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Journal of Science and Technology in the Tropics 57
respective media supplemented with 5% Fetal Bovine Serum (FBS), 100 IU mL -1
penicillin and 100 mg mL -1 streptomycin. The cultures were maintained at 37 °C in
an incubator with 5% CO 2
.
Cytotoxicity and MTT assay
Cells were seeded in a 96 well microplate at 3 × 105 cells mL -1 and then incubated
at 37 °C in 5% CO 2
atmosphere. After 24 hours, the medium was removed and
replaced with fresh medium containing test compounds at various concentrations
(serial dilution) between 0 and 30 mg mL -1 . 72 hours later, the cells were tested with
the MTT (3-[4,5-dimethylthioazol-2yl]-2-5-diphenyltetrazolium bromide) assay to
evaluate the viability through its metabolic activity. 20 mL of MTT (5 mg mL -1 ) in
PBS solution was added to each well. Then, the plates were further incubated for 3
hours in the dark. During incubation period, viable cells convert MTT to a waterinsoluble
formazan dye. The plates were then centrifuged (Centrifuged 5810R,
Eppendorf) at 400 × g and 4 °C. All the remaining supernatant was then removed
and 100 mL of dimethylsulphoxide (DMSO, Fisher Scientific) was added to each
well. The plates were reincubated, as done earlier, but for an hour to dissolve the
crystals of formazan formed. The formation of the dye is proportional to the number
of viable cells, which was detected using a microplate spectrophotometer (mQuant
Universal Microplate Spectrophotometer, BIOTEK instrument, Inc) at wavelength
of 570 nm. The mean absorbance for each compound concentration was expressed
as a percentage of control untreated well absorbance and plotted versus compound
dose. IC 50
values represent the concentration that reduced the mean absorbance at
570 nm to 50% of those in the untreated control wells.
RESULTS AND DISCUSSION
Anthraquinones are known to be a homogeneous group of constituents in the
Morinda species. Anthraquinone derivatives, including emodin, physcion, aloeemodin,
rhein, and chrysophanol, are nowadays well recognized as important
biologically active components [13]. In this report, the anthraquinones tested
include modified and rearranged anthraquinone groups. They reveal an interesting
trend of cytotoxic effect on HT-29 cell line with IC 50
values between 4.5 and
10.0 mg mL -1 . However, these cytotoxic results show significant dissimilarity in
inhibition effects probably due to the nature of the substituents and the substitution
pattern of the anthraquinone skeleton. Structurally, these include damnacanthal(1),
nordamnacanthal(2), 2-ethoxy-1-hydroxyanthraquinone (3), as well as rubiadin(4),
1-hydroxy-2-methylanthraquinone (5) and rubiadin-1-methyl ether (6). The
cytotoxic results of these compounds are summarized in Table 1.