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.     

Receptor binding and transactivation activities of red clover isoflavones and their metabolites

Red clover extracts contain a variety of isoflavones, which have affinity toward estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), androgen receptor (AR), and progesterone receptor (PR). Upon ingestion, they undergo various metabolic transformations. For a complete evaluation of red clover extracts and possible health benefits, the resulting metabolites should also be investigated. Biochanin A, formononetin, genistein, daidzein, dihydrobiochanin A, dihydroformononetin, dihydrogenistein, dihydrodaidzein, 3'-hydroxygenistein, 6-hydroxydaidzein, 6-hydroxydesmethylangolensin, equol, O-desmethylangolensin, angolensin, and p-ethylphenol were tested for their transactivation potential toward ERalpha, AR, and PR in yeast. Competitive binding assays with radiolabeled 17beta-estradiol, 17alpha-methyltrienolone or progesterone assessed binding to the respective ERalpha and ERbeta, AR, and PR. The compounds showed only weak binding affinity to AR and PR, with IC(50) values being greater (i.e., lesser affinity) than 10(-5)M for the respective receptor. So far, beneficial health effects have been attributed to the production of equol. We propose that other metabolites can also contribute to these effects. However, more detailed information for the formation of these metabolites in humans and for bioavailability data are required to confirm our assumptions.     

Oxidative metabolism and genotoxic potential of major isoflavone phytoestrogens

The soy isoflavones daidzein, genistein and glycitein are extensively metabolized by rat liver microsomes to a variety of catechol metabolites. Hydroxylated metabolites of daidzein and genistein have also been demonstrated in incubations with human hepatic microsomes and in the urine of humans after ingestion of soy food. Although the microsomal metabolism of formononetin and biochanin A is dominated by demethylation to daidzein and genistein, respectively, catechols of the parent isoflavones and of the demethylation products are also formed. Thus, oxidative metabolism appears to be common among isoflavones and may have implications for their biological activities. As genistein but not daidzein exhibits clastogenic activity in cultured mammalian cells, the role of oxidative metabolism for the genotoxicity of isoflavones is of particular interest.     

Isoflavone Content in Subterranean Clover Germplasm from Sardinia

Subterranean clover (Trifolium subterraneum) is an important pasture legume, and Sardinia is known as a major centre of diversification of this species. As other legumes, this clover produces biologically active flavonoids including the subclass of isoflavones that are natural phytoestrogens with positive health effects. Present sources of isoflavones for medical/nutraceutical treatments are red clover (Trifolium pratense) and soybean (Glycine max). This study assessed the content and composition of flavonoids in 14 subterranean clover genotypes from Sardinia, grown ex‐situ in comparison with two red clover ecotypes, to acquire information on the potential of the species as an alternative source of isoflavones for possible exploitation. Twenty compounds were tentatively identified across the two clovers after HPLC and LC/ESI‐MS analyses, including clovamide, four flavonols, and 15 isoflavones. Most compounds were present as glucosides or glucosyl malonates. Subterranean clover extracts mainly comprised of derivatives of the isoflavones genistein, biochanin A, and formononetin. Compared to red clover, subterranean clover had higher content of total isoflavones and lower concentration of total flavonols. The isoflavone concentration in subterranean clover was higher than literature data for soybean or red clover. The existing genotypic variation warrants the possibility of selecting varieties with high isoflavone concentration for nutraceutical or pharmaceutical purposes.     

Quantification of isoflavones in red clover by high-performance liquid chromatography

An RP-HPLC method for the determination of daidzein, genistein, formononetin and biochanin A in red clover (Trifolium pratense L.) was developed and validated. The compounds are quantified after hydrolytic extraction using an internal standard. On a base-deactivated C(18) column good separation of the analytes, also from accompanying substances, and excellent peak shape are achieved by gradient elution with aqueous sulfuric acid and acetonitrile. The method was applied to the analysis of different red clover cultivars.     

The effects of soy isoflavones on obesity

Over the last decades, the prevalence of obesity and related diseases has increased rapidly in the Western world. Obesity is a disorder of energy balance and is associated with hyper-insulinemia, insulin resistance, and abnormalities in lipid metabolism, and it is one of the most important risk factors in the development of Type II diabetes, cardiovascular disease, atherosclerosis, and certain cancers. Because of the lower frequency of these diseases in Asian countries, attention has been turned toward the Asian diet, which consists highly of soy and soy-based products. The health benefits associated with soy consumption have been linked to the content of isoflavones, the main class of the phytoestrogens. As a result of their structural similarities to endogenous estrogens, isoflavones elicit weak estrogenic effects by competing with 17beta-estradiol (E2) for binding to the intranuclear estrogen receptors (ERs) and exert estrogenic or antiestrogenic effects in various tissues. The estrogenic activities of soy isoflavones are thought to play an important role in their health-enhancing properties. Additionally, the isoflavones have been proved to exert non-ER-mediated effects through numerous other pathways. Genistein, daidzein, and glycitein are the principal isoflavones in soy. Genistein is the most thoroughly examined of these, because it is the most prevalent isoflavone in soy and the most active of these compounds, because of its higher binding affinity for the ER. Genistein and daidzein can be obtained in high levels in humans under certain nutritional conditions, and epidemiologic and laboratory data suggest that these compounds could have health benefits in human obesity. This review will focus on the latest results of research on isoflavones and their effect on obesity in cell cultures, rodents, and humans.     

Chemical Studies on “Clover Sickness”

Two new isoflavonoids were isolated from red clover as germination inhibitors for the same plant and their structures were determined as a glucoside of biochanin A (7-d-β-glucosyl-5,7-dihydroxy-4′-methoxyisoflavone) (II) and its 5-malonate (I), respectively. Besides these compounds the following substances were also isolated as inhibitors: trifolirhizin (III), ononin (IV), daidzein (V) and its 7-glucoside (VI), formononetin (VII), genistein (VIII) and biochanin A (IX).     

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.     

Identification of isoflavone metabolites dihydrodaidzein, dihydrogenistein, 6'-OH-O-dma, and cis-4-OH-equol in human urine by gas chromatography-mass spectroscopy using authentic reference compounds.

The metabolic products of daidzein and genistein, the principal isoflavones of soy, were examined. Six volunteers included soy into their normal diet for a 2-week period and urine samples were analyzed before and after soy consumption. Isolation and characterization of the urinary metabolites were carried out with absorption chromatography on Sephadex LH-20 and gas chromatography-electron ionization mass spectrometry (GC-EIMS). The structures of the isoflavones isolated were confirmed by using authentic reference compounds. Dihydrogenistein, 6'-OH-O-desmethylangolensin, and cis-4-OH-equol were identified, in addition to known isoflavonoids daidzein, genistein, glycitein, and the known metabolites equol, O-desmethylangolensin, and dihydrodaidzein, by comparing the retention times and the spectra of the urinary compounds with those of the synthesized reference standards. The mammalian lignans enterolactone and enterodiol were also identified. Derivatization of the isoflavones for GC-MS was examined by comparing two silylating reagents, N, O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) and pyridine:hexamethyldisilazan:trimethylchlorosilane (QSM), both used for the derivatization of these compounds. The silylation experiments revealed significant differences in the compositions of the derivatization products. Some corrections were made concerning the earlier published data of dihydrogenistein and 6'-OH-O-dma.     

Metabolic O-demethylation does not alter the influence of isoflavones on the biophysical properties of membranes and MRP1-like protein transport activity

Multidrug resistance transporter MRP1 could be effectively inhibited by some flavonoids. The influence of the two pairs of isoflavones: formononetin/daidzein and biochanin A/genistein on the efflux of fluorescent substrate of MRP1-like protein from erythrocytes and biophysical properties of lipid membranes has been compared. Compounds in each pair differ by the substituent in position 4' of B ring of isoflavone molecule. In the process of O-demethylation, CH(3) group (present in formonetin and biochanin A) is replaced by hydrogen (daidzein, genistein). Inhibition of MRP1-like protein transport activity by methylated and demethylated isoflavones was very similar. Their influence on lipid thermotropic properties and fluidity of lipid bilayer was not also significantly different.     

Inhibition of angiogenesis and inflammation by an extract of red clover (Trifolium pratense L.)

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 μg/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.     

Phyto-oestrogens in herbage and milk from cows grazing white clover, red clover, lucerne or chicory-rich pastures

A grazing experiment was carried out to study the concentration of phyto-oestrogens in herbage for cattle and in milk during two periods (May and June). Forty-eight Danish Holstein cows were divided into four groups with four treatment diets; white clover, red clover, lucerne and chicory-rich pastures. Each experimental period lasted 15 days. Herbage samples from the first day and individual milk samples from the last day of the experimental period were analysed for phyto-oestrogens using LC-MS technique. The total concentration of phyto-oestrogens was 21 399 mg/kg dry matter (DM) for red clover and 238 to 466 mg/kg DM for the other three herbages mainly due to a much higher concentration of biochanin A, formononetin and glycitein in red clover. In the milk, the total concentration of phyto-oestrogens was 253 to 397 μg/l for red clover milk and 56 to 91 μg/l in the milk from the other three treatments. This was especially due to a higher concentration of equol, daidzein and formononetin in the red clover milk. The concentration of biochanin A was significantly higher in milk from the red clover treatment in May while no differences were observed in June. Enterodiol was similar across treatments while the concentration of enterolactone was significantly lower for red clover milk compared with the other treatments. Of the tested pastures, red clover appears to have the highest concentration and to be the best source of phyto-oestrogens, especially equol, in bovine milk.     

Identification of isoflavones in Acca sellowiana and two Psidium species (Myrtaceae)

Several types of isoflavonoid-like immunoreactivity were detected in water-ethanolic extracts from Acca sellowiana (Berg) Burret, Psidium guajava L. and Psidium littorale Raddi (Myrtaceae) leaves. Chromatographic mobility of the immunoreactive substances was compared to that of authentic standards, revealing a spectrum of isoflavonoid metabolites in both genera. Aglycones as well as glycosides were detected, namely daidzin, genistin, daidzein, genistein, formononetin, biochanin A, prunetin, and several incompletely characterized isoflavones. Subsequent HPLC–MS study verified the identities of the main immunoreactive isoflavones and found several others, namely glycitein, glycitin, ononin, sissotrin, including the malonylated and acetylated glucosides. It is concluded that the isoflavonoid metabolic pathway is present in the Myrtaceae family.     

Isoflavones stimulate estrogen receptor-mediated core histone acetylation

The isoflavones genistein and daidzein and the daidzein metabolite equol have been reported to interact with estrogen receptors (ERs). Some studies indicate that they behave clinically like estrogen in some estrogen-deficiency diseases. However, the detailed molecular mechanism used by these compounds to create beneficial effects in patients with estrogen-related diseases has not been clarified. Using histone acetyltransferase (HAT) assay, we found that equol, genistein, and AglyMax had significant effects on ERalpha-mediated histone acetylation. Although 17beta-estradiol (E2)-dependent HAT activity of steroid receptor coactivators 2 (SRC2) and p300 mediated by ERbeta could be detected, it was weaker than that mediated by ERalpha. Equol, genistein, AglyMax, and daidzein all markedly stimulated ERbeta-mediated histone acetylation. On the other hand, anti-estrogenic compounds ICI 182,780 (ICI) and tamoxifen (TA) did not have an effect on HAT activity mediated by either ERalpha or ERbeta. Our data indicate that estrogenic ligands exert their effects by elevating histone acetylation and coactivator activity of ER, and suggest that the risk of estrogen-related diseases might be reduced by a sufficient amount of genistein or AglyMax supplements.     

Metabolism of the soy isoflavones daidzein, genistein and glycitein in human subjects. Identification of new metabolites having an intact isoflavonoid skeleton

Epidemiological studies have associated high soy intake with a lowered risk for certain hormone-dependent diseases. Soy and soy foods are rich sources of isoflavones, which have been shown to possess several biological activities. In this study, the metabolism of soy isoflavones daidzein, genistein and glycitein was investigated in human subjects. The aim was to find and identify urinary phase I metabolites of isoflavones, which have an intact isoflavonoid skeleton, and which might possess some bioactivity. Six volunteers included three soy bars per day into their normal western diet for a 2-week period. Daily urine samples were collected before, and after the supplementation period. Urine samples were hydrolyzed with Helix pomatia, extracted with diethyl ether, purified with Sephadex LH-20 chromatography, and analyzed as trimethylsilyl derivatives using gas chromatography–mass spectrometry (GC–MS). The structures of the isoflavone metabolites were identified using authentic reference compounds. The metabolites, for which authentic reference compounds were not available, were identified by the interpretation of mass spectra. Several new isoflavone metabolites were identified, and the presence of previously reported metabolites confirmed. The metabolic pathways of daidzein, genistein and glycitein are presented on the basis of the identification of the metabolites in human urine after soy supplementation.     

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.     

Inhibition of angiogenesis and inflammation by an extract of red clover (Trifolium pratense L.)

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 μg/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.     

Lactofen induces isoflavone accumulation and glyceollin elicitation competency in soybean

Lactofen, the active ingredient of the soybean disease resistance-inducing herbicide, Cobra, induces large accumulations of isoflavone conjugates and aglycones in soybean tissues. The predominant isoflavones induced in cotyledon tissues are daidzein (and its conjugates) and formononetin and glycitein aglycones. The latter two isoflavones are usually present only at very low levels in soybean seedling tissues. In leaves, the predominant lactofen-induced isoflavones are daidzein and formononetin aglycones and the malonyl-glucosyl conjugate of genistein. Isoflavone induction also occurs in cells distal to the point of treatment, but is only weakly systemic. Lactofen also induces elicitation competency, the capacity of soybean cells to accumulate the pterocarpan phytoalexin glyceollin in response to glucan elicitors from the cell wall of the pathogen Phytophthora sojae. Comparison of the activity of a series of diphenyl ether herbicides demonstrated that while all diphenyl ethers tested induced some degree of elicitation competency, only certain ones induced isoflavone accumulation in the absence of glucan elicitor. As a group the diphenyl ethers are thought to inhibit protoporhyrinogen oxidase, eventually leading to singlet oxygen generation. Another singlet oxygen generator, rose bengal, also induced elicitation competency, but little isoflavone accumulation. It is hypothesized that diphenyl ether-induced activated oxygen species mimic some aspects of hypersensitive cell death, which leads to elicitation competency in infected tissues.