目的 构建一套基于质量标志物（quality marker，Q-Marker）理论、兼顾化学属性及药效相关性的满药功效关联质量评价方法，并以核桃楸皮抗肝癌药效评价为例验证其适用性。方法 按照功效导向的核心药效部位界定、物质基础解析与候选Q-Marker物质库构建、候选成分药效代表性验证、体内有效性与安全性评价的递进路径，建立质量评价方法。以核桃楸皮为例，采用CCK-8、划痕和Transwell实验筛选抗肝癌药效部位；采用UPLC-Q-TOF-MS技术解析物质成分；结合前期谱效关系、网络药理学、分子对接分析、单体及配伍实验筛选并验证候选Q-Marker；采用H22荷瘤小鼠评价体内药效与安全性；采用HPLC方法对从核桃楸皮中筛选的Q-Marker进行定量分析。结果 所构建方法形成了“药效定位-成分解析-功效验证-体内确证”的多层次证据链。在核桃楸皮应用中，醋酸乙酯部位在0.6 mg/mL时对HepG2细胞增殖的抑制率为92.6%，且对细胞迁移和侵袭的抑制作用较强；共鉴定没食子酸、丁香酸、槲皮素、鞣花酸和山柰酚等17种成分；综合化学结构、可测性、前期谱效关系、网络药理学及体外药效结果，筛选丁香酸、鞣花酸和山柰酚为候选Q-Marker，其配伍组分可表征醋酸乙酯部位76.3%的体外抗肿瘤活性；H22荷瘤小鼠实验中，醋酸乙酯部位高剂量组抑瘤率为60.42%，与模型组相比，高剂量组血清白细胞介素-2（interleukin-2，IL-2）水平显著升高至（227.85±1.92）pg/mL，IL-6和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）水平分别降低至（71.76±2.86）pg/mL和（384.19±4.74）pg/mL（P＜0.01），脾脏指数和胸腺指数分别升高至（3.965±1.594）和（3.079±1.366）mg/g（P＜0.01），并改善肿瘤组织病理改变。结论 构建了基于Q-Marker理论的满药功效关联质量评价方法，并以核桃楸皮为例验证了方法的可行性。优选出的3种候选Q-Marker可作为核桃楸皮抗肝癌质量控制的重点指标，为满药及相关制剂由单一成分控制向功效关联的多成分质量评价转变提供参考。

Objective To establish an efficacy-linked quality evaluation method for Manyao based on Q-Marker theory, comprehensively considering chemical properties and pharmacodynamic correlation, and to verify its applicability by evaluating the anti-hepatocellular carcinoma efficacy of Cortex Juglandis Mandshuricae. Methods The quality-evaluation system was constructed following the progressive workflow: definition of core pharmacodynamic fractions guided by efficacy, analysis of material basis and establishment of the candidate Q-Marker library, verification of pharmacodynamic representativeness for candidate components, as well as in vivo efficacy and safety evaluation. Taking Cortex Juglandis Mandshuricae as the research object, CCK-8, wound-healing and Transwell assays were adopted to screen its anti-hepatocellular-carcinoma active fraction. UPLC-Q-TOF-MS was used for chemical-component identification. Candidate Q-Markers were screened and validated by combining spectrum-effect relationship, network pharmacology, molecular docking, monomer and compatibility experiments. H22 tumor-bearing mice were utilized to assess in vivo antitumor efficacy and biosafety. HPLC was used to quantify the selected Q-Markers from Cortex Juglandis Mandshuricae. Results The established method formed a multi-layered evidence chain consisting of pharmacodynamic localization, component identification, efficacy validation and in vivo confirmation. For Cortex Juglandis Mandshuricae, the ethyl acetate fraction inhibited HepG2 cell proliferation by 92.6% at 0.6 mg/mL and showed strong inhibitory effects on cell migration and invasion. Seventeen constituents, including gallic acid, syringic acid, quercetin, ellagic acid, and kaempferol, were identified. Based on chemical structure, measurability, previous spectrum-effect relationship analyses, network pharmacology, and in vitro pharmacodynamic results, syringic acid, ellagic acid, and kaempferol were selected as candidate Q-Markers. Their combination accounted for 76.3% of the in vitro antitumor activity of the ethyl acetate fraction. In H22 tumor-bearing mice, the high-dose ethyl acetate fraction achieved a tumor inhibition rate of 60.42%. Compared with the model group, serum IL-2 levels increased to (227.85 ± 1.92) pg/mL, while IL-6 and TNF-α levels decreased to (71.76 ± 2.86) pg/mL and (384.19 ± 4.74) pg/mL, respectively (P < 0.01). The spleen and thymus indices increased to (3.965 ± 1.594) and (3.079 ± 1.366) mg/g, respectively (P < 0.01), and histopathological changes in tumor tissues were ameliorated. Conclusion This study constructs an efficacy-associated quality evaluation approach for Manchu medicinal herbs on the basis of Q-Marker theory, whose feasibility is verified via the case of Cortex Juglandis Mandshuricae. The three selected candidate Q-Markers can serve as core indicators for the anti-hepatocellular-carcinoma quality control of Cortex Juglandis Mandshuricae. This research provides a reference for shifting from single-component-based control to efficacy-linked, multi-component quality evaluation of Manyao and their related preparations.

R285

辽宁省科技厅揭榜挂帅（科技攻关专项）（2021JH1/10400058）