Reaction of Paprika Carotenoids, Capsanthin and Capsorubin, with

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Reaction of Paprika Carotenoids Capsanthin and Capsorubin with Reactive Oxygen Species Azusa Nishino, Hiroyuki Yasui, and Takashi Maoka J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01706 • Publication Date (Web): 27 May 2016 Downloaded from http://pubs.acs.org on June 1, 2016

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Journal of Agricultural and Food Chemistry

Reaction of Paprika Carotenoids Capsanthin and Capsorubin with Reactive Oxygen Species

Azusa Nishino, † Hiroyuki Yasui, ‡ and Takashi Maoka*,§

†

Institute of Health Sciences, Ezaki Glico Co., Ltd, 4-6-5 Utajima, Nishiyodogawa-ku,

Osaka 555-8502, Japan ‡

Department of Analytical and Bioinorganic Chemistry, Division of Analytical and

Physical Chemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan §

Research Institute for Production Development, 15 Shimogamo, Morimoto Cho,

Sakyoku, Kyoto 606-0805, Japan

Corresponding author *T.M. Tel.: +81 75 781 1107; fax: +81 75 781 1118. E-mail address: [email protected] Supporting Information

1

ACS Paragon Plus Environment


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ABSTRACT:

2

The reaction of paprika carotenoids, capsanthin and capsorubin, with reactive oxygen

3

species (ROS) such as superoxide anion radical (.O2-), hydroxyl radical (.OH), and

4

singlet oxygen (1O2) was analyzed by LC/PDA ESI-MS and ESR spectrometry.

5

Capsanthin formed both the 5,6-epoxide and 5,8-epoxide by reaction with .O2- and .OH.

6

Furthermore, capsanthin also formed 5,6- and 5,8-endoperoxide on reaction with 1O2.

7

The same results were obtained in the case of capsanthin diacetate. On the other hand,

8

capsorubin showed higher stability against these ROS. Capsorubin formed 7,8-epoxide

9

on reaction with .O2- and .OH and 7,8-endoperoxide on reaction with 1O2.

10 11

KEYWORDS:

Capsanthin;

Capsorubin;

12

endoperoxide; Reactive oxygen species;

Capsanthin

13 14 15 16 17

2


epoxide;

Capsanthin

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18

INTRODUCTION

19 20

Paprika (Capsicum annuum) is a good source of carotenoids and used widely as a

21

vegetable and food colorant. The red carotenoids are mainly capsanthin and capsorubin,

22

1-4

23

1), and their carotenoids account for 30-80% of the total carotenoids in fully ripe fruits.

24

1-4

25

Recently, Maeda et al. reported that these carotenoids showed effects to prevent and

26

improve obesity-related insulin resistance.15 Furthermore, it was reported that

27

capsanthin could take up peroxynitrite into its molecule by the formation of

28

nitrocapsanthin.16

along with capsanthin 3,6-epoxide5 and capsanthone6 as minor components (Figure

These carotenoids showed excellent anti-oxidative7-10 and anti-cancer11-14 activities.

29

It is known that carotenoids inhibit singlet oxygen (1O2) through physical quenching

30

and chemical reactions. 17 For example, β-carotene formed endoperoxides, epoxides and

31

apocarotenoids by the reaction with 1O2. 18-21 Very recently, we investigated reaction of

32

astaxanthin with 1O2, superoxide anion radical (.O2-), and hydroxyl radical (.OH), using

33

LC/PDA ESI-MS and ESR spectrometry.22 As the results, we found that astaxanthin

34

formed astaxanthin endoperoxides on reaction with 1O2 and formed astaxanthin

35

epoxides on reaction with .O2-, and .OH. 22

3



36

However, there have been few reports on the reaction of capsanthin and capsorubin

37

with reactive active oxygen species (ROS). In order to clarify the chemical scavenging

38

mechanism of these carotenoids with ROS, we investigated reaction products of these

39

paprika carotenoids with .O2-, .OH, and

40

spectrometry. This paper reports the scavenging effect of .O2-, .OH, and 1O2 with

41

capsanthin and capsorubin by ESR spin-trapping analysis and the reaction products of

42

these paprika carotenoids with these ROS. Furthermore, the reaction mechanisms of

43

ROS with capsanthin and capsorubin are discussed.

1

O2 using LC/PDA ESI-MS and ESR

44 45

MATERIALS AND METHODS

46

Chemicals. Hematoporphyrin, riboflavin, and hydrogen peroxide were purchased from

47

Wako Pure Chemicals (Osaka, Japan). 2,2,6,6-Tetramethyl-4-piperidone (TMPD) was

48

purchased from Aldrich (Milwaukee, WI, USA). 5,5-Dimethyl-1-pyrroline-N-oxide

49

(DMPO) was purchased from Labotec (Tokyo, Japan). β-Carotene, zeaxanthin,

50

capsanthin, and capsorubin were prepared from paprika according to the method

51

described previously.9,10,12 Astaxanthin was prepared from Paracoccus carotinifaciens.23

52

Capsanthin diacetate and astaxanthin diacetate were prepared from capsanthin and

53

astaxanthin, respectively by acetylation with acetic anhydride in pyridine at room

4


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temperature. syn- and anti-Capsanthin 5,6-epoxide and capsanthin 5,8-epoxide were

55

prepared from capsanthin by epoxydation with m-chloroperbenzoic acid as described

56

below.

57

Apparatus. The ESR spectra were recorded at room temperature on a JEOL

58

JES-RFR30 spectrometer (Tokyo, Japan). The LC/MS and MS/MS analysis of

59

carotenoids was carried out using a Waters Xevo G2S Q TOF mass spectrometer

60

(Waters Corporation, Milford, CT, USA) equipped with an Acquity UPLC system. The

61

1

62

spectrometer (Agilent Technologies, Santa Clara, CA, USA) in CDCl3 with TMS as an

63

internal standard.

H NMR (500 MHz) spectra were measured with a Varian UNITY INOVA 500

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Reaction of Capsanthin with ROS. According to the method described previously,22

65

after the addition of 200 µL of 8 mM H2O2 solution to 200 µL of 22 µg/mL capsanthin

66

CH3CN solution, .OH was generated by UV-A irradiation using SUPERCURE-203S

67

(SAN-Ei ELECTORIC) at room temperature. In a similar manner to that described

68

above, .O2- and 1O2 were generated by the addition of 200 µL of 25 µM of riboflavin or

69

200 µL of 250 µM hematoporphyrin, respectively to 200 µL of 22 µg/mL of capsanthin

70

CH3CN solution. At regular intervals of UV-A irradiation, reaction products were

71

analyzed by LC/PDA-ESI-MS.

5



72

ESR spin-trapping analysis. ESR spectra were recorded at room temperature on a

73

JEOL JES-RFR30 spectrometer (Tokyo, Japan) using an aqueous quartz flat cell

74

(Labotec, Tokyo, Japan). DMPO was used as a .O2- and .OH-trapping agent, and TMPD

75

was used as 1O2, respectively. The .O2- and .OH were generated by addition of either the

76

100 µL of 25 µM of riboflavin, or 100 µL of 8 mM H2O2 solution and 10 µL of 250 mM

77

DMPO to 100 µL of 14 nmol /mL capsanthin CH3CN solution by UV-A irradiation. In a

78

similar manner described above, 1O2 was generated by the addition of both the 100 µL

79

of 250 µM hematoporphyrin and 10 µL of 500 mM TMPD to 100 µL of 14 nmol /mL

80

capsanthin CH3CN solution by UV-A irradiation. ESR spectra were started

81

simultaneously to measure after UV-A irradiation. The all spin-trapped ESR spectra

82

were monitored between the third and fourth signals from the low magnetic field due to

83

the external standard, Mn(II) doped MnO. As the similar manner described above,

84

reaction of capsanthin acetate and capsorubin (14 nmol /mL of both in CH3CN solution)

85

with ROS were also performed.

86

Analysis of Reaction Products of Capsanthin with ROS Using LC/MS and

87

MS/MS. The electro-spray ionization (ESI) time-of-flight (TOF) MS spectra were

88

acquired by scanning from m/z 100 to 1,500 with a capillary voltage of 3.2 kV, cone

89

voltage of 40 eV, and source temperature of 120 °C. Nitrogen was used as a nebulizing

6


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gas at a flow rate of 30 L/h. MS/MS spectra were measured with a quadrupole-TOF

91

MS/MS instrument with argon as a collision gas at a collision energy of 30 V.

92

UV-Visible (UV/vis) absorption spectra were recorded from 200 to 600 nm using a

93

photodiode-array detector (PDA). An Acquity 1.7 µm BEH UPLC C18 column (Waters

94

Corporation) was used as a stationary phase and 90%MeOH as a mobile phase, at a

95

flow rate of 0.2 mL/min for the HPLC system. In a similar manner to that described

96

above, reaction of capsanthin diacetate (27 µg/mL in CH3CN solution) and capsorubin

97

(34 µg/mL in CH3CN solution) with ROS were performed.

98

Capsanthin diepoxide. ESI TOF MS m/z 639.4217 (M+Na+, calcd for C40H56O5Na,

99

639.4025)；UV/vis 405~420 nm (in 90% MeOH).

100

Capsanthin 5,6-epoxide. ESI TOF MS m/z 623.4080 (M+Na+, calcd for C40H56O4Na,

101

623.4076)；UV/vis 463 nm (in 90% MeOH).

102

Capsanthin 5,8-epoxide. ESI TOF MS m/z 623.4080 (M+Na+, calcd for C40H56O4Na,

103

623.4076)；UV/vis 448 nm (in 90% MeOH).

104

Capsanthone 5,6-epoxide. ESI TOF MS m/z 621.3920 (M+Na+, calcd for C40H54O4Na,

105

621.3920)；UV/vis 463 nm (in 90% MeOH).

106

Capsanthin diendoperoxide. ESI TOF MS m/z 671.3937 (M+Na+, calcd for

107

C40H56O7Na, 671.3948)；UV/vis 446 nm (in 90% MeOH).

7



108

Capsanthin 5,6-endoperoxide. ESI TOF MS m/z 639.4056 (M+Na+, calcd for

109

C40H56O5Na, 639.4025); UV/vis 463 nm (in 90% MeOH).

110

Capsanthin 5,8-endoperoxide. ESI TOF MS m/z 639.4003 (M+Na+, calcd for

111


112

Capsanthin diacetate diepoxide. ESI TOF MS m/z 723.4258 (M+Na+, calcd for

113

C44H60O7Na, 723.4237)；UV/vis 405~420 nm (in 90% MeOH).

114

Capsanthin diacetae 5,6-epoxide. ESI TOF MS m/z 707.4280 (M+Na+, calcd for

115


116

Capsanthin diacetate 5,8-epoxide. ESI TOF MS m/z 707.4280 (M+Na+, calcd for

117


118

Capsanthin diacetate diendoperoxide. ESI TOF MS m/z 755.4145 (M+Na+, calcd for

119

C44H60O9Na,755.4135)；UV/vis 446 nm (in 90% MeOH).

120

Capsanthin diacetate 5,6-endoperoxide. ESI TOF MS m/z 723.7229 (M+Na+, calcd for

121


122

Capsanthin diacetate 5,8-endoperoxide. ESI TOF MS m/z 723.7229 (M+Na+, calcd for

123


124

Capsorubin 7,8-epoxide. ESI TOF MS m/z 639.4003 (M+Na+, calcd for C40H56O5Na,

125

639.4025); UV/vis 424 nm (in 90% MeOH).

8


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Capsorubin 7,8-endoperoxide. ESI TOF MS m/z 665.3962 (M+Na+, calcd for

127


128

Preparation of capsanthin 5,6-epoxides and 5,8-epoxides from capsanthin by

129

epoxydation with m-chloroperbenzoic acid. According to the method described by

130

Khachik et al,24 m-chloroperbenzoic acid 3.6 mg in 2 mL dichlorometane was added to

131

a solution of capsanthin 2 mg in 2 mL dichlorometane. The solution was stand for 20

132

min at room temperature in darkness. After the time, the reaction mixture was

133

partitioned with 5% solution of NaHCO3 and ether. The ether layer was removed and

134

evaporated. The reaction products was submitted to preparative HPLC. HPLC

135

separation provided syn-capsanthin 5,6-epoxide (0.8 mg) and anti-capsanthin

136

5,6-epoxide (0.4 mg) and capsanthin diepoxides (0.2 mg mixtures). These spectral data

137

were identical with those of published data by Deli at al.

138

were prepared by epoxide-furanoxide rearrangement

139

5,6-epoxides. Preparative HPLC was performed on a Shimadzu LC-6AD with a

140

Shimadzu SPD-6AV spectrophotometer (Shimadzu Corporation, Kyoto, Japan) set at

141

450 nm. The column used was a 250 X 10 mm i.d., 10 µm Cosnosil 5C18-II (Nacalai

142

Tesque, Kyoto, Japan) and a 250 X 10 mm i.d., 10 µm Cosnosil 5SL-II (Nacalai Tesque,

143

Kyoto, Japan). Spectral data of syn-capsanthin 5,6-epoxide, anti-capsanthin 5,6-epoxide,

26

25

Capsanthin 5,8-epoxides

with HCl from capsanthin

9



144

and capsanthin 5,8-epoxides were available for Supporting Information.

145 146

RESULTS AND DISCUSSION

147 148

Scavenging effect of ROS with capsanthin and capsorubin. Figure 2 shows the

149

scavenging effect of 1O2 (A) and .OH (B) with capsanthin and capsorubin. Astaxanthin,

150

astaxanthin diacetae, β-carotene, and zeaxanthin were used as positive controls. It was

151

reported that capsanthin and capsorubin quenched

152

peroxidation caused by free radicals.8,9

153

also revealed that capsanthin, capsanthin acetate, and capsorubin quenched not only 1O2

154

but also scavenged .OH. The order of quenching activity for 1O2 is astaxanthin diacetate

155

>capsorubin >astaxanthin >capsanthin diacetate> capsanthin> β-carotene> zeaxanthin.

156

It was reported that the 1O2 quenching activity of carotenoids depends on the number of

157

conjugated polyenes, polyene chain structures, and functional groups, especially

158

conjugated carbonyl groups.7,10,27

159

group improved carotenoid stability.22

160

conjugated double bonds including two conjugated carbonyl groups. Capsorubin has

161

eleven conjugated double bonds including two conjugated carbonyl groups. Capsanthin

1

O27,10 and inhibited lipid

In the present investigation, the ESR study

It was also reported that acetylation of the hydroxyl Astaxanthin and its acetate have thirteen

10


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and its acetate has eleven conjugated double bonds including one conjugated carbonyl

163

group. On the other hand, β-carotene and zeaxanthin have eleven conjugated double

164

bonds without a conjugated carbonyl group. The results of the present investigation are

165

markedly consistent with this hypothesis.

166

On the other hand, capsanthin and capsorubin showed excellent scavenging activity

167

for .OH. Contrary to 1O2, astaxanthin and its acetate hardly showed scavenging activity

168

for .OH. The presence of the 3-hydroxy-κ-end group with a conjugated carbonyl group,

169

which is a characteristic structural moiety of paprika carotenoids, may enhance the

170

quenching activity of .OH.

171

Reaction products of capsanthin with .O2- and .OH. Structures of reaction products of

172

capsanthin with ROS are shown in Figure 1. Figure 3 shows HPLC chromatograms of

173

reaction products of capsanthin with .O2- by UV-A irradiation of riboflavin solution.

174

After 1 min of UV-A irradiation, reaction products assigned as capsanthin 5,6-epoxide

175

and capsanthin 5,8-epoxide were detected on HPLC. On UV-A irradiation, the amount

176

of these reaction products was increased and amount of capsanthin was decreased

177

time-dependently. After 6 min of UV-A irradiation, the HPLC peak of capsanthin

178

completely disappeared and the amount of reaction products reached a maximum. Then,

179

the reaction products were markedly decreased and the reaction solution was bleached.

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The structures of reaction products were assigned to capsanthin diepoxides (Peaks 1

181

and 2), capsanthone 5,6-epoxide (Peak 3), capsanthin 5,6-epoxide (Peaks 4 and 5), and

182

capsanthin 5,8-epoxide (Peak 6) based on the ESI-TOF MS, MS/MS, and UV/vis

183

spectral data.

184

Both reaction products of peaks 4 and 5 showed the molecular formula of C40H56O4,

185

which corresponded to the molecular formula of capsanthin epoxide. The MS/MS

186

spectrum of sodium adduct ions [M+Na]+ of these compounds showed the characteristic

187

product ions [M+Na-92]+ (elimination of toluene moiety from polyene chain) and

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[M+Na-106]+ (elimination of xylene moiety from polyene chain), which were

189

characteristic product ions of carotenoids in EI and ESI MS.28,29 Both compounds

190

showed an absorption maximum at 463 nm. These spectral data were in agreement with

191

those of capsanthin 5,6-epoxide. 25, 30 In order to confirm the structure of these reaction

192

products, capsanthin 5,6-epoxides were prepared from capsanthin by epoxydation with

193

m-chloroperbenzoic acid, as shown in Supporting Information. Peaks 4 and 5 were

194

identical

195

5,6-epoxide, respectively, on comparison with retention times in HPLC, MS, MS/MS,

196

and UV/vis spectra data. Therefore the structures of reaction products of peaks 4 and 5

197

were identified as capsanthin 5,6-epoxides.

with

semi-synthetic

syn-capsanthin

5,6-epoxide

12


and

anti-capsanthin

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198

Reaction products of peak 6 showed the molecular formula of C40H56O4, consistent

199

with the molecular formula of capsanthin epoxide. Its absorption maximum was at 448

200

nm. The hypsochromic shift of about 15 nm from capsanthin 5,6-epoxide suggested that

201

these compounds were furanoid rearrangement products of capsanthin 5,6-epoxide, 26,31

202

namely capsanthin 5,8-epoxide. These compounds were identical to semi-synthetic

203

capsanthin 5,8-epoxides prepared from capsanthin 5,6-epoxides based on acid catalysis

204

on comparison of the retention times of HPLC and MS, MS/MS, and UV/vis spectral

205

data.

206

Both reaction products of peaks 1 and 2 showed the molecular formula of C40H56O5,

207

which corresponded to the molecular formula of capsanthin diepoxide. These absorption

208

maxima were at 405~420 nm. Therefore, these compounds were assigned to capsanthin

209

diepoxide. Epoxidation positions in capsanthin could not be determined.

210

Peak 3 showed the molecular formula of C40H54O4. Its absorption maximum was at

211

463 nm. These data suggest that this compound is a didehydro derivative of capsanthin

212

5,6-epoxide. Therefore, this reaction product was tentatively identified as capsanthone

213

5,6-epoxide. This compound was suggested to form from capsanthin 5,6-epoxide

214

through oxidation of the hydroxy group at C-3'. Superoxide anion radical might oxidize

215

the hydroxy group in carotenoids to ketone.

13



216

As well as .O2-, the reaction of capsanthin with .OH hydroxyl radical generated by

217

UV-A irradiation in hydrogen peroxide solution provided capsanthin diepoxide,

218

capsanthone 5,6-epoxide, capsanthin 5,6-epoxide, and capsanthin 5,8-epoxide.

219

Reaction products of capsanthin with 1O2. Figure 4 shows the HPLC chromatograms

220

of the reaction products of capsanthin with 1O2 by UV-A irradiation in hematoporphyrin

221

solution. After 3 min of UV-A irradiation, reaction products assigned as capsanthin

222

diendoproxide and capsanthin 5,6-endoperoxide were detected in HPLC. The amounts

223

of these reaction products were increased and amount of capsanthin was decreased with

224

UV-A irradiation time-dependently. After 6 min of UV-A irradiation, the amount of the

225

reaction products reached a maximum. Then, the reaction products were markedly

226

decreased and the reaction solution was bleached. Both reaction products of peaks 2 and

227

3 showed the molecular formula of C40H56O5 and an absorption maximum at 463 nm.

228

These spectral data indicated that the dioxetane moiety was attached at the double bond

229

of C-5 to C-6 in capsanthin. Therefore, the structure of these compounds was assigned

230

as capsanthin 5,6-endoperoxide. Compounds of peak 2 and 3 were suggested to be

231

stereoisomers of C-5 and C-6. Reaction products of peak 1 showed the molecular

232

formula of C40H56O7, which indicated that two more oxygen atoms were attached to

233

capsanthin 5,6-endoperoxide. It absorption maximum was at 446 nm. The

14


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hypsochromic shift of the UV/vis spectrum of 15 nm from capsanthin 5,6-endoperoxide

235

comprised with the loss of one conjugated double bond from capsanthin

236

5,6-endoperoxide by attaching a dioxetane moiety.26,31

237

compound was tentatively assigned as capsanthin diendoperoxide.

238

Reaction products of capsanthin diacetate with .O2-, .OH, and 1O2. Results similar to

239

those for capsanthin were obtained in the case of capsanthin diacetate with these ROS.

240

Namely, capsanthin diacetate epoxides were obtained on reaction with .OH and .O2-,

241

while capsanthin diacetate endoperoxides were produced on reaction with 1O2. It was

242

found that oxidation products of capsanthin diacetate were more stable than those of

243

free capsanthin. Acetylation might protect the oxidation hydroxy groups of capsanthin

244

from ROS and inhibit rapid degradation of the capsanthin molecule.

245

Reaction products of capsorubin with .O2- and .OH. Figure 5 shows the HPLC

246

chromatograms of the reaction products of capsorubin with .O2-. Reaction products

247

assigned as capsorubin epoxide were increased with UV-A irradiation time-dependently.

248

After 12 min of UV-A irradiation, the amount of reaction products reached a maximum.

249

Then, the reaction solution was bleached. The major reaction products (Peaks 1 and 2)

250

showed the molecular formula of C40H56O5, which corresponded to the formula of

251

capsorubin epoxide. The epoxidation position of capsorubin in these compounds was

Therefore, the structure of this

15



252

estimated from the UV/vis absorption spectra. These compounds showed an absorption

253

maximum at 424 nm. The hypsochromic shift of the UV/vis spectrum of 45 nm from

254

capsorubin indicated that these compounds lacked two conjugated double bonds (double

255

bond of a carbonyl group at C-6 and a double bond at C-7 to C-8 in the polyene chain)

256

from eleven conjugated double bonds of capsorubin by epoxidation.31 Therefore, the

257

epoxidation position of these compounds was assigned to a double bond at C-7 to C-8.

258

Reaction products of capsorubin with 1O2. Figure 6 shows the HPLC chromatograms

259

of the reaction products of capsorubin with

260

astaxanthin,22 capsorubin hardly formed oxidation products on exposure to 1O2. The

261

compound showed the molecular formula of C40H56O6, which corresponded to the

262

molecular formula of capsorubin endoperoxide, being slightly generated after 10 min of

263

UV-A irradiation. After 15 min of UV-A irradiation, capsorubin and its reaction product

264

were markedly decreased. Capsorubin showed greater stability on exposure to singlet

265

oxygen than β-carotene, capsanthin, or astaxanthin.22 Therefore, capsorubin showed a

266

strong quenching effect for 1O2.

1

O2. Contrary to capsanthin and

267

Previously, we investigated the reaction of astaxanthin and its acetate with .O2-, .OH,

268

and 1O2 by LC/PDA ESI-MS and ESR spectrometry.22 As the results, astaxanthin

269

endoperoxides were identified as major reaction products of astaxanthin with 1O2, and

16


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270

astaxanthin epoxides were found to be major reaction products of astaxanthin with .O2-

271

and .OH. The same results in this study were obtained in the case of astaxanthin acetate.

272

The ESR spin-trapping study revealed that astaxanthin and its acetate take up

273

superoxide anion radical by the formation of their epoxide through peroxide and oxide

274

radicals.22

275

In the present investigation, we found that capsanthin and capsorubin quenched not

276

only 1O2 but also scavenged .OH by the ESR spin-trapping study. Furthermore, we

277

found that capsanthin formed capsanthin epoxides by the reaction with .O2- and .OH and

278

also formed capsanthin endoperoxides by the reaction with 1O2. Contrary to astaxanthin,

279

capsanthin formed epoxide and endoperoxide more effectively and stably. It is

280

well-known that .O2- and 1O2 are electrophilic. Thus, they react with the electron-rich

281

double bond in carotenoids. The electron density of the double bond at C-5 (5') to C-6

282

(6') in the β-end group of astaxanthin is suggested to be lower than in parts of double

283

bonds because of the presence of the electron withdrown effect of the carbonyl group at

284

C-4 (4'). On the other hand, the electron density of the double bond at C-5 to C-6 in the

285

β-end group of capsanthin is suggested to be higher than that astaxanthin.

286

Therefore, .O2- and 1O2 might facilely attack the double bond at C-5 to C-6 in

287

capsanthin and form 5,6-epoxide and 5,6-endoperoxide, respectively. Then, these

17



288

5,6-epoxide and 5,6-endoperoxides are converted to the corresponding 5,8-epoxide and

289

5,8-endoperoxide by acid catalisis.22

290

capsanthin with .OH can be explained as follow: A hydroxyl radical was attached at C-5

291

of capsanthin. Then, an epoxide ring was formed by intra-molecular homolytic

292

substitution reaction.22 Capsorubin showed more stability against the attack of ROS,

293

especially 1O2. Capsorubin has a long linier conjugated double bond system in its

294

molecule. Capsorubin might exclusively quench 1O2 through a physical quenching

295

system. Therefore, capsorubin showed the strongest quenching effect on 1O2 among the

296

carotenoids.10 Therefore, we conclude that the reaction of carotenoids with ROS might

297

depend on their structure, such as the conjugated polyene system and functional

298

groups.32,33

The mechanism of epoxide ring formation in

299

In conclusion, capsanthin and capsorubin formed their epoxides and endoperoxides on

300

reaction with .O2-, .OH, and 1O2. The results indicate that capsanthin and capsorubin

301

could trap .O2- and .OH by the formation of their epoxides. Furthermore, it was found

302

that capsanthin and capsorubin could quench 1O2 through not only a pysical quenching

303

system but also chemical direst reaction by the formation of their endoperoxide.

304 305

ASSOCIATED CONTENT

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Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website

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at DOI:

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ESI TOF MS and MS/MS data of reaction products of capsanthin, capsanthin acetate,

310

and capsorubin. UV/vis, ESI TOF MS, MS/MS and 1H NMR data of syn- and

311

anti-capsanthin 5,6-epoxide. UV/vis, ESI TOF MS and MS/MS data of capsanthin

312

5,8-epoxide. Preparation scheme of capsanthin 5,6-epoxides and capsanthin

313

5,8-epoxides from capsanthin by m-chloroperbenzoic acid (PDF).

314

AUTHOR INFORMATION

315

Corresponding author

316

*T.M. Tel.: +81 75 781 1107; fax: +81 75 781 1118. E-mail address:

317

[email protected]

318

ACKNOWLEGEDMENTS

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We thank to Dr Yumiko Yamano, Kobe Pharmaceutical University for valuable

320

suggestion to this work.

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REFERENCES

325

(1) Almela, L.; Lopez-Roca, J.M.; Candela, M.E.; Alcazar N.D. Carotenoid composition

326

of new cultivates of red pepper for paprika. J. Agric. Food Chem. 1991, 39, 1606-1609.

327

(2) Minguez-Mosquera, M.I.; Hornero-Mendez, D. Separation and quantification of the

328

carotenoid pigments in red peppers (Capsicum annuum L.), paprika and oleoresin by

329

reversed-phase HPLC. J. Agric. Food. Chem. 1993. 41, 1616-1620.

330

(3) Minguez-Mosquera, M.I.; Hornero-Mendez, D. Comparative study of the effect of

331

paprika processing on the carotenoids in paprika (Capsicum annuumn) on the Bola and

332

Agridulce varieties. J. Agric. Food. Chem. 1994. 42, 1555-1560

333

(4) Deli, J.; Matus, Z.; Toth, G. Carotenoid composition in the fruits of Capsicum

334

annum Cv. Szentesi kosszarv during ripening. J. Agric. Food Chem. 1996, 44, 711-716.

335

(5) Deli, J.; Molnár, P.; Matus, Z.; Tóth, G.; Steck, A.; Pander, H. Reisolation of

336

carotenoid 3,6-epoxides from red paprika (Capsicum annuum). Helv. Chim. Acta 1996,

337

79, 1435–1443.

338

(6) Deli, J.; Matus, Z.; Molnár, P.; Tóth, G.; Steck, A.; S. Pander, H. Isolation of

339

capsanthone

340

(Capsicum annuum). Chimia 1995, 49, 69-71.

((all-E,3R,5’R)-3-hydroxy-β,κ-carotene-3’,6’-dione)

20


from

paprika

Page 21 of 41


341

(7) Hirayama, O.; Nakamura, K.; Hamada, S.; Kobayashi, K. Singlet oxygen quenching

342

ability of naturally occurring carotenoids. Lipids 1994, 29, 149–150.

343

(8) Matsufuji, H.; Nakamura, H.; Chino, M.; Takeda, M. Antioxidant activity of

344

capsanthin and fatty acid esters in paprika (Capsicum annuum). J. Agric. Food Chem.

345

1998, 46, 3468–3472.

346

(9) Maoka, T.; Goto, Y.; Isobe, K.; Fujiwrara, Y.; Hashimoto, K.; Mochida, K.

347

Antioxidative activity of capsorubin and related compounds from paprika (Capsicum

348

annuum). J. Oleo Sci. 2001, 50, 663–665.

349

(10) Nishino, A.; Ichihara, T.; Takaha, T.; Kuriki, T.; Nihei, H.; Kawamoto, K.; Yasui,

350

H.; Maoka, T. Accumulation of paprika carotenoids in human plasma and erythrocytes.

351

J. Oleo Sci. 2015, 64, 1135-1142.

352

(11) Murakami, A.; Nakashima,M.; Koshiba, T.; Maoka, T.; Nishino, H.; Yano, M.;

353

Sumida, T.; Kim., O.; Koshimizu, K.; Ohigashi, H. Modifying effects of carotenoids on

354

superoxide and nitric oxide generation from stimulated leukocyte. Cancer Lett. 2000,

355

149, 115–123.

356

(12) Maoka, T.; Mochida, K.; Kozuka, M.; Ito, Y.; Fujiwara, Y.; Hashimoto, K.; Enjo,

357

F.; Ogata, M.; Nobukuni, Y.; Tokuda, H.; Nishino, H. Cancer chemopreventive activity

358

of carotenoids in the fruits of red paprika Capsicum annuum L. Cancer Lett. 2001, 172,

21



359

103–109.

360

(13) Maoka, T.; Mochida, K.; Kozuka, M.; Enjo, F.; Kuchide, M.; Nobukuni, Y.;

361

Tokuda, H.; Nishino, H. Chemopreventive activity of paprika extract and capsanthin on

362

nitric oxide or peroxynitrite induced carcinogenesis (in Japanese). Syokuhin Rinsyou

363

Eiyo 2006, 1, 7–14.

364

(14) Molnár, J.; Serly, J.; Pusztai, R.; Vincze, I.; Molnár, P.; Horváth, G.: Deli, J.;

365

Maoka, T.; Zalatnai, A.; Tokuda, H.; Nishino, H. Putative supramolecular complexes

366

formed by carotenoids and xanthophylls with ascorbic acid to reverse multidrug

367

resistance in cancer cells. Anticancer Research 2012, 32, 507-518.

368

(15) Maeda, H.; Saito, S.; Nakamura, N,; Maoka, T. Paprika pigments attenuate

369

obesity-Ibduced Inflammation in 3T3-L1 adipocytes, ISRN Inflammation, 2013, Article

370

ID 763758, 9 pages

371

(16) Tsuboi, M.; Etoh, E.; Kato, K.; Nakatugawa, H.; Kato, H.; Maejima, Y.; Gaku, M.;

372

Matumoto, M.; Mori, H.; Hosokawa, M,; Miyashita, K.; Tokuda, H,; Suzuki, N.;

373

Maoka, T. Nitrocapsanthin and nitrofucoxanthin, respective products of capsanthin and

374

fucoxanthin reaction with peroxynitrite. J. Agric. Food. Chem. 2011, 59, 10872-10578.

375

(17) Footes, C.S.; Denny, R.W. Chemistry of singlet oxygen. VII. Quenching by

376

β-carotene. J. Am. Chem. Soc. 1968, 90, 6233-6235. 22


Page 22 of 41

Page 23 of 41


377

(18) Yamauchi, R.; Tsuchihashi, K.; Kato, K. Oxidation producta of β-carotene during

378

the peroxidation of methyl linolate in the bulk phase. Biosci. Biotech. Biochem. 1998,

379

62, 1301-1306.

380

(19) Martin, H.D.; Ruck, C.; Schmidt, M. Sell, S.; Beutner, S.; Mayse, B.; Walsh, R.

381

Chemistry of carotenoid oxidation and free radical reactions. Pure Appl. Chem. 1999,

382

71, 2253-2262.

383

(20) Fiedor, J.; Fiedor, L.; Haeβner, R.; Scheer, H. Cyclic endoperoxides of β-carotene,

384

potential pro-oxidants, as products of chemical quenching of singlet oxygen. Biochem.

385

Biophy. Acta. 2005, 1709, 1-4.

386

(21) Mordi, R. C.; Walton, J. C.; Burton, G. W., Hughes, L.; Krith, I. U.; David, L.A.;

387

Douglase, M. J. Oxidative degradation of beta-carotene and brta-apo-8’-carotenal.

388

Tetrahedron 1995, 49, 911-928.

389

(22) Nishino, A.; Maoka, T. Yasui, H. Analysis of reaction products of astaxanthin

390

and its acetate with active oxygen species using LC/PDA ESI-MS and ESR

391

spectrometry. Tetrahedron Lett. 2016, 57, 1967-1970.

392

(23) Tsubokura, A.; Yoneda, H.; Mizuta, H. Paracoccus carotinifaciens sp. nov., a new

393

aerobic gram-negative astaxanthin-producing bacterium. J. Syst. Bacteriol. 1999, 49,

23



394

277-282.

395

(24) Khachik, F.; Steck A.; Niggli, U. A.; Pfander, H. Partial synthesis and structural

396

elucidation of the oxidative metabolites of lycopene inentified in tomato paste, tomato

397

juice and human serum. J. Agric. Food Chem. 1998, 46, 4885-4874.

398

(25) Deli, J.; Molnár, P.; Matus, Z.; Tóth, G.; Steck, A.; Pfander, H. Partial synthesis and

399

characterization of capsokarpoxanthins and 3,6-epoxcapsanthins, Helv. Chim. Acta 1998,

400

81, 1242–1253.

401

(26) Eugster, C. H. Chemical derivatization: Microscale tests for the presence of

402

common functional group in carotenoids. in Carotenoids Volume 1A: Isolation and

403

analysis. Birkhäuser, Basel; 1995, pp 71-80.

404

(27) Shimidzu, N.; Goto, M.; Miki, W. Carotenoids as singlet oxygen quenchers in

405

marine organism. Fish. Sci. 1996, 62, 134-137

406

(28) Frassantio, R.; Cantonati,M.; Flaim, G.; Manchini, I.; G. Guella, G. A new method

407

for the identification and the structural characterization of carotenoid esters in

408

freshwater microorganisms by liquid chromatography-electrospray tandem mass

409

spectrometry. Rapid Commun Mass Spectrom. 2008, 22, 3531-3539.

410

(29) Weesepoel, Y.; Vincken, J-P.; Pop, P. M.; Liu, K.; Gruppen, H. Sodiation as a tool

411

for enhancing the diagnostic value of MALDI-TOF/TOF-MS spectra of complex

24


Page 24 of 41

Page 25 of 41


412

astaxanthin ester mixtures from Haematococcus pluvialis. J. Mass. Spectrum. 2013, 48,

413

862-874.

414

(30) Britton, G.; Liaaen-Jensen, S.; Pfander, H. Eds. Carotenoid Hand Bock,

415

Carotenoids Hand Book, Birkhäuser, Basel, Switerland, 2004.

416

(31) Britton, G.; UV/Visible spectroscopy in Carotenoids (Britton, G.; Liaaen-Jensen,

417

S.; Pfander, H. Eds. Birkhäuser, Basel; 1995; Vol. 1B, pp13-62.

418

(32) Woodall, A.A; Jackson, M.J.; Gruszecki, W.; Small, A.; Sielewiesuik, J.; Symons,

419

M.C.R.; Britton, G. Structure and properties of carotenoids in relation to function.

420

FASEB J. 1995, 9, 1551-1558.

421

(33) Woodall, A.A; Lee, S.W.; Weesie, R.J.; Jackson, M.J.; Britton, G. Oxidation of

422

carotenoids by free radicals: relationship between structure and reactivity. Biochem.

423

Biophys. Acta. 1997, 1366, 33-40.

424 425 426 427

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Figure legends Figure 1. Structures of carotenoids in this study Figure 2. Quenching effect on 1O2 (A) and scavenging effect on .OH (B) with capsanthin, capsanthin diacetate, and capsorubin. The vertical axis shows the relative rate (%) of the ESR signal intensity due to singlet oxygen (A) and hydroxyl radical (B) compared with the control group (100 %). Astaxanthin, astaxanthin diacetate, β-carotene, and zeaxanthin were used as positive controls. Significance compared with the control group: * p

Reaction of Paprika Carotenoids, Capsanthin and Capsorubin, with

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