why do we expect flavonoids to function as antioxidants in...
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Why do we expect flavonoids to function as antioxidants in vivo?
Catherine Rice-Evans PhD, DSc, FRCPathAntioxidant Research Group
Wolfson Centre for Age-Related DiseasesGuy’s, King’s & St. Thomas’s School of
Biomedical SciencesKing’s College, University of London
FLAVONOIDS: FOCUS OF MUCH CURRENT NUTRITIONAL
AND THERAPEUTIC INTEREST
§ CARDIOPROTECTIONRole for flavonoid-rich dietary components in
reduction in risk of cardiovascular disease
§ NEUROPROTECTIONAnthocyanin-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 wineteaschocolatefruit
• Flavonol - fruit vegetables
• Hydroxycinnamates -most fruit & some vegetables
OH OH
Anthocyanidin
e.g. cyanidinmajor constituents of darkred fruit berries e.g. raspberries
+
OH
HO
OH
HO
OHOH
O
OH
O
OH
OH O
OH
OHO
OH
Flavonol
e.g. quercetinonion, cranberry, red applemany fruit and vegetables
OHOH
Hydroxycinnamate
e.g. caffeic acidmost fruit especially tomato, applesome vegetables e.g. egg plantgrains
COOH
OHOH
OH
HO
OH
O
Flavanol
e.g. epicatechinred wine, green tea,as procyanidins in apple, chocolate
Flavanone
e.g. hesperetinCitrus fruit, orange
O
OOH
HO
OHOCH3
SMALL DIFFERENCES IN STRUCTUREà LARGE CHANGES IN BIOLOGICAL ACTIVITIES
Number and specific positions of OH groups / nature of substitutions determine whether flavonoids function as:
antioxidant, antiantioxidant, anti--inflammatory, inflammatory, cytotoxic cytotoxic or or antimutagenic antimutagenic agents in vitro or in vivo.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 species-H-donating abilities• Transition metal chelators – catechol
requirement?• Scavengers of reactive nitrogen speciesnitric 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-ring2,3-unsaturation in C-ring4-carbonyl group
Bors et al. 1990; Rice-Evans et al. 1996; QUERCETIN
OHO
OH OOH
OHOH
A C
B
SCREENING FLAVONOIDS FOR ANTIOXIDANT ACTIVITY: INFLUENCE OF B-RING STRUCTURE
Reduction Antioxidantpotentials activityE7 TEAC
CATECHOLSquercetin 0.33 4.7
epicatechin 0.57 2.4MONOHYDROXY B-RING
kaempferol 0.75 1.3hesperetin 0.72 0.9
ALKYLPEROXYL RADICAL 1.06VITAMIN C 0.25
Jovanovic et al. 1998; Rice-Evans et al. 1996
STRUCTURAL DETERMINANTS OF CYTOTOXICITY
§Ease of oxidation –catechol vs monophenolic
§lipophilicity
O
OH
OH
OH
OH
HO
O
O
OH
O
O
OH
HO
O
O
O
O
O
HO
OH
OH
O
O
O
OH
O
OH
OH
Damage through adductformation with proteins,
GSH, RNA and DNA
-quinone
Quercetin-5-quinone methide
Quercetin-7-quinone methide
o
OXIDATION OF QUERCETIN
Quercetin
STRUCTURAL DEPENDENCE OF PEROXIDATIVE METABOLISM OF FLAVONOIDS – monophenolic B-ring
FlavOH + ferryl radical à FlavO Phenoxyl
radical
FlavO + GSH à GS Thiyl radical
GS + O2 à Reactive oxygen species
à 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
• UDP-glucuronosyl transferases
• Catechol-O-methyl transferases
• Sulfotransferases
• Hydrolases
• Esterases
• Cytochrome P450s
OTHERS:• Glutathione-S transferases• Quinone reductases
Absorption and Biotransformation of Dietary Flavonoids In Vivo
Monomericunits
OligomericFlavonoids
Stomach
Small Intestine
jejunum
ileum
Phase I and IImetabolism
Colon
Liver
Phenolic acids
glucuronides
glucuronides Kidney
Urine
O-methylated
SulphatesPortalvein
Furthermetabolism
Renal excretionof glucuronides
Oligomerscleaved cells
SKIN AND BRAIN
Gut microflora
Flavonoid
POTENTIAL MOLECULAR SITES OF METABOLIC MODIFICATION
•glucuronidation•sulphation
•methylation• oxidation
•cleavage
OHO
OHOH
OHOH
EFFECTS OF METABOLISM ON FLAVONOID STRUCTURES –IMPLICATIONS FOR BIOLOGICAL PROPERTIES
OH
OH O
OH
OCH3
OH
3‘-O-methyl-epicatechin
OH
OH O
OH
OCH3
OH
3‘-O-methyl-epicatechin
epicatechin-7-β-D-glucuronide
O
OH
OH
OH
OCOO
H
OH
HOH
H
OH
H
OH
O
H
epicatechin-7-β-D-glucuronide
O
OH
OH
OH
OCOO
H
OH
HOH
H
OH
H
OH
O
H
epicatechin-7-β-D-glucuronide
O
OH
OH
OH
OCOO
H
OH
HOH
H
OH
H
OH
O
H
4‘-O-methyl-epicatechin-7-β-D-glucuronide
O
OH
OH
OCH3
OCOO
H
OH
HOH
H
OH
H
OH
O
H
4‘-O-methyl-epicatechin-7-β-D-glucuronide
O
OH
OH
OCH3
OCOO
H
OH
HOH
H
OH
H
OH
O
H
4‘-O-methyl-epicatechin-7-β-D-glucuronide
O
OH
OH
OCH3
OCOO
H
OH
HOH
H
OH
H
OH
O
H
epicatechin
A C
B12
3
45
6
78
1'
2'3'
4'
5'
6'
O
OH
OH
OH
OH
HO
epicatechin
A C
B12
3
45
6
78
1'
2'3'
4'
5'
6'
O
OH
O H
OH
OH
HO
STRUCTURAL FACTORS INFLUENCING INTRACELLULAR ANTIOXIDANT PROPERTIES
• Reduction potentials of resulting conjugates
• Cellular access and partition coefficients
• Intracellular/extracellular metabolism and structural modifications
3'-hydroxylation
4‘-demethylation
No demethylation
Kaempferol Quercetin
QuercetinTamarixetin
Isorhamnetin
O
OHOOH
HO
OH
O
OHOOH
HO
OHOCH3
O
OHOOH
HO
OCH3
OH
OHO
OH OOH
OHOH
OHO
OH OOH
OHOH
Isorhamnetin
O
OHOOH
HO
OCH3
OH
FLAVONOIDS CAN BE EXTENSIVELY METABOLISED BY cytP450s -> metabolites with modified biological activities–human liver microsomes II
Breinholt et al. 2002
O
OH
OH
OH
OH
HO
O
O
OH
O
OH
OH
HO
O
CH3
O
OH
OH
OH
OH
HO
O
O
O
OH
OH
OH
HO
O
Glucose
O
OH
O
OMe
OH
HO
O
H
O
OH
O
O
OH
HO
O
O
O
O
O
OH
HO
O
CH3
??
O
OH
OH
OH
OH
HO
O
S Glutathione
GSH, cys,protein thiol ??
3OMeQ
Q
STRUCTURAL CONSEQUENCES OF INTRACELLULAR METABOLISM
Q
3OMeQ
demethylation
GSH, cys,protein thiol
O
OH
OH
OMe
OH
HO
O
quercetin
3’-O-methyl quercetin
4’-O-methyl quercetin
glucuronide/ glucoside
AU
(320
nm
)
nm
250
550 51.0?
55.4Querc
56.8?
61.13´OMeQ
0.000
0.004
0.00851.02
55.43
RT (min)
Quercetin
0.000
0.004
0.008
55.47
56.80
61.07
3´-O-Me-quercetin
50.00 54.00 58.00 62.00
320
300 340 380 420 460 500
m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
371.8
317.7
393.7447.9
O
OH
O
OH
OH
HO
O
CH3
[M + H+]+ : 317.7
RT: 55.43303.7
444.5304.7
472.3
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
300 340 380 420 460 500
m/z
O
OH
OH
OH
OH
HO
O
[M + H+]+ : 303.7
RT: 61.07
Quercetin
Spencer et al. 2002
COLONIC BIOTRANSFORMATION
WHAT’S HAPPENING IN THE COLON?
Majority of ingested flavonoids undergo colonic metabolism
G. Gibson, University of Reading UK
Rutin
OHO
OH
OH
O
OH
O-Rutinose
Deglycosylation
Pathway of the colonic degradation of rutin -implications for properties of in vivo metabolites
Ring fission, water elimination,dehydroxylation
HO
COOH
3-(3-hydroxyphenyl)-propionic acid
Dehydoxylation
COOHHO
3-hydroxyphenyl-acetic acid
COOHHO
HO
3,4-dihydroxyphenyl-acetic acid
Absorption from the colon
HO
COOHHO
Protocatechuic acid
HO
CONH
COOH
3-hydroxyhippuric acid
OHO
OH
OH
O
OH
OH
Quercetin
ß-Oxidation
+ glycination
Further degradation
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?
§§ on what we mean by ‘on what we mean by ‘antioxidation’antioxidation’
§§ on the extent and structural on the extent and structural consequences of conjugation consequences of conjugation and metabolismand metabolism
IT DEPENDS:
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
ANTHOCYANIN GLYCOSIDES
100 nM; 147 nMAnthocyanin –berry juices
NARINGENIN /HESPERETIN GLUCURONIDE< 4 uM
Flavanone –grapefruit/orange
EPICATECHINSULPHATE +GLUCURONIDE
4 uM; 0.26 uM; 0.7 uM
Procyanidin:Chocolate/cocoa
METHYL + SULPHATE +GLUCURONIDE100 nM
Flavan-3-ol:Wine catechins
Donovan et al., Keen et al., Baba et al., Ameer et al, Miyazawa et al.
Caspase-3ApoptoticStimulus
Pro-caspase-3p-nitroaniline
acetyl-Asp-Glu-Val-Aspp-nitroanilide
405 nm
0.00
0.05
0.10
0.15
0.20
0.25
H2O2 (50 µM)
EC
Glu
c
MeE
C
EC
Con
trol
******
H2O
2(5
0 µM
)
Incr
ease
in A
bsor
banc
e (4
05 n
m)
over
con
trol c
ells
All 30µM§METHYLATED METABOLITE
Lower H-donating potential –modified catechol group
Similar protective effects against oxidative stress-induced cell death§GLUCURONIDE METABOLITE
Marginally lower H-donating potential
No protective effects against oxidative stress-induced cell death- inaccessibility or substituted A-ring? SPENCER et al. 2001
IN VIVO METABOLITE FORMS VERSUS CELLULAR OXIDATIVE STRESS
PROTECTION OF NEURONS FROM OXIDATIVE STRESS-INDUCED CELL DEATH BY EPICATECHIN
III
IVIII
I Control neurons II Neurons exposed to oxidative stressIII Control neurons treated with epicatechinIV 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