Species discrimination of Radix Bupleuri through the simultaneous determination of ten saikosaponins by high performance liquid chromatography with evaporative light scattering detection and electrospray ionization mass spectrometry

A simple, rapid and robust high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method was established for the species discrimination and quality evaluation of Radix Bupleuri through the simultaneous determination of ten saikosaponins, namely saikosaponin-a, -b(1), -b(2), -b(3), -b(4), -c, -d, -g, -h, and -i. These compounds were chromatographed on an Ascentis(?) Express C18 column with a gradient elution of acetonitrile and water containing 0.1% acetic acid at a flow rate of 1.0 mL/min. Saikosaponins were monitored by ELSD, which was operated at a 50°C drift tube temperature and 3.0 bar nebulizer gas (N(2)) pressure. The developed method was validated with respect to linearity, intra- and inter-day accuracy and precision, limit of quantification (LOQ), recovery, robustness and stability, thereby showing good precision and accuracy, with intra- and inter-assay coefficients of variation less than 15% at all concentrations. Furthermore, a high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) method was developed to certify the existence of ten saikosaponins, as well as to confirm the reliability of ELSD. The extraction conditions of saikosaponins from Radix Bupleuri were also optimized by investigating the effect of extraction methods (sonication, reflux and maceration) and various solvents on the extraction efficiencies for saikosaponins. Sonication with 70% methanol for 40 min was found to be simple and effective for extraction of major saikosaponins. This analytical method was applied to determine saikosaponin profiles in 20 real samples consisting of four Bupleurum species, namely B. falcatum, B. chinense, B. sibiricum and the poisonous B. longiradiatum. It was found that three major saikosaponin-a, -c and -d were the major constituents in B. falcatum, B. chinense, and B. longiradiatum, while one major saikosaponin (saikosaponin-c) was not identified from B. sibiricum. In addition, no saikosaponin-b(3) was detected in B. longiradiatum samples, indicating that the toxic B. longiradiatum may be tentatively distinguished from officially listed Bupleurum species (B. falcatum and B. chinense) based on their saikosaponin profiles. Overall the simultaneous determination of ten saikosaponins in Radix Bupleuri was shown to be a promising tool to adopt for the discrimination and quality control of closely related Bupleurum species.     

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...     

红柴胡调查中的新发现

基于野外调查及形态学、解剖学、细胞学和植物化学的研究,在黑龙江省发现了一种新的柴胡属植物,该植物与红柴胡混生于草甸草原、灌丛和柞树林等群落之中,其不同于红柴胡的特点是,根黑色,根头稀具纤维,叶略宽;根中具木纤维群,叶下表皮下方无栅栏组织;染色体数目2n=26或20,具二型性;柴胡皂甙较多而挥发油较少。该植物与传统中药北柴胡相似,且分布广,数量大,是黑龙江省的一种新的柴胡资源。     

精恶唑禾草灵对不同品种柴胡茎秆力学特性的影响

为应用茎秆生物力学性质指标研究不同剂量除草剂对柴胡安全性的影响,以南柴胡和北柴胡茎秆为试验材料,精恶唑禾草灵为试验除草剂,分别在拔节期和开花期利用非金属材料万能试验机对茎秆基部第2节间的最大荷载、应力、应变、弹性模量、拉伸强度、剪切强度等力学指标进行测定和统计分析.结果表明:北柴胡的茎杆直径、最大荷载、抗拉强度等均高于南柴胡,最大荷载高了约13%,抗拉强度与茎杆直径呈线性相关关系(R=0.9194);南柴胡和北柴胡拔节期的茎杆拉伸强度和剪切强度均高于开花期;柴胡茎经中低剂量(0-1500ml/hm~2)的精恶唑禾草灵处理后,其抗拉强度和剪切强度无明显变化,但高剂量(3000ml/hm~2)的精恶唑禾草灵施用后显著降低了柴胡茎杆的抗拉强度和剪切强度,会直接导致柴胡抗倒伏能力降低,造成柴胡产量和品质下降.该结果可为柴胡田除草剂的安全施用及药材机械化收割技术的研发提供参考依据.     

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.     

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。     

银州柴胡根的发育解剖学研究

本研究采用常规石蜡切片结合荧光显微镜技术对银州柴胡根的发育解剖学进行了研究。结果表明:（1）银州柴胡根顶端分生组织由原分生组织及其衍生的初生分生组织组成。原生分生组织细胞体积小、排列紧密、细胞质浓厚、细胞核大而明显，具有典型的分生组织的特点；（2）初生分生组织由根冠原、表皮原、皮层原和中柱原组成。在根发育过程中，表皮、皮层和维管柱共同组成其初生结构。银州柴胡根初生木质部为二原型或三原型，外始式；同时在根表皮细胞的径向壁观察到径向壁的细胞壁加厚；（3）在根次生生长过程中，位于初生木质部和初生韧皮部之间的原形成层恢复分裂能力产生维管形成层，维管形成层不断地向外产生次生韧皮部，向内产生次生木质部；同时位于根内皮层内方的中柱鞘细胞恢复分裂能力产生木栓形成层，木栓形成层向外形成木栓层，向内形成栓内层。在维管形成层和木栓形成层分裂的过程中，在次生韧皮部和中柱鞘组织中产生形态大小不同的分泌道，均为次生的裂生型分泌道。研究认为，银州柴胡根的结构类似于药典收录的北柴胡和红柴胡根的结构特点，但其根表皮细胞径向壁加厚、木纤维的分布、分泌道的大小和数量等有别于柴胡属其它植物，可作为柴胡属植物重要的分类鉴定依据。     

基于染色体计数和ITS序列初步探讨横断山区柴胡属植物(伞形科)的系统发育

横断山区是中国柴胡属Bupleurum植物的分布中心。本文对横断山区6个种2变种进行了染色体记数报道,其中4个种2变种是首次报道。对横断山区的10个种4个变种、中国北方（河北和黑龙江）的3个种的nrDNA ITS进行测序,同时从GenBank里面下载同属的来自非洲和地中海西部的16个nrDNA ITS序列数据,结合染色体数目变化结果,初步探讨了横断山区柴胡属植物的系统发育。结果表明横断山区可能是现代柴胡属植物的频度中心和多样分布中心之一。它们的祖先种可能是非洲北部的木本柴胡属植物B.fruticosum,或者是地中海西部的柴胡属植物,推测是通过中东和高加索扩散而形成的,其中与非洲南部特有种B.mundtii的亲缘关系也较近;染色体基数演化趋势是：8是较原始基数,6和7是次生基数,其染色体异基数变异和多倍化可能是物种形成、进化以及向外扩散的主要方式;在ITS系统发育树中,中国柴胡属植物染色体基数为8的种类聚为一支,染色体基数为6和7的种类聚为了一支,不支持舒璞等（1998）关于中国柴胡属的属下分类系统。结合已有的形态学、细胞学、孢粉学证据和ITS系统发育树,建议窄竹叶柴胡B.marginatum var.stenophyllum独立成种。     

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．     

北柴胡种子内生菌群落结构与多样性

为研究北柴胡种子内生菌的群落结构与多样性.采用Illumina Miseq高通量测序技术,分别对山西(BC_1)、黑龙江(BC_2)、河北(BC_3)和内蒙古(BC_4)的四个产地(4份)北柴胡种子的16S RNA V3-V4区和ITS1区扩增片段进行测序,并对内生细菌和内生真菌群落结构和多样性进行分析.结果表明,BC...     

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。     

Phylogenetic inference of the genus Bupleurum (Apiaceae) in Hengduan Mountains based on chromosome counts and nuclear ribosomal DNA ITS sequences

pleurum of Apiaceae in China. This paper reports chromosome numbers of six species and two varieties of Bupleurum, and for four species and two varieties their chromosome numbers are reported for the first time. The phylogeny of Bupleurum was investigated based on the ITS region of the nuclear ribosomal DNA (nrDNA) of 14 taxa from the Hengduan Mountains, 3 taxa from the North China (Hebei and Heilongjiang), and 16 taxa from Africa and the Mediterranean region. Varia-tions in chromosome numbers and the ITS sequences were used to infer phylogenetic relationships between Bupleurum species in Hengduan Mountains. The results showed that the Hengduan Mountains might represent one of the frequency and diversity centers for Bupleurum. The ancestors of Bupleurum species in the Hengduan Mountains may be related to the woody B. fruticosum in North Africa, or the species in the western Mediterranean region. It is postulated that the ancestral population migrated into Hengduan Mountains through the Middle East and the Caucasus. Furthermore, the neo-endemic B. mundtii in South Africa appeared to be a close relative of the species in the Hengduan Mountains. In the trend of basic chromosome number evolution, x = 8 should be regarded as the ancestral basic number, while x = 6, 7 as the derived ones. The Bupleurum species in the Hengduan Moun-tains have been undergoing changes in the basic chromosome numbers or the ploidy level. The ITS phylogenetic tree showed that the Chinese species were divided into two clades: one with the basic chromosome number x = 8, and the other with x = 6, 7. The results rejected the previous infrageneric classification of Bupleurum in China. We further suggested to raise B. marginatum var. stenophyllum to species rank based on the combined evidence from morphology, karyology, pollen morphology, and the ITS phylogenetic tree.     

DNA barcoding Chinese medicinal Bupleurum

We tested 4 markers, namely nuclear internal transcribed spacer 2 (ITS2), psbA-trnH, matK, and rbcL, to evaluate these candidate DNA barcodes for distinguishing Bupleuri radix (Chaihu) from its adulterants. 51 plant samples of Bupleurum representing 19 species were collected from different areas in China. Amplification and sequencing were attempted for all the 4 candidate barcode regions, whose validity was assessed in terms of the success rate of PCR amplification and sequencing, differential intra- and inter-specific divergences, DNA barcoding gap and the ability to discriminate species. The results showed that ITS2 had the best performance in identifying Bupleurum with an identification efficiency of 73.68%, which, after combining with psbA-trnH, increased to 83.33%. We further evaluated the efficiency of ITS2 for discriminating the species of Bupleurum using a large database from GenBank, which archived data of 223 samples from 74 species, and ITS2 successfully discriminated 64.13% of the samples at the species level. In conclusion, the ITS2 can serve as a potentially useful barcode for Bupleurum species, with psbA-trnH as a supplementary locus.     

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.     

黄土高原半干旱区柴胡种植模式

采用大田试验,设计冬小麦收获后复播大豆、大豆套作柴胡(小麦-大豆/柴胡)和冬小麦收获后复播柴胡(小麦-柴胡)及玉米套作柴胡(玉米/柴胡)3种种植模式,探讨黄土高原半干旱区适宜柴胡的"粮药"栽培模式.结果表明:与玉米/柴胡和小麦-柴胡种植模式相比,小麦-大豆/柴胡种植模式经济产量最高,生态效益最好;不同柴胡品种中,万柴胡(原产地温度相对较高)产量最高,左柴胡(原产地温度相对较低)最低,表明柴胡适宜从高温地区引种到低温地区栽培,可提高产量.小麦-大豆/柴胡种植模式为最佳"粮药"栽培模式,其柴胡产量比玉米/柴胡和小麦-柴胡种植模式分别增产11.6%和16.8%.     

北柴胡根的发育解剖学研究

应用常规石蜡切片方法对北柴胡（Bupleurum chinense DC．）1年生主根的中部和2年生主根的根尖和中部进行了横向与纵向解剖观察,研究根的发育过程,并对其1年生与2年生主根中部的结构进行了比较分析。结果表明,北柴胡根的发育包括原分乍组织、初生分生组织、初生结构和次生生长4个发育阶段。原分生组织由3群原始细胞组成,其细胞具有典型分生组织的细胞学特征;初生分生组织包括根冠原、表皮原、皮层原和中柱原。初生结构由表皮、皮层和中柱组成。初生木质部为二原型。次生生长主要是依靠维管形成层和木栓形成层的活动来完成。其木栓形成层由中柱鞘细胞恢复分裂能力而形成。2年生北柴胡主根的基本结构与1年生的相同,但与1年生的相比,其周皮、次生韧皮部和木质部面积均增加。不同点具体表现在：2年生主根的周皮增厚;分泌道数量增加;次生木质部中木纤维成群分布,在横切面上连接成一个圆环,其维管射线明显。多为多列射线。分泌道主要分布在中柱鞘薄壁组织和次生韧皮部中,均是裂生方式发生,属次生分泌道,初生结构中无分泌道存在。     

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...     

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.     

狭叶柴胡大小孢子发生和雌雄配子体发育的研究

用石蜡切片法、半薄切片法对狭叶柴胡的大、小孢子发生和雌、雄配子体发育进行观察研究。结果显示:(1)狭叶柴胡花药壁的发育为双子叶型,腺质绒毡层;小孢子母细胞减数分裂过程中的胞质分裂为同时型,四分孢子为正四面体形;成熟的花粉粒为3-细胞型,具3个萌发孔。(2)倒生型胚珠,单珠被,薄珠心;大孢子母细胞常为一个雌性孢原直接发育而成,大孢子四分体呈线型,多数情况为合点端一个大孢子分化为功能大孢子;由功能大孢子发育为蓼型成熟胚囊。(3)八核胚囊时期,珠心基部和两侧的一些珠心细胞留存较久,成为珠心座细胞。此外,珠被内表皮细胞发育为珠被绒毡层。     

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.