International Journal of Molecular Sciences Article Free Radical Scavenging andand CellularCellular AntioxidantAntioxidant Properties of Astaxanthin Janina DoseDose 11, Seiichi Matsugo 2,, Haruka Haruka Yokokawa Yokokawa 2,, Yutaro Yutaro Koshida Koshida 2,, Shigetoshi Shigetoshi Okazaki Okazaki 33, , Ulrike Seidel 1,, Manfred Eggersdorfer 4,, Gerald Gerald Rimbach Rimbach 11 andand Tuba Tuba Esatbeyoglu Esatbeyoglu 1,1,** Received: 12 October 2015; Accepted: 8 January 2016; Published: January 14 January 2016 2016 Academic Editor: Esra Capanoglu 11 InstituteInstitute of of Human Human Nutrition Nutrition and and Food Food Science, Science, University University of of Kiel, Kiel, Hermann-Rodewald-Straße Hermann-Rodewald-Straße 6, 6, D-24118D-24118 Kiel, Kiel, Germany; Germany; dose@foodsci [email protected] (J.D.); (J.D.); seidel [email protected]@foodsci.uni-kiel.de (U.S.); (U.S.); [email protected]@foodsci.uni-kiel.de (G.R.) (G.R.) 2 SchoolSchool of of Natural Natural System, System, Kanazawa Kanazawa Univer University,sity, Kakuma-machi, Kakuma-machi, Kanazawa Kanazawa 920-1192, 920-1192, Japan; Japan; [email protected]@se.kanazawa-u.ac.jp (S.M.); (S.M.); haruside [email protected]@gmail.com (H.Y.); (H.Y.); [email protected] [email protected] (Y.K.) (Y.K.) 3 MedicalMedical Photonics Photonics Research Research Center, Center, Hamamatsu Hamamatsu Univ Universityersity School School of of Medicine, Medicine, Handamachi Handamachi 1-20-1, 1-20-1, Higashi-ku,Higashi-ku, Hamamatsu, Hamamatsu, Shizuoka Shizuoka 431- 431-3192,3192, Japan; Japan; [email protected] [email protected] 4 DSMDSM Nutritional Nutritional Products, Products, P.O. P.O. Box Box 2676, 2676, 4002 4002 Base Basel,l, Switzerland; Switzerland; [email protected] [email protected] ** Correspondence:Correspondence: esatbeyoglu@f [email protected];oodsci.uni-kiel.de; Tel.: Tel.: +49-431-880-5333 +49-431-880-5333 Abstract: Astaxanthin is a coloring agent which is used as a feed additive in aquaculture nutrition. Recently, potentialpotential healthhealth benefitsbenefits ofof astaxanthinastaxanthin have been discusseddiscussed which may be partly related to its its free free radical radical scavenging scavenging and and antioxidant antioxidant properties. properties. Our Our electron electron spin spin resonance resonance (ESR) (ESR) and andspin spintrapping trapping data suggest data suggest that synthetic that synthetic astaxanthi astaxanthinn is a potent is a free potent radical free scavenger radical scavenger in terms inof termsdiphenylpicryl-hydrazyl of diphenylpicryl-hydrazyl (DPPH) (DPPH)and galvinox and galvinoxylyl free freeradicals. radicals. Furthermore, Furthermore, astaxanthinastaxanthin dose-dependently quenched singlet oxygen as determined by photon counting. In addition to free radical scavenging and singlet oxygenoxygen quenchingquenching properties, astaxanthin induced the antioxidantantioxidant enzyme paroxoanase-1, enhanced enhanced glutathione glutathione conc concentrationsentrations and and prevented prevented lipid lipid peroxidation peroxidation in incultured cultured hepatocytes. hepatocytes. Present Present results results suggest suggest th that,at, beyond beyond its its coloring coloring properties, syntheticsynthetic astaxanthin exhibits free radicalradical scavenging,scavenging, singlet oxygen quenching,quenching, and antioxidantantioxidant activitiesactivities which couldcould probablyprobably positivelypositively affectaffect animalanimal andand humanhuman health.health. Keywords: astaxanthin; free radical scavenging;scavenging; antioxidant; electron spinspin resonanceresonance spectroscopyspectroscopy 1. Introduction Introduction 1 1 1 Astaxanthin (3,3 ′-dihydroxy--dihydroxy-ββ,β′,β-carotene-4,4-carotene-4,4′-dione,-dione, for for chemical chemical structure structure see see Figure Figure 1)1 )is is a axanthophyll xanthophyll carotenoid carotenoid [1] [1 ]which which naturally naturally occurs occurs in in algae, algae, krill, krill, trout, trout, crayfish, crayfish, and and salmon. Astaxanthin isis widely usedused in aquaculture nutritionnutrition asas aa coloringcoloring agentagent [[2].2]. In addition to its coloring properties, astaxanthinastaxanthin maymay alsoalso affectaffect immuneimmune statusstatus andand reproductionreproduction [[3,4].3,4]. Figure 1. Chemical structure of astaxanthin. Astaxanthin has has two two chiral chiral centers centers at positions at positions 3 and 3 3and'. The 3'. astaxanthin The astaxanthin stereoisomer stereoisomer -3S,3S1- is-3S the,3S main′- is formthe main found form in wild found salmon in wild [5]. salmon Most astaxanthin [5]. Most asta usedxanthin in aquaculture used in aquaculture nutrition is produced nutrition syntheticallyis produced whichsynthetically yields three which different yields stereoisomers, three different including stereoisomers,3S, 31S; 3R including, 31S; and 3R 3S,,3 13R′S[1; ].3R In, 1981,3′S; and Widmer 3R, 3et′R 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 www.mdpi.com/journal/ijmswww.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 103 2 of 14 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(3,5,5-trimethyl-2-cyclohexene-1,4-dione) was obtained with yields up to 50% via yieldsa seve [n-step6]. synthesis. Over a Wittig reaction of two Astaxanthinequivalents C15-phosphonium has also significant salt with applications C10-dialde inhyde, the nutraceuticalastaxanthin was industry obtained[ 7with]. Various yields up health benefits,to 50% including yields [6]. cardiovascular disease and cataract prevention, have been associated with astaxanthinAstaxanthin consumption has also [3,8 significant–10]. applications in the nutraceutical industry [7]. Various health Althoughbenefits, including it has been cardiovascular suggested thatdisease potential and cataract health prevention, benefits of astaxanthinhave been associated are, at least with 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, systematicmediated studies by its are free scarce. radical scavenging [11], antioxidant [12,13], and gene-regulatory properties In[14,15], previous systematic studies, studies free radicalare scarce. scavenging activity of astaxanthin has been determined by FRAP (ferric reducingIn previous antioxidant studies, free power) radical [16 scavenging], TEAC (trolox activity equivalent of astaxanthin antioxidant has been capacity)determined [17 by], and ORACFRAP (oxygen (ferric radical reducing absorbance antioxidant capacity) power) [16], [18] assays,TEAC (trolox respectively. equivalent Furthermore, antioxidant thecapacity) decolorization [17], of theand 2,2-diphenyl-1-picrylhydrazyl ORAC (oxygen radical absorbance (DPPH) radicalcapacity) may [18] be assays, used to respectively. assess the free Furthermore, radical scavenging the activitydecolorization of antioxidants, of the taking 2,2-diphenyl-1-picrylhydrazyl into account that alcohols (DPPH) as solvents radical may may be lead used to to an assess overestimation the free of its freeradical radical scavenging scavenging activity activity of antioxidants, [19–21]. The taking different into account antioxidant that alcohols test systems, as solvents as describedmay lead to in the literature,an overestimation differ in terms of ofits underlyingfree radical methodology,scavenging activity reagents, [19–21]. wavelength, The different pH, antioxidantetc. It is suggested test 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 Inantioxidant addition activities to these ratherin vitro indirect [22]. methods, electron spin resonance spectroscopy (ESR) combined with spinIn trapping addition has to beenthese establishedrather indirect as amethods, robust method electron for spin the resonance direct assessment spectroscopy of free (ESR) radical reactionscombined [23]. with Therefore, spin trapping in this has study, been weestablished applied as ESR, a robust as wellmethod as photonfor the direct counting assessment methods, of to determinefree radical the free reactions radical [23]. scavenging Therefore, and in singletthis study, oxygen we applied quenching ESR, activity as well ofas astaxanthin.photon counting Furthermore,methods, to determine we studied the the free cellular radical antioxidant scavenging activityand singlet of astaxanthin oxygen quenching in cultured activity hepatocytes. of We addressedastaxanthin. the question, if and to what extend astaxanthin may induce enzymatic antioxidant defense mechanisms,Furthermore, includingwe studied paraoxoanase-1. the cellular antioxidant Furthermore, activity we determinedof astaxanthin cellular in cultured glutathione levelshepatocytes. in response We to addressed an astaxanthin the question, treatment, if and since