# Comparative Study of _Plantago media_ Extracts in the Treatment of _Acanthamoeba_ sp. Trophozoites
## 1 Introduction  
_Plantago media_ L., the horary plantain, belongs to the Plantagiaceae family, and is a representative of the genus _Plantago_ L., which contains 242 species of herbaceous plants, or, less often, semi-shrubs, characterized by a cosmopolitan occurrence [1, 2]. _P. media_ (the leaves were the commonly used part) had several applications in folk medicine in the past and, now, many of its medical activities have been scientifically proven, such as its antioxidant, antimuoral, cytotoxic, anti-allergenic, mycostatic, anti-inflammatory, and tyrosine kinase inhibitor properties [3]. Several other _Plantago_ species are known medicinal plants, such as _P. major, P. asiatica, P. lancelata,_ and _P. ovata_[4], and some of them havemonographs in pharmacopoeias, e.g., _P. lanceolata_, and _P. ovata_[5]. Plants of the genus _Plantago_ can be used in medicine due to their anti-inflammatory, analgesic, anti-cancer, antiviral, and anti-ulcer effects; they also have expectorant and diuretic properties [4; 6; 7; 8; 9]. The main groups of compounds with a wide spectrum of biological activity that can be found in _P. media_ are phenylethanol glycosides [10; 11; 12], iridoid glucosides [13; 14], phenolic acids [15; 16], flavonoids [15; 17], and polysaccharides [12; 18]. In the micropropagated plants of _P. media_, there were mainly phenylethanol glycosides present, and no flavonoids were detected [19]. The phenylethanol glycosides occurring in _Plantago_ are represented mainly by acetoside (verbascoside) and also its positional isomer--isoacetoside, which is a water-soluble, low-molecular secondary metabolite [10; 11; 20]. The phenylethanol glycosides are associated with a wide range of biological and pharmacological activities [21; 22; 23]. Acteoside and isoacetoside are known for their prominent antiviral activity and, as suggested by recent literature reports, are likely to possess an inhibitory effect against SARS-CoV-2 [24]. Acteoside has been recognized as an effective scavenger of free radicals and an inhibitor of lipid peroxidation [25]. It exhibits neuroprotective activity through cholinergic, anti-inflammatory, and antioxidant mechanisms [26]. In addition, it shows an antiproliferative effect in many cancer cells [27]. What is particularly interesting is that the pronounced antiprotozoan activity against _Leishmana donovani_ and trypanicidal activity against _Trypanosoma brucei_ rhodesiense have been reported for acetoside and its isomer--isoacetoside [21]. More recently, acetoside was shown to inhibit _Leishmana amazonensis_ by the inhibition of an enzyme arginase of extracellular promastigotes [28] and intracellular amastigotes [29]. The opportunistic pathogens of _Acanthamoeba_ species have become recognized as dangerous agents leading to serious diseases, including granulomatous amoebic encephalitis (GAE), amoebic keratitis (AK), and amoebic pneumitis (AP). The single-cell free-living organisms commonly occur in the environment and are found in soil, air, and fresh and salt water. It is easy to become infected because their place of existence can also be air-conditioning systems, showers, sanitary appliances, swimming pools, and dialysis units. They were discovered in bottled mineral water and contact lens fluids. _Acanthamoeba_ trophozoites are metabolically and productively active in the appropriate conditions; however, they have the ability to transform into dormant cysts in a stressful environment [30]. The treatment of amoebic diseases is usually long-lasting and not very effective. Plant-derived natural products remain a promising source for drug development [30; 31; 32; 33; 34; 35]. The report on the use of an acteoside-containing traditional medicinal plant species _Stachytarpheta cyanensis_ (Rich.) [PERSON] from the family Verbenaceae (such as Plantaginaceae, classified within the order Lamiales) against a protozoan parasite _Leishmana_[28] has prompted us to investigate _Plantago media_ for anti-acanthamoeba activity.