Separation,Purification And Anti-hbv Activities Of Ganoderma Lucidum Polysacharides

Ganoderma lucidum,a kind of large fungi,is usually the dry sporophre when it comes down to the medical field.The distribution of Ganoderma lucidum is wide and its ingredients are extremely complicated.Component contents of Ganoderma lucidum coming from different producing areas show some differences.The main active component of Ganoderma lucidum is polysaccharide and triterpenoid,both of which have many pharmacological activities,such as hepatoprotective activity,anti-tumor effect,immunomodulatory effects,anti-aging activity,etc.Different preparations and compound prescriptions of Ganoderma lucidum have been widely used for the chronic hepatopathy of various etiologies,for instance,chronic hepatitis B,hepatic fibrosis,liver cancer,etc.So far,scholars have mainly foucused on Ganoderma lucidum's clinical research,at home or abroard,lacking of its pharmacological experiments in vitro.In this study,HepG2.2.15 cell model was used to detective the anti-HBV activity of Ganoderma lucidum by MTT and ELISA method.Three parts are involved in this dissertation as bellow:Part1.Extraction,isolation and purification of polysacharides of Ganoderma lucidum.In this part,crude polysaccharide,as the anti-HBV active component according to the experiment in vitro,was obtained by using 80%alcohol deposit,and the yield of which was 2.8%.Then,the crude polysaccharide,with the final concentration of ethanol adjusted to 20%,40%,60%,80%successively,was studied by step precipitation after deproteinization and decoloration.So we got all levels of alcohol precipitated polysaccharides and their anti-HBV activities were studied.The result showed that the 60%level exhibits relatively good anti-HBV activity,therefore,it was further isolated by DEAE cellulose-52 and SephadexG-100 column chromatography.Two main fractions,GLP-1 and GLP-2,were gained and proved to be homogeneous by gel column chromatography,agarose gel electrophoresis and high performance gel permeation chromatography.Part2.A primary research of the physicochemical properties and the structure of Ganoderma lucidum polysaccharides.GLP-1 is a kind of white powder and GLP-2 is light yellow glittering flake-like solid.They were all dissolved easily in water,but insoluble in the organic solvents like ethanol,acetone and etc.The molecular weights of GLP-1 and GLP-2 were tested by high performance gel permeation chromatography(HPGPC)and monosaccharide compositions of GLP-1 and GLP-2 were determined by using sugar nitriles acylation method.The results showed that GLP-1 with molecular 95279Da consisted of arabinose,xylose,mannose,glucose and galactose with the molar ratio of 1:1.3:3.0:15.0:2.6,and GLP-2 with molecular 95279Da consisted of rhamnose,arabinose,xylose,mannose,glucose and galactose with the molar ratio of 2.2:1:3.8:25.4:1.3:2.6.Infrared spectroscopy showed that both GLP-1 and GLP-2 had characteristic absorption peaks of polysaccharide and furanosides might be contained in GLP-2.Experiments of congo red indicated that GLP-1 probably contained triple-helical structure,while GLP-2 did not have.Part3.The screening of polysacharides of Ganoderma lucidum that anti hepatitis B virus in vitro.In this part,HepG2.2.15 cellular model was established and different drugs,including ethanol extract,water extract,all levels of alcohol deposited polysaccharides,and homogeneous polysaccharides GLP-1 and GLP-2,were administrated.Cytotoxicity was measured by MTT colormetric method with IC50 as index,and antigen inhibitory effect was measured by enzymelinked immunosorbent assay(ELSA)with TC50 as index.Finally,the therapy effects of different drugs were assessed with the value of therapy index(TI=TC50/IC50).Results showed that the T1 values of HBsAg and HBeAg of ethanol extract were 2.04 and 1.07 respectively,which of water extract were 2.15 and 0.67.Thus,water extract has better anti-HBV effect.Among all levels of alcohol precipitated polysaccharides,60%ethanol deposited polysaccharides,with the TI values as 5.95 and 1.25 respectively,showed the best anti-HBV activity.Polysacharide GLP-1,with the TI values as 4.79 and 2.52 respectively,showed better anti-HBV effect.