Essential Oil of Cymbopogon nardus (L.) Rendle: A Strategy to Combat Fungal Infections Caused by Candida Species

Background: The incidence of fungal infections, especially those caused by Candida yeasts, has increased over the last two decades. However, the indicated therapy for fungal control has limitations. Hence, medicinal plants have emerged as an alternative in the search for new antifungal agents as they present compounds, such as essential oils, with important biological effects. Published data demonstrate important pharmacological properties of the essential oil of Cymbopogon nardus (L.) Rendle; these include anti-tumor, anti-nociceptive, and antibacterial activities, and so an investigation of this compound against pathogenic fungi is interesting. Objective: The aim of this study was to evaluate the chemical composition and biological potential of essential oil (EO) obtained from the leaves of C. nardus focusing on its antifungal profile against Candida species. Methods: The EO was obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). Testing of the antifungal potential against standard and clinical strains was performed by determining the minimal inhibitory concentration (MIC), time-kill, inhibition of Candida albicans hyphae growth, and inhibition of mature biofilms. Additionally, the cytotoxicity was investigated by the IC50 against HepG-2 (hepatic) and MRC-5 (fibroblast) cell lines. Results: According to the chemical analysis, the main compounds of the EO were the oxygen-containing monoterpenes: citronellal, geranial, geraniol, citronellol, and neral. The results showed important antifungal potential for all strains tested with MIC values ranging from 250 to 1000 μg/mL, except for two clinical isolates of C. tropicalis (MIC > 1000 μg/mL). The time-kill assay showed that the EO inhibited the growth of the yeast and inhibited hyphal formation of C. albicans strains at concentrations ranging from 15.8 to 1000 μg/mL. Inhibition of mature biofilms of strains of C. albicans, C. krusei and C. parapsilosis occurred at a concentration of 10× MIC. The values of the IC50 for the EO were 96.6 μg/mL (HepG-2) and 33.1 μg/mL (MRC-5). Conclusion: As a major virulence mechanism is attributed to these types of infections, the EO is a promising compound to inhibit Candida species, especially considering its action against biofilm.

The retention time (t R ) of citronellal was 11.2 min by GC-FID analysis. The equation and R 2 value obtained from the analytical curve for citronellal were y = 571,529.9016x´102,555.3281 and 0.99976. The concentration of citronellal in the EO (GC-FID) was determined by means of the external standardization method as 282.5 mg/mL. Considering the relative density of commercial EO at 25˝C-0.897 g/mL (Sigma-Aldrich, St. Louis, MO, USA, 2016), the concentration of citronellal in the EO can be expressed as 31% (m/m). This value is consistent with the value obtained in the GC-MS analysis (28%).  1 Arithmetic retention indices [20] relative to C7-C30 n-alkanes calculated [21]; 2 Arithmetic retention indices [20,21].
Unlike conventional antimicrobial drugs, the literature does not present a standard of MIC values (sensitive and resistant) for natural products against Candida species. A study performed by Webster et al. [26] that evaluated antifungal activity of 14 medicinal plant extracts, found MIC values equal to, or lower than, 1000 µg/mL. The authors believe that values equal to or lower than 1000 µg/mL confirm sensitivity.
The values observed in this study were satisfactory, as the EO exhibited inhibitory action against 90% of the strains tested. Although some strains were inhibited with the highest concentration evaluated (1000 µg/mL), these data are relevant, since most of the strains are resistant to fluconazole (MIC > 64 µg/mL), the main drug used in the medical practice [27].
Interestingly, the MIC values (500 to 250 µg/mL) of the EO against the C. krusei ATCC clinical isolate are promising, due to the fact that this species presents intrinsic resistance to azoles [28].
The study carried out by Nakahara et al. [12] demonstrated that the EO inhibited filamentous fungus from the environment, however, the methodology used to determine the MIC was different from this study.
Recent research by Trindade et al. [24] showed the antifungal activity of the EO against ATCC and clinical strains of C. albicans and C. tropicalisi, with MIC values ranging from 32 to 64 g/mL. The differences found are expected because factors, such as climate, region, and the time of harvest of C. nardus, in addition to the extraction method, can directly affect the characteristics and concentration of chemical compounds [29].
The assay used to determine the MFC showed that the fungicidal properties of EO against Candida species were capable of killing the fungal cells using the concentrations evaluated in this study.
The antifungal activity of terpenoids, one of the major groups of volatile secondary metabolites, is known in the pharmaceutical field [17]. Thus, the antifungal activity of the EO in this present study may be related to the monoterpenes identified in the GC-MS assay.
The anti-Candida potential of the terpenes, geraniol, and citronellol has been investigated previously, with effective inhibitory activity against C. albicans [18] and filamentous fungi of the Aspergillus species [30]. In addition, Mesa-Arango et al. [31] showed that oxygenated monoterpenes in the citral chemotype, such as geraniol, citral and citronellal, have antifungal activity against C. parapsilosis, C. krusei, Aspergillus flavus, and Aspergillus fumigatus.

Minimal Inhibitory Concentration and Minimal Fungicidal Concentration of Citronellal
The MIC and MFC of citronellal are shown in Table 3. Citronellal showed antifungal activity against C. albicans ATCC, C. krusei (ATCC and clinical strain), and C. glabrata (ATCC and clinical strain). The species C. tropicalis, C. parapsilosis, C. orthopsilosis, and C. albicans clinical strains were resistant to citronellal, with MIC > 1000 µg/mL. Thus, in this present investigation, EO had better antifungal activity compared to citronellal, probably owing to synergisms among the chemical compounds present in the EO.

Inhibition on Candida albicans Hyphae Growth
The results exhibited that the EO was able to inhibit the transition of C. albicans from yeast to the hyphal form. Microscopic observation of EO-treated fungal cells revealed an absence of filamentous cells in concentrations ranging from 1000 to 15 µg/mL (after 12 and 24 h) ( Figure 1).

Inhibition on Candida albicans Hyphae Growth
The results exhibited that the EO was able to inhibit the transition of C. albicans from yeast to the hyphal form. Microscopic observation of EO-treated fungal cells revealed an absence of filamentous cells in concentrations ranging from 1000 to 15 μg/mL (after 12 and 24 h) ( Figure 1). Some therapeutic approaches are used to combat C. albicans, including blocking the transformation of yeast cells to filaments. This morphological change is considered to be a virulence factor, and, the biological mechanism has been explored using several active ingredients against this fungal species [32].
The ability of C. albicans to form hyphae is a risk factor in infections because hyphae play an important role in further tissue invasion due to their ability to adhere to host epithelial and endothelial cells [1]. Therefore, the results of this work are promising, since the EO was able to inhibit this morphological transition.
Leite et al. [33] showed the action of citral, a mixture of two geometric isomers known as neral and geranial, that are found at high concentrations in the EO used in this study. These authors found that these components were able to inhibit pseudohyphae, chlamydospores, and blastoconidia at the concentration of 128 μg/mL over 48 h.
The ability to EO to interfere in hyphal formation was demonstrated for different species of fungi. Chen et al., [8] for example, proved that the oil was able to promote deformities in the hyphal structure of the fungus Alternaria.

Time-Kill Assay
The results showed that the EO inhibited the fungal growth of the different Candida species in a similar manner (Figures 2-7). The cell growth was constant until 24 h. After this, an exponential growth-CFU/mL-was noted which remained proportional for a long time. The strains CK-ATCC 6258, CK4, CG-ATCC 2001, GG3, CP1, CO-ATCC 96141, and CO1 presented a superior inhibitory behavior than amphotericin-B over 48 h.
The current literature does not present data on time-kill assays evaluating this EO against Candida species. Thus, this study has an innovator character. Ahmad and Viljon [25] observed synergic activity of EOs from the genus Cymbopogon with silver ions against fungi. Some therapeutic approaches are used to combat C. albicans, including blocking the transformation of yeast cells to filaments. This morphological change is considered to be a virulence factor, and, the biological mechanism has been explored using several active ingredients against this fungal species [32].
The ability of C. albicans to form hyphae is a risk factor in infections because hyphae play an important role in further tissue invasion due to their ability to adhere to host epithelial and endothelial cells [1]. Therefore, the results of this work are promising, since the EO was able to inhibit this morphological transition.
Leite et al. [33] showed the action of citral, a mixture of two geometric isomers known as neral and geranial, that are found at high concentrations in the EO used in this study. These authors found that these components were able to inhibit pseudohyphae, chlamydospores, and blastoconidia at the concentration of 128 µg/mL over 48 h.
The ability to EO to interfere in hyphal formation was demonstrated for different species of fungi. Chen et al., [8] for example, proved that the oil was able to promote deformities in the hyphal structure of the fungus Alternaria.

Time-Kill Assay
The results showed that the EO inhibited the fungal growth of the different Candida species in a similar manner (Figures 2-7). The cell growth was constant until 24 h. After this, an exponential growth-CFU/mL-was noted which remained proportional for a long time. The strains CK-ATCC 6258, CK4, CG-ATCC 2001, GG3, CP1, CO-ATCC 96141, and CO1 presented a superior inhibitory behavior than amphotericin-B over 48 h.
The current literature does not present data on time-kill assays evaluating this EO against Candida species. Thus, this study has an innovator character. Ahmad and Viljon [25] observed synergic activity of EOs from the genus Cymbopogon with silver ions against fungi.

Biofilm
Biofilms produced by C. albicans, C. parapsilosis, and C. krusei were treated with EO (10ˆMIC). The EO showed expressive anti-biofilm activity at different concentrations (Table 4). The percentage of inhibition of biofilms by EO is demonstrated in Figure 8. The EO showed high inhibition of biofilms, mainly against C. albicans ATCC (97.7%), CA3 (82.0%), C. parapsilosis ATCC (93.6%), CP1 (86.2%), C. Krusei ATCC (65.0%), and CK4 (48.5%). Table 4. Anti-biofilm effect of the essential oil of C. nardus against C. albicans, C. krusei, and C. parapsilosis.   The anti-biofilm potential of the EO was the main result of the present study. The elimination and control of fungal biofilm is very hard, due to several types of molecular, structural, and specifically physiological interactions [4]. Additionally, the mechanism of resistance presented by planktonic cells, and the quorum-sensing processes (signaling molecules), the production of specific enzymes and natural mutations can explain the increased resistance of biofilm to antimicrobial agents used in the clinical practice [1].

Strains EO (mg/mL)
Biofilm is an important virulence factor and the treatment for its control is concentration dependent and can be high to inhibit the biofilm. The MIC to act against microbial biofilm ranges from 10-100 times higher than that necessary against the planktonic form [34].
The current results show that the EO was able to eliminate mature biofilm of C. albicans and C. parapsilosis at a concentration of 10× MIC. The scientific literature does not present any study of this EO against mature biofilm. Thus, these results are important and contribute to control strategies to eradicate mature biofilm.

Cytotoxic Evaluation
The concentrations of the EO and citronellal that inhibited cell vitality by 50% (IC50) are shown in Table 5. Citronellal demonstrated a higher IC50 than the EO. The EO exhibited inhibitory effects against HepG-2 and MRC-5 with IC50 values of 96.6 μg/mL and 33.1 μg/mL, respectively. The anti-biofilm potential of the EO was the main result of the present study. The elimination and control of fungal biofilm is very hard, due to several types of molecular, structural, and specifically physiological interactions [4]. Additionally, the mechanism of resistance presented by planktonic cells, and the quorum-sensing processes (signaling molecules), the production of specific enzymes and natural mutations can explain the increased resistance of biofilm to antimicrobial agents used in the clinical practice [1].
Biofilm is an important virulence factor and the treatment for its control is concentration dependent and can be high to inhibit the biofilm. The MIC to act against microbial biofilm ranges from 10-100 times higher than that necessary against the planktonic form [34].
The current results show that the EO was able to eliminate mature biofilm of C. albicans and C. parapsilosis at a concentration of 10ˆMIC. The scientific literature does not present any study of this EO against mature biofilm. Thus, these results are important and contribute to control strategies to eradicate mature biofilm.

Cytotoxic Evaluation
The concentrations of the EO and citronellal that inhibited cell vitality by 50% (IC 50 ) are shown in Table 5. Citronellal demonstrated a higher IC 50 than the EO. The EO exhibited inhibitory effects against HepG-2 and MRC-5 with IC 50 values of 96.6 µg/mL and 33.1 µg/mL, respectively. On comparing the results, the EO was less cytotoxic to HepG-2 than to MRC-5 cell lines. This can be explained by the metabolizer action of HepG-2. Although lower IC 50 values were found, it is important to stress that amphotericin-B, the gold standard in the treatment of fungal diseases, has toxic effects, such as nephrotoxicity, and has an acute reaction after intravenous infusions [5]. In vivo tests must be performed because factors, such as immune response, metabolism, and the pharmacokinetics of the EO, are important.

Extraction of the Essential Oil from the Leaves of C. nardus
Fresh leaves of C. nardus (150 g) were submitted to hydrodistillation using a Clevenger-type apparatus attached to a round bottom flask (3 L) with 1500 mL of deionized water. The residual water in the EO was separated from the sample by freezing. The yield of the EO was 0, 7% (w/w). The EO was stored under refrigeration until chemical analysis and biological tests.

Gas Chromatography-Mass Spectrometry
Gas chromatography-mass spectrometry (GC-MS) analysis was performed using an Agilent ® GC-7890B/MSD-5977A gas chromatograph (mass detector: electron impact ionization; mass quadrupole analyzer, (Agilent ® , Santa Clara, CA, USA) fitted with a HP-5ms capillary column 5% diphenyl-polydimethylsiloxane (30 mˆ0.25 mm, film thickness 0.25 µm-Agilent ® ). Helium was used as the carrier gas at a flow rate of 1.00 mL/min (8.2 psi) and linear velocity of 36.6 cm/s. Injector temperature: 250˝C; injection volume: 1 µL; splitting ratio: 1:100; oven temperature program: 60-246˝C (3˝C/min, 62 min); transfer line temperature: 280˝C; detector temperature: 150˝C; and ionization energy: 70 eV. EO was solubilized in hexane (chromatographic grade; Tedia ® , Fairfield, OH, USA) 1:100 (v/v). The identification of the EO components was based on the comparison of acquired mass spectra (from chromatogram peaks) with reference spectra of the NIST mass-spectral library version 2.0, 2012 (243,893 compounds) and data from the literature. Furthermore, arithmetic retention indices [20] were calculated as described in [21] by linear interpolation relative to the retention times (t R ) of a series of n-alkanes (C7-C30); the obtained values were compared with published retention index values [21]. Relative amounts of EO components were calculated based on the chromatogram peak area normalization method.

Determination of Minimum Inhibitory Concentration
The evaluation of the antifungal activity by determining the MIC was performed by the microplate dilution technique according to the protocol described in the M27-A3 document [35] with modifications. The concentration of the EO and citronellal was 7.8 to 1000 µg/mL. The EO was dissolved in 10% methanol and 2% Tween 80. A quantity of 0.1 mL was placed in a 96-well microtiter plate containing Roswell Park Memorial Institute (RPMI) 1640 medium. Each well was inoculated with 0.1 mL of a suspension containing 2.5ˆ10 3 CFU/mL of yeast.
Amphotericin B (AmB) (Sigma-Aldrich ® ) and fluconazole (FLU) (Sigma-Aldrich ® ) were used as the positive controls. Additional controls also included the culture medium, yeast growth, EO, and solvent. The microplates were incubated at 37˝C for 48 h. After incubation, 20 µL of an aqueous 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) was added, the plates were incubated at 37˝C for 2 h [32], and absorbance of the samples was measured by spectrophotometer (Biospectro, SP22, Curitiba, Brazil). All tests were performed in triplicate.
According to obtained results of the EO MIC determination, the more sensitive strains (one ATCC and one clinical strain of each species) were selected to evaluate the antifungal activity of citronellal.

Determination of Minimum Fungicidal Concentration
An aliquot from each well that showed antifungal activity was plated in Petri dishes containing Sabouraud Dextrose Agar (SDA)-DIFCO, to determine the minimum fungicidal concentration (MFC). The assays were carried out in triplicate. MFC was defined as the lowest concentration of the EO and citronellal that allowed no visible growth on the solid medium [32].

Inhibition of C. albicans Hyphae Growth
A microassay was developed to evaluate the inhibition effect on the growth of fungal strains. Growth of C. albicans (ATCC 10231) from a 48 h culture was transferred to a microplate with RPMI 1640 medium supplemented with fetal bovine serum (FBS) to obtain a final concentration of 2.5ˆ10 3 yeast/mL. EO was added to the growth medium at concentrations ranging from 7.5 to 1000 µg/mL, and the cultures were incubated for 12 and 24 h at 37˝C. The hyphal formation of C. albicans was observed through an inverted light microscope. Amphotericin B (16 µg/mL) was used as a positive control [32].

Time-Kill Assay
The time-kill assay was performed according to Santos-Filho et al. [36], with modifications. This assay tested one ATCC strain and one clinical strain of each Candida species (CA ATCC 90028, CA3, CK ATCC 6258, CK4, CG ATCC 2001, CG3, CT ATCC 13803, CT3, CP ATCC 22019, CP1, CO ATCC, and CO1). In brief, Sabouraud Dextrose broth (SDB)-DIFCO, containing 2.5ˆ10 3 CFU/mL of Candida spp. and 2ˆMIC of EO were incubated at 37˝C and aliquots of 100 µL were removed at different time intervals (0, 1, 2, 4, 8, 12, 24, 36 and 48 h). The aliquots were then diluted in a buffer solution of sterile PBS 1:100, twice. Each EO-cell suspension was spread onto Sabouraud plates and colonies were counted after 48 h incubation at 37˝C. Amphotericin B was used as a positive control. Negative controls were established with cell suspensions without the addition of EO.

Biofilm Assay
The biofilm adhesion method was performed as described by Pitangui et al. [37], with modifications. The CA ATCC 90028, CA3, CK ATCC 6258, CK4, CP ATCC 22019, and CP1 strains were selected for the biofilm assay. Initially, 100 µL of inoculum (5.0ˆ10 8 cells/mL), suspended in 0.9% saline solution, was added to the wells of microplates (96 wells) and incubated in a shaker at 80 rpm at 37˝C for 2 h. After the pre-adhesion period, the supernatant was removed and 100 µL of RPMI medium was added to each microplate well. Incubation continued at 37˝C for 48 h with the RPMI renewed after 24 h. The supernatant was then removed, and the wells were washed with 100 µL of 0.9% saline solution. Next, 100 µL of EO (10ˆMIC) were added to each microplate well. The microplates were incubated again for 24 h at 37˝C. Subsequently, the EO was removed and the wells were washed with sterile saline solution (to eliminate the drug carryover effect). Solvent, medium culture, and yeast growth were established as controls with the colorimetric indicator 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl(phenylamino)]-2H-tetrazolium hydroxide (XTT ® , Sigma-Aldrich ® , Saint Louis, MO, USA).

Cytotoxic Assay
The cytotoxic assay [38] consisted of collecting the cells using a solution of trypsin/ ethylenediaminetetraacetic acid (EDTA, Vitrocell ® ), centrifuging the solution (2000 rpm for 5 min) and counting the number of cells in a Neubauer chamber followed by adjustment of the cell concentration to 7.5ˆ10 4 cells/mL in DMEM. Then, 200 µL of this suspension was placed in each well of a 96-well microplate to obtain a concentration of 1.5ˆ10 4 cells/well, and the microplates were then incubated at 37˝C in 5% carbon dioxide for 24 h to facilitate cell attachment to the plate. The serial dilutions of EO were prepared to obtain concentrations from 3.90 to 1000 µg/mL. These dilutions were added to the cells after the removal of the medium and the non-adherent cells. Then, the cells were incubated for an additional 24 h. The cytotoxicity of the compounds was determined by adding 30 µL of resazurin and reading on a microplate reader (BioTek ® , Winoosky, VT, USA) after 6 h of incubation using a microplate and excitation emission filters with wavelengths of 530 and 590 nm, respectively. The IC 50 was defined as the highest concentration of compound that allowed a viability of at least 50% of the cells. All experiments were performed in triplicate. A solution of 5% dimethyl sulfoxide (DMSO) was used as the control.

Conclusions
According to the results of this study, it is possible to conclude that the EO from C. nardus is a promising source of active molecules with antifungal properties. The biological assays reported in this investigation show that the EO inhibits ATCC and clinical strains of Candida species, including those with resistance to drugs employed in medical practice. Additional to this simple inhibitory activity, the EO is able to inhibit and control the main virulence factors attributed to the Candida species used in this study, such as the formation and proliferation of hyphae of C. albicans and, more importantly, the eradication of mature biofilms. Moreover, the EO exhibits better antifungal action than citronellal, probably due to some synergistic effect among the EO components.