## 4 Discussion
However, the most significant discrepancy is observed in rosmarinic acid. As the in silico analysis proposed, this compound is a potent inhibitor compared to the other tested compounds; yet the result is inconsistent with the in vitro data. There is a possibility that _Acanthamoeba_ is able to degrade or metabolize rosmarinic acid, leading to the reduction in the effective concentration. Nonetheless, this study only focuses on the interaction with a single profilin protein and a single molecule, which could not explain other factors that are present at the cellular level. [PERSON] et al. (2022) reported low inhibitory potency of rosmarinic acid isolated from _Rinorea yaundensis_ and _Salvia triloba_ against _A. castellanti_, as a 90% viable amoeba was still observed after treatment (100 mg/mL, overnight) [46]. [PERSON] et al. (2022) proposed that anti-amoebic activity from the _Eryngium_ species does not come from the polyphenol contents but rather from its triterpenoid saponin content [30]. This genus has been extensively studied for its oleanane triterpenoid saponins as the dominant component [47; 48]. This is consistent with the study by [PERSON] et al. (2019) that pentacyclic triterpenoids, unsolic acid, and its derivatives are potent anti-amoebic agents that induce programmed cell death via the mitochondrial pathway [49]. The observed anti-amoeba activity in _E. planum_, _L. flos-cuculi_, and _K. elatine_ cell biomass extract probably arises from the synergistic interactions of their constituent compounds. Studies on other medicinal plants, such as _Chaenomeles japonicus_[50] and _Plantago media_[51], show similarly strong inhibitory effects on _Acanthamoeba_ trophozoites. For example, _C. japonica_ extracts inhibited 95.4% of _Acanthamoeba_ trophozoites at 5 mg/mL after three days, an effect attributed to pentacyclic triterpenoids and polyphenols [50]. _Plantago media_ extracts, rich in phenylethanol glycosides, exhibited strong inhibition (63.89%) at 5 mg/mL [51]. The alkaloid and pentacyclic triterpenoid content of _Pericampylus glaucus_ presented potent inhibition to _A. triangularis_. At 100 mg/mL, the alkaloid extract demonstrated potent anti-amoebic activity, inhibiting 99% of trophozoite growth over 72 h. Notably, periglaucine A (the major alkaloid constituent), exhibited the same level of inhibition (99%) within 24 h at the same concentration. Furthermore, the isolated betulinic acid (pentacyclic triterpenoid) inhibited 94.6% of trophozoites' survival within 48 h [52]. These findings suggest that the chemical composition of plant extracts plays a crucial role in their anti-amoebic potential. The combination of in vitro studies with molecular docking (in silico) approaches can aid in predicting target proteins and ligands, as well as their interactions, which are important for understanding bioactivity properties. Sterol 14-demethylase is an enzyme that regulates ergosterol synthesis, which is crucial for fungal cell permeability, fluidity, and activity of membrane-associated proteins. Therefore, many antifungal agents were developed to target this protein [53]. The antibacterial property was evaluated against dihydroproteate synthase (PDB 1 AD4 and 5V7A) and gyrase B (PDB 4 URN and 1 KZN) of Gram-positive and Gram-negative bacteria, respectively. These proteins have been studied as targets for antibacterial treatments [35]. Dihydroproteate synthase is an enzyme that catalyzes 7,8-dihydroproteate production from _para_-aminobenz acid and dihydrotperin pyrophosphate, which are crucial for bacterial growth [54]. Gyrase B catalyzes the cleavage of ATP in the ATP hydrolysis reaction and is coupled with gyrase A, which is involved in DNA breakage during negative supercoiling of plasmid and chromosomal DNA [55]. The molecular docking findings suggested that rosmarinic acid and chlorogenic acid, which are present in _E. planum_, are potent antimicrobial inhibitors with the possibility of targeting proteins responsible for growth and genetic multiplication (Table 12).