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Fast Regeneration of Carotenoids from Radical Cations by Isoflavonoid Dianions: Importance of the Carotenoid Keto Group for Electron Transfer Rui-Min Han,† Chang-Hui Chen,† Yu-Xi Tian,† Jian-Ping Zhang,† and Leif H. Skibsted*,‡ Department of Chemistry, Renmin UniVersity of China, Beijing 100872, People’s Republic of China and Food Chemistry, Department of Food Science, Faculty of Life Sciences, UniVersity of Copenhagen, RolighedsVej 30, DK-1058 Frederiksberg C, Denmark ReceiVed: July 31, 2009; ReVised Manuscript ReceiVed: September 30, 2009
Electron transfer to radical cations of β-carotene, zeaxanthin, canthaxanthin, and astaxanthin from each of the three acid/base forms of the diphenolic isoflavonoid daidzein and its C-glycoside puerarin, as studied by laser flash photolysis in homogeneous methanol/chloroform (1/9) solution, was found to depend on carotenoid structures and more significantly on the deprotonation degree of the isoflavonoids. None of the carotenoid radical cations reacted with the neutral forms of the isoflavonoids while the monoanionic and dianionic forms of the isoflavonoids regenerated the oxidized carotenoid. Electron transfer to the β-carotene radical cation from the puerarin dianion followed second order kinetics with the rate constant at 25 °C k2 ) 5.5 × 109 M-1 s-1, zeaxanthin 8.5 × 109 M-1 s-1, canthaxanthin 6.5 × 1010 M-1 s-1, and astaxanthin 11.1 × 1010 M-1 s-1 approaching the diffusion limit and establishing a linear free energy relationship between rate constants and driving force. Comparable results found for the daidzein dianion indicate that the steric hindrance from the glucoside is not important suggesting the more reducing but less acidic 4′-OH/4′-O- as electron donors. On the basis of the rate constants obtained from kinetic analyses, the keto group of carotenoids is concluded to facilitate electron transfer. The driving force was estimated from oxidation potentials, as determined by cyclicvoltametry for puerarin and daidzein in aqueous solutions at varying pH conditions, which led to the standard reduction potentials E° ) 1.13 and 1.10 V versus NHE corresponding to the uncharged puerarin and daidzein. For pH > pKa2, the apparent potentials of both puerarin and daidzein became constants and were E° ) 0.69 and 0.65 V, respectively. Electron transfer from isoflavonoids to the carotenoid radical cation, as formed during oxidative stress, is faster for astaxanthin than for the other carotenoids, which may relate to astaxanthins more effective antioxidative properties and in agreement with the highest electron accepting index of astaxanthin. Introduction The positive effects on human health of a high intake of carotenoids through a diet rich in fruits and vegetables are often assigned to the functions of carotenoids as antioxidants.1 β-Carotene and certain other carotenoids have provitamin A activity, and lutein and zeaxanthin have been shown to protect the retina of the eye against light-induced oxidative stress, but otherwise antioxidative functions of carotenoids are still uncertain.2-6 In biological systems, many factors affect the efficiency of antioxidants, including the radical scavenging capacity of the individual antioxidant, the actual distribution of antioxidants in heterogeneous environment, the influence of antioxidants on membrane structure, and the interaction with other antioxidants present.7-9 It has long been known that carotenoids may interact with other antioxidants in membranes and protect against development of cardio-vascular diseases and cancer.10 However, the molecular basis of carotenoid interaction and possible synergism with other types of antioxidants like polyphenols have not been studied in any detail, although there are some indications of electron transfer between β-carotene radical cations formed from oxidative stress and polyphenols at water/ * To whom correspondence should be addressed: Tel: +45 3528 3221. Fax: +45 3528 3344. E-mail:
[email protected]. † Renmin University of China. ‡ University of Copenhagen.
lipid interfaces.11,12 Results of theoretical calculations recently became available that predict the relative ease of one-electron oxidation of carotenoids to form the carotenoid radical cation and which accordingly also predict the relative efficiency of carotenoids as antioxidants.13 In order to study the mechanism of electron transfer to carotenoids, we have selected four wellcharacterized and structurally related carotenoids including β-carotene as the parent compound together with zeaxanthin, canthaxanthin, and astaxanthin for a systematic study of the effects of hydroxyl- and keto group substitution in β-carotene on interaction with polyphenolic antioxidants, cf. Scheme 1. Daidzein and its C-glycoside puerarin with only two phenolic groups were selected as relative simple plant phenols for the kinetic studies of reduction of carotenoid radical cations, since the radical dynamics of the isoflavonoids skeleton recently has been characterized.14 Materials and Methods Chemicals. β-Carotene, zeaxanthin, and astaxanthin were from the same source as previously reported.15 Canthaxanthin sealed in ampules under argon was supplied by Roche A/S (Hvidovre, Denmark) and used as-received.16 Puerarin and daidzein (>98%) were purchased from Huike Plant Exploitation Inc., (Shanxi, China) and used as received. Water was supplied by a Milli-Q apparatus (Millipore Corp., Billerica, MA). Methanol of high-performance liquid chromatography (HPLC)
10.1021/jp907349x 2010 American Chemical Society Published on Web 12/03/2009
Fast Regeneration of Carotenoids from Radical Cations
J. Phys. Chem. A, Vol. 114, No. 1, 2010 127
SCHEME 1: Molecular Structures of (a) β-Carotene, (b) Zeaxanthin, (c) Canthaxanthin, (d) Astaxanthin, (e) Puerarin, and (f) Daidzein
TABLE 1: Standard Reduction Potential of Carotenoid Radical Cations versus NHE at 25°C, Absorption Maximum (λmax) of Carotenoid Radical Cation in near Infrared Spectral Region, The Potential Difference between Carotenoid and Puerarin/ Daidzein Dianion ∆E (V), Gibbs Free Energy ∆G (kJ · mol-1) and ∆∆G (kJ · mol-1, relative to ∆G for of β-carotene) of the Electron Transfer Reaction between Carotenoid Radical Cations and Puerarin or Daidzein Dianions car•+/puerarin2-
E°/V(ref) carotenoid β-carotene zeaxanthin canthaxanthin astaxanthin
car•+/daidzein2-
aqueous33
CH2Cl215,18
λmax/nm20,27
∆Ee/V
∆G°/kJ · mol-1
∆Ee/V
∆G°/kJ · mol-1
∆∆G°/kJ · mol-1
1.06a 1.03a 1.04a 1.03a
0.84b 0.85b 0.95c 0.97b
940 940 860d 850
0.15 0.16 0.26 0.28
-14.48 -15.44 -25.09 -27.02
0.19 0.20 0.30 0.32
-18.34 -19.30 -28.95 -30.88
0 -0.96 -10.61 -12.54
a In Triton X-100 micelles. b Corrected from value relative to Ag/AgCl electrode by addition of 0.22 V. c Corrected from value relative to saturated calomel electrode by addition of 0.24 V. d In ditert-butyl peroxide/benzene (7/3 v/v). e E′(puerarin2-) ) 0.69 V, E′(daidzein2-) ) 0.65 V.
grade (Caledon Laboratories, Georgetown, Ontario, Canada) was used as received. Chloroform (>99.0%, Beijing Chemical Plant) was purified before use by passing through an alumina column (Wusi Chemical Reagent Ltd., Shanghai, China). The solution of puerarin- and puerarin2- were prepared by addition 1 or 2 equiv. tetramethylammonium hydroxide (CH3)4N+OH- (97%, Sigma, Louis, USA) to puerarin neutral solutions and similar procedures were used for daidzein. Other chemicals were all of analytical grade. Determination of Oxidation Potentials. Cyclic voltammetry (CV) was performed on a CHI 660B electrochemical analyzer (CH Instruments Inc., Austin, TX, USA) with a three-electrode configuration,17 for which the solutions of puerarin at a concentration of 2.0 × 10-5 M in Britton-Robinson(B-R) buffer were used (pH ) 2-12). For the less water-soluble daidzein (2.0 × 10-5 M), the B-R buffer containing 5% dimethylsulfoxide was used to increase the solubility. The working electrode was a glassy carbon piece (diameter, 4 mm), the reference electrode was of the Ag/AgCl type (KCl-saturated), and the counter electrode was a platinum wire. The ionic strength was adjusted to 0.1 M with NaCl. Laser Flash Photolysis. Submicrosecond time-resolved absorption spectra were obtained at room temperature (25 ( 1 °C). The excitation laser pulses at 500 nm (2 mJ/pulse, 7 ns, 10 Hz) was obtained by an optical parametric oscillator (OPO, Quanta-Ray MOPO-SL, Spectra Physics, Mountain View, CA, USA) driven by an Nd3+:YAG pumped laser (Quanta-Ray PRO230; Spectra Physics). The optical path length of the flow cuvette used for laser flash photolysis was 1 cm. The anaerobic condition
was achieved by bubbling the solution with high-purity argon for ∼30 min. Near-infrared (NIR) kinetics at individual wavelengths was recorded with a xenon lamp probe (CW, 350 W) and a photodiode detector (S8890-02, Hamamatsu, Hamamatsu City, Japan). Results The rate of electron transfer from puerarin or daidzein to each of the four carotenoids β-carotene, zeaxanthin, canthaxanthin, and astaxanthin were determined in methanol/chloroform (1/9, v/v). The rate of electron transfer between β-carotene and puerarin has previously been found to depend strongly on the charge of puerarin corresponding to the three isoflavonoid acid/ base forms.11 The change in oxidation potentials for puerarin and daidzein corresponding to the standard reduction potentials of their radicals upon deprotonation is accordingly important for calculating and comparing the differences in the driving force between carotenoid radical cations and various acid/base forms of the isoflavonoids. The oxidation potentials of the carotenoids corresponding to the standard reduction potentials of their radical cations are available in literature as shown in Table 1. The reported potentials in CH2Cl2 are used for the calculations for the kinetic study of the electron transfer rather than the values reported for aqueous solution due to the consistency between the absorption maximum of the carotenoid radical cations in the NIR spectral region, and the potentials determined in CH2Cl2 rather than those determined in micelles in aqueous solution.15,18-21 The oxidation potential of puerarin and daidzein in aqueous solution were determined by cyclic voltammetry for 2 < pH
10, the apparent potential is E′ ) 0.47 V for puerarin and 0.43 V for daidzein as seen from in Figure 2, parts a and b, which are ascribed to the oxidation potential of puerarin and daidzein dianions and are corrected to 0.69 and 0.65 V versus NHE, respectively. The 7-phenolate and the 4′-phenol coexist at an intermediate pH interval between pKa1 ) 7.20 (7-OH) and pKa2 ) 9.84 (4′OH) for puerarin and between pKa1 ) 7.47 and pKa2 ) 9.65 for daidzein, and it is not possible using the present experimental methods to differentiate between the initial oxidation of the 7-phenolate (i) as shown in eq 10 from the initial oxidation of
Figure 1. Cylic voltammogram of 2.0 × 10-5 M (a) puerarin and (b) daidzein at varying pH in aqueous solution of ionic strength 0.1 adjusted with NaCl at a scan rate of 100 mV · s-1 at room temperature (∼25°). For daidzein, 5% dimethylsulfoxide was used to increase the solubility.
Fast Regeneration of Carotenoids from Radical Cations
J. Phys. Chem. A, Vol. 114, No. 1, 2010 129 SCHEME 2: Acid/Base Equilbria of Puerarin in Aqueous Solutiona
Figure 2. Oxidation potential of 4′-hydroxyl (O) and glucoside (0) for (a) puerarin (2 × 10-5 M) and (b) daidzein (2 × 10-5 M) in aqueous solution at varying pH as determined by cyclic voltametry at room temperature (∼25°).
the 4′-phenol at the same pH or in oxidation parallel processes. However, oxidation of the 7-phenolate (i) leads to a fast intramolecular electron transfer with an rate constant ∼105 s-1 from the 7-phenoxyl radical (ii) to the 4′-phenoxyl radical (iii) as also formed by direct oxidation of the 4′-phenol in eq 7. Both the two reaction paths correspond accordingly to the same overall oxidation process.
The deviation from the Nernst equation observed for both puerarin and daidzein with the slope of ∼0.05 rather than the theoretical value of 0.06 as found for catechol-containing flavonoids such as catechin and quercetin,24,25 may be due to the acid/base equilibria shown in Scheme 2. The most reducing form of puerarin (and daidzein) monoanion is the HO(7)-P-(4′)O- form, which is present in extremely low concentration, and kinetic aspects related to the acid/base equilibria may become important. From the bond dissociation energy (BDE) reported in ref 22 the equilibrium constant of the following equation:
HO(7) - P - (4′)O• h •O(7) - P - (4′)OH
(11)
is calculated to be K7,4′RAD ) 1.4 × 10-6, and if the deprotonated 7-OH is oxidized it will rearrange also complicating the electrode kinetics. In Scheme 3, the acid/base equilibria are combined with the one-electron oxidation reactions showing the most important reaction paths. For puerarin, a new peak at higher potential appears for 8.5 < pH < 12, which shows the same pH-dependence for E′ as the initial oxidation at lower potential shown in Figure 2a. Notably, this new peak at higher potential is not observed for daidzein (vide infra) and could accordingly be ascribed to the involvement of glucoside moiety of puerarin. From the measured E′ at 8 < pH < 12 shown in Figure 2, a value of E° ) 1.25 ( 0.02 V
a K ) 8.3 × 10-22, is calculated from the difference of the deprotonation energy (DE) for 7-OH and 4′-OH from ref 11.
versus Ag/AgCl is estimated with R ) 0.047 ( 0.002 V by extrapolating to pH ) 0 and corrected to yield the value of E° ) 1.47 V versus NHE. Reduction of Carotenoid Radical Cations by Isoflavonoids. Upon direct photoexcitation to the optically allowed S2 excited state with 500 nm laser pulse, carotenoids will form radical cations in an electron withdrawing solvent such as chloroform.15,26-28A mixture of chloroform and methanol was used in which both carotenoids (5 × 10-6 M) and isoflavonoids (∼10-5 M) are sufficiently soluble. The kinetics of the decay of the carotenoid radical cations initially formed by nanosecond laser flash photolysis was recorded in real time by NIR detection as previously described.11 It was found that the kinetics of the four carotenoids was hardly affected by the presence of neutral puerarin or daidzein as shown for astaxanthin in Figure 3. The decay of carotenoid radical cations were in contrast significantly accelerated by the presence of monoanions and especially dianions of puerarin and daidzein as seen in Figure 3 for astaxanthin. In Figure 4, the kinetics for decay of the four carotenoid radical cations in the presence of excess puerarin and daidzein dianions are presented. The relatively slow decay of the carotenoid radical cation in the absence of isoflavonoids (Figure 3) has been found for β-carotene to follow second-order kinetics.11 For the dianion form of puerarin and daidzein, carotenoid radical cations decay monoexponentially, and the pseudofirst order rate constant for decay depends on the concentration of (excess) puerarin or daidzein, as shown in Figure 5 for β-carotene radical cation and puerarin anion. The initial carotenoid radical cation concentration is low compared to the puerarin dianion concentration given the ∼10% quantum yield for its formation from the S2 excited state ensuring the pseudofirst order reaction conditions.15 The pseudofirst order rate constant depends not strictly linearly on the concentration of puerarin or daidzein dianions, as shown in Figure 5 for β-carotene radical cation and puerarin dianion. This kind of nonlinear deviation for the higher puerarin dianion concentration may be ascribed to association between the carotenoid radical
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Han et al.
SCHEME 3: One-Electron Oxidation/Reduction of Acid/Base Forms of Puerarin in Aqueous Solution
cation and the puerarin dianion prior to or in parallel with electron transfer as presented in the following equation,29
Car•+ + Pue2- h {Car•+, Pue2-} f Car + Pue•-
(12)
In order to simplify the kinetic analysis, the pseudofirst order rate constants were, however, converted to second-order rate constants from the initial slope of the pseudofirst order rate constants as a function of puerarin dianion concentration as shown in Figure 5. The second-order rate constants for reaction between the three other carotenoid radical cations and puerarin/
Figure 3. Time trace of astaxanthin radical cation at 900 nm measured by transient absorption spectroscopy following a laser flash photolysis of astaxanthin (5 × 10-6 M) in methanol/chloroform (1/9) at 25° in presence of 1.2 × 10-5 M neutral, mono- and dianionic forms of (a) puerarin and (b) daidzein.
Figure 4. Time trace of carotenoid radical cations measured by transient absorption spectroscopy following laser flash photolysis of solutions of carotenoid (5 × 10-6 M) in methanol/chloroform (1/9) at 25° in presence of 1.2 × 10-5 M dianion of (a) puerarin and (b) daidzein.
daidzein dianions collected in Table 2 were based on experiments with low dianion concentration. A similar kinetics analysis was not attempted for the puerarin/daidzein monoanions due to the complicated acid/base equilibria as shown in Scheme 2 with simultaneous presence of neutral, mono- and dianionic forms during reaction with the carotenoid radical cation. Discussion Structural aspects of interactions between different antioxidants are important for understanding antioxidant synergism. Carotenoids are known to be regenerated from their radical cations formed during oxidative stress by tocopherols and tocotrienols, depending on their structures.16 More recently, flavonoids also have been found to regenerate β-carotene from its radical cations as anionic forms of the isoflavonoid puerarin and of the flavonoid baicalin, found to reduce the radical cation.11,12 For regeneration of carotenoids from their radical cations by other carotenoids, an antioxidant hierarchy has been established in which astaxanthin ranks as the least efficient, while lycopene, β-carotene, and zeaxanthin are among the most efficient and comparable with the tocopherols.16,30 The low rank in this hierarchy of especially astaxanthin is remarkable, since astaxanthin has been found to interact with R-tocopherol in salmon,31 which is being considered as a protector in carcinogensis5,32 and is demonstrated in a model system to be an efficient antioxidant.33,34
Figure 5. Observed pseudofirst order rate constant for electron transfer from puerarin dianion to β-carotene radical cation. From the initial slope, a second order rate constant k2 ) (5.5 ( 0.3) × 109 l · mol-1 · s-1 is calculated.
Fast Regeneration of Carotenoids from Radical Cations
J. Phys. Chem. A, Vol. 114, No. 1, 2010 131
TABLE 2: Second Order Rate Constants for Electron Transfer from Puerarin Dianion (1.2 × 10-5 M) and Daidzein Dianion (1.2 × 10-5 M) to Carotenoid Radical Cation in Methanol/Chloroform (1/9) and Activation Free Energy ∆G# (kJ · mol-1) at 25° puerarin2carotenoid a
β-carotene zeaxanthina canthaxanthinb astaxanthina a
k (l · mol-1 · s-1)
daidzein2∆G#/kJ · mol-1
k (l · mol-1 · s-1)
∆G#/kJ · mol-1
17.4 16.3 11.3 10.0
(5.8 ( 0.3) × 10 (8.3 ( 0.2) × 109 (5.7 ( 0.1) × 1010 (9.2 ( 0.1) × 1010
17.3 16.4 11.6 10.4
(5.5 ( 0.1) × 10 (8.5 ( 0.1) × 109 (6.5 ( 0.1) × 1010 (11.1 ( 0.1) × 1010 9
9
Reaction followed at 950 nm. b Reaction followed at 900 nm.
Figure 6. Activation free energy ∆G# as a function of ∆∆G°, reaction free energy relative to reaction with β-carotene radical cation, for electron transfer to carotenoid radical cations from puerarin or daidzein dianion. From linear regression, the relation ∆G# ) 0.55 · ∆∆G° + 17.0 is obtained.
Astaxanthin contains two hydroxyl and two keto groups which make this carotenoid more hydrophilic than the parent β-carotene, a feature which has been associated with the antioxidative properties of astaxanthin. In order to explore the roles of these two functional groups, we have accordingly selected the four carotenoids seen in Scheme 1, in which hydroxyl and keto groups vary systematically facilitating structural comparison. The reduction of the radical cations of these carotenoids by the isoflavonoid dianions followed second-order kinetics and the second-order rate constants are collected in Table 2. The second-order rate constants are approaching the diffusion limit, which may indicate that some preassociation is involved prior to electron transfer as shown in eq 12. The reaction is an electron transfer and the driving force for the reaction may be calculated from the standard reduction potential for the carotenoid radical cations and for the isoflavonoid radicals shown in Table 1. Since the potentials for the carotenoid in dichloromethane need a correction not available for specific solvation effects to be comparable with the potential determined for puerarin and daidzein dianions in aqueous solution, the ∆G° calculated for the electron transfer reactions are in Table 1, expressed relative to the value for β-carotene as ∆∆G°. The activation free energy, ∆G#, is calculated using transition state theory from the secondorder rate constants and shown in Table 2.35 These thermodynamic data together establish a free energy linear relationship between ∆G# and ∆∆G° with the slope 0.55 ( 0.04 V as seen in Figure 6. The linearity indicates that a common mechanism is operative for the different carotenoids, and the intermediate value of the slope indicate that both the reactants and the products have important contributions to the structure of the transition state for electron transfer. A similar analysis for 13 vitamin E analogues and the stable 2,6-ditert-butyl-4-(4-methoxylhexyl)phenoxyl radical led to a slope of 0.32 ( 0.04.36 From a comparison of the four carotenoids, it is seen that the astaxanthin radical cation as the most oxidizing radical is reacting with the highest rate and faster than the radicals of both canthaxanthin and zeaxanthin. Furthermore, canthaxanthin reacts faster than zeaxanthin, suggesting the keto-group to be
the more important group for electron transfer than the hydroxyl. The presence of both hydroxyl and keto groups as in astaxanthin therefore yields the highest rate of reaction. As for the isoflavonoids, the rate of puerarin dianion reacting with carotenoid radical cations is quite similar to that of daidzein dianion, and the presence of glucoside in puerarin is not resulting in any steric hindrance. The 4′-phenolate group, which notably is the more reducing in both isoflavonoids, may be identified as electron donor. The π-bond of the keto group of the carotenoid is polarized according to Oδ- ) Cδ+ accelerating the interaction with the phenolate groups of the isoflavonoids. Astaxanthin is found to be an efficient antioxidant despite its low rank in the regeneration hierarchy among the carotenoids as shown both experimentally and based on theoretical calculations.21,37,38 On the basis of the high electron accepting index, astaxanthin was recently classified as the best antiradical substance among the carotenoids, while canthaxanthin, zeaxanthin and especially β-carotene were concluded to be less effective.38 This classification relates, however, to carotenoids as “antireductants” rather than as an antioxidants, and under conditions of oxidative stress, the reverse ordering applies with β-carotene as the best antiradical substance due to its highest relative electron donating index among these carotenoids.38 Plant phenols, like isoflavonoids, are good antioxidants and, as such, also good antiradical substance.39 The major finding of the present study is that when carotenoids under oxidative stress act as electron donors and antioxidants rather than electron acceptors and “antireductants”, the carotenoid radical cation formed is efficiently reduced by the isoflavonoids, acting as good electron donors. Moreover, the rate for this electron transfer depends on the driving force as calculated from the standard reduction potential, and the rate is approaching the diffusion limit for the radical cation of astaxanthin, the carotenoid with the highest electron accepting index. Acknowledgment. This work has been supported by grantsin-aid from Natural Science Foundation of China (Nos. 20673144 and 20803091) and from the Ministry of Science and Technology of China (Nos. 2009CB220008 and 2006BAI08B04-06). Continuing support from LMC (U-10 grant), Centre for Advanced Food Studies to the Food Chemistry group at University of Copenhagen, is gratefully acknowledged. References and Notes (1) Key, T. J.; Thorogood, M.; Appleby, P. N.; Burr, M. L. Br. Med. J. 1996, 313, 775. (2) El-Agamey, A.; Cantrell, A.; Land, E. J.; McGarvey, D. J.; Truscott, T. G. Photochem. Photobiol. Sci. 2004, 3, 802. (3) Gerster, H. J. Am. Coll. Nutr. 1997, 16, 109. (4) McNulty, H. P.; Byun, J.; Lockwood, S. F.; Jacob, R. F.; Mason, R. P. Biochim. Biophys. Acta 2007, 1768, 167. (5) Hosokawa, M.; Okada, T.; Mikami, N.; Konishi, I.; Miyashita, K. Food Sci. Biotechnol. 2009, 18, 1. (6) Edge, R.; McGarvey, D. J.; Truscott, T. G. J. Photochem. Photobiol. 1997, 41, 189.
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