Chemical Compounds and Antimicrobial Assessment of the Essential oil from Lippia micromera Harvested in Cuba

Abstract

Background/Objective:

Anti-infective agents derived from plants have become important and favorable resources against diseases. In particular, essential oils have emerged as promising antimicrobial natural alternatives. The objective of this study is to evaluate the potentialities of Lippia micromera Schauer essential oil (EO-Lm) against a panel of bacteria, fungi, and protozoal parasites. Methods: Leaves of L. micromera were collected, the essential oils were obtained by hydrodistillation and analyzed by gas chromatography – mass spectrometry (GC-MS). Biological antimicrobial assays were performed and the cytotoxicity effect was determined on the MRC-5 cell line. Results: A total of 29 components (99.0% of the oil) were identified, which the main chemical compounds were thymol (36.0%), carvacrol (28.8%), p-cymene (13.2%), estragole (5.2%), γ-terpinene (4%), (E)-β-caryophyllene (3.7%) and myrcene (1.6%). No activity was observed against Escherichia coli, Staphylococcus aureus, Candida albicans, Trypanosoma cruzi, or Leishmania infantum. However, against Plasmodium falciparum, Trypanosoma brucei, and Leishmania amazonensis EO-Lm displayed IC₅₀ values of 7.5, 51.8 and 26.2 μg/mL, respectively. In addition, no cytotoxicity was observed at the maximum working concentration (64 µg/mL) on the MRC-5 cell line. Conclusions: This study shows, for the first time, the chemical composition and the antimicrobial activity of EO-Lm harvested in Cuba. In particular, the relevant activity observed on P. falciparum should be corroborated in further experiments using animal models.

Introduction

Phytochemicals and their associated bioactivities are presently a topic of enormous attention in the food, cosmetics, and pharmaceutical industries. ¹ Among them, higher plants have been utilized for treatment of numerous human maladies and to alleviate the proliferation of communicable diseases. Anti-infective agents derived from plants are particularly favorable because they are generally composed of a diverse range of biologically active components, ² and they are largely considered safer than synthetic agents.^3,4 There are an estimated 500,000 species of higher plants. However, very few of them have been investigated for their phytochemical compositions or their biological activities. ⁴ Nevertheless, a considerable number of drugs on the market have either been obtained from plants directly or have structures based on the phytochemicals, either from semi-synthetic derivatization or de novo synthesis. ⁵

Although plant-derived products such as essential oils (EOs) are frequently ignored in drug discovery, they do possess some desirable characteristics that include: (i) EOs are complex mixtures of relatively hydrophobic compounds, (ii) could contain as many as 250 components, (iii) synergism could be result of interaction between compounds or targeted different proteins/enzyme/pathways, and (iv) some of the most commonly found compounds are terpenes and phenylpropanoids.^6–9 Today, more than 3000 EOs have already been documented, but only around 300 have been shown to be commercially important in various industrial sectors. ¹⁰ However, there are concerns within the scientific community regarding the gap in understanding between the compositions and biological activities of EOs and their underlying mechanisms. In this context, we have been focused on antimicrobial assessment of EOs from Cuban plants to identify potential natural products with antibacterial, antifungal, or antiprotozoal activities.^11,12

The aim of this work was to provide new insights into the antimicrobial potential of Lippia micromera Schauer essential oil (EO-Lm). The genus Lippia (Verbenaceae family) comprises 250 species, most of which are distributed in the Neotropical ecozone as Central and South America and tropical Africa. ¹³ The plant has found culinary use as a seasoning as well as in traditional herbal medicine for treatment of pain, inflammation, gastrointestinal and genitourinary ailments, and respiratory diseases. ¹⁴ Lippia micromera is a strong aromatic shrub with many branches and tiny white flowers with yellow throats (see Figure 1). The origin of the plant has not been established, but it is believed to range from northern South America through the Caribbean and Central America. ¹⁵ The species is well known for culinary seasoning in northern South America, Central America, and the Caribbean, where the people especially tend to eat with meat in soup and gravy. ¹⁶ As a therapeutic, the plant displays spasmolytic muscle-relaxant, antibacterial, antimycotic, and antioxidative properties. ¹⁷

Figure 1.

Photograph of Lippia micromera Schauer Taken by Authors in the Natural Habitat.

Materials and Methods

Plant Material

The vegetal sample of L. micromera was collected in September 2014 in “La Quirubina” (22°57′27″N 82°35′47″W, 99 m elevation), Caimito Municipality, Artemisa Province, Cuba. A specimen was deposited in the National Botanic Garden, Havana, Cuba, and authenticated by M. Sc. Eldys Bécquer. The voucher specimen number of HFC-88350 was assigned. The leaves were selected and hydrodistilled for 4 h using a Clevenger's apparatus under laboratory conditions to give the essential oil, which was stored in a sealed amber vial at 4°C until analysis and screening.

Gas Chromatographic Analysis

The essential oil of L. micromera was analyzed by GC-MS as previously reported. ¹⁸ Identification of the components in the essential oil was based on comparison of mass spectra and retention index (RI) values.

Hierarchical Cluster Analysis and Principal Component Analysis

Hierarchical cluster analysis (HCA) was carried out to examine the similarity of studied EO-Lm with those reported in literature,^19–24 based on the percentages of 13 major components (Table 1). The eight essential oil samples were treated as operational taxonomic units (OTUs) and Pearson correlation was used to measure similarity. The unweighted pair-group method with arithmetic average (UPGMA) was used to define the clusters. Principal component analysis (PCA, type Pearson Correlation) was used to determine the interrelationship of essential oil components and to verify the HCA analysis. Both analyses (HCA and PCA) were performed using XLSTAT v. 2018.1.1.62926 (Addinsoft, Paris, France).

Table 1.

Major Components and Percentages of Lippia micromera Used in the Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA).

Compounds	This work	Escobar et al	Pino et al	Rojas et al	Scotto et al	Tucker et al	Ruiz-Duran et al	do Nascimento et al
α-Thujene	0.9	0.0	0.1	1.5	1.5	3.8	1.3	0.0
Myrcene	1.6	3.1	0.7	1.7	2.3	3.4	2.0	0.5
α-Terpinene	0.7	4.0	0.4	2.9	1.9	4.9	2.0	0.4
p-Cymene	13.2	13.1	7.2	10.2	18.7	11.5	26.8	3.0
γ-Terpinene	4.0	12.5	4.5	14.5	14.6	22.5	5.4	2.8
Estragole	5.2	0.0	0.7	0.0	0.0	0.0	0.0	0.0
Thymyl methyl ether	0.8	14.0	0.0	10.6	10.3	1.5	26.3	1.3
Thymol	36.0	29.1	0.2	33.7	8.3	7.1	17.8	62.8
Carvacrol	28.8	1.0	42.2	0.2	23.1	26.5	0.0	0.0
Thymyl acetate	0.2	0.0	0.0	1.6	0.0	0.2	5.7	7.2
(E)-β-Caryophyllene	3.7	1.9	12.1	7.0	8.0	3.0	1.7	8.8
trans-α-Bergamotene	0.2	0.0	9.3	0.2	0.3	0.3	0.0	0.2
α-Humulene	0.5	0.0	4.0	1.0	1.4	0.4	0.0	1.5
Caryophyllene oxide	1.1	0.5	9.0	0.9	1.9	0.0	0.9	1.1

Escobar et al, 2010, Colombia, ¹⁹ Pino et al, 1998, Cuba, ²⁰ Rojas et al, 2009, Venezuela, ²¹ Scotto et al, 2016, 2017, French Guiana,^22,23 Tucker et al, 1993, Virgin Islands, ²⁴ Ruiz-Duran et al, 2023, Colombia, ²⁵ do Nascimento et al, 2021, Pará, Brazil (Lippia thymoides, syn. Lippia micromera var. tonsilis). ²⁶

Antimicrobial Assessment and Cytotoxic Effects

The biological assays were performed using a 96-well plate format ²⁷ using protocols developed in our laboratories.^28–30 Each assay was carried out in duplicate. The EO-Lm was diluted in dimethylsulfoxide (DMSO) to create a stock solution (equivalent to 20 mg/mL). It was subsequently serially diluted to give concentrations ranging from 0.25 to 64 μg/mL using an automated liquid-handling workstation (Beckman Coulter Biomek 3000), in which the final DMSO concentration was < 1%. Antibacterial assays were carried out on Gram-negative Escherichia coli (ATCC8739) and Gram-positive Staphylococcus aureus (ATCC6538) cultured in Mueller Hinton broth (MHB; Sigma-Aldrich, St. Louis, MO, USA) medium; while for the antifungal experiment, the yeast Candida albicans (B59630) was used in RPMI medium. In all cases, plates with different EO concentrations (0.25 to 64 µg/mL) were seeded with 5 × 10³ CFU/well and incubated for 17 h at 37°C. Viability was determined fluorometrically by the addition of resazurin (Sigma-Aldrich, St. Louis, MO, USA), which an additional incubation for 30 min or 4 h under same conditions for bacteria or yeast cultures, respectively. ²⁷ Fluorescence was measured using a Tecan GENios Multifunction Fluorimeter (Tecan Group, Maennedorf, Switzerland) at 530 nm excitation and emission of 590 nm.

Antiplasmodial activity was determined on P. falciparum (K1 strain) cultured in human erythrocytes A + in RPMI-1640 culture medium, supplemented with 0.5% (w/v) Albumax at 37°C and an atmosphere of 3% O₂, 4% CO₂, and 93% N₂. ³¹ Suspensions of infected human red blood cells with 1% parasitemia and 2% hematocrit were added to each well along with EO-Lm at the serially-diluted concentrations. The plate was incubated under the same conditions. After 72 h of incubation, the plate was frozen at −20°C and parasite multiplication was measured after mixing in a new plate 20 µL of the hemolyzed parasite suspension with 100 µL of Malstat reagent (Flow Incorporated, USA). The plate was incubated at room temperature for 15 min and 20 µL of nitro blue tetrazolium chloride (NBT; Sigma Aldrich, St. Louis, MO, USA) at 2 mg/mL/phenazine ethosulfate (PES; Sigma Aldrich, St. Louis, MO, USA) at 0.1 mg/mL solution was then added. Finally, the plate was incubated at room temperature in the dark for an additional 2 h and the absorbance was read at 655 nm in a Biorad 3550-UV microplate reader. ³²

Antitrypanosomal screening was carried out using trypomastigotes of T. brucei (Squib-427), cultured in Hirumi-9 medium supplemented with 10% inactivated fetal calf serum (FCSi) at 37°C and 5% CO₂. ³³ A culture of 1.5 × 10⁴ trypomastigotes was added to each well with the different EO concentrations. After 72 h of incubation under the same conditions, parasite growth was determined fluorometrically by adding resazurin for 24 h at 37°C and measurement was carried out as previously described. On the other hand, 4 × 10⁴ amastigotes of T. cruzi (Tulahuen CL2) were added to in 4 × 10³ MRC-5 cells maintained in minimal essential medium (MEM; Life Technologies, USA) supplemented with 20 mM L-glutamine, 16.5 mM sodium bicarbonate and 5% of FCSi with the different dilutions of EO-Lm. After an initial seven-day incubation under the previous conditions, parasite growth was assessed by adding the β-galactosidase substrate chlorophenol red β-D-galactopyranoside (Sigma Aldrich, St. Louis, MO, USA). ³⁴

Antileishmanial activity screening against the intracellular amastigote form of L. infantum (MHOM/MA(BE)/67) and promastigotes of L. amazonensis (MHOM/77BR/LTB0016) were also carried out. For L. infantum, 3 × 10⁴ peritoneal macrophages from BALB/c mice (PMM) obtained by peritoneal washing in RPMI medium (Sigma, St. Louis, MO, USA) and antibiotics (100 µg of streptomycin/mL, 100 U of penicillin/mL; Sigma, St. Louis, MO, USA) were infected with amastigotes obtained from an infected hamster at a density of 15 parasites per cell. The plate was incubated for 48 h at 37°C and 5% CO₂ and the pre-diluted concentrations of EO-Lm were added. The plates were then incubated under the same conditions over a 120-h period. In the experiment with L. amazonensis, a monolayer of PMM (obtained after 2 h at 37°C and 5% CO₂ after seeded for at 10⁶/mL) was infected with stationary-phase promastigotes at a 4:1 parasite/macrophage ratio for 4 h. Cells were washed to remove free parasites and the EO-Lm test concentrations were added. The plates were incubated under the same conditions for an additional 48 h. ³⁵ In both cases, after the period of incubation with the test products, the supernatant was discarded and the cells were fixed with methanol, stained with 10% Giemsa and examined microscopically (Motic, Japan) under immersion oil. The total parasite burden was assessed based on the number of infected macrophages and the number of amastigotes inside the macrophages.

In all cases, negative (100% parasite growth) and positive (a reference drug) control were included. Included reference drugs were: chloramphenicol (Sigma-Aldrich, Bornem, Belgium) for E. coli, erythromycin (Sigma-Aldrich, Bornem, Belgium) for S. aureus, miconazole (Janssen Pharmaceuticals, Beerse, Belgium) for C. albicans, and chloroquine (donated by the Special Programme for Research and Training in Tropical Diseases from the World Health Organization (WHO-TDR)) for P. falciparum, suramine (donated by WHO-TDR) for T. brucei, benznidazol (donated by WHO-TDR) for T. cruzi, miltefosine (donated by WHO-TDR) for L. infantum and pentamidine (Richet, Buenos Aires, Argentina) for L. amazonensis. In each case, percent inhibition for each concentration of EO-Lm was determined based on the untreated controls. The median inhibitory concentration (IC₅₀) was determined in each antimicrobial assay from lineal dose-response curves.

For cytotoxicity evaluation, the MRC-5 cell line was used and viability was assessed fluorometrically after 72 h of incubation with tested products by the resazurin method. The median cytotoxic concentrations (CC₅₀) were obtained from lineal dose-response curves. Finally, the selectivity index (SI) was calculated by CC₅₀/IC₅₀.

Results

Essential Oil Composition

Leaves of L. micromera were collected in Artemisa Province, Cuba, and the EO was obtained by hydrodistillation. Gas-chromatography coupled with a mass spectrometric detector (GC-MS) was used to study the composition of the EO and compound identification was based on chromatographic (linear retention indices, LRI) and spectroscopic (mass spectral comparison with databases) criteria. In the EO-Lm, a total of 29 compounds were identified that represented 99.0% of the total oil composition (Table 2). The major chemical constituents of the EO were thymol (36.0%), carvacrol (28.8%), p-cymene (13.2%), estragole (5.2%), γ-terpinene (4%), (E)-β-caryophyllene (3.7%) and myrcene (1.6%). The remaining components showed concentrations below 1%.

Table 2.

Chemical Compositions (Percentages) of the Leaf Essential Oils of Lippia micromera Harvested in Artemisa, Cuba.

RT (min)	RIcalc	RIref	Compound	%
7.11	935	930	α-Thujene	0.9
9.23	981	979	1-Octen-3-ol	0.3
9.74	992	990	Myrcene	1.6
10.81	1015	1017	α-Terpinene	0.7
11.29	1026	1024	p-Cymene	13.2
12.81	1058	1059	γ-Terpinene	4.0
14.74	1099	1096	Linalool	0.3
16.91	1144	1145	Ipsdienol	0.1
18.36	1175	1177	Terpinen-4-ol	0.7
18.92	1186	1188	α-Terpineol	0.2
19.47	1198	1196	Estragole (= Methyl chavicol)	5.2
21.09	1227	1235	Thymyl methyl ether	0.8
21.48	1236	1244	Carvacryl methyl ether	0.1
22.47	1255	1252	Geraniol	0.1
23.72	1290	1290	Thymol	36.0
24.41	1307	1299	Carvacrol	28.8
26.55	1355	1352	Thymyl acetate	0.3
27.32	1372	1372	Carvacryl acetate	0.3
29.29	1420	1419	(E)-β-Caryophyllene	3.7
29.87	1434	1434	trans-α-Bergamotene	0.2
30.59	1451	1454	α-Humulene	0.5
31.52	1474	1479	γ-Muurolene	tr
32.49	1498	1500	Pentadecane	tr
32.84	1506	1505	β-Bisabolene	tr
33.41	1522	1523	δ-Cadinene	tr
35.53	1572	1578	Spathulenol	tr
35.75	1581	1583	Caryophyllene oxide	1.1
36.69	1605	1608	Humulene epoxide II	0.1
46.25	1875	1875	1-Hexadecanol	0.1
			Monoterpene hydrocarbons	20.3
			Oxygenated monoterpenoids	67.7
			Sesquiterpene hydrocarbons	4.4
			Oxygenated sesquiterpenoids	1.1
			Benzenoid aromatics	5.2
			Others	0.3
			Total identified	99.0

RT = Retention time in minutes. RI_calc = Retention index calculated with respect to a homologous series of n-alkanes on an HP-5 ms column using the linear equation of van den Dool and Kratz. ³⁶ RI_ref = Reference retention index obtained from the Adams database. ¹⁸ % = percentage based on total ion current (TIC) peak integrations. tr = trace (< 0.05%).

Most phytochemical investigations of Lippia species have been aimed at essential oil analyses. However, L. micromera has been relatively understudied in this respect. ¹³ Nevertheless, with the purpose of comparing the essential oil compositions of L. micromera, multivariate analyses were carried out between the studied EO and those reported in literature from other geographic locations. Hierarchical cluster analysis (HCA) showed two well-defined groups (Figure 2). There is a group rich in thymol and a group rich in carvacrol. A principal component analysis (PCA) corroborates the HCA and clearly shows the correlations between the EOs with their major components (Figure 3). Note that EO-Lm (this work) has a strong correlation with both thymol and carvacrol.

Figure 2.

Dendrogram acquired by agglomerative hierarchical cluster analysis (HCA) based on the major essential oil components from Lippia micromera harvested in Artemisa, Cuba, and those reported in scientific literature, based on Pearson correlation, and using the unweighted pair-group method with arithmetic average (UPGMA). (Escobar et al, 2010, Colombia, ¹⁹ Pino et al, 1998, Cuba, ²⁰ Rojas et al, 2009, Venezuela, ²¹ Scotto et al, 2016, 2017, French Guiana,^22,23 Tucker et al, 1993, Virgin Islands, ²⁴ Ruiz-Duran et al, 2023, Colombia, ²⁵ do Nascimento et al, 2021, Pará, Brazil ²⁶ ).

Figure 3.

The two-dimensional plot (biplot) of the first two principal components (PC1 and PC2) from principal component analysis (PCA, type Pearson Correlation) of Lippia micromera based on 13 major essential oil components (Escobar et al, 2010, Colombia, ¹⁹ Pino et al, 1998, Cuba, ²⁰ Rojas et al, 2009, Venezuela, ²¹ Scotto et al, 2016, 2017, French Guiana,^22,23 Tucker et al, 1993, Virgin Islands, ²⁴ Ruiz-Duran et al, 2023, Colombia, ²⁵ do Nascimento et al, 2021, Pará, Brazil ²⁶ ). The blue oval encompasses the carvacrol-rich group and the red oval encompasses the thymol-rich group.

Antimicrobial Assessment and Cytotoxic Effects

In order to assess the antimicrobial potentialities of EO-Lm, a wide collection of microbes was tested, including: bacteria (Escherichia coli and Staphylococcus aureus), fungus (Candida albicans), and protozoal parasites (Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei, Leishmania amazonensis, and Leishmania infantum). In general, no activity was observed against the bacteria or the fungus at the highest tested concentration (64 µg/mL). However, antiprotozoal activity was observed (Table 3). Recently, an analysis of differential antimicrobial effects of EOs and their main components on bacteria, fungi, and protozoans demonstrated that protozoans are more susceptible to EOs. ³⁷ The most relevant activity was observed against causal agent of malaria, P. falciparum (K1 strain).

Table 3.

Antiprotozoal Assessment of the Essential oil from Lippia micromera Harvested in Artemisa, Cuba.

Products	IC50 (μg/mL)
	Plasmodium Falciparum	Trypanosoma cruzi	Trypanosoma brucei	Leishmania amazonensis	Leishmania infantum
Essential oil	7.5 ± 0.4	> 64	51.8 ± 2.6	26.2 ± 1.3	> 64
Reference drug	0.020 ± 0.001	0.82 ± 0.04	0.050 ± 0.002	1.30 ± 0.06	3.68 ± 0.20

IC₅₀: Median inhibitory concentration with a 95% confidence interval obtained from nine independent assays carried out in duplicate. Reference drug: pentamidine for L. amazonensis. Reference drug used are: chloroquine for P. falciparum, benznidazol for T. cruzi, suramine for T. brucei, pentamidine for L. amazonensis, and miltefosine for L. infantum.

In consideration of the cytotoxicity tests on the MRC-5 cell line, no cytotoxicity was observed at the maximum working concentration (64 µg/mL). Similar results have been reported for EO-Lm collected in Colombia, in which a safety profile was confirmed on different cell lines. For example, against the Vero cell line the CC₅₀ was 93.6 µg/mL; while on THP-1 did not display cytotoxic effects at 100 µg/mL. ¹⁹ Thus, the selectivity index (SI) values showed that EO-Lm exhibited a selective antiplasmodial effect that was higher than 9.

Discussion

Essential Oil Composition

The number of constituents and the relative percentages of compounds above 1% showed variation based on geographical collection site. Previously, the leaf essential oil obtained from L. micromera grown in Cuba showed 34 components, which carvacrol was the main component with 42%. ²⁰ In contrast, EO-Lm from plants collected in Colombia displayed thymol as the major component (29.1%), while carvacrol was observed in only 1.0%. ¹⁹ These differences in composition could be due to predominant chemotypes. It is known that EO composition depends not only on plant genetic factors,^38,39 but also includes various factors that depend on physiological and environmental conditions. In particular, growth periods, seasons, ⁴⁰ geographical location, ⁴¹ harvesting technique, ⁴² edaphic factors, ⁴³ herbivory, ⁴⁴ and climate^41,43 could affect the qualitative composition, as well as the accumulation of active components that cause quantitative differences. ⁴⁵

Interestingly, the HCA shows that the two samples from Cuba (this work and Pino et al ²⁰ have very dissimilar compositions (only 17% similarity). The samples from Colombia ¹⁹ and from Venezuela ²¹ are very similar (96% similarity) and dominated by thymol. Likewise, the samples from French Guiana^22,23 and from the Virgin Islands ²⁴ have similar compositions (86% similarity), but are rich in carvacrol.

The PCA biplot clearly shows the separation between the EO sample points and reveals that the compounds carvacrol and thymol are the major contributors to the overall cluster separation. The PCA of the composition data showed that the two principal components, PC1 and PC2, explained 82.27% of the phytochemical variation in the L. micromera samples. The PC1 accounted for 51.42% of the variation and showed a positive correlation with both carvacrol and thymol. The PC2 explained 30.86% of the variation and had a positive correlation with carvacrol, but a negative correlation with thymol. The samples can be divided into two groups, the carvacrol-rich group (23.1-42.2% carvacrol) and the thymol-rich group (17.8-62.8% thymol).

Antimicrobial Assessment and Cytotoxic Effects

Overall, the antimicrobial screening demonstrated that the most relevant activity observed was on P. falciparum, the causal agent of malaria. This parasitic disease continues to be a major cause of morbidity and mortality. In 2015, the World Health Organization (WHO) estimated that 3.2 billion people were at risk of becoming infected with malaria, with 214 million worldwide cases, leading to 438,000 mortalities. Ninety percent of the deaths occurred in the WHO African region. ⁴⁶ Artemisinin-based combination therapies are the currently available treatment options. Although these have proven useful, they have led to drug resistance. ⁴⁷ Thus, the search for low-toxicity and effective compounds against malaria is necessary. With this finding, the strain of P. falciparum used in this study was K1, which is resistant, in vitro, to chloroquine, pyrimethamine, and sulfadoxine. ⁴⁸ This observation emphasizes the importance the observed antiplasmodial activity of EO-Lm.

No previous reports of antiplasmodial activity were found for EO-Lm. However, Lippia is a genus that has showed potential antiplasmodial activity. In this sense, EOs from Lippia sidoides Cham., ⁴⁹ Lippia origanoides Kunth, ²⁹ and Lippia javanica (Burm.f.) Spreng. ⁵⁰ showed IC₅₀ values of 10.5, 14.4, and 8.0 µg/mL against P. falciparum, respectively. Note, however, that the antiplasmodial activities of Lippia essential oils are lower than that for chloroquine.

In addition, the observed inhibitory antiplasmodial effects of EO-Lm in this study could be attributable to the main essential oil components. Modern methods of phytochemical analysis have led to isolation and identification of numerous biologically active components from medicinal plants, a valuable contribution to contemporary medicine. In general, the biological activities, including antimicrobial activities, of medicinal plants are due to their secondary metabolites, which are found is smaller quantities than the primary metabolites such as lipids, proteins, and carbohydrates. ⁵¹ In particular, the main compounds thymol (36.0%) and carvacrol (28.8%) showed antiplasmodial activity with IC₅₀ values of 4.5 μg/mL ⁴⁹ and 6.3 μg/mL, ²⁸ respectively. Recently, Kumar and collaborators highlighted the activity of thymol against chloroquine-sensitive (NF54) and -resistant (K1) P. falciparum strains, displaying IC₅₀ values of 12 µM (1.8 μg/mL) on both strains. ⁵² In addition, the other minor bioactive compounds could contribute to the antiplasmodial activity. For example, estragole ⁴⁹ and (E)-β-caryophyllene ⁵³ also displayed activity on P. falciparum with IC₅₀ values of 30.7 and 12.8 µg/mL, respectively. Furthermore, components in trace amounts may contribute to the biological activity through synergistic effects.^54,55

Related to kinetoplastid protozoal parasites, divergent results were also observed. While T. cruzi and L. infantum were not sensitive, T. brucei and L. amazonensis displayed some level of activity. Escobar and collaborators reported that EO-Lm from plants cultivated in Colombia (thymol chemotype) displayed antiparasitic activity against T. cruzi and L. chagasi with IC₅₀ values of 60.7 and 51.8 μg/mL, respectively, ¹⁹ while the EO from plants in French-Guyana (carvacrol chemotype) the antileishmanial activity seems negligible against L. guyanensis.^22,23 Thus, our results together with previous scientific reports suggest that EO-Lm could demonstrate some effects on Trypanosomatidae, although some factors could affect the activity such as the chemotype of the EO. Additionally, the antileishmanial and antitrypanosomal activities of EO-Lm are lower than the reference drugs (positive controls). Nevertheless, it is likely that the parasites of this family are not highly susceptible to EO-Lm and the different effects are due to species specificity.

In the same way, different results have been reported for compounds present in EO-Lm related with their antikinetoplastic activities. For example, thymol showed an IC₅₀ of 17.3 μg/mL against L. donovani, ⁵³ 21.2 μg/mL against L. mexicana and 16.6 μg/mL against T. cruzi, ³⁷ while carvacrol displayed an IC₅₀ value of 13.1 μg/mL against L. donovani, ⁵³ 79.7 μg/mL against L. mexicana, and 36.4 μg/mL against T. cruzi. ³⁷ Previously, we did not observe activity of carvacrol on T. brucei, T. cruzi, or L. infantum, although an IC₅₀ value of 28.8 µg/mL was obtained on L. amazonensis. ²⁸ Escobar and collaborators reported that carvacrol and thymol were active on promastigotes of L. chagasi (IC₅₀ = 28 and 65.2 µg/mL), but both compounds were inactive on the amastigote form (IC₅₀ > 30 µg/mL). ¹⁹ Therefore, similar to EO-Lm, it appears that the activities of the main compounds could also be species/stage-dependent. In the absence of toxicity on mammalian cell lines in this study and those reported in the literature, we can suggest the safety of EO-Lm and indicates a selectivity of the EO for P. falciparum parasites.

As mentioned previously, the main identified compounds in the EO-Lm were terpenoids, which are organic compounds containing isoprene subunits. In general, the mechanism of action proposed for terpenoids such as thymol and carvacrol can be related to membrane disruption. ⁵⁶ Both thymol and carvacrol are hydrophobic, enabling them to disintegrate cell membranes, increasing permeability, releasing cellular components, or interacting directly with critical intracellular sites.^57,58 However, Andrade-Ochoa and collaborators suggest that external structures and cell membrane characteristics in the different microbial groups are determining factors for their susceptibilities to EOs and major EO constituents. ³⁷ Nevertheless, the diverse compounds in the EO-Lm could led to multiple mechanisms of action affecting not only plasma membrane, but also intracellular organelles and other metabolic pathways.

Currently, one of the most pressing challenges in malaria therapy is the development of drug resistance, which fewer effective treatment options would remain available. ⁵⁹ However, essential oils are multicomponent mixtures with up to several hundred individual components. These diverse compounds with different functional groups can exhibit multiple-target activities, which would hinder development of resistance. ⁶⁰ In line with the mentioned perspective, our research is ongoing to promote products that have demonstrated comparable activities in both sensitive and resistant strains. However, the main limitation of this pharmacological perspective is that our studies were carried out in vitro; additional in-vivo research is needed.

Conclusions

This study shows, for the first time, the essential oil components and the antimicrobial activity of EO-Lm harvested in Cuba. The chemical composition and comparison with EO-Lm reported in the literature demonstrated that the studied chemotype was thymol, but with a high concentration of carvacrol. Additional EOs compositions from other geographical locations in its natural range are needed to more completely define the chemotypes of this plant. In addition, the most relevant activity was observed on P. falciparum and in vitro cytotoxicity assays suggested no or low toxicity. In this sense, further research is needed to examine potential antimalarial effect of EO-Lm in infected mice, as well as to evaluate the development of resistance to the EO-Lm.