Eco-friendly isolation and purification of oleuropein via three-phase high-speed counter-current chromatography and preparative HPLC.

Oleuroppein is one of the bioactive secoiridoids found in the leaves of Olea europaea and possesses health-promoting properties, thus requiring practical purification guidelines. This study reports a two-step chromatographic strategy for isolating and purifying oleuropein. First, an optimised high-speed counter-current chromatography (HSCCC) protocol by means of a novel three-phase solvent system (water/acetonitrile/n-hexane/dichloromethane, 5:5:5:1, v/v) was developed to fractionate olive leaf extracts. The HSCCC step enriched oleuropein but did not achieve analytical-grade purity, likely due to HSCCC's lower chromatographic resolution (fewer theoretical plates) compared to HPLC. Therefore, the HSCCC fraction containing oleuropein was further purified by preparative high-performance liquid chromatography (HPLC). The HSCCC-prep-HPLC system yielded an oleuropein fraction that was 94.7 % pure by analytical HPLC. Mass spectrometric analysis and co-chromatography with established standards were used to support the compound's structural identity and purity. This combined approach, by taking advantage of the ability of HSCCC to handle crude extract mixtures in combination with the high resolution of HPLC, avoided the limitations that were inherent with HSCCC itself. As a result, a high-purity oleuropein isolate suitable for quality-control tests or bioactivity studies could be obtained, thus demonstrating the effectiveness of the combination of HSCCC and preparative HPLC in isolating near-pure oleuropein and related natural products from complex botanical samples.