## 3 Results and Discussion
### Influence of Water Addition on the Resultant Effect of the Mixture's Antioxidant Activity
In the studies aimed at examining the effect of metal ions and hydrogen ions on the antioxidant properties of the binary mixture, it was decided to assume that the system containing water was the reference system. Such a procedure seems reasonable because when examining the influence of a specific factor (metal ion or hydrogen ion concentration), its specific amount was introduced into the tested system/systems in the form of aqueous solutions. It should be emphasized here that the concentrations of metal ions that can be found in plants were used in the experiments (assuming extraction of the entire amount of metal from 1 g of the plant with 50 mL of the extractant) [14]. Comparing the position of the experimental and theoretical curves in each figure, it can be seen that, as in previous studies, these curves do not coincide and that the experimental curve is under the theoretical curve. As is known, this indicates antagonism, which is confirmed by the data in Table 5.  
As for the effect of metal ions, a greater one is observed for iron ions. In the systems containing mentioned iron ions, higher \(F\) values and lower \(p\) values are observed, respectively, compared to the same values for the reference system (with water). This confirms the increase in the antagonistic antioxidant effect in the chlorogenic acid/kaempferol mixture, and moreover, this effect is most pronounced with a higher content of chlorogenic acid (for a mixture containing 80 \(\mu\)L acid and 20 \(\mu\)L kaempferol, the greatest difference between the experimental and theoretical value is observed, \(F\) = 60.97). In the case of systems with copper ions, the antagonism effect is also visible, but compared to the reference system, it seems to be slightly smaller (lower \(F\) values). It is difficult to clearly explain the observed resultant antagonistic effect of the mixture in the presence of metal ions. It seems to be the result of many phenomena. On one hand, the observed poorer performance of the mixture may result from the ability of both metals to reduce as a result of attaching an electron from the antioxidant, which may inhibit the radical neutralization reaction. On the other hand, these metals can be complexed by the ABTS cation radical itself as well as by the antioxidants themselves [32]. As the literature data show [33], ABTS complexes have an intense color, which can cause falsification of absorbance measurements and cause apparently poorer antioxidant properties [33]. Transition metal complexes with polyphenolic compounds are also known from the literature. Moreover, in most cases, these complexes usually turn out to be more active than the parent compounds [34]. On the other hand, less antagonism in the presence of copper ions may result from a greater (than in systems with iron) predominance of the formation of complexes with better antioxidant properties [35]. In addition to metal ions, the presence and concentration of hydrogen ions also influence the observed antagonism in the mixture containing chlorogenic acid and kaempfer. Greater differences between the theoretical and experimental values are observed for systems with pH = 1 (greater \(F\) values than in the reference system). This is not surprising because, as already mentioned, ABTS is a method in which the neutralization of the cation radical occurs after the ionization and deprotonation of the active groups in the antioxidant. As such, it is a pH dependent reaction and the ionization potential of phenolic compounds decreases with an increasing pH value. Thus, in an acidic environment, the poorer antioxidant properties observed for both single antioxidants and their mixtures may be the result of hindered ionization of the antioxidant and poorer antioxidant properties.