Odontites linkii subsp. cyprius Ethanolic Extract Indicated In Vitro Anti-Acanthamoeba Effect

This study aims to investigate three endemic ethanolic leaf extracts from Cyprus for anti-<i>Acanthamoeba</i> activities: <i>Odontites linkii</i> subsp. <i>cyprius</i> (Boiss.) Bolliger, <i>Ptilostemon chamaepeuce</i> subsp. <i>cyprius</i> (Greuter) Chrtek & B. Slavík, and Quercus <i>alnifolia</i> Poech. Screening for radical scavenging activity, total phenolic content (TPC), and total flavonoid content (TFC) were performed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABST) methods, Folin-Ciocalteu method, and aluminum chloride method, respectively. An antibacterial-susceptibility test (AST) was performed using a broth microdilution assay to estimate the minimum inhibitory concentration (MIC) using iodonitrotetrazolium chloride (INT). Trypan blue (0.5%) was used to assess in vitro anti-<i>Acanthamoeba</i> cell viability of the ethanolic leaf extracts after 24-, 48-, and 72-h exposure-screening of ethanolic leaf extracts with liquid chromatography-mass spectrometry (LC-MS) for known compounds with biological activity. The ethanolic leaf extract of <i>Odontites linkii</i> subsp. <i>cyprius</i> demonstrated the highest anti-<i>Acanthamoeba</i> activity, with an inhibitory concentration (IC₅₀) of 7.02 mg/mL after 72 h. This extract also showed an in vitro minimum inhibitory concentration (MIC) of 0.625 mg/mL against <i>Enterococcus faecalis</i>, a common nosocomial pathogen. The LC-MS analysis revealed the presence of bioactive iridoid compounds in <i>O. linkii</i> subsp. <i>cyprius</i>, further highlighting its potential as a source for new drug compounds. The ethanolic extract of <i>O</i>. <i>linkii</i> subsp. <i>cyprius</i> demonstrated a dose-dependent and time-dependent anti-<i>Acanthamoeba</i> effect in vitro. This study is the first to report the presence of iridoid compounds and anti-<i>Acanthamoeba</i> activities in the ethanolic extract of <i>O. linkii</i> subsp. <i>cyprius.</i> These promising findings highlight the potential of plant extracts, particularly <i>O. linkii</i> subsp. <i>cyprius</i>, as a source for new drug compounds for <i>Acanthamoeba</i> infections.