| Objective:The extraction,separation and purification,structure characterization of some components of Isatis root polysaccharides,and the study of different components of Isatis root polysaccharides by sugar spectrum.There are few researches on the structure analysis of Isatis root polysaccharides,so this article is based on the extraction,separation and purification.On this basis,the structure of the separated homogeneous components is characterized.And customized DEAE chromatographic columns,using ELSD and MALLS two detectors to explore the sugar spectrum of Isatis root polysaccharide,to promote the quality standards of Isatis indigotica Fort.Methods:Crude Isatis root polysaccharides(IRPS),was extracted by water extraction and alcohol precipitation.Crude Isatis root polysaccharides(IRPS)was isolated and purified by DEAE FF 16/10.Then the physicochemical properties,monosaccharide composition,relative content and molecular weight of different components of Isatis root polysaccharides were studied by phenol sulfuric acid method,m-hydroxybiphenyl method,Coomassie brilliant blue method,HPAEC-PAD,HPGPC-RID and MALLS.The homogeneous components IRPS1-1,IRPS1-4 and IRPS2-2 were further characterized by UV,IR,NMR,HPAEC-PAD,MALLS,methylation combined with GC-MS,atomic force microscopy(AFM)and scanning electron microscopy(SEM).A customized DEAE-2SW(4.6 × 250 mm,5μm)column was used to detect the crude polysaccharide spectrum of Isatis root polysaccharides by ELSD and MALLS.Different components of Isatis root polysaccharides were separated on the same chromatogram to improve the quality standard of Isatis indigotica Fort.Results:1.The extraction rate of crude Isatis root polysaccharides was 2.5%.IRPS was separated and purified by DEAE,and thirteen fractions were named IRPS1-1,IRPS1-2,IRPS1-3,IRPS1-4,IRPS2-1,IRPS2-2,IRPS3-1,IRPS3-2,IRPS3-3,IRPS4-1,IRPS5-1,IRPS5-2,IRPS6-1.The yield was 26.30%,0.61%,1.03%,0.61%,9.82%,1.58%,1.82%,2.00%,3.82%,19.09%,3.28%,1.39%,2.06%,respectively.2.The neutral sugar,acid sugar and protein content of IRPS 1-1,IRPS 1-2,IRPS 1-3,IRPS1-4,IRPS2-1,IRPS2-2,IRPS3-1,IRPS3-2,IRPS3-3,IRPS4-1,IRPS5-1,IRPS5-2,IRPS6-1 was 2.43%,24.51%,34.57%,29.31%,24.71%,21.07%,41.43%,31.62%,26.18%,45.34%,26.96%,17.25%,18.97%;4.90%,3.62%,4.28%,5.90%,6.43%,5.20%,12.80%,10.73%,12.72%,16.10%,18.03%,8.14%,11.21%;4.93%,15.75%,15.76%,12.84%,7.95%,9.00%,7.47%,9.09%,7.20%,7.52%,13.78%,15.76%,17.51%.After deproteinization by sevag method,the content of crude polysaccharide protein is still high,and there may be glycoprotein structure linked with polysaccharide,but the protein content of most polysaccharide components is reduced after DEAE column chromatography.3.The monosaccharide compositions of the 13 Isatis root polysaccharides were rhamnose,arabinose,galactose,glucose,xylose and mannose,and some of them contained glucuronic acid and galacturonic acid.The corresponding molar ratio of IRPS1-1 is 0.093:36.360:6.210:36.78:9.385:11.175;that of IRPS1-2 is 0.289:37.832:5.703:48.548:3.384:4.245;that of IRPS1-3 is 0.862:46.578:2.956:45.813:1.259:2.531;that of IRPS1-4 is 0.257:30.844:8.901:48.985:2.456:8.557;that of IRPS2-1 is 6.752:22.774:31.388:30.113:3.123:5.849;that of IRPS2-2 is 0.579:20.279:11.259:61.028:0.951:5.904;that of IRPS3-1 is 3.062:30.404:27.029:33.380:0.849:3.347:0.675:1.253;that of IRPS3-2 is 6.114:35.458:27.392:22.963:0.802:1.402:1.533:4.335;that of IRPS3-3 is 2.313:28.228:18.887:34.896:1.216:1.685:1.468:11.305;that of IRPS4-1 is 6.121:18.340:11.189:6.630:1.878:1.507:1.673:52.664;that of IRPS5-1 is 22.865:25.711:12.639:12.761:2.569:1.114:1.287:21.054;that of IRPS5-2 is 10.806:31.571:12.214:37.104:2.179:0.049:1.543:6.387;that of IRPS6-1 is 7.361:19.838:3.536:58.111:0.258:1.842:1.637:7.418.The monosaccharide composition and content of each component were similar,but slightly different.After the concentration of eluent increased to 0.1 M,the isolated polysaccharides appeared glucuronic acid and galacturonic acid.4.The results showed that the weight average molecular weight of 13 Isatis root polysaccharides were 2.855 × 105,9.320 × 105,2.064 × 105,1.960 × 105,7.717 × 104,1.061 ×105,1.445 × 105,2.683 × 105,7.236 × 104,5.412 × 104,1.641 × 105,1.290 × 105,1.173 × 105 Da.The polydispersity of 13 Isatis root polysaccharides were 1.076,8.139,1.898,1.153,1.031,1.453,1.073,1.900,1.060,1.084,1.113,1.041,1.064.IRPS1-1,IRPS2-1,IRPS3-1,IRPS3-3,IRPS4-1,IRPS5-2,IRPS6-1 are uniformly distributed,IRPS1-3,IRPS1-4,IRPS2-2,IRPS3-2,IRPS5-1 are narrow distribution,IRPS1-2 is wide distribution.The hydrodynamic radii rh of 13 Isatis root polysaccharides are 37.004,292.677,248.685,273.676,182.642,287.582,264.609,266.844,250.437,210.951,265.463,274.377 and 253.989 nm,respectively.Radii rn are 74.2,94.0,98.3,90.9,62.8,46.9,29.9,80.5,54.5,65.8,79.9,63.8 and 65.6 nm,respectively.5.IRPS1-1,IRPS1-4 and IRPS2-2 had a single absorption peak of polysaccharides at 195 nm,and no characteristic absorption peak of carotenoids was found in the wavelength range of 350 nm～550 nm.There was no obvious absorption peak at 260 nm,indicating that the three polysaccharides did not contain nucleic acid or trace nucleic acid,and there was a weak absorption peak at 260 nm～280 nm,indicating that IRPS1-1,IRPS1-4 and IRPS2-2 were glycoprotein complexes.IRPS1-1 had the typical absorption peaks of 3415,2929,1638,1413 cm-1 and the ring skeleton structure of pyran ring;IRPS1-4 had the characteristic absorption peaks of 3440,2931,1635 cm-1 and the ring skeleton structure of furan ring;IRPS2-2 has the characteristic absorption peaks of 3426,2928,1634 and 1420 cm-1,and has the ring skeleton structure of pyran ring.IRPS1-1,IRPS1-4,and IRPS2-2 are homogeneous polysaccharides,the weight average molecular weight,monosaccharide composition and ratio are as described above.IRPS1-1 contains β pyranose,α-glycosidic bonds,T-α-Arap and α-1,5-Arap glycosidic bonds,1→4 glycosidic bonds C-2 and C-6 substitutions,1→6 Glycosidic bond C-5 substitution;IRPS1-4 contains β-furanose and α-glycosidic bond,The C-2,C-5,C-4 and C-6 positions are substituted in the 1→4 glycosidic bond,and the C-5 in the 1→6 glycosidic bond is substituted,There is an absorption peak at C-2 of α-1,4-GalpA;IRPS2-2 contains β-pyranose,β-glycosidic bonds,and a signal peak at the C-6 position of rhamnose.There are 8 glycosidic bonds in IRPS1-1,namely T-Arap,1,4-Arap,1,3,4-Arap,1,6-Galp,1,3-Galp,1,4,6-Galp,1,4-Glcp and 1,6-Glcp;IRPS1-4 contains 4 types of glycosidic bonds,namely 1,3-Araf,T-Araf,1,6-Galf,and T-Galf;IRPS2-2 contains two types of glycosidic bonds,namely 1,5-Araf and 1,4-Arap.IRPS1-1 is a chain like multi filament branching structure with smooth surface;IRPS1-4 is a granular stacking and interweaving structure with smooth surface;IRPS2-2 is a chain like multi filament branching structure with rough surface.IRPS1-1 is a branched chain structure.These macromolecular chains intertwine to form a network like structure;IRPS1-4 is a network of branched filaments with scattered fusiform structure;IRPS2-2 presents a small dot like structure,accompanied by a fusiform aggregation structure.6.In this study,we used custom DEAE-2SW(4.6 × 250 mm,5 μm)column,evaporation light scattering detector(ELSD)to detect 8 parts of Isatis root polysaccharides,and 8 parts of Isatis root polysaccharides by multi angle laser light scattering detector(MALLS),and then the quantitative determination of polysaccharides was obtained.Conclusion:In this study,HPAEC-PAD analysis method without derivatization,high sensitivity and good separation effect were established,which can be used for the determination of monosaccharide composition and content of IRPS,and MALLS method for direct measurement of weight average molecular weight of IRPS without establishing molecular weight standard curve.In addition,the structures of IRPS1-1,IRPS1-4 and IRPS2-2 were preliminarily characterized.The saccharide spectrum of Isatis root polysaccharides was obtained by using a customized column.The different components of Isatis root polysaccharides were separated on the same chromatogram to improve the quality standard of Isatis indigotica Fort and promote the qualitative and quantitative analysis of polysaccharides. |
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