Separation And Physiological Function Analysis Of Korean Pine Proteins And Its Regulation Effect Of Glucose And Lipid Metabolism

Diabetes mellitus(DM)is a common endocrine disease,and its prevalence is increasing year by year.The synthetic drugs often used in the clinical treatment of diabetes have some detrimental side effects.Many studies have proved that natural products not only have hypoglycemic effect,but also have certain safety.Some plant proteins were proved functional foods base materials for decreasing hyperglycaemia and hyperlipemia.In this research,we studied the Korean pine protein systematically,isolated the functional protein with the regulatory effect on glycolipid metabolism,and analyzed its mechanism.In order to lay a theoretical foundation for the development of medicine food homology food and new health food.In this study,Korean pine nuts were used to extract protein using a freeze-thaw combined with ultrasonic method.The parameters for freeze-thaw and ultrasonic extractions were optimized using single factors and a Box-Behnken response surface method.SDS-PAGE,amino acid analyzer and shotgun method were used to analyze the molecular weight,amino acid composition and protein composition of Korean pine protein.A diabetic mice model induced using a high fat diet-STZ was established.The effects of Korean pine protein on fasting blood glucose,blood lipids,insulin levels,antioxidant capacity,activities of enzymes of glucose metabolism,liver morphology and other indexes were investigated.To evaluate the effect of Korean pine protein on glucose and lipid reduction in diabetic mice induced using high fat diet-STZ.Combined with the results of high-throughput screening of different genes in the liver of experimental mice,GO and KEGG analysis were carried out,and the key genes in the pathway of glucose metabolism and lipid metabolism were verified by q PCR to reveal the mechanism of glucose and lipid metabolism regulation.Korean pine protein was obtained by Box-Behnken response surface optimization,optimum extraction conditions were as follows: freezing for 24 h at-40 ?,a solid-liquid ratio of 1:90 g/m L,ultrasonic bath power of 500 W with an extraction time of 104 min,an extraction temperature of 45 ? and use of a dilute alkali solution at p H 11.6.With these conditions,the highest crude protein yield was23.68±0.70%.SDS-PAGE analysis showed that most proteins were 20?50 k Da,including 5 major proteins with molecular weights of 48.9,37.5,35.9,23.6 and 20.3k Da.The essential amino acid accounted for 34.3 g/100 g and nonessential amino acid accounted for 69.4 g/100 g,and the most prevalent amino acids were Glx(Glu + Gln),Arg,Asx(Asp + Asn)and Leu at 17.8±0.1?16.6±0.2?9.54±0.00 and 8.49±0.1 g/100 g,respectively.The major protein Q9C7F7 with the molecular function of lipid binding in Korean pine protein.The biological pathways were closely related to metabolism,mainly involving carbohydrate metabolism,lipid metabolism and amino acid metabolism pathway.Animal test results show that Korean pine protein could significantly improve the symptoms of drinking more,excess urinatione,and weight loss in DM mice,reduce the fasting blood glucose,improve OGTT,and increase liver glycogen and muscle glycogen storage.The Korean pine protein also had a good protective effect of organs,especially the liver,significantly improve the enzyme activities of the HK,PK,SDH and LDH,reduce the TC,TG,and LDL-C,increase the content of HDL-C,and improve glycolipid metabolism.It also improved the activities of GSH-Px,T-SOD and CAT enzymes,and reduced MDA level.High throughput screening and targeted analysis of differential genes in the liver of mice among the Korean pine protein group,DM model group and control group were carried out using gene chip technology.It showed that 2050 genes were significantly different between the DM model group and the control group,including 1064 up-regulated genes and 986 down-regulated genes;1191 genes were significantly different between the model group and the Korean pine protein group,including 563 up-regulated genes and 628 down-regulated genes.The results of KEGG analysis and classification showed that Korean pine protein had the most significant effect on lipid metabolism and glucose metabolism pathway of diabetic mice induced by high fat diet and STZ.The key genes of glycolipid metabolism,Adipoq,Pck1,Adipor2,PPARa,cyp4a12 a,Gck,prkcz,g6 pc,adpgk,have positive regulatory effect on PPAR,AMPK signal pathway,gluconeogenesis pathway,steroid hormone biosynthesis,arachidonic acid metabolism signal pathways.In this study,the isolated and purified Korean pine protein both had hypoglycemic and hypolipidemic actions.The protein enzymolysis products obtained by simulating gastrointestinal environment were analyzed.The modulations of the protein on glucose and lipid metabolism in diabetic mice induced using high fat diet and STZ were evaluated.The research opened up a new way for the development and utilization of the bioactive substances of Korean pine,and provided a theoretical basis for further research on the development of natural,high-efficiency,low toxicity,no side effects hypoglycemic,hypolipidemic and diabetic syndrome prevention functional foods.