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Revista argentina de microbiología

versión On-line ISSN 1851-7617

Rev. argent. microbiol. v.41 n.4 Ciudad Autónoma de Buenos Aires oct./dic. 2009

 

ARTÍCULO ORIGINAL

Antibacterial and antioxidant activities of the essential oil of Artemisia echegarayi Hieron. (Asteraceae)

A. Laciar1*, M. L. Vaca Ruiz1, R. Carrizo Flores2, J. R. Saad2

1Área Microbiología,
2Área de Química Orgánica-INTEQUI-CONICET. Universidad Nacional de San Luis. Chacabuco y Pedernera. 5700 San Luis. Argentina

*Correspondence. E-mail: alaciar@unsl.edu.ar

ABSTRACT

Artemisia echegarayi Hieron. (Asteraceae) is commonly known in Argentina as “ajenjo”. Many studies report high efficacy of essential oils against food-borne pathogenic bacteria. The antimicrobial activity and minimal inhibitory concentration of A. echegarayi essential oil were evaluated against seven bacterial species of significant importance in food hygiene, by using the disc diffusion assay and the micro-well dilution method, respectively. Volatile components of the extract were analyzed by gas chromatography-mass spectrometry and major components were determined. Furthermore, the essential oil was tested for its antioxidant activity. The essential oil inhibited the growth of gram-positive and gram-negative tested bacteria, with the exception of Proteus mirabilis. A. echegarayi essential oil presented the lowest minimal inhibitory concentration against Listeria monocytogenes and Bacillus cereus. Two terpenes, thujone and camphor, were identified from this essential oil as the principal constituents responsible for antibacterial activity. The oil showed a free radical scavenging activity equivalent to 50% of the reference compound. These preliminary studies showed promising results since this essential oil may provide an alternative to promote its use as a natural food additive.

Keywords: Artemisia echegarayi; Essential oil; Antibacterial activity; Antioxidant activity.

RESUMEN

Actividad antibacteriana y antioxidante del aceite esencial extraído de Artemisia echegarayi Hieron. (Asteraceae). Artemisia echegarayi Hieron. (Asteraceae), conocida como “ajenjo”, es una planta típica de la región de Cuyo (Argentina). En este trabajo se evaluó la actividad antimicrobiana in vitro y la concentración inhibitoria mínima del aceite esencial extraído de sus partes aéreas frente a especies bacterianas que con frecuencia contaminan los alimentos. Se utilizaron las técnicas de difusión con discos en agar y microdilución en placa respectivamente. Además, se determinó la actividad antioxidante de este aceite esencial in vitro por espectrofotometría. En general, tanto las bacterias gram-positivas como las gram-negativas fueron inhibidas por este aceite, con excepción de Proteus mirabilis. Listeria monocytogenes y Bacillus cereus resultaron ser las bacterias más sensibles. El análisis por croma-tografía en fase gaseosa y espectrometría de masa permitió la identificación cualitativa y cuantitativa de los componentes mayoritarios del aceite esencial del ajenjo. Entre ellos, la tuyona y el alcanfor se destacaron como los principales responsables de la actividad antibacteriana observada. Los datos preliminares obtenidos en el presente estudio sugieren que el aceite esencial de Artemisia echegarayi representa una alternativa para promover su empleo como aditivo natural en alimentos.

Palabras clave: Artemisia echegarayi; Aceite esencial; Actividad antibacteriana; Actividad antioxidante.

INTRODUCTION

The beneficial health effects of extracts from many types of plants have been acknowledged for centuries (13).
Essential oils (EOs) are aromatic and volatile oily liquids obtained from plant material such as leaves, bark or fruit (22). Although the antibacterial properties of EOs have long been known, the recent interest in alternative naturally derived antimicrobials has led to a renewed scientific interest in these substances. Many studies in vitro have reported high efficacy of EOs against food-borne pathogenic bacteria (11, 18, 29).
Nowadays, the excessive use of synthetic antimicrobial compounds in food manufacture as additive agents is well known, many of which are suspected for their residual toxicity. Several EOs offer potential applications in food preservation, and the use of EOs in the food industry can help reduce the addition of chemical preservatives as well as the intensity of heat treatments, resulting in foods which are more naturally preserved and richer in organic properties (16).
Because of the extraction mode, mostly by distillation from aromatic plants, the EOs contain a variety of volatile molecules such as terpenes and terpenoids, phenol-derived aromatic components and aliphatic components. In particular, the antioxidant capacity of these compounds has been described to promote their use as natural food additives (1).
Artemisia echegarayi Hieron. (Asteraceae) is commonly known as “ajenjo” in the Region of Cuyo, Argentina. This is a plant of about one meter high, with ashgrey green colour leaves and clustered in spherical chapters (capitula) of 34-64 flowers. A. echegarayi grows on slopes of the Central Andes region especially in the provinces of Mendoza, San Juan and La Rioja, at a height of 2000-3000 m above sea level. Nowadays, there is not much information of the bioactivity of A. echegarayi essential oil (EO). However, there are many reports on the bioactivities of essential oils of other species from the genus Artemisia. Several of them are used in folk medicine, for example: Artemisia douglassiana is used as gastric cytoprotector (20, 31) and topical bactericidal agent on skin burns (26). A. annua is known by its antimalarial, anti-inflammatory and anti-carcinogenic properties (19).
The purpose of the study presented here was to gain insight on the antioxidant and antimicrobial activities of A. echegarayi EO against a range of food-borne bacteria, including gram-negative and gram-positive bacteria. Moreover, volatile components of the extract were analyzed by gas chromatography-mass spectrometry (GCMS) and the major components were determined.

MATERIAL AND METHODS

Plant material
Artemisia echegarayi Hieron. (Asteraceae) aerial parts were collected in Potrerillos, province of Mendoza, Argentina, in February 2008. The plant was identified by Ing. Luis Del Vitto, and a voucher specimen is kept at San Luis University (UNSL) herbarium, Argentina (N° 492).

Extraction of essential oil
Fresh aerial parts (5,000 g) were cut into small pieces and subjected to steam-distillation at 96 °C for 3 h using a Clevengertype apparatus. The oil obtained was dried over anhydrous sodium sulphate (21).

Chemical characterization of the essential oil
The A. echegarayi EO composition was determined by GCMS through comparison of the major signals with an MS library (20). Retention times and mass spectral data were checked with those obtained from authentic samples and/or from the MS instrument library. Relative percentages of the major components were calculated by integrating the registered peaks. GC-MS experiments were performed on an ion trap GCQ-Plus (Finnigan, ThermoQuest, Austin, TX, USA) instrument with MS-MS program using a silica capillary column Rtx ® -5MS (30 m x 0.25 mm ID, 0.25 μm). The carrier gas was helium (40 cm/s -1 ). The port temperature was 200 °C in the splitless mode with 1.0 ml injection volume. The initial GC temperature was maintained at 40 °C for 2 min, then increased to 210 °C at 2 °C/min, and maintained at this temperature up to 120 min. For the analysis of low resolution MS, the ion trap mass detector was set in full scan mode from m/z 50 to m/z 450. For the product analysis (CID), the precursor was selected using tandem mass spectrometry (MS/MS) scan standard function, with 0.5 Da peak-widths for the parent ion and dynamically programmed scans, as described previously (10).

Microorganisms
A total of 8 bacteria were selected for this study. Two strains of Listeria monocytogenes were obtained from the Pasteur Institute, France, culture collection: CLIP 74903 and CLIP 74904, while Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium and Proteus mirabilis were isolated at the UNSL laboratory. These bacteria were chosen in order to represent the diversity of species responsible for food-borne diseases.

Antimicrobial activity
Disk diffusion assay. The antimicrobial activity of A. echegarayi EO was determined by the standard disc diffusion technique (CLSI) (25, 31). A population of approximately 108 CFU/ml of each strain was inoculated on duplicate plates containing Müeller Hinton Agar (MH) (Britania, Argentina) using sterile cotton swabs. The plates were allowed to dry for 5 min at room temperature. Sterilised paper discs (Britania, Argentina) of 6 mm diameter were used. Ten microlitres of A. echegarayi EO were added to impregnate paper disks and allowed to dry for 15 min. Commercial gentamicin discs (10 μg, Britania, Argentina) were used as positive control. The discs were then placed aseptically over the surface of the bacterial cultures on MH plates and incubated at 37 °C for 24 h. After incubation, the inhibition zones around paper discs were measured accurately using a metric ruler. The assays were carried out on duplicate MH plates for each strain. The experiments were replicated at least twice.
Determination of Minimal Inhibitory Concentration (MIC).
The MICs of A. echegarayi EO and gentamicin (antibiotic reference) were determined by the microplate method (microwell dilution) according to the CLSI method (Clinical and Laboratory Standard Institute, 2005) (formerly NCCLS) (8), in MH broth (Britania, Argentina) pH 7.2 supplemented with 0.01% (w/v) of 2,3,5-triphenyltetrazolium chloride (TTN) as visual indicator of bacterial growth. The inoculum of each strain was prepared from 18 h broth culture and adjusted to the tube 0.5 of Mc Farland scale (108 bacterial cells). Then, they were diluted 100 times. The EO was dissolved in 20% Tween 80 and then diluted with phosphate buffer saline (PBS) to the highest concentration to be tested (75 μg/ml), and then serial two-fold dilutions were made in concentration ranges from 75 to 2.4 μg/ml. In addition, gentamicin dilutions were prepared in a concentration range from 128 to 0.25 μg/ml. The 96-well plates were prepared by dispensing into each well 95 μl of nutrient broth and 5 μl of the inoculum (final concentration of 104 CFU/ml). One hundred microlitre aliquot from the stock solutions of the EOs and their serial dilutions initially prepared was transferred into six consecutive wells. The final volume in each well was 200 μl. The plates were covered with sterile plate sealer and then incubated at 37 °C for 24 h. MIC was defined as the lowest concentration of the EO in the medium in which there was no visible growth after incubation (no red colour signifying live growth). It is established that TTN, a water-insoluble, colorless compound, can be reduced to water-insoluble red formazan by a variety of organisms. TTN reduction is used as a quantitative method in the evaluation of tissue viability (33). The experiments were replicated at least twice.

Thin-Layer chromatography (TLC)
Merck F254 plates, 10 x 10 cm, 1mm thick were used. A. echegarayi EO was applied and the chromatogram developed using n-hexane-ethyl acetate (95:5) as solvent. TLC plates were run in duplicate. Spots and bands were visualized by H2SO4 spray reagent. Thujone (Sigma-Aldrich) and camphor (Sigma-Aldrich) were used as standards. TLC plates were dried overnight in a sterile room for complete removal of solvent.

Bioautography
Plates TLC were covered with 1-2 mm layer of soft medium (BHI with 0.6% agar) containing 0.1% (w/v) TTN (tetrazolium red) and an aliquot of an overnight culture of L. monocytogenes (CLIP 74903) (108 CFU/ml) and P. mirabilis (108 CFU/ml). The plates were placed in a sterile tray, sealed to prevent the thin agar layer from drying, and incubated at 37 °C for 24 h. Where microbial growth has been inhibited, an uncoloured area can be seen on the deep pink-red background. The plates were run in duplicate.

Antioxidant activity
2, 2'-diphenylpicrylhydrazyl (DPPH) assay
The hydrogen atom or electron donation ability of the corresponding oils and some pure compounds were measured from the bleaching of purpled coloured methanol solution of DPPH. This spectrophotometer assay uses stable DPPH as reagent (5, 20). Five hundred microlitres of this solution were added to 500 μl of EO methanol solution in 1 cm path length disposable microcuvette. After a 30 min-incubation period at room temperature, the absorbance at 517 nm was read against a blank. Inhibition free radical DPPH in percent (I %) was calculated in the following way:

I%= (ABlank- ASample)/ ABlank × 100
Where ABlank is the absorbance of the control reaction (containing all reagents except the test compound), and ASample is the absorbance of the test compound. Rutine, a quercetin glucoside was used as reference compound. The assay was repeated twice.

RESULTS

The EO extracted from leaves of A. echegarayi plant by hydrodistillation presented light blue colour, fragrant smell and density of 0.915 g/ml and a yield of 0.56 g/kg of plant material. The A. echegarayi EO on the GC-MS analysis resulted in the identification of 15 constituents representing 92.48% (Table 1). The major components were 3-thujanone (49.25%) and thujone (10.72%). These components have the following chemical groups in their structure: hydrocarbons, alcohols, ketones and esthers.

Table 1. Chemical composition of A. echegarayi EO (by GC(1) and GC-MS(2) analysis)

A. echegarayi EO showed antibacterial activity against all tested bacteria, except for P. mirabilis. Generally, grampositive bacteria were more sensitive to A. echegarayi EO than gram-negative bacteria, and L. monocytogenes CLIP 74903 and B. cereus were among the most sensitive with MIC of = 2.4 μg/ml (Table 2).

Table 2: Antibacterial activity of A. echegarayi EO

To obtain some information on the active components, A. echegarayi EO was analyzed by TLC on silica gel plates and assayed by bioautography. Figure 1 shows the appearance of the chromatogram after treatment with L. monocytogenes, indicating the localization of the bacterial inhibition zone.


Figure 1. Thin layer chromatography plate of A. echegarayi essential oil. (A) visual appearance. (B) Listeria monocytogenes (CLIP 74903) bioautography overlay. Arrows indicate regions of inhibition growth visualized with tetrazolium red. (1) A. echegarayi essential oil. (2) Thujone (standard). (3) Camphor (standard).

The A. echegarayi EO presented moderate antioxidant activity (50% of free radical DPPH inhibition). Its antioxidant activity was lower than that of quercetin, a powerful natural antioxidant (100%).

DISCUSSION

The use of EOs in food industry can be an alternative to satisfying the increasing consumers' demand for freshtasting, ready-to-eat, minimally-processed foods and also to developing “novel” food products (e.g. less acidic, or with lower salt content) (17).
Determination of the inhibition zones by means of the disc diffusion method showed that the EO of an Argentine native plant, A. echegarayi, exhibited an antibacterial effect against gram-positive and gram-negative foodborne pathogen tested bacteria. The minor susceptibility of gram-negative bacteria may be attributed to an outer membrane surrounding the cell wall which restricts diffusion of hydrophobic compounds through the lipopolysaccharide. Moreover, the periplasmic space contains enzymes, which are able to break down foreign molecules introduced from outside (12). This can be confirmed analyzing the chemical composition of the A. echegarayi EO; therefore we could conclude that the antibacterial activity following TLC separation of EO A. echegarayi was found to be attributed mainly to the presence of two major constituents, thujone and camphor. They cause leaking of cell contents due to alterations on the membrane permeation system (6).
In the present study, an interesting finding has been that all strains tested, except P. mirabilis, showed MICs ranging from ≤ 2.4 to 18.7 μg/ml. These values confirm the existence of a significant activity of A. echegarayi EO against gram-positive and gram-negative bacteria considering Barbiéri's reports which suggested that if the EO displayed a MIC lower than 100 μg/ml, the antimicrobial activity is good (3).
L. monocytogenes CLIP 74903 was more susceptible than L. monocytogenes CLIP 74904. These results are in agreement with those reported by Barbiére Holetz, who reported that within bacterial species, EO efficacy was dependent on the strain and in some cases on the strain origin (27).
Moreover, the results can be explained considering the efficacy of the main components of EO isolated from aerial parts of A. echegarayi. The chemical composition was investigated by GC-MS and the results showed high contents of terpenes: 3-thujanone, thujone, borneol and camphor were the main components, whose contents were 49.25%, 10.72%, 5.26% and 5.07% respectively. Among these, thujone and camphor are responsible for the antimicrobial properties reported here. In this respect, they have also shown to be responsible for antibacterial activity of EOs from different Artemisia species. For example, thujone and camphor have been identified as the major compounds with antibacterial activity in Artemisia absinthium, Artemisia scoparia and Artemisia sieberiri EOs (14, 24).
Terpenes are naturally occurring substances produced by a wide variety of plants and animals. A broad range of the biological properties of terpenoids has been described, including cancer chemopreventive effects, antioxidant, antimicrobial, antifungal, antiviral, antihyperglycemic, antiinflammatory, and antiparasitic activities (23).
Identification of terpenoid constituents as principal agents in this plant is consistent with the results of a number of early studies on other plants. The antibacterial activity of terpenoids is generally believed to involve actions on phospholipid membranes, where partitioning results in destabilisation and disorder culminating in ion leakage in bacteria and disruption of membrane-dependent energygenerating processes in eukaryotic microorganisms (28).
The EOs extracted by distillation of aromatic plants, contain a variety of volatile molecules such as terpenes and terpenoids, phenol- derived aromatic components and aliphatic components. In vitro physicochemical assays characterize most of them as antioxidants (4, 30). Thus, in this work, terpenes, particularly those with activated methylene groups in their molecules, could probably be the reason of the antioxidant activity shown by A. echegarayi EO. From this, it was inferred that it could be beneficial for human health in line with recent findings and common belief that many diseases are due to an overload of oxidative stress reactions following excessive consumption of fat, sugar, meat, etc. Antioxidants are believed to be directly antimutagenic (7) and anticarcinogenic due to their radical scavenging properties (9, 15). However, recent work shows that in eukaryotic cells, essential oils can act as prooxidants by intermediate of terpenes affecting the cellular redox status. This may play a significant “protective” role by removing damaged cells by apoptosis (2, 32).
Because A. echegarayi EO was inhibitory in small quantities to selected pathogenic microorganisms, these preliminary results suggest it may provide alternatives to conventional additives in food products. Furthermore, this EO may add prooxidant effects and thus, could be employed in traditional and modern medical domains.

Acnowledgements: We thank the UNSL (Projects 7301 and 8802), CONICET (PIP 6228) for the financial support of this study.

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Recibido: 24/02/09
Aceptado: 24/09/09