FLAVONOIDS: BIOCHEMISTRY, BIOPHYSICS, MEDICINE - Tarakhovsky Yu. S. - 2013

CHAPTER 2. MECHANISMS OF FLAVONOID ACTION

2.3. Flavonoids as Metal Chelators

Flavonoids readily bind Metal Ions and form coordination complexes with them. Since many metals—primarily transition metals such as Iron and copper ions—act as initiators of Lipid Peroxidation and promote free radical generation, Metal ion binding represents a crucial contribution of flavonoids to cellular defense against oxidative stress. Thus, the antioxidant action of flavonoids is determined not only by their ability to scavenge free radicals directly through immediate interaction, but also by their capacity to bind (chelate) and remove metal ions that trigger free radical formation.

According to numerous researchers, metal chelation is the most effective pathway for the suppression of peroxidation processes by flavonoids. Furthermore, upon complexation with transition metals such as Fe(II), Fe(III), Cu(I), Cu(II), and others, the free-radical-neutralizing capacity of flavonoids is enhanced. This effect is achieved because flavonoid-metal complexes exhibit superoxide dismutase activity. These Reactions Involving the flavonoid-metal complex and the superoxide anion radical can be written as follows:

(1) Me⁽ⁿ⁺¹⁾⁺- Flav + O₂^•- —> Meⁿ⁺Flav + O₂,

(2) Meⁿ⁺- Flav + O₂^•- + 2H⁺—> Me⁽ⁿ⁺¹⁾⁺- Flav + H₂O₂,

where Me represents transition metals: Fe (n = 2); Cu (n = 1), and others.

These processes have been detected not only in vitro, but also in animal experiments. For instance, Cu-rutin complexes effectively protected the lung tissue of animals exposed to asbestos dust against oxidation. The Antioxidant Properties of flavonoids with other metals have also been investigated, such as morin complexes with Pd(II) and Pt(II). It was demonstrated that complexation increases the capacity of morin to scavenge superoxide radicals and inhibit lipid peroxidation. Notably, the Pt(II) complex exhibited higher activity than the Pd(II) complex. Morin complexes with La(III), Gd(III), and Lu(III) ions display antibacterial activity against Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus comparable to the efficacy of penicillin. Quercetin complexes with trivalent rare-earth metals (La, Nb, Eu, Gd, Tb, Dy, Tm, Y) exhibit cytotoxicity against tumor Cells. Their ability to bind to DNA molecules has also been demonstrated, which likely contributes to the antitumor activity of these complexes. High antioxidant activity and DNA-binding capacity have likewise been discovered in naringenin-2-hydroxybenzoyl hydrazone complexes with trivalent rare-earth metals Y and Eu, as well as hesperetin-4-benzoyl hydrazone complexes with trivalent lanthanides, particularly Nd. Metal complexes of rutin, quercetin, catechin, and other flavonoids have shown high activity in animals, cells from various Organs, and subcellular fractions, which not only highlights the broad Biological Significance of these complexes but also opens up promising Prospects for their biomedical application.

2.3.1. Interaction of flavonoids with metals

Potentially, flavonoid molecules can possess multiple metal-binding sites, the locations of which are determined by the presence of adjacent pairs of hydroxyl or carbonyl groups. For instance, the pair of 3'- and 4'-hydroxyl groups in ring B can participate in binding. This particular pair of hydroxyls is often referred to as the catechol group, although this moiety is present not only in catechins but also in certain other flavonoids, such as quercetin, taxifolin, and others. In addition, metal binding can involve the 3-hydroxyl and 4-carbonyl groups of ring C, or the 5-hydroxyl and 4-carbonyl groups belonging to rings A and C, respectively.

The catechol group is regarded by some researchers as the most probable metal-binding center. Metal coordination at this position can be facilitated in alkaline media (pH 10) due to hydroxyl deprotonation. This binding site was investigated using the quercetin-Cu(II) ion complex as an example. In this complex, the quercetin-to-copper(II) molar ratio was 2:1 (Fig. 51).

Class="center">Fig. 51. Complex of quercetin with divalent copper. Quercetin:copper ratio = 2:1. The copper atom interacts with the hydroxyls of the catechol groups, thereby binding two quercetin molecules. In the proposed scheme, the quercetin molecules are situated in orthogonal planes.

Not all flavonoids possess a catechol group. Moreover, the number of hydroxyl groups in the molecules of certain flavonoids is quite limited, which simplifies the structural analysis of their complexes. For example, the naringenin glycoside-copper complex features only a single metal cation binding site located between the 5-hydroxyl and 4-carbonyl groups (Fig. 52). It was found that complexation with copper enhances the antioxidant properties of naringenin. Furthermore, this substance exhibits increased cytotoxicity against malignant tumor cells and amplified anti-inflammatory activity.

Fig. 52. Complex of naringenin with divalent copper. Naringenin:Cu(II) ratio = 1:1. Naringenin is dissolved in ethyl alcohol; hence, two ethanol molecules are incorporated into the complex. The Structure of the metal complex was determined using infrared, ultraviolet, and visible spectroscopy, as well as NMR data.

However, in certain cases, the precise Location OF THE metal atom and the Stoichiometry of the complexes are difficult to determine, and this issue remains a subject of debate. When quercetin interacts with iron ions of various valencies, The formation of complexes with different binding stoichiometries is hypothesized. Calculations indicate that among the three possible binding sites (Fig. 53, a–c), the highest binding energy for iron ions in the quercetin molecule is observed in the complex involving the 3-hydroxyl and 4-carbonyl groups (Fig. 53a). These are followed by the 4-carbonyl and 5-hydroxyl groups (Fig. 53b), and the 3'- and 4'-hydroxyl groups (Fig. 53c).

Fig. 53. Proposed structure of quercetin-iron complexes with various binding stoichiometries: (a–c) - quercetin/iron stoichiometry 1:1;

(d) - 2:1; (e) - 3:2; (f) - 3:1.

Binding stoichiometry depends on the oxidation state of iron and can yield ratios of 1:1, 1:2, 2:3, and 1:3 for Fe(II) and Fe(III) (Fig. 53). Thus, the formation of iron complexes containing one, two, or three quercetin molecules is theoretically possible. As mentioned in previous chapters, owing to the Fenton reaction, the interconversion between Fe(II) and Fe(III) forms of iron is significantly facilitated in the presence of hydrogen peroxide, which is generated within The Cell during various redox reactions occurring in Mitochondria and, partially, in The Endoplasmic reticulum. Therefore, iron ions with varying degrees of oxidation may potentially be present intracellularly. At the same time, one must account for the fact that due to the presence of reducing agents such as NADH reductase, ascorbic and citric acids, and thioredoxin, iron cations in vivo predominantly exist in the reduced form (Fe²⁺), whereas the oxidized form of iron (Fe³⁺) is virtually absent.

2.3.2. Lipophilicity of metal complexes

The formation of flavonoid complexes with iron cations can exert a profound effect on their lipophilicity and interaction with the phospholipid bilayer. For instance, calculations of the octanol/Water partition coefficient (C log P) demonstrate that the 1:1 quercetin-iron complex is less hydrophobic than the free quercetin molecule, whereas lipophilicity increases substantially in the 2:1, 3:2, and 3:1 quercetin-iron complexes (Table 6).

Table 6. Calculated values of the octanol/water partition coefficient (C log P) for quercetin and its iron complexes.

Note. Data were obtained using Chem3DUltra 9.0 software (Chemical Office software package, Cambridge Soft). A C log P value > 1 indicates increased lipophilicity of the substance.

Calculations indicate that the Formation of the quercetin-iron complex does not hinder the interaction of this flavonoid with the phospholipid bilayer. According to the calculations, this interaction may be enhanced in metal complexes with a quercetin-to-iron ratio of 2:1, 3:2, or 3:1, whereas the lipophilicity of the

1:1 metal complex is lower than that of free quercetin.

Experimental measurements also demonstrate an increase in flavonoid lipophilicity in the presence of iron, which is reflected by an increase in the partition coefficient of these substances in the octanol/water system (Fig. 54). However, the obtained log P values are substantially lower than the calculated C log P values obtained for quercetin/iron complexes > 1:1.

Fig. 54. Dependence of the partition coefficient (log P) in the octanol/water system on the iron(II)/flavonoid ratio for quercetin (a) and taxifolin (b).

2.3.3. Stoichiometry of Metal Complexes

Experimental studies on the interaction of various flavonoid-metal complexes show that their stoichiometry strongly depends on proton concentration. Under mildly acidic or neutral pH conditions, the coordination number can be higher than in alkaline media. A pH of 6 is optimal for complex formation (Table 7).

Table 7. Stoichiometry of flavonoid-metal complexes.

At high proton concentrations (pH 3), hydroxyl groups do not dissociate, which prevents the formation of metal complexes. The formation of quercetin-iron complexes can be analyzed using the visible and ultraviolet absorption spectra of quercetin. It is well known that quercetin exhibits two absorption maxima: at 373 nm (band 1) and 255 nm (band 2). Band 1 corresponds to electronic transitions of the B-ring, whereas band 2 is associated with transitions of the A-ring of quercetin. Upon titration of quercetin with increasing concentrations of iron, the maximum at 373 nm decreases, while a new maximum at 425 nm appears and increases, which is attributed to the formation of the metal complex (Fig. 55).

Fig. 55. Determination of the quercetin:Fe(II) stoichiometry by titration. A - set of absorption spectra of 20 μM quercetin (1) and quercetin-iron complexes at concentrations of 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM (numbers 2-6, respectively). B - dependence of absorbance at 425 nm on iron concentration. The inflection point of the curve occurs at 10 μM iron, which corresponds to a quercetin/iron stoichiometry of 2:1.

The obtained set of curves features a single isosbestic point at 398 nm, indicating a unified equilibrium and reversible process for the formation of both chromophores. The dependence of absorbance at 425 nm on iron concentration can be approximated by a straight line with an inflection point at an iron concentration of 10 μM. Given that the quercetin concentration in the experiment was 20 μM, we obtain a quercetin:Fe stoichiometry of 2:1. The spectra of quercetin-iron(II) complexes obtained by mixing the components at various molar ratios do not exhibit an isosbestic point, which is characteristic of this method of component mixing (Fig. 56). The resulting dependence of absorbance on the molar ratios of the components shows a maximum at a ratio of 3:2.

Fig. 56. Determination of the quercetin/Fe(II) stoichiometry by the molar ratio method. A - absorption spectra of quercetin (1) and quercetin-iron complexes obtained by mixing quercetin and iron in molar ratios of 8:2, 6:4, 4:6, 2:8, and 1:9 (numbers 2-6, respectively). B - dependence of absorbance at 425 nm on the quercetin/iron ratio. The maximum of the curve corresponds to a quercetin/iron ratio of 6:4.

The partition coefficient of polyphenols in the water-octanol system (log P), which determines their lipophilicity and ability to insert into the hydrophobic region of the phospholipid bilayer of Introduction/36.html">Biological Membranes, correlates with their antioxidant activity and ability to interrupt lipid peroxidation processes in both the polar and hydrophobic Regions of the membrane, depending on the depth of insertion of these molecules into the bilayer.

The ability of flavonoids to penetrate the hydrophobic region of the membrane decreases for molecules with a larger number of hydroxyl groups. The propensity of flavonoids to insert into the bilayer can be influenced by salt concentration and the presence of negatively charged Lipids in the membrane. Using catechins as an example, it has been demonstrated that even different stereoisomers of these molecules can exert distinct effects on the Physical Properties of the bilayer.

Numerous studies indicate that soy isoflavones also reduce the risk of breast Cancer through protection against the action of 4-HNE. Specifically, it was found that genistein is able to modulate the expression of Glutathione S-transferase in human breast epithelial cells, which is disrupted by the toxic action of lipid peroxidation products, particularly 4-HNE. This reduces the likelihood of cellular DNA damage observed in the presence of 4-HNE, thereby preventing neoplastic growth in these Tissues and tumor development.