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）传承至今已有数千年历史，但直至近数十年，其在亚洲以外地区的应用才愈发广泛。人们始终对包括传统中医药在内的多款主流补充替代疗法的有效性、合法性及安全性存在诸多顾虑。已知部分传统中医药的成分包含濒危及受贸易限制的动植物物种衍生物，因此违反了《濒危物种国际贸易公约（Convention on International Trade in Endangered Species, CITES）》相关法规。色谱研究已在部分传统中医药中检出重金属与植物毒素，且已有多例不良反应报告。开发针对传统中医药等药用产品的筛查技术，既有利于生物多样性保护，也能保障公众安全。针对质体trnL基因的p-loop区域以及线粒体16S核糖体RNA基因，研究团队从15份不同剂型的传统中医药样本中生成了超过49000条扩增子序列读段，这些样本包括粉剂、片剂、胶囊、胆片以及草本茶饮。本研究证实，第二代DNA高通量测序（High-Throughput Sequencing, HTS）可作为一种有效的手段，对复杂传统中医药中的有机成分进行基因核查。将DNA序列数据与参考数据库比对后，共检出68个不同的植物科属，其中包括麻黄属（Ephedra）和细辛属（Asarum）等具有潜在毒性的类群。同样，研究还检出了多个动物科属，其中部分类群被列为易危、濒危或极危物种，包括亚洲黑熊（Ursus thibetanus）与赛加羚羊（Saiga tatarica）。研究还在多款传统中医药样本中检出了牛科（Bovidae）、鹿科（Cervidae）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，通过高通量测序开展的深度测序技术，是对高加工度传统中医药产品进行基因核查的高效且经济的手段，有助于监管此类产品的合法性与安全性，尤其是在植物参考数据库进一步完善的情况下。