目的 建立HPLC指纹图谱，区分白头翁Pulsatilla chinensis与其常见混伪品；建立正品白头翁HPLC指纹图谱和抗氧化活性间的谱效关系，辨识并验证白头翁潜在抗氧化质量标志物（quality markers，Q-Marker）。方法 分别建立18批正品白头翁及其常见混伪品的HPLC指纹图谱，通过相似度计算区分正品及其混伪品；采用UPLC-QTOF-MS/MS技术表征正品白头翁化学成分；建立正品白头翁HPLC指纹图谱和清除1,1-二苯基-2-苦肼自由基（1,1-diphenyl-2-picrylhydrazyl，DPPH）间的谱效关系模型，筛选出潜在的抗氧化Q-Marker，测定含量；以脂多糖促RAW264.7细胞氧化应激损伤模型，评估潜在Q-Marker的生物效应。结果 正品白头翁与其常见混伪品指纹图谱差异明显，相似度为0.667～0.321；18批正品白头翁的HPLC指纹图谱，相似度为0.994～0.959，共有峰21个；经质谱表征解析了正品白头翁中的21个化学成分；药效学实验表明各组正品白头翁均具有良好的抗氧化能力，谱效相关性分析结果显示17号和21号峰与抗氧化活性关系密切；经对照品比对确定17号和21号峰分别为白头翁皂苷B4和白头翁皂苷D，质量分数分别为15.99%～8.86%和2.55%～0.97%；抗氧化活性验证结果表明，白头翁皂苷B4和白头翁皂苷D显著降低脂多糖诱导的RAW264.7 细胞NO和ROS水平（P＜0.001），且二者配伍时不同程度的优于单一成分。结论 所建立的HPLC指纹图谱方法能够准确鉴定正品白头翁药材，整合药效学实验，能够科学辨识白头翁Q-Marker用于白头翁的质量控制。建立了一套系统的评价白头翁质量的方法，为有效成分含量较低、难于质控的中药材的品质评价提供参考。

Objective The HPLC fingerprint was established to distinguish Baitouweng (Pulsatillae Radix) from its common adulterants. The spectrum-effect relationship between the HPLC and antioxidant effects was established to identify and validate the antioxidant quality markers of Pulsatillae Radix. Methods The HPLC fingerprints of 18 batches of authentic Pulsatillae Radix and its common adulterants were established, and similarity calculations were conducted to distinguish the genuine samples and its adulterants. The chemical composition of Pulsatillae Radix was characterized by the UPLC-QTOF-MS/MS method. A spectrum-effect relationship model between HPLC fingerprints and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assays of authentic Pulsatillae Radix was established to preliminarily screen potential antioxidant quality markers, then the contents of quality markers were determined. Furthermore, a lipopolysaccharides-induced RAW264.7 cell oxidative stress model was utilized to evaluate the biological effects of potential antioxidant quality markers of Pulsatillae Radix. Results The fingerprints of authentic Pulsatillae Radix and its common adulterants were obviously different, and the similarity was between 0.667 and 0.321. Analysis of the HPLC fingerprint of these 18 batches revealed a similarity ranging from 0.994 to 0.959, with a total of 21 common peaks identified. Mass spectrometry characterization and analysis identified 21 chemical constituents among the authentic Pulsatillae Radix samples. Pharmacological experiments demonstrated that all authentic Pulsatillae Radix samples exhibited excellent antioxidant capabilities. The spectrum-effect relationship analysis indicated a close relationship between peaks 17 and 21 with antioxidant activity. Upon comparison with reference standards, peaks 17 and 21 were identified as Pulsatilla saponin B4 and Pulsatilla saponin D, with contents ranging from 15.99% to 8.86% and 2.55% to 0.97%, respectively. Antioxidant activity verification results showed that Pulsatilla saponin B4 and Pulsatilla saponin D significantly reduced the levels of NO and ROS induced by lipopolysaccharides in RAW264.7 cells (P＜0.0001). Furthermore, their combination exhibited varying degrees of superiority over individual components. Conclusion The study of the HPLC fingerprinting method can accurately identify authentic Pulsatillae Radix samples, integrating pharmacological experiments to discern quality markers. The study’s systematic evaluation method can serve as a reference for assessing the quality of low-potency ingredient, difficult-to-control traditional Chinese medicine.

R286.2

黑龙江省自然科学基金联合指导计划（LH2020H069）；哈尔滨商业大学青年学术骨干支持计划（2020CX12）；黑龙江省创新人才青年计划（UNPSYCT-2018134）；黑龙江中医药管理局中医药科研课题（ZHY2024-313）；龙江科技英才春雁支持计划团队项目（2022CYCX0079）