Article
pubs.acs.org/JAFC

Reaction of Paprika Carotenoids, Capsanthin and Capsorubin, with
Reactive Oxygen Species
Azusa Nishino,† Hiroyuki Yasui,‡ and Takashi Maoka*,§†

Institute of Health Sciences, Ezaki Glico Co., Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka 555-8502, Japan
Department of Analytical and Bioinorganic Chemistry, Division of Analytical and Physical Chemistry, Kyoto Pharmaceutical
University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan§
Research Institute for Production Development, 15 Shimogamo, Morimoto Cho, Sakyoku, Kyoto 606-0805, Japan‡S Supporting Information*

ABSTRACT: The reaction of paprika carotenoids, capsanthin and capsorubin, with reactive oxygen species (ROS), such as
superoxide anion radical (·O2−), hydroxyl radical (·OH), and singlet oxygen (1O2), was analyzed by LC/PDA ESI-MS and ESR
spectrometry. Capsanthin formed both the 5,6-epoxide and 5,8-epoxide by reaction with ·O2− and ·OH. Furthermore, capsanthin
also formed 5,6- and 5,8-endoperoxide on reaction with 1O2. The same results were obtained in the case of capsanthin diacetate.
On the other hand, capsorubin showed higher stability against these ROS. Capsorubin formed 7,8-epoxide on reaction with ·O2−
and ·OH and 7,8-endoperoxide on reaction with 1O2.
KEYWORDS: capsanthin, capsorubin, capsanthin epoxide, capsanthin endoperoxide, reactive oxygen species■■

INTRODUCTION
Paprika (Capsicum annuum) is a good source of carotenoids
and used widely as a vegetable and food colorant. The red
carotenoids are mainly capsanthin and capsorubin,1−4 alongwith capsanthin 3,6-epoxide5 and capsanthone6 as minor
components (Figure 1), and their carotenoids account for
30−80% of the total carotenoids in fully ripe fruits.1−4 These
carotenoids showed excellent antioxidative7−10 and anticancer11−14 activities. Recently, Maeda et al. reported thatthese carotenoids showed eﬀects to prevent and improveobesity-related insulin resistance.15 Furthermore, it wasreported that capsanthin could take up peroxynitrite into its
molecule by the formation of nitrocapsanthin.16
It is known that carotenoids inhibit singlet oxygen (1O2)
through physical quenching and chemical reactions.17 For
example, β-carotene formed endoperoxides, epoxides, andapocarotenoids by the reaction with 1O2.18−21 Very recently,
we investigated reaction of astaxanthin with 1O2, superoxideanion radical (·O2−), and hydroxyl radical (·OH), using LC/
PDA ESI-MS and ESR spectrometry.22 As the results, we foundthat astaxanthin formed astaxanthin endoperoxides on reaction
with 1O2 and formed astaxanthin epoxides on reaction with ·O2− and ·OH.22
However, there have been few reports on the reaction ofcapsanthin and capsorubin with reactive active oxygen species(ROS). In order to clarify the chemical scavenging mechanismof these carotenoids with ROS, we investigated reaction
products of these paprika carotenoids with ·O2−, ·OH, and1O2 using LC/PDA ESI-MS and ESR spectrometry. This paper
reports the scavenging eﬀect of ·O2−, ·OH, and 1O2 withcapsanthin and capsorubin by ESR spin-trapping analysis andthe reaction products of these paprika carotenoids with these
ROS. Furthermore, the reaction mechanisms of ROS withcapsanthin and capsorubin are discussed.
© XXXX American Chemical Society

MATERIALS AND METHODS
Chemicals. Hematoporphyrin, riboﬂavin, and hydrogen
peroxide were purchased from Wako Pure Chemicals (Osaka,
Japan). 2,2,6,6-Tetramethyl-4-piperidone (TMPD) was purchased from Aldrich (Milwaukee, WI, USA). 5,5-Dimethyl-1pyrroline-N-oxide (DMPO) was purchased from Labotec(Tokyo, Japan). β-Carotene, zeaxanthin, capsanthin, andcapsorubin were prepared from paprika according to themethod described previously.9,10,12 Astaxanthin was preparedfrom Paracoccus carotinifaciens.23 Capsanthin diacetate andastaxanthin diacetate were prepared from capsanthin andastaxanthin, respectively, by acetylation with acetic anhydridein pyridine at room temperature. syn- and anti-Capsanthin 5,6epoxide and capsanthin 5,8-epoxide were prepared from
capsanthin by epoxydation with m-chloroperbenzoic acid as
described below.
Apparatus. The ESR spectra were recorded at room
temperature on a JEOL JES-RFR30 spectrometer (Tokyo,
Japan). The LC/MS and MS/MS analysis of carotenoids was
carried out using a Waters Xevo G2S Q TOF mass
spectrometer (Waters Corporation, Milford, CT, USA)equipped with an Acquity UPLC system. The 1H NMR (500MHz) spectra were measured with a Varian UNITY INOVA
500 spectrometer (Agilent Technologies, Santa Clara, CA,
USA) in CDCl3 with TMS as an internal standard.
Reaction of Capsanthin with ROS. According to themethod described previously,22 after the addition of 200 μL of
8 mM H2O2 solution to 200 μL of 22 μg/mL capsanthin
CH3CN solution, ·OH was generated by UV-A irradiation
Received: April 15, 2016
Revised: May 26, 2016
Accepted: May 27, 2016

A

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Figure 1. Structures of carotenoids in this study.

detector (PDA). An Acquity 1.7 μm BEH UPLC C18 column(Waters Corporation) was used as a stationary phase and 90%
MeOH as a mobile phase, at a ﬂow rate of 0.2 mL/min for the
HPLC system. In a similar manner to that described above,
reaction of capsanthin diacetate (27 μg/mL in CH3CN
solution) and capsorubin (34 μg/mL in CH3CN solution)with ROS were performed.
Capsanthin Diepoxide. ESI TOF MS m/z 639.4217 (M+Na+, calcd for C40H56O5Na, 639.4025) ; UV/vis 405−420 nm(in 90% MeOH).
Capsanthin 5,6-Epoxide. ESI TOF MS m/z 623.4080 (M+Na+, calcd for C40H56O4Na, 623.4076) ; UV/vis 463 nm (in
90% MeOH).
Capsanthin 5,8-Epoxide. ESI TOF MS m/z 623.4080 (M+Na+, calcd for C40H56O4Na, 623.4076) ; UV/vis 448 nm (in
90% MeOH).
Capsanthone 5,6-Epoxide. ESI TOF MS m/z 621.3920 (M+Na+, calcd for C40H54O4Na, 621.3920) ; UV/vis 463 nm (in
90% MeOH).
Capsanthin Diendoperoxide. ESI TOF MS m/z 671.3937(M+Na+, calcd for C40H56O7Na, 671.3948) ; UV/vis 446 nm(in 90% MeOH).
Capsanthin 5,6-Endoperoxide. ESI TOF MS m/z 639.4056(M+Na+, calcd for C40H56O5Na, 639.4025); UV/vis 463 nm(in 90% MeOH).
Capsanthin 5,8-Endoperoxide. ESI TOF MS m/z 639.4003(M+Na+, calcd for C40H56O5Na, 639.4025); UV/vis 448 nm(in 90% MeOH).
Capsanthin Diacetate Diepoxide. ESI TOF MS m/z
723.4258 (M+Na+, calcd for C44H60O7Na, 723.4237) ; UV/
vis 405−420 nm (in 90% MeOH).
Capsanthin Diacetae 5,6-Epoxide. ESI TOF MS m/z
707.4280 (M+Na+, calcd for C44H60O6Na, 707.4288); UV/vis
463 nm (in 90% MeOH).

using SUPERCURE-203S (SAN-Ei ELECTORIC) at room
temperature. In a similar manner to that described above, ·O2−
and 1O2 were generated by the addition of 200 μL of 25 μM of
riboﬂavin or 200 μL of 250 μM hematoporphyrin, respectively,
to 200 μL of 22 μg/mL of capsanthin CH3CN solution. At
regular intervals of UV-A irradiation, reaction products wereanalyzed by LC/PDA-ESI-MS.
ESR Spin-Trapping Analysis. ESR spectra were recordedat room temperature on a JEOL JES-RFR30 spectrometer(Tokyo, Japan) using an aqueous quartz ﬂat cell (Labotec,
Tokyo, Japan). DMPO was used as a ·O2− and ·OH-trapping
agent, and TMPD was used as 1O2, respectively. The ·O2− and ·OH were generated by addition of either the 100 μL of 25 μM
of riboﬂavin, or 100 μL of 8 mM H2O2 solution and 10 μL of
250 mM DMPO to 100 μL of 14 nmol/mL capsanthin CH3CN
solution by UV-A irradiation. In a similar manner describedabove, 1O2 was generated by the addition of both the 100 μL of
250 μM hematoporphyrin and 10 μL of 500 mM TMPD to 100
μL of 14 nmol/mL capsanthin CH3CN solution by UV-A
irradiation. ESR spectra were started simultaneously to measure
after UV-A irradiation. The all spin-trapped ESR spectra weremonitored between the third and fourth signals from the low
magnetic ﬁeld due to the external standard, Mn(II) doped
MnO. As the similar manner described above, reaction ofcapsanthin acetate and capsorubin (14 nmol/mL of both inCH3CN solution) with ROS were also performed.
Analysis of Reaction Products of Capsanthin with ROS
using LC/MS and MS/MS. The electro-spray ionization (ESI)
time-of-ﬂight (TOF) MS spectra were acquired by scanningfrom m/z 100 to 1,500 with a capillary voltage of 3.2 kV, cone
voltage of 40 eV, and source temperature of 120 °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 energyof 30 V. UV−visible (UV/vis) absorption spectra wererecorded from 200 to 600 nm using a photodiode-array
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Capsanthin Diacetate 5,8-Epoxide. ESI TOF MS m/z
707.4280 (M+Na+, calcd for C44H60O6Na, 707.4288); UV/vis
448 nm (in 90% MeOH).
Capsanthin Diacetate Diendoperoxide. ESI TOF MS m/z
755.4145 (M+Na+, calcd for C44H60O9Na,755.4135) ; UV/vis
446 nm (in 90% MeOH).
Capsanthin Diacetate 5,6-Endoperoxide. ESI TOF MS m/
z 723.7229 (M+Na+, calcd for C44H60O7Na, 723.4237); UV/vis
463 nm (in 90% MeOH).
Capsanthin Diacetate 5,8-Endoperoxide. ESI TOF MS m/
z 723.7229 (M+Na+, calcd for C44H60O7Na, 723.4237); UV/vis
448 nm (in 90% MeOH).
Capsorubin 7,8-Epoxide. ESI TOF MS m/z 639.4003 (M+Na+, calcd for C40H56O5Na, 639.4025); UV/vis 424 nm (in
90% MeOH).
Capsorubin 7,8-Endoperoxide. ESI TOF MS m/z 665.3962(M+Na+, calcd for C40H56O6Na, 655.3975); UV/vis 424 nm(in 90% MeOH).
Preparation of Capsanthin 5,6-Epoxides and 5,8Epoxides from Capsanthin by Epoxydation with mChloroperbenzoic Acid. According to the method describedby Khachik et al.,24 m-chloroperbenzoic acid, 3.6 mg in 2 mLdichlorometane, was added to a solution of capsanthin, 2 mg in
2 mL dichlorometane. The solution stood for 20 min at room
temperature in darkness. After the time, the reaction mixture
was partitioned with 5% solution of NaHCO3 and ether. The
ether layer was removed and evaporated. The reaction products
were submitted to preparative HPLC. HPLC separation
provided syn-capsanthin 5,6-epoxide (0.8 mg) and anticapsanthin 5,6-epoxide (0.4 mg) and capsanthin diepoxides(0.2 mg mixtures). These spectral data were identical withthose of published data by Deli at al.25 Capsanthin 5,8-epoxides
were prepared by epoxide-furanoxide rearrangement26 with
HCl from capsanthin 5,6-epoxides. Preparative HPLC was
performed on a Shimadzu LC-6AD with a Shimadzu SPD-6AV
spectrophotometer (Shimadzu Corporation, Kyoto, Japan) set
at 450 nm. The columns used were a 250 × 10 mm i.d., 10 μm
Cosnosil 5C18-II (Nacalai Tesque, Kyoto, Japan) and a 250 ×
10 mm i.d., 10 μm Cosnosil 5SL-II (Nacalai Tesque, Kyoto,
Japan). Spectral data of syn-capsanthin 5,6-epoxide, anticapsanthin 5,6-epoxide, and capsanthin 5,8-epoxides areavailable in the Supporting Information.

Figure 2. Quenching eﬀect on 1O2 (A) and scavenging eﬀect on ·OH(B) with capsanthin, capsanthin diacetate, and capsorubin. The verticalaxis shows the relative rate (%) of the ESR signal intensity due tosinglet oxygen (A) and hydroxyl radical (B) compared with the control
group (100%). Astaxanthin, astaxanthin diacetate, β-carotene, andzeaxanthin were used as positive controls. Signiﬁcance compared withthe control group: * p < 0.05, p < 0.01, p < 0.001.

two conjugated carbonyl groups. Capsanthin and its acetate
have 11 conjugated double bonds including one conjugatedcarbonyl group. On the other hand, β-carotene and zeaxanthinhave 11 conjugated double bonds without a conjugatedcarbonyl group. The results of the present investigation aremarkedly consistent with this hypothesis.
On the other hand, capsanthin and capsorubin showed
excellent scavenging activity for ·OH. Contrary to 1O2,
astaxanthin and its acetate hardly showed scavenging activity
for ·OH. The presence of the 3-hydroxy-κ-end group with aconjugated carbonyl group, which is a characteristic structural
moiety of paprika carotenoids, may enhance the quenchingactivity of ·OH.
Reaction Products of Capsanthin with ·O2− and ·OH.
Structures of reaction products of capsanthin with ROS are
shown in Figure 1. Figure 3 shows HPLC chromatograms ofreaction products of capsanthin with ·O2− by UV-A irradiationof riboﬂavin solution. After 1 min of UV-A irradiation, reaction
products assigned as capsanthin 5,6-epoxide and capsanthin
5,8-epoxide were detected on HPLC. On UV-A irradiation, theamount of these reaction products was increased and amount of
capsanthin was decreased time-dependently. After 6 min of UVA irradiation, the HPLC peak of capsanthin completely
disappeared and the amount of reaction products reached amaximum. Then, the reaction products were markedly
decreased and the reaction solution was bleached.

■

RESULTS AND DISCUSSION
Scavenging Eﬀect of ROS with Capsanthin andCapsorubin. Figure 2 shows the scavenging eﬀect of 1O2(A) and ·OH (B) with capsanthin and capsorubin. Astaxanthin,astaxanthin diacetae, β-carotene, and zeaxanthin were used as
positive controls. It was reported that capsanthin andcapsorubin quenched 1O27,10 and inhibited lipid peroxidation
caused by free radicals.8,9 In the present investigation, the ESR
study also revealed that capsanthin, capsanthin acetate, andcapsorubin quenched not only 1O2 but also scavenged ·OH.
The order of quenching activity for 1O2 is astaxanthin diacetate> capsorubin > astaxanthin > capsanthin diacetate > capsanthin> β-carotene > zeaxanthin. It was reported that the 1O2
quenching activity of carotenoids depends on the number ofconjugated polyenes, polyene chain structures, and functionalgroups, especially conjugated carbonyl groups.7,10,27 It was also
reported that acetylation of the hydroxyl group improved
carotenoid stability.22 Astaxanthin and its acetate have 13
conjugated double bonds including two conjugated carbonylgroups. Capsorubin has 11 conjugated double bonds including
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Figure 3. HPLC chromatograms of reaction products of capsanthin with superoxide anion radical. Peaks 1 and 2: capsanthin diepoxide; peak 3:
capsanthone 5,6-epoxide; peak 4, syn-capsanthin 5,6-epoxide; peak 5: anti-capsanthin 5,6-epoxide; and peak 6: capsanthin 5,8-epoxide. HPLC
condition ACQUITY UPLC system (Waters); column: BEH Shield RP18 (1.7 μm, 2.1 × 100 mm); mobile phase: 90% MeOH; column
temperature: 40 °C; ﬂow rate: 0.2 mL/min; detection: 450 nm.

Figure 4. HPLC chromatograms of reaction products of capsanthin with singlet oxygen. Peak 1: capsanthin diendoperoxide and peaks 2 and 3:
capsanthin 5,6-endoperoxide. HPLC conditions were described in Figure 3.

ESI MS.28,29 Both compounds showed an absorption maximum
at 463 nm. These spectral data were in agreement with those of
capsanthin 5,6-epoxide.25,30 In order to conﬁrm the structure ofthese reaction products, capsanthin 5,6-epoxides were preparedfrom capsanthin by epoxydation with m-chloroperbenzoic acid,as shown in Supporting Information. Peaks 4 and 5 wereidentical with semisynthetic syn-capsanthin 5,6-epoxide andanti-capsanthin 5,6-epoxide, respectively, on comparison withretention times in HPLC, MS, MS/MS, and UV/vis spectra
data. Therefore, the structures of reaction products of peaks 4
and 5 were identiﬁed as capsanthin 5,6-epoxides.
Reaction products of peak 6 showed the molecular formulaof C40H56O4, consistent with the molecular formula of

The structures of reaction products were assigned tocapsanthin diepoxides (peaks 1 and 2), capsanthone 5,6epoxide (peak 3), capsanthin 5,6-epoxide (peaks 4 and 5), andcapsanthin 5,8-epoxide (peak 6) based on the ESI-TOF MS,
MS/MS, and UV/vis spectral data.
Both reaction products of peaks 4 and 5 showed themolecular formula of C40H56O4, which corresponded to themolecular formula of capsanthin epoxide. The MS/MS
spectrum of sodium adduct ions [M+Na]+ of these compounds
showed the characteristic product ions [M+Na-92]+ (elimination of toluene moiety from polyene chain) and [M+Na106]+ (elimination of xylene moiety from polyene chain),which were characteristic product ions of carotenoids in EI andD

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Figure 5. HPLC chromatograms of reaction products of capsorubin with superoxide anion radical. Peaks 1 and 2: capsorubin epoxides. HPLC
conditions were described in Figure 3.

capsanthin epoxide. Its absorption maximum was at 448 nm.
The hypsochromic shift of about 15 nm from capsanthin 5,6epoxide suggested that these compounds were furanoid
rearrangement products of capsanthin 5,6-epoxide,26,31 namely
capsanthin 5,8-epoxide. These compounds were identical tosemisynthetic capsanthin 5,8-epoxides prepared from capsanthin 5,6-epoxides based on acid catalysis on comparison of theretention times of HPLC and MS, MS/MS, and UV/vis
spectral data.
Both reaction products of peaks 1 and 2 showed themolecular formula of C40H56O5, which corresponded to themolecular formula of capsanthin diepoxide. These absorption
maxima were at 405−420 nm. Therefore, these compounds
were assigned to capsanthin diepoxide. Epoxidation positions in
capsanthin could not be determined.
Peak 3 showed the molecular formula of C40H54O4. Itsabsorption maximum was at 463 nm. These data suggest that
this compound is a didehydro derivative of capsanthin 5,6epoxide. Therefore, this reaction product was tentatively
identiﬁed as capsanthone 5,6-epoxide. This compound wassuggested to form from capsanthin 5,6-epoxide throughoxidation of the hydroxy group at C-3′. Superoxide anion
radical might oxidize the hydroxy group in carotenoids toketone.
As well as ·O2−, the reaction of capsanthin with ·OH
hydroxyl radical generated by UV-A irradiation in hydrogen
peroxide solution provided capsanthin diepoxide, capsanthone
5,6-epoxide, capsanthin 5,6-epoxide, and capsanthin 5,8epoxide.
Reaction Products of Capsanthin with 1O2. Figure 4
shows the HPLC chromatograms of the reaction products ofcapsanthin with 1O2 by UV-A irradiation in hematoporphyrinsolution. After 3 min of UV-A irradiation, reaction products
assigned as capsanthin diendoproxide and capsanthin 5,6endoperoxide were detected in HPLC. The amounts of thesereaction products were increased and amount of capsanthin was
decreased with UV-A irradiation time-dependently. After 6 minof UV-A irradiation, the amount of the reaction products
reached a maximum. Then, the reaction products weremarkedly decreased and the reaction solution was bleached.

Both reaction products of peaks 2 and 3 showed the molecular
formula of C40H56O5 and an absorption maximum at 463 nm.
These spectral data indicated that the dioxetane moiety was
attached at the double bond of C-5 to C-6 in capsanthin.
Therefore, the structure of these compounds was assigned as
capsanthin 5,6-endoperoxide. Compounds of peak 2 and 3 weresuggested to be stereoisomers of C-5 and C-6. Reaction
products of peak 1 showed the molecular formula of C40H56O7,
which indicated that two more oxygen atoms were attached tocapsanthin 5,6-endoperoxide. Its absorption maximum was at
446 nm. The hypsochromic shift of the UV/vis spectrum of 15
nm from capsanthin 5,6-endoperoxide comprised with the lossof one conjugated double bond from capsanthin 5,6endoperoxide by attaching a dioxetane moiety.26,31 Therefore,
the structure of this compound was tentatively assigned as
capsanthin diendoperoxide.
Reaction Products of Capsanthin Diacetate with ·O2−,·OH, and 1O2. Results similar to those for capsanthin wereobtained in the case of capsanthin diacetate with these ROS.
Namely, capsanthin diacetate epoxides were obtained onreaction with ·OH and ·O2−, while capsanthin diacetate
endoperoxides were produced on reaction with 1O2. It was
found that oxidation products of capsanthin diacetate weremore stable than those of free capsanthin. Acetylation mightprotect the oxidation hydroxy groups of capsanthin from ROS
and inhibit rapid degradation of the capsanthin molecule.
Reaction Products of Capsorubin with ·O2− and ·OH.
Figure 5 shows the HPLC chromatograms of the reaction
products of capsorubin with ·O2−. Reaction products assigned
as capsorubin epoxide were increased with UV-A irradiation
time-dependently. After 12 min of UV-A irradiation, theamount of reaction products reached a maximum. Then, thereaction solution was bleached. The major reaction products(peaks 1 and 2) showed the molecular formula of C40H56O5,
which corresponded to the formula of capsorubin epoxide. The
epoxidation position of capsorubin in these compounds wasestimated from the UV/vis absorption spectra. These
compounds showed an absorption maximum at 424 nm. The
hypsochromic shift of the UV/vis spectrum of 45 nm from
capsorubin indicated that these compounds lacked two
E

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Figure 6. HPLC chromatograms of reaction products of capsorubin with singlet oxygen. Peaks 1 and 2: capsorubin endoperoxides. HPLC conditions
were described in Figure 3.

withdrawn eﬀect of the carbonyl group at C-4 (4′). On theother hand, the electron density of the double bond at C-5 to
C-6 in the β-end group of capsanthin is suggested to be higher
than that of astaxanthin. Therefore, ·O2− and 1O2 might facilelyattack the double bond at C-5 to C-6 in capsanthin and form
5,6-epoxide and 5,6-endoperoxide, respectively. Then, these
5,6-epoxide and 5,6-endoperoxides are converted to thecorresponding 5,8-epoxide and 5,8-endoperoxide by acidcatalysis.22 The mechanism of epoxide ring formation incapsanthin with ·OH can be explained as follows: A hydroxyl
radical was attached at C-5 of capsanthin. Then, an epoxide ring
was formed by intramolecular homolytic substitution reaction.22 Capsorubin showed more stability against the attack of
ROS, especially 1O2. Capsorubin has a long linear conjugateddouble bond system in its molecule. Capsorubin might
exclusively quench 1O2 through a physical quenching system.
Therefore, capsorubin showed the strongest quenching eﬀect
on 1O2 among the carotenoids.10 Therefore, we conclude thatthe reaction of carotenoids with ROS might depend on theirstructure, such as the conjugated polyene system and functionalgroups.32,33
In conclusion, capsanthin and capsorubin formed their
epoxides and endoperoxides on reaction with ·O2−, ·OH, and1
O2. The results indicate that capsanthin and capsorubin could
trap ·O2− and ·OH by the formation of their epoxides.
Furthermore, it was found that capsanthin and capsorubin
could quench 1O2 through not only a pysical quenching systembut also chemical direct reaction by the formation of theirendoperoxide.

conjugated double bonds (double bond of a carbonyl group at
C-6 and a double bond at C-7 to C-8 in the polyene chain)from 11 conjugated double bonds of capsorubin by
epoxidation.31 Therefore, the epoxidation position of thesecompounds was assigned to a double bond at C-7 to C-8.
Reaction Products of Capsorubin with 1O2. Figure 6
shows the HPLC chromatograms of the reaction products ofcapsorubin with 1O2. Contrary to capsanthin and astaxanthin,22
capsorubin hardly formed oxidation products on exposure to1
O2. The compound showed the molecular formula ofC40H56O6, which corresponded to the molecular formula ofcapsorubin endoperoxide, being slightly generated after 10 minof UV-A irradiation. After 15 min of UV-A irradiation,
capsorubin and its reaction product were markedly decreased.
Capsorubin showed greater stability on exposure to singlet
oxygen than β-carotene, capsanthin, or astaxanthin.22 Therefore, capsorubin showed a strong quenching eﬀect for 1O2.
Previously, we investigated the reaction of astaxanthin and its
acetate with ·O2−, ·OH, and 1O2 by LC/PDA ESI-MS and ESR
spectrometry.22 As the results, astaxanthin endoperoxides wereidentiﬁed as major reaction products of astaxanthin with 1O2,
and astaxanthin epoxides were found to be major reaction
products of astaxanthin with ·O2− and ·OH. The same results inthis study were obtained in the case of astaxanthin acetate. The
ESR spin-trapping study revealed that astaxanthin and its
acetate take up superoxide anion radical by the formation of
their epoxide through peroxide and oxide radicals.22
In the present investigation, we found that capsanthin andcapsorubin quenched not only 1O2 but also scavenged ·OH by
the ESR spin-trapping study. Furthermore, we found that
capsanthin formed capsanthin epoxides by the reaction with ·O2− and ·OH and also formed capsanthin endoperoxides by thereaction with 1O2. Contrary to astaxanthin, capsanthin formed
epoxide and endoperoxide more eﬀectively and stably. It is wellknown that ·O2− and 1O2 are electrophilic. Thus, they reactwith the electron-rich double bond in carotenoids. The electron
density of the double bond at C-5 (5′) to C-6 (6′) in the β-end
group of astaxanthin is suggested to be lower than in parts ofdouble bonds because of the presence of the electron■

ASSOCIATED CONTENT

S Supporting Information*

The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.6b01706.
ESI TOF MS and MS/MS data of reaction products ofcapsanthin, capsanthin acetate, and capsorubin. UV/vis,
ESI TOF MS, MS/MS, and 1H NMR data of syn- andanticapsanthin 5,6-epoxide. UV/vis, ESI TOF MS, andMS/MS data of capsanthin 5,8-epoxide. Preparation
F

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scheme of capsanthin 5,6-epoxides and capsanthin 5,8epoxides from capsanthin by m-chloroperbenzoic acid(PDF)

AUTHOR INFORMATION

Corresponding Author*Tel.: +81 75 781 1107; Fax: +81 75 781 1118; E-mail address:
maoka@mbox.kyoto-inet.or.jp.
Notes

The authors declare no competing ﬁnancial interest.

■■

ACKNOWLEDGMENTS
We thank Dr. Yumiko Yamano, Kobe Pharmaceutical
University, for valuable suggestions to this work.
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DOI: 10.1021/acs.jafc.6b01706
J. Agric. Food Chem. XXXX, XXX, XXX−XXX