Antifungal and antioxidant activity of fatty acid methyl esters from vegetable oils

Fatty acid methyl esters (FAMEs) were obtained from vegetable oils of soybean, corn and sunflower. The current study was focused on evaluating the antifungal activity of FAMEs mainly against Paracoccidioides spp., as well as testing the interaction of these compounds with commercial antifungal drugs and also their antioxidant potential. FAMEs presented small IC50 values (1.86-9.42 μg/mL). All three FAMEs tested showed antifungal activity against isolates of Paracoccidioides spp. with MIC values ranging from 15.6500 μg/mL. Sunflower FAMEs exhibited antifungal activity that extended also to other genera, with an MIC of 15.6 μg/mL against Candida glabrata and C. krusei and 31.2 μg/mL against C. parapsilosis. FAMEs exhibited a synergetic effect with itraconazole. The antifungal activity of the FAMEs against isolates of Paracoccidioides spp. is likely due to the presence of methyl linoleate, the major compound present in all three FAMEs. The results obtained indicate the potential of FAMEs as sources for antifungal and antioxidant activity.


INTRODUCTION
Antioxidant compounds are capable of inhibiting or delaying lipid oxidation, which is associated with the appearance of degenerative or chronic diseases. Thus, antioxidants act to prevent or avoid incidence of cancer, diabetes, atherosclerosis, coronary heart disease and the aging process (Rufino et al. 2011). Natural substances with known antioxidant potential are phenolic compounds (flavonoids and phenolic acids), vitamins A, C, E and carotenoids which, in different proportions and quantities, can be obtained from foods of plant origin, such as vegetables, fruits, teas, herbs, beans and oil seeds (Ara andNur 2009, Arnao et al. 2001). Several studies have suggested that the development of chronic or degenerative diseases can be decreased with the regular consumption of vegetables or fruits (Chu et al. 2002). Therefore, in recent years, the search for natural antioxidants, especially of plant origin, has greatly increased (Lima et al. 2010). MARIA E.A. PINTO et al. Paracoccidioidomycosis (PCM) is a systemic mycosis caused by fungi of the genus Paracoccidioides, and two species are known, P. brasiliensis and P. lutzii. PCM is a disease limited to Latin America and is a common cause of deaths from fungal diseases. PCM is the eighth most important cause of mortality from chronic infectious diseases reaching rates of 1.65 deaths per 10 6 habitants (Bocca et al. 2013, Coutinho et al. 2002, Prado et al. 2009). There are some drugs available to treat PCM; itraconazole therapy is the first choice to control the mild to moderate clinical forms but this therapy is not easily available in most of the endemic regions. Consequently, sulfamethoxazoletrimethoprim (SMX-THT) is a useful option; this drug is freely distributed by the Brazilian Ministry of Health. The main disadvantage of SMX-THT is the need for long-term treatment (more than 12 months) in moderate and severe cases which can lead patients to abandon treatment (Brummer et al. 1993, Paniago et al. 2003, Travassos et al. 2008. Amphotericin B therapy is the best choice for severe cases of PCM, but toxicity, mainly nephrotoxicity, is related to this drug which will sometimes require discontinuation of this therapy (de Oliveira et al. 2015, Ferreira 2009, Shikanai-Yasuda 2015. Relapses, a common event in PCM patients, associated with toxicity represent an unresolved problem in the conventional therapeutic approach (Travassos et al. 2008, Travassos andTaborda 2012). Due to these facts it is necessary to research new drugs that are safer, more effective and cheaper and with shorter periods of therapy for the treatment of PCM.
Fatty acids are the most abundant component of oils, the most commonly found being stearic, palmitic, oleic, linoleic and linolenic acids (Cabral 2005). The antibacterial and antifungal properties of vegetable oils are reported in the literature, these being especially attributed to the presence of fatty acids (Desbois andSmith 2010, Erdemoglu andKusmenoglu 2003). However, some works done with methyl esters showed their potential as antifungals (Abdelillah et al. 2013, Agoramoorthy et al. 2007, Chandrasekaran et al. 2011, Golebiowiski et al. 2013, Lima et al. 2011, but these works are still scarce, so it is important to test these compounds. Fatty acid methyl esters (FAMEs) are obtained from vegetable oils. This work is focused on evaluating the antifungal activity of FAMEs from vegetable oils against Paracoccidioides spp., and also antioxidant potential by the scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals.

PREPARATION OF METHYL ESTERS
Vegetable oils (1 g) were refluxed with 1.0 mol/L methanolic sodium hydroxide solution for 30 min and then extracted with ethyl ether. The aqueous phase was acidified with 1.0 mol/L hydrochloric acid solution and extracted with ethyl ether. The  Hahn and Hamdan (2000). The resulting suspensions were diluted in RPMI 1640 (1 : 10) to obtain the final inoculum with 1-5 × 10 5 yeasts/ mL (Cruz et al. 2013). For Candida spp. and Cryptococcus spp., the inoculum was obtained with a final concentration of 0.5-2.5 × 10 3 cells/mL for susceptibility testing (CLSI 2008).

ANTIFUNGAL ACTIVITY
The antifungal activity was evaluated by the minimal inhibitory concentration (MIC) in accordance with the guidelines from the CLSI M27-A3 document (CLSI 2008). The MIC for Paracoccidioides was performed according to the method described by Johann et al. (2010). FAMEs were tested in the range of 2000-7.81 μg/mL, amphotericin B was tested in the range of 1-0.008 μg/mL and SMX-THT (4.49 × 10 −3 to 2.3 μmol/mL) was tested in MARIA E. A. PINTO et al. the range of 600-4.6 μg/mL. The MIC values correspond to the lowest concentrations that did not allow for the detection of any visual fungal growth. All assays were performed in triplicate and repeated at least once.

ASSESSMENT OF DRUG INTERACTIONS
The assessment of drug interactions of FAMEs and positive controls (amphotericin B, SMX-THT and itraconazole) were performed according to the described methods (Pyun andShin 2006, White et al. 1996), using eight serial dilutions of each compound. These dilutions were added to 96-well plates in several combinations of concentrations of the two compounds tested. After that, a suspension of Pb-18, done according to the MIC test, was added to all wells of the plate and cultured for 14 days. The fractional inhibitory concentration (FIC) was obtained by dividing the MIC of the positive control in the presence of FAMEs by the MIC of the positive control alone. The FIC of FAMEs was calculated in the same way. The FIC index (FICI) was obtained by adding both FICs. Synergistic activity was observed when the FICI was ≤ 0.5, an indifferent effect in the range 0.5 < FICI < 2.0 and an antagonistic effect when the FICI was > 2.0.

CELLULAR REACTIVE OXYGEN SPECIES (ROS)
The P. brasiliensis Pb-18 was cultured in YPD agar during days at 37 ºC. Microplate wells received 100 μL inoculum containing 1 x 10 5 cfu/mL in RPMI medium without phenol red, and then the probes were added. Briefly, the probe for reactive oxygen species (ROS) (2',7'-dichlorofluorescein diacetate) and reactive nitrogen species (RNS) (dihidrorhodamine 123) were prepared in methanol and PBS, respectively, at 100 μM and 20 μL were distributed to each well for a final concentration of 10 μM. Hydrogen peroxide (4M) was included as a control. The drug treatment solutions were evaluated at the respective MIC, in a final volume of 200 μL. The plates were incubated at 37 ºC in the dark. After 120 hour, the fluorescence was measured with a fluorometer (Biotek Synergy 2 SL Luminescence Microplate Reader/US) using excitation at 485 nm and emission wavelengths of 530 nm.

STATISTICAL ANALYSIS
Student's t test was utilized to evaluate the statistical difference between the control group and the group exposed to FAMEs. To evaluate the statistical difference between the three FAMEs tested we used the Newman-Keuls test and ANOVA (1 way) to cellular ROS. A P value < 0.05 was considered statistically significant. The analyses were performed using Prisma 5.0 software.

ANTIOXIDANT ACTIVITY
In this study, FAMEs obtained from vegetable oils were investigated by GC-MS. The analysis by GC-MS revealed a high percentage of unsaturated methyl esters (83.95-88.33%) as compared to saturated methyl esters (11.67-16.05%). In samples, methyl oleate and methyl linoleate were detected as the major unsaturated methyl esters. Methyl palmitate was the most abundant among the saturated methyl esters, followed by methyl stearate (Table I).
The scavenging effect on DPPH radicals of FAMEs from vegetable oils is presented in Figure  1. FAMEs, BHT and AA revealed an inhibitory effect in a concentration-dependent manner. FAMEs were more effective in scavenging DPPH radicals than AA at a concentration of 1 μg/mL and BHT at concentrations of 1 and 10 μg/mL. FAMEs showed IC 50 values of 1.86, 9.42 and 3.33 μg/mL for soybean, corn and sunflower oils, respectively, lower than BHT (16.36 μg/mL; P < 0.05), but higher than AA (1.62 μg/mL; P < 0.05). FAMEs obtained from Annona cornifolia seeds also presented antioxidant activity, with IC 50 = 3.83 μg/mL (Lima et al. 2012). Soybean and sunflower FAMEs were more active than the FAMEs of Annona cornifolia.

ANTIFUNGAL ACTIVITY
The antifungal activity of FAMEs obtained from vegetable oils was evaluated against 18 fungal strains of clinical interest (Table II). Results showed that all FAMEs tested showed antifungal activity against the isolates of Paracoccidioides tested, with values between 15.6 and 500 µg/mL. Paracoccidioides brasiliensis Penguin showed more susceptibility to soybean and sunflower FAMEs with an MIC value of 15.6 µg/mL, but this isolate was less susceptible to corn FAMEs (MIC = 500 µg/mL). For P. lutzii, isolate 1578 was most susceptible to the FAMEs tested in the present study. Moreover, FAMEs (soybean, corn and sunflower) showed better activity than SMX-THT (P = 0.0001) against the isolates of P. brasiliensis Pb-18, Pb-608, Pb-1017, Pb-9673 and P. lutzii 01. SMX-THT is used in the treatment of PCM, mainly in endemic regions where other therapy (such as itraconazole) is not easily available; consequently, therapy consisting of SMX-THT is a useful option (de Oliveira et al. 2015, Shikanai-Yasuda 2015. The difference in antifungal activity between different FAMEs, against 11 isolates of Paracoccidioides spp., is not statistically significant (P < 0.05). MIC values found for SMX-THT and amphotericin B against the isolates of Paracoccidioides were statistically significant when compared with FAMEs (P = 0.0001).
It is interesting to note that when we tested the susceptibility of other fungus genera we observed that only sunflower FAMEs showed activity under the conditions tested in the present study (Table II). This antifungal activity (sunflower FAMEs) was more pronounced against C. krusei, C. glabrata and C. parapsilosis with MIC values of 15.6, 15.6 and 31.2 µg/mL, respectively.
Few works reporting the biological activity of FAMEs against pathogenic fungi are found in the literature. Twelve clinical strains of the fungus P. brasiliensis were susceptible to FAMEs from seeds of A. cornifolia, with MICs in the range between 3.4 and 55.5 µg/mL (Lima et al. 2011). The FAME extract of Excoecaria agallocha presented antifungal activity against Candida albicans, C. krusei, C. parapsilosis and Candida tropicalis (Agoramoorthy et al. 2007).  Golebiowiski et al. (2013) reported the antifungal activity of FAMEs from Calliphora vomitoria against entomopathogenic fungi, protecting this ectoparasite against entomopathogenic microorganisms present in its habitat. Furthermore, these authors showed that when individual FAMEs were tested they presented weak antifungal activity, but the mixture of FAMEs found in living forms of C. vomitoria was more active. These experiments are in agreement with those of the present study where the mixture of FAMEs presented antifungal activity.
In the present work we also tested the antifungal activity of standards of the methyl esters which were present in the FAMEs (Table III). Of these methyl esters tested only methyl linoleate showed antifungal activity against P. brasiliensis (Pb-18), with an MIC value of 62.5 µg/mL (Table III). It is interesting to note that sunflower FAMEs had a higher percentage of methyl linoleate (65.93%) and also presented better antifungal activity, with 54.54% of isolates of Paracoccidioides tested with an MIC value ≤ 62.5 µg/mL. The other two FAMEs tested also followed this pattern; soybean FAMEs containing 55.67% methyl linoleate showed antifungal activity with an MIC value ≤ 62.5 µg/mL for 36.36% of isolates of Paracoccidioides tested and corn FAMEs (48.38% methyl linoleate) with 27.27% of isolates tested with an MIC value ≤ 62.5 µg/mL. Probably, methyl linoleate is responsible for the antifungal activity of the FAMEs studied, because it is also present in a greater concentration in all the FAMEs tested (Table I). In present work we also observed the production of reactive oxygen (ROS) by methyl linoleate, sunflower and corn FAMEs (Figure 2) with production significantly compared to the control group (P. brasiliensis 18 without treatment) (P < 0.05). Methyl linoleate is able to produce hydroperoxide (Yamamoto et al. 1984) and probably this property is responsible for the antifungal activity of this compound in the present work. Therefore the ROS production could be related with antifungal activity of sunflower and corn FAMEs and methyl linoleate compound   present in these FAMEs could be responsible for ROS production.
Methyl linoleate has already been tested against inhibition of the in vitro proliferation of human tumor cell lines; this compound showed antiproliferative activity with an IC 50 of 250 µg/ mL, but not induction of differentiation. This is an interesting finding, because 5-fluorouracil is also a known antitumor compound that does not induce differentiation and is currently used as an antifungal (Lampronti et al. 2003).
In the present work we also tested the antifungal activity of standards of fatty acids (Table III) because these compounds were present in the oils (sunflower, soybean and corn oils) that originated the FAMEs. Although two of these compounds (oleic acid and linoleic acid) exhibited interesting antifungal activity against Pb-18, the oils did not present activity (data not shown).
To explore the possibility of developing more powerful combination therapies of FAMEs with other antifungal drugs, a checkerboard micro-titer test was carried out. FAMEs showed an antagonistic effect with amphotericin B and an indifferent effect with SMX-THT (Table IV). FAMEs showed a synergetic effect with itraconazole. This illustrates one important result, because it could lead to the development of a new combination therapy using lower concentrations of itraconazole in a shorter treatment time, which would reduce the side effects of patients. Itraconazole interferes in ergosterol synthesis, leading to cell membrane perturbation (Odds et al. 2003). Alteration in membrane permeability could allow increased concentrations of FAMEs to penetrate the cell, resulting in cell death.

CONCLUSIONS
In the search for new drugs with antifungal and antioxidant properties, FAMEs (obtained from vegetable oils of soybean, corn and sunflower) might be good candidates: these esters presented better radical-scavenging activity and IC 50 values than BHT, a commercial antioxidant. All three FAMEs tested showed antifungal activity against isolates of Paracoccidioides spp. with MIC values ranging from 15.6-250 µg/mL. Sunflower FAMEs also exhibited antifungal activity that extended to other genera, with an MIC of 15.6 μg/mL against Candida glabrata and C. krusei and 31.2 μg/mL against C. parapsilosis. FAMEs exhibited a synergetic effect with itraconazole. The antifungal activity of the FAMEs against isolates of Paracoccidioides spp. is likely due to the presence of methyl linoleate, the major compound present in all three FAMEs. This work provided knowledge, for the first time, of the antioxidant and antifungal activity of FAMEs obtained from vegetable oils and indicates the potential of FAMEs as a source of antifungal and antioxidant activity.