Structural Characterization And Biological Activity Of Protein-anthocyanin Complexes From Purple Sweet Potato

Purple sweet potato is a special breed of sweet potato,which is rich in anthocyanins and protein.Anthocyanins can form complexes with protein via covalent bonding or non-covalent bonding due to anthocyanins compounds to have high protein affinity.In this paper,the ultrasound-assisted extraction method was used to extract anthocyanins and protein-anthocyanins complexes from purple sweet potato cultivar Eshu No.12.Anthocyanins were extracted with 60%ethanol,and anthocyanins-protein complexes were extracted with water and precipitated by ammonium sulfate solution of 60%saturation.Purple sweet potato protein was further purified from the complexes by DEAE-52 ion exchange chromatography and Sephadex G-75 gel filtration chromatography.The Chromatograph HPLC-DAD-ESI-MS/MS was used to determine compositions of anthocyanins,and protein was indentified by Peptide Mass Finger print?PMF?.Kinetics anthocyanins degradation and polymeric color index during storage and heat were determined.Additionally,the anti-inflammatory activity and underlying mechanism of anthocyanins-protein complexes were investigated in lipopolysaccharide?LPS?-induced RAW264.7 macrophage cells.The prevention and antidiabetic effect of protein-anthocyanins complexes was evaluated,compared to anthocyanins extract.The main conclusions are as follows:1.Thirteen anthocyanins were identified in the purple sweet potato cultivar Eshu No.12 throught HPLC-DAD-ESI-MS/MS analysis.But Only 3 kinds of anthocyanins were found in PSPFA-E,this indicated that PSPAP-E contains abundant anthocyanins.SDS-PAGE analysis exhibited four bands with molecular weights of 58,22,18 and 12kDa,respectively.A total of 17 proteins were indentified using Triple-TOF-MS analysis,which mainly included biologically active enzymes and Sporamin proteins.2.The total and monomeric anthocyanin contents decreased during storage,following first-order reaction kinetics.The values of total anthocyanin half-life time(t_1/2)were found to be 228.8,48.1 and 32.6 d at 4,20 and 35?.Moreover,we found that with the same glycosides,the half-life of cyanidin was lower than that of peonidin.In the case of the same anthocyanidin,the t_1/2/2 of acylated anthocyanins is higher than that of non-acylated anthocyanins,and the t_1/2/2 of diacyl anthocyanin is higher than that of monoacyl anthocyanins.Besides,the brown index and polymeric color index increased with the extension of storage time and the increase of temperature.Our results suggested that strong negative correlation between the anthocyanin content and polymeric color index in purple sweet potato,which could be expressed by exponential relationship.The difference in the color of the model system is not obvious.3.The kinetic data indicated a first-order reaction for the degradation of purple sweet potato anthocyanins with t_1/2/2 of 9.7,12.4 and 4.7 h at pH 3.0,pH 5.0 and pH 7.0respectively.The polymeric color formation followed zero-order kinetics,progressively increasing with pH as heating time increased from 0 to 60 h.The reaction rate constants for polymeric color formation were 0.0150,0.0155 and 0.0284h^-11 at pH 3.0,pH 5.0 and pH 7.0,respectively.Changes in color of purple sweet potato extract were assessed using the CIELAB system.L*,a*,b*and?E were significantly changed during heat treatment?p<0.05?.Depending on the time and pH,changes in anthocyanins could be due to condensation reactions and degradation reaction,which may lead to a perceptible color change.4.The anti-inflammatory activity and underlying mechanism of PSPAP-E were investigated in LPS-induced RAW264.7 cells.Our results demonstrated that PSPAP-E significantly reduced LPS-induced nitric oxide?NO?,tumor necrosis factor??TNF-??,and reactive oxygen species?ROS?production,and reduced LPS-mediated induction of mRNA expression of inducible nitric-oxide synthase?iNOS?,tumor necrosis factor alpha?TNF-??.Meanwhile,PSPAP-E significantly promoted the expression of heme oxygenase 1?HO-1?and nuclear factor erythroid-2 related factor 2?Nrf2?.Further,PSPFA-E and PSPAP-E inhibited both LPS-induced activation of JNK,and p38 and the nuclear translocation of NF-?B and AP-1,but there is no difference between PSPFA-E and PSPAP-E.These results support that PSPFA-E and PSPAP-E may be a promising candidate for inflammation treatment.5.Streptozocin?STZ?-induced diabetic IRC mice were used to investigate the antidiabetic effect of PSPA-E and PSPAP-E in a dose of 200 mg/kg BW by gavage for 12 weeks.The results showed that PSPA-E and PSPAP-E significantly lowered fasting blood glucose levels,improved oral glucose tolerance of diabetic ICR mice.Meanwhile,PSPA-E and PSPAP-E markedly increased serum concentrations of insulin and reduced glucagon in diabetic ICR mice.In addition,serum total cholesterol?TC?,triglycerides?TG?,low density lipoprotein cholesterol?LDL-c?levels were sighnificantly reduced in PSPA-E and PSPAP-E supplemented mice as compared to the model group.PSPA-E and PSPAP-E significantly increased high density lipoprotein cholesterol?HDL-c?levels and AST/ALT radio.Morever,the comparison of MDA,Cu-Zn SOD,T-AOC and GSH-Px in liver shows that PSPFA-E and PSPAP-E significantly decreased MDA level,and the Cu-Zn SOD,T-AOC and GSH-Px in liver are significantly raised than the model group?P<0.05?.We can find that PSPA-E and PSPAP-E can repair the liver in diabetic mice caused by STZ in a certain excent.