Study On The Preparation And Antitumor Activity Of Peptides From Mylopharyngodon Piceus Muscle

In this paper,the enzymatic hydrolysis process for preparing antitumor peptide was optimized by single factor and orthogonal experiment using black carp(Mylopharyngodon piceus)as raw material.Then,Sephadex G-15 was used to separate the two-step enzymatic hydrolysates.In addition,Nano-LC-ESI-Q-Orbitrap-MS/MS was used to identify the components with the strongest antitumor ability,and the peptides matching the characteristics of antitumor peptides were selected for synthesis.The tumor cell proliferation inhibition experiment was carried out to verify the effect,and the structure-activity relationship between amino acid composition and antitumor ability was explored.In order to provide some theoretical basis for the practical application of health food and polypeptide drugs.The main research results are as follows:(1)The basic composition meat of black carp was determined,and it was proved that meat of black carp is a kind of meat resource with high protein and low fat.One-step enzymatic hydrolysis meat of black carp by alkaline protease was conducted,and the antioxidant capacity and proliferation inhibition rate were taken as the evaluation indexes.It was found that the two-step enzymatic hydrolysis of compound protease had the best antioxidant activity and higher proliferation inhibition rate of cancer cells.Single factor experiment and orthogonal experiment were used to optimize the experimental conditions of the two-step enzymatic hydrolysis for the preparation of active peptide of black carp fish:solid-liquid ratio,2:10(g:m L);p H,8.0;volume of enzyme,6 000 U/g;temperature,50?;hydrolysis time,3 h.Under these conditions,a two-step enzymatic hydrolysate was prepared,which exhibited strong DPPH·scavenging ability with the IC₅₀ value of 9.52 mg/m L,at 10 mg/m L,the proliferation inhibition rate of Hep G₂ cells was 80.97%.(2)According to the molecular weight distribution of the two-step enzymatic hydrolysates was determined.Sephadex G-15 chromatographic column was used to separate and purify the two-step enzymatic hydrolysates.The amino acid content of each separated component was determined.The antioxidant activity and antitumor ability were determined.The amino acid sequences of the components with the strongest antitumor ability were identified by Nano-LC-ESI-Q-Orbitrap-MS/MS technology.The results showed that the two-step enzymatic hydrolysates were mostly composed of oligopeptides with molecular weights ranging from 130 to 1 400 Da.After separation and purification by Sephadex G15,four active components were obtained.Among them,fraction 4 was rich in hydrophobic and acidic amino acids and had the strongest antioxidant activity and antitumor ability,with IC₅₀ value of 3.17 mg/m L,which was 3.0 times higher than that before separation and purification.Under the same conditions,the proliferation inhibition rate of Hep G₂ cells was increased to 92.54%.From fraction 4,15 peptides with ALC>95%was identified through Nano–LC–ESI–Q–Orbitrap–MS/MS,and oligopeptides with potential antitumor effects were synthesized.(3)To verify 15 peptides inhibitory mechanism on Hep G₂ cells,the oligopeptides of LPPW were subjected to AO/EB double fluorescence staining,caspase-9concentration changes in cell,cell cycle and apoptosis.It was found that after treatment with 0,0.5,2.5 and 5.0 mg/m L polypeptide solution,the proportion of damaged cells gradually increased after AO/EB fluorescence double staining.The content of intracellular Caspase-9 increased simultaneously.The proportion of G₀/G₁ phase cells in the control group was(38.89±2.74)%,and the proportion of G₀/G₁ phase cells treated with polypeptide was(45.95±4.32)%(83.94±0.91)%and(90.88±0.93)%,respectively.The apoptosis rates of Hep G₂ cells were 3.8%,7.4%,13.7%and 19.4%,respectively,suggesting that LPPW could induce Hep G₂ cell apoptosis in a dose dependent manner...