Efficient preparative isolation and identification of walnut bioactive components using high-speed counter-current chromatography and LC-ESI-IT-TOF-MS

Preparative isolation of complex mixtures of compounds from walnut polar extracts was established by a combination of high-speed counter-current chromatography (HSCCC) and electrospray ionization-ion trap-time of flight mass spectrometry (ESI-IT-TOF-MS). Compounds were isolated after a solvent optimisation selection based on solute distribution in a biphasic solvent system. Isolation was achieved through one or two successive HSCCC runs, and final purification on Sephadex LH-20. Isolated compounds included ellagitannins (1-11), gallic acid (12), dicarboxylic acid glucosides (13-15), hydrojuglone glucoside (16), catechin (17), procyanidin B2 (18), and megasterone glucosides (19-20). Praecoxin D (4) was isolated for the first time from walnut, while praecoxin A methyl ester (5) and glansreginin A n-butyl ester (14) are newly identified compounds. The purity and identity of isolated compounds were confirmed by NMR and HPLC-ESI-MS/MS. These results provided a foundation for in depth characterisation of walnut compounds and offered an efficient strategy for isolation of potentially health-relevant phytochemicals from walnuts.