Industry-ready purification of multi-target acetylcholinesterase inhibitors from Gardenia jasminoides fruit: UNIFAC-guided consecutive-CCC and integrated bioactivity validation

This work establishes an industry-oriented strategy for discovering and producing bioactive constituents from Gardenia jasminoides fruit. Target-directed affinity affinity ultrafiltration combined with liquid chromatography-mass spectrometry enabled rapid identification of acetylcholinesterase-binding compounds, which were structurally confirmed through molecular docking and molecular dynamics simulations. UNIFAC -guided optimization improved solvent system design in consecutive-counter-current chromatography, markedly reducing organic solvent consumption while maintaining stable stationary phase retention. In combination with semi-preparative HPLC, eight neuroprotective compounds with > 90 % purity were obtained. These compounds significantly alleviated microglial inflammatory responses and reduced APP and BACE1 expression in vitro , indicating multi-target contributions to cognitive health support. The integrated workflow demonstrates strong scalability, operational stability, and green processing advantages, positioning G. jasminoides -derived constituents as promising functional food and nutraceutical ingredients and providing technical support for industrial-scale production. • Affinity UF-LC-MS and MD identified AChE-binding compounds in G. jasminoides fruit. • UNIFAC model optimized biphasic system composition, enhancing separation efficiency. • Consecutive-CCC and semi-preparativeHPLC isolated high-purity compounds. • The isolated compounds demonstrate neuroprotective potential. • The workflow enables scalable production of plant-based cognitive health ingredients.