Next Article in Journal
A Narrative Review: Analysis of Supplemental Parenteral Nutrition in Adults at the End of Life
Next Article in Special Issue
Special Issue “Drug Discovery of Antiprotozoal Agents”
Previous Article in Journal
Paclitaxel and Therapeutic Drug Monitoring with Microsampling in Clinical Practice
Previous Article in Special Issue
Immunomodulatory, Antioxidant, and Anti-Inflammatory Activities of Green Synthesized Copper Nanoparticles for Treatment of Chronic Toxoplasma gondii Infection
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Pharmaceuticals
Volume 17
Issue 1
10.3390/ph17010064
pharmaceuticals-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Eugenol-Rich Essential Oil from Pimenta dioica: In Vitro and In Vivo Potentialities against Leishmania amazonensis

by
Lianet Monzote
1,2,*,
Laura Machín
3,
Adiel González
1,
Ramón Scull
3,
Yamilet I. Gutiérrez
3,
Prabodh Satyal
4,
Lars Gille
5 and
William N. Setzer
2,4,6,*
1
Parasitology Department, Center of Research, Diagnostic and Reference, Institute of Tropical Medicine “Pedro Kouri”, Havana 17100, Cuba
2
Research Network Natural Products against Neglected Diseases (ResNetNPND), 48149 Munster, Germany
3
Department of Pharmacy, Institute of Pharmacy and Food, Havana University, Havana 13600, Cuba
4
Aromatic Plant Research Center, 230 N 1200 E, Suite 100, Lehi, UT 84043, USA
5
Institute of Pharmacology and Toxicology, Department of Biomedical Sciences, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria
6
Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA
*
Authors to whom correspondence should be addressed.
Pharmaceuticals 2024, 17(1), 64; https://doi.org/10.3390/ph17010064
Submission received: 1 November 2023 / Revised: 16 December 2023 / Accepted: 21 December 2023 / Published: 29 December 2023
(This article belongs to the Special Issue Drug Discovery of Antiprotozoal Agents)
Browse Figures
Versions Notes

Abstract

:
Pimenta dioica L. is one the most recognized species with diverse biological activities. In this study, in vitro activity and in vivo efficacy of essential oil from P. dioica (EO-Pd) was evaluated. The main compound was also included in the animal studies and its in silico prediction related to biological activities, molecular ligands, drug likeness, and ADME (absorption, distribution, metabolism, and excretion) properties are listed. The chemical composition analyzed by GC-MS retrieved 45 components, which the most abundant compound was the eugenol (80.1%). The EO-Pd was able to inhibit the growth of L. amazonensis (IC50 = 9.7 ± 0.7 and 11.3 ± 2.1 µg/mL, promastigotes and amastigotes, respectively). The cytotoxicity assay showed a CC50 of 104.5 ± 0.9 µg/mL and a selectivity index of 9. In the model of cutaneous leishmaniasis in BALB/c mice, the effect of EO-Pd and eugenol was observed after treatment at 30 mg/kg by intralesional route with 5 administrations every 4 days. In the in silico predictions, some targets that justified the antileishmanial activity of eugenol and good drug like properties for this compound, were obtained. This study showed for first time the potential of EO-Pd to inhibit L. amazonensis, which could be linked to the activity of major compound eugenol.

1. Introduction

Pimenta dioica L. (Family Myrtaceae) is a plant characterized by its high essential oil (EO) content, which has been used in worldwide traditional medicine to alleviate the symptoms of microbial infections, including Caribbean region [1,2]. During the last decade, some biological activities have been demonstrated for essential oil of P. dioica (EO-Pd), including antioxidant [3], antimicrobial [4] and antiparasitic, which have been described against ectoparasites [5], mosquitoes [6] and pinewood nematode [7]. Among the most important component isolated from the EO-Pd, eugenol has been described, which composes 60–90% of the oil [8]. In general, the described biological effects of EO-Pd have been explained on assumption of the bioactive compound eugenol [8].
There have been no previous reports regarding the activity of EO-Pd against the Leishmania parasite. However, some reports have shown the potentialities of EOs rich in eugenol (EO-RE) against different Leishmania species [9,10]. Currently, leishmaniasis is a neglected tropical disease endemic primarily to low- and middle-income countries, for which there has been inadequate development of affordable, safe, and efficacious therapies [11]. Cutaneous leishmaniasis (CL) is the most common presentation of leishmaniasis, with global estimates of 0.7 to 1.2 million cases per year. Available antileishmanial therapies to CL are significantly limited (low efficacy, toxicity, adverse side effects, drug resistance, length of treatment, and cost), so there is an urgent need to discover new compounds. Therefore, various successful approaches have been explored to search for antileishmanial drugs as therapeutic compounds and natural products may also contribute to the development of new drugs based on their chemical structures [12,13]. In recent years, an increased interest in EOs as alternative therapies in the treatment of leishmaniasis has been presented in original articles and reviewed as promising natural products [14,15].
Following our program of investigation related with new antileishmanial agents, we evaluated the potential in vitro activity and in vivo efficacy of EO-Pd. In addition, the main compound was also evaluated against the experimental CL, as well as, its in silico predictions with respect to biological activities, molecular ligands, drug likeness and ADME properties, are also listed.

2. Results

2.1. Chemical Characterization of EO

To characterize the EO-Pa, the chemical composition was analyzed by GC-MS. Detected compounds are listed in Table 1, in which 45 components were identified representing 99.7% of EO. The chromatogram is shown in Supplementary Figure S1. In this oil, the phenolic compounds were the most abundant with 85.1%, follow by 6.5% of monoterpenes hydrocarbons, 3.6% of oxygenated monoterpenoids, 2.6% of oxygenated sesquiterpenoids, 1.8% of sesquiterpene hydrocarbons and 0.1% of other components type. In correlation with previous reports, the most abundant compound was eugenol (80.1%).
Agglomerative hierarchical cluster analysis (HCA) is a common type of clustering used to group objects in clusters based on their similarities. The method starts by treating individual data points as a single cluster, then it is continuously merged based on similarity until one large cluster containing all the objects is obtained. The result is a hierarchical tree (dendrogram) reflecting the dissimilarities of the objects. In this study, HCA was carried out using the concentrations of the most abundant chemical components of EO-Pd from this work and 16 previously reported compositions (Figure 1). The HCA shows three well-defined groupings based on chemical compositions. Group I is dominated by eugenol (65.8–88.8%), Group II has lower concentrations of eugenol (44.5–55.5%) as well as relatively high concentrations of myrcene (0.1–22.4%), and Group III, a single sample, is low in eugenol (8.6%), but rich in myrcene (44.1%), 1,8-cineole (18.8%), and limonene (11.7%). Thus, the EO-Pd in this work falls into the eugenol-rich Group I cluster.

2.2. In Vitro Assay

The effect of EO-Pd on growth of promastigotes and intracellular amastigotes of L. amazonensis was quantitatively determined. On both parasite forms, EO-Pd caused a similar IC50 (p > 0.05); which was statistically (p < 0.05) superior to Pentamidine® (Table 2). However, SI retrieved the same value, since CC50 effect of EO-Pd was 10 times higher (p < 0.05) in comparison with Pentamidine®.

2.3. In Vivo Assay

In addition to in vitro experiments, an in vivo evaluation of the EO-Pd and eugenol after administration by intralesional route was also carried out. In the groups treated with these products, infection control was observed (Figure 2a,b). However, eugenol displayed a quick and efficient decrease of lesion size and parasite burden (p < 0.01) compared with animals treated with placebo or Pentamidine®, as well as with respect to untreated mice. The higher efficacy of eugenol can be appreciated in photographs of infected and uninfected footpads (Figure 2c) and after reduction of infection calculation (Figure 2d). In addition, throughout the observation period (10 weeks p.i.), animals showed no death or signs of toxicity and increase in weight body (Figure 2d).

2.4. In Silico Predictions

The main compound of EO-Pd, eugenol, was used for PASS and ADME prediction studies using web tools. Table 3 presents the results of PASS prediction study, in which seven different activities and 31 ligand interactions were obtained with Pa > 0.7. The analysis of bioavailability and ADME properties of eugenol are compiled in Table 4. Physicochemical parameters used to predict drug-likeness properties showed that comply the rules (Lipinski or rule of five (RO5), Ghose, Veber and Egan); except to Muegge rule with one violation. In general, drug likeness values of eugenol compounds were same or better than pentamidine.

3. Discussion

Pimenta is an important myrtaceous genus that encompasses 15 species, mostly found in the American Caribbean area, and commonly used for several medicinal purposes [16,17]. Pimenta dioica is one the most recognized species from this genus, which has been used to relieve indigestion in Cuban traditional medicine [18]. However, the widespread and diverse uses of this plant, have stimulated several studies in the last two decades on systematic investigations of the potential evidence-based medical use by the population that have been inherited from ancestors among different cultures. With this finding, more scientific studies are necessary to delineate the potential medicinal value of P. dioica, their main constituents or enriched products [8].
This research article presents the chemical composition analysis of the leaf essential oil of P. dioica (EO-Pd) grown in NBG located in Havana, Cuba. The chemical profile of the studied oil was generally consistent with those previously reported in literature from Jamaica [7], India [3,6], Mauritius [19] and Egypt [2], which eugenol was highlighted as the main component. Nevertheless, a fruit essential oil from the Dominican Republic [20] showed a higher percent of methyleugenol. In Mexico, both methyleugenol and eugenol chemotypes of the fruit essential oil have been reported [4,5]. As shown in Figure 2, the eugenol-rich EO-Pd constitutes the most common chemotype for the leaf essential oil, although it is likely that chemical composition of EO-Pd depends on geographical location, climatic and edaphic conditions, seasonality, as observed in other EOs [21,22].
The study of in vitro antileishmanial potentialities of the EO-Pd showed good results related with it activity and selectivity. In the scientific literature, EO-RE from other plants has been also shown potential in vitro antileishmanial activity. For example, EO-RE from Syzygium aromaticum L. (eugenol content = 59.7%) cause an IC50 of 21 µg/mL and 15.24 µg/mL against promastigotes and intracellular amastigotes of L. donovani, respectively, and presented no adverse cytotoxic effects against murine macrophages at 200 µg/mL [9]. Le and collaborators reported that EO-RE from Ocimum gratissimum L. (eugenol content = 86.5%) displayed an IC50 of 4.85 nL/mL against L. mexicana without cytotoxicity at the highest tested concentration (50 nL/mL) on WI38 and J774 mammalian cells [10]. Nevertheless, only a few published studies have demonstrated that EO-RE have antiparasitic activity [23], which constitutes a contribution of this present study.
On the other hand, the profile of in vitro antileishmanial activity of eugenol was summarized by Le and collaborators in 2017. In the cited study, the pure compound was effective on promastigotes of L. amazonensis and L. mexicana with an IC50 value of 12.7 µg/mL [24] and 2.72 µg/mL [25], respectively. In this study, values in same range were obtained for EO-Pd (IC50 = 9.7 µg/mL). However, contradictory results (did not display activity) were described for different L. infantum/chagasi strains [24], causal agents of Visceral Leishmaniasis (VL). It is known that for leishmaniasis, specificities related to individual species should be considered, in particular with respect to variation in drug susceptibility between CL and VL species [26].
Although international criteria to develop natural products against Leishmania are not clear at all, specifically the proposed route map for the discovery and pre-clinical development of new drugs for CL treatment recommend that products should be able to kill amastigotes of parasite (Emax = 90%) and show a SI > 5 [26]. Then, biological in vitro parameters obtained for EO-Pd justify the subsequent exploration on animal models. This led us to investigate the effect of EO-Pd and eugenol on experimental CL.
The majority of CL cases in humans are uncomplicated, with localized single lesions. Available drugs are often administered via systemic routes, exposing patients to unacceptable levels of systemic toxicity. Currently, for New World CL, most patients are treated by systemic route, even though it has already been recommended the use of intralesional treatment by Pan-American Health Organization [27], with the aim to reduce side effects and a rapid circulation clearance [27,28]. In addition, an accelerated healing of the cutaneous lesions and a high percentage of cure with lower cost are also benefits compared to conventional therapy [28].
The efficiency of EO-Pd and eugenol was observed after treatment of BALB/c mice infected with L. amazonensis by intralesional route at 30 mg/kg. Both groups treated with the studied products, showed remarkably smaller CL lesions in comparison with control animals: (i) with no treatment; (ii) treated with placebo; and (iii) treated with reference drug. In addition, the Leishmania parasite burden, measured by microtitration method, also displayed significant differences between tested products with control groups. Finally, reduction of infection (taking into account the lesion size and the parasite burden compared to untreated animals) demonstrated the superior activity by both tested products compared with reference drugs.
It is important to mention that, in this study, the treatment was initiated when the infection has already been established (week four post-infection), which the disease in untreated animal has already progressed to a stage where ulcers and scars can be avoided. In contrast, treated mice with EO-Pd and eugenol displayed a complete epithelization of the skin (as shown in Figure 2c). Secondly, a single dose of 30 mg/kg the EO-Pd or eugenol was administered, although this is the first time that the effect of these products is evaluated. In this sense, Caridha and collaborators suggest using a maximum dose of 30 mg/kg as part of the route map for the discovery and pre-clinical development of new drugs for CL cutaneous leishmaniasis [26], and we have been obtained promising results of Cuban EOs administered to experimentally infected mice with L. amazonensis, such as the EO from Artemisia absinthium L. [29], Melaleuca leucadendra L. [30], and Pluchea carolinensis (Jacq.) G. Don. [31]. These results could suggest that in vitro results were potentially correlated with the in vivo studies in BALB/c mice infected with L. amazonensis, a species that causes difficult-to-treat CL in Latin America [32].
Other generic relevant international criteria for drug development against leishmaniasis, suggest that a potential product should display no acute toxicity in the animal studies [33]. In this study, although no formal in vivo safety studies were performed at this stage, careful observation could be informative. In this sense, the animals were also checked daily, for signs of morbidity and death according to the international guidelines (WHO/OECD), which increase of body weight were observed and manifestation of toxicity was not observed. Nevertheless, toxicity studies should be designed related to safety of evaluated products, such as acute toxicity of EO-Pd and eugenol.
In particular, in the animal model, eugenol showed higher efficacy than the EO-Pd. For centuries, natural substances from plants have been used to control and treat diseases. However, many large pharmaceutical companies have been focused on pure compounds isolated from plants to develop modern pharmaceutical agents or generate large libraries of synthetic compounds to the detriment of natural products-based drug discovery research [34,35]. Eugenol is a phenylpropanoid largely used as flavoring agent for food and displays many pharmacological actions [23]. In particular, the observed antileishmanial action of eugenol could be attributed to several mechanisms. Furthermore, pharmacological properties and probable cellular ligands were analyzed using in silico prediction programs using web tools.
In the obtained activities by PASS analyses, antiprotozoal/antiparasitic effects were not listed, although some of them could contribute to skin diseases healing, including CL. Among then, antieczematic (Pa = 0.868), mucomembranous protector (Pa = 0.835) and antiseptic (Pa = 0.722) could be mentioned. However, when ligand interaction prediction was analyzed, several targets for the antileishmanial activity were obtained. First one, related with mitochondrial inhibition (ubiquinol-cytochrome-c reductase inhibitor: Pa = 0.825), which experimental data demonstrated that eugenol-rich EO from S. aromaticum cause mitochondrial membrane potential and reactive oxygen species generation [9]. Other relevant ligands showed relation with membrane fluidity (membrane permeability inhibitor: Pa = 0.781) and apoptosis promotion (apoptosis agonist: Pa = 0.743 and MAP kinase stimulant: Pa = 0.735), mechanisms recently proposed by Hughes et al., (2023) in promastigotes of L. mexicana [25]. The exact mechanism of the antiparasitic action of eugenol is unknown; although has been studied on bacteria, fungi [36], and protozoa [25,37]. In general, different mechanisms have been described to explain the activity of eugenol, including: (i) changes and cell morphology of cytoplasmatic membrane which increases membrane nonspecific permeability and affects the transport of ions and ATP; (ii) through increase of intracellular Reactive Oxygen Species (ROS) production which induces the inhibition of the growth of cell, disruption of the cell membrane and DNA damage resulting in cell decomposition and death; and (iii) inhibition of some enzymes such as protease, histidine carboxylase, amylase, and ATPase [36]. In particular, concerning Leishmania parasites, Hughes and collaborators have recently demonstrated that eugenol decreases lipid droplets without impacting membrane integrity and induces morphological alterations resulting in rounding and swelling promastigotes [25]. In this sense, further studies related to antileishmanial mechanisms of action should be addresses to confirm these predicted targets.
To promote eugenol as an antileishmanial potential lead, it should have acceptable physicochemical properties and pharmacokinetic profiles, which were analyzed through predicted drug-likeness properties and ADME characteristics [38]. In general, analysis of the ADME parameters showed good drug-like properties, which suggest that eugenol can be developed as an oral drug. Therefore, this compound was predicted to have good bioavailability and satisfied the drug likeliness parameters according to Lipinski (RO5), Ghose, Veber, Egan and Muegge rules. Thus, these theoretical data showed that eugenol presents drug-likeness properties the same or better than the currently used reference drug in antileishmanial therapy obtained from the Swiss ADME web tool, which provides free access to fast and robust models to compute the pharmacokinetics properties, drug-likeness and therapeutics chemistry of a molecule [38]. Recently, Daina and Zoete summarized the applications of this web tool in virtual screening to successfully support the discovery of bioactive small molecules [39]. In particular, the utility of the Swiss ADME tool has been shown in the components of EOs [40] and other natural compounds [41,42].
An interesting prediction of eugenol was related with penetration of eugenol through the Blood–Brain Barrier (BBB), which could suggest that eugenol can also exert its action in the Central Nervous System (CNS). In this sense, this compound could be promoted against other protozoal parasites, such as Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii, and may be suitable for treating the stages that compromise the CNS. In fact, the potential effect of eugenol against Plasmodium falciparum with an impact on cerebral malaria in an experimental murine model were recently reported [43]. In fact, accumulating studies have shown the beneficial effects of eugenol under various neuropathological conditions [44].

4. Materials and Methods

4.1. Plant Material

Aerial samples of P. dioica (Figure 3) were collected during the dry season (March) from the National Botany Garden of Havana (NBG; Havana, Cuba), located at 23°07′59.88″ N, 82°21′59.76″ W. A plant specimen was authenticated by MSc. Eldys Bécquer and deposited in the Herbarium of Cuban Flora (HFC), under the voucher number HFC-88584 of respective NBG. Vegetal material, collected early in the morning, was transported to Institute of Pharmacy and Food (Havana, Cuba) in plastic containers.

4.2. Essential Oil Extraction, Chemical Characterization, and Main Compound

In the same day, fresh leaves were selected, rinsed with abundant water, manually crushed into small pieces, and deposited in the flask of a Clevenger type apparatus. Immediately, the EO was obtained by hydrodistillation over a 5-h period. The essential oil was then chemically characterized by gas-chromatography coupled with a mass spectrometric detector (GC-MS) using a Shimadzu GC-MS-QP2010 Ultra Equipment (Shimadzu Scientific Instruments, Columbia, MD, USA), which was operated in the electron impact mode with electron energy = 70 eV, scan range = 40–400 atomic mass units, scan rate = 3.0 scans/s, and GC-MS solution software v. 4.45. The GC column was a ZB-5 fused silica capillary column (5% phenyl-polymethylsiloxane stationary phase, film thickness of 0.25 μm, a length of 30 m, and an internal diameter of 0.25 mm; Phenomenex, Torrance, CA, USA) and the carrier gas was helium with a column head pressure of 552 kPa, flow rate of 1.37 mL/min, injector temperature of 250 °C and the ion source temperature of 200 °C. The GC oven temperature program was programmed at 50 °C for initial temperature, and increased at a rate of 2 °C/min until 260 °C. The EO was prepared at 5% in a solution of CH2Cl2, which 0.1 μL was injected with a splitting mode (30:1). Finally, the essential oil components were identified according to their retention indices (RI) determined by reference to a homologous series of n-alkanes, and by comparison of their mass spectral fragmentation patterns with those reported in the literature [45], and data stored in our in-house Sat-Set library [46].
A sample of EO-Pd was used to perform the biological assays at 20 mg/mL. Additionally, eugenol (99% purity; Sigma-Aldrich, St. Louis, MO, USA) was dissolved at 40 mM. In both cases, dimethyl sulfoxide (DMSO; AppliedChem, Panreas, Germany) was used as the solvent.

4.3. Hierarchical Cluster Analysis

A total of 16 previously reported EO-Pd compositions [2,3,6,47,48,49,50,51,52,53,54,55] in addition to this present work were used for the Hierarchical Cluster Analysis (HCA). The 11 most abundant EO components (myrcene, p-cymene, limonene, 1,8-cineole, linalool, chavicol, eugenol, methyl eugenol, β-caryophyllene, α-humulene, and α-cadinol) were used to establish the chemical relationship between the EO samples using the XLSTAT software, version 2018.1.1.62926 (Addinsoft™, Paris, France). Euclidean distance was used to measure dissimilarity, and Ward’s method was used for cluster definition.

4.4. Parasite, Cells, Animals, and Reference Drug

Leishmania amazonensis (Strain code: MHOM/77BR/LTB0016) used in this study were isolated by aspiration with a needle from mouse lesions and cultivated at 26 °C in Schneider’s medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (HFBS; Sigma-Aldrich) and antibiotics (100 μg of streptomycin/100 U of penicillin solution, Sigma-Aldrich). When promastigotes were obtained, passage was carried out every 3 or 5 days and used for experiments before to 10 in vitro passages.
Murine macrophage cells were also used, which were obtained from mouse peritoneum, washed with RPMI medium (Sigma, St. Louis, MO, USA) and antibiotics (100 μg of streptomycin/mL and 100 U of penicillin/mL; Sigma, St. Louis, MO, USA) at the moment of use from normal BALB/c mice.
Female BALB/c mice were used to obtain peritoneal macrophages and as animal model of CL. Mice were purchase from the National Center of Laboratory Animals Production (CENPALAB, Havana, Cuba) with their respective certificate of quality and were maintained under standard conditions with food and water ad libitum during the study. All of the experimental procedures involving animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, Eighth Edition, and were approved by the Ethics Committee (CEI-IPK 44-20), Havana, Cuba.
As reference drug, Pentamidine® (Richet, Buenos Aires, Argentina) was used. The compound was diluted in distilled water at 10 mg/mL.

4.5. In Vitro Antileishmanial and Cytotoxicity Assays

For antipromastigote viability test, a 96-well plate was used, which 100 µL of medium (Schneider’s medium + HFBS + antibiotics) was distributed in each well. In the last lane of the plate, additional 96 µL and 4 µL of stock solution of EO-Pd was added and six 1:2 serial dilutions were performed between lanes H to C. Then, 100 µL of exponentially growing parasites of L. amazonensis (106 promastigotes/mL) in medium were added to each well (final concentration of DMSO was 1%), except lane A that was used as medium control; while lane B constituted the negative control (100% of growing). The plates were then sealed with Parafilm® and incubated at 26 °C for 72 h. Afterward, 10 µL of resazurin solution (3 mM/PBS; Sigma-Aldrich) was added to each well and the plates were incubated for an additional 4 h. The absorbance was determined with a plate reader (Molecular Devices, San Jose, CA, USA) with a test wavelength of 560 nm and a reference wavelength of 600 nm from which median Inhibitory Concentrations (IC50) were calculated. The IC50 values were determined from linear dose–response curves fit to the data using a linear equation model. Each concentration of products was carried out in triplicate, and experiments were repeated three times. The results were expressed as means ± Standard Deviation (SD).
For the amastigote model, peritoneal macrophages were distributed in a 24-well plate with sterile slides at 106 cells/mL in RPMI medium with 10% of HFBS and antibiotics. After 2 h of incubation at 37 °C and 5% CO2, the supernatant was eliminated and macrophages were infected with stationary-phase promastigotes of L. amazonensis at a 4:1 parasite/macrophage ratio for 4 h, at same conditions. Then, the free parasites were removed and four different concentrations of each product were added in a final volume of 1 mL (final concentration of DMSO was 1%). The plates were then incubated under the same conditions over 48 h [56]. Finally, the supernatant was discarded, cells were fixed with methanol, stained with Giemsa and microscopically examined. Total parasite burden was determined according to the number of infected macrophages and the number of amastigotes inside the macrophages of 25 cells [57]. IC50 values were determined from linear dose–response curves fit to the data using a linear equation model and results were expressed as means ± SD of three replicates.
Cytotoxicity was determined on uninfected peritoneal macrophages. Briefly, cells were collected and seeded at 3 × 105 macrophages/mL in a 96-well plate (except lane A) and incubated at 37 °C and 5% CO2. After 2 h, the medium was removed and 50 µL of medium (RPMI + HFBS + antibiotics) was distributed in each well. In the last lane of the plate, additional 48 µL and 2 µL of products was added and six 1:2 serial dilutions were performed between lanes H to C. Then, 50 µL of medium were added to each well (final concentration of DMSO was 1%), except lane A that was used as medium control; while lane B constituted the negative control (100% of viability). The plate was incubated at the same conditions for 72 h and viability was determined with 15 µL of MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma-Aldrich) at 5 mg/mL. After 4 h, formazan crystals were dissolved with 100 µL of DMSO and the optical density was measured at 570 nm and at 630 nm as reference wavelength [58]. In this case, median Cytotoxic Concentrations (CC50) were calculated from linear dose–response curves and results was expressed as means ± SD of three replicates, where lane A was used as medium control and lane B as 100% of macrophage viability.
Finally, Selectivity Indices (SI) were calculated through the ratio of CC50 (macrophages)/IC50 (amastigotes of L. amazonensis).

4.6. In Vivo Antileishmanial Assay

At day 0, female healthy BALB/c mice were infected in the right hind footpad with 5 × 106 stationary-phase promastigotes of L. amazonensis by the subcutaneous route in a volume of 50 μL. The animals were maintained in standard conditions until 4 weeks post-infection (p.i.), which mice were randomly distributed into four groups (n = 8 in each group): Group 1, EO-Pd (30 mg/kg); Group 2, eugenol (30 mg/kg); Group 3, Pentamidine® (4 mg/kg); Group 4, placebo (solvent used to dilute studied products: 30% DMSO:70% saline solution [59]); Group 5, control (untreated). Treatments with EO-Pd and eugenol were applied every four days to a total of five doses by the intralesional route, while Pentamidine® was administered by intraperitoneal route for 15 days. During the experimental period, animals were observed daily, body weight and the lesion size (mean of the differences between infected and uninfected footpads) were determined weekly using a technical bascule (SCALTEC, Göttingen, Germany) and a caliper was used to measure footpad swelling and lesion diameter. In addition, parasite burden was determined at the end of treatment (6 weeks p.i.) and in the final point (10 weeks p.i.) through the culture microtitration method in 96-well plates using excised tissues of subcutaneous infected area [60].

4.7. In Silico Predictions

The chemical structure of eugenol was constructed using BIOVIA Draw Program 2018 (Version 18.1.0.1601, Dassault Systémes). After that, SMILES of structure were inserted in the online server for a Prediction of Activity Spectra for Substances (PASS; accessed on 28 July 2022) and automatically potential bioactivity predictions were obtained. Activities with Pa > 0.7 (probability of the substance to be active) were selected. In parallel, SMILES was also used to obtain its physicochemical properties and Absorption, Distribution, Metabolism, Excretion (ADME) from SwissADME online server (accessed on 28 July 2022) [38] and compare with pentamidine as reference drug. In all cases, data were exported to Excel Microsoft 2010, store in a spreadsheet and analyzed.

4.8. Statistical Analysis

In the in vitro assays, respective IC50 and CC50 were compared using Mann–Whitney. In the in vivo assay, a Variance Analysis Test, accompanied by a Post Hoc Test (Fisher’s least significant difference (LDS test) or planned comparison) was applied for comparison among groups [61]. In both cases, Statistica Program for Windows (Version 13.1, StatSoft, Inc. 2016) was used and a p < 0.05 were considered statistical differences.

5. Conclusions

This study showed for first time the potentialities of the EO-Pd to inhibit L. amazonensis (promastigotes and intracellular amastigotes) growth with a selective activity and demonstrated the antileishmanial effect in a murine model of experimental CL. This interesting result could be linked to the activity of the major component eugenol, which displayed a potent in vivo efficacy and good drug-like properties that could contribute to the discovery of new effective drugs against leishmaniasis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph17010064/s1, Figure S1: Gas chromatogram of Pimenta dioica essential oil.

Author Contributions

Conceptualization, L.M. (Lianet Monzote); methodology, L.M. (Lianet Monzote), L.M. (Laura Machín), A.G., R.S., Y.I.G., P.S. and W.N.S.; formal analysis, L.M. (Lianet Monzote), L.G. and W.N.S.; investigation, L.M. (Lianet Monzote), L.M. (Laura Machín), A.G., R.S., Y.I.G., P.S., L.G. and W.N.S.; resources, L.M., L.G. and W.N.S.; data curation, L.M. (Lianet Monzote), A.G., Y.I.G., L.G. and W.N.S.; writing—original draft preparation, L.M. (Lianet Monzote); writing—review and editing, L.M. (Lianet Monzote), L.M. (Laura Machín), A.G., R.S., Y.I.G., P.S., L.G. and W.N.S.; visualization, L.M. (Lianet Monzote), L.G. and W.N.S.; supervision, L.M. (Lianet Monzote) and L.G.; project administration, L.M. (Lianet Monzote) and L.G.; funding acquisition, L.M. (Lianet Monzote) and L.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partially funded by Austrian Science Fund (FWF), grant number P-27814-B22.

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Ethics Committee of Institute of Tropical Medicine Pedro Kourí (CEI-IPK 44-20, date of approval: 14 July 2020).

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are available in the article and Supplementary Materials.

Acknowledgments

The award of an Ernst Mach scholarship to Lianet Monzote by the Austrian Exchange Service is gratefully acknowledged.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Contreras-Moreno, B.Z. Chemical Composition of Essential Oil of Genus Pimenta (Myrtaceae). In Potential of Essential Oils; El-Shemy, H., Ed.; IntechOpen Limited: London, UK, 2018; p. 21. [Google Scholar]
  2. Ismail, M.M.; Samir, R.; Saber, F.R.; Ahmed, S.R.; Farag, M.A. Pimenta Oil as A Potential Treatment for Acinetobacter baumannii Wound Infection: In Vitro and In Vivo Bioassays in Relation to Its Chemical Composition. Antibiotics 2020, 9, 679. [Google Scholar] [CrossRef] [PubMed]
  3. Padmakumari Amma, K.P.; Rani, M.P.; Sasidharan, I.; Sreekumar, M.M. Comparative chemical composition and in vitro antioxidant activities of essential oil isolated from the leaves of Cinnamomum tamala and Pimenta dioica. Nat. Prod. Res. 2013, 27, 290–294. [Google Scholar] [CrossRef] [PubMed]
  4. Lorenzo-Leal, A.C.; Palou, E.; López-Malo, A.; Bach, H. Antimicrobial, Cytotoxic, and Anti-Inflammatory Activities of Pimenta dioica and Rosmarinus officinalis Essential Oils. Biomed. Res. Int. 2019, 2019, 1639726. [Google Scholar] [CrossRef]
  5. Martinez-Velazquez, M.; Castillo-Herrera, G.A.; Rosario-Cruz, R.; Flores-Fernandez, J.M.; Lopez-Ramirez, J.; Hernandez-Gutierrez, R.; Lugo-Cervantes, C. Acaricidal effect and chemical composition of essential oils extracted from Cuminum cyminum, Pimenta dioica and Ocimum basilicum against the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Parasitol. Res. 2011, 108, 481–487. [Google Scholar] [CrossRef] [PubMed]
  6. Narayanankutty, A.; Kuttithodi, A.M.; Alfarhan, A.; Rajagopal, R.; Barcelo, D. Chemical Composition, Insecticidal and Mosquito Larvicidal Activities of Allspice (Pimenta dioica) Essential Oil. Molecules 2021, 26, 6698. [Google Scholar] [CrossRef] [PubMed]
  7. Park, I.K.; Kim, J.; Lee, S.G.; Shin, S.C. Nematicidal Activity of Plant Essential Oils and Components from Ajowan (Trachyspermum ammi), Allspice (Pimenta dioica) and Litsea (Litsea cubeba) Essential Oils Against Pine Wood Nematode (Bursaphelenchus Xylophilus). J. Nematol. 2007, 39, 275–279. [Google Scholar] [PubMed]
  8. Zhang, L.; Lokeshwar, B.L. Medicinal properties of the Jamaican pepper plant Pimenta dioica and Allspice. Curr. Drug Targets. 2012, 13, 1900–1906. [Google Scholar] [CrossRef]
  9. Islamuddin, M.; Sahal, D.; Afrin, F. Apoptosis-like death in Leishmania donovani promastigotes induced by eugenol-rich oil of Syzygium aromaticum. J. Med. Microbiol. 2014, 63, 74–85. [Google Scholar] [CrossRef]
  10. Le, T.B.; Beaufay, C.; Nghiem, D.T.; Mingeot-Leclercq, M.P.; Quetin-Leclercq, J. In Vitro Anti-Leishmanial Activity of Essential Oils Extracted from Vietnamese Plants. Molecules 2017, 22, 1071. [Google Scholar] [CrossRef]
  11. Pacheco-Fernandez, T.; Markle, H.; Verma, C.; Huston, R.; Gannavaram, S.; Nakhasi, H.L.; Satoskar, A.R. Field-Deployable Treatments for Leishmaniasis: Intrinsic Challenges, Recent Developments and Next Steps. Res. Rep. Trop. Med. 2023, 20, 61–85. [Google Scholar] [CrossRef]
  12. Gervazoni, L.F.O.; Barcellos, G.B.; Ferreira-Paes, T.; Almeida-Amaral, E.E. Use of Natural Products in Leishmaniasis Chemotherapy: An Overview. Front. Chem. 2020, 8, 579891. [Google Scholar] [CrossRef] [PubMed]
  13. Cohen, A.; Azas, N. Challenges and Tools for In Vitro Leishmania Exploratory Screening in the Drug Development Process: An Updated Review. Pathogens 2021, 10, 1608. [Google Scholar] [CrossRef] [PubMed]
  14. Luna, E.C.; Luna, I.S.; Scotti, L.; Monteiro, A.F.M.; Scotti, M.T.; de Moura, R.O.; de Araújo, R.S.A.; Monteiro, K.L.C.; de Aquino, T.M.; Ribeiro, F.F.; et al. Active Essential Oils and Their Components in Use against Neglected Diseases and Arboviruses. Oxid. Med. Cell. Longev. 2019, 2019, 6587150. [Google Scholar] [CrossRef] [PubMed]
  15. De Lara da Silva, C.E.; Oyama, J.; Ferreira, F.B.P.; de Paula Lalucci-Silva, M.P.; Lordani, T.V.A.; de Lara da Silva, R.C.; de Souza Terron Monich, M.; Teixeira, J.J.V.; Lonardoni, M.V.C. Effect of essential oils on Leishmania amazonensis: A systematic review. Parasitology. 2020, 147, 1392–1407. [Google Scholar] [CrossRef]
  16. Chaverri, C.; Cicció, J.F. Leaf and fruit essential oil compositions of Pimenta guatemalensis (Myrtaceae) from Costa Rica. Rev. Biol. Trop. 2015, 63, 303–311. [Google Scholar] [CrossRef]
  17. Morton, J.F. Atlas of Medicinal Plants of Middle America: Bahamas to Yucatan; Charles C Thomas Pub Ltd.: Springfield, IL, USA, 1981. [Google Scholar]
  18. Ramos, A.; Visozo, A.; Piloto, J.; Garc, A.; Rodr, C.A.; Rivero, R. Screening of antimutagenicity via antioxidant activity in Cuban medicinal plants. J. Ethnopharmacol. 2003, 87, 241–246. [Google Scholar] [CrossRef]
  19. Mahomoodally, F.; Aumeeruddy-Elalfi, Z.; Venugopala, K.N.; Hosenally, M. Antiglycation, comparative antioxidant potential, phenolic content and yield variation of essential oils from 19 exotic and endemic medicinal plants. Saudi J. Biol. Sci. 2019, 26, 1779–1788. [Google Scholar] [CrossRef]
  20. Achimón, F.; Brito, V.D.; Pizzolitto, R.P.; Ramirez Sanchez, A.; Gómez, E.A.; Zygadlo, J.A. Chemical composition and antifungal properties of commercial essential oils against the maize phytopathogenic fungus Fusarium verticillioides. Rev. Argent. Microbiol. 2021, 53, 292–303. [Google Scholar] [CrossRef]
  21. Moore, B.D.; Andrew, R.L.; Külheim, C.; Foley, W.J. Explaining intraspecific diversity in plant secondary metabolites in an ecological context. New Phytol. 2014, 201, 733–750. [Google Scholar] [CrossRef]
  22. Sangwan, N.S.; Farooqi, A.H.A.; Shabih, F.; Sangwan, R.S. Regulation of essential oil production in plants. Plant Growth Regul. 2001, 34, 3–21. [Google Scholar] [CrossRef]
  23. De Morais, S.M.; Vila-Nova, N.S.; Bevilaqua, C.M.; Rondon, F.C.; Lobo, C.H.; de Alencar Araripe Noronha Moura, A.; Sales, A.D.; Rodrigues, A.P.; de Figuereido, J.R.; Campello, C.C.; et al. Thymol and eugenol derivatives as potential antileishmanial agents. Bioorg. Med. Chem. 2014, 22, 6250–6255. [Google Scholar] [CrossRef] [PubMed]
  24. Grecco, S.S.; Costa-Silva, T.A.; Sousa, F.S.; Cargnelutti, S.B.; Umehara, E.; Mendonça, P.S.; Tempone, A.G.; Lago, J.H.G. Neolignans isolated from twigs of Nectandra leucantha Ness & Mart (Lauraceae) displayed in vitro antileishmanial activity. J. Venom. Anim. Toxins Incl. Trop. Dis. 2018, 24, 27. [Google Scholar] [PubMed]
  25. Hughes, K.; Le, T.B.; Van Der Smissen, P.; Tyteca, D.; Mingeot-Leclercq, M.P.; Quetin-Leclercq, J. The Antileishmanial Activity of Eugenol Associated with Lipid Storage Reduction Rather Than Membrane Properties Alterations. Molecules 2023, 28, 3871. [Google Scholar] [CrossRef] [PubMed]
  26. Caridha, D.; Vesely, B.; van Bocxlaer, K.; Arana, B.; Mowbray, C.E.; Rafati, S.; Uliana, S.; Reguera, R.; Kreishman-Deitrick, M.; Sciotti, R.; et al. Route map for the discovery and pre-clinical development of new drugs and treatments for cutaneous leishmaniasis. Int. J. Parasitol. Drugs Drug Resist. 2019, 11, 106–117. [Google Scholar] [CrossRef]
  27. Pan American Health Organization. Leishmaniasis in the Americas: Treatment Recommendations; PAHO: Washington, DC, USA, 2018. [Google Scholar]
  28. Roatt, B.M.; de Oliveira Cardoso, J.M.; De Brito, R.C.F.; Coura-Vital, W.; de Oliveira Aguiar-Soares, R.D.; Reis, A.B. Recent advances and new strategies on leishmaniasis treatment. Appl. Microbiol. Biotechnol. 2020, 104, 8965–8977. [Google Scholar] [CrossRef]
  29. Monzote, L.; Piñón, A.; Scull, R.; Setzer, W.N. Chemistry and leishmanicidal activity of the essential oil from Artemisia absinthium from Cuba. Nat. Prod. Commun. 2014, 9, 1799–1804. [Google Scholar] [PubMed]
  30. Monzote, L.; Scherbakov, A.M.; Scull, R.; Satyal, P.; Cos, P.; Shchekotikhin, A.E.; Gille, L.; Setzer, W.N. Essential Oil from Melaleuca leucadendra: Antimicrobial, Antikinetoplastid, Antiproliferative and Cytotoxic Assessment. Molecules 2020, 25, 5514. [Google Scholar] [CrossRef] [PubMed]
  31. García, M.; Scull, R.; Satyal, P.; Setzer, W.N.; Monzote, L. Chemical Characterization, Antileishmanial Activity, and Cytotoxicity Effects of the Essential Oil from Leaves of Pluchea carolinensis (Jacq.) G. Don. (Asteraceae). Phytother. Res. 2017, 31, 1419–1426. [Google Scholar] [CrossRef]
  32. Teixeira, R.R.; Rodrigues Gazolla, P.A.; Borsodi, M.P.G.; Castro Ferreira, M.M.; Andreazza Costa, M.C.; Costa, A.V.; Cabral Abreu Grijó, B.; Rossi Bergmann, B.; Lima, W.P. Eugenol derivatives with 1,2,3-triazole moieties: Oral treatment of cutaneous leishmaniasis and a quantitative structure-activity relationship model for their leishmanicidal activity. Exp. Parasitol. 2022, 238, 108269. [Google Scholar] [CrossRef]
  33. Katsuno, K.; Burrows, J.N.; Duncan, K.; Hooft van Huijsduijnen, R.; Kaneko, T.; Kita, K.; Mowbray, C.E.; Schmatz, D.; Warner, P.; Slingsby, B.T. Hit and lead criteria in drug discovery for infectious diseases of the developing world. Nat. Rev. Drug Discov. 2015, 14, 751–758. [Google Scholar] [CrossRef]
  34. Cheuka, P.M.; Mayoka, G.; Mutai, P.; Chibale, K. The Role of Natural Products in Drug Discovery and Development against Neglected Tropical Diseases. Molecules 2016, 22, 58. [Google Scholar] [CrossRef] [PubMed]
  35. Rishton, G.M. Natural Products as a Robust Source of New Drugs and Drug Leads: Past Successes and Present Day Issues. Am. J. Cardiol. 2008, 101, S43–S49. [Google Scholar] [CrossRef]
  36. Marchese, A.; Barbieri, R.; Coppo, E.; Orhan, I.E.; Daglia, M.; Nabavi, S.F.; Izadi, M.; Abdollahi, M.; Nabavi, S.M.; Ajami, M. Antimicrobial activity of eugenol and essential oils containing eugenol: A mechanistic viewpoint. Crit. Rev. Microbiol. 2017, 43, 668–689. [Google Scholar] [CrossRef] [PubMed]
  37. Chudin, A.A.; Zlotnikov, I.D.; Krylov, S.S.; Semenov, V.V.; Kudryashova, E.V. Allylpolyalkoxybenzene Inhibitors of Galactonolactone Oxidase from Trypanosoma cruzi. Biochemistry 2023, 88, 131–141. [Google Scholar] [CrossRef] [PubMed]
  38. Daina, A.; Michielin, O.; Zoete, V. SwissADME: A free web tool to evaluate the pharmacokinetics, drug-likeness, and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717. [Google Scholar] [CrossRef] [PubMed]
  39. Daina, A.; Michielin, O.; Zoete, V. SwissTargetPrediction: Updated data and new features for efficient prediction of protein targets of small molecules. Nucleic Acids Res. 2019, 47, W357–W364. [Google Scholar] [CrossRef] [PubMed]
  40. Supandi, S.; Wulandari, M.S.; Samsul, E.; Azminah, A.; Purwoko, R.Y.; Herman, H.; Kuncoro, H.; Ibrahim, A.; Silfi Ambarwati, N.S.; Rosmalena, R.; et al. Dipeptidyl peptidase IV inhibition of phytocompounds from Artocarpus champeden (Lour.) Stokes: In silico molecular docking study and ADME-Tox prediction approach. J. Adv. Pharm. Technol. Res. 2022, 13, 207–215. [Google Scholar] [PubMed]
  41. Dey, D.; Biswas, P.; Paul, P.; Mahmud, S.; Ema, T.I.; Khan, A.A.; Ahmed, S.Z.; Hasan, M.M.; Saikat, A.S.M.; Fatema, B.; et al. Natural flavonoids effectively block the CD81 receptor of hepatocytes and inhibit HCV infection: A computational drug development approach. Mol. Divers 2023, 27, 1309–1322. [Google Scholar] [CrossRef]
  42. Foudah, A.I.; Alqarni, M.H.; Alam, A.; Salkini, M.A.; Ross, S.A.; Yusufoglu, H.S. Phytochemical Screening, In Vitro and In Silico Studies of Volatile Compounds from Petroselinum crispum (Mill) Leaves Grown in Saudi Arabia. Molecules 2022, 27, 934. [Google Scholar] [CrossRef]
  43. Pontes, K.A.O.; Silva, L.S.; Santos, E.C.; Pinheiro, A.S.; Teixeira, D.E.; Peruchetti, D.B.; Silva-Aguiar, R.P.; Wendt, C.H.C.; Miranda, K.R.; Coelho-de-Souza, A.N.; et al. Eugenol disrupts Plasmodium falciparum intracellular development during the erythrocytic cycle and protects against cerebral malaria. Biochim. Biophys. Acta Gen. Subj. 2021, 1865, 129813. [Google Scholar] [CrossRef]
  44. Zhu, J.; Park, S.; Kim, C.H.; Jeong, K.H.; Kim, W.J. Eugenol alleviates neuronal damage via inhibiting inflammatory process against pilocarpine-induced status epilepticus. Exp. Biol. Med. 2023, 248, 722–731. [Google Scholar] [CrossRef] [PubMed]
  45. Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed.; Allured Publishing: Carol Stream, IL, USA, 2007. [Google Scholar]
  46. Satyal, P. Development of GC-MS Database of Essential Oil Components by the Analysis of Natural Essential Oils and Synthetic Compounds and Discovery of Biologically Active Novel Chemotypes in Essential Oils. Ph.D. Thesis, University of Alabama in Huntsville, Huntsville, AL, USA, 2015. [Google Scholar]
  47. Andrade, B.F.; Ferreira, V.R.F.; Cardoso, G.P.; Andrade, M.P.D.; Ramos, A.L.S.; Cardoso, M.G.; Ramos, E.M. Allspice (Pimenta Dioica Lindl) leaves essential oil as a potential antioxidant and antimicrobial source for use in mechanically deboned poultry meat. Braz. J. Food Technol. 2023, 26, e2022125. [Google Scholar] [CrossRef]
  48. Ashokkumar, K.; Murugan, M.; Vellaikumar, S.; Dhanya, M. Essential oil composition of allspice [Pimenta dioica (L.) Merill.] leaf from the Western Ghats, India. J. Curr. Opin. Crop. Sci. 2022, 3, 168–172. [Google Scholar]
  49. Gomes, P.R.B.; Marinho, S.C.; Everton, G.O.; Silva, E.F.; Fontenele, M.A.; Lyra, W.S.; de Freitas, A.C.; Hunaldo, V.K.L.; Souza, R.D.; Louzeiro, H.C.; et al. Chemical composition and larvicidal activity of the essential oil of Pimenta dioica leaves. Bol. Latinoam. Caribe. Plant Med. Aromat. 2022, 21, 207–214. [Google Scholar] [CrossRef]
  50. Bruno Marongiu, A.P.; Porcedda, S.; Casu, R.; Pierucci, P. Comparative Analysis of Supercritical CO2 Extract and Oil of Pimenta dioica Leaves. J. Essent. Oil Res. 2005, 17, 530–532. [Google Scholar] [CrossRef]
  51. Marques, C.S.; Pinheiro, P.F.; Villanova, J.C.O.; Bernardes, P.C. Comparative chemical evaluation and antibacterial activity of Pimenta dioica essential oil obtained by different extraction methods. Open Sci. Res. III 2022, 3, 242–255. [Google Scholar]
  52. Mérida-Reyes, M.S.; Muñoz-Wug, M.A.; Oliva-Hernández, B.E.; Gaitán-Fernández, I.C.; Simas, D.L.R.; Ribeiro da Silva, A.J.; Pérez-Sabino, J.F. Composition and Antibacterial Activity of the Essential Oil from Pimenta dioica (L.) Merr. from Guatemala. Medicines 2020, 7, 59. [Google Scholar] [CrossRef] [PubMed]
  53. Sarathambal, C.; Rajagopal, S.; Viswanathan, R. Mechanism of antioxidant and antifungal properties of Pimenta dioica (L.) leaf essential oil on Aspergillus flavus. J. Food Sci. Technol. 2021, 58, 2497–2506. [Google Scholar] [CrossRef]
  54. Youssef, F.S.; Labib, R.M.; Gad, H.A.; Eid, S.; Ashour, M.L.; Eid, H.H. Pimenta dioica and Pimenta racemosa: GC-based metabolomics for the assessment of seasonal and organ variation in their volatile components, in silico and in vitro cytotoxic activity estimation. Food Funct. 2021, 12, 5247–5259. [Google Scholar] [CrossRef]
  55. Pino, J.A.; Rosado, A. Chemical Composition of the Leaf Oil of Pimenta dioica L. from Cuba. J. Essent. Oil Res. 1996, 8, 331–332. [Google Scholar] [CrossRef]
  56. Caio, T.S.E.; Lima, M.D.; Kaplan, M.A.C.; Nazareth, M.M.; Rossi-Bergmann, B. Selective effect of 2′,6′-dihydroxy-4′-methoxychalcone isolated from Piper aduncum on Leishmania amazonensis. Antimicrob. Agents Chemother. 1999, 43, 1234–1241. [Google Scholar]
  57. Delorenzi, J.C.; Attias, M.; Gattass, C.R.; Andrade, M.; Rezende, C.; da Cunha Pinto, A.; Henriques, A.T.; Bou-Habib, D.C.; Saraiva, E.M. Antileishmanial activity of an indole alkaloid from Peschiera australis. Antimicrob. Agents Chemother. 2001, 45, 1349–1354. [Google Scholar] [CrossRef] [PubMed]
  58. Sladowski, D.; Steer, S.J.; Clothier, R.H.; Balls, M. An improve MTT assay. J. Immunol. Methods 1993, 157, 203–207. [Google Scholar] [CrossRef] [PubMed]
  59. Monzote, L.; Pastor, J.; Scull, R.; Gille, L. Antileishmanial activity of essential oil from Chenopodium ambrosioides and its main components against experimental cutaneous leishmaniasis in BALB/c mice. Phytomedicine 2014, 21, 1048–1052. [Google Scholar] [CrossRef]
  60. Buffet, P.A.; Sulahian, A.; Garin, Y.J.F.; Nassar, N.; Derouin, F. Culture microtitration: A sensitive method for quantifying Leishmania infantum in tissues of infected mice. Antimicrob Agents Chemother. 1995, 39, 2167–2168. [Google Scholar] [CrossRef]
  61. Pereira, D.G.; Afonso, A.; Medeiros, F.M. Overview of Friedman’s Test and Post-hoc Analysis. Commun. Stat. Simul. Comput. 2015, 44, 2636–2653. [Google Scholar] [CrossRef]
Pharmaceuticals 17 00064 g001
Figure 1. Dendrogram obtained from the hierarchical cluster analysis of Pimenta dioica leaf essential oil compositions.
Figure 1. Dendrogram obtained from the hierarchical cluster analysis of Pimenta dioica leaf essential oil compositions.
Pharmaceuticals 17 00064 g001
Pharmaceuticals 17 00064 g002
Figure 2. Effect of essential oil from Pimenta dioica in BALB/c mice infected with Leishmania amazonensis. (a) The results of lesion size and (b) parasite burden are expressed as mean ± standard deviation. (c) Photographs of footpads at week 10 post-infection. At this time, (d) percent of reduction of infection was calculated (multiply lesion size and parasite burden of treated group divided by same data of untreated animals). Statistical differences with respect to placebo, untreated groups, and the reference drug Pentamidine® are represented as * (p < 0.05) and ** (p < 0.01).
Figure 2. Effect of essential oil from Pimenta dioica in BALB/c mice infected with Leishmania amazonensis. (a) The results of lesion size and (b) parasite burden are expressed as mean ± standard deviation. (c) Photographs of footpads at week 10 post-infection. At this time, (d) percent of reduction of infection was calculated (multiply lesion size and parasite burden of treated group divided by same data of untreated animals). Statistical differences with respect to placebo, untreated groups, and the reference drug Pentamidine® are represented as * (p < 0.05) and ** (p < 0.01).
Pharmaceuticals 17 00064 g002
Pharmaceuticals 17 00064 g003
Figure 3. Photo of Pimenta dioica in the natural habitat at National Botanic Garden, Havana, Cuba (Photographs taken by the authors during the collection of the plant).
Figure 3. Photo of Pimenta dioica in the natural habitat at National Botanic Garden, Havana, Cuba (Photographs taken by the authors during the collection of the plant).
Pharmaceuticals 17 00064 g003
Table 1. Peak assignment for gas chromatography—mass spectrometry profiles of the essential oil obtained by hydrodistillation from Pimenta dioica leaves collected from the National Botanic Garden, Havana, Cuba.
Table 1. Peak assignment for gas chromatography—mass spectrometry profiles of the essential oil obtained by hydrodistillation from Pimenta dioica leaves collected from the National Botanic Garden, Havana, Cuba.
RI 1Compound%
873(3Z)-Hexenol0.1
8801-Hexanoltr 2
924α-Thujene0.3
930α-Pinene0.3
971Sabinenetr
987Myrcene0.2
1000α-Phellandrene0.6
1006δ-3-Carene0.1
10121,4-Cineoletr
1013α-Terpinene0.2
1021p-Cymene3.6
1026β-Phellandrene0.6
10281,8-Cineole2.3
1059γ-Terpinene0.1
1090Terpinolene0.6
1104Linalool0.1
1150Unidentified0.1
1180Terpinen-4-ol0.8
1189p-Cymen-8-ol0.2
1193α-Terpineol0.2
1201Methyl chavicol (=Estragole)tr
1203cis-Sabinol0.1
1253Chavicoltr
1286Thymoltr
1290Carvacroltr
1360Eugenol85.1
1390β-Elemene0.1
1395Vanillin0.1
1416β-Caryophyllene0.7
1450α-Humulene0.4
1484β-Selinene0.2
1494α-Selinene0.3
1525δ-Cadinene0.1
1581Caryophyllene oxide0.8
1590Viridiflorol0.1
1602Ledol0.1
1608Humulene epoxide II0.3
1613Unidentified0.2
1616Junenol0.1
16281-epi-Cubenol0.1
1641τ-Cadinol0.2
1643τ-Muurolol0.1
1647α-Muurolol0.1
1654α-Cadinol0.3
1658Selin-11-en-4β-ol0.6
Total identified99.7
1 Retention Index determined with respect to a homologous series of n-alkanes on a ZB-5 column. 2 Traces (concentration < 0.05%). Bold letters indicate main compound.
Table 2. In vitro antileishmanial activity, cytotoxic effects, and selectivity of the leaf essential oil of Pimenta dioica (collected from the National Botanic Garden, Havana, Cuba) and reference drug.
Table 2. In vitro antileishmanial activity, cytotoxic effects, and selectivity of the leaf essential oil of Pimenta dioica (collected from the National Botanic Garden, Havana, Cuba) and reference drug.
ProductsIC50 1 ± SD 2 (µg/mL)CC50 3 ± SD (µg/mL)SI 4
PromastigotesAmastigotes
P. dioica EO9.7 ± 0.711.3 ± 2.1104.5 ± 0.99
Pentamidine 52.6 ± 0.91.3 ± 0.111.7 ± 1.79
1 Median inhibitory concentration. 2 Standard deviation. 3 Median cytotoxic concentration. 4 Selectivity index (CC50 macrophage / IC50 amastigotes of L. amazonensis). 5 Reference drug.
Table 3. Prediction of biological activities and ligand interactions by the PASS online webserver of eugenol, the main compound identified in the essential oil from Pimenta dioica collected in National Botanic Garden, Havana, Cuba (organized by Pa value).
Table 3. Prediction of biological activities and ligand interactions by the PASS online webserver of eugenol, the main compound identified in the essential oil from Pimenta dioica collected in National Botanic Garden, Havana, Cuba (organized by Pa value).
Prediction Eugenol
Pa 1Pi 2
Biological Activity:Carminative0.9410.001
Antimutagenic0.8780.003
Antieczematic0.8680.008
Mucomembranous protector0.8350.011
Preneoplastic conditions treatment0.8030.004
Anesthetic general0.7420.005
Antiseptic0.7220.005
Ligand interactions:Aspulvinone dimethylallyltransferase inhibitor0.9370.004
Chlordecone reductase inhibitor0.9020.005
Feruloyl esterase inhibitor0.8810.005
Caspase 3 stimulant0.8730.004
JAK2 expression inhibitor0.8730.004
Linoleate diol synthase inhibitor0.8630.004
CYP2E1 substrate0.8560.004
Membrane integrity agonist0.8660.020
CYP2E substrate0.8500.004
Vanillyl-alcohol oxidase inhibitor0.8400.002
CYP2A substrate0.8410.005
Ubiquinol-cytochrome-c reductase inhibitor0.8250.024
G-protein-coupled receptor kinase inhibitor0.8070.012
Beta-adrenergic receptor kinase inhibitor0.8070.012
MMP9 expression inhibitor0.7970.003
Gluconate 2-dehydrogenase (acceptor) inhibitor0.7970.017
Membrane permeability inhibitor0.7810.013
5 Hydroxytryptamine release stimulant0.7730.017
Apoptosis agonist0.7430.011
Cardiovascular analeptic0.7360.005
MAP kinase stimulant0.7350.004
CYP2C8 inhibitor0.7330.004
Fatty-acyl-CoA synthase inhibitor0.7240.010
CYP2C substrate0.7250.015
TP53 expression enhancer0.7240.021
Respiratory analeptic0.7150.014
CYP1A2 substrate0.7090.008
HMOX1 expression enhancer0.7050.008
CYP1A substrate0.7010.011
CYP2C12 substrate0.7340.051
CDP-glycerol glycerophosphotransferase inhibitor0.7170.049
1 Probability to be active (Pa > 0.7). 2 Probability to be inactive.
Table 4. Drug-likeness and ADME properties predicted by in silico studies using SwissADME online webserver of eugenol, the main compound identified in the leaf essential oil from Pimenta dioica collected from the National Botanic Garden, Havana, Cuba.
Table 4. Drug-likeness and ADME properties predicted by in silico studies using SwissADME online webserver of eugenol, the main compound identified in the leaf essential oil from Pimenta dioica collected from the National Botanic Garden, Havana, Cuba.
Predicted ParameterEugenolPentamidine
Physico-Chemical Properties:
Molecular weight164.2340.4
Hydrogen bond acceptors24
Hydrogen bond donors14
Number of rotatable bonds310
Topological polar surface area (Å)29.5118.2
Molar refractivity49.1100.7
Absorption Parameters:
Consensus LogS−2.46−3.26
Consensus Log P2.252.72
Solubility classSolubleSoluble
Drug Likeness Prediction:
Lipinski (RO5)00
Ghose00
Veber00
Egan00
Muegge10
Bioavailability
Bioactivity score0.550.55
Synthetic accessibility1.582.38
Distribution Parameters Prediction
Log Kp (cm/seg)−5.69−6.56
GI AbsorptionHighHigh
BBB PermeantYesNo
Metabolism Parameters Prediction
P-Glycoprotein substrateNoNo
CYP1A2, CYP2C19,
CYP2C9, CYP2D6 and
CYP3A4 inhibitors		No,
except to CYP1A2		No,
except to CYP2C9 and CYP2D6
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Monzote, L.; Machín, L.; González, A.; Scull, R.; Gutiérrez, Y.I.; Satyal, P.; Gille, L.; Setzer, W.N. Eugenol-Rich Essential Oil from Pimenta dioica: In Vitro and In Vivo Potentialities against Leishmania amazonensis. Pharmaceuticals 2024, 17, 64. https://doi.org/10.3390/ph17010064

AMA Style

Monzote L, Machín L, González A, Scull R, Gutiérrez YI, Satyal P, Gille L, Setzer WN. Eugenol-Rich Essential Oil from Pimenta dioica: In Vitro and In Vivo Potentialities against Leishmania amazonensis. Pharmaceuticals. 2024; 17(1):64. https://doi.org/10.3390/ph17010064

Chicago/Turabian Style

Monzote, Lianet, Laura Machín, Adiel González, Ramón Scull, Yamilet I. Gutiérrez, Prabodh Satyal, Lars Gille, and William N. Setzer. 2024. "Eugenol-Rich Essential Oil from Pimenta dioica: In Vitro and In Vivo Potentialities against Leishmania amazonensis" Pharmaceuticals 17, no. 1: 64. https://doi.org/10.3390/ph17010064

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop