目的 采用指纹图谱技术结合化学计量学分析方法，探究不同发酵程度建曲中化学成分的差异性，筛选差异性标志物，为规范建曲炮制工艺、建立饮片质量标准提供参考。方法 采用Waters SunFire C₁₈（250 mm×4.6 mm，5 μm）色谱柱，以乙腈-0.1%乙酸水溶液为流动相梯度洗脱，体积流量1.0 mL/min，检测波长280 nm，柱温30℃，进样体积10 μL，建立不同发酵时间建曲HPLC指纹图谱，采用中药色谱指纹图谱相似度评价软件（2004A版）进行相似度评价，标定共有峰并进行指认及归属；以共有峰峰面积为指标，利用层次聚类分析法（hierarchical cluster analysis，HCA）对不同发酵时间建曲进行区分，再借助正交偏最小二乘-判别分析法（orthogonal partial least square-discriminant analysis，OPLS-DA）筛选出不同发酵程度建曲间差异性标志物；建立多指标成分含量测定方法，对多个差异性标志物进行定量分析。结果 不同发酵时间建曲指纹图谱相似度大于0.900，共标定46个共有峰，指认并归属21个成分；HCA结果显示9个不同发酵时间建曲可聚类为4个发酵阶段；OPLS-DA结果表明不同发酵阶段建曲间差异性标志物略有不同，芹菜素、新橙皮苷、峰28、柚皮苷、橙皮苷和木犀草素为不同发酵程度建曲的主要差异性标志物；对5个已知差异性标志物进行定量研究，发酵过程中，柚皮苷、新橙皮苷质量分数呈下降趋势，分别从0.048 5%、0.046 4%下降至0.014 8%、0.001 8%，芹菜素、木犀草素质量分数呈上升趋势，由0.007 8%、0.029 3%上升至0.019 8%、0.085 2%，橙皮苷质量分数在0.307 9%～0.341 6%波动；以5个差异性标志物含量为指标，进行热图聚类分析，结果与HCA结果一致。结论 建立的方法可有效区分不同发酵时间建曲，为建曲的质量控制及评价提供参考。

Objective Fingerprint technology combined with chemometrics analysis method was used to study the changes of chemical components and screen the differential markers in Jianqu with different degrees of fermentation, so as to lay a foundation for standardizing processing technology and establishing the quality standard of Jianqu. Methods The fingerprint of jianqu fermented in different times was established by a Waters SunFire C₁₈ column(250 mm×4.6 mm, 5 μm)by gradient elution, and the mobile phase consisted of acetonitrile and 0.1% acetic acid solution, the flow rate was 1.0 mL/min, the wavelength was 280 nm and the column temperature was 30℃. Similarity evaluation was carried out using the raditional Chinese medicine chromatographic fingerprint similarity evaluation system (2004A version), the common peaks identification and attribution were carried out. The common peaks area was used as indicators, and hierarchical cluster analysis (HCA) was used to distinguish Jianqu fermented in different times, and then orthogonal partial least square discriminant analysis (OPLS-DA) was used to screen the differential markers in Jianqu fermentation. The content of differential markers was determined by HPLC. Results The similarity of fingerprint of Jianqu fermented in different times was greater than 0.900, 46 common peaks were calibrated, and 21 components were identified and attributed; HCA clustered Jianqu fermented in nine different times into Jianqu fermented in four different stages. The results of OPLS-DA showed that there were slight differences in the differential markers between Jianqu fermented in different stages; Apigenin, neohesperidin, peak 28, naringin, hesperidin and luteolin were the main differential markers in Jianqu with different degrees of fermentation; The contents of 5 differential markers were determined, and the contents of naringin and neohesperidin showed a decreasing trend from 0.048 5% and 0.046 4% to 0.014 8% and 0.001 8%, respectively; the contents of apigenin and luteolin increased from 0.007 8% and 0.029 3% to 0.019 8% and 0.085 2%, respectively; The content of hesperidin fluctuated between 0.307 9% and 0.341 6%. The content of five differential markers was used as indicators to conduct heat map cluster analysis which can also effectively distinguish Jianqu fermented in different times. Conclusion The method established in this study can effectively distinguish Jianqu fermented in different times and provide reference for quality control and evaluation of Jianqu.

R283.6

中药建曲发酵工艺优化及质量标准提升研究(22B360004);河南中医药大学博士基金项目(RSBSJJ2019-09)