Bioassay-Guided Separation ofCentipeda minimaUsing Comprehensive Linear Gradient Centrifugal Partition Chromatography

Abstract

A comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) was developed for the bioassay-guided isolation of natural compounds. The gradient solvent system consisted of three different ternary biphasic solvents types:n-hexane-acetonitrile-water (10:2:8,v/v), ethyl acetate-acetonitrile-water (10:2:8,v/v), and water-saturatedn-butanol-acetonitrile-water (10:2:8,v/v). The lower phase of then-hexane-acetonitrile-water (10:2:8,v/v) was used as the stationary phase, while its upper phase, as well as ethyl acetate-acetonitrile-water (10:2:8), and water-saturatedn-butanol-acetonitrile-water (10:2:8,v/v) were pumped to generate a linear gradient elution, increasing the mobile phase polarity. We used the gradient CPC to identify antioxidant response elements (AREs), inducing compounds fromCentipeda minima, using an ARE-luciferase assay in HepG2 cells, which led to the purification of the active molecules 3-methoxyquercetin and brevilin A. The developed CPC solvent systems allow the separation and isolation of compounds with a wide polarity range, allowing active molecule identification in the complex crude extract of natural products.