Regeneration of Î²-Carotene from Radical Cation by Eugenol

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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

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Journal of Agricultural and Food Chemistry

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

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clove oil in the Marcus theory inverted region for electron transfer

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

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

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

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C, Denmark

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E-mail addresses of authors:

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Hui-Ting Chang: [email protected]

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Hong Cheng: [email protected]

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Rui-Min Han: [email protected]

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Peng Wang: [email protected]

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Jian-Ping Zhang: [email protected]

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Leif H. Skibsted: [email protected]

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*To whom correspondence should be addressed

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L. H. Skibsted,

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

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J.-P. Zhang

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

ACS Paragon Plus Environment


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Abstract

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

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

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

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

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

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

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

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

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

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

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

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

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Introduction

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

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

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

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

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capacity of such oils4.

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

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

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

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

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

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carotenoids both in plant oils and in food emulsions:

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Car•+ + ϕ-OH → Car + ϕ-O• + H+

(1)

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

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

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

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

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



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

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

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reaction in the actual reaction medium.

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

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

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

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

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

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

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

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

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kET

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

(2)

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

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

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

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

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

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

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phenols like quercetin and the tea catechins9,12.

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

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

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

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

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

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

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

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

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

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plant oils and in food emulsions made from such oils.

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Materials and Methods

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

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

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

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

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

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



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

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

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

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solutions.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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

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a thermostated room (23 ± 1°C).

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Results and Discussion

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

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

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

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

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emulsions under various conditions17.

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

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

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

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

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

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

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

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hv Car → Car•+ + e−solvent

(3)

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

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

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

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

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

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

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followed by a secondary bleaching.

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

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

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

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Car•+ + ϕ-O− → Car + ϕ-O•

(4)

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

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fitting:

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∆OD900 nm = a + b⋅exp(−t/τ)

(5)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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yields a protection similar to the protection by eugenol.

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

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

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reaction:

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ϕ-O• + e− + H+ → ϕ-OH

(6)

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

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

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

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

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

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carotene.

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

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

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

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

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

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

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

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

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

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as defined by the Marcus theory for electron transfer13.

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

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

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

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

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

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non-vitamin antioxidants22.

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

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

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

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

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

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

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

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

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

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

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

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

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

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protection of β-carotene by clove oil.

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Acknowledgements

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

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21673289).

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Notes

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The authors have declared no conflict of interest.

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References

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Scheme 1

333 334

Eugenol

Isoeugenol

335 336

β-carotene

337 338 339 340

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Scheme 2

342 343 344 345 346 347

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

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

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

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

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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

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

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of Figure 2.

Protection (%)

eugenol 43

isoeugenol 29

eugenol/isoeugenol 46

clove oil 36

360

18


Page 19 of 23

361


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



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


Page 21 of 23


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



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


Page 23 of 23

437


Table of Content:

438

439

23


Regeneration of Î²-Carotene from Radical Cation by Eugenol

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