目的 基于ITS序列進(jìn)行分子鑒定，采用指紋圖譜、重金屬元素定量與化學(xué)計(jì)量法相結(jié)合的方法，評(píng)價(jià)不同產(chǎn)地忍冬茶藨子葉狀層菌Phylloporia lonicerae的質(zhì)量，為其進(jìn)一步開發(fā)利用提供依據(jù)。方法 分別提取采自4個(gè)不同產(chǎn)地的忍冬茶藨子葉狀層菌的基因組DNA，擴(kuò)增ITS序列后測(cè)序，并構(gòu)建系統(tǒng)發(fā)育樹；采用HPLC法，流動(dòng)相為0.1%冰乙酸-乙腈進(jìn)行梯度洗脫，柱溫30 ℃，體積流量0.8 mL/min，檢測(cè)波長(zhǎng)280 nm，對(duì)12批忍冬茶藨子葉狀層菌建立指紋圖譜及銅、砷、鎘、汞、鉛5種重金屬元素含量測(cè)定，運(yùn)用聚類分析（hierarchical cluster analysis，HCA）、主成分分析（principal component analysis，PCA）和正交偏最小二乘法-判別分析（orthogonal partial least squares-discriminant analysis，OPLS-DA）對(duì)忍冬茶藨子葉狀層菌進(jìn)行化學(xué)計(jì)量分析。結(jié)果 構(gòu)建了基于ITS序列的系統(tǒng)發(fā)育樹，12批忍冬茶藨子葉狀層菌屬于同一個(gè)種；建立了12批忍冬茶藨子葉狀層菌的指紋圖譜，相似度為0.829～0.992，共標(biāo)定15個(gè)共有峰，指認(rèn)了5個(gè)成分，ICP-MS測(cè)定結(jié)果表明不同產(chǎn)地忍冬茶藨子葉狀層菌中重金屬元素含量存在明顯差異；HCA分析12批忍冬茶藨子葉狀層菌明顯分為2類；PCA得到4個(gè)主成分的累積方差貢獻(xiàn)率為86.751%；OPLS-DA表明銅、砷可作為忍冬茶藨子葉狀層菌的特征元素。忍冬茶藨子葉狀層菌中共有峰2、3和13（原兒茶醛）可能是影響藥材質(zhì)量的差異標(biāo)志物。結(jié)論 建立的忍冬茶藨子葉狀層菌HPLC指紋圖譜及5個(gè)重金屬元素含量測(cè)定方法的專屬性強(qiáng)，且準(zhǔn)確、可靠，結(jié)合化學(xué)計(jì)量法可用于忍冬茶藨子葉狀層菌的藥材鑒別和質(zhì)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

國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目（2019YFC1711200）；山東省重點(diǎn)研發(fā)計(jì)劃項(xiàng)目（2021CXGC010508）