Complexation and Reduction/Oxidation
Reactions of Selected Flavonoids with
Iron and Iron Complexes: Implications
on In-Vitro Antioxidant Activity
OH
OH
HO
O
OH
OH
O
Quercetin
1
A quote by Dr. Barry Halliwell from
the American Journal of Medicine1:
“It is difficult these days to open a medical journal and not
find some paper on the role of ‘reactive oxygen species’ or
‘free radicals’ in human disease.”
“These species have been implicated in over 50 diseases.
This large number suggests that radicals are not something
esoteric, but that they participate as a fundamental component
of tissue injury in most, if not all, human disease.”
Despite a vast volume of research on flavonoids as antioxidants,
the mechanism of their action is incomplete2.
1.
2.
Halliwell, B. American Journal of Medicine. 1991, 91(3), 14.
Burda S. and Wieslaw O. J. Agric. Food Chem. 2001, 49, 2774-2779.
2
Reactive Oxygen Species (ROS)
O2
e
-
dioxygen
+
.
O2 - H
e-
superoxide anion
O2
e-
H+
2-
.-
[O + O ]
.
-
3H+
.
hydroxyl
radical
H+
2H+
oxide
H2O2
hydrogen peroxide
H2O + HO
water
2O2-
H+
HO2
hydroperoxide
peroxide
2-
e-
HO2
• ROS are a minor product
of the oxidative respiratory
chain (~1-2%), mostly in
the form of superoxide.
• Excess production of ROS
may result from iron
overload and inflammation
or immune responses.
2OH-
hydroxide
2H2O
water
3. Kaim w. and Schwederski B. “Bioinorganic Chemistry: Inorganic Elements in
the Chemistry of Life.” J. Wiley and Sons, 1994, New York.
3
ROS Induced Damage
R
R
O2
H2O +
R
H
.
R
R
.
Hydrogen Abstraction
1. Initiation
R
OO
OH
R
R
R
+
R
OO
R
2. Propagation
+
R
.
R
.
R
.
OOH
R
R
3. Termination
+
R
.
.
R
R
R
R
• Lipid peroxidation
• DNA scission/crosslinking
• Protein disruption and
disintegration
– Above damage has been
correlated to Alzheimer’s
and Parkinson’s disease,
cancer, arthritis, diabetes,
Lupus and many other age
related degenerative
diseases4.
Lipid crosslinkage
R
4. Pieta P. J. Nat. Prod. 2000, 63, 1035-1042.
4
Natural ROS Defenses
2O2
.-
+ 2H
+
SOD
H2O2 + O2
catalase
2H2O2
2H2O + O2
glutathione
peroxidase
2GSH + R-OOH
GSSG + R-OH + H2O
5
Hydroxyl Radical and The Fenton Reaction
• H2O2 + e-  HO• + HO• Fe(II)  Fe(III) + e-
E°’ = 0.30 V, S.H.E., pH 7.0
E°’ = depends on complex
• Fe(II) + H2O2  Fe(III) + HO• + HO– The impact of Ferrous salts on H2O2 reduction was
discovered over 100 years ago.5
– The Fenton reaction in form above, including the hydroxyl
radical, was suggested over 75 years ago.6
5. H.J.H. Fenton. J. Chem. Soc. 1894, 65, 889.
6. F. Haber and J.J. Weiss. Proc. Roy. Soc. London, Ser. A. 1934, 147, 332.
6
Peroxy-FeEDTA and the Fenton
Reaction
-
[FeIIIEDTA-O2H]2- + e
[FeIIEDTA-O2H]3-
[FeIIEDTA-O2H]3- + H+
[FeIIIEDTA]- + HO + HO
.
-
7
Antioxidant Activity
–
–
–
–
Enhance or mimic antioxidant enzymes.
Direct scavenging of ROS.
Repair damaged cellular components.
Pro-oxidant metal deactivation.
* The activity of a potential antioxidant is limited by the
thermodynamic constants for its reactions involving
complexation and reduction/oxidation.
8
Fenton Metal Deactivation
[FeII(ATP)L] + H2O2
No Reaction
+L
FeIIATP+ H2O2
.
FeIIIATP + HO + HO
-
+L (antioxidant)
ATP
(pro-oxidant ligand
displacement)
FeIIL + H2O2
No Reaction
Quercetin deactivates the Fe-ATP complex7, although the
precise mechanism is still unclear. The use of a strong
chelate, like EDTA, should provide additional insight.
7. F. Cheng and K. Breen. Biometals. 2000, 13, 77-83.
9
Flavonoid Structure
OH
OH
OH
HO
B
1'
O
7
A
A
OH
O
OH
Quercetin
OH
Taxifolin
O
6'
C
OH
3
OH
5
4
5
OH
5'
2
6
3
B
1'
O
7
6'
C
6
1
8
5'
2
O
4'
2'
Flavone
4'
2'
1
8
HO
3'
3'
Base Structure
O
OH
O
HO
O
OH
HO
O
OH
3'
3'
Flavanonol
8
1
1'
O
7
2
A
B
8
5'
C
1
1'
O
7
6'
2
A
4'
2'
Flavonol
4'
2'
OH
B
OH
OH
Kaempferol
O
OH
Myricetin
O
5'
6'
C
6
6
5
OH
4
OH
5
HO
O
HO
O
O
HO
Baicalein
3'
Flavanone
8
1
1'
O
7
2
A
C
6
4'
2'
3
OH
Isoflavone
B
8
5'
6'
1
O
O
OH
2
A
C
6
3'
1'
5
5
OH
O
7
Chrysin
O
4'
HO
O
HO
O
O
OH
B
5'
2'
OH
6'
OH
O
Morin
OH
OH
O
Galangin
10
Flavonoid Facts
• Flavonoids are found in higher vascular plants, particularly
in the flower, leaves and bark. They are especially
abundant in fruits, grains and nuts, particularly in the skins.
• Beverages consisting of plant extracts (beer, tea, wine, fruit
juice) are the principle source of dietary flavonoid intake.
A glass of red wine has ~200 mg of flavonoids.
• Typical flavonoid intake ranges from 50 to 800 mg/day,
which is roughly 5, 50 and 100 times that of Vitamins C,
and E, and carotenoids respectively.
4. P. Pieta.
11
Experimental Design
• Observe Metal-Flavonoid binding interactions via shifts
in the visible spectrum of the flavonoid when in the
presence of the metal.
• Investigate the electrochemical behavior of the
FeEDTA, and peroxy-FeEDTA complexes for the
purpose of assaying flavonoid antioxidant activity and
elucidating flavonoid antioxidant mechanisms.
• Measure the proton, metal and mixed-ligand binding
constants for the flavonoids using potentiometry.
• Correlate constants and observations to published
antioxidant efficiency data for structure activity
relationships and mechanism elucidation.
12
UV-visible Spectrophotometry
2.5
Ca, Naringenin
Absorbance (AU)
2
1:3 (M:L)
1.5
1
1:1 (dashed), 0:1 (solid)
0.5
0
200
250
300
350
400
450
Wavelength (nm)
3.5
FeII, Quercetin
Absorbance (AU)
3
2.5
1:3
2
1:1
1.5
0:1
1
• HP 8453 UV-vis diode
array. 25 mM Metal, 2575 mM flavonoid,
unbuffered and at pH 7.4
with 10 mM HEPES,
60/40 vol%
water/dioxane.
• Flavonoid-metal
interaction is easily
observed via shifts in the
visible spectrum.
0.5
0
200
250
300
350
400
450
500
550 Wavelength (nm)
13
FeII
FeIII CuII CaII ZnII
Quercetin
+
+
+
-
+ 7.4
Galangin
+
+
+
-
+ 7.4
Fisetin
+
+
+
-
+ 7.4
Chrysin
-
-
-
-
-
Naringenin -
-
-
-
-
Iron is the most abundant physiological transition metal; copper
is second. Ca is the fifth most abundant element (by mass,
behind O, C, H, and N) in the human body at ~ 1 kilogram
present. Both Ca and Zn are commonly implicated in pro- and
anti- oxidant processes.
14
Chelators
HO
Structure Activity
Relationship suggests
that the 4-keto, 3hydroxy moiety is
important for chelation.
Non-chelators
OH
O
OH
OH
O
HO
Fisetin
O
OH
OH
Chrysin
OH
HO
O
O
OH
OH
OH
HO
O
Quercetin
OH
OH
O
O
Naringenin
O
OH
HO
Galangin
O
This is in agreement
with numerous other
studies indicating the
importance of the 3hydroxy group.8
Catechol moiety cannot
be discounted without
testing a flavonoid that
lacks the 3-hydroxy
group.
8. A. Arora et. al. Free Radical Biology and Medicine. 1998, 24(9)1355-1363.
15
Voltammetry
O
N
O
N
O
Gamry PC4 Potentiostat
with CMS100 framework
and CMS130 voltammetry
software
Fe
O
O
O
O
O
FeIIIEDTA + e-
FeIIIEDTA + e-
FeIIEDTA
FeIIEDTA
Conditions:
-0.20 mM Fe(NO3)3
-0.10 M NaNO3
-20 mM HEPES pH 7.4
-25 mV/s, carbon disk
-Ag/AgCl reference
-Pt wire counter
electrode
16
Why EDTA?
• Its involvement in the Fenton reaction is
well studied, and its binding constants,
including very hard-to-find peroxy-mixedligand species, are readily available.
• Although not physiologically present, it is a
commonly used model for an amine and
carboxylate containing metal chelate.
• And it’s cheap too!
17
F e ( III)ED TA
% fo mr a toi n re al tvi e to Fe
100
80
60
(HO)2-FeEDTA
40
FeHEDTA
20
0
0
4
8
12
pH
F e ( II)ED TA
100
% fo mr a toi n re al tvi e to Fe
HO-FeEDTA
FeEDTA
FeEDTA
Fe
80
-0.1 mM FeII/III
-0.1 mM EDTA
60
(HO)2-FeEDTA
HO-FeEDTA
40
FeHEDTA
20
0
0
4
8
12
pH
Hyperquad Speciation and Simulation software (HySS) by Peter Gans
Formation Constants obtained from Robert M. Smith and Arthur E. Martell
18
19
Nernst Equation
E = E0 - 0.059 x log
[FeIIEDTA][OH-]
[FeIIIEDTA-OH]
E = 0.059(log[OH-]) + E0 - log
[FeIIEDTA]
[FeIIIEDTA-OH]
20
FeEDTA E1/2 pH Dependence
E1/2 (V vs Ag/AgCl)
0
-0.05
FeEDTA
-0.1
-0.15
FeEDTAOH/FeEDTA-(OH)2
-0.2
Linear (FeEDTAOH/FeEDTA-(OH)2)
-0.25
-0.3
y = -0.0849x + 0.5574
R2 = 0.986
-0.35
0
5
10
15
pH
21
FeIIEDTA + H2O2
FeIIIEDTA + e-
.
-
FeIIIEDTA + HO + HO
FeIIEDTA
H2O2 + e-
.
-
HO + HO
Conditions:
-0.20 mM FeEDTA
-0.10 M NaNO3
-20 mM HEPES, 7.4
-9.5 mM H2O2
-25 mV/s, C disk
-Ag/AgCl reference
-Pt wire counter
electrode
The electrocatalytic current (EC’) is highly dependant on pH,
[H2O2] and [EDTA].
22
1:1:540
1:1:140
1:1:40
1:1:10
1:1:10
Conditions:
-0.10 mM Fe(NO3)3
-0.10 mM EDTA
-1.0-54 mM H2O2
-0.10 M NaNO3
-20 mM HEPES pH 7.4
-25 mV/s, carbon disk
-Ag/AgCl reference
-Pt counter electrode
-ratios are labeled
according to
Fe:EDTA:H2O2
23
FeIIIEDTA, H2O2 Speciation
pH 7.4
% fo m
r a toi n re al tvi e to Fe
100
80
FeEDTA
60
HOO-FeEDTA
40
HO-FeEDTA
20
0
4
6
8
pH
100
% fo m
r a toi n re al tvi e to Fe
Conditions:
-0.10 mM FeEDTA (1:1)
-4.0 mM H2O2 (top), 14 mM
H2O2 (bottom).
pH 7.4
HOO-FeEDTA
FeEDTA
80
10
60
40
20
HO-FeEDTA
0
4
6
8
pH
10
24
1:10:540
1:10:140
1:10:40
1:10:10
Conditions:
-0.10 mM Fe(NO3)3
-1.0 mM Na2EDTA
-0.10 M NaNO3
-1.0-54 mM H2O2
-20 mM HEPES pH 7.4
-25 mV/s, carbon disk
-Ag/AgCl reference
-Pt counter electrode
-ratios are labeled
according to
Fe:EDTA:H2O2
25
1:1:40
1:10:40
1:1:10
1:10:10
Another way of looking at the data is
that at relatively low excesses of
H2O2, the EC’ current is nearly
independent of the Fe:EDTA ratio.
Conditions:
-0.10 mM Fe(NO3)3
-0.10/1.0 mM EDTA
-1.0/4.0 mM H2O2
-0.10 M NaNO3
-20 mM HEPES pH 7.4
-25 mV/s, carbon disk
-Ag/AgCl reference
-Pt counter electrode
-ratios are labeled
according to
Fe:EDTA:H2O2
26
140
1:1:540
120
1:1:140
80
60
40
0.8
0.4
0
-0.4
1:10:540
20
1:10:140
0
-0.8
-20
-1.2
current ( m A)
100
Conditions:
-0.10 mM Fe(NO3)3
-0.10/1.0 mM EDTA
-1.0-54 mM H2O2
-0.10 M NaNO3
-20 mM HEPES pH 7.4
-25 mV/s, carbon disk
-Ag/AgCl reference
-Pt counter electrode
-ratios are labeled according
to Fe:EDTA:H2O2
potential (V)
At a relatively high excess of H2O2, the EC’ current exhibits a
drastic dependence on the Fe:EDTA ratio. In contrast to the
EC’ dependence on [H2O2], the effects of the Fe:EDTA ratio
on the EC’ current could not be explained by speciation
calculations. Kinetic factors may be important.
27
FeEDTA, Quercetin Composite
0.20 mM
Fe(III)EDTA (1:1)
6
4
3
2
1
0
0.8
0.4
0
-0.4
current (m A, relative)
5
0.20 mM quercetin
sum composite
0.20 mM
Fe(III)EDTAquercetin (1:1:1)
experimental 0.20
mM Fe(III)EDTAquercetin (1:1:1)
-1
-0.8
potential (V, absolute)
28
29
Quercetin shifts the formal
reduction potential, but what
about the speciation of the
peroxy-FeEDTA complex?
30
Formation Constant Refinement
• Collect the experimental titration curve.
• Simulate a titration curve using the same
experimental concentrations and estimated
formation constants.
• Use non-linear least squares regression analysis to
minimize the difference between the experimental
data (pHexp) and the simulated curve (pHcalc).
• When the curves match, the formation constants
have been determined.
• The curve fitting process provides a statistical
evaluation of the data through sigma and Chisquare values.
31
Potentiometric Titrations
•An ion selective electrode is used to monitor the concentration of a
species as a titrate involved in competitive binding with another
species which is added as a titrant.
• Denver Instruments
Titrator 280 auto titrator
• Fisher Isotemp 1016D
water bath
• Accumet Model 20 pH
Meter
• Denver Instruments semimicro glass pH Ag/AgCl
reference combination
electrode.
• 0.50-2.0 mM Flavonoid
• 0.10 M NaNO3 ionic
strength
• 0.05 M NaNO3 titrant
(standardized daily)
• CO2 scrubbed water, N2
purged headspace
• 60/40 vol% H2O/dioxane
32
OH
Speciation and pH: data from c:\my documents\research\data\flavonoid ka's\fisetin 121101.ppd
100
90
11
HO
80
10
70
OH
60
9
O
pH
% formation relative to H
OH
50
40
8
pka
7
11.906
6
11.773
30
20
10
0
Fisetin
residuals in pH for selected data. Unweighted rms=2.86e-02
0.2
0.1
0.0
-0.1
-0.2
9.965
8.405
0
10
20
30
40
point number
50
60
70
sigma
1.54
chi2
11.9
33
Speciation and pH: data from C:\My Documents\chrysin 092402.ppd
100
HO
10
80
9
70
60
8
50
7
40
Chrysin
OH
O
pH
% formation relative to Chry
90
6
5
pka
4
11.406
30
20
10
0
residuals in pH for selected data. Unweighted rms=3.19e-02
7.983
0.1
0.0
-0.1
0
20
40
60
80
point number
100
120
sigma
1.62
chi2
73
34
Speciation and pH: data from c:\my documents\mark's\research\data\flavonoid ka's\galangin 121301.ppd
100
11
90
10
70
OH
9
60
OH
pH
% formation relative to H
80
HO
50
Galangin
pka
8
40
O
30
11.694
7
20
10
10.684
6
0
8.232
residuals in pH for selected data. Unweighted rms=6.32e-03
0.02
0.0
-0.02
0
10
20
30
40
point number
50
60
sigma
0.53
chi2
10.7
70
35
OH
Speciation and pH: data from c:\my documents\mark's\research\data\flavonoid ka's\kaempferol 121201.ppd
100
11
HO
O
90
10
70
OH
O
Naringenin
9
60
50
8
pka
pH
% formation relative to H
80
40
11.324
30
7
10.034
20
10
6
0
8.238
residuals in pH for selected data. Unweighted rms=4.01e-02
0.2
0.1
0.0
-0.1
-0.2
0
10
20
30
40
point number
50
60
70
sigma
1.61
chi2
7.74
36
OH
80
HO
OH
10
70
OH
OH
9
O
Morin
60
8
50
40
pH
% formation relative to Mor
Speciation and pH: data from c:\my documents\mark's\research\data\flavonoid ka's\morin 121401.ppd
100
11
90
7
pka
11.642
30
6
11.851
20
5
10
0
10.555
residuals in pH for selected data. Unweighted rms=3.79e-02
8.860
0.1
0.0
5.702
-0.1
0
20
40
60
80
point number
100
120
140
sigma
3.7
chi2
21.8
37
OH
OH
Speciation and pH: data from c:\my documents\mark's\research\data\flavonoid ka's\quercetin 022602b.ppd
100
10
HO
90
9
OH
70
8
OH
60
7
50
40
Quercetin
pka
6
30
11.948
5
20
O
pH
% formation relative to H
80
12.378
10
4
0
11.211
residuals in pH for selected data. Unweighted rms=9.67e-02
0.5
9.667
0.0
8.331
-0.5
0
10
20
30
40
50
point number
60
70
80
90
sigma
chi2
2.5
4.9
38
quercetin morin
naringin
galangin
chrysin
Fisetin
pk1
8.331
5.702
8.238
8.232
7.983
8.405
pk2
9.667
8.860
10.034
10.684
11.406
9.965
pk3
11.211
10.555
11.324
11.694
pk4
11.948
11.642
pk5
12.378
11.851
11.773
11.906
39
Flavonoid Potentiometric Titration Curve
11.000
10.000
9.000
pH
8.000
7.000
6.000
Q only
Q:Zn(II) 3:1
5.000
Q:Zn(II) 1:1
Q:Fe(II) 3:1
4.000
Q:Ca(II) 1:1
3.000
0.00
0.20
0.40
0.60
0.80
1.00
NaOH added (m l 0.0501 M)
40
Work in Progress
• Complete spectroscopic studies in order
reveal SAR.
• Extend the EC’ assay to other flavonoids.
• Obtain FeEDTA-flavonoid mixed ligand
binding constants.
41
pH 7.4
% fo m
r a toi n re al tvi e to Fe
100
FeEDTA
80
Q = quercetin
Fe = ferric FeIII
Q-FeEDTA
60
40
20
k=
HO2-FeEDTA
HO-FeEDTA
[FeEDTA-H2Q]
[FeEDTA][H2Q]
=1013
0
4
6
8
10
pH
pH 7.4
% fo m
r a toi n re al tvi e to Fe
100
k=
FeEDTA
80
[FeEDTA-H2Q]
[FeEDTA][H2Q]
=1010
HO2-FeEDTA
60
Assuming 0.1 mM
FeIIIEDTA, 14 mM H2O2,
and 0.1 mM quercetin
40
20
Q-FeEDTA
HO-FeEDTA
0
4
6
8
pH
10
42
Summary
• The mechanism of Flavonoid antioxidant activity
by metal chelation is most likely two-fold:
– Flavonoids that posses large enough affinity constants
for the mixed FeEDTA-flavonoid complex formation
disfavor the speciation of the highly reactive FeEDTAperoxy complex.
– The newly formed FeEDTA-flavonoid complex shifts
the metal based electrochemistry beyond the range for
Fenton redox cycling.
43
Acknowledgements:
Coworkers:
Cheng Group
Tom Brandt
Jessica Poindexter
Terry Hyatt
Rob Bobier
Kevin Breen
Ryan Hutcheson
Chemistry department
...and for moral support:
The Engelmanns
Financial:
National Institute of Health
Renfrew scholarship
44