Extraction,Isolation,Purification And Structural Identification Of Polysaccharides From Curculigo Orchioides

Curculigo orchioides Gaertn.is one of the traditional Chinese medicines in the treatment of osteoporosis.In the previous study,the anti-osteoporosis activity of polysaccharides(CO50,CO70,CO90 and COB)from C.orchioides was studied,and the results showed that CO50,CO70,CO90 and COB could significantly improve the bone density and bone mass of femur and vertebra in ovariectomized rats,as well as improve the bone microstructure,indicating that the cude polysaccharides from C.orchioides had anti-osteoporosis activity.Therefore,in this paper,we further studied the polysaccharide of C.orchioides,and the homogenized polysaccharides were obtained by isolation and purification.Its primary structure and advanced structure were characterized and described,which provided a theoretical basis for the follow-up study on the mechanism of anti-osteoporosis of polysaccharide of C.orchioides.Firstly,four crude polysaccharides CO50,CO70,CO90 and COB were obtained by water or alkali extraction and graded alcohol precipitation from the rhizome of C.orchioides.The content of sugar was51.68%,50.35%,55.72% and 57.69% by phenol sulfuric acid method,respectively.The crude polysaccharides were successively purified using chromatography on DEAE-52 anion exchange column and Sephadex G-75 gel filtration column,and eight polysaccharides COP50-1,COP50-4,COP70-1,COP70-2,COP70-3,COP90-1,COP90-2 and COBP-2 were finally obtained.The purity of polysaccharides was verified by specific rotation method and high-performance gel permeation chromatography.The results showed that they were all homogeneous polysaccharides,with relative molecular weights of 3.1 k Da,28.1 k Da,3.1 k Da,4.7 k Da,10.6k Da,6.5 k Da,6.2 k Da,8.1 k Da,respectively.Subsequently,the monosaccharide composition of polysaccharides was analyzed by PMP pre-column derivatization combined with high performance liquid chromatography method.The types and proportions of sugar residues in homogeneous polysaccharides were analyzed by methylation combined with GC-MS.The linkage mode of sugar residues was determined by nuclear magnetic resonance(NMR)technology,and the primary structure of homogeneous polysaccharides were confirmed.The results showed that COP50-1,COP50-4,COP70-1,COP70-2,COP70-3,COP90-1,COP90-2 and COBP-2 were heteropolysaccharides consist of combinations of various types of sugar residues.The structures of eight homogeneous polysaccharides from the rhizome of C.orchioides have not been reported.Congo red test showed that these homogeneous polysaccharides did not have three helical structures,and scanning electron microscopy conformed that these homogeneous polysaccharides had different morphological characteristics.