Regeneration of β-Carotene from Radical Cation by Eugenol

Jan 6, 2017 - Isoeugenol, and Clove Oil in the Marcus Theory Inverted Region for ... For eugenol/isoeugenol mixtures and clove oil, kinetic control by...
0 downloads 0 Views 2MB Size
Subscriber access provided by Fudan University

Article

Regeneration of #-carotene from radical cation by eugenol, isoeugenol and clove oil in the Marcus theory inverted region for electron transfer Hui-Ting Chang, Hong Cheng, Rui-Min Han, Peng Wang, Jian-Ping Zhang, and Leif H. Skibsted J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04708 • Publication Date (Web): 06 Jan 2017 Downloaded from http://pubs.acs.org on January 7, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23

Journal of Agricultural and Food Chemistry

1

Regeneration of β-carotene from radical cation by eugenol, isoeugenol and

2

clove oil in the Marcus theory inverted region for electron transfer

3

4

Hui-Ting Chang,1 Hong Cheng,1 Rui-Min Han,1 Peng Wang,1 Jian-Ping Zhang,1,* Leif H. Skibsted2,*

5

6

1

Department of Chemistry, Renmin University of China, Beijing, 100872, China

7

2

Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg

8

C, Denmark

9 10

E-mail addresses of authors:

11

Hui-Ting Chang: [email protected]

12

Hong Cheng: [email protected]

13

Rui-Min Han: [email protected]

14

Peng Wang: [email protected]

15

Jian-Ping Zhang: [email protected]

16

Leif H. Skibsted: [email protected]

17

18

*To whom correspondence should be addressed

19

L. H. Skibsted,

20

Phone: +45 3533 3221; E-mail: [email protected];

21

J.-P. Zhang

22

Phone: +86-10-62516604; E-mail: [email protected] 1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

23

Page 2 of 23

Abstract

24 25

Rate of regeneration of β-carotene by eugenol from the β-carotene radical cation, an initial bleaching

26

product of β-carotene, was found by laser flash photolysis and transient absorption spectroscopy to

27

be close to the diffusion limit in chloroform/methanol (9:1, v:v) with a second-order rate constant (k2)

28

of 4.3×109 L⋅mol−1⋅s−1 at 23 °C. Isoeugenol, more reducing with a standard reduction potential 100

29

mV lower than eugenol, was slower with k2 = 7.2×108 L⋅mol−1⋅s−1. Regeneration of β-carotene

30

following photobleaching was found 50% more efficient by eugenol, indicating that for the more

31

reducing isoeugenol, the driving force exceeds the reorganization energy for electron transfer

32

significantly in the Marcus theory inverted region. For eugenol/isoeugenol mixtures and clove oil,

33

kinetic control by the faster eugenol determines the regeneration, with a thermodynamic back-up of

34

reduction equivalent through eugenol regeneration by the more reducing isoeugenol for the mixture.

35

Clove oil accordingly is a potential protector of provitamin A for use in red plan oils.

36

37

Keywords: carotenoid regeneration; photobleaching; eugenol; electron transfer; inverted region.

38

39

40

41

2

ACS Paragon Plus Environment

Page 3 of 23

42

Journal of Agricultural and Food Chemistry

Introduction

43

44

Vitamin A deficiency is a serious nutritional problem worldwide especially for children1,2. Plant oils

45

like red palm oil, which are rich in α- and β-carotene are important as an affordable dietary source of

46

provitamin A3. Carotenes are, however, like all carotenoids sensitive to light and prooxidants like

47

metal ions, and β-carotene degradation during food processing and storage may deplete the vitamin

48

capacity of such oils4.

49

50

Oxidative degradation of carotenoids involves free radicals, and the carotenoid radical cation is the

51

initial product of carotenoid scavenging of lipid peroxyl radicals, and is a photoproduct of

52

carotenoids exposed to light in the presence of electron acceptors5,6. Carotenoids have been found to

53

be regenerated by tocopherols and tocotrienols and by plant phenols, ϕ-OH, from the carotenoid

54

radical cations7,8. Such regeneration has now been recognized as important in the protection of

55

carotenoids both in plant oils and in food emulsions:

56

57

Car•+ + ϕ-OH → Car + ϕ-O• + H+

(1)

58

59

The regeneration process occurs in homogeneous lipid systems like plant oils by lipophilic phenols

60

like the tocopherols and the tocotrienols4. For heterogeneous systems, the regeneration is associated

61

with the interface between the lipid and the aqueous phase like in cell membranes, and regeneration

62

is known to be facilitated by deprotonation of the water-soluble plant phenols9. The regeneration

63

efficiency depends most importantly on the spatial separation of the carotenoid and the phenol, the 3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 23

64

viscosity of the lipid and the driving force for the electron transfer of the reaction of eq.(1)10,11. For

65

optimal conditions, the electron transfer is fast and approaching the diffusion limit for a bimolecular

66

reaction in the actual reaction medium.

67

68

An important finding has, however, been that the rate of the regeneration through electron transfer

69

from the deprotonated plant phenol to the carotenoid radical cation shows a maximum for the

70

combination of carotenoid and plant phenolate for which the driving force corresponds to the

71

reorganization energy according to the Marcus theory for electron transfer9,12. On the basis of

72

Marcus’s nonadiabatic electron transfer mechanism, the rate of electron transfer depends on V, a

73

matrix element, and on λ, the reorganization energy, which have been determined to have the value

74

5.85 cm−1 and 0.41 eV, respectively for β-carotene reacting with plant phenols in the presence of one

75

equivalent of base in chloroform/methanol (9:1, v:v) at room temperature9:

76

77

kET

 ( ∆G° + λ ) 2  4π 3 2 V exp−  =  h 2λ RT  4λ RT 

(2)

78

79

The rate constant for the reaction between the cation of β-carotene and the plant phenolates are

80

increasing for increasing driving force, −∆G°, as long as the driving force is less than the

81

reorganization energy. The maximum rate is obtained when the driving force matches the

82

reorganization energy. However, the rate decreases with the further increase in driving force in the

83

so-called inverted region as described by the Marcus theory13−15. A decreasing regeneration rate has

4

ACS Paragon Plus Environment

Page 5 of 23

Journal of Agricultural and Food Chemistry

84

now been demonstrated for several carotenoids for an increasing number of the most reducing plant

85

phenols like quercetin and the tea catechins9,12.

86

87

In order to apply this new knowledge to problems related to protection of provitamin A in plant oils,

88

clove oil was selected as a phenol-rich food ingredient soluble in vegetable oil16−18. Eugenol (Scheme

89

1), the main component of clove oil (from Syzigium aromaticum), has a reduction potential for the

90

phenoxyl radical slightly smaller than the potential for syringic acid which is the phenol found to

91

show the most efficient reduction of the β-carotene radical cation19. Isoeugenol (Scheme 1), found in

92

other plants like Ylang-Ylang (Gananga odorata), and more reducing than eugenol, was together

93

with eugenol studied for their efficiencies in regenerating β-carotene18,19. The two isomers were

94

studied separately and in combination and compared with clove oil using real-time methods for fast

95

radical kinetics with the perspective of using clove oil for protection of provitamin A carotenes in

96

plant oils and in food emulsions made from such oils.

97

98

Materials and Methods

99

100

Sample preperation: All-trans-β-carotene (β-Car) was purchased from Sigma-Aldrich (St. Louis,

101

MO), and the β-Car was purified by recrystallization in n-hexane/acetone mixture and the purity

102

checked by HPLC was 98%. Eugenol (99%) was also purchased from Sigma-Aldrich. Isoeugenol

103

(≥97%) was purchased from J&K (Scientific Ltd., Beijing, China). The pure clove oil was purchased

104

from O′plants (Australia). Methanol (>99.0%, Beijing Chemical Works, Beijing, China) was used as

105

received. Chloroform (>99.0%, Beijing Chemical Works, Beijing, China) was purified by being 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 23

106

passed through an alumina column before use (AR, Tianjin Fuchen Chemical Plant, Tianjin, China).

107

Solutions of phenolates were prepared by addition of one equivalent of tetramethylammonium

108

hydroxide (97%, Sigma-Aldrich) to the phenols dissolved in neutral chloroform/methanol (9:1, v/v)

109

solutions.

110

111

UV-Visible absorption spectroscopy: UV-visible absorption spectra were measured on a Cary50

112

spectrophotometer (Varian Inc., Palo Alto, CA), using 1.0 cm quartz cells.

113

114

Laser Flash Photolysis and Transient Absorption Spectroscopy: The nanosecond laser flash

115

photolysis and transient absorption spectroscopy apparatus was described in detail elsewhere11.

116

Excitation laser pulses (7 ns, 10 Hz) at the wavelength of 532 nm were supplied by a Nd3+:YAG

117

laser (Quanta-Ray PRO-230; Spectra Physics, Mountain View, CA), and pulse energy was

118

attenuated to 3 mJ/pulse. The optical path length of the flow cuvette was 5 mm. The desired probe

119

wavelengths (400-1050 nm) were provided by a laser-driven white light source (LDLS-EQ-1500,

120

Energetic technology Inc., Woburn, MA). Kinetics were detected with a photodiode (model S3071,

121

Hamamatsu Photonics, Hamamatsu, Japan) attached to a spectrograph (SP2500i, Princeton

122

Instruments, NJ), when both the excitation light and the probe light were focused on the optical

123

sample cuvette during experiment, and the signals were stored and averaged with a digital storage

124

oscilloscope (bandwidth 500 MHz; Teledyne LeCroy HDO 4054, Chestnut Ridge, NY) connected to

125

a personal computer. The original collected data provided single wavelength kinetics and parameter

126

fitting was based on Matlab 5.3 (Mathworks) software. The concentration of β-carotene was

127

5.0×10−5 mol⋅L−1 and the concentration of eugenol or isoeugenol was 1.0×10−4 mol⋅L−1, and for clove

128

oil an amount to yield the same concentration of eugenol was used. The concentration of

6

ACS Paragon Plus Environment

Page 7 of 23

Journal of Agricultural and Food Chemistry

129

tetramethylammonium hydroxide was 1.0×10−4 mol⋅L−1. All of the measurements were carried out in

130

a thermostated room (23 ± 1°C).

131

132

Results and Discussion

133

134

Eugenol is an aromatic plant phenol, used in bakery products and spiced beverages, and the main

135

component of clove oil also used after chemical transformations as a fragrance16. Eugenol is a

136

versatile compound, as it also exhibits antibacterial properties used in dentistry and is an

137

antioxidant18. The antioxidant properties have recently been shown to protect β-carotene in

138

emulsions under various conditions17.

139

140

The interaction of eugenol or the closely related isomer isoeugenol with β-carotene in solution was

141

studied by steady state and time-resolved absorption spectroscopy. The UV-visible absorption

142

spectra of β-carotene and eugenol/isoeugenol was found additive in their mixtures in

143

chloroform/methanol (9:1, v:v), also in the presence of base, as may be seen in Figure 1. Clove oil or

144

eugenol/isoeugenol mixtures at the same total phenol concentration showed a similar behavior.

145

146

Upon light exposure, β-carotene form the radical cation with a characteristic absorption in the near

147

infra region with a concomitant bleaching of the β-carotene visible absorption:

148

7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 23

149

150

hv Car → Car•+ + e−solvent

(3)

151

152

The formation and decay of the β-carotene radical cation is seen in Figure 2(a) as the time trace of

153

rise and decay of the 900 nm absorption together with the time trace for bleaching and recovery for

154

510 nm absorption in Figure 2(b). The decay of the β-carotene radical cation is in the µs time regime

155

as is also the recovery of β-carotene color following bleaching. The decay of the radical cation is

156

accelerated by the presence of eugenol, isoeugenol, their mixture or clove oil. In the presence of the

157

plant phenols, the bleaching is partly reversible, while in their absence the initial bleaching is

158

followed by a secondary bleaching.

159

160

The time trace of the 900 nm absorbance could for up to 80 µs be described by a single exponential,

161

see Figure 3, corresponding to the reaction of β-carotene radical cation with excess of eugenol or

162

isoeugenol (ϕ-OH) in the presence of the equivalent concentration of base:

163

164

Car•+ + ϕ-O− → Car + ϕ-O•

(4)

165

166

The lifetime, τ, for the β-carotene radical cation at 23 °C obtained from single exponential decay

167

fitting:

168

8

ACS Paragon Plus Environment

Page 9 of 23

169

Journal of Agricultural and Food Chemistry

∆OD900 nm = a + b⋅exp(−t/τ)

(5)

170

171

in the presence of 1.0×10−4 mol⋅L−1 eugenol or isoeugenol or the equimolar mixture of the two

172

isomers at the same total concentration are presented in Table 1 together with the lifetime for the β-

173

carotene radical cation in the presence of clove oil to provide the same eugenol concentration. For

174

eugenol and isoeugenol the lifetime converted to second-order rate constants for the reaction of eq.(4)

175

are included in Table 1. From Figure 2 it may be seen that the decay for the equimolar

176

eugenol/isoeugenol mixture has the same time profile, as has also the decay in the presence of clove

177

oil with the same eugenol concentration. Eugenol reacts faster by a factor of 6 with the β-carotene

178

radical cation compared to isoeugenol. Clove oil reacts with the same rate as eugenol, when the clove

179

oil is present in the amount required to give the same eugenol concentration. An equimolar mixture

180

of eugenol and isoeugenol reacts with the same rate as eugenol at the same total phenol concentration.

181

182

The protection of β-carotene against photobleaching, as calculated from the ∆OD of Figure 2

183

following regeneration by eugenol, isoeugenol, eugenol/isoeugenol mixture, shows that eugenol

184

provides 50% more regeneration than isoeugenol, see Table 2. Clove oil protects β-carotene with an

185

efficiency similar to the efficiency of eugenol. The equimolar mixture of eugenol and isoeugenol

186

yields a protection similar to the protection by eugenol.

187

188

Isoeugenol is more reducing than eugenol due to extension of the conjugation from the aromatic ring

189

into the carbon side chain, see Scheme 1. The standard reduction potential corresponding to the

190

reaction:

9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 23

191

192

ϕ-O• + e− + H+ → ϕ-OH

(6)

193

194

has been determined in aqueous solutions to have the value at 25 °C of E° = +0.75 V for eugenol and

195

of E° = +0.66 V for isoeugenol, respectively, versus the standard hydrogen electrode19. For

196

conditions of pH = 7 for comparison of the potentials with potentials known for other plant phenols20,

197

the potential gets the value E′ = +0.34 V for eugenol and E′= +0.25 V for isoeugenol, respectively.

198

Clearly isoeugenol is the most reducing, but still reacts with the slowest rate in regeneration of β-

199

carotene.

200

201

For a series of plant phenols, the rate constant for the reaction of eq.(4) has been determined for

202

identical conditions as used for eugenol, isoeugenol and clove oil in the present study. The driving

203

force for the regeneration reaction of eq.(4) may be calculated from the difference in reduction

204

potential between the β-carotene radical cation, +1.06 V10, and the potential for the neutral phenoxyl

205

radical, to yield +0.72 V for eugenol and +0.81 V for isoeugenol, respectively. In Figure 4, the

206

second order rate constants for eugenol and isoeugenol are included in order to compare eugenol and

207

isoeugenol with the other plant phenols for which rate data now are available. For a potential

208

difference larger than approximately +0.4 V, the rate decreases for increasing potential difference

209

and accordingly increasing driving force. Eugenol and isoeugenol are clearly in this inverted region

210

as defined by the Marcus theory for electron transfer13.

211

10

ACS Paragon Plus Environment

Page 11 of 23

Journal of Agricultural and Food Chemistry

212

The structure of eugenol and isoeugenol is very similar, although the standard reduction potential of

213

their phenoxyl neutral radicals are different by approximately 100 mV with isoeugenol being the

214

more reducing. It is very encouraging that this difference and resulting decrease in rate of electron

215

transfer to the β-carotene radical cation can be accommodated by the Marcus theory as is seen from

216

Figure 4, supporting the use of this theoretical framework for electron transfer between vitamin and

217

non-vitamin antioxidants22.

218

219

Currently there is an increasing interest in the stability of carotenoids in foods in relation both to

220

their storage stability and their health effect23,24. Dietary carotenoids like β-carotene also seem

221

important for protection against free radicals in the stomach during food digestion25. As for a more

222

practical use of the results of the present study, clove oil has been shown to be very efficient in

223

regenerating β-carotene and could find use in foods based on red palm oil as a natural protector of

224

provitamin A against light-induced degradation. The acceptable daily intake (ADI) of eugenol and

225

isoeugenol is 5 mg kg-1 and 2 mg kg-1, respectively. The use at the level required for antioxidative

226

protection present no toxicological problems26−28. The observation that mixtures of eugenol and

227

isoeugenol yield the same protection as eugenol could indicate a fast regeneration of eugenol, as the

228

reactant regenerating β-carotene, by the more reducing isoeugenol, as seen Scheme 2. The

229

regeneration of β-carotene is kinetically controlled as seen from Table 2, but the backup with

230

reductant equivalents is a thermodynamic factor. Other components in clove oil seem to have no

231

effect on the regeneration of β-carotene as the content of eugenol was found fully to account for the

232

protection of β-carotene by clove oil.

233

234 11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

235

Page 12 of 23

Acknowledgements

236

237

This work has been supported by the Natural Science Foundation of China (No. 21673288 and

238

21673289).

239

240

Notes

241

242

The authors have declared no conflict of interest.

243

244

References

245

246

1. WHO. Guidline: Vitamin A supplementation in infants and children 6–59 months of age. Geneva:

247

World Health Organization. 2011.

248

2. Akhtar, S., Ahmed, A., Randhawa, M. A., Atukorala, S., Arlappa, N., Ismail, T. Prevalence of

249

vitamin A deficiency in Soth Asia: Cause, outcomes, and possible remedies. J. Health Pop. and Nutr,

250

2013, 31, 413–423.

251

3. Ayu, D. F.; Andarwulan, N.; Hariyadi, P.; Purnomo, E. H. Effect of tocopherols, tocotrienols, β-

252

Carotene, and chlorophyll on the photo-oxidative stability of red palm oil. Food Sci. Biotechnol.

253

2016, 25, 401−407.

254

4. Schroeder, M. T.; Becker, E. M.; Skibsted, L. H. Molecular Mechanism of Antioxidant

255

Synergism of Tocotrienols and Carotenoids in Palm Oil. J. Agric. Food Chem. 2006, 54, 3445−3453. 12

ACS Paragon Plus Environment

Page 13 of 23

Journal of Agricultural and Food Chemistry

256

5. Böhm, F.; Edge, R.; Truscott, G. Interactions of dietary carotenoids with activated (singlet)

257

oxygen and free radicals: potential effects for human health. Mol. Nutr. Food Res. 2012, 56,

258

205−216.

259

6. Skibsted, L. H. Carotenoids in antioxidant networks. Colorants or radical scavengers. J. Agric.

260

Food Chem. 2012, 60, 2409−2417.

261

7. Mortensen, A.; Skibsted, L. H. Relative stability of carotenoid radical cations and homologue

262

tocopheroxyl radicals. A real time kinetic study of antioxidant hierarchy. FEBS Lett. 1997, 417,

263

261−266.

264

8. Edge, R.; Land, E. J.; McGarvey, D.; Mulroy, L.; Truscott, T. G. Relative one-electron reduction

265

potentials of carotenoid radical cations and the interactions of carotenoids with the vitamin E

266

radical cation. J. Am. Chem. Soc. 1998, 120, 4087−4090.

267

9. Cheng, H.; Han, R. M.; Zhang, J. P.; Skibsted, L. H. Electron transfer from plant phenolates to

268

carotenoid radical cations. Antioxidant interaction entering the Marcus theory inverted region. J.

269

Agric. Food Chem. 2014, 62, 942−949.

270

10. Burke, M.; Edge, R.; Land, E. J.; Mcgarvey, D. J; Truscott, T. G. One-electron reduction

271

potentials of dietary carotenoid radical cations in aqueous micellar environment. FEBS Lett. 2001,

272

500, 132−136.

273

11. Song, L. L.; Liang, R.; Li, D. D.; Xing, Y. D.; Han, R. M.; Zhang, J. P.; Skibsted, L. H. β-

274

Carotene radical cation addition to green tea polyphenols. Mechanism of antioxidant antagonism in

275

peroxidizing liposomes. J. Agric. Food Chem. 2011, 59, 12643−12651.

276

12. Cheng, H.; Han, R. M.; Lyu, M. K.; Zhang, J. P.; Skibsted, L. H. Regeneration of β‑carotene

277

from the radical cation by tyrosine and tryptophan. J. Phys. Chem. B 2015, 119, 6603−6610.

278

13. Marcus, R. A. Electron transfer reactions in chemistry. Theory and experiment. Rev. Mod. Phys.

279

1993, 65, 599−610.

280

14. Turró, C.; M. Zaleski, J.; M. Karabatsos, Y.; G. Nocera, D. Bimolecular electron transfer in the 13

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 14 of 23

281

Marcus inverted region. J. Am. Chem. Soc. 1996, 118, 6060−6067.

282

15. Fukuzumi, S.; Ohkubo, K.; Imahori, H.; M. Guldi, D. Driving force dependence of

283

intermolecular electron-transfer reactions of fullerenes. Chem.-Eur. J. 2003, 9, 1585−1593.

284

16. Mihara, S.; Shibamoto, T. Photochemical reactions of eugenol and related compounds: synthesis

285

of new flavor chemicals. J. Agric. Food Chem. 1982, 30, 1215−1218.

286

17. Guan, Y. G.; Wu, J. N.; Zhong, Q. X. Eugenol improves physical and chemical stabilities of

287

nanoemulsions loaded with β-carotene. Food Chem. 2016, 194, 787–796.

288

18. Chaieb, K.; Hajlaoui, H.; Zmantar, T.; Kahla-Nakbi, A. B.; Rouabhia, M.; Mahdouani, K.;

289

Bakhrouf, A. The chemical composition and biological activity of clove essential oil, Eugenia

290

caryophyllata (Syzigium aromaticum L. Myrtaceae): A short review. Phytother. Res. 2007, 21, 501–

291

506.

292

19. Guha, S. N.; Priyadarsini, K. I. Kinetic and redox characteristics of phenoxyl radicals of eugenol

293

and isoeugenol: A pulse radiolysis study. Inter. J. Chem. Kine. 2000, 32, 1097−4601.

294

20. Simić, A.; Manojlović, D.; Šegan, D.; Todorović, M. Electrochemical behavior and antioxidant

295

and prooxidant activity of natural phenolics. Molecules 2007, 12, 2327−2340.

296

21. Cheng, H.; Liang, R.; Han, R. M.; Zhang, J. P.; Skibsted, L. H. Efficient scavenging of β-

297

carotene radical cations by antiinflammatory salicylates. Food Funct. 2014, 5, 291−294.

298

22. Skibsted, L. H. Vitamin and non-vitamin antioxidants and their interaction in food. J. Food Drug

299

Anal. 2012, 20, 355−358.

300

23. Yi, J.; Andersen, M. L.; Skibsted, L. H. Interaction between tocopherols, tocotrienols and

301

carotenoids during autoxidation of mixed palm olein and fish oil. 2011, 127, 1792−1797.

302

24. Rodriguez-Amaya, D. B. Status of carotenoid analytical methods and in vitro assays for the

303

assessment of food quality and health effects. Curr. Opin. Food Sci. 2015, 1, 56−63.

304

25. Sy, C.; Dangles, O.; Borel, P.; Caris-Veyrat, C. Stability of bacterial carotenoids in the presence

305

of iron in a model of the gastric compartment-comparison with dietary reference carotenoids. Arch. 14

ACS Paragon Plus Environment

Page 15 of 23

Journal of Agricultural and Food Chemistry

306

Biochem. Biophys. 2015, 572, 89−100.

307

26. Opdyke, D.L.J. Monographs on fragrance and raw materials: eugenol. Food and Cosmetic

308

Toxicol. 1975, 13, 545–547.

309

27. Sousa, G.D.; Teng, S.; Salle-siri, R.; Pery, A.; Rahmani, R. Prediction of the metabolic clearance

310

of benzophenone-2, and its interaction with isoeugenol and coumarin using cryopreserved human

311

hepatocytes in primary culture. Food Chem Toxicol. 2016, 90, 55–63.

312

28. Atsumi, T.; Fujisawa, S.; Tonosaki, K. A comparative study of the antioxidant/prooxidant

313

activities of eugenol and isoeugenol with various concentrations and oxidation conditions.

314

Toxicology in Vitro. 2005, 19, 1025–1033.

315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 15

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

332

Page 16 of 23

Scheme 1

333 334

Eugenol

Isoeugenol

335 336

β-carotene

337 338 339 340

16

ACS Paragon Plus Environment

Page 17 of 23

341

Journal of Agricultural and Food Chemistry

Scheme 2

342 343 344 345 346 347

348

17

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 18 of 23

349

Table 1 Lifetime, τ in µs, for β-carotene radical cation in presence of 1.0×10−4 mol⋅L−1 eugenol or

350

isoeugenol in chloroform/methanol (9:1, v:v) under pseudo first-order conditions at 23 °C as

351

detected from decay of absorbance at 900 nm together with second-order rate constant, k2, for the

352

reaction of eugenol and isoeugenol with the β-carotene radical cation. The lifetime, τ, for an

353

equimolar mixture of eugenol and isoeugenol and for clove oil is determined similarly.

Sample

τ / µs

k2 / L⋅mol−1⋅s−1

Eugenol Isoeugenol Mixture Clove oil

4.7 27.9 4.9 5.4

4.26×109 7.17×108

354 355 356 357

Table 2 Regeneration of β-carotene by eugenol, isoeugenol, clove oil and equimolar

358

eugenol/isoeugenol mixture all at the same total phenol concentration for the experimental conditions

359

of Figure 2.

Protection (%)

eugenol 43

isoeugenol 29

eugenol/isoeugenol 46

clove oil 36

360

18

ACS Paragon Plus Environment

Page 19 of 23

361

Journal of Agricultural and Food Chemistry

Figures

362 1.0

363 364 365

Absorbance

0.8

β-Car β-Car-base β-Car-eug β-Car-eug-base

(a)

β-Car β-Car-base β-Car-isoeug β-Car-isoeug-base

(b)

β-Car β-Car-base β-Car-oil β-Car-oil-base

(c)

β-Car β-Car-base β-Car-eug-isoeug β-Car-eug-isoeug-base

(d)

0.6 0.4 0.2

366

0.0 1.0

367 368 369 370

Absorbance

0.8 0.6 0.4 0.2 0.0

300

400

500

600

300

Wavelength / nm

400

500

600

Wavelength / nm

371 372

Figure 1: UV-vis absorption spectra of (a) β-carotene (5.0×10−5 mol⋅L−1) in absence or presence of

373

tetramethylammonium hydroxide (1.0×10−4 mol⋅L−1) in chloroform/methanol (9:1, v:v) alone or

374

together with eugenol (eug) (1.0×10−4 mol⋅L−1); (b) isoeugenol (isoeug) replacing eugenol; (c) clove

375

oil (oil) replacing eugenol at the same phenol concentration; (d) an equimolar mixture replacing

376

eugenol at total concentration of 1.0×10−4 mol⋅L−1.

377 378 379 380 381 382 19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 20 of 23

383

1.2

(a)

900 nm

1.0

∆OD

0.8 β-Car only β-Car+eug+base β-Car+isoeug+base β-Car+oil+base β-Car+eug+isoeug+base

0.6 0.4 0.2 0.0

0.0

β-Car only β-Car+eug+base β-Car+isoeug+base β-Car+oil+base β-Car+eug+isoeug+base

(b)

510 nm

384

-0.4 386 387 388

∆OD

385

-0.8

-1.2

389

-1.6 390 391

0

20

40 60 80 100 Delay time / µs

120

140

392

Figure 2: Transient absorption kinetics of β-carotene in chloroform/methanol (9:1,v:v) following

393

laser flash photolysis (wavelength 532 nm, pulse width 7 ns) at 23 °C in the absence or presence of

394

eugenol, isoeugenol, their equimolar mixture or clove oil together with one equivalent of base

395

relative to the phenol. Concentration of β-carotene was 5.0×10−5 mol⋅L−1, and concentration of

396

eugenol or isoeugenol was 1.0×10−4 mol⋅L−1.

397

398 20

ACS Paragon Plus Environment

Page 21 of 23

Journal of Agricultural and Food Chemistry

399

400

401

β-Car+eugenol+base β-Car+isoeugenol+base

1.0 402

0.8

404

∆OD

403

0.6

900 nm

0.4

405

0.2

406

0.0 0

407

20

40 Delay time / µs

60

80

408

Figure 3: Absorption decay at 900 nm following laser flash photolysis of β-carotene in

409

chloroform/methanol (9:1, v:v) at 23 °C with fitting to a single exponential decay curve for

410

experimental details, see Fig. 2

411

412

413

414

415

416

417 21

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 22 of 23

418

420

421

422

423

424

10.0

log k2 / L⋅⋅mol −1⋅s−1

419

Syringic Acid

Caffeic Acid Puerarin/daidzein

p-Coumaric Acid

9.5

Vanillic Acid

eugenol

Rutin Quercetin Tyrosine

9.0

m-Hydroxybenzoic Acid

isoeugenol

8.5 Salicylic Acid Tea catechins

8.0 0.0

0.2

0.4

0.6

0.8

1.0

E/V

425

426

Figure 4: Logarithm of the second-order rate constant (k2) for electron transfer to β-carotene radical

427

cation from plant phenolates as depending on the potential difference E = E′(Car•+) − E′(ϕ-O•). Data

428

from references 9, 10, 11, 12, 20, 21.

429

430

431

432

433

434

435

436 22

ACS Paragon Plus Environment

Page 23 of 23

437

Journal of Agricultural and Food Chemistry

Table of Content:

438

439

23

ACS Paragon Plus Environment