Purification And Identification Of Antioxidant Peptides From Defatted Walnut Meal Hydrolysate And Its Memory Improvement Effect

The aim of this study was to isolate memory enhancing peptide from defatted walnut meal hydrolysate(DWMH) and identify novel antioxidant and potential memory enhancing peptides by Ultra performance liquid chromatography-tandem mass spectrometry(UPLC-ESI-MS/MS). Defatted walnut meal was hydrolyzed with pancreatin. Antioxidant activity in vitro and memory enhancing effect in vivo of DWMH were evaluated. The hydroxyl scavenging capacity and oxygen radical absorbance capacity(ORAC) of DWMH were comparable to those of glutathione(GSH). DWMH exhibited stronger protective effect on H2O2-induced PC12 cells than GSH and cerebrolysin(Cere). DWMH also reduced the generation of the intracellular reactive oxygen species(ROS) and apoptosis rate of PC12 cells. Furthermore, DWMH could shorten the latency and total distance to the platform in Morris water maze test. It also prolonged the latency of the mice to enter the dark area and reduced the electric shock number in Dark/light avoidance test. Therefore, DWMH could improve the learning and memory performance of the D-galatose-treated mice, which might be related to its good antioxidant and free radical-scavenging activities. Then DWMH was subjected to SP-825 macroporous absorption resin and concentrated of hydrophobic fragments with the best protective effect on H2O2-injured PC12 cells. The active fraction was further separated by medium pressure liquid chromatography preparation system. The purified fraction was identified using UPLC-ESI-MS/MS and obtained 77 peptides. Six peptides with molecular weights of 833.86 Da, 962.98 Da, 1377.40 Da, 710.74 Da, 967.99 Da, 1124.18 Da were synthetized as WSREEQ, WSREEQE, WSREEQEREE, ADIYTE, ADIYTEEAG, ADIYTEEAGR, respectively. The peptides possessed protective effects against oxidative apoptosis and the ORAC values were higher than GSH. These findings are significant for the discovery and development of natural neuroprotective agent from defatted walnut meal, and the identified peptides may have potential applications as a functional food to improve learning and memory.