## 3 Results and Discussion
### Content of TPC Using the Folin-Ciocalteu Reagent Method
#### 3.1.1 Extracts Obtained Using Shaking Solvent Extraction (SSE)  
Extracts made by shaking them with a solvent in various time variants were subjected to a preliminary analysis, and the content of total polyphenols was determined using the Folin-Ciocalteu reagent method in each version of the obtained apple leaf extracts (Table 1). Based on the results obtained, the most favourable time variant was selected, in which the polyphenol content was the highest, and further determinations were made only for extracts shaken at that time. The amount of polyphenols in water extracts decreased with increasing shaking time. This relationship was observed for all tested leaf cultivars of apple leaves. However, the greatest losses of these compounds were observed in Ligol leaf extract (over 9 mg GAE/ 1 g of DW) compared to shaking for 1 min and 90 min. For extracts from the leaves of the Gala and Gloster cultivars, these losses amounted to approximately 4 mg GAE/1 g of DW. The decrease in the content of polyphenols in water extracts with increasing extraction time was most likely related to the oxidation of phenolic compounds. The longer time of oxygen exposure to the extract, the lower the polyphenol content. The best solution for this solvent was to use the shortest extraction possible. The content of polyphenols in water-methanol extracts in relation to shaking time differed between different cultivars. In the case of the extract from apple leaves of the Ligol and Gloster cultivars, the content of phenolic compounds increased and reached its maximum after 30 min of shaking; further extraction resulted in a decrease in the amount of polyphenols in the extract. The opposite was the case with the Gala extract, where, from the beginning of extraction, the content of polyphenols decreased with increasing shaking time. This difference could be due to the different polyphenol composition of the leaves of different apple cultivars. Phenolic compounds found in the Gala cultivar may have been more sensitive to conditions such as oxygenation or solar radiation compared to the Ligol and Gloster cultivars. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Type of Solvent** & **Apple Cultivar** & **Shaking Time [min]** & **TPC** \\ \hline \multirow{6}{*}{Ligol} & & 1 & 47.9 \(\pm\) 1.8 \\ \cline{3-4}  & & 30 & 42.0 \(\pm\) 0.5 \\ \cline{3-4}  & & 60 & 39.5 \(\pm\) 1.3 \\ \cline{3-4}  & & 90 & 39.4 \(\pm\) 1.2 \\ \cline{3-4}  & & 1 & 37.6 \(\pm\) 0.9 \\ \cline{3-4}  & & 30 & 34.4 \(\pm\) 0.4 \\ \cline{3-4}  & & 60 & 34.2 \(\pm\) 0.6 \\ \cline{3-4}  & & 90 & 33.9 \(\pm\) 1.7 \\ \cline{3-4}  & & 1 & 32.9 \(\pm\) 0.8 \\ \cline{3-4}  & & 30 & 29.6 \(\pm\) 1.2 \\ \cline{3-4}  & & 60 & 29.0 \(\pm\) 1.1 \\ \cline{3-4}  & & 90 & 28.6 \(\pm\) 1.2 \\ \hline \multirow{6}{*}{Ligol} & & 1 & 78.5 \(\pm\) 1.7 \\ \cline{3-4}  & & 30 & 80.9 \(\pm\) 2.6 \\ \cline{3-4}  & & 60 & 78.5 \(\pm\) 3.8 \\ \cline{3-4}  & & 90 & 76.0 \(\pm\) 2.3 \\ \cline{3-4}  & & 1 & 65.6 \(\pm\) 3.6 \\ \cline{3-4}  & & 30 & 60.6 \(\pm\) 0.4 \\ \cline{3-4}  & & 60 & 58.8 \(\pm\) 3.2 \\ \cline{3-4}  & & 90 & 58.8 \(\pm\) 2.3 \\ \cline{3-4}  & & 1 & 58.5 \(\pm\) 1.6 \\ \cline{3-4}  & & 30 & 66.7 \(\pm\) 1.2 \\ \cline{3-4}  & & 60 & 61.5 \(\pm\) 2.4 \\ \cline{3-4}  & & 90 & 57.7 \(\pm\) 2.2 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The content of total polyphenols (TPC) in extracts obtained with shaking solvent extraction (SSE). Water-ethanol extracts were characterised by an increase in the content of phenolic compounds at up to 30 min of extraction by shaking and then, depending on the cultivar of apple leaves, a smaller or greater decrease in the content with an increase in the shaking time. The increase in the content of total polyphenols in water and ethanol extracts between 1 min and 30 min was from approximately 6.5 mg GAE/1 g of DW in the Gala cultivar to 10 mg GAE/1 g of DW in the apple leaf extract of the Ligol cultivar.