肠道微生物代谢产物雌马酚的生成及其主要影响因素北大核心CSCD

雌马酚是大豆苷原的微生物代谢产物,被认为是大豆异黄酮发挥生理作用的关键,但仅有33%-50%的人群能够产生雌马酚。最新研究表明氢气在雌马酚生成过程中起着非常重要的作用。目前此类研究尚处于起步阶段,本文总结了以往的研究结果,对雌马酚的生成、生物学作用及影响雌马酚生成的因素、氢气与雌马酚之间的关系作了较为详细的介绍。     

大豆素代谢产物——雌马酚研究进展

雌马酚是大豆异黄酮的主要组份之一大豆素（Dai）的代谢产物,人群中约有30％～50％能将大豆素转化为雌马酚,影响因素目前尚不完全清楚,最重要的因素是肠道菌群。雌马酚较其原型具有更为有效的生物学作用,因此受到普遍关注。研究和开发雌马酚的生物活性,在多种常见慢性病的预防与控制方面具有重要的理论和实际意义。     

微生物转化影响不同内源雌激素水平大鼠粪便雌马酚含量和菌群结构

雌马酚(Equol)是肠道中特定细菌转化大豆异黄酮的产物,与其前体大豆苷元(Daidzein)相比,雌马酚具有更强的生物学活性。【目的】研究口服雌马酚产生菌对大鼠转化大豆苷元能力的可能促进作用及内源雌激素对大鼠肠道菌群的可能影响。【方法】使用平均体重为211±9g的卵巢摘除和假手术雌性大鼠各30只,分别随机分为5组,并灌胃蒸馏水、雌二醇、大豆苷元、雌马酚和大豆苷元+雌马酚产生菌ZX7。【结果】从灌胃第2天开始,接受大豆苷元后大鼠粪样中始终具有较高水平的雌马酚,显著高于对照和雌二醇组(P<0.01);灌胃大豆苷元+雌马酚产生菌ZX7的大鼠和直接灌胃雌马酚的大鼠在粪样雌马酚含量上十分接近;DGGE图谱的PCA分析显示,卵巢摘除大鼠和假手术大鼠粪便菌群存在明显差异;大鼠粪便拟杆菌门细菌数量与粪样中雌马酚水平显著正相关。【结论】大鼠肠道固有菌群中可能存在能够将大豆苷元转化为雌马酚的细菌,利用外源菌株改变大鼠雌马酚产生能力具有一定的可行性,不同的内源雌激素水平可能影响大鼠肠道菌群结构,拟杆菌门细菌可能在大豆苷元的生物转化过程中起着十分重要的作用。     

172名上海地区成人饮食及尿雌马酚和代谢产物的调查研究

目的确定上海地区成人雌马酚代谢表型及雌马酚的生理范围;把握由于大豆异黄酮负荷而产生的雌马酚产生者比例;调查雌马酚表型和食物摄取频率及有关激素间的关系。方法应用现状调查方法,筛选出172名居住在上海市区健康成年男女。填写问卷获得研究对象日常饮食频率,检测研究对象血清获得血液激素浓度,采用HPLC法分析负荷大豆异黄酮前后尿中雌马酚等大豆异黄酮24 h排泄量,统计产雌马酚者比例及其与摄食频率和激素的关系。结果负荷前雌马酚生理范围0~33.74μmol/24 h,产雌马酚者比例为30.2%,负荷大豆异黄酮后比例提高至53.5%。产雌马酚者与非产雌马酚者之间日常食品摄取频率的差别无统计学意义(P>0.05)。产Eq者血中游离雌二醇的浓度较非产Eq者低(P<0.05)。结论在通常膳食条件下,约有1/3上海成人尿液中能检测到雌马酚,但负荷大豆异黄酮后,约有1/2能产生雌马酚。     

肠道微生物代谢产物—S-雌马酚与 人类健康关系研究进展

大豆异黄酮是一种年销量很大、市场普及率较高的保健品。具有抗氧化、抗肿瘤等功能,对延缓女性衰老和改善更年期综合征有很好效果。但研究表明,大豆异黄酮的保健功能主要归功于其肠道代谢产物S-雌马酚。S-雌马酚是豆类食品在肠道内经特定微生物代谢后产生的一种高度稳定小分子,其与雌激素的结构和功能高度相似,能与β-雌激素(ER-β)受体结合。具有防治更年期综合征、心血管疾病和多种雌素依赖性癌症的功能。到目前为止,雌马酚的功能及其与人类健康之间的关系还有待进一步研究。本文结合国内外最新研究进展,着重介绍雌马酚的生理功能及其与各类疾病的研究进展,展望雌马酚在各类疾病防治中可能发挥的重要作用。     

重组酿酒酵母催化二氢大豆苷元生产雌马酚

雌马酚是大豆异黄酮的代谢产物,是一种天然的选择性雌激素受体调节剂,稳定性和生物学活性高。为实现雌马酚的微生物合成,采用模块途径工程策略,构建编码雌马酚合成关键酶基因 orf-1、orf-2和orf-3 的表达载体,成功用于转化酿酒酵母BY4741,得到工程菌株。结果表明,工程菌株有效表达了外源基因,并可将大豆异黄酮代谢中间体二氢大豆苷元转化为雌马酚。为构建从头合成雌马酚的微生物细胞工厂提供了重要科学参考。     

产S-雌马酚梭菌C1三代全基因组测序及功能基因筛选鉴定

【目的】挖掘产S-雌马酚梭菌C1转化大豆苷元产生S-雌马酚的功能基因,为梭菌C1的S-雌马酚转化机制研究提供参考,并为利用合成生物学方法生产S-雌马酚提供新基因资源。【方法】利用GridION测序平台,对梭菌C1进行第三代全基因组测序、基因组组装和功能注释等分析,从C1菌全基因组中筛选和鉴定参与S-雌马酚生物转化的功能基因。【结果】C1全基因组大小为3 035 113 bp,预测编码3 166个基因,包含53个tRNA、15个rRNA、4个ncRNA和1个基因岛。通过生物信息学分析,发现C1-07020基因编码蛋白与已报道的Lactococcus sp.20-92大豆苷元还原酶具有44.8%的氨基酸序列相似性和相同的3个功能保守结构域,体外蛋白功能验证表明,C1-07020具有大豆苷元还原酶功能。此外,C1菌中没有发现与已知产S-雌马酚菌相似的功能基因簇或大豆苷元还原酶以外的其他功能基因。【结论】在C1中鉴定到一个新的产S-雌马酚功能基因,并发现了C1可能具备特殊的产S-雌马酚机制,实验所获基础数据可为进一步挖掘产S-雌马酚新功能基因、了解S-雌马酚的生成机制及体外产S-雌马酚基因资源...     

人粪样中雌马酚含量调查及其与菌群结构相关性研究

目的 调查人粪样中大豆素和雌马酚的含量及其与年龄和性别的关系;了解人粪样中雌马酚含量高低与菌群结构的关系.方法 采用高效液相色谱(HPLC)对来自杭州的125份粪样进行大豆素和雌马酚含量检测,并使用生物统计学软件SPSS进行统计学分析;使用PCR-DGGE对粪样中雌马酚含量的高低与菌群结构的关系进行初步研究.结果 HPLC检测结果表明,尽管粪样中雌马酚含量的高低与性别关系不大,但却与年龄大小存在很大的相关性,41 ～50岁年龄组的粪样中雌马酚含量明显高于其他年龄组.PCR-DGGE结果表明,粪样中雌马酚含量的高低与菌群结构无明显相关性.结论 人粪样中雌马酚含量的高低与年龄大小有很强的相关性.     

雌马酚产生细菌及其雌马酚合成代谢机制

大豆食品中通常富含染料木素和大豆苷元等异黄酮素,人和动物肠道中的某些细菌具有将异黄酮素代谢转化为S-雌马酚的能力。到目前为止,S-雌马酚被认为是一种具有潜在健康调节作用的化合物。啮齿类动物均具备产雌马酚的能力,但不同人群之间存在差异,产雌马酚细菌是否存在可能是造成这种差异的重要原因;不同产雌马酚细菌的代谢机制可能不同,并影响机体最终产雌马酚的能力。本文对已知的各种产雌马酚细菌及其细菌的雌马酚合成机制进行综述,以期为进一步了解雌马酚产生个体差异、雌马酚代谢转化效率、体外雌马酚的发酵生产,以及临床产雌马酚细菌的应用等提供理论参考。     

(S)-雌马酚对肠道菌群的体外调控作用及其可代谢性研究

[目的]研究(S)-雌马酚对人体肠道菌群的体外调控作用和人体肠道菌群对(S)-雌马酚的代谢衍生作用。[方法]采用人体肠道菌群体外批量发酵、细菌16S rRNA基因高通量测序、气相色谱、液相色谱和质谱等检测(S)-雌马酚与人体肠道菌群体外相互作用。[结果]体外添加(S)-雌马酚对总体人肠道菌群结构和短链脂肪酸产量影响不明显。添加0.45 mmol/L (S)-雌马酚组与对照组相比,未检测到相对丰度发生显著变化的细菌;添加0.90 mmol/L (S)-雌马酚组与对照组相比,显著增加了肠杆菌科(Enterobacteriaceae)等条件致病菌的相对丰度,减少了潜在益生菌粪球菌属(Coprococcus)的比例。代谢分析发现,发酵培养液中(S)-雌马酚的浓度降低了约15%−30%,推测可能被微生物进一步降解或衍生修饰。[结论]从体外调控肠道菌群的角度判断,0.45 mmol/L (S)-雌马酚相对较安全,而0.90 mmol/L (S)-雌马酚可能会破坏肠道菌群平衡。(S)-雌马酚可以被人体肠道菌群进一步代谢,其特定代谢产物的结构与功能及其体内生物安全性有待进一步研究。     

Equol,an Isoflavone Metabolite,Regulates Cancer Cell Viability and Protein Synthesis Initiation via c-Myc and eIF4G

Epidemiological studies implicate dietary soy isoflavones as breast cancer preventives, especially due to their anti-estrogenic properties. However, soy isoflavones may also have a role in promoting breast cancer, which has yet to be clarified. We previously reported that equol, a metabolite of the soy isoflavone daidzein, may advance breast cancer potential via up-regulation of the eukaryotic initiation factor 4GI (eIF4GI). In estrogen receptor negative (ER−) metastatic breast cancer cells, equol induced elevated levels of eIF4G, which were associated with increased cell viability and the selective translation of mRNAs that use non-canonical means of initiation, including internal ribosome entry site (IRES), ribosome shunting, and eIF4G enhancers. These mRNAs typically code for oncogenic, survival, and cell stress molecules. Among those mRNAs translationally increased by equol was the oncogene and eIF4G enhancer, c-Myc. Here we report that siRNA-mediated knockdown of c-Myc abrogates the increase in cancer cell viability and mammosphere formation by equol, and results in a significant down-regulation of eIF4GI (the major eIF4G isoform), as well as reduces levels of some, but not all, proteins encoded by mRNAs that are translationally stimulated by equol treatment. Knockdown of eIF4GI also markedly reduces an equol-mediated increase in IRES-dependent mRNA translation and the expression of specific oncogenic proteins. However, eIF4GI knockdown did not reciprocally affect c-Myc levels or cell viability. This study therefore implicates c-Myc as a potential regulator of the cancer-promoting effects of equol via up-regulation of eIF4GI and selective initiation of translation on mRNAs that utilize non-canonical initiation, including certain oncogenes.     

Equol, a natural estrogenic metabolite from soy isoflavones: convenient preparation and resolution of R- and S-equols and their differing binding and biological activity through estrogen receptors alpha and beta

Equol is a metabolite produced in vivo from the soy phytoestrogen daidzein by the action of gut microflora. It is known to be estrogenic, so human exposure to equol could have significant biological effects. Equol is a chiral molecule that can exist as the enantiomers R-equol and S-equol. To study the biological activity of racemic (+/-)-equol, as well as that of its pure enantiomers, we developed an efficient and convenient method to prepare (+/-)-equol from available isoflavanoid precursors. Furthermore, we optimized a method to separate the enantiomers of equol by chiral HPLC, and we studied for the first time, the activities of the enantiomers on the two estrogen receptors, ERalpha and ERbeta. In binding assays, S-equol has a high binding affinity, preferential for ERbeta (K(i)[ERbeta]=16 nM; beta/alpha=13 fold), that is comparable to that of genistein (K(i)[ERbeta]=6.7 nM; beta/alpha=16), whereas R-equol binds more weakly and with a preference for ERalpha (K(i)[ERalpha]=50 nM; beta/alpha=0.29). All equol isomers have higher affinity for both ERs than does the biosynthetic precursor daidzein. The availability and the in vitro characterization of the equol enantiomers should enable their biological effects to be studied in detail.     

Equol is a novel anti-androgen that inhibits prostate growth and hormone feedback

Equol (7-hydroxy-3[4'hydroxyphenyl]-chroman) is the major metabolite of the phytoestrogen daidzein, one of the main isoflavones found abundantly in soybeans and soy foods. Equol may be an important biologically active molecule based on recent studies demonstrating that equol can modulate reproductive function. In this study, we examined the effects of equol on prostate growth and LH secretion and determined some of the mechanisms by which it might act. Administration of equol to intact male rats for 4-7 days reduced ventral prostate and epididymal weight and increased circulating LH levels. Using binding assays, we determined that equol specifically binds 5alpha-dihydrotestosterone (DHT), but not testosterone, dehydroepiandrosterone, or estrogen with high affinity. Equol does not bind the prostatic androgen receptor, and has a modest affinity for recombinant estrogen receptor (ER) beta, and no affinity for ERalpha. In castrated male rats treated with DHT, concomitant treatment with equol blocked DHT's trophic effects on the ventral prostate gland growth and inhibitory feedback effects on plasma LH levels without changes in circulating DHT. Therefore, equol can bind circulating DHT and sequester it from the androgen receptor, thus altering growth and physiological hormone responses that are regulated by androgens. These data suggest a novel model to explain equol's biological properties. The significance of equol's ability to specifically bind and sequester DHT from the androgen receptor have important ramifications in health and disease and may indicate a broad and important usage for equol in the treatment of androgen-mediated pathologies.     

The small molecule SI113 hinders epithelial-to-mesenchymal transition and subverts cytoskeletal organization in human cancer cells

The small molecule SI113 is an inhibitor of the kinase activity of SGK1, a key biological regulator acting on the PI3K/mTOR signal transduction pathway. Several studies demonstrate that this compound is able to strongly restrain cancer growth in vitro and in vivo, alone or in associative antineoplastic treatments, being able to elicit an autophagic response, either cytotoxic or cytoprotective. To elucidate more exhaustively the molecular mechanisms targeted by SI113, we performed activity-based protein profiling (ABPP) proteomic analysis using a kinase enrichment procedure. This technique allowed the identification via mass spectrometry of novel targets of this compound, most of them involved in functions concerning cell motility and cytoskeletal architecture. Using a glioblastoma multiforme, hepatocarcinoma and colorectal carcinoma cell line, we recognized an inhibitory effect of SI113 on cell migration, invading, and epithelial-to-mesenchymal transition. In addition, these cancer cells, when exposed to this compound, showed a remarkable subversion of the cytoskeletal architecture characterized by F-actin destabilization, phospho-FAK delocalization, and tubulin depolimerization. These results were definitely concordant in attributing to SI113 a key role in hindering cancer cell malignancy and, due to its negligible in vivo toxicity, can sustain performing a Phase I clinical trial to employ this drug in associative cancer therapy.     

A New Design of Mental State Classification for Subject Independent BCI Systems

BackgroundBrain Computer Interface (BCI) systems have been widely used to develop sustainable assistive technology for people suffering from neurological impairments. A major limitation of current BCI systems is that they are based on Subject-dependent (SD) concept. The SD based BCI system is time consuming and inconvenient for physical or mental disables people and also not suitable for limited computer resources. In order to overcome these problems, recently subject-independent (SI) based BCI concept has been introduced to identify mental states of motor disabled people but the expected outcome of the SI based BCI has not been achieved yet. Hence this paper intends to present an efficient scheme for SI based BCI system. The goal of this research is to develop a method for classifying mental states which can be used by any user. For attaining this target, this study employs a supervised spatial filtering method with four types of feature extraction methods including Katz Fractal Dimension, Sub band Energy, Log Variance and Root Mean Square (RMS) and finally the obtained features are used as input to Linear Discriminant Analysis (LDA) classification model for identifying mental states for SI BCI system.ResultsThe performance of the proposed design is evaluated in several ways such as considering different time window length; different frequency bands; different number of channels. The mean classification accuracy using Katz feature is 84.35% which is the maximum output compare to other features that outperforms the existing methods.ConclusionsOur proposed design will help to make a new technology for development of real-time SI based BCI systems that can be more supportive for the motor disabled patients.     

Estrogenicity of the isoflavone metabolite equol on reproductive and non-reproductive organs in mice

Equol, a metabolite of the phytoestrogen daidzein, is present at significant levels in some humans who consume soy and in rodents fed soy-based diets. Equol is estrogenic in vitro, but there have been limited studies of its activity in vivo. We evaluated equol effects on reproductive and non-reproductive endpoints in mice. Ovariectomized age-matched (30-day-old) female C57BL/6 mice were fed phytoestrogen-free diets and given a racemic mixture of equol by daily injections (0, 4, 8, 12, or 20 mg [kg body weight](-1) day(-1)) or in the diet (0, 500, or 1,000 ppm) for 12 days. Mice were killed, and serum concentrations of total and aglycone equol were measured. Total serum equol concentrations ranged from 1.4 to 7.5 microM with increasing doses of injected equol, but uterine weight increased significantly only at 12 and 20 mg (kg body weight)(-1) day(-1). Dietary equol at 500 or 1,000 ppm produced total serum equol concentrations of 5.9 and 8.1 microM, respectively, comparable with those in rodents consuming certain high-soy chows; the proportion of equol present as the free aglycone was much lower with dietary administration than injections, which may be a factor in the greater biological effects induced by injections. Dietary equol did not significantly increase uterine weight. Increasing dietary and injected equol doses caused a dose-dependent increase in vaginal epithelial thickness. Uterine epithelial proliferation was increased by equol injections at 8-20 mg (kg body weight)(-1) day(-1) and 1,000 ppm dietary equol. Neither dietary nor injected equol decreased thymic or adipose weights. In conclusion, equol is a weak estrogen with modest effects on endpoints regulated by estrogen receptor alpha when present at serum levels seen in rodents fed soy-based diets, but quantities present in humans may not be sufficient to induce estrogenic effects, although additive effects of equol with other phytoestrogens may occur.     

Photoimmune protective effect of the phytoestrogenic isoflavonoid equol is partially due to its antioxidant activities

Topical application of lotions containing the phytoestrogenic isoflavonoid equol have been reported to protect mice against UV radiation-induced inflammation, immune suppression and photocarcinogenesis. The photoimmune protective property was shown to depend on equol's activation of oestrogen receptor signalling in the skin. However, isoflavones are also recognised for their antioxidant properties in biological systems. As endogenous cutaneous antioxidant enzymes including the inducible stress protein haem oxygenase (HO)-1, have photoprotective efficacy, this study in the Skh:hr-1 hairless mouse seeks evidence for an antioxidant role for equol in contributing to its photoimmune protection. Oxidative stress has been measured as UVA-induced lipid peroxidation in the mouse skin, and was dose-dependently inhibited by topical equol. Inhibition of the UVA (320-400 nm)-inducible HO activity significantly reduced the level of equol protection against lipid peroxidation, thereby attributing a component of equol's lipid protection capacity to this stress enzyme. It was consistent that topical equol enhanced the level of HO induction by UVA irradiation in both skin and liver. Subsequently, the dose-dependent protection by topical equol lotions against solar simulated UV radiation induced immunosuppression, measured by the contact hypersensitivity reaction, was found also to be partially reduced by the inhibition of HO activity. Therefore, in addition to the activation by equol of oestrogenic signalling pathways for photoprotection, this isoflavonoid also provides UV-protective antioxidant effects that depend partially on HO-1 induction.     

Dietary daidzein decreases food intake accompanied with delayed gastric emptying in ovariectomized rats

ABSTRACT

We previously found that equol, a metabolite of intestinal bacterial conversion from soy isoflavone daidzein, has female-specific anorectic effects. In the present study, we used seven-week-old female ovariectomized (OVX) Sprague Dawley rats to test the hypothesis that the anorectic effect of dietary daidzein may be attributed to delayed gastric emptying. Results suggest that dietary daidzein delays gastric emptying and that it has an anorectic effect with residual gastric contents, but not without gastric contents. Dietary equol significantly decreased daily food intake in the OVX rats without sleeve gastrectomy, but not in those with sleeve gastrectomy, suggesting that the accumulation of food in the stomach is required for the anorectic effect of equol to occur. These results support the hypothesis that the anorectic effect of dietary daidzein is attributed to delayed gastric emptying.     

Quantitative analysis of urinary daidzein and equol by gas chromatography after solid-phase extraction and high-performance liquid chromatography

Daidzein and its main metabolite equol are isoflavone phytoestrogens. Several studies have suggested that intake of an isoflavone-rich diet may prevent hormone-related cancer and estrogen-related disorders (cardiovascular disease, osteoporosis and menopausal symptoms). To better understand the role of isoflavones in preventing such severe disease, several methods have been developed to measure these compounds in biological fluids. However, the analytical procedures to measure isoflavones are often time-consuming and require highly skilled technicians. In this paper we describe a method for urinary daidzein and equol measurement that combines solid phase extraction and HPLC purification before gas chromatographic determination. The specificity of the method was confirmed by the gas chromatography-mass spectrometry technique. The mean recovery of daidzein and equol was 94.6% and 97.0%, respectively. The repeatability of the method was in the range of 2.0-7.4% for daidzein and 1.3-4.9% for equol. A linear relationship between observed and expected values was found in the dilution (r2=0.9983 for daidzein; r2=0.9982 for equol) and addition (r2=0.9984 for daidzein; r2=0.9989 for equol) assays. The method is suitable to measure changes in the urinary excretion of isoflavones and to investigate urinary isoflavonoids as biomarkers of isoflavone exposure.     

Equol, a metabolite of the soybean isoflavone daidzein, inhibits neoplastic cell transformation by targeting the MEK/ERK/p90RSK/activator protein-1 pathway

Daidzein and genistein are isoflavones found in soybean. Genistein is known to exhibit anticarcinogenic activities and inhibit tyrosine kinase activity. However, the underlying molecular mechanisms of the chemopreventive activities of daidzein and its metabolite, equol, are not understood. Here we report that equol inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal cells by targeting the MEK/ERK/p90RSK/activator protein-1 signaling pathway. TPA-induced neoplastic cell transformation was inhibited by equol, but not daidzein, at noncytotoxic concentrations in a dose-dependent manner. Equol dose-dependently attenuated TPA-induced activation of activator protein-1 and c-fos, whereas daidzein did not exert any effect when tested at the same concentrations. The TPA-induced phosphorylation of ERK1/2, p90RSK, and Elk, but not MEK or c-Jun N-terminal kinase, was inhibited by equol but not by daidzein. In vitro kinase assays revealed that equol greatly inhibited MEK1, but not Raf1, kinase activity, and an ex vivo kinase assay also demonstrated that equol suppressed TPA-induced MEK1 kinase activity in JB6 P+ cell lysates. Equol dose-dependently inhibited neoplastic transformation of JB6 P+ cells induced by epidermal growth factor or H-Ras. Both in vitro and ex vivo pull-down assays revealed that equol directly bound with glutathione S-transferase-MEK1 to inhibit MEK1 activity without competing with ATP. These results suggested that the antitumor-promoting effect of equol is due to the inhibition of cell transformation mainly by targeting a MEK signaling pathway. These findings are the first to reveal a molecular basis for the anticancer action of equol and may partially account for the reported chemopreventive effects of soybean.