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ADIL EL HADRI Y COLS. AN. R. ACAD. NAC. FARM.

2.5. Chemicals

1,8-cineole, a-thyjone, ß-thyjone, a-humulene, trans-caryophyl-
lene, tamoxifen and different solvents were purchased from Sigma-

Aldrich, Madrid, Spain.

2.6. Measurement of viability using the MTT Assay

Cytotoxicity was assessed using the microtitration colorimetric method
of MTT reduction test as described by Mosmann in 1983 (19). The cells
were trypsinized, counted and placed in 96-well plates at optimal plating
density of each cell line which was determined over a range from 5x104
to 10x104. After 24 h, the cells were treated with serial dilutions of the
samples. Each sample was initially dissolved in dimethyl sulphoxide
(DMSO) and further diluted in medium to produce different concentra-
tions. One hundred microliters/ well of each dilution were added to the
plates in six replicates to obtain the final concentrations ranging from 12.5
to 800 µg/ml for whole essential oil, their active fractions, subfractions and
sub-subfractions, from 1.56 to 100 µg/ml for the commercially available
identified constituents (1,8-cineole, ß-thyjone, a-humulene, trans-
caryophyllene). All compounds were dissolved in DMSO and the final con-
centration of DMSO in the culture medium was maintained at 0.5% (v/v).
Cytotoxic activity of these compounds was expressed as the concentration
of drugs inhibiting cell growth by 50% (IC50). Fluorescence was measured
on the automated 96-well plate reader at 550 nm. Tamoxifen (Sigma-
Aldrich, Madrid, Spain) was used as positive control.

2.7. Statistical analysis

All experiments were carried out in triplicate. Data were expressed
as means ± S.D. Differences were evaluated by the one-way analysis
of variance (ANOVA) test completed by a multicomparison Dunett’s
test. Differences were considered significant at (*P < 0.05). The IC50
was calculated by a nonlinear regression curve with the use of Prism
GraphPad Prism version 5.0 software for Windows. The dose-response
curve was obtained by plotting the percentage of inhibition versus the
concentrations.

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