JOURNAL OF MEDICINAL FOOD

J Med Food 00 (0) 2019, 1–8

# Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2018.4294

Effects of Low Temperature-Aged Garlic on Exercise Performance

and Fatigue in Mice
Kyung A. Hwang,1,* Yu-Jin Hwang,1 In-Guk Hwang,1 Wan Heo,2 and Young-Jun Kim2
1
Department of Agrofood Resources, National Institute of Agricultural Sciences, Rural Development Administration,
Jeollabuk-do, Korea.
2
Department of Food and Biotechnology, Korea University, Sejong, Korea.
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ABSTRACT We developed low temperature-aged garlic (LTAG) to remove its unique and spicy flavor and evaluated the
anti-fatigue properties of LTAG against exercise-induced fatigue in mice. In the results, the treadmill running time to
exhaustion in the mice fed LTAG was prolonged compared with the control. There was significant difference in blood
parameters of glucose, lactate, lactate dehydrogenase (LDH), and free fatty acid (FFA) concentration between the LTAG-fed
mice and the control. In addition, LTAG effectively increased the content of glycogen and creatine kinase and the activity of
antioxidant enzymes in the muscle. The mechanism underlying the anti-fatigue activity of LTAG is hypothesized to involve
increase in postexercise tissue glycogen accumulation to improve the aerobic and anaerobic exercise capacity. LTAG may
have an ergogenic effect on endurance exercise while decreasing the levels of FFA, LDH, and lactate, which are associated
with the anti-fatigue effect. Thus, LTAG has potential as a pharmacological anti-fatigue agent.

KEYWORDS:  aged garlic  creatine kinase  glycogen  lactate  physical fatigue

INTRODUCTION factors leading to fatigue.8 High levels of oxidative stress

lead to the excessive generation of reactive oxygen species
F atigue is tiredness resulting from a physical or mental
exertion wherein the physical and mental capacities are
reduced; this is classified as physical and mental fatigue.
(ROS), which are highly reactive molecules that cause lipid
peroxidation in membrane structures and damage cellular
structures. The release of ROS could result in lipid perox-
Physical fatigue, such as that caused by exercise, is the
idation in the mitochondrial membrane. Damaged mito-
feeling of lethargy owing to the accumulation of a variety of
chondria were observed to reduce cellular respiration and
substances in the blood.1,2 Mental fatigue, such as that caused
adenosine triphosphate (ATP) generation, and are suggested
by extended cognitive activity, emotional conflict, anxiety, or
to be a primary cause of fatigue.9 Several studies have
malaise, refers to a transient state of lethargy, somnolence, or
demonstrated that exogenous antioxidants can reduce the
decreased attention.3,4 Such fatigue deteriorates the perfor-
contribution of exercise-induced oxidative stress.10,11 Re-
mance of the body and can lead to illnesses such as liver
cently, researchers have been seeking natural components
dysfunction, diabetes, and gastrointestinal diseases.5,6
that improve athletic ability and accelerate the elimination
There are various theories regarding the mechanisms
of fatigue.12–14
underlying physical fatigue, including exhaustion, clogging,
Garlic (Allium sativum L.) is one of the most popular
and the radical theory, among others. The exhaustion and
vegetables worldwide; it is often used as a spice in Asian
radical theories have attracted the most interest. The ex-
and Mediterranean foods and has long been used in tradi-
haustion theory refers to the exhaustion of various energy
tional medicine because of its diverse health benefits, in-
sources, including glucose and glycogen, during exercise,
cluding antibacterial,15 anticancer,16 antioxidant,17,18 and
resulting in physical fatigue.7 The radical theory proposes
hypotensive19 properties. Garlic has also been the subject of
that intense exercise can lead to an imbalance between the
study because of its endurance and stamina-enhancing ef-
body’s oxidative and antioxidative systems; it has previ-
fects.20 The major components involved in garlic’s stamina-
ously been demonstrated that oxidative stress is one of the
enhancing effect are scordinin and allicin.21 Allithiamine,
the combination of allicin and vitamin B1, breaks down
Manuscript received 12 July 2018. Revision accepted 26 May 2019. carbohydrates to produce energy, stimulates metabolism, is
effective in alleviating fatigue,22 and has a stronger effect
Address correspondence to: Kyung A. Hwang, PhD, Department of Agrofood Resources,
National Institute of Agricultural Sciences, RDA, 166 Nongsaengmyeong-ro, Iseo-myeon,
than natural vitamin B1.23 Hence, if we take water-soluble
Wanju-gun, Jeollabuk-do 55365, Republic of Korea, E-mail: kah366@korea.kr vitamins and garlic together after exercise, they will have a

1
 2 HWANG ET AL.
great effect in reducing fatigue.21 However, because of the
strong aroma and pungent taste of raw garlic, it gives a sense
of rejection when consumed. To overcome these factors,
research methods have been developed to remove or conceal
the strong flavor and spicy taste while maintaining the
physiological activity of garlic. The typical method is heat
treatment or modifying the aging period.24–26 Several stud-
ies have reported that aged garlic extract protects against
fatigue.27,28 Morihara et al.27 reported the ameliorating ef-
fect of aged garlic extract on physical fatigue through the
turnover of aerobic glucose metabolism, attenuate oxidative
stress, and increase oxygen supply based on vasodilation.
Ushijima et al.28 compared anti-fatigue effect of raw garlic
juice, heated garlic juice, processed garlic powder, and aged
garlic extract. Although several studies have evaluated the
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anti-fatigue effect of aged garlic, there have been few
studies on the anti-fatigue effects of low temperature-aged
garlic (LTAG) extract and the anti-fatigue mechanism of
LTAG extract remain unclear. Therefore, this study aimed
to evaluate the anti-fatigue properties of LTAG extract on
exercise and endurance capacity in mice.
MATERIALS AND METHODS
Preparation of LTAG extract
Garlic was purchased from Uiseong-Gun, Gyeongsangbuk-
do, Korea. It was stored in a sealed container and the aging
treatment was performed under the following conditions: the
aged garlic at 60C for 60 days was peeled and lyophilized,
then pulverized with a Philips mini blender (HR 2860; Ya-
horng Electronic Co., China) for 10 min. Subsequently, 5 g
LTAG powder was added to 200 mL of 70% EtOH and ul-
trasonic extraction was performed for 30 min. After standing
at room temperature for 1 h, the supernatant was recovered.
This was repeated three times and then filtered (Whatman
No. 2 filter paper). The 70% EtOH extract of LTAG was
concentrated under vacuum at 45C (EYELA N-1000; To-
kyo Rikakikai Co., Ltd., Tokyo, Japan), and then the con-
centration lyophilized with a Bondiro Lyophpride freeze
dryer (Ilshin Lab Co. Ltd., Yangju, Korea) at -70C under
reduced pressure (<20 Pa). The dry residue was stored at
-70C. For the raw garlic extract, the same process was used
except for the aging process using the same batch of garlic.
Reagents
Assay kits used for the determination of blood glucose,
lactate dehydrogenase (LDH), lactate, free fatty acid (FFA),
glycogen, and creatine kinase (CK) were purchased from
Abcam (Cambridge, United Kingdom). All other reagents
used in this study were of analytical grade.
Animals and treatment
The 6-week-old male ICR mice were purchased from
Orient Bio, Inc. (Seongnam, Korea). All animal experiments
were approved according to the guidelines of the Institu-
tional Animal Care and Use Committee of the National
Institute of Agricultural Sciences (NIAS201602), and all
procedures were conducted in accordance with the Animal
Experiments Guidelines of the National Institute of Agri-
cultural Sciences. The mice were housed under a controlled
temperature (23C – 3C) with a relative humidity of 40–
60% and 12 h light/dark cycles. Food (rodent diet 5L79;
Orient Bio, Inc.) and water were provided ad libitum.
After 1 week of acclimation, the animals were divided
randomly into six groups (n = 10) as follows: (a) normal
group (administered phosphate-buffered saline [PBS] with
no exercise); (b) control group (administered PBS with ex-
ercise); (c) low-dose raw garlic extract (200 mg/kg)-treated
group with exercise (raw 200); (d) high-dose raw garlic
extract (500 mg/kg)-treated group with exercise (raw 500);
(e) low-dose LTAG extract (200 mg/kg)-treated group with
exercise (LTAG 200); and (f) high-dose LTAG extract
(500 mg/kg)-treated group with exercise (LTAG 500). The
mice in each group were orally administered with the extract
or PBS for 28 days.
The mice in each group were forced to run on the tread-
mill for 4 weeks. The pattern of loaded exercise consisted of
forced running as follows: During the first week, 20 m/min
for 5 min, 25 m/min for 5 min, and 30 m/min for 5 min;
during the second week, 20 m/min for 5 min, 25 m/min for
10 min, and 30 m/min for 5 min; during the third week,
20 m/min for 5 min, 25 m/min for 15 min, and 30 m/min for
15 min; during the fourth and final week, the speeds used for
determination of the exhaustion were 30 m/min during the
50 min.
Exhaustive treadmill test
The exhaustive treadmill test evaluated the effects of
LTAG extract on exercise durability in mice. After 28 days
of treatment, 5 mice from each group were subjected to the
exhaustive treadmill capacity test. At 30 min after the final
treatment, the mice were placed individually onto tread-
mills. The exhaustive treadmill capacity test increased the
treadmill speed from 15 to 40 m/min every 3 min, and the
running time was monitored until the mouse failed to follow
the speed increase on three consecutive occasions and lag
occurred, at which point the total running time was imme-
diately recorded.29
Analysis of the biochemical parameters of blood
and gastrocnemius muscle
The effect of LTAG on the levels of glucose, FFA, LDH,
and lactate in the blood was evaluated immediately after
exercise. At 1 h after the last oral administration with the
extract or PBS, the mice underwent a 60-min treadmill ex-
ercise. After the treadmill exercise, the mice were killed
with CO2. Blood samples were collected from the abdomi-
nal aorta with heparinized syringes. Immediately after blood
collection, the gastrocnemius muscle was isolated and rap-
idly frozen in liquid nitrogen, then stored at -70C before
tissue analysis. The plasma was prepared by centrifugation
at 850 g at 4C for 15 min (GZ-1730MR; Gyrozen Co. Ltd.,
Daejeon, Korea). The concentration of blood glucose, FFA,
 AGED GARLIC IMPROVES ANTI-FATIGUE EFFECT 3

FIG. 1. Effect of LTAG extract on (A) body weight and (B) food intake in mice. Data are expressed as mean – SEM (n = 10). LTAG, low
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temperature-aged garlic; SEM, standard error of the mean.

lactate, and LDH activity in plasma and glycogen and CK in
muscle were determined by detection kits according to the
instructions.
Quantitative real-time polymerase chain reaction
and analyses of antioxidant gene expression
The total RNA was isolated from muscle tissue using an
RNA extraction kit (Qiagen, Valencia, CA, USA) according
to the manufacturer’s instructions. cDNA was synthesized
from 500 ng of total RNA using M-MLV Reverse Tran-
scriptase (Promega, Madison, WI, USA). The real-time
polymerase chain reaction (RT-PCR) reaction was per-
formed using 2 · SYBR Green Master mix (Qiagen) and
specific primers for the indicated antioxidant enzyme-related
genes: superoxide dismutase (SOD) and glutathione peroxi-
dase (GPx). All results were normalized to those of house-
keeping gene, glyceraldehyde 3-phosphate dehydrogenase.
Statistical analysis
Statistical analyses were performed using the SPSS v12.0
software (SPSS, Inc., Chicago, IL, USA). Data are re-
presented as the mean – standard error of the mean from
three independent experiments, unless stated otherwise.
Differences between groups were analyzed by one-way
analysis of variance test followed by Tukey’s post hoc least
significant difference test and P < .05 was considered to
indicate a significant difference.
Effect of LTAG treatment on the exhaustive
treadmill test
We selected an exhaustive running test to assess the de-
gree of physical fatigue, wherein the length of the exhaus-
tive running time indicates the degree of fatigue. The effects
of raw garlic and LTAG extract on the running time of mice
are presented in Figure 2. The groups treated with LTAG
showed a significant increase in their running time com-
pared with the control group. The running time of 200 and
500 mg/kg LTAG groups increased by 65.4% and 104.1%,
respectively. The results indicated that the LTAG extracts
intake could increase exercise endurance in mice and that
LTAG extracts had anti-fatigue effects.
Effect of LTAG treatment on plasma biochemistry
Biochemical analysis results of the plasma from each
group are presented in Figure 3. The plasma glucose levels
were lower in the 500 mg/kg LTAG-treated group than in
the control group. The plasma glucose levels in the raw
garlic and 200 mg/kg LTAG-treated groups tended to be

RESULTS
Effect of LTAG treatment on body weight
and food intake
We evaluated the general characteristics of the mice by
LTAG extract treatment, with body weight, observing be-
havior, and food intake. As presented in Figure 1, body
weight was unaltered in all groups over the duration of 4
weeks. In addition, daily intake of diet did not differ in the FIG. 2. Effect of LTAG extract on exhaustive treadmill test perfor-
normal, control, and garlic treatment groups, and no no- mance in mice. Data are expressed as mean – SEM (n = 5). Different
ticeable side effects were observed. letters (a–c) above the bars indicate significant differences at P < .05.
 4 HWANG ET AL.
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FIG. 3. Effect of LTAG extract on (A) blood glucose, (B) free fatty acid, (C) LDH activity, and (D) lactate in mice plasma. Data are expressed
as the mean – SEM (n = 5). Different letters (a–c) above the bars indicate significant differences at P < .05. LDH, lactate dehydrogenase.
slightly lower than those in the control group; however, no
statistically significant differences were observed (Fig. 3A).
There was no significant difference in the plasma FFA
level between the raw garlic or LTAG-treated groups and
the control group, with the exception of the 500 mg/kg
LTAG group, which exhibited significantly lower FFA
levels than those in the control group (Fig. 3B).
During periods of extended and vigorous exercise, excess
lactate accumulates in the body. Lactate can be used as an
indicator of intense exercise and fatigue.30 The effects of raw
garlic and LTAG extract on the LDH activity and lactate
content after running are giving in Figure 3C and D. Raw
garlic and LTAG extract significantly decreased the LDH
activity and lactate content. We found that plasma LDH
activity was reduced by 51.6% and 49.3%, 50.9% and
47.3%, in the 200 and 500 mg/kg raw garlic, 200 and
500 mg/kg LTAG groups, respectively, as compared with
that in the control group (Fig. 3C). Plasma lactate levels were
reduced by 14.1%, 24.9%, 21.9%, and 34.1%, in the 200 and
500 mg/kg raw garlic, 200 and 500 mg/kg LTAG groups,
respectively. Raw garlic and LTAG extract treatment re-
duced lactate levels in a dose-dependent manner (Fig. 3D).
Effect of LTAG treatment on gastrocnemius
muscle parameters
The importance of muscle glycogen levels in endurance
exercise has previously been verified, and the depletion of
muscle glycogen is a factor in fatigue and exhaustion.31
As given in Figure 4A, after vigorous exercise, the muscle
tissue glycogen content in the LTAG-treated groups was
higher than that in the control group. Muscle glycogen levels
significantly increased by *1.4-fold upon LTAG treatment.
Muscle CK is known to be an accurate indicator of muscle
damage.32 As given in Figure 4B, the CK levels in mice
significantly increased in the LTAG extract-treated mice
compared with that in the control mice. There were no
significant changes in the CK levels for the raw garlic
extract-treated groups.
Effect of LTAG treatment on antioxidant enzyme
expression in muscle
ROS are produced during normal metabolism and per-
form a variety of physiological functions; however, exces-
sive oxygen production through intense exercise can cause
peroxidation of the membrane lipids and lead to intracellular
tissue and DNA damage.33
We assessed whether increased exercise endurance by
LTAG treatment were associated with antioxidant enzymes
such as SOD and GPx using RT-PCR and western blot. As
indicated in Figure 5 and Supplementary Figure S1, raw
garlic and LTAG treatment significantly increased SOD and
GPx expression compared with the control group. Both
LTAG 200 and 500 mg/kg groups were significantly dif-
ferent from control group, and there was no significant
difference between the two concentrations.
 AGED GARLIC IMPROVES ANTI-FATIGUE EFFECT 5

FIG. 4. Effect of LTAG extract on gastrocnemius muscle (A) glycogen and (B) creatine kinase in mice. Data are expressed as mean – SEM
(n = 5). Different letters (a–c) above the bars indicate significant differences at P < .05.
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DISCUSSION Various biochemical parameters, including lactate, am-

In this study, the effects of LTAG extract on exercise
performance and fatigue were investigated in mice. To
confirm the anti-fatigue effects of LTAG extract and its
mechanisms, the mice were subjected to an exhaustive
running test and biochemical parameters related to fatigue,
including glucose, FFA, LDH activity, lactate, tissue gly-
cogen, CK, SOD, and GPx were determined.
The length of the exhaustive running time indicates the
degree of fatigue.29,34 As a result of the exhaustive running
test, we confirmed that mice treated with LTAG extract had
longer exhaustive running times than the control mice.
The occurrence of fatigue during intense exercise is clo-
sely associated with the depletion of glycogen, both in the
liver and muscle.35 Glycogen storage is an important energy
source, particularly during exercise, and serves a central role
in maintaining glucose homeostasis by supplementing the
blood glucose.36 Fatigue occurs when the stored glycogen is
almost exhausted. Thus, glycogen is used as an accurate
marker for fatigue, and increased glycogen is closely asso-
ciated with improved endurance and anti-fatigue effects.37
In this study, the muscle glycogen concentrations in the
mice of the LTAG-treated groups significantly increased
compared with those of the control group (Fig. 4A).
monia, glucose, and CK, are important markers of muscle
fatigue during exercise.38 Lactate is an endogenous metab-
olite of the anaerobic reaction produced by the reduction of
pyruvate, and its concentration in body fluids increases
during intense exercise.39 Conversely, human tissues per-
form optimally within a narrow pH range; thus, if the pH is
lowered because of increased lactate in the muscle and
blood, and acidification occurs, mitochondrial function is
disturbed and the muscle energy production capacity is in-
hibited. Consequently, muscle contraction becomes diffi-
cult, and thus muscular strength is weakened. After the
administration of LTAG extract, plasma lactate levels were
significantly decreased compared with that of the control
group (Fig. 3D). Therefore, LTAG extract decreased gly-
cogen utilization and lactate production in mice during ex-
ercise, suggesting that the anti-fatigue effect of LTAG
extract is associated with improvements in energy metabo-
lism activation.
In addition, LDH regulates lactate formation and con-
version through reducing pyruvate during anaerobic meta-
bolic processes in muscle. LDH is a specific enzyme in the
blood used as an index to evaluate the energy metabolic
pathways mobilized during exercise. It allows evaluation of
metabolic function adaptation during energy metabolism,

FIG. 5. Effect of LTAG extract on the mRNA expression the antioxidant enzymes (A) SOD and (B) GPx in the gastrocnemius muscle of mice.
Data are expressed as mean – SEM (n = 5). Different letters (a–c) above the bars indicate significant differences at P < .05. GPx, glutathione
peroxidase; SOD, superoxide dismutase.
 6 HWANG ET AL.
exercise intensity, exercise duration, muscle rigidity, fatigue
recovery, and excessive training, and is used to evaluate
histological damage in muscle tissue and assess physical
fitness.40 In this study, we confirmed that the LTAG extracts
decreased blood lactate levels by decreasing blood LDH
activity, which is increased by exercise (Fig. 3C, D).
Through this mechanism, it is considered that LTAG ex-
tracts prevent the depletion of energy sources required for
exercise, thereby reducing the excessive accumulation of
fatigue substances such as lactate and alleviating muscle
fatigue.
CK is known to be an accurate indicator of muscle
damage. The function of CK is to add a phosphate group to
creatine and convert it into the high-energy molecule
phosphocreatine, which is then burned as a fast energy
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source by the cells.32 However, the normal function of CK is
not associated with what happens to CK in the event of
muscle damage. During muscle degeneration, muscle cells
are dissolved and their contents enter the bloodstream. Most
of the body’s CK is present in muscle. When the muscle
damage has occurred or is occurring,41 muscle CK comes
out into blood. Therefore, muscle CK is decreased and blood
CK is increased in muscle damage status. As given in
Figure 4B, the muscle CK levels in mice were significantly
decreased compared with the control, whereas the blood CK
levels were significantly increased (Supplementary Fig. S2).
There were no significant differences between the high and
low LTAG treatment groups for these two biochemical pa-
rameters. Combined, these data suggest that the adminis-
tration of LTAG extract can alleviate fatigue in mice during
exercise.
ROS serve a role in exercise-induced protein oxidation
and contribute to muscle fatigue.8 Muscle cells contain two
main classes of endogenous cellular defense mechanisms to
eliminate ROS. The primary antioxidant enzymes include
SOD and GPx. SOD helps the superoxide radicals break
down into H2O2 and O2.42 GPx reduces H2O2 or organic
hydroperoxides to water and alcohol, respectively.43 These
antioxidant defense mechanisms are attenuated in chronic
fatigue and other disease states.44 Therefore, enhancement
of these defense mechanisms could assist in overcoming
fatigue. As presented in Figure 5, SOD and GPx expression
levels in the muscle tissue of the LTAG-treated group were
higher than those in the control group. The results support
the hypothesis that LTAG can promote the activity of these
antioxidant enzymes and exert an anti-fatigue effect.
The mitochondrion plays an important role in oxidative
stress.45 Decreased SOD and GPx activity leads to mito-
chondrial DNA (mtDNA) damage.46,47 The mtDNA copy
number is thought to be a representative marker of mito-
chondrial function.48 Mitochondrial transcription factor A
(TFAM) is a key transcription factor for the regulation of
mitochondrial gene transcription and a direct regulator of
mtDNA duplication.49 Nuclear respiratory factor 1 (NRF-1)
initiates the synthesis of mitochondrial proteins, such as
mitochondrial import proteins, heme biosynthesis proteins,
components of the electron transport chain complexes, cy-
tochrome c, and TFAM as a positive regulator of tran-
scription.50 Thus, mitochondrial function in muscles
contributes to exercise-induced fatigue. To evaluate mito-
chondrial function, we confirmed mtDNA copy number,
mRNA expression of TFAM and NRF-1 were measured. In
this study, we found LTAG could improve mitochondrial
function by restoring the mtDNA content and increasing the
mRNA expression of NRF-1 and TFAM (Supplementary
Fig. S3), thereby suppressing oxidative stress and generating
more ATP for energy supplement. This might be a potential
mechanism of the anti-fatigue effects of LTAG.
According to Moreno et al.,51 the amadori compound
obtained in the first step of the Maillard reaction of the aged
garlic extract has antioxidant activity. And also, Cardelle-
Cobas et al.52 have also reported that amadori compounds
produced from the browning reaction of garlic and onion
have antioxidant properties. In this respect, functionalities
have been reported on the components of garlic during the
aging process, and they are thought to affect the anti-fatigue
effect through oxidative stress inhibition. However, it is
necessary to carry out an in-depth study on the aged garlic
compounds that exert the anti-fatigue effect in the future.
In conclusion, this study confirmed that LTAG extract
exhibits anti-fatigue effects by reducing plasma LDH ac-
tivity, lactate concentration, and muscle glycogen utiliza-
tion, and that it also enhances exercise performance in mice.
The bioactive compounds of the extract and the mechanism
underlying the anti-fatigue effects are yet to be elucidated,
but this study provided evidence to support the anti-fatigue
effect of LTAG. Thus, LTAG could be a useful anti-fatigue
agent and an ergogenic aid.
ACKNOWLEDGMENTS
This study was supported by the ‘‘Research Program for
Agricultural Science & Technology Development (Project
No. PJ01094202),’’ National Institute of Agricultural Sci-
ences, Rural Development Administration, Republic of
Korea.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
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