Antimicrobial activity and chemical composition of essential oils from Aloysia polystachya ( Griseb . ) Moldenke grown in Brazil

Aloysia polystachya (Griseb.) Moldenke (Verbenaceae) is considered as a medicinal plant by Argentineans and Brazilians, being popularly used as sedative, eupeptic and carminative agent. The high amount of carvone produced by this species could justify its use in the treatment of some digestive disorders. The aim of this work was to evaluate the effect of the time of leaf harvesting on the composition of A. polystachya essential oil. The antimicrobial activity of this essential oil was evaluated against Escherichia coli, Staphylococcus aureus, three Candida strains and Trichophyton rubrum. Essential oils from dry and fresh leaves obtained by hydrodistillation were analyzed by gas chromatography (GC) and GC-mass spectrometry (GC-MS). The chemical profile of A. polystachya essential oils varied according to day-time harvesting of the plant material though they were basically composed of carvone and limonene. The antifungal activity of the essential oils against clinical isolates of Tricophyton rubrum, Candida albicans, C. krusei and C. famata was validated.


INTRODUCTION
Fungal infection processes are very common especially in individuals living in tropical regions, with the genus Candida being considered as the main opportunistic infectous agent affecting mucosae, digestive and respiratory systems.Fungal resistance, antifungal toxicity, drug interactions and unavailability of conventional antifungals demand a rapid development of innovative formulations to treat the emerging fungal infections.
Plants of the Verbenaceae family, which encompasses 100 genera growing in tropical and subtropical regions worldwide, typically produce antimicrobial essential oils (Alea et al., 1997;Pascual et al., 2001;Oliveira et al., 2006;Khokra et al., 2008).Important examples are the essential oil of Lippia grandis, which is effective against Staphylococcus aureus; Enterococcus faecalis and Escherichia coli, which are rich in carvacrol, thymol and qcymene (Sarrazin et al., 2012); the oil of Vitex agnuscastus, which is potent against Aspergillus niger because of the presence of 1,8-cineole and α-pinene (Stojkovic et al., 2011); and the oil of Lippia sidoides, which is rich in thymol, carvacrol and β-caryophyllene, an active agent against Pseudomonas aeruginosa and Staphylococcus aureus (Veras et al., 2011).
The anxiolytic activity of hydroalcoholic extracts from Aloysia polystachya (Griseb.)Moldenke (Verbenaceae) leaves have been reported (Mora et al., 2005;Hellion-Ibarrola et al., 2006).According to Mora et al. (2005), the presence of carvone may be responsible for the activity on the nervous system and also for the digestive and carminative activity recommended by the etnopharmacological research.
The aims of this study were to determine the chemical composition of A. polystachya essential oil, the effect of leaf harvesting time the composition of the essential oil and its in vitro antimicrobial activity against E. coli, S. aureus, Candida albicans, Candida krusei, and Tricophyton rubrum.

MATERIALS AND METHODS
A. polystachya leaves were collected at 9:00 AM, 12:00 AM and 03:00 PM from plants found in Ribeirão Preto, São Paulo, Brazil.Taxonomic identification was confirmed by botany specialist (L.Rossi) at the Herbarium of the Botanical Institute, São Paulo, where a voucher sample was deposited with the reference number HPM 1213. A. polystachya essential oil was extracted from fresh picked leaves and from leaves dried under 45°C for 2 days through hydrodistillation using a Clevenger-type apparatus (Gottlieb and Magalhães, 1960).The obtained essential oils analyzed with gas chromatography (GC) and GC-mass spectrometry (MS).

GC and GC-MS
The identification of volatile constituents was performed by using a Hewlett-Packard 6890 gas chromatograph, equipped with a HP-5975 mass selective detector and capillary column HP-5MS (30 m 0.25 mm 0.25 µm).GC and GC-MS were carried out by using split/splitless injection, with injector set at 220°C, column set at 60°C, heat ramp of 3°C.min -1 , final temperature of 240°C and the flame ionization detector (FID) set at 250°C.Helium was used as carrier gas at 1 ml.min -1 .The GC-MS electron ionization system was set at 70 eV.A sample of the essential oil was solubilized in ethyl acetate for analyses.Retention indices (RI) were determined by co-injection of hydrocarbon standards.The oil components were identified by comparison with data from literature (Adams, 2007) and the profiles from the Nist-05 library and by co-injection of authentic standards, when available.

Fungal strains
The strains used in the assays were reactivated in Sabouraud (Difco ) solid medium at 28°C for 2 to 14 days.Strains of C. albicans (2), C. krusei (2) C. famata (1) and T. rubrum (2) were investigated.The strains of C. albicans, C. fanata and C. krusei were isolated from a patient attending the Electro Bonini Clinical Center, whereas C. krusei was a field strain.C. albicans (ATCC10231) and C. krusei (ATCC6528) were used as control.
The clinical isolate T. rubrum (ATCC MYA-3108) and a mutant strain designated Tru MDR2 were grown according to previously Pina et al. 5413 described methods (Fachin et al., 2006).The mutant strain is a transformant of the MYA-3108 strain whose TruMDR2 gene encoding the ABC-type transporter, involved in drug multi-resistance, was attenuated.

Bacterial strains
The study included 4 bacterial strains, 2 ATCCs, E. coli (25922) and S. aureus (6538) and 2 clinical isolates from the oropharyngeal tract of patients in the Clinical Center Electro Bonini of the University of Ribeirão Preto -UNAERP, stored at -80°C.Previous to the assays, the strains were reactivated in Brain Heart Infusion (Difco ) liquid medium at 37°C overnight.

Antifungal assays
Inocula were prepared by using suspension of Candida blastoconidia or Tricophyton conidia, which were transferred to a tube containing sterile saline (0.9% NaCl) for inoculum turbidity of 10 5 and optical density determined spectrophotometrically (Spectronic Genesys 2) at 530 nm to a transmittance of 70-75%.For the microdilution method, the inocula were adjusted in saline to 2.5 to 5 × 10 3 cfu/ml (Candida) and 5 × 10 4 cfu/ml (Tricophyton) and quantified by spectrophotometry.Suspensions were diluted 50 times in culture medium RPMI 1640 with L-glutamine without sodium bicarbonate (Sigma Chemical Company ) and buffered at pH 7.0 with 0.165 M MOPS (Sigma).The minimal inhibitory concentration (MIC) was determined as described by the Clinical Laboratory Standards Institute, (CLSI, protocol M-27-A2 with modifications, 2008) and the results were visually evaluated.For minimum fungicidal concentration (MFC) determinations, cultures of Candida and Tricophyton strains from multiwell plates were subcultured in solid Sabouraud for 2 and 7 days, respectively.
A clinical T. rubrum isolate (ATCC -MYA 3108) was obtained from a patient treated in the University Hospital of the Faculty of Medicine of Ribeirão Preto, USP -University of São Paulo.The mutant strain TruMDR2 was obtained from isolate MYA3108 by disruption of the TruMDR2 gene (Fachin et al., 2006) using standard techniques of manipulation and growth previously described by the same authors (Fachin et al., 2001).Susceptibilities of MYA 3108 and the TruMDR2 mutant were confirmed by MIC values of the plant extracts.According to the M38-A microdilution protocol proposed by the Clinical Laboratory Standards Institute (CLSI, 2008).The mycelial formation of Trichophyton rubrum, which had been obtained by growing the strains in Sabouraud plates at 28°C for 15 days was harvested by sterile scraping, mixed with sterile saline and filtered through glass wool.The resulting filtrate was transferred to a sterile tube and adjusted spectrophotometrically at 530 nm to a transmittance of 70 to 75%.The conidial suspensions were diluted 1:50 in MOPS -buffered RPMI 1640 (Sigma), which corresponded to twice the density needed for the test, 3 × 10 5 to 5 × 10 5 cfu ml -1 .Each microdilution well containing 100 l of crude extract (diluted twice) was inoculated with 100 l of the conidial inoculum suspensions (diluted twice), resulting in a final volume of 200 l in each well.Growth and sterility controls were included for each isolate and extract or fraction tested.Microliter trays were incubated at 28°C and MICs recorded after 7 days of incubation.The volume of essential oil tested in the experiments was 75 to 0.42 µl/mL.The MIC value of 100 was defined as the lowest concentration of extract or fraction producing total growth inhibition.Amphotericin and terbinafine were used as reference standards.

Antibacterial assays
MIC determinations as described by the M7-A6 protocol of the Clinical Laboratory Standard Institute (CLSI, 2008) were conducted in Luria-Bertan (LB) culture medium (Difco ) and the results visually

RESULTS AND DISCUSSION
The MIC values determined for essential oils from fresh and dried leaves of A. polystachya were 28.2 µL.mL -1 for E. coli, and 14.1 µL.mL -1 for S. aureus ATCC (Table 1), and both values were smaller than that of Gentamycin used as reference drug.
The essential oil extracted from A. polystachya showed significant activity against C. albicans and C. krusei (ATCC and clinical) when compared to the antibiotic amphotericin B (Table 2).Both Candida species were more sensitive to the oil extracted from fresh leaves.As the main difference in oil composition between fresh and dried leaves is the presence of limonene oxide, cis-pinocarveol, mirtenal, trans-carveol, cis-carveol and trans-carvone oxide, it is possible that those compounds may be responsible for enhancing antifungal activity even at low concentrations (1%).
There are studies reporting these substances as effective antimicrobials even in at low concentrations (Mazutti et al., 2008;Sabulal et al., 2006).Essential oils from fresh and dried leaves of A. polystachya tested against the clinical isolate of T. rubrum (MYA-3108) showed a MIC of 0.45 L/ml, whereas the value for terbinafin was 1.86 g/ml.Different MIC values were obtained when the extract of fresh (MIC, 0.11 µL/mL) or dried (MIC, 0.23 µL/mL) leaves were tested against the mutant strain, but both were lower than that of the reference antifungal (0.90 µL/mL) (Table 2).
The main chemical difference between A. polystachya and C. carvi oils is the optical activity of limonene, dextrorotatory in the former (Sedláková et al., 2003) and levorotatory in the latter.Limonene is a carvone precursor and the enantiomorph S-(-)-limonene is mainly present in Mentha species (Chauhan et al., 2009) being toxic to most microorganisms.Carvone occurs, both as R-(+) and S-(-) and also as a racemic mixture.Its isomers show considerable differences in sensorial, chemical and biological properties.R-(+)-carvone is widely used in the manufacture of aromas and fragrances (Bauer et al., 1990) and has antifungal activity (Ulbert et al., 1999).Leaves collected by 9:00 AM produced the highest ratio of oil, that is 5.3% (Table 4).Leaves collected in the afternoon yielded reduced percentage of oil.Oil extracted from dry leaves harvested at 9:00 AM contained higher amount of carvone (80.40%), while the highest quantity (20.2%) of limonene was determined in oil from leaves harvested by 12:00 PM.Brant et al. (2008) reported that the yield of A. triphylla oil varies according to the seasons, with early fall being the best period for harvesting.In general, Aloysia species comprises chemotypes with qualitative and quantitative variations due to several variables (Gil et al., 2007).Environment interactions occurring during the day may influence directly or indirectly the pathways of secondary metabolism resulting in both qualitative and quantitative variations in the production of essential oils.Cabanillas et al. (2003), studying A. polystachya native to Argentina reported that the species has two chemotypes, one with predominance of carvone Means followed by the same letter do not show significant differences (Scott-Knott, p < 0.05).
and the second with -thujone, a substance with a confirmed neurotoxic activity (Hold et al., 2000;Ratra et al., 2001).
No -thujone was detected in the essential oils extracted from leaves of A. polystachya grown in Ribeirão Preto, a finding validating it as a safe product to be used in antifungal phyto-formulation.

Table 1 .
CIMs of essential oils extracted from A. polystachya against E. coli and S. aureus compared to reference antibiotic.

Table 2 .
MICs of essential oils extracted from A. polystachya against Candida sp. and T. rubrum compared to reference antibiotics.
evaluated.For determining minimal bactericidal concentration (MBC), cultures of all strains tested were subcultured in solid brain heart infusion (BHI) medium for 24 h.

Table 3 .
Volatile constituents of A. polystacya essential oils.
b percent fraction of total integrated chromatogram area.

Table 4 .
Proportion of essential oils extracted from dried leaves A. polystachya harvested at different times during the day.