Preparation,Modification And Biological Activity Of Mulberry Leaves Polysaccharides

Mulberry leaves polysaccharides,one of the main active components of mulberry leaves,has many biological activities such as antioxidant,anti-tumor,anti-virus and immunological regulation.The mulberry leaves are often used to extract alkaloids and flavonoids,however,the polysaccharides in residues with rich content and strong activity are not developed and utilized,resulting in a huge waste of resources.In this paper,mulberry leaves polysaccharides(MLP)was prepared by water-extraction and alcohol-precipitation method.Furthermore,four components of MLP-1,MLP-2,MLP3 and MLP-4 were separated from MLP by DEAE-52 cellulose column chromatography.Fourier transform infrared spectroscopy(FT-IR),gel chromatography(GPC)and gas chromatography(GC)were used to characterize the structure of mulberry leaves polysaccharides.Selenized mulberry leaves polysaccharides(MLP-Se)was prepared by selenization modification of mulberry leaves polysaccharides with nitric acid-sodium selenite method.The preparation process of MLP-Se was optimized by response surface methodology(RSM)based on single factor experiment.The effects of selenization modification on the molecular structure and physicochemical properties of mulberry leaves polysaccharides were investigated by SEM-EDS,GPC,particle size and zeta-potential,atomic force microscopy(AFM),FT-IR and TG.The scavenging activity against three kinds of free radicals,inhibitory activity against α-glucosidase and cytotoxicity of mulberry leaves polysaccharides before and after selenization modification were also studied.The main conclusions are as follows.(1)Structural characterization of polysaccharides and its purified components from mulberry leaves.The polysaccharides content was 45.87%,88.46%,78.18%,69.42%and 67.35%,respectively.FT-IR analysis showed that MLP and four purified components had typical characteristic absorption peaks of polysaccharides,and contained pyranose ring structure.GPC analysis showed that the molecular weights of the four purified components were 6.14 kDa,8.08 kDa,10.25 kDa and 2017.55 kDa,respectively.GC analysis showed that the four purified components were composed of rhamnose,arabinose,xylose,mannose,glucose and galactose,but with different molar ratios of monosaccharides.(2)Preparation and structural characterization of selenized mulberry leaves polysaccharides.The optimized process were mass ratio of Na2SeO3 to MLP 1.27(g/g),reaction temperature 85℃ volume fraction of nitric acid 0.54%,reaction time 7 h.Under these conditions,the selenium content of MLP-Se was 3.147 mg/g.SEM-EDS analysis showed that the apparent morphology of MLP was changed by selenization and there was selenium in MLP-Se.GPC analysis showed that the molecular weight of MLP and MLP-Se were 2.099×103 kDa and 2.385×103 kDa,respectively,and selenization increased the molecular weight of MLP.Particle size and zeta-potential analysis showed that the grain size of mulberry leaves polysaccharides decreased and the absolute value of zeta potential increased after selenization,and selenization improved the stability of MLP in solution system.FT-IR analysis showed that MLP was selenized successfully,and selenium and polysaccharides were bound by C-O-Se and Se=O.AFM analysis showed that selenization modified the apparent structure and intermolecular force of MLP.TG analysis showed that the thermal stability of MLP decreased slightly after selenization modification.(3)Free radical scavenging activities of mulberry leaves polysaccharides and selenized mulberry leaves polysaccharides.MLP-1,MLP-2 and MLP-4 showed the highest scavenging activity against DPPH free radical,ABTS free radical and hydroxyl free radical,respectively.The IC50 values of DPPH free radical scavenging by MLP-1 and MLP-1-Se were 1.0957 mg/mL and 0.3011 mg/mL,respectively.The IC50 value of ABTS free radical scavenging by MLP-2 and MLP-2-Se was 0.5365 mg/mL and 0.2190 mg/mL,respectively.The IC50 values of hydroxyl free radical by MLP-4 and MLP-4-Se were 2.1604 mg/mL and 0.7425 mg/mL,respectively.The free radical scavenging activity of polysaccharides from different components varied significantly,and the free radical scavenging activity of mulberry leaves polysaccharides was improved after selenization.(4)Inhibition of α-glucosidase activity of mulberry leaves polysaccharides and selenized mulberry leaves polysaccharides.Only MLP-1 showed significant inhibitory activity against α-glucosidase.The IC50 values of α-glucoglyase inhibition by MLP-1 and MLP-1-Se were 503.8 μg/mL and 109.9 μg/mL,respectively.The results showed that selenification could effectively improve the α-glucosidase inhibitory activity of MLP-1.The kinetics of α-glucosidase inhibition showed that the inhibition of α-glucosidase by MLP-1 and MLP-1-Se was non-competitive reversible inhibition,the decomposition rate of substrate was reduced by reducing the activity of enzyme.MLP-1-Se had better inhibition effect on the reaction system,and the substrate reacted more slowly with the enzyme.The Km of MLP-1 and MLP-1-Se were 1.3941 mmol/L and 1.1958 mmol/L,respectively.(5)Cytotoxicity of mulberry leaves polysaccharides and selenized mulberry leaves polysaccharides.When the concentration of MLP and MLP-Se was 1000 μg/mL,the survival rate of PC-12 cells was 96.39%and 87.20%,respectively.Selenized mulberry leaves polysaccharides showed no obvious biological toxicity.