Complexation and ReductionOxidation Reactions of Selected Flavonoids with Iron and Iron Complexes: I

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Complexation and Reduction/Oxidation Reactions of
Selected Flavonoids with Iron and Iron Complexes
Implications on In-Vitro Antioxidant Activity
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.
Despite a vast volume of research on flavonoids
as antioxidants, the mechanism of their action is
incomplete2.

• Halliwell, B. American Journal of Medicine.
  1991, 91(3), 14.
• Burda S. and Wieslaw O. J. Agric. Food Chem.
  2001, 49, 2774-2779.

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Reactive Oxygen Species (ROS)

• 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.

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 and disintegration
• Above damage has been correlated to Alzheimers
  and Parkinsons disease, cancer, arthritis,
  diabetes, Lupus and many other age related
  degenerative diseases4.

4. Pieta P. J. Nat. Prod. 2000, 63, 1035-1042.
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Natural ROS Defenses
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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.
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Peroxy-FeEDTA and the Fenton Reaction
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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
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.
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Flavonoid Structure
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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
  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.

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UV-visible Spectrophotometry
Ca, Naringenin

• HP 8453 UV-vis diode array. 25 mM Metal, 25-75
  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.

13 (ML)
11 (dashed), 01 (solid)
FeII, Quercetin
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11
01
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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.
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Chelators
Non-chelators
Structure Activity Relationship suggests that the
4-keto, 3-hydroxy moiety is important for
chelation. This is in agreement with numerous
other studies indicating the importance of the
3-hydroxy 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.
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Voltammetry
Gamry PC4 Potentiostat with CMS100 framework and
CMS130 voltammetry software
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
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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 a
  commonly used model for an amine and carboxylate
  containing metal chelate.
• And its cheap too!

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HO-FeEDTA
FeEDTA
(HO)2-FeEDTA
FeHEDTA
-0.1 mM FeII/III -0.1 mM EDTA
FeEDTA
Fe
(HO)2-FeEDTA
HO-FeEDTA
FeHEDTA
Hyperquad Speciation and Simulation software
(HySS) by Peter Gans Formation Constants
obtained from Robert M. Smith and Arthur E.
Martell
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Nernst Equation
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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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11540
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 FeEDTAH2O2
11140
1140
1110
1110
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FeIIIEDTA, H2O2 Speciation
pH 7.4
FeEDTA
Conditions -0.10 mM FeEDTA (11) -4.0 mM H2O2
(top), 14 mM H2O2 (bottom).
HOO-FeEDTA
HO-FeEDTA
pH 7.4
HOO-FeEDTA
FeEDTA
HO-FeEDTA
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110540
110140
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 FeEDTAH2O2
11040
11010
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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 FeEDTAH2O2
1140
11040
1110
11010
Another way of looking at the data is that at
relatively low excesses of H2O2, the EC current
is nearly independent of the FeEDTA ratio.
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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 FeEDTAH2O2
11540
11140
110540
110140
At a relatively high excess of H2O2, the EC
current exhibits a drastic dependence on the
FeEDTA ratio. In contrast to the EC dependence
on H2O2, the effects of the FeEDTA ratio on
the EC current could not be explained by
speciation calculations. Kinetic factors may be
important.
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Quercetin shifts the formal reduction potential,
but what about the speciation of the
peroxy-FeEDTA complex?
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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.

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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 semi-micro 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

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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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pH 7.4
Q quercetin Fe ferric FeIII
FeEDTA
Q-FeEDTA
HO2-FeEDTA
HO-FeEDTA
pH 7.4
FeEDTA
HO2-FeEDTA
Assuming 0.1 mM FeIIIEDTA, 14 mM H2O2, and 0.1
mM quercetin
Q-FeEDTA
HO-FeEDTA
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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
  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.

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Acknowledgements
Coworkers
Cheng Group Tom Brandt Jessica Poindexter Terry
HyattRob Bobier Kevin Breen Ryan
Hutcheson Chemistry department
...and for moral support
The Engelmanns
Financial
National Institute of Health
Renfrew scholarship