A method combining enzyme assay-guided high-performance liquid chromatography (HPLC) micro-fractionation and elution-extrusion counter-current chromatography (EECCC) was developed to screen and separate aldose reductase (AR) inhibitory activities from those of the ethyl acetate (EtOAc) fraction of Nardostachys chinensis. Under the target-guidance of HPLC micro-fractionation, two phenolic compounds, three caffeoylquinic acid derivatives and two sesquiterpene were isolated by high-speed countercurrent chromatography (HSCCC) using elution modes of extrusion-elution. A one-step HSCCC isolation method was developed, which included a solvent system of n-hexane-EtOAc-methanol-water at a ratio of 2:8:3:7 (v/v/v/v). The chemical structures of the isolated compounds were determined using (1)H- and (13)C-nuclear magnetic resonance spectrometry. The compounds inhibiting AR in the EtOAc fraction of 70 % ethanol extracts of N. chinensis were identified as chlorogenic acid (2) and 1,5-di-O-caffeoylquinic acid (6). Our results indicate that the combined method of HPLC micro-fractionation and EECCC is fast, efficient, and reproducible for systematically isolating AR inhibitory compounds from complex natural products.
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