Evaluation of Antioxidant and Cytotoxic Activities of Vernonia
amygdalina Del. Leaves
Poppy Anjelisa Zaitun Hasibuan
1*
, Urip Harahap
1
, Panal Sitorus
2
, Denny Satria
2
1
Department of Pharmacology,
2
Department of Pharmaceutical Biology
Faculty of Pharmacy, University of Sumatera Utara, Medan, 20155
*Presenting author
Keywords: Antioxidant, Cytotoxic, Vernonia amygdalina Del., leaves, fraction.
Abstract: The excessive production of oxygen free radicals and the unbalanced mechanism of antioxidant protection
results in the onset of many diseases such as breast cancer. Antioxidant activity was determined by 1-1-
diphenyl-2-picrylhydrazil (DPPH) method and cytotoxic activity was determined with MTT method towards
T47D cell line. Antioxidant activity from n-hexane, ethylacetate, ethanol fractions and quercetine as positive
control with DPPH assay measured as IC
50
were 297.33 ± 0.46; 177.99 ± 0.32; 37.92 ± 1.03 and 2.32 ± 0.01
µg/mL respectively. Cytotoxic activity from n-hexane, ethylacetate, ethanol fractions and doxorubicin as
positive control with MTT assay measured as IC
50
were 327.89 ± 1.13; 64.92 ± 0.72; 1591.75 ± 37.05 and
1.82 ± 0.05 µg/mL respectively. The results reveal that fractions of Vernonia amygdalina Del. leaves have
antioxidant and cytotoxic activities. Our further study is to asses anticancer mechanism of Vernonia
amygdalina Del. leaves.
1 INTRODUCTION
Oxidation is a natural process in living organisms.
Free radicals producing by metabolism process or
enviromental sources which interact with biological
system. Reactive species are atoms or molecules
which have instability and relatively reactive. The
uncontrolled production of free radicals and the
unbalanced mechanism process of antioxidant
protection cause of many degenerative diseases,
such as heart diseases, cancer, Alzheimer’s,
diabetes, and aging (Jamuna, et el., 2012; Nagmoti,
et al., 2012; Rosidah, et al., 2008; Yang, et al.,
2004).
There are some factors that influence the
risk of breast cancer such as the length period of
exposure to hormones, dietary factors, reproductive
factors and first pregnancy at an advanced age, and
lack of physical activity, hormone replacement
therapy on chronic use, as well as congenital genetic
factors associated with breast cancer like the
presence of gene mutations and radiation during
breast development, (Barnett, et. al., 2008).
Vernonia amygdalina Del. from family of
Asteraceae come from West Africa. Several studies
find some chemical constituents such as flavonoids,
sesquiterpene lactones, fatty acids and steroidal
saponins (Ohigashi, et al., 1991; Igile, et al., 1994;
Sinise, et al., 2015; Igile, et al., 1995; Jisaka, et al.,
1992; Quasie, et al., 2016; Erasto, et al., 2007) and
indicated some of pharmacological activity such as
anti-malaria, anti-inflammation, anti-tumor, anti-
obesity, and other activities (Eyong, et al., 2011;
Adedapo, et al., 2008; Egedigwe, et al., 2016;
Atangwho, et al., 2012; Erasto, et al., 2008;
Adaramoye, et al., 2008; Luo, et al., 2010; Sonibare,
et al., 2009). The aim of this study was to evaluation
of antioxidant and cytotoxic activities of n-hexane,
214
Hasibuan, P., Harahap, U., Sitorus, P. and Satria, D.
Evaluation of Antioxidant and Cytotoxic Activities of Vernonia amygdalina Del. Leaves.
DOI: 10.5220/0008360202140217
In Proceedings of BROMO Conference (BROMO 2018), pages 214-217
ISBN: 978-989-758-347-6
Copyright
c
2018 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
ethylacetate and ethanol fraction of Vernonia
amygdalina Del. leaves.
2 MATERIALS AND METHODS
2.1 Plant and Chemicals Material
Fresh leaves of Vernonia amygdalina Del. was
collected from Faculty of Pharmacy, University of
Sumatera Utara, Medan, Indonesia. Vernonia
amygdalina Del. was identified in Herbarium
Medanense, Department of Biology, Faculty
Mathematic and Natural Sciences, University of
Sumatera Utara and the voucher specimen was
deposited in herbarium (No.
1712/MEDA/2017). Chemicals used were distilled
water, DMSO (Sigma), 1,1-diphenyl-2-
picrylhydrazyl (DPPH) (Sigma), [3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium
bromide] (MTT) (Sigma).
2.2 Preparation of Fractions
The air-dried and powdered leaves of Vernonia
amygdalina Del. (500 g) were repeatedly
fractionated by maceration method with n-hexane
(3x3 d, 7.5 L). The powder was dried in the air and
fractionated with ethylacetate and continued with
ethanol 96% (3x3 d, 7.5 L) at room temperature with
occasional stirring. The filtrate was collected, and
then evaporated to give a viscous fraction and then
freeze dried to dry (Satria, et al., 2015; Anggraeni, et
al., 2015; Hasibuan, et al., 2015; Harahap, et al.,
2018).
2.4 Free Radical Scavenging Activity
Test
The free radical scavenging activity was measured
by 1,1-diphenyl-2-picrylhydrazyl (DPPH•). 0.2mM
solution of DPPH• in methanol was prepared and
100μl of this solution was added to various
concentrations of fractions. After 60 minutes,
absorbance was measured at 516 nm. Quercetin was
used as the standard. All the tests were performed in
triplicate and percentage of inhibition was calculated
by comparing the absorbance values of the control
and test samples (Rosidah, et al., 2018; Satria, et al.,
2017; Dalimunthe, et al., 2018).
2.5 Cytotoxicity Assay
The cells were treated with n-hexane, ethylacetate,
ethanol fractions and doxorubicin. In this test, T47D
cell line was grown in RPMI 1640 medium, medium
containing 10% Fetal Bovine Serum (Gibco), 1%
penicillin-streptomycine (Gibco), and fungizone
0.5% (Gibco) in a flask in a humidified atmosphere
(5% CO
2
) at 37
o
C. The inoculums seeded at
1x10
4
cells/mL at an optimal volume of 0.1 mL per
well. After 24 h incubation, the medium was
discharged and treated by fractions and doxorubicin.
After incubation 24 h, the cells were incubated with
0.5 mg/mL MTT for 4 h in 37
o
C. Viable cells
reacted with MTT to produce purple formazan
crystals. After 4 h, SDS 10% as stopper (Sigma) in
0.01N HCl (Merck) was added to dissolve the
formazan crystals. The cells were incubated for 24 h
in room temperature and protected from light. After
incubation, the cells were shaken, and absorbance
was measured using ELISA reader at λ 595 nm. The
data which were absorbed from each well were
converted to percentage of viable cells
(Hasibuan, et
al., 2016; Harahap, et al., 2018).
2.6 Statistical Analysis
Data was expressed as mean ± SD. All statistics
were analyzed using the SPSS 21 software.
3 Results and Discussion
3.1 Antiradical Activity
Antiradical power of the plant samples was
measured in term of hydrogen donating ability using
DPPH which is a stable, nitrogen-centered free
radical and produces deep purple colour in methanol
solution. Antioxidants either transfer an electron or a
hydrogen atom to DPPH, thus neutralizing its free
radical character (Pan, et al., 2008). DPPH test,
which is based on the ability of DPPH, a stable free
radical, to decolorize in the presence of antioxidants,
is a direct and reliable method for determining
radical scavenging action. The DPPH assay has been
largely used as a quick, reliable and reproducible
parameter to search the in vitro general antioxidant
acitivity of pure compounds as well as plant extracts
(Koleva,et al., 2002). The reducing capacity of
compounds could serve as indicator of potential
antioxidant property (Meir,et al.,1995). It is very
important to point out that a low IC
50
value reflects a
high antioxidant activity of the fraction, since the
concentration necessary to inhibit the radical
oxidation in 50% is low. Antioxidant activity from
Evaluation of Antioxidant and Cytotoxic Activities of Vernonia amygdalina Del. Leaves
215
n-hexane, ethylacetate, ethanol fractions and
quercetine as positive control with DPPH assay
measured as IC
50
were 297.33 ± 0.46; 177.99 ± 0.32;
37.92 ± 1.03 and 2.32 ± 0.01 µg/mL respectively.
3.2 Inhibitory Concentration 50% (IC
50
)
MTT method was used to determine cell viability
after incubation for 24 h. In every treatment
fractions and doxorubicin was shown to inhibit cells
growth. The IC
50
from n-hexane, ethylacetate,
ethanol fractions and doxorubicin as positive control
were 327.89 ± 1.13; 64.92 ± 0.72; 1591.75 ± 37.05
and 1.82 ± 0.05 µg/mL respectively.The cytotoxicity
estimate of natural product is related to content of
active compound in these plants including Vernonia
amygdalina Del. Flavonoids and
triterpenoids/steroids estimated as active compounds
(Yadav, et al., 2012; Igile, et al., 1994; Quassie, et
al., 2016). Doxorubicin is one of chemotherapeutic
agent showed strong activity on T47D cell lines with
IC
50
value of 1.82 ± 0.05 µg/mL. T47D cells line
underwent resistant to doxorubicin pass through to
p53 mutation (Di Leo, et al., 2007; Vassade, et al.,
2005).
ACKNOWLEDGEMENTS
We gratefully thank to Directorate of Higher
Education, Ministry of Research Technology and
High Education, Indonesia through “Hibah
Penelitian Dasar Unggulan Perguruan Tinggi”
Research Grant 2018 for financial support in the
study.
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