[36] FLAVONOIDS AS ANTIOXIDANTS 343

OH OH

"0 a a

~ 7 3 7
-0 -0 4 6
VIII IX

Using the spin-stabilization technique, one can also obtain the kinetic
parameters (Vmax, Kin, etc.) in enzymatic systems.16,26

Acknowledgments
This work was supported by National Institutes of Health Grants GM29035 and
RR01008.

26 W. Korytowski, T. Sarna, B. Kalyanaraman, and R. C. Scaly, Biochim. Biophys. Acta

924, 383 0987).

[36] F l a v o n o i d s as Antioxidants: D e t e r m i n a t i o n of
R a d i c a l - S c a v e n g i n g Efficiencies
By WOLF BORS, W E R N E R HELLER, CHRISTA M I C H E L ,
a n d M A N F R E D SARAN

Introduction
Flavonoids are phenolic compounds widely distributed in plants, with
over 4000 individual substances known and the list constantly expand-
ing. 1-3 This multiplicity is not surprising in view of the structural diversity
of flavonoids (Scheme I). Based on just a few backbone structures (chal-
cones, flavanols, flavanones, flavones, flavonols, flavylium salts or antho-
cyanidins, isoflavonoids, neo-, and biflavanoids), various hydroxylation,
methoxylation, sulfation, and/or glycosylation patterns exist. Consider-
l j. Kfihnau, World Rev. Nutr. Diet. 7,4, 117 (1976).
2 j. B. Harborne, in "Plant Flavonoids in Biology and Medicine" (V. Cody, E. Middleton,
and J. B. Harborne, eds.), p. 15. Alan R. Liss, New York, 1986.
3 j. B. Harborne (ed.), "Flavonoids: Advances in Research." Chapman & Hall, London,
1988.

Copyright © 1990by AcademicPress. Inc.

METHODS IN ENZYMOLOGY.VOL. 186 All fightsof reproductionin any formreserved.
344 ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS [36]

3'
Chalcone

0
Flava n-3-o[ Flavanone

Anthocyonidin

0 0
Flavonol Flavone

Neoflavane Isofiavone
SCHEME I. Structures of flavonoids.

ing the biosynthetic pathways of these substances 4 and their evolutionary

development, a major function for the intensely colored flavonols and
anthocyanidins in flower petals seems to be a visual signal for pollinating
insects (or hummingbirds), whereas catechins and other flavanols, owing
to their astringent character, probably evolved as feeding repellants. 5
Isoflavonoid derivatives constitute one of the most important groups of
plant-protective phytoalexins.6

4 W. Heller and G. Forkmann, in "Flavonoids: Advances in Research" (J. B. Harborne,

ed.), p. 399. Chapman & Hall, London, 1988.
5 j. B. Harborne, Biochem. Syst. Ecol. 5, 7 (1977).
6 D. A. Smith and S. W. Banks, Phytochemistry 25, 979 (1986).
[36] FLAVONOIDS AS ANTIOXIDANTS 345

The antioxidative effect of flavonoids has been of interest for a consid-

erable time. In the early 1930s, the discovery of their vitamin C-sparing
activity 7 led to the short-lived proposal of "vitamin P." 8 After a hiatus of
more than I0 years, a flurry of activity lasted throughout the 1950s and
into the 1960s. l'9,t° In these studies, probable mechanisms and structure-
activity relationships were put forward as an explanation for the role of
flavonoids as food protectants. Owing to the fact that flavonoids are rap-
idly degraded in the digestive system to inactive compounds, 1,1~interest
in their in vivo antioxidative effects in mammals ebbed. Only after the
recognition that the antioxidative and lipid peroxidation-inhibiting poten-
tial predominantly resides in the radical-scavenging capacity rather than
the chelation of metals 12,13 did the problem of the radical chemistry of
flavonoid aglycones become a topic of interest.
While a general capability of flavonoids to scavenge superoxide was
proposed repeatedly,14-18 the specific scavenging of hydroxyl,~9 superox-
ide, 2° and peroxy121 radicals may have been an overinterpretation of the
results. In all cases, the suggestions were derived from inhibition studies
after more or less nonspecific radical generation in lipid peroxidation,
enzymatic or chemical systems, with additional problems arising from the
nonspecific assay methods which were used in these studies. As shown
below, flavonoids do react rapidly with hydroxyl radicals, which is, how-
ever, of no surprise because of the generally high reactivity of this radical
with aromatic compounds. For 02% in contrast, even for the very effi-
cient flavonol radical scavengers kaempferol and quercetin, only very low
rate constants were found.
7 C. A. B. Clementon and L. Andersen, Ann. N. ¥. Acad. Sci. 136, 339 (1966).
8 A. Bentsath, S. Rusznyak, and A. Szent-Gy6rgyi, Nature (London) 139, 326 (1937).
9 R. E. Hughes and H. K. Wilson, Med. Chem. 14, 285 (1977).
10 L. R. Dugan, in "Autoxidation in Food and Biological Systems" (M. G. Simic and
M. Karel, eds.), p. 261. Plenum, New York, 1980.
" A. M. Hackett, in "Plant Flavonoids in Biology and Medicine" (V. Cody, E. Middleton,
and J. B. Harborne, eds.), p. 177. Alan R. Liss, New York, 1986.
12 C. G. Fraga, V. S. Martino, G. E. Ferraro, J. F. Coussio, and A. Boveris, Biochem.
Pharmacol. 36, 717 (1987).
13A. K. Ratty and N. P. Das, Biochem. Med. Metab. Biol. 39, 69 (1988).
14j. Baumann, G. Wurm, and F. yon Bruchhausen, Arch. Pharm. (Weinheim, Ger.) 313,
330 (1980).
15 I. Ueno, M. Kohno, K. Haraikawa, and I. Hirono, J. Pharmacobio-Dyn. 7, 798 (1984).
16 U. Takahama, Photochem. Photobiol. 42, 89 (1985).
~7M. Damon, F. Michel, C. Le Doucen, and A. Crastes de Paulet, Bull. Liaison Groupe
Polyphenols 13, 569 (1986).
18 U. Takahama, Plant Cell Physiol. 28, 953 (1987).
19 S. R. Husain, J. Cillard, and P. Cillard, Phytochemistry 26, 2489 (1987).
2o j. Robak and R. J. Gryglewski, Biochem. Pharmacol. 37, 837 (1988).
2~ j. Torel, J. Cillard, and P. Cillard, Phytochemistry 25, 383 (1986).
346 ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS [36]

We limited our present study on the reactivity of flavonoids with selec-

tively generated radicals to a number of commercially available hydroxyl-
ated and methoxylated flavonoid aglycones. 22 Sulfate ester derivatives are
a recently recognized subgroup, 23 and the multitudinous C- and O-glyco-
sylated sugar moieties have no bearing on the reactivities of the remaining
phenolic h y d r o x y groups. The hydroxyl groups are considered to be of
prime importance for the radical-scavenging properties.
To generate specific types of radicals, pulse radiolysis is a uniquely
suited method. 24,25 Data evaluation after fast kinetic spectroscopy of the
buildup a n d / o r d e c a y of transient absorption leads to kinetic parameters
for the primary radical attack, the stability of the secondarily formed
radicals, as well as for the redox potentials of these radicals.Z6 It is the
radical-scavenging property which is the focus of this chapter, whereas
the radical-generating function as studied by Pardini and co-workers, 27
pharmacological aspects, interactions with various enzymes, and cyto-
toxic and mutagenic/antimutagenic activities 2s,29 are not covered.

Methods

Radiolytic and Photolytic Principles o f Radical Generation

To test whether a certain substance acts as radical scavenger, the
clearest evidence comes from the determination of reaction rate constants
with a set of different specifically produced radicals. In our investigation
we used hydroxyl (. OH), azide (N3-), superoxide (O2-), linoleic acid
peroxyl ( L O O . ) , tert-butoxyl (t-BuO.), and sulfite radicals (SO3-:).
F o r m y l m e t h y l radical (. C H 2 - - C H = O ) was reacted with some flavonoids
in an earlier study, 3° and it was assumed that at pH 13.5 this radical at
least partially exists as the ethylene oxy radical ( C H 2 = C H O .).31 All

22E. Wollenweber and V. H. Dietz, Phytochemistry 20, 896 (1981).

23D. Barron, L. Varin, R. K. Ibrahim, J. B. Harborne, and C. A. Williams, Phytochemistry
27, 2375 (1988).
24K.-D. Asmus, this series, Vol. 105, p. 167.
25W. Bors, M. Saran, C. Michel, and D. Tait, in "Advances on Oxygen Radicals and
Radioprotectors" (A. Breccia, C. L. Greenstock, and M. Tamba, eds.), p. 13. Lo Scara-
beo, Bologna, 1984.
L. G. Forni and R. L. Willson, this series, Vol. 105, p. 179.
27W. F. Hodnick, E. B. Milosavljevic, J. H. Nelson, and R. S. Pardini, Biochem. Pharma-
col. 37, 2607 (1988).
B. Havsteen, Biochem. Pharmacol. 32, 1141 (1983).
z9 E. Middleton, Trends Pharmacol. Sci. 5, 335 (1984).
30S. Steenken and P. Neta, J. Phys. Chem. 86, 3661 (1982).
31S. Steenken, J. Phys. Chem. 83, 595 (1979).
[36] FLAVONOIDS AS ANTIOXIDANTS 347

these radicals are oxidizing species and are assumed to form aroxyl radi-
cals with phenolic compounds. 32 Hydrated electrons (eaq) and formate
radicals (CO2-) are reducing radicals and react only with a few flavonoid
structures.
Spectral observation of radical reactions with flavonoids is generally
assisted by the strong absorption characteristics of both the parent com-
pounds and their respective aroxyl radicals. The 15-photomultiplier array
used at our institute, 33 which allows analysis of spectral changes within a
130 nm spectral region at a time resolution of 500 nsec after just one pulse,
could be optimally applied to determine whether different absorption
bands show identical kinetic behavior. These data are especially impor-
tant for establishing structure-activity relationships. The aroxyl radicals
are preferentially generated with N3" radicals rather than • OH radicals,
because it cannot be ruled out that after attack of the latter radical highly
unstable hydroxycyclohexadienyl radical derivatives are formed, which
may exhibit different spectral and kinetic properties. 34 The strong pH
dependence of the absorption spectra of flavonoids also has to be consid-
ered. Owing to the various dissociable phenolic hydroxyl groups,35-38 both
the transient spectra of the radicals and the kinetics of their formation
( e . g . , N3" radicals react with phenolate anions rather than with undissoci-
ated phenols) 39 can change drastically.
To determine whether substances act as antioxidants, the stability of
the radical formed after scavenging should be known. A very reactive
secondary radical would propagate rather than interrupt a chain reaction!
Furthermore, effective antioxidants have been shown to react in a 1 : 2
stoichiometry,4° that is, one antioxidant molecule reacts with two radical
species, the second reaction being a radical-radical recombination pro-
cess. This type of reaction has thus far been observed only for aliphatic
peroxyl radicals reacting with phenolic and arylamine antioxidants 4° and

32 M. Erben-Russ, W. Bors, and M. Saran, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem.
Med. 52, 393 (1987).
33 M. Saran, G. Vetter, M. Erben-Russ, R. Winter, A. Kruse, C. Michel, and W. Bors, Rev.
Sci. Instrum. 58, 363 (1987).
G. E. Adams and B. D. Michael, Trans. Faraday Soc. 63, 1171 (1967).
35 N. P. Slabbert, Tetrahedron 33, 821 (1977).
P. K. Agrawal and H. J. Schneider, Tetrahedron Lett. 24, 177 (1983).
37 j. A. Kennedy, M. H. G. Munro, H. K. J. Powell, L. J. Porter, and L. Y. Foo, Aust. J.
Chem. 37, 885 (1984).
O. S. Wolfbeis, M. Leiner, P. Hochmuth, and H. Geiger, Ber. Bunsenges. Phys. Chem.
88, 759 (1984).
39 Z. B. Alfassi and R. H. Schuler, J. Phys. Chem. 89, 3359 (1985).
4o C. E. Boozer, G. S. Hammond, C. E. Hamilton, and J. N. Sen, J. Am. Chem. Soc. 77,
3233 (1955).
348 ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS [36]
with t~-tocopherol. 41,42 Recently, we obtained kinetic evidence that re-
combination of kaempferol and quercetin aroxyl radicals with linoleic
acid peroxyl radicals takes place with rate constants exceeding 10a M -~
s e e - 1.32
It is easily understood that with increasing stability of an antioxidant-
derived aroxyl radical, a recombination reaction becomes more and more
likely:
PhOH + ROO. --->PhO" + ROOH (I)
PhO. + R O 0 . -* ROO--Ph(~---O) (2)
Consequently, to classify a certain substance as an antioxidant, the fol-
lowing points have to be known: (i) rate constants with different types of
radicals; (ii) stability (and decay kinetics) of the "antioxidant radical";
and (iii) stoichiometry of the radical-scavenging process. Pulse radiolysis
yields data for the first two points, while unequivocal evidence of the
stoichiometry can only be obtained from product identification.
Rate constants of reaction with the photolytically produced t-BuO.
radical were, with one exception, 43 determined in the so-called crocin
assay (see below). 44 The • OH radicals simultaneously formed are scav-
enged by the presence of 20% tert-butanol, which in addition helps to
dissolve the flavonoid aglycones. Only substances not absorbing in the
wavelength region of crocin (hmax 440 nm), such as flavanols and
flavanones, can be measured by this method.
Another problem with flavonoid aglycones is their rather poor solubil-
ity in water. This can be overcome by dissolving the substances in alka-
line solutions and titrating back to the desired pH value, taking care to
avoid autoxidation of the substances 27 bykeeping the solution constantly
under nitrogen.

Determination of Reaction Rate Constants

Pseudo-First-Order Reactions. If a considerable excess of substrate
concentration over radical concentration (pulse dose) can be achieved, a
concentration-dependent plot of the first-order rate constant should yield
a straight line intersecting the origin. At the same time, a value for the
second-order rate constant is obtained by division with the substrate con-
centration. Rate constants with highly reactive radicals (. OH, N3", e~q)
can usually be obtained by this method.

41 j. Tsuchiya, E. Niki, and Y. Kamiya, Bull. Chem. Soc. Jpn. 56, 229 (1983).
42 j. Winterle, D. Dulin, and T. Mill, J. Org. Chem. 49, 491 (1984).
43 M. Erben-Russ, C. Michel, W. Bors, and M. Saran, J. Phys. Chem. 91, 2362 (1987).
44 W. Bors, C. Michel, and M. Saran, Biochim. Biophys. Acta 796, 312 (1984).
[36] FLAVONOIDS AS ANTIOXIDANTS 349

Competitive Radical Decay Processes. When the radical concentra-

tion approaches that of a poorly soluble substrate, direct kinetic evalua-
tion of the data is no longer feasible. In that case, competitive bimolecular
decay of the radicals dominates. To obtain reaction rate constants under
such conditions, kinetic modeling of parallel and sequential reactions, as
they occur for the various radical-generating systems, is required. Combi-
nations of differential equations are run, and optimized rate constants are
obtained as variables after iterative approximation of the experimental
results with the theoretical model. Rate constants with 02- (C. Michel,
unpublished results), t-BuO .,43 SO3 ~-,45 and LOO. 32 have been deter-
mined by this procedure. The kinetic model for LOO. generation and
reactions turned out to be the most complex one.
Competitive Radical Attack. Evaluation of the parallel attack of radi-
cals at a substrate and a reference compound (competitor) is usually
applied, if one of the substances is optically transparent at the respective
wavelength. 46,47 The relative rate constants with t-BuO, in the crocin
assay are always obtained from competition plots by observing the
bleaching of the strong absorption of crocin with a molar absorptivity of
133.500 M -~ cm -1.44 Using 3 × 109 M -~ sec -~,43 the absolute rate constant
of crocin with t-BuO., as the reference value, relative rate constants can
be converted to absolute ones.

Radical Chemistry of Flavonoids

Rate Constants o f Flavonoid Aroxyl Radical Formation and Decay

Owing to the low solubility of flavonoids, conditions for pseudo-first-
order reactions with radical species can rarely be achieved for flavonoid
aglycones. Therefore, kinetic modeling calculations have to be employed.
Table I gives a compilation of all presently known rate constants of
flavonoid aglycones with three different types of radicals. Both • OH and
N3" are highly electrophilic radicals and consequently show little differ-
ence in their diffusion-controlled rate constants. The values for t-BuO.
are 20- to 80-fold lower, with the only exception of the flavonol quercetin,
whose rate constant was obtained directly by pulse radiolysis. 43 This

45 M. Erben-Russ, C. Michel, W. Bors, and M. Saran, Radiat. Environ. Biophys. 26, 289
(1987).
G. E. Adams, J. W. Boag, J. Currant, and B. D. Michael, in "Pulse Radiolysis"
(M. Ebert, J. P. Keene, A. J. Swallow, and J. H. Baxendale, eds.), p. 131. Academic
Press, New York, 1965.
47 W. Bors, C. Michel, and M. Saran, in "CRC Handbook of Methods for Oxygen Radical
Research" (R. A. Greenwaid, ed.), p. 181. CRC Press, Boca Raton, Florida, 1985.
350 ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS [36]

TABLE I
REACTION RATE CONSTANTS OF FLAVONOIDS WITH STRONGLY OXIDIZING RADICALS

Flavonoid
Rate c o n s t a n t
Substitution pattern ( x 108 M -x sec -I)

Trivial n a m e OH OCH3 • OH a N3 .b t - B u O .c

Dihydrochalcones
Phloretin 4,2',4',6' -- -- -- 0.4
Flavanols
(+)-(2R,3S)-Catechin 3,5,7,3 ',4' -- 66 50 1.35
( -)-(2R,3R)-Epicatechin 3,5,7',3',4' -- 64 51 --
Flavanones
Dihydrofisetin 3,7,3',4' -- 45 56 --
Eriodictyol 5,7,3',4' -- -- 31 --
Dihydroquercetin 3,5,7,3 ',4' -- -- 24 1.0
Hesperetin 5,7,3' 4' -- 58 0.7
Flavylium salts (anthocyanidins)
Pelargonidin chloride 3,5,7,4' ~ 45 62 --
Cyanidin chloride 3,5,7,3 ',4' ~ -- 30 --
Flavones
Apigenin 5,7,4' -- -- 48 --
Luteolin 5,7,3',4' -- -- 41 --
Acacetin 5,7 4' -- 28 1.3
Flavonols
Fisetin 3,7,3 ',4' -- -- 52 --
Kaempferol 3,5,7,4' -- 46 88 --
Quercetin 3,5,7,3',4' -- 43 66 25.0 d
Morin 3,5,7,2',4' -- -- 73
Kaempferid 3,5,7 4' -- 65 --

o D e t e r m i n e d pulse-radiolytically from the buildup of aroxyl radical absorption [M.

E r b e n - R u s s , W. Bors, a n d M. Saran, Int. J. Radiat. Biol. 52, 393 (1987)]; the values for
dihydrofisetin a n d pelargonidin chloride have not previously been published.
b D e t e r m i n e d pulse-radiolytically from the buildup of aroxyl radical absorption [W. Bors
and M. Saran, Free Radical Res. Commun. 2, 289 (1987)].
c Data obtained in the crocin a s s a y [W. Bors, C. Michel, and M. Saran, Biochim.
Biophys. Acta 796, 312 (1984)l and recalculated using the reference value for crocin o f
3 × 109M -1 sec -l [M. E r b e n - R u s s , C. Michel, W. Bors, and M. Saran, J. Phys. Chem.
91, 2362 (1987)].
d D e t e r m i n e d pulse-radiolytically from the buildup of aroxyl radical absorption [M.
E r b e n - R u s s , C. Michel, W. Bors, and M. Saran, J. Phys. Chem. 91, 2362 (1987)].

points to flavonols having structural features which may facilitate the

attack of more discriminately reacting radicals such a s t - B u O .. T h e in-
vestigation was therefore expanded to include reactions with some
other oxidizing radicals and the two flavonols kaempferol and quercetin
(Table II).
[36] FLAVONOIDS AS ANTIOXIDANTS 351

TABLE II
REACTION RATE CONSTANTS OF FLAVONOLSWITH OTHER
OXIDIZING RADICALS

Rate constant (× 10a M -~ sec -1)

Flavonol (SCN)2 : a SO3 : b LOO.c 02 ~ d

Kaempferol 8.5 4.0 0.34, 0.42 0.0055

Quercetin • 4.0 2.5 0.18, 0.15 0.0009

Determined pulse-radiolytically from the kinetics of

(SCN)2 ~ decay, substrate bleaching, as well as the buildup
of aroxyl radical absorption [M. Erben-Russ, W. Bors, and
M. Saran, Int. J. Radiat. Biol. 52, 393 (1987)].
b Determined pulse-radiolytically from the buildup of aroxyl
radical absorption [M. Erben-Russ, C. Michel, W. Bors,
and M. Saran, Radiat. Environ. Biophys. 26, 289 (1987)].
c Value dependent on method of radical generation, with the
first one for a mixture of LOO. radical isomers and the
second for 13-LOO. radicals specifically [M. Erben-Russ,
W. Bors, and M. Saran, Int. J. Radiat. Biol. 52, 393 (1987)].
d Determined pulse-radiolytically from kinetic modeling of
the 02- decay at pH 7.5; at higher pH no reaction at all was
observed (C. Michel, unpublished results)~
e Exceptionally high values of 3.1 x 109M -l sec -~ (and 1.8 x
109M- i sec- 1for catechin) were determined for the reaction
with CH2~CHO'/'CH2--CH~O radicals at pH 13.5
IS. Steenken and P. Neta, J. Phys. Chem. 86, 3661 (1982)].

Table I I I c o m p i l e s the d e c a y rate c o n s t a n t s o f flavonoid a r o x y l radi-

cals after g e n e r a t i o n b y N3" radicals at p H I 1.5, w h i c h in all cases repre-
sent s e c o n d - o r d e r d i s m u t a t i o n p r o c e s s e s . T h e m o s t striking feature is the
wide range o f d e c a y rates, and this should be v i e w e d in c o n t e x t with the
r a d i c a l - s c a v e n g i n g v e r s u s antioxidative capacity. While b o t h k a e m p f e r o l
and q u e r c e t i n are highly efficient radical s c a v e n g e r s , only the quercetin
a r o x y l radical d e c a y s slowly e n o u g h to m a k e this flavonol a p o t e n t antiox-
idant. L o o k i n g at the structures o f the m o s t stable a r o x y l radicals, t h o s e
with d e c a y rates o f 105 to 106 M -1 sec -l, it is evident that w i t h o u t e x c e p -
tion all c o n t a i n the 3 ' , 4 ' - c a t e c h o l structure. All o t h e r phenolic c o m -
p o u n d s f o r m far less stable a r o x y l radicals.

Transient Spectra o f R a d i c a l I n t e r m e d i a t e s
T h e w i d e v a r i e t y o f flavonoid structures (see S c h e m e I) and h y d r o x y l -
ation p a t t e r n s offers a u n i q u e o p p o r t u n i t y to study the influence o f sub-
strate substitution p a t t e r n o n s h a p e and intensity o f transient spectra.
352 ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS [36]

TABLE III
SECOND-ORDER DECAY RATE CONSTANTS AND SPECTRAL PARAMETERS
OF FLAVONOID AROXYLRADICALSa

Molar Absorption
Rate constant absorptivity maximum
Substance b 2k (x 106 M -1 sec -I) (M -1 cm -l) (nm)

(+)-Catechin 0.6 10,000 310

Eriodictyol 0.4 8,900 315
Dihydroquercetin 0.1 7,600 315
Pelargonidin chloride 210 19,500 685
Apigenin 170 8,100 360
Luteolin 0.2 8,100 475
Acacetin 500 6,600 325
Fisetin 0.3 4,100 600
Kaempferol 140 24,000 550
Quercetin 3.4 15,600 530
Morin 63 17,600 525
Kaempferid 370 8,000 480

Aroxyl radicals were generated by attack of N3" radicals at pH 11.5 [W. Bors and
M. Saran, Free Radical Res. Commun. 2, 289 (1987)].
b For substitution pattern of substances see Table I.

This was best exemplified in the comparison of aroxyl radical spectra

generated by N3" attack. 48 The study revealed three structural principles
governing the formation of transient spectra (with one exceptional case):
flavan-3-ols and flavanones, which possess a saturated 2,3-bond, show
only the semiquinone radical of the B ring; flavones, with either the
3-hydroxyl group (the flavonol fisetin) or the 5-hydroxyl group (e.g., the
flavone luteolin), show transient spectra of intermediate strength and sim-
ilar shape; flavonols which also possess a 5-hydroxyl group show the
strongest absorption of the aroxyl radical, in wavelength regions remote
from the parent phenol absorption. These substances are thus best suited
for competition experiments. The exception is pelargonidin chloride, a
flavylium salt. Cyanidine chloride, the corresponding B-ring catechol ana-
log, forms a weakly absorbing water adduct,I a pseudo-base, where the
conjugation to the heterocyclic ring is interrupted (Scheme II). As ex-
pected, the aroxyl radical exhibits only the semiquinone structure of the B
ring.

W. Bors and M. Saran, Free Radical Res. Commun. 2, 289 (1987).

[36] FLAVONOIDS AS ANTIOXIDANTS 353

+ ~ r ,.OH ~-OH
HO.[~O ~ OH-. HO..~0,,~..~
~I~ O H "~OH
OH OH
SCHEMEII. Reaction of flavylium salt to form a water adduct.

Structural Principles for Effective Radical Scavenging by Flavonoids

On the basis of the presently available spectral and kinetic evidence on
the formation and decay of the flavonoid aroxyl radicals, a structure-
activity relationship can be derived. As shown in Scheme III, three struc-
tural groups are important determinants for radical scavenging and/or
antioxidative potential: (a) the o-dihydroxy (catechol) structure in the B
ring, which is the obvious radical target site for all flavonoids with a
saturated 2,3-bond (flavan-3-ols, flavanones, cyanidine chloride) [whether
semiquinone formation itself occurs with flavonols cannot be determined
because of spectral overlaps and would require electron paramagnetic
resonance (EPR) investigations; however, the catechol moiety does con-
fer a higher stability to aroxyl radicals and obviously participates in elec-

OH 0

b
Ho. o °H
"~r~-~OH
OH 0

C H O ~ ~

% ...O...H

SCHEMEIII. Structural groups for radical scavenging.

354 ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS [36]

O-H. o-H.

-0 0 - 0~ / " 'O..r~
0 ~ Ib-
OHO H O.H..O_...H"

excitation

o-H,° - o-H

0 0 -0 0

o ~ H o' o...
_
H' O,H 0 H

radical formation

o-H. O-H o-H.

-0 0 -0 0

OH 0 014'
• ..... [ ! H'0
O.H...O... o,H.9...H"

SCHEME IV. Reactions of quercetin showing principal radical structures involving three
ring systems.

tron delocalization]; (b) the 2,3-double bond in conjugation with a 4-oxo

function, which are responsible for electron delocalization from the B
ring; and (c) the additional presence of both 3- and 5-hydroxyl groups for
maximal radical-scavenging potential and strongest radical absorption
(from a kinetic standpoint, the 3- and 5-hydroxyl groups are equivalent
owing to their hydrogen bonds with the keto group). 35
In Scheme IV, using quercetin as an example, we attempted to draw
the principal mesomeric structures for the flavonol in its doubly-dissoci-
ated state which, at pH 11.5, is to be expected based on the dissociation
sequence of 7 - - O H > 4 ' - - O H > 5 - - O H . 49 For the aroxyl radicals
formed by electron transfer to N3" we assume that attack at the most
likely site is kinetically indistinguishable. Dipolar structures and espe-

49 T. J. Mabry, K. R. Markham, and M. B. Thomas (eds.), " T h e Systematic Identification of

Flavonoids," Part 2. Springer-Verlag, Berlin, 1970.
[37] FLUORESCENCE MEASUREMENTS OF TOCOPHEROL 355

cially the involvement of the 7- and 4'-hydroxyl groups in the excited

state (see top part of Scheme IV), have already been noted) s Taking these
mesomeric structures as guide, we present three principal radical struc-
tures involving all three ring systems.

Conclusions
The radical chemistry of flavonoids not only is of interest from a
kinetic or mechanistic point of view but also offers considerable insight
into structural relationships of these highly evolved plant components.
First of all, the consistently high rate constants for attack by different
types of radicals (see Tables I and II) demonstrate the effective radical-
scavenging capabilities of most flavonoids. Second, as shown in Scheme
IV, owing to extensive electron delocalization as a prerequisite for radical
stabilization, multiple mesomeric structures exist for aroxyl radical spe-
cies of flavonoids. From Scheme IV it can also be predicted which struc-
tural principles are necessary for optimal antioxidative capacity. By the
method of pulse radiolysis, as described here, it was possible to obtain the
data necessary to confirm the hypothesis. This might allow construction
of even better flavonoid antioxidants, were it not for nature itself which
has already optimized the biosynthesis along these lines. 4

Acknowledgments
The dedicated assistance during the pulse-radiolytic experiments by Alf Kruse is greatly
appreciated. Michael Erben-Russ determined a number of the quoted data (as reported in his
Ph.D. thesis). Matthias Born and Attain Furch were very helpful during discussion of the
manuscript.

[37] F l u o r e s c e n c e M e a s u r e m e n t s of I n c o r p o r a t i o n a n d
H y d r o l y s i s of T o c o p h e r o l a n d T o c o p h e r y l E s t e r s
in B i o m e m b r a n e s
By VALERIAN E. KAGAN, RUMYANA A. BAKALOVA,
ELENA E. SERBINOVA, and TSANKO S. STOYTCHEV

In qualitative terms, the stabilizing action of vitamin E (a-tocopherol)

in biomembranes can be ascribed to four effects, namely, (1) interaction
with lipid peroxide radicals, (2) specific rearrangement in the membrane
lipid bilayer (i.e., restriction of molecular mobility), (3) protection of

Copyright © 1990 by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 186 All rights of reproduction in any form reserved.