基于元基因组学的中药制剂物种成分分析方法及其与化学成分分析方法的整合研究

Compared with herbal materials and Traditional Chinese medicine (TCM) extracts, the Chinese export of TCM preparations remains low in recent year. One possible reason for above phenomenon is the lack of a standardized method to assess the quality of TCM preparations. The quality evaluation of TCM preparations mainly includes the analysis of chemical ingredients and biological ingredients. To date, more researches were focused on chemical ingredients while few studies were reported for biological ingredients. Biological ingredient analysis is very important for preparations composed of powder of TCM materials. The biological ingredient analysis for TCM preparations could be abstracted as the identification of multiple species from a biological mixture. The metagenomic approach has been considered as one of the most effective methods for multiple species analysis from a biological mixture, which would also be helpful for the analysis of biological ingredients in TCM preparations. Simultaneous identification for diverse species, including the prescribed species, adulterants, toxic species, protected species, and even the biological impurities introduced through production process, could be achieved by selecting appropriate DNA biomarkers. By this approach, it is prospective to offer assessment accordance for the safety and legality of TCM preparations. Based on the results of biological ingredients analysis of Liuwei Dihuang Wan by high-throughput sequencing previously, this project intends to analyze biological ingredients of TCM preparations extensively by metagenomic method. In addition, in order to establish a comprehensive and effective method for quality evaluation of TCM preparations, an integrated approach for biological ingredient analysis method and chemical ingredient analysis method based on HPLC fingerprint was explore.

与中药材和中药提取物相比，多年来中药制剂的出口额总在低位徘徊，主要原因之一在于其质量评价体系不完善。中药制剂质量评价主要分为化学成分分析和物种成分分析，目前对于前者研究较多，而对于后者报道很少。物种成分分析对于药材直接细粉入药的制剂质量评价非常重要。中药制剂物种成分分析的实质是对包含多个生物物种混合体系的物种鉴定。认识、分析生物混合体系结构和功能最有效、最重要的方法之一是元基因组学方法。利用元基因组学方法，选择合适的DNA分子标记，可在对中药制剂处方药材鉴定的同时鉴别其所含的混伪品、有毒或受保护的动植物成分，以及生产过程引入的生物杂质，从而为中药制剂的安全性和合法性提供评价依据。本项目拟在前期基于基因测序分析六味地黄丸物种成分基础上，将元基因组学方法广泛应用于中药制剂物种成分分析，并探讨其与基于HPLC指纹图谱化学成分分析方法的整合途径，以期建立一种全面、有效的中药制剂成分分析新方法。

随着高通量测序技术的迅速发展，中药制剂生物成分的评价方法也在不断完善。本项目以四种代表性中药制剂（八珍益母丸、大活络丸、牛黄降压丸和右归丸）为研究对象，结合高通量测序技术和多种DNA条形码方法对其物种成分进行了全面的分析，同时对本方法的普适性、灵敏性和可靠性进行了系统评价。基于ITS2和trnL条形码，从4种中药制剂中检测到处方植物成分种类介于77.8%-100%之间。ITS2在检测处方植物成分方面显示出比trnL更高的可靠性，但是两个条形码的结合可以提供更高的灵敏度和可靠性。在多组学的大数据时代，本项研究使得中药制剂处方药材分析方法向前迈进了一步，为实现中药制剂质量控制的数字化和现代化奠定了基础。同时，本项目以六味地黄丸为研究对象，建立了一种基于生物成分和化学成分整合分析评价中药制剂质量的方法。化学成分来源于HPLC指纹图谱，生物成分来源于基于ITS2的高通量测序，对不同成分的相对丰度进行归一化计算，然后通过随机森林法和邻近算法（KNN）法对整合方法进行优化和评价。将此整合方法与仅基于化学或生物成分的评价方法进行比较，发现（1）对不同厂家和不同批次的中药制剂进行分类时，整合方法评价指标全面，准确度较高；（2）通过选择特征成分进行样品分类的准确性比选择所有成分进行分类的准确度高。合理选择代表性的生物和化学特征，相互补充，以评估中药制剂成分的一致性和差异性，有助于确保中药制剂的有效性，安全性和合法性。此外，项目研究过程中，根据需要分别建立了DNA条形码数据库：http://holmes.tcm.mesh.wuhansoftware.com/和中药制剂网络药理数据库：http://watson.tcm.mesh.wuhansoftware.com/，可为科研工作者提供开放性服务。