Iron-binding properties of plant phenolics and cranberry's bio-effects

Abstract

The health benefits of cranberries have long been recognized. However, the mechanisms behind its function are poorly understood. We have investigated the iron-binding properties of quercetin, the major phenolic phytochemical present in cranberries, and other selected phenolic compounds (chrysin, 3-hydroxyflavone, 3',4'-dihydroxy flavone, rutin, and flavone) in aqueous media using UV/vis, NMR and EPR spectroscopies and ESI-Mass spectrometry. Strong iron-binding properties have been confirmed for the compounds containing the "iron-binding motifs" identified in their structures. The apparent binding constants are estimated to be in the range of 10(6) M(-1) to 10(12) M(-2) in phosphate buffer at pH 7.2. Surprisingly, quercetin binds Fe(2+) even stronger than the well known Fe(2+)-chelator ferrozine at pH 7.2. This may be the first example of an oxygen-based ligand displaying stronger Fe(2+)-binding affinity than a strong nitrogen-based Fe(2+)-chelator. The strong Fe-binding properties of these phenolics argue that they may be effective in modulating cellular iron homeostasis under physiological conditions. Quercetin can completely suppress Fenton chemistry both at micromolar levels and in the presence of major cellular iron chelators like ATP or citrate. However, the radical scavenging activity of quercetin provides only partial protection against Fenton chemistry-mediated damage while Fe chelation by quercetin can completely inhibit Fenton chemistry, indicating that the chelation may be key to its antioxidant activity. These results demonstrate that quercetin and other phenolic compounds can effectively modulate iron biochemistry under physiologically relevant conditions, providing insight into the mechanism of action of bio-active phenolics.

Chart 1

Structures and atom numbering of the flavonoids and ferrozine.

Fig. 1

Titration of quercetin by Fe²⁺ in 20 mM KPB buffer, pH 7.2. Top to bottom: 10 μM quercetin in the presence of 0, 2.5, 5.0, 7.5, 10, 20 and 30 μM Fe²⁺. Inset, titration curve: absorbance of the quercetin–Fe²⁺ complex at 425 nm versus Fe²⁺ concentrations, with 10 μM quercetin initially.

Fig. 2

Titration of quercetin with Fe³⁺ in 20 mM KPB buffer, pH 7.2. From top to bottom: 10 μM quercetin in the presence of 0, 2, 4, 6, 8, 10, 15, 20, 25 and 30 μM Fe³⁺. Inset, titration curve: absorbance at 430 nm versus Fe³⁺ concentrations, with 10 μM quercetin initially.

Fig. 3

Electrospray mass spectra of solutions of Fe²⁺/Fe³⁺quercetin and (10 μM each, 1 : 1) in methanol/water (1 : 1, v/v). (A), with Fe²⁺, the inset is the isotopic pattern of the peak at m/z = 375.1; (B), with Fe³⁺, the inset is the isotopic pattern of the peak at m/z = 658.

Fig. 4

EPR spectra of quercetin, Fe²⁺/Fe³⁺ and EDTA in MeOH/H₂O (v/v : 50/50) under anaerobic conditions except for (a). (a) quercetin (0.6 mM) + Fe³⁺ (0.3 mM), in air; (b) quercetin (0.6 mM) + Fe³⁺ (0.3 mM); (c) quercetin (0.4 mM) + Fe²⁺ (0.3 mM); (d) EDTA (0.6 mM) + Fe³⁺ (0.3 mM). Spectra (a), (b) and (c) have been amplified 10-fold.

Fig. 5

Titration of 3-hydroxyflavone (A), 3′,4′-dihydroxyflavone (B), chrysin (C) and rutin (D) with Fe²⁺ in 20 mM KPB, pH 7.2. Insets: titration curves. (A), from top to bottom: 10 μM 3-hydroxyflavone in the presence of 0, 2, 4, 6, 8, 10, 12 and 14 μM Fe²⁺, respectively. (B), from top to bottom: 10 μM 3′4′-dihydroxyflavone in the presence of 0, 2, 4, 6, 8, 10 and 12 μM Fe²⁺, respectively. (C), from top to bottom: 40 μM chrysin in the presence of 0, 10, 15, 20, 30, 40 and 50 μM Fe²⁺, respectively. (D), from bottom to top: 10 μM rutin in the presence of 0, 2, 4, 6, 8, 10, 12 and 14 μM Fe²⁺, respectively.

Fig. 6

¹H NMR spectra of quercetin (5 mM) and the titration with Zn²⁺ in d⁶-DMSO/D₂O (v/v : 50/50), 50 mM Tris-HCl, pH* 7.20. (a) quercetin only; (b) to (f), with the addition of 0.25, 0.50, 0.75, 1.0 and 2.0 mol eq. of Zn²⁺, respectively.

Fig. 7

¹H-NMR spectra of quercetin (5 mM) and the titration with Ga³⁺ in d⁶-DMSO/D₂O (v/v : 50/50), 50 mM Tris-HCl, pH* 7.20. (a) quercetin only; (b) to (e), with the addition of 0.25, 0.50, 1.0 and 2.0 mol eq. of Ga³⁺, respectively.

Chart 2

Proposed structures for the complexes formed between quercetin and Fe³⁺ or Fe²⁺ under ESI-Mass conditions.

Fig. 8

Competition between ferrozine and quercetin for Fe²⁺ in 20 mM KPB, pH 7.2. Addition of quercetin into (ferrozine)₃–Fe²⁺: (a) 30 μM ferrozine only; (b) addition of 10 μM Fe²⁺. Then addition of 10 μM quercetin (c) and the kinetics of the competition was monitored in 5 min (d), 1 h (e), 2 h (f), 3 h (g), 4 h (h) and 5 h (i).

Fig. 9

Absorbance of malonaldehyde–TBA complex at 532 nm at various Fe²⁺ concentrations in the absence (a) or presence (b) of 10 μM quercetin.

Fig. 10

Effect of quercetin (Q) on Fe-promoted 2-deoxyribose degradation in the presence of ATP or citrate in 20 mM KPB at pH 7.4, 100 μM ascorbate and 200 μM H₂O₂. (A), with increasing concentration of Fe²⁺, [ATP] = 25 μM, [citrate] = 25 μM and [quercetin] = 25 μM; (B), with increasing concentration of quercetin, [ATP] = 25 μM, [citrate] = 25 μM and [Fe²⁺] = 25 μM.

Fig. 11

Effect of EDTA on the ability of quercetin to prevent 2-deoxyribose degradation in 20 mM KPB at pH 7.2. (a) 25 μM EDTA with Fe²⁺; (b) 25 μM EDTA with Fe²⁺ in the presence of 10 μM quercetin; (c) Fe²⁺ in the presence of 10 μM quercetin. [H₂O₂] = 200 μM.