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

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.