Tetrahedron Letters 57 (2016) 1967–1970

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Analysis of reaction products of astaxanthin and its acetate withreactive oxygen species using LC/PDA ESI-MS and ESR spectrometry
Azusa Nishino a, Takashi Maoka b,⇑, Hiroyuki Yasui ca

Institute of Health Sciences, Ezaki Glico Co., Ltd, 4-6-5 Utajima, Nishiyodogawa-ku, Osaka 555-8502, Japan
Research Institute for Production Development, 15 Shimogamo, Morimoto Cho, Sakyoku, Kyoto 606-0805, Japanc
Department of Analytical and Bioinorganic Chemistry, Division of Analytical and Physical Chemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi,
Yamashina-ku, Kyoto 607-8414, Japanba r t i c l ei n f oArticle history:
Received 22 January 2016
Revised 21 March 2016
Accepted 24 March 2016
Available online 24 March 2016
Keywords:
Astaxanthin
Reactive oxygen species
Astaxanthin-endoperoxide
Astaxanthin-epoxide
Astaxanthin peroxide radicala b s t r a c t
Reaction products of astaxanthin and its acetate with hydroxy radical, superoxide anion radical, and singlet oxygen were analyzed by LC/PDA ESI-MS and ESR spectrometry. Astaxanthin epoxides were found to
be major reaction products of astaxanthin with superoxide anion radical and hydroxyl radical. On theother hand, astaxanthin endoperoxides were identified as major reaction products of astaxanthin withsinglet oxygen. The same results were obtained in the case of astaxanthin acetate. The ESR study revealed
that astaxanthin and its acetate take up superoxide anion radical by the formation of their epoxidethrough peroxide and oxide radicals.
Ó 2016 Elsevier Ltd. All rights reserved.

Astaxanthin, which is widely distributed in marine bacteria,green algae, crustaceans, fish, and birds,1 is an excellent antioxidant not only for quenching singlet oxygen but also for lipid peroxidation.2–4 Recently, Hama et al. reported that astaxanthin could
scavenge hydroxyl radical in a liposome system.5 Furthermore, it
was reported that astaxanthin could take up peroxynitrite into
its molecule by the formation of nitroastaxanthin.6,7 However,there are few reports on the reaction products of astaxanthin withreactive oxygen species. In order to elucidate the chemical
scavenging mechanism of astaxanthin with reactive oxygenspecies, we investigated reaction products of astaxanthin and its
acetate with hydroxyl radical (OH), superoxide anion radical(O2 ), and singlet oxygen (1O2) using both LC/PDA ESI-MS andESR spectrometry. This Letter reports the reaction products ofastaxanthin and its acetate with reactive oxygen species and thesereaction mechanisms.
Hydroxyl radical, O2 , and 1O2 were generated by UV-A irradiation of hematoporphyrin, riboflavin, and hydrogen peroxide (H2O2)
solution, respectively.8 An acetonitrile (CH3CN) solution ofastaxanthin or its acetate was added to these reactive oxygen
species-generation systems. At regular intervals of UV-A irradiation, the reaction products were analyzed by LC/PDA ESI-MS8 andESR spin-trapping.9⇑ Corresponding author. Tel.: +81 75 781 1107; fax: +81 75 781 1118.
E-mail address: maoka@mbox.kyoto-inet.or.jp (T. Maoka).
http://dx.doi.org/10.1016/j.tetlet.2016.03.078
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.

In the case of the reaction of astaxanthin acetate with hydroxyl
radical, reaction products detected by HPLC conditions shown inFigure 1 were increased with UV-A irradiation time-dependently.
The amount of reaction products reached a maximum with 6 minof UV-A irradiation. Then, astaxanthin acetate and oxidation products were markedly decreased and the reaction solution wasbleached. Figure 1A shows the HPLC profiles of the reaction
product of astaxanthin acetate with hydroxyl radical with 6 minof UV-A irradiation. Three reaction products (Peaks 1–3) weredetected on HPLC. They were assigned to astaxanthin acetate triepoxide,10 astaxanthin acetate 5,8-epoxide (or 7,8-epoxide),11and astaxanthin acetate 5,6-epoxide12 based on the MS, MS/MS,and UV–Vis spectral data. ESI-TOF MS of Peak 312 showed molecular formula of C44H56O7. This formula clearly indicated that one
oxygen atom was attached to astaxanthin acetate. Its UV–Vis
showed an absorption maximum at 452 nm, consisted with theloss of two conjugated double bonds from 13 conjugated double
bonds of astaxanthin acetate, which shows absorption maximum
at 478 nm.13 The MS/MS spectrum of sodium adduct ions [M+Na]+of this compound showed m/z 627 [M+Na-92]+ (elimination oftoluene moiety from polyene chain) and m/z 613 [M+Na-106]+(elimination of xylene moiety from polyene chain), which werediagnostic fragments in EI and ESI MS of carotenoids,14,15 alongwith m/z 659 [M+Na-AcOH]+ and m/z 599 [M+Na-2AcOH]+. These
spectral data indicated that an oxygen atom was attached to the5,6-double bond and formed an epoxide ring in astaxanthin

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A. Nishino et al. / Tetrahedron Letters 57 (2016) 1967–1970

Figure 1. (A) HPLC of reaction products of astaxanthin acetate with OH for 6 min. Peak 1: astaxanthin acetate tri-epoxide, Peak 2: astaxanthin acetate 5,8-epoxide (or 7,8epoxide), Peak 3: astaxanthin acetate 5,6-epoxide, Peak 4: astaxanthin acetate, Peaks 5 and 6: astaxanthin acetate cis isomers. (B) HPLC of reaction products of astaxanthin
acetate with 1O2 for 6 min. Peak 1: hematoporphyrin, Peak 2: astaxanthin acetate 5,6-endoperoxide, Peak 3: astaxanthin acetate 5,8-endoperoxide, Peak 4: astaxanthin
acetate, Peaks 5 and 6: astaxanthin acetate cis isomers. HPLC condition ACQUITY UPLC system (Waters), column:BEH Shield RP18 (1.7 lm, 2.1  100 mm), mobile phase:
MeOH, column temperature: 40 °C, flow rate: 0.2 ml/min, detection: 450 nm.

acetate. Peak 211 has the same molecular formula (C44H56O7) asthat of astaxanthin acetate-5,6-epoxide. Its UV–Vis showed anabsorption maximum at 430 nm. The hypsochromic shift of theUV–Vis spectrum of 22 nm from astaxanthin acetate 5,6-epoxide
indicated that this compound lacked one conjugated double bond(double bond C7 to C8) from astaxanthin acetate 5,6-epoxide.13Therefore, the structure of astaxanthin acetate 5,8-epoxide or7,8-epoxide was postulated for this compound. ESI-TOF MS of Peak
110 showed the molecular formula of C44H56O9. Its UV–Vis showed
an absorption maximum at 400–424 nm. These data were compatible with the structure of astaxanthin acetate tri-epoxide.
However, substituted positions of oxygen atoms could not be specified from these spectral data. Astaxanthin acetate 5,6-epoxide,5,8-epoxide (or 7,8-epoxide), and tri-epoxide were also formed asmajor reaction products of astaxanthin acetate with O2 .
In the case of the reaction of astaxanthin acetate with 1O2, astaxanthin acetate 5,6-endoperoxide16 was obtained as major reaction
product (Fig. 1B). This compound showed the molecular formulaof C44H56O8, which indicated that two oxygen atoms were attached
to astaxanthin acetate. This compound showed the same UV–Visspectrum as that of astaxanthin acetate 5,6-epoxide. These spectral
data indicated that the dioxetane moiety was attached to C-5 andC-6 positions in astaxanthin acetate. MS/MS spectral data also support this structure. Furthermore, astaxanthin acetate-5,8-endoperoxide17 was also detected. This compound showed the same MS
spectral data as those of astaxanthin acetate-5,6-endoperoxide
except for the hypsochromic shift of the UV–Vis spectrum of
22 nm from astaxanthin acetate 5,6-endoperoxide. These data arein agreement with the structure of astaxanthin acetate-5,8endoperoxide for this compound. The structure of astaxanthin
acetate-7,8-endoperoxide was also considered for this compound.
Similar results as those for astaxanthin acetate were obtained in
the case of free-form astaxanthin with these reactive oxygen species. Namely, astaxanthin epoxides were obtained by the reaction

1619

17

116185RO 318OO

18'

20
15

3'

8'
15'

1'
6'

11'

Astaxanthin R=H 20'
Astaxanthin acetate R=Ac

OR

5'

19'

17'

16'

O
ORO
RO
Astaxanthin 5,6-epoxide R=H
Astaxanthin acetate 5,6-epoxide R=AcO

O
ORO

ROO

RO
O

Astaxanthin 5,8-epoxide R=H
Astaxanthin acetate 5,8-epoxide R=Ac

OO Astaxanthin 5,6-endoperoxide R=H
Astaxanthin acetate 5,6-endoperoxide R=AcO
OROORROOOO Astaxanthin 5,8-endoperoxide R=H
Astaxanthin acetate 5,8-endoperoxide R=Ac

Figure 2. Structures of astaxanthin and its acetate and their major reaction
products with reactive oxygen species.

of OH and O2 and astaxanthin endoperoxide were produced by
the reaction with 1O2. It was found that oxidation products ofastaxanthin acetate were more stable than those of free astaxanthin. It was assumed that acetylation might protect the oxidationof hydroxy groups of astaxanthin from reactive oxygen speciesand inhibit the rapid degradation of the astaxanthin molecule.

1969A. Nishino et al. / Tetrahedron Letters 57 (2016) 1967–1970

Mn2+ (3rd)

Mn2+ (4th)8.69 mT0-2.5
min0-2.5min

aHγ=0.15 mT
aHβ=1.06 mT
aN=1.46 mT
gDMPO-OOAsx=2.00
aHβ=1.57 mT
aN=1.42 mT
gDMPO-OAsx=2.00

aHγ=0.12 mT
aHβ=1.16 mT
aN=1.40 mT
gDMPO-OOH=2.00
aHβ=1.48 mT
aN=1.48 mT
gDMPO-OH=2.00

5.0-7.5min

aHγ=0.15 mT
aHβ=1.06 mT
aN=1.46 mT
gDMPO-OOAsx=2.00
aHβ=1.57 mT
aN=1.42 mT
gDMPO-OAsx=2.00

12.5-15.0min

aHγ=0.15 mT
aHβ=1.06 mT
aN=1.46 mT
gDMPO-OOAsx=2.00
aHβ=1.57 mT
aN=1.42 mT
gDMPO-OAsx=2.00

aHγ=0.12 mT
aHβ=1.16 mT
aN=1.40 mT
gDMPO-OOH=2.00
aHβ=1.48 mT
aN=1.48 mT
gDMPO-OH=2.00

2.5-5.0minFigure 3. ESR spectra due to 8 lg/mL of astxanthin acetate in acetonitrile (100 lL), 0.025 mM of riboflavin solution (100 lL), and 250 mM of 5,5-dimethyl-1-pyrroline-Noxide (DMPO) (10 lL). After 2.5 min of UV-A irradiation (upper), after 7.5 min of UV-A irradiation (middle), after 15 min of UV-A irradiation (lower). ESR spectra due to boththe DMPO-OOAsx (astaxanthin acetate-OO) and DMPO-OAsx (astaxanthin acetate-O) were observed. Initially, ESR signal of DMPO-OOAsx was dominant, and timedependently ESR signal of DMPO-OAsx was increased. On the other hand, only ESR spectra due to both the DMPO-OOH and DMPO-OH were observed without astxanthinacetate, indicating the formation of O2 followed by OH in the control RF/UV-A system. Left column: control (riboflavin+DMPO), Right column: riboflavin+astaxanthin
acetate+DMPO.

.

O2-.

OO -RO

RO+OO
1O2.

OO
O(H3O)

O..

OOH

ROOO.

ROSHi.
OH

RO

H+O

ROOO.

O

OH

RO
O.

+ OH.

+ OHO

RO
O

SHi+ H2O

ROO

Figure 4. Reaction schemes of astaxanthin and its acetate with reactive oxygen species. R = H astaxanthin, R = Ac astaxanthin acetate. SHi; intra-molecular homolytic
substitution reaction.

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A. Nishino et al. / Tetrahedron Letters 57 (2016) 1967–1970

Structures of astaxanthin and its acetate and their major reaction
products with reactive oxygen species are shown in Figure 2.
In order to reveal the reaction mechanism of astaxanthin and its
acetate with these reactive oxygen species, these reactions weremonitored with the ESR spin-trapping method.9 Astaxanthin acetate peroxide radical (AsxOO) and astaxanthin acetate oxide radical(AsxO) were detected during the reaction of astaxanthin acetatewith O2 by the ESR spin-trapping method, as shown in Figure 3.
Namely, a DMPO-OOAsx signal was observed in ESR for the0–2.5 min reaction of astaxanthin acetate with O2 . The intensityof the DMPO-OOAsx signal reached it maximum with a 5 min reaction. Subsequently, the DMPO-OOAsx signal was decreased andDMPO-OAsx signal was observed for the 12.5–15.0 min reaction,
as shown in Figure 3. These results indicate that astaxanthin acetate takes up O2 together with the formation of corresponding
peroxy radical. Subsequently, astaxanthin acetate alkoxy radical(AsxO) was formed with the production of OH from peroxy radical. Eventually, epoxide was formed from alkoxy radical, as shownin Figure 4. The ESR spectrum showed that astaxanthin (acetate)scavenged OH directly. Although signals of alkoxy and/or peroxy
radicals were not observed in ESR spin-trapping during the reaction of astaxanthin (acetate) with OH, the formation of epoxideby this reaction could be considered as shown in Figure 4. Namely,an OH radical was attached at C-5 of astaxanthin (acetate). Then,an epoxide ring was formed by the intra-molecular homolytic
substitution reaction.
It was reported that carotenoids inhibit 1O2 through a physicalquench18 and chemical reaction.19 For example, b-carotene reactedwith 1O2 and formed b-carotene-endoperoxide and apo-b-carotenals.19 In the present study, we found that astaxanthin and its acetate take up 1O2 by the formation of endoperoxide. On the otherhand, astaxanthin and its acetate take up O2 by the formation ofepoxide through astaxanthin peroxide radical as shown in Figure 4.
Because of their unstability, there are few reports on astaxanthin
endoperoxides and astaxanthin epoxides. Recently, Weesepoel
et al., reported that epoxy apo-9- and epoxy-apo-13-palmitoyl
astaxanthinone were the formed from palmitoyl astaxanthin withreaction of hypochlorous acid/hypochlorite (HOCl/OCl ).20 In thepresent study, we could detect astaxanthin endoperoxides, astaxanthin epoxides, and astaxanthin peroxy and alkoxy radicals asreaction products of reactive oxygen species by LC/PDA ESI MS
and the ESR method. This is the first Letter on the detection ofastaxanthin (its acetate) endoperoxides, epoxides, and peroxy
and alkoxy radicals.

Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.tetlet.2016.03.
078.
References and notes
1. Carotenoids Volume 3: Biosynthesis and Metabolism; Britton, G., Liaaen-Jensen, S.,
Pfander, H., Eds.; Birkhäuser: Basel, 1998.
2. Miki, W. Pure Apple Chem. 1991, 63, 141–146.
3. Shimizu, N.; Goto, M.; Miki, W. Fisheries Sci. 1996, 62, 134–137.
4. Maoka, T.; Etho, H. Some Biological Functions of Carotenoids in Japanese Food.
In Functional Foods of the East; Shi, J., Ho, C-T., Shahidi, F., Eds.; CRC press: BocaRaton, 2010; pp 85–97.

5. Hama, S.; Takahashi, K.; Inai, Y.; Shirota, K.; Sakamato, R.; Yamada, A.;
Tsuchidya, H.; Kanamura, K.; Yamashita, E.; Kogure, K. J. Pharm. Sci. 2012, 101.
2909-2016.
6. Yoshioka, R.; Hayakawa, T.; Ishizuka, K.; Kulkarni, A.; Terada, Y.; Maoka, T.;
Etoh, H. Tetrahedron Lett. 2006, 47, 3637–3640.
7. Hayakawa, T.; Kulkarni, A.; Terada, Y.; Maoka, T.; Etoh, H. Biosci. Biotechnol.
Biochem. 2008, 72, 2716–2722.
8. After the addition of 200 lL of 8 mM H2O2 solution to 200 lL of 70 lg/mL ofastaxanthin or astaxanthin-diacetate CH3CN solution, OH was generated byUV-A irradiation using SUPERCURE-203S (SAN-Ei ELECTORIC) at roomtemperature. In a similar manner to that described above, O2 and 1O2 weregenerated by the addition of 200 lL of 0.025 mM of riboflavin and 200 lL of0.25 mM hematoporphyrin, respectively, to 200 lL of 70 lg/mL of astaxanthinor astaxanthin-diacetate CH3CN solution, with UV-A irradiation at roomtemperature. At regular intervals of UV-A irradiation, reaction products wereanalyzed by LC/PDA-ESI-MS. The electro-spray ionization (ESI) time-of-flight(TOF) MS spectra were acquired by scanning from m/z 100 to 1500 with acapillary voltage of 3.2 kV, cone voltage of 40 eV, and source temperature of120 °C. Nitrogen was used as a nebulizing gas at a flow rate of 30 L/h. MS/MS
spectra were measured with a quadrupole-TOF MS/MS instrument with argon
as a collision gas at a collision energy of 30 V. UV–Visible (UV–Vis) absorption
spectra were recorded from 200 to 600 nm using a photodiode-array detector(PDA). An Acquity 1.7 lm BEH UPLC C18 column (Waters Corporation) wasused as a stationary phase and 90% MeOH as a mobile phase, at a flow rate of
0.2 mL/min for the HPLC system.
9. ESR spectra were recorded at room temperature on a JEOL JES-RFR30
spectrometer (Tokyo, Japan) using an aqueous quartz flat cell (Labotec,Tokyo, Japan). 5,5-Dimethoxy-1-pyrroline N-oxide (DMPO) was used ashydroxyl radical and superoxide anion radicals-trapping agent and 2,2,6,6tetramethyl-4-piperidone (TMPD) was used as a singlet oxygen-trapping agent.
Hydroxyl radical or O2 were generated by addition of both the 100 lL of 8 mM
H2O2 or 100 lL of 0.025 mM of riboflavin solution and 10 lL of 250 mM DMPO
to 100 lL of 8.8 lg/mL astaxanthin or 7 lg/mL astaxanthin acetate CH3CNsolution. In a similar manner to that described above, 1O2 was generated by theaddition of both the 100 lL of 0.25 mM hematoporphyrin and 10 lL of 500 mM
TMPD to 100 lL of 8.8 lg/mL astaxanthin or 7 lg/mL astaxanthin acetate
CH3CN solution. ESR spectrum measurements started after UVA irradiation. All
spin-trapped ESR spectra were monitored between the third and fourth signals
from the low magnetic field due to the external standard, Mn(II) doped MnO.
10. Astaxanthin acetate tri-epoxide, ESI TOF MS m/z 751.3843 (M+Na+, calcd for
C44H56O9Na, 751.3822), m/z 729.4012 (M+H+, calcd for C44H57O9, 729.4003);
Product ions of MS/MS (precursor ion M+Na+) m/z 691 (M+Na-AcOH), 659 M+Na-toluene), 645 (M+Na-xylene), 631 (M+Na-2AcOH); UV–Vis 400–424 nm(in 90% MeOH).
11. Astaxanthin acetate 5,8-epoxide (or 7,8-epoxide), ESI TOF MS m/z 719.3909(M+Na+, calcd for C44H56O7Na, 719.3924), m/z 697.4113 (M+H+, calcd for
C44H57O7, 697.4104); Product ions of MS/MS (precursor ion M+Na+) m/z 659(M+Na-AcOH), 627 M+Na-toluene), 613 (M+Na-xylene), 599 (M+Na-2AcOH);
UV–Vis 430 nm (in 90% MeOH).
12. Astaxanthin acetate 5,6-epoxide, ESI TOF MS m/z 719.3909 (M+Na+, calcd for
C44H56O7Na, 719.3924), m/z 697.4113 (M+H+, calcd for C44H57O7, 697.4104);
Product ions of MS/MS (precursor ion M+Na+) m/z 659 (M+Na-AcOH), 627 M+Na-toluene), 613 (M+Na-xylene), 599 (M+Na-2AcOH); UV–Vis 452 nm (in90% MeOH).
13. Britton, G. UV/Visible Spectroscopy In Carotenoid Hand Bock; Britton, G., LiaaenJensen, S., Pfander, H., Eds.; Birkhäuser: Basel, 1995; Vol. 1B, pp 13–62.
14. Frassantio, R.; Cantonati, M.; Flaim, G.; Manchini, I.; Guella, G., G RapidCommun. Mass Spectrom. 2008, 22, 3531–3539.
15. Weesepoel, Y.; Vincken, J.-P.; Pop, P. M.; Liu, K.; Gruppen, H. J. Mass. Spectrum.
2013, 48, 862–874.
16. Astaxanthin acetate 5,6 endperoxide, ESI TOF MS m/z 735.3866 (M+Na+, calcd
for C44H56O8Na, 735.3873), m/z 713.4024 (M+H+, calcd for C44H57O8,713.4053); Product ions of MS/MS (precursor ion M+Na+) m/z 675 (M+NaAcOH), 643 M+Na-toluene), 629 (M+Na-xylene), 615 (M+Na-2AcOH); UV–Vis
452 nm (in 90% MeOH).
17. Astaxanthin acetate 5,8 endperoxide, ESI TOF MS m/z 735.3866 (M+Na+, calcd
for C44H56O8Na, 735.3873), m/z 713.4024 (M+H+, calcd for C44H57O8,713.4053); Product ions of MS/MS (precursor ion M+Na+) m/z 675 (M+NaAcOH), 643 M+Na-toluene), 629 (M+Na-xylene), 615 (M+Na-2AcOH); UV–Vis
430 nm (in 90% MeOH).
18. Foot, C. S.; Denny, R. W. J. Am. Chem. Soc. 1968, 90, 6233–6235.
19. Yamauchi, R.; Tsuchihashi, K.; Kato, K. Biosci. Biotech. Biochem. 1998, 62, 1301–1306; Fieder, J.; Fieder, L.; Winkler, J.; Scherz, A.; Schert, H. Photochem.
Photobiol. 2001, 74, 64–71; Fieder, J.; Fieder, L.; Kammbinber, N.; Scherz, A.;
Schert, H. Photochem. Photobiol. 2002, 76, 145–152; Fieder, J.; Fieder, L.;
Haebner, R.; Schert, H. Biochim. Biophys. Acta 2005, 1709, 1–4.
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2014, 62, 10254–10263.