| Traditional Chinese Medicine theory believes that Astragalus membranaceus can enhance Qi-tonifying effect after honey processing.Our previous studies showed that polysaccharides were important active substances in the effect of reinforcing Qi of Astragalus membranaceus and the relative molecular weight and monosaccharide composition ratio of polysaccharides also changed before and after honey processing.In this study,novel high-molecular-weight polysaccharides were extracted and purified from honey-processed Astragalus and Astragalus and their structural characteristics were further elucidated and compared.Furthermore,their effects on probiotics and metabolites(SCFAs,lactic acid and pyruvate)in vitro were further evaluated in order to enrich the experimental basis of processing mechanism of Astragalus.Methods: The crude polysaccharides HAPS and APS were obtained from the same batch of honey-processed Astragalus and Astragalus by heating reflux extraction,alcohol precipitation,removing protein by enzyme-Sevage method and freeze-drying.Six high purity polysaccharides HAPS2 a,APS2a,HAPS2 b,APS2b,HAPS3 a and APS3 a were obtained by further isolation and purification by DEAE-52 anion exchange column,FOcurose c L-6B and HW-65 F exclusion column.The purity and relative molecular weight of polysaccharides were determined by UV scanning and HPLC-GFC-ELSD.Their structures were characterized by UPLC/Q-TOF-MS,Fourier-transform infrared spectroscopic(FT-IR)analysis,methylation analysis,gas chromatography-mass spectrometry(GC-MS),NMR analysis,triple helix structure determination.Four kinds of probiotics were cultured in vitro with the above homogeneous polysaccharides as the only carbon source of the culture medium.The concentration of probiotic was determined by enzyme labeling instrument and the molecular weight of degraded polysaccharide was determined by HPLC-GFC-ELSD.The monosaccharide composition of degraded polysaccharides and the metabolites(SCFAs,lactic acid and pyruvate)were determined by UPLC/Q-TOF-MS.Results: Three high molecular weight honey-processed Astragalus polysaccharides(HAPS2a,HAPS2 b,HAPS3a)and Astragalus polysaccharides(APS2a,APS2 b,APS3a)were all highly branched acidic heteropolysaccharides containing β-configuration and α-configuration glycosidic bonds.The molecular weights of HAPS2 a and APS2 a were 10009.7 k Da and 16844.7 k Da,respectively,which were composed of Man,Rha,Glc A,Gal A,Glc,Gal and Ara and their corresponding molar percentages were 0.22: 1.47: 0.27: 0.22: 2.44: 50.76: 44.62 and 0.90: 10.05: 1.10: 8.76: 2.05: 33.52: 43.62,respectively.They were different in the molar ratios of Rha,Gla and Gal A.The backbond of HAPS2 a was consisted of 1,3,4-β-Galp and 1,6-α-Galp while APS2 a was 1,3,4-α-Galp,1,3,4-β-Rhap,1,6-α-Galp and 1,3-α-Rhap.The molecular weights of HAPS2 b and APS2 b were 5345.7 k Da and 7664.8 k Da,respectively,which were composed of Man,Rha,Glc A,Gal A,Glc,Gal,Xyl,Ara and Fuc and their corresponding molar percentages were 0.92: 9.27: 0.26: 45.79: 2.02: 15.75: 25.01: 0.98 and 1.82: 21.41: 1.82: 51.08: 2.17: 11.34: 9.26: 2.91,respectively.They were different in the molar percentages of Rha,Gal and Gal A.The backbond of HAPS2 b was consisted of 1,2,3,4-α-Galp,1,3,4,6-α-Galp and 1,2,4-α-Galp A while APS2 b was 1,2,3,4-α-Galp A,1,3,4,6-α-Galp A and 1,2,4-α-Galp A.The molecular weights of HAPS3 a and APS3 a were 2463.5 k Da and 3373.2 k Da,respectively,which were composed of Man,Rha,Glc A,Gal A,Glc,Gal,Ara and Fuc and their corresponding molar percentage were 0.4: 13.8: 0.69: 33.24: 2.37: 22.66: 26.18: 0.66 and 0.25: 14.47: 1.16: 49.55: 0.79: 11.87: 21.47: 0.44,respectively.They were different in the molar percentages of Gal and Gal A.The backbond of HAPS3 a and APS3 a were 1,2,3-β-Rhap,1,4-α-Galp A,1,2,6-β-Galp,1,4-β-Galp and 1,2-α-Galp A.The results of in vitro culture of four kinds of probiotics showed that HAPS2 a,HAPS2b,APS2 b and HAPS3 a could obviously promote the growth of BL,while only HAPS2 a and HAPS3 a could significantly promote the growth of LGG.The above six polysaccharides could obviously promote the growth of BO and BT.The main metabolites were lactic acid,acetic acid,propionic acid,butyric acid and pyruvate.However,the content of butyric acid was higher in APS2 a,APS2b and APS3 a groups while the content of lactic acid produced by LGG was higher in HAPS2 a,HAPS2b and HAPS3 a groups.Conclusion: In this study,homogenous high-molecular-weight honey-processed Astragalus polysaccharides and Astragalus polysaccharides were successfully isolated and purified.These polysaccharides were differed in molecular weight,monosaccharide composition and glycosidic bonds before and after honey processing.HAPS2 a and HAPS3 a could promote the growth of LGG and BL more significantly than APS2 a and APS3 a,respectively.The differences between the effects of honey-processing Astragalus polysaccharides and Astragalus polysaccharides on probiotics were related to the differences of chemical structure caused by honey processing. |
