## 3 Results and Discussion
### Influence of Temperature on the Resultant Effect of the Mixture's Antioxidant Activity  
As noted above, Figure 1 shows changes in the percentage of inhibition as a function of concentration (volume) obtained for systems containing single antioxidants (chlorogenic acid or kaempfer) and their binary mixtures. The experiments were carried out at three different temperatures, i.e., 15 \({}^{\circ}\)C (see Figure 1A), 25 \({}^{\circ}\)C (see Figure 1B) and 35 \({}^{\circ}\)C (data in Figure 1C). As can be seen from the presented relationships, regardless of the temperature used, an increase in antioxidant properties (increase in % of inhibition) is observed for individual antioxidants with increasing concentration (volume) of the antioxidant in the measuring system. This is not surprising because the more antioxidant, the better the antioxidant properties. As for temperature, according to the literature [22], temperature is one of the important factors affecting antioxidant activity. As it results from the presented data, the temperature affects the % inhibition values obtained for systems containing single antioxidants. At a higher temperature, higher % inhibition values are observed for the same measurement system than at a lower temperature. \begin{table}
\begin{tabular}{c c c c c c c} \hline
**Solvent** & **System Number** & **Volume Ratio (r/r)** & **\(F\)-Values** & **\(p\)-Values** & **(le-lc)-Values** & **Observed Effect** \\ \hline methanol & 7 & 20/80 & 13.83 & 0.0205 & negative & antagonism/additivism \\  & 8 & 50/50 & 11.03 & 0.0293 & negative & antagonism/additivism \\  & 9 & 80/20 & 21.07 & 0.0101 & negative & antagonism \\ \hline methanol with water (100 \(\mu\)L) & 7 & 20/80 & 29.21 & 0.0056 & negative & antagonism \\ \hline  & 8 & 50/50 & 33.96 & 0.0043 & negative & antagonism \\  & 9 & 80/20 & 35.19 & 0.0040 & negative & antagonism \\ \hline  & & & & & & \\ \end{tabular}
\end{table} 
Table 4: Statistical significance (\(F\) and \(p\) values) of the difference between the experimental inhibition percent (I\({}_{\rm e}\)) and calculated inhibition percent (I\({}_{\rm c}\)) for the binary mixtures of chlorogenic acid and kaempfer at three different volume ratios and in the system with methanol and with methanol in the presence of water addition, together with the difference (I\({}_{\rm e}\)-I\({}_{\rm c}\)) and with the observed effect (the resultant) antioxidant effect of the antioxidants in the mixture (_Crrit_ = 7.71). \begin{table}
\begin{tabular}{c c c c c c} \hline
**Factors** & **Volume Ratio (r/r)** & **\(F\)-Values** & **\(p\)-Values** & **(le-lc)-Values** & **Observed Effect** \\ \hline water & 20/80 & 29.21 & 0.0056 & negative & antagonism \\  & 50/50 & 33.96 & 0.0043 & negative & antagonism \\  & 80/20 & 35.19 & 0.0040 & negative & antagonism \\ \hline  & & & & & \\ \hline  & 20/80 & 27.03 & 0.0065 & negative & antagonism \\ Fe\({}^{3+}\) & 50/50 & 58.64 & 0.0015 & negative & antagonism \\  & 80/20 & 60.97 & 0.0014 & negative & antagonism \\ \hline  & 20/80 & 28.80 & 0.0058 & negative & antagonism \\ Cu\({}^{2+}\) & 50/50 & 18.05 & 0.0132 & negative & antagonism \\  & 80/20 & 17.89 & 0.0134 & negative & antagonism \\ \hline  & & & & & & \\ \end{tabular}
\end{table} 
Table 5: Statistical significance (\(F\) and \(p\) values) of the difference between the experimental inhibition percent (I\({}_{\rm e}\)) and calculated inhibition percent (I\({}_{\rm c}\)) for the binary mixtures of chlorogenic acid and kaempfer at three different volume ratios and in the systems with different additives, together with the difference (I\({}_{\rm e}\)-I\({}_{\rm c}\)) and with the observed effect (the resultant) antioxidant effect of the antioxidants in the mixture (_Crrit_ = 7.71). In the literature, any changes in antioxidant properties resulting from an increase in temperature are most often explained by changes in the oxidation initiation process (acceleration with increasing temperature) and/or changes in the mechanism of antioxidant action [23].