Zhou Qian-Ru, Ma Yun-Ying, Lv Hu-Qiang, Lu Zhao-Cen, Wang Li-Sheng, Liang Jun-Song, Li Jing-Jian
Guangxi Key Laboratory of Drug Discovery and Optimization, School of Pharmacy, Guilin Medical University, Guilin, 541004, China.
College of Pharmacy, Guilin Medical University, Guilin, 541004, China.
Heliyon. 2024 Sep 27;10(19):e38700. doi: 10.1016/j.heliyon.2024.e38700. eCollection 2024 Oct 15.
Difengpi, derived from the air-dried stem bark of and enlisted in the Chinese Pharmacopoeia for its therapeutic effect against common ailments, confronts challenges due to dwindling wild resources and intentional substitution with potentially harmful botanical relatives. The imperative need to authenticate this herbal remedy has led to the development of robust methods. Here, we integrated chloroplast mini-barcoding and high resolution melting (HRM) analysis to distinguish Difengpi from the reported adulterants. We assembled the complete chloroplast (cp) genomes of and substituted close relatives and , and conducted an in-depth comparative analysis to screen divergent regions for exploiting as DNA mini-barcodes. Despite the conservativeness characterizing the whole cp genomes among these species, we identified some highly variable regions with promising potential as molecular markers for species identification. Subsequently, we designed DNA mini-barcodes and subjected them to HRM analysis to assess their efficacy in species discrimination. Melting profiles unveiled that mini-barcodes designed from four divergence regions -, -, - and - exhibited substantial discriminatory power, distinctly differentiated from , and . We tested ten commercially available Difengpi products from online stores and local traditional markets using these four mini-barcodes. All ten samples clustered closely with the reference with genotype confidence higher than 93 %, indicating the presence of the claimed species in these samples, sans any reported toxic adulterants. Consequently, we simulated Difengpi mimic samples utilizing , and . and subjected them to evaluate the practicability of these mini-barcodes. The outcomes confirmed the precision of the four mini-barcodes in accurately discerning the mimic samples. In conclusion, integrating taxon-specific DNA mini-barcodes with HRM analysis is an efficient strategy for the authentication of species identity within commercial herbal products.
地枫皮来源于干燥的茎皮，被《中国药典》收录用于治疗常见疾病，由于野生资源减少以及有意用可能有害的近缘植物替代，面临诸多挑战。对这种草药进行鉴定的迫切需求促使了可靠方法的发展。在此，我们整合了叶绿体微条形码和高分辨率熔解（HRM）分析来区分地枫皮与已报道的掺假品。我们组装了地枫皮及其替代近缘种和的完整叶绿体（cp）基因组，并进行了深入的比较分析以筛选出可作为DNA微条形码的差异区域。尽管这三个物种的整个cp基因组具有保守性，但我们仍鉴定出一些具有很大潜力作为物种鉴定分子标记的高度可变区域。随后，我们设计了DNA微条形码并对其进行HRM分析以评估它们在物种鉴别中的功效。熔解曲线表明，从四个差异区域——、、和设计的微条形码具有显著的鉴别能力，能明显区分地枫皮与、和。我们使用这四个微条形码测试了从在线商店和当地传统市场购买的十种市售地枫皮产品。所有十个样品都与参考地枫皮紧密聚类，基因型置信度高于93%，表明这些样品中存在所声称的物种，且没有任何报道的有毒掺假品。因此，我们利用和模拟了地枫皮模拟样品，并对其进行评估以检验这些微条形码的实用性。结果证实了这四个微条形码在准确辨别模拟样品方面的准确性。总之，将特定分类群的DNA微条形码与HRM分析相结合是鉴定商业草药产品中物种身份的有效策略。
