Alternating isocratic and step gradient elution high-speed counter-current chromatography for the isolation of minor phenolics from Ormocarpum kirkii bark

•Two new flavones and one biflavanone glycosides were isolated from Ormocarpum kirkii by HSCCC.•Application of several solvent systems afforded 14 compounds with different polarities.•Structures of compounds were identified by HPLC-UV, ESI-MS, 1D and 2D NMR.•The present study shows that HSCCC is a useful and fast method for natural product isolation.

A total of 14 compounds were isolated from the ethanol bark extract of O. kirkii S. Moore (Fabaceae) by alternating isocratic and step gradient elution high-speed counter-current chromatography (HSCCC) methods, using several solvent systems with reference to the polarity of compounds being purified. The extract was successively fractionated with generic solvent systems including n-hexane-ethanol-water (4:2:2) and ethyl acetate-water (1:1). Resulting fractions were further purified using the following preparative gradient elution consisting of ethyl acetate-n-butanol-water (X:Y:10), (X:Y = 9:1 (I); 8:2 (II); 7:3 (III); 6:4 (IV); 5:5 (V); 4:6 (VI) 3:7 (VII) and n-hexane- ethyl acetate-methanol-water (1:X:1:1), X = 1, 2, 2.5, 3 solvent systems. Two flavone glycosides, apigenin-6-C-β-d-glucopyranosyl-4′-O-[β-d-glucopyranosyl-(1 → 5)]-β-d-apiofuranoside (1) and apigenin-6-C-β-d-glucopyranosyl-4′-O-β-d-apiofuranoside (2), and one biflavanone diglycoside 7,7″-di-O-β-d-glucosylliquiritigeninyl-(I-3,II-3)-naringenin (4) were isolated as new compounds along with other 11 known ones. The structures of the isolated compounds were identified by HPLC-UV, ESI-MS, 1D and 2D NMR and comparison with literature data. Thus, over common traditional chromatographic methods, the present study shows that HSCCC is a useful and fast method for natural product research with no losses and lower solvent use.