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VOL. 76 (3), 343-356, 2010 CYTOTOXIC ACTIVITY OF a-HUMELENE AND...

(Merck) with heptane (100) as the mobile phase. The plates were re-
vealed by sparing with vanillin/ sulfuric acid reagent and subsequent
heating at 105 ºC. Three subfractions were obtained (F1.1, F1.2 and
F1.3); the subfractions F1.1 and F1.2 showed a cytotoxic activity in the
three cell lines (Table 2). In the same way, the two active subfractions
were fractionated again with TLC analysis in silica plates with
toluene/ethyl acetate (97/3) as mobile phase, and two sub-subfractions
F1.1.1 and F1.2.1 were selected from the subfractions F1.1 and F1.2
respectively.

2.3. Gas Chromatography-Mass Spectrometry (GC-MS) analysis

GC-MS analysis of the essential oil S. officinalis and of its active
fractions was carried out using a Hewlett-Packard 7890 gas chromato-
graph equipped with a methylpolysiloxane capillary column (30 m
length, 0.25 mm internal diameter, 0.25 film thickness) coupled to a
Hewlett Packard 5975C mass spectrometer. Ionization of the sample
components was performed in electron impact mode (EI, 70 eV). The
carrier gas was helium (5psi, 0.7ml/min) and the temperature pro-
gram was 70 ºC for 2min, then 6 ºC/min to 270 ºC and held constant
for 20 min. 1 µl of a hexane solution at 0.2-1% was injected in the
split mode. Volatile components were identified from their retention
data and their mass spectra, which were compared with reference data
(Wiley mass spectral library). Relative amount was estimated from to-
tal ion current peak area.

2.4. Cancer cell lines

The colorectal adenocarcinoma human cell line (HCT-116), human
breast cancer cell (MCF-7) and murine macrophage cell line (RAW264.7)
were cultured in RPMI-1640 medium (+)L-glutamin, supplemented with
10% foetal bovine serum, 1% penicillin/streptomycin, 2 mg of gentam-
icin, 1% sodium pyruvate 100 mM (Invitrogen-Gibco, Madrid, Spain)
and 1% D-(+) glucose solution (45%) (Sigma-Aldrich, Madrid, Spain).
Cells were cultured in a humidified atmosphere at 37 ºC in 5% CO2. The
cultures were passed twice a week by trypsinization using a trypsin-
EDTA (0.05%) solution (Invitrogen-Gibco, Madrid, Spain).

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