Isolation,purification,structural Characterization Of Saccharides From Cibotium Barometz And Its Anti-osteoporosis Activity

This paper gives a further study on the anti-osteoporosis activities of Cibotium barometz crude saccharides in vivo.Meanwhile,the C.barometz crude saccharides were isolated and purified to obtain homogeneous saccharide,and their structures were also studied.The water-soluble saccharides of C.barometz were extracted with hot water and precipitated by adding ethanol,named as CB50,CB70 and CB90,respectively.While,the water extraction residues of C.barometz were extracted with alkali solution and the supernatant was precipitated to obtain the alkaline soluble crude saccharides,denoted as CBB.The results of our previous in vivo anti-osteoprosis assay has demonstrated that four parts of C.barometz crude saccharides exhibited positive anti-osteoprosis effects in ovariectomy-induced osteoporosis rat.Moreimportantly,due to the significant anti-osteoprosis effects of CBB,the femur and lumbar vertebrae of rats were scanned via a Micro-CT scanner to further evaluate the effects of CBB on trabecular microarchitecture in the present study.The results revealed that treatment with CBB prevented the deterioration of trabecular bone microarchitecture in OVX rats by increasing trabecula number(Tb.N)and trabecula thickness(Tb.Th),decreasing the trabecula separation(Tb.Sp),and returning the trabecular structures from rod to plate form.Based on the positive anti-osteoprosis effects in vivo,four parts of C.barometz crude saccharides were isolated,purified to obtain homogeneous saccharide,and their structure and anti-osteoporosis activities were investigated to determine the exact biological active ingredient(s)for C.barometz crude saccharides.The four parts of C.barometz crude saccharides were deproteined,dialyzed,and isolated via a DEAE-52 Cellulose chromatography column.Eluents with saccharide were collected,concentrated,and further purified using a Sephadex G-75 gel permeation chromatography column to obtain homogeneous saccharides,denoted as CBBP-1,CBBP-2,CBBP-3,CBP70-1-1 and CBP70-1-2,respectively.CBBP-1,CBBP-2,CBBP-3,CBP70-1-1 and CBP70-1-2 were all confirmed as having no nucleic acids and protein using a UV spectrometry,and the determination of specific rotation and High-performance gel permeation chromatography(HPGPC)implid that there were all homogeneous saccharide and the their molecular weight were found to be 1445 Da,10662 Da,122975 Da,12724 Da and 3611 Da,respectively.The PMP-pre-column HPLC method and methylation-GC-MS method were adopted to analyze the monosaccharide and sugar residues composition,and the NMR studies were used to obtain the linkage sites and sequence among the sugar residues.Thus,the primary structure of all homogeneous saccharide was obtained.CBBP-1 was identified a novel oligo-glucomannan and CBBP-2,CBBP-3,CBP70-1-1 and CBP70-1-2 were all identified heteropolysaccharides.The Congo-red assay,electronic circular dichroism spectroscopy and scanning electron microscope were used to investigate the advanced structure of homogeneous saccharide.Results of electronic circular dichroism spectroscopy(ECD)indicated that the homogeneous saccharide does not possess a triple helix structure.In addition,morphological analysis revealed that CBBP-1 had an irregular sheet structure,and its surface is smooth;CBBP-2 had a fragmented and flaky structure.CBBP-3 had a neuron-like morphology,with a number of dendritic branches;CBP70-1-1 is shown a sheet that is irregular in size and shape,and its surface with sharp protuberances;CBP70-1-2 also had an irregular sheet structure,and its surface is smooth gurgitation.Up to now,the structure of all the homogeneous saccharide has not been reported in the literature.