## 3 Results
### Effect of the Propolis Extracts on IL-6, IL-10, and TNF-\(\alpha\) Secretion by BMDM Macrophages  
Since the three propolisis extraction methods resulted in three extracts with distinct compositions, the next step was to determine whether these three extracts differ in their effects on cytokine production by LPS-stimulated macrophages. BMDM in cell culture were activated with LPS for two hours, followed by treatment with one of the three extracts, PPF, MPE, or PSDE, at concentrations of 1, 10, 25, 50, 100, or 300 \(\mu\)g/mL for 20 h.  
As expected, LPS stimulation of BMDM significantly increased the secretion of IL-6, IL-10, and TNF-\(\alpha\) compared to cytokine levels observed in the negative control (Figure 2). When we compared the effects of propolisis extracts on the LPS-stimulated macrophages with the control (no propolisis), we observed that the PPF extract increased IL-6 and IL-10 secretion at almost all concentrations tested, though with no significant effect on TNF-\(\alpha\) (Figure 2A-C). \begin{table}
\begin{tabular}{l c c c c} \hline \hline \multicolumn{1}{c}{**Chemical Marker**} & \multirow{2}{*}{**Regression Curve**} & \multirow{2}{*}{**r**} & **LOD** & **LOQ** \\ \cline{3-5}  & & & & \(\mu\)g/mL & \(\mu\)g/mL \\ \hline Caffeic acid & y = 32,558x - 11,096 & 1.00 & 0.59 & 1.79 \\ \(p\)-Coumaric acid & y = 52,979x - 2033 & 0.99 & 1.18 & 3.57 \\
3,5 Dicaffeoylquinic acid & y = 18,942x - 44,655 & 0.99 & 6.41 & 19.43 \\
4,5 Dicaffeoylquinic acid & y = 15,827x - 13,446 & 0.99 & 7.15 & 21.68 \\ Aromadendran-4-O\({}^{\prime}\)-methyl-ether & y = 23,304x - 4193 & 0.99 & 0.49 & 1.47 \\ Drupanin & y = 26,870x - 36,385 & 0.99 & 1.40 & 4.24 \\ Chrysin & y = 66,663x - 3433.7 & 0.99 & 1.35 & 4.08 \\ Galangin & y = 34,798x - 17,489 & 0.99 & 3.04 & 9.20 \\ Artepillin C & y = 19,313x - 725,866 & 0.99 & 8.03 & 24.34 \\ Baccharin & y = 49,810x - 39,607 & 0.99 & 0.85 & 2.58 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Linearity, quantitation, and detection limits obtained for the isolated propolisis compounds investigated in this study. Data obtained based on HPLC/DAD methodology [36] (\(n\) = 3 samples analyzed for each product). \begin{table}
\begin{tabular}{l c c c c} \hline \hline \multicolumn{3}{c}{**Total Phenolic Compounds**} & \multicolumn{3}{c}{**Total Flavonoid Compounds**} \\ \cline{2-5}
**Propolis Extracts** & **Mean (mg GAE/g) \(\pm\) SD** & **RSD (\%)** & **Mean (mg QUE/g) \(\pm\) SD** & **RSD (\%)** \\ \hline PPF & 53.05 \(\pm\) 1.30 & 2.37 & 18.11 \(\pm\) 1.3 & 3.53 \\ PSDE & 123.24 \(\pm\) 1.90 & 1.53 & 45.48 \(\pm\) 0.5 & 1.17 \\ MPE & 49.45 \(\pm\) 1.30 & 2.60 & 23.17 \(\pm\) 0.6 & 2.42 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Total amounts of phenolic and flavonoid compounds, expressed as amounts of gallic acid (GAE) and quercetin (QUE), respectively, in the three propolisis extracts investigated using spectrophotometric methods. See Table 1 for definitions of the propolisis formulation abbreviations. In the case of PSDE, the cytokine behavior varied with the concentration used. Low concentrations (1, 10, and 25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) increased IL-6 and IL-10 production, while TNF-\(\alpha\) was not significantly affected. On the other hand, at the highest concentration (300 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), this extract increased TNF-\(\alpha\) and reduced IL-10 (Figure 2D-F). MPE did not significantly affect the secretion of the pro-inflammatory cytokines IL-6 and TNF-\(\alpha\). However, at low concentrations (1, 10, and 25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), this extract increased IL-10 secretion (Figure 2G-I). Effects of Isolated Propolis Compounds on IL-6, IL-10, and TNF-\(\alpha\) Secretion by Macrophages  
We also investigated cytokine production induced by selected propolis components present in different proportions in the extracts. Caffeic acid significantly increased IL-6 and IL-10 at the highest concentrations (100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), while TNF-\(\alpha\) levels were not significantly affected (Figure 3A-C).