| Decolourization of polysaccharides is one of the crucial procedures that affects their structure.Here,Grifola frondosa polysaccharide(GFP)was decolourized with H2O2 and AB-8 macroporous resin.Then,two main fractions,named DGFP and SGFP,were obtained by purification with Sepharose CL-4B.The total carbohydrate contents of DGFP and SGFP were determined to be 95.26%and 92.02%by the phenol-sulfuric acid method.The molecular weights of two polysaccharides were determined as 6.306 × 106(±0.410%)Da and 1.174 × 107(±0.299%)Da by SEC-MALS.Monosaccharide analysis indicated that DGFP was composed of glucose,mannose,and galactose(molar ratio of 32.20:1.00:1.75),while SGFP consisted entirely of glucose.Despite a backbone→4)-α-Glcp-(1→in two polysaccharides,reducing ends Rα→3)-α-Glcp and Rβ→4)-β-Glcp were observed in DGFP by NMR analysis.The results suggested that decolourization with H2O2 might alter the structure of polysaccharide and generate new reducing ends.In vitro antioxidant results implied that DGFP might be a more promising natural antioxidant than SGFP.By optimizing the conditions for the purification of Grifola frondosa polysaccharide by analytical SEC,a method that can be used for the refinement of complex sample components was successfully established.Four polysaccharide subfractions with different molecular weight distributions were purified from Grifola frondosa polysaccharide,and the stability and feasibility of purification were analyzed.Sulfated Grifola frondosa polysaccharide(S-GFP)was prepared by the chlorosulfonic acid-pyridine method.The degrees of substitution of S-GFP were determined as 0.516 and 0.511 by IC analysis and elemental analysis.HPGPC analysis indicated that sulfation and degradation could exist concurrently during the sulfation reaction.S-GFP is a heteropolysaccharide composed of galactose,glucose and mannose.The substitution position of S-GFP was C-6 characterized by FT-IR and NMR analysis,which confirmed the successful synthesis of sulfated polysaccharide.The results implied that S-GFP is a non-competitive inhibitor of α-glucosidase with an IC50 value of 3.353 μg/mL.After interacting with α-glucosidase,S-GFP quenched the endogenous fluorescence of α-glucosidase and increased the polarity of fluorescent microenvironment.The interaction between S-GFP and α-glucosidase changed the backbone structure of the protein,resulting in an increase in the molecular diameter of the enzyme and an enhanced scattering effect.CD analysis showed that the treatment of S-GFP changed the conformation of α-glucosidase,increased the content of α-helix structure,and destroyed the hydrogen bond system in the secondary structure of enzyme.S-GFP could significantly reduce the levels of HB sAg and HB eAg in HepG2.2.15 cells.When the concentration of S-GFP was 200 μg/mL,the inhibition rates of extracellular HBsAg and HBeAg after 6 days of treatment were 33.0%and 34.1%,respectively.The relative levels of extracellular HBV DNA were 69.81%,28.67%and 5.27%,while the relative levels of intracellular HBV DNA increased 2.71,3.26 and 3.37-fold,respectively.S-GFP could inhibit the synthesis of intracellular M-HBs and S-HB s,and increase the level of intracellular L-HB s.The lack of M-HBs and S-HBs could not form a complete envelope protein,causing the accumulation of L-HB s in the endoplasmic reticulum.At the same time,HBV DNA synthesized by reverse transcription of pgRNA cannot be secreted to the outside of the cell due to the lack of the correct envelope protein,causing its accumulation in the cell.Under the co-culture of MG132 or Bortezomib with S-GFP,the levels of M-HBs and S-HBs envelope proteins showed an increasing trend.The results show that the M-HBs and S-HBs envelope proteins could be degraded in cells through the proteasome pathway,and the degradation process could be blocked by proteasome inhibitors.In L-HBs-deficient Huh7 cells transfected with M-HBsAg gene,S-GFP could still inhibit the synthesis of MHBs and S-HBs,and reduce the levels of glycosylated and aglycosylated antigens. |
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