Amoebicidal activity of Plantago lanceolata extract and expression of three genes in Acanthamoeba castellanii trophozoites

Abstract

This study investigated the therapeutic potential of an ethanolic leaf-stem extract from Turkish Plantago lanceolata against Acanthamoeba castellanii. The extract exhibited amoebicidal activity against A. castellanii trophozoites at varying concentrations (125, 62.5, 31.25, 15.625, 7.81, 3.9 and 1.95 mg/mL). The cytotoxicity of the extract in HeLa cells was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The DNA protective potential of the extracts against DNA damage was investigated. The phytochemical constituents of the extract were identified and characterized using Gas Chromatography-Mass Spectrometry (GC-MS). RT-qPCR analysis quantified the transcriptional modulation of oxidative stress-responsive genes (SOD, CAT) and the pseudocyst formation marker CSII in A. castellanii trophozoites following exposure to P. lanceolata extract. The viability and number of Acanthamoeba trophozoites were evaluated through hemocytometer counting coupled with the Trypan Blue exclusion technique. IC₅₀ values of A. castellanii trophozoites were 2.6, 4.2 and 14.45 mg/mL at the 72nd, 48th and 24th hours, respectively. MTT viability assays demonstrated dose-dependent cytotoxicity in HeLa cells following 72-h exposure to P. lanceolata extract (12.5, 6.25, 3.125, 1.562, 0.781, 0.391, 0.195 mg/mL), yielding an IC₅₀ of 0.85 mg/mL. The extract exhibited protective effects against DNA damage induced by hydroxyl radicals at 15.625, 7.81 and 3.9 mg/mL concentrations. Furthermore, it was determined that the extract showed a strong inhibitory effect on CSII and almost as much effect on CAT and SOD genes as the specific inhibitors of CAT and SOD genes. The obtained data indicate that this extract may serve as a potential therapeutic agent for both the prevention and treatment of acanthamoebiasis.