目的 基于ITS序列进行分子鉴定，采用指纹图谱、重金属元素定量与化学计量法相结合的方法，评价不同产地忍冬茶藨子叶状层菌Phylloporia lonicerae的质量，为其进一步开发利用提供依据。方法 分别提取采自4个不同产地的忍冬茶藨子叶状层菌的基因组DNA，扩增ITS序列后测序，并构建系统发育树；采用HPLC法，流动相为0.1%冰乙酸-乙腈进行梯度洗脱，柱温30 ℃，体积流量0.8 mL/min，检测波长280 nm，对12批忍冬茶藨子叶状层菌建立指纹图谱及铜、砷、镉、汞、铅5种重金属元素含量测定，运用聚类分析（hierarchical cluster analysis，HCA）、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）对忍冬茶藨子叶状层菌进行化学计量分析。结果 构建了基于ITS序列的系统发育树，12批忍冬茶藨子叶状层菌属于同一个种；建立了12批忍冬茶藨子叶状层菌的指纹图谱，相似度为0.829～0.992，共标定15个共有峰，指认了5个成分，ICP-MS测定结果表明不同产地忍冬茶藨子叶状层菌中重金属元素含量存在明显差异；HCA分析12批忍冬茶藨子叶状层菌明显分为2类；PCA得到4个主成分的累积方差贡献率为86.751%；OPLS-DA表明铜、砷可作为忍冬茶藨子叶状层菌的特征元素。忍冬茶藨子叶状层菌中共有峰2、3和13（原儿茶醛）可能是影响药材质量的差异标志物。结论 建立的忍冬茶藨子叶状层菌HPLC指纹图谱及5个重金属元素含量测定方法的专属性强，且准确、可靠，结合化学计量法可用于忍冬茶藨子叶状层菌的药材鉴别和质量控制。

Objective Based on the method of ITS sequences, HPLC fingerprint, heavy metals quantitation and chemometric analysis, the quality of Phylloporia lonicerae from different regions was evaluated to provide a basis for its further development and utilization.Methods Genomic DNA was extracted from samples collected in four different regions, and ITS sequences were amplified and sequenced to construct a phylogenetic tree. HPLC method was used with 0.1% glacial acetic acid-acetonitrile as mobile phase for gradient elution. The column temperature was 30 ℃, the flow rate was 0.8 mL/min, and the detection wavelength was 280 nm. The fingerprint of 12 batches of P. lonicerae was established and the contents of five heavy metal elements including copper, arsenic, cadmium, mercury and lead were determined. Chemometrics analysis of P. lonicerae was performed by hierarchical cluster analysis (HCA), principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). Results The phylogenetic tree based on ITS sequence was constructed, and the 12 batches of P. lonicerae were belong to the same species. The fingerprints of 12 batches of P. lonicerae were established. The similarity was 0.83—0.99, and a total of 15 common peaks were calibrated, and five components were identified. The results of ICP-MS determination showed that there were significant differences in the content of heavy metal elements in P. lonicerae from different regions. HCA analysis showed that 12 batches of P. lonicerae were obviously divided into two categories. PCA obtained a cumulative variance contribution rate of 86.751% for four principal components. OPLS-DA showed that copper and arsenic could be used as characteristic elements of P. lonicera. The common peaks 2, 3 and 13 (protocatechuic aldehyde) in P. lonicerae may be the difference markers that affect the quality of medicinal materials.Conclusion The established HPLC fingerprint of P. lonicerae and the determination method of five heavy metal elements content have strong specificity, and are accurate and reliable. Combined with chemometric method, it can be used for the identification and quality control of P. lonicerae.

R286.2

国家重点研发计划项目（2019YFC1711200）；山东省重点研发计划项目（2021CXGC010508）