The Separation And Effects On Lipid Metabolism Of Hypolipidemic Peptides From Pinus Koraiensis Pine Nuts

Hyperlipidemia is a chronic syndrome caused by dyslipidemia,which seriously threatens human health.In recent years,many studies have shown that bioactive peptides derived from food proteins have a significant hypolipidemic effect,and have long-lasting efficacy and few side effects.As a natural active substance that can effectively improve blood lipid balance in vivo,hypolipidemic active peptides have attracted the attention of the majority of scholars.In this paper,protein from pinus koraieneis pine nuts was used as the raw material,and was prepared by enzymatic method and in vitro hypolipidemic experiments to obtain pine nut hypolipidemic peptides,which were isolated and activity-tracked.Animal experiments were conducted to establish a high-fat model to explore the effects of pine nut hypolipidemic peptide on lipid metabolism in mice.The optimal enzyme for preparing hypolipidemic peptides was selected as the alkaline protease by binding cholate and inhibiting the solubility of cholesterol micelles experiments,and the amount of enzyme,substrate concentration,enzymatic temperature,and enzymolysis p H were determined using the degree of hydrolysis as an indicator.On the basis of single-factor experiments,response surface Box-Behnken center combination test design was conducted to optimize the process parameters.The optimal conditions for the preparation of pine nut,hypolipidemic peptides were p H 8.0,temperature 53 ℃,and enzyme amount 8800U/g,The substrate concentration was 1.2%,and the actual average degree of hydrolysis under this optimized condition was 43.15%.Using cholate binding capacity and inhibition of the solubility of cholesterol micelles as indicators,the pineal peptides with an enzymatic hydrolysis time of 1 to 6 h were tested in vitro for hypolipidemic activity.Finally,it was determined that the pineal hypolipidemic peptides with 4 h of enzymolysis had the highest hypolipidemic activity in vitro.As a follow-up experimental sample.The active peptides of pine nuts were prepared at different stages by enzymatic method.According to the degree of hydrolysis,pinein peptides with an enzymatic hydrolysis time of 1 to 6 h were used for in vitro hypolipidemic activity experiments.The experimental results showed that the solubility of chondroitin in micelles was significantly inhibited at all stages.of which pinein peptides for 4 h was the best inhibitory effect,up to 56.72%;pinein peptides had higher bile salt binding capacity,of which the enzymatic hydrolysis time was 2⁴h.Pine nut peptides are not ideal for inhibiting HMG-Co A reductase.Considering comprehensively,the pine nutrient-reducing peptides that were digested for 4 h were selected as follow-up experimental samples,and the two factors such as the solubility inhibition rate of cholesterol micelles and binding capacity of cholate were selected for separation and activity tracking.Using the ability to bind cholate and inhibiting the solubility of cholesterol micelles as indicators,the pineal peptides with an enzymatic hydrolysis time of 1 to 6 h were screened by in vitro hypolipidemic activity.The final determination of pineal hypolipidemic peptides after 4 hours of enzyme hydrolysis was the highest in hypolipidemic activity,as a follow-up experiment sample.The pineal lipid-lowering peptide was fractionated by DA201-C macroporous resin,DEAE-52 ion exchange chromatography,and Sephadex-25 gel chromatography to obtain the most active component P₇₅-B-b,its inhibition rate of the cholesterol micelle solubilit and binding strength of sodium glycocholate reached 86.5% and 63.45%,respectiveely.The P₇₅-B-b component was analyzed by ESI-MS.The results showed that the ion peak was mainly distributed below 1000m/z,the peak with the highest abundance was 518.52m/z,and it was presumed to be about 3-5 amino acids.The amino acid composition analysis showed that P₇₅-B-b has a higher content of hydrophobic amino acids and is rich in arginine and proline,which may be an important reason for its higher hypolipidemic activity in vitro.A high-fat animal model was established to study the hypolipidemic effect of pine el peptides in vivo.The results showed that pine nut hypolipidemic peptides could significantly relieve the increase of body weight,reduce the liver index,regulate the lipid balance in high-fat mice,and relieve the impaired liver function caused by the high-fat model.Middle and high doses of pine nutrient-lowering peptide could effectively increase the excretion of bile acids and reduce the cholesterol level in mice.By analyzing and measuring the activity of cholesterol metabolism enzymes and the expression of key genes in mice,the potential mechanism of pineal hypolipidemic peptides in regulating lipid metabolism was elucidated.The results of enzyme activity test showed that pine nut hypolipidemic peptides could significantly promote the activity of CYP7A1 enzyme and significantly reduce the activity of ACAT2 enzyme,but it has no obvious effect on the activity of HMG-Co AR and LCAT enzymes.Real-time fluorescence quantitative PCR results show that pine nut hypolipidemic peptide could significantly upregulate the expression of CYP7A1 and PPARα in cholesterol catabolism andsignificantly upregulate the expression of LDL-R that regulates cholesterol reverse transport,and also significantly downregulate the level of ACAT2 gene that regulates cholesterol absorption metabolism.However,there was no significant effect on the expression of HMG-Co AR and SREBP2 genes regulating cholesterol anabolism.It is speculated that pine nut hypolipidemic peptides can achieve lipid-lowering effects by promoting the decomposition and reverse transport of cholesterol and inhibiting the absorption of cholesterol in vivo.