ARTICLE
pubs.acs.org/JAFC

Nitrocapsanthin and Nitrofucoxanthin, Respective Productsof Capsanthin and Fucoxanthin Reaction with Peroxynitrite
Makoto Tsuboi,† Hideo Etoh,†,‡ Kyuki Kato,‡ Hiroki Nakatugawa,‡ Hideki Kato,‡ Yasunori Maejima,†
Gaku Matsumoto,§ Hironobu Mori,§ Masashi Hosokawa,|| Kazuo Miyashita,|| Harukuni Tokuda,^
Nobukuni Suzuki,^ and Takashi Maoka*,#†

Graduate of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan§
Research Institute, Kagome Co. Ltd., 17 Nishitomiyama, Nasushiobara-shi, Tochigi 329-2762, Japan
Faculty of Fisheries Science, Hokkaido University, 3-1-1 Minato Hakodate, Hokkaido 041-8611, Japan^
Department of Complementary and Alternative Medicine, Clinical R&D, Graduate School of Medical Science, Kanazawa University,
13-1 Takara-machi, Kanazawa 920-8640, Japan#
Research Institute for Production and Development, Shimogamo-morimotocho, Sakyo-ku, Kyoto 606-0805, Japan)‡

ABSTRACT: The in vitro reactivity of capsanthin (1) and fucoxanthin (2) with peroxynitrite was investigated, and the reaction
products produced by scavenging with peroxynitrite were analyzed. (140 Z)-Nitrocapsanthin (3) and 12-nitrocapsanthin (4) wereisolated from the products of the reaction of capsanthin with peroxynitrite. Similarly, (14Z)-15-nitrofucoxanthin (5), (11Z)-11nitrofucoxanthin (6), and (14Z,90 Z)-15-nitrofucoxanthin (7) were obtained from the reaction of peroxynitrite reaction
with fucoxanthin. Capsanthin and fucoxanthin inhibited the nitration of tyrosine by peroxynitrite. Furthermore, nitrocapsanthins(3 and 4) and nitrofucoxanthins (5 and 6) exhibited an inhibitory eﬀect on EpsteinBarr virus early antigen activation in Rajicells and an antiproliferative eﬀect on human pancreatic carcinoma. Moreover, nitrocapsanthins (3 and 4) inhibited carcinogensis ofmouse skin tumors initiated by 7,12-dimethylbenz[a]anthracene (DMBN).
KEYWORDS: Capsanthin, fucoxanthin, nitrocapsanthin, nitrofucoxanthin, reaction with peroxynitrite’ INTRODUCTION
Carotenoids show an excellent capacity to quench singlet
oxygen (1O2) and inhibit lipid peroxidation. However, theycannot 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 thereaction of reactive nitrogen species with carotenoids.1 Peroxynitrite (ONOO), which is one of the reactive nitrogen species,
is formed from superoxide (O2) and nitric oxide (NO•) in vivo
and is a highly reactive oxidant that causes nitration of thearomatic ring of free tyrosine and protein tyrosine residues.
Furthermore, peroxynitrite was found to induce various forms ofoxidative damage such as low-density lipoprotein (LDL) oxidation, lipid peroxidation, and DNA strand breakage.2
It has been reported3 that β-carotene is an eﬀective scavengerof nitrogen dioxide (NO2), peroxynitrous acid (ONOOH), andperoxynitrite and that the nitrogen atoms derived from nitrogendioxide were tightly bound to the β-carotene molecule.3 Thissuggested that carotenoids scavenge reactive nitrogen species.
Thus, we have studied the reaction of carotenoids such as
β-carotene, astaxanthin, and lutein with peroxynitrite46 toclarify the mechanism whereby reactive nitrogen species arescavenged by carotenoids and reported the formation of nitrocarotenoids by this reaction.
Capsanthin (1), a major carotenoid in red paprika, has
excellent antioxidative79 and anticancer activities.1012 Fucoxanthin (2), a major carotenoid in brown algae, has also beenr 2011 American Chemical Society

noted to exhibit biological activities such as radical scavenging
and singlet oxygen quenching13 and anticarcinogenic,14,15
antidiabetic,16 and antiobesity17 potential. In this study, weinvestigated the products of the reaction of capsanthin (1) andfucoxanthin (2) with peroxynitrite (Figure 1). Furthermore, theinhibitory eﬀect of the nitration of tyrosine with capsanthin andfucoxanthin and the antitumor-promoting activity of nitrocapsanthins and nitrofucoxanthins were studied.

’ MATERIALS AND METHODS
General Experimental Procedures. UVvisible (UV/vis) spectra were recorded with a Hitachi U-2001 in diethyl ether (Et2O).
Positive ion fast atom bombardmentmass spectrometry (FAB-MS)
spectra were recorded using a JEOL JMS-700 110A mass spectrometerwith m-nitrobenzyl alcohol as a matrix. The 1H NMR (500 MHz) and13
C NMR (125 MHz) spectra were measured with a Varian UNITYINOVA 500 spectrometer in CDCl3 with TMS as an internal standard.
High-performance liquid chromatography (HPLC) was performed ona Shimadzu LC-6AD with a Shimadzu SPD-6AV spectrophotometer.
Reagents. Capsanthin (1) and fucoxanthin (2) were prepared from
paprika and brown algae, respectively, according to the method described
previously.9,11,13 Peroxynitrite, 12-O-tetradecanoylphorbol-13-acetate
Received: June 8, 2011
Accepted: September 7, 2011
Revised:
September 6, 2011
Published: September 07, 2011
10572

dx.doi.org/10.1021/jf203493k | J. Agric. Food Chem. 2011, 59, 10572–10578

Journal of Agricultural and Food Chemistry

ARTICLE

Figure 1. Structures of carotenoids.
(TPA), 7,12-dimethylbenz[a]anthracene (DMBA), and L-tyrosine werepurchased from Sigma Chemical Co. (St. Louis, MO).

Reaction of Capsanthin and Fucoxanthin with Peroxynitrite. According to the previous methods,46 capsanthin or fucoxanthin(400 mg) was dissolved in 50 mL of tetrahydrofuran (THF). To this,trifluoroacetic acid (TFA) was added to make up the final concentration to
0.2%, before the addition of 16 mL of peroxynitrite in NaOH solution (final
concentration, 6.8 mM). Then, the solution was allowed to react for 1 min.
To the above mixture, 300 mL of CHCl3 and 300 mL of H2O were added so
as to separate the reaction products into organic and aqueous phases. The
whole procedure was performed three times. The organic layer was driedover anhydrous sodium sulfate and concentrated. This organic concentrate
was then subjected to HPLC. For HPLC separation, a 250 mm  4.6 mm i.d.,
5 μm, Develosil C30-UG-5 column (Nomura Chemical Co., Ltd., Seto,
Japan) was used and kept at 40 C. The mobile phase used was a MeCN/
H2O (82:18 v/v), at a flow rate of 1 mL/min. A more specific separation
procedure was performed using a 250 mm  4.6 mm i.d., 5 μm, TSK gel
ODS 100Z column (Tosoh Corp., Tokyo, Japan), kept at 40 C with amobile phase of MeCN/H2O (75:25 v/v), at a flow rate of 1 mL/min.
(140 Z)-150 -Nitrocapsanthin (3). UV/vis λ max (Et2O) nm 359, 456.
HR FAB MS m/z 630.4159 [M + H+] (calcd for C40H56O5N,
630.4161). 1H NMR and 13C NMR (Table 1).
10573

dx.doi.org/10.1021/jf203493k |J. Agric. Food Chem. 2011, 59, 10572–10578

Journal of Agricultural and Food Chemistry

ARTICLE

Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data for (140 Z)-150 -Nitrocapsanthin (3) and 12-Nitrocapsanthin (4) and 1H(500 MHz) Data for (14Z)-15-Nitrofucoxanthin (5), (11Z)-11-Nitrofucoxanthin (6), and (14Z,90 Z)-15-Nitrofucoxanthin (7) in
CDCl3a
3

4

5

mult
no.

δ 13C

δ 1H

mult
δ 13C

δ 1H

(J in Hz)α 1.77overlapped

37.1

7

mult
δ 13C

mult

δ 1H

(J in Hz)δ 13C

α 1.50overlapped(J in Hz)δ 1H

(J in Hz)α 1.50overlapped

α 1.50overlappedoverlapped

37.1

2

48.3

β1.48

dd (12, 12)

β 1.48dd (12, 12)β ∼1.36

overlapped

β ∼1.36

overlapped

β ∼1.36

3

64.9

4.00

m

64.8

4.00

m

64.2

3.82

m

64.1

3.82

m

3.82

m

4

42.5

α 2.39 dd (17, 6)

42.6

α 2.39dd (17, 6)

41.6

α 2.35dd (15, 6)

41.5

α 2.35dd (15, 6)

α 2.35dd (15, 6)

β 2.04dd (17, 9)

β 1.79dd (15, 9)

β 1.79dd (15, 9)

β 1.79dd (15, 9)

5

126.9

6

137.5

7

126.9

α 1.77 overlapped

8

128.1

9

139.2

10

48.3

dd (17, 9)

35.8

mult

δ 1H

1

β 2.06127.4
d (16)

132.3
137.7

35.8

66.1

137.6
6.13

47.1

66.9
6.46

d (15)

41.0

47.2

66.1
66.7
2.61

d (18)

3.67

d (18)

6.24

d (16)

130.9

6.16

d (10)

11

130.7

12

136.0

13

149.9

14

125.4

5.96

d (12)

136.0

6.25

d (11)

123.0

15

130.3

8.09

d (12)

132.0

6.65

m

150.3

16

28.7

1.07

s

28.8

1.08

s

25.0

0.97

17
18

30.2
21.6

1.07
1.73

s
s

30.2
21.7

1.08
1.74

s
s

28.1
21.1

1.05
1.22

19

13.7

2.02

s

13.5

2.13

s

12.0

20

14.1

2.18

s

16.4

2.00

s

15.2

10

44.0

20

50.8

20

a

(J in Hz)

6

41.4

6.20

d (15)

124.0

6.15

d (11)

127.7

7.16

d (11)

128.3

6.95

dd (15, 11) 129.4

7.99

d (11)

126.4

6.74

dd (15, 11)

141.6

6.41

d (15)

152.4

144.7

6.82

d (15)

128.4

141.6

148.6

α 2.00 overlappedoverlapped

50.8

α 2.00

m
m

45.3

2.61

d (18)

3.67

d (18)

7.31

s

143.1

141.8

7.91

7.16

d (11)

6.74

dd (15, 11)

s

6.82

d (15)

6.49

s

129.7
6.49

s

146.1

6.80

d (11)

127.5

6.57

dd (14.11)

s

24.9

0.98

s

0.97

s

s
s

28.1
21.1

1.05
1.22

s
s

1.05
1.23

s
s

1.97

s

13.9

1.72

s

1.96

s

1.80

s

14.8

1.86

s

1.80

s

35.1
β 1.71d (18)d (18)

197.8

137.2

44.0
β 1.71

198.1

2.67
3.73

35.1
α 2.00overlapped

β 1.41dd (15, 15)45.4

α 2.00overlapped

α 1.99overlapped

β 1.41dd (15, 15)

β 1.40overlapped

30

70.3

4.52

m

70.3

4.51

m

67.8

5.38

m

67.9

5.38

m

5.38

m

40

45.2

α 2.96 dd (15, 8)
β 1.49 overlapped

45.3

α 2.96β 1.49moverlapped

45.2

α 2.29β 1.51dd (15, 6)
overlapped

45.2

α 2.29β 1.51dd (15, 6)
overlapped

α 2.29β 1.51dd (15, 6)
overlapped

50

59.0

59.0

72.6

72.7

60

203.0

202.9

117.8

117.6

70

122.0

6.51

d (15)

121.3

6.47

d (15)

203.0

80

146.4

7.33

d (15)

146.7

7.33

d (15)

103.2

6.06

s

6.59

s

90

140.1

100

130.9

6.57

d (11)

140.2

6.53

d (11)

127.7

6.13

d (11)

128.7

6.14

d (11)

6.01

d (11)

110
120

138.0
139.8

6.75
6.65

dd (15, 11) 125.0
d (15)
141.4

6.67
6.55

dd (15, 11)
d (15)

130.8
136.8

6.88
6.38

dd (15, 11)
d (15)

127.0
136.5

6.69
6.36

dd (15, 11)
d (15)

7.00
6.33

dd (15, 11)
d (15)

130

142.1

140

121.4

6.41

s

133.9

6.36

d (11)

125.1

5.93

d (12)

131.4

6.28

d (11)

5.93

d (12)

150

145.1

129.8

6.67

m

131.2

8.12

d (12)

137.4

6.92

dd (14, 11)

8.12

d (12)

160

25.1

1.38

s

25.8

1.37

s

29.2

1.39

s

29.2

1.39

s

1.37

s

170

25.8

1.22

s

25.1

1.21

s

32.0

1.07

s

32.0

1.07

s

1.09

s

180

21.2

0.85

s

21.2

0.85

s

31.2

1.35

s

31.2

1.35

s

1.40

s

190
200

13.0
15.2

1.98
1.77

s
s

12.9
12.8

1.97
1.98

s
s

11.7
14.2

1.86
2.18

s
s

14.1
13.1

1.83
2.02

s
s

1.84
2.18

s
s

CH3

21.4

2.04

s

21.4

2.05

s

2.04

s

CO

170.4

134.4

202.6
6.05

s

103.3

134.1

137.2

134.1

135.7

140.9

170.4

s, singlet; d, doublet; m, multiplet.
10574

dx.doi.org/10.1021/jf203493k |J. Agric. Food Chem. 2011, 59, 10572–10578

Journal of Agricultural and Food Chemistry

ARTICLE

12-Nitrocapsanthin (4). UV/vis λ max (Et2O) nm 346, 456. HR FAB
MS m/z 630.4168 [M + H+] (calcd for C40H56O5N, 630.4161). 1H
NMR and 13C NMR (Table 1).
(14Z)-15-Nitrofucoxanthin (5). UV/vis λ max (Et2O) nm 314, 451.
HR FAB MS m/z 704.4169 [M + H]+ (calcd for C42H58O8N,
704.4171). 1H NMR and 13C NMR (Table 1).
(11Z)-11-Nitrofucoxanthin (6). UV/vis λ max (Et2O) nm 334, 456.
HR FAB MS m/z 704.4168 [M + H]+ (calcd for C42H58O8N,
704.4171). 1H NMR and 13C NMR (Table 1).
(14Z,90 Z)-15-Nitrofucoxanthin (7). UV/vis λ max (Et2O) nm 334,
453. HR FAB MS m/z 704.4167 [M + H]+ (calcd for C42H58O8N,
704.4171). 1H NMR (Table 1).

Inhibition of Nitration of Tyrosine with Peroxynitrite by
Carotenoids. Capsanthin or fucoxanthin (0.2 mg) was dissolved in
1 mL of methanol (MeOH). L-Tyrosine (0.36 mg) was added to thissolution. In the control group, L-tyrosine (0.36 mg) was dissolved in
1 mL of MeOH. 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 thisassay. The solution was subsequently allowed to react for 5 min at room
temperature. After that time, the content of nitrotyrosine, 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 mm  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 absorbance ofthe sample solution (in MeOH) and ascorbic acid solution (in H2O).
In Vitro Antitumor-Promoting Activity. In Vitro EpsteinBarr Virus (EVA) Early Antigen 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 Cin a medium containing n-butyric acid (4 nmol), TPA (32 pmol), andvarious amounts of test compounds. Smears were made from the cellsuspension, and we employed an indirect immunofluorescence technique. Details of the in vitro assay on EBV-EA induction have beenreported previously.11,18
Antiproliferative Effects of Human Pancreatic Carcinoma (MIA
PaCa-2). The MIA PaCa-2 (PaCa) cell line was established in 1975
from pancreatic tumor tissue of a 65 year old Caucasian male obtained
from DS Pharma Biochemical Co. (Osaka, Japan). The cells were
cultured in suspension at 37 C in DMEM medium supplemented with
10% fetal bovine serum in a humidified atmosphere containing 5% CO2
in air. Nunclon 3.5 cm cell culture dishes from Nunc Co. (Kamstrupvej,
Denmark) were used. PaCa cancer cells in the exponential growth phasewere seeded and incubated with different concentrations of the carotenoids. For antiproliferation evaluation, the PaCa cells were incubatedwith the various concentrations of the test substances, and the effect on
the cells was assessed after 3 days. For determination of the antiproliferation effects on surviving cells, the cell counting method was used.
At the end of each experiment, the visible round-form cells were counted
within a fixed range by macroscopic observation. All results are expressed as the number of round-form cells as a percentage of all visible
cells.19

In Vivo Two-Stage Carcinogensis Assay for Nitrocapsanthins on Mouse Skin Papillomas Initiated by DMBA
and Promoted by TPA. The animals (specific pathogen-free female
ICR 6 week old mice) were divided into three experimental groups, eachwith 10 mice. The backs of the mice were shaved with surgical clippers,
and they were treated topically with 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. Group 1 (control group)

Figure 2. (A) HPLC of products of reaction of capsanthin withperoxynitrite. Peaks: 1, capsanthin; 3, (140 Z)-150 -nitrocapsanthin; and4, 12-nitrocapsanthin. (B) HPLC of products of reaction of fucoxanthinwith peroxynitrite. Peaks: 2, fucoxanthin; 5, (14Z)-15-nitrofucoxanthin;6, (11Z)-11-nitrofucoxanthin; and 7, (14Z,90 Z)-15-nitrofucoxanthin.
received TPA treatment alone, and groups II and III received the topicalapplication of 12-nitrocapsanthin (4) (85 nmol) and (140 Z)-150 -nitrocapsanthin (3) (85 nmol) in acetone, respectively, 1 h before the TPA
treatment. The incidence and numbers of papillomas were monitored
weekly for 20 weeks.
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.

’ RESULTS AND DISCUSSION
Structure of Products of Capsanthin and Fucoxanthin
Reaction with Peroxynitrite. Capsanthin (1) was reacted withperoxynitrite, and the reaction products were analyzed by
HPLC, as shown in Figure 2A. Two major reaction products,
compounds 3 and 4, were obtained.
Compound 3 showed absorption maxima at 359 and 456 nm.
Its molecular formula was determined as C40H55O5N by
HRFAB-MS, and it demonstrated a NO2-substituted capsanthin
structure. This structure was also characterized from 1H and 13C
NMR including 2D NMR experiments. The partial structure of
the end group and the polyene chain of compound 3 werecharacterized by 1H NMR and 13C NMR including 1H1H
correlation spectroscopy (COSY), nuclear Overhauser eﬀect
spectroscopy (NOESY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation(HMBC) experiments. The downﬁeld shift of the 13C NMR
10575

dx.doi.org/10.1021/jf203493k |J. Agric. Food Chem. 2011, 59, 10572–10578

Journal of Agricultural and Food Chemistry
signal at C-150 (δ 145.1, quaternary carbon) along with disappearance of a methine proton at the C-150 position in 1H NMR as
compared with capsanthin clearly indicated that a nitro group
was attached to the C-15 position of capsanthin. Furthermore,
the change in the coupling pattern and the downﬁeld shifts of
the 1H NMR signals at H-15 (δ 8.09) and H-140 (δ 6.41) ascompared with capsanthin supported the substitution position of
the nitro group at C-150 . The steric structure was conﬁrmed by
NOESY correlations between CH3-16/17 and H-7, CH3-19 andH-7/11, CH3-20 and H-11/15, CH3-190 and H-70 /110 , and CH3200 and H-110 /14. Therefore, the structure of 3 was determined
to be (140 Z)-150 -nitrocapsanthin (3), as shown in Figure 1.
A high intensity cis peak in the UV/vis spectrum (about 80%
AB/AII) of 3 was also in agreement with this structure.20
Compound 4 showed absorption maxima at 346 and 456 nm.
Its molecular formula was also determined to be C40H55O5N by
HRFAB-MS, compatible with the formula of nitrocapsanthin.
The 1H and 13C NMR signals of 4 were assigned based on
1
H1H COSY, NOESY, HSQC, and HMBC experiments. The
appearance of a quaternary carbon at δ 152.4 (C-12) in the 13C
NMR spectrum, along with the disappearance of a methine
proton at the C-12 position in 1H NMR as compared withcapsanthin clearly indicated that a nitro group was attached to the
C-12 position of capsanthin. Furthermore, the change in the
coupling pattern from double doublet to doublet and the downﬁeld shifts of the 1H NMR signals at H-11 (δ 7.99) as comparedwith capsanthin also supported the substitution position of thenitro group at C-12. The trans geometry of the polyene chain in(4) was determined based on NOESY data, as shown in Figure 1.
Disappearance of the cis peak in UV/vis spectrum also supports
this geometry.20 Therefore, the structure of 4 was determined tobe 12-nitrocapsanthin (Figure 1). Employing a similar method tothat described above, fucoxanthin (2) was reacted with peroxynitrite, and the reaction products were analyzed by HPLC, as
shown in Figure 2B.
Compound 5 showed absorption maxima at 314 and 451 nm.
Its molecular formula was determined to be C42H57O8N by
HRFAB-MS, and it demonstrated an NO2-substituted fucoxanthin structure. This structure was also characterized from 1Hand 13C NMR including 2D NMR experiments. The partial
structure of the end group and the polyene chain of compound 5
was characterized by 1H NMR including 1H1H COSY andNOESY experiments. The disappearance of a methine proton at
the C-15 position and in 1H NMR and the change in the coupling
pattern from double doublet to doublet at H-150 as comparedwith fucoxanthin clearly indicated that a nitro group was attachedto the C-15 position of fucoxanthin. The marked downﬁeld shiftof quaternary carbon at C-15 (δ 150.3) was in agreement withthis structure. The steric structure was conﬁrmed by NOESY
correlations between H-7 and H-11, CH3-19 and H-11, CH3-20
and H-11, CH3-190 and H-110 , and CH3-200 and H-110 /150 , asshown in Figure 1. Therefore, the structure of 5 was determined
to be (14Z)-15-nitrofucoxanthin (5). A high-intensity cis peakin the UV/vis spectrum (about 80% AB/AII) of 5 was also inagreement with this structure.20
Compound 6 showed absorption maxima at 334 and 456 nm.
Its molecular formula was determined to be C42H57O8N by
HRFAB-MS, and it demonstrated an NO2-substituted fucoxanthin structure. This structure was also characterized from 1Hand 13C NMR including 2D NMR experiments. The disappearance of a methine proton at the C-11 position and in 1H NMR
and the change in the coupling pattern from double to singlet at

ARTICLE

H-10 (δ 7.31) and H-12 (δ 7.91) as compared with fucoxanthin
clearly indicated that a nitro group was attached to the C-11
position of fucoxanthin. The marked downﬁeld shift of quaternary carbon at C-11 (δ 141.6) was in agreement with thisstructure. NOESY correlations between CH3-20 and H-10 and
between H-7 and H-10 indicated the (11Z) conﬁguration.
Therefore, the structure of 6 was determined to be 11-cis-11nitrofucoxanthin (6), as shown in Figure 1.
Compound 7 also showed a molecular formula of C42H57O8N
by HRFAB-MS. 1H NMR was similar to that of (14Z)-15nitrofucoxanthin (3) except for H-70 , H-100 , and H-110 . The
marked downﬁeld shift of H-70 (0.54 ppm) as compared withthat of 5 suggested that this compound was a 90 -cis isomerof 3. NOESY correlations between H-190 and H-100 and between H-70 and H-110 were in agreement with this structure.
Therefore, 7 was determined to be (14Z,90 Z)-15-nitrofucoxanthin (Figure 1).
Other minor reaction products were obtained. Because of thesmall amount of samples, complete structural characterization
could not be accomplished. From the partial assignment of 1H
NMR, they may be 12- and 120 -nitrofucoxanthins.
Inhibition of Nitration of Tyrosine with Peroxynitrite by
Carotenoids. Capsanthin inhibited the nitration of tyrosine byperoxynitrite. Capsanthin suppressed the formation of nitrotyrosine to about 10.5% 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 fucoxanthin (11.5%).
These results indicate that capsanthin and fucoxanthin are able
to capture peroxynitrite to form nitrocarotenoids and inhibit thenitration of tyrosine.
It was reported that phenolic compounds such as p-coumaricacid and pelargonidin could scavenge peroxynitrite by theformation of nitro-p-coumaric acid and nitro-pelargonidin, respectively, and protect against the nitration of tyrosine.2123
Similar results were reported for tocopherols.24,25 Our investigations also indicated that carotenoids could take up peroxynitrite
through the formation of nitrocarotenoids.
Antitumor-Promoting Activity of Nitrocapsanthins andNitrofucoxanthin. It is well-known that peroxynitrite and nitricoxide are strong initiators of carcinogenesis.26,27 Previously, we
reported that paprika carotenoids such as capsanthin inhibitedthe carcinogenesis initiated by peroxynitrite and promotedwith TPA using a two-stages mouse carcinogenesis model.12
This result suggested that capsanthin inhibited peroxynitriteinitiated carcinogenesis by the uptake of peroxynitrite andnitrocarotenoids themselves and that this might have an anticarcinogenic effect.
Thus, antitumor-promoting activities of nitrocapsanthins andnitrofucoxanthin were investigated. The in vitro antitumorpromoting activity of nitrocapsanthins (3 and 4) and nitrofucoxanthins (5 and 6) was examined using the EpsteinBarr virus(EBV) activation assay in Raji cells. The results are shown in
Table 2. Nitrocapsanthins (3 and 4) and nitrofucoxanthins(5 and 6) showed inhibitory eﬀects on the EBV-EA inductionof Raji cells without signiﬁcant cytotoxicity (more than 60%
viability of Raji cells) in this assay. Among them, nitrofucoxanthins (5 and 6) showed slightly higher activity than fucoxanthin (2). Furthermore, nitocapsanthins (3 and 4) andnitrofucoxanthins (5 and 6) inhibited the proliferation of humanpancreatic cancer cells, as shown in Table 3. These results
showed that nitrocarotenoids themselves have anticarcinogenic
activity.
10576

dx.doi.org/10.1021/jf203493k |J. Agric. Food Chem. 2011, 59, 10572–10578

Journal of Agricultural and Food Chemistry

ARTICLE

Table 2. Relative Rate of EBV-EA Activationa with Respect to
the Positive Control (100%) in the Presence of Carotenoids(16)
concn (mol ratio/TPA)b

1000

500

compds

100

10

IC50

310

valuescapsanthin (1)c5.5 (>60)

29.4

78.1

100

12-nitrocapsanthin (4)7.4 (>60)

30.9

79.7

100

346

(140 Z)-150 -nitrocapsanthin (3)4.6 (>60)

26.9

76.8

100

305

fucoxanthin (2)3.1 (>60)

26.8

76.6

100

296

(11Z)-11-nitrofucoxanthin (6)(14Z)-15-nitrofucoxanthin (5)1.3 (>60)
2.6 (>60)

23.6
25.0

72.0
74.2

95.0
100

275
282

a

Values represent the percentage relative to the positive control values(100%). b TPA concentration was 20 ng (32 pmol)/mol. c Values inparentheses are percentage viability of Raji cells. There are signiﬁcantdiﬀerences (P < 0.01) in inhibitory capacity in treatment groups ofcompounds 15 as compared with the control group.

Table 3. Relative Ratio of Antiproliferative Eﬀects on Human
Pancreastic Carcinoma (MIA PaVa-2) with Respect to the
Positive Control (100%) in the Presence of Carotenoids(16)a% to control group
concn

1 mM

compds

0.1 mMvaluescapsanthin (1)9012-nitrocapsanthin (4)

90

100
100

(140 Z)-150 -nitrocapsanthin (3)

80

100

fucoxanthin (2)7090(11Z)-11-nitrofucoxanthin (6)6090(14Z)-15-nitrofucoxanthin (5)

80

100

a

There are signiﬁcant diﬀerences (P < 0.01) in inhibitory capacity in thetreatment groups of compounds 16 at 1 mM as compared with thecontrol group.

Furthermore, the inhibitory eﬀects of nitrocapsanthins on
two-stages mouse skin carcinogenesis were investigated. The
incidence (%) of papilloma-bearing mice and the average number of papillomas per mouse are presented and compared with acontrol group in Figure 3. When 12-nitrocapsanthin (4)(85 nmol) and (140 Z)-150 -nitrocapsanthin (3) (85 nmol) wereapplied before each TPA treatment, they markedly delayed theformation of papillomas and reduced the number per mouse,as shown in Figure 3. In the positive control group, the ﬁrst
papillomas appeared within 5 weeks of promotion, and in groups
treated with 12-nitrocapsanthin (4) and (140 Z)-150 -nitrocapsanthin (3), the ﬁrst papillomas appeared at 6 and 8 weeks,
respectively. After 11 weeks of promotion, the control group
showed a 100% incidence of papillomas, while in groups treatedwith 12-nitrocapsanthin (4) and (140 Z)-150 -nitrocapsanthin (3),only 50 and 30% of mice bore papillomas, respectively. After 20
weeks of promotion, 7.9 papillomas were found per mouse in thecontrol group, whereas only 5.9 and 4.1 papillomas were foundper mouse in the groups treated with 12-nitrocapsanthin (4)(85 nmol) and (140 Z)-150 -nitrocapsanthin (3), respectively.
These results indicate that nitrocapsanthins (3 and 4) also had

Figure 3. Inhibitory eﬀects of casanthin on DMBA-induced mouse skincarcinogenesis. (A) Percentage of mice bearing papillomas and (B)average numbers of papillomas per mouse. Group I, DMBA (390 nmol) +TPA (1.7 nmol) (b). Group II, 12-nitrocapsanthin (4) (85 nmol) +peroxynitrite (390 nmol) + TPA (1.7 nmol) (9). Group III, (140 Z)-150 nitrocapsanthin (3) (85 nmol) + peroxynitrite (390 nmol) + TPA(1.7 nmol) (2).

inhibitory eﬀects on two stages of mouse skin carcinogenesis as
well as capsanthin.11
In conclusion, capsanthin and fucoxanthin could take up
peroxynitrite through the formation of nitrocapsanthins andnitrofucoxanthins, respectively, and inhibit the nitration oftyrosine by peroxynitrite. Furthermore, nitrocapsanthins andnitrofucoxanthins exhibited antitumor-promoting activity.
Therefore, carotenoids such as capsanthin and fucoxanthin
may have the potential to reduce the risk of disease induced byreactive nitrogen species. Paprika and sea weeds, which containcapsanthin (1) and fucoxanthin (2) as major carotenoids,
10577

dx.doi.org/10.1021/jf203493k |J. Agric. Food Chem. 2011, 59, 10572–10578

Journal of Agricultural and Food Chemistry
respectively, might be valuable foods as prevention for active
nitrogen-induced disease.

’ AUTHOR INFORMATION
Corresponding Author*Tel: +81-75-781-1107. Fax: +81-75-791-7659. E-mail: maoka@
mbox.kyoto-inet.or.jp.
Funding Sources

This work was supported in part by a Grant-in-Aid from the
Ministry of Education, Culture, Sports, Science and Technology(Grant No. 20580126).

’ REFERENCES(1) Maoka, T.; Hideo Etoh, H. Some biological functions ofcarotenoids in Japanese food. In Functional Foods of the East; Shi, J.,
Ho, C.-T., Shahidi, F., Eds.; CRC Press: Boca Raton, 2010; pp 8597.
(2) Scheidegger, R.; Pande, A. K.; Bounds, P. L.; Koppenol, W. H.
The reaction of peroxynitrite with zeaxanthin. Nitric Oxide: Biol. Chem.
1998, 2, 8–16.
(3) Kikugawa, K.; Hiramoto, K.; Tomiyama, S.; Asano, Y. β-Carotene eﬀectively scavenges toxic nitrogen oxides: Nitrogen dioxide andperoxynitrous acid. FEBS Lett. 1997, 404, 175–178.
(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.
(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 oflutein with peroxynitrite. Tetrahedron Lett. 2010, 51, 676–678.
(7) Hirayama, O.; Nakamura, K.; Hamada, S.; Kobayashi, K. Singlet
oxygen quenching ability of naturally occurring carotenoids. Lipids
1994, 29, 149–150.
(8) Matsufuji, H.; Nakamura, H.; Chino, M.; Takeda, M. Antioxidant
activity of capsanthin and fatty acid esters in paprika (Capsicum
annuum). J. Agric. Food Chem. 1998, 46, 3468–3472.
(9) Maoka, T.; Goto, Y.; Isobe, K.; Fujiwrara, Y.; Hashimoto, K.;
Mochida, K. Antioxidative activity of capsorubin and related compounds
from paprika (Capsicum annuum). J. Oleo Sci. 2001, 50, 663–665.
(10) Murakami, A.; Nakashima, M.; Koshiba, T.; Maoka, T.; Nishino,H.; Yano, M.; Sumida, T.; Kim., O.; Koshimizu, K.; Ohigashi, H.
Modifying eﬀects of carotenoids on superoxide and nitric oxide generation from stimulated leukocyte. Cancer Lett. 2000, 149, 115–123.
(11) Maoka, T.; Mochida, K.; Kozuka, M.; Ito, Y.; Fujiwara, Y.;
Hashimoto, K.; Enjo, F.; Ogata, M.; Nobukuni, Y.; Tokuda, H.; Nishino,
H. Cancer chemopreventive activity of carotenoids in the fruits of red
paprika Capsicum annuum L. Cancer Lett. 2001, 172, 103–109.
(12) Maoka, T.; Mochida, K.; Kozuka, M.; Enjo, F.; Kuchide, M.;
Nobukuni, Y.; Tokuda, H.; Nishino, H. Chemopreventive activity of
paprika extract and capsanthin on nitric oxide or peroxynitrite induced
carcinogenesis (in Japanese). Syokuhin Rinsyou Eiyo 2006, 1, 7–14.
(13) Sachindra, N. M.; Sato, E.; Maeda, H.; Hosokawa, M.; Niwano,Y.; Kohno, M.; Miyashita, K. Radical Scavenging and singlet oxygen
quenching activity of marine carotenoid fucoxanthin and its metabolites.
J. Agric. Food Chem. 2008, 56, 8516–8522.
(14) Das, S. K.; Hashimoto, T.; Shimizu, K.; Yoshida, T.; Sakai, T.;
Sowa, Y.; Komoto, A.; Kanazawa, K. Fucoxanthin induces cell cycle
arrest at G0/G1 phase in human colon carcinoma cells through upregulation of p21WAF1/Cip1. Biochim. Biophys. Acta 2005, 1726,
328–335.
(15) Satomi, Y.; Nishino, H. Fucoxanthin, a natural carotenoid,
induces G1 arrest and GADD45 gene expression in human, cancer cells.
In Vivo 2007, 21, 305–309.

ARTICLE(16) Maeda, H.; Hosokawa, M.; Sashima, T.; Miyashita, K. Dietarycombination of fucoxanthin and ﬁsh oil attenuates the weight gainof white adipose tissue and decreases blood glucose in obese/diabetic
KK-Ay mice. J. Agric. Food Chem. 2007, 55, 7701–7706.
(17) Maeda, H.; Hosokawa, M.; Sashima, T.; Funayama, K.; Miyashita,
K. Fucoxanthin from edible seaweed, Undaria pinnatiﬁda, shows antiobesity
eﬀect through UPCi expression in white adipose tissues. Biochem. Biophys.
Res. Commun. 2005, 332, 392–397.
(18) Tsushima, M.; Maoka, T.; Katsuyama, M.; Kozuka, M.;
Matsuno, T.; Tokuda, H.; Nishino, H.; Iwashima, A. Inhibitory eﬀectof 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) Yunis, A. A.; Arimura, G. K.; Russin, D. J. Human pancreatic
carcinoma (MIA PaCa-2) in continous culture: Sensitivity to asparaginase. Int. J. Cancer 1997, 19, 128–135.
(20) Britton, G. UV/Visible Spectroscopy. In Carotenoids; Britton,
G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkh€auser Verlag: Basel, 1995;
Vol. 1B, pp 1362.
(21) Niwa, T.; Doi, U.; Kato, Y.; Osawa, T. Antioxidative propertiesof phenolic antioxidants isolated from corn steep liquor. J. Agric. Food
Chem. 2001, 49, 177–182.
(22) 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.
(23) Tsuda, T.; Kato, Y.; Osawa, T. Mechanism for the peroxynitrite
scavenging activity by anthocyanins. FEBS Lett. 2000, 484, 207–210.
(24) P., A.; Goaa, 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.
(25) Mortion, L. W.; Ward, N. C.; Croft, K. D.; Puddey, I. B.
Evidence for the nitoration of γ-tocopherol in vivo: 5-Nitro-γ-tocopherol is elevated in the plasma of subjects with coronary heart disease.
Biochem. J. 2002, 364, 625–628.
(26) Halliwell, B. Free radicals, antioxidants, and human disease:
Curiosity, cause or consequence? Lancet 1994, 344, 721–724.
(27) Wink, D. A.; Kasprak, K. S.; Maragos, C. M.; Elespuru, R. K.;
Misra, M.; Dunams, T. M.; Cebula, T. A.; Koch, W. H.; Andrews, A. W.;
Allen, J. S.; Keﬀer, L. K. DNA deaminating activity and genotoxicity of
nitric oxide and its progenitors. Science 1991, 254, 1001–1003.

10578

dx.doi.org/10.1021/jf203493k |J. Agric. Food Chem. 2011, 59, 10572–10578