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Development of Singlet Oxygen Absorption Capacity (SOAC) Assay Method. Measurements of the SOAC Values for Carotenoids and #-Tocopherol in Aqueous Triton X-100 Micellar Solution Kazuo Mukai, Aya Ouchi, Nagao Azuma, Shingo Takahashi, Koichi Aizawa, and Shin-ichi Nagaoka J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04329 • Publication Date (Web): 06 Jan 2017 Downloaded from http://pubs.acs.org on January 12, 2017
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Journal of Agricultural and Food Chemistry
1
(J. Agric. Food Chem.)
(Revised Manuscript, Second revision)
January 5, 2017
2 3
Development of Singlet Oxygen Absorption Capacity (SOAC) Assay Method.
4
Measurements of the SOAC Values for Carotenoids and α-Tocopherol in Aqueous Triton
5
X-100 Micellar Solution
6 7
Kazuo Mukai,†* Aya Ouchi,† Nagao Azuma,† Shingo Takahashi,‡ Koichi Aizawa,‡ and
8
Shin-ichi Nagaoka†
9 10
†
11
Japan
12
‡
13
Japan
Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790-8577,
Research & Development Division, Kagome Co. Ltd., Nasushiobara-shi, Tochigi 329-2762,
14 15
*To whom correspondence should be addressed. Tel: 81-89-927-9588. Fax: 81-89-927-9590.
16
E-mail: (K.M.)
[email protected] 17 18
(Received:
September
, 2016)
19 20
ABSTRACT: Recently a new assay method was proposed for quantification of the singlet
21
oxygen absorption capacity (SOAC) of antioxidants (AOs) and food extracts in homogeneous
22
organic solvents. In the present study, second-order rate constants (kQ) for the reaction of
23
singlet oxygen (1O2) with eight different carotenoids (Cars) and α-tocopherol (α-Toc) were
24
measured in aqueous Triton X-100 (5.0 wt %) micellar solution (pH 7.4, 35 oC), which was
25
used as a simple model of biomembranes. The kQ and relative SOAC values were measured
26
using UV-vis spectroscopy. The UV-vis absorption spectra of Cars and α-Toc were measured
27
in both micellar solution and chloroform, to investigate the effect of solvent on the kQ and
28
SOAC values. Furthermore, decay rates (kd) of 1O2 were measured in 0.0, 1.0, 3.0 and 5.0
29
wt % micellar solutions (pH 7.4), using time-resolved near-infrared fluorescence spectroscopy,
30
to determine the absolute kQ values of the AOs. Results obtained demonstrate that the kQ
31
values of AOs in homogeneous and heterogeneous solutions vary notably depending on (i)
32
polarity (dielectric constant (ε)) of the reaction field between AOs and 1O2, (ii) local
33
concentration of AOs, and (iii) mobility of AOs in solution. Further, the kQ and relative SOAC 1
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values obtained for the Cars in heterogeneous micellar solution differ remarkably from those
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in homogeneous organic solvents. Measurements of kQ and SOAC values in micellar solution
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may be useful for evaluating the 1O2-quenching activity of AOs in biological systems.
37 38
KEYWORDS: singlet oxygen, quenching rate, carotenoids, α-tocopherol, SOAC value,
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kinetic study, micellar solution, endoperoxide
40 41
INTRODUCTION
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Singlet oxygen (1O2) is well-known as a representative reactive oxygen species generated
43
in biological systems, and reacts with various biological targets including lipids, sterols,
44
proteins, DNA and RNA.1,2 Chemical reactions with 1O2 often induce degradation of
45
biological systems. Carotenoids and phenolic antioxidants are widely present in foods and
46
plants in high concentrations3-9 and may function as efficient 1O2 quenchers in biological
47
systems.10-14
48
Kinetic studies of the quenching reaction of 1O2 with many natural antioxidants (AOs)
49
(such as carotenoids, vitamin E homologues, and polyphenols) were recently performed in our
50
laboratory.15-18 Specifically, the overall rate constants, kQ (= which are the sum of the physical
51
quenching (kq) and chemical reaction (kr) rates; see eq 1), for reaction of AOs with 1O2 were
52
measured in ethanol/chloroform/D2O (50:50:1, v/v/v) (hereafter abbreviated as “mixed
53
solvent”) and ethanol solutions at 35oC, using a competition reaction method, where
54
endoperoxide (EP) was used as a singlet oxygen generator and 2,5-diphenyl-3,4-benzofuran
55
(DPBF) as an UV-vis absorption probe (Scheme 1). kQ
56 57
1
O2 + AO → physical quenching (kq) + chemical reaction (kr)
(1)
58 59
The second-order rate constants, kQ (S) and kQ (t1/2), were determined by analyzing the
60
first-order rate constant (S) and half-life (t1/2) of the decay curve of DPBF, respectively, and
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were in good accordance with each other (Figure 1).15-18 Further, kQ (S) and kQ (t1/2) values
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were measured for vegetable, fruit, and edible oil extracts.18,19 Based on these results, a new
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assay method was proposed for quantification of the singlet oxygen absorption capacity
64
(SOAC) of natural AOs and food extracts.15-17 The relative SOAC value was defined in the
65
following equation:
66 2
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Relative SOAC value
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= {(t1/2AO - t1/2Blank)/(t1/2α-Toc - t1/2Blank)} × {[α-Toc]/[AO]} = kQAO/kQα-Toc
(2)
69 70
where [α-Toc] and [AO] denote the molar concentrations (mol/L) (or weight concentrations
71
(g/L)) of α-tocopherol (α-Toc) and AOs, respectively. α-Toc was used as a standard
72
compound.15,16 All measurements were performed in homogeneous organic solvents using a
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UV-vis spectrophotometer.
74
To ascertain the validity of the SOAC method proposed for the reaction of 1O2 with
75
natural AOs in organic solvents (mixed solvent and ethanol),15-17 measurements were
76
performed for eight different Cars (Table 1) and α-Toc in Triton X-100 (5.0 wt %) micellar
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solution (0.02 M phosphate buffer, pH 7.4) at 35 oC. Aqueous micellar solution was used as a
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simple model of biomembranes. UV-vis absorption spectra were measured for the eight Cars
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and α-Toc in both Triton X-100 micellar solution and chloroform, to investigate the polarity
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of the reaction field between the Cars (and α-Toc) and 1O2 in micellar solution. Furthermore,
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decay rate constants (kd) of 1O2 were measured in aqueous Triton X-100 (0.0, 1.0, 3.0, and 5.0
82
wt %) micellar solutions (pH 7.4) by time-resolved near-infrared fluorescence
83
spectroscopy.20,21 The kd values in Triton X-100 micellar solution are necessary to determine
84
the absolute values of kQ (S) and kQ (t1/2) (see eqs 3 and 4). Comparisons of the kQ (S) (and
85
SOAC) values obtained for the various Cars and α-Toc in homogeneous organic solvents and
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heterogeneous micellar solution were performed to elucidate the effect of the reaction field on
87
the 1O2-quenching activity of various AOs.
88 89
MATERIALS AND METHODS
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Materials. Lutein (Lut), β-cryptoxanthin (β-Cry), zeaxanthin (Zea), and capsanthin (Cap)
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were obtained from Extrasynthese (Genay, France). α- and β-carotene (α- and β-Car) and
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lycopene (Lyc) were obtained from Wako Chemicals, Japan. Astaxanthin (Ast) was obtained
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from Funakoshi Co. Ltd., Japan. D-α-tocopherol (α-Toc) and DPBF were obtained from Eisai
94
Food Chemicals Co. Ltd., Japan and Tokyo Kasei Chemicals, Japan, respectively.
95
3-(1,4-Epidioxy-4-methyl-1,4-dihydro-1-naphthyl)propionic acid (endoperoxide, EP) was
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obtained from Wakenyaku Co. Ltd., Japan. The UV spectrum of EP indicated that the powder
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sample contains 95% EP and 5% EP-precursor unreacted.15
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Preparation of Micellar Solutions Containing Carotenoids (or α-Tocopherol), DPBF,
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and Endoperoxide. Triton X-100 was selected as the surfactant for this study, because the 3
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solubilities of AOs, DPBF, and EP in Triton X-100 micellar solution are generally higher than
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those in sodium dodecyl sulfate (SDS) and cetyl trimethyl ammonium bromide (CTAB)
102
solutions. Further, Triton X-100 is a neutral compound, while SDS and CTAB molecules
103
have negative and positive charges, respectively.
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Triton X-100 (5.0 wt %) micellar solution (0.02 M phosphate buffer (KH2PO4-Na2HPO4),
105
pH 7.4) containing 9.06×10-5 M β-Car was prepared as follows; β-Car (2.43 mg) was
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dissolved in tetrahydrofuran (or chloroform) (~3 ml) and injected (using a fine needle) into
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50.0 mL of Triton X-100 micellar solution. The solution was stirred in a water bath (T =
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20oC) and a stream of nitrogen gas was used to remove the organic solvent. Triton X-100
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micellar solution containing DPBF ([DPBF] = 1.90×10-4 M) was prepared in a similar manner,
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and 1.00 mL of DPBF solution was added to 1.00 mL of β-Car solution. Triton X-100
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micellar solution containing EP ([EP] = 2.98×10-3 M) was also prepared in a similar manner.
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The production of 1O2 due to the thermal decomposition of EP occurs at temperature ≥
113
25oC.15 Therefore, samples were prepared by adding 1.00 mL of EP solution to 2.00 ml of
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solution containing DPBF and an AO in a quartz cuvette at ~20oC to avoid decomposition of
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EP, and measurements of the UV-vis absorption spectra were then started at 35oC. It took
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about 5 min to prepare solutions of six cuvettes. About 3 min was necessary before the
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solution temperature in the cuvette rose from ~20oC to 35oC. As solubility of Lyc in Triton
118
X-100 (5.0 wt %) micellar solution was low, we were unsuccessful in preparing the solution
119
of Lyc usable to our measurement.
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Measurements of the Rate Constants (kQ) and UV-Vis Absorption Spectra of
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Carotenoids and α-Tocopherol. Rate constants (kQ) were measured in Triton X-100 (5.0
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wt %) micellar solution using a Shimadzu UV-vis spectrophotometer (UV-1800), equipped
123
with a six-channel cell-positioner and an electron-temperature control unit (CPS-240A).15-17
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All measurements were performed at 35.0 ± 0.5oC under nitrogen atmosphere, to avoid
125
degradation of AOs and DPBF. All measurements were carried out in a sealed system using a
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cuvette with a sealing cap to avoid water evaporation.
127 128
UV-vis absorption maxima (λmax) of the eight Cars and α-Toc were measured in chloroform and aqueous Triton X-100 (5.0 wt %) micellar solution (pH 7.4).
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Analyses of the Rate Constants (kQAO (S) and kQAO (t1/2)) and SOAC Values. The
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second-order rate constants (kQAO (S) and kQAO (t1/2)) for the reaction of 1O2 with AOs were
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determined according to eqs 3 and 4, respectively, as previously reported.15-17
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SBlank/SAO = 1 + {kQAO (S) [AO]}/kd
(3)
t1/2AO/t1/2Blank = 1 + {kQAO (t1/2) [AO]}/kd
(4)
134 135 136 137
SBlank and SAO denote the slopes of the first-order plots (i.e., ln (Absorbance) versus time
138
plots) of the disappearance of DPBF in the absence and presence of AO, respectively, and
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t1/2Blank and t1/2AO denote the half-lives of DPBF in the absence and presence of AO,
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respectively (Figure 1). kd (= 1.47 × 105 s-1) is the rate of natural deactivation of 1O2 in the
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Triton X-100 (5.0 wt %) micellar solution.14 The measurement of kd value is described in the
142
following section.
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Measurement of the Natural Decay Rate Constant (kd) of Singlet Oxygen in Aqueous
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Triton X-100 Micellar Solution. Measurement of kd value of 1O2 was performed, using a
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time-resolved near-infrared fluorescence spectrophotometer (Hamamatsu C-7990-01)
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operating in single-photon-counting mode, as reported in previous studies.20,21 1O2 was
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generated with 532 nm Nd:Yag laser excitation of rose Bengal in air-saturated Triton X-100
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(0.0, 1.0, 3.0, and 5.0 wt %) micellar solutions. Time-resolved-profiles of
149
phosphorescence measured at room temperature are shown in Figure 2A.
1
O2
150 151 152
RESULTS 1
O2-Quenching Rates (kQ (S) and kQ (t1/2)) and SOAC Values for Carotenoids and
153
α-Tocopherol in Triton X-100 (5.0 wt %) Micellar Solution. Measurements of kQ (S), kQ
154
(t1/2), and SOAC values were performed for eight different Cars (Table 1) and α-Toc in Triton
155
X-100 (5.0 wt %) micellar solution (pH 7.4), by varying the concentrations of the Cars and
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α-Toc.15,16
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β-Car shows an UV-vis absorption at 370 ~ 530 nm in micellar solution (Figure 1A).
158
Figure 1B shows an example of the reaction between DPBF and EP in the absence ((a) Blank)
159
and presence of AOs ((b) [α-Toc] = 5.11×10-4 M, (c) [β-Car] = 1.21×10-5 M, (d) [β-Car] =
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1.81×10-5 M, (e) [β-Car] = 2.41×10-5 M, (f) [β-Car] = 3.02×10-5 M) in micellar solution (pH
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7.4) at 35℃. The disappearance of DPBF during the chemical reaction with 1O2 was
162
monitored at λmax = 416 nm. The baseline correction in c-f was performed by using the
163
absorption at 416 nm of β-Car and Lambert-Beer’s law (i.e., Absorbance (at 416 nm) = ε (at
164
416 nm) × [β-Car] × l (l = 1 cm)) (Figure 1C).15,16 The first-order decay rate constants (SBlank,
165
Sα-Toc, Sβ-Car) were calculated by analyzing the decay curve of DPBF (Figure 1D). The analysis 5
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of the decay curve was performed at ~10 < t < ~40 min.15,16 This is an important condition to
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obtain an accurate rate constant (kQ) for AOs. The values of half-life (t1/2α-Toc, t1/2β-Car, t1/2Blank)
168
were calculated carefully according to the method described in a previous work.15
169
Plots of SBlank/Sβ-Car and t1/2β-Car/t1/2Blank versus [β-Car] are shown in Figure 1E and F,
170
respectively. The kQβ-Car (S) and kQβ-Car (t1/2) values obtained by using eqs 3 and 4 are 4.23×109
171
and 4.47×109 M-1s-1, respectively, showing fair agreement with each other. As the
172
measurements were performed for one concentration of α-Toc and four concentrations of
173
β-Car, four sets of relative SOAC values were determined using eq 2. The relative SOAC
174
values obtained for β-Car (15.6, 17.0, 17.0, 21.0, average 17.7) also show a considerable
175
agreement with each other.
176
Similar measurements were performed for seven Cars. The kQ (S), kQ (t1/2), and relative
177
SOAC values obtained for Ast, β-Car, Cap, Zea, α-Car, Lut, and β-Cry are summarized in
178
Table 1, showing reasonable agreement between the kQAO (S)/kQα-Toc (S) and relative SOAC
179
values. These results indicate that the definition of eq 2 is useful for the estimation of the
180
SOAC values of Cars and α-Toc in micellar solution.
181
As reported in previous studies, the analysis of the decay curve of DPBF caused by the
182
reaction of 1O2 with DPBF in organic solvents (such as mixed solvent and ethanol) was
183
performed at ~5 < t < ~60 min.15-17 Analysis of the decay curve of DPBF in micellar solution,
184
on the other hand, was performed within a narrower time range (~10 < t < ~40 min) (Figure
185
1D). The difference in time ranges is the reason for the insufficient agreement between the
186
kQAO (S)/kQα-Toc (S) and relative SOAC values compared with those in organic solvents.
187
Decay Rate Constant (kd) of Singlet Oxygen in Aqueous Triton X-100 Micellar
188
Solution. Measurement of the kd of 1O2 in Triton X-100 (5.0 wt %) micellar solution is
189
necessary to obtain absolute kQ (S) and kQ (t1/2) values for each AO, as eqs 3 and 4 indicate. kd
190
value was measured in micellar solution, using time-resolved near-infrared fluorescence
191
spectroscopy.20,21 The relative SOAC values (which are independent of the kd value) of Cars
192
in micellar solution, on the other hand, were determined using eq 2.
193
kd value were measured in Triton X-100 (0.0, 1.0, 3.0, and 5.0 wt %) micellar solutions
194
(0.02 M phosphate buffer, pH 7.4). Figure 2A shows phosphorescence decay curves of 1O2
195
observed at ~ 1270 nm by using photosensitization from rose Bengal in Triton X-100 ((a) 0.0
196
and (b) 5.0 wt %) micellar solutions. The fitted curves were added to confirm that the
197
phosphorescence decays are well-characterized by simple first-order decays. kd values of 1O2
198
were determined to be 2.03×105, 1.46×105, 1.45×105, and 1.47×105 s-1 in 0.0, 1.0, 3.0, and 5.0 6
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wt % Triton X-100 micellar solutions, respectively. The kd value decreases in the presence of
200
Triton X-100 micelles, and exhibits a constant value in micellar solutions containing 1.0, 3.0,
201
and 5.0 wt % (Figure 2B). Using the kd value obtained in 5.0 wt % micellar solution, absolute
202
kQ (S) and kQ (t1/2) values were determined for each Car and α-Toc.
203
The kd value of 1O2 in H2O solution was previously measured by Kanofsky.
22
The
204
reported rate (kd = 2.38 × 105 s-1 at 25 oC) is similar to that obtained in the present work (2.03
205
× 105 s-1 at 25 oC). Zinukov et al.23 measured the temperature dependence of the kd of 1O2 in
206
H2O. The result indicates that kd is nearly temperature independent (3.39 × 105 - 3.00 × 105
207
s-1) at 10 to 70 oC, and then decreases rapidly to 2.08 × 105 s-1 at 95 oC. Addition of a 1.0 wt %
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Triton X-100 solution (at 24 oC) leads to a decrease in kd from 3.23 × 105 to 2.17 × 105 s-1.
209
Absolute kd values reported by Zinukov et al. 23 are 1.59 and 1.49 times larger than those
210
obtained in the present work, respectively. However, the ratios of kd (i.e., kd (in H2O)/kd (in
211
1.0 wt % micelle) = 3.23/2.17 = 1.49 and 2.03/1.46 = 1.39) are similar to each other, although
212
the reason why the values obtained in the present work are smaller than those reported by
213
Zinukov et al. is not clear at present.
214
UV-Vis Absorption Spectra of Carotenoids and α-Tocopherol in Organic Solvents
215
and Micellar Solution. UV-vis absorption spectra for various Cars have previously been
216
measured in organic solvents.
217
measured in both Triton X-100 micelle solution (5.0 wt %, pH 7.4) and chloroform. Values of
218
UV-vis absorption maxima (λmax) obtained for the eight investigated Cars and α-Toc are
219
summarized in Table 2, together with those measured in mixed solvent and ethanol.15-17 As
220
listed in Table 2, the λmax values of the eight Cars in ethanol are 9 - 12 nm smaller than those
221
in chloroform. The λmax values of the eight Cars in mixed solvent show intermediate values
222
between those in ethanol and chloroform. The λmax values of seven Cars in micellar solution
223
are 8 - 13 nm and 2 - 5 nm larger than those in ethanol and mixed solvent, respectively. On
224
the other hand, the λmax values of the eight Cars in micellar solution are similar (-3 - +3 nm) to
225
those in chloroform. These results suggest that Cars in micellar solution are localized to
226
regions that exhibit an environment similar to that of chloroform.
24,25
In the present work, UV-vis absorption spectra were
227
It is well known that the energy of the π-π* transition in organic molecules decreases with
228
increasing polarity of the solvent used for measurement, that is, the λmax value exhibits a red
229
shift. However, the λmax values of the eight Cars in ethanol, which has higher polarity than
230
chloroform, are 9 - 12 nm smaller than those in chloroform. The spectral shift of the Cars
231
depends on the polarizability rather than the polarity of the solvent, and the UV-vis absorption 7
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maximum shifts to longer wavelengths, as the refractive index of the solvent increases.24
233
The eight Cars investigated exhibit different molecular structures containing different
234
numbers of polar groups. Specifically, (i) Lyc, β-Car, and α-Car do not contain any polar
235
groups (such as OH and C=O groups), (ii) β-Cry has one OH group, (iii) Zea and Lut have
236
two OH groups, (iv) Cap has two OH groups and one C=O group, and (v) Ast has two OH
237
and two C=O groups (see Table 1). Consequently, one may expect that the eight Cars are
238
localized to regions within the micellar solution that exhibit different polarities. However, as
239
described above, all of the investigated Cars seem to localize to environments within the
240
micellar solution that are similar to that of chloroform.
241
As α-Toc contains a polar phenolic OH group, one may expect the chroman ring to be
242
localized to regions that exhibit a polarity similar to that of ethanol (i.e., the regions nearer to
243
the micelle surface), as reported by Fukuzawa for unilamellar liposomes.26 However, the λmax
244
value of α-Toc in micellar solution (298.0 nm) is also similar to that in chloroform (296.5 nm),
245
and is red-shifted from that in ethanol (292.0 nm), as observed for the Cars (Table 2). These
246
results suggest that the chroman ring of the α-Toc molecule also localized to a region of the
247
micellar solution with an environment similar to that of chloroform.
248 249
DISCUSSION
250
Comparison of the 1O2-Quenching Rate Constants (kQAO (S)) of Carotenoids and
251
α-Tocopherol in Homogeneous and Heterogeneous Solutions. The kQ values of several
252
Cars in unilamellar dipalmitoyl phosphatidylcholine (DPPC) liposomes were previously
253
measured by Fukuzawa et al.27,28 and Cantrell et al.29 In the present study, 1O2-quenching rates
254
(kQAO (S) (hereafter abbreviated as “kQAO” for simplicity)) were measured for seven Cars and
255
α-Toc in Triton X-100 micellar solution. The kQAO values obtained in micellar solution are
256
summarized in Table 3, together with those in DPPC liposome, mixed solvent, and
257
ethanol.15-17,27-29
258
The kQCar values increased in the following order:
259 260
kQCar (liposome) < kQCar (micelle) < kQCar (mixed solvent) < kQCar (EtOH)
(5)
261 262
On the other hand, the kQα-Toc values increased in the order:
263 264
kQα-Toc (mixed solvent) < kQα-Toc (EtOH) < kQα-Toc (micelle) 8
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where the value of kQα-Toc in liposome, kQα-Toc (liposome), which is used for comparison in eq
267
6, was not reported by Cantrell et al.29
268
The kQAO values were previously measured for eleven tocopherol derivatives, as well as
269
for five palm oil extracts and one soybean extract in mixed solvent and ethanol.18 The kQ
270
(EtOH) values were found to correlate linearly with the kQ (mixed solvent) values. The ratios
271
of the rate constants (kQ (EtOH)/kQ (mixed solvent)) were estimated to be 1.72 ± 0.04, i.e.:
272 273
kQ (EtOH) = 1.72 kQ (mixed solvent)
(7)
274 275
This result suggests that the ratios for pure AOs and food extracts, which contain many types
276
of AOs and the other compounds, are intrinsically constant, and do not depend on the types of
277
AOs present. Similar results were also obtained for six Cars and sixteen different phenolic
278
AOs including polyphenols.17 The average ratio of the rate constants (1.79) was similar to that
279
reported in eq 7 (i.e., 1.72).
280
In the present work, measurements of kQAO (micelle) values were performed for seven
281
Cars and α-Toc in micellar solution (Table 3). The kQAO (micelle) values were plotted against
282
kQAO (mixed solvent) and kQAO (EtOH), respectively (Figures 3A and B). The ratios of kQCar
283
(micelle) to kQCar (mixed solvent) and kQCar (EtOH) for Cars were calculated to be 0.530 ±
284
0.034 and 0.286 ± 0.012, respectively, except for α-Toc (see Supplementary Table S1).
285 286
kQCar (micelle) = 0.530 kQCar (mixed solvent)
(8)
kQCar (micelle) = 0.286 kQCar (EtOH)
(9)
287 288 289 290 291
On the other hand, the ratios of kQα-Toc (micelle) to kQα-Toc (mixed solvent) and kQα-Toc (EtOH) for α-Toc are 2.41 and 1.54, respectively (eqs 10 and 11).
292 293
kQα-Toc (micelle) = 2.41 kQα-Toc (mixed solvent)
(10)
kQα-Toc (micelle) = 1.54 kQα-Toc (EtOH)
(11)
294 295 296 297
These results indicate that the values of the ratios for α-Toc (2.41 and 1.54) are quite different 9
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from those for Cars (0.530 and 0.286). The
Reason
Why
Carotenoids
Show
Lower
1
O2-Quenching
Activity
in
300
Heterogeneous Solutions Than That in Homogeneous Solutions. As Cars and α-Toc are
301
lipophilic, they are expected to localize within micelle. Consequently, the local concentrations
302
of these AOs in Triton X-100 micellar solution (5.0 wt %) should become ~ 20 times larger
303
than in homogeneous ethanol solution (or mixed solvent), if one assumes that the volume that
304
Triton X-100 molecules (5.0 wt %) occupy in micellar solution is 5.0 % of the total volume.
305
As the 1O2 molecule is also lipid soluble,30 it is also expected to react with these AOs inside of
306
the micelle. Therefore, if the polarity of the reaction field between 1O2 and AOs in micellar
307
solution is similar to that of ethanol, the kQ values of the AOs observed in micelles should
308
become ~ 20 times larger than those in ethanol. To our surprise, however, the ratios (kQCar
309
(micelle)/kQCar (EtOH) and kQα-Toc (micelle)/kQα-Toc (EtOH)) observed were only 0.286 and
310
1.54, respectively, as determined from eqs 9 and 11.
311
Gruszka et al.31 reported that the 1O2-quenching rate constants (kQ) of α-, β-, γ-, and
312
δ-Tocs increase linearly with increasing solvent dielectric constant (ε) (that is, polarity). For
313
example, kQ values (3.13×108 and 1.03×108 M-1s-1) of α- and δ-Tocs in acetonitrile (ε = 35.94)
314
are 2.59 and 7.46 times larger than those (1.21×108 and 1.38×107 M-1s-1) in CCl4 (ε = 2.2),
315
respectively. Similar solvent effects were obtained for β- and γ-Tocs, and ubiquinol-10.
316
As the dielectric constants (ε) of ethanol, chloroform, and D2O are 24.58, 4.806, and 78.30,
317
respectively, an average εav value for the mixed solvent (ethanol/chloroform/D2O = 50:50:1.
318
v/v/v) was estimated using the following relation: εav = (50×24.58 + 50×4.806 + 1×78.30)/101
319
= 15.32.17 The estimated εav value of the mixed solvent is smaller than that of ethanol, but
320
larger than that of chloroform. In fact, kQAO (EtOH) values of Cars and α-Toc (ε = 24.58) are
321
larger than the corresponding kQAO (mixed solvent) values (εav = 15.32), as indicated in eqs 5
322
and 6 (Table 3).
323
As described in the Results section, the eight Cars investigated in this study contain
324
different numbers of polar OH and CO groups. Therefore, one might expect that the Cars are
325
located at the position having different polarities (i.e., different ε values) in micellar solution.
326
However, the measured λmax values of the Cars suggest that all are localized to environments
327
with polarities similar to that of chloroform. In fact, the relative kQCar (micelle) values of Cars
328
are similar to those in homogeneous solutions, as eqs 8 and 9 indicate (Table S1).
329
As reported by Gruszka et al.,31 the values of kQCar (CHCl3) and kQα-Toc (CHCl3) should be
330
several times (or a few times) smaller than those of kQCar (EtOH) and kQα-Toc (EtOH), because 10
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chloroform exhibits a smaller dielectric constant than ethanol. Even after taking into account
332
the above decrease of kQCar and kQα-Toc in chloroform, the small values of the observed ratios
333
(i.e., 0.286 and 1.54), which are much smaller than the expected ratio of ~ 20, cannot be
334
explained. The significant decrease in the kQAO values of Cars and α-Toc may be due to
335
suppression of the mobility of AO molecules in the heterogeneous micellar solution.
336
Fukuzawa et al.27,28 measured the 1O2-quenching rates (kQ/kd) of three Cars (β-Car,
337
canthaxanthin (Can), and Ast) in DPPC liposome. 1O2-quenching experiments were carried
338
out using both a water-soluble 1O2-sensitizer (rose Bengal) and a lipid-soluble sensitizer
339
12-(1-pyrene)dodecanoic acid (PDA) in DPPC liposomes. The kQ/kd values (eq 3) were
340
independent of the site of 1O2-generation, and all three Cars showed similar kQ values.
341
Specifically, the kQCar (liposome) values determined using rose Bengal as a 1O2-sensitizer
342
were 5.2×109 M-1s-1 for β-Car, 4.5×109 for Can, and 4.6×109 for Ast, where kQCar (liposome)
343
was calculated using the kd value in tert-butanol (3.0×104 sec-1) (Table 3).
344
Cantrell et al.29 measured the rate constants (kQCar (liposome)) for the quenching of 1O2 by
345
six Cars in unilamellar DPPC liposomes. The kQCar (liposome) values for Lyc, β-Car, Can, Ast,
346
Zea, and Lut obtained by the reaction with 1O2 produced by rose Bengal photosensitization
347
are listed in Table 3. The kQCar (liposome) values for β-Car and Can are similar to those
348
reported by Fukuzawa et al.27,28 On the other hand, kQCar (liposome) values for Ast (5.9×108
349
M-1s-1), Zea (2.3×108), and Lut (1.1×108) were an order of magnitude smaller than those of
350
β-Car (2.3×109 M-1s-1) and Can (2.3×109). The former (Ast, Zea, and Lut) each contain two
351
OH groups, while the latter (β-Car and Can) contain no OH groups.
352
As listed in Table 3, the Cars generally show kQ values similar to each other in both
353
homogeneous organic solvents and heterogeneous micellar solution. The ratio between the
354
maximum and minimum kQCar values is less than twice in both the organic solvents and
355
micellar solution. Furthermore, values of the ratios (kQCar (micelle)/kQCar (mixed solvent) and
356
kQCar (micelle)/kQCar (EtOH)) are constant (0.530 and 0.286), respectively, as eqs 8 and 9
357
indicates.
358
On the other hand, remarkable differences were observed for the ratios of the kQCar values
359
in DPPC liposomes and micellar solution. kQCar values of Cars (β-Car, Ast, Zea, and Lut) in
360
micellar solution are 1.8, 12, 25, and 50 times larger than those in liposome. kQCar value for
361
β-Car (which does not contain an OH group) in liposome is similar to that in micellar solution.
362
However, the kQCar values for Ast, Zea, and Lut (each of which contains two OH groups) in
363
liposome are one to two orders of magnitude smaller than those in micellar solution. 11
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364
Remarkable decrease of the kQCar values in liposome may be due to decrease of mobility of
365
the Car molecules caused by hydrogen bond formation between the DPPC and the Car
366
molecules (Ast, Zea, and Lut), as all 3 Cars contain two OH groups. On the other hand, kQβ-Car
367
for β-Car (which does not contain any OH groups) in liposome is similar to that in micellar
368
solution. These results suggest that β-Car, Lyc, and Can, which do not contain any OH groups,
369
may exhibit higher 1O2-quenching activity than Ast, Zea, and Lut in biological systems.
370
As eqs 9 and 11 indicate, the values of the ratios (kQCar (micelle)/kQCar (EtOH) = 0.286) for
371
Cars are smaller than that for α-Toc (kQα-Toc (micelle)/kQα-Toc (EtOH) = 1.54). The value of the
372
latter is about 5.4 times larger than the former, and suggests that the mobility of the seven
373
Cars, which exhibit rigid molecular structures, should be more suppressed than that of α-Toc
374
in micellar solution, resulting in greater decreases in kQCar than kQα-Toc. Furthermore,
375
differences in the ratios (0.286 and 1.54) between Cars and α-Toc may also be due to
376
differences in the rigidity of the regions at which Cars and α-Toc molecules are localized.
377
However, details are not clear at present.
378
Fukuzawa et al.,27,28 indicated that notable variations of the kQ values of AOs in
379
homogeneous ethanol and heterogeneous liposome solutions may be explained by (i) polarity
380
(dielectric constant (ε)) of reaction field between AOs and 1O2, (ii) local concentration of AOs,
381
and (iii) mobility of AOs in solutions. The results obtained in the present study also support
382
the above explanation, although large differences were observed for the kQ values of AOs
383
between micellar and liposome solutions.
384
Relative SOAC Values for Carotenoids in Heterogeneous Micellar Solution Differ
385
from Those in Homogeneous Organic Solvents. As eq 2 indicates, the relative rate
386
constants (kQAO/kQα-Toc) of AOs are equal to the relative SOAC values. In fact, good agreement
387
between the relative rate constants and the relative SOAC values was previously reported for
388
eleven tocopherol derivatives and six edible oil extracts.18 The relative SOAC (EtOH) values
389
obtained in ethanol were found to correlate linearly with the SOAC (mixed solvent) values in
390
mixed solvent (see Figure 5 in ref 18). The ratio of SOAC (EtOH) to SOAC (mixed solvent)
391
was estimated to be 0.98 ± 0.02. This result indicates that the relative SOAC values of various
392
AOs and food extracts in mixed solvent and ethanol do not depend on the solvents used, and
393
are essentially constant, if α-Toc is used as a standard compound. The solubility of each of the
394
AOs and food- and plant-extracts in organic solvents will be different from each other.
395
However, the relative SOAC values will be constant in different solvents.
396
In the present study, the relative SOAC values were measured for seven Cars and α-Toc in 12
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397
micellar solution. The results obtained are listed in Table 1, together with the SOAC values in
398
mixed solvent and ethanol reported in previous studies.15-17 The relative SOAC (EtOH) values
399
for six of the Cars are similar to the corresponding SOAC values in mixed solvent. However,
400
the SOAC values in micellar solution were different from those in organic solvents. The
401
relative SOAC values were found to increase in the following order:
402 403
Relative SOAC (micelle) value < SOAC (mixed solvent) value
404
~ SOAC (EtOH) value
(12)
405 406
As listed in Table 1, good agreement between the kQAO (micelle)/kQα-Toc (micelle) ratio and
407
the relative SOAC (micelle) values was obtained in micellar solution (Table 1). For example,
408
using eqs 9 and 11, the relative SOAC (micelle) value was estimated from the kQCar (EtOH)
409
and kQα-Toc (EtOH) values.
410 411
Relative SOAC (micelle) value = kQCar (micelle)/kQα-Toc (micelle)
412
= 0.286 kQCar (EtOH)/1.54 kQα-Toc (EtOH) = (1/5.38) × {kQCar (EtOH)/kQα-Toc (EtOH)}
413
= (1/5.38) × Relative SOAC (EtOH) value
(13)
414 415
This result suggests that the relative SOAC value in micellar solution is ~ 5.4 times smaller
416
than that in ethanol. Similarly, the SOAC value in micellar solution is ~ 4.5 times smaller than
417
that in mixed solvent. In fact, the SOAC values observed for Cars in micellar solution are 3.7
418
~ 6.2 and 3.5 ~ 5.9 times smaller than the corresponding SOAC values in mixed solvent and
419
ethanol, respectively, demonstrating good agreement (Table 1).
420
As described above, relative SOAC values were measured for only seven of the
421
investigated Cars in micellar solution. In the future, it will be necessary to measure the SOAC
422
values for phenolic AOs (such as tocopherol homologues and polyphenols) and vitamin C (Vit
423
C) in micellar solution (at pH 7.4). As the kQAO values (i.e., SOAC values) of the phenolic
424
AOs (especially polyphenols) and Vit C show notable pH dependence because of the
425
dissociation of the proton (H+) from the OH group as reported by Bisby et al.,32 notable
426
differences between the relative SOAC values in micellar solution and organic solvents will
427
be observed.
428
Generally, natural AOs are localized to heterogeneous reaction fields, including lipophilic
429
and hydrophilic phases (i.e., at heterogeneous biological tissues such as plasma, blood, heart, 13
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430
liver, brain etc.). Therefore, it will be important to evaluate the kQ and SOAC values in
431
heterogeneous micellar solution to elucidate the 1O2-quenching activity of AOs in biological
432
systems. As preparation of micellar solution is easier than that of liposomes and the solubility
433
of AOs in micellar solution is higher than that in liposomes, use of Triton X-100 micellar
434
solution is preferred to measure the kQ and SOAC values for various types of AOs.
435 436
■ ASSOCIATED CONTENT
437
Supporting Information
438
Supplementary Table S1 showing the kQAO (S) values for antioxidants (AOs) in micellar
439
solution (pH 7.4), mixed solvent, and ethanol at 35.0oC, and the ratios (kQAO (S)
440
(micelle)/kQAO (S) (mixed solvent) and kQ (S) (micelle)/kQ (S) (ethanol)). This material is
441
available free of charge via the Internet at http://pubs.acs.org.
442 443
■ AUTHOR INFORMATION
444
Corresponding Author
445
*Tel: 81-89-927-9588. Fax: 81-89-927-9590. E-mail:
[email protected].
446
Funding
447
This work was partly supported by JSPS KAKENHI Grant Number 15k07431.
448
Notes
449
The authors declare no competing financial interest.
450 451
■ ACKNOWLEDGMENT
452
We are very grateful to Ms. Yuko Iwasaki-Kino of Kagome Co. Ltd. for the measurement of
453
UV-vis absorption spectra of six carotenoids in chloroform. We are also very grateful to Prof.
454
Junji Terao for his continuous encouragement throughout this work.
455 456
■ REFERENCES
457
(1) Davies, M. J.; Truscott, R. J. W. Photo-oxidation of proteins and its role in
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cataractogenesis. J. Photochem. Photobiol.. B: Biol. 2001, 63, 114-125.
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Biophys. Res. Commun. 2003, 305, 761-770.
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content of fruits and vegetables: an evaluation of analytic data. J. Am. Diet. Assoc. 1993, 93, 14
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(4) Holden, J. M.; Eldridge, A. L.; Beecher, G. R.; Buzzard, I. M.; Bhagwat, S.; Davis, C. S.;
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an update of database. J. Food Compos. Analysis 1999, 12, 169-196.
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in Japan. Food Sci. Technol. Res. 2007, 13, 247-252.
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(6) Graham, H. N. Green tea composition, consumption and polyphenol chemistry. Prev. Med.
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flavonoids and phenolic acids. Free Radic. Biol. Med. 1996, 20, 933-956.
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(8) Sookwong, P.; Nakagawa, K.; Yamaguchi, Y.; Miyazawa, T.; Kato, S.; Kimura, F.;
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Miyazawa, T. Tocotrienol distribution in foods: Estimation of daily tocotrienol intake of
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Japanese population. J. Agric. Food Chem. 2010, 58, 3350-3355.
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(9) Finley, J. W.; Kong, A. N.; Hintze, K. J.; Jeffery, E. H.; Ji, L. L.; Lei, X. G. Antioxidants in
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foods: State of the science important to the food industry. J. Agric. Food Chem. 2011, 59,
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6837-6846.
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(10) Foote, C. S.; Denny, R. W. Chemistry of singlet oxygen VII. Quenching by β-carotene. J.
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Am. Chem. Soc. 1968, 90, 6233-6235.
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(11) Di Mascio, P.; Kaiser, S.; Sies, H. Lycopene as the most efficient biological carotenoid
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singlet oxygen quencher. Arch. Biochem. Biophys. 1989, 274, 532-538.
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(12) Sies, H.; Stahl, W.; Sundquist, A. R. Antioxidant functions of vitamins. Vitamin E and C,
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β-carotene, and other carotenoids. Ann. N. Y. Acad. Sci. 1992, 669, 7-20.
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(13) Di Mascio, P.; Sundquist, A. R.; Devasagayam, T. P. A.; Sies, H. Assay of lycopene and
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other carotenoids as singlet oxygen quenchers. Methods Enzymol. 1992, 213, 429-438.
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(14) Wilkinson, F.; Helman, W. P.; Ross, A. B. Rate constants for the decay and reactions of
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the lowest electronically excited singlet state of molecular oxygen in solution. An expanded
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and revised compilation. J. Phys. Chem. Ref. Data 1995, 24, 663-1021.
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(15) Ouchi, A.; Aizawa, K.; Iwasaki, Y.; Inakuma, T.; Terao, J.; Nagaoka, S.; Mukai, K.
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Kinetic study of the quenching reaction of singlet oxygen by carotenoids and food extracts in
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solution. Development of a singlet oxygen absorption capacity (SOAC) assay method. J.
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Agric. Food Chem. 2010, 58, 9967-9978.
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(16) Aizawa, K.; Iwasaki, Y.; Ouchi, A.; Inakuma, T.; Nagaoka, S.; Terao, J.; Mukai, K.
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Development of singlet oxygen absorption capacity (SOAC) assay method. 2. Measurements 15
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of the SOAC values for carotenoids and food extracts. J. Agric. Food Chem. 2011, 59,
497
3717-3729.
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(17) Mukai, K.; Ouchi, A.; Takahashi, S.; Aizawa, K.; Inakuma, T.; Terao, J.; Nagaoka, S.
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Development of singlet oxygen absorption capacity (SOAC) assay method. 3. Measurements
500
of the SOAC values for phenolic antioxidants. J. Agric. Food Chem. 2012, 60, 7905-7916.
501
(18) Mukai, K.; Ishikawa, E.; Ouchi, A.; Nagaoka, S.; Suzuki, T.; Izumisawa, K.; Koike, T.
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Kinetic study of the quenching reaction of singlet oxygen by α-, β-, γ-, δ-tocotrienols and
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palm oil and soybean extracts in solution. Biosci. Biotechnol. Biochem. 2014, 78, 2089-2101.
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(19) Iwasaki, Y.; Takahashi, S.; Aizawa, K.; Mukai, K. Development of singlet oxygen
505
absorption capacity (SOAC) assay method. 4. Measurements of the SOAC values for
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vegetable and fruit extracts. Biosci. Biotechnol. Biochem. 2015, 79, 280-291.
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(20) Ohara, K.; Kikuchi, K.; Origuchi, T.; Nagaoka, S. Singlet oxygen quenching by trolox C
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in aqueous micelle solutions. J. Photochem. Photobiol. B: Biol. 2009, 97, 132-137.
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(21) Nagaoka, S.; Ohara, K.; Takei, M.; Nakamura, M.; Mishima, M.; Nagashima, U. UV
510
protechtion and singlet-oxygen quenching activity of intramolecularly hydrogen-bonded
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hydroxyanthraquinone derivatives found in aloe. J. Photochem. Photobiol. A: Chem. 2011,
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225, 106-112.
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(22) Kanofsky, J. R. Quenching of singlet oxygen by human plasma. Photochem. Photobiol.
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1990, 51, 299-303.
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(23) Zinukov. S. V.; Kamalov, V. F.; Koroteev, N. I.; Krasnovskii, A. A. Jr. Nanosecond
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measurements of photosensitized luminescence of singlet molecular oxygen in aqueous
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solutions saturated by air: effect of temperature and detergent presence. Opt. Spectrosc.
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(USSR), 1991, 70, 460-462.
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(24) Britton, G. Chapter 2, UV/Visible Spectroscopy. Carotenoids, Vol. 1B: Spectroscopy, G.
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Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkhauser Verlag Basel, Switzerland, 1995;
521
pp. 13-62.
522
(25) Beutner, S.; Bloedorn, B.; Frixel, S.; Blanco, I. H.; Hoffmann, T.; Martin, H.-D.; Mayer,
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B.; Noack, P.; Ruck, C.; Schmidt, M.; Schülke, I.; Sell, S.; Ernst, H.; Haremza, S.; Seybold,
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G.; Sies, H.; Stahl, W.; Walsh, R. Quantitative assessment of antioxidant properties of natural
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colorants and phytochemicals: carotenoids, flavonoids, phenols and indigoids. The role of
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β-carotene in antioxidant functions. J. Sci. Food Agric. 2001, 81, 559-568.
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(26) Fukuzawa, K. Dynamics of lipid peroxidation and antioxidation of α-tocopherol in
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membranes. J. Nutr. Sci. Vitaminol. 2008, 54, 273-285. 16
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(27) Fukuzawa, K. Singlet oxygen scavenging in phospholipid membranes. Methods Enzymol.
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2000, 319, 101-110.
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(28) Fukuzawa, K.; Inokami, Y.; Tokumura, A.; Terao, J.; Suzuki, A. Rate constants for
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quenching singlet oxygen and activities for inhibiting lipid peroxidation of carotenoids and
533
α-tocopherol in liposomes. Lipids 1998, 33, 751-756.
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(29) Cantrell, A.; McGarvey, D. J.; Truscott, T. G.; Rancan, F.; Böhm, F. Singlet oxygen
535
quenching by dietary carotenoids in a model membrane environment. Arch. Biochem. Biophys.
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2003, 412, 47-54.
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(30) Oxygen and Ozone (1981) in Solubility Data Series (Battino, R., ed.), Vol. 7, pp. 1-40,
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Pergamon Press, Oxford.
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(31) Gruszka, J.; Pawlak, A.; Kruk, J. Tocochromanols, plastoquinol, and other biological
540
prenyllipids as singlet oxygen quenchers – determination of singlet oxygen quenching rate
541
constants and oxidation products. Free Radic. Biol. Med. 2008, 45, 920-928.
542
(32) Bisby, R. H.; Morgan, C. G.; Hamblett, I.; Gorman, A. A. Quenching of singlet oxygen
543
by trolox C, ascorbate, and amino acids: Effects of pH and temperature. J. Phys. Chem. A
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1999, 103, 7454-7459.
545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 17
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562 563 564 565 566 567 568 569 570 571 572 573 574 575 576
Figure Captions.
577 578
Scheme 1.
579 580
Figure 1. (A) Absorption spectrum of β-carotene in Triton X-100 (5.0 wt %) micellar solution
581
(0.02 M buffer, pH 7.4). The concentration of β-carotene is 1.81×10-5 M. (B) Change in
582
absorbance of DPBF at 416 nm upon the reaction of DPBF with 1O2 in the absence and
583
presence of sample (α-tocopherol or β-carotene) in Triton X-100 (5.0 wt %) micellar solution
584
(0.02 M buffer, pH 7.4) at 35oC. [DPBF]t = 0 = 6.33×10-5 M and [EP]t = 0 = 9.92×10-4 M. The
585
values of [α-Toc]t = 0 and [β-Car]t = 0 are shown in panel C. (C) Change in absorbance of DPBF,
586
where the correction of baseline due to β-carotene was performed (see text). (D) Plot of ln
587
(Absorbance) versus t. (E) Plot of SBlank/Sβ-Car versus [β-Car]. (F) Plot of t1/2β-Car /t1/2Blank versus
588
[β-Car].
589 590
Figure 2. (A) Time-evolution of 1O2 phosphorescence produced at ~ 1270 nm through
591
photosensitization from rose Bengal in 0.0 wt % (open circles) and 5.0 wt % (dots) Triton
592
X-100 micellar solution. The fitted curves (0.0 wt %: dashed line and 5.0 wt %: solid line)
593
were added to confirm that the phosphorescence decays after subtraction of background
594
counts due to dark current are well-characterized by single exponential decays. 18
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595
Journal of Agricultural and Food Chemistry
(B) Plot of decay rate (kd) of singlet oxygen versus [wt %] of Triton X-100 micellar solution.
596 597
Figure 3. (A) Plot of kQ (S) (Micelle) versus kQ (S) (Mixed solvent) for seven carotenoids and
598
α-tocopherol. (B) Plot of kQ (S) (Micelle) versus kQ (S) (EtOH) for six carotenoids and
599
α-tocopherol.
19
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600 601 602
Table 1. The kQAO (S) and kQAO (t1/2) Values for Antioxidants (AOs) in Triton X-100 (5.0 wt %) Micellar Solution (pH 7.4) at 35.0oC, Relative Rate Constants (kQAO (S)/kQα-Toc (S)), and Relative SOAC Values Antioxidant (AO)
α-Tocopherol (α-Toc) Lycopene (Lyc) (No OH group) a Astaxanthin (Ast) (2 OH, 2 C=O) β-Carotene (β-Car) (No OH) Capsanthin (Cap) (2 OH, 1 C=O) Zeaxanthin (Zea) (2 OH) α-Carotene (α-Car) (No OH) Lutein (Lut) (2 OH) β-Cryptoxanthin (β-Cry) (1 OH) 603 604 605 606 607 608 609 610
Page 20 of 27
kQAO (S)/M-1s-1 b (SBlank/SAO plot) micelle (3.16±0.04)×108 c low solubility
kQAO (t1/2)/M-1s-1 b (t1/2AO/t1/2Blank plot) micelle (3.28±0.22)×108 c low solubility
kQAO (S) /kQα-Toc (S) micelle 1.00 -----
Relative SOAC value micelle 1.00 low solubility
Relative SOAC value d mixed solvent 1.00 123
Relative SOAC value e EtOH 1.00 low solubility
(7.11±0.49)×109
(7.43±0.52)×109
23.0
109
low solubility
(4.23±0.49)×109
(4.47±0.49)×109
13.4
95.8
71.1
(4.54±0.41)×109
(4.55±0.68)×109
14.4
99.3
78.6
(5.83±0.07)×109
(6.26±0.06)×109
18.4
92.8
87.0
(5.70±0.59)×109
(6.57±0.74)×109
18.0
92.4
83.7
(5.53±0.63)×109
(6.04±0.72)×109
17.5
73.8
74.5
(4.40±0.23)×109
(4.30±0.27)×109
13.9
26.5-31.0 (av 28.8) 15.6-21.0 (av 17.7) 13.8-19.2 (av 16.0) 24.1-26.9 (av 25.1) 8.84-20.6 (av 15.2) 9.03-16.7 (av 12.7) 15.2-17.9 (av 16.1)
67.6
68.0
a
Numbers of polar OH and C=O groups included in carotenoid molecules. The kQ value was calculated using the kd value (= 1.47×105 s-1) measured in Triton X-100 (5.0 wt %) micellar solution at 25oC in the present work. c Values are expressed as mean ± SD (n = 4). d Relative SOAC values in mixed solvent reported in ref 16. e Relative SOAC values in ethanol reported in ref 17. b
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611 612 613
614 615 616 617 618 619 620 621 622 623 624 625 626 627
Journal of Agricultural and Food Chemistry
Table 2. UV-Vis Absorption Maxima (λmax) of the Carotenoids in Micelle, Chloroform, Mixed Solvent, and Ethanol Solutions, and Difference (∆λmax) between the λmax Values in Micelle and Each Solvent Carotenoid
λmax/nm
λmax/nm
α-Tocopherol Lycopene Astaxanthin β-Carotene Capsanthin Zeaxanthin α-Carotene Lutein β-Cryptoxanthin
micelle 298.0 a insoluble 488 b 462 483 464 455 455 464
CHCl3 296.5 a 483 b 488 464 485 462 458 456 461
{λmax (micelle) - λmax (CHCl3)}/nm +1.5 ----0 -2 -2 +2 -3 -1 +3 (∆λmax = -3 ~ +3 nm)
λmax/nm c mixed solvent d 292.5 a 479 b 486 459 481 459 453 452 459
{λmax (micelle) - λmax (mixed solvent)}/nm +5.5 ----+2 +3 +2 +5 +2 +3 +5 (∆λmax = +2 ~ +5 nm)
a
Experimental error is ± 0.5 nm. Experimental error is ± 1.0 nm. c λmax values in mixed solvent reported in ref 15. d Mixed solvent: ethanol/chloroform/D2O (50:50:1, v/v/v). e λmax values in ethanol reported in ref.17. b
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λmax/nm e EtOH 292.0 a 473 b 479 452 475 452 446 446 452
{λmax (micelle) - λmax (EtOH)}/nm +6.0 ----+9 +10 +8 +12 +9 +9 +12 (∆λmax = +8 ~ +13 nm)
Journal of Agricultural and Food Chemistry
628 629 630
Table 3. The kQAO Values for Antioxidants (AOs) in Triton X-100 (5.0 wt %) Micellar Solution (pH 7.4), Liposome, Mixed Solvent, and Ethanol, and the Ratios (kQAO (liposome)/kQAO (micelle)) Antioxidant
α-Tocopherol Lycopene Astaxanthin β-Carotene Capsanthin Zeaxanthin α-Carotene Lutein β-Cryptoxanthin Canthaxanthin 631 632 633 634 635 636 637 638 639 640 641 642
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kQAO a (pH 7.4) /M-1s-1 micelle present work 3.16×108 low solubility 7.11×109 4.23×109 4.54×109 5.83×109 5.70×109 5.53×109 4.40×109
kQAO
kQAO
/M-1s-1 liposome Fukuzawa et al. b
/M-1s-1 liposome Cantrell et al. c
4.6×109 5.2×109
2.4×109 5.9×108 2.3×109
1/12.1 1/1.84
2.3×108
1/25.3
1.1×108
1/50.3
4.5×109
kQAO (liposome) c /kQAO (micelle) ratio
2.3×109
a
In this Table, “kQAO (S)” was abbreviated as “kQAO” for simplicity, as described in the Discussion section. b kQAO (liposome) values reported in refs 27 and 28. c kQAO (liposome) values reported in ref 29. d kQAO (mixed solvent) and kQAO (EtOH) values reported in ref 17.
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kQAO
kQAO
/M-1s-1 mixed solvent Mukai et al. d 1.31×108 1.38×1010 1.18×1010 1.08×1010 1.06×1010 1.05×1010 9.76×109 9.24×109 7.31×109
/M-1s-1 EtOH Mukai et al. d 2.06×108 low solubility low solubility 1.71×1010 1.79×1010 1.82×1010 1.92×1010 1.76×1010 1.54×1010
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643 644 645 646 647 648 649 650 651 652 653 654 655 656
Journal of Agricultural and Food Chemistry
Table of Contents Graphic: Development of Singlet Oxygen Absorption Capacity (SOAC) Assay Method. Measurements of the SOAC Values for Carotenoids and α-Tocopherol in Aqueous Triton X-100 Micellar Solution KAZUO MUKAI,†* AYA OUCHI,† NAGAO AZUMA,† SHINGO TAKAHASHI,‡ KOICHI AIZAWA,‡ and SHIN-ICHI NAGAOKA†
kQ Carotenoid + 1O2 → Carotenoid + 3O2 657 658 659 660 661 662 663 664 665 Carotenoid 666 667
Measurements of Singlet Oxygen Absorption Capacity (SOAC) Values for Carotenoids and α-Tocopherol in Micellar Solution
668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 23
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Journal of Agricultural and Food Chemistry
694 695 696 697 698 699 700 701 702 703 704 705 706 707 708
O O
Carotenoid
1
O2
Physical Quenching (kq) + (kQ = kq + kr) Chemical Reaction (kr)
CH2CH2COOH
EP
kF
kd
O
DPBF
Scheme 1
λmax = 416 nm
3
O2
Product
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Journal of Agricultural and Food Chemistry
(B)
(A)
3.5
2.5
a) Blank -4 b) [α-Toc ] = 5.11×10 M
-5
[β-Carotene] = 1.81×10 M in Micellar Solution (0.02 M Buffer, pH 7.4)
3.0
-5
c) [β-Carotene] = 1.21×10 M -5
d) [β-Carotene] = 1.81×10 M
Absorbance
Absorbance
2.0
1.5
1.0
e) [β-Carotene] = 2.41×10-5 M
2.5
f) [β-Carotene] = 3.02×10-5 M
2.0
f)
1.5
e) d)
1.0
c)
0.5
b)
0.5
a) 0
0 300
709
400
500
600
Wavelength / nm
(C)
0
30
60
90
120
Time / min
(D) 1
2.0 [ DPBF ] = 6.33×10-5 M [ EP ] = 9.92×10 -4 M in Micellar Solution (0.02 M Buffer, pH 7.4) at 35 ℃
0
a) Blank b) [α-Toc ] = 5.11×10-4 M
ln (Absorbance)
Absorbance
1.5
-5
c) [β-Carotene] = 1.21×10 M -5
d) [β-Carotene] = 1.81×10 M
1.0
b) f)
e) [β-Carotene] = 2.41×10-5 M f) [β-Carotene] = 3.02×10-5 M
f) b) d)
-1
e) a) Blank -4 b) [α-Toc ] = 5.11×10 M
0.5 a)
0
0
(E)
-2
d) e)
30
60
90
Time / min
30
2.0
1.5
1.5
1.0
60
90
120
Time / min
(F)
2.0
a)
-5
e) [β-Carotene] = 2.41×10 M f) [β-Carotene] = 3.02×10-5 M
0
120
t1/2β-Carotene / t1/2 Blank
SBlank / Sβ-Carotene
710
c)
c)
c) [β-Carotene] = 1.21×10-5 M d) [β-Carotene] = 1.81×10-5 M
1.0
0.5
0.5 kQβ-Carotene (S) = 4.23×109 M-1s-1
kQβ-Carotene (t1/2) = 4.47×109 M-1s-1 0
0 0
711 712
1 2 [β-Carotene] / 10 -5 M
0
3
1 2 [β-Carotene] / 10 -5 M
Figure 1
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3
Journal of Agricultural and Food Chemistry
(A)
713
(B) 25
15
d
5
k / 10 s
-1
20
10 5 0
714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729
0
1
2
3 wt %
4
5
6
Figure 2
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Journal of Agricultural and Food Chemistry
(A) Ast Zea α-Car Lut
6
9
-1 -1
kQ (S) / 10 M s (Micelle)
8
4
β-Car
2
0
Slope = 0.530
α-Toc 0
1.0
0.5 kQ (S) / 10
730
(B)
Cap
β-Cry
10
M s (Mixed solvent)
7
Zea
6
Lut
α-Car
5
Cap
β-Cry 4
β-Car
9
-1 -1
kQ (S) / 10 M s (Micelle)
1.5
-1 -1
3 2
Slope = 0.286 1
α-Toc 0
0
0.5
1.0 10
731 732 733 734 735 736 737 738 739 740
1.5
2.0
2.5
-1 -1
kQ (S) / 10 M s (EtOH)
Figure 3
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