subjects completed the study with good compliance. Urinary isoflavones levels suggested good compliance of subjects with supplementation. Whole soy and purified daidzein had no significant effect on body weight, body mass index (BMI), waist and hip circumferences, waist to hip ratio (WHR), body fat percentage, fat mass, and free fat mass. CONCLUSION: Six-month consumption of whole soy and purified daidzein at provided dosage had no improvement on body weight and composition compared with isocaloric milk placebo among prehypertensive equol-producing postmenopausal women. This trial is registered with ClinicalTrials.gov NCT01270737.

Interaction effects between genes involved in the AKT signaling pathway and phytoestrogens in gastric carcinogenesis: a nested case-control study from the Korean Multi-Center Cancer Cohort.

SCOPE: To investigate whether genes involved in AKT/nuclear factor kappa B signaling and/or gene-environment interactions between the genes and phytoestrogens may be susceptible factors for gastric cancer. METHODS AND RESULTS: The representative single nucleotide polymorphisms (SNPs) identified during the primary analysis (screening a total of 622 SNPs within ± 5 kbp of the 51 target gene locations) were further investigated in 317 matched case-control sets. The summary odds ratios (ORs) and 95% confidence intervals (CIs) for gastric cancer were calculated. Interaction effects between the SNPs and phytoestrogen biomarkers (genistein, daidzein, equol, and enterolactone) were computed. CDK1 rs4145643, FAS rs6586161, and FAS rs1468063 in the AKT signaling pathway presented significant genetic effects on gastric cancer (OR = 0.81 (95% CI: 0.66-0.99) for CDK1 rs4145643; OR = 1.27 (95% CI: 1.03-1.58) for FAS rs6586161; OR = 1.29 (95% CI: 1.03-1.56) for FAS rs1468063; Cochran Q statistics > 0.10). Risk alleles of FAS rs6586161, FAS rs1468063, MAP3K1 rs16886448, and MAP3K1 rs252902 showed significant interaction effects with enterolactone (p(interaction) < 0.05). CONCLUSION: CDK1 and FAS genes involved in AKT signaling and influenced by anti-carcinogenic property of phytoestrogens can play a role as susceptible genetic factors in gastric carcinogenesis. FAS and MAP3K1 genes significantly interact with enterolactone, thereby modifying the individual's risk for gastric cancer.