| Dendrobium officinale?D.officinale?is a herbaceous plant in orchidaceae.The dry stem is used as medicine.It is a special medicine in China to nourish yin and clear away heat,tonify stomach and promote fluid.It has been reported that the main active ingredients of D.oficinale including polysaccharides,amino acids,anthraquinones and flavonoids.It is known that polysaccharide is the main active component.Pharmacological studies have demonstrated that polysaccharides extracted from D.officinale possessed various biological activities,such as antioxidant,immunological,anti-tumour,hypoglycaemic activities.Polysaccharide is macromolecular compound with complex structure,which is one of the basic materials in organism.Polysaccharides have low toxicities and few side effects with development prospect.In recent years,many researchers have carried out some studies about D.officinale polysaccharides,and great progress has been gain.With the deepening of research,the purification of polysaccharides and the structure-active relationship catch more and more attention.During the past twenty years,DEAE cellulose-52 chromatography have been used to isolate the crude polysaccharide with water elution and NaCl elution,and Sephacryl S-300 chromatography were used for further purification of the polysaccharides of D.officinale.The purified and homogeneous polysaccharides obtained were conducted for chemical characterization and biological study.However,there is some flawed phenomenon on the study of D.officinale polysaccharides.For example,the polysaccharides reported previously were isolated by DEAE cellulose-52 chromatography with water elution.However,few attentions were put on the polysaccharides from NaCl elution.According to the isolated principle of DEAE cellulose-52 chromatography?Ion exchange?,there may be some difference between the polysaccharides from water elution and the NaCl elution,which would cause the difference in bioactivity.During the past twenty years,it has been found that most of the polysaccharides from NaCl elution possessed various bioactivities.The salt-soluble polysaccharide is an important part of the study of D.officinale and is the necessary part to explore the specificity of D.officinale.In this regard,detailed work is needed to study the structural characteristics and the biological capacities of salt-soluble polysaccharide from D.officinale.ObjectiveTo isolate and purify salt-soluble polysaccharide from D.officinale by DEAE cellulose-52 chromatography and Sephacryl S-300 chromatography and two new polysaccharides are obtained.Therefore,the aims of this study are to preliminarily characterize the structure of the polysaccharide fractions isolated from the stem of D.officinale and to evaluate the immunomodulatory and antioxidant activity of these fractions in vitro.Methods1.The water extraction and alcohol precipitation method was used to gain crud polysaccharides and the polysaccharides were deproteinized by the Sevag method.The polysaccharides were furtherly purified by DEAE-52 eluted with water,0.1,0.3,and 1.0 mol/L NaCl solutions.The polysaccharides from 0.1 M NaCl elution were further purified by Sephacryl S-300 chromatography to gain purified sault-souled polysaccharides?SDOPA and SDOPB?.2.HPGPC was employed to determine the molecular weights of SDOPA and SDOPB,and the monosaccharide composition of the two the samples was determined using HPLC.FT-IR spectroscopy,methylation and NMR spectra were used to analyse the structural characterization of SDOPA and SDOPB.3.The MTT method,neutral red uptake assay and Griess test were utilized to evaluate the effects of the polysaccharides on proliferation,phagocytosis and the NO production in RAW 264.7 macrophages,respectively.The MTT assay was used to evaluate the effects of the polysaccharides on proliferation of splenocytes.4.The effects of D.officinale sault-soulded polysaccharides on the viability of H2O2-induced macrophages were determined using an MTT assay.The Hoechst 33258 was used to analyze the effects of D.officinale salt-soluble polysaccharides on morphological changes of H2O2-induced RAW 264.7 cells,which was observed under a phase-contrast microscope.Results1.The crud polysaccharides were isolated by DEAE-52 and Sephacryl S-300 chromatography to gain two salt-soluble polysaccharides?SDOPA and SDOPB?.The transfer rate of crude polysaccharide?DOP?,SDOP,SDOPA and SDOPB was 18.89%?21.29%?19.34%and 6.59%,and the yield of crude polysaccharide?DOP?,SDOP,SDOPA and SDOPB was 18.89%,4.02%,0.78%and 0.27%.2.The main sugar constituents of D.officinale,DOP,SDOP,SDOPA and SDOPB were mannose and glucose,which were in the molar ratio of 2.7:1.0,3.6:1.0,4.5:1.0,5.7:1.0,4.6:1.0.The average molecular weights of SDOPA and SDOPB were determined to be 3.94 × 105 Da Da and 3.62 × 105 kDa,respectively.The dispersion coefficients were determined to be 1.74 and 1.77.The FT-IR spectra indicate that SDOPA and SDOPB mainly contained ?-type glycosidic linkages with O-acetyl groups.The combination of the methylation analysis and NMR spectra demonstrated that both SDOPA and SDOPB were glucomannans with O-acetyl groups and had a backbone consisting of 1,4-linked ?-D-Manp and 1,4-linked ?-D-Glcp.3.The results demonstrated that the polysaccharides of D.officinale?SDOPA,SDOPB and DOPA?could promote macrophage proliferation and activate morphological changes in the macrophages.The polysaccharides?6.25-50 ?g/mL?significantly enhanced the phagocytosis of RAW 264.7 cells in a dose-dependent manner?P<0.05?.In addition,D.officinale polysaccharides?6.25-50 ?g/mL?increased the NO production of the RAW 264.7 cells in a dose-and time-dependent manner?P<0.05?.In addition,the polysaccharides of D.officinale?SDOPA,SDOPB and DOPA?could significantly promote the proliferation of splenocytes?6.25-50 ?g/mL?and T-lymphocytes?6.25-50 ?g/mL??P<0.05?.SDOPA?12.5-50 ?g/mL?and SDOP?12.5-50 ?g/mL?could significantly enhance the proliferation of B-lymphocytes?P<0.05?.However,SDOPB had no promoted effect on B-lymphocytes.4.H2O2-mediated macrophages were employed in this study to examine the antioxidant activity of D.officinale polysaccharides.RAW 264.7 cells treated with 500 ?M H2O2 for 2 h served as the control in the remaining studies.The preliminary experiments demonstrated that pretreatment with DOPA.SDOPA and SDOPB significantly promoted the viability of RAW 264.7 cells induced by H2O2?P<0.05?.The results showed that pretreatment with D.officinale polysaccharides significantly decreased the apoptosis induced by H2O2 and protected cell morphology and structure from H2O2-mediated oxidative lesions.ConclusionTwo purified and homogeneous polysaccharide fractions?SDOPA,SDOPB?are isolated and purified by DEAE cellulose-52 and Sephacryl S-300 chromatography.The isolated and purified method is proved to be reliable.SDOPA and SDOPB are glucomannans with O-acetyl groups and have a backbone consisting of 1,4-linked ?-D-Manp and 1.4-linked ?-D-Glcp.D.officinale polysaccharides have significant immunomodulatory effects on the immune responses mediated by macrophages and lymphocytes,and exert significant protective effects against H2O2-induced oxidative injury in RAW 264.7 macrophages.Thus,the D.officinale polysaccharides have antioxidant and immunomodulatory activities.The preliminary result of the structural characterization and bioactivity of salt-soluble polysaacharide of can provide information for the deep research and the analyisi of structural-relationship of D.officinale polysaccharides. |
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