| The chemical composition of natural products represented by traditional Chinese medicine is complex,and systematic knowledge of the chemical constituents of traditional Chinese medicine has always been a key area of research on traditional Chinese medicine.In this study,the extract of Epimedium sagittatum was studied,and a method for the systematic separation of the chemical constituents of Epimedium sagittatum based on online two-dimensional chromatography was designed to provide reference for the study of chemical constituents of traditional Chinese medicine.The extracts of Epimedium were used as the research object,and the extracts were separated and analyzed by full-gradient HPLC on C18column and Xion column.Epimedium extract was analyzed under three different chromatographic conditions,chromatographic separation information was collected,and XTool chromatographic expert software was introduced to optimize the one-dimensional separation conditions to determine the optimal one-dimensional separation conditions-Preservative Chromatography Platform（PCP）for enrichment mode for one-dimensional preparation,determination of separation reproducibility,optimal sample loading and cutting procedure,and application of this method for one-dimensional separation preparation The two components obtained by one-dimensional separation are further separated by two-dimensional preparative separation conditions optimized by XTool chromatography expert software to obtain high-purity components and monomers;the obtained monomer components are passed through UV,HPLC,and nuclear magnetic resonance.Means for structural characterization.By analyzing the chromatographic separation of the filler and the solvent system,a total of 44 visible peaks were analyzed by full gradient HPLC with C18 filler and acetonitrile-water as mobile phase,and all of them were flavonoids by UV detection.The chromatographic peak information was imported into XTool chromatography expert software for one-dimensional separation condition method simulation and optimization;the optimized conditions were linearly amplified to the preparative chromatography factory,and PCP was used for one-dimensional separation preparation,and the optimal sample loading was determined to be1000 mg.The cutting procedure was determined for one-dimensional separation to obtain 18components;the obtained 18 components were prepared by two-dimensional separation conditions optimized by XTool chromatography expert software,and 21 high-purity components were obtained,wherein the content was higher.The six monomeric compounds were characterized by UV,HPLC and NMR.The six flavonoids of Epimedium were identified as radix A（34 mg）and radix B（12.4 mg）.C（52.9 mg）,icariin（53.3 mg）,arrowside A（14.5mg）,and baicalin-I（45 mg）.The purity was 98.15%,98.60%,98.15%,98.55%,98.17%,and98.99%,respectively.The results showed that the application of the above method basically realized the efficient,rapid and systematic separation of Epimedium flavonoids,and provided new ideas and methods for the extraction and separation of complex traditional Chinese medicines. |
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