Isolation,Purification,Structural Characterization And Anti-osteoporosis Activities Of Saccharides From Morinda Officinalis How

In this article,crude saccharides were extracted and isolated from the roots of Morinda officinalis How.Then the crude saccharides were purified by column chromatography and their structures were characterized.At the same time,its anti-osteoporosis activities in vivo and in vitro were also detected.Fistly,the water-soluble saccharides and alkali-soluble saccharides of Morinda officinalis were extracted by hot water and alkaline solution,respectively.MO50,MO70,MO90 and MOB were obtained by graded alcohol precipitation.The sugar content of the four saccharides were 68.8%,71.3%,54.3 and 32.1% using phenol-sulfuric acid colorimetric method.After removal of proteins of MO70 and MO90 by Sevage method,isolation and further purification by DEAE-cellulose 52 and Sephacryl S-100 HR column chromatography,three purified saccharides MOW70-1,MOP70-2 and MOW90-1 were obtained.MOW70-1,MOP70-2 and MOW90-1 were verified by high performance gel permeation chromatography as homogeneous saccharides with molecular weights of 1248 Da,3109 Da and 2172 Da,respectively.The structures were identified by high performance anion exchange chromatography,infrared spectroscopy,methylation analysis and NMR.The results indicated that three saccharides were composed of(2→1)-linked-β-D-Fruf and terminated with glucose residue as an inulin-type fructan.Ultraviolet scanning spectrum showed that all saccharides had no protein and nucleic acid The results of scanning electron microscope showed that three saccharides exhibited different micro-morphology and circular dichroism spectroscopy indicated that there were triple-helix structures in MOW70-1,MOP70-2 and MOW90-1.The anti-osteoporosis activity in vivo was studied using the ovariectomized model of rats,and bone mineral density(BMD),bone mineral content(BMC),biomechanical properties,biochemical index in urine and serum,Micro-CT microstructure and parameters were applied for evaluation.The results showed that M.officinalis saccharides could significantly improve the bone mineral density and bone mineral content of femur and lumbar vertebra of OVX rats,especially the enhancement effect in the MO50 group for the whole and distal femur BMD,MO50 and MO90 group for the lumbar vertebra BMC was stronger than the positive group E2,and increase the biomechanical properties,bone turnover markers and repair the damaged bone microstructure in ovariectomized rats,which indicating that M.officinalis saccharides play a role in reversing bone degeneration in ovariectomized rats.The results of osteogenic activity indicated that three saccharides could increase cell proliferation of MC3T3-E1 cells especially for MOP70-2.Further studies showed that MOP70-2 exhibited promising osteogenic activity in osteoblastic MC3T3-E1 cells,attributed to increasing alkaline phosphatase activity and mineralization in a concentration-dependent manner and up-regulating osteogenic differentiation-related marker genes.Experimental results showed that M.officinalis saccharides exerted its anti-osteoporosis activities by promoting bone formation and inhibiting bone resorption and MO50 and MO90 exhibited most significant effect.