Journal of Electroanalytical Chemistry 756 (2015) 212–221

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Journal of Electroanalytical Chemistry

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The electrochemical approach to antioxidant activity assay of metalcomplexes with dipicolylamine ligand, containing2,6-di-tert-butylphenol groups, based on electrochemical DPPH-test

V.Yu. Tyurin a,⁎, A.А. Moiseeva a, D.B. Shpakovsky a, E.R. Milaeva a,b

a Lomonosov Moscow State University, Department of Chemistry, 119991 Moscow, Russiab Institute of Physiologically Active Compounds of Russian Academy of Sciences, 142432 Chernogolovka, Russia

⁎ Corresponding author.E-mail address: tyurin@med.chem.msu.ru (V.Y. Tyurin

http://dx.doi.org/10.1016/j.jelechem.2015.07.0241572-6657/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 29 April 2015Received in revised form 30 June 2015Accepted 18 July 2015Available online 26 July 2015

Keywords:Metal complexesRedox properties2,6-Di-tert-butylphenolAntioxidant activityCyclic voltammetryRotating disk electrode2,2-Diphenyl-1-picrylhydrazyl

The novel electrochemical approach to antioxidant activity assay based on the reaction with stable radical2,2′-diphenyl-1-picrylhydrazyl (DPPH) monitored by the rotating disk electrode (RDE) method was developed.This method has advantages in comparison with usual spectrophotometrical assay since it can be applied tocolored compounds and in a wide range of concentrations. The electrochemical behavior of Co(II), Mn(II),Fe(II), Cu(II), Zn(II), and Ni(II) complexes with di-(2-picolyl)amine ligand L(1) containing antioxidant2,6-di-tert-butylphenol pendantwas studied by cyclic voltammetry (CV), and their radical scavenging propertieswere examined using the electrochemical DPPH-test. CV curves for these compounds contain the peaks of ligandandmetal oxidation/reduction. The possible schemes of redox transformationswere proposed. Itwas shown thatthe antioxidant capacity of these complexes depends strongly upon their redox properties, and themetal natureplays key role. Thus, the Co(II), Zn(II), andNi(II) complexes are less active than free ligand. But the easily oxidizedFe(II) complex shows high antioxidant activity which is comparable with the efficiency of the well-knownantioxidant Trolox. In this case the synergetic antioxidant effect is observed due to the presence of an essentialorganic radical scavenger and redox-active metal. The feasible mechanism of antioxidant activity is discussed.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Reactive oxygen species (ROS) such as superoxide radical-anionO2•−,

hydroxyl HO•, peroxyl radicals LOO, and lipid hydroperoxides LOOH thatemerge as a result of the respirative cycle of oxidative phosphorylationmay attack intracellular biomolecules (proteins, lipids, and nucleicacids), giving rise to single-strand and double-strand breaks. Whenthe cellular defense system is overwhelmed by an excessive generationof ROS a situation of oxidative stress occurs, that may eventually causecell aging, and leads to cardiovascular diseases, mutagenic changes,and cancerous tumor growth [1–4].

The aerobic organisms developed complex defense network againstoxidative stress during the course of evolution. From the view oftheir mechanistical functions the antioxidants may be classifiedinto preventing, scavenging, repair and de novo antioxidants [5].The preventing antioxidants function as the “first line defense” bysuppressing the formation of ROS. The scavenging antioxidants removeactive species rapidly before they attack biologically essential mole-cules. These scavenging antioxidants act as the “second line defense”

).

in vivo. Phenolic compounds (flavonoids, phenolic acids, stilbenes,tocopherols etc.), that quench free radicals by donating a hydrogenatom and/or electron to free radicals are the typical representatives ofthis group.

Negative influence of ROS on living organisms is the reason of a sig-nificant interest in compounds exhibiting antioxidant properties and inmethods used for the estimation of such properties. There are numerousmethods of antioxidant activity assay based on their ability to quenchstable radicals or reduce metal ions [6]. One of the most popular assaysis a reaction with a stable radical 2,2′-diphenyl-1-picrylhydrazyl(DPPH) which is monitored spectrophotometrically [7,8]. The antioxi-dant capacity of the examined compounds is determined from the ki-netic of this reaction [9–11]. DPPH is dissolved in alcohol (ethanol,methanol) solutions to give an absorbance band at 515–517 nm, andthe addition of antioxidants decreases the absorbance of the DPPH to astable value (Adetect). This method is simple and widely used in recentyears in various modifications but, on the other hand, has a number ofserious drawbacks. First, it cannot be applied to colored compoundswhich have an absorption band close to 515 nm. Second, high concen-trations of DPPH in the reactionmixture give absorbance beyond the ac-curacy of spectrophotometric measurements. Except these, there aremany contradictions between the results obtained by different groups[12]. The discussed relationships confirm the necessity of standardizing

Table 1The potentials of reduction (ERed) and oxidation (Eox) (vs. Ag/AgCl/KCl (sat.)) obtained byCV on Pt or GC electrodes at 100 mV/s for 1–7 (CH3CN, 0.5 · 10−3 Bu4NBF4).

Compound M EpRed, V EpOx, V

GC Pt GC Pt

1 L – −2.10 – 0.97 1.121.52 1.68

^ 1.632 Co −1.48 −1.48 1.15 1.01/0.81, 0.59

−1.81 1.63 1.50/1.04−2.10

3 Mn −2.02 −1.30/−1.07 1.08/0.98 1.06/1.00−2.37 −1.54/−1.50 1.38 1.40/1.30

1.65 1.744 Fe −1.71 – 0.45/0.21 –

−2.08 0.92/0.561.65

5 Cu −0.17/-0.08 −0.20/−0.07 1.19 –−1.87/−0.36 −0.78/−0.36 1.67

6 Zn −2.05 – 1.27 –−2.46 1.56

7 Ni −0.96/−0.84 −0.99 1.18 1.19/0.97−1.71 −1.42 1.41/0.84 1.71−1.93 1.71

213V.Y. Tyurin et al. / Journal of Electroanalytical Chemistry 756 (2015) 212–221

the DPPH method and show the complexity of spectrophotometricalestimating antioxidant activity, even in the case of very simple DPPH/antioxidant systems [13,14].

During the last years, electrochemical methods are more widely ap-plied to antioxidant activity evaluation, because for the analysis of anti-oxidants in colored or complex samples, they are more advantageousthan spectrometric. Cyclic voltammetry with glassy carbon (GC) elec-trode has been used for antioxidant capacity assay of blood plasma, tis-sue homogenates and some plant extracts [15]. The voltammetricapproach of antioxidant activity estimation based on the decrease ofthe electroreduction oxygen current in the presence of the antioxidantwas proposed in Refs. [16–18], and the further development of the the-ory of this process has been carried out in Ref. [19]. The analogous ap-proach was proposed in Refs. [20–22]. The cyclic voltammetricresponse of the O2/O2

•− redox couple was used in order to study theinteraction of the dihydropyridine derivatives with superoxide. Ascanning electrochemical microscope (SECM) was applied in orderto characterize the electrode mechanism of the O2/O2

•− couple indimethylsulphoxide (DMSO) [21].

The potentiometric method of antioxidant activity determinationbased on the sharp change of electrode potential for the ferricyanide/ferrocyanide mediator system after addition of antioxidant was devel-oped in Refs. [23–25]. A method was proposed and tested concerningthe characterization of antioxidants by means of their reaction withelectrogenerated HO• radicals in galvanostatic assayswith simultaneousO2 evolution [26–28]. A new approach to detect free radicals using anelectrochemical procedure, in which the radicals destroy a well-defined molecular layer on an electrode, was proposed in Ref. [29].Antioxidant activity and free radical scavenging ability of curcuminwere investigated using two electrochemical methods and electronspin resonance (ESR) technique [30].

The number of transferred electrons during oxidation of moleculeswith antioxidant capacity was approximately obtained by means ofthe slope of CUPRAC calibration plots, comparedwith that for the Trolox[31]. Independent determinations of these numbers were made bybipotentiometric titrations. Both sets of data are in reasonable agree-ment between each other. In Ref. [32] the new electrochemical assayof total antioxidant power (TAP) measurements was proposed. In thiscase TAPmeasure is the integral of the product of current and exponentfrom the potential alongnormal or differential pulse voltammetric peak.An amperometric flow injection (FI) method suitable for evaluation of‘total antioxidant capacity’ (TAC) was presented in Ref. [33]. In thismethod, a carrier stream of a solution of DPPH continuously flowsthrough an electrochemical cell and the sample zone containingantioxidant(s) is injected into the carrier stream therein reduction reac-tion of DPPH occurring within the sample zone. The decreased amountof the radical in the sample zone leads to a drop of the amperometricsignal at the modified-glassy carbon electrode.

Recently we proposed the novel approach to monitoring the reac-tion with DPPH by cyclic voltammetry (CV) and rotating disk electrode(RDE) techniques [34–38]. In Ref. [39] this method was successfullyapplied to study of substituted tetraphenyporphyrines antioxidantactivity.

In thisworkwe studied redox and antioxidant properties of biogenicmetal (Cu, Fe, Co, Mn, Zn, Ni) complexes 2–7 containing the [N-(3,5-di-tert-butyl-4-hydroxybenzyl)-N,N-di-(2-pyridylmethyl)]amine (L or 1)[40]. The metal complexes 2–7 could be described as polytopic agentssince theirmolecules possess of both organic antioxidant phenol groupscapable of hydrogen atom transferring and transition metal center thatis capable of electron transfer. Thesemetal-based antioxidants are of in-terest because of the next reasons: (a) the alteration of the chemicalstructures of metal complexes by changing metal or ligand nature;(b) the stabilization of phenoxyl radical formed by the assistance ofmetal ion in the intramolecular redox process; and (c) the attachmentof functional end-group in order to regulate the solubility and transportfunction of complexes [41].

The redox and antioxidant properties are tightly connected [42]. Theintroduction of appropriate metal may open newways for increasing ofthe antioxidant efficiency of such polyfunctional compounds and eluci-date the possible role of redox center in mechanism of their activity.Earlier, the synthesis of these complexes and the antioxidant activityassay using spectrophotometrical DPPH-test and CUPRAC method wasreported [40]. In this work the redox behavior and antioxidant proper-ties of compounds 1–7 were studied by means of CV and RDE.

2. Experimental

The compounds 1–4 and 6, and 7were synthesized as described ear-lier in Ref. [40]. The complex 5 (N-(3,5-di-tert-butyl-4-hydroxybenzyl)-N,N-di-(2-piridylmethyl)amine copper (II) dichloride) was obtained asfollows: the mixture of ligand 1 (210 mg, 0.5 mmol) and CuCl2·2H2O(85 mg, 0.5 mmol) in 6 ml of acetone was stirred at r.t. for 30 min.The resulting green precipitate was filtered and washed with acetone.Yield 210 mg, 76%, m.p. 205–210 °С (dec.). Elemental analysis, %:С27H35N3OCuCl2 found С 58.75, H 6.39, N 7.63, calculated С 58.53, H6.40, N 7.56. IR (KBr, cm−1), 3623 (OH), 3018–2869 (СH), 1708, 1608,1442. MS, m/z: 515 [C27H35N3OCuCl]+. UV, λmax, nm (lg ε), (EtOH)257.5 (4.1); 683.0 (2.2); (CH2Cl2) 229.0 (3.6), 262.5 (3.6), 759.5 (2.7).

All electrochemical measurements were carried out under argon atroom temperature. Cyclic voltammetry experiments were performedin classical three-electrode cell in CH3CN solution with 0.05 MBu4NBF4 as supporting electrolyte using a model IPC-Win potentiostat.The number of electrons transfered were determined by comparingwith the height of Fc2+/Fc3+ wave for the same concentration and byrotating disk electrode method (RDE) as well. A platinum or glassycarbon (GC) working electrodes with diameter 3 and 2 mm, corre-spondingly, platinum wire auxiliary electrode and aqueous Ag/AgCl/KCl (sat.) reference electrode were used. The same cell and electrodes

E, mV

-0.08

-3000 -2000 -1000 0 1000 2000

-0.04

0

0.04

0.08

I, mA

Fig. 2. Cyclic voltammogram of Co complex 2 on GC electrode (CH3CN, sweep rate100 mV/s, C = 10−3 M, 0.5 · 10−3 M n-Bu4NBF4, vs. Ag/AgCl/KCl).

-3000 -2000 -1000 0 1000 2000

E, mV

-0.02

-0.01

0

0.01

0.02

0.03

0.04I, mA

Fig. 1.Cyclic voltammogramof compound1 onGC electrode (CH3CN, sweep rate 100mV/s,C = 10−3 M0.5 · 10−3 M n-Bu4NBF4, vs. Ag/AgCl/KCl).

214 V.Y. Tyurin et al. / Journal of Electroanalytical Chemistry 756 (2015) 212–221

were used for measurements carried out by RDE method. The rate ofrotating was 2800 min−1. The solvents were routinely distilled anddried prior to use [43].

The antioxidant activity assay was performed using the electro-chemical DPPH-test [34–39] The reaction rate with DPPH was moni-tored by the rotating RDE technique, using the change of DPPHreduction limiting current value I (time of reaction from 60 to300 min, at various compound/DPPH ratios). The antioxidant efficiencywas expressed by twoways: the evaluation of the percentage of remain-ing DPPH concentration ((antioxidant efficiency) AOE = [(C0 − Cfin.) /C0] × 100%, where C0 is the initial concentration, Cfin.— thefinal concen-tration of DPPH when the kinetic curve becomes constant). In the caseof compounds 1 and 4 the antioxidant capacity also was estimated bydetermining of reaction stoichiometry, i.e., thenumber of radical speciesquenched by one molecule of antioxidant.

3. Results and discussion

3.1. Redox behavior of compounds 1–7

The experimental redox potential data are summarized in Table 1.

Scheme 1. Redox transfo

As it is seen the electrochemical behavior of compounds 1–7depends strongly upon electrode nature. The ligand 1 on GC-electrodeundergoes three-step oxidation (Fig. 1). The first two one-electronpeaks correspond to oxidation of ligand according to the proposedknown Scheme 1a [44]. The first step results in N-centered radical-cation formation followed by deprotonation, and the second one isassigned to one-electron phenol group oxidation. Since CH3CN maycontain some traces of water, the nucleofilic attack leads to C–C bondcleavage. The two-electron irreversible peak at the most anodic poten-tials is assigned to phenol group oxidation. Another possible pathwayof oxidation may involve the resonance structure participation(Scheme 1). CV in cathodic range contains two-electron wave corre-sponding the pyridine fragments reduction. At the same time CV of 1on Pt electrode shows two diffusion-controlled peaks at anodic range.The first one at Epa1 = 1.12 V corresponds to one-electron N-centeredoxidation and results in the radical-cation formation. The two-electronirreversible wave at Epa2= 1.68 V is assigned to phenol group oxidationaccording to ECE-mechanism (Scheme 1b).

The oxidation of complexes Co (2), Cu (5), and Zn (6) undergoes twosteps on both GC and Pt electrodes and also may be described by theScheme 1b (Fig. 2). However, oxidation of Mn(II) 3 and Ni(II) 7 com-plexes depends strongly on the electrode nature: CV of compounds 3

rmations of ligand 1.

-3000 -2000 -1000 0 1000 2000

E, mV

-0.04

-0.02

0

0.02

0.04

I, mA

Fig. 4.Cyclic voltammogramof Fe complex4 onGC electrode (CH3CN, sweep rate 100mV/s,C = 10−3 M, 0.5 · 10−3 M n-Bu4NBF4, vs. Ag/AgCl/KCl).

215V.Y. Tyurin et al. / Journal of Electroanalytical Chemistry 756 (2015) 212–221

and 7 shows three peaks at anodic range onGC electrode. The reversibil-ity of first peak in the case of Mn(II) complex 3 points out the high sta-bility of radical-cation formed (Fig. 3). At the same time the oxidation ofcomplexes 3 and 7 proceeds in two steps on Pt according Scheme 1b.Only in the case of the Fe(II) complex 4 one-electron peak appears onGC-electrode at Ера=0.45 Vwhichwas assigned to Fe(II)/Fe(III) transi-tion (Table 1). The quasi reversible character of this wave indicates thatthe structural rearrangement of complex takes place. The one-electronquasi reversible peak of N-centered aminogroup oxidation and irrevers-ible two-electron peak of phenol group oxidation are observed at moreanodic range (Fig. 4).

The cathodic behavior of compounds 2–7 is more complex. CV ofcomplex 2 on GC electrode contains three peaks in cathodic range(Fig. 2). The first one at Epc1 = −1.48 V is attributed to Co(II)/Co(I) transition [47,48]. The next wave corresponds to pyridine frag-ments reduction that results in radical-anion formation (Scheme 2). Atthemost cathodic potentials reduction Co(I)/Co(0) takes place. It shouldbe mentioned that the deposition of Co and peak of desorption onreverse scan were not observed: probably, the formation of anionicform of ligand stabilizes the Co(0) [45,46]. The only peak of Co(II)/Co(I) at Epc1 = −1.48 V transition appeared on Pt electrode.

The reduction of Mn(II) complex 3 on GC electrode undergoestwo steps: the pyridine fragments reduction followed by Mn(II)/Mn(0) transition (Scheme 3). The black deposition on electrode surfacein this case points out the complex destruction. The redox behavior of 3on Pt electrode is different: two peaks at the cathodic range are ob-served as well. But in this case the redox potential values are substan-tially shifted to more positive range (ΔE ≈ 0.7–0.8 V) that cannot beexplained by the influence of electrode nature (Epc1 = −1.30/1.07 V,Epc2 =−1.54/−1.50 V) only. Therefore, we can conclude that the dra-matic change of reduction mechanism is observed. The elucidation ofcathodic peaks nature in this case demands further investigations andwill be the subject of the next study. It should be mentioned that theoxidation intermediates of 3 formed in anodic range are more stableon Pt electrode (the degree of wave reversibility is higher).

CV of Fe complex 4 on GC electrode also contains two peaks incathodic range: the first one, at Epc = −1.7 В is assigned to pyridinereduction and the second one at Epc = −2.08 V corresponds to Fe(II)/Fe(I) transition (Scheme 4).

The character of Cu complex reduction depends strongly on the elec-trode nature. CV of compound 5 on GC electrode contains two peaksin cathodic range (Fig. 5a). The one-electron reversible peak at

E, mV

-0.08

-0.04

0

0.04

0.08

I. mAa b

-

-

-

-3000 -2000 -1000 0 1000 2000

Fig. 3. Cyclic voltammograms of Mn complex 3 on GC (a) and Pt (b) electrodes (CH3C

Ерс = −0.17/−0.08 V is assigned to Cu(II)/Cu(I) transition. In this casethe Cu(I)/Cu(0) reduction as well as the disproportionation 2Cu(I) →Cu(II) + Cu(0) is not observed. The absence of desorption peak on thereverse scan points out the high stability of Cu(I) intermediates formed.The two-electron peak at Ер = −1.87 V is assigned to simultaneouspyridine fragments reduction (Scheme 5a). On the reverse scanafter Ер = −2.2 V the reoxidation peak of low intensity appears at−0.36 V.

But on the Pt electrode the metal-centered reduction of the Cu(II)complex undergoes two steps. The of Cu(II)/Cu(I) transition atЕр = −0.20 В on Pt has a quasireversible character because of the ac-companying rate-limiting structural changes. The peak atmore cathodicpotential corresponds to Cu(I)/Cu(0) transition. On the reverse scan,after Cu(I)/Cu(0) potential, the characteristic triangular peak of

E, mV

0.06

0.04

0.02

0

0.02

0.04

0.06

I, mA

-2000 -1000 0 1000 2000

N, sweep rate 100 mV/s, C = 10−3 M, 0.5 · 10−3 M n-Bu4NBF4, vs. Ag/AgCl/KCl).

Scheme 2. Redox transformations of Co(II) complex.

Scheme 3. Redox transformations of Mn(II) complex.

Scheme 4. Redox transformations of Fe(II) complex.

216 V.Y. Tyurin et al. / Journal of Electroanalytical Chemistry 756 (2015) 212–221

desorption appears that suggests the complex destruction (Fig. 5b).Potential-controlled electrolysis at Ер = −1.0 V during 15 s leads tothe increase of height of this peak. It should be mentioned that thepeak of low intensity precedes the peak of desorption. This wave maybe assigned to reduction product of disproportionation reactionCu(0)+ Cu(II)= 2Cu(I) with another ligand environment (Scheme 5b).

The CV of compound 6 also contains two peaks in cathodic range(Fig. 6) corresponding to successive pyridine cycle and Zn(II)/Zn(0) reduction at E = −2.46 V (Scheme 6a). The slight reversibilityof this wave provides an evidence of Zn(0) state stabilization by the an-ionic ligand.

Voltammogram of Ni(II) complex 7 on GC electrode containsquasireversible peak of Ni(II)/Ni(I) reduction and two irreversiblepeaks at close potentials (Fig. 7a). Probably, Ni(II) reduction leads tothe change of complex geometry and the conjugation between pyridinerings results in their successive reduction (Scheme 6b). The fact that the

E, mV

-0.12

-0.08

-0.04

0

0.04

0.08

I, mA

-0

-0

0

0

0

Ia b

-3000 -2000 -1000 0 1000 2000

Fig. 5. Cyclic voltammogram of complex 5 on GC (a) and Pt (b) electrodes (CH3CN,

correspondingwave obtained by RDEmethod has two-electron charac-ter confirms this suggestion.

The redox behavior of 7 on Pt electrode is different. The peak of lowintensivity at cathodic range assigned to Ni(II)/Ni(I) transition atEpc1 = −0.99 V has an irreversible character (Fig. 7b). Second peakcorresponding pyridine fragments reduction is shifted to less negativepotentials (Epc2 = −1.42 V) comparing GC electrode (ΔE ≈ 300 mV)(Table 1). In this case the change of electrode nature leads to dramaticchange of redox mechanism.

Thus, we can conclude that in Zn(II) andMn(II) complexes the stabi-lization of metal low valent state takes place as a result of pyridine frag-ments reduction at less negative potentials. Тhe «anionic» character ofligand influences metal redox properties that leads to complex destruc-tion. The nature of electrode surface influence the mechanism of redoxprocesses. In general, the intermediates with low-valent state (I) and(0) are more stable on a more inert GC-electrode.

E, mV

.04

.02

0

.02

.04

.06

, mA

-2000 -1000-1500 -500 5000

sweep rate 100 mV/s, C = 10−3 M, 0.5 · 10−3 M n-Bu4NBF4, vs. Ag/AgCl/KCl).

Scheme 5. Redox transformations of Cu(II) complex.

217V.Y. Tyurin et al. / Journal of Electroanalytical Chemistry 756 (2015) 212–221

3.2. Study of antioxidant activity

The antioxidant activity of compounds 1–7was estimated using hy-drogen transfer reaction from phenol group to DPPH (Scheme 7a)which was monitored by the electrochemical methods.

It should be mentioned that this simple scheme considering the re-action as single step hydrogen transfer, in most cases doesn't reflectthe true mode of action. In works [9,10] the kinetics of three knownphenolic antioxidant (2,6-di-tert-butyl-4-methylphenol (BHT), eugenoland isoeugenol) interactionwith DPPHwas studied spectrophotometri-cally and complex mechanism including few different pathways wasproposed. It was also shown that propofol (2,6-di-isopropylphenol) re-action with DPPH proceeds through dipropofol formation which isquickly oxidized to corresponding quinone derivative [49]. The mecha-nismof antioxidant Trolox (TrOH) reactionwith DPPHwas investigatedin buffered hydroalcoholic media by using a stopped-flow system [50].It was shown that phenoxyl-type radical TrO• produced in the firststep of reaction may interact with remaining DPPH or might disappearthrough dimerization or disproportionation reactions involving

-3000 -2000 -1000 0 1000 2000

E, mV

-0.04

-0.02

0

0.02

0.04

I, mA

Fig. 6. Cyclic voltammogram of Zn complex 6 on GC electrode (scan rate 100 mV/s, C =10−3 M, 0.5 · 10−3 M n-Bu4NBF4, vs. Ag/Ag/Cl).

resonant forms. The stoichiometry of reaction was determined as ap-proximately 2. The pathways of the DPPH reaction with (+)-catechinin alcoholic solvents were studied [51]. It was concluded that once theintermediate o-quinone is formed the reaction proceeds in two path-ways, either the o-quinone reacts with catechin to form dimer whichis further oxidized to oligomers of higher molecular weights, or the A-ring of the o-quinone reacts further with another DPPH radicals toform the adduct of oxidized form of catechin with DPPH. Thus, we canconclude that in most cases the reaction mechanism includes not onlythe hydrogen atom transfer to DPPH from phenol moiety but also fur-ther transformations of radical intermediates formed.Moreover, antiox-idants may react through electron transfer concerted with, or followedby, proton transfer. An alternative mechanism assumes that ionizationof the phenol group precedes electron exchange and it was named se-quential proton loss electron transfer (SPLET) [52–55].

The cyclic voltammogramofDPPHdisplays two one-electron revers-ible peaks corresponding to oxidation and reduction of this radical(Scheme 7b) [56–58]. In this case the height of peak current is describedby the Randles–Shevchik equation [59]

Imax ¼ 2;72 � 105n3=2AD1=2C0v1=2; ð1Þ

where n — the number of transferred electrons, A — the electrode sur-face area, cm2, F — Faraday constant, D — diffusion coefficient, cm2/s,ν — potential scan velocity, V/s, and C0 — concentration of depolarizer,mol/cm3.

As it follows from the Eq. (1), at specified electrode surface area andpotential sweep rate, the ratio of radical reduction and oxidation peakcurrents during the reaction is equal to the concentration ratio:

I=I0 ¼ C=C0: ð2Þ

Here, I is a peak current for given concentration C of DPPH; I0 is apeak current for initial concentration C0 of DPPH.

Another possible approach to estimate the rate of reaction withDPPH is the application of rotating disk electrode (RDE). In this casethe diffusion current value Id can be found from Levich equation:

Id ¼ 0:62nAFD2=3ω1=2ν−1=6C0 ð3Þ

where n — number of transferred electrons, A — the electrode surfacearea, cm2, F — Faraday constant, D — diffusion coefficient, cm2/s, ω —the rate of rotating (rad/s), ν — kinematic viscosity of solution, cm2/s,and C0 — the depolarizer concentration, mol/cm3.

Thus, both these methods (CV and RDE) may be applied in order tomonitor the reaction rate and to obtain the quantitative characteristicsof antioxidant activity. However, the rotating disk provides constant

Scheme 6. Redox transformations of Ni(II) complex.

218 V.Y. Tyurin et al. / Journal of Electroanalytical Chemistry 756 (2015) 212–221

flow on electrode surface and regular stirring of solution thatmakes thisapproachmore preferable. Owing to this reason themonitoring of reac-tion with DPPH was completed using RDE technique in our work. Thevarious time of reaction (from 1 to 5 h) and different molar ratios ofcompound/DPPH concentrations (from 1:1 to 1:10) were applied.

The data obtained demonstrate the strong dependence of reactionrate in presence of compounds 2–7 upon metal nature. The Zn(II),Ni(II), and Co(II) complexes are practically inactive in reaction withDPPH at concentration ratio [DPPH]/[compound]= 1:1 (the initial con-centration C0 of DPPH decreases very slightly even when the time of re-action is 24 h). Thus, these compounds belong toweak antioxidants andfurther investigation of their properties seems unpromising. But in thecase of ligand 1 and Mn(II), Cu(II), and Fe(II) complexes the significantdecrease of DPPH concentration in presence of these compounds is ob-served. In this case the antioxidant activity may be quantitatively esti-mated using the percentage of DPPH remained:

AOE ¼ 1 − Cfin:=C0� � � 100 %ð Þ; ð4Þ

where C0 — initial concentration of DPPH, and Cfin.— concentration, de-termined from Eq. (2) for the moment of time when reaction reachedthe plateau.

Thus, the higher is AOA value the most antioxidant efficiency showsthe compound under study (Fig. 8). It could be seen that the Cu complexexhibits the activity lower than that of ligand 1. This fact could be ex-plained by the interaction of metal ion vacant d-orbitals with the elec-tron pair of nitrogen atom that results in decrease of complexreductive properties. At the same time the activity of Mn(II) complexis higher than that of ligand. Presumably, it can be caused by themetal ion stabilization of phenoxyl radical which is formed in the reac-tion with DPPH. The fact that the first peak in anodic range on CVA is

E, mV

-0.04

-0.02

0

0.02

0.04

0.06

I, mAa

-3000 -2000 -1000 0 1000 2000

Fig. 7. Cyclic voltammogram of Ni complex 7 on GC (a) and Pt (b) electrodes (swee

reversible supports this assumption. The form of kinetic curves in thepresence of 1 and Mn(II) complex is similar that points out the essen-tially same mechanism of action. Therefore, in the case of Cu(II) andMn(II) complexes the incorporation of metal ion doesn't lead to thechange of reaction mechanism. But in the case of Fe(II) complex 4 thesituation is different. At ratio [DPPH]/[compound 4] = 1:1 the DPPHconcentration almost immediately falls to zero, and it is impossible toobtain the kinetic curve. Thus, we can conclude that Fe(II) complex ex-hibits the high antioxidant activity and properties of this compoundwere studied more thoroughly at various concentrations.

The kinetic curves of DPPH reaction with compound 4 for a [DPPH]/[compound 4] ratios 2:1 and 10:1 are presented in Fig. 9.

It can be seen that the rate of reaction substantially depends on thecompound concentration. The highest reaction rate is observed at theinitial period (during approximately first 3–5 min). The rate of reactionat ratio [DPPH]/[compound] = 10:1 decreases more gradually. It isknown that BHT belongs to the class of slow antioxidants (the timeneeded to reach the steady state of reaction≈ 5 h), although stoichiom-etry of this reaction σ≈ 2.8 [7]. This fact indicates that the real mecha-nism of reaction consists of few possible pathways. In the case ofcomplex 4 the combination of 2,6-di-tert-butylphenol, dipicolylaminefragment and Fe(II) ion intends even more complex mode of action. Itwas obtained that at ratio [DPPH]/[compound] = 10:1 the AOE valueis 48.1(%) (Fig. 8). Thus, the EC50 of compound 4 is ≈12.5 μM. Thisvalue is comparable with EC50 of Trolox commonly used as a standard.As it was shown earlier [8] we can easily obtain the stoichiometry ofthe reaction as:

σ ¼ 100= 2 � EC50ð Þ≈ 4:

E, mV

-0.08

-0.04

0

0.04

0.08

0.12

I, mAb

-2000 -1000 0 1000 2000

p rate 100 mV/s, C = 10−3 M, 0.5 · 10−3 M n-Bu4NBF4, vs. Ag/AgCl/KCl (sat.)).

Scheme7.DPPH reactionwith phenolic antioxidants (a) and DPPH redox transformations(b).

Fig. 8. The antioxidant efficiency values AOE = (1 − C / C0) ∗ 100(%) at various [DPPH]/[compound] ratios:

1 ligand L, 1:4; 1a— ligand L, 1:1; 1b — ligand L, 4:1;2 Fe complex 4, 2:1; 2a— Fe complex 4, 10:1;3 Cu complex 5, 1:4; 3a— Cu complex 5, 1:1;4 Mn complex 3, 1:1; 4a— Mn complex 3, 1:2;5 DPA, 1:1; 5 — DPA, 1:2; 6 — TrOH, 10:1; (DPPH concentration 10−4 M).

219V.Y. Tyurin et al. / Journal of Electroanalytical Chemistry 756 (2015) 212–221

Thus, one molecule of Fe(II) complex can quench 4 or more DPPHradicals. In order to elucidate the possible mechanism of reaction westudied the interaction of ligand 1, FeCl2 and dipicolylamine (DPA)with DPPH (Fig. 10). For the FeCl2 the reaction proceeds also very fast,but stoichiometry σ = 1. On the other hand, the ligand 1 reacts slowlyand the time needed to reach the steady state (plateau) at concentra-tions ratio [DPPH]/[compound] = 1:1 is almost 5 h. But in the case of1 the value σ ≈ 3 is almost the same, as in the case of BHT. The DPAshows antioxidant activity as well, but the reaction proceeds with alow rate and σ ≈ 0.5 (Fig. 10).

Therefore, we may conclude that the fast step of reaction at initialperiod of time is associated with the DPPH reduction by Fe(II), and thefollowing comparatively slow process of propagation is assigned to li-gand 1 interaction with DPPH. This assumption let us to suppose thepossible action mechanism of complex 4.

4. Discussion

The research demonstrates that the antioxidant and redox proper-ties of compounds 1–7 are strongly connected. The easy oxidized com-plex 4 interacts with DPPH very fast and stoichiometry of reaction ismore than 4. Since the rate of process is highest on the initial stage (ap-proximately 3–5 min) we can propose that the first step of reaction isthe reduction of DPPH due to electron transfer from Fe(II) ion(Scheme 8). The next possible step is the phenol fragment reactionwith another molecule of DPPH that leads to the formation of corre-sponding radical which exists in two forms [9]. This radical is able toreact with another DPPH resulting in quinonoid structure formation.Another possible step is the attack of twoDPPH radicals on one ofmeth-ylene groups in dipicolylamine fragment leading to final product. Thus,

Scheme 8. Possible pathways of DPP

this possible scheme involves fourDPPH radicals quenchingbyonemol-ecule of complex 4 that is agreed with stoichiometry derived from ki-netic data. Moreover, this consequence of stages explains the ratedependence on time: very fast interaction in initial period of reactioncaused by reduction of DPPH, and relatively slow propagation reactionfurthermore.

These data were compared with the results obtained earlier bymeans of spectrophotometric DPPH test and CUPRAC method [40].The data derived by these methods (EC50 values, rate constants k inDPPH reaction and Cu2+ reducing activity in Trolox-equivalents) dem-onstrate that themost active reducing agent is complex 4, probably dueto the presence of readily oxidizable Fe(II). This conclusion coincidewith the results of electrochemical DPPH test. Some differences inEC50 values obtained by spectrophotometric and electrochemical ap-proaches may be connected with different experimental conditions(solvent, time of reaction etc.)

5. Conclusions

The electrochemical behavior and antioxidant properties ofpolytopic complexes 2–7were studied by means of cyclic voltammetry

H reaction with Fe(II) complex.

Fig. 9.Kinetic curves of DPPH reactionwith: 1— Fe complex 4, [DPPH]/[compound]— 2:1;2 — TrOH, 10:1; 3 — Fe complex 4, 10:1 (DPPH concentration 10−4 M).

220 V.Y. Tyurin et al. / Journal of Electroanalytical Chemistry 756 (2015) 212–221

and rotating disk electrode methods, and the schemes of oxidation/reduction were proposed. It was shown that the electrochemicalcharacteristics and antioxidant activity are strongly connected: themost easily oxidized complex 4 demonstrates highest antioxidant ef-ficiency. Comparison with the data derived by CUPRAC method andspectrophotometrical DPPH-test [40] shows that these results corre-late: the Fe(II) complex demonstrates the high activity in all tests.The scheme of reaction between complex 4 and DPPH explainingthe stoichiometry observed is given. We can conclude that the elec-trochemical approach to antioxidant activity assay allows one to ob-tain verified results, to elucidate the possible mechanism of actionand can be applied for study of antioxidant properties of compoundsin the case when investigation by spectrophotometrical DPPH-test isrestricted.

Acknowledgments

This work was supported by the RFBR 15-03-03057-a.

Fig. 10. Kinetic curves of DPPH reaction with: 1 — compound 1, [DPPH]/[compound] =1:4; 2 — compound 1, [DPPH]/[compound] = 1:1; 3 — DPA, 1:2; 4 — compound 1,[DPPH]/[compound] = 4:1 (DPPH concentration 10−4 M).

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