In-Vitro Evaluation of 52 Commercially-Available Essential Oils Against Leishmania amazonensis

Leishmaniasis is a neglected tropical disease caused by members of the Leishmania genus of parasitic protozoa that cause different clinical manifestations of the disease. Current treatment options for the cutaneous disease are limited due to severe side effects, poor efficacy, limited availability or accessibility, and developing resistance. Essential oils may provide low cost and readily available treatment options for leishmaniasis. In-vitro screening of a collection of 52 commercially available essential oils has been carried out against promastigotes of Leishmania amazonensis. In addition, cytotoxicity has been determined for the essential oils against mouse peritoneal macrophages in order to determine selectivity. Promising essential oils were further screened against intracellular L. amazonensis amastigotes. Three essential oils showed notable antileishmanial activities: frankincense (Boswellia spp.), coriander (Coriandrum sativum L.), and wintergreen (Gualtheria fragrantissima Wall.) with IC50 values against the amastigotes of 22.1 ± 4.2, 19.1 ± 0.7, and 22.2 ± 3.5 μg/mL and a selectivity of 2, 7, and 6, respectively. These essential oils could be explored as topical treatment options for cutaneous leishmaniasis.


Introduction
Leishmaniasis is a collection of parasitic diseases caused by several Leishmania species [1,2]. The disease is transmitted by several members of Phlebotominae sand flies. The genera Lutzomyia (New World) and Phlebotomus (Old World) are the only hematophagous vectors of the diseases. Leishmaniasis is considered by the World Health Organization to be a neglected tropical disease [3]. Depending on the Leishmania species, the disease can manifest itself in three main forms: cutaneous, mucocutaneous, and visceral. There are currently around 12-15 million people worldwide infected by Leishmania spp. with an estimated 350 million people at risk of acquiring leishmaniasis. Unfortunately, at the present time, there are no vaccines or prophylactic medicines available to prevent the disease. Current chemotherapeutic treatment options include sodium stibogluconate, meglumine antimoniate, conventional (deoxycholate) and liposomal amphotericin B, pentamidine isethionate, miltefosine and paromomycin. However, these agents suffer from severe side effects, poor efficacy, limited availability, high cost, or developing resistance [2,4]. Furthermore, treatment of leishmaniasis is often hampered by limited access to medicines and treatment options in developing countries where the disease is prevalent [5,6].
Essential oils are complex mixtures of volatile phytochemicals with numerous and varied biological properties [7,8]. Essential oils, particularly those that are readily available, may provide low cost treatment options for leishmaniasis. The antiparasitic activities of essential oils and their components have been demonstrated and previously reviewed [9,10]. Commercially available EOs have been screened for activity against a variety of human pathogens [11][12][13][14][15][16][17] as well as for cytotoxic activity [12,16]. In this work, we carried out in-vitro antileishmanial and cytotoxic screening of a selection of 52 commercially available essential oils against the promastigote form of Leishmania amazonensis and peritoneal macrophages from BALB/c, respectively. Promising antileishmanial essential oils were further screened against intracellular amastigotes.

Results and Discussion
A total of 52 commercially-available essential oils were screened for in-vitro antileishmanial activity against the promastigote form of L. amazonensis, as well as for cytotoxic activity against mouse peritoneal macrophages, the host cells for the amastigote form of the parasite. The antileishmanial and cytotoxic activities of the essential oils are summarized in Table 1. Of the essential oils tested, three showed notable antileishmanial activity and selectivity, frankincense oil (from the oleogum resin of Boswellia spp.), coriander oil (from the seeds of Coriandrum sativum L.), and wintergreen oil (from the leaves of Gualtheria fragrantissima Wall.). These three products were also active against the intracellular amastigote form of L. amazonensis ( Table 2).
The cluster analysis revealed four clusters based on biological activities ( Figure 1). Cluster 1 is made up of essential oils that showed good antileishmanial activity with little or no cytotoxicity. The very large cluster 2 can be described as essential oils that showed relatively weak antileishmanial activity and relatively weak cytotoxic activity, cluster 3 is composed of essential oils with both moderate antileishmanial activity but also with moderate cytotoxic activity, and cluster 4 is made up of essential oils that were very cytotoxic.
Likewise, the unfavorable bioactivity profile of lemongrass (Cymbopogon flexuosus (Nees) Will. Watson) essential oil in this study is likely due to the abundance of geranial (49.9%) and neral (23.4%). However, Machado and co-workers found that citral-rich C. citratus as well as citral did not exhibit cytotoxic effects on either primary bovine aortic endothelial cells or RAW 264.7 macrophage cells [28]. Similarly, Santin and co-workers found both C. citratus essential oil and citral to be antileishmanial against promastigotes (IC 50 1.7 and 8.0 µg/mL, respectively) and amastigotes (IC 50 3.2 and 25.0 µg/mL, respectively) of L. amazonensis, but with lower cytotoxicity against J774G8 macrophage cells [33].
As has been appreciated, many pure components present in the tested EOs have exhibited some level of antileishmanial activity. In fact, it is known that the compounds present in the EOs can act synergistically in mixtures, increasing the intrinsic effects of the purified components. Therefore, we suggest the potential use of EOs as mixtures. In addition, we have also taken into account that the tested EOs are commercially available in their present forms, reducing the cost of a therapeutic alternative, which constitutes one of main drawbacks of current antileishmanial treatments. The notable antileishmanial effects and moderate cytotoxicities in the case of mixtures of compounds could be further explored in animal models of cutaneous leishmaniasis by intralesional or topical routes.

Essential Oils
The essential oils were obtained from commercial sources, dōTERRA International (Pleasant Grove, UT, USA), Ameo Essential Oils (Zija International, Lehi, UT, USA), Mountain Rose Herbs (Eugene, OR, USA), or Albert Vielle (Vallauris, France) and were previously analyzed by gas chromatography-mass spectrometry.

Parasites
Leishmania amazonensis parasites (Reference strain MHOM/77/BR/LTB0016) were kindly provided by the Department of Immunology, Oswaldo Cruz Foundation (FIOCRUZ), Brazil. The parasites were routinely isolated from mouse lesions (BALB/c mice) and maintained as promastigotes at 26 • C in Schneider's medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (HFBS) (Sigma-Aldrich, St. Louis, MO, USA) and 100 U of penicillin and 100 µg streptomycin per mL as antibiotics. Parasite cultures were passaged every 3-4 days, but no longer used after the tenth passage after isolation from mice.

In-vitro Anti-promastigote Screening
Screening against L. amazonensis promastigotes was carried out as previously described [34][35][36]. Into each well in a 96-well plate, 50 µL medium (Schneider's medium + HFBS + antibiotics) was added. Into the wells of column 2 and 7, 48 µL medium + 2 µL sample (dimethylsulfoxide solutions of essential oil, 20 mg/mL) were added. Five two-fold serial dilutions were carried out down each column to give final test concentrations of 12.5, 25, 50, 100 and 200 µg/mL. Exponentially growing parasites (4 × 10 5 ) in medium were added to each well. Dimethylsulfoxide (DMSO) served as the negative control and pentamidine (Richet, Buenos Aires, Argentina) was used as a positive control. The plates were sealed with Parafilm ® and incubated at 26 • C for 72 h. Afterward, 20 µL of a solution (5 mg/mL) of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. The plates were incubated for an additional 4 h, the supernatant was removed, and the formazan crystals were dissolved with 100 µL DMSO. The absorbance of each well was determined with a plate reader (Molecular Devices, San Jose, CA, USA) with a test wavelength of 560 nm and a reference wavelength of 630 nm from which median inhibitory concentrations (IC 50 ) were calculated [37,38]. The IC 50 values were determined from linear dose-response curves fit to the data using a linear equation model. Each test was carried out in triplicate, and the results expressed as means ± standard deviations (SD).

Mouse Peritoneal Macrophage Cytotoxicity Screening
The median cytotoxic concentrations (CC 50 ) of the essential oils on mouse peritoneal macrophages (the host cells of Leishmania parasites) were determined as previously described [34][35][36]. Macrophages were collected from the peritoneal cavities of normal BALB/c mice and suspended in ice-cold RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with antibiotics. The cells were seeded in 96-well plates (3 × 10 5 cells/well) and incubated at 37 • C for 2 h. Non-adherent cells were removed. Into the wells of column 2 and 7, 2 µL of essential oil solutions (as above) and 48 µL medium (supplemented with 10% HFBS and antibiotics) were also added. Two-fold serial dilutions down each lane were carried out to give final concentrations of 12.5-200 µg/mL. The macrophages were incubated for 72 h, after which the cytotoxicity was determined using the MTT assay (as above), using 15 µL/well. The CC 50 was calculated for each compound in the same manner as the IC 50 and selectivity indices (SI) for each essential oil were determined (CC 50 for macrophages / IC 50 for promastigotes).
Essential oils with an IC 50 < 100 µg/mL and a SI ≥ 5 were selected for further evaluation against L. amazonensis amastigotes [34].

In-vitro Intracellular Anti-amastigote Screening
Median inhibitory concentrations (IC 50 ) of the active essential oils on L. amazonensis amastigotes were carried out as previously described [34][35][36]. The peritoneal macrophages, obtained from BALB/c mice (as above), were seeded in 24-well culture plates at 10 6 cells/mL. The plates were incubated at 37 • C under a 5% CO 2 atmosphere for 2 h. Non-adherent cells were removed, and stationary-phase L. amazonensis promastigotes were added (4:1 promastigote/macrophage ratio), and incubation continued for an additional 4 h. Free parasites were removed and 1000 µL RPMI completed medium was added to each well. Into the first well, 990 µL medium and 10 µL essential oil solution were added. Four two-fold dilutions were carried out to give final concentrations of 12.5, 25, 50 and 100 µg/mL. The plate was incubated at 37 • C under a 5% CO 2 atmosphere for 48 h. The cell monolayers were fixed in absolute methanol, stained with Giemsa, and evaluated using a light microscope. The number of intracellular amastigotes was determined by counting 25 macrophages per sample. The results are expressed as percent reduction of infection rate (% infected macrophages × number amastigotes per infected macrophage) compared to that of the controls. The IC 50 values were determined from the concentration-response linear curves. Each evaluation was carried out in triplicate and the results expressed as means ± SD.

Hierarchical Cluster Analysis
The chemical compositions of the commercial essential oils along with the antileishmanial and cytotoxic activities were used in an agglomerative hierarchical cluster (AHC) analysis. The essential oil compositions of the 52 commercially-available essential oils and the bioactivities were used to determine the associations between the essential oils and their activities using XLSTAT Premium, version 2018.5.53172 (Addinsoft, Paris, France). Dissimilarity was determined using Euclidean distance, and clustering was defined using Ward's method.

Conclusions
This study has shown that the essential oils of frankincense, coriander, wintergreen, and birch have notable antiparasitic activities against Leishmania amazonensis with an acceptable SI with respect to cytotoxicity against mouse peritoneal macrophages. However, essential oils are complex mixtures of compounds, and the biological activities cannot necessarily be attributed to individual components; there are apparent synergistic and/or antagonistic interactions as well. Nevertheless, essential oils containing appreciable concentrations of α-pinene, linalool, or methyl salicylate should be investigated for antiparasitic activity.