Electrochemical and Density Functional Theory Study on the Reactivity of Fisetin

Electrochemical and Density Functional Theory Study on the Reactivity of Fisetin

(Parte 1 de 3)

Electrochemical and Density Functional Theory Study on the Reactivity of Fisetin and Its Radicals: Implications on in Vitro Antioxidant Activity

Zoran S. Markovic,† Slavko V. Mentus,‡ and Jasmina M. Dimitric Markovic*,‡

Department of Biochemical and Medical Sciences, State UniVersity of NoVi Pazar, Vuka Karadzica b, NoVi Pazar 36300, Republic of Serbia, and Faculty of Physical Chemistry, UniVersity of Belgrade, Studentski trg 12-16, 10 Belgrade, Republic of Serbia

ReceiVed: July 24, 2009; ReVised Manuscript ReceiVed: NoVember 2, 2009

Antioxidative properties of naturally occurring flavon-3-ol, fisetin, were examined by both cyclic voltammetry and quantum-chemical based calculations. The three voltametrically detectable consecutive steps, reflected the fisetin molecular structure, catecholic structural unit in the ring B, C3-OH, and C7-OH groups in the rings C and A. Oxidationpotentialvalues,used as quantitativeparameterin determiningits oxidationcapability, indicated good antioxidative properties found with this molecule. Oxidation of the C3′C4′ dixydroxy moiety at the B ring occurred first at the lowest positive potentials. The first oxidation step induced fast intramolecular transformationsin which the C3 hydroxy group disappeared and the product of this transformationparticipated in the second oxidation step. The highest potential of oxidation was attributed to the oxidation of C7 hydroxy group. The structural and electronic features of fisetin were investigated at the B3LYP/6-311++G** level of theory. Particularly, the interest was focused on the C3′ and C4′-OH sites in the B ring and on C3-OH site in the C ring. The calculated bond dissociation enthalpy values for all OH sites of fisetin clearly indicated the importance of the B ring and C3′ and C4′-OH group. The importance of keto-enol tautomerism has also been considered. The analysis also included the Mulliken spin density distribution for the radicals formed after H removal on each OH site. The results showed the higher values of the BDE on the C7-OH and C3-OH sites.

Introduction

A variety of naturally occurring and synthetic antioxidant moleculeshave been shown to quench free radicals,reduce lipid peroxidation, and detoxify hydrogen peroxide through nonenzymatic defense mechanisms. These compounds include both dietary factors, such as flavonoids, vitamins C and E, hydroquinones and various sulfhydryl compounds, as well as sodium benzoate, butylated hydroxytoluene, butylated hydroxyanisole, and others. Because of their beneficial actions there has been an increasedinterestin the applicationof antioxidantsto medical treatment since they can significantly inhibit or postpone oxidative processes with products that are often very toxic.1-4

In the past years the chemical behavior of flavonoids as antioxidantshas become the subject of intense experimentaland theoretical research. Flavonoid family is the vast and major group among the phenolics with more than several thousands known compounds.The numerousinvestigationsprovidedsome circumstantial evidence that flavonoids exhibit biological activity, including antiallergic, antiviral, antimutagenic, anti-inflammatory, vasodilatory, and inhibitory actions.1 They also play multiple roles in the ecology of plants.5-8 They are responsible for the colors of fruits (e.g., the red or blue of grape and berry skins)and vegetables;they act as attractorsfor pollinatinginsects and as catalysts in the light phase of the photosynthesis. They are involved in the UV protection of plants and protection of plants against microbial invasion;9,10 they are chelating agents of transition metal ions and also reducing agents.

Overwhelmingly, all the mentioned activities of flavonoids are generally related to their pronounced antioxidant activity, which arises from their ability to scavenge free radicals.1,4,1-19 Potentially, oxygen radical species can damage almost all types of biologically important molecules: lipids (causing lipid peroxidation), amino acids, carbohydrates, and nucleic acids (causing mutations). An imbalance between antioxidants and reactive oxygen species results in oxidative stress which is implicated in many diseases suggesting that free radicals participate as fundamental components in large majority, if not all, human diseases.1,12 Antioxidant molecules present in those systems maintain an important balance between the formation of reactive oxygen species and their removal. There are two possible reaction pathways through which flavonoids and other phenolic compounds scavenge free radicals: (1) rapid donation of the hydrogen atom to a radical form, forming a new radical, more stable than the initial one (mechanism leading to the direct O-H bond breaking) and (2) the chain-breaking mechanism, by which the primary antioxidant donates an electron to the free radical present in the system (e.g., lipid or some other radical), leading to indirect H abstraction

The first mechanism is governed by the O-H bond dissociation enthalpy (BDE), which is the molecular property used in the assessment of possible radical scavenging potential of the molecule. It is calculated as the difference in heat of formation between the molecule (fisetin in this case) and its radicals,

* To whom correspondence should be addressed. E-mail: markovich@ ffh.bg.ac.rs. Phone/fax: + 381 1 187 133.

† State University of Novi Pazar. ‡ University of Belgrade.

10.1021/jp907071v 2009 American Chemical Society Published on Web 12/02/2009 implicatingits correspondenceto the OH bond-breakingenergy. The second mechanism is governed by a one-electron transfer process with both the ionization potential and reactivity of the radical-cation ROH+• playing importantroles. Whatevermechanism is involved the formed radical species (RO•, phenoxide radical in the case of flavonoids) needs to be relatively stable, so that reactions 1 and 2 could be thermodynamically favorable in the sense that it is easier to remove a hydrogen atom from ROH than from HOH and should slowly react with other molecules without toxic effects such is the oxidative stress.

Fisetin is a naturally occurring flavone-3-ol commonly found in strawberries and other fruits and vegetables. Because of its specificstructuralfeaturesit is consideredas a potent antioxidant capable of effective free radical scavenging under in vivo. Medical interest in it arises from its numerous beneficial effects, among which the most striking are involved with stimulating signaling pathways that enhance long-term memory.20,21

The objective of this study was to experimentally and theoretically investigate the antioxidant activity of fisetin. Because of its electrochemical activity, antioxidant properties were correlated with redox potential values22,23 and evaluated by applying cyclic voltammetry (CV). For theoretical calculations of the BDE, density functional (DFT) and semiempirical methods are used. Those methods often give good results with relatively reasonable computational cost still leaving the most reliable methodology to be established.24-34 In this paper the results of bond order, BDE, highest occupied molecular orbital (HOMO), and Mulliken spin density for fisetin are presented. The structure-activity relationship is also examined in the light of these results. Unlike most theoretical investigations which are focused only on the B-ring, particularly the catechol moiety, in this paper the attention is focused to the DFT interpretation of the reactivity of all OH groups in fisetin and the radicals formed after H-removal from this molecule. Keto-enol tautomerism before H-abstraction is also discussed for explaining the role of 3-OH group.

Experimental Section

Chemicals. The following substances were used: fisetin

(Merck), sodium chloride (Merck, USA), acetic acid (Merck, USA), ethanol (Uvasol, Merck, USA), sodium hydroxide (Merck, USA). All the chemicals were used as received.

Supporting Electrolytes. Acetate buffered solutions (pH 2.0-7.0) of constant ionic strength,adjusted by sodium chloride (5 × 10-2 M), were used.The solutionswere obtainedby mixing acetic acid (5 × 10-2 M) and sodium hydroxide (1.5 M). Stock solution of fisetin (1 × 10-2 M) was prepared in ethanol. This solution was diluted to the concentration of 1 × 10-3Mb y addition of the buffers.

CV Measurements. CV measurements were carried out by

PAR EG&G Model 273 potentiostat/galvanostat. A threeelectrode system, consistingof a rotating disk shaped measuring glassy carbon electrode (A ) 0.196 cm2), a saturated calomel electrode (SCE) as reference one, and a platinum foil as an auxiliary electrode, was used. Glassy carbon was a suitable electrode material for electrochemical investigations inside a potential range -0.6 to 1.2 V vs SCE because of its poor catalyticactivityfor water oxidationand reduction.Nevertheless, at potentialsclose to 1.2 V vs SCE the currentof water oxidation became measurable part of the background current. To make the difference between the background current and the current of fisetin electrochemical reactions simultaneous plotting of those two currents was performed. Another possible source of complications, peroxide formation by oxygen reduction, was eliminated by purging electrolyte solution by nitrogen stream.

Because of the ability of fisetin oxidation products to adsorb on the electrode surface, prior to each measurement the glassy carbon electrode was polished by alumina (0.05 µm) and carefully rinsed with bidistilled water. The equilibration period was at least10 s. The CV measurementswereperformedwithout stirring. Cyclovoltammograms were recorded in a range of potentials from -0.600 V to + 1.200 V, at a potential sweep rate ranging from 20 to 200 mV/s. The cell volume was 10 cm3. The voltammograms of the supporting electrolyte buffer solutionswere recorded first, after which an aliquot of the fisetin solutions were added, and the voltammograms recorded under the same conditions.

Computational Method. All calculations were conducted using Gaussian 0335 with the B3LYP hybrid functional36-38 The triple split valence basis set 6-311+G (d,p) was used. This polarized basis set adds p functions to hydrogen atoms in additionto the d functionsand diffusefunctionson heavy atoms. The suitability of this level of theory for studies of bond dissociation enthalpy involving quercetin and taxifolin has already been evidenced elswhere.39 The geometrical parameters for fisetin and its 3-OH, 3′-OH, 4′-OH, and 7-OH radicalspecies were optimizedin vacuum.Vibrationalanalysisand naturalbond orbital (NBO) analysis were performed for all structures. Obtained zero-point energies were used to correct all energetic terms. All calculated structures were verified to be local minima (all positive eigenvalues for ground-state structures) by frequency calculations. The bond dissociation enthalpy (BDE) for fisetin was calculated using the equation where Hfis, Hfis , and H present the enthalpies of fisetin, fisetin radical, and hydrogen,respectively.The ionizationpotential(IP) of fisetin was determined by optimizing neutral and cationic species.

Potential energy surfaces were obtained in relation with the torsion angle τ between the rings B and C, defined by the C3-C2-C1′-C2′ atoms (Figure 1). The torsion angle τ was scanned in steps of 15° without constraints on all other geometrical parameters. The effects of the following torsion angles rotations were also studied: C2-C3-OH, C6-C7-OH, C2′-C3′-OH, and C3′-C4′-OH. Afterward,the structureswere further optimized without any constrain around each potential minimum.

Results and Discussion

Electrochemical Activity of Fisetin. Fisetin is a tetrahydroxyflavone molecule possessing structural features known as

Figure 1. Atomic numbering of fisetin. Reactivity of Fisetin and Its Radicals J. Phys. Chem. A, Vol. 113, No. 51, 2009 14171

very important for high antioxidant activity and scavenging radical activity.1,12 Those activitiesreside mainly in its hydroxyl groups at C3, C3′,C 4′, and C7 and are enhanced with carbonyl group at C4, a double bond between C2 and C3 conjugated with the 4-oxo group enabling higher electron delocalization (Figure 1).

Previously, the electrochemical oxidation of fisetin is studied by Golabi and Irannejad40 with the aim to modify GC electrode for determination of NADH and ascorbic acid. They confirmed that the oxidation products kept adsorbed on the GC surface modifiing its electrocatalytic behavior.

By the obtained cyclic voltammograms it is possible to suppose that the oxidation of fisetin proceeds in three poorly resolved steps. The final oxidation products obviously stay on the electrode surface and block its electrochemical activity toward fisetin dissolved in the electroyte, since the peak heights decrease with the repetition of polarization cycles (Figure 2). Therefore, the electrode surface is polished before each recording, and the first voltammogram was only considered.

By enlarging steply the anodic potential values, from 0.6 V upward, only the first oxidation step shows reversible cathodic peak, which is significantly reduced in surface compared to anodic one. This confirms conclusions published previously41 that one deals with the oxidation of catehcholic group, followed by intramolecular rearrangement.

The oxidation peak potentials and peak currents are pH dependent (Table 1). The pattern of fisetin oxidation potential change at different pH values is shown in Figure 3. Starting from the low pH values (Table 1) and proceeding upward, the peak potentialsshift toward more negativevalues. This indicates the participation of protons in the electrochemical steps, i.e., that the deprotonation acompanies the deelectronation. Deprotonation facilitates oxidation due to the shift of the molecular charge to negative value.42 The plot of the Ep vs pH for the firts oxidation peak (Figure 4) has the slope of 52 mV per pH unit, which closely corresponds to the same number of protons and electrons involved in electrode process. Obtained results indicate two-electron oxidation of fisetin in the pH range observed,followingthe mechanismof the eHHe type established for hydroquinones by Driebergen.43 According to numerous substantialand exstensiveinvestigationson antioxidantactivities of different flavonoids42 the first oxidation wave (Ep (pH 4) ≈ 0.40 V) is possible to correlate to the oxidation of the catecholic group in the B ring, which involves the elimination of two electrons.

Figure 2. Two consecutive cyclovoltammograms of fisetin (c ) 1 × 10-3 M) at pH 4.0 (polarization rate 100 mV/s).

pH V (mV/s) Ep (V) Ip (µA) Ep (V) Ip (µA)

Figure 3. CV curves of fisetin (c ) 1 × 10-3 M) at pH 2 and pH 7 (polarization rate 100 mV/s).

14172 J. Phys. Chem. A, Vol. 113, No. 51, 2009 Markovic et al.

The product of the first oxidation step, o-quinone, undergoes fast intramolecularrearrangement(EC mechanism)givinga new oxidizable species, with renewed catehol unit, in the B ring, on the account of transformation of C3 hydroxyl group into keto group. From the fact that reverse cathodic peak is much smaller than the corresponding first anodic peak, one may conclude that the intramolecular rearrangement is very fast. The ratio anodic vs cathodic peak height tends to reach unity on enlarging the scaning rate, confirming that one deals here with the EC type of electrochemical processes.

In the second anodic oxidation step (Ep (pH 4) ≈ 0.80 V) primarily participates the catechol formed by intramolecular rearrangement of o-quinone. It is not excluded that a part of o-quinone, which does not catch up to undergo rearrangement, participates with its C3 hydroxyl group in the second oxidation step too, but the similarity in energies of these two molecular states does not allow the two mutually resolved corresponding peaks in the cyclovoltammogram.

The third oxidation step observed at a potential of nearly 1

V may be attributed to the oxidation of C7 hydroxyl group. This may be confirmed by the similarity of this oxidation potential with the oxidation potential of flavone containing hydroxyl group only in the A ring: either 5,5-dihydroxyflvone or 7-hydroxyflavone.42 This step is difficult to examine electrochemically because it occurs at potentials adjacent to the potential of water oxidation.

On the basis of the presented results it is possible to suppose the oxidation pathway of fisetin (Scheme 1) is similar to that of quercetin44 and other flavonols, 3-OH flavones, with similar hydroxylation pattern.45

UV-Vis SpectrochemicalInvestigation.Structuralchanges are also evident in electronic spectra of fisetin at different pH values. By increasing the pH and inducing ionization of the OH groups,absorptionbands I and I (Figure5 bands designated on the spectra) belonging to the cinnamoyl (B+C ring) and benzoyl (A+B ring) moieties, respectively, show moderate red shift and broadering. This behavior is a consequence of the sucessive deprotonation as well as the coexisting mixture of ionic and neutral species in the solution.

DFT Study on the Reactivity of Fisetin. To determine the preferred relative positions of the rings B and C, conformational space of the structure 1 (Figure 6) is investigated as a function of torsional angle τ (C3-C2-C1′-C2′) between those rings. The minimization procedure for the fisetin structure, performed at the B3LYP level with the 6-311+G** basis set, yields a planar conformation (τ ) 180°) as the more stable one. In this structure,the moleculeis completelyconjugatedand all hydroxyl groups are oriented in a way to form the maximum number of hydrogen bonds. The present results concur with the literature data on theoretical study of quercetin structure obtained using RHF/STO-3G46 and B3LYP/6-311++G**25 methods, which both report the planar structure of quercetin molecule.

The DFT results are presented in Figure 7. By removal of the torsional angle constraint, the conformational absolute minimum is found at τ ) 180.0° followed by a relative minimum at τ ) 0° with energy difference of only 0.91 kcal mol-1. The potential energy maximum lies at τ ) 90°, and the intercorvesion barrier between the two minima is about 5.53 kcal mol-1, which is almost equal to that for quercetin.25

The ionization potential value of 166.5 kcal mol-1 for the structure 1a of fisetin (Figure 5) is very similar to that of the quercetin molecule.25 On the basis of the ionization potential values and structural similarities between these two molecules one can also expect similar parameters of their antioxidant activities (A). The experimental values of the half-wave potentials of the first oxidation wave, E1/2, confirm this assumption.47 In favor to this also goes somewhathigher dipole moment of the fisetin structure 1a (3.89 D) in comparison to the value for quercetin.25

The results of fisetin NBO analysis indicate strongly localized double bonds at the C2-C3 and C4-O positions of the ring C. The bond order values suggest a highly independent electronic delocalization in the rings B and A, which is also one of the main structural features implicated in antioxidant activity of flavon-3-olsand other flavonoids.The molecularplanarstructure 1 presented in Figure 6 enables such electronic delocalization in fisetin molecule. The C2-C1′ bond lies in the chromone plane, because the C4-C3-C2-C1′ torsional angle is 180°, and its length is 1.465 Å. The order of this bond, obtained by the NBO analysis, is close to 1 (Table 2) indicating very small conjugation across all the rings of the π system. The double bonds in the ring C around the carbonyl group indicate a crossconjugated system48 in which the delocalization is allowed only between C and A or C and B rings but not between rings A and B. This fact is indirectly confirmed by the investigation in

SCHEME 1: Proposed Oxidation Pathway of Fisetin in Aqueous Buffered Solutions Figure 5. Electronic spectra of fisetin at different pH values.

Reactivity of Fisetin and Its Radicals J. Phys. Chem. A, Vol. 113, No. 51, 2009 14173

whichbiophenolsmoleculeswere foundto be neithercompletely planar nor conjugated.48

In the molecule structure 1 (Figure 6) three hydrogen bonds are present, between the C3-OH and C4-O carbonyl groups, between ortho hydroxyl groups in the B ring, and between the C3-OH and C6′. Generally all hydrogen bonds have stabilizing effects. Additional stabilization of the C3-OH group is established by the formation of the hydrogen bond between oxygen of the C3-OH group and hydrogen on the C6′. This finding is in agreement with previous results for quercetin.49 The conformations lacking these bonds (Figure 8) are less stable with respect to the absolute minimum by 9.8, 4.04, and 13.81 kcal mol-1, respectively. The hydrogen bond lacking between the C3-OH and C4-O carbonyl group (1.973 Å) has higher stabilizing effect than that between the C3′-OH and C4′-OH hydroxyl groups (2.166 Å).

(Parte 1 de 3)

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