Composition and Antimicrobial Activity of the Essential Oil from Aloysia sellowii

Os óleos essenciais obtidos das partes aéreas de Aloysia sellowii, coletadas em duas diferentes localidades (A e B) do sul do Brasil, foram analisadas por CG, CG-EM, e através de cromatografia gasosa com fases quirais (CGC). Dos quarenta e oito componentes identificados, representando cerca de 93 e 91% dos óleos das regiões A e B, respectivamente, 1,8-cineol, β-pineno, sabineno e β-(Z)-santalol foram os principais componentes. Um “screening” preliminar das duas amostras de óleos de A. sellowii mostrou atividade frente a Artemia salina (LC50 = 2,85 μg mL), além de exibirem atividade antimicrobiana frente a bactérias Gram-positivas e Gram-negativas e frente a leveduras (MIC 1,7-16 mg mL).


Introduction
Aloysia (Verbenaceae) is a genus that comprises about 30 species widespread all over the Americas, mainly in the South and Central America countries. 1Aloysia sellowii (Briq.)Moldenke [= Lippia affinis (Briq.)], 2 known as "garupá", "cidrozinho" or "erva de sepultura" is a large aromatic shrub up to 2-4 meters in height, found throughout the greater part of Southern of Brazil (Rio Grande do Sul), Uruguay, Paraguay and Argentina. 3,4 n Rio Grande do Sul aerial part of this plant is traditionally used by local people as diuretic, stomach ache agent and as a remedy for colds, influenza, and for the treatment of respiratory disorders. 3,4 ere are few studies published about the chemical composition of the essential oils of this genus in spite of their interesting aromas.A. gratissima 5 and A. triphylla 2,6 are the most widely studies species.
In our continuing research on the essential oils of Rio Grande do Sul aromatic plants, 7,8 we have investigated the chemical composition of the essential oil of A. sellowii, its antimicrobial activity and the biological screening by brine shrimp lethality assay, which is considered an efficient tool for preliminary evaluation of the pharmacological activity. 9In addition, we report on the enantiomeric distributions of the chiral monoterpene constituents in A. sellowii.

Results and Discussion
The leaf oils of A. sellowi were obtained by hydrodistillation in 1.2-1.4% yield.Its constituents were identified by GC-MS using an electron-impact ionization technique and by co-chromatography with authentic samples.Twenty-eight compounds, making up 92.75 % of the oil from Sample A (Santa Maria), and thirty-four compounds, making up 91.04 % of the oil from Sample B (Livramento), were identified.Both oils displayed some qualitative and quantitative differences (see Table 1).The major constituent of the sample A was 1,8-cineole (43.30%), which is present in only 1.16% in sample B. The oil of sample B was richer in sesquiterpenoids than the oil of sample A, and was characterized by a high content of sabinene (12.62%) and β-santalol (Z) (12.88%), which are not present in sample A. The enantiomeric composition of the chiral monoterpene constituents were analyzed on a dual column system, 10 using two 25 m fused silica columns coated with modified cyclodextrins (2,6-Me-3-Pe-β-CD and 6-Me-2,3-Pe-β-CD) as the chiral stationary phases.Of the chiral monoterpenes identified in the oils of A. sellowi, the main constituents were: (-)- sabinene, (+)-β-pinene, (+)-α-pinene, and (+)-terpinen-4-ol (see Table 2).The essential oils were screened for biological activity using the brine shrimps lethality test. 11Samples A and B displayed high toxicity (LC50 = 2.85 μg mL -1 ) in the brine shrimp bioassay.Antimicrobial activity of samples A and B was determined by the broth micro dilution method, 12,13 to determine the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC).Table 3 shows that sample B exhibited a more potent antifungal activity against S. cerevisiae than sample A, probably due to the presence of carvacrol in sample B (> 5 %). 14Yet for antibacterial activity, our results indicate that S. epidermidis (MIC = 1.7 mg mL -1 , samples A and B) was found to be the most sensitive while S. aureus, M. luteus, K. pneumoniae, E. coli and S. setubal were the most resistant bacteria (MIC = 6.7-20 mg mL -1 ).A chemical-composition and antibacterial activity comparison in our results (Table 3) demonstrates that the compounds present in greatest proportions as 1,8-cineole (> 40 % in sample A and only 1.16 % in sample B) 15 are not necessarily responsible for the greatest share of total activity.Thus, the activity of the less abundant components as carvacrol, terpinen-4-ol, linalool, βpinene, R (+)-limonene, myrcene 16 should be considered or at least the activity could be attributed to a synergistic effect among components.Although the MICs and the MLCs results varied between test organisms, in most cases the MIC and MLC are equivalent, indicating an antimicrobial action of the oils.To our knowledge, the antimicrobial activity of A. sellowii essential oils is reported here for the first time.

Plant material
The leaves of about 6-10 individuals of a wild population of Aloysia sellowii (to give a representative sample) were collected in two localities of Rio Grande do Sul (Santa Maria and Livramento), in three successive years at flowering time (October-November 2001-2003, one collection/year).Specimens were identified by Prof. Gilberto Zanetti (Dept. of Biology, University of Santa Maria, RS, Brazil).Voucher specimens (HDFI 166-170) were deposited at the Herbarium of the University of Santa Maria, RS, Brazil.

Essential oil isolation and chemical analysis
Fresh plant leaves were subjected to hydro distillation for 4h using a modified Clevenger-type apparatus, followed by exhaustive extraction of the distillate with diethyl ether.After removal of the solvent, the yield of the crude oils was 1.4 % and 1.2 % (m/v) for sample A and B, respectively.The physical properties for sample A and B were: d 25: 0.92; ηd 25: 1.48, [α] D 2 5 : + 9.54 ( in Hexane, c =0.22) and d 25: 0.89; ηd 25: 1.50, [α] D 2 5 : -17.9 (in hexane, c = 0.18), respectively.The essential oils were submitted to GC analysis in a Varian 3800 Gas Chromatograph equipped with a capillary fused silica column (25 m x 0.25 mm; film thickness 0.2 μm) coated with SE-54 (The GC conditions used were: carrier gas H 2 (1 mL min -1 ); injector split/splitless 220 o C; FID Detector 280 °C; column temperature 50 o C to 250 o C at 4 o C min -1 .GC-MS analyses were performed on a HP 5973-6890 GC-MSD system operating in the EI mode at 70 eV, equipped with a HP-5 crosslinked capillary column (30 m x 0.25 mm; film thickness 0.2 μm).The temperature of the column and the injector were the same as those from GC.The identification of the components of the oils was based on comparison of the retention times and Kovats Indices ( Ir i = 100.n+ 100.∆n.tr i -tr n / tr m -tr n ) 17 on both columns and mass spectra with those of NBS/NIST Library 18 and those described by Adams. 19The chiral monoterpene constituents of A. sellowii oils were identified by peak enrichment in enantioselective capillary GC with two fused capillary columns, 25 m x 0.25 mm, film thickness 0.2 μm, coated with heptakis-(6-O-methyl-2,3-di-O-pentyl)-β-cyclodextrin and octakis-(6-O-methyl-2,3-di-O-pentyl)-γ-cyclodextrin, each diluted with the polysiloxane OV-1701 (1:1), using a Varian 3800, equipped with a flame ionization detector (FID), and hydrogen as the carrier gas.All runs were performed with a temperature program from 35 °C for 15 min and 35 °C to180 °C at 3 °C min -1 .

Toxicity assays
The preliminary biological evaluation of the essential oils of A. sellowii was determined using the brine shrimp lethality test according to Meyer et al. 11 with some modifications. 9The brine shrimp eggs were hatched in artificial sea water (3.8 g sea salt per liter of water) and, after hatching (2 days old), the shrimp larvae were used for experimental bioassay.The oil was dissolved in DMSO and them added to the saline solution containing 3% of tween 20.Emulsification was assisted by ultrasound and the dilutions of this stock solution were made with saline solution containing tween 20.Then, shrimp larvae were placed in multiwelled culture plate and test solution was added to each well (in triplicate).Saline solution with 3% tween 20 and DMSO was used as a negative control (LC50>1000 μg mL -1 ), while potassium dichromate (LC50= 20 μg mL -1 ) and cinchonine (LC50= 100 μg mL -1 ) were used as standard positive controls. 9Survivors were counted after 24h and the LC50 values were determined in μg mL -1 , using the Finney probit analysis 20 obtained values with 95% confidence intervals.

Antibacterial assays
The antibacterial activities of the oils of samples A and B were assayed using the broth micro dilution method.A collection of eight microorganisms were used, including three Gram-positive bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, three Gram-negative bacteria: Klebsiella pneumoniae, Escherichia coli and Salmonella setubal, and two yeasts: Saccharomyces cerevisiae and Candida albicans.Standard strains of microorganisms were obtained from American Type Culture Collection (ATCC), and standard antibiotics chloramphenicol and nistatine were used in order to control the sensitivity of the microbial test. 21The minimal inhibitory concentration (MIC) was determined on 96 well culture plates by a micro dilution method using a microorganism suspension at a density of 105 CFU mL -1 with Casein Soy Broth incubated for 24 hours at 37 °C for bacteria, and Sabouraud Broth incubated for 72 hours at 25 o C for yeasts.The cultures that did not present growth were used to inoculate plates of solid medium (Muller Hinton Agar and Sabouraud Agar) in order to determine the minimal letal concentration (MLC).Proper blanks were assayed simultaneously and samples were tested in triplicate.Technical data have been described previously. 12,13

Table 1 .
Percentage composition of the essential oils of Aloysia sellowii

Table 3 .
Antibacterial activity of the oils of A. sellowii (minimal inhibitory concentration (MIC) and the minimal letal concentration (MLC) in mg mL-1 ) a ATCC (American Type Culture Collection); b Mean of 3 replicates in mg mL -1 .

Table 2 .
Enantiomeric distribuitions (%) of monoterpenes in the A. sellowii essential oils determined by co-injection with authentic material