No abstract

Antioxidant mechanisms of curcumin, bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, have been studied by laser flash photolysis and pulse radiolysis. The keto−enol−enolate equilibrium of the heptadienone moiety of curcumin determines its physicochemical and antioxidant properties. In neutral and acidic aqueous solutions (from pH 3 to 7), the keto form dominates, and curcumin acts as an extraordinarily potent H-atom donor. The reaction rate constant with the methyl radical (3.5 ± 0.3) × 109 M-1 s-1 is close to diffusion control in 40% aqueous DMSO at pH 5. The tert-butoxyl radical reacts with curcumin in acetonitrile solutions at a diffusion controlled rate, k = (7.5 ± 0.8) × 109 M-1 s-1. The apparent site of reaction is the central CH2 group in the heptadienone link, which has two labile hydrogens. This is supported by comparing the reaction patterns of curcumin and dehydrozingerone (DHZ) (“half-curcumin”, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one). DHZ does not react with the methyl radical, indicating that the presence of the labile hydrogens is crucial for the H-atom donating ability of curcumin. The tert-butoxyl radical reacts with DHZ at almost an order of magnitude lower rate (1.1 ± 0.1) × 109 M-1 s-1, clearly abstracting an H-atom from the phenolic OH group. The reaction mechanism of curcumin changes dramatically above pH 8, where the enolate form of the heptadienone link predominates. As a consequence, the reaction of the methyl radical diminishes completely in alkaline media, and the phenolic part of the molecule takes over as (electron donor) reaction site. The electron donating ability of curcumin is assessed from the measurements of one-electron-transfer equilibria of DHZ radicals. Reduction potential of the DHZ phenoxyl radical, E(pH = 6.5) = 0.83 ± 0.06 V, and E(pH = 13.0) = 0.47 ± 0.06 V vs NHE, which may be expected for an ortho-methoxy-substituted phenoxyl radical, indicate only moderate electron-donating ability. The importance of H-atom donation vs electron donation in free radical scavenging and antioxidant mechanisms of curcumin is discussed.

Model phenoxyl and more complex flavonoid radicals were generated by azide radical induced oneelectron oxidation in aqueous solutions. Spectral, acid-base and redox properties of the radicals were investigated by the pulse radiolysis technique. The physicochemical characteristics of the flavonoid radicals closely match those of the ring with the lower reduction potential. In flavonoids which have a 3,s-dihydroxyanisole (catechins), or a 2,4-dihydroxyacetophenone (hesperidin, rutin, quercetin)-like A ring and a catechol-or 2-methoxyphenol-like B ring, the antioxidant active moiety is clearly the B ring [reduction potential difference between the model phenoxyls is AE(A-B ring models) > 0.1 V]. In galangin, where the B ring is unsubstituted phenyl, the antioxidant active moiety is the A ring. Even though the A ring is not a good electron donor, E7 > 0.8/NHE V, it can still scavenge alkyl peroxyl radicals, E7 = 1.06 V, and the superoxide radical, E7 > 1.06 V. Quercetin is the best electron donor of all investigated flavonoids (measured El,,, = 0.09 V, and calculated E, = 0.33 V). The favourable electron-donating properties originate from the electron donating 0 -3 hydroxy group in the C ring, which is conjugated to the catechol (B ring) radical through the 2,3-double bond. The conjugation of the A and B rings is apparently minimal, amounting to less than 2.5% of the substituent effect in either direction. Thus, neglecting the acid-base equilibria of the A ring, and using those of the B ring and the measured values of the reduction potentials at pH 3,7 and 13.5, the pH dependence of the reduction potentials of the flavonoid radicals can be calculated. In neutral and slightly alkaline media (pH 7-9), all investigated flavonoids are inferior electron donors to ascorbate. Quercetin, E7 = 0.33 V, and gallocatechins, E, = 0.43 V, can reduce vitamin E radicals (assuming the same reduction potential as Trolox C radicals, E, = 0.48 V). Since all investigated flavonoid radicals have reduction potentials lower than E7 = 1.06 V of alkyl peroxyl radicals, the parent flavonoids qualify as chain-breaking antioxidants in any oxidation process mediated by these radicals. OH OH Flavan-3-01s RS'= HI (f)-Catechin RS'= OH, (-) -E p ig a I loca t ec h i n OH Flavanones R3=R7=R4'=OH, Di h ydroq ue rce t in (Tax if oli n)

One-electron oxidation of uric acid generates the urate radical, which was studied in aqueous solution by pulse radiolysis and oxygen-uptake measurements. Acid-base properties of the uric acid radical were determined, i.e., p£al = 3.1 ± 0.1 and pXa2 = 9.5 ± 0.1. The reaction of the radical with oxygen was too slow to be measured, k < 10~2 dm3 mol-1 s'1. The one-electron-redox potential vs NHE, £7 = 0.59 V, was derived from the pH dependence of the redox potential, which was fitted through the values measured at pH 7 and 8.9 and those previously determined at pH 13. Rapid reactions of uric acid with oxidizing species and peroxy radicals were indicative of uric acid as a possible water-soluble physiological antioxidant. Rapid reaction of uric acid with the guanyl radical indicates that uric acid may also act as a repair agent of oxidative damage to DNA bases.Free-radical processes are increasingly invoked in many deleterious biological effects,1 such as replicative inactivation of DNA,2 mutation,3 carcinogenesis,4 atherosclerosis,5 arthritis,6 and aging.7 The generation of free radicals in biosystems exposed to ionizing radiations2 or in systems undergoing autoxidation5 has been firmly established by numerous direct observations of free radicals and their reactions in appropriate model systems.8 The involvement of free radicals in normal physiology,9 pathophysiology,10 and aging,7 although lacking direct proof, is backed by strong indirect evidence. Direct monitoring of free radicals in vivo is much more complex than detecting their presence in model biosystems,11 which is in any case considerably limited. Consequently, in addition to indirect-measurement approaches in vivo, comparison to model systems is crucial in assessing free-radical processes in biological media. In this work, the energetics, kinetics, and mechanisms of the antioxidant properties of uric acid were investigated by pulse and steady-state radiolysis in model aqueous solutions.Various sulfhydryls and antioxidants may act as efficient free-radical scavengers and repair agents of specific-free-radical damage to biomolecules.4 On the basis of these properties and the effects they exhibit, therefore, these two classes of compounds are considered to play an important role in anticarcinogenesis and chemical protection from ionizing radiations.4 Their role in the * The author to whom the correspondence should be addressed. f This work is based on the Ph.D.