## 4 Discussion  
Currently, with the increasing rate of conventional therapy-resistant parasites, low drug efficacy, prominent adverse effects, and heavy costs, it is urgent to search for new anti-parasitic agents [15; 16]. As previously highlighted, aromatic plant EO has low density and rapid diffusion across cell membranes, and this aspect may improve in targeting EO active components to endoparasites [17]. _L. luisieri_ EO evidenced on its chemical composition oxygen containing monoterpenes (75.7%) in large amounts, with 1,8-cineole (18.9%) being the major compound and representing necrodane derivatives (36%). _L. viridis_ presented \(\alpha\)-pinene (9.2%), 1,8-cineole (29.7%), linalool (9.0%), and camphor (10.0%) as major compounds. The assessment of _L. luisieri_ and _L. viridis_ inhibitory concentration at 50% (IC\({}_{50}\)) showed promising anti-_Leishmania_ activity, both against cutaneous and visceral strains, _L. infantum_, _L. major_, and _L. tropica_. A more prominent effect was observed in _L. luisieri_ (IC\({}_{5}\) = 31-63 \(\mu\)g/mL), while _L. viridis_ showed a high IC\({}_{50}\) value (263 \(\mu\)g/mL) as well as its compound, \(\alpha\)-pinene (IC\({}_{50}\) = 161 \(\mu\)g/mL). This fact might be related with the higher content on oxygen containing monoterpenes, and somehow, with necrodane derivatives. The other tested compounds did not reveal any activity. Therefore, it is feasible to suppose that the major compounds present in EO did not seem to be accountable for EO-stated effects. _L. luisieri_ EO revealed a promising potential, as it obtained data in accordance with that already described research by [PERSON] [18]. In this study, _L. luisieri_ EO also promoted phosphatidylinositol exposure, MMP depolarization, and G0/G1 phase cell cycle arrest leading to cell death [18]. These features have been stated as playing a pivotal role in protists drug-induced death, such as _Leishmania_ species [19]. Taking SEM and TEM imaging advantages, some of the above highlighted facts could be  
Figure 4: Representative dot plots showing JC-1 staining of _Leishmania infantum_ promastigotes exposed to _Lazunda luisieri_ essential oil. _L. infantum_ promastigotes were incubated at 26 \({}^{\circ}\)C for 24 h in the absence ((**A**) 3 h; (**B**) 24 h) or presence of _L. luisieri_ essential oil ((**C**) 3 h; (**D**) 24 h) at IC\({}_{50}\) concentrations. JC-1 staining was performed and samples were analyzed by flow cytometry. J-aggregates (blue) reflect functioning mitochondria and monomers are indicative of compromised mitochondria (pink). linked to _L. luisieri_ EO-induced morphologic changes. Indeed, among treated cells, it was not possible to identify an intact kinetoplast (swelled), only an increment in cell and organelle volume, cytoplasm clearing, and disorganization was found, as already stated in other studies [20; 21; 22]. Further, low density and rapid diffusion across cell membranes can raise EO in targeting parasites organelles, as well as inducing membrane destabilization [23]. This also suggests the existence of autophagic process with the consequent formation of autophagosomes [24], which seems to be involved in abnormal membrane structures' breakdown and recycling (supposing an intense intracellular organelles remodeling process, EO-irreversibly damaged). Similarly, other reports have already stated this change in drugs-treated kinetoplasticids [25; 26]. Mitochondrial changes were also stated, mainly on its matrix, arising complex structures, or otherwise, a less electrodes matrix, and mitochondrion swelling. Those alterations were also previously stated by [PERSON] et al. [27] using _L. brasiliensis_, and by [PERSON] et al. [28] and [PERSON] et al. [29] using _L. amazonensis_. These data may be explained by the ability of inducing mitochondrial potential depolarization, which may promote apoptosis as corroborated by the annexin-V values reached [30].