In vitro amoebicidal and antioxidant activity of Helianthemum ledifolium (L.) Mill. varieties

Purpose: Acanthamoeba spp. are opportunistic protozoa causing Acanthamoeba keratitis (AK), an ocular infection frequently associated with contact lens use. Given the limitations and adverse effects of current treatments, this study aimed to discover natural, safe, and cost-effective therapeutic alternatives. Specifically, the purpose was to investigate the in vitro amoebicidal and antioxidant activities of methanol and water extracts from three varieties of Helianthemum ledifolium (L.) Mill.. Method: The study evaluated methanol and water extracts obtained from the aerial parts of three H. ledifolium varieties (H. ledifolium var. ledifolium, H. ledifolium var. microcarpum, and H. ledifolium var. lasiocarpum). Amoebicidal activity was tested in vitro against Acanthamoeba castellanii. In addition, total phenolic and flavonoid contents were calculated using Folin-Ciocalteu and AlCl3 reagents. Antioxidant activities were evaluated by measuring radical scavenging activities using the DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) assays. Findings: The amoebicidal activity against Acanthamoeba castellanii showed a dose- and time-dependent effect. Methanol extracts exhibited superior amoebicidal potency compared to water extracts, even at lower concentrations, and also had the highest total phenolic and flavonoid contents in relation to antioxidant radical scavenging activities. The most toxic effect was observed in the H. ledifolium var. lasiocarpum methanol extract, with an LD50 of 0.0034 mg/mL at 72 hours. The H. ledifolium var. microcarpum water extract exhibited the fastest effect LT50 = 0.5603 hours. Conclusion: These results suggest that H. ledifolium species represent promising candidates for the development of natural antiparasitic agents. The superior anti-Acanthamoeba activity of methanol extracts is strongly related to their significantly richer total phenolic and flavonoid contents compared to aqueous extracts, indicating that methanol is a much more effective solvent for obtaining potential bioactive compounds. H. ledifolium taxa have the potential to be an important natural resource for future antiparasitic drug research, especially in the context of increasing drug resistance and treatment failure.