柴胡属药用植物的分子鉴定及市售柴胡药材的质量调查  

The molecular identification of Bupleurum medicinal species and the quality investigation of Bupleuri Radix

作　　者：袁伯川 李文东[2] 马永生[1] 周姗[1] 朱林峰[1] 林瑞超[3] 刘颖[1] 

机构地区：[1]北京中医药大学生命科学学院,北京100102 [2]北京市药品检验所,北京102206 [3]北京中医药大学中药品质评价研究中心,北京100102

出　　处：《药学学报》2017年第1期162-171,共10页Acta Pharmaceutica Sinica

基　　金：国家科技重大专项(2014zx09304307001)

摘　　要：柴胡是最常用的大宗中药材之一,在我国有着两千多年的药用历史。但是目前柴胡药材品种混乱,有25种、8变种、3变型的柴胡属药用植物在不同地区和中药材市场作为柴胡使用,通过传统方法鉴别极为困难。为快速准确地对柴胡属为数众多的药用植物进行鉴定,本文从全国9省份14居群采集了168株柴胡属药用植物,扩增获得了长度为600~606 bp的ITS序列;通过DNAMAN比对分析找到86个变异位点,并确定了19种ITS单倍型(TH1~TH19);利用MEGA 5.0进行K2P(Kimura 2-parameter)遗传距离分析,发现种间遗传距离显著大于种内遗传距离;利用邻接(neighbor joining,NJ)法构建系统发育树,显示各物种聚类关系清晰,因此建立了基于ITS序列的柴胡属药用植物分子鉴定方法。利用该方法对来自全国5个主要中药材市场的52份柴胡药材进行了分子鉴定,并进一步利用HPLC法测定各药材中柴胡皂苷a、柴胡皂苷c及柴胡皂苷d的含量,应用ANOVA和LSD T检验进行统计学分析,从而对市售柴胡药材的质量进行评价。本文不仅对柴胡属药用植物的快速准确鉴定具有重要意义,而且对于掌握市场上柴胡药材的流通现状及质量评价具有指导意义。Bupleuri Radix is one of the most frequently used herbal medicines in China with a 2000-year medicinal history. However, the use of Bupleuri Radix is very confused. Twenty-five species and eight varieties of Bupleurum have been used as Bupleuri Radix in different regions of China. It is very difficult to identify these Bupleurum species using traditional morphological method. In order to establish a fast and effective method to identify these Bupleururn species, we collected 168 Bupleurum medicinal plants from 14 populations of 9 provinces, and amplified their ITS sequences. 168 ITS sequences with a full length of 600-606 bp were obtained. DNAMAN analyzing results showed that 86 variable sites were present in these sequences and 19 haplotypes （TH1-TH19） were determined. After calculating K2P distance and analyzing an NJ tree, we established a molecular identification method based on ITS sequence. Using this method,52 samples of Bupleuri Radix were identified successfully. Furthermore, we tested saikosaponin a, c, d contents in these Bupleuri Radix by HPLC and analyzed the results by ANOVA and LSD T test to evaluate the quality of Bupleuri Radix. This method is significant for effective identification of Bupleurum medicinal plants, and quality control of Bupleuri Radix in the market.

关 键 词：柴胡 ITS 分子鉴定 柴胡皂苷 质量评价 

分 类 号：R931[医药卫生—生药学]