Ethyl acetate–n-butanol gradient solvent system for high-speed countercurrent chromatography to screen bioactive substances in okra

• A gradient HSCCC solvent system is established for screening of bioactive components.
• Anti-cancer components is isolated by HSCCC with MTT detection.
• Anti-cancer compounds is screened out from okra.
• Carolignan is a minor anti-cancer compound with strong activity.
• Hydroxy phenethyl trans-ferulate is a major anti-cancer compound with middle activity.

High-speed countercurrent chromatographic separation (HSCCC) possesses the property of zero-loss of sample, which is very useful for the screening of bioactive components. In the present study, the ethyl acetate–n-butanol gradient HSCCC solvent system composed of n-hexane–ethyl acetate–n-butanol–water was investigated for the screening of bioactive substances. To screen the antiproliferative compounds in okra extract, we used the stationary phase ethyl acetate–n-butanol–water (1:1:10) as the stationary phase, and eluted the antiproliferative components by 6-steps of gradient using mobile phases n-hexane–ethyl acetate (1:2), n-hexane–ethyl acetate (1:4), n-hexane–ethyl acetate (0:4), n-butanol–ethyl acetate (1:4) n-butanol–ethyl acetate (1:2), n-butanol–ethyl acetate (2:2), and n-butanol–ethyl acetate (2:1). The fractions collected from HSCCC separation with the gradient solvent system were assayed for antiproliferative activity against cancer cells. Bioactive components were identified: a major anti-cancer compound, 4′-hydroxy phenethyl trans-ferulate, with middle activity, and a minor anti-cancer compound, carolignan, with strong activity. The result shows that the gradient solvent system is potential for the screening of bioactive compounds from natural products.