赤芍(PRR)因其广泛的药理作用，在我国临床实践中被广泛用于活血化瘀。然而，抗氧化作用的“质量标志物”（Q-markers）仍然未知。基于多种策略探索抗氧化活性的Q-标志物，为基于特定药效学的PRR质量评价提供参考。首先，通过超高效液相色谱-四极杆飞行时间串联质谱（UHPLC-Q-TOF MS/MS）获得并鉴定了15批PRR的“指纹”图谱，并提取了共同峰。同时采用MTT法评价15批PRR对H2O2诱导的HT-22细胞氧化应激的影响。通过超氧化物歧化酶 (SOD)、谷胱甘肽 (GSH) 和丙二醛 (MDA) 商业试剂盒同时研究 PRR 的抗氧化活性。通过灰色关联分析（GRA）和偏最小二乘判别分析（PLS-DA）构建共同峰和抗氧化指数之间的关系，以识别预选的Q标记。其次，通过实验验证初步成分对氧化应激下的HT-22细胞的保护作用。最后，为进一步验证抗氧化Q标记物的有效性，应用网络药理学探索潜在靶点，并利用分子对接技术评价PRR潜在活性成分与抗氧化靶点的结合能力。 三十七通过 UHPLC-Q-TOF MS/MS 定性鉴定了 15 批 PRR 的共同峰。 MTT法检测MDA、SOD、GSH指标显示PRR可减轻H2O2对HT-22细胞造成的氧化损伤。通过谱效相关分析筛选出8种潜在的抗氧化成分：芍药苷、没食子芍药苷、白花苷、1,2,3,4,6-邻五没食子酰葡萄糖、苯甲酰芍药苷、松香素、齐墩果酸、异鼠李素-3-邻橙皮苷。这些初步成分均显示出对细胞氧化应激的显着保护作用（P < 0.05）。白介素6（IL-6）、蛋白激酶B（AKT1）和肿瘤坏死因子（TNF）被预测为PRR的主要潜在靶点，并且PRR的潜在活性成分与各成分之间呈现出良好的结合能力。基于综合多模式策略，八个成分被确定为 PRR 的抗氧化 Q 标记物。版权所有 © 2024 Li、Zhao、Wang、Peng、Tang、Sun、Yang、Liu 和 Liu。

Paeoniae Radix Rubra (PRR) has been used widely to promote blood circulation and eliminate blood stasis in China clinical practice owing to its extensive pharmacological effects. However, the "quality markers" (Q-markers) of the antioxidant effects remains unknown.To explore the Q-markers of antioxidant activity based on multiple strategies, which would provide reference for the quality evaluation of PRR based on specific pharmacodynamic-oriented.Firstly, the "fingerprint" profiles of 15 batches of PRR were acquired and identified by ultrahigh performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF MS/MS) and the common peaks extracted. Meanwhile, the MTT assay was used to evaluate the effect of 15 batches of PRR on H2O2-induced oxidative stress in HT-22 cells. The antioxidant activity of PRR was investigated simultaneously by superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) commercial kits. The relationship between common peaks and antioxidant indexes were constructed by grey relational analysis (GRA) and partial least squares-discriminant analysis (PLS-DA) for the identification of preselected Q-markers. Secondly, experimental verification was conducted to investigate the protective effect of the preliminary components on HT-22 cells undergoing oxidative stress. Finally, for the further validation of effectiveness of antioxidant Q-markers, network pharmacology was applied to explore potential targets, and the molecular docking technology was used to value the binding ability of the potential active components of PRR to the antioxidant targets.Thirty-seven common peaks from 15 batches of PRR were identified qualitatively by UHPLC-Q-TOF MS/MS. The MTT assay showed that PRR could reduce the oxidative damage induced by H2O2 upon HT-22 cells according to the index of MDA, SOD and GSH. Eight potential antioxidant components were screened by spectrum-effect correlation analysis: paeoniflorin, galloylpaeoniflorin, albiflorin, 1,2,3,4,6-o-pentagalloylglucose, benzoylpaeoniflorin, pinocembrin, oleanic acid, and isorhamnetin-3-o-nehesperidine. Each of these preliminary components showed significant protections on cellular oxidative stress (P < 0.05). Interleukin-6 (IL-6), protein kinase B (AKT1), and tumor necrosis factor (TNF) were predicted to be the major potential targets of PRR, and the good binding ability were presented between the potential active components of PRR and each target as a whole.Eight components were identified as the antioxidant Q-markers of PRR based on an integrated multimodal strategy.Copyright © 2024 Li, Zhao, Wang, Peng, Tang, Sun, Yang, Liu and Liu.