Extraction and separation of flavonoids from Malus hupehensis using high-speed countercurrent chromatography based on deep eutectic solvent

In the present experiment, a green and highly efficient extraction method for flavonoids established on deep eutectic solvents (DESs) was investigated by using the response surface methodology. The DES-based high-speed countercurrent chromatography (HSCCC) solvent systems were developed for the separation of high purity compounds from the DES extract of Malus hupehensis for the first time. Under the optimal conditions (liquid-to-solid ratio of 26.3 mL/g, water content of 25.5%, and extraction temperature of 77.5°C), the yield of flavonoids was 15.3 ± 0.1%, which was superior to that of the methanol extraction method. In accordance with the physical property of DES-based HSCCC solvent systems and K values of target compounds, DES-based HSCCC solvent systems composed of choline chloride/glucose-water-ethyl acetate (ChCl/Glu-H2O-EAC, 1:1:2, v/v) was selected for the HSCCC separation. Thus, five flavonoids (two novel compounds 1-2, 6´´-O-coumaroyl-2´-O-glucopyranosylphloretin and 3´´´-methoxy-6´´-O-feruloy-2´-O-glucopyranosylphloretin; three know compounds 3-5, namely, avicularin, phloridzin, and sieboldin) were efficiently separated from Malus hupehensis. DESs are the environment friendly and highly efficient solvents as the components of extraction solvent and HSCCC solvent system, and can be re-utilized many times. However, ethyl acetate can be soluble with a few hydrogen bond donors, such as urea, carboxylic acid and polyol, through the shake flask test. It is the great difficulty for the efficient and rapid separation of target compounds from the DESs extract because of the DESs residual in the HSCCC fractions. ChCl and Glu are the great choices of DESs without this problem. In addition, K values increased with the increase of the molar ratio of ChCl/Glu and the content of water, which could effectively guide us to choose the suitable DES-based HSCCC solvent system. The twice HSCCC separation results indicated that DES was the valuable and green solvent for the HSCCC separation of pure compounds from the extract for the first time, and showed the recycle superiority of DES-based HSCCC solvent system.