The Virtual Free Radical School

Flavonoids
and their free radical reactions
Wolf Bors, Christa Michel, Kurt Stettmaier
Inst. Strahlenbiol., GSF Research Center
D-85764 Neuherberg, Germany
ph.: (+49-89) 3187-2508
fax: (+49-89) 3187-2818
e-mail: bors@gsf.de

Flavonoids

Society For Free Radical Biology and Medicine

W. Bors et al. 1

Flavonoids - Contents
Introduction
Structure, occurrence and nomenclature
flavonoids in general
flavan-3-ols (catechins)
proanthocyanidins (condensed tannins)
Functions - in vitro
radical scavengers
redox properties
mechanisms of radical interactions

Flavonoids

Society For Free Radical Biology and Medicine

W. Bors et al. .2

Flavonoids - Introduction

Flavonoids comprise a large group of secondary plant

metabolites. Presently more than 5000 individual compounds are
known, which are based on very few core structures. Their
multitude derives mainly from the various hydroxylation patterns
(up to six hydroxy groups) and ether substitution by simple
methylation or diverse mono- and di-saccharides
(Harborne JB, ed.: The Flavonoids. Advances in Research, Chapman & Hall, 1988)

Their function in plants themselves most likely involves screening

of UV light, in situ radical scavenging, anti-feeding effects
(astringency), etc. Proanthocyanidins mostly occur in green tea
(Camellia sinensis), grape seeds and skin (Vitis vinifera), or cacao
(Theobroma cacao). The distinct occurrence of flavonoids makes
them good candidates for taxonomic studies.

Flavonoids

Society For Free Radical Biology and Medicine

W. Bors et al. .3

Flavonoids - Introduction
Since the early fifties, their antioxidant potential with regard

to food preservation has been a focus of research.

Numerous papers exist on their inhibition of lipid
peroxidation. Due to their structural variety, the flavonoids
offer themselves also for detailed structure-activity
relationship (SAR) studies.
(Bors W et al. (1990) Meth. Enzymol. 186:343.)

The antioxidant effect of flavonoids can reside both in their

radical-scavenging activity or in their metal-chelating

properties, of which the former may dominate.

(Bors W et al. (1996) in: Handbook on Antioxidants, Cadenas & Packer, eds, Dekker, New York,
pp. 409.)

Flavonoids

Society For Free Radical Biology and Medicine

W. Bors et al. 4

Core structures and nomenclature

OH
B
O

The nomenclature of flavonoids

proper is straight-forward with the
aromatic ring A condensed to the
heterocyclic ring C and the
aromatic ring B most often
attached at the C2 position. The
various substituents are listed
first for the A and C ring and - as
primed numbers - for the B ring
(note that the numbering for the
aromatic rings of the openchained precursor chalcones is
reversed).
(Harborne JB, ed. (1988) The Flavonoids.
Advances in Research. Chapman & Hall.)

Chalcone

O
C

O
OH

OH

Flavanone

Flavan-3-ol

Dihydroflavonol

+
O
OH

OH

Anthocyanidin

Flavon-3-ol

Flavone

Isoflavone
Neoflavone

Flavonoids

Society For Free Radical Biology and Medicine

W. Bors et al. .5

Procyanidin dimers

Flavan-3-ols
OH

OH
2'

HO
7

6 A
5

3 ' 4'

OH

OH

B 5'

HO

6'

HO

2
4

OH

OH

OH

OH
OH

OH

OH

(+ )-ca te ch in (C AT )

(-)-ep icate ch in (E C )

F
HO

OH
O

HO

OH

O
C

OH

OH

HO

OH
OH

OH

OH

OH

OH

HO

OH

pro cyan id in A 2 (P C -A )

OH

OH

OH

(-)-e pica techin g allate (E C G )

(-)-ep iga llo ca te ch in (E G C )

HO

OH
HO

OH

OH

OH

C
OH

OH

O
C

OH

OH

HO

OH

OH

F
OH

OH

OH

OH

OH

(-)-ep ig allocate ch in ga lla te (E G C G )

p ro cy an idin B 2 (P C -B )

The nomenclature for the flavan-3-ols is rather confusing, as it is based both on the actual
structure, the chemical identification, and derivations thereof. For the proanthocyanidin
oligomers, a highly systematic nomenclature exists, based on the structures of the
monomers and the attachment sites.
(Kaul (1996) Pharmazie uns Zeit. 25:175.)
Flavonoids

Society For Free Radical Biology and Medicine

W. Bors et al. 6

Radical scavenging - flavonoids

Scavenging rate constants of flavonoids
for oxidizing radicals
Substance (trivial name)
(substitution pattern)

Rate constant (x108 M -1s-1)

OH

N3

t-BuO

66

50

1.35

64

51

210

52

2.65

dihydrofisetin (3,7,3,4-tetra-OH)

67

56

eriodictyol (5,7,3,4-tetra-OH)

117

47

0.8

dihydrokaempferol (3,5,7,4-tetra-OH)
dihydroquercetin (3,5,7,3,4-penta-OH)

58
103

89
43

0.95
1.0

Flavylium salts (anthocyanidins)

pelargonidine chloride (3,5,7,4-tetra-OH)

45

62

Flavones
apigenin (5,7,4-tri-OH)

135

48

3.0

130

41

5.7

141
51

88
66

6.0
6.6

Flavanols
(+)-catechin (3,5,7,3,4-penta-OH)
(-)-epicatechin ( - - )
Flavanones, Dihydroflavonols
naringenin (5,7,4-tri-OH)

luteolin (5,7,3,4-tetra-OH)
Flavonols (3-hydroxyflavones)
kaempferol (3,5,7,4-tetra-OH)
quercetin (3,5,7,3,4-penta-OH)

Flavonoids

The reaction of flavonoid

aglycones with the electrophilic
radicals OH or N3 are at the
diffusion-controlled limits but
within the same region for
almost all investigated compounds. Except for rutin and
hydroxyethylrutoside, very few
studies with flavonoid glycosides exist - in fact, the antioxidant principle is based on
the number and position of the
various hydroxy groups. Other
oxidizing radicals, e.g. t-BuO,
O2-, ROO, etc. also react
effectively with flavonoids, all
forming the same transient
aroxyl radicals. (Bors W et al.
(1992) in: Free Radicals and the Liver,
Csomos & Feher, eds, Springer, Berlin,
pp. 77.)

Society For Free Radical Biology and Medicine

W. Bors et al. 7

Radical scavenging - catechins

(Bors W et al. (1999) FRBM 27:1413.)

(Plumb et al. (1998) Free Rad Res. 29, 351.)
(Yokozawa et al. (1998) Biochem Pharm.
56:213 .
Flavonoids

k
l

30

20

j
i

10

TEAC

e
d

a
c
b

rel.units

-1 -1

k [10 M s ]

Only for the flavan-3-ols could a

clear-cut SAR be established, in
which case a linear increase of
the rate constants with OH
correlate with the number of
reactive hydroxy groups (e.g. the
number of catechol or pyrogallol
moieties). This correlation was
not observed with N3 as it only
reacts with dissociated phenols.
Linear correlation is also seen in
TEAC and DPPH assays.

40

3
2
1/IC50(DPPH)

1
0

10

15

10 15 20 25 30

20

25

30

number of adjacent aromatic hydroxyl groups

Society For Free Radical Biology and Medicine

W. Bors et al. 8

Transient flavonoid radicals

Absorbance [mAU]

Flavanols

300

400

Eriodictyol

Luteolin

Flavones

Dihydro fisetin

Fisetin

Flavonols

Dihydro quercetin

Quercetin

500

600

700

Wavelength

|----| 2G

The transient spectra observed with the pulseradiolytic technique reflect the B ring semiquinones for all compounds with a saturated C
ring (2,3-single bond). Desaturation leads to
expanded delocalization of the odd electron
over the whole three-ring system.

EPR spectra with HRP/H2O2 reveal

the same structure of B ring semiquinones. However, flavonol radicals
(e.g. quercetin) are unstable and
convert into other unresolved radicals
structures.

(Bors W et al. (1992) in: Free Radicals and the Liver,

Cosmos & Feher, eds., Springer, Berlin, pp. 77.)

(Bors W et al. (1993) in: Free Radicals, Poli et

al., eds., Birkhuser, pp. 374.)

Flavonoids

Society For Free Radical Biology and Medicine

W. Bors et al. 9

Transient flavonoid radicals

HRP/H2O2

2+

HRP/H2O2+Zn

CAT

HRP/H2O2
EGCG
0

EC
93 s

EGC

ECG
152 s

|-----|

EGCG

5G

EPR spectra of flavan-3-ols have also been studied

using
the
spin
stabilization
technique.
(Kalayanaraman, Meth. Enzymol. 186:333, 1990)
The results show the limitation of that method, as it
seems not to improve the signal of the pyrogallol
semiquinones and only enhances the catechol
semiquinone signal of epicatechin gallate (ECG).
(Bors W et al. (2000) Arch Biochem Biophys. 374:347.)
Flavonoids

TA
|-----| 0
5G

For catechins, the propensity for oligomerization is reflected in the EPR spectra
as well, leading from well-resolved signals
to single line signals of polymers.
(Bors W et al. (2000) Arch Biochem Biophys.
374:347.)

Society For Free Radical Biology and Medicine

W. Bors et al. 10

R ea ctio n sch em e o f in tera ctio n s b etw een fla v o n o id s a n d a sco rb a te

a fter in itia tio n o f ra d ica l rea ctio n s w ith a zid e ra d ica ls

[0]
[1]

OH + N3

F lO H

[2]

[3]
[4]

N 3 + F lO

+ A sc

F l= O + A sc

[8]

F lO

+ H

2 F lO

[7a,b]

N3

F lO

[6]

N 3 + A scH

[5a,b]

+ A sc
+ A sc

[9]

F lO

[10]

2 A sc

+ H

F lO

F lO

A sc

3 N2

F lO

F lO H + F l= O

F lO

N3 + OH

+ N3

+ N3

+ A scH

F lO

F l= O + A scH

A scH

+ DHA

+ DHA

an d D H A tho se o f asco rb ate; reaction s [5 a,b] and [7a,b ]

rep resent th e tw o un iv alen t redo x eq uilibria, reactio ns [8 ] an d [9 ] are tw o rad ical/rad ical
elim in atio n reaction s, th e direction d eterm in ed b y th e resp ective red u ctio n p oten tials.

As antioxidants, flavonoids are by definition

capable of electron-transfer reactions.
Among those, the reaction with ascorbate
has been studied in detail, with kinetic
modeling of the complex scheme allowing
the determination of the respective redox
potentials. (Bors W et al. (1995) FRBM 19:45.)
Flavonoids

|-------| 5G

F lO H /F lO , F lO , F l= O d en o te the d isso ciatio n an d u niv alen t o xid atio n step s o f th e

flav on o ids; A scH , A sc

+ DHA

+ H+

Redox properties

+ H+

In the case of dihydroquercetin (c), whose

optical semiquinone spectrum is superimposed over that of ascorbate (a) and its
radical, EPR spectra describe the
reducing power of dihydroquercetin
vis-a-vis the ascorbate radical (note
that the signal intensity of the latter
radical is much higher).
(Bors W et al. (1997) J Magnet Reson Anal. 3:149.)

Society For Free Radical Biology and Medicine

W. Bors et al. 11

Oxidation of Quercetin and formation of ROS

O

OH

Mechanism of radical interactions

- flavonols

OH
OH

disprop.

OH
OH

O
O2

reduction

disprop.

O 2O

O
O

Formation of flavonoid aroxyl radicals is an essential

step after initial scavenging of an oxidizing radical.
The stability of the aroxyl radicals strongly depends
on their bimolecular disproportionation reaction and
electron delocalization. Furthermore, the quinones
formed from the resp. semiquinones behave quite
distinctly from the flavonoids proper and the flavan-3ols: in the first case, quinones can undergo (futile)
redox cycling, with O2 - formed in a pro-oxidant effect.
(Metodiewa et al. (1999) FRBM 26:107.)

OH
OH

Flavonoids

Society For Free Radical Biology and Medicine

W. Bors et al. 12

Mesom eric structures of quercetin radicals and

quinone m ethides
O
O

O
H

HO

O
OH

O
O

OH

O
O

O
H

HO

O
O

Mechanism of radical
interactions - flavonols

OH
O

OH

The quinone methides, e.g. those of

flavonols are prone to nucleophilic
attack, which with DNA might lead to
pro-mutagenic adducts.
(Boersma et al. (2000) Chem Res Toxicol 13:185;
Awad et al. (2001) Chem Res Toxicol. 14:398.)

O
O

HO

O
O

O
O

OH

OH

O
O

OH
O

OH

Flavonoids

OH

Society For Free Radical Biology and Medicine

W. Bors et al. 13

Mechanism of radical interactions - catechins

OH
OH
HO

O
O

In contrast to the potentially prooxidant flavonoid quinones, those of

catechins (flavan-3-ols) preferentially
react via phenolic coupling reactions
(SN2) to form dimers and oligomers,
each retaining its original number of
reactive
hydroxy
groups,
i.e.
enhancing its antioxidant capacity
until a level is reached when the
oligomers become insoluble and
precipitate.
(Bors W et al. (2001) Antiox Redox Signal. 3:995.)

OH

EGCG
OH

O
OH

OH
OH

[O]
OH

O
HO

O
O

O
OH

O
OH

EGCG quinone

OH
OH
+ EGCG

OH

OH
OH

HO

H O
HO

OH H

H
H

HO

H
OH

HO
HO

OH

HO

OH
OH

HO

OH

OH

OH

HO

HO

O OH

OH
OH

H
O
O

OH

OH
OH

O
OH

OH H

H
OH
OH

HO

OH

EGCG dimer
2',4 adduct
NMR sensitive

Flavonoids

Society For Free Radical Biology and Medicine

EGCG dimer
2',2" gallyl adduct
NMR insensitive

W. Bors et al. 14

Summary and Conclusions

Flavonoids in general scavenge oxidizing (electrophilic) radicals

preferentially via their B-ring catechol or pyrogallol units at diffusioncontrolled rates.
In proanthocyanidins, due to oligomerization, several catechol/
pyrogallol sites exist for radical attack.
All flavonoid aroxyl (semiquinone) radicals decay by second-order
kinetics, i.e. bi-molecular disproportionation, forming quinones
(quinone methides) and the parent hydroxy compound.
Quinones and quinone methides of flavonols especially may have prooxidant activities due to either futile redox cycling or nucleophilic
attack.
Quinones of proanthocyanidins preferentially dimerize via phenolic
coupling reactions, thereby enhancing the antioxidant potential with
each oligomerization step.

Flavonoids

Society For Free Radical Biology and Medicine

W. Bors et al. 1