Antioxidant activity of oils extracted from orange (Citrus sinensis) seeds

Abstract

Due to the increasing production of food in the world with consequent increase of the production of waste, the importance of developing researches for its use is noticed. Thus, the interest in vegetable oils with bioactive compounds, such as the ones extracted from fruit seeds, is growing. Therefore, the present study aims to characterize the oils extracted from seeds of Hamlin, Natal, Pera-rio and Valencia orange varieties (Citrus sinensis), as to the levels of total carotenoids, total phenolic compounds, tocopherols and phytosterols, as well as to determine their antioxidant activity. The orange seed oils presented important content of total carotenoids (19.01 mg/kg), total phenolic compounds (4.43 g/kg), α-tocopherol (135.65 mg/kg) and phytosterols (1304.2 mg/kg). The antioxidant activity ranged from 56.0% (Natal) to 70.2% (Pera-rio). According to the results it is possible to conclude that the orange seed oils can be used as specialty oils in diet, since they contain considerable amounts of bioactive compounds and antioxidants.

Full text

An Acad Bras Cienc (2016) 88 (2)
Anais da Academia Brasileira de Ciências (2016) 88(2): 951-958
(Annals of the Brazilian Academy of Sciences)
Printed version ISSN 0001-3765 / Online version ISSN 1678-2690
http://dx.doi.org/10.1590/0001-3765201620140562
www.scielo.br/aabc
Antioxidant activity of oils extracted from orange (Citrus sinensis) seeds
NEUZA JORGE, ANA CAROLINA DA SILVA and CAROLINE P.M. ARANHA
Departamento de Engenharia e Tecnologia em Alimentos, Universidade Estadual de São Paulo, Rua
Cristóvão Colombo, 2265, Jardim Nazareth, 15054-000 São José do Rio Preto, SP, Brasil
Manuscript received on November 25, 2014; accepted for publication on February 24, 2015
ABSTRACT
Due to the increasing production of food in the world with consequent increase of the production of
waste, the importance of developing researches for its use is noticed. Thus, the interest in vegetable oils
with bioactive compounds, such as the ones extracted from fruit seeds, is growing. Therefore, the present
study aims to characterize the oils extracted from seeds of Hamlin, Natal, Pera-rio and Valencia orange
varieties (Citrus sinensis), as to the levels of total carotenoids, total phenolic compounds, tocopherols and
phytosterols, as well as to determine their antioxidant activity. The orange seed oils presented important
content of total carotenoids (19.01 mg/kg), total phenolic compounds (4.43 g/kg), -tocopherol (135.65
mg/kg) and phytosterols (1304.2 mg/kg). The antioxidant activity ranged from 56.0% (Natal) to 70.2%
(Pera-rio). According to the results it is possible to conclude that the orange seed oils can be used as
specialty oils in diet, since they contain considerable amounts of bioactive compounds and antioxidants.
Key words: agro-industrial waste, phytosterols, tocopherols, vegetable oils.
Correspondence to: Neuza Jorge
E-mail: njorge@ibilce.unesp.br
INTRODUCTION
The citrus industry has great potential for growth,
and is one of the most competitive agricultural
sectors. Orange varieties such as Hamlin, Natal,
Pera-rio, and Valencia belong to the species Citrus
sinensis (L.) Osbeck, which is mainly characterized
for its sweet taste. The flowers and fruits of
these oranges are smaller, and have thinner peel
and albedo (Koller 2006). Orange is extensively
processed in order to obtain natural juices, pulps,
candies, and extracts for manufacturing industries.
However, wastes generated by the processing
of orange, represent approximately 50% of the
fruits (Hernández-Montoya et al. 2009). With
the intention of adding value to those wastes and
minimizing environmental impacts, the use of these
sub-products is essential, since they present great
potential for application in food, pharmaceutical,
and cosmetic industries (Schieber et al. 2001).
The waste may contain compounds that are
capable of providing benefi ts to health, by prevent-
ing diseases or even favoring the functioning of
the body, which are called bioactive compounds.
Such substances perform several functions from a
biological point of view, such as antioxidant activity,
modulation of detoxifi cation enzymes, stimulation
of the immune system, reduction of platelet
aggregation, antibacterial and antiviral activities
(Schmidt and Pokorný 2005). The vegetable oils are
considered sources of these compounds, especially

An Acad Bras Cienc (2016) 88 (2)
952 NEUZA JORGE, ANA CAROLINA DA SILVA and CAROLINE P.M. ARANHA
carotenoids, phenolic compounds, tocopherols, and
phytosterols.
The seeds of fruits such as oranges (Citrus
sinensis) are shown to be promising sources of oils,
rich in carotenoids, phenolic compounds, tocoph-
erols, and phytosterols. For this reason, a study to
quantify the presence of these compounds in oils
extracted from the mentioned fruits is important,
in order to use them as specialty oils (Malacrida et
al. 2012).
MATERIALS AND METHODS
PLANT MATERIALS
In the present study, seeds of the orange varieties
Hamlin, Natal, Pera-rio, and Valencia were used,
since they are the most employed types in the pro-
duction of juice in the state of São Paulo, Brazil.
Three batches of waste of each variety were
acquired between June and August, in the harvest
of 2010, from Indústria Suco Cítrico Cutrale -
Uchôa, São Paulo, Brazil. Immediately after receipt
of the waste, the seeds were manually separated
from peels and pulp, and dried for approximately
96 h, on trays, at room temperature for moisture
reduction (< 10%). The batches of each variety of
orange seeds, which weighed around 800-850 g,
homogenized, packed in rigid plastic polypropylene
packaging, sealed with screw caps and properly
labeled for further analysis.
EXTRACTION OF OILS FROM THE SEEDS
The oils were obtained by pressing in vegetable
oil extractor; brand Brazil Metal Wilhelms (Porto
Alegre, Brazil), at room temperature, with initial
rotation of 25 Hz and final rotation of 60 Hz,
approximately. After extraction, the oils were
placed in amber glass bottles with nitrogen gas and
stored at -18 °C for further analysis.
ANTIOXIDANT PROPERTIES
Total carotenoids were determined by spectropho-
tometry, according to the method described by
Rodriguez-Amaya (1999). The quantification
was performed in a UV-vis spectrophotometer
(Shimadzu, Kyoto, Japan), with wavelength
interval from 300 nm to 550 nm. Quantifi cation
was measured by absorption wavelength of
maximum absorbance and absorptivity value of
2592 in petroleum ether, expressed as milligrams
of -carotene per kilogram.
The total phenolic compounds were extracted
from the oil samples according to the procedure
described by Parry et al. (2005) and quantifi ed by
spectrophotometry, using Folin-Ciocalteu reagent
(Singleton and Rossi 1965), through calibration
curve with gallic acid as standard. The levels of
total phenolic compounds in oils were expressed
as grams of gallic acid equivalents (GAE) per
kilogram of oil.
The determination of tocopherols was per-
formed according to the method AOCS Ce 8-89
(2009), by high performance liquid chromatogra-
phy (HPLC), using a Varian model 210-263 (Varian,
Palo Alto, California, USA), with fluorescence
detector. The quantification was carried out by
external standardization and the values were
determined based on peak areas and expressed in
values of each isomer, separately, in milligrams per
kilogram.
The contents of phytosterols extracted from
the seeds were determined by gas chromatography,
with previous saponification of the samples.
Saponification was performed according to the
method published by Duchateau et al. (2002).
For the determinations of phytosterols, the
method AOCS Ch 6-91 (2009) was used, with
adaptations. The analyses were carried out in a gas
chromatograph, model CG 2010-Plus (Shimadzu,
Tokyo, Japan), equipped with a fl ame-ionization
detector, split-splitless injector, and an autosampler.
The compounds were separated in a fused silica
capillary column (Restek RTX 5, Shimadzu), 30 m
length, 0.25 mm internal diameter, and 0.25 m fi lm
thickness. The programming of the temperature

An Acad Bras Cienc (2016) 88 (2)
ORANGE SEEDS OIL 953
column was initiated at 100 °C, for 2 min, heated at
15 °C/min until 260 °C, and kept isothermal for 35
min. The temperatures used in the injector and in
the detector were 280 and 320 °C, respectively. The
carrier gas was hydrogen with linear velocity of 40
ml/min. Samples of 1.0 L were injected with split
ratio of 1:50. Sterols (cholesterol, campesterol,
stigmasterol, -sitosterol, and stigmastanol) were
identifi ed by comparison with the retention time of
pure standards (Supelco, Bellefonte, Pennsylvania,
USA), analyzed under the same conditions of
the samples. The quantification of each isomer
was performed by internal standardization
(5-cholestano-3β-ol), based on the peak areas.
The antioxidant activity of the oils was
determined according to the method described by
Kalantzakis et al. (2006). This method consists
in evaluating the scavenging activity of the free
radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•).
To determine the antioxidant activity of the oils,
1 g of oil was diluted with 10 ml of ethyl acetate.
From this solution, 1 ml was added to 4 ml of a
DPPH• solution, in 10-4 mol/l ethyl acetate, and
was vigorously shaken in vortex, for 10 sec. After
30 min in the dark, the mixture absorbance was
measured at 517 nm. A control sample (without
oil) was prepared and the absorbance was equally
measured. The levels of absorbance obtained were
converted to percentage of antioxidant activity
(AA).
The effi cient concentration (EC), defi ned as
the suffi cient concentration to obtain 50% of the
maximum effect estimated in 100% (expressed
in kilograms of oil per kilogram of DPPH•), was
graphically determined. To do so, oil samples
were diluted with ethyl acetate in concentrations
of 10, 25, 50, 75, and 100 mg/ml. Measurements
of the absorbance of reaction mixtures (1 ml of
solution sample and 4 ml of DPPH• in ethyl acetate)
were performed at 517 nm at 0 and 30 min. The
antiradical effi ciency (AE) of oils was determined
according to Equation 1 (Brand-Williams et al.
1995).
AE = 1/EC50 (1)
STATISTICAL ANALYSES
The results obtained from analytical determinations,
in triplicate, were submitted to analysis of variance,
and differences between means were tested at 5%
probability, by Tukey test (Gacula Jr et al. 2009),
using ESTAT program, version 2.0.
RESULTS AND DISCUSSION
The levels of total carotenoids, total phenolic
compounds, and tocopherols in the oils of Hamlin,
Natal, Pera-rio, and Valencia seeds are shown in
Table I.
The oil of Hamlin orange seeds presented the
lowest amount of total carotenoids (11.64 mg/kg,
TABLE I
Total carotenoids, total phenolic compounds and α-tocopherol of oils extracted from
orange seeds.
Varieties TC (mg/kg) TPC (g/kg) α-tocopherol (mg/kg)
Hamlin 11.64 ± 0.54c3.79 ± 0.03d 135.50 ± 0.29b
Natal 18.47 ± 0.78b4.80 ± 0.01b134.07 ± 0.21c
Pera-rio 26.69 ± 0.27a 4.91 ± 0.01a137.43 ± 0.16a
Valencia 19.24 ± 0.93b4.21 ± 0.04c135.63 ± 0.49b
The results represent the mean ± standard deviation of the analyses performed in triplicate.
Means followed by the same letter in the lines do not differ by Tukey test (p < 0.05).
TC - total carotenoids are expressed as -carotene, TPC - total phenolic compounds are
expressed as GAE equivalents.

An Acad Bras Cienc (2016) 88 (2)
954 NEUZA JORGE, ANA CAROLINA DA SILVA and CAROLINE P.M. ARANHA
expressed as -carotene) and the oil of Pera-rio
orange presented the highest amount, 26.69 mg/
kg. In Table I, it can be observed that the oils of
Natal and Valencia orange seeds did not differ
signifi cantly by Tukey test (p > 0.05), with values
of 18.47 mg/kg and 19.24 milligrams of -carotene
per kilogram, respectively.
Malacrida et al. (2012) evaluated Pera-rio
orange seed oils, and found levels of 0.13 mg/
kg and 0.19 mg/kg of lutein and -carotene,
respectively. The corn germ oil presented 5 mg/
kg of total carotenoids (Moreau et al. 2007). The
above mentioned values are lower than the ones
presented by orange seed oils in the present study.
Xu et al. (2008) found total carotenoids
concentration of 0.08, 2.92, and 0.72 mg/ml
(expressed as -carotene) in lemon, ponkan, and
Hamlin orange juices, respectively.
The content of total carotenoids in oils is
affected by the maturation stage of the fruits and
by their extraction and storage conditions. Oils
extracted from ripe fruits may present higher
amounts of carotenoid pigments, while those
obtained from partially ripe fruits have higher
chlorophyll concentration (Ramadan and Moserl
2003).
All oils showed important levels of total
phenolic compounds: the highest content is present
in the oils of Pera-rio orange seeds, 4.91 g/kg
(expressed as GAE), followed by Natal, Valencia,
and Hamlin, 4.80, 4.21, and 3.79 g/kg, respectively.
Malacrida et al. (2012) studied the phyto-
chemicals and antioxidant activity of citrus seed
oils. The level of total phenolic compounds of Pera-
rio orange seed oil was determined by using the
Folin-Ciocalteu reagent under the same analytical
conditions, and the results were expressed as gallic
acid equivalents per kilogram of oil. According
to the results, this oil obtained 1.15 g/kg of total
phenolic compounds. The levels of phenolic
compounds in the orange seed oils from this study
were higher than those found in the oils analyzed in
the mentioned study.
When analyzing lemon, ponkan, and Hamlin
orange juices, Xu et al. (2008) found concentrations
of total phenolic compounds of 751.82, 830.32,
and 1499.71 g/kg, respectively, using the Folin-
Ciocalteu reagent.
In soybean, sunfl ower, corn, canola, and rice
oils, extracted by cold extraction, the quantities of
total phenolic compounds were found from 126 g/
kg to 148 g/kg, in caffeic acid equivalents (Siger et
al. 2008) and from 0.16 mg/kg to 0.40 g/kg in olive
oil, in gallic acid equivalents (Nakbi et al. 2010).
The quantifi cation of these substances is infl u-
enced by the nature of the compound, the method of
extraction employed, as well as by the presence of
interfering elements, such as waxes, terpenes, and
chlorophyll. A satisfactory method for extraction
of phenolics present in the samples has not been
developed yet.
According to Table I, only -tocopherol was
detected in the oils extracted from seeds of all
orange varieties. Due to the fact that -tocopherol
presents higher biological activity as vitamin E, the
oils analyzed may present vitamin activity.
Anwar et al. (2008) determined the compo-
sition of tocopherols in oils extracted from citrus
species seeds (C. paradisi, C. sinensis, C. reticu-
lata) and also obtained -tocopherol as the main
tocopherol, 380, 220, and 557.82 mg/kg, respec-
tively.
The orange seed oils presented higher amounts
of total tocopherols than babaçu oil (60-130 mg/
kg), similarly to that of palm stearin (100-700 mg/
kg), and lower amounts regarding soybean oil
(600-3370 mg/kg) (Codex Alimentarius Commis-
sion 2009).
The phytosterols are of great interest due
to their antioxidant activity and their impact
on health. They are the main components of the
unsaponifiable matter in oils. The analysis of
sterols provides information about the quality of
the oil. Phytosterols are some of the constituents
of the cell wall in vegetables. When ingested,

An Acad Bras Cienc (2016) 88 (2)
ORANGE SEEDS OIL 955
they reduce the absorption of cholesterol by the
intestine, due to their similarity with the cholesterol
molecule. In the last decades, purifi ed phytosterols
and phytostanols have been added to several foods
for the obtainment of functional foods that perform
hypocholesterolemic activity after ingested. The
daily intake of 1.6-2.0 g/day of phytosterols and
phytostanols, incorporated into these foods, is
capable of reducing cholesterol absorption by the
intestine in up to 30%, in addition to lowering
the level of plasma LDL-cholesterol in 8-10%
(Marangoni and Poli 2010). The results on the
amount of phytosterols of orange seed oils are
displayed in Table II.
TABLE II
Amount of phytosterols of oils extracted from orange seeds.
Phytosterols (mg/kg) Varieties
Hamlin Natal Pera-rio Valencia
Cholesterol 23.0 ± 0.0c36.9 ± 0.0a15.1 ± 0.0d25.7 ± 0.0b
Campesterol 60.2 ± 0.0d81.7 ± 0.0a76.5 ± 0.0b77.4 ± 0.0c
-sitosterol 1205.3 ± 0.0b1215.4 ± 0.0a1203.1 ± 0.0c1196.5 ± 0.0d
Totals 1288.5 1334.0 1294.7 1299.6
The results represent the mean ± standard deviation of the analyses performed in triplicate.
Means followed by the same letter in the lines do not differ by Tukey test (p < 0.05).
Only cholesterol, campesterol, and -sitosterol
were detected in the samples analyzed.
The oil of Natal orange seeds presented higher
amounts of the three phytosterol isomers: 1215.4,
81.7, and 36.9 mg/kg of -sitosterol, campesterol,
and cholesterol, respectively. This oil was the one
that presented higher level of total phytosterols
(1334.0 mg/kg), followed by Valencia (1299.6 mg/
kg), Pera-rio (1294.7 mg/kg), and Hamlin (1288.5
mg/kg) seed oils (Fig. 1).
When studying bitter melon, kalahari melon,
kenaf, pumpkins, and roselle oils, Nyam et al.
(2009) found levels of total phytosterols of 4643,
6416.90, 3675.60, 2740, and 7575.60 mg/kg,
respectively. Nehdi et al. (2010), when studying
Phoenix canariensis seed oils, reported 3360.70
mg/kg of total phytosterols. In comparison to the
percentage of total phytosterols in palm oil (270-
800 mg/kg), the orange seed oils contain lower
amounts of these compounds (Codex Alimentarius
Commission 2009).
Arena et al. (2007) obtained 100.40 mg/
kg of total phytosterols for pistachio seed oils.
Cheikh-Rouhou et al. (2008) found levels of total
phytosterols in Nigella sativa and Pinus halepensis
seed oils of 281 mg/kg and 735 mg/kg. Such values
are lower than those of orange seed oils.
It is important to note that the content of phy-
tosterols detected in samples may vary depending
on the chromatographic conditions employed, such
as the programming of the temperature column, the
temperatures used in the injector and in the detec-
tor, the carrier gas speed, among other.
The antioxidant activity (%), the value of
necessary concentration of the oil, in order to reduce
the free radicals (EC50) in 50%, and the antiradical
effi ciency, are presented in Table III.
It can be observed that all the orange seed
oils showed DPPH• radical scavenging activity.
However, the oil of Pera-rio orange seeds was the
most effective, presenting antioxidant activity of
70.2%. The other oils presented antioxidant activity
of 59.9, 58.9, and 56.0%, in Valencia, Natal, and
Hamlin orange varieties, respectively.
The amount of oil necessary to decrease the
initial concentration of DPPH• by 50% (EC50)
ranged from 35.08 kg to 38.31 kg of oil per kilo-
gram of DPPH•.

An Acad Bras Cienc (2016) 88 (2)
956 NEUZA JORGE, ANA CAROLINA DA SILVA and CAROLINE P.M. ARANHA
The oils of Pera-rio orange seeds presented
the highest value for antiradical effi ciency (2.79),
which was determined by using EC50 value. The
other oils studied obtained similar values: 2.61,
2.68, and 2.69, for Valencia, Natal, and Hamlin
varieties, respectively.
Figure 1 - Chromatograms of phytosterolsof oils extracted from orange seeds: Hamlin (a), Natal (b), Pera-rio (c) and Valencia (d).

An Acad Bras Cienc (2016) 88 (2)
ORANGE SEEDS OIL 957
Malacrida et al. (2012), while evaluating the
antioxidant activity of citrus oils, found the highest
antioxidant activity from the oil obtained from
Pera-rio orange seeds (54.2%). The orange seed
oil presented higher DPPH• scavenging activity
among the analyzed oils reaching 47.6% of the
DPPH• in the reaction mixture. The orange oil also
showed the best EC50 (10.75 g oil/g DPPH•) and
antiradical effi ciency (9.30 x 10-2). The levels of
antioxidant activity and EC50 in the orange seed
oils were higher than in the oils analyzed in the
mentioned study.
The antioxidant activity was signifi cantly cor-
related to the level of -tocopherol (r = 0.81), which
indicates that the oils with higher concentrations of
-tocopherol presented higher radical scavenging
activity.
CONCLUSIONS
All oils analyzed in this study presented consider-
able amounts of carotenoids, phenolic compounds,
-tocopherol, and phytosterols, thus they may be
good sources of these compounds. The oils analyzed
showed free radical scavenging capacity. The
antiradical effi ciency of the oils analyzed followed
a decreasing order: Pera-rio > Hamlin = Natal >
Valencia. Such fact may add value to wastes from
orange processing, increasing the viable sources
for obtainment of specialty oils.
ACKNOWLEDGMENTS
To Conselho Nacional de Desenvolvimento
Científico e Tecnológico – CNPq, (process nº
555870/2010-3) for their fi nancial support.
RESUMO
Devido ao aumento da produção mundial de alimentos
com consequente aumento de produção de resíduos,
verifi ca-se a importância do desenvolvimento de
pesquisas para o aproveitamento dos mesmos. Assim, o
interesse em óleos vegetais com compostos bioativos,
como os obtidos a partir de sementes de frutas, está
crescendo. Portanto, o presente estudo teve como
objetivo caracterizar os óleos extraídos a partir de
sementes de laranja (Citrus sinensis), das variedades
Hamlin, Natal, Pera-rio e Valencia, e determinar os
níveis de carotenoides totais, compostos fenólicos totais,
tocoferóis e fi toesteróis bem como determinar as suas
atividades antioxidantes. Os óleos de sementes de laranja
apresentaram conteúdo importante de carotenoides totais
(19,01 mg/kg), compostos fenólicos totais (4,43 g/kg),
-tocoferol (135,65 mg/kg) e fi toesteróis (1304,2 mg/
kg). A atividade antioxidante variou de 50,0% (Natal) a
70,2% (Pera-rio). De acordo com os resultados obtidos,
é possível concluir que os óleos das sementes de laranja
podem ser usados como óleos especiais na dieta, uma
vez que contêm quantidades substanciais de compostos
bioativos e antioxidantes.
Palavras-chave: resíduo agroindustrial, fi tosteróis, toco-
feróis, óleos vegetais.
TABLE III
Antioxidant activity, EC50 and antiradical effi ciency of oils extracted from orange seeds.
Varieties Antioxidant activity (%) EC50
(kg/kg) Antiradical effi ciency
Hamlin 58.9 ± 0.7b37.19 2.69 × 10-2
Natal 56.0 ± 0.5b37.25 2.68 × 10-2
Pera-rio 70.2 ± 0.4a 35.08 2.79 × 10-2
Valencia 59.9 ± 1.0c38.31 2.61 × 10-2
The results represent the mean ± standard deviation of the analyses performed in triplicate.
Means followed by the same letter in the column do not differ by Tukey test (p < 0.05).
EC50 is defi ned as the suffi cient concentration to obtain 50% of the maximum effect estimated in
100% (expressed in kilograms of oil per kilogram of DPPH•).

An Acad Bras Cienc (2016) 88 (2)
958 NEUZA JORGE, ANA CAROLINA DA SILVA and CAROLINE P.M. ARANHA
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