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1
Complexation and Reduction/Oxidation
Reactions of Selected Flavonoids withIron and Iron Complexes: Implications
on In-Vitro Antioxidant Activity
O
O
OH
OH
OH
OH
OH
Quercetin
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2
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.
1. Halliwell, B. American Journal of Medicine. 1991, 91(3), 14.
2. Burda S. and Wieslaw O. J. Agric. Food Chem. 2001, 49, 2774-2779.
Despite a vast volume of research on flavonoids as antioxidants,
the mechanism of their action is incomplete2.
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Reactive Oxygen Species (ROS)
ROS are a minor productof 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.
O2
2O2-
O2.-
O22-
[O2- + O.-]
e-
e-
e-
e-
HO2.H
+
H+
3H+
2H+
HO2-
H2O + HO.
2OH-
H2O2
2H2O
H+
H+
dioxygen
oxide
peroxide
superoxide anion
hydroxide water
water hydroxyl
hydrogen peroxidehydroperoxide
radical
3. Kaim w. and Schwederski B. Bioinorganic Chemistry: Inorganic Elements in the
Chemistry of Life. J. Wiley and Sons, 1994, New York.
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ROS Induced Damage
Lipid peroxidation
DNA scission/cross-linking
Protein disruption anddisintegration Above damage has been
correlated to Alzheimersand Parkinsons disease,
cancer, arthritis, diabetes,Lupus and many other agerelated degenerativediseases4.
R
R
.OH
H
H2O +
R
R
.
R
R
O2
.R
R
OO.
R
R
OO.
R
R
+
R
R
OOH
+
R
R
. R
R.+
R
R
R
R
1. Initiation
2. Propagation
3. Termination
Lipid crosslinkage
Hydrogen Abstraction
4. Pieta P. J. Nat. Prod.2000
, 63, 1035-1042.
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Natural ROS Defenses
2 O2
. - 2 H + H 2 O 2 + O 2S O D
2 H 2 O 2 2 H 2 O + O 2
c t l s
2GSH+ R-OOH
gl t t ir i s
GSSG+ R-OH+H2O
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Hydroxyl Radical and The Fenton Reaction
H2O2 + e- p HO + HO- E = 0.30 V, S.H.E., pH 7.0
Fe(II)p Fe(III) + e- E = depends on complex
Fe(II) + H2O2 p Fe(III) + HO + HO-
The impact of Ferrous salts on H2O2 reduction wasdiscovered over 100 years ago.5
The Fenton reaction in form above, including the hydroxylradical, was suggested over75 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.
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Peroxy-FeEDTA and the Fenton
Reaction
[F IIIEDT -O2H]2-+-
[F IIEDT -O2H] -
[F IIEDT -O2H]-+H+ [F IIIEDT ]-+HO
.+ HO
-
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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.
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Fenton Metal Deactivation
FeIIATP+ H2O2 FeIIIATP + HO
.+ HO
-
ATP
FeIIL + H2O2
(antioxidant)+L
+L
(pro-oxidant ligand
[FeII(ATP)L] + H2O
2
displacement) No Reaction
No Reaction
7. F. Cheng and K. Breen.B
iometals.2000
, 13, 77-83.
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.
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Flavonoid Structure
O
A
B
C
1
2
3
45
6
7
8 1'
2'
3'
4'
5'
6'
O
OH
A
B
C
1
2
5
6
7
8 1'
2'
3'
4'
5'
6'
O
O
A
B
C
1
2
3
5
6
7
8 1'
2'
3'
4'
5'
6'
O
O
A
B
C
1
2
5
6
7
8 1'
2'
3'
4'
5'
6'
O
O
OH
A
B
C
1
2
5
6
7
8 1'
2'
3'
4'
5'
6'
4
3
Bas Str ct r
Flavanonol
Flavone
Flavanone
Flavonol
A
B
C
1
2
5
6
7
8
1'
2'
3'
4'
5'
6'
O
O
Isoflavone
O
O
OH
OH
OH
OH
OH
O
O
OH
OH
OH
OH
OH
O
O
OH
OH
OH
OH
O
O
OH
OH
OH
OH
OH
OH
Quercetin Taxifolin
Kaempferol M
ricetin
O
O
OH
OH
OH
O
O
OH
OH
O
O
OH
OH
OH
OH
OH
O
O
OH
OH
OH
Baicalein Chr
sin
Morin alan
in
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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.
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Experimental Design
Observe Metal-Flavonoid binding interactions via shiftsin the visible spectrum of the flavonoid when in thepresence of the metal.
Investigate the electrochemical behavior of the
FeEDTA, and peroxy-FeEDTA complexes for thepurpose of assaying flavonoid antioxidant activity andelucidating 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 activityrelationships and mechanism elucidation.
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UV-visible Spectrophotometry
HP 8453 UV-vis diodearray. 25 QM Metal, 25-75 QM flavonoid,unbuffered and at pH 7.4
with 10 mM HEPES,60/40 vol%water/dioxane.
Flavonoid-metalinteraction is easilyobserved via shifts in thevisible spectrum.
Wavelen th (nm)200 250 300 350 400 450 500 550
Absorbance(A
U)
0
0.5
1
1.5
2
2.5
3
3.5
Wavelen
th (nm)200 250 300 350 400 450
Absorbance(AU)
0
0.5
1
1.5
2
2.5
FeII, Quercetin
Ca, Naringenin
1:3 (M:L)
1:10:1
1:3
1:1 (dashed), 0:1 (solid)
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FeII FeIII CuII CaII ZnII
Quercetin + + + - +7.4
Galangin + + + - +7.4
Fisetin + + + - +7.4
Chrysin - - - - -
Naringenin - - - - -
Iron is the most abundant physiological transition metal; copperis 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.
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O
O
OH
OH
OH
OH
OH
QuercetinO
O
OH
OH
OH
Naringenin
O
O
OH
OH
Chrysin
O
O
OH
OH
OH
OH
Fisetin
O
O
OH
OH
OH Galangin
Chelators Non-chelators Structure ActivityRelationship suggests
that the 4-keto, 3-hydroxy moiety is
important for chelation.
This is in agreement
with numerous other
studies indicating theimportance of the 3-
hydroxy group.8
Catechol moiety cannot
be discounted withouttesting a flavonoid that
lacks the 3-hydroxy
group.
8. A. Arora et. al. Free RadicalBiology and Medicine. 1998, 24(9)1355-1363.
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Voltammetry
FeII IEDTA
-1
-0.5
0
0.5
1
-0.6-0.300.30.6
potential (V)
curre
nt(A)
FeIII DTA + e- FeII DTA
FeIII DTA + e- FeII DTA
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
Gamry PC4 Potentiostat
with CMS100 framework
and CMS130 voltammetry
software
Fe
N
O
N
OO
O
O
O
O
O
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Why EDTA? Its involvement in the Fenton reaction is
well studied, and its binding constants,
including very hard-to-find peroxy-mixed-ligand species, are readily available.
Although not physiologically present, it is acommonly used model for an amine andcarboxylate containing metal chelate.
And its cheap too!
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Fe(III)EDTA
0 4 8 12
pH
0
20
40
60
80
100
%f
ormation
relativetoFe
FeHEDTA
FeEDTAHO-FeEDTA
(HO)2-FeEDTA
Hyperquad Speciation and Simulation software (HySS) by Peter Gans
Formation Constants obtained from Robert M. Smith and Arthur E. Martell
-0.1 mM FeII/III
-0.1 mM EDTAFe(II)EDTA
0 4 8 12
pH
0
20
40
60
80
100
%formationrelativetoFe
Fe
FeHEDTA
FeEDTA
HO-FeEDTA
(HO)2-FeEDTA
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0.20 mM Fe(III)EDTA (1:1) unbuffered
-2
0
2
4
6
8
10
-1.2-0.7-0.20.3
potential (V)
current(
A)
pH = 4.1
pH = 6.1
pH = 7.2
pH = 7.7
pH = 8.2
pH = 9.1
pH = 9.6pH = 10.1
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Nernst Equation
E=E0 - 0.059 x log[FeIIEDTA][OH-]
[F
e
IIIE
DTA-OH]
E=0.059(log[OH-]) + E0 - log[FeIIEDTA]
[FeIIIEDTA-OH]
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FeED E1/2 pH Dependence
y = 0.0849 0.55742 = 0.9860.35
-0.3
-0.25
-0.2
-0.15
-0.1
-0.05
0
0 5 10 15
pH
E1/2
(VvsAg/AgCl)
FeEDTA
FeEDTA-
OH/FeEDTA-(OH)2
L nea (FeEDTA-
OH/FeEDTA-(OH)2)
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-
5
- .5- .3- .0.0.30.5
volt ge (V)
u
ent(
,relatve)
H2O
2+ e- HO
.+ HO
-
FeIIIEDTA + e- FeIIEDTA
FeIIIEDTA + HO.+ HO
-FeIIEDTA + H
2O
2
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].
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-
40
60
80
100
120
140
-1.
-
.8-
.400.40.8
pote tial
c
rre
t
1:1:540
1:1:140
1:1:40
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
EC' Current Dependence onrelative [H2O2]
0
20
40
60
80100
120
140
0 100 200 300 400 500 600
[H2O2]excess relativeto FeEDTA
current(A
)
1:1:10
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4 6 8 10pH
0
20
40
60
80
100
%
formationrelativeto
Fe
HOO-FeEDTA
FeEDTA
HO-FeEDTA
pH7.4
Conditions:
-0.10 mM FeEDTA (1:1)
-4.0 mM H2O2(top), 14 mM
H2O2 (bottom).
4 6 8 10
pH
0
20
40
60
80
100
%
formationrelativetoFe HOO-FeEDTA
FeEDTA
HO-FeEDTA
pH 7.4
FeIIIEDTA, H2O2 Speciation
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-5
0
5
10
15
20
-1.2-0.8-0.400.40.8
potential (V)
current(
A)
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
EC Current De enden e nre at ve [H2O2]
0
2
4
6
8
10
12
14
16
18
0 100 200 300 400 500 600
[H2O2] excess, re at ve t 1:10 FeED
current(A
)
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-2
-1
0
1
2
3
4
5
6
7
8
-1.2-0.8-0.400.40.8
pote tial ( )
c
e
t(
A)
1:1:40
1:10:40
1:1:10
1:10:10
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 labeledaccording to
Fe:EDTA:H2O2
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.
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-
1
1
1
-1.- .- ...
potent al V)
urrent
Q
)
1:1:540
1:1:140
1:10:140
1:10:540
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
At a relatively high excess of H2O2, the EC current exhibits adrastic 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.
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FeEDT , Q erceti mp site
-1
0
1
2
3
4
5
6
-0.8-0.400.40.8
p tential V, a s l te)
current
Q
A,
relative)
0.20 mMFe(III)E A(1 1)
0.20 mM quercetin
sum composite
0.20 mM
Fe(III)E A-
quercetin(1 1 1)
e perimental 0.20
mM Fe(III)E
A-quercetin(1 1 1)
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FeIIIEDT /Q H2O2 Catalytic
-5
0
5
10
15
20
25
30
-0.8-0.400.40.8
potential (V)
current(
)
"blank"-9.5 mM H2O2, 95
mM NaNO3, 20 mM
HEPES pH 7.2
blank + 0.19 mM
Fe(III)EDTA (1:1)
blank + 0.19 mM
Fe(III)EDTA-quercetin
(1:1:1)
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Quercetin shifts the formal
reduction potential, but what
about the speciation of theperoxy-FeEDTA complex?
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Formation Constant Refinement
Collect the experimental titration curve.
Simulate a titration curve using the sameexperimental concentrations and estimatedformation constants.
Use non-linear least squares regression analysis tominimize the difference between the experimentaldata (pHexp) and the simulated curve (pHcalc).
When the curves match, the formation constantshave been determined.
The curve fitting process provides a statisticalevaluation of the data through sigma and Chi-square values.
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Potentiometric Titrations
Denver InstrumentsTitrator280 auto titrator
Fisher Isotemp 1016Dwater bath
Accumet Model 20 pH
Meter Denver Instruments semi-
micro glass pH Ag/AgClreference combinationelectrode.
0.50-2.0 mM Flavonoid 0.10 M NaNO3 ionic
strength
0.05 M NaNO3 titrant(standardized daily)
CO2 scrubbed water, N2purged headspace
60/40 vol% H2O/dioxane
An ion selective electrode is used to monitor the concentration of aspecies as a titrate involved in competitive binding with anotherspecies which is added as a titrant.
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resi uals inpHfor selecteddata.Unwei hted rms 2.86e-02
0 10 20 30 40 50 60 70point number
-0.2-0.1
0.0
0.1
0.2
SpeciationandpH:datafrom c:\mydocuments\research\data\flavonoid ka's\fisetin121101.ppd
0
10
20
30
40
50
60
70
80
90
100
%f
ormationrelativetoH
pH
6
7
8
9
10
11
O
OH
OH
OH
OH
Fisetin
pka
11.906
11.773
9.965
8.405
sigma 1.54
chi2 11.9
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residuals in pH for selected data. Unweighted rms=3.19e-02
0 20 40 60 80 100 120point number
-0.1
0.0
0.1
Speciation and pH: data from C:\My Documents\chrysin 092402.ppd
0
10
20
30
40
50
60
70
80
90
100
%
formationrelativetoChry
pH
4
5
6
7
8
9
10
O
OH
OH
Chrysin
pka
11.406
7.983
sigma 1.62
chi2 73
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residuals inpHfor selecteddata.Unwei hted rms 6.32e-03
0 10 20 30 40 50 60 70point number
-0.02
0.0
0.02
peciationandpH:datafrom c:\mydocuments\mark's\research\data\flavonoid ka's\ alan in121301.ppd
0
10
20
30
40
50
60
70
80
90
100
%f
ormationrelativetoH
pH
6
7
8
9
10
11
O
OH
OH
OH
Galan in
pka
11.694
10.684
8.232
sigma 0.53
chi2 10.7
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residuals in pH for selected data. Unweighted rms=4.01e-02
0 10 20 30 40 50 60 70point number
-0.2
-0.1
0.0
0.1
0.2
ciation and pH: data from c:\my documents\mark's\research\data\flavonoid ka's\kaempferol 121201.ppd
0
10
20
30
40
50
60
70
80
90
100
%f
ormationrelativetoH
pH
6
7
8
9
10
11O
O
OH
OH
OH Naringenin
sigma 1.61
chi2 7.74
pka
11.324
10.034
8.238
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residuals inpHfor selecteddata.Unwei hted rms 3.79e-02
0 20 40 60 80 100 120 140point number
-0.1
0.0
0.1
ciationandpH:datafrom c:\mydocuments\mark's\research\data\flavonoid ka's\morin121401.ppd
0
10
20
30
40
50
60
70
80
90
100
%f
ormationrelativetoMor
pH
5
6
7
8
9
10
11
O
OH
OH
OH
OH
OH
Morin
sigma 3.7
chi2 21.8
pka
11.642
11.851
10.555
8.860
5.702
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residuals inpHfor selecteddata.Unwei hted rms 9.67e-02
0 10 20 30 40 50 60 70 80 90point number
-0.5
0.0
0.5
0
10
20
30
40
50
60
70
80
90
100
%fo
rmationrelativetoH
pH
4
5
6
7
8
9
10
O
OH
OH
OH
OH
OH
Quercetin
sigma 2.5
chi2 4.9
pka11.948
12.378
11.211
9.667
8.331
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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 11.773
pk4 11.948 11.642 11.906
pk5 12.378 11.851
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Flav noid Potentio etric itration Curve
3.
4.
.
6.
7.
8.
9.
10.
11.
. . .40 0.60 0.80 1.
Na H added l . 5 )
pH
Q on
Q:Zn( )3:1
Q:Zn( )1:1
Q:Fe( )3:1
Q:Ca( )1:1
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Work in Progress Complete spectroscopic studies in order
reveal SAR.
Extend the EC assay to other flavonoids.
Obtain FeEDTA-flavonoid mixed ligand
binding constants.
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4 6 8 10
pH
0
20
40
60
80
100
%f
ormationre
lativetoFe
4 6 8 10
pH
0
20
40
60
80
100
%f
ormationrelativetoFe
FeEDTA
HO2-FeEDTA
Q-FeEDTA
HO-FeEDTA
Q-FeEDTA
HO2-F
eEDTA
HO-FeEDTA
FeEDTA
pH 7.4
pH 7.4
[FeEDTA][H2Q]
[FeEDTA-H2Q]
k = =1010
[FeEDTA][H2Q]
[FeEDTA-H2Q]
k = =1013
Assuming 0.1 mM
FeIIIEDTA, 14 mM H2O2,
and 0.1 mM quercetin
Q = quercetin
Fe = ferric FeIII
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Summary The mechanism of Flavonoid antioxidant activity
by metal chelation is most likely two-fold:
Flavonoids that posses large enough affinity constantsfor the mixed FeEDTA-flavonoid complex formation
disfavor the speciation of the highly reactive FeEDTA-
peroxy complex.
The newly formed FeEDTA-flavonoid complex shifts
the metal based electrochemistry beyond the range for
Fenton redox cycling.
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Acknowledgements:
Cheng Group
Tom Brandt
Jessica Poindexter
Terry HyattRob Bobier
Kevin Breen
Ryan Hutcheson
Chemistry department
National Institute of Health
Coworkers:
Financial:
Renfrew scholarship
...and for moral support:
The Engelmanns