In vitro Antifungal Activity of Baccharis trimera Less (DC) Essential Oil against Dermatophytes

Purpose: To identify the main components of the essential oil (EO) of Baccharis trimera Less and investigate their in vitro antifungal activity against seven fungal strains that cause onychomycosis. Methods: The chemical composition of EO was determined using gas chromatography, and its minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and antifungal activity were compared with those of terbinafine and ketoconazole. Scanning electron microscopy (SEM) was used to investigate morphological changes in the strains of interest. Results: Twenty compounds, with β -pinene being the major constituent (23.4 %), were identified in EO. EO exhibited fungicide potential, with MFC values in the range of 0.06 to 125 µg mL −1 , which were lower than those of the reference drugs against Trichophyton rubrum CCT 5507 URM1666 and Microsporum canis ATCC 32903. MIC range for the compounds was from 0.03 to 125 µg mL −1 for five strains of the fungi evaluated. For Trichophyton mentagrophytes ATCC 11481 and Epidermophyton floccosum CCF-IOC 3757, MIC was ≥ 1000 µg mL −1 . Flattening, distortions, and shrinkage were observed in the SEM images of structures of the five fungal species that were subjected to the action of the EO. Conclusion: The results indicate that EO has antifungal activity against filamentous fungi and may be developed as an alternative for the treatment of onychomycosis.


INTRODUCTION
Mycosis represents an important public health concern and its incidence has been increasing in recent years, being a frequent cause of visits to dermatologists [1].This increase has numerous causes such as susceptible population, immunocompromised individuals, fact associated with the practice of sports, wearing shoes with poor ventilation, use of public pools, and manicure instruments [2,3].
Treatment of onychomycosis is still considered a challenge owing to the anatomical characteristic of the nail substrate, chronicity of the disease, and relapse cases [12].Many antifungal drugs available in the market are not effective either because of their improper use or due to the development of resistance mechanisms of the fungus [13,14].
In this context, many studies have been carried out in search of new antimicrobial products, including products that make use of medicinal plants [12,[15][16][17][18], such as Baccharis trimera Less (DC) [19,20].Being a part of the Asteraceae family [21,22], B. trimera has numerous pharmacological activities popularly attributed to their leaves, such as antiinflammatory, digestive [23], antioxidant [24], antiulcer [23,25], and antimicrobial [19,20] properties that can be helpful in the treatment of diabetes [26], rheumatism, and hepatobiliary disorders [27].Therefore, the aim of this study was to investigate the in vitro antifungal activity of the essential oil of B. trimera against the main strains of filamentous fungi that cause onychomycosis.

B. trimera essential oil
The essential oil of the leaves of B. trimera (lot BATRI0111), which was extracted by vapor entrainment, was obtained from Laszlo Aromatology Company LTDA, Brazil.

Gas chromatography
To qualitatively and quantitatively characterize the main chemical constituents of this essential oil, an aliquot (50 µL) of oil was subjected to high-resolution gas chromatography (HP 5890) equipped with flame ionization detector.A BP-1 (SGE) 25 m × 0.25 mm column was used, with a temperature gradient of 60 °C/min and 3 °C/min to 220 °C, along with an injector (split of 1/50) at 220 °C and detector at 220 °C.The carrier gas used was hydrogen (2 mL/min) and the injection volume was 1 µL.The samples were diluted to 0.5 % in chloroform.Characterization of the peaks was performed by calculating the Kovats retention index and compared with the literature data [28].

Microbiological
screening was performed according to the technique described by Souza et al [29] by using sterile 24-well plates (Sarstedt, Germany).A 2 mm fungal fragment was inoculated onto a plate containing Sabouraud dextrose agar (SDA) with the essential oil of B. trimera (1,000 µg mL-1 β-pinene).Likewise, a positive control (SDA with fungal fragment in the absence of essential oil) and negative control (SDA with essential oil in the absence of fungal fragment) were prepared.Subsequently, the plates were incubated at 28 °C ± 2 °C for 7 days.

Determination of minimum inhibitory concentration (MIC)
The MIC was determined for the essential oil of B. trimera according to the protocol M38-A of The National Committee for Clinical Laboratory Standards (CLSI) [30], adapted as required by Almeida et al [31].All analyses were performed in triplicates.
For the preparation of the fungal suspension, fungal cultures obtained after 7 days of incubation in tubes with slanted SDA were used.The cultures were washed thrice by adding 2 mL of 0.85 % sterile saline and 20 µL of Tween-80 solution/dimethyl sulfoxide analytical grade (1: 1, v/v).The suspension formed [30] was analyzed by employing a spectrophotometer (Libra S12, Biochrom, UK) using quartz cuvette with an optical path of 1.0 cm and the transmittance was adjusted in the range of 68 -70 % at a fixed wavelength of 530 nm [31].The suspension was subsequently diluted at a ratio of 1: 50 (v/v) with RPMI-1640 medium (Sigma, USA) buffered with 3 (N-morpholino) propanesulfonic acid (MOPS, JTBaker, Germany).A 100 µL aliquot of this final solution was transferred to the microtiter plate and used for MIC determination.
The essential oil of B. trimera was solubilized in RPMI-1640 buffered with MOPS and evaluated at eight concentrations in the range from 7.8 to 1,000 µg mL −1 of β-pinene.Subsequently, 100 µL of each dilution of terbinafine, ketoconazole, and essential oil were transferred to the 96-well microplate.The fungal viability test was performed by adding 200 µL of RPMI-1640 buffered with MOPS containing the fungal inoculum.After these steps, the culture was homogenized for 2 min and incubated at 28 °C ± 2°C for 7 days.

Minimum fungicidal concentration (MFC)
After the determination of MIC, MFC was determined according to Magagnin et al [32].Aliquots of 10 µL of the medium without fungal growth were transferred to new 96-well microplate containing 200 µL of Sabouraud dextrose broth without any antifungals.

Scanning electron microscopy (SEM)
Untreated fungal fragments and fungal fragments treated with terbinafine, ketoconazole, or essential oil of B. trimera were analyzed by SEM.The fungal fragments that were subjected to SEM were prepared according to Martinelli and Santos [33] and Mio et al [34] using the Karnovsky solution (2.5 % glutaraldehyde and 2.5 % formaldehyde in 0.05 M sodium cacodylate buffer and 0.001 M calcium chloride; pH of 7.2) for 24 h, a solution of 1 % osmium tetroxide for 1 h in the dark, and solutions of acetone (25 %, 50 %, 70 %, 90 %, and 100 %).Subsequently, the resulting fragment was fixed on aluminum stubs with the aid of a double-sided carbon tape and brought to the metallizer (Balzers FL-9496/Fürstentum Liechtenstein), where they were subjected to a gold steam bath for 180 s.
The fungal samples were subjected to scanning electron microscope (JSM-5310, JEOL) under the conditions of 25-Kv power and working distance of 17 mm.The analyzes were performed at the Electron Microscopy Center Scanning the Federal University of Rio de Janeiro and the Department of Physics of the Pontifical Catholic University of Rio de Janeiro.

RESULTS
The essential oil of B. trimera subjected to gas chromatography showed monoterpenes carquejyl acetate and β-pinene as the major constituents (Figure 1 and Table 1), with βpinene exhibiting the highest percentage (23.4%).Peaks under 0.1 % were not considered.
Preliminary assay of the fungal fragments revealed that T. mentagrophytes ATCC 9533; T. mentagrophytes ATCC 11480, T. rubrum CCT 5507 URM 1666, M. canis ATCC 32903, and M. gypseum ATCC 14683 showed no visible fungal growth when exposed to B. trimera oil (1,000 µg mL-1 β-pinene).However, T. mentagrophytes ATCC 11481 and E. floccosum CCF-IOC 3757 presented some growth.Thus, it was possible to observe the respective antifungal activity of the essential oil against five strains tested.The MIC values for the essential oil of B. trimera and the reference drugs (terbinafine and ketoconazole) are shown in Table 2.The electron micrographs of the fungal strains (exposed or not exposed to the reference drugs and essential oil of interest) are presented in Figure 2.
The analysis of electron micrographs allowed us to morphologically identify the fungal structures and perceive the morphological changes in the fragments of T. rubrum CCT 5507 URM 1666, T. mentagrophytes ATCC 9533, M. gypseum ATCC 14683, and M. canis ATCC 32903, which were subjected to the essential oil of B. trimera.The abnormalities identified suggested the "flattening" of the fungal structures caused by this essential oil.

DISCUSSION
The constituents considered as chemical markers of the essential oil of this species were identified as carquejyl acetate and carquejol; however, these were present in small percentages [19,20].Nevertheless, the percentage of the chemical constituents of the essential oil analyzed is different from that reported in other works [19,35], It should be noted that there are factors related to the essential oil can cause changes in its chemical composition.These include the time of collection and drying of the plant material, soil types, genetic factors, climate, and extractive processes [36].
In B. trimera oil, monoterpenes were identified, and among them, β-pinene and carquejila acetate were found at high levels.This class of substances present in the Asteraceae family is related to antifungal activity [37], because they may increase the concentration of lipid peroxides such as hydroxyl, alkoxyl, and alkoperoxyl radicals and lead to cell death [36].Therefore, further experiments were developed according to the percentage of β-pinene because it is the major constituent for this B. trimera oil and has been reported to possess antifungal activities [35].It is important to emphasize that there is a possibility of synergism between different molecules of the essential oil, which could be responsible for the pharmacological activity [38].Sharma and Tripathi [39] indicated that essential oil acts on the hyphae presumably causing leakage of the cytoplasm, less stiffness and integrity of the cell wall, and collapse and death of the mycelium.In general, essential oil, being hydrophobic, could penetrate the mitochondrial membrane and cause cell damage, allowing easier release of vital cell components, besides interfering with the enzymatic and cellular respiration system [36].
It should be noted that the essential oil of B. trimera (MFC = 0.06 µg mL −1 ) was more potent than the drugs terbinafine and ketoconazole (MFC = 0.19 and 4.00 µg mL −1 , respectively) against T. rubrum and also exhibited lower value of MFC (0.98 µg mL −1 ) than ketoconazole (8.01 µg mL −1 ) against M. canis.In other words, the antifungal activity of the essential oil against the two fungal strains, T. rubrum and M. canis, was higher, when compared with that of ketoconazole.These data are in accordance with those reported by Rodriguez et al [40], who stated that plants of the genus Baccharis L. are used in Argentina and other South American countries for the treatment of fungal diseases.
Although the MIC values of the essential oil for some fungal strains were higher than those of terbinafine and ketoconazole, with the increase in recurrence, development of fungal resistance, and high number of adverse reactions caused by the current treatment available, the use of this vegetable substance may be an alternative way to treat onychomycosis and/or act as an adjuvant therapeutic [2].
The electron micrographs of the untreated T. mentagrophytes presented tubular form with branches, similar to that described by Park et al [41], indicating the effect of terpenes on this strain.After being exposed to terbinafine, ketoconazole, and essential oil of B. trimera, distortions and shrinkages in the hyphal morphology were observed.Comparison of the electron micrographs of the strains exposed to essential oil with those not exposed to any substance of interest revealed the potential damage of the fungal structures, exhibiting results similar to those obtained with the reference drugs.This finding demonstrated the effectiveness of this oil against the fungal strains analyzed.

CONCLUSION
The essential oil of B. trimera has antifungal effect against two of the three genera of filamentous fungi.Further studies on the safety and toxicity of the oil is required.

Figure 1 :
Figure 1: Chromatographic profile of the essential oil of B. trimera.

Figure 2 :
Figure 2: Electron micrographs of the fragments of T. rubrum CCT 5507 URM 1666 (A), T. mentagrophytes ATCC 9533 (B), M. canis ATCC 32903 (C), and M. gypseum ATCC 14683 (D).The magnification of the images corresponds to 3,500x, with the exception of images D III, whose magnification is 4,000x.Fungal fragments: not exposed to any substance of interest (I), exposed to ketoconazole (II), exposed to terbinafine (III), and exposed to the essential oil of B. trimera (IV)."a" indicates hyphae and no changes in tubular appearance."b" denotes hyphae/mycelium puckered and/or compacted."c" indicates hyphae/mycelium forming a compact mass and/or contorted and rough."d" denotes macroconidia."e" indicates macroconidia and rough

Table 1 :
Chemical profile of the essential oil of B. trimera

Table 2 :
In vitro susceptibility of the filamentous dermatophytes to the essential oil of B. trimera¸ terbinafine and ketoconazole NE: not established (for strains with MIC values above the determined working range)