藏药铁棒锤DNA条形码、指纹图谱及主要成分含量测定研究

易国旗; 李梦佳; 孟明坤; 张艺; 刘悦

doi:10.13748/j.cnki.issn1007-7693.20242548

藏药铁棒锤DNA条形码、指纹图谱及主要成分含量测定研究

DNA Barcoding, Fingerprinting and Main Components Quantification of Tibetan Medicine “Tiebangchui”

摘要

摘要:
目的对多基原藏药铁棒锤进行DNA条形码分子鉴定，并通过HPLC指纹图谱结合化学模式识别以及主要成分定量对不同基原的铁棒锤进行质量评价。
方法采用DNA条形码对藏药铁棒锤2个基原，即铁棒锤Aconitum pendulum Busch和伏毛铁棒锤Aconitum flavum Hand-Mazz.进行分子鉴定研究。采用ZORBAX Eclipse XDB-C₁₈色谱柱，0.01%冰醋酸(三乙胺调pH 6.5)(A)-乙腈(B)为流动相，梯度洗脱；检测波长235 nm；流速1.0 mL·min⁻¹；进样量10 μL；柱温30 ºC进行指纹图谱研究。采用“中药色谱指纹图谱相似度评价系统(2012版)”进行图谱分析，对共有峰进行主成分分析(principal component analysis，PCA)和正交偏最小二乘法-判别分析(orthogonal partial least squares discriminant analysis，OPLS-DA)，并对其主要成分进行含量测定。
结果 DNA条形码分子鉴定能够通过ITS2序列中的变异位点、遗传距离以及系统发育邻接树是否聚为一支实现多基原藏药铁棒锤准确鉴定。20批藏药铁棒锤样品指纹图谱中共对比出15个共有峰。通过对照品比对指认了其中3个共有成分，并建立了其含量测定方法。指纹图谱相似度范围为0.853~0.997。PCA得到5个主成分的累积方差贡献率为83.931%；OPLS-DA结果表明藏药铁棒锤按基原可分为2类，并筛选出7个差异标志物。指认出的苯甲酰乌头原碱、乌头碱、3-脱氧乌头碱的质量分数为0.1068~1.1269、1.8983~28.4839、0.2763~7.2596 mg·g⁻¹。
结论本研究建立的方法稳定可行，可为藏药铁棒锤基原鉴定以及质量控制提供参考。

Abstract:
OBJECTIVE To conduct molecular identification of DNA barcoding for the polybasic proto-Tibetan medicine “Tiebangchui” and perform quality evaluation of its different origins through the combination of HPLC fingerprint and chemical pattern recognitions, along with the quantification of main components.
METHODS DNA barcoding was used to study the molecular identification of 2 basigens of the Tibetan medicine “Tiebangchui”, namely Aconitum pendulum Busch and Aconitum flavum Hand-Mazz.. HPLC fingerprinting was performed on a ZORBAX Eclipse XDB-C₁₈ column with 0.01% acetic acid(pH=6.5 adjusted by triethylamine)(A)-acetonitrile(B) as the mobile phase in a gradient elution with the detection wavelength of 235 nm, the flow rate was 1.0 mL·min⁻¹, the injection was 10 μL, and the column temperature was 30 ºC. Fingerprint were analyzed using the “Similarity Evaluation System for Traditional Chinese Medicine Chromatography Fingerprint(2012Edition)”. Based on the fingerprint, principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were performed, and the contents of the main components were quantified.
RESULTS The molecular identification of DNA barcodes could be based on the variant sites in the ITS2 sequences, genetic distances, and whether the phylogenetic neighbour joining tree was clustered into a single unit as the basis for the identification of the multibasic original Tibetan medicines of A. pendulum and A. flavum. A HPLC fingerprint overlay map was established for 20 batches of “Tiebangchui” samples, and 15 common peaks were identified. Three chromatographic peaks were identified through comparison with the control samples, and the quantification method was established. The similarity of the fingerprint spectrum was 0.853–0.997. Five principal component factors were determined by PCA, and the cumulative variance contribution rate was 83.931%, and OPLS-DA results indicated that A. pendulum and A. flavum could be classified into 2 categories, and a total of 7 differential markers were screened. The contents of the 3 recognized components benzoyl aconitine, aconitine and 3-deoxyaconitine were 0.1068–1.1269, 1.8983–28.4839, and 0.2763–7.2596 mg·g⁻¹, respectively.
CONCLUSION The method established in this study is stable and feasible, and can provide a reference for the identification and quality control of Tibetan medicine “Tiebangchui”.

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