DNA条形码技术在国家药品抽验中的应用研究

Application of DNA barcoding technology to national drug sampling inspection

国家药品抽验工作中发现中成药红金消结胶囊、红金消结片和柴黄颗粒处方中部分饮片来源复杂,存在掺伪现象,但常规检验方法难以确定混伪品基原物种。故本研究收集其处方中部分中药,包括柴胡、八角莲、黑蚂蚁和鼠妇虫饮片及相关药材样品184份,采用DNA条形码技术进行基原物种鉴定。结果显示,115份市售柴胡类药材及饮片获得111条ITS2序列,其中71份为柴胡(Bupleurum chinense)、3份为狭叶柴胡(B.scorzonerifolium)、31份为同属混伪品,另检出非同属伪品臭椿(Ailanthus altissima)1份、防风(Saposhnikovia divaricata)2份、一枝黄花(Solidago decurrens)3份;22份市售八角莲药材样本获得21条ITS2序列,其中15份为混伪品南方山荷叶(Diphylleia sinensis)、6份为八角莲(Dysosma versipellis)等鬼臼属植物;22份市售黑蚂蚁饮片基因组DNA降解均未能获得COI序列;38份市售鼠妇虫饮片样本获得24条COI序列,其中9份为平甲虫(寻常卷甲虫Armadillidium vulgare)、11份为光滑鼠妇(Porcellio laevis)、2份为中华蒙潮虫(Mongoloniscus sinensis),另有2份不能判定物种。本研究证实中药材DNA条形码分子鉴定技术适用于中成药处方投料的基原鉴定,有助于建立中药材、中药饮片、中成药生产流通使用全过程追溯体系。

Adulterants and counterfeits were found in some of the commercial traditional Chinese medicine(TCM)decoctions in Hongjin Xiaojie Jiaonang,Hongjin Xiaojie Pian,and Chaihuang Keli during the national drug sampling inspection.However,it was difficult to determine the species of the adulterants and counterfeits by conventional testing methods.Therefore,a total of 184 samples of the TCM decoctions and raw materials belong to the prescriptions of above mentioned traditional Chinese patent medicines,including Bupleuri Radix,Bajiaolian,Heimayi,and Shufuchong,were collected and authenticated by DNA barcoding technology.111 ITS2 sequences were obtained from 115 commercial TCM decoctions and raw materials of Bupleuri Radix,among which 71 were Bupleurum chinense,three were B.scorzonerifolium,and 31 were closely related species in the same genus.In addition,counterfeits derived from different genera,such as Ailanthus altissima(one sample),Saposhnikovia divaricate(two samples),and Solidago decurrens(three samples),were also detected.21 ITS2 sequences were obtained from 22 commercial TCM raw materials of Bajiaolian,among which 15 were Diphylleia sinensis and six were Dysosma versipellis and other species in genus Dysosma.For 22 Heimayi samples,PCR amplification of COI sequence was failed due to genomic DNA degradation.Among 38 Shufuchong samples,24 COI sequences were obtained and only nine of them were the genuine species(Armadillidium vulgare)recorded in the Chinese Pharmacopoeia,11 were Porcellio laevis,two were Mongoloniscus sinensis,and two samples could not be identified due to the limitation of database.This study demonstrates that DNA barcoding technology is suitable for the species authentication of the decoctions of traditional Chinese patent medicine prescription.It is a conductive way for the establishment of traceability system for the whole TCM industrial chain.