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Copyright@ Ichrak Ben Amor | Biomed J Sci & Tech Res | BJSTR. MS.ID.006434. 32121
Research Article
ISSN: 2574 -1241
Antioxidant Capacity and Antitumoral Activity of
Citrus Paradisi Essential Oil
Bochra Gargouri1, Ichrak Ben Amor1*, Ezzedine Ben Messaoud2, Asma Elaguel2, Ahmed
Bayoudh3, Imen Kallel2 and Hammadi Attia1
1Unité de Biotechnologie et Pathologies (LR11ES45), Institut Supérieur de Biotechnologie de Sfax, 3029, Université de Sfax, Sfax,
Tunisia
2Laboratoire de recherche Toxicologie- Microbiologie Environnementale et Santé, Faculté des Sciences de Sfax, Université de Sfax, Sfax,
Tunisia
3Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d’Ingénieurs de Sfax, Université de Sfax, Tunisia
*Corresponding author: Ichrak Ben Amor, Unité de Biotechnologie et Pathologies (LR11ES45), Institut Supérieur de Biotechnologie
de Sfax, 3029, Université de Sfax, Sfax, Tunisia
DOI: 10.26717/BJSTR.2021.40.006434
Introduction
Phytomedicine has been of increasing interest in recent years.
Over 65 % of the world’s population relies on traditional and medical
approaches to treating diseases [1]. The return to phytotherapy
is more than using an herb to treat an illness or a crude herbal
considered essential resources for researchers to prove and develop
new drugs. Generally, using plants in the treatment of disease has
a long history [2,3]. Thus plants have been primary resources
for producing traditional drugs effective in treating cancer and
oils have bioactivity against a host of bacteria, fungi, viruses
[5,6]. Currently, essential oils of medicinal plants are increasingly
ARTICLE INFO ABSTRACT
Received: November 24, 2021
Published: December 01, 2021
Citation: Bochra Gargouri, Ichrak
Ben Amor, Ezzedine Ben Messaoud,
Asma Elaguel, Ahmed Bayoudh, et al.,
Antioxidant Capacity and Antitumoral
Activity of Citrus Paradisi Essential Oil.
Biomed J Sci & Tech Res 40(2)-2021.
BJSTR. MS.ID.006434.
Abbreviations: CPEo: Citrus Paradisi
Essential Oil; DPPH: Diphenyl Picryl Hy-
drazine; ABTS: Azino-Bis-3-Ethylbenzo-
thiazoline-6-Sulfonic Acid; FRAP: Fer-
ric-Reducing Antioxidant Power; TCA:
Trichloroacetic Acid; FCS: Foetal Calf Se-
rum; SD: Standard Deviation
Background: The present study was focused on the study of essential oil extraction
from the zest of grapefruit (Citrus paradisi) and the determination of antioxidant and
antitumor activities.
Methods: Grapefruit essential oil was extracted by hydro distillation, grapefruit
essential oil was analyzed for antioxidant potential in the chemical system by the
DPPH, ABTS, FRAP test, and scavenging activity of nitric oxide. The cytotoxicity study
of grapefruit essential oil was assessed using MTT assay against HeLa and MCF-7 tumor
cells.
Results: The extraction yield obtained after optimization was 1.32 %. Antioxidant
activities were studied using chemical systems. The results have been very interesting
since we got 54 %, 83 %, 66 % respectively with DPPH, ABTS, FRAP, and a notable
essential oil was observed in HeLa and MCF-7 tumor cells.
Conclusion: The data serve as the basis for the consumption of Citrus paradisi
cells, and with antitumor potential.
Keywords: Citrus Paradisi; Essential Oil; Antioxidant Activity; Antitumor Activity;
MTT; HeLa; MCF-7
Copyright@ Ichrak Ben Amor | Biomed J Sci & Tech Res | BJSTR. MS.ID.006434.
Volume 40- Issue 2 DOI: 10.26717/BJSTR.2021.40.006434
32122
antiviral, antifungal, antioxidant, and antitumor potential [7].
Essential oils are also used in other areas of economic interest,
such as cosmetics, perfumes, and aromatherapy [7,8]. Fragrant and
aromatic plants such as members of the Asteraceae, Lamiaceae,
Rutaceae, and Verbenaceae produce essential oils, which historically
have been important in traditional medicines [5]. Citrus species
vascular protectant, antispasmodic, analgesic, antipyretic, anti-
Citrus peel essential oils have also been searched for their natural
antioxidant and antimicrobial properties [12,13]. Furthermore,
studies have shown the synergistic effect of two or more ingredients
of essential oils against various human pathogens. It is necessary to
know more about Citrus paradisi essential oil (CPEo) antioxidant,
cytotoxic, and antitumor activity. Therefore, the DPPH, ABTS, FRAP,
and NO• techniques studied the antioxidant potential in a chemical
system. Furthermore, to check the antitumor activity, MTT was
realized using HeLa and MCF-7 tumor cells.
Material and Methods
Samples
In this study, the grapefruit (citrus paradisi) was collected in
March 2018 from Sfax, Tunisia. Sfax is one of the biggest coastal
cities in Tunisia. It is located in the eastern part of the country. The
climate in this area is arid to semiarid with irregular and torrential
precipitations [14]. After the collection, it was washed, peeled off
and cut into small pieces.
Hydrodistillation
The zest of citrus paradisi was hydro distilled using a Clevenger-
type apparatus to recuperate the essential oils for 2h to produce
the volatile constituents. The distilled essential oils were dried over
anhydrous sodium sulfate; then separated from the distillate by
liquid-liquid extraction using cyclohexane solvent. The recuperated
oils were stored at + 4 °C.
Antioxidant Activity: Chemical System
DPPH Radical Scavenging Assay: Different aliquots from
the stock solution (200µl in 1 ml Me OH) of essential oils were
mixed with 500µ
volume brought to 1mL. The mixtures were vigorously shaken
and allowed to stand in the dark for 30min at room temperature.
The absorbance was measured by spectrophotometry (LKB
BIOCHROM® ULTROSPECPUS 4054 UV/VIS) at 517 nm against a
control sample without DPPH. The percentage of radical scavenging
activity was calculated using the following equation:
DPPH scavenging effect (%) = (A0- A1)/A0 × 100
A0: The absorbance of the control at 30min
A1: The absorbance of the sample at 30min.
ABTS+ Radical Scavenging Effect: The antiradical activity
was performed by the ABTS+ free radical decolorization
assay as developed by Re, et al. [15]. The 2,2-azino-bis-3-
ethylbenzothiazoline-6-sulfonic acid (ABTS) was prepared as an
aqueous stock solution (7mM). The ABTS radical cations (ABTS+)
were produced by the reaction of the ABTS stock solution with
2.5mM of ammonium persulfate methanolic solution. First,
the reaction mixture is incubated in the dark for 16h at room
temperature. Then, the solution is diluted to an absorbance of 0.7
± 0.02 at 734nm to form the working reagent. Next, the reaction
mixtures containing 100μl of the sample at different concentrations
and 900μL of reagent were incubated at 30 °C for 6min. Finally, the
antioxidant power of each sample was expressed as the inhibition
percentage calculated according to the following formula:
ABTS+ scavenging effect (%) = (A0- A1)/A0 × 100
A0: the absorbance of the control at 6min
A1: the absorbance of the sample at 6min.
Ferric-Reducing Antioxidant Power (FRAP): This method is
based on the plant’s ability to reduce ferric iron (Fe3+) to ferrous iron
(Fe2+). The mechanism is known to be a marker of electron donor
activity [16]. For 1mL of the sample at different concentrations,
2.5mL of a solution phosphate buffer (0.2M, pH 6.6) and 2.5mL of
1 % K3Fe (CN) 6 potassium ferricyanide solution were added. The
mixture is incubated at 50 °C for 20min and then cooled to room
temperature. Then, 2.5mL of 10 % trichloroacetic acid (TCA) is
added to stop the reaction, and then the tubes are centrifuged at
3000rpm for 10min. 2.5ml of the supernatant are then added
to 2.5mL of distilled water and 500μL of a 0.1 % solution of iron
trichloride (FeCl3, 6H2O) [16]. The absorbance reading is performed
against a blank at 700 nm using a spectrophotometer. Ascorbic acid
is used as a positive control. The increase in the absorption capacity
of the components indicates the increase in the reduction of iron.
Scavenging Activity of Nitric Oxide (NO·): NO scavenging
activity of the essential oils was determined as previously described
incubated with 0.5mL of sodium nitrite (0.01mg/mL in 100mM
sodium citrate pH 5) at 37 °C for 2h. After incubation, 0.5mL of
Griess reagent was added, and the absorbance was read at 540nm
using a spectrophotometer (Pharmacia, Uppsala, Sweden). The
equation obtained the percentage of RNS scavenging:
NO· Scavenging effect (%) = (A0- A1)/A0 × 100
A0: The absorbance of the control
A1: The absorbance of the sample.
Copyright@ Ichrak Ben Amor | Biomed J Sci & Tech Res | BJSTR. MS.ID.006434.
Volume 40- Issue 2 DOI: 10.26717/BJSTR.2021.40.006434
32123
Cytotoxicity Activity
Cell Lines and Culture Conditions: In this study, cancerous
cells were used: HeLa and MCF-7. HeLa is a transformed cell line
expressing the HPV18 virus (human papillomavirus) [18]. MCF-7
(Michigan Cancer Foundation-7) was isolated from a 69 years old
woman with metastatic disease [19]. Hela and MCF-7 cell lines
were supplied by ATCC (Manassas, VA, USA). All cells were grown in
RPMI 1640 medium (Gibco) supplemented with 10 % (v/v) foetal
(Nunc). They are incubated at 37 °C, 95 % air and 5 % CO2.
MTT: For the test of cytotoxicity activity, the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
(Sigma-Aldrich, St. Louis, MO, USA) test was done [20]. First,
HeLa or MCF-7 cells in 96-well plates were exposed to different
concentrations of CPEo (3 wells for each concentration) and
incubated for 48h at 37 °C. Then, 20μl of MTT was added to each
well. After 4h of incubation at 37 °C, the supernatant was removed,
180µL of DMSO/Methanol (1V/1V) were added to each well to
solubilize the formazan crystals. Finally, the plates were shaken
15min at room temperature, and the absorbance was detected at
570nm with a spectrophotometric plate reader.
Statistical Analysis
The statistical studies are carried out using the program SPSS
(19.0). The t-Student test carried out the comparison between the
averages. The results are represented as mean ± standard deviation
(SD).
Results
The Yield of Grapefruit Essential Oils
mass of essential oil obtained and the mass of the plant material.
Y (%) = A/A1 ×100
A: Quantity of extracts recovered in g.
A1: Quantity of dry vegetable matter used for extraction
expressed in g.
CPEo yield was 1.32g Eo / 100g dry matter. This result proves
that the zest of the fruit was rich in Eo.
Antioxidant Effects
DPPH Radical Scavenging Assay: The curve relating to DPPH
radical scavenging shows that the inhibition percentage of the free
radical increases with the increase of concentration, either for
BHT or CPEo (Figure 1). In effect, Citrus paradisi essential oil has
such having an important antioxidant activity.
Figure 1: Antiradical activity against the radical DPPH in the percentage of inhibition CPEo. Aliquots of various concentrations
of CPEo (0, 0.2, 0.4, 0.6, 0.8, 1mg/mL) and standard BHT were mixed with DPPH and incubated in the dark. The antioxidant
activity of CPEo was measured using a spectrophotometer at 517nm. Results were expressed as mean inhibition percentage (%)
± standard deviations (n = 3). BHT was used as the reference standard.
Copyright@ Ichrak Ben Amor | Biomed J Sci & Tech Res | BJSTR. MS.ID.006434.
Volume 40- Issue 2 DOI: 10.26717/BJSTR.2021.40.006434
32124
ABTS+ Radical Scavenging Effect: The results illustrated in
the curve show that the percentage of inhibition gradually increases
with the concentration of grapefruit essential oil. At a concentration
of 1mg/mL, the inhibition rate reaches 83 % (Figure 2).
Figure 2: Anti-radical activity against the radical ABTS in percentage of inhibition of CPEo. Various concentrations of CPEo
(0 to 1mg/mL) and acid ascorbic were mixed with FRAP reagents. The reduction of ferric ion (Fe3+) to ferrous form (Fe2+) by
CPEo produces an intense blue light revealed as a change in absorption at 700nm. Results were expressed as mean inhibition
percentage (%) ± standard deviations (n = 3). Ascorbic acid at various concentrations (0 to 1mg/mL) was used as standard.
Ferric-Reducing Antioxidant Power (FRAP): The FRAP assay
is usually used to measure the capacity of the sample to reduce the
antioxidant activity of CPEo, with values equal to 66 % (Figure 3).
Figure 3: Inhibition (%) of standard Vit C and CPEo by Ferric Reducing Antioxidant Power (FRAP) assay. Various concentrations
of CPEo (0 to 1mg/mL) and acid ascorbic were mixed with FRAP reagents. The reduction of ferric ion (Fe3+) to ferrous form
(Fe2+) by CPEo produces an intense blue light revealed as a change in absorption at 700nm. Results were expressed as mean
inhibition percentage (%) ± standard deviations (n = 3). Ascorbic acid at various concentrations (0 to 1mg/mL) was used as
standard.
NO· Scavenging Assay
The capacity of citrus paradisi essential oils to scavenge NO
was also measured. CPEo was checked for its inhibitory effect on
nitric oxide production. The inhibition on free radical percentage
increases with elevated concentrations of essential oil and Vitamin
C (Figure 4).
Copyright@ Ichrak Ben Amor | Biomed J Sci & Tech Res | BJSTR. MS.ID.006434.
Volume 40- Issue 2 DOI: 10.26717/BJSTR.2021.40.006434
32125
Figure 4: Inhibition (%) of standard Vit C and CPEo by Ferric Reducing Antioxidant Power (FRAP) assay. Various concentrations
of CPEo (0 to 1mg/mL) and acid ascorbic were mixed with FRAP reagents. The reduction of ferric ion (Fe3+) to ferrous form
(Fe2+) by CPEo produces an intense blue light revealed as a change in absorption at 700nm. Results were expressed as mean
inhibition percentage (%) ± standard deviations (n = 3). Ascorbic acid at various concentrations (0 to 1mg/mL) was used as
standard.
Cytotoxicity Activity
The MTT test studied the cytotoxic effect on HeLa and MCF-7
cells. Cells were cultured in 96-well plates for 48 h, in the presence
and absence of the different concentrations of CPEo. The percentage
of cytotoxicity was calculated. Our results showed an antitumor
potential of the grapefruit essential oils on both cancer cells: Hela
and MCF-7 according to the concentrations used: from 0.78 to 25μg
/ mL (Figure 5).
Figure 5:
(a) Study the cytotoxicity of the essential oil of the grapefruit outer wall in the HeLa
(b) and MCF-7 cell lines.
Cytotoxic activity of citrus paradisi essential oil on cancer cell lines. MTT assessed cell viability. The percent growth reduction
was calculated from the extinction difference between treated cell culture and the control. Results are the means of three
repetitions.
Discussion
The current study was designed to extract the essential oil
from grapefruit. It was obtained by hydro distillation, which is the
oldest method and the most used because it is very easy to achieve.
in essential oil, and their yields varied from one plant species to
Copyright@ Ichrak Ben Amor | Biomed J Sci & Tech Res | BJSTR. MS.ID.006434.
Volume 40- Issue 2 DOI: 10.26717/BJSTR.2021.40.006434
32126
another, ranging from 0.2 to 2.0 % [21]. Other work has shown
that yield of Citrus paradisi essential oil collected in September in
southern Taiwan was attending 0.37 %, and others were collected
in October from Iran were 0.85 % [22,23]. So the difference in
EOs yields may be related to the plant’s origin, the environmental
conditions, time of collection, and extraction method [24]. The CPEo
analyzed in this work showed potent radical scavenging activity.
We have demonstrated that CPEo has an interesting antioxidant
activity highlighted by the DPPH, ABTS+, FRAP, and NO. Our results
agree with the literature, which showed that citrus EO possesses an
anti-free radical activity DPPH of 17.7 to 64 % [7]. Other studies on
grapefruit seed oil showed a high antioxidant potential (61 %) [25].
Furthermore, the effect of citrus paradisi essential oils on ABTS free
radical was determined and exhibited a higher radical scavenging
activity. Our results corroborate with other studies showing a
higher FRAP values in the peel of grapefruit [26]. The superoxide
anion assay is commonly used to evaluate the superoxide anion
radical-scavenging ability of plant extract. Therefore, CPEo was
checked for their inhibitory effect on nitric oxide production in
the present study, attending 47 %. Our results agree with studies
from 36.83 % to 50.31 % in peels [27]. The antitumor potential
of CPEo was tested on HeLa and MCF-7 tumor cells. Our results
showed a considerable antitumor activity dependent on essential
oil concentration. This result agrees with the work of Zu et al.,
which shows that essential oil from grapefruit exhibited an anti-
proliferative effect on MCF-7 cells [28]. Furthermore, Monajemi et
al. studied the impact of different concentrations of essential oils of
other species of the citrus family as Citrus limon, Citrus medica, and
decrease in viability in a dose-dependent manner in both tumor
cells [29].
Conclusion
and fragrance properties and numerous aromatherapeutic and
medicinal applications. Accordingly, the essential oils of Citrus
species exhibited intense antioxidant activity. In addition, the
essential oil of Citrus paradisi showed an antitumor effect against
both cancers cells, HeLa and MCF-7.
Acknowledgment
This work is dedicated to the memory of Pr. Saloua Lassoued
Elamri. We will never forget her, and she is always in our hearts.
Conict of Interest
The authors declare that they have no competing interests.
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ISSN: 2574-1241
DOI: 10.26717/BJSTR.2021.40.006434
Ichrak Ben Amor. Biomed J Sci & Tech Res
