| Objective: The purpose of this study was to isolate and identify peptides with antioxidant activity from hydrolysate of tartary buckwheat albumin hydrolyzed by multiple protease.The protective effects on oxLDL-induced oxidative damage model of human umbilical vein endothelial cells(HUVEC)were analyzed.Methods: 1.Alkaline protease,papain,pepsin and trypsin were screened using the degree of hydrolysis(DH)as the indicator to determine the best protease,and the conditions of alkaline protease hydrolysis were optimized by single factor test and response surface method to determine the optimal hydrolysis conditions.2.Hydroxyl radical scavenging activity was used as an indicator.The tartary buckwheat protein hydrolysate was isolated and purified by ultrafiltration,ion exchange chromatography,gel chromatography,RP-HPLC to obtain the best antioxidant peptide,and the amino acid sequences of peptides were identified by N-terminal sequencing and Q-Tof mass spectrometry.Finally,the antioxidant activity of the purified peptide was comprehensively analyzed from four aspects: hydroxyl radical scavenging activity,DPPH scavenging activity,reducing power and lipid peroxidation inhibitory activity.3.HUVEC were incubated with ox-LDL of different concentrations for 24 h to screen for the optimal concentration of ox-LDL that could lead to oxidantive damage of cells.Experimental group: blank control group,ox-LDL model group,P3 peptide(low concentration group,high concentration group),GSH positive drug control group.The cell viability was detected by MTT assay;malondialdehyde(MDA)content and lactate dehydrogenase(LDH)activity in each cell culture medium were measured;reactive oxygen species(ROS),nitric oxide(NO)and superoxide dismutase(SOD)were measured in each group of cells.The expression of LOX-1,NF-kB,ICAM-1 and VCAM-1 in each group was detected by Western blot.Results: 1.The alkaline protease was identified as tartary buckwheat protein hydrolyzing protease.The optimal enzymatic hydrolysis process of alkaline protease was pH 9.1,temperature 45°C,time 3.5h,and the degree of hydrolysis obtained under this condition reached 27.52%.2.Three antioxidant peptides(P1,P2 and P3)were successfully isolated from tartary buckwheat albumin hydrolysate,and their sequences were Gly-Glu-Val-Pro-Trp(GEVPW),Tyr-Met-Glu-Asn-Phe(YMENF),and Ala-Phe-Tyr-Arg-Trp(AFYRW),and their molecular weights were 586.65 Da,702.79 Da and 741.85 Da,respectively.The same sequence of active peptides was not retrieved in BIOPEP-UWM(Bioactive Peptide Database).P3 peptide(AFYRW)exhibits the strongest antioxidant activity.Its IC50 values for scavenging hydroxyl radicals(OH·)and DPPH· free radicals were 0.65 mM and 0.64 mM,respectively.In addition,P3 peptide exhibited the strongest lipid peroxidation inhibition ability and the highest reducing power.3.The ox-LDL concentration was set to 100 ?g/mL to cause the cell oxidative damge.The P3 peptide could increase the cell viability of oxidatively damaged HUVECs,especially at 1 ?g/mL,the P3 peptide could increase cell viability to 84.27%.Pretreatment of HUVEC with P3 peptide(0.25?g/mL,1?g/mL)could reduce the content of ROS,MDA and LDH in HUVEC culture medium damaged by ox-LDL,and the levels of SOD and NO in HUVECs damaged by ox-LDL could also be increased.Western blot results showed: Compared with the ox-LDL oxidative damage model group,P3 peptide(0.25?g/mL,1?g/mL)and positive control GSH pretreated HUVEC could reduce the expression of LOX-1,NF-kB,ICAM-1,VCAM-1,and the high concentration group(1?g/mL)and GSH group were the most obvious,and the difference was statistically significant(P<0.05).Conclusion: The antioxidant peptides(P1,P2 and P3)were successfully prepared,and all three peptides have good antioxidant activity in vitro.P3 peptide had protective effect on oxidative damage of HUVEC induced by ox-LDL. |
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