Isolation, Purification, Structural Characterization And Their Bioactivities Of Saccharides From Achyranthes Bidentata

This article systematically studied on the extraction, isolation, purification, and structural characterization of saccharides from Achyranthes bidentata Blume, the bioactivities were also conducted. The saccharides were obtained by hot water extraction followed by alchol precipitation, and then further successively purified, structural features were elucidated by chemical methods, spectroscopic methodology and chromatography method. The bioactivities in vivo were also systematically investigated. 4 crude saccharides AB50、AB70、AB90、ABB were obtained though hot water extraction, alkali extraction and alchol precipitation. Their contents were estimated as 62.7%, 70.3%, 31.6%, 21.6% using phenol-sulfuric acid colorimetric method. The crude saccharides obtained above were primary purified by using Sevage reagent to remove proteins, then dialyzed and lyophilized. 6 purifed saccharides ABW50-1, ABP50-2, ABW70-1, ABP70-2, ABW90-1, ABP90-2 were obtained using DEAE-Cellulose-52 and Sephacryl S-100 gel-filtration column. The homogeneous distribution and relative molecular weight were carried out by high performance gel permeation chromatography(HPGPC) and UV spectra, and results indicated that 6 purified saccharieds were homogeneous saccharieds and relative molecular weight were estimated as 1260 Da, 5118 Da, 1316 Da, 3406 Da, 1074 Da and 2976 Da, respectively. The MALDI-TOF-MS measurements were also applied to further confirm the accurate degree of polymerization(DP) of oligosaccharide ABW50-1 and ABW90-1, the results were in good agreement with that in HPGPC. Their structural features were elucidated by a combination of monosaccharide analysis, infrared spectroscopy, methylation, gas chromatography-massspectrometry(GC-MS) analysis, and 1D, 2D NMR studies. The data obtained indicate that 5 homogeneous saccharieds were fructan, which all composed of(2→1)-linked-β-D-fructofuranosyl(Fruf), and(2→6)-linked-β-D-Fruf residues and terminal with ← 2)-β-D-Fruf. In addition, the microstructure and conformation changes were investigated by scanning electron microscope(SEM), atomic force microscope(AFM), circular dichroism spectroscopy(CD), and Congo-red assay(CR). The bioactivities of A. bidentata saccharides were investiged using ovariectomized rat as model, and bone mineral density(BMD), biomechanical assay, elisa test, Micro-CT test were employed. Results indicated that A. bidentata saccharides could significantly increase the bone mineral density(BMD) and bone mineral content(BMC), and improve the biomechanical properties in ovariectomized rats. The bone volume/ total volume(BV/TV), trabecular connectivity density(Conn-Dens), trabecular number(Tb.N) and trabecular thickness(Tb.Th) of OVX rats were all significantly increased. A. bidentata saccharides improved the trabecular structure of OVX rats, while the trabecular separation(Tb.Sp) and structure model index(SMI) decreased, which suggested that A. bidentata saccharides could reverse the trabecular structural damage. Data indicated that the anti-osteoporosis effect of A. bidentata saccharides was by promoting bone formation.