Structure Characterization, Chemical Modification And Activity Of Exopolysaccharides From Punica Granatum L. NJUSTb1

Endophytes which are ubiquitous in plants,can induce a lot of secondary metabolites the same as their host plants.Over the past decades,endophytes have become an important source of new drug research and development.The previous studies indicates that the exopolysaccharides of endophytes from medicinal and/or edible plants have multiple biological activities,such as antitumor,immunomodulatory,sterilization and antioxidation etc.In this study,we studied the isolation,purification,structure elucidation,chemical modification,immunomodulatory bioactivity and hydroscopicity and moisture retentive ability of the exopolysaccharides of endophytic fungus NJUSTb1 isolated from the leaves of Punica granatum L.The results were showed as follows:1.Endophytic fungus NJUSTb1 was fermented in large scale.The fermentation liquor was centrifuged to remove cells and the supernate was pooled.The supernate was then precipitated by 4 volumes ethanol.The protein in precipitate was removed by Sevage method,and then dialyzed.Dialyzate was freez-dried under reduced pressure.The yield of EPS was 900 mg/L.After purified by Sephadex G-200,two exopolysaccharides(EPS-1 and EPS-2)were obtained.Due to the content of EPS-2 was less than 4%,EPS-1 was investigated here.The MW of EPS-1 was 343 kDa.Composition analysis revealed that in EPS and EPS-1,protein content was 0.65% and 0.19%,respectively,polysaccharide content was 95.98% and 98.66%.EPS and EPS-1 had no uronic acid.2.The chemical structure of EPS-1 was invistigated by various methods.UV spectrum showed that there was no nucleic acid in EPS and EPS-1.The results of monosaccharide composition showed that EPS-1 was mainly composed of glucose,little mannose and galactose.The IR spectrum showed that the peaks of ESP-1 were in agreement with the typical absorption peaks of polysaccharides.Methylation reaction,Periodate oxidation,Smith degradation,GC-MS and NMR analasis indicated that the backbone of EPS-1 were composed of four monosaccharides unit ?4)-?-D-Glcp-(1?6)-?-D-Glcp-(1?4)-?-D-Glcp-(1?4)-?-D-Glcp-(1?.SEM analysis indicated that EPS-1 was aggregated in the form of filaments,and the accumulation was dishevelled.There were spherical aggregates.Congo red reaction indicated that EPS-1 had not triple helical structure.3.Chlorosulfonic acidic pyridine method and solvent media process were used to modify EPS-1,respectively.The IR spectrum showed that sulfated polysaccharide S-EPS1 which was blue and carboxymethyl polysaccharide C-EPS1 which was white were obtained.The degrees of substitution(DS)of S-EPS1 and C-EPS1 were 1.228 and 0.903,respectively.The immunomodulatory experiments in vitro indicated EPS,S-EPS1 and C-EPS1 significantly promoted proliferation of RAW 264.7 cells at the concentrations of 31.25-500 ?g/mL.EPS-1 had no effect on cell grow at the concentration of 31.25-500 ?g/mL.EPS could significantly promote production of NO,TNF-? and IL-6 within a certain concentration range.When the concentrations were 31.25-500 ?g/mL,EPS-1,S-EPS1 and C-EPS1 had no effect on the production of NO and IL-6.When the concentrations were 250 ?g/mL and 500 ?g/mL,EPS-1 could significantly promote TNF-? production.S-EPS1 and C-EPS1 could significantly promote TNF-? production at the concentrations of 500 ?g/mL.The hydroscopicity of S-EPS1 and C-EPS1 was better than EPS-1.The moisture retention effect of S-EPS1 was better than EPS-1.The sulfated derivatives showed a potential promoting effects on hydroscopicity and moisture retentive ability of EPS-1.