MDGC-MS analysis of essential oils from Protium heptaphyllum (Aubl.) and their antifungal activity against Candida specie

Protium heptaphyllum is found in the Amazon region, and in various Brazilian states and South American countries. Also Known as almecega, it produces an oil resin used in traditional medicine as analgesic, anti-inflammatory, cicatrizant and expectorant, it is rich in pentacyclic triterpenes and essential oil. The main objective of this study was to analyze the chemical composition of P. heptaphyllumresin (OEPh) over different extraction times and to evaluate their antifungal activity against Candida species, obtained from gardeners with onychomycosis, using the disk diffusion method. The OEPh was obtained by hydrodistillation and analyzed by Multidimensional Gas Chromatography coupled with Mass Spectrometry (MDGC / MS). Candida species were obtained from lesions on the nails of horticulturist from a community garden in the city of Teresina, Piauí, Brazil. The antifungal activity in concentrations of 1000 μg/L, 500 μg/L and 250 μg/L, PROTOCOL M44-A2 (CLSI 2009) OEPh was tested. The main constituents identified were: l-limonene, α-terpineol, p-cineol, o-cymene and α-phellandrene, however, its composition varies significantly with extraction time. All species, except C. rugosa, were inhibited with halo (≥ 14 mm) at 1000 μg / L. C. krusei is naturally resistant to the drug fluconazole, but when tested with OEPh the clinical species (case 9) demonstrated sensitivity in three dilutions (halo ≤ 10 ≥ 14) and the standard strain was inhibited at concentration of 1000 μg/Lg / L (halo 14mm). A similar situation also occurred with the standard strain of C. parapsilosis (halo ≥ 11mm). OEPh has considerable antifungal activity, which merits further investigation for alternative clinical applications, since this species is widely distributed in our community, and it presents good yields, and also has important therapeutic applications.


INTRODUCTION
Protium heptaphyllum (Aubl.)Marchand is a tree of the Burseraceae family, aromatic, evergreen or semi-deciduous, of humid sandy soil or dry and that reaches 10-20 m in height (Lorenzi & Matos, 2008).These species are widely spread throughout tropical and subtropical regions, mainly in the Amazon Rainforest.
P. heptaphyllum is found in the Amazon region and in some Northeastern states, such as Bahia, Ceara, Piaui, as well as in other countries of South America (Colombia, Paraguay, Suriname and Venezuela) (Citó et al. 2003;Lorenzi & Matos, 2008).It's recognized by the use of its leaves and bark as hemostatic, healing and anti-inflammatory, being used by the native people also as a nasal decongestant.Its stalk exudes an oily resin called almecega or breu-branco, which hardens on contact with air, of white-greenish color, pleasant aroma and rich in essential oils (Lorenzi & Matos, 2008).Contraceptive, antineoplastic, healing, expectorant and antimicrobial action were also related others authors (Oliveira et al. 2005, Rüdiger et al. 2007).
Due to the high cost of antifungal agents, the side effect reactions observed and the occurrence of resistance of some species to antifungal agents, the use of essential oils from plants has been target of studies given its potential as natural antifungal agents.Onychomycosis is a fungal infection of nail which affects about 6 to 8% of the adult population worldwide (Amartya et al. 2013).The yeasts, including Candida spp.account for 2-10 % of fungal nail infections (Thomas et al. 2010).
In view of the popular knowledge of the antimicrobial activity of almecega and the absence of studies on the antifungal activity of this material in clinical isolates, this study was conducted to analyze the chemical composition of the essential oil of Protium heptaphyllum and evaluate their antifungal activity against yeast species, obtained from lesions of workers with onychomycosis in a community garden located in the neighborhood "Dirceu", in the City of Teresina, Piaui, Brazil.

Plant Material
The resin was collected at a private property in the municipality of Timon, Maranhão, Brazil, in December 2012 and kept under refrigeration until the time of extraction of the oil.The specie was identified in the Herbarium Graziela Barroso of the Federal University of Piaui, Brazil, and a voucher specimen was registered under No. 28730.

Extraction
The resin was subjected to hydrodistillation in a Clevenger type system.Six successive fractions were collected at intervals of one hour.In another system was collected a fraction after 6 continuous hours (6h cont), this fraction was used for the antifungal tests, due its higher yield.The extracted oils were dried with sodium sulfate (Na 2 SO 4 ), weighed and stored under refrigeration (5°C).
In the second dimension similar column and analysis methods were used, GC-MS.The conditions of MS were: ion detector quadrupole operating in electron impact and impact energy of 70 eV; scan speed 1,000; and the fragments found in the range of 45 to 650 Da.
The identification of the essential oil components was performed by comparing the retention indices, interpretation of the fragmentation pattern of the respective mass spectra, comparison with the database of the analysis system (being considered only the spectra with index of equal similarity or greater, at 95% ) and with literature data.The pattern solution of n-alkanes (C 8 -C 20 ) was used for the calculation of the index of Kolvats, and this value was compared to the one described in the literature (Adams 2007, Costa et al. 2010).

Antifungal activity
To evaluate the antifungal activity of the essential oil of P. heptaphyllum resin, we used disk diffusion tests that were performed in the Research Laboratory of the University Center UNINOVAFAPI, according to the protocol M44-A2 (CLSI 2009).
A suspension was prepared for each species of yeast with fresh culture of 24h of clinical isolates of: Candida krusei, C. albicans, C. parapsilosis, C. metapsilosis, C. rugosa, C. guillermondii.The identified agents came from lesions of onychomycosis of workers in a community garden located at Dirceu district, city of Teresina, State of Piaui, Brazil (after being approved by the research ethics committee 0207.0.043.000-10).25 μL of each yeast suspension was inoculated on Petri dishes containing culture medium Mueller-Hinton (Difco TM , USA), with glucose at 2% and methylene blue (MGB agar).Three oil concentrations were used, being 1000 μg/L, 500 μg/L and 250 μg/L.
Withdrew 20 μL of each concentration, placed in sterile and standardized discs with a diameter of 6 mm, which were then deposited on the surface of the MGB medium.For quality control with strains of C.parapsilosis ATCC 22019 and C. krusei ATCC 6258, and as negative control, we used solvent acetone/water at 15%.All tests were carriedout in triplicate.
The descriptive analysis of the categorical variables was carried out by reading the absolute and relative frequencies (%), while in the numeric variables, the average position and standard deviation variability (SD) measurements were taken.

Performance and Analysis of Chemical Constituents
The MDGC/GCMS-2010 is a MDGC system that offers excellent performance and highly reproducible separation with "multi-deans switching" technology, supporting the analysis of complex matrices, such as rocks extract samples, oils (petroleum), aromas and optical isomers.In this research work, we did not identified significant changes in the oil composition when using similar polarity column,however we focused especially on the separation and quantification power in the first dimension (MDGC) and identification in the second dimension (GCMS).The analysis of the essential oil of P. heptaphyllum resin by MDGC-MS allowed the identification of 26 constituents, predominantly monoterpenes (Table 1).The constituents present in greater abundance were: l-limonene, α-terpineol, p-cineole and o-cymene (Figure 1 concentration of α-terpineol increases, as well as other compounds that did not appear in the first hours begin to emerge.This fact is explained by the secondary reactions that may occur in the system due to the constant heating. Research on antifungal effects of DLlimonene against various yeast species was presented by Ünal et al. (2012).More specifically, these authors demonstrated that DL-limonene at a concentration of 0.20% (w V-1) inhibited cell growth, ethanol formation, and sugar utilization by S. cerevisiae.
The α-terpineol is a major component of the essential oil of many plants and has been reported to enhance the permeability of skin to lipid-soluble compounds (Williams & Barry, 1991).Antimicrobial effect of linalool and α-terpineol against periodontopathic and cariogenic bacteria was evaluated by Park et al. (2012).The α-terpineol has also been described to have anti-inflammatory properties (Held et al. 2007), antibacterial (Kotan et al. 2007) and antifungal activities (Pitarokili et al. 2002).Hassan et al. (2010)showed that α-terpineol also has a potential anticancer agent.The p-cineole is responsible for several activities, amongst which, *Indice de Kolvats (Adams, 2007).lc/a = low concentration or absent.it is used in the treatment of skin infections (Hammer et al. 2002).Recently, Santin et al. (2014) showed that the major compounds identified in the essential oil Origanum vulgare, are; thymol, 4-terpineol, α-terpinene which are responsible for antifungal activity against the yeast M. pachydermatis.
In the world market, much of the α-terpineol comes from synthetic route that uses pinene and turpentine as precursor substances (Baptistella et al. 2009) and other synthetic routes from l-limonene and nerol have been studied.The α-terpinil trifluoroacetate when derivative from d-limonene, through the addiction of Markvnikov using trifluoroacetic acid can be converted into d-αterpineol by hydrolysis (Yuasa &Yuasa, 2006), as illustrated in (Figure 2).
The chemical composition of the oil from the resin of Protium varies according to time of year and the region where the material was collected.Previous studies performed with the same resin, but acquired from other sources showed that the chemical composition of the oil undergoes significant changes.Citó et al. (2003) found as major compounds β-terpinilacetate (23.2%), limonene (18.2%) and β-ocimene (11.2%), in resin from Teresina (Piaui , Brazil), while the oil obtained by Marques et al. (2010) in Cruzeiro do Sul, State of Acre (Brazil) showed p-cymene (39.93% ), n-tetradecane (13.38%) and dihydro-4-carene (11.69%).The (Table 2) shows the major components identified in the essential oil of P. heptaphyllum collected in different regions.
The differences in the composition of essential oils extracts of P. heptaphyllum may occur due to other factors including in traspecific genetic differences in plants, seasonal and environmental influences such as temperature, humidity, soil, and time of collection.The time and method of storage of the resin is an important aspect to be considered, especially in view of the essential oils consisting mainly of monoterpenes and light compounds (volatiles, heat-unstable and photolabile) (Mattana et al. 2015;Rüdiger et al. 2007).

Antifungal Activity
So far there are no reports in the literature on the use of OEPh for the treatment of onychomycosis, which is considered a crucial disease in public health due to its high incidence and worldwide prevalence, representing more than 50 % of all onychopathies (Bokhari et al. 1999, Thomas et al. 2010, Zotti et al. 2011).Performance evaluation of disk diffusion method for determining the antifungal activity of OEPh, against yeasts agents of onychomycosis are shown in (Table3).
It was observed that all the species, except C. rugosa, were inhibited with halo ≥ 14 mm at 1000 µg/L.The C. krusei(case 9), C. guilliermondii(case 54) and C. albicans (cases 71, 89) were inhibited in all three concentrations (halo 12 ≤ Χ ≥ 14 mm).Fluconazole is an antifungal that has good clinical activity against most Candida spp.However,C.krusei is naturally resistant to this drug, but when tested with OEPh the clinical species showed sensitivity in three dilutions, and standard strain was inhibited at 1000 µg/L (halo ≥ 14mm).A similar situation also occurred with the standard strain of C. parapsilosis, which at the same concentration showed halo Χ ≥ 11mm.Comparing the performance of OEPh and fluconazole in relation to other Candida species, the drug Fluconazole is more reactive than the tested oil (halo 20 ≤ ≥35 mm).
Values of inhibition halo equal to or greater than 8 mm or 10 mm are indicative of the presence of inhibitory activity (Fenner et al. 2006, Lima et al. 2006, Aibinu et al. 2007, Dutta et al. 2007, Nascimento et al. 2007, Packer & Luz, 2007, Scorzoni et al. 2007, Pozzati et al. 2008, Menezes et al. 2009, Almeida et al. 2011, Cavalcanti et al. 2012).These values do not predict clinical response, but are associated to products with pharmacological potential in studies of drug screening (Cavalcanti et al. 2012).
Although the disk diffusion test is one of the most used and accepted for the search for new drugs, the results obtained in tests with this method are difficult to interpret.The substances used to  dilute the compounds may constitute relevant factors affecting the result of inhibition of the isolates evaluated.For example, the dimethyl sulfoxide (DMSO), acetone and ethyl and methyl alcohols are organic solvents often cited in the specialized literature on plant products; however, the correct evaluation of the influence of the diluent on the result of inhibition obtained is required.
In this study we investigated solvents such as ethanol, methanol and acetone (data not shown) verifying that only the last of these diluents showed no inhibitory activity itself against clinical isolates and standard strains.Thus, the study was conducted by employing acetone in water (15%) as diluent.Moreover, the characteristics of the products evaluated, such as volatility, viscosity, hydrophobicity are factors of impact on the results.Thus, the different products have physico-chemical properties that interfere with diffusion in the agar.Lima et al. (1993) evaluated the antifungal activity of the essential oil of various species of plants, against 16 species of dermatophytes (Epidermophyton, Microsporum, Trichophyton), and 80% of these plants showed antifungal activity, except the P .heptaphyllum,which presented β-terpinolene, L-phelkandrene e α-pinene as majority constituents.It is important to note that in this study, these constituents are in low relative abundance (L-phelkandrene -1.49%, α-pinene-1.17%) or absent (β-terpinolene), demonstrating the importance of the collected region in composition and also that these compounds can not be responsible for such activity.
The essential oil obtained from the resin of Protium heptaphyllum when analyzed by MDGC-MS and showed as major constituents l-limonene, α-terpineol and p-cineol.The analysis of the fractions collected at intervals of 1 hour showed that the abundance of the main components decrease, while the α-terpineol increases over time due to secondary reactions when subjected to heating.As compared to literature data, our study evidenced some differences in the chromatographic profile as well as in quantitative composition of essential oil P. heptaphyllum.
This study presented considerable antifungal activity of Protium oil resin, inhibiting species of Candida, by disk diffusion method, with diameter of halos ( Χ > 14mm), which cause a broad spectrum of invasive superficial infections and nail infections that are public health problems.
Though the literature data reports the antifungal activity of some constituents identified in this study, there is still a need for more detailed studies of OEPh, including activity in vivo for possible alternative clinical applications, since this specie is widely distributed in our community, it presents good yield and also important therapeutic applications.
FIGURE 1.Chemical structure of the main components of the essential oil of P. heptaphyllum.

TABLE 1 .
Extraction time influence on the chemical composition (%) of the essential oil of P. heptaphyllum resin.

TABLE 2 .
Literature review of major constituents of essential oil of P. heptaphyllum.

TABLE 3 .
Performance evaluation (halo inhibition, mm) of the disk diffusion method for determination of antifungal activity of the essential oil of P. heptaphyllum against yeast species Χ , average; (-) null average due to the absence halo; standard deviation (SD) calculated for tests performed in triplicate.