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In vitro and in vivo antioxidant activities of a flavonoid isolated from celery

(Apium graveolens L. var. dulce)

Article · November 2013

DOI: 10.1039/c3fo60273g · Source: PubMed

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In vitro and in vivo antioxidant activities of a

flavonoid isolated from celery (Apium graveolens
Cite this: Food Funct., 2014, 5, 50
L. var. dulce)
Peng Li, Jia Jia, Daihui Zhang, Jingli Xie,* Xueshu Xu and Dongzhi Wei
Published on 15 November 2013. Downloaded on 18/06/2014 16:28:07.

Received 16th July 2013
Accepted 11th October 2013
DOI: 10.1039/c3fo60273g
www.rsc.org/foodfunction
In the present article, a flavonoid was separated and purified from celery leaf through ethanol extraction,
column chromatography and crystallization. The product was identified as apiin by LC/ESI-MS, and its
antioxidant activities were evaluated in vitro, including by 1,1-diphenl-2-picrylhyrazyl free radical (DPPHc),
superoxide radical (O2
c) and hydroxyl radical (OHc) scavenging assays. IC50 values were 68.0 mg ml
1 in
the DPPH assay, 0.39 mg ml
1 in the O2
c assay and 48.0 mg ml
1 in the OHc assay. The antioxidant
activities were investigated in vivo with the use of mice models. All data were measured including the
contents of maleic dialdehyde (MDA) and lipofuscin (LPF), the activities of superoxide dismutase (SOD),
glutathione peroxidase (GSH-Px) and catalase (CAT), and the total antioxidant capacity (TAOC), in the
serum, brain, heart, liver and kidney. Results showed that apiin had a remarkable scavenging activity on
MDA and LPF, promoted TAOC and significantly enhanced the activities of SOD, GSH-Px and CAT.

non-nutrients, such as volatile oils (a-selinene, butylphalide,

1. Introduction
Reactive oxygen species (ROS) generated by NADPH oxidase
during oxidative phosphorylation, are normal components of
healthy cells. ROS are also mediators of the rst defensive
actions of cells and are involved in phagocytosis, apoptosis and
detoxication.1 Furthermore, ROS are toxic and can have a
mutagenic effect on all types of cells via the oxidation of DNA,
membrane lipids and proteins.2 Fortunately, there exist some
natural scavenging systems, for instance, antioxidant enzymes
and polyphenols, which can destroy ROS.3–8 Phenolic
compounds promote human health through reducing oxidative
phenomena and decrease the risk of several diseases.9 Flavo-
noids are one of the main subclasses of phenolic compounds,
comprised of a wide distribution of phytochemicals consisting
of two aromatic rings linked by three carbons in an oxygenated
heterocycle. Studies have shown that avonoids are associated
with a low risk of these ROS related diseases and damage,10 and
research has focused on the development of techniques to
obtain safe and effective antioxidants from natural plants.
Celery (Apium graveolens L. var. dulce) is a herbaceous biennial
plant in the family Apiaceae, originating from the coasts of
western and northern Europe.11 It is distinguished as a low fat
food and a source of digestible carbohydrates, proven rich in
bioactive compounds such as vitamins, free amino acids,
minerals and a high amount of dietary bers, in addition to
State Key Laboratory of Bioreactor Engineering, Department of Food Science and
Technology, East China University of Science and Technology, P. O. Box 283, East
China University of Science and Technology, 130 # Meilong Rd, Shanghai 200237,
P. R. China. E-mail: jlxie@ecust.edu.cn; Fax: +86-21-64252563; Tel: +86-21-64252563
neocoidilde, celerin), organic acids (chlorogenic acid, caffeic
acid), bergapten, niacin, inositol etc.12 Among these
compounds, apigenin, one of the main bioactive components in
celery, belongs to a class of avones, existing in the form of the
avonoid, apiin. Apigenin can relax rat thoracic aorta,
predominantly by means of suppressing Ca2+ inux through
both voltage- and receptor-operated calcium channels, which
contributes to its antihypertensive activity. Furthermore, api-
genin has genotoxic and radioprotective effects on radiation-
induced chromosome aberrations in human lymphocytes B.
Under the protection of apigenin, the frequency of cytokinesis-
block micronuclei can be decreased.13
However, in comparison to the attention on the biological
activity of apigenin, there are few reports on the systematic
investigation of the antioxidant activity of apiin in vitro and in
vivo. In particular there are few reports on the effects of apiin on
enzymatic activities in vivo, such as superoxide dismutase
(SOD), catalase (CAT) and glutathione (GSH), which play an
important role in the antioxidant systems of living organisms
that counteract reactive species to reduce the damage they
cause. Consequently, the objective of this study was to extract,
separate and identify this avonoid from celery, and then
determine its antioxidant activities through various in vitro
methods and in vivo models.
2. Material and methods
2.1. Materials
Leaves of Apium graveolens L. var. dulce were purchased from a
local supermarket. 1,1-Diphenyl-2-picrylhydrazyl was purchased

50 | Food Funct., 2014, 5, 50–56 This journal is © The Royal Society of Chemistry 2014

Paper
from Sigma-Aldrich, China (Shanghai, China). Rutin, sodium
carboxymethyl cellulose (Na-CMC), and pyrogallol acid were
purchased from Beijing Chemical Reagents Company (Beijing,
China). Other materials used were safranine T, ethylene
diamine tetraacetic acid (EDTA), trishydroxymethylamino-
methane (Tris), and silica gel (75–150 mm), obtained from
Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Assay
kits for MDA, LPF, SOD, GSH-Px, CAT and TAOC were obtained
from Nanjing Jiancheng Institute of Bioengineering (Nanjing,
China). All solutions were freshly prepared with distilled water.
Stock solutions of test extracts and compounds (0.5 mg ml
1)
were prepared in anhydrous ethanol. Appropriate blanks were
used for the individual assays.
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2.2. Preparation and separation of the plant extracts
The preparation of plant extracts was performed according to
the method of Kähkönen,14 with some modications. The leaves
of celery were dried at 60  C, powdered and extracted twice by
70% (v/v) ethanol solution (dried powder : ethanol solution was
1/15 (w/w)) at 80  C for 4 h. Aer ltration, the ltrate was
concentrated under vacuum by rotary evaporation to give the
sample S1, ready for further extraction. 5 ml S1 was subjected to
RP-HPLC on an Ultimate 3000 C18 semi-prep column (10 mm 
150 mm; DIONEX, USA), and eluted with a gradient mobile
phase (ethyl acetate : methanol (90 : 10) / ethyl aceta-
te : methanol (50 : 50) / methanol) within 50 minutes at a ow
rate of 36 ml h
1, while being monitored at 215 nm. Fractions
were collected automatically before being checked by silica gel
thin-layer chromatography (TLC). The TLCs were developed in
ethyl acetate : methanol (90 : 10) and the thin-layer was visual-
ised under UV 365 nm aer spraying with a solution of AlCl3 in
ethanol. The fractions with Rf ¼ 0.4 were combined, dried and
recrystallized from ethanol at 4  C.
2.3. Identication of avonoid by LC/ESI-MS
LC/ESI-MS was used for avonoid detection and identica-
tion15,16 with a reversed-phase BDS Hypersil C18 (3 mm particle
size; 150  2.1 mm, i.d.) column. The solvent system was an
acetonitrile–water mixture (A) and formic acid (B), in the
following gradient: 0 min, 20% A; 10 min, 25% A; 15 min, 100%
A. Elution was performed at a solvent ow rate of 0.2 ml min
1.
Detection was accomplished with a Thermo Finnigan Deca XP
MAX LCQ ion trap mass spectrometer equipped with an ESI
interface (San Jose, CA, USA) (source voltage 3.5 kV; capillary
temperature 320  C). The mass spectrometer was operated in
negative-ion mode, with a scan range from m/z 100–800 (scan
rate 1.2 scans s
1).
2.4. Determination of antioxidant activity in vitro
2.4.1. DPPH scavenging assay. The scavenging activity with
respect to the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH),
was determined by a modied method according to Wu17 and
Luo.18 Briey, 1 ml ethanol solution of DPPH (100 mM) was
added to 1 ml extract in different concentrations. The samples
were incubated at room temperature in the dark for 30 min and
the optical absorbance was measured at 517 nm. The control
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sample was 1 ml of 100 mM DPPH mixed with 1 ml of ethanol.
The percent scavenging ratio (%) was calculated by (1
As/Ac) 
100 (where As refers to the absorbance of the extract and Ac
refers to the absorbance of the control). Rutin was used as a
positive control. Triplicate experiments were carried out and a
dose response curve was plotted to determine the IC50, which is
dened as the concentration required to obtain a scavenging
capacity of 50%. A lower IC50 value corresponds to a greater
antioxidant activity.
2.4.2. Superoxide radical-scavenging assay. In this experi-
ment, the superoxide radical was generated by the autoxidation
of pyrogallol acid (PA) and the scavenging radical activity was
determined by the method described by Marklund and Mar-
klund.19 Pyrogallol acid (PA) is readily auto-oxidized at pH 8.2,
generating superoxide radicals and an intermediate product
which exhibits a strong absorbance at 320 nm. At the beginning
of the PA auto-oxidization, the observed absorbance is linear
with respect to time. However, when the antioxidant inhibits
the superoxide radical, the generation velocity of the interme-
diate product is slowed down. The activity was calculated from
the tempered slope of the resulting absorbance–time curve. The
reaction was carried out in a tube with 2.8 ml Tris–HCl buffer
(200 mM, pH 8.2). The mixture was incubated at 25  C for
10 min and 0.1 ml extract solution in varying concentrations
was added before preheating. The reaction was started by the
addition of 0.1 ml PA solution (3 mM, preheated to 25  C) into
the tube and the reaction process was measured against
distilled water at 320 nm. The absorbance was recorded every
30 s during the rst 4 min. The slope (DA min
1) of the time
response curve represented the value of the oxidation velocity
(V). The control sample was made using 0.1 ml distilled water
instead of extract solution. The percent inhibition activity (%)
was calculated by (1
Vs/Vc)  100 (where Vs refers to the
oxidation velocity in the sample containing extract, and Vc refers
to oxidation velocity in the control sample). Rutin was used as a
positive control.
2.4.3. Hydroxyl radical-scavenging assay. The hydroxyl
radical scavenging activity was determined according to the
method of Zhong,20 with some modications. Hydroxyl radicals,
generated by the reaction of a Fe(II) complex with H2O2 in the
presence of acid, can easily cross cell membranes, and readily
react with biomolecules including carbohydrates, proteins,
lipids, and DNA in cells, causing tissue damage or cell death.
Hydroxyl radical scavengers have the ability to quench hydroxyl
radicals that can lead to fading in the colour of safranine T. The
absorbance of safranine T was detected during a xed reaction
period at 520 nm. The hydroxyl radicals were generated by the
Fenton reaction model system and the scavenging activity was
determined as follows. The reaction was performed in a mixture
of 0.5 ml Na2HPO4–NaH2PO4 (200 mM, pH 7.4), 0.1 ml safra-
nine T solution in ethanol (520 mg ml
1), 0.5 ml EDTANa2–Fe2+
(10 mM), 3.5 ml extract in various concentrations and 0.4 ml
H2O2 (6%, V/V). The mixtures were incubated at 40  C for
30 min and the optical absorbance was measured at 520 nm
relative to a corresponding blank. The control sample was made
up from 0.9 ml distilled water instead of EDTANa2–Fe2+ and
H2O2, and another 3.5 ml distilled water in place of the extract
Food Funct., 2014, 5, 50–56 | 51

Food & Function
solution. The percent inhibition activity (%) was calculated by
(As
Ao)/(Ac
Ao)  100 (where As refers to the absorbance of
the sample containing extract, Ac refers to the absorbance of the
control sample, and Ao refers to the absorbance of the original
sample). Rutin was used as a positive control.
2.5. Determination of antioxidant activity in vivo
2.5.1. Experimentation on animals. The antioxidant
activity in vivo was determined according to the method of
Zhang,21 with some modications. Male ICR mice (8 weeks old,
29.9  1.3 g) were purchased from Sino-British Sippr/B&K Lab
Animal Ltd. (Shanghai, China). The experiment was carried out
under the “Guidelines and regulations for the Care and Use of
Laboratory Animal” of the Science and Technology Commission
Published on 15 November 2013. Downloaded on 18/06/2014 16:28:07.
of the Shanghai Municipality, China. The mice were randomly
divided into three groups of ten mice each. Group 1 (control)
was only given suspending agent (0.4 ml 0.5% Na-CMC)
by gavage, while groups 2 and 3 were given apiin and rutin at
50 mg kg
1 dissolved in 0.4 ml 0.5% Na-CMC by gavage,
respectively. Administration was performed over 28 consecutive
days. Aer overnight fasting, blood samples were collected by
retro orbital puncture and processed to obtain serum, at which
point the mice were killed by decapitation. Four organs (brain,
heart, liver and kidney) were excised from the animals, and
tissue homogenates were processed for biochemical analysis.
2.5.2. Biochemical analysis. The biochemical assays were
carried out according to the instructions of kits purchased from
Sino-British Sippr/B&K Lab Animal Ltd. (Shanghai, China).
Serum samples were obtained by centrifuging the whole blood
at 2000 rpm at 4  C for 10 min. For biochemical assays, the
organ homogenates were prepared in 0.1 g ml
1 wet weight of
PBS solution (50 mM, pH 7.4). Samples were centrifuged at
3000 rpm at 4  C for 10 min, and the supernatants were then
subjected to measurement of MDA, SOD, GSH-Px, CAT and
TAOC levels by spectrophotometric methods, while LPF deter-
mination was performed with the use of a GENios Pro Scanning
Autoreader (TECAN, Switzerland). Lipid peroxidation was esti-
mated in terms of Thiobarbituric Acid Reactive Species (TBARS),
using MDA as a standard. The MDA level was quantied with 2-
thiobarbituric acid and expressed as nmol ml
1 or nmol mg
1
protein. LPF was evaluated by uorescence at an excitation
wavelength of 365 nm and an emission wavelength of 435 nm,
and was expressed as kRFU ml
1 or kRFU mg
1 protein. SOD
activity was analyzed by the xanthine/xanthine oxidase method;
GSH-Px activity was detected with 5,50 -dithiobis-p-nitrobenzoic
acid; CAT was detected with hydrogen peroxide and ammonium
molybdate; TAOC was measured by a ferric reducing/
antioxidant activity assay. The above four biochemical param-
eters were expressed as U ml
1 or U mg
1 protein. Protein
content was assayed according to Bradford using bovine serum
albumin as the standard.
2.6. Statistical analysis
The experimental results are expressed as the mean  SD of
parallel measurements. The signicance of differences between
sample means was calculated by the Independent-Samples t test
52 | Food Funct., 2014, 5, 50–56
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using SPSS 12.0. A p value of <0.05 was regarded as a signicant
difference and p < 0.01 was regarded as a very signicant
difference.
3. Results and discussion
3.1. LC/MS analysis of avonoid
Negative ion electrospray LC/MS analysis of the celery avonoid
is shown in Fig. 1. The Chromatogram obtained from the LC/
ESI/MS/MS experiment for the ion at m/z 563.2 gave a single
peak at a retention time of 4.52 min (Fig. 1A), and the corre-
sponding MS/MS spectrum showed a fragment ion at 269.2
(Fig. 1B), which represents the fragmentation of apigenin. The
mass difference between the molecular ions and the aglycone
ions was 294 Da, as expected for the loss of the apiosylglucosyl
derivative. The results indicated that the puried celery avo-
noid was apigenin-7-apisoylglucoside, namely apiin.
3.2. Antioxidant activity in vitro
3.2.1. DPPH-scavenging activities. DPPH is characterized
as a stable free radical, owing to the delocalization of the spare
electron over the whole molecule, resulting in a deep violet color
at 520 nm.22 The scavenging activities of the apiin towards
DPPH compared with those towards rutin are shown in Fig. 2,
where it can be seen that the DPPH scavenging activity of apiin
is dose dependent. Furthermore, the DPPH scavenging activity
of apiin (IC50 68.0 mg ml
1) was less than that of rutin (IC50
45.6 mg ml
1), which has been proved to be a potent avonoid
exhibiting highly efficient physiological activity.23 Additionally,
the extracted apiin showed a stronger scavenging activity than
that of vitamin E according to Wu and Ng,24 who reported the
IC50 of vitamin E to be 172.21 mg ml
1, suggesting that the
extracted apiin exhibited remarkable free radical scavenging
activity.
3.2.2. Superoxide radical-scavenging activities. The super-
oxide radical, arising either through metabolic processes or
from oxygen activation by physical irradiation, is considered as
the primary ROS. Superoxide radicals can further interact with
other molecules to generate secondary ROS (e.g., hydroxyl
radicals, hydrogen peroxide and singlet oxygen), either directly
or more usually through enzyme or metal catalyzed processes.25
It can be seen from the superoxide radical-scavenging activities
of apiin shown in Fig. 3 that the scavenging rate of apiin
correlated well with increasing concentration. Under the same
conditions, the IC50 of rutin was 0.19 mg ml
1, which indicates
that the superoxide radical-scavenging activity of rutin was a
little higher than that of apiin with an IC50 of 0.39 mg ml
1. The
excellent superoxide radical-scavenging activity of rutin may be
due to its greater amount of hydroxyl substituents,26 more
specically its 40 -OH and 70 -OH, which are not present in apiin.
However, the extracted apiin demonstrated a stronger super-
oxide radical-scavenging activity than that of vitamin E
according to Wu and Ng,24 who reported the IC50 of vitamin E to
be 0.42 mg ml
1. Therefore, apiin can be considered as a good
scavenger of superoxide radicals.
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Fig. 1 LC/MS analysis of the celery flavonoid, apiin. (A) HPLC profile of the purified celery flavonoid; the peak was identified as apiin. (B) ESI/MS/
MS spectrum of the purified celery flavonoid.
Fig. 2 DPPH radical-scavenging activities of apiin, with rutin being
used as a control. Data were presented as the percentage of DPPH
radical scavenging, mean  SD (n ¼ 3).
3.2.3. Hydroxyl radical-scavenging activities. The hydroxyl
radical, known as the most reactive radical, can attack and
damage almost every bio-macromolecule in living cells. The
best characterized biological damage caused by the hydroxyl
radical is its capacity to stimulate lipid peroxidation, which
occurs when hydroxyl radicals are generated close to
membranes, attacking the fatty acid side chains of the
membrane phospholipids.25 The results of the hydroxyl radical-
scavenging activities are illustrated in Fig. 4. Apiin exhibited
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Fig. 3 Superoxide radical-scavenging activities of apiin, with rutin
being used as a control. Data were presented as the percentage of
superoxide radical scavenging, mean  SD (n ¼ 3).
hydroxyl radical-scavenging activity in a concentration depen-
dant manner, in the range of 5–100 mg ml
1. In this assay,
although apiin acted as a powerful radical scavenger, the
increase of scavenging activity gradually slowed down when the
concentration was increased from 75 mg ml
1 to 100 mg ml
1.
The same result was also found in the assay for rutin. As well as
quenching the hydroxyl radical, the avonoid can also simul-
taneously reduce Fe3+ into Fe2+, and Fe2+ can further react with
H2O2 to form OHc and Fe3+, which means that the avonoid can
Food Funct., 2014, 5, 50–56 | 53

Food & Function
Fig. 4 Hydroxyl radical-scavenging activities of apiin, and rutin was
used as a control. Data were presented as the percentage of hydroxyl
radical scavenging, mean  SD (n ¼ 3).
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also promote the formation of hydroxyl radicals. Therefore, in
low concentration, the avonoid was able to quench the
hydroxyl radicals, however at high concentration, the avonoid
would be promoting the formation of hydroxyl radicals, thus
explaining why the plots of the lines in Fig. 4 became at at high
concentrations of extract.
3.3. Antioxidant activity in vivo
The antioxidant properties of apiin were further conrmed with
the use of in vivo assays. Brain tissue contains high concentra-
tions of polyunsaturated fatty acids, catecholamines and
oxidative metabolites, which are targets of reactive oxygen
species. However, there are few antioxidant enzymes and
nonenzymatic endogenous antioxidants in brain tissue, which
results in the brain being under tremendous oxidative stress
and aggravated neuropathic apoptosis. Meanwhile, it is known
that polyunsaturated fatty acids easily form peroxides in the
presence of oxygen and that this induces the production of free
radicals, which attack membrane phospholipids and initiate
lipid peroxidation. This peroxidative effect on membrane
lipids and the low density of lipoproteins are directly related to
the pathogenesis of atherosclerosis. As this condition worsens,
coronary disease is induced and when ischemic reperfusion
injury occurs, more radicals are produced. Hence, this can be
considered as a vicious spiral which can cause signicant
damage to the heart.27 Furthermore, the liver is the biggest
endocrine organ, involved in food digestion and detoxica-
tion. Many injurants, including medicaments and chemicals,
are transformed by the liver to be non-toxic, and a great deal of
radicals are produced at the same time. When the amount of
free radicals exceeds the ability of endogenous antioxidants,
injury will denitely occur and the function of the liver will be
affected.28 The kidney is one of the primary battleelds with
respect to the production and removal of free radicals. The
glomerulus and its cells (epithelial cells, interstitial cells and
Sertoli’s cells) are capable of producing radicals. In cases of
Bright’s disease (acute or chronic nephritis), neutrophils,
macrophages, plasma cells and eosinophils can cause
enhanced oxidative stress.29 Thus, the kidney is also an organ
capable of mass producing free radicals and needs more
antioxidants. Therefore, the serum, brain, heart, liver and
kidney of mice were employed to evaluate the antioxidant
54 | Food Funct., 2014, 5, 50–56
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properties of the extracted apiin. Six biochemical parameters
were utilized in the in vivo experiments, which were the
concentrations of maleic dialdehyde (MDA) and lipofuscin
(LPF), the activities of superoxide dismutase (SOD), gluta-
thione peroxidase (GSH-Px) and catalase (CAT), and the total
antioxidant capacity (TAOC).
MDA is one of the most frequently used indicators for lipid
peroxidation, and it can also be an indicator of cell membrane
injury, though aldehydes are a highly toxic molecule and should
be considered as more than just a marker of lipid perox-
idation.30 Fig. 5A shows the comparison of MDA content of the
serum, brain, heart, liver and kidney of the three different
groups, aer the administration of the extracted apiin and
rutin, and it can be seen that the MDA content was generally
decreased. MDA in the mice’s liver was reduced signicantly by
over 24% compared with the control (p < 0.05). However, the
decrease of MDA in the kidney was not as obvious, at less than
10% compared with the control. Furthermore, the ability of
apiin to inhibit the generation of MDA was basically equal to
that of rutin, which has been proven to have a strong antioxi-
dant activity. The results of the present study have demon-
strated that apiin extracted from celery can inhibit the
generation of MDA in serum and brain, heart, liver and kidney
tissue. LPF is the product of the peroxidation of unsaturated
fatty acids, and may be symptomatic of membrane damage, in
addition to damage to mitochondria and lysosomes. Patho-
logical accumulation of LPF is associated with Alzheimer’s
disease, Parkinson’s disease, certain lysosomal diseases,
acromegaly, denervation atrophy, lipid myopathy and chronic
obstructive pulmonary disease.31 As shown in Fig. 5B, the
strongest inhibition of LPF generation by apiin from celery
appeared in the brain (25%), followed by the serum (20%),
but no signicant reduction was observed in the heart, liver
or kidney.
Superoxide dismutase (SOD) catalyzes the dismutation of
superoxide into oxygen and hydrogen peroxide. SOD decreases
the generation of reactive oxygen species and oxidative stress
and thus restrains endothelial activation, indicating the
modulation of factors which govern adhesion molecule
expression and leukocyte–endothelial interactions. As such, it is
an important antioxidant defense, present on nearly all cells
that are exposed to oxygen.32 Catalase (CAT) is a common
enzyme found in nearly all living organisms, and it can catalyze
the decomposition of hydrogen peroxide into water and oxygen.
Catalase has one of the highest turnover numbers of all
enzymes. One molecule of catalase can convert millions of
molecules of hydrogen peroxide into water and oxygen per
second. Catalase can also oxidize different toxins, such as
formaldehyde, formic acid, phenols, and alcohols.33,34 GSH-Px is
the general name of an enzyme family with peroxidase activity,
whose main biological role is to protect the host organism from
oxidative damage. The biochemical function of GSH-Px is to
reduce lipid hydroperoxides to their corresponding alcohols,
and to reduce free hydrogen peroxide to water.35 Thus, SOD,
GSH-Px and CAT are the most important antioxidant enzymes to
inhibit free radical formation, and are usually used as
biomarkers to indicate ROS production.
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Fig. 5 Comparison of (A) MDA and (B) LPF content, and the activities of (C) SOD, (D) GSH-Px, (E) CAT and (F) TAOC in the serum, brain, heart, liver
and kidney of mice administered with the extracted apiin and rutin; suspending agent (0.4 ml 0.5% Na-CMC) was used as a control. Values are
mean  SD (n ¼ 3). *p < 0.05, **p < 0.01 compared with the control.
As can be seen from the results shown in Fig. 5C–E, the
activities of SOD, CAT and GSH-Px were remarkably improved
in the serum, brain, heart, liver and kidney aer administra-
tion of the extracted apiin. Compared with the control, the
activities of SOD, GSH-Px and CAT were found to be very
signicantly (p < 0.01) increased in the serum (26%, 19% and
57%), brain (32%, 27% and 14%), heart (23%, 17% and 8%)
liver (14%, 16% and 17%) and kidney (15%, 17% and 11%).
All of the results demonstrated that the apiin extracted
from celery was capable of enhancing the activities of SOD,
CAT and GSH-Px, consequently representing protection
against oxidative tissue damage by apiin. The enhanced
activities of the antioxidant enzymes may provide an effective
defense from the damaging effects of free radicals. As repor-
ted, the enhanced activities of the antioxidant enzymes were
partially due to the increased mRNA expression of these
enzymes.36
The total antioxidant activities (TAOC) of apiin and rutin are
shown in Fig. 5F. The observed increase of TAOC promoted by
apiin was 6% (p < 0.05) in serum, and about 13%, 16% and 17%
in the brain, heart and liver, respectively, but no signicant
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improvement was observed in the kidney. In addition, apiin
showed a lower TAOC relative to that of rutin.
The results of the treatment of mice with apiin showed that
the apiin extracted from celery can assist in the prevention of a
brain oxidative state, and simultaneously promote the activity of
antioxidant enzymes to provide additional protection for the
heart, liver and kidney. It has clearly been demonstrated that
apiin has signicant antioxidant activity against various anti-
oxidant systems in vivo.
4. Conclusions
In conclusion, the present study has demonstrated that apiin
extracted from Apium graveolens L. var. dulce possesses free
radical scavenging activities as well as excellent antioxidant
activity in ICR mice, by protecting several important organs
from oxidative stress. These antioxidant activities could have
contributed, at least partially, to effects underpinning certain
traditional claims of the therapeutic benets of celery. In view of
the potential use of celery in the health food industry, its
Food Funct., 2014, 5, 50–56 | 55

Food & Function
therapeutic benets and bioactive compounds behoove further
investigations.
Acknowledgements
This work was supported by “the Fundamental Research Funds
for the Central Universities”, and “National Major Science and
Technology Projects of China (no. 2012ZX09304009)”.
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