Isolation, Purification,Structural Characterization And Anti-osteoporosis Effects Of Saccharides From Two Kinds Of Kidney Reinforcing And Bone Strengthening Traditional Chinese Medicine

The saccharides of two kinds of kidney reinforcing and bone strengthening traditional Chinese medicine(Achyranthes bidentata Blume and Morinda officinalis How)were extracted,isolated and purified in this study.Furthermore,their primary and advanced structures were characterized and their anti-osteoporosis effects were also investigated.3 kinds of crude saccharides were finally obtained though hot water extraction,alkali extraction and alcohol precipitation.Combining the methods of ion exchange chromatography and gel chromatography,the crude saccharides were isolated and purified.The primary structures of homogeneous saccharides were elucidated by chemical methods,spectroscopic methodology and chromatography method.In addition,the anti-osteoporosis activity of homogeneous saccharides from Achyranthes bidentata Blume was evaluated by the osteoporosis model of zebrafish induced by glucocorticoid.The crude saccharides ABPB,MO50 and MOPB were isolated and purified by the ion exchange chromatography column(DEAE-Cellulose-52)and gel chromatography column(Sephacryl S-100)systematically.6 kinds of homogeneous saccharides were obtained finally,which were labeled as ABPB-1,ABPB-2,ABPB-3,MOW50-1,MOP50-2 and MOPB-2.Specific optical rotation and high performance gel permeation chromatography(HPGPC)showed that ABPB-1,ABPB-2,ABPB-3,MOW50-1,MOP50-2 and MOPB-2 were homogeneous with the relative molecular weight of 2319 Da,25680 Da,77231 Da,1810 Da,2503 Da and 94994 Da,respectively.Combining the results of monosaccharide composition,infrared spectroscopy(IR),gas chromatography-mass spectrometry(GC-MS)and NMR spectrums,MOW50-1 and MOP50-2 were determined as inulin-type fructan,ABPB-1 was determined as mixed levan and ABPB-2,ABPB-3,MOPB-2 were determined as acidic heteropolysacchairdes.Congo-red assay,circular dichroism spectra(CD)and scanning electron microscope(SEM)were used to detect the advanced structure of homogeneous saccharides.The results of SEM showed that the configuration of these 6 kinds of homogeneous saccharides were different,which may be related to their primary structure,molecular weight and monosaccharide composition.The CD analysis also revealed that there were no triple helix structure existed in these 6 kinds of homogeneous saccharides.The 3 days post fertilization(3-dpf)zebrafish embryos were exposed to the glucocorticoid(prednisolone)to induce the osteoporosis model of zebrafish embryo.The anti-osteoporosis effects of ABPB-3 and ABW50-1 were determined by the results of relative fluorescence intensity(RFI)of zebrafish skull.As expected,both ABW50-1 and ABPB-3 enhanced the RFI of zebrafish skull with dose dependence significantly,and did not show any side effects,indicating that ABPB-3 and ABW50-1 have prominent therapeutic effects on the osteoporosis induced by glucocorticoid.