Study On The Purification Of Bioactive Peptides From Male Silkworm Proteinand Its Cell Carbohydrate Metabolismon AMPK Signaling Pathway

As a traditional tonic,male silkworm moth has anti-fatigue,anti-aging,immune regulation,and other effects.The content of protein in silkworm moth is rich,its hydrolysis into peptides can solve the part of people’s problem of allergies after eating.So,usage of the silkworm moth peptides to develop related functional food will bring better health benefits and market prospects.In this paper,male silkworm moth protein was used as raw material to prepare male silkworm hydrolysates.The use of enzyme hydrolysis of male silkworm moth peptides,using ultrafiltration,Sephadex G-25 gel and preparation RP-HPLC in turn purified antioxidant peptides from male silkworm hydrolysates,finally identified its structure.Additionally,the effect of male silkworm hydrolysateson the carbohydrate metabolism of skeletal muscle cells was studied.The mechanism of energy metabolism by AMPK signaling pathway was proved.The main studies are as follows: 1.Isolation and purification of male silkworm hydrolysates(MSH)and evaluation of its antioxidant activityThe male silkwormproteinwashydrolyzed by compound enzyme(Alcalase alkaline protease and Daniske alkaline protease).And then,the molecular weight of MSH wasmainly concentrated in 1000~3000 Da.The fractionsof MSH-Ⅰ(MW< 1 k Da),MSH-Ⅱ(MW 1~3 k Da)and MSH-Ⅲ(MW> 3 k Da)were obtained by ultrafiltration of 3 k Da and 1 k Da ultrafiltration membranes.The value of ORAC and DPPH scavenging capacity of ultrafiltrationfractions were determined as following order: MSH-Ⅱ>MSH-Ⅰ>MSH-Ⅲ.There was no significant statistical difference between MSH-Ⅰ and MSH-Ⅱ in ORAC(P>0.05).The Sephadex G-25 gel chromatography was used to separatemale silkworm hydrolysates fractions(MW< 3 k Da).Male silkworm hydrolysates could be separated into two fractions P1 and P2,among which the second peak had the highest antioxidant activity.Through amino acid analysis,fractions P2 was rich in antioxidative amino acids,including methionine,tyrosine,lysine,histidine.HPLC-DPPH was a method which used to rapidly screen and identify of antioxidants from male silkworm moth peptides.Then,the component with high antioxidant activity was collected by preparation HPLC.fractions P2-A and P2-B were preparated by Sun Fire Prep C18 Columnand XBridge CSH Prep C18 Column.The antioxidant activity of P2-B was much higher than that of P2-A(P<0.05),the O RAC value of P2-B was up to 1384.64 μmol TE/g.Thepurity of P2-B reached 90% by HPLC analysis.MALDI-TOF-MS analysis showed that the molecular weight of P2-B was 792.55 Da.P2-B was probably composed of these kinds of amino acids,including serine,glycine,alanine,valine,histidine and arginine.2.By the cell experimental evaluation ofmale silkmoth hydrolysatesregulation of carbohydrate metabolismWe found that MSH and its various fractions MSH-Ⅰ,MSH-Ⅱ,MSH-Ⅲ couldpromote the expression of L6 skeletal muscle cells in glucose,but thedifferencewas insignificant among groups(P>0.05).The effect of glucose uptake of skeletal muscle cells treated with different times(6,12,18,24,30,36 h)and various concentrations(0.1,0.2,0.4,0.8,1.6 mg/m L)were studied.We found that the optimum condition for promoting glucose uptake was 0.4 mg/m L MSH treated skeletal muscle cells for 24 h.Skeletal muscle cells were incubated with different treatments(0.4 mg/m L MSH,insulin and AICAR)increased the glucose intake of skeletal muscle cells.After being preincubated with 25 μmol/L Compound C(the AMPK inhibitor)for 30 minutes,and then skeletal muscle cells treated with MSH for 24 hours,the insulin group and the AICAR group were considered as the positive and negative controls.The results showed that Compound C could inhibit the stimulating effect of glucose uptake by MSH and AIC AR in skeletal muscle cells(P<0.05),but didn’t affect insulin to stimulate glucose uptake(P>0.05).By RT-PCR detection of AMPKα m RNA expression and Western blot detecting AMPKα,p-AMPKα,GLUT4 expression,the results showed that MSH could increase skeletal muscle cells AMPKα m RNA and protein expression,ratio of p-AMPKα/AMPKα and m-GLUT4 expression also increased significantly.It is speculated that MSH may activate AMPKα to phosphorylation,increase the translocationof GLUT4 to the cell membrane,promote glucose uptake in skeletal muscle cells,thus regulate the level of intracellular carbohydrate metabolism.Comprehensive analysis,MSHthrough AMPK signaling pathway to promote cell glucose uptake.