Syagrus coronata seed oils have antimicrobial action against multidrug-resistant Staphylococcus aureus

Syagrus coronata seed oils have antimicrobial action against multidrug-resistant Staphylococcus aureus Cibele Maria Alves da Silva Bessa, Rodrigo Santana do Nascimento, Renata Carla Corrêa Alves*, José Matias Anselmo, Ana Paula Sant'Anna da Silva, Alexandre Gomes da Silva, Vera Lúcia de Menezes Lima, Josean Fechine Tavares, Luís Cláudio Nascimento da Silva, Márcia Vanusa da Silva and Maria Tereza dos Santos Correia


INTRODUCTION
Bacteria, with their increasing drug resistance and their capacity to spread around the world, have become the most complex threats to a global public health system that is increasingly in need of effective antimicrobial treatments (Gould et al., 2012).Among human and animal pathogens, Staphylococcus aureus is of particular concern due to its ability to express a variety of virulence factors that facilitate cell adhesion, immune evasion, host cell damage, and provoke symptoms of disease (Du Toit et al., 2014).Furthermore, S. aureus strains have developed increased resistance to antimicrobial agents.In fact, methicillin-resistant S. aureus (MRSA) and multidrug-resistant S. aureus (MDRSA) have been found to be the major cause of hospital-acquired infections (Davis et al., 2013).
The use of plants or their derived products (extracts, oils, infusions, etc.) to treat infections is an age-old practice in many parts of the world, especially in developing countries such as Brazil, where folk medicine is widely used for a variety of diseases (Nascimento et al., 2013).These plant materials apparently have less toxicity compared to synthetic drugs, which make them attractive candidates for drug development.Brazil is the fifth-largest country in the world and is characterized by a huge biological and cultural diversity.Amongst Brazilian biomes, one in particular stands out for being exclusively Brazilian: the Caatinga, which occupies a large portion of the Brazilian Northeast.The Caatinga is marked by an accentuated dryness (rainfall is usually less than 900 mm/year) and is, therefore, considered a semi-arid region.It supports a great diversity of plant species (Albuquerque et al., 2012).As a result of the environmental conditions to which they are exposed, Caatinga plants have developed interesting chemical features, some of which have been described as excellent weapons against microorganisms (Castelo Branco Rangel de Almeida et al., 2012;Oliveira et al., 2012;Da Silva et al., 2013).
Paradoxically, the Caatinga ecosystem harbors many under-utilized plant species with biotechnological and economic potential.Syagrus coronata (Mart.)Becc.(Areaceae), a palm species native to the Brazilian semiarid and cerrado regions, is a good example of this situation.This species is popularly known as licuri or ouricuri and its derived products have played a vital role in the diet and subsistence economy of traditional communities of the Brazilian Northeast region.Nevertheless, few scientific reports have been published regarding the biomedical activity of S. coronata.Recent studies have demonstrated that crude extracts or fractions of this plant have anti-Leishmania amazonensis (Rodrigues et al., 2011), antimicrobial (Hughes et al., 2013), and antioxidant (Belviso et al., 2013) activities.Specifically, oils from S. coronata have been evaluated for use as biodiesel (Teixeira da Silva de La Salles et al., 2010) and topical emulsion (Leal et al., 2013).
This study provides the chemical characterization and reports the anti-S.aureus activity of two oils from seeds of S. coronata.The first seed oil is a commercial available and is extracted by traditional rural inhabitants Bessa et al. 311 of Catimbau National Park, a national park of Brazil for Caatinga preservation.The second is an essential oil extracted in our laboratory.This is the first report of the chemical profile of an essential oil from S. coronata and antimicrobial activity of both materials.

Plant
Samples of fruits were collected at Catimbau National Park (Pernambuco, Brazilian Northeast) in mature fruit stage, during the month of March 2013.The identification of this material was made by Dr. Alexandre Gomes da Silva, and a voucher specimen (IPA 86950) was deposited at the Agronomic Institute of Pernambuco (IPA/PE).The seeds were removed from mature fruits and dried (at 33°C ) in an open area with active ventilation until constant weight was attained (three weeks).Lastly, the seeds were ground using a household grinder.

Extraction and analysis of the essential oil from S. coronata seeds (SCEO)
Samples of S. coronata seeds (250 g) were submitted to hydrodistillation for 4 h, in a Clevenger-type apparatus.The oils were dried over anhydrous Na2SO4.The oils were stored at 4°C until further analysis.All experiments were done in triplicate and results were expressed in terms of dry mass.The main constituents were analyzed by GC/MS, which were performed in the EI mode on a Hewlett Packard-6890 GC system with a fused capillary column (30 m × 0.25 mm × 0.25 µm, HP-5MS, Crossbond 5% phenyl/95% dimethylpolysiloxane) directly coupled to a Hewlett Packard 5973 selective mass detector.The mass spectrometer was operated at 70 eV.The constituents of the essential oils were identified by comparison of their mass spectral pattern and retention indices (RI) with those reported in the literature (Adams, 2009).

S. coronata seed oil (SCO) and its fatty acid composition
The commercial oil from seeds of S. coronata was kindly provided by traditional rural inhabitants of Catimbau National Park in March 2013.Fatty acid methylation was performed by the saponification and esterification procedure described by Metcalfe et al. (1966).Dosage of methyl esters was achieved using gas chromatography coupled with mass spectrometry (GC/MS).A GC/MS/QP 2010 Shimadzu instrument was used, equipped with a capillary column of type HP5 MS, 30 mm long by 250 μm internal diameter; the thickness of the film was 0.250 μm.The temperature of the injector was 250°C.Helium was used as the carrier gas at a flow rate of 0.8 mL/min, the injection mode was Split 50:1 and the temperature *Corresponding author.E-mail: mtscorreia@gmail.com.Tel: +55 (81) 2126-8547.Fax: +55 (81) 2126-8576.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License program was set at 150 to -240°C (5°C/min).

Isolation, identification and resistance profile of S. aureus isolates
Sixteen S. aureus strains were isolated from samples processed in the microbiology laboratories of referral health care institutions in Recife (Pernambuco, Brazil) between September and December 2012.The isolates were cultured on sheep blood agar and the phenotypic identification of S. aureus was based on colony morphology, Gram stain, positive plasma coagulase reaction (slide and tube test) and growth in mannitol salt agar (positive colonies changed the medium color from red to yellow).
The antibiotic-susceptibility profile of isolates was performed using a disc diffusion assay on Müeller-Hinton agar (MHA) according to the recommendations of CLSI (2011).In brief, each S. aureus isolate was grown overnight on Mueller-Hinton agar at 37°C and the colonies were suspended in sterile saline water equivalent to 0.5 McFarland standard.The suspension (100 μL) was spread over a medium plate and an antibiotic disk was applied aseptically onto the surface.Afterwards, the plates were incubated at 37°C for a period of 24 h.The antibiotics used were erythromycin, clindamycin, oxacillin, penicillin, linezolid, tetracycline, vancomycin, chloramphenicol and gentamicin.The multiple antibiotic resistance (MAR) index was calculated as previously described by Krumperman (1983) using the formula MAR = x/y, where "x" is the number of antibiotics to which the isolate demonstrated resistance and "y" is the total number of antibiotics tested.

Determination of minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The antimicrobial activity was determined using broth microdilution assay against all sixteen S. aureus strains identified in this work and a standard S. aureus strain (UFPEDA 02), which was provided by the Culture Collection from Department of Antibiotics, Federal University of Pernambuco (UFPEDA).Solutions of both oils used in the antimicrobial assays were obtained according to the following procedure: 400 μL of the SCEO or SCO were mixed with 40 μL of Tween 80 and 5 mL of sterile water (q.s. f.) in a sterile tube and shaken using a vortex (Fanem).After 5 min, solutions with a final concentration of 80 μL/mL were obtained from both samples, SCEO or SCO.
MIC was determined by the microdilution method (CLSI, 2011).Twofold serial dilutions of each solution containing SCEO or SCO (40 to 0.002 μL/mL) were prepared in Müeller-Hinton broth (MHB) and 10 μL of bacterial suspension (approximately 1.5 × 10 8 CFU/ml) were added.The samples were incubated for 24 h at 37°C.Resazurin solution (0.01%) was used as an indicator by color change visualization: any color changes from purple to pink were recorded as bacterial growth.The lowest concentration at which no color change occurred was taken as the MIC.Afterwards, cultures were seeded in MHA and incubated for 24 h at 37°C to determine the minimum bactericidal concentration (MBC), which corresponds to the minimum concentration of the sample that eliminated the bacteria.

Statistical analysis
All tests were performed in triplicate.Statistical analysis was performed using the Student's t-test.Differences were considered significant at p<0.05.The correlation indices were calculated using the Pearson coefficient (ρ).

Fatty acid composition of SCO
The fatty acid composition of S. coronata seed oil is shown in Table 2.In total, 19 fatty acids were identified in this oil, which correspond to 99.84% of the total.A predominance of saturated fatty acids was observed (72.3%), while unsaturated fatty acids represented 27.49% of total fatty acid content (23.9% for monounsaturated, and 3.6% for polyunsaturated fatty acids).The most represented fatty acids were dodecanoic acid (41.58%) and 9-octadecenoic acid (23.81%), followed by tetradecanoic acid (9.68%), hexadecanoic acid (7.19%) and octanoic acid (5.32%).All fatty acids with odd numbers of carbon atoms (C7, C9, C11, C13 and C15) were found in trace concentrations.It is noteworthy that medium-chain fatty acids accounted for 51.44% of total fatty acid content, namely hexanoic, heptanoic, octadecanoic, nonanoic, decanoic, undecanoic and dodecanoic acids.The levels of saturated fatty acids were approximately three times higher than unsaturated fatty acids.

Antimicrobial activity of SCEO
The essential oil from S. coronata seeds showed very strong activity against the standard S. aureus strain (UFPEDA 02) and also against both MRSA and MSSA strains (Table 4).The values of MIC ranged from 0.002 μL/mL to 0.08 μL/mL.The growth of the S. aureus UFPEDA 02 was inhibited by 0.002 μL/mL of SCEO.Among the clinical isolates, the majority (68.75%) were sensitive to concentrations between 0.002 and 0.01 μL/mL.Regarding the MBC values, a variation of 0.002 to 0.312 μL/mL was observed, as well as a strong correlation between MIC and MBC values (ρ= 0.89).The MBC/MIC ratios ranged from 1 to 4, thus SCEO is a bactericidal agent (Pankey and Sabath, 2004).Finally, a weak correlation was observed between the MAR indexes and MIC (ρ = 0.17) or MBC (ρ = 0.01) values, indicating that there is no relationship between the SCEO efficacy and the multidrug-resistance profile of S. aureus strains.A weak correlation between SCEO and chloramphenicol was also detected (ρ values of -0.18 and -0.19 for MIC and MBC, respectively), revealing that SCEO was effective against S. aureus strains less sensitive to the drug's action.

Antimicrobial activity of SCO
The oil obtained from S. coronata also showed a strong anti-S.aureus activity (Table 4).The SCO, at a concentration of 0.16 μL/mL, inhibited the growth of 41.18% of the strains (including UFPEDA 02).The remaining strains were sensitive to oil at 0.625 μL/mL (11.76%), 1.25 μL/mL (23.53%) and 2.5 μL/mL (23.53%).The MBC values of SCO ranged from 0.16 to μL/mL; however, 47.05% of isolates were killed by 2.5 μL/mL of SCO.Both bactericidal and bacteriostatic effects were observed for SCO (MBC/MIC ratios ranged from 1 to 16), but bactericidal action was more prominent (for 81.25% of strains).The MIC and MBC values were strongly related (ρ = 0.78), while these values were moderately correlated with MAR indices of clinical isolates (ρ = 0.49 for MIC/MAR correlation and ρ = 0.56 for MBC/MAR correlation).Unlike SCEO, the MIC and MBC values found for the SCO were substantially correlated to chloramphenicol (ρ = 0.43 for MIC; ρ = 0.58 for MBC).

DISCUSSION
S. aureus is an extremely versatile, worldwide pathogen, which is able to cause from superficial to deep-seated skin infections that can lead to sepsis (Du Toit, 2014).This bacterium has an exceptional capacity to acquire resistance to antibiotics (Gould et al., 2012).These combined features make S. aureus the most important pathogen in the Twenty-first Century and point to the urgent need for new anti-S.aureus agents.In the present study, we reports the antimicrobial action of S. coronata seed oils against MRSA and MSSA S. aureus strains and the chemical composition and fatty acid content of SCEO and SCO, respectively.While both oils showed antimicrobial activity, SCEO was more active (15.6 to 250 times greater) than SCO.Their MIC values were moderately correlated between themselves (ρ = 0.43).Nevertheless, while SCEO is a more effective bactericidal agent, SCO showed both bactericidal and bacteriostatic actions.The antimicrobial activity of compounds derived from S. coronata has been evaluated for aqueous and methanol extracts from different tissues (leaves, inflorescence, nut-shell, liquid and solid endosperm nuts).
In that study, only the extracts obtained from inflorescence tissue showed antimicrobial activity by inhibiting S. aureus (including strains with antibiotic resistance) and Bacillus cereus.The authors did not report the chemical characterization of these active extracts (Hughes et al., 2013).
To the best of our knowledge, the composition of S. coronata essential oil and its biological activity have not been reported before.Only one study on the volatile fraction of S. coronata is known (Belviso et al., 2013).The study's authors evaluated the volatile fraction of raw and roasted seeds.A total of 59 volatile compounds were identified in S. coronata (34 in raw and 55 in roasted) belonging to 8 chemical classes.Among these, 30 compounds were found in both raw and roasted seeds.Carboxylic acids (such as octanoic and hexanoic acids) prevailed in raw S. coronata seeds, while after roasting, Strecker aldehydes (δ-lactones and alkyl pyrazines) were the most abundant.These data corroborate with our results, which showed that the essential oil of S. coronata seeds is also predominantly composed of fatty acids such as octanoic, dodecanoic and hexanoic acids.
Although less active than SCEO, the seed oil of S. coronata also showed a significant anti-S.aureus activity.SCO is predominantly composed of saturated fatty acids, with lauric acid (dodecanoic acid) the main constituent.The levels of saturated fatty acids were approximately three times higher than unsaturated fatty acids.Saturated fatty acids with medium chain length, such as lauric acid, have been found to be major components of other oils from Arecaceae plants, such as Syagrus oleraceae, Syagrus romanzoffiana and Acrocomia aculeate (Coimbra and Jorge, 2011).This study demonstrated that S. coronata seed oil is a rich source of medium-chain fatty acids, which could be suitable for biomedical applications (cosmetic and pharmaceutical industries), as showed by Leal et al. (2013).Our data are in agreement with the work of Bauer et al. (2013), which showed that saturated fats with high levels of medium-chain fatty acids (such as lauric and myristic fatty acids) are prevalent in kernel and fruit oil of S. coronata collected in Bahia, Brazil.These authors commented that this composition is very similar to coconut oil.The presence of saturated chains made a biodiesel derived from S. coronata less viscous and more stable to oxidation and these physico-chemical properties showed that it has good potential for use in engines (Iha et al., 2014).
In this study, the strong anti-staphylococcal properties of S. coronata seed oils were demonstrated.These are promising results that encourage further research on the toxicological and pharmacological aspects of this species, as well the determination of the action mechanisms involved.Such research would clarify these substances' suitability in any potential application as antimicrobial agents for therapy, food practices and/or cosmetic industry.

Table 1 .
Chemical composition of essential oil from S. coronata seeds.

Table 2 .
Fatty acid composition of S. coronata seed oil.

Table 4 .
Anti-S.aureus activity of oil and essential oil from seeds of S. coronata.MBC values are expressed in µL/mL; b MIC and MBC values are expressed in µg/ml; MRSA: Methicillin-resistant S. aureus strain; MSSA: Methicillin-sensitive S. aureus strain.UFPEDA02: Standard S. aureus strain provided by the Culture Collection UFPEDA.
a MIC and