Keywords

1 Introduction

Besides being an important component of the plant defense system against pathogenic attacks and environmental stress, the secondary metabolism of plants provides a useful range of natural products (Piasecka et al. 2015). Due to their biological activities, the secondary metabolites of plants have been increasingly used as medicinal substances and food additives for therapeutic, aromatic and culinary purposes. The characteristics and concentration of secondary molecules and the biosynthesis by a plant are defined by the identity of the species and genetic, ontogenic, morphogenetic, physiological, developmental, and environmental factors. This suggests that various taxonomic groups of plants have adaptive physiological responses to deal with stress and defensive stimuli (Yang et al. 2018; Isah 2019).

Terpenes and terpenoids (the oxygenated derivatives of terpenes) are chemical compounds that represent the majority of molecules in the composition of essential oils (EOs) (Matos et al. 2019). This class of molecules is characterized by a different number of isoprene (C5H8) units (Blowman et al. 2018). Depending on the number of these units, terpenes can be categorized into hemiterpenes , monoterpenes , sesquiterpenes , diterpenes , triterpenes , among others (Rubulotta and Quadrelli 2019; Sharma et al. 2021). They can also be divided into groups such as acyclic, monocyclic, and bicyclic (Blowman et al. 2018). The terpenoid is a type of terpene that has oxygen attached to its structure (Sharma et al. 2021).

Essential oils, which are one of the substance types formed by terpenes, are widely used and studied for their pharmacological, biological, and permeation enhancing properties. However, several terpenes and EOs are sensitive to environmental conditions and may undergo volatilization and chemical degradation (Matos et al. 2019). Essential oils are natural products with a complex composition and are used in different ways, namely, through inhalation, topical application onto the skin, and oral consumption. There are, therefore, three main routes of ingestion or application: the skin system , the olfactory system , and the gastrointestinal system . Understanding these routes is important to clarify the mechanisms of action of EOs (Koyama and Heinbockel 2020).

The biological and pharmacological activities of EOs investigated so far include antibacterial (Ács et al. 2018), antifungal (Mutlu-Ingok et al. 2020), antiviral (Brochot et al. 2017), antileishmanial (Oliveira et al. 2020), antioxidant (Menezes Filho et al. 2020), cytotoxic (Contini et al. 2020), and anti-inflammatory (Saldanha et al. 2019) activities.

Leishmaniasis is a collection of diseases caused by parasitic protozoa of more than 20 species of Leishmania. The disease has three main forms: the tegumentary (most common form), the visceral (most severe form), and the mucocutaneous (most disabling form). Humans are contaminated by these parasites by the bite of infected female phlebotomine sandflies (WHO 2021). The clinical manifestations of leishmaniasis are quite mutable and can range from localized skin lesions to dissipation of life-threatening visceral disease (Meira and Gedamu 2019). Currently, more than 1 billion people worldwide are in endemic areas of leishmaniasis and are at risk of infection (WHO 2021).

The first-line drugs for the treatment of leishmaniasis are antimonials. In resistant cases, pentavalents , amphotericin B deoxycholate , liposomal amphotericin B , and paromomycin are used as secondary options. However, these drugs have their use limited because of side effects, high costs, induction of resistance in parasites, and administration in hospitalized patients (Albuquerque et al. 2020). Therefore, research for new compounds is needed. In this sense, EOs have been increasingly investigated for their effectiveness against species of the genus Leishmania, to serve as an alternative for the treatment of leishmaniasis (Mahmoudvand et al. 2016; Sharifi-Rad et al. 2018; Rottini et al. 2019; Macêdo et al. 2020; Ferreira et al. 2020; Vandesmet et al. 2020; Gomez et al. 2021).

Therefore, this review seeks to understand the action of EOs against Leishmania species, parasites that cause vector-borne diseases known as leishmaniasis and which represent a serious public health problem.

2 Methodology

2.1 Database Search

Articles were searched through consultations in the Scopus© database (https://www.scopus.com/). As keywords, the descriptors “Essential oil AND Leishmania” were used, only in the English language.

2.2 Inclusion and Exclusion Criteria

Only scientific articles that addressed specific information about the potential of EOs extracted from different plant species against Leishmania spp. and published in the last 10 years (2011–2021) were selected. Regarding the exclusion criteria, review articles, e-books, book chapters, editorials, course completion works, dissertations, theses, abstracts published in congress proceedings, and articles on the potential of extracts, isolated chemical compounds, EOs commercialized without identification of the species, non-active EOs, and fixed oils against Leishmania spp. were discarded.

2.3 Data Screening and Information Categorization

Initially, 186 scientific articles were identified and selected in the Scopus© database. After applying the exclusion criteria, 72 documents that did not fit the theme of this review were discarded (Fig. 13.1). Finally, 114 articles containing data on the potential of EOs against Leishmania spp. were included (Fig. 13.1). The information collected in the articles was categorized into: (1) “Essential oils against Leishmania spp.”; (2) “Terpenes”; (3) “Mechanisms of action”; (4) “Other compounds present in essential oils”; and (5) “Other applications”. Further details about the species, active concentration of essential oils, evolutionary form of Leishmania spp., major constituents, and mechanism of action were also organized and presented in a table.

Fig. 13.1
figure 1

Flowchart of selection of scientific documents included in this review

Full size image

3 Results

Of 186 articles, 114 met the inclusion criteria and were selected for data extraction (Table 13.1). Of the 114 studies, 111 are in vitro (97.4%), 2 in vivo/in vitro (1.7%), and 1 in vivo (0.9%) assays of EOs with leishmanicidal activity. The Leishmania species most used in the assays were: L. amazonensis, used in 54 (47.4%) of the studies, L. infantum, in 33 (28.9%) of the studies, and L. major, in 21 (18.4%) of the studies. Table 13.1 presents the EOs of plant species from 74 genera belonging to 26 families, among which the most frequent were Lamiaceae with 14 genera (18.9%), Asteraceae with 9 genera (12.1%), and Myrtaceae with 8 genera (10.8%).

Table 13.1 Antileishmanial activity of aromatic species
Full size table

Of the 114 studies included in the review, 100 (87.7%) performed the chemical characterization of the EOs and 14 (12.3%) did not. Carvacrol was the major constituent most present in the EOs, being reported in 8 studies (7%), followed by thymol, cited in 7 studies (6.1%), and α-pinene and 1,8-cineole cited in 5 studies (4.3%) each.

Seventeen articles (14.9%) performed tests to verify the possible mechanism of action of the EOs, while 97 (85.1%) did not. In this sense, among the studies that investigated the mechanism of action, the ones by Demarchi et al. (2015) and Demarchi et al. (2016) with the EO of Tetradenia riparia stood out with the best result in terms of IC50 (0.03 μg/ml). The leishmanicidal potential of T. riparia EO against L. amazonensis was explained by the oil’s ability to modify the ultrastructure of promastigotes, suggesting an autophagic process with chromatin condensation; presence of blebbings and nuclear fragmentation; decreased macrophage infection rate by amastigotes; and, finally, inhibition of granulocyte and macrophage colony-stimulating factor, interleukin-4 (IL-4), IL-10 and tumor necrosis factor. Other EOs are also noteworthy for their ability to inhibit parasites at low concentrations, such as those from Origanum compactum (IC50 = 0.02 μg/mL), Ferula communis (IC50 = 0.05 μg/mL), and Teucrium polium (IC50 = 0.09 μg/mL) against L. infantum isolates.

4 Discussion

4.1 Essential Oils Against Leishmania spp.

The genus Leishmania is a group of flagellated parasites comprising more than 20 different species distributed in the subgenus Leishmania or Viannia, whose main vectors are phlebotomine sandflies of the genus Lutzomyia and Phlebotomus (Espinosa et al. 2018). Members of the genus Leishmania differentiate from proliferative promastigotes in the insect vector gut into infective metacyclic promastigotes in the foregut of the insect. The parasites are inoculated by the vector as flagellated promastigotes into the mammalian host, where they infect macrophages, differentiating into amastigote forms (Rocha et al. 2005).

Leishmania parasites can be divided according to their clinical forms and manifestations, geographic distribution, and reservoir. Leishmania (L.) amazonensis, L. mexicana L, L. (L.) tropica, and L. (V.) guyanensis are more prevalent in South America and are characterized by causing multiple or individual ulcerative lesions, a condition called cutaneous leishmaniasis. In addition to L. (V.) braziliensis, which can cause mucocutaneous changes, L. infantum and L. donovani cause the most serious conditions called visceral leishmaniasis, which include, but are not limited to persistent fever, splenomegaly, and weight loss (Burza et al. 2018).

The main anti-Leishmania therapeutic methods involve the use of pentavalent antimonials, amphotericin B, paromomycin, pentamidine, and miltefosine; however, there is great resistance to treatment adherence due to their high toxicity and side effects, in addition to the financial impact in more poor regions (Roatt et al. 2020). There is also concern about the development of resistant strains and variable response to treatment depending on the parasite species. In Brazil, strains of L. infantum resistant to miltefosine have been isolated in patients whose treatment was unsuccessful. According to Roatt et al. (2020), this finding suggests a natural resistance to this drug because ince it had not yet been used in the country (Carnielli et al. 2019).

The exploration of the plant kingdom is one of the only options for the development of therapeutic agents with high safety and cost-benefit profile for various health problems, as highlighted by Bekhit et al. (2018). The investigation of new compounds that can be used in the treatment of leishmaniasis begins with ethnobotanical studies, which provide information about the medicinal properties of various plant species based on the knowledge disseminated in traditional communities.

Ethnobotanical studies and the investigation of the therapeutic potential of plants make it possible to track new bioactive molecules with the potential to become new drugs in the future. Passero et al. (2021) list 216 species distributed in 76 genera that present contributions to the experimental treatment of leishmaniasis, opening a wide range of options for investigations in the field. A review published by Rocha et al. (2005) found about 239 chemically defined natural molecules reported in the literature which were evaluated for anti-Leishmania activity, including alkaloids, terpenes, various lactones, flavonoids, diterpenes, steroids, lipids, carbohydrates, proteins, coumarins, phenylpropanoids, and depsides. Recently, a review published by Fampa et al. (2021) highlighted about 30 volatile compounds that were also evaluated for their anti-Leishmania activity.

4.2 Terpenes

According to the data obtained, analyses show that, among the different compounds that constitute EOs, terpenes are the most abundant, present both as sesquiterpenes and monoterpenes. The anti-Leishmania activity of compounds present in EOs can be attributed to their lipophilic character. Several studies indicate that these substances act by breaking the microbial cytoplasmic membrane, making it permeable, affecting polarization and compromising biological barriers and the enzyme matrix (Cristani et al. 2007).

The EO of Myrciaria plinioides leaves was effective against L. amazonensis promastigotes and presented an IC50 value of 14.16 ± 7.40 μg/mL; however, the activity against L. infantum promastigotes was less pronounced, with an IC50 value of 101.50 ± 5.78 μg/ml (Kauffmann et al. 2019). The anti-Leishmania activity was attributed to the presence of the sesquiterpenes spathulenol (1) and caryophyllene oxide (2), which represent 36.32% of the total components that can cause alterations in the mitochondrial membrane potential, in addition to modification of the redox index, inhibition of cellular isoprenoid biosynthesis, and changes in the plasma membrane (Santos et al. 2008; Rodrigues et al. 2013; Monzote et al. 2014c).

The EO of Lantana camara was able to cause 100% inhibition of proliferation of L. amazonensis at concentrations above 3 μg/mL, and about 90% inhibition in L. chagasi at the concentration of 250 μg/mL (Machado et al. 2012b). The presence of germacrene-D (3) in the composition of the EO was considered to be responsible for the inhibitory effect on the growth of promastigote cultures. This hypothesis is based on the activity of amphotericin B, which is able to act as an antifungal and antiparasitic agent, as suggested by tests in germacrene-D (3). It is noteworthy that Biavatti et al. (2001) observed a toxic effect of the EO in tests using brine shrimp and mammalian cells in vitro. However, the authors mentioned that this effect was not related to the presence of germacrene-D (3), as it did not present a toxic effect in the same models.

Another terpene with anti-Leishmania activity widely cited in the literature is pinene (4,10). More than 40 components were found through gas chromatography analysis in the EO of propolis, with 36.17% of α-pinene (4). In in vitro tests, pinene (4,10) was effective against the promastigotes and amastigotes of L. major and L. infantum, with IC50 of 5.29 μg/mL and 3.67 μg/mL for promastigotes, and 7.38 μg/mL and 4.96 μg/ml for amastigotes of L. major and L. infantum, respectively. Furthermore, the EO exhibited synergistic activity with amphotericin B, inhibiting the growth of Leishmania by more than 98%. Although the activity was attributed to its major compound, the authors did not rule out a synergy of pinene (4,10) with the less expressive components present in the EO (Jihene et al. 2020).

In tests performed by Dias et al. (2013), the EO of Syzygium cumini showed good activity against the promastigote forms of L. amazonensis. At all concentrations and time points analyzed, significantly higher mortality was observed in the treatment than in the control groups, leading to the conclusion that S. cumini EO has leishmanicidal rather than leishmanistatic activity. The greatest efficacy was seen within 24 hours of exposure, with an IC50 of 36 mg/L. Although the author did not perform specific tests to determine the mechanisms of action through which the EO acts, leishmanicidal activity was attributed to the lipophilic characteristic of the EO, mentioned above. With 31.85% of α-pinene (4) in its composition, its action can be compared to that of the EO of Cinnamodendron dinisii , which has 35.41% α-pinene (4) (Andrade et al. 2016). Although C. dinisii EO has a higher concentration of pinene (4,10) in its composition, its activity was lower than that of S. cumini EO. It is possible that the minor compounds in these species interfere in the action of pinene (4,10).

Bouyahya et al. (2019) tested the EO of leaves and fruits of Pistacia lentiscus , obtaining an IC50 of 11.28 and 8 μg/mL, respectively, against L. infantum, 17.52 and 21.42 μg/mL against L. major, and 23.5 and 26.2 μg/mL against L. tropica. Both EOs presented better results than the standard drug glucantime and although they were obtained from P. lentiscus, both presented major compounds at different concentrations. In the EO of the leaves, was in higher concentration, 33.46%, while α-pinene (4) represented only 19.20%. The EO of the fruits presented 20.46% of α-pinene (4), and the second compound with the highest concentration was limonene (5), corresponding to 18.26%. This shows that the composition of EO can change according to the part of the plant from which it is extracted.

It is known that besides varying according to the part of the plant from which it is extracted, the composition of the EO can be altered by environmental factors such as climate, time of collection, and geographic location (Do Carmo et al. 2012; Essid et al. 2015; Bouyahya et al. 2019). Variability is also present in plants of same genus but different species. This is the case of Artimisia plants studied by Mathlouthi et al. (2018). In their tests, they showed a remarkable anti-Leishmania activity, with an IC50 of 2.20 μg/mL and 1.20 μg/mL for Artemisia campestres and Artemisia herba-alba, respectively, both against the promastigote forms of L. major. Artemisia herba-alba had β-thujone (8) (29.4%) and 1,8-cineole (9) (14.8%), with only a small fraction of β-pinene (10) (2.3%), while A. campestres had β-pinene (10) (32%) and limonene (5) (17.3%), but β-thujone (8) was absent.

Although many EOs have shown better results than the isolated compounds, several factors may be involved in these processes. In a study by Do Carmo et al. (2012), the EO of Piper duckei showed a lower result than its major compound, trans-caryophyllene, against L. amazonensis promastigotes. The IC50 was 46 μg/mL for the EO, and 96 μg/mL for the isolated compound. The authors reported that, during the experiments, it was possible to observe that the purity of trans-caryophyllene is an important factor for the activity against L. amazonensis. The oxidation of trans-caryophyllene to its corresponding oxides affects the results; depending on the level of oxidation, activity may not be observed. Another plant of the Piper genus, Piper cernuum Vell, also had caryophyllene (11) in its composition (16%). In in vitro tests with macrophages infected with L. amazonensis, the isolated compound reached greater efficiency in reducing parasite infection in macrophages at concentrations of 2 and 10 μg/mL, leading to infection rates of 105 ± 16 and 101 ± 7, respectively, both lower than values obtained with amphotericin B (34 ± 5 ​​at 0.1 μg/mL), but superior to those obtained with the EO (131 ± 15 at 2 μg/mL and 115 ± 13 at 10 μg/mL). According to Capello et al. (2015), the effect of the EO may be associated with bioactive sesquiterpenes present in its composition.

In a research carried out by Essid et al. (2015), compounds of the EOs extracted from F. communis, T. polium, and Pelargonium graveolens exhibited strong inhibitory activity against the growth of promastigote forms of L. major and L. infantum, with IC50 values <1 μg/mL. Their main constituents were β-caryophyllene (11), carvacrol (12) and citronellol (13) respectively. In tests with the isolated compounds, β-caryophyllene (11) was the most active, with an IC50 of 1.06 ± 0.37 μg/mL for L. infantum and 1.33 ± 0.52 μg/mL for L. major. Carvacrol (12) had an IC50 of 7.35 ± 1.78 g/mL for L. infantum and 9.15 ± 0.12 g/mL for L. major. Very low activity was recorded for citronellol (13). It is interesting to note that the isolated compounds showed lower activity than the EO.

According to Carvalho et al. (2017), EOs are more effective than their individual chemical constituents. Their bioactivity depends on the additive and synergistic action of the components. The EO of Cymbopogon citratus and its major constituents citral (14) (neral (15) 40% + geranial (16) 60%) and myrcene (6,7) were tested against L. infantum by Machado et al. (2012a), resulting in IC50 values of 25 μg/mL for the EO, 42 μg/mL for citral (14), and 164 μg/mL for myrcene (6,7), thus showing the best result for the EO. In a work carried out by Moreira et al. (2017), the EO of Vernonia polyanthes Less presented an IC50 of 19.4 μg/mL against L. infantum, lower than the IC50 of zerumbone (17) (9 μg/mL), a monoterpene present in the EO.

On the other hand, in the work by Leal et al. (2013), the EOs of Piper brachypodom and Piper var. brachypodom presented trans-ß-caryophyllene (11) as the major component (20.2%). The results showed that the EOs were more active against L. infantum promastigotes (IC50 23.43 and 23.68 μg/mL, respectively). However, none of these EOs was active against the intracellular forms of this protozoan. Trans-ß-caryophyllene (11) had an IC50 of 24.02 μg/mL against L. infantum promastigotes, a result slightly lower than that obtained for the EOs, but it was active against amastigote forms, with an IC50 of 53.39 μg/mL. The author stated that it is much more difficult for components to reach intracellular forms because they need to penetrate barriers and reach the place where the parasite is alive, as opposed to free forms in whose case the product can act directly on the parasite. Considering the similarity of the results, it is possible to say that the action of Piper EOs is due to its major constituent, and that the constituents with lower expression possibly acted negatively, preventing the action of the EOs in the intracellular forms of L. infantum.

According to Cristani et al. (2007), the activity of monoterpernes such as carvacrol (12) and thymol (18) results from the disturbance of the lipid fraction of the plasma membrane of microorganisms, as bacteria. Other studies point to the same type of interaction in parasites and claim that terpenes are responsible for the hydrophobic characteristic of EOs, allowing their diffusion across the cell membrane of parasites such as Leishmania and affecting intracellular metabolic pathways and organelles (Andrade et al. 2016).

In a work carried out by de Medeiros et al. (2011), the incubation of L. amazonensis promastigotes with Lippia sidoides EO and its main constituent thymol (18) efficiently inhibited the growth of the parasite. IC50/48 h values ​​were 44.38 and 19.47 μg/mL for EO and thymol (18), respectively. The treatment of intracellular amastigotes with the EO at concentrations of 25, 50 and 100 μg/mL caused a significant decrease in the survival rate of the parasites, with an IC50 value of 34.4 μg/mL. The authors also pointed out that, while thymol (18) had low selectivity against promastigotes and showed toxicity to mammalian macrophages, the EO showed low toxicity to mammalian cells, a fact attributed to the protective effect of other constituents.

Study conducted by Farias-Junior et al. (2012) brought the first analysis of the anti-Leishmania properties of L. sidoides EO, in which carvacrol (12) instead of thymol (18), was the main constituent. It was demonstrated that the carvacrol-rich (12) EO had an IC50 lower than that of the EO whose main constituent was thymol (18) against L. chagasi promastigotes. Although it is logical to attribute such activity to carvacrol (12), the EO also had 6% of thymol (18), and thus there is a possibility of a synergistic effect between thymol (18) and carvacrol (12) to explain the greater anti-Leishmania effect observed in this EO.

Essid et al. (2015) suggest that the inhibitory activity of carvacrol (12) is enhanced in the presence of its isomer thymol (18) and its precursors γ-terpene and p-cymene (19), as demonstrated by Lambert et al. (2001). In their studies, the EOs of F. communis, T. polium, and P. graveolens reduced by more than 90% the number of parasites in a dose-dependent manner, in the case of L. infantum and L. major, presenting anti-Leishmania activity greater than amphotericin B. The authors highlight that the mechanism of action of the EOs may involve changes in the mitochondrial membrane.

The relationship between carvacrol (12) and p-cymene (19) was also suggested by Bouyahya et al. (2017a). In their study, the EO of O. compactum extracted from different plant phases (vegetative, flowering and post flowering) showed effective action against three Leishmania species in a dose-dependent manner, being the EO obtained in the flowering phase the most active against the three parasites tested. The author also speculated that the involved mechanisms of action may include induction of apoptosis, disruption of the electron transport chain, and inhibition of DNA topoisomerase (Castro et al. 1992).

Monzote et al. (2011) brought another perspective to the action of carvacrol (12). Treatment of L. amazonensis-infected murine macrophages with the EO of Chenopodium ambrosioides L. proved to inhibit parasite growth. The authors attribute this activity to ascaridol (20) and also mention that the toxicity exhibited by the sample could have been caused by the different compounds present in the EO or by the interaction between them. This hypothesis was formulated from the study of Monzote et al. (2009) that showed that ascaridol (20) forms a highly reactive carbon-centered free radical. The authors suggested that, through its phenolic hydroxyl group, carvacrol (12) serves to attenuate the cytotoxic activity of ascaridol (20) by eliminating the free radical (Dapkevicius et al. 2002; Guimarães et al. 2010).

The EOs of Lippia gracilis Schauer genotypes 106 and 110 were analyzed and tested against L. chagasi promastigotes, resulting in IC50 values ​​of 86.32 μg/mL− 1 and 77.26 μg/mL−1, respectively (de Melo et al. 2013). The authors also showed that thymol (18) and carvacrol (12), the main compounds of the EOs, which also had exhibitory activity, and the latter (IC50 of 2.3 μg/mL−1) had similar performance to amphotericin B (0.51 μg/mL−1).

Both compounds were also found in the EO of Z. multiflora, which showed a significant anti-Leishmania effect on the promastigote forms of L. tropica. Furthermore, it was shown that the promastigote forms of L. tropica without treatment were able to infect 84.1% of macrophages, while promastigotes treated with Z. multiflora EO had potency to infect only 11.3% (Dezaki et al. 2016).

Thymoquinone (21), the major compound (43.4%) of the EO of Nigella sativa L. (Ranunculaceae), showed an inhibitory capacity for parasitic growth of L. tropica promastigotes, with IC50/72 h of 1.16 mg/mL, and L. infantum, with IC50/72 h of 1.47 mg/mL, while the EO presented IC50/72 h values of 9.3 mg/mL for L. tropica and 11.7 mg/mL for L. infantum (Mahmoudvand et al. 2015a). An assay was also carried out to evaluate the inhibition of the infection in macrophages: the promastigotes of L. tropica were able to infect only 13 and 27.3%, and those of L. infantum infected only 16.3 and 33.6% of the murine macrophages when treated with thymoquinone (21) and the EO of N. sativa, respectively. However, despite the results showing the high anti-Leishmania potential of thymoquinone (21), this coumpound was more cytotoxic compared to EO (Mahmoudvand et al. 2015a).

Forty-four compounds were detected through GC–MS in the EO of Pluchea carolinensis; selin-11-en-4α-ol (22) (51%) was the major compound (García et al. 2017). In this study, in vitro assays for antiparasitic evaluation of the EO showed the ability to inhibit 100% of the growth of promastigote and amastigote forms of L. amazonensis at concentrations of 100 and 200 μg/mL, with a lower IC50 on amastigote (6.2 ± 0.1 μg/ mL) than promastigote (24.7 ± 7.1 μg/mL) forms. In in vivo models of cutaneous leishmaniasis in BALB/c mice, no mortality or weight loss was observed in the treated groups. The administration of the EO of P. carolinensis demonstrated to control the size of the lesions and parasite load of animals infected with L. amazonensis. The authors of the work suggest that the results found in vitro and in vivo on the anti-Leishmania effect of EO may be due to the major compound selin-11-en-4α-ol (22), but indicate the need to reiterate analyses with the isolated compound to elucidate its mechanism of action.

Because the intracellular forms of Leishmania species complete part of their cell cycle inside macrophages, it is important to establish the selectivity index (SI) of the EO and its components (Moreira et al. 2019). More toxic compounds must be more selective for protozoa than host cells. SI values greater than 1 are considered more selective for activity against parasites, and values lower than 1 are considered more selective for activity against cells.

In their studies, Moreira et al. (2019) established the SI ratio for the EO of Casearia sylvestris SW. and its major compound (22.2%) E-caryophyllene (23), with values of 2.9 and 5.8, respectively. This was an interesting result, as both were moderately toxic against BALB/c mouse macrophages. The EO presented an IC50 of 29.8 μg/mL on L. amazonensis promastigotes, better than the result for E -caryophyllene (23) (49.9 μg/mL). On amastigote forms, E-caryophyllene (23) had a better result (10.7 μg/mL) than the EO (14 μg/mL) (Fig. 13.2).

Fig. 13.2
figure 2figure 2

Structural representation of the compounds presented in this section

Full size image

4.3 Mechanisms of Action

4.3.1 Morphological Changes

Chemical analyses revealed 97.9% of 𝛼-bisabolol (24) in the constitution of the EO of Vanillosmopsis arborea (Colares et al. 2013). The compound and the EO showed efficiency in inhibiting the growth of L. amazonensis promastigotes with IC50/24 h of 4.95 𝜇g/mL and 7.35 𝜇g/mL, respectively. The parasites showed alterations such as severe cell damage with loss of 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. In addition, the SI, especially for intracellular amastigotes, showed that the compound (9383) was less toxic than the EO (11,526) (Colares et al. 2013). The apoptotic mechanism can be seen in Fig. 13.3.

Fig. 13.3
figure 3

The apoptotic mechanism. (1) The products pass through the parasite membrane passively, due to their lipophilic characteristics; (2) Then, several authors have observed that volatile compounds act by inhibiting the cell cycle in the G0/G1 phase; (3) Phenotypic alterations, such as DNA fragmentation , chromatin condensation and nuclear disorganization , have also been reported, whose images can be found in the studies mentioned in this section; (4) Depolarization of the mitochondrial membrane, which plays a crucial role as a therapeutic target in protists such as Leishmania, is the main mechanism promoted by these compounds. (5) Together, these pathways share characteristics responsible for the release of factors that activate the apoptosis cascade, which despite being a programmed process, is the main form of cell death induced by chemical agents

Full size image

The above results corroborate the findings of Hajaji et al. (2018), in which 𝛼-bisabolol (22) isolated from the EO of Matricaria recutita L. showed SI values of 5.5 and 6.7 for L. amazonensis and L. infantum amastigotes, respectively, and IC50 of 16.0 ± 1.2 and 9.5 ± 0.1 μg/mL on L. amazonensis and L. infantum promastigotes , respectively. The researchers demonstrated the ability of the compound to affect plasma membrane permeability without causing necrotic effects, and to activate a programmed cell death process by cellular enhancement of phosphatidylserine externalization and membrane damage, with an apoptosis percentage of 21.66 (IC50) and 40% (IC90) for L. amazonensis and 17 (IC50) and 20% (IC90) for L. infantum after 24 h of treatment.

The EO of Cryptocarya aschersoniana was rich in limonene (5) (42%) and had remarkable activity against L. amazonensis promastigotes (IC50 = 4.46 μg/mL) in the study by Andrade et al. (2018b). However, it was highly toxic to mouse macrophages, with a CC50 of 7.71 μg/mL. According to the authors, compounds with CC50 below 10 μg/mL are highly toxic, above 10 and below 100 μg/mL are moderately toxic, and above 100 and below 1000 μg/mL are non-toxic. This type of classification allows evaluating the cytotoxicity of a compound and understanding the mechanisms of action of different substances in their interactions with tissues. The authors recognized that, as this was an in vitro test, it did not replicate the actual architecture of the living tissue in which the underlying cells could repair the damage suffered (Andrade et al. 2018b).

The EO from Vernonia brasiliana (L.) Druce was rich in terpenes, with the major component being β-caryophyllene (11) (Mondêgo-Oliveira et al. 2021). The EO showed activity against L. infantum promastigotes, with IC50 of 39.01 μg/mL and SI of 1.61, being more toxic to parasites than to DH82 cells. Although the IC50 of the standard drug miltefosine was higher (2.54 μg/mL), it was more toxic to DH82 cells, with an SI of 0.55. When tested in combined therapy, there was an antagonistic effect. According to the author, this shows that although both products are bioactive against Leishmania, this does not mean that the products will act synergistically. The mechanisms of action of V. brasiliana EO were tested and, after 72 hours in contact with L. infantum promastigotes at IC50 of 39.01 μg/mL, important structural changes were observed, with decreased mitochondrial membrane potential and increased reactive species of oxygen (ROS) production, inducing a late apoptosis.

Although little research has been carried out to identify the mechanisms of action by which EOs and their constituents act, a general analysis of the findings suggests disturbances in the plasma membrane of Leishmania causing significant morphological alterations that can induce apoptosis. In the work by Machado et al. (2012a), C. citratus EO induced the death of L. infantum promastigotes in which depolarization of the mitochondrial potential was observed, involving cell-cycle arrest at the G0/G1 phase and nuclear disorganization, with chromatin condensation. In a study by Aloui et al. (2016), A. campestres presented β-pinene (10) (32.95%) and was active against L. infantum promastigotes (IC50 = 44 μg/mL). Furthermore, the EO increased the proportion of cells in the subG0/G1 phase, indicating DNA degradation in promastigotes, suggesting alterations of the apoptotic type.

The EO of Myrcia ovata caused growth inhibition of L. amazonensis, with a considerable difference at 20 and 30 mg/mL compared to the untreated control (Amorim Gomes et al. 2020). Both concentrations caused 100% inhibition with IC50/96 h of 8.69 mg/mL. The authors observed that after incubation for 3 days with 10 mg/mL of EO, the parasites showed accumulation of lipid bodies, nucleolus disorganization, and the appearance of structures suggestive of autophagosome; and after 4 days of treatment with 5 mg/mL, the parasites showed mitochondrial enlargement (Amorim Gomes et al. 2020). This effect was attributed to the main constituents of the EO geranial (16) and neral (15). The effect of citral (14), which is a mixture of geranial (16) and neral (15) isomers, already tested on L. amazonensis, caused ultrastructural changes that included mitochondrial damage and presence of two or more flagella in the parasites, among other effects (Santin et al. 2009).

Neral (15) (cis-citral) and geranial (16) (trans-citral) together represented about 81% of the EO of C. citratus, which was able to kill 65% of L. infantum and L. major promastigotes and 80% of L. tropica promastigotes at a concentration of 50 μg/ml (Machado et al. 2012a). In turn, at the same concentration, citral (14) killed about 45% of L. infantum and L. tropica promastigotes, and about 60% of L. major promastigotes. Furthermore, none of them showed cytotoxicity in bovine aortic endothelial cells and macrophage lineage in the MTT test (Machado et al. 2012a).

The investigations of Sen et al. (2010) showed that promastigotes treated with EO and citral (14) showed prominent ultrastructural effects such as the appearance of aberrant-shaped cells with cell body septation, cytoplasmic disorganization , increased cytoplasmic clearance and loss of intracellular content, presence of autophagosomal structures, characterized by intense cytoplasmic vacuolization, in addition to irregular surface with blebs formation and rupture of the membrane. Another factor highlighted is the presence of membrane vesicles in the flagellar pocket, characteristic of an exocytosis process, and it is possible that they resulted from the secretion of abnormal lipids, which accumulate as a consequence of the effect of citral (14). Cymbopogon citratus EO and citral (14) further promoted sustained mitochondrial membrane depolarization, which is a typical feature of metazoan apoptosis and has been observed to play a key role in drug-induced death in protists such as Leishmania. The authors also noted the presence of myelin-like figures as multilamellar bodies, where the nuclear chromatin was organized similarly to the nucleus of apoptotic cells, with disruption of the nuclear membrane. The authors’ main hypothesis is that EO and citral (14) may have a passive entry and accumulate in the cell membranes of the parasite, leading to an increase in membrane permeability and formation of structures known as autophagosomes (Rodrigues et al. 2002) that are probably involved in an intense process of remodeling of intracellular organelles irreversibly damaged by the EO and citral (14).

Islamuddin et al. (2014a) showed that camphor (25) (52.06%) was the major component in the chemical composition of the EO of Artemisia annua leaves. The EO exhibited IC50 of 14.63 ± 1.49 μg/mL and 7.3 ± 1.85 μg/mL against L. donovani promastigotes and amastigotes, respectively. In their evaluations, the authors reported changes in cell morphology, shrinkage in promastigotes that became round in shape, with ruptured flagella and no motility. The apoptosis mechanism was also recognized by the externalization of phosphatidylserine in the cell membrane, evidenced by increased annexin V binding. The authors also observed DNA fragmentation in apoptotic cells, showing an increased proportion of cells in the subG0/G1 phase when treated with A. annua EO. Also at the intracellular level, treatment with EO was able to cause depolarization of the parasite’s mitochondrial membrane, leading to permeabilization of the inner mitochondrial membrane and consequent release of apoptotic factors.

Monzote et al. (2014b) demonstrated that the EO of Bixa orellana presented activity against the intracellular amastigote form of L. amazonensis, with IC50 of 8.1 μg/mL and SI of 7, and cytotoxic concentration sevenfold higher for the host cells than for the parasites. The EO also showed the ability to control the progression of established cutaneous leishmaniasis in BALB/c mice, with significant differences in lesion size and parasite load between animals treated with EO compared to controls, with no deaths observed after 14 days of application intraperitoneal of the EO. According to the authors, the geranylgeraniol (26) present in the composition of the EO (9.1%) may be associated with such activity, since it has been reported that this compound promotes alterations in the mitochondrial structure, including swelling and formation of circular cristae (Vannier-Santos and Castro 2009). In addition, the compound has also been observed to cause kinetoplast DNA disorganization (Vannier-Santos and Castro 2009) as well as increased superoxide anion production, leading to apoptosis (Lopes et al. 2012).

In the study of the antiparasitic action of the EO of Lavandula luisieri, Machado et al. (2019) observed an effect on cell viability in promastigotes of L. infantum, with IC50/24 h equal to 63 μg/mL, L. tropica, with IC50/24 h equal to 38 μg/mL, and L. major, with IC50/48 h equal to 31 μg/mL. In the MTT test, no toxicity was observed at the doses tested (CC50 > 200 μg/mL; SI > 3.17). The authors suggest that the action of the EO is linked to oxygenated monoterpenes (75.7%) in its chemical composition, and necrodane derivatives as major compounds (36%). The effects of the EO were verified from image analysis in Scanning Electron Microscope (SEM) and Transmission Electron Microscopy (TEM) , in which round and aberrant shapes, cell body septation, disorganization of cytoplasmic organelles, and many autophagosomal structures featured by intense cytoplasmic vacuolization were observed in L. infantum promastigotes. The EO was able to induce mitochondria swelling and mitochondrial membrane disorganization indicated by the presence of complex invaginations and formation of concentric membranous structures. These data can be explained by the ability to induce depolarization of the mitochondrial potential, which can promote apoptosis (Arnoult et al. 2002). The arrest of cells in the G0/G1 phase was also detected, with a reduction in the number of cells in the S and G2/M phases; the authors suggested that this may have occurred due to an decrease in mitochondrial membrane potential and since this reduces the energy available.

The analysis of the EO of Eremanthus erythropappus conducted by Amorim Gomes et al. (2020) revealed the presence of 13 constituents, corresponding to 94.22% of its composition, with 85.98% of α-bisabolol (22). The authors verified a percentage of inhibition of L. amazonensis promastigotes of 35% under concentrations of 5 and 10 mg/mL of E. erythropappus EO, and almost 100% inhibition using concentrations higher than 20 and 30 mg/mL after 96 h of treatment, with IC50/96 h of 9.53 mg/ml. The ultrastructural analysis showed that after 3 days of incubation with 10 mg/mL of EO, the parasites showed accumulation of lipid bodies, demonstrating a possible mechanism of action of the compound.

De Medeiros et al. (2011) also pointed out that the treatment with L. sidoides EO induced remarkable changes in the morphology of the parasites , particularly the accumulation of large lipid droplets in the vicinity of the plasma membrane. At high EO concentrations, membrane disruption, increased lipid electron density, and loss of cytoplasmic content, alterations compatible with loss of cell viability and cell death by necrosis (Menna-Barreto et al. 2009), were also observed. Furthermore, characteristics such as parasite swelling, presence of wrinkled or ruptured membranes, and loss of cytoplasmic material in promastigotes were present, supporting the deleterious effects of EO on the plasma membrane so widely disseminated in the literature. The hypothesis of the authors is that the constituents of the EO penetrate into the cell and impair the ergosterol biosynthesis pathway, and they may also react directly with the membrane through their reactive hydroxyl portion. Thus, the extensive membrane damage may be due to a combined effect of the two events (Nafiah et al. 2011).

Subsequently, Monzote et al. (2014a) demonstrated that NADH- and succinate-dependent reduction of cytochrome-C was inhibited in mitochondrial fractions of L. amazonensis and liver mitochondria from BALB/c mice in the presence of C. ambrosioides EO and its pure major compounds, carvacrol (12) and thymol (18).

Their findings suggested that such reduction was not specifically sensitive to EO in Leishmania mitochondria, however, the existence of other more sensitive and more selective targets, such as mitochondrial membrane potential, was not ruled out. The authors could not establish whether the loss of mitochondrial membrane potential was a primary effect of EO (directly influencing mitochondrial functions) or arose subsequent to other cellular effects triggering apoptosis via mitochondria. Furthermore, they suggested that other parasite damages caused by EO such as free radical-triggered DNA or protein-alterations, or parasite-specific transporters such as the P2 amino-purine transporter (De Koning 2001), DNA triggered by free radicals or protein alterations, or parasite-specific transporters, such as the P2 amino-purine transporter (De Koning 2001), could contribute to specific killing of Leishmania.

Tasdemir et al. (2019) performed tests with both carvacrol (12) and thymol (18), the main constituents of the EO of Origanum onites, reporting for the first time their effect on L. donovani amastigotes. The authors suggested that the EO permeates the cell membrane and kills parasites by affecting the cytoplasmic metabolic pathways or organelles, and not by compromising the integrity of the parasite’s membrane, as presented by several studies in this section. They reached this conclusion based on a flow cytometry study performed by Santoro et al. (2007) and also highlighted the importance of the presence of the hydroxyl group in the bioactivity of phenolic compounds such as carvacrol (12) and thymol (18) (Dorman and Deans 2000; Ultee et al. 2002).

4.3.2 Immunological Changes

The evaluation of the EO of Pseudotrachydium kotschyi revealed the presence of Z-α-trans-bergamotol (27) (23.25%), durylaldehyde (28) (16.07%), and α-bergamotene (29) (10.48%) (Ashrafi et al. 2020a). It was observed that the EO had anti-Leishmania potential at a concentration of 5000 μg/mL and suggested that these compounds are involved in the biological activities of the oil, for it was observed that EO was able to protect macrophages against infection by promastigotes. Their data indicated that EO exerts anti-Leishmania activity by affecting the levels of TNF-α and TGF-β1 in macrophages. These cytokines were determined in Leishmania-infected macrophages after treatment with EO. The immunological mechanism can be seen in the Fig. 13.4.

Fig. 13.4
figure 4

Modulation of the immune response by essential oils and their compounds. (1) Parasitophorous vacuoles (PV) contain a plasma membrane and may represent a specific adaptation to minimize the toxic effects of reactive nitrogen intermediates generated by the host cell. (2) The modulation of the response occurs in cells with a T-helper type 1 (Th1) cytokine profile. (3)This profile is associated with the production of cytokines such as IFN-γ, IL-12 and TNF. (4) These cytokines lead to the activation of anti-Leishmania activities, mainly through the activation of the inducible nitric oxide synthase (iNOS) enzyme. (5) The production of nitric oxide (NO) induces an oxidative explosion in infected cells. (6) This oxidative explosion is associated with the process of loss of the parasite’s mitochondrial membrane potential, however, it can also cause extensive nuclear DNA fragmentation in axenic and intracellular amastigotes

Full size image

The EO of Artemisia absinthium inhibited the in vitro growth of L. amazonensis promastigotes and amastigotes, with IC50 of 14.4 ± 3.6 μg/mL and 13.4 ± 2.4 μg/mL, respectively (Monzote et al. 2014c). The activity in vivo was evaluated in a model of cutaneous leishmaniasis in BALB/c mice, where control of lesion size and parasite burden was observed. Furthermore, no evidence of mortality in the treated groups or weight loss greater than 10% were observed during the study. The authors suggested that Artemisia EO may improve Th1 immune responses and microbicide activation of macrophages.

Nunes et al. (2021) found 69.76% of hydrocarbon sesquiterpenes in the EO of Eugenia piauhiensis Vellaff. (Myrtaceae), with 23.5% being γ-elemene (30) and 11.94% (E)-β-caryophyllene (23). The EO and the isolated compound γ-elemene (30) presented greater activity against amastigote (EC50 = 4.59 ± 0.07 μg/mL and 8.06 ± 0.12 μg/mL, respectively) than promastigote (IC50 = 6.43 ± 0.18 μg/mL and 9.82 ± 0.15 μg/mL, respectively) forms of L. amazonensis. The authors suggested that this difference could be indicative of immunomodulatory activity and macrophage activation, as experiments of macrophage infection models in vitro revealed increased levels of TNF-α, IL-12, NO, and ROS in the supernatant of L. amazonensis-infected macrophages, suggesting an activation of the Th1 (not Th2) profile, a mechanism that has been the objective of anti-Leishmania drugs.

In the work by Carvalho et al. (2017), the EO of Myracrodruon urundeuva , rich in myrcene (6,7) (α-myrcene (6) 37.23% and β-myrcene (7) 42.46%), caused morphological changes such as cells with rounded or completely spherical shapes, with the presence of cell debris, typical of cell lysis. Furthermore, the results obtained against both forms of L. amazonensis (IC50 = 205 μg/mL for promastigotes; 104.5 μg/mL for axenic amastigotes; 44.5 μg/mL for intracellular amastigotes) suggest an increase in the phagocytic capacity of macrophages. According to the authors, this increase can be triggered by immunomodulatory mechanisms. One way to assess this activity is by determining the NO content. NO production is stimulated by protective cytokines, such as IFN-γ, and is extremely reactive, causing damage to the parasite’s proteins and DNA. However, their tests with the EO of M. urundeuva did not promote NO production, suggesting that phagocytosis was not stimulated by immunomodulatory mechanisms.

In line with the immunomodulator role of NO, Jihene et al. (2020) showed that Leishmania-infected macrophages produced 36.8% more NO than uninfected ones. Furthermore, uninfected macrophages treated with 14.76, 7.38 and 3.69 μg/mL of propolis EO produced 50.4%, 38.1% and 25% respectively more NO than control cells. Macrophages infected and treated with EO showed a significant increase in NO levels, reaching 230% at the highest concentration.

The EO of Nectranda hihua , composed mainly of sesquiterpenes (89%), especially bicyclogermacrene (31) (28.1%), showed activity against intracellular L. infantum amastigotes (IC50 = 0.2 ± 1.1 mg/mL). The SI values ​​were 249.4 and 149.0 for murine fibroblasts and macrophages, respectively, reflecting the oil’s highly selective action on amastigote forms. The EO of Nectranda gardneri was active in intracellular amastigotes of L. infantum and L. amazonensis (IC50 = 2.7 ± 1.3 and 2.1 ± 1.06 mg/mL, respectively), with low cytotoxicity. This EO was also composed mainly of sesquiterpenes (85.4%), with intermediol (32) being the main component (58.2%) (Bosquiroli et al. 2017). The authors observed that the EO of the two species induced a significant increase in NO production by L. amazonensis-infected cells, however, in the case of L. infantum, only the EO from N. gardneri was active, suggesting that the anti-Leishmania activity of the EO may be associated with this important mechanism (Olekhnovitch and Bousso 2015).

Bosquiroli et al. (2015) demonstrated the inhibition of proliferation of intracellular amastigotes 24 h after the EO of Piper angustifolium was added to infected cells. The infection rate decreased in a range of 88.1 to 100% from the lowest to the highest concentration, with an IC50 of 1.43 μg/mL for L. infantum and low cytotoxicity for mammalian cells compared to amphotericin B , although the latter is more active. A significant increase in NO release was found after treatment with the EO at concentrations of 6.25 and 12.5 μg/mL; however, at concentrations of 25 and 50 μg/mL, the EO did not induce a significant increase in NO release, showing an atypical result that may be due to the presence of certain compounds in the EO.

The EO of Curcuma longa expressed anti-Leishmania action against promastigote and amastigote forms of L. amazonensis (Teles et al. 2019). The concentration of 125 μg/mL generated a decrease of 80.73% of promastigote and 40.75% of amastigote forms in infected cells. In terms of possible mechanisms of action, the authors evaluated the production of nitrite, an indirect measure to quantify NO. They found that the EO inhibited the production of NO in macrophages. Thus, the authors suggested the existence of other possible mechanisms involved in the activity of C. longa EO against intracellular amastigotes yet to be investigated.

4.3.3 Antioxidants

Since the loss of membrane balance can lead to the entry of ions into the cells, causing polarization changes; verifying the antioxidant capacities of EOs may serve to detetct this activity. According to Bouyahya et al. (2017b), antioxidant tests serve to express mechanisms of action involving polarization and chemical behavior in the presence of the product being tested.

Ahmed et al. (2011) found the compound camphor (25) (13.82%) in the composition of the EO of Thymus hirtus sp. Algeriensis and verified its anti-Leishmania activity. They found an IC50 of 0.43 μg/mL for L. major promastigotes and 0.25 μg/mL for L. infantum promastigotes. The composition and anti-Leishmania activity of the EO of Ruta chalepensis was investigated in the same study, highlighting the presence of 84.28% of 2-undecanone, and inhibitory action only against L. infantum promastigotes. Their tests to assess antioxidant potential through DPPH free radical scavenging showed a low antioxidant power for the EO, suggesting that anti-Leishmania activity was not correlated with antioxidant activity of the EO.

High concentrations of camphor (25) (36.82%) and compounds such as 𝛼-thujone (33) (7.65%) and 𝛽-thujone (8) (7.21%) were found in the EO of A. herba-alba (Aloui et al. 2016). The EO was tested against promastigote forms of L. infantum, revealing inhibitory power with an IC50 of 68 𝜇g/mL. Antioxidant capacity by DPPH radical scavenging, with an IC50 of 9.1 mg/mL, and intense reducing capacity by the of ferric reducing antioxidant power (FRAP) assay, with a result of 27.48 mM Fe2+, were also observed. The effect of the EO on the cell membrane assessed through measurement of lactate dehydrogenase showed no induction of cytolysis even after prolonged incubation time (72 h). Flow cytometric analysis of L. infantum promastigotes detected DNA degradation by the increase in the proportion of cells in the sub-G0/G1 phase between the applied doses, followed by a decrease in the number of cells in the S and G2/M phases. Annexin V/7-ADD staining showed that treatment with the EO caused the parasites to express apoptotic profiles without inducing necrosis.

4.3.4 Enzymatic Activity

According to the study by Marques et al. (2011), the EO of Piper claussenianum leaves was rich in sesquiterpenes , with nerolidol (34) being the major component (81%), and caused 62.17% inhibition in the levels of arginase activity. Pretreatment of L. amazonensis promastigotes with the EO reduced the percentage of macrophage infection by 42.7%, and the treatment of already infected macrophages promoted a reduction of 31.25% of the infected cells. Cytotoxicity of the EO in macrophage and fibroblast cell lines was absent at concentrations ranging from 40 to 0.56 mg/mL. The authors also performed treatment with the EO of P. claussenianum and INF-γ together, which provided an increase in NO production of 20.5% in cells infected with Leishmania. Such production was considered by the authors as a useful strategy for infection control by inhibiting arginase activity levels in the parasite.

In parasites of the genus Leishmania, arginase activity is essential for the growth of the protozoans (Vincendeau et al. 2003; Roberts et al. 2004) in addition to being associated with cytotoxic processes and immunological mechanisms due to the role in NO synthesis (Kanyo et al. al. 1996; Da Silva et al. 2002). Thus, arginase activity is a potential target of anti-Leishmania pharmacological compounds.

Oxygenated monoterpenes , especially 1,8-cineole (9) (23.6%) and camphor (25) (18.7%), were predominant in the EO of Rosmarinus officinalis L. (Bouyahya et al. 2017c). In chemical analyses of the EO of Melaleuca leucadendra L. (Myrtaceae), there was 61% of 1,8-cineole (9) (Monzote et al. 2020b). In their assays with L. amazonensis, the authors demonstrated that 1,8-cineole (9) had an IC50 value ​​of 68.3 ± 3.4 μg/mL and no cytotoxicity against macrophages at 200 μg/mL. Despite this, the authors did not associate the antiprotozoal activity to the compound, suggesting that the activity of the EO may result from complex interactions between its constituents, and that even components in smaller amounts can play a critical role.

In a computational analysis of the structure and binding of 1,8-cineole (9) isolated from Croton nepetifolius EO in relation to the enzyme L. infantum trypanothione reductase (LiTR), in the structural representation of LiTR coupled to 1,8-cineole (9), favorable interactions of different types were formed, as Van der Waals, hydrophobic and hydrogen bonds, with participation of 7 residues (Gly197; Tyr221; Arg222), and the ligand established H bonding interaction with Gly196 within a radius of 3.68 Å (Morais et al. 2019). Turkano et al. (2018) demonstrated the RT inhibition of the compound 2-(diethylamino)ethyl 4-((3-(4-nitrophenyl)-3-oxopropyl)amino)benzoate with the participation of the residues Tyr221, Gly197, ​​Asn254, Arg222, and Arg228, which are essential for LiTR inactivation, suggesting a possible mechanism of action against the Leishmania species tested.

4.4 Other Compounds Present in Essential Oils

Although most results point to terpenes as the main constituents present in EOs, other compounds, such as phenylpropanoids, have shown strong anti-Leishmania activity. One of the main representatives of this class is eugenol (35).

In a research carried out by Moemenbellah-Fard et al. (2020), 33 components were identified in the EO of Syzygium aromaticum , and among the main ones, eugenol (35) (65.41%), trans-caryophyllene (12.06%), eugenol acetate (9.85%), and caryophyllene oxide (2) (3.0%) stood out. The EO and eugenol (35) were tested as for their antiparasitic activity against L. major promastigotes, reaching IC50 values of 654 μg/mL and 517 μg/mL, respectively, and against L. tropica promastigotes, with IC50 of 180 μg/mL and 233 μg/mL, respectively.

In the studies by Islamuddin et al. (2013), the EO of S. aromaticum revealed a concentration of 59.75% of eugenol (35) and 29.24% of eugenyl acetate (36). The authors found an anti-Leishmania effect against intracellular promastigote and amastigote forms of L. donovani, with IC50 of 21 mg/mL and 15.24 mg/mL, respectively. In this study, it was indicated that EO-induced cell death occurred due to loss of membrane integrity, with evidence indicating late apoptosis. The authors also reported that EO-treated promastigotes exhibited a hypodiploid peak in subG0/G1, and the parasites presented reduced DNA content, thus confirming the occurrence of DNA fragmentation and induction of apoptosis. It is noteworthy that the mechanisms of action presented were similar to those presented by terpene-rich EOs.

Analysis of the EO of Ocimum gratissimum identified the presence of 86.5% of eugenol (35) (Le et al. 2017). This EO had its anti-Leishmania activity against L. mexicana tested using concentrations of 25 and 50 nL/mL, with IC50 of 4.85 nL/mL. Cytotoxicity tests showed survival of more than 80% of the analyzed mammalian cells after 72 hours, at the maximum concentration used (Le et al. 2017).

Methyl-eugenol (37) was reported as the major compound (33.89%) of the EO of C. nepetifolius, followed by E -caryophyllene (23) (21.23%) and 1,8-cineole (9) (10.44%). According to Morais et al. (2019), these compounds were likely responsible for the anti-Leishmania activity of the EO at concentrations of 100, 50, 25, 12.5, and 6.25 μg/mL against L. amazonensis (IC50 = 9.87 ± 2.21 μg/mL) and L. braziliensis (IC50 = 9.08 ± 2.59 μg/ml). In addition, at the concentration of 100 μg/mL, the EO presented toxicity against macrophages statistically similar to amphotericin B. It is important to note that, although the largest fractions of these EOs are phenylpropanoids, there are terpenes in considerable concentrations present in their composition.

This is the case of the EO of leaves of Scheelea phalerata Mart. ex Spreng (Arecaceae). The EO had phytol (38) as a major compound in percentages of 36.7% and 26.1% in plants collected in the dry and rainy seasons, respectively; the EO extracted in the rainy season also presented 18.7% of palmitic acid (39), as found in the work of Oliveira et al. (2020). Nevertheless, only the EO extracted in the rainy season had an effect against L. amazonensis promastigotes (IC50 = 165.05 ± 33.26 μg/mL). The authors suggested the role of compounds produced in this season in the inhibitory effect on parasites, emphasizing a synergistic action between the main components of the EO, phytol (38) and palmitic acid (39), since the EO extracted during the dry season showed a higher concentration of phytol (38) but no anti-Leishmania activity. Another hypothesis addressed in the study was based on the possibility that other compounds present in the EO are capable of altering the activity of phytol (38) by the formation of compounds, promoting the inactivation of the molecule.

The compound methyl chavicol, also called estragole (40), was found in the EO of Tagetes lucida Cav., constituting approximately 97% of the oil. The EO was tested against L. tarentolae promastigotes, resulting in an IC50 of 61.4 ± 2.4 μg/mL, and against L. amazonensis promastigotes, with an IC50 of 118.8 ± 1.2 μg/mL. Estragole (40) proved to be more effective than the EO, with an IC50 of 28.5 ± 1.0 μg/mL and 25.5 ± 3.3 μg/mL for L. tarentolae and L. amazonensis, respectively (Monzote et al. 2020a). The authors observed that the EO promoted inhibition of oxygen consumption in L. tarentolae at the maximum tested concentration of 100 μg/mL; however parasites treated with estragole (40) remained with normal oxygen consumption, suggesting that the EO targets the mitochondria of protozoa. Furthermore, estragole (40) was able to cause mitochondrial rupture. The authors suggested that the molecule acts as a mitochondrial uncoupler, although it is only a weak inhibitor of mitochondrial electron transfer in Leishmania.

4.5 Other Applications

Other forms of application for EOs have been explored, as in the case of EO eluted in nanoemulsions and nanogel. These mixtures can be used topically, improving the pharmacodynamic profiles of the product. In the study by Ghanbariasad et al. (2021a), the EO from Citrus sinensis, whose major compound was limonene (5) (71.26%), was used against L. tropica and L. major promastigotes, and IC50 values of 151.13 μg/mL and 108.31 μg/mL, respectively, were observed. Then, the nanogel based on C. sinensis nanoemulsion was prepared to improve its stability. According to the author, the advantage of converting nanoemulsions into nanogels is the increase in viscosity, which promotes the accumulation of the solution and improves the hydration of the application site. The nanometric dispersion of the EO and the better hydration lead to better penetration of the EO in to the locality. It is suggested that this type of application could also prevent the entry of environmental pathogens into the lesion, reducing the chance of secondary infection. In tests, the viability against L. major and L. tropica was reduced to less than 10% when used at a concentration of 9.15 mg, which was a better result than that obtained with EO alone in topical application (Ghanbariasad et al. 2021a).

4.6 Perceptions, Conclusions and Perspectives

Although EOs are presented as important candidates in the search for new anti-Leishmania drugs, we observed that some steps are still needed, especially considering that most studies did not perform the in vivo analyses necessary to identify the main characteristics of the compounds (bioavailability, pharmacokinetics, pharmacodynamics etc.) in new pharmacological approaches. The investigation of compounds in in vivo assays is essential to leverage new therapeutic hypotheses, since many compounds are discarded for not showing results in vivo or in vitro, as discussed by Brito et al. (2013). However, the authors emphasize that the mechanisms of action and interaction of drugs in humans are often discovered after their indication and use.

Another important highlight is that the evaluations presented in this section used the promastigote form to screen the most prominent compounds, probably due to handling, cost and duration of the tests. However, it is important to mention that studies conducted with amastigotes cultivated in macrophages are considered the best choice for evaluating the potential of the compounds in initial evaluation models, although, in experimental stages of sandflies, for example, there is no difference between promastigotes and amastigotes as to the development of the infection, as observed by Fampa et al. (2021) in L. donovani. This condition is important, considering that the morbidity and mortality associated with Leishmania is caused by this evolutionary form (Brito et al. 2013).

This question is evident in the studies by Tasdemir et al. (2019) who found discrepancies between the efficacy of thymol (18) and carvacrol (12) in vitro and in vivo, with reduced effects in animals. The authors attributed this result to non-ideal pharmacokinetics and physicochemistry, such as very fast absorption, low solubility, low bioavailability and elimination rate, considered the main obstacles in the development of drugs from the EO and its volatile components (Wang et al. 2009; Nagoor Meeran et al. 2017).

Despite the importance of the initial investigation of compounds, it is important to mention that some authors leave clues about the steps to follow after their studies, through the elucidation of some mechanisms of action. They cited, for example, the release of NO or the observation of the ultrastructural effects of compounds on the parasites. Although there are cost and equipment limitations, it is important to set a path for future investigations of active substances, minimizing secondary studies aimed at screening mechanisms, which are important due to the phenotypic and genotypic differences presented by the Leishmania species used in the bioassays.

Another alternative is presented by Andrade-Ochoa et al. (2021) who, based on the varied chemical structures and biological activities exhibited by the compounds, suggested the use of in silico methodologies to identify different therapeutic targets for EO constituents. Analyses performed by Ogungbe and Setzer (2013) provided evidence of the interaction of different structural types of terpenoids with certain targets in Leishmania that may support new phytochemical investigations and synthetic modifications in compounds or the synthesis of new antiparasitic structures.

It is possible to conclude that the anti-Leishmania activity of EOs stems from to the lipophilic character of their constituents, such as terpenes and phenylpropanoids, which can passively cross the membranes and disturb the osmotic balance of the cells. This may partly explain why many of the EOs have a certain degree of toxicity for mammalian cells. Given the few studies that have tested the mechanisms of action of EOs, research aimed at elucidating these bioactivities is necessary.