Extraction and preparation of 5-lipoxygenase and acetylcholinesterase inhibitors from Astragalus membranaceus stems and leaves

In this study, an efficient method that employs 5-lipoxygenase and acetylcholinesterase as biological target molecules in receptor–ligand affinity ultrafiltration–liquid chromatography was developed for the screening of enzyme inhibitors derived from the Astragalus membranaceus stems and leaves. The effects of the extraction time, number of extraction cycles, ethanol concentration, and liquid–solid ratio on the total yield of the target compounds were investigated using response surface methodology, and the bioactive components were isolated using a combination of semi-preparative high-performance liquid chromatography and high-speed countercurrent chromatography via a two-phase solvent system consisting of n-hexane–ethyl acetate–methanol–water (1:6:2:6, v/v/v/v). Subsequently, 10 naturally-occurring bioactive components in the Astragalus membranaceus stems and leaves, including wogonin, ononin, isoquercitrin, calycosin-7-glucoside, 3-hydroxy-9,10-dimethoxyptercarpan, hyperoside, 7,2′-dihydroxy-3′,4′-dimethoxyisoflavan, baicalein, calycosin, and soyasaponin, were screened using affinity ultrafiltration to determine their potential effects against Alzheimer's disease. Consequently, all target compounds had purities higher than 95.0%, and the potential anti-Alzheimer's disease effect of the obtained bioactive compounds was verified using molecular docking analysis. Based on the results, the back-to-back screening of complex enzyme inhibitors and separation of the target bioactive compounds using complex chromatography could provide a new approach to the discovery and preparation of natural active ingredients.