Why do we expect flavonoids to
function as antioxidants in vivo?
Catherine Rice-Evans PhD, DSc, FRCPath
Antioxidant Research Group
Wolfson Centre for Age-Related Diseases
Guy’s, King’s & St. Thomas’s School of
Biomedical Sciences
King’s College, University of London
FLAVONOIDS:
FOCUS OF MUCH CURRENT NUTRITIONAL
AND THERAPEUTIC INTEREST
 CARDIOPROTECTION
Role for flavonoid-rich dietary components in
reduction in risk of cardiovascular disease
 NEUROPROTECTION
Anthocyanin-rich fruit associated with
protection against age-related decline in
cognitive function
 CHEMOPREVENTION
Flavonoids: naturally occurring low molecular wt
phenols consisting of 2 benzene rings linked via a
heterocyclic pyrone or pyran ring -> patterns and
substitutions comprising the sub-classes:
• Anthocyanin - berries
• Flavanone - citrus
• Flavanol
- red wine
teas
chocolate
fruit
• Flavonol
- fruit
vegetables
• Hydroxycinnamates most fruit & some vegetables
Flavonol
Flavanol
e.g. quercetin
e.g. epicatechin
onion, cranberry, red apple
many fruit and vegetables
red wine, green tea,
as procyanidins in apple, chocolate
OH
OH
Flavanone
OH
OH
HO
O
e.g. hesperetin
HO
Citrus fruit, orange
O
OH
OH
OH
O
OH
OCH3
Anthocyanidin
HO
O
e.g. cyanidin
OH
Hydroxycinnamate
e.g. caffeic acid
major constituents of dark
red fruit berries e.g. raspberries
OH
OH
O
most fruit especially tomato, apple
some vegetables e.g. egg plant
grains
OH
COOH
+
HO
O
OH
OH
OH
OH
SMALL DIFFERENCES IN STRUCTURE
 LARGE CHANGES IN BIOLOGICAL ACTIVITIES
Number and specific positions of OH groups /
nature of substitutions determine whether
flavonoids function as:
antioxidant, anti-inflammatory, cytotoxic
or antimutagenic agents in vitro or in vivo.
•
•
•
•
Antioxidant/pro-oxidant activities
Enzyme induction / inhibition
Cell proliferation / growth inhibition
Lipophilicity / polarity - cellular access
PROTECTIVE PROPERTIES OF FLAVONOIDS
AGAINST OXIDATIVE STRESS ARE
STRUCTURE-DEPENDENT
• Scavengers of reactive oxygen speciesH-donating abilities
• Transition metal chelators – catechol
requirement?
• Scavengers of reactive nitrogen species
nitric oxide, peroxynitrite etc – nitration or
oxidation?
• Non-antioxidant mechanisms - modulation
of signaling pathways, gene expression
STRUCTURAL REQUIREMENTS FOR
H-DONATING ANTIOXIDANT ACTIVITY:
ortho-dihydroxy substitution in B-ring
2,3-unsaturation in C-ring
HO
4-carbonyl group
A
OH
OH
B
O
C
OH
OH
Bors et al. 1990; Rice-Evans et al. 1996;
O
QUERCETIN
SCREENING FLAVONOIDS FOR ANTIOXIDANT ACTIVITY:
INFLUENCE OF B-RING STRUCTURE
Reduction
potentials
CATECHOLS
quercetin
epicatechin
MONOHYDROXY B-RING
kaempferol
hesperetin
ALKYLPEROXYL RADICAL
VITAMIN C
Jovanovic et al. 1998; Rice-Evans et al. 1996
E7
Antioxidant
activity
TEAC
0.33
0.57
4.7
2.4
0.75
0.72
1.3
0.9
1.06
0.25
STRUCTURAL DETERMINANTS OF
CYTOTOXICITY
Ease of oxidation –
catechol vs monophenolic
lipophilicity
OXIDATION OF QUERCETIN
Quercetin-7-quinone methide
OH
O
Quercetin
OH
O
O
OH
HO
O
OH
O
Damage through adduct
formation with proteins,
GSH, RNA and DNA
OH
OH
OH
O
OH
O
O
HO
O
O
O
HO
O
O
OH
Quercetin-5-quinone methide
OH
OH
O o-quinone
STRUCTURAL DEPENDENCE OF PEROXIDATIVE
METABOLISM OF FLAVONOIDS – monophenolic B-ring
FlavOH + ferryl radical
 FlavO
.
Phenoxyl
radical
.
FlavO + GSH
.
GS
+ O2
.
 GS Thiyl radical
 Reactive oxygen species
 GSSG
Galati et al. 2002
WHAT’S HAPPENING IN VIVO?
STRUCTURAL CHANGES ON ABSORPTION
Influence of conjugation and
metabolism on structural
parameters governing biological
properties
MAJOR METABOLIZING ENZYMES:
small intestine / liver / colon
• Glucosidases
• Hydrolases
• UDP-glucuronosyl
transferases
• Esterases
• Cytochrome P450s
• Catechol-O-methyl
transferases
• Sulfotransferases
OTHERS:
• Glutathione-S transferases
• Quinone reductases
Absorption and Biotransformation
of Dietary Flavonoids In Vivo
SKIN AND
BRAIN
Oligomers
cleaved
Stomach
Oligomeric
Flavonoids
cells
Monomeric
units
jejunum
O-methylated
Phase I and II
metabolism
Further
metabolism
Portal
vein
Small Intestine
Sulphates
glucuronides
ileum
Liver
glucuronides
Colon
Flavonoid
Phenolic acids
Kidney
Renal excretion
of glucuronides
Gut microflora
Urine
POTENTIAL MOLECULAR SITES OF METABOLIC
MODIFICATION
OH
OH
•glucuronidation
•sulphation
O
HO
OH
OH
•methylation
• oxidation
•cleavage
EFFECTS OF METABOLISM ON FLAVONOID STRUCTURES –
IMPLICATIONS FOR BIOLOGICAL PROPERTIES
OH
epicatechin
3'
2'
HO
B
1
8
O
7
A
2
OCH3
H
4'
OH
5'
1'
OH
6'
C
O
3
6
OH
4
5
3‘-O-methyl-epicatechin
OH
O
OH
OH
OH
4‘-O-methyl-epicatechin-7--D-glucuronide
epicatechin-7--D-glucuronide
OH
OH
COO
O
H
OH
H
OH
H
OH
H
OH
H
OH
OH
O
H
O
O
OCH3
COO
OH
H
OH
H
H
OH
O
O
H
OH
OH
STRUCTURAL FACTORS INFLUENCING
INTRACELLULAR ANTIOXIDANT PROPERTIES
• Reduction potentials of resulting
conjugates
• Cellular access and partition coefficients
• Intracellular/extracellular metabolism and
structural modifications
FLAVONOIDS CAN BE EXTENSIVELY METABOLISED BY cytP450s
-> metabolites with modified biological activities–human liver microsomes II
Kaempferol
HO
Quercetin
OH
OH
OH
O
3'-hydroxylation
OH
OH
HO
O
OH
O
OH
Tamarixetin
O
OH
Quercetin
OH
OH
OCH3
HO
HO
O
O
4‘-demethylation
OH
OH
OH
OH
O
Isorhamnetin
OCH3
O
Isorhamnetin
OCH3
OH
OH
HO
HO
O
OH
OH
O
O
No demethylation
OH
OH
O
Breinholt et al. 2002
STRUCTURAL CONSEQUENCES OF
INTRACELLULAR METABOLISM
OH
quercetin
OH
HO
Q
O
OH
??
OH
3OMeQ
OMe
HO
O
O
HO
O
glucuronide/
glucoside
O
CH3
O
O
GSH, cys,
protein thiol
OH
OH
HO
O
O
S
OH
OH
O
??
GSH, cys,
protein thiol
OH
O
OH
O
OH
O
OH
O
OH
O
OH
O
O
O
O
HO
O
HO
CH3
O
HO
OH
demethylation
OH
OH
OH
OH
OH
O
H
O
O
3’-O-methyl quercetin
HO
HO
OMe
OH
OH
OH
O
4’-O-methyl quercetin
3OMeQ
OH
Q
OH
Glucose
OH
O
Glutathione
303.7
RT: 55.43
OH
100
Quercetin
550
51.0
?
55.4
Querc
56.8
?
61.1
3´OMeQ
nm 320
Relative Abundance
OH
250
[M + H+]+ : 303.7
OH
60
40
O
OH
O
444.5
304.7
472.3
20
0
Quercetin
0.008
HO
80
300
340
380
420
100
0.000
0.008
3´-O-Me-quercetin
56.80
61.07
0.004
55.47
0.000
54.00
RT (min)
58.00
62.00
Relative Abundance
AU
(320 nm)
55.43
50.00
500
m/z
51.02
0.004
460
60
O
CH3
OH
HO
80
O
OH
317.7
OH
O
[M + H+]+ : 317.7
40
393.7
447.9
20
0
Spencer et al. 2002
RT: 61.07
371.8
300
340
380
420
m/z
460
500
COLONIC BIOTRANSFORMATION
WHAT’S HAPPENING IN THE COLON?
Majority of ingested flavonoids undergo
colonic metabolism
G. Gibson, University of Reading UK
Pathway of the colonic degradation of rutin implications for properties of in vivo metabolites
OH
OH
OH
OH
HO
Deglycosylation
HO
O
Further degradation
O
OH
OH
O-Rutinose
OH
Quercetin
O
Rutin
Dehydoxylation
O
HO
HO
Ring fission,
HO
water elimination,
3,4-dihydroxyphenyl- dehydroxylation
COOH
COOH
HO
Protocatechuic acid
acetic acid
HO
COOH
COOH
3-(3-hydroxyphenyl)propionic acid
HO
3-hydroxyphenylacetic acid
HO
β-Oxidation
Absorption
from the colon
+ glycination
NH
CO
COOH
3-hydroxyhippuric acid
MAJOR COLONIC METABOLITES
 3,4-dihydroxyphenyl acetic acid
 3-(3-hydroxyphenyl)propionic acid
 3-(4-hydroxyphenyl)propionic acid
 Hydroxybenzoates
SO DO WE EXPECT FLAVONOIDS TO
BE ANTIOXIDANTS IN VIVO?
IT DEPENDS:
 on what we mean by ‘antioxidation’
 on the extent and structural
consequences of conjugation
and metabolism
BIOAVAILABILITY AND METABOLISM OF
FLAVONOIDS
• Less bioavailable than ascorbate and
tocopherols
• MODIFIED by metabolism on absorption
• Less extensively absorbed and circulating
levels in vivo much lower
PLASMA LEVELS OF FLAVONOID CONJUGATES
Flavan-3-ol:
Wine catechins
Procyanidin:
Chocolate/cocoa
100 nM
4 uM; 0.26 uM;
0.7 uM
Flavanone –
grapefruit/orange
< 4 uM
Anthocyanin –
100 nM; 147 nM
berry juices
METHYL + SULPHATE
+GLUCURONIDE
EPICATECHIN
SULPHATE +
GLUCURONIDE
NARINGENIN
/HESPERETIN
GLUCURONIDE
ANTHOCYANIN
GLYCOSIDES
Donovan et al., Keen et al., Baba et al., Ameer et al, Miyazawa et al.
IN VIVO METABOLITE FORMS VERSUS CELLULAR OXIDATIVE STRESS
Caspase-3
acetyl-Asp-Glu-Val-Asp
p-nitroanilide
Apoptotic
Stimulus
Pro-caspase-3
p-nitroaniline
405 nm
0.20
All 30mM
METHYLATED METABOLITE
Lower H-donating potential –
modified catechol group
0.15
***
0.10
***
Similar protective effects
against oxidative stressinduced cell death
GLUCURONIDE METABOLITE
0.05
EC Gluc
MeEC
EC
H2O2 (50 mM)
0.00
Control
Increase in Absorbance (405 nm)
over control cells
0.25
H2O2 (50mM)
Marginally lower H-donating
potential
No protective effects against
oxidative stress-induced cell
death- inaccessibility or substituted
A-ring? SPENCER et al. 2001
PROTECTION OF NEURONS FROM OXIDATIVE STRESSINDUCED CELL DEATH BY EPICATECHIN
I
II
III
IV
I Control neurons
II Neurons exposed to oxidative stress
III Control neurons treated with epicatechin
IV Neurons pretreated with epicatechin prior to oxidative stress
Schroeter et al. 2000
CONCLUSIONS:
※ BIOACTIVITY OF FLAVONOIDS in vivo MAY NOT DEPEND
ON THEIR ACTIVITIES AS DIRECT SCAVENGERS OF
REACTIVE OXYGEN OR NITROGEN SPECIES PER SE
※ BUT RATHER ON THE INFLUENCE OF THEIR IN
VIVO FORMS ON THE MODULATION OF ENZYME /
PROTEIN FUNCTIONS, INTRACELLULAR CELL
SIGNALLING AND RECEPTOR ACTIVITIES