Preparation Of Beta-casomorphin-7 And Its Prevention On Alcoholic Fatty Liver

1 Preparation(Separation, Purification and Identification) of Beta-casomorphin-7Objective To investigate the best condition to prepare Beta-casomorphin-7(β-CM-7)from casein. To set up a method to separate, purify and identifyβ-CM-7. Methods Casein was hydrolyzed by pepsin for 120 minutes. The enzymatic hydrolysis was filtrated by ultrafiltrate membrane, and analyzed by SDS-PAGE.β-CM-7 was identificated in the enzymatic hydrolysis by RP-HPLC in external and internal standard method, withβ-CM-7 as the standard. The enzymatic hydrolysis of the casein was chromatographed by Sephadex-G-15.β-CM-7 in the eluate from Sephadex-G-15 was detected by RP-HPLC both qualitative and quantitative methods. The fractions containingβ-CM-7, eluted from Sephadex-G-15, was chromatographed by DEAE-Sepharose Fast Flow, each fraction of which was determined if there wasβ-CM-7 by RP-HPLC both qualitative and quantitative methods. Resultsβ-CM-7 could be preparated from casein by enzymatic hydrolysis of pepsin for 120 minutes. The enzymatic hydrolysis was chromatographed by Sephadex-G-15,β-CM-7 emerged in the 6th fraction identified by RP-HPLC, its content was 20.5μg·mL -1. The 6th fraction eluted from Sephadex-G-15 was chromatographed by DEAE-Sepharose Fast Flow,β-CM-7 emerged in the 3th fraction identified by RP-HPLC, its content was 29.5μg·mL-1. Conclusion Casein enzymatic hydrolysis by pepsin could produceβ-CM-7.β-CM-7 could be isolated by Sephadex-G-15 and DEAE-Sepharose Fast Flow.β-CM-7 could be identificated by RP-HPLC both qualitative and quantitative methods. 2 Preventive effects of beta-casomorphin-7 on Alcoholic fatty liver and its MechanismsObjective To establish the mice models of Alcoholic fatty liver(AFL).To investigate the preventive effects and the mechanism of beta-casomorphin-7(β-CM-7) on AFL. Methods The hepatocytes of Kunming infant mice were isolated, cultured by trypsin digestion and induced by ethanol. Various concentrations ofβ-CM-7 were added to the cultured hepatocytes for 24h. The viabilities of hepatocytes were measured by MTT. In this study, mice were drenched with liquor to make the model of AFL. 60 Kunming mice were randomly divided into six groups: control group, model group, HGP group,β-CM-7Ⅰgroup,β-CM-7Ⅱgroup andβ-CM-7Ⅲgroup. Except the control group, the other groups were given alcoholic intragastric administration. The alcohol was infused chronically, in the first 4 weeks, 30% alcohol at 10 mg·kg-1, in the next 4 weeks, 40% alcohol at 11 mg·kg-1, and in the last 4 weeks, 50% alcohol at 12 mg·kg-1, twice a day. The control group was given equal physiological saline. In the same time, HGP group was given HGP at 0.3g·kg-1,β-CM-7Ⅰ,ⅡandⅢgroups were givenβ-CM-7 at 2.0×10-1g·L-1, 2.0×10-2g·L-1 and 2.0×10-3g·L-1 respetively, in 250μl once. The whole experimental time was 12 weeks. At the end of the experiment, the mice were killed to collect the samples of serum and liver. The body and liver were weighed, and liver indexes(liver weight/ body weight) were counted. The contents of ALT,AST,TG,TC,LDL-C and HDL-C in serum were examined. The activities of SOD and GSH-PX, and the content of MDA were measureed in the liver. The slice of liver tissue was made and dyed by HE and Oil Red O to observe the pathologic changes. Resultsβ-CM-7 had the protective effect on primary infant mouse hepatocyte injured by ethanol. In the model group, the activities of ALT, AST and the contents of TG, TC, LDL-C in mice serum were remarkably increased, the content of HDL-C in mice serum was remarkably decreased. Liver histopathological examination showed microvesicular steatosis were seen throughout the liver tissue.β-CM-7 reduced the liver index(LI) of mice. LI of β-CM-7Ⅰg roup was remarkably lower than that in the model group(P<0.05). These showedβ-CM-7 had the protective effect on liver cells. The activities of AST and ALT inβ-CM-7Ⅰgroup were significantly lower than the model group(P<0.01).β-CM-7 accelerated the fat metabolism of liver cells. The contents of TG and LDL-C inβ-CM-7Ⅰg roup were remarkably decreased and the content of HDL-C was significantly increased(P<0.01) than that of the model group.β-CM-7 inhibits the lipid peroxidation(LPO) of fat in the liver.β-CM-7 enhanced the activities of SOD and GSH-PX and reduced the content of MDA. Liver histopathological examination showedβ-CM-7 could ameliorate hepatic steatosis. Conclusions AFL animal model was sucessefully copied.β-CM-7 has the preventive effect on AFL in mice.β-CM-7 could operate with modulation of fat metabolism and reduction of LPO for the prevention of AFL.