Comprehensive separation of major compositions from Sophora japonica var. violacea by counter-current chromatography using a liquid-liquid extraction strategy

In the present study, a liquid-liquid extraction strategy for sample pretreatment and solvent system selection was established to assist comprehensive separation of major active compositions from natural product by counter-current chromatography. The two-phase solvent systems composed of n-hexane–ethyl acetate–methanol–water at the ratio of 10:0:8:2, 5:5:5:5 and 3:5:3:5 (v/v) were used for sample pretreatment, in which the major compositions were almost entirely distributed in the upper phase of two-phase solvent systems. After extraction, the enriched components FI, FV and FVI were obtained. According to this result, the two-phase solvent systems were slightly adjusted to lower polarities and used for HSCCC separation. As a result, 62 mg of compound 6 was isolated from 200 mg FI. 15 mg of compound 4 and 13 mg of compound 5 were isolated from 70 mg FV. 15 mg of compound 3, 51 mg of compound 1 and 16 mg of compound 2 were isolated from 200 mg FVI. The purities of all isolated compounds were over 93%. Their structures were identified as p-hydroxybenzoic acid, kaempferol-3-O-β-d-glucopyranoside, kaempferol 7-O-α-l-rhamnopyranoside, formononetin, 4′-O-methyl kaempferol, and dibutyl phthalate by 1H NMR and 13C NMR.