高效液相色谱(HPLC)技术鉴定中国南方大豆品种异黄酮主要组分

采用高效液相色谱(HPLC)技术检测中国南方六个省份的249份大豆品种异黄酮主要组分含量.结果显示大豆籽粒中可检测出6种主要的异黄酮组分,分别为大豆甙(Daidzin)、甲氧基黄豆甙原(Glycitin)、染料木甙(Genistin)、丙二酰基大豆甙(Malonyldaidzin)、丙二酰基黄豆甙原(Malonylglycitin)和丙二酰基染料木甙(Malonylgenistin).各组分中以丙二酰基(Malonyl)异黄酮组分含量最高(61.2%),且各组分间相关极显著.大豆品种间异黄酮含量变异较大,变异系数达49.6%.来自江苏省的品种海门红黄豆乙异黄酮含量最高(4932.3μg/g),品种宝应等西风含量最低(367.1μg/g).不同省份间异黄酮含量差异极显著,来自浙江省的大豆品种平均含量最高(2717.2μg/g),来自安徽省的平均含量最低(1181.8μg/g).异黄酮含量与生育期呈极显著正相关(r=0.319* * *),与百粒重呈显著正相关(r=0.132*),而与脂肪含量(r=-0.45* * *)和蛋白质含量(r=-0.136)呈负相关.     

大豆籽粒发育过程中异黄酮合成相关酶基因的表达模式与异黄酮积累的相关分析

利用高效液相色谱法和实时定量PCR方法,分别测定了2个异黄酮含量显著差异的大豆品种鲁黑豆2号(LHD2)和南汇早黑豆(NHZ)在子粒发育过程中的异黄酮含量变化以及异黄酮合成相关酶基因的表达模式变化,试图分析异黄酮积累与各基因表达量变化的相关关系。结果表明在大豆子粒发育过程中,异黄酮含量逐渐升高,而不同异黄酮合成相关酶基因的表达趋势不同,CHS7、CHS8、CHR、CHI1A和IFS2的表达趋势与异黄酮积累模式基本一致,而IFS1和CHI1B1的表达趋势与异黄酮积累模式相反。IFR的表达模式在2个大豆品种中存在相反的趋势,在LHD2中与异黄酮组分积累趋势相反,而在NHZ中与异黄酮组分积累趋势相同。结果还表明,同一基因家族中不同基因在子粒发育过程中的表达量也存在差异。查尔酮合酶基因家族中CHS7和CHS8以及查尔酮异构酶基因家族的CHI1A的表达水平相对其他成员较高,异黄酮合酶基因家族中IFS2的表达量显著高于IFS1的表达量,预示这些基因家族在大豆子粒异黄酮积累过程中存在功能分化。此外,各基因表达模式与异黄酮积累的相关分析结果表明,不同基因表达模式与异黄酮积累的相关性在2个品种中也不尽相同。LHD2中CHS7、CHS8和IFS2在子粒发育过程中的表达量变化与不同异黄酮组分呈显著正相关,CHI1B1基因的表达量变化与不同异黄酮组分呈显著负相关。而在NHZ中,IFR在子粒发育过程中的表达量变化与多个异黄酮组分呈显著正相关。这预示了不同大豆品种异黄酮含量差异的潜在遗传基础。各异黄酮合成相关酶基因表达量变化的相关分析表明,在2个品种中,苯丙氨酸水解酶PAL1与4CL,4CL与CHS2以及CHS1与IFS2基因的表达量均呈现显著正相关。表明这些基因可能通过协同作用共同调控异黄酮的合成与积累。这些结果为今后利用基因工程提高大豆异黄酮含量奠定了基础。     

大豆异黄酮神经保护作用研究进展

对大豆异黄酮神经保护作用的研究进展进行综述,旨在为大豆异黄酮功能食品开发以及其他食品功能组分的神经保护作用研究提供科学依据。     

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。     

黄淮海大豆异黄酮含量分析与特异种质遴选

以黄淮海生态区181份栽培大豆与32份野生大豆为材料,采用高效液相色谱法测定其籽粒异黄酮及组分含量,分析该地区大豆籽粒异黄酮含量遗传变异,遴选高异黄酮特异种质,为相关基因克隆表达、RIL群体构建和专用型品种选育提供资源。结果表明,供试栽培大豆异黄酮含量在1462.6-6115.5 μg/g,平均为3558.2 μg/g,最大差异可达4.2倍;供试野生大豆异黄酮含量在3896.1-7440.4 μg/g,平均为5182.4 μg/g,最大差异可达1.9倍。可见,黄淮海生态区大豆资源异黄酮及组分含量存在较大遗传变异,且野生大豆异黄酮平均含量显著高于栽培大豆。从供试资源中遴选出异黄酮含量超过6000 μg/g特异种质4份（超过7000 μg/g种质1份）。     

代表性大豆种质异黄酮主要组分含量鉴定

采用HPLC检测方法,对100份不同来源的代表性大豆种质进行异黄酮含量分析,结果显示大豆种质中主要含有6种异黄酮组分,分别为黄豆苷（D）、黄豆黄苷（GL）、染料木苷（G）、丙二酰基黄豆苷（MD）、丙二酰基黄豆黄苷（MGL）和丙二酰基染料木苷（MG）,且异黄酮含量在不同生态区间和品种间均存在显著差异,变异丰富;南方产区大豆品种的异黄酮总含量最高（2465.48ug/g）,黄淮大豆产区次之（2308.48ug/g）,北方大豆产区最低（1705.89ug/g）;6 种主要异黄酮组分含量的变异系数变化范围为33.44%～52.03%。相关分析表明异黄酮总含量与各主要组分含量之间均呈极显著正相关,与脂肪含量呈极显著负相关（r = -0.323**）。在此基础上,筛选出高异黄酮含量的大豆种质2份,分别为平顶黑豆（4459.91 ug/g）和PI-567479（4073.95 ug/g）,低异黄酮含量的大豆种质2份,分别为茶色豆（857.74 ug/g）和牡丰1号（922.82 ug/g）,可以用于大豆异黄酮育种或遗传研究。     

双歧杆菌发酵对豆芽汁中大豆异黄酮含量及组分影响的研究

目的探讨双歧杆菌发酵对豆芽汁中大豆异黄酮含量及组分影响.方法采用HPLC法检测波长260 nm,测定豆芽汁中金省异黄素、大豆黄素的含量.结果表明通过发酵处理后,豆浆中总大豆异黄酮浓度没有明显的变化(P＞0.05);大豆异黄酮甙元的浓度较未经过发酵处理的豆浆组明显增加(P＜0.05);而大豆异黄酮糖甙的浓度却明显降低(P＜0.05).结论豆浆中的大豆异黄酮糖甙在双歧杆菌的β葡萄糖苷酶作用下水解为异黄酮甙元,异常酮甙元的浓度显著增加.     

西南大豆种质资源的化学评价及其与田间霉变抗性的相关性研究

以西南地区不同来源的15个大豆种质资源为研究对象,研究大豆主要化学成分与其田间霉变抗性间的相关性。采用高效液相色谱法测定大豆中12种异黄酮含量,气质联用法测定大豆中19种脂肪酸含量,分光光度法测定大豆皂苷、可溶性多糖、蛋白含量,并进行聚类及相关性分析。结果表明,15个大豆种质资源依据化学成分含量被分为5个类别,C103等材料的品质性状优良,具有较好的田间霉变抗性,可作为优质大豆种质资源进一步开发利用。棕榈酸、反亚油酸和芥酸等脂肪酸组分含量高的大豆材料其霉变程度更高,A环C-6位甲氧基化的GL型异黄酮(黄豆黄素、黄豆黄苷、乙酰黄豆黄苷、丙二酰黄豆黄苷)及异黄酮苷元含量高的大豆具有更大的抗霉变潜力。     

传统大豆发酵食品中大豆异黄酮的生理保健机能的研究进展

大豆异黄酮属于黄酮类化合物,是一类具有重要生物活性的化合物,在大豆和传统大豆发酵食品中含量丰富。近年来研究发现大豆异黄酮在预防癌症、骨质疏松症、心血管疾病和改善妇女更年期综合症等方面具有广泛的生理活性,因此引起了国内外学者的广泛关注。大豆异黄酮的研究,不仅为功能性大豆制品的开发提供理论基础,也为人们合理的膳食提供参考。综述近年来国内外对大豆异黄酮生理保健机能的研究进展。     

大豆异黄酮生物合成关键酶及其代谢工程研究进展

异黄酮是一类具有C-6/C-3/C-6骨架的二次代谢产物,具有抗氧化和抗肿瘤活性。异黄酮与黄酮类物质具有相似的苯丙烷生物合成途径。天然的绝大部分异黄酮分布在豆科植物中,目前在大豆中已经发现了超过12个异黄酮(苷)。大豆异黄酮的生物合成主要涉及三个关键的酶查尔酮合酶(CHS)、查尔酮异构酶(CHI)和异黄酮合酶(IFS)。总结了大豆异黄酮的提取分离方法和生物合成途径,着重综述了CHI、CHS、IFS生物学特征和功能及异黄酮的代谢工程研究。     

Bioflavonoids as poisons of human topoisomerase II alpha and II beta

Bioflavonoids are human dietary components that have been linked to the prevention of cancer in adults and the generation of specific types of leukemia in infants. While these compounds have a broad range of cellular activities, many of their genotoxic effects have been attributed to their actions as topoisomerase II poisons. However, the activities of bioflavonoids against the individual isoforms of human topoisomerase II have not been analyzed. Therefore, we characterized the activity and mechanism of action of three major classes of bioflavonoids, flavones, flavonols, and isoflavones, against human topoisomerase IIalpha and IIbeta. Genistein was the most active bioflavonoid tested and stimulated enzyme-mediated DNA cleavage approximately 10-fold. Generally, compounds were more active against topoisomerase IIbeta. DNA cleavage with both enzyme isoforms required a 5-OH and a 4'-OH and was enhanced by the presence of additional hydroxyl groups on the pendant ring. Competition DNA cleavage and topoisomerase II binding studies indicate that the 5-OH group plays an important role in mediating genistein binding, while the 4'-OH moiety contributes primarily to bioflavonoid function. Bioflavonoids do not require redox cycling for activity and function primarily by inhibiting enzyme-mediated DNA ligation. Mutagenesis studies suggest that the TOPRIM region of topoisomerase II plays a role in genistein binding. Finally, flavones, flavonols, and isoflavones with activity against purified topoisomerase IIalpha and IIbeta enhanced DNA cleavage by both isoforms in human CEM leukemia cells. These data support the hypothesis that bioflavonoids function as topoisomerase II poisons in humans and provide a framework for further analysis of these important dietary components.     

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.     

Inhibition of lipoxygenase by soy isoflavones: evidence of isoflavones as redox inhibitors

Hydroperoxides, the products of lipoxygenase mediated pathways, play a major role in the manifestation of chronic inflammatory diseases. Soy isoflavones act as antioxidants due to their ability to scavenge free radicals. Isoflavones inhibit the activity of soy lipoxygenase-1 and 5-lipoxygenase, from human polymorph nuclear lymphocyte in a concentration dependent manner. Spectroscopic and enzyme kinetic measurements have helped to understand the nature and mechanism of inhibition. Genistein is the most effective inhibitor of soy lipoxygenase 1 and 5-lipoxygenase with IC(50) values of 107 and 125 microM, respectively. Genistein and daidzein are noncompetitive inhibitors of soy lipoxygenase 1 with inhibition constants, K(i), of 60 and 80 microM, respectively. Electron paramagnetic resonance and spectroscopic studies confirm that isoflavones reduce active state iron to ferrous state and prevent the activation of the resting enzyme. A model for the inhibition of lipoxygenase by isoflavones is suggested.     

Behavioral effects of endocrine-disrupting substances: phytoestrogens

A major source of endocrine-disrupting substances, usually not considered in laboratory animal experiments, is the diet used in research investigations. Soy represents the main protein source in almost all natural-ingredient commercially available formulated diets. Soy-derived isoflavones are the most abundant and in many ways the most studied phytoestrogens, and phytoestrogens (isoflavones) are known endocrine disruptors. Research is reviewed that identifies the physiological and behavioral endocrine-disrupting effects of dietary phytoestrogens (isoflavones) in animal diets, including most of the isoflavones, which are in a glycoside form and biologically inactive, and those in the gastrointestinal tract, which are biologically active. The isoflavones genistein and daidzein have similar molecular weights and structural characteristics to that of 17-beta estradiol, which may enable them to exert estrogenic and antiestrogenic properties are described and characterized. Daidzein can be further metabolized to the potent and abundant molecule equol, which in rodents is produced in very large amounts and represents the major circulating metabolite among all biologically active isoflavones. Equol has the unique and important ability to specifically bind 5 alpha-dihydro-testosterone, and to act in turn to inhibit the action of this potent androgen. The specific influence of dietary soy phytoestrogens on consumptive, learning and memory, and anxiety-related behaviors is identified. Regulatory behaviors such as food and water intake, adipose deposition and leptin, and insulin levels affected by dietary isoflavones are also discussed.     

Diets high in selenium and isoflavones decrease androgen-regulated gene expression in healthy rat dorsolateral prostate

Background  

High dietary intake of selenium or soybean isoflavones reduces prostate cancer risk. These components each affect androgen-regulated gene expression. The objective of this work was to determine the combined effects of selenium and isoflavones on androgen-regulated gene expression in rat prostate.     

Analysis of the estrogenic components in kudzu root by bioassay and high performance liquid chromatography

The estrogenic activity of the Chinese herb kudzu root was investigated by a recombinant yeast screening assay (YES). Isoflavones are the main components in the plant, of which puerarin is the most abundant one. The kudzu root extract was separated into four fractions according to the polarity. The crude extract and its sub-fractions, except the water fraction, showed clear estrogenic activity and the potencies were in the range of 10(-3) to 10(-1)g/l. The ligand potency was used to compare the estrogenic activity of these fractions. The crude extract and its sub-fractions were further analyzed by high performance liquid chromatography (HPLC) to correlate the activity and the active components. Bioassay and chemical analysis showed that theoretical estrogenic activity expressed as equivalent 17beta-estradiol concentration or the cumulative effects are comparable to that experimentally determined by YES. The results showed that the high content of isoflavones as well as the high estrogenic activity could make kudzu root extract an interesting candidate for hormone replacement therapy.     

Isoflavones: their effects on cardiovascular risk and functions

PURPOSE OF REVIEW: Isoflavones - plant polyphenols with oestrogenic activity - are becoming widely distributed in foods and sold as nutriceuticals. Their similarity to steroidal oestrogens has stimulated research into potential cardiovascular benefits. The two major areas, those of lipid lowering and of improved vascular function, are reviewed. RECENT FINDINGS: A major plasma cholesterol lowering effect has not been sustained in recent publications. Relatively minor reductions in LDL-cholesterol or non-HDL-cholesterol have generally been reported, but studies in which no reduction was found have also been reported. Fewer publications have dealt with effects on vasculature, but these have been more consistent. Arterial compliance, a parameter of the elasticity or distensibility of large arteries, is improved by as much as has been reported for estrogens. Vasodilatory effects on the microcirculation with isoflavones or their metabolites may provide opportunities for pharmacological intervention. The metabolism of isoflavones is reviewed briefly in order to highlight its complexity. SUMMARY: Although research has failed to demonstrate substantial cholesterol lowering with dietary isoflavones, consumption of legumes is generally to be encouraged as part of the overall strategy for lowering plasma lipids. The favourable effects of ingested or infused isoflavones on several parameters of arterial function suggest an alternative explanation for the apparent low prevalence of coronary disease in populations that eat legumes as a staple food. A better understanding of the pharmacokinetics and bioavailabilty of individual isoflavones is needed if definitive studies are to be designed.     

The characterisation of structural and antioxidant properties of isoflavone metal chelates

Isoflavone metal chelates are of interest as isoflavones act as oestrogen mimics. Metal interactions may enhance isoflavones biological properties so understanding isoflavone metal chelation is important for the commercial application of isoflavones. This work aimed to determine if isoflavones, daidzein (4',7-dihydroxyisoflavone) and genistein (4',5,7-trihydroxyisoflavone) could chelate with metals as isoflavone chelates. Biochanin A (4'-methoxy-5,7-dihydroxyisoflavone) was also examined for it's ability to chelate with Cu(II) and Fe(III). This study found daidzein does not chelate with Cu(II) and Fe(III) but genistein and biochanin A chelate with a 1:2 M/L stoichiometry. The copper and iron chelates were synthesised and characterised by elemental analysis, FTIR, thermogravimetric analysis (TGA) and electrospray ionisation mass spectrometry (ESI-MS). These studies indicated a 1:2 M/L stoichiometry and suggested the isoflavones bind with the metals at the 4-keto and the 5-OH site. 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition assays showed that copper isoflavone chelates have higher antioxidant activity than free isoflavones while the iron isoflavone chelates showed pro-oxidant activity compared to the free isoflavone. Synergistic DPPH studies with 0.02 mM ascorbic acid revealed copper chelates exhibit reduced antioxidant activity versus free isoflavones whereas the iron chelates showed lower pro-oxidant activity except at 1.0 mM.     

Determination of Seed Storage Proteins and Total Isoflavones in Wild and Cultivated Soybeans

Two soybean components namely, storage proteins and isoflavone content in a wild and three cultivated soybean genotypes were characterized and compared. The storage proteins, β-conglycinin and glycinin were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and two major storage proteins and their subunits were characterized using mass spectrometry. The three isoflavones, aglycon and the nine conjugated forms were separated by HPLC (high performance liquid chromatography) and identified by comparison of retention time, ultraviolet and mass spectral analyses. Comparison between the number of 2D-PAGE protein spots of the storage protein subunits and HPLC area of twelve isoflavones was also evaluated. The analysis of proteins and isoflavones from the wild genotype and the three cultivated genotypes suggested possible interactions between proteins and isoflavones. The same wild genotype, which showed significant statistical differences in β-conglycinin and glycinin protein profiles also revealed considerable reduction in total isoflavones (> 55%) content.     

高效液相色谱法测定大豆乳清提取物中大豆异黄酮的含量

建立了大豆乳清提取物中大豆异黄酮含量的高效液相色谱测定方法。采用Nova-Pak C₁₈（3.9×150 mm,4 μm）色谱柱;以甲醇：0.4%磷酸=30：70（v/v）为流动相分析染料木苷和黄豆苷;流速为0.7 mL·min^-1;柱温为30℃;检测波长为260 nm。试验结果表明,大豆乳清提取物中的大豆异黄酮含量为72.5%,其组成以染料木苷和黄豆苷为主,二者比例接近1∶1,苷元型大豆异黄酮未检出。染料木苷和黄豆苷的平均回收率分别为98.1%和98.4%,相对标准偏差（RSD）分别为0.7%（n=5）和0.8%(n=5)。该方法快速、准确、重复性好。