Active Essential Oils and Their Components in Use against Neglected Diseases and Arboviruses
The term neglected diseases refers to a group of infections caused by various classes of pathogens, including protozoa, viruses, bacteria, and helminths, most often affecting impoverished populations without adequate sanitation living in close contact with infectious vectors and domestic animals. The fact that these diseases were historically not considered priorities for pharmaceutical companies made the available treatments options obsolete, precarious, outdated, and in some cases nonexistent. The use of plants for medicinal, religious, and cosmetic purposes has a history dating back to the emergence of humanity. One of the principal fractions of chemical substances found in plants are essential oils (EOs). EOs consist of a mixture of volatile and hydrophobic secondary metabolites with marked odors, composed primarily of terpenes and phenylpropanoids. They have great commercial value and were widely used in traditional medicine, by phytotherapy practitioners, and in public health services for the treatment of several conditions, including neglected diseases. In addition to the recognized cytoprotective and antioxidative activities of many of these compounds, larvicidal, insecticidal, and antiparasitic activities have been associated with the induction of oxidative stress in parasites, increasing levels of nitric oxide in the infected host, reducing parasite resistance to reactive oxygen species, and increasing lipid peroxidation, ultimately leading to serious damage to cell membranes. The hydrophobicity of these compounds also allows them to cross the membranes of parasites as well as the blood-brain barrier, collaborating in combat at the second stage of several of these infections. Based on these considerations, the aim of this review was to present an update of the potential of EOs, their fractions, and their chemical constituents, against some neglected diseases, including American and African trypanosomiasis, leishmaniasis, and arboviruses, specially dengue.
Neglected diseases (ND) are a group of infections caused by various classes of infectious and parasitic pathogens, including protozoa, viruses, bacteria, and helminths. They most often affect low-income populations, with higher prevalence in tropical and subtropical countries. According to the World Health Organization (WHO), they are a set of 17 diseases, including leishmaniasis, African and American trypanosomiasis, leprosy, tuberculosis, leptospirosis, onchocerciasis, and schistosomiasis, affecting more than one billion people around the world and considered a threat to public health in 149 countries [1, 2].
The fact that these diseases are not considered priorities by large medical and pharmaceutical companies results in low investment in research and development of new drugs, causing the few available treatment options to become obsolete, precarious, outdated, and even in some cases nonexistent .
These diseases are known for the lack of attention paid to them by political, social, and health entities. The worsening of social and health conditions in a great part of the world’s population has accelerated in recent years according to studies providing data regarding current social policies [4–6].
According to some studies in Latin American countries, Brazil has the highest rate of neglected diseases, and a large part of the Brazilian population both above and below the poverty line suffers from some of these diseases [7–9].
Several neglected diseases are arboviruses, that is, viral diseases transmitted through the saliva of contaminated arthropods . Thus, they spread with worrisome epidemiological speed because of human exposure to the insect vectors [11, 12]. In some cases, a single vector can transmit more than one disease, as is the case of the vector Aedes aegypti that may transmit dengue, Zika, and chikungunya.
According to some authors [13, 14], 80% of the Brazilian population lives in urban areas. Demographic expansion and climatic conditions create an attractive environment for the reproduction of arbovirus vectors, rapidly affecting a large part of the population living in these urban centers . Due to the absence of many natural predators, disease-bearing arthropods reproduce more easily in urban centers.
The use of plants for medicinal, religious, and cosmetic purposes dates back to the emergence of humanity. This historical interest promoted the development of several areas of inquiry invested in understanding and unraveling the potential of substances produced by plants as a result of their adaptive evolution [15, 16].
One of the principal fractions of chemical substances found in plants are essential oils (EOs). EOs consist primarily of a mixture of volatile hydrophobic secondary metabolites with marked odors and great commercial value; EOs may be thought of as fingerprints of the plants where they are found [17, 18]. They include aromatic alcohols, acids, esters, phenolics, ketones, aldehydes, and hydrocarbons. In plants, EOs act as protective agents against predators and attractants for pollinators [19, 20].
The main chemical compounds found in EOs are terpenoids and phenylpropanoids [21, 22] that are widely used as bioactive molecules in biology, agronomy, medicine, and pharmaceutical sciences. Among the medical-pharmaceutical activities of these compounds are antitumor, antihelmintic, and larvicidal [23–25] and insecticidal [26–32] activities, as well as activity against arbovirus vectors [33, 34].
Despite the recognized cytoprotective and antioxidative activities of these compounds, in many cases larvicidal, insecticidal, and antiparasitic activities are associated with the induction of oxidative stress in the parasite, increasing the level of nitric oxide production by the infected host, reducing parasite resistance to reactive oxygen species, and generating high levels of free radicals and increasing lipid peroxidation, ultimately leading to serious damage to cell membranes and the killing of parasites [35–40].
Based on these considerations, in this review an update of the potential of EOs, their fractions, and their components that are active against some neglected diseases, including American and African trypanosomiasis, leishmaniasis, and arboviruses, specially dengue is presented.
To compose the database, the authors did a systematical search in the following databases: PubMed, Web of Science, Medline, SciELO, Cochrane Library, and NAPRALERT. The following search terms were combined and screened: essential oils, name of the neglected disease, and/or the parasite name. No language restrictions were applied. The period considered was the last 10 years, without however disregarding some articles of revision, and some classical literatures were considered important.
1.1. Essentials Oils Active against Trypanosoma cruzi
Chagas disease (CD), also known as American trypanosomiasis, is a potentially life-threatening disease caused by the protozoan Trypanosoma cruzi . According to the World Health Organization, CD affects about 7 million people worldwide and is endemic in 21 Latin American countries. It is estimated that CD leads to more than 7000 deaths per year and there are about 25 million people at risk for infection. Transmission to humans usually occurs through the excrement of infected vector insects (haematophagous triatomines) that normally become active at night, feeding on mammalian blood by biting exposed skin and defecating close to the bite. Transmission can also occur through transfusion of infected blood, congenital transmission, and (less frequently) organ transplantation or laboratory accidents .
Although it is an infectious disease with more than 100 years since its discovery, the morbidity and mortality associated with CD remains high. This may be attributed to several factors, including the absence of effective vaccines and/or medical treatments. Currently, only two drugs are available for CD treatment: benznidazole (BZ) and nifurtimox (NFX). Unfortunately, these drugs carry high toxicity and cause severe side effects that often interrupt treatment . Therefore, there is a need for new therapeutic alternatives for CD that are more effective and less toxic for both acute and chronic phases.
Therefore, the use of natural products is an important strategy to identify new trypanocidal compounds, and the literature already described promising studies with the use of EOs, found in some species of plants in many tropical countries .
The literature described that EOs have been used since the Middle Ages by Arabs for their antiseptic, bactericidal, virucidal, and fungicidal properties. They are also used for embalming and preservation of foods and as drugs with antimicrobial, analgesic, sedative, anti-inflammatory, spasmolytic, and local anesthesic properties . However, little was known about their antiparasitic applications, with the first reports appearing only at the end of the 20th century and beginning of the 21st century. Chagas disease was no different, and only in recent decades has there been an increase in the number of publications describing the use of EOs and/or their constituents with inhibitory activity against trypanosomatid protozoa [48–50].
Holetz and coworkers produced one of the first reports of the effects of the essential oil of Ocimum gratissimum on Herpetomonas samuelpessoai, a nonpathogenic trypanosomatid that shares important antigens with T. cruzi and can be easily cultured. This trypanosomatid also induced humoral and cell-mediated immune responses, showing itself to be a good model for study of the biology of trypanosomatids and a suitable model for screening new trypanocidal drugs. The EO of Ocimum gratissimum inhibited protozoal growth in a dose-dependent manner with values between 100 and 91 μg/mL, at concentrations from 20 to 250 μg/mL [51, 52].
In 2006, Santoro and colleagues investigated the effects of EOs from Origanum vulgare and Thymus vulgaris on growth and ultrastructure of evolutionary forms of T. cruzi. EO from oregano showed an /24 h value of 175 μg/mL against epimastigotes and induced trypomastigote lysis with /24 h of 115 μg/mL. Thyme EO presented an /24 h of 77 μg/mL for epimastigotes and 38 μg/mL for trypomastigotes. Thymol (1) (Figure 1), the primary constituent of thyme, was also tested and showed an /24 h value of 62 μg/mL for epimastigotes and an /24 h value of 53 μg/mL for trypomastigotes. These /24 h results were similar to those obtained after treatment in vitro of the parasites with the reference drug benznidazole (BZ) (/24 h approximately 50 μg/mL) .
In another study, the Santoro group evaluated the EOs of three species, clove (Syzygium aromaticum L.), basil (Ocimum basilicum L.), and yarrow (Achillea millefolium L.) and some of their primary constituents (eugenol (2) and linalool (3)) (Figure 2). Treatment with EOs and their constituents showed that they inhibited T. cruzi growth, with clove EO being the most effective. Its /24 h value was 99.5 μg/mL for epimastigotes and 57.5 μg/mL for trypomastigotes. Incubation of T. cruzi with eugenol (2) (the main constituent of clove) gave an /24 h value of 246 μg/mL for epimastigotes and 76 μg/mL for trypomastigotes. This suggested that the chemical constituents of clove EO had a synergistic effect that increased trypanocidal activity. Linalool (3) (the primary constituent of basil) was also tested and gave /24 h values of 162.5 μg/mL for epimastigotes and 264 μg/mL for trypomastigotes. Chamazulene (4) (Figure 2), one of the primary constituents of yarrow EO, was not evaluated in their study .
Menna-Barreto and coworkers evaluated the potential trypanocidal effect of the oleaginous ethanolic extract of Pterodon pubescens seeds, its fractions, and geranylgeraniol, among other components isolated from the hexane fraction. The ethanolic extract was fractionated, yielding three subfractions including geranylgeraniol (5) (Figure 3). The EO of P. pubescens, its fractions, and geranylgeranyl induced dose-dependent lysis in T. cruzi trypomastigotes. Geranylgeraniol (5) was the most active compound, with an /24 h value of 15.3 μg/mL .
Escobar and coworkers evaluated the antiprotozoal activities of Colombian Lippia spp. EO and their major components. Nineteen EO samples were extracted from the aerial parts of five different species by microwave radiation-assisted hydrodistillation and were characterized by GC-FID and gas chromatography–mass spectrometry (GC-MS). Geraniol (6), neral (7), limonene (8), nerol (9), carvacrol (10), p-cymene (11), γ-terpenes (12), carvone (13), and thymol (1) were major components identified. Nine oils were active against epimastigotes from T. cruzi with values in the range of 5.5 to 32.2 μg/mL. Six EOs were active against amastigotes interiorized in Vero cells with ranging from 12.2 to 53.0 μg/mL. In addition, the authors evaluated seven of the major EO components, including thymol (1), carvacrol (10), geranial, and p-cymene (11) (Figure 4) which were active against epimastigotes, with values ranging from 0.3 to 28.1 μg/mL. Thymol (1) and S-carvone (13) were the most active against intracellular amastigote-infected Vero cells, with values of 3.6 and 6.0 μg/mL, respectively .
In a study by Rojas and coworkers, the authors evaluated the in vitro activity of EOS from 10 plants against T. cruzi, their cytotoxic activities, and their modulatory activities in a nitric oxide assay. The evaluated plants were Mentha piperita L. (mentha), Rosmarinus officinalis L (romero), Chenopodium ambrosioides L. (paico), Eucalyptus globulus Labill (eucalipto), Artemisia absinthium L. (ajenjo), Melissa officinalis L (toronjil), Minthostachys setosa Brig (muña), Cymbopogon citratus (hierba luisa), Aloysia triphylla (cedrón), and Mentha spicata L. (hierba buena). Of these, EOs from Cymbopogon citratus and Aloysia triphylla were the most active against epimastigote forms, inhibiting T. cruzi growth with values of 63.09 and 96.49 μg/mL, respectively. There was no significant variation in the concentration of nitric oxide, and no cytotoxicity was evident .
Rojas and colleagues also evaluated the in vivo anti-T. cruzi effects of Cymbopogon citratus and Aloysia triphylla EOs in Balb/c mice. C. citratus EO administered at 250 mg/kg/day reduced parasitemia peaks from 113.92 ± 25.66 to 74.60 ± 12.37 trypomastigotes/mL. Reduction in the number of amastigotes and number of inflammatory infiltrates in the heart was also observed. When administered at 100 mg/kg/day, no reduction in the number of trypomastigotes was observed (77.40 ± 14.93 trypomastigotes/mL) . Aloysia triphylla EO produced significant reduction in parasitemia (85.4%) with a peak of 250 mg/kg/day. They also observed a reduction in the number of amastigotes and inflammatory infiltrates in the heart .
Santos et al. evaluated Piper malacophyllum EO against diverse microorganisms including T. cruzi. Among the 28 compounds identified in the EO, (+) camphor (14) was the major constituent (32.8%) followed by camphene (15) (17.8%) (Figure 5). P. malacophyllum EO showed activity against epimastigote forms of T. cruzi with an value of 311.82 μg/mL. This was considered a low activity value compared to that of other EOs described in the literature .
EOs from Lippia sidoides, Lippia origanoides, Chenopodium ambrosioides, Ocimum gratissimum, Justicia pectoralis, and Vitex agnus-castus were tested against T. cruzi by Borges and coworkers. All EOs inhibited epimastigote growth and reduced cell viability of trypomastigote forms in a dose-dependent manner. C. ambroside (main constituent – terpinolene (16) (69.9%)), L. origanoides (main constituent – carvacrol (10) (37.3%)), and L. sidoides (main constituent – thymol (1) (78.4%)) (Figures 1, 4, and 6) were the most effective against epimastigote forms ( values of 21.3, 26.2, and 28.9 μg/mL, respectively). The less active were EOs obtained from J. pectorales, O. gratissimum, and V. agnus-castus with values of 56.8, 71.1, and 157.1 μg/mL, respectively. On the other hand, L. sidoides, O. gratissimum (38.4% – eugenol (2) as the main constituent), C. ambrosioides, and L. origanoides were found to be more effective against trypomastigotes with LC50/24 h values of 10.3, 11.5, 28.1, and 39.7 μg/mL, respectively .
Esperandim et al. investigated the activity in vitro of the Piper cubeba EO against trypomastigote and amastigote forms of T. cruzi and promastigote forms of Leishmania amazonensis. GC-MS analysis of the essential oils demonstrated than the main components were sabinene (17) (19.99%), eucalyptol (18) (11.87%), γ-terpineol (19) (6.36%), α-pinene (20) (5.82%), and camphor (14) (5.61%) (Figure 7). The in vitro activity against trypomastigotes and amastigotes of T. cruzi increased with increasing concentrations, giving values of 45.5 and 87.9 μg/mL, respectively. The EO was not active against L. amazonensis .
In 2014, Azeredo and coworkers evaluated the in vitro antitrypanocidal activity of eight different EOs (Cinnamomum verum, Citrus limon, Cymbopogon nardus, Corymbia citriodora, Eucalyptus globulus, Eugenia uniflora, Myrocarpus frondosus, and Rosmarinus officinalis) against T. cruzi. Of these, Cinnamomum verum EO was the most effective against T. cruzi epimastigotes (), followed by Myrocarpus frondosus () and Eugenia uniflora (). No other evaluated EOs presented significant activity. In addition, C. verum EO showed /24 h values of 5.05 μg/mL and 20 μg/mL against T. cruzi metacyclic trypomastigotes and intracellular amastigotes, respectively. GC-MS analysis of C. verum EO showed (E)-cinnamaldehyde (21) (81.52%) (Figure 8) and eugenol (2) (16.68%) as the main components .
Monzote and colleagues evaluated the Chenopodium ambrosioides EOs and their principal components against Leishmania infantum, Plasmodium falciparum, Trypanosoma brucei, and T. cruzi. C. ambrosioides EO showed activity against evaluated protozoa, with values ranging between 0.2 and 6.4 μg/mL. Against T. cruzi, the value was 1.9 μg/mL .
In 2015, Andrade et al. evaluated the trypanocidal activity of EOs from Cinnamodendron dinisii Schwacke (canellaceae) and Siparuna guianensis Aublet (Siparunaceae). Both EOs showed low trypanocidal activity, with /24 h values of 209.30 μg/mL for S. guianensis and 282.93 μg/mL for C. dinisii. The /24 h of the reference drug benznidazol was 12.8 μg/mL .
The EO from Latana camara leaves was tested against Leishmania braziliensis and T. cruzi by Barros and coworkers. They found that L. camara EO was more effective against L. braziliensis than T. cruzi with IC50 values of 72.31 and 201.94 μg/mL to promastigote and epimastigote forms, respectively. The authors also evaluated the composition of this EO by CG-MS and found that the main constituents were (E)-caryophyllene (22) (23.75%), bicyclogermacrene (23) (15.80%), and germacrene D (24) (11.73%) (Figure 9) [66, 67].
In 2016, a review published by Barros de Alencar and coworkers summarized many reports that identified the activity of diterpenes against NDs, including Chagas disease. The compounds and classes of compounds that were identified as most promising were abietic acid (25) derivatives, with values between <20 μM and > 150 μM (Olmo et al., 2015); 12-hydroxy-11,14-diketo-6,8,12-abietatrien-19,20-olide (26) isolated from Salvia cuspidate with value of 5 μg/mL (Lozano et al., 2015); 5-epi-icetexone (27) (10 mg/kg/day, i.p.) used for 5 days, capable of reversing infection in Swiss mice (Lozano et al., 2016); and aphidicolin (28) derivatives obtained by semisynthesis exhibited high potency and selectivity against parasitic amastigote forms (Santos et al., 2016). Two of these derivatives gave values of 0.6 μM (28 A) and 0.78 μM (28 B), with selectivity for both (Figure 10) [68, 69].
Other semisynthetic compounds were evaluated by Varela and colleagues. The EO obtained from the areal parts (leaves) of Piper malacophylum (Piperaceae) presented two alkenyphenol derivatives as primary compounds: gibbilimbols A (29) and B (30), evaluated against promastigote/amastigote forms of Leishmania infantum and trypomastigote/amastigote forms of T. cruzi. Gibbilimbol B (30) showed better trypanocidal activity and lower toxicity to mammalian cells. These results encouraged researchers to prepare several quite simple synthetic analogues. Among these, the compound n-octyl-4-hydrozybenzylamine (LINS03003) (31) was the most potent, with values of 5.5 μM against amastigote forms of T. cruzi, with a higher activity than that of the natural prototype (Figure 11) .
Spurred on by the previous results, Varela’s group isolated a new natural product from the P. malacophylum EO — compound 5-[(3E)-oct-3-en-1-il]-1,3-benzodioxole (32) (Figure 12), and a new set of five compounds were synthesized. The isolated compound was evaluated against L. infantum and T. cruzi and presented potential against promastigote/trypomastigote/amastigote forms of T. cruzi, with values of 32–83 μM and low toxicity ( to mammalian cells). The synthetic compounds possessed promising antiparasitic activity; however, the compounds were considered to be cytotoxic. Compound LINS03011 (33) (Figure 12) was the most active compound with values of 13.3 mM against amastigotes, comparable to the phenolic prototype LINS03003 (31), but with better selectivity index () and less toxicity .
Souza and coworkers evaluated the chemical composition and the in vitro effects of the EOs from the leaves of Eugenia brejoensis (EbEO), Hyptis pectinata (HpEO), Hypenia salzmannii (HsEO), Lippia macrophylla (LmEO), and seeds of Syagrus coronata (ScEO) against T. cruzi. GC-MS allowed the identification of 114 of 162 compounds. Among these, hydrocarbon sesquiterpenes were the most abundant found in EbEO (94.51%), HfEO (69.84%), HpEO (77.88), and HsEO (56.16%). The LmEO fraction had monoterpenes as its principal constituents (98.08%). The EOs presented values between 29 and 110.6 mg/mL for epimastigote forms, 17.39–182.49 mg/mL for trypomastigote forms, and 12.5–408.33 mg/mL for amastigote forms of T. cruzi. The most active was EbEO, and the least active was ScEO. EbEO had as its main constituents δ-cadinene (34) (15.88%), trans-caryophyllene (22) (9.77%), and α-muurolol (35) (9.42%) (Figure 13) .
Villamizar et al. analyzed the trypanocidal effects of Piper aduncum EO (PaEO) and its main constituents against various forms of T. cruzi. GC-MS identified nerolidol and linalool (3) as the major PaEO components. PaEO showed against metacyclic trypomastigotes and /24 h values of 9 μg/mL against amastigotes forms. Among the two main constituents isolated and identified, linalool (3) (Figure 2) was the best, showing trypanocidal activity with an /24 h value of 306 ng/mL .
A recent study by Junior and colleagues evaluated the efficacy of the EO of Syzygium aromaticum (SaEO) administrated orally, alone, and in combination with benznidazole (BZ) in mice infected with T. cruzi IV strain. GC-MS analysis identified eugenol (2) (82.2%) and γ-caryophyllene (36) (13.0%) (Figure 14) as the main components of SaEO. Treatment with SaEO promoted reduction in 1/5 parameters derived from the parasitemia curve when compared with untreated control groups. The groups treated with BZ and BZ + SaEO had reductions in 4/5 of these parameters, with similar profiles of the parasitemia curve. In addition, the animals treated with BZ and BZ + SaEO presented lower patency periods compared with animals treated only with SaEO, as well as lower positivity of blood cultures compared with the untreated control group. With respect to cure rates, SaEO showed 12.5% (1/8), BZ showed 25.0% (1/4), and BZ + SaEO showed 37.5% (3/8), considered a very interesting result .
1.2. Essential Oils and Components with Anti-Trypanosoma brucei Activity
Trypanosoma brucei (Trypanosomatid family) is the principal protozoan responsible for causing the type of trypanosomiasis known as sleeping sickness (or human African trypanosomiasis). It is classified as a neglected disease and is transmitted by tsetse flies (Glossina spp., Diptera: Glossinidae).
In humans, this infection is caused by two subspecies of T. brucei (T. brucei gambiense, predominant in the West Africa, and T. brucei rhodesiense, predominant in the East Africa). There is also T. brucei brucei that is infective only in animals. It is endemic to Sub-Saharan Africa. Despite the fact that it has been declining over the last two decades, T. brucei nevertheless affects approximately 30,000 people annually (as well as about 60 million people - distributed in 36 countries - that inhabit areas at risk), with high mortality rates, mainly in rural populations that have little access to necessary treatments, causing substantial socioeconomic impact [74–86].
Human African trypanosomiasis, after parasite inoculations in the human host (or animal, mutatis mutandis), evolves in two stages. The primary stage is a hemolymphatic phase, or the parasitic cell replication stage, followed by a secondary meningoencephalitic phase, in which the parasites cross the blood-brain barrier and invade the central nervous system [86, 87]. This stage of the disease is characterized by a symptomatology including fever, headache, swelling of the lymph nodes, skin rashes, splenomegaly, confusion, sensory disturbances, motor coordination deficiency, and sleep disturbances (characteristic of the disease). It can be fatal if not properly treated .
The primary treatment is chemotherapy [89–91]; however, only four drugs are approved for human use: suramin, pentamidine, melarsoprol, and eflornithine . The former two alternatives are used in the first stage of the disease, while the later two are used only in the last stage, representing alternatives of increased toxicity of the treatment [46, 88–92]. However, as was observed for the treatment of the other NDs, the treatment of T. brucei includes only a few effective drugs, and those that exist carry substantial toxicities, high costs, absence of sustainable production, and development of resistance by the protozoa [93–97]. There is an urgent need for the discovery and development of new drugs that are more efficient and secure. This need is reflected in the growing interest of many research groups in the search for new compounds with anti-T. brucei potential [98–106].
In many cases, traditional medicine has been the only alternative available for treatments based on natural plant products, and the treatment of T. brucei infections is no exception [64, 104, 107–114]. Natural plant products are excellent sources of safe and effective compounds to combat human African trypanosomiasis [88, 107, 115]. These natural bioactive products are for the most part plant secondary metabolites, including several classes of promising bioactive compounds, among which are EOs [92, 116–118].
As a reflection of the increasing interest in EOs for use against T. brucei evaluation, a number of investigators have studied their potential, from determination of vegetal sources in various plant parts (aerial or radicular systems) to acquisition of species and types of essential oils.
In parallel with the increase in the use of EOs obtained from various parts of plants (aerial parts, fruits, flowers, and/or root systems) to combat sleeping sickness, several investigators worldwide have expanded their studies into the potential of these EOs and their main constituent chemicals against T. brucei.
One of these studies (Monzote and colleagues ) evaluated the EO from Chenopodium ambrosioides L. (CaEO), an aromatic plant with known biological potential from the Americas and Africa . The plant’s anti-Leishmania sp. activities have already been identified . The main CaEO components carvacrol (10) (62%), ascaridole (37) (22%), and caryophyllene oxide (38) (5%) (Figure 15) demonstrated substantial growth inhibition of T. brucei parasites (), with activity comparable to that of the standard drug suramin ( of 0.05 ± 0.05 μg/mL). These results, demonstrating the broad antiprotozoal spectra of CaEO, are an example of the potential of their use in endemic areas where more than one infectious species is present.
EOs from Cymbopogon species have aroused interest of many research groups because of their antitrypanosomal activities, as the example of their growth inhibitory activity against T. brucei [121, 122]. Kpoviessi et al.  evaluated the EOs of fresh leaves from four Cymbopogon species consisting primarily of monoterpenes and sesquiterpenes (C. citrates – with 29 compounds, C. giganteus – with 53 compounds, C. nardus – with 28 compounds, and C. schoenantus – with 41 compounds). The principal identified constituents were as follows: from C. citrates: geranial (6), neral (7), β-pinene (39), cis-geraniol (or nerol) (9), cis-verbenol (40), and geranyl acetate (41); from C. giganteus: trans-p-mentha-1(7),8-dien-2-ol (42), trans-carveol (43), trans-p-mentha-2,8-dienol (44), cis-p-mentha-2,8-dienol (45), cis-p-mentha-1(7),8-dien-2-ol (46), limonene (8), cis-carveol (47), cis-carvone (48), 4,4-dimethylandrost-5-en-3-one (49) and trans-carene-4,5-epoxy (50); from C. nardus: β-citronellol (51), elemol (52), limonene (8), α-cadinol (53), β-elemene (54), cubenol (55), germacrene-D (24), geranyl acetate (41), δ-cadinene (34), and δ-cadinol (56); and from C. schoenantus: piperitone (57), (+)-2-carene (58), limonene (8), elemol (52), β-eudesmol (59), trans-p-mentha-2,8-dienol (44), cis-p-mentha-2,8-dienol (45), and τ-eudesmol (60) (Figure 16).
These authors measured the cytotoxicity against WI38 and CHO cells and in vitro activity against T. brucei. All EOs evaluated were active, with values of 5.71 ± 1.40 μg/mL for C. nardus, 2.10 ± 0.89 μg/mL for C. schoenantus, 1.83 ± 0.13 μg/mL for C. citrates, and 0.25 ± 0.11 μg/mL for C. giganteus. The reference drug suramin had an of 0.11 ± 0.02 μg/mL. C. giganteus EO was the most active oil and demonstrated the largest selectivity index (SI of >200, as well as SI of 21.73 for C. citrates, 8.75 for C. nardus, and 23.81 for C. schoenantus). The anti-T. brucei evaluation of some of the components from these EOs demonstrated high potentialities: β-myrcene (62) (), citronellal (63) (), R(+)-limonene (8) (), citral (64) (), and β-citronellol (51) () (Figure 16). Of these, limonene (8) was the principal component of three of the four EOs, including C. giganteus (the most active), probably tightly influencing their antitrypanosomal activities.
The composition of essential oils of C. giganteus was highly rich in oxygenated monoterpenes that should greatly contribute their potential against T. brucei. The results showed that these compounds should be analyzed separately with greater depth in order to identify new alternatives for the treatment of human African trypanosomiasis.
Kpoviessi et al.  analyzed the EOs from the aerial parts (seeds, leaves, and stems) of Ocimum gratissimum in various vegetative stages. Their analysis of the principal EO components from O. gratissimum revealed 47 constituents (the majority of them hydrocarbons and a minority of oxygenated compounds). Of these, p-cymene (11), thymol (1), γ-terpinene (64), β-myrcene (61), and α-thujene (65) (Figure 17) were the most abundant. O. gratissimum EO demonstrated moderate anti-T. brucei activity ( of 27.23 ± 3.74 μg/mL), as also previously observed with many of their principal compounds  as observed with thymol (1) ( of 22.86 μg/mL) and p-cymene (11) ( of 76.32 μg/mL). Thus, the primary isolated components did not possess strong anti-T. brucei activity and did not act synergistically (demonstrating an interesting selectivity index of 410 for the leaves and seeds portions). Conversely, the ethanolic extract of O. gratissimum demonstrated values less than 15 μg/mL, leading us to believe that other bioactive compounds that were not present in the EO were responsible for the anti-T. brucei activity, justifying its continued use in traditional medicine.
Nibret and Wink  evaluated anti-T. brucei activity and cytotoxicity against human leukemic cells HL-60 in several plants with biological potentialities well-known in traditional Ethiopian medicine, including Hagenia abyssinica (female flower), Leonotis ocymifolia (aerial parts), and Moringa stenopetala (seeds). The results were compared to those of the standard drug diminazene aceturate ( and ). CG-MS revealed 20 components of H. abyssinica (including ledol (66), valeranone (67), palustrol (68), E-15-heptadecenal (69), α-phellandren-8-ol (70), and verbenol (40) as primary compounds) and 68 components for L. ocymifolia (including caryophyllene oxide (38), palmitic acid (71), carotol (72), camphor (14), hexahydrofarnesyl acetone (73), estragole (74), and linalool (3) as the primary compounds) and for M. stenopetala, including benzyl isothiocyanate (75) and isobutyl isothiocyanate (76) as primary compounds. M. stenopetala EO demonstrated greater antitrypanosomal activity ( of 5.03 μg/mL), but with a lower selectivity index (). Among the isolated compounds, the best results were observed with benzyl isothiocyanate (75) ( and ), cinnamaldehyde (21) ( and ), styrene oxide (78) ( and ), and (−)-α-cedrene (79) ( and ). Cinnamaldehyde (21) and styrene oxide (78) (Figure 18) were the compounds with the best combined results ( and SI). Nevertheless, they observed that benzyl isothiocyanate (75) was the majority component of M. stenopetala EO and was therefore responsible for the anti-T. brucei potentiality of this plant. Despite a low selectivity index, the compound can be utilized as a basis for developing new antitrypanosomial drugs.
Keetia leucantha, a plant used in traditional medicine of West African countries, was the object of study of Bero and colleagues . They investigated the chemical composition of KlOE and their anti-T. brucei activities. The plant is used empirically in the treatment of sleeping sickness . K. leucantha EO demonstrated values of 20.9 ± 12.6 μg/mL for bloodstream forms of T. brucei brucei (compared to for the standard drug suramin). Analysis of the secondary metabolites contained in the OE returned 42 components, including n-hexadecanoic acid (43.46%) (80), oleic acid (9.51%) (81), phytol (5.68%) (82), tetradecanoic acid (83) (5.19%), and phytone (84) (4.3%) (Figure 19). Some of these compounds were also evaluated and presented moderate to weak activities, confirming the moderate activity of KlOE against T. brucei. The values of the isolated EO components were as follows: phytol (82) (), oleic acid (81) (), and n-hexadecanoic acid (80) (). The best values were observed with the minority components: geranylacetone (85) (), α-ionone (86) (), and β-ionone (87) () (Figure 19). These components were evaluated against procyclic forms of T. brucei, and they were found to be even less active. According to the authors, these results can be an indication of the mechanisms of action of inhibition of T. brucei brucei growth. The authors suggest inhibition of glycolysis as a possible mechanism of action, as this is the main pathway of ATP synthesis for the bloodstream forms that do not possess the Krebs cycle or mitochondrial respiratory chain coupled to ATP. This phenomenon may explain their greater sensitivities compared with procyclic forms, highlighting the inhibitory activity of β-ionone (87) against glyceraldehyde-3-phosphate dehydrogenase (32.6% ± 3.4% at 20 μg/mL); however, the inhibitory effect was not significant for α-ionone (86) (0.00% ± 7.2% at 20 μg/mL) or phytol (82) (6.8% ± 0.7% at 20 μg/mL).
Another well-known plant in traditional medicine, Mentha crispa, was evaluated for EO activity by Sousa et al. . They studied four activities against T. brucei bloodstream forms, as well as their cytotoxicity against human leukemic cells (HL-60). Phytochemical analysis identified major EO components, including rotundifolone (88) (58.11%), limonene (8) (10.58%), myrcene (61) (7.79%), germacrene (24) (6.55%), cis-β-ocimene (89) (5.01%), and β-pinene (39) (4.43%), as well as four other monoterpenic analogues: (+)-limonene epoxide (90), (−)-limonene epoxide (90), (−)-perillyl alcohol (91), and (−)-perillyl aldehyde (92) (Figure 20). The isolated compounds and the EO demonstrated dose-dependent activity in their minimum inhibitory concentration (MIC) values, varying from 1 to 100 μ/mL, and their GI50, varying from 0.3 to 13.3 μg/mL, compared to the standard drug suramin (, , ). Greater potentialities were observed for McEO (, , ), rotundifolone (88) (, , ), and (−)-perillyl aldehyde (92) (Figure 20) (, , ). These compounds were only slightly less active than the reference drug. The presence of rotundifolone (88) as the major component in the McEO should be the primary compound responsible for its high anti-T. brucei activity.
Structurally comparing these major components with other analogues, the presence of an α,β-unsaturated carbonyl appeared to favor anti-T. brucei activity, because when these components were absent there was significant loss of activity. The change in the position of this α,β-unsaturated carbonyl, and the change of position of an additional unsaturation outside of the ring, appeared to strongly influence the cytotoxicity in HL-60, where (−)-perillyl aldehyde (92) showed less toxicity than did rotundifolone (88), but with a better selectivity index .
Habila et al.  investigated the potential activity against T. brucei brucei of EOs from four plants (Cymbopogon citratus – leaves, Eucalyptus citriodora – leaves, Eucalyptus camaldulensis – leaves, and Citrus sinensis – fruit peels). CG-MS of EOs demonstrated an abundance of cyclobutane (93) (96.08%) in C. sinensis; eucalyptol (18) (75.04%), bicyclo[3.1.1]hept-2-ene (94) (10.27%), benzene (95) (6.23%), and cyclohexene (96) (4.92%) in E. camaldulensis; 6-octenal (97) (77.11%) and 6-octen-1-ol (98) (14.09%) in E. citriodora; and citral (63) (38.32%) and 2,6-octadienal (99) (35.05%) and 2,6-octadien-1-ol (100) (26.63%) in C. citratus (Figure 21).
The death of the parasites was observed after the use of 0.4 g/mL of the EOs. The EO most capable of killing parasites quickly was the C. citratus EO (3 min). EOs from E. citriodora and E. camaldulensis killed parasites in 4 minutes, and C. sinensis EO killed parasites in 5 min. In smaller doses, the time of death increased to 6 min (0.2 g/mL) and 15 min (0.1 g/mL) for C. sinensis, 8 min (0.2 g/mL) and 17 min (0.1 g/mL) for E. camaldulensis, 8 min (0.2 g/mL) and 22 min (0.1 g/mL) for E. citriodora, and 4 min (0.2 g/mL) and 11 min (0.1 g/mL) for C. citratus. Thus, their activities appeared to be dose-dependent and the four EOs demonstrated powerful activity, causing the death of protozoa in short periods of time, compared with that of the reference drug (diminaveto) that promoted the death of the parasites in equivalent times.
Gutiérrez et al.  evaluated a species of the Piper genus, a well-known source of bioactive compounds . Piper ossanum (leaves) was collected in two locations in Cuba (Bauta and Ceiba). Chromatograms of the EOs demonstrated the presence of 43 components for the Bauta species (PoB) and 39 for the Ceiba species (PoC). In both, the primary components were piperitone (57), camphene (15), camphor (14), and viridiflorol (101) (Figure 22), with PoB being more abundant (20.07% for PoB, and 19.01% for PoC). Some quantitative differences were observed in PoB and PoC EO constituents: camphor (14) (13.87% for PoB and 9.41% for PoC), viridiflorol (101) (12.97% for PoB and 18.80% for PoC), and camphene (15) (7.41% for PoB and 5.39% for PoC). PoB has as minor components p-cymene (11), sylvestrene (102), (Z)- and (E)-β-ocimene (89), terpinen-4-ol (103), cubebol (104), and humulene epoxide (105); PoC possessed small quantities of β-phellandrene (106), γ-muurolene (107), spathulenol (108), and globulol (109) (Figure 22). The values of the sensitivity of T. brucei against the EOs showed no significant difference in terms of values (8.1 μg/mL for PoB and 8.4 μg/mL for PoC), suggesting that the various compositions of their constituents were not decisive for a differentiation of their potential, classified as weak when compared to the standard drug suramin (). Both EOs demonstrated low toxicity in a human fetal lung fibroblast cell line (MRC-5) (4.2 μg/mL for PoB and 0.95 μ/mL for PoC). Evaluation of the T. brucei activity of the main compounds of each EO is necessary in order to identify more promising compounds.
Strychnos spinosa leaves are widely utilized in African trypanosomiasis treatment . Hoet et al.  studied the cytotoxicity and the anti-T. brucei brucei activity of SsEO leaves and 16 of their components. SsEO leaves contained more than 100 constituents, including palmitic acid (71) (34.3%), linalool (3) (16%), (E)-phytol (82) (6.7%), and (E)-geraniol (6) (4%). SsEO showed moderately activity () and weak selectivity index (). Of the pure components tested, the prominent compounds were (E)-nerolidol (110) (Figure 23) (, ), linalool (3) (, ), (E)-geranylacetone (85) (, ), β-ionone (87) (, ), (E)-phytol (82) (, ), and α-terpineol (111) (Figure 23) (, ). Among these, only linalool (3) and (E)-phytol (82) were found in abundance in SsEO, leading to the conclusion that interaction with other compounds decreased the activity of the EO. In addition, the presence in minimal quantities of the more active compounds (identified as minority components) prevented the EOs from exerting their potential effects. Palmitic acid (71), the primary component for this species, was inactive and highly toxic ().
Preliminary results  demonstrated good antitrypanosomal activity of dichloromethane extracts of S. spinosa (), causing us to speculate that there are active components that are not EO constituents that should be evaluated more intensively. The better results observed with linalool (3) and (E)-nerolidol (110) reflect the need for further studies of these substances and for investigation of the potential of other oxygenated terpenes to identify alternatives for human African trypanosomiasis treatment.
Mulyaningsih et al.  investigated the Kadsura longipedunculata EO that had 50 identified components. The EO fraction was composed of 75% sesquiterpenes and 22.63% monoterpenes, including δ-cadinene (34) (21.79%), camphene (15) (7.27%), borneol (112) (6.05%) (Figure 24), cubenol (55) (5.12%), and δ-cadinol (56) (5.11%). KlEO and camphene (15) were evaluated against bloodstream forms of T. brucei brucei and human hepatocellular liver carcinoma (HepG2); however, the results were not encouraging. KlEO had an of 50.52 ± 0.029 μg/mL and an of 136.96 μg/mL. The of the pure compounds were 80.66 ± 0.87 μg/mL for camphene (15) and 70.00 ± 1.28 μg/mL for borneol (112), both unfavorable cytotoxicity profiles. These results indicate that the active substances of K. longepedunculata useful against human African trypanosomiasis treatment were not part of their EO constituents.
Hamdan and coworkers  evaluated the EOs of two Egyptian species of Citrus sp., C. jambhiri and C. pyriformis. For both species, 94 compounds were identified, with limonene (8) the major compound (92.48% for C. jambhiri and 75.56% for C. pyriformis). Both EOs had poor activity against bloodstream forms of T. b. brucei, with values of 72.47% ± 0.87 μg/mL for C. jambhiri and of 71.29 ± 0.38 μg/mL for C. pyriformis, much less active than the standard drug diminazene aceturate ().
Cruz et al.  evaluated the Eugenia uniflora EO from leaves and identified 87 compounds (spathulenol (108) – 15.8%, α-copaene (113) – 10.96%, muurola-4,10-dien-1β-ol (114) – 9.3%, caryophyllene oxide (38) – 8.93%, allo-aromadendrene (115) – 5.5%, and nootkatone (116) – 5.17%) (Figure 25). The oxygenated sesquiterpenes (54.09%) were the most abundant class of compounds. An in vitro T. b. brucei evaluation demonstrated moderate activity of EuEO () with a weak selectivity index (). The reference drug suramin had an of 0.16 ± 0.03 μg/mL.
Petrelli et al.  investigated the EO of Erigeron floribundus aerial parts, containing 85 constituents. The primary class of constituents was sesquiterpenes (60.4% - being 38.5% oxygenated and 21.9% hydrocarbons). The main components were caryophyllene oxide (38) (12.4%), spathulenol (108) (12.2%), (E)-β-farnesene (117) (Figure 26) (5.5%), and (E)-caryophyllene (22) (4.2%), in addition to limonene (8) (8.8%). EfEO was evaluated for its antitrypanosomal activity against T. brucei and cytotoxicity in a human breast adenocarcinoma cell line (A375). There was moderate activity () with a low selectivity index value (). Their main constituent, caryophyllene oxide (38), was also assayed and showed up inactive, reflecting the low antitrypanosomal activity of the EO. However, the evaluation of another potential major component, limonene (8), demonstrated promising activity ( and ), being the main contributor to the expression of EO potentiality. The authors concluded that this difference of activity in favor of limonene (8) could be attributed to the degree of its unsaturation, in addition to its exocyclic methylene group. The occurrence of a possible binding site with SH groups of proteins may be an indicator of their mechanism of action. However, this speculation requires more detailed investigation.
EOs from aerial parts of a variety of plant species were analyzed by Costa et al. , including Cymbopogon citratus L. (leaves), Distichoselinum tenuifolium (aerial parts), Juniperus oxycedrus (leaves and berries), Lavandula luisieri (influorescences), Lavandula viridis (aerial parts), Lippia graveolens (aerial parts), Mentha cervina (leaves), Mentha x piperita (leaves), Origanum virens (leaves), Rosmarinus officinalis (leaves), Seseli tortuosum (aerial parts), Syzygium aromaticum (floral buttons), Thymbra capitata (aerial parts), Thymus capitellatus (aerial parts), Thymus mastichina (leaves), and Thymus zygis sylvestris (leaves).
CG-MS allowed identification of the main components, including geranial (6) (45.7%), neral (7) (32.5%), and β-myrcene (61) (11.5%) for C. citratus; β-myrcene (61) (84.6%) and limonene (8) (2.2%) for D. tenuifolium; α-pinene (118) (54.7%), germacrene D (24) (10.4%), and β-myrcene (61) (17.8%) for the berries of J. oxycedrus; α-pinene (118) (65.5%), Δ-3-carene (119) (5.7%), β-phellandrene (106) (3.2%), and β-myrcene (61) (2.7%) for leaves of J. oxycedrus; 1,1,2,3-tetramethyl-4-hidroxymethyl-2-cyclopentene (120) (2.4%), 2,3,4,4-tetramethyl-5-methylene-cyclopent-2-enone (121) (5.2%), trans-α-necrodyl acetate (122) (16.0%), lyratyl acetate (123) (3.5%), 1,8-cineole (124) (18.9%), lavandulyl acetate (125) (7.2%), linalool (3) (3.1%), and α-pinene (118) (2.3%) for L. luisieri; 1,8-cineole (124) (29.7%), camphor (14) (10.0%), α-pinene (118) (9.2%), linalool (3) (9.0%), selina-3,7(11)-diene (126) (6.6%), Z-α-bisabolene (127) (6.3%), borneol (112) (2.7%), and camphene (15) (2.7%) for L. viridis; thymol (1) (19.8%), ρ-cymene (11) (16.9%), 1,8-cineole (124) (6.6%), caryophyllene oxide (38) (5.7%), linalool (3) (5.4%), Δ-3-carene (119) (4.3%), α-terpineol (111) (3.6%), myrcene (61) (3.4%), E-caryophyllene (22) (2.4%), and trans-sabinene hydrate (17) (2.3%) for L. graveolens; pulegone (128) (74.8%), isomenthone (129) (10.6%), and limonene (8) (5.4%) for M. cervina; menthol (130) (44.0%), menthofuran (131) (10.9%), menthone (132) (9.8%), menthyl acetate (133) (7.8%), 1,8-cineole (124) (5.8%), neo-menthol (134) (4.0%), neo-isomenthol (135) (2.9%), and pulegone (128) (2.4%) for M. x piperita; carvacrol (10) (68.2%), γ-terpinene (64) (7.9%), p-cymene (11) (7.4%), β-myrcene (61) (2.4%), and thymol (1) (2.1%) for O. virens; β-myrcene (61) (32.0%), 1,8-cineole (124) (13.7%), camphor (14) (11.9%), α-pinene (118) (11.1%), limonene (8) (6.6%), ρ-cymene (11) (3.8%), camphene (15) (3.4%), and linalool (3) (2.1%) for R. officinalis; α-pinene (118) (27.4%), β-pinene (39) (16.0%), limonene (8) (10.0%), γ-terpinene (64) (9.3%), (Z)-β-ocimene (89) (8.0%), β-myrcene (61) (3.0%), camphene (15) (2.1%), and sabinene (17) (2.0%) for S. tortuosum; eugenol (2) (85.3%) and humulene (136) (6.8%) for S. aromaticum; carvacrol (10) (74.6%), p-cymene (11) (5.5%), E-caryophyllene (22) (3.9%), γ-terpinene (64) (3.6%), and linalool (3) (2.8%) for T. capitata; 1,8-cineole (124) (58.6%), borneol (112) (10.0%), camphene (15) (6.5%), α-pinene (118) (4.5%), sabinene (17) (3.0%), and β-pinene (39) (2.0%) for T. capitellatus; 1,8-cineole (124) (67.4%), linalool (3) (4.3%), β-pinene (39) (4.0%), α-terpineol (111) (3.5%), α-pinene (118) (3.0%), and sabinene (17) (2.4%) for T. mastichina; and geranyl acetate (41) (44.5%), geraniol (6) (33.1%), and camphor (14) (3.9%) for T. zygis sylvestris (Figure 27).
Of all these EOs, noteworthy findings were as follows: JoEO berries ( and ), CcEO ( and ), and LlEO ( and ). Some pure compounds were also evaluated, and the best IC50 values were obtained with α-pinene (118) () and citral (63) (). α-Pinene (118) is the main component of the most active EO (Juniperus oxycedrus), contributing to its high anti-T. brucei activity. Because the same result was not observed with JoEO leaves, the authors speculated that the synergistic effects on the most promising species potentiated their effects, suggesting that their interactions with germacrene D (24) and β-myrcene (61) should be further investigated .
Cheikh-Ali et al.  evaluated the EO from Aframomum sceptrum rizhomes. AsEO consists of 75 compounds, including β-pinene (39) (12.71%), caryophyllene oxide (38) (10.03%), cyperene (137) (5.99%), 3,4-dimethylcyclohex-3-ene-1-carbaldehyde (138) (3.40%), α-pinene (118) (2.78%), β-caryophyllene (22) (2.33%), α-terpineol (111) (2.12%), D-limonene (8) (1.89%), 1,8-cineole (124) (1.84%), and α-caryophyllene (or humulene) (136) (1.1%) (Figure 28). The minimum lethal concentration (MLC) against T. brucei brucei was determined for the EO () and for some pure compounds, β-pinene (39) () and caryophyllene oxide (38) (). When compared to the reference drug pentamidine (), this EO and their components showed very good anti-T. brucei brucei activities.
Herrmann et al.  obtained the Carlina acaulis root EO and identified carlina oxide (139) (Figure 29) as its main component. Evaluation of the antitrypanosomal activity of carlina oxide against T. b. brucei demonstrated promising results (), with great selectivity index values (). In addition to the pure compound (main component of the C. acaulis EO), the authors analyzed the hexane, dichloromethane, and methanol extracts, and the two former (with considerable amount of carlina oxide (139)) were more active ( () and 4.5 μg/mL (), respectively) than was the latter (, ). When compared to the reference drug suramin ( and ), carlina oxide (139) showed greater activity and better selectivity index. The authors suggested that the mechanism of action of carlina oxide (139) occurred through trypanothione reductase inhibition. The presence of the furan ring and the triple bond were considered by the authors to be important structural features for the activity and should be considered for the design of new drugs.
Kamte et al.  investigated the EOs of several herb species from the Apiaceae family from the Mediterranean zone (Siler montanum Crantz subsp. siculum – flowering aerial parts, Sison amomum L. – flowering aerial parts, Echinophora spinosa L. – roots and flowering aerial parts, Kundmannia sicula (L.) DC. – inflorescences, Crithmum maritimum L. – flowering aerial parts, Helosciadium nodiflorum (L.) Koch – whole, Pimpinella anisum L. – seeds, Heracleum sphondylium subsp. ternatum (Velen.) Brummit – seeds, and Trachyspermum ammi (L.) Sprague – seeds). The EOs from five of these plants were potent inhibitors of T. brucei (SaEO – 4.3 ± 0.7 μg/mL, EsEO roots – 2.7 ± 0.6 μg/mL, EsEO aerial parts – 4.0 ± 1.6 μg/mL, CmEO– 5.0 ± 0.8 μg/mL, and HnEO – 10.7 ± 4 μg/mL), and some of their main components were evaluated in isolation. The great potentiality of these EOs was attributed to the presence of the main constituents, considering as well the great importance of synergistic effects with minority constituents. For SaEO, the main constituents were sabinene (17) (54.4%), β-phellandrene (106) (16.6%), and germacrene D (24) (6.7%). For EsEO roots, they were myristicin (41.3%) (140), terpinolene (141) (22.2%), and (Z)-falcarinol (142) (23.3%); for EsEO aerial parts, they were α-phellandrene (143) (47.2%), p-cymene (11) (25.6%), β-phellandrene (106) (8.3%), E,E-2,6-dimethyl-1,3,5,7-octatetraene (144) (6.3%), and α-pinene (118) (5.5%) (Figure 30); for CmEO, they were limonene (8) (38.4%), γ-terpinene (64) (19.9%), and sabinene (17) (12.4%); and for HnEO, they were myristicin (140) (49.1%), (Z)-β-ocimene (89) (19%), limonene (8) (7.8%), terpinolene (141) (7.1%), and germacrene D (24) (6.0%). With the exception of CmEO, all other active EOs showed significant selectivities against T. brucei, as well as good selectivity index values. For example, in their cytotoxicity evaluation performed in Balb3T3 rat fibroblasts, they found for S. amomum, 2.1 and 3.7 for E. spinosa roots and aerial parts, respectively, and >9.1 for H. nodiflorum.
The isolated components from the EOs were also evaluated by Kamte et al. . Some of these showed more activity and selectivity than did their respective EOs. The most active constituents against T. brucei were terpinolene (141) (0.035 ± 0.005 μg/mL, ), α-pinene (118) (1.0 ± 0.3 μg/mL, ), β-ocimene (89) ( of 1.1 ± 0.5 μg/mL, ), p-cymene (11) ( of 4.5 ± 1.0 μg/mL, ), limonene (8) ( of 5.6 ± 1.6 μg/mL, ), sabinene (17) ( of 6.0 ± 1.5 μg/mL, ), α-phellandrene (143) ( of 24 ± 8 μg/mL, ), and myristicin (140) ( of 74 ± 4 μg/mL, ). As observed for the previous values, the high anti-T. brucei potentiality of terpinolene (141) should be responsible for the great value found in EsEO roots, containing these compounds as its primary constituents.
Comparison of the chemical structure of terpinolene (141) with other structurally similar compounds (γ-terpinene (64), α-phellandrene (143), limonene (8), and p-cymene (11)) highlighted the great importance of the presence of a double endocyclic and a double exocyclic bound, the latter linking the cyclohexene ring to a dimethylated carbon for the inhibition of T. brucei growth. This can be used as scaffold for drug design of new anti-T. brucei agents.
Petrelli et al.  evaluated EOs of many parts of Smyrnium olusatrum L. (roots, leaves, flowers, and fruits), known to be rich in furan ring-containing sesquiterpenes as the main constituents. CG-MS analysis identified a domain of oxygenated sesquiterpenes (68.2% - 74.5%), followed by monoterpene hydrocarbons (13.2–22.2%) and sesquiterpene hydrocarbons (3.1-14.1%). The EO from the fruits resulted in the identification of 66 constituents, including β-acetoxyfuranoeudesm-4(15)-ene (145) (31.2%), curzerene (146) (23.8%), isofuranodiene (147) (6.6%), β-phellandrene (106) (6.2%), and α-pinene (118) (5.4%). Among the flower EOs, they found 47 compounds, including curzerene (146) (30.5%), myrcene (61) (18.2%), furanoeremophil-1-one (148) (12.1%), germacrone (149) (10.4%), and isofuranodiene (147) (9.8%) (Figure 31). They identified 45 compounds from roots EO, including curzerene (146) (39.7%), furanoeremophil-1-one (148) (24.4%), β-phellandrene (106) (14.4%), and isofuranodiene (147) (5.8%). The EO from leaves produced 43 constituents, including furanoeremophil-1-one (148) (30.0%), curzerene (146) (24.1%), germacrone (149) (9.7%), β-pinene (39) (9.5%), and isofuranodiene (147) (4.8%).
These SoEOs showed effectiveness against T. brucei brucei with satisfactory SIs. The most powerful EO was obtained from fruits (, SI = 29), followed by the EO from flowers (, SI = 23) and from leaves (, ), and at least active was the EO from roots (, ). Some pure compounds were also tested, including isofuranodiene (147) (, ), β-acetoxyfuranoeudesm-4(15)-ene (145) (, ), and germacrone (149) () which had the best profiles. Taking into account that curzerene (146) is a thermal degradation product of isofuranodiene (147) [136, 137], and considering the abundance of these two constituents, it may be considered that isofuranodiene (147) is the major component of all SoEO and is therefore responsible for their antitrypanosomal activities. The SoEO from fruit had as its principal component β-acetoxyfuranoeudesm-4(15)-ene (145), also contributing to its superior potential over the other components.
Structurally, the absence of the furan ring in germacrone (149), as opposed to isofuranodiene (147) and β-acetoxyfuranoeudesm-4(15)-ene (145) structures (Figure 20), can be the main explanation of its inactivity. In addition, the more potent compound isofuranodiene (147) was already known for its inhibitory activity against dihydrofolate reductase, an important enzyme in purine biosynthesis and therefore in DNA synthesis. This may be the mechanism of action for its inhibition of T. brucei brucei growth .
Muhd Haffiz and colleagues  evaluated the anti-T. brucei brucei activity of Cymbopogon nardus L. (whole plant) EO and fractions, using pentamidine (, ) as the standard drug. CG-MS revealed that the majority of the constituents of CnEO were oxygenated monoterpenes (79%), sesquiterpenes (12.3%), oxygenated sesquiterpenes (2.6%), and monoterpene hydrocarbons (1.9%), including citronellal (62) (35.5%), geraniol (6) (28%), and citronellol (51) (11%). In the biological evaluation, C. nardus EO exhibited strong antitrypanosomal activity and high SI (, ).
These EOs were fractionated into 7 fractions, then combined, and then refracted into 8 subfractions, and finally their activities were determined. In the great majority of cases, the fractions and subfractions showed great potential for inhibition of T. b. brucei growth with low cytotoxicity. The most active fractions were as follows: subfraction 4 (, ), subfraction 6 (, ), subfraction 7 (, ), and subfraction 8 (, ). The major constituents were γ-eudesmol (150) (Figure 32) and α-cadinol (53) for subfractions 4 and 6 and elemol (52) for subfractions 7 and 8.
Essential oils from aromatic and medicinal plants from Cameroon with known antitrypanosomal properties were evaluated by Kamte et al. . The authors identified chemical compositions and evaluated the anti-T. brucei potential of the EOs and constituents from Azadirachta indica (leaves), Aframomum melegueta (seeds), Aframomum daniellii (leaves), Clausena anisata (leaves), Dichrostachys cinerea (seeds), and Echinops giganteus (roots).
For AiEO, 13 compounds were identified, with abundance of sesquiterpene hydrocarbons (97.4%), especially germacrene B (151) (74.0%) and γ-elemene (152) (18.3%) (Figure 33); for AmEO, 59 compounds were identified, with abundance of oxygenated monoterpenes (83.3%), of which 1,8-cineole (124) (58.5%), α-terpineol (111) (19.4%), and β-pinene (39) (7.1%) were the main constituents; for AdEO, 57 compounds were identified, with greater abundance of monoterpene hydrocarbons (59.8%), where sabinene (17) (43.9%), (E)-caryophyllene (22) (16.6%), and β-pinene (39) (5.8%) were the most abundant; for CaEO, 47 compounds were identified, with abundance of phenylpropanoids (84.0%), where (E)-anethole (153) (64.6%) and (E)-methyl isoeugenol (2) (16.1%) (Figure 33) were the main constituents; for EgEO, 35 compounds were identified, with abundance of sesquiterpenes (94.3%-54.7% of sesquiterpene hydrocarbons and 39.6% of oxygenated sesquiterpenes), where silphiperfol-6-ene (154) (23.0%), presilphiperfolan-8-ol (155) (22.7%), presilphiperol-7-ene (156) (7.8%), cameroonan-7-α-ol (157) (7.1%), and caryophyllene (22) (6.3%) were the most abundant (Figure 33); and for D. cinereal, 49 compounds were identified, and the class of oxygenated monoterpenes (50.6%) was the primary, and geraniol (6) (18.2%), terpinen-4-ol (103) (7.5%), linalool (3) (4.0%), and umbellulone (158) (3.8%) were the most representative constituents. Representative amounts of other classes of compounds were also observed, especially ligustrazin (159) (5.1%), elemicin (160) (3.0%), and decanoic acid (161) (2.8%) (Figure 33).
The in vitro evaluation of these EOs demonstrated inactivity of DcEO, AmEO, and CaEO (), and moderate activity of AdEO (), EgEO (), and AiEO (), when compared to standard drug suramin (). The SIs of the three more active EOs were also evaluated and were >13.1, >9.52, and >6.57, respectively .
Some of the main constituents of A. melegueta and A. daniellii EOs were also evaluated. 1,8-Cineole (124) and terpinen-4-ol (103) (main compounds of AmEO) were inactive (), while sabinene (17) (), (E)-caryophyllene (22) (), and β-pinene (39) (), main components of AdEO, showed moderate to good anti-T. brucei activities .
1.3. Essentials Oils Active against Leishmaniasis
Leishmaniasis is a group of noncontagious infectious diseases caused by at least 20 parasitic protozoa belonging to the family Trypanosomatidae (genus Leishmania). It is estimated that at least 12 million people in more than 100 countries are infected, and at least another 350 million people are at risk in endemic areas [139, 140]. This set of diseases can be divided into two major groups of distinct diseases, tegumentary leishmaniasis (subdivided into cutaneous, diffuse cutaneous, and mucosal), and visceral leishmaniasis, usually more severe, with high mortality [139, 141].
The life cycle of Leishmania sp. begins with the blood supply of a phlebotomine that infects itself through the blood of an infected mammalian, ingesting amastigote forms of the parasite that are internalized in macrophages. In the intestine of the vector-insect, the amastigote form becomes a metacyclic promastigote form. When the insect is fed again and it bites a new mammalian, virulent promastigote forms are inoculated in the new host. In the bloodstream, they are internalized by macrophages, differentiating again into amastigotes, thus completing the cycle [140, 141].
The few therapeutic options available for treatment include a class of drugs that is more than 60 years old, the pentavalent antimonials (meglumine antimoniate and sodium stibogluconate). More recently, for the treatment of refractory and resistant cases, pentamidine, miltefosine, amphotericin B, and paromomycin are used .
In the same way as observed for the trypanosomiases described above, the scarcity of drugs for leishmaniasis necessitated the use of plants, and parts of plants, as therapeutic alternatives in several parts of the world.
Between 2000 and 2018, the literature reported the use of EOs from 142 plant species (30 families) and 44 isolated compounds that were evaluated against various Leishmania species (Leishmania amazonensis, Leishmania braziliensis, Leishmania aethiopica, Leishmania donovani, Leishmania chagasi, Leishmania infantum, Leishmania guyanensis, Leishmania panamensis, Leishmania mexicana, Leishmania tropica, and Leishmania major) and forms (promastigote, axenic amastigote, and intracellular amastigote).
The most studied families were Lamiaceae, Piperaceae, and Asteraceae, with 35, 29, and 14 species evaluated, respectively. Fifty-one of the EOs (especially from plant species the families Lamiaceae and Asteraceae) and 13 isolated compounds showed values less than or equal to 10 μg/mL against at least one species of Leishmania (promastigote or amastigote forms) and were considered active based on hit selection criteria for visceral leishmaniasis . In addition, four EOs and one isolated compound showed quantified activity in nanomolar concentrations, considered strongly active (Tables 1 and 2 and Figure 34) (see supplementary material Figures S1 and S2). In our review, some of the most active EOs and pure compounds were detailed for their major constituents (for EOs), efficacy ( values), selectivity index (SI), and morphological and ultrastructure alterations in the parasite, as well as for their mechanism of action. For inactive extracts and chemical entities (), and for those in which only the parasite was sensitive (moderate activity; ), see supplementary material (Table S1).
EOs from 10 plants issued from the Sned region of Tunisia, an endemic region for various forms of Leishmania, showed antileishmanial activities against L. major and L. infantum promastigote forms. In general, for all tested extracts, L. infantum species were more sensitive than were L. major, and only two EOs were more effective against the parasites than were murine macrophagic cells. EOs from Thymus hirtus sp. Algeriensis, rich in oxygen-containing monoterpenes, especially linalool (3) (17.62%) and camphor (14) (13.82%), and from Ruta chalepensis, rich in 2-undecanone (162) (Figure 35) (84.28%), showed against L. infantum promastigote forms of 0.25 and 1.13 μg/mL, respectively . In addition, only Thymus hirtus sp. Algeriensis EO showed leishmanicidal activity against L. major (). On the other hand, for these two EOs, SI values (calculated as ) ranging from 0.19 to 1.57 showed inadequate antileishmanial activity by hit selection . Two other EOs (from T. articulata and Lavandula multifida) were inactive against L. major promastigotes, and for the remaining six EOs, values ranged from 0.30 to 2.23 μg/mL, but with high levels of cytotoxicity () .
EOs from sixteen plant species belonging to various families, purchased from QUINARI Cosmetic and Fragances Inc. (Maringá, Pr, Brazil) or collected in Lavras, MG (Brazil), were evaluated for their biological potential against L. amazonensis promastigote forms after 24 h of exposure . None of the EOs tested showed desirable values. For the four most effective EOs (from Matricaria chamomilla, Cordia verbenaceae, Siparuna guianensis, and Cinnamodendron dinisii species), the values ranged from 48.55 to 64.75 μg/mL and were considered moderately active or inactive. Furthermore, for all EOs tested, SI were not satisfactory (). From the chemical composition of the evaluated EOs and the and SI values, the authors compared these leishmanicidal activities and cytotoxicities with those of EOs from other plant species. Literature reports that EOs from Matricaria chamomilla showed no leishmanicidal activity against L. donovani specie. Another EO from Lantana camara, with similar major constituents (farnesene (117) derivatives as major constituents), showed significant leishmanicidal activity against L. amazonensis (), but with high cytotoxicity [145, 146]. In another example, EO from Bulnesia sarmientoi, with 48.29% of guaiol (163) (Figure 35) as its major constituent, was inactive against L. amazonensis (). However, EO from Endlicheria bracteolate, with 72.12% of guaiol (163), had an of 7.93 μg/mL against the same Leishmania species and a similar value of SI (1.91) . Finally, they observed that the concentration of linalool (3) in the EO from Salvia sclarea was insufficient to promote leishmanicidal activity for this EO, since Kuźma et al. demonstrated that linalool-rich EO from the leaves of Croton cajucara promoted morphological changes in L. amazonensis promastigotes treated at concentrations of 15 ng/mL .
In order to explore ethnopharmacological data of copaiba oils obtained from Peru, Maranhão, and Amazonas (states in Brazil) and French Guiana which are used in folk medicine to treat leishmaniasis [149–152], Santos and coworkers investigated the antiproliferative effect of EOs from eight kinds of Brazilian copaiba against promastigote and amastigote forms of L. amazonensis . All copaiba oils showed some level of activity with values ranging between 5.0 and 22.0 μg/mL. The most active EO (from Copaifera reticulata, ) demonstrated effects against axenic and intracellular amastigote forms, with values of 15.0 and 20 μg/mL, respectively. With respect to cytotoxicity, this same EO showed a tolerable value of SI (8.0) to promastigote forms and an inadequate value (2.5) to axenic amastigote forms. Nevertheless, studies revealed that copaiba oil from Copaifera reticulata did not alter the viability of peritoneal macrophages at a concentration of 500 μg/mL, and the treatment at 500 mg/kg with EOs from Copaifera reticulata and Copaifera multijuga did not induce behavioral alterations, lesions, or bleeding in the stomachs of mice [154, 155].
These results involving copaiba EOs from various species led the same authors to investigate the in vivo antileishmanial activity against L. amazonensis of an EO from Copaifera martii, also using electron microscopy, biochemical analysis, and flow cytometry . Infected mice were treated with copaiba oil for 30 days. After oral treatment (100 mg/kg/day), oral and topical (1 mg/mm2) treatments reduced the average lesion size in relation to the control group (75% and 72.5% reduction, respectively); however, no significant difference was observed when compared to the reference drug glucantime (79.5% reduction). Nevertheless, topical and subcutaneous (100 mg/kg/day) treatments did not show significant reduction in average lesion size, and the subcutaneous treatment caused injuries to the animal.
Histopathological and mutagenicity evaluations showed no genotoxic effects or changes in various organs in animals treated with copaiba EO compared with control animals. In addition, the following microscopic aspects of the parasite were observed: (i) promastigote forms of L. amazonensis treated with concentrations of 14 μg/mL () and 70 μg/mL (IC90) showed ultrastructural changes and swollen mitochondria, and (ii) an increase in plasma membrane permeability and depolarization of the mitochondrial membrane potential were observed in axenic amastigotes treated with 100 and 200 μg/mL (Figure 1). These results supported the notion that copaiba oil may be the source of a new drug; however, its mechanism of action still needs to be clarified .
The antileishmanial effects of copaiba EOs from Copaifera reticulata and Copaifera martii species against L. amazonensis led another research group to investigate the biological potential of four commercial oils from Copaifera ssp. C1-C4, β-caryophyllene-, sesquiterpene- and diterpene-rich fractions were obtained from distillation . Diterpene-rich EOs (C2 and C3) were more active against L. amazonensis promastigotes. Sesquiterpene-rich EOs C1 and C4 were more potent against intracellular amastigotes, with of 2.9 and 2.3 μg/mL, respectively. In comparison with the same activity of EO from Copaifera reticulata , C1 and C4 EOs were 6.9- and 8.7-fold more potent, respectively. For all the compared EOs containing β-caryophyllene (22) as their major constituent, the good value (1.3 μg/mL or 6.4 μM) demonstrated by the isolated sesquiterpene suggested that this compound may directly influence the antileishmanial activity of the EOs assayed. In addition, SI values of 29.3, 40.1, and 48.9 for C1, C4, and β-caryophyllene (22), respectively, demonstrated a better ratio of efficacy to toxicity. Finally, the C4 sesquiterpene-rich fraction showed a similar antileishmanial activity against amastigote forms, probably due to the same concentration of β-caryophyllene (22) in both the EO and the fraction, and C4 diterpene-rich fraction was not active in low concentrations .
EOs from four plant species reported in traditional medicine in Ethiopia, Artemisia absinthium L., Artemisia abyssinica, Echinops kebericho MESFIN, and Satureja punctata, were tested against promastigote and axenic amastigote forms of L. donovani and L. aethiopica. The results demonstrated that two EOs from Artemisia absinthium L. and Echinops kebericho MESFIN possessed strong antileishmanicidal activity against promastigote ( ranging from 0.24 to 42.00 μg/mL) and axenic amastigote forms ( ranging from 0.0097 to 0.15 μg/mL) of both Leishmania species. In relation to the other two species tested, Artemisia abyssinica was inactive against the two forms of parasite, and Satureja punctata showed high cytotoxicity. Unfortunately, the great number of constituents (including mono- and sesquiterpenes, aliphatic and aromatic hydrocarbons, carotenoid derivatives, and oxygenated structures) that appeared to have no specific cellular targets could explain the inadequate SI values for the most active oils [158, 159].
In a recent study, Silva and colleagues evaluated the antileishmanial activity of the EO from leaves of Ocimum canum Sims. that is commonly used as an infusion or syrup for the treatment of a number of diseases. The biological results revealed that OcEO was rich in monoterpenes such as thymol (1) (42.15%), p-cymene (11) (21.17%), and γ-terpinene (64) (19.81%), demonstrating moderate activity against promastigote and intracellular amastigote forms of L. amazonensis, with values of 17.4 and 13.1 μg/mL, respectively. These values were more potent than those of the reference drug pentamidine, with satisfactory SI values (18.1 and 24.0, respectively) . Electron microscopy showed that OcEO was associated with several ultrastructural alterations in promastigote forms of the parasite, including autophagosome-like structures that were multivesicular and had lipid bodies that could result in cell death, discontinuity of the nucleus membrane, and exocytic activity by the flagellar pocket .
EO from leaves and flowers of Achillea millefolium, species used as antihelmintic and trypanocidals, was evaluated in vitro against L. amazonensis. Significant activity was observed against promastigote and intracellular amastigote forms, with values of 7.8 and 6.5 μg/mL and acceptable SI values of 9.2 and 11.0, respectively . Unfortunately, the major constituents of AmEO were not determined. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis of the treated promastigote forms revealed morphological alterations in the shape and size of the parasite as well as ultrastructural alterations, including changes in the flagellar membrane, abnormal membrane structures, rupture of the plasma membrane, atypical vacuoles, myelin-like figures, and vesicles that resembled autophagic vacuoles. The mechanism of action remains unclear; however, the morphological changes observed in the parasite may be related to inhibition of two parasite enzymes: serine-protease and squalene synthase (BPQ-OH) .
Rosa et al. investigated the leishmanicidal effects of linalool-rich EO from leaves of Croton cajucara and purified linalool (3) . Both EO and purified linalool (3) showed potent antileishmanial activity, with values of 8.3 and 4.3 ng/mL against promastigote forms and values of 22.0 and 15.5 ng/mL against the amastigote form of the parasite, respectively. SI values were not determined, but for both, the concentration of 15.0 ng/mL presented no cytotoxic effects against mammalian cells. Mitochondrial swelling and important alterations in the organization of the nuclear and kinetoplast chromatins decrease in the association between macrophages and parasites, and increases in nitric oxide levels in infected macrophages could explain the extreme toxicity of the CcEO for L. amazonensis .
Some studies reported that linalool (3) (oxygenated monoterpene) and eugenol (2) (phenolic compound), chemical constituents in many EOs from various plant species, as well as eugenol- and linalool-rich EOs, were fatal for several species of Leishmania [162–165]. Dutra and coworkers explored the biological potential of these purified compounds in L. infantum chagasi promastigotes and axenic amastigote forms at baseline and during the infection of peritoneal mouse macrophages . The estimated values for eugenol (2) and linalool (3) against axenic amastigotes forms were 220 and 550 μg/mL, respectively. The value for eugenol (2) against promastigote forms was 500 μg/mL, and for linalool (3), the value for the last parasite form was not determined. For both compounds, some of the mechanisms associated with leishmanicidal activity were confirmed: the two derivatives were able to enhance the activities of protein kinases PKC and PKA, linalool (3) decreased parasite oxygen consumption, and eugenol (2) reduced the parasite resistance to reactive oxygen species .
Santim et al. (2009) evaluated the effect of EO from Cymbopogon citratus and its major constituent, citral (63), against L. amazonensis. The EO showed good activity against promastigote and amastigote forms, with values of 1.7 and 3.2 μg/mL, respectively, a good SI value (14.7) for promastigotes and a tolerable value of 8.7 μg/mL against promastigote forms, with a tolerable SI of 6.3. SEM and TEM analysis revealed aberrant morphologies of the amastigote forms exposed to the EO, including disintegration of the parasite, mitochondrial swelling, exocytic projections in the flagellar pocket, swollen mitochondria, and rupture of the plasma membrane, characterized by the presence of extracellular materials .
Rodrigues and colleagues [139, 169] evaluated the antileishmanial potential of EOs from Eugenia uniflora L. and Syzygium cumini (L.) Skeels, and α-pinene (118), major constituents of EuEO. α-Pinene (118) was more effective than was the EO from the plant, without desirable potency ( against various forms of the parasite). The isolated compound showed moderate values (16.1 and 15.6 μg/mL against axenic and intracellular amastigote forms, respectively) and low cytotoxicity (). The mechanism of action for monoterpene was mediated by immunomodulatory activity (increase in phagocytic and lysosomal activities) and elevated NO levels. However, EO from E. uniflora showed significant antileishmanial activity, with values of 3.04 and 1.92 μg/mL against promastigote and amastigote forms, respectively, and good SI values of 14.9 and 23.9, respectively. In relation to the mechanism of action, this EO activity was not mediated by nitric oxide production, suggesting that macrophage activation may be involved in the biological activity, as demonstrated by increases in both phagocytic capacity and lysosomal activity [168, 169].
Bosquiroli et al. evaluated the antileishmanial activity of EO from Piper angustifolium. PaEO rich in sesquiterpenes such as spathulenol (108) (23.78%) and caryophyllene oxide (38) (13.06%) reduced the number of intracellular amastigotes of L. infantum with an value of 1.43 μg/mL. It was more toxic to NIH/3T3 fibroblasts and murine macrophage J774.A1, with good SI values of 33.72 and 22.5, respectively. For PpEO, the mechanism of action may be associated with increased NO release after treatment at low concentrations .
EOs obtained from the aerial parts of four Moroccan plant species (Lamiaceae) were screened against various Leishmania species [171–173]. The EOs from Lavandula stoechas, with fenchone (166) (31.81%), camphor (14) (29.60%), and terpineol (111) (13.1%) as major constituents, exhibited strong leishmanicidal effects against two Leishmania species, whereas L. major was the most sensitive strain with an of 0.9 μg/mL, followed by L. infantum () and L. tropica (). For the EO from Origanum compactum, three phenological stages of the plant were analyzed, and the oxygenated monoterpenes were the major constituents at the three phenological stages (49.4% at vegetative stage, 62.975% at flowering stage, and 61.379% at postflowering stage). In lower quantities, other chemical classes such as sesquiterpene hydrocarbons, oxygenated sesquiterpenes, alcohols, ketones, and acids were also identified. At low concentrations, these EOs showed significant cytotoxicity against three Leishmania species, while the cytotoxic effects are not significantly variable with increasing concentrations. The L. infantum species was the most sensitive strain, with values ranging from 0.02 to 0.12 μg/mL at the three phenological stages. The other two EOs from Mentha pulegium and Rosmarinus officinalis were studied by the same authors, and both monoterpene-rich EOs were potent against L. major, L. infantum, and L. tropica species, showing values ranging from 0.36 to 2.6 μg/mL. Unfortunately, for all EOs studied, the SI values were not determined, and the relationship between efficacy and toxicity could not be measured [171–173].
Essid et al. evaluated the antileishmanial activity of EOs from twelve medicinal plants from Northern Tunisia. The first group of EOs isolated from Ferula communis, Teucrium polium, and Pelargonium graveolens species displayed strong inhibitory activities against the promastigote forms ofL. major and L. infantum, showing values ranging from 0.05 to 0.28 μg/mL. The second group contained five EOs belonging to Thymus hirtus, Artemisia campestris, Myrtus communis, Artemisia herba-alba, and Alvia officinalis, exhibiting good leishmanicidal activities with values ranging from 1 to 10 μg/mL against the same species of the parasite, but with lower activity when compared with amphotericin B. In the third group, EOs from Nigella sativa, Laurus nobilis, Rosmarinus officinalis, and Eucalyptus globulus showed values up to 10 μg/mL. Purified β-caryophyllene (22), camphor (14), and carvacrol (10) were screened against the same parasite species and showed less activity when compared with the most active EOs classified as the first group. In addition, the most active EOs and purified compounds showed low cytotoxicity to macrophage cells, presenting ideal values of SI () .
An in vivo study was performed with the EO extracted from the leaves of Artemisia annua, which has camphor (14) (52.06%), β-caryophyllene (22) (10.95%), and 1,8-cineole (124) (5.57%) as major constituents. AaEO presented an value of 7.3 μg/mL against amastigote forms of L. donovani, but no cytotoxic effects against mammalian macrophages even at 200 μg/mL. In addition, intraperitoneal administration of the AaEO (200 mg/kg/b.w.) to infected BALB/c mice reduced the parasite burden by almost 90% in the liver and spleen, with significant reduction in weight. There was no hepatic or renal toxicity, as demonstrated by normal levels of serum enzymes .
Drmachi et al. (2016) evaluated the immunomodulatory effects of EO from Tetradenia riparia against L. amazonensis species. They found that TrEO induced 50% death of amastigote forms of the parasite after 24 h incubation at a concentration of 30 μg/mL. In addition, the oil did not present cytotoxicity in murine macrophages at the same concentration, with cell viability >95%. They evaluated the modulatory effects of TrEO on cytokine levels in peritoneal fluid cells that were infected with L. amazonensis. They observed inhibition of some of the most critical cytokines for parasite growth and the establishment of infection, including granulocyte-macrophage colony-stimulating factor, interleukin-4 (IL-4), IL-10, and tumor necrosis factor (TNF). The parasite inhibited interferon-γ and IL-12, and the EO blocked this inhibition, indicating that these cytokines were critical for activating mechanisms associated with the death and elimination of the parasite .
EO from Chenopodium ambrosioides, containing carvacrol (10) (62.36%) and ascaridole (37) (22.54%) as major constituents, was investigated against L. amazonensis. CaEO showed values of 3.7 and 4.6 μg/mL against promastigote and amastigote forms, respectively, and cytotoxicity was approximately 15-fold higher for peritoneal macrophages than for both parasite forms . For the promastigote form, they observed synergistic activity in conjunction with pentamidine , and other results showed similar activity against promastigote and amastigote forms of L. donovani, with values of 4.45 and 5.1 μg/mL, respectively . An in vivo study demonstrated significant reduction in the size of the lesions in animals treated with the EO during the first 15 days of treatment. They also observedsignificant suppression of the number of parasites in the infected footpads, as compared with the burden in the footpads of the other treated mice, and oral administration at a dose of 30 mg/kg slowed the infection in an experimental model .
In a subsequent study, the authors observed a significant reduction in lesion size in BALB/c mice treated with the EO from C. ambrosioides compared with untreated animals at doses of 30 and 150 mg/kg by intralesional and oral routes. This activity was superior to that of the reference drugs glucantime, amphotericin B, and pentamidine, or the two major constituents carvacrol (10) and ascaridole (37) [179, 180]. This ability to control disease progression of cutaneous leishmaniasis was also observed with EO from Bixa orellana, at dose of 30 mg/kg over 14 days . Finally, recent studies suggested that CaEO caused a breakdown of mitochondrial membrane potential and a modification of redox indexes , and the isolated compounds carvacrol (10), ascaridole (37), and caryophyllene oxide (38) mediated their leishmanicidal activity via various mitochondrial targets, including the electron transport chain, thiol depletion, and production of superoxide radical, causing significant impairment of mitochondrial coupling .
The chemical analysis of EO from Vanillosmopsis arborea Baker showed that α-bisabolol (165) was its main constituent (97.9%). Both VaEO and α-bisabolol (165) efficiently inhibited the growth of L. amazonensis promastigotes, with values of 7.35 and 4.95 μg/mL, respectively . In addition, α-bisabolol (165) was more effective against promastigotes and intracellular amastigotes, and the cytotoxic assay against intracellular amastigotes showed that the pure compound () was less toxic than was VaEO (). These results suggested that both α-bisabolol (165) and VaEO increased cell permeability to exogenous compounds, since some sesquiterpenes can induce changes in membranes, allowing microorganisms to enter the cells, thus augmenting the microbial permeability to antimicrobial agents. The ultrastructural analysis of the parasite showed an elongated cell body and the presence of a well-defined kinetoplast and nucleus. The promastigotes treated with α-bisabolol (165) for 24 hours at 30 μg/mL showed severe cell damage with loss of parasite morphology, discontinuity of the nuclear membrane, increased mitochondrial volume and kinetoplast, and presence of vesicles with an electrondense display with lipid inclusion in the plasma membrane. However, in the same conditions of analysis for VaEO, they observed increased volumes of flagellar pockets with consequent breakage, increased volumes and changes in mitochondrial kinetoplasts, abnormal condensation of chromatin in the nucleus, discontinuity of the nuclear membrane, lipid inclusions in the presence of electrondense vesicles, and visualization of the inclusion of a lipid envelope within the plasma membrane, with the consequent loss of parasite morphology .
Another group evaluated the effect of (−)-α-bisabolol (165) against promastigote forms of L. infantum. In general, this sesquiterpene showed a very similar percentage of inhibition of the parasite when compared with the reference drug pentamidine (Figure 36). The value of for the compound was 10.99 μg/mL, and the presence of a hydroxyl in its structure explains its high capacity to inhibit L. infantum promastigote form. In addition, the very low toxicity in vivo supported the therapeutic use of (−)-α-bisabolol (165) to treat leishmaniasis caused by L. infantum [184, 185].
Xavier et al. explored the leishmanicidal properties of synthesized compounds derived from the oxygenated monoterpenes eugenol (2), thymol (1), and carvacrol (10), by molecular hybridation with nitrated Morita-Baylis-Hillman adducts (MBHAs) . The general structure and values of the most potent hybrid compounds against L. amazonensis promastigote forms are shown in Figure 37. Hybrid compounds 180, 181, and 182 exhibited the best antileishmanial activity, with values ranging from 4.71 to 8.75 μg/mL, and (except for derivative 3, which was not determined). In addition, the compounds substituted with nitro groups at the ortho position were more bioactive than the meta and para nitroaryl isomers. Experimental and in silico studies indicated that the mechanism of action was connected to nitro group reduction that generated RNO−• .
Ten chemical constituents of EOs, including nine monoterpenes and one phenylpropanoid, were investigated as antileishmanial agents against promastigote forms of L. amazonensis . Among the monoterpenes evaluated, the positional isomers carvacrol (10) and thymol (1) that presented a phenolic moiety were the most active compounds, showing moderate antiparasitic activity, with values of 25.4 and 26.8 μg/mL, respectively. This result suggests that the position of the hydroxyl group in the aromatic ring did not influence the antileishmanial activity. For eugenol (2), a hydrogen bond between hydroxyl and o-methoxyl groups reduced the release of protons by the OH group, and the acidity of this group may play an important role in the potency of the compounds. In addition, for this series of compounds, the absence of aromatic hydroxyls caused compounds to be less potent, as observed for p-cymene (11) and menthol (130) with values of up to 198.9 μg/mL.
In the same study, the value found for carvacrol (10) was different from values reported in the literature for L. chagasi and L. amazonensis promastigotes (  and 28.0 , and 15.3 μg/mL , respectively). Drug-parasite incubation (24-72 h) and assay methods may explain these differences between values of . For thymol, similar activity was observed by de Medeiros et al., who reported an value of 22.6 μg/mL. values ranging from 9.8 to 65.2 μg/mL were found for other species of Leishmania [56, 188, 189]. Both eugenol (2) and thymol (1) compounds showed low cytotoxicity at concentrations of 50 and 100 μg/mL, where the percentage of viability on L929 fibroblasts ranged from 46.1 to 64.5%.
1.4. Essentials Oils Active against Arboviruses, Especially Dengue
Arboviruses (arthopod-borne viruses) are viral diseases transmitted to humans by arthropods (insect and arachnid). Among the vectors, one that stands out is the mosquito Aedes aegypti, which every year transmits diseases to more than 700 million people worldwide. According to the World Health Organization, it causes more death than any other arboviral disease, killing about 2.5% of all affected children .
Aedys aegypti is a carrier of various diseases, including dengue fever, dengue haemorrhagic fever, chikungunya, yellow fever, and even Zika virus . Yellow fever, dengue, and Zika virus have their greatest focus in tropical and subtropical areas , while dengue haemorrhagic fever has a greater number of reports in Asia, Thailand, and Indonesia .
In recent decades, the spread of dengue has increased worldwide, and in 2012 it was considered the most important viral disease in the world, with more than 100 countries and more than 2.5 billion people (about 40% of the world population) at risk of infection by some mosquito-borne disease. Of this total population, approximately 50-100 million are infected annually, of which 500 thousand cases are considered of greater severity [204, 205]
The mosquito transmits arboviruses through the blood meals of the female. The life cycle consists of egg, larva, pulp, and adult. Eggs are deposited on the walls of any reservoirs containing water, and the ideal temperature range for hatching is 28-33°C; larval and pupal stages take place in the water. The transition phase from larval to pupa lasts 24 hours, and the pupal phase lasts 48 hours until the adult mosquito appears. When adults appear, it takes a few seconds for the stiffening of chitin that allows them to fly .
Treatment of dengue, chikungunya, and Zika virus are symptomatic, and vaccines are not yet available. Currently, the only effective way to control these diseases is by breaking the disease transmission cycle, controlling the mosquito population through the use of biological and/or synthetic insecticides and repellents.
In several public health programs, various larvicides and insecticides currently used are effective in eliminating mosquitoes and larvae, including the organophosphates temephos  and pyrethroids . However, their continuous use has allowed some mosquitoes to become resistant , and the insecticides are not selective, causing damage to other organisms, including humans [205, 208–210]. Temephos, for example, was monitored in 16 countries between 1960 and 2000. It was observed that the agent was active from 48 to 72 hours in water, and it interfered with the viability of other living organisms (in addition to A. aegypti), causing serious environmental impact .
Currently, in Brazil temephos 1% and Bti (an efficient bioinsecticide) are both used. Bti does not present the same environmental impact as does temephos; however, its high cost makes its general use impossible .
In this scenario, it is necessary to identify new larvicides with various modes of action to reduce the development of mechanisms of resistance to pesticides, thereby bolstering public health system efforts .
The use of biological agents has been gaining great prominence, reaching the point of a single review article compiling the larvicidal activity of 361 EOs from 269 plant species. Among these, more than 60% were considered active, with .
Knowing that it is easier to interfere with the proliferation of Aedes aegypti in the larval phase than in the adult phase , several EOs are being studied and have already shown to have relevant larvicidal activity, with advantages over synthetic insecticides. Among these advantages, the following stand out: lower risks of allergy, less irritability to the skin, less encephalopathy in children , more pleasant odors , less toxicity to humans, animals, plant life and other organisms , and biodegradable properties . In these terms, EOs are superior for the control of arboviruses .
In this context, several authors reported activity of EOs and their phytoconstituents in the control of Aedes aegypti adults and larvae. A summary of these studies is presented in Table 3.
Most of the studies, while identifying the constituents of the EO, restricted the experiments of biological activity to the oil itself, with few studies evaluating the activity of each individual substance. In some cases, only the activity of the compounds with the highest concentration was evaluated. This is primarily due to the high cost of these compounds, when they are available for purchase .
The monoterpenes were the most tested compounds, especially oxygenated monoterpenes, corresponding to 38% () of the compounds shown in Table 3. The monoterpene hydrocarbon class presented the best activity. This group included 3-carene (119) ( and 25.3 μg/mL), α-terpinene (182) (), and limonene (8) (), among others.
Knowing the isolated constituents of the EOs and their activity allowed the execution of chemical analysis and quantitative structure-activity relationship (QSAR), which identifies characteristics related to the structure of the compound that describe the biological activity, making possible the design of new promising compounds.
One study conducted a consensus principal component analysis (CPCA) and principal component analysis (PCA), identifying characteristics related to hydrophobicity (LogS, DRY, and H2O) of thymol (1) and carvacrol (10) derivatives that were important to explain the activities of the compounds against larva of Aedes aegypti . Another study performed CPCA and PCA in a series of 55 substances isolated from EOs, obtaining similar results. They highlighted the importance of hydrophobicity (DRY and H2O) for the activity of these compounds against the larva of Aedes aegypti; however, steric characteristics were also important to explain this activity, indicating that these characteristics were crucial to the activity of these substances, based on the evaluated compounds .
In this work, a large number of papers that dealt with the use of EOs, their fractions, and/or their chemical constituents as treatment options for some neglected diseases were reviewed. Despite the full knowledge that mankind makes use of plants for medicinal purposes from its origin, the absence of new effective drugs, with fewer side effects that can be used against resistant strains of the parasites, promotes a strong demand for natural products, especially for the poorest and most affected populations that are continuously under risk of these diseases. Several EOs and chemical constituents were identified, which have biological activities equipotential to those of the few commercial drugs available, with good margins of safety, suggesting that popular use cannot be neglected.
As examples for isolated active compounds against T. cruzi, the isolated compounds thymol (1) and linalool (3) showed similar activity when compared with benznidazole and quantified activity in nanomolar concentrations, respectively. In addition, semisynthetic aphidicolin (28) derivatives (diterpenes) exhibited high potency and selectivity against the same parasite.
For EOs with anti-T. brucei activity, the isolated compounds rotundifolone (88) and (−)-perillyl aldehyde (92) were slightly less active than the reference drug suramin, and carvacrol (10), ascaridole (37), and caryophyllene oxides (38) showed strong potential to growth inhibition of the parasite, with activity comparable to the same standard drug. In addition, some EOs and isolated compounds demonstrated promising antitrypanosomal activity and high selectivity index values, specially the compounds carlina oxide (139) (isolated from Carlina acaulis roots), linalool (3) (which exhibited potential activity against different trypanosome species), and C. nardus EO (rich in oxygenated monoterpenes), which demonstrated values ranged from 0.31 to 2.5 μg/mL and high selectivity index (>40).
For antileishmanial activity, several EOs or isolated compounds were active against the parasite (). Some of them promoted morphological alterations in the parasite, but few showed a great ratio of efficacy to toxicity. Linalool-rich EO (from Croton cajucara) and purified linalool (3) showed potent antileishmanial activity at nanomolar concentration, promoting morphological changes in the parasite, as well as decreased parasite oxygen consumption and enhances the activities of the protein kinases and increases in nitric oxide levels in infected macrophages, confirming some of the mechanisms associated with leishmanicidal activity. Another isolated compound identified as α-bisabolol (165) (main constituent of EO from Vanillosmopsis arborea) efficiently inhibited the growth of promastigotes and amastigotes forms of the same parasite, inducing several ultrastructural alterations and severe cell damage in the parasite, where the very similar percentage of inhibition (compared with pentamidine) and very low toxicity in vivo supported their therapeutic use to treat leishmaniasis.
Other EOs rich in spathulenol (108) and caryophyllene oxide (38) (from Piper angustifolium) and citral (63) (from Cymbopogon citratus) showed potential activity against promastigote and amastigote forms of the parasite and good SI values.
For control of Aedes aegypti adults and larvae, some monoterpene hydrocarbon EOs, specially 3-carene (119), α-terpinene (182), and limonene (8), exhibited the best activity.
In this sense, knowledge of the chemical structures of these phytochemicals may allow them to serve as scaffolds for rational drug design, suggesting chemical modifications to increase activity, bioavailability, and toxicity, among other characteristics, allowing the design of new and more active compounds.
Conflicts of Interest
The authors declare no conflict of interests regarding the publication of this paper.
The authors acknowledge the financial support from the Brazilian agency CNPq and from State University of Paraiba (UEPB). F.J.B.M.J. is granted by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) PQ #308590/2017-1. E.C.L. and R.S.A.A. are granted by FAPESQ-CNPQ (fellowships).
Figure S1: plant families and number of OEs evaluated between the years 2000 and 2018 against at least one species of Leishmania. Figure S2: plant families and the number of active EOs (IC50 ≤ 10 μg/mL) against at least one Leishmania species and parasite form. Table S1: inactive or moderated active EOs against Leishmania species. (Supplementary Materials)
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