基于UPLC-Q-Orbitrap-MS和分子对接技术的青盐方药效物质基础分析  

Analysis of Pharmacodynamic Material Basis of Qingyan Formula Based on UPLC-Q-Orbitrap-MS and Molecular Docking

作　　者：邹钊 李佳珊 徐颖[2] 杨一博 徐盼瑜 杜寒倩 朱卫丰[1] 林娜[2] ZOU Zhao;LI Jiashan;XU Ying;YANG Yibo;XU Panyu;DU Hanqian;ZHU Weifeng;LIN Na(Jiangxi University of Chinese Medicine,Nanchang 330004,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]江西中医药大学,南昌330004 [2]中国中医科学院中药研究所,北京100700

出　　处：《中国实验方剂学杂志》2022年第22期159-166,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：中国中医科学院科技创新工程项目(CI2021A03802);国家自然科学基金项目(81774203,82004248)。

摘　　要：目的:采用血清药物化学方法研究青盐方的入血成分,并考察入血成分与雌激素受体(ER)的结合能,确认青盐方在大鼠体内的药效物质基础。方法:采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱法(UPLC-Q-Orbitrap-MS)比较青盐方70%乙醇提取物、各单味药70%乙醇提取物、空白血清、青盐方70%乙醇提取物给药后血清的指纹图谱差异,根据质谱的保留时间、相对分子质量,以及一级、二级离子碎片,判定青盐方在大鼠体内的入血成分,流动相选择0.1%甲酸水溶液(A)-乙腈(B)梯度洗脱(0~5 min,2%~20%B;5~10 min,20%~50%B;10~15 min,50%~80%B;15~25 min,80%~95%B;25~26 min,95%~2%B;26~30 min,2%B),流速0.3 mL·min^(-1),进样量5μL,电喷雾离子源,检测范围m/z 150~2000,正、负离子扫描模式。采用分子对接技术表征入血成分与ERα、ERβ的结合能,进一步确认青盐方药效物质基础。结果:口服青盐方后,从血清中检测出30种入血成分,其中9种为原型成分,21种为代谢产物。经鉴定9种原型成分分别为水晶兰苷、车叶草苷、麦角甾苷、松果菊苷、β-蜕皮甾酮、尿囊素、去乙酰基车叶草苷酸、甜菜碱、咖啡酸,21种代谢产物包括有机酸、氨基酸、胆碱类等。上述9种原型成分与ERα的结合能分别为-6.7、-8.9、-6.0、-5.7、-5.3、-4.9、-7.3、-3.3、-6.3 kcal·mol^(-1)(1 kcal≈4184 J),与ERβ的结合能分别为-6.6、-7.2、-7.7、8.0、-7.4、-5.5、-6.9、-3.6、-6.4 kcal·mol^(-1)。结论:水晶兰苷等9种入血的原型成分是青盐方在体内发挥雌激素样作用的活性成分,可为该方质量标准的制定及后续研发提供实验依据。Objective:To study the constituents migrating to blood of Qingyan formula by serum pharmacochemistry,and investigate the binding energy between these constituents and estrogen receptor(ER),so as to confirm the pharmacodynamic material basis of Qingyan formula in rats.Method:Ultra-high performance liquid chromatography-quadrupole electrostatic field-orbital trap high resolution mass spectrometry(UPLC-Q-Orbitrap-MS)was used to determine the constituents migrating to blood of Qingyan formula in rats by comparing the fingerprint differences of 70%ethanol extract of Qingyan formula,70%ethanol extract of each single drug in this formula,blank serum and serum after administration of 70%ethanol extract of Qingyan formula,according to the retention time,relative molecular weight and the primary and secondary ion fragments provided by MS.Mobile phase was 0.1%formic acid aqueous solution(A)-acetonitrile(B)for gradient elution(0-5 min,2%-20%B;5-10 min;20%-50%B;10-15 min,50%-80%B;15-25 min,80%-95%B;25-26 min,95%-2%B;26-30 min,2%B),the flow rate was 0.3 mL·min^(-1) and the injection volume was 5μL,electrospray ionization was used with detection range of m/z 150-2000,positive and negative ion scanning modes.Molecular docking technology was used to characterize the binding energy of constituents migrating to blood with ERαand ERβ,and to confirm the material basis of this formula.Result:After oral administration of Qingyan formula,30 components were detected in serum,of which 9 were prototype components and 21 were metabolites.Nine prototype components were identified as monotropein,asperuloside,verbascoside,β-ecdysone,allantoin,deacetyl asperuloside acid,echinacoside,betaine and caffeic acid,21 metabolites mainly included organic acids,amino acids,cholines and so on.The binding energies of the above 9 prototype components with ERαwere-6.7,-8.9,-6.0,-5.7,-5.3,-4.9,-7.3,-3.3,-6.3 kcal·mol^(-1)(1 kcal≈4184 J),and the binding energies of them with ERβwere-6.6,-7.2,-7.7,8.0,-7.4,-5.5,-6.9,-3.6,-6.4 kcal·mol^(-1),respective

关 键 词：青盐方 血清药物化学 分子对接 原型成分 指纹图谱 雌激素受体(ER) 超高效液相色谱-四极杆-静电场轨道阱高分辨质谱法(UPLC-Q-Orbitrap-MS) 

分 类 号：R22[医药卫生—中医基础理论] R28[医药卫生—中医学] R969.1[理学—分析化学] O657[理学—化学]