Evaluation of Antioxidant and Cytotoxic Activities of Vernonia

Amygdalina Del. Leaves

Poppy Anjelisa Zaitun Hasibuan

, Urip Harahap

, Panal Sitorus

, Denny Satria

Department of Pharmacology,

Department of Pharmaceutical Biology

Faculty of Pharmacy, University of Sumatera Utara, Medan, 20155

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

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

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,

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

Hasibuan, P., Harahap, U., Sitorus, P. and Satria, D.

Evaluation of Antioxidant and Cytotoxic Activities of Vernonia amygdalina Del. Leaves.

DOI: 10.5220/0009844800002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

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).

2.3 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).

2.4 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

) at 37

C. The inoculums seeded at

1x10

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

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).

2.5 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

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

n-hexane, ethylacetate, ethanol fractions and

quercetine as positive control with DPPH assay

measured as IC

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

)

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

from n-hexane, ethylacetate,

ethanol fractions and doxorubicin as positive control

were 327.89 ± 1.13; 64.92 ± 0.72; 1591.75 ± 37.05

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

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

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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