Major isoflavonoid contents of the 1-year-cultivated phytoestrogen-rich herb, Pueraria mirifica

Pueraria mirifica is a tuberous plant enriched with active phytoestrogens. There is no established information about the factors influencing isoflavonoid storage in the tubers. We investigated the tuberous storage of the major isoflavonoids of 1-year-old plants. Four cultivars of P. mirifica were cultivated in the same field trial during the same period to establish a unique plant age and differentiation under the same environment and soil conditions. The tubers collected from the 1-year-old plants in the summer, rainy season and winter were submitted to an HPLC analysis with a gradient system comprising 0.1% acetic acid and acetonitrile. Five major isoflavonoids, puerarin, daidzin, genistin, daidzein and genistein, were adopted as standards. P. mirifica tubers of different cultivars collected in the same season exhibited significant differences in individual and total isoflavonoid contents, showing chemovariety. P. mirifica tubers of the same cultivar collected from different seasons also exhibited significant differences in individual and total isoflavonoid contents, showing the influence of season. In conclusion, the tuberous storage of major isoflavonoids in 1-year-cultivated plants was greatly diverse and was strongly influenced by the season and plant genetics.     

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.     

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     

不同采集时间野葛不同部位总黄酮和葛根素含量比较

临床常用药材葛根来源于野葛（Pueraria lobata（Willd．）Ohwi]的干燥根,含有多种异黄酮化合物,市场需求量较大,资源比较紧张。过度无序的采挖不仅使其野生资源面临枯竭的危险,也造成了产地区域生态环境的破坏。有研究表明,野葛地上部位含有的成分与葛根基本相同。为寻求利用野葛地上藤茎部位替代葛根药用的可行性,作者用紫外分光光度法和高效液相色谱法分析了不同时间采集的野葛不同部位的总黄酮和葛根素含量,为进一步扩大和开发野葛的药用新资源提供参考依据。     

超声提取和超声水解法从野葛根中提取纯化葛根素和大豆苷元

采用超声法从野葛根中提取葛根异黄酮,再将提取物在盐酸水溶液中超声水解结合有机溶剂萃取法从野葛根中分离纯化葛根素和大豆苷元。葛根素收得率为1．2％,纯度为97．8％;大豆苷元收得率为0．5％,纯度为98．2％。超声法从野葛根中提取分离葛根异黄酮活性成分葛根素和大豆苷元具有省时、节能、提取率和产品纯度高的优点。     

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.     

野葛离体培养生产异黄酮类化合物研究进展

野葛含有大量的异黄酮类化合物,其中葛根素是葛属植物特有药用成分。该文综述了野葛细胞和器官培养生产异黄酮类化合物的研究现状以及各种影响葛根素等异黄酮类化合物产生的因素,并评述葛根素的应用前景。     

Molecular characterization of the C‐glucosylation for puerarin biosynthesis in Pueraria lobata

C‐glycosyltransferases (CGTs) are important enzymes that are responsible for the synthesis of the C‐glycosides of flavonoids and isoflavonoids. Flavonoid CGTs have been molecularly characterized from several plant species; however, to date, no gene encoding an isoflavonoid CGT has been reported from any plant species. A significant example of an isoflavonoid C‐glycoside is puerarin, a compound that contributes to the major medicinal effects of Pueraria lobata. Little is known about the C‐glucosylation that occurs during puerarin biosynthesis. One possible route for puerarin synthesis is via the C‐glucosylation of daidzein. This study describes the molecular cloning and functional characterization of a novel glucosyltransferase (PlUGT43) from P. lobata. Biochemical analyses revealed that PlUGT43 possesses an activity for the C‐glucosylation of daidzein to puerarin; it shows activity with the isoflavones daidzein and genistein, but displays no activity towards other potential acceptors, including flavonoids. To validate the in vivo function of PlUGT43, the PlUGT43 gene was over‐expressed in soybean hairy roots that naturally synthesize daidzein but that do not produce puerarin. The expression of PlUGT43 led to the production of puerarin in the transgenic soybean hairy roots, confirming a role for PlUGT43 in puerarin biosynthesis.     

鄂西产三种野生葛总黄酮含量的紫外分光光度法测定

鄂西产三种野生葛总黄酮含量的紫外分光光度法测定李石生刘欣邓京振赵守训（中国药科大学天然药物化学教研室,南京２１００３８）Ｄｅｔｅｒｍｉｎａｔｉｏｎｏｆｔｏｔａｌｆｌａｖｏｎｏｉｄｉｎ３ｗｉｌｄｓｐｅｃｉｅｓｏｆＰｕｅｒａｒｉａｆｒｏｍＷｅｓｔＨｕｂ...     

The Genetic Transformation of Pueraria as Medicinal Plant by Agrobacterium rhizogenes

Pueraria radix (the dried root of Pueraria plant) is known as a traditional Chinese drug. Hairy roots of Pueraria lobata (Willd.) Ohwi, P.lobata var. montana and P. phaseoloides (Roxb.) Benth. transformed by Agrobacterium rhizogenes (Riker et al .) Conn R1601 were developed directly from the surface of sterile leaves in vitro . The transformation frequency was 16.6%, 16.2% and 26.6%, respectively. All hairy roots in the three species displayed the typical phenotypes of rapid growth, highly branched and plagiotropism, and also exhibited hormone autotrophy and resistance to kanamycin.The genetic transformations were confirmed by opine paper electrophoretic analysis, rol gene PCR amplification and molecular hybridization.     

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.     

吐温-80对野葛毛状根生长及异黄酮含量的影响

将不同浓度的吐温-80添加到野葛毛状根悬浮培养液中,研究在一定的作用时间内其对毛状根生长及次生代谢物合成与分泌的影响。结果表明,采用2％浓度处理较为适宜,不仅可以提高毛状根内葛根素的含量,而且有利于培养液中葛根素、大豆甙元及总异黄酮的积累,与对照相比,其含量可分别提高24．2％、50％和46．7％。在该浓度下连续处理毛状根72h后,发现毛状根仍生长旺盛,其生长量已是对照的1．5倍。但不同时间的连续处理对毛状根及培养液中几种异黄酮物质的积累与释放作用不同,其中以处理48h最有利于培养液中总异黄酮的累积,其含量是毛状根中的38倍。     

粉葛主要营养物质、异黄酮成分的积累规律及其相关性分析

为确定粉葛的最佳采收时期及各部位的应用价值，探索粉葛主要营养物质与异黄酮成分的积累规律及相关关系，该研究以广西藤县粉葛为材料，采用测试盒法、酶重量法、索氏抽提法、高效液相色谱法等生理生化技术，测定了成熟期不同部位异黄酮成分的含量及不同生长时期粉葛块根主要营养物质、异黄酮成分的含量，并分析了各营养物质与异黄酮成分积累的相互关系。结果表明：(1)在成熟期，粉葛不同部位的染料木素含量差异均不显著，但叶片的大豆苷元含量显著小于中藤，根头和藤的总异黄酮、葛根素、大豆苷的含量显著大于叶片和块根，并且根头和藤的葛根素含量均超过1.00%。(2)不同生长时期，粉葛的淀粉、多糖、粗蛋白、可溶性蛋白的积累在11月和12月达到最大，不溶性膳食纤维的积累在12月最小，可溶性膳食纤维在8月和12月的积累量最大，总异黄酮和葛根素在8月和9月的积累量最大。(3)总异黄酮、葛根素的积累与淀粉、多糖的积累呈显著负相关，总异黄酮、葛根素的积累与不溶性膳食纤维的积累呈显著正相关。综上结果认为：粉葛的根头、藤蔓含有丰富的总异黄酮、葛根素、大豆苷，具有很好的药用开发价值；粉葛药用的最佳采收期为8月和9月，粉葛食用的最佳采收期为...     

野葛异黄酮合酶基因的分离与功能验证

异黄酮是野葛（Pueraria lobata）中的主要活性成分,而异黄酮合酶（IFS）是催化异黄酮生物合成的第一步关键酶,尽管野葛的IFS基因已被分离,但其功能还未得到任何验证。本研究以中国安徽省郎溪县的野葛为材料,利用RT-PCR技术成功克隆到野葛IFS基因,命名为PlIFS,PlIFS开放阅读框大小为1566 bp,编码521个氨基酸,将该基因克隆到GAL1启动子控制下的酵母表达载体pESC-TRP上,得到重组质粒pESC-TRP-PlIFS,通过LiAc/ssDNA/PEG方法将其转化进酿酒酵母（Saccharomyces cerevisiae）WAT11中进行异源表达,并在酵母体内对其活性进行验证,结果显示PlIFS能催化甘草素生成大豆苷元,表现出异黄酮合酶活性特征。荧光定量PCR分析显示,PlIFS基因主要在野葛的根中表达,这与活性物质异黄酮主要在野葛根中的积累模式一致。     

葛根资源遗传多样性和性状关联分析

通过研究葛根资源的遗传多样性和葛根表型性状与分子标记的关联分析,为葛根的分子育种和指纹图谱构建提供理论依据。鉴定分析了127份不同来源葛根资源的10个表型性状,并用ISSR标记研究127份葛根资源的遗传多样性,对ISSR标记与表型性状进行关联分析。表型性状鉴定结果显示葛根资源的10个性状变异大、多样性较好。利用ISSR标记获得多态性条带109条,平均每个引物扩增5.73条、平均Nei's基因多样性0.2085、平均Shannon's指数0.3378,最远遗传距离为0.46。ISSR分子标记聚类分析将127份资源聚为两大类,群体结构分析和PCo A分析结果类似将127份资源分为2个亚群。GLM分析发现3个与茸毛性状关联标记,MLM分析未发现与表型性状关联的标记。本研究收集的资源遗传多样性较好,葛根分子标记聚类结果与地域关系不大,综合GLM和MLM关联分析结果,本试验未发现与表型关联位点。     

The effects of Pueraria mirifica extract,diadzein and genistein in testosterone-induced prostate hyperplasia in male Sprague Dawley rats

Molecular Biology Reports - Pueraria mirifica (PM) is a medicinal plant native to Thailand contained high amount of phytoestrogen and possesses anticancer activity. This study reports the effect of...     

Preparation,Structural Analysis and Antioxidant Activity of Polysaccharides and Their Derivatives from Pueraria lobata

Pueraria lobata polysaccharides (PLPs) were obtained by hot water extraction using Pueraria lobata as raw material. Structural analysis revealed that PLPs may have a repetitive backbone units of →4) -α-D-Glcp (1→4-α-D-Glcp (1→. Phosphorylated Pueraria lobata polysaccharides (P-PLPs), carboxymethylated Pueraria lobata polysaccharides (CM-PLPs) and acetylated Pueraria lobata polysaccharides (Ac-PLPs) were obtained by chemical modifications of PLPs, respectively. The physicochemical properties and antioxidant activities of these four Pueraria lobata polysaccharides were studied in comparison. In particular, the clearance rate of P-PLPs exceeded 80 %, and was expected to achieve the same effect as V_c. The results showed that the effects of different chemical modifications on the antioxidant activity of PLPs varied greatly.     

Seasonal variations in the isoflavonoids of radix Puerariae

The variations in isoflavonoid content in Radix Puerariae, the root of Pueraria lobata (Wild) Ohwi, have been examined in order to determine the optimum time to harvest the herb. Total isoflavonoid contents have been analysed by a UV spectrophotometric method whilst HPLC with photodiode array detection has been used to monitor the contents of seven major individual isoflavonoids, namely, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, daidzin, genistin, formononetin-7-glucoside and daidzein. Ninety-six samples of Radix Puerariae of different ages and harvested in different months were collected and analysed. The results clearly showed that 3-year-old roots harvested in January have the highest yields of isoflavonoid compounds. Moreover, the results obtained by both methods have a strong correlation. This suggested that the simple and fast UV spectrophotometric method could be used for monitoring the phytochemical quality of Radix Puerariae.     

金钟藤和葛藤在干旱与复水条件下的生理比较

对外来入侵种金钟藤和本地种葛藤进行干旱胁迫和水分恢复处理,比较了两种植物抗氧化酶(SOD、CAT)活性、丙二醛(MDA)含量及叶绿素荧光参数的变化。研究表明,干旱胁迫下两种植物SOD、CAT活性均高于复水后,金钟藤高于相同处理下的葛藤;金钟藤在干旱与复水条件下的MDA含量无差异,而葛藤干旱下高于复水后,不同处理下葛藤均高于金钟藤;干旱条件下金钟藤比葛藤有更强的抗氧化能力,膜脂过氧化水平明显较低,MDA与SOD、CAT呈明显的负相关。两种植物在复水后Fv/Fm、ETR与ΦPSⅡ值均显著高于干旱时,葛藤变化程度略大于金钟藤。复水后金钟藤的NPQ值增加而葛藤降低,且干旱时葛藤大于金钟藤,复水后金钟藤大于葛藤。综合比较,金钟藤比葛藤对干旱的生理适应能力强,这可能是其入侵性的重要特征。     

野葛藤茎的异黄酮类化学成分

从野葛Pueraria lobata(Willd.)Ohwi藤茎中分离得到5个异黄酮类化合物,分别鉴定为:大豆甙元(daidzein),芒柄花异黄酮(formononetin),6,7-二甲氧基-3’,4’-次甲二氧基异黄酮(6,7-dimethoxy-3’,4’-methlenedioxyisoflavone),大豆甙(daidzin)和葛根素(puerarin).