Anti-Trichophyton Activity of Protocatechuates and Their Synergism with Fluconazole

Dermatophytosis and superficial mycosis are a major global public health problem that affects 20–25% of the world's population. The increase in fungal resistance to the commercially available antifungal agents, in conjunction with the limited spectrum of action of such drugs, emphasises the need to develop new antifungal agents. Natural products are attractive prototypes for antifungal agents due to their broad spectrum of biological activities. This study aimed to verify the antifungal activity of protocatechuic acid, 3,4-diacetoxybenzoic, and fourteen alkyl protocatechuates (3,4-dihydroxybenzoates) against Trichophyton rubrum and Trichophyton mentagrophytes and to further assess their activities when combined with fluconazole. Susceptibility and synergism assays were conducted as described in M38-A2 (CLSI), with modifications. Three strains of Trichophyton rubrum and three strains of Trichophyton mentagrophytes were used in this work. The pentyl, hexyl, heptyl, octyl, nonyl, and decyl protocatechuates showed great fungicidal effects, with minimum inhibitory concentration (MIC) values ranging from 0.97 to 7.8 mg/L. Heptyl showed a synergistic activity (FIC index = 0.49), reducing the MIC of fluconazole by fourfold. All substances tested were safe, especially the hexyl, heptyl, octyl, and nonyl compounds, all of which showed a high selectivity index, particularly in combination with fluconazole. These ester associations with fluconazole may represent a promising source of prototypes in the search for anti-Trichophyton therapeutic agents.


Introduction
Superficial fungal infections are a major global public health problem that affects 20-25% of the population worldwide [1]. Among these diseases, dermatophytosis, or tinea, is one of the most frequent fungal infections. This infection is caused by dermatophyte species that belong to the Trichophyton, Microsporum, or Epidermophyton genera [2]. These dermatophytes commonly invade different keratinophilic regions of the body, causing tinea corporis, tinea cruris, tinea pedis, tinea manus, tinea capitis, tinea barbae, and tinea unguium [3]. Dermatophyte infections can lead to either mild or severe symptoms, depending on the immunological response of the host [4]. Several patient groups also seem to be especially at risk of infection, including individuals with uncontrolled diabetes, AIDS, renal diseases, psoriasis, and types of immunosuppression, such as transplant recipients and patients on long-term corticosteroid therapy [5].
There is an urgent need to find new sources of substances with antidermatophytic activity because the treatment of 2 Evidence-Based Complementary and Alternative Medicine dermatophytosis is long and expensive, particularly in the case of onychomycosis. Furthermore, the spectrum of the available drugs is limited, such drugs may induce adverse effects, and several reports of antifungal resistance have been published [6][7][8][9]. For this reason, various antifungal agents have been introduced into clinical practice, among them, amorolfine, ciclopirox, griseofulvin, terbinafine, itraconazole, fluconazole, and more recently, voriconazole [10,11]. However, efforts should be concentrated on the discovery and development of novel, safer, and effective antidermatophytic agents.
Protocatechuic acid (3,4-dihydroxybenzoic) is a phenolic compound produced by the secondary metabolism of plants. It is naturally present in almost all plant materials, including food, fruits, and vegetables [12][13][14]. Together with its natural and synthetic derivatives, it has been associated with a broad spectrum of biological actions and is known to have antioxidant, proapoptotic [15], anti-inflammatory, antiglycative [16], and antimelanogenic [17] functions. However, the major interest in protocatechuic acid and its derivatives is due to its antimicrobial properties. It has been reported that protocatechuic acids have activity against susceptible and antibiotic-resistant Campylobacter spp. and Helicobacter pylori [18,19]. Furthermore, it has been shown thatoctyl 3,4-dihydroxybenzoate has fungicidal activity against Saccharomyces cerevisiae [20].
Thus, considering the broad spectrum of protocatechuates and the need for the discovery of new antifungal agents, this study aimed to investigate the antifungal activity of a synthetic homologous series of n-alkyl protocatechuates against Trichophyton rubrum and Trichophyton mentagrophytes. This study also aimed to investigate the chemical characteristics of the compounds responsible for the biological activity of dermatophytes, including the importance of free hydroxyl radicals and the size of the carbon side chain.

Compounds Synthesis.
Synthetic compounds of protocatechuic acid were prepared as described by de Faria et al. [21], with minor modifications. Briefly, a 3 mL solution of N, Ndicyclohexylcarbodiimide (DCC, 1 mmol) in -dioxane was added to a cooled (5 ∘ C) solution of 0.2 mmol protocatechuic acid (1) (Sigma-Aldrich, St. Louis, MO, USA) and 20 mmol of -alkyl alcohols in 6 mL of -dioxane. The solution was stirred for 48 h and the solvent was removed under reduced pressure. The residue was partitioned 3 times with EtOAc and filtered. The filtrate was washed successively with a saturated aqueous citric acid solution (3 times) and saturated aqueous NaHCO 3 (3 times), dried over anhydrous MgSO 4 , and evaporated under reduced pressure. The crude products were purified over a silica gel column (0.06-0.20 mm, ACROS Organics, USA) and eluted isocratically with CHCl 3 /MeOH (98 : 2) to produce esters 2−15. Their structures were then established by 1 H and 13 C NMR spectral analysis. For the synthesis of compound 16, protocatechuic acid (20 mmol) was dissolved in dried pyridine (5.0 mL) and anhydride acetic (5.0 mL) under a hydrogen atmosphere. The mixture was then stirred for 48 h at room temperature, dried under reduced pressure, and purified by column chromatography with a mixture of CHCl 3 /MeOH (85 : 15) to produce product 16. The NMR spectroscopic data for compound 16 were compatible with it being 3,4-diacetoxybenzoic acid.

Microorganisms.
To evaluate its antifungal activity, six species of dermatophytes were tested: two clinical strains of Trichophyton rubrum (Tr1 and Tr2), Trichophyton rubrum ATCC MYA 3108, two clinical strains of Trichophyton mentagrophytes (Tm1 and Tm2), and Trichophyton mentagrophytes ATCC 40131 (Tm3). The microorganisms were obtained from the collection of the Clinical Mycology Laboratory of the Department of Clinical Analyses at the School of Pharmaceutical Sciences of Universidade Estadual Paulista (UNESP). The strains were cultivated on Sabouraud dextrose agar (Difco, BD Biosciences) and incubated at 28 ∘ C for 7-15 days. For all experiments, the strains were cultivated on Potato Dextrose Agar (Difco, BD Biosciences) and incubated at 28 ∘ C as described above or until sporulation.

Dilution of Test Substances.
The dilution of the synthetic compounds was performed with DMSO (Synth, Diadema, Sao Paulo, Brazil) as described by Scorzoni et al. [22]. The concentrations of the compounds on 96-well plates (TPP, Trasadigen, Switzerland) ranged from 500 mg/L to 0.97 mg/L. The antifungal drugs were diluted according to the CLSI M38-A2 document [23]. Stock solutions of Fluconazole (Sigma-Aldrich, St. Louis, MO, USA) were prepared, considering its power. Serial twofold dilutions were prepared according to the recommendations of Zhang and collaborators [24], with some modifications.

Minimum Inhibitory Concentration (MIC).
The antifungal activity tests were performed using the broth microdilution method as described in M38-A2, a document produced by the Clinical and Laboratory Standards Institute (CLSI, 2008) [23], with modifications. The medium used was RPMI 1640 with L-glutamine (Sigma-Aldrich, St. Louis, MO, USA) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 2% glucose. The cell suspension was prepared in a 0.85% saline solution. The suspension of conidia was then transferred to small sterile test tubes where they remained for 40 minutes to separate the microconidia, which were lighter and therefore present in the supernatant. The separated microconidia were then counted with a hematocytometer, and their concentration was adjusted to obtain a final concentration ranging from 2.5 × 10 3 to 5 × 10 3 CFU/mL. These suspensions were diluted in RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) and inoculated on 96-well plates (TPP, Trasadigen, Switzerland) that had been previously prepared with the compounds diluted at concentrations from 250 to 0.48 mg/L. Positive (100 L of RPMI medium with 100 L of inoculum) and negative (200 L of RPMI) controls were included in all experiments. The plates were incubated with agitation at 35 ∘ C for 7 days. The MIC reading was performed by spectrophotometry at 490 nm. For fluconazole, the MIC was defined as the concentration that produced a 50% inhibition of fungal growth.

Minimum Fungicide Concentration (MFC).
A qualitative analysis of the fungal viability was performed by transferring a portion of the wells to a plate with Sabouraud (Difco, BD Biosciences) medium and incubating it at 35 ∘ C for the time determined for each fungal agent. The MFC was defined as the lowest extract concentration that did not allow the growth of any fungal colonies on the solid medium after the incubation period [25]. A visual reading was performed to confirm the death or growth inhibition provided by fluconazole and the sixteen semisynthetic substances derived from protocatechuic acid.
2.6. Synergistic Activity. The drug activity was assessed using a checkerboard method derived from a standardised procedure established by the National Committee for Clinical Laboratory Standards (M38-A2) [23]. Briefly, the test was performed on the same medium used for susceptibility testing. Volumes of 50 L of each drug in a concentration four times the final concentration were dispensed in 96-well plates (TPP, Trasadigen, Switzerland). To each well, 100 L of the fungal suspension was added to produce a final concentration of 5.0 × 10 3 CFU/mL.
As a negative control, we used 200 L of RPMI, while as a positive control, we used 100 L of RPMI medium with 100 L of inoculum. The plates were incubated at 35 ∘ C, and the reading was completed after 168 hours. We conducted visual and spectrophotometric readings at 490 nm. To determine the effect of combinatorial fractions, we calculated the fractional inhibitory concentration (FIC). The FIC was calculated by taking the MIC of the substance in combination/MIC of the substance alone. The sum of the fractional inhibitory concentration (FIC) of each substance consists of the fractional inhibitory concentration index: (the MIC of drug A in combination/the MIC of drug A alone) + (the MIC of drug B in combination/the MIC of drug B alone). A synergistic relationship was defined as FIC index ≤0.5, an additive relationship was defined as 0.5 < FIC index ≤ 1.0, an indifferent relationship was defined as 1.0 < FIC index ≤ 4.0, and an antagonistic relationship was defined as FIC index >4.0 [26][27][28].

Cytotoxicity
Assay. The cytotoxicity of sixteen semisynthetic compounds derived from protocatechuic acid was assessed using a sulphorhodamine B assay in NOK (oral human keratinocyte) cell lines obtained from the American Type Culture Collection (Manassas, VA, USA). The strains were maintained in bottles appropriate for cell culture with keratinocyte serum free medium (Gibco, Life Technologies) and incubated in standard conditions of 37 ∘ C and 5% CO 2 . Cell concentrations ranging from 2.5 to 5.0 × 10 4 cells/mL were used for the formation of cell monolayers. The concentrations of pure substances were kept in contact with the cells for 24 hours. After the incubation period, the cells were treated with the sulphorhodamine B reagent (Sigma-Aldrich, St. Louis, MO, USA) as previously described by Skehan et al. [29], with some modifications.

Statistical Analysis.
All experiments were performed in triplicate. Statistical analysis was performed with a ttest or one-way ANOVA with GraphPad Prism 5 software (Version 5, USA). values < 0.05 were considered statistically significant.

Synergistic
Activity. The activity of the combination of fluconazole with pentyl, hexyl, heptyl, octyl, and nonyl protocatechuates was evaluated and classified as antagonistic, indifferent, additive, and synergistic based on the FIC index, as shown in Table 2. The FIC index was calculated based on the results of the checkerboard test.
The combinations were tested in clinical isolates of Trichophyton rubrum (Tr1) and Trichophyton mentagrophytes (Tm1) and in the reference strain Trichophyton mentagrophytes ATCC 40131 (Tm3).    Evidence-Based Complementary and Alternative Medicine  In the clinical isolate Tm1, the combination of fluconazole and all protocatechuates acids tested showed a conservation of the fluconazole MIC (1.0 mg/L) ( > 0.05) and a 130fold reduction in the MIC values of the protocatechuates for substances 7, 9, and 11 (0.03 mg/L) ( < 0.01) and in 63.33 times for compound 8 and 10 (0.03 mg/L) ( < 0.01). All combinations were classified as additive for Tm1, with an FIC index value of 1.0. However, for the strain ATCC 40131 (Tm3), a conservation in the fluconazole MIC (0.5 mg/L) was observed when combined with compounds 7 and 11 ( > 0.05). The combination of the activity between these compounds was classified as additive, with an FIC index equal to 1.0. A reduction in the MIC values of two-, four-, and twofold, respectively, for substances 8, 9, and 10 ( < 0.05) was observed when combined with fluconazole. A fourfold reduction (0.12 mg/L) in the MIC value was observed when associated with these compounds ( < 0.05). The associations between fluconazole and compounds 8 and 10 were classified as additive and had an FIC index equal to 0.72, whereas the association with compound 9 instead had a synergistic activity, with an FIC index equal to 0.48.

Cytotoxicity Assay.
The compounds pentyl (7), hexyl (8), heptyl (9), octyl (10), and nonyl (11) were evaluated in a cytotoxicity assay in NOK cells. These substances showed high values for IC 50 after reaching the necessary concentration to produce 50% lethality of 57 mg/L for cells treated with compound 7, 66.5 mg/L for cells treated with compound 8, 78.1 mg/L for cells treated with compound 9, 54 mg/L for cells treated with compound 10, and 52.1 mg/L for cells treated with compound 11. These results showed that these compounds had a low toxicity in human oral keratinocytes ( Table 3). The selectivity index (SI) was calculated for all six strains of Trichophyton rubrum and Trichophyton mentagrophytes. A selectivity index greater than 10 indicates a substance with a higher selectivity for fungi. Considering all six strains, compounds 9 and 11 had SI values greater than 10.
The SI values for compounds 9 and 11 ranged from 20.0 to 80.5 and 13.2 to 26.7, respectively.

Discussion
It is estimated that superficial mycoses affect approximately 25% of the world's population [30]. Due to the high rate of recurrence of superficial fungal infections and the increasing problem of antifungal resistance, especially against the azole family of drugs, new treatment alternatives with fungicidal activity are sorely needed [31][32][33]. In this context, the anti-Trichophyton spp. activity of protocatechuic acid, 3,4diacetoxibenzoic acid, and fourteen alkyl protocatechuates (3,4-dihydroxybenzoates) were evaluated on their own or in combination with fluconazole. In the sixteen compounds studied, significant antifungal activity was found. Remarkable results were observed in six compounds against the two species with pentyl, hexyl, heptyl, octyl, nonyl, and decyl compounds. These results showed that increasing the length of the side chain by up to 9 carbons enhanced both the hydrophobicity and thus the antifungal activity of the compounds. However, the addition of more than 9 carbons leads to a reduction in the antifungal activity. The same protocatechuic acid esters were used by de Faria et al. [21]. However, in their work, the antioxidant activity of those compounds was evaluated. They also reported that the alkylation process increased the hydrophobicity of these compounds, resulting in an increased inhibition of the oxidative process. Nihei and collaborators [20] evaluated the series of alkyl 3,4dihydroxybenzoates (protocatechuates) and their fungicidal activity against the yeast Saccharomyces cerevisiae. Nonyl and octyl, 3,4-dihydroxybenzoates obtained the lowest values of MIC. In addition, the anti-Saccharomyces activity was also correlated with the hydrophobicity of the carbon chain. Thus, by analysing the structure-activity relationship of these compounds, we can conclude that the carbon chain has a key role not only in antioxidant activity, as described by de Faria and collaborators [21], but also in the antifungal activity against Trichophyton spp.
Due to the lack of new classes of drugs or different molecular targets, drug combinations can be considered a strategy for therapy [34]. Several studies have proposed the use of natural compounds in combination with drugs to establish a new strategy for the treatment and prevention of certain diseases [35][36][37][38]. Different combinations of statins and some antifungal drugs were tested against four dermatophyte species (Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis, and Microsporum gypseum). Most of the synergistic activity was found with the combination of statins with terbinafine and the different azoles [39]. Our results showed additive and synergistic activity of the protocatechuic acid derivatives with fluconazole against Trichophyton rubrum and Trichophyton mentagrophytes. We observed a synergistic activity of fluconazole associated with the heptyl derivative (9) when tested against Tm3, with a reduction in the MIC value of fluconazole and heptyl by fourand eightfold, respectively; an additive activity was observed for Tm1 and Tm3 with this combination. For the isolate Tr1, we observed additive activity with the association of pentyl, heptyl, octyl, and nonyl with fluconazole. For this isolate, the combination of a hexyl derivative (8) with this azole was found to not produce a change in its antifungal activity, such that this association was considered indifferent. All other combinations produced additive activity against the isolates tested. The great advantage of using phenolic compounds in combination with conventional therapies is that they can increase the susceptibly of microorganisms compared to the usual drugs and therefore are associated with reduced toxicity [38].
Palafox-Carlos and collaborators [40] reported an antioxidative synergistic effect between phenolic acids present in mango (Mangifera indica L.) and protocatechuic acid, gallic acid, vanillic acid, and chlorogenic acid. The authors found that the association showed a synergistic effect and that the gallic and protocatechuic acids presented higher antioxidative capacities. Jayaraman and collaborators [38] studied the combination of seven antibiotics and six phytochemical compounds, including protocatechuic acid, against isolates of Pseudomonas aeruginosa and found that the combination of protocatechuic acid and sulfamethoxazol showed synergistic activity against all bacterial isolates, with a fractional inhibitory concentration index of 0.25 to 0.5.
The efficacy of fluconazole can be improved using combination therapy [34]. Aala and collaborators [41] found good activity between allicin in combination with ketoconazole or fluconazole demonstrating a synergistic or additive interaction against dermatophytes. Galgóczy and coauthors [42] evaluated the in vitro antifungal activity against strains of dermatophytes, combining the protein (PAF) of Penicillium chrysogenum with fluconazole (FCZ). PAF and FCZ acted synergistically and/or additively on all of the tested fungi except Microsporum gypseum, for which no interactions were detected [42].
When the cytotoxicity of the substances was evaluated, high IC 50 values were found. The IC 50 indicates the concentration of the compound that is necessary to kill 50% of the cell line. The selective index was also calculated using the ratio of the IC 50 and MFC. For all of the isolates of the two species tested, most IS values of the substances were higher than 10, and the best results were found for the heptyl and nonyl derivatives. The IS values indicate that the concentrations tested caused injury to the fungal cells but no toxicity to the human cells [43][44][45].
Compounds not recognised as antifungal agents may cause potent inhibition of growth when used in combination with fluconazole. Our results suggest that the combination of substances can act with synergistic or additive activity in the treatment of dermatomycosis. More studies related to these combinations should be performed to identify a possible mechanism of action and to provide a verification of their in vivo activity. The evaluation of drug combinations defines a strategy for the discovery of new therapies, leading to a future clinical evaluation. In addition to the potent antidermatophytic capabilities, the protocatechuate derivatives presented a low toxicity to keratinocytes, demonstrating that the topical use of these novel compounds may represent a promising new option for the treatment of superficial mycoses.

Conclusion
In summary, new therapeutic trials are important to unravel biological data and thus result in the discovery of new combination drug. Overall, our results may contribute to a database of information on test susceptibility and synergism of dermatophytes in vitro, targeting the development and optimization of antifungal drugs. In the present work the esters of protocatechuic acid as well as their associations with commercial drug could be a promising therapeutic approach. More studies are needed to determine the mechanism of action of these substances providing the molecular basis of understanding.