Isolation,Purification,Structural Analysis And Biological Activities Of Water-soluble Polysaccharide From The Root Of Glehnia Littoralis

Glehniae radix,a dry root,belongs to Glehnia littoralis Fr.Schmidtex Miq.As aChinese folk medicine,Glehniae radix was used for expelling phlegm and arresting coughing.It has been reported to possess pharmacological actions of antioxidant,anti-inflammatory,immunomodulatory and neurogenic promotion.Studies showed that polysaccharides from Glehniae radix exhibit activities of antioxidant,improving immunomodulatory and anticancer.Previous studies on polysaccharides of Glehniae radix were mainly focused on crude polysaccharides for the optimization of extraction method,total carbohydrate content determination and the biological effect investigation.There has been no reports until now concerning isolation,purification and structural elucidation of purified polysaccharides from Glehniae radix.Hence,the exploitation and utilization of Glehniae radix polysaccharides could be significant and worth to study further.All results were summarized as follows.1)Isolation,purification and physical and chemical properties of GRPGlehniae radix were milled into powder and defatted with of 80%ethanol.Then the powder was extracted twice with distilled water.The aqueous extracts was then precipitated with ethanol at the final concentration of 80%（v/v）.The precipitate was collected and dried to obtain the crude polysaccharide.The crude polysaccharide was redissolved in distilled water and purified with a Cellulose DEAE-52 and Sephadex G-75 gel filtration.The main fraction was collected and lyophilized to obtain white pure polysaccharide named GRP（Glehniae radix polysaccharide）for next analyses.GRP was homogeneous,with a mean molecular weight of 1.33×10⁴ Da,which determined by high-performance size-exclusion chromatography-refractive index detector analysis,the total sugar content of GRP was 95.87%and the protein content of GRP was 0.22%.2)structural analysis of GRPGRP was composed of only glucose monomer as detected by GC of the alditol acetate derivatives and HPLC of the 1-Phenyl-3-methyl-5-pyrazalone derivatives.The FT-IR analysis suggested that GRP was mainly composed of alpha（α）glycosidic bond-linked glucopyranose（Glcp）.GRP had slight absorption at 280 nm and 260 nm in the UV spectrum,indicating the few protein and nucleic acid exist in GRP.The periodate oxidation and Smith degradation indicated that GRP included（1→6）–linkage（terminal）,（1→4）–linkage and（1→3）–linkage with the approximate molar ratio of 14.3:1.0:1.2.The fully methylated product of GRP was hydrolazed with acid,then conwerted into alditol acetates,and analyzed by GC-MS.Four types of glucose derivatives in a molar ratio of 12.8:1.1:1:1.3 according to the peak areas were as follows:1,6–Linked Glcp,1,3–Linked Glcp,1,4,6–Linked Glcp and terminal Glcp.Partial acid hydrolysis indicated GRP had a possible（1→3）,（1→6）–linked backbone,and（1→6）-linked and terminal glucoses submitting at the C-4position.The date of ¹H NMR and ¹³C NMR indicated GRP had four kinds ofα–glycosal linkages.The ¹H and ¹³C NMR chemical shifts of GRP were assigned according to ¹H,¹H COSY,¹H,¹³C HMBC and ¹³C DEPT spectra.On the basis of the above-mentioned results,it can be concluded that GRP is comcosed of repeating unit the possible structure as follow.（?）3)biological activities in vitro of GRPThe biological activities of GRP upon splenic lymphocyte,RAW264.7 cells,A549 cell proliferation and nitric oxide（NO）,IL-2 production,were investigated in vitro.The results showed that GRP exhibited inhibition against A549 cells proliferation and NO production in RAW264.7 cells,and promotion for mouse spleen lymphocytes and RAW264.7 cells proliferation,which suggested that GRP may have potential immunoregulation,anti-inflammatory and anti-tumor activity.A homogeneous polysaccharide named as GRP was obtained from Glehniae radix for the first time.Then the isolation,purification,structural analysis and biological activities of water-soluble polysaccharide from the root of Glehnia littoralis have been studied.There has been no reports until now concerning isolation,purification and structural elucidation of purified polysaccharides from Glehniae radix.However,the exploitation and utilization of Glehniae radix polysaccharides could be significant and worth to study further.All results were summarized as follows.