合成大豆异黄酮的植物基因工程研究

异黄酮是主要存在于豆科植物中的一种具有广谱抗菌活性的黄酮类物质。经过多年生理生化研究发现，异黄酮，特别是作为大豆异黄酮代表的染料木素和大豆黄素，具有良好的预防骨质疏松、更年期综合症、心脑血管疾病和多种癌症的保健效果，是大豆及豆制品中重要的营养成分之一，已有多年被用作保健品功能成分的历史。异黄酮是植物苯丙烷类次生代谢途径的一类产物，随着近年来对植物次生代谢途径研究的不断深入，特别是合成异黄酮的关键酶——异黄酮合酶（isoflavone synthase, IFS）基因的成功克隆，异黄酮合成途径已经得到完全解析。研究者随即展开了合成异黄酮的基因工程研究，在研究过程中逐渐了解到这条代谢途径受到了复杂的分子调控。本文将结合本课题组的工作，综述基因工程合成异黄酮的研究历史与成果，重点深入分析和讨论异黄酮生物合成所受到的分子调控。     

高异黄酮大豆突变体的筛选及其特性初步研究

大豆是异黄酮的主要来源,且是唯一在营养学上有意义的自然食物来源,为提高日常大豆异黄酮的摄入量,培育高异黄酮大豆植株,采用120 Gy60Co-γ射线对大豆品系ZN01干种子进行辐射诱变处理,筛选获得高异黄酮大豆突变株SHIS-01(5‰),并对其成分及特性进行初步研究。结果表明,除黄豆苷元外,突变体的黄豆苷、大豆苷、染料木苷、丙二酰黄豆苷、丙二酰染料木苷、丙二酰黄豆黄素苷、染料木苷元等组分含量均显著高于野生型。此外,除膳食纤维外,高异黄酮大豆突变体的营养成分与野生型基本相同。突变体的糊化特性和质构特性与野生型差异较大,可能由膳食纤维含量的变化引起。突变体SHIS-01稳定的高大豆异黄酮含量特性,为今后高异黄酮大豆的生物育种拓宽了思路,也为进一步研究大豆异黄酮的加工利用提供了良好的试验材料。     

施氮量对间作小麦蚕豆根系分泌大豆异黄酮的影响

通过盆栽和水培试验,采用小麦蚕豆间作、蚕豆单作、小麦单作3种种植方式,研究了不同生育期不同氮水平(低氮、常规氮和高氮)处理下,单、间作小麦和蚕豆根系大豆异黄酮分泌量的变化,为进一步探明间作增产和控病机制提供依据。结果表明,随作物生育期推移,小麦根系分泌大豆异黄酮数量明显减少,蚕豆根系大豆异黄酮的分泌量先增加后减少。随施氮量增加,小麦蚕豆根系大豆异黄酮分泌量均减少,且多达显著水平。与低氮处理相比,常规氮和高氮处理下,单、间作小麦大豆异黄酮分泌量分别显著减少,且多达显著水平。间作可以显著提高作物大豆异黄酮的分泌量,但间作优势仅在低氮和常规氮处理下明显,高氮处理下,单、间作小麦和蚕豆根系分泌大豆异黄酮数量差异不显著,并且这种间作效应会随生育期的推移逐渐消失。总之,间作种植和施氮量均影响作物根系大豆异黄酮分泌量,且低施氮量的影响更明显。     

大豆异黄酮的保健功效、生物合成及种质发掘与遗传育种

大豆异黄酮系一类植物次生代谢物,属于二元酚类植物雌激素,具有显著的保健功能,为了更好地推动高异黄酮大豆生物育种及其产业化,本文综述了大豆异黄酮的研究进展,包括大豆类黄酮作用机制和保健功、产品开发和安全评估、生物合成、种质筛选鉴定与遗传育种。     

大豆异黄酮生物合成与调控的分子机制研究进展

大豆异黄酮,是由肉桂酰辅酶A产生的3-苯基衍生物,属植物次生代谢产物。它具有降血压、缓解更年期综合症等诸多功效,被称为"植物雌激素"。本文在阐述影响大豆子粒中异黄酮含量的QTL位点的基础上,重点综述了异黄酮生物合成途径中的多种酶和相关基因,以及调控该途径中相关酶系基因表达的转录因子和相关基因,并分析探讨了该领域一些亟待解决的问题和未来发展的方向,以期为深入研究大豆异黄酮合成的分子机制及高异黄酮大豆分子育种提供理论参考。     

甘肃省大豆中异黄酮及组分与品质的相关性分析

利用高效液相色谱法(HPLC),对甘肃省种植的86份栽培品种和86份地方品种的大豆异黄酮、粗脂肪和蛋白质含量进行了检测,分析了大豆异黄酮与大豆主要品质性状的相关性。结果表明,总异黄酮含量与蛋白质含量呈负相关,与脂肪含量呈显著负相关;粗脂肪含量与大豆苷、大豆苷元、染料木苷和黄豆黄苷4种组分含量呈负相关。大豆总异黄酮含量与大豆苷、大豆苷元、染料木素和染料木苷含量呈极显著正相关,相关系数分别为0.520、0.268、0.462、0.969。     

大豆中6种异黄酮的高效液相色谱同步测定方法研究

本文建立了大豆中大豆苷、大豆黄苷、染料木苷、大豆素、大豆黄素和染料木素6种大豆异黄酮的高效液相色谱同步测定方法.大豆样品经80％甲醇提取,用反相C18柱分离,紫外检测器检测.结果表明,6种异黄酮线性范围为0.096～4.8 μg/mL,相关系数大于0.999,在豆粕中的添加回收率在80％～120％,变异系数小于10％.利用建立的方法检测了国内400个不同品种大豆中的异黄酮含量,同时分析了发酵、膨化、浸提等不同加工工艺对大豆异黄酮含量的影响,这些数据的获得将为大豆品种的选育及不同加工工艺的大豆产品在畜禽饲料中的高效利用提供基本参数.     

大豆异黄酮植物雌激素调节大鼠睾丸间质细胞分泌睾酮的机理

通过检测cAMP的浓度、cAMP依赖性的蛋白激酶A(PKA)活性、睾酮的含量及芳香化酶活性的变化，体外研究了大豆异黄酮调节大鼠睾丸间质细胞分泌睾酮的机制。结果显示：大豆异黄酮能够提高cAMP的含量和PKA的活性，同时抑制了芳香化酶的活性，最终导致间质细胞分泌睾酮增加。提示大豆异黄酮植物雌激素促进睾酮的分泌主要是通过激活间质细胞cAMP／PKA信号通路，并且抑制芳香化酶活性，从而降低睾酮向雌二醇的转化而实现的。     

大豆连作及其根茬腐解物对大豆根系分泌物中异黄酮类物质—黄豆甙元(daidzein)的影响

本试验通过收集正茬、重茬7年、重茬7年并经MB-生物制剂调控和加入根茬腐解物(20g kg-1土)的正茬、重茬、调控的大豆根系分泌物,采用高效液相色谱检测方法,对根系分泌物中的大豆异黄酮—黄豆甙元(daidze in)进行了检测。结果在正茬根系分泌物中没有检测到该物质,在重茬、调控根系分泌物中检测到了该物质,其检测出的量为重茬>调控。在加入根茬腐解物的各处理的根系分泌物中均检测到了大豆异黄酮,其检测出的量为重茬>正茬>调控。加入根茬腐解物的处理与不加根茬腐解物的相应的处理相比,在其根系分泌物中检测到的大豆异黄酮的量均有显著提高。     

4种中国大豆萌芽过程活性物质及抗氧化能力变化规律

为探究大豆萌芽过程中生物活性物质及抗氧化能力变化规律,以黑河43、黔豆7号、徐豆14和中黄13大豆为研究对象,对其萌芽过程中总多酚、总黄酮、大豆异黄酮和γ-氨基丁酸(GABA)含量进行动态监测,同时评价其抗氧化能力。结果表明,大豆经萌芽处理后,GABA大量积累,4种大豆GABA含量峰值分别为萌芽前的30、37、6.8和6.5倍,黔豆7号对GABA的富集最显著;总多酚、总黄酮及异黄酮的含量均有所上升,抗氧化能力有所增强,其中,黔豆7号总黄酮和异黄酮含量最高,说明其抗氧化能力也最强。相关性分析表明,4种大豆抗氧化能力与总多酚、总黄酮、大豆异黄酮含量显著相关。综上,萌芽是提高大豆功能性组分及抗氧化能力的有效手段;黔豆7号是富集生物活性物质的最佳品种,可用于功能性产品的开发。本研究为萌芽大豆的开发利用提供了科学依据,为后续的进一步研究奠定了理论基础。     

Identification of urinary metabolites of the red clover isoflavones formononetin and biochanin A in human subjects

Dietary supplements manufactured from red clover are widely marketed to provide the beneficial health effects of isoflavones without changing the original diet. In this study the metabolism of formononetin and biochanin A, the principal isoflavones of red clover, was studied in human subjects. Seven women ingested four red clover dietary supplements, and the metabolites of the isoflavones were identified in their urine samples. The structures of trimethylsilyl derivatives of the metabolites were established by GC-MS. New reduced metabolites of formononetin (dihydroformononetin and angolensin) and biochanin A (dihydrobiochanin A and 6'-hydroxyangolensin) were identified in urine samples using authentic reference compounds. Possible metabolic pathways are presented for the red clover isoflavones formononetin and biochanin A.     

Characterization of isoflavones and their conjugates in female rat urine using LC/MS/MS

Isoflavone phytoestrogens found in soybeans are the most widely studied phytochemicals in human diets and soy infant formulas. The health benefits of the isoflavones daidzein and genistein have been reported, and concerns about potential adverse effects have also been raised. However, the results of direct analysis of isoflavones and their metabolites in biological fluids after consumption of soy-containing diets are scarce. This study describes an LC/MS/MS method for the analysis of isoflavones and their metabolites in the urine of female rats fed diets made with soy protein isolate. Five isoflavones (daidzein, genistein, glycitein, dihydrodaidzein, and O-desmethylangolensin) were identified by comparison with authentic standards. Seventeen conjugates of isoflavones were characterized in the urine, the most unusual being genistein 5-glucuronide and four glucuronide conjugates of reductive metabolites of daidzein. The application of LC/MS/MS to analyze isoflavone metabolites is simple and sensitive, and appears to be an excellent method for determining the bioavailability and metabolism of food phytochemistry.     

Isoflavone aglycon and glucoconjugate content of high- and low-soy U.K. foods used in nutritional studies

The isoflavone aglycon and glucoconjugate content of commercially prepared and "home-prepared" high- and low-soy foods selected for use in an on-going nutritional study were measured by LC-MS. The daidzin, daidzein, 6"-O-malonyldaidzin, 6"-O-acetyldaidzin, genistein, genistin, 6"-O-malonylgenistin, 6"-O-acetylgenistin, glycitin, glycitein, 6"-O-malonylglycitin, and 6"-O-acetylglycitin content are expressed in terms of individual isoflavones, total isoflavone equivalents, and milligrams of isoflavones per portion served. Soybeans (774 mg x kg(-1) total isoflavones) and soybean-containing foods had the highest isoflavone content of the foods examined. The low-soy foods all contained very low concentrations (<8 mg x kg(-1) total isoflavones) of the isoflavone aglycons and glucoconjugates. High- and low-soy 11 day rotating menus were constructed from the analyzed foods to deliver 100.0 and 0.5 mg of isoflavones per day, respectively.     

Group B saponins in soy products in the U.S. Department of Agriculture--Iowa State University isoflavone database and their comparison with isoflavone contents

Isoflavones in soy protein foods are thought to contribute to the cholesterol-lowering effect observed when these products are fed to humans. The group B saponins are another ethanol-soluble phytochemical fraction associated with soy proteins and isoflavones and have also been associated with cholesterol-lowering abilities. We measured the group B soyasaponin concentrations in a variety of soy foods and ingredients in the U.S. Department of AgricultureIowa State University Isoflavone Database. We compared the isoflavone and soy saponin concentrations and distributions in intact soybeans, soy ingredients, and retail soy foods. Group B saponins occur in six predominant forms. There appears to be no correlation between saponin and isoflavone concentrations in intact soybeans ranging from 5 to 11 mumol isoflavones/g soybean and from 2 to 6 mumol saponin/g soybean. Depending upon the type of processing, soy ingredients have quite different saponins/isoflavones as compared to mature soybeans. In soy foods, the saponin:isoflavone ration ranges from 1:1 to 2:5, whereas in soy protein isolates, the ratio is approximately 5:3. Ethanol-washed ingredients have very low saponins and isoflavones. These very different distributions of saponins and isoflavones in soy products may affect how we view the outcome of feeding trials examining a variety of protective effects associated with soy consumption.     

Regulative actions of dietary soy isoflavone on biological antioxidative system and lipid metabolism in rats

Male Sprague-Dawley rats, 4 weeks of age, were fed purified diets either with or without 0.2% soy isoflavones rich powder for 5 weeks to elucidate their direct functions such as antioxidative action and regulation of lipid metabolism. Dietary soy isoflavones decreased serum lipid peroxide level in rats. Levels of liver and serum alpha-tocopherol were higher in the rats fed isoflavone than in those fed isoflavones-free diet. Thus, dietary soy isoflavones exhibited mild antioxidative function in this animal experiment. Isoflavone metabolites from diet may act as scavengers of reactive oxygen species. Dietary soy isoflavones lowered hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity, although liver cholesterol level was not modulated. However, the levels of serum cholesterol and triglyceride decreased by consumption of soy isoflavones. Therefore, dietary soy isoflavones may exhibit hypocholesterolemic and hypolipidemic functions. Moreover, dietary soy isoflavones lowered hepatic Delta6 desaturase activity. Reflecting this observation, Delta6 desaturation indices ((18:2(n = 6) + 18:3(n = 6))/20:4(n = 6)) of tissue lipids tended to be lower in rats fed isoflavones than in those fed isoflavones-free diet. This action may contribute to the prevention of inflammatory response by imbalance of eicosanoids. These observations suggest that the positive intake of soy isoflavones may reduce the risk of some cardiovasucular diseases through their radical scavenging function and hypocholesterolemic action.     

Pharmacokinetics of a slow-release formulation of soybean isoflavones in healthy postmenopausal women

Pharmacokinetic studies of soybean isoflavones have shown that following oral ingestion, the two major isoflavones, daidzin and genistin, are hydrolyzed in the intestine, rapidly absorbed into the peripheral circulation, and eliminated from the body with a terminal half-life of 7-8 h. These characteristics make maintenance of steady-state plasma isoflavone concentrations difficult to attain unless there is repeated daily ingestion of foods or supplements containing isoflavones. In an attempt to sustain more constant plasma isoflavone concentrations, a new slow-release formulation of a soybean isoflavone extract was prepared by microencapsulation with a mixture of hydroxypropylcellulose and ethylcellulose to alter its dissolution characteristics. In vitro experiments confirmed slow aqueous dissolution of isoflavones from this formulation when compared with the conventional isoflavone extract. The pharmacokinetics of this slow-release isoflavone extract was studied in 10 healthy postmenopausal women after oral administration of a single capsule containing the equivalent of 22.3 mg of genistein and 7.47 mg of daidzein expressed as aglycons. A comparison of the key pharmacokinetic parameters obtained in this study with those established in extensive studies performed previously in this laboratory indicated that the mean residence time of genistein and daidzein increased 2-fold with microencapsulation. These findings are indicative of a decreased rate of absorption, consistent with the observed slow in vitro dissolution rate. These findings show that it is feasible to employ polymer matrices that slow the aqueous dissolution for preparing sustained-release formulations of soy isoflavones. Further studies to optimize such formulations are warranted.     

Effects of solvent polarity and acidity on the extraction efficiency of isoflavones from soybeans (Glycine max)

Soy isoflavones have been correlated with beneficial health effects. The predominant chemical forms of isoflavones present may affect their biological activities. Choosing the solvent system that can accurately quantify the amounts of individual isoflavones present in these products is paramount. Our objectives were to compare frequently used solvent systems and to evaluate the effects of polarity and acidity on the recovery of isoflavones from soybeans. Isoflavones were extracted from pulverized Manokin soybeans using six solvent systems, which are the combinations of three polarity levels (83% acetonitrile, 80% methanol, and 58% acetonitrile) and two acidity levels (nonacidified and acidified). The pulverized soybean was stirred for 2 h in each solvent system before filtration and concentration using rotary evaporation. The extract was resuspended in 16% acetonitrile and analyzed by high-performance liquid chromatography. Recoveries of pure standards were evaluated with all solvent systems. Solvents with a higher polarity extracted a significantly higher amount of total isoflavones. For individual isoflavones, 58% acetonitrile (highest polarity) extracted either the highest amounts or no less than other solvents, while 83% acetonitrile (lowest polarity) extracted either the lowest amounts or no more than other solvents except for the aglycone form. Acidification significantly reduced the recovery of the malonylglucoside form and the total isoflavones. The recovery study revealed that acidification favored the chemical transformations of isoflavones during the extraction. Among the six solvent systems examined, 58% acetonitrile aqueous solution without acid was the best for extraction of isoflavones from soybeans.     

Isoflavone conjugates are underestimated in tissues using enzymatic hydrolysis

Many health effects of soy foods are attributed to isoflavones. Isoflavones upon absorption present as free form, glucuronide, and sulfate conjugates in blood, urine, and bile. Little is known about the molecular forms and the relative concentrations of soy isoflavones in target organs. Acid hydrolysis or enzymatic hydrolysis (glucuronidases and sulfatases) was used to study isoflavone contents in the heart, brain, epididymis, fat, lung, testis, liver, pituitary gland, prostate gland, mammary glands, uterus, and kidney from rats fed diets made with soy protein isolate. The heart had the lowest isoflavone contents (undetectable), and the kidney had the highest (1.8 +/- 0.6 nmol/g total genistein; 3.0 +/- 1.1 nmol/g total daidzein). Acid hydrolysis released 20-60% more aglycon in tissues than enzymatic digestion (p < 0.05), and both hydrolysis methods gave the same level of isoflavones in serum. Approximately 28-44% of the total isoflavone content within the liver was unconjugated aglycon, and the remainder was conjugated mainly as glucuronide. The subcellular distribution of total isoflavones was 55-60% cytosolic and 13-16% in each of the nuclear, mitochondrial, and microsomal fractions. These results demonstrated that (1) soy isoflavones distribute in a wide variety of tissues as aglycon and conjugates and (2) the concentrations of isoflavone aglycons, which are thought to be the bioactive molecules, are in the 0.2-0.25 nmol/g range, far below the concentrations required for most in vitro effects of genistein or daidzein.     

Correlations of oil and protein with isoflavone concentration in soybean [Glycine max (L.) Merr.

Twelve isoflavones were detected by high-performance liquid chromatography in seeds of 17 soybean [Glycine max (L.) Merrill] cultivars grown at three locations. 6' '-O-Malonyldaidzin and 6' '-O-malonylgenistin together constituted 71-81% of total isoflavones, which ranged in concentration from 2038 to 9514 microg/g and averaged 5644 microg/g across locations and cultivars. The total as well as several individual isoflavones had a moderate negative correlation with oil across locations and cultivars. Six cultivars had a moderate or strong negative correlation of total isoflavones with oil. Five cultivars had a moderate or strong positive correlation of total isoflavones with protein. These results suggest that judicious selection of germplasm for soybean breeding may facilitate development of soybean lines with desirable isoflavone concentrations.