通过三相高速逆流色谱和制备型高效液相色谱（preparative HPLC）实现环保的橄榄苦苷分离与纯化

《Journal of Chromatography A》：Eco-friendly isolation and purification of oleuropein via three-phase high-speed counter-current chromatography and preparative HPLC

本研究采用高效旋流色谱（HSCCC）结合制备性高效液相色谱（HPLC），通过优化三相溶剂系统（水/乙腈/n-己烷/二氯甲烷，5:5:5:1，v/v）成功分离纯化橄榄叶中的oleuroppein，最终获得94.7%纯度的样品，验证了联用技术的有效性。

　　
olive leaf extracts is a significant advancement in natural product purification technology. Oleuropein, a bioactive secoiridoid compound, has garnered attention for its potential applications in nutraceuticals and pharmaceuticals. The research team from the Chinese Academy of Sciences developed a two-step purification strategy combining high-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (HPLC). This integrated approach addresses the inherent limitations of each technique while achieving high-purity oleuropein suitable for quality control and bioactivity studies.

The purification process begins with ethanol extraction, a sustainable method that effectively isolates oleuropein-rich fractions from olive leaf matrices. Subsequent HSCCC utilises a novel three-phase solvent system (water/acetonitrile/n-hexane/dichloromethane, 5:5:5:1, v/v) to separate complex phenolic mixtures. This reversed-phase HSCCC configuration leverages centrifugal forces to maintain three liquid phases within the column, enabling efficient separation without solid supports that could cause irreversible adsorption. The three-phase system demonstrates superior selectivity compared to conventional two-phase systems, particularly for structurally similar compounds like ligstroside and luteolin-glucoside. Initial HSCCC purification achieves an 88.5% pure fraction, which is then refined through preparative HPLC to reach 94.7% purity.

Key technical innovations include the solvent system optimization for HSCCC and the gradient elution parameters in preparative HPLC. The three-phase solvent combination effectively balances the polarity requirements of oleuropein while maintaining operational stability. Post-HSCCC, HPLC employs an acid-water/acetonitrile gradient to further resolve and concentrate the target compound. This sequential purification strategy combines the scalability of HSCCC with the high-resolution separation capabilities of HPLC.

Mass spectrometry (MS) plays a critical role in verifying both structural identity and purity. Oleuropein's characteristic [M-H]? ion at m/z ~539 and fragmentation patterns serve as definitive identifiers. The MS validation process confirms the absence of major contaminants, ensuring compliance with nutraceutical standards. This analytical rigor is particularly important when the final product is intended for human consumption or as a reference standard in bioactivity studies.

The research addresses a long-standing challenge in olive phenolic extraction - achieving high purity while minimizing processing complexity. Traditional methods like supercritical CO2 extraction or microwave-assisted extraction often require multiple purification steps, which can compromise yield and increase costs. The proposed methodology integrates green extraction principles with advanced chromatographic techniques, aligning with sustainable industrial practices.

From an industrial perspective, this approach offers several advantages. HSCCC handles large sample volumes with high recoveries, making it suitable for initial purification of crude olive extracts. The subsequent HPLC step provides the necessary precision for final product specification. This combination reduces the environmental impact compared to methods requiring toxic solvents or high energy inputs. The scalable nature of HSCCC allows for process intensification, where successive purification stages can be sequentially applied without significant loss of material.

The study also highlights the importance of methodological validation. Co-chromatography with reference standards and offline MS analysis establish confidence in the purification process. This comprehensive quality control ensures the final product meets stringent purity requirements for food additives or pharmaceutical intermediates. The 94.7% purity achieved represents a significant improvement over previous reports, which often struggled to exceed 90% purity without excessive purification steps.

In terms of application potential, the purified oleuropein serves multiple functions. As a nutraceutical ingredient, it must meet regulatory purity thresholds for safety and efficacy. In pharmaceutical research, high-purity standards are essential for bioactivity assays and mechanism studies. The established methodology provides a template for isolating other secoiridoid compounds from olive byproducts, enhancing the economic value of olive processing waste.

The technical contributions include methodological advancements in both HSCCC and HPLC operations. The three-phase solvent system expands the application scope of counter-current chromatography, demonstrating its adaptability to complex mixtures. Preparative HPLC parameters optimised for this system improve separation efficiency and reduce solvent consumption. These methodological developments could have broader implications for natural product purification beyond olive leaf extracts.

Future research directions might focus on process optimization for industrial scale-up. Current limitations include the need for offline MS verification, which could be streamlined through integrated LC-MS systems. Additionally, exploring alternative three-phase solvent systems with lower environmental impact might further enhance sustainability. The methodology could also be adapted for other plant matrices containing structurally similar bioactive compounds.

This work exemplifies the integration of green chemistry principles with advanced separation technologies. By combining HSCCC's sample tolerance and solvent versatility with HPLC's analytical precision, researchers achieve a balance between process efficiency and product quality. The successful isolation of 94.7% pure oleuropein validates the feasibility of this hybrid approach for high-value natural product recovery.

In summary, the study presents a robust purification protocol for oleuropein that addresses both technological and economic constraints. The sequential application of HSCCC and preparative HPLC leverages the complementary strengths of each technique, overcoming the resolution limitations of standalone HSCCC. The implementation of mass spectrometry for quality assurance establishes a reliable standardization process. This methodology not only advances the field of olive byproduct valorization but also provides a generalizable framework for isolating polar secondary metabolites from complex botanical sources.