Research On The Characterization And Biological Activity Of Polysaccharides From Three Kinds Of Late-maturing Apple

Apple polysaccharides was obtained from three kinds of late-maturing apple: Granny Smith,Pink Lady and Fuji apple.Polysaccharides content,monosaccharide composition and molecular weight were determined by phenol-sulfuric acid method,gas chromatography and high performance gel permeation(HPGPC)chromatography,seperately.Granny Smith,Pink Lady and Fuji apple polysaccharides was further characterized using fourier transform infrared spectroscopy(FTIR)and differential scanning calorimetry(DSC).The difference of yield,antioxidant activity and inducing apoptosis in gastric and leukemia cells of polysaccharides was compared.Pink Lady apple polysaccharides was purified by chromatography of Cellulose DEAE-52 to get pure fractions.An investigation of the difference of characterization and antioxidant activity of half pure and pure product was carried out.The results were as follows:(1)Compared with the freeze-thaw method and Sevage method,TCA method was more suitable for apple polysaccharides protein removal.When TCA final concentration was 6% more protein and less polysaccharides were removed.Granny Smith apple polysaccharides' s yield(7.00 ± 0.38)% and content(70.15 ± 1.23)%)were greater than those of Pink Lady((4.31±0.45)% and(67.32±2.37)%)and Fuji apple((5.80±0.57)% and(68.55 ± 0.39)%).Taking yield into account,Granny Smith apple polysaccharides was more suitable for the industrial preparation.The molecular weight of Granny Smith apple polysaccharides(1177.06 kDa)were greater than that of Pink Lady(111.28 kDa)and Fuji apple polysaccharides(327.78 kDa).Glucose and galacturonic acid,galacturonic acid,glucuronic acid were the main monosaccharide of Granny Smith,Pink Lady and Fuji apple polysaccharides,respectively.Physical and chemical characteristics of the three were similar and their representational structure was slightly different.Glycoside bond configurations of Granny Smith,Pink Lady and Fuji apple polysaccharide was ?-configuration while thermal characteristics of the three were significantly different.(2)In the range of certain concentration,Granny Smith,Pink Lady and Fuji apples polysaccharides could reduce Fe3+ and scavenge DPPH·,ABTS+·,·OH There was a positive correlation between the antioxidant capacity and the concentrations of apples polysaccharides.Given the above four aspects of the antioxidant system,a comprehensive evaluation was obtained that antioxidant activity of Pink Lady apple polysaccharides was higher than the Granny Smith and Fuji apple polysaccharides,but they were weaker than Trolox.Therefore,Pink Lady apple polysaccharides was an important potential biological response modifiers.(3)The activity of promoting apoptosis in MKN-45 and NB4 cells of Granny Smith apple polysaccharides was stronger than that of the Pink Lady and Fuji apple polysaccharides by the MTT cytotoxicity screening test.Moreover,Granny Smith apple polysaccharides had much toxicity in NB4 cells than MKN-45 cells.MKN-45 and NB4 cells apoptosis rate was significantly increased with its concentration increased using AO/EB staining and flow cytometry.Granny Smith apple polysaccharides at concentration of 10mg/mL,MKN-45 and NB4 cells were significantly inhibited proliferation effect.SOD activity was reduced and MDA content and LDH activity increased with its concentration increasing when different concentrations of Granny Smith apple polysaccharides were added to MKN-45 and NB4 cells.Besides,Granny Smith apple polysaccharides had different degrees of damage in MKN-45 and NB4 cells by cell protease activity detection.Therefore,Granny Smith apple polysaccharides had much potential applying in the field of treatment of leukemia cells.(4)Pink Lady apple polysaccharides was further purified by chromatography of Cellulose DEAE-52 to get pure fractions.The content of the polysaccharides components,monosaccharide composition and molecular weight were respectively determined by phenol sulfuric acid method,gas chromatography,high efficiency and gel permeation chromatography(HPGPC),and polysaccharides components were characterized using Fourier transform infrared spectroscopy(FTIR)and differential scanning calorimetry(DSC).Two kinds of single products(PPS-1 and PPS-2)were gained when the sample amount of PPS was 200 mg and the DEAE-52 column was eluted with 0.1 M,0.2 M NaCl aqueous solution in turn.And the polysaccharides content of PPS,PPS-1 and PPS-2 were 67.32%,89.74% and 67.32% respectively,the molecular weight of PPS,PPS-1 and PPS-2 were 111.28 kDa,52.79 kDa,258.39 kDa seperately.Besides,the monosaccharide composition of PPS,PPS-1 and PPS-2 was the same.But the mole percentage of galacturonic acid in PPS,arabinose and galacturonic acid in PPS-1,galactose and galacturonic acid in PPS-2 were 51.6%,69.9% and 81.3%,respectively.FTIR analysis showed that PPS,PPS-1 and PPS-2 had polysaccharides characteristic absorption peak.The thermal property of the three fractions were consistent with their chemical composition.Antioxidant test revealed that reducing power and removing DPPH·,ABTS+· ability of PPS was higher than PPS-1 and PPS-2 and there was no significant difference in removing ·OH.Since PPS was simple-prepared and had high antioxidant activity compared with PPS-1 and PPS-2,it could be used as the pink lady value-added products in functional food,medicine and other related fields.In summary,it was verified that three apple polysaccharides semi-pure product and Pink Lady apple polysaccharides pure products had characteristic structure and physicochemical properties of polysaccharides.In addition,TCA method was suitable for removing protein.The yield,polysaccharide content and the activity of promoting apoptosis in MKN-45 and NB4 cells of Granny Smith apple polysaccharides was greater than the other apple polysaccharides among the three kinds of late-maturing apple.Afterwords,the yield and polysaccharide content of Pink Lady apple polysaccharides were less and its antioxidant activity was greater than the other apple polysaccharides.Suitable separating conditions of Pink Lady apple polysaccharides was obtained and its anoxidant activity was higher than the pure products.