Panax ginseng Fraction F3 Extracted by Supercritical Carbon Dioxide Protects against Oxidative Stress in ARPE-19 Cells

Abstract

In our previous work, the ethanolic extract of Panax ginseng C. A. Meyer was successively partitioned using supercritical carbon dioxide at pressures in series to yield residue (R), F1, F2, and F3 fractions. Among them, F3 contained the highest deglycosylated ginsenosides and exerted the strongest antioxidant and anti-inflammatory activities. The aim of this study was to investigate the protective effects of P. ginseng fractions against cellular oxidative stress induced by hydrogen peroxide (H₂O₂). Viability of adult retinal pigment epithelium-19 (ARPE-19) cells was examined after treatments of different concentrations of fractions followed by exposure to H₂O₂. Oxidative levels (malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), and reactive oxygen species (ROS)) and levels of activity of antioxidant enzymes were assessed. Results showed that F3 could dose-dependently protected ARPE-19 cells against oxidative injury induced by H₂O₂. F3 at a level of 1 mg/mL could restore the cell death induced by H₂O₂ of up to 60% and could alleviate the increase in cellular oxidation (MDA, 8-OHdG, and ROS) induced by H₂O₂. Moreover, F3 could restore the activities of antioxidant enzymes suppressed by H₂O₂. In conclusion, F3 obtained using supercritical carbon dioxide fractionation could significantly increase the antioxidant capacity of P. ginseng extract. The antioxidant capacity was highly correlated with the concentration of F3.

Keywords: Panax ginseng; hydrogen peroxide-induced oxidative damage; retinal pigment epithelium; supercritical carbon dioxide fractionation.

Figure 1

Cytotoxic effect of H₂O₂ on adult retinal pigment epithelium-19 (ARPE-19) cells. Cell viability of ARPE-19 cells, following different concentrations of H₂O₂ exposure, measured by trypan blue exclusion test. Dose (A); and (B) time effect of H₂O₂ (100 μM) on ARPE-19 cells. Results are expressed as percentages of control, and each value represents the mean ± SD of three independent experiments.

Figure 2

Effects of P. ginseng supercritical CO₂ extracted fractions on cell viability of H₂O₂-induced cytotoxicity in ARPE-19 cells. ARPE-19 cells were treated with or without a series of concentrations of P. ginseng supercritical CO₂ fractions for 2 h, and then treated with H₂O₂ for another 24 h. (A–C) Hoechst 33342 staining; (D–F) Quantitative analysis of panel (A–C). * p < 0.05, ** p < 0.01, *** p < 0.001 versus H₂O₂ group. Results are expressed as percentages of control, and each value represents the mean ± SD of three independent experiments.

Figure 3

Effects of P. ginseng supercritical CO₂ extracted fraction F3 on H₂O₂-induced cellular oxidation in ARPE-19 cells. ARPE-19 cells were pretreated with or without a series of concentrations of F3 for 2 h, and then treated with 100 μM H₂O₂ for another 24 h. (A) Intracellular reactive oxygen species levels were measured by flow cytometry analysis; (B) malondialdehyde (MDA); and (C) 8-hydroxydeoxyguanosine (8-OHdG) contents were measured on a microplate reader (VersaMax). MDA and 8-OHdG levels in the H₂O₂ group increased significantly compared with the untreated group. F3 attenuated the H₂O₂-induced changes in MDA and 8-OHdG. ** p < 0.01 versus untreated group. ^## p < 0.01, ^### p < 0.001 versus H₂O₂ group. Each value represents the mean ± SD of three independent experiments.

Figure 4

Effect of P. ginseng supercritical CO₂ extracted fraction F3 on cellular antioxidant enzymes: (A) superoxide dismutase (SOD); (B) catalase (CAT); and (C) glutathione peroxidase (GPx) activities in ARPE-19 cells subjected to H₂O₂-induced oxidative stress. ARPE-19 cells were pretreated with or without a series of concentrations of F3 for 2 h, and then treated with 100 μM H₂O₂ for another 24 h. Antioxidant enzymes activities were determined using commercially available kits following the manufactures’ instructions. ** p < 0.01 versus untreated group, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus H₂O₂ group. Each value represents the mean ± SD of three independent experiments.

Figure 4

Effect of P. ginseng supercritical CO₂ extracted fraction F3 on cellular antioxidant enzymes: (A) superoxide dismutase (SOD); (B) catalase (CAT); and (C) glutathione peroxidase (GPx) activities in ARPE-19 cells subjected to H₂O₂-induced oxidative stress. ARPE-19 cells were pretreated with or without a series of concentrations of F3 for 2 h, and then treated with 100 μM H₂O₂ for another 24 h. Antioxidant enzymes activities were determined using commercially available kits following the manufactures’ instructions. ** p < 0.01 versus untreated group, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus H₂O₂ group. Each value represents the mean ± SD of three independent experiments.