Kinetic study of the quenching reaction of singlet oxygen by carotenoids and food extracts in solution. development of a singlet oxygen absorption capacity (SOAC) assay method

Article abstract of DOI:10.1021/jf101947a

A kinetic study of the quenching reaction of singlet oxygen ( ¹O₂) with eight kinds of carotenoids and α-tocopherol was performed in ethanol/chloroform/D₂O (50:50:1, v/v/v) solution at 35 °C. The overall rate constants, k_Q (= k_q + k_r, physical quenching + chemical reaction), for the reaction of carotenoids with ¹O₂ were measured, using the competition reaction method, where endoperoxide was used as a singlet oxygen generator, 2,5-diphenyl-3,4-benzofuran (DPBF) as an UV-vis absorption prove, and α-tocopherol as a standard compound. The rate constants, k_Q (S) and k_Q (t_1/2), were determined by analyzing the first-order rate constant (S) and the half-life (t_1/2) of the decay curve of DPBF with carotenoids, respectively, showing good accordance with each other. Similar measurements were performed for tomato and carrot extracts. From the results, a new assay method that can quantify the singlet oxygen absorption capacity (SOAC) of antioxidants, including carotenoids, α-tocopherol, and vegetable extracts, has been proposed.

Full text of DOI:10.1021/jf101947a

J. Agric. Food Chem. 2010, 58, 9967–9978 9967
DOI:10.1021/jf101947a
Kinetic Study of the Quenching Reaction of Singlet Oxygen
by Carotenoids and Food Extracts in Solution. Development
of a Singlet Oxygen Absorption Capacity (SOAC) Assay Method
#
A
YA
O
UCHI,† KOICHI
A
IZAWA,§ YUKO
HIN ICHI
I
WASAKI,§ TAKAHIRO
†
I
NAKUMA,§ JUNJI
,†
T
ERAO
,
S
-
N
AGAOKA
,
AND
K
AZUO
M
UKAI
*
†Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790-8577, Japan,
§Research Institute, Kagome Company Ltd., 17 Nishitomiyama, Nasushiobara-shi, Tochigi 329-2762,
Japan, and #Department of Food Science, Graduate School of Nutrition and Bioscience,
The University of Tokushima, Tokushima 770-8503, Japan
A kinetic study of the quenching reaction of singlet oxygen (1O2) with eight kinds of carotenoids and
R-tocopherol was performed in ethanol/chloroform/D2O (50:50:1, v/v/v) solution at 35 °C. The overall rate
constants, kQ (= kq þ kr, physical quenching þ chemical reaction), for the reaction of carotenoids with 1O2
were measured, using the competition reaction method, where endoperoxide was used as a singlet
oxygen generator, 2,5-diphenyl-3,4-benzofuran (DPBF) as an UV-vis absorption prove, and R-toco-
pherol as a standard compound. The rate constants, kQ (S) and kQ (t1/2), were determined by analyzing
the first-order rate constant (S) and the half-life (t1/2) of the decay curve of DPBF with carotenoids,
respectively, showing good accordance with each other. Similar measurements were performed for
tomato and carrot extracts. From the results, a new assay method that can quantify the singlet oxygen
absorption capacity (SOAC) of antioxidants, including carotenoids, R-tocopherol, and vegetable extracts,
has been proposed.
KEYWORDS: Singlet oxygen; quenching rate; endoperoxide; carotenoids; β-carotene; lycopene;
astaxanthin; foods; SOAC value; kinetic study
INTRODUCTION
where the former results in energy transfer and de-excitation of the
singlet state but no chemical change in the energy acceptor. The
latter results in oxidation of the target. Di Mascio et al. reported
that carotenoids generally show very high activity for the quenching
of singlet oxygen. For instance, the second-order rate constants (kQ)
(∼1010 M-1 s-1) for carotenoids (18, 19) are about 2-3 orders of
magnitude larger than those for R-tocopherol (20,21), ubiquinol-10
(22), catechins (23), and flavone derivatives (24). Measurement
of the quenching rate of singlet oxygen by carotenoids in liposome
Carotenoids have attracted much attention because of their high
antioxidant capacity and their abundance in the human diet (1, 2).
Vegetables and fruits contain considerable amounts of carotenoids
(3-5). Several epidemiolologic studies show an inverse association
between serum/adipose β-carotene levels and coronary heart disease
risk (6). Furthermore, there is now growing evidence that carotenoids
may possess inhibitory effects against cancer, although the human
epidemiology remains inconclusive (7-11). An inverse relationship
between β-carotene ingestion and the incidence of certain types of
cancer, such as lung and intestinal tract cancer, has been reported (12).
Dietary consumption of the carotenoid lycopene (mostly from
tomato products) has been associated with a lower risk of prostate
cancer in men (13-15). The cancer preventive effects often have been
attributed to antioxidant actions (16). Carotenoids have been recog-
nized to be efficient singlet oxygen quenchers (17-19).
1
has also been performed, indicating that carotenoids inhibit O2-
dependent lipid peroxidation in liposome (25). On the other hand,
the activities of carotenoids to trap chain-carrying peroxyl radicals
were much less than that of R-tocopherol (26).
In recent years, the method to assess the total oxygen radical
absorpton capacity (ORAC) of foods and plants has been esta-
blished (27-30). On the other hand, a singlet oxygen absorption
capacity (SOAC) assay method to assess the total quenching
activity of singlet oxygen by carotenoids and phenolic anti-
oxidants included in foods and plants has not been established.
Lipid peroxyl radical (LOO•) and singlet oxygen (1O2) are well-
known as two representative reactive oxygen species (ROS)
generated in biological systems. 1O2 reacts with many kinds of
biological targets including lipids, sterols, proteins, DNA, and
RNA (31,32), as well as peroxyl radical. Reactions with 1O2 occur
mainly by chemical reaction, inducing the degradation of bio-
logical systems. Carotenoids are widely present in vegetables and
Measurement of the quenching rates kQ (= kq þ kr, physical
quenching þ chemical reaction) of singlet oxygen with several
carotenoids in solution has been performed by Di Mascio
et al. (18,19), using a near-IR 1O2 luminescence method (reaction 1)
k
f
s Q
1O2 þ Carotenoid
physical quenching ðkqÞ
þ chemical reaction ðkrÞ
ð1Þ
*Author ro whom correspondence should be addressed (phone
81-89-927-9588; fax 81-89-927-9590; e-mail mukai@chem.sci.ehime-u.ac.jp).
© 2010 American Chemical Society
Published on Web 08/20/2010
pubs.acs.org/JAFC

9968 J. Agric. Food Chem., Vol. 58, No. 18, 2010
Ouchi et al.
Table 1. UV-Vis Absorption Maxima (λmaxi (i = 1-5)) and Molar Extinction Coefficients (εi (i = 1-5)) of the Carotenoids 1-8 and Used Compounds in Ethanol/
Chloroform/D2O (50:50:1, v/v/v) Solution
molecule
λmax1/nm (ε1/M-1 cm-1
)
λmax2/nm (ε2/M-1 cm-1
)
λmax3 and λmax4/nm (ε3 and ε4/M-1 cm-1
)
λ5/nm (ε5/M-1 cm-1
)
lycopene (Lyc, 1)
479 (160000)a
511 (140000)
452 (110000)
298 (40500)
413 (40900)a
astaxanthin (Ast, 2)
β-carotene (β-Car, 3)
capsanthin (Cap, 4)
zeaxanthin (Zea, 5)
R-carotene (R-Car, 6)
lutein (Lut, 7)
486 (124000)a
459 (133000)a
481 (106000)a
459 (129000)a
453 (138000)a
452 (126000)a
459 (95100)a
237 (1930)
413 (35600)a
413 (56800)a
413 (32900)a
413 (55200)a
413 (64700)a
413 (61100)a
413 (43500)a
485 (117000)
277 (21500)
292 (18700)
278 (21900)
270 (24000)
269 (26000)
262 (22700)
485 (114000)
481 (123000)
480 (113000)
485 (82400)
293 (2980)a
β-cryptoxanthin (Crp, 8)
R-tocopherol (R-Toc)
endoperoxide (EP)
EP-precursor
236 (1630)
290 (7670)
413 (20900)b
281 (6350)
315 (7950)
239 (9130)
264 (25600)
DPBF
413 (20900)b
a Average of values obtained by measurements repeated three to five times. The values of ε were not varied by the dilution of the solution, indicating that the carotenoids 1-8
are completely dissolved in the solution. Experimental errors in the values of ε were estimated to be 5%. b Average of values obtained by measurements repeated five times.
fruits in high concentrations (3-5) and may function as quenchers
of O2 in biological systems (16, 18, 19, 26). Consequently, the
through the reaction of EP-precursor with 1O2 produced in H2O2 solution
including Na2MoO4 (see reaction 2) (36).
1
development of a SOAC method is very important.
35°C
1
1
EP-Precursorþ O2ðNa2MoO4; H2O2Þ f EP
s
EP-Precursorþ O2
f
In previous works, to clarify the structure-activity relation-
ship in the quenching reaction of 1O2 by natural phenolic
antioxidants, we measured the second-order rate constant (kQ)
for the reaction of 1O2 with many phenolic antioxidants such as
vitamin E homologues (21), biological hydroquinones (22), and
polyphenols (23, 24) in ethanol solution (reaction 1), using the
competition reaction method, where endoperoxide (EP) was used
as a generator of singlet oxygen and 2,5-diphenyl-3,4-benzofuran
(DPBF) was used as an indicator of the singlet oxygen quenching
capacity.
ð2Þ
Tomato and carrot extracts were prepared according to a procedure
similar to that used by Wu et al. (37). The method is as follows: 1.00 g of
freeze-dried powder sample from tomato (or carrot) was mixed with 5 g of
sea sand. Sample and sand were transferred to an 11 mL extraction cell and
were extracted with ethanol/chloroform/D2O (50:50:1, v/v/v) three times,
using an ASE-200 accelerated solvent extractor (DionexCorp., Sunnyvale,
CA). The extracts were combined, and the volume was adjusted to 25.0 mL
with the same solvent in a volumetric flask. This solution was used to
measure the SOAC value.
Measurements of UV-Vis Absorption Spectra and Molar Ex-
tinction Coefficients of Carotenoids 1-8 and DPBF. Carotenoids
(3-4 mg) were dissolved into 200 mL of ethanol/chloroform/D2O solu-
tion. Then, each of 1.00, 2.00, or 3.00 mL of the above solution was diluted
to 10.0 mL, and the measurement of UV-vis spectrum was performed for
each solution. The values of molar extinction coefficients (ε1) at λmax1 for
the three solutions showed good accordance with each other. The experi-
mental errors in ε1 values were <5%, indicating that carotenoids were
completely dissolved in the solution. Similar measurements were repeated
three times for each carotenoid, and the value of ε1 was determined.
Measurement of UV-vis absorption spectrum of DPBF was also per-
In the present work, first, the measurements of UV-vis
absorption spectra were performed for eight kinds of carotenoids
1-8 (lycopene (Lyc, 1), astaxanthin (Ast, 2), β-carotene (β-Car, 3),
capsanthin (Cap, 4), zeaxanthin (Zea, 5), R-carotene (R-Car, 6),
lutein (Lut, 7), β-cryptoxanthin (β-Cry, 8)), to determine the values
of wavelengths of absorption maxima (λmax) and correct molar
extinction coefficients (ε) in ethanol/chloroform/D2O (50:50:1, v/v/v)
solution. Second, the stability of carotenoids 1-8 in ethanol/
chloroform/D2O solution was ascertained by the measure-
ment of UV-vis absorption spectra at 35 °C. Third, the
quenching rates (kQ) of 1O2 by carotenoids 1-8, R-tocopherol,
and two kinds of food extracts (from tomato and carrot) were
measured in ethanol/chloroform/D2O at 35 °C, by using the
above competition reaction method (21-24). R-Tocopherol
was used as a standard compound. Furthermore, the chemical
i
formed five times in ethanol/chloroform/D2O solution. The values of λmax
and the average values of εi (εAv ) obtained for carotenoids 1-8 and DPBF
i
are summarized in Table 1.
Measurements of Rate Constants (kQ). Measurements of rate
constants (kQ) were performed in ethanol/chloroform/D2O (50:50:1, v/v/v)
solution, by using a Shimadzu UV-vis spectrophotometer (UV-1800),
equipped with a six-channel cell positioner and an electron-temperature
control unit (CPS-240A). The rate constants, kQ (S) and kQ (t1/2), were
determined by analyzing the first-order rate constant (S) and the half-life
(t1/2) of the decay curve of DPBF with carotenoids, respectively, as
described under Results and Discussion. All measurements were per-
formed at 35.0 ( 0.5 °C.
1
reaction of carotenoids 1-8 with O2 in solution was studied
1
spectrophotometrically, by reacting the carotenoids with O2
at 35 °C. From the results, the method to assess the total SOAC
of the antioxidants included in foods and plants has been
proposed.
MATERIALS AND METHODS
Materials. Lutein, β-cryptoxanthin, zeaxanthin, and capsanthin were
obtained from Extrasynthese (Genay, France). R- and β-carotene and
lycopene were obtained from Wako Chemicals, Japan. Astaxanthin was
Measurements of UV-vis absorption spectra were performed under
nitrogen atmosphere, to avoid the degradation of carotenoids 1-8,
vegetable extracts, R-tocopherol, and DPBF. A closed system (that is, a
cuvette with a sealing cap) was used to avoid loss of solvent, because the
solvents show high vapor pressures at 35 °C.
obtained from Funakoshi Co. Ltd., Japan. -R-Tocopherol (Eisai Food
D
Chemicals Co. Ltd., Japan) and DPBF (Tokyo Kasei Chemicals, Japan)
are commercially available. Na2MoO4 2H2O (KANTO Chemical Co.,
The production of 1O2 due to the thermal decomposition of EP occurs
at 25 °C. Consequently, sample preparation was performed by adding
1.00 mL of EP solution to 2.00 mL of solution including DPBF and
an antioxidant in a quartz cuvette at ∼20 °C to avoid the decomposition
of EP, and measurements of the UV-vis absorption spectra were then
started at 35 °C. We took about 5 min to prepare solutions of six cuvettes.
3
Inc., Japan) is also commercially available. Sea sand was obtained from
Wako Chemicals, Japan.
3-(4-Methyl-1-naphthyl)propionic acid (EP-precursor) was prepared
according to a published procedure (33-35). 3-(1,4-Epidioxy-4-methyl-
1,4-dihydro-1-naphthyl)propionic acid (endoperoxide, EP) was prepared

Article
J. Agric. Food Chem., Vol. 58, No. 18, 2010 9969
About 3 min was necessary before the solution temperature in the cuvette
rose from ∼20 to 35 °C.
In eq 3, SBlank and SR-Toc are slopes of the first-order plots (that is,
ln(absorbance) versus t plots) of disappearance of DPBF in the
absence or presence of R-tocopherol, respectively (see Figure 2B).
kd is the rate of natural deactivation of 1O2 in the solvent.
As described above, eq 3 was derived from the steady-state
approximation to 1O2 (40, 41), that is, by assuming that
RESULTS AND DISCUSSION
UV-Vis Absorption Spectra and Stability of Carotenoids 1-8 in
Solution. The wavelengths of absorption maxima (λmax) of
UV-vis absorption spectra were reported for many carote-
noids (38, 39). However, carotenoids for which molar extinction
coefficients (ε) have been reported are very limited (38). One
reason for this is that carotenoids, except for β-carotene, are
costly, and at least several milligrams of carotenoids is necessary
to determine the ε value. Measurements of the quenching rate
(kQ) for phenolic antioxidants (such as vitamin E analogues,
polyphenols, and biological hydroquinones) were performed in
ethanol (21-24). However, the solubility of carotenoids is low in
organic solvents, including ethanol, and depends on the mole-
cular structure of carotenoids. Carotenoids are soluble in chloro-
form. However, the degradation of DPBF occurs in chloroform.
Therefore, measurements of the kQ values were performed in
ethanol/chloroform/D2O (50:50:1, v/v/v) solution. This solution
was used by Di Mascio et al. (18, 19) and Beutner et al. (39) to
measure the kQ values of carotenoids.
- d½1O2ꢁ=dt ¼ - kf ½EPꢁ þ kDPBF½DPBFꢁ½1O2ꢁ
þ kQ½R-Tocꢁ½1O2ꢁ þ kd½1O2ꢁ ¼ 0
ð4Þ
where kf and kDPBF are the formation rate constant of 1O2 from
EP at 35 °C and the second-order rate constant for the chemical
reaction of DPBF with 1O2, respectively. As shown in Figure 2B,
the first-order plots indicate straight lines at ∼5 < t < ∼60 min.
At t < ∼5 min, the plots deviate from straight lines, because it
takes about 5 min for the solution temperature in quartz cuvette
to increase from 20 to 35 °C and for the steady-state approxima-
tion (eq 4) to be fulfilled. This approximation will be broken at
t > ∼60 min, because the formation rate of 1O2 (kf[EP]) in eq 4
decreases gradually at t > ∼60 min due to the consumption of
EP. Therefore, the analysis of the decay curve was performed
at ∼5 < t < ∼60 min. This is an important condition to obtain a
correct rate constant (kQ) for antioxidants.
The measurements of UV-vis absorption spectra were care-
fully performed for carotenoids 1-8 in ethanol/chloroform/D2O,
and the values of λmax and ε obtained are summarized in Table 1.
The values of ε are important to determine the concentrations of
carotenoids used for the reaction and to obtain the correct rate
constant (kQ). As listed in Table 1, absorption spectra of car-
otenoids 1-8 overlap with that of DPBF at λmax = 413 nm.
Therefore, the correction of baseline due to the absorption of
carotenoids 1-8 at 413 nm is necessary (see Figure 1). The εAv
values of carotenoids 1 -8 at 413 nm are also listed in Table 1.
It is important to investigate the stability of carotenoids 1-8
at 35 °C in ethanol/chloroform/D2O solution because the correc-
tions of the baseline due to the absorption of carotenoids at 413 nm
are necessary for the analysis of the rate constant (kQ), as described
below. Therefore, the measurements of the time dependence of the
absorbance of carotenoids at 413 nm were performed at 35 °C in
solution under nitrogen atmosphere. As shown in Figure 1A, line e,
the change of the absorbance of β-carotene (1.41 ꢀ 10-5 M) was
negligible at t=0-260 min. Similar results were obtained for the
other carotenoids, indicating that these carotenoids are stable in
ethanol/chloroform/D2O at 35 °C (data are not shown).
A plot of SBlank/SR-Toc versus the concentration of R-tocopherol
([R-Toc]) is shown in Figure 2C. The rate constant (kQ) was
calculated by using the value of kd in ethanol/chloroform/D2O
(50:50:1, v/v/v) (kd=3.03 ꢀ 105 s-1) (18, 19, 39). The kQ value
obtained is 1.24 ꢀ 108 M-1 s-1. Similar measurements were
repeated three times, and the individual and average kQ values
are listed in Table 2.
Similarly, solutions containing EP (4.56 ꢀ 10-4 M), DPBF
(5.65 ꢀ 10-5 M), and various amounts of β-carotene (0-1.41 ꢀ
10-5 M) in ethanol/chloroform/D2O were reacted at 35 °C (see
Figure 1A). The disappearance of DPBF was measured at 413 nm.
However, as the absorption of β-carotene overlaps with that of
DPBF at 413 nm (see Figure 1A), the correction of the baseline is
necessary for each decay curve. As shown in Figure 1A, the
measurement of the baseline was performed by observing the time
dependence of the absorption of β-carotene (1.41 ꢀ 10-5 M) at
413 nm (see line e in Figure 1A), where 1.41 ꢀ 10-5 M is the
highest concentration of β-carotene used for the measurement.
The correction of the baseline for each decay curve of DPBF was
performed by taking the absorbance of this baseline into account
and using Lambert-Beer’s equation (absorbance = ε[β-Car]).
The decay curves corrected are shown in Figure 1B. The ln-
(absorbance) versus t plot is shown in Figure 1C, indicating that
the first-order plots are straight lines at ∼5 < t < ∼60 min, as
Overall Rate Constants (kQ) for the Reaction of 1O2 with
r-Tocopherol and Carotenoids 1-8. Singlet oxygen was gene-
rated by the thermal decomposition of the endoperoxide (EP)
(see eq 2) (21-24). The UV absorption spectra of EP-precursor
and EP show absorption maxima at λmax (ε) = 290 (7670),
281 (6350), and 239 nm (9130 M-1 cm-1) and λmax (ε)=236 nm
(1630 M-1 cm-1), respectively, in ethanol/chloroform/D2O (50:50:1,
v/v/v) solution (see Table 1). The result of the measurement of the
UV spectrum of EP indicates that the powder sample of EP includes
95.6% EP and 4.4% EP-precursor unreacted.
observed for R-tocopherol. A plot of SBlank/Sβ-Car versus [β-Car]
β-Car
is shown in Figure 1D. The kQ
value obtained is 1.10 ꢀ
1010 M-1 s-1. Similar measurements were performed for β-carotene
three times. As listed in Table 2, the kQ
β-Car
values obtained are
nearly equal to each other.
Similar measurements were performed for carotenoids 1, 2,
and 4-8 in ethanol/chloroform/D2O solution. SBlank/SCarotenoid
versus [Carotenoid] plots are shown in Figure 3A. Measurements
were repeated three times for each carotenoid, to obtain the
correct kQ values. The individual kQ values obtained and the
average values of kQ (kQ (av)) for carotenoids 1-8 are summar-
ized in Table 2, together with those obtained for R-tocopherol.
The experimental error in kQ value for each carotenoid was (5%
at maximum.
DPBF was used as the UV-vis absorption probe. Figure 2A
shows an example of the reaction between DPBF (7.95 ꢀ 10-5 M)
and EP (4.68 ꢀ 10-4 M) in the absence or presence of R-toco-
pherol ((0-9.46) ꢀ 10-4 M) in ethanol/chloroform/D2O solution
at 35 °C. By the reaction, the disappearance of DPBF at λmax
=
1
413 nm due to the chemical reaction between DPBF and O2
produced was observed. The overall rate constant kQ (= kq þ kr)
for the reaction of 1O2 with R-tocopherol was determined by eq 3
(Stern-Volmer plot), as reported in previous works (40, 41).
The kQ values of carotenoids 1-8 decrease in the order lyco-
pene > astaxanthin > β-carotene ∼ capsanthin ∼ zeaxanthin >
R-carotene > lutein > β-cryptoxanthin in ethanol/chloroform/D2O.
SBlank=SR-Toc ¼ 1 þ ðkQR-Toc=kdÞ½R-Tocꢁ
ð3Þ

9970 J. Agric. Food Chem., Vol. 58, No. 18, 2010
Ouchi et al.
Figure 1. (A) Change in absorbance of DPBF at 413nmduring thereaction of DPBFwith1O2 inthe absence (line a) and presence (lines b-d) of β-carotene
in ethanol/chloroform/D2O at 35 °C. [DPBF]t=0 = 5.65 ꢀ 10-5 M and [EP]t=0 = 4.56 ꢀ 10-4 M. The values of [β-Car]t=0 are shown in A. Lines e and f indicate
the time dependence of absorbance of β-carotene (1.41 ꢀ 10-5 M) at 413 nm in the absence and presence of EP, respectively, where DPBF is not included.
(B) ChangeinabsorbanceofDPBF, wherethe correctionofbaselineduetoβ-carotenewasperformed(seetext). (C) Plot of ln(absorbance) versust. (D) Plot
of SBlank/Sβ-Car versus [β-Car]. (E) Plot of t1/2β-Car/t1/2Blank versus [β-Car].
However, the difference among the kQ values is not remarkable.
The value of lycopene is only 1.89 times larger than that of
β-cryptoxanthin, although the value of lycopene is 105 times larger
than that of R-tocopherol.
Measurements of the kQ values have been reported for many
carotenoids. However, for instance, the kQ values reported for
β-carotene in solutions vary from 5 ꢀ 109 to 30 ꢀ 109 M-1 s-1 (18).
The variety may be due to the difference in the experimental
methods used, solvent, temperature, etc. Di Mascio et al. (18, 19)
and Beutner et al. (39) reported the kQ values of carotenoids 1-3
and 5-8 obtained by measuring the photoemission of 1O2 at
1270 nm in ethanol/chloroform/D2O (50:50:1, v/v/v). The values
are listed in Table 2. The order of the kQ values for carotenoids 1-3
and 5-8 is the same as that obtained in the present work. However,
the value of lycopene is 5.2 times as large as that of β-cryptoxanthin,
and the kQ values of carotenoids 1-3 and 5-8 obtained in the

Article
J. Agric. Food Chem., Vol. 58, No. 18, 2010 9971
Figure 2. (A) Change in absorbance of DPBF at 413 nm during the reaction of DPBF with 1O2 in the absence and presence of R-tocopherol in ethanol/
chloroform/D2O at 35 °C. [DPBF]t=0 = 7.95 ꢀ 10-5 M and [EP]t=0 = 4.68 ꢀ 10-4 M. The values of [R-Toc]t=0 are shown in A. (B) Plot of ln(absorbance) versus t.
(C) Plot of SBlank/SR-Toc versus [R-Toc]. (D) Plot of t1/2R-Toc/t1/2Blank versus [R-Toc].
persent work are 1.5-4.1 times larger than those of the correspond-
ing carotenoids reported by Di Mascio et al. The reason for such a
difference is not clear at present.
Measurement of the Rate Constant (kQ) Based on the Half-Life
(t1/2) of DPBF. It will be better to use t1/2R-Toc instead of SR-Toc to
assay the kQ value (that is, the SOAC value) of antioxidants and
vegetable extracts in solution, because the analysis of t1/2R-Toc is
more direct and easier than that of SR-Toc. Furthermore, the use of
the half-life (t1/2R-Toc) is better than that of the first-order rate
constant (SR-Toc) to define the SOAC value for vegetable extracts.
The decay rate (SR-Toc) of DPBF due to the chemical reac-
tion with 1O2 decreases with increasing the concentration of
R-tocopherol, as shown in Figure 2A. The kQ values were
determined by using eq 3. As the decay of DPBF is a first-order
reaction, there exists the following relationship between SR-Toc
and half-life (t1/2R-Toc) at ∼5 < t < ∼60 min:
Chemical Reaction of 1O2 with Carotenoids 1-8. The measure-
ment of the rate constant (kr) for the chemical reaction of
β-carotene with 1O2 was performed by a few investigators (42-44),
showing that the kr values (3.66 ꢀ 107, 3.8 ꢀ 106, and 2.0 ꢀ
106 M-1 s-1) are much smaller than the kq values (2.99 ꢀ 1010,
7.00 ꢀ 109, and 1.2 ꢀ 1010 M-1 s-1) in n-hexane, CCl4, and
acetonitrile/benzene (4:1, v/v) solution, respectively. β-Carotene
1
quenches O2 through a very efficient physical reaction. On the
other hand, cyclic endoperoxides of β-carotene were obtained as
products of chemical quenching of 1O2, if the generation of 1O2 was
performed by using a photosensitizer (45, 46). However, measure-
ments of the kr values and the 1O2-quenching products for the other
carotenoids have not been performed, as far as we know.
R-Toc
A1=2 ¼ A0 expð- SR-Toc ꢀ t1=2
Þ
ð5Þ
R-Toc
A0 is the absorbance of DPBF at t0=5 min, and (t1/2
þ t0) is
In the present work, the chemical reaction of carotenoids 1-8
with 1O2 in ethanol/chloroform/D2O solution was studied spec-
the time at which A1/2=A0/2 (see Figure 2A). We can easily obtain
1
the following relationship from eq 5:
trophotometrically, by reacting the carotenoids with O2 gener-
ated by the thermal decomposition of the EP at 35 °C. As shown
R-Toc
ð6Þ
t1=2
¼ ln 2=SR-Toc
1
in Figure 1A, line f, the chemical reaction between O2 and
β-carotene is very slow, and a measurable change in the absorbance
of β-carotene was not observed at 413 nm. Similarly, by reacting
carotenoids 1-8 with 1O2 for 2-4 h at 35 °C, no changes of
UV-vis spectra of carotenoids were observed (data are not shown).
Consequently, the kQ values obtained for carotenoids are thought
to be due to physical quenching (kq), that is, kQ ≈ kq.
By substituting eq 6 into eq 3, we can obtain eq 7a
t1=2R-Toc=t1=2
¼ 1 þ ðkQR-Toc=kdÞ½R-Tocꢁ
ð7aÞ
Blank
R-Toc
where t1/2Blank and t1/2
are the half-lives in the absence and
presence of R-tocopherol, respectively. Equation 7a indicates that

9972 J. Agric. Food Chem., Vol. 58, No. 18, 2010
Ouchi et al.
Sample
Sample
Table 2. Second-Order Rate Constants (kQ
and kQ
(av)) and
Relative Rate Constants (kQSample (Av) f kQSample (av)) for the Reaction of
1O2 with Carotenoids 1-8 and R-Tocopherol in Ethanol/Chloroform/D2O
(50:50:1, v/v/v)
kQSample/M-1 s-1
Sample
kQ
/
kQSample (av) a/ Di Mascio et al. b kQSample (av)/
sample
M-1 s-1
M-1 s-1
(Beutner et al.) c kQR-Toc (av)
R-tocopherol (R-Toc)
1.24 ꢀ 108
1.33 ꢀ 108
1.36 ꢀ 108
1.31 ꢀ 108
8.5ꢀ 107
1.00
lycopene (Lyc, 1)
1.43 ꢀ 1010
1.31 ꢀ 1010
1.41 ꢀ 1010
1.38 ꢀ 1010
1.18 ꢀ 1010
1.08 ꢀ 1010
1.06 ꢀ 1010
1.05 ꢀ 1010
9.76 ꢀ 109
9.24 ꢀ 109
7.31 ꢀ 109
9.4 ꢀ 109
105
(8.8 ꢀ 109)
astaxanthin (Ast, 2)
β-carotene (β-Car, 3)
capsanthin (Cap, 4)
zeaxanthin (Zea, 5)
R-carotene (R-Car, 6)
lutein (Lut, 7)
1.16 ꢀ 1010
1.23 ꢀ 1010
1.14 ꢀ 1010
7.3 ꢀ 109
90.1
(9.0 ꢀ 109)
1.10 ꢀ 1010
1.04 ꢀ 1010
1.11 ꢀ 1010
4.2 ꢀ 109
82.4
80.9
80.2
74.5
70.5
55.8
(8.4 ꢀ 109)
9.36 ꢀ 1010
1.16 ꢀ 1010
1.07 ꢀ 1010
1.04 ꢀ 1010
1.02 ꢀ 109
1.11 ꢀ 1010
3.0 ꢀ 109
5.7 ꢀ 109
2.4 ꢀ 109
1.8 ꢀ 109
1.08 ꢀ 1010
8.82 ꢀ 109
9.66 ꢀ 109
9.16 ꢀ 109
9.14 ꢀ 109
9.43 ꢀ 109
Figure 3. (A) Plot of SBlank/SCarotenoid versus concentrations of carote-
noids (lycopene, astaxanthin, capsanthin, zeaxanthin, R-carotene, lutein,
versus concentra-
β-cryptoxanthin (Crp, 8) 7.04 ꢀ 109
7.25 ꢀ 109
Blank
and β-cryptoxanthin). (B) Plot of t1/2Carotenoid/t1/2
tions of carotenoids.
7.63 ꢀ 109
a Experimental errors in the rate constants (kQ
(av)) were estimated to be
Sample
For instance, the value of t1/2R-Toc for [R-Toc]=9.46 ꢀ 10-4 M is
81.4 min, when the correction is performed by using eq 9. On
the other hand, if the correction is not performed, the value of
<5%. b Values reported by Di Mascio et al. (18, 19). c Values reported by Beutner
et al. (39).
R-Toc
Blank
t1/2R-Toc obtained is 97.6 min, showing a larger value. The correc-
the kQ
value can be obtained from t1/2R-Toc/t1/2
versus
R-Toc
R-Toc
tion is very important to obtain a correct t1/2
value. In
[R-Toc] plot. In fact, t1/2
concentration of R-tocopherol, as shown in Figure 2D.
increases linearly with increasing
conclusion, the kQ value can be calculated from t1/2R-Toc/t1/2
versus [R-Toc] plot, as shown in Figure 2D. The kQR-Toc (t1/2) value
(1.13 ꢀ 108 M-1 s-1) obtained by using eq 7a is nearly equal to the
kQR-Toc (S) value (1.24 ꢀ 108 M-1 s-1) obtained by using eq 3 (see
Table 3).
Blank
If the concentration of R-tocopherol is low ([R-Toc]=0, 1.89 ꢀ
R-Toc
10-5, 3.78 ꢀ 10-5, 5.67 ꢀ 10-4 M) and the value of t1/2
is
<60 min (see Figure 2A), we can directly determine the half-life
from the decay curve of DPBF. However, if the concentration of
R-tocopherol is large (7.57 ꢀ 10-4 or 9.46 ꢀ 10-4 M), the time (t)
at which A1/2=A0/2 (that is, the seeming half-life) is >60 min. In
such a case, we cannot directly determine the t1/2R-Toc value from
the decay curve of DPBF, because the decay does not follow the
first-order kinetics, as described above. The half-life must be
calculated in the following way.
A similar analysis of the half-life (t1/2β-Car) was performed for
β-Car
β-carotene (see Figure 1B). The kQ
(t1/2) value (1.16 ꢀ 1010
M-1 s-1) obtained is nearly equal to the kQβ-Car (S) value (1.10 ꢀ
1010 M-1 s-1) (see Figure 1 and Table 3).
Carotenoid
Blank
Similarly, t1/2
and t1/2
values were calculated
for the carotenoids 1, 2, 4-8. The t1/2Carotenoid/t1/2Blank versus
[Carotenoid] plots are shown in Figure 3B, indicating the linear
There exists the following relationship between Am/n and
Blank
correlation between t1/2Carotenoid/t1/2
As summarized in Table 3, the kQ
and [Carotenoid].
(t1/2) values obtained
(S) values obtained by
R-Toc
tm/n
.
Car
R-Toc
Car
Am=n ¼ A0 expð- SR-Toc ꢀ tm=n
Þ
ð8Þ
are close to the corresponding kQ
the analyses of SCar values. The result indicates that the
R-Toc
where Am/n=(m/n)A0 and (tm/n
þ t0 (t0=5 min)) is the time at
analysis based on half-life is reliable. The values of the ratio
Car
Car
which Am/n=(m/n)A0. By substituting eq 6 into eq 8, we can obtain
(kQ
(t1/2)/kQ
(S)) are listed in the last column of
eq 9, that is, we can estimate t1/2R-Toc from tm/n
.
R-Toc
Table 3.
Development of Singlet Oxygen Absorption Capacity (SOAC)
Assay Method. There are the following relationships (eqs 10
R-Toc
R-Toc
t1=2
¼ tm=n
ðlnð1=2Þ=lnðm=nÞÞ
ð9Þ

Article
J. Agric. Food Chem., Vol. 58, No. 18, 2010 9973
Sample
Sample
Blank
Table 3. Second-Order Rate Constants (kQ
(S) and kQ
(t1/2))
versus
Obtained from SBlank/SSample versus [Sample] and t1/2Sample/t1/2
[Sample] Plots, Respectively, and the Ratios of the Rate Constants (kQ
Sample
(t1/2)/kQSample (S))
kQSample (S)/M-1 s-1 kQSample (t1/2)/M-1 s-1a kQSample (t1/2)/
sample
(SBlank/SSample plot) (t1/2Sample/t1/2Blank plot) kQSample (S)
R-tocopherol
1.24 ꢀ 108
1.33 ꢀ 108
1.36 ꢀ 108
av: 1.31 ꢀ 108
1.13 ꢀ 108
1.22 ꢀ 108
1.52 ꢀ 108
av: 1.29 ꢀ 108
0.911
0.917
1.07
av: 0.984
lycopene
1.43 ꢀ 1010
1.31 ꢀ 1010
1.41 ꢀ 1010
av: 1.38 ꢀ 1010
1.32 ꢀ 1010
1.16 ꢀ 1010
1.29 ꢀ 1010
av: 1.26 ꢀ 1010
0.923
0.885
0.914
av: 0.913
astaxanthin
β-carotene
capsanthin
zeaxanthin
R-carotene
lutein
1.16 ꢀ 1010
1.23 ꢀ 1010
1.14 ꢀ 1010
av: 1.18 ꢀ 1010
1.09 ꢀ 1010
1.18 ꢀ 1010
1.03 ꢀ 1010
av: 1.10ꢀ 1010
0.940
0.959
0.904
av: 0.932
1.10 ꢀ 1010
1.04 ꢀ 1010
1.11 ꢀ 1010
av: 1.08 ꢀ 1010
1.16ꢀ 1010
1.01 ꢀ 1010
9.96 ꢀ 109
1.00
0.971
0.892
av: 0.976
av: 1.06 ꢀ 1010
9.36 ꢀ 109
1.04 ꢀ 1010
1.16 ꢀ 1010
1.09 ꢀ 1010
av: 1.07 ꢀ 1010
1.11
1.00
1.16 ꢀ 1010
1.07 ꢀ 1010
av: 1.06 ꢀ 1010
1.02
av: 1.01
1.04 ꢀ 1010
1.02 ꢀ 1010
1.11 ꢀ 1010
av: 1.05 ꢀ 1010
1.06 ꢀ 1010
1.00 ꢀ 1010
9.56 ꢀ 109
1.03
0.985
0.861
av: 0.961
av: 1.01 ꢀ 1010
Figure 4. (A) Change in absorbance of DPBF at 413 nm during the
1
reaction of DPBF with O2 in the absence or presence of sample
1.08 ꢀ 1010
9.85 ꢀ 109
8.03 ꢀ 109
9.01 ꢀ 109
av: 8.96 ꢀ 109
0.912
0.910
8.82 ꢀ 109
9.66 ꢀ 109
0.933
av: 0.918
(R-tocopherol or β-carotene) in ethanol/chloroform/D2O at 35 °C.
[DPBF]t=0 = 6.94 ꢀ 10-5 M and [EP]t=0 = 4.52 ꢀ 10-4 M. The values of
[R-Toc]t=0 and [β-Car]t=0 are shown in Figure A. (B) Change in
absorbance of DPBF, where the correction of baseline due to β-carotene
was performed (see text).
av: 9.76 ꢀ 109
9.16 ꢀ 109
9.14 ꢀ 109
9.43 ꢀ 109
av: 9.24 ꢀ 109
9.30 ꢀ 109
9.01 ꢀ 109
8.83 ꢀ 109
av: 9.05 ꢀ 109
1.01
0.985
0.936
av: 0.979
Equation 11 indicates that the half-life (t1/2Sample) of the sample
increases linearly with increasing concentration of the sample and
depends on the product of kQSample and [Sample], if the concen-
tration of R-tocopherol is constant. From eq 11, we can derive
eq 12 and define the relative SOAC value as follows:
β-cryptoxanthin
7.04 ꢀ 109
7.25 ꢀ 109
7.63 ꢀ 109
av: 7.31 ꢀ 109
7.55 ꢀ 109
7.01 ꢀ 109
6.27 ꢀ 109
av: 6.94 ꢀ 109
1.07
0.967
0.822
av: 0.949
a Experimental errors in the rate constants (kQSample (t1/2) (av)) were estimated to
be <10%.
relative SOAC value ðgiven in molar units ðM ¼ mol=LÞÞ
Blank
Blank
¼ fðt1=2Sample - t1=2
Þ=ðt1=2R-Toc - t1=2
Þg
ꢀ½R-Tocꢁ=½Sampleꢁ ¼ kQSample=kQ
ð12Þ
R-Toc
Sample
and 7b) between the half-lives (t1/2
concentrations ofcarotenoid sample ([Sample]) and R-tocopherol
([R-Toc]), respectively.
and t1/2R-Toc) and the
Equation 12 indicates that the SOAC value corresponds to the
ratio (kQSample/ kQR-Toc) of the quenching rate of singlet oxygen
(kQSample) by sample to that (kQR-Toc) by R-tocopherol. R-Toco-
pherol is used as a standard compound of SOAC assay.
t1=2Sample=t1=2
¼ 1 þ ðkQSample=kdÞ½Sampleꢁ
ð10Þ
ð7bÞ
Blank
The measurement of the SOAC value was performed for
β-carotene and astaxanthin. Figure 4A shows an example of the
reaction between DPBF (6.94 ꢀ 10-5 M) and EP (4.52 ꢀ 10-4 M)
in the absence ((a) Blank) and presence of antioxidants ((b) [R-
Toc]=5.00 ꢀ 10-4 M, (c) [R-Toc]=1.00 ꢀ 10-3 M, (d) [β-Car]=
3.24 ꢀ 10-6 M, (e) [β-Car]=6.46 ꢀ 10-6 M, (f) [β-Car]=9.70 ꢀ
10-6 M) in ethanol/chloroform/D2O solution at 35 °C. The
disappearance of DPBF at λmax =413 nm due to the chemical
t1=2R-Toc=t1=2
¼ 1 þ ðkQR-Toc=kdÞ½R-Tocꢁ
Blank
By eliminating kd from eqs 7b and 10, we can obtain the following
relationship:
Blank
Blank
ðt1=2Sample - t1=2
Þ=ðt1=2R-Toc - t1=2
Þ
Sample
R-Toc
1
¼ kQ
½Sampleꢁ=kQ
½R-Tocꢁ
ð11Þ
reaction with O2 was observed. The correction of the baseline

9974 J. Agric. Food Chem., Vol. 58, No. 18, 2010
Ouchi et al.
Table 4. Half-Lives (t1/2) and Employed Concentrations of R-Tocopherol, Sample, and DPBF, Relative SOAC Values, and Relative Rate Constants (kQSample (S)/
kQR-Toc (S)) in Ethanol/Chloroform/D2O
sample
t1/2R-Toc/min ([R-Toc]/M)
t1/2Sample/min ([Sample]/M)
t1/2Blank/min ([DPBF]/M)
16.6 (6.94 ꢀ 10-5
relative SOAC valuea
kQSample (S) /kQR-Toc (S)
β-carotene
50.5 (5.00 ꢀ 10-4
)
35.0 (3.26 ꢀ 10-6
58.7 (6.46 ꢀ 10-6
76.0 (9.70 ꢀ 10-6
)
)
)
)
83.8
96.1
90.3
82.4
84.1 (10.0 ꢀ 10-4
)
35.0 (3.26 ꢀ 10-6
58.7 (6.46 ꢀ 10-6
76.0 (9.70 ꢀ 10-6
)
)
)
84.1
96.5
90.7
av = 90.3
astaxanthin
33.5 (2.00 ꢀ 10-4
54.7 (5.00 ꢀ 10-4
)
33.5 (2.80 ꢀ 10-6
55.7 (5.59 ꢀ 10-6
74.2 (8.39 ꢀ 10-6
)
)
)
15.8 (6.99 ꢀ 10-5
)
71.4
80.7
78.7
90.1
)
33.5 (2.80 ꢀ 10-6
)
)
81.3
91.7
55.7 (5.59 ꢀ 10-6
74.2 (8.39 ꢀ 10-6
)
89.5
av = 82.2
a Experimental errors in the relative SOAC values (av) were estimated to be <10%.
Table 5. Half-Lives (t1/2) and Employed Concentrations of R-Tocopherol, Sample (Tomato and Carrot Extracts) and DPBF, and Relative SOAC Values in Ethanol/
Chloroform/D2O
vegetable extract
t1/2R-Toc/min ([R-Toc]/(g/L))
t1/2Sample/min ([Sample]/(g/L))
t1/2Blank/min ([DPBF]/M)
relative SOAC value
tomato-1
50.8 (2.17 ꢀ 10-1
)
23.3 (5.33 ꢀ 10-1
25.1 (8.00 ꢀ 10-1
32.6 (1.60)
)
)
18.2 (5.55 ꢀ 10-5
)
0.0633
0.0572
0.0598
42.8 (2.67)
0.0612
av = 0.0604
tomato-2
25.3 (7.16 ꢀ 10-2
)
37.0 (2.67)
47.7 (4.00)
76.2 (8.00)
118.5 (13.3)
21.8 (6.07 ꢀ 10-5
)
0.0638
0.0651
0.0625
0.0643
av = 0.0639
carrot
55.6 (2.16 ꢀ 10-1
)
29.3 (1.07)
33.6 (1.60)
48.0 (3.20)
63.6 (5.33)
18.2 (5.55 ꢀ 10-5
)
0.0503
0.0501
0.0528
0.0497
av = 0.0507
was performed by using the value of ε at 413 nm of β-carotene (see
Table 1). The values of half-life (t1/2R-Toc, t1/2β-Car, t1/2Blank) were
calculated according to the method described in a previous
section (see Table 4). As the measurements were performed for
two different concentrations of R-tocopherol and three concen-
trations of β-carotene, we can determine six sets of relative SOAC
values, using eq 12, as listed in Table 4. The relative SOAC values
(83.8-96.5, av=90.3) obtained for β-carotene are similar to each
other and are close to the ratio (kQβ-Car/kQR-Toc=82.4, Table 2) of
the quenching rateconstant ofβ-carotene to that ofR-tocopherol,
as expected from eq 12.
follows. The preparation of tomato and carrot extracts was
described under Material and Methods. For example, the toma-
to-1 extract prepared from 1.00 g of freeze-dried powder was
dissolved in 25 mL of ethanol/chloroform/D2O (50:50:1, v/v/v)
solution. From this solution, four concentrations of tomato
extract (tomato-1, see Table 5) were prepared, where the con-
centration of tomato-1 extract was defined as grams per liter
because we cannot use the molar concentration (M=mol/L) for
tomato extracts. Similarly, the concentration of R-tocopherol as a
standard sample was expressed as grams per liter. The concen-
tration of R-tocopherol used for the measurement was 5.03 ꢀ
10-4 M, that is, 2.17 ꢀ 10-1 g/L (see Table 5).
A similar measurement was performed for astaxanthin. The six
sets of relative SOAC values obtained are listed in Table 4. The
The tomato-1 extract shows an UV-vis absorption spectrum
at 400-550 nm, suggesting that high concentrations of carote-
noids are included in tomato-1 (see Figure 5A) (5). Decay curves
relative SOAC values (71.4-91.7, av=82.2) obtained for astax-
Ast
anthin are similar to each other and are close to the ratio (kQ
kQ
/
R-Toc
1
= 90.1, Table 2) of the quenching rate constant of
of the absorbance of DPBF due to the reaction with O2 for
astaxanthin to that of R-tocopherol. The result indicates that
the definition of eq 12 is useful for the estimation of the SOAC
value of the carotenoids.
Application of SOAC Assay Method to Vegetable Extracts.
Application of SOAC method to vegetable extracts (tomato and
carrot) was performed. The preliminary results obtained are as
tomato-1 extract are shown in Figure 5B. Baseline corrections
were performed by using an absorbance at 413 nm of UV-vis
absorption spectrum in Figure 5A, and the decay curves corrected
are shown in Figure 5C. ln [Absorbance] versus t plots are shown
in Figure 5D, indicating that the decay of DPBF for tomato-1
extract also follows first-order kinetics at ∼5 < t < ∼60 min.

Article
J. Agric. Food Chem., Vol. 58, No. 18, 2010 9975
Figure 5. (A) Absorption spectrum of tomato-1 extract in ethanol/chloroform/D2O. The concentration of tomato extract is 2.67 g/L. (B) Change in absorbance
of DPBF at 413 nm during the reaction of DPBF with 1O2 in the absence or presence of sample (R-tocopherol and tomato extracts) in ethanol/chloroform/D2O
at 35 °C. [DPBF]t=0 = 5.55 ꢀ 10-5 M and [EP]t=0 = 4.35 ꢀ 10-4 M. The values of [R-Toc]t=0 and [Tomato]t=0 are shown in B. (C) Change in absorbance of
DPBF, where the correction of baseline due to tomato extract was performed (see text). (D) Plot of ln(absorbance) versus t. (E) Plot of SBlank/STomato versus
concentration of tomato extract. (F) Plot of t1/2Tomato/t1/2Blank versus concentration of tomato extract.
Table 6. Rate Constants (kQSample (S) and kQSample (t1/2) (in L g-1 s-1 Units)) Obtained from SBlank/SSample versus [Sample] (in g/L Units) and t1/2Sample/t1/2
Blank
versus [Sample] Plots, Respectively, the Ratios (kQSample (t1/2)/kQSample (S)), and Relative SOAC Values for the Reaction of 1O2 with Vegetable Extracts in Ethanol/
Chloroform/D2O
kQSample (S)/(L g-1 s-1
(SBlank/SSample plot)
)
kQSample (t1/2)/(L g-1 s-1
(t1/2Sample/t1/2Blank plot)
)
kQSample (t1/2) /
kQSample (S)
relative
SOAC value
vegetable extract
tomato-1
tomato-2
carrot
1.69 ꢀ 104
1.56 ꢀ 104
1.41 ꢀ 104
1.53 ꢀ 104
1.37 ꢀ 104
1.20 ꢀ 104
0.905
0.878
0.851
av = 0.0604
av = 0.0639
av = 0.0507

9976 J. Agric. Food Chem., Vol. 58, No. 18, 2010
Ouchi et al.
Figure 6. (A) Absorption spectrum of carrot extract in ethanol/chloroform/D2O. The concentration of carrot extract is 5.33 g/L. (B) Change in absorbance of
DPBF at 413 nm during the reaction of DPBF with 1O2 in the absence or presence of sample (R-tocopherol and carrot extracts) in ethanol/chloroform/D2O at
35 °C. [DPBF]t=0 = 5.55 ꢀ 10-5 M and[EP]t=0 = 4.35 ꢀ 10-4 M. The valuesof [R-Toc]t=0 and [Carrot]t=0 are shown in B. (C) Changeinabsorbanceof DPBF,
where the correction of baseline due to carrot extract was performed (see text). (D) Plot of ln(absorbance) versus t. (E) Plot of SBlank/SCarrot versus
concentration of carrot extract. (F) Plot of t1/2Carrot/t1/2Blank versus concentration of carrot extract.
The values of t1/2Tomato, t1/2R-Toc, and t1/2Blank obtained are listed
in Table 5.
Table 6). Furthermore, the linear dependence of SBlank/SSample
and t1/2Sample/t1/2Blank values on [Tomato] suggests that the effects
of the interactions between carotenoids included in tomato
(mainly lycopene and β-carotene (5)) and among the carotenoids
and many compounds included in solution are negligible.
A similar measurement was repeated again by varying the
concentrations of tomato extract ([Tomato]) and R-tocopherol
S
Blank/STomato and t1/2Tomato/t1/2Blank versus [Tomato] (by g/L
unit) plots are shown in Figure 5, panels E and F, respectively.
Blank
Both the SBlank/SSample and t1/2Sample/t1/2
values increase
linearly with increasing concentration of tomato-1 extract
([Tomato]), and the plots show similar slopes, that is, similar
rate constants (kQTomato (S) and kQTomato (t1/2)), where the unit of
kQTomato is L g-1 s-1. The kQTomato (S) and kQTomato (t1/2) values
obtained are 1.69 ꢀ 104 and 1.53 ꢀ 104 L g-1 s-1, respectively (see
([R-Toc]). Sample of tomato-2 extract was prepared similarly,
Tomato
and the measurements of the rate constants (kQ
(S) and
Tomato
kQ
(t1/2)) were performed again (data are not shown).

Article
J. Agric. Food Chem., Vol. 58, No. 18, 2010 9977
The two rate constants obtained for tomato-2 were similar to each
other, as listed in Table 6. Furthermore, the rate constants obtained
for tomato-2 were similar to those for tomato-1, as listed in Table 6,
indicating that both the methods of sample preparation and
measurement of the rate constants are reliable for the assay of
singlet oxygen quenching activity of vegetable extracts.
for their helpful discussions. We are also grateful to Takashi
Origuchi and Miyabi Fujii of Ehime University for their kind help
in the preparation of endoperoxide (EP).
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Blank
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Blank
Blank
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ACKNOWLEDGMENT
We are very grateful to Prof. Kunihiko Tajima of Kyoto
Institute of Technology, Prof. Tateaki Ogata of Yamagata Uni-
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