Studies On Protective Effect And Antioxidant Mechanism Of Black Rice Anthocyanins On Hepatic Injury Of Mice Induced By CCl₄

In the last 20 years, active oxygen and free radical have become the hot topics of modern life science, and appraisal and selection of natural resources with strong antioxidant activities have become the new trends of biology, medical science and food science and technology. As a good and important rice resource, black rice is an important source for the development of natural antioxidants due to rich natural anthocyanins in pericap and seed coat of brown rice. Free radical can result in liver injury, and how to prevet from liver injury is still a world-wide problem presently. Thus, it's greatly important and meaningfull to select medication or food protecting liver from injurying. In this paper. BRA (Black rice anthocyanins, BRA)was seperated and purified through macroporous resin, meanwhile, the main anthocyanin components in BRA were identified by HPLC. With sub-acute hepatic injury of mice by CCl₄ established, the potential protection of BRA was demonstrated. Otherwise, with the research of antioxidant activities of BRA in vitro and protective effect of BRA on L-02 injured by CCl₄, the protective effect and antioxidant mechanism of BRA on hepatic injury of mice induced by CCl₄ was further discussed. The main results were as follows:1. Study on purification and identification of BRAThe absorption and purification effects of BRA were studied, and the main anthocyanin components of BRA were identified by HPLC. AB-8 is the best resin for BRA due to its better adsorption and desorption capability. The optimal technics parameters of AB-8 were as follows: the pH of solution is 2.0; anthocyanins concentration of the sample solution is 1.0mg /ml, adsorption flow rate was 1ml /min; desorption flow rate was 1.0ml/min, with 70% ethanol as desorptioner. The resin has good reutilization uses which desorption capability has no significant changes after using 5 times (P>0.05), with only 2.58% reduction after using 7 times. After purified with AB-8, the total antioxidant capability of BRA enhanced 3.99 times. With HPLC(High performance Liquid Chromatography, HPLC) analysis, the total anthocyanins content in BRA was 44.62%, with 36.98%C3G(Cyanidin-3-glucoside, C3G), 4.88% Pn3G (Peondidin-3-glucoside, Pn3G), 2.76% Pg3G (Pelargonindin-3-glucoside, Pg3G), as a rate of 82.88%. 10.94%, 6.18% in total anthocyanins content in BRA.2. The protection and the effect of oxidation-response of BRA on sub-acute hepatic injury of mice induced by CCl₄The protective effect and antioxidant mechanism of BRA on sub-acute hepatic injury of mice induced by CCl₄ was investigated through measuring liver enzymes activities in mice serum and the activities of oxidative and anti-oxidative system in mice, and pathological changes in liver tissue were observed by H&E. In the mice treated with BRA(in dosages of 0.40g/kg bw, 0.80g/kg bw, 1.60g/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 activity of antioxidant enzymes, such as SOD,GSH-Px were enhanced (P<0.05); the total antioxidant capacity (T-AOC) were significantly increased (P<0.05); the content of 8-OHdG was decreased (P<0.05); and the pathology Damage of the liver induced by CCl₄ , such as Balloon-like change, fatty degeneration, inflammatory infiltration, was improved obviously. BRA could improve the antioxidant activity and protect mice from sub-acute hepatic injury mice induced by CCl₄.3. Eliminating free radicals and antioxidant activities of BRAThe antioxidant activities of CBRA (Crude extract of black rice anthocyanins, CBRA) and BRA were measured in different systems, as well as C3G Pn3G Pg3G. BRA showed good reductive capability, antioxidant activities, and abilities of scavenging DPPH·. And BRA showed better total antioxidant capability and scavenging DPPH·capability than CBRA. TAC (total antioxidant capacity, TAC) of 0.025g/L BRA was 1.993mmol/g, 3.99 times of CBRA. Considering inhibiting DPPH·capacities. IC₅₀ of CBRA was 22μg/ml, while IC₅₀ of BRA was 22μg/ml .TAC of C3G, Pg3G, Pn3G with 0.1g/L were 1.299, 0.173, 0.112. Considering inhibiting DPPH·capacities, IC₅₀ of C3G Pg3G, Pn3G were 2.50μg/ml,14.00μg/ml 25.40μg/ml.4. The protection of BRA on L-02 injuried by CCl₄The protective effect of BRA and 3 anthocyanin monomers on L-02 injured by CCl₄ were determined in the view of oxidation-response. To establish liver cell injury model, the concentration of CCl₄ was 20mmol/L and the time was 6 h. In the model group, the L-02 cell survival rate were decreased to 45.68%, the liver enzyme activities were enhanced, the activities of antioxidant system were decreased and the content of MDA, NO were increased significantly (P<0.05). The injury of L-02 was improved by BRA. Compared to cell in model group, the cell survival rate were increased, and the more the concentration, the higher the survival rate. BRA had capability of improving the release of ALT and AST. The three anthocyanins of C3G Pg3G and Pn3G in BRA could still protect L-02 from injurying by CCl₄ at following aspect. Firstly, cell survival rate could be increased and showed dosage-effect relation. The cell survival rate in C3G group could be up to 88.56%. 94.89% higher than that in model group. Secondly. ALT and AST could be inhibited. With a dosage of 80μg/ml. the activity of AST and ALT in C3G group decreased to 66.56U/L and 53.18U/L, with percentages of 84.22, 66.95 in Pg3G group and 91.23, 65.00 in Pn3G group. With the same dosage, the effect of C3G, Pg3G, and Pn3G on AST. ALT activities in L-02 could be expressed by a inequation: C3G>Pg3G>Pn3G.5. Oxidation-response effect and protection mechanism of BRA on L-02 injured by CCl₄The oxidation-response effect of BRA and 3 anthocyanin monomers on L-02 injured by CCl₄ were studied. BRA could restrain the level of NO and improve the antioxidant activities in L-02. 80μg/ml BRA could significantly decrease the content of NO. 20μg/ml BRA could significantly decrease the content of MDA. BRA could increase the content of GSH. the level of GSH in L-02 added BRA with a dosage of 120μg/ml reached almost the same level in normal group without injury induced by CCl₄ (P>0.05). Meanwhile, BRA could improve the activities of SOD, CAT (P<0.05), and presented dosage-response relation. The three anthocyanins of C3G Pg3G, and Pn3G in BRA could still protect L-02 from injurying by CCl₄ at following aspect. Firstly, C3G at a dosage of 80μg/ml could restrain the level of NO (P<0.01). Secondly, the content of MDA could be decreased. The level of MDA in C3G and Pg3G group at a dosage of 40μg/ml decreased by 23.73% and 3.96%. Thirdly, the level of GSH could be improved. 40μg/ml C3G and 80μg/ml Pg3G could improve the content of GSH. while Pn3G could not. With the same dosage, the effect of C3G Pg3G, and Pn3G on GSH level in L-02 could be expressed by a inequation: C3G>Pg3G>Pn3G. Fourthly, the activities of SOD and CAT could be enhanced. 80μg/ml C3G, Pg3G Pn3G signifieantly increased the activities of SOD and CAT. and the activities of SOD in C3G group was 1.54 times of that in Pg3G group and 1.93 times of that in Pn3G group. The activity of CAT in C3G group was 1.28 times of that in Pg3G group and 1.42 times of that in Pn3G group. The effect of C3G Pg3G Pn3G on activities of antioxidant enzymes in L-02 could be expressed by a inequation C3G>Pg3G>Pn3G. The main innovations of this paper were as followsIt is the first time to discover and demonstrate that BRA could protect liver of mice from damage induced by CCl₄. BRA can signifiantly decrease liver enzymes activities, improve pathological injury. The mechanism of BRA on protection of mice injured by CCl₄ is related to the antioxidant activity of BRA. with functions of improving antioxidant enzymes activities, decreasing the content of MDA.Make sure that the main anthocyanins monomers in BRA are C3G Pg3G Pn3G. These three anthocyanins monomers present strong antioxidant activities, and can protect L-02 from injury by CCl₄, because of the function of increasing L-02 survival rate, decreasing liver enzymes activities, and improving antioxidant abilities of oxidant-antioxidant system in L-02. What's more, as a main basic, C3G show the strongest antioxidant activities, thus-demonstrating the main protection mechanism of BRA on liver-injury induced by CCl₄ in the view of cell level.The study focused on the cutting edge problems of modern life science, and was valuable in enriching free radical biology and rice resource theory. The results can also play an important role in explpiting and utilizing good rice resources, enlarging planting areas of famous and excellent crops such as black rice, adusting and optimizing agricultural structures , improving agricultural efficiency, promoting deep processing and comprehensive utilization of specialty rice, improving people's dietary structure, and promoting sale of specialty agricultural products of China in the international market.