目的 建立HPLC-ABTS-DAD-ESI-TOF/MS在线筛选、鉴别柴银颗粒抗氧化活性成分的方法，并探讨抗氧化活性成分指纹图谱联合多成分定量用于柴银颗粒质量评价的可行性。方法 采用Phenomenex Luna C₁₈色谱柱（250 mm×4.6 mm，5.0 μm），以甲醇-0.2%甲酸水溶液为流动相，梯度洗脱，体积流量为0.8 mL/min，柱温为25 ℃，在280 nm下实现柴银颗粒提取物的色谱分析。柱后通过三通与ABTS溶液混合进入反应线圈，ABTS体积流量为0.6 mL/min，反应管内径为0.25 mm，长度为10 m。通过检测ABTS在734 nm下产生倒峰所对应的色谱峰，筛选出抗氧化成分。结果 在柴银颗粒中筛选出44种抗氧化成分，采用DAD-ESI-Q-TOF/MS初步鉴别出37种。活性验证表明，连翘酯苷A、木犀草苷、黄芩苷、绿原酸、葛根素均具有较好的抗氧化活性，其中，连翘酯苷A半数清除浓度（IC₅₀）为（11.08±1.21）μmol/L，优于阳性对照抗坏血酸。对该5种活性成分进行了含量测定。以筛选出的抗氧化成分为共有峰，建立了柴银颗粒抗氧化成分指纹图谱；相似度分析表明，不同批次样品相似度均在0.99以上。结论 建立的抗氧化成分在线筛选、鉴别方法分析速度快、灵敏度高，可以快速辨识柴银颗粒中的抗氧化成分。由此建立的抗氧化活性成分指纹图谱，把化学成分与生物活性相关联，为中成药的整体质量评价研究提供方法与思路支持。

Objective An on-line HPLC-ABTS-DAD-ESI-TOF/MS method was established to screen and identify the antioxidant components of Chaiyin Granules (柴银颗粒, CG), then the feasibility of the quality evaluation of CG based on the fingerprint of antioxidant components combined with multi-component quantification was discussed. Methods A Phenomenex Luna C₁₈ column (250 mm×4.6 mm, 5.0 μm) with gradient elution (methanol-0.2% formic acid aqueous solution) at a flow rate of 0.8 mL/min, column temperature of 25 ℃ was used. The HPLC chromatogram of CG extract was detected at 280 nm. The eluates were mixed with the ABTS solution through a tee joint and entered the reaction coil. The flow rate of ABTS was 0.6 mL/min, the inner diameter of the reaction tube was 0.25 mm, and the length was 10 m. The antioxidant components were screened out by detecting the chromatographic peak corresponding to the inverted peak generated by ABTS at 734 nm. Results Forty-four antioxidant components were screened from CG, and 37 components were preliminarily identified by DAD-ESI-Q-TOF/MS. The activity verification showed that forsythoside A, luteoloside, baicalin, chlorogenic acid and puerarin all had suitable antioxidant activities, and the IC₅₀ of forsythoside A was (11.08 ±1.21) μmol/L, which was better than the positive control ascorbic acid. The contents of the five active components were determined. The fingerprint for the antioxidant components of CG was established, taking the screened antioxidant components as common peaks. Similarity analysis showed that the similarity of different batches of samples was all above 0.99. Conclusion The on-line screening and identification method of antioxidant components established in this study is fast and sensitive, which can quickly identify antioxidant components in CG. The fingerprint of antioxidant components established by this study associates chemical elements with biological activity, which provides methods and ideas support for the overall quality evaluation of Chinese patent medicines.

R283.6

山东省重点研发计划（重大科技创新工程）（2021CXGC010508）;山东省泰山学者项目（tstp20221138）;财政部和农业农村部：国家现代农业产业技术体系（CARS-21）