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。     

水提取法分离大豆异黄酮苷和苷元

本文建立了一种仅用水作为溶剂分离大豆异黄酮苷和苷元的方法。采用水加热提取的方法分离大豆异黄酮苷和苷元,分别对所用溶剂,提取温度,提取时间和物料比进行优化。实验上清液干燥后的固形物中大豆异黄酮苷含量为32．24％,苷元含量仅为3．05％,沉淀中大豆异黄酮苷元含量为72．03％,苷含量仅为4．28％。该方法经济,简单,绿色无毒,适合工业生产。     

黑龙江省野生和栽培大豆异黄酮与其组分相关性分析

利用高效液相色谱法（HPLC）检测了黑龙江省556份不同生态区以及不同类型大豆的异黄酮、大豆苷和染料木苷含量,其中野生大豆243份,栽培大豆313份。结果表明：野生大豆异黄酮含量高于栽培大豆,同时筛选出高异黄酮含量种质3份,低异黄酮含量种质2份。异黄酮、大豆苷和染料木苷含量三者问的相关分析表明,大豆异黄酮含量与大豆苷含量及染料木苷含量、大豆苷含量与染料木苷含量均呈极显著正相关。     

加工方法对毛豆中大豆异黄酮苷元含量的影响及其对糖尿病小鼠的降血糖降血脂活性研究

本文比较不同处理方式下毛豆中大豆异黄酮苷元的含量,继而对冻干条件下处理的毛豆样品进行降血糖降血脂活性的研究。采用高效液相色谱(HPLC)法测定通过热烫、煮沸、冷冻、冻干、烘干从毛豆中提取出的大豆异黄酮苷元的含量。HPLC测定条件如下:色谱柱:Hypersil C18(250 mm×4.6 mm,5μm),流动相:甲醇-0.3%磷酸溶液(48∶52),流速:0.7 mL/min,测定波长:260 nm,柱温:25℃,进样量:20μL。留一组健康小鼠作为空白,将造模成功的ICR小鼠随机分为3组,分别用生理盐水,消渴丸,冻干毛豆,对其连续灌胃4周,4周后处死,测定其血糖和血脂指标。结果显示,冻干条件下,大豆异黄酮苷元含量最多(115.45μg/g),并且冻干的毛豆能够显著改善糖尿病小鼠的血糖和血脂水平。     

大豆异黄酮浸种对盐胁迫大豆幼苗的生理效应

大豆异黄酮( Soybean isoflavones)是在大豆生长过程中形成并在成熟种子和叶片中积累较多的一类具有生物活性的次生代谢物,通常可作为人们日常生活中的一类营养保健品.研究了外源大豆苷或染料木苷溶液(0.01 mg/L)浸种处理对盐胁迫栽培大豆(N23674品种)和滩涂野大豆(BB52种群)及其经逐代耐盐性筛选的杂交后代(4076株系,F5)幼苗叶片伤害率、光合作用、Na+含量和Na+/K+值、活性氧清除酶活性及内源大豆异黄酮含量等生理指标的影响.结果表明:盐胁迫下,两种外源大豆异黄酮浸种处理均可显著抑制叶片相对电解质渗透率和硫代巴比妥酸反应物(TBARS)含量的上升及净光合速率(Pn)的下降,降低Na+含量和Na+/K+值,增强超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性,提高内源大豆异黄酮含量,从而表现对盐害的缓解效应,其中对耐盐性较弱的栽培大豆N23674品种效应更明显.这为大豆异黄酮在大豆耐盐育种、化学调控和盐碱地种植利用等提供了理论依据.     

茉莉酸甲酯和ABA对野葛毛状根中异黄酮含量的影响

10～ 10 0 μmol·L-1茉莉酸甲酯 (MJ)可提高野葛毛状根培养液中葛根素和大豆甙元的水平 ,促进毛状根内总异黄酮含量的增加。而用 0 .5～ 2 .0mg·L-1ABA处理后 ,无论对野葛毛状根还是培养液中葛根素、大豆甙元的含量仅略有提高 ,但对总异黄酮的合成与分泌等则有显著的促进。 10 0 μmol·L-1MJ和 1mg·L-1ABA处理 2 4～ 72h ,可促进毛状根内葛根素和培养液中总异黄酮的水平 ,以处理 4 8h的增加量最大     

光照对大豆幼苗组织中异黄酮含量和分布的影响

利用高效液相色谱（ＨＰＬＣ）测定了不同光照处理的大豆（Ｇｌｙｃｉｎｅｍａｘ（Ｌ．）Ｍｅｒｒｉ．）幼苗不同组织的异黄酮类含量。子叶中最高,叶片和根部相对较少。子叶的异黄酮以大豆甙和染料木甙及其丙二酰基结合体为主,且在光照条件下,异黄酮含量随光照时间的增加而显著升高;相反,黑暗中的异黄酮含量随苗龄的增加呈下降趋势;当子叶由黑暗转为光照处理以后,异黄酮含量同样随光照时间的增加而升高。在叶片和根部异黄酮含量和种类也因光照条件的不同而有很大差异。光照条件下,叶片中以染料木甙及其丙二酰结合体和黄酮芦丁为主,且随时间增加呈上升趋势;黑暗中的黄化叶片,则以大豆甙和丙二酰结合体为主,但随时间的变化不明显。在幼苗根部,黑暗条件下几乎检测不出异黄酮的存在;光照条件下,则可检测到５种异黄酮,其中以大豆甙元及其衍生物占主要部分。实验证实了光照对大豆异黄酮的积累有明显的促进作用     

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.     

西南地区三种天麻变型巴利森苷类成分含量比较

本文对西南地区三种天麻变型的巴利森苷类成分含量进行了分析。结果表明西南地区不同天麻变型巴利森苷类含量间差异较大,其中四川广元红天麻巴利森苷A、C含量最高,分别为8. 770 mg/g和3. 827 mg/g;四川北川乌天麻巴利森苷B、E含量最高,分别为11. 461 mg/g和21. 010 mg/g,均与其他天麻材料间含量差异极显著;四川地区3种天麻变型巴利森苷B、E含量显著高于云南、贵州地区天麻变型,巴利森苷A、C含量在3地区间差异不显著。综合来看,西南地区天麻变型巴利森苷类含量整体差异较大,其中巴利森苷类含量以四川地区最高,其含量与天麻变型种类无关,总体上以四川北川的乌天麻巴利森苷类成分最高。     

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）。     

Identification of a naturally‐occurring 8‐[α‐D‐glucopyranosyl‐(1→6)‐β‐D‐glucopyranosyl]daidzein from cultivated kudzu root

Introduction – Kudzu root (Radix puerariae) is a rich source of isoflavones that are effective in preventing osteoporosis, heart disease and symptoms associated with menopause. The major isoflavonoids in kudzu root extracts were reported as puerarin, daidzin and daidzein. Recently, an unknown isoflavonoid (compound 1) was detected from one‐year‐old kudzu root cultivated in Vietnam. Objective – To identify a novel compound 1 in kudzu root extract and determine the structure of the compound by ESI⁺ TOF MS‐MS, ¹H‐, ¹³C‐NMR and enzymatic hydrolysis. Methodology – Samples were prepared by extraction of one‐year‐old kudzu root with 50% ethanol and the isoflavonoids were purified using recycling preparative HPLC. Unknown compound 1 was detected using UV‐light at 254 nm in TLC and HPLC analyses. The molecular weight of 1 was determined using a TOF mass spectrometer equipped with an electrospray ion source. The structure of 1 was determined from the ¹³C and ¹H NMR spectra recorded at 100.40 and 400.0 MHz, respectively. Results – ESI⁺ TOF MS‐MS analysis shows that 1 is a puerarin diglycoside. The interglycosidic linkage of diglycoside determined by ¹H‐, ¹³C‐NMR, and enzymatic hydrolysis suggests that 1 has a glucosyl residue linked to puerarin by an α‐1,6‐glycosidic bond. This compound is the first naturally‐occurring 8‐[α‐D ‐glucopyranosyl‐(1→6)‐β‐D ‐glucopyranosyl]daidzein in kudzu root. The concentration of glucosyl‐α‐1,6‐puerarin in kudzu root was 2.3 mg/g as determined by HPLC. Conclusion – The results indicate that puerarin diglycoside is one of the major isoflavonoids in kudzu root and has a significant impact on the preparation of highly water‐soluble glycosylated puerarin. Copyright © 2009 John Wiley & Sons, Ltd.     

In vitro isoflavonoid production in callus from different organs of Pueraria lobata (Wild.) Ohwi

Pueraria lobata (Wild.) Ohwi is a medicinal plant producing large amounts of isoflavonoid glycosides. Here, the ability of in vitro callus cultures to synthesize isoflavonoids was tested. Callus cultures have been initiated from different explants of in vitro germinated plants using modified MS medium. Roots, leaves and stem segments were the best sources of callus tissue. The isoflavonoid profile and content was determined by means of chromatographic methods. Callus from all organs contained isoflavonoid aglycones: genistein and daidzein and daidzein glycosides: daidzin, puerarin and 3'-methoxypuerarin. The differences between each kind of explant were observed in both the total amount of isoflavonoids and in the proportion of individual compounds. The highest content was in root callus, followed by leaf- and stem callus.     

Identification of isoflavone glycosides in Pueraria lobata cultures by tandem mass spectrometry

Isoflavones in the methanolic extracts of kudzu (Pueraria lobata) callus, suspension and root cultures were compared in order to develop an experimental system in which puerarin (daidzein 8-C-glucoside) and other isoflavones could be synthesised in vitro. Quantitative variation of puerarin and other known isoflavones was estimated in kudzu culture extracts using HPLC-UV. The highest and lowest amounts of puerarin (14.56 and 0.33 mg/g) were present in in vitro root cultures and leaf tissue-derived callus cultures, respectively. A total of 48 isoflavone metabolites were detected in extracts of kudzu root cultures by HPLC-MS/MS, and the structures of 33 of them were tentatively assigned. Amongst these, 12 isoflavone C-glycosides were identified. Hydroxyderivatives of puerarin in several isomeric forms were detected, some of which have not been previously reported in kudzu root. The molecular weights, interpretation of characteristic fragment ions obtained from HPLC-MS/MS and comparison with reported data allowed the putative identification of the isoflavone metabolites.     

Correlation of antioxidant activity and major isoflavonoid contents of the phytoestrogen-rich Pueraria mirifica and Pueraria lobata tubers.

The antioxidant activity of wild Pueraria mirifica collected from 28 of the 76 provinces of Thailand and Pueraria lobata collected from China were assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. P. mirifica tuberous extracts showed weak antioxidant activity in comparison with alpha-tocopherol. Six plant samples exhibited stronger antioxidant activity than the mean value of the P. mirifica population. In addition, the mean value of the P. mirifica population indicated significantly lower antioxidant activity than P. lobata. The analysis of the antioxidant activity of isoflavonoids revealed that puerarin and daidzein exhibited the same level of antioxidant activity as alpha-tocopherol. The results showed convincingly that puerarin and daidzein in the plant tubers may play an important role in antioxidant activity. The correlation analysis between antioxidant activity and major isoflavonoid contents of plant tubers indicated a significant correlation only with puerarin and a significant lack of correlation with daidzin, daidzein and genistein.     

Regulation of isoflavone production in hydroponically grown Pueraria montana (kudzu) by cork pieces, XAD-4, and methyl jasmonate

A mini-hydroponic growing system was employed for seedlings of kudzu vine (Pueraria montana) and contents of isoflavones (daidzein, genistein, daidzin, genistin, and puerarin) from shoot and root parts of seedlings were analyzed quantitatively. In addition, exogenous cork pieces, polymeric adsorbent, XAD-4, and universal elicitor, methyl jasmonate (MeJA), were used to regulate the production of these isoflavones. It was shown that cork pieces up-regulate the production of daidzein and genistein up to seven- and eight-fold greater than the levels obtained for control roots. In contrast, levels of glucosyl conjugates, daidzin and genistin, decrease up to five- and eight-fold, respectively. Cork treatment also induces the excretion of the root isoflavone constituents into the growth medium. Minimal levels of isoflavones are absorbed by the cork pieces. XAD-4 stimulates the production of glucosyl conjugates, daidzin and genistin, in root parts about 1.5-fold greater than that obtained in control roots. These are the highest amounts of daidzin and genistin that are observed (5.101 and 6.759 mg g⁻¹ dry weight, respectively). In contrast to these two adsorbents, MeJA increases the accumulation of isoflavones in shoot rather than in root parts of seedlings, about three- to four-fold over control levels, with the exception of genistein. These studies reveal new observations on the regulation of isoflavone production in hydroponically grown Pueraria montana plants by two adsorbents (cork pieces and XAD-4) and MeJA elicitor.     

葛藤与葛根中异黄酮类成分的比较

分析比较了野葛〔Ｐｕｅｒａｒｉａｌｏｂａｔａ（Ｗｉｌｄ．）Ｏｈｗｉ〕的藤（葛藤）和根（葛根）的主要异黄酮类活性成分。从葛藤中首次分离得到３个化合物,经化学方法和光谱鉴定,证实为大豆甙元（Ａ）、大豆甙（Ｂ）和葛根素（Ｃ）。采用双波长薄层扫描法测定葛藤与葛根中上述３种异黄酮化合物的含量,葛藤中大豆甙元、大豆甙和葛根素的含量分别为０．１９５％,３．９３３％和２．４８１％;葛根中则分别为０．０５９％,０．７１４％和４．３１５％。研究结果为葛藤新药源的开发利用提供了科学依据     

Isoflavonoids in the Rutaceae family: 1. Fortunella obovata, Murraya paniculata and four Citrus species

Several types of compounds with immunoreactivity similar to isoflavonoids were detected in water: ethanol extracts of leaves of Fortunella obovata Hort. ex Tanaka, Murraya paniculata Jack. and four Citrus species, namely C. aurantium L, C. grandis Osbeck, C. limonia Osbeck., and C. sinensis Osbeck (Rutaceae). The chromatographic mobilities of the immunoreactive substances were compared with those of authentic standards, revealing a spectrum of isoflavonoid metabolites in all plants studied. Aglycones as well as glycosides were recognized, namely daidzin, genistin, daidzein, genistein, formononetin, biochanin A, prunetin, and several incompletely characterized isoflavonoids. A subsequent HPLC-MS study verified the identities of the main immunoreactive isoflavonoids and established the identities of several others, viz. glycitein, glycitin, ononin and sissotrin, including the malonylated and acetylated isoflavonoid glucosides. The estimated content of the individual immunoreactive entities ranged from a few microg to about 2 mg/kg (dry weight). It is concluded that the isoflavonoid metabolic pathway is present throughout the Rutaceae family.     

Isolation of human intestinal bacteria metabolizing the natural isoflavone glycosides daidzin and genistin

Fecal bacteria from a healthy individual were screened for the specific bacteria involved in the metabolism of dietary isoflavonoids. Two strains of bacteria capable of producing primary and secondary metabolites from the natural isoflavone glycosides daidzin and genistin were detected. The metabolites were identified by comparison of their HPLC/mass, 1H NMR and UV spectra with those of standard and synthetic compounds. Both Escherichia coli HGH21 and the gram-positive strain HGH6 converted daidzin and genistin to the their respective aglycones daidzein and genistein. Under anoxic conditions, strain HGH6 further metabolized the isoflavones daidzein and genistein to dihydrodaidzein and dihydrogenistein, respectively. The reduction of a double bond between C-2 and C-3 to a single bond was isoflavonoid-specific by strain HGH6, which did not reduce a similar bond in the flavonoids apigenin and chrysin. Strain HGH6 did not further metabolize dihydrodaidzein and dihydrogenistein. This is the first study in which specific colonic bacteria that are involved in the metabolism of daidzin and genistin have been detected.     

A process for high-efficiency isoflavone deglycosylation using Bacillus subtilis natto NTU-18

In order to produce isoflavone aglycosides effectively, a process of isoflavone hydrolysis by Bacillus subtilis natto NTU-18 (BCRC 80390) was established. This process integrates the three stages for the production of isoflavone aglycosides in one single fermenter, including the growth of B. subtilis natto, production of β-glucosidase, deglycosylation of fed isoflavone glycosides. After 8 h of batch culture of B. subtilis natto NTU-18 in 2 L of soy medium, a total of 3 L of soy isoflavone glucoside solution containing 3.0 mg/mL of daidzin and 1.0 mg/mL of genistin was fed continuously over 34 h. The percentage deglycosylation of daidzin and genistin was 97.7% and 94.6%, respectively. The concentration of daidzein and genistein in the broth reached 1,066.8 μg/mL (4.2 mM) and 351 μg/mL (1.3 mM), respectively, and no residual daidzin or genistin was detected. The productivity of the bioconversion of daidzein and genistein over the 42 h of culture was 25.6 mg/L/h and 8.5 mg/L/h, respectively. This showed that this is an efficient bioconversion process for selective estrogen receptor modulator production.     

Isoflavonoids production in callus culture of Pueraria tuberosa, the Indian kudzu

Isoflavonoid contents of different plant parts and callus tissues of the Indian Kudzu, Pueraria tuberosa (Roxb.ex.Willd.) DC are presented. The initial cultures were slow growing, associated with browning of the tissues. The production of four isoflavonoids (puerarin, genistin, genistein and daidzein) in the callus cultures of P. tuberosa was studied by manipulating the plant growth regulators and sucrose concentration in the medium. Organogenesis was not recorded in callus on any of these treatments. Tuber and stem accumulated puerarin, a glycoside of daidzein, at high amounts, 0.65% and 0.054% respectively. However, the daidzein content of the callus tissues grown on Murashige and Skoog medium containing BA (20.9 microM) and sucrose (60 gl(-1)) was significantly higher (0.056%) than in vivo plant material (0.02%) and other comparable culture systems like Genista and Pueraria lobata.