## 3 Results
The anti-_Acanthamoeba_ activity observed in the callus extract can be partly attributed to the presence of ferulic acid and _p_-coumaric acid, as demonstrated in their isolated form in Table 6. This supports the idea that these phenolic acids contribute substantially to the extract's bioactivity. Table 8 evaluates the anti-amoebic activity of phenylethanolamine glycoside, namely acetoside (Figure 2A), by measuring the impact on _Acanthamoeba_ trophozoite growth over three days at different concentrations (Table 8). Inhibition increases with higher concentrations, especially by day 3. Inhibition becomes more pronounced over time. Even at the lowest dose (0.01 mg/mL), growth inhibition rises from 6.52% (day 1) to 43.33% (day 3). Aceto-side exhibited inhibitory activity against _Acanthamoeba_ trophozoites, with a stronger and consistent dose- and time-dependent effect. These results support the potential role of phenylethanolamine glycosides in developing plant-based anti-_Acanthamoeba_ treatments. The extract of _Kickxia elatine_ exhibits a dose-dependent inhibition of _Acanthamoeba_ growth (Table 9). The inhibition pattern is not strictly linear across time. The highest inhibition is achieved on day 3 at 10 mg/mL (80.12%), indicating a sustained and strong inhibitory effect at the highest dose. The extract is a promising candidate for anti-_Acanthamoeba_ activity, though higher concentrations are required for sustained inhibition. _Lychnis flos-cuculi_ shows the strongest inhibitory effect, reaching 87.52% inhibition on day 3 at 10 mg/mL. _K. elatine_ also performs well, reaching 80.12% inhibition. _E. planum_ does not inhibit growth; instead, it stimulates _Acanthamoeba_ growth. Acteoside (Table 8; Figure 2A) and the whole callus extract of _K. elatine_ (Table 9) demonstrate significant inhibition of trophozoite growth, confirming that the active con stituent, acteoside, likely contributes to the observed biological activity of the extract. The isolated compound and the full extract show increased effectiveness over time, which was particularly evident by day 3. This pattern indicates a cumulative anti-amoebic effect, whether from individual glycosides or the complex mixture within the extract. Given that acteoside is present in _K. elatine_ and shows individual anti-amoebic properties, its presence likely contributes significantly to the strong inhibitory effects observed with the whole callus extract. The strong anti-_Acanthamoeba_ activity seen in the _K. elatine_ callus extract (Table 9) mirrors the effects of acteoside when tested individually (Table 8). The datasets confirm that phenylethanol gigycoside is a key contributor to the extract's bioactivity. The dose- and time-dependent inhibition observed in both tables reinforces the idea that _K. elatine_ is a promising source of natural anti-amoebic compounds. Molecular docking is an in silico analysis of protein-ligand interactions to provide insight into its binding affinity at the active site and potency. This approach has been used to help with drug design and discovery, or to provide an understanding of disease treatment [41]. Table 10 and Figure 3 present the docking results between selected compounds against the _Acanthamoeba_ profilin 1B protein. This protein is a crucial regulator of the actin cytoskeleton, which participates in cellular activities, including filament assembly [33]. Based on the docking analysis, chlorogenic acid and rosmarinic acid have the most negative \(\Delta\)G (\(-\)4.58 kcal/mol) and a low inhibitory constant (437.19 and 483.33 uM, respectively), suggesting a strong binding affinity of these compounds for the profilin protein. \(p\)-coumaric acid was proposed to give strong binding affinity and high potency with \(-\)4.33 kcal/mol and a 667.05 uM inhibitory constant. Meanwhile, ferulic acid and acteoside showed moderate to low binding affinity for the _Acanthamoeba_ profilin protein. As shown in Table 11, the callus cultures are more effective than suspension cultures in inhibiting bacterial growth.