The estrogenic activity of certain phytoestrogens in the Siberian sturgeon Acipenser baeri

Various phytoestrogens such as formononetin, daidzein, genistein and equol were synthesized. Their purity was assessed by various analytical techniques including melting point determination, thin-layer chromatography (TLC), infra-red spectra (i.r. spectra), nuclear magnetic resonance (1H- and 13C-NMR) and gas chromatography coupled with mass spectrometry (GC-MS). The estrogenic activity of these compounds, as well as biochanin A and coumestrol, was biologically tested by the induction of vitellogenin secretion in yearling sturgeon and compared to the activity of estradiol-17 beta. Pure daidzein, biochanin A, genistein, equol and coumestrol all had estrogenic activity as assessed by their induction of hepatic synthesis of vitellogenin when administrated intraperitoneally to yearling Siberian sturgeon. Coumestrol seemed to be the most potent compound, inducing the most vitellogenin secretion with the lowest dose administered. Formononetin was inactive when administered by the intraperitoneal route. All the phytoestrogens tested were considerably less potent than estradiol-17 beta.     

Relationship between estrogen receptor-binding and estrogenic activities of environmental estrogens and suppression by flavonoids

In this study, we investigated the estrogenic activity of environmental estrogens by a competition binding assay using a human recombinant estrogens receptor (hERbeta) and by a proliferation assay using MCF-7 cells and a sulforhodamine-B assay. In the binding assay, pharmaceuticals had a stronger binding activity to hERbeta than that of some phytoestrogens (coumestrol, daidzein, genistein, luteolin, chrysin, flavone, and naringenin) or industrial chemicals, but phytoestrogens such as coumestrol had a binding activity as strong as pharmaceuticals such as 17alpha-ethynylestradiol (EE), tamoxifen (Tam), and mestranol. In the proliferation assay, pharmaceuticals such as diethylstilbestrol, EE, Tam, and clomiphene, and industrial chemicals such as 4-nonylphenol, bisphenol A, and 4-dihydroxybiphenyl had a proliferation-stimulating activity as strong as 17beta-estradiol (ES). In addition, we found that phytoestrogens such as coumestrol, daidzein, luteolin, and quercetin exerted a proliferation stimulating activity as strong as ES. Furthermore, we examined the suppression of proliferation-stimulating activity, induced by environmental estrogen, by flavonoids, such as daidzein, genistein, quercetin, and luteolin, and found that these flavonoids suppressed the induction of the proliferation-stimulating activity of environmental estrogens. The suppressive effect of flavonoids suggests that these compounds have anti-estrogenic and anti-cancer activities.     

Phytoestrogen content of purified, open- and closed-formula laboratory animal diets.

BACKGROUND AND PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Interaction of phytoestrogens and other environmental estrogens with prostaglandin synthase in vitro

The phytoestrogens daidzein, genistein, equol and coumestrol were found to stimulate microsomal prostaglandin H synthase (PHS) in vitro in a concentration-dependent manner when PHS-activity was measured by arachidonic acid-dependent oxygen uptake. These compounds were co-oxidized by PHS and the conversion of parent compounds was measured by HPLC analysis. The stimulation of PHS-cyclooxygenase by these compounds was partially reversed at high concentrations probably due to their antioxidant properties causing inhibition. In contrast, the monomethyl ethers of daidzein and genistein, formononetin and biochanin A, had little or weakly inhibitory effect on PHS, and appear to be no or poor co-substrates for PHS. Compared to the equine estrogen equilin, its metabolite d-equilenin was poorly metabolized by PHS and inhibited rather than stimulated PHS-cyclooxygenase activity in vitro. The resorcylic acid lactones zearalenone and zeranol, on the other hand, were surprisingly good inhibitors of PHS-cyclooxygenase. Furthermore, zeranol inhibited both the arachidonic acid and the hydrogen-peroxide-dependent oxidation of DES in contrast to indomethacin which inhibited only cyclooxygenase-dependent co-oxidation of DES. The results of this in vitro study are discussed in the context of data on synthetic and steroidal estrogens and support the idea that PHS-activity may be modulated by interaction with certain estrogenic compounds.     

Acute effects of three isoflavone class phytoestrogens and a mycoestrogen on cerebral microcirculation

Phytoestrogens and mycoestrogens are naturally occurring plant and fungus secondary metabolites with estrogen-like structure and/or actions. We aimed to check the hypothesis that phytoestrogens and mycoestrogens, due to their ability to elicit cerebral vasodilation, can induce acute increases in brain blood perfusion. For this purpose, we continuously recorded cerebrocortical perfusion by laser-Doppler flowmetry in anesthetized rats receiving intracarotid infusions (1 mg/kg) of one of the following estrogenic compounds: biochanin A, daidzein, genistein or zearalanone. We have shown the ability of two isoflavone class phytoestrogens (daidzein and biochanin A) and the mycoestrogen zearalanone to induce acute increases in brain blood flow when locally infused into the cerebral circulation of anesthetized rats. The isoflavone genistein failed to induce a significant increase in brain perfusion. No concomitant changes in blood pressure were recorded during the cerebral effects of the estrogenic compounds. Therefore, these microcirculatory effects were due to direct actions of the estrogenic compounds on the cerebrovascular bed.     

湖南八大公山的植物区系及其在植物地理学上的意义

湖南八大公山自然保护区是华中地区面积大且植物保存较完好的地点。土著种子植物计162科、709属、1775种。全部属按15个地理分布型进行分析,全部种按更细的分布型类别（中国特有分为10个亚型）进行分析。结果表明,本地热带分布的科属虽为数不少,但温带分布的科属更显优势,北温带科属比较集中,同时中国、东亚、及东亚一北美特有属也很集中。本山中国特有种计1120种,华中特有种为347种。植物区系的古老、残遗及特有性突出,可视为中国（华中）有典型代表性的山地。     

Daidzein, coumestrol and zearalenone affect lipogenesis and lipolysis in rat adipocytes

Daidzein, coumestrol and zearalenone - compounds called phytoestrogens, considered as active biological factors affecting many important physiological and biochemical processes appeared to be also significant regulators of adipocyte metabolism. In our experiments the influence of daidzein (0.01, 0.1 and 1 mM), coumestrol (0.001, 0.01 and 0.1 mM), zearalenone (0.01, 0.1 and 1 mM) and estradiol (0.01, 0.1 and 1 mM) on basal and insulin-stimulated (1 nM) lipogenesis from glucose and acetate was tested in adipocytes isolated from growing (160 +/- 5 g b.w) male Wistar rats. All tested compounds significantly attenuated glucose conversion to lipids. In the case of daidzein and coumestrol, this effect was probably due to inhibition of glycolysis. Daidzein (0.01, 0.1 and 1 mM), coumestrol (0.01 and 0.1 mM) and zearalenone (0.01, 0.1 and 1 mM) affected also basal and epinephrine-stimulated (1 microM) lipolysis. Daidzein (0.01 and 1 mM) augmented basal glycerides breakdown in adipocytes. The epinephrine-induced lipolysis was dependent on daidzein concentration and its stimulatory (0.1 mM) or inhibitory (1 mM) influence was observed. Zearalenone changed lipolysis only at the concentration of 1 mM and its effect was contradictory in the absence or presence of epinephrine (the stimulatory or inhibitory effect, respectively). Results obtained in experiments with inhibitors (insulin, 1 nM and H-89, 50 microM) and activators (dibutyryl-cAMP, 1 mM and forskolin, 1 microM) of lipolysis allowed us to assume that daidzein augmented basal lipolysis acting on PKA activity. The inhibitory effect of daidzein and zearalenone on epinephrine-induced lipolysis is probably due to restriction of HSL action. The influence of coumestrol on glycerides breakdown was less marked. Estradiol augmented only epinephrine-stimulated lipolysis.     

Expression profiling of the estrogen responsive genes in response to phytoestrogens using a customized DNA microarray

Here, we examined phytoestrogens, isoflavones (genistein, daidzein, glycitein, biochanin A and ipriflavone), flavones (chrysin, luteolin and apigenin), flavonols (kaempferol and quercetin), and a coumestan, a flavanone and a chalcone (coumestrol, naringenin and phloretin, respectively) by means of a DNA microarray assay. A total of 172 estrogen responsive genes were monitored with a customized DNA microarray and their expression profiles for the above phytoestrogens were compared with that for 17beta-estradiol (E2) using correlation coefficients, or R values, after a correlation analysis by linear regression. While R values indicate the similarity of the response by the genes, we also examined the genes by cluster analysis and by their specificity to phytoestrogens (specific to genistein, daidzein or glycitein) or gene functions. Several genes were selected from p53-related genes (CDKN1A, TP53I11 and CDC14), Akt2-related genes (PRKCD, BRCA1, TRIB3 and APPL), mitogen-activated protein kinase-related genes (RSK and SH3BP5), Ras superfamily genes (RAP1GA1, RHOC and ARHGDIA) and AP-1 family and related genes (RIP140, FOS, ATF3, JUN and FRA2). We further examined the extracts from two local crops of soy beans (Kuro-daizu or Mochi-daizu) by comparing the gene expression profiles with those of E2 or phytoestrogens as a first step in utilizing the expression profiles for various applications.     

The effects of dietary phytoestrogens on aromatase activity in human endometrial stromal cells

Dietary phytoestrogens have been reported to inhibit aromatase activity in placental microsomes, but the effects in the human endometrium are unknown. Aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, has recently been shown to be expressed in the endometrium of women with endometriosis and is thought to play a role in the pathophysiology of this disease. Therefore, the objective of this study was to screen dietary phytoestrogens for their ability to inhibit aromatase activity in human endometrial stromal cells (ESC) and identify potential novel therapeutic agents for the treatment of endometriosis. The inhibition of aromatase activity by direct interaction with the dietary phytoestrogens genistein, daidzein, chrysin, and naringenin was tested in a cell free assay. Furthermore, test compound effects on aromatase activity in ESC cultures were also examined. Genistein and daidzein were inactive in the human recombinant aromatase assay whereas naringenin and chrysin inhibited aromatase activity. However, genistein (1 nM to 1 mM) stimulated aromatase activity in ESC whereas other phytoestrogens had no effect. Immunopositive aromatase cells were demonstrated in genistein-treated ESC but not in untreated control cultures. Taken together, our data suggest that genistein can increase aromatase activity in ESC likely via increased enzyme expression.     

Phytoestrogens directly inhibit TNF-α-induced bone resorption in RAW264.7 cells by suppressing c-fos-induced NFATc1 expression

TNF-α-induced osteoclastogenesis is central to post-menopausal and inflammatory bone loss, however, the effect of phytoestrogens on TNF-α-induced bone resorption has not been studied. The phytoestrogens genistein, daidzein, and coumestrol directly suppressed TNF-α-induced osteoclastogenesis and bone resorption. TRAP positive osteoclast formation and resorption area were significantly reduced by genistein (10(-7) M), daidzein (10(-5) M), and coumestrol (10(-7) M), which was prevented by the estrogen antagonist ICI 182,780. TRAP expression in mature TNF-α-induced osteoclasts was also significantly reduced by these phytoestrogen concentrations. In addition, in the presence of ICI 182,780 genistein and coumestrol (10(-5) -10(-6) M) augmented TNF-α-induced osteoclast formation and resorption. However, this effect was not observed in the absence of estrogen antagonist indicating that genistein's and coumestrol's ER-dependent anti-osteoclastic action normally negates this pro-osteoclastic effect. To determine the mechanism mediating the anti-osteoclastic action we examined the effect of genistein, coumestrol, and daidzein on caspase 3/7 activity, cell viability and expression of key genes regulating osteoclast differentiation and fusion. While anti-osteoclastic phytoestrogen concentrations had no effect on caspase 3/7 activity or cell viability they did significantly reduce TNF-α-induced c-fos and NFATc1 expression in an ER dependent manner and also inhibited NFATc1 nuclear translocation. Significant decreases in NFκB and DC-STAMP levels were also noted. Interestingly, constitutive c-fos expression prevented the anti-osteoclastic action of phytoestrogens on differentiation, resorption and NFATc1. This suggests that phytoestrogens suppress TNF-α-induced osteoclastogenesis via inhibition of c-fos-dependent NFATc1 expression. Our data provides further evidence that phytoestrogens have a potential role in the treatment of post-menopausal and inflammatory bone loss directly inhibiting TNF-α-induced resorption.     

反相高效液相层析法测定新鲜植物材料中的植物雌激素含量

通过适当的样品处理方法,游离的和结合的植物雌激素［大豆素,雌马酚,染料木素,芒柄花素,香豆雌酚和美皂异黄酮］被从新鲜植物材料的提取物中分离出来,并在不同的紫外光波长下,可被ＨＰＬＣ法定量测定,根据滞留时间和标准品的添加,而鉴别出植物雌激素的层析波峰。本方法的测定灵敏度为２ｐｐｍ。白三叶草样品的加样回收率在８０％－１００％之间（平均回收率变异系数为５．４％）。通过比较游离植物雌激素的含量测定,本方法     

The influence of coumestrol,zearalenone, and genistein administration on insulin receptors and insulin secretion in ovariectomized rats

The influence of phytoestrogens (genistein and coumestrol) and mycoestrogen (zearalenone) on insulin secretion, liver insulin receptors and some aspects of lipid and carbohydrate metabolism were investigated in this study. Ovariectomized rats were injected s.c. with the above mentioned compounds in the amount of 1 mg for three days. Coumestrol and zearalenone caused a significant increase in uterus weight, similar to the effects observed after estrone action, while this effect was not observed after the genistein injection. Blood insulin level was not changed after phyto- or mycoestrogen treatment. However, coumestrol and genistein significantly decreased the binding capacity of liver insulin receptors. These changes corresponded with alterations in glucose and free fatty acids profiles in blood, as well as with glycogen content in liver. The effects observed after genistein and coumestrol injections differed from those noticed in rats treated with zearalenone or estrone. On the basis of these results we conclude that metabolic effects of high doses of coumestrol and genistein in ovariectomized rats are partly mediated by changes in insulin sensitivity of the liver and that the action of plant estrogens on metabolism is, at least to the some degree, independent of their estrogen activity.     

THE INFLUENCE OF COUMESTROL,ZEARALENONE, AND GENISTEIN ADMINISTRATION ON INSULIN RECEPTORS AND INSULIN SECRETION IN OVARIECTOMIZED RATS

ABSTRACT

The influence of phytoestrogens (genistein and coumestrol) and mycoestrogen (zearalenone) on insulin secretion, liver insulin receptors and some aspects of lipid and carbohydrate metabolism were investigated in this study. Ovariectomized rats were injected s.c. with the above mentioned compounds in the amount of 1?mg for three days. Coumestrol and zearalenone caused a significant increase in uterus weight, similar to the effects observed after estrone action, while this effect was not observed after the genistein injection. Blood insulin level was not changed after phyto- or mycoestrogen treatment. However, coumestrol and genistein significantly decreased the binding capacity of liver insulin receptors. These changes corresponded with alterations in glucose and free fatty acids profiles in blood, as well as with glycogen content in liver. The effects observed after genistein and coumestrol injections differed from those noticed in rats treated with zearalenone or estrone. On the basis of these results we conclude that metabolic effects of high doses of coumestrol and genistein in ovariectomized rats are partly mediated by changes in insulin sensitivity of the liver and that the action of plant estrogens on metabolism is, at least to the some degree, independent of their estrogen activity.     

Dose-response effects of phytoestrogens on the activity and expression of 3beta-hydroxysteroid dehydrogenase and aromatase in human granulosa-luteal cells

There is evidence that certain phytoestrogens can inhibit key steroidogenic enzymes although most studies have been carried out on microsomal or purified enzyme preparations, some using cell lines. This study was designed to test the hypothesis that low doses of phytoestrogens, at concentrations that would be attained through the diet, could inhibit 3beta-hydroxysteroid dehydrogenase (HSD) and/or aromatase in primary cultures of human granulosa-luteal (GL) cells and that this effect was due to a decrease in the expression of these proteins. Based on published evidence, eight compounds were selected for investigation and these included the flavones apigenin and quercetin, the isoflavones genistein, biochanin A and daidzein, the lignans, enterodiol and enterolactone, and the mycotoxin zearalenone. Human GL cells were cultured for 48 h in the presence of these phytoestrogens at concentrations ranging from 0.01 to 100 microM and after addition of fresh media the conversion of pregnenolone to progesterone or androstenedione to oestradiol over a 4h period was measured. Biochanin A was the only phytoestrogen that displayed any dose-dependent inhibition of 3beta-HSD, others showing inhibition at doses >/=10 microM. Apigenin and quercetin only inhibited aromatase/17beta-HSD at high doses as did genistein, biochanin A and daidzein. The lignans had weak inhibitory effects on aromatase/17beta-HSD, whilst zearalenone showed potent inhibition at 0.1 microM. Phytoestrogens did not exert any significant effects on protein expression of 3beta-HSD or aromatase as determined by Western blots. It is concluded that steroidogenic enzymes are inhibited by phytoestrogens in primary cultures of human GL cells but these cells are less sensitive to the effects of phytoestrogens than cell-free systems. This may be due to poor lipid solubility or cellular metabolism. We have also shown for the first time that phytoestrogens do not act by inhibiting the cellular concentration of 3beta-HSD and aromatase even though exposure time would have allowed for changes in gene expression.     

Effect of mycorrhization on the isoflavone content and the phytoestrogen activity of red clover

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.     

The effects of ACTH, phytoestrogens and estrogens on corticosterone secretion by gander adrenocortical cells in breeding and nonbreeding seasons

The aim of this study was to investigate the effects of ACTH, phytoestrogens (genistein, daidzein, biochanin A and coumestrol), and animal estrogens (estradiol and estrone) on corticosterone secretion by isolated adrenocortical cells of the ganders in breeding (April) and nonbreeding seasons (July). ACTH stimulated corticosterone output in the breeding season. In July (photorefractoriness and postbreeding molt) ACTH had no effect on corticosterone production. Coumestrol reduced corticosterone secretion by the cells obtained in nonbreeding season. Other examined phytoestrogens did not affect corticosterone production. Estrogens showed differentiated effects. Estradiol stimulated the corticosterone output in breeding season; estrone inhibited corticosterone release in July. The season can probably affect sensitivity of isolated gander adrenal cells, especially to ACTH. It seems that goose adrenocortical cells, in contrast to the mammalian cells, can be weakly sensitive to phytoestrogens.     

Lack of mutagenicity of some phytoestrogens in the salmonella/mammalian microsome assay

8 phytoestrogens were tested for mutagenicity using a variation of the Salmonella/mammalian microsome (or Ames) assay. Zearalenone is a mycotoxin produced by a grain contaminant, Fusarium graminearum (Gibberella zeae) and the isomers of zearalanol are reduced derivatives of this compound. The remaining compounds are all flavonoids which occur naturally at relatively high concentrations in many plants, particularly legumes. 4 of these flavonoids (daidzein, genistein, formononetin and biochanin-a) are isoflavones and the 5th, coumestrol, is a coumestan. Each compound was tested at several concentrations ranging from 1--500 micrograms per plate. The microsomal fracton was obtained from Aroclor 1254 (a PCB)-induced rat livers. None of the compounds tested was mutagenic to Salmonella strains TA1538, TA98 or TA100 at any concentration.