Screening Of Novel Antioxidant Peptides In Duck Embryos And Protection Mechanism Against Oxidative Damage In Cells

Duck embryo eggs are not only rich in nutrients,but also have certain functional effects,which play an important role in human nutrition and health.Duck eggs as the main raw material of health food development prospects are very broad.Duck embryo eggs had a certain antioxidant activity,but the specific substances for this function were not clear.At the same time,duck embryo eggs contain a large number of peptides,which have not been studied.The dynamic changes of endogenous antioxidants during incubation of duck embryo eggs were studied.The duck embryo eggs incubated for 15 days（presented high total antioxidant activities）were degreased to explore the relationship between the antioxidant activity and the molecular weight range of duck embryo peptides.The antioxidant activity of different molecular fragments of ZT1（≤3 k Da）,ZT2（≤10 k Da）and ZT3（≤30 k Da）in duck embryo and their protective effect on H₂O₂ induced oxidant damage of Hep G2 cells were investigated.The peptides with the strongest antioxidant activity（≤3 k Da）were separated by ion exchange column chromatography,and the peptides were identified by LC-MS/MS for further functional annotation by bio-informatics prediction and hot spot recognition.（Ⅰ）Changes of endogenous antioxidants in duck embryo eggs during incubation have been studied.The antioxidant substances,including vitamin E（VE）,glutathione（GSH）,reduced glutathione（reduced GSH）and oxidized glutathione（GSSG）increased during the early incubation period and decreased in the middle and late periods;meanwhile,the total antioxidant capacity（T-AOC）of duck embryo eggs increased during early incubation and then decreased,reaching the highest level on Day 15.The change trends of GSH,reduced GSH and GSSG were approximately the same as that of T-AOC which suggesting that they may represent be the key factors determining the T-AOC of duck embryo eggs.In addition,the contents of superoxide dismutase（SOD）,vitamin C（VC）,catalase（CAT）,and glutathione peroxidase（GSH-px）showed increasing trends during the incubation period.The content of malonic dialdehyde（MDA）presented a peak on Day 18.（Ⅱ）Our previous work showed that the peptides from duck eggs with incubated for 15 days present high total antioxidant activities;however,the relationship between the antioxidant activity and the molecular weight range of these peptides,and the detailed characteristics are still unknown.Here,we explored the antioxidant activities of different segments,ZT1,ZT2 and ZT3 derived from duck embryo peptides and their protective effects on H₂O₂-induced oxidative damage in Hep G2 cells.We showed that these peptides present no cytotoxicity on Hep G2 cells.Moreover,ZT1 exhibited stronger abilities to scavenge diphenyl picryl hydrazinyl radical,superoxide anion,hydroxyl radical and hydrogen peroxide radical,as well as a stronger inhibitory effect on H₂O₂-induced oxidative stress by reducing intracellular reactive oxygen species accumulation than ZT2 and ZT3.The activities of catalase and glutathione peroxidase and the total superoxide dismutase increased in a concentration-dependent manner.（Ⅲ）The peptides were isolated from ZT1 by ultra filtration and ion exchange column chromatography,then subjected to LC-MS/MS to identify the sequences following by function annotation,bioinformatics prediction and hot-spot motif recognition.As a result,413 potential functional peptides are identified,with some compounds exhibiting more than one function.（Ⅳ）A total of 132 antioxidant peptides were obtained by screening the antioxidation peptide motifs,of which included 5 peptides selected by GO annotation.The antioxidant function of the five peptides annotated by GO was verified in vitro.The results showed that all the five peptides had strong antioxidant activity,among which LA18,RK14 and TD12 had obvious effects.（Ⅴ）Western blot analysis studied the effect of TD12 on the levels of Bcl-2,cleaved caspase-3 and cleaved PARP protein.The results indicated that TD12 can inhibit cell apoptosis by regulating the mitochondrial apoptosis pathway in cells.The above results show that duck embryo eggs produce abundant endogenous antioxidants during incubation,and the highest antioxidant activity can be observed at the 15 th day during incubation.In addition to the role of endogenous antioxidants,the peptides derived from duck embryo eggs also exhibit strong antioxidant activity and protect Hep G2 cells from oxidative damage induced by H₂O₂.Functional annotation,bio-informatics prediction and hot spot recognition have found that duck embryo eggs contain multiple functional peptides.This work is helpful to explore the bioactive substances of duck embryo and further improve the utilization value of duck eggs or other food raw materials.