Anti-Trichomonas vaginalis activity of essential oils extracted from Caatinga Myrtaceae species and chemical composition of Eugenia pohliana DC.

Abstract The current trichomoniasis treatment is restrict to 5-nitroimidazole drugs and the emergence of resistant isolates points the need for new therapeutical alternatives. In this study the anti-Trichomonas vaginalis activity of essential oils obtained from Myrtaceae occurring in Caatinga, a plant family with potential antiparasitic activity, was showed. The essential oils varied in their capacity to kill ATCC and fresh clinical T. vaginalis isolates, which was associated with heterogeneity and different patterns of endosymbiosis. Essential oils caused moderate to strong cytotoxicity against mammalian cells, but essential oil of Eugenia pohliana (EOEp) exhibited promising selectivity index towards vaginal epithelial cells. A checkerboard assay revealed a synergistic effect when EOEp and metronidazole were associated, indicating different mechanisms of action. The GC/MS analysis demonstrated the volatile composition of EOEp, with δ-cadinene as majoritary component. This molecule seems to contribute to the trichomonacidal effect and shows potential for the prospection of new antiparasitic compounds. Graphical Abstract


Introduction
Although trichomoniasis is the most common non-viral sexually transmitted infection worldwide, it is still a neglected disease (Rigo et al. 2022). The infection, caused by Trichomonas vaginalis, can have serious health consequences and is also an important cofactor in the transmission and acquisition of HIV (Mercer and Johnson 2018). Moreover, the therapy is restricted and there are failures due to the emergence of resistant T. vaginalis clinical isolates (Kirkcaldy et al. 2012). In this manner, it is important to search new compounds capable of improving the treatment of trichomoniasis.
Essential oils (EO's) are a group of natural products represented by complex mixtures of volatile compounds that have demonstrated numerous pharmacological properties, including a potential activity against T. vaginalis (Eldin and Badawy 2019; Dai et al. 2016;Farias et al. 2019). In a recent review, the Myrtaceae family has been identified as holder of plants with trichomonicidal potential in its extracts and fractions (Mehriardestani et al. 2017). However, EO's obtained from these species still need to be validated as trichomonicidal agents. Herein, we aimed to investigate the anti-T. vaginalis activity of EO's from Brazilian Caatinga Myrtaceae species and to characterize the essential oil of the unexplored Eugenia pohliana DC. in order to identify potential therapeutic options for the treatment of trichomoniasis.

Results and discussion
2.1. Anti-Trichomonas vaginalis activity of the essential oils Among the seven EO's tested, those belonged to Eugenia gracillima, Eugenia pohliana, Myrciaria floribunda, and Psidium myrsinites (EOEg, EOEp, EOMf, and EOPm) presented marked anti-T. vaginalis activity, inhibiting ATCC 30236 trophozoites proliferation by 100% ( Figure S1). The anti-T. vaginalis activity of EOEg, EOEp, EOMf, and EOPm has not been previously reported in literature. However, these species are used in folk medicine in the Brazilian Northeast (Pernambuco state) to treat inflammatory diseases and have already demonstrated activity against other parasitic protozoa (Sampaio et al. 2019;Vandesmet et al. 2020). Table 1 shows results of EOEp and EOPm tested against fresh clinical isolates (TV-LACM22, TV-LACM15, and TV-LACH4) and ATCC 30236. EOPm presented the best results of minimal inhibitory concentration (MIC) and half of the maximum inhibitory concentration (IC 50 ) for all fresh clinical isolates, except for TV-LACM22. These differences of values could be related to heterogeneity and the presence of the endosymbionts Mycoplasma hominis and Trichomonasvirus among the isolates, which impacts metabolic pathways and upregulates the expression of genes involved in cytoadherence, cytotoxicity, and host immune evasion by the flagellated (Dess ı et al. 2019).

Cytotoxic activity of the essential oils
The EO's cytotoxicity results against MRC-5 and HMVII mammalian cell lines, as well as the hemolytic effect on human erythrocytes are presented in Table 1. In general, EO's showed low selectivity index (SI) against the cell lines used in the study. However, EOEp showed values above 1.0 for HMVII, the vaginal epithelial cell line (1.17) and erythrocytes (1.14), while OEMf showed SI of 1.05 for human erythrocytes. The SI compares the selectivity of compounds to cells or parasites. Values above 1.0 indicate greater selectivity for parasites, while values below 1.0 suggest greater selectivity for host cells (Moreira et al. 2019).
EOEp and EOPm presented moderate cytotoxicity to HMVII cells (301.9 and 148.8 ug/mL, respectively), the TV target site of infection, thus indicating that these could be promising oils in the sense of not causing damage to the host cells (R ıos et al. 2008). Our data suggest that despite showing favorable anti-T. vaginalis activity, EO's demonstrate low compatibility with mammalian cells. This issue should be further explored by using encapsulation approaches that can be employed in order to reduce the cytotoxicity of EO's (Sherry et al. 2013).

Synergistic effect with metronidazole
TV-LACM15 isolate was used to test the association between EOEp or EOPm and MTZ ( Figure S2). The checkerboard assay showed a synergistic effect, a fractional inhibitory concentration index (FICI) 0.5, at the highest metronidazole concentration (4 Â IC 50 , 0.72 lg/mL) associated with EOEp at all concentrations tested. As for EOPm, a synergistic effect was observed at the highest EO concentration (4 Â IC 50 , 651.2 lg/mL) and MTZ concentrations from 1 = 2 Â IC 50 to 4 Â IC 50 . These results demonstrate that the association between EOEp and MTZ allows the reduction of EOEp concentration required to cause parasite death. This suggests different mechanisms of action by which MTZ and the EO lead to trophozoite death, in order to prevent MTZ resistance evasion pathways.

Chemical composition of Eugenia pohliana essential oil
Eugenia pohliana is still a species that lacks phytochemical investigations. Considering the promising results in cytotoxicity and synergism assays with metronidazole, EOEp was characterized by Gas Chromatography-Mass Spectrometry (GC/MS) ( Figure S3). The EOEp showed a yield of 0.91% and chemical characterization revealed a total of 39 compounds, representing 99.94% of the oil. The main compounds identified were d-cadinene (20.23%), bicyclogermacrene (13.73%), and epi-a-muurolol (10.90%), all sesquiterpenes (Table S1). In fact, sesquiterpenes-rich EO's are evidenced as potential antiparasitic agents (Moreira et al. 2017). Although little is known about the biochemical mechanisms by which this class of compounds acts on parasitic protozoa, it is known that the lipophilic character of terpenes allows them to interact and permeate cell membranes. Thus, these compounds disrupt membranes and interact with intercellular structures, impairing their functions and leading to cell death due to increased permeation of cytoplasmic components (Barros et al. 2016).

Conclusions
This study demonstrated that essential oils from Caatinga Myrtaceae species are potential trichomonacidal agents. Although EO's have shown strong to moderate cytotoxicity towards mammalian cells, EOEp presented favorable selectivity to the parasite and a synergistic effect upon the association with metronidazole was found, highlighting the need for strategies to reduce toxicity and improve safety of this oil. This is the first report of the volatile constituents of Eugenia pohliana DC. leaves and the presence of d-cadinene may be responsible for its trichomonacidal activity. One possible direction for future studies is to focus on isolate compounds and on characterization of the mechanism of action of these essential oils.

Disclosure statement
No potential conflict of interest was reported by the authors.