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. Halliwell, B. American Journal of Medicine. 1991, 91(3), 14.

2
2. Burda S. and Wieslaw O. J. Agric. Food Chem. 2001, 49, 2774-2779.
 Reactive Oxygen Species (ROS)
O2
dioxygen
• ROS are a minor product
e-
of the oxidative respiratory
.
O2 - H
+
HO2
. chain (~1-2%), mostly in
superoxide anion
e-
the form of superoxide.
O2 2- H+ -
H+ • Excess production of ROS
HO2 H2 O 2
e-
peroxide hydroperoxide hydrogen peroxide
may result from iron
overload and inflammation
3H+
e-
2-
[O + O ]
.-
H2O + HO
.
hydroxyl
or immune responses.
water
radical

2H+ H+
2O2- 2OH- 2H2O
oxide hydroxide 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 R

O2
H2 O + 1. Initiation
• Lipid peroxidation
R R .
H
. Hydrogen Abstraction
R
OO
. • DNA scission/cross-
OH
linking
R R R R
• Protein disruption and
+ + 2. Propagation disintegration
R R R R . – Above damage has been
.
OO OOH correlated to Alzheimer’s
R and Parkinson’s disease,
R
3. Termination
cancer, arthritis, diabetes,
. R Lupus and many other age
+
R
related degenerative
R
R . R diseases4.
Lipid crosslinkage
R

4. Pieta P. J. Nat. Prod. 2000, 63, 1035-1042. 4

 Natural ROS Defenses

.- + SOD
2O2 + 2H 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- E°’ = 0.30 V, S.H.E., pH 7.0
• Fe(II)  Fe(III) + e- 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
II . -
Fe ATP+ H2O2 III
Fe ATP + 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

OH

HO O HO O
3' 3'
2' 4'
Base Structure 2' 4' Flavone
1 B 8 1 B OH OH
8 1'
O 1'
5' O 5' Quercetin
7 OH O OH O Taxifolin
7
2 6' 2 6'
A C A C OH
6 6 3
3
OH OH
5 4 5

O
HO O HO O
OH

3' 3'
2' 4'
Flavanonol
2' 4' Flavonol OH OH

1 B 8 1 B OH O
Kaempferol
OH O
Myricetin
8 1'
O 1'
5' O 5'
7 7
2 6' 2 6'
A C A C
6 6
OH OH
5 4 5 HO O HO O
O

HO
3' Baicalein Chrysin
2' 4' OH O OH O
Flavanone Isoflavone
8 1 B 8 1
1' OH
O 5' O
7 7
2 6' 2
A C A C 3'
6 6 1'
3 4'
HO O HO O
5 5
B OH

O O 2' 5'

6' OH OH
Morin Galangin
OH O OH O

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
Ca, Naringenin
2.5

2 • HP 8453 UV-vis diode

1.5 1:3 (M:L) array. 25 M Metal, 25-
Absorbance (AU)

1
1:1 (dashed), 0:1 (solid)
75 M flavonoid,
0.5
unbuffered and at pH 7.4
0
with 10 mM HEPES,
200 250 300 350 400 450 Wavelength (nm)
60/40 vol%
3.5
water/dioxane.
3 FeII, Quercetin • Flavonoid-metal
2.5
1:3 interaction is easily
2
observed via shifts in the
Absorbance (AU)

1.5 1:1
1 0:1 visible spectrum.
0.5

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 Non-chelators Structure Activity
Relationship suggests
OH

HO O
OH
that the 4-keto, 3-
OH
hydroxy moiety is
O
Fisetin HO O
important for chelation.
OH

OH
OH O
Chrysin This is in agreement
HO O with numerous other
OH studies indicating the
importance of the 3-
OH

OH O
Quercetin
HO O
hydroxy group.8

HO O OH O
Naringenin Catechol moiety cannot
be discounted without
OH
testing a flavonoid that
Galangin
OH O
lacks the 3-hydroxy
group.

8. A. Arora et. al. Free Radical Biology and Medicine. 1998, 24(9)1355-1363. 15
 O
Voltammetry
N
O N
O
Fe Gamry PC4 Potentiostat
O O O
O with CMS100 framework
O
and CMS130 voltammetry
III software
Fe EDTA

FeIIIEDTA + e- FeIIEDTA
1
Conditions:
-0.20 mM Fe(NO3)3

current ( A)
0.5 -0.10 M NaNO3
-20 mM HEPES pH 7.4
0
-25 mV/s, carbon disk
-0.5
-Ag/AgCl reference
III
Fe EDTA + e - II
Fe EDTA -Pt wire counter
-1 electrode
0.6 0.3 0 -0.3 -0.6
16
potential (V)
 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 a
commonly used model for an amine and
carboxylate containing metal chelate.
• And it’s cheap too!

17
 Fe(III)EDTA
100
HO-FeEDTA
80 FeEDTA
%formationrelativetoFe

60
(HO)2-FeEDTA
40
FeHEDTA
20

0
0 4
pH
8 12
-0.1 mM FeII/III
100
Fe(II)ED
TA -0.1 mM EDTA
FeEDTA
80
Fe
%formationrelativetoFe

60
(HO)2-FeEDTA
40 HO-FeEDTA

20 FeHEDTA

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
0.20 mM Fe(III)EDTA (1:1) unbuffered

10

current ( A)
pH = 4.1 4
pH = 6.1
pH = 7.2
pH = 7.7
2
pH = 8.2
pH = 9.1
pH = 9.6
pH = 10.1
0

-2
0.3 -0.2 -0.7 -1.2
potential (V)
19
 Nernst Equation
[FeIIEDTA][OH-]
E = E0 - 0.059 x log
[FeIIIEDTA-OH]

[FeIIEDTA]
E = 0.059(log[OH-]) + E0 - log
[FeIIIEDTA-OH]

20
 FeEDTA E1/2 pH Dependence

0
-0.05
E1/2 (V vs Ag/AgCl)

FeEDTA
-0.1
-0.15 FeEDTA-
OH/FeEDTA-(OH)2
-0.2
Linear (FeEDTA-
-0.25 OH/FeEDTA-(OH)2)

-0.3 y = -0.0849x + 0.5574

R2 = 0.986
-0.35
0 5 10 15
pH

21
 6
. -
FeIIEDTA + H2O2 FeIIIEDTA + HO + HO
5

current ( A, relative)
Conditions:
3 -0.20 mM FeEDTA
-0.10 M NaNO3
FeIIIEDTA + e- FeIIEDTA 2 -20 mM HEPES, 7.4
-9.5 mM H2O2
1
-25 mV/s, C disk
-Ag/AgCl reference
0
-Pt wire counter
.
H2O2 + e- HO + HO
-
electrode
-1
0.5 0.3 0.1 -0.1 -0.3 -0.5
voltage (V)

The electrocatalytic current (EC’) is highly dependant on pH,

[H2O2] and [EDTA].
22
 140

120
1:1:540
100

Conditions:

current ( A)
80
1:1:140
60
-0.10 mM Fe(NO3)3
40

20
-0.10 mM EDTA
1:1:40
-1.0-54 mM H2O2
1:1:10 0

-20 -0.10 M NaNO3

0.8 0.4 0 -0.4 -0.8 -1.2

potential (V) -20 mM HEPES pH 7.4

1:1:10
EC' Current Dependence on relative [H2O2] -25 mV/s, carbon disk
140
-Ag/AgCl reference
120 -Pt counter electrode
100
current ( A)

80
60
-ratios are labeled
40 according to
20
0
Fe:EDTA:H2O2
0 100 200 300 400 500 600
23
[H2O2] excess, relative to FeEDTA
 FeIIIEDTA, H2O2 Speciation
100
pH 7.4

80
FeEDTA Conditions:
%formationrelativetoFe

60 -0.10 mM FeEDTA (1:1)

HOO-FeEDTA
40
-4.0 mM H2O2 (top), 14 mM
H2O2 (bottom).
20 HO-FeEDTA

0
4 6 8 10
pH
pH 7.4
100
HOO-FeEDTA
80 FeEDTA
%formationrelativetoFe

60

40

20
HO-FeEDTA
0
4 6 8 10 24
pH
 20

1:10:540
15

1:10:140

A)
10

Conditions:

current (
1:10:40
5

1:10:10 -0.10 mM Fe(NO3)3

0
-1.0 mM Na2EDTA
0.8 0.4 0 -0.4 -0.8 -1.2
-5 -0.10 M NaNO3
potential (V) -1.0-54 mM H2O2
EC' Current Dependence on relative [H 2O2] -20 mM HEPES pH 7.4
18 -25 mV/s, carbon disk
16
14
-Ag/AgCl reference
-Pt counter electrode
current ( A)

12
10
8
6
4 -ratios are labeled
2
0
according to
0 100 200 300 400 500 600 Fe:EDTA:H2O2 25
[H2O2] excess, relative to 1:10 FeEDTA
 8

1:1:40 7 Conditions:
1:10:40
6 -0.10 mM Fe(NO3)3
5
-0.10/1.0 mM EDTA

current ( A)
4
1:1:10 3
-1.0/4.0 mM H2O2
1:10:10 2 -0.10 M NaNO3
1
-20 mM HEPES pH 7.4
0

-1
-25 mV/s, carbon disk
-2 -Ag/AgCl reference
0.8 0.4 0 -0.4 -0.8 -1.2
-Pt counter electrode
potential (V)

-ratios are labeled

Another way of looking at the data is according to
that at relatively low excesses of Fe:EDTA:H2O2
H2O2, the EC’ current is nearly
independent of the Fe:EDTA ratio. 26
 140
Conditions:
1:1:540 -0.10 mM Fe(NO3)3
120
-0.10/1.0 mM EDTA
100
-1.0-54 mM H2O2
1:1:140 80 -0.10 M NaNO3

current ( A)
60 -20 mM HEPES pH 7.4
40 -25 mV/s, carbon disk
1:10:540 20
-Ag/AgCl reference
-Pt counter electrode
1:10:140 0

0.8 0.4 0 -0.4 -0.8 -1.2

-20
-ratios are labeled according
potential (V)
to Fe:EDTA:H2O2

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

6 0.20 mM
Fe(III)EDTA (1:1)
5

current ( A, relative)
4 0.20 mM quercetin

sum composite
2 0.20 mM
Fe(III)EDTA-
1 quercetin (1:1:1)

experimental 0.20
0 mM Fe(III)EDTA-
quercetin (1:1:1)
-1
0.8 0.4 0 -0.4 -0.8
potential (V, absolute)

28
 III
Fe EDTA/Q H2O2 Catalytic

30

"blank"-9.5 mM H2O2, 95
mM NaNO3, 20 mM 25
HEPES pH 7.2
blank + 0.19 mM 20
Fe(III)EDTA (1:1)

current ( A)
15
blank + 0.19 mM
Fe(III)EDTA-quercetin
(1:1:1) 10

-5
0.8 0.4 0 -0.4 -0.8
potential (V)
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 Chi-square 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 • 0.50-2.0 mM Flavonoid

280 auto titrator
• Fisher Isotemp 1016D water
bath
• Accumet Model 20 pH Meter
• Denver Instruments semi-
micro glass pH Ag/AgCl
reference combination
electrode.
• 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 OH

90 11
HO
80
10
70
OH
%formation relative to H

60
9 Fisetin
O
50

pH
40 8
pka
30

20
7
11.906
10
6 11.773
0
residuals in pH for selected data. Unweighted rms=2.86e-02
0.2
0.1
9.965
0.0
-0.1 8.405
-0.2
0 10 20 30 40 50 60 70
point number

sigma 1.54
chi2 11.9 33
 Speciation and pH: data from C:\My Documents\chrysin 092402.ppd
100
HO
90
10
80
9
70
% formation relative to Chry

Chrysin
60 8 OH O

50 7

pH
40
6
30
5
pka
20

10 4 11.406
0
residuals in pH for selected data. Unweighted rms=3.19e-02
7.983
0.1
0.0
-0.1

0 20 40 60 80 100 120 sigma 1.62

point number

chi2 73

34
 Speciation and pH: data from c:\my documents\mark's\research\data\flavonoid ka's\galangin 121301.ppd
100
11
90 HO

80 10
70 OH
%formation relative to H

60 9 OH O
Galangin
50

pH
40
8 pka
30
7 11.694
20

10 6
10.684
0
residuals in pH for selected data. Unweighted rms=6.32e-03 8.232
0.02

0.0

-0.02 sigma 0.53

0 10 20 30 40 50 60 70
point number
chi2 10.7

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

80 10

70 Naringenin
OH O
9
%formation relative to H

60

50
pka

pH
8
40
11.324
30
7
20 10.034
10 6
0
8.238
residuals in pH for selected data. Unweighted rms=4.01e-02
0.2
0.1
0.0
-0.1 sigma 1.61
-0.2
0 10 20 30 40 50 60 70
point number chi2 7.74
36
 OH

Speciation and pH: data from c:\my documents\mark's\research\data\flavonoid ka's\morin 121401.ppd

100
11 HO
90 OH

80 10
OH

70 Morin
OH O
9
%formation relative to Mor

60

50
8 pka

pH
40 7
11.642
30
6
20 11.851
10 5
10.555
0
residuals in pH for selected data. Unweighted rms=3.79e-02
0.1
8.860
0.0
5.702
-0.1

0 20 40 60 80 100 120 140

point number 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
80
OH
70 8
OH O
Quercetin
%formation relative to H

60
7
50

pH
40 6 pka
30
5 11.948
20

10
4
12.378
0
residuals in pH for selected data. Unweighted rms=9.67e-02 11.211
0.5
9.667
0.0

-0.5
8.331
0 10 20 30 40 50 60 70 80 90
point number
sigma 2.5
chi2 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 11.773

pk4 11.948 11.642 11.906

pk5 12.378 11.851

39
 Flavonoid Potentiometric Titration Curve

11.000

10.000

9.000

8.000
pH

7.000

6.000
Q only
Q:Zn(II) 3:1
5.000
Q:Zn(II) 1:1

4.000 Q:Fe(II) 3:1

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
100

80 FeEDTA Q-FeEDTA Q = quercetin

Fe = ferric FeIII
%formationrelativetoFe

60

40
[FeEDTA-H2Q]
20
HO2-FeEDTA
k= =1013
HO-FeEDTA [FeEDTA][H2Q]
0
4 6 8 10
pH
pH 7.4
100 [FeEDTA-H2Q]
FeEDTA k= =1010
80 [FeEDTA][H2Q]
%formationrelativetoFe

60 HO2-FeEDTA

40
Assuming 0.1 mM
FeIIIEDTA, 14 mM H2O2,
20 Q-FeEDTA
HO-FeEDTA
and 0.1 mM quercetin
0
4 6 8 10 42
pH
 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 FeEDTA-
peroxy 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
...and for moral support:
Rob Bobier
Kevin Breen The Engelmanns
Ryan Hutcheson
Chemistry department
Financial:
National Institute of Health
Renfrew scholarship
44