基于化学指纹-细胞代谢组学相关性分析淫羊藿及巫山淫羊藿促成骨分化的药效物质基础

Pharmacodynamic Substances in Promoting Osteogenic Differentiation of Epimedii Folium and Epimedii Wushanensis Folium Based on Chemical Fingerprint-cell Metabolomics Correlation Analysis

目的:确定淫羊藿及巫山淫羊藿促进成骨分化的药效物质基础,并建立一种基于化学指纹与细胞代谢组学相关性分析中药药效物质基础的方法。方法:采用超高效液相-四极杆-静电场轨道阱高分辨质谱法(UPLC-Q-Exactive Orbitrap-MS)分析4种基原15批淫羊藿和3批巫山淫羊藿药材的化学指纹,通过偏最小二乘法-判别分析(PLS-DA)对不同基原淫羊藿及巫山淫羊藿化学指纹峰面积进行统计学分析;运用细胞增殖与活性检测法(CCK-8)和酶联免疫吸附测定法(ELISA)分别检测淫羊藿及巫山淫羊藿对MC3T3-E1成骨前体细胞增殖活力及成骨细胞碱性磷酸酶(ALP)活性的影响,采用超高效液相色谱-四极杆-飞行时间串联质谱法(UPLC-Q-TOF-MS/MS)分析不同基原淫羊藿及巫山淫羊藿对MC3T3-E1细胞代谢组学的影响。构建细胞代谢组峰表,并引入平均药效作用指标mean Y0,采用PLS及Pearson相关性分析计算各组mean Y0和化学指纹的相关性,依据变量重要性投影(VIP)值>1筛选淫羊藿促成骨分化的药效成分,依据各组的mean Y0评价淫羊藿及巫山淫羊藿的药效作用。结果:各基原淫羊藿及巫山淫羊藿化学指纹可完全分离。与空白组比较,不同基原淫羊藿及巫山淫羊藿组MC3T3-E1细胞活力均明显增加,淫羊藿(甘肃)、朝鲜淫羊藿、柔毛淫羊藿组均可明显增强MC3T3-E1细胞ALP活性(P<0.05)。细胞代谢组学结果显示,空白组与模型组有明显的分离趋势,不同基原淫羊藿及巫山淫羊藿与模型组距离有差异,表明不同基原淫羊藿及巫山淫羊藿促成骨分化作用具有差异。化学指纹-细胞代谢组学整合分析筛选出9个成分与药效关系密切,包括双藿苷B、朝藿定C、淫羊藿苷、宝藿苷Ⅰ、yinyanghuo B、β-脱水淫羊藿素、木兰花碱、隐绿原酸和槲皮素,以朝鲜淫羊藿促成骨分化的药效作用最强。结论:该研究确定了不同基原淫羊藿及巫山淫羊藿促成骨分化的药效物质基础多为黄酮类、生物碱类和有机酸类成分,为中药药效相关成分筛选及中药疗效预测提供思路与方法。

Objective:To determine the pharmacodynamic substance basis of Epimedii Folium(EF)and Epimedii Wushanensis Folium(EWF)in promoting osteogenic differentiation,and to establish a method to analyze the material basis of Chinese materia medica based on the correlation between chemical fingerprint and cellular metabolomics.Method:The chemical fingerprints of 15 batches of EF with 4 species and 3 batches of EWF were analyzed by ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap highresolution mass spectrometry(UPLC-Q-Exactive Orbitrap-MS),and partial least squares-discriminant analysis(PLS-DA)was used to analyze the peak areas of chemical fingerprints of samples.The effects of different samples on proliferative activity of MC3T3-E1 osteoblast precursors,as well as the activity of alkaline phosphatase(ALP)in osteoblasts were detected by cell counting kit-8(CCK-8)and enzyme-linked immunosorbent assay(ELISA).At the same time,UPLC-quadrupole-time-of-flight mass spectrometry(UPLC-QTOF-MS/MS)was used to analyze the effects of different samples on the metabolomics of MC3T3-E1 cells,then metabolic peak table of osteogenic differentiation cells was constructed,and pharmacodynamic index mean Y0 was introduced into the peak table.PLS was used to calculate mean Y0 of each group,and the mean Y0 was added to the peak table of chemical fingerprint to construct the correlation between chemical fingerprint and cell metabolome,the pharmacodynamic components of EF and EWF that promote bone differentiation were screened according to variable importance in the projection(VIP)value>1.The pharmacodynamic effects of EF and EWF were evaluated according to the mean Y0 of each group.Result:The chemical fingerprints of EF with different origins and EWF were completely separated.Compared with the blank group,the activity of MC3T3-E1 cells in EF and EWF groups was significantly increased,the activity of ALP in the Epimedium brevicornu(Gansu province),E.koreanum and E.pubescens groups was significantly increased(P<0.05).The results of cell metabolomics showed that the blank group and the model group had an obvious trend of separation.EF with different origins and EWF had different distance from the model group,indicating that EF with different origins and EWF had different effect on promoting osteogenic differentiation.Chemical fingerprint-cell metabolomics integration analysis screened 9 components closely related to the efficacy of EF and EWF,including diphylloside B,epimedin C,icariin,baohuosideⅠ,yinyanghuo B,β-anhydroicaritin,magnoflorine,cryptochlorogenic acid and quercetin.E.koreanum had the strongest effect on promoting osteogenic differentiation.Conclusion:This study determined that the material basis of EF and EWF promoting osteogenic differentiation were mostly flavonoids,alkaloids and organic acids,which provided ideas and methods for the screening of pharmacodynamic components and the prediction of therapeutic effect of Chinese materia medica.