Protective Effects Of Wheat Germ Protein Hydrolysates On Oxidative Injured Cell And Characterization Of Related Peptides

Wheat germ, as a by-product of wheat processing, has a relatively high protein contentand high nutritional properties. Latest studies have confirmed that wheat germ protein hadcertain sequence of various physiological activities. To improve the antioxidant activityevaluation system and the application direction of WGPIH, the biological effects of WGPIHon oxidative stress in cells was evaluated.Firstly, the antioxidant activities of different WGPIH were studied. The H₂O₂-inducedinjury on PC12cells as a model was established. The cell viability was decreased to about50%when PC12cells were treated for2h by200μmol/L H₂O₂after PC12cells were culturedfor48h. A dose-dependent increase in antioxidant activities of WGPIH1^WGPIH5wasshowed in three antioxidant models in vitro and in H₂O₂-induced injury on PC12cells model.The results showed that WGPIH5（enzymolysis time was240min） possessed the highestantioxidant activity. The oxidation of linoleic acid was significantly inhibited. The IC₅₀valuefor ferrous iron-chelating activity was0.37±0.05mg/mL. The TEAC value reached0.81±0.01mmol/L Trolox. The cell viability was observed to increased by32.85%and the LDH leakagewas observed to decrease by4.33%compared to the viability of the H₂O₂-stressed PC12cells.Secondly, the composition of amino acids and the molecular weight distribution profileswere measured. The composition of essential amino acid of WGPIH5reached30.17%and thecomposition of hydrophobic amino acids was high. The percent of relative molecular weightbetween180Da and2000Da reached86.72%.Thirdly, in the present study, PC12cells were used as an oxidative stress model toinvestigate the protective effects of WGPIH against H₂O₂-induced cytotoxicity and apoptosis,as well as its underlying mechanisms. The cytotoxicity was assessed by morphologicalobservation, the activities of antioxidant enzymes and malondialdehyde （MDA） production.The morphological alteration was observed by phase-contrast microscopy and the micro-structure of the PC12cell surface was observed by SEM. PC12cells kept normalmorphology without congregating and notable cell number diminishing by WGPIH5（1mg/mL）. What’s more, the number of apoptotic bodies on the surface of PC12cell decreasedby WGPIH5（1mg/mL）. The results demonstrated that pretreatment of PC12cells with1mg/mL WGPIH5, prior to H₂O₂exposure, significantly increased the survival of cells and theactivities of catalase （CAT） and superoxide dismutase （SOD）, and reduced the content ofMDA. The apoptosis was monitored by flow cytometric analysis. The presence of WGPIH5attenuated8.10%the apoptotic portion Sub-G1DNA contents and decreased11.20%the（Annexin V-positive cell） apoptosis. The accumulation of intracellular Ca²⁺and the reductionin mitochondrial membrane potential were also inhibited by WGPIH treatment. In conclusion,it is confirmed that WGPIH5possesses protective effects in PC12cells against H₂O₂-inducedinjury.Finaly, WGPIH5was fractionated by ethanol elution from a macroporous adsorptionresin （DA201-C） to get better quality. WGPIH5was separated into five fractions （WGPIH5-a to WGPIH5-e）.35%ethanol fractions （WGPIH5-b） exhibited the highest antioxidant activity.The cell viability was observed to increase by33.51%. WGPIH5-b was separated bySuperdex Peptide10/300GL further. WGPIH5-b was separated into five fractions （WGPIH5-b1to WGPIH5-b5） and the antioxidant activity of WGPIH5-b2was the highest. The cellviability was observed to increase by34.43%. WGPIH5-b2was then identified by MALDI-TOF-TOF MS/MS. Their molecular weight of WGPIH5-b2was1221Da and2168Da. Then,the amino acid sequences were His-Trp-Pro-Leu-His-Lys-Val-His-Ala-Pro （HWPLHKVHAP）and Pro-Lys-Lys-Ser-Asp-Arg-Cys-Phe-Thr-Leu-Asp-Lys-Cys-Ala-His-Thr-Tyr-Asn （PKKSDRCFTLRKCAHTYN）, respectively.