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Postmenopausal osteoporosis is a common disorder accompanied with estrogen deficiency in women. Plants containing phytoestrogens and amino acids have been used in the osteoporosis treatment. The present study aims to evaluate the estrogen-like activity of the Cicer arietinum extract (CAE) and its ability to inhibit osteoclastogenesis process. These achieved by investigating the binding of its active phytoestrogens (genistein, daidzein, formononetin and biochanin A) to the estrogen receptors (ER) α and β of rats and human in silico. In addition, in vivo study on ovariectomized (OVX) rats is performed. For in vivo study, twenty four rats were divided into four groups (n= 6). Group I is the sham control rats which administered distilled water. Groups II, III, and IV are OVX groups which administered distilled water, CAE (500 mg/kg), and alendronate; respectively. The docking study revealed that the phytoestrogens docked into the protein active site with binding energies comparable with that of estrogens (estriol and β-estradiol) which means the similarity between the estrogenic contents of CAE and the ensogenous ones. Additionally, in vivo study revealed that CAE reverse TRAP5b and RANKL levels that drastically increased in the untreated OVX group. But, it trigger upregulation of OPG, enhance the OPG/RANKL ratio and modulate the bone and uterus alterations of OVX group. Phytoestrogens and the bone-protective amino acids contents of CAE could be responsible for their estrogen-like effect and antiosteoporotic activity. These results concluded that CAE is an attractive candidate for developing a potential therapeutic cheap agent used as an alternative to the synthetic estrogen replacement therapy. Further, in vivo validation is required for its clinical application.

Hop ( Humulus lupulus L.), as a key ingredient for beer brewing, is also a source of many biologically active molecules. A notable compound, 8-prenylnaringenin (8-PN), structurally belonging to the group of prenylated flavonoids, was shown to be a potent phytoestrogen, and thus, became the topic of active research. Here, we overview the pharmacological properties of 8-PN and its therapeutic opportunities. Due to its estrogenic effects, administration of 8-PN represents a novel therapeutic approach to the treatment of menopausal and post-menopausal symptoms that occur as a consequence of a progressive decline in hormone levels in women. Application of 8-PN in the treatment of menopause has been clinically examined with promising results. Other activities that have already been assessed include the potential to prevent bone-resorption or inhibition of tumor growth. On the other hand, the use of phytoestrogens is frequently questioned regarding possible adverse effects associated with long-term consumption. In conclusion, we emphasize the implications of using 8-PN in future treatments of menopausal and post-menopausal symptoms, including the need for precise evidence and further investigations to define the safety risks related to its therapeutic use., Competing Interests: The authors declare no conflict of interest.

Recently, the palbociclib/letrozole combination therapy was granted accelerated US FDA approval for the treatment of estrogen receptor (ER)-positive breast cancer. Since the underlying metabolic effects of these drugs are yet unknown, we investigated their synergism at the metabolome level in MCF-7 cells. As xenoestrogens interact with the ER, we additionally aimed at deciphering the impact of the phytoestrogen genistein and the estrogenic mycotoxin zearalenone. A global metabolomics approach was applied to unravel metabolite and pathway modifications. The results clearly showed that the combined effects of palbociclib and letrozole on cellular metabolism were far more pronounced than that of each agent alone and potently influenced by xenoestrogens. This behavior was confirmed in proliferation experiments and functional assays. Specifically, amino acids and central carbon metabolites were attenuated, while higher abundances were observed for fatty acids and most nucleic acid-related metabolites. Interestingly, exposure to model xenoestrogens appeared to counteract these effects., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The biologic effects of estrogens are transduced by two estrogen receptors (ERs), ER α and ER β , which function in dimer forms. The ER α / α homodimer promotes and the ER β / β inhibits estrogen-dependent growth of mammary epithelial cells; the functions of ER α / β heterodimers remain elusive. Using compounds that promote ER α / β heterodimerization, we have previously shown that ER α / β heterodimers appeared to inhibit tumor cell growth and migration in vitro. Further dissection of ER α / β heterodimer functions was hampered by the lack of ER α / β heterodimer-specific ligands. Herein, we report a multistep workflow to identify the selective ER α / β heterodimer-inducing compound. Phytoestrogenic compounds were first screened for ER transcriptional activity using reporter assays and ER dimerization preference using a bioluminescence resonance energy transfer assay. The top hits were subjected to in silico modeling to identify the pharmacophore that confers ER α / β heterodimer specificity. The pharmacophore encompassing seven features that are potentially important for the formation of the ER α / β heterodimer was retrieved and subsequently used for virtual screening of large chemical libraries. Four chemical compounds were identified that selectively induce ER α / β heterodimers over their respective homodimers. Such ligands will become unique tools to reveal the functional insights of ER α / β heterodimers., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Stilbenes have been reported to be phytoestrogen compounds owing to its structural similarity to the estrogenic agent diethylstilbestrol. To find new stilbene-derivative phytoestrogens, isolation of stilbene-rich R. undulatum was performed and led to identify six new compounds (1-5 and 28), one newly determined absolute configurations compound (27) together with 21 previously reported compounds (6-26). The structures of compounds were determined on the basis of extensive spectroscopic methods including 1D and 2D NMR and CD spectra data. All the isolated compounds were tested for their estrogenic activities in HepG2 cells transiently transfected with ERα, ERβ and ERE-reporter plasmid. Among them, stilbene-derivatives, piceatannol 3'-O-β-d-xylopyranoside (12), cis-rhaponticin (16) and rhapontigenin 3'-O-β-d-glucopyranoside (17), showed the more potent binding affinity for estrogen receptors than 17β-estrodiol., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Alfalfa and red clover are the most widespread and most important perennial legumes, primarily used as a high-quality feed for livestock. Both alfalfa and red clover, as well as some other plant species from Fabaceae family, are a rich natural source of phytoestrogens, nonsteroidal compounds with an estrogenic activity whose beneficial effects in the prevention and treatment of many diseases are demonstrated in numerous scientific studies., Objectives: Nowadays, various herbal preparations are present on the world market and their use is constantly increasing, as well as the growing demands of consumers for environmentally sound and health-safe production of plant species used as sources of active substances. Because of their widespread distribution, the possibility of organic breeding, and also significant increases in surface area under genetically modified crops in most EU countries, alfalfa and red clover have become more interesting alternative sources of phytoestrogens. The most common phytoestrogens in these forage legumes are genistein, daidzein, glycitein, formononetin, biochanin, coumestrol, kaempferol and apigenin. The content of these substances is significantly influenced by a number of factors including genotype, environment, production technology, plant maturity stage, and individual plant parts., Conclusion: Available evidence suggests that forage legumes represent high promising sources of health-promoting phytoestrogens. Due to numerous favorable features, they can find commercial application in different industries such as pharmaceutical, nutraceutical, cosmetic, and agriculture., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Phytoestrogens are plant compounds with estrogenic effects found in many foods. We have previously reported phytoestrogen activity of blackcurrant anthocyanins (cyanidin-3-glucoside, cyanidin-3-rutinoside, delphinidin-3-glucoside, and delphinidin-3-rutinoside) via the estrogen receptor (ER)α. In this study, we investigated the participation of ERβ in the phytoestrogen activity of these anthocyanins. Blackcurrant anthocyanin induced ERβ-mediated transcriptional activity, and the IC 50 of ERβ was lower than that of ERα, indicating that blackcurrant anthocyanins have a higher binding affinity to ERβ. In silico docking analysis of cyanidin and delphinidin, the core portions of the compound that fits within the ligand-binding pocket of ERβ, showed that similarly to 17β-estradiol, hydrogen bonds formed with the ERβ residues Glu305, Arg346, and His475. No fitting placement of glucoside or rutinoside sugar chains within the ligand-binding pocket of ERβ-estradiol complex was detected. However, as the conformation of helices 3 and 12 in ERβ varies depending on the ligand, we suggest that the surrounding structure, including these helices, adopts a conformation capable of accommodating glucoside or rutinoside. Comparison of ERα and ERβ docking structures revealed that the selectivity for ERβ is higher than that for ERα, similar to genistein. These results show that blackcurrant anthocyanins exert phytoestrogen activity via ERβ., Competing Interests: The authors declare no conflict of interest.

A great interest has been developed last decades on the role of phytoestrogens as an alternative to hormone replacement therapy. In fact, numerous studies have shown the effects of soy isoflavones on climacteric symptomatology by significantly reducing the frequency and intensity of hot flashes, as well on cardiovascular health. The aim of this paper was to review the last studies, mainly randomized controlled trials, on the effect of isoflavones on climacteric symptoms and their impact on women's quality of life, as well as on other health markers. Following a review of meta-analyzes and recent clinical trials, evidence has been obtained of the efficacy of isoflavones use in reducing hot flashes, improving the quality of life, lowering the cardiovascular risk by optimizing lipid profile, and Improving bone mineral density and other markers of bone resorption. Most of the authors concluded on the favorable effect of regular consumption of soy isoflavones on the health and quality of life of peri and postmenopausal women.

Prenylated flavonols are known as phytoestrogen and have good bioactivties. However, their abundances in nature are pretty low. It is required to find an efficient synthesis technique. Icariin is a prenylated flavonol glycoside with low cost. It can be used to synthesize different prenylated flavonols. A combination of cellulase and trifluoacetic acid hydrolysis could effectively remove rhamnose and glucose from icariin. Icaritin, anhydroicaritin and wushanicaritin were the leading prenylated flavonol products. Their affinities to estrogen receptors α and β were predicted by docking study. The weak affinity of wushanicaritin indicated that prenyl hydroxylation impaired its affinity to estrogen receptor β. The prenyl cyclization led to a loss of affinity to both receptors. The interactions between icaritin and ligand binding cavity of estrogen receptor β were simulated. π-π stacking and hydrophobic forces were predicted to be the dominant interactions positioning icaritin, which induced the helix (H12) forming an activated conformation.

Botanical dietary supplements contain multiple bioactive compounds that target numerous biological pathways. The lack of uniform standardization requirements is one reason that inconsistent clinical effects are reported frequently. The multifaceted biological interactions of active principles can be disentangled by a coupled pharmacological/phytochemical approach using specialized ("knock-out") extracts. This is demonstrated for hops, a botanical for menopausal symptom management. Employing targeted, adsorbent-free countercurrent separation, Humulus lupulus extracts were designed for pre- and postmenopausal women by containing various amounts of the phytoestrogen 8-prenylnaringenin (8-PN) and the chemopreventive constituent xanthohumol (XH). Analysis of their estrogenic (alkaline phosphatase), chemopreventive (NAD(P)H-quinone oxidoreductase 1 [NQO1]), and cytotoxic bioactivities revealed that the estrogenicity of hops is a function of 8-PN, whereas their NQO1 induction and cytotoxic properties depend on XH levels. Antagonization of the estrogenicity of 8-PN by elevated XH concentrations provided evidence for the interdependence of the biological effects. A designed postmenopausal hop extract was prepared to balance 8-PN and XH levels for both estrogenic and chemopreventive properties. An extract designed for premenopausal women contains reduced 8-PN levels and high XH concentrations to minimize estrogenic while retaining chemopreventive properties. This study demonstrates the feasibility of modulating the concentrations of bioactive compounds in botanical extracts for potentially improved efficacy and safety.

Within this study, a new method enabling monitoring of various estrogenic substances potentially occurring in milk and dairy products was proposed. Groups of compounds fairly differing in physico-chemical properties and biological activity were analyzed: four natural estrogens, four synthetic estrogens, five mycoestrogens, and nine phytoestrogens. Since they may pass into milk mainly in glucuronated and sulfated forms, an enzymatic hydrolysis was involved prior to the extraction based on the QuEChERS methodology. For the purification of the organic extract, a dispersive solid-phase extraction (d-SPE) with sorbent C18 was applied. The final analysis was performed by ultra-high-performance liquid chromatography (UHPLC) coupled with triple quadrupole tandem mass spectrometry (MS/MS). Method recovery ranged from 70 to 120% with a relative standard deviation (RSD) value lower than 20% and limits of quantification (LOQs) in the range of 0.02-0.60 μg/L (0.2-6.0 μg/kg dry weight) and 0.02-0.90 μg/kg (0.2-6.0 μg/kg dry weight) for milk and yogurt, respectively. The new procedure was applied for the investigation of estrogenic compounds in 11 milk samples and 13 yogurt samples from a Czech retail market. Mainly phytoestrogens were found in the studied samples. The most abundant compounds were equol and enterolactone representing 40-90% of all estrogens. The total content of phytoestrogens (free and bound) was in the range of 149-3870 μg/kg dry weight. This amount is approximately 20 times higher compared to non-bound estrogens.

Phytoestrogens are plant-derived compounds that functionally and structurally mimic mammalian estrogens. Phytoestrogens have broad inhibitory activities toward several steroidogenic enzymes, such as the 17β-hydroxysteroid dehydrogenases (17β-HSDs), which modulate the biological potency of androgens and estrogens in mammals. However, to date, no crystallographic data are available to explain phytoestrogens binding to mammalian 17β-HSDs. NADP(H)-dependent 17β-HSD from the filamentous fungus Cochliobolus lunatus (17β-HSDcl) has been the subject of extensive biochemical, kinetic and quantitative structure-activity relationship studies that have shown that the flavonols are the most potent inhibitors. In the present study, we investigated the structure-activity relationships of the ternary complexes between the holo form of 17β-HSDcl and the flavonols kaempferol and 3,7-dihydroxyflavone, in comparison with the isoflavones genistein and biochanin A. Crystallographic data are accompanied by kinetic analysis of the inhibition mechanisms for six flavonols (3-hydroxyflavone, 3,7-dihydroxyflavone, kaempferol, quercetin, fisetin, myricetin), one flavanone (naringenin), one flavone (luteolin), and two isoflavones (genistein, biochanin A). The kinetics analysis shows that the degree of hydroxylation of ring B significantly influences the overall inhibitory efficacy of the flavonols. A distinct binding mode defines the interactions between 17β-HSDcl and the flavones and isoflavones. Moreover, the complex with biochanin A reveals an unusual binding mode that appears to account for its greater inhibition of 17β-HSDcl with respect to genistein. Overall, these data provide a blueprint for identification of the distinct molecular determinants that underpin 17β-HSD inhibition by phytoestrogens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Phytochemicals are botanical compounds used in dermatology applications as cosmeceuticals to improve skin health. Resveratrol and equol are two of the best-known polyphenolic or phytoestrogens having similar chemical structures and some overlapping biological functions to 17β-estradiol. Human skin gene expression was reviewed for 28 different biomarkers when resveratrol, 4' acetoxy resveratrol (4AR), R -equol, racemic equol or S -equol were tested. Sirtuin 1 activator (SIRT 1) was stimulated by resveratrol and 4AR only. Resveratrol, R -equol and racemic equol were effective on the aging biomarkers proliferating cell nuclear factor (PCNA), nerve growth factor (NGF), 5α-reductase and the calcium binding proteins S100 A8 and A9. Racemic equol and 4AR displayed among the highest levels for the collagens, elastin and tissue inhibitor of the matrix metalloproteinase 1 (TIMP 1). S -equol displayed the lowest level of effectiveness compared to the other compounds. The 4AR analog was more effective compared to resveratrol by 1.6-fold. R -equol and racemic equol were almost equal in potency displaying greater inhibition vs. resveratrol or its 4' analog for the matrix metalloproteinases (MMPs), but among the inflammatory biomarkers, resveratrol, 4AR, R -equol and racemic equol displayed high inhibition. Thus, these cosmeceuticals display promise to improve dermal health; however, further study is warranted to understand how phytochemicals protect/enhance the skin.

With increasing interests of phytoestrogens for their potential applications, a rapid and simple tool for screening these phytochemicals is still required. In this study, a simple assay to detect phytoestrogens was developed based on the competition binding between the tested samples and the fluorescently labelled oestrogen (E2) to the human ligand binding domain of oestrogen receptor (LBD-ER) that was immobilised on the magnetite nanoparticles (MNPs). The 40-kDa LBD-ER peptide was produced in an Escherichia coli system. The synthesised 68.7-nm MNPs were silanised and subsequently covalently linked to the C-terminus of LBD-ER peptide. The LBD-ER immobilised MNPs demonstrated the specific binding for the standard E2 with the equilibrium dissociation constant of 9.56 nM and the binding capacity of 0.08 pmol/1 mg of the MNPs. The LBD-ER immobilised MNPs could evaluate oestrogenic activity of the extracts of Asparagus racemosus and Curcuma comosa , the reported phytoestrogenic plants, but not progesterone (P4) and Raphanus sativus extract, the negative controls. The results of this work clearly demonstrated a potential assay for detecting phytoestrogens of crude plant extracts, which is simple and easily adapted to a high throughput format.

Pueraria candollei var. mirifica or White Kwao Krua (WKK) is a phytoestrogen-rich plant widely used among women to improve climacteric symptoms. Additionally, the tuberous roots of this plant have been added as an active ingredient for skin rejuvenation and breast enlargement effects in various functional foods. However, most of the products on the market containing WKK have not been sufficiently standardized with respect to the active compound or identical marker. To control the quality of these plant materials, an enzyme-linked immunosorbent assay (ELISA) using anti-isomiroestrol antibodies was established for the determination of isomiroestrol, an identical marker in WKK. Polyclonal and monoclonal antibodies against isomiroestrol were generated and their specificity characterized in this study. Monoclonal antibody 12C1 showed higher specificity to isomiroestrol and was thus selected to develop the ELISA. Based on the validation analysis and the tested performance of the developed ELISA in variably sourced WKK samples, the assay can provide an alternative approach that is reliable and highly sensitive for the quantitative analysis of isomiroestrol in plant., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Isoflavones (genistein, daidzein, and glycitein) are bioactive compounds with mildly estrogenic properties and often referred to as phytoestrogen. These are present in significant quantities (up to 4-5 mg·g -1 on dry basis) in legumes mainly soybeans, green beans, mung beans. In grains (raw materials) they are present mostly as glycosides, which are poorly absorbed on consumption. Thus, soybeans are processed into various food products for digestibility, taste and bioavailability of nutrients and bioactives. Main processing steps include steaming, cooking, roasting, microbial fermentation that destroy protease inhibitors and also cleaves the glycoside bond to yield absorbable aglycone in the processed soy products, such as miso, natto, soy milk, tofu; and increase shelf lives. Processed soy food products have been an integral part of regular diets in many Asia-Pacific countries for centuries, e.g. China, Japan and Korea. However, in the last two decades, there have been concerted efforts to introduce soy products in western diets for their health benefits with some success. Isoflavones were hailed as magical natural component that attribute to prevent some major prevailing health concerns. Consumption of soy products have been linked to reduction in incidence or severity of chronic diseases such as cardiovascular, breast and prostate cancers, menopausal symptoms, bone loss, etc. Overall, consuming moderate amounts of traditionally prepared and minimally processed soy foods may offer modest health benefits while minimizing potential for any adverse health effects.

Curcuma comosa (C. comosa) is widely used in traditional medicine as a dietary supplement for health promotion in postmenopausal women in Thailand. It contains several diarylheptanoids, which are considered to be a novel class of phytoestrogens. However, the diarylheptanoids isolated from the plant rhizome are shown to have low oral bioavailability and faster elimination characteristics. The aim of this study was to investigate the permeation behavior of the active compounds of diarylheptanoids. The effects of binary vehicle systems and permeation enhancers on diarylheptanoids permeation and accumulation within the skin were studied using side-by-side diffusion cells through the porcine ear skin. Among the tested binary vehicle systems, the ethanol/water vehicle appeared to be the most effective system for diarylheptanoids permeation with the highest flux and shortest lag time. The presence of transcutol in the vehicle system significantly increased diarylheptanoid's permeation and accumulation within the skin in a concentration-dependent manner. Although the presence of terpenes in formulation decreased the flux of diarylheptanoids, it raised the amount of diarylheptanoids retained within the skin substantially. Based on the feasibility of diarylheptanoid permeation, C. comosa extract should be further developed into an effective transdermal product for health benefits and hormone replacement therapy.

Soy, a major component of the diet for centuries contains the largest concentration of isoflavones, a class of phytoestrogens. A variety of health benefits are associated with the consumption of soy primarily because of the isoflavones genistein, daidzein, and glycitein with a potential protective effect against a number of chronic diseases. Owing to the pharmaceutical and nutraceutical properties allied with isoflavonoids and their use in functional foods, there is a growing interest in these compounds. This review throws light on the chemistry, and significant pharmacological and biopharmaceutical aspects of soy isoflavones. This article critically describes the mechanisms of action, infers conclusions and shows opportunity for future research.

With growing interests of phytoestrogens, many natural phytochemicals extracted from diverse plant species have been explored for their estrogenic-like activities and potential applications. In this work, a simple and rapid separation of phytoestrogenic compounds from crude plant extracts was purposed using magnetic nanoparticles (MNPs) of Fe 3 O 4 immobilized with the ligand-binding domain (LBD) of estrogen receptor alpha (ERα). The recombinant LBD-ERα peptide of 40 kDa was produced and subsequently covalently linked to MNPs. One milligram of the LBD-ERα-immobilized MNPs demonstrated a specific binding to the standard 17β-estradiol (E2) of 3.37 nmol and 91.3-100 % of the bound E2 were subsequently recovered. LBD-ERα-immobilized MNPs could separate phytoestrogens of 4.6 nmol E2-equivalent activity from a 1-mg crude extract of Asparagus racemosus. The produced MNPs showed no separation yield when were applied to the negative controls, the crude extract of radish (Raphanus sativus), and the standard progesterone (P4). Thin-layer chromatography demonstrated a single phytochemical band of the separated phytoestrogens, which exhibited the activity to promote MCF-7 cell proliferation at 4.7 folds greater than the crude A. racemosus extract. The results of this work demonstrated a simple method to specifically separate phytoestrogens from crude plant extracts via the LBD-ERα-immobilized MNPs.

Advantages of the supercritical fluid (SCF) process compared to the conventional solution stirring method (CSSM) in the preparation of daidzein-hydroxypropyl-β-cyclodextrin (HPβCD) complexes were investigated. Formation of daidzein/ HPβCD inclusion complexes was confirmed by Fourier transformed-infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD) and scanning electron microscopy (SEM). Particle size, inclusion yield, drug solubility and dissolution of daidzein/HPβCD complexes were evaluated. Compared to CSSM, the SCF process resulted in higher inclusion yield and higher solubility. Also, extended dissolution of daidzein from the SCF processed HPβCD inclusion complexes was observed, with only 22.94 % released in 45 min, compared to its rapid release from those prepared by CSSM, with 98.25 % drug release in 15 min. This extended release of daidzein from SCF prepared inclusion complexes was necessary to avoid drug precipitation and improve drug solubilisation in the gastrointestinal tract. The results showed that the SCF process is a superior preparation method for daidzein-hydroxypropyl-β-cyclodextrin complexes.

Gold nanoparticles conjugated with drug substances are used in diagnostics and therapies. Apart from the combinations involving gold nanoparticles conjugated with drug substances through linkers, a direct bonding is also known. In our paper the example of such a direct bonding between gold nanoparticles and genistein (AuNPs-GE) is presented. This conjugate was obtained in a one-pot synthesis and the formation of AuNPs-GE was monitored in terms of color change and UV-Vis spectroscopy. It has been shown that genistein reduces Au 3+ ions to spherical Au 0 nanocrystallites and acts as a stabilizing agent. The efficiency of the purification of the conjugate from free genistein was controlled by the capillary electrophoresis. Gold nanoparticles are homogeneously shaped and have a narrow range of size from 14 to 33nm and the size of the nanoparticles modified with genistein is around 64.64±0.41nm, as measured by the TEM and DSL techniques, respectively. The zeta potential of the gold nanoparticles modified with genistein is -19.32±0.82mV and suggests a high stability of the nanoparticles and lower toxicity for the normal cells. The identity of genistein on the gold nanoparticles was proved by the electrochemistry, NMR and Raman spectroscopy. The mechanism of the conjugate forming has been proposed. The coverage of gold nanoparticles with genistein 5.09% (m/m) has been calculated from the TGA analysis. Moreover, it has been proved that the obtained conjugate is characterized by a high cytotoxic activity towards cancer cells, as observed in the cell line test., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Natural organic compounds such as phytoestrogens and phytosterols found in various plants, as well as mycotoxins produced by fungi, can be found in aquatic environments. The aim of this study was to investigate the occurrence of three different classes of natural estrogenic compounds, i.e., phytoestrogens, phytosterols and mycotoxins, in estuarine water samples from the Ave River estuary. For that, water samples were collected at five sampling points distributed along the estuary at low tide, during 1 year, processed by solid-phase extraction (SPE) and analyzed by gas chromatography coupled to mass spectrometry (GC-MS). To correlate the presence of phytoestrogens and phytosterols in the estuarine water, local flora was collected on riverside. Trace elements content and physicochemical parameters such as nutrients and dissolved oxygen were also determined seasonally at each sampling point, to give insights for the evaluation of water quality and anthropogenic pressure. Both phytoestrogens and phytosterols showed a seasonal variation, with the highest values observed in spring and summer and the lowest in winter. Daidzein (DAID) was found up to 404.0 ng L(-1) in spring and coumestrol (COUM) was found up to 165.0 ng L(-1) in summer. The mycotoxin deoxynivalenol (DON) was ubiquitously determined with values ranging from 59.5 to 642.4 ng L(-1). Nutrients and metals distribution and concentration varied among sampling stations and seasons. This study revealed for the first time the presence of mycotoxins, various classes of phytoestrogens and stigmasterol (STG) in estuarine water from the Ave River (Portugal), and the evaluation of the water quality confirmed that this estuary is still highly impacted by anthropogenic activities.

Isoflavones genistein and daidzein are nonsteroidal phytoestrogens occurring mainly in soybean foods. These phytoestrogens possess estrogenic properties and show a variety of health benefits as anti-inflammatory agents. However, the mechanism of their action has not been identified in detail. The aim of this study is to characterize the antioxidant powers of genistein, daidzein and daidzein metabolite-equol through their activities to scavenge superoxide anion radical (O(•) 2 (-) ), hydroxyl radical (HO(•) ), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH(•) ) and hydrogen peroxide (H2 O2 ) using chemiluminescence and spectrophotometry techniques. Potassium superoxide in dimethyl sulphoxide (DMSO) and 18-crown-6 ether were used as a source of O(•) 2 (-) . Hydroxyl radicals were produced using the Fenton reaction. In free radical assays, genistein had the IC50 values (an amount of antioxidant concentration required to decrease the initial radical concentration by 50%) 0.391 ± 0.012 mM for O(•) 2 (-) , 0.621 ± 0.028 mM for HO(•) and 1.89 ± 0.16 mM for DPPH(•) . The IC50 values for daidzein for these free radicals were 1.924 ± 0.011 mM, 0.702 ± 0.012 mM and 2.81 ± 0.03 mM, respectively. Equol was the most active the free radical scavenger with IC50  = 0.451 ± 0.018 mM for HO(•) and IC50  = 1.36 ± 0.11 mM for DPPH(•) . All tested compounds exerted a significant effect on the H2 O2 : IC50  = 18.1 ± 1.1 μM for genistein, IC50  = 2.1 ± 0.5 μM for daidzein, and IC50  = 1.06 ± 0.2 μM for equol. These findings show that genistein, daidzein and equol are effective free radical scavengers and possess high antioxidant power in vitro. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Effects of phytoestrogen isoflavones in Berseem clover on hormonal balance during early pregnancy and fertility of heifers were studied. Holstein heifers (n=26) were divided into two equal homogenous groups. Heifers in the first group (Clover-fed group) were fed Egyptian clover (Trifolium alexandrinum) as oestrogenic roughage; whereas the heifers in the second group (Silage-fed group) were fed maize silage. Concentrations of four isoflavone aglycones (genistein. Daidzein, biochanin A and formononetein) were determined in the two roughages. Treatment lasted for 20 consecutive weeks (5 months) during which blood samples were collected biweekly for determining the metabolic profile of heifers. Heifers were subjected to oestrous synchronisation using a double prostaglandin F2α (PGF2α) based protocol, 14days apart (week 10 and 12), and were artificially inseminated 12h following detection of overt signs of oestrus. Concentrations of serum oestradiol (E2) and progesterone (P4) on the day of insemination (Day 0) and on Days 7, 14 and 21 post-insemination (early pregnancy) were determined and P4 to E2 ratio was calculated. Clover had greater total isoflavone content than maize silage. The dominant isoflavone detected in Berseem clover was biochanin A, whereas the least was formononetein. Metabolic profile was not affected (P>0.05) by the type of roughage and was in the same trend in both experimental groups. The overall mean concentration of serum E2 was greater (P<0.05) in the clover-fed group than in the silage-fed group. The overall mean concentration of serum P4 was less (P<0.05) in the clover-fed group than in the silage-fed group. During the period of early pregnancy (from Day 7-21 post-insemination) the concentration of serum P4 increased in the silage-fed group, however, no change was observed in the clover-fed group. The overall mean of P4 to E2 ratio was greater (P<0.001) in the silage-fed group compared with that in the clover-fed group. Heifers fed clover had a lesser (P=0.054) conception rate and a greater (P=0.062) percentage of heifers returning to oestrus than silage-fed heifers. In conclusion, isoflavones in Berseem clover apparently disturbed hormonal balance during early pregnancy leading to a reduction in the fertility of heifers., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objectives: The current study aimed at exploring the secondary metabolites content of Erythrina crista-galli aqueous methanol extract and assessing its phytoestrogenic and cytoprotective activities., Methods: Isolation of the compounds was carried out using conventional chromatographic techniques. The structures of the isolated compounds were elucidated based on the UV, NMR spectral data along with their mass-spectrometric analyses. The phytoestrogenic activity was evaluated in-silico and in vitro using the Arabidopsis thaliana pER8: GUS reporter assay and the proliferation-enhancing activity of MCF-7 cells., Key Findings: Phytochemical investigation of E. crista-galli aqueous methanol extract resulted in the isolation and identification of five flavonoids. The plant extract and its fractions showed significant estrogenic activities compared to controls., Conclusion: Five flavonoids were identified from E. crista-galli aqueous methanol extract. To the best of our knowledge, among these flavonoids, apigenin-7-O-rhamnosyl-6-C-glucoside was isolated for the first time from nature. Moreover, luteolin-6-C-glucoside was isolated for the first time from this plant. The plant revealed promising phytoestrogenic activities. This gives rationale to some of its pharmacological properties and suggests additional phytoestrogenic effects, which have not been reported yet.

Humulus lupulus (hop plant) has long been used in traditional medicine as a sedative and antimicrobial agent. More recently, attention has been devoted to the phytoestrogenic activity of the plant extracts as well as to the anti-inflammatory and chemopreventive properties of the prenylated chalcones present. In this study, an Italian sample of H. lupulus cv. "Cascade" has been investigated and three new compounds [4-hydroxycolupulone (6), humudifucol (7) and cascadone (8)] have been purified and identified by means of NMR spectroscopy along with four known metabolites. Notably, humudifucol (7) is the first prenylated dimeric phlorotannin discovered in nature. Because structurally related phloroglucinols from natural sources were found previously to inhibit microsomal prostaglandin E2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO), the isolated compounds were evaluated for their bioactivity against these pro-inflammatory target proteins. The prenylated chalcone xanthohumol inhibited both enzymes at low μM concentrations.

It is believed that breast cancer stem cells (BCSCs), like normal stem cell counterparts, have the capacity of self-renewal and differentiation. Simultaneously, estrogen receptor (ER)-negative (-) BCSCs are affected by surrounding differentiated ER-positive (+) tumor cells by virtue of paracrine signaling within the tumor micro-environment. Genistein (GEN), as a sort of phytoestrogen, can act on ER+ breast cancer cells but the role of GEN in the differentiation of neighboring ER- BCSCs has not been defined. Transwell co-culture system was utilized so as to elaborate the interaction between well-differentiated ER+ breast cancer cells (MCF-7) and ER- breast cancer stem/progenitor cells (mammospheres derived from MDA-MB-231 cells). GEN-induced differentiation of BCSCs was analyzed by mammospheres formation assay, flow cytometry and RT-PCR after a 3 day solo-culture or co-culture. We find that GEN sized 2 µM, and 40 nM, effectively promotes morphological alteration of mammospheres, reduces the ratio of subset of CD44+/CD24-/ESA+ cells and upregulates the expression of differentiated cell markers of mammospheres in co-culture system, but not in solo-culture condition. Besides, we demonstrate that the differentiation-inducing effect of GEN on mammospheres is associated with PI3K/Akt and MEK/ERK signaling pathways which are activated by amphiregulin released from ER+ cancer cells. These results indicate that GEN was able to induce the differentiation of breast cancer stem/progenitor cells through interaction with ER+ cancer cells by a paracrine mechanism.

Formononetin is an O-methylated isoflavone that is isolated from the root of Astragalus membranaceus, and it has antitumorigenic effects. Our previous studies found that formononetin triggered growth-inhibitory and apoptotic activities in MCF-7 breast cancer cells. To further investigate the potential effect of formononetin in promoting cell proliferation in estrogen receptor (ER)-positive cells, we used in vivo and in vitro studies to elucidate the possible mechanism. ERα-positive cells (HUVEC, MCF-7) were treated with formononetin. The CCK8 assay, Hoechst 33258, and flow cytometry were used to assess cell proliferation and apoptosis. mRNA levels of ERα, Bcl-2, and miR-375 were quantified using real-time polymerase chain reaction. ERα, p-Akt, and Bcl-2 expression was determined using Western blot. Compared with the control, low formononetin concentrations (2-6 μM) stimulated ERα-positive cell proliferation (HUVEC, MCF-7). The more sensitive HUVEC cells were used to study the relevant signaling pathway. After treatment with formononetin, ERα, miR-375, p-Akt, and Bcl-2 expression was significantly upregulated. The proliferative effect of formononetin was also blocked by a miR-375 inhibitor or raloxifene pretreatment. Additionally, in the in vivo studies, uterine weight in ovariectomized mice treated with formononetin increased significantly, but the weight dramatically decreased with raloxifene or miR-375 inhibitor pretreatment before formononetin. This study demonstrated that formononetin promoted ERα-positive cell proliferation through miR-375 activation and this mechanism is possibly involving in a miR-375 and ERα feedback loop., (© 2015 Wiley Periodicals, Inc.)

A sensitive and robust method using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for quantitation of 13 phytoestrogens and related metabolites in rat serum samples. A new type of column, the Kinetex core-shell C18 column, was applied for rapid separation of the target analytes in 10min. Two enzymes, sulfatase H-1 and gulcuronidase H-5 from Helix pomatia were compared on the efficiency of releasing the conjugated forms of the target analytes to their free forms in serum samples. The method detection limit (MDL) defined as three times the signal to noise ratio in spiked serum matrix-based solutions was in the range of 0.1-3.5ng/mL. The linear dynamic calibration was in the broad range of 0.2-500ng/mL for all target compounds. Thirty-two rat serum samples from the rats that were fed with diets containing either casein or soy protein isolates with various amounts of isoflavones for 8 weeks were analyzed for the target analytes with the developed method. Nine target analytes were detected in the serum samples. Those detectable compounds are all the metabolites of the dietary isoflavones, suggesting that the diet isoflavones were mostly metabolized to their metabolites in rat., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

An enormous technological progress has resulted in an explosive growth in the amount of biological and chemical data that is typically multivariate and tangled in structure. Therefore, several computational approaches have mainly focused on dimensionality reduction and convenient representation of high-dimensional datasets to elucidate the relationships between the observed activity (or effect) and calculated parameters commonly expressed in terms of molecular descriptors. We have collected the experimental data available in patent and scientific publications as well as specific databases for various agrochemicals. The resulting dataset was then thoroughly analyzed using Kohonen-based self-organizing technique. The overall aim of the presented study is to investigate whether the developed in silico model can be applied to predict the agrochemical activity of small molecule compounds and, at the same time, to offer further insights into the distinctive features of different agrochemical categories. The preliminary external validation with several plant growth regulators demonstrated a relatively high prediction power (67%) of the constructed model. This study is, actually, the first example of a large-scale modeling in the field of agrochemistry., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The effects of germination and elicitation on (iso)flavonoid composition of extracts from three edible lupine species (Lupinus luteus, Lupinus albus, Lupinus angustifolius) were determined by RP-UHPLC-MS(n). The total (iso)flavonoid content of lupine increased over 10-fold upon germination, with the total content and composition of isoflavonoids more affected than those of flavonoids. Glycosylated isoflavones were the most predominant compounds found in lupine seedlings. Lesser amounts of isoflavone aglycones, including prenylated ones, were also accumulated. Elicitation with Rhizopus oryzae, in addition to germination, raised the content of isoflavonoids further: the total content of 2'-hydroxygenistein derivatives was increased considerably, without increasing that of genistein derivatives. Elicitation by fungus triggered prenylation of isoflavonoids, especially of the 2'-hydroxygenistein derivatives. The preferred positions of prenylation differed among the three lupine species. The change in isoflavone composition increased the agonistic activity of the extracts towards the human estrogen receptors, whereas no antagonistic activity was observed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Seven prenylated 6a-hydroxy-pterocapans and five prenylated 6a,11a-pterocarpenes with different kinds of prenylation were purified from an ethanolic extract of fungus-treated soybean sprouts. The activity of these compounds toward both human estrogen receptors (hERα and hERβ) was determined in a yeast bioassay and the activity toward hERα was additionally tested in an U2-OS based hERα CALUX bioassay. In the yeast bioassay, compounds with chain prenylation showed in general an agonistic mode of action toward hERα, whereas furan and pyran prenylation led to an antagonistic mode of action. Five of these antagonistic compounds had an agonistic mode of action in the U2-OS based hERα CALUX bioassay, implying that these compounds can act as SERMs. The yeast bioassay also identified 8 ER subtype-selective compounds, with either an antagonistic mode of action or no response toward hERα and an agonistic mode of action toward hERβ. The ER subtype-selective compounds were characterized by 6a-hydroxy-pterocarpan or 6a,11a-pterocarpene backbone structure. It is suggested that either the extra D-ring or the increase in length to 12-13.5Å of these compounds is responsible for an agonistic mode of action toward hERβ and, thereby, inducing ER subtype-selective behavior., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: A novel soy protein aggregate enriched with isoflavones (SPA-IS), a mixture of soy protein and isoflavones (Mix), isoflavones (IS), and the soy protein were obtained to investigate the preventive effects on osteoporosis induced by retinoic acid in Kunming mice., Results: The serum osteocalcin levels in the Mix and SPA-IS groups decreased compared with the model group (mice showing retinoic acid-induced osteoporosis) (P < 0.05). The trabecular analysis results showed an increased preventive effect of the SPA-IS group over the Mix group, the IS group, and the soy protein group. The results of both left tibial maximum load and the 4th lumbar structural strength differ between the IS and the SPA-IS groups., Conclusion: The SPA-IS exhibited obvious oestrogenic activities on retinoic acid-induced osteoporosis in Kunming mice compared to Mix, IS, and soy protein. The results suggest that there is a potential use for SPA-IS in the treatment of osteoporosis induced by intake of retinoic acid. The improvement of bone indicators might be attributed to the formation of aggregate particles and the improvement of IS solubility., (© 2015 Society of Chemical Industry.)

Menopause jeopardizes the integrity of brain and makes it vulnerable to various diseases, both of psychiatric and degenerative nature. Exogenous estrogen supplementation confers neuroprotection but the results of Women's Health Initiative (WHI), Million Women Study (MWS) and incidence of endometrial cancer, breast cancer and venous thromboembolism reported with estrogen use have engendered doubts over its clinical translation for postmenopausal neurological disorders. Scientific community and general public have started recognizing the protective potential of phytochemicals in climacteric medicine. These phytochemicals are plant-derived, non-steroidal bioactive estrogenic compounds. Emerging preclinical studies have suggested that these phytochemicals display potential benefits in mitigating postmenopausal depression, anxiety, cerebral ischemia and cognitive dysfunction. Thus, the aim of present review is: a) to give an overview of neuroprotective action of estrogen, b) to address the chemical and pharmacological features of various classes of phytoestrogens, and c) to present preclinical and clinical evidence of effect of phytoestrogens on climacteric neurobiology with their possible mechanisms of action.

Low doses of endocrine disrupting chemicals (EDCs) used in combination may act in a manner different from that of individual compounds. The objective of the study was to examine in vitro effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 pM) and genistein (500 nM) on: 1) progesterone (P4) and estradiol (E2) secretion (48 h); 2) dynamic changes in aryl hydrocarbon receptor (AhR) mRNA and protein expression (1, 3, 6, 24 and 48 h); 3) dynamic changes in estrogen receptor β (ERβ) mRNA and protein expression (1, 3, 6, 24 and 48 h); and 4) induction of apoptosis in porcine granulosa cells derived from medium follicles (3, 6 and 24 h). TCDD had no effect on P4 or E2 production, but potentiated the inhibitory effect of genistein on P4 production. In contrast to the individual treatments which did not produce any effects, TCDD and genistein administered together decreased ERβ and AhR protein expression in granulosa cells. Moreover, the inhibitory effect of TCDD on AhR mRNA expression was abolished by genistein. The treatments did not induce apoptosis in the cells. In summary, combined effects of low concentrations of TCDD and genistein on follicular function of pigs differed from that of individual compounds. The results presented in the current paper clearly indicate that effects exerted by low doses of EDCs applied in combination must be taken into consideration when studying potential risk effects of EDCs on biological processes.

Belamcandae chinensis rhizoma, is a rhizome of Iris domestica (syn. Belamcanda chinensis). Under the Chinese name she gan, it is extensively used in Traditional Chinese Medicine and other East Asian phytotherapy systems. Recently, the monograph of Belamcandae chinensis rhizoma has been included in the European Pharmacopeia. This review provides a comprehensive summary and systematizes the literature data on ethnobotanical uses, chemical constituents and biological effects of Belamcandae chinensis rhizoma and its components. The main group of phytochemicals identified in the dried rhizoma are polyphenols such as isoflavones, xanthone glycosides, stilbenes, simple phenols and quinones. Another characteristic class of substances are triterpenopid iridals. The most typical traditional usage of Belamcandae chinensis rhizoma is for healing respiratory diseases but most of pharmacological research so far has been focused on isoflavones and their estrogenic properties. In pharmacological research, it has been mainly considered as a source of tectorigenin--a phytoestrogene with therapeutic potential in hormone-dependent cancer. The most active isoflavones are tectoridin, tectorigenin and irigenin. The available literature indicates that Belamcandae chinensis rhizoma can prevent excessive oxidation of biomolecules based on various antioxidant mechanisms: transition metal ions reduction, inhibition of lipid peroxidation, free radicals scavenging. The other biological activities proven by a number of in vitro studies include: antimutagenic,anti-inflammatory, anti-angiogenic, hypoglycemic. In conclusion, the knowledge about Belamcandae chinensis rhizoma has been growing rapidly in the recent years,but there are still significant gaps in our understanding of its bioactivity, therapeutic value, and roles played by each of the numerous phytochemicals.

An enantioselective synthesis of the indole diterpenoid natural product paspaline is disclosed. Critical to this approach was the implementation of stereoselective desymmetrization reactions to assemble key stereocenters of the molecule. The design and execution of these tactics are described in detail, and a thorough analysis of observed outcomes is presented, ultimately providing the title compound in high stereopurity. This synthesis provides a novel template for preparing key stereocenters in this family of molecules, and the reactions developed en route to paspaline present a series of new synthetic disconnections in preparing steroidal natural products.

During pregnancy, human exposure to endogenous estrogens and xenoestrogens (such as lignans) may comprehensively impact the gestational maintenance and fetal growth. We measured the concentrations of 5 lignans and the profile of 13 estrogen metabolites (EMs) in the urine samples of 328 pregnant women and examined their associations with birth outcomes. We found significantly positive associations between gestational age and urinary matairesinol (MAT), enterodiol (END) and enterolactone (ENL), as well as 16-hydroxylation pathway EMs. There were consistently positive relationships between END and the 16-hydroxylation pathway EMs. The positive relationships of MAT, END and ENL exposures with the length of gestation were mainly in the low exposure strata of the levels of these EMs. This study reveals that MAT, END and ENL as well as 16-hydroxylation pathway EMs are associated with birth outcomes, and that there are interactive relationships between lignans and 16-hydroxylation pathway EMs with birth outcomes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Ethnopharmacological Relevance: Sambucus williamsii Hance (SWH), which belongs to the Caprifoliaceae family distributed in various regions of China, Korea and Japan, has been used as a folk medicine for treatment of bone and joint diseases in China for thousands of years. In previous studies, SWH was shown to possess anti-osteoporosis, healing fracture, anti-inflammatory and analgesic activities. Our previous studies showed that SWH extract effectively suppressed ovariectomy-induced increase in bone turnover and improved bone mineral density and bone biomechanical strength in rats as well as in mice. An 8-O-4' norlignan, (7R,8S)-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-propanediol (PPD) was previously isolated and identified as the bioactive ingredient in SWH. The present study aimed to characterize the bone protective effects as well as its mechanism of actions in osteoblasts., Materials and Methods: Bone protective actions of PPD on different cells were determined by proliferation assay, alkaline phosphatase (ALP) activity assay, calcium deposition as well as real-time reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, estrogen receptor (ER) antagonist ICI182,780 and mitogen-activated protein kinase kinase (MEK) inhibitor U0126 blocking assays, competitive ER radioligand binding assay, ERE-dependent luciferase reporter assay and immunoblotting were used to determine if PPD activated ER and if the effects of PPD on osteoblastic functions were ER dependent., Results: PPD exerted anabolic effects in osteoblasts and its effects were abolished by co-incubation with ICI182,780 or U0126. PPD induced mRNA expressions of Runx2, ALP, osteocalcin, and increased the ratio of osteoprotegerin/receptor activator of nuclear factor κB (OPG/RANKL). PPD failed to bind to either ERα or ERβ and did not activate ERE-luciferase activity via ER. PPD induced the phosphorylation of extracellular regulated kinases (ERK) and its effect was completely abolished by U0126. It also induced the phosphorylation of ERα at serine 118., Conclusion: These data show that PPD is a bioactive compound in SWH that exerts oestrogen-like actions in osteoblast-like cells via ligand-independent, estrogen response element (ERE)-independent and mitogen-activated protein (MAP) Kinase-mediated rapid nongenomic ER signaling pathway., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Phytoestrogens are plant-derived hormonally-active compounds known to cause varied reproductive, immunosuppressive and behavioral effects in vertebrates. In this study, biodegradation of luteolin, a common phytoestrogen, was investigated during incubation with endophytic fungus Phomopsis liquidambari. The optimum concentration of luteolin as sole carbon source supplied in culture was 200 mg L(-1), which allowed 97 and 99 % degradation of luteolin by P. liquidambari in liquid culture and soil conditions, respectively. The investigation of the fungal metabolic pathway showed that luteolin was first decomposed to caffeic acid and phloroglucinol. These intermediate products were degraded to protocatechuic acid and hydroxyquinol, respectively, and then rings were opened by ring-cleavage dioxygenases. Two novel genes encoding the protocatechuate 3,4-dioxygenase and hydroxyquinol 1,2-dioxygenase were successfully cloned. Reverse-transcription quantitative polymerase chain reaction demonstrated that expression levels of mRNA of these two genes increased significantly after P. liquidambari was induced by the intermediate products caffeic acid and phloroglucinol, respectively. These results revealed that P. liquidambari can biodegrade luteolin efficiently and could potentially be used to bioremediate phytoestrogen contamination.

Phytoestrogens have structures similar to endogenous steroids and may induce or inhibit the response of hormone receptors. The objectives of the present study were to compare the effects of long-term vs. short-term grassland management in organic and conventional dairy production systems, compare organic and conventional production systems and assess seasonal variation on phytoestrogen concentrations in bulk-tank milk. The concentrations of phytoestrogens were analyzed in bulk-tank milk sampled three times in two subsequent years from 28 dairy farms: Fourteen organic (ORG) dairy farms with either short-term or long-term grassland management were paired with 14 conventional (CON) farms with respect to grassland management. Grassland management varied in terms of time since establishment. Short-term grassland management (SG) was defined as establishment or reseeding every fourth year or more often, and long-term grassland management (LG) was defined as less frequent establishment or reseeding. The proportion of red clover (Trifolium pretense L.) in the herbage was positively correlated with milk concentrations of the mammalian isoflavone equol. Therefore, organically produced bulk-tank milk contained more equol than conventionally produced milk, and milk from ORG-SG farms had more equol than milk from ORG-LG farms. Milk produced during the indoor-feeding periods had more equol than milk produced during the outdoor feeding period, because pastures contained less red clover than fields intended for silage production. Organically produced milk had also higher concentrations of the mammalian lignan enterolactone, but in contrast to equol, concentrations increased in the outdoor-feeding periods compared to the indoor-feeding periods. There were no indications of fertility problems on ORG-SG farms who had the highest red clover proportions in the herbage. This study shows that production system, grassland management, and season affect milk concentrations of phytoestrogens. However, compared to soy products, milk concentrations of phytoestrogens are low and future studies are required to investigate if the intake of phytoestrogens from dairy products has physiological effects in humans.

Legumes are an excellent source of macronutrients and phytochemicals as isoflavones. The aim of this work was to develop a new analytical method for determining five isoflavone compounds, three of which are aglycons, namely daidzein, genistein, biochanin A, and two of which, daidzin and genistin, are glycosilated, in lentils and other pulses, using an effective clean-up system and UHPLC-MS/MS (triple quadrupole) method. The recoveries obtained by spiking the lentil samples with a standard mixture of isoflavones at three levels of fortification (5, 25 and 100 μg kg(-1)) were in the range of 54.4-111.1%, 68.6-91.1%, and 84.4-114%, respectively. The method was applied to analyse 48 lentil samples from central Italy and pulses for determining the isoflavone content, which was found to range from 1.1 to 95.6 μg kg(-1)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Activation of estrogen receptor-β (ERβ) is an important mechanism for colon cancer prevention. Specific sorghum varieties that contain flavones were shown to activate ER in non-malignant colonocytes at low concentrations. This study aimed to determine positive interactions among estrogenic flavonoids most relevant in sorghum. Apigenin and naringenin were tested separately and in combination for their ability to influence ER-mediated cell growth in non-malignant young adult mouse colonocytes (YAMC). Sorghum extracts high in specific flavanones and flavones were also tested. Apigenin reduced ER-mediated YAMC cell growth comparable to physiological levels of estradiol (E₂, 1 nM) at 1 μM; naringenin had similar effect at 10 μM. However, when combined, 0.1 μM apigenin plus 0.05 μM naringenin produced similar effect as 1 nM E₂; these concentrations represented 1/10th and 1/200th, respectively, of the active concentrations of apigenin and naringenin, demonstrating a strong enhanced action. A sorghum extract higher in flavones (apigenin and luteolin) (4.8 mg g(-1)) was more effective (5 μg mL(-1)) at activating ER in YAMC than a higher flavanone (naringenin and eriodictyol) (28.1 mg g(-1)) sorghum extract (10 μg mL(-1)). Enhanced actions observed for apigenin and naringenin were adequate to explain the level of effects produced by the high flavone and flavanone sorghum extracts. Strong positive interactions among sorghum flavonoids may enhance their ability to contribute to colon cancer prevention beyond what can be modeled using target compounds in isolation.

Consumers nowadays are playing an active role in their health-care. A special case is the increasing number of women, who are reluctant to use exogenous hormone therapy for the treatment of menopausal symptoms and are looking for complementary therapies. However, food supplements are not clearly regulated in Europe. The EFSA has only recently begun to address the issues of botanical safety and purity regulation, leading to a variability of content, standardization, dosage, and purity of available products. In this study, isoflavones (puerarin, daidzin, genistin, daidzein, glycitein, genistein, formononetin, prunetin, and biochanin A) from food supplements (n = 15) for menopausal symptoms relief are evaluated and compared with the labelled information. Only four supplements complied with the recommendations made by the EC on the tolerable thresholds. The intestinal bioavailability of these compounds was investigated using Caco-2 cells. The apparent permeability coefficients of the selected isoflavonoids across the Caco-2 cells were affected by the isoflavone concentration and product matrix.

Use of soy supplements to inhibit cancer cell growth is increasing among patients due to the perception that phytoestrogens in soy inhibit carcinogenesis via induction of apoptosis. Genistein, the most prevalent phytoestrogen in soy, is a potent endocrine disruptor and tyrosine kinase inhibitor (TKI) that causes apoptosis in many cells types. Chemotherapeutic TKIs limit cancer cell growth via the same mechanisms. However, TKIs such as Sunitinib cause cardiotoxicity in a significant number of patients. Molecular interactions between Sunitinib and dietary TKIs like genistein have not been examined in cardiomyocytes. Significant lethality occurred in mice treated with Sunitinib and fed a phytoestrogen-supplemented diet. Isolated cardiomyocytes co-treated with genistein and Sunitinib exhibited additive inhibition of signaling molecules important for normal cardiac function and increased apoptosis compared with Sunitinib alone. Thus, dietary soy supplementation should be avoided during administration of Sunitinib due to exacerbated cardiotoxicity, despite evidence for positive effects in cancer., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Phytoestrogens, which can bind with estrogen receptor and produce estrogen-like effects, are a kind of nonsteroidal compound in plant. Phytoestrogens chemically include isoflavones, coumarins, lignans and other compounds. Phytoestrogens are selective estrogen receptor modulator, and have therapeutical effects on breast cancer, prostate cancer, cardiovascular disease, menopausal symptoms, osteoporosis and other disease, however, do not produce stimulatory hyperplasia effects on uterus, mammary glands and other tissues and organs with positive estrogen receptor. Long-term exposure or excessive use of phytoestrogens maybe affects male reproductive system and hematopoietic function of fetus. Some questions need to be further studied, such as evaluation criteria on biological activity, adverse effects, and action mechanism of phytoestrogen. This review covers plant sources, chemical structure, pharmacological activity and safety of phytoestrogens. It will provide a useful reference for intensive research and rational utilization the phytoestrogens.

Formononetin (1), a plant-derived phytoestrogen, possesses bone protective properties. To address the potential therapeutic efficacy and mechanism of action of 1, we investigated its antiosteoclastogenic activity and its effect on nuclear factor-kappaB ligand (RANKL)-induced bone-marrow-derived macrophages (BMMs). Compound 1 markedly inhibited RANKL-induced osteoclast differentiation in the absence of cytotoxicity, by regulating the expression of osteoprotegerin (OPG) and RANKL in BMMs and in cocultured osteoblasts. Compound 1 significantly inhibited RANKL-induced tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T cell expressed and secreted (RANTES), and macrophage inflammatory protein-1α (MIP-1α) in a concentration-dependent manner. These effects were accompanied by a decrease in RANKL-induced activation of the NF-κB p65 subunit, degradation of inhibitor κBα (IκBα), induction of NF-κB, and phosphorylation of AKT, extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK). NF-κB siRNA suppressed AKT, ERK, JNK, and p38 MAPK phosphorylation. Furthermore, 1 significantly suppressed c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), key transcription factors during osteoclastogenesis. SP600125, a specific inhibitor of JNK, reduced RANKL-induced expression of phospho-c-Jun, c-Fos, and NFATc1 and inhibited osteoclast formation. These results suggested that 1 acted as an antiresorption agent by blocking osteoclast activation.

Scope: Hops (Humulus lupulus L.) produce unique prenylflavonoids that exhibit interesting bioactivities. This study investigates the interactions between selected prenylflavonoids and breast cancer resistance protein (BCRP/ABCG2), an efflux transporter important for xenobiotic bioavailability and multidrug resistance (MDR)., Methods and Results: ABCG2-inhibitory activity of xanthohumol (XN), isoxanthohumol (IX), 6-prenylnaringenin (6-PN), 8-prenylnaringenin (8-PN), and 6,8-diprenylnarigenin (6,8-diPN) was evaluated using mitoxantrone accumulation and vesicular transport assays. XN, IX, and 8-PN were tested for a substrate-type relationship with ABCG2 using ATPase and bidirectional transport assays. The prenylflavonoids exhibited significant ABCG2-inhibitory activities in mitoxantrone accumulation and vesicular transport assays. In the ATPase assay, XN, IX, and 8-PN inhibited baseline and sulfasalazine-stimulated ATPase activities with IC50 of 2.16-27.0 μM. IX and 8-PNalso displayed bell-shaped activation curves in Ko143-suppressed membranes, indicating a substrate-type relationship. For IX, efflux ratios of 1.25 ± 0.21 and 9.18 ± 0.56 were observed in wild type and ABCG2-overexpressing MDCKII cell monolayers, respectively. The latter was reduced to 1.25 ± 0.15 in the presence of the ABCG2-specific inhibitor Ko143, demonstrating an ABCG2-mediated efflux of IX. Additionally, evidence was shown for the involvement of ABCG2 in the efflux of 8-PN and/or its sulfate conjugate., Conclusion: Prenylflavonoids are potent inhibitors of ABCG2 and therefore implicated in ABCG2-mediated food/herb-drug interactions and MDR. ABCG2-mediated efflux of prenylflavonoids may represent one mechanism that regulates prenylflavonoid bioavailability., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aim of this study was to determine the flavonoid profile of Lupinus mexicanus germinated seed extract (PE) and to evaluate its effect as a phytoestrogen on the morphometric parameters of CA3 hippocampal neurons of ovariectomized rats (OVX). L. mexicanus seeds, germinated for 48 h, were homogenized and macerated using an 80% ethanol solution. The extract was analyzed by HPLC/MS-MS. Thirty young Wistar strain female rats (200±10 g) were randomly distributed into four groups: sham operated (S) treated with dimethyl sulfoxide (vehicle); ovariectomized and treated with 1250 μg of PE extract (OVX-PE); ovariectomized and treated with 5 μg estradiol benzoate (OVX-EB); and ovariectomized and vehicle treated (OVX). All substances were injected subcutaneously daily for 28 days. On day 29, the animals were sacrificed, perfused, and fixed to obtain the brains for histological processing. Each brain was cut and stained with hematoxylin and eosin. The thickness of the stratum oriens (SO), the nuclear diameter, and the neuronal density were measured in the hippocampus CA3 area. Nine different flavonoids and one non-identified compound were detected. The histological analysis demonstrated that the thickness of the SO was higher in the OVX-EB and S groups than in the OVX-PE and OVX groups (p⟨0.05); in addition, the nuclear diameters of the neurons in the OVX-EB and S groups were higher compared with the other groups (p⟨0.05). The OVX group had the highest cellular density among groups (p⟨0.05). Based on our results, the PE obtained did not have beneficial effects on CA3 hippocampal neurons.