Pure standard of α-pinene, β-pinene, myrcene, limonene, terpinen-4-ol, sabinene hydrate, thymol, germacrene D, caryophyllene oxide, were purchased from Sigma Aldrich (Milan, Italy). They were selected as representative of the different chemical classes of the essential oil components for the calculation of response factors towards an internal standard (n-decane) to correct the potential shift of flame ionization detector (FID) response towards the different classes of compounds . Standard solutions at different concentrations of each of them containing 5 μg/μl of n-decane as internal standard were dissolved in cycloexane. Solvents were all HPLC grade from Riedel de Haen (Seelze, Germany).
Plant material and essential oil preparation
Aerial parts of M. communis, S. thymbra and T. herba-barona were collected in Jerzu, Cagliari and Osini (Sardinia, Italy), respectively, in three different periods, before (sample A), during (sample B) and after flowering (sample C) to investigate how the variability of the EO could affect the biological activity.
They were botanically identified and registered with the specimen numbers 514 (Myrtus), 1,079 (Satureja) and 1,065 (Thymus) at the General Herbarium of Dipartimento di Scienze della Vita e dell’Ambiente, Macrosezione Botanica e Orto botanico, University of Cagliari (CAG). EOs from fresh plant materials were obtained in agreement with the European Pharmacopoeia VII ed.: fresh crushed aerial parts were submitted to steam distillation for five hours. The resulting EOs were left to stabilize for one hour.
Successively, each sample (A, B, C) of the essential oil of S. thymbra was fractionated on silica gel column chromatography using petroleum ether (PE) with increasing amount of ethyl acetate (EtOAc), giving seven fractions, three of them in significant amounts (fractions A1-A3, B1-B3, C1-C3, see Results).
Essential oil analyses
GC analyses were carried out with a Thermo Electron TRACE GC Ultra (Rodano, Italy) provided with a FID detector. GC/MS analysis was carried out with a Thermo Electron TRACE GC Ultra coupled to a Trace DSQ mass spectrometer operating in Electron Impact mode.
GC-FID-MS analyses were carried out on a Mega5 column (5% phenyl methyl polysiloxane) 25 m, 0.25 mm i.d., 0.25 mm film thickness, from MEGA (Milan – Italy). GC and GC-MS conditions: injection mode: split; split ratio: 1: 20. Temperatures: injector: 220°C, transfer line: 230°C; ion source: 230°C; carrier gas: He, flow-rate: 1.0 ml/min in constant flow-mode. MS detector operated in electron impact ionization mode (EI) at 70 eV, scan rate was 1,111 amu/s and mass range of 35–350 m/z. Temperature program: from 50°C (1 min) to 220°C (5 min) at 3°C/min.
The components were identified by comparison of both their linear retention indices (I
T), calculated versus a C8-C25 hydrocarbon mixture, and their mass spectra to those of authentic samples or with data from the literature . The FID response factors were used for the semi-quantitative determination of the volatile components.
The chloroquine (CQ) -sensitive (D10) and the CQ-resistant (W2) strains of P. falciparum were sustained in vitro as described by Trager and Jensen . Parasites were maintained at 5% haematocrit (human type A-positive red blood cells) in RPMI 1640 (EuroClone, Pero, Milan, Italy) medium with the addition of 1% AlbuMaxII (Invitrogen, Monza, Italy), 0.01% hypoxantine 20 mM Hepes (EuroClone, Pero, Milan, Italy), 2 mM glutamine (EuroClone, Pero, Milan, Italy). The viability and parasitaemia of cultured parasites was evaluated by light microscopy analysis of Giemsa-stained blood smears. The parasitaemia was maintained within 1% and 4% diluting the cultures with uninfected erythrocytes in complete medium at 5% haematocrit. All cultures were maintained at 37°C in a standard gas mixture consisting of 1% O2, 5% CO2, 94% N2.
The EOs and fractions were dissolved in DMSO and then diluted with a medium to achieve the required concentrations (final DMSO concentration <1%, which is non-toxic to the parasite). Asynchronous cultures with parasitaemia of 1–1.5% and 1% final haematocrit were aliquoted into the plates and incubated for 72 hrs at 37°C. Parasite growth was determined spectrophotometrically (OD: 650 nm) by measuring the activity of the parasite lactate dehydrogenase (pLDH), according to a modified version of Makler’s method in control and drug-treated cultures . EOs and fractions were tested at 1–100 μg/ml. Anti-plasmodial activity is expressed as the 50% inhibitory concentrations (IC50), each IC50 value is the mean ± standard deviation (sd) of at least three separate experiments performed in duplicate. Chloroquine was used as positive reference compound at 0.8-100 ng/ml and 8–1000 ng/ml on D10 and W2 strains, respectively.
Plasmepsin II inhibition assay
Pro-plasmepsin (Pro-PLM) II was prepared according to the procedure previously described , with slight modifications . Protein was diluted to the final concentration of 0.5 mg/ml in 50% glycerol and stored at −20°C. Pro-PLM II was activated by addition of one tenth volume of 100 mM sodium acetate buffer pH 4.7 following by incubation at 37°C for 90 min. The enzyme activity of PLM II was evaluated spectrophotometrically at 300 nm by following the cleavage of the chromogenic substrate Lys-Glu-Phe-Val-Phe-NPhe-Ala-Leu-Lys (where NPhe is para-nitro-phenylalanine), as described .
Satureja thymbra EO was tested at 1–50 μg/ml dissolved in DMSO (final concentration <1% of the sample volume). IC50 values (mean ± sd of three experiments in triplicate) were obtained using Graph Pad Prism 4.
Cytotoxicity was evaluated in human neonatal dermal fibroblasts (Cell applications, inc. San Diego, CA, USA, cat number 106-05n). Cell proliferation was followed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. Fibroblasts (2 × 104/ml) were grown in DMEM (Dulbecco’s modified Eagle’s medium) containing 10% foetal calf serum, 1% penicillin/streptomycin, and 1% L- glutamine. The compounds to be tested were added to culture medium dissolved in DMSO 48 hrs after plating. The final concentration of the vehicle in control and test medium was 0.1% of the incubation volume. The effect of compounds (10–100 μg/ml) on cell proliferation was assayed at 24–48 hrs after treatment. Each concentration was tested in triplicate in three separate experiments.
Anopheles gambiae, molecular form M, originated from Yaoundé, Cameroon and maintained since more than one decade under the conditions of the insectary in IRD Dakar Senegal (at temperature 28 ± 2°C and relative humidity 80% + −5%) were used . All mosquitoes were 100% susceptible to pyrethroids, DDT, bendiocarb and fenitrothion. To measure the ability of the EO to kill adult mosquitoes, bottle test bioassays were conducted according to the method of Brogdon and McAllister , with acetone as solvent. Twenty-five mosquitoes, aged three days from the susceptible Dakar strain, were used. For each EO, solutions were prepared at concentrations of 8.3 μg/ml, 16.6 μg/ml, 24.91 μg/ml, 32.22 μg/ml, 41.52 μg/ml, 49.88 μg/ml, 58.13 μg/ml and 66.44 μg/ml. Four replicates were set up for each concentration, using 1 ml of solution/bottle. Deltamethrin 12.5 μg/ml was used for positive control while the negative control was acetone. Mortality was assessed every 15 min. With practice, the mortality of mosquitoes in the control bottle at two hours should be zero. In most cases, mortality of up to 3% in the control bottles may be ignored. The data were corrected using the Abbot’s formula  for mortalities in the control ranging 3-10%. A dose-mortality line depending on the exposure time was developed.
The larvicidal activity was performed according to the guidelines for laboratory and field testing of mosquito larvicides published by WHO (who/cds/whopes/gcdpp/2005.13), with minor modifications.
A laboratory colony of An. gambiae larvae (susceptible strain) was used for the larvicidal activity of EO collected at the three stages (samples A, B and C). Twenty-five third instars larvae were kept in 500 ml glass beaker containing aqueous suspension of EO at dilution from 14 to 75%. Four replicates were set up for each dilution. The negative control was exposed to water. Larval mortality was assessed after 24 hrs of exposure by probing the larvae with needle and moribund larvae were counted as dead. A dose-mortality line was recorded and the lethal concentration of EO needed to kill 50% (LC50) of larvae was determined.
Statistical analyses were performed with Graph Pad Prism 4 software, using t test or one-way analysis of variance followed by Bonferroni’s post hoc test. The significance was set at p <0.05.