Antifungal and cytotoxicity activities of Anacardium othonianum extract

Antifungal and cytotoxicity activities of Anacardium othonianum extract Flávia Maria Lígia Marangoni Jordão Curado, Ana Paula Gazolla, Rita Cassia Nascimento Pedroso, Letícia Pereira Pimenta, Pollyanna Francielli de Oliveira, Denise Crispim Tavares, Márcio Luis Andrade e Silva, Wilson Roberto Cunha, Rosemeire Cristina Linhari Rodrigues Pietro, Ana Helena Januário, Patrícia Mendonça Pauletti, Juliana de Fátima Sales and Fabiano Guimarães Silva*

Dermatomycoses are common dermatological conditions of the skin, hair and nails caused by superficial fungal infections, affecting more than 20-25% of the people in the world, mainly in tropical and subtropical regions.These diseases alter the individuals' quality of life and are considered a public health issue (Havlickova et al., 2008;Simonnet et al., 2011).The etiologic agents of dermatomycoses comprise dermatophytes, yeasts and non-dermatophytic filamentous fungi.Epidemiological studies have verified that most patients with superficial fungal infections had dermatophytes or yeast depending on the geographic location (Silva et al., 2014;Kemna and Elewski, 1996).The treatment available for dermatomycoses includes both topical and systemic antifungal drugs, such as the allylamines (terbinafine), triazoles (fluconazole, itraconazole and voriconazole), imidazoles (ketoconazole) and griseofulvin (Meis and Verweij, 2001).The resistance to the available therapeutic agents, for example, in the case of terbinafine, has led to the need for new drugs (Mukherjee et al., 2003).Plant crude extracts have demonstrated antifungal activity in many studies (Kuete et al., 2009;Mbaveng et al., 2008).
Antifungal effects have not been reported for A. othonianum.Therefore, herein, the authors described the antifungal activities of the crude ethanolic extract (EE); the n-hexane (HF), EtOAc (EF), n-BuOH (BF), and hydromethanolic (HMF) fractions; and isolated compounds of interest against the following microorganisms: Candida albicans and Trichophyton rubrun.Additionally, the cytotoxicities of these compounds were also evaluated.

General
1 H-and 13 C-NMR spectra were recorded in CD3OD on Bruker 400 and 500 NMR spectrometers using tetramethylsilane (TMS) as an internal standard.A positive-ion mode HRESIMS spectrum was obtained on a Bruker Daltonics HRMS ultrOTOF-Q-ESI-TOF instrument using electrospray ionization.Analytical and preparative HPLC analyses were performed on Shimadzu LC-20AD and LC-6AD systems, with a degasser DGU-20A5, an SPD-M20A series PDA detector or an SPD-20A series UV-VIS detector, a CBM-20A communication bus module, and a Reodyne manual injector or SIL-20A autosampler.A SHIMADZU Shim-pack ODS (particle diameter = 5 μm, 250 × 4.60 mm, and 250 × 20 mm) column equipped with a pre-column were used in the HPLC separations.The MeOH employed was of HPLC grade (J.T. Baker), and ultrapure water was obtained from Millipore Direct-Q UV3 system.Sample preparation was accomplished using silica gel 90 reverse-phase ODS (Fluka, 230-400 mesh).

Plant
The leaves of A. othonianum Rizz.were obtained from the Brazilian Cerrado in the Goiás State in the city of Montes Claros de Goiás (17°48′15.9ʹʹS and 50°54′19.5ʹʹW), in September 2012.A voucher specimen (HJ3793) was deposited in the Herbarium Jataiense Germano Guarim Neto of the Universidade Federal de Goiás, Brazil (Herbarium HJ).

Chromatographic studies
Chromatographic separations of the samples were carried out on a Phenomenex Gemini C18 (Octadecylsilane) (5 μm, 250 x 4.60 mm) column with a pre-column.For the experiments, the elution conditions were as follows: methanol/water (0.1% acetic acid) gradient from 5 to 100% methanol for 30 min, then elution with 100% methanol for 10 min, oven at 40°C, and flow = 1.0 mL/min.The analysis also includes 3 min to regress to the initial conditions and 15 min of equilibration.The chromatogram wavelength was set at 254 nm, and UV data were recorded between 190 and 500 nm.The 1-mg samples (EE, EF, BF and 1-4) were weighed and dissolved in 1 mL of methanol.The sample solutions were filtered and then transferred to a vial.

Antifungal assay
The following microorganisms were employed for evaluation of the antifungal activity: Candida albicans (ATCC 64548) and Trichophyton rubrum (Tr1, earlier identified by Profa.Dra.Ana Marisa Fusco Almeida).The T. rubrum strain was preserved and cultured in Petri dishes having Sabouraud agar.It was incubated and subcultured for 7 days at 28°C.The strain was suspended in 0.85% saline solution, counted in a Neubauer chamber and final concentration of 5.0 x 10 3 CFU/mL was achieved by dilution in RPMI medium (Ghannoum et al., 2006).The strain C. albicans was cultivated in RPMI-1640.The inoculum was adjusted to 0.5 McFarland scale to yield a cell concentration of 1×10 6 -5×10 6 yeast/mL (CLSI, 2008a).The extracts, fractions and compounds 1-4 were resuspended in dimethyl sulfoxide (DMSO) to concentrations of 4 mg/mL and subsequently diluted in RPMI medium with Lglutamine (pH 7.2) with 0.165 mol/L morpholine propane sulfonic acid (MOPS) complemented with 2% glucose.Amphotericin B (Sigma Chemical Co.Saint-Louis, USA) was prepared in DMSO and tested in the concentrations of 0.00625 to 32 μg/mL.Minimum inhibitory concentration (MIC) values for each sample were obtained in triplicate using document M-38 A2 adapted to 96-well microplates for T. rubrum (CLSI, 2008b).Each well contained 100 μL of a twofold serially diluted sample and 100 μL of RPMI medium, with 100 μL being transferred to the next well, sequentially.Then, 100 μL of the T. rubrum inoculum was added to obtain a final sample volume of 200 μL with concentrations ranging from 1250 to 2.44 μg/mL.For amphotericin B, 100 μL of the twofold sample dilution was added to 100 μL of the inoculum.The microplates were incubated in an orbital shaker at 120 rpm and 28°C for 7 days.The MIC was determined as the minimum concentration of the tested sample for which no growth was visualized, and then 30 μL of a 0.01% resazurin aqueous solution (Sigma-Aldrich) was added to determine the microorganism viability.The minimum inhibitory concentration (MIC) values against C. albicans for each sample were determined in triplicate using the reference method M-27 A3 (CLSI, 2008a) with modifications.Each well contained 100 μL of a twofold serially diluted sample and 100 μL of RPMI medium, with 100 μL being transferred to the next well, consecutively.Then, 100 μL of the C. albicans inoculum was added to obtain a final sample volume of 200 μL with concentrations ranging from 1250 to 2.44 μg/mL.For amphotericin B, 100 μL of the twofold drug dilution was added with 100 μL of the microorganism.The orbital shaker at 120 rpm and 35°C for 2 days was used to incubate the microplates.Then, the viability of C. albicans was determined by the addition of a 2.0 % 2,3,5-triphenyltetrazolium chloride aqueous solution (Sigma-Aldrich).The MIC was characterized as the smallest concentration of the tested sample for which no development was observed.

Cytotoxicity assay
The cytotoxicity was measured using the in vitro Toxicology Colorimetric Assay Kit (XTT; Roche Diagnostics) according to manufacturer´s instructions, using a normal human lung fibroblasts (GM07492A), and as previously described (Alvarenga et al., 2015).

Statistical analysis
Inhibitory concentration at 50% cell growth inhibition (IC50) was obtained with the GraphPad Prism 5 software.One-way ANOVA was used for the comparison of means (p ˂ 0.05).

RESULTS AND DISCUSSION
The effects of the crude ethanolic extract from the leaves of A. othonianum (EE) on the growth of the selected fungi are shown in Table 1.The lowest MIC value was obtained for C. albicans (MIC= 78.12 μg/mL), and the EE showed no cytotoxicity.Once, Suffness and Pezzuto (1990), extracts that present IC 50 values lower than 30 μg/mL indicated cytotoxic action.In addition, the EE displayed a MIC of 312.5 μg/mL against T. rubrum.
Among the four fractions, including the n-hexane (HF), EtOAc (EF), n-BuOH (BF), and hydromethanolic (HMF) fractions, achieved by liquid-liquid partitioning of the EE, the EtOAc fraction (EF) displayed the highest antifungal activity, with MIC values of 4.88 and 39.06 μg/mL against C. albicans and T. rubrum, respectively, and no cytotoxicity was observed for this fraction.
According to these results, the EF fraction exhibits promising activity.The n-BuOH fraction (BF) also exhibited potential antifungal activity against C. albicans and T. rubrum (MIC= 19.53 and 39.06 μg/mL, respectively) and did not significantly inhibit the growth of the normal cell line.
When compared with the EE, the n-hexane fraction (HF) provided inferior antifungal activity against C. albicans (MIC ˃ 1250 μg/mL).However, HF displayed the same MIC value against T. rubrum (MIC= 312.5 μg/mL), but HF was considered cytotoxic when compared with the  other fractions and the crude extract, with an IC 50 value of 235.3 μg/mL.Although, this IC 50 compared with the reference (Suffness and Pezzuto, 1990) did not indicate promising cytotoxic activity.The hydromethanolic fraction (HMF), when compared with the EE, displayed an equal MIC value against C. albicans.Meanwhile, the result obtained for the HMF against T. rubrum was superior (MIC= 156.25 μg/mL) to that of EE, and no cytotoxic effects were observed for HMF.Thus, the HF and HMF fractions are not promising antifungal agents against the strains tested in this work when compared with the EF and BF fractions.
Regarding the antifungal activities of the compounds, only 1 displayed significant inhibitory effects against C. albicans, with a MIC value of 19.53 μg/mL.Compounds 2 and 3 presented significant inhibitory effects against T. rubrum TR1 (MIC values of 9.76 and 39.06 μg/mL, respectively).Again, no cytotoxicity was observed for the isolated compounds (IC 50 ˃ 400 μM).However, the isolated compounds displayed weaker antifungal activities when compared with amphotericin B, which was used as a positive control (Table 1).Additionally, compounds 1-4 have previously been shown to possess antifungal activities (Mbaveng et al., 2008;Leal et al., 2009;Kuete et al., 2009;Soares et al., 2014).Flavonoids have been found to be active against a wide range of microorganisms; their mechanism of action is possibly due to their capacity to complex with proteins and with bacterial cell walls (Cowan, 1999).
The structures of gallic acid (2) and ethyl 3,4,5trimethoxybenzoate (4) were quite related, differing in the occurrence of three free hydroxyl substituents and an acidic moiety in 4 versus 3 methoxy groups and an ethyl acetate group in 4. The presence of three methoxyl groups in the latter compound might cause a decrease in the activity, suggesting that the three free OH groups could play a role in the antifungal activity.This finding is in accordance with the data obtained by Leal et al. (2009).Additionally, the mechanism of action of phenolic compounds comprises enzyme inhibition (Cowan, 1999).
Protocatechuic acid (3) and gallic acid (2) also showed related chemical structures, diverging only in the occurrence of two hydroxyl moieties in 3, which decreased the activity.Once more, these results suggested that the presence of the three free OH groups appears to be necessary for antifungal activity.
Regarding the chemical composition of the bioactive extract (EE) and fractions (EF and BF) (Figure 2a-c), an HPLC-DAD analysis was performed using a C18 column.The chromatograms were recorded using a methanol/water (0.1% acetic acid) gradient from 5 to 100% methanol over 30 min, then elution with 100% methanol for 10 min.These conditions were similarly employed to evaluate the presence of compounds 1-4 in the samples, based on an evaluation of retention times (λ = 254 nm) and UV spectra from a DAD detector of the compounds previously isolated from EF (Figure 2d).
Compounds 1-4 were recognized in different proportions (Table 2) in the ethanol extract from the leaves of A. othonianum (EE) and the EtOAc fraction (EF) (Figure 2a and b).Additionally, compound 4 was not detected in the n-BuOH fraction (BF) (Figure 2c).Conversely, the amentoflavone (1) was always present at a higher concentration relative to 2, 3 and 4. Based on the data obtained from the chromatogram and MIC values of EF, BF and compounds 1-3, when compounds 1-3 were combined in the fractions, the antifungal activity was improved, at least in the case of C. albicans.
Thus, the data obtained suggest that the antifungal activity of A. othonianum may be mainly attributed to the effects of amentoflavone (1), gallic acid (2) and protocatechuic acid (3).Frequently plant extracts showed better activity than the isolated compounds, this situation can be explained by synergy effects (Wagner and Ulrich-Merzenich, 2009).Although, in this study the isolated compounds displayed increased antifungal activity, when compared with the crude extract (EE).

Conclusions
To the best of the authors' knowledge, the antifungal activity of A. othonianum and the occurrence of compounds 1-4 are reported for the first time in this work.Though, the antifungal activities of compounds 1-4 have been documented previously.In conclusion, the antimicrobial activity of the crude extract of the leaves of A. othonianum may be due to the occurrence of compounds 1-3.The evaluated extract, together with fractions EtOAc (EF) and n-BuOH (BF) and compounds 1-3, could be beneficial for the research of new antifungal medications.Nevertheless, the toxicological and pharmacological studies of the analyzed samples will check this proposition.

Figure 1 .
Figure 1.Chemical structures of the isolated compounds.
the lower area (%); b not detected.

Figure 2 .
Figure 2. HPLC-DAD chromatogram of (a) the crude ethanol extract from A. othonianum leaves, (b) the EtOAc fraction and (c) the n-BuOH fraction and (d) UV-DAD spectra of compounds 1-4.Chromatographic conditions:methanol/water (0.1% acetic acid) linear gradient from 5 to 100% methanol over 30 min and 100% methanol for 10 min, including 3 min to return to the initial condition and 15 min of equilibration, oven at 40°C, and detection at λ = 254 nm.The flow-rate was 1.0 mL/min.

Table 1 .
Minimum inhibitory concentration (MIC) values and cytotoxic activities obtained for the crude ethanolic extract of A. othonianum, fractions and isolated compounds.