Three-tiered authentication of herbal Traditional Chinese Medicine ingredients used in women's health provides progressive qualitative and quantitative insight - part 2/4

Traditional Chinese Medicine herbal products are increasingly used in Europe but prevalent authentication methods have significant gaps in detection. In this study, three authentication methods were tested in a tiered approach to improve accuracy on a collection of 51 TCM plant ingredients obtained on the European market. We show the relative performance of conventional barcoding, metabarcoding and standardized chromatographic profiling for TCM ingredients used in one of the most diagnosed disease patterns in women, endometriosis. DNA barcoding using marker ITS2 and chromatographic profiling are methods of choice reported by regulatory authorities and relevant national pharmacopeias. HPTLC was shown to be a valuable authentication tool, combined with metabarcoding, which gives an increased resolution on species diversity, despite dealing with highly processed herbal ingredients. Conventional DNA barcoding as a recommended method was shown to be an insufficient tool for authentication of these samples, while DNA metabarcoding yields an insight into biological contaminants. We conclude that a tiered identification strategy can provide progressive qualitative and quantitative insight in an integrative approach for quality control of processed herbal ingredients.传统中药（Traditional Chinese Medicine, TCM）草本产品在欧洲的使用日益广泛，但主流的真伪鉴定方法在检测方面存在显著短板。本研究采用分层测试策略，对三种真伪鉴定方法进行评估，以提升对欧洲市场购得的51份TCM植物原料样本的鉴定准确率。本研究对比了常规条形码测序（barcoding）、宏条形码测序（metabarcoding）与标准化色谱指纹图谱（chromatographic profiling）三种方法，针对用于治疗女性最常见确诊病症之一——子宫内膜异位症（endometriosis）的TCM原料的相对鉴定性能。采用ITS2标记的DNA条形码测序以及色谱指纹图谱法，是监管机构与各国药典推荐的首选鉴定方法。高效薄层色谱（High Performance Thin Layer Chromatography, HPTLC）与宏条形码测序联用时，可有效提升物种多样性的分辨能力，即便针对经过高度加工的草本原料也能发挥出色的鉴定作用。尽管常规DNA条形码测序作为官方推荐方法，但其对本次研究样本的鉴定能力并不充分；而DNA宏条形码测序则可揭示样本中的生物污染物信息。综上，分层鉴定策略可通过整合式分析逐步提供样本的定性与定量信息，从而实现对加工草本原料的质量管控。