## 3 Results
### Effect of Extracted Polyphenols from Si3 Treatment on Cancer Cell Line Growth  
Extracted polyphenols, from both Leccino and Istarska Bjelica cultivars, showed high cytotoxic activity in all three tested cancer cell lines. In general, all cancer cells showed higher growth inhibition when treated with polyphenols from plants with Si treatment than from those without Si treatment. This growth inhibition was significant for all three cancer cell lines (Figure 5). Significant differences were recorded between extracts from non-Si treated plants, as well as between extracts from Si treated plants. Differences were also clear between genotypes from either Si treated or non-treated plants. The most resistant cell line was HeLa, with IC\({}_{50}\) values of 239.689 \(\upmu\)g/mL for Leccino, 141.433 \(\upmu\)g/mL for Leccino with Si, 175.611 \(\upmu\)g/mL for Istarska Bjelica, and 79.034 \(\upmu\)g/mL for Istarska Bjelica with Si. The HCT116 cell line proved to be the most sensitive to all tested extracts, especially to the plant extracts treated with Si (IC\({}_{50}\) values of 95.497 \(\upmu\)g/mL for LE, 51.446 \(\upmu\)g/mL for LExSi3, 70.142 \(\upmu\)g/mL for IB, and 36.075 \(\upmu\)g/mL for IBxSI3). The highest growth inhibition was observed for polyphenol extract with Si treatment from the Istarska Bjelica cultivar in all tested cell lines. This result is interesting because the Istarska Bjelica cultivar generally has lower polyphenol concentrations and lower concentrations of accumulated silicon. A healthy cell line, human retinal pigment epithelial-I (RPE1), showed very similar growth inhibition to the HCT116 and U2 OS cell lines, although there were significant differences between the polyphenols isolated from Si-treated and untreated plants.  
Figure 4: Interaction of Si foliar treatment and cultivar on Cu, Zn, and Ca content in olive leaves. Different lowercase letters represent statistically significant differences between mean values, for each variable (Cu, Zn, Ca), at \(p\leq 0.05\) obtained by a two-way ANOVA and Tukey’s test.  
Since the polyphenols isolated from the plants treated with Si had a significantly higher oleuppein content, its cytotoxic effect was also tested to determine whether it was responsible for the excellent cytotoxic activity. Oleuppein showed significant cytotoxic activity towards the HeLa cell line, comparable with polyphenols from Si-treated Leccino plants but significantly weaker than those extracted from Istarska Bjelica with Si. In the HCT116 and U2 OS cell lines, oleuppein inhibited cell growth to the same extent as the LE control but had a weaker cytotoxic effect than the extracts of the IB control and the Si-treated plants. Oleuppein showed slightly lower cytotoxicity on the healthy cell line RPE1 compared to extracts from Si-treated plants but slightly higher compared to extracts from non-Si-treated plants. In summary, oleuppein has better cytotoxic activity than polyphenols extracted from non-Si-treated plants but weaker activity compared to polyphenols from Si-treated plants (Figure 5). Those findings strongly indicate that oleuppein undoubtedly has a significant impact but that other polyphenols (especially in Si-treated plants) are also responsible for the exceptional biological activity.  
Figure 5: Cytotoxic activity of extracted polyphenols from non-Si treated Leccino and Istarska Bjelica plants (LE and IB), Si-treated Leccino and Istarska Bjelica plants (LExi3 and I2 Sci3), and oleuppein on HeLa, HCT116, U2 OS, and RPE1 cell lines as assessed by the MTS cell proliferation assay. IC\({}_{50}\) values are means of three independent experiments, and SE values are labelled as error bars. Statistically significant differences are marked with *** \(p<0.001\) and *** \(p<0.0001\).