柴胡属药用植物的分子鉴定及柴胡药材的质量考察

发布时间：2019-06-04 12:17

【摘要】：各种柴胡的混用滥用会导致柴胡质量的不可控制以及相应的用药安全的问题,对不同种柴胡属药用植物的鉴定就显得尤为重要,建立一种快速准确的新鉴定方法迫在眉睫。本论文试图通过DNAbarcoding技术建立对柴胡属常见药用植物的分子鉴定新方法,并以此为基础,通过HPLC手段考察不同种不同产地柴胡的皂苷含量,从而达到质量评价的作用。本文首先通过小样本扩增不同种柴胡的4种候选DNA条形码序列,ITS、psbA-trnH、matK、rbcL,比较这4种DNA条形码的鉴定效率,从而筛选出最佳DNA条形码。进而大样本量的扩增该最佳DNA条形码序列,建立起序列数据库,对这些数据进行变异位点、遗传距离、聚类、"单倍型-物种"4个层次的分析,以建立起柴胡属常见药用植物的分子鉴定方法。在对柴胡药材的质量评价方面,本文通过HPLC技术分析柴胡药材的柴胡皂苷含量,并通过SAS 8.0进行统计学分析,从而评价不同种柴胡的优劣,不同产地柴胡的优劣。最终将建立起来的分子鉴定及质量评价方法用于对市售柴胡药材的评价,从而了解目前柴胡市场信息,给出合理的建议。本文取得了如下研究结果:(1)描述并总结了 7种柴胡属常见药用植物的性状及粉末显微特征,总结了柴胡属内常见的药用植物的特点。(2)分析比较了各种DNA条形码对不同种柴胡的序列扩增效率、测序成功率、遗传距离分析、聚类分析、"单倍型-物种"对应关系,筛选出了适合柴胡属常见药用植物鉴定的最佳DNA条形码,即:ITS序列。(3)成功建立了用于鉴定常见柴胡属药用植物的分子鉴定方法。本实验通过对大样本量的柴胡属药用植物的ITS序列进行扩增,获得了 7种常见药用柴胡的19种ITS序列单倍型,统计得到了 86个变异位点。以此为基础,进行了变异位点、遗传距离、聚类、"单倍型-物种"四个层次的分析,从而确定了 ITS序列能够成功对柴胡属常见药用植物进行鉴定,并建立了基于19种ITS序列单倍型检索的"单倍型-物种"的鉴定方法。(4)以柴胡皂苷含量为标准评价了各类柴胡的质量。柴胡样品中的三种柴胡皂苷SSa、SSd、SSc的含量有着密切的正相关关系。按照柴胡皂苷的含量高低,不同种柴胡的优劣顺序为:窄竹叶柴胡银州柴胡北柴胡锥叶柴胡狭叶柴胡。此外,本实验亦评价了不同产地柴胡的皂苷含量,发现山西与甘肃所产柴胡质量较优,而宁夏所产次之。(5)对市售柴胡药材进行了质量评价。本论文收集了河北安国、河南禹州、安徽亳州、广东广州、四川荷花池五大药材市场的柴胡药材,利用(3)中建立的分子鉴定方法,对市售柴胡药材进行了物种鉴定,明确了目前市场上柴胡以窄竹叶柴胡和北柴胡为主,银州柴胡亦有一定份额,狭叶柴胡、锥叶柴胡、三岛柴胡较少。以《中国药典》规定的柴胡皂苷a、d含量为评价标准,这五大药材市场柴胡药材合格率为100%,但不同种柴胡的皂苷含量差异较大,窄竹叶柴胡能达到4%以上,而锥叶柴胡仅为0.4%左右。
[Abstract]:The misuse of the various bupleurum can lead to the incontrol of the quality of the bupleurum and the corresponding safety of the medication. The identification of the medicinal plants of the genus Bupleurum is of particular importance, and the establishment of a rapid and accurate new method of identification is urgent. This paper attempts to establish a new method for molecular identification of the common medicinal plants of the genus Bupleurum by means of DNA barcoding, and on the basis of which, the content of the soap in different places of origin is investigated by means of HPLC, so as to achieve the effect of quality evaluation. In this paper, four candidate DNA bar code sequences, ITS, psbA-trnH, matK and rbcL of different kinds of Bupleurum chinense were amplified by small sample, and the identification efficiency of the four DNA barcodes was compared, and the optimal DNA barcode was selected. And then a large sample size is used for amplifying the optimal DNA barcode sequence, a sequence database is established, the data is subjected to analysis of the mutation sites, the genetic distance, the cluster and the "haplotype-species", so as to establish a molecular identification method for the common medicinal plants of the genus Bupleurum. In the aspect of quality evaluation of Bupleurum chinense, this paper analyzes the content of Chaihu in Bupleurum chinense by HPLC, and makes a statistical analysis by SAS 8.0, so as to evaluate the advantages and disadvantages of the different kinds of bupleurum, and the quality of the bupleurum in different places. And finally, the established molecular identification and quality evaluation method is used for evaluating the commercial radix bupleuri medicinal materials, so as to obtain the reasonable proposal of the present radix bupleuri market information. The following results were obtained: (1) The characters and the characteristics of the common medicinal plants of the genus Bupleurum were described and summarized, and the characteristics of the common medicinal plants in the genus Bupleurum were summarized. (2) The sequence amplification efficiency, the success rate of the sequencing, the genetic distance analysis, the cluster analysis and the "haplotype-species"-corresponding relation of the various DNA barcodes on the different kinds of bupleurum are compared, and the optimal DNA barcode suitable for the identification of the common medicinal plants of the genus Bupleurum is selected, that is, the ITS sequence. (3) The molecular identification method for identifying the medicinal plants of the genus Bupleurum is successfully established. In this experiment, the ITS sequence of the medicinal plants of Bupleurum belonging to the genus Bupleurum was amplified, and 19 kinds of ITS sequence haplotypes of 7 common medicinal bupleurum were obtained, and 86 mutation sites were obtained. On the basis of this, the analysis of four levels, such as mutation site, genetic distance, cluster and "haplotype-species", was carried out, so that it was determined that the ITS sequence could successfully identify the common medicinal plants of the genus Bupleurum, and established the identification method of the "haplotype-species" based on the haplotype search of the 19 kinds of ITS sequences. And (4) the quality of the various bupleurum is evaluated by using the content of the chaihu soap as the standard. There is a close positive correlation between the contents of three kinds of the Chinese Chaihu soap, SSa, SSd and SSc in the Bupleurum chinense sample. According to the content of the chaihu, the advantages and disadvantages of the different kinds of bupleurum are as follows: In addition, the experiment also evaluated the soap content of Bupleurum in different places, and found that the quality of the bupleurum produced by Shanxi and Gansu was better, and the second in Ningxia. And (5) carrying out quality evaluation on the commercial radix bupleuri medicinal materials. In this paper, the Chinese medicinal materials of the five medicinal materials of Anguo, Henan, Anhui, Guangdong, Guangzhou and Sichuan were collected, and the molecular identification methods established in (3) were used to identify the medicinal materials of Bupleurum chinense. It is clear that the radix bupleuri on the market is mainly composed of the narrow bamboo leaves, the bupleurum root and the northern bupleurum, and the radix bupleuri of the silver state also has a certain share, and the radix bupleuri, the bupleurum root and the three islands of the bupleurum are less. The content of Chaihu was 100%, but the content of Chaihu was more than 4%, while the root of Chaihu was only about 0.4%.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284.1

【参考文献】