## 3 Results and Discussion  
### Extraction of Wild Olive Leaves  
The results of the HPLC analysis of wild olive leaves after the use of three extraction methods: low-frequency ultrasound solvent extraction, supercritical CO2 extraction, and MAE extraction, are shown in Table 2 and Figure 1. \begin{table}
\begin{tabular}{c c c} \hline \hline GAPDH & Forward & ACCCACTCCTCCACCTTTGAC \\ \cline{2-3}  & Reverse & CATACCAGGAAATGCTTGACAA \\ \hline \multirow{2}{*}{IL-6} & Forward & CCCCCGAGAAGATTCCA \\ \cline{2-3}  & Reverse & TCAATTCGTTCTGAGAGGTGAGT \\ \hline \multirow{2}{*}{IL-8} & Forward & CTGGCCCTGCTCTCTTG \\ \cline{2-3}  & Reverse & CCTTGGCAAAACTGCACCTT \\ \hline \multirow{2}{*}{IL-10} & Forward & TGAACAACGTCCACCCACCTT \\ \cline{2-3}  & Reverse & CCTGAAGGCATCTCGGAGAT \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Primers used in qPCR for quantification of selected genes. F—forward primers, R—reverse primers. GAPDH—glyceraldehyde-3-phosphate dehydrogenase, IL-6—interleukin 6, IL-8—interleukin 8, IL-10—interleukin 10. Figure 1: HPLC chromatograms of three extraction methods; (**A**) low-frequency ultrasound solvent extraction; (**B**) supercritical CO\({}_{2}\) extraction, (**C**) microwave-assisted extraction. Peaks: (1) hydroxytyrosol, (2) 3,4-hydroxybenzoic acid, (3) tyrosol, (4) catechin, (5) 4-hydroxybenzoic acid, (6) coffee acid, (7) \(p\)-coumaric acid, (8) \(t\)-ferulic acid, (9) \(o\)-coumaric acid, (10) oleupropein, (11) oleupropein aglycone, (12) pinoresinol, (13) luteolin, (14) apigenin, (15) kaempfer. \begin{table}
\begin{tabular}{c c c c c} \hline  & **Solvent Extraction** & \
\begin{tabular}{c} **Supercritical CO\({}_{2}\)** \\ **Extraction** \\ \end{tabular} &
\begin{tabular}{c} **MAE** \\ **Extraction** \\ \end{tabular} & **Retention Time** \\ \hline Compound & \multicolumn{3}{c}{Concentration (\(\mu\)g/mL)} & (min) \\ \hline hydroxytyrosol & 9.72 \(\pm\) 0.48 a & 1.41 \(\pm\) 0.02 b & 0.16 \(\pm\) 0.01 c & 18.10 \\ \hline
3,4-hydroxybenzoic acid & 1.70 \(\pm\) 0.09 a & 0.20 \(\pm\) 0.01 c & 0.89 \(\pm\) 0.05 b & 19.86 \\ \hline tyrosol & 3.04 \(\pm\) 0.34 b & 3.51 \(\pm\) 0.38 b & 17.76 \(\pm\) 1.05 a & 22.42 \\ \hline catechin & 1.91 \(\pm\) 0.15 a & 0.74 \(\pm\) 0.05 b & n.d. & 24.86 \\ \hline
4-hydroxybenzoic acid & 1.24 \(\pm\) 0.09 c & 4.84 \(\pm\) 0.16 a & 2.96 \(\pm\) 0.11 b & 25.27 \\ \hline coffee acid & 0.93 \(\pm\) 0.11 a & 0.47 \(\pm\) 0.03 ab & n.d. & 28.66 \\ \hline \(p\)-coumaric acid & 8.60 \(\pm\) 1.17 a & 1.78 \(\pm\) 0.13 b & 6.49 \(\pm\) 0.61 a & 33.46 \\ \hline \(t\)-ferulic acid & 1.56 \(\pm\) 0.18 a & 0.72 \(\pm\) 0.08 b & n.d. & 35.04 \\ \hline \(o\)-coumaric acid & n.d. & 0.53 \(\pm\) 0.08 b & 1.91 \(\pm\) 0.15 a & 37.73 \\ \hline oleupropein & 71.97 \(\pm\) 3.77 a & 2.05 \(\pm\) 0.13 c & 13.62 \(\pm\) 0.77 b & 39.57 \\ \hline oleupropein aglycone & 20.62 \(\pm\) 1.89 a & 0.97 \(\pm\) 0.08 b & 20.60 \(\pm\) 1.47 a & 40.38 \\ \hline pinoresinol & n.d. & 3.34 \(\pm\) 0.58 a & n.d. & 44.57 \\ \hline luteoline & 4.40 \(\pm\) 0.62 a & 0.37 \(\pm\) 0.04 b & n.d. & 48.40 \\ \hline apigenine & 2.70 \(\pm\) 0.10 a & 0.33 \(\pm\) 0.03 b & n.d. & 51.77 \\ \hline kaempfer & 0.84 \(\pm\) 0.07 a & 0.18 \(\pm\) 0.20 b & n.d. & 52.14 \\ \hline \end{tabular}
\begin{tabular}{c c} Values represent the mean \(\pm\) standard deviation of two measurements. Different letters in a row indicate significant differences (one-way ANOVA and the post-hoc [PERSON]’s new multiple range test, with \(p\leq\) 0.05). n.d.—not detectable compound.  
\end{table} 
Table 2: HPLC analysis of wild olive leaf extracts. The focus of this study was on the effects of the extraction methods on oleuppein content. The results showed that the highest concentration of oleuppein was obtained after low-frequency ultrasound solvent extraction (71.97 ug/mL) (Table 2, Figure 1A), compared to supercritical CO\({}_{2}\) extraction (2.05 ug/mL) (Table 2, Figure 1B), and MAE extraction (13.62 ug/mL) (Table 2, Figure 1C).