Studies On Structure-activity Relationship Of Dendrobium Huoshanense Polysaccharides Against Protein Nonenzymatic Glycosylation Process Using Sulfated Modification

Dendrobium huoshanense C.Z. Tang et S.J. Cheng belonging to Orchidanceaefamily, is a famous traditional Chinese medicine in China. Polysaccharides havebeen reported tobe the principal bioactive constituents of this plant, exhibitingsome important functions of anti-oxidant, immunoregulation and anti-cataract. Theaim of this study was to investigate the inhibitory effects of sulfatedD.huoshanense polysaccharides on protein nonenzymatic glycation. Accordingly,the relationships between sulfated structures of polysaccharides and activitiesagainst protein nonenzymatic glycation were elucidated. The main results wereobtained as follows:(1) Purification and Characterization of Polysaccharides: After extractionand purification, two kinds of homogenous polysaccharide fractions were obtainedfrom the crude polysaccharides of Dendrobium huoshanense (DHP), namedDHP-D1, DHP-D2respectively. The molecular weights of DHP-D1and DHP-D2were determined as3.2×103Da and8.09×106Da, respectively. DHP-D1was mainlycomposed of glucose, arabinose, galactose in the ratio of1.023:0.023:0.021, alongwith trace of mannose and xylose. DHP-D2consisted of galactose, glucose,arabinose, mannose, xylose and rhamnose in the ratio of0.896:0.723:0.2:0.072:0.031:0.028. Structure analysis showed that the backbone of DHP-D1consisted of1,4-α-D-Glcp,1,6-α-D-Glcp,1,5-α-L-Araf and1,6-α-D-Glap where1-α-Glcp and atrace of1-β-D-Xylp were attached to the C-6position of1,4-α-D-Glcp. What’smore,1-α-Glcp also was attached to the C-6position of1,3-α-D-Manp. DHP-D2was mainly composed of1,6-β-D-Galp,1,4-β-D-Glcp,1,6-β-D-Glcp,1,5-α-L-Arafand1,6-α-D-Glcp where1-α-D-Manp,1-α-D-Xlyp and3-α-L-Rhap were attachedto the C-3position of1,6-β-D-Galp.(2) Sulfation of DHP-D1and DHP-D2: The chlorosulfonic acid-pyridinemethod was empolyed to modify the polysaccharides from Dendrobiumhuoshanense. The effects of solvent type, solvent amount, ratio and dosage ofesterification reagent, reaction time and reaction temperature on substitution degree(DS) were investigated in turn. Based on the results of single-factor-test, thesulfation conditions were further optimized by using response surface methodology. The maximum DS of1.473was obtained under an optimal reaction conditionincluding DHP of100mg, formamide dosage of5mL, esterification reagent dosageof6mL, volume ratio of pyridine to chlorosulfonic acid of2:1, reaction time of160minutes, and the reaction temperature of60℃. By applying this sulfatedreaction conditions, the sulfated modification of DHP-D1and DHP-D2wereperformed by changing the reaction time, respectively, giving a series of sulfatedderivatives with different DS. For DHP-D1, the DS was increased with theprolonging of reaction time. However, negative effects on DHP-D2sulfation wereobserved when the reaction time was too long. The positioned sulfationpolysaccharide derivatives were prepared by the strategies of positioning protectedhydroxy.6-primary hydroxyl was found to be easier sulfated, and the sulfatedderivatives of more substitution were obtained.(3) Inhibitory Actions on Protein Nonenzymatic Glycation: The model ofprotein non-enzymatic glycosylation in vitro was employed to evaluate thebioactivities of DHP-D1, DHP-D2and their sulfated derivatives. Amino guanidinewas used as the positive control. Results showed that all agents exhibited inhibitoryactions on protein non-enzymatic glycosylation in a dose-dependent manner. Forthe actions of polysaccharides against Amadori products formation, the inhibitoryactivities of all agents increased with the culture proceeding from0to21days andthen decreased to different level. Moreover, DHP-D1, DHP-D2and their sulfatedderivatives showed stronger inhibitory response on Amadori products formationthan that of aminoguanidine. For the actions of polysaccharides against Dicarbonylcompounds formation, all test samples displayed this ability in a dose-dependentmanner. The maximum inhibition rates of each agent at different concentrationwere observed at culture time of28day. For the actions of polysaccharides againstAGEs formation, we found that the capability of all test agents was consistent withthose of inhibiting Dicarbonyl compounds formation.(4) Relationships between Structures and Inhibitory Activities on ProteinNonenzymatic Glycation: Results showed that sulfated modification couldenhance the inhibitory actions of DHP-D1and DHP-D2on protein nonenzymaticglycation. The inhibitory activity was enhanced with the increase of sulfate groupnumbers attached to polysaccharides. Moreover, the derivatives with sulfate grouplinked to C-6position of polysaccharides had higher activity than those of2,2’,4-Osulfated polysaccharide derivatives.