Study On The Molecular Structure Modification Of Mori Fructus Polysaccharides And Its Hepatoprotective Activity On Chemical Liver Injury In Mice

Chemical liver injury,caused by chemical hepatotoxic substances,which can eventually develop into cirrhosis and liver cancer,and seriously threatens human health.At present,many hepatoprotective drugs on the market,but most of them are too expensive and poisonous to extremely exert in clinical application.Therefore,developing natural drugs with good hepatoprotective effects and low side effects is an urgent problem to be solved.Our previous study found that Mori.Fructus crude polysaccharides play an important role in reducing liver damage,which indicated M.Fructus crude polysaccharides have good liver protection.However,the effect of M.Fructus refined polysaccharides on hepatoprotection,the best configuration and the structure-activity relationship are unclear,which seriously hinder the comprehensive understanding of M.Fructus polysaccharides and obstruct the development of new medicines research for M.Fructus polysaccharides.In order to solve the above problems,this study taked M.Fructus as the research object,used water extract-alcohol extraction method to obtain M.Fructus crude polysaccharides,further separated and purified by DEAE-52 cellulose and Sephadex G-100 column chromatography,and obtained three relatively uniform polysaccharides components?MFP-1,MFP-2,and MFP-3?.Then,oxidative degradation,sulfation,and carboxymethylation modification were separately selected to study on hepatoprotective effects in vitro of MFP-1,MFP-2 and MFP-3,and then the pre and post M.Fructus polysaccharides were analyzed by HPLC precolumn derivatization and infrared spectroscopy.The derivatives were subjected to primary structure analysis.Finally,the hepatoprotective effect of MFP-1 and its carboxymethylated derivatives on acute alcoholic liver injury model,ConA acute liver injury and subacute alcoholic liver injury in mice were also researched.The main research contents and results are as follows:1.Extraction,isolation and purification of M.Fructus polysaccharides.The M.Fructus crude polysaccharides was obtained by alcohol sedimentation,and then separated and purified by DEAE-52 cellulose column chromatography using distilled water,0.1,0.5 M NaCl solution as eluent,fllowed by Sephadex G-100 column,the elution fractions were collected,dialysised and freeze-dryed,obtained M.Fructus seminal polysaccharides?MFP-1,MFP-2 and MFP-3?.The elution curve showed a single peak shape,which can prove that each polysaccharides component is homogeneous polysaccharides.2.The physicochemical properties and structure characterization analysis of M.Fructus polysaccharides were determined by chemical and instrumental analysis methods.The research contents and results were listed as follows:?1?The neutral polysaccharide fraction?MFP-1?of molecular weight 20.93 kDa contains with 6.9%sulfate,2.3%uronic acid and 75.3%polysaccharides content,and mainly comprises Gal,Glc,Ara,Rha and Man?26.8?20.4?8.7?5?1?.?2?The molecular weight of MFP-2was 56.2 kDa.MFP-2 containing 12.9%sulfate,22.2%uronic acid and 83.7%polysaccharides content,and was mainly composed of Ara,Gal,Rha,Glc and GalA?34.2?38.2?8?17.5?15.1?.?3?The molecular weight of MFP-3 was 118.76 kDa,MFP-3 contains 1.8%sulfate,61.7%uronic acid and 74.3%polysaccharides content,and mainly contains Glc,Gal,Rha,GalA,Ara and GlcA?47.4?34.9?36.1?33.1?19.9?4.1?.Using Infrared spectral data analysis,three polysaccharides showed strong broad absorption peaks near 3400 cm^-1 and 2940 cm^-11 shows they be characteristic absorption peaks of polysaccharides.The broad absorption peak at 1039 cm^-1 is the stretching vibration of the pyran ring,which can be assumed that the polysaccharides are a pyran ring structure.MFP-3 exhibits an absorption peak at 897 cm^-1,suggesting that it contain a?-glycosidic bond.In addition,three polysaccharides did not change color in the case of iodine solution,indicating that they did not contain continuous?-?1?4?Glc bond type.3.Oxidated,sulfated and carboxymethylated modification of M.Fructus polysaccharides,respectively yielded 9 products:Oxidated polysaccharides?O-MFP-1,O-MFP-2 and O-MFP-3?,sulfated polysaccharides?S-MFP-1,S-MFP-2 and S-MFP-3?and carboxymethylated polysaccharides?C-MFP-1,C-MFP-2 and C-MFP-3?.After the oxidative degradation,the molecular weight of MFP-1 was reduced from 20930 Da to7460 Da,the yield was 30.4%;the molecular weight of MFP-2 was reduced from51,230 Da to 30,310 Da,the yield was 17.7%;the MFP-3 molecular weight from118,760 Da to 4189 Da,the yield was 21.3%.After sulfated modification,the DS of S-MFP-1,S-MFP-2 and S-MFP-3 were 0.64,0.39 and 0.55,respectively.The yields were 45.9%,33.4%and 52.1%,respectively.After carboxymethylation,the DS of C-MFP-1,C-MFP-2 and C-MFP-3 were 0.71,0.46 and 0.21,respectively.The yields were 74.2%,68.7%and 73.6%,respectively.After infrared spectroscopy analysis,all of 9 samples appeared characteristic absorption peaks near 3440 cm^-1 and 2927 cm^-1,indicating that the modified sample retained the polysaccharides structure.Sulfate modification products?S-MFP-1,S-MFP-2 and S-MFP-3?showed absorption peaks near 820 cm^-1,indicating that they were partially sulfated.Carboxymethylation products?C-MFP-1,C-MFP-2 and C-MFP-3?showed new peaks at 1617 cm^-1 and 1423 cm^-1,indicating that they had been partially carboxymethylated.It is noteworthy that C-MFP-1showed a new absorption peak at 848.21 cm^-1,explaining that C-MFP-1 may contain?-glucopyranose.4.M.Fructus polysaccharides and their modified products were evaluated on ADH activity.In this study,the activation rate of ADHwas used as an index to determine activities of polysaccharides.Results shown that C-MFP-1(6.59±0.030mg·mL^-1)had the higher activation rate of ADH,which may be related its structural stability and the?-pyranose present.5.Hepatoprotective function of M.Fructus polysaccharides and their carboxymethylated modified products on chemical liver injury in mice.In this study,the model of acute alcoholic liver damage in mice,the ubacute alcoholic liver injuried mice model,and the acute liver injuried mice model induced by con A was duplicated to evaluate the protective effect of M.Fructus polysaccharides and its carboxymethylated modified products?MFP-1 and C-MFP-1?on chemical liver injury.Followed,MFP-1 and C-MFP-1 were detected by AST,ALT,LDH,TG,LDL-C,and TBIL in serum,MDA,SOD and GSH-PX in liver tissue and observation of pathological sections to determine the better polysaccharides.The results showed that there were significant differences effects between the C-MFP-1 group and the model group in biochemical indexes on the alcoholic liver injury model.It can be seen that reduction of AST,ALT and LDH,enhancement of SOD and GSH-PX content and decrease of MDA can increased the ability of the body to scavenge reactive oxygen free radicals and reduce the lipids produced by alcohol.C-MFP-1 can also improve cell deformation,cell rupture and other pathological phenomena.