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2008, 112, 11456–11461 Published on Web 08/26/2008
One-Electron Reduction of Superoxide Radical-Anions by 3-Alkylpolyhydroxyflavones in Micelles. Effect of Antioxidant Alkyl Chain Length on Micellar Structure and Reactivity Artur M. S. Silva,† Paulo Filipe,‡ Raquel S. G. R. Seixas,† Diana C. G. A. Pinto,† Larry K. Patterson,§ Gordon L. Hug,§ Jose´ A. S. Cavaleiro,† Jean-Claude Mazie`re,| Rene´ Santus,⊥ and Patrice Morlie`re*,| Department of Chemistry and OOPNA, UniVersity of AVeiro, 3810-193 AVeiro, Portugal, Faculdade de Medicina de Lisboa, Clı´nica de Dermatologia, 1699 Lisboa, Portugal, Radiation Laboratory, UniVersity of Notre Dame, Notre Dame, Indiana 46556, INSERM, ERI 12, 80054 Amiens, France, Faculte´ de Me´decine et de Pharmacie, UniVersite´ de Picardie Jules Verne, 80036 Amiens, France, Laboratoire de Biochimie, CHU Amiens Nord, 80054 Amiens, France, INSERM, U 697, 75475 Paris, France, and De´partement RDDM, Muse´um National d’Histoire Naturelle, 75231 Paris, France ReceiVed: July 4, 2008; ReVised Manuscript ReceiVed: August 4, 2008
In micellar solutions, one-electron reduction of •O2- radical-anions by 3-alkylpolyhydroxyflavones (FnH) with alkyl chains of n ) 1, 4, 6, 10 carbons produces phenoxyl radicals (•Fn) identical to those obtained by one-electron oxidation by •Br2- radical-anions or by repair of tryptophan radicals. In cetyltrimethylammonium bromide (CTAB), F1H localizes in the Stern layer, and alkyl chains of other FnH solubilize in the hydrophobic interior, interacting with cetyl tails. This interaction produces more compact micelles with lower intramicellar fluidity, as suggested by the increase in the pseudo-first-order rate constant of •Fn formation (k1) from ∼390 s-1 for n ) 1 to 610 s-1 for n ) 10, leading to an intramicellar bimolecular rate constant of 1 × 105 M-1 s-1. Additionally, •F1 and •F4 decay by intermicellar bimolecular reaction (2k ) 20 and 2 × 105 M-1 s-1, respectively) whereas other •Fn radicals are stable over seconds due to increased localization with regards to the Stern layer. In contrast, the thick uncharged hydrophilic palisade layer and the compact hydrophobic core of Triton X100 micelles are responsible for a much higher microviscosity resulting in a decrease in k1 from ∼15.6 s-1 for n ) 1 to 9.6 s-1 for n ) 10. I. Introduction •O -, •OH 2
and H2O2 Reactive oxygen species (ROS) such as produced in cellular metabolism or arising from environmental sources (chemicals, UV light) contribute to major diseases such as atherosclerosis, cancer and aging.1 To counter adverse mechanisms involved, inherent cellular enzymes such as superoxide dismutase, catalase, peroxidases or DNA glycosylases may eliminate or transform ROS. However, small potent antioxidant molecules, available in large quantities in fruits and vegetables, are also excellent ROS scavengers. These include vitamins (ascorbic acid and R-tocopherol), carotenoids and flavonoids and are essential to good health.1 In practice, the protection afforded by such antioxidants against the oxidative stress and its consequences for human health is limited by their systemic distribution and accessibility to sites of oxidation in cells.2,3 Another important factor that limits the efficacy of antioxidants is their bioavailability. Extremely low levels of albumin* Corresponding author: Mailing address: Laboratoire de Biochimie, INSERM ERI12, CHU Amiens Nord, place Victor Pauchet, 80054 Amiens Cedex 01, France. Tel: +33 3 22 66 86 69. Fax: +33 3 22 66 89 17. E-mail:
[email protected]. † University of Aveiro. ‡ Faculdade de Medicina de Lisboa. § University of Notre Dame. | INSERM, Universite ´ de Picardie Jules Verne, and CHU Amiens Nord. ⊥ INSERM and Muse ´ um National d’Histoire Naturelle.
10.1021/jp805889g CCC: $40.75
bound conjugates, i.e., metabolites of dietary phenolic antioxidants, are detected in plasma even with oral supplementation.4 Hence, the synthesis of derivatives which are effectively transferred from the digestive lumen into the blood stream before extensive conjugation and excretion may help to ameliorate the limitations of such naturally occurring antioxidants. This may be approached in one manner by modifying the hydrophobic character of such agents. The present study explores the antioxidant properties of four novel 3-alkylpolyhydroxyflavones, hereafter specified as FnH, in which the hydroxyl group at C-3 position on the chromone ring, a major site of conjugation, has been replaced by alkyl chains of increasing length with n ) 1, 4, 6 and 10 carbons, much less susceptible to conjugation.4,5 In terms of their biological application, it may be suggested that the alkyl chain length should strongly modulate FnH partition in the multiple polar and nonpolar environments encountered in cells and consequently enhance their inhibitory effect on ROS related damage. The objective of this study has been to study FnH antioxidant and redox properties in model media which mimic the microenvironments that may be relevant to antioxidant activity in biological structures. The present investigation was carried out by pulse radiolysis in cationic cetyltrimethylammonium bromide (CTAB) and nonionic (Triton X100) buffered (pH 7) micellar solutions. In the presence of formate ions, this technique allows 2008 American Chemical Society
Letters
Figure 1. (A) Absorbance spectra of F1H in micelles and in various solvents at 25 °C (1 mM aqueous NaOH, buffered (pH 7) 10 mM CTAB, buffered (pH 7) 4.6 mM TX100 and ethanol). Inset: structure of the 3-alkylpolyhydroxyflavones. (B) Transient absorbance spectra of the •F1 radical obtained by one-electron oxidation of 200 µM F1H by •O2- and •Br2- radical-anions after pulse radiolysis of O2-saturated buffered (pH 7) solutions containing 0.1 M formate and 10 mM CTAB or 4.6 mM TX100 and of N2O-saturated buffered (pH 7) solutions of 10 mM CTAB containing 0.1 M Br-. The radiolytic dose was 10 Gy in all cases.
for selective and full conversion of the primary species of water radiolysis into •O2- radical-anions,6 the most abundant ROS produced by cellular oxidation.1 For purposes of identification, the radicals resulting from the oxidation of FnH by the •O2radical-anions, were compared to those generated by oxidation of these flavones with selective oxidizing •Br2- radical-anions or by repair of neutral indolyl radicals (•Trp) produced by oneelectron oxidation of the aromatic amino acid, tryptophan.7,8 Because increasing the chain length from 1 to 4, 6 and 10 carbons must increase the hydrophobicity of FnH, we have examined effects of the alkyl chain length on the rates of formation and decay of the phenoxyl radicals to elucidate possible changes in micellar structures induced by these molecules. The kinetics have been interpreted in terms of location in micelles. II. Experimental Methods The four 3-alkylpolyhydroxyflavones (FnH) (Figure 1A) were synthesized following well established methods of polyhydroxychromone synthesis.5 All other chemicals were of analytical grade and were used as received from suppliers. D,L-tryptophan (Trp), L-tyrosine (Tyr), Triton X100 (TX100), and cetyltrimethylammonium bromide (CTAB), were purchased from Sigma (St Louis, MO). Spectroscopic grade dimethylsulfoxide (DMSO), absolute ethanol and methanol were supplied by Merck (Darmstad, Germany). The phosphate buffer (pH 7) was prepared in pure water obtained with a reverse osmosis system
J. Phys. Chem. B, Vol. 112, No. 37, 2008 11457 from Ser-A-Pure Co. The water exhibits a resistivity of >18 Mohms cm-1 and a total organic content of 4 are much better inhibitors of lipid peroxidation and of cell injury than is quercetin, the most effective natural flavonoid antioxidant. Acknowledgment. This work was supported by the FrancoPortuguese exchange programs GRICES-INSERM 2005-2006 and Pessoa 07958NF. Notre Dame Radiation Laboratory is supported by the Office of Basic Energy Sciences of the U.S. Department of Energy. This is document NDRL-4764 from the Notre Dame Radiation Laboratory. P.F. thanks the “Sociedade Portugesa de Dermatologia e Venerologia” for a travel grant.
Letters References and Notes (1) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and Medicine, 2nd ed.; Clarendon Press: Oxford, U.K., 1989. (2) Giordani, A.; Martin, M.-E.; Beaumont, C.; Santus, R.; Morlie`re, P. Photochem. Photobiol. 2000, 72, 746. (3) Halliwell, B. Free Rad. Res. 1999, 31, 261. (4) Mullen, W.; Edwards, C. A.; Crozier, A. Br. J. Nutr. 2006, 96, 107. (5) Santos, C. M. M.; Silva, A. M. S.; Cavaleiro, J. A. S. Eur. J. Org. Chem. 2003, 457, 5–4585. (6) Klug, D.; Rabani, J.; Fridovich, I. J. Biol. Chem. 1972, 247, 4839. (7) Redpath, J. L.; Santus, R.; Ovadia, J.; Grossweiner, L. I. Int. J. Radiat. Biol. 1975, 27, 201. (8) Butler, J.; Land, E. J.; Prutz, W. A.; Swallow, A. J. Biochim. Biophys. Acta 1982, 705, 150. (9) Infelta, P. P.; Gra¨tzel, M. J. Phys. Chem. 1979, 70, 179. (10) Patterson, L. K.; Lilie, J. A. Int. J. Radiat. Phys. Chem 1974, 6, 129. (11) Hug, G. L.; Wang, Y.; Schoneich, C.; Jiang, P. Y.; Fessenden, R. W. Radiat. Phys. Chem. 1999, 54, 559. (12) Schuler, R. H.; Patterson, L. K.; Janata, E. J. Phys. Chem. 1980, 84, 2088. (13) Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross, A. B. J. Phys. Chem. Ref. Data 1988, 17, 513. (14) Jovanovic, S. V.; Steenken, S.; Simic, M. G.; Hara, Y. Antioxidant properties of flavonoids: reduction potentials and electronic transfer reactions
J. Phys. Chem. B, Vol. 112, No. 37, 2008 11461 of flavonoid radicals. In FlaVonoids in Health and Disease; Rice-Evans, C., Packer, L., Eds.; Marcel Dekker: New York, 1998; pp 137. (15) Fernandez, M. S.; Fromherz, P. J. Phys. Chem. 1977, 81, 1755. (16) Roy, D.; Karmakar, R.; Mondal, S. K.; Sahu, K.; Bhattacharyya, K. Chem. Phys. Lett. 2004, 399, 147. (17) Filipe, P.; Morlie`re, P.; Patterson, L. K.; Hug, G. L.; Mazie`re, J.C.; Mazie`re, C.; Freitas, J. P.; Fernandes, A.; Santus, R. Biochim. Biophys. Acta 2002, 1572, 150. (18) Patterson, L. K.; Gra¨tzel, M. J. Phys. Chem. 1975, 79, 956. (19) Tachyia, M. Chem. Phys. Lett. 1975, 33, 289. (20) Sassoon, R. E.; Hug, G. L. J. Phys. Chem. 1993, 1993, 7823. (21) Gra¨tzel, M.; Thomas, J. K. J. Am. Chem. Soc. 1973, 95, 6885. (22) Panda, D.; Khatua, S.; Datta, A. J. Phys. Chem. B. 2007, 1648. (23) Almgren, M.; Grieser, F.; Thomas, J. K. J. Am. Chem. Soc. 1979, 101, 279. (24) Chauvet, J.-P.; Viovy, R.; Santus, R.; Land, E. J. J. Phys. Chem. 1981, 85, 3449. (25) Robson, R. J.; Dennis, E. A. J. Phys. Chem. 1977, 81, 1075. (26) Chauvet, J.-P.; Viovy, R.; Land, E. J.; Santus, R.; Truscott, T. G. J. Phys. Chem. 1983, 87, 592. (27) Farhataziz; Ross, A. B. Nat. Stand. Ref. Data Ser. Nat. Bur. Stand. (US) 1977, 59, 83. (28) Turro, N. J.; Gra¨tzel, M.; Braun, A. M. Angew. Chem., Int. Ed. 1980, 19, 675.
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