Antioxidant Activity of Moringa oleifera Extracts

Abstract

Moringa oleifera have been evaluated for its antioxidant activity. M. oleifera leaves were extracted with methanol, ethyl acetate, dichloromethane and n-hexane. The antioxidant activity of extracts were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity assay and an improved 2,2’-azino-bis-[3-ethylbenzothiazoline sulphonate] (ABTS) radical cation decolorization assay in vitro. Trolox was used as standard with IC50 5.89 μg/mL in DPPH assay and 3.06 μg/mL in ABTS assay. The methanol extract showed the highest free radical scavenging activity with IC50 value of 49.30 μg/mL in DPPH assay and 11.73 μg/mL in ABTS assay. This study provided that M. oleifera leaves possess antioxidant.

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Wiwit Denny Fitriana et al.
297
* Corresponding author. Tel/Fax : +62-31-5943353/5928314
Email address : fatma@chem.its.ac.id
Antioxidant Activity of Moringa oleifera Extracts
Wiwit Denny Fitriana1,2, Taslim Ersam1, Kuniyoshi Shimizu3, and Sri Fatmawati1,*
1Department of Chemistry, Sepuluh Nopember Institute of Technology (ITS)
Jl. Arief Rahman Hakim, Sukolilo, Surabaya 60111, Indonesia
2Faculty of Mathematics and Natural Sciences, Universitas Pesantren Tinggi Darul Ulum, Jombang 61481, Indonesia
3Department of Agro-environmental Science, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
Received November 20, 2015; Accepted May 4, 2016
ABSTRACT
Moringa oleifera have been evaluated for its antioxidant activity. M. oleifera leaves were extracted with
methanol, ethyl acetate, dichloromethane and n-hexane. The antioxidant activity of extracts were evaluated by 1,1-
diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity assay and an improved 2,2’-azino-bis-[3-
ethylbenzothiazoline sulphonate] (ABTS) radical cation decolorization assay in vitro. Trolox was used as standard
with IC50 5.89 μg/mL in DPPH assay and 3.06 μg/mL in ABTS assay. The methanol extract showed the highest free
radical scavenging activity with IC50 value of 49.30 μg/mL in DPPH assay and 11.73 μg/mL in ABTS assay. This
study provided that M. oleifera leaves possess antioxidant.
Keywords: Moringa oleifera leaves; antioxidant; DPPH; ABTS
ABSTRAK
Aktivitas antioksidan dari ekstrak daun kelor (Moringa oleifera) telah diteliti. Daun kelor diekstrak daunnya
dengan metanol, etil asetat, dikolorometana, dan n-heksana. Pengujian aktivitas antioksidan dari masing-masing
ekstrak dilakukan dengan metode pengukuran penangkapan radikal oleh 1,1-difenil-2-pikrilhidrazil (DPPH) dan
metode penghilangan radikal kation oleh 2,2’-azino-bis-[3-etilbenzotiazolin sulfonat] (ABTS) secara in vitro. Trolox
digunakan sebagai kontrol positif dengan nilai IC50 5,89 μg/mL uji DPPH dan 3,06 μg/mL pada uji ABTS. Ekstrak
metanol daun kelor menunjukkan nilai aktivitas paling tinggi dengan nilai IC5 0 = 49,30 μg/mL pada uji DPPH dan
IC50 = 11,73 μg/mL pada uji ABTS. Penelitian ini menjadi bukti ilmiah bahwa daun kelor memiliki aktivitas
antioksidan yang tinggi.
Kata Kunci: Moringa oleifera; antioksidan; daun kelor; DPPH; ABTS
INTRODUCTION
Free radical and reactive oxygen species are well
known as inducers of cellular and tissue pathogenesis
which is causing some diseases like diabetes, cancer,
inflammatory and also cardiovascular. Free radical
reactions take place in the human body and food
systems can causing injury and death [1]. Free radicals
are one of the main factors which necessary to cause
DNA mutation, which is involve in the initiation stage of
carcinogenesis [2]. Reactive Oxygen Species (ROS) are
constantly produced in human body by normal metabolic
system. An over-production of reactive oxygen can occur
the imbalance of defense system. Therefore,
investigations of antioxidants are needed which focused
on natural compounds from natural sources.
The most widely used synthetic antioxidants in food
are butylated hydroxytoluene (BHT) or butylated
hydroxyanisole (BHA). Both of them are very effective as
antioxidants but their use in food products is not
popular anymore due to their instability and also due to
a suspected action as promoters of carcinogenesis. For
this reason, there is another interest in the studies of
natural healthy (non-toxic) additives as potential
antioxidants [3]. The total antioxidant capacity values
should include methods applicable to both lipophilic
and hydrophilic antioxidants, with regards to similarity
and differences of both hydrogen atom transfer and
electron transfer mechanism [4]. Some methods have
been used to evaluate antioxidant activities of natural
compounds by using stable free radical DPPH and
ABTS [5].
Most of the antioxidant compounds derived from
plant source have wide variety and chemical
properties. The antioxidant characteristic is based on
its ability to trap free radicals. Moringa oleifera
(Moringaceae; Indonesian name: kelor) is an
ornamental plant native in tropical and subtropical

Indones. J. Chem., 2016, 16 (3), 297 - 301
Wiwit Denny Fitriana et al.
298
areas, and commonly cultivated in all region of Indonesia
as a vegetable for cooking purposes. All parts of this
plant had been reported to have variously biological
activities such as reducing hyperglycemia [6] anti-
inflammatory, anti-diabetic, antimicrobial, anticancer and
antioxidant [7]. Kelor Leaves extracts were also found
have antioxidant activities with linoleic acid, α-tocopherol
and sunflower oil [8]. In Asia and Africa, the leaves is
recommended as a supplement because of rich in
nutrients for breastfeeding mothers and infant [9]. Nitrile
compounds, mustard oil glycosides, benzyl glycosides,
phenolic glycosides, flavonoid glycosides, thiocarbamate
glycosides and amino acids had been isolated from this
plant [7]. The antioxidant activities extract MeOH of M.
oleifera leaves showed the IC50 = 1.60 ± 0.03 mg/mL in
DPPH assay and IC50 = 1.02 ± 0.06 mg/mL in ABTS
assay) [10]. M. oleifera leaves contain of natural source
of polyphenol that potential to have antioxidant. The
purpose of this paper is to evaluate the antioxidant
activity of various extract of M. oleifera leaves.
EXPERIMENTAL SECTION
Materials
Plant materials is M. oleifera leaves from Jombang,
East Java, Indonesia. Solvent methanol, ethyl acetate,
dichloromethane, n-hexane, DMSO and ethanol were
purchased from Wako Pure Chemical Industries Japan.
The DPPH and ABTS reagent were purchased from
Wako (Japan). Trolox (6-hydroxy-2,5,7,8-tetramethyl
chroman-2-carboxylic acid; Sigma Aldrich) was used as
antioxidant standard. All other reagents were of
analytical grade.
Instrumentation
Incubator EYELA SLI-400 used to process
incubation of sample. The reaction was monitored by
spectrophotometer (UV Jasco V-530, Japan).
Procedure
Plant and extraction
The leaves of M. oleifera were collected during
December to February, 2013 from Jombang, East Java.
The leaves of M. oleifera dried in room temperature and
ground into powder. Twenty grams of leaf powder were
extracted with 250 mL of solvent (methanol, n-hexane,
ethyl acetate, and dichloromethane). The liquid extracts
were filtered with filter paper. The filtrates were
evaporated to remove the solvent and get four crude
extracts.
Antioxidant assay
DPPH radical scavenging assay. The radical
scavenging activity of M. oleifera extracts against the
DPPH radical was determined by the method of Brand
Williams with slightly modified by Dudonńe et al. [11-
12]. Determination procedures were as follow: 1 mL of
6 × 10−5 M DPPH radical solution (prepared daily) was
mixed with 33.33 μL of methanolic solutions of M.
oleifera extracts (maximum dissolved concentration).
After 20 min incubation for at 37 °C, absorbance
decrease of the mixture was monitored at 515 nm (As).
During reduction by the antioxidant, the solution colour
changed from violet to yellow pale. DPPH radicals have
an absorption maximum at 515 nm. Blank samples with
33.33 μL of methanol in the above DPPH radical
solution were prepared and measured daily at same
wavelength (Ab). Trolox was used as positive control.
The experiment was carried out in triplicate. Radical
scavenging activity was calculated using the following
formula.
 
Ab -As
Inhibition rate % = ×100
Ab
 
 
  (1)
The 50% inhibitory concentration (IC50) was expressed
as the quantity of the extracts to react with a half of
DPPH radicals.
Radical scavenging by ABTS radical. ABTS assay
was mostly based on the methods described previously
[13]. The ABTS method was used based on the ability
of antioxidant molecules to quench the long live
ABTS∙+. The ABTS∙+, in which the oxidant, was
generated by peroxydisulfate oxidation 2,2ʹ azino - bis
(3-ethylbenzothiazoline-6-sulfonic acid). Briefly, the
ABTS radical solution was prepared with 5 mL of 7 mM
ABTS in ammonium aqueous solution, and then 88 μL
of 140 mM potassium persulfate (K2S2O8) was added
then allowed to stand at room temperature for 12-16 h
to yield a dark blue solution. This solution was
subsequently diluted with ethanol 99.5% before use
which gave an absorbance of 0.7 ± 0.02 at 734 nm as
working solution. One milliliter of working solution was
mixed with 10 μL of M. oleifera extract (maximum
dissolved concentration) and shaken well for 10 sec;
after 4 min of incubation at 30 °C, the absorbance of
the reaction mixture was measured at 734 nm to give
As values. Trolox was used as positive control. Ethanol
99.5% was used as a blank (absorbance was Ab). The
antioxidative activity of the M. oleifera extracts
calculated by determining the decrease in absorbance
at different concentrations by using equation 1.
Statistical analysis
Values of experimental results shown in figures
were the mean of at least three determinations (±
standard deviation).

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RESULT AND DISCUSSION
Antioxidant from natural source can improve the
antioxidant system in body for scavenging free radicals.
An interest in antioxidant from natural sources increasing
faster than synthetic sources. Phenolic compounds
which naturally present in M. oleifera plant can reduce
the risk of many diseases and its effects which
correlated with the antioxidant compounds. Recently,
there are some reports about M. oleifera leaves which
rich in phenolic compounds such as flavonoids, gallic
acid, quercetin and kaempferol as antioxidant activity
[14]. M. oleifera is a one of Indonesian traditional plant
that has multipurpose biological activities. M. oleifera
Fig 1. DPPH inhibition of M. oleifera extracts at a
concentration of 319.45 μg/mL; values are mean SD of
three independent experiments in triplicate at each
concentration. MeOH, methanol extract; EtOAc, ethyl
acetate extract; DM, dichloromethane extract; Hx,
hexane extract
Fig 2. DPPH inhibitory activity of M. oleifera methanol
extract
extracts were evaluated for antioxidant activity by using
DPPH and ABTS assays.
DPPH Radical Scavenging Assay
The relatively stable DPPH radical had been used
widely to test the ability of compounds to act as free
radical scavengers or hydrogen donors. This capability
was used to evaluate antioxidant activity. Compounds
with radical scavenger capacity are able to reduce
DPPH radical using donor hydrogen atom to DPPH
free radical based on the type and concentration of
sample. Interaction of antioxidant compound with
DPPH is based on transfer electron or hydrogen atom
to DPPH radical and convert it to 1-1,diphenyl-2-
picrylhydrazyl. The result of reduction DPPH radicals
causes discoloration from purple color to yellow pale
color which indicates the scavenging activity. The
decrease of absorbance of DPPH radicals was
measured at 515 nm. The result of antioxidant DPPH
from M. oleifera various extracts is shown at Fig. 1.
The percentage of DPPH inhibition of various
extracts; MeOH, ethyl acetate, dichloromethane, and n-
hexane were 90.59 0.39, 36.19 0.55, 17.67 0.53,
22.45 0.22, respectively, while trolox as a standart
had antioxidant activity of 96.61 0.02. The IC50 value
for M. oleifera extracts determined by linear regression.
IC50 value for the IC50 value of methanol extracts was
49.30 μg/mL, dichloromethane extract was 1035.57
μg/mL, ethyl acetate extract was 444.10 μg/mL, n-
hexane extract was 715.21 μg/mL. The activity of M.
oleifera methanol extract as shown in Fig. 2. The
methanol extract had the highest antioxidant activity
among the other leaves extract due to these extract
may contain many phenolic compounds that
contributed of antioxidant activity. Trolox was used as
positive control of antioxidant with IC50 5.89 μg/mL.
ABTS Radical Scavenging Assay
ABTS is frequently used by the food industry and
also agricultural researchers to measure the
antioxidant capacities of foods. ABTS assay is used to
measure the relative ability of antioxidant to scavenge
the ABTS with compared with Trolox standard. The
ABTS solution was prepared by reacting a strong
oxidizing agent potassium persulfate with ABTS salt.
Reduction of blue-green ABTS radical colored solution
is measured in long wave 734 nm absorption spectrum.
The blue ABTS radical cation was converted back to
colorless neutral form during this reaction. Fig. 3
showed the ABTS scavenging activity of M. oleifera
leaves extracts.
The percentage of ABTS inhibition of various
extracts MeOH, ethyl acetate, dichloromethane, and

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Fig 3. ABTS inhibition of M. oleifera extracts at a
concentration of 99.00 μg/mL; values are mean SD of
three independent experiments in triplicate at each
concentration. MeOH, methanol extract; EtOAc, ethyl
acetate extract; DM, dichloromethane extract; Hx,
hexane extract
Fig 4. ABTS inhibitory activity of M. oleifera methanol
extract
Table 1. Antioxidant activity of M. oleifera extracts
Samples IC50 DPPH
(μg/mL)
IC50 ABTS
(μg/mL)
Methanol extract 49.30 11.73
Ethyl acetate extract 444.10 241.33
Dichloromethane extract 1035.57 159.06
n-hexane extract 715.21 163.79
Trolox 5.89 3.06
n-hexane were 89.40 0.73, 20.0 40.83, 31.37 0.57,
30.94 1.07, respectively, while trolox as a standard
had antioxidant activity of 94.99 0.58. The methanol
extract had the highest activity among the other leaves
extract.
The IC50 value for M. oleifera extracts have been
determined as shown in Fig. 4, and it gave IC50 value of
11.73 μg/mL. Trolox was used as standard antioxidant
with IC50 3.06 μg/mL. IC50 value for the
dichloromethane extract was 159.06 μg/mL, ethyl
acetate extract was 241.33 μg/mL, n-hexane extract
was 163.79 μg/mL (Table 1). The extract can be said
has active antioxidant, if IC50 value of extract
≤ 100 µg/mL, whereas inactive antioxidant if IC50 value
of extract ≥ 200 µg/mL [15].
MeOH extract was exhibited fine scavenging
abilities against DPPH and ABTS radicals with
concentration dependent manner. Thus, the MeOH
extract was exhibited a high ABTS radical scavenging
efficiency rather than the DPPH radicals, which could
be indicated to a different mechanism. In the DPPH
assay, the scavenging action may be due to the
hydrogen donating ability, whereas scavenging of
ABTS radical is due to scavenging of proton radicals
induced through donation of electrons [16].
The antioxidant activity of DPPH and ABTS from
M. oleifera extracts is depends on the solvent used in
the extraction. The different compounds can be
extracted with different solvent due to different
solubility. There is correlation between antioxidant
activities with total phenolic compound. MeOH extract
should have total phenolic compound higher than the
other extracts. The polyphenol compounds such us
quercetin and kaempferol should be exist in the leaves
[10]. Methanol is the highest polar solvent among the
others solvent which can pull out more polyphenol
compounds. The previous study reported that methanol
extract of C. spinosa buds showed result of rich in
flavonoids including several quercetin and kaempferol
glycosides. They demonstrated to possess strong
antioxidant / free radical scavenging effectiveness [17].
Further investigation about toxicity and in vivo assay for
clinical purposes is needed for clarify the safety of M.
oleifera methanol extracts.
CONCLUSION
Antioxidant activies of M. oleifera extracts with
various solvents (methanol, ethyl acetate,
dichloromethane, and n-hexane) were determined
using DPPH and ABTS methods. MeOH extracts
showed the highest antioxidant activity both in DPPH
free radical scavenging and ABTS assay in vitro. This
finding provides scientific evidence for the Indonesian
traditional people way, which used M. oleifera leaves
as one of nutrition food to prevent diseases. This study
also indicated that M. oleifera leaves can be used as
antioxidant source.

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ACKNOWLEDGEMENT
This work was supported by a grant from research
project for international research collaboration and
scientific publication 2014, no of contract
07555.13/IT2.7/PN.01.00/2014, Directorate General of
Higher Education, Ministry of Education and Culture,
Indonesia.
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