Introduction

Nowadays a man is exposed to life under constant stress, including oxidative stress (Sies 2020). In general, stress should be defined as a process evoked by environmental factors (the so-called stressors), causing a threat to the body balance or affecting it significantly (Salleh 2008). On the other hand, oxidative stress should be treated as an imbalance between oxidation and reduction reactions in the body (Betteridge 2000; Pizzino et al. 2017). It occurs when reactive oxygen and nitrogen species, including free radicals, are stressors. Oxidative stress is a condition that can lead to faster aging of body tissues and cells, which in turn can cause many serious diseases (Forman and Zhang 2021).

Substances that prevent formation of reactive oxygen and nitrogen species, neutralize these forms or eliminate the damage caused by them are antioxidants (Neha et al. 2019). Despite the fact that many substances with antioxidant properties are known today, research is still being carried out to expand their resources. This is justified by the fact that many antioxidants, especially synthetic ones, are believed to possess a mutagenic activity (Gulcin 2020). Therefore, antioxidants derived from plant products, including polyphenolic compounds, whose antioxidant properties result mainly, but not only, from the presence of hydroxyl groups in their structure become very popular (Biela et al. 2020; Olszowy-Tomczyk and Wianowska 2023a). It should be noted here that the active substances in the plant do not occur individually, but in the form of mixtures. It cannot be ruled out that the accumulation of a large variety of compounds with different properties, including antioxidants, in such a mixture, may cause that the presence of some ingredients affect the properties of others.

As reported in the literature the antioxidant properties of a mixture are not always the sum of the properties of its individual components. There is also a negative (antagonistic) or positive (synergistic) mutual influence of the mixture components on its resultant antioxidant effect (Dawidowicz et al. 2021). Moreover, in addition to its active ingredients, a plant can contain components that, despite not possessing antioxidant properties, they can contribute to an improper assessment of antioxidant properties of the extract obtained from it (e.g., metal ions, hydrogen ions or water) (Olszowy-Tomczyk and Wianowska 2023b).

Taking the above into account, the main aim of the study was to investigate how not only one antioxidant affects another in the mixture, but also to check whether the presence of ingredients without antioxidant properties affects the antioxidant effect of this mixture. Simple measurement systems containing quercetin and kaempferol, as well as quercetin and chlorogenic acid were selected for the study (the structural formulas of the compounds mentioned are shown in Fig. 1). The choice of these antioxidants was dictated by the fact that all these compounds are part of the daily diet. They are consumed by the body mainly in the form of vegetables, fruits and drinks. Quercetin can be found, among others, in onions, grapes, blueberries, cherries, broccoli and citrus fruits (David et al. 2016). Kaempferol occurs in large amounts in onions, broccoli, chives, tea, grapes, tomatoes and strawberries. Chlorogenic acid, on the other hand, is a bioactive phenolic compound found in potatoes, eggplants, apples, plums and coffee beans (Somerset and Johannot 2008; Wianowska and Gil 2019). Moreover, quercetin and kaempferol occur side by side, among others: in apples, asparagus, broccoli, kale, onions, spinach, dill, fennel and chives (Dabeek and Marra 2019); while, quercetin and chlorogenic acid can be found together in apples, elderberries, grapes, plums and peaches (Alverez-Parrilla et al. 2005, Maslin et al. 2022; Nile et al. 2013)., Additionally, a decision was made to examine whether the presence of metal ions, hydrogen ions, water and another solvent affects the resultant antioxidant effects of the binary mixture containing the above-mentioned antioxidants. Antioxidant properties were determined after a 60-min neutralization reaction of the colored ABTS cation radical with the antioxidant(s).

Fig. 1
figure 1

Chemical structures of the examined compounds

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Experimental

Materials and methods

Chemicals

Chlorogenic acid, kaempferol, quercetin, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and potassium persulfate (di-potassium peroxdisulfate) were purchased from Sigma Aldrich (Poznań, Poland). Copper (II) chloride nine hydrate, disodium hydrogen phosphate dehydrate, iron (III) sulfate (VI) pentahydrate, phosphoric acid (V), sodium dihydrogen phosphate dihydrate, ethanol (EtOH), methanol (MeOH) were purchased from the Polish Chemical Plant POCh (Gliwice, Poland).Water was purified using a Milli-Q system from Millipore (Millipore, Bedford, MA, USA).

Antioxidant properties of individual substances and their binary mixtures

The antioxidant properties of chlorogenic acid, kaempferol and quercetin as well as of the binary mixture of quercetin with chlorogenic acid and quercetin with kaempferol were investigated at various volume ratios of reagents in the mixture. For this purpose, methanolic solutions of chlorogenic acid, kaempferol and quercetin were prepared with a concentration of 0.06 mg/mL. Binary mixtures were prepared by mixing individual components using different volume compositions. The volumes of reagent solutions (antioxidants and radicals) used in the systems containing only single antioxidants and their binary mixtures are presented in Table 1.

Table 1 Volumes of solutions used for the determination of the antioxidant properties of chlorogenic acid with quercetin and kaempferol with quercetin, and their binary mixtures
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Impact of solvent

Influence of the solvent (methanol or ethanol) on the antioxidant properties of chlorogenic acid, kaempferol and quercetin as well as of the binary mixture of quercetin with chlorogenic acid and quercetin with kaempferol was investigated at various volume ratios of reagents in the mixture. For this purpose there were prepared, methanolic and ethanolic solutions of chlorogenic acid, kaempferol and quercetin with a concentration of 0.06 mg/mL, which were mixed at different volume ratios. The effect of ethanol was determined using the same volumes of components of the tested systems as in methanol, with the difference that ethanol was the solvent of all the solutions used to form the systems. The volumes of reagent solutions (antioxidants and radicals) used in one-component systems (containing a single antioxidant) and two-component systems (being a binary mixture) are presented in Table 1.

Influence of the addition of water, metals and hydrogen ions on the antioxidant properties of the substances and their binary mixtures

The effect of the addition of water, metal ions and hydrogen ions on the antioxidant properties of kaempferol, chlorogenic acid, quercetin as well as of the binary mixtures of quercetin with chlorogenic acid and quercetin with kaempferol was determined by adding 100 μl of the tested factor (water or an aqueous solution of copper (II) or iron (III) ions with the ion concentrations of 0.001 mg/mL and 0.022 mg/mL, respectively, or buffer with a specific pH) to the systems containing the solutions of: antioxidant/antioxidants and the radical cation. The components volumes of the measuring systems containing both single antioxidants and their binary mixtures are presented in Table 2.

Table 2 Volumes of solutions used for the determination of the impact of chosen factors on the antioxidant properties of chlorogenic acid and kaempferol, and their binary mixtures
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Preparation of the radical cation

Antioxidant properties of the tested antioxidants and their mixtures were determined using the ABTS method (Gulcin et. al 2019; Taslimi et al. 2020). The ABTS cation radical used in the experiments was prepared as a result of the reaction of 2,2'-azinobis(3-ethylbenzenothiazoline-6-sulfonate) (ABTS) (5 mL c = 7 mM) with potassium persulfate (K2S2O8) (88 µl c = 140 mM K2S2O8). The generation of the cation radical lasted 12 h at room temperature in the absence of light (Nenadis et al. 2004). Before each measurement, the radical cation was diluted with a solvent to an absorbance of 0.7 ± 0.02 at a wavelength of 744 nm. The absorbance changes were monitored at 744 nm using a UV Probe-2550 spectrophotometer. Measurements were performed in the systems containing specific volumes of antioxidants or binary mixtures and solutions of factors differentiating the tested systems (accurate volumetric compositions of the measuring systems are presented in Tables 1 and 2). Antioxidant properties were determined as % inhibition, which was calculated using the following equation:

$$I\left( \% \right) = (1 - \frac{{A_{60} }}{{A_{0} }}) \cdot 100\%$$

where: A0 and A60 are the values of ABTS●+ absorbance at 0 and 60 min of the radical neutralization reaction, respectively.

Statistical analysis

All results are presented as mean values of three independent measurements ± standard deviation (SD). The one-way analysis of variance (ANOVA) and Fisher coefficient (F) value were used to assess the influence of experimental factors on the antioxidant activity. The statistical analysis was performed using Excel (Microsoft Excel 2010).

If the calculated value of F (Fcal) exceeds the tabular value F (Ftab), this indicates a statistically significant effect of the given parameter. To determine the significance of each Fisher coefficient, the p values were used. The values were considered to be significantly different when the result of the compared parameters differed at the p = 0.05 significance level.

Results and discussion

Figures 2, 3, 4 and 5 show the antioxidant properties expressed as % inhibition for the systems containing:

  • Variable amount of chlorogenic acid (dashed line with crosses, Figs. 2A, 3A, C, 4A, C and 5A, C,E,G, I. Variable amount of quercetin (dotted line with squares)

  • Variable amount of kaempferol (dashed line with diamonds—Figs. 2B, 3B, D, 4B, D and 5B, D, F, H, J)

  • A mixture composed of quercetin and chlorogenic acid (dashed-dotted line with dots in Figs. 2A, 3A, C, 4A, C and 5A, C, E, G, I) and quercetin and kaempferol (dashed -dotted line with dots in Figs. 2B, 3B, D, 4B, D and 5B, D, F, H, J). Additionally, all figures include a curve called the "calculated curve". It shows the antioxidant properties that the mixture should have, assuming that they are the sum of the antioxidant activities of the antioxidants included in it. It should be noted here that the summed values represent the % of inhibition obtained in the single-component systems containing the same amount of antioxidant as in the binary mixture. In all figures the x-axis represents the amount of the added ingredient. The upper x-axis should be read from right to left. Each of the presented graphs was drawn on the basis of the data obtained in various measurement systems, which were differentiated according to the type of antioxidants (Fig. 2), the used solvent (Fig. 3), the presence of water (Fig. 4), metal ions and hydrogen ions (Fig. 5). In the experiments there were used methanol or ethanolic solutions of the tested antioxidants with a concentration of 0.06 mg/mL, which were mixed in various volume proportions, obtaining binary mixtures (20/80; 50/50; 80/20 v/v—see Table 1).

Fig. 2
figure 2

Antioxidant activity changes assessed by the ABTS method for the systems containing different volumes of: quercetin solutions (dotted line with squares) with chlorogenic acid solutions (dashed line with crosses) (A) and quercetin solutions (dotted line with squares) with kaempferol solutions (dashed line with diamonds) (B), and binary mixtures (dashed-dotted line with dots). The solid line with triangles corresponds to the expected activity values for the tested pairs of compounds. The experimental values are the mean values for n = 5

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Fig. 3
figure 3

Antioxidant activity changes assessed by the ABTS method for the systems containing different volumes of methanolic (A, B) and ethanolic (C, D) solutions of: quercetin (dotted line with squares) with chlorogenic acid (dashed line with crosses) and quercetin (dotted line with squares) with kaempferol (dashed line with diamonds), and binary mixtures (dashed-dotted line with dots). The solid line with triangles corresponds to the expected activity values for the tested pairs of compounds. The experimental values are the mean values for n = 5

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Fig. 4
figure 4

Antioxidant activity changes assessed by the ABTS method for the systems containing different volumes of methanolic (A, B) and methanolic with water addition (C, D) solutions of: quercetin (dotted line with squares) with chlorogenic acid (dashed line with crosses) and quercetin (dotted line with squares) with kaempferol (dashed line with diamonds), and binary mixtures (dashed-dotted line with dots). The solid line with triangles corresponds to the expected activity values for the tested pairs of compounds. The experimental values are the mean values for n = 5

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Fig. 5
figure 5

Changes in antioxidant activity assessed by the ABTS method for systems containing different volumes of methanolic solutions of quercetin (dotted line with squares) with chlorogenic acid (dashed line with crosses) and quercetin (dotted line with squares) with kaempferol (dashed line with diamonds), and binary mixtures (dashed-dotted line with dots) with the addition of water (A, B), ions iron (C, D), copper ions (E, F), buffer with pH = 1 (G, H) and buffer with pH = 3 (I, J). The solid line with triangles corresponds to the expected activity values for the tested pairs of compounds. The experimental values are the mean values for n = 5

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For better understanding of the presented relationships, four points are used in Fig. 2A(a, b, c, d). The point "a" corresponds to the % inhibition value obtained for the system containing 50 μl of chlorogenic acid (i.e., value 22.62%, measurement system No. 2 in Table 1); while, the point "b" indicates the % inhibition in the measurement system containing 50 μl of quercetin (i.e., the value 41.82%, measurement system no. 5 in Table 1). After summing up these values, the value of 64.44% is obtained, which is presented in the figure with the point "d" positioned on the "calculated curve". This is the so-called the expected value (Ic) that the mixture should have, assuming the additivity of the antioxidant properties of its components. However, the actual (experimental) % inhibition (Ie) obtained for the mixture containing 50 μl of chlorogenic acid and 50 μl of quercetin is lower and equals 57.18% (point "c" in Fig. 2 A, measurement system no. 11 in Table 1).

Tables 3, 4, 5, 6 and 7 present the results of statistical analyses obtained for the differences between the inhibition percentage determined experimentally (Ie) and the theoretical inhibition percentage (Ic), which is the sum of the inhibition percentage determined for the individual antioxidants (for a solution in which the amount of the ingredient, i.e., chlorogenic acid, kaempferol and quercetin corresponds to the amount present in the mixture). The tables contain the data obtained for % inhibition determined at various volume ratios of antioxidants in their mixture. In the interpretation of the results, it was assumed that the lack of a significant difference between the Ie and Ic values indicates the additive antioxidant effect of the mixture. However, statistically significant differences (Fcal > Fcrit) indicate antagonistic or synergistic antioxidant effects of the mixture. In the case of the first of the two mentioned effects, the difference between Ie and Ic has a negative value, while for the second effect it has a positive value.

Table 3 Statistical significance (F and p values) of the difference between the experimental inhibition percent (Ie) and calculated inhibition percent (Ic) for the binary mixtures of the chlorogenic acid with quercetin, and kaempferol with quercetin at three different volume ratios together with the difference (Ie-Ic) and with the observed effect (the resultant antioxidant effect of the antioxidants in the mixture (Fcrit = 7.71)
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Table 4 Statistical significance (F and p values) of the difference between the experimental inhibition percent (Ie) and calculated inhibition percent (Ic) for the binary mixtures of chlorogenic acid with quercetin and kaempferol with quercetin at three different volume ratios and in different solvent together with the difference (Ie-Ic) and with the observed effect (the resultant) antioxidant effect of the antioxidants in the mixture (Fcrit = 7.71)
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Table 5 Statistical significance (F and p values) of the difference between the experimental inhibition percent (Ie) and calculated inhibition percent (Ic) for the binary mixtures of chlorogenic acid with quercetin and kaempferol with quercetin at three different volume ratios and in the system with methanol and with methanol in the presence of water addition together with the difference (Ie-Ic) and with the observed effect (the resultant) antioxidant effect of the antioxidants in the mixture (Fcrit = 7.71)
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Table 6 Statistical significance (F and p values) of the difference between the experimental inhibition percent (Ie) and calculated inhibition percent (Ic) for the binary mixtures of chlorogenic acid with quercetin and kaempferol with quercetin at three different volume ratios and in the systems with metal ions additives (Fe 3 3+ or Cu 2 +) together with the difference (Ie-Ic) and with the observed effect (the resultant) antioxidant effect of the antioxidants in the mixture (Fcrit = 7.71)
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Table 7 Statistical significance (F and p values) of the difference between the experimental inhibition percent (Ie) and calculated inhibition percent (Ic) for the binary mixtures of chlorogenic acid with quercetin and kaempferol with quercetin at three different volume ratios and in the systems with hydrogen ions additives (pH = 1 or pH = 3) together with the difference (Ie-Ic) and with the observed effect (the resultant) antioxidant effect of the antioxidants in the mixture (Fcrit = 7.71)
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Antioxidant properties of mixtures containing chlorogenic acid and quercetin, and kaempferol and quercetin

Figure 2 shows the antioxidant properties in the systems containing single antioxidants (chlorogenic acid and quercetin—Fig. 2A as well as kaempferol and quercetin—Fig. 2B) and their binary mixtures. For the systems containing single components, an increase in antioxidant properties is observed with the increasing in the amount of a given antioxidant in the tested measurement system. This is nothing unusual because a greater amount of a given antioxidant in the measurement system, the greater the antioxidant properties. It seems more important here to pay attention to the theoretical and experimental curves. Comparing the data in Fig. 2A and 2B, and more precisely the course of the experimental and theoretical curves, it can be seen that in both cases the courses are different and the experimental curve is below the theoretical curve. This is confirmed by the data in Table 3, which as mentioned above, show the statistical significance (F and p values) of the difference between the experimental percentage of inhibition (Ie) and the calculated percentage of inhibition (Ic) for these binary mixtures. Antioxidant properties are not the result of the additive effect of antioxidants in the mixture. In most cases, the experimental values are smaller than the calculated ones (negative value of the Ie-Ic difference), which may indicate antagonism. The data in Table 3 this observation in most cases.

As shown by the data in Table 3, the mixture containing chlorogenic acid and quercetin has an antagonistic effect regardless of the volume ratio of antioxidants in the mixture. While the mixture containing kaempferol and quercetin exhibits both antagonistic and additive effects. In the case of the first mixture, it was noticed that the antagonism is greater the greater the amount of chlorogenic acid is (increasing F values and smaller p values are observed); while, the kaempferol/quercetin mixture has an antagonistic effect when both antioxidants are present in the mixture at the same volume ratio. The antagonistic effect of the mixture containing chlorogenic acid and quercetin was found in the paper published by of Corrigan et al. (Corrigan et al. 2023) but the authors did not attempt to explain the antioxidant behavior of this mixture. In the literature the antagonism between antioxidants in a mixture is most often explained based on the involvement of a strong antioxidant in the rebuilding of a weaker one (Uduwana et al. 2023). Due to this, the participation of the former in the neutralization of the colored radical is limited. In both mixtures, quercetin has stronger properties (which can be observed comparing the % inhibition obtained in the systems containing single antioxidants). Therefore, rebuilding of chlorogenic acid by quercetin may explain the antagonism found in a mixture containing these two antioxidants. However, this hypothesis does not explain the antioxidant activity (additive and antagonistic) observed for the kaempferol/quercetin mixture.

The presented data can also indicate that the observed antioxidant antagonism in the mixtures of tested antioxidants does not result from interactions between the individual components of the mixture, but from differences in the kinetics of the reaction of a given antioxidant with the ABTS cation radical. It seems that in the binary systems availability of ABTS cation radicals for one antioxidant molecules in the presence of the other antioxidant, is reduced which results in antagonism.

Summing up, it can be undoubtedly stated that even in the seemingly simple measurement systems (containing only two antioxidants), antioxidant properties of mixtures are difficult to predict because they are rarely the sum of the antioxidant activities of the components that constitute them. The presence of one component affects the properties of the other one in different ways.

Influence of solvent on the antioxidant properties of the tested binary mixtures

Figure 3 presents the changes in % inhibition as a function of concentration (volume) in the systems containing single antioxidants (chlorogenic acid or kaempferol or quercetin) and their binary mixtures in the systems where methanol or ethanol was used as the reaction solvent. It should be noted that the data in Fig. 3A and B are a replica of the data in Fig. 2. The repetition was intentional for better interpretation of the results, and the system containing methanol was taken as the reference one. As can be seen from the presented results, in the ethanol systems containing single components the antioxidant properties are greater than in the analogous methanol ones. As follows from the literature, the solvent affects the antioxidant properties of a substance because it determines the ionization and deprotonation potential of the antioxidant and consequently, its reducing capacity (Bakhouche et al. 2015). Comparing the course of the theoretical and experimental curves, it can be seen that in the system containing a mixture of kaempferol and quercetin dissolved in ethanol, the curves begin to converge. This proves the additive effect of these antioxidants which is confirmed by the data in Table 4. This additivism was found for this pair of antioxidants in methanol, however it is more visible (lower F and higher p values are observed) in ethanol. As reported in the literature (Ludwig 1999; Borowski et al. 2003), both solvents used in the research are structurally different because they are composed of different clusters joined by hydrogen bonds. There is a thermodynamic equilibrium between them. In methanol, hepta, hexa, penta, tetra and trimers predominate, while monomeric forms are present in small amounts. In contrast, ethanol consists maily of pentamers. It is possible that the different structure of the solvent influences the reaction kinetics of the neutralization of the ABTS radical and therefore, the mutual antioxidant effect of the mixture.

The observed effects may be related to different solubilities of the examined antioxidants in methanol or ethanol (Razmara et al. 2010); Wang et al. 2017). All of the tested antioxidants are more soluble in ethanol. This may be the reason for the better antioxidant properties observed in the measurement systems containing single antioxidants. Better solubility in ethanol can also justify the change in the observed antioxidant effect of the mixture containing kaempferol and quercetin from antagonism to addivism (observed for the volume ratio 50/50). Nevertheless, the antioxidant effect of the mixture is difficult to explain clearly. This is the result of the interactions of various factors related to not only solubility of the components and their interactions with each other but also to the structure of the solvents, which undoubtedly affects the kinetics of the cation radical neutralization reaction.

Influence of the addition of water on the antioxidant properties of the examined antioxidants and their binary mixtures

Water is a widely available solvent as a lot of moisture occurs also in the air. Even apparently dry plant material used for extraction can contain water. Therefore, in this study a decision was made to investigate the effect of water on the antioxidant properties of individual antioxidants and their binary mixtures. 100 μl of water was used in the tests, which constitutes just over 3% of the volume of the entire measurement system. Such research seems to be justified because the influence of water on the antioxidant properties of phenolic antioxidant (BHT) is commonly known in the literature (Dawidowicz and Olszowy 2013). The research results showed that the presence of water, even at such a low concentration in the measurement system, can have a significant impact on the observed antioxidant properties.

As noted above, Fig. 4 shows the effect of water addition on the antioxidant properties of the tested antioxidants and their binary mixtures. As can be seen from the presented relationships, the presence of water in the measurement system affects both the antioxidant properties of individual antioxidants (especially kaempferol and chlorogenic acid) and those observed in the system containing their binary mixtures. In the presence of water, chlorogenic acid shows better antioxidant properties (compare the dashed curves with the crosses in Fig. 4A and C); while, kaempferol has worse antioxidant properties (compare the curves in Fig. 4B and D). This fact can be explained based on the solubility of both antioxidants. Kaempferol is practically insoluble in water therefore, in the presence of water, due to its lower solubility, it is characterized by worse antioxidant properties. In turn, the solubility of chlorogenic acid in water is 40 mg/mL; while, in alcohol it is about 25 mg/mL.

It is important to compare the theoretical and experimental curves, in the presence of water it is noticed that these curves are closer to each other. This indicates the decreasing antagonism in the system and even addivism (in the case of the kaempferol–quercetin pair). This observation is confirmed by the data in Table 5 (lower F values and higher p values).

It is difficult to explain clearly the cause of the changes observed in the antioxidant effect of the tested antioxidant pairs in the presence of water. It is possible that this is the result of many factors: influence of water on individual components, influence of water on their dissociation, as well as the fact that water causes the disintegration of solvent clusters, which favors a better transfer of electron and/or hydrogen from the antioxidant to the radical, and thus better antioxidant properties.

Influence of the addition of metal ions on the antioxidant properties of the examined antioxidants and their binary mixtures

Figure 5A-F shows the antioxidant properties expressed as % inhibition in a function of volume for the systems containing single antioxidants: chlorogenic acid, quercetin and kaempferol, and the binary mixtures: chlorogenic acid and quercetin (Fig. 5A,C,E) and kaempferol and quercetin (Fig. 5B,D,F) in the systems containing: the addition of water (Fig. 5A and B), iron ions (Fig. 5C and D), and copper ions (Figs. 5E, F) (detailed volumetric components for all water systems are presented in Table 2). The use of systems differing in the presence of metal ions in research seems to be justified because besides antioxidants, other components such as metal ions can be also present in plant extracts treated as natural sources of antioxidants.

It should be emphasized here that Fig. 5A and B is a replica of Fig. 4C and D. This repetition is intentional to ensure clarity of the discussion. For individual pairs of antioxidants, the water containing system was taken as the reference one. Such a procedure seems justified because, when examining the influence of the presence of metal ions, a specific amount of metal ions was introduced into the tested system(s) in the form of aqueous solutions. It should be noted here that the concentrations of both metal ions used in the tests correspond to their content in plants (assuming the extraction of the entire amount of metal from 1 g of the plant with 50 mL of extractant) (Dawidowicz and Olszowy 2013).

It seems that metal ions, because they do not have antioxidant properties themselves, should not affect the antioxidant properties of the extract. As can see from the presented data, nothing could be further from the truth. While the presence of metal ions does not affect the antioxidant properties of individual antioxidants, it affects the observed antioxidant effect of the mixture evidently. Comparing the position of the experimental and theoretical curves in each figure, it can be seen that as in the previous studies, these curves do not overlap and the theoretical curve is above the experimental one, which indicates clearly that the mixture has worse antioxidant properties than would result from summing up the antioxidant properties of the components creating it. This is particularly visible in the system containing chlorogenic acid and quercetin (regardless of the metal ion) and in the system containing kaempferol and quercetin (in the presence of Fe3+, in the system containing 80 μl of kaempferol). The data in Table 6 (F and p values) confirm antagonism for the mentioned systems.

It is difficult to explain clearly what is responsible for the observed antagonistic behavior of the tested antioxidant mixtures. It seems to be a result of several effects due to from the possible formation of metal complexes with both the tested antioxidants (Mucha et al. 2021) and the cation radical (Deng et al. 2009) as well as the reduction of metal ions by the tested antioxidants, which themselves undergo oxidation (Nabavi et al. 2017). The last two arguments seem to be more reliable because, according to the literature, after complexation with metals phenolic compounds exhibit better antioxidant properties (Kalinowska et al. 2022), which should rather reduce antagonism. Based on the data obtained in the systems containing single antioxidants, it seems that the mentioned effects are less visible. Hence, the antioxidant properties in the systems containing single antioxidants are almost the same, regardless of whether metal ions are present or absent in the examined system.

Influence of hydrogen ions addition on the antioxidant properties of the examined antioxidants and their binary mixtures

Plant extracts can differ not only in the qualitative and quantitative compositions of the antioxidants, but also they can contain natural acids, the presence of which can affect their antioxidant properties. Moreover, this factor is very rarely taken into account when assessing the antioxidant properties of such mixtures as extracts. The problem of different pH values of the extract and its effect on antioxidant properties also seems to be quite important because researchers very often use solvents with different pH for extraction.

Figure 5G-J shows the antioxidant properties expressed as % inhibition as a function of volume for the systems containing single antioxidants: chlorogenic acid, quercetin and kaempferol and binary mixtures: chlorogenic acid and quercetin (Fig. 5A, G, I) and kaempferol and quercetin (Fig. 5B, H, J) in the systems containing: the addition of water (Fig. 5A and B), buffer with pH = 1 (Fig. 5G and H) and buffer with pH = 3 (Fig. 5 I, J). (Detailed volumetric components for all water systems are shown in Table 2). The system containing water, as in the case of metal ions, was taken as the reference one. As can be seen from the data presented in Fig. 5, the presence of different concentrations of hydrogen ions in the tested measurement systems affects the antioxidant properties observed in them. In the single-component systems (containing single antioxidants), the greatest impact is observed for those containing chlorogenic acid and quercetin. In the case of the systems containing a buffer with pH = 1, the antioxidant properties are worse than in the analogous systems with water; while, the systems with a buffer with pH = 3 exhibit better antioxidant properties. This is not surprising because, as reported in the literature, pH affects the kinetics of the reaction between the phenolic antioxidants and the ABTS cation radical. The researchers also found a clear relationship between the antioxidant properties observed in this method and the pH of the reaction environment—greater properties are estimated in the systems with a higher pH. The ABTS method is classified as a SET method in which an electron and/or hydrogen is transferred from an antioxidant to a cation radical (Sadeer et al. 2020). The ionization potential of active antioxidant groups, which determines its antioxidant activity, depends on pH—it decreases with the increasing pH.

Comparing the antioxidant properties in the two-component systems, it can be found that the largest changes are observed in the system of chlorogenic acid and quercetin for pH = 1 (which is also confirmed by the F and p values in Table 7 for this mixture). At this pH, there are greater differences between the theoretical and experimental curves for this mixture, which is also visible as a change in the observed antioxidant effect from additivism (for the systems with water) to high antagonism. In the case of the kaempferol and quercetin mixture, pH does not have such a significant impact on the antioxidant properties of the mixture. In most cases, regardless of the hydrogen ion concentration, additivism is observed for this mixture. Antagonism is only observed for the system containing much more kaempferol than quercetin of the system at a high concentration of hydrogen ions (pH = 1). It seems that this results from the poorer ionization of the weaker antioxidant (in this case kaempferol) in the system in which its quantity predominates; and hence, the worse properties of the entire mixture are observed.

It is worth noting here that buffer solutions with pH = 1 and pH = 3 were used in the experiments. The application of buffers with a higher pH value may result in unreliable data, which will undoubtedly complicate the interpretation of the antioxidant effect of the mixture. There are two reasons for this effect. One of them is the fact that the reaction of the tested antioxidant with a cation radical is pH-dependent (which results from the reaction mechanism, the SET mechanism). In general, as pH increases, the ability to donate electrons through deprotonation increases. The second reason may be the instability of the ABTS cation radical itself at higher pH. According to the literature, as the pH of the reaction mixture increases, the concentration of the ABTS radical cation gradually decreases and it is rather unstable at neutral pH, especially alkaline pH (Zheng et al. 2016).

Undoubtedly, the presence of hydrogen ions in the measurement system affects its antioxidant properties. This is related to both the influence of hydrogen ions on individual antioxidants and the mutual influence of antioxidants.

Conclusions

The paper presents and discusses the antioxidant properties of chlorogenic acid, kaempferol and quercetin and binary mixtures containing: chlorogenic acid and quercetin as well as kaempferol and quercetin. Additionally, the influence of such factors as the reaction environment, the presence of water and metal ions or hydrogen ions concentration on these properties was examined.

The following conclusions can be drawn from the data presented:

  • The examined pairs of antioxidants show different resultant antioxidant effects. In the mixture of chlorogenic acid and quercetin, the antagonistic effect predominates, and in the mixture of kaempferol and quercetin, the additive effect predominates.

  • The magnitude of the observed resultant effect is influenced not only by the mutual volume ratio of antioxidants in the mixture but also by the above-mentioned factors

  • Clearly determination of the cause of the observed antioxidant effect is difficult because this is a result of the action of a specific factor on a given antioxidant and antioxidants on each other in the presence of this factor.

The results obtained in the seemingly simple measurement systems, indicate that the assessment of antioxidant properties is not easy. The obtained results and the conclusions drawn from them can be useful in the extension of food, pharmaceutical and cosmetic industries. The increasing social awareness of natural antioxidants and their mixtures will bring tangible benefits in every area of human life and improve the quality of life.