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Boletín de la Sociedad Chilena de Química

versión impresa ISSN 0366-1644

Bol. Soc. Chil. Quím. v.46 n.3 Concepción set. 2001

http://dx.doi.org/10.4067/S0366-16442001000300011 

STUDY OF THE IRON CATECHIN COMPLEXES IN DIMETHYL
SULPHOXIDE. REDOX CHEMISTRY AND INTERACTION WITH
SUPEROXIDE RADICAL ANION IN THIS MEDIUM.

MARIO E. BODINI *, M. A. DEL VALLE , RICARDO TAPIA & , FEDERICO
LEIGHTON# , AND LORENA GONZALEZ.

* Departamento Química Inorgánica ,& Departamento Química Orgánica ,
Facultad de Química , # Laboratorio de Citología , Facultad de Ciencias Biológicas ,
Pontificia Universidad Católica de Chile. Vicuña Mackenna 4860 . Santiago . Chile.

* Author to whom correspondence should be addressed.

(Received: March 13, 2001 - Accepted: June 18, 2001)

SUMMARY

The redox chemistry of catechin (catH2) and its iron complexes has been studied in dimethyl sulphoxide. In the absence of base a one-to-one iron(II)-catechin complex is formed which exhibits oxidation processes at 0.28 , 0.66, and 0.92 V vs SCE. These processes correspond to the oxidation of Fe(II) to Fe(III) , the formation of the quinonic form of the catechol moiety and the oxidation of another hydroxy group to a radical. In the presence of base a stable 1:1 complex is formed with oxidation processes that show up at +0.25, +0.64, and +0.88 V vs SCE. The voltammetric and spectroscopic characterization of the species produced after the oxidation processes is described. Upon interaction of the complex with superoxide radical anion in dimethyl sulphoxide, the basic character of this radical anion causes the formation of the monoanion of catechin leading to a more stable complex of iron(II). The protonated superoxide disproportionates to molecular oxygen and peroxide, causing the oxidation of the metal ion. The addition of a second equivalent of superoxide oxidizes bound catechin to the corresponding semiquinone. The formation of hydroxy radicals through Fenton chemistry does not take place because peroxide is consumed and the metal ion remains as a stable iron(III) complex.

KEYWORDS: catechin, antioxidants, iron complexes, superoxide, dimethyl sulphoxide.

RESUMEN

La química redox de catequina (CatH2) y sus complejos de hierro se ha estudiado en dimetilsulfóxido. En ausencia de base se forma un complejo 1:1 de hierro (II)-catequina el cual presenta oxidaciones a 0,28 , 0,66 y 0,92 V vs E.C.S. Estos procesos corresponden a la oxidación de Fe (II) a Fe (III), la formación de la forma quinónica del residuo catecólico y la oxidación del sistema hidroxílico del anillo principal. En presencia de base un complejo estable 1:1 se forma, el cual presenta oxidaciones a +0,25, +0,64 y 0.88 V vs E.C.S. . La caracterización voltamétrica y espectroscópica de las especies formadas luego de las oxidaciones se describe. Luego de la interacción el complejo con el radical anión superóxido en dimetilsulfóxido, el carácter básico de este radical induce la formación del monoanión de catequina con la consiguiente formación del complejo estable de Fe (II) . El superóxido protonado dismuta a oxígeno molecular y peróxido, produciendo la oxidación del ión metálico. La adición de un segundo equivalente de superóxido oxida la catequina coordinada a la especie semiquinónica. La formación de radicales OH mediante la reacción de Fenton no se verifica porque el peróxido se ha consumido y el ión metálico permanece estable como complejo de Fe (III) .

PALABRAS CLAVES: catequina, antioxidantes, complejos de hierro (II), superóxido, dimetil sulfóxido

INTRODUCTION

The role of antioxidants has been established for some time as effective scavengers of very deleterious free radicals 1-3 These species are characterized by having one unpaired electron and, consequently, are particularly reactive toward any molecule that might lose an electron to stabilize it. As a consequence of this process, the latter species gets oxidized and generates new radicals that could initiate a decomposition process to afford substances that can be harmful for the organism. That is the case of LDL cholesterol oxidation whose product gets deposited on the internal walls of arteries to cause arteriosclerosis4,5. Consequently, coronary heart disease develops. Many other diseases are caused by the action of free radicals, such as arthritis, cataract, macular degeneration, cancer, aging, etc. 6

An important source of these radicals is oxygen reduction that takes place in foodstuff "combustion" to release the energy stored in their molecules. The superoxide radical anion is produced in this process and its reactivity must be neutralized to avoid its deleterious effect.

This radical is a very effective nucleophile and can act as a strong Brønsted base via formation of HOO.7 . In water, superoxide ion is rapidly converted to dioxygen and hydroperoxide ion:8

0000002 O2-. + H2O ® O2 + HOO- + HO- K= 2.5 x 108 atm

Such a proton-driven disproportionation means that superoxide can deprotonate acids much weaker than water. In dimethyl sulfoxide the HOO. disproportionation rate is very small.9 Further reactions of this radical generate other radical species, such as hydroperoxide and the most dangerous of all, the hydroxyl radical, OH...

In commenting on the defense of an organism against the deleterious effect of superoxide we must mention the enzyme superoxide dismutase, which is a catalyst for the disproportionation of two HOO. radicals to yield molecular oxygen and hydrogen peroxide.

Another source of antioxidant substances is the content of oxidizable compounds such as flavonoids found in foodstuff. These compounds are present in fruits and vegetables. They are incorporated into our body by direct ingestion or by consumption of products made from them, like tea or wine.10-12

It is also very important to mention at this point that the presence of metal ions, like iron, is a factor to be considered in the lipid peroxidation process.10 If hydrogen peroxide is produced in any step of oxygen reduction, the presence of iron(II) induces the formation of hydroxyl radicals through a Fenton reaction. These metal ions are found in fruits and, as a consequence of that, in a product like wine where significant amounts of iron, copper, manganese and zinc are present.13,14

The negative effect of these ions, particularly in the case of iron, can be avoided if the antioxidants being used possess chelating capacity and bind to the metal thereby precluding the formation of the deleterious hydroxyl radical through Fenton chemistry.15 Some antioxidants found in the above-mentioned fruits or vegetables are flavonoids having a catechol residue in their structure which upon being oxidized to the corresponding semiquinone and quinone forms can coordinate metal ions16. On the other hand, these complexes might exhibit different oxidation potentials and their antioxidant activity could be affected.17

Catechin is one of the most frequently found flavonoids in biological fluids, and it exhibits a catechol residue in its structure. Furthermore, it is known that iron complexes are formed in aqueous medium18 which can be transported within cells. In this medium they can interact with radical species, such as superoxide radical anion, produced after food metabolism.

In this work we have studied the redox chemistry of this flavonoid and the corresponding iron(II) and iron(III) complexes in dimethyl sulfoxide as an aprotic medium to mimic lipidic biological membranes and the interaction of these complexes with superoxide radical anion has been assesed.

EXPERIMENTAL

Equipment

A three-electrode potentiostat (Bioanalytical systems Model CV-27) was used for cyclic voltammetry and a high-capacity potentiostat BAS SP-2 was used for controlled-potential electrolyses. A current integrator based on operational amplifiers was used as a coulometer in the controlled-potential electrolyses. Cyclic voltammograms were recorded on a Hewlett-Packard Model 7004-A X-Y recorder. The electrochemical cell was equipped with a BAS glassy carbon working electrode (area 4,6 mm2), a platinum coil auxiliary electrode, and a Ag/AgCl reference electrode filled with an aqueous tetramethylammonium chloride solution and adjusted to 0.00 V vs SCE.. The latter was contained in a Pyrex tube with a soft-glass cracked tip; this electrode was placed inside a Luggin capillary. For controlled-potential electrolysis a BAS reticulated glassy carbon working electrode was employed and the potentials to be used were determined by cyclic voltammetry. A Milton Roy Model Spectronic 3000 diode array spectrophotometer was used for UV-visible spectrophotometric measurements.

Reagents

Reagents for the investigation and synthesis included Fe(ClO4)2. 6H2O , Fe(ClO4)3. 6H2O (G. Frederick Smith), tetrabuthylammonium hydroxide (Bu4NOH,1.0 M in methanol ,Aldrich), and tetramethylammonium chloride (Aldrich). Tetraethylammonium perchlorate was synthesized by neutralization of an aqueous solution of tetraethylammonium hydroxide (Aldrich) with concentrated perchloric acid (Merck). The resulting solid was recrystallized from ethanol. Catechin was obtained from Aldrich and was used without further purification.

catechin

Aldrich "Gold Label" solvents were used in all the experiments. High-purity argon was used to deaerate solutions.

Since the solvation water of Fe(ClO4)2 . 6 H2O and Fe(ClO4)3 . 6 H2O interferes with the stoichiometric formation of the complexes under study, [FeII(DMU)6][ClO4] 2 and [FeIII(DMU)6][ClO4 ]3 {DMU= dimethylurea} were used in all experiments in which the iron complexes were formed in situ.19

The superoxide radical anion was prepared by controlled-potential electrolysis of an oxygen saturated solution in dimethylsulphoxide at -1.1 V vs S.C.E. and its concentration was determined by the measurement of the anodic current in its cyclic voltammogram. Knowing that an oxygen saturated solution in DMSO is 2.1 mM20 we can assume the presence of that superoxide concentration at the surface of the electrode after the reduction process . This conclusion is based on the fact that the cathodic peak current for oxygen reduction has the same value as the anodic peak current for superoxide oxidation in the cyclic voltammogram of an oxygen saturated solution. .

RESULTS AND DISCUSSION

The voltammetric behavior of catechin and its iron complexes in dimethyl sulfoxide has been studied. Figure 1 shows cyclic voltammograms of 3 mM solutions of this flavonoid in the absence and in the presence of one and two equivalents of tetrabuthyl ammonium hydroxide. Catechin by itself exhibits two oxidation processes at 0.92 and 1.14 V vs SCE when glassy carbon is used as the working electrode (Fig 1A) .

In the presence of one equivalent of base the first oxidation process splits and the resulting solution presents anodic peaks at -0.23, 0.93 and 1.16 V vs SCE (fig 1B). Addition of a second equivalent of base yields the dianion of catechin that shows an anodic peak at -0.2 V vs S.C.E. with a peak current twice as large as in the presence of only one equivalent of base (fig 1C).

Controlled-potential electrolysis of a solution of catechin in the presence of two equivalents of base has been done at 0.00 V vs SCE and residual current is attained after two equivalents of charge have been transfered . These conditions have been chosen for this electrolysis because the oxidation peak is well separated from the second process and no overlapping occurs. Therefore, this result indicates that a quinoid structure is produced in this process. It has been determined that, in aqueous solution, other flavonoids undergo oxidation of the catechol group on ring B.21

Previous work done in our group on quercetin iron complexes in aprotic medium clearly shows this oxidation of the catechol group22. For catechin, the peak observed at 1.24 V vs S.C.E probably corresponds to the oxidation of the 5,7 dihydroxy substituent on the A ring. This conclusion is based on the results reported for the electrochemical behaviour of a flavonoid like 5,7-dihydroxyflavone.21 (chrysin).

Fig.1. Cyclic voltammograms in dimethyl sulfoxide (0.1 M tetraethylammonium perchlorate , TEAP) of a) a 3 mM solution of catechin , b) a 3 mM solution of catechin plus 3 mM TBAOH and c) a 3 mM solution of catechin plus 6 mM TBAOH. The scan rate was 0.1 Vs-1 and a glassy carbon electrode (area 4,6 mm2).

 

Fig.2. UV-Visible spectra of the dimethyl sulfoxide solutions that are : a) ( - - - -) a 0.3 mM solution of catechin ;b) (¾¾¾) a 0.3 mM solution of catechin plus 0.3 mM TBAOH , c) ( - . - . -) a 0.3 mM solution of catechin plus 0.6 mM TBAOH and d) (. . . . . ) a 0.3 mM solution of catechin dianion after controlled-potential electrolysis at 1,00 V vs S.C.E. Path length= 0.1 cm.

The UV- visible spectra of a pure catechin solution and of catechin solutions with one and two equivalents of added base are illustrated in Figure 2. After controlled-potential electrolysis at 0.00 V vs S.C.E. of a solution with two equivalents of added base the UV-vis spectrum shows a new maximum at 380 nm which corresponds to the quinone produced in this process. as it can be observed in figure 2 D. This maximum is shown by other quinones, like 3,5-ditertbuthyl-o-benzoquinone and quercetin in DMSO 22,23.. Therefore, the following set of equations describe the oxidation of catechin:

The neutralization of acidic protons of catechin shifts the oxidation potentials to lower values making the processes easier:

The monoanion is oxidized at -0.23 V to the corresponding protonated semiquinone which disproportionates yielding quinone and catechin:

CatH¾¾0.23V ® e- + CatSQH· (1)
2 CatSQH· ® CatQ + CatH2 (2)

The presence of protonated catechin after the oxidation of the monoanion is responsible for the oxidation peak observed at 0,93 V vs S.C.E. in the cyclic voltammogram of figure 1B.

The oxidation potential for the monoanion and the dianion of catechin are practically the same because the dianion is a strong base and in the presence of water (both residual and generated in the neutralization process) the monoanion must be the predominant species in solution:

Cat= + H2O ® CatH- + OH- (3)

On the other hand, protonated semiquinone must rapidly lose a proton to form the semiquinone radical CatSQ.- (eq 4).

CatSQH· ® CatSQ.- + H+ (4)

Finally, the semiquinone radical CatSQ.- is oxidized at the same potential.

CatSQ.¾0.25 v ® e- + CatQ (5)

In the presence of iron(II) oxidation peaks are observed at 0.34, 0.90 and 1.13 V vs SCE. and the corresponding cyclic voltammogram is illustrated in Figure 3A. A change of color to light green is observed in the presence of iron(II) and the spectrum shows an absorption maximum at 700 nm (Figure 3B). This spectrum is illustrated in figure 3B .The use of the mole-ratio method indicated that a 1:1 complex is formed in this medium.

Formation of the iron-catechin complex is easier in the presence of base. In this case the cyclic voltammogram shows oxidation processes at 0.60, 1.02 and 1.24 V vs SCE.

Fig.3. A) Cyclic voltammogram in dimethyl sulfoxide (0.1 M tetraethylammonium perchlorate , TEAP) of a 3 mM solution of catechin plus 3 mM FeII(DMU)6(ClO4) 2. The scan rate is 0.1 V s-1 and B) UV-visible spectra of the dimethyl sulfoxide solutions that are ( - . . - . . -) 3 mM in catechin and (¾¾¾) 3 mM catechin plus 3 mM FeII(DMU)6(ClO4) . Path length= 0.1 cm.

Fig.4. UV-visible spectrum of a 3 mM solution of the monoanion of catechin plus 3 mM FeII(DMU)6(ClO4) 2 after exhaustive controlled-potential electrolysis at 0.70 V vs S. C.E.. Path length= 0.1 cm.

The first is a one-electron process corresponding to the oxidation of iron(II) to iron(III), as confirmed by controlled-potential electrolysis done at 0.8 V vs S.C.E. which shows that residual current is attained after one equivalent of charge has been transfered. If the complex is prepared from iron(III) and catechin, the resulting complex exhibits the same spectrum as that shown by the solution prepared by controlled-potential electrolysis., again confirming the production of this species in the first oxidation process. A 1:1 stoichiometry is obtained by use of the mole-ratio method for the iron(III) - catechin complex.

Controlled potential electrolysis at potentials adequate to oxidize bound catechin indicates that the first process generates the corresponding quinone of the catechol moiety on ring B. The spectrum of the resulting solution shows the absorption maxima of the oxidized catequin to the quinone form, as can be observed in Figure 4 . The absorption maximum at 330 nm confirms that the quinone form is present.

The oxidation of the catechol moiety to the o-quinone form releases iron(III) because the coordinating ability of this functional group is very poor. Therefore, iron(III) is present as the solvated ion after this oxidation process.

The interaction of the iron(II) complexes with superoxide radical anion was studied in dimethyl sulfoxide. The superoxide was generated by controlled-potential electrolysis at - 1.10 V vs SCE of an oxygen-saturated solution. The concentration of superoxide was adjusted by controlling the number of coulombs used in its generation and its concentration was determined voltammetrically by measuring its anodic peak current. The stabillity of this species was established by monitoring the voltammetric behaviour of the resulting solution, which did not change for two hours. The cyclic voltammogram and the UV - visible spectrum of the solution after the interaction of the iron(II) - catechin complex with one equivalent of superoxide are illustrated in Figures 5 and 6. The cyclic voltammogram clearly shows that no iron(II)-catechin complex is present after the reaction. It is known that, after its action as a base, the neutral protonated superoxide disproportionates to yield peroxide and molecular oxygen. 8

[FeII (Cat -)]+ + O2- ® [FeII (Cat 2-)]+ + HO2
2 [HO2] ® H2O2 + O2

Fig.5. Cyclic voltammograms of 3 mM solutions of the iron(II) - catechin complex after reaction with A) one equivalent of superoxide and B) two equivalents of superoxide in dimethyl sulfoxide. Glassy carbon was used as the working electrode

Fig.6. UV - vis spectra of 3 mM solutions of the iron(II) - catechin complex after reaction with A) one equivalent of superoxide and B) two equivalents of superoxide in dimethyl sulfoxide. Path length= 0.1 cm.

Notice that the comparison of the voltammetric and spectroscopic behaviour of these solutions, compared with those of iron-catechin solutions electrolyzed at the different oxidation potentials, indicate that oxidation of the 1:1 complex by the superoxide radical anion is due to the presence of oxygen and peroxide, according to the following reactions:

2 Fe(II)(cat--)+ +O2 ® [Fe(III) (cat-)+ OO Fe(III) (cat-)+]

[Fe(III) (cat-)2+ OO Fe(III) (cat-)2+] ® 2 [Fe(III)O(cat=)]+ + H2O

and

2 Fe(II) (cat--)+ + HOOH ® 2Fe(III)(OH)(cat-)]-

2 Fe(III)(OH)(cat-)]-- ® 2 [ Fe(III)O(cat=)]+ + H2O

The addition of a second equivalent of superoxide to the iron(III) complex leads to the oxidation of bound catechin to the corresponding semiquinone, as can be inferred from the cyclic voltammogram and spectrum of the resulting solution illustrated in Figures 5B and 6B.

From these results we can conclude that catechin in an aprotic medium forms 1:1 complexes both with iron(II) and iron(III) either as the neutral species or as the corresponding anion. On the other hand, the acidic flavonoid protonates the superoxide radical anion and the presence of the metal complex catalyzes its disproportionation. The appearance of peroxide would be extremely noxious in the organism in the presence of iron(II), because it would generate hydroxyl radicals through Fenton chemistry. In this case, peroxide would be consumed in the oxidation of the iron(II) species to the corresponding iron(III) complex. This iron(III) species can interact with superoxide, and bound catechin is oxidized to the corresponding semiquinone by the action of molecular oxygen and peroxide originated in superoxide disproportionation.

Additional equivalents of superoxide lead to decomposition products that are difficult to identify and which are currently being investigated in our group.

The oxidizing ability of this iron(II) - catechin monoanion complex would improve the capacity of this flavonoid to deactivate superoxide radical anion in an aprotic medium , because the potential for the oxidation of the iron(II) complex to the iron(III) species is lower than that of catechin by itself. On the other hand, the presence of acidic protons in flavonoids like quercetin or catechin generates the neutral species HO2., which disproportionates to molecular oxygen and peroxide. The oxidizing power of these species is consumed first converting iron(II) to iron(III) and then oxidizing some bound flavonoid. Neither free iron(II) nor peroxide would be present to generate the deleterious hydroxy radicals through Fenton chemistry.

ACKNOWLEDGEMENT

The authors are grateful to the Catholic University of Chile for financial support of this work through the PUC - PBMEC 98 research project. The valuable comments and suggestions of Prof. Donald T. Sawyer are deeply appreciated.

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