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.

Figure 1. Photographs of four TCM samples genetically audited in this study using high-throughput sequencing.

See Table 1 for a detailed list of all samples and listed package ingredients. From left to right; (A) Bear Bile crystals (TCM-015), (B) Saiga Antelope Horn powder (TCM-011), (C) Yatong Yili Wan capsules (TCM-016), and (D) Babao Ching Hsin San powder (TCM-026).

Figure 2. MEGAN phylogram of plant components in Yatong Yili Wan capsules (TCM-016).

The data was generated using trnL c/h fusion primers and HTS using the Roche GS Junior. 2123 reads were queried against GenBank and parsed through MEGAN, SAP and QIIME (see Methods). The assignments of both MEGAN and SAP (with posterior support) are shown. Size of red node labels is proportional to number of sequence reads at each taxonomic level.