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Potential protective effects of oral administration of

allicin on acrylamide-induced toxicity in male mice
Cite this: Food Funct., 2013, 4, 1229
Published on 20 May 2013. Downloaded by Temple University on 28/10/2014 02:59:36.

Lulu Zhang,†a Enting Wang,†a Feng Chen,b Haiyang Yana and Yuan Yuan*a

Received 5th February 2013
Accepted 19th May 2013
DOI: 10.1039/c3fo60057b
www.rsc.org/foodfunction
Acrylamide (AA) forms during the heating of starchy foods at high temperature, and is regarded as a
potential genotoxic carcinogen. However, with the worldwide concern about the carcinogenicity of AA,
how to reduce the toxicity of AA has become a hot research topic. In this study, we further discussed
the effects of oral administration of allicin on AA-induced toxicity by determining the hematological,
biochemical and immunological parameters in the serum, kidney, liver, and brain of male mice. Our data
showed that the orally administered allicin of 5, 10, and 20 mg kg
1 bw d
1 could significantly decrease
thiobarbituric reactive substances (TBARS) and myeloperoxidase (MPO) levels, and simultaneously
remarkably increased the superoxide dismutase (SOD) activity, glutathione S-transferase (GST) and
glutathione (GSH) levels in the kidney, liver, and brain of the AA-treated mice. Furthermore, oral
administration of allicin not only significantly decreased aspartate aminotransferase (AST), alanine
aminotransferase (ALT), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), tumor necrosis factor
a (TNF-a), interleukin (IL)-1b, interleukin (IL)-6, reactive oxygen species (ROS), and 8-hydroxy-
desoxyguanosine (8-OHdG), but also increased interleukin (IL)-10 in the serum of AA-treated mice.
Therefore, it was concluded that oral administration of allicin had a significant in vivo protective effect
against the AA induced toxicity.

Introduction mediated mechanism,7 more evidence tends to indicate that the

Acrylamide (AA) is a chemical monomer that is widely used in
polyacrylamide synthesis and a variety of other chemicals in
industry. Polyacrylamide is mainly used in the textile, paper and
cosmetics industries, as a occulent in the treatment of waste-
water, as a soil conditioner, and in ore processing and gel
electrophoresis.1 In 2002, a large amount of AA was identied in
baked and fried carbohydrate-rich foods such as French fries,
potato chips, bread, and cereals. AA has been shown to have
neurotoxic, genotoxic, carcinogenic, developmental, and
reproductive toxicities on animals. The detection of AA in food
immediately raised wide concern about human health, and
debate on how to reduce the potential toxicity of AA in food.2–4
Two major metabolic pathways for AA have been reported.5,6
One pathway is conjugation with glutathione to form the
urinary metabolites N-acetyl-S-(3-amino-3-oxypropyl) cysteine
and N-acetyl-S-(2-carbamoylethyl) cysteine. The second pathway
is epoxidation to glycidamide by the participation of cyto-
chrome P450 2E1. Although some studies have shown that AA
carcinogenicity may be related to its regulation to a hormone-
a
College of Quartermaster Technology, Jilin University, Changchun, China, 130062.
E-mail: yuanyuan1024@gmail.com; Tel: +86-431-87836376
b
Department of Food, Nutrition, and Packaging Sciences, Clemson University,
Clemson, USA 29634
† The rst two authors contributed equally to the work.
genotoxicity of AA predominantly results from the oxidative
biotransformation to its epoxide derivative glycidamide (GA),
and subsequent formation of glycidamide–DNA adducts.8–12
Although the exact mechanism of the induced toxicity of AA
is not completely clear, cumulative data showed that oxidative
stress played a critical role in its induced toxicity, which is
closely related to the activity of CYP2E1. Exogenous antioxi-
dants have been investigated to decrease or protect against
oxidative damage, and exert their protective effects in part by
inhibition of CYP2E1, thereby preventing the formation of GA
and glycidamide–DNA adducts.13–15 Recently, there have been
growing interests in exploiting natural antioxidants, due to their
natural origin, cost effectiveness and lesser side effects. Allicin,
as the most abundant thiosulnate found in garlic, is thought
to be a bioactive component of garlic.16 It is a natural antioxi-
dant that can not only scavenge oxygen free radicals and
hydroxyl radicals, but also prevent the lipid peroxidation of liver
homogenates, which leads to lipid hydroperoxide formation
oen, occurring in response to oxidative stress.17 The mecha-
nism of the antioxidant activity of allicin may rely on its ability
to scavenge oxygen free radicals.18,19 Besides, allicin is also
responsible for the characteristic odor and has a protective
effect on some immune-mediated diseases.20 Zhu et al. found
that allicin exerted neuroprotection against spinal cord I/R
injury in rabbits, which may be associated with the improve-
ment of mitochondrial functions.21 In our previous study, we

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Food & Function
found for the rst time that allicin was effective in preventing
the AA-induced hepatocyte damage both in vitro and in vivo.22 It
was hypothesized that allicin might be able to protect the
damages in the kidney, liver and brain that could be induced by
AA.23 In the light of the aforementioned medical properties of
allicin, this study was carried out to investigate the possible
protective properties of allicin against hematological, immu-
nological toxicities and various oxidative stresses in the kidney,
liver and brain of AA intoxicated mice.
Materials and methods
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Materials
AA (2-propene amide) (CAS: 79-06-1, purity > 99.8%) was
purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Allicin
(CAS: 539-86-6, purity > 99%) was purchased from Beijing Shiji
Aoke Biotechnology Co., Ltd (Beijing, China). Allicin was dis-
solved in normal saline (nal concentrations at 0.5, 1 and 2 mg
mL
1). AA was diluted in normal saline to give a nal concen-
tration of 5 mg mL
1. The allicin and AA solutions' dosing
volumes were determined based on each animal's body weight,
on a basis of a volume of 0.2 mL for a mouse weighing 20 g. The
dose of AA was selected on the basis of its effectiveness in
inducing clastogenicity in mice.24
Animals and experimental design
Fiy male BALB/c mice, weighing 20  5 g, were provided by the
Laboratory Animals Center of Jilin University (Changchun,
China). The research was carried out in accordance with the
Guidelines for Animal Experimentation of Jilin University
(Changchun, China). Animals were housed (10 mice in each
cage) in an air-conditioned room at 22  2  C and 30  10%
relative humidity. The animals were observed for general
condition for 7 days during the quarantine and acclimation
period to conrm that there were no abnormalities.
Aer a quarantine period of 7 days, 50 mice were randomly
divided into ve groups each consisting of ten animals. Group I
was treated with saline by oral gavage for 14 consecutive days
and was denoted as the control group. Group II was intra-
gastrically given saline for 7 consecutive days. On the 8th day,
aer the oral gavage of saline, the mice were intraperitoneally
injected with AA solution (50 mg kg
1 bw d
1) for another 7 days
and denoted as Group AA. Groups III, IV, and V were intra-
gastrically given allicin (5, 10 and 20 mg kg
1 bw d
1), respec-
tively, for 7 days (once daily).25 On the 8th day, Groups III–V were
intragastrically given allicin (5, 10 and 20 mg kg
1 bw d
1) and
intraperitoneally injected with a single dose of AA (50 mg kg
1
bw d
1) for another 7 days. The body weight of the animals was
measured daily and the doses of AA and allicin were recorded
according to the body weight of the animals. On the 15th day,
the animals were sacriced within 24 h aer the last treatment,
the whole blood of mice was collected in heparinized test tubes
and centrifuged at 2500g for 15 min at 4  C to separate serum,
and the serum was stored at
70  C in a freezer for further
analysis. The kidney, liver and brain were excised immediately
from the mice, washed thoroughly with ice-cold normal saline.
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The tissues were homogenized with 10% pre-chilled normal
saline in a tissue homogenizer, and then centrifuged at 2500g
for 10 min at 4  C. The supernatant was used for subsequent
biochemical analyses.
Biochemical analysis
The activities of SOD, AST, ALT, LDH and GST, levels of TBARS,
GSH, BUN and MPO, as well as protein content were determined
using commercial kits obtained from Beijing Dingguo Chang-
sheng Biotechnology Co., Ltd. (Beijing, China) according to the
manufacturer's instructions. ROS, 8-OHdG, and cytokine,
including TNF-a, (IL)-1b, (IL)-6, and (IL)-10, were determined
using ELISA kits (R & D, America) according to the manu-
facturer's instructions. Each measurement was carried out in
three independent experiments.
Determination of ROS, 8-OHdG, and cytokine content in
serum
The contents of ROS, 8-OHdG, and cytokine were determined
using the commercial ELISA kits, which were selected based on
their high degree of sensitivity, specicity, inter- and intra-assay
precision and use of small amounts of serum sample during the
assay. The samples that were treated with streptavidin–horse-
radish peroxidase (HRP) were added to plate wells precoated
with mouse monoclonal anti-cytokine antibodies. Then a
substrate containing 3,30 ,5,50 -tetramethylbenzidine (TMB) was
added and catalyzed by the peroxidase, resulting in the color
change of the substrate to blue. The reaction was terminated by
the addition of sulphuric acid, which changes the blue color of
the substrate to yellow. The intensity of the color was measured
at 450 nm using a spectrophotometer. Briey, the concentra-
tions of the standard solution were prepared for every ELISA
assay according to the instructions. About 10 mL of serum and
40 mL of sample diluent were added to the tested sample wells
and were precoated with mouse monoclonal anti-cytokine, anti-
ROS and anti-OHdG antibodies. These wells were treated with
HRP at 37  C for 60 min. The wells were then washed ve times.
A substrate containing TMB was added before the wells were
incubated at 37  C for another 15 min in the dark. The reaction
was terminated by adding 50 mL of the stop solution to each
well. The absorbance was measured at 450 nm. The contents of
TNF-a, (IL)-1b, (IL)-6 and (IL)-10 were expressed as picograms
per milliliter. The level of 8-OHdG was expressed as nanograms
per milliliter. The level of ROS was expressed as units per
milliliter.
Determination of AST and ALT activity in serum
AST and ALT were used as the biochemical markers for the early
acute hepatic damage. Briey, about 25 mL of matrix liquid and
5 mL of serum were added to the testing sample well. The
mixture was incubated at 37  C for 30 min. 25 mL of 2,4-dini-
trophenylhydrazine solution was added to the testing sample
wells and incubated at 37  C for 20 min. Finally, 250 mL of NaOH
solution with a concentration of 0.4 mol L
1 was added to the
wells. Aer thoroughly mixing and incubating for 15 min, the
This journal is ª The Royal Society of Chemistry 2013

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absorbance was measured at 510 nm. Activities of the AST and
ALT were expressed as units per liter.
Determination of the LDH level in serum
The measurement of LDH activity in serum was based on the
principle that LDH catalyzes the oxidation of NAD to NADH. In
brief, about 25 mL of matrix liquid, 20 mL of serum and 5 mL of
coenzyme I were added to the testing sample wells. The mixture
was incubated at 37  C for 15 min. Then, 25 mL of 2,4-dini-
trophenylhydrazine solution was added to the testing sample
wells and incubated at 37  C for 15 min. Finally, 250 mL of NaOH
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solution with a concentration of 0.4 mol L
1 was added to the
wells. Aer 15 minutes of thorough mixing and incubation, the
absorbance was determined at a wavelength of 450 nm. The
activity of LDH was expressed as units per liter.
Determination of the BUN content level in serum
The BUN content in serum was determined using commercial
kits according to the manufacturer's instructions. Briey, about
2 mL of serum and 25 mL of enzyme buffer were mixed and
incubated at 37  C for 10 min. Finally, 2 mL of working solution
containing a chromogenic agent and alkaline solution of
sodium hypochlorite were added. The mixture was incubated at
95  C for 40 min and was determined at a wavelength of 640 nm.
The content of BUN was expressed as milligrams per liter.
Determination of protein, SOD activity, GSH content, and
TBARS in tissue supernatant
The detection of protein, SOD activity, GSH content, and TBARS
was based on the methods described in the study of Zhang
et al.22
Determination of MPO content in tissue homogenates
The principle of the assay was based on using 4-amino-
antipyrine–phenol solution as the substrate for MPO-mediated
oxidation by H2O2. Briey, 20 mL of tissue homogenate was
added to 3.2 mL of reaction mixture and incubated at 37  C for
30 min. Finally, 50 mL of working solution was added to the
mixture, which was incubated at 60  C for 10 min and deter-
mined at a wavelength of 460 nm. The content of MPO was
expressed as units per gram.
Table 1 Effects of allicin on the activity of AST, ALT and LDH, as well as levels of BUN, ROS, and 8-OHdG in serum of AA-treated micea
AA (50 mg kg
1 Allicin (5 mg kg
1 bw d
1) + Allicin (10 mg kg
1 bw d
1) + Allicin (20 mg kg
1 bw d
1) +
Control bw d
1) AA (50 mg kg
1 bw d
1)
AST (U L
1) 9.63  0.57* 20.88  1.01# 15.52  0.79*#

1
ALT (U L ) 5.41  1.00* 14.38  0.94 #
9.91  0.89*#
LDH (U L
1) 329.36  7.59* 470.63  10.22# 402.52  11.56*#
BUN (mg L
1) 84.16  1.60* 120.67  2.07# 99.80  2.39*#
ROS (U mL
1) 155.03  4.85* 290.29  4.94# 241.25  4.94*#
8-OHdG (ng mL
1) 11.15  0.66* 27.09  0.34# 21.85  0.74*#
a
All values are means SD (n ¼ 10). (*) indicates statistically signicant differences between the groups treated with AA ( p < 0.05). (#) indicates
statistically signicant differences with the control ( p < 0.05).
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Determination of GST activity in tissue supernatant
GST ability was assayed spectrophotometrically using 1-chloro-
2,4-dinitro-benzene (CDNB) as the substrate as described in the
manufacturer's instructions. GST has the ability to catalyze the
reaction of glutathione (GSH) and CDNB. The ability of GST is
linear with the concentration of reaction substrate in a period of
time. Briey, 30 mL of matrix liquid and 25 mL of serum were
mixed and incubated at 37  C for 30 min. Then, 2 mL of working
solution was added and centrifuged at 3000  g for 10 min at
4  C to separate the supernatant. Finally, 2 mL of supernatant
was added to 4.5 mL of reaction mixture and determined at a
wavelength of 412 nm. The ability of GSH was expressed as units
per milligram protein.
Statistical analysis
Statistical analysis was performed using SPSS 11.5 soware
(Chicago, USA). The signicance of difference was calculated by
the one-way ANOVA test, and the results with p < 0.05 were
considered to be statistically signicant. Graphs were drawn
with OriginPro 7.5 soware (OriginLab Corporation, North-
ampton, MA, USA).
Results
Hematological parameter changes
Effects of AA alone and supplementation of allicin during the
AA exposure on some hematological proles are shown in Table
1. The activities of AST and ALT are two important reliable
markers for the function of the liver. Both the activities of AST
and ALT signicantly increased in the AA-treated group
compared with those of the control group ( p < 0.05). Aer the
addition of allicin, the activities of AST and ALT decreased along
with the increase of concentration of allicin. The treatment with
20 mg kg
1 bw d
1 of allicin for 14 days reduced the AST activity
by 50.1%, whereas the AST activity was reduced by 46.1% and
25.7% aer the treatment with 10 and 5 mg kg
1 bw d
1 of
allicin, respectively. By contrast, the treatments with 20, 10, and
5 mg kg
1 bw d
1 of allicin for 14 days reduced the ALT activ-
ities by 55.7%, 51.2%, and 30.1%, respectively.
The LDH activity in serum, as an indicator of an overall
tissue damage, was dramatically elevated to 470.63  10.22 U
L
1, compared to the value of 329.36  7.59 U L
1 in the control
group. The groups treated with 20 mg kg
1 bw d
1 of allicin
AA (50 mg kg
1 bw d
1) AA (50 mg kg
1 bw d
1)
11.26  1.14* 10.30  1.38*
6.95  0.94* 6.37  0.76*
357.11  7.36*# 336.57  11.95*
92.67  2.16*# 87.50  1.81*
212.15  4.94*# 190.06  8.90*
16.78  0.66*# 13.83  0.66*
Food Funct., 2013, 4, 1229–1236 | 1231

Food & Function
showed a signicant decrease in elevation of serum LDH values
( p < 0.05), with the value of 336.57  11.95 U L
1. In addition,
there were no signicant differences among the groups treated
with 10, and 5 mg kg
1 bw d
1 of allicin.
The BUN level is an important reliable marker for kidney
damage induced by AA. As shown in Table 1, mice in the AA
treated group showed a signicant increase of the BUN level in
the serum (120.67  2.07 mg L
1) compared with the value of
84.16  1.60 mg L
1 of the control group ( p < 0.05). On the other
hand, serum BUN of the groups treated with 5, 10, and 20 mg
kg
1 bw d
1 of allicin had values of 99.8  2.39, 92.67  2.16,
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and 87.50  1.81 mg L
1, respectively, which were signicantly
lower than those in the AA group ( p < 0.05).
The ROS and 8-OHdG levels are widely used as indicators of
oxidative DNA damage. We measured the ROS and 8-OhdG
levels in the serum, of which the results are shown in Table 1.
The ROS level in the AA treated group (290.29  4.94 U mL
1)
was signicantly higher than that in the control group (155.03 
4.85 U mL
1, p < 0.05). But this effect was diminished signi-
cantly by the allicin treatment ( p < 0.05, Table 1). The ROS levels
in the allicin treated group (190.06  8.90, 212.15  4.94, and
241.25  4.94 U mL
1 at doses of 20, 10 and 5 mg kg
1 bw d
1,
respectively) were signicantly lower than those in the AA
treated group ( p < 0.05). The ndings indicated that the free
radicals released in the serum were effectively scavenged aer
the allicin treatment.
The lowest level of 8-OHdG (11.15  0.66 ng mL
1) was
observed in the control group. The 8-OHdG levels signicantly
increased in the AA group compared with those in the control
group ( p < 0.05). The highest level of 8-OHdG (27.09  0.34 ng
mL
1) was observed when the mice were treated with AA alone.
The 8-OHdG level then decreased accordingly with the increase
of concentration of allicin from 5 to 20 mg kg
1 bw d
1. The
administration of allicin at concentrations of 5, 10, and 20 mg
kg
1 bw d
1 reduced the 8-OHdG levels by 19.34%, 38.06%, and
48.87%, respectively, as compared with that of the AA group.
Immunological parameter changes
The cytokine contents including the TNF-a, (IL)-1b, (IL)-6, and
(IL)-10 were determined from the mouse serum. As shown in
Fig. 1A, the content of (IL)-6 (81.22  2.08 pg mL
1) in the serum
was drastically enhanced aer the treatment with AA, which was
double the value of the control group (45.76  1.56 pg mL
1).
Through the administration of allicin with a dose of 5, 10, and
20 mg kg
1 bw d
1, the content of (IL)-6 was effectively reduced
( p < 0.05), while the treatment of 20 mg kg
1 bw d
1 of allicin
effectively reduced the (IL)-6 level (52.36  2.08 pg mL
1) close
to that of the control group.
The (IL)-1b level (Fig. 1B) was found to be elevated in the
serum of the AA treated group (88.93  1.72 pg mL
1) compared
to that of the control group of 44.08  0.85 pg mL
1. Aer
administration of allicin, the (IL)-1b levels signicantly decreased
in the AA-treated group compared with that of the control group
( p < 0.05). Administration of allicin with a concentration of 20 mg
kg
1 bw d
1 was found to be very effective in causing a drastic
decrease of the (IL)-1b level ( p < 0.05).
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Signicant enhancement of the TNF-a (432.09  6.67 pg
mL
1, Fig. 1C) in the level of serum was observed in the AA
group compared with that of the control group (229.12 
6.68 pg mL
1). Aer the administration with allicin, the levels of
TNF-a were reduced along with the increase of allicin concen-
tration. When the mice were treated with 20 mg kg
1 bw d
1 of
allicin, its level of TNF-a (250.95  8.82 pg mL
1) was nearly
back to the level of the control group.
The (IL)-10 level (Fig. 1D) was found to be signicantly
decreased in the serum of the AA treated group (98.45  3.30 pg
mL
1) compared with that of the control group (209.85 
4.75 pg mL
1). The serum (IL)-10 levels of the groups treated
with 5, 10, and 20 mg kg
1 bw d
1 of allicin showed values of
134.87  3.30, 168.65  7.26, and 196.10  7.14 pg mL
1,
respectively, which were signicantly higher than those in the
AA group ( p < 0.05).
Biochemical parameter changes in the kidney, liver and brain
of AA intoxicated mice
To determine the effect of allicin on oxidative damage in the
kidney, liver and brain of the AA-treated mice, the activities of
the major antioxidant enzymes such as SOD and GSH, GST
content, TBARS and MPO content were determined. As shown
in Table 2, AA induced a signicant decrease in the activities of
SOD, levels of GSH and GST content compared with those in the
control group ( p < 0.05). In contrast, the SOD activity, the GSH
level and GST content signicantly increased in the groups
administered with allicin compared with those in the AA group
( p < 0.05). The TBARS and MPO content signicantly increased
in the AA-treated group compared with those of the control
group ( p < 0.05). The TBARS and MPO content markedly
decreased in mice orally treated with allicin compared with
those of the AA group ( p < 0.05).
Discussion
As reported, the cytotoxic and genotoxic activities of AA were
related to its causative decrease of the oxidative defense system
and increase of ROS in the cells.26 Many previous investigations
demonstrated that garlic organosulfur compounds prevented
toxicities induced by cyanide, sodium nitrite, carbon tetra-
chloride, ethanol and sodium arsenite.27–31 Their toxic mecha-
nisms entailed oxidative stress and impairment in the
antioxidant defense system. In our previous study, we found for
the rst time that allicin was effective in preventing the AA-
induced hepatocyte damage in vitro and in vivo.22 Based on this
fact, it was hypothesized that allicin might also have the ther-
apeutic potential in prevention of AA-induced toxicity on some
other tissues.
The hepatic enzymes, such as ALT and AST, were used as the
biochemical markers for the detection of early acute hepatic
damage. Their increased levels in serum indicated the
increased permeability and damage and/or necrosis of hepato-
cytes.32 In our present study, AA caused a signicant increase in
the activities of AST and ALT (Table 1), which was attributed
to the severe damage of the tissue membrane. Aer an
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Fig. 1 Effects of allicin on AA-induced alterations of IL-6 (A), IL-1b (B), IL-a (C), and IL-10 (D) in mice serum. Data are presented as the mean  SD for 10 animals in each
group (n ¼ 10). (*) indicates statistically significant differences between the groups treated with AA ( p < 0.05). (#) indicates statistically significant differences with the
control ( p < 0.05).
administration of allicin, the activities of AST and ALT
decreased in the serum of mice. Similarly, the increased level of
LDH, which is an intracellular enzyme in serum, is an indicator
of cell damage.33 These ndings suggested allicin was effective
in preventing the AA-induced hepatocyte damage. Similarly,
allicin signicantly prevented the increase of BUN levels in
serum among the AA-treated mice, suggesting the allicin
potently protected against the kidney toxicity induced by AA.
Besides, allicin could signicantly decrease the level of 8-OHdG
in the AA-treated mice, suggesting the allicin had a protective
effect against the oxidative DNA damage induced by AA.
AA can promote the release of intracellular ROS.34 In the
present study, we also found that the treatment with AA induced
a signicant increase in intracellular generation of ROS. The
ROS are much more reactive than molecular oxygen and can
cause severe damage to nucleic acids, cell membranes, and
proteins.35 ROS are also known to attack other cellular compo-
nents, such as lipids, leaving behind reactive species that in
turn can couple to DNA bases.36 Besides, ROS are able to activate
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Food & Function
nuclear factor kappa B (NFkB) and its controlled cytokine.37–39
Keiss et al. showed that garlic powder extracts could reduce LPS-
induced production of proinammatory cytokines interleukin
IL-1 and tumor necrosis factor TNF-a, whereas the expression of
the anti-inammatory cytokine IL-10 was unchanged. Garlic
may indeed promote an anti-inammatory environment by
cytokine modulation in human blood that leads to an overall
inhibition of activity in the surrounding tissue.40
Our data about cytokine showed that AA activated inam-
matory cells and subsequently amplied the inammatory
response by releasing various cytokines. When the mice were
intragastrically given allicin, the contents of TNF-a, (IL)-1b and
(IL)-6 markedly decreased and the (IL)-10 level increased in
mice serum. These results suggested that allicin could alleviate
tissue injury caused by AA through suppressing inammatory
response.
ROS include superoxide anions, hydrogen peroxides, and
hydroxyl radicals, which are generated by the incomplete
reduction of oxygen.41 Our results showed that these ndings
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Table 2 Effects of allicin on the activity of SOD and GST, levels of MPO, GSH, and MDA in tissue of AA-treated micea

AA (50 mg kg
1 Allicin (5 mg kg
1 bw d
1) + Allicin (10 mg kg
1 bw d
1) + Allicin (20 mg kg
1 bw d
1) +
Control bw d
1) AA (50 mg kg
1 bw d
1) AA (50 mg kg
1 bw d
1) AA (50 mg kg
1 bw d
1)

SOD (U mg
1)
Liver 113.03  4.13* 63.61  9.91# 73.36  6.83*# 99.73  6.21*# 107.69  7.44*
Kidney 63.50  2.93* 19.12  0.57# 30.62  1.47*# 45.68  2.75*# 58.94  3.39*
Brain 100.28  4.92* 58.38  8.89# 67.01  6.51*# 88.95  7.41*# 94.94  5.01*

GST (U mg
1)
Liver 58.48  2.42* 23.69  1.54# 39.41  1.01*# 44.85  3.6*# 56.19  1.22*
Kidney 23.93  1.73* 8.64  1.09# 12.63  1.06*# 19.03  0.4*# 21.96  1.09*
23.72  1.37* 7.31  0.41# 11.46  1.44*# 18.57  1.36*# 22.83  2.13*
Published on 20 May 2013. Downloaded by Temple University on 28/10/2014 02:59:36.

Brain

MPO (U g
1)
Liver 0.63  0.01* 1.15  0.03# 0.99  0.03*# 0.81  0.01*# 0.68  0.03*
Kidney 0.23  0.02* 0.74  0.04# 0.45  0.04*# 0.32  0.03*# 0.27  0.03*
Brain 0.11  0.02* 0.39  0.02# 0.28  0.02*# 0.22  0.03*# 0.13  0.02*

GSH (nmol mg
1)

Liver 5.84  0.42* 2.42  0.10# 2.34  0.13*# 4.63  0.12*# 5.66  0.14*
Kidney 3.00  0.46* 0.76  0.04# 1.18  0.15*# 2.31  0.07*# 2.76  0.35*
Brain 2.82  0.18* 0.67  0.07# 1.03  0.18*# 2.12  0.08*# 2.67  0.39*

MDA (nmol mg
1)

Liver 1.61  0.09* 6.15  0.33# 3.73  0.15*# 2.52  0.29*# 1.81  0.14*
Kidney 0.75  0.06* 1.85  0.05# 1.27  0.03*# 1.02  0.07*# 0.89  0.06*
Brain 0.75  0.04* 4.23  0.17# 2.85  0.22*# 1.22  0.17*# 0.84  0.06*
a
All values are means SD (n ¼ 10). (*) indicates statistically signicant differences between the groups treated with AA ( p < 0.05). (#) indicates
statistically signicant differences with the control ( p < 0.05).

were consistent with the study of Xie et al.42 that allicin can
scavenge oxygen free radicals and hydroxyl radicals. Free radi-
cals seem to trigger the accumulation of leukocytes in the tissue
involved, and thus cause further injury through activated
neutrophils. It has been shown that activated neutrophils
secrete myeloperoxidase enzyme (MPO) and liberate oxygen
radicals.43,44 As reported, MPO, which is an endogenous lyso-
somal enzyme that removes H2O2 and catalyzes the formation
of toxic hypochlorous acid, increased signicantly due to AA
toxicity.45 Our data indicated that allicin treatment prevented
increased MPO activity in the mice suffered by the AA-induced
toxicity. Accordingly, allicin blocked the neutrophil inltration
into the injured tissues. As a result of decreased neutrophil
inltration, tissues produced less free radicals. It has been
shown that the protective effect of allicin against tissue damage
was partly mediated by the inhibition of inammatory
responses. MPO and its oxidative products play key roles in the
lipid peroxidation in liver damage. Some previous studies
showed the correlation between the levels of neutrophil and
TBARS, the latter was an indicator of free radical-mediated lipid
peroxidation damage.46 Our data showed that the oral treatment
with allicin caused signicantly decreased TBARS compared
with the AA-treated group (Table 2).
The human body has an effective antioxidant defense system
against free radicals and ROS induced damage, in which the
endogenous enzymatic and non-enzymatic antioxidants such as
GSH, SOD and GST play an important role.47 As reported, AA
could inhibit GST activity and deplete the GSH level.48 GSH is as
an essential intracellular reducing substance for the
maintenance of thiol groups on intracellular proteins and
antioxidant molecules in living organisms.49 GSH is an impor-
tant constituent of intracellular protective mechanisms against
various noxious stimuli including oxidative stresses.50 GST plays
a vital role in the liver by eliminating toxic compounds by
conjugating them with GSH.51 However, once the balance
between ROS production and antioxidant defenses is lost,
oxidative stress will consequently occur, which through a series
of biological events deregulates the cellular functions leading to
various pathological conditions.52 SOD, GST and other antioxi-
dant enzymes constitute a mutually supportive team of defense
against ROS. SOD is a metalloproteinase to detoxify superoxide
anions as an efficient dismutative mechanism and it is the rst
enzyme involved in the antioxidant defense.53 Allicin could
increase glutathione S-transferase activity and the level of
glutathione remarkably.42 Our ndings suggested that a single
dose of AA given to mice by injection i.p. aer 14 days caused
depletion of GSH, and reduced activities of antioxidant enzymes
SOD and GST in the liver, brain and kidney compared with
those in the control group ( p < 0.05, Table 2), which is consis-
tent with that Zhu et al. concluded from experiments in rabbit.21
Administration of allicin signicantly elevated the levels of GSH
and GST compared with those in the AA-treated group.
Conclusion
In conclusion, this study further investigated the effects of
allicin on AA-induced toxicity in mice brain, liver, kidney and
tested tissues by evaluating hematological, biochemical and

1234 | Food Funct., 2013, 4, 1229–1236 This journal is ª The Royal Society of Chemistry 2013

Paper
immunological markers. Our data demonstrated that allicin
possessed a powerful protective capacity for the AA-treated
mice. Therefore, taking food rich in or supplemented with
allicin might be health benecial for the individuals who are at
risk of AA toxicity.
Acknowledgements
This work was nancially supported by the “National Natural
Science Foundation (31000750)”, China Postdoctoral Science
Foundation Special Funded Project (201104527), and Fund for
Published on 20 May 2013. Downloaded by Temple University on 28/10/2014 02:59:36.
Distinguished Young Scholars of Heping Campus of Jilin
University (4305050102Q9). Accordingly, the authors gratefully
acknowledge the fund supports.
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