Electron Transfer from Plant Phenolates to Carotenoid Radical

9 Jan 2014 - For β-carotene, the reorganization energy has values of 0.41 ± 0.04 and 0.40 .... Trivista spectrograph monochromator (Princeton Instru...
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Electron Transfer from Plant Phenolates to Carotenoid Radical Cations. Antioxidant Interaction Entering the Marcus Theory Inverted Region Hong Cheng,† Rui-Min Han,† Jian-Ping Zhang,*,† and Leif H. Skibsted*,‡ †

Department of Chemistry, Renmin University of China, Beijing 100872, China Food Chemistry, Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark



ABSTRACT: β-Carotene, lycopene, and zeaxanthin are maximally regenerated by plant phenolates from their radical cations formed during laser flash photolysis in 9:1 (v/v) chloroform/methanol for a driving force corresponding to the reorganization energy according to the Marcus theory. For β-carotene, the reorganization energy has values of 0.41 ± 0.04 and 0.40 ± 0.04 eV for the plant phenols in the presence of 1 and 2 equiv of base, respectively, at 23 °C. For a driving force lower than the reorganization energy, regeneration of the carotenoids is less efficient as is seen for m-hydroxybenzoic acid, vanillic acid, and pcoumaric acid. For a driving force above the maximum rate as determined to have kET = 6.3 × 109 L·mol−1·s−1 for syringic acid and β-carotene, the reaction becomes gradually slower and regeneration less efficient as is seen for the more reducing caffeic acid, rutin, and quercetin corresponding to an inverted region for the rate of electron transfer. Lycopene and zeaxanthin show a similar behavior for the same series of plant phenols with slightly lower reorganization energy, in agreement with the lower reduction potential of their radical cations, while, for the ketocarotenoids astaxanthin and canthaxanthin, fast reactions with a solvent of radical cations inhibit regeneration from being detected. Intermediate reducing plant phenols accordingly yield maximal protection of carotenoids against photobleaching in foods and beverages. KEYWORDS: carotenoid regeneration, photobleaching, plant phenolate, electron transfer, inverted region

1. INTRODUCTION Regeneration of lipophilic carotenoids from their radical cations at water/lipid interfaces by the more hydrophilic (iso)flavonoids and their glycosides or by other plant phenols has been recognized as important for the antioxidant synergism often observed between carotenoids and polyphenols in heterogeneous food systems.1,2 Carotenoids (Car’s) have been identified as radical scavengers in membranes where they reduce lipid peroxyl and certain other radicals through electron transfer (ET) in processes crucial for oxidative stability of unsaturated lipids and other sensitive nutrients3 Car + ROO• → Car •+ + ROO−

For liposomes as models for membranes, antioxidant synergism has been demonstrated especially when oxidation is initiated in the lipid phase of such heterogeneous systems using in situ spectral analysis of lipid oxidation products. A regeneration of the carotenoid by plant phenolates at the water/lipid interface by ET fully accounts for this synergism, since the regenerated carotenoid now repeatedly may scavenge radicals in the lipids of the membrane at the expense of water-soluble plant phenols not in direct contact with the peroxidizing lipids.2,7 Such regeneration by flavonoids also seems important for protection of eye function by yellow carotenoids.1,2 The ET between plant phenolates and carotenoid radical cations is very efficient, with rates for the more oxidizing carotenoid radical cations approaching the diffusion limit. For a series of four carotenoids, including astaxanthin as one of the least reducing carotenoids and accordingly with a strongly oxidizing carotenoid radical cation, the rate for ET from plant phenolates has moreover been found to depend on the driving force following a linear free energy relationship.7 The driving force is based on electrochemical potentials, and it is accordingly expected that ET to carotenoid radical cations from green tea catechins, as some of the most reducing flavonoids, will be even faster. In contrast, ET is not observed to the β-carotene radical cation from the catechins but replaced by addition reactions between β-carotene and the catechins

(1)

or by addition of radicals4 Car + R• → Car−R•

(2)

while only for the hydroxyl radical5,6 has hydrogen atom transfer (HAT) been confirmed Car + OH• → Car( −H)• + H 2O

(3)

Carotenoid radical cations are more oxidizing than most phenoxyl radicals, and the regeneration of the carotenoids has been followed by time-resolved transient absorption spectroscopy to provide second-order rate constants for ET from plant phenolates to carotenoid radical cations in homogeneous solution for an increasing number of plant phenol/carotenoid combinations: Car •+ + φ‐O− → Car + φ‐O• © 2014 American Chemical Society

Received: Revised: Accepted: Published:

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July 25, 2013 January 8, 2014 January 9, 2014 January 9, 2014 dx.doi.org/10.1021/jf404725v | J. Agric. Food Chem. 2014, 62, 942−949

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without any detectable regeneration of β-carotene from its radical cation.8 It is accordingly of interest to study ET to carotenoid radical cations from a series of plant phenolates, with decreasing reduction potentials resulting in increasing driving force for ET. Such a kinetic study should further explore whether the fundamental ET reaction of eq 4 in an antioxidant interaction in membranes will enter the inverted region for increasing driving force where the rate of ET according to the Marcus theory decreases. Such an inverted region for the rate of ET now has been recognized for other types of bimolecular ET reactions and may for antioxidant regeneration have important biological consequences for controlling oxidative stress and also for design of functional foods with sensitive nutrients.9−19 We have selected β-carotene, lycopene, zeaxanthin, astaxanthin, and canthaxanthin as a series of carotenoids of general food interest for our studies together with the selection of plant phenols shown in Figure 1, all of which are known to be important plant-based antioxidants in human nutrition or used for antioxidant protection of food emulsions. A large difference in antioxidative activity has been noted for rather small changes in molecular structure in homologue series of natural plant phenols also with a positive effect on human health as taylored by increasing hydrophobicity.20,21

2. MATERIALS AND METHODS 2.1. Chemicals. all-trans-β-Carotene (β-Car) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). all-trans-Lycopene was extracted from tomato juice and purified according to the procedure described by Zhang et al.22 Astaxanthin, canthaxanthin, and zeaxanthin sealed in ampules under argon were supplied by Roche A/S (Hvidovre, Denmark). m-Hydroxybenzoic acid (≥99%), vanillic acid (≥97%), p-coumaric acid (≥98%), syringic acid (≥95%), caffeic acid (≥98%), and rutin (≥94%) were also purchased from Sigma-Aldrich. Quercetin (≥98%) was purchased from Fluka Biochemika (Buchs, Switzerland). Methanol of high-performance liquid chromatography (HPLC) grade (J&K Scientific Ltd., Beijing, China) was used as received. Chloroform (>99.0%, Beijing Chemical Works, Beijing, China) was purified before use by being passed through an alumina column (AR, Tianjin Fuchen Chemical Plant, Tianjin, China). Solutions of phenolates were prepared by addition 1 or 2 equiv of tetramethylammonium hydroxide (97%, Sigma-Aldrich) to the phenols dissolved in neutral chloroform/methanol (9:1, v/v) solutions. 2.2. Laser Flash Photolysis. The laser flash photolysis apparatus has been described elsewhere.8 Briefly, the excitation laser pulses at 532 nm (4 mJ/pulse, 7 ns, 10 Hz) were supplied by a Nd3+:YAG laser (Quanta-Ray PRO-230; Spectra Physics, Santa Clara, CA), and the probe light was provided by a xenon lamp (CW 300 W). The optical path length of the flow cuvette (∼30 mL) used for laser flash photolysis was 5 mm. The near-infrared kinetics probed at 900 nm for β-carotene radical cation (β-Car•+) was detected with a photodiode (model S3071, Hamamatsu Photonics, Hamamatsu, Japan) attached to a Trivista spectrograph monochromator (Princeton Instruments, Trenton, NJ), and the kinetic traces were stored and averaged with a digital storage oscilloscope (bandwidth 600 MHz; Teledyne LeCroy WaveSurfer 64Xs, Chestnut Ridge, NY) connected to a personal computer. For kinetic analyses, the time evolution profiles of the optical density change (ΔOD) upon pulsed excitation were fitted to a two- or three-exponential model function as previously described.2,8 The concentration of β-carotene was 50 μM. Solution samples were used immediately after preparation under reduced light conditions. All of the measurements were carried out in a thermostated room (23 ± 1 °C). The decay kinetics for carotenoid radical cations was found independent of dissolved oxygen upon a comparison between airsaturated and degassed solutions.

Figure 1. Molecular structures of (a) m-hydroxybenzoic acid, (b) vanillic acid, (c) syringic acid, (d) p-coumaric acid, (e) caffeic acid, (f) rutin, (g) quercetin, (h) lycopene, (i) β-carotene, (g) zeaxanthin, (k) canthaxanthin, and (l) astaxanthin.

3. RESULTS AND DISSCUSSION Carotenoids modulate oxidative stress in membranes and cellular structures through ET to oxidizing radicals.3 The fate of carotenoid radical cations formed under such conditions of oxidative stress have previously been followed in real time kinetic studies using fast spectroscopic techniques for homogeneous solutions, and their effect on lipid oxidation 943

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Figure 2. Normalized transient absorption probed at 900 nm for chloroform/methanol (9:1, v/v) solutions of (a) caffeic acid (100 μM) and (b) syringic acid (100 μM) with β-carotene (50 μM) following 7 ns flash excitation at 532 nm, and parameter fitting for caffeic acid (c) and syringic acid (d) using two exponentials for addition of 1 equiv of base and three exponentials for addition of 2 equiv of base.

Table 1. Second-Order Rate Constants for the Reaction of Radical Cations of β-Carotene, Lycopene, and Zeaxanthin with Phenolic Acids in the Presence of 1 or 2 equiv of Base to Plant Phenol Based on Experiment As Shown in Figure 2 for βCarotene, All at 23 °C β-carotene

lycopene −1 −1

k4/ L·mol ·s

a

a

compd

E /V

m-hydroxybenzoic adid vanillic acid p-coumaric acid syringic acid caffeic acid rutin quercetin

0.01 0.11 0.17 0.35 0.39 0.61 0.74

1 equiv of base 8.0 2.8 3.7 6.3 5.0 3.4 2.0

× × × × × × ×

108 109 109 109 109 109 109

k4/L·mol−1·s−1

k4/L·mol ·s a

2 equiv of base

E /V

× × × × × × ×

−0.07 0.03 0.09 0.27 0.31 0.53 0.66

7.2 2.0 3.3 4.2 4.2 2.0 1.4

zeaxanthin −1 −1

108 109 109 109 109 109 109

1 equiv of base 5.7 1.2 1.8 2.0 1.1 1.9 1.3

× × × × × × ×

108 109 109 109 109 109 109

a

2 equiv of base

E /V

× × × × × × ×

−0.02 0.08 0.14 0.32 0.36 0.58 0.71

5.9 1.3 1.9 1.6 1.5 1.8 1.2

108 109 109 109 109 109 109

1 equiv of base 1.3 2.1 3.2 3.3 3.0 3.2 2.5

× × × × × × ×

109 109 109 109 109 109 109

2 equiv of base 1.3 2.3 3.1 3.1 1.9 2.7 2.1

× × × × × × ×

109 109 109 109 109 109 109

⊖ ⊖ ⊖ E = E⊖ Car•+ − Ephenol with Ephenol based on ref 16 and ECar•+ based on ref 17.

have been evaluated in liposomes as a model for membranes.2,7 Carotenoids have thus been found to form an antioxidant network according to their tendency of reduction of their oneelectron-oxidized forms but also to undergo regeneration reactions by other antioxidants such as polyphenols when available as electron donors, leading to antioxidant synergism between different carotenoids and between carotenoids and polyphenols.7,14 Carotenoid radical cations were generated as in our previous studies using laser flash photolysis in homogeneous chloroform/methanol (9:1, v/v) as an electron-withdrawing solvent.2 The fate of the β-carotene radical cation was followed using transient absorption spectroscopy at 900 nm in the absence or presence of one of the plant phenols shown in Figure 1 with and without base added as shown in Figure 2 for caffeic acid and syringic acid. The β-carotene radical decays only slowly even in the presence of a plant phenol in the absence of base. However, addition of 1 or 2 equiv of base in the presence of a plant phenol induces rapid decay, which for 1 equiv of base is described by a biexponential decay curve and for 2 equiv of base requires a triexponential fitting as can be seen in Figure 2. Similar results were obtained for the other plant phenols

investigated (data not shown), and an analysis of the numerical values for the multiexponential decay curves showed in all cases that one exponential term dominated with a larger amplitude contribution. Other minor components in the exponential decay curves may be due to interference from the laser or to parallel or sequential reactions as previously noted for reactions of β-carotene radical cations with (iso)flavonoids and their glycosides.2 The rate constant for the dominant decay reaction of β-carotene radical cations by each of the seven plant phenols investigated can be found in Table 1 for conditions with both 1 and 2 equiv of base added after conversion to second-order rate constants. Similar results were obtained for lycopene and zeaxanthin, for which the second-order rate constants are also presented in Table 1. For none of these carotenoids did the decay of the radical cation induced by base alone interfere with the reduction of the radical cation by the plant phenolate. In contrast, under alkaline conditions, the radical cation of astaxanthin and canthaxanthin decayed rapidly. The firstorder rate constants for decay of carotenoid radical cations formed by laser flash photolysis in the absence of phenolates are for all five carotenoids collected in Table 2. For astaxanthin and canthaxanthin, the observed first-order rate constants are 944

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second-order rate constants and are included in Table 1 for further discussion. The second-order rate constants calculated from the observed pseudo-first-order rate constants and included in Table 1 for further discussion are all based on the conditions used for the experiment of Figure 2. The plant phenols were found not to react directly with the β-carotene radical cation despite a large driving force. The values of E of Table 1 are based on the reduction potentials determined in aqueous solution of pH 7.016

Table 2. Observed First-Order Rate Constants for Decay of Carotenoid Radical Cation in 9:1 (v/v) Chloroform/ Methanol with Tetramethylammonium Hydroxide as Base Following 532 nm Photolysis at 23 °C kobsd/s−1 compd (10 μM)

[OH−] = 20 μM

β-carotene lycopene zeaxanthin canthaxanthin astaxanthin

× × × × ×

1.7 1.3 4.7 5.1 5.9

4

10 104 104 105 105

[OH−] = 40 μM 2.5 1.4 5.1 7.0 1.5

× × × × ×

104 104 104 105 106

φ‐O• + e− + H+ → φ‐OH

(5)



and E = 1.06 V for reduction of the radical cation of βcarotene, 0.98 V for lycopene, and 1.03 V for zeaxanthin as determined for aqueous conditions.17 However, deprotonation of the plant phenols studied (Figure 1) resulted in each case in a rapid reaction even following reaction with only 1 equiv of base as for m-hydroxybenzoic acid:

larger by more than a factor of 10 than for zeaxanthin, lycopene, and especially β-carotene. Accordingly, under the actual experimental conditions, the reaction of the radical cations of the two ketocarotenoids with the plant phenols became slower than the reaction with the solvent. This observation of a rapid reduction of these carotenoid radicals by the alkaline solvent is in agreement with the previous identification of the keto group as important for electron transfer to astaxanthin and canthaxanthin radical cations.7 The first-order rate constants for decay of the carotenoid radical cations were assigned to the reaction of eq 4 in which a carotenoid is regenerated by ET from the phenolate.2,7 Notably, the phenolate (≥100 μM) was for all experimental conditions in large excess compared to the radical cations, which were only formed as a minor fraction of the total concentration of the carotenoid (50 μM) during laser flash photolysis. This large excess provides pseudo-first-order conditions for the reactions with phenolates following the laser flash photolysis. The pseudo-first-order rate constants determined as shown in Figure 2 for β-carotene by exponential fitting were found to depend linearly on the concentration of excess plant phenol, in agreement with a second-order reaction as shown in Figure 3 for caffeic acid and syringic acid reacting

The deprotonation of the neutral phenols is suggested to facilitate formation of an encounter complex with the radical cation. The deprotonation of the plant phenol will mainly occur at the more acidic carboxylic group (pKa ≈ 3) rather than at the phenol group (pKa ≈ 8). However, the equilibrium, although shifted to the left

may be important for ET to the radical cation in the encounter complex. The values of the second-order rate constants for ET from the plant phenol to the radical cation of β-carotene, lycopene, and zeaxanthin increase in the chloroform/methanol binary solvent for increasing driving force to a certain rate close to the diffusion limit for bimolecular reactions;18 see Figure 4 for βcarotene, Figure 5 for lycopene, and Figure 6 for zeaxanthin. However, for a continuing increase in driving force, the rate constants decrease for all these carotenoid radical cations. Such a bell-shaped dependence on the driving force for the rate of ET was predicted by the Marcus theory for ET9 and was later confirmed experimentally for a number of processes, including

Figure 3. Dependence of the observed first-order rate constant for reduction of β-carotene (50 μM) radical cation on the concentration of caffeic acid and syringic acid in the presence of 1 equiv of base in chloroform/methanol (9:1, v/v) solution at 23 °C. Linear regression for up to 4.0 × 10−4 M provides the second-order rate constants of Table 1.

with β-carotene for up to 400 μM phenolate. For a higher concentration of plant phenolate, deviations from this simple second-order reaction were noted with indications of rate saturation at higher phenol concentrations (data not shown), as also noted previously for the puerarin dianion reacting with carotenoid radical cations.7 The pseudo-first-order rate constants determined for lycopene and zeaxanthin by the same procedure as for β-carotene were likewise converted to

Figure 4. Logarithm of the second-order rate constant for electron transfer to β-carotene radical cation from plant phenolate in the presence of 1 or 2 equiv of base in chloroform/methanol (9:1, v/v) at ⊖ 23 °C as a function of the driving force for ET (E = E⊖ β‑Car•+ − Ephenol). For identification of phenols, see Table 1. 945

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Figure 5. Logarithm of the second-order rate constant for electron transfer to lycopene radical cation from plant phenolate in the presence of 1 or 2 equiv of base in chloroform/methanol (9:1, v/v) at ⊖ 23 °C as a function of the driving force for ET (E = E⊖ β‑Car•+− Ephenol). For identification of phenols, see Table 1.

Figure 7. Marcus plot for reduction of β-carotene radical cation by plant phenolate in chloroform/methanol (9:1, v/v) at 23 °C in the presence of 1 (a) and 2 (b) equiv of base.

decreases for decreasing E⊖ for the carotenoid, but is independent of the charge on the phenolate. In contrast, V decreases with increasing charge on the phenolate. The regeneration of β-carotene following laser flash photolysis shows a pattern similar to the bell-shaped relationship seen in Figure 4 for the rate of ET. The highest degree of protection of the β-carotene, defined as

Figure 6. Logarithm of the second-order rate constant for electron transfer to zeaxanthin radical cation from plant phenolate in the presence of 1 or 2 equiv of base in chloroform/methanol (9:1, v/v) at ⊖ 23 °C as a function of the driving force for ET (E = E⊖ Zea•+ − Ephenol). For identification of phenols, see Table 1.

protection = ΔOD(absence of phenol) − ΔOD(presence of phenol) × 100 ΔOD(absence of phenol) (9)

ET from phenolate ions.10−14 Accordingly, we analyzed the rate data on the basis of the Marcus theory.9,14 The rate of ET is controlled by the driving force (−ΔG°ET), a matrix coupling element (V), and the reorganization energy (λ) for the reaction:14,19 kET =

⎧ (ΔG° + λ)2 ⎫ 4π 3 2 ⎨ ⎬ − exp V h2λRT ⎩ 4λRT ⎭

in which ΔOD is the relative steady-state absorbance at 500 nm following bleaching of β-carotene, is found for caffeic acid and syringic acid, as can be seen in Figures 8 and 9. Both for less

(8)

The plot of Figure 7 is based on a reorganization of eq 8 and yields λ = 0.41 ± 0.03 and 0.40 ± 0.03 eV from the slope of the linear regression lines for the reorganization energy for ET from the plant phenols to the β-carotene radical cation in the presence of 1 or 2 equiv of base, respectively.23 The matrix coupling element has the value V = 5.85 cm−1 for the plant phenols with 1 equiv of base added and V = 5.10 cm−1 for 2 equiv of base. For lycopene, similar plots gave λ = 0.33 ± 0.04 eV and V = 4.53 cm−1 and λ = 0.34 ± 0.05 eV and V = 4.40 cm−1 for 1 and 2 equiv of base, respectively. For zeaxanthin the values were λ = 0.39 ± 0.04 eV and V = 6.03 cm−1 and λ = 0.38 ± 0.04 eV and V = 5.24 cm−1 for 1 and 2 equiv of base, respectively. The values for the relative coupling elements represent the coupling between each of the carotenoid radical cations and the plant phenolates investigated. For the three carotenoids, the driving force, −ΔGET ° , increases for the same series of phenolates reacting with radical cations as β-carotene > zeaxanthin > lycopene, in agreement with the increasing maximal rate for ET observed in experiments (β-carotene > zeaxanthin > lycopene) and an increasing calculated reorganization energy, λ, in agreement with the Marcus theory. λ

Figure 8. Fraction of β-carotene regenerated by plant phenol in the presence of 1 or 2 equiv of base for 50 μM β-carotene and 100 μM phenol in chloroform/methanol (9:1, v/v) following 7 ns flash photolysis at 532 nm and 23 °C as a function of the driving force for ⊖ ET (E = E⊖ β‑Car•+ − Ephenol). For identification of phenols, see Table 1.

reducing and for more reducing plant phenol than caffeic acid and syringic acid, the degree of protection decreases. For both lycopene and zeaxanthin, the same similarity between the dependence of protection of carotenoid and rate of regeneration of carotenoid was found. This dependence of protection of β-carotene through regeneration of β-carotene 946

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solvent radical formed in the initial photobleaching of βcarotene in a 1:1 ratio.24 For the chloroform/methanol mixture as solvent, CHCl3•− as the initially formed solvent radical was previously found to be converted to CHCl2• prior to reaction with β-carotene, resulting in the secondary and slower bleaching.24 The reactions of Scheme 1 show the coupling between the primary photobleaching and the slower bleaching of β-carotene Scheme 1 Figure 9. Slow bleaching of β-carotene in chloroform/methanol (9:1, v/v) at 23 °C following laser flash photolysis at 532 nm as monitored at 500 nm in the absence or presence of plant phenol and 2 equiv of base.

through ET according to eq 4 was further confirmed by inspection of the spectral changes of the β-carotene/phenolate solutions subjected to photolysis; see Figure 10. β-Carotene is bleached by irradiation at 532 nm, a wavelength where none of the plant phenols absorb light, to a certain degree depending on the nature of the plant phenol present in reactions providing no spectral evidence for formation of β-carotene/phenol adducts as was seen for catechins.8 The recovery of β-carotene following photolysis as followed by transient absorption at 500 nm (Figure 10 d) is seen to occur on a time scale similar to that of the decay of the radical cation (Figure 2) with a half-life around 1 μs corresponding to k ≈ 106 s−1. The fast regeneration of βcarotene is, however, followed by a slower bleaching as is evident in Figure 9, in agreement with previous findings.24 The maximal protection of β-carotene approaches 50% as is seen for caffeic acid and syringic acid. The protection of lycopene by the same plant phenols was found to be slightly better, while the protection of zeaxanthin was found to be comparable to the protection of β-carotene. This observation is in agreement with the detection of a slow secondary bleaching of β-carotene in electron-withdrawing solvents initiated by a

in chloroform/methanol in the presence of plant phenolates. The maximal protection against photobleaching of β-carotene provided by the plant phenolates reacting most efficiently in ET reactions only approaches 50% as is seen in Figure 8. This observation seems to indicate that the plant phenolates are not involved in the secondary bleaching, but only in the primary bleaching. The β-carotene radical cation formed in the primary bleaching reaction may react with a plant phenolate to form a precursor complex for further reaction. Such precursor complexes are often indicated in ET reactions, and their formation may, for some combinations of electron donors and electron acceptors, be rate determining, while, for other combinations, the subsequent reaction of the encounter complex controls the rate.19 For the ET from the plant phenolate to the carotenoid radical cations, the reaction of the encounter complex seems to be rate determining; see Figures

Figure 10. Steady-state absorption spectra of β-carotene (50 μM) in the presence of 100 μM m-hydroxybenzoic acid (a), syringic acid (b), or caffeic acid (c) in chloroform/methanol (9:1, v/v) at 23 °C in the presence of 2 equiv of base before and after laser flash photolysis at 532 nm for the indicated time. (d) Normalized recovery of β-carotene bleaching followed at 500 nm for 10 μs in the presence of m-hydroxybenzoic acid, syringic acid, and caffeic acid each in the presence of 2 equiv of base. 947

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4−6, since the rate depends on the driving force for ET and does not level off for increasing driving force, but continues to decrease. Other factors such as an increasing edge-to-edge distance of the encounter complex may, however, also contribute to the decreasing rate for the increasing driving force for the actual series of plant phenols. A reaction competitive with ET from the phenolate in the encounter complex to the radical cation formed during the primary bleaching may explain the correlation between kET and the degree of protection of β-carotene. Such a competing reaction may involve a reversible adduct formation as shown in Scheme 1, which for some strongly reducing plant phenols may involve further rearrangements as was seen for the tea catechins and β-carotene.8 For the plant phenolates investigated in the present study, adducts formed with carotenoids may not react further as they are not seen as stable products. However, it remains unexplained why the phenolate dianion reacts with a rate comparable to or even slower than the phenolate monoanion; see Table 1. However, a single negative charge seems to facilitate formation of the encounter complex, and intramolecular proton transfer equilibria as shown in eq 7 may be involved. Such equilibria may specifically be involved for caffeic acid as a simple plant phenol with two phenol groups in the ortho position, decreasing the rate for the reactions of the cations of lycopene and zeaxanthin with the anions of caffeic acid. The small difference in the matrix coupling constant corresponds to a large difference in rates between the reaction of monoanions and dianions. Notably, the rates are comparable for a driving force below the reorganization energy for the two series of phenolates for all three carotenoids, while the difference increases above the reorganization energy, with the monoanion reacting faster; see Figure 4. Regeneration of lipophilic carotenoids from their radical cations as formed in membranes during oxidative stress by hydrophilic plant phenolates seems to involve an encounter complex, the formation of which depends on electrostatic forces. This encounter complex may react through at least two reaction channels. One reaction involves ET and leads to immediate regeneration of the carotenoids, while the other leads to adduct formation and for some polyphenols to further degradation of the carotenoids. The ET channel is fast, but for increasing driving force above the reorganization energy for ET, inverted region effects as described by the Marcus theory are recognized. As a consequence, the most reducing phenolic antioxidants may not be the most efficient in regeneration of carotenoids and may even show antioxidant antagonism.1,21 This aspect may also be worth considering when foods or beverages colored by carotenoids need an antioxidant protection agent against light-induced bleaching.



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ACKNOWLEDGMENTS

Dr. Ran Liang is thanked for helpful comments.

ABBREVIATIONS USED Car, carotenoid; β-Car, β-carotene; Lyc, all-trans-lycopene; Zea, zeaxanthin; ET, electron transfer



REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86-10-62516604. *E-mail: [email protected]. Phone: +45 3533 3221. Funding

Support from the Danish Research Council for Technology and Production to L.H.S. as Grant 09-065906/FTP: Redox Communication in the Digestive Tract and from the Fundamental Research Funds for The Central Universities to J.-P.Z. is acknowledged. Notes

The authors declare no competing financial interest. 948

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