Deep Sequencing of Plant and Animal DNA Contained within Traditional Chinese Medicines Reveals Legality Issues and Health Safety Concerns

Traditional Chinese medicine (TCM) has been practiced for thousands of years, but only within the last few decades has its use become more widespread outside of Asia. Concerns continue to be raised about the efficacy, legality, and safety of many popular complementary alternative medicines, including TCMs. Ingredients of some TCMs are known to include derivatives of endangered, trade-restricted species of plants and animals, and therefore contravene the Convention on International Trade in Endangered Species (CITES) legislation. Chromatographic studies have detected the presence of heavy metals and plant toxins within some TCMs, and there are numerous cases of adverse reactions. It is in the interests of both biodiversity conservation and public safety that techniques are developed to screen medicinals like TCMs. Targeting both the p-loop region of the plastid trnL gene and the mitochondrial 16S ribosomal RNA gene, over 49,000 amplicon sequence reads were generated from 15 TCM samples presented in the form of powders, tablets, capsules, bile flakes, and herbal teas. Here we show that second-generation, high-throughput sequencing (HTS) of DNA represents an effective means to genetically audit organic ingredients within complex TCMs. Comparison of DNA sequence data to reference databases revealed the presence of 68 different plant families and included genera, such as Ephedra and Asarum, that are potentially toxic. Similarly, animal families were identified that include genera that are classified as vulnerable, endangered, or critically endangered, including Asiatic black bear (Ursus thibetanus) and Saiga antelope (Saiga tatarica). Bovidae, Cervidae, and Bufonidae DNA were also detected in many of the TCM samples and were rarely declared on the product packaging. This study demonstrates that deep sequencing via HTS is an efficient and cost-effective way to audit highly processed TCM products and will assist in monitoring their legality and safety especially when plant reference databases become better established.传统中医药（Traditional Chinese Medicine, TCM）已有数千年的应用历史，但直至近数十年，其在亚洲以外地区的应用才逐渐普及。目前，人们持续对多款热门补充替代疗法药物（包括各类中医药）的有效性、合法性与安全性提出质疑。部分中医药制剂的成分已知包含濒危且受贸易限制的动植物物种衍生物，因此违反《濒危野生动植物种国际贸易公约》（Convention on International Trade in Endangered Species, CITES）相关法规。色谱研究已在部分中医药制剂中检出重金属与植物毒素，且不良反应病例屡见不鲜。开发针对中医药等药用物质的筛查技术，既符合生物多样性保护的需求，也契合公共安全的宗旨。本研究以质体trnL基因的p-loop区域以及线粒体16S核糖体RNA基因为靶点，从15份形态涵盖粉剂、片剂、胶囊剂、胆膏片以及花草药茶的中医药样本中，获得了超过49000条扩增子序列读段。本研究证实，第二代DNA高通量测序（High-Throughput Sequencing, HTS）是对复杂中医药制剂内有机成分进行遗传合规核查的有效手段。将DNA序列数据与参考数据库比对后，共检出68个不同的植物科，其中包括麻黄属（Ephedra）、细辛属（Asarum）等具有潜在毒性的属类。同理，研究还检出了多个动物科，其下辖类群被列为易危、濒危或极危物种，包括亚洲黑熊（Ursus thibetanus）以及赛加羚羊（Saiga tatarica）。多个样品中还检出牛科（Bovidae）、鹿科（Cervidae）以及蟾蜍科（Bufonidae）的DNA，但这些成分极少在产品包装上标注。本研究表明，借助高通量测序技术开展深度测序，是对高加工度中医药产品进行合规核查的高效且经济的手段，尤其在植物参考数据库进一步完善后，该方法将有助于监管中医药产品的合法性与安全性。