Electrochemical Oxidability of Antioxidants: Synergism and Antagonism in Mixes.

AIMS: To investigate electrochemical oxidability of antioxidants to reveal synergistic and antagonistic effects in mixes.
MATERIALS AND METHODS: Electrochemical oxidability of some widely used antioxidants, including uric acid, glutathione, trolox, ascorbic acid, gallic acid, Mexidol, and potassium fenozan, was investigated by the amperometric approach.
RESULTS: All obtained electrochemical oxidability values correlate well with antioxidant activity parameters of the same compounds measured by other methods. The measurements of the electrochemical oxidability for binary mixes of substances were tested to reveal any synergistic actions. The experimental results and calculated values overlap for various combinations of tested mixes. It testifies to the absence of interaction between them (both synergism and antagonism) in the oxidation process.
CONCLUSIONS: The constants of oxidation were defined for different antioxidants and mixes by amperometric approach. Most mixes of probed compounds revealed absence of interaction between them in oxidation process. In some cases (mainly with glutathione and ascorbic acid), antagonism takes place, deteriorating effects of their joint application. Apparently, a partial reduction of glutathione by ascorbic acid leads to excess of the measured value over calculated value.

INTRODUCTION

Different electrochemical methods are widely used for the determination of redox properties of different biological objects, including dietary supplements, blood plasma, and so on.[12345678910111213] One of them is the amperometric method developed by Jashin et al.[6] The recent works[7814] showed the possibility to determine the total content of antioxidants in different natural objects. It is especially important regarding possible prevention of oxidative stress using antioxidants.[15] Biryukov[16] paid attention to features of determination of antioxidant concentration by amperometric method. It was shown that this method is quite suitable for the measurement of electrochemical oxidability of compounds but can give essential errors in case of measurements of the total antioxidant content. It is considerably expressed in mixes containing components with notably different values of electrochemical oxidation constant (this parameter correlates to radical scavenging activity). There are few results of oxidizing characteristic determination and their comparison with the antioxidant parameters received by other methods.[17] Thus, there is still less information for evaluating the interaction between antioxidants regarding their redox properties. It is necessary to determine such parameters for drug and food supplement. The electrochemical methods are the most appropriate for this task because the redox processes are basic and are precisely revealed by amperometric and voltamperometric methods.

This work aimed to determine the oxidation constants of some widely used bio-antioxidants and their binary mixes, and to evaluate the synergistic or antagonistic interaction in mixes by the amperometric approach.

MATERIALS AND METHODS

Reagents

The following substrates were used in the experiment [Figure 1]: uric acid (UA), glutathione reduced (Gl) and glutathione oxidized (Gl ox.), trolox (Tr) (a water-soluble analog of α-tocopherol), ascorbic acid (AA), gallic acid (GA), Mexidol (M), and potassium fenozan (FP). All these substances are used as oxidation inhibitors in different biological systems, food additives, and medicines, separately and in mixtures. As shown in Figure 1, all tested substances are of different structure, but they possess significant well-known antioxidant properties. Most of these substances are used as standard antioxidants as well.

Figure 1

Structural formulas of tested compounds: gallic acid (GA), trolox (Tr), Mexidol (M), potassium fenozan (FP), ascorbic acid (AA), uric acid (UA), glutathione reduced (Gl)

Apparatus

The measurements were taken on the device “Zvet-yauza-01-AA” (NPO Khimavtomatika ltd, Moscow, Russia) in which the amperometric method was carried out.[9] This method is based on the measurement of the electric current arising during electrochemical oxidation of investigated substance on a surface of a glassy carbon anode with electric potential of +1.3 V.[9] The electrochemical oxidation proceeding under scheme R–ОН → R–О• + e– + H+ can be used as a model for the measurement of free radical absorption activity. Capture of free radicals is carried out according to the reaction: R–ОН → R–О• + Н•. Both reactions include the rupture of the same bond, О–Н or S–H. In this case, the ability of the same type of antioxidants to capture free radicals directly correlates with the oxidability of these compounds on a working electrode of the amperometric detector.[10111213] Oxidation current depending on time registers when probe passes through an electrochemical cell. The signal of amperometric detector is recalculated as integral on current curve vs. time (area under current curve S = ʃ i × dt) and represented as nА·s. After measuring oxidation constants for the individual antioxidants (k) and knowing their concentration Ci, we calculate the areas under a current curve for binary mixes as Sc and to compare them with experimentally measured values (Se). Theoretical oxidation values were calculated for binary mixes as i = k1·C1 + k2·C2, where C1 and C2 are concentrations of components, and k1 and k2 are oxidation constants. Time of integration is identical for different substances and is defined by time of passing of probe through electrode spacing. The standard deviation (SD) of the measured Se values was not more than 5%.[918] Experiments were performed at least five times. Statistical analysis was performed with the help of MS Excel 2010 Software (Microsoft, USA). Results are presented as mean ± SD.

RESULTS

Dependence of oxidizing parameter (S) on concentration C of tested substances is shown in Figure 2. Oxidability is expressed in nanoamperes per second by amperometric approach. Direct linear dependence on the concentration is observed for all samples [Figure 2].

Figure 2

Dependence of the oxidizing parameter (S) on concentration (C) for six different antioxidants: 1, gallic acid (GA); 2, trolox (Tr); 3, uric acid (UA); 4, ascorbic acid (AA); 5, Mexidol (M) and potassium fenozan (FP); 6, glutathione reduced (Gl); (n = 5)

On the basis of these results, the constants of oxidation, K = S/C (nA·s/µM), were calculated. The highest oxidation coefficient with K = (384±16) nA·s/µM was shown for gallic acid [Table 1].

Table 1

Oxidation coefficients of individual antioxidants measured by amperometric method (n = 5, mean ± standard deviation)

The measurements of electrochemical oxidability for 15 different combinations of binary antioxidant mixes (1:1) revealed a wide range of values. The concentration of all antioxidants in mixes was identical (2.5 µM/L). Comparative diagrams of calculated (Sc = C (Ki+ Kj)) and experimentally measured Se values with SDs are provided in Figure 3. Most of the values correlate well.

Figure 3

Diagrams of calculated Sc (red columns) and experimentally measured Se (green columns) oxidability values of binary mixes (1:1) of tested substances at concentration of 2.5 µM (n = 5, bar mean ± standard deviation [SD])

DISCUSSION

Antagonism or synergism appears due to the differences in the chemical structure, and hence, different mechanisms underlying unique oxidation–reduction properties are seen. Obtained results revealed diversity in oxidizing properties of antioxidants [Table 1]. As shown in Figure 2, all antioxidants were oxidized in a dose-dependent manner. However, oxidized glutathione gives a response at the level of measurement noise (~5 nA) and therefore its oxidability was not significant and not shown. This clearly proved that electrochemical oxidability of these compounds depends on the molecular structure of specific groups, which is supported by literature.[11] In particular, oxidability of phenolic antioxidants depends on level of screening of hydroxyl groups in a benzene ring and on ortho-effect of two hydroxyl groups or hydroxyl and carbonyl groups. The screening of OH group leads to essential increase in efficiency of antioxidants in comparison with unshielded phenol. It explains the differences in oxidability and antioxidant properties of phenolic substances. Tertiary butyl deputies in ortho-situation increase electronic density on OH group, reducing energy of its dissociation.[1920] Antioxidant activity of complex drug, especially plant extracts, also strongly depends on total phenolic content. High oxidability of gallic acid is explained by the existence of three electron donor OH deputies in its molecule. This O–H bond revealed durability or energy of dissociation of approximately DOH = 347 kJ/mol,[20] and electrochemical potential of oxidation on the glassy carbon anode equals 0.4 V.[21] From this point of view, dissociation energy of trolox (the feeblest O–H bond) is higher[20]; therefore, oxidability under the same conditions is less. There is not enough information about dissociate energy and parameters of oxidation for non-phenolic antioxidants, such as uric acid. Uric acid has rather low potential of oxidation. Uric acid is oxidized at potential about 0.65 V on the graphite electrode in water media. The resulted product of two-electronic oxidation is allantoin.[22] Well-known ascorbic acid is a derivative of a monosaccharide and is a strong reducer. In the presence of oxygen, ascorbate is oxidized quickly with the formation of dehydroascorbic acid. However, in case of electrochemical oxidation, there is destruction of O–H bonds in a molecule of ascorbic acid. At the same time, the molecule loses two hydrogen atoms. The potential of ascorbic acid oxidation on a graphite electrode is approximately 0.384 V.[23] Considerable reduction of electrochemical oxidation potential is achieved, when different modifiers of an electrode surface are used.[24] Mexidol and potassium fenozan belong to spatial complicated phenols as well as monophenols, and they are feeble inhibitors and reducers. Phenolic and benzoic acid derivatives also possess radical scavenging potential because of the electron-donating substituent. This process becomes even more significant in the evaluation of oxidizing and antioxidant properties in complex mixes. Glutathione is an important coenzyme for glutathione peroxidase activity. It also provides protection to sulfhydryl groups of proteins against oxidation, and at the same time, glutathione itself is oxidized to a disulfide. The relation of reduced and oxidized glutathione is an index of oxidative stress.[252627] Chemically modified electrodes, functioning on the principles of electrocatalysis, revealed decrease in oxidation potential for glutathione for up to 0.6 V.[25] In case of glassy carbon electrodes, oxidation occurs with big overvoltage, and oxidability of glutathione declines.[2527] The values of oxidation coefficients received in this work correlate well with the antioxidant parameters of the same compounds measured by other methods.[2829] For example, comparison of oxidability and antiradical activity by chemiluminescence method showed correlation coefficient of approximately 0.97.[28] Other methods also showed rather good correlation of oxidability and antioxidant activity for some tested antioxidants.[29]

Electrochemical oxidability of different antioxidant mixes (1:1) was assessed for finding possible interaction between antioxidants (synergism or antagonism) or absence of mutual influence. The results revealed that calculated and experimental values match each other within the errors of measurements for majority of tested mixes [Figure 3]. It is explained with independent electrochemical oxidation of these antioxidants and absence of any interaction between separate substances during oxidation. However, exception is made by mixes gallic acid and glutathione reduced, uric acid and glutathione reduced, Mexidol and ascorbic acid, and glutathione reduced and ascorbic acid, for which the measured S values are noticeably less than the calculated one. It is directly linked to the existence of chemical antagonism for these couples of antioxidants, resulting in interaction and formation of hardly oxidizing substrates. Regarding mixes with ascorbic acid, it can be partly explained by decay of ascorbic acid during measurements. The combination of Mexidol with glutathione reduced and Mexidol with trolox shows excess measured values over calculated values. It demonstrates, apparently, increasing oxidation of these components in the presence of each other or formation of the intermediate strong reducer (synergistic interaction of both components). Similar results were obtained for mixes with double the concentration for each of the separate antioxidants, i.e., approximately 5.0 µM. In case of mixes of antioxidants with oxidized glutathione, no significant changes were observed, except for mixture with ascorbic acid, where the moderate (8%–10%) excess of Se over Sс took place. It is due to partial reduction of oxidized glutathione by ascorbic acid. It should be noted that ascorbic acid has relatively low stability in solution, which could affect results in mixes with ascorbic acid in all experiments.

CONCLUSION

We revealed absence of interaction between most of the antioxidants in oxidation process. Antagonism takes place mainly in the presence of glutathione and ascorbic acid, deteriorating effects of their joint application. Opposite, mixes with Mexidol demonstrates gain of oxidability of components, i.e. synergistic antioxidant effect. Obtained results could be used for the prognosis of individual antioxidants and mixture applications in the food, pharmaceutical, and cosmetic industries.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.