ITS sequence analysis used for molecular identification of the Bupleurum species from northwestern China

Radix Bupleuri (Chaihu), a famous Traditional Chinese Medicine, is derived from the dried roots of Bupleurum chinense DC. and B. scorzonerifolium Willd. But nowadays, other nine species and varieties from genus Bupleurum are also used as Radix Bupleuri in northwestern China. The authentic identification of dried roots of B. chinense and B. scorzonerifolium, however, is difficult to just base on the appearance and morphology. A molecular genetic method was developed to help discriminate the original species of Radix Bupleuri. The ITS sequences ( approximately 600 bp) were amplified by the PCR technique from genomic DNA isolated from all the collected samples. According to analyzing the information given by ITS sequences, the conclusion can be made that ITS sequence could serve as markers for authentication of Radix Bupleuri.     

Efficient production of saikosaponins in Bupleurum falcatum root fragments combined with signal transducers

An efficient system to produce saikosaponins (saikosaponin-a and -d) in Bupleurum falcatum adventitious root fragments combined with signal transducers was developed. The roots are heterogeneous in terms of size and shape and sometimes form aggregates during cultivation. When the roots were cut to lengths of about 5 mm using a scalpel and cultivated, the root fragments did not form the aggregates, and root growth and saikosaponin production were not inhibited. After screening various signal transducers, it was clear that methyl jasmonate (MeJA) markedly promoted saikosaponin production. By comparing the effect of MeJA and related substances on saikosaponin production, we conclude that both the pentenyl and carboxylmethyl group of MeJA play an important role in the promotion of saikosaponin production. Addition of both 100 microM MeJA and 20 mM CaCl2 to the medium stimulated the content of saikosaponin in the root, with levels reaching 31.7 mg/g-dry root for 15 days of cultivation. A large amount of root fragments were prepared using a blender and cultivated (23 g-dry root/l) with 400 microM MeJA and 20 mM CaCl2, resulting in a high concentration of saikosaponins (747.3 mg/l).     

十六种晋产北柴胡柴胡皂苷含量分析

本实验分别以高效液相色谱法和分光光度法测定了山西省十三个产地的16种北柴胡中柴胡皂苷a、d及柴胡总皂苷的含量.结果表明,在16种晋产北柴胡中,柴胡皂苷a的质量分数为0.038％～0.369％,柴胡皂苷d的质量分数为0.036％～0.681％,柴胡总皂苷的质量分数为0.354％～3.949％;12种晋产北柴胡中的柴胡皂苷a、10种中的柴胡皂苷d和13种中的柴胡总皂苷含量分别超过了0.1％、0.2％和1.0％.     

Localization and dynamic change of saikosaponin in root of Bupleurum chinense

Anatomical,histochemlcal and phytochemical methods were used to investigate the structure,the localization and content changes of total saikosaponin and saikosaponin-a of the roots of Bupleurum chinense DC.at different developmental stages.Results showed that saikosaponin was mainly distributed in pericycle and primary phloem in the young root;but In the mature root,it was mainly distributed in vascular camblum and secondary phloem.During the whole growth period from the pre-blossom,blossom,fruit,and fruit mature periods until the pre-withering period,it was in the fruit mature period that both the total saikosaponin content and the saikosaponin-a content reached the highest level.So the last 20 d of October was considered as the right collecting season for the drug of B.chinense.In addition,the quality of 1-year-old drug was better than that of 2-year-old drug due to its higher saikosaponin content.On the other hand,judging from the external characteristics of the drug,the one with an acerose taproot and more lateral roots was of better quality.The results offered theoretical bases for selecting medicinal material of high quality and determining the most appropriate harvesting stage and part of B.chinense.     

Purification of saponin compounds in Bupleurum falcatum by solvent partitioning and preparative LC

Saponin compounds (saikosaponin c, a, and d) in Bupleurum falcatum were partially purified by solvent partitioning of the herbal extract using diethyl ether, distilled water, n-butanol, and acetone. After separation of the saponins by preparative LC, the purity of each saikosaponin was more than 94%. The identities of purified individual saikosaponins were confirmed by TLC, analytical LC, and fast-atom bombardment mass spectrometry.     

Purification of Saponin Compounds in Bupleurum falcatum by Solvent Partitioning and Preparative LC

Saponin compounds (saikosaponin c, a, and d) in Bupleurum falcatum were partially purified by solvent partitioning of the herbal extract using diethyl ether, distilled water, n-butanol, and acetone. After separation of the saponins by preparative LC, the purity of each saikosaponin was more than 94%. The identities of purified individual saikosaponins were confirmed by TLC, analytical LC, and fast-atom bombardment mass spectrometry.     

东北柴胡属细胞分类学研究Ⅰ．6种柴胡的核型分析

本文报道了国产６种柴胡的核型,发现在肇东五里木存在一种与日本产三岛柴胡Ｂ．ｆａｌｃａｔｕｍ具有相同核型特征的柴胡,暂称其五里木居群。柞柴胡Ｂ．ｋｏｍａｒｏｖｉａｎｕｍＬｉｎｃｚ．２ｎ＝８＝２ｍ＋６ｓｍ（２ＳＡＴ）．红柴胡Ｂｓｃｏｒｚｏｎｅｒｉｆｏｌｉｕ,Ｗｉｌｌｄ．２ｎ＝１２＝１０ｍ（２ＳＡＴ）＋２ｓｍ．北柴胡ＢｃｈｉｎｅｎｓｅＤＣ．２ｎ＝２４＝１４ｍ＋１０ｓｍ（２ＳＡＴ）．锥叶柴胡Ｂ．ｂｉｃａｕｌｅＨｅｌｍ２ｎ＝３２＝２０ｍ＋１２ｓｍ．大叶柴胡Ｂ．ｌｏｎｇｉｒａｄｉａｔｕｍＴｕｒｃｚ．２ｎ＝１２＝１０ｍ（２ＳＡＴ）＋２ｓｍ五里木居群２ｎ＝２６＝１６ｍ＋１０ｓｍ（２ＳＡＴ）文章最后根据核型特征分析了东北地区各种柴胡间的演化关系．     

Two New Species of the Genus Bupleurum (Umbelliferae) from China

Bupleurum is a genus largely distributed in temperate regions of the Northern Hemisphere. In China 36 species, 17 varieties and 7 forms have hitherto been reported and most of them are used as Chinese traditional drugs under the name of Chai-Hu. Chai-Hu is one of the most popular drugs used ever since the ancient time and is prescribed principally in the treatment of fevers and influenza. In this article, the authors report two new species, B. kunmingense Y. Li et S. L. Pan and B. polyclonum Y. Li et S. L. Pan which were discovered in Yunnan Province based on morphological studies and preliminary phytochemical tests. The ultraviolet spectrums and thin layer chromatograms of the essential oil and the crude saikosaponins of the two new species are similar to those of B. chinense DC., the standard material medica of Chai-Hu. Moreover, two new saikosaponin spots located between saikosaponin a and c in TLC were discovered in the two new species, and this could be served as a chemical evidence for identification purpose. In the histochemistry examination, the reaction of saikosaponin with color developing agent in parenchyma of the roots of the two new species is obviously more significant than that of B. chinense DC. and this phenomenon has also been proved to be true by TLC of the crude saikosaponin extracts of these two new species. It is suggested that the two new species be used as a substitute ofhigh quality for Chai-Hu.     

柴胡属植物中柴胡皂甙的资源利用

本文用高效液相色谱法(HPLC),分析了国产19种柴胡及市售商品柴胡中柴胡皂甙a、c、d(Saikosaponin a、c、d)的含量。结果表明19种柴胡根中均含上述三种成分,但44个样品的含量不同,其中北柴胡以河南信阳的含量较高,南柴胡以陕西延安的为好,多种柴胡的茎叶含有上述三种成分。但含量较根部低,少数种未检出。     

北柴胡不定根培养及茉莉酸甲酯处理对柴胡皂苷含量的影响

目的:建立北柴胡不定根培养体系,明确茉莉酸甲酯(MeJA)处理对北柴胡不定根中柴胡皂苷含量积累的影响。方法:利用固体和液体相结合的组织培养技术培养北柴胡不定根;分别以不同浓度的MeJA处理不定根不同时间,利用HPLC测定处理后不定根中柴胡皂苷含量的积累变化。结果:培养了北柴胡不定根;MeJA处理对北柴胡不定根中柴胡皂苷含量的积累有明显促进作用,当MeJA浓度为200μmol/L时,柴胡皂苷含量最高,为0.45%;以200μmol/L MeJA处理北柴胡不定根26 d时,柴胡皂苷含量最高,为0.51%。结论:北柴胡不定根培养结合MeJA诱导,可做为柴胡皂苷次生代谢合成途径及其积累规律研究的有效技术体系。     

柴胡类药材的柴胡皂甙分析

柴胡类药材的柴胡皂甙分析李光慧罗燕燕王瑛袁昌齐王年鹤（北京临床药学研究所,北京１０００３５）（江苏省中国科学院植物研究所,南京２１００１４）ＡｎａｌｙｓｉｓｏｆｓａｉｋｏｓａｐｏｎｉｎｓｉｎｍｅｄｉｃｉｎａｌＢｕｐｌｅｕｒｕｍｓｐｐ．ＬｉＧｕａｎｇ...     

Saikosaponin C induces endothelial cells growth, migration and capillary tube formation

Saikosaponin C is one of the saikosaponins that are consisted in a Chinese herb, Radix Bupleuri. Recently, saikosaponins have been reported to have properties of cell growth inhibition, inducing cancer cells differentiation and apoptosis. However, saikosaponin C had no correlation with cell growth inhibition. In this study, we investigated the role of saikosaponin C on the growth of endothelial cells and angiogenesis. We found that saikosaponin C yielded a potent effect on inducing human umbilical vein endothelial cells (HUVECs) viability and growth. In addition to inducing endothelial cells growth, saikosaponin C also induced endothelial cells migration and capillary tube formation. The gene expression or activation of matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF) and the p42/p44 mitogen-activated protein kinase (MAPK, ERK) that correlated with endothelial cells growth, migration and angiogenesis were also induced by saikosaponin C. From these results, we suggest that saikosaponin C may have the potential for therapeutic angiogenesis but is not suitable for cancer therapy.     

Phenethyl alcohol glycosides and isopentenol glycoside from fruit of Bupleurum falcatum.

Investigation on the constituents of the fruit of Bupleurum falcatum L. resulted in the isolation of the three new glycosides, phenethyl alcohol 8-O-beta-D-glucopyranosyl-(1-->2)-O-beta-D-apiofuranosyl-(1-->6)-b eta-D- glucopyranoside, phenethyl alcohol 8-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside and isopentenol 1-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside along with five known glycosides, icariside D1, icariside F2, saikosaponin a, saikosaponin c and saikosaponin d. The structures of these compounds were elucidated on the basis of interpretation of chemical and spectral data.     

Production of saikosaponins by tissue culture of Bupleurum falcatum L.

The studies for production of saikosaponins by tissue culture of Bupleurum falcatum L. were carried out to produce saikosaponins with several kinds of media and plant hormones. Among the media and plant hormones studied, Gamborg's B-5 [23] medium containing 0.5 ppm kinetin (k) and 1.0 ppm 3-indolebutyric acid (IBA) was the most effective medium and hormone for production of saikosaponins. The highest content of saikosaponin-d in the dried cells was 0.26%, which was similar to a concentration of Bupleuri Radix.Abbreviations MS medium used by Murashige and Skoog [22] - G medium used by Gamborg (B 5) [23] - W medium used by White [24] - NN medium used by Nitsch and Nitsch [25] - k Kinetin - BAP 6-Benzylaminopurine - 2,4 D 2,4-Dichlorophenoxyacetic acid - NAA -Naphtylacetic acid - IBA 3-Indolebutyric acid - IAA 3-Indoleacetic acid - ssd saikosaponin-d - PM Production medium - dw Dry weight     

中药柴胡不同采收期的皂甙含量

中药柴胡不同采收期的皂甙含量潘泽惠庄体德周雪林林湘（江苏省中国科学院植物研究所,南京２１００１４）（黑龙江双鸭山矿务局师范学校,双鸭山１５５１２５）ＯｎｓｅａｓｏｎａｌｃｈａｎｇｅｓｏｆｔｈｅｔｏｔａｌｓａｐｏｎｉｎｓｉｎＣｈｉｎｅｓｅｔｒａｄｉｔ...     

中国柴胡属染色体数目和核型研究

采用植物染色体常规压片技术,报道了中国柴胡属(Bupleurum L .)13种5变种24个居群的染色体数目,其中4种5变种为首次报道,同时报道了5种1变种的细胞核型.除天山柴胡两个居群、紫花鸭跖柴胡一个居群和北柴胡一个居群是多倍体外,其它均为二倍体.主要结论如下:(1)秦岭柴胡和细茎有柄柴胡处理为独立的种更为合适;紫花阔叶柴胡作为大叶柴胡变种的处理是合理的;黄花鸭跖柴胡处理为紫花鸭跖柴胡的变种是不合理的.(2)紫花鸭跖柴胡和北柴胡可能都是还没有被完全认识的复合类群,需要进一步研究.(3)x=7可能是柴胡属的原始基数.     

A New Species of the Genus Bupleurum (Umbelliferae) from China

Chai-Hu is one of the most popular Chinese traditional drugs used ever since the ancient time and is prescribed principally in the treatment of fevers and influenza. In this article, the authors report a new species, Bupleurum luxieuse Y. Li et S. L. Pan which was discovered in Yunnan Province and used as Chinese drug under the name of Chai-Hu. The morphology was analysed and preliminary phytochemical tests of B. luxiense were carried out. The thin layer chromatograms and gas chromatograms of the essential oil and the qualitative analysis of the saikosaponin of this new species are similar to those of Bupleurum chinense DC., a standard material medica of Chai-Hu. Moreover, the roots of B. luxiense is discovered to have saikoside 2 times more than B. chinense. The results suggest that the new species be used as a substitute of high quality for Chai-Hu.     

Visual detection of saikosaponins by on-membrane immunoassay and estimation of traditional Chinese medicines containing Bupleuri radix

The purpose of this study was to describe the simple, rapid, and environmental-cost effective determination method for saikosaponins in complicated samples like Bupleuri radix and traditional Chinese medicines (TCM). Saikosaponin standards, extracts of Bupleuri radix and TCM, were applied to a polyethersulphone (PES) membrane and developed by acetonitrile-water (1:4, by volume). Saikosaponin a (SSa), SSc, and SSd were visually detected by an immunostaining method (called Eastern blotting technique) using a monoclonal antibody (MAb) against SSa. At least 62.5 ng of SSa, SSc, and SSd were clearly detectable individually. These coloring spot areas of saikosaponins on PES membrane were calculated by using the NIH Imaging software and three saikosaponins can be analyzed quantitatively between 62.5 ng and 1.0 microg. Saikosaponins in Bupleuri radix and TCM were determined and these results of SSa and total saikosaponin concentrations were in good agreement with those from the ELISA analysis.     

北柴胡不定根系生物转化合成皂苷研究

通过在北柴胡不定根系培养体系中添加目的产物柴胡皂苷的前体物质和生物合成促进物质以及进行高糖渗透胁迫试验,探讨了利用不定根系生产柴胡皂苷的条件和方法。结果表明,在1/2 MS培养基中添加硫酸镁、硝酸钾、丙酮酸钠以及高糖渗透胁迫均有利于柴胡皂苷的合成。1/2 MS培养基+硫酸镁0.37 g/L+硝酸钾5.7 g/L+丙酮酸钠2.0 mmol/L+蔗糖50 g/L是最适合柴胡皂苷转化合成的培养基,此条件下使柴胡皂苷的含量提高了9.74倍。