Ultrasound-assisted dynamic extraction coupled with parallel countercurrent chromatography for simultaneous extraction, purification, and isolation of phytochemicals: application to isoflavones from red clover.

A novel procedure comprising ultrasonic-assisted dynamic extraction (UADE) coupled with two countercurrent-chromatography systems (parallel countercurrent chromatography, PCCC) was developed. This technique offers the possibility for simultaneous extraction, solvent configuration, purification, and isolation of target compounds. This new approach was applied to the fractionation and purification of isoflavones from red clover. The two-phase solvent system was prepared by automating the equipment. The lower aqueous phase of the two-phase solvent system was used as the UADE solution and as the mobile phase for PCCC. After the extraction and purification steps, the purified sample was pumped into the countercurrent chromatography 1 (CCC1) column for the first isolation step. During CCC1 separation, the sample was enriched and purified and then pumped into the CCC2 column for the second isolation step. After completion of the first cycle of UADE-PCCC steps, the second-cycle experiments were performed. Using this sequence, five target compounds, daidzein, prunetin, genistein, irilone, and maackiain, with >95.31 % purity were successfully extracted and isolated using the two-phase solvent system of n-hexane-ethyl acetate-ethanol-water (0.623:1.00:0.99:1.246, v/v). With this instrumental setup, scientific and systematic extraction and isolation of natural products was achieved, and this technique has great potential for industrial application. Graphical Abstract Simplified schematic of instrumental setup of UADE combined with two HSCCC instruments.