段亚萍;骆骄阳;豆小文;何柳;李坤伦;杨世海;杨美华;
DUAN Ya-ping;LUO Jiao-yang;DOU Xiao-wen;HE Liu;LI Kun-lun;YANG Shi-hai;YANG Mei-hua;College of Traditional Chinese Medicine,Jilin Agricultural University;Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences,Peking Union Medical College;

• 1:吉林农业大学中药材学院
• 2:中国医学科学院北京协和医学院药用植物研究所

摘要(Abstract)：

随着雷公藤药材在临床应用比较广泛,其市场上的伪品较多,传统技术不能有效的鉴别该中药的真伪。该实验拟基于DNA条形码和化学指纹图谱方法对雷公藤属药材进行准确的鉴定及质量评价。采用DNA条形码技术、隐马尔可夫模型的HMMer注释方法和K2P模型进行遗传距离分析,并结合最近距离法、相似性搜索法、构建NJ树等方法对雷公藤的DNA序列进行鉴定和评估;采用UPLC-PDA方法建立雷公藤药材指纹图谱,评价不同来源的药材指纹图谱的相似度;采用LC-MS/MS方法检测雷公藤主要成分的含量。结果表明:药材基原物种雷公藤的ITS2序列种内最大K2P遗传距离均远小于其与混伪品的种间最小K2P遗传距离;应用相似性搜索法分析结果表明ITS2序列初步鉴定雷公藤药材及其混伪品;在NJ聚类树上雷公藤药材及其混伪品聚类较为清晰,初步分析27份样品中有12种为雷公藤正品。化学指纹谱研究表明特征1区可以较为直观的区分雷公藤伪品;LC-MS/MS,HCA-热图分析表明不同产地雷公藤药材6种有效成分的含量差异显著,可评价雷公藤药材的质量。该实验对雷公藤属的药材准确鉴定及质量评价具有指导意义。
With the extensive clinical application of Tripterygium wilfordii,there are many counterfeit products on the market. Traditional technology can not effectively identify the authenticity of the traditional Chinese medicine. Therefore,a strategy of accurate identification and quality evaluation of Tripterygium based on DNA barcode and chemical fingerprint spectrum was established. Based on DNA barcode technology,HMMer annotation method of hidden Markov model and K2 P model were used to analyze genetic distance.BLAST1,nearest distance and phylogenetic tree(NJ-tree) methods were used to assess the identification efficiency of the ITS2 barcode. The fingerprint of 27 T. wilfordii was established by UPLC-PDA method,and the similarity of the fingerprint of different sources was evaluated. The main components of T. wilfordii were determined by LC-MS/MS. The results revealed that the intraspecific genetic distances of T. wilfordii were lower than the interspecific genetic distances between T. wilfordii and its adulterants. The results of similarity search showed that ITS2 sequence was used to identify T. wilfordii and its adulterants. The clustering of T. wilfordii and its adulterants was clear in the tree of NJ cluster,and 12 of 27 samples were identified as true T. wilfordii. The chemical fingerprint spectrum research indicates that the feature one region can distinguish the false product of tripterygium glycosides more intuitively. The cluster analysis of HCA-thermal map showed that the contents of six active components of T. wilfordii from different habitats were significantly different,which could be used to evaluate the quality of T. wilfordii. This paper is of guiding significance for the accurate identification and quality evaluation of Tripterygium medicinal plants.

关键词(KeyWords)： 雷公藤;DNA条形码;指纹图谱;UPLC;LC-MS/MS;HCA-热图分析
Tripterygium wilfordii;DNA barcoding;fingerprint;UPLC;LC-MS/MS;HCA-cluster analysis

Abstract:

Keywords:

基金项目(Foundation): 中国医学科学院医学与健康科技创新工程(2017-I2M-1-013,2016-I2M-3-010)

作者(Author): 段亚萍;骆骄阳;豆小文;何柳;李坤伦;杨世海;杨美华;
DUAN Ya-ping;LUO Jiao-yang;DOU Xiao-wen;HE Liu;LI Kun-lun;YANG Shi-hai;YANG Mei-hua;College of Traditional Chinese Medicine,Jilin Agricultural University;Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences,Peking Union Medical College;

Email:

DOI: 10.19540/j.cnki.cjcmm.20180510.005

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