table1_Species Quantification in Complex Herbal Formulas—Vector Control Quantitative Analysis as a New Method.doc

Product mislabeling and/or species fraud in Traditional Chinese Medicine (TCM) not only decrease TCM quality, but also pose a potential health issue to the end user. Up to now, methods to control TCM quality have been developed to detect specific metabolites or identify the original species. However, species quantification in complex herbal formulas is rarely concerned. Here, we reported a simple Vector Control Quantitative Analysis (VCQA) method for flexible and accurate multiplex species quantification in traditional Chinese herbal formulas. We developed PCR-based strategy to quickly generate the integrated DNA fragments from multiple targeted species, which can be assembled into the quantitative vector in one round of cloning by Golden Gate ligation and Gateway recombination technique. With this method, we recruited the nuclear ribosomal DNA Internal Transcribed Spacer (ITS) region for the quantification of Ligusticum sinense “Chuanxiong,” Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav., Notopterygium incisum K. C. Ting ex H. T. Chang, Asarum sieboldii Miq., Saposhnikovia divaricata (Turcz.) Schischk., Nepeta cataria L., Mentha canadensis L., and Glycyrrhiza uralensis Fisch. ex DC. in ChuanXiong ChaTiao Wan, a classic Chinese herbal formula with very long historical background. We found that, firstly, VCQA method could eliminate the factors affecting such as the variations in DNA extracts when in combination with the use of universal and species-specific primers. Secondly, this method detected the limit of quantification of A. sieboldii Miq. in formula products down to 1%. Thirdly, the stability of quality of ChuanXiong ChaTiao Wan formula varies significantly among different manufacturers. In conclusion, VCQA method has the potential power and can be used as an alternative method for species quantification of complex TCM formulas.

传统中药材（Traditional Chinese Medicine，TCM）存在产品错标或物种欺诈行为，不仅会降低中药材品质，还会对终端使用者构成潜在健康风险。迄今为止，已有针对中药材质量管控的方法被开发，用于检测特定代谢物或鉴别原物种。然而，复杂复方中药中的物种定量分析却鲜有涉及。本研究报道了一种简便的载体控制定量分析（Vector Control Quantitative Analysis，VCQA）方法，可灵活且精准地对复方中药中的多种物种进行定量分析。我们开发了基于聚合酶链式反应（Polymerase Chain Reaction，PCR）的策略，可快速从多种目标物种中扩增得到整合DNA片段，并通过Golden Gate连接与Gateway重组技术，经一轮克隆即可组装至定量载体中。依托该方法，我们选取核糖体DNA内部转录间隔区（Internal Transcribed Spacer，ITS）作为靶标，对经典古方川芎茶调丸中的川芎（Ligusticum sinense 'Chuanxiong'）、白芷[Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav.]、羌活（Notopterygium incisum K. C. Ting ex H. T. Chang）、细辛（Asarum sieboldii Miq.）、防风[Saposhnikovia divaricata (Turcz.) Schischk.]、荆芥（Nepeta cataria L.）、薄荷（Mentha canadensis L.）以及甘草[Glycyrrhiza uralensis Fisch. ex DC.]进行定量分析。研究结果显示：其一，结合通用引物与物种特异性引物的使用，VCQA方法可消除DNA提取过程中产生的波动等影响因素；其二，该方法对复方制剂中细辛的定量限可低至1%；其三，不同厂商生产的川芎茶调丸制剂，其质量稳定性存在显著差异。综上，VCQA方法具备可观的应用潜力，可作为复杂复方中药物种定量分析的替代方法。