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Molecular authentication of commercial “Qian-hu” through the integration of nrDNA internal transcribed spacer 2 and nucleotide signature

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Abstract

Background

Peucedani Radix, also known as “Qian-hu” is a traditional Chinese medicine derived from Peucedanum praeruptorum Dunn. It is widely utilized for treating wind-heat colds and coughs accompanied by excessive phlegm. However, due to morphological similarities, limited resources, and heightened market demand, numerous substitutes and adulterants of Peucedani Radix have emerged within the herbal medicine market. Moreover, Peucedani Radix is typically dried and sliced for sale, rendering traditional identification methods challenging.

Materials and methods

We initially examined and compared 104 commercial “Qian-hu” samples from various Chinese medicinal markets and 44 species representing genuine, adulterants or substitutes, utilizing the mini barcode ITS2 region to elucidate the botanical origins of the commercial “Qian-hu”. The nucleotide signature specific to Peucedani Radix was subsequently developed by analyzing the polymorphic sites within the aligned ITS2 sequences.

Results

The results demonstrated a success rate of 100% and 93.3% for DNA extraction and PCR amplification, respectively. Forty-five samples were authentic “Qian-hu”, while the remaining samples were all adulterants, originating from nine distinct species. Peucedani Radix, its substitutes, and adulterants were successfully identified based on the neighbor-joining tree. The 24-bp nucleotide signature (5′-ATTGTCGTACGAATCCTCGTCGTC-3′) revealed distinct differences between Peucedani Radix and its common substitutes and adulterants. The newly designed specific primers (PR-F/PR-R) can amplify the nucleotide signature region from commercial samples and processed materials with severe DNA degradation.

Conclusions

We advocate for the utilization of ITS2 and nucleotide signature for the rapid and precise identification of herbal medicines and their adulterants to regulate the Chinese herbal medicine industry.

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Data availability

The sequences of each haplotype in this study have been submitted to GenBank of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov; see Table S1). Voucher samples were deposited in the School of Pharmaceutical Sciences and Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University (Yunnan, China), and detailed information was listed in Table S1.

Abbreviations

ITS2:

Internal transcribed spacer 2

NJ:

Neighbor-joining

CTAB:

Hexadecyltrimethylammonium bromide

HMM:

Hidden Markov model

MEGA:

Molecular evolutionary genetics analysis

NCBI:

National Centre for Biotechnology Information

K2P:

Kimura-2-parameter

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Acknowledgements

We thank two anonymous reviewers for valuable, constructive comments that helped improve the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 31960048), the Ten Thousand Talents Program of Yunnan (YNWRQNBJ-2019-208), the Department of Science and Technology of Yunnan Province (Grant No. 202201AT070118) and Gaoligong Mountain, Forest Ecosystem, Observation and Research Station of Yunnan Province (Grant No. 202205AM070006).

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Authors and Affiliations

  1. School of Pharmaceutical Science and Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University, Kunming, 650500, China

    Junmei Niu, Xinyue Wang, Shilin Zhou, Jiarui Yue & Jing Zhou

  2. Yunnan Academy of Forestry and Grassland, Kunming, 650201, China

    Zhenwen Liu

  3. Yunnan College of Modern Biomedical Industry, Kunming Medical University, Kunming, 650500, China

    Jing Zhou

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  1. Junmei Niu
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Contributions

ZL and JZ conceived and designed the study, as well as revised the manuscript. JN, XW, JY, and SZ collected samples and conducted the experiments. JN analyzed the data and authored the manuscript. All authors have reviewed and approved the final manuscript.

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Correspondence to Zhenwen Liu or Jing Zhou.

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Niu, J., Wang, X., Zhou, S. et al. Molecular authentication of commercial “Qian-hu” through the integration of nrDNA internal transcribed spacer 2 and nucleotide signature. Mol Biol Rep 51, 639 (2024). https://doi.org/10.1007/s11033-024-09557-8

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