Studies On Structures And Anti-cataract Activities Of Polysaccharids From Dendrobium Nobile Lindl.

Dendrobium nobile Lindl. (Chinese name"Jin-Chai-Shi-Hu") is a species of Orchidaceae with distribution in southeast Asia, southwest and south of China and has been recorded in Chinese Pharmacopoeia (2010 Edition). The chemical structures of several low molecular weight compounds from D. nobile have been elucidated, such as alkaloids, bibenzyls, stilbenoids, glycosides, sesquiterpenes, fluorenones and phenanthrenes. In contrast, the polysaccharides of D. nobile were much less investigated, even though polysaccharides have been proved to be one of major active constituents of Dendrobium plants. In order to investigate the chemical structural features, solution behavior and biological activities of the polysaccharides obtained from D. nobile, the isolation, purification, structural elucidation, solution behavior and anti-cataract tests of polysaccharides from D. nobile were carried out in this paper.The hot-water extracts from the dried stems of D. nobile were precipitated with 4 volume ethanol to give the crude polysaccharide DNP-W. Further, DNP-W was fractionated on DEAE–Cellulose anion-exchange column and purified by gel filrtaiton chromatography to obtain seven homogeneous polysaccharide fractions: DNP-W1A, DNP-W1B, DNP-W2, DNP-W3, DNP-W4, DNP-W5 and DNP-W6. The structural features of seven homogeneous polysaccharides were revealed by a combination of chemical and instrumental analysis, including IR, GC, GC-MS, ESI-MS, methylation analysis, periodate oxidation, partial acid hydrolysis, and NMR. The results showed that DNP-W1A had the backbone consisting of 1,6-linked and 1,3-linkedα-D Manp residues with branches at O-3 and O-2 of 1,6-linkedα-D-Manp residues. The branches were composed of terminalβ-D-Glcp residues, 1,4-linkedβ-D-Glcp residues, 1,6-linkedα-D-Galp residues, and terminalα-D-Galp residues. DNP-W1B possessed a backbone of a disaccharide of [→4)-β-Glcp-(1→6)-β-Glcp-(1→], with 33% branches at O-4 of (1→6)-linkedβ-D-Glcp residues. The side chains contained arabinosyl and galactosyl residues. DNP-W2 was a 2-O-acetyl galactomannoglucan and had a backbone consisting of (1→4)-linkedβ-D-Glcp, (1→6)-linkedβ-D-Glcp, and (1→4)-linkedβ-D-Manp residues, with branches at O-6 of (1→4)-linkedβ-D-Glcp andβ-D-Manp residues. The branches were composed ofα-D-galp residues. The acetyl groups were substituted at O-2 of (1→4)-linked Manp residues. DNP-W3 was a rhamnoarabinogalactan and had the backbone consisting of 1,3-linkedβ-D-Galp residues with branches at O-4. The branches were composed of 1-linkedβ-L-Arap and 1,4-linkeα-L-Rhap residues. DNP-W4 was a complex heteropolysaccharide and possessed a backbone compoesd of (1→4)-linkedβ-D-Glcp, (1→6)-linkedβ-D-Glcp, and (1→6)-linkedβ-D-Galp residues, with substitutes at O-4/6 of Glcp residues and O-3 of Galp residues. The branches composed of terminal Manp residues, (1→6)-linkedβ-D-Manp, (1→3)-linkedβ-D-Glcp,β-D-Glcp,β-D-Galp, (1→4)-linkedα-D-GalAp, (1→2)-linkedα-L-Rhap, and Xylp residues. DNP-W5 and DNP-W6 were pectic polysaccharides with the backbone of a disaccharide of [→4)-α-GalAp-(1→2)-α-Rhap-(1→]. The side chains of DNP-W5 contained galactosyl, mannosyl, glucosyl, and xylosyl residues substituted at O-4 of the Rhap and O-3 of the GalpA residues. The side chains of DNP-W6 were attached at the O-4 of Rhap residues, including terminal Manp,β-(1→4)-linked Glcp residues,β-(1→6)-linked Manp residues, terminal Galp,β-(1→3) andβ-(1→6)-linked Galp (galactan) residues.The anti-cataract activities of polysaccharide fractions from the stems of D. nobile were assessed through streptozotocin(STZ)-induced cataract model in Sprague-Dawley (SD) rats. The results indicated that all the polysaccharide fractions from D. nobile exhibited anti-cataract activities to some extent. Among tested polysaccharide samples, DNP-W and DNP-W1A presented higher anti-cataract activities and there was a dose–response relationship between concentration of polysaccharides and anti-cataract activities. According to DNP-W and DNP-W1A treatment groups, at the end of experiment, the level of lenticular opacification from rats was much lighter than that of other polysaccharide treatment groups, the added value of body weight was between 150 and 190%, and the blood sugar decreased over 25%. The mean activities of the antioxidant enzymes and GSH content were significantly higher in lenses of rats from DNP-W and DNP-W1A treatment groups than in lenses of rats from model group. Conversely, the mean levels of MDA, H₂O₂, advanced glycation end products and protein carbonyl content in lenses was significantly lower in DNP-W and DNP-W1A group rats than in model group rat lenses. Together with the results of biochemical indicator in anti-cataract activity experiments and structural characteristics of seven homogeneous polysaccharide fractions, the biochemical mechanism of protective effect in rat lenses appeared to occur by maintaining the antioxidant defense system and decreasing protein glycation. And the chemical structure basis for anti-cataract activities is related to mannose content and its glycosyl linkage types.The conformation and solution behavior of DNP-W1A were investigated with scanning electron microscope (SEM), X-ray, differential scanning calorimetry (DSC), size-exclusion chromatography-Laser light scattering (SEC-LLS), atomic force microscopy (AFM) and Congo-red reaction. The results of SEM,X-ray and DSC indicated that the physical structure state of DNP-W1A was noncrystal, and its vitrification temperature was 85℃. The AFM observation showed that the molecular chain morphology of DNP-W1A was different size islands and random coils in solution. DNP-W1A was proved to be the hemi-flexible with the technique of SEC-LLS, Congo-red reaction and intrinsic viseosity, and the conformation could be changed by temperature.