Studies On Regulation Lipid Metabolism Of Bio-activity Peptides From Chlorella Pyrenoidosa And Its Mechanism

Microalgae is recognized as novel green sources of multiple components like proteins,carbohydrates,lipids and pigments for food,pharmaceutical,cosmetic and energy products.Chlorella pyrenoidosa,as a single-cell algae,has rich nutritional value and medicinal value.Extensive research has been carried out on the bioactive components,such as polysaccharides,peptides,glycoproteins,etc.This paper focused on the antioxidant and lipid metabolism regulation of peptides,as well as its mechanism,in order to further expand the medicinal value of Chlorella pyrenoidosa.A new protocol is established to maximize extract amounts of protein,which is involved in ethanol soaking,enzyme digest,ultrasonication and homogenization techniques.Under the optimized conditions,72.4% of protein was extracted from the microalgae Chlorella pyrenoidosa and the extraction rate was 47.15%.Compared with the effect of decoloring on its oxidation activity,the protein without decoloring has a strong ability to remove free radicals.The freeze-dried decoloring proteins were dissolved in water separately hydrolyzed with four proteases(pepsin,papain,trypsin,and alcalase)under controlled conditions,The hydrolysis degree and molecular weight distribution of them were measured.The results showed that pepsin enzymatic hydrolysis produced the lowest hydrolysis degree of polypeptide,and the corresponding polypeptide molecular weight <1000 Da only accounted for 9.03%.The hydrolysis degree of polypeptide obtained by Alclase hydrolysis was the highest,and the molecular weight <1000 Da was 14.06%.The antioxidant activity results showed that papain hydrolysis had the highest ABST free radical scavenging rate(1000 ?g/mL 95.76%),which was similar to the positive control Vc at 10 ?g/mL.The PL inhibition ability of each crude protein and hydrolysates was measured by copper soap method,the order of PL inhibitory capacities is Alcalase hydrolysate > pepsin hydrolysate> trypsin hydrolysate > papain hydrolysate > protein.In 3T3-L1 differentiation model and OAinduced LO2 model,the inhibition rate of triglyceride and lipid accumulation was used as biochemical indexes of lipid metabolism.The scavenging ability of triglycerides and lipids was measured.The results showed that the TG scavenging rate of trypsin hydrolysates and Alcalase hydrolysates to triglyceride(TG)in 3T3-L1 cells at the same concentration was 26.47% and 24.29%,respectively,while papain hydrolysates and Alcalase hydrolysates had a better capacity of scavenging lipid in LO2 cells.which have a certain connect with the inhibition of 3T3-L1 differentiation.Based on these results,and the Alcalase hydrolysates was selected to further studied.After ultrafiltration,it was found that the molecular weight <5k Da(Ae)in Alcalase hydrolysates had a most inhibition rate of PL activity(75.73%).After the separation of Ae components by gel filtration chromatography Sephadex G-25,four components(AI,AII,AIII and AIV)were obtained.Peptides SISISVAGGGR,LLVVYPWTQR,SDDPHTFGQGTK,SRQLTLYPGAER,KNGAPAEK and KQTALVELVK was obtained by LCMS/MS analysis based on MASCOT.A novel lipid inhibition peptide LLVVYPWTQR(PP1)(MW 1274.53 Da)was obtained using Peptide Ranker(http://bioware.ucd.ie)and Pep-site(http://pepsite2.russelllab.org/).Its lipid inhibition effects indicated that the synthetic peptide PP1 exhibits a good inhibitory effect against porcine pancreatic lipase(PL)(47.95%)at 200 ? g/mL,which could be attributed to its hydrogen binding into catalytic sites of PL(Ser153,Asp177 and His264)by docking analysis.Furthermore,in 3T3-L1 cells,the synthetic PP1 remarkedly decreased the accumulation of intracellular triacylglycerol(27.9%,600 ?g/mL),which carried a similar consequence as the positive drug simvastatin(24.1%,10 ?M).Western blot revealed that PP1 inhibited the lipid accumulation and fatty acid synthesis in 3T3-L1 adipocytes in two pathways,primarily: activating the AMPK pathway and down-regulating adipogenic-specific proteins,including C/EBP?,SREBP-1c,and PPAR?,in 3T3-L1 cells.Further animal-level studies have shown that Ae can effectively reduce the symptoms of fatty liver and reduce the growth of body weight.Compared to the model group,Ae reduced body weight in mice by 22.98%,slightly less than orlistat(33.17%)in the positive control group.At the same time,it can effectively alleviate the symptoms of fatty liver,reduce the accumulation of lipids,catalyze the decomposition of TG and TC;lipid synthesis can be prevented by inhibiting the expression of C/EBP-?.Western blot analysis shows that Ae alleviated chronic HFD-induced inflammation by reducing the secretion of inflammatory cytokines in liver by increasing the expression of NF-?B.Using a 16 S rRNA sequencing technique,the results shows Ae increased the ratio of Firmicutes to Bacteroidetes to 2.04 in HFD-fed obese mice.Taken together,these results suggest that Ae could protected mice from NAFLD by increasing the abundance of beneficial gut microbiota and is mainly involved in the metabolism of the ae regulated COG class at the metabolic level,including biosynthesis of amino acids,biosynthesis of amino acid transmission,and biosynthesis,transport and decomposition metabolism of secondary metabolites.KEGG pathway analysis also proved that Ae was closely related to lipid metabolism,carbon metabolism.These findings may provide institutional insights into the role of Ae in the development and progress of the NAFLD.Simulated human digestion,TPe(the hydrolysate MW<5k Da)was obtained by ultrafiltration,after pepsin and trypsin hydrolysis at 37?.After the separation of Ae components by gel filtration chromatography Sephadex G-25,four components(TP-I,TP-II,TP-III and TP-IV)were obtained.Four peptides VVPEGVR?GSGFCGSSR?NPDGEPR and RDAEEWFHAK was obtained by LCMS/MS analysis based on MASCOT.A novel lipid inhibition peptide RDAEEWFHAK(TP1)was obtained using Peptide Ranker(http://bioware.ucd.ie)and Pep-site(http://pepsite2.russelllab.org/).Its lipid inhibition effects indicated that the synthetic peptide TP1 exhibits a good inhibitory effect against porcine pancreatic lipase(PL)(40.2%)at 200 ?g/mL,which could be attributed to three hydrogen bindings into catalytic sites of PL(Ser153,Asp177 and His264)by docking analysis.Furthermore,in 3T3-L1 cells,the synthetic TP1 remarkedly decreased the accumulation of intracellular triacylglycerol(23.59%,600 ?g/mL),which carried a similar consequence as the positive drug simvastatin(24.1%,10 ?M).In vivo activity study of TPe,it was found that TPe slowed the body weight gain in mice fed with high-fat diets,reduced the accumulation of lipids in the liver,catalyzed the decomposition of TG and TC,as well as reduced the level of blood lipids.It can also be achieved by activating the SIRTI/FOXP3 signaling pathway.