phytoestrogens and formononetin

Phytoestrogens are naturally occurring nonsteroidal phenolic plant compounds that, due to their molecular structure and size, resemble vertebrate steroids estrogens. This review is focused on plant flavonoids isoflavones, which are ranked among the most estrogenic compounds. The main dietary sources of isoflavones for humans are soybean and soybean products, which contain mainly daidzein and genistein. When they are consumed, they exert estrogenic and/or antiestrogenic effects. Isoflavones are considered chemoprotective and can be used as an alternative therapy for a wide range of hormonal disorders, including several cancer types, namely breast cancer and prostate cancer, cardiovascular diseases, osteoporosis, or menopausal symptoms. On the other hand, isoflavones may also be considered endocrine disruptors with possible negative influences on the state of health in a certain part of the population or on the environment. This review deals with isoflavone classification, structure, and occurrence, with their metabolism, biological, and health effects in humans and animals, and with their utilization and potential risks.

Topics: Animals; Equol; Genistein; Humans; Isoflavones; Phytoestrogens

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.. 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.. 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.

Topics: Animals; Biological Products; Biopharmaceutics; Fabaceae; Genistein; Humans; Isoflavones; Phytoestrogens; Plant Leaves; Plant Preparations; Trifolium; Vegetables

This study was undertaken to address the vascular effects of isolated isoflavones as potential contributors to their cardioprotective properties, focusing on biochanin and formononetin.. In a randomized, double-blind trial, 80 healthy subjects, 46 men and 34 women, 45 to 75 years of age, received isoflavones enriched in either biochanin or formononetin (precursors of genistein and daidzein; 80 mg/d) crossed over randomly with placebo in two 6-week periods. The end points were measured at baseline and after each intervention and included large artery stiffness (systemic arterial compliance and pulse wave velocity), endothelial function in conduit arteries (flow-mediated vasodilation), 24-hour ambulatory blood pressure, and total peripheral resistance. Isoflavone intervention significantly reduced arterial stiffness with improved systemic arterial compliance (P=0.04; repeated-measures ANOVA, Bonferroni correction) attributable to a reduction in total peripheral resistance (P=0.03) and a corresponding reduction in central pulse wave velocity (P=0.02) compared with placebo. Isoflavones did not affect blood pressure (P=0.5) or flow-mediated vasodilation (P=0.44). Improvements seemed limited to formononetin-enriched isoflavones (adjusted P=0.06). Formononetin treatment also reduced circulating vascular adhesion cell molecule-1 (P<0.01).. In normotensive men and postmenopausal women, red clover isoflavones enriched in formononetin reduced arterial stiffness and total vascular resistance but had no effect on blood pressure. These effects may partly explain the lower cardiovascular risk in populations eating isoflavone-rich diets.

Topics: Aged; Arm; Blood Pressure; Cross-Over Studies; Diet; Double-Blind Method; Endothelium, Vascular; Female; Genistein; Humans; Isoflavones; Male; Middle Aged; Phytoestrogens; Plant Preparations; Postmenopause; Reference Values; Regional Blood Flow; Trifolium; Vascular Cell Adhesion Molecule-1; Vascular Resistance; Vasodilation

The antioxidant and anti-inflammatory features of Formononetin, an isoflavone constituent extracted from traditional Chinese medicine, have been reported. The present study investigated that whether Formononetin plays a benefit on hyperoxic ALI.. C57BL/6 mice were exposed to hyperoxia for 72 h to produce experimental hyperoxic ALI model. Formononetin or vehicle was administrated intraperitoneally. Samples from the lung were collected at 72 h post hyperoxia exposure for further study. Pulmonary microvascular endothelial cells isolated from the lung of C57BL/6 mice were used for in vitro study.. Formononetin pretreatment notably attenuated hyperoxia-induced elevating pulmonary water content, upregulation of proinflammatory cytokine levels and increasing infiltration of neutrophil in the lung. Western blot analyses showed that Formononetin enhanced the expression of nuclear factor erythroid-2-related factor 2 (Nrf2) which is a key transcription factor regulating the expression of heme oxygenase-1 (HO-1). Formononetin increased HO-1 expression and activity compared with vehicle-treated animals. Moreover, Formononetin reversed hyperoxia-caused the reduction of M2 macrophage polarization. However, pretreatment of a HO-1 inhibitor reduced the protective effect of Formononetin on hyperoxic ALI. Cell study showed that the Formononetin-induced upregulation of HO-1 was abolished when the Nrf2 was silenced.. Formononetin pretreatment reduces hyperoxia-induced ALI via Nrf2/HO-1-mediated antioxidant and anti-inflammatory effects.

Topics: Acute Lung Injury; Animals; Disease Models, Animal; Endothelial Cells; Heme Oxygenase-1; Hyperoxia; Isoflavones; Lung; Membrane Proteins; Mice; NF-E2-Related Factor 2; Phytoestrogens

Formononetin is a natural isoflavone compound found mainly in Chinese herbal medicines such as astragalus and red clover. It is considered to be a typical phytooestrogen. In our previous experiments, it was found that formononetin has a two-way regulatory effect on endothelial cells (ECs): low concentrations promote the proliferation of ECs and high concentrations have an inhibitory effect. To find a specific mechanism of action and provide a better clinical effect, we performed a structural transformation of formononetin and selected better medicinal properties for formononetin modifier J1 and J2 from a variety of modified constructs. The MTT assay measured the effects of drugs on human umbilical vein endothelial cell (HUVEC) activity. Scratch and transwell experiments validated the effects of the drugs on HUVEC migration and invasion. An in vivo assessment effect of the drugs on ovariectomized rats. Long-chain non-coding RNA for EWSAT1, which is abnormally highly expressed in HUVEC, was screened by gene chip, and the effect of the drug on its expression was detected by PCR after the drug was applied. The downstream factors and their pathways were analysed, and the changes in the protein levels after drug treatment were evaluated by Western blot. In conclusion, the mechanism of action of formononetin, J1 and J2 on ECs may be through EWSAT1-TRAF6 and its downstream pathways.

Topics: Animals; Apoptosis; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Isoflavones; Phytoestrogens; Rats; Rats, Sprague-Dawley; RNA-Binding Protein EWS; TNF Receptor-Associated Factor 6

Epidermal growth factor receptor (EGFR) activating mutations play crucial roles in the tumorigenesis of human non-small cell lung cancer (NSCLC). The mechanism regarding how EGFR signaling regulates myeloid cell leukemia sequence 1 (Mcl-1) protein stability and ubiquitination remains undefined.. MTS assay was used for natural product library screening. The effect of formononetin (Formo) on NSCLC cells was determined by MTS assay and soft agar assay. Molecular modeling was performed to analyze the potential different binding modes between Formo and EGFR WT or mutants. Mcl-1 protein level and the inhibitory effect of Formo on EGFR signaling were examined by immunoblot, in vitro kinase assay, in vitro pulldown and ATP competition assays, co-immunoprecipitation assay, ubiquitination analysis, in vivo xenograft model, and immunohistochemical staining.. Formo was identified as an EGFR inhibitor by a 98 commercially available natural product screening. Formo suppresses WT and mutant EGFR kinases activity in vitro, ex vivo, and in vivo. Molecular modeling indicates that Formo docks into the ATP-binding pocket of both WT and mutant EGFR. Formo inhibits EGFR-Akt signaling, which in turn activates GSK3β and promotes Mcl-1 phosphorylation in NSCLC cells. Treatment with Formo enhances the interaction between Mcl-1 and SCF. This study highlights the importance of promoting ubiquitination-dependent Mcl-1 turnover might be an alternative strategy to enhance the anti-tumor efficacy of EGFR-TKI.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; ErbB Receptors; Female; Humans; Isoflavones; Lung Neoplasms; Mice; Mice, Nude; Models, Molecular; Myeloid Cell Leukemia Sequence 1 Protein; Phytoestrogens

Many breast cancer patients suffer from obvious side effects induced by chemotherapy. Formononetin (FM), one kind ingredient of Chinese herbal medicine, has been suggested to inhibit MCF-7 breast cancer cells. And recently metformin (MET) has gained more attention as a potential anti-cancer drug. The aim of this study was to investigate the synergistic effects of FM and MET on the proliferation of MCF-7 cells and to clarify the possible molecular mechanism involved. MCF-7 cells were treated with various concentrations of FM (40 and 80 μM) or FM (40 and 80 μM) combined with MET (150 μM) for 48 h. Cell proliferation was tested by an methyl tetrazolium (MTT) (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay. The percentage of apoptotic cells was measured by flow cytometry. The expression level of b-cell lymphoma/leukemia-2 (bcl-2) mRNA was examined by RT-PCR, while the expression levels of phosphorylated extracellular signal-regulated kinases (p-ERK1/2) and bcl-2 protein were detected by Western blotting. Compared with untreated cells, 40 μM and 80 μM FM efficiently inhibited proliferation and increased apoptosis in MCF-7 cells. Additionally, 40 μM and 80 μM FM greatly downregulated bcl-2 mRNA expression when compared with untreated cells. Furthermore, the protein expression of bcl-2 and p-ERK1/2 was significantly reduced by 40 μM and 80 μM FM. The cytotoxic effect of FM was more remarkable when 150 μM MET was added. Taken together, the combinational use of FM and MET enhanced cell growth inhibition, and the induction of apoptosis in MCF-7 cells mediated by the ERK1/2 signaling pathway.

Topics: Apoptosis; Breast Neoplasms; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; Female; Growth Inhibitors; Humans; Hypoglycemic Agents; Isoflavones; MCF-7 Cells; Metformin; Phytoestrogens

Formononetin is one of the main active components of traditional Chinese medicine red clover (Trifolium pratense L). Prior studies have demonstrated that formononetin is a typical phytoestrogen and exhibits an estrogen-like effect that protects the cardiovascular system with minimal side effects. In this study, we further investigated the role of formononetin in human umbilical vein endothelial cells (HUVECs) and its potential molecular mechanisms. We founded that formononetin promotes proliferation and migration of HUVECs as assessed by the MTT and wound healing assays. Meanwhile, insulin-like growth factor 1 (IGF-1) and intercellular adhesion molecule 1 (ICAM-1) protein levels were increased in a dose-dependent manner. Our findings illustrated that formononetin exhibits a protective effect on HUVECs, and the molecular mechanisms may correlate with IGF-1R and ICAM-1, as well as upregulation of vascular endothelial growth factor (VEGF) and activation of extracellular signal-regulated kinase (ERK).

Topics: Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Human Umbilical Vein Endothelial Cells; Humans; Isoflavones; Phytoestrogens; Trifolium

In the present study, we demonstrated the anti-catabolic effects of formononetin, a phytoestrogen derived from herbal plants, against interleukin-1β (IL-1β)-induced severe catabolic effects in primary rat chondrocytes and articular cartilage. Formononetin did not affect the viability of primary rat chondrocytes in both short- (24 h) and long-term (21 days) treatment periods. Furthermore, formononetin effectively antagonized the IL-1β-induced catabolic effects including the decrease in proteoglycan content, suppression of pericellular matrix formation, and loss of proteoglycan through the decreased expression of cartilage-degrading enzymes like matrix metalloproteinase (MMP)-13, MMP-1, and MMP-3 in primary rat chondrocytes. Moreover, catabolic oxidative stress mediators like nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, and prostaglandin E

Topics: Animals; Cartilage, Articular; Cells, Cultured; Chondrocytes; Drug Antagonism; Inflammation; Interleukin-1beta; Isoflavones; Metabolism; Osteoarthritis; Phytoestrogens; Rats

Formononetin (FN), a typical phytoestrogen has attracted substantial attention as a novel agent because of its diverse biological activities including, osteogenic differentiation. However, the molecular mechanisms underlying osteogenic and myogenic differentiation by FN in C2C12 progenitor cells remain unknown. Therefore the objective of the current study was to investigate the action of FN on myogenic and osteogenic differentiation and its impact on signaling pathways in C2C12 cells. FN significantly increased myogenic markers such as Myogenin, myosin heavy chains, and myogenic differentiation 1 (MyoD). In addition, the expression of osteogenic specific genes alkaline phosphatase (ALP), Run-related transcription factor 2(RUNX2), and osteocalcin (OCN) were up-regulated by FN treatment. Moreover, FN enhanced the ALP level, calcium deposition and the expression of bone morphogenetic protein isoform (BMPs). Signal transduction pathways mediated by p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-related kinases (ERKs), protein kinase B (Akt), Janus kinases (JAKs), and signal transducer activator of transcription proteins (STATs) in myogenic and osteogenic differentiation after FN treatment were also examined. FN treatment activates myogenic differentiation by increasing p38MAPK and decreasing JAK1-STAT1 phosphorylation levels, while osteogenic induction was enhanced by p38MAPK dependent Smad, 1/5/8 signaling pathways in C2C12 progenitor cells.

Topics: Animals; Cell Differentiation; Cell Survival; Dose-Response Relationship, Drug; Isoflavones; Janus Kinase 1; Mice; Muscle Development; Osteogenesis; p38 Mitogen-Activated Protein Kinases; Phytoestrogens; Signal Transduction; Smad1 Protein; Smad5 Protein; Smad8 Protein; STAT1 Transcription Factor; Stem Cells

Isoflavones have an affinity for estrogen receptors (ERs) including beneficial affinity for ERβ. Widely used soy is a source of poorly absorbed isoflavones glycosides. Red clover contains mostly easily absorbed free aglycones. Red clover sprouts were cultivated under different conditions (white light, UVA or UVB for 12 or 24 h a day at 18 or 25 °C) to maximise the content of isoflavones, especially of high affinity for ERβ. The affinity of isoflavones to ERs was evaluated by molecular modelling and isothermal titration calorimetry (ITC). The richest source of isoflavones, especially formononetin were sprouts cultivated for 10 days under continuous white light and at 25 °C (562 mg/100 g of fresh mass). Formononetin and the above sprouts has been shown to have a high affinity for ERβ. Red clover sprouts can be considered as a source of phytoestrogens with high biological activity and as a dietary supplement reducing menopausal symptoms.

Topics: Calorimetry; Dietary Supplements; Isoflavones; Models, Molecular; Phytoestrogens; Temperature; Trifolium

Phyto-oestrogens such as isoflavones are natural compounds that can profoundly affect reproductive function. In the present study, we tested whether including isoflavone compounds (genistein, biochanin A, formononetin) in the maturation medium would affect the outcomes for ovine oocytes in vitro. Each isoflavone compound was evaluated at five concentrations (0, 2.5, 5, 10, 25µgmL-1) and the entire protocol was repeated four times. Cumulus-oocyte complexes were randomly allocated to the treatments, then fertilised and cultured in vitro. Compared with control (0µgmL-1), the lower concentrations of isoflavone (2.5, 5 and 10µgmL-1) had no detectable effect on the rates of cleavage or embryo development, or on embryo total cell counts (TCC). However, the highest concentration (25µgmL-1) of all three isoflavones exerted a variety of effects (P<0.05): genistein decreased cleavage rate, blastocyst rate and blastocyst efficiency (blastocysts produced per 100 oocytes); biochanin A decreased cleavage rate and blastocyst efficiency; and formononetin decreased blastocyst rate and blastocyst efficiency. Biochanin A (25µgmL-1) reduced embryo TCC specifically at the hatched blastocyst stage (P<0.05). We conclude that the presence of isoflavones at 25µgmL-1 during IVM decreases the cleavage rate and inhibits blastocyst hatching.

Topics: Animals; Cumulus Cells; Dose-Response Relationship, Drug; Embryonic Development; Female; Fertilization; Genistein; In Vitro Oocyte Maturation Techniques; Isoflavones; Oocytes; Phytoestrogens; Sheep

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.

Topics: Alendronate; Animals; Bone Density Conservation Agents; Calcium-Binding Proteins; Cicer; Disease Models, Animal; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression Regulation; Genistein; Humans; Isoflavones; Membrane Glycoproteins; Molecular Docking Simulation; Osteogenesis; Osteoporosis; Osteoprotegerin; Ovariectomy; Phytoestrogens; Phytotherapy; Protein Structure, Secondary; RANK Ligand; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Peptide

Radix Astragali, has long been used to alleviate allergic diseases (ADs). Formononetin is one of the major active components in Radix Astragali, but its mechanism on ADs is not definitively known. The fluorescein isothiocyanate isomer-induced atopic contact dermatitis mouse model and poly I:C or lipopolysaccharide-treated HaCaT cells were used to examine thymic stromal lymphopoietin (TSLP)/interleukin (IL)-33 production and expression of E-cadherin. After administration of formononetin, TSLP/IL-33 levels decreased both in vitro and in vivo, while E-cadherin was increased in vivo and restored in vitro. Furthermore, small interference RNA silencing of E-cadherin resulted in elevated levels of TSLP, whereas the inhibitory effect of formononetin on TSLP was no longer observed. In addition, TSLP resulted in no detectable changes in delocalization or protein expression of E-cadherin in HaCaT cells. These results indicated that formononetin showed a protective effect in ADs, which was correlated with decreasing TSLP/IL-33 production via regulation of E-cadherin.

Topics: Animals; Astragalus propinquus; Cadherins; Cells, Cultured; Cytokines; Dermatitis, Allergic Contact; Disease Models, Animal; Drugs, Chinese Herbal; Epithelial Cells; Humans; Interleukin-33; Isoflavones; Mice; Mice, Inbred BALB C; Phytoestrogens; RNA, Small Interfering; Thymic Stromal Lymphopoietin

Isoflavones are a class of polyphonic compounds present in legumes and are called phytoestrogens because of their estrogen-like activity. Estrogen influences the behavior of mammary epithelial cells (MECs) during pregnancy and lactation. In this study, we investigated the direct influences of isoflavones and their metabolites in milk production ability of MECs.. Mouse MECs were cultured with prolactin and dexamethasone (glucocorticoid analog) to induce milk production ability. Subsequently, lactating MECs were treated with each isoflavone. Coumestrol, biochanin A, genistein, and formononetin decreased the intracellular and secreted β-casein. On the other hand, p-ethylphenol, daidzein, and equol did not significantly influence β-casein production at any concentration. Coumestrol, biochanin A and genistein down-regulated the mRNA expression of whey acidic protein (WAP), lactoferrin and α-lactalbumin. In contrast, p-ethylphenol, daidzein and equol up-regulated β-casein and/or WAP with α-lactalbumin. Furthermore, coumestrol and genistein down-regulated the expression of prolactin receptor and signal transducer and activator of transcription 5 (STAT5) accompanied by a decrease in STAT5 phosphorylation.. Isoflavones and their metabolites influence the milk production ability of MECs through different interactions with prolactin/STAT5 signaling. Simultaneous intake of multiple isoflavones by consumption of legumes may induce promotive or adverse effects on lactating MECs.

Topics: Animals; Caseins; Cells, Cultured; Coumestrol; Epithelial Cells; Female; Genistein; Isoflavones; Mammary Glands, Animal; Mice; Mice, Inbred ICR; Milk; Phenols; Phosphorylation; Phytoestrogens; Prolactin; STAT5 Transcription Factor

The bone regeneration and healing effect of formononetin was evaluated in a cortical bone defect model that predominantly heals by intramembranous ossification. For this study, female Balb/c mice were ovariectomised (OVx) and a drill-hole injury was generated in the midfemoral bones of all animals. Treatment with formononetin commenced the day after and continued for 21 d. Parathyroid hormone (PTH1-34) was used as a reference standard. Animals were killed at days 10 and 21. Femur bones were collected at the injury site for histomorphometry studies using microcomputed tomography (μCT) and confocal microscopy. RNA and protein were harvested from the region surrounding the drill-hole injury. For immunohistochemistry, 5 µm sections of decalcified femur bone adjoining the drill-hole site were cut. μCT analysis showed that formononetin promoted bone healing at days 10 and 21 and the healing effect observed was significantly better than in Ovx mice and equal to PTH treatment in many aspects. Formononetin also significantly enhanced bone regeneration as assessed by calcein-labelling studies. In addition, formononetin enhanced the expression of osteogenic markers at the injury site in a manner similar to PTH. Formononetin treatment also led to predominant runt-related transcription factor 2 and osteocalcin localisation at the injury site. These results support the potential of formononetin to be a bone-healing agent and are suggestive of its promising role in the fracture-repair process.

Topics: Animals; Bone Regeneration; Core Binding Factor Alpha 1 Subunit; Cortical Bone; Disease Models, Animal; Fabaceae; Femur; Fractures, Bone; Isoflavones; Mice, Inbred BALB C; Osteocalcin; Osteogenesis; Ovariectomy; Parathyroid Hormone; Phytoestrogens; Phytotherapy; Plant Extracts; Wound Healing

Compounds with estrogenic potencies and their adverse effects in surface waters have received much attention. Both anthropogenic and natural compounds contribute to overall estrogenic activity in freshwaters. Recently, estrogenic potencies were also found to be associated with cyanobacteria and their blooms in surface waters. The present study developed and compared the solid phase extraction and LC-MS/MS analytical approaches for determination of phytoestrogens (8 flavonoids - biochanin A, coumestrol, daidzein, equol, formononetin, genistein, naringenin, apigenin - and 5 sterols - ergosterol, β-sitosterol, stigmasterol, campesterol, brassicasterol) and cholesterol in water. The method was used for analyses of samples collected in stagnant water bodies dominated by different cyanobacterial species. Concentrations of individual flavonoids ranged from below the limit of detection to 3.58 ng/L. Sterols were present in higher amounts up to 2.25 μg/L. Biological potencies of these phytoestrogens in vitro were characterized using the hERα-HeLa-9903 cell line. The relative estrogenic potencies (compared to model estrogen - 17β-estradiol) of flavonoids ranged from 2.25E-05 to 1.26E-03 with coumestrol being the most potent. None of the sterols elicited estrogenic response in the used bioassay. Estrogenic activity was detected in collected field water samples (maximum effect corresponding to 2.07 ng/L of 17β-estradiol equivalents, transcriptional assay). At maximum phytoestrogens accounted for only 1.56 pg/L of 17β-estradiol equivalents, contributing maximally 8.5% of the total estrogenicity of the water samples. Other compounds therefore, most likely of anthropogenic origin such as steroid estrogens, are probably the major drivers of total estrogenic effects in these surface waters.

Topics: Cholestadienols; Cholesterol; Cyanobacteria; Estradiol; Estrogens; Estrone; Fresh Water; Genistein; HeLa Cells; Humans; Isoflavones; Phytoestrogens; Phytosterols; Receptors, Estrogen; Sitosterols; Sterols; Tandem Mass Spectrometry; Water; Water Pollutants, Chemical

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.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Astragalus propinquus; Breast Neoplasms; Cell Proliferation; Estrogen Receptor alpha; Female; Human Umbilical Vein Endothelial Cells; Humans; Isoflavones; MCF-7 Cells; Mice; MicroRNAs; Phytoestrogens; Signal Transduction

The amount of secondary metabolites in plants can be enhanced or reduced by various external factors. In this study, the effect of strontium ions on the production of phytoestrogens in soybeans was investigated. The plants were treated with Hoagland's solution, modified with Sr(2+) with concentrations ranging from 0.5 to 3.0 mM, and were grown for 14 days in hydroponic cultivation. After harvest, soybean plants were separated into roots and shoots, dried, and pulverized. The plant material was extracted with methanol and hydrolyzed. Phytoestrogens were quantified by HPLC. The significant increase in the concentration of the compounds of interest was observed for all tested concentrations of strontium ions when compared to control. Sr(2+) at a concentration of 2 mM was the strongest elicitor, and the amount of phytoestrogens in plant increased ca. 2.70, 1.92, 3.77 and 2.88-fold, for daidzein, coumestrol, genistein and formononetin, respectively. Moreover, no cytotoxic effects were observed in HepG2 liver cell models after treatment with extracts from 2 mM Sr(2+)-stressed soybean plants when compared to extracts from non-stressed plants. Our results indicate that the addition of strontium ions to the culture media may be used to functionalize soybean plants with enhanced phytoestrogen content.

Topics: Cations, Divalent; Cell Survival; Chromatography, High Pressure Liquid; Coumestrol; Genistein; Glycine max; Hep G2 Cells; Humans; Hydroponics; Isoflavones; Methanol; Phytoestrogens; Plant Extracts; Plant Roots; Plant Shoots; Solvents; Strontium

Epidemiological studies indicate lower prevalences of breast and prostate cancers and cardiovascular disease in Southeast Asia where vegetarianism is popular and diets are traditionally high in phytoestrogens. This study assessed plasma isoflavones in vegetarian and non-vegetarian Malaysian men according to age. Daidzein, genistein, equol (a daidzein metabolite), formononetin, biochanin A, estrone, estradiol and testosterone were measured by validated liquid chromatography tandem mass spectrometry (LCMSMS). Plasma isoflavone and sex hormone concentrations were measured in 225 subjects according to age (18-34, 35-44 and 45-67 years old). In all age groups, vegetarians had a higher concentration of circulating isoflavones compared with non-vegetarians especially in the 45-67 year age group where all isoflavones except equol, were significantly higher in vegetarians compared with omnivores. By contrast, the 18-34 year group had a significantly higher concentration of daidzein in vegetarians and significantly higher testosterone and estrone concentrations compared with non-vegetarians. In this age group there were weak correlations between estrone, estradiol and testosterone with some of the isoflavones. This human study provides the first Malaysian data for the phytoestrogen status of vegetarian and nonvegetarian men.. 流行病学研究表明东南亚乳腺癌、前列腺癌和心血管疾病的发病率较低，素食 在这些地区备受欢迎，传统饮食中富含植物雌激素。本研究根据年龄评估了马 来西亚素食和非素食男性血浆中异黄酮的含量。通过高效液相色谱-串联质谱 法（LCMSMS）测定血浆中大豆异黄酮、染料木素、雌马酚（一种大豆异黄 酮的代谢产物）、芒柄花素、鹰嘴豆芽素A、雌酮、雌二醇和睾酮的浓度。按 照年龄（18-34 岁、35-44 岁和45-67 岁）测定了225 名研究对象的血浆异黄酮 和性激素的浓度。在所有年龄组，尤其是45-67 岁组，素食者循环异黄酮浓度 高于非素食者，在45-67 岁组，除雌马酚外的所有异黄酮，素食者均显著高于 杂食者。相反，在18-34 岁组，素食者的大豆异黄酮、睾酮和雌酮的浓度显著 高于非素食者。在这个年龄组中，雌酮、雌二醇和睾酮与一些异黄酮之间有弱 的相关性。本研究提供了马来西亚素食和非素食男性植物雌激素状态的第一手 资料。.

Topics: Adolescent; Adult; Aged; Aging; Cross-Sectional Studies; Dairy Products; Diet, Vegetarian; Eggs; Equol; Estradiol; Estrone; Genistein; Humans; Isoflavones; Malaysia; Male; Middle Aged; Phytoestrogens; Testosterone

Recent studies have shown that immune system plays a major role in pathophysiology of postmenopausal osteoporosis. Previously we have shown that phytoestrogens like daidzein and medicarpin exhibit immunoprotective effects, by virtue of which they alleviate bone loss. With this background, methoxyisoflavones like formononetin (formo) and isoformononetin (isoformo) that have been studied for preventing bone loss in ovariectomized rats were tested for their immunomodulatory effects in estrogen-deficient bone loss mice model.. Adult Balb/c mice (N = 8/group) were given oral dose of formo and isoformo at 10 mg/kg body weight, post ovariectomy (Ovx) daily for 6 weeks. Animals were autopsied and long bones were harvested to study bone microarchitecture. Peripheral blood mononuclear cells were isolated for fluorescence-activated cell sorting and RNA analysis. Serum was collected for enzyme-linked immunosorbent assay.. It was observed that formo and isoformo treatment to Ovx mice led to significant restoration of Ovx-induced deterioration of trabecular microarchitecture. Pro-osteoclastogenic subset Th17 and B cells were decreased in formo/isoformo-treated Ovx mice in comparison with vehicle-treated Ovx group. Formo and isoformo treatment to Ovx mice also led to decreased expression of Th17 diffentiation factors and promoted T-regulatory cell differentiation. Formo was more effective in enhancing the FOXP3 expression compared with isoformo. IL-17A-induced osteoclastogenesis and inhibition of osteoblast apoptosis were also suppressed by formo and isoformo treatment, with formo having a more potent effect.. Our study demonstrates the immunomodulatory activity of methoxyisoflavones, formo, and isoformo, which translate into improved skeletal parameters, thereby preventing Ovx-induced bone loss.

Topics: Animals; Apoptosis; B-Lymphocytes; Cell Differentiation; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Humans; Isoflavones; Leukocytes, Mononuclear; Lymphopoiesis; Menopause; Mice; Mice, Inbred BALB C; Osteoblasts; Osteogenesis; Osteoporosis, Postmenopausal; Ovariectomy; Phytoestrogens; Th17 Cells

Dietary supplements high in isolated isoflavones are commercially available for human consumption primarily to alleviate menopausal symptoms in women. The isoflavone composition, quantity and importantly their estrogenic potency are poorly standardised and can vary considerably between different products. The aim of this study was to analyse the isoflavone composition of 11 dietary supplements based on soy or red clover using the HPLC/MS/MS technique. Furthermore, we investigated the transactivational potential of the supplements on the estrogen receptors (ER), ERα and ERβ, performing luciferase reporter gene assays. As expected, we found that the isoflavone composition varies between different products. The measured total isoflavone contents in various supplements were mostly comparable to those claimed by the manufacturers in their product information. However expressing the isoflavone content as isoflavone aglycone equivalents, soy-based supplements had a clearly lower quantity compared to the manufacturer information. All supplements transactivated more or less ERα and ERβ with a preference for ERβ. The transactivational efficiency exceeded partly the maximal 17β-estradiol induced ER activation. While the different soy-based supplements revealed similar transactivation potential to both ERs, red clover-based supplements differed considerably. We conclude that different commercial dietary supplements based on soy or red clover vary in their isoflavone composition and quantity. They are estrogenically active, although especially the red clover-based supplements show considerable differences in their estrogenic potential to ERα and ERβ. Thus, different isoflavone-rich products cannot be necessarily compared regarding possible biological effects.

Topics: Capsules; Dietary Supplements; Estrogen Receptor alpha; Estrogen Receptor beta; Food Inspection; Food Labeling; Genes, Reporter; Genistein; Germany; Glycine max; Glycosides; HEK293 Cells; Humans; Isoflavones; Phytoestrogens; Recombinant Proteins; Response Elements; Transcriptional Activation; Trifolium

Naturally occurring phytoestrogens may mimic biogenic estrogens and modulate endocrine action in vertebrates. Little is known, however, about their temporal and spatial variability in the environment and the biological effects associated with exposures. The present study assessed the environmental presence of phytoestrogens in human-impacted and relatively pristine areas. The response in larval and sexually mature fathead minnows to environmentally relevant concentrations of 3 common phytoestrogens (genistein, daidzein, and formononetin), both singly and in mixture, was also quantified. Phytoestrogens were only present in the human-impacted surface waters. When detected, mean concentrations were low (± standard deviation) in an urban lake: 1.4 ± 0.5 ng/L, 1.6 ± 0.7 ng/L, and 1.1 ± 0.2 ng/L for genistein, daidzein, and formononetin, respectively, and in treated wastewater effluent: 1.6 ± 0.4 ng/L, 1.8 ± 1.3 ng/L, and 2.0 ng/L. Biochanin A was detected twice, whereas zearalenone and coumestrol were never detected. No clear temporal trends of aqueous phytoestrogen concentration were evident. Larval survival was significantly reduced in genistein, formononetin, and mixture treatments, whereas adult male fish only exhibited subtle changes to their anatomy, physiology, and behavior. Daidzein-exposed adult females produced greater quantities of eggs. The present study indicates that genistein, daidzein, and formononetin are likely attenuated rapidly and are unlikely to cause widespread ecological harm in the absence of other stressors.

Topics: Animals; Cyprinidae; Female; Genistein; Isoflavones; Larva; Male; Phytoestrogens; Water Pollutants, Chemical

Phytoestrogens are endocrine active compounds derived from plants, including the isoflavones genistein and daidzein, and their methylated derivatives biochanin A and formononetin. These compounds have been detected at the µg/L level in the effluents of plant-processing industries and municipal treatment plants and at the ng/L level in surface waters worldwide. The present study assessed the persistence of genistein and daidzein in natural aquatic systems, specifically riverine samples. Initial concentration, temperature, sample location, and time of sample collection varied. Genistein and daidzein were found to be readily biodegradable at all tested concentrations, at both 10 °C and 20 °C, in samples collected during different seasons, and in samples from 3 different rivers. In addition, organismal responses in larval and sexually mature fathead minnows (Pimephales promelas) were quantified following exposure to microbiologically degraded phytoestrogens (genistein, daidzein, and formononetin). Products of the microbiological degradation of parent phytoestrogens did not affect larval survival, growth, or predator avoidance. Female adult fathead minnows exposed to these degradation products produced significantly fewer eggs than those exposed to a control, but no other morphological, physiological, or behavioral changes were observed with male or female minnows. The present research suggests that although phytoestrogens are not likely to persist in aquatic systems, they may pseudo-persist if discharges are continuous; in addition, caution should be exercised with respect to high-concentration effluents because of the potentially antiestrogenic effects of phytoestrogen degradates.

Topics: Animals; Cyprinidae; Female; Genistein; Isoflavones; Larva; Male; Phytoestrogens; Rivers; Water Microbiology; Water Pollutants, Chemical

Worldwide, cervical cancer (CC) is the third most common malignancy in women, and it remains a leading cause of cancer-related death of women. Genomic studies indicate that phosphoinositide 3-kinase (PI3K)/AKT signaling is one of the most frequently deregulated pathways in several human cancers, including CC. This signaling pathway has an important role in cancer cell proliferation, survival, motility, and metabolism, and therefore could be an attractive therapeutic target. In a previous study, we used a sensitive and high-speed homogeneous assay for the detection of kinase activity and for screening of PI3K/AKT signaling inhibitors in a high-throughput screening (HTS) format and then obtain formononetin, as an O-methylated isoflavone existed in a number of plants and herbs like Astragalus membranaceus. We showed that formononetin inhibited the phosphorylation of AKT and induced the apoptosis of CC cell line HeLa in a dose-dependent manner. Furthermore, formononetin suppressed xenograft tumor growth in nude mice. Our results indicated that formononetin may be used as an anti-cancer drug for cervical cancer in the future.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Proliferation; Female; Flow Cytometry; HeLa Cells; Humans; Isoflavones; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Experimental; Phosphatidylinositol 3-Kinases; Phytoestrogens; Proto-Oncogene Proteins c-akt; Signal Transduction; Uterine Cervical Neoplasms; Xenograft Model Antitumor Assays

Prostate cancer is one of the most prevalent malignant cancers in men. The isoflavone formononetin is a main active component of red clover plants. In the present study, we assessed the effect of formononetin on human prostate cancer DU-145 cells in vitro, and elucidated possible mechanisms. DU-145 cells were treated with different concentrations of formononetin and cell proliferation was assessed by MTT assay, cell apoptosis by Hoechst 33258 and flow cytometry, and protein levels of RASD1, Bcl-2 and Bax by Western blotting. The results showed that formononetin inhibited the proliferation of DU-145 cells in a dose-dependent manner. DU-145 cells treated with different concentrations of formononetin displayed obvious morphological changes of apoptosis under fluorescence microscopy. In addition, formononetin increased the proportion of early apoptotic DU-145 cells, down-regulated the protein levels of Bcl-2 and up-regulated those of RASD1 and Bax. The level of RASD1 reached its maximum at 48 h post-treatment, and rapidly decreased thereafter. Together, we present evidence that formononetin triggered cell apoptosis through the mitochondrial apoptotic pathway by up-regulating RASD1.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Proliferation; Flow Cytometry; Humans; In Vitro Techniques; Isoflavones; Male; Phytoestrogens; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; ras Proteins; Tumor Cells, Cultured

The study aimed to determine the effects of red clover (Trifolium pratense) silage with high phyto-oestrogen content on ewe performance during their first breeding season. Red clover silage containing formononetin, biochanin A, genistein, and daidzein was fed to 10 nulliparous ewes of the prolific Finnish Landrace breed before, during and after the breeding season, for a total of 5 months. A control group of 10 ewes was fed with grass silage. The mean numbers of foetuses per pregnancy were 2.1±0.7 and 2.2±0.8 for the red clover and control groups, respectively. The total mass of the uterus with its contents was significantly greater in ewes of the red clover group compared with those of the control group. This difference was mainly explained by the greater volume of foetal fluids. Serum progesterone concentration in the red clover group was significantly lower over the entire period analysed than in the control group. In conclusion, the fecundity of the ewes was not reduced by red clover feed with high phyto-oestrogen concentrations. The volume of foetal fluids increased that could increase the risk for vaginal prolapse before the term.

Topics: Animals; Female; Fertility; Isoflavones; Phytoestrogens; Pregnancy; Sheep; Silage; Trifolium

Dietary phytoestrogens are metabolized or converted in the gastrointestinal tract of ruminants, only limited knowledge exists on the extent and location of this conversion in vivo. The objective of this study was to quantify the gastro-intestinal metabolism of phytoestrogens in lactating dairy cows fed silages with different botanical composition. Four lactating rumen cannulated Norwegian Red cattle were assigned to a 4 × 4 Latin square with 1 cow per treatment period of 3 wk. The 4 treatment silages were prepared from grasslands with different botanical compositions: organically managed short-term timothy (Phleum pratense L.) and red clover (Trifolium pratense L.) ley (2 yr old: ORG-SG); organically managed long-term grassland with a high proportion of unsown species (6 yr old; ORG-LG); conventionally managed perennial ryegrass (Lolium perenne L.) ley (CON-PR); and conventionally managed timothy ley (CON-TI). The herbages were cut, wilted, and preserved with additive in round bales, fed as a mix of the first and third cut at 90% of ad libitum intake, and contributed to 70% of the total dry matter intake. Milk, feed, omasal digesta, urine, and feces were collected at the end of each period and analyzed for the concentrations of phytoestrogens by using a liquid chromatography-tandem mass spectrometry technique. Concentration of total isoflavones was highest in ORG-SG and lowest in CON-TI silage, whereas the content of total lignans was highest in the grass silages. The isoflavones were extensively metabolized in the rumen on all diets, and the recovery of formononetin and daidzein in omasum, mainly as equol, averaged 0.11 mg/mg. The apparent intestinal metabolism was less severe as, on average, 0.29 mg/mg of the omasal flow was recovered in feces. The plant lignans were also strongly degraded in the rumen. However, the flow of lignans to omasum and excretion in feces were, on average, 7.2- and 5.2-fold higher, respectively, than the intake of the plant lignans matairesinol and secoisolariciresinol, known as precursors of mammalian lignans. Thus, excretion to milk could not be directly related to intake, implying that plant lignans other than matairesinol and secoisolariciresinol in forage are precursors for enterolactone production in the rumen and for its content in milk. Equol followed mainly the flow of large particles out of the rumen, whereas the mammalian lignans were distributed between phases proportional to dry matter flow. The main metabolism of

Topics: Animals; Cattle; Chromatography, Liquid; Diet; Feces; Female; Furans; Isoflavones; Lactation; Lignans; Lolium; Mass Spectrometry; Milk; Omasum; Phleum; Phytoestrogens; Poaceae; Rumen; Silage; Trifolium

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.

Topics: Chemokine CCL2; Interleukin-6; Isoflavones; JNK Mitogen-Activated Protein Kinases; Macrophages; Mitogen-Activated Protein Kinases; Molecular Structure; NF-kappa B; Osteoblasts; Osteoclasts; p38 Mitogen-Activated Protein Kinases; Phytoestrogens; Proto-Oncogene Proteins c-fos; RANK Ligand; Signal Transduction

The frequent development of multidrug resistance (MDR) hampers the efficacy of available anticancer drugs in treating cervical cancer. In this study, we aimed to use formononetin (7-hydroxy-4'-methoxyisoflavone), a potential herbal isoflavone, to intensify the chemosensitivity of human cervical cancer HeLa cells to epirubicin, an anticancer drug. The reactive oxygen species (ROS) levels were correlated with MDR modulation mechanisms, including the transporter inhibition and apoptosis induction. Our results revealed that formononetin significantly enhanced the cytotoxicity of epirubicin. Co-incubation of epirubicin with formononetin increased the ROS levels, including hydrogen peroxide and superoxide free radicals. Epirubicin alone markedly increased the mRNA expression of MDR1, MDR-associated protein (MRP) 1, and MRP2. In contrast, formononetin alone or combined treatment decreased the mRNA expression of MRP1 and MRP2. This result indicates that efflux transporter-mediated epirubicin resistance is inhibited at different degrees by the addition of formononetin. This isoflavone significantly intensified epirubicin uptake into HeLa cells. Apoptosis was induced by formononetin and/or epirubicin, as signified by nuclear DNA fragmentation, chromatin condensation, increased sub-G1 and G2/M phases. The cotreatment triggered the mitochondrial apoptotic pathway indicated by increased Bax-to-Bcl-2 expression ratio, loss of mitochondrial membrane potential, and significant activation of caspase-9 and -3. In addition, extrinsic/caspases-8 apoptotic pathway was also induced by the cotreatment. N-acetyl cysteine abrogated these events induced by formononetin, supporting the involvement of ROS in the MDR reversal mechanism. This study pioneered in demonstrating that formononetin may potentiate the cytotoxicity of epirubicin in HeLa cells through the ROS-mediated MRP inhibition and concurrent activation of the mitochondrial and death receptor pathways of apoptosis. Hence, the circumvention of pump and non-pump resistance using formononetin and epirubicin may pave the way for a powerful chemotherapeutic regimen for treating human cervical cancer.

Topics: Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; bcl-2-Associated X Protein; Caspases; Cell Survival; Drug Synergism; Epirubicin; Female; Flow Cytometry; HeLa Cells; Humans; Isoflavones; Membrane Potential, Mitochondrial; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Phytoestrogens; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; RNA, Neoplasm; Uterine Cervical Neoplasms

Formononetin has shown a variety of pharmacologic properties including anti-inflammatory effect. In the present study, we analyzed the role of formononetin in acute lung injury induced by lipopolysaccharide (LPS) in mice. The cell counting in the bronchoalveolar lavage fluid (BALF) was measured. The animal lung edema degree was evaluated by wet/dry weight ratio. The superoxidase dismutase (SOD) activity and myeloperoxidase (MPO) activity was assayed by SOD and MPO kits, respectively. The levels of inflammatory mediators, tumor necrosis factor-α (TNF-α) and IL-6,were assayed by enzyme-linked immunosorbent assay method. Pathological changes of hung tissues were observed by HE staining. Peroxisome proliferator-activated receptor (PPAR)-γ gene expression was measured by real-time PCR. The data showed that treatment with the formononetin group markedly attenuated inflammatory cell numbers in the BALF, increased PPAR-γ gene expression and improved SOD activity and inhibited MPO activity. The histological changes of the lungs were also significantly improved by formononetin compared to LPS group. The results indicated that formononetin has a protective effect on LPS-induced acute lung injury in mice.

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Edema; Inflammation; Interleukin-6; Isoflavones; Lipopolysaccharides; Lung; Male; Mice; Mice, Inbred BALB C; Peroxidase; Phytoestrogens; PPAR gamma; Pulmonary Edema; Superoxide Dismutase; Tumor Necrosis Factor-alpha

Daidzein (Daid) has been implicated in bone health for its estrogen-'like' effects but low bioavailability, unfavorable metabolism and uterine estrogenicity impede its clinical potential. This study was aimed at assessing isoformononetin (Isoformo), a naturally occurring methoxydaidzein, for bone anabolic effect by overcoming the pitfalls associated with Daid.. Sprague-Dawley ovariectomized (OVx) rats with established osteopenia were administered Isoformo, 17β-oestradiol (E2) or human parathyroid hormone. Efficacy was evaluated by bone microarchitecture using microcomputed tomography and determination of new bone formation by fluorescent labeling of bone. Osteoblast apoptosis was measured by co-labeling of bone sections with Runx-2 and TUNEL. Biochemical markers of bone metabolism were measured by ELISA. Plasma and bone marrow levels of Isoformo and Daid were determined by LC-MS-MS. Rat bone marrow stromal cells were harvested to study osteoblastic differentiation by Isoformo and Daid. New born rat pups were injected with Isoformo and Daid to study the effect of the compounds on the expression of osteogenic genes in the calvaria by real time PCR.. In osteopenic rats, Isoformo treatment restored trabecular microarchitecture, increased new bone formation, increased the serum osteogenic marker (procollagen N-terminal propeptide), decreased resorptive marker (urinary C-terminal teleopeptide of type I collagen) and diminished osteoblast apoptosis in bone. At the most effective osteogenic dose of Isoformo, plasma and bone marrow levels were comprised of ~90% Isoformo and the rest, Daid. Isoformo at the concentration reaching the bone marrow achieved out of its most effective oral dosing induced stromal cell mineralization and osteogenic gene expression in the calvaria of neonatal rats. Isoformo exhibited uterine safety.. Our study demonstrates that Isoformo reverses established osteopenia in adult OVx rats likely via its pro-survival effect on osteoblasts. Given its bone anabolic and anti-catabolic effects accompanied with safety at uterine level we propose its potential in the management of postmenopausal osteoporosis.

Topics: Animals; Apoptosis; Biomarkers; Bone and Bones; Bone Density Conservation Agents; Bone Diseases, Metabolic; Bone Resorption; Calcification, Physiologic; Female; Isoflavones; Metabolism; Osteoblasts; Osteogenesis; Osteoporosis; Ovariectomy; Phytoestrogens; Phytotherapy; Plant Extracts; Rats; Rats, Sprague-Dawley; Stromal Cells; Uterus

The role of peroxisome proliferator-activated receptors (PPARs) as anti-inflammatory mediators has been established, and the fact that some isoflavones are dual agonists of PPARα/γ indicates the involvement of PPARα and/or PPARγ in the anti-inflammatory action of certain isoflavones. However, the dependency of isoflavones on PPARs in their anti-inflammatory action has not been demonstrated. Here, we report the dependency of an isoflavone biochanin A and the independency of another isoflavone genistein in relation to PPARγ to ameliorate the cytokine secretion profile of lipopolysaccharide (LPS)-stimulated mouse RAW264.7 macrophages. A total amount of 10 µmol/l of biochanin A or genistein significantly suppressed the secretion of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) in LPS-induced RAW264.7 cells, whereas another two isoflavones, formononectin and daidzein, only significantly suppressed the secretion of IL-6. Their anti-inflammatory efficiencies were not in correspondence with their PPARα/γ agonist activities. Inhibition of PPARγ activity by its antagonist GW9662 significantly reversed the anti-inflammatory effect of biochanin A but not genistein, which demonstrated the dependency of biochanin A and the independency of genistein on PPARγ in their anti-inflammatory actions. Meanwhile, the PPARγ-dependency of biochanin A was further confirmed by the result that the suppression of LPS-induced NF-κB activation by biochanin A was reversed following GW9662 co-treatment. Moreover, inhibition of PPARα activity by its antagonist MK886 did not significantly reverse the anti-inflammatory effects of biochanin A and genistein, indicating that their anti-inflammatory properties were PPARα-independent.

Topics: Anilides; Animals; Anticarcinogenic Agents; Cell Line; Genistein; Humans; Indoles; Interleukin-6; Isoflavones; Lipopolysaccharides; Lipoxygenase Inhibitors; Macrophages; Mice; NF-kappa B; Phytoestrogens; PPAR alpha; PPAR gamma; Tumor Necrosis Factor-alpha

The risks of hormone replacement therapy have led to a search for new alternatives such as phytoestrogens, plant compounds with estrogen-like biological activity. Isoflavones are the phytoestrogens most extensively studied and can be found in soybean, red clover and other plants. Due to this estrogen-like activity, phytoestrogens can have some effect on atherosclerosis. Human umbilical vein endothelial cells (HUVEC) have been extensively used to study the biology and pathobiology of human endothelial cells and most of the knowledge acquired is due to experiments with cultures of these cells.. To evaluate the effects of the phytoestrogen extracts from Glycine max soy bean, genistein, formononetin, biochanin A and daidzein, as well as a mixture of these extracts (Mix), on expression of adhesion molecules, VCAM-1, ICAM-1 and E-selectin, by endothelial cell HUVEC, stimulated with lipopolysaccharide.. HUVEC were cultured in medium EBM(2), pretreated with isoflavones for 24 and 48 h and then stimulated with lipopolysaccharide; in addition, isoflavones were added, after stimulation by lipopolysaccharide, to HUVEC. We evaluated the production of VCAM-1, ICAM-1 and E-selectin on cell surface, by cell-based enzyme immunoassay, and of sVCAM-1, sICAM-1 and sE-selectin in culture supernatant, by ELISA.. Genistein, formononetin, biochanin A and daidzein, as well as the Mix were able to reduce VCAM-1, ICAM-1 and E-selectin on cell surface and in culture supernatant. Conclusion Isoflavones extracted from Glycine max soy bean, in vitro, presented antiatherogenic effects, reducing the expression of adhesion molecules and acting as preventive agents as well as therapeutic agents.

Topics: Cells, Cultured; Drug Therapy, Combination; E-Selectin; Genistein; Glycine max; Human Umbilical Vein Endothelial Cells; Humans; Intercellular Adhesion Molecule-1; Isoflavones; Phytoestrogens; Time Factors; Vascular Cell Adhesion Molecule-1

Formononetin (Formo) prevents ovariectomy (Ovx)-induced bone loss in rats. However, there are no reports on the curative effects of Formo. The objective of this study was to investigate the ability of Formo in restoring trabecular microarchitecture and promoting new bone formation in osteopenic rats.. Adult Sprague-Dawley rats were ovariectomized and left for 90 days for osteopenia to develop. After 90 days, Formo (10.0 mg kg d) was given orally for the next 12 weeks to Ovx rats in a therapeutic protocol. Sham-operated, Ovx + vehicle, and Ovx + parathyroid hormone (PTH) groups served as controls. Trabecular microarchitecture, osteoid formation, bone turnover/resorption markers, and bone osteoprotegerin-to-receptor activator for nuclear κB ligand ratio were studied. One-way analysis of variance was used to test significance of effects.. Formo treatment significantly restored the lost trabecular microarchitecture in the femurs and tibia of osteopenic Ovx rats and promoted new bone formation. Formo was devoid of any uterine estrogenicity. Serum levels of type I collagen N-terminal propeptide, which is a reliable marker of bone formation, were increased in Ovx rats treated with Formo compared with Ovx + vehicle group, and the levels were comparable with those in the sham group. Formo prevented the Ovx-induced increase in bone turnover markers, including serum osteocalcin and urinary type I collagen degradation product. Furthermore, Formo-treated Ovx rats had an increased bone osteoprotegerin-to-receptor activator for nuclear κB ligand ratio compared with the Ovx + vehicle group.. Daily oral administration of Formo for 12 weeks has a substantial anabolic effect, thus raising the possibility of its use in postmenopausal osteoporosis.

Topics: Animals; Bone Diseases, Metabolic; Bone Remodeling; Female; Femur; Isoflavones; Osteogenesis; Osteoprotegerin; Ovariectomy; Peptide Fragments; Phytoestrogens; Procollagen; RANK Ligand; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tibia

Topics: Animals; Bone Diseases, Metabolic; Female; Isoflavones; Ovariectomy; Phytoestrogens

Formononetin is one of the main active components of red clover plants, and considered as a phytoestrogen. Its pharmacological effects in vivo may be either estrogenic or anti-estrogenic, mainly depending upon the estrogen levels. Our recent studies suggested that formononetin inactivated IGF1/IGF1R-PI3K/Akt pathways and decreased cyclin D1 mRNA and protein expression in human breast cancer cells in vitro and in vivo. In the present study, we further investigated the molecular mechanisms involved in the induced apoptosis effect of formononetin on breast cancer cells. Our results suggested that formononetin inhibited the proliferation of ER-positive MCF-7 cells and T47D cells. In contrast, formononetin could not inhibit the cell of growth of ER-negative breast cancer cells such as MDA-MB-435 S cells. We further found that formononetin activated MAPK signaling pathway in a dose-dependent manner, which resulted in the increased ratio of Bax/Bcl-2, and induced apoptosis on MCF-7 cells. However, when MCF-7 cells were pretreated with p38MAPK inhibitor SB203580 before formononetin, apoptosis induced by formononetin was significantly attenuated. Thus, we conclude that the induced apoptosis effect of formononetin on human breast cancer cells were related to Ras-p38MAPK pathway. Considering that red clover plants are widely used clinically, our results provide the foundation for future development of formononetin for treatment of ER-positive breast cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; Isoflavones; p38 Mitogen-Activated Protein Kinases; Phytoestrogens; Proto-Oncogene Proteins c-bcl-2; ras Proteins; Receptors, Estrogen

Formononetin is a novel herbal isoflavonoid isolated from Astragalus membranaceus, a medicinal plant that possesses antitumorigenic properties. Our previous findings demonstrated that formononetin initiates growth-inhibitory and pro-apoptotic activities in human colon cancer cells. In the present study, we aimed to further examine the potential of formononetin in controlling angiogenesis and tumor cell invasiveness in human colon cancer cells and tumor xenografts. The results showed that formononetin downregulated the expression of the key pro-angiogenic factors, including vascular endothelial growth factor (VEGF) and matrix metalloproteinases. We also discovered that the invasiveness of metastatic colon cancer cells was alleviated following drug treatment. The potential anti-angiogenic effect of formononetin was examined in nude mouse xenografts. The tumor size and the number of proliferating cells were reduced in the tumor tissues obtained from the formononetin-treated group. The serum VEGF level was also reduced in the drug-treated animals when compared to the controls. These findings suggest that formononetin inhibits angiogenesis and tumor cell invasion, and thus support its use in the treatment of advanced and metastatic colon cancers.

Topics: Animals; Astragalus propinquus; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colonic Neoplasms; Disease Progression; Female; Humans; Isoflavones; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neovascularization, Pathologic; Phytoestrogens; Plant Extracts; Plant Roots; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

We developed and validated three different sample preparation and extraction methods followed by HPLC-MS/MS (negative electrospray ionization) analysis for the quantification of estrogenic isoflavones (formononetin, daidzein, equol, biochanin A, and genistein) and coumestrol in red clover, soil, and manure. Plant and manure samples were solid-liquid extracted, whereas soil was extracted with accelerated solvent extraction. Absolute recoveries were between 80 and 93%, 20 and 30%, and 14 and 91% for plant, soil, and manure samples, respectively. Relative recoveries ranged from 75 to 105% for all matrices, indicating that isotope-labeled internal standards (¹³C₃-formononetin, ¹³C₃-daidzein, ¹³C₃-equol, ¹³C₃-biochanin A, and ¹³C₃-genistein) were capable to compensate for losses during analysis. The limits of detection in red clover, soil, and manure were 3-9 μg/g(dryweight(dw)), 0.6-8.2 ng/g(dw), and 34.2 ng/g(dw) to 17.0 μg/g(dw), respectively. Formononetin was the most dominant compound in red clover plants (up to 12.5 mg/g(dw)) and soil (up to 3.3 μg/g(dw)), whereas equol prevailed in manure (up to 387 μg/g(dw)).

Topics: Chromatography, High Pressure Liquid; Coumestrol; Equol; Genistein; Isoflavones; Manure; Phytoestrogens; Soil; Tandem Mass Spectrometry; Trifolium

We evaluated the vasorelaxation effects of formononetin, an isoflavone/phytoestrogen found abundantly in Astragalus mongholicus Bunge, on rat isolated aorta and the underlying mechanisms involved. Cumulative administration of formononetin, genistein, daidzein and biochanin A relaxed phenylephrine-preconstricted aorta. Formononetin and biochanin A caused a similar magnitude of relaxation whereas daidzein was least potent. Mechanical removal of endothelium, L-NAME (100 microM) and methylene blue (10 microM) suppressed formononetin-induced relaxation. Formononetin increased endothelial nitric oxide (NO) synthase (eNOS), but not inducible NO synthase, activity with an up-regulation of eNOS mRNA and p-eNOS(Ser1177) protein expression. In endothelium-denuded preparations, formononetin-induced vasorelaxation was significantly reduced by glibenclamide (3 microM) and iberiotoxin (100 nM), and a combination of glibenclamide (3 microM) plus iberiotoxin (100 nM) abolished the relaxation. In contrast, formononetin-elicited endothelium-independent relaxation was not altered by ICI 182,780 (10 microM, an estrogen receptor (ER alpha/ER beta) antagonist) or mifepristone (10 microM, a progesterone receptor antagonist). In single aortic smooth muscle cells, formononetin caused opening of iberiotoxin-sensitive Ca(2+)-activated K(+) (BK(Ca)) channels and glibenclamide-sensitive adenosine triphosphate (ATP)-dependent K(+) (K(ATP)) channels. Thus, our results suggest that formononetin caused vascular relaxation via endothelium/NO-dependent mechanism and endothelium-independent mechanism which involves the activation of BK(Ca) and K(ATP) channels.

Topics: Animals; Aorta, Thoracic; Cardiovascular Diseases; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; In Vitro Techniques; Isoflavones; Male; Muscle, Smooth, Vascular; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Osmolar Concentration; Phytoestrogens; Phytotherapy; Rats; Rats, Sprague-Dawley; RNA, Messenger; Up-Regulation; Vasodilation; Vasodilator Agents

Osteoarthritis is a multifactorial disease characterized by loss of articular cartilage and subchondral plate thickening. Therefore, biochemical analysis of the underlying bone tissue has provided important information for treatment of osteoarthritis. In this study, we determined the potential role of formononetin, a phytoestrogen isolated from Astragalus membranaceus to alter the expression of metabolic markers and cytokine production of human normal osteoblasts (Obs) and osteoarthritis subchondral osteoblasts (OA Obs). Human OA Obs and normal Obs were cultured for 3days, 7days or 14days in the present medium only or were treated with various doses of formononetin. Cells were analyzed for viability by WST-8 assay, alkaline phosphatase (ALP) activity, osteogenic markers (osteocalcin (OCN) and type I collagen (Col I)) and cytokines (interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), bone morphogenic protein-2 (BMP-2)). The level of IL-6, VEGF, BMP-2, OCN and Col I was increased in OA Obs compared with normal Obs. Formononetin dose-dependently decreased ALP, IL-6, VEGF, BMP-2, OCN and Col I in OA Obs, while markedly increased ALP, VEGF, BMP-2, OCN and Col I in normal Obs. Interestingly, formononetin markedly increased the expression of VEGF and BMP-2 for 3days of culture and significantly increased OCN and Col I at 14days in human normal Obs. The remodeling effect of formononetin on osteogenic markers and cytokines of inflammatory mediators was more striking in OA Obs as well. Taken together, these results could suggest that formononetin has biphasic positive effects on normal Obs and OA Obs by modifying their biological synthetic capacities.

Topics: Aged; Alkaline Phosphatase; Astragalus propinquus; Bone and Bones; Bone Morphogenetic Proteins; Cell Proliferation; Cells, Cultured; Collagen Type I; Cytokines; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Humans; Interleukin-6; Isoflavones; Osteoarthritis; Osteoblasts; Osteocalcin; Phytoestrogens; Reverse Transcriptase Polymerase Chain Reaction; Vascular Endothelial Growth Factor A

To observe the estrogenic effect of formononetin and its effect on the expressions of atrial estrogen receptor subtypes alpha and beta (ERalpha and ERbeta).. 50 femal rats were randomly divided into five groups: sham group, model group, nilestriol group, formononetin groups of low and high dose. Rats in sham group were cut a piece of fat before closing the abdomen, the others were ovariectomized. Vaginal exfoliated cell were observed from the fifth day to the tenth after operation to test if the model is successful. The sham and model group were given nomal saline in 10 mL/kg by gavage, the remaining three groups were given nilestriol 2.5 mg/(kg x w), low [20 mg/(kg x d) land high dose [100 mg/(kg x d)) of formononetin by gavage respectively. In the 8th week, vaginal exfoliated cell were observed, then decapitated the rats, removed the uterus, weighed and take wright staining microscopy. The relative expressions of ERalpha and ERbeta of right atrium were detected by RT-PCR.. The vaginal cells exhibit a change of estrus after had been fed with high dose of formononetin after 8 weeks. Formononetin increase the uterus coefficient and the expression of atrial ERbeta (P < 0.01), but it dose not have any effect on the expression of ERalpha (P > 0.05).. Formononetin have estrogenic effect in ovariectomized rats, and it can markedly upregulate the expression of rats' atrial ERbeta.

Topics: Administration, Oral; Animals; Disease Models, Animal; Estriol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Heart Atria; Isoflavones; Ovariectomy; Phytoestrogens; Quinestrol; Random Allocation; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Trifolium; Uterus

Research on the phytoestrogenic effect and its possible mechanism of formononetin.. To evaluate the estrogenic effect and mechanisms of formononetin through the test of its influence on proliferation and ER subtype expression of T47D cells.. The proliferation rates of T47D cells treated with 1 x 10(-7) -1 x 10(-6) mol x L(-1) formononetin were not increased. On the influence of ICI182, 780, the proliferation rates of T47D cells treated with 1 x 10(-7) 1 x 10(-6) mol x L(-1) formononetin were decreased. Formonenetin could induce the augment of ERalpha expression significantly of T47D.. Formonenetin has phytoestrogenic effect Formonenetin can not accelerate ER(+) T47D cell proliferation. But the expression level of ERalpha subtype in T47D cells change significantly with certain concentrations of formonenetin.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; Isoflavones; Phytoestrogens; Receptors, Estrogen

Antioxidant and estrogenic effects of formononetin on ovariectomized mice have been investigated in the present study. The adult female Kunming mice were divided into 5 groups: sham-operated group, ovariectomized group, stilbestrol replacement therapy group (0.20 mg/kg day), low-dose formononetin group (0.05 g/kg day) and high-dose formononetin group (0.5 g/kg day). The mice in the latter 4 groups were ovariectomized. The drug was given by oral administration for 6 months. Estrogenic effect was determined by the change of uterine weight, and oxidant effects were determined by the content of SOD, GSH-Px, CAT and MDA. The intake of formononetin increased the uterine weight of the mice significantly as well as the content of SOD, GSH-Px, CAT, and reduced MDA in body. Formononetin had obvious antioxidant effects and estrogenic effect, and the estrogenic effect was not dosage-related.

Topics: Administration, Oral; Animals; Antioxidants; Catalase; Diethylstilbestrol; Dose-Response Relationship, Drug; Estrogens, Non-Steroidal; Female; Glutathione Peroxidase; Isoflavones; Lipid Peroxidation; Malondialdehyde; Mice; Organ Size; Ovariectomy; Phytoestrogens; Plant Extracts; Superoxide Dismutase; Trifolium; Uterus

A UPLC-UV/ELSD method has been developed for analysis of major triterpenoids and formononetin in ACTAEA RACEMOSA L. (family Ranunculaceae) samples. The best results were obtained with an Acquity UPLC BEH C18 (100 mmx2.1 mm, i. d., 1 microm) column system using gradient elution with a mobile phase consisting of water and acetonitrile:methanol (7:3) at a constant flow rate of 0.3 mL/min. Owing to their low UV absorption, the triterpene saponins were detected by evaporative light scattering. Within 5.5 minutes, three main triterpenoid glycosides [cimiracemoside A, 23- EPI-26-deoxyactein, and actein] and an isoflavonoid, formononetin, could be separated, with detection limits of 5, 5, 10, and 0.01 microg/mL, respectively. The method was successfully used to analyze different Actaea racemosa market products as well as to distinguish between two other ACTAEA species. There was a significant variability in the amounts of the selected triterpene glycosides for the products containing black cohosh and rhizomes of black cohosh. The isoflavone formononetin was not detected in the samples analyzed. LC-MS coupled with the electrospray ionization (ESI) interface method is described for the identification of formononetin and triterpenoid glycosides in plant samples and dietary supplements that claim to contain black cohosh and different species of Actaea.

Topics: Chromatography, Liquid; Cimicifuga; Dietary Supplements; Isoflavones; Phytoestrogens; Rhizome; Scattering, Radiation; Triterpenes

As a new sample preparation technique, pressurized liquid extraction (PLE), in combination with reversed-phase liquid chromatography (RP-LC) and photodiode-array (PDA) detection were used for the isolation and determination of phytoestrogenic isoflavones in hydrolyzed extracts obtained from aerial parts of five Trifolium L. (clover) species. To optimize the effectiveness of PLE procedure, variable extraction parameters: methanol and acetone (or their 75% aqueous solutions), as extraction solvents, temperatures (75, 100 and 125 degrees C) and the changeable number of static extraction cycles were tested. Additionally, two other micropreparative techniques: ultrasound-assisted extraction (UAE), and conventional solvent extraction (CSE), under optimized conditions, were also used and compared. Optimum extraction efficiency, expressed in the highest yield of biochanin A, formononetin, daidzein and genistein from plant material, with PLE, using methanol-water (75:25, v/v) as an extraction solvent, an oven temperature of 125 degrees C and three 5-min static extraction cycles, was obtained.

Topics: Chromatography, High Pressure Liquid; Genistein; Isoflavones; Phytoestrogens; Solvents; Trifolium; Ultrasonics

Chemical compounds, including plant-based phytoestrogens, can function as hormone mimics and alter endocrine signaling in wildlife. In the present study, the waste streams from 19 plant-processing industries, including biofuel manufacturers, were sampled and analyzed for the phytoestrogens genistein, daidzein, coumestrol, formononetin, biochanin A, and zearalenone, via liquid chromatography/mass spectrometry. Eight of these industries contained phytoestrogens at environmentally relevant levels (≥1,000 ng/L), with the highest at approximately 250,000 ng/L. The influent and effluent streams of three municipal wastewater treatment plants receiving flow from some of these industries also were sampled and analyzed for the same phytoestrogens. It appeared that aerobic biological treatment, such as activated sludge, was able to remove these compounds from the liquid stream. Nevertheless, the effluent stream from one of the wastewater treatment plants had a phytoestrogen concentration above 1,000 ng/L. Results of the present study indicate the need for caution when designing facilities to treat the effluents from biofuel and other plant-processing industries.

Topics: Biofuels; Chromatography, Liquid; Coumestrol; Environmental Monitoring; Genistein; Industrial Waste; Isoflavones; Mass Spectrometry; Phytoestrogens; Sewage; Water Pollutants, Chemical

Following a lead obtained from stem-bark extract of Butea monosperma, two structurally related methoxyisoflavones; cajanin and isoformononetin were studied for their effects in osteoblasts. Cajanin had strong mitogenic as well as differentiation-promoting effects on osteoblasts that involved subsequent activation of MEK-Erk and Akt pathways. On the other hand, isoformononetin exhibited potent anti-apoptotic effect in addition to promoting osteoblast differentiation that involved parallel activation of MEK-Erk and Akt pathways. Unlike genistein or daidzein, none of these two compounds appear to act via estrogen receptors in osteoblast. Once daily oral (by gavage) treatment for 30 consecutive days was given to recently weaned female Sprague-Dawley rats with each of these compounds at 10.0 mg kg(-1) day(-1) dose. Cajanin increased bone mineral density (BMD) at all skeletal sites studied, bone biomechanical strength, mineral apposition rate (MAR) and bone formation rate (BFR), compared with control. BMD levels at various anatomic positions were also increased with isoformononetin compared with control however, its effect was less potent than cajanin. Isoformononetin had no effect on the parameters of bone biomechanical strength although it enhanced MAR and BFR compared with control. Isoformononetin had very mild uterotrophic effect, whereas cajanin was devoid of any such effect. Our data suggest that cajanin is more potent than isoformononetin in accelerating peak bone mass achievement. To the best of our knowledge, this work represents the first attempt to elucidate structure-activity relationship between the two methoxylated isoflavones regarding their effects in osteoblasts and bone formation.

Topics: Aging; Analysis of Variance; Animals; Apoptosis; Bone and Bones; Bone Density; Bone Marrow Cells; Calcification, Physiologic; Cell Differentiation; Cell Proliferation; Cells, Cultured; Diethylstilbestrol; Enzyme Inhibitors; Estrogen Antagonists; Estrogen Receptor Modulators; Estrogens; Female; Genistein; Isoflavones; MAP Kinase Signaling System; Osteoblasts; Osteogenesis; Phytoestrogens; Rats; Rats, Sprague-Dawley; Uterus

The intake of isoflavones and the resulting equol contents of both plasma and milk of the same red clover-fed cows are reported for the first time in cyclic change-over design study. Cows were fed four different red clover silages and two timothy-meadow fescue silages as controls. The red clover silages contained daidzein, formononetin, biochanin A and genistein, whereas the timothy-meadow fescue silages contained no isoflavones. We found a strong association (y = 0.071x+2.75, R 2 0.71) between the formononetin intake (x) and equol concentration (y) in the plasma, while the formononetin intake and milk equol concentration were weakly associated (y = 0.0035x+0.358, R 2 0.20). This means that a small part of the total formononetin in the silage is secreted into milk as equol. The mean equol contents in plasma and milk of cows fed red clover silage diets were in the range of 4.6-8.4 mg/l and 458-643 microg/l, respectively, while the respective values for the control diets were in the range of 0.8-1.5 mg/l and 171-287 microg/l. We showed that shorter growing periods of red clover resulted in higher silage formononetin contents and plasma and milk equol contents, suggesting that the equol content of milk can be manipulated by varying the harvesting strategy of red clover. We conclude that milk equol is derived from the formononetin of red clover silage and that milk from red clover-fed cows can be considered as a source of equol in human nutrition.

Topics: Animal Nutritional Physiological Phenomena; Animals; Cattle; Equol; Female; Food Analysis; Isoflavones; Milk; Phytoestrogens; Silage; Trifolium

Antiangiogenic compounds are gaining more and more interest as a new approach in the prevention and treatment of cancer and inflammatory diseases. The objective of this study was the evaluation of the antiangiogenic effect of a red clover extract (RCE) used in food supplements for menopausal complaints as well as of its main isoflavones in an in vivo system, the chorioallantoic membrane assay of fertilized hen's eggs. At a dosage of 250 microg/pellet the red clover extract showed excellent inhibition of angiogenesis. The antiangiogenic activity of the non-methylated isoflavones daidzein and genistein was higher than that of the methylated compounds formononentin and biochanin A. The results demonstrate that RCE is not only suitable for menopausal complaints, but might also be a powerful chemopreventive agent against chronic diseases e.g. which have a high incidence especially in elderly female.

Topics: Angiogenesis Inhibitors; Animals; Chick Embryo; Chorioallantoic Membrane; Genistein; Inflammation; Isoflavones; Methylation; Phytoestrogens; Plant Extracts; Trifolium

Plant-derived phytoestrogens have bone protective effects, but the molecular mechanism behind these effects remains unclear. This study is aimed at fully characterizing the fracture healing process of formononetin, and investigating the mechanism underlying angiogenesis in calluses of a rat fracture model. Femoral fractures were produced in 2-month-old Sprague-Dawley rats. A 20 microg/kg or 200 microg/kg dose of formononetin was orally administrated once a day during the healing period of 21 days. The results showed that in the early stage of chondrogenesis (days 3), formononetin significantly increased the number of vessels, and expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2/flk-1) compared with control. However, the larger dose of formononetin had no significant difference on expression of VEGF and VEGFR-2/Flk-1 compared with that of the smaller dose of formononetin. After 7 days of administration, formononetin markedly induced differentiation of mesenchymal stem cells in the fracture site. After 14 days, gene expression of mesenchymal progenitors such as alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN) and collagen type I (Col I), indicating osteogenic differentiation, was markedly stimulated by formononetin compared with control. These results suggest that formononetin promotes early fracture healing through angiogenesis activation in the early stage of fracture repair, and osteogenesis acceleration in the later stages, and thus may be beneficial for fracture healing.

Topics: Animals; Astragalus propinquus; Bony Callus; Cell Differentiation; Chondrogenesis; Collagen Type I; Disease Models, Animal; Femur; Fractures, Bone; Isoflavones; Male; Mesenchymal Stem Cells; Neovascularization, Physiologic; Osteocalcin; Osteopontin; Phytoestrogens; Phytotherapy; Plant Extracts; Plant Roots; Rats; Rats, Sprague-Dawley; Receptors, Vascular Endothelial Growth Factor; Vascular Endothelial Growth Factor A; Wound Healing

Exposure to xenoestrogens in humans and animals has gained increasing attention due to the effects of these compounds on reproduction. The present study was undertaken to investigate the influence of low-dose dietary phytoestrogen exposure, i.e. a mixture of genistein, daidzein, biochanin A and formononetin, on the establishment of testosterone production during puberty in male goat kids.. Goat kids at the age of 3 months received either a standard diet or a diet supplemented with phytoestrogens (3-4 mg/kg/day) for approximately 3 months. Plasma testosterone and total and free triiodothyronine (T3) concentrations were determined weekly. Testicular levels of testosterone and cAMP were measured at the end of the experiment. Repeated measurement analysis of variance using the MIXED procedure on the generated averages, according to the Statistical Analysis System program package (Release 6.12, 1996, SAS Institute Inc., Cary, NC, USA) was carried out.. No significant difference in plasma testosterone concentration between the groups was detected during the first 7 weeks. However, at the age of 5 months (i.e. October 1, week 8) phytoestrogen-treated animals showed significantly higher testosterone concentrations than control animals (37.5 nmol/l vs 19.1 nmol/l). This elevation was preceded by a rise in plasma total T3 that occurred on September 17 (week 6). A slightly higher concentration of free T3 was detected in the phytoestrogen group at the same time point, but it was not until October 8 and 15 (week 9 and 10) that a significant difference was found between the groups. At the termination of the experiment, testicular cAMP levels were significantly lower in goats fed a phytoestrogen-supplemented diet. Phytoestrogen-fed animals also had lower plasma and testicular testosterone concentrations, but these differences were not statistically significant.. Our findings suggest that phytoestrogens can stimulate testosterone synthesis during puberty in male goats by increasing the secretion of T3; a hormone known to stimulate Leydig cell steroidogenesis. It is possible that feedback signalling underlies the tendency towards decreased steroid production at the end of the experiment.

Topics: Animals; Cyclic AMP; Diet; Genistein; Goats; Isoflavones; Male; Phytoestrogens; Sexual Maturation; Testis; Testosterone; Triiodothyronine

To observe the effects of Astragalus injection, astragaloside IV and formononetin on proliferation and Akt phosphorylation of basal-like human breast carcinoma cell lines MDA-MB-468 and MDA-MB-231.. The effects of different concentrations of Astragalus injection, astragaloside IV and formononetin on proliferation of breast cancer cell lines were assayed by methyl thiazolyl tetrazolium (MTT) assay, and their effects on phospho-Akt were assayed by in-cell Western blot method.. The results of the MTT assay showed that the best concentrations of Astragalus injection, astragaloside IV, formononetin and astragaloside IV plus formononetin were 1 g/mL, 80 microg/mL, 40 microg/mL and 10 microg/mL plus 40 microg/mL respectively. After 1- or 2-day culture, Astragalus injection, astragaloside IV, formononetin and astragaloside IV plus formononetin decreased the expressions of p-Akt (Thr 308) and p-Akt (Ser 473) in MDA-MB-468 cells. Formononetin and astragaloside IV plus formononetin down-regulated the expression of p-Akt (Thr 308) protein in MDA-MB-231 cells after 1- and 2-day culture, but had no effects on the expression of p-Akt (Ser 473) protein in MDA-MB-231 cells.. Astragalus injection, astragaloside IV and formononetin can inhibit proliferation of breast cancer cell lines MDA-MB-468 and MDA-MB-231, and the antiproliferation effects vary according to their concentrations. And the antiproliferation mechanisms may be related to their down-regulation effects on Akt phosphorylation.

Topics: Apoptosis; Astragalus Plant; Breast Neoplasms; Cell Proliferation; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Isoflavones; Oncogene Protein v-akt; Phosphorylation; Phytoestrogens; Saponins; Triterpenes

Aryl hydrocarbon receptor (AhR) activation affects the cell cycle and drives cells to apoptosis. Thus, selective AhR modulators (SAhRMs) have previously been implicated in cancer therapy and prevention, particularly for hormone-dependent cancers. In the present study, isoflavones a remedy used to ameliorate menopausal complaints were tested for their potential in transactivating AhR in order to investigate the biological function of red clover isoflavones. The results were compared to the transactivation potentials of other flavonoids and plant-derived indole compounds. We found that the isoflavones biochanin A and formononetin were potent AhR agonists in vitro, with EC(50) values of 2.5 x 10(-7) and 1.3 x 10(-7)mol/l, respectively. These isoflavones are 10 times more potent compared to the indole compounds indole-3-carbinol and diindolylmethane, publicised as powerful AhR agonists with EC(50) values of 5.8 x 10(-6) and 1.1 x 10(-6)mol/l, respectively. Because activated AhR crosstalks with estrogen receptor alpha, future risk-benefit assessments of isoflavones should take into consideration their AhR transactivating potential.

Topics: Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Estrogens; Genistein; Humans; Isoflavones; Ligands; Phytoestrogens; Receptors, Aryl Hydrocarbon; Saccharomyces cerevisiae; Trifolium

Red clover, known for its estrogenic activity due to its isoflavones content (biochanin A, genistein, daidzein and formononetin), was inoculated with the arbuscular mycorrhizal fungus Glomus mosseae. Once the symbiotic fungus was well established, plants were harvested and we determined the root and shoot dry weight as well as the P-content. In roots and leaves the levels of biochanin A, genistein, daidzein and formononetin were quantified by reversed-phase HPLC and the estrogenic activity of the leaves was measured by a transactivation assay using a yeast two-plasmid system. Mycorrhization increased the levels of biochanin A in the root and the shoot and reduced the levels of genistein in the shoot of red clover. The levels of the other isoflavones were not affected. The shoot biomass of mycorrhizal plants more than doubled compared with non-mycorrhizal control plants, and this growth-stimulating effect of arbuscular mycorrhiza did not affect the estrogenic activity of red clover. In a control P treatment, the biomass of red clover was greatly enhanced. However, the estrogenic activity was reduced. These results suggest that, in contrast to an enhanced shoot biomass production after P application with a reduced estrogenic activity, with arbuscular mycorrhiza the shoot biomass of red clover can be enhanced without a negative effect on estrogenic activity.

Topics: Biomass; Genistein; Glomeromycota; Isoflavones; Mycorrhizae; Phosphates; Phytoestrogens; Plant Leaves; Plant Roots; Trifolium

To study the estrogenic activity of formononetin in vitro.. We have established a highly sensitive bioassay system by placing estrogen-responsive elements upstream of the luciferase reporter gene, and used this assay to determine the estrogenic activity of formononetin. Cell growth was measured by the MTT (3-(4,5-dimethylthioazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and MG-63 cell function was studied by measuring alkaline phosphatase activity.. Formononetin activated expression of the estrogen-responsive reporter gene in human breast cell line MCF-7 in a concentration-dependent manner (0.5-500 microM), and this activation was inhibited by estrogen antagonist (ICI 182780 at 100 nM). Furthermore, it induced the proliferation of MCF-7 breast cancer cells and MG-63 osteosarcoma cells, and it also increased the alkaline phosphatase activity in MG-63 cells.. Formononetin is a phytoestrogen that exhibits variable degrees of estrogen receptor agonism in different test systems.

Topics: Biological Assay; Cell Differentiation; Cell Proliferation; Estrogens; Genes, Reporter; Humans; Isoflavones; Models, Biological; Osteoblasts; Phytoestrogens; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Phyto-oestrogens are polyphenolic non-steroidal plant compounds with oestrogen-like biological activity. Phyto-oestrogens have many biological effects including oestrogen agonist/antagonist properties. However, the effect of phyto-oestrogens on allergic responses remains unclear. In this study we investigated whether formononetin, a phyto-oestrogen, and its metabolites, daidzein and equol, affect production of interleukin-4 (IL-4), a pro-inflammatory cytokine closely associated with allergic immune response, in primary CD4+ T cells and EL4 T lymphoma cells. Formononetin, daidzein and equol significantly enhanced IL-4 production from both CD4+ T cells and EL4 cells in a dose-dependent manner. Formononetin, daidzein and equol also enhanced IL-4 gene promoter activity in EL4 cells transiently transfected with IL-4 gene promoter constructs, but this effect was impaired in EL4 cells transfected with an IL-4 promoter construct deleted of P4 site carrying nuclear factor of activated T cells (NF-AT) and activator protein-1 (AP-1) binding sites. In addition, formononetin, daidzein and equol increased AP-1 DNA binding activities while did not affect NF-AT DNA binding activities. The enhancing effects on IL-4 production and AP-1 DNA binding activities were abrogated by specific inhibitors for phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC) and p38 mitogen-activated protein kinase (MAPK), indicating that formononetin, daidzein and equol might enhance IL-4 production by increased activation of AP-1 through the PI3-K/PKC/p38 MAPK signalling pathway. These results suggest that phyto-oestrogens and some of their metabolites may increase allergic responses via the enhancement of IL-4 production in T cells.

Topics: Animals; CD4-Positive T-Lymphocytes; Cells, Cultured; DNA-Binding Proteins; Equol; Humans; Interleukin-4; Isoflavones; Lymphocyte Activation; Lymphoma, T-Cell; Mice; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phytoestrogens; Protein Kinase C; T-Lymphocyte Subsets; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Tumor Cells, Cultured

In an international context of promoting scientific research on food safety, the interest in molecules having potential hormonal disrupting effects is growing. While industrial endocrine disruptors (phthalates, alkylphenols, PCBs, etc.) have been studied for several years, natural compounds like phytoestrogens remain less investigated. Accordingly, a research project was initiated with its main objectives to develop efficient analytical methods for a wide range of phytoestrogens in various food matrices, and to evaluate their occurrence in food products. Electrospray ionization with tandem mass spectrometric (MS/MS) analysis of isoflavones (genistein, daidzein, equol, formononetin, biochanin A), lignans (enterolactone, enterodiol), and coumestans (coumestrol) was investigated. This study revealed the formation of a large number of fragment ions in both positive and negative modes, corresponding to specific cleavages of the hydroxyl, carbonyl, and/or methoxy groups, and to Retro-Diels-Alder reactions. An LC/ESI-MS/MS method was developed consistent with the 2002/657/EC European decision criteria. An extraction and clean-up method was developed for milk samples. The identification limit for the proposed method appears to be under 1 ng/mL. The developed methodology was applied to various milk samples, and the occurrence of isoflavones (particularly equol) was demonstrated in the concentration range 1-30 ng/mL. The efficiency of the proposed analytical method permitted evaluation of a new and promising approach to a global risk assessment of natural estrogenic active substances including phytoestrogens and their metabolites.

Topics: 4-Butyrolactone; Animals; Cattle; Chromans; Chromatography, Liquid; Coumestrol; Equol; Estrogens, Non-Steroidal; Genistein; Ions; Isoflavones; Lignans; Milk; Molecular Structure; Phytoestrogens; Plant Preparations; Spectrometry, Mass, Electrospray Ionization

Phytoestrogens are dietary estrogenic contaminants capable of inducing vitellogenin synthesis in rainbow trout and Siberian sturgeon. A competitive-binding assay on their hepatic estrogen receptors (ER) was performed to determine the relative affinity of phytoestrogens compared to estradiol (E(2)). Phytoestrogen concentrations used were 1000 times higher than for E(2), except for genistein and formononetin. For each compound, the competition with 50%-bound labelled E(2) (DC(50)) was considered in order to classify phytoestrogens according to their affinity for ER. The affinities are compared for each species. In rainbow trout, estradiol (DC(50): 7 nM)>formononetin (DC(50): 260 nM)>genistein (DC(50): 570 nM)>equol (DC(50): 5.3 microM)>daidzein (DC(50): 9 microM)>biochanin A (DC(50): 100 microM). In sturgeon, estradiol (DC(50): 5 nM)>genistein (DC(50): 220)>formononetin (DC(50): 1 microM)>equol>(DC(50): 8.3 microM)>daidzein>(DC(50): 80 microM)>biochanin A (DC(50): 100 microM). These results demonstrate that phytoestrogens, mimicking estradiol, can disturb the endocrine system by competing for ER. Also, the higher sensitivity to genistein observed in vivo in Siberian sturgeon (vitellogenin synthesis), compared to rainbow trout, is not due to a higher affinity of genistein for the hepatic ER. Thus, the metabolism of phytoestrogen could be species dependent and affect sensitivity.

Topics: Animals; Binding, Competitive; Estradiol; Estrogens, Non-Steroidal; Female; Fishes; Genistein; Isoflavones; Liver; Oncorhynchus mykiss; Phytoestrogens; Plant Preparations; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Vitellogenins

There is widespread concern that fetal exposure to hormonally active chemicals may adversely affect development of the reproductive tract. Therefore, the present study was performed to develop the necessary analytical methods and test the hypothesis that dietary phytoestrogens can be quantified in second trimester human amniotic fluid. Amniotic fluid samples (n=59) from women (n=53) undergoing routine amniocentesis between 15 and 23 weeks of gestation were analyzed by gas chromatography/mass spectrometric (GC/MS). Analytes included the phytoestrogens daidzein, genistein, formononetin, biochanin A, and coumestrol. Dietary phytoestrogens were quantified in 96.2% of second trimester amniotic fluid samples tested. The mean (+/- standard deviation (S.D.)) concentration of daidzein and genistein in amniotic fluid was 1.44 +/- 1.34 and 1.69 +/- 1.48 ng/ml with maximum levels of 5.52 and 6.54 ng/ml, respectively. Second trimester amniotic fluid contains quantifiable levels of dietary phytoestrogens and thus is a marker of mid pregnancy fetal exposure.

Topics: Adult; Amniocentesis; Amniotic Fluid; Coumestrol; Diet; Estrogens, Non-Steroidal; Female; Gas Chromatography-Mass Spectrometry; Genistein; Humans; Isoflavones; Middle Aged; Phytoestrogens; Plant Preparations; Pregnancy; Pregnancy Trimester, Second

-Estrogens are known to induce cardioprotective effects by inhibiting smooth muscle cell (SMC) growth and neointima formation. However, the use of estrogens as cardioprotective agents is limited by carcinogenic effects in women and feminizing effects in men. If noncarcinogenic and nonfeminizing estrogenlike compounds, such as natural phytoestrogens, afford cardioprotection, this would provide a safe method for prevention of cardiovascular disease in both men and women. Therefore, we evaluated and compared in human aortic SMCs the effects of phytoestrogens (formononetin, genistein, biochanin A, daidzein, and equol) on 2.5% fetal calf serum-induced proliferation (3H-thymidine incorporation and cell number), collagen synthesis (3H-proline incorporation), and total protein synthesis (3H-leucine incorporation) and on PDGF-BB (25 ng/mL)-induced migration (modified Boydens chambers). Moreover, the effects of phytoestrogens on PDGF-BB (25 ng/mL)-induced mitogen-activated protein kinase (MAP kinase) activity in SMCs was also studied. Phytoestrogens inhibited proliferation, collagen and total protein synthesis, migration, and MAP kinase activity in a concentration-dependent manner and in the following order of potency: biochanin A>genistein>equol>daidzein>formononetin. In conclusion, our studies provide the first evidence that in human aortic SMCs phytoestrogens inhibit mitogen-induced proliferation, migration and extracellular matrix synthesis and inhibit/downregulate MAP kinase activity. Thus, phytoestrogens may confer protective effects on the cardiovascular system by inhibiting vascular remodeling and neointima formation and may be clinically useful as a safer substitute for feminizing estrogens in preventing cardiovascular disease in both women and men.

Topics: Adult; Animals; Aorta, Thoracic; Calcium-Calmodulin-Dependent Protein Kinases; Cattle; Cell Division; Cell Movement; Cells, Cultured; Chromans; Culture Media; DNA; Dose-Response Relationship, Drug; Equol; Estrogens, Non-Steroidal; Female; Genistein; Humans; Isoflavones; Kinetics; Male; Muscle, Smooth, Vascular; Phytoestrogens; Plant Preparations; Protein Biosynthesis

Phytoestrogens exert estrogenic effects on the central nervous system, induce estrus, and stimulate growth of the genital tract of female animals. Over 300 plants and plant products, including some used in laboratory animal diets, contain phytoestrogens. Therefore, the source and concentration of phytoestrogens in rodent diets were determined.. Twelve rodent diets and six major dietary ingredients were assayed for phytoestrogens (daidzein, genistein, formononetin, biochanin A, and coumestrol), using high-performance liquid chromatography. Three rodent diets recently formulated to reduce phytoestrogen content also were assayed.. Formononetin, biochanin A, and coumestrol were not detected. Soybean meal was the major source of daidzein and genistein; their concentrations were directly correlated to the percentage of soybean meal in each diet.. High, variable concentrations of daidzein and genistein are present in some rodent diets, and dietary phytoestrogens have the potential to alter results of studies of estrogenicity. Careful attention should be given to diet phytoestrogen content, and their concentration should be reported. A standardized, open-formula diet in which estrogenic substances have been reduced to levels that do not alter results of studies that are influenced by exogenous estrogens is recommended.

Topics: Animal Feed; Animals; Animals, Laboratory; Coumestrol; Estrogens, Non-Steroidal; Female; Food, Formulated; Genistein; Glycine max; Isoflavones; Phytoestrogens; Plant Preparations; Rodentia

Phytoestrogens can exhibit agonistic actions on estrogen-dependent gene expression in breast cancer cells. Since several different phytoestrogens may be found within a single dietary plant source, we sought to investigate whether estrogen-dependent gene expression may be further influenced by the collective treatment of breast cancer cells with multiple phytoestrogens. Accordingly, we transfected MCF-7 breast cancer cells with estrogen-responsive reporters followed by treatment with one of four phytoestrogens (genistein, daidzein, formononetin, and equol) or a combination of these in the absence of estradiol. Our results demonstrated clear-cut agonistic effects of phytoestrogens on estrogen-dependent gene expression. Moreover, combinatorial treatment consistently stimulated reporter activity above that observed for individual phytoestrogens. Inasmuch as the phytoestrogens tested are frequently found together in food sources, these combinatorial responses may more accurately reflect the consequences of in vivo exposure.

Topics: Breast Neoplasms; Chromans; Drug Interactions; Equol; Estrogens; Estrogens, Non-Steroidal; Gene Expression; Genistein; Humans; Isoflavones; Luciferases; Phytoestrogens; Plant Preparations; Transfection; Tumor Cells, Cultured

This study is a summary of Finnish investigations of the phytoestrogen content of legume plants, red clover, white clover, alfalfa, and goat's rue. In addition to the chemical analyses, biological studies were performed. Uterine weight of immature rats was used as an indicator of the estrogenic effect of the fodder used. All red clover varieties studied contained estrogenic isoflavones, especially formononetin and biochanin-A. The phytoestrogen content varied from 1.0% to 2.5% of dry matter. The biological study of white clover showed a clear estrogenic effect not visible through chemical analysis. Alfalfa contains small quantities of formononetin and biochanin-A, but 25-65 ppm coumestrol in dry matter. The estrogenic effect of alfalfa was obvious in the biological study. Goat's rue did not contain any known phytoestrogens, and the biological study was completely negative.

Topics: Animal Feed; Animals; Coumestrol; Estrogens, Non-Steroidal; Fabaceae; Female; Finland; Genistein; Isoflavones; Organ Size; Phytoestrogens; Plant Preparations; Plants, Medicinal; Rats; Uterus

Eight Swedish Finewool Landrace ewes, ovariectomized 5 months earlier and kept on nonestrogenic hay, were each fed 3.5 kg red clover silage, corresponding to 6.1 g phytoestrogens (of which 3.5 g was formononetin) per day, for 14 days in November (short days). In January (short days), two groups (3 each) of these ewes received one or two 17 beta-estradiol sc implants. In May (long days), one of two new groups (4 each) of these ewes was reexposed to phytoestrogens for another 14 days while the other served as a control. Physical examination of ewes for changes in reproductive organs was carried out two or three times per week during each feeding/treatment, and continued until observed changes disappeared. Clinically significant changes occurred in the reproductive organs of ewes fed red clover. Vulva color changed from pale to pink and red, and there were enlargements of the vulva, uterus, and udder. In addition, teat length and circumference increased, and secretion of milky fluid began. These changes were similar, but more pronounced during treatment with 17 beta-estradiol, particularly teat circumference. The changes in vulva were more dramatic in May than in November and resembled those observed in ewes treated with estradiol. Our data show that a daily intake of 3.5 g formononetin for 14 days caused the increase of teat size and changes in the color of the vulva and in uterus weight in ovariectomized ewes.

Topics: Animals; Drug Implants; Estradiol; Estrogens, Non-Steroidal; Fabaceae; Female; Genitalia, Female; Isoflavones; Ovariectomy; Phytoestrogens; Plant Preparations; Plants, Medicinal; Progesterone; Seasons; Sheep; Silage

It is becoming increasingly apparent that dietary factors may play a role in the etiology of hormone dependent neoplasias. It has been hypothesized that estrogens play some role in the etiology of benign prostatic hyperplasia (BPH) in the canine. The presence of estrogen receptor binding activity in a fraction of canine urine purified by high performance liquid chromatography (HPLC) that did not correspond to estriol, estradiol, estrone or any of their primary metabolites was observed in the present study. We used thermospray-mass spectrometry and GC-MS to identify the phytoestrogens daidzein, equol, formononetin and genistein in HPLC purified fractions of urine obtained from male beagles. Using the same techniques we also confirmed the presence of daidzein and genistein in the commercial diet fed to these same dogs. Using the immature rat uterine cytosol estrogen receptor assay, relative binding affinities of 0.08, 1.1, less than 0.01 and 3.9% were obtained for daidzein, equol, formononetin and genistein, respectively when compared to estradiol (100%). In conclusion, phytoestrogens are present in urine of male beagles. Moreover, the commercial diet fed to these dogs contains isoflavones which can be converted to equol by intestinal microflora. These results suggest the need for investigations of phytoestrogens (e.g. equol) excreted into the urine daily and its relationship to the incidence and severity of BPH in the dog.

Topics: Animal Feed; Animals; Binding, Competitive; Chromans; Chromatography, High Pressure Liquid; Dogs; Equol; Estrogens; Estrogens, Non-Steroidal; Gas Chromatography-Mass Spectrometry; Genistein; Isoflavones; Male; Phytoestrogens; Plant Preparations; Prostatic Hyperplasia; Receptors, Estrogen; Reference Values