Chemical composition and antifungal activity of essential oil and fractions extracted from the leaves of Laurus nobilis L . cultivated in Southern Brazil

Chemical composition and antifungal activity of essential oil and fractions extracted from the leaves of Laurus nobilis L. cultivated in Southern Brazil Carla M. M. Fernandez-Andrade, Maurício F. da Rosa, Édela Boufleuer, Fabiana Borges Padilha Ferreira, Camila Cristina Iwanaga, José E. Gonçalves, Diógenes A. G. Cortez, Cleide Viviane Buzanello Martins, Giani Andrea Linde, Márcia R. Simões, Viviane S. Lobo and Zilda C. Gazim*


INTRODUCTION
Infectious diseases caused by fungi are responsible for morbidity and mortality in thousands of hospitalized and immune compromised individuals annually (Lemke et al., 2005;Alangaden, 2011).Therefore, the development of novel antifungal drugs is of vital importance.Patients with human immunodeficiency virus infection (HIV infection/ AIDS) comprise a highly susceptible group, and the number of opportunistic infections reported for this group has increased dramatically (Omoruyi et al., 2014).Cryptococcosis, a systemic mycosis caused by yeasts of the Cryptococcus genus, most commonly Cryptococcus gattii and Cryptococcus neoformans, is the third most prevalent disease in people with HIV infection/ AIDS (Gullo et al, 2013;Maziarz and Perfect, 2016).These agents are responsible for cryptococcal meningitis, a disease most commonly diagnosed in sub-Saharan Africa, where it may kill more people each year than tuberculosis.Globally, one million new cases of cryptococcosis are estimated to occur in HIV-positive individuals annually, resulting in nearly 624,700 deaths, most due to meningitis (Park et al., 2009).
Additionally, the Candida yeasts are of clinical importance, causing opportunistic infections.Candidemia (disseminated hematogenous infection) or deep-seated infection in normally sterile body sites of immunosuppressed patients cause high morbidity and mortality, and also increase medical costs by increasing the duration of hospitalization (Patterson, 2005;Alangaden, 2011;Menezes et al., 2012).C. albicans is one of the major causes of infection of skin and mucosal surfaces, it can infect any organ and in cases of infections in the bloodstream can lead to death, if left untreated (Noble and Johnson., 2007;Duggan et al., 2015).Another species of great importance is C. parapsilosis, which has recently emerged as the second most commonly isolated species in candidemia, infects groups such as neonates, transplant patients, and individuals receiving parenteral nutrition.C. parapsilosis has the ability to form biofilms with high affinity for intravascular and parenteral nutrition devices, being more prevalent than C. albicans in patients using such devices (Trofa et al., 2008;Menezes et al., 2012).C. tropicalis is increasingly isolated from patients with hematologic malignancies, and its presence is predictive for infection causing mucositis and neutropenia.C. krusei is the fifth most common species in immunocompromised patients, with high mortality rates because of resistance to commonly used antifungal drugs such as fluconazole (Pfaller et al., 2008;Sipsas et al., 2009;Alangaden, 2011).
Microbial resistance develops through naturally occurring mutations in fungal cells during prolonged antifungal treatment, resulting in selection of the most resistant strain (Pfaller, 2012).Resistance to drugs is a major concern worldwide because of the limited number of antifungal drug classes, and because the number of patients requiring antifungal treatment is increasing (Maubon et al., 2014).Because of the pressing need for novel therapies to treat the fungal infections, researchers have directed their studies toward the discovery of natural substances with greater efficacy and lower toxicity (Pina et al., 2012;Santos and Novales, 2012).
Secondary metabolism in plants produces many compounds that have complex chemical structures, many of these substances have been reported to have antimicrobial properties as essential oils (EOs) (Edris, 2007).
EOs are important natural products, being multifunctional, well accepted by consumers, and safer than synthetic additives.Thus, they have been targeted for research on natural food preservation, crop protection, pharmaceutical applications, and cosmetic production (Bakkali et al., 2008;Okoh et al., 2010).
Laurus nobilis L. is a tree belonging to the Lauraceae family, native to Asia.The plant is popularly known as laurel, and is cultivated in south and southeast Brazil (Marques, 2001;Lorenzi and Matos, 2008).Laurel is an aromatic spice, commonly used to season recipes owing to its aroma.Laurel leaf is also used in folk medicine as infusions or decoctions, being considered a gastric stimulant as well as a treatment for rheumatic disorders.It is also used externally for rheumatism, and as an antiseptic for dandruff and lice (Joly, 1993;Marques, 2001;Lorenzi and Matos, 2008).
This present study was designed to evaluate the antifungal activity of EO and fractions extracted from the leaves of L. nobilis cultivated in Southern Brazil.

Plant material
Fresh leaves of L. nobilis L. were collected in January 2014, in the city of Pérola, Paraná, Brazil (23º 50' 56.6" S 53º 41' 06.2" W, 20 m), identified by Msc.Mayara Lautert and Camila Vanessa Buturi, as sample number 1615, and were deposited at the Herbarium of the State University of Western Paraná.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License apparatus for 2 h (Fiorini et al., 1997).EO was collected, dried over sodium sulfate, filtrated, and stored in amber-colored vials at 4°C.After total evaporation of the solvent, the EO was weighed to calculate oil yield (%).

GC-MS analysis
Analysis of EO was carried out in a gas chromatograph (Agilent 7890 B) coupled to a mass spectrometer (Agilent 5977 A) equipped with an Agilent HP-5MS capillary column (30 m × 0.250 mm × 0.25 μm), using the following conditions: injector temperature of 250°C, injection volume 1 μL at a ratio of 1:30 (split mode), initial column temperature of 50°C, heated gradually to 260°C at 3°C/min rate.The carrier gas (helium) flow was set at 1 mL/min.The temperatures of the transfer line, ion source, and quadrupole were 250, 230 and 150°C, respectively (Derwich et al., 2009;Moghtader and Salari, 2012).Mass spectra were obtained with a scan range of 40 to 500 m/z and a solvent delay time of 3 min, and compounds were identified based on comparison of their retention indices (RI), obtained using various n-alkanes (C8-C25).In addition, their electron ionization (EI) mass spectra were compared with the NIST 11.0 library spectra according to Adams (2007).

Determination of the minimum inhibitory concentration (MIC)
Minimum inhibitory concentrations (MIC) of EO were determined against C. albicans ATCC 18804, C. krusei ATCC 20298, C. parapsilosis ATCC 20019, C. tropicalis ATCC 750, C. gattii L21/01, and C. neoformans H99.An 80 mg/mL of the EO solution was prepared, diluted with 2% polysorbate 80 (tween 80) in Muller Hinton Broth with the addition of 2% glucose for yeasts.The culture medium (100 μL) was distributed into the wells of a microdilution plate, and then 200 μL EO solution was added to the second well.Following homogenization, this was transferred to the third well, and so on until the tenth.Thus, the final concentrations obtained were 40.00, 20.00, 10.00, 5.00, 2.50, 1.25, 0.62 and 0.31 mg/mL.A microbial suspension was prepared in saline with turbidity equivalent to 0.5 on the McFarland scale (1 × 108 UFC/mL).Next, the 1:50 yeast suspension was diluted to 1:20 in Mueller Hinton Broth modified for fungi to yield 1 × 105 UFC/mL inoculums.Hundred microliters of the suspensions was inoculated in triplicate into each well containing the various EO concentrations.Well 1 was used as sterility control.The toxicity control was well 11 with 2% polysorbate 80 in culture medium.Well 12 was used as the growth control, where microbial suspension was added to the culture medium.Microplates were incubated at 35°C for 24 h in aerobic conditions.MICs were determined by examining the plates.The lowest concentration of EO causing complete inhibition of growth (CLSI, 2008) was reported.The same procedure was performed with fractions of the essential oil, using an initial solution of 20 mg/ml and the fluconazole was used as positive control.

Statistical analysis
The data were subjected to analysis of variance (ANOVA) and comparisons of means by Tukey's test at a 5% significance level.

RESULTS AND DISCUSSION
Hydrodistillation of laurel leaves resulted in a 0.66% yield of EO.The yield obtained is in accordance with that reported by Lira et al. (2009), who obtained a yield between 0.3 and 1.2% during their 15-month study.
The results for MIC tests of EO and fractions are presented in Table 2.In order to compare the results, the values found in this study were compared with the classification values proposed by Aligiannis et al. (2001) and Duarte et al. (2005) for plant materials, based on MIC results.This classification system categorizes materials as strong inhibitors; MIC up to 0.5 mg/mL, moderate inhibitors; MIC between 0.6 and 1.5 mg/mL, and weak inhibitors; MIC above 1.6 mg/mL.In the present study, EO demonstrated low inhibition of Candida strains, moderate inhibition of C. neoformans (MIC 0.62 mg/mL), and high inhibition of C. gattii (MIC 0.31 mg/mL).FR1 moderately inhibited C. gattii (MIC 1.25 mg/mL).
FR3 moderately inhibited C. krusei (MIC 1.25 mg/mL) and C. neoformans (MIC 0.62 mg/mL), and strongly    Erturk et al. (2006) showed the antifungal activity of laurel EO against C. albicans, with an MIC of 2.4 mg/mL.Peixoto et al. (2017) evaluated the antifungal activity of EO of laurel collected in Brazil, and found isoeugenol (53.5%) and myrcene (16.6%) as major constituents.The EO showed activity against C. albicans strains (MIC 0.25 mg/mL), C. tropicalis (MIC 0.50 and 0.25 mg/mL), C. krusei and C. glabrata (MIC 0.5 mg/mL).The EO evaluated in the present study exhibited a higher MIC (10.00 and 5.00 mg/mL) for Candida strains; the differences in biological activities between these findings and the literature may be attributable to the differences in chemical composition of EO of laurel that directly influences its antimicrobial activity.

Microorganisms
Monoterpenes and sesquiterpenes with aromatic rings and phenol groups are capable of forming hydrogen bonds with the active sites of target enzymes, and this is the main mode of antimicrobial action of EO.Other compounds such as alcohols, aldehydes, and esters also contribute to antimicrobial activity (Belletti et al., 2004).The antifungal activity of the fractions can be attributed to the presence of terpenes.Linalool, the major constituent in FR 4 and FR5, was screened for activity against Candida isolates by Marcos-Arias et al. ( 2011), who reported findings against C. albicans (MIC, 0.30-2.50mg/mL), C. tropicalis (0.60-2.50 mg/mL), C. parapsilosis (0.30 mg/mL), and C. krusei (0.60 mg/mL).The eugenol in FR1 and FR2, and terpinen-4-ol present in FR3-5 were also investigated by Marcos-Arias et al. (2011); against C. albicans eugenol had an MIC in the range of 0.60-2.50mg/mL, and terpinen-4-ol in the range of 0.60-5.00mg/mL; for C. tropicalis, the eugenol and terpinen-4ol MIC range was 0.60-1.20 mg/mL; for C. parapsilosis, the MIC of both substances was 0.60 mg/mL; and for C. krusei, both had an MIC of 1.20 mg/mL.In general, the MIC values reported for the fractions in this study against Candida species are close to those found by Marcos-Arias et al. (2011), considering that they evaluated pure substances, while the fractions in this present study are a mixture of terpenes, which may or may not have synergistic effects.Hsu et al. (2013) determined the MIC against Candida species for linalool, and found it to be1.23 to 4.93 mg/mL for C. albicans, and 2.47 mg/mL for C. tropicalis.These values are comparable to those found in the present study for FR4 and FR5, in which linalool is the major compound.
1,8-Cineole is present in all fractions, and is the major compound in FR2 and FR3 (76.88 and 84.24%, respectively); its antifungal activity was investigated by Adegoke et al. (2000) against Candida tropicalis yeast.It was found to have an MIC of 0.16 mg/mL against C. tropicalis yeast, with an activity superior to the that recorded for the fractions containing 1,8-cineole in the present study.However, these authors investigated the use of pure substances.Hammer et al. (2003) determined the MIC for 1,8-cineole against isolates of C. albicans (40.00 mg/mL) and C. parapsilosis (80.00 mg/mL).Pattnaik et al. (1997) analyzed the antifungal action of oxygenated linalool and 1,8-cineole monoterpenes, and found linalool to possess activity against C. albicans (MIC 0.20 mg/mL).C. albicans was resistant to 1,8-cineole at MIC up 5.00 mg/mL.This present study documented lower linalool activity in the fractions containing it as the major compound.Fractions containing 1.8-cineole had higher antifungal activity against C. albicans and C. parapsilosis in comparison with the results of the studies mentioned above; this may be explained by the fact that fractions are a mixture of antifungal substances acting in synergy.
To our knowledge, this is the first investigation documenting antifungal activity of EO and fractions extracted from fresh leaves of laurel against strains of C. parapsilosis, C. gattii, and C. neoformans.EO extracted and analyzed in this study, as well as its fractions, possess antifungal properties.The presence and proportion of the EO constituents are related to biological properties of laurel.However, further studies on toxicity of these substances and in vivo experiments are necessary to confirm the results here presented.

Table 1 .
Chemical composition of essential oil and fractions obtained from the leaves of Laurus nobilis.

Table 2 .
Minimum inhibitory concentration (MIC) of essential oil and fractions of L. nobilis (mg/mL).