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

doi:10.15302/J-FASE-2017157

Front. Agr. Sci. Eng.

2017, Vol. 4

Issue (3) : 353-357 https://doi.org/10.15302/J-FASE-2017157

RESEARCH ARTICLE

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

Jikun WANG¹, Jing DU², Meng CAO¹, Lu YAO², Suhua XIE², Jiafu CHEN², Xingbo ZHAO¹(

)

¹. National Engineering Laboratory for Animal Breeding; Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture; College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
². Research Institute of Beijing Tongrentang Co., Ltd., Beijing 100079, China

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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

Corresponding Author(s): Xingbo ZHAO

Just Accepted Date: 10 May 2017 Online First Date: 05 June 2017 Issue Date: 12 September 2017

Cite this article:

Jikun WANG,Jing DU,Meng CAO, et al. Molecular authentication of the traditional Chinese medicine Tongren Dahuoluo Wan and its alternative formulation[J]. Front. Agr. Sci. Eng. , 2017, 4(3): 353-357.

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https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2017157
https://academic.hep.com.cn/fase/EN/Y2017/V4/I3/353

Fig.1 Gel electrophoresis of DNA extracted from Tongren Dahuoluo Wan pills and animal bones. M, 100 bp DNA Ladder marker; 1, Tongren Dahuoluo Wan (original); 2, Tongren Dahuoluo Wan (alternative); 3, zokor bone; 4, leopard bone.

Fig.2 PCR identification of patent medicines of Tongren Dahuoluo Wan original and alternative pills using species-specific mtDNA primers. 1, Tongren Dahuoluo Wan (original with leopard); 2, internal positive control (leopard); 3, Tongren Dahuoluo Wan (alternative with zokor); 4, internal positive control (zokor); 5, leopard bone; 6, zokor bone; 7, blank control. L1–L5, specific primers for leopard; Z1–Z4, specific primers for zokor.

Fig.3 Phylogenetic clustering of the leopard and zokor medicines using the COI sequences. Red circles represent the Eospalax baileyi individuals, the blue triangles represented those from Panthera pardus, and the solid node represents the medicine sequences. The numbers at each node represent the posterior probability support values.

Fig.4 Strategy for molecular authentication of animal products used in Chinese traditional medicines

1	Ping H. Experimental study on anti-inflammatory effect of Sailong Bone. Liaoning Journal of Traditional Chinese Medicine, 2000, 27(11): 524–526 (in Chinese)
2	Zhai J Y, Chen X, Jin R, Yue F, Liu D, Li J S. Effect of Zaizao Wan (Sailong bone replacement of leopard bone) against cerebral ischemia. Chinese Journal of Experimental Traditional Medical Formulae, 2016, 22(6): 124–129 (in Chinese)
3	Lee D G, Kang H W, Park C G, Ahn Y S, Shin Y. Isolation and identification of phytochemicals and biological activities of Hericium ernaceus and their contents in Hericium strains using HPLC/UV analysis. Journal of Ethnopharmacology, 2016, 184: 219–225 https://doi.org/10.1016/j.jep.2016.02.038 pmid: 26924563
4	Shellie R A, Marriott P J, Huie C W. Comprehensive two-dimensional gas chromatography (GC×GC) and GC×GC-quadrupole MS analysis of Asian and American ginseng. Journal of Separation Science, 2003, 26(12–13): 1185–1192 https://doi.org/10.1002/jssc.200301404
5	Puchert T, Lochmann D, Menezes J C, Reich G. Near-infrared chemical imaging (NIR-CI) for counterfeit drug identification—a four-stage concept with a novel approach of data processing (Linear Image Signature). Journal of Pharmaceutical and Biomedical Analysis, 2010, 51(1): 138–145 https://doi.org/10.1016/j.jpba.2009.08.022 pmid: 19766424
6	Cao M, Wang J, Yao L, Xie S, Du J, Zhao X. Authentication of animal signatures in traditional Chinese medicine of Lingyang Qingfei Wan using routine molecular diagnostic assays. Molecular Biology Reports, 2014, 41(4): 2485–2491 https://doi.org/10.1007/s11033-014-3105-x pmid: 24445529
7	Li M, Au K Y, Lam H, Cheng L, Jiang R W, But P P H, Shaw P C. Identification of Baiying (Herba Solani Lyrati) commodity and its toxic substitute Xungufeng (Herba Aristolochiae Mollissimae) using DNA barcoding and chemical profiling techniques. Food Chemistry, 2012, 135(3): 1653–1658 https://doi.org/10.1016/j.foodchem.2012.06.049 pmid: 22953906
8	Xiang H, Gao J, Yu B, Zhou H, Cai D, Zhang Y, Chen X, Wang X, Hofreiter M, Zhao X. Early Holocene chicken domestication in northern China. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(49): 17564–17569 https://doi.org/10.1073/pnas.1411882111 pmid: 25422439
9	Kalmár T, Bachrati C Z, Marcsik A, Raskó I. A simple and efficient method for PCR amplifiable DNA extraction from ancient bones. Nucleic Acids Research, 2000, 28(12): E67 https://doi.org/10.1093/nar/28.12.e67 pmid: 10871390
10	Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 2013, 30(12): 2725–2729 https://doi.org/10.1093/molbev/mst197 pmid: 24132122
11	Linacre A. The use of DNA from non-human sources. Forensic Science International: Genetics Supplement Series, 2008, 1(1): 605–606 https://doi.org/10.1016/j.fsigss.2007.10.108
12	Whiting M, Williams V, Hibbitts T. In: Alves R R N, Rosa I L, eds. Animals in traditional folk medicine. Springer, 2013, 421–473
13	Coghlan M L, Haile J, Houston J, Murray D C, White N E, Moolhuijzen P, Bellgard M I, Bunce M. Deep sequencing of plant and animal DNA contained within traditional Chinese medicines reveals legality issues and health safety concerns. PLoS Genetics, 2012, 8(4): e1002657 https://doi.org/10.1371/journal.pgen.1002657 pmid: 22511890
14	Rohland N, Hofreiter M. Comparison and optimization of ancient DNA extraction. BioTechniques, 2007, 42(3): 343–352 https://doi.org/10.2144/000112383 pmid: 17390541
15	Leonard J A, Wayne R K, Cooper A. Population genetics of ice age brown bears. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(4): 1651–1654 https://doi.org/10.1073/pnas.040453097 pmid: 10677513
16	Hofreiter M, Rabeder G, Jaenicke-Despr�s V, Withalm G, Nagel D, Paunovic M, Jambrĕsić G, Pääbo S. Evidence for reproductive isolation between cave bear populations. Current Biology, 2004, 14(1): 40–43 https://doi.org/10.1016/j.cub.2003.12.035 pmid: 14711412
17	Rådström P, Knutsson R, Wolffs P, Lövenklev M, Löfström C. Pre-PCR processing: strategies to generate PCR-compatible samples. Molecular Biotechnology, 2004, 26(2): 133–146 https://doi.org/10.1385/MB:26:2:133 pmid: 14764939
18	Cheng K T, Tsay H S, Chen C F, Chou T W. Determination of the components in a Chinese prescription, yu-ping-feng san, by RAPD analysis. Planta Medica, 1998, 64(6): 563–565 https://doi.org/10.1055/s-2006-957515 pmid: 9776663
19	Chang S, Kalok W, But P, Su W W, Pangchui S. Molecular authentication of the Chinese herb Huajuhong and related medicinal material by DNA sequencing and ISSR markers. Journal of Food and Drug Analysis, 2010, 18(18): 161–170
20	Reunova G D, Kats I L, Muzarok T I, Zhuravlev IuN. Polymorphism of RAPD, ISSR and AFLP markers of the Panax ginseng C. A. Meyer (Araliaceae) genome. Russian Journal of Genetics, 2010, 46(8): 1057–1066 https://doi.org/10.1134/S1022795410080053 pmid: 20873202
21	Lin T C, Yeh M S, Cheng Y M, Lin L C, Sung J M. Using ITS2 PCR-RFLP to generate molecular markers for authentication of Sophora flavescens Ait. Journal of the Science of Food and Agriculture, 2012, 92(4): 892–898 https://doi.org/10.1002/jsfa.4667 pmid: 22413146
22	Kim J, Jo B H, Lee K L, Yoon E S, Ryu G H, Chung K W. Identification of new microsatellite markers in Panax ginseng. Molecules and Cells, 2007, 24(1): 60–68 pmid: 17846499
23	Hayashi K, Hashimoto N, Daigen M, Ashikawa I. Development of PCR-based SNP markers for rice blast resistance genes at the Piz locus. Theoretical and Applied Genetics, 2004, 108(7): 1212–1220 https://doi.org/10.1007/s00122-003-1553-0 pmid: 14740086
24	Cheng X, Su X, Chen X, Zhao H, Bo C, Xu J, Bai H, Ning K. Biological ingredient analysis of traditional Chinese medicine preparation based on high-throughput sequencing: the story for Liuwei Dihuang Wan. Scientific Reports, 2014, 4(1): 5147 https://doi.org/10.1038/srep05147 pmid: 24888649

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