基于UPLC-QTOF-MS/MS和TCMIP v2.0辨识胆南星防治中风的质量标志物

Analysis of Q-markers of Arisaema Cum Bile Acting on Stroke Based on UPLC-QTOF-MS/MS and TCMIP v2.0

目的:基于超高效液相色谱-四极杆-飞行时间质谱法(UPLC-QTOF-MS/MS)及中医药整合药理学研究平台(TCMIP)v2.0,探索胆南星防治中风的潜在质量标志物。方法:采用UPLC-QTOF-MS/MS,流动相0.1%甲酸水溶液(A)-0.1%甲酸乙腈溶液(B)梯度洗脱(0~3 min,0.2%~5%B;3~5 min,5%~8%B;5~8 min,8%~10%B;8~14 min,10%~25%B;14~18 min,25%~50%B;18~20 min,50%~70%B;20~21 min,70%~98%B;21~23 min,98%B;23~24 min,98%~0.2%B;24~26 min,0.2%B),流速0.5 mL·min^(-1),电喷雾离子源(ESI),在正、负离子模式下分别进行扫描并采集高质量MS/MS数据;扫描范围m/z 50~1500。利用UNIFI 1.8建立胆南星药材化学成分的本地数据库。借助高分辨质谱仪提供的化合物精确相对分子质量和二级质谱等信息,与本地数据库匹配,并结合文献和对照品的相应信息,完成对胆南星化学成分的定性鉴别;利用TCMIP v2.0收集胆南星化学成分对应的候选靶标谱和中风的疾病基因集,进行“疾病-方剂”关联分析,通过拓扑特征值筛选核心靶标;利用DAVID 6.8进行核心靶标的京都基因与基因组百科全书(KEGG)通路富集分析;并根据质量标志物“五原则”结合文献报道对胆南星防治中风质量标志物进行预测,得到作用于这些目标基因的核心成分,运用Cytoscape 3.8.0绘制“药材-活性成分-目标基因-通路”网络图。应用AutoDock Vina 1.2.2对候选成分与关键靶点进行分子对接计算和验证。结果:在正、负离子模式下初步鉴定了76个化学成分;通过“疾病-方剂”关联分析得到胆南星防治中风的85个核心靶标,将核心靶标导入KEGG数据库,筛选出与中风相关通路31条,目标基因23个,作用于目标基因的核心成分9个;结合质量标志物成分可测性、特征性和传递性,预测出胆南星防治中风可能的质量标志物4个。结论:胆南星防治中风的可能质量标志物为没食子酸、芹菜素-6,8-二-C-葡萄糖苷、芹菜素、胆酸,这4个成分的作用靶点可能是雌激素受体α(ESR1)。

Objective:To predict the possible quality markers(Q-markers)of Arisaema Cum Bile in the prevention and treatment of stroke based on ultra performance liquid chromatography-quadrupole-time-offlight tandem mass spetrometry(UPLC-QTOF-MS/MS)and Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine(TCMIP)v2.0.Method:UPLC-QTOF-MS/MS was employed with the mobile phase of 0.1%formic acid aqueous solution(A)-0.1%formic acid acetonitrile solution(B)for gradient elution(0-3 min,0.2%-5%B;3-5 min,5%-8%B;5-8 min,8%-10%B;8-14 min,10%-25%B;14-18 min,25%-50%B;18-20 min,50%-70%B;20-21 min,70%-98%B;21-23 min,98%B;23-24 min,98%-0.2%B;24-26 min,0.2%B),the flow rate of 0.5 mL·min^(-1)and electrospray ionization(ESI).High quality MS/MS data were scanned in positive and negative ion modes with scanning range of m/z 50-1500.A local database of the chemical constituents in Arisaema Cum Bile was established by UNIFI 1.8.Then the chemical constituents in Arisaema Cum Bile were characterized by matching with the local database and comparing with the reference substances and literature information.TCMIP v2.0 was used to obtain the targets corresponding to the identified components of Arisaema Cum Bile and stroke,and the"disease-formula"correlation analysis was carried out to screen the core targets by topological eigenvalues.DAVID 6.8 was used for enrichment analysis of Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway of core targets.According to the"five principles"of Q-markers and combined with literature reports,the Q-markers of Arisaema Cum Bile in the prevention and treatment of stroke were predicted,and the core components acting on these target genes were obtained.Cytoscape 3.8.0 was employed to draw the network diagram of"medicinal materials-active ingredients-target genes-pathways".Finally,AutoDock Vina 1.2.2 was used to calculate and verify the molecular docking between the candidate components and the key targets.Result:A total of 76 chemical components was identified in positive and negative ion modes,85 core targets were collected for Arisaema Cum Bile in the prevention and treatment of stroke.A total of 31 stroke-related pathways,23 target genes and 9 main active components of Arisaema Cum Bile acting on these genes were screened,and then we determined 4 possible Q-markers for Arisaema Cum Bile in the prevention and treatment of stroke according to the"five principles".Conclusion:The possible Q-markers of Arisaema Cum Bile for stroke are gallic acid,apigenin-6,8-di-C-glucoside,apigenin and cholic acid,and the target of these four components may be estrogen receptor alpha(ESR1).