Equol and Resveratrol Improve Bone Turnover Biomarkers in Postmenopausal Women: A Clinical Trial.

Estrogen deficiency is a major cause of loss of postmenopausal bone mineral density (BMD). This study aimed to evaluate the effects of equol and resveratrol on bone turnover biomarkers in postmenopausal women. Sixty healthy postmenopausal women were randomly assigned to receive 200 mg fermented soy containing 10 mg equol and 25 mg resveratrol or a placebo for 12 months. Whole-body BMD and bone turnover biomarkers, such as deoxypyridinoline (DPD), tartrate-resistant acid phosphatase 5b (TRACP-5b), osteocalcin, and bone-specific alkaline phosphatase (BAP), were measured at baseline and after 12 months of treatment. At the end of treatment, DPD, osteocalcin, and BAP significantly improved in the active group (p < 0.0001 for all) compared to the placebo group. Conversely, TRACP-5b levels were unaffected by supplementation (p = 0.051). Statistically significant changes in the concentrations of DPD (p < 0.0001), osteocalcin (p = 0.0001), and BAP (p < 0.0001) compared to baseline were also identified. Overall, the intervention significantly increased BMD measured in the whole body (p = 0.0220) compared with the placebo. These data indicate that the combination of equol and resveratrol may positively modulate bone turnover biomarkers and BMD, representing a potential approach to prevent age-related bone loss in postmenopausal women.

Equol exerts a protective effect on postmenopausal osteoporosis by upregulating OPG/RANKL pathway.

BACKGROUD: Estrogen deficiency is the leading cause of postmenopausal osteoporosis(PMOP) and phytoestrogens soy isoflavones (SI) have been shown to improve PMOP. Equol (Eq), an in vivo metabolite of phytoestrogens soy isoflavones (SI), has a more stable structure and stronger biological activity than its parent compound and has the greatest estrogenic activity. However, there are few studies on the therapeutic effect of Eq on PMOP. PURPOSE: To explore the therapeutic effect and mechanisms of Eq on POMP. METHODS: Osteoblast-like cells ROS1728 were cultured with different doses of Eq, estradiol (E2), separately. The effect of Eq on the proliferation, apoptosis, cell cycle of osteoblasts were detected by CCK-8 and flow cytometry, and the expression of OPG/RANK/RANKL signaling pathway of osteoblasts was detected by Quantitative real-time PCR (qRT-PCR) and Western blot (WB), and RNA silencing technology were carried out to explore the receptors through which Eq plays a role. Then PMOP rat model was established and treated by Eq or E2 to further verification of the effect and mechanism of Eq on PMOP. RESULT: Eq promoted the proliferation and inhibited the apoptosis of osteoblasts and increased the proportion of osteoblasts in the S phase and G2/M phase in a dose-dependent manner. Mechanistically, Eq treatment upregulated the expression of OPG and OPG/RANKL ratio in osteoblasts and this regulatory effect was mainly mediated through the ERß receptor. Furthermore, in vivo study, Eq improved microstructure and BMD of the femur of PMOP rat model, which imitated the osteoprotective effect of E2. Moreover, the Eq or E2 treatment increased serum levels of Ca, 1,25(OH)2D3, bone Gla-protein(BGP), and Type I procollagen (PC1), and reduced serum levels of phosphorus (P), parathyroid hormone(PTH), pyridinol (PYD), tartrate-resistant acid phosphatase (TRAP) and urinary level of deoxypyridinoline (DPD) in the treatment OVX group compared with the untreated OVX group. Meanwhile, Eq or E2 markedly induced the mRNA and protein expression of OPG and OPG/RANKL ratio. CONCLUSION: Eq can combine with ERß and exert a protective effect on PMOP by upregulating OPG/RANKL pathway.

6-methoxydihydroavicine, the alkaloid extracted from Macleaya cordata (Willd.) R. Br. (Papaveraceae), triggers RIPK1/Caspase-dependent cell death in pancreatic cancer cells through the disruption of oxaloacetic acid metabolism and accumulation of reactive oxygen species.

BACKGROUND: Many extracts and purified alkaloids of M. cordata (Papaveraceae family) have been reported to display promising anti-tumor effects by inhibiting cancer cell growth and inducing apoptosis in many cancer types. However, no evidence currently exists for anti-pancreatic cancer activity of alkaloids extracted from M. cordata, including a novel alkaloid named 6­methoxy dihydrosphingosine (6-Methoxydihydroavicine, 6-ME) derived from M. cordata fruits. PURPOSE: The aim of this study was to investigate the anti-tumor effects of 6-ME on PC cells and the underlying mechanism. METHODS: CCK-8, RTCA, and colony-formation assays were used to analyze PC cell growth. Cell death ratios, changes in MMP and ROS levels were measured by flow cytometry within corresponding detection kits. A Seahorse XFe96 was employed to examine the effects of 6-ME on cellular bioenergetics. Western blot and q-RT-PCR were conducted to detect changes in target molecules. RESULTS: 6-ME effectively reduced the growth of PC cells and promoted PCD by activating RIPK1, caspases, and GSDME. Specifically, 6-ME treatment caused a disruption of OAA metabolism and increased ROS production, thereby affecting mitochondrial homeostasis and reducing aerobic glycolysis. These responses resulted in mitophagy and RIPK1-mediated cell death. CONCLUSION: 6-ME exhibited specific anti-tumor effects through interrupting OAA metabolic homeostasis to trigger ROS/RIPK1-dependent cell death and mitochondrial dysfunction, suggesting that 6-ME could be considered as a highly promising compound for PC intervention.

Phytoestrogens (Resveratrol and Equol) for Estrogen-Deficient Skin-Controversies/Misinformation versus Anti-Aging In Vitro and Clinical Evidence via Nutraceutical-Cosmetics.

The overarching theme for this review is perspective. Superfoods (a marketing term for fruits and vegetables, etc.) have a positive connotation, while many superfoods contain phytoestrogens, a term that is alarming to the public and has a negative connotation because phytoestrogens are endocrine-disruptors, even though they are strong antioxidants that have many health benefits. To understand phytoestrogens, this paper provides a brief summary of the characteristics of: (a) estrogens, (b) estrogen receptors (ER), (c) estrogen-deficient skin, (d) how perspective(s) get off track, (e) phytoestrogen food sources, and (f) misconceptions of phytoestrogens and food safety, in general, that influence person(s) away from what is true. Finally, a brief history of cosmetics to nutraceuticals is covered plus the characteristics of phytoestrogens, resveratrol and equol on: (g) estrogen receptor binding, (h) topical and oral dosing, and (i) in vitro, molecular mechanisms and select clinical evidence, where both phytoestrogens (resveratrol and equol) demonstrate promising applications to improve skin health is presented along with future directions of nutraceuticals. Perspective is paramount in understanding the controversies associated with superfoods, phytoestrogens, and endocrine-disruptors because they have both positive and negative connotations. Everyone is exposed to and consumes these molecules everyday regardless of age, gender, or geographic location around the world, and how we understand this is a matter of perspective.

Roles of Phytoestrogen in the Pathophysiology of Intracranial Aneurysm.

Background and Purpose: The incidences of intracranial aneurysm and aneurysmal subarachnoid hemorrhage are high in postmenopausal women. Although population-based studies suggest that hormone replacement therapy is beneficial for postmenopausal women with intracranial aneurysms, estrogen replacement may no longer be recommended for the prevention of chronic diseases given its association with adverse outcomes, such as cancer and ischemic stroke. The isoflavone daidzein and its intestinal metabolite equol are bioactive phytoestrogens and potent agonists of estrogen receptors. Given their estrogenic properties, we investigated whether the isoflavones daidzein and equol are protective against the formation and rupture of intracranial aneurysms in a mouse model of the postmenopausal state. Methods: We induced intracranial aneurysms in ovariectomized adult female mice using a combination of induced systemic hypertension and a single injection of elastase into the cerebrospinal fluid. We fed the mice with an isoflavone-free diet with/without daidzein supplementation, or in a combination of intraperitoneal equol, or oral vancomycin treatment. We also used estrogen receptor beta knockout mice. Results: Both dietary daidzein and supplementation with its metabolite, equol, were protective against aneurysm formation in ovariectomized mice. The protective effects of daidzein and equol required estrogen receptor-ß. The disruption of the intestinal microbial conversion of daidzein to equol abolished daidzein's protective effect against aneurysm formation. Mice treated with equol had lower inflammatory cytokines in the cerebral arteries, suggesting that phytoestrogens modulate inflammatory processes important to intracranial aneurysm pathogenesis. Conclusions: Our study establishes that both dietary daidzein and its metabolite, equol, protect against aneurysm formation in ovariectomized female mice through the activation of estrogen receptor-ß and subsequent suppression of inflammation. Dietary daidzein's protective effect required the intestinal conversion to equol. Our results indicate the potential therapeutic value of dietary daidzein and its metabolite, equol, for the prevention of the formation of intracranial aneurysms and related subarachnoid hemorrhage.

Depletion of dietary phytoestrogens reduces hippocampal plasticity and contextual fear memory stability in adult male mouse.

Introduction: Phytoestrogens are non-steroidal estrogen analogues and are found primarily in soy products. They have received increasing attention as dietary supplements for estrogen deficiency and as modulators of endogenous estrogen functions, including cognition and emotion. In addition to modifying the levels of circulating sex hormones, phytoestrogens also exert direct effects on estrogen and androgen receptors in the brain and thus effectively modulate the neural circuit functions.Objective: The aim of this study was to investigate the long-term effects of low phytoestrogen intake (∼6 weeks) on the hippocampal plasticity and hippocampus-dependent memory formation in the adult C57BL/6 male mice.Methods and Results: In comparison to mice on a diet with normal phytoestrogen content, mice on low phytoestrogen diet showed a significant reduction in the phosphorylation of NR2B subunit, a molecular correlate of plasticity in the Schaffer collateral-CA1 synapse. We observed a profound decrease in long-term potentiation (LTP) in the ventral hippocampus, whereas no effect on plasticity was evident in its dorsal portion. Furthermore, we demonstrated that acute perfusion of slices with an estrogen analogue equol, an isoflovane metabolized from daidzein produced by the bacterial flora in the gut, was able to rescue the observed LTP deficit. Examining potential behavioral correlates of the plasticity attenuation, we found that mice on phytoestrogen-free diet display decreased contextual fear memory at remote but not at recent time points after training.Conclusions: Our data suggests that nutritional phytoestrogens have profound effects on the plasticity in the ventral hippocampus and ventral hippocampus-dependent memory.

Therapeutic Potential of Equol: A Comprehensive Review.

Equol (4',7-isoflavandiol), is a phytoestrogenic compound, which is synthesized from parent molecule diadzein by intestinal bacterial flora. It is among one of the most extensively researched molecule due to its high affinity towards estrogen receptors. Its enantiomeric form S-equol has been explored in the treatment of estrogen/androgen mediated diseases. Various therapeutic applications such as anti-cancer, cardioprotective, antidiabetic, antiosteoporosis, anti-ageing, and neuroprotective efficacy are attributed to it. This review explored major studies related to biochemistry and pharmacological applications of equol for human health.

Dietary daidzein decreases food intake accompanied with delayed gastric emptying in ovariectomized rats.

We previously found that equol, a metabolite of intestinal bacterial conversion from soy isoflavone daidzein, has female-specific anorectic effects. In the present study, we used seven-week-old female ovariectomized (OVX) Sprague Dawley rats to test the hypothesis that the anorectic effect of dietary daidzein may be attributed to delayed gastric emptying. Results suggest that dietary daidzein delays gastric emptying and that it has an anorectic effect with residual gastric contents, but not without gastric contents. Dietary equol significantly decreased daily food intake in the OVX rats without sleeve gastrectomy, but not in those with sleeve gastrectomy, suggesting that the accumulation of food in the stomach is required for the anorectic effect of equol to occur. These results support the hypothesis that the anorectic effect of dietary daidzein is attributed to delayed gastric emptying.

Selective Estrogen Receptor ß Agonists: a Therapeutic Approach for HIV-1 Associated Neurocognitive Disorders.

The persistence of HIV-1 associated neurocognitive disorders (HAND) in the post-cART era, afflicting between 40 and 70% of HIV-1 seropositive individuals, supports a critical need for the development of adjunctive therapeutic treatments. Selective estrogen receptor ß agonists, including S-Equol (SE), have been implicated as potential therapeutic targets for the treatment of neurocognitive disorders. In the present study, the therapeutic efficacy of 0.2 mg SE for the treatment of HAND was assessed to address two key questions in the HIV-1 transgenic (Tg) rat. First, does SE exhibit robust therapeutic efficacy when treatment is initiated relatively early (i.e., between 2 and 3 months of age) in the course of viral protein exposure? Second, does the therapeutic utility of SE generalize across multiple neurocognitive domains? Treatment with SE enhanced preattentive processes and stimulus-response learning to the level of controls in all (i.e., 100%) HIV-1 Tg animals. For sustained and selective attention, statistically significant effects were not observed in the overall analyses (Control: Placebo, n = 10, SE, n = 10; HIV-1 Tg: Placebo, n = 10, SE, n = 10). However, given our a priori hypothesis, subsequent analyses were conducted, revealing enhanced sustained and selective attention, approximating controls, in a subset (i.e., 50%, n = 5 and 80%, n = 8, respectively) of HIV-1 Tg animals treated with SE. Thus, the therapeutic efficacy of SE is greater when treatment is initiated relatively early in the course of viral protein exposure and generalizes across neurocognitive domains, supporting an adjunctive therapeutic for HAND in the post-cART era. Graphical Abstract HIV-1 transgenic (Tg) and control animals were treated with either 0.2 mg S-Equol (SE) or placebo between 2 and 3 months of age (Control: Placebo, n = 10, SE, n = 10; HIV-1 Tg: Placebo, n = 10, SE, n = 10). Neurocognitive assessments, tapping preattentive processes, stimulus response learning, sustained attention and selective attention, were conducted to evaluate the utility of SE as a therapeutic for HIV-1 associated neurocognitive disorders (HAND). Planned comparisons between HIV-1 Tg and control animals treated with placebo were utilized to establish a genotype effect, revealing prominent neurocognitive impairments (NCI) in the HIV-1 Tg rat across all domains. Furthermore, to establish the utility of SE, HIV-1 Tg animals treated with SE were compared to control animals treated with placebo. Treatment with 0.2 mg SE ameliorated NCI, to levels that were indistinguishable from controls, in at least a subset (i.e., 50-100%) of HIV-1 Tg animals. Thus, SE supports an efficacious, adjunctive therapeutic for HAND.

Equol Pretreatment Protection of SH-SY5Y Cells against Aß (25-35)-Induced Cytotoxicity and Cell-Cycle Reentry via Sustaining Estrogen Receptor Alpha Expression.

ß-amyloid formation in the brain is one of the characteristics of Alzheimer's disease. Exposure to this peptide may result in reentry into the cell cycle leading to cell death. The phytoestrogen equol has similar biological effects as estrogen without the side effects. This study investigated the possible mechanism of the neuron cell-protecting effect of equol during treatment with Aß. SH-SY5Y neuroblastoma cells were treated with either 1 µM S-equol or 10 nM 17ß-estradiol for 24 h prior to 1 µM Aß (25-35) exposure. After 24 h exposure to Aß (25-35), a significant reduction in cell survival and a reentry into the cell cycle process accompanied by increased levels of cyclin D1 were observed. The expressions of estrogen receptor alpha (ERα) and its coactivator, steroid receptor coactivator-1 (SRC-1), were also significantly downregulated by Aß (25-35) in parallel with activated extracellular signal-regulated kinase (ERK)1/2. However, pretreatment of cells with S-equol or 17ß-estradiol reversed these effects. Treatment with the ER antagonist, ICI-182,780 (1 µM), completely blocked the effects of S-equol and 17ß-estradiol on cell viability, ERα, and ERK1/2 after Aß (25-35) exposure. These data suggest that S-equol possesses a neuroprotective potential as it effectively antagonizes Aß (25-35)-induced cell cytotoxicity and prevents cell cycle reentry in SH-SY5Y cells. The mechanism underlying S-equol neuroprotection might involve ERα-mediated pathways.

Effect of Equol on Vasocontractions in Rat Carotid Arteries Treated with High Insulin.

Previous research has indicated that high insulin affects vascular function. Equol is an active metabolite of daidzein, an isoflavone produced from soy by intestinal microbial flora, with beneficial effects on the vascular system. This study investigated whether equol was beneficial for vascular function under high insulin conditions. Using organ culture techniques, rat carotid arteries were treated for 23 ± 1 h with a vehicle, high insulin (100 nM), or equol (100 µM) plus high insulin (100 nM). Vascular isometric forces were measured by the organ bath technique. In each endothelium-intact ring, the contractions induced by high-K+, noradrenaline, or by serotonin (5-HT) were similar for the vehicle, insulin, and equol + insulin treatments. Contractions induced by a selective 5-HT2A receptor agonist (TCB2) increased with insulin treatment (vs. vehicle), but less so with equol + insulin. Under basal conditions, a selective 5-HT2B receptor agonist (BW723C86) did not induce contraction; following precontraction by a thromboxane analog, it induced contraction but not relaxation. These responses were similar across the three treatments. Acetylcholine-induced relaxations were also similar for the three treatments. In the endothelium-denuded preparations, 5-HT-induced contraction was augmented with insulin treatment (vs. vehicle) but less so by equol + insulin treatment. These differences in 5-HT-induced contractions were eliminated by iberiotoxin, a large-conductance calcium-activated K+ channel (BKCa) inhibitor. These results suggest that equol exerts a preventive effect on the enhancement of 5-HT-induced contraction by high insulin (possibly mediated by the 5-HT2A receptor), and that these effects may be attributed to the activation of BKCa channels in vascular smooth muscle.

Opposing effects of S-equol supplementation on metabolic and behavioral parameters in mice fed a high-fat diet.

Phytoestrogens, such as daidzein and genistein, may be used to treat various hormone-dependent disorders. Daidzein can be metabolized by intestinal microbes to S-equol. However, not all individuals possess bacteria producing this metabolite, resulting in categorization of equol vs nonequol producers. Past human and rodent studies have suggested that supplementation of this compound might yield beneficial metabolic and behavioral effects. We hypothesized that administration of S-equol to diet-induced obese male and female mice would mitigate potential diet-induced metabolic and comorbid neurobehavioral disorders. To test this possibility, we placed 5-week-old C57 mice on a high-fat diet (HFD) to mimic the diet currently consumed by many Western adults. Animals were randomly assigned to S-equol supplementation (10 mg/kg body weight) or vehicle control group. After 4 weeks on HFD with or without S-equol supplementation, metabolic and behavioral phenotyping was performed. Although the initial hypothesis proposed that S-equol treatment would improve metabolic and neurobehavioral outcomes, this supplementation instead exacerbated aspects of HFD-induced metabolic disease, as indicated by suppressed physical activity in treated individuals, reduced energy expenditure in treated males, and serum chemistry changes (hyperglycemia in treated individuals; hyperinsulinemia and hypoleptinemia in treated males). Conversely, S-equol individuals exhibited less anxiety-like and depressive-like behaviors, as evidenced by increased exploratory time in the elevated plus maze by treated males and increased time spent mobile in the tail suspension test for treated individuals. In summary, S-equol may be beneficial in mitigating depression and anxiety disorders in individuals, but for indeterminate reasons, supplementation may worsen facets of metabolic disorders in obese individuals.

The phytoestrogens daidzein and equol inhibit the drug transporter BCRP/ABCG2 in breast cancer cells: potential chemosensitizing effect.

PURPOSE: The soy isoflavone genistein has been described to up-regulate breast cancer resistance protein (BCRP) and, thus, enhance chemoresistance in breast cancer cells. The aim of this work was to assess the effect of long- and short-term incubation with daidzein, the second most abundant soy isoflavone and its metabolite equol on the expression and activity of P-glycoprotein, multidrug resistance-associated proteins 1 and 2 (MRP1 and MRP2) and BCRP in breast cancer cells. METHODS: MCF-7 and MDA-MB-231 cells were treated with phytoestrogen concentrations within the range achieved in individuals with a high isoflavone intake. Transporter expression was evaluated at protein and mRNA level through western blot and qRT-PCR, respectively. Transporter activity was determined using doxorubicin, mitoxantrone and carboxy-dichlorofluorescein as substrates. RESULTS: Daidzein (5 µM) up-regulated MRP2- and down-regulated MRP1 protein expressions in MCF-7 and MDA-MB-231 cells, respectively. Both effects were ER-dependent, as determined using the antagonist ICI 182,780. The decrease in MRP1 mRNA in MDA-MB-231 cells indicates a transcriptional mechanism. On the contrary, MRP2 induction in MCF-7 cells takes place post-transcriptionally. Whereas changes in the transporter expression had a minor effect on the transporter activity, acute incubation with daidzein, R-equol and S-equol led to a strong inhibition of BCRP activity and an increase in the IC50 of BCRP substrates. CONCLUSIONS: In contrast to previous reports for genistein, daidzein and equol do not provoke a major up-regulation of the transporter expression but instead an inhibition of BCRP activity and sensitization to BCRP substrates.

Equol Decreases Hot Flashes in Postmenopausal Women: A Systematic Review and Meta-Analysis of Randomized Clinical Trials.

Soy isoflavones may benefit some, but not all, menopausal women, and the ability of the women to produce equol may be the major determinant of effectiveness. We assessed the efficacy of soy isoflavones and equol for alleviating menopausal symptoms, especially vasomotor symptoms, in postmenopausal women who were equol producers and nonproducers by using systematic review and meta-analysis of randomized clinical trials (RCTs). We searched 12 English, Korean, and Chinese language scientific and medical databases. We selected all available RCTs that assessed the effect of equol, either equol itself or soy isoflavone in equol producers, on menopausal symptoms in peri- or postmenopausal women. The primary outcome was the effect on hot flashes. The severity of hot flashes was determined by the scores, and sensitivity and risk of bias analyses were conducted. Other outcomes of the review, but not meta-analysis, included depression and adverse events. Six studies (779 total subjects) met all criteria for the systematic review, 5 of those could be included in the meta-analysis (728 total subjects). Two studies included in the meta-analysis reported no statistically significant benefits of equol; the other three did report significant benefits of equol. Meta-analysis revealed a significant benefit of equol for lowering hot flash scores and revealed a generally low risk of bias. In conclusion, this study found that supplementing equol to equol nonproducers significantly lowered the incidence and/or severity of hot flashes in menopausal women.

Lack of combination effects of soy isoflavones and taxane chemotherapy of castration-resistant prostate cancer.

BACKGROUND: Patients with cancer, including prostate cancer, often use dietary supplements, such as soy or isoflavones, before, during, or after therapy. There is little information about possible interactions between supplements and cancer chemotherapy. There are some reports suggesting enhancement by genistein of taxane chemotherapy for castrate-resistant prostate cancer (CRPC). METHODS: We investigated whether physiologically attainable concentrations of soy isoflavones (≤10 µM) interact with taxanes on growth inhibition of CRPC cells in vitro and in vivo in nude mice exposed via the diet, on microtubule disassembly in vitro, and on P-glycoprotein-mediated drug efflux in 22Rv1 cells and CYP3A4 activity in microsomes. RESULTS: Genistein, daidzein, and equol did not affect growth of VCaP, 22Rv1, C4-2, and PC-3 CRPC cells or growth inhibition of these cells by docetaxel and cabazitaxel. These isoflavones did not inhibit microtubule disassembly in vitro or inhibit the microtubule effects of taxanes and genistein did not bind substantially to microtubules. Genistein considerably inhibited P-glycoprotein-mediated drug efflux in 22Rv1 cells and CYP3A4 activity in microsomes. However, dietary supplementation with genistein at 250 and 500 ppm did not affect the tumor growth inhibiting effect of docetaxel on 22Rv1 cells xenografted in nude mice. CONCLUSIONS: Our results with relevant cell models and clinically achievable concentrations of soy isoflavones do not support the notion that genistein or other soy isoflavones can enhance the effects of taxane chemotherapy in CRPC cell and xenograft models. Yet, the inhibitory effects of genistein on drug efflux in 22Rv1 cells and on microsomal CYP3A4 activity raise the possibility that genistein can affect taxane effects on CRPC cells in other circumstances than those we studied, which merits further research.

Employing different types of phytoestrogens improve bone mineralization in bisphenol A stimulated osteoblast.

AIMS: Phytoestrogens and xenoestrogens act as agonists/antagonists in bone formation and differentiation. Strong bones are depending of the ability of osteoblasts to form new tissue and to mineralize the newly formed tissue. Dysfunctional or loss of mineralization leads to weak bone and increased fracture risk. In this study, we reported the effect of different types of phytoestrogens (daidzein, genistein and equol) on mineralization in hFOB 1.19 cells stimulated with bisphenol A (BPA). MAIN METHODS: Cell mineralization capacity of phytoestrogens was investigated by evaluating calcium, phosphate content and alkaline phosphatase activity. Bone related markers, osteocalcin and osteonectin, responsible in maintaining mineralization were also measured. KEY FINDINGS: BPA is significantly interfering with bone mineralization in hFOB 1.19 cells. However, the enhanced mineralization efficacy of daidzein and genistein (particularly at a dose of 5 and 40 µg/mL, respectively) was evidenced by increasing calcium and phosphate content, higher ALP activity, compared to the untreated BPA group. The quantitative analyses were confirmed through morphological findings. Osteocalcin and osteonectin levels were increased in phytoestrogens-treated cells. These findings revealed the potential effect of phytoestrogens in reverting the demineralization process due to BPA exposure in hFOB 1.19 cells. SIGNIFICANCE: We found that osteoblast differentiation and mineralization were maintained following treatment with phytoestrogens under BPA exposure.

Effects of Equol Supplement on Bone and Cardiovascular Parameters in Middle-Aged Japanese Women: A Prospective Observational Study.

OBJECTIVE: To examine changes in the bone and cardiovascular parameters and tolerability in middle-aged Japanese women taking equol supplement for a year. DESIGN: This was a prospective observational study. SUBJECTS AND SETTING: Participants were 74 women receiving outpatient care at Hamasite Medical Clinic, Minato-ku, Tokyo, from 2013 to 2015. INTERVENTIONS: Participants received per oral equol-containing supplement, 10 mg/day. OUTCOME MEASURES: The primary outcome measures were percent changes in bone and cardiovascular parameters after 1 year supplementation with equol. The secondary measures included factors affecting the parameter changes and adverse effects associated with equol use for a year. RESULTS: Reduction in arterial stiffness was observed after 12 months of equol supplement (1402.3 cm/s vs.1367.3 cm/s, p < 0.001). Significant reductions in respective parameters were observed in women with moderate and high risk for arteriosclerosis (median [95% confidence interval]: -3.2% [-5.79 to -0.74]; -12.65% [-18.52 to -4.28]; respectively); hypertriglyceridemia -45.53% [-70.24 to -5.58]; bone resorption risk (-15.15% [-23.71 to 1.56]; and bone fracture risk -26.68% [-76.43 to -5.99]. All 15 women with high baseline parathyroid hormone levels had achieved a median of 50% [-54.11 to -31.69] reduction from their baseline values. These associations were further confirmed in the results of multiple linear regression analysis. There were no reported adverse events or abnormal findings in the blood chemistry, Pap smear, mammography, and transvaginal ultrasound during periodic follow-ups. CONCLUSION: One year equol supplement was tolerable and induced improvement of certain bone and cardiovascular parameters, especially in higher risk groups. Further controlled studies are needed to explore long-term equol use for wellbeing of middle-aged women.

1H Nuclear Magnetic Resonance Based Metabolomics Approach Reveals the Metabolic Mechanism of (-)-5-Hydroxy-equol against Hepatocellular Carcinoma Cells in Vitro.

1H nuclear magnetic resonance (NMR)-based metabolomics can rapidly detect metabolic shift under various stimulus; thus, it facilitated the dissection of the therapeutic mechanisms of compounds. (-)-5-Hydroxy-equol is an isoflavone metabolite that be obtained by microbial biotransformation. In the current work, the effect of (-)-5-hydroxy-equol on hepatocellular carcinoma (HCC) cells and its mechanism have been explored based on 1H NMR-based metabolomics approach. Our results revealed that (-)-5-hydroxy-equol can significantly inhibit the proliferation, migration, and invasion of SMMC-7721 cells and inhibit the proliferation of HepG2 cells. Metabolomics revealed that 17 differential metabolites involving in amino acid metabolism and energy metabolism were significantly changed inside and outside of the cells after treatment of (-)-5-hydroxy-equol. Specifically, (-)-5-hydroxy-equol at a concentration of 30 µM significantly decreased the concentrations of pyruvate, glutamate, and glucose. Because glycometabolism is a crucial feature of cancer-specific metabolism, we further verified enzymes and proteins that are closely relevant to glycometabolism. Our results indicated that (-)-5-hydroxy-equol-modulated glycolysis in HCC through the inhibition of activities of hexokinase, phosphofructokinase, and pyruvate kinase, and the expression of pyruvate kinase M2. This study revealed that metabolomic analysis integrating with further verifications at the biochemical level can facilitate understanding the anti-HCC mechanisms of (-)-5-hydroxy-equol.

Combination of Dietary Ahiflower Oil and Equol Enhances Long-Chain Polyunsaturated Fatty Acid Levels in Rainbow Trout Tissues.

Equol and Ahiflower oil have been shown to increase either eicosapentaenoic acid (EPA, 20:5n-3) or docosahexaenoic acid (DHA, 22:6n-3) levels in tissues of rainbow trout when applied individually. Thus, we investigated whether the combination of an Ahiflower oil-based diet and equol might increase both, EPA and DHA levels, in rainbow trout. Rainbow trout (87.1 ± 0.3 g) were fed with five diets for 8 weeks. A diet based on a blend of fish and vegetable oils (FV) served as a reference diet. The four experimental diets contained a blend of Ahiflower oil and vegetable oils (AV). The AV-diets were supplemented with equol by 0.0%, 0.1%, 0.2%, and 0.3% DM of the diet (AV-C, AV-EQ1, AV-EQ2, and AV-EQ3). The dietary treatments did not affect growth performance of fish and chemical nutrient composition of the whole body samples. Fish fed with the equol diets showed dose-dependently increased liver weights and 18:0 liver levels. The content of EPA showed no consistent pattern between tissues but all AV-groups were characterized by higher liver EPA values than FV. DHA values of AV-EQ2 and AV-EQ3 were similar to FV in fillet, tended to be the highest in the whole body and were significantly higher in liver compared to FV. In contrast, mRNA steady state levels of fatty acyl desaturase 2a (delta-6) [fads2a(d6)] were not affected by the dietary treatments. In conclusion, the combination of dietary Ahiflower oil and equol (0.2% and 0.3%) seems to affect the fatty acid metabolism of rainbow trout positively to increase DHA tissue levels.

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).