Biochemical and Biophysical Research Communications 282, 11611168 (2001) doi:10.1006/bbrc.2001.4705, available online at http://www.idealibrary.

com on

Flavonoid B-Ring Chemistry and Antioxidant Activity: Fast Reaction Kinetics

Ananth Sekher Pannala,* Tom S. Chan, Peter J. OBrien, and Catherine A. Rice-Evans* ,1
*Wolfson Centre for Age Related Diseases, GKT School of Biomedical Sciences, Kings College London, St. Thomass Street, London SE1 9RT, United Kingdom; and Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 2S2, Canada

Received March 19, 2001

Rapid scavenging of the model stable radical cation, ABTS , has been applied to screen for the antioxidant activity of avonoids. The reaction follows two distinct phases. For compounds with a monophenolic B-ring there is a rapid initial phase of reduction of ABTS within 0.1 s with no further change in the subsequent 2.9 s. In contrast, compounds with a catechol-containing B ring follow a fast initial scavenging phase with a slow secondary phase. Flavonoids with an unsubstituted B ring do not react within this time scale. The ndings suggest that the structure of the B ring is the primary determinant of the antioxidant activity of avonoids when studied through fast reaction kinetics. 2001 Academic Press Key Words: avonoid; hydroxycinnamate; anthocyanidin; catechin; ABTS radical cation; structureactivity relationship; antioxidant activity; TEAC.

Long-lived free radicals with high extinction coefcients are very useful in one-electron transfer reactions. 2,2-Azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) can donate an electron to generate a relatively long-lived radical cation (ABTS ) (Scheme I) (1, 2) with absorption maxima at 645, 734, and 815 nm. Scavenging of ABTS has been applied to the screening of various compounds, both lipophilic and hydrophilic, and food products for their antioxidant activities (3 8). ABTS reacts at a rate of k 10 8 with hydroxyl, cysteinyl, glutathione, thiocyanate, and bromide radicals to yield the corresponding radical cation as demonstrated by pulse radiolysis (1). It has also
Abbreviations used: TEAC, Trolox equivalent antioxidant capacity; ABTS, 2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt; ECG, epicatechin gallate; EGC, epigallocatechin; EGCG, epigallocatechin gallate; SAR, structure activity relationship. 1 To whom correspondence should be addressed. Fax: 44 20 7848 6143. E-mail: catherine.rice-evans@kcl.ac.uk.

been demonstrated that the ABTS formed reacts with ascorbic acid at neutral pH at a rate constant of 8 10 6 M 1 s 1 and in acidic pH k 10 5 M 1 s 1 (1). Flavonoids are a class of compounds that have been demonstrated to be potent antioxidants based on their phenolic hydroxyl groups. Antioxidant activity has been attributed to their electron-donating ability. Structure-activity relationship (SAR) studies of avonoids have shown that the o-dihydroxy structure in the B ring and the 2,3 double bond in conjugation with the 4 oxo function in the C ring (as in avones) are essential for effective free radical scavenging activity (6, 9 12). The presence of a 3-hydroxyl group in the heterocyclic ring also increases the radical-scavenging activity of avonols, while additional hydroxyl groups at positions 5 and 7 of the A ring appear to be less important. These structural features contribute to increasing the stability of the aroxyl radical, i.e., the antioxidant capacity of the parent compound. An additional hydroxyl group on the B ring is reported to increase the antioxidant activity (9). The ABTS assay initially developed for screening the compounds for their antioxidant activity was based on the activation of metmyoglobin with hydrogen peroxide in the presence of ABTS to produce the radical cation, in the presence or absence of the antioxidants under investigation (3). More recently Re et al. (7) have described a modied ABTS assay based on the decolorization of the preformed radical cation. This method involved formation of the radical cation through oxidation by potassium persulfate, exposure of the antioxidant under investigation to the radical cation for a dened time period, and spectrophotometric measurement of the extent of the radical quenched. A method has been devised for measuring the relative activities of avonoids and phenolic acids as electron donors by measuring their rapid reaction with ABTS rather than their reactivity over longer timescales, during which slower reacting functional hydroxyl groups might also participate in the reaction.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS absorbance change is triggered as soon as the cuvette is lled which triggers a signal to the spectrophotometer. The dead time-volume for mixing is 8 ms. Absorbance changes are monitored every 0.1 s for a duration of 3 s at 734 nm. Prior to the testing with the antioxidants, a baseline is obtained by monitoring the change in absorbance between ABTS and ethanol. This reading is used as the basal value for calculating the antioxidant activity of the compounds. Subsequently three concentrations of the antioxidants (1, 5, and 10 M, nal concentration) are tested for their antioxidant activity. Stock solutions of the antioxidants (1 mM) are prepared in ethanol and diluted subsequently with ethanol to give an initial concentration of 2, 10, and 20 M. As the antioxidants are mixed in the cuvette with the ABTS solution, the nal concentrations are half the initial concentrations. Results are expressed in terms of the stoichiometric factor and Trolox equivalent antioxidant capacity (TEAC). Stoichiometric factor is calculated on the basis of extent of ABTS scavenged by the antioxidant. Results are calculated at 0.1 s and 3 s. The extent of radical cation present at 0.1 s and 3 s is calculated from the absorbance recorded and the extinction coefcient (7). The concentration of the ABTS reacted is then plotted against the concentration of the antioxidant applied. The ratio of the concentration of the antioxidant to the concentration of the ABTS is expressed as the stoichiometric factor. TEAC is calculated based on the percentage scavenging of the radical cation by the avonoid relative to that by Trolox. Percentage scavenging of ABTS is calculated from the absorbance values at 0.1 s and 3 s compared to the base value at 0 s. To calculate TEAC value, percentage scavenging of the radical cation is plotted against the concentration of the avonoid, which exhibits a linear relationship. A similar plot is also obtained with Trolox. The ratio of the slope obtained from the avonoid percentage inhibition graph with the slope obtained from Trolox is the antioxidant activity expressed as TEAC.

SCHEME I.

Formation of ABTS radical cation.

The purpose of the rapid assay described here is to identify the polyphenolic compounds which are more effective reductants and the structural features of the molecules underlying these effects. MATERIALS AND METHODS
Chemicals. Trolox, ABTS (2,2-azinobis-(3-ethylbenzthiazoline6-sulfonic acid) diammonium salt), potassium persulphate, and gallic acid were purchased from Sigma-Aldrich Chemical Company (Poole, Dorset). Hydroxycinnamates (p-, m-, and o-coumaric acids, caffeic acid, chlorogenic acid, ferulic acid, and sinapic acid), anthocyanidins (malvidin, delphinidin, cyanidin, and pelargonidin), avonols (quercetin, kaempferol, and galangin), avones (rutin, luteolin, apigenin, chrysin, and disometin), avanones (naringenin, taxifolin, and hesperetin), and catechins (catechin, epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) were obtained from Extrasynthe ` se (Lyon, France). Rathburn Chemicals Ltd. (Walkerburn, Scotland) supplied HPLC grade ethanol. ABTS decolorization assay. The assay was carried out by interacting the antioxidants with the ABTS radical cation prepared as described in Re et al. (7). A stock solution of 7 mM ABTS was prepared in water. To this solution potassium persulphate (2.45 mM nal concentration) was added and the solutions allowed to react for a duration of 12 h at room temperature in the dark. ABTS and potassium persulphate react stoichiometrically at 1:0.5 leading to an incomplete oxidation to generate ABTS . The radical thus generated is stable in the dark at room temperature for two days (7). The ABTS radical cation solution was diluted in ethanol to obtain an absorbance of 0.70 0.02 at 734 nm. The nal concentration of the radical cation was calculated to be 80 M ( 16000 M 1.cm 1, at 734) (7). The interaction between antioxidants and ABTS was carried out by stop-ow kinetics (SFA20, Hi-Tech Scientic, Salisbury, UK). The SFA20 is a two-syringe stop-ow kinetics system capable of mixing two solutions in a spectrophotometer (Hewlett-Packard 8453). One syringe of the SFA20 is lled with the antioxidant/ethanol solution and the other syringe with ABTS solution. Solutions are withdrawn into the driving syringes followed by rapid mixing of the solutions in the cuvette by pushing the base plate. Measurement of

RESULTS The structures of the families of phenolic compounds studied, avonol, avone, avanone, hydroxycinnamate, anthocyanidin, and avanol are shown in Figs. 1a1d. The different families show close structural similarities with major variations in the nature of the C ring, and individual family members have varying numbers of hydroxyl groups in the B and C rings. The rapid rates of reaction with the ABTS radical cation are assessed by the spectrophotometric monitoring of the change in absorbance at 734 nm every 0.1 s for a total duration of 3 s. Figure 2a shows the reaction between ABTS and increasing concentrations of Trolox (1, 5, and 10 M) relative to the ABTS control. For all concentrations the reaction appears to be complete within the rst measured time point (0.1 s) with no further reaction taking place in the subsequent 2.9 s. The extent of scavenging, or reduction of ABTS, is proportional to the concentration of Trolox used. Interaction of avonoids with the radical cation was assessed at 1, 5, and 10 M concentrations as a function of time. The mode of inhibition exhibited two distinct types of reaction depending on the structure of the B ring (Figs. 2b and 2c). For compounds with a single hydroxyl group in the B ring, the reaction ap-

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FIG. 1.

Chemical structures of avonoids.

pears to be rapid and complete within the 0.1 s (Fig. 2b). This is particularly the case for naringeninthe avanone with a single 4-hydroxyl group in the B ring, apigenina avone with a single 4-hydroxyl group in the B ring, p-coumaric acid (4-hydroxycinnamic acid) and pelargonidinthe anthocyanidin with a single 4hydroxyl group in the B ring, as well as the standard compound for comparison, Trolox. Other compounds react very rapidly in the initial 0.1 s followed by slow secondary phase in the subsequent 2.9 s (Fig. 2c).

These include catechol-containing compounds (quercetin, luteolin, catechin, and epicatechin), trihydroxy B ring structures such as delphinidin, EGC and EGCG and hindered phenols such as ferulic acid (4-hydroxy,3methoxy cinnamic acid). The extent of scavenging of the radical cation by the phenolics relative to that scavenged by Trolox, represented by the TEAC value, is reported in Table 1. The catechol-containing B ring structures, especially the trihydroxy compounds such as EGC, delphinidin, and

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FIG. 2. (a) Scavenging of ABTS radical cation by Trolox. }, control; , 1 M Trolox; , 5 M Trolox; E, 10 M Trolox. (b) Reaction of compounds (10 M) exhibiting a fast initial phase within 0.1 s with no signicant change in subsequent 2.9 s. }, control; , naringenin; , apigenin; , p-coumaric acid; I, pelargonidin; F, Trolox. (c) Reaction of compounds (10 M) exhibiting a fast initial phase within 0.1 s and a slow secondary phase in the subsequent 2.9 s. }, control; , ferulic acid; , epicatechin; , delphinidin; F, ECG; E, EGCG.

the avanol gallate esters are the most potent electrondonating compounds according to the TEAC values of the various avonoid classes tested. This class of compounds shows a reactivity with ABTS greater than or close to that of Trolox after 3 s of reaction. As shown in Table 1, most of the catechol-rich structures demonstrate their reactivity essentially within 0.1 s, namely, EGC, EGCG, luteolin, quercetin, caffeic acid, and gallic acid. Other catechol-containing phenolics continue an extended reaction to 3 s such as taxifolin, ECG, catechin, epicatechin, delphinidin, and cyanidin. The potency of the antioxidant activity is also shown in terms of stoichiometric ratio (Table 1), which represents the concentration of ABTS reacted per unit antioxidant concentration (M). The stoichiometric factor is calculated by comparing the concentration of ABTS reacted (based on the extinction coefcient) with the concentration of the antioxidant applied. The concentration of the radical cation scavenged is plotted against the concentration of the antioxidant applied. The slope represents the stoichiometric factor, as shown in Fig. 3 for Trolox and three representative phenolics: sinapic acid (4-hydroxy,3,5-dimethoxy cinnamic acid)a hindered phenol, chlorogenic acid (the quinic acid ester of 3,4-dihydroxy cinnamic acid)a catechol compound, and pelargonidinan anthocyanidin with a monohydroxyl group in the 4-position of the B ring. The ratios of the stoichiometric factor observed for each compound relative to that for Trolox, are given in Table 1 obtained for reaction at 0.1 s and 3 s. Hence, by denition, the stoichiometric factor is relative to and is consistent with the TEAC values obtained. For the majority of compounds the stoichiometric value is between 1 and 2 at 0.1 s, depending on the reactivity of the B ring. However, stoichiometric factors do not take into account the kinetics of the reaction. Since the majority of the reaction of the avonoids occurs within the rst 0.1 s as indicated in Table 1, an alternative approach incorporating the reaction time was used to calculate the antioxidant activity, as described by Lebeau et al. (13). The kinetics of the reaction were calculated by plotting the reciprocal of the concentration of the ABTS reacted against time at 0.1 s for each concentration of the compound. The slope of this graph is taken as the rate constant of the reaction. The rate constant obtained from this graph was then plotted against the ratio of the concentration of antioxidant to that of ABTS reacted. The slope ( r 2 0.95) gave the parameter Z dened as radical scavenging activity (13). The ratio of the Z value obtained for each compound to that obtained for Trolox, the relative radical scavenging activity, is given in Table 1. The Z value is time-dependent.

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TEAC Values and Relative Stoichiometric Factors at 0.1 s and 3 s

TEAC Compounds Trihydroxy compounds EGCG EGC ECG Delphinidin Gallic acid Catechols Luteolin Quercetin Taxifolin Catechin Epicatechin Cyanidin Caffeic acid Chlorogenic acid 3,4-dihydroxy compound Kaempferol Hindered phenols Hesperetin Ferulic acid Sinapic acid Malvidin Monophenolic compounds p-Coumaric acid Pelargonidin Apigenin Naringenin Unsubstituted B ring Chrysin Trolox
a b

Ratio stoichiometric factor relative to Trolox 3s 0.1 s 3s

Z value ratio 0.1 s E 7 (V) 0.43 a 0.42 a 0.60 a 0.33 a 0.50 a 0.57 a 0.57 a 0.54 b 0.75 a 0.72 a 1.00 a 1.00 a

0.1 s

2.07 1.77 1.64 1.16 1.21 1.04 1.01 0.65 0.96 0.83 0.69 0.82 0.8 0.61 0.18 0.62 0.95 0.63 0.48 0.46 0.16 0.11 0.08 1

2.24 1.88 2.02 1.62 1.24 1.01 1.21 0.95 1.4 1.44 0.94 0.92 0.98 0.83 0.45 1.18 1.12 0.79 0.53 0.51 0.16 0.12 0.09 1

2.46 1.98 1.84 1.16 1.31 1.19 1.05 0.68 1.04 0.96 0.72 0.96 0.65 1.04 0.21 0.77 0.96 0.64 0.50 0.76 0.17 0.13 0.11 1

2.77 2.19 2.39 1.69 1.42 1.19 1.35 1.02 1.65 1.72 1.02 1.10 0.96 1.26 0.47 1.46 1.18 0.84 0.59 0.84 0.19 0.15 0.12 1

2.03 1.67 1.53 1.15 1.04 1.06 0.97 0.68 0.77 0.66 0.72 0.66 0.65 0.90 0.21 0.47 0.95 0.63 0.25 0.67 0.17 0.13 0.11 1

From reference 9. From reference 15.

DISCUSSION The three structural categories of the B ring: catechol-containing compounds (including the trihydroxy compounds), hindered phenols containing methoxy groups, and the monophenolic B ring structures display different radical scavenging properties. The catechol group in the B ring of avonoids is among the major structural considerations underlying antioxidant activity due to the favourable reduction potential (12, 14, 15). Furthermore, phenolics containing three adjacent hydroxyl groups such as delphinidin and EGC are more effective than their dihydroxyl counterparts, cyanidin, and epicatechin, respectively, illustrating the greater oxidizability of these specic trihydroxy structures. The predominant mechanism of action is probably via donation of a single electron to the radical cation resulting in the formation of a semiquinone, which can donate a further electron to form

the quinone (Scheme II). For example, it has been demonstrated by EPR spectroscopy that the spin distribution during oxidation of quercetin remains entirely on the B ring favouring the donation of two electrons leading to the formation of an ortho-quinone (16). This has also been related to the total conjugation of the quercetin molecule over the B and C rings. However, in the case of taxifolin which lacks this extended conjugation, the spin distribution was also reported to be on ring B emphasising the ready electron-donating ability of the catechol group leading to its oxidation (16). Compounds containing a 4-monohydroxyl group on the B ring are less potent antioxidants, the mechanism of action probably being via the formation of a phenoxyl radical (17, 18) (Scheme III). This is especially the case for phenolics in which there is no conjugation with the C-ring (i.e., the C-ring is either saturated or unsaturated at the 2,3 position but lacking a 4-carbonyl group),

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FIG. 3. Calculation of stoichiometric factor depending on the concentration of ABTS radical cation scavenged.

or for avones lacking a hydroxyl group in the 3-position. Thus, apigenin and naringenin have minimal reactivity possibly due to the relatively slow for-

SCHEME II. Potential oxidation products of catechols

mation of the phenoxyl radical compared to the case with hindered phenols. The position of the hydroxyl group in hydroxycinnamates plays an important role in determining the antioxidant activity (19). A para hydroxyl group enhances the antioxidant activity, while there is little or no effect when present in the meta or ortho positions. Hence, as predicted, m-coumaric acid demonstrates no activity while p-coumaric acid is relatively more reactive, with about 50% of the value of Trolox at 100 ms and 3 s. Kaempferol, a avonol with a single 4-hydroxyl group in the B ring, has a relatively high activity, compared with other monohydroxyl compounds studied which is most likely due to the potential for conjugation between the 4-hydroxyl group and the 3-hydroxyl group through the conjugated C ring. The phenoxyl radical that is formed could in theory abstract an electron from the radical cation to generate the di-cation and the phenolate. However, Chan et al. (17), based on their observation of the reaction between monophenolics and NADH to generate NAD , have suggested that the predominant mechanism of action for these compounds is via the formation of a phenoxyl radical. It has also recently been demonstrated that the phenoxyl radicals formed from

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SCHEME III. Possible mechanism of action of monophenolic compounds.

apigenin and naringenin are capable of oxidising GSH to GS . However, avonoids with a catechol group in the B-ring, quercetin, and luteolin, form semi-quinone radicals which are not capable of oxidising GSH (20). The presence of a hindered phenol on the B ring via the presence of a methoxyl group greatly enhances the electron-donating properties in the 4- or 4-position. For example, the hydroxycinnamates, ferulic (3methoxy, 4-hydroxycinnamic) acid, and sinapic (3,5dimethoxy, 4-hydroxycinnamic) acid are approximately twice as effective in scavenging the ABTS radical cation, in relation to the equivalent monophenolic structures (4-hydroxycinnamic acid), at 3 s. The more hindered the phenol, the more rapid the reaction, as shown by sinapic acid and by malvidin, in which the

phenolic group is hindered by the presence of two methoxyl groups. This increases the rate of the reaction and stabilises the formation of the phenoxyl radical such that the reaction is almost complete at 100 ms (Scheme IV). In contrast, ferulic acid shows a biphasic trend in scavenging the radical cation, a fast initial phase followed by a continued and extensive reaction between 100 ms and 3 s (Fig. 2c). It is of interest to note that ferulic acid dimer is a far more potent antioxidant when compared to the monomer (21). The low activity of hesperetin is accounted for by the location of the hydroxyl group in the B-ring at the 3-position akin to a m-hydroxyphenolic structure, with little electrondonating potential. In contrast, the 4-position would show more rapid formation of the phenoxyl radical as

SCHEME IV. Reaction of hindered phenols with ABTS radical cation.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 7. Re, R., Pellegrini, N., Proteggente, A., Pannala, A. S., Yang, M., and Rice-Evans, C. A. (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 12311237. 8. van den Berg, R., Haenen, G. R. M. M., van den Berg, H., and Bast, A. (1999) Applicability of an improved Trolox equivalent antioxidant capacity (TEAC) assay for evaluation of antioxidant capacity measurements of mixtures. Food Chem. 66, 511517. 9. Pietta, P-G. (2000) Flavonoids as antioxidants. J. Nat. Prod. 63, 10351042. 10. Lien, E. J., Ren, S., Bui, H. H., and Wang, R. (1999) Quantitative structure activity relationship analysis of phenolic antioxidants. Free Radic. Biol. Med. 26, 285294. 11. Bors, W., and Saran, M. (1987) Radical scavenging by avonoid antioxidants. Free Radic. Res. Commun. 2, 289 294. 12. Jovanovic, S. V., Steenken, S., Hara, Y., and Simic, M. G. (1996) Reduction potentials of avonoid and model phenoxyl radicals. Which ring in avonoids is responsible for antioxidant activity. J. Chem. Soc. Perkin Trans. 2, 24972504. 13. Lebeau, J., Furman, C., Bernierm, J-L., Duriez, P., Teissier, E., and Cotelle, N. (2000) Antioxidant properties of di-tertbutylhydroxylated avonoids. Free Radic. Biol. Med. 29, 900 912. 14. Jovanovic, S. V., Steenken, S., Tosic, M., Marjanovic, B., and Simic, M. G. (1994) Flavonoids as antioxidants. J. Am. Chem. Soc. 116, 4846 4851. 15. Jovanovic, S. V., Steenken, S., Simic, M. G., and Hara, Y. (1998) Antioxidant properties of avonoids: Reduction potentials and electron transfer reactions of avonoid radicals. In Flavonoids in health and disease (Rice-Evans, C. A., and Packer, L., Eds.), pp. 137161, Marcel-Dekker Inc., New York, NY. 16. van Acker, S. A. B. E., de Groot, M. J., van den Berg, D-J., Tromp, M. N. J. L., den Kelder, G. D-O., van der Vijgh, W. J. F., and Bast, A. (1996) A quantum chemical explanation of the antioxidant activity of avonoids. Chem. Res. Tox. 9, 13051312. 17. Chan, T., Galati, G., and OBrien, P. J. (1999) Oxygen activation during peroxidase catalysed metabolism of avones or avanones. Chemico-Biol. Interac. 222, 1525. 18. Galati, G., Chan, T., Wu, B., and OBrien, P. J. (1999) Glutathione-dependent generation of reactive oxygen species by the peroxidase-catalysed redox cycling of avonoids. Chem. Res. Tox. 12, 521525. 19. Pannala, A. S., Razaq, R., Halliwell, B., Singh, S., and RiceEvans, C. A. (1998) Inhibition of peroxynitrite dependent tyrosine nitration by hydroxycinnamates: Nitration or electron donation? Free Radic. Biol. Med. 24, 594 606. 20. Galati, G., Moridani, M. Y., Chan, T. S., and OBrien, P. J. (2001) Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: Glutathione oxidation and conjugation. Free Radic. Biol. Med. 30, 370 382. 21. Garcia-Conesa, M. T., Plumb, G. W., Waldron, K. W., Ralph, J., and Williamson, G. (1997) Ferulic acid dehydrodimers from wheat bran: Isolation, purication, and antioxidant properties of 8-O-4-diferulic acid. Redox Report 3, 319 323.

demonstrated by the fast reaction at 0.1 s. Under these conditions, there is a possibility that hindered phenols might regenerate the radical cation by interacting with ABTS itself. For the avonoids studied, hydroxyl groups at positions 5 and 7 on the A ring are common features, with variations in the number and position of hydroxyl groups in the B ring and in the 3-position on the C ring. Under the time-scale of these fast reaction conditions, there is negligible contribution to the antioxidant activity from the slower acting A-ring meta-hydroxyl groups as demonstrated from the reaction of chrysin, a avone with an unsubstituted B-ring. The method of analysis described here using a model free radical in a chemical system can provide a fast and reproducible assay to screen compounds and plant extracts to give an indication of their relative abilities to act as electron-donating antioxidants, based on the chemistry of the B-ring. (It is of course, conceivable that the reaction occurs more rapidly than the rst measured value at 0.1 s). Indeed, the antioxidant activity observed for each compound is reected in the reported reduction potentials. ACKNOWLEDGMENT
The authors wish to thank Dr. Paul Talalay (Johns Hopkins University, Baltimore, MD, USA) for his valuable contribution towards the discussion.

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