基于DNA metabarcoding技术的如意金黄散处方成分鉴定研究

DNA metabarcoding identification of prescription ingredients in traditional medicine Ruyi Jinhuang San

中药的有效性和安全性是国内外关注的热点,本研究以如意金黄散为例,基于DNA metabarcoding技术,尝试从投料药材真伪角度评价中药的有效性和安全性.首先,收集如意金黄散处方成分药材及其混伪品样品161份,从已发表文献中下载密切相关物种序列154条,构建"如意金黄散DNA条形码鉴定数据库",该数据库包含10属119个物种.收集不同批次的如意金黄散样品3份,以ITS2序列为条形码,使用Illumina HiSeq平台进行PE250深度测序,利用本研究构建的"如意金黄散DNA条形码鉴定数据库"进行投料药材基原物种识别,结果表明,基于DNA metabarcoding技术可检测到除厚朴外的全部处方成分,不同药材的测序量存在显著差异;检测到苍术药材的混伪品朝鲜苍术和天南星药材的混伪品虎掌南星,提示如意金黄散临床使用的有效性和安全性存在潜在风险;不同批次鉴定结果具有稳定性和可重复性.本研究表明,基于DNA metabarcoding技术的如意金黄散处方成分鉴定方法符合中药复杂体系特点,有望成为中成药鉴定的新方法,并为中成药的有效性和安全性评价提供参考.

The efficacy and safety of traditional Chinese medicine are global concerns. Here, we selected a traditional medicinal powder Ruyi Jinhuang San as a research subject and employed DNA metabarcoding technology to identify its ingredients. First, 161 specimens constituting the ingredients of Ruyi Jinhuang San and their adulterants were collected. In addition, 154 ITS2 sequences belonging to the closed related species of these ingredients were extracted from the published literatures. First, a DNA barcode reference library of Ruyi Jinhuang San, comprising 119 species belonging to 10 genera, was constructed. Second, three different batches ofRuyi Jinhuang San were collected, and ITS2 was selected as a DNA barcode. PCR products were sequenced with the Illumina HiSeq sequencing platform using PE250 libraries. Taxonomy assignments were completed with the help of the above-mentioned DNA barcode reference library. The results indicated that all ingredients of Ruyi Jinhuang San can be successfully detected except Houppo. The medicine Cangzu had been contaminated with Atractylodes koreana. The medicine Tiannanxing had been substituted with Pinellia pedatisecta. The contamination and substitution of medicinal ingredients are potential threats to consumer safety and medicinal effectiveness. The authentication results of different samples are repeatable and stable. In conclusion, DNA metabarcoding is a valuable technology to validate the efficacy and safety of traditional Chinese medicine and has the potential to be a new method for identifying traditional Chinese medicine ingredients.