Selective isolation of Artepillin C from Brazilian green propolis by countercurrent chromatography

The selective isolation by countercurrent chromatography of artepillin C, a prenylated derivative of p-coumaric acid, directly from the ethanol extract of Brazilian green propolis in a single step was developed using a two-phase system containing organic solvent and a basic aqueous solution, based on the ionization ability of carboxyl groups in a basic medium, which allows the selective extraction of compounds such as artepillin C to the aqueous phase, in the form of a salt. The solvent system hexane - 5 % aqueous Na₂CO₃ was first optimized with different proportions of ethyl acetate, with the hexane - ethyl acetate ratio 1:1 - 5 % aqueous Na₂CO₃ being selected. Fractionation of the ethanol extract of green propolis with this solvent system yielded artepillin C with a purity of 72 %. However, the high pH obtained with the Na₂CO₃ solution also ionized other acidic derivatives (triterpenic acids and other phenolic derivatives) and the base was replaced by 4 % NaHCO₃. The composition of the organic phase was re-optimized, with a ratio of hexane - ethyl acetate (8:2) and 4 % aqueous NaHCO₃ (1:1, v/v). Fractionation of the ethanol extract of green propolis with this new optimized solvent system provided artepillin C with a purity of 81 %, ensuring an excellent separation from the other compounds in the extract. The lower basicity of the bicarbonate solution was not able to ionize other acidic compounds, and the lower concentration of ethyl acetate prevented the loading of salt into the organic phase.