Development of a cell-based assay coupled HPLC micro-fractionation technology for identification of anticancer...

Natural products represent one of the vital sources of anticancer drugs. However, the rapid screening of anticancer active compounds from complex extracts continues to pose a significant challenge. Although microplate-based high-resolution inhibition profiling have demonstrated their effectiveness in rapidly pinpoint individual bioactive components in extracts, the majority of these assays rely on chemical or enzymatic reactions. This study presents a new analytical screening method combining cellular assays and HPLC micro-fractionation to identify anticancer compounds in complex plant extracts. The method development involved optimizing 96-well plate configurations and DMSO transfer volumes for the cell-based assay using standard natural molecules and an artificial mixture. The optimized method was applied to profile anticancer compound in ethyl acetate extracts of Eomecon chionantha and Tacca plantaginea. Through repeated chromatographic separation and structural elucidation, we isolated two anticancer compounds from E. chionantha and eight from T. plantaginea, including three new molecules. Our method overcomes the toxicity associated with organic solvents used in HPLC fractionation on cell models and optimizes the volume of DMSO required for transferring materials into cell culture plates, enabling the profiling of anticancer molecules through the widely used MTT assay.