| Dendrobium officinale.Kimum et Migo is a valuable traditional Chinese medicine of nourishing yin.According to the record of China Pharmacopeia(2010 version),D.nobile,D.chrysotoxum,D.fimbriatum,and the related species of Dendrobium genus are all officially used as Shihu,and D.officinale is listed separately which indicate it is very important in filed of "ShiHu".There are many modern studies on D.officinale in this country,many researchers have carried out some studies about D.officinale polysaccharides,and great progress has been gain.Many studies have shown that many plant's water-soluble polysaccharide has good biological activity,for example,enha-nceimmunity,anti-tumor,antioxidant,antihypertension,fatigue resistance,etc.With the deepening of research,the purification of polysaccharides,pharmacological activity and the structure-active relationship catch more and more attention.At present,most researchers used DEAE cellulose to separate crude polysaccharide preliminary by the purified water,and then further purified by gel chromatography,obtain homogenous segments of polysaccharide,and get the uniform polysaccharide clips for structural characterization and biological activity research.And there are protocorm of D.officinale and the stem of D.officinale as the raw materials of research.For different materials,different researchers get polysaccharide information differences.The proportion of water-soluble polysaccharide in the crude is largest.So,this research do drug efficacy study and structural research into polysaccharide of stem of D.officinale.And look forward to establish a basis for further mechanism research.Objective1.To obtain the homogeneous polysaccharide fragment D.officinale by using different separation column to separation and purification,and to study its physical and chemical properties.2.To identify the structures of homogeneous polysaccharides by using the instrument detection of HPLC.GPC.IR.NMR and the chemistry method of methylation.3.The cytotoxicity,phagocytic activity,product of NO and lymphopoiesis are taken as indexs to study the immunocompetence of homogeneous polysaccharides of D.officinale,which expect to provide scientific basis for looking the ingredient that accomplish immune effect.Methods1.Used petroleum ether to degrease,80%ethanol to remove small molecules,sevage method to move protein,water extract and alcohol precipitate to gain curde polysaccharide.Used the DEAE-52 anion exchange resin to obtain the water-solution polysaccharide,and used the Sephacryl S-300 gel resin to gain the homogeneous polysaccharides of D.officinale.2.Used the TFA-PMP hydrolyzation and derivation to obtain monosaccharide.And the monosaccharide composition and proportion be analysed by HPLC.Used coomassie brilliant blue method to determine the content of protein,and viscometer measure viscosity.3.Used the method of gel test and freeze-thaw appraisal WDOPA and WDOPB uniformity4.Molecular weight of the homogeneous polysaccharides of D.officinale was measured by GPC.According to chemical techniques,including total hydrolysis,methylation analysis,in combination with instrumental techniques of FT-IR and NMR studies,speculate the possible structures of the homogeneous polysaccharides of D.officinale.5.The stimulation effect of pollysaccharides of D.officinale.on normal mice lymphocyte proliferation and macrophage RAW264.7 were assayed by MTT method,the phagocytic ability of macrophage RAW264.7 was evaluated with neutral red dye phagocytosis and the stimulation effect of polysaccharides of D.officinale.on NO production by macrophage RAW264.7 was measured by Griess reagents.And compare the different effect between the curde polysacc-haride and homogeneous polysaccharides of D.officinale.Results1.Two polysaccharides fractions(WDOPA and WDOPB)were successfully purified from the crude polysaccharide,which were WDOPA and WDOPB.2.From the raw medicinal material of D.officinale to WDOPA and WDOPB,the transfer rate of DOP,WDOP,WDOPA and WDOPB were 18.98%,51.40%,25.4%and 13.12%,respectively.And the microbial recovery rate were 18.98%,9.75%,2.45%,1.28%,respectively;The polysaccharide content of DOP?WDOP?WDOPA and WDOPB were 70,44%?85.51%?95.36%?94.74%,respectively.And the protein content of them were 4.15%,2.32%,0.83%,0.75%,respectively;Viscosity were 0.37,0.30,0.28,0.26 mL/mg,respectively.It can elucidate protein content decreases gradually,polysaccharide content is increasing and viscosity decline in the process of purification step by step.3.The WDOPA and WDOPB had average molecular weights(Mws)of 386.98KD and 374.11KD,and mainly consisted of mannose and glucose,in a molar ratio of 10.75:1 and 8.82:1 respectively.The result indicated that both WDOPA and WDOPB possessed a backbone of(1?4)-linked ?-D Manp and(1?4)-linked ?-D Glcp with branches of the T-?-D Manp.The possible structures of WDOPA and WDOPB are indicated as follows:WDOPA:M:?-D-MannopyranoseG:?-D-Glucopyranose-4M'-:?4)-?-D-Mannopyranose-(1?-4G1-:?4)-?-D-Glucopyranose-(1?4.WDOPA and WDOPB significantly(P<0.01 or P<0.05)enhanced natural killer cell-mediated cytotoxicity when the concentration were in the 6.25-50 ?g/mL,while WDOP achieve the same effect only in the 50 ?g/mL.WDOPA and WDOPB could promote the ability of macrophage RAW264.7 in phagocytizing neutral red(P<0.01)when the concentration were in the 3.125-50 ?g/mL,while WDOP achieve the same effect in 25-50 ?g/mL.WDOP?WDOPA?WDOPB could significantly increase the nitric oxide production of macrophages(P<0.01)at 48h when they were in the same concentration at 12-48h.Relatively speaking,the low dosage could increase the nitric oxide production of macrophages and could keep the effect for a long time.WDOP?WDOPA?WDOPB could significantly(P<0.05)promote splenocyte,T-lymphocyte and B-lymphocytes proliferation when the concentration in the 6.25-50 ?g/mL,and the promoted ability was stronger as the increase of the dose.Conclusion1.The purification method which was established is feasible.It can gain the water-soluble polysaccharide fragments of relatively high yield?high purity and good homogenization.2.Structure characterization of WDOPA,WDOPB were study indicated that both WDOPA and WDOPB possessed a backbone of(1?4)-linked ?-D Manp and(1?4)-linked ?-D Glcp.And the proportion of(1?4)-linked ?-D Manp almost be 850%.3.The immunological effects of the purified polysaccharides of D.officinale are better than the unpurified polysaccharides.Combine with the conclusions of structure analytical,which show that in the process of immune activity,(1?4)-linked,?-D Manp may play a major role.4.The study provides scientific basis for development and utilization of polysaccharides of D.officinale in the medicine fields.And it provides the reference for a later deeper study. |
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