Isolation,Purification,Structure Identification And Biological Activity Of Pumpkin Polysaccharide

Pumpkin,（Cucurbita moschata（Duch.Ex Lam.）Duch.Ex Poiret）is a kind of plant that can be used as both medicine and food,which belongs to the fruit of annual herb of Cucurbita coniensis.It has many functions such as antioxidant,antibacterial,enhancing fertility,anti-cancer and hypoglycemic activity.Polysaccharide is an important active ingredient of pupmkin.It has been found that the molecular weight of pumpkin polysaccharide is mainly in the range of 16-200 KDa.It is mainly composed of rhamnose,arabinose,glucose and galactose and contains various types of glycosidic bonds such asβ-1→4,β-1→3,β-1→2 andα-1→6.But there is no report on the primary structural repeating units,advanced structure,and immune-enhancing effect and the mechanism of its action on spleen cells from mice.The hybrid variety of Zhenliqing pumpkin is a local vegetable cultivar,its flesh is dense and delicate in taste and tastes like chestnuts.In our previous study,the polysaccharide content in Zhengliqing pumpkin was higher（55.52±2.29 mg/g）than it in Dongsheng pumpkin（37.6±1.56mg/g）.Also the free radical scavenging ability of its aqueous extract was better than that of Dongsheng pumpkin,which shows that Zhenliqing pumpkin is worthy for in-depth research and development.Therefore,two pumpkin polysaccharide fractions were isolated and prepared form Dongsheng pumpkin and Zhenliqing pumpkin,and its physicochemical properties and structure were characterized by various modern analytical tools.The antioxidant and immunomodulatory activities of the two polysaccharide fractions were compared by chemical antioxidant assay and mouse spleen cell model.This study can provide the basis for the further development and utilization of Zhenliqing Pumpkin.The main research contents are as follows:（1）Extraction,purification and physicochemical property analysis of polysaccharides from Dongsheng pumpkin and Zhenliqing pumpkin.After the hot water extraction,the crude polysaccharides DPP and ZPP obtained from Dongsheng pumpkin and Zhenliqing pumpkin were flocculent in yellow and creamy white with the yields of 29.56%and 17.86%,respectively.Then DPP and ZPP were purified with DEAE-FF and DEAE-52.Three polysaccharide fractions and two polysaccharide fractions were obtained,respectively,in which the water-washed fractions were the main component with the highest yields（28.38%and 34.07%）.Then the main fraction were further purified by Sephacryl^TMS-400 dextran gel chromatography column,and the purified polysaccharide fractions（DPP1 and ZPP1）of Dongsheng pumpkin and Zhenliqing pumpkin were obtained.The polysaccharide contents and protein contents were 77.94%and 79.27%as well as 10.63%and 10.89%.Both of them are mainly composed of glucose.（2）Structural analysis of DDP1 and ZPP1.The structures of DPP1 and ZPP1were studied by various modern analytical techniques,including IR,methylation combined with GC-MS,Congo red experiment,HPSEC-MALLS-RI,DLS,GC,XRD and AFM,etc.Results showed that sugar residues of DPP1 and ZPP1 were:1,4-Glcp and 1,6-Glcp;T-Glcp,1,4-Glcp,1,6-Glcp and 1,3,4,6-Glcp,with the molar ratios34.8:1 and 1.3:1.1:1.3:1,respectively.DPP1 and ZPP1 all had the characteristic absorption peaks of general polysaccharides.DPP1 had bothα-andβ-glycosidic pyranose rings,but ZPP1 just hadβ-glycosidic pyranose rings.Both DPP1 and ZPP1were homogeneous components with molecular weights of 1.738×10⁴Da and4.029×10⁶Da.The particle size distribution in both DPP1 and ZPP1 were relatively uniform,with average particle size in 197.5 nm and 85.54 nm.DPP1 had a three-stranded helical structure,but ZPP1 didn’t.Both of them intertwined molecules in an irregular agglomerated state,and the height of the molecular chain ranges were from 0.5 to 6 nm.But ZPP1 had better dispersibility and stability in aqueous solution.The solid surface of DPP1 was loose with honeycomb structure,while ZPP1 was tightly with aggregated lamellar structure.Both of them were non-crystalline structures.（3）Comparative study on antioxidant activity and immunomodulatory activity of DPP1 and ZPP1 in vitro.A comparative study on the scavenging ability of DPP1 and ZPP1 on ABTS⁺·and·OH were performed by chemical antioxidant method.The results showed that both DPP1 and ZPP1 had certain scavenging ability for ABTS⁺·and·OH in the concentration range of 0.315-5 mg/m L,and the scavenging effect of ZPP1 was stronger than that of DPP1.The immunomodulatory activities of the DPP1 and ZPP1 were studied comparatively using a mouse splenocyte model.The results showed that DPP1 and ZPP1 significantly increased the viability of splenocytes.When stimulated for 12 h,both of them have the strongest stimulating effect on splenocytes.But the stimulating effect gradually decreased with the increased stimulation time and dose,especially when the stimulation time was greater than 24 h and the concentration was higher than 200μg/m L,DPP1 could significantly inhibit the viability of splenocytes.At 12 h of stimulation,both DPP1 and ZPP1 could promote the proliferation of T and B splenic lymphocytes,but the effect of ZPP1 was significantly superior to DPP1.At concentration of 800μg/m L,ZPP1 could increase the secretion of IL-2,IFN-γand Ig G,but DPP1 had no effect on them.Both DPP1and ZPP1 could significantly upregulate the m RNA expression of i NOS、ERK、TLR2、TRAF-6 and My D88,revealing the possible molecular mechanisms of immunomodulation by DPP1 and ZPP1 were that DPP1 and ZPP1 could combined with TLR2,which could activated the adaptor protein-My D88 to act with TRAF6.It could promote the expression of ERK and further enhance the secretion of cytokines to exert immunomodulatory effects.