The Structural Characterization Of Flaxseed Peptides Of Cholesterol-lowering And Its Cholesterol Reduction Mechanism In HepG2 Cells

Flaxseed protein was enzymolyzed to prepare flaxseed protein peptide with cholesterol lowering activity,and its purification and structure were identified.Flaxseed peptide was synthesized by chemical solid-phase synthesis method,and its cholesterol lowering activity was analyzed.The mechanism of chemical solid-phase synthesis of flaxseed peptide on human hepatoma cell line HepG2 was preliminarily explored,so as to provide theoretical basis for further processing and utilization of flaxseed protein and flaxseed peptide.The main results are as follows:1.Flaxseed protein was hydrolyzed by Protease M to prepare flaxseed protein peptide with cholesterol lowering activity.The flaxseed protein peptide was purified and its structure was identified.The corresponding flaxseed peptide was synthesized by chemical solid phase synthesis to analyze its cholesterol lowering activity.The results showed that the cholesterol lowering activity of flaxseed protein hydrolysate peptide was the highest,47.57%,when flaxseed protein hydrolysate reached 4 h.Ultrafiltration technology was used to separate them into five components with molecular weight ?3 kDa,3-5 kDa,5-10 kDa,10-30 kDa and >30 kDa.It was found that the component with molecular weigh ?3 kDa had the highest cholesterol lowering activity of 70.96%.After adsorption with macroporous resin and elution with 75% ethanol,the cholesterol lowering activity of the component with molecular weight ?3 kDa was the highest,which was 79.84%.Nine components were separated by RP-HPLC,and four components with high peak value were collected.Among them,F9 component had the highest cholesterol lowering activity(85.72%).Finally,six flaxseed peptides were identified by MALDI-TOF-MS/MS from the F9 fraction,and their sequences were Ile-Ile-Pro-Ala-Phe(IIPAF),Leu-Asn-Phe-Phe(LNFF),Leu-Leu-Gly-Thr-Leu(L LGTL),Ile-Ile-Phe(IIF),Ile-Pro-Pro-Phe(IPPF)and Leu-Leu-Gly-Ala(LLGA).The inhibition rates of cholesterol micelle solubility were 82.81%,77.77%respectively 88.02%,80.31%,93.47% and 87.06%.2.We used western blot and dot blot to identify the flaxseed peptide IPPF which has the highest cholesterol-lowering activity after chemical-solid phase synthesis.Western blot analysis showed that there was a positive reaction band between 17?26 kDa of flaxseed protein,which proved that the peptide IPPF existed in flaxseed's protein sequence.In addition,dot blot showed that there was IPPF in both flaxseed protein and flaxseed's proteolytic peptides with different hydrolysis degree.When the hydrolysis time was 4 h and the degree of hydrolysis was 14.25%,the relative content of IPPF was the higher in the protease-hydrolyzed peptides of flaxseed with cholesterol-lowering activity.3.We used human hepatocellular carcinoma cell line HepG2 as a model to investigate the cholesterol-lowering mechanism in HepG2 cells of flaxseed peptide IPPF which synthesized by chemical-solid phase.The results showed that compared with the control group,the levels of TC and TG in HepG2 cells were significantly decreased when the concentration of flaxseed peptide IPPF was 200 ?g/m L,400 ?g/m L and 800 ?g/m L in a dose-dependent manner.When the concentration of flaxseed peptide IPPF was 800 ?g/m L,the expressions of SREBP-2,LDLR,HMGCR,LXR-? and CYP7A1 mRNA were significantly up-regulated.