Chemical Composition and In Vitro and In Silico Antileishmanial Evaluation of the Essential Oil from Croton linearis Jacq. Stems

Croton linearis Jacq. is an aromatic shrub that has been utilized in traditional medicine in the Bahamas, Jamaica, and Cuba. Recent studies have revealed the antiprotozoal potential of its leaves. The present work is aimed to identify the volatile constituents of essential oil from the stems of C. linearis (CLS-EO) and evaluate its in vitro antileishmanial activity. In addition, an in silico study of the molecular interactions was performed using molecular docking. A gas chromatographic–mass spectrometric analysis of CLS-EO identified 1,8-cineole (27.8%), α-pinene (11.1%), cis-sabinene (8.1%), p-cymene (5.7%), α-terpineol (4.4%), epi-γ-eudesmol (4.2%), linalool (3.9%), and terpinen-4-ol (2.6%) as major constituents. The evaluation of antileishmanial activity showed that CLS-EO has good activity on both parasite forms (IC50Promastigote = 21.4 ± 0.1 μg/mL; IC50Amastigote = 18.9 ± 0.3 μg/mL), with a CC50 of 49.0 ± 5.0 μg/mL on peritoneal macrophages from BALB/c mice (selectivity index = 2 and 3 using the promastigote and amastigote results). Molecular docking showed good binding of epi-γ-eudesmol with different target enzymes of Leishmania. This study is the first report of the chemical composition and anti-Leishmania evaluation of CLS-EO. These findings provide support for further studies of the antileishmanial effect of this product.


Introduction
Neglected tropical diseases (NTDs) are a major public problem in the health systems of many countries, causing significant morbidity and mortality. Among these, leishmaniasis is a parasitic disease caused by an obligate intracellular parasite of genus Leishmania, which is transmitted to humans by the bite of infected female phlebotomine sandflies. This disease has wide clinical spectra, with three main forms being recognized: visceral (also known as kala-azar, which is the most severe form of the disease), cutaneous (the most common), and mucocutaneous [1,2].

Antileishmanial Activity and Cytotoxicity
In the present study, the effect of CLS-EO was evaluated against the promastigote and amastigote forms of L. amazonensis for the first time. Additionally, the cytotoxicity was assayed on peritoneal macrophages from BALB/c mice. CLS-EO showed good inhibitory activity against both parasite forms of L. amazonensis, with IC 50 values of 21.4 ± 0.1 µg/mL and 18.9 ± 0.3 µg/mL, respectively (Table 2). However, CLS-EO exhibited toxic effects on peritoneal macrophages from BALB/c mice, and the calculated SI indicates low selectivity of this product ( Table 2).

Molecular Docking
After the in vitro evaluation, a molecular docking study was performed, aiming to predict the effects of the main compounds of CLS-EO ( Figure 2) on several important enzymatic targets of Leishmania. The docked poses for each compound were evaluated, selecting the ones with the lowest ∆G dock (Table 3). After docking score normalization to account for molecular weights was carried out, trypanothione synthetase (TryS) and 14α demethylase (Cyp51) stood out as the protein targets most susceptible (DS norm < −6 kcal/mol) to the CLS-EO compounds. Table 3 also highlights epi-γ-eudesmol as the most potentially active metabolite, which presented better docking parameters for more than one target. In particular, for TryS and ArgI, the docking scores were superior to the commercial antileishmanial drugs used as standards. In addition to epi-γ-eudesmol, some other CLS-EO metabolites showed docking affinities to the Leishmania target enzymes. These included terpinen-4-ol, 1,8-cineole, and αpinene for Cyp51 and cis-sabinene, α-pinene, terpinen-4-ol, linalool, and p-cymene for trypanothione reductase (TryR). As declared before, TryS was the best target for CLS-EO metabolites, with all main compounds presenting DS norm values lower than −6 kcal/mol, except for 1,8-cineole. In addition, α-terpineol, linalool, and epi-γ-eudesmol docked well to ArgI ( Table 3).
The docking simulation of the ArgI-ligand (epi-γ-eudesmol) complexes exhibited an H-bonding interaction with Asp141 in a radius of 4.15 Å. This interaction with Asp141 occupies the same position as Asp128, which is responsible for coordinating the Mn 2+ cofactor in the active site [33]. As shown in Figure 3, the oxygen atom of the hydroxyl group engaged in coordination with a Mn 2+ ion.

Figure 6.
Interaction between epi-γ-eudesmol and the sterol 14α demethylase (Cyp51) target, characterizing the amino acid residues of the catalytic site involved in the complex stabilization.

Figure 6.
Interaction between epi-γ-eudesmol and the sterol 14α demethylase (Cyp51) target, characterizing the amino acid residues of the catalytic site involved in the complex stabilization.

Discussion
The studied CLS-EO showed a yield higher than other EOs from stems of Croton species, including Croton heliotropiifolius Kunth (0.17%) [21], Croton rhamnifolioides Pax & Figure 6. Interaction between epi-γ-eudesmol and the sterol 14α demethylase (Cyp51) target, characterizing the amino acid residues of the catalytic site involved in the complex stabilization.
Among the major components identified in CLS-EO, only α-pinene coincides with those reported for other species of the genus, such as Croton antanosiensis Leandri, Croton adenocalyx A. DC., Croton argyrophylloides Muell. Arg., Croton zambesicus Muell. Arg., and Croton. sakamaliensis Leandri; it is a compound that is considered, together with β-caryophyllene and β-pinene, to be a chemotaxonomic marker [44].
The chemical composition of the CLS-EO described in this study is very similar to the EO of leaves reported by Amado et al. in 2020 [45], with 48 compounds being found in both oils. The % relative abundance presented a comparable chemical profile, although there were differences observed in some compounds according to plant tissue, such as α-pinene (stems 11.05% vs. leaves 1.52%), p-cymene (leaves 3.37% vs. stems 5.72%), β-elemene (leaves 2.75% vs. stems 0.66%), epi-γ-eudesmol (leaves 2.75% vs. stems 0.66%), and hinesol (leaves 5.65% vs. stems 2.98%). In contrast, noticeable differences were observed with respect to other reports of leaf oils collected in the same geographical location [28]. These findings could be related to rainfall and other climatic characteristics of the habitat where this species grows (coastal xeromorphic scrub). Comparing the results with reports in the literature, differences and similarities among the EOs of leaves and stems from Croton species have been observed [37,39,44,46]. The differences in oil content and composition may be attributed to several factors, such as physiological variations, environmental conditions (climate, pollution, diseases, pests, and edaphic factors), geographic variation, genetic factors, season and harvest period, and others [47,48]. In this sense, it would be interesting, in future studies, to evaluate the effects of environmental parameters on the quality of EOs from leaves and stems of C. linearis growing in the Siboney-Juticí Ecological Reserve with prospects for future standardization.
Previously, we reported the antileishmanial activity of the EO from leaves of C. linearis, which was also active on both forms of L. amazonensis, with similar IC 50 values: IC 50Promastigote = 20.0 µg/mL and IC 50Amastigote = 13.8 µg/mL [28]. Other Croton species have shown activity on Leishmania spp. EOs from C. argyrophylloides, C. jacobinensis, Croton nepetifolius Baill., and Croton sincorensis Mart. displayed activity against promastigotes of L. chagasi, L. braziliensis, and L. amazonensis, with an IC 50 range between 9.1 and 27.0 µg/mL [49]. The EO from berries of Croton macrostachyus Hochst. ex Del. was effective against L. donovani and L. aethiopica promastigotes (IC 50 = 0.1 µL/mL and 0.2 µL/mL, respectively) and the axenic amastigote stages (IC 50 = 20.0 nL/mL and 6.66 nL/mL, respectively) [50]. Croton cajucara Benth leaf essential oil and its purified component 7hydroxycalamenene showed in vitro activity against L. chagasi promastigotes, with IC 50 values of 66.7 µg/mL and 11.4 µg/mL, respectively [20]. Thus, the potential of the Croton genus as a source of metabolites with antileishmanial effects could be highly suggested.
EOs and their constituents can act on parasites of the genus Leishmania in several ways: (i) affecting the layers of polysaccharides, fatty acids, and phospholipids in the autophagosomal structures, cytoplasmatic/mitochondrial/nuclear membranes, and chromatin; (ii) interrupting specific metabolic pathways for lipids and proteins; or (iii) increasing reactive oxygen species that cause DNA damage that leads to parasite death through necrosis or apoptosis [56].
The determination of the cytotoxicity is important to evaluate the selectivity of natural products as future antiprotozoal candidates. In this study, a low selectivity of CLS-EO was revealed. In contrast, the EO from leaves presented a better selectivity (SI > 5) [28]. EOs obtained from four Croton spp. showed lower cytotoxicity on the monocytic cell line AMJ2-C11 than the reference drug at 100 µg/mL [49]. C. cajucara EO did not display toxicity against mouse peritoneal macrophages at concentrations up to 500 µg/mL [20], while oils extracted from Croton pallidulus Baill., Croton ericoides Baill., and Croton isabelli Baill. showed significant cytotoxicities on the Vero cell line [57].
Nevertheless, the in vitro cytotoxicity of CLS-EO could be corroborated in animal models due to the more complex in vivo situation; a metabolic transformation of toxic molecules to nontoxic ones in multicellular organisms may occur, and thus toxicity might dramatically change under these conditions [58].
A biomolecular target is considered to be a protein or nucleic acid with biological activity (an enzyme, receptor, transcription factor, ion channel, transport protein, proteinprotein interface, or ribonucleic acid (RNA)) that is linked to a disease or infection and can be modified by a small molecule or drug [59]. The primary criterion for a biological macromolecule (protein/enzyme/nucleic acid) being a target is that it should be essential for the survival of the organism or pathogen [60]. Several metabolic pathways are currently under study, including the metabolism of fatty acids, sterols, glucose, glycolipids, etc. [61]. Enzymes that play a role in the metabolic pathway of parasites are considered potential targets for the development of new antiparasitic compounds, including targets of the trypanothione, sterol, and polyamine biosynthetic pathways of Leishmania [62].
Low binding free energies were obtained for several of the evaluated compounds from CLS-EO by docking studies with Cyp51, TryR, TryS, and ArgI Leishmania enzyme targets. Our in silico molecular docking study was consistent with the previous experimental results regarding the low activity of the main CLS-EO compound (1,8-cineole). This directed us to more deeply explore the potential interactions and the binding profile with these enzymes of epi-γ-eudesmol, the most potentially active among the main CLS-EO compounds.
In Leishmania, polyamines are important molecules that possess antioxidant activity and are possibly involved in controlling reactive oxygen species induced apoptosis [63]. Polyamine biosynthesis enzymes are promising drug targets for the treatment of leishmaniasis, Chagas disease, and African sleeping sickness. Arginase (ArgI), which is a metallohydrolase, is the first enzyme involved in polyamine biosynthesis, and it converts arginine into ornithine and urea. Ornithine is used in the polyamine pathway that is essential for cell proliferation and ROS detoxification by trypanothione. In Leishmania species, arginase regulates parasite growth, differentiation, and infectivity [64]. Other studies demonstrated that coordination with the Mn 2+ ion in the active binding site of ArgI is important to inhibit this target [65]. Other favorable interactions of different types were formed, such as Van der Waals and π-alkyl interactions with 12 residues (Ser150, Asn143, Ala140, Ile142, Glu197, Gly155, Glu288, Asp245, Asp137, Thr257, His139, His154, and His114) in the active site [66]. That is why those interactions were monitored for epi-γ-eudesmol as the most promising of the main CLS-EO compounds.
The enzyme TryR participates in polyamine-dependent redox metabolism and performs antioxidant functions to protect the parasite against oxidative damage [67]. The bifunctional TryS catalyzes the biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids [34].
In 2019, Feitosa et al. [68] identified the key residues responsible for the inhibitory process to be Thr335 and Thr51, mainly through hydrophobic interactions, analogous to the results obtained in this study. Other important interactions involve two amino acid residues, Cys52 and Cys57, reported by Baiocco et al. [35] as amino acids that participate in the redox reactions of this enzyme and maintain a hydrophobic interaction with epi-γ-eudesmol.
Another key enzyme of Leishmania spp. is sterol 14α demethylase (Cyp51), which catalyzes the removal of the 14α-methyl group from precursors during ergosterol biosynthesis [69]. Unlike mammals, which can accumulate cholesterol from the diet, the blocking of ergosterol production in fungi and protozoa is lethal; it affects cytokinesis, stops cell growth, and eventually leads to the collapse of the cellular membrane [70].
Molecular docking studies with enzyme Cyp51 confirm that most of the interactions are of a hydrophobic nature. The interactions were found to be in accordance with those reported by Hargrove et al. [36] and Sheng et al. [71], who demonstrated that the interactions with the enzyme Cyp51 fundamentally occur by means of der van Waals interactions and π-π stacking.
Even though the chemical composition studies revealed that 1,8-cineole is the metabolite with the highest concentration in CLS-EO, the molecular docking studies showed that epi-γ-eudesmol, despite having a lower concentration, could be responsible for the antileishmanial activity. In addition, epi-γ-eudesmol had relatively lower binding energies compared to the antileishmanial control drugs, and the main interactions with the studied enzymes were found to be of a different nature, including van der Waals, hydrogen bonds, π-σ, π-alkyl, etc. This molecular docking study suggests that it is not necessarily the metabolite with the highest concentration that is responsible for the antiprotozoal activity. However, it provides important indications regarding the possible mechanism of action of the main metabolites present in CLS-EO and on which enzymes they might be acting. Further investigations with respect to the main components identified in CLS-EO should be conducted to better understand the possible mechanisms of action against Leishmania spp.
In addition, the lipophilic nature of EOs permits easy diffusion through cell membranes, and they may then act directly on the parasite or stimulate cellular mechanisms for its elimination. One of these mechanisms is mediated by a significant increase in nitric oxide (NO) production in infected macrophages, which together with reactive oxygen species participates in the destruction of phagocytosed microbes [62]. Some components present in CLS-EO showed activity against amastigotes of Leishmania spp. The monoterpene linalool (3.91%), isolated from the leaves of C. cajucara, exhibited its action through the mechanism described above [20], and α-pinene (11.05%) purified from the essential oil of Syzygium cumini L. produced an increase in NO levels as well as immunomodulatory activity by increasing phagocytic and lysosomal activity [10].

Plant Material
Fresh aerial parts of C. linearis were collected in the Siboney-Juticí Ecological Reserve, Santiago de Cuba, Cuba (at 19.958488 N, −75.692820 W) in September 2020. A voucher specimen was deposited in the Herbarium "Jorge Sierra Calzado" of the Eastern Center of Ecosystems and Biodiversity (BIOECO, Santiago de Cuba, Cuba) under registration 21 659 after authentication by the botanist Ing. Felix Acosta Cantillo.

Extraction and Analysis of the Essential Oil
The EO was extracted from fresh stems of C. linearis (CLS-EO) by a hydrodistillationcohobation method in a classic Clevenger-type apparatus, using 400 g of the sample and 1.6 L of distilled water for 3 h. Then, the EO was collected manually and dried with anhydrous sodium sulfate (Sigma-Aldrich, St. Louis, MO, USA). CLS-EO was stored in an amber bottle and kept in a refrigerator at 4 • C until analysis. The yield was calculated and expressed in % (v/w). The chemical composition was determined by gas chromatographymass spectrometry (GC-MS) using a Shimadzu GCMS-

Antipromastigote Assay
A stock solution of CLS-EO was diluted in 100% DMSO at 20 mg/mL. The assay was carried out under the same methodology reported previously [28]. Serial 1:2 dilutions were carried out to obtain final concentrations between 12.5 and 200 µg/mL, and parasites (2 × 10 5 promastigotes/mL) were treated for 72 h at 26 • C. The cellular viability was determined by a colorimetric assay with 20 µL of 3-[4,5-dimethylthiazol-2-yl]2,5diphenyltetrazolium bromide (MTT; SIGMA, St. Louis, MO, USA). After 4 h of incubation, the supernatant was eliminated, tetrazolium salt was dissolved with 100 µL of DMSO, and the microplate was read in an ELISA microplate reader (Sirio S Reader, 2.4-0, Italy) at 540 nm and 620 nm as reference wavelengths [72]. The median inhibitory concentration (IC 50 ) was determined by a dose-response linear regression analysis. Each experiment was performed in duplicate, and the results were expressed as means ± standard deviations.

Antiamastigote Assay
Peritoneal macrophages from BALB/c mice were collected and seeded with a density of 10 6 /mL in 24-well plates and incubated at 37 • C and 5% CO 2 atmosphere for 2 h. Nonadherent cells were removed. Cells were infected with promastigotes (in the stationary phase) at a parasite/macrophage ratio of 4:1 and incubated for 4 h under the same conditions. Afterwards, the assay was carried out under the same methodology reported previously [28]. Serial 1:2 dilutions were performed to test concentrations between 12.5 and 100 µg/mL and were incubated for 48 h. Then, the supernatant was removed, and monocultures were fixed with methanol and stained with Giemsa. For each sample, the number of intracellular amastigotes and the percentage of infected macrophages were determined in 25 macrophages by counting at a microscope at 100× under immersion oil. The results were expressed as the percentage of reduction of the infection rate (which was obtained by multiplying the percentage of infected macrophages by the number of amastigotes per infected macrophage) in comparison with the negative controls. The IC 50 was determined by a dose-response linear regression analysis. Each experiment was performed in duplicate, and the results were expressed as means ± standard deviations. In both experiments, Pentamidine ® (Richet, Buenos Aires, Argentina) at 10 mg/mL was used as a reference drug.

Cytotoxic Assay
The median cytotoxic concentration (CC 50 ) was determined on peritoneal macrophages from BALB/c mice, which were collected and washed with RPMI 1640 medium (SIGMA) supplemented with antibiotics (200 IU of penicillin and 200 µg of streptomycin per milliliter). The assay was carried out under the same methodology as reported previously [28]. The cellular viability was determined by a colorimetric assay with MTT, as previously described, but using 15 µL of MTT solution/well. The CC 50 was determined by a dose-response linear regression analysis. Each experiment was performed in duplicate, and the results were expressed as means ± standard deviations. The selectivity index (SI) was calculated by the following formula: CC 50 /IC 50 .

Molecular Docking Studies
Molecular modeling was performed using the high-performance computing capabilities of the cluster of the Universidad de Oriente, Cuba (HPC-UO) (https://portal.uo.hpc. cu/website/ (accessed on 6 November 2022)). Molecular docking studies of the CLS-EO metabolites against Leishmania target proteins were performed using AutoDock 4.2 software [73]. The 3D structures of the main compounds identified in the study ( Figure 2) were constructed using the programs included in the ChemOffice 17.1 software [74].
The crystal structures of the target proteins were obtained from the Protein Data Bank (PDB ID) with the codes sterol 14α demethylase (3L4D), trypanothione reductase (2JK6), trypanothione synthetase (2VOB), and arginase I (4ITY), which are considered relevant and interesting molecular targets in Leishmania spp. [75].
The assay parameter was the Lamarckian genetic algorithm (GA) with the following conditions: population amount, 100; maximum number of evals, 2,500,000; with maximum number of generations, 27,000; and other parameters were taken by default. The induced coupling geometric regions were determined with AutoDockTools 1.5.6 [73]. AutoDock requires precalculated grid maps, one for each atom type present in the ligand being docked, as it stores the potential energy arising from the interaction with the macromolecule. This grid must surround the region of interest (active site) in the macromolecule (Table 4). The docked conformations of each ligand were ranked into clusters based on the binding energy. During the molecular docking, interactions (∆G dock = ∆G vdW + ∆G elec + ∆G hbond + ∆G desolv +∆G tors ) were evaluated by molecular mechanics (MM) in AutoDock4 [71]. The top ranked conformations were visually analyzed; Discovery Studio Visualizer v.20.1.0.19295 [76] was used to plot the bonding and nonbonding interactions of the ligand with the receptor in the receptor-ligand complex. Docking calculations were validated by redocking the co-crystallized ligands in the receptor structures [77]. However, in some target structures, no inhibitors were present and the known antileishmanial drugs pentamidine [78] and miltefosine [79], taken from DrugBank, were used. They were built and docked. Due to the high molecular weights of the control compounds, we accounted for the recognized bias and homogenized the energy value using the formula DS norm = 5.71 × ∆G dock /MW 1/3 , where DS norm is the normalized docking score, ∆G dock is the docking energy from the molecular docking program, and 5.71 is a scaling constant to make the average DS norm values comparable to ∆G dock [80].

Conclusions
This study is the first report of the chemical composition and antileishmanial evaluation of CLS-EO. The chemical composition of this oil revealed high concentrations of monoterpene hydrocarbons and oxygenated monoterpenes, with 1,8-cineole as the main compound. CLS-EO showed activity on the promastigote and amastigote forms of L. amazonensis, with low values of SI. The biological activity can be attributed to the synergistic interactions of the EO components. A molecular docking analysis confirmed the stability of the complexes between epi-γ-eudesmol and the different target enzymes of Leishmania, which implies that the antileishmanial action could occur by different mechanisms of action whose main component would be this metabolite.
In conclusion, these findings complete a series of studies about the pharmacological potentialities of C. linearis essential oils. In addition, this study provides support for the further exploration of the main components from CLS-EO as antileishmanial agents and may contribute to research of new candidates for this NTD.