Antioxidant Properties of Sweet Orange Peels in Several Fractions of
Methanolic Extract
Suhartomi
1
, Kristin Natalia Gulo
1
, Albert Daniel Saragih
2
, Ahmad Raif Martinus
1
, Refi Ikhtiari
1,2*
1
Laboratory of Biomolecular Chemistry, Graduate School of Biomedical Science,
Universitas Prima Indonesia
2
Laboratory of Materials Engineering, Faculty of Industrial Engineering,
Universitas Prima Indonesia
Emails: (suhartomi, kristin, albert, ahmadraif, refiikhtiari)@unprimdn.ac.id
Keywords: Citrus sinensis, methanol, dichloromethane, n-hexane, Hydrogen peroxide, DPPH
Abstract: Citrus sinensis is a fruit that is widely consumed over the world and has potent natural antioxidant activity.
This study was aimed to determine the potential of antioxidants from each partition of sweet orange peel
methanol extract. The sweet orange peel methanol extract was made by the maceration method followed by
fractionation through a separating funnel with n-hexane and dichloromethane solvents. Antioxidant activity
of the methanolic extract was measured by H
2
O
2
and DPPH methods. Results showed that the most potent
H
2
O
2
inhibition activity was the fraction of n-hexane (IC
50
: 91.33 µg/ml), followed by dichloromethane
(IC
50
: 174 µg/ml), and methanol (IC
50
: 189.63 µg/ml). While the most potent DPPH inhibitory activity was
the n-hexane fraction (IC
50
: 709.60 µg/ml), followed by the methanol fraction (IC
50
: 790.64 µg/ml) and the
dichloromethane fraction (IC
50
: 895.58 µg ml). The fraction of n-hexane has higher antioxidant potential
than the fraction of dichloromethane and methanol. These antioxidant properties might be related to
bioactive compounds in the methanolic extracts which will be investigated further. However, the research
results on several fractions might provide a preliminary study on citrus-based drug preparation in the
pharmacy industry for antioxidants application.
1 INTRODUCTION
Free radicals are types of high reactivity molecules
that have unpaired electrons and last only for a very
short time (usually 10
-9
to 10
-12
seconds) before they
react with other molecules and take or donate
electrons to achieve stability. The most damaging
main radicals in biological systems are oxygen
radicals (sometimes called oxidative oxygen species),
especially superoxide, O
2
*, hydroxyl, OH *, and per
hydroxyl, O
2
H *. Normally free radicals are formed
in the body that are useful in cell signaling and
especially the signaling process for cell apoptosis in
damaged cells. However, these substances can also
cause damage to nucleic acids, proteins, and lipids in
cell membranes and plasma lipoproteins which then
cause disorders such as cancer, atherosclerosis, and
coronary artery disease, as well as autoimmune
disease (Bander, 2015). More than half (54%) of
deaths occurred during 2016 (56.9 million) due to
the top 10 causes. Ischemic heart disease and stroke
are the biggest killers, accounting for 15.2 million
deaths in 2016 due to a combination of these
diseases. Besides these two diseases are also the two
most common causes of death in the world over the
past 15 years (Top 10 causes of death, 2018).
Imbalance of oxidant and antioxidant lead to the
impairment of signaling process, reduction-oxidation
reaction control and/or molecular damage (Sies,
2015). The antioxidant is a chemical compound that
prevents the oxidation of another chemical
compound, Several phytochemicals that have potent
antioxidant activity include tannin, flavonoid, and
phenolic acid. The human body uses an antioxidant
defense system to neutralize excessive Reactive
Oxygen Species (ROS). This system consists of
enzymatic and non-enzymatic antioxidants. Some
antioxidant enzymes found to protect against ROS
are superoxide dismutase, catalase, and glutathione
peroxidase, in addition to that many small non-
enzymatic molecules, are widely distributed in
biological systems and are capable of cleaning free
radicals. These non-enzymatic molecules include
Suhartomi, ., Gulo, K., Saragih, A., Martinus, A. and Ikhtiari, R.
Antioxidant Properties of Sweet Orange Peels in Several Fractions of Methanolic Extract.
DOI: 10.5220/0009515503710378
In Proceedings of the International Conference on Health Informatics and Medical Application Technology (ICHIMAT 2019), pages 371-378
ISBN: 978-989-758-460-2
Copyright
c
2020 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
371
glutathione, tocopherol (vitamin E), vitamin C, β-
carotene, and selenium (Shalaby and Shanab, 2013).
Antioxidants are categorized in two synthetic and
natural groups, which are mostly substituted
phenolic compounds (Akbarirad et al., 2016). The
antioxidant content of plant ingredients acts as a free
radical scavenger and helps convert radicals into less
reactive species. Various free radical eradicating
antioxidants found in food sources such as fruit,
vegetables, and tea (Kumar, 2014; Zouaghi, Najar
dan Abderrabba, 2018). While the synthetic
compounds that have antioxidant properties must be
non-toxic, have high activity at low concentrations
(0.01-0.02%), and can be concentrated on the
surface of fat or other oil phases, due to the non-
protein nature of synthetic compounds. The
synthetic antioxidant is relatively stable and usually
can penetrate cells, so it can be given orally
(Akbarirad et al., 2016).
Citrus sinensis is a fruit that is widely consumed
throughout the world and has strong natural
antioxidant activity. Citrus plants are a group of
plants originating from the Rutaceae family (Rafiq et
al., 2016). The genus Citrus (Citrus L. of Rutaceae)
is one of the world's most common fruit plants and is
consumed mostly as a fresh product or juice because
of its unique nutritional value and taste. The most
popular in Europe and North America are grapefruits
(Citrus paradisi), lemons (Citrus limon), limes
(Citrus aurantifolia) and sweet oranges (Citrus
sinensis). The level of consumption of citrus fruits or
juices is found to be inversely related to the
incidence of several diseases.
The health benefits of citrus fruits are mainly
related to the presence of bioactive compounds, such
as phenolics (for example, flavanone glycosides,
hydroxylic acids), vitamin C, and carotenoids.
Although these fruits are mainly used for dessert,
they are also a source of essential oils due to their
aromatic compounds. For example, the taste of lime
is used in drinks, snacks, cakes, and desserts. Many
authors have reported antioxidant and radical
properties of essential oils and in some cases,
applications that are directly related to food
(Guimarães et al., 2010).
Citrus plants promise various nutritional benefits
as well as human life. The processing of Citrus by-
products has the potential to represent a rich source
of phenolic compounds and dietary fiber, as they are
found in the skin. This orange fruit residue, which is
generally disposed of as waste in the environment,
can act as a potential nutraceutical resource. Because
of their low cost and easy availability, the waste can
offer significant low nutritional food supplements.
Utilization of bioactive citrus-rich residues can
provide an efficient, inexpensive, and
environmentally friendly platform for the production
of new nutraceuticals or for enhancements that
already exist (Rafiq et al., 2016).
Many studies have been conducted on the orange
peel, especially sweet orange (Citrus sinensis) to
examine its effects on health including antioxidants,
antibacterial, and anti-inflammatory which are
compatible with ascorbic acid, ciprofloxacin, and
aspirin respectively. This could be related to the
content of alkaloids, flavonoids, tannins, saponins,
and steroids on extra sweet orange peels
(Omodamiro dan Umekwe, 2013).
Flavonoid is a secondary metabolite that was
found widely among plants and has some
pharmacology properties. Mechanism of antioxidant
that was had by donor the hydrogen ion or
transferring single electron of flavonoid into reactive
oxygen species and forms chelate complex
(Banjarnahor and Artanti, 2014).
Previous studies conducted by Selvi et al. (2016)
who conducted phytochemical screening and
evaluation of antioxidant activity in Citrus sinensis
skin extracts with several types of solvents reported
the presence of alkaloids, flavonoids, saponins and
other phenolic compounds that contribute to their
antioxidant effects. Other studies conducted by Park
et al. (2014) which aims to evaluate the antioxidant
effect of orange peel extract and flesh with various
variations show that the extract with acetone solvent
has the best antioxidant effect on both types of
extract with IC
50
value of orange peel extract is
781.9 µg / mL (Park, Lee and Park, 2014; Selvi,
Kumar and Bhaskar, 2016).
The level of orange consumption was reversely
correlation with the incidence of some diseases.
There are several health benefits of sweet orange
due to the presence of some bioactive compounds,
like phenolate, vitamin c, and carotenoid. However,
this fruit was used as a dessert, this fruit is riched by
essential oil due to its aromatic compound. Several
studies have reported this antioxidant activity
against free radicals as a food additive (Guimarães et
al., 2010). Based on the information above, this
study was aimed to determine the potential of
antioxidants from each partition of sweet orange
peel methanol extract.
ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology
372
2 RESEARCH METHODS
2.1 Identification of Sample
The sample of sweet orange peel was obtained from
the traditional market in Medan followed by an
assessment of species identification at Herbarium
Medan of North Sumatera University.
2.2 Extraction Process
Extract quality is influenced by several factors such
as plant parts used as starting material, solvents used
for extraction, extraction procedures, and ratio of
plant to solvent. etc. From the laboratory scale to the
pilot scale all parameters are optimized and
controlled during extraction. Extraction techniques
separate plant metabolites that can dissolve through
the use of selective solvents (Gupta et al., 2012).
Different solvent systems are available to extract
bioactive compounds from natural products.
Extraction of hydrophilic compounds using polar
solvents such as methanol, ethanol or ethyl acetate.
For extraction of more lipophilic compounds,
dichloromethane or a mixture of
dichloromethane/methanol in a ratio of 1: 1 is used.
In some instances, extraction with hexane is used to
remove chlorophyll (Sasidharan et al., 2011).
Since the target compound may be non-polar to
polar and thermally labile, the appropriateness of the
extraction method must be considered. Various
methods, such as sonification, heating by reflux,
soxhlet extraction and others are usually used for
extraction of plant samples. Besides, plant extracts
are also prepared by maceration or percolation of
fresh green plants or dry powder of plant material in
water and/or organic solvent systems (Sasidharan et
al., 2011).
Sweet Orange peels were washed, dried and
mesh into simplicia powder. Amount of 218.46 gram
of simplicia powder macerated using 1 liter of
methanol for 24 hours and the residue was re-
macerated for the next 2nd and 3rd day using 1 liter
of methanol. While the filtrate from each maceration
was evaporated using a rotary evaporator at 40oC.
The concentrated form of the filtrate was named
Methanol Extract of Sweet Orange Peel. The
Methanol extract of sweet orange peel was then
stratified by using separation funnel.
In the first step of the separation process, 10 gr
of methanol extract of sweet orange peels was added
and homogenized. The mixture was added 50 ml of
n-hexane in the separation funnel then shook at the
same time, the air from the funnel was then released
slowly. After it formed 2 layers, the polar solvent
(lower part) was withdrawn from funnel’s stopcock
and the hexane layer (upper part) was poured out
from the funnel's stopper, at the same time remain
polar solvent was separated again by same technique
until they become colorless solvent.
In the second step of the separation process
against the remaining polar solvent using
dichloromethane was done by the same procedure.
Finally, we obtained the n-hexane partition,
dichloromethane partition and methanol partition
(Andersen and Markham, 2006; Zouaghi, Najar and
Abderrabba, 2018).
Each partition and ascorbic acid as positive
control were dissolved into various concentrations
by DMSO. A stock solution was 1,000 µg/mL
serially diluted by using DMSO into some
concentration including 50 µg/mL, 100 µg/mL, 250
µg/mL, and 500 µg/mL.
The variations of these concentrations were done
by making the mother solution first by mixing 50 mg
of each fraction of sweet orange peel extract with 50
ml of DMSO so that the mother solution is found
with a concentration of 1000 µg / mL. Then the
mother solution was successively pipetted as much
as 12.5ml, 6.25ml, 2.5 ml, and 1.25 ml into a 25 ml
volumetric flask and added a DMSO solution to 25
ml so that a solution concentration of 50 μg / mL,
100 μg / mL, 250 μg was found. µg / mL, and 500
µg / mL. Making variations in the concentration of
ascorbic acid as positive control is done in the same
way (Park, Lee and Park, 2014).
2.3 H
2
O
2
Assay
Hydrogen peroxide (H2O2) scavenging activity has
been widely used to determine natural antioxidant
activity by measuring H2O2 reduction and detected
by spectrophotometer at a wavelength of 230 nm.
The main drawback of this method is the possibility,
disruption of secondary metabolites that are in the
absorbance range. Optimum conditions for
determining antioxidant activity by this method at
37oC and pH 7 (Fernando dan Soysa, 2015).
Hydrogen peroxide (40 Mm) solution is prepared
in a phosphate buffer (pH 7.4). Samples with
different concentrations from each treatment group
were added to hydrogen peroxide solution (0.6 ml,
40 Mm). The absorbance of hydrogen peroxide was
measured using spectrophotometry at a wavelength
of 230 nm with a blank solution in the form of a
phosphate buffer without hydrogen peroxide.
Percent inhibition by hydrogen peroxide with
Antioxidant Properties of Sweet Orange Peels in Several Fractions of Methanolic Extract
373
Vitamin C (Ascorbic Acid) is calculated by this
equation:
% Inhibition = [(A0-A1) / A0] x 100
Where A0 is the absorbance of the control, and
A1 is the absorbance of the sample and standard
groups (Rekha and Bhaskar, 2013).
2.4 DPPH Assay
A fast, simple and inexpensive method for
measuring the antioxidant capacity of foods involves
the use of free radicals 2,2-Diphenyl-1-
picrylhydrazyl (DPPH). DPPH is widely used to test
the ability of compounds to act as free radical
cleaners or hydrogen donors and to evaluate the
antioxidant activity of foods. It has also been used to
measure antioxidants in complex biological systems
in recent years. The DPPH method can be used for
solid or liquid samples and is not specific to certain
antioxidant components, but applies to the overall
antioxidant capacity of the sample. A measure of
total antioxidant capacity helps to understand the
functional properties of food (Shalaby dan Shanab,
2013).
The amount of 1 ml of each fraction and ascorbic
acid as a group of the sample were added by 1 ml of
0.05 mM DPPH methanolic followed by incubation
in the dark chamber for 30 minutes. The absorbance
of the sample was measured at 518 nm and the
percent of inhibition was calculated by using the
following formulation (2).
Percent of Inhibition = ([a - b]/a) x 100%
a : absorbance of control
b : absorbance of sample
While the absorbance of control was the same as
the group of sample however it was not added by
either fraction or ascorbic acid. Those were repeated
in triplicate (Selvi, Kumar and Bhaskar, 2016; Okoh
et al., 2017).
2.5 Data Analysis
All data analysis in this research is done by software
IBM SPSS Statistics 25. Univariate analysis is
intended to describe the central tendency and
distribution of each variable. The variables in this
study include the percent inhibition as an antioxidant
potential through percent inhibition and IC
50
values
presented in tables and diagrams. Percent of
inhibition was expressed analyzed based on the
normality of data distribution. Percent inhibition that
distributes normally was expressed as Mean ± SD
and analyzed by one way ANOVA followed by Post
Hoc test Tukey HSD. Meanwhile, percent inhibition
that did not distribute normally was expressed as
Median (Min-Max) and analyzed by the Kruskal-
Wallis test and followed by the Mann-Whitney test.
3 RESULTS AND DISCUSSION
3.1 Determination of Orange Fruit
The sample of orange sweet fruit was collected from
the traditional market in Medan, North Sumatera.
The fresh fruit was then identified as a result of plant
determination as well as the taxonomy below:
Kingdom: Plantae
Division: Spermatophyta
Class: Dicotyledoneane
Ordo: Rustales
Family: Rutaceae
Genus: Citrus
Species: Citrus sinensis (L.)
Local Name: Sweet Orange (Jerukmanis)
Citrus sinensis (L.) belongs to Rutaceae family
fruit. It was widely consumed among humans over
the world that had various health benefits include
antioxidants, anti-bacterial, and anti-inflammation.
Several types of citrus within the Rutaceae family
are grapefruit (Citrus paradisi), lemon (Citrus
limon), and lime (Citrus aurantifolia). On the other
hand, sweet orange peel is also riched by various
content of phytochemicals include alkaloid,
flavonoid, tannin, saponin, and steroid (Guimarães et
al., 2010; Omodamiro and Umekwe, 2013; Rafiq et
al., 2016).
3.2 DPPH Scavenging Activity of
Various Fraction from Sweet
Orange Peel Methanol Extract
Evaluation of the antioxidant activity by DPPH
assay from various Fraction from Sweet Orange Peel
Methanol extract was shown as percent inhibition.
Due to the difference from data distribution in
percent inhibition of hexane fraction and other
fraction, then percent inhibition of hexane fraction
was analyzed by non-parametric test while the others
were the parametric test. The result of the analysis
was shown in the Table 1.
ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology
374
Table 1: Percent Inhibition Against DPPH of Various
Fraction from Sweet Orange Peel (Citrus sinensis)
Methanol Extract and Ascorbic Acid as Positive Control
Group
Conc.
(µg/ml)
Percent Inhibition (%)
MF* HF** DF* AA*
1000
47.22
(46.96-
47.96)
a
53.37 ±
0.90
a
57.71
(54.39-
57.77)
a
77.38
(76.81-
84.15)
a
500
45.32
(44.91-
59.03)
a
45.15 ±
2.42
b
47.46
(33.90-
56.24)
a,b
62.55
(61.91-
64.87)
b
200
16.52
(15.11-
16.53)
b
34.74 ±
2.90
c
40.14
(23.25-
46.55)
b
52.10
(52.03-
61.31)
c
100
12.30
(11.08-
13.88)
c
25.20 ±
3.75
d
35.53
(25.64-
42.21)
b,c
45.44
(32.03-
49.50)
d
50
8.83
(2.45-
9.25)
d
15.02 ±
0.97
e
23.48
(22.53-
27.69)
c
15.00
(22.86-
34.29)
d
Conc. = Concentration; MF = Methanol Fraction; HF =
Hexane Fraction; DF = Dichloromethane; AA=
Ascorbic Acid. * Data was expressed as Median (Min-
Max). The different of the small letter in the same
column shows significant at P < 0.05 by Kruskal-Wallis
and Mann-Whitney test. ** Data was expressed as
Mean ± SD. The different of the small letter in the
same column shows significant at P < 0.05 by Post Hoc
Test Tukey HSD.
Based on data analysis in the table 1, there was
no significant difference in DPPH scavenging
among methanol fraction in the concentration higher
than 500 µg/ml. Whereas hexane fraction at various
concentrations showed a significant difference in
DPPH scavenging activity. However, fraction
dichloromethane showed a significant difference of
percent inhibition in DPPH scavenging among 1000
µg/ml, 200 µg/ml, and 50 µg/ml. As a positive
control, ascorbic acid showed a significant
difference in DPPH scavenging activity at all
concentrations, except at least 2 lower
concentrations (100 µg/ml and 50 µg/ml).
Based on literatures, studies on Citrus aurantium
(bitter orange) showed that the ethanol and aqueous
media were comparatively more effective in
extracting the antioxidant components. The total
phenol content of the extracts ranged from 2.5 to
22.5 mg/g and 5.0 to 45.0 mg/g of pulp and peel
fragments, respectively. The fruit components
exhibited proton radical, oxyradical, and hydroxyl
radical scaveng- ing abilities and were effective in
preventing lipid peroxidation. Their analysis showed
positive association between total phenolics and
different antioxidant assays (Divya et.el. 2016).
Furthermore, the analysis was followed by linear
regression to determine IC
50
for each group of the
sample. The IC
50
value of each sample as shown in
the Table 2.
Table 2: The IC
50
Value against DPPH of Various
Fraction from Sweet Orange (Citrus sinensis) Peels
Methanol Extract and Ascorbic Acid as Positive Control.
Group of
Sample
Equation R
2
IC
50
(µg/m
l)
Methanol
fraction
Y = 21.537 +
0.036
0.819 790.64
Hexana fraction
Y = 28.712 +
0.030x
0.652 709.60
D
ichlorometane
fraction
Y = 9.699 +
0.045x
0.738 895.58
Ascorbic acid
Y = 33.564 +
0.050x
0.715 328.72
Based on data in table 2, the lowest IC
50
value
was shown by ascorbic acid (328.72 µg/ml),
followed by hexane fraction (709.60 µg/ml),
methanol fraction (790.64 µg/ml), and
dichloromethane fraction (895.58 µg/ml). The
higher the IC
50
value means the higher the
concentration of the sample needed to decompose
some of the free radicals tested.
The results of this study indicate that the results
contradict with reported by Guimaraes et al. (2010)
which states that the polar fraction of Citrus sinensis
orange peel contains flavonoids at 0:21 ± 3.97 mg
CE/g extract with IC
50
values against DPPH
amounted 0:31 ± 4.99 mg/ml. The difference in the
results of this research could be due to the solvent
and the way the extraction was carried out, in this
study the fractionation process was carried out using
separation and by using solvents while the research
conducted by Guimaraes et al. (2010) carried out by
stirring the orange peel powder samples for 12 hours
at 25
o
C at a speed of 150 rpm using a methanol
solvent (Guimarães et al., 2010).
Other research conducted by Ghasemi et al.
(2009) on several samples of Citrus sinensis variant
extracted by methanol solvent percolation method
showed similar results with this study where the
total flavonoid content in sweet orange peel with
Washinton Navel Variant contained 23.2 mg QE/gr
extract powder with IC
50
against DPPH 1.1 mg/ml
whereas, in other variants namely, the variant shows
a lower total flavonoid content of 2.1 mg QE/gr
Antioxidant Properties of Sweet Orange Peels in Several Fractions of Methanolic Extract
375
extract powder with IC
50
to DPPH only 1.7 mg/ml
(Ghasemi, Ghasemi dan Ebrahimzadeh, 2009).
As a comparison result, Omodamiro and
Umekwe (2013) states that the flavonoid which is
owned by the ethanol extract of sweet orange peel
with maceration using ethanol in the ratio of 1: 4 (g /
ml) for 18 hours showed that the content of total
flavonoids of the extract is only 2.5 ± 0:04 % and
the extract has antioxidant activity through the
inhibition of nitric oxide IC
50
value of 1100 pg/ml
and anti-lipid peroxidation IC
50
of 1000 ug/ml. Other
studies that can be a comparison of IC50 content of
several fruit peel samples from the genus Citrus are
shown in the Table 3 (Omodamiro dan Umekwe,
2013).
Table 3: Comparison of IC
50
content of several fruit peel
samples from the genus Citrus
Sampel
DPPH (IC
50
)
(µg/ml)
Sour Orange (SO) 742.7
Citrus macrophylla (CM) 946.4
Citrus carrizzo (CC) 985.4
Citrus volkameriana (CV) 585.4
Mandarin Cleopatra
(MCL)
934.03
Citumelo swingle (Citru) 1095.1
Lemon rangpur (LR) 782.05
Poncirus trifoliate (PT) 827.49
However, another studies by Park et.al. 2014
reported antioxidant activity of orange (Citrus
auranthium) flesh (OF) and peel (OP) extracted with
acetone, ethanol, and methanol. Antioxidant
potential was examined by measuring -diphenyl-1-
picrylhydrazyl (DPPH). The results suggested that
acetone is the best solvent for the extraction of
antioxidant compounds from OF and OP.
Furthermore, the high antioxidant activity of OP,
which is a by-product of orange processing, suggests
that it can be used in nutraceutical and functional
foods.
3.3 H
2
O
2
Scavenging Activity of
Various Fraction from Sweet
Orange Peel Methanol Extract
By assessing the H
2
O
2
inhibitory activity of each
fractionation result of methanol extract of sweet
orange peel (Citrus sinensis (L.)), the data is
presented in table 4. The data of H
2
O
2
inhibitory
activity of each fraction and positive control in the
form of ascorbic acid were analyzed for normality in
advance by the Shapiro-Wilk test and percent
inhibition data owned by methanol extract fraction
of sweet orange peel (Citrus sinensis (L.)) and acidic
acid. abnormally distributed ascorbate. Then the data
analysis continued to assess the differences in
percent inhibition held by each concentration in each
fraction and positive control. The results of the
analysis of differences in each group of positive
fractions and controls are shown in Table 4.
Table 4: Percent Inhibition Against H
2
O
2
of Various
Fraction from Sweet Orange Peel (Citrus sinensis)
Methanol Extract and Ascorbic Acid as Positive Control
Group.
Conc.
(µg/ml
)
Percent Inhibition (%)
MF* HF** DF* AA*
1000
90.81
(56.12-
92.03)
a
97.42
(89.95-
99.45)
a
68.54
(67.36-
69.14)
a
90.81
(56.12
-
92.03)
a
500
56.68
(50.72-
61.43)
a, b
95.05
(94.82-
95.30)
a
60.20
(59.66-
60.20)
b
56.68
(50.62
-
61.43)
a
200
55.09
(55.03-
55.26)
b
53.20
(51.61-
53.40)
b
55.09
(55.03-
55.26)
c
37.95
(37.95
-
42.71)
b
100
49.63
(49.58-
50.16)
c
49.28
(32.86-
49.28)
c
49.63
(49.58-
50.16)
d
34.26
(31.89
-
34.26)
c
50
39.29
(38.76-
44.21)
d
45.99
(45.48-
47.12)
c
39.29
(38.76-
44.21)
e
24.47
(24.46
-
26.43)
d
Conc. = Concentration; MF = Methanol Fraction; HF =
Hexane Fraction; DF = Dichloromethane; AA=
Ascorbic Acid. * Data was expressed as Median (Min-
Max). The different of the small letter in the same
column shows significant at P < 0.05 by Kruskal-Wallis
and Mann-Whitney test. ** Data was expressed as
Mean ± SD. The different of small letters in the same
column shows significant at P < 0.05 by Post Hoc Test
Tukey HSD.
Based on the analysis of the sample groups of the
methanol, n-hexane, and dichloromethane fractions,
ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology
376
with a difference in percent inhibition of hydrogen
peroxide at concentrations ≥ 500 μg/ml, 200 μg/ml,
100 μg/ml, and 50 μg/ml. Whereas in the positive
control group in the form of ascorbic acid it was
found that the difference in inhibition of hydrogen
peroxide was at concentrations ≥ 500 μg/ml, 200
μg/ml, and ≤ 100 μg/ml.
To compare antioxidant activity through percent
inhibition of hydrogen peroxide in each fraction and
positive control, an analysis was performed using
linear regression to determine the IC
50
of each
fraction of sweet orange peel (Citrus sinensis (L.))
and positive control. The statistical analysis of the
antioxidants of fractions are presented in Table 5.
Table 5. IC50 Inhibition of Hydrogen Peroxide from Each
Sweet Orange (Citrus sinensis (L.)) Skin Fraction and
Ascorbic Acid.
Group of
Sample
Equation R
2
IC
50
(µg/m
l)
Methanol
fraction
Y = 43.363 +
0.035x
0.669 189.63
Hexane
fraction
Y = 44.520 +
0.060x
0.776 91.33
Dichlorometha
ne fraction
Y = 45.824 +
0.024x
0.828 174
Ascorbic acid
Y = 26.634 +
0.55x
0.846 42.48
From the statistical data, it was showed that the
IC
50
value of each fraction of sweet orange peel
(Citrus sinensis (L.)) and ascorbic acid as a positive
control that the smallest IC
50
value was 42.48 µg/ml
namely ascorbic acid while the most IC
50
value was
in the methanol fraction that is 189.63 µg/ml. So that
the antioxidant activity through IC
50
value inhibition
of hydrogen peroxide was found that the most potent
antioxidant effect is owned by ascorbic acid as a
positive control and the weakest is the methanol
fraction. While the antioxidant activity of each
orange peel fraction from the strongest to the
weakest was the fraction of n-hexane (91.33 µg/ml),
dichloromethane (174 μg/ml), and methanol (189.63
μ/ml).
4 CONCLUSION
The most potential hydrogen peroxide inhibition
activity was the fraction of n-hexane, followed by
dichloromethane, and methanol. While the most
potent DPPH inhibitory activity was the n-hexane
fraction, followed by the methanol fraction, and the
dichloromethane fraction. Hence, the fraction of n-
hexane is the best antioxidant potential instead of
dichloromethane and methanol. This research result
might give valuable preliminary information on the
efficacy of Citrus sinensis extracts for pharmacology
industry applications.
ACKNOWLEDGEMENTS
This research was fully supported by the Ministry of
Research, Technology and Higher Education by
Funding Contract No. 7/E/KPT/2019 and No.
T/63/L1.3.1/PT.01.03/2019.
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