Three-tiered authentication of herbal Traditional Chinese Medicine ingredients used in women's health provides progressive qualitative and quantitative insight - part 1/4

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.中药（Traditional Chinese Medicine, TCM）草本制品在欧洲的应用愈发广泛，但当前主流的真伪鉴定方法在检测层面存在显著短板。本研究针对欧洲市场采集的51种TCM植物原料样本，采用分层检测策略对三种真伪鉴定方法进行评估，以提升鉴定准确性。本研究针对用于女性最常见确诊疾病类型之一——子宫内膜异位症治疗的TCM原料，对比分析了传统DNA条形码（conventional barcoding）、宏条形码（metabarcoding）与标准化色谱指纹图谱（standardized chromatographic profiling）三种方法的相对性能。使用ITS2标记的DNA条形码技术与色谱指纹图谱法，是监管机构与各国药典推荐的首选鉴定方案。尽管本次研究涉及的样本多为高度加工的草本制品，但联合宏条形码技术的高效薄层色谱（High Performance Thin Layer Chromatography, HPTLC）被证实为极具价值的真伪鉴定工具，可实现更高分辨率的物种多样性解析。作为官方推荐方法的传统DNA条形码技术，在本次样本的鉴定中被证实存在明显局限；而DNA宏条形码技术则可揭示样本中的生物污染物信息。本研究最终得出结论：采用分层鉴定策略，可通过整合性分析路径为加工草本制品的质量控制提供逐步深化的定性与定量分析依据。