Transcript The Virtual Free Radical School Flavonoids and their free radical reactions Wolf Bors, Christa Michel, Kurt Stettmaier Inst.

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: [email protected]
Flavonoids
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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
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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.
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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
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Core structures and nomenclature
A
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
B
A
O
C
O
O
O
O
OH
Flavanone
OH
Flavan-3-ol
Dihydroflavonol
O
O
O
O
+
O
OH
OH
Anthocyanidin
Flavon-3-ol
Flavone
O
O
O
Isoflavone
Neoflavone
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Procyanidin dimers
Flavan-3-ols
OH
OH
OH
OH
2'
HO
7
8
O
B
B 5'
HO
6'
O
O
HO
OH
2
6 A
5
OH
3 ' 4'
A
C
O
3
4
OH
OH
OH
OH
OH
OH
(+)-ca te ch in (C A T )
D
(-)-ep icate ch in (E C )
HO
E
OH
HO
F
OH
OH
HO
O
O
C
O
OH
OH
HO
OH
OH
O
OH
O
pro cyan id in A 2 (P C -A )
OH
OH
OH
OH
OH
(-)-e pica tec hin g allate (E C G )
(-)-ep iga llo ca te ch in (E G C )
OH
B
OH
O
HO
OH
HO
OH
A
C
OH
OH
O
O
C
E
OH
OH
O
HO
OH
OH
D
O
F
OH
OH
OH
OH
OH
(-)-ep ig allocate ch in ga lla te (E G C G )
p ro cyan 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.)
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Radical scavenging - flavonoids
Scavenging rate constants of flavonoids
for oxidizing radicals
Substance (trivial name)
(substitution pattern)
Rate constant (x10 8 M -1s-1)
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)
58
89
0.95
dihydroquercetin (3,5,7,3’,4’-penta-OH)
103
43
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
luteolin (5,7,3’,4’-tetra-OH)
130
41
5.7
Flavonols (3-hydroxyflavones)
kaempferol (3,5,7,4’-tetra-OH)
141
88
6.0
51
66
6.6
Flavanols
(+)-catechin (3,5,7,3’,4’-penta-OH)
(-)-epicatechin ( - “ - )
Flavanones, Dihydroflavonols
naringenin (5,7,4’-tri-OH)
quercetin (3,5,7,3’,4’-penta-OH)

OH

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.)
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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
9
6
i
h
5
10
e
d
f
a
c
b
0
TEAC
4
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
g
3
2
1/IC50(DPPH)
1
0
0
0
5
10
5
15
10 15 20 25 30
20
25
30
number of adjacent aromatic hydroxyl groups
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Transient flavonoid radicals
Absorbance [mAU]
Flavanols
300
400
Eriodictyol
Luteolin
Flavones
Dihydro fisetin
Fisetin
Flavonols
Dihydro quercetin
Quercetin
500
600
700
|----| 2G
Wavelength
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., Birkhäuser, pp. 374.)
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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.)
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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
2

—
N3
—
F lO
[6]
2 F lO
[7a,b]
—
N 3 + A scH
[5a,b]
+ A sc

F lO


—
+ H
+

F l= O + A sc
[8]
+ A sc
[9]
F lO
[10]
2 A sc
—

—
+ A sc
—
—
+ H
+

N3 + OH
—

F lO

F lO

A sc

3 N2

F lO

F lO H + F l= O

F lO
—

+ H+
+ N3
—


—
+ N3
—
+ A scH

F lO

F l= O + A scH

A scH
+ DHA
—
+ DHA

—
—
an d D H A th o se o f asco rb ate; reactio n s [5 a,b ] an d [7 a,b ]
rep resen t th e tw o u n iv alen t red o x eq u ilib ria, reactio n s [8 ] an d [9 ] are tw o rad ical/rad ical
elim in atio n reactio n s, th e d irectio n d eterm in ed b y th e resp ectiv e red u ctio n p o ten 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
c
|-------| 5G
—
F lO H /F lO , F lO , F l= O d en o te th e d isso ciatio n an d u n iv alen t o x id atio n step s o f th e
flav o n o id s; A scH , A sc
b
+ DHA
—
+ H+
a
—
—
—
Redox properties
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.)
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Oxidation of Quercetin and formation of ROS
O
O
O
OH
Mechanism of radical interactions
- flavonols
OH
OH
O
R
disprop.
O
O
O
O
OH
OH
O
O2
reduction
disprop.
O 2O
O
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
O
Flavonoids
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Mesom eric structures of quercetin radicals and
quinone m ethides
O
O
O
H
O
O
HO
O
O
O
O
H
O
OH
OH
H
O
O
O
O
H
O
O
HO
O
O
O
O
H
Mechanism of radical
interactions - flavonols
OH
O
OH
H
O
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
O
H
O
O
HO
O
O
O
O
O
H
OH
O
H
OH
O
O
O
OH
O
OH
Flavonoids
OH
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Mechanism of radical interactions - catechins
OH
OH
HO
O
O
OH
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.)
EGCG
OH
O
OH
OH
OH
[O]
OH
O
HO
O
O
O
EGCG quinone
OH
O
OH
OH
OH
+ EGCG
OH
OH
OH
O
HO
H O
OH
H
OH
OH
H
OH
HO
H
H
H
O
O
OH
OH
O OH
OH
OH
HO
HO
H
H
O
HO
H
OH H
O
H
O
HO
OH
O
HO
HO
HO
H
O
O
OH
OH
OH
O
OH
OH H
H
H
OH
OH
HO
OH
EGCG dimer
2',4 adduct
NMR sensitive
Flavonoids
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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
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