Isolation, Purification, Structural Identification And Anti-Alzheimer’s Disease Research Of Polysaccharides From Polygala Tenuifolia

Polygala tenuifolia Willd.is a traditional Chinese medicine to treat amnesia.It has been reported that P.tenuifolia can improve learning and memory abilities,and has anti-Alzheimer’s disease(AD)and antineuroinflammation effects.However,there are few researches on the structure and anti-AD activity of polysaccharides from P.tenuifolia.Therefore,in this study,we systematically isolated and purified P.tenuifolia polysaccharides,and evaluated the anti-AD activity of crude polysaccharides from P.tenuifolia in vivo.This study will provide a theoretical basis for elucidating the anti-AD active constituents in crude polysaccharides of P.tenuifolia.Firstly,four kinds of crude polysaccharides named PT50,PT70,PT90,and PTB were gained by water and alkali extraction and alcohol precipitation from P.tenuifolia.A rat model of Alzheimer’s disease(AD)was successfully constructed by brain localization injection of 50 μg lipopolysaccharide(LPS).The effects of four kinds of crude polysaccharides on the learning and memory ability of AD rats were evaluated by water maze and other behavioral experiments.The results showed that crude polysaccharides from P.tenuifolia could significantly improve the learning and memory ability of AD rats,and their effect were similar to that of positive drug huperzine A(HA).Based on the good anti-AD activity of crude polysaccharide from P.tenuifolia in vivo,isolation and purification of polysaccharide from P.tenuifolia by expension system.Four crude polysaccharides were deproteinized by Sevag method,and DEAE-cellulose 52 anion exchange column and Sephadex G-75 gel column were used for separation and purification after concentration,dialysis and freeze drying.Finally,eight polysaccharides were obtained as follows: PTP50-1-1,PTP50-1-2,PTP50-1-3,PTP70-1-3,PTP70-3,PTBP-1-2,PTBP-1-3,PTBP-2-1.The results of high performance gel permeation chromatography(HPGPC)and specific rotatory spectrophotometry showed that the above eight polysaccharides were homogenous,and their molecular weights were: 36.32 k Da,16.07 k Da,12.04 k Da,3.21 k Da,10.17 k Da,12.25 k Da,7.15 k Da,and 34.98 k Da,respectively.Combined with monosaccharide composition analysis,absolute configuration analysis,infrared spectroscopic analysis(IR),methylation analysis,gas chromatography-mass spectrometry(GC-MS)analysis and nuclear magnetic resonance(NMR)analysis,the primary structures of the above eight homogeneous polysaccharides were inferred.Among them,PTP50-1-1 was glucan,and the other seven polysaccharides were heteropolysaccharides.The advanced structures of the eight polysaccharides were investigated by Congo red experiment and scanning electron microscopy.The Congo red experiment showed that none of the eight polysaccharides had a three-strand helix structure.The scanning electron microscopy analysis showed that PTP50-1-1 was a connected rectangle with cracks on the surface.PTP50-1-2 was irregular sheet.The surface is rough,PTP50-1-3 is irregular scale,PTP70-1-3 is granular,PTP70-3 is irregular fragment,PTBP-1-2 is flake,PTBP-1-3 is irregular flake,PTBP-2-1 is porous strip.The structures of the eight homogeneous polysaccharides have not been reported in the literature.