Isolation,Purification And Structure Analysis Of Enzymatic Hydrolysate Of Gynamosidase From Curculigo Orchioides Gaertn

Curculigo is the rhizome of Curculigo orchioides Gaertn,an amarylaceae plant.It has the functions of tonifying kidney and helping yang,benefiting essence and strengthening bones.It is mainly used for kidney yang deficiency,vaginal internal injuries and muscle and bone pain.Curculigo polysaccharide can activate mouse peritoneal macrophages to secrete cytokines.Studies have shown that polysaccharides mediate immune responses by binding their specific oligosaccharide fragments to receptors.Degradation of polysaccharides from Curculigo chinensis into suitable low molecular weight oligosaccharide fragments is of great significance not only for the study of the functional effects and mechanism of the polysaccharides,but also for the clearer structure of the polysaccharides.In the early stage of the research group,preliminary in vitro immunological activity screening of the enzymatic hydrolysate of Curculigo latifolia was carried out to obtain an immunologically active enzymatic mixture.In this paper,the immunosuppressed polysaccharides of Curculigo latifolia were isolated and purified to obtain active oligosaccharide components.The main results are as follows:1.The crude polysaccharide of Curculigo latifolia was isolated and purified by DEAE-cellulose and Sephadex G-50 gel column chromatography to obtain pure polysaccharide X-2,which has a sugar content of 84%.The X-2 monosaccharide composition was determined by HPLC-PMP method and consisted of Man and Glc in a molar ratio of Man:Glc=17:1.2.Curculigo pure polysaccharide X-2 is enzymatically hydrolyzed with glucanase: 50% enzyme,52 °C,5 h,pH 6.0,desalted and Sephadex LH-20 gel column chromatography.Separation and purification,mainly obtained five components of XP1 ~ XP5.The X-2 enzymatic mixture,XP4,XP3 monosaccharide composition are Man and Glc,and the molar ratios are: Man: Glc = 9:1,2:1,6:1.Mass spectrometry,methylation and GC-MS,1D and 2D NMR analysis showed that XP5 is a monosaccharide,the XP4 structure is [α-Glc-(1→6)-α-Man] disaccharide,and the XP3 structure is [α-Man-(1→4)-α-Glc-(1→6)-α-Man].Atomic force microscopy(AFM)observed that X-2 is a single-molecular chain of filamentous fractal structure with irregular arrangement.Compared with X-2,its enzymatic products are not intertwined with each other.The lumpy filament structure is also a random,multi-branched single molecular chain.3.Curculigo pure polysaccharide X-2,enzymatic hydrolysis mixture XP,oligosaccharide XP1 ~ XP5 were analyzed for in vitro immunological activity,and the results of stimulating macrophage secretion of cytokines TNF-α,IL-1 and IL-2 were known.X-2,enzymatic hydrolysis mixture XP and oligosaccharide XP1~XP5 can significantly induce the secretion of TNF-α by macrophages in a dose-dependent manner.The induction ability of oligosaccharides was lower than that of X-2,and most of them were high and medium doses.A very significant difference is presented.Both X-2 and its enzymatic hydrolysates stimulated the secretion of interleukin IL-1 and IL-2 by macrophage RAW264.7.The high dose group of oligosaccharide XP3 showed significant difference,and XP3 induced macrophage secretion of IL-1.The ability is higher than X-2.Comprehensive analysis showed that the immunological activity of oligosaccharide XP3 was better.