Preparative separation of flavonoid glycosides and flavonoid aglycones from the leaves of Platycladus orientalis by REV-IN and FWD-IN high-speed counter-current chromatography

Abstract

Three flavonoid glycosides and four flavonoid aglycones were isolated and purified for the first time from the leaves of Platycladus orientalis via two different elution modes of high-speed counter-current chromatography. First, the fractions containing the flavonoid glycosides and flavonoid aglycones were obtained via polyamide column chromatography to remove the nonflavonoids; afterwards, a reverse-inlet (REV-IN) elution mode was applied to purify the flavonoid glycosides using a two-phase solvent system composed of ethyl acetate–ethanol–acetic acid–water (4 : 1 : 0.25 : 5, v/v); then, a forward-inlet (FWD-IN) elution mode was applied to purify the flavonoid aglycones with a two-phase solvent system composed of chloroform–methanol–water (4 : 3 : 2, v/v). In total, three flavonoid glycosides, myricetrin (1, 10.2 mg), quercetin-3-O-α-L-rhamnoside (2, 25.8 mg), and kaempferol-3-O-α-L-rhamnoside (3, 9.3 mg), and four flavonoid aglycones, quercetin (4, 10.5 mg), kaempferol (5, 4.5 mg), amentoflavone (6, 13.8 mg) and myricetin (7, 14.7 mg), were successfully obtained from the enriched fractions of flavonoid glycoside and flavonoid aglycone by the REV-IN mode HSCCC and FWD-IN mode HSCCC, respectively, structures of which were elucidated by HR-ESI-MS and ¹H and ¹³C NMR spectroscopy. The results demonstrate that the combination of polyamide column chromatography and HSCCC is effective for the rapid enrichment and separation of flavonoid glycosides and flavonoid aglycones from the leaves of P. orientalis.