Essential Oils from Leaves of Medicinal Plants of Brazilian Flora: Chemical Composition and Activity against Candida Species

Background: The biotechnological potential of medicinal plants from Brazilian Caatinga and the Atlantic Forest has not been extensively studied. Thus, screening programs are important in prospecting for compounds for developing new drugs. The purpose of this study was to determine the chemical composition and to evaluate the anti-Candida activity of essential oils from leaves of Hymenaea courbaril var. courbaril, Myroxylon peruiferum, and Vismia guianensis. Methods: The oils were extracted through hydrodistillation and their chemical compositions were analyzed by gas chromatography coupled with mass spectrometry. Antifungal activity against C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, and C. krusei was evaluated by determining the minimal inhibitory (MIC) and fungicidal (MFC) concentrations. Results: The major compounds of the oils were caryophyllene oxide and trans-caryophyllene for H. courbaril; spathulenol, α-pinene, and caryophyllene oxide for M. peruiferum; and caryophyllene oxide and humulene epoxide II for V. guianensis oil. The oils showed antifungal activity against all the strains tested, and the MIC values ranged between 0.625 and 1.25 μL/mL and MFC from 0.625 to 2.5 μL/mL. Conclusion: The essential oils from the species studied have the potential to be evaluated as clinical applications in the treatment of candidiasis.


Introduction
Candidiasis is the most frequent infection caused by opportunistic fungi, with a very extensive spectrum ranging from mild manifestations (e.g., a colonization of mucosal tissues) to systemic infections with the invasion of different organs [1]. Candida albicans is the most common causative agent in humans, but the species Candida tropicalis, Candida parapsilosis, Candida glabrata, and Candida krusei are also commonly associated with serious infections [2][3][4]. In addition, C. glabrata and C. krusei are described as having intrinsic resistance to some of the more conventional antifungal drugs [5,6].
Azole antifungal agents are the most used in therapeutic regimens for the treatment of candidiasis. However, these antibiotics are not always successful because of the significant incidence of microorganism resistance [7]. This has stimulated the search for new antifungal agents in order to increase the number of alternatives to treat infections and minimize the impact of resistance.
Natural products have been traditionally used to treat many diseases, including fungal infections, and plant compounds have been explored by the pharmaceutical industry for use as antimicrobial agents or as models for the synthesis of new drugs [8]. Essential oils, which consist in liquid, are volatile, and are rarely colored mixtures of compounds soluble in organic solvents, are among these potential alternatives [9,10]. The essential oils are usually constituted by some components at high concentrations (20-70%), being deemed majoritary compounds, while the other components are present in trace amounts [11].
Plants from Caatinga and Atlantic Forest have also been used to obtain natural medicines by Brazilian local populations [12]. However, despite the rich flora of these biomes, the biotechnological potential of plants as sources of antifungal agents has not been extensively studied. Thus, screening programs are very important in order to prospect for compounds for developing new antimicrobial drugs.
This study was conducted with essential oils from three medicinal plants of Brazilian flora. Hymenaea courbaril var. courbaril (Fabaceae) has a broad distribution being found in Brazil at Atlantic and Amazon Forests, Caatinga, Cerrado, and Pantanal. It has several medicinal uses; for example, the stem bark decoction or syrup can be used against coughs and anemia, and leaf infusion can be used against urinary tract disorders [13,14]. Myroxylon peruiferum (Fabaceae) is found at the Brazilian Atlantic Forest and Cerrado and has heavy wood that is resistant to deterioration. Its bark is used as an anti-inflammatory, and the fruits are used to alleviate earache [15,16]. The leaves of Vismia guianensis (Hypericaceae) are used against problems in spine and kidneys as well as to alleviate pains in general [17]. This plant can be found in the Amazon and Atlantic Forests [13].
The aim of this work was to determine the chemical composition of essential oils from leaves of H. courbaril var. courbaril, M. peruiferum, and V. guianensis and to evaluate the antifungal activity of them against Candida species.

Plant Materials
Leaves of V. guianensis were collected in December 2012, at the nature reserve of Camaçari

Extraction of Essential Oils
The powders of leaves were separately submitted to hydrodistillation in a Clevenger-type apparatus for 4 h. The essential oils obtained were then dried over an anhydrous sodium sulfate and stored in sealed vials protected from the light at −20 • C. The essential oil emulsions used in antifungal assays were prepared as follows: 400 µL of essential oil plus 40 µL of Tween 80 and 4.56 mL of sterile distilled water were combined and shaken for 5 min using a vortex, obtaining emulsions with a final concentration of 80 µL/mL.

Chromatography Analyses
Gas chromatography (GC) analyses were performed in order to determine the relative proportions of the components of the oils. GC analyses were carried on a Thermo TraceGC Ultra (Thermo Scientific, Milan, Italy) equipped with a flame ionization detector (FID) (Thermo Scientific, Milan, Italy) and a VB-5 fused silica capillary column (ValcoBond 30 m × 0.25 mm i.d.; film thickness: 0.25 mm) (Valco Instruments Company Inc. , Houston, TX, USA). Nitrogen was employed as a carrier gas at a flow rate of 1 L/min and 30 psi inlet pressure. The oven temperature program was initially 40 • C, was held for 2 min, was increased to 230 • C at 4 • C/min, and was then held for 5 min. The injector and detector temperatures were set to 250 • C and 280 • C, respectively. The sample (1 µL; 2 mg/mL in n-hexane) was injected splitless. The relative amount of each component was estimated from the corresponding peak area and expressed as a percentage of the total area of the chromatogram. Analyses were carried out in triplicate.
The identification of the compounds present in the oils was performed by GC coupled with mass spectrometry (GC-MS). These analyses were carried out using an Agilent 5975C Series GC/MSD (Agilent Technologies, Palo Alto, CA, USA) quadrupole instrument equipped with an Agilent J&W non-polar DB-5 fused silica capillary column (30 m × 0.25 mm i.d.; film thickness: 0.25 µm) (Agilent Technologies, Palo Alto, CA, USA). For each sample, 1 µL was injected in split mode (50:1) with the injector temperature set to 250 • C. GC oven temperature was set to 40 • C, was held for 2 min, was increased to 230 • C at 4 • C/min, and was then held for 5 min. Helium (He) was used as a carrier gas at a flow of 1 mL/min, maintained at a constant pressure of 7.0 psi. MS Source and quadrupole temperatures were set to 230 • C and 150 • C, respectively. Mass spectra were taken at 70 eV (in EI mode) with a scanning speed of 1.0 scans from m/z 35-350. The identification of the individual components was carried out by comparison with previously reported values of retention indices, obtained by co-injection of oil samples and a set of C 9 -C 30 linear hydrocarbons, and calculated according to the equation of Van den Dool and Kratz [18]. Subsequently, the MS data acquired for each component were matched with the data available in the mass spectral library of the GC-MS system (MassFinder 4, Dr. Hochmuth scientific consulting, Hamburg, Germany); NIST08 Mass Spectral Library (ChemSW Inc. Fairfield, CA, USA); Wiley Registry™ of Mass Spectral Data 9th Edition (Wiley, Hoboken, NJ, USA) and with other published mass spectral data [19]. Analyses were carried out in triplicate.

Antifungal Assays
Strains of C. tropicalis (URM-6741), C. krusei (URM-6391), C. albicans (URM-6543), C. parapsilosis (URM-6557), and C. glabrata (URM-6393) were obtained from the University Recife Mycologia (URM) from the Departamento de Micologia of Universidade Federal de Pernambuco. The yeasts were grown overnight at 30 • C in Petri plates containing Sabouraud dextrose agar (Merck, Darmstadt, Germany), and inocula for use in the assays were prepared by diluting scraped cell mass in 0.85% NaCl, adjusting to 0.5 in McFarland. Cell suspensions were finally diluted to 10 6 CFU/mL for use in the assays.
The minimal inhibitory concentrations (MICs) of the essential oils were determined through the microdilution technique. Firstly, 100 µL of Sabouraud dextrose broth (Merck, Darmstadt, Germany) were taken into the wells of a 96-well microtiter plate. The first well of each row was used as a sterility control and contained only culture medium. Next, 100 µL of the emulsion of oil emulsion (80 µL/mL) were placed in the second well of a row and two-fold serial dilution was then performed until a concentration of 0.04 µL/mL was reached. In each well, aliquots of 10 µL of the microorganism inoculum were dispensed. In parallel, a negative control containing only microorganism and medium was run to assure the viability of the yeast strains. It was also performed a positive control using the antifungal fluconazole (Sigma-Aldrich, St. Louis, MO, USA). Assays were performed in triplicate and plates were incubated at 30 • C for 48 h. After this period, 10 µL of the dye 2,3,5-triphenyl tetrazolium chloride (0.5%, w/v) was added in each well to detect bacterial growth. The MIC was defined as the lowest oil concentration that promoted inhibition of yeast growth.
After the determination of the MIC, aliquots from the wells where there was growth inhibition, as well as from positive and negative controls, were subcultured on Petri plates containing Sabouraud dextrose agar (Merck). After 24 h at 30 • C, the microbial growth was observed and the number of CFU was counted. The minimal fungicide concentration (MFC) was defined as the lowest oil concentration that reduced the number of CFU in 99.9% in comparison with the initial inoculum.
According to the MFC/MIC ratio, the antifungal activity was classified as follows: if MFC/MIC = 1 or 2, the effect was considered fungicidal; if MFC/MIC ≥ 4, the effect was defined as fungistatic.

Results
The yields of oil extraction were 0.975%, 0.276%, and 0.306% for H. courbaril, M. peruiferum, and V. guianensis, respectively. The chemical composition of the essential oils can be seen in Table 1. A total of 62 compounds were identified in the essential oils. The oils of the species H. courbaril, M. peruiferum, and V. guianensis were rich in mono and sesquiterpenes. The trans-caryophyllene, δ-cadinene and caryophyllene oxide were present in all the three oils. a Constituents listed in order of elution on a non-polar DB-5 column. b Retention indices (RI) calculated from retention times in relation to those of a series of C 9 -C 30 n-alkanes on a 30 m DB-5 capillary column. c Values taken from Adams [19]. d Value taken from Bos et al. [20].
All tested Candida species were sensitive to the essential oils and MIC values ranged from 0.625 to 1.25 µL/mL ( Table 2). C. albicans and C. krusei were more sensitive to the V. guianensis oil, while C. glabrata and C. parapsilosis were more sensitive to the H. courbaril oil. All the oils showed fungicidal activity and all Candida strains were sensitive to the positive control fluconazole (Table 2). According to MFC/MIC ratios, the effect of all oils on the Candida species can be considered fungicidal.

Discussion
As mentioned before, mono and sesquiterpenes were the main compounds identified in the materials used in the present work, which is commonly reported for essential oils [21]. Andrade et al. [22] evaluated an essential oil from M. peruiferum bark and found a chemical composition highly distinct from that found for the leaf oil. None of the majoritary compounds of the leaf oil was present in the bark oil, and two of the major compounds of the bark oil (α-copaene and δ-Cadinene) were detected in the leaf oil but in small concentrations. A similar situation is observed when comparing the composition of an essential oil from H. courbaril ripe fruit peel [23], and the leaf oil of H. courbaril var. courbaril that we described. The main components of the fruit oil were α-copaene, spathulenol, and β-selinene (11.1%, 10.1%, and 8.2%, respectively), which were only present in small concentrations in the leaf oil (0.41%-1.99%).
All essential oils showed antifungal activity. The concentration range at which the essential oils were active was close to that reported for the essential oil of Myrtus communis leaves against Candida species [24]. The mechanisms of antifungal activity of essential oils seem to involve the disruption of cell membrane structure causing leakage and cell death, blocking of membrane synthesis, and cellular respiration as well as inhibition of the spore germination [25]. An essential oil from Coriandrum sativum leaves, containing the mono-and sesquiterpene hydrocarbons decanal, trans-2-decenal, 2-decen-1-ol, and cyclodecane as major compounds, was active against C. albicans probably by binding to the membrane ergosterol, by increasing ionic permeability, and by decreasing proteolytic activity [26]. The authors also demonstrated that this oil did not interfere with cell wall biosynthesis pathways and was most active than isolated fractions, which suggest a synergistic effect between the oil components.
The antifungal activity of H. courbaril var. courbaril, M. peruiferum, and V. guianensis oils can be linked to the major compounds caryophyllene oxide and trans-caryophyllene. Caryophyllene oxide was proven to be an antifungal agent against C. albicans [27], and Vieira et al. [28] showed that essential oil from Piper diospyrifolium, rich in trans-caryophyllene, was active against clinical isolates of C. albicans and C. parapsilosis. Caryophyllene oxide also showed antifungal activity against dermatophytes compared to ciclopirox olamine and sulconazole [29].
Antifungal activity has been also reported for oils containing other compounds found in the oils analyzed here. Spathulenol and bicyclogermacrene (both found in M. peruiferum oil) were the main constituents of the essential oil from leaves of Croton argyrophylloides, which was active against C. albicans, Candida tropicalis, and Microsporum canis [30]. An essential oil from the rhizome of Ferula hermonis containing mainly α-pinene (also present in M. peruiferum oil) was active against Candida albicans, Candida lactis-condensi, Saccharomyces cerevisiae, Tricophyton mentagrophytes, Microsporum gypseum, Aspergillus niger, Aspergillus fumigates, and Penicillium purpurogenum [31]. The authors also reported that the compounds α-pinene and spathulenol were active against C. albicans when tested separately.

Conclusions
The essential oils from three plants commonly used in Brazilian folk medicine were effective antifungal agents against Candida species. The antifungal activity of the oils is probably linked to the action of compounds such as caryophyllene oxide and trans-caryophyllene.