Study On Functional Activity Of Walnut Peptides

In this paper,the functional activity of walnut peptides(WPs)were studied and the cell proliferation activity,antitumor activity and ACE-inhibitory activity of WPs were examined.The WPs was divided by ultrafiltration,the sequence of the peptides was identified by UPLC-MS/MS.The main conclusions are as followings:1.The molecular weight(MW)distribution of WPs by ultrafiltration.After ultrafiltration,the WPs were divided into four parts,the peptide yield of F1(MW<10 KDa)was 15.71%and the peptide content was 69.21%,the peptide yield and peptide content of F2 fraction(5 KDa-10 KDa)were 22.93%and 67.43%,the peptide yield and peptide content of F3 fraction(1 KDa-5 KDa)were 29.24%and 75.31%,the peptide yield and peptide content of F4 fraction(<1 KDa)were 32.11%and 63.18%.The results of molecular weight distribution of WPs showed that the molecular weight of walnut protein decreased significantly compared with those not fermented and the SSF is a feasible and efficient method for preparation of walnut peptides.2.The ACE-inhibitory activity of walnut peptides.The ACE-inhibitory activity results showed that the IC50 values of F1,F2,F3 and F4 were 330 ?g/mL,263.8?g/mL,102.1 ?g/mL and 39.76 ?g/mL.The fraction corresponding to MW<1 kDa exhibited the highest ACE-inhibitory activity and very good fits with sigmoid curves and reproducible IC50 values were obtained.The inhibition type of F4 was noncompetitive inhibition.3.The cell proliferation activity of walnut peptides.According to the cell proliferation activity of different fractions,the part of F3 showed the highest proliferation activity.The fractions of F1,F2,F3 and WPs exerted proliferative efficacy in 3T3-L1 cells but has no cytotoxic effect on it.The fraction of F4 exerted cytotoxic and antiproliferative efficacy in 3T3-L1 cells.The fraction of F3 effected on 3T3-L1 cell growth kinetics,it could represent a new and interesting alternative for animal cell culture processes.In addition,the selected walnut peptide fraction allowed the formulation of a low-serum medium,based on DMEM basic medium,which did not compromise cell growth;on the contrary the cell death rate was strongly reduced in this medium in comparison with the peptide.Besides,the morphologic changes of 3T3-L1 by F4 was also examined,3T3-L1 cells treated with F4 revealed a disrupted cell membrane.4.The antitumor activity of walnut peptides.The antitumor activity was examined by MTT method.The peptide mixture had a proliferative efficacy on SGC-7901 cells and S180 cells,and the four fractions of WPs exerted cytotoxic and antiproliferative efficacy in the two cells but F2 had a proliferative efficacy on S180 cells.SGC-7901 cells and S180 cells were treated with F4,the IC50 value of SGC-7901 cells was 0.648 mg/mL and the IC50 value of S180 cells was 0.932 mg/mL.In addition,F4 would reduce the number of S180 cells and revealed a disrupted cell membrane,the cell had a heterogeneity size.5.The molecular weight and amino acid sequence of F4 peptide.The walnut peptides were purified sequentially by ultrafiltration and RP-HPLC,the sequence of the F4 peptide with the highest biological activity was identified to be Val-Gln-Thr-Leu(VQTL,459.351 Da)and Leu-Gly-Tyr-Glu-Asn(LGYEN,594.276 Da).