Modification And Activity Analysis Of Low Molecular Weight Guar Gum Polysaccharide

Guar gum is known as the most effective and water-soluble natural polymer,currently used as additive with stable source and low price.In this study,the procedures for the acquisition of uniform low molecular weight guar polysaccharides included enzymolysis and ultrafiltrationa.After being chemically modified,the basic composition,structure analysis and activity evaluation were carried out to provide the basis for the study of the structure-activity of modified polysaccharides.Based on the activity evaluation,the affinity column was prepared to purify the active ingredient,to obtain the high anticoagulant active components,which could provide reference for the development and utilization of guar gum.The first part,preparation and analysis of LGP.The basic composition of guar gum was analyzed,and the crude polysaccharide was deproteinized and purified by DEAE-Sephacel column chromatography to obtain neutral polysaccharide.The procedures for the acquisition of LGP included enzymolysis and ultrafiltration,HPGPC showed the relative molecular mass was 1.12×104Da,and the gas chromatographic analysis showed mannose and galactose at a molar ratio of 1.96:1.00.The structure of LGP was identified by IR,1D NMR and 2D NMR.The sugar chain structure of LGP was introduced as follows:repeat?4)-?-D-Manp-(1?unit as the main chain skeleton,a-D-Galp-(1?as branched which linked to mannose O-6.The second part,chemical modification of LGP and structure analysis of the products.Three sulfated polysaccharides S-LGP1,S-LGP2 and S-LGP3 were modified by chlorosulfonic acid-pyridine method.The degrees of substitution were 0.50,0.64 and 1.20.In addition to the basic composition analysis,the IR spectra showed that the sulfated groups were successfully bound,and the 13C NMR showed that the sulfation substitution sites occurred mainly at the C2 of mannose and the C6 of galactose.Ac-LGP1,Ac-LGP2,Ac-LGP3 and Ac-LGP4 were obtained by acetic anhydride method.The degree of substitution was 0.07,0.11,0.13 and 0.08.After the basic composition analysis,the IR spectra showed acetyl groups have been successfully bound,13C NMR shows that the acetylation substitution site occurs at the C6 of mannose and the C4 of galactose.The third part,activity assessment of modified polysaccharides S-LGP and Ac-LGP.The antioxidant activity showed that the modified polysaccharide showed a significant difference of antioxidant capacity than that of LGP,and concentrations in the experimental concentration range was positively correlated,especially the sulfated polysaccharide showed higher activity more than that of Ac-LGP.The study of anticoagulant activity showed that LGP and Ac-LGP had no obvious anticoagulant activity;S-LGP1 had only a significant effect on the thrombin time;S-LGP2 and S-LGP3 had significant effect on the activated partial thromboplastin time and thrombin time,little effect on prothrombin time.The fourth part,purification of active ingredients by self-made affinity column and evaluation of anticoagulant activity:the reaction conditions of epichlorohydrin activated crosslinked 4B agarose gel were optimized:reaction time was 2 h,2 mg/g NaBH4,2 mL/g ECH,1 mL/g DMSO,epoxy density was 102.0 ?mol/g under the optimum conditions.Optimization of thrombin cross-linking reaction conditions:reaction time was 10 h,reaction temperature was 30 ? and thrombin adsorption rate were 73.21%under the optimum conditions.S-LGP2 was purified by affinity column to obtain the sample(S-LGP2a)with a yield of 16.53%.Analysis of anticoagulant activity showed that for activated partial thromboplastin time and thrombin time,S-LGP2a had a significantly higher activity compared to S-LGP2.