Combined cloud point extraction and high-speed counter-current chromatography for enrichment and separation of three flavonoid aglycones from Scutellaria baicalensis Georgi

An efficient strategy integrating cloud point extraction (CPE) and high-speed counter-current chromatography (HSCCC) was developed to separate active flavonoids from Scutellaria baicalensis Georgi. The liquid stationary phase advantage of HSCCC enabled the direct injection of the surfactant-rich phase, preventing the irreversible adsorption of surfactants on solid carriers and avoiding contamination, thereby establishing a rapid and efficient extraction and enrichment methodology. By optimizing the surfactant type and concentration, solid–liquid ratio, extraction pressure, and extraction time, CPE additive type and concentration, equilibrium temperature, and extraction time, the optimal CPE process was obtained. Based on the partition coefficient (KD) values of the target compounds, a solvent system composed of trichloromethane: tetrachloromethane: methanol: water (1:4:3:2, v/v/v/v) was selected from various tested systems, as it provided ideal retention behaviors for the separation. Baicalein (I), wogonin (II), and oroxylin A (III) were successfully isolated from Scutellaria baicalensis with HPLC purities of 96.21%, 94.91%, and 96.68%, respectively. Their chemical structures were further confirmed by NMR and ESI-MS. A rapid and efficient method for enrichment, separation, and preparation of high-purity monomeric compounds was achieved by applying CPE and HSCCC. This comprehensive method provides a new solution for the extraction and separation of natural compounds.