Purification,structure Characterization,sulfated Modification And Activities Of Water-soluble Polysaccharides From Polygonatum Ordoratum(Mill.)Druce

Polygonum odoratum,belonging to the family of Polygonatum ordoratum(Mill.)Druce,is widely distributed in north and southwest of China.Polygonum odoratum is a kind of medicine and food homologous plant,which exhibited many activity,including hyperglycemic,antitumor,antioxidant,immunomodulation and regulating glucose and lipid metabolism effects.And there were many various active substances such as polysaccharides,steroidal saponins,flavonoids and volatile oils in this herb.As one of the most important parts,polysaccharides have been confirmed to show many pharmacological activities.At present,there are still some shortages in the structural research of polysaccharides from Polygonatum ordoratum(Mill.)Druce,such as the primary structure,high-level structure and macrostructure studies were still not well understood.Furthermore,the research on the activity of polysaccharides from Polygonatum ordoratum(Mill.)Druce still focused on hypoglycemic,anti-oxidation and antitumor activities and many studies have shown that the biological activity of polysaccharides from Polygonatum ordoratum(Mill.)Druce was less than the synthetic drugs.The research on the modification of polysaccharides from Polygonatum ordoratum(Mill.)Druce is an effective method to improve the biological activities and explore new biological activities.Therefore,In the study,the extraction conditions of crude polysaccharide from Polygonatum ordoratum(Mill.)Druce(PORPs)were optimized.And the chemical compositions,structure characterization and antioxidant properties of PORPs were studied.Then a purified watersoluble polysaccharide,designated as PORP-1,was isolated from Polygonatum ordoratum(Mill.)Druce and the primary structure,high-level structure and macrostructure were determined.Finally,PORP-1 was modified by means of sulfate modification and two sulfated polysaccharide named SPORP-2 and SPORP-1 were obtained with a DS of 0.41 and 0.54.The antioxidant activity of modified polysaccharides in vitro was also studied.In the meanwhile,the relationship between antioxidant activity and structure of polysaccharides was further explored.Finally,the antibacterial activity of PORP-1 and sulfated polysaccharides(SPORP-1)were studied and the antibacterial curve of SPORP-1 was obtained.In addition,the antibacterial mechanism of SPORP-1 was investigatived.The main findings are listed as follows:(1)The influences of extraction conditions were firstly studied.According to the results of response surface analysis,the optimal conditions including extraction temperature,ratio of liquid to solid and extraction time were 70.50 oC,40.23 m L/g and 1.14 h,respectively.And the extraction yield of PORPs was 5.61±0.12% under the conditions.The chemical compositions of PORPs including the total sugar content,the total protein content and the content of uronic acid were 76.26±1.53%,0.21±0.09% and 2.27%,respectively.The monosaccharide composition was consisted of mannose,glucuronic acid,galacturonic acid galactose,glucose,arabinose and fucose with a molar ratio of 18.30:0.81:1.46:41.83:1.53:1.06.Furthermore,the FT-IR analysis revealed that POPRs contained-COOH and ?-type glycosidic linkages with pyranose ring.The AFM results suggested that PORPs were dome-shaped particles with particle diameter ranging from 70 to 240 nm.In addition,SEM image showed there was a dense,smooth and wrinkled sheet-like structure through aggregation in PORPs.Moreover,PORPs also exhibited free radical scavenging ability in vitro and the scavenging rate of hydroxyl free radical reached 41.98 ± 2.10%.This study may provide a theoretical guide for the application of PORPs.(2)PORPs was purified by DEAE-52 cellulose columns and two fractions including PORP-1 and PORP-2 were obtained.PORP-2 was not homogenous while PORP-1 owned only one peak in HPGPC with an average molecular weight of 4025 Da.Therefore,PORP-1 was collected for further study.The results of FT-IR analysis revealed that POPRs may contain-COOH and ?,?-type glycosidic linkages with pyranose ring.The monosaccharide composition was consisted of Man,Glc A,Gal A,Glc,Ara and Fuc with a molar ratio of 18.30:0.81:1.46:41.83:1.53:1.06.In addition,periodate oxidation–Smith degradation reaction and NMR analysis revealed that the main glycosidic bonds of PORP-1 were comprised of ?6)-?-D-Glcp-(1?,?-Glcp-(1?,?4,6)???D?Glcp(1?,?4)-?-D-Man(1?,?3)?-D-Manp-(1?.The Congo-red test and Circular dichroism results showed that PORP-1 owned a symmetrical space structure in solution instead of triple helix conformation.The AFM results suggested that PORPs were dome-shaped particles with particle diameter ranging from 123 to 202 nm.In addition,SEM image showed there was a dense,smooth and wrinkled network structure through aggregation in PORP-1.(3)Sulfated modification was carried out to modify PORP-1 and two sulfated derivatives were prepared,designated as SPORP-1 and SPORP-2 with degree of substitution(DS)of 0.54 and 0.41.The maximum of DPPH,ABTS,superoxide and hydroxyl radicals scavenging rate of SPORP-1 were highly improved from 40.95±2.10%,28.99±2.11%,30.52±0.91% and 41.97±2.10% to 77.43±1.48%,70.44±1.74%,66.43±1.93% and 68.23±1.73% compared with PORP-1.Based on correlation analysis,the DS,uronic acid content and different monosaccharide compositions might be closely associated with the antioxidant activity.Finally,the relationship between antioxidant activity and the structure of polysaccharides was established by combining the antioxidant mechanism,antioxidant activity of PORP-1,SPORP-1 and SPORP-2 polysaccharide structures.(4)The antibacterial experiment was carried out to explore the antibacterial activity and mechanism of PORP-1 and SPORP-1.The results showed that PORP-1 and SPORP-1 had antibacterial activity against Staphyloccocus aureus and Bacillus subtilis.However,there was no significant antibacterial effect on Pseudomonas aeruginosa and Escherichia coli.The inhibition zone diameters of PORP against Bacillus subtilis and Staphyloccocus aureus were 9.17±0.75 and 6.77±0.81 mm.The inhibition zone diameters of SPORP-1 against Bacillus subtilis and Staphyloccocus aureus were 13.63±0.79 and 12.25±0.85 mm.It was found that the minimum inhibitory concentration values of SPORP-1 against Bacillus subtilis and Staphylococcus aureus were 25 mg/m L and 50 mg/m L.And the inhibitory effect of SPORP on the bacterias was dose-dependent.Futhermore,SPORP-1 had an antibacterial effect on the colony index period and stable period of Bacillus subtilis and Staphylococcus aureus.In addition,SPORP-1 could exert antimicrobial effects by damaging the cell wall of Bacillus subtilis which resulted in the release of electrolytes and cell components including proteins and electrolytes.