目的 探究不同煎煮时间对山豆根Sophorae Tonkinensis Radix et Rhizoma功效物质的动态变化及其抗炎与免疫活性的影响，为山豆根临床合理煎煮工艺优化提供科学依据。方法 采用HPLC法，对不同煎煮时间（0.5、1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5、5.0 h）山豆根样品构建指纹图谱，并对其7种指标性成分进行定量分析，探究山豆根功效物质随煎煮时间变化的动态规律。同时，采用环氧合酶-2（cyclooxygenase-2，COX-2）试剂盒测定不同煎煮时间山豆根样品的抗炎作用；此外，运用蛋白质印迹法（Western blotting）分析不同煎煮时间山豆根样品对小鼠骨髓来源巨噬细胞（bone-marrow-derived macrophages，BMDMs）中干扰素调节因子3（interferon regulatory factor 3，IRF3）及干扰素基因刺激因子（stimulator of interferon genes，STING）蛋白磷酸化水平的影响。结果 不同煎煮时间山豆根样品指纹图谱共确定了15个共有峰，指认了7种成分，分别为金雀花碱（峰1）、氧化苦参碱（峰2）、氧化槐果碱（峰3）、苦参碱（峰8）、槐果碱（峰9）、三叶豆紫檀苷（峰10）和高丽槐素（峰14）。7种功效物质含量随煎煮时间呈现3种变化趋势：其一，先增后减——氧化苦参碱（2.5 h达峰值）、氧化槐果碱（1.0 h达峰值）及高丽槐素（2.5 h达峰值）均在特定时间点达到峰值后随煎煮时间延长而逐渐降低；其二，持续增加——苦参碱、槐果碱及三叶豆紫檀苷的含量随煎煮时间延长呈稳定上升趋势；其三，金雀花碱的含量在整个煎煮过程中含量波动较小，基本保持稳定。体外抗炎活性测定结果表明，随着煎煮时间的延长，半数抑制浓度（50% inhibitory concentration，IC₅₀）呈下降趋势，且IC₅₀与三叶豆紫檀苷、苦参碱、槐果碱的含量均呈现显著负相关关系。Western blotting结果显示，山豆根提取物对cGAS-STING通路的调控作用呈现明显的煎煮时间依赖性。结论 山豆根中7个指标成分（金雀花碱、氧化苦参碱、氧化槐果碱、苦参碱、槐果碱、三叶豆紫檀苷、高丽槐素）含量及抗炎、免疫活性与煎煮时间呈现显著关联；研究揭示了其“煎煮时间-成分-活性”的动态关联规律，为山豆根临床合理应用提供科学依据。

Objective To investigate the dynamic changes of functional substances in Shandougen (Sophorae Tonkinensis Radix et Rhizoma, STRR) under different decoction times and their effects on anti-inflammatory and immunomodulatory activities, so as to provide a scientific basis for optimizing the clinical decoction process of STRR. Methods This study employed HPLC to establish fingerprints and quantitative analysis of its seven indicator components of STRR samples at different decoction times (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 h), so as to investigate the dynamic changes of STRR functional substances with decoction time. The anti-inflammatory effects of the STRR samples were determined using a cyclooxygenase-2 (COX-2) kit. Additionally, Western blotting was used to analyze the effects of STRR samples on the phosphorylation levels of interferon regulatory factor 3 (IRF3) and stimulator of interferon genes (STING) in mouse bone-marrow-derived macrophages (BMDMs). Results A total of 15 common peaks were identified in the fingerprints of STRR samples under different decoction times, among which seven components were identified: cytisine (peak 1), oxymatrine (peak 2), oxysophocarpine (peak 3), matrine (peak 8), sophocarpine (peak 9), trifolirhizin (peak 10), and maackiain (peak 14). The contents of the seven functional substances exhibited three changing trends with the extension of decoction time. Firstly, an initial increase followed by a decrease—oxymatrine (peaking at 2.5 h), oxysophocarpine (peaking at 1.0 h), and maackiain (peaking at 2.5 h) all reached their peak values at specific time points and then gradually decreased as the decoction time prolonged; Secondly, a continuous increase—the contents of matrine, sophocarpine, and trifolirhizin showed a steady upward trend with the extension of decoction time; Thirdly, cytisine exhibited minimal fluctuations in content throughout the decoction process, remaining relatively stable. In vitro anti-inflammatory activity assays showed that the 50% inhibitory concentration (IC₅₀) decreased with prolonged decoction time, and IC₅₀ was significantly negatively correlated with the contents of trifolirhizin, matrine, and sophocarpine. Western blotting results showed that the regulation of the cGAS-STING pathway by STRR extracts was significantly dependent on the decoction time. Conclusion The contents of the seven indicator components (cytisine, oxymatrine, oxysophocarpine, matrine, sophocarpine, trifolirhizin, and maackiain), along with the anti-inflammatory and immunomodulatory activities of STRR exhibited a significant correlation with decoction time. This study reveals the dynamic correlation rule of ‘decoction time-component-activity’, which provides a scientific basis for the rational clinical application of STRR.

R283.6

国家重点研发计划(2022YFD1600302);国家自然科学基金联合基金项目(U23A20519);广西岐黄学者资助项目(GXQH202401)