基于DNA分子技术的中药传统散剂、丸剂处方成份动物源药材鉴定研究

There are no effective methods for the identification of biological ingredients in TCM preparations. TCM powders and pills are blends of several traditional Chinese medicinal material powders. The degrees of DNA destroyed or degraded for TCM powders and pills are relatively lower than other type of traditional Chinese medicines. Animal medicinal materials contain a lot of endangered species and are frequently substituted and contaminated. Here, we use TCM powders and pills as a breakthrough and animal medicinal materials for a starting point to construct the methods for the identification of animal medicinal materials in TCM preparations biological ingredients. First, we collect 10 kinds of TCM powders and pills including Zixue San, DaHuoluo Wan, etc. These TCM powders and pills comprise animal medicinal materials like Lingyang Jiao, Lu Rong, etc. Then, we enrich the reference COI sequences library for these animal medicinal materials, design 100-300 bp mini-COI, and optimize the DNA extraction and PCR amplification methods. We further complete the DNA extraction and PCR amplification of the TCM powders and pills, and then use Illumina platform for High-throughput DNA sequencing. We use FLASH software to merger pair-end reads, use Trimmomatic software to filter low quality reads, use UCHIME software to remove chimeras, use QIIME software to divide OUTs. The Blast software and HMM software are used to complete the species designation for OUTs with the help of the reference COI sequences library. The Alpha and Beta methods are employed to compare the species diversities between intra-samples and inter-samples. The stability, repeatability and accuracy of this research strategies and methods will be investigated. The strategy used in this study will perform animal biological ingredient analysis of TCM powders and pills to guarantee the quality of these kinds of TCM medicines.

中成药鉴定是中药鉴定学的难点。本项目以DNA破坏相对较小的散剂和丸剂为突破口，以掺伪造假突出的动物源药材为切入点，尝试建立散剂和丸剂动物源药材DNA分子鉴定体系。收集紫雪散、大活络丸等10种散剂和丸剂，涉及羚羊角、鹿茸等动物源药材，丰富其COI序列数据库，设计100-300bp的微COI序列引物，优化低丰度DNA提取和PCR扩增方法，完成样品DNA提取和微COI序列扩增，基于Illumina平台进行高通量测序，使用FLASH软件Merge Pair-End测序数据，Trimmomatic软件过滤低质量序列，UCHIME软件去除嵌合体，QIIME软件划分OUT，运用Blast和HMM完成OUT物种分类，通过Alpha和Beta完成样品内和批次间物种多样性比较，考察方法稳定性、重现性和准确性。本项目的顺利实施将实现中药散剂和丸剂动物源药材的准确鉴定，保障中药疗效和质量安全。

中成药处方成分鉴定是中药鉴定学的难点，中成药处方成分含混伪品导致了一系列严重安全事件。基于此，本项目以掺伪造假突出的动物源药材为研究重点，以DNA破坏较小的原粉入药中成药为研究对象，完成通心络胶囊、清肺消炎丸、同仁大活络丸等38种中成药的样品收集，涉及地龙、水蛭、金钱白花蛇等33种动物源药材；对水蛭、地龙、金钱白花蛇等具有较多混伪品的药材开展专类研究，进一步丰富中国动物药材DNA 条形码数据库（TCM-BOL）；为降低计算负载，基于NCBI Entrez从GenBank获得3564430条COI序列，基于CD-Hit v4.6.8构建了简化率为91.6%的COI简化数据库；提取91350条全长COI序列，筛选出3对mini-barcode引物。通过对SDS、CTAB提取方法和柱式、磁珠DNA纯化试剂盒的系统比较，建立了中成药DNA提取方案和质量分级标准。基于Illumina平台进行高通量测序，每个样品的平均测序量为8.02 G。采用shotgun metagenomics和DNA metabarcoding两种策略对不同质量DNA的中成药开展研究，建立相应的数据分析流程：基于Trimmomatic v0.38进行低质量序列去除，基于COI简化数据库进行reads富集，每个样品富集的COI平均reads为1.02 M。基于metaspades v3.140，CAP3和megahit v1.2.9进行短序列组装，基于Quast v5.0.2进行组装质量评估，基于CodonCode Aligner v9.01进行序列注释，基于Uchime v4.2去除chimeras序列。基于Bowtie2 v2.4.1进行短序列mapping，基于samtools v1.10进行测序深度和覆盖度计算，去除测序深度≤3和覆盖度≤95%的COI序列。基于TCM-BOL数据库，结合GenBank和BOLD数据库进行物种鉴定，结果表明：在38种中成药中，除珍珠、牛黄、穿山甲外，豹骨、地龙、水蛭等23种药材可以进行动物来源鉴定，共发现15种替代品或混伪品，其中水蛭、地龙、海马、乌梢蛇的混伪品较多，鹿茸仅检测到其替代品驯鹿。本项目的顺利实施为中成药动物源药材的准确鉴定提供了新的技术手段，为提升中药品质、保障中药疗效和质量安全提供基础。