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

Abstract

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.

Author Summary

Chemicals derived from plants and animals are widely used in traditional Chinese medicine (TCM), and it is commonplace for remedies to contain a complex list of ingredients. Due to their heterogeneous origins, and subsequent processing into pills and powders, it can be difficult for the biological origin of ingredients within each remedy to be reliably determined. In this study, we have, for the first time, used a second-generation DNA sequencing method to analyse TCM remedies and determine their animal and plant composition. Using this deep-sequencing approach we identified plant species that are known to contain toxic chemicals and identified animal DNA from species that are currently endangered and protected by international laws. Consumers need to be made aware of legal and health safety issues that surround TCMs before adopting them as a treatment option. More widespread testing of complementary medicines using the DNA methods developed herein represents an efficient and cost-effective way to audit their composition.