Astaxanthin is a coloring agent which is used as a feed additive in aquaculture nutrition. free radical scavenging, singlet oxygen quenching, and antioxidant activities which could probably positively impact animal and human health. . In 1981, Widmer  synthesized astaxanthin from your educt 6-It is usually suggested that a combination of numerous assays should be used in assessing antioxidant activities . In addition to these rather indirect methods, electron Betanin cost spin resonance spectroscopy (ESR) combined with spin trapping has been established as a robust method for the direct assessment of free radical reactions . Therefore, Betanin cost in this study, we applied ESR, as well as photon counting methods, to determine the free radical scavenging and singlet oxygen quenching activity of astaxanthin. Furthermore, we analyzed the cellular antioxidant activity of astaxanthin in cultured hepatocytes. We addressed the question, if and to what lengthen astaxanthin may induce enzymatic antioxidant defense mechanisms, including paraoxoanase-1. Furthermore, we decided cellular glutathione levels in response to an astaxanthin treatment, since glutathione is the most important endogenous cytosolic antioxidant centrally involved in redox signaling and stress response. Overall, this manuscript aims at providing comprehensive information in terms of the free radical scavenging, antioxidant, and cell signaling modifying properties of astaxanthin. 2. Results and Conversation We applied ESR and spin trapping in order to directly quantify the free radical scavenging activity of astaxanthin. Astaxanthin dose-dependently scavenged DPPH (Physique 2A) and galvinoxyl (Physique 2B) free radicals. However, astaxanthin did not scavenge superoxide anion free radicals (data not shown). Accordingly, astaxanthin did not inhibit xanthine oxidase, which generates superoxide anion free radicals (Physique 2C). When singlet oxygen is relaxed to the ground state, oxygen photon emission is usually observed. Singlet oxygen quenching activity of astaxanthin as a function of emission spectrum, concentration, and decay curve is usually given in Physique 2D. As shown in Physique 2D, astaxanthin dose-dependently quenched singlet oxygen as determined by photon counting. Open in a separate window Open in a separate window Physique 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 M DPPH and the given concentration of astaxanthin. All values are means + SD (experiments Betanin cost performed in triplicate); (B) Numerous astaxanthin concentrations were mixed with 500 M galvinoxyl. Changes in the radical transmission intensity are shown on the right side of the physique. All values are means + SD (three impartial 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 impartial experiments performed Rabbit Polyclonal to VEGFR1 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. In our cell culture studies, astaxanthin did not exhibit any significant cytotoxicity in concentrations of up to 20 M in Huh7, PON1-Huh7, and HepG2 as summarized in Physique 3. TritonX was used as a control to induce cytotoxicity as determined by the neutral reddish assay. Open in a separate window Physique 3 Effects of astaxanthin on cell viability in Huh7, PON1-Huh7, and HepG2 cells Betanin cost after 24 h incubation. Data are means + SD of at least two experiments performed in triplicate. Supplementation of cultured PON1-Huh7 cells with 20 M synthetic astaxanthin resulted in a moderate induction of paraoxoanse-1 (PON1) (Physique 4A). Furthermore, synthetic astaxanthin dose-dependently increased cellular glutathione (GSH) levels in HepG2 cells (Physique 4B). The increase in cellular GSH was not accompanied by an increase in Nrf2 transactivation as shown in Physique 4C. Curcumin and resveratrol were used as positive controls, respectively. Open in a separate window Physique 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 M astaxanthin. PON1 transactivation was measured after 48 h incubation of the cells with synthetic astaxanthin. Curcumin (Curc; 20 M) 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..