Free Radical Scavenging and Cellular Antioxidant Properties of Astaxanthin

Abstract

Astaxanthin is a coloring agent which is used as a feed additive in aquaculture nutrition. Recently, potential health benefits of astaxanthin have been discussed which may be partly related to its free radical scavenging and antioxidant properties. Our electron spin resonance (ESR) and spin trapping data suggest that synthetic astaxanthin is a potent free radical scavenger in terms of diphenylpicryl-hydrazyl (DPPH) and galvinoxyl free radicals. Furthermore, astaxanthin dose-dependently quenched singlet oxygen as determined by photon counting. In addition to free radical scavenging and singlet oxygen quenching properties, astaxanthin induced the antioxidant enzyme paroxoanase-1, enhanced glutathione concentrations and prevented lipid peroxidation in cultured hepatocytes. Present results suggest that, beyond its coloring properties, synthetic astaxanthin exhibits free radical scavenging, singlet oxygen quenching, and antioxidant activities which could probably positively affect animal and human health.

Keywords: antioxidant; astaxanthin; electron spin resonance spectroscopy; free radical scavenging.

Figure 1

Chemical structure of astaxanthin.

Figure 2

Scavenging effects of astaxanthin on DPPH radical (A), galvinoxyl radical (B), xanthine oxidase inhibition (C), and quenching of singlet oxygen (D). (A) The reaction mixture contained 500 μM DPPH and the given concentration of astaxanthin. All values are means + SD (experiments performed in triplicate); (B) Various astaxanthin concentrations were mixed with 500 µM galvinoxyl. Changes in the radical signal intensity are shown on the right side of the figure. All values are means + SD (three independent experiments performed in triplicate); (C) The reaction mixture contained 5 U/mL xanthine oxidase in 50 mM potassium phosphate buffer and the given astaxanthin concentrations. Allopurinol was used as a positive control. All values are means + SD (three independent experiments performed in triplicate); (D) Singlet oxygen quenching activity of astaxanthin as a function of concentration (5.4 × 10⁻⁷, 1.1 × 10⁻⁶, 2.1 × 10⁻⁶, 4.2 × 10⁻⁶, and 8.1 × 10⁻⁶ M astaxanthin), wavelength (left), and time (right). Photo emission was determined by a photon complex after laser irradiation at 532 nm in the presence of the test sample. Two independent experiments performed in duplicate.

Figure 2

Scavenging effects of astaxanthin on DPPH radical (A), galvinoxyl radical (B), xanthine oxidase inhibition (C), and quenching of singlet oxygen (D). (A) The reaction mixture contained 500 μM DPPH and the given concentration of astaxanthin. All values are means + SD (experiments performed in triplicate); (B) Various astaxanthin concentrations were mixed with 500 µM galvinoxyl. Changes in the radical signal intensity are shown on the right side of the figure. All values are means + SD (three independent experiments performed in triplicate); (C) The reaction mixture contained 5 U/mL xanthine oxidase in 50 mM potassium phosphate buffer and the given astaxanthin concentrations. Allopurinol was used as a positive control. All values are means + SD (three independent experiments performed in triplicate); (D) Singlet oxygen quenching activity of astaxanthin as a function of concentration (5.4 × 10⁻⁷, 1.1 × 10⁻⁶, 2.1 × 10⁻⁶, 4.2 × 10⁻⁶, and 8.1 × 10⁻⁶ M astaxanthin), wavelength (left), and time (right). Photo emission was determined by a photon complex after laser irradiation at 532 nm in the presence of the test sample. Two independent experiments performed in duplicate.

Figure 3

Effects of astaxanthin on cell viability in Huh7, PON1-Huh7, and HepG2 cells after 24 h incubation. Data are means + SD of at least two experiments performed in triplicate.

Figure 4

Effects of astaxanthin on transactivation of paraoxonase-1 (PON1) (A), cellular glutathione (GSH) levels (B) and transactivation of Nrf2 (C) in cultured hepatocytes. (A) PON1-Huh7 cells were seeded at a density of 0.15 × 10⁶ cells/well into 24 well plates and incubated for 24 h at 37 °C. Cells were treated with 1 and 20 µM astaxanthin. PON1 transactivation was measured after 48 h incubation of the cells with synthetic astaxanthin. Curcumin (Curc; 20 µM) was used as a positive control; (B) HepG2 cells were seeded at a density of 0.15 × 10⁶ cells/well into 24 well plates and incubated for 24 h. Cells were treated with 1 and 5 µM astaxanthin and incubated for an additional 24 h. Resveratrol (Res; 25 µM) was used as a positive control; (C) Huh7 cells were seeded at a density of 0.15 × 10⁶ cells/well into 24 well plates and incubated for 24 h at 37 °C. Cells were transfected for 24 h. Nrf2 transactivation was measured after 24 h incubation of the cells with 1 and 20 µM astaxanthin. Curcumin (Curc; 20 µM) was used as a positive control. All values are means + SEM (two independent experiments performed in triplicate), statistical significant differences between THF-control cells and astaxanthin supplemented cells are indicated as * p < 0.05, *** p < 0.001; one-way ANOVA LSD (Figure 4A) and Dunnett-T (Figure 4B).

Figure 5

Impact of astaxanthin on the ability of human HepG2 cells to prevent cumene hydroperoxide/hemin stimulated lipid peroxidation. HepG2 cells were seeded at a density of 0.1 × 10⁶ cells/well into a 96-well plate and incubated for 24 h. Cells were treated with two concentrations of astaxanthin (10 and 20 µM) for an additional 24 h. Cells were rinsed and loaded with C11-BODIPY for 30 min and then exposed to cumene hydroperoxide (8 mM)/hemin (8 µM) for 1 h. Control cells were treated with cell culture medium only. Simultaneously, basal levels of lipid peroxidation were assessed for each treatment concentration (data not shown) and control. Lipid peroxidation was determined by examining the ratio of green (oxidized) and the sum of green and red (oxidized + non-oxidized) emissions using a fluorometric plate reader. Data are means + SD of at least two independent experiments performed in duplicate. ** p < 0.01, one-way ANOVA Games–Howell.