Antifungal Action of the Dillapiole-rich Oil of Piper aduncum against Dermatomycoses Caused by Filamentous Fungi

Aims and Study Design : Piper aduncum L. is a Brazilian plant with many biological properties attributed to its dillapiole-rich essential oil. Despite the development of antibiotics, bacterial and fungal infections are still a public health issue in the medical field. This study measured the antimicrobial activity of the dillapiole-rich essential oil of P. aduncum against pathogenic skin microorganisms. Place of Study: Faculty of Pharmacy and Graduate Program in Pharmaceutical Science, Federal Original Research Article Ferreira et al.; BJMMR, 15(12): 1-10, 2016; Article no.BJMMR.26340 2 University of Pará, Brazil. This work was performed in 2014. Methodology: Gas Chromatography (GC) and Gas Cromatography-Mass spectrometry (GC-MS) have analyzed the oil and its dillapiole-rich fraction. The determination of Minimum Inhibitory Concentration (MIC) and Minimum fungicidal concentration (MFC) values was carried out by microdilution method and counting of formed colonies. Results: For the strains of Trichophyton mentagrophytes (ATCC 9533 and clinical isolate), the oil and its dillapiole-rich fraction exhibited MIC values of 500 μg/ml while the MFC values were 1,500 μg/ml for the oil and 1,000 μg/ml for the fraction rich in dillapiole. For clinical isolates of T. rubrum and Epidermophyton floccosum, MIC values of 500 μg/ml and MFC 1,500 μg/ml were equal for the oil and the dillapiole-rich fraction, respectively. For clinical isolates of Microsporum canis and M. gypseum, the MIC and MFC values were 250 μg/ml and 500 μg/ml, respectively. For strains of Aspergillus fumigatus (ATCC 40152 and clinical isolate), the oil and its dillapiole-rich fraction have shown the same MIC value of 3.9 μg/ml while the MFC values were 7.8 μg/ml for the strain ATCC 40152, and 15.6 μg/ml for the clinical isolate. The oil and dillapiole-rich fraction did not show antibacterial activity against the strain of Staphylococcus aureus ATCC 6538 and its clinical isolate Conclusion: The dillapiole-rich essential oil of P. aduncum and its dillapiole-rich fraction demonstrates significant antifungal activity against dermatophytes, filamentous fungi and potent antifungal activity against non-dermatophyte filamentous fungi.


INTRODUCTION
Despite the development of antibiotics, bacterial and fungal infections are still a public health issue in the medical field. The presence of numerous strains resistant to current drugs continues to represent a significant challenge. Medicinal Herbals have been used for this purpose, for many centuries. More recently, there has been an increased interest in natural products due to their availability, reduced side effects, toxicity, and greater biodegradability, if compared with available antibiotics [1]. In this regard, the essential oils of plants can offer a significant therapeutic potential when used alone or in combination with traditional antibiotics.
The leaf extract and essential oil of P. aduncum were active against protozoa parasites and diverse microorganisms, including pathogenic bacteria and fungi, showing properties as antimicrobial, molluscicidal and cytotoxic [7]. Also, the oil of P. aduncum showed to be toxic and insecticide against Ostrinia nubilalis (a lepidopteran corn borer), Anopheles marajoara and Aedes aegypti (the mosquitoes that transmit malaria and dengue), and Solenopsis saevissima (a painful fire ant). These biological actions were attributed to the dillapiole, its main constituent [8][9][10]. Dillapiole was also reported acting as a synergist of natural insecticides, including carbamates, organochlorines, pyrethrum, tenulin, and azadirachtin [11,12]. The oil of P. aduncum showed antifungal activity against Crinipellis perniciosa and Fusarium solani f. sp. Piperis, two phytopathogenic fungi, which infects the cocoa and pepper crops in the Brazilian Amazon [13,14]. The toxicological evaluation of the oil of P. aduncum had low toxicity in mice (LD 50 2,400±191.7 mg/kg) [15].
Diseases caused by dermatophytes generate a large public health problem, leading to reduced quality of life of people affected and the high cost of treatment with synthetic antimicrobials that, in turn, have some drawbacks such as high toxicity, side effects and the emergence of resistant strains [7,16]. Dermatomycosis is a common injury that affects humans and animals. It is often caused by dermatophytes of the genera Epidermophyton, Microsporum, and Trichophyton. Usually, it infects skin, nails and scalp, causing lesions characterized by skin irritation, peeling, redness, swelling and inflammation [17,18].
Thus, in view of the significant biological action demonstrated by P. aduncum oil, in the present work we decided to evaluate this oil and dillapiole, its main constituent, as antimicrobial agents with therapeutic alternative of low cost and low toxicity, against the microorganisms Candida albicans, Trichophyton mentagrophytes, T. rubrum, Microsporum canis, M. gypseum, Epidermophyton flocossum, and Aspergillus fumigatus. Also, the oil composition of P. aduncum and its dillapiole-rich fraction were analyzed by GC and GC-MS.

Plant Material
The aerial parts (leaves and thin stems) of P. aduncum were collected in the city of Santo Antonio do Tauá, the state of Pará, Brazil, in a particular agronomic experiment with plant matrices. Specimens were deposited in the Herbarium of the Emilio Goeldi Museum, the city of Belém, the state of Pará, Brazil, under the number MG150678.

Plant Processing and Extraction of the Essential Oil
The plant material was air dried for 48 h, ground and submitted to hydrodistillation using a Clevenger-type apparatus (100 g, 3 h). The oil was dried over anhydrous sodium sulfate, and its percentage content was calculated based on the plant dry weight. The moisture content of the plant was calculated using a balance with moisture measurement by infra-red. The oil was kept in an amber vial and stored at 5ºC before Gas Chromatography (GC) and Gas Chromatography-Mass Spectrometry (GC-MS) analysis and the bioassays. The procedure was performed in triplicate.

Oil Fractionation (Purification of
Dillapiole) The oil (10 g) was submitted to fractionation on silica (230-400 mesh, Merck, 200 g) chromatographic column (80 x 3 cm) using nhexane and n-hexane-ethyl acetate (10%) as elution solvent and the separated fractions were monitored by thin-layer chromatography (revealed by vanillin/H 2 SO 4, 110°C). Gas Chromatography-Flame Ionization Detection (GC-FID) obtained the content of dillapiole in the oil and the purified fractions. Also, the minor compounds found with the dillapiole in the purified fraction were identified by GC-MS.

Analysis of the Oil Composition and Dillapiole
The analyses were performed on a GC-MS Thermo Focus DSQ II, under the following conditions: DB-5ms (30 m x 0.25 mm; 0.25 mm film thickness) fused-silica capillary column; programmed temperature: 60-240°C (3ºC/min); injector temperature: 250ºC; carrier gas: Helium, adjusted to a linear velocity of 32 cm/s (measured at 100°C); injection type: split (2 µl of a 1:1000 hexane solution); split flow was adjusted to yield a 20:1 ratio; septum sweep was a constant 10 ml/min; Electronic Impact-Mass Spectrometry (EI-MS): electron energy, 70 eV; temperature of ion source and connection parts: 200°C. The quantitative data regarding the volatile constituents were obtained by peak area normalization using a GC-FID Thermo Focus operated under similar conditions of the GC-MS, except for the carrier gas, which was nitrogen. The retention index was calculated for all the volatiles constituents using an n-alkane (C8-C40, Sigma/Aldrich) homologous series. Individual components were identified by comparison of both mass spectrum and GC retention data with authentic compounds which were previously analyzed and stored in a private library, as well as with the aid of commercial libraries containing retention indices and mass spectra of volatile compounds commonly found in the essential oils [19,20].

Culture media
The culture media used for the antibacterial activity were Mueller-Hinton broth, Mueller-Hinton agar (Merck, Germany), and mannitol agar (HiMEDIA, India); and for the antifungal activity were RPMI 1640 with glutamine without bicarbonate, phenol red indicator, MOPS pH 7 buffer, Saboraund dextrose agar (SDA), and Potato agar (HiMEDIA, India).

Strains of microorganisms tested
The strains were from American Type Culture Collection (ATCC), provided by the Instituto

Antibacterial assay
The bacterium was cultured on Mueller-Hinton agar and mannitol agar (on Petri dishes). Then the dishes were incubated at 35ºC (24 h) for growth analysis. The inoculum was prepared using 3-4 colonies of bacteria transferred to a tube containing 1 ml of Mueller-Hinton broth and making some adjustments to achieve an approximate concentration of 1 x 10 8 CFU/ml, consistent with the 0.5 McFarland scale (from 0.09 to 0.11). For this step, a turbidimeter apparatus (Densimat-Biomerieux) was used. The suspension was incubated for 1 h to reach an exponential bacterial growth and, then, a serial dilution was made to obtain the inoculum 1 x 10 3 CFU/ml, according to Clinical and Laboratory Standards Institute [21].

Antifungal assay
For filamentous fungi, the inoculum was prepared based on the M38-A2 standard of Clinical and Laboratory Standards Institute [22]. The fungi were cultured in Petri dishes containing potato dextrose agar and kept in an oven at room temperature: T. mentagrophytes (21 days), T. rubrum and E. floccosum (30 days), and M. canis and M. gypseum (14 days). Intending to the exponential growth of conidia of A. fumigatus was used agar saboraund dextrose, at 37°C, for seven days. The inoculum was prepared by washing the conidia with RPMI medium (5 ml), and the obtained suspension was transferred to a test tube for the settling of larger particles, for 5 min. Then, the suspension was vortexed (Phenix, AT 56 model) for 15 min. The turbidity of the suspension was adjusted to McFarland scale (turbidimeter Densimat, Biomerieux), 0.4 to 5 x 10 6 CFU/ml. For A. fumigatus was made a dilution (1:50) in RPMI medium, resulting in 0.4 to 5 x 10 4 CFU/ml. For yeast, the inoculum preparation was based on the M27-A3 standard of Clinical and Laboratory Standards Institute [23]. Candida albicans was cultured on Petri plates containing Sabouraud dextrose agar and kept in an oven at 35°C (24 h). About five colonies of C. albicans were suspended in RPMI medium (5 ml), and the suspension was vortexed (15 min). The density of cells was adjusted to McFarland standard scale to 1 x 10 6 to 5 x 10 6 CFU/ml. The inoculum was incubated (1 h) to achieve the exponential growth of fungi and, then, dilutions 1:50 and 1:20 were made with RPMI 1640 liquid medium, resulting in a concentration of 1 x 10 3 to 5 x 10 3 CFU/ml.

Determination of Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC)
The broth microdilution method was used [21]. The dilution of oil and concentrate of dillapiole was done in solution of Tween 20 (0.5%) to obtain the test concentration (20 µl/ml) and, then, with serial dilution using MHA and SDA for the preparation of other concentrations: Bacterial strains ranging from 125 to 2,000 µg/ml; and fungal strains ranging from 1.9 to 2,000 µg/ml [7]. The test was performed in 96 well plates, where each well received 90 µl of the particular concentration of the oil and concentrate of dillapiole, 90 µl of the MHA or SDA media, and 20 µl of the inoculum. The control of microbial growth, control of sterility of the medium and the solvent control were done simultaneously. Each well kept a final volume of 200 µl. The inhibition of growth of bacteria and fungi was revealed by addition of sterile resazurin solution (20 ul, 0.02%, w/v) (Sigma), after incubation (24h, 36 ± 1°C). Then the plates were incubated for another 3 h. The MIC, which is defined as the lowest concentration of oil capable of inhibiting the growth of microorganisms, was determined by the permanence of blue coloration (resazurin) in the wells. The wells that showed no apparent growth were selected to evaluate the MBC and MFC, which were determined by the absence of microbial growth on plates containing MHA and SDA media, respectively [21,22]. The halo formed around the disk was measured using a digital caliper and compared to standards antimicrobial discs: Chloramphenicol (250 µg/ml) for bacteria and amphotericin B (100 µg/ml) for fungi. The MIC is the lowest concentration of antimicrobial agent that prevents the visible growth of a microorganism in a sensibility test of broth microdilution. The MBC was obtained by seeding method, by removing an aliquot of 10 ul of the contents of the wells, followed by spreading on Petri plates previously prepared with Mueller-Hinton agar and incubated at 35°C (24 h).

Statistical Analysis
Data were statistically analyzed by ANOVA (One-way ANOVA), with correction using Tukey's test. The MIC, MBC, and MFC data were expressed as mean ± standard error of the mean (SEM). The probability accepted as indicative of the existence of significant differences was p <0.05.

Oil and Purification of Dillapiole
The oil showed a yield of 3.0% and it was analyzed by GC and GC-MS. Twenty-four constituents were identified, and they are listed in Table 1. Dillapiole was the main component in the oil, with 85.9%. The oil was submitted to purification process using column chromatography, and the content of dillapiole reached 98.4%.

Antibacterial Activity
The oil and dillapiole-rich fraction did not show antibacterial activity against the strain of S. aureus ATCC 6538 and its clinical isolate, at the concentration range used (125-2,000 µg/ml). Confirming our data, another dillapiole-rich oil of P. aduncum from Ecuador did not show antibacterial activity against S. aureus ATCC 29213 [4]. But, another sample of P. aduncum oil, containing dillapiole as the principal constituent, showed antibacterial activity against S. aureus ATCC 25923 and S. epidermidis ATCC 12228, with MIC and MBC values between 250 and 1000 µg/ml [7]. On the other hand, antibacterial properties against various microorganisms have been attributed to the ethanol extract of P. aduncum, due the presence of p-hydroxybenzoic acid, chalcones and chromenes [24,25]. RICalc. = Calculated retention index; RILit. = Retention index of literature (Adams, 2007) Studies of antimicrobial activity of plants have shown controversies. Certain factors may explain these variations, such as extracts of different compositions, different techniques used, the culture media used, changes in pH, the growth conditions (aerobic and anaerobic), the incubation time of the cultured microorganisms, as well as the inoculum size. Also, different strains tested show variation in susceptibility to the various extracts as the origin, seasonality and the plant constituents [26]. Studies have reported that essential oils are more active against fungi than against gram-positive bacteria, estimating that 60% of them have antifungal effects while 35% exhibit antibacterial properties. This information corroborates our findings [27].

Antifungal Activity
The results of the antifungal assay against the dermatophytes filamentous fungi are shown in Table 2 and Fig. 1. The MIC values for the oil and dillapiole-rich fraction were lower compared to MFC values, indicating fungicidal action against the tested microorganisms, in both products.

Fig. 1. Antifungal activity of the oil and concentrate of dillapiole against dermatophyic filamentous fungi
The antifungal activity has demonstrated similar MIC and MFC values for T. mentagrophytes, T. rubrum, and E. flocossum, as well as between M. canis and M. gypseum. However, the strains of the genera Trichophyton and Epidermophyton showed higher MIC and MFC values, when compared to the strains of the genus Microsporum. The explanation for these results would be the hypothesis that anthropophilic species, such as T. mentagrophytes, T. rubrum, and E. flocossum, are more resistant to the antifungal agents. They are better adapted to man and, therefore, exhibit higher values of MIC and MFC than the zoophylic (M. canis) and geophilic (M. gypseum) species. Also, the results suggest that dilapiolle would be responsible for the antifungal activity of P. aduncum oil, since their content is quite high (85.9%), reflecting on the similarity of MIC and MFC values found in oil and dillapiole-rich fraction (99.48%), in almost all tests. This fact is confirmed by the activity observed in the fungus T. mentagrophytes ATCC and its clinical isolate, whose MFC values for the dillapiole-rich fraction are smaller (1,000 µg/ml) than in the oil P. aduncum (1,500 µg/ml).
The oil of P. aduncum was previously submitted to antifungal action against the strains of T. mentagrophytes ATCC 16066, showing an MIC value of 500 µg/ml [4], therefore, comparable to our results. A dilapiolle-rich fraction (95-98.5%) of P. aduncum oil presented fungicidal action on basidiospores and germination of Clinipellis perniciosa, a fungus that attacks cacao plantations, with MIC value of 0.6 mg/ml [9,13]. An extract of P. aduncum, harvested in Minas Gerais, Brazil, was active against strains of T. rubrum, with MIC and MFC values of 0.31 µg/ml and 0.62 µg/ml, respectively. But, the main constituents of the extract were sesquiterpenes, such as germacrene D, (E)-nerolidol and δcadineno [28].
The greater effectiveness of the P. aduncum oil against the filamentous fungi, compared to bacteria and yeasts, can be attributed to its volatility and lipophilic character, which will permeate the membrane of filamentous fungi, allowing its absorption and diffusion [29]. A probable mechanism of action of essential oils occurs in the cell walls of fungi, which is mainly composed of chitin, glucans, and glycopeptides. The oil act in the destruction of components of the cell wall and cell membrane and it causes leakage of their constituents into the cytoplasm that, in turn, suffer a consequent coagulation [1].
The results of antifungal activity for the oil and the dillapiole-rich fraction against A. fumigatus, a non-dermatophyte filamentous fungus, are in Table 3 and Fig. 2. Again, it was demonstrated the fungicidal action of these products, presenting MIC values lower than that of MFC values.
The results of the tests against nondermatophyte filamentous fungi (e.g. A. fumigatus), as well as against dermatophyte filamentous fungi (e.g. T. rubrum), showed similar values of MIC and MFC for the oil and the dillapiole-rich fraction, suggesting once again that dillapiole is responsible for the antifungal activity of the P. aduncum oil.   [31]. Also, in the oil of Thymus caespititius and α-terpineol, its primary constituent, with MIC ranged from 1.25 to 2.5 µg/ml and 0.32 to 0.64 µg/ml, respectively [32].
Furthermore, it has been observed a difference in MIC and MFC values, among dermatophyte filamentous fungi (e.g., T. rubrum), and nondermatophyte filamentous fungi (e.g., A. fumigatus). This difference is attributed to the higher pathogenicity of virulence factors of the dermatophytes, which it is related to the accession process and invasion of host tissue, making that the fungi can be more specialized and, therefore, more pathogenics. The fungi T. mentagrophytes and T. rubrum can express specific fibrillar adhesins that recognize and bind to mannose and galactose residues of the cell surface. These structures are responsible for making the correct anchoring of these fungi in the host cells and prevent them from being disconnected by external aggressions, such as the act of scratching [33].
Regarding the invasion process, dermatophytes stand out for their lipolytic capacity, with the release of extracellular enzymes when there is contact with the host tissue lipids, facilitating the invasion process. The mannans, a particular component of the glycoprotein of the cell wall of fungi, promote the suppression of the inflammatory response and cell proliferation of the host organism [34].
The oil and concentrate of dilapiolle showed no antifungal activity against C. albicans, with MIC and MFC values above 2,000 µg/ml. Previously the oil and the ethanol extract of P. aduncum were tested against C. albicans, but only the oil showed antifungal activity in concentrations above 2,000 µg/ml [35]. Therefore, with a similar result to that presented in this work.
The high effectiveness of essential oils against filamentous fungi, when compared to yeasts and bacteria can be attributed to the lipophilic character and the volatility of these products, as well as the nature of the lipophilic and aerobic membranes of filamentous fungi, allowing the oil to be absorbed and diffused across the membrane [29]. According to established criteria, it is possible to evaluate the effectiveness of compounds tested in antifungal studies. Holetz et al. [36] consider that the MIC value less than 100 µg/ml indicates good antifungal activity, MIC value 100-500 µg/ml has moderate activity, MIC value 500-1,000 µg/ml has weak activity and MIC value above 1,000 µg/ml is considered inactive. Aligiannis et al. [37] and Duarte et al. [34] adopted the following values: strong inhibitor, MIC up to 500 µg/ml; moderate inhibitor, MIC from 600 to 1,500 µg/ml; and weak inhibitor, MIC above 1,500 µg/ml.

CONCLUSION
Thus, considering the results and the criteria described in the mentioned literature, the oil of P. aduncum and the dillapiole-rich fraction showed good antifungal activity for dermatophyte filamentous fungi and potential antifungal activity for non-dermatophyte filamentous fungi. This significant activity can be explained by the high lipophilicity of the oil, which preferentially promotes the passage of dillapiole and other liposoluble substances through the cell walls of fungi over the water-soluble substances.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.