A strategy based on isocratic and linear-gradient high-speed counter-current chromatography for the comprehensive separation of platycosides from Platycodi radix

Abstract

Platycosides, the generally recognized main active constituents of Platycodi radix, have been studied extensively for their wide pharmacological activities. Herein, we have successfully developed an efficient method for the enrichment and comprehensive isolation of platycosides from Platycodi radix by MCI resin column chromatography (CC) and two different modes of high-speed counter-current chromatography (HSCCC). MCI resin CC was the preferable enrichment operation for platycosides from the 70%-ethanol extract of Platycodi radix and rendered target platycosides when eluted by 60% aqueous methanol solution. As for the separation, two different modes, including isocratic HSCCC and linear-gradient HSCCC, were applied together to separate the platycosides using a mixture of ethyl acetate, n-butanol and water coupled with evaporative light scattering detection, for the first time. Isocratic HSCCC was applied to separate crude platycosides from Platycodi radix using ethyl acetate–n-butanol–water (1 : 1 : 2, v/v), yielding seven pure platycosides (compounds 1–6, 8) and two fractions of enriched mixtures of compounds 7, 9, 10, and 11. Linear-gradient HSCCC was employed to rapidly separate compounds 7, 9, 10, and 11 by constantly changing the proportions of ethyl acetate and n-butanol in the ethyl acetate–n-butanol–water solvent system. Finally, platycoside E (1), deapio-platycodin D₃ (2), platycodin D₃ (3), deapio-platycodin D₂ (4), platycodin D₂ (5), platycodin D (6), polygalacin D₂ (7), polygalacin D (8), and three tautomers, namely 2′′-O-acetylplatycodin D (9) and 3′′-O-acetylplatycodin D (9′), 2′′-O-acetylpolygalacin D₂ (10) and 3′′-O-acetylpolygalacin D₂ (10′), and 2′′-O-acetylpolygalacin D (11) and 3′′-O-acetylpolygalacin D (11′), were obtained from 300 mg of crude platycosides from Platycodi radix.