Flavonoid Structure

1

Complexation and Reduction/Oxidation Reactions of Selected Flavonoids with Iron and Iron Complexes: Implications

on In-Vitro Antioxidant Activity

O

O

OH

OH

OH

OH

OH

Quercetin

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 somethingesoteric, but that they participate as a fundamental componentof 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.

3

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.

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.

4

ROS Induced Damage

• Lipid peroxidation• DNA scission/cross-

linking• 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.

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.

5

Natural ROS Defenses

2O2.- + 2H+ H2O2 + O2

SOD

2H2O2 2H2O + O2

catalase

2GSH + R-OOH

glutathioneperoxidase

GSSG + R-OH + H2O

6

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.

7

Peroxy-FeEDTA and the Fenton Reaction

[FeIIIEDTA-O2H]2- + e- [FeIIEDTA-O2H]3-

[FeIIEDTA-O2H]3- + H+ [FeIIIEDTA]- + HO. + HO-

8

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.

9

Fenton Metal Deactivation

FeIIATP+ H2O2 FeIIIATP + HO. + HO

-

ATP

FeIIL + H2O2

(antioxidant)+L

+L

(pro-oxidant ligand

[FeII(ATP)L] + H2O2

displacement) No Reaction

No Reaction

7. F. Cheng and K. Breen. Biometals. 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.

10

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

Base Structure

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 Myricetin

O

O

OH

OH

OH

O

O

OH

OH

O

O

OH

OH

OH

OH

OH

O

O

OH

OH

OH

Baicalein Chrysin

Morin Galangin

11

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.

12

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.

13

UV-visible Spectrophotometry• HP 8453 UV-vis diode

array. 25 M Metal, 25-75 M 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.

Wavelength (nm)200 250 300 350 400 450 500 550

Abs

orba

nce

(AU)

0

0.5

1

1.5

2

2.5

3

3.5

Wavelength (nm)200 250 300 350 400 450

Abs

orba

nce

(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)

14

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 antioxidant processes.

15

O

O

OH

OH

OH

OH

OH

QuercetinO

O

OH

OH

OH

Naringenin

O

O

OH

OHChrysin

O

O

OH

OH

OHOH

Fisetin

O

O

OH

OH

OH Galangin

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.

16

Voltammetry

FeIIIEDTA

-1

-0.5

0

0.5

1

-0.6-0.300.30.6

potential (V)

curr

ent (

A)

FeIIIEDTA + e- 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

Gamry PC4 Potentiostatwith CMS100 frameworkand CMS130 voltammetrysoftware

Fe

N

O

N

OO

O

O

O

O

O

17

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!

18

Fe(III)EDTA

0 4 8 12pH

0

20

40

60

80

100%

form

ation

relat

ive to

Fe

FeHEDTA

FeEDTA HO-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 12pH

0

20

40

60

80

100

% fo

rmat

ion re

lative

to F

e

Fe

FeHEDTA

FeEDTA

HO-FeEDTA

(HO)2-FeEDTA

19

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)

curr

ent (

A)

pH = 4.1pH = 6.1pH = 7.2pH = 7.7pH = 8.2pH = 9.1pH = 9.6pH = 10.1

20

Nernst Equation

E = E0 - 0.059 x log[FeIIEDTA][OH-]

[FeIIIEDTA-OH]

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

[FeIIIEDTA-OH]

21

FeEDTA E1/2 pH Dependence

y = -0.0849x + 0.5574R2 = 0.986-0.35

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0 5 10 15

pH

E 1/2

(V v

s Ag

/AgC

l)

FeEDTA

FeEDTA-OH/FeEDTA-(OH)2

Linear (FeEDTA-OH/FeEDTA-(OH)2)

22

-1

0

1

2

3

4

5

6

-0.5-0.3-0.10.10.30.5

voltage (V)

curr

ent (

A, r

elat

ive)

H2O2 + e- HO. + HO-


FeIIIEDTA + HO. + HO-FeIIEDTA + H2O2

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].

23

-20

0

20

40

60

80

100

120

140

-1.2-0.8-0.400.40.8

potential (V)

curr

ent (

A)

1:1:540

1:1:140

1:1:40

1:1:10


-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 on relative [H2O2]

0

20

40

60

80

100

120

140

0 100 200 300 400 500 600

[H2O2] excess, relative to FeEDTA

curr

ent (

A)

1:1:10

24

4 6 8 10pH

0

20

40

60

80

100

% fo

rmat

ion re

lative

to F

e

HOO-FeEDTA

FeEDTA

HO-FeEDTA

pH 7.4

Conditions:-0.10 mM FeEDTA (1:1)-4.0 mM H2O2 (top), 14 mM H2O2 (bottom).

4 6 8 10pH

0

20

40

60

80

100

% fo

rmat

ion re

lative

to F

e HOO-FeEDTAFeEDTA

HO-FeEDTA

pH 7.4

FeIIIEDTA, H2O2 Speciation

25

-5

0

5

10

15

20

-1.2-0.8-0.400.40.8

potential (V)

curr

ent (

A)

1:10:540

1:10:140

1:10:40

1:10:10


-1.0 mM Na2EDTA-0.10 M NaNO3

-1.0-54 mM H2O2



EC' Current Dependence on relative [H2O2]

02468

1012141618

0 100 200 300 400 500 600

[H2O2] excess, relative to 1:10 FeEDTA

curr

ent (

A)

26

-2

-1

0

1

2

3

4

5

6

7

8

-1.2-0.8-0.400.40.8

potential (V)

curr

ent (

A)

1:1:40

1:10:40

1:1:10

1:10:10


-0.10/1.0 mM EDTA-1.0/4.0 mM H2O2

-0.10 M NaNO3



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.

27

-20

0

20

40

60

80

100

120

140

-1.2-0.8-0.400.40.8

potential (V)

curr

ent (

A)

1:1:540

1:1:140

1:10:140

1:10:540


-0.10/1.0 mM EDTA-1.0-54 mM H2O2

-0.10 M NaNO3



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.

28

FeEDTA, Quercetin Composite

-1

0

1

2

3

4

5

6

-0.8-0.400.40.8

potential (V, absolute)

curr

ent (

A,

rel

ativ

e)

0.20 mMFe(III)EDTA (1:1)

0.20 mM quercetin

sum composite0.20 mMFe(III)EDTA-quercetin (1:1:1)

experimental 0.20mM Fe(III)EDTA-quercetin (1:1:1)

29

FeIIIEDTA/Q H2O2 Catalytic

-5

0

5

10

15

20

25

30

-0.8-0.400.40.8

potential (V)

curr

ent (

A)

"blank"-9.5 mM H2O2, 95mM NaNO3, 20 mMHEPES pH 7.2

blank + 0.19 mMFe(III)EDTA (1:1)

blank + 0.19 mMFe(III)EDTA-quercetin(1:1:1)

30

Quercetin shifts the formal reduction potential, but what about the speciation of the peroxy-FeEDTA complex?

31

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.

32

Potentiometric Titrations

• 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

•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.

33

residuals in pH for selected data. Unweighted rms=2.86e-02

0 10 20 30 40 50 60 70point number

-0.2-0.10.00.10.2

Speciation and pH: data from c:\my documents\research\data\flavonoid ka's\fisetin 121101.ppd

0

10

20

30

40

50

60

70

80

90

100%

form

atio

n re

lativ

e to

H

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

34


0 20 40 60 80 100 120point number

-0.10.00.1

Speciation and pH: data from C:\My Documents\chrysin 092402.ppd

0

10

20

30

40

50

60

70

80

90

100

% fo

rmat

ion

rela

tive

to C

hry

pH

4

5

6

7

8

9

10

O

OH

OHChrysin

pka

11.406

7.983

sigma 1.62

chi2 73

35


0 10 20 30 40 50 60 70point number

-0.02

0.0

0.02

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

0

10

20

30

40

50

60

70

80

90

100%

form

atio

n re

lativ

e to

H

pH

6

7

8

9

10

11

O

OH

OH

OH

Galangin

pka

11.694

10.684

8.232

sigma 0.53

chi2 10.7

36


0 10 20 30 40 50 60 70point number

-0.2-0.10.00.10.2

Speciation 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

% fo

rmat

ion

rela

tive

to H

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

37


0 20 40 60 80 100 120 140point number

-0.1

0.0

0.1

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

0

10

20

30

40

50

60

70

80

90

100

% fo

rmat

ion

rela

tive

to M

or

pH

5

6

7

8

9

10

11

O

OH

OH

OH

OH

OH

Morin

sigma 3.7chi2 21.8

pka

11.642

11.851

10.555

8.860

5.702

38


0 10 20 30 40 50 60 70 80 90point number

-0.5

0.0

0.5

Speciation and pH: data from c:\my documents\mark's\research\data\flavonoid ka's\quercetin 022602b.ppd

0

10

20

30

40

50

60

70

80

90

100

% fo

rmat

ion

rela

tive

to H

pH

4

5

6

7

8

9

10

O

OH

OH

OH

OH

OH

Quercetin

sigma 2.5chi2 4.9

pka

11.948

12.378

11.211

9.667

8.331

39

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

40

Flavonoid Potentiometric Titration Curve

3.000

4.000

5.000

6.000

7.000

8.000

9.000

10.000

11.000

0.00 0.20 0.40 0.60 0.80 1.00

NaOH added (ml 0.0501 M)

pH

Q only

Q:Zn(II) 3:1

Q:Zn(II) 1:1

Q:Fe(II) 3:1

Q:Ca(II) 1:1

41

Work in Progress

• Complete spectroscopic studies in order reveal SAR.

• Extend the EC’ assay to other flavonoids.• Obtain FeEDTA-flavonoid mixed ligand

binding constants.

42

4 6 8 10pH

0

20

40

60

80

100%

form

ation

relat

ive to

Fe

4 6 8 10pH

0

20

40

60

80

100

% fo

rmat

ion re

lative

to F

e

FeEDTA

HO2-FeEDTA

Q-FeEDTA

HO-FeEDTA

Q-FeEDTA

HO2-FeEDTA

HO-FeEDTA

FeEDTA

pH 7.4

pH 7.4

[FeEDTA][H2Q][FeEDTA-H2Q]

k = =1010

[FeEDTA][H2Q][FeEDTA-H2Q]

k = =1013

Assuming 0.1 mMFeIIIEDTA, 14 mM H2O2, and 0.1 mM quercetin

Q = quercetinFe = ferric FeIII

43

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.

44

Acknowledgements:

Cheng GroupTom BrandtJessica PoindexterTerry HyattRob BobierKevin BreenRyan HutchesonChemistry department

National Institute of Health

Coworkers:

Financial:

Renfrew scholarship

...and for moral support:The Engelmanns