The Functional Walnut Peptide With Anti-amyloid ?1-42 Aggregation Activity And Its In Vitro/In Vivo Absorption And Metabolism Study

Oxidative stress plays an important role in the progression of Alzheimer's disease.Antioxidants have become a new direction for the prevention and treatment of AD.It can be applied to alleviate the clinical symptoms of AD patients by reducing the intracellular free radical production,reducing the level of oxidative stress and inhibiting the formation of A? deposition.Walnut is well recognized as health food that benefits our brain while the mechanism remains unknown.Walnut protein is considered to be one of its important active component whose antioxidant activity has been proved.In recent paper,walnut peptide was identified from walnut protein.Then the antioxidant activity,anti-A?1-42 aggregation activity and in vivo absorption and metabolism was evaluated.Walnut protein was used as the raw material to prepare the hydrolysate,from which 8 peptides were identified by LC-MS/MS.Amongs those walnut peptides,peptide WDQW(WW4)was selected as a potential antioxidant.Furthermore,WDQWCIW(WW7)was designed based on WW4.Theses two peptides were synthesized for the further study.Results from antioxidant experiments revealed that WW7(7.26 ± 0.92 ? mol Trolox/? mol sample)possessed not only the same oxygen free radical scavenging capacity as WW4(6.14 ± 0.60 ? mol Trolox/?mol sample)but also a stronger DPPH· radical scavenging capacity(IC50 66.39 ± 0.98? g/m L)than WW4.Both of them presented more favorable antioxidant activity than GSH.The E22G-mCherry Hek 293 cell model was used to evaluate the anti-A?1-42 aggregation activity of WW4 and WW7 via Incucyte Zoom long time live cell analysis system and the Amnis imaging flow cytometers detection.It was demonstrated by these two methods that only WW7 peptide presented a significant anti-A?1-42 aggregation activity.Besides,the cell viability,cell cycle and reactive oxygen species of E22G-mCherry Hek 293 cells were detected to evaluate the protective effect of WW7 and WW4 on A?-induced cell death and oxidative stress.WW7 peptide was demonstrated to have a significant reversal effect on the increasing of ROS level,S phase block of cell cycle and the decreasing of cell viability.However,the protective effet was not observed with the treatment of WW4.Result from the intracellular absorption and metabolism of WW4 and WW7 revealed that WW7 with excellent stability owned a lower absorption and metabolism rate than that of WW4.Low bioactivity of WW4.was due to its rapidly metabolized by lysosomes.The in vivo absorption and metabolism and the capability to cross blood brain barrier of WW7 and WW4 were later analyzed by tracing the distribution of FITC labeled WW4 and WW7 in BALB/c nude mouse via in vivo imaging instrument.Results from the in vivo imaging showed that WW7 had the capability to cross blood brain barrier.The distribution of WW7 in other organs was followed the order of heart < spleen < liver < lung < kidney < stomach-gut was also larger than WW4.Besides,WW7 with longer metabolic time exhibited a higher stability than that of WW4 in the in vivo study.In conclusion,WW7 peptide,designed based on walnut peptide WW4 was proved to be a potential functional food for AD prevention and treatment because of its favorable antioxidant activity,anti-A?1-42 aggregation activity,high in vivo stability and the capability to cross blood brain barrier.