Anti-oxidant activity and cytotoxicity of ethanolic extracts from rhizome of Musa acuminata

doi:10.4236/ns.2011.34037

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Vol.3, No.4, 291-294 (2011) Natural Science

http://dx.doi.org/10.4236/ns.2011.34037

Anti-oxidant activity and cytotoxicity of ethanolic

extracts from rhizome of Musa acuminata

Kps Adinarayana1*, Ajay P. Babu2

1Department of Anatomy, Andhra Medical College, Visakhapatnam, India; *Corresponding Author: [email protected]

2Bio-Lab, Research Gateway for Biosciences, Visakhapatnam, India; [email protected]

Received 21 January 2011; revised 20 February 2011; accepted 10 March 2011.

ABSTRACT

In the present study, antioxidant activities of

rhizome of Musa acuminata were investigated.

Free radical scavenging assay (DPPH) and re-

ducing power of the ethanolic extract of banana

rhizome resulted in potential antioxidant activi-

ties. A relatively high percentage of antioxidant

activity by DPPH assay (81.41% at 200 μg/ml)

which was comparable to that of the standard,

ascorbic acid at 100 μg/ml was observed. With

the gallic acid as standard the extract showed a

relatively low reductive potential, however, when

tested for cytotoxicity at the highest concentra-

tion of the tested dose (256 μg/ml), the maxi-

mum rate of inhibition observed was 50.32%.

The present work indicates that the ethanolic

extract of Musa acuminata exhibits significant

antiproliferative and antioxidant activities.

Keywords: Antioxidant; Antiproliferative; DPPH;

Free Radical; MTT Assay

1. INTRODUCTION

It has been reported in literature that the role of free

radicals in many disease conditions was due to the reac-

tive oxygen species generated from various biochemical

reactions [1]. These are capable of damaging crucial

biomolecules and have been the major causative factor

of many chronic and degenerative diseases including

atherosclerosis, diabetes mellitus, cancer, Parkinson’s

disease and immune dysfunction [2,3]. Primary sources

of naturally occurring antioxidants are whole grains,

fruits and vegetables. Plant source food antioxidants like

vitamin C, vitamin E, carotnenes, phenolic acids, phytate

and phytoestrogens have been recognized as having the

potential to reduce risk. Moreover, antioxidants from

natural sources such as medicinal plants and vegetables

have shown to protect against oxidative stress [4]. Com-

pounds such as gallates, have strong antioxidant activity,

while others such as the mono-phenols are weak anti-

oxidants. Phenolic compounds, in particular, which are

widely distributed in many fruits, vegetables and me-

dicinal plants accounted for their antioxidant capacity of

many plants [5].

Antioxidants play major role in neutralizing the ef-

fects of free radicals and are known to be effective in

preventing the free radical formation by scavenging or

promotion of their decomposition [6]. Free radicals pre-

sent in the biological system which may oxidize nucleic

acids, proteins, lipids or DNA are entrapped by the anti-

oxidant compounds like phenolic acids, peroxide, hy-

droperoxide or lipid peroxyl inhibiting the oxidative

mechanism that lead to degenerative diseases [7,8]

Different methods published in the literature for the

determination of antioxidant activity of foods involve

electron spin resonance (ESR) and chemiluminiscence

methods. These methods measure the free radical-sca-

venging activity of antioxidants against free radicals like

the 1,1-diphenyl-2-picry;hydrazyl (DPPH) radical, the

superoxide anion (O2), the hydroxyl radical (OH), or the

peroxyl radical (ROO). The malondialdehyde (MDA) or

thiobarbituric acid-reactive-substance (TBARS) assays

[7] have been used extensively since the 1950’s to esti-

mate the peroxidation of lipids in membrane and bio-

logical systems. The ABTS [2,2’-azinobis (3-ethylben-

zothiazoline-6-sulfonic acid)] radical cation [8] has been

used to screen the relative radical-scavenging abilities of

flavonoids and phenolics [9]. Vinson et al. have meas-

ured phenoilics in fruits and vegetables calorimetrically

using the Folin-Ciocalteau reagent and determined their

antioxidant capacity by inhibition of low density lipo-

protein oxidation mediated by Cupric ions [10].

In the present study, we investigated the antioxidant

and anticancer activities of the crude extracts of rhizome

of Musa acuminata.

K. Adinarayana et al. / Natural Science 3 (2011) 291-294

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





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-

K. Adinarayana et al. / Natural Science 3 (2011) 291-294

293

Figure 1. DPPH radical scavenging activity of ethanolic ex-

tract of banana rhizome as compared to the standard Ascorbic

acid.

Figure 2. Reducing power of ethanolic extract of banana rhi-

zome as compared to Gallic acid.

tively low reductive potential than the standard. This

indicates that the reducing capacity of rhizome may

serve as an indicator of its potential antioxidant activity.

3.1. MTT Assay

Mean OD values of the extract was corrected by

subtracting with the mean OD of blanks. Relative

percent inhibition activity is expressed as:

%inhibition = 100 – (corrected mean OD of sample ×

100/corrected mean OD of control)

A gradual decrease in the viability of HeLa cells was

observed in a dose-dependent manner (Table 1 and Fig-

ure 3). At the highest concentration of the tested dose

(256 μg/ml), the maximum rate of inhibition observed

was 50.32%. The morphology of the cells treated with

the extract appeared significantly different when com-

pared to untreated control cells, which could probably

due to the growth inhibitory and cell death initiating

ability of the studied ethanolic extract of banana rhi-

zome.

Table 1. Optical Density (OD) and inhibitory data of ethanolic

extract of banana rhizome at various concentrations.

Cpd conc.

(µg/ml)

Observed

ODa

corrected

ODb % viabilityc % inhibitiond

2 0.853 0.803 89.88 10.12

4 0.817 0.767 85.85 14.15

8 0.783 0.733 82.12 17.88

16 0.738 0.688 77.08 22.92

32 0.660 0.610 68.31 31.69

64 0.580 0.530 59.35 40.65

128 0.505 0.455 50.95 49.05

256 0.494 0.444 49.68 50.32

aMean of triplicates, OD at 570 nm; bCorrected OD= observed – blank OD,

Blank OD: 0.05, Control OD: 0.893; c% viability = corrected OD of sample

× 100/control OD of cell culture; d% inhibition = 100- % viability.

Figure 3. Dose-response curve of the given sample on the

growth of HeLa cells cultured in vitro.

4. CONCLUSIONS

The studied ethanolic extract of banana rhizome has

relatively low reducing power and moderate DPPH

radical scavenging activity. However, an increase in

dose-dependent treatment of extracts when exposed to

carcinoma of cervix (HeLa) cells was observed. The

results reported in this paper suggest that the ethanolic

extract of banana rhizome could be helpful in addition to

the basic medicine in treatment of a few diseases. Fur-

ther works are needed to be carried out to isolate, iden-

tify and characterize the potential antioxidant or anti-

proliferative compound(s) in the extract for potential

clinical use.

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