Food Chemistry 135 (2012) 2119–2127

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Protective effects of sweet orange (Citrus sinensis) peel and their bioactive
compounds on oxidative stress
Zong-Tsi Chen a, Heuy-Ling Chu b, Charng-Cherng Chyau c, Chin-Chen Chu d, Pin-Der Duh b,⇑
a
Department of Medicinal Chemistry, Chia Nan University of Pharmacy and Science, 60 Erh-Jen Road, Section 1, Pao-An, Jen-Te District, Tainan, Taiwan, ROC
b
Department of Food Science and Technology, Chia Nan University of Pharmacy and Science, 60 Erh-Jen Road, Section 1, Pao-An, Jen-Te District, Tainan, Taiwan, ROC
c
Research Institute of Biotechnology, Hungkuang University, 34 Chung-Chie Road, Shalu County, Taichung, Taiwan, ROC
d
Department of Anesthesiology, Chi-Mei Medical Center, Tainan, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history: Protective effects of sweet orange (Citrus sinensis) peel and their bioactive compounds on oxidative stress
Received 23 February 2012 were investigated. According to HPLC-DAD and HPLC-MS/MS analysis, hesperidin (HD), hesperetin (HT),
Received in revised form 18 May 2012 nobiletin (NT), and tangeretin (TT) were present in water extracts of sweet orange peel (WESP). The cyto-
Accepted 3 July 2012
toxic effect in 0.2 mM t-BHP-induced HepG2 cells was inhibited by WESP and their bioactive compounds.
Available online 15 July 2012
The protective effect of WESP and their bioactive compounds in 0.2 mM t-BHP-induced HepG2 cells may
be associated with positive regulation of GSH levels and antioxidant enzymes, decrease in ROS formation
Keywords:
and TBARS generation, increase in the mitochondria membrane potential and Bcl-2/Bax ratio, as well as
Cytoprotection
Hesperidin
decrease in caspase-3 activation. Overall, WESP displayed a significant cytoprotective effect against oxi-
Oxidative stress dative stress, which may be most likely because of the phenolics-related bioactive compounds in WESP,
Sweet orange peel leading to maintenance of the normal redox status of cells.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction derived from natural sources on modulation of ROS have recently

Oxidative stress, defined as imbalance of pro-oxidants and anti-
oxidants in the organism, is a well-known key phenomenon in
chronic disease (Tapia et al., 2004). As they are major pro-oxidants,
reactive oxygen species (ROS) are a family of active molecules con-
taining free radicals, and are involved in the modulation of biolog-
ical cell function. However, excessive ROS bring about oxidative
stress and attack cellular biomolecules such as lipid, protein and
DNA. The oxidative stress-induced damage may disrupt cellular
function and membrane integrity, thereby leading to cell death
(Shieh et al., 2010). To avoid redox imbalance and oxidative DNA
damage, living cells have a biological defense system composing
of a wide array of non-enzymatic and enzymatic antioxidants that
convert ROS to harmless species (Huang, Ou, & Prior, 2005). Thus, a
main protective strategy against oxidative damage that is able to
induce cell injury is through the induction of non-enzymatic and
enzymatic antioxidants in cells by substances that show cytopro-
tective effect. Epidemiological studies have provided convincing
evidence that diets rich in fruits, vegetables and good sources of
antioxidants such as polyphenolic compounds are associated with
a lower risk of chronic diseases (Ness & Powles, 1997). Therefore,
the investigations regarding biological effects of compounds
⇑ Corresponding author. Tel./fax: +886 6 2668618.
E-mail address: ipdduh@mail.chna.edu.tw (P.-D. Duh).
received a great deal of attention (Cantin, Moreno, & Gogorcena,
2009).
Citrus fruits have commercial importance due to their nutri-
tional value and special flavor. It is estimated that the world pro-
duction of citrus fruits in 2007–2008 reached 72 million tons;
among which, the major commercially important orange fruit ac-
counted for almost 45 million tons (Khan, Abert-Vian, Fabiano-Tix-
ier, Dangles, & Chemat, 2010). Around 34% of these products were
used for juice production, yielding about 44% of peels of byprod-
ucts (Li, Lo, & Ho, 2006). Therefore, large amounts of peels are pro-
duced each year. The citrus peel residue is the primary waste
fraction. However, it is also a source of molasses, pectin, and limo-
nene and is usually dried and sold as cattle feed, mixed with dried
pulps (Bocco, Cuvelier, Richard, & Berset, 1998). In addition, citrus
peel is also an interesting source of phenolic compounds, including
phenolic acids, polymethoxylated flavones, and glycosylated flava-
nones, which have been extensively studied (Bocco et al., 1998;
Huang & Ho, 2010; Li et al., 2006). Extraction of these bioactive
compounds will thus enhance the value of products from citrus
peel. Therefore, citrus peel should be thoroughly studied for possi-
ble utilisation. Traditionally, orange peel was processed to obtain
valuable fractions for applications in food, drugs, and cosmetics
(Li et al., 2006). In addition, biological effects of citrus peels have
been reported. Tonkan (Citrus tankan Hayata) peel and Ponkan
(Citrus reticulate Blanco) peel, which contain polymethhoxy flav-
ones, displayed anti-inflammatory activity (Huang et al., 2010).

0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.07.041
2120 Z.-T. Chen et al. / Food Chemistry 135 (2012) 2119–2127
As far as sweet orange (Citrus sinensis) peel is concerned, it is found
to have a good total radical antioxidative potential and can be used
as antioxidants in food and medicinal preparations (Anagnosto-
poulou, Kefalas, Papageorgiou, Assimopoulou, & Boskou, 2006).
Moreover, hydroxylated polymethoxyflavones and methylated
flavonoids in sweet orange peel were identified (Li et al., 2006).
Apart from these, the effectiveness of sweet orange peels in protec-
tion against cell injury in a cellular model system caused by oxida-
tive stress has not been previously established. Thus, the present
study aimed to investigate the protective effects of sweet orange
peel and their bioactive compounds on cytotoxicity induced by
oxidative stress.
2. Materials and methods
2.1. Sample preparation
The sweet orange (Citrus sinensis L.) peel, donated by Ma Dou
Agricultural Association, Tainan, Taiwan, were dried by oven at
50 °C, and then ground to a fine powder. The peel powder (100 g)
was extracted with boiling water (1000 ml) and stirred for
40 min. The extract was filtered and the residue was re-extracted
under the same conditions. The combined filtrate was freeze-dried.
The dehydrated powder was suspended in water and called crude
water extracts of sweet orange peel (WESP). To yield soluble frac-
tions of n-hexane, ethyl acetate, and n-butanol, 10 g of WESP was
successively liquid–liquid partitioned with n-hexane, ethyl acetate,
and n-butanol. The collected soluble fractions n-hexane, ethyl ace-
tate, and n-butanol were evaporated to dryness under reduced
pressure and called HESP, EESP, and BESP, respectively.
2.2. High-performance liquid chromatography (HPLC) analysis of
flavonoid compounds in sweet orange peel with diode array and
electrospray ionization mass spectrometric (MS) detection
2.2.1. HPLC analysis
Each extract (20 ll) was analysed using a series 1260 Infinity
high-performance liquid chromatography (HPLC) system (Agilent
Technologies, Santa Clara, CA, USA) equipped with ChemStation
software (version: B.04.03), a model G1379B degasser, a model
G1312B binary gradient pump, a model G1329B autosampler, a
model G1316A column oven, and a model G1315D diode array
detection system. The Symmetry C18 analysis column (2.1 
150 mm; 3.5 lm particle size, Waters) and a precolumn [Sceurity-
Guard C18 (ODS) 4 mm  3.0 mm ID, Phenomenex Inc., Torrance,
CA, USA] was placed in a column oven set at 30 °C. Gradient elution
using solvent A (water, containing 0.1% formic acid, FA) and solvent
B (acetonitrile, containing 0.1% FA) as the mobile phase at a flow
rate of 0.3 mL/min was applied. The gradient conditions were:
0–5 min, 10% B, 5–10 min linear from 10% to 15% B; 10–15 min,
linear from 15% to 35% B; 15–20 min, isocratic at 35% B; 20–
25 min, linear from 35% to 45% B; 25–35 min, linear from 45% to
50% B; 35–45 min isocratic at 50% B, 45–50 min, linear from 50%
to 90% B; 50–55 min held at 90% B then returned to 10% B in
5 min. The injection volume was 20 ll. The absorption spectra of
eluted compounds were scanned within 200–600 nm using the
in-line diode array detector (DAD) monitored at 290, 300, 340
and 365 nm for the UV–Vis absorption.
2.2.2. HPLC-MS/MS analysis
The compounds having been eluted and separated were further
identified with an Agilent 6420 Triple Quadrupole Mass Spectrom-
eter equipped with Mass Hunter software (version:B.01.04) (Agi-
lent Technologies, Santa Clara, CA, USA). The system was
operated in electrospray ionization (ESI) with positive and negative
ionization modes. Nitrogen was used both as a drying gas at a flow
rate of 9 l/min and as nebulising gas at a pressure of 35 psi. The
drying gas temperature was 325 °C, and a potential of 4000 V
was applied across the capillary. The fragmentor voltage was
120 V, and the collision voltage was 20 V. Quadrupole 1 filtered
the calculated m/z of each compound of interest, whilst quadrupole
2 scanned for ions produced by nitrogen collision of these ionised
compounds in the range 100–800 m/z at a scan time of 500 ms/cy-
cle. The identification of flavonoid compounds was carried out by
comparing their retention times and mass spectra provided by
ESI-MS and ESI-MS/MS with those of authentic standards when
available.
2.2.3. Quantification of flavonoids
The content of the four flavonoids was determined by compar-
ison of the HPLC peak areas of the four compounds with those of
reference standards under a wave length of 300 nm. The calibra-
tion curves were constructed by plotting the mean peak areas
against the concentrations of standards.
2.3. Measurement of HepG2 cells viability
HepG2 cells (ATCC No.: CRL-11997) were purchased from Biore-
sources Collection and Research Center (Shin-chu, Taiwan) and cul-
tured in minimum essential medium (MEM) containing 10% fetal
bovine serum, 2 mM glutamine, and maintained in humidified 5%
CO2/95% air at 37 °C. After cells were cultured with WESP, HESP,
EESP, and BESP in the range of 0–1000 lg/ml for 24 h, cell viability
was determined by colorimetric measurement of the reduction
product of (3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) (Yu et al., 2007). In addition, after cells were cul-
tured with WESP in the range of 50–500 lg/ml, and hesperidin
(HD), hesperetin (HT), and nobiletin (NT) at 10 lg/ml in the pres-
ence of 0.2 mM t-BHP or not for 6 h, cell viability was determined
by MTT (Yu et al., 2007). Triplicate samples were run for each set
and averaged.
2.4. Total polyphenolics and total flavonoid content assay
The concentration of phenolic compounds and total flavonoid in
WESP were measured according to previous report and calculated
using gallic acid and rutin as standards, respectively (Yu et al.,
2007). Triplicate samples were run for each set and averaged.
2.5. Lactate dehydrogenase (LDH) assay
HepG2 cells (5  104 cells/ml) were treated as described above.
The cells were cultured with WESP in the range of 50–500 lg/ml,
and hesperidin (HD), hesperetin (HT), and nobiletin (NT) at
10 lg/ml in the presence of 0.2 mM t-BHP or not for 6 h. At the
end of the incubation, 50 ll of media from each well was trans-
ferred to a new plate, and 50 ll of LDH reagent was added and
incubated for 30 min in dark at room temperature. The absorbance
was read at 490 nm using ELISA reader (Thermo Electron Corpora-
tion, Marietta, OH, USA). The LDH leakage was estimated from the
ratio between the LDH activity in the medium and that of the
whole cell content (Alia et al., 2006). Triplicate samples were run
for each set and averaged.
2.6. Measurement of intracellular reactive oxygen species (ROS)
Intracellular reactive oxygen species were determined by using
the 20 ,70 -dichlorofluorescin diacetate (H2DCFDA) fluorescence
probe. Prior to tert-butyl-hydroperoxide (t-BHP) stimulation,
HepG2 cells were cultured with H2DCFDA (10 lM). After 20 min
of incubation, various concentrations of WESP, HD, HT, and NT
 Z.-T. Chen et al. / Food Chemistry 135 (2012) 2119–2127
were added to cells for 30 min, and then incubated with t-BHP
(0.2 mM) for 4 h. After incubation, reactive oxygen species pro-
duced from cells was determined using a Bio-Tek FLx800 micro-
plate fluorescence reader (Winooski, VT, USA) with excitation
and emission wavelengths of 485 and 535 nm, respectively. Tripli-
cate samples were run for each set and averaged.
2.7. Measurement of lipid peroxidation products
Molonaldehyde (MDA) was determined by the method of Buege
and Aust (1978). In brief, 1 ml of the medium was mixed with 1 ml
of 7.5% (w/v) cold trichloroacetic acid (TCA) to precipitate proteins
and then centrifuged at 157.39g. The supernatant was reacted with
1 ml of 0.8% (w/v) thiobarbituric acid (TBA) in boiling water for
45 min. Lipid peroxidation products were estimated by measuring
the concentration of thiobarbituric acid reaction substances
(TBARS) in fluorescence at 530 nm excitation/552 nm emission.
Triplicate samples were run for each set and averaged.
2.8. Evaluation of glutathione
Intracellular GSH levels were determined after staining cell
with chloromethylfluorescein-diacetate (CMF-DA) (Han, Moon,
You, & Park, 2009). CMF-DA passes freely through cell membranes,
but once inside the cell, are transformed into cell-impermeant
products and reacted with the thiol group of GSH. After HepG2
cells were pretreated with WESP in the range of 50–500 lg/ml,
and HD, HT, and NT at 10 lg/ml, 0.2 mM t-BHP was added to the
medium and incubated at 37 °C for 2 h. After incubation, cells were
washed with PBS and treated with CMF-DA (5 mM) for 30 min.
Then, cells were washed with PBS and intracellular GSH was de-
tected by using a Bio-Tek FLx800 microplate fluorescence reader
(Winooski, VT, USA) with excitation and emission wavelengths of
485 and 528 nm, respectively. Triplicate samples were run for each
set and averaged.
2.9. Evaluation of antioxidant enzyme activities
Glutathione peroxidase (GPx) activity was determined as previ-
ously described (Yu et al., 2007). Briefly, 0.1 ml of supernatant was
mixed with 0.8 ml of 100 mM potassium phosphate buffer (1 mM
EDTA, 1 mM NaN3, 0.2 mM NADPH, 1 unit/ml GR, and 1 mM GSH,
pH 7.0) and incubated for 5 min at room temperature. Thereafter,
the reaction was initiated after adding of 0.1 ml of 2.5 mM hydro-
gen peroxide (H2O2). GPx activity was calculated by the change of
the absorbance at 340 nm for 5 min. The catalase (CAT) activity
was determined by the method of (Yu et al., 2007). The 50 ll
homogenate was mixed with 950 ll 0.02 M H2O2 was incubated
at room temperature for 2 min. The CAT activity was calculated
by the change of the absorbance at 240 nm for 3 min. SOD activity
was determined by the method of Yu et al. (2007). The 50 ll of
homogenate was mixed with 100 ll of Tris–HCl buffer (pH 8.2,
50 mmol/l) and distilled water added to a final volume of 980 ll,
then 20 ll of pyrogallol (0.2 mmol/l) was added. The absorbance
was measured at 420 nm, and the SOD activity of sample was re-
ported as unit/mg protein. Triplicate samples were run for each
set and averaged.
2.10. Evaluation of the mitochondrial membrane potential
The mitochondrial membrane potential of HepG2 cells was
determined by a JC-1 mitochondrial membrane potential assay
kit (Cayman Chemical Company, Ann Arbor, MI, USA). HepG2 cells
were incubated in 100 ll medium at 37 °C. After the cells were pre-
treated with WESP in the range of 50–500 lg/ml, and HD, HT, and
NT at 10 lg/ml, the samples were exposed to 0.2 mM t-BHP for 2 h.
2121
Following incubation, the JC-1 dye staining solution was added to
the cells and incubated for 30 min. The fluorescence was detected
using a Bio-Tek FLx800 microplate fluorescence reader (Winooski,
VT, USA) with excitation and emission wavelengths of 560 and
595 nm, respectively. Triplicate samples were run for each set
and averaged.
2.11. Cytosolic caspase-3 activity determination
The caspase-3 activity was determined by a fluorometric assay
kit (Promega-Corporation, Madison, WI, USA). After HepG2 cells
were pretreated with WESP in the range of 50–500 lg/ml, and
HD, HT, and NT at 10 lg/ml for 30 min, the samples were exposed
to 0.2 mM t-BHP for 4 h. The substrate Ac-DEVD-AMC was cleaved
by caspase-3 and fluorescence was detected using a Bio-Tek
FLx800 microplate fluorescence reader (Winooski, VT, USA) with
excitation and emission wavelengths of 485 and 530 nm, respec-
tively. Triplicate samples were run for each set and averaged.
2.12. Western blot
Cells were treated with the extracts for 24 h. After treatment,
cells were collected and lysed in the ice-cold lysis buffer and kept
on ice for 30 min. After centrifugation at 7553.25g for 10 min at
4 °C, the supernatants were collected and the protein contents
were determined by using the BCA protein assay kit (Pierce, Rock-
fold, IL, USA). Each sample, which contained 100 lg proteins, was
separated on 4% SDS–polyacrylamide gels. After electrophoresis,
gels were transferred to nitrocellulose paper. After washing with
distilled water, the membrane was blocked with 5% skimmed milk
in PBST (0.1% v/v Tween-20 in PBS, pH 7.2) for 30 min, and then
immunoblotted with primary antibody at 4 °C for overnight and
then with secondary antibody for 1 h, and visualised using an en-
hanced chemiluminescence (ECL) kit (Amersham, NJ, USA). The
expression levels of Bcl-2 and Bax and b-action proteins were
determined by densitometry and analysed. Triplicate samples were
run for each set and averaged.
2.13. Statistical analysis
Statistical analysis involved use of the Statistical Analysis Sys-
tem software package. Analysis of variance was performed by AN-
OVA procedures. Significant differences between means were
determined by Duncan’s multiple range tests at a level of p < 0.05.
3. Results
The yields of different solvent extracts from sweet orange peel
were determined. The yield of crude water extracts of sweet or-
ange peel was 39.2%. The efficiency of the solvents on the extrac-
tion was in the order n-butanol (3.2%) > ethyl acetate (0.45%) > n-
hexane (0.23%). Obviously, the yields of extracts increased with
increasing polarity of solvent.
The cell viability in the presence of WESP, BESP, EESP, and HESP
for 24 h, respectively, in the range of 0–1000 lg/ml, decreased
with increasing the concentration of extracts (data not shown).
Of the four extracts, the cell survival in the presence of WESP ran-
ged from 0 to 1000 lg/ml was >85%, indicating that WESP showed
no cytotoxicity to HepG2 cells. However, the proliferation of cells
was <80% for BESP, EESP, and HESP at 200 lg/ml, respectively, indi-
cating that BESP, EESP, and HESP significantly affected the survival
of HepG2 cells (p < 0.05). Therefore, WESP was selected as the
samples in the following study.
Polyphenolic compounds are associated with biological activity
and play an important role in preventing oxidative damage,
2122 Z.-T. Chen et al. / Food Chemistry 135 (2012) 2119–2127
therefore, the levels of polyphenolic compounds and flavoniods in
WESP were determined. The data obtained showed that the levels
of polyphenolic compounds and flavonoids in WESP were 49.04
and 29.86 mg/g, respectively. The major compounds in WESP were
analysed by HPLC-DAD and HPLC/ESI-MS/MS. Fig. 1 shows the
HPLC-MS total ion and HPLC-DAD chromatograms of WESP. Table
1 shows the chromatographic and spectral data of four major flavo-
noids of WESP. These four flavonoids were identified as hesperidin
(1), hesperetin (2), nobiletin (3) and tangeretin (4) on the basis of
m/z value of ions detected in ESI mass spectra, retention time (tR)
of HPLC chromatograms and UV–Vis spectra of the compound cor-
responding to the chromatographic peak of reference standards,
and their structures are shown in Fig. 1. The UV–visible and MS
spectral data of these four flavonoids were very similar to those re-
ported in the literature (Kim et al., 2011).
In addition, by calculating from the standard curves of hesperi-
din, hesperetin, nobiletin, and tangeretin, the content of hesperi-
din, hesperetin, nobiletin, and tangeretin in WESP was 30.89 mg/
g, 0.97 mg/g, 0.44 mg/g, and 0.32 mg/g, respectively. As shown in
Fig. 1, hesperidin is the main constituent. However, hesperetin,
nobiletin, and tangeretin, which are bioactive compounds, are
clearly present in WESP. The molecular structure of the four com-
pounds shows that hesperetin is a flavonone, and hesperidin, a
Fig. 1. Total ion chromatogram (top), HPLC-DAD chromatogram (bottom) of WESP, and chemical structures of the identified compounds in WESP. Peak 1: hesperidin, peak 2:
hesperetin, peak 3: nobiletin, and peak 4: tangeretin.
flavonone glycoside, while both nobiletin and tangeretin are poly-
methoxyflavones (PMFs). In addition, they are known for their bio-
logical activity. Therefore, nobiletin, a representative of PMFs, as
well as hesperidin and hesperetin were selected as reference com-
pounds for the following experiments.
To evaluate whether WESP and their bioactive compounds pro-
tected HepG2 cells from oxidative stress induced by 0.2 mM t-BHP,
we first determined the effects of WESP and their bioactive com-
pounds on the growth of HepG2 cells induced by 0.2 mM t-BHP.
As shown in Fig. 2A, 0.2 mM t-BHP significantly decreased the via-
bility of cells (p < 0.05), while treatment of HepG2 cells with differ-
ent concentrations of WESP, HD, HT, and NT significantly protected
cells against t-BHP-induced cytotoxicity (p < 0.05). WESP at 50 lg/
ml, and HD, HT, and NT at 10 lg/ml significantly increased cell via-
bility up to 116.57%, 96.58%, 113.61%, and 81.46%, respectively,
from 56.21% (p < 0.05). Clearly, WESP, HD, HT, and NT exhibited
comparable protection against t-BHP-induced cytotoxicity.
LDH was widely used as a marker to study the toxicity of toxi-
cants. It was found that HepG2 cells treated with 0.2 mM t-BHP
showed greater LDH release compared with the control, indicating
an unequivocal cell damage in HepG2 (Fig. 2B). However, evident
decrease in LDH release from cells is observed after exposure to
different concentrations of WESP, HD and HT at 10 lg/ml. LDH
 Z.-T. Chen et al. / Food Chemistry 135 (2012) 2119–2127 2123

Table 1
Chromatographic and spectral data of flavonoids in WESP.

Peak Compound tR (min) UV–vis kmax (nm) [M + H]+ MS/MS (m/z) [M  H] MS/MS (m/z)
1 Hesperidin 17.94 284, 329 609 301, 285, 242, 215
2 Hesperetin 22.52 287, 329 301 285, 257, 242, 215, 199
3 Nobiletin 28.26 270, 334 403 388, 373, 357, 355, 327, 301
4 Tangeretin 30.90 270, 325 373 358, 343, 328, 325, 300, 297

(A) tration-dependent manner. HD, HT, and NT also significantly de-

creased the ROS generation in t-BHP-induced HepG2 cells
140 WESP t-BHP 0.2mM
HD
(p < 0.05). The above results demonstrate that WESP, HD, HT, and
a a a b
HT NT act as scavengers of the ROS generated by t-BHP in HepG2 cells.
120 NT * * * a
* In order to evaluate the possible mechanism involved in the
Cell viability (% of control)

*
protective effect of WESP and their bioactive compounds on cyto-
100
a toxicity. MDA formation in t-BHP-induced HepG2 cells was deter-
* mined. As shown in Table 2, t-BHP-induced HepG2 cells pretreated
80
with different concentrations of WESP, HD, HT, and NT signifi-
60 # cantly reduced the amount of MDA (p < 0.05), as compared with
t-BHP-induced HepG2 cells, indicating that WESP, HD, HT, and NT
40 exhibited a remarkable protective activity on oxidative stress of li-
pid peroxidation.
20 Table 2 also shows the effect of WESP and their bioactive com-
pounds on glutathione (GSH) levels of HepG2 cells induced by
0
Control t-BHP 0.2mM 50 200 500 10 0.2 mM t-BHP. 0.2 mM t-BHP significantly decreased the GSH lev-
Concentration (µg/ml) els of the cells (p < 0.05), however, the GSH levels increased with
(B) increasing concentrations of WESP, as compared with t-BHP-in-
160 duced HepG2 cells. This finding reveals that WESP may positively
WESP regulate the GSH content in t-BHP-induced HepG2 cells. HT also
HD
140 positively regulated the GSH content in t-BHP-induced HepG2
HT t-BHP 0.2mM
NT cells, but the effect of HD and NT was not significant (p > 0.05).
120 # a
To further characterize the antioxidant enzymes of HepG2 cells
LDH release(%)

a in culture exposed to t-BHP, the activities of CAT, SOD, and GPx of

100
a a*
a a
80 * *
60
40
20
0 activity.
Control t-BHP 0.2mM 50
Concentration (µg/ml)
Fig. 2. Effects of water extracts of sweet orange peel (WESP), hesperidin (HD),
hesperetin (HT) and nobiletin (NT) on cell viability (A) and LDH leakage (B) in
0.2 mM t-BHP-induced HepG2. The cells were treated with WESP, HD, HT, and NT,
respectively, and exposure to 0.2 mM t-BHP for 6 h. Data are presented by
means ± SD (n = 3). #p < 0.05 compared with the control group and ⁄p < 0.05
compared with 0.2 mM t-BHP-induced cells alone. Bars with different lower case
letters in the different concentrations of WESP and HD, HT, and NT at 10 lg/ml
indicate significant difference, respectively (p < 0.05).
release is also influenced by 10 lg/ml of NT, but not significantly
(p > 0.05). Thus, it is clear that pretreatment with WESP and their
bioactive compounds protected HepG2 cells against t-BHP-induced
cytotoxicity.
To understand whether the observed cytoprotective effect of
WESP and their bioactive compounds is attributed to the reduction
of oxidative stress, we next determine the effect of the samples on
intracellular ROS generation of HepG2 cells exposed to t-BHP. As
shown in Table 2, ROS generation increased significantly when
the cells were treated with 0.2 mM t-BHP, indicating that 0.2 mM
t-BHP had a strong effect on ROS generation. When the cells were
pretreated with WESP, ROS generation was decreased in a concen-
t-BHP-induced HepG2 cells were determined. Table 2 shows the ef-
* fects of WESP, HD, HT, and NT on antioxidant enzymes in HepG2
*
cells induced by 0.2 mM t-BHP. WESP, at 200 and 500 lg/ml, sig-
nificantly raised CAT and GPX activities (p < 0.05), as compared
with cells treated with t-BHP. For SOD activity, WESP in the range
of 50–500 lg/ml did not alter SOD activity. HD and NT at 10 lg/ml
significantly raised CAT and GPx activity (p < 0.05). The activities of
SOD and GPx were raised by 10 lg/ml of HT, but did not affect CAT
200 500 10
Fig. 3 shows the effect of WESP, HD, HT and NT on the ratios of
Bcl-2/Bax in 0.2 mM t-BHP-induced HepG2 cells. As expected, the
ratio of Bcl-2/Bax in HepG2 cells treated with 0.2 mM t-BHP was
0.68 compared with the control (1.0). The ratios of Bcl-2/Bax in-
creased with increasing concentrations of WESP, and were signifi-
cantly raised to 1.73, 1.39 and 1.27 for HD, HT, and NT (p < 0.05),
respectively. Clearly, WESP, HD, HT, and NT markedly prevented
the decrease in the expression of Bcl-2 protein, whereas they
markedly suppressed the increase of Bax expression in t-BHP-in-
duced HepG2 cells.
Many studies noted that mitochondria play an important role in
death signal transduction. Thus, we further explored the effect of
WESP and their bioactive compounds on t-BHP-induced mitochon-
dria in HepG2 cells, and the mitochondrial membrane potential
(DWm) of HepG2 cells was determined. As shown in Fig. 4A,
0.2 mM t-BHP significantly decreased the DWm of HepG2 cells,
implying that t-BHP induced mitochondria damage. Treatment of
HepG2 cells with WESP, HD, HT and NT plus t-BHP significantly in-
creased the DWm (p < 0.05), compared with HepG2 cells induced
by 0.2 mM t-BHP. These data suggest that WESP, HD, HT, and NT
may exert the protective effects on t-BHP-induced HepG2 cells
against oxidative stress via prevention of mitochondrial
dysfunction.
2124 Z.-T. Chen et al. / Food Chemistry 135 (2012) 2119–2127

Table 2
The effects of water extracts of sweet orange peels (WESP) on reactive oxygen species (ROS), malonaldehyde (MDA), glutathione (GSH), and enzyme activity in t-BHP-induced
HepG2 cells.

Sample ROS (%) TBARS (%) GSH (%) Activity (nmol/min/ mg protein) SOD (l/mg protein)
GPX CAT
Control 29.42 ± 2.09 100.00 ± 17.68 100.00 ± 12.91 5.43 ± 0.38 1493.86 ± 9.35 0.39 ± 0.01
t-BHP 0.2 mM 100.0 ± 7.89a 156.47 ± 6.63a 54.08 ± 2.29a 3.26 ± 0.12a 1016.95 ± 21.44a 0.23 ± 0.01a
WESP 50 lg/ml 73.64 ± 9.38b,A 131.18 ± 7.86b,A 74.48 ± 0.29b,A 3.58 ± 0.76 A 1061.05 ± 176.80 A 0.22 ± 0.01 A
WESP 200 lg/ml 41.88 ± 7.69b,B 130.13 ± 6.73b,A 78.70 ± 6.24b,B 4.88 ± 0.17 b,A 1341.15 ± 12.88 b,A 0.29 ± 0.08 A
WESP 500 lg/ml 41.69 ± 4.56b,B 139.26 ± 11.05b,A 98.34 ± 0.59b,B 5.32 ± 0.06 b,A 1345.57 ± 15.04 b,A 0.27 ± 0.03 A
HD 10 lg/ml 55.62 ± 7.50b,AB 133.94 ± 13.07b,A 46.06 ± 6.64B 4.71 ± 0.01b,B 1324.71 ± 72.74 b,A 0.22 ± 0.01 A
HT 10 lg/ml 49.40 ± 1.88b,B 115.89 ± 16.61b,A 68.05 ± 1.81b,A 5.04 ± 0.25 b,B 1103.10 ± 73.37 b,A 0.33 ± 0.02b,A
NT 10 lg/ml 68.30 ± 4.85b,A 110.18 ± 14.59b,A 35.82 ± 0.86b,C 6.18 ± 0.21 b,A 1351.00 ± 76.11 b,A 0.21 ± 0.01 A

The data are displayed as means ± SD (n = 3). The cells were exposed to 0.2 mM t-BHP for 4, 2, 2, and 2 h for determination of ROS, TBARS, GSH, and enzyme activity,
respectively. ap < 0.05, compared with control. bp < 0.05, compared to treatment with 0.2 mM t-BHP. Values with different uppercase letters in different concentrations of
WESP, and with different uppercase letters in the same concentration of HD, HT, and NT indicate significantly different, respectively (p < 0.05). HD, hesperidin; HT, hesperidin;
NT, nobiletin.

2.5
(A)
WESP 120
HD t-BHP 0.2mM
WESP
HT
2.0 HD
NT

Mitochondrial membrane potential

a 100 HT
NT
a *a t-BHP 0.2mM
b
Bcl-2/Bax (fold)

1.5 * * 80 a
a
(% of control)

b * a a ab
*
c * 60 * * a *
1.0
* # *
#
40
0.5

20

0.0
Control t-BHP 0.2mM 50 200 500 10 0
Control t-BHP 0.2mM 50 200 500 10
Concentration (µg/ml)
Concentration (µg/ml)
Fig. 3. Effects of water extracts of sweet orange peel (WESP), hesperidin (HD),
hesperetin (HT) and nobiletin (NT) on the protein levels of Bcl-2 and Bax in HepG2
(B)
WESP
cells induced by 0.2 mM t-BHP. The cells were treated with WESP, HD, HT, and NT
HD
Intracellular Caspase 3 production (%)

respectively, and exposure to 0.2 mM t-BHP for 2 h. Data are presented by 300 HT t-BHP 0.2mM
means ± SD (n = 3). #p < 0.05 compared with the control group and ⁄p < 0.05 NT a
#
compared with 0.2 mM t-BHP-induced cells alone. Bars with different lower case
letters in the different concentrations of WESP and HD, HT, and NT at 10 lg/ml
indicate significant difference, respectively (p < 0.05).
200 b
b
To understand the effect of WESP on t-BHP-induced apoptotic a **
a
pathway in HepG2 cells, the caspase-3 activity was tested. As *
a *
shown in Fig. 4B, the caspase-3 activity was significantly increased 100
after 0.2 mM t-BHP treatment (p < 0.05), compared with the con-
*
trol. However, WESP in the range of 50–500 lg/ml and HD, and
HT at 10 lg/ml markedly decreased the activation of caspase-3 in
t-BHP-induced HepG2 cells, indicating that t-BHP-induced cas- 0
Control t-BHP 0.2mM 50 200 500 10
pase-3 activation was significantly inhibited by WESP, HD, and
Concentration (µg/ml)
HT (p < 0.05).
Fig. 4. Effects of water extracts of sweet orange peel (WESP), hesperidin (HD),
hesperetin (HT) and nobiletin (NT) on mitochondrial membrane potential (A) and
caspase-3 activity (B) in HepG2 celll induced by 0.2 mM t-BHP. The cells were
4. Discussion treated with WESP, HD, HT, and NT respectively, and exposure to 0.2 mM t-BHP 2 h
and 4 h for determination of mitochondrial membrane potential and caspase-3
In this study, an intracellular system was employed to test the activity, respectively. Data are presented by means ± SD (n = 3). #p < 0.05 compared
cytotoxic effect from various solvent extracts of sweet orange peel. with the control group and ⁄p < 0.05 compared with 0.2 mM t-BHP-induced cells
alone. Bars with different lower case letters in the different concentrations of WESP
On the basis of the data obtained, the sample extracted from water and HD, HT, and NT at 10 lg/ml indicate significant differences, respectively
showed no significant cytotoxicity. However, the relatively weak (p < 0.05).
polar solvent extracts show significant cytotoxic effects on HepG2
cells. This observation implies that cytotoxicity of the extracts from
sweet orange peel increased with decreasing polarity of solvent. for subsequent experiments that aim to evaluate the cytoprotec-
For this reason, water extracts of sweet orange peel were chosen tive effects against oxidative damage.
 Z.-T. Chen et al. / Food Chemistry 135 (2012) 2119–2127
In the case of cell viability oxidatively damaged by t-BHP, the
WESP showed significant protective activity (Fig. 2A). In the case
of t-BHP-induced LDH release (Fig. 2B), the WESP showed the same
trend. Numerous studies noted that t-BHP, an organic hydroperox-
ide, induces an array of cellular dysfunctions, including generation
of peroxyl radicals, peroxidation of membrane lipids, glutathione
and protein thiol deletion, and DNA damage, and eventually lead-
ing to cell death (Garcia-Alonso, Ros, & Periago, 2006). However,
WESP prevented t-BHP-induced cell death, evidenced by MTT test
and LDH assay, indicating that they are cytoprotective. Moreover,
HD, HT, and NT, which are major compounds present in WESP, dis-
played cell protection, resulting from increase in MTT and decrease
in LDH leakage. These protective effects of HD, HT, and NT may in
part be responsible for the cytoprotection of WESP.
Oxidative stress can be defined as an imbalance between the
oxidant and antioxidant system. Under normal circumstances the
levels of ROS are low enough to be removed by the natural defense
systems of the cell. However, when ROS is induced by oxidants to
such an extent that cellular defenses are overwhelmed, the cells
are exposed to oxidative stress, consequently leading to cell injury
(Alia, Romos, Mateos, Bravo, & Goya, 2005). Thus, phytochemical or
antioxidant therapy is therefore regarded as a promising strategy
to prevent cells from oxidative damage (Kaliora, Dedoussis, &
Schmidt, 2006). In addition, apoptosis of the cells can be induced
by ROS, leading to pathological cell death. According to the results,
0.2 mM t-BHP treatment induced ROS generation. As expected,
treatment with WESP, HD, HT, and NT decreased ROS generation.
These results suggest that the protective effect of WESP, HD, HT,
and NT on the cytotoxicity of HepG2 cells may in part be attributed
to the scavenging ROS, consequently preventing t-BHP-induced
oxidative damage in HepG2 cells.
Lipid peroxidation is responsible for the degradation of unsatu-
rated fatty acids and cholesterol in lipid bilayers, and has been sug-
gested to play an important role in the development of toxicity
(Rosa et al., 2008). In addition, peroxidation of the cell membrane
phospholipids and an accumulation of lipid peroxides may alter
membrane fluidity and permeability, leading to disruption of
membrane structure and function (Garcia-Alonso et al., 2006). In
other words, cell injury death was associated with lipid peroxida-
tion. According to the data obtained, t-BHP induced significant
MDA formation (Table 2), indicating that development of lipid per-
oxides occurred in t-BHP-induced HepG2 cells, consequently caus-
ing cells death. WESP, HD, HT, and NT showed a remarkable and
comparable antioxidant activity against t-BHP-induced oxidative
stress, showing a clear protective effect on lipid peroxidation. This
finding revealed that WESP and their bioactive compounds, HD,
HT, and NT, were able to preserve the integrity of biological mem-
branes from the detrimental oxidative process caused by free rad-
icals in vitro (Rosa et al., 2008).
Glutathione (GSH), widely distributed in animal tissues, plants
and microorganisms, is well known to function both as a reductant
and as a nucleophile due to its side-chain sulfhydryl (SH) residue in
cysteine of GSH (Anderson, 1985). Numerous studies showed that
GSH levels may be induced to increase by some extracts and natu-
rally occurring phenolic compounds (Yu et al., 2007). In an attempt
to further explain the observed cytoprotective effect of WESP, we
determined the GSH levels in t-BHP-induced HepG2 cells co-incu-
bated with WESP and their bioactive compounds. According to the
data obtained, t-BHP was significantly cytotoxic to HepG2 cells,
accompanied by ROS generation, lipid peroxidation and a marked
depletion in GSH levels. Indeed, severe GSH depletion leaves cells
more vulnerable to oxidative damage and is normally associated
with calcium homeostasis disruption, which ultimately causes cell
death (Lima et al., 2007). However, treatment with WESP, HD, HT
and NT prevented decrease in GSH levels induced by t-BHP. Clearly,
the protection against GSH depletion was probably the highly
2125
relevant effect of WESP providing an effective cellular reducing
agent, and thereby leading to the detoxification of xenobiotics.
Several antioxidant enzymes such as SOD, CAT and GPx exist in
the cells that convert ROS into less noxious compounds. These anti-
oxidant enzymes provide the first line of defense against superox-
ide and hydrogen peroxides (Masella, Benedetto, Vari, Filesi, &
Giovannini, 2005). Changes in the activity of these enzymes can
be considered as biomarkers of the antioxidant response (Esmaeili,
Sonboli, & Noushabadi, 2010). In the present study, when HepG2
cells were incubated with t-BHP, GSH levels and antioxidant en-
zymes such as SOD, CAT and GPx were reduced, but ROS generation
and lipid peroxidation were significantly increased. Pretreatment
of cells with WESP resulted in suppression of ROS generation and
lipid peroxidation, probably as a result of elevation of GSH levels
as well as CAT and GPX activity. We speculated that an increase
in activity of catalase and GPx as well as enhancement of GSH lev-
els in t-BHP-induced HepG2 cells by WESP clearly indicated a po-
sitive response of the cell defense system against oxidative
condition. Many reports have noted that polyphenols are able to
activate the cellular antioxidative system by modulating the
expression of some antioxidant enzymes. In addition, phytochemi-
cals have also been shown to stimulate synthesis of antioxidant en-
zymes and detoxification systems at the transcriptional level,
through antioxidant response elements (Masella et al., 2005). In
contrast, the activation status of a variety of signalling molecules
and transcriptional factors are up-regulated by ROS (Kao et al.,
2010). In the present study, the activity of CAT and GPx was posi-
tively modulated by WESP, indicating that WESP may activate the
cellular antioxidative enzymes. Although transcriptional effects of
WESP have not been examined, we speculated that WESP not only
reduced oxidative stress directly through scavenging free radicals
and restoring antioxidants, but also inactivated signalling mole-
cules and transcriptional factors, leading to attenuation of oxida-
tive stress.
The fact that apoptosis is associated with the generation of ROS
in several experimental models has been proven. Excessive ROS
generation ultimately causes apoptotic or necrotic cell death. As
mentioned above, 0.2 mM t-BHP induced excessive ROS generation
in HepG2 cells, leading to HepG2 cell death. We then investigated
whether the protective effects of WESP against t-BHP-induced
HepG2 cell death are regulated by its anti-apoptotic activity.
Expression of proteins involved in apoptosis is taken as a marker
regarding the execution of molecular actions. For instance, proteins
of Bcl-2 family such as Bcl-2 and Bax are major regulators of the
intrinsic mitochondria-medicated pathway (Papi et al., 2008).
The ratios of Bal-2/Bax in a cell may determine the propensity of
the cell to undergo apoptosis, with an excess of Bcl-2 resulting in
cell survival and an excess of Bax resulting in cell death (Nagata,
1997). Indeed, Bcl-2 blocks apoptosis by inhibiting the release of
mitochondrial protein and phosphatidylserine exposure, thus,
overexpression of Bcl-2 is associated with a diminished apoptotic
response (Papi et al., 2008). Hence, we evaluated the effect of
WESP, HD, HT, and NT on Bcl-2 and Bax expression. As shown by
Western blot analysis, WESP, HD, HT, and NT caused an up-regula-
tion of Bcl-2 protein and a down-regulation of Bax in t-BHP-in-
duced HepG2 cells, causing increase in ratio of Bcl-2/Bax.
Obviously, WESP, HD, HT, and NT affected the protein expression
of Bcl-2 and Bax in the mitochondria of t-BHP-induced HepG2 cells.
This observation may be responsible for the amelioration of de-
crease in mitochondrial membrane potential in t-BHP-induced
HepG2 cells treated with WESP, HD, HT, and NT, thereby prevent-
ing cell death.
In general, a change in the externalization of membrane phos-
phatidylserine follows the reduction of mitochondrial membrane
potential (DWm). Therefore, detection of DWm may provide an
early indication of the initiation of cellular apoptosis (Weng, Yang,
2126 Z.-T. Chen et al. / Food Chemistry 135 (2012) 2119–2127
Ho, & Yen, 2009). In addition, apoptosis is accompanied by the
signs of mitochondrial dysfunction, including a loss of inner mito-
chondrial transmembrane potential and the release of soluble
intermembrane proteins, including cytochrome c (Park et al.,
2003). In the current study, t-BHP showed a reduced DWm
(Fig. 4A). This result might suggest that apoptosis was indeed ini-
tiated by treatment with t-BHP. However, treatment of HepG2 cells
with WESP, HD, HT, and NT significantly increased DWm compared
with cells induced by t-BHP, indicating that the apoptosis of t-BHP-
induced HepG2 cells was ameliorated by WESP, HD, HT, and NT.
Apoptosis is generally associated with activation of a caspase
cascade and the family of Bcl-2 proteins. Two separable pathways
including intrinsic and extrinsic pathway leading to caspase activa-
tion have been characterized (Johnstone, Ruefli, & Lowe, 2002). For
the intrinsic pathway, the disruption of mitochondrial membrane
causes the release of cytochrome c to the cytosol. Cytochrome c
functions with Apaf-1 to induce caspase-9, thereby initiating cas-
pase-3, which precedes the onset of apoptosis (Johnstone et al.,
2002; Park et al., 2003). Once caspase-3 is activated, it might result
in DNA fragmentation (Park et al., 2003). Thus, caspase activation is
the initiating trigger of the apoptosis pathway. In the present
study, we find that t-BHP-induced rise in oxidative stress is associ-
ated with increased caspase-3, which supports the role of caspase-
3 in t-BHP-induced apoptosis. From Fig. 4B, however, treatment of
HepG2 cells with WESP, HD, HT, and NT significantly decreased the
activity of caspase-3. We suggest that the cleavage of initiator and
effector caspase was blocked by WESP, HD, HT and NT. This prob-
ably explains the cytoprotective effect of WESP and their bioactive
compounds on t-BHP-stimulated in vitro cell death may be attrib-
uted partly to the suppression of apoptosis of HepG2 cells.
Many studies have shown that naturally occurring compounds
such as polyphenolic compounds exert remarkable biological
activity. Peel of citrus fruits contains phenolic compounds. The lev-
els of polyphenolic compounds and flavonoids in WESP were 49.04
and 29.86 mg/g, respectively. These levels are relatively higher
compared with the results of our previous studies, which showed
levels of polyphenolics at 36.3 and 21.6 mg/g for napiergrass (Pen-
nisetum purpureum S.) and roasted coffee residues (Yu et al., 2007).
We suggest that the high levels of polyphenolics in WESP may con-
tribute to the cytoprotective effect on t-BHP-induced HepG2 cells.
In addition, both HPLC and LC-MS/MS analyses of WESP identified
compounds including hesperidin, hesperetin, nobiletin, and tang-
ertin. Hesperidin and hesperetin are flavanones, while nobiletin
and tangeretin are polymethoxylated flavones. Flavonones have
been reported to possess a wide range of biological and pharmaco-
logical activities. For instance, hesperidin, a flavanone glycoside,
improves vascular integrity, decreases capillary permeability and
is given as a supplement to patients with blood vessel fragility
(Londono-Londonono et al., 2010). In addition, hesperidin was re-
ported to be less active than hesperetin, a flavanone, when evalu-
ated at 10 lg/ml, suggesting a role of sugar moiety (Londono-
Londonono et al., 2010). Nobiletin, a polymethoxyflavone, also
showed immunomodulatory, antiatherogenic activity and other
biological activities (Huang et al., 2010). In the present study,
HD, HT, and NT significantly inhibited oxidative stress in HepG2
cells induced by t-BHP in various experimental models tested. This
may in part be responsible for the ameliorating effect of WESP on
the oxidative damage of HepG2 cells induced by t-BHP. In addition,
besides HD, HT, and NT, sweet orange peel contains other bioactive
compounds, such as polymethoxyflavones (Li et al., 2006), phenolic
acids, flavonoids, limonoids, and fiber (Huang et al., 2010). In the
present study, according to the HPLC analysis, there are other
peaks present in WESP, suggesting that other uncharacterized bio-
active compounds may be present in WESP. Collectively, these
unidentified compounds may be as a result of synergy between
compounds, thereby leading to the protective effect of WESP
against oxidative stress in t-BHP-induced HepG2 cells.
To the best of our knowledge, this study is the first report on the
evaluation of cytoprotective effect of WESP in t-BHP-induced
HepG2 cells. The possible mechanism is that WESP displayed sig-
nificant scavenging ROS in t-BHP-induced HepG2 cells. The de-
crease in ROS generation appeared in parallel with the decrease
in lipid peroxidation, as well as with up-regulation of GSH levels
and antioxidant enzyme activity. Moreover, the direct scavenge
of ROS by WESP may regulate the expression of Bcl-2 family pro-
teins, mitochondrial function and caspase activity. These appear
to be associated with the explanation of why WESP can prevent
the cytotoxicity of HepG2 cells induced by t-BHP. In addition, most
likely WESP exerted their protective action on oxidative damage in
t-BHP-induced HepG2 cells because of the phenolics-related bioac-
tive compounds in WESP. Further investigation in vivo is under
way.
Acknowledgments
This research work was supported by research grants from the
National Science Council of the Republic of China NSC 99-2313-B-
041-002-MY3.
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