Cytotoxicity of A. Malaccensis oil on tumor cells was measured by microculture tetrazolium (MTT)
assay [12]. A 96-well microplate were seeded with 300 μl of medium containing 1 x 10-3 cells of HCT
116 in suspension and incubated for at least 24 h prior to treatment. The cells were treated with the
supercritical extracted oil sample at concentration of 25μg/ml. DMSO was used as a negative control and
suramin was as a positive control. All tested samples were performed in triplicate and repeated three
times. After 48 h incubation in 5% CO at 37ºC, (Sigma) tetrazolim salt was added as cytotoxicity
indicator for cell viability. The absorbance reading was recorded at 590 nm and at reference wavelength
of 650 nm with a scanning Multiskan Ascent micro plate reader (Thermolab system 354, Finland). Values
are expressed as the percentage of mean cell viability relative to the untreated cultures. The same
experiment was repeated using the fractions oil at the same concentration of 25 μg /ml and at 2.5, 5, 10,
15, 20 and 25 μg/ml and incubated for 48 h in 5% CO2 at 37ºC, in order determine it respective IC50
values.