目的 采用半定量分析方法对制首乌与9个补虚药配伍后成分含量及DPPH自由基清除能力变化进行研究。方法 以UPLC-DAD建立制首乌多成分半定量分析方法，对制首乌与9味常用补虚药（当归、熟地黄、白芍、党参、黄芪、甘草、麦冬、枸杞子、墨旱莲）配伍后成分含量变化进行分析。采用DPPH法分别测定单味药以及配伍药对的自由基清除能力，绘制量-效曲线并计算半数清除浓度（EC₅₀）。采用多元统计分析方法建立制首乌中多成分含量与DPPH自由基清除能力间的量-效回归模型，筛选量-效关系中影响显著的化学标志物，并通过质谱进行定性分析。结果 线性范围、准确度、精密度、重复性及稳定性5项方法学验证结果表明，半定量分析方法可用于制首乌中12个成分在配伍前后的含量对比分析。含量对比分析结果表明，制首乌与不同药物配伍后，12个成分的含量均发生了不同程度的变化，且与墨旱莲配伍后制首乌中有33%的成分含量显著降低（P<0.05）、42%的成分含量显著升高外（P<0.05），与另8味药配伍后制首乌中至少50%的成分含量显著降低（P<0.05）。DPPH自由基清除能力实验结果显示，制首乌DPPH自由基清除能力高于其他9味中药，配伍后9个制首乌药对的DPPH自由基清除能力低于制首乌，但高于相应的配伍药物。量-效回归正交偏最小二乘法（orthogonal projections to latent structures，OPLS）模型中R_X²、R_Y²及Q²值分别为0.841、0.981及0.962，筛选出4个量-效关系化学标志物，分别为反式-2，3，5，4'-四羟基二苯乙烯-2-O-β-D-吡喃葡萄糖苷（trans-THSG）、大黄素甲醚、顺式-2，3，5，4'-四羟基二苯乙烯-2-O-β-D-吡喃葡萄糖苷（cis-THSG）、大黄素-8-O-β-D-吡喃葡萄糖苷（EG）。结论 建立的多成分半定量分析方法可用于何首乌在药对配伍过程中多成分含量变化的对比分析，trans-THSG、大黄素甲醚、cis-THSG、EG是影响制首乌在上述药对中发挥DPPH自由基清除作用的化学标志物，可为制首乌药对配伍机制的深入研究提供参考。

Objective To study the changes of ingredients content and DPPH free radical scavenging activity of Polygoni Multiflori Radix Praeparata (PMRP) combined with nine tonic herbs by semi-quantitative analysis. Method The semi-quantitative analysis method of multiple components was established by UPLC-DAD, and the method was used to analyze the changes of components of PMRP while it was compatible with nine tonic herbs[Danggui (Angelicae Sinensis Radix), Shudihuang (Rehmanniae Radix Praeparata), Baishao (Paeoniae Radix Alba), Dangshen (Codonopsis Radix), Huangqi (Astragali Radix), Gancao (Glycyrrhizae Radix et Rhizoma), Maidong (Ophiopogonis Radix), Gouqizi (Lycii Fructus), and Mohanlian (Ecliptae Herba)]. DPPH method was used to determine the free radical scavenging activity of single drug and herb pair. The dose-effect curves were drawn and the half scavenging concentration (EC₅₀) of each sample was calculated. Then, a quantity-effect regression model was established by multivariate statistical analysis. The chemical markers of quantity-effect relationship were screened in the model and qualitatively analyzed by mass spectrometry. Result The results of five validation methods, namely linearity and range, accuracy, precision, repeatability and stability, showed that the proposed semi-quantitative analysis method was suitable for the quantitative analysis of 12 components in PMRP. The results of content analysis showed that the content of 12 components changed in varying degrees. The content of 33% of 12 components in PMRP was decreased significantly (P < 0.05) and the content of 42% of 12 components was increased significantly (P < 0.05) after being combined with Ecliptae Herba. In combination with eight other medicines, at least 50% of 12 components were significantly lower (P < 0.05) than in PMRP. The results of antioxidant test showed that the DPPH free radical scavenging ability of PMRP was higher than that of other nine medicines, DPPH free radical scavenging ability of 9 herb pairs was lower than that of PMRP, but higher than that of nine corresponding tonic herbs. A quantity-effect OPLS regression model was established, the value of R_X², R_Y² and Q² of the model was 0.841, 0.981 and 0.962, respectively. Four chemical markers of quantity-effect relationship were selected and were qualitatively analyzed, namely trans-2,3,5,4'-tetrahydroxy styrene-2-O-β-D-glucoside, physcion (trans-THSG), cis-2,3,5,4'-tetrahydroxy styrene-2-O-β-D-glucoside (cis-THSG), emodin-8-O-β-D-glucoside (EG). Conclusion The semi-quantitative analysis method established in this paper can be used to study the changes of ingredients content of PMP while it was compatible with nine tonic herbs. And trans-THSG, physcion, cis-THSG, EG were the chemical markers of quantity and DPPH free radical scavenging activity of PMP in 9 herb pairs.

R286.02

国家重点研发计划项目（2017YFC1703400）；天津市科技计划项目（17ZXMFSY00030）；天津市卫生健康委员会、天津市中医药管理局中医中西医结合科研课题青年项目（2019025）；天津市中医药管理局中医中西医结合科研课题面上项目（13024）；天津市卫生健康委员会中医药重点领域科研项目（2019002）