Study On Structural Characterization Of Mori Fructus Polysaccharides By Graded Alcohol Precipitated With Weak Polarity And Its Anti Acute-alcoholic Liver Injury Effect

Mori Fructus?MF?is a mature fruit ear of Morus alba L.It contains abundant nutrients and chemical active ingredients,which exhibits a variety of medicinal values.It is also included in the first part of the Chinese Pharmacopoeia 2015 edition and the catalogue of medicinal and food homologous raw materials?2017?Mori Fructus polysaccharides?MFP?is one of the high content of MF.Some studies have shown that MFPs have many biological activities,such as antioxidant,immune regulation,hypoglycemic and so on.In addition,the previous study in our group also found that MFPs have a good protective effect on alcoholic liver injury,which may be a potential substance for the treatment of alcoholic liver injury.Polysaccharides belong to a class of macromolecule polymers with large molecular weight and extremely complex structure.Their structure directly or indirectly affects their biological activities.However,most structures of polysaccharides are not yet clear,the different separation and purification methods may also obtain polysaccharides of different quality,which greatly affects the biological activities,efficacy and structure-activity relationship of polysaccharides.To understand the MFPs more comprehensively,and the effects of different MFPs on alcoholic liver injury.crude polysaccharides of MF obtained by graded alcohol precipitated with weak polarity were studied to explore the potential and effective MFPs with anti-alcoholic liver injure effects,to provide the material basis for the field of anti-alcoholic and hepatoprotective medicine and health products and to promote the development and utilization of MFPs.But due to the heavy workload and the limited time of postgraduate stage,this paper chose MFPs by graded alcohol precipitated with weak polarity as the material research basis.In this study,the refined MFPs?R-MFPs?were obtained by graded alcohol precipitated with weak polarity,and then purified by column chromatography.The structure of R-MFPs were characterized from three aspects:physicochemical properties,molecular weight and monosaccharide composition.To obtain the different derivatives of MFPs,the R-MFPs were modified by different chemical modification methods.Taking the MFPs and their derivatives as the research object,an alcohol dehydrogenase activity?ADH?model was established in vitro.The MFPs and derivatives with better ADH activity were screened according to the activation rate of ADH activity in vitro.Finally,based on the screening results,an animal model of acute alcoholic liver injury was established to systematically evaluate the protective effect of MFPs and its derivatives on acute alcoholic liver injury through various biochemical indicators and pathology.The main results were as follows:?1?Extraction,separation and purification of MFPsTwo homogeneous neutral MFPs?MFPs-70-1,MFPs-90-1?and two homogeneous acid MFPs?MFPs-70-2,MFPs-90-2?were obtained by crushing,degreasing,hot water extraction,70%and 90%alcohol precipitation,deproteinization,purifying with DEAE-52 cellulose column and SephadexG-100 gel column chromatography from MF dry products.?2?Preliminary characterization of the structure of MFPsMFPs-70-1 contains 87.64%total sugar,1.29%protein,6.26%glucuronic acid and molecular weight was 316 kDa.It was composed of It was composed of mannose11.0%,rhamnose 5.4%,galacturonic acid 4.7%,glucose 33.5%and xylose 45.5%;MFPs-90-1 contains 91.33%total sugar,protein 0.32%,glucuronic acid 5.53%,molecular weight was 398 kDa,and was composed of mannose 7.2%,rhamnose1.4%,glucose 28.6%and xylose 62.8%;MFPs-70-2 contains 60.27%total sugar,1.06%protein,11.95%glucuronic acid and 1584 kDa,which was composed of which is composed of mannose 18.8%,rhamnose 4.6%,glucuronic acid 6.2%,galacturonic acid 1.5%,glucose 34.7%,xylose 25.5%and arabinose 8.7%;MFPs-90-2 contains71.41%total sugar,0.32%protein,10.46%glucuronic acid and with 229.1 kDa molecular weight,which is composed of mannose 5.8%,rhamnose 15.4%,glucuronic acid 3.0%,galacturonic acid 2.9%,glucose 36.5%,xylose 21.2%,arabinose 15.1%.Infrared spectroscopy revealed that MFPs-70-1,MFPs-70-2,MFPs-90-1 and MFPs-90-2 may all had pyran ring structure.In addition,MFPs-70-2 and MFPs-90-2may also contain?-glycoside bonds.?3?Molecular Modification of MFPsMFPs-70-1,MFPs-90-1,MFPs-70-2 and MFPs-90-2 were sulfated to obtain S-MFPs-70-1,S-MFPs-70-2,S-MFPs-90-1and S-MFPs-90-2,respectively.The degree of substitution were 0.33,0.58,0.55,and 0.40,and the yields were 22.76%,27.93%,21.15%and 23.05%,respectively.Four groups of sulfated derivatives retained the original sugar structure,and strong absorption peaks in S-MFPs-70-1,S-MFPs-70-2,S-MFPs-90-1,S-MFPs-90-2 at 116.32 cm^-1,1149.62 cm^-1,1147.21cm^-1 and 1143.27 cm^-1,respectively.This was the S=O structure,indicating that the sulfation modification of MFPs was successful.C-MFPs-70-1,C-MFPs-70-2,C-MFPs-90-1 and C-MFPs-90-2 were obtained by carboxymethylated.The degree of substitution were 0.49,0.52,0.41,0.33,and the yields were 17.23%,30.68%,27.56%,28.33%,respectively.Four groups of carboxymethylated derivatives also retained the original polysaccharides structure by infrared spectroscopy.in addition,the signal of-COO-of C-MFPs-70-1,C-MFPs-90-1,C-MFPs-70-2 and C-MFPs-90-2 all had enhanced near 1600¹400 cm^-1,which indicated that the carboxymethylation of MFPs were successful.?4?Activation of ADH activity in vitroThe ADH activity of MFPs with their sulfated and carboxymethylated derivatives in vitro were studied.Investigated the activation effect of MFPs and their derivatives on ADH activity,found that the unmodified groups MFPs-70-1,MFPs-90-1,MFPs-70-2,MFPs-90-2 and sulfated derivatives S-MFPs-70-1,S-MFPs-90-1,S-MFPs-70-2 and S-MFPs-90-2 all had activation effects on ADH.the ADH activation rates of MFPs-70-1 and MFPs-90-1 in the unmodified group were the highest?P<0.01?that was 58.14%and 58.47%,respectively.Therefore,MFPs-70-1 and MFPs-90-1 had relatively good effects on ADH activation in vitro.?5?Protective effect of acute alcoholic liver injury in vivoBased on the results of ADH activity activation rate,MFPs-70-1,MFPs-90-1,S-MFPs-70-1 and S-MFPs-90-1 were selected as the research objects and established the animal model of acute alcoholic liver injury in mice.The results showed that each group of MFPs and their sulfated derivatives(50 mg·kg^-1·BW^-1)could control the weight loss,inhibit the increase the levels of AST,ALT,TG in blood and MDA in liver?P<0.05?,meanwhile,the levels of SOD and GSH-Px in liver increased?P<0.01?.It indicated that MFPs?MFPs-70-1,MFPs-90?by graded alcohol precipitated with weak polarity and its sulfated derivatives?S-MFPs-70-1 and S-MFPs-90-1?could improve the antioxidant capacity of mice and inhibit the production of lipid peroxides so that protected the liver of mice from damage by ethanol.Compared with the model group,the liver index,the levels of AST,ALT,TG of serum index and MDA of liver content in S-MFPs-90-1 group were significantly decreased?P<0.05?by 17.15%,32.34%,21.29%,28.46%.While SOD,GSH-Px were significantly increased?P<0.01?by 44.40%,112.61%,respectively.Showing a relatively prominent antioxidant effect.By observing pathological sections,it were found that the hepatocytes in model group were severely disrupted and deformed,the cells swelled and spacing increased,and the nucleus exposed;In each administration group,the degree of hepatocyte disruption,cell swelling and spacing decreased,and the hepatocyte injury were repaired and improved with different degrees.According to the physiological indexes and pathological sections of serum and liver in the model group,the animal model of acute alcoholic liver injury was successfully established.The MFPs?MFPs-70-1,MFPs-90-1?by graded alcohol pr-ecipitated with weak polarity and their sulfated derivatives?S-MFPs-70-1,S-MFPs-90-1?had protective effects on acute alcoholic liver injury.Among which S-MFPs-90-1 might be relatively effective.However,the results against the activation of ADH in vitro,this might be due to the good antioxidant effect of S-MFPs-90-1 and the complex metabolic of alcohol in the liver.In conclusion,the MFPs by grad-ed alcohol precipitated with weak polarity?MFPs-70-1,MFPs-90-1?and their sulfated derivatives?S-MFPs-70-1,S-MFPs-90-1?had protective effects on alcohol-induced acute liver injury in mice,which might be a potential anti-alcoholic and hepatoprotective substances.