Preparation,bioactivity Analysis And Homeostasis Of Buckwheat Glycopeptides

Glycopeptides not only have a wide range of biological activities but can also play an important role in molecular biology.However,due to the complex binding of oligosaccharides and polypeptides in glycopeptides,artificial synthesis is highly difficult.Many kind of glycopeptides are generally present in animals and plants,the extraction of functional glycopeptides from natural organisms has become an important way.In recent years,domestic and foreign researchers have mainly focused on the synthesis and enrichment of glycopeptides,while less attention has been paid to the acquisition and functional activity of natural glycopeptides.Thus,glycopeptide research still has great potential for development and exploration value.Buckwheat is rich in high-quality nutrients,and the polypeptides,polyphenols,and polysaccharides extracted from buckwheat that was reported by domestic and foreign research scholars have antioxidant,anti-inflammatory,and immune-enhancing effects,but there are few reports on the extraction and utilization of glycopeptides in buckwheat.Based on the above background,microbial fermentation method and separation and purification technology were firstly used to prepare buckwheat glycopeptide(BG)with hypoglycemic activity based on the goal ofα-glucosidase(AGase)inhibition.And the structural characteristics and enzyme inhibition dynamics of BG were further explord.Subsequently,the chemical antioxidant capacity of BG was evaluated based on DPPH·,ABTS~+·,·OH clearance rate and reduction power,and a RAW 264.7 mouse macrophage oxidative stress model was established to explore the antioxidant effect of BG at the cellular level.Finally,the steady-state study was carried out to improve the bioavailability of BG.Reverse evaporation was used to prepared buckwheat glycopeptide liposomes(BGL),whose encapsulation process was optimized by response surface experiment.Subsequently,the stability,in vitro sustained release properties and biological activity of BGL were further investigated.The main conclusions were as follows:1.Crude buckwheat glycopeptid with high AGase inhibition rate was prepared by fermentation of Lactobacillus plantarum and Streptococcus thermophilus which were selected from 6 strains.The optimum fermentation conditions determined by the response surface experiment were as follows:inoculum volume 2%,p H 7.5,material to liquid ratio 1:14.8 and fermentation time 2.8 days.Under these conditions,the inhibition rate of AGase was 70.83%.2.Sephadex G-25 was used to separate the crude buckwheat glycopeptide fermentation solution to obtain three main separation components I1,I2 and I3.The yield of the three components was 38.24%,5.82%and 14.96%,and the IC50 of AGase were 14.71±0.33,1.72±0.02 and 8.54±0.37 mg/m L,respectively.I2 was further separated by RP-HPLC to obtain BG with a purity of 94.17%,whose final yield was 1.27%and the IC50 of AGase was0.62±0.04 mg/m L.3.The molecular weight of BG was determined by Tricine-SDS-PAGE electrophoresis to be 5.8 k Da.Fourier transform infrared spectroscopy of BG had the characteristic peaks of the amide band I,which suggested that the secondary structure composition was estimated as70.51%β-folding,18.95%α-helices and 10.54%β-Corner.The type of glycopeptide bond in BG was O-glycopeptide bond.Amino acid analysis showed that the amino acid content of BG was 29.75%of the total mass,with hydrophobic amino acids accounting for 62.91%of the total amino acid content.Among them,Ile was the highest proportion which accounted for24.68%of the total amino acid content.According to rheological analysis,BG had the characteristics of shear thinning and was a non-Newtonian liquid.DSC indicated that thermal denaturation temperature of BG was 106.38℃,the enthalpy of melting was 92.76 J/g.The results of the enzyme inhibition kinetics study showed that BG exhibited a mixed-type noncompetitive inhibition of non-competitive and anti-competitive inhibition.4.BG showed the highest antioxidant activity among the isolated components,Vitamin C equivalent antioxidant capacity(VCEAC)was used to indicate that DPPH·、ABTS+·、·OH scavenging ability and reducing power of BG were 12.12±0.27、153.82±5.04、901.95±39.30 and 109.23±1.18μg/mg,respectively.In the RAW 264.7 macrophage oxidative stress model,BG with the concentration of 200μg/m L had a significant(P<0.05)effect on all measured antioxidant indicators compared with the model group,which significantly(P<0.05)promoted the viability of oxidatively damaged cells by 13.80%,reduced the levels of intracellular reactive oxygen species(ROS)by 40.63%and malondialdehyde(MDA)by11.92 nmol/mgprog.Meanwhile,the activities of superoxide dismutase(SOD)and catalase(CAT)were increased by 23.48 and 15.71 U/mgprot at this concentration,respectively.5.In vitro stability results indicated that temperature(20～80)has a weak effect on the stability of BG,whose AGase inhibitory activity and DPPH free radical scavenging ability were 85.60±3.96%and 87.69±1.31%of the original activity at 100°C,respectively.Conversely,BG was extremely unstable in alkaline solutions,and the inhibition rate of AGase and the clearance rate of DPPH·were significantly(P<0.05)reduced to 79.81±5.41%and66.79±3.06%of neutral condition at p H=9.6.Egg yolk lecithin was selected from five phospholipids to prepare BGL,and the optimum embedding conditions determined by the response surface experiment were as follows:the phospholipid to cholesterol mass ratio 4.2:1,lipid to drug ratio 7.5:1 and aqueous phase volume 1 m L.Experiments carried out under these conditions led to the encapsulation rate of 72.64%for BGL.In vitro stability results indicated that the structural integrity of the BGL was easily destroyed under high temperatures(100℃).Conversely,BGL showed a broad-spectrum p H(2～10)activity when the treatment time was 1 h and there was no significant effect(P>0.05)on leakage rate of BGL after repeated freezing and thawing five times.BGL had sustained-release characteristics in the simulated gastric and intestinal systems in vitro,and the cumulative release rate of 24 h was 84.50±2.30%and 60.17±1.42,respectively.The establishment of the liposome embedding system was beneficial to improve the stability of BG,and showed a good protective effect on its AGase inhibition and antioxidant activity,which provides a theoretical basis for the development and application of buckwheat glycopeptide liposomes.