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

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-speci ﬁ c primers were veri ﬁ ed for discriminating the two species. For the patent medicine authentication, we set up a molecular diagnostic assay to resolve the dif ﬁ culties of low concentration of target DNAs and presence of PCR-inhibitory substances in this complex medicine, and successfully con ﬁ rmed leopard or zokor content using the nine pairs of species-speci ﬁ c 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.


Introduction
Tongren Dahuoluo Wan is a traditional Chinese medicines (TCMs) produced by Beijing Tongrentang which is a famous time-honored brand in the TCM industry. Leopard (Panthera pardus) bone has been regarded as a key element of Tongren Dahuoluo Wan. Due to its specific use for treating wind-cold-dampness arthralgia-syndrome, Tongren Dahuoluo Wan has been extensively produced and widely used. However, leopard has been listed in Appendix I of CITES (Convention on International Trade in Endangered Species) and the Red List of IUCN (International Union for the Conservation of Nature), which lists species that are the most endangered and threatened with extinction. With an increasing international demand for Tongren Dahuoluo Wan, compliance with legality, and hence the CITES status, Beijing Tongrentang developed an alternative Tongren Dahuoluo Wan, which replaced the bone of leopard with zokor (Eospalax baileyi), and previous studies reported the rational substitute of the bone content using the zokor for the leopard, based on medical bioactivities which included anti-inflammatory effect, dissipation of blood stasis effect and antioxidant capacity [1,2] . Therefore, it is of great importance to be able to authenticate Tongren Dahuoluo Wan with this alternative formulation.
Even though morphometrics is reliable for distinguishing leopard bones from zokor bones, it is not applicable to identifying the sources of ingredients in processed medicine. Over recent decades, chemo-profiling techniques, such as high performance liquid chromatography [3] , gas chromatography [4] and near-infrared spectroscopy [5] have been widely applied to medical ingredient identification. By addressing the detection of certain compounds, these methods have complemented the limitations of morphological identification, yet cannot reliably provide species authentication. Therefore, molecular diagnostic assays have been increasingly utilized for authenticating particular species signatures in TCMs [6,7] .
Since highly processed patent medicines usually contains poor nuclear DNAs, the high copy number of mitochondrial DNA (mtDNA) is a suitable target for species testing [8] . Therefore, we set up a testing system for discrimination of leopard and zokor contents. Our testing system includes a series of species-specific primers designed for mtDNA 12S rRNA, COI and Cytb genes, and an internal positive control as indicator to monitor the PCR-inhibitory substances in Tongren Dahuoluo Wan formulations.

Materials
Leopard (P. pardus) bones, zokor (E. baileyi) bones and Tongren Dahuoluo Wan pills were provided by the Research Institute of Beijing Tongrentang Co., Ltd.
Traditionally, Tongren Dahuoluo Wan consists of 50 ingredients; detail information is listed in Table S1.

DNA extraction from bones and patent pills
Briefly, bones were ground into powder and incubated overnight at 55°C with lysis buffer (10 mmol$L -1 Tris-HCl pH 8.0, 200 µg$mL -1 Proteinase K, 0.1 mol$L -1 EDTA pH 8.0, 0.5% SDS), followed by centrifugation for 15 min at 12000 g and taking the supernatant for phenol-chloroform extraction [9] . DNA was detected by 0.7% agarose gel electrophoresis. DNA extraction from Tongren Dahuoluo Wan formulations was based on the protocol of Cao et al. [6] .

Primers
Nine pairs of species-specific primers were designed according to mitochondrial genome sequences of leopard (NC_010641) and zokor (JN540033). Primers specific for leopard and zokor were designed based on their SNP sites detected in 12S rRNA, COI and Cytb gene regions, including five pairs of species-specific primers for leopards and four pairs of species-specific primers for zokors. PCR primer information was listed in Table S2.

PCR amplification and sequencing
The PCR reaction mixture (25 μL) included 2.5 μL of 10 Â Taq PCR buffer, 0.5 μL of Taq DNA polymerase, 0.5 μL primers (10 μmol$L -1 ), 0.5 μL of dNTPs (10 mmol$L -1 ), 20 μL of distilled water and 1 μL of DNA template. In addition, 10 ng PCR product of zokor DNA (preserved in our laboratory) were added to the PCR mixture and used as the internal positive control. The reaction was performed under the following conditions: 95°C 5 min, and 35 cycles of 95°C 30 s, 55-70°C 30 s and 72°C 30 s, at last 72°C 5 min. The products were then analyzed by 1.5% agarose gel electrophoresis.

Phylogenetic cluster analysis
The COI sequences of zokor and leopard were used as anchors to blast homologous sequences in GenBank. The targeted sequences were defined as species E. baileyi or P. pardus, and potential incorrect sequences were excluded manually. The extracted data sets are listed in Table S3. Consensus NJ (Neighbor-Joining) trees were constructed by 1000 bootstraps and depicted using MEGA6 software [10] .

DNA extraction from animal bones and pills
Total DNA was extracted from animal bones in large amounts but was degraded, and from the Tongren Dahuoluo Wan pills, the DNA extracted was badly degraded (Fig. 1).

PCR identification of patent pills using species-specific primers
The internal positive control generated target fragments, indicating no PCR inhibitory substances were present in bone DNA extracts. All species-specific primers generated correct amplicons, showing evidence of accurate authentication of the medicine (Fig. 2).

Sequence verification
For the accuracy of the authentication, all PCR fragments were subsequently sequenced. For the 12S rRNA fragment, the zokor or leopard medicines gave the corresponding complete identical sequence (NC_010641 for leopard, and JN540033 for zokor), while the leopard medicine had one polymorphic site in the COI fragment and 10 sites in the Cytb fragments, and the zokor medicine had one polymorphic site in the COI fragment and 10 sites in the Cytb fragments (Table S4; Table S5). A Phylogenetic tree for zokor or leopard medicine respectively was constructed using COI sequences which were joined to form an integrated sequence. The two medicines were separately clustered with GenBank sequences, including 56 alignments of E. baileyi and 20 of P. pardus (Table S3). Both phylogenetic consensus trees resulted in two apparent clusters, which represented E. baileyi and P. pardus, respectively (Fig. 3). The sequences generated by zokor specific primers was found in the E. baileyi clade (Fig. 3a), while the sequences generated by leopard specific primers were found in the P. pardus clade (Fig. 3b). The results clearly verified the validity and fidelity of the medicine identification.

Discussion
For CITES member states to enforce legislation and to prosecute cases of illegal trade, a reliable method of species identification is needed [11] . TCM preparations are of different types, including pills, capsules, powders and tablets [12] , and their chemical composition is quite complex, which makes it difficult to identify animal origins by traditional taxonomic and chromatographic methods [13] . Molecular authentication of particular species signatures in TCMs using DNA markers can meet the requirements for correctness and accuracy.
Molecular authentication of TCM suffers from a series of technical problems, mainly arising from DNA extraction, PCR assay and impacts the resulting authenticity, as illustrated in Fig. 4. DNAs preserved in TCMs are actually  complex and in various states of degradation, therefore it is crucial to preserve the available quantity as much as possible for further enzymatic manipulation. The extraction method needs to avoid overly aggressive treatments, such as high temperatures or use of strong detergents [14] . So far, the phenol-chloroform method has proven to be a simple and effective technique for extracting DNA from complex samples [6,9] . In addition, concentration of DNA using membranes [15] or binding DNA to silica [16] can be an effective but expensive alternative for TCM DNA extraction. The sensitivity and kinetics of PCR may be dramatically reduced by PCR-inhibitory substances (e.g., plant polyphenol) [17] in TCMs, so purification of TCM DNAs using QIAquick TM Purification Kit (Qiagen, Hilden, Germany) and setting the internal positive control to monitor the presence or absence of the PCR-inhibitory factors [12,13] is necessary. We also used a blank control to monitor false positive results. When both the sample and the internal positive control are negative, the TCM DNA contains PCR-inhibitory substances and the DNA extract needs to be purified.
In recent years, various molecular markers have been developed for authentication of TCMs, including random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR) and restriction fragment length polymorphism (RFLP) [18][19][20][21] . In addition to poor reproducibility, RAPD, ISSR and RFLP can be easily affected by DNA degradation, thus they are not suitable for identification of highly processed samples. Simple sequence repeats [22] have also been used in authenticating medicinal materials, but the products amplified from each allele are usually short fragments, subsequently complicating reliability scores by standard agarose gel electrophoresis [23] . Next-generation sequencing technology is a powerful tool in identifying biological ingredients in TCMs [13,24] . The advantage of quality evaluation of TCM preparations based on metagenomic approach via high-throughput sequencing is that it not only detects the prescribed species but also any contaminating species. However, it is also restricted by DNA degradation during TCM processing or the lack of reference sequence in the database.
Species-specific primers to authenticate target species (e.g., E. baileyi) in TCMs are simple and rapid, with low thresholds and high efficiency, and the increasing availability of mitogenome sequences deposited in EMBL or GenBank will enable a wide range of species-specific primers to be designed.

Conclusions
In this study, a range of mtDNA SNPs were detected between E. baileyi and P. pardus. With the specific primers designed from SNP sites and an internal positive control as an indicator to monitor PCR-inhibitory substances, the animal origin of contents of Tongren Dahuoluo Wan formulations were precisely identified. In conclusion, molecular diagnostic assays are considered invaluable tools for authenticating particular species signatures of TCMs.  (Tables S1-S5).