The Protection Of Blueberries Anthocyanins Against Chemical-induced Liver Injury And The Underlying Mechanisms Exploration

Blueberry anthocyanins are one kind of important bioactivators in blueberries, having many kinds of significant physiological functions and broad prospects of development and application. In order to gain insight into the comprehensive utilization of blueberry anthocyanins, we studied blueberries in the northeast and identified its anthocyanins monomers structures on the basis of isolation and purification of blueberry anthocyanins. With acute hepatic injury of mice by CCl4model established, the potential protection of blueberry anthocyanins was demonstrated. Otherwise, with the research of antioxidant activities of blueberry anthocyanins in vitro and protective effect of blueberry anthocyanins on L-02injured by CCl4. The protective effect and antioxidant mechanism of blueberry anthocyanins on hepatic injury of mice induced by CCl4was further discussed; Results were as follows:(1) The optimum enzyme assisted extracting condition of anthocyanin compounds from blueberry:0.5%trifluoroacetic acid-methanol as the extraction solvent, solid-liquid ratio of1:15, p H2.5, added3%of the amount of enzyme, hydrolysis temperature70℃, the enzymatic reaction time1h. Under these conditions, the yield of blueberry anthocyanins was5.65mg/g, measured by RP-HPLC method.Amberlite XAD-7macroporous adsorptive resin had good adsorption and desorption capability. The optimum dynamic adsorption condition of Amberlite XAD-7was as follows:blueberry anthocyanins concentration2.5mg/mL, pH value2.5, velocity of flow1.0mL/min. The optimum desorption condition was as follows:methanol concentration70%, velocity of flow1.0mL/min, desorption volume10BV. Under these parameters, the maximum dynamic adsorption quantity of Amberlite XAD-7resin for blueberry anthocyanin was up to18.5mg/g, dynamic desorption rate was87.15%.Three main anthocyanins monomers were got by the preparation of HPLC, through the structure identification, No.1component was Malvidin-3-galactoside (M3G), with the purity of95.49%; No.3component was Cyanidin-3-glucoside(C3G), with the purity of98.57%; No.6component was Cyanidin-3-rutinoside(C3R), with the purity of96.34%. The three monomers were respectively made up12.76%,60.68%and15.87%of total anthocyanin contents.(2)Blueberry anthocyanins have certain antioxidant activity, whose effect was better than than vitamin C in the same concentration in DPPH radical scavengingassay and β-carotin/linolic acid oxidation inhibiting activity. The order of antioxidant activity of three blueberry anthocyanins monomers was C3G>C3R>M3G.While the values result of IC50was showed that cyanidin-3-glucoside(C3G) exhibited the strongest response in alkyl peroxide and DPPH radical scavenging. All the results turned out that cyanidin-3-glucoside was one of the most active antioxidant ingredients in blueberry anthocyanin.(3) When the mice was treated with blueberry anthocyanins(in dosages of0.5g/kg bw,1.0g/kg bw,2.0g/kg bw), the activity of ALT was significantly decreased compared with model group (P<0.05), AST was also decreased(P<0.05); MDA, the product of lipid peroxidation was decreased(P<0.05); Meanwhile, the activities of antioxidant enzymes, such as SOD and GSH-Px were enhanced (P<0.05); The pathology damage of the liver indueed by CCl4, such as balloon-like change, fatty degeneration.inflammatory infiltration were all improved obviously.(4) To establish liver cell injury model, the concentration of CCl4was20mmol/L, and the time was6h. In the model group, the L-02cell survival rate were decreased to45.68%, while the liver enzyme activities were enhanced and the content of MDA was increased significantly(P<0.05). The injury of L-02was retarded by blueberry anthocyanins. Compared to cell in model group, the cell survival rate was increased, and the liver enzyme was reduced, the more the concentration, the higher the survival rate(P<0.05). Results showed the obvious dose-response relationship. Furthermore, liver damage protection effect of the three main ingredients of blueberry anthocyanins (C3G, C3R and M3G) on L-02cells were investgated. The results showed that C3G had the best effect to improve the level of cell survival and cell anti-oxidation, and there were significant difference between the others(P<0.05), Cyanidin-3-glucoside(C3G) was the most effective anti-damage active monomer.Result of cell colony formationinhibitory assay, cell cycle progress and Annexin V-FITC/PI staining analysis showed that Cyanidin-3-glucoside protected L-02cells by reducing cell necrosis. Contents change of cell apoptosis regulation protein Caspase-3detected by western blotting demonstrated that the cell necrosis caused by injury was Caspase-dependent in L-02cells, while necrosis of cells might be via mitochondria pathway.