1. 1 Complexation and Reduction/Oxidation Reactions of Selected Flavonoids with Iron and Iron Complexes: Implications on In-Vitro Antioxidant Activity

2. 2 A quote by Dr. Barry Halliwell from the American Journal of Medicine 1 : “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 incomplete 2.

3. 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. 3. Kaim w. and Schwederski B. “Bioinorganic Chemistry: Inorganic Elements in the Chemistry of Life.” J. Wiley and Sons, 1994, New York.

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

5. 5 Natural ROS Defenses

6. 6 Hydroxyl Radical and The Fenton Reaction H 2 O 2 + e -  HO + HO - E°’ = 0.30 V, S.H.E., pH 7.0 Fe(II)  Fe(III) + e - E°’ = depends on complex Fe(II) + H 2 O 2  Fe(III) + HO + HO - –The impact of Ferrous salts on H 2 O 2 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. 7 Peroxy-FeEDTA and the Fenton Reaction

8. 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. 9 Fenton Metal Deactivation 7. F. Cheng and K. Breen. Biometals. 2000, 13, 77-83. Quercetin deactivates the Fe-ATP complex 7, although the precise mechanism is still unclear. The use of a strong chelate, like EDTA, should provide additional insight.

10. 10 Flavonoid Structure

11. 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. 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. 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. Fe II, Quercetin Ca, Naringenin 1:3 (M:L) 1:1 0:1 1:3 1:1 (dashed), 0:1 (solid)

14. 14 Fe II Fe III Cu II Ca II Zn II 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.

15. 15 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. 16 Voltammetry Conditions: -0.20 mM Fe(NO 3 ) 3 -0.10 M NaNO 3 -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

17. 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. 18 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 Fe II/III -0.1 mM EDTA Fe FeHEDTA FeEDTA HO-FeEDTA (HO) 2 -FeEDTA

19. 19

20. 20 Nernst Equation

21. 21

22. 22 Conditions: -0.20 mM FeEDTA -0.10 M NaNO 3 -20 mM HEPES, 7.4 -9.5 mM H 2 O 2 -25 mV/s, C disk -Ag/AgCl reference -Pt wire counter electrode The electrocatalytic current (EC’) is highly dependant on pH, [H 2 O 2 ] and [EDTA].

23. 23 1:1:540 1:1:140 1:1:40 1:1:10 Conditions: -0.10 mM Fe(NO 3 ) 3 -0.10 mM EDTA -1.0-54 mM H 2 O 2 -0.10 M NaNO 3 -20 mM HEPES pH 7.4 -25 mV/s, carbon disk -Ag/AgCl reference -Pt counter electrode -ratios are labeled according to Fe:EDTA:H 2 O 2 1:1:10

24. 24 HOO-FeEDTA FeEDTA HO-FeEDTA pH 7.4 Conditions: -0.10 mM FeEDTA (1:1) -4.0 mM H 2 O 2 (top), 14 mM H 2 O 2 (bottom). HOO-FeEDTA FeEDTA HO-FeEDTA pH 7.4 Fe III EDTA, H 2 O 2 Speciation

25. 25 1:10:540 1:10:140 1:10:40 1:10:10 Conditions: -0.10 mM Fe(NO 3 ) 3 -1.0 mM Na 2 EDTA -0.10 M NaNO 3 -1.0-54 mM H 2 O 2 -20 mM HEPES pH 7.4 -25 mV/s, carbon disk -Ag/AgCl reference -Pt counter electrode -ratios are labeled according to Fe:EDTA:H 2 O 2

26. 26 1:1:40 1:10:40 1:1:10 1:10:10 Conditions: -0.10 mM Fe(NO 3 ) 3 -0.10/1.0 mM EDTA -1.0/4.0 mM H 2 O 2 -0.10 M NaNO 3 -20 mM HEPES pH 7.4 -25 mV/s, carbon disk -Ag/AgCl reference -Pt counter electrode -ratios are labeled according to Fe:EDTA:H 2 O 2 Another way of looking at the data is that at relatively low excesses of H 2 O 2, the EC’ current is nearly independent of the Fe:EDTA ratio.

27. 27 1:1:540 1:1:140 1:10:140 1:10:540 Conditions: -0.10 mM Fe(NO 3 ) 3 -0.10/1.0 mM EDTA -1.0-54 mM H 2 O 2 -0.10 M NaNO 3 -20 mM HEPES pH 7.4 -25 mV/s, carbon disk -Ag/AgCl reference -Pt counter electrode -ratios are labeled according to Fe:EDTA:H 2 O 2 At a relatively high excess of H 2 O 2, the EC’ current exhibits a drastic dependence on the Fe:EDTA ratio. In contrast to the EC’ dependence on [H 2 O 2 ], the effects of the Fe:EDTA ratio on the EC’ current could not be explained by speciation calculations. Kinetic factors may be important.

28. 28

29. 29

30. 30 Quercetin shifts the formal reduction potential, but what about the speciation of the peroxy-FeEDTA complex?

31. 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 (pH exp ) and the simulated curve (pH calc ). 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. 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 NaNO 3 ionic strength 0.05 M NaNO 3 titrant (standardized daily) CO 2 scrubbed water, N 2 purged headspace 60/40 vol% H 2 O/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. 33 pk a 11.906 11.773 9.965 8.405 sigma1.54 chi 2 11.9

34. 34 pk a 11.406 7.983 sigma1.62 chi 2 73

35. 35 pk a 11.694 10.684 8.232 sigma0.53 chi 2 10.7

36. 36 sigma1.61 chi 2 7.74 pk a 11.324 10.034 8.238

37. 37 sigma3.7 chi 2 21.8 pk a 11.642 11.851 10.555 8.860 5.702

38. 38 sigma2.5 chi 2 4.9 pk a 11.948 12.378 11.211 9.667 8.331

39. 39 quercetinmorinnaringingalanginchrysinFisetin pk 1 8.331 5.7028.2388.2327.9838.405 pk 2 9.667 8.86010.03410.68411.4069.965 pk 3 11.211 10.55511.32411.69411.773 pk 4 11.948 11.64211.906 pk 5 12.378 11.851

40. 40

41. 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. 42 FeEDTA HO 2 -FeEDTA Q-FeEDTA HO-FeEDTA Q-FeEDTA HO 2 -FeEDTA HO-FeEDTA FeEDTA pH 7.4 Assuming 0.1 mM Fe III EDTA, 14 mM H 2 O 2, and 0.1 mM quercetin Q = quercetin Fe = ferric Fe III

43. 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. 44 Acknowledgements: Cheng Group Tom Brandt Jessica Poindexter Terry Hyatt Rob Bobier Kevin Breen Ryan Hutcheson Chemistry department National Institute of Health Coworkers: Financial: Renfrew scholarship...and for moral support: The Engelmanns