摘要
阐明中药复方的质量标志物对于了解中药复方的作用机制、质量标准提升使用和中药新药二次开发至关重要.近年来的研究表明,表面等离子体共振(SPR)技术在中药生物质量标志物鉴定领域具有广阔的应用前景.该研究建了一种基于SPR垂钓技术来分析安宫牛黄丸的质量标志物.首先,利用UPLC-Q-TOF-MS分析安宫牛黄丸主要化学成分71个,并采用网络药理学对鉴定化学成分预测包括STAT3在内的安宫牛黄丸抗脑卒中的潜在靶点;其次,构建STAT3蛋白芯片,采用SPR垂钓系统对安宫牛黄丸提取物进行垂钓回收,分析鉴定其潜在的活性成分,包括黄连碱、巴马汀、表小檗碱、小檗碱、甲基黄连碱、去亚甲基小檗碱、药根碱、四氢黄连碱、黄芩素、黄芩苷甲酯10种成分;第三,采用SPR筛选和分子对接技术确定10个活性成分对STAT3靶点的亲和动力学参数KD分别为44.7、44、58.1、51.3、39.7、32.1、49.2、69.1、19.7、24.9 pmol·L-1,分子对接结果良好.首次利用STAT3靶点芯片联合UPLC-Q-TOF-MS有效鉴定安宫牛黄丸化学成分及与STAT3靶点配体成分,提示STAT3可作为安宫牛黄丸构建生物质量评价的潜在质量标志物.
Abstract
Elucidating the quality markers(Q-markers)of traditional Chinese medicines is essential for understanding the mechanisms of action and promoting the rational use of traditional Chinese medicines as well as for developing traditional Chinese medicine-derived drugs.Studies have shown that surface plasmon resonance(SPR)is promising in this field.This study proposed a method based on pull-down with SPR chips to predict the Q-markers of Angong Niuhuang pills(AGNHP).Firstly,71 main chemical components of AGNHP were analyzed by UPLC-Q-TOF-MS,and then network pharmacology was employed to predict the potential targets of AGNHP against stroke.Secondly,the STAT3 protein chip was constructed,and the extract of AGNHP was recovered by pull-down of the SPR system for STAT3 ligand.The potential active ingredients were collected,enriched,and identified as coptisine,palmatine,epiberberine,berberine,worenine,demethyleneberberine,jatrorrhizine,tetrahydrocoptisine,baicalein,and baicalin methyl ester.Next,the affinity constants of the 10 active ingredients were determined as 44.7,44,58.1,51.3,39.7,32.1,49.2,69.1,19.7,and 24.9 μmol·L-1,respectively.The molecular docking results showed that the 10 compounds could compete for binding with STAT3.This is the first report that SPR combined with UPLC-Q-TOF-MS is reliable and feasible for determining the active ingredients of AGNHP at the molecular level from complex systems.STAT3 could be used as a potential target for the biological quality evaluation of AGNHP.
基金项目
国家自然科学基金项目(82274087)
福建省科技厅引导性项目(2022Y0037)
福建中医药大学-漳州片仔癀药业股份有限公司合作课题(HX2021006)
维普
万方数据