基于小分子-蛋白质相互作用的甘草质量标志物筛选及活性验证

Screening of quality markers and activity verification of Glycyrrhizae Radix et Rhizoma based on small molecule compound-protein interaction

为了解决甘草质量控制成分与功效关联性不强的问题,该研究通过荧光淬灭法检测甘草药材中小分子化学成分与小鼠各脏器总蛋白的相互作用,筛选潜在的活性成分;以HPLC检测甘草中27种化学成分并计算其在34批甘草药材中的缺失率;结合成分有效性和可测性原则共同筛选甘草潜在质量标志物(Q-marker)。再采用微塑料诱导RAW264.7巨噬细胞损伤模型,以CCK-8和Griess法检测细胞活力和一氧化氮含量,采用ELISA法等检测炎症因子(TNF-α、IL-1β、IL-6、CRP)和氧化应激标志物(SOD、MDA、GSH)水平,验证质量标志物的活性。发现甘草中不同化学成分与各脏器蛋白之间的相互作用强度各不相同,体现出不同的脏器选择性,并筛选出18个活性较强的成分;结合成分HPLC色谱峰信噪比和批间稳定性,最终筛选出芹糖甘草苷、甘草苷、芹糖异甘草苷、异甘草苷、甘草素、异甘草素以及甘草酸铵等7个化学成分作为甘草药材的潜在质量标志物。体外实验表明,甘草质量标志物可剂量依赖性缓解微塑料诱导的RAW264.7细胞损伤,抑制细胞炎症因子分泌,减轻细胞氧化应激,且在总剂量相同的条件下,各化学成分合用与单用相比,能够协同增强抗炎、抗氧化作用。该研究确定了与甘草抗炎抗氧化功效相关的质量标志物,可为提高甘草质量控制标准提供参考依据。

In order to solve the problem of weak correlation between quality control components and efficacy of Glycyrrhizae Radix et Rhizoma,this study detected the interaction between small molecular chemical components of Glycyrrhizae Radix et Rhizoma and total proteins of various organs of mice by fluorescence quenching method to screen potential active components.The 27 chemical components in Glycyrrhizae Radix et Rhizoma were detected by HPLC and their deletion rates in 34 batches of Glycyrrhizae Radix et Rhizoma were calculated.Combined with the principle of component effectiveness and measurability,the potential quality markers(Q-markers) of Glycyrrhizae Radix et Rhizoma were screened.RAW264.7 macrophage injury model was induced by microplastics.The cell viability and nitric oxide content were detected by CCK-8 and Griess methods.The levels of inflammatory factors(TNF-α,IL-1β,IL-6,CRP) and oxidative stress markers(SOD,MDA,GSH) were detected by the ELISA method to verify the activity of Q-markers.It was found that the interaction strength between different chemical components and organ proteins in Glycyrrhizae Radix et Rhizoma was different,reflecting different organ selectivity and 18 active components were screened out.Combined with the signal-to-noise ratio of the HPLC chromatographic peaks and between-run stability of the components,seven chemical components such as liquiritin apioside,liquiritin,isoliquiritin apioside,isoliquiritin,liquiritigenin,isoliquiritigenin and ammonium glycyrrhizinate were finally screened as potential Q-markers of Glycyrrhizae Radix et Rhizoma.In vitro experiments showed that Q-markers of Glycyrrhizae Radix et Rhizoma could dose-dependently alleviate RAW264.7 cell damage induced by microplastics,inhibit the secretion of inflammatory factors,and reduce oxidative stress.Under the same total dose,the combination of various chemical components could synergistically enhance anti-inflammatory and antioxidant effects compared with the single use.This study identified Q-markers related to the anti-inflammatory and antioxidant effects of Glycyrrhizae Radix et Rhizoma,which can provide a reference for improving the quality control standards of Glycyrrhizae Radix et Rhizoma.