Extraction,isolation,purification And Structural Identification Of Polysaccharides From Acorus Tatarinowii

Acorus tatarinowii Schott is the most common traditional Chinese medicine among the prescriptions for the treatment of Alzheimer’s disease(AD).However,the active ingredients of A.tatarinowii are still unclear.From the early study of our research group,the crude polysaccharides from A.tatarinowii(AT50,AT70,AT90 and ATB)were tested for anti-AD activity in vivo.The results suggested that AT50,AT70 and ATB significantly improved the learning and memory ability and alleviated oxidative stress response of AD mice,which indicated that the crude polysaccharides from A.tatarinowii exhibited the effect of anti-AD.Therefore,in this paper,in order to provide theoretical basis for further study on the anti-AD mechanism and structure-activity relationships of A.tatarinowii polysaccharides,the polysaccharides were systematically isolated,purified and structurally identified from the rhizome of A.tatarinowii.Firstly,the rhizome of A.tatarinowii was extracted with hot water at80 ℃.The crude polysaccharides of water extract were obtained by gradational precipitation with 50%,70%,and 90% ethanol,respectively.The residue after water extraction was extracted with sodium hydroxide solution(0.3 M).The alkaline extraction was precipitated with 75%ethanol to obtain crude polysaccharide in alkaline faction.The crude polysaccharides were deproteinized by Sevag method,then concentrated,dialyzed and lyophilized to obtain the crude polysaccharides AT50,AT70,AT90 and ATB,successively.Finally,eight refined polysaccharides,named ATP50-2-1,ATP50-2-2,ATP50-3,ATP50-4-1,ATP70-2,ATPB-2,ATPB-3-1 and ATPB-3-2 were separated and purified from the crude polysaccharide samples by a DEAE-Cellulose 52 ion exchange column and a Sephadex G-75 gel column in turn.In addition,according to the results of specific rotation spectrophotometry and high performance gel permeation chromatography(HPGPC),these eight polysaccharides were homogeneous polysaccharides,with the relative molecular weights of 76.4k Da,20.1 k Da,87.4 k Da,35.5 k Da,8.3 k Da,24.1 k Da,88.9 k Da and 22.0k Da,respectively.The monosaccharide composition of these eight polysaccharides was analyzed using the 1-phenyl-3-methyl-5-pyrazolone(PMP)pre-column derivatization and high performance liquid chromatography(HPLC)method.What’s more,absolute configuration determination,infrared spectrum analysis,methylation analysis,gas chromatography-mass spectrometry(GC-MS)analysis and nuclear magnetic resonance(NMR)spectrum analysis were conducted to identify the primary structures of the polysaccharides.The results showed that all of them are heteropolysaccharides,composed of various sugar residues,and their structures have not been reported.Furthermore,according to the results of scanning electron microscope analysis,the advanced structure and morphology of the above eight polysaccharides are different.The results above provided beneficial evidence for the further study on the antiAD mechanism and structure-activity relationships of A.tatarinowii polysaccharides.