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.     

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.     

大豆异黄酮抗肿瘤效应研究进展(综述)

大豆异黄酮是大豆生长过程中形成的一类次生代谢产物,对人体具有多种生理功能。研究表明,大豆异黄酮具有抗肿瘤、预防肿瘤作用,是一种很有潜力的癌症化学预防剂,能显著降低多种恶性肿瘤的发病率。现将近几年大豆异黄酮抗肿瘤效应及其机制的研究进展综述如下,以期为大豆异黄酮的深入研究及开发利用提供参考。     

大豆异黄酮微生物转化研究进展

大豆异黄酮是大豆在其生长过程中形成的一类次生代谢产物,具有抗氧化、抗癌、减少骨质流失、降低心脑血管发病率等多种生理功能。目前已知,被摄人机体的大豆异黄酮将被肠道微生物菌群转化为具有更高、更广生物学活性的不同产物。因此,大豆异黄酮对人体的有益调节作用强弱并不简单取决于摄人机体的净含量的多少,更在于被摄人机体的大豆异黄酮将如何被肠道菌群转化。本文从大豆异黄酮的组成与功能、大豆异黄酮体内吸收、代谢及微生物转化、转化产物的活性以及高效合成等方面进行了系统综述,对大豆异黄酮微生物生物转化研究现状和存在问题进行分析总结,并对今后发展趋势进行展望,旨在推动高活性大豆异黄酮微生物转化产物的研究与开发。     

Genistein and daidzein induce cell proliferation and their metabolites cause oxidative DNA damage in relation to isoflavone-induced cancer of estrogen-sensitive organs

The soy isoflavones, genistein (5,7,4'-trihydroxyisoflavone) and daidzein (7,4'-dihydroxyisoflavone), are representative phytoestrogens that function as chemopreventive agents against cancers, cardiovascular disease, and osteoporosis. However, recent studies indicated that genistein and/or daidzein induced cancers of reproductive organs in rodents, such as the uterus and vulva. To clarify the molecular mechanisms underlying the induction of carcinogenesis by soy isoflavones, we examined the ability of genistein, daidzein, and their metabolites, 5,7,3',4'-tetrahydroxyisoflavone (orobol), 7,3',4'-trihydroxyisoflavone (7,3',4'-OH-IF), and 6,7,4'-trihydroxyisoflavone (6,7,4'-OH-IF), to cause DNA damage and cell proliferation. An E-screen assay revealed that genistein and daidzein enhanced proliferation of estrogen-sensitive breast cancer MCF-7 cells, while their metabolites had little or no effect. A surface plasmon resonance sensor showed that binding of isoflavone-liganded estrogen receptors (ER) to estrogen response elements (ERE) was largely consistent with cell proliferative activity of isoflavones. Orobol and 7,3',4'-OH-IF significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in human mammary epithelial MCF-10A cells, while genistein, daidzein, and 6,7,4'-OH-IF did not. Experiments using isolated DNA revealed a metal-dependent mechanism of oxidative DNA damage induced by orobol and 7,3',4'-OH-IF. DNA damage was enhanced by the addition of endogenous reductant NADH, formed via the redox cycle. These findings suggest that oxidative DNA damage by isoflavone metabolites plays a role in tumor initiation and that cell proliferation by isoflavones via ER-ERE binding induces tumor promotion and/or progression, resulting in cancer of estrogen-sensitive organs.     

Molecular mechanisms of action of the soy isoflavones includes activation of promiscuous nuclear receptors. A review

Consumption of soy has been demonstrated to reduce circulating cholesterol levels, most notably reducing low-density lipoprotein (LDL) cholesterol levels in hypercholesterolemic individuals. The component or components that might be responsible for this effect is still a matter of debate or controversy among many researchers. Candidate agents include an activity of soy protein itself, bioactive peptides produced during the digestive process, or the soy isoflavones. Although soy intake may provide other health benefits including preventative or remediative effects on cancer, osteoporosis and symptoms of menopause, this review will focus on isoflavones as agents affecting lipid metabolism. Isoflavones were first discovered as a bioactive agent disrupting estrogen action in female sheep, thereby earning the often-used term 'phytoestrogens'. Subsequent work confirmed the ability of isoflavones to bind to estrogen receptors. Along with the cholesterol-lowering effect of soy intake, research that is more recent has pointed to a beneficial antidiabetic effect of soy intake, perhaps mediated by soy isoflavones. The two common categories of antidiabetic drugs acting on nuclear receptors known as peroxisome proliferator activated receptors (PPARs) are the fibrates and glitazones. We and others have recently asked the research question 'do the soy isoflavones have activities as either "phytofibrates" or "phytoglitazones"?' Such an activity should be able to be confirmed both in vivo and in vitro. In both the in vivo and in vitro cases, this action has indeed been confirmed. Further work suggests a possible action of isoflavones similar to the nonestrogenic ligands that bind the estrogen-related receptors (ERRs). Recently, these receptors have been demonstrated to contribute to lipolytic processes. Finally, evaluation of receptor activation studies suggests that thyroid receptor activation may provide additional clues explaining the metabolic action of isoflavones. The recent advances in the discovery and evaluation of the promiscuous nuclear receptors that bind many different chemical ligands should prove to help explain some of the biological effects of soy isoflavones and other phytochemicals.     

High-performance liquid chromatographic methods for the determination of topoisomerase II inhibitors

Various methods for separating eleven different types of topoisomerase II (TOPO-2) inhibitors, including epipodophyllotoxins, anthracyclines, anthracenediones, anthrapyrazoles, anthracenebishydrazones, indole derivatives, aminoacridines, benzisoquinolinediones, isoflavones, bisdioxopiperazines and thiobarbituric acids, are summarized. Proper sample preparation and storage is critical to the successful analysis of some TOPO-2 inhibitors due to difficulties associated with adsorption, instability and complex biological components. Solid-phase and liquid–liquid extractions are widely used to separate TOPO-2 inhibitors from biological samples, although simple deproteinization followed by direct analysis of the supernatant is preferable to extraction based on its speed and simplicity. High-performance liquid chromatography (HPLC) is the favored method for the bioanalysis of TOPO-2 inhibitors. UV or diode array detection is generally employed for early pharmacokinetic studies, while fluorescence or electrochemical detection is used more frequently for analytes with fluorescent or oxidative–reductive properties. For analyses requiring highly sensitive and/or specific detection, electrospray mass spectrometry (ESI-MS or ESI-MS–MS) provides a suitable alternative. A comprehensive compilation of the HPLC techniques currently used to separate TOPO-2 inhibitors will aid the future development of analytical methods for new TOPO-2 inhibitors.     

Metabolism of isoflavones in human subjects

Isoflavones form a group of plant compounds that occur mainly in legumes, soy being the most important source in human diet. The high levels of isoflavones in the diet have been associated with a lowered risk for hormone-dependent diseases, including breast and prostate cancers, osteoporosis and cardiovascular disease. The metabolism of isoflavones in humans has been studied to a certain extent, but detailed studies are lacking. This paper reviews the current knowledge on metabolism of isoflavones and presents some preliminary results of a comprehensive soy feeding study, in which the phase I metabolites of soy isoflavones, daidzein, genistein and glycitein, were identified by GC-MS. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Determination of clozapine, desmethylclozapine and clozapine N-oxide in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid–liquid extraction with n-hexane–isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 μl of 0.1 M dibasic phosphate (pH 2.2 with 25% H₃PO₄). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile–0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250×4.60 mm I.D. analytical column packed with 5 μm C₆ silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were ≤13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from −19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.     

Soy isoflavones and cognitive function

There is growing interest in the physiological functions of soy isoflavones, especially in whether they affect cognitive function and have beneficial effects on neurodegenerative diseases. Here we review the recent evidence from clinical and experimental studies supporting a role for soy isoflavones in cognitive function. Soy isoflavones may mimic the actions and functions of estrogens on brain, and they have been shown to have positive effects on the cognitive function in females; however, studies on their effects on spatial memory have not provided consistent results in males. Although data from humans, cultures, and animal models are currently insufficient for elucidating the metabolism of soy isoflavone actions on cognitive function and the nervous system, we suggest two putative pathways; (1) an estrogen receptor-mediated pathway and (2) via the inhibition of tyrosine kinase, in particular by genistein, which is one of the soy isoflavones. Although soy isoflavones appear to have a positive effect on brain function, further research is needed to determine not only the efficacy but also the safety of soy isoflavones on the nervous system and cognitive function.     

Soy,Red Clover,and Isoflavones and Breast Cancer: A Systematic Review

Background

Soy and red clover isoflavones are controversial due to purported estrogenic activity and possible effects on breast cancer. We conducted a systematic review of soy and red clover for efficacy in improving menopausal symptoms in women with breast cancer, and for potential impact on risk of breast cancer incidence or recurrence.

Methods

We searched MEDLINE, Embase, the Cochrane Library, and AMED from inception to March 2013 for human interventional or observational data pertaining to the safety and efficacy of soy and red clover isoflavones in patients with or at risk of breast cancer.

Results

Of 4179 records, we included a total of 131 articles: 40 RCTs, 11 uncontrolled trials, and 80 observational studies. Five RCTs reported on the efficacy of soy for hot flashes, showing no significant reductions in hot flashes compared to placebo. There is lack of evidence showing harm from use of soy with respect to risk of breast cancer or recurrence, based on long term observational data. Soy intake consistent with that of a traditional Japanese diet (2-3 servings daily, containing 25-50mg isoflavones) may be protective against breast cancer and recurrence. Human trials show that soy does not increase circulating estradiol or affect estrogen-responsive target tissues. Prospective data of soy use in women taking tamoxifen does not indicate increased risk of recurrence. Evidence on red clover is limited, however existing studies suggest that it may not possess breast cancer-promoting effects.

Conclusion

Soy consumption may be associated with reduced risk of breast cancer incidence, recurrence, and mortality. Soy does not have estrogenic effects in humans. Soy intake consistent with a traditional Japanese diet appears safe for breast cancer survivors. While there is no clear evidence of harm, better evidence confirming safety is required before use of high dose (≥100mg) isoflavones can be recommended for breast cancer patients.     

Analysis of amphetamine-type stimulants and their metabolites in plasma, urine and bile by liquid chromatography with a strong cation-exchange column-tandem mass spectrometry

The aim of this work was to develop and validate a method for analysing amphetamine-type stimulants (ATSs) and their metabolites in plasma, urine and bile by liquid chromatography with a strong cation-exchange column-tandem mass spectrometry, and to apply it to the pharmacokinetic study of ATSs. 3,4-Methylenedioxymethamphetamine, methamphetamine, ketamine and their main metabolites, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, p-hydroxymethamphetamine, amphetamine and norketamine, were simultaneously quantified by the new method (50-5000 ng/ml). The coefficients of variation and the percent deviations for the eight compounds were in the range of 0.2 to 5.3% and -9.4 to +12.8%, respectively. The recoveries were over 90% in all biological samples tested. This method was effective for the separation and the identification of ATSs and their main metabolites having amine moieties in plasma, urine and bile, and was applicable to pharmacokinetic analysis of methamphetamine, ketamine and their main metabolites in biological samples. This analytical method should be useful for the pharmacokinetic analysis of ATSs.     

Soy isoflavones' osteoprotective role in postmenopausal women: mechanism of action

Ovarian hormone deficiency is a major risk factor for osteoporosis. Current therapies emphasize the use of antiresorptive agents, such as estrogen, calcitonin, and bisphosphonates. These therapies are associated with certain risks and side effects making compliance a major obstacle. Recent findings suggest that a class of synthetic and naturally occurring compounds, selective estrogen receptor modulators, e.g. raloxifene and soy isoflavones can offer attractive alternatives. Evidence for bone-sparing effects of isoflavones relies mainly on animal findings supported by a limited number of human studies. These observations suggest that isoflavones exert their effects on bone by stimulating bone formation and at the same time suppressing bone resorption. However, the precise osteoprotective mechanism of isoflavones remains uncertain and awaiting further clarification. From a clinical point of view, larger and longer duration studies are warranted to enable us to draw clear conclusions in regards to the role of isoflavones on bone.     

Diet and cancer prevention: Dietary compounds,dietary MicroRNAs,and dietary exosomes

Cancer is one of main health public problems worldwide. Several factors are involved in beginning and development of cancer. Genetic and internal/external environmental factors can be as important agents that effect on emerging and development of several cancers. Diet and nutrition may be as one of important factors in prevention or treatment of various cancers. A large number studies indicated that suitable dietary patterns may help to cancer prevention or could inhibit development of tumor in cancer patients. Moreover, a large numbers studies indicated that a variety of dietary compounds such as curcumin, green tea, folat, selenium, and soy isoflavones show a wide range anti‐cancer properties. It has been showed that these compounds via targeting a sequence of cellular and molecular pathways could be used as suitable options for cancer chemoprevention and cancer therapy. Recently, dietary microRNAs and exosomes have been emerged as attractive players in cancer prevention and cancer therapy. These molecules could change behavior of cancer cells via targeting various cellular and molecular pathways involved in cancer pathogenesis. Hence, the utilization of dietary compounds which are associated with powerful molecules such as microRNAs and exosomes and put them in dietary patterns could contribute to prevention or treatment of various cancers. Here, we summarized various studies that assessed effect of dietary patterns on cancer prevention shortly. Moreover, we highlighted the utilization of dietary compounds, dietary microRNAs, and dietary exosomes and their cellular and molecular pathways in cancer chemoprevention.     

Dietary soy isoflavone-aglycone lowers food intake in female rats with and without ovariectomy

Objective: Estrogens downregulate eating behavior, and soy isoflavones are known to be estrogenic agents. We aimed to examine whether the estrogenic property of soy isoflavones can affect food intake and body weight. Methods and Procedures: Seven‐week‐old male, female, and ovariectomized (OVX) Sprague‐Dawley rats were given free access to a diet containing 100–300 mg total isoflavone/kg diet, or to a control diet, either with or without concurrent administration of estradiol by subcutaneous implantation. Results: Dietary soy isoflavone was shown to lower food intake in female rats, whether or not the animals had undergone ovariectomy. Administration of estradiol lowered the food intake in male rats and in OVX female rats. The decrease in weekly food intake in female rats led to a reduction in their weekly gain in body weight. Dietary soy isoflavone significantly increased the concentration of serum isoflavones, especially equol (a metabolite of daidzein), regardless of gender or ovariectomy. Dietary soy isoflavone did not affect either serum estradiol concentration or uterine and didymus weights, but estradiol administration improved the uterine atrophy in OVX rats, and decreased the didymus weight in male rats. Discussion: Soy isoflavone lowers the food intake in female rats, but not in the male animals. Contrary to the hypothesis currently in vogue, the reduction in food intake caused by soy isoflavone may not be a purely estrogenic effect. This follows from the finding that the effects of soy isoflavones on food intake and on the reproductive organs differ from the corresponding effects produced by estrogen.     

Bioavailability of isoflavones

Isoflavones are disease protective components of soybeans. Isoflavone metabolism and bioavailability are key to understanding their biological effects. Isoflavone glucuronides, dominant biotransformation products in humans that are more hydrophilic than isoflavone aglycones, activate human natural killer cells in vitro but are less toxic to NK cells than the parent aglycones. Gut microbial isoflavone metabolites have also been identified, but remain to be well characterized. Gut transit time (GTT) seems to be a significant determinant of isoflavone bioavailability because women with more rapid GTT (<40 h) experienced 2-3-fold greater absorption of isoflavones than did women with longer GTT (>65 h). Isoflavone metabolism varies a great deal among individuals, thus limiting the quantitative value of urine or plasma isoflavones as biomarkers of soy ingestion. Defining and lessening interindividual variation in isoflavone bioavailability, and characterizing health-related effects of key isoflavone metabolites are likely to be crucial to further understanding of the health benefits of isoflavones.     

饲喂雌鼠大豆异黄酮对后代雌性生殖性状的影响

目的研究雌性小鼠饲喂异黄酮后对其后代雌鼠生殖性状的影响。方法分别在雌鼠从断奶到妊娠前和妊娠期间两个不同阶段的日粮中添加不同剂量的大豆异黄酮（0、50、400 mg/kg）。记录F1代的出生窝产仔数、出生窝重、雌鼠的阴道最早开张时间和最早见栓时间,以及后代雌鼠性成熟期和体成熟期生殖器官重量、血清雌激素的含量。结果雌鼠妊娠期饲喂含50、400 mg/kg大豆异黄酮的饲料后F1代的窝产仔数显著高于对照组（P〈0.05）;雌鼠妊娠期采食400 mg/kg大豆异黄酮饲料,其后代雌鼠初次配种时间明显延迟（P〈0.05）;45、65日龄体重显著低于对照组,65日龄子宫重显著低于对照组,卵巢重显著高于对照组;血清中雌激素含量显著低于对照组（P〈0.05）,妊娠期间采食50 mg/kg大豆异黄酮饲料,其F1代雌鼠仅窝产仔数和45日龄时血清中雌激素含量受到影响;而雌鼠从断奶到怀孕期间饲喂含大豆异黄酮饲料的F1代在各项指标与对照组均差异无显著性。结论雌鼠妊娠期间饲喂含400 mg/kg大豆异黄酮的饲料会显著影响其雌性后代的初情期、生殖器官发育、血清雌激素含量等生殖生理性状,而在非妊娠期饲喂含大豆异黄酮饲料对F1代无显著影响。     

Determination of two neuropeptide growth factor antagonists, [d-Arg,d-Phe,d-Trp,Leu]-substance P and [Arg, d-Trp,N-MePhe]-substance P(6–11), by high-performance liquid chromatography with electrochemical detection

n-MePhe⁸]-substance P(6–11) (G) are currently undergoing preclinical evaluation as potential anticancer agents and clinical trials are planned for G in the near future. A reversed-phase high-performance liquid chromatographic separation has been developed which is both sensitive (limit of detection 250 pg/263 fmol for G; 500 pg/330 fmol for D) and selective, based on electrochemical detection of the two tryptophan residues present in each peptide. Two ion-pairing agents were included in the isocratic mobile phase to eliminate adsorption of the peptides onto the analytical column. Extensive sample clean-up procedures have been developed for plasma, tissue and tumour based on solid-phase extraction. Precision and accuracy of each assay was 91.3 ± 16.9% (between-day) for G and 99.3 ± 16.9% (between-day) for D. The assays were able to detect the intact peptides and a number of their metabolites in plasma, liver and the WX 322 SCLC human xenograft in nude mice for at least 6 hr after administration of therapeutic and pharmacological doses.