## 4 Discussion  
_Acanthamoeba_ infections present a persistent clinical challenge. Therefore, the search for effective, less toxic, and, ideally, novel treatments is crucial. Chemotherapy has been the gold standard for treating _Acanthamoeba_ infections. Yet, animal-based and plant-based products have been reported to be a promising alternative for novel anti-amoebic agents [42]. This study investigated the anti-amoebic potential of _E. planum_, _L. flos-cuculi_, and _K. elatine_ callus culture extracts against _Acanthamoeba_ trophozoites. The findings demonstrate a species-dependent effect, ranging from stimulation (_E. planum_) to strong inhibition (_L. flos-cuculi_). Contrary to expectations, _E. planum_ callus extract stimulated trophozoite proliferation at all tested concentrations over the three-day experiment. The growth stimulation increased with both dose and time, reaching 59.35% stimulation at 10 mg/mL on day 3. These results align with previous studies showing that flavonoid-rich extracts can have unexpected proliferative effects on microorganisms. The stimulatory effect may be attributed to phenolic acids and flavonoids, which, in _Erngium_ species, have been linked to antioxidant and immunomodulatory properties rather than antimicrobial activity [30]. Additionally, studies on _Acanthamoeba_ suggested that this organism has a vast range of oxidative stress-counteracting machinery, making it resistant to most oxidative-based therapy [43; 44]. Other studies confirm that _Erngium_ extracts possess various pharmacological effects, including diuretic, expectorant, and anti-inflammatory properties, but their anti-amoebic potential remains questionable [30]. In contrast, _L. flos-cuculi_ callus culture extract exhibited the strongest growth inhibition, achieving 87.52% inhibition at 10 mg/mL on day 3. The inhibitory effect was dose- and time-dependent, with higher concentrations yielding more significant reductions in trophozoite counts [8]. _K. elatine_ callus culture extract demonstrated moderate inhibition of trophozoite growth, reaching 80.12% inhibition at 10 mg/mL on day 3. However, the inhibition pattern was inconsistent--while higher doses resulted in greater inhibition over time, lower doses  
Figure 7: Three-dimensional representations of (**A**) acteoside, (**B**) azelaic acid, (**C**) chlorogenic acid, (**D**) \(p\)-coumaric acid, (**E**) ferulic acid, (**F**) leucoscopetoside A, and (**G**) rosmarinic acid conformations at the binding pocket of gyrase B (Gram-negative, PDB 1 KZN). Figures were generated using BIOVIA Discovery Studio 2025.  
sometimes exhibited fluctuating effects. Compared to _L. flos-cuculi_, _K. elatine_ appears to have a weaker but still significant amoebicidal effect. Previous studies highlight the presence of flavonoids and iridoid glycosides in _K. elatine_[20; 21; 24], which may contribute to its anti-amoebic potential. The observed effects can be contextualized within a broader scope of plant-derived anti-amoebic agents. In silico anti-amoebic analyses were conducted between the tested compounds and _Acanthamoeba_ profilin 1B protein. This protein is involved in the synthesis and degradation of actin microfilaments, which makes it crucial for cell shaping. Thus, it is one of the anti-_Acanthamoeba_ targets [34]. Comparative analyses, including in silico and in vitro studies on chlorogenic and _p_-cumaric acids, further support the potential of plant-derived compounds as anti-amoebic agents. Both compounds exhibited potent inhibition against _Acanthamoeba_ and possessed high predicted binding affinity for the profilin protein. In parallel, _Lonicera japonica_ and its major active constituent, chlorogenic acid, significantly reduced the viability by 49.2% after 48 h of 1.5 mg/mL fraction treatment and 100% reduction in the cysts/trophozoite ratio after 24 h of treatment with 1 mg/mL chlorogenic acid [45]. Furthermore, there are agreements between both analyses on ferulic acid and acetoside as moderate and the least potent anti-_Acanthamoeba_ activities.