A new consecutive high-performance counter-current chromatography approach based on co-current and overlap mode for isolating active compounds from olive leaves.

Conventional consecutive separation methods based on overlapping injection modes produce interactions between samples as the separation time increases. This will lead to a reduction in the purity of the target due to the gradual saturation of the stationary phase distribution. To enhance the purity of the sample preparation and separation efficiency, we devised a consecutive separation method under high-performance counter-current chromatography (HPCCC) utilizing co-current elution and overlapping injection. The method was used for the separation and preparation of olive leaves for their main bioactive polyphenolic constituents. Six HPCCC separations were conducted in reversed-phase mode using a two-phase solvent system consisting of petroleum ether/ethyl acetate/water (1:40:40, v/v). Finally, 302.7 mg of luteolin-4'-O-β-D-glucoside and oleuropein were isolated from 720 mg of crude sample with purities of 93.3% and 96.2%, respectively. The optimization strategy outlined in this study can be used to enhance the extraction of bioactive components from olive leaves.