Trypanocidal Essential Oils: A Review

Trypanosomiases are diseases caused by parasitic protozoan trypanosomes of the genus Trypanosoma. In humans, this includes Chagas disease and African trypanosomiasis. There are few therapeutic options, and there is low efficacy to clinical treatment. Therefore, the search for new drugs for the trypanosomiasis is urgent. This review describes studies of the trypanocidal properties of essential oils, an important group of natural products widely found in several tropical countries. Seventy-seven plants were selected from literature for the trypanocidal activity of their essential oils. The main chemical constituents and mechanisms of action are also discussed. In vitro and in vivo experimental data show the therapeutic potential of these natural products for the treatment of infections caused by species of Trypanosoma.


Introduction
Trypanosomiases are insect-borne parasitic diseases of humans and animals caused by flagellate protozoans of the genus Trypanosoma. Occurring mainly in Latin America and Africa, where they are considered endemic, and they are of great importance to human health [1,2]. According to Britannica Academic [3], the life cycle of trypanosomes includes one stage in the blood or tissue of a vertebrate host and another stage in an invertebrate vector. Furthermore, approximately 20 Trypanosoma species are known, but only two species cause disease in humans, Trypanosoma cruzi and Trypanosoma brucei. T. cruzi is the cause of American trypanosomiasis, also called Chagas disease, which is prevalent throughout the Americas. It is spread by Triatominae insects, commonly called "kissing bugs". In the bloodstream, the parasite multiplies and can reach the heart, liver and spleen, where it can cause extensive damage. T. brucei is responsible for African trypanosomiasis or sleeping sickness, most commonly found in equatorial Africa. Human African trypanosomiasis takes two forms depending on the parasite involved, which are both transmitted by tsetse flies (Glossina spp.). Sleeping sickness in eastern and southern sub-Saharan Africa is an acute form caused by the subspecies T. brucei rhodesiense. Trypanosomiasis in the central and western regions of Africa is a slow-progressing form caused by T. brucei gambiense. Both trypanosomes invade the brain, causing mental deterioration, coma and death if left untreated [4,5]. There is also Surra, which is a trypanosomiasis caused by T. evansi, that affects wild and domestic animals mainly in Africa, Asia and South America, causing serious economic losses due to mortality and morbidity [6]. However, despite being a disease that predominantly affects animals, cases of surra have been reported in humans in the Asian region, where the main symptoms observed were fever and drowsiness, without invasion of the parasite in the central nervous system [7]. Figure 1 illustrates the worldwide distribution of the main species causing human trypanosomiasis.
Molecules 2020, 25, x FOR PEER REVIEW 2 of 20 the central nervous system [7]. Figure 1 illustrates the worldwide distribution of the main species causing human trypanosomiasis. Essential oils and their constituents present a broad spectrum of pharmacological activities, such as antinociceptive [8,9], anti-inflammatory [10], antitumor [11] and antiulcer [12] activities. The various pharmacological properties of essential oils and their constituents against Trypanosoma sp. are well studied [13].
The purpose of the current review is to provide a comprehensive summary of the literature on essential oils as potential trypanocidal drugs against Trypanosoma spp. In this study, available data could be used as an updated source of the progress or success for identification of trypanocidal compounds.

Essential Oils with Trypanocidal Activity
Forty-six articles in the literature were found that describe trypanocidal activity of essential oils, totaling seventy-seven plants studied; however, three species were not bioactive. Among trypanocidal essential oils, forty-one were active against T. cruzi, thirty-four against T. brucei and seven had activity against T. evansi.
The trypanocidal models/methods employed (Tables 1-3) for the in vitro and in vivo testing of trypanocidal activity of essential oils were as follows: in vitro testing with dimethyl sulfoxide (DMSO) microdilution was used for 95.1% of the oils, dissolution in 5% ethanol was used for 2.4% of the oils, and dissolution in 5% acetone was used for 2.4% of the essential oils; evaluation with colorimetric test MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used for 46.7% of the oils. The resazurin test that evaluates proliferation of trypanosomes in an in vivo test was used for 6.7% of the studies.
Administration routes for parasitemia-inducing forms and blood collection for evaluation were by intraperitoneal (i.p.) and tail vein (c.d.) routes, respectively. The route of administration for essential oils was oral gavage (p.o.). In 15.5% of cases, parasitemia was evaluated through blood samples and stained using a panoptic method, while hematoxylin and eosin (HE) was used in 26.3% of cases. Scanning and transmission electron microscopy was used for 10% of cases, while Essential oils and their constituents present a broad spectrum of pharmacological activities, such as antinociceptive [8,9], anti-inflammatory [10], antitumor [11] and antiulcer [12] activities. The various pharmacological properties of essential oils and their constituents against Trypanosoma sp. are well studied [13].
The purpose of the current review is to provide a comprehensive summary of the literature on essential oils as potential trypanocidal drugs against Trypanosoma spp. In this study, available data could be used as an updated source of the progress or success for identification of trypanocidal compounds.

Essential Oils with Trypanocidal Activity
Forty-six articles in the literature were found that describe trypanocidal activity of essential oils, totaling seventy-seven plants studied; however, three species were not bioactive. Among trypanocidal essential oils, forty-one were active against T. cruzi, thirty-four against T. brucei and seven had activity against T. evansi.
The trypanocidal models/methods employed (Tables 1-3) for the in vitro and in vivo testing of trypanocidal activity of essential oils were as follows: in vitro testing with dimethyl sulfoxide (DMSO) microdilution was used for 95.1% of the oils, dissolution in 5% ethanol was used for 2.4% of the oils, and dissolution in 5% acetone was used for 2.4% of the essential oils; evaluation with colorimetric test MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used for 46.7% of the oils. The resazurin test that evaluates proliferation of trypanosomes in an in vivo test was used for 6.7% of the studies.
Administration routes for parasitemia-inducing forms and blood collection for evaluation were by intraperitoneal (i.p.) and tail vein (c.d.) routes, respectively. The route of administration for essential oils was oral gavage (p.o.). In 15.5% of cases, parasitemia was evaluated through blood samples and stained using a panoptic method, while hematoxylin and eosin (HE) was used in 26.3% of cases. Scanning and transmission electron microscopy was used for 10% of cases, while measurement with a Neubauer camera was used in 25.3% of cases. ELISA was used for 15.3% of cases with an absorbance wavelength of 492-600 nm.

Essential Oils with Trypanocidal Activity against T. cruzi
The essential oils of the species Cinnamodendron dinisii Schwacker and Siparuna guianensis Aublet [14] presented in vitro trypanocidal activity against T. cruzi at concentrations of 282.93 and 209.30 µg/mL, respectively, dissolved in DMSO and analyzed using a colorimetric assay (MTT) [15,16]. The study presented by Andrade et al. [14] suggested that the trypanocidal activity of these essential oils may be associated with oxygenated terpenes found in small quantities in both plant species. Trypanocidal activity of oxygenated terpenes had already been reported in a previous study [17]. Barros et al. (2016) [18] highlighted the in vitro trypanocidal activity of the Lantana camara L. essential oil against T. cruzi at a concentration of 291.94 µg/mL dissolved in DMSO. Analysis of the essential oil of the species revealed that (E)-caryophyllene and bicyclogermacrene represent the principal components of this oil. Thus, (E)-caryophyllene may be partly responsible for the trypanocidal activity of the species. The study also highlighted that other chemical compounds in this species may present activity against T. cruzi [18].
The Cinnamomum verum J. Presl. essential oil presented in vitro anti-T. cruzi activity at concentrations of 24.13, 20.0 and 5.05 µg/mL and was dissolved in DMSO [19]. Inhibitory activity was assessed by Thiazolyl Blue Tetrazolium Bromide (MTT) colorimetric assay [15,16]. Analysis of the essential oils of the species revealed that (E)-cinnamaldehyde and eugenol were the principal constituents, suggesting that its trypanocidal activity may be related to these components. The study also pointed out that (E)-cinnamaldehyde was not effective against T. cruzi epimastigotes at low concentrations of up to 300 µM.
Essential oils from Xylopia frutescens var. ferruginea Aubl. and Xylopia laevigata (Mart.) [20] presented in vitro trypanocidal activity against T. cruzi at concentrations below 30 and 15 µg/mL, respectively. Analysis of the essential oils of both species revealed very similar chemical compositions, differing in the concentration of some constituents, yet the main components of both were bicyclogermacrene, (E)-caryophyllene and germacrene D. The trypanocidal activity was attributed to the high concentration of these components. Recent work has demonstrated the trypanocidal properties of these constituents [20,21].
As reported by   [22], the in vitro trypanocidal activity of the species Thymus vulgaris L. must be associated with the presence of thymol (80.4%). The author does not rule out the possibility that other components may be more active against T. cruzi than thymol. As a result of this study, IC 50 /24 h values equal to 77 µg/mL for epimastigotes and 38 µg/mL for trypomastigotes were found; each essential oil was dissolved in DMSO for the trypanocidal activity assays.
The essential oil of Aloysia triphylla (L'Hér.) Britton [23] presented in vivo trypanocidal activity against T. cruzi at doses of 100 and 250 mg/kg when dissolved in DMSO and administered via an orogastric tube. Analysis of the essential oil of this species revealed citral, a mixture of two isomeric monoterpene aldehydes, geranial, and neral [24] (this last compound was the principal component, suggesting that the trypanocidal activity is related to this constituent). In a previous study,   [17] reported trypanocidal activity attributed to this compound when investigating the in vitro trypanocidal activity of the essential oil of Cymbopogon citratus (DC.) Stapf against T. cruzi at concentrations of 126.5 and 15.5 µg/mL.
The Achillea millefolium L., Syzygium aromaticum L., and Ocimum basilicum L. essential oils [25] presented in vitro anti-T. cruzi activity at concentrations of 145.5 and 228 µg/mL, 57.5 and 99.5 µg/mL, and 102 and 467.5 µg/mL, respectively, after serial dilution in DMSO. The main chemical components of S. aromaticum L., O. basilicum L., and A. millefolium L. are eugenol, linalool, and chamazulene, respectively, which were attributed with the trypanocidal activity. Previous studies have shown the trypanocidal activity of S. aromaticum L. [26], O. basilicum L. [27], and A. millefolium L. [28] essential oils. The essential oil of Piper cubeba L. [29] presented in vitro trypanocidal activity against T. cruzi at concentrations of 45.5 and 87.9 µg/mL, being serially diluted in DMSO and evaluated by the MTT colorimetric assay [15,16]. Analysis of the essential oil of this species revealed that sabinene, eucalyptol, 4-terpineol, β-pinene and camphor were the main components and were considered responsible for the observed trypanocidal activity.
The essential oils of Chenopodium ambrosioides L., Justicia pectoralis Jacq., and Vitex agnus-castus L. [13] present in vitro anti-T. cruzi activity at concentrations of 21.3, 56.8, and 155.8 µg/mL, respectively. Each was dissolved in DMSO and further analyzed by colorimetric assay (MTT) [15,16]. The compound 1,8-cineole is the largest constituent of the V. agnus-castus essential oil, and terpinolene is a major component of the essential oils obtained from J. pectorals and C. ambrosioides.
Bay et al. [33] investigated the trypanocidal activity of the essential oils of four species of Annonaceae (Bocageopsis multiflora, Duguetia quitarensis, Fusaea longifolia and Guatteria punctata) against trypomastigote and intracellular amastigote forms of T. cruzi. The trypanocidal action results indicated that the essential oils were active, and that the essential oil of G. punctata was the most active (the main constituents for this oil were germacrene D, (E)-nerolidol and (E)-caryophyllene), with an IC 50 = 0.029 µg/mL, which presented the highest selectivity index (SI) and was 34 times more effective than benznidazole (1 µg/mL).
Gutierrez et al. 2016 [35], examined the chemical analysis, antimicrobial activity and cytotoxic effects of OEs from Piper aduncum var. ossanum that was harvested from two locations, Bauta and Ceiba, Artemisa Province, Cuba. Both OEs showed the same activity against T. cruzi (approximately 8 µg/mL).
Recently, the inhibitory properties and cellular effects of the essential oils of L. alba and their main bioactive terpenes and the synergy between them were shown against the strains of T. cruzi. The L. alba OEs had significant differences in their chemical composition and trypanocidal performance (p = 0.0001). Citral chemotype oils showed greater trypanocidal activity than carvone essential  [38] verified the leishmanicidal and anti-T. cruzi potential as well as the cytotoxicity of the Alpinia speciosa K. Schum. essential oil. A. speciosa presented 1,8-cineole (28.46%), camphor (17.10%) and sabinene (9.95%) as the main constituents. The cytotoxic activity of the essential oil showed a low value, while the antipromastigote and antiepimastigote activities showed values that were considered clinically relevant, with values below 500 µg/mL.
Zanusso Júnior et al. (2018) [39] investigated the activity of the EO of Syzygium aromaticum (main compounds are eugenol and β-caryophyllene) alone and in association with benznidazole (BZ) in mice infected with T. cruzi AM14 strain (TcIV) (considered resistant to BZ in vivo). When compared to untreated animals, experiments with the EO of S. aromaticum alone promoted a reduction of the parasitemia. However, the animals treated with BZ alone or in association showed a more significant reduction in parasitemia.
The study presented by Nibret and Wink (2010) [46] was compared to that of Costa et al. (2013) [21] where the analysis of the essential oil of Cinnamomum verum J. Presl was made against the T cruzi species. (E)-cinnamaldehyde was effective against T. brucei trypomastigotes in low concentrations The essential oil from the species Keetia leucantha (K. Krause) Bridson [47] presented in vitro anti-T. brucei activity at a concentration of 20.9 µg/mL. Analysis of the essential oil suggested α-ionone, β-ionone, and ursolic acid were the compounds that presented the best inhibitory activity against T. brucei, although analysis of the oil revealed that the principal constituents of the oil were n-hexadecanoic acid and phytol, which presented IC 50 values of >100 and 19.1 µg/mL, respectively.
The Ocimum gratissimum L. essential oil [48] was active against T. brucei (in vitro) at a concentration of 1.66 µg/mL when dissolved in DMSO and evaluated using the MTT colorimetric assay [15,16]. Analysis of the essential oil of this species revealed that sabinene, eucalyptol, 4-terpineol, β-pinene and camphor are the main components and are considered responsible for the trypanocidal activity.
In another study, the essential oil of Aframomum sceptrum (Oliv. and D. Hanb.) K. Schum. [49] presented in vitro anti-T. brucei activity at a concentration of 1.51 µg/mL. Analysis of the essential oils of this species revealed the constituents β-pinene and caryophyllene oxide, to which the trypanocidal activity was attributed.
The Kadsura longipedunculata Finet el Gagnep (Nanwuweizi) essential oil [50] presents in vitro anti-T. brucei activity at the concentration of 50.52 µg/mL. This evaluation was made using the resazurin dye test [51] to evaluate trypanosome proliferation. Analysis of the essential oils revealed δ-cadinene and camphene as the principal components, which were attributed to the trypanocidal properties.
De   [52] investigated the EO of Mentha crispa L. and its main constituents (rotundifolone and four related p-menthane monoterpenes) against T. brucei trypomastigotes. The essential oil, the compounds rotundifolone and perillyl aldehyde presented a dose-dependent action and identical 50% growth inhibitory concentration (GI 50 ) of 0.3 µg/mL. Petrelli et al. (2016) [53] evaluated the biological activity exhibited by the essential oil obtained from the aerial parts of Croton floribundus Spreng against T. brucei in vitro. The proliferation of T. brucei was inhibited with IC 50 values of 33.5 µg/mL from the essential oil and 5.6 µg/mL from the active component limonene.
Gutierrez et al. 2016 [35], also examined the chemical analysis, antimicrobial activity and cytotoxic effects of OEs of Piper aduncum var. ossanum against T brucei, and showed an activity similar to T cruzi (approximately 8 µg/mL).
A study by Sobeh et al. (2016) [54] analyzed the composition of the Eugenia uniflora L. essential oil, to which T. brucei was highly susceptible, with an IC 50 of 11.20 µg/mL and an SI of 6.82.
According to Carmo et al. (2015) [61] and Baldissera et al. (2014) [62], the essential oil from the species Achyrocline satureioides (Lam.) DC. (Macela) presented bioactivity in vivo against T. evansi at a dose of 1.5 mL/kg after oral administration for five consecutive days. For hematological analysis, blood samples were collected on the fifth day. The chemical compounds with higher proportions in the essential oil were the terpenes α-pinene and β-caryophyllene, and the trypanocidal activity was attributed to these compounds.
The essential oil of Aniba canelilla (HBK) Mez, popularly known as "bark-precious" (precious bark), presented as a major constituent 1-nitro-2-phenylethane, a rare molecule in plants, and methyleugenol, and the oil presented results against T. evansi. Methyleugenol was slightly more active than 1-nitro-2-phenylethane, and in vitro studies showed that the oil extracted from the stems of A. canelilla can be considered a potential natural treatment for trypanosomiasis [63]. Baldissera et al. (2013) [64] investigated the in vitro activity of the essential oils of andiroba (Carapa guaianensis) and aroeira (Schinus molle) against T. evansi trypanosomes using conventional (at concentrations of 0.5%, 1.0%, and 2.0%) and nanoemulsion forms (at concentrations of 0.5% and 1.0%). The tests were performed in duplicate and the numbers of parasites evaluated after 1, 3 and 6 h. The trypanocidal activity of essential oils was dose-dependent and, after 6 h of experiment, no living trypanosomes were observed. For the nanoemulsion oils, the activity was dose-dependent after 1 h of interaction, but after 3 h no alive parasites were observed. Baldissera et al. (2017) [65] verified the trypanocidal activity in vitro of EOs of Lippia alba and Lippia origanoides against T. evansi trypomastigotes. The lower concentration of EOs (0.5%) eliminated the parasites after 6 h of interaction. Experiments in vivo with mice also were performed. The animals were treated with EOs at a dose of 1.5 mL/kg for five days and, despite not showing curative efficacy, the treated mice with EO of L. origanoides showed increase in longevity when compared to the control group. Table 3 shows essential oils with trypanocidal activity against T. evansi.

Proposed Mechanisms of Action for the Trypanocidal Activity of Essential Oils
Essential oils from aromatic plants and their major components have been studied for their antimicrobial activities, with significant anthelmintic and antiprotozoal activity [66]; nevertheless, their mechanism of action remains poorly studied. Therefore, identification of the active components of EOs and understanding their mechanisms of action are essential.
The activity of some EOs could be associated with the lipophilic characteristics of their constituents. Lipophilic molecules can cross the cell membrane, and once inside the cells, these molecules can interact with a variety of proteins, inactivating enzymes and affecting cellular activities [67]. Depolarization of the mitochondrial membrane is associated with changes in calcium channels and ROS generation that can trigger cell death by apoptosis and necrosis [68,69] (Figure 2).
During apoptosis, there are alterations such as cytoplasmic blebbing, cell volume reduction, loss of mitochondrial membrane potential, condensation of nuclear chromatin, and DNA fragmentation [71]. Such characteristics were observed from the essential oils of Cinnamodendron dinisii [14], Siparuna guianensis [14], Cinnamomum verum [19], Lippia dulcis [30], Achyrocline satureioides [61,62], Lippia sidoides [13], Lippia origanoides [13,30,65], Chenopodium ambrosioides [13], Justicia pectorales [13], Lippia citriodora [30], Lippia pedunculosa [31] and Lippia alba [30]. However, due to the large number of components and potential synergistic and/or antagonistic interactions between them, it is likely that in addition to membranes, essential oils can also act against other cellular targets ( Figure 2). Despite the various pharmacological properties attributed to essential oils, and prospects for clinical application, especially in the area of psychopharmacology due to the relaxing effects of essential oils traditionally used for therapeutic purposes, there are few reports of toxicological studies of essential oils. Therefore, it is not possible to describe the possible side effects. Thus, it is important to advance in the knowledge about the therapeutic safety of these natural products [72].

Methodology
The present study was based on works published on essential oils with trypanocidal activity in experimental models. The search was performed on the PubMed Scientific Database (Home-PubMed-NCBI) in November 2019. For the selection of data in English, search terms related to the Despite the various pharmacological properties attributed to essential oils, and prospects for clinical application, especially in the area of psychopharmacology due to the relaxing effects of essential oils traditionally used for therapeutic purposes, there are few reports of toxicological studies of essential oils. Therefore, it is not possible to describe the possible side effects. Thus, it is important to advance in the knowledge about the therapeutic safety of these natural products [72].

Methodology
The present study was based on works published on essential oils with trypanocidal activity in experimental models. The search was performed on the PubMed Scientific Database (Home-PubMed-NCBI) in November 2019. For the selection of data in English, search terms related to the theme were used, such as "essential oils" and "Trypanosoma" or "trypanocidal". Articles that are literature reviews or contain only oil constituents were not included in the review.

Conclusions
The data presented demonstrate the importance of essential oils as natural products to be investigated for the development of new therapeutic options in the face of neglected diseases, especially trypanosomiasis. It is necessary to evaluate the main chemical components of these oils in experimental models in vitro and in vivo, in addition to researching their mechanism of trypanocidal action. In addition, the therapeutic safety of these products must be evaluated through toxicological studies. The abundance of essential oils in several tropical countries and low production costs make them interesting compounds to be researched as new pharmacological tools for use in the treatment of neglected diseases. The study will also support the discovery of new essential oils and/or chemical constituents, as well as analogous compounds with trypanocidal activity.