Extraction and characterization of polysaccharides from blackcurrant fruits and its inhibitory effects on acetylcholinesterase.

Microwave assisted aqueous two-phase system (MA-ATPS) was used to simultaneously extract two polysaccharides from blackcurrant. Under the suitable ATPS (ethanol/(NH)SO, 26.75 %/18.98 %) combining with the optimal MA conditions (liquid-to-material ratio 58.5 mL/g, time 9.5 min, temperature 60.5 °C, power 587 W) predicted by response surface methodology, the yields of the top/bottom phase polysaccharides were 13.08 ± 0.37 % and 42.65 ± 0.89 %, respectively. After purification through column chromatography, the top phase polysaccharide (PRTP) and bottom phase polysaccharide (PRBP) were obtained. FT-IR, methylation and NMR analyses confirmed that the repeating unit in the backbone of PRTP was →2, 5)-α-L-Araf-(1 → 3)-α-D-Manp-(1 → 6)-β-D-Galp-(1 → 6)-α-D-Glcp-(1 → 4)-α-L-Rhap-(1 → 4)-α-D-GalAp-(1→, while the possible unit in PRBP was →4)-α-L-Rhap-(1 → 3)-α-D-Manp-(1 → 6)-β-D-Galp-(1 → 6)-α-D-Glcp-(1 → 2, 5)-α-L-Araf-(1 → 4)-α-D-GalAp-(1→. PRBP with relatively low molecular weight exhibited better stability, rheological property, free radical scavenging and acetylcholinesterase (AChE) inhibitory activities than PRTP. PRTP and PRBP were reversible mixed-type inhibitors for AChE, and the conformation of AChE was changed after binding with the polysaccharides. Molecular docking, fluorescence and isothermal titration calorimetry assays revealed that PRTP and PRBP quenched the fluorescence through static quenching mechanism, and the van der Waals interactions and hydrogen bonding played key roles in the stability of polysaccharide-enzyme complexes. This study provided a theoretical basis for blackcurrant polysaccharides as AChE inhibitors to treat Alzheimer's disease.