| In this thesis,albumin was extract from corn germ meal and enzymatic hydrolysised to prepare sober and hepatoprotective peptides with alcohol dehydrogenase(ADH)activation rate and hydroxyl radical(·OH)inhibition as indicator.The peptide fractions APF4 with MW<1k Da was obtained by ultrafiltration and purified and fractionated by chromatography.The effect of APF4 on acute alcoholism mice and acute and chronic alcohol-induced liver injury in mice was studied.The main conclusions obtained were as follows:The albumin was extracted under the conditions of 40?,extraction time 30 min and solid-liquid ratio(g:m L)1:20 using corn germ meal as raw material;The neutral protease was selected as the optimal protease using the ADH activation rate and ·OH inhibition rate as index among acid protease,neutral protease and alcalase protease enzyme;The optimum enzymatic hydrolysis conditions obtained by the single factor experiment and response surface experiment were as follows: temperature 50.63?,p H 6.94,amount of enzyme 4012.7U/g,time 1.99 h,substrate concentration 1mg/m L.The corn germ meal albumin peptides mixture prepared were separated by ultrafiltration membrane with molecular weight of 3 k Da and 1 k Da,and four fractions were obtained: APF1(MW>3k Da),APF2(MW<3k Da),APF3(3k Da>MW>1k Da),APF4(MW<1k Da).The effect of peptides mixture and the four fractions on blood alcohol concentration in mice was detected and APF4 exhibited best compared to the alcohol model group(P<0.01).The five fractions F1,F2,F3,F4,F5 were obtained by chromatography and fractions F5 had higher ·OH inhibition rate in 32.10% than any other four fractions.Administrated 50%(v/v)ethanol solution in mice,the maximum drunk dose was 12 m L/kg bw;In the acute alcoholism model in mice,AFP4 at high dose of 800mg/kg bw could significantly prolong the loss time and shorten the recovery time of righting reflex in mice,decreased the drunk rate,and the APF4(400mg/kg bw and 800mg/kg bw)could significantly reduced blood ethanol content in mice(P<0.01);Pretreatment with APF4 in differet dose increased the levels of hepatic aldehyde dehydrogenase(ALDH)and decreased the content of hepatic cytochrome P4502E1(CYP2E1)in mice,APF4 at the dose of 800mg/kg bw was very significantly effective(P<0.01).The results showed that APF4 had a good effect on the metabolism of ethanol,and the effect of high dose APF4(800mg/kg bw)was best of all.In the acute alcohol-induced liver injury in mice,APF4 at high dose decreased the activity of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and the lever of liver triglyceride(TG)and malondialdehyde(MDA)(P<0.01),and increased total superoxide dismutase(T-SOD)activity and reduced glutathione(GSH)level(P<0.01),but there was no significant change in catalase(CAT)activity(P>0.05);H&E staining showed that the pathological changes of liver tissue were improved by AFP4 and the high dose group(800mg/kg bw)recovered the structure of hepatocellular cord significantly,liver sinus space returned to normal,showing complete cell structure.The results suggested that APF4 had protective effect on alcohol-induced liver injury in mice.In the chronic alcohol-induced liver injury in mice,APF4 at high dose decreased the activity of ALT,AST and the lever of liver TG and MDA(P<0.01),and increased T-SOD activity and reduced GSH level(P<0.01),but there was no significant change in CAT activity(P>0.05).H&E staining showed that AFP4 had a dose-effect relationship on the effect of protect the liver injury induced by alcoholism.The APF4 high dose group(800mg/kg bw)had the best effect,significantly reduced the number of lipid droplets around the nucleus of liver cells,and the structure of hepatocytes was clear with integral cell structure and cytoplasm homogeneity.The central vein was radial arrangement around the center veins.The results showed that APF4 had the same protective effect on both acute and chronic alcohol-induced liver injury in mice.APF4 could be used as a long-term dose of liver disease therapy. |
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