Molecular authentication of the traditional Chinese medicine Tongren Dahuoluo Wan and its alternative formulation

Abstract

Tongren Dahuoluo Wan has been a popular traditional Chinese medicine in international pharmaceutical markets for hundreds of years. Leopard bone powder is the key element in its formulation. However, the leopard has been listed for wildlife conservation, which limits the use of the leopard bone supplies. Therefore, an alternative formulation which substitutes leopard bone with zokor bone in the formula of Tongren Dahuoluo Wan is now manufactured. To develop a simple and reliable molecular method for authenticating the two patent medicines, mitochondrial nucleotide polymorphic sites of 12S rRNA, COI and Cytb genes were screened in leopard and zokor bones, and nine pairs of species-specific primers were verified for discriminating the two species. For the patent medicine authentication, we set up a molecular diagnostic assay to resolve the difficulties of low concentration of target DNAs and presence of PCR-inhibitory substances in this complex medicine, and successfully confirmed leopard or zokor content using the nine pairs of species-specific primers. We recommend a common technical strategy for authentication of species origins in traditional Chinese medicine, and discuss the experimental solutions for technical problems of molecular diagnostic assays.

Keywords

Tongren Dahuoluo Wan / molecular diagnostic assay / Eospalax baileyi / Panthera pardus / species-specific primers

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Supplementary materials

The online version of this article at http://dx.doi.org/10.15302/J-FASE-2017157 contains supplementary materials (Tables S1–S5).

Acknowledgements

The research was supported by projects of Beijing Municipal Science & Technology Commission (D08080203640903) and National Natural Science Foundation of China (31672379). We thank Dr. Hai Xiang (Institute of Genetics and Developmental Biology, CAS) and Dr. Langqing Liu (Wageningen University) for assistance with phylogenetic cluster analysis.

Compliance with ethics guidelines

Jikun Wang, Jing Du, Meng Cao, Lu Yao, Suhua Xie, Jiafu Chen, and Xingbo Zhao declare that they have no conflict of interest or financial conflicts to disclose.

This article does not contain any studies with human or animal subjects performed by any of the authors.

RIGHTS & PERMISSIONS

The Author(s) 2017. Published by Higher Education Press. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0)