Preliminary Study On Cholesterol-Lowering Effect And Mechanism Of Flaxseed Proteolytic Peptides Ile-Pro-Pro-Phe

Flaxseed protein has the same nutritional value as soy protein and is a high-quality plant protein resource.The main use of flaxseed in China is the production of oil,the protein content of the by-product flaxseed cake after oil extraction is 32%-49%,and most of the flaxseed cake is used as feed,resulting in a large amount of waste of high-quality plant protein resources,so it is particularly important to extract protein from flaxseed cake and deep processing and utilization.In addition,long-term consumption of cholesterollowering drugs can cause various adverse side effects,so it is necessary to seek foodborne cholesterol-lowering active ingredients that are safe,effective,and free of toxic side effects.To this end,in the early stage of our research group with the purpose of efficient use of flaxseed protein,using low-temperature defatted flaxseed cake as raw material,extracting flaxseed protein,enzymatically hydrolyzing,separating and purifying the enzymatic digest with the inhibition rate of cholesterol micelle solubility,that is,cholesterol-lowering activity in vitro,and screening and identifying 6 kinds of flaxseed protein enzymatic hydrolysis peptides,but its cholesterol-lowering effect and mechanism in the body are not clear.In this study,six kinds of flaxseed proteolytic peptides were synthesized by chemical solid-phase synthesis,and their purity,molecular weight,cholesterol-lowering activity in vitro,and their interaction with cholesterol molecules were explored,on this basis,flaxseed proteolytic peptides with the highest cholesterol-lowering activity in vitro were selected for animal experiments to explore their cholesterol-lowering effect and mechanism in vivo.The main findings are as follows:1.Six kinds of flaxseed proteolytic peptides Ile-Pro-Pro-Phe(IPPF),Ile-Ile-Pro-AlaPhe(IIPAF),Leu-Asn-Phe-Phe(LNFF),Leu-Leu-Gly-Thr-Leu(LLGTL),Leu-Leu-GlyThr-Leu(LLGTL),Ile-Ile-Phe(IIF)and Leu-Leu-Gly-Ala(LLGA),analyzed their purity and molecular weight,and found that the purity of six chemical solid phase synthetic flaxseed proteolytic peptides was higher than 96%,and their actual relative molecular weight was consistent with the theoretical relative molecular weight,indicating that the target peptide with higher purity was obtained by chemical solid-phase synthesis.The inhibition rate of cholesterol micellar solubility was further analyzed,and it was found that the cholesterol micelle solubility inhibition rate was 93.47%,82.81%,77.77%,88.02%,80.31%,and 87.06%,respectively,among which the cholesterol micellar solubility inhibition rate of flaxseed proteolytic peptide IPPF(flaxseed peptide IPPF)was the highest93.47%.Therefore,the interaction and binding stability of flaxseed peptide IPPF were further analyzed by molecular docking and molecular dynamics simulation methods,and the results showed that flaxseed peptide IPPF could bind to cholesterol molecules and had good stability.For this reason,flaxseed peptide IPPF was selected to further explore its cholesterol-lowering effect and mechanism in vivo.2.The cholesterol-lowering effect of flaxseed peptide IPPF was explored through animal experiments,and it was found that after giving gastric flaxseed peptide IPPF 42days(6 weeks)to mice on a high-cholesterol diet,it was possible to increase food intake,body weight,and kidney index of mice on the high-cholesterol diet,reduce liver index,reduce serum total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C)levels,increase serum high-density lipoprotein cholesterol(HDL-C)levels,and reduce liver TC,TG levels,increase the excretion of TC,TG,total bile acids(TBA)in feces,improve the degree of hepatic steatomosis,and can reduce oxidative stress caused by high-cholesterol diets by reducing malondialdehyde(MDA)levels,increasing superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and catalase(CAT)levels,indicating that flaxseed peptide IPPF plays a cholesterol-lowering effect.3.Further metabolomics technology and real-time PCR technology based on ultraperformance liquid chromatography-tandem time-of-flight mass spectrometry(UHPLC/QTOF-MS)preliminarily explored the cholesterol-lowering mechanism of flaxseed peptide IPPF in the body,and found that after giving high-cholesterol diet mice gavage flaxseed peptide IPPF,20 cholesterol-related metabolites such as glycine,palmitic acid,and cholesterol in the liver changed significantly.Biomarkers of amino acid metabolism and lipid metabolism;The effect of flaxseed peptide IPPF on the expression level of genes involved in liver cholesterol metabolism was explored by real-time PCR,and the results showed that flaxseed peptide IPPF inhibited the absorption of liver cholesterol by reducing the expression of NPC1L1 m RNA,a gene related to cholesterol absorption in the liver of mice on a high-cholesterol diet.It promotes cholesterol efflux by increasing the expression of genes CYP7A1 related to bile acid synthesis,cholesterol efflux genes ABCG5 and ABCG8,and LDLR m RNA genes related to LDL-C clearance.