Development of a Singlet Oxygen Absorption Capacity (SOAC) Assay

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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

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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

36

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,

39

kinetic study, micellar solution, endoperoxide

40 41

INTRODUCTION

42

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

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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

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following equation:

66 2


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Relative SOAC value

68

= {(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

73

UV-vis spectrophotometer.

74

To ascertain the validity of the SOAC method proposed for the reaction of 1O2 with

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natural AOs in organic solvents (mixed solvent and ethanol),15-17 measurements were

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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

79

and α-Toc in both Triton X-100 micellar solution and chloroform, to investigate the polarity

80

of the reaction field between the Cars (and α-Toc) and 1O2 in micellar solution. Furthermore,

81

decay rate constants (kd) of 1O2 were measured in aqueous Triton X-100 (0.0, 1.0, 3.0, and 5.0

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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

93

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

96

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,

99

and Endoperoxide. Triton X-100 was selected as the surfactant for this study, because the 3



100

solubilities of AOs, DPBF, and EP in Triton X-100 micellar solution are generally higher than

101

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.

104

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

106

dissolved in tetrahydrofuran (or chloroform) (~3 ml) and injected (using a fine needle) into

107

50.0 mL of Triton X-100 micellar solution. The solution was stirred in a water bath (T =

108

20oC) and a stream of nitrogen gas was used to remove the organic solvent. Triton X-100

109

micellar solution containing DPBF ([DPBF] = 1.90×10-4 M) was prepared in a similar manner,

110

and 1.00 mL of DPBF solution was added to 1.00 mL of β-Car solution. Triton X-100

111

micellar solution containing EP ([EP] = 2.98×10-3 M) was also prepared in a similar manner.

112

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

114

solution containing DPBF and an AO in a quartz cuvette at ~20oC to avoid decomposition of

115

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

117

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.

120

Measurements of the Rate Constants (kQ) and UV-Vis Absorption Spectra of

121

Carotenoids and α-Tocopherol. Rate constants (kQ) were measured in Triton X-100 (5.0

122

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

124

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

126

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).

129

Analyses of the Rate Constants (kQAO (S) and kQAO (t1/2)) and SOAC Values. The

130

second-order rate constants (kQAO (S) and kQAO (t1/2)) for the reaction of 1O2 with AOs were

131

determined according to eqs 3 and 4, respectively, as previously reported.15-17

132 4


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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,

140

respectively (Figure 1). kd (= 1.47 × 105 s-1) is the rate of natural deactivation of 1O2 in the

141

Triton X-100 (5.0 wt %) micellar solution.14 The measurement of kd value is described in the

142

following section.

143

Measurement of the Natural Decay Rate Constant (kd) of Singlet Oxygen in Aqueous

144

Triton X-100 Micellar Solution. Measurement of kd value of 1O2 was performed, using a

145

time-resolved near-infrared fluorescence spectrophotometer (Hamamatsu C-7990-01)

146

operating in single-photon-counting mode, as reported in previous studies.20,21 1O2 was

147

generated with 532 nm Nd:Yag laser excitation of rose Bengal in air-saturated Triton X-100

148

(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

156

α-Toc.15,16

157

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] =

160

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

161

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



166

of the decay curve was performed at ~10 < t < ~40 min.15,16 This is an important condition to

167

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


Page 6 of 27

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199

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 %

208

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



232

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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


(6)

Page 9 of 27


265 266

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



298 299

Page 10 of 27

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


Page 11 of 27


331

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



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


Page 12 of 27

Page 13 of 27


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



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

458

cataractogenesis. J. Photochem. Photobiol.. B: Biol. 2001, 63, 114-125.

459

(2) Davies, M. J. Singlet oxygen-mediated damage to proteins and its consequences. Biochem.

460

Biophys. Res. Commun. 2003, 305, 761-770.

461

(3) Mangels, A. R.; Holden, J. M.; Beecher, G. R.; Forman, M. R.; Lanza, E. Carotenoid

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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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284-296.

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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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Douglass, L. W.; Gebhardt, S.; Haytowitz, D.; Schakel, S. Carotenoids content of U.S. foods:

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an update of database. J. Food Compos. Analysis 1999, 12, 169-196.

467

(5) Aizawa, K.; Inakuma, T. Quantitation of carotenoids in commonly consumed vegetables

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in Japan. Food Sci. Technol. Res. 2007, 13, 247-252.

469

(6) Graham, H. N. Green tea composition, consumption and polyphenol chemistry. Prev. Med.

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1992, 21, 334-350.

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(7) Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Structure-antioxidant activity relationships of

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flavonoids and phenolic acids. Free Radic. Biol. Med. 1996, 20, 933-956.

473

(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.

480

Am. Chem. Soc. 1968, 90, 6233-6235.

481

(11) Di Mascio, P.; Kaiser, S.; Sies, H. Lycopene as the most efficient biological carotenoid

482

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.

485

(13) Di Mascio, P.; Sundquist, A. R.; Devasagayam, T. P. A.; Sies, H. Assay of lycopene and

486

other carotenoids as singlet oxygen quenchers. Methods Enzymol. 1992, 213, 429-438.

487

(14) Wilkinson, F.; Helman, W. P.; Ross, A. B. Rate constants for the decay and reactions of

488

the lowest electronically excited singlet state of molecular oxygen in solution. An expanded

489

and revised compilation. J. Phys. Chem. Ref. Data 1995, 24, 663-1021.

490

(15) Ouchi, A.; Aizawa, K.; Iwasaki, Y.; Inakuma, T.; Terao, J.; Nagaoka, S.; Mukai, K.

491

Kinetic study of the quenching reaction of singlet oxygen by carotenoids and food extracts in

492

solution. Development of a singlet oxygen absorption capacity (SOAC) assay method. J.

493

Agric. Food Chem. 2010, 58, 9967-9978.

494

(16) Aizawa, K.; Iwasaki, Y.; Ouchi, A.; Inakuma, T.; Nagaoka, S.; Terao, J.; Mukai, K.

495

Development of singlet oxygen absorption capacity (SOAC) assay method. 2. Measurements 15



496

of the SOAC values for carotenoids and food extracts. J. Agric. Food Chem. 2011, 59,

497

3717-3729.

498

(17) Mukai, K.; Ouchi, A.; Takahashi, S.; Aizawa, K.; Inakuma, T.; Terao, J.; Nagaoka, S.

499

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.

502

Kinetic study of the quenching reaction of singlet oxygen by α-, β-, γ-, δ-tocotrienols and

503

palm oil and soybean extracts in solution. Biosci. Biotechnol. Biochem. 2014, 78, 2089-2101.

504

(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

506

vegetable and fruit extracts. Biosci. Biotechnol. Biochem. 2015, 79, 280-291.

507

(20) Ohara, K.; Kikuchi, K.; Origuchi, T.; Nagaoka, S. Singlet oxygen quenching by trolox C

508

in aqueous micelle solutions. J. Photochem. Photobiol. B: Biol. 2009, 97, 132-137.

509

(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

511

hydroxyanthraquinone derivatives found in aloe. J. Photochem. Photobiol. A: Chem. 2011,

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225, 106-112.

513

(22) Kanofsky, J. R. Quenching of singlet oxygen by human plasma. Photochem. Photobiol.

514

1990, 51, 299-303.

515

(23) Zinukov. S. V.; Kamalov, V. F.; Koroteev, N. I.; Krasnovskii, A. A. Jr. Nanosecond

516

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.

518

(USSR), 1991, 70, 460-462.

519

(24) Britton, G. Chapter 2, UV/Visible Spectroscopy. Carotenoids, Vol. 1B: Spectroscopy, G.

520

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,

523

B.; Noack, P.; Ruck, C.; Schmidt, M.; Schülke, I.; Sell, S.; Ernst, H.; Haremza, S.; Seybold,

524

G.; Sies, H.; Stahl, W.; Walsh, R. Quantitative assessment of antioxidant properties of natural

525

colorants and phytochemicals: carotenoids, flavonoids, phenols and indigoids. The role of

526

β-carotene in antioxidant functions. J. Sci. Food Agric. 2001, 81, 559-568.

527

(26) Fukuzawa, K. Dynamics of lipid peroxidation and antioxidation of α-tocopherol in

528

membranes. J. Nutr. Sci. Vitaminol. 2008, 54, 273-285. 16


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529

(27) Fukuzawa, K. Singlet oxygen scavenging in phospholipid membranes. Methods Enzymol.

530

2000, 319, 101-110.

531

(28) Fukuzawa, K.; Inokami, Y.; Tokumura, A.; Terao, J.; Suzuki, A. Rate constants for

532

quenching singlet oxygen and activities for inhibiting lipid peroxidation of carotenoids and

533

α-tocopherol in liposomes. Lipids 1998, 33, 751-756.

534

(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.

536

2003, 412, 47-54.

537

(30) Oxygen and Ozone (1981) in Solubility Data Series (Battino, R., ed.), Vol. 7, pp. 1-40,

538

Pergamon Press, Oxford.

539

(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

544

1999, 103, 7454-7459.

545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 17



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


Page 18 of 27

Page 19 of 27

595


(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



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

20


Page 21 of 27

611 612 613

614 615 616 617 618 619 620 621 622 623 624 625 626 627


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

21


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)


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

Page 22 of 27

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.

22


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

Page 23 of 27

643 644 645 646 647 648 649 650 651 652 653 654 655 656


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



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

24


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Page 25 of 27


(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

25


3


(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

26


Page 26 of 27

Page 27 of 27


(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

27


Development of a Singlet Oxygen Absorption Capacity (SOAC) Assay

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