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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.

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 (O2c) and hydroxyl radical (OHc) scavenging assays. IC50 values were 68.0 mg ml1 in
the DPPH assay, 0.39 mg ml1 in the O2c assay and 48.0 mg ml1 in the OHc assay. The antioxidant
activities were investigated in vivo with the use of mice models. All data were measured including the
Received 16th July 2013
Accepted 11th October 2013
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
DOI: 10.1039/c3fo60273g
serum, brain, heart, liver and kidney. Results showed that apiin had a remarkable scavenging activity on
www.rsc.org/foodfunction 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 neocoidilde, celerin), organic acids (chlorogenic acid, caffeic
Reactive oxygen species (ROS) generated by NADPH oxidase acid), bergapten, niacin, inositol etc.12 Among these
during oxidative phosphorylation, are normal components of compounds, apigenin, one of the main bioactive components in
healthy cells. ROS are also mediators of the rst defensive celery, belongs to a class of avones, existing in the form of the
actions of cells and are involved in phagocytosis, apoptosis and avonoid, apiin. Apigenin can relax rat thoracic aorta,
detoxication.1 Furthermore, ROS are toxic and can have a predominantly by means of suppressing Ca2+ inux through
mutagenic effect on all types of cells via the oxidation of DNA, both voltage- and receptor-operated calcium channels, which
membrane lipids and proteins.2 Fortunately, there exist some contributes to its antihypertensive activity. Furthermore, api-
natural scavenging systems, for instance, antioxidant enzymes genin has genotoxic and radioprotective effects on radiation-
and polyphenols, which can destroy ROS.3–8 Phenolic induced chromosome aberrations in human lymphocytes B.
compounds promote human health through reducing oxidative Under the protection of apigenin, the frequency of cytokinesis-
phenomena and decrease the risk of several diseases.9 Flavo- block micronuclei can be decreased.13
noids are one of the main subclasses of phenolic compounds, However, in comparison to the attention on the biological
comprised of a wide distribution of phytochemicals consisting activity of apigenin, there are few reports on the systematic
of two aromatic rings linked by three carbons in an oxygenated investigation of the antioxidant activity of apiin in vitro and in
heterocycle. Studies have shown that avonoids are associated vivo. In particular there are few reports on the effects of apiin on
with a low risk of these ROS related diseases and damage,10 and enzymatic activities in vivo, such as superoxide dismutase
research has focused on the development of techniques to (SOD), catalase (CAT) and glutathione (GSH), which play an
obtain safe and effective antioxidants from natural plants. important role in the antioxidant systems of living organisms
Celery (Apium graveolens L. var. dulce) is a herbaceous biennial that counteract reactive species to reduce the damage they
plant in the family Apiaceae, originating from the coasts of cause. Consequently, the objective of this study was to extract,
western and northern Europe.11 It is distinguished as a low fat separate and identify this avonoid from celery, and then
food and a source of digestible carbohydrates, proven rich in determine its antioxidant activities through various in vitro
bioactive compounds such as vitamins, free amino acids, methods and in vivo models.
minerals and a high amount of dietary bers, in addition to
2. Material and methods
State Key Laboratory of Bioreactor Engineering, Department of Food Science and 2.1. Materials
Technology, East China University of Science and Technology, P. O. Box 283, East
China University of Science and Technology, 130 # Meilong Rd, Shanghai 200237, Leaves of Apium graveolens L. var. dulce were purchased from a
P. R. China. E-mail: jlxie@ecust.edu.cn; Fax: +86-21-64252563; Tel: +86-21-64252563 local supermarket. 1,1-Diphenyl-2-picrylhydrazyl was purchased

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from Sigma-Aldrich, China (Shanghai, China). Rutin, sodium sample was 1 ml of 100 mM DPPH mixed with 1 ml of ethanol.
carboxymethyl cellulose (Na-CMC), and pyrogallol acid were The percent scavenging ratio (%) was calculated by (1  As/Ac) 
purchased from Beijing Chemical Reagents Company (Beijing, 100 (where As refers to the absorbance of the extract and Ac
China). Other materials used were safranine T, ethylene refers to the absorbance of the control). Rutin was used as a
diamine tetraacetic acid (EDTA), trishydroxymethylamino- positive control. Triplicate experiments were carried out and a
methane (Tris), and silica gel (75–150 mm), obtained from dose response curve was plotted to determine the IC50, which is
Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Assay dened as the concentration required to obtain a scavenging
kits for MDA, LPF, SOD, GSH-Px, CAT and TAOC were obtained capacity of 50%. A lower IC50 value corresponds to a greater
from Nanjing Jiancheng Institute of Bioengineering (Nanjing, antioxidant activity.
China). All solutions were freshly prepared with distilled water. 2.4.2. Superoxide radical-scavenging assay. In this experi-
Stock solutions of test extracts and compounds (0.5 mg ml1) ment, the superoxide radical was generated by the autoxidation
were prepared in anhydrous ethanol. Appropriate blanks were of pyrogallol acid (PA) and the scavenging radical activity was
used for the individual assays. determined by the method described by Marklund and Mar-
klund.19 Pyrogallol acid (PA) is readily auto-oxidized at pH 8.2,
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2.2. Preparation and separation of the plant extracts generating superoxide radicals and an intermediate product
The preparation of plant extracts was performed according to which exhibits a strong absorbance at 320 nm. At the beginning
the method of Kähkönen,14 with some modications. The leaves of the PA auto-oxidization, the observed absorbance is linear
of celery were dried at 60  C, powdered and extracted twice by with respect to time. However, when the antioxidant inhibits
70% (v/v) ethanol solution (dried powder : ethanol solution was the superoxide radical, the generation velocity of the interme-
1/15 (w/w)) at 80  C for 4 h. Aer ltration, the ltrate was diate product is slowed down. The activity was calculated from
concentrated under vacuum by rotary evaporation to give the the tempered slope of the resulting absorbance–time curve. The
sample S1, ready for further extraction. 5 ml S1 was subjected to reaction was carried out in a tube with 2.8 ml Tris–HCl buffer
RP-HPLC on an Ultimate 3000 C18 semi-prep column (10 mm  (200 mM, pH 8.2). The mixture was incubated at 25  C for
150 mm; DIONEX, USA), and eluted with a gradient mobile 10 min and 0.1 ml extract solution in varying concentrations
phase (ethyl acetate : methanol (90 : 10) / ethyl aceta- was added before preheating. The reaction was started by the
te : methanol (50 : 50) / methanol) within 50 minutes at a ow addition of 0.1 ml PA solution (3 mM, preheated to 25  C) into
rate of 36 ml h1, while being monitored at 215 nm. Fractions the tube and the reaction process was measured against
were collected automatically before being checked by silica gel distilled water at 320 nm. The absorbance was recorded every
thin-layer chromatography (TLC). The TLCs were developed in 30 s during the rst 4 min. The slope (DA min1) of the time
ethyl acetate : methanol (90 : 10) and the thin-layer was visual- response curve represented the value of the oxidation velocity
ised under UV 365 nm aer spraying with a solution of AlCl3 in (V). The control sample was made using 0.1 ml distilled water
ethanol. The fractions with Rf ¼ 0.4 were combined, dried and instead of extract solution. The percent inhibition activity (%)
recrystallized from ethanol at 4  C. was calculated by (1  Vs/Vc)  100 (where Vs refers to the
oxidation velocity in the sample containing extract, and Vc refers
2.3. Identication of avonoid by LC/ESI-MS to oxidation velocity in the control sample). Rutin was used as a
positive control.
LC/ESI-MS was used for avonoid detection and identica- 2.4.3. Hydroxyl radical-scavenging assay. The hydroxyl
tion15,16 with a reversed-phase BDS Hypersil C18 (3 mm particle radical scavenging activity was determined according to the
size; 150  2.1 mm, i.d.) column. The solvent system was an method of Zhong,20 with some modications. Hydroxyl radicals,
acetonitrile–water mixture (A) and formic acid (B), in the generated by the reaction of a Fe(II) complex with H2O2 in the
following gradient: 0 min, 20% A; 10 min, 25% A; 15 min, 100% presence of acid, can easily cross cell membranes, and readily
A. Elution was performed at a solvent ow rate of 0.2 ml min1. react with biomolecules including carbohydrates, proteins,
Detection was accomplished with a Thermo Finnigan Deca XP lipids, and DNA in cells, causing tissue damage or cell death.
MAX LCQ ion trap mass spectrometer equipped with an ESI Hydroxyl radical scavengers have the ability to quench hydroxyl
interface (San Jose, CA, USA) (source voltage 3.5 kV; capillary radicals that can lead to fading in the colour of safranine T. The
temperature 320  C). The mass spectrometer was operated in absorbance of safranine T was detected during a xed reaction
negative-ion mode, with a scan range from m/z 100–800 (scan period at 520 nm. The hydroxyl radicals were generated by the
rate 1.2 scans s1). Fenton reaction model system and the scavenging activity was
determined as follows. The reaction was performed in a mixture
2.4. Determination of antioxidant activity in vitro of 0.5 ml Na2HPO4–NaH2PO4 (200 mM, pH 7.4), 0.1 ml safra-
2.4.1. DPPH scavenging assay. The scavenging activity with nine T solution in ethanol (520 mg ml1), 0.5 ml EDTANa2–Fe2+
respect to the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), (10 mM), 3.5 ml extract in various concentrations and 0.4 ml
was determined by a modied method according to Wu17 and H2O2 (6%, V/V). The mixtures were incubated at 40  C for
Luo.18 Briey, 1 ml ethanol solution of DPPH (100 mM) was 30 min and the optical absorbance was measured at 520 nm
added to 1 ml extract in different concentrations. The samples relative to a corresponding blank. The control sample was made
were incubated at room temperature in the dark for 30 min and up from 0.9 ml distilled water instead of EDTANa2–Fe2+ and
the optical absorbance was measured at 517 nm. The control H2O2, and another 3.5 ml distilled water in place of the extract

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solution. The percent inhibition activity (%) was calculated by using SPSS 12.0. A p value of <0.05 was regarded as a signicant
(As  Ao)/(Ac  Ao)  100 (where As refers to the absorbance of difference and p < 0.01 was regarded as a very signicant
the sample containing extract, Ac refers to the absorbance of the difference.
control sample, and Ao refers to the absorbance of the original
sample). Rutin was used as a positive control.
3. Results and discussion
2.5. Determination of antioxidant activity in vivo 3.1. LC/MS analysis of avonoid
2.5.1. Experimentation on animals. The antioxidant Negative ion electrospray LC/MS analysis of the celery avonoid
activity in vivo was determined according to the method of is shown in Fig. 1. The Chromatogram obtained from the LC/
Zhang,21 with some modications. Male ICR mice (8 weeks old, ESI/MS/MS experiment for the ion at m/z 563.2 gave a single
29.9  1.3 g) were purchased from Sino-British Sippr/B&K Lab peak at a retention time of 4.52 min (Fig. 1A), and the corre-
Animal Ltd. (Shanghai, China). The experiment was carried out sponding MS/MS spectrum showed a fragment ion at 269.2
under the “Guidelines and regulations for the Care and Use of (Fig. 1B), which represents the fragmentation of apigenin. The
Laboratory Animal” of the Science and Technology Commission mass difference between the molecular ions and the aglycone
Published on 15 November 2013. Downloaded on 18/06/2014 16:28:07.

of the Shanghai Municipality, China. The mice were randomly ions was 294 Da, as expected for the loss of the apiosylglucosyl
divided into three groups of ten mice each. Group 1 (control) derivative. The results indicated that the puried celery avo-
was only given suspending agent (0.4 ml 0.5% Na-CMC) noid was apigenin-7-apisoylglucoside, namely apiin.
by gavage, while groups 2 and 3 were given apiin and rutin at
50 mg kg1 dissolved in 0.4 ml 0.5% Na-CMC by gavage,
respectively. Administration was performed over 28 consecutive 3.2. Antioxidant activity in vitro
days. Aer overnight fasting, blood samples were collected by
3.2.1. DPPH-scavenging activities. DPPH is characterized
retro orbital puncture and processed to obtain serum, at which
as a stable free radical, owing to the delocalization of the spare
point the mice were killed by decapitation. Four organs (brain,
electron over the whole molecule, resulting in a deep violet color
heart, liver and kidney) were excised from the animals, and
at 520 nm.22 The scavenging activities of the apiin towards
tissue homogenates were processed for biochemical analysis.
DPPH compared with those towards rutin are shown in Fig. 2,
2.5.2. Biochemical analysis. The biochemical assays were
where it can be seen that the DPPH scavenging activity of apiin
carried out according to the instructions of kits purchased from
is dose dependent. Furthermore, the DPPH scavenging activity
Sino-British Sippr/B&K Lab Animal Ltd. (Shanghai, China).
of apiin (IC50 68.0 mg ml1) was less than that of rutin (IC50
Serum samples were obtained by centrifuging the whole blood
45.6 mg ml1), which has been proved to be a potent avonoid
at 2000 rpm at 4  C for 10 min. For biochemical assays, the
exhibiting highly efficient physiological activity.23 Additionally,
organ homogenates were prepared in 0.1 g ml1 wet weight of
the extracted apiin showed a stronger scavenging activity than
PBS solution (50 mM, pH 7.4). Samples were centrifuged at
that of vitamin E according to Wu and Ng,24 who reported the
3000 rpm at 4  C for 10 min, and the supernatants were then
IC50 of vitamin E to be 172.21 mg ml1, suggesting that the
subjected to measurement of MDA, SOD, GSH-Px, CAT and
extracted apiin exhibited remarkable free radical scavenging
TAOC levels by spectrophotometric methods, while LPF deter-
activity.
mination was performed with the use of a GENios Pro Scanning
3.2.2. Superoxide radical-scavenging activities. The super-
Autoreader (TECAN, Switzerland). Lipid peroxidation was esti-
oxide radical, arising either through metabolic processes or
mated in terms of Thiobarbituric Acid Reactive Species (TBARS),
from oxygen activation by physical irradiation, is considered as
using MDA as a standard. The MDA level was quantied with 2-
the primary ROS. Superoxide radicals can further interact with
thiobarbituric acid and expressed as nmol ml1 or nmol mg1
other molecules to generate secondary ROS (e.g., hydroxyl
protein. LPF was evaluated by uorescence at an excitation
radicals, hydrogen peroxide and singlet oxygen), either directly
wavelength of 365 nm and an emission wavelength of 435 nm,
or more usually through enzyme or metal catalyzed processes.25
and was expressed as kRFU ml1 or kRFU mg1 protein. SOD
It can be seen from the superoxide radical-scavenging activities
activity was analyzed by the xanthine/xanthine oxidase method;
of apiin shown in Fig. 3 that the scavenging rate of apiin
GSH-Px activity was detected with 5,50 -dithiobis-p-nitrobenzoic
correlated well with increasing concentration. Under the same
acid; CAT was detected with hydrogen peroxide and ammonium
conditions, the IC50 of rutin was 0.19 mg ml1, which indicates
molybdate; TAOC was measured by a ferric reducing/
that the superoxide radical-scavenging activity of rutin was a
antioxidant activity assay. The above four biochemical param-
little higher than that of apiin with an IC50 of 0.39 mg ml1. The
eters were expressed as U ml1 or U mg1 protein. Protein
excellent superoxide radical-scavenging activity of rutin may be
content was assayed according to Bradford using bovine serum
due to its greater amount of hydroxyl substituents,26 more
albumin as the standard.
specically its 40 -OH and 70 -OH, which are not present in apiin.
However, the extracted apiin demonstrated a stronger super-
2.6. Statistical analysis oxide radical-scavenging activity than that of vitamin E
The experimental results are expressed as the mean  SD of according to Wu and Ng,24 who reported the IC50 of vitamin E to
parallel measurements. The signicance of differences between be 0.42 mg ml1. Therefore, apiin can be considered as a good
sample means was calculated by the Independent-Samples t test 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


Fig. 3 Superoxide radical-scavenging activities of apiin, with rutin
used as a control. Data were presented as the percentage of DPPH
being used as a control. Data were presented as the percentage of
radical scavenging, mean  SD (n ¼ 3).
superoxide radical scavenging, mean  SD (n ¼ 3).

3.2.3. Hydroxyl radical-scavenging activities. The hydroxyl hydroxyl radical-scavenging activity in a concentration depen-
radical, known as the most reactive radical, can attack and dant manner, in the range of 5–100 mg ml1. In this assay,
damage almost every bio-macromolecule in living cells. The although apiin acted as a powerful radical scavenger, the
best characterized biological damage caused by the hydroxyl increase of scavenging activity gradually slowed down when the
radical is its capacity to stimulate lipid peroxidation, which concentration was increased from 75 mg ml1 to 100 mg ml1.
occurs when hydroxyl radicals are generated close to The same result was also found in the assay for rutin. As well as
membranes, attacking the fatty acid side chains of the quenching the hydroxyl radical, the avonoid can also simul-
membrane phospholipids.25 The results of the hydroxyl radical- taneously reduce Fe3+ into Fe2+, and Fe2+ can further react with
scavenging activities are illustrated in Fig. 4. Apiin exhibited H2O2 to form OHc and Fe3+, which means that the avonoid can

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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-
Fig. 4 Hydroxyl radical-scavenging activities of apiin, and rutin was idation.30 Fig. 5A shows the comparison of MDA content of the
used as a control. Data were presented as the percentage of hydroxyl
serum, brain, heart, liver and kidney of the three different
radical scavenging, mean  SD (n ¼ 3).
groups, aer the administration of the extracted apiin and
rutin, and it can be seen that the MDA content was generally
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decreased. MDA in the mice’s liver was reduced signicantly by


also promote the formation of hydroxyl radicals. Therefore, in
over 24% compared with the control (p < 0.05). However, the
low concentration, the avonoid was able to quench the
decrease of MDA in the kidney was not as obvious, at less than
hydroxyl radicals, however at high concentration, the avonoid
10% compared with the control. Furthermore, the ability of
would be promoting the formation of hydroxyl radicals, thus
apiin to inhibit the generation of MDA was basically equal to
explaining why the plots of the lines in Fig. 4 became at at high
that of rutin, which has been proven to have a strong antioxi-
concentrations of extract.
dant activity. The results of the present study have demon-
strated that apiin extracted from celery can inhibit the
3.3. Antioxidant activity in vivo generation of MDA in serum and brain, heart, liver and kidney
The antioxidant properties of apiin were further conrmed with tissue. LPF is the product of the peroxidation of unsaturated
the use of in vivo assays. Brain tissue contains high concentra- fatty acids, and may be symptomatic of membrane damage, in
tions of polyunsaturated fatty acids, catecholamines and addition to damage to mitochondria and lysosomes. Patho-
oxidative metabolites, which are targets of reactive oxygen logical accumulation of LPF is associated with Alzheimer’s
species. However, there are few antioxidant enzymes and disease, Parkinson’s disease, certain lysosomal diseases,
nonenzymatic endogenous antioxidants in brain tissue, which acromegaly, denervation atrophy, lipid myopathy and chronic
results in the brain being under tremendous oxidative stress obstructive pulmonary disease.31 As shown in Fig. 5B, the
and aggravated neuropathic apoptosis. Meanwhile, it is known strongest inhibition of LPF generation by apiin from celery
that polyunsaturated fatty acids easily form peroxides in the appeared in the brain (25%), followed by the serum (20%),
presence of oxygen and that this induces the production of free but no signicant reduction was observed in the heart, liver
radicals, which attack membrane phospholipids and initiate or kidney.
lipid peroxidation. This peroxidative effect on membrane Superoxide dismutase (SOD) catalyzes the dismutation of
lipids and the low density of lipoproteins are directly related to superoxide into oxygen and hydrogen peroxide. SOD decreases
the pathogenesis of atherosclerosis. As this condition worsens, the generation of reactive oxygen species and oxidative stress
coronary disease is induced and when ischemic reperfusion and thus restrains endothelial activation, indicating the
injury occurs, more radicals are produced. Hence, this can be modulation of factors which govern adhesion molecule
considered as a vicious spiral which can cause signicant expression and leukocyte–endothelial interactions. As such, it is
damage to the heart.27 Furthermore, the liver is the biggest an important antioxidant defense, present on nearly all cells
endocrine organ, involved in food digestion and detoxica- that are exposed to oxygen.32 Catalase (CAT) is a common
tion. Many injurants, including medicaments and chemicals, enzyme found in nearly all living organisms, and it can catalyze
are transformed by the liver to be non-toxic, and a great deal of the decomposition of hydrogen peroxide into water and oxygen.
radicals are produced at the same time. When the amount of Catalase has one of the highest turnover numbers of all
free radicals exceeds the ability of endogenous antioxidants, enzymes. One molecule of catalase can convert millions of
injury will denitely occur and the function of the liver will be molecules of hydrogen peroxide into water and oxygen per
affected.28 The kidney is one of the primary battleelds with second. Catalase can also oxidize different toxins, such as
respect to the production and removal of free radicals. The formaldehyde, formic acid, phenols, and alcohols.33,34 GSH-Px is
glomerulus and its cells (epithelial cells, interstitial cells and the general name of an enzyme family with peroxidase activity,
Sertoli’s cells) are capable of producing radicals. In cases of whose main biological role is to protect the host organism from
Bright’s disease (acute or chronic nephritis), neutrophils, oxidative damage. The biochemical function of GSH-Px is to
macrophages, plasma cells and eosinophils can cause reduce lipid hydroperoxides to their corresponding alcohols,
enhanced oxidative stress.29 Thus, the kidney is also an organ and to reduce free hydrogen peroxide to water.35 Thus, SOD,
capable of mass producing free radicals and needs more GSH-Px and CAT are the most important antioxidant enzymes to
antioxidants. Therefore, the serum, brain, heart, liver and inhibit free radical formation, and are usually used as
kidney of mice were employed to evaluate the antioxidant 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 improvement was observed in the kidney. In addition, apiin
activities of SOD, CAT and GSH-Px were remarkably improved showed a lower TAOC relative to that of rutin.
in the serum, brain, heart, liver and kidney aer administra- The results of the treatment of mice with apiin showed that
tion of the extracted apiin. Compared with the control, the the apiin extracted from celery can assist in the prevention of a
activities of SOD, GSH-Px and CAT were found to be very brain oxidative state, and simultaneously promote the activity of
signicantly (p < 0.01) increased in the serum (26%, 19% and antioxidant enzymes to provide additional protection for the
57%), brain (32%, 27% and 14%), heart (23%, 17% and 8%) heart, liver and kidney. It has clearly been demonstrated that
liver (14%, 16% and 17%) and kidney (15%, 17% and 11%). apiin has signicant antioxidant activity against various anti-
All of the results demonstrated that the apiin extracted oxidant systems in vivo.
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 4. Conclusions
activities of the antioxidant enzymes may provide an effective
defense from the damaging effects of free radicals. As repor- In conclusion, the present study has demonstrated that apiin
ted, the enhanced activities of the antioxidant enzymes were extracted from Apium graveolens L. var. dulce possesses free
partially due to the increased mRNA expression of these radical scavenging activities as well as excellent antioxidant
enzymes.36 activity in ICR mice, by protecting several important organs
The total antioxidant activities (TAOC) of apiin and rutin are from oxidative stress. These antioxidant activities could have
shown in Fig. 5F. The observed increase of TAOC promoted by contributed, at least partially, to effects underpinning certain
apiin was 6% (p < 0.05) in serum, and about 13%, 16% and 17% traditional claims of the therapeutic benets of celery. In view of
in the brain, heart and liver, respectively, but no signicant the potential use of celery in the health food industry, its

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therapeutic benets and bioactive compounds behoove further 16 L. Lin, S. Mukhopadhyay, R. J. Robbins and J. M. Harnly, J.
investigations. Food Compos. Anal., 2007, 20, 361–369.
17 J. H. Wu, Y. T. Tung, S. Y. Wang, L. F. Shyur, Y. H. Kuo and
S. T. Chang, J. Agric. Food Chem., 2005, 53, 5917–5921.
Acknowledgements 18 X. D. Luo, M. J. Basile and E. J. Kennelly, J. Agric. Food Chem.,
2002, 50, 1379–1382.
This work was supported by “the Fundamental Research Funds
19 S. Marklund and G. Marklund, Eur. J. Biochem., 1974, 47,
for the Central Universities”, and “National Major Science and
469–474.
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