Antifungal and cytotoxic activity of purified biocomponents as carvone , menthone , menthofuran and pulegone from Mentha spp

Medicinal plants are attractive sources in the search of bioactive compounds in the treatment of infectious diseases. Considering that the infectious agent often develops resistance to existing treatments rapidly, the searches for such compounds are a never-ending process. One such infection, caused by Candida spp. is candidiasis, which is a public health problem. Additionally, many strains have resistance to traditional therapies. Therefore, we tested the antifungal activity of four compounds present in Mentha spp. with promising antifungal precedents. We measured inhibition of growth by microdilution, disruption of biofilm viewed by electron microscopy, inhibition of germ tube formation by optical microscopy and toxicity on HaCaT cells. Tests showed that the compounds tested had antifungal activity with a minimum inhibitory concentration of 0.5 mg/mL, at least, 50% of biofilm inhibition in the 0.5 mg/mL concentration, an inhibition of polymorphism to 86% and the changes in the cell envelope of yeast (SEM) and cell viability above 50% among the Candida strains tested. Therefore, the compounds exhibit promising antifungal properties and provide a reasonable therapeutic window to be used in association with other traditional antimitotic.

morphological transition where the yeast cell forms filaments that aids in infection of the host (Jacobsen et al., 2012); and (2) biofilm formation, which reduces the susceptibility of microorganisms to various immunological and chemotherapeutic modes of action, contributing to persistent infection (Chandra and Mukherje, 2015).
In order, to combat the increase of microorganisms resistant, it becomes necessary to continually develop new classes of antifungal compounds.Plant extracts provide a promising source of molecules, due to the long history of mutual tolerance and co-regulation between plants and fungi (Saharkhiz et al., 2012).We chose to focus our attention on the essential oil of Mentha spp.because it has been shown to contain a number of bioactive compounds, including those that may have antimicrobial activity (Schelz et al., 2006;Mkaddem et al., 2009;Zore et al., 2011).
We evaluated carvone, menthone, menthofuran and pulegone present in Mentha spp.against yeasts of the genus Candida and how they affect growth and survival, germ tube formation and biofilm formation and maintenance.In addition, we evaluated the toxicity of these compounds on HaCaT cells.

MATERIALS AND METHODS
Planktonic anti-Candida assay, minimum fungicidal concentration (MFC) and inhibition of germ tube formation were performed with three independent experiments; biofilm and cytotoxicity assay were performed with two independent experiments in triplicate.All tests were performed include positive control (without compounds) and negative control (without cell suspension).

Planktonic anti-Candida assay
The assay planktonic anti-Candida (Table 1) was made according to the CLSI, 2008-M27-A3 reference method.In summary, yeast was inoculated in Sabouraud Dextrose Agar (SDA) culture medium and incubated aerobically at 37° C for 24 h.After that, was diluted to 2.5×10 3 CFU/mL in RPMI culture medium and measured at A530.In microtiter plates, were made dilution series with compounds in RPMI (concentration range from 8 to 0.125 mg/mL) and added the cell.Fluconazole was tested (concentration range 64 to 0.5 µg/mL).The plates were incubated for 48 h at 37°C in aerobic conditions, followed by visual reading to evaluate cell growth.The concentrations that no showed cell growth was considered minimum inhibitory concentration (MIC).

Minimum fungicidal concentration (MFC)
The fungal viability after incubation with test compounds (concentration range 16 to 0.125 mg/mL) was performed by plating an aliquot of the MIC plate well contents to SDA plates and incubating at 37°C for 48 h.MFC was determined as the lowest concentration of a given compound that did not allow the growth of any fungal colony on SDA after the incubation period.The read was made visually (Gullo et al., 2012).

Inhibition of germ tube formation
The compounds were tested at sub-MIC concentrations (Table 2).Candida albicans MYA-2876 cells were grown on SDA for 24 h at 37°C under aerobic conditions and adjusted to 2.5×10 7 CFU/mL in PBS (1x) and Neubauer chamber under optical microscope (magnification 400x).The cell suspension was added a 1:1 mixture of RPMI culture medium (with diluted compounds) and fetal bovine serum.The cells count that forming germ tubes were conducted using an optical microscope at 400 x magnifications at intervals of 2, 4 and 6 h of aerobic incubation at 37°C and compared to positive control.Values were calculated as percentage (Consolaro et al., 2005).

Inoculum adjustment
C. albicans MYA-2876 were incubated in yeast extract peptone dextrose (YPD) liquid medium for 24 h in orbital shaker (30 rpm) at 30°C.An aliquot of 7 mL of the inoculum was centrifuged at 10.8 g for 4 minutes.This process was performed twice with a phosphate buffered saline (PBS) (1x) wash step.The pellet was resuspended in 7 mL of RPMI.Cell concentration was determined using a Neubauer chamber under optical microscope (magnification 400x); the suspension was diluted to 1.0×10 6 CFU/mL in RPMI (Pierce et al., 2008).

Biofilm formation
The cell suspension (100 µL) was added to a sterile microtiter plate with U-shaped wells.The cell suspension were incubated for 2 h under agitation (100 rpm) at 37°C.After incubation, the plate was washed 3 times with saline (0.9%).Solutions of the compounds (concentration range 16 to 0.125 mg/mL) were added to the wells.The cells were incubated for 24 h at 37°C under aerobic conditions.After incubation period, were compared to the positive control (Da Silva et al., 2010).

Mature biofilm
The cell suspension (100 µL) was added to a sterile microtiter plate with U-shaped wells.The cells were incubated aerobically at 37°C for 24 h.Solutions of the compounds (concentration range 16 to 0.125 mg/mL) were added to the wells.The suspension cell with compounds was incubated for 24 h at 37°C under aerobic conditions.After incubation period, were compared to the positive control (Pierce et al., 2008).

Scanning electron microscopy (SEM) of the biofilm
The biofilm formation and mature biofilm samples (in 0.5 mg/mL concentration) were grown in culture slides (BD Falcon).After the incubation period, the supernatant was discarded and biofilm fixed with glutaraldehyde (2.0%) for 30 min followed by drying ambient temperature.Specimens were dehydrated with increasing concentrations of ethanol (50, 70, 90 and 100%) for 10 min each.
After that, specimens were dried, metallized with gold and observed by SEM (JEOL JSM 5600LV) to view the biofilm structure in the compounds presence.The images were selected randomly.

Cytotoxicity of the compounds
HaCaT cells (epithelial cells from normal human keratinocytes immortalized but not transformed) were grown in RPMI/FBS and adjusted to 6.5×10 4 CFU/mL in Neubauer chamber under optical microscope (magnification 400x).Posteriorly, 100 µL of the cells was added to microtiter plate and incubated for 24 h at 37°C and 5% CO2.Subsequently, the compounds (concentration range 16 to 0.125 mg/mL) were added in the cells and incubated for 24 h under the same conditions.The cells were fixed with trichloroacetic acid (TCA) 10% and incubated for 1 h at 4°C.The plates were washed with distilled water (3x), dried at ambient temperature and stained with sulforhodamine B (SRB, 0.4) dissolved in 1% aqueous acetic acid, washed 4 times with distilled water, and then treated with SRB in 10 μM TRIZMA pH 8.0.The cell suspension were measured at A530 in microplate reader to evaluate cell viability after treatment with the compounds (Endo et al., 2010) and reported as IC50%, the concentration of the compound that induces 50% lysis or cell death (Liu et al., 2016).

Statistical analysis
The biofilm and cytotoxicity tests were analyzed by the Dunnett statistical test (ANOVA) using the software Bioestat 5.0; a p<0.05 was considered statistically significant.

Planktonic anti-Candida assay
The compounds tested showed inhibitory activity against nearly all Candida spp.for the concentration range tested, with the exception of menthone for C. tropicalis CBS 94 (Table 1).Carvone and pulegone showed inhibitory activity at lower concentrations compared to menthone and menthofuran.A commercially available antifungal agent tested was Fluconazole, to compare the results of inhibition obtained by isolated compounds of essencial oils obtained from Mentha spp.Fluconazole showed inhibitory activity only in some strains tested.

Minimum fungicidal concentration (MFC)
Carvone and pulegone expressed MFC for the strains tested in lower concentrations compared to the other compounds.The concentration of 2 mg/mL of carvone and 1 mg/ml of pulegone showed most fungicidal activity inhibiting most species tested (Table 1).

Inhibition of germ tube formation
Pulegone showed the greatest efficiency inhibiting until 86% germ tube formation compared to other compounds and positive control as shown in Table 2.

Action of compounds on biofilm
The compounds tested prevented at least 50% adhesion, inhibited progress of biofilm formation, and damaged of mature biofilms at low concentrations: 0.250 mg/mL for carvone and pulegone; 1 mg/ml for menthone and 0.5 mg/mL for menthofuran compared to the positive control (Figure 1).SEM assay was made to view the action of the compounds on biofilm.The images obtained showed changes in cells as pore formation, roughness and presence of leakage of cellular contents after treatment with the compounds.The results of biofilm formation are shown in Figure 2 and mature biofilm in Figure 3.

Cytotoxicity of the purified compounds
Cytotoxicity evaluation of the compounds in HaCaT cells using menthone and menthofuran showed cell viability above 50% at concentrations 4 to 0.125 mg/mL; carvone and pulegone showed cell viability above 50% at concentrations of 8 to 0.125 mg/mL compared to positive control (Figure 4).

DISCUSSION
In planktonic anti-Candida assays, all compounds showed antifungal activity, however, carvone and pulegone inhibited the growth of most strains in less concentrations compared to other compounds.These results indicate a greater antifungal potential of carvone and pulegone against these species.The results obtained with the commercial antifungal Fluconazole did not exhibited antifungal activity against some species tested, including clinical isolates.These results corroborate with Ramesh et al. (2010) demonstrating that clinical isolates strains of Candida spp.were resistant to Fluconazole.In this sense, the results obtained was satisfactory because the compounds showed antifungal activity in species that Fluconazole did not showed, suggesting that such compounds may be a possible source for new drugs, especially as adjuvant.Minimum fungicidal concentration was performed to confirm cell death in MIC through observation of no colony growth in solid medium and classify fungistatic or fungicidal activity by compounds (Gullo et al., 2012).Carvone and pulegone expressed MFC for the strains tested in lower concentrations demonstrating most fungicidal character compared to the other compounds.
We also observe that the compounds tested acted in virulence factors of Candida spp.such as hyphae and biofilm.The germ tube formation is the beginning of the growth of hyphae (Ellepola and Saramayake, 2001).We observed that compounds reduced germ tube formation.These results are important because this form is related to the invasiveness of the fungus to the host tissue (Jacobsen et al., 2012;Mayer et al., 2013).In biofilm tests, the compounds reduced biofilm formation and deconstructed the mature biofilm at low concentrations tested.Considering that biofilms are associated with approximately 80 of infections caused by Candida spp.(Tsang et al., 2012), such compounds can be promise as therapeutic agents against this virulence factor.
The antifungal action of the compounds can be attributed to their chemical class.Other members of the terpenoids are known to destabilize cell membranes and increase cellular permeability which permit the disruption and death of the microorganisms (Zore et al., 2011).The SEM assay was performed to evaluated the action of the compounds in C. albicans MYA-2876 biofilms.The images obtained showed changes in cells as the presence of pores and ridges in the cell envelope and extravasation of cellular contents confirming this hypothesis.
With the purpose to evaluate the therapeutic applicability of the compounds, we performed cytotoxicity assays on HaCaT epithelial cells.The compounds showed results with cell viability above 50% in the most part concentrations tested although high concentrations of some neared the 50% viability limit.The low cytotoxicity of compounds isolated from Mentha spp.has also been demonstrated by Amaral et al. (2015) who evaluated cytotoxicity of rotundifolone, a major constituent of the essential oil of Mentha x villosa against tumor cell lines HCT-116 and SF-295.The data obtained in the initial cytotoxicity assays indicate a potential therapeutic window for these compounds isolated to be used as antifungal agents.

Conclusion
The compounds tested modify the pathogenic Candida spp.fungus in many ways, including inhibition of growth, formation and maintenance of biofilms, as well as germ tube formation.Although each compound and strain has their unique profile of action or resistance, the low cytotoxicity and therapeutic effect of these compounds make them worthwhile candidates for complementing antifungal clinical protocols.

Table 1 .
MIC and MFC of the compounds tested against Candida spp.

Table 2 .
Inhibition of germ tube formation expressed in percentage.