| Corbicula fluminea(C.fluminea),a member of the family Cyrenidae originated in Eastern Asia,is considered as a kind of both functional food and medicine in traditional Chinese medicine for hepatic protection.Nutritious as it is,C.fluminea is reported to be abundant in protein,glycogen,animo acid,taurine,vitamins and microelements.C.fluminea processed into dry powder from Taiwan was used in this thesis.Its protein content is as high as 56.59 ± 0.50 %,and the protein hydrolysates,obtained by hydrolysis using several proteases,were used for the further experiments.The bioactive peptides were purified and identified,and evaluated by its protective effect on ethanol-injured LO2 cell.Five proteases(alkaline protease,dispase,papain,pancreatin and flavourzyme)were selected to hydrolyze the C.fluminea protein respectively,and different hydrolysates of the C.fluminea were obtained.DPPH free radical scavenging assay showed that different hydrolysates of C.fluminea had certain DPPH free radical scavenging ability.ORAC experiments showed that the ORAC values of the hydrolysates of pancreatin and flavourzyme were 2.54 ± 0.18 and 2.59 ± 0.06 mmol Trolox/g,respectively,which were significantly higher than those of other hydrolysates.The results of MTT assay and ALT,AST activity assay showed that the cell viability decreased to 53.49 ± 5.33% when the ethanol concentration was 0.8 mol/L.And at the same time,the activities of ALT and AST in the culture medium were also significantly higher than that of the blank group.Therefore,it was certified that the ethanol-induced LO2 cell injury model was established successfully.The cell experiments revealed that compared with the model group,the cell viabilities of hydrolyzates pre-treated group were significantly increased.Besides,the hydrolysates of pancreatin and flavourzyme had the best protective effect.Both of them could inhibit the secretion of ALT and AST in LO2 cells and alleviate the damage of LO2 cells induced by ethanol.PCH(hydrolysate obtained by pancreatin)and FCH(hydrolysate obtained by flavourzyme)contained 17 amino acids,of which the ratios of essential amino acids to total amino acids were 38.67% and 37.90%,respectively.And 75.69% of the amino acids from total amino acids of PCH and 75.06% from total amino acids of FCH have anti-oxidative capacity.Molecular weight distribution measured by HPLC method showed that 53.37% and 86.93% of the peptides had a molecular weight of less than 1000 Da in PCH and FCH,respectively.After PCH and FCH were separated and purified by Sephadex G-15,the components with the best anti-oxidative capacity conducted by ORAC were selected for UPLC-MS/MS experiments.Six peptides from C.fluminea were identified and their sequences were: RRKC(RC-4),DAPF(DF-4),LVYP(LP-4),MASR(MR-4),ELY(EY-3),and YFLP(YP-4).The six peptidess were obtained by solid-phase synthesis under the standard Fomc strategy.And the purity was above 99%.The ORAC values of MR-4,YP-4,EY-3,and LP-4 were significantly higher than those of GSH.The ORAC values of peptides YP-4 and LP-4,the top two,were 2.25 ± 0.07 and 2.18 ± 0.28 mol Trolox/mol,respectively.Peptides had no significant effect on the proliferation of LO2 cells within a certain concentration range.MR-4,YP-4,DF-4,EY-3,and LP-4 all markedly increased the viabilities of the LO2 cells,among which the protective effects of YP-4 and LP-4 on ethanol-induced LO2 cells were the best.Moreover,both of YP-4 and LP-4 showed the inhibitory ability to the decrease of mitochondrial membrane potential(MMP,??m)induced by ethanol and attenuated the mitochondrial dysfunction.The two peptides also could inhibit ethanol-induced reactive oxygen species(RPS)formation in LO2 cells.That indicated the protective effect of YP-4 and LP-4 on ethanol-induced LO2 injury. |
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