远志HPLC指纹图谱及rbcL序列分子鉴定研究  

HPLC fingerprint and molecular identification of rbcL sequence of Polygala tenuifolia

作　　者：郑玉霄 杨晨[1,2] 张彬 李鹏辉 龚年春 李超 李跃辉 ZHENG Yuxiao;YANG Chen;ZHANG Bin;LI Penghui;GONG Nianchun;LI Chao;LI Yuehui(Hunan University of Chinese Medicine,Changsha 410208,China;Hunan Academy of Traditional Chinese Medicine,Changsha 410013,China;Hunan Provincial Center for Drug Evaluation and Adverse Drug Reaction Monitoring,Changsha 410013,China)

机构地区：[1]湖南中医药大学,湖南长沙410208 [2]湖南省中医药研究院,湖南长沙410013 [3]湖南省药品评审与不良反应监测中心,湖南长沙410013

出　　处：《中草药》2024年第2期588-595,共8页Chinese Traditional and Herbal Drugs

基　　金：湖南省自然科学基金科药联合项目(2021JJ80045)。

摘　　要：目的建立14批不同产地远志药材HPLC指纹图谱并利用化学计量学方法对其进行质量评价。采用DNA分子鉴定技术对远志药材进行物种鉴定,为远志药材质量控制提供依据。方法选用Agilent ZORBAX SB-C18色谱柱(250 mm×4.6 mm,0.5μm),流动相为乙腈-0.05%磷酸溶液,梯度洗脱,体积流量1.0 mL/min,柱温30℃,检测波长316 nm,进样量10μL,运用软件“中药色谱指纹图谱相似度评价系统”(2012版)对14批远志药材进行相似度评价,确认共有峰,对测定结果进行聚类分析和主成分分析,并结合正交偏最小二乘-判别分析对样品进行模式识别。提取14批远志以及白薇、瓜子金样品的DNA进行序列分析和物种鉴定,并从NCBI数据库下载混淆品的序列,借助MEGA-X计算遗传距离并构建系统发育树。结果建立了14批远志药材的HPLC指纹图谱,标定了20个共有峰,相似度均大于0.918,化学计量学分析将14批远志药材分为3类。经DNA分子鉴定,14批远志药材均为远志科植物远志Polygala tenuifolia,遗传距离和NJ树分析可将远志与混淆品区分开。结论所建立的HPLC指纹图谱稳定可靠,可为远志的质量评价提供参考。rbcL序列能区分远志及其混淆品,可用于远志药材的鉴定。Objective To establish HPLC fingerprints of 14 batches of Polygala tenuifolia from different habitats,evaluate their quality by chemometric methods,and identify the species of P.tenuifolia by DNA molecular identification technology,so as to prove a basis for the quality control of P.tenuifolia.Methods The analysis was performed on Agilent ZORBAX SB-C18chromatographic column(250 mm×4.6 mm,0.5μm)with mobile phase consisted of acetonitrile-0.05%phosphoric acid solution for gradient elution,with flow rate of 1.0 m L/min,column temperature of 30℃,detection wavelength of 316 nm,injection volume of 10μL.Similarity evaluation was performed by Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System(2012 edition)to confirm the common peaks.The clustering analysis(CA),principal component analysis(PCA),and orthogonal partial least squares-discriminant analysis(OPLS-DA)method were carried out.The DNA of 14 batches of P.tenuifolia,Cynanchum atratum and P.japonica was extracted for sequence analysis and species identification,and the sequences of adulterants were downloaded from NCBI database.The genetic distance was calculated by MEGA-X and the phylogenetic tree was constructed.Results The HPLC fingerprints of 14 batches of P.tenuifolia were established,20 common peaks were calibrated,their similarities were all greater than 0.918 and the 14 batches of P.tenuifolia were divided into three groups by chemometric analysis.The 14 batches of P.tenuifolia were identified by DNA molecular identification,and genetic distance and NJ tree analysis could distinguish P.tenuifolia from adulterants.Conclusion The HPLC fingerprint established in this paper is stable and reliable,which can provide reference for the quality evaluation of P.tenuifolia.The rbcL sequence can distinguish P.tenuifolia and its adulterants,and can be used for the identification of P.tenuifolia.

关 键 词：远志 HPLC指纹图谱 DNA分子鉴定 RBCL序列 西伯利亚远志糖A5 远志(口山)酮Ⅲ 3 6′-二芥子酰基蔗糖 

分 类 号：R286.2[医药卫生—中药学]