Isolation,Purification,Structural Characterization And Primary Biological Activities Of Polysaccharides From Ficus Carica

Objective:This paper is aimed to systematically extract,isolate and purify novel polysaccharides from Ficus carica（F.carica）and investigate their properties,structure characterization,sulfated modifacations and bioactivities.Methods:By using ultrasound-assisted hot water,alkali,simulated gastric extraction methods,followed by alcohol precipitation,Sevag method for deproteinization,dialysis and lyophilization,crude polysaccharides were obtained from F.carica.The crude polysaccharides were further purified by DEAE Sepharose Fast Flow and Sephadex G-75 column chromatography to get pure polysaccharides.The homogeneity of polysaccharides was determined by specific rotation spectrophotometry,UV scanning and HPGPC.The properties were determined by phenol-sulfuric,Biuret reagent and sulfuric acid carbazole methods.The primary structures of pure polysaccharides were determined by classical chemical methods including complete acid hydrolysis and methylation,combined with instrumental analysis such as FT-IR,HPAEC-PAD,GC-MS,HPLC,1D and 2D NMR.The sulfated polysaccharide was obtained by chlorosulfonic acid-pyridine method.Furthermore,the advanced structure of each polysaccharide was characterized by the Congo red method,scanning electron microscopy（SEM）and atomic force microscopy（AFM）.The radical scavenging assays and zebrafish antioxidant assays were used to evaluate the antioxidant activities in vitro and in vivo.The immunomodulatory activities were analyzed by the proliferation of mouse splenocytes,the viability of RAW264.7 macrophages and phagocytic functions.Results:Five crude polysaccharides were obtained from F.carica through three kinds of extraction methods,which were designated as FCPW50,FCPW80,FCPB80,FCPA50 and FCPA70.The crude polysaccharides FCPW80,FCPB80 and FCPA70were further purified by DEAE Sepharose Fast Flow and Sephadex G-75 column chromatography to get four pure polysaccharides which were designated as FCPW80-2,FCPB80-1,FCPB80-2 and FCPA70-1,respectively.The results of purity identification indicated that the four polysaccharides had high purity and contained no uronic acid and no protein.The carbohydrate contents of FCPW80-2,FCPB80-1,FCPB80-2 and FCPA70-1 were 95.64%,95.01%,96.6%and 97.6%by the phenol-sulfuric method.The average molecular weights of these four polysaccharides were1.21×10⁵ Da,2.25×10⁴ Da,1.03×10⁵ Da and 2.19×10⁴ Da according to HPGPC.The results of structure analysis indicated that the backbone of FCPW80-2 was composed of（1→5）-α-L-Ara,（1→4,6）-β-D-Gal,（1→3,6）-β-D-Man,which branched at O-4 and O-6.The branches consisted of（1→3）-linkedβ-L-Rha,（1→4）-linkedα-D-Glc,terminated with（1→）-β-D-Glc.The backbone of FCPB80-1 was composed of（1→6）-β-D-Gal,（1→5）-α-L-Ara,（1→3,6）-β-D-Man,（1→2,6）-β-D-Man,which both branched at O-6.The branches consisted of（1→6）-β-D-Gal,（1→6）-α-D-Glc and（1→4）-α-D-Glc,terminated with（1→）-α-D-Glc and（1→）-α-D-Man.The backbone of FCPB80-2was composed of（1→6）-α-D-Glc,（1→2）-β-D-Man,（1→5）-α-L-Ara and（1→3,6）-β-D-Man,which branched at O-6.The branches consisted of（1→4）-β-L-Rha and（1→6）-β-D-Gal,terminated with（1→）-α-D-Glc.The backbone of FCPA70-1 was composed of（1→6）-β-D-Gal,（1→5）-α-L-Ara,（1→4）-β-D-Gal and（1→3,6）-β-D-Gal,which branched at O-6.The branches consisted of（1→4）-β-L-Rha,（1→6）-α-D-Glc and（1→4）-α-D-Glc terminated with（1→）-α-D-Glc.The sulfated polysaccharide（FCPW80-M2）was obtained by chlorosulfonic acid-pyridine method,which was to be proved successful by FT-IR spectra.The DS value was calculated as 1.70 and the content of sulfur content was 16.2%.The average molecular weight was 9.86×10⁴ Da.The results of Congo red assay indicated that there was no triple helical comformation in the four pure polysaccharides.The in vitro antioxidant assays indicated that four pure polysaccharides including FCPW80-2,FCPB80-1,FCPB80-2 and FCPA70-1 exhibited DPPH and ABTS^+·radical scavenging activitites and FCPA70-1 showed the highest antioxidant activity.The in vivo zebrafish antioxidant assays showed that FCPW80-2,FCPB80-2,FCPA70-1 and FCPW80-M2 exhibited good antioxidant abilities and the sulfated polysaccharide FCPW80-M2 showed higher antioxidant activity compared with FCPW80-2.The results of the immunomodulatory assay showed that polysaccharides exhibited moderate immunomodulatory activity and the sulfated polysaccharide obviously showed higher immunomodulatory potency.Conclusion:Four pure polysaccharides were obtained from F.carica and their primary structures were also elcudiated.The sulfated polysaccharide（FCPW80-M2）was successfully prepared from FCPW80-2 and its properties were also investigated.The bioactivity assays indicated that the polysaccharides from F.carica showed antioxidant and immunomodulatory activity and the sulfated polysaccharide（FCPW80-M2）obviously showed higher activity compared with FCPW80-2.