Isolation,Structural Characterization And Antioxidant Activity Of Polysaccharides From Dendrobium Officinale

Dendrobium officinale Kimura and Migo,which belongs to Dendrobium Sw of Orchidaceae,is an edible medicinal plant.As the valuable traditional Chinese herbs,Dendrobium officinale has the functions of maintaining stomach tonicity,nourishing body,clearing within fire,moistening lung and so on.Polysaccharides from Dendrobium officinale were found to be the main active components,and its stems are the main active part.In view of Dendrobium officinale being a valuable and its resource shortage,it was significant that the nutritional polysaccharides from stems were analyzed.In this paper,the crude polysaccharide was extracted from Dendrobium officinale powders by hot water and separated by ethanol.After trying many isolation method,two purification process were determined to get two kinds of pure Dendrobium officinale polysaccharides named as DOP-1 and DOP-2.Combining chemical analysis and spectral analysis techniques,the composition and structural information of DOP-1 and DOP-2 were identified.In addition,their antioxidant activity were investigated.Part I Preparation of DOP-1 and DOP-2 from Dendrobium officinaleIn this part,the crude polysaccharide was extracted by hot water and separated by ethanol from powders of Dendrobium officinale stems,which was yield from Yandang Mountain in Zhejiang province.In order to get refined polysaccharides with high efficiency,methods including freeze-thawing,ultrafiltration,H2O2 decoloration,and dialysis were used to purify the crude polysaccharide,Two kinds of purification process including methods of were determined to get two kinds of polysaccharides DOP-1 and DOP-2 with different molecular weight,respectively.The total carbohydrate content of DOP-1 and DOP-2 were determined as 99.47% and 97.46% by phenol-sulfuric acid method.The weight-average molecular weight of the DOPs from HPGPC analysis were calculated as 492 k Da and 33 k Da,respectively.Uronic acid and protein were not detected in either DOP-1 or DOP-2 by UV spectra analysis and the Bradford test.In addition,uronic acid was not detected by m-hydroxydiphenol method.Total phenolic content of DOP-1 and DOP-2 were detected as 147.25 and 918.67 mg/kg,respectively.All the results suggested that DOP-1 and DOP-2 have good traits and high purity,which met the requirement of the following structural identified experiments.Part II Structural characterization of Dendrobium officinale polysaccharidesChemical analysis and spectral analysis techniques were employed to determine the structural information of DOP-1 and DOP-2.Infrared spectroscopy(IR)scanning demonstrated the characteristic absorption peaks,and some main organic groups could also be detected.1-Phenyl-3-methyl-5-pyrazolone(PMP)method was used to determine the monosaccharide compositions.The type and proportion of sugar residues were analyzed by chemical analyses including periodate oxidation,Smith degradation,and methylation analysis.1D(1H,13C)and 2D(HSQC,HMBC)NMR spectral was used to further determine the sequence of sugar residues.The FT-IR spectrum showed the characteristic absorption peaks of O-acetyl groups and mannose,indicating that DOP-1 and DOP-2 were acetylated polysaccharides and contained mannose.The result of monosaccharide compositions suggested that DOP-1 and DOP-2 comprised D-Glcp and D-Manp in the proportions of 1.00:1.38 and 1.00:4.06,respectively.Periodate oxidation and Smith-degradation analyze showed that the non-reducing terminal residues or(1→6)-linked residues were in small amounts,the main component was the(1→4)-linked or(1→4,6)-linked residue that oxidized to produce erythritol.Methylation analysis revealed that DOP-1 was made up with(1→4)-linked Glcp and(1→4)-linked Manp with the ratio of 1.00:1.78,DOP-2 was composed of(1→4)-linked Glcp,(1→4)-linked Manp,and(1→6)-linked Glcp with the ratio of 1.90:14.10:1.00.the results analyze of FT-IR spectrum,Smith-degradation,and NMR spectrum demonstrated that the acetyl groups of DOP-1 and DOP-2 were linked to 2-O-or 3-O-position of 1,4-β-D-Manp,the degree of acetylation was about 50% and 30%,respectively.The main repeated unit of DOP-2 is proposed for its low molecular weight,low viscosity and good solubility,which is better for NMR spectroscopy characterization.The main repeated unit of DOP-2Part Ⅲ Antioxidant activity of Dendrobium officinale polysaccharidesIn this part,in vitro radical scavenging ability and protecting effect of oxidative injury Hep G2 of DOP-1 and DOP-2 were investigated.DPPH,hydroxyl and superoxide radical scavenging assays were used to evaluate the in vitro antioxidant activity.DOP-2 exhibited stronger hydroxyl radical scavenging ability and more excellent superoxide anion radical scavenging ability at high concentration,which was partly attribute to its lower molecular weight,lower viscosity,the lower degree of acetylation,and higher molar proportion of mannose than DOP-1.Additionally,the oxidative damaged Hep G2 cells which was induced by H2O2 was used to investigate the influence of cell viability.The result showed that DOP-1 and DOP-2 had strong protective effect from oxidative injury.The cell viability was over 90% when concentration was at 800 μg/m L What’s more,the intercellular ROS was decreased after treating with DOPs and presented in a concentration-dependent manner.