International Journal of
Molecular Sciences Article
Antioxidant Free Radical Scavenging and Cellular Antioxidant Properties of Astaxanthin 2 2 2 3 Janina Dose Dose 11,, Seiichi Janina Seiichi Matsugo Matsugo 2,, Haruka Haruka Yokokawa Yokokawa 2,, Yutaro Yutaro Koshida Koshida 2,, Shigetoshi Shigetoshi Okazaki Okazaki 3, , Ulrike Seidel 1, Manfred Eggersdorfer 44,, Gerald Gerald Rimbach Rimbach 11 and andTuba Tuba Esatbeyoglu 1,1,**
2016 Received: 12 October 2015; Accepted: 8 January 2016; Published: January 14 January 2016 Academic Editor: Editor: Esra Capanoglu Academic 11
Institute InstituteofofHuman HumanNutrition Nutritionand andFood FoodScience, Science,University UniversityofofKiel, Kiel,Hermann-Rodewald-Straße Hermann-Rodewald-Straße6,6, D-24118 D-24118Kiel, Kiel,Germany; Germany;
[email protected] [email protected](J.D.); (J.D.);
[email protected] [email protected](U.S.); (U.S.);
[email protected] [email protected](G.R.) (G.R.) 2 School SchoolofofNatural NaturalSystem, System,Kanazawa KanazawaUniversity, University,Kakuma-machi, Kakuma-machi,Kanazawa Kanazawa920-1192, 920-1192,Japan; Japan;
[email protected] (S.M.);
[email protected] (H.Y.);
[email protected] (Y.K.)
[email protected] (S.M.);
[email protected] (H.Y.);
[email protected] (Y.K.) 33 Medical MedicalPhotonics PhotonicsResearch ResearchCenter, Center,Hamamatsu HamamatsuUniversity UniversitySchool Schoolof ofMedicine, Medicine,Handamachi Handamachi1-20-1, 1-20-1, Higashi-ku, Higashi-ku,Hamamatsu, Hamamatsu,Shizuoka Shizuoka431-3192, 431-3192,Japan; Japan;
[email protected] [email protected] 44 DSM DSMNutritional NutritionalProducts, Products,P.O. P.O.Box Box2676, 2676,4002 4002Basel, Basel,Switzerland; Switzerland;
[email protected] [email protected] ** Correspondence: Correspondence:
[email protected];
[email protected];Tel.: Tel.:+49-431-880-5333 +49-431-880-5333
Abstract: nutrition. 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 Recently, potential health benefits of astaxanthin to its free free radical radical scavenging scavengingand andantioxidant antioxidantproperties. properties. electron resonance OurOur electron spinspin resonance (ESR)(ESR) and and trapping data suggest that synthetic astaxanthin is a free potent free scavenger radical scavenger spin spin trapping data suggest that synthetic astaxanthin is a potent radical in terms in of terms of diphenylpicryl-hydrazyl (DPPH) and galvinoxyl free radicals. Furthermore, astaxanthin 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 quenching properties, astaxanthin induced the antioxidant antioxidant enzyme paroxoanase-1, paroxoanase-1, enhanced enhancedglutathione glutathioneconcentrations concentrationsand andprevented prevented lipid peroxidation lipid peroxidation in in cultured hepatocytes. Present resultssuggest suggestthat, that,beyond beyondits its coloring coloring properties, synthetic cultured hepatocytes. Present results synthetic astaxanthin exhibits free radical radical scavenging, scavenging, singlet oxygen quenching, quenching, and antioxidant antioxidant activities activities which which could could probably probably positively positively affect affect animal animal and and human humanhealth. health. Keywords: astaxanthin; free radical scavenging; antioxidant; electron electron spin spin resonance resonance spectroscopy spectroscopy
1. Introduction 1. Introduction Astaxanthin (3,31 -dihydroxy-β,β1 -carotene-4,41 -dione,for forchemical chemicalstructure structuresee seeFigure Figure1)1)is isa Astaxanthin (3,3′-dihydroxy-β,β′-carotene-4,4′-dione, axanthophyll xanthophyllcarotenoid carotenoid[1][1]which whichnaturally naturallyoccurs occursinin algae, algae, krill, krill, trout, trout, crayfish, crayfish, and and salmon. salmon. Astaxanthin is widely used in aquaculture nutrition as a coloring agent [2]. In addition to its coloring Astaxanthin is widely used in aquaculture nutrition as a coloring agent [2]. In addition to its coloring properties, properties, astaxanthin astaxanthin may may also also affect affect immune immune status status and and reproduction reproduction[3,4]. [3,4].
Figure 1. 1. Chemical of astaxanthin. astaxanthin. Figure Chemical structure structure of
Astaxanthinhas hastwo two chiral centers at positions and 3'.astaxanthin The astaxanthin stereoisomer -3S,3S′is Astaxanthin chiral centers at positions 3 and33'. The stereoisomer -3S,3S1- is the main the main form found in wild [5]. Most astaxanthin used in aquaculture nutrition synthetically is produced form found in wild salmon [5].salmon Most astaxanthin used in aquaculture nutrition is produced synthetically which yieldsstereoisomers, three different stereoisomers, 3S,313′S; 3′S; and 3R, 3′R which yields three different including 3S, 31S; 3R,including 31S; and 3R, R [1].3R, In 1981, Widmer et al.[1]. [6] In 1981, Widmer et al. [6] synthesized astaxanthin from the educt 6-oxo-isophorone Int. J. Mol. Sci. 2016, 17, 103; doi:10.3390/ijms17010103 doi:10.3390/ijms17010103
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synthesized astaxanthin from the educt 6-oxo-isophorone (3,5,5-trimethyl-2-cyclohexene-1,4-dione) via a Int. J. Mol. Sci. 2016, 17, 103 2 of 13 seven-step synthesis. Over a Wittig reaction of two equivalents C15-phosphonium salt with C10-dialdehyde, astaxanthin was obtained with yields up to 50% [6]. (3,5,5-trimethyl-2-cyclohexene-1,4-dione) via yields a seven-step synthesis. Over a Wittig reaction of two equivalents C15-phosphonium salt with C10-dialdehyde, astaxanthin wasindustry obtained[7]. withVarious yields up Astaxanthin has also significant applications in the nutraceutical health to 50% yields [6]. cardiovascular disease and cataract prevention, have been associated with benefits, including Astaxanthin has also significant applications in the nutraceutical industry [7]. Various health astaxanthin consumption [3,8–10]. benefits, including cardiovascular disease and cataract prevention, have been associated with Although it has been suggested that potential health benefits of astaxanthin are, at least partly, astaxanthin consumption [3,8–10]. mediated by its free radical scavenging [11], antioxidant [12,13], and gene-regulatory properties [14,15], Although it has been suggested that potential health benefits of astaxanthin are, at least partly, systematic studies are scarce. mediated by its free radical scavenging [11], antioxidant [12,13], and gene-regulatory properties In previous studies, free radical scavenging activity of astaxanthin has been determined by FRAP [14,15], systematic studies are scarce. (ferric reducing antioxidant power) [16], TEAC (trolox antioxidant [17], In previous studies, free radical scavenging activityequivalent of astaxanthin has been capacity) determined by and ORAC (oxygen absorbance capacity) [18] assays, respectively. Furthermore, decolorization FRAP (ferricradical reducing antioxidant power) [16], TEAC (trolox equivalent antioxidant the capacity) [17], ORAC (oxygen radical absorbance capacity)may [18]beassays, Furthermore, the of theand 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical used torespectively. assess the free radical scavenging decolorization of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical may be used to assess the free activity of antioxidants, taking into account that alcohols as solvents may lead to an overestimation of radical scavenging activity of antioxidants, taking into account that alcohols as solvents may lead toin the its free radical scavenging activity [19–21]. The different antioxidant test systems, as described an overestimation of its free radical scavenging activity [19–21]. The different antioxidant test literature, differ in terms of underlying methodology, reagents, wavelength, pH, etc. It is suggested systems, as described in the literature, differ in terms of underlying methodology, reagents, that a combination of various assays should be used in assessing antioxidant activities in vitro [22]. wavelength, pH, etc. It is suggested that a combination of various assays should be used in assessing In addition to these rather indirect methods, electron spin resonance spectroscopy (ESR) combined antioxidant activities in vitro [22]. with spinIn trapping as amethods, robust method theresonance direct assessment of free radical additionhas to been these established rather indirect electronfor spin spectroscopy (ESR) reactions [23]. Therefore, in this study, we applied ESR, as well as photon counting methods, combined with spin trapping has been established as a robust method for the direct assessment of to determine the free radical[23]. scavenging and oxygen quenching free radical reactions Therefore, in singlet this study, we applied ESR,activity as well of asastaxanthin. photon counting methods, to determine thethe free radicalantioxidant scavengingactivity and singlet oxygen quenching activity of Furthermore, we studied cellular of astaxanthin in cultured hepatocytes. astaxanthin. We addressed the question, if and to what extend astaxanthin may induce enzymatic antioxidant Furthermore, including we studied the cellular antioxidant activity of astaxanthin in cultured defense mechanisms, paraoxoanase-1. Furthermore, we determined cellular glutathione hepatocytes. We addressed the question, if and to what extend astaxanthin may induce enzymatic levels in response to an astaxanthin treatment, since glutathione is the most important endogenous antioxidant defense mechanisms, including paraoxoanase-1. Furthermore, we determined cellular cytosolic antioxidant centrally involved in redox signaling and stress response. glutathione levels in response to an astaxanthin treatment, since glutathione is the most important Overall, thiscytosolic manuscript aims atcentrally providing comprehensive information in terms of the free radical endogenous antioxidant involved in redox signaling and stress response. scavenging, antioxidant, and cell signaling modifying properties of astaxanthin. 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 Discussion
2. Results and Discussion We applied ESR and spin trapping in order to directly quantify the free radical scavenging applied ESR and spin trapping in order to directly quantify the(Figure free radical scavenging activity ofWe astaxanthin. Astaxanthin dose-dependently scavenged DPPH 2A) and galvinoxyl activity of astaxanthin. Astaxanthin dose-dependently scavenged DPPH (Figure 2A) and galvinoxyl (Figure 2B) free radicals. However, astaxanthin did not scavenge superoxide anion free radicals (data (Figure 2B) free radicals. However, did astaxanthin did xanthine not scavenge superoxide free radicals not shown). Accordingly, astaxanthin not inhibit oxidase, whichanion generates superoxide (data not shown). Accordingly, astaxanthin did not inhibit xanthine oxidase, which generates anion free radicals (Figure 2C). When singlet oxygen is relaxed to the ground state, oxygen photon superoxide anion free radicals (Figure 2C). When singlet oxygen is relaxed to the ground state, emission is observed. Singlet oxygen quenching activity of astaxanthin as a function of emission oxygen photon emission is observed. Singlet oxygen quenching activity of astaxanthin as a function spectrum, concentration, and decay curve is given in Figure 2D. As shown in Figure 2D, astaxanthin of emission spectrum, concentration, and decay curve is given in Figure 2D. As shown in Figure 2D, dose-dependently quenched singlet oxygen as determined by photon by counting. astaxanthin dose-dependently quenched singlet oxygen as determined photon counting. 140
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Figure 2. Cont.
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Figure 2. Scavenging effects of astaxanthin on DPPH radical (A), galvinoxyl radical (B), xanthine
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 oxidase quenching of singlet (D). (A) The reaction mixture 500 µM 500inhibition μM DPPH(C), andand the given concentration ofoxygen astaxanthin. All values are means + SD contained (experiments DPPH and the given concentration of astaxanthin. All values are means + SD performed performed in triplicate); (B) Various astaxanthin concentrations were mixed with(experiments 500 μM galvinoxyl. in triplicate); (B)the Various concentrations mixed µMAll galvinoxyl. in Changes in radicalastaxanthin signal intensity are shown on were the right side with of the500 figure. values are Changes means + SD (three experiments in triplicate); (C) The contained the radical signalindependent intensity are shown onperformed the right side of the figure. Allreaction values mixture are means + SD (three 5 U/mL experiments xanthine oxidase in 50 in mM potassium buffer and contained the given5 astaxanthin independent performed triplicate); (C) phosphate The reaction mixture U/mL xanthine concentrations. Allopurinol was used as a positive control. All values are means + SDAllopurinol (three oxidase in 50 mM potassium phosphate buffer and the given astaxanthin concentrations. independent experiments performed in triplicate); (D) Singlet oxygen quenching activity of was used as a positive control. All values are means + SD (three independent experiments performed 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 in triplicate); (D) Singlet oxygen quenching activity of astaxanthin as a function of concentration astaxanthin), wavelength (left), and time (right). Photo emission was determined by a photon (5.4 ˆ 10´7 , 1.1 ˆ 10´6 , 2.1 ˆ 10´6 , 4.2 ˆ 10´6 , and 8.1 ˆ 10´6 M astaxanthin), wavelength (left), and complex after laser irradiation at 532 nm in the presence of the test sample. Two independent time (right). Photo emission was determined by a photon complex after laser irradiation at 532 nm in experiments performed in duplicate. 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
In our cell culture studies, did not exhibit any concentrations of up to 20 astaxanthin μM in Huh7, PON1-Huh7, and significant HepG2 as cytotoxicity summarized in in concentrations Figure 3. of upTritonX to 20 µM in Huh7, PON1-Huh7, and HepG2 as summarized in Figure 3. TritonX was used as a was used as a control to induce cytotoxicity as determined by the neutral red assay. control to induce cytotoxicity as determined by the neutral red assay.
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Figure 3. 3. Effects Effects of of astaxanthin astaxanthin on on cell cell viability viability in in Huh7, Huh7, PON1-Huh7, PON1-Huh7, and and HepG2 HepG2 cells cells after after 24 24 h h Figure incubation. Data are means + SD of at least two experiments performed in triplicate. incubation. Data are means + SD of at least two experiments performed in triplicate.
Supplementation of of cultured PON1-Huh7 cells with 20 μM synthetic astaxanthin resulted ininaa of cultured cultured PON1-Huh7 PON1-Huh7cells cellswith with20 20μM µMsynthetic syntheticastaxanthin astaxanthinresulted resultedin Supplementation moderate induction ofofparaoxoanse-1 paraoxoanse-1 (PON1) (Figure 4A). Furthermore, astaxanthin a moderateinduction inductionof paraoxoanse-1(PON1) (PON1) (Figure (Figure 4A). 4A). Furthermore, Furthermore, synthetic astaxanthin moderate dose-dependently increased cellular glutathione (GSH) levels in HepG2 cells (Figure 4B). The dose-dependently increased cellular glutathione (GSH) levels in HepG2 cells (Figure 4B). The increase dose-dependently increased cellular glutathione (GSH) levels in HepG2 cells (Figure 4B). The increase in cellular GSH was not accompanied by an increase in Nrf2 transactivation as shown in in cellular wasGSH not accompanied by an increase Nrf2 transactivation as shown in increase inGSH cellular was not accompanied by aninincrease in Nrf2 transactivation as Figure shown4C. in Figure 4C. 4C. and Curcumin and resveratrol resveratrol were used controls, as positive positive controls, respectively. respectively. Curcumin resveratrol were used as positive respectively. Figure Curcumin and were used as controls, B B Glutathione Glutathione [µmoL/1*10 [µmoL/1*106 6HepG2 HepG2cells] cells]
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Figure Figure 4. 4. Effects Effects of of astaxanthin astaxanthin on on transactivation transactivation of of paraoxonase-1 paraoxonase-1 (PON1) (PON1) (A), (A), cellular cellular glutathione glutathione cultured hepatocytes. (GSH) levels (B) and transactivation of Nrf2 (C) in cultured hepatocytes. (A) PON1-Huh7 (GSH) levels (B) and transactivation of Nrf2 (C) in cultured hepatocytes. (A) PON1-Huh7 cells cells were were 6 ˝ 6 cells/wellinto into2424 24 well plates and incubated forh24 24 h at atC. 37Cells °C. Cells Cells ataaadensity densityofof of0.15 0.15 10cells/well seeded ˆ ××1010 well plates andand incubated for 24 at 37 were 6 cells/well into well plates incubated for h 37 °C. seeded at at density 0.15 were treated with 1 and 20 μM astaxanthin. PON1 transactivation was measured after 48the h treated with 1 and 20 µM astaxanthin. PON1 transactivation was measured after 48 h incubation of 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 cells with synthetic astaxanthin. Curcumin (Curc; 20 µM) was used as a positive control; (B) HepG2 incubation of the cells with synthetic astaxanthin. Curcumin (Curc; 20 μM) was used as a positive cells/well into 24-well 24-well plates and control; (B) HepG2 cells wereofseeded seeded at6 aacells/well density of of 0.15 1066plates cells were seeded at acells density 0.15 ˆ 10 into 24 ××well and incubated for plates 24 h. Cells cells/well into and control; (B) HepG2 were at density 0.15 10 were treated and were 5 µM treated astaxanthin for an additional 24 h. Resveratrol (Res; incubated forwith 24 h. h.1Cells Cells were treated with 11and andincubated μM astaxanthin astaxanthin and incubated incubated for an an additional additional incubated for 24 with and 55 μM and for 6 cells/well 25 µM) was used as a positive control; (C) Huh7 cells were seeded at a density of 0.15 ˆ 10 24 h. Resveratrol (Res; 25 μM) was used as a positive control; (C) Huh7 cells were seeded at a density 24 h. Resveratrol (Res; 25 μM) was used as a positive control; (C) Huh7 cells were seeded at a density into 24 well and incubated forplates 24 h atand 37 ˝incubated C. Cells were transfected forCells 24 h. were Nrf2 transfected transactivation of 0.15 0.15 1066plates cells/well into 24 24 well well plates and incubated for 24 24 h at at 37 37 °C. °C. Cells were transfected for for cells/well into for h of ×× 10 was measured after 24 h incubation of the cells with 1 and 20 µM astaxanthin. Curcumin (Curc; 24 h. Nrf2 transactivation was measured after 24 h incubation of the cells with 1 and 20 μM 24 h. Nrf2 transactivation was measured after 24 h incubation of the cells with 1 and 20 μM 20 µM) was used as a positive control. All values means +control. SEM (two experiments astaxanthin. Curcumin (Curc; 20 20 μM) was was used as asare positive control. Allindependent values are are means means SEM astaxanthin. Curcumin (Curc; μM) used aa positive All values ++ SEM performed in triplicate), statistical significant differences between THF-control cells and astaxanthin (two independent experiments performed in triplicate), statistical significant differences between (two independent experiments performed in triplicate), statistical significant differences between supplemented cells areastaxanthin indicated as * p < 0.05, ***cells p
