Isolation,Purification,Structural Characterization And Anti-osteoporosis Activity In Vivo Of Polysaccharides From Curculigo Orchioides Gaertn

This study systematically evaluated the extraction,isolation,purification,structural characterization and anti-osteoporosis activity in vivo of polssaccharides from the dried rhizomes of Curculigo orchioides Gaertn.First,the water-soluble polysaccharides and alkali-soluble polysaccharides of C.orchioides Gaertn were extracted by hot water and alkaline solution,respectively.The polysaccharides obtained by hot water were precipitated by fractional alcohol precipitation to obtain three polysaccharide fractions with different molecular weights.The four different precipitates were re-dissolved and deproteinated by the Sevag method,dialyzed against distilled water,and concentrated,and freeze-dried to obtain the CO50,CO70,CO90,and COB polysaccharides.CO70 and CO90,which had higher solubility,higher sugar content,and anti-osteoporotic activity in vivo,were subjected to separation with DEAE-Cellulose 52,and then the separated main peak was separated and purified over a gel Sephadex G-75 column to obtain COP70-1,COP70-3 and COP90-1.COP70-1,COP70-3 and COP90-1 were verified by high-performance gel permeation chromatography as homogeneous polysaccharides with molecular weights of 100647 Da,53399 Da and 4609 Da,respectively.The observation of no absorption at 260 nm and 280 nm in the UV spectra indicated the absence of protein and nucleic acids.The primary structures of the three homogeneous polysaccharides were analyzed by complete acid hydrolysis-pre-column PMP derivatization HPLC analysis,ion chromatography analysis,D/L absolute configuration analysis of monosaccharides,complete methylation,GC-MS analysis,and nuclear magnetic resonance analysis.The results showed that COP70-1 was found to be composed of(1?4)-linked 3-O-acetyl-?-D-Manp,(1?4)-linked ?-D-Glcp,(1,3?6)-linked ?-D-Manp,and terminated with ?-D-Glcp.COP70-3 was composed of 7 kinds of monosaccharides,including(1?5)-linked ?-L-Araf,(1,3?5)-linked ?-L-Araf,(1,2?5)-linked ?-L-Araf,terminated with ?-L-Araf,(1?3)-linked ?-L-Rhap,(1?4)-linked ?-D-Manp,(1,3?6)-linked ?-DManp,(1?6)-linked ?-D-Galp,(1,3?6)-linked ?-D-Galp,(1?3)-linked ?-D-Galp A,(1?6)-linked ?-D-Glcp,(1?3)-linked ?-D-Galp A,terminated with ?-D-Glcp A.COP90-1 was found to be composed of(1?4)-linked ?-D-Manp,(1?4)-linked 3-Oacetyl-?-D-Manp,and(1?4)-linked ?-D-Glcp,(1,3?6)-linked ?-D-Manp,and terminated with ?-D-Manp and ?-D-Glcp.The results of Congo red and circular dichroism analysis indicated that COP70-3 and COP90-1 do not exist in a triple-helix structure.The results of scanning electron microscopy(SEM)show that the micromorphology of COP70-1 was uniform spherical particle and agglomeration in some places due to the hygroscopicity of polysaccharide,COP70-3 was a nonhomogeneous lamellar structure,and COP90-1 was homogeneous with an inerratic ellipsoid shape.The three homologous polysaccharides were not reported in the literature.In this paper,the ovariectomized primary osteoporosis model of rats was used to study the anti-osteoporotic activity of polysaccharides CO50,CO70,CO90 and COB.Body weight and organ coefficient test results showed that Curculigo orchioides polysaccharides can effectively inhibit the trend of the weight gain of ovariectomized rats.CO70 and CO90 have a certain effect on the uterine atrophy of ovariectomized rats.CO50,CO70,CO90 and COB could alleviate the effect of ovariectomy on the organs of rats.The results also showed that polysaccharides of Curculigo orchioides had no side effects on rats in the long-term administration.BMD and BMC test showed that all parts of polysaccharides were able to increase BMD and BMC of femur and lumbar vertebrae in ovariectomized rats,among which CO70 and CO90 improved better.Elisa test results showed that polysaccharides from Curculigo orchioides can inhibit the bone resorption and bone formation in ovariectomized rats,regulate the biochemical indexes of bone metabolism in serum and urine,and adjust the bone metabolism to normal level,and the effect of CO50 was the most significant.Threepoint bending experiments and compression experiments showed that Curculigo orchioides polysaccharides significantly improved the biomechanical properties of femur and lumbar vertebrae and increased bone strength and bone toughness,reduced the risk of fracture.The result of Micro-CT scanning and three-dimensional reconstruction showed that Curculigo orchioides polysaccharides significantly improved the microstructure of trabecular bone of femur and lumbar vertebrae,increased trabecular bone mass,trabecular bone thickness,reduced trabecular bone separation,improved structural model index,degeneration of bone structure,the effect of CO70 and CO90 was more effective than that of 17?-estradiol.Based on the above results,four Curculigo orchioides polysaccharides were effective for osteoporosis in ovariectomized rats,and CO70 and CO90 were more effective.