基于ITS2序列的川贝母中成药的鉴定

Identification of Chinese patent medicines containing Fritillariae cirrhosae bulbus using ITS2 region

润肺圣药川贝母资源匮乏,为国家三级保护植物.近年雾霾天气频发使得其资源需求加大,导致以川贝母入药的中成药掺假现象严重.中成药成分复杂,单利用普通的DNA条形码鉴定流程无法对其一次性完成鉴定,因此需结合单克隆分析逐一鉴定.本研究首先建立了包含贝母属208条ITS2序列的数据库;收集了市售20份川贝中成药,其中包含15份蛇胆川贝胶囊,4份川贝末胶囊和1份蛇胆川贝散.扩增其ITS2序列,对PCR产物进行克隆测序,对测序结果进行BLAST比对;同时本实验还选取了3份蛇胆川贝胶囊进行高通量测序,利用单克隆和二代测序相结合的混合测序方法对该3份中成药中川贝母再次鉴定.基于二代测序数据对中成药组分解析存在数据冗余现象,而单克隆结果与选取的克隆数量有关,二者结合可相互弥补缺陷.单克隆结果数据表明,15份蛇胆川贝胶囊中3份含有平贝母,10份含有伊贝母及14份含有黄花贝母等非标签成分,4份川贝末胶囊中有2份检测到川贝母,2份检测到平贝母,但同时3份中还检测到黄花贝母;1份蛇胆川贝散中仅检测到伊贝母与黄花贝母.二代测序结果表明,3份蛇胆川贝胶囊中主要检测出平贝母、伊贝母等非标签成分.通过比对二者序列,发现无论克隆还是二代测序数据均表明混合测序的3份蛇胆川贝胶囊中均不含川贝母;本研究表明,基于单克隆和二代测序辅助的方法可以准确对川贝母中成药进行鉴定.目前川贝母中成药掺假现象严重,平贝母和伊贝母是川贝母中成药的主要掺伪品,应加强对中成药市场的监管.

Fritillariae cirrhosae bulbus （BM CPMs）, which is used as an effective treatment for moistening lung, has been listed in the third level of protection category due to its decline in the wild. The demand has greatly increased since 2013 along with the growing concem over the high frequency of fog and haze. Shedanchuanbei particles are one of the adulterants or substituents that frequently occur in Chinese patent medicines containing BM CPMs. In this research, we first established a BM database comprising 208 ITS2 sequences from 25 species of Fritillaria and 1 adulterant. We then collected 20 commercial BM CPMs, selecting 20 CPMs for cloning experiments, which included 15 batches of shedanchuanbei particles, 4 batches of chuanbeimo particles, and 1 batch of shedanchuanbei powder. Results of cloning experiments revealed that among 15 batches of shedanchuanbei particles, 3 batches contained F. ussuriensis bulbus, 10 batches contained F. pallidiflorae bulbus, and 14 batches contained F. verticillate. Moreover, among 4 batches of chuanbeimo particles, only 2 batches contained BM CPM, but these batches were adulterated with E ussuriensis bulbus and F. verticillate. Both E pallidiflorae bulbus and F. verticillate were detected in shedanchuanbei powder. Three batches of shedanchuanbei particles were also analyzed using high-throughput sequencing, which detected no BM CPM. This study presents the seriousness of the herbal adulterant problem and the feasibility of the TA cloning with high-throughput sequencing for identifying CPM.