A macroporous frit capillary electrophoresis method for discovery of tumor-targeted components from complex natural...

The discovery of antitumor components in complex natural medicine mixtures is often hampered by the time-consuming, labor-intensive, and poorly reproducible nature of conventional screening methods, which may also fail to adequately simulate physiological conditions. To address these limitations, a macroporous frit capillary electrophoresis (MFCE) method was established, in which tumor tissue, serving as the interaction phase, is physically intercepted by a macroporous frit formed from packed silica beads. This critical step ensures effective tissue interception and mass transfer. By detecting and analyzing key capillary electrophoretic parameters, tissue-compound interactions can be directly monitored, thereby enabling qualitative and quantitative analysis of the interactions between complex mixtures and tumor tissue. The MFCE was applied to qualitatively and quantitatively investigate the interactions between Aidi injection and A549 tumor tissue. Five interactive compounds were identified by integrating MFCE with CE-MS/MS analyses, and the results were consistent with previous reports, confirming the method's reliability. Subsequently, MFCE was used to evaluate the interactions of both ethyl acetate and water extracts of Xiao-Ai-Fei Honey Ointment with HT-29 tumor tissues. Integrated analysis using MFCE, CE-MS/MS, and LC-MS/MS identified four and five interacting compounds from the ethyl acetate and water extracts, respectively. Enabled by stable tissue immobilization and the optimized frit architecture, the binding kinetic parameters (k_d, K, k_a, k') for these compounds were quantified by non-linear chromatography. The biological relevance of these findings was confirmed through in vitro cell viability assays and in vivo xenograft models, which demonstrated that the identified interactive components possess potent antitumor activity, thereby validating the reliability and accuracy of the MFCE method. MFCE thus provides a rapid, reliable strategy to directly monitor tumor tissue-compound interactions, linking the method's design, operational principle, and biological relevance, thereby laying a foundation for multi-targeted anticancer compound discovery.