| Alcoholic liver disease?ALD?,a disease of liver injury caused by long-term excessive drinking,has become one of the global public health problems.Fatty liver disease is a typical representative of ALD.Long-term development will lead to hepatitis,cirrhosis and even hepatocellular carcinoma,which will seriously affect people's life and health.Through the unremitting efforts and practice of a large number of scientific and technological researchers,various effective treatment methods for ALD have emerged,among which antioxidants and liver transplantation are the most commonly used in clinic.Their emergence has brought good news to the majority of alcoholic liver disease patients.However,with the development of science and technology and the improvement of people's living needs,most of the treatment methods also slowly reflect their shortcomings.Therefore,exploration and innovation,search for safe,efficient and high economic value of treatment methods has become one of the common goals of the majority of researchers nowadays,but also an inevitable trend of the development of the times.Many studies have found that many natural active substances have many biological activities,among which polysaccharides have become the focus of attention in the fields of medicine and food because of their advantages of safety,high efficiency and low toxicity.In our previous practice,we found that the polysaccharides in Mori Fructus?MF?not only have the reported activities of anti-oxidation,anti-cancer and anti-fatigue,but also had good activity of ethanol dehydrogenase in vitro,which was a potential natural active substance to protect liver.However,the relationship between the polysaccharides and the anti-alcoholic liver injury activity was still unclear,which seriously restricts the development of MF polysaccharides?MFP?.Therefore,in-depth study of MFP and its relationship with anti-alcoholic liver injury activity are of great significance for the development and application of liver-protecting drugs and related health food.To further explore the relationship between MFP and anti-alcoholic liver injury activity.On the basis of previous studies,our group continued to take MFP as the research object,purify it by ethanol fractionation,and study its anti-alcoholic liver injury activity from physical and chemical properties,structural characterization,molecular modification,in vitro activity and in vivo activity.However,due to the objective factors such as the workload and the number of samples,this paper mainly chooses the method of fractional purification with low concentration ethanol to separate and purify MFP,and studies their physical and chemical properties,structural characterization and molecular modification.Then,the acute alcoholic liver injury model of mice was established by studying the activity of ethanol dehydrogenase in vitro,and the anti-acute alcoholic liver injury activity of MFP and its derivatives was studied.Finally,the following results were obtained:1.Two components of MF crude polysaccharides?MFPA,MFPB?were obtained from MF by hot water extraction and fractional purification with low concentration ethanol?30%,50%,V/V?.MFPA and MFPB were further separated and purified by DEAE-52 cellulose chromatography.Four components,MFPA1,MFPA2,MFPB1 and MFPB2,were obtained,with the yield of 13.78%,8.68%,9.79%and 4.38%,respectively.MFPA1,MFPA2,MFPB1 and MFPB2 all contain a small amount of protein?0.73%,0.72%,1.29%,6.07%?and glucuronic acid?3.92%,15.87%,6.11%,11.76%?.Among them,MFPA1 and MFPB1 are mainly neutral polysaccharides.The content of polysaccharides is 93.44%and 89.68%,respectively.MFPA2 and MFPB2were mainly acidic polysaccharides.The contents were 70.56%and 69.32%respectively.2.The Mw and Mn of MFPA1,MFPA2,MFPB1 and MFPB2 were determined and analyzed by high performance gel chromatography.The Mw was 177 kDa,638kDa,165 kDa and 380 kDa respectively,and the kDa was 115 kDa,393 kDa,108 kDa and 239 kDa respectively.Their chromatographic peaks were single,which was confirmed that MFPA1,MFPA2,MFPB1 and MFPB2 were homogeneous polysaccharides.MFPA1 was mainly composed of Man,Rha,Glc and Xyl.MFPB1was mainly composed of Man,Rha,GalA,Glc and Xyl.MFPA2 and MFPB2 were mainly composed of Man,Rha,GlcA,GalA,Glc,Xyl and Arb.Ultraviolet spectroscopy and infrared spectroscopy showed that MFPA1,MFPA2,MFPB1 and MFPB2 contained only a small amount of protein and nucleic acid.The molecule contained pyranose ring and?-glycoside bond,and all had characteristic absorption peaks of polysaccharides.It was confirmed that MFPA1,MFPA2,MFPB1 and MFPB2were polysaccharides.3.MFP derivatives,namely S-MFPA1,S-MFPA2,S-MFPB1,S-MFPB2,C-MFPA1,C-MFPA2,C-MFPB1 and C-MFPB2,were sulfated and carboxymethylated by concentrated sulfuric acid method and solvent method respectively.The yields were23.90%,21.25%,24.03%,18.17%,30.01%,28.93%,25.69%and 18.55%,respectively.The sulfate content of S-MFPA1,S-MFPA2,S-MFPB1 and S-MFPB2 were 6.13%,12.18%,6.12%and 5.95%respectively.Their degree of substitution were 0.39,1.01,0.38 and 0.37,respectively.The degree of substitution of C-MFPA1,C-MFPA2,C-MFPB1 and C-MFPB2 were 0.66,0.73,0.32 and 0.41,respectively.4.The activity of ethanol dehydrogenase in vitro was screened by Waller-Hoch method for MFPA1,MFPA2,MFPB1,MFPB2,S-MFPA1,S-MFPA2,S-MFPB1,S-MFPB2,C-MFPA1,C-MFPA2,C-MFPB1 and C-MFPB2.The results showed that MFPA1,MFPB1,S-MFPA1 and S-MFPB1 had good activity of ethanol dehydrogenase in vitro.The activation rates of alcohol dehydrogenase in vitro by MFPA1,MFPB1,S-MFPA1 and S-MFPB1 were 68.51±0.32%,64.41±0.54%,56.90±1.37%and45.30±1.51%respectively at the concentration of 5 mg·mL-1,which may be potential active substances for the prevention and treatment of alcoholic liver injury.5.To further explore the effect of MFP on alcoholic liver injury.In this study,MFPA1,MFPB1,S-MFPA1 and S-MFPB1 were used as experimental objects through the activity of ethanol dehydrogenase in vitro.The effects of MFPA1,MFPB1,S-MFPA1 and S-MFPB1 on the liver of mice with acute alcoholic liver injury were evaluated by constructing acute alcoholic liver injury model and observing the body weight,liver index,AST,ALT,TG,SOD,GSH-Px,MDA and liver histopathological sections of mice in different experimental groups.The results showed that MFPA1,S-MFPA1,MFPB1 and S-MFPB1 could effectively enhance the free radical scavenging ability and increase the level of lipid peroxidation,which had different protective effects on the liver of mice with acute alcoholic liver injury.MFPA1 has the best effect.In this group,the liver index of mice was 4.11±0.13%,serum ALT,AST activity and TG level were 33.14±0.47 U·L-1,33.74±0.86 U·L-1 and 3.15±0.12 mmol·L-1,and SOD,GSH-Px activity and MDA content in liver of mice were 117.38±0.90 U·mL-1,75.14±3.25 U·L-1 and 1.57±0.02 nmol·mL-1,respectively.There was no significant difference between these indexes and blank control group and positive drug control group.The morphological structure and integrity of hepatocytes in this group were the best.It was similar to blank control group and positive drug control group.MFPA1 was an important potential active substance to protect alcoholic liver injury.This studied provides an important scientific basis and application value for MFP as a potential drug for the treatment of acute alcoholic liver injury. |
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