The cytotoxicity and protective effects of Astragalus membranaceus extracts and butylated hydroxyanisole on hydroxyl radical-induced apoptosis in fish erythrocytes

Erythrocytes play an essential role in transporting O_2 and CO_2 for respiration in fish. However,erythrocytes continuously suffer from reactive oxygen species(ROS)-induced oxidative stress and apoptosis. Thus, it is essential to expand our knowledge of how to protect erythrocytes against ROS-induced oxidative stress and apoptosis in fish. In this study, we explored the cytotoxicity and the effects of butylated hydroxyanisole(BHA), ethyl ether extracts, ethyl acetate extracts, acetone extracts(AE), ethanol extracts, and aqueous extracts of Astragalus membranaceus(EAm) on hydroxyl radical(·OH)-induced apoptosis in carp erythrocytes. The rat hepatocytes and carp erythrocytes were incubated with different concentrations of BHA or EAm(0.125 to 1 mg/mL). The toxicity in rat hepatocytes and carp erythrocytes was then measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay and a haemolysis assay,respectively. The carp erythrocytes were treated with BHA or EAm in the presence of 40 μmol/L FeSO4 and20 μmol/L H_2 O_2 at 37℃, except for the control group. Oxidative stress and apoptosis parameters in the carp erythrocytes were then evaluated using the commercial kit. The results indicated that at high concentrations, BHA and EAm could induce toxicity in rat hepatocytes and fish erythrocytes. However, BHA was more toxic than EAm at the same concentrations. Moreover, the toxicity order of BHA and EAm in the fish erythrocytes approximately agreed with that for the rat hepatocytes. Butylated hydroxyanisole and EAm suppressed the ·OH-induced phosphatidylserine exposure and DNA fragmentation(the biomarkers of apoptosis) by decreasing the generation of ROS, inhibiting the oxidation of cellular components, and restoring the activities of antioxidants in carp erythrocytes. Of all of the examined EAm, the AE showed the strongest effects. The effects of AE on superoxide anion, H_2 O_2, met-haemoglobin and reduced glutathione levels, as well as glutathione reductase activity and apoptosis were equivalent to or stronger than those of BHA. These results revealed that the AE of Astragalus membranaceus could be used as a potential natural antioxidant or apoptosis inhibitor in fish erythrocytes.

Inhibition of lipid oxidation in foods and feeds and hydroxyl radical-treated fish erythrocytes:A comparative study of Ginkgo biloba leaves extracts and synthetic antioxidants

This study explored the effects of butylated hydroxytoluene(BHT) and ethoxyquin(EQ) and ethyl ether extracts, ethyl acetate extracts(EAE), acetone extracts, ethanol extracts and aqueous extracts of Ginkgo biloba leaves(EGbs) on lipid oxidation in a linoleic acid emulsion, fish flesh and fish feed and in hydroxyl radical(·OH)-treated carp erythrocytes. The linoleic acid, fish flesh and fish feed were incubated with BHT,EQ and EGbs at 45℃ for 8 d, respectively, except for the control group. The lipid oxidation in the linoleic acid emulsion, fish flesh and fish feed was then measured by the ferric thiocyanate method or thiobarbituric acid method. The carp erythrocytes were treated with BHT, EQ. or EGbs in the presence of40 μmol/L FeSO_4 and 20 μmol/L H_2 O_2 at 37℃ for 6 h,except for the control group. Oxidative stress and apoptosis parameters in carp erythrocytes were then evaluated by the commercial kit. The results showed that BHT, EQ and EGbs inhibited lipid oxidation in the linoleic acid emulsion, fish flesh and fish feed and·OH-induced phosphatidylserine exposure and DNA fragmentation(the biomarkers of apoptosis) in carp erythrocytes. Furthermore, BHT, EQ and EGbs decreased the generation of reactive oxygen species(ROS),inhibited the oxidation of cellular components and restored the activities of enzymatic antioxidants in·OH-treated carp erythrocytes. Of all examined EGbs, EAE showed the strongest effects. The effects of EAE on lipid oxidation in the linoleic acid emulsion and on superoxide anion and malonaldehyde levels,catalase activity and apoptosis in. OH-treated carp erythrocytes were equivalent to or stronger than those of BHT. Moreover, these results indicated that the inhibition order of EGbs on the generation of ROS and oxidation of cellular components in fish erythrocytes approximately agreed with that for the food and feed materials tested above. And, the antioxidative and anti-apoptotic effects of EGbs were positively correlated with their flavonoid content. Taken together, these results revealed that the fish erythrocyte system can be used as an experimental model to evaluate lipid oxidation in food and feed ingredients. The EAE can be used as a potential natural antioxidant or apoptosis inhibitor. The inhibition effects of EGbs on lipid oxidation and apoptosis may be due to the presence of flavonoid compounds.