Extraction, Purification, Structural Characterization And Immune Activity In Vivo Of Yellow Pear Residue Polysaccharides

Gaoping yellow pears are a special local product of Shanxi Province.Gaoping is known as the town of pears and has a 1500-year history of pear planting.Gaoping yellow pears are well-known for their large size,strong sweet and delicate taste,suitable mouth feel and ease for storage,and are rich in vitamins,minerals,sugars and moisture.However,the development of the pear juice processing industry has left an increasing amount of pear residues,which are rich in polysaccharides,the important bioactive substances for organisms.Polysaccharide also have biological functions of immunity regulation,antioxidation,antitumor,hypolipidemic and hypoglycemic effects.Therefore,pear residues can be used as a main source to extract polysaccharides.In this study,starting from Shanxi Gaoping yellow pear residues and using modern extraction and purification technology,we primarily analyzed the polysaccharide extraction process from yellow pear residues as well as the isolation & purification,relative molecular weights,composition and basic structures of the monosaccharides.Also the immunomodulatory and antioxidant capacities in vivo were evaluated.The findings will offer some reference for further exploitation and utilization of yellow pear residues.The results are as follows:(1)From the residues of Gaoping yellow pears,polysaccharides were extracted by either the water bath alcohol precipitation method or the ultrasound-assisted method.Then with the polysaccharide yield as the target,the two methods were optimized via both single-factor assays and RSM assays.The optimal conditions of the water bath alcohol precipitation method were found to be material-liquid ratio = 1:35,temperature at 67 ? and extraction time of 3.7 h,under which the polysaccharide yield averaged from three paralell assays was 56.15 mg/g.The optimal conditions of the ultrasound-assisted method were material-liquid ratio = 1:13,ultrasonic time= 60 min,ultrasonic power = 132 W,and ultrasonic temperature at 57 ?.Then three repeated experiments were conducted under these conditions,and the average polysaccharide yield was 62.38 mg/g,which was higher than that of the water bath alcohol precipitation method.In comparison,the ultrasound-assisted extraction method outperformed with higher efficiency,convenience and less time consumption,and thus was used to extract polysaccharides from yellow pear residues.(2)The crude polysaccharides were deproteinized via the papain +Sevag method,which resulted in a protein removal rate of 75% and polysaccharide loss rate of only 24.71%.Thus,the combined method can utmost remove proteins in an easy and time-saving way,while ensuring the polysaccharide yield.The H2O2 method yielded the highest decolorization rate of 78.56% and the lowest polysaccharide loss rate,and it was operationally easy.After chromatography on the DEAE-52 cellulose column,the deproteinized,decolorized and dialyzed pear residue polysaccharides were isolated into 3 peaks,indicating the polysaccharides consisted of 3different portions.Since the other two portions were minor,only LPB-C was used in the subsequent experiments.The curve of LPB-C after passing the Sephadex G-100 gel column showed a single symmetrical peak,and UV spectra showed a characteristic absorption peak at 225 nm,but not at 260 or280 nm.It was suggested the polysaccharides after the chromatography purification did not contain nucleic acids,proteins or other impurity,and the LPB-C after freeze-thaw and centrifugation did not precipitate.All the three methods confirm LPB-C was pure polysaccharides with simple distribution of relative molecular mass.(3)The intrinsic viscosity [?] of LPB-C was measured to be 1.11 cm3/g,and its viscosity-averaged molecular weight was determined by the Mark-Houwink equation to be 8.47×104 g/mol.Then the super-high-performance liquid chromatography images of the yellow pear residue polysaccharide derivatives were compared with the mixed standard monosaccharides.Based on the peak appearance time,it was determined the yellow pear residue polysaccharides contained four monosaccharides ofmannose,rhamnose,glucose and xylose,mass ratio of which was determined to be 3.3: 2.3: 10.6: 23.2 according to the peak areas.Infra-red spectra of LPB-C showed characteristic absorption peak of polysaccharides,which were deduced to be pyran polysaccharides containing ?-glucosidic bond.(4)In conclusions,mice were injected intraperitoneally with 40 mg/kg CY for 5 days to construct an immunosuppressive mice model in this study.The immune indexes(except the MDA level)in the model group decreased to different degrees,indicating the model was established successfully.On this basis,the immunosuppressive mice were gavaged with different concentrations of LPB-C to study their effects on the immune indexes.It was found the thymus index and spleen index significantly increased after 15 days of continuous intragastric administration.The carbon clearance ability gradually rose with the increase of LPB-C concentration and the phagocytic index in high dose group was the most significant.The earlap swelling rate obviously increased,suggesting LPB-C could restore the inhibited DTH reaction in mice and promote the cellular immune function.The secretion of serum TNF-?,IFN-? and IL-4 in the treated mice increased significantly,which can effectively kill and inhibit tumor cells,inhibit virus replication,and regulate various hematopoietic cells.Meanwhile,the activities of SOD,CAT and GSH-Px in the liver,heart and kidney were all markedly elevated,while the MDA levels decreased,indicating LPB-C could improve the antioxidant ability of immunosuppressive mice.