Evaluation of inhibitory and antioxidant activities of free essential oils and nanoemulsions on trichomonas vaginalis

Trichomoniasis, an infection caused by Trichomonas vaginalis, is the most commom non-viral sexually transmitted infection worldwide. Due to Metronidazole’s (MTZ) resistance to standard drugs, this study aimed to evaluate the efficacy of free and nanostructured essential oils (EO) as Cymbopogon flexuosus (lemongrass), Eucalyptus globulus (eucalyptus) and Ocimum basilicum (basil), besides antioxidant activities against T. vaginalis . The free and nanostructured EOs were tested for in vitro activity against Trichomonas vaginalis ; trophozoites were treated with different concentrations (ranging from 0.25% to 1.5%) in order to establish the MIC and IC 50 values. Oxidative stress was also evaluated by measuring lipid peroxidation by the detection of thiobarbituric acid reactive substances (TBARS). The MIC observed for lemongrass was 1.5% with free oil and 1.0% with nanoemulsion, while for basil it was 1.5% with free and nanostructured oils. Significant antiparasitic activities were observed in 12 and 24 hours exposure, respectively, inhibiting 100% of trophozoites. Antioxidant activity was also observed in these oils, which, either in free or nanostructured form, were able to reduce lipid peroxidation.


INTRODUCTION
Trichomoniasis is the most commom non-viral sexually transmited infeccion in the world.
This infection is caused by the flagellated protozoan Trichomonas vaginalis (MUZNY, 2018). This parasite is considered the main agent responsible for the occurrence of vaginitis in women, and has alsobeen associated to infertility, cervical cancer, pelvic inflammatory disease, preterm birth, and low birth weight. Besides, it has been indicated that it is a risk factor for human immunodeficiency virus acquisition and transmission (KISSINGER, 2015).
Since 1959, metronidazole (MTZ) has been the drug of choice for the treatment of trichomoniasis. A 2 g single oral dose or 500 mg twice daily for 7 days is the recommended treatment. The latter regimen is suggested for HIV-infected or recidives patients. Side effects such as nausea vomiting and epigastric pain represent a barrier for patients finishing the protocol of treatment, which cause the emergence of resistant strains (DUNNE et al., 2003;CUDMORE et al., 2004).
Despite the known efficacy of MTZ, drug resistance has increased since 1962. Faced with several reports os resistance, natural products have been important sources of novel therapeutic options.
The literature demonstrates the remarkable performance of essential oils (EOs), derived from the secondary metabolism of plants, showing the many biological activities of this compounds (BILIA et al., 2014). Due to their lipophilic and chemical characteristics, EOs can be incorporated into nanostructures. Nanotecnology is an alternative to facilitate and improve their absorption and distribution to target cells, besides reducing volatility and instability through their incorporation into nanocapsules (COUVREUR; VAUTHIER, 2006;DANIELLI et al., 2013;SALVIA-TRUJILLO et al., 2015;CHANG et al., 2015). Thus, the aim of this study was to evaluate the efficacy of essential oils of Cymbopogon flexuosus (lemongrass), Eucalyptus globulus (eucalyptus) and Ocimum basilicum (basil) against T. vaginalis, both free and nanostructured forms.

Trichomonas vaginalis recovered and Method of culture
Samples were collected at the Clinic of Gynecology and Obstetrics in the Infant and Maternal Department of the School of Medicine (FAMED) of the Universidade Federal de Pelotas (UFPel), during routine medical appointments. Swab was to isolate and keep the axenic culture, trophozoites were inoculated in TYM (trypticase-yeast-extract-maltose) medium, pH 6.0, supplemented with 10% inactivated bovine serum at 37 ºC (DIAMOND, 1957). Isolates were sub-cultured every 24 hours in new culture tube containing complete TYM medium and maintained at laboratory.

Ethics
All procedures performed in this study involving human participants were in accordance with the ethical standards of the Ethics Committee of the School of Medicine, Federal University of Pelotas (Committee Approval number 873.180) and National Commission on Ethics in Research (amendment 1.838.829).

In vitro assay
The culture samples were collected and viable trophozoites were counted in a Neubauer cellcounter chamber, in triplicate, to adjusted the final density to 2.6x10 5 trophozoites/ml culture. The tests were performed at 37 ºC at atmosphere of 5% CO 2 , in nonoplicate, in 96 wells microtiter plates following SENA-LOPES et al., (2018), adapted. From the stock solution with free and nanoemulsion oils (100%), dilutions in TYM medium were prepared at double the concentration desired for testing.
Free and nanostructured oils were added to each well containing 100 μL of medium with T. vaginalis trophozoites to reach a final concentration of 0.25%, 0.5%, 1.0%, and 1.5%. The viability of the trophozoites in the culture plate was evaluated by 0.4% trypan blue dye exclusion and quantified in a cell-counter chamber using a hemocytometer. Also, theanalysis determined the IC50 (half the maximum inhibitory concentration) and evaluated the minimal inhibitory concentration (MIC) of the free oils or nanoemulsions that were able to inativate 100% of viable trophozoites at the screening.

Essential oil
The essential oils of lemongrass (C. flexuosus) and eucalyptus (Eucalyptus globulus) were commercially obtained respectively from FERQUIMA Indústria e Comércio de Oliveira, and basil

Characterization of the essential oils
The characterization of free essential oils of lemongrass, eucalyptus and basil were performed at the Nanotechnology Laboratory of the Fransciscana University of Santa Maria-RS (UFN), following the method by Hussain et al. (2008), adapted (GODOI et al., 2017;DA SILVA GÜNDEL et al., 2018a, 2018b.

Development of nanoemulsions
Nanoemulsions were developed by the Nanotechnology Laboratory (UFN) under high agitation method with homogenizator to incorporate the lemongrass, eucalyptus or basil EOs. The blank nanoemulsion was developed using a medium chain triglyceride, derived from caprylic and capric acids (GODOI et al., 2017;DA SILVA GÜNDEL et al., 2018a, 2018b.

Characterization of nanoemulsions
The physicochemical characterization of the formulations was evaluated at the same laboratory by determining the mean droplet size, polydispersity index, zeta potential and pH at the same day of production (DA SILVA GÜNDEL et al., 2018a).

Statistical analysis
The results of the in vitro tests are presented as percentages and were analyzed by an analysis of variance (ANOVA) followed by comparison with the Tukey test using the Graph Pad Prism 7.04 software. The significance level was p < 0.05.
The efficacy obtained with the free and nanostructured oils, MTZ and controls used for the in vitro assays can be observed at   The TBARS assay showed that trophozoites at negative control (NC) increased lipoperoxide levels. Nevertheless, when free EO or nanoemulsions containing these oils were added, as well as Tween 80, peroxidation activity was reduced, showing significant difference when compared to the NC, as shown in the graphic (Figure 3). Disciplinarum Scientia. Série: Naturais e Tecnológicas, Santa Maria, v. 21, n. 1, p. 139-153, 2020.

DISCUSSION
Several reports over the years have shown T. vaginalis resistance when exposed to MTZ, even at low levels (DUNNE et al., 2003;CUDMORE et al., 2004;KIRKCALDY et al., 2011). Studies which aimed to evaluate biological activity, like our research, emphasize the increase of the search for new metabolites with therapeutic value considering the large biodiversity within the Brazilian territory and the potential of natural products in the treatment of diseases (BASSO et al., 2005). Our study found promising results using lemongrass and basil EO, in addition to the new data with nanoemulsion.
In this work, the compounds identified by gas chromatography for C. flexuosus corresponded to the literature. The lemongrass was composed mainly of an isomeric misture of two compounds, alfacitral (geranial) and beta-citral (neral) (KAKARLA; DEEPAK, 2009), and this is the first report of anti-T.
vaginalis activity for C. flexuosus. In addition to a complex natural mixture, where the synergism between these chemical components could be linked to their promising pharmacological potential (BAKKALI et al., 2008;GALINDO et al., 2010). Thus, they act through different mechanisms of action in several targets within the cells, as in the cell proteins present in the cytoplasmatic membrane (BURT et al., 2004).
Our study found higher trophozoites inactivation of T. vaginalis when treated with lemongrass in both presentations, as detected by Adukwu et al., (2016) against four strains of Acinetobacter baumannii, including two multi-drug-resistant, which showed suscetibility when exposed to C.

flexuosus, being an alternative for resistant infections. The higher bioactivity of nanostructured clove
EOs when compared to free oil and amikacin, was also evidenced in the present study with lemongrass incorporated into nanostructures, presenting similar activity to free oil and MTZ, in lower concentration (ANWER et al., 2004). This highlights the potencial of natural products associated to nanoemulsion droplets, enhancing the biological properties of the components to the target.
Nanotechnology has been used as an alternative to improve the activity of EOs. In our study, this formulation probably contributed for a higher celular absorption and bioefficacy, avoiding the decomposition of these oils due to enviromental factors, thus maintaining stability and decreasing volatility, which can justify the higher activity presented by lower concentrations of lemongrass nanoemulsion when exposed to trophozoites (BILIA et al., 2014). This nanoemulsion was able to reduce the MIC of lemongrass, when compared to free oil, as observed with a nanostructured drug in another study (BALDISSERA et al., 2013;FUENTEFRIA et al., 2014;BAJERSKI et al., 2016). The protection offered by nanostructures to bioactive content enhance the delivery to target, resulting in more effectivenessthan other convencional forms (LOVELYN et al., 2011).
Studies have suggested that the use of drug system delivery in nano size may increase passive cellular absorption, probably through the association of porins and phenolic compounds. This interaction promotes cell breaking due to leakage of ions and others cell contents, increasing the Disciplinarum Scientia. Série: Naturais e Tecnológicas, Santa Maria, v. 21, n. 1, p. 139-153, 2020. 148 bioactivity and consequently the therapeutic efficacy of oils when nanostructured (DONSÌ et al., 2012;SALVIA-TRUJILLO et al., 2015), which justifies the better activity of lemongrass nanoemulsion observed in our study against T. vaginalis.
Nanostructured systems are prepared from differents materials with dimension ranging from 0.1 to 500 nm. This size and biocompatibility could lead to better penetration into the membrane of the trophozoites and facilitate EO activity, which could explain the significant inhibitory activity observed in our tests. The surface charge of nanoemulsions droplets, also known as zeta potential, is the electrical characteristics of the delivery system drug (MCCLEMENTS et al., 2011). Controlling the charge is a way to control the interactions with the infectuous agent, since the microorganisms have the opposite charges to the nanoemulsions, and when in contact with the oil incorporated into these droplets, there will be electrostatic attraction (ZIANI et al., 2011;CHANG et al., 2015). The electrostatic attraction could be discarded in this study, beacause the nanoemulsion has negative charge and the trophozoites membrane is made up mainly by sialic acid, which contributes to the negative charge of the protozoan (SILVA-FILHO et al., 1986).
The performance of basil against T. vaginalis is in accordance with the literature. The same growth inhibitory ability of the EO observed in our research was reported earlier. That study suggests that the antiprotozoal activity observed is probably related to the capacity of basil to inhibit the proteolytic activity of cysteine proteinase, an enzyme present in the life cycle of several protozoan, and the same may occur with T. vaginalis (ELDIN et al., 2015). In our study, the growth inhibition was dose and time dependent and no statistically significant difference was detected when compared to MTZ.
However, there are no reported studies using nanoemulsions containing O. basilicum with anti -Trichomonas activity for comparison. This is the first time that the activity of this nanostructured oil was reported against this protozoan. This activity can also be justified by the characteristics characteristics of the EO, which has low density and hydrophobicity that facilitate the delivery of active compounds into specific target cells in parasites (MONZOTE et al., 2012). The literature states that basil oil is composed of terpenes (linalool and geraniol) and phenylpropanoids (methyl chavicol, eugenol, methy leugenol and methyl cinnamate). This varying concentration or presence of major compounds between the species can be justified by environmental factors and the geography of the place where the plant was obtained (ÖZCAN et al., 2005;PANDEY et al., 2014).
Anti-trichomonas activity revealed that no growth was observed after 24h of incubation of lemongrass and basil. The mechanism of action of EOs against microorganisms has not been understood

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The promising pharmacological potential of EOs against parasites could be linked to major compounds or to the synergism within this complex natural mixture, acting through diferents mechanism of action in several targets within the cells, such as the proteins present in the cytoplasmatic membrane (BURT et al., 2004;BAKKALI et al., 2008). The literature suggests that this relationship of bioactives compounds contributes to avoid the emergence of new resistant strains, due to the different receptors that the oil can affect during all stages of the lifecycle of a parasite (ANDRÉ et al., 2018).
Basil and lemongrass oils were identified in this study as potent antioxidants with ability to improve lipid and antioxidant status. EOs can be considered good sources of natural compounds with significant antioxidant activity, which can also be attributed to their main constituents. The capacity of phenolic compounds of donating hydrogen or an electron and removing oxygen from free radicals is responsible for this biological property (SINGH;NISHI, 2015). The reduction of lipid peroxidation observed for the treatments with free and nanostructured oils is confirmed by earlier reports. It is well known that lemongrass is a source of bioactive compounds, flavonoids and vitamins which could inhibit the chain reactions of lipid oxidation, thus justifying the antioxidant activity obtained (IBRAHIM; FERIAL, 2013).

CONCLUSIONS
Lemongrass and basil are promissing alternatives for the treatment of trichomoniasis. Furthermore, lemongrass showed that the incorporation of oils into nanoemulsions is an attractive option in the developing of new biologically active natural drugs, to promote therapeuthic efficacy in the treatment of this sexually transmitted infection.