Chemical composition and antimicrobial activity of the essential oil from the leaves and flowers of Aloysia gratissima

Volatile oils from leaves and flowers of Aloysia gratissima were investigated for their chemical composition and antimicrobial activity against the bacteria Bacilus subtilis, Staphylococcus aureus, Salmonella choleraesuis, Pseudomonas aeruginosa, Streptococcus pneumoniae and the Candida albicans yeast. The minimum inhibitory concentrations (MIC) of the oils were determined by the micro-dilution method, while the chemical composition was determined by GC-MS (gas chromatography mass spectrometry). The fresh leaves and inflorescence were subjected to hydrodistillation for 120 min using a Clevenger-type apparatus, and the essential oil was tested against microorganisms. High concentrations of sesquiterpenes were observed for the inflorescence, and monoterpenes were observed for the leaves. The main compounds of the inflorescence essential oil were E-caryophyllene, germacrene B, guaiol and bulnesol, while in the leaves the main compounds were trans-pinocamphone, trans-pinocarveyl acetate, and guaiol. The essential oil from the leaves showed an effect against P. aeruginosa and S. pneumonia, and the essential oil of the inflorescence showed an effect against P. aeruginosa, S. pneumonia, and Candida albicans.


INTRODUCTION
In the last decades, essential oils have been of great interest as natural products.They have been screened for their potential uses as alternatives in the treatment of many infectious diseases (Kelen and Tepe, 2008).
Aloysia (Cham) Steudel (Santos et al. 2011).It is a bush used in folk medicine for the treatment of bronchial infections, lung diseases, bladder disorders, and as an antispasmodic.It is also used as an antimicrobial agent and as a flavoring for infusions and meat (Pinto et al., 2007;Santos et al., 2009).
It grows spontaneously and is commonly pollinated by insects, especially bees, during its flowering.It is used in folk medicine for the treatment of bronchial infections, lung diseases, bladder disorders, and as an antispasmodic.It is further used as an astringent, antimicrobial agent, stimulant and tranquilizer, as well as in the treatment of headaches, respiratory infection (inhalation), indigestion, earache and colic in babies.Due to its aromatic properties, it is also used as a flavoring for infusions and meat, and pot-pourri (Silva et al.,2007).
Studies have demonstrated antibacterial activity of the crude extracts and oil from the leaves of Aloysia species (Vandresen et al.,2010).There is no report that investigated the antimicrobial activity of volatile compounds of this plant.The aim of this study was to identify the chemical composition and evaluate the antimicrobial activity of leaves and inflorescence oils of A. gratissima from southeast of Brazil against different microorganisms.

Plant material
Aloysia gratissima (Gillies et Hook) Tronc.fresh leaves and inflorescence were collected at Lavras, Minas Gerais state, Brazil.The county of Lavras is situated at 918.87 m altitude, 21º14´S latitude and 45 º 00´W longitudes.The plant was identified based on morphological features, and the voucher specimen (number 19810) has been deposited in the herbarium of the Department of Biology at the Federal University of Lavras (UFLA).

Essential oil extraction
The fresh leaves and inflorescence parts (120g and 60g, respectively) of A. gratissima were subjected to hydrodistillation for 120 min using a Clevenger-type apparatus.The essential oil was dried over anhydrous magnesium sulphate (MgSO 4 ) and preserved in a sealed vial at 4ºC for further analysis.

Gas chromatography (GC-FID) and Gas chromatography-mass spectrometry (GC-MS) analysis
The chemical analysis of the essential oil was performed using Agilent Technologies CG-6890A and MS5975 with quadruple detector operating at 70eV in electron ionization mode, equipped with HP-5 fused silica capillary column (300 × 0.25 mm i.d.; 0.25 µm film thickness).The carrier gas helium (He) was used at a constant flow rate of 1.0 mL min -1 .The thermal program was 60-240°C at a rate of 3°C per min.The injected volume was 1,0 µL in split ratio 30:1.The injector and detector temperatures were kept at 220ºC and 250ºC, respectively.The amount of each compound was expressed as a relative percentage of the total area of the chromatograms.

Identification procedure
The identification of the oil components was established from their GC retention indices, relative to C 8 -C 32 n-alkanes (Sigma, St Louis, MO, USA), by comparison of their MS spectra with those reported in the literature (Adams, 2007), and by computer matching with the Agilent Chemstation mass spectra and NIST/EPA/NHI (NIST, 2010) libraries.

Microorganisms
Antimicrobial activity was investigated using a panel which included laboratory control strains from the American Type Culture Collection (ATCC) and

antimicrobial assay
The antimicrobial assays were carried out at the Microbiology Division of CPQBA UNICAMP, Campinas, São Paulo State, Brazil.Culture media for preservation and activity tests with bacteria and C. albicans were, respectively, Muller-Hinton broth and RPMI-1640.Prior to the assays, microorganism strains were grown overnight at 36ºC in the respective media.Inocula for the assays were prepared by diluting scraped cell mass in 0.85% NaCl solution, adjusted to McFarland scale 0.5 and confirmed by spectrophotometrical reading at 600 nm.Cell suspensions were finally diluted to 10 4 UFC mL -1 for use in the activity assays.Minimum Inhibitory Concentration (MIC) tests were carried out according to CLSI (2002CLSI ( e 2005)), on a tissue culture test plate (96 wells).Stock solutions from the essential oils were prepared in 0.1% Tween 80 in distilled water at 4 mg mL -1 .An aliquot of 100 µL was transferred into the first well, and serial dilutions were performed so that concentrations in the range of 1.0 -0.0156 mg mL -1 were obtained.Each oil was tested in duplicate.Chloramphenicol and nystatin (Merck) were used as the reference antimicrobial control.
The inoculum was added to all wells and the plates were incubated at 36 ºC for 24-48h.Antibacterial activity was detected by adding 20 µL of 0.5% TTC (triphenyl tetrazoluim chloride, Merck) aqueous solution.MIC was defined as the lowest concentration of the compounds that inhibited visible growth, as indicated by TTC staining (dead cells are not stained by TTC).After the incubation period, changes in the RPMI-1640 medium color were verified from pink (original color) to yellow, for anti-C-albicans activity evaluation.The change indicates a medium acidification by the yeast growth.

Chemical compositions
The extraction yields of essential oil shows that the inflorescence (0.56%) of A. gratissima contained more oil than their leaves (0.35%).The essential oil from inflorescence is viscous, white and becomes solid in low temperatures.Otherwise, essential oil from leaves is limpid yellow and liquid.
The essential oils were obtained by hydrodistillation from fresh parts of A. gratissima and subsequently analyzed by GC-MS.The identified compounds with their relative percentage are reported in Table 1.In total, 29 compounds were identified in the leaves (97.3% of the total oil) and 21 compounds in the inflorescence (86.6% of the total oil).There were 15 monoterpenes compounds on leaves (56.0%) and seven monoterpenes on inflorescences (11.7%).The leaves' essential oil presented more monoterpenes, while the essential oil from inflorescence was dominated mainly by sesquiterpenes, being 30.4% non-oxygenated and 44.5% oxygenated, which corresponds to 74.9% of total sesquiterpenes.On the other hand, there is 41.3% of total sesquiterpenes on the leaves, 18.7% non-oxygenated and 22.6% oxygenated (Figure 1 and Table 1).The difference amount between mono and sesquiterpenes can be seen in Eucalyptus oleosa.The observed difference may be due to the species, seasonal variations and organ (Marzouf et al., 2011).
The results on chemical composition of the essential oil from the leaves of A. gratissima differed partially from those reported previously in other literature (Zygadlo, 1995;Ricciardi et al., 2000;Trovati et al., 2009).On the other hand, our findings were in accordance to those obtained by (Sartoratto and Augusto, 2003), who also identified the major presence of trans-pinocarveyl acetate (9.3%) and trans-pinocamphone (10.9%), though in a smaller quantity than the ones obtained in this current research.

antimicrobial activity
The essential oils showed variable activities against tested bacteria.The results on antimicrobial activity oils against different microorganisms are summarized in Table 2.The essential oil from leaves showed activity against P. aeruginosa (MIC 0.8 mg mL -1 ) and S. pneumoniae (MIC 0.6 mg mL -1 ), similar to the reference antibiotic used in the assays.
The essential oil activity of the flower was more effective than of the leaf, and was especially pronounced against gram-negative bacterium P. aeruginosa, gram-positive bacterium S. pneumonia, and the yeast C. albicans.The positive controls used in this assay were chloramphenicol and nystatin for the bacteria and yeast respectively.Our results showed that flower oil had antimicrobial activity higher than the positive antibacterial standard chloramphenicol and nystatin.The inflorescence oil was five times more effective against P. aeruginosa (MIC 0.15 mg mL -1 ) than chloramphenicol, which needed 0.85 mg mL -1 to suppress the bacterial growth.When we evaluated it for S. pneumoniae, the MIC from inflorescence oil with the smallest inhibitory dose was 0.02 mg mL -1 , which means that its inhibition is three times more effective than the reference antibiotic concentration (0.06 mg mL -1 ).For the yeast C. albicans, there was an inhibition 2 ½ times lower, with MIC 0.02 mg mL -1 , with 0.05 mg/mL necessary for nystatin.The main compounds of flower oil were E-caryophyllene, germacrene B, guaiol and bulnesol (49.0%).The antimicrobial activity of an essential oil is attributed mainly to its major compounds; however the synergistic or antagonistic effect of a minor component of the complex mixture has to be considered.Others studies showed antimicrobial activity with oil containing at least one of these major oil compounds as the principal component (Hisham et al., 2006;Mevya et al., 2007;Asfaha et al., 2008;Ho et al., 2009).The results showed that Gram-positive bacteria S. pneumoniae was more sensitive than Gram-negative FIGURE 1. Percentage of compounds in essential oil from leaves and inflorescence of Aloysia gratissima-Total Sesquiterpene total (TS); Oxygenated sesquiterpene (OS); sesquiterpene (S); total oxygenated monoterpene (TOM).
P. aeruginosa and S. choleraesuis probably because they possess an outer membrane surrounding the cell wall, which restricts diffusion of hydrophobic compounds through its lipopolysaccharide covering.Without this barrier, the membrane in gram-positive bacteria can be permeated more easily (Marzoug et al., 2011).
Otherwise, the essential oil from leaves showed an activity similar to chloramphenicol (0.85 mg mL -1 ) against P. aeruginosa (0.80 mg mL -1 ) only.It showed no activity against the other five microorganisms, when compared to the reference antimicrobials values.The essential oils of flowers had better antimicrobial activities than that of leaves.This is probably because the essential oils of flowers were full of sesquiterpenes.It´s known that sesquiterpenes usually possess antimicrobial activity (Chen et al., 2011).
These results corroborate the importance of ethnopharmacology on studies that are guided by folk culture, such as the use of A. gratissima for treatments against pneumonia and bronchial affections.Nevertheless, pharmacological research and specific clinics are still necessary for making feasible the use of the essential oil of A. gratisima as a phytotherapeutic.

aCKNOWlEDGEMENTS
The authors are thankful to CNPq/Brazil (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for financial support and for research fellowships (JEBPP).CAPES/Brazil (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil) is acknowledged for a Ph.D. fellowship (FMS) and FAPEMIG/Brazil (Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brazil) for financial support.

TaBlE 1 .
Chemical composition (%) of A. gratissima leaves and inflorescence essential oil, plants were cultivated on field.
nd: not detected.

TaBlE 2 .
Minimum inhibitory concentration (MIC -mg/mL) of Aloysia gratissima leaves and inflorescence essential oil, plants were cultivated on field.