## 2 Materials and Methods
### Molecular Docking  
Molecular docking was carried out to understand the interaction between target compounds and _Acanthamoeba_ profilin and identify possible targets for antimicrobial properties from extracts of _E. planum_, _K. elatine_, and _L. flos-cuculi_ cell biomass. Acteoside, chlorogenic acid, \(p\)-couamic acid, ferulic acid, and rosmarinic acid were chosen for in silico analysis  
Figure 1: Unstained (**A**,**B**) and hematoxylin-stained trophozoites (**C**,**D**) from the culture collection. _Acanthamoeba_ spp. cysts observed under light microscope (**E**–**H**).  
based on the compounds used in this study (Figure 2). Chemical structures were drawn using ChemSketch software (2024.2.0; Advance Chemistry Development, Inc., Toronto, ON, Canada). On the other hand, acteoside, leucoscopetoside A, rosmarinic acid, chlorogenic acid, \(p\)-coumaric acid, and ferulic acid were chosen based on prior research for an antibacterial and antifungal computational assay [12; 15; 23]. Chemical structures were acquired from the open chemistry database NCBI PubChem ([[https://pubchem.ncbi.nlm.nih.gov/](https://pubchem.ncbi.nlm.nih.gov/)]([https://pubchem.ncbi.nlm.nih.gov/](https://pubchem.ncbi.nlm.nih.gov/)) (accessed on 28 March 2025)) and RCSB Protein Data Bank ([[https://www.rcsb.org/](https://www.rcsb.org/)]([https://www.rcsb.org/](https://www.rcsb.org/)) (accessed on 28 March 2025)).  
_Acanthamoeba castellanti_ profiling 1B (PDB 1 ACF) was used for anti-_Acanthamoeba_ docking [33]. Profilin 1B is a common anti-_Acanthamoeba_ target, as it is a crucial protein for cell shaping through the synthesis and degradation of actin microfilaments [34]. The chosen proteins for antibacterial investigation from bacteria, Gram-positive and Gram-negative, were dihydropyteonate synthase (PDB 1 AD4, 5V7A) and gyrase B (PDB 4 URN, 1 KZN). These proteins have been previously assessed as targets for antibacterial treatments [35]. Additionally, sterol 14-demethylase (PDB 5 TZ1) was chosen as the protein target for antifungal examination against _C. albicans_. Sterol 14-demethylase regulates ergosterol production, which is crucial for fungal cell permeability, fluidity, and the efficacy of membrane-associated proteins, in which some antifungal agents were developed to specifically target it [36].  
The selected target proteins were then subsequently brought to the PyMOL Molecular Graphics System, Version 3.0 Schrodinger LLC. (New York, NY, USA), for water molecules, ions, and complex co-structure removal [37]. Using AutoDockTools in MGLTools 1.5.7 [38], polar hydrogen atoms, Kollman charges, and Gasteiger charges were assigned to target proteins and then converted into PDBQT format. At the active site, a 60 \(\times\) 60 \(\times\) 60 cubic grid box was visualized for each protein. Simulations were carried out in AutoDock 4.2.6 [39] with the Lamarckian genetic algorithm for docking parameters. Post-docking visualization and analysis were performed using BIOVIA Discovery Studio 2025 [40].  
Figure 2: Chemical structures of compounds (**A**) acteoside, (**B**) ferulic acid, (**C**) \(p\)-coumaric acid, (**D**) chlorogenic acid, and (**E**) rosmarinic acid used in the in vitro and in silico study of anti-_Acanthamoeba_. The structures were created using ChemSketch 2024.2.0.