Essential Oils of Rosmarinus officinalis and Eucalyptus globulus Cultivated in the Mountainous Region of the State of Rio de Janeiro (Brazil): Chemical Profile and Antileishmanial Activity

The present study aimed to characterize the chemical profile of essential oils of Rosmarinus officinalis (EORo) and Eucalyptus globulus (EOEg) cultivated in the mountainous region of the state of Rio de Janeiro, Brazil, and to verify the leishmanicidal potential, as well as to determine the cytotoxicity in vitro in mammalian cells. EORo had α-Pinene (37.99%), Cineol (21.48%), D-Verbenone (5.78%) and Limonene (3.28%) as major constituents. The EOEg had as major components Cineol (75.52%), Limonene (8.82%) and o-Cymeno (7.81%). The EOEg had a lower IC 50 value of 14.03 ± 2.08 µg/mL, while EORo had an IC 50 value of 31.12 ± 4.6 µg/mL, both during the 24-hr exposure period. In the 48-hr period, the IC 50 value was almost twice as high with rosemary oil, while in eucalyptus oil there was no significant difference when exposed in this period. The selectivity of EOs was demonstrated in murine cells, showing almost 30 times less toxic to the cell than to the parasite. It can be concluded that this is a pioneering study on the potential of these essential oils against the leishmania parasite. Furthermore, the low toxicity effect on the host cell encourages future studies and in vivo applications.


INTRODUCTION
Leishmaniasis is a spectrum of infectious parasitic diseases caused by protozoa of the genus Leishmania.The disease is considered as Neglected by the World Health Organization (WHO) because it mainly affects low-income people, there is a lack of investment in research, and few interest from the pharmaceutical industries in the development of new and more effective treatments. [1]eishmaniasis is endemic in 98 (sub)tropical countries, with about 12 million people infected and more than 350 million people at risk. [2]Approximately 95% of Cutaneous Leishmaniasis (CL) cases occur in the Americas, the Mediterranean region, the Middle East and Central Asia.In 2020, more than 85% of new cases of CL occurred in 10 countries, including the Old World and the Americas. [2]e use of chemotherapy drugs is still the main option for the treatment of leishmaniasis.Pentavalent antimony is the first choice in the treatment of infection, although greater resistance has been shown in some endemic areas. [3,4]As a second treatment step, miltefosine, amphotericin B and their liposomal formulations have also been used.Several disadvantages limit the use of these chemotherapeutics, such as high cost, serious adverse effects (e.g.cardiotoxicity and hepatotoxicity) and increased parasite resistance. [4,5]Thus, it is necessary and urgent to develop new drugs and therapeutic alternatives for the treatment of leishmaniasis.
Natural products have been used for the treatment of several diseases over the centuries. [6]In this context, plants have been one of the main sources of drug discovery and development of new compounds for several diseases due to the production of secondary metabolites and bioactive molecules, [7,8] such as Essential Oils (EOs).Chemically, EOs are characterized as a complex mixtures of low molecular weight compounds, being highly volatile and responsible for aromas. [9]The volatile compounds can be easily extracted from secretory glands present on the surface of leaves and flowers or inside roots and stems. [10]Different plant species have shown EOs with potential biological activities, including antiprotozoal, [11,12] which makes the evaluation of its leishmanicidal activity interesting.
Secondary metabolites represent a chemical interface between plants and the surrounding environment, and the biosynthesis of these compounds is often affected by environmental conditions.Thus, in this work, essential oils extracted by hydrodistillation from Rosmarinus officinalis and Eucalyptus globulus cultivated in the mountainous region of the state of Rio de Janeiro (Brazil) were obtained and the a effect were evaluated in vitro.
Rosmarinus officinalis Linnaeus (Lamiaceae), is a woody perennial herb, routinely known as Rosemary, native to the Mediterranean region, but is now cultivated all over the world.It is used as an ornamental and aromatic plant, condiment but also has a pharmacological potential.5] Eucalyptus globulus Labill. is an evergreen tree, native to Australia and widely cultivated for the pulp industry. [16]Belonging to the Myrtaceae family, eucalyptus is commonly used to treat asthma and bronchitis.Recent studies have demonstrated antimicrobial, antifungal, anthelmintic and antidiabetic properties for leaf extracts and essential oils. [17]erefore, the objective of this study was to characterize the chemical profile of essential oils produced from plants grown in the mountainous region of the state of Rio de Janeiro, Brazil, and evaluate the potential of the antileishmania activity/selectivity, as well as to determine the in vitro cytotoxicity of Rosmarinus officinalis (EORo) and Eucalyptus globulus (EOEg) essential oils.

Plant Material and Essential oil
Rosmarinus officinalis and Eucalyptus globulus were grown at Nova Friburgo, State of Rio de Janeiro, Brazil, and the EOs were produced by Entrefolhas Óleos Essenciais e Produtos Naturais LTDA, in July 2019.Nova Friburgo has a tropical climate of altitude, with cool and dry winters and pleasant and humid summers.The average temperature of the municipality is 18°C, relative humidity of 80% and annual precipitation of 1279.8mm. [18]

Essential Oil Chemical Identification
The analyses were performed using a gas chromatograph (GC/ MS from Thermo Scientific, Bremen, Germany), model TRACE 1310 / spectrometer TSQ-9000 mass model, with the TriPlus RSH self-sampling under the chromatographic analysis conditions described in Table 1.The data was acquired in scan mode (SCAN) in the range of 40 to 550 Da m/z, and cutting the solvent made at 5.5 min.The characterization of the oils and the attribution of the data was performed from the Thermo software Scientific Chromeleon Chromatography Data System (CDS) (version 7.2.10),The characterization of the oils and the attribution of the data was performed from the Thermo software Scientific Chromeleon Chromatography Data System (CDS) (version 7.2.10), and the compounds were experimentally identified by comparison of spectra, with the databases of the library NIST 05 and based on the comparison of the retention indices of the compounds, calculated from a pattern of n-alkanes from C7 to C24, with the indices available in the literature.

In vitro leishmanicidal Assay
Leishmanicidal assays were performed with L. amazonensis promastigotes (2 × 10 6 /mL) according to Rodrigues et al. [19] Parasites were distributed into 96 wells plates, 1 x 10 5 parasites per well.EOs were diluted in dimethylsulfoxide (DMSO) and 199 medium at maximal DMSO concentration of 0.05%.Then, EOs were submitted to serial dilution 125 to 0.5 μg/mL.As a positive control, the parasites were cultured without extracts and/ or DMSO.Pentamidine (Sigma) was diluted at 15.6 to 0.5 μg/ mL concentrations and was used as the standard drug.All tests were performed in three independent experiments.Plates were incubated for 24 hr or 48 hr of 26°C, until the analysis of parasites viability with Presto Blue (Invitrogen) 10μL/well.After 2hr of incubation with the reagent, plates were analyzed at 530 nm with plate reader equipment (BioTek, Winooski, VT, United States).
The percentage of growth inhibition was calculated as follow: The EOs were always tested in triplicates of each concentration, and the parameters for calculating the 50% inhibitory concentration (IC 50 ) were obtained by the Origin Lab 8.0 software, applying nonlinear regression to a sigmoidal curve.

Raw 264.7 macrophages Cytotoxic Assays
The cytotoxic effects of the EOs were evaluated in Raw 264.7 cells, according to Lall et al. with modifications. [20]Macrophages were seed in RPMI1640 medium supplemented with 10% FBS in 96-weel plates culture at 1 x 10 5 macrophages per well.Cells were treated with EOs at 125 to 0.5 μg/mL concentrations for 24 hr at 37°C in the presence of 5% CO 2 .Then, Presto Blue (10 μL/well) was added, and after 2hr the cells viability was analyzed as described in promastigote assays.The 50% cytotoxic concentration (CC 50 ) was calculated with the Origin Lab 8.0 software and the selectivity indices (SI) were calculated according to Rodrigues et al. [19] Statistical Analysis

RESULTS AND DISCUSSION
The chemical composition determined by GC/ MS are presented in Table 2 and 3.The chromatograms can be seen in the Figures 1  and 2. In the essential oil of Rosmarinus officinalis (EORo), it was possible to identify fifty-three compounds, representing 99.75% of the total detected constituents.The major components were α-Pineno (37.99%),Cineol (21.48%)D-Verbenone (5,78%) and Limonene (3.28%).Approximately 150 compounds have been described in the work carried out essential oils of Rosmarinus officinalis, and the majority compounds are 1,8-cineole (Cineiol), α-pinene, camphor, bornyl acetate, borneol, camphene, α-terpineol, limonene, β-pinene, β-caryophyllene and myrcene. [15]hus, the compounds found in our study corroborate the data from the literature and even the compound Verbenone was described in some works. [21,22]So, the chemical composition of essential oils depends on the species studied age, variety, the part collected, origin, climate, soil, agrochemicals used, stocking time, preparation and other factors, [15] and Rosmarinus from different part of the world differ from each other quantitatively.
Considering the studies already conducted with Eucaliptus, it can be observed that the essential oil of this species are usually rich in monoterpenes and in some cases sesquiterpenes and when we evaluate the chemical composition, these oils are complex mixtures ranging from 20 to 80 compounds, differing in their concentrations. [16]However, the common characteristic among studies is that the majority compound is 1,8-cineole (Cineol), and in most of these studies, cineol represents more than 80% of the chemical composition.In this study, cineol was around 75%, followed by compounds Limonene, o-Cineno and γ-Terpineno.In a study with essential oil extracted from Eucaliptus globulous in the state of São Paulo, Brazil, the chemical composition was 1,8 Cineol (83.89%), (+)-limonen (8.16%), α-pineno (4.15%), o-cymeno (2.93%), and γ-terpinene (0.87%).( 23) Thus, 1.8 cienol is the majority compound but minority compounds may alter among some components already described in the genus.
In Figure 3A, treatment with the two essential oils tested showed a dose-dependent inhibition effect on the proliferation of L. amazonensis promastigotes.The EOEg had a lower IC 50 value of 14.03 ± 2.08 µg/mL, followed by the EORo with an IC 50 value of 31.12 ± 4.6 µg/mL, all during the 24-hr exposure period.In the 48-hr exposure period, the IC 50 value was about twice as high with rosemary oil, showing no significant difference as compared to EOEg at that time (Figure 3B).
Essential oils were also evaluated for their potential toxic effects on the host cell, the macrophage strain Raw 264.7.The 50% cytotoxic concentration (CC 50 ) was evaluated in the 24-hr treatment period, with CC 50 values of 79.96 ± 7.16 and 908.60 ± 14.2 µg/mL.In this case, the host cell toxicity values were five to almost thirty times higher than the 50% parasite inhibition concentration (Table 4).The efficiency of the EO activity was evaluated by the Selectivity Index (SI).The SI results for L. amazonensis showed that EORo was 29.19 times less toxic to the macrophage than to the protozoan.While the reference drug used (Pentamidine) had a lower SI of 9.39 (  [24] As for the EO of Rosmarinus officinalis, in this same study, the values were 89.7 ± 2.0 (IC 50 ), 83.4 ± 7.3 (CC 50 ) µg/mL and SI<1.

Eucalyptus globulus
Compound RT (min) Relative area (%) RI (Experim.)RI (Literat.)Zauli, et al.: Essential oils with Antileishmanial Activity oil had no activity against L. major promastigotes (IC 50 282 µg/ mL). [27]e potential leishmanicidal effects of EOEg and Ro observed in this work may be due to the presence in greater amounts of Cineol compounds in the case of EOEg and α-Pinene in the case of EORo.Santana et al., [28] demonstrated that these compounds inhibited the survival of intracellular parasites of L. amazonensis in a dose-dependent manner with IC 50 of 48.4 μg/mL and 37 μg/mL, respectively.In contrast, the pure compound 1,8 Cineol had no in vitro effect against L. infantum, L. tropica and L. major. [29]Leishmanicidal activity has also been described in the literature with compounds found here in smaller amounts, such as Limonene, [30] and Cimeno, [28] against different species of the parasite.

CONCLUSION
The results of this study showed the variety of chemical composition of the essential oils of Rosmarinus officinalis and Eucalyptus globulus collected in this Brazilian region, highlighting the compounds Cineol and α-Pineno.Appreciably, essential oils are complex mixtures of compounds, and biological activities cannot necessarily be attributed to individual components.Furthermore, it revealed the in vitro anti-Leishmania effect against promastigotes of the L. amazonensis species, with a high selectivity index, that is, a low cytotoxic effect against the host cell.This enables future studies and possible in vivo applications.

Figure 3 :
Figure 3: Effects of EOEg, EORo, and Pentamidine on the survival of L. amazonensis promastigotes.The cell viability of promastigotes was evaluated after (A) 24 hr or (B) 48 hr of incubation with different concentrations of EOEg, EORo, and Pentamidine.The IC 50 was obtained through non-linear regression of means and the percentage of growth inhibition was calculated as described in material and methods.Cultures were tested in triplicates, and the results shown are the average of at least three independent experiments.

Table 4 :
In vitro antileishmanial activity, cytotoxicity and selectivity index.The concentration needed to inhibit 50% of the Leishmania promastigotes (IC 50 ) viability, as well as the selectivity index (SI), which was calculated by the ratio between the CC 50 and IC 50 values.

Table 1 : Instrumental parameters of analysis.
Zauli, et al.: Essential oils with Antileishmanial Activity

Table 4 )
. Demonstrating then, the selectivity of the oils tested against this Leishmania species, one of the Eucalyptus and Rosmarinus have been little reported in the world and absent in Brazil.A study carried out with commercial EO produced in the USA and France was cited with EO from Eucalyptus radiata with an IC 50 value of 164.7 ± 8.3 µg/mL, CC 50 of 100.2 ± 8.4 µg/mL and SI<1 versus L. amazonensis.