The Sensitivity Modifying Activity of Nerolidol and α-Bisabolol Against Trichophyton spp

Abstract

Trichophyton spp. is one of the main causative agents of dermatophytosis such as tinea ungium and tinea pedis. Resistance to antifungal drugs is a significant clinical problem in dermatophytosis. The main molecular mechanism of antifungal resistance to conventional therapy in dermatophytes is the expression of efflux pumps. Efforts aimed at improving the efficacy of current antifungals such as griseofulvin are relevant. Given this, sesquiterpenes such as α-bisabolol and nerolidol found in essential oils represent promissing alternatives. Griseofulvin sensitivity modulation activity in T. rubrum, T. interdigitale H6, and T. interdigitale Δmdr2 (mutant strain of T. interdigitale) promoted by α-bisabolol and nerolidol were investigated. The minimum inhibitory concentration (MIC) of the test drugs were determined by microdilution. Subsequently, the effect of the drugs tested on plasma membrane functionality (K⁺ release) was analyzed. The MIC of griseofulvin was determined at sub-inhibitory sesquiterpene concentrations (modulation assay). An association study was performed with griseofulvin and sesquiterpenes (checkerboard). α-bisabolol was more potent than nerolidol; presenting lower MIC values. All of the fungi were sensitive to griseofulvin, starting at 8 µg/mL. With the exception of griseofulvin, all of the test drugs increased K⁺ release (p < 0.05). Nerolidol modulated the sensitivity of all strains to griseofulvin; α-bisabolol sensitivity modulation was limited to T. interdigitale H6 and T. interdigitale Δmdr2. In association with griseofulvin: nerolidol and α-bisabolol respectively presented synergism and additivity. Finally, the results of our study suggest using α-bisabolol and nerolidol compounds as potential antifungal agents and griseofulvin sensitivity modulators for Trichophyton spp.

Introduction

Dermatophytosis are fungal infections that affect keratinized tissues such as the skin, hair and nails of humans and animals; it is the most prevalent type of infection worldwide. The principal agent of dermatophytosis is T. rubrum, which commonly causes chronic onychomycosis, recurrent tinea pedis and tinea corporis [2, 29]. Allylamines, azoles, cyclopirox, amorolfine and griseofulvin are currently available for the treatment of these infections. In fact, griseofulvin is known as a drug standard for therapy of dermatophytosis [27]. The efficacy of griseofulvin has been decreased over the years, larger doses and longer treatment are commonly required. These conventional therapies often present acquisition of fungal resistance resulting in decreased antifungal susceptibility [28].

In dermatophytes, efflux pumps are most prevalent mechanism of resistance in dermatophytes. ABC (ATP-binding cassette) transporters are proteins that cause drug efflux, conferring multidrug resistance (MDR) that in turn reflects in the high number of therapeutic failures [11, 17]. It is reported that Trichophyton spp. strains, when exposed to differing antifungal classes increase mRNA transcription levels for the mdr2 gene, which encodes an ABC transporter in dermatophytes conferring multidrug resistance [8, 13]. In this context, natural products appear as a source of molecules with potential antifungal activity or which modulate the sensitivity of pathogenic fungi [5]. However, α-bisabolol and nerolidol in combination with griseofulvin have not yet been investigated. Thus, our study investigated the antifungal and modulatory activity of α-bisabolol and nerolidol on Trichophyton spp. griseofulvin sensitivity.

The monocyclic sesquiterpene alcohol α-bisabolol (C₁₅H₂₆O) is found in essential oils of a variety of plants such as Matricaria chamomilla, Eremanthus erythropappus and Salvia runcinata [18]. A number of pharmacological and biological effects have been reported for α-bisabolol including antimicrobial [10] and antileishmanial activity [24] Nerolidol is an aliphatic sesquiterpene alcohol (C₁₅H₂₆O) present in the essential oil of Piper gaudichaudianum, P. claussenianum, Baccharis dracunculifolia and Melaleuca leucadendra [18]. Nerolidol has insecticidal [26] and acaricide activity [7]. In the scientific literature, the plasma membrane is the main cellular target involved in antifungal activity of essential oils and their terpene components. They damage microorganism membranes, cause proton pump collapse, cytoplasm granulation, and electron transport chain breakdown [9, 21].

Materials and Methods

Chemicals

The drugs α-bisabolol, nerolidol, griseofulvin and chlorpromazine were purchased from Sigma-Aldrich® (Brazil). Emulsions were freshly prepared for the tests by dissolving first in dimethylsulfoxide (DMSO), and sterilized distilled water to obtain a concentration of 1024 µg/mL. From this concentration, dilutions were performed to achieve a concentration of 1 µg/mL using RPMI 1640 medium.

Fungi

The strain T. rubrum LM 4 was provided by the Laboratory of Mycology of the Department of Pharmaceutical Sciences, Federal University of Paraíba (Brazil). T. interdigitale strain H6 (ATCC MYA-3108) was isolated from clinical specimens at the University Hospital of Ribeirão Preto Medical School, São Paulo University, Brazil, and its Δmdr2 strain derivative was obtained by an inactivation method, as previously described (Fachin, et al., 2006). The fungi were grown in potato dextrose agar (Difco®) at 28° C for 7 days to obtain the fungi inocula in sterile saline (0.85% NaCl). Turbidity of the final inocula was adjusted to 10⁶ conidia/mL, at a wavelength of 520 nm, and transmission adjusted to 70% in a UV-5100 Spectrophotometer [4, 25].

Minimum Inhibitory Concentration (MIC)

MIC values of the drugs-test were determined against dermatophyte fungi by microdilution technique using 96 well flat bottom micro-titer plates [4, 25]. To each row of the plate was added 100 µL of the diluted test drugs in RPMI 1640 (Sigma-Aldrich® -Brazil). To each well of the plate was added 100 µL of a previously prepared inoculum diluted in RPMI 1640 at a ratio of 1:50. A fungal control was performed by replacing the test drug using sterile saline (growth control). A sterility control and DMSO were also performed. The plates were sealed and incubated at 28° C for 7 days. MIC was the lowest concentration of drugs capable of inhibiting observed fungal growth in the wells.

Release of K⁺

The concentration of free potassium (K⁺) ions in the fungal inoculum was measured after exposure to MIC of the test drugs at 28 °C. After the incubation period (4 h), extracellular K⁺ concentration was measured using turbidimetry procedures, K + dosing kits; (Doles ®, Goiânia, Brazil) and a UV 5100 spectrophotometer. Control experiments without test drugs were performed in a similar form. Sodium dodecyl sulfate (SDS) 2% was used as a reference base capable of inducing K⁺ release [31].

Antifungal Activity Modulatory Effects

To assess whether the drugs tested modulate the activity of the antifungal agents when used against the test strains, we applied the method proposed by Coutinho et al. [6]. Using microdilution technique, the MIC values of griseofulvin in the absence or presence of sub inhibitory concentrations (1/4MIC) of α-bisabolol, nerolidol or chlorpromazine were obtained. Chlorpromazine was used as a positive control as an efflux pump inhibitor [12]. For reading, the plates were sealed and incubated at 28 °C for up to 7 days.

Drug Association Study (Checkerboard)

Dilutions of the test drug solutions at (1/8MIC, 1/4MIC, 1/2MIC, MIC, 2 × MIC 4 × MIC and 8 × MIC) were made in RPMI 1640. An aliquot of 50 µL of griseofulvin was then added to the wells of the plate in a vertical sense, and then 50 µL of a specific α-bisabolol or nerolidol dilution was added in the horizontal direction of the plate. Finally, 100 µL of inoculum was added. The plates were sealed and incubated at 28 °C for 7 days for MIC readings. In the context of evaluating the activity of the drug associations, the fractional inhibitory concentration index (FICI) was calculated as the sum of: FIC_A + FIC_B, where A represents α-bisabolol or nerolidol; and B represents griseofulvin. The FIC_A = (MIC_A combined)/(MIC_A alone), while the FIC_B = (MIC_B combined)/(MIC_B alone). The FICI was interpreted in the following way: synergism (< 0.5), additivity (0.5-1.0), indifference (> 1.0 and < 4.0), or antagonism (> 4.0) [15].

Statistical Analysis

MIC determinations were performed in triplicate and given that the results obtained were the same in the replicates the results expressed in modal values. The K⁺ liberation test results were expressed as mean ± standard deviation (SD). Statistical evaluation of the results was done using the unpaired t-test to determine significant differences, with a value of p < 0.05.

Results and Discussion

In order to evaluate the sensitivity of the T. rubrum LM 4, T. interdigitale H6, and T. interdigitale Δmdr2 fungal strains, the MIC values for α-bisabolol, nerolidol, griseofulvin and chlorpromazine (Table 1) were determined. α-bisabolol was more potent than nerolidol and presented lower MIC values (32-fold). T. interdigitale Δmdr2 was the most sensitive of the strains tested. MICs were recorded for griseofulvin and chlorpromazine respectively at 8 µg/mL and 0.5 µg/mL, which remained constant for the three fungal strains evaluated. There was no fungal growth inhibition from DMSO, and all of the strains grew in the absence of drugs.

Table 1.

Values of minimum inhibitory concentration (MIC) of α-bisabolol, nerolidol, griseofulvin and chlorpromazine against Trichophyton spp

Strains	MIC (µg/mL)a
	α-bisabolol	Nerolidol	Griseofulvin	Chlorpromazine
Trichophyton rubrum LM 4	16	512	8	0.5
Trichophyton interdigitale H6	16	512	8	0.5
Trichophyton interdigitale Δmdr2	8	256	8	1

^aModal value of three experiments

Both α-bisabolol and nerolidol interfered in the functionality of the plasma membrane (Fig. 1), and induced greater losses of K⁺ in relation to the control (p < 0.05). Both the sesquiterpenes and griseofulvin induced lesser K⁺ releases relative to the 2% SDS lysis agent (p < 0.05). However, nerolidol exhibited more effect than α-bisabolol. The effect of griseofulvin on the membrane was similar to the control.

Fig. 1.

Rate of K⁺ from Trichophyton rubrum LM 4 in the absence (control) and presence of SDS, griseofulvin, α-bisabolol and nerolidol. *Compared with control (p < 0.05). #Compared with SDS (2%) (p < 0.05)

The in vitro antifungal activity of α-bisabolol against T. tonsurans, T. mentagrophytes, T. rubrum, and M. canis strains has been reported in the literature. There are also reports of nerolidol presenting antifungal activity against dermatophytes such as T. mentagrophytes and M. gypseum [3, 16]. It has been reported in the literature that α-bisabolol inhibits ergosterol biosynthesis by blocking the fecosterol formation from zimosterol [23]. A study by Park et al. [19] with nerolidol and other terpenes evidenced loss of membrane integrity, with destruction and disorganization of cytoplasmic organelles in hyphae in T. mentagrophytes. However, we verified that α-bisabolol and nerolidol interfere in the cell membrane functionality of T. rubrum LM4, as evidenced by intracellular (K⁺) components released to the external environment.

Considering the interference in the functioning of the membrane structures, ABC transporter activity may be disrupted by α-bisabolol and nerolidol. We thus investigated sesquiterpene sensitivity modifying activity on fungal strains: either expressing (T. interdigitale H6), or not expressing mdr2 genes (T. interdigitale Δmdr2). Nerolidol modulated the sensitivity of the fungi to griseofulvin, with a strong decrease in MIC in the presence of nerolidol. α-bisabolol did not modulate the sensitivity of T. interdigitale H6 to griseofulvin. Both α-bisabolol and nerolidol modulated T. interdigitale Δmdr2 sensitivity, even without it expressing the ABC transporter. The results are summarized in Table 2. We observed that chlorpromazine, a recognized blocker of efflux pumps, does not modify T. rubrum LM 4 sensitivity to griseofulvin, which may be due to its non-expression of ABC transporter proteins. Nerolidol and α-bisabolol modulated the sensitivity of T. interdigitale H6 and T. interdigitale Δmdr2. This is suggestive that such modulation may not involve the activity of ABC transporters encoded by the mdr2 genes.

Table 2.

Minimum inhibitory concentration (MIC) of griseofulvin alone and combined with sub-inhibitory concentrations (1/4MIC) of α-bisabolol, nerolidol and chlorpromazine against Trichophyton spp

Strains	MIC alonea	Combined MICa
		α-bisabolol	Nerolidol	Chlorpromazine
T. rubrum LM 4	8	8	0.5	8
T. interdigitale (H6)	8	1	1	2
T. interdigitale (Δmdr2)	8	1	1	8

^aModal value of three experiments. MIC: µg/mL

Phenothiazine is a tricyclic compound, and clinically its derivatives (such as chlorpromazine) are widely used as antipsychotic and antihistaminic compounds. They are potent inhibitors of K⁺ transport and Ca²⁺ channels [20] whose most likely mechanism is interference in calcium-dependent energy generation (ATP hydrolysis) with inhibition of efflux pump activity [14]. The literature reports their capacity to inhibit MDR efflux pumps in certain strains of bacteria, as well as their use as pharmacological tools to indirectly identify the involvement of efflux pumps in fungal resistance.

Using the T. rubrum LM 4 strain, association studies were performed between griseofulvin, α-bisabolol, and nerolidol in order to verify the relationships between them. The MIC of Griseofulvin, when combined with nerolidol, was 8x less when compared to its isolated MIC. α-bisabolol decreased the MIC value of griseofulvin by 4x. The result of these associations is that nerolidol presents synergistic interactions with griseofulvin (FICI: 0.25); and α-bisabolol exhibits additive interactions (FICI: 0.75).

Due to reports of pathogenic resistance, the general lack of new drugs, and to discoveries of various and differing molecular targets; and since natural and antimicrobial combination products have recently been studied and reported to increase antibiotic activity, combination drugs are now considered a potential strategy for therapy [21, 22, 30]. Studies involving associations between antifungal drugs and natural products have been described and the association between terpenes and antifungals often presents favorable results. Ahmad et al. [1] reported that both thymol and carvacrol present synergism with fluconazole in Candida spp. (whether sensitive or resistant to fluconazole), sensitizing fungal cells to the drug and decreasing their Cdr1 and Mdr1 efflux pump activity. Yet to date, there are no reports in the literature of studies associating α-bisabolol or nerolidol with griseofulvin.

In conclusion, the sesquiterpenes α-bisabolol and nerolidol exhibit antifungal activity and griseofulvin sensitivity modulation for strains of Trichophyton spp., affecting plasma membrane functionality. The modulation may not involve interference with (mdr2 gene encoded) ABC transporter activity. Nerolidol presents synergistic interactions with griseofulvin; α-bisabolol exhibits additive interactions; and plasma membrane interference may help promote the positive effects of these associations. The results of this study were satisfactory and offer new perspectives for treatment of dermatophytosis, especially for T. rubrum infections.