In Vitro Studies on Phytochemical Content, Antioxidant, Anticancer, Immunomodulatory , and Antigenotoxic Activities of Lemon, Grapefruit, and Mandarin Citrus Peels

Abstract

Background:
In recent years, there has been considerable research on recycling of agroindustrial waste for production of bioactive compounds. The food processing industry produces large amounts of citrus peels that may be an inexpensive source of useful agents.

Objective:
The present work aimed to explore the phytochemical content, antioxidant, anticancer, antiproliferation, and antigenotxic activities of lemon, grapefruit, and mandarin peels.

Materials and methods:
Peels were extracted using 98% ethanol and the three crude extracts were assessed for their total polyphenol content (TPC), total flavonoid content (TFC), and antioxidant activity using DPPH (1, 1diphenyl2picrylhydrazyl). Their cytotoxic and mitogenic proliferation activities were also studied in human leukemia HL60 cells and mouse splenocytes by CCK8 assay. In addition, genotoxic/ antigenotoxic activity was explored in mouse splenocytes using chromosomal aberrations (CAs) assay.

Results:
Lemon peels had the highest of TPC followed by grapefruit and mandarin. In contrast, mandarin peels contained the highest of TFC followed by lemon and grapefruit peels. Among the extracts, lemon peel possessed the strongest antioxidant activity as indicated by the highest DPPH radical scavenging, the lowest effective concentration 50% (EC50= 42.97 ?g extract/ mL), and the highest Trolox equivalent antioxidant capacity (TEAC=0.157). Mandarin peel exhibited moderate cytotoxic activity (IC50 = 77.8 ?g/mL) against HL60 cells, whereas grapefruit and lemon peels were ineffective antileukemia. Further, citrus peels possessed immunostimulation activity via augmentation of proliferation of mouse splenocytes (Tlymphocytes). Citrus extracts exerted noncytotoxic, and antigenotoxic activities through remarkable reduction of CAs induced by cisplatin in mouse splenocytes for 24 h.

Conclusions:
The phytochemical constituents of the citrus peels may exert biological activities including anticancer, immunostimulation and antigenotoxic potential.

Full text

Asian Pacic Journal of Cancer Prevention, Vol 17, 2016 3559
10.14456/apjcp.2016.134/APJCP.2016.17.7.3559
In Vitro Study of Biological Activities of Lemon, Grapefruit, and Mandarin Citrus Peels
Asian Pac J Cancer Prev, 17 (7), 3559-3567
Introduction
Oxidative stress is dened as a disturbance between the
formation of reactive oxygen species (ROS, pro-oxidants)
and their elimination by antioxidant defenses mechanism.
This disturbance is the primary cause of several health
problems such as cancer, heart diseases, aging and
neurodegenerative diseases (Alfadda and Sallam, 2012).
ROS are produced from endogenous (internal) and /
or exogenous (external) sources. Endogenous ROS is
generated from physical and mental stress, restriction
in blood supply to the tissues, microbial infection,
cancer, and aging. Exogenous ROS is produced from
radiation, environmental pollutions, pharmaceuticals
and industrial chemicals (Klaunig et al., 2010). ROS
can damage macromolecules (DNA, protein, lipid),
leading to alternations in genetic material that may cause
cancer (Reuter et al., 2010). In this sense, exploring the
Genetics and Cytology Department, National Research Centre, Cairo, Egypt *For correspondence: kawthar_diab@yahoo.com;
ka.diab@nrc.sci.eg
Abstract
Background: In recent years, there has been considerable research on recycling of agro-industrial waste for
production of bioactive compounds. The food processing industry produces large amounts of citrus peels that
may be an inexpensive source of useful agents. Objective: The present work aimed to explore the phytochemical
content, antioxidant, anticancer, antiproliferation, and antigenotxic activities of lemon, grapefruit, and mandarin
peels. Materials and Methods: Peels were extracted using 98% ethanol and the three crude extracts were assessed
for their total polyphenol content (TPC), total avonoid content (TFC), and antioxidant activity using DPPH (1,
1-diphenyl-2-picrylhydrazyl). Their cytotoxic and mitogenic proliferation activities were also studied in human
leukemia HL-60 cells and mouse splenocytes by CCK-8 assay. In addition, genotoxic/ antigenotoxic activity was
explored in mouse splenocytes using chromosomal aberrations (CAs) assay. Results: Lemon peels had the highest
of TPC followed by grapefruit and mandarin. In contrast, mandarin peels contained the highest of TFC followed
by lemon and grapefruit peels. Among the extracts, lemon peel possessed the strongest antioxidant activity as
indicated by the highest DPPH radical scavenging, the lowest effective concentration 50% (EC50= 42.97 µg extract/
mL), and the highest Trolox equivalent antioxidant capacity (TEAC=0.157). Mandarin peel exhibited moderate
cytotoxic activity (IC50 = 77.8 µg/mL) against HL-60 cells, whereas grapefruit and lemon peels were ineffective
anti-leukemia. Further, citrus peels possessed immunostimulation activity via augmentation of proliferation of
mouse splenocytes (T-lymphocytes). Citrus extracts exerted non-cytotoxic, and antigenotoxic activities through
remarkable reduction of CAs induced by cisplatin in mouse splenocytes for 24 h. Conclusions: The phytochemical
constituents of the citrus peels may exert biological activities including anticancer, immunostimulation and
antigenotoxic potential.
Keywords: Antioxidant assay - chromosomal aberration assay - cell viability - HL-60 cells - in vitro mouse splenocytes
RESEARCH ARTICLE
In Vitro Studies on Phytochemical Content, Antioxidant,
Anticancer, Immunomodulatory , and Antigenotoxic Activities
of Lemon, Grapefruit, and Mandarin Citrus Peels
Kawthar AE Diab
antimutagenic/anticancer compounds from plant sources
that are capable of repairing genomic changes are given
a vast signicance to protect human beings from human
health problems (Roleira et al., 2015).
Citrus fruits are one of the most popular food crops in
the world for their nutritional and therapeutic values. As
per recent information in 2013, the world production of
citrus fruits reached 135 million tons that harvested over
9.6 million hectares. Egyptian citrus fruits contributed to
4.09 million tons of the world production that harvested
over 1.75 million hectares in 2013. The mandarin, lemon,
and limes, and grapefruit are represented 28.6, 15.1
and 8.4 million tons of global production, respectively
(FAOSTAT, 2015). Large quantities of citrus peel wastes
are generated during citrus-juice processing industry. The
citrus by-products are considered a low-priced source of
bioactive compounds that can be economically exploited
to boost the national income in the industrial area (Liu et

Kawthar AE Diab
Asian Pacic Journal of Cancer Prevention, Vol 17, 2016
3560
al., 2012). Polymethoxyavones (PMFs) are a class of
novel avonoids containing two or more methoxy groups
that are entirely found in citrus peels, mainly orange peels
(Wang et al., 2008). Chemopreventive activity of PMFs
against the incidence of cancer was reported in vivo and
in vitro systems (Fan et al., 2007; Lee et al., 2011; Wang
et al., 2014).
The phytochemical content and antioxidant activity
of citrus peels were evaluated in various regions of the
world. However, little information is available on the
antioxidant activity and bioactive content of Egyptian
citrus species that cultivated under organic agriculture.
Also, there has been very restricted data into cytotoxic,
immunomandatory and antigenotoxic activities of the
citrus peels in particular lemon, mandarin and grapefruit
peels. Therefore, the present work was designed to explore
the following endpoints. (1) total polyphenol and avonoid
content; (2) antioxidant capacity by DPPH; (3) in vitro
cytotoxic effect in human leukemia HL-60 cells and mouse
splenocytes; (4) mitogenic proliferation response in mouse
splenocytes; (5) chromosomal aberrations (CAs) in vitro
mouse splenocytes.
Materials and Methods
Chemicals and Regents
Folin-ciocalteu reagent (FCR); chlorogenic acid (CA);
2, 2-diphenyl-2-picrylhydrazyl (DPPH), sodium nitrate
(NaNO3); sodium carbonate (Na2CO3); aluminum chloride
(AlCl3); dimethyl sulfoxide (DMSO); cisplatin (CDDP);
penicillin and streptomycin were obtained from Nacalai
Tesque (Kyoto-Japan). Concanavalin (Con A); colchicine;
6- hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic
acid (Trolox); (+) - Catechin were purchased from Sigma-
Aldrich (St Louis, MO, United States). Cell counting
kit (CCK-8) assay, fetal bovine serum (FBS), RPMI-
1640 medium were supplied from Dojindo Laboratories
(Kumamoto, Japan), Biowest (Nuaillé, France) and Wako
Chemicals Industries (Osaka, Japan) respectively.
Extraction process
Eureka lemon (Citrus limon), grapefruit (Citrus
paradise) and Baladi mandarin (Citrus reticulata) were
collected from Research and Production Station of
National Research Centre, El-Nubaria district, El-Behira
Governorate, Egypt. The citrus trees were planted in sandy
soil under organic agricultural conditions. The citrus peels
were dried and ground into powder by electric a mill. The
dried peels were submerged into 98% ethanol (EtOH) in
round bottom ask equipped with a condenser at room
temperature. The extraction procedures were repeated
four times and the liquid extracts were collected, ltrated
and evaporated using vacuum rotary evaporator at 40°C,
giving a brown oily residue. The three extracts had a
pleasant odor owing to presence part of the essential oil
in the infusions.
Phytochemical Content
Determination of Total Polyphenol Content (TPC)
TPC was determined using FCR method as described
by Herald et al. (2012), and chlorogenic acid (CA) is used
as reference standard. Briey, the extracts were dissolved
in DMSO to prepare different concentrations ranging
from 0.156- 20 mg/mL. Aliquot of the diluted extract
(25 µl) was mixed with 125 µl of FCR (1:10 diluted with
Milli-Q water) and 125 µl of Na2CO3 (10% w/v) in a well
of the 96 well at-bottomed microplate. The mixture was
kept in the dark at room temperature with intermittent
shaking for 10 min. The mixture reaction (200 µl) was
transferred into a new well of a 96 well microplate and
allowed to stand for 5 min. The absorbance of the mixture
was measured using microplate reader (SH-1000, Corona
Electronics, Ibaraki, Tokyo, Japan) at 600 nm versus the
blank. The blank sample had the same mixture but FCR
was replaced with Milli-Q water. A calibration curve of
CA was prepared under the same conditions as described
above in the range from 0.0625 to 2 mg/mL. The amount
of TPC was expressed as mg CA equivalent per mg of
the sample (mg CAE/mg sample) through the calibration
curve of CA.
Determination of Total Flavonoid Content (TFC)
TFC was determined using the AlCl3 colorimetric
method as described by Herald et al. (2012), and catechin
was used as the reference standard. Briey, the diluted
extract (25 µl) was mixed with Milli-Q water (125 µl) and
5% NaNO2 (7.5 µl). The mixture reaction was allowed to
stand for 6 min followed by addition 10% AlCl3 solution
(15 µl). The reaction was left to stand for 5 min before
the addition 1 N NaOH (50 µl) and Milli-Q water (275
µl). Then, an aliquot of the reaction mixture (200 µl/well)
was transferred into 96 well microplate. The absorbance
of the mixture was measured at 510 nm versus the same
mixture which containing Milli-Q water (15µl) instead of
AlCl3 as a blank. A calibration curve of (+) catechin was
prepared under the same conditions as described above in
the range from 0.0156 to 1 mg/mL. The amount of TFC
was expressed as mg (+) catechin equivalent per mg of
the sample (mg CE/mg sample) through the calibration
curve of (+)catechin.
Determination of antioxidant activity using DPPH
DPPH radical scavenging activity was determined
according to the procedures described by Herald et al.
(2012). Briey, 10 µl of the diluted extract at different
concentrations (2.5-1000 µg/mL) was mixed with 90 µl of
70% EtOH, 100 µl of 0.1M sodium acetate buffer (pH 5.5)
and 50 µl of DPPH solution (nal concentration was 0.5
mM in 100% EtOH). The mixture was shaken vigorously
and kept at room temperature for 30 min. Subsequently,
the mixture reaction (200 µl/well) was transferred into
a 96 well microplate. The optical density (OD) of the
mixture was determined at 517 nm against the blank
which containing 50 µl of 100% EtOH as a substitute of
DPPH. The inhibition of the DPPH radical scavenging
was calculated according to the following formula: DPPH
radical scavenging activity (%) = [1 - (OD of sample - OD
of blank)/ (OD of control - OD of blank)] × 100.
EC50 value (μg extract/mL) is the effective concentration
of the plant extract able to scavenge 50% of DPPH radical.
Trolox is used as a standard antioxidant reference to
convert the DPPH scavenging capacity of each sample to

Asian Pacic Journal of Cancer Prevention, Vol 17, 2016 3561
10.14456/apjcp.2016.134/APJCP.2016.17.7.3559
In Vitro Study of Biological Activities of Lemon, Grapefruit, and Mandarin Citrus Peels
the Trolox equivalent antioxidant capacity (TEAC). Trolox
was prepared by the same procedures described above in
the concentration ranging from 2.5 to 1000 μg/mL. TEAC
value was calculated as the ratio between EC50 of Trolox
(μg/mL) and EC50 of extract (μg/mL).
Human cell culture and Treatment
The human promyelocytic leukemia HL-60 cells
were grown in RPMI-1640 medium supplemented with
10% heat-inactivated FBS, penicillin (100 units/mL)
and streptomycin sulfate (100 mg/mL). The cells were
placed in 100 mm-diameter disposable Petri-dish and
maintained in an incubator (5% CO2 and 90 % humidity)
at 37OC. The cell passage was performed twice a week to
keep the cell viability and exponential cell growth. The
extracts were dissolved in DMSO as a stock solution (50
mg/mL) and diluted in RPMI-1640 medium to prepared
different concentrations ranging from 0.5 to 500 µg/
mL. The maximum nal concentration of DMSO in the
medium was less in 1%.
Isolation of mouse splenocytes
Mouse splenocytes were prepared as described
previously (Ko and Joo, 2010). Briey, the spleen was
excised from an adult male mouse (3-months-old inbred
Swiss strain, National Research Centre, Dokki, Cairo,
Egypt) and transferred into a sterile cell strainer (40µm)
over a petri-dish containing the RPMI-1640 medium.
The spleen was gently crushed through the sieve using
the plunger end of the syringe. The resulting cells were
centrifuged at 1000 rpm for 10 min at room temperature.
The cell pellets were resuspended in 1mL of erythrocyte
lysis buffer (144 mM NH4Cl, 1.7 mM Tris Base, pH 7.2)
at room temperature for 5 min. Then, 9 mL of phosphate
buffer (PBS) were added to stop the lysis followed by
centrifugation at 1000 rpm for 5 min. The cell pellets were
washed twice with PBS (0.14 M NaCl, 2.68 mM KCl,
10.14 mM Na2HPO4, 1.76 mM KH2PO4, and pH 7.2).
The cells were resuspended in complete medium (RPMI-
1640 medium supplemented with 10% heat-inactivated
FBS, penicillin (100 units/mL) and streptomycin sulfate
(100 mg/mL) and counted using a hemocytometer. The
experimental animals were conducted under the guidelines
for the Care and Use of Laboratory Animals in Scientic
Research approved by ethical committee at National
Research Centre.
In vitro cytotoxicity/viability assay
The cell viability was determined colorimetrically
with CCK-8 as described previously (Diab et al., 2015).
Cytotoxicity of citrus peel extracts was evaluated in two
types of cells, human leukemia HL-60 cells, and primary
murine splenocytes. HL-60 cells (50 × 10 3cell/100 µl/
well in a 96 well plate) were grown in medium containing
different concentrations of extracts (0.5-500 µg/mL) for
24 h.
Mouse splenocytes (1× 105 cell/100 µl/ well in a 96
well plate) were grown in complete medium supplemented
with different concentration of plant extracts (20-500µg/
mL) in the absence of
Con A for 48 h.
After the end of incubation, a volume of 20 µl of
CCK-8 was added per well, and the plate was incubated
in the CO2 incubator for 3 h. CCK-8 contains the
tetrazolium salt WST-8 [2-(2-Methoxy-4-nitrophenyl)-
3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium,
monosodium salt] which reduced by mitochondrial
dehydrogenases to generate a water-soluble yellow-color
product (formazan). The quantity of formazan is directly
proportional to the number of living cells (Tsukatani
et al., 2011). The samples absorbance was measured
at wavelength 450 nm against blank which containing
medium only. The cell viability was calculated according
to the following formula: (%) = (OD of sample− OD of
blank)/ (OD of control - OD of blank) × 100.
In vitro mitogen proliferation assay
Proliferation response of mouse splenocytes to
mitogen was determined using the CCK-8 colorimetric
assay. Mouse primary splenocytes are composed of about
90% of lymphocytes (50 % B cells and 45% T cells) and
up to 10% other immune cells such as macrophage and
dendritic cells (Małaczewska et al., 2016). Con A is a
plant mitogen for T-lymphocytes. Mouse splenocytes
(3×105 cell/100 µl/ well in a 96 well plate) were grown in
complete mediums supplemented by plant extracts (50-
500 µg/mL) in the presence or absence of Con A (5 μg/
mL). After 48 h, 20 µl of CCK-8 was added per well, and
the plate was further incubated for 3 h. The absorbance of
the samples was measured at 450 nm using a microplate
reader. The immunoproliferation was expressed as
stimulation index according to the following formula:
Stimulation index = O.D. of Con A- stimulated cells / O.D.
of non-stimulated cells (Krifa et al., 2014).
Assessment of genotoxicity and antigenotxicity
Chromosomal aberration assay
Mouse splenocytes were cultured at a density of
5×106cell/mL in 60 mm petri dish. Following 24 h of
incubation, the cells were treated with plant extracts alone
(100 µg /mL), CDDP alone (10 µg/mL) or combination of
both (plant extract+ CDDP) for 24 h. A negative control
(non-treated cells) was also evaluated. Two hours before
harvest, the cells incubated with colchicine (200 µg/mL)
to arrest the cells at the metaphase stage. The cells were
harvested by centrifugation at 1500 rpm for 10 min at
room temperature. The cell pellets were resuspended in
hypotonic solution (0.075 M KCl) for 20 min at 37°C
followed by centrifugation at 1500 rpm for 10 min. The
cell pellets were xed with a freshly prepared ice-cold
xative (3:1 volume of methanol: glacial acetic acid)
followed by centrifugation for at 1500 rpm for 5 min.
The cell pellets were washed twice with the xative,
and the drops of cells suspension were dropped onto a
clean microscopic slide. After drying, the slides were
stained with 10% Giemsa in phosphate buffer (0.06 M
Na2HPO4 and 0.06 M KH2PO4, pH 6.8) for 10 min,
washed with distilled water, air-dried. At least 500 well-
spread metaphases were analyzed per concentration
under a light microscope at 2000X magnication for
chromosomal aberration. The reduction rate (%) was
calculated according the following equation: Reduction

Kawthar AE Diab
Asian Pacic Journal of Cancer Prevention, Vol 17, 2016
3562
(%) =[number of aberrant cells in (A) -number of the
aberrant cells in (B)]/ number of the aberrant cells in
(A)- number of the aberrant cells in (C)] ×100 (Diab and
Elshafey 2011) . Where (A) represents positive control
(cells treated with CDDP alone); (B) represents culture
treated with plant extract + CDDP; (C) represent negative
control (non-treated cells)
Data Analysis
The experiments were replicated at least three times
with quadruplicate wells in each concentration. The results
were computerized and analyzed using Statistical Package
for Social Science (SPSS Inc, version 17, Chicago, IL,
USA). One-way analysis of variance (ANOVA) and
Duncan’s New Multiple-range test with a condence
interval of 95% were used to determine the differences
among the means. The P value <0.05 were considered as
statistically signicant. Cell viability and proliferation data
were subjected to Student’s t-test for independent samples
with equal or unequal variances. Linear regression analysis
calculated both EC50 (median effective concentration)
and IC50 (median inhibitory concentration). The Pearson
correlation coefficients (r) were conducted between
DPPH, TPC, and TFC.
Results and Discussion
Determination of TPC and TFC
FCR is a mixture of phosphomolybdate and
phosphotungstate that commonly used for quantication
of polyphenol compounds in plant extracts. This assay
is widely used for its simplicity, popularity, low-cost,
and reproductively. In this reaction, the movement of a
single electron from polyphenol was caused a reduction
of FCR to produce blue colored phosphotungstic-
phosphomolybdenum complex. However, quantication
of TPC interferes with any oxidation substances in plant
sample that react with FC reagent in an inhibitory, additive
or enhancing manner (Singleton et al., 1999; Cicco et
al., 2009).
Data presented in Table (1) illustrated that, among
the citrus extracts, the lemon peel possessed the highest
level of TPC (142.63±18.74 µg CAE/mg extract) that
represented 2.3 and 2.6-fold increases compared with the
values for grapefruit and mandarin peel respectively. It was
reported that the citrus peels are characterized by the high-
level content of polyphenol antioxidants compared to their
segment and seeds (Abeysinghe et al., 2007; Guimarães
et al., 2010). The accumulation of polyphenol compounds
in the outer epidermal cells was contributed in protection
the plant from UV damage, pathogenic microorganisms
and insect attacks (Mierziak et al., 2014).
The aluminum chloride colorimetric assay is widely
used for quantication of TFC in plant extracts. In this
reaction, the presence of NaNO2 in alkaline medium
resulted in nitration the aromatic ring that has a catechol
group with its 3 or 4 positions unsubstituted or sterically
hindrance. The reaction between aluminum chloride and
carbonyl and hydroxyl groups of avones and avonols
produce a yellow stable complex which turned instantly
to a red-color complex after addition of NaOH (Pękal and
Pyrzynska, 2014).
As shown in Table (1), the order of TFC was arranged
in descending order as follows: mandarin peel > lemon
peel ≥ grapefruit peel. Similarity, the variations in TPC
and TFC were recorded in 21 varieties of 11 citrus peels.
Among all of them, lemon peel had the highest level
of TPC (1882±65 µg of gallic acid/g extract) and the
lowest amount of TFC. While tangelo and mandarin
peels (5615±93µg/g extract and 5237±68µg /g extract,
respectively) possessed the highest level of TFC which
expressed in μg quercetin/g extract (Ramful et al., 2010).
The diversity in TPC and TFC are related to agriculture
factors such as nitrogen and nutrient supply, mulching,
irrigation, light exposure, temperature, cultural methods,
and fruit ripening level (Ghasemi et al., 2009; Ramful et
al., 2010; Oboh and Ademosun, 2012). Moreover, this
variation is associated with the extraction procedures
such as the solvent type and its concentration , solvent/
solid ratio, extraction time and temperature , and pH
value (Li et al., 2006). According to Hegazy and Ibrahium
(2012), alcoholic solvents (ethanol and methanol) are
the superior solvents for extraction of polyphenol/
avonoids compounds from orange peels among the other
solvents such as hexane, acetone, dichloromethane, and
ethylacetate. In fact, that the ethanol have hydrophilic
(OH group), and hydrophobic (hydrocarbon portion) ends
that have a propensity for extraction polar and non-polar
compounds respectively. Hence, the addition of water
increases the polarity of ethanol which reected its high
afnity to extract polyphenols from high, mid, and low
ends of polarity (Spigno et al., 2007).
Determination of antioxidant activity by DPPH
The stable radical DPPH molecule is characterized
by the presence of an odd, unpaired electron in its outer
orbital which responsible for the visible dark purple. In
this reaction, DPPH molecule is reduced by hydrogen-
donating antioxidant compounds and became stable,
non-radical (diamagnetic) molecule and decolorized to
yellow-colored diphenyl-picrylhydrazine (Kedare and
Singh, 2011). The major characteristics of DPPH are its
simplicity, rapidity, and accuracy. However, many factors
can be inuenced on the DPPH assay, for example, the
interaction between antioxidants, reaction time and
interference compounds (Kedare and Singh, 2011).
Indeed, the antioxidant compounds can be classied as be
hydrophilic (water-soluble), hydrophobic (lipid-soluble)
and bound (insoluble) to cell walls that cannot react
with DPPH. Hydrophilic and hydrophobic antioxidant
compounds react with DPPH at different rates, and the
reaction will not reach the nishing point in a reasonable
reaction time. For this reason, the amount of plant sample
necessary to react with one-half of the DPPH is selected
as an endpoint for quantication the antioxidant activity
(Kedare and Singh, 2011).
The present study showed that, the three extracts
were differently exhibited DPPH antiradical activity in a
concentration-dependent manner (Figure 1). As shown in
Table (1), Trolox was potently radical scavenging activity
with the minor EC50 value (6.5 µg extract/mL) as compared
with the citrus peel extracts. Lemon peel extract had the

Asian Pacic Journal of Cancer Prevention, Vol 17, 2016 3563
10.14456/apjcp.2016.134/APJCP.2016.17.7.3559
In Vitro Study of Biological Activities of Lemon, Grapefruit, and Mandarin Citrus Peels
Table 1. Measurement of Phytochemical Content and Antiradical Scavenging Activity of Citrus Peels
Samples TPC TFC Antioxidant activity
(µg CAE/mg extract) (µg CE/mg extract) EC50 value (µg extract/ml) TEAC value
Lemon Peel Extract 142.63±18.74a16.24±5.28a42.967 0.157
Grapefruit Peel Extract 59.68±30.75b15.17±1.64a>1000 0.005
Mandarin Peel Extract 52.83±11.32b19.36± 1.79ab >1000 0.006
Trolox (Standard) ------ ------- 6.5 ----------
The Data having different superscript letters in each column are signicantly different from one another as calculated by ANOVA (Duncan’ test, (p<0.05)
Table 2. Pearson’s Correlation Boefcients between
TPC, TFC and DPPH Assays of Citrus Peels
Correlation Lemon Peel
Extract
Grapefruit
Peel Extract
Mandarin
Peel Extract
TPC & TFC 0.963** 0.609 0.514
TPC &DPPH –0.829* –0.664 –0.858
TFC &DPPH –0.703 –0.997* –0.882
*Signicant at p<0.05; **Signicant at p<0.01
higher DPPH radical scavenger, lower EC50 (42.97 µg
extract/mL) and higher TEAC value indicating its greater
antioxidant activity. Whereas, grapefruit peel extract
possessed the lower DPPH, higher EC50 values (>1000 µg
extract/mL) and lower TEAC values reecting its lower
antioxidant activity. It is known that, the plant extract,
having superior antioxidant activity, is characterized by
its greater antiradical activity with lower EC50 and higher
TEAC values (Fernandes de Oliveira et al., 2012).
Pearson’s correlation analysis
Table (2) shows the interrelationship between TPC,
TFC and antioxidant activity for citrus peels. The present
study recorded positive correlation between TPC and
TFC of three extracts veried that avonoids represented
the primary fraction of polyphenol compounds. Indeed,
phenolic compounds consist of simple phenols (phenolic
acid) and complex phenol (polyphenols), depending on
the number of phenol subunits attached to it. Simple
phenols are low-molecular weight compounds that include
only one phenol subunit. Polyphenol compounds are
intermediate (avonoids) or high (condensed tannins,
lignans, and stilbenes) molecular weight compounds
having more than one phenol subunit in their chemical
conFigureuration (Landete, 2012).
Interestingly, the negative relationships between TPC
and DPPH for the citrus peel extracts were shown in Table
(2). This relationship attributed to two possible reasons.
Firstly, FCR reacts with both phenolic and non-phenolic
compounds such as vitamin C, lipid, amino acids (Georgé
et al., 2005). Secondly, the high concentration of ethanol
(98%) is inadequate to release hydrophilic phenolic
compounds which responsible for antioxidant activity
(Naczk and Shahidi, 2006).
Further, the negative correlations between TFC and
DPPH for the three extracts were reported in Table (2).
Similar ndings reported negative correlations between
TPC and DPPH for orange (Citrus sinensis L) peel
extract and its fractions (Diab et al., 2015). This negative
Figure 2. Cytotoxic Activity of Citrus Peel Extracts
Against Human Leukemia Promyelocytic HL-60 Cells.
The cells (50 ×104cells/mL), grown in a 96-well plate,
were incubated with different concentrations of citrus
peels for 24h. Then, CCK-8 (20 µL) were added to the
cells for 3 h. Data are represented as mean % ± SD
Figure 1. Antiradical Activity of Citrus Peel Extract
and Trolox. Data are represented as mean % ± SD
0
10
20
30
40
50
60
70
80
90
100
0 100 200 300 400 500 600 700 800 900 1000
DPPHRadicalScaveningAc1vity(%)
Concentra1on(µg/ml)
Trolox Lemon Grapefruit Mandarin
0
10
20
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0 100 200 300 400 500 600 700 800 900 1000
DPPHRadicalScaveningAc1vity(%)
Concentra1on(µg/ml)
Trolox Lemon Grapefruit Mandarin
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0 100 200 300 400 500 600 700 800 900 1000
DPPHRadicalScaveningAc1vity(%)
Concentra1on(µg/ml)
Trolox Lemon Grapefruit Mandarin
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0 100 200 300 400 500 600 700 800 900 1000
DPPHRadicalScaveningAc1vity(%)
Concentra1on(µg/ml)
Trolox Lemon Grapefruit Mandarin
0
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90
100
0 50 100 150 200 250 300 350 400 450 500
CellViability(%)
Concentra2on(µg/ml)
LemonPeelExtract(IC50>500µg/mL)
GrapefruitpeelExtract(IC50=195.2±0.11µg/mL)
MandrinePeelExtract(IC50=77.8±1.4µg/mL)


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0 50 100 150 200 250 300 350 400 450 500
CellViability(%)
Concentra2on(µg/ml)
LemonPeelExtract(IC50>500µg/mL)
GrapefruitpeelExtract(IC50=195.2±0.11µg/mL)
MandrinePeelExtract(IC50=77.8±1.4µg/mL)


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0 50 100 150 200 250 300 350 400 450 500
CellViability(%)
Concentra2on(µg/ml)
LemonPeelExtract(IC50>500µg/mL)
GrapefruitpeelExtract(IC50=195.2±0.11µg/mL)
MandrinePeelExtract(IC50=77.8±1.4µg/mL)


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40
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0 50 100 150 200 250 300 350 400 450 500
CellViability(%)
Concentra2on(µg/ml)
LemonPeelExtract(IC50>500µg/mL)
GrapefruitpeelExtract(IC50=195.2±0.11µg/mL)
MandrinePeelExtract(IC50=77.8±1.4µg/mL)


Figure 3. Effect of Citrus Peel Extracts on cell viability
in Non-stimulated Primary Mouse Splenocytes. The
cells (1×106 cells/mL), grown in a 96-well plate, were incubated
with different concentrations of citrus peels for 48 h. thereafter,
the cells were incubated with 20 µL of CCK-8 for 3 h. Data are
represented as mean % ± SD. *P<0.05; **P<0.01 compared to
control culture
0
20
40
60
80
100
120
LemonPeelExtract GrapefruitPeelExtract MandarinPeelExtract
CellViability(%)
Control 20µg/mL 50µg/mL 100µg/mL 200µg/mL 300µg/mL 500µg/mL
****
**
*
**
*
**
0
20
40
60
80
100
120
LemonPeelExtract GrapefruitPeelExtract MandarinPeelExtract
CellViability(%)
Control 20µg/mL 50µg/mL 100µg/mL 200µg/mL 300µg/mL 500µg/mL
****
**
*
**
*
**
0
20
40
60
80
100
120
LemonPeelExtract GrapefruitPeelExtract MandarinPeelExtract
CellViability(%)
Control 20µg/mL 50µg/mL 100µg/mL 200µg/mL 300µg/mL 500µg/mL
****
**
*
**
*
**
0
20
40
60
80
100
120
LemonPeelExtract GrapefruitPeelExtract MandarinPeelExtract
CellViability(%)
Control 20µg/mL 50µg/mL 100µg/mL 200µg/mL 300µg/mL 500µg/mL
****
**
*
**
*
**
0
20
40
60
80
100
120
LemonPeelExtract GrapefruitPeelExtract MandarinPeelExtract
CellViability(%)
Control 20µg/mL 50µg/mL 100µg/mL 200µg/mL 300µg/mL 500µg/mL
****
**
*
**
*
**
0
20
40
60
80
100
120
LemonPeelExtract GrapefruitPeelExtract MandarinPeelExtract
CellViability(%)
Control 20µg/mL 50µg/mL 100µg/mL 200µg/mL 300µg/mL 500µg/mL
****
**
*
**
*
**

Kawthar AE Diab
Asian Pacic Journal of Cancer Prevention, Vol 17, 2016
3564
0
25.0
50.0
75.0
100.0
Newly diagnosed without treatment
Newly diagnosed with treatment
Persistence or recurrence
Remission
None
Chemotherapy
Radiotherapy
Concurrent chemoradiation
10.3
0
12.8
30.0
25.0
20.3
10.1
6.3
51.7
75.0
51.1
30.0
31.3
54.2
46.8
56.3
27.6
25.0
33.1
30.0
31.3
23.7
38.0
31.3
0
25.0
50.0
75.0
100.0
Newly diagnosed without treatment
Newly diagnosed with treatment
Persistence or recurrence
Remission
None
Chemotherapy
Radiotherapy
Concurrent chemoradiation
10.3
0
12.8
30.0
25.0
20.3
10.1
6.3
51.7
75.0
51.1
30.0
31.3
54.2
46.8
56.3
27.6
25.0
33.1
30.0
31.3
23.7
38.0
31.3
correlation may be due to synergistic or antagonistic
interaction among the bioactive compounds in the crude
extracts (Wang et al., 2011). It was found that chemical
con Figureuration of avonoids are closely associated
with their antioxidant activity (Heim et al., 2002). For
example, the presence of catechol (ortho-dihydroxy)
structure in the B-ring provide the avonoid with highly
antiradical scavenging activity (Bors et al., 1990). Further,
the presence of the 2, 3- unsaturation in combining with a
4-oxo function participates in electron transferring from
the B-ring to C-ring (Rice-Evans, 2001).
In vitro cytotoxic assay
A-Human leukemia HL-60 cells
The majority of chemotherapy drugs is not only
cytotoxic to the cancer cells but also is toxic to healthy
cells and has immune suppressive side effects. Therefore,
the discovery of novel compounds that possess not only
cytotoxic activity against cancer cells but also non-toxic
to healthy cells and modulating the immune response has
become an important goal of research in the biomedical
sciences (Sak, 2012). The present study showed that, all
the tested extracts were clearly decreased the cell viability
in a concentration-dependent manner in HL-60 cells
(Figure 2). According to Atjanasuppat et al. (2009), the
cytotoxic activity of the extracts has classied into four
groups according to their IC50 value. Those are active
extract (≤20µg/ml), moderately active extract (>20-100
μg/ml), weakly active extract (>100-1000 µg/ml), inactive
extract (>1000 µg/ml). In view of that mandarin peel had
a moderate anticancer activity (IC50= 77.8 ± 1.4 µg/mL).
While grapefruit peel (IC50 = 195.2 ± 0.11 µg/mL) and
lemon peel (IC50 > 500 µg/mL) exhibited a weak cytotoxic
toward HL-60 cells. The anticancer activity of citrus peels
was reported either in the form of single molecules as or as
a mixture of molecules (Manassero et al., 2013; Wang et
al., 2014; Rawson et al., 2014). For example, the essential
oils of lemon and grapefruit peel exhibited moderated to
weak cytotoxicity toward human prostate (PC-3), lung
(A549) and breast (MCF-7) tumor cell lines (Zu et al.,
2010). Moreover, ethanolic extract from orange peel
and its fractions exhibited a weak to moderate cytotoxic
activity toward HL-60 cells (Diab et al., 2015).
B-In vitro mouse splenocytes cytotoxicity
The in vitro cytotoxicity can predict the level of
acute toxicity (oral and intervenous) in animal studies.
Subsequently, the number of animals can be reduced for
in vivo toxicity assay (Ukelis et al., 2008). As depicted
in Figure (3), negative controls of all extracts have a cell
viability of 100%. In non-stimulated mouse splenocytes,
all extracts were increased cell viability in a concentration-
independent manner, indicating their potential non-
cytotoxic and proliferative activities. Cell viability reached
its maximum after treatment the splenocytes with lemon,
grapefruit, and mandarin peel extracts at concentrations
300, 200, and 500 μg/mL, respectively. Interestingly,
insignicant reductions in cell viability were observed
after treatment the splenocytes with 20 μg/mL of lemon
Table 3. Protective Activity of Citrus Peel Extracts against Cisplatin-induced Chromosomal Aberrations in
Mouse splenocytes
Treatment Gap Br/Frag Del M. A Including gaps Excluding gaps
% % % % Mean% ± S.E R(%) Mean %±S.E R(%)
Control Treatments
Negative (non-treatment) 1.6 2 0.2 ---- 3.80 ± 0.58a2.20 ± 0.37a
Positive (CDDP,10 µg/mL) 410.4 2.2 0.8 17.40 ± 0.51f13.40 ± 0.51e
Lemon Peel Extract (LPE)
LPE (100 µg/mL) 0.4 2.8 0.2 0.2 3.60 ± 0.40a3.20 ± 0.37a
LPE (100 µg/mL)+CDDP 1.8 4.6 1 0.6 8.00 ± 0.55bc69.1 6.20 ± 0.66bc 62.2
LPE (50 µg/mL)+ CDDP 3.8 90.2 ---- 13.00 ± 1.38e32.4 9.20 ± 0.86d37.5
Grapefruit Peel Extract (GPE)
GPE (100 µg/mL) 1.2 2.6 0.2 ---- 4.00 ± 0.77a2.80±0.80a
GPE (100 µg/mL)+CDDP 1.8 41 0.6 7.40 ± 1.50b73.5 5.60±0.93b69.6
GPE (50 µg)+CDDP 3.4 61.8 0.4 11.60 ± 0.68de 42.6 8.20±0.66cd 46.4
Mandarin Peel Extract(MPE)
MPE ( 100 µg/mL) 0.4 2 0.8 ---- 3.20 ± 0.73a2.80 ± 0.58a
MPE (100 µg/mL)+CDDP 0.8 50.8 ---- 6.60 ± 0.40b79.4 5.80 ± 0.66b69.8
MPE (50 µg/mL)+CDDP 1.2 7.8 0.8 0.4 10.20 ± 1.28cd 52.9 9.00 ± 1.18d39.2
Total 500 metaphases were examined per each treatment; Data are expressed as mean%± S.E; Br=Break; Fra= Fragment; Del= Deletion; M.A
metaphases with more than one type of aberrations; R = Reduction rate; The Data having different superscript letters in each column are signicantly
different from one another as calculated by ANOVA (Duncan’ test, p<0.05)
Figure 4. Effect of Citrus Peel Extracts on Proliferation
of Mouse Splenocytes. The cells (3×106 cells/mL), grown
in a 96-well plate, were incubated with different concentrations
of citrus peels with and without Con A (5 µg/mL) for 48 h.
thereafter, the cells were incubated with 20 µL of CCK-8 for 3
h. Data are represented as mean % ± SD. * P<0.05; ** P< 0.01
compared to control culture
LemonPeelExtract GrapefruitPeelExtract MandarinPeelExtract
0
0.2
0.4
0.6
0.8
1
1.2
1.4
S"mula"onIndex
Control 50µg/mL 100µg/mL 200µg/mL 300µg/mL 500µg/mL
**
**
*******
**
***
*
*
*
*
LemonPeelExtract GrapefruitPeelExtract MandarinPeelExtract
0
0.2
0.4
0.6
0.8
1
1.2
1.4
S"mula"onIndex
Control 50µg/mL 100µg/mL 200µg/mL 300µg/mL 500µg/mL
**
**
*******
**
***
*
*
*
*
LemonPeelExtract
GrapefruitPeelExtract MandarinPeelExtract
0
0.2
0.4
0.6
0.8
1
1.2
1.4
S"mula"onIndex
Control 50µg/mL 100µg/mL 200µg/mL 300µg/mL 500µg/mL
**
**
*******
**
***
*
*
*
*
LemonPeelExtract
GrapefruitPeelExtract
MandarinPeelExtract
0
0.2
0.4
0.6
0.8
1
1.2
1.4
S"mula"onIndex
Control 50µg/mL 100µg/mL 200µg/mL 300µg/mL 500µg/mL
**
**
*******
**
***
*
*
*
*
LemonPeelExtract GrapefruitPeelExtract
MandarinPeelExtract
0
0.2
0.4
0.6
0.8
1
1.2
1.4
S"mula"onIndex
Control 50µg/mL 100µg/mL 200µg/mL 300µg/mL 500µg/mL
**
**
*******
**
***
*
*
*
*
LemonPeelExtract GrapefruitPeelExtract MandarinPeelExtract
0
0.2
0.4
0.6
0.8
1
1.2
1.4
S"mula"onIndex
Control 50µg/mL 100µg/mL 200µg/mL 300µg/mL 500µg/mL
**
**
*******
**
***
*
*
*
*

Asian Pacic Journal of Cancer Prevention, Vol 17, 2016 3565
10.14456/apjcp.2016.134/APJCP.2016.17.7.3559
In Vitro Study of Biological Activities of Lemon, Grapefruit, and Mandarin Citrus Peels
(95%), grapefruit (96%), and mandarin (97%) peels. This
data indicated that the lowest concentration did not induce
cytotoxic and proliferative activities in mouse splenocytes.
The obtained results did not permit to measure median
lethal concentration (LC50), indicating that citrus peels
had LC50 values > 1000 μg/mL. This data are in line with
the nding of Hosseinimehr et al. (2003) who found that
bitter orange peel was non-toxic in male mouse at the
dose 1000 mg/kg.
In vitro muse splenocytes proliferation assay
Exploration the natural products that stimulate or
suppress lymphocyte proliferation response is considered
the rapidly growing area of autoimmunity, inammation
and cancer immunotherapy (Duong et al., 2011).
Lymphocytes are key effector cells of humoral and
cell-mediated immune responses mediated by B and T
lymphocytes, respectively (Kawai et al., 2006). Several
assays have been applied for measuring the growth pattern
of murine lymphocytes. The mitogenic activity has used
lipopolysaccharide and Con A as immunostimulant agents
for B and T-lymphocytes, respectively (Colic et al., 2002;
Ang et al., 2014). This activity represents an early stage
of the immune response and has evaluated as the rst
screening of immunomodulatory activity (de Melo et
al., 2011). In the present study, the incubation of mouse
splenocytes (T-lymphocytes) with citrus extracts in the
presence or absence of Con A were used as a model for
their vital role in cellular mediated immunity (Duong et al.,
2011). As shown in Figure (4), the three extracts increased
the proliferation of mouse splenocytes in a concentration-
independent manner compared to control cultures. Lemon
peel extract exerted a signicant immunostimulation
activity at all tested concentrations that reached its highest
level after treatment splenocytes with the concentration
200 μg/mL. Interestingly, grapefruit and mandarin peels
had insignicant increase in the stimulation index at a
low concentrations 100 and 50 μg/mL, respectively. The
maximum proliferation index was recorded for grapefruit
and mandarin at the concentration 500 μg/mL (p<0.01).
These data suggested that citrus peels had mitogenic
activity and stimulated the proliferation of T-lymphocytes
response to Con A through their bioactive compounds
of the extract. Similar results obtained by Tanaka et
al. (1999) reported that auraptene isolated from citrus
peel (Citrus natsudaidai Hayata), have been exerted an
augmentation activity on mouse splenocytes stimulating
response to Con A. Immunomodulatory properties of
plants are closely associated to polyphenol compounds.
Polyphenols containing OH group at R2 position (e.g.,
caffeic acid and chlorogenic acid) have been possessed
lesser potent immunostimulating activity than polyphenol
containing other groups at the same position (Chiang et
al., 2003; Cuevas et al., 2013). The immunostimulation by
plant extracts is believed to be a promising way to prevent
and cure disease (Kumar et al., 2012).
It is noteworthy that, the citrus peels exhibited a
weak to moderate cytotoxic/antiproliferative activity
against human leukemia HL-60 cells (cells undergoing
mitosis). As well, citrus peels exerted their potential
non-cytotoxic and proliferative effects toward non-
stimulated mouse splenocytes (cells in resting stage of
mitosis) or stimulated cells (cells undergoing mitosis).
This means that, polyphenol compounds are not only
toxic to cancerous cells, but also are non-toxic or are less
toxic to normal cells.
In vitro chromosomal aberrations (CAs) assay
CAs assay has used as a marker for DNA damage of
cancer risk in humans. Mouse splenocytes are the most
sensitive indicator of genetic damage in both in-vivo
and in-vitro model (Steiblen et al., 2005). In the present
study, genotoxic effect of citrus peels was examined at the
concentration 100 µg/mL in mouse splenocytes for 24 h.
The results showed that the tested extracts did not induce
a dramatic increase in the frequency of structural CAs
compared to the negative and positive control cultures.
These data indicated their non-genotoxicity and supported
their potential uses as chemopreventive agents. Similarity,
the methanol extract of citrus peel (Citrus aurantium var.
amara) cannot induce micronuclei in mouse bone marrow
cells at the doses of 200 and 400 mg/kg (Hosseinimehr et
al., 2003; Hosseinimehr and Karami, 2005). Further, the
essential oil of bitter orange did not possess genotoxic
activity at the doses from 0.1% to 0.5% in the Drosophila
wing spot test (Demir et al., 2009).
Table (3) shows the antigenotoxic activity of the
tested citrus peels at two concentration (50, 100 µg/
mL) against chemotherapeutic agent CDDP. It found
that CDDP was induced a remarkable increase in the
occurrence of CAs, mainly breaks/fragments (10.4%)
compared with control culture (2.0%). This implied that
CDDP exerts its genotoxic activity by reacting with the
N7- position of purine base in the DNA molecule which
forms DNA adducts (Eastman, 1999; Tanida et al., 2012).
The intrastrand and interstrand cross-linked DNA adducts
interfered with DNA replication and transcription causing
DNA damage and inhibiting cell proliferation (Eastman,
1999; Attia, 2010). Further studies demonstrated that the
antitumor activity of CDDP is correlated to its genotoxic
activity on tumor cells. Besides, the toxicity of CDDP in
the healthy cells is associated with the production of ROS
(Basu and Krishnamurthy, 2010; Attia, 2010)
The protective activity of the citrus extracts was
examined by simultaneous treatment of extracts (50, 100
µg/mL) and CDDP (10 µg/mL) to mouse splenocytes.
Data showed that the citrus peels were drastically reduced
the frequency of CAs induced by CDDP for 24 h. The
reduction rate ranged from 32.4% to 69.1% for the lemon
peel, from 42.6% to 73.5% for the grapefruit peel and
from 52.9% to 79.4% for the mandarin peel. Similar
results showed that bitter orange peel extract exhibited
antimutagenic activity against cyclophosphamide and
radiation in mouse bone marrow cells (Hosseinimehr
and Karami, 2005; Hosseinimehr et al., 2003). Further,
bitter orange (Citrus aurentium) peel oil decreased
somatic mutation in the wings of Drosophila melanogaster
induced by potassium dichromate, cobalt chloride, ethyl
methane-sulfonate and N-ethyl-N-nitrosourea (Demir et
al., 2009). Further, citrus avonoid (naringin) inhibited
mutagenesis in Salmonella typhimurium strain TA100
NR- induced by N-methyl-N’-nitro-N-nitrosoguanidine

Kawthar AE Diab
Asian Pacic Journal of Cancer Prevention, Vol 17, 2016
3566
(Francis et al., 1989).
These data suggested that citrus peels exert their
antigenotoxicity in a desmutagenic manner. Indeed,
antimutagens classied into desmutagens or biomutagens.
Desmutagenic compounds exert their action extracellularly
to inactivate the mutagenic chemicals before they attack
DNA molecule mutagens. Demutagenic agents are
frequently administrated before or simultaneous with
the mutagenic agents. In the other hand, biomutagens
are administrated after mutagenic compounds and act
intracellularly by inhibition the xation of mutations (De
Flora and Ferguson 2005; Słoczyńska et al., 2014). Taken
together, the bioactive compounds presented in citrus peels
exert their anticancer activity through the antimutagenic
mechanism. Earlier reports describing citrus peels as
powerful antioxidants provide protection against diverse
mutagens through elimination ROS generated from
mutagens and prevent mutation-related diseases in human
(Alfadda and Sallam, 2012; Wang et al., 2014; Rawson et
al., 2014; Diab et al., 2015)
In conclusion, this study exhibited a weak to moderate
antitumor activity of the tested citrus peels in HL-60
cells. The same extracts increased the cell viability and
stimulation index of mouse splenocytes in absence or
presence of Con A indicating their non-cytotoxicity and
immunostimulation activity. The three citrus extracts
exerted their antimutagenic activity through reduction
of CAs induced by CDDP in mouse splenocytes in
a desmutagenic manner (simultaneous treatment).
Further biological experiments are required for a better
understanding of the mechanistic studies of citrus peels
as chemopreventive agents. Extra antioxidant assays
and quantication of phytochemical content (such as
saponins, tannins) are needed for increased knowledge
about the antioxidant mechanism of crude extracts and
their fractionations and the isolated pure compounds.
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