9 Anti-oxidative, anti-tumor-promoting, and anti-carcinogensis activities of nitroastaxanthin and nitrolutein, the reaction products of astaxanthin and lutein with peroxynitrite

Article abstract of DOI:10.3390/md10061391

Astaxanthin captured peroxynitrite to form nitroastaxanthins. 15-Nitroastaxanthin was a major reaction product of astaxanthin with peroxynitrite. Here, the anti-oxidative, anti-tumor-promoting, and anti-carcinogensis activities of 15-nitroastaxanthin were investigated. In addition to astaxanthin, 15-nitroastaxanthin showed excellent singlet oxygen quenching activity. Furthermore, 15-nitroastaxanthin showed inhibitory effects of in vitro Epstein-Barr virus early antigen activation and two-stage carcinogensis on mouse skin papillomas. These activities were slightly higher than those of astaxanthin. Similar results were obtained for the 15-nitrolutein, a major reaction product of lutein with peroxynitrite.

Full text of DOI:10.3390/md10061391

Mar. Drugs 2012, 10, 1391-1399; doi:10.3390/md10061391
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Anti-Oxidative, Anti-Tumor-Promoting, and Anti-Carcinogensis
Activities of Nitroastaxanthin and Nitrolutein, the Reaction
Products of Astaxanthin and Lutein with Peroxynitrite
Takashi Maoka 1,*, Harukuni Tokuda 2, Nobutaka Suzuki 2, Hideaki Kato 3 and Hideo Etoh 3
1
Research Institute for Production Development, 15 Shimogamo-morimoto-cho, Sakyo-ku,
Kyoto 606-0805, Japan
2
Department of Complementary and Alternative Medicine, Clinical R&D,
Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi,
Kanazawa 920-8640, Japan; E-Mails: htokuda@med.kanazawa-u.ac.jp (H.T.);
pcam@med.kanazawa-u.ac.jp (N.S.)
3
Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan;
E-Mails: hideakick7@hotmail.com (H.K.); acheto@ipc.shizuoka.ac.jp (H.E.)
* Author to whom correspondence should be addressed; E-Mail: maoka@mbox.kyoto-inet.or.jp;
Tel.: +81-75-781-1107; Fax: +81-75-791-7653.
Received: 29 March 2012; in revised form: 7 June 2012 / Accepted: 13 June 2012 /
Published: 18 June 2012
Abstract: Astaxanthin captured peroxynitrite to form nitroastaxanthins. 15-Nitroastaxanthin
was a major reaction product of astaxanthin with peroxynitrite. Here, the anti-oxidative,
anti-tumor-promoting, and anti-carcinogensis activities of 15-nitroastaxanthin were
investigated. In addition to astaxanthin, 15-nitroastaxanthin showed excellent singlet oxygen
quenching activity. Furthermore, 15-nitroastaxanthin showed inhibitory effects of in vitro
Epstein-Barr virus early antigen activation and two-stage carcinogensis on mouse skin
papillomas. These activities were slightly higher than those of astaxanthin. Similar results
were obtained for the 15-nitrolutein, a major reaction product of lutein with peroxynitrite.
Keywords: carotenoid; 15-nitroastaxanthin; 15-nitrolutein; anti-carcinogensis activity;
anti-oxidative activity

Mar. Drugs 2012, 10
1392
1. Introduction
Carotenoids have an excellent capacity to quench singlet oxygen (1O2) and inhibit lipid
−
peroxidation. However, they cannot scavenge superoxide anion radicals (O2 ), hydroxy radicals (OH·),
or hydrogen peroxide (H2O2). On the other hand, there have been few reports on scavenging and/or the
reaction of reactive nitrogen species with carotenoids [1]. Peroxynitrite (ONOO−), a reactive nitrogen
−
species, is formed from superoxide (O2 ) and nitric oxide (NO·) in vivo, and is a highly reactive
oxidant that causes nitration of the aromatic ring of free tyrosine and protein tyrosine residues [2].
Furthermore, peroxynitrite was found to induce various forms of oxidative damage such as low-density
lipoprotein (LDL) oxidation, lipid peroxidation, and DNA strand breakage [2].
Astaxanthin is a marine carotenoid, which is distributed in marine bacteria, algae, crustaceans, and
fish. Astaxanthin is an excellent antioxidant and shows biological functions including anti-carcinogensis,
anti-inflammatory, anti-diabetic, and anti-obesity activities [1]. Astaxanthin also inhibits reactive
nitrogen species-induced inflammation [3]. Previously, we studied the reaction of astaxanthin with
peroxynitrite in vitro and reported the formation of nitroastaxanthins by the reaction of astaxanthin
with peroxynitrite [4,5]. This result suggested that astaxanthin could scavenge peroxynitrite to uptake
peroxynitrite into its molecule by the formation of nitroastaxanthins. Lutein, a major carotenoid in
green algae and higher plants, also showed radical scavenging, anti-carcinogenic, anti-diabetic, and
anti-obesitic activities [1]. Lutein also formed nitroluteins and lutein-oxazine by the reaction with
peroxynitrite in vitro [6]. Therefore, we assumed that astaxanthin and lutein could inhibit tyrosine
1
nitration induced by peroxynitrite and that nitroastaxanthin and nitrolutein themselves may have O2
quenching and anti-carcinogensis activities.
In this study, we investigated the inhibitory effect of tyrosine nitration with peroxynitrite by
1
astaxanthin and lutein. Furthermore, O2 quenching, anti-tumor-promoting, and anti-carcinogensis
activities of 15-nitroastaxanthin and 15-nitrolutein (Figure 1), the major reaction products of
astaxanthin and lutein with peroxynitrite, were studied.
Figure 1. Structures of carotenoids and nitrocarotenoids.
O
OH
OH
HO
HO
O
Astaxanthin
Lutein
NO2
NO2
HO
HO
O
O
OH
15-Nitroastaxanthin
OH
15-Nitrolutein

Mar. Drugs 2012, 10
1393
2. Results and Discussion
2.1. Inhibition of Tyrosine Nitration with Peroxynitrite by Astaxanthin and Lutein
Peroxynitrite induces nitration of the aromatic ring of free tyrosine and protein tyrosine residues
and causes various diseases [2].
Astaxanthin suppressed the formation of nitrotyrosine to about 10.0% of that of the control group.
This effect was almost the same as that of γ-tocopherol (9.2%). A similar result was also obtained in
the case of lutein (12.5%) These results indicate that astaxanthin and lutein are able to capture
peroxynitrite to form nitroastaxanthin and nitrolutein and inhibit the tyrosine nitration.
It was reported that phenolic compounds such as p-coumaric acid, pelargonidin [7–9], and
tocopherols [10] could scavenge peroxynitrite by the formation of nitro-p-coumaric acid,
nitro-pelargonidin, and nitro-tocopherols, respectively, and protect against tyrosine nitration.
Capsanthin and fucoxanthin also inhibited the nitration of tyrosine to form nitrocarotenoids [11]. These
results indicated that carotenoids, with long polyene chains, could inhibit the tyrosine nitration to
form nitrocarotenoids.
Mechanism of nitration of carotenoid with proxynitrite is uncertain. However, we assumed that this
reaction might be a radical addition of nitroradical (NO2) with carotenoid.
It was reported that NO2 was formed from proxynitrite as following mechanism [12].
ONOO− + ONOOH→ONOO− + ·NO2 + OH−
Therefore, we assumed that ·NO2, generated from proxynitrite, took out hydrogen from carotenoid
to form carotenoid radical. Then, NO2 reacted with carotenoid radical to form nitrocarotenoid.
Carotenoids might have higher reactivity with proxynitrite and/or NO2 than phenol compounds such as
tyrosine. Therefore, carotenoid could inhibit nitration of tyrosine.
2.2. Quenching Effects of 1O2 by 15-Nitroastaxanthin and 15-Nitrolutein
1O2 quenching activity of nitrocarotenoids was examined by the methylene blue-sensitized
1
photooxidation method [13–15]. The IC50 values of O2 quenching activity were as follows;
astaxanthin 7.0 μM, 15-nitroastaxanthin 20.0 μM (Table 1), and β-carotene 100 μM [15]. As well as
1
astaxanthin, 15-nitroastaxanthin also showed excellent O2 quenching activity. This activity was
slightly weaker than that of astaxanthin and higher than that of β-carotene. It is reported that
carotenoids with more than eleven conjugated double bonds show excellent quenching activity for 1O2
and that the 1O2 quenching activity of carotenoids depends on the number of conjugated double bonds,
polyene chain structures and functional groups [13–17]. 15-Nitroastaxanthin has the same conjugated
double bonds system as that of astaxanthin. Therefore, 15-nitroastaxanthin exhibited strong 1O2 quenching
1
activity. 15-Nitrolutein also showed O2 quenching activity (IC50 value 71.5 μM). This activity was
slightly weaker than that of lutein (IC50 value 60.5 μM) and higher than that of β-carotene (100 μM).
These results indicated that not only carotenoids, but also nitro-carotenoids, could scavenge 1O2.
1
Concerning the O2 quenching activity of carotenoids [13–17], astaxanthin and 15-nitroastaxanthin
showed strong activity and lutein and 15-nitrolutein exhibited moderate activity among the carotenoids
1O2 quenching activity investigated.

Mar. Drugs 2012, 10
Table 1. 1O2 quenching activity (%) a of carotenoids and nitrocarotenoids.
1394
Concentration
100 (μM)
10 (μM)
1 (μM)
IC50 (μM)
Compounds
Astaxanthin
93.8
91.8
75.8
72.2
65.5
48.5
38.5
36.5
30.8
5.0
7.0
15-Nitroastaxanthin
Lutein
20.0
60.5
71.5
1.0
0.8
15-Nitrolutein
a Triplicate assays were performed for each compound in this assay system.
2.3. Anti-Tumor-Promoting and Anti-Carcinogensis Activities of 15-Nitroastaxanthin and 15-Nitrolutein
The in vitro anti-tumor-promoting activity of 15-nitroastaxanthin and 15-nitrolutein was examined
using an Epstein-Barr virus early antigen (EBV-EA) activation assay in Raji cells [18,19]. This assay
was used to estimate the anti-tumor-promoting effects of several natural carotenoids [18,19]. The
results are shown in Table 2. Both 15-nitroastaxanthin and 15-nitrolutein showed inhibitory effects on
the EBV-EA induction of Raji cells without significant cytotoxicity (more than 60% viability of Raji
cells) in this assay. 15-Nitroastaxanthin showed slightly higher activity than astaxanthin. Furthermore,
15-nitrolutein also showed slightly higher activity than lutein. These results showed that nitrocarotenoids
themselves have anti-tumor-promoting activity.
a
Table 2. Relative rate of Epstein-Barr virus early antigen (EBV-EA) activation with
respect to the positive control (100%) in the presence of carotenoids and nitrocarotenoids.
Concentration (mol ratio/TPA) b
Compounds
1000
500
100
values
78.0
10
IC50 (nmol)
Astaxanthin
5.0 (70) c
4.1 (60) c
2.9 (70) c
1.6 (60) c
29.0
28.5
24.5
23.3
100
100
307
300
283
277
15-Nitroastaxanthin
Lutein
76.9
75.4
97.6
95.4
15-Nitrolutein
74.1
a
Values represent the percentage relative to the positive control values (100%). Triplicate assays were
b
c
performed for each compound in this assay system; TPA concentration was 20 ng (32 pmol); Values in
parentheses are percentage viability of Raji cells; There were significant differences (P < 0.01) in the
carotenoids and nitrocarotenoids treatment groups compared to the control group.
Next, the inhibitory effects of astaxanthin and 15-nitroastaxanthin on two-stage mouse skin
carcinogenesis [19] were investigated. The average number of papillomas per mouse and the
incidence (%) of the mice bearing papillomas in the astaxanthin, 15-nitroastaxanthin, and control
groups are shown in Figure 2.
When astaxanthin and 15-nitroastaxanthin were applied before each 12-O-tetradecanoylphorbol-13-
acetate (TPA) treatment, they markedly delayed the formation of papillomas and reduced the average
numbers of papillomas per mouse compared with the control group. Among them, 15-nitroastaxanthin
showed slightly higher inhibitory activity than astaxanthin, as shown in Figure 2.

Mar. Drugs 2012, 10
Figure 2. Inhibitory effects of astaxanthin and 15-nitroastaxanthin on 7,12-dimethylbenz
1395
[a]anthracene (DMBA) induced mouse skin carcinogenesis. (A) Average number of
papillomas per mouse; (B) Percentage of mice bearing papillomas. ♦-♦ DMBA (390 nmol)
+ 12-O-tetradecanoylphorbol-13-acetate (TPA) (1.7 nmol); ■-■ Astaxanthin (85 nmol)
+ DMBA (390 nmol) + TPA (1.7 nmol);
(390 nmol) + TPA (1.7 nmol).
▲-▲ 15-Nitroastaxanthin (85 nmol) + DMBA
A
B

Mar. Drugs 2012, 10
1396
Similar results were obtained for lutein and 15-nitrolutein, as shown in Figure 3. 15-Nitrolutein
showed slightly higher inhibitory activity than lutein. These results indicated that nitrocarotenoids
themselves showed anti-carcinogenesis effect.
Figure 3. Inhibitory effects of lutein and 15-nitrolutein on DMBA induced mouse skin
carcinogenesis. (A) Average number of papillomas per mouse; (B) Percentage of mice
bearing papillomas. ♦-♦ DMBA (390 nmol) + TPA (1.7 nmol); ■-■ Lutein (85 nmol)
+ DMBA (390 nmol) + TPA (1.7 nmol);
(390 nmol) + TPA (1.7 nmol).
▲-▲ 15-Nitrolutein (85 nmol) + DMBA
A
B

Mar. Drugs 2012, 10
1397
Previously, we reported that capsanthin and fucoxanthin could take up peroxynitrite through the
formation of nitrocapsanthins and nitrofucoxanthins, respectively, and inhibit tyrosine nitration by
peroxynitrite. Nitrocapsanthins and nitrofucoxanthins also exhibited anti-tumor-promoting activity. In
the present study, similar results were obtained for astaxanthin and lutein [11]. Therefore, it was
assumed that carotenoids could inhibit peroxynitrite-induced damages such as, oxidation of lipids and
protein, DNA stanrard scission, and induction of carcinogenesis.
3. Experimental Section
3.1. Preparation of 15-Nitroastaxanthin and 15-Nitrolutein
15-Nitroastaxanthin and 15-nitrolutein were prepared by reaction of astaxanthin and lutein with
peroxynitrite according to the method previously reported [4,6].
3.2. Inhibition of Tyrosine Nitration with Peroxynitrite by Carotenoids
Astaxanthin or lutein (0.2 mg) was dissolved in 1 mL of methanol (MeOH).
L-Tyrosine (0.36 mg)
was added to this solution. In the control group, -tyrosine (0.36 mg) was dissolved in 1 mL of MeOH.
L
Then, 20 μL of peroxynitrite (final concentration: 6.8 mM) was added to the solution. The same
procedure was carried out for γ-tocopherol (0.15 mg), which was used as a positive control for this
assay. The solution was subsequently allowed to react for 5 min at room temperature. After that, the
nitrotyrosine content, a product of the reaction between tyrosine and peroxynitrite, was quantified
using HPLC. The following HPLC systems were employed for the quantification of nitrotyrosine.
Column: Develosil ODS-UG-5 (20.0 × 250 i.d. mm; mobile phase: phosphate-buffer (50 mM)/MeOH
(97:3 v/v), flow rate: 1 mL/min, column temp: 40 °C, detection: 250 nm. Inhibition of the nitrotyrosine
generated was measured by comparing with the absorbance of the sample solution (in MeOH).
3.3. Measurement of 1O2 Quenching Activity
1O2 quenching activity was examined by measuring methylene blue-sensitized photooxidation of
linoleic acid [15]. Forty microliters of 0.05 mM methylene blue, 10 μL of 2.4 M linoleic acid with or
without 40 μL carotenoid (final concentration 1–100 μM, each dissolved in EtOH) were added to
micro glass vials (5.0 mL). The vials were tightly closed with a screw cap and a septum, and the
mixtures were illuminated at 7000 lux at 22 °C for 3 h in corrugated cardboard. Then, 50 μL of the
reaction mixture was removed and diluted to 1.5 mL with EtOH, and absorbance at 235 nm was
measured to estimate the formation of conjugates dienes. The value in the absence of carotenoid was
1
determined and O2 repression activity was calculated relative to this reference value. Activity is
indicated as the IC50 value representing the concentration at which 50% inhibition was observed [15].
3.4. In Vitro Epstein-Barr Virus Early Antigen (EBV-EA) Activation Induction Effect
EBV genome-carrying lymphoblastoid cells (Raji cells) derived from Burkitt’s lymphoma were
cultivated in RPMI-1640 medium with 10% fetal bovine serum (FBS). The Raji cells were incubated
for 48 h at 37 °C in a medium containing n-butyric acid (4 nmol), TPA (32 pmol), and various

Mar. Drugs 2012, 10
1398
amounts of test compounds. Smears were made from the cell suspension, and we employed an indirect
immunofluorescence technique. Details of the in vitro assay for EBV-EA induction have been reported
previously [18].
3.5. In Vivo Two-Stage Carcinogensis Assay on Mouse Skin Initiated by DMBA and Promoted by TPA
The animals (specific pathogen-free female ICR-6-week old mice) were divided into three
experimental groups, each with 10 mice. The backs of the mice were shaved with surgical clippers, and
they were treated topically with 7,12-dimethylbenz[a]anthracene (DMBA) (100 μg, 390 nmol) in
acetone (0.1 mL) as the initiator. One week after initiation, papilloma formation was promoted twice a
week by the application of TPA (1 μg, 1.7 nmol) in acetone (0.1 mL) to the skin. The control group
received TPA treatment alone. Experimental groups received topical application of carotenoids
(85 nmol) in acetone (0.1 mL), 1 h before the TPA treatment. The incidence and numbers of
papillomas were monitored weekly for 20 weeks [11,19].
Experiments involving mice were conducted in accordance with Kanazawa University, Institute for
Experimental Animals and use Committee Guidelines, under the jurisdiction of the Ministry of
Education, Culture, Sports, Science and Technology.
4. Conclusions
Astaxanthin and lutein could take up peroxynitrite through the formation of nitroastaxanthin and
nitrolutein, respectively, and inhibit tyrosine nitration by peroxynitrite. Furthermore, 15-nitroastaxanthin
1
and 15-nitrolutein exhibited O2 quenching, anti-tumor-promoting, anti-carcinogenesis activities.
Therefore, astxanthin and lutein may have the potential to reduce the risk of disease induced by
reactive nitrogen species and prevent active nitrogen-induced disease.
Acknowledgements
We wish to thank S. Shindo and A. Osawa, Department of Food and Nutrition, Nihon Women’s
University for measurements of 1O2 quenching activity.
References
1. Maoka, T.; Etoh, 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: Boca Raton, FL, USA, 2010;
pp. 85–97.
2. Halliwell, B. Free radicals, antioxidants, and human disease: Curiosity, cause or consequence?
Lancet 1994, 344, 721–724.
3. Lee, S.-J.; Bai, S.-K.; Lee, K.-S.; Namkoong, S.; Na, H.-J.; Ha, K.-S.; Han, J.-A.; Yim, S.-V.;
Chang, K.; Kwon, Y.-G.; et al. Astaxanthin inhibits nitric oxide production and inflammatory gene
expression by suppressing IκB kinase-dependent NF-κB activation. Mol. Cells 2003, 16, 97–105.
4. Yoshioka, R.; Hayakawa, T.; Ishizuka, K.; Kulkarni, A.; Terada, Y.; Maoka, T.; Etoh, H.
Nitration reaction of astaxanthin and β-carotene by peroxynitrite. Tetrahedron Lett. 2006, 47,
3637–3640.

Mar. Drugs 2012, 10
1399
5. Hayakawa, T.; Kulkarni, A.; Terada, Y.; Maoka, T.; Etoh, H. Reaction of astaxanthin with
peroxynitrite. Biosci. Biotechnol. Biochem. 2008, 72, 2716–2722.
6. Tsuboi, M.; Etoh, H.; Yomoda, Y.; Kato, K.; Kato, H.; Kulkarni, A.; Terada, Y.; Maoka, T.; Mori, H.;
Inakuma, T. Nitration reaction of lutein with peroxynitrite. Tetrahedron Lett. 2010, 51, 676–678.
7. Niwa, T.; Doi, U.; Kato, Y.; Osawa, T. Antioxidative properties of phenolic antioxidants isolated
from corn steep liquor. J. Agric. Food Chem. 2001, 49, 177–182.
8. Kato, Y.; Ogino, Y.; Aoki, T.; Uchida, K.; Kawakishi, S.; Osawa, T. Phenolic antioxidants prevent
peroxynitrite-derived collagen modification in vitro. J. Agric. Food. Chem. 1997, 45, 3004–3009.
9. Tsuda, T.; Kato, Y.; Osawa, T. Mechanism for the peroxynitrite scavenging activity by
anthocyanins. FEBS Lett. 2000, 484, 207–210.
10. Goss, S.P.A.; Hogg, N.; Kalyanaraman, B. The effect of α-tocopherol on the nitration of
γ-tocopherol by peroxynitrite. Arch. Biochem. Biophys. 1999, 363, 333–340.
11. Tuboi, M.; Etoh, H.; Kato, K.; Nakatsugawa, H.; Kato, H.; Maejima, Y.; Natsumoto, G.; Mori, H.;
Hosokawa, M.; Miyashita, K.; et al. Nitrocapsanthin and nitrofucoxanthin, respective products of
capsanthin and fucoxanthin reaction with peroxynitrite. J. Agric. Food Chem. 2011, 59, 10572–10578.
12. Groves, J.T.; Marla, S.S. Peroxynitrite-induced DNA stanrard scission mediated by a magnanese
porphyrin. J. Am. Chem. Soc. 1995, 117, 9578–9579.
13. Hirayama, O.; Nakamura, K.; Hamada, S.; Kobayashi, Y. Singlet oxygen quenching ability of
naturally occurring carotenoids. Lipid 1994, 29, 149–150.
14. Kobayashi, M.; Sakamoto, Y. Singlet oxygen quenching ability of astaxanthin esters from the
green alga Haematococcus pluvialis. Biotechnol. Lett. 1999, 21, 265–269.
15. Osawa, A.; Ishii, Y.; Sasamura, N.; Morita, M.; Kasai, H.; Maoka, T.; Shindo, K. Characterization
and antioxidative activities of rare C50 carotenoids-sarcinaxanthin, sarcinaxanthin monoglucoside,
and sarcinaxanthin diglucoside-obtained from Micrococcus yunnanensis. J. Oleo Sci. 2010, 59,
653–659.
16. Miki, W. Biological functions and activities of animal carotenoids. Pure Appl. Chem. 1991, 63,
141–146.
17. Shimidzu, N.; Goto, M.; Miki, W. Carotenoids as singlet oxygen quenchers in marine organisms.
Fish. Sci. 1996, 62, 134–137.
18. Tsushima, M.; Maoka, T.; Katsuyama, M.; Kozuka, M.; Matsuno, T.; Tokuda, H.; Nishino, H.;
Iwashima, A. Inhibitory effect of natural carotenoids on Epstein-Barr virus activation activity of a
tumor promoter in Raji cells. A screening study for anti-tumor promoters. Biol. Pharm. Bull.
1995, 18, 227–233.
19. Maoka, T.; Mochida, K.; Kozuka, M.; Ito, Y.; Fujiwara, Y.; Hashimoto, K.; Enjo, F.; Ogata, M.;
Nobukuni, Y.; Tokuda, H.; et al. Cancer chemopreventive activity of carotenoids in the fruits of
red paprika Capsicum annuum L. Cancer Lett. 2001, 172, 103–109.
Samples Availability: Available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open-access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).