coumestrol and formononetin

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 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

Cadmium is a serious environmental pollution threats to the planet. Its accumulation in plants affects many cellular functions, resulting in growth and development inhibition, whose mechanisms are not fully understood. However, some fungi forming arbuscular mycorrhizal symbiosis with the majority of plant species have the capacity to buffer the deleterious effect of this heavy metal. In the present work we investigated the capacity of Rhizophagus irregularis (syn. Glomus irregularis) to alleviate cadmium stress in Medicago truncatula. In spite of a reduction in all mycorrhizal parameters, plants colonized for 21 days by R. irregularis and treated by 2 mg kg⁻¹ cadmium displayed less growth inhibition in comparison to plants grown without cadmium. Cadmium strongly increased the accumulation of some isoflavonoids and their derivates: formononetin, malonylononin, medicarpin 3-O-β-(6'-malonylglucoside), medicarpin and coumestrol. Interestingly, in plants colonized by R. irregularis we noticed a strong reduction of the cadmium-induced accumulation of root isoflavonoids, a part for medicarpin and coumestrol. Moreover, transcripts of chalcone reductase, a protein that we reported previously as being down-regulated in R. irregularis-colonized M. truncatula roots, revealed a similar expression pattern with a strong increase in response to cadmium and a reduced expression in cadmium-treated mycorrhizal roots.

Topics: Alcohol Oxidoreductases; Biomass; Cadmium; Coumestrol; Down-Regulation; Gene Expression Regulation, Plant; Glomeromycota; Glucosides; Isoflavones; Medicago truncatula; Mycorrhizae; Plant Proteins; Plant Roots; Plant Shoots; Pterocarpans; Soil; Symbiosis

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

Isoflavones and coumestrol (COU) are estrogenic compounds that are naturally produced by plants (e.g., red clover, soybeans). Although these compounds have been extensively studied in food and feed, only little is known about their environmental fate. Therefore, we investigated the occurrence of isoflavones (formononetin, daidzein, equol, biochanin A, and genistein) and COU over 3.5 years in red clover, manure, and soil of a grassland field with and without manure application, as well as their emission via drainage water. Isoflavones were regularly quantified in plant (≤ 15 × 10(6) ng/g(dry weight (dw))), manure (≤ 230 × 10(3) ng/g(dw)), soil (≤ 3.4 × 10(3) ng/g(dw)), and drainage water samples (≤ 3.6 × 10(3) ng/L). In contrast, COU was observed only in manure and soil. Cumulative isoflavone loads emitted via drainage water were around 0.2 × 10(-3) kg/ha/y, which is very little compared to the amounts present in red clover (105-220 kg/ha/y), manure (0.5-1.0 kg/ha/y), and soil (0.1-5.1 kg/ha/y). Under good agricultural practice, no additional emission of isoflavones into drainage water was observed after manure application. With calculated 17β-estradiol equivalents up to 0.46 ng/L in drainage water, isoflavones can constitute a dominant and ecotoxicological relevant portion of the total estrogenicity in small rural river catchments.

Topics: Coumestrol; Drainage, Sanitary; Ecotoxicology; Environmental Monitoring; Isoflavones; Manure; Poaceae; Rivers; Soil; Switzerland; Trifolium; Water

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

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

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

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

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

Topics: Animals; Coumestrol; Estrogens; Female; Genistein; Glycine max; Isoflavones; Mice; Uterus

The interactions of phytoestrogens with estrogen receptors were studied in the human breast cancer cell line, MCF-7. The compounds tested were coumestrol, genistein, and formononetin and the mycotoxins, zearalenone and its reduced derivative, zearalenol. All but formononetin compete for binding of [3H]-estradiol to unfilled cytoplasmic estrogen receptor or unfilled nuclear estrogen receptor sites. Relative binding affinities are zearalenol HMP (high melting point isomer) greater than zearalenol LMP (low melting point isomer) greater than zearalenone = coumestrol greater than genistein greater than formononetin. Dissociation constants estimated from competition curves show that binding affinities are high. In contrast to estradiol, phytoestrogens bind only weakly to sex steroid-binding globulin; they also do not bind to corticosteroid-binding globulin. These compounds translocate the cytoplasmic estrogen receptor and bind to unfilled nuclear estrogen receptors in whole cells. Bound nuclear receptors are then processed in a manner similar to estradiol in a step which rapidly decreases total cellular estrogen receptors. The phytoestrogens are also biologically active; they can markedly enhance tumor cell proliferation. In sum, phytoestrogens interact with the estrogen receptors of human breast cancer cells in culture and, therefore, may affect estrogen-mediated events in these cells.

Topics: Breast Neoplasms; Cell Line; Coumarins; Coumestrol; Flavonoids; Humans; Isoflavones; Mycotoxins; Receptors, Estrogen; Sex Hormone-Binding Globulin; Zearalenone; Zeranol