Molecular Identification of Chinese Materia Medica and Its Adulterants Using ITS2 and psbA-trnH Barcodes: A Case Study on Rhizoma Menispermi


Rhizoma Menispermi, derived from the rhizoma of Menispermum dauricum DC., is one of the most popular Chinese medicines. However Rhizoma Menispermi is often illegally mixed with other species in the herbal market, including Aristolochia mollissimae Hance, which is toxic to the kidneys and potentially carcinogenic. The use of DNA barcoding to authenticate herbs has improved the management and safety of traditional medicines. In this paper, 49 samples belonging to five species, including 34 samples of M. dauricum, from different locations and herb markets in China were collected and identified using DNA barcoding. The sequences of all 34 samples of Rhizoma Menispermi are highly consistent, with only one site variation in internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA and no variations in the psbA-trnH region. The intra-specific genetic distance is much smaller than inter-specific one. Phylogenetic analysis shows that both sequences allow the successful identification of all species. Nearest distance and BLAST1 methods for the ITS2 and psbA-trnH regions indicate 100% identification efficiency. Our research shows that DNA barcoding can effectively distinguish Rhizoma Menispermi from its adulterants from both commercial and original samples, which provides a new and reliable way to monitor commercial herbs and to manage the modern medicine market.

Share and Cite:

Yang, P. , Li, X. , Zhou, H. , Hu, H. , Zhang, H. , Sun, W. , Wang, Y. and Yao, H. (2014) Molecular Identification of Chinese Materia Medica and Its Adulterants Using ITS2 and psbA-trnH Barcodes: A Case Study on Rhizoma Menispermi. Chinese Medicine, 5, 190-198. doi: 10.4236/cm.2014.54023.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Shan, B.E., Ren, F.Z., Liang, W.J., Zhang, J., Li, Q.X. and Zeng, Y.P. (2006) Isolation and Purification of Antitumor Component in Rhizoma Menispermi and Analysis of Its Activity. Carcinogenesis, Teratogenesis & Mutagenesis, 3, No. 007.
[2] Zhao, B., Chen, Y., Sun, X., Zhou, M., Ding, J., Zhan, J.J. and Guo, L.J. (2012) Phenolic Alkaloids from Menispermum dauricum Rhizome Protect against Brain Ischemia Injury via Regulation of GLT-1, EAAC1 and ROS Generation. Molecules, 17, 2725-2737.
[3] Lin, M., Xia, B., Yang, M., Gao, S., Huo, Y. and Lou, G. (2013) Characterization and Antitumor Activities of a Polysaccharide from the Rhizoma of Menispermum dauricum. International Journal of Biological Macromolecules, 53, 7276.
[4] Hoang, M.L., Chen, C.H., Sidorenko, V.S., He, J., Dickman, K.G., Yun, B.H., Moriya, M., Niknafs, N., Douville, C., Karchin, R., Turesky, R.J., Pu, Y.S., Vogelstein, B. and Papadopoulos, N. (2013) Mutational Signature of Aristolochic Acid Exposure as Revealed by Whole-Exome Sequencing. Science Translational Medicine, 5, 197ra102.
[5] Chen, H. (2010) The Difference and the Reasonable Application of Menispermum dauricum and Sophorae tonkinensis. Chinese Medicine Modern Distance Education of China, 8, 60-61.
[6] Miller, S.E. (2007) DNA Barcoding and the Renaissance of Taxonomy. Proceedings of the National Academy of Sciences of the United States of America, 104, 4775-4776.
[7] Chen, S.L., Yao, H., Han, J.P., Liu, C., Song, J.Y., Shi, L.C., Zhu, Y.J., Ma, X.Y., Gao, T., Pang, X.H., Luo, K., Li, Y., Li, X.W., Jia, X.C., Lin, Y.L. and Leon, C. (2010) Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species. PLoS One, 5, e8613.
[8] Yao, H., Song, J., Liu, C., Luo, K., Han, J.P., Li, Y., Pang, X.H., Xu, H.X., Zhu, Y.J., Xiao, P.G. and Chen, S.L. (2010) Use of ITS2 Region as the Universal DNA Barcode for Plants and Animals. PLoS One, 5, 13102.
[9] Li, D.Z., Gao, L.M., Li, H.T., Wang, H., Ge, X.J., Liu, J.Q., Chen, Z.D., Zhou, S.L., Chen, S.L., Yang, J.B., Fu, C.X., Zeng, C.X., Yan, H.F., Zhu, Y.J., Sun, Y.S., Chen, S.Y., Zhao, L., Wang, K., Yang, T. and Duan, G.W. (2011) Comparative Analysis of a Large Dataset Indicates That Internal Transcribed Spacer (ITS) Should Be Incorporated into the Core Barcode for Seed Plants. Proceedings of the National Academy of Sciences of the United States of America, 108, 19641-19646.
[10] Kress, W.J., Wurdack, K.J., Zimmer, E.A., Weigt, L.A. and Janzen, D.H. (2005) Use of DNA Barcodes to Identify Flowering Plants. Proceedings of the National Academy of Sciences of the United States of America, 102, 8369-8374.
[11] Shaw, J., Lickey, E.B., Schilling, E.E. and Small, R.L. (2007) Comparison of Whole Chloroplast Genome Sequences to Choose Noncoding Regions for Phylogenetic Studies in Angiosperms: The Tortoise and the Hare III. American Journal of Botany, 94, 275-288.
[12] White, T.J. (1990) Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In: PCR Protocols, a Guide to Methods and Applications, 315-322.
[13] Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 28, 2731-2739.
[14] Ross, H.A., Murugan, S. and Li, W.L. (2008) Testing the Reliability of Genetic Methods of Species Identification via Simulation. Systematic Biology, 57, 216-230.
[15] Xin, T.Y., Yao, H., Luo, K., Xiang, L., Ma, X.C., Han, J.P., Lin, Y.L., Song, J.Y. and Chen, S.L. (2012) Stability and Accuracy of the Identification of Notopterygii Rhizoma et Radix Using the ITS/ITS2 Barcodes. Yao Xue Xue Bao, 47, 1098-1105.
[16] Luo, K., Ma, P., Yao, H., Xin, T.Y., Hu, Y., Zhen, S.H., Huang, L.F., Liu, J. and Song, J.Y. (2012) Identification of Gentianae Macrophyllae Radix Using the ITS2 Barcodes. Yao Xue Xue Bao, 47, 1710-1717.
[17] Pang, X.H., Song, J.Y., Xu, H.B. and Yao, H. (2012) Using ITS2 Barcode to Identify Ephedraeherba. China Journal of Chinese Materia Medica, 37, 1118-1121.
[18] Hou, D.Y., Song, J.Y., Shi, L.C., Ma, X.C., Xin, T.Y., Han, J.P., Xiao, W., Sun, Z.Y., Cheng, R.Y. and Yao, H. (2013) Stability and Accuracy Assessment of Identification of Traditional Chinese Materia Medica Using DNA Barcoding: A Case Study on Flos Lonicerae Japonicae. BioMed Research International, 2013, Article ID: 549037.
[19] Chiou, S.J., Yen, J.H., Fang, C.L., Chen, H.L. and Lin, T.Y. (2007) Authentication of Medicinal Herbs Using PCRAmplified ITS2 with Specific Primers. Planta Medica, 73, 1421-1426.
[20] Li, J.L., Wang, S., Yu, J., Wang, L. and Zhou, S.L. (2013) A Modified CTAB Protocol for Plant DNA Extraction. Chinese Bulletin of Botany, 48, 72-78.
[21] Xu, X.L., Shi, L.C., Song, J.Y., Han, J.P., Yao, H., Chen, S.L., et al. (2012) Molecular Identification of Sophorae Tonkinensis Radix et Rhizoma Original Plant and Its Adulterants Based on ITS2 DNA Barcode. World Science and Technology/Modernization of Traditional Chinese Medicine and Materia Medica, 14, 1147-1152.
[22] Yancy, H.F., Zemlak, T.S., Mason, J.A., Washington, J.D., Tenge, B.J. and Nguen, N.T. (2008) Potential Use of DNA Barcodes in Regulatory Science: Applications of the Regulatory Fish Encyclopedia. Journal of Food Protection, 71, 210-217.
[23] Kool, A., Boer, H.J., Kruger, A., Rydberg, A., Abbad, A., Bjork, L. and Martin, G. (2012) Molecular Identification of Commercialized Medicinal Plants in Southern Morocco. PLoS One, 7, e39459.
[24] Newmaster, S.G., Grguric, M., Shanmughanandhan, D., Ramalingam, S. and Ragupathy, S. (2013) DNA Barcoding Detects Contamination and Substitution in North American Herbal Products. BMC Medicine, 11, 222.
[25] Chase, M.W., Cowan, R.S., Hollingsworth, P.M., Berg van den, C., Madrinan, S., Petersen, G. and Seberg, O. (2007) A Proposal for a Standardised Protocol to Barcode All Land Plants. Taxon, 56, 295-299.
[26] Song, J.Y., Shi, L.C., Li, D.Z., Sun, Y.Z., Niu, Y.Y., Chen, Z.D., Luo, H.M., Pang, X.H., Sun, Z.Y., Liu, C., Lv, A.P., Deng, Y.P., Larson-Rabin, Z., Wilkinson, M. and Chen, S.L. (2012) Extensive Pyrosequencing Reveals Frequent IntraGenomic Variations of Internal Transcribed Spacer Regions of Nuclear Ribosomal DNA. PLoS One, 7, e43971.
[27] Chen, X.C., Liao, B.S., Song, J.Y., Pang, X.H., Han, J.P. and Chen, S.L. (2013) A Fast SNP Identification and Analysis of Intraspecific Variation in the Medicinal Panax Species Based on DNA Barcoding. Gene, 530, 39-43.

Copyright © 2021 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.