Study On The Cholesterol-lowering Effect Of Flaxseed Peptide

Hyperlipidemia is the main factor for cardiovascular diseases such as atherosclerosis and coronary heart disease.Reduction of blood lipid level is the main method to prevent hyperlipidemia.The important strategy of reducing the plasma cholesterol level is regarded as lowering choleterol.In the experiment,the enzymolysis process of flaxseed peptide was optimized and graded preparation was carried out.Then this study investigated the effect and mechanism of the inhibition of flaxseed peptide,to evaluation of the Cholesterol absorption inhibiting ability of flaxseed peptide with cholesterol-lowering experiment in vitro.And a monolayer Caco-2 cell model was further established,to explore its effect on the absorption and transport of cholesterol in Caco-2 cells.Finally,through the induction of hyperlipidemia rat by high fat diet,the cholesterol-lowering effect and mechanism of flaxseed peptide in vivo were explored to provide a basis for the development and utilization of flaxseed.The main findings are as follows:1.The preparation of flaxseed cholesterol lowering peptide by hydrolysis of flaxseed protein isolate with proteasem was studied.The optimal preparation process was determined by single factor experiment and orthogonal experiment.The enzymatic hydrolysate of flaxseed was separated by ultrafiltration technology and its cholesterol lowering activity was evaluated.The results showed that the optimum preparation conditions were as follows:1.5%of enzyme,2.0%of substrate,50? of enzymolysis temperature and 3 hours of enzymolysis time.Under these conditions,the inhibition rate of cholesterol micelle solubility(cmsir)of enzymolysis peptide was 53.19%,and the percentage of components with molecular weight distribution of ?1kDa was the highest,reaching 65.54%.The ultrafiltration separation results showed that components with relative molecular mass ?1kDa have the strongest cholesterol-lowering activity,and the inhibition rate of cholesterol micelle solubility is 72.39%.The amino acid analysis results showed that the total hydrophobic amino acid content of the components whose molecular weight was less than or equal to 1 kDa after ultrafiltration was 15.97%higher than that before ultrafiltration,and the ratio of lysine to arginine was lower than that before ultrafiltration significantly,which may be the main reason why the cholesterol lowering activity of the components was stronger than that before ultrafiltration.2.To study the cholesterol-lowering effect of linseed peptide molecular weight ?1 kDa ultrafiltration components in vitro.The results showed that the linseed peptide had a certain scavenging effect on cholesterol,with a certain dose relationship.The scavenging rate of 2.5mg/ml linseed peptide reached 68.24%;the binding ability of different concentrations of linseed peptide to bile salts was certain,and the binding rates of 12mg/ml linseed peptide to sodium taurocholate,sodium cholate hydrate and sodium glycine cholate reached 52.05%and 6%,respectively 5.12%and 68.29%respectively;linseed peptide had inhibitory effect on pancreatic lipase and cholesterol esterase,with IC50 values of 6.20mg/ml and 4.57mg/ml.In addition,the flaxseed peptide had a certain inhibition on the formation of cholesterol micelles,and the inhibition rate of 12 mg/ml flaxseed peptide reached 57.18%.Based on the above results,it can be concluded that the flaxseed peptide plays a role in lowering cholesterol in vitro by removing cholesterol,combining with cholic acid salt,inhibiting the activities of pancreatic lipase and cholesterol esterase,and inhibiting the formation of cholesterol micelles.3.Caco-2 monolayer model was used to study the effect of linseed peptide with molecular weight?1 kDa on cholesterol absorption and transport.The results showed that after cultured 21 days,the TEER value of Caco-2 cell monolayer model reached 529.76?·cm2,and the alkaline phosphatase of AP and BL side was asymmetric distribution.Caco-2 cells basically covered the culture plate,and the model was established successfully,which can be used as an in vitro model to study the absorption and transport of small intestine in day 21.When the concentration of linseed peptide was 1.2,2.4 and 4.8mg/ml,it had no significant toxicity to Caco-2 cells;when the concentration of linseed peptide was 1.2,2.4 and 4.8mg/mL.The absorption and transport of cholesterol in Caco-2 cells were inhibited in a dose-dependent manner by linseed peptide.Flaxseed peptide may play a role in lowering cholesterol by inhibiting the absorption and transport of cholesterol in Caco-2 cells.4.The mechanism of lowering cholesterol of linseed peptide was studied by using hyperlipidemia rat model.32 male SD rats were fed with high-fat diet.The dosage of flaxseed peptide was 200mg/kg and 800mg/kg,and the experimental period was 4 weeks.The results showed that the low and high dosage linseed peptide could significantly reduce the body weight of hyperlipidemia rats;the low and high dosage linseed peptide could effectively reduce the serum TC,TG and LDL-C levels,improve the HDL-C level significantly;and the low and high dosage linseed peptide could significantly reduce the NPC1L1 level.The results showed that linseed peptide has a strong cholesterol lowering effect.Its mechanism was that it could inhibit the absorption and transport of cholesterol and enhance the conversion and efflux of cholesterol by increasing the levels of ABCG5/8 and CYP7A1 in rat liver and down regulating the expression of NPC1L1.