# Comparative Study of _Plantago media_ Extracts in the Treatment of _Acanthamoeba_ sp. Trophozoites
## 4 Discussion  
_Acanthamoeba_ species have become recognized as dangerous agents leading to diseases including granulomatous amoeba encephalitis (GAE), amoebic keratitis (AK), and amoebic pneumitis (AP). The various synthetic anti-_Acanthamoeba_ substances are often ineffective. Regarding the anti-parasitic activity, a plethora of raw materials and groups of bioactive compounds with amoebicidal or amoebistatic properties were studied including abietane diterpenoids from _Salvia sclarea_ L. (with the highest activity of ferruginol--72%) [45]; essential oil from _Peteroaulon polystachyum_ D.C. [46]; extracts from _Artemisia argyi_ [PERSON] [47] and _Artemisia annua_ L. with the artemisinin as the most active compound [34]; _Lonicera japonica_ Thunb. extract with its major constituent chlorogenic acid [48]; _Buddleja cordata_ Tunht. extract rich in laminin belonging to flavones [49]; a brew of green tea (_Camelia sinensis_ ([PERSON] [PERSON]) rich in flavans [50]; extracts of _Eryngium_ species [33;51]; _Rubus chamaenorus_ L. extract with ellagic acid as the leading compound; _Pueraria lobata_ (Wild.) Ohwi. extract with active isoflavunes--puerarin and daidzein [32];  
Figure 4: Correlations between IC\({}_{50}\) values and the content of phenylethanol glycosides (**a**), acetoside (**b**), phenolic acids (**c**), flavonoids in all materials (**d**), flavonoids without consideration of in vitro-derived materials (**e**), and phenolics sum (phenylethanoid glycosides + phenolic acids + flavonoids) (**f**). \({}^{*}\) The Pearson correlation coefficients are statistically significant at two-tailed \(p<0.05\). extracts of _Teucrium_ species [52]; _Centaurea_ species, _Rharponticum pulchrum_ Fisch. et Mey. and _Tanacetum vulgar_ L. extracts with sesquiterpene lactones [53]; _Lychnis flos-cuculi_ L. extracts with phytoedysones [54]; and _Chaenomeles japonica_ (Thunb.) Lindl. ex Spach extract with a high content of pentacyclic triterpenoids (mainly unsolic, oleanolic, and betulinic acids) [55]. Only amoebicidal substances are useful in the treatment of _Acanthamoeba_ infections. Amoebostatic (change of trophozoites into cysts) substances are not suitable for use in therapy. The transformation of amoeba into cysts under the influence of the administered preparation is very unfavorable for the patient. The resulting cysts can survive in the patient's tissues for a very long time and, under conditions favorable for the amoeba, they can cause the invasion to resume. Our task was to find a substance that would kill the trophozoites, but would not cause cyst formation. In the current study, no encapsulation (transformation of amoeba into cysts) was observed upon application of the tested extracts from _Plantago_. In the present study, the highest anti-amoebic activity of the water fractions of _Plantago media_ was recorded for concentrations of 5 and 10 mg/mL on the third and fourth day of treatment of trophozoites. Leaf extracts from the crop with the highest content of acetoside had an anti-amoebic effect from the lowest concentration of 1 mg/mL and achieved the lowest IC\({}_{50}\) value among the tested samples, while extracts from infloresences and in vitro roots with the lowest content of acetoside also showed the highest IC\({}_{50}\) index. _Plantago Lancolata_ leaf extracts had a lower concentration of acetoside than _P. media_ leaves and reached the lowest IC\({}_{50}\) result, although only on the fourth day of the experiment, which, in turn, may be associated with a different profile of phenolic acids in these species. The presence of flavonoids in the composition does not seem to affect the strength of the antiprotozoan effect, which is confirmed by the particularly good result of this effect of in vitro shoot extracts devoid of these components.