基于UHPLC-Q-Exactive Orbitrap MS技术藏药冠心宁颗粒化学成分及作用机制研究

Study on the Chemical Composition and Mechanism of Action of Tibetan Medicine Guanxinning Granules Based on UHPLC-Q-Exactive Orbitrap MS Technology

关键词:  冠心宁颗粒  冠心病  LC-MS  网络药理学  分子对接  Elisa实验

Key words:  Guanxinning granules  Coronary artery disease  LC-MS  network pharmacology  molecular docking  Elisa experiment

摘要:

目的：系统识别鉴定藏药冠心宁颗粒主要化学成分并研究其治疗冠心病的作用机制。方法：采用高效液相-四级杆-静电场轨道阱高分辨质谱(UHPLC-Q-Exactive Orbitrap MS)技术对藏药冠心宁颗粒的主要化学成分进行分析,结合组方药材化学成分、一级色谱峰、二级碎片离子、标准品质谱等信息鉴定化合物。使用生物信息学方法,利用SwissTargetPrediction数据库预测化合物靶点,并利用GeneCards、DisGeNet、OMIM和DRUGBANK数据库收集冠心病相关靶点。利用STRING数据库对预测的靶标蛋白进行相互作用分析,并进行GO和KEGG富集分析,分析预测主要作用通路和靶点。利用AutodockTools1.5.6软件对主要成分和核心靶点进行分子对接验证,并利用ELISA实验验证藏药冠心宁颗粒对血清中与冠心病密切相关的蛋白的影响。结果：冠心宁颗粒中共鉴定出73个化合物,主要为酚类(23个)、黄酮类(15个)、萜类(11个)、有机酸类(10个)、生物碱(6个),筛选出活性化合物44个。从数据库筛选出活性成分的作用靶点476个,其中63个与冠心病相关靶点有交集。通过PPI、GO和KEGG分析表明该方主要通过调控ACE、AKT1、ESR1、SERPINE1、HIF1A、IFNG、NFKB1、NR3C1、PPARG、PTGS2等蛋白,作用于脂质与动脉粥样硬化、糖尿病并发症中的AGE?RAGE信号通路、内分泌抵抗、流体剪切应力与动脉粥样硬化等信号通路来发挥治疗冠心病的功效。分子对接结果表明,主要活性成分与靶点之间通过氢键、范德华力、疏水作用相互结合,具有良好的亲和力。ELISA实验表明,冠心宁颗粒可降低大鼠血清中的ACE(P<0.05),升高大鼠血清中的SERPINE1(P<0.0001)。结论：本研究对藏药冠心宁颗粒的主要成分进行了系统的分析鉴定,运用网络药理学方法探讨了冠心宁颗粒治疗冠心病的有效成分和作用机制,并进行了初步验证。这些结果为冠心宁颗粒的进一步临床应用、药理学和质量控制研究提供了基础理论依据。

Abstract:

Objective Systematic identification and characterisation of the main chemical components of the Guanxinning Granules and study of its mechanism of action in the treatment of coronary heart disease. Methods: High performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) was used to analyse the main chemical components of the Guanxinning Granules, and the compounds were identified by combining the information of chemical composition of the herbal medicines of the composition group, the primary chromatographic peaks, the secondary fragmentation ions, and the standard mass spectra. Using bioinformatics methods, the SwissTargetPrediction database was used to predict compound targets, and GeneCards, DisGeNet, OMIM and DRUGBANK databases were used to collect coronary heart disease-related targets. The predicted target proteins were analysed for interaction analysis using the STRING database, and GO and KEGG enrichment analyses were performed to analyse and predict the main pathways of action and targets. AutodockTools1.5.6 software was used to validate the molecular docking of the main components and core targets, and Elisa assay was used to verify the effect of Tibetan medicine Guanxinning Granules on serum target proteins closely related to coronary heart disease. RESULTS: A total of 73 compounds were identified in Guanxinning Granules, mainly phenols (23), flavonoids (15), terpenoids (11), organic acids (10) and alkaloids (6), and 44 active compounds were screened. From the database, 476 targets of action of the active ingredients were screened, of which 63 intersected with targets related to coronary heart disease. The PPI, GO and KEGG analyses indicated that the formula mainly acted on AGE-RAGE signaling in lipids and atherosclerosis, diabetic complications by modulating proteins such as ACE, AKT1, ESR1, SERPINE1, HIF1A, IFNG, NFKB1, NR3C1, PPARG, PTGS2 and so on pathway, endocrine resistance, fluid shear stress and atherosclerosis to exert therapeutic effects on coronary heart disease. The results of molecular docking showed that the main active ingredients and the targets were combined with each other through hydrogen bonding, van der Waals force, and hydrophobic interaction with good affinity.Elisa assay showed that Guanxinning Granules could reduce ACE in rat serum ( P<0.05 ) and elevate SERPINE1 in rat serum ( P<0.0001 ). Conclusion: In this study, the main components of the Guanxinning Granules were systematically analysed and identified, and the effective components and mechanism of action of Guanxinning Granules for the treatment of coronary heart disease were explored and preliminarily verified by using network pharmacology methods. These results provide a basic theoretical basis for further clinical application, pharmacological and quality control studies of Guanxinning Granules.