Journal of Dietary Supplements

ISSN: 1939-0211 (Print) 1939-022X (Online) Journal homepage: http://www.tandfonline.com/loi/ijds20

Orange Peel Extracts: Chemical Characterization,

Antioxidant, Antioxidative Burst, and Phytotoxic
Activities

Ochuko L. Erukainure, Osaretin A.T. Ebuehi, M. Iqbal Chaudhary, M. Ahmed

Mesaik, Ahmed Shukralla, Aliyu Muhammad, Moses Z. Zaruwa & Gloria N.
Elemo

To cite this article: Ochuko L. Erukainure, Osaretin A.T. Ebuehi, M. Iqbal Chaudhary, M.
Ahmed Mesaik, Ahmed Shukralla, Aliyu Muhammad, Moses Z. Zaruwa & Gloria N. Elemo
(2016): Orange Peel Extracts: Chemical Characterization, Antioxidant, Antioxidative Burst, and
Phytotoxic Activities, Journal of Dietary Supplements, DOI: 10.3109/19390211.2016.1150932

To link to this article: http://dx.doi.org/10.3109/19390211.2016.1150932

Published online: 01 Mar 2016.

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 Journal of Dietary Supplements, Early Online:1–10, 2016
Copyright C Taylor & Francis Group, LLC
DOI: 10.3109/19390211.2016.1150932

ARTICLE

Orange Peel Extracts: Chemical Characterization,

Antioxidant, Antioxidative Burst, and Phytotoxic
Activities

Ochuko L. Erukainure1,2,3 , Osaretin A.T. Ebuehi4 , M. Iqbal Chaudhary2 ,

M. Ahmed Mesaik3,5 , Ahmed Shukralla3 , Aliyu Muhammad3,6 , Moses
Downloaded by [Orta Dogu Teknik Universitesi] at 12:27 09 March 2016

Z. Zaruwa7 , & Gloria N. Elemo1

1
Nutriton and Toxicology Division, Federal Institute of Industrial Research, Lagos,
Nigeria, 2 H.E.J. Research Institute of Chemistry, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi, Pakistan, 3 Dr. Panjwani Center for
Molecular Medicine and Drug Research, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi, Pakistan, 4 Department of
Biochemistry, College of Medicine, University of Lagos, Lagos, Nigeria, 5 Tabouk
Medical College, University of Tabuk, Tabuk, Saudi Arabia, 6 Department of
Biochemistry, Ahmadu Bello University, Zaria, Nigeria, 7 Department of Chemistry,
Adamawa State University, Mubi, Nigeria

ABSTRACT. The search for novel drugs and alternative medicine has led to increased
research in medicinal plants. Among such plants is the orange fruit. Its peels have been
utilized for long as an active ingredient in most traditional medicines. This study aims
at investigating the chemical properties of the hexane and dichloromethane (DCM) ex-
tracts of orange peel as well as their biological potentials. Blended peels were extracted
with n-hexane and n-dichloromethane, respectively. The resulting extracts were sub-
jected to gas chromatography mass spectrometry (GCMS) characterization. The ex-
tracts were also assayed for free radical scavenging ability against 1,1 – diphenyl – 2
picrylhydrazyl (DPPH), antioxidative burst via measuring luminol – amplified chemi-
luminescence response in human blood, and phytotoxicity against lemna minor. GCMS
analysis revealed a predominance of fatty acid methyl esters in the hexane extract, while
the DCM extract had more ketone metabolites. The DCM extract had significant (p <
.05) higher free radical scavenging and antioxidative burst activities compared to the
hexane. Both extracts revealed a significantly (p < .05) high phytotoxicity activity. Re-
sults from this study indicated that solvent type played a vital a role in the extraction of
secondary metabolites, which are responsible for the observed biological activities. The
higher activities by the DCM extract can be attributed to its constituents as revealed
by GCMS analysis. There is great need to explore the phytotoxicity potentials of both
extracts as natural herbicides.

Address correspondence to: Ochuko L. Erukainure, Nutrition and Toxicology Division, Federal Institute of
Industrial Research, PMB 21023, Ikeja, Lagos, Nigeria. (E-mail: loreks@yahoo.co.uk)
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ijds

1
 2 Erukainure et al.

KEYWORDS. antioxidative burst, phytotoxicity, scavenging activity, secondary

metabolites

INTRODUCTION
Sweet orange (Citrus sinensis L.) is the most commonly grown tree fruit in the
world, constituting about 60% of the total citrus world production (Hegazy &
Ibrahium, 2012; Morton, 1987). Its production is usually used to address indus-
trial production of juice, thereby leading to huge amounts of residues, primarily the
peels (Hegazy & Ibrahium, 2012). Over time there have been increasing interests in
the utilization of these peels for both nutritional and medicinal purposes. The peel
has been reported to be edible and mostly consumed when there was a scarcity of
resources and maximal nutritional value was desired (Erukainure, Ajiboye, Davis,
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Obabire & Aliyu, 2012). It has an increased vitamin C and fiber contents but with
high concentrations of pesticides (Anon, 2005). However, increased dietary vita-
min A is required when consuming orange peel due to the presence of citral, an
aldehyde that antagonizes the action of vitamin A (Ensminger, 1983).
The medicinal properties of orange peel have been documented in several
studies. It has been used for centuries in the traditional Chinese medicine to treat
indigestion and to improve inflammatory syndromes of the respiratory tract (Yung-
Sheng & Su-Chen, 2010). Erukainure et al. (2012) reported the antioxidant poten-
tial of orange peel oil in laboratory rodent, indicating its protective effect against
oxidative stress-mediated ailments. These reported medicinal properties can be
attributed to the phytochemical constituents of the peel. Flavonoids, consisting
mainly of polymethoxylated flavonoids (PMFs), terpenoids, such as limonene and
linalool, and other volatile oils make up the major phytochemical constituents of
orange peel. The chemopreventive potential of these secondary metabolites espe-
cially PMFs in antimutagenic and antitumor properties has been reported (Li, Pan,
Tan, Wang & Shahidi, 2009).
The phytotoxic properties of orange peel methanolic extract and essential oil
have been reported (Ribeiro & Lima, 2012). This observed effect has been de-
scribed as a tremendous benefit over the use of synthetic herbicides. These peels are
readily available and often regarded as waste, thus a cheaper and healthier method
in weed control.
This article aims at reporting the gas chromatography mass spectrometry
(GCMS) characterization of the n-hexane and n-dichloromethane (DCM) extracts
of orange peel, as well as their biological potentials which cover for antioxidant,
antioxidative burst, and phytotoxic activities.

MATERIALS AND METHODS

Plant Materials
About 50 g of sweet orange peels were collected from a local fruit vendor in Lagos,
Nigeria. They were sorted, air dried, and blended to fine a smooth powder before
◦
being subjected to soxhlet extraction for 3 hr using n-hexane as the solvent at 80 C.
◦
After extraction, the extract was concentrated and stored in glass vials at 4 C for
 Orange Peel Extracts: Chemical Characterization 3

further analysis. After hexane extraction, the peels were subjected to another ex-
traction using n-dichloromethane (DCM) as the solvent for 3 hr at the same tem-
◦
perature. The extract was concentrated and stored in glass vials at 4 C for further
analysis.

GCMS Analysis
The extracts were subjected to GCMS analysis to identify their chemical con-
stituents.

Instrumentation
GC: Agilent 6890 N gas chromatograph, flame ionization detector (FID) at 220, N2
at 1.0ml/min, ZB-5 HT capillary column (30 m × 0.53 mm ID; 0.32 mm), split ratio
1:30 injector temperature of column 260◦ C, temperature of column maintained at
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70◦ C for 3 min and then raised to 235◦ C (5◦ C/min) followed by five minutes at
260◦ C.
GC-MS: Hewlett Packard 6890 gas chromatograph combined with a Jeol JMS-
HX 110 mass spectrometer with source at 270◦ C at 70 eV. Injector was set at 270◦ C
with splitting ratio 1:30. A mass spectral survey was performed using the NIST mass
spectral program 2008. The concentrations of the identified compounds were cal-
culated using area normalization over FID response method.

Free Radical Scavenging Assay

The free radical scavenging ability of the extracts against DPPH (1,1-diphenyl-2
picrylhydrazyl) free radical was evaluated as described by Ursini, Maiorino, Moraz-
zoni, Rover & Pifferi (1994). Briefly, the extracts (50–200 μl) were diluted in 3 ml
ethanol and mixed with 3 ml DPPH solution, respectively. The final concentration
of DPPH solution was 100 μM. The reaction mixture was shaken, and incubated in
the dark. The absorbance of the solution was measured against a blank at 517 nm
after 30 min. Percentage inhibition of DPPH was calculated using the following
equation:
%Inhibition = [(A0 − A1 ) /A0 ] × 100, (1)

where A0 is the absorbance of the blank sample and A1 the absorbance of the tested
sample. Gallic acid was used as the standard antioxidant.

Luminol-Amplified Chemiluminescence Assay

Luminol-amplified chemiluminescence assay was conducted on human whole
blood from a healthy volunteer as described by Helfand, Werkmeister & Roder
(1982). Briefly, whole blood (1:20) suspended in modified Hank’s solution (MHS)
was incubated for 30 min with serial concentrations of the respective extracts. All
the dilutions were made using phosphate buffer saline. MHS with cells and no ex-
tract was run as control (+C). Then Zymosan (1 mg/ml) (Sigma-Aldrich,Buchs,
Switzerland) was added, followed by 25 μl (10−5 M) of luminol (G-9382 Sigma-
Aldrich). Total chemiluminescence (CL) was recorded with a luminometer (Lab
system Luminoskan RS, Helsinki, Finland). The luminometer was set to measure
the resulting light emission in 96-well plate for a period of 50 min in repeated
 4 Erukainure et al.

scan mode with 50 scans, 30 s interval, and one second point measuring time. This
method was adopted due to its sensitivity, reproducibility, and availability.

Phytotoxicity Assay
The Lemna minor phytotoxicity assay was adopted for this study as described by
Atta-ur-Rehman (1991). Briefly, Lemna minor was harvested from a local pond in
Karachi, Pakistan. The pond water was collected, filtered, and autoclaved and used
as growth media. Three dilutions of 5,000 μg/ml, 500 μg/ml, and 50 μg/ml were pre-
pared from a stock solution of the sample extracts (100 mg/ml) in DMSO. A 4 ml
quantity from each dilution was added to a 50 ml beaker and allowed to evaporate
till dryness. Fifteen milliliters of growth media were added into each beaker. The
pH was adjusted to 5.5 and 10 lemna minor were added. The volume was made
to 20 ml with the growth media so that it contained 1,000, 100 and 10 μg/ml of
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the sample extracts. Glyphosate (50 μg/ml) was used as the positive control, while
growth media served as negative control. All the beakers were covered with alu-
minum foil to minimize evaporation. The plants were observed daily for 7 days. The
lemna fronds were counted in all beakers and mortality calculated with the formula
below:
100 − number of f r onds in test samples
%Mor talit y = × 100. (2)
Number of f r onds in negative contr ol

Statistical Analysis
To address the biological variability, each set of experiments was repeated at least
three times (n = 3). Differences between the groups were analyzed by one-way
analysis of variance (ANOVA) with the aid of SPSS software (SPSS Inc., Chicago,
IL, USA) standard version 17. The p values of <.05 were considered statistically
significant for differences in mean using the least of significance difference, and
data were reported as mean + standard deviation.

RESULTS
GCMS characterization of the extracts revealed the presence of fatty acid methyl
esters as the most predominant compounds in the hexane extract of the sample
as depicted in Figure 1(a) and Table 1. While the DCM extract was observed to
contain more ketones as shown in Figure 1(b) and Table 2.
Free radical scavenging assay of the extracts against DPPH revealed a rather
lower significant (p > .05) activity compared to the standard as shown in Figure 2.
However, the DCM extract was observed to display significantly (p < .05) higher
activity than the hexane extract.
As seen in Figure 3, at 100 μg/ml DCM fraction showed significant (p < .05) po-
tency in the suppression of reactive oxygen species (ROS) production from whole
blood than hexane fraction. None of the concentration of the extracts was able to
cause 50% inhibition.
As depicted in Figure 4, both extracts showed significant (p < .05) phytotoxic
activity at the highest concentration, 1,000 μg/ml. Little or no activity was observed
at lower concentrations.
 Orange Peel Extracts: Chemical Characterization 5
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FIGURE 1. (a) GCMS chromatogram of hexane extract of orange peel. (b) GCMS chro-
matogram of dichloromenthane (DCM) extract of orange peel.
 6 Erukainure et al.

TABLE 1. Identified compounds in n – hexane extract of orange peel

S/N Compounds Peak No. Similarity Index (%) Proportion (%)

1. 2,6,9,11 – Dodecatetraenal, 694 68 10.06

2,6,10-trimethyl-
2. Hexadecanoic acid, 779 82 12.01
15-methyl-,methyl ester
3. á-D-Glucopyranose,4-O-á-D- 806 90 12.03
galactopyranosyl-
4. Bicyclo[2.2.1] heptane, 2- 1109 78 16.23
ethylidene-1,7,7-trimethyl-,(Z)-
5. Tetradecanoic acid, 12 – methyl-, 1434 76 21.04
methyl ester, (S)-
6. 1,2,3,4,5 –Cyclopentanepentol 1731 80 25.34
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DISCUSSION
From time immemorial, plants have been the basis for medical treatments and still
widely practiced to date. The search for novel drugs and alternative medicine has
led to increased research in medicinal plants. Among such plants is the orange fruit.
Its peels have been utilized for a long time as an active ingredient in most traditional
medicines. In this study, the hexane and DCM extracts of orange peel were charac-
terized using GCMS to identify the chemical constituents. Its biological activities,
which cover free radical scavenging, antioxidative burst, and phytotoxicity, were
also investigated.
Hexane and DCM are well known organic solvents used for extracting nonpo-
lar natural compounds from plant materials. In this study, orange peel was first
extracted with hexane, and followed by DCM. The identified compounds in the
hexane extract were mainly methyl esters of fatty acids (Table 1). Several studies
have documented the use of hexane in oil extraction (Topallar & Gecgel, 2000),
and this explains the presence of the observed methyl esters. The observed fewer
methyl esters in the DCM fraction can be attributed to the initial extraction with

TABLE 2. Identified compounds in n-dicholoromethane extract of orange peel

S/N Compounds Peak No. Similarity Index (%) Proportion (%)

1. 4 – Acetoxy – 3 – 336 98 4.02

methoxystyrene
2. 3 – Methyl – hepta – 1,6 – dien 469 78 6.21
– 3 – ol
3. Hexadecanoic acid, methyl 699 80 9.92
ester
4. Tridecanoic acid 724 94 10.03
5. (R) – (-) – 14 – Methyl – 8 – 783 89 10.05
hexadecyn – 1 – ol
6. 1,15 – Pentadecanediol 820 74 11.01
7. 1 – Pentadecyne 981 85 14.23
8. Phthalic acid, 4 – cyanophenyl 1129 70 15.67
octyl ester
9. Cyclohexene, 4 – (4 – 1338 90 17.34
ethylcyclohexyl) – 1 – pentyl –
 Orange Peel Extracts: Chemical Characterization 7

FIGURE 2 . DPPH scavenging activities of extracts of orange peel. Data = mean ± SD;
n = 3. ∗ Statically significant (p < .05).
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hexane, thereby leading to a high concentration of ketone metabolites in the frac-

tion (DCM) (Table 2). Most of these metabolites are insoluble in hexane.
DPPH assay has been widely applied in several studies to evaluate antioxidant
activities (Brad-Williams, Cuvelier & Berset, 1995). The extracts were observed
to exhibit a rather low scavenging activity below 50% radical scavenging activity
(Figure 2), contradicting earlier reports by Hegazy & Ibrahium (2012), which stated
a variable antioxidant activity for different fractions of orange peel against DPPH.
Hexane extract exhibited the lowest; this can be attributed to the observed pres-
ence of fatty acid methyl esters, which generates free radicals on oxidation (lipid
peroxidation).
The free radical scavenging abilities of the extracts were further affirmed in an-
tioxidative burst assay in whole blood (Figure 3). The hexane extract also exhibited
little or no activity. However, the DCM fraction exhibited a rather moderate activ-
ity. This activity demonstrates the potential of the extract to inhibit reactive oxygen
species and scavenge free radicals: superoxide, hydrogen peroxide, etc. (Okokon,

FIGURE 3 . Luminol-amplified chemiluminescence assay results from whole blood using

orange peel extracts. Data = mean ± SD; n = 3. ∗ Statically significant (p < .05).
 8 Erukainure et al.

FIGURE 4. Phytotoxic activity of orange peel extracts. Data = mean ± SD; n = 3.

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Nwafor, Charles, Dar & Choudhary, 2013). This activity may be attributed to the
presence of ketone metabolites such as 4 – Acetoxy – 3 – methoxystyrene, 3 –
Methyl – hepta – 1,6 – dien – 3 – ol, (R) – (-) – 14 – Methyl – 8 – hexadecyn –
1 – ol, Cyclohexene, 4 – (4 – ethylcyclohexyl) – 1 – pentyl – , etc., as revealed by
GC-MS analysis. The presence of these compounds has been reported in most es-
sential oils with documented antioxidant activities (Cai et al., 2012).
Crop weeds have been described as the main problem in agriculture (FAO,
2009). The use of synthetic herbicides is the principal method to control it (Hong,
Xuan, Eiji & Khanh, 2004). Although these products have brought unquestionable
gains to agriculture, they often contaminate the environment, which is a threat to
human and livestock health (Silva, Novato-Silva, Faria & Pinheiro, 2005). There-
fore, there is a need for an alternative natural herbicide to replace the synthetics
which must be environmentally friendly. In this present study, orange peel extracts
inhibited the growth of Lemna minor indicating a phytotoxic potential at the high-
est concentration (1,000 μg/ml). Ribeiro & Lima (2012) also reported phytotoxic
effects of orange peel. This is of great benefit as orange peel is readily available and
often discarded as waste. Therefore, its use as a natural herbicide will not only aid
in curbing weeds but also reduce environmental pollution. The observed activity
of the extracts can also be attributed to the secondary metabolites revealed by the
GCMS.

CONCLUSION
Results from this study indicate that solvent type plays a vital role in the extraction
of secondary metabolites that are responsible for biological activities. The higher
activities by the dichloromenthane extract can be attributed to its constituents as
revealed by GCMS analysis. There is great need to explore the phytotoxicity po-
tentials of both extracts as natural herbicides.

Declaration of Interest: The authors report no conflict of interest. The authors

alone are responsible for the content and writing of this paper.
 Orange Peel Extracts: Chemical Characterization 9

ABOUT THE AUTHORS

Ochuko L. Erukainure, MSc, Nutritional Biochemistry, Federal Institute of Indus-
trial Research, Oshodi, Lagos, Nigeria. Osaretin A.T. Ebuehi, PhD, Biochemistry,
University of Lagos, Lagos, Nigeria. M. Iqbal Chaudhary, PhD, Organic Chemistry,
International Center for Chemical and Biological Sciences, University of Karachi,
Karachi, Pakistan. M. Ahmed Mesaik, PhD, Microbiology, Tabouk Medical Col-
lege, University of Tabuk, King Fahad Road, Tabuk 71491, Saudi Arabia. Ahmed
Shukralla, MSc, International Center for Chemical and Biological Sciences, Uni-
versity of Karachi, Karachi, Pakistan. Aliyu Muhammad, PhD, Biochemistry, Ah-
madu Bello University, Zaria, Nigeria. Moses Z. Zaruwa, PhD, Pharmacology,
Adamawa State University, Mubi, Nigeria. Gloria N. Elemo, PhD, Human Nu-
trition, Federal Institute of Industrial Research, Oshodi, Lagos, Nigeria.
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FUNDING
One of the authors, Ochuko L. Erukainure, is thankful to the Third World
Academy of Science (TWAS) for financial support/ICCBS-TWAS fellowship at
the H.E.J. Research Institute of Chemistry, ICCBS, University of Karachi, Karachi,
Pakistan.

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