基于超高效液相色谱-串联质谱的不同加工干燥方法下的杜仲代谢组学分析

Metabolomics Analysis of Eucommiae Cortex Obtained by Different Processing and Drying Methods Based on Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

考察了不同加工干燥方法对杜仲整体代谢产物的影响。分别制备新鲜杜仲、堆置“发汗”、烫泡“发汗”和阴干杜仲样品,进行超高效液相色谱-串联质谱代谢组学分析。通过主成分分析(PCA)、正交偏最小二乘法判别分析(OPLS-DA)等多元统计分析方法,筛选出不同加工干燥方法下杜仲的差异代谢物,比较其相对含量。结果显示,4个组的杜仲样品在PCA得分图中可明显区分。与新鲜杜仲相比,杜仲加工干燥后有170种以上的代谢物发生显著变化,其中超过80%的代谢物表达上调。3种加工干燥方法之间,堆置“发汗”杜仲的上调代谢物数量较多,且有利于木脂素和香豆素类、环烯醚萜类、醌类成分的合成;阴干有利于酚酸类成分的保留,烫泡“发汗”则有利于黄酮类成分的保留。通过比较不同加工干燥方法下杜仲的差异代谢物,为探究杜仲药材的品质形成机制及优选其加工方法提供了科学依据。

The effects of different processing and drying methods on overall metabolites of Eucommiae Cortex were investigated.First of all,the rough skin on the surface of fresh Eucommiae Cortexes were scraped off,some of them were stored in the refrigerator at-80℃,and the rest were treated with three kinds of different processing and drying methods,including stacking“sweating”,scalding“sweating”and drying in the shade.Secondly,three kinds of processed and dried Eucommiae Cortex,including fresh Eucommiae Cortex samples,were freeze-dried,crushed and then extracted with 70%methanol.The metabolites of Eucommiae Cortex were analyzed by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)with electrospray ionization(ESI)ion source,and positive or negative ion mode was used for data collection.The collected data were processed in a series of ways,including peak extraction,peak integration,normalization,etc.After that,the molecular weights of metabolites were obtained according to the first-order mass spectrometry information.And the characteristic ion fragments of metabolites were obtained with the second-order mass spectrometry information,so as to realize the structural analysis of modern metabolites.By searching Metware database(MWDB),human metabolome database(HMDB),MassBank and other databases,the qualitative analysis of metabolites in Eucommiae Cortex was carried out.Finally,the metabolite data were analyzed by principal component analysis(PCA),orthogonal partial least squares discriminant analysis(OPLS-DA)and other multivariate statistical analysis.On the basis of variable importance in the projection(VIP)obtained by OPLS-DA model and fold change(FC),the different metabolites in Eucommiae Cortex obtained by different processing and drying methods were selected.The relative contents of metabolites were characterized by relative peak area,and the relative contents of different metabolites were compared.Results showed that the Eucommiae Cortex samples of the four groups could be clearly distinguished in the PCA score diagram.More than 170 metabolites in Eucommiae Cortex obtained by three kinds of processing and drying methods changed significantly,compared with those in fresh Eucommiae Cortex.And more than 80%of the metabolites were up-regulated.Among the Eucommiae Cortexes obtained by three kinds of processing and drying methods,the number of up-regulated metabolites of stacking“sweating”Eucommiae Cortex was higher,followed by that of drying in the shade.In addition,stacking“sweating”was conducive to the retention of lignans,coumarins,iridoids and quinones,drying in the shade was conducive to the retention of phenolic acids,and scalding“sweating”was conducive to the retention of flavonoids.By comparing the different metabolites of Eucommiae Cortex with different processing and drying methods,a scientific basis was obtained for exploring the quality formation mechanism of Eucommiae Cortex and optimizing its processing methods.