## 3 Results and Discussion
### Biological Potential of WOLE-S vs. Pure Oleuppein
Different capital letters in a column, for each concentration and sample separately, indicate significant differences (one-way ANOVA and post-hoc [PERSON]’s new multiple range test, with \(p\leq\) 0.05). In general, the highest inhibitory effect of WOLE-S and pure oleupopein on cell proliferation was achieved against T24 cells (Table 3b). Two concentrations of WOLE-S and pure oleupopein (0.5 and 1 g/L) were tested after different incubation times (4, 24, 48, and 72 h) on all cancer cell lines. The antiproliferative effect of both samples was dose-dependent in all tested cells. Both concentrations of the samples showed high inhibitory activity against T24 cancer cells, already after 24 h of incubation (83.61% WOLE-S and 81.12% pure oleupopein). The antiproliferative effect of both samples remained high even after 48 and 72 h of incubation (Table 3b). Table 3a,c,d shows that in other cancer cell lines tested (MD-MBA-231, A1235, and A549), the antiproliferative effect of pure oleupopein was significantly higher compared to WOLE-S at both concentrations and after all incubation times tested. Compared to T24 cells, the antiproliferative effect of WOLE-S against A1235, A549, and MD-MBA 231 cells was generally moderate (the average rate was 35.48% at a concentration of 1 g/L after 72 h of incubation). In contrast, the antiproliferative activity of pure oleupopein against A1235, A549, and MD-MBA 231 cells was high (the average rate was 79.57% at a concentration of 1 g/L after 72 h of incubation). Except T24 at a concentration of 1.00 g/L after 24 h and 48 h of incubation, A1235 at 1.00 g/L after 4 h of incubation, and A549 at 1.00 g/L and 0.50 g/L after 24 h of incubation, in all other tested cell line/concentration/incubation time combinations oleupopein showed significantly higher inhibition of cell proliferation than WOLE-S. Regarding the incubation time, in the majority of the combinations, the highest inhibition by oleupopein was achieved after 72 h. However, in T24 cells, that was after 48 h of incubation with oleupopein 0.50 g/L, and in the A549 line after 48 h of incubation with oleupopein 1.00 g/L. Moreover, in A549 cells, with oleupopein 1.00 g/L, we got significantly better results after 48 h and 24 h compared to 72 h. Incubation for 4 h resulted in the weakest effect in all combinations. The results obtained are interesting because, on the one hand, 1 g/L of WOLE-S contains only 0.072 g/L of oleupopein incorporated into the plant matrix on one hand and on the other hand, the average rate of antiproliferative activity of pure oleupopein is only 2-3 times higher than that of WOLE-S against all cancer cell lines tested (for T24 cells, their activity was very similar at a concentration of 1 g/L). Considering that the amount of 1 g/L pure oleupopein is approximately 14 times higher than the concentration of oleupopein in 1 g/L WOLE-S, one might expect a much higher antiproliferative activity of pure oleupopein compared to WOLE-S. Moreover, their antiproliferative activity against T24 cells at a concentration of 1 g/L was similar after 24, 48, and 72 h. This could be due to the fact that both samples were very effective against T24 cells and reached a maximum of their activity after 24 h of incubation. [PERSON] et al. [26] identified 74 phenolic compounds in olive leaf extract whose synergistic effect is expected, but it is impossible to characterise the exact mechanisms between so many  
Figure 2: Analysis of IL-6, IL-8, and IL-10 expression in A549 cells treated for 2 h with WOLE-S and oleupopein. Statistically significant results were denoted with asterisks (* for \(p<0.05\), ** for \(p<0.01\), *** for \(p<0.0001\)). biologically active compounds in the olive leaf plant matrix. Also, the effect of the plant matrix could cause a change in the stability of antioxidants from wild olive leaves in MTT media after 72 h of incubation. To the best of our knowledge, this is the first report on the antiproliferative activity of WOLE-S and pure oleupreein against A1235 cancer cells.