| The word of Dendrobium was first speared in a book of shanhaijing.Dendrobium candidum Wall.Ex Lindl.,as a kind of medicinal Dendrobium,has the effect of beneficial stomach fluid,runfei,ziyin,and so on.As from the Chinese pharmacopoeia,2010 edition,Dendrobium candidum as item respectively,shows the importance of the medicinal.Fresh or dry stems of Dendrobium candidum were used.Polysaccharide as one of the important indexes in Dendrobium candidum,the content of crude polysaccharide in DOP can reach 20%～35%generally,of which the mass fraction of water-soluble polysaccharide was high.And studies have shown that the activity of polysaccharide was affected on monosaccharide composition,molecular weight,solubility and viscosity,then the specific correlation of the polysaccharide activity with molecular weight was not confirm.Currently,people used column chromatography and ultrafiltration membrane to separate of polysaccharides,and the above two methods operation numerous cable and the economic cost was high.In addition,the degradation can separate segments of different molecular weight of polysaccharides,but free degradation can reduce the molecular weight of polysaccharides.Different concentrations of ethanol can isolate different segments of molecular weight of polysaccharides,and the method was simple and easy to operate,economic and efficient characteristics.So this study by alcohol sinking method to separating Dendrobium candidum stem segments of different molecular weight of polysaccharides,and to study the relationship between the molecular weight and concentration of alcohol,and to study the Dendrobium candidum polysaccharide molecular weight and its in vitro activity(antitumor and antioxidant)relationship.Objectives:This paper take Dendrobium candidum as raw materials,and use the method of water extract-alcohol precipitation to get the coarse polysaccharide DOP,and using step by step alcohol to sink separation,the resulting alcohol concentration was 40%,50%,60%,70%of polysaccharide separation four fragments in different molecular weight of polysaccharide,again by the HW-65F to purify the fragments,then to obtain DOP1、DOP2、DOP3、DOP4.To study the chemistry of Dendrobium candidum coarse polysaccharide and the separation of polysaccharides fragments,and to study the vitro antitumor activity and antioxidant activity.To explore the molecular weight,viscosity of polysaccharides in Dendrobium candidum on the effects of its biological activity.The research of polysaccharide in Dendrobium candidum is aim to provide the basis for the product research and development.Methods:1、The method of water extract-alcohol precipitation was used to extract Dendrobium candidum crude polysaccharide,the fragments of different molecular weight polysaccharide were isolated from crude polysaccharide by the method of step by step alcohol sinking.Toyopearl HW-65F gel resin was used to purify the separation polysaccharide of Dendrobium candidum.2、Ultraviolet spectrophotometry was used to detect the content of polysaccharide of coarse polysaccharide and each separate segments.Hydrolysate monosaccharide composition in the separation polysaccharides of Dendrobium candidum were analyzed by the method of High performance liquid chromatography(HPLC).The method of coomassie brilliant blue was used to determine the binding protein content in the Dendrobium candidum coarse polysaccharide and the separation of polysaccharides.3、The viscosity of coarse polysaccharide and the separation of the polysaccharides were determined by ubbelohde capillary viscometer.4、Efficient gel permeation chromatography(HPGPC)was used to the determinate the separation polysaccharides molecular weight of Dendrobium candidum.5、Antineoplastic activity was studied,Dendrobium candidum coarse polysaccharide and the separation of polysaccharides on human hepatocellular carcinoma HepG2 cells growth inhibition was determined by the method of MTT.The scavenging effect on super oxygen radicals and hydroxyl free radical was investigated of the activities of Dendrobium candidum coarse polysaccharide and the separation polysaccharides.Results:1、Alcohol sinking method by alcohol concentration 40%,50%,40%,70%,the separation of polysaccharides,the corresponding rate was 12.40%,5.00%,12.40%and 5.00%respectively.Each part polysaccharide,respectively by the HW-65F get corresponding segments of polysaccharide purified DOP1,DOP2,DOP3,DOP4,fragments of the polysaccharide yield was 6.97%,4.74%,0.58%and 6.97%respectively.2.DOP and the separation polysaccharides of DOP1,DOP2,DOP3,DOP4 measured their purified total sugar content and binding protein content were 87.6%、75.726、81.6%、98.4%、90.0%and 4.28%,1.68%,1.12%,0.03%,1.12%activities.Dendrobium candidum coarse polysaccharide and the separation of polysaccharides are mainly consists of D-mannose and d-glucose,and two kinds of polysaccharide content had some differences.The content of coarse polysaccharide ratio was 6.72,the content ratio of separation of polysaccharide were 6.32,8.67,8.34,8.82,respectively.3.Ubbelohde capillary viscometer measured the viscosity of DOP and the separation polysaccharides of DOP1,DOP2,DOP3,DOP4 were 8.8×102 mL/g,6.3×102 mL/g,4.4×102 mL/g,3.4×102 mL/g.4.Efficient gel permeation chromatography(HPGPC)measured DOP polysaccharides molecular weight was 826.474 kDa,the molecular weight of the separate polysaccharide DOP1,DOP2,DOP3,DOP4 were 999.137 kDa,657.206 kDa,243.072 kDa,49.984 kDa.5、The vitro antitumor activity,according to a study in the drug 48 h,absorbance values were significantly less than the negative control group.In the concentration range of 25 ug/mL to 400 ug/mL,the inhibition rate increased along with the increase of drug concentration,shows that the Dendrobium candidum coarse polysaccharide and the separation of polysaccharides have good inhibitory effect on the growth of hepG2 human liver cells.6、In the study of vitro antioxidant activity on Dendrobium candidum coarse polysaccharide and the separation of polysaccharides,we found that Dendrobium candidum coarse polysaccharide and the separation of polysaccharides in reagent concentration in the range of 0.5 mg/mL-5 mg/mL super oxygen anion clearance had relevance to the concentration,the higher concentration,the greater clearance.When the drug concentration reached 5 mg/mL,DOP1 and DOP4 clearance rates were more than 50%.The larger and smaller molecular weight of polysaccharides fragments had stronger superoxide anion scavenging effects,and the medium molecular weight polysaccharides were relatively weak.In another sides,DOP、DOP1、DOP3 scavenging effect was relatively strong,and DOP2 and DOP4 scavenging effect were relatively weak in cleared to hydroxyl.Conclusion:1、The method of step by step alcohol sink separation can separate Dendrobium candidum coarse polysaccharide,with the result of high concentration of ethanol precipitation segments of the smaller molecular weight polysaccharides,low concentration of ethanol precipitation segments of the larger molecular weight polysaccharides.And each separated polysaccharide pieces have certain difference in total sugar content,monosaccharide composition,protein content and intrinsic viscosity,etc.Indicate that the method could be used to separate different segments in different molecular weight of polysaccharides.2、Compare the polysaccharide separation of molecular weight distribution and the intrinsic viscosity,the larger molecular weight polysaccharides fragments,the greater the viscosity,showe that the viscosity is related to the molecular weight of polysaccharides in Dendrobium candidum.3、From the in vitro activity studies of Dendrobium candidum coarse polysaccharide and the separation of polysaccharideound,we know that low molecular weight polysaccharides has stronger proliferation inhibit effect on HepG2.For the ultra oxygen anion scavenging effect is characterized by large molecular weight and molecular weight smaller fragments polysaccharide removal effects were stronger.As to hydroxyl function,large molecular weight of DOP,DOP1 and medium molecular weight polysaccharides had stronger scavenging effects.Show that the antioxidant activity of Dendrobium candidum polysaccharide is relate to the size of molecular weight.4、Step by step alcohol sink method can use to separate Dendrobium candidum polysaccharide yield is higher,and simple operation,low economic cost and strong feasibility. |
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