K. Adinarayana et al. / Natural Science 3 (2011) 291-294
Copyright © 2011 SciRes. OPEN ACCESS
292
2. MATERIALS AND METHODS
2.1. Plant Material
The rhizome of Musa acuminata was collected from
local farm in Visakhapatnam. The material was cleaned,
washed, dried and carefully powdered.
2.2. Chemicals
Folin-Ciocalteu reagent, Trichloroacetic acid (TCA)
and ascorbic acid were purchased from Merck, Mumbai,
India. Butylated hydroxytoluene (BHT) was purchased
from Sigma Chemical Co. (USA). 1, 1-diphenyl-2-
picrylhydrazyl (DPPH), Gallic acid and MTT (3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide)
were obtained from Hi Media Laboratories Pvt. Ltd,
Mumbai, India.
2.3. Extraction
Fresh rhizomes of Musa acuminata were dried under
shade and the dried material was powdered using mortar
and pestle. Ten grams of the powder was packed in filter
paper and introduced in the extraction unit of Soxhlet
extractor and extracted with ethanol for 48 h. The extract
was concentrated and subjected to anti-cancer and anti-
oxidant activities.
2.4. Determination of the DPPH Scavenging
Activity
1, 1-Diphenyl-2-picrylhydrazyl free radical scaveng-
ing assay (DPPH) was carried out according to the fol-
lowing procedure [11]. One ml of ethanolic extracts of
rhizome and standard (Ascorbic acid) at various concen-
trations (10, 50, 100, 150 and 200 μg/ml) were added to
3 ml of 0.004% DPPH in ethanol and the reaction mix-
ture was shaken vigorously. These solution mixtures
were kept in dark for 30 min and optical density was
measured at 517 nm using LT-29 labtronics spectropho-
tometer. Ethanol with DPPH was used as blank. The %
scavenging activity was calculated using the formula:
Percentage of inhibition of DPPH activity100
AB
where A = optical density of the blank and B = optical
density of the sample.
2.5. Determination of Reducing Power
Reducing power of the extract was determined as re-
ported [12]. The rhizome extract (10, 50, 100, 150 and
200 μg/ml) was mixed with 2.3 ml of phosphate buffer
(0.2 M, pH 6.6) and 2.5 ml of 1% potassium ferricyanide
K3[Fe(CN)6]. The mixture was incubated at 37˚C for 20
min. 10% Trichloroacetic acid (2.5 ml) was added to the
mixture and centrifuged for 10 min at 1 000 rpm; the
supernatant (2.5 ml) was mixed with 2.5 ml of distilled
water and 0.5 ml of 0.1% FeCl3. After standing for 10
min, the absorbance was measured at 700 nm. High ab-
sorbance of the reaction mixture indicates high reducing
power. All experiments were repeated at least three
times.
2.6. Cancer Cell Culture
Carcinoma of cervix (HeLa) cells were maintained in
Dulbecco’s modified Eagles medium (DMEM) supple-
mented with 4.5 g/L glucose, 2 mM L-glutamine and 5%
fetal bovine serum (FBS) (growth medium) at 37˚C in
5% CO2 incubator.
2.7. MTT Assay
The MTT assay developed by Mosmann [13] was
modified and used to determine the inhibitory effects of
test compounds on cell growth in vitro. In brief, the
trypsinized cells from T-25 flask were seeded in each
well of 96-well flat-bottomed tissue culture plate2 at a
density of 5 × 103 cells/well in growth medium and cul-
tured at 37˚C in 5% CO2 to adhere. After 48hr incuba-
tion, the supernatant was discarded and the cells were
pretreated with growth medium and were subsequently
mixed with different concentrations of extract (2, 4, 8,
16, 32, 64, 128 and 256 µg/ml) in triplicates to achieve a
final volume of 100 µl and then incubated for 48 hr. The
extract was prepared as 2.0 mg/ml concentration stock
solutions in dimethyl sulfoxide (DMSO). The final con-
centration of DMSO in the culture was within 0.2%.
Culture medium and solvent were used as controls. Each
well then received 5 µl of fresh MTT (0.5mg/ml in PBS)
followed by incubation for 2hr at 37˚C. The supernatant
growth medium was removed from the wells and re-
placed with 100 µl of DMSO to solubilize the colored
formazan product. After 30 min incubation, the absorb-
ance (OD) was read at a wavelength of 570 nm on an
ELISA reader, Anthos 2020 spectrophotometer.
3. RESULTS AND DISCUSSION
The results of the DPPH scavenging activity of the
extract (Figure 1) shows that it possesses relatively high
percent antioxidant activity (81.41% at 200 g/ml) which
was comparable to that of the standard, ascorbic acid at
100 μg/ml. All concentrations of the studied extract
demonstrated a dose-dependent DPPH radical scaveng-
ing activity.
The results of the reductive potential (Figure 2) of the
extract and that of the gallic acid standard showed that
the ethanolic extract of banana rhizome possess a rela-