Leaves from Artemisia arborescens were collected in Sardinia (Italy), identified and voucher specimens deposited in the herbarium of the Institute of Botany and Botanical Garden, University of Cagliari, Italy. Up to 1500 g of fresh leaves were distilled in a Clevenger-type apparatus for 5 h, the essential oil was dried over anhydrous sodium sulfate and stored at 4°C until use. For the experiments, the oil was dissolved in dimethyl sulfoxide (DMSO) and therefore diluted in the medium. To avoid toxicity or interference by the solvent, the maximum concentration of DMSO in the test medium was 1%.
Virus and cells
African green monkey kidney cells (Vero) were obtained from the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia (Brescia, Italy). Cells were grown in RPMI 1640 (Gibco) supplemented with 10% fetal calf serum (FCS, Gibco) and penicillin, streptomycin and fungizone (100 U/ml, 100 μg/ml, and 2.5 μg/ml, respectively). Overlay medium for the plaque assays of HSV consisted of Modified Eagle Medium (MEM) without phenol red (Gibco) plus 2% FCS containing antibiotics as described above and 0.5% agarose.
The strains of HSV type 1 (HSV-1 strain F) and HSV type 2 (HSV-2 strain G) used in this study were obtained from the American Type Culture Collection (ATCC), Rockville, Md. HSV-1 and HSV-2 were propagated in Vero cells. Virus titers were determined by plaque assay in Vero cells and are expressed as plaque forming units (PFU)·ml-1. The viruses were stored at -70°C until use.
Cellular toxicity of A. arborescens essential oil was tested in vitro according to a cell viability assay previously reported [25, 26]. Monolayers of Vero cells in 96-multiwell plates were incubated with the essential oil at concentration of 1000 – 15.6 μg/ml in RPMI 1640 for 48 h and the medium replaced with 50 μl of a 1 mg/ml solution of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma) in RPMI without phenol red (Sigma). Cells were incubated at 37°C for 3 h, the untransformed MTT removed and 50 μl of acid-isopropanol (HCl 0.04 N in isopropanol) was added to each well. After a few minutes at room temperature to ensure that all crystals were dissolved, the plates were read using an automatic plate reader with a 570 nm test wavelength and a 690 nm reference wavelength.
Maximum Non Toxic Dose (MNTD) was determined microscopically by the observation for morphological changes of cells at 24, 48 and 72 hours of incubation.
Plaque reduction assay
A. arborescens essential oil was first tested for antiviral activity against HSV-1 and HSV-2 by a plaque reduction assay with monolayer cultures of Vero cells grown in RPMI. Cells were infected with 200–250 PFU of HSV-1 or HSV-2. After 1 h adsorption at 37°C plates were washed and medium replaced with MEM containing agarose 0.5%, FCS 2% and different concentrations of essential oil. After 72 h incubation monolayers were fixed with 10% formaldehyde in phosphate buffered saline (PBS), nutrient agar was removed, and cells stained with a 1% solution of crystal violet in methanol 70%.
Some experiments were also performed incubating about 200–250 PFU of HSV-1 and HSV-2 with A. arborescens essential oil at concentrations of 100 – 0.19 μg/ml at 37°C or 4°C for varying time periods up to 2 h. Viruses were then adsorbed at 37°C on Vero cells for 1 h, cells were washed and medium replaced with MEM containing agarose 0.5% and FCS 2%. After 72 h incubation at 37°C monolayers were fixed and processed as described above.
The IC50 values were calculated by regression analysis of the dose response curves generated from the data.
Inhibition of plaque development assay
Reduction of plaque development assays were performed as previously described  with some modification. Monolayers of Vero cells were infected with about 100 PFU of HSV-1 or HSV-2 for 3 h at 37°C. Cells were then washed and the medium was replaced with nutrient agar containing 100, 50, 25, 12.5 and 6.25 μg of essential oil per ml and 10 μg/ml of HSV-1 and -2 neutralizing antibody (Chemicon International Inc., Temecula, CA) to ensure that plaque development was actually due to cell-to cell virus spread. After 48, 72 and 96 h, the plates were fixed with 10% formaldehyde in PBS for 30 min, the nutrient agar overlay was removed, and the cells were stained with 1% solution of crystal violet in 70% methanol for 30 min. The stained monolayers were then washed and plaque diameter was measured with a digital caliper (Mitutoyo, Japan). Reduction of plaque size by 50% was considered positive inhibition. At least 30 plaques were measured per well. Plaques < 0,2 mm in diameter were considered abortive and therefore were not counted.
Yield reduction assay
Monolayers of Vero cells grown in 6-well plates were infected by adsorption of HSV-1 or HSV-2 at a multiplicity of infection (MOI) of 1 plaque forming unit per cell (PFU/cell) for 1 h at 37°C. Cells were washed with warm medium and A. arborescens essential oil at concentrations ranging between 100 and 3.12 μg/ml in minimum essential medium with 2% FCS was added immediately after adsorption. At 24 h after virus inoculation, cells in the culture medium were lysed by freezing and thawing (three times), and supernatant consisting of culture medium and cell lysate was obtained by centrifugation at 400 × g for 10 min at 4°C. Virus titer was determined by plaque forming assay in Vero cells as described above.
Vero monolayers grown in 6-well plates were prechilled at 4°C for 15' and infected with HSV-1 or HSV-2 diluted in serum-free MEM to 200 PFU/ml for varying time periods up to 3.0 h at 4°C in the presence or absence of serial dilutions of A. arborescens essential oil (40, 20, 10, 5 and 2.5 μg/ml). Unadsorbed virus was then removed and cells overlaid with nutrient agar. After 72 h cells were fixed and stained as described above.
Penetration assays were performed using published procedures with modifications . Briefly, about 200 PFU of HSV-1 or HSV-2 were adsorbed on Vero cells grown on 6-well plates for 3 h at 4°C. The medium was replaced with pre-warmed fresh medium containing A. arborescens essential oil (final concentrations 40, 20, 10, 5 and 2.5 μg/ml) and the temperature was abruptly increased to 37°C to maximize penetration of virus. Penetration proceeded for various time period (30 min., 1 h, 1.5 h and 2 h). Monolayers were then treated with PBS, pH 3 for 1 min to neutralize any remaining attached virus and after several washes with serum-free medium cells were overlaid with MEM-0.5% agarose to quantitate surviving virus versus time of essential oil exposure.
Post-attachment virus neutralization assay
Post-attachment virus neutralization assays were carried out using published procedures with modifications [29, 30]. About 250 PFU of HSV-1 and HSV-2 in 0.5 ml of MEM were adsorbed to Vero cells for 2 h at 4°C. Cells were then washed, medium replaced with DMEM containing the essential oil of A. arborescens (100 – 12.5 μg/ml) and incubated for 2 h at 4°C. Cell monolayers were again washed and overlaid with DMEM containing 0.5% agarose and incubated at 37° until plaques were fully developed. As a control, HSV-1 and HSV-2 were incubated with serial dilutions of the essential oil for 2 h at 4°C prior to adsorption to cells (pre-attachment neutralization). Cells were fixed and stained as described above, and the number of plaques obtained with control HSV-1 and HSV-2 pretreated with the essential oil was compared with the number of plaques obtained when the essential oil was added after adsorption.
Antibacterial and antifungal activity
A. arborescens essential oil was tested for its antibacterial activity by twofold dilution method in Mueller Hinton Agar (Difco Laboratories) according to standard procedures against five Gram positive (S. aureus, S. epidermidis, S. faecalis, S. agalactiae and B. subtilis) and six Gram negative species (E. coli, P. aeruginosa, K. pneumoniae, S. marcescens, S. typhi and P. mirabilis) isolated from clinical specimens. The antifungal activity was evaluated against C. albicans ATCC E10231 in Sabouraud Dextrose agar. For the evaluation of antimicrobial activity, concentrations of essential oil ranging between 500 and 3.9 μg/ml were employed.