Preparation Of Lupin Antioxidant Peptides And Its Effect On The Protection Against Oxidative Damage In HepG2 Cells

In this study,lupin protein was prepared from lupin flour using a method of alkali extraction and acid precipitation,following by hydrolysis and ultrafiltration to acquire lupin peptides.Subsequently,the antioxidant activity of lupin peptides was evaluated by using a combination of chemical methods together with an in vitro cell experiment to screen out the lupin peptide with highest antioxidant activity,which might provide a theoretical evidence for the development and application of lupin active peptide.Firstly,lupin protein hydrolystes were produced using Alcalase,Pepsin and Trypsin.The antioxidant and ACE inhibitory activities were analyzed using chemical methods.The results showed that hydrolysis of proteins resulted in a significant increases in bioactivities.In general,Alcalase hydrolysis had the highest antioxidant activity,whilst the hydrolysates from Trypsin displayed the highest ACE inhibitory at 30 min proteolysis.The hydrolystes were subsequently fractionated into three fractions,namely LPH1(MW>10 KDa),LPH2(MW3-10KDa)and LPH3(MW<3KDa),and the antioxidant and ACE inhibitory activities of the each fractions were analyzed.It was revaled that LPH3 had the highest antioxidant and ACE inhibitory activities.Analysis of amino acid components showed that the high antioxidant and ACE inhibitory activities of LPH3 were related to the adequate hydrophobic amino acids and aromatic amino acids.Secondly,the protection against H2O2-induced oxidative damage to HepG2 cells of lupin peptide was investigated using in vitro cell experiments.The cytotoxic effect or proliferatin effect of the lupin peptides on HepG2 cells was carried out and the results revaled that the three fractions in the concentrations of 4,6 and 8 mg/mL did not show cytotoxicity or proliferation of HepG2 cells.Based on the cell viability,the oxidatively damaged HepG2 cell model was established by H2O2,and the optimal injury concentration and time of H2O2 were 300 μmol/L and 4 h,respectively,when the HepG2 cell viability reduced to approximately 47.68± 3.75%.Through this model,the protection against oxidative damaged effects of the three fractions were investigated.It was showed that the three fractions all improved the cell viability of damaged cells and the LPH3 fracton had the strongest protective effect on cells.Finally,based on the results of chemical mthod and cell experiment,the LPH3 fraction was screened out to carry out the research on cellular protective mechanism.A variety of cell specific markers were analyzed and the results indicated that LPH3 treatment inhibited the intracellulare accumulation of ROS and reduced the levels of MDA as well as increased the activities of super oxide dismutase(SOD)and glutachione peroxidase(GSH-PX),compared with the oxidatively damaged cells.The changes in the expressions of genes about oxidative strees and apoptotic in the HepG2 cells were detected by qPCR.The results revaled that LPH3 treatment significantly up regulated the expression of SOD1,GPX1,GCLM,SLC7A11 and SRXN1.Moreover,the administration of LPH3 also down regulated the expression of CASP3 and p53 and up regulated the Bcl-2 level.These results suggested that LPH3 protected cells from oxidative damage by inhibiting oxidative stress and blocking mitochondrial apoptosis pathway.