Hindawi

Journal of Food Biochemistry

Volume 2023, Article ID 6663895, 12 pages
https://doi.org/10.1155/2023/6663895

Research Article
Antifatigue Potential of Loquat Leaf Extract in Physical Stress in
C2C12 Myotubes and In Vivo Models

Hee-Yun Kim,1 Soonsik Kang,2 Kyunghwon Min,2 Minson Kweon,3 Jungeun Kim,4
Su-Young Choi,3 Chang-Ju Kim,5 Hyung-Min Kim,2 and Hyun-Ja Jeong 1,6
1
BioChip Research Center, Hoseo University, 20 Hoseo-ro, 79Beon-gil, Baebang-eup, Asan 31499, Republic of Korea
2
Department of Science in Korean Medicine, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea
3
COSMAX NBT, INC., Seongnam 13487, Republic of Korea
4
COSMAX NS, INC., Seongnam 13486, Republic of Korea
5
Department of Physiology, College of Medicine, Kyung Hee University, Seoul 02447, Republic of Korea
6
Department of Food Science & Technology and Research Institute for Basic Science, Hoseo University, 20 Hoseo-ro, 79 Beon-gil,
Baebang-eup, Asan 31499, Republic of Korea

Correspondence should be addressed to Hyun-Ja Jeong; hjjeong@hoseo.edu

Received 18 February 2023; Revised 26 October 2023; Accepted 4 November 2023; Published 13 November 2023

Academic Editor: Nizar Tlili

Copyright © 2023 Hee-Yun Kim et al. Tis is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Recent studies suggest that oxidative stress could be one of the mechanisms contributing to fatigue. Te purpose of the present
study was to determine the antifatigue potential of loquat leaf (Eriobotrya japonica Lindl., EJ) and its component, chlorogenic acid
(CA) in C2C12 myotubes and treadmill stress test (TST), and forced swimming test (FST) in animal models. EJ and CA reduced
levels of metabolic factors associated with fatigue and enhanced catalytic properties of superoxide dismutase and catalase in
C2C12 myotubes. In the FST and TST, EJ and CA decreased immobility time and prolonged exhaustion time. Te administration
of EJ and CA reduced the levels of the metabolic factors associated with fatigue and augmented the levels of the substances which
relieve fatigue. Tese results prove that EJ alleviates fatigue by increasing antioxidative activity. Terefore, we suggest that EJ may
be a potential candidate for management of fatigue as a health functional food.

1. Introduction capability which occurs due to the accumulation of meta-

bolic products in the blood which induces fatigue [2].
Fatigue is a term used to describe a decrease in physical Psychological fatigue occurs due to emotional conficts,
performance and is related to an increase in the actual/ anxiety, or boredom [3]. Currently, there are several theories
perceived difculty in performing a task or exercise. It is regarding the mechanisms which cause fatigue. Te meta-
a common symptom of many physical, neurological, and bolic causes of fatigue include a decrease in phosphocreatine
psychiatric disorders, including depression, human immu- levels, accumulation of protons (acidemia), depletion of
nodefciency virus infection, Parkinson’s disease, cancer, glycogen in the muscles and decrease in blood glucose
multiple sclerosis, aging, and other potential physical ill- concentrations, and increase in specifc amino acids in the
nesses [1]. Fatigue can be categorized into three types, plasma [4]. Also, fatigue is caused by the accumulation of
namely, pathological, physiological, and psychological fa- lactate through the activation of lactate dehydrogenase
tigue [2]. Pathological fatigue is often experienced by people (LDH) and the production of cortisol by the adrenocorti-
with chronic illnesses such as those with liver dysfunction, cotropic hormone in the anterior pituitary gland, whereas it
diabetes, and gastrointestinal diseases and can severely is improved by energy production through glycolysis and the
impair functional activity and quality of life [3]. Physio- citrate cycle which is activated by hexokinase and citrate
logical fatigue is a state of reduced mental or physical synthase. A rapidly emerging concept that has recently
2 Journal of Food Biochemistry

received attention is the role of the free radicals in fatigue of a standard solution of linear alkanes C8–C20 in chro-
and the benefts of antioxidants in relieving fatigue [5, 6]. matograph gas was injected into the same chromatographic
Te increased oxidative stress during excessive exercise column with the same conditions of analysis as in the case of
induces fatigue by increasing the release of free radicals and basil volatile oil. After confrmation (based on MS spectra) of
infammatory cytokines [7, 8]. Terefore, it can be inferred the linear alkane structures separated by the gas chro-
that symptoms of fatigue can improve through the con- matographic analysis, we determined the retention time for
sumption of antioxidants that increase superoxide dismutase each component of the mixture (Supplementary Table 1).
(SOD) and catalase activities and regulate infammatory
cytokines.
Terapeutic drugs used to relieve fatigue provide tem- 2.3. C2C12 Myoblasts Cell Culture. Murine myoblast C2C12
porary benefts and are also associated with side efects. Te cells were grown in Dulbecco’s modifed eagle medium
antifatigue efect of natural antioxidants has been reported (DMEM; Gibco BRL, Grand Island, NY, USA) containing
in [9]. Terefore, components of natural products are worth 10% fetal bovine serum and antibiotics at 37°C in a 5% CO2
investigating, as they are associated with fewer side efects atmosphere. To diferentiate the C2C12 cells, 80% of the
and could be efective in treating or preventing fatigue. confuent culture was replaced with DMEM containing 2%
In traditional medicine, loquat leaf (Eriobotrya japonica horse serum, and the medium was replaced with fresh
Lindl., EJ) is used to treat infammatory diseases [10] and has medium every other day for 4 days.
neuroprotective, antiallergic, antioxidant, and antiarthritic
properties [11–14]. Furthermore, EJ contains many anti- 2.4. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
oxidants such as chlorogenic acid (CA), quercetin-3-sam- Bromide (MTT) Assay. Te MTT tetrazolium assay was used
bubioside, euscaphic acid, and ursolic acid [15]. In a study by to check cytotoxicity by analyzing the intracellular metabolic
Jung et al. [15], EJ and CA demonstrated antioxidative and activity in which MTT is converted into a purple formazan
antiinfammatory properties. Terefore, in this study, we product. Te diferentiated C2C12 cells (3 × 105/well) were
hypothesized that EJ might exhibit antifatigue potential plated on a 24-well plate, stabilized for 30 min, and treated
through its antioxidative actions. We used the C2C12 with WEJ, EEJ, and CA for 24 h at 37°C. After changing to
myotubes, the treadmill stress test (TST), and the forced a fresh medium, the plates were treated with MTT (0.5 mg/
swimming test (FST) to investigate the antifatigue potential ml) for 4 h. Insoluble formazan crystals were solubilized in
of EJ and CA and its mechanism. Tese results can be dimethyl sulfoxide. Te amount of formazan was quanti-
utilized as basic experimental data for the development of tated by absorbance at 570 nm using an enzyme-linked
health nutraceuticals and herbal medicines to combat immunosorbent assay (ELISA) reader.
fatigue.

2.5. ELISA. Te produced contents of cytokines were

2. Materials and Methods quantitated by the ELISA method [16].
2.1. Preparation of EJ Extracts. EJ extracts were provided by
COSMAX NBT, INC. (Seongnam, Republic of Korea). Te 2.6. Animals. Te ICR mice (male, 4 weeks old), purchased
EJ solution was extracted using water (EJ water extract, WEJ) from the Dae–Han Experimental Animal Center (Chung-
or ethanol (EJ ethanol extract, EEJ). Each of the extracted buk, Republic of Korea), were stabilized in individual cages
solutions was fltered, concentrated, mixed with dextrin, and for one week at room temperature (20–23°C), with 50–60%
spray-dried. humidity and a 12 h light and 12 h dark cycle. Te mice were
treated in accordance with the current law and animal care
standards provided in the Guide for the care and use of
2.2. Ultrahigh Performance Liquid Chromatography (UHPLC) laboratory animals approved by the Animal Ethics Com-
and Mass Spectrometry (MS) Analysis. Liquid chromatog- mittee of Kyung Hee University (KHUASP (SE)-20-263).
raphy/MS (LC/MS) analyses were carried out using an LTQ
Orbitrap XL Fourier transform mass spectrometer (Termo
Fisher Scientifc, Inc., Waltham, MA, USA) coupled to an 2.7. FST. In the FST, time spent by the mice swimming or
Accelar ultrahigh pressure liquid chromatography system being immobile is measured and increased immobility time
(Termo Fisher Scientifc, Inc.). Te chromatographic indicates fatigue. Mice were subjected to an FST initially, to
separation of metabolites was carried out using an ACQ- determine the diferences between the individual mice. Te
®
UITY UPLC BEH C18 column and mobile phases A (water
with 0.1% formic acid) and B (acetonitrile with 0.1% formic
mice were divided into 7 groups based on the immobility
time: normal, control (distilled water, D.W.), WEJ (25, 50,
acid). Each compound was detected with a photodiode array and 100 mg/kg), EEJ (50 mg/kg), and CA (1 mg/kg).
at 200 ∼ 500 nm. Te MS analysis was performed with According to the previous study [11], the concentrations of
polarity switching with the following parameters for the MS/ WEJ were determined to be 25, 50, and 100 mg/kg. Te CA
MS scan: m/z range of 150−1,500. High-resolution mass (1 mg/kg) concentration was also determined according to
spectra were acquired on the LTQ Orbitrap XL (Supple- an earlier study [17]. WEJ, EEJ, and CA were orally ad-
mentary Figure 1). To determine the Kovats retention in- ministered to the mice daily for 28 days. Te FST was then
dices, we proceeded as follows: a sample of 2 μl of a mixture carried out on the last day as previously reported [18].
Journal of Food Biochemistry 3

2.8. TST. Te TST was conducted as previously reported infammatory cytokines, interleukin (IL)-6, and tumor ne-
[19]. Briefy, the treadmill speed was started at 0 m/min and crosis factor (TNF)-α compared with the unstimulated cells.
slowly increased (10 m/min at 10 min, 16 m/min at 10 min, Treatment with WEJ, EEJ, or CA signifcantly decreased the
and 21 m/min at 10 min). On the 28th day, the mice were levels of IL-6, and TNF-α increased by H2O2 stimulation
made to exercise until exhaustion (10 m/min at 5 min and (Figures 1(d) and 1(e), P < 0.05). WEJ dose-dependently
40 m/min at 30 min). Te oral administration of D.W., WEJ, regulated the fatigue-related biochemical indicators. Fur-
EEJ, and CA was carried out daily for 28 days. thermore, WEJ, EEJ, or CA showed no cytotoxicity to C2C12
myotubes (Figure 1(f )).
2.9. Analysis of Fatigue-Related Biochemical Indicators in
Serum, Muscle, or Liver. Blood, muscle (extensor digitorum 3.2. Efect of EJ on Oxidation-Related Biochemical Indicators
longus (EDL), tibialis anterior (TA), soleus, and gastroc- in H2O2-Stimulated C2C12 Myotubes. Oxidative stress is
nemius muscles), or liver were collected immediately after known to be one of the factors triggering fatigue. To investigate
performing FST and TST. Te serum was obtained by the antioxidative efect of EJ, the activities of SOD and catalase
centrifugation of blood of the mice, and muscle and liver were analyzed using the respective assay kits. Te stimulation
samples were quickly collected. A glycogen colorimetric with H2O2 signifcantly decreased the activities of SOD and
assay kit (Biovision Inc., Milpitas, California, USA) was used catalase compared with unstimulated cells (Figures 2(a) and
to measure the levels of glycogen. Te levels of citrate 2(b), P < 0.05). However, treatment with WEJ, EEJ, or CA
synthase were analyzed using a commercially available kit showed a signifcant increase in the activities of SOD and
(MyBiosource Inc., San Diego, California, USA). Te levels catalase which were decreased by H2O2 (Figures 2(a) and 2(b),
of lactate, SOD, malondialdehyde (MDA), and catalase were P < 0.05). In addition, WEJ remarkably increased the activ-
quantitated using assay kits (DoGenBio, Seoul, South ities of SOD and catalase (Figures 2(a) and 2(b), P < 0.05).
Korea). Te levels of free fatty acids and cortisol were also MDA is a direct indicator of oxidative stress and a biomarker
quantitated using assay kits (Abcam, Cambridge, UK and of fatigue. In H2O2-stimulated C2C12 myotubes, WEJ, EEJ, or
Sigma-Aldrich Co., St. Louis, MO, USA), respectively. A CA treatment decreased the levels of MDA signifcantly
DRI CHEM NX500 analyzer (Fujiflm, Tokyo, Japan) was (Figure 2(c), P < 0.05).
used to quantify the levels of creatine kinase (CK), alanine
transaminase (ALT), aspartate aminotransferase (AST),
blood urea nitrogen (BUN), glucose, and LDH. 3.3. Efect of EJ on Immobility and Exhaustion Time in Animal
Models. Next, to investigate the antifatigue efect of EJ, we
conducted experiments in vivo in mice [18]. In the FST, the
2.10. Statistical Analysis. For all statistical analyses, the SPSS immobility time of the mice administered WEJ, EEJ, or CA
12.0 (IBM Corp., Armonk, NY, USA) software was utilized. was signifcantly reduced compared with the control group
To verify the normality of the data, the Shapiro–Wilk test was at 28 days (Figure 3(a), P < 0.05). In addition, WEJ re-
used. An independent t-test was performed for a comparison markably decreased the immobility time (Figure 3(a),
between the normal (or blank) and control groups, and a one- P < 0.05). Furthermore, the antifatigue efect of EJ was also
way ANOVA test was performed for a comparison of three or investigated by performing the TST. Te time to exhaustion
more groups such as the control and drug groups. In vivo of the mice administered with WEJ, EEJ, or CA was sig-
(n � 5/group) and in vitro data were expressed as mean- nifcantly prolonged compared with the control group
± standard error mean (SEM). In vitro data were indicated as (Figure 3(b), P < 0.05). Tere was no signifcant change in
the results of three independent experiments. A P value less the body weight of the WEJ, EEJ, or CA groups compared to
than 0.05 meant that an efect was observed. the control group (Supplementary Figure 2).

3. Results
3.4. Efect of EJ on Metabolic Factors Associated with Fatigue
3.1. Efect of EJ on Fatigue-Related Biochemical Indicators in after FSTand TST. We investigated the antifatigue efects of
H2O2-Stimulated C2C12 Myotubes. Initially, to investigate EJ by analyzing metabolic factors associated with fatigue.
the antifatigue efects of EJ, the levels of fatigue-related Following exercise, the levels of lactate, LDH, MDA, or
biomarkers in H2O2-stimulated C2C12 myotubes were de- cortisol increased in the serum. Te levels of lactate, LDH,
termined. Te levels of LDH and CK were signifcantly MDA, and cortisol were quantitated in the serum of the mice
augmented by H2O2 stimulation. However, the levels that administered with WEJ, EEJ, or CA after performing the FST
were increased by H2O2 stimulation were remarkably de- and TST. As seen in Figure 4, the serum levels of lactate,
creased by treatment with WEJ, EEJ, or CA (Figures 1(a) and LDH, MDA, and cortisol in the control group were re-
1(b), P < 0.05). Glycogen levels were signifcantly augmented markably augmented compared with the normal group
by treatment with WEJ, EEJ, or CA, compared with the levels (P < 0.05). Te lactate, LDH, MDA, and cortisol levels in
seen with H2O2 stimulation (Figure 1(c), P < 0.05). the serum of the mice administered WEJ or CA remarkably
Te infammatory responses induced by the in- decreased signifcantly compared to the control group
fammatory cytokines are closely related to fatigue, and their (Figure 4, P < 0.05). EEJ remarkably decreased the serum
high levels are indicative of chronic fatigue. In this study, levels of LDH, MDA, and cortisol (Figure 4). Te lactate
H2O2 stimulation remarkably augmented the secretion of levels were decreased by EEJ, but not signifcantly (Figure 4).
4 Journal of Food Biochemistry

400 1.0 #
#
300 * *
* *
*
LDH (U/L)

CK (mg/dL)
200 0.5
*
* *
* *
100

0 0.0
H2O2 - + - + + + + + H2O2 - + - + + + + +
WEJ (μg/ml) - - 100 25 50 100 - - WEJ (μg/ml) - - 100 25 50 100 - -
EEJ (μg/ml) - - - - - - 50 - EEJ (μg/ml) - - - - - - 50 -
CA (μg/ml) - - - - - - - 1 CA (μg/ml) - - - - - - - 1

(a) (b)
5 2 #

4
* * *

IL-6 levels (ng/ml)

Glycogen (μg/ml)

* *
* * *
3 *
# 1
2

0 0
H2O2 - + - + + + + + H2O2 - + - + + + + +
WEJ (μg/ml) - - 100 25 50 100 - - WEJ (μg/ml) - - 100 25 50 100 - -
EEJ (μg/ml) - - - - - - 50 - EEJ (μg/ml) - - - - - - 50 -
CA (μg/ml) - - - - - - - 1 CA (μg/ml) - - - - - - - 1

(c) (d)
2 # 1.5
TNF-α levels (ng/ml)

*
* * *
* 1.0
Cell viability

0.5

0 0.0
H2O2 - + - + + + + + H2O2 - + - + + + + +
WEJ (μg/ml) - - 100 25 50 100 - - WEJ (μg/ml) - - 100 25 50 100 - -
EEJ (μg/ml) - - - - - - 50 - EEJ (μg/ml) - - - - - - 50 -
CA (μg/ml) - - - - - - - 1 CA (μg/ml) - - - - - - - 1

(e) (f)

Figure 1: Efect of EJ on fatigue-related biochemical indicators in H2O2-stimulated C2C12 myotubes. C2C12 cells were diferentiated by
culture in DMEM containing 2% horse serum for 4 days. Diferentiated C2C12 cells were treated with WEJ (25, 50, and 100 μg/ml), EEJ
(50 μg/ml), or CA (1 μg/ml) and then stimulated with H2O2 (1 mM) for 24 h. Levels of (a) LDH, (b) CK, and (c) glycogen were measured.
Levels of (d) IL-6 and (e) TNF-α were analyzed by the ELISA method. (f ) Cell viability was measured by a MTT assay. Results show the
mean ± SEM of data from three separate experiments with duplicate samples. WEJ, EJ water extract; EEJ, EJ ethanol extract; and CA,
chlorogenic acid. #P < 0.05, signifcantly diferent from the unstimulated C2C12 myotubes. ∗ P < 0.05, signifcantly diferent from the H2O2-
stimulated C2C12 myotubes.

Te levels of fatigue-related blood biochemical in- 3.5. Efect of EJ on Factors Related to Improvement in Fatigue
dicators were measured after the FST and TST. Te serum after the FSTand TST. Te glucose, free fatty acids, glycogen,
levels of BUN, ALT, AST, and CK in the control mice were and citrate synthase contents were measured after the FST
remarkably augmented when compared with the normal and TST to investigate whether EJ increased the levels of the
group, but WEJ, EEJ, or CA-administration resulted in factors associated with improvement in fatigue. As shown in
a signifcant decrease in these levels compared to the control Figure 5(a), the glucose levels of the control group were
group (Table 1, P < 0.05). remarkably augmented compared with the normal group.
Journal of Food Biochemistry 5

3 1.5

Catalase activity (mU/ml)

SOD activity (mU/ml)
2 1.0

* *
* *
1 # 0.5 *
* * * *
#

0 0.0
H2O2 - + - + + + + + H2O2 - + - + + + + +
WEJ (μg/ml) - - 100 25 50 100 - - WEJ (μg/ml) - - 100 25 50 100 - -
EEJ (μg/ml) - - - - - - 50 - EEJ (μg/ml) - - - - - - 50 -
CA (μg/ml) - - - - - - - 1 CA (μg/ml) - - - - - - - 1
(a) (b)
100 #

*
* * *
MDA (μM)

50 *

0
H2O2 - + - + + + + +
WEJ (μg/ml) - - 100 25 50 100 - -
EEJ (μg/ml) - - - - - - 50 -
CA (μg/ml) - - - - - - - 1
(c)

Figure 2: Efect of EJ on oxidation-related biochemical indicators in H2O2-stimulated C2C12 myotubes. C2C12 cells were diferentiated by
culture in DMEM containing 2% horse serum for 4 days. Diferentiated C2C12 cells were treated with WEJ (25, 50, and 100 μg/ml), EEJ
(50 μg/ml), or CA (1 μg/ml) and then stimulated with H2O2 (1 mM) for 24 h. Activities of (a) SOD, (b) catalase, and (c) levels of MDA were
analyzed with each kit. Results show the mean ± SEM of data from three separate experiments with duplicate samples. WEJ, EJ water extract;
EEJ, EJ ethanol extract; and CA, chlorogenic acid. #P < 0.05, signifcantly diferent from the unstimulated C2C12 myotubes. ∗ P < 0.05,
signifcantly diferent from the H2O2-stimulated C2C12 myotubes.

180 4000
* * *
*
3000 *
Immobility time (s)

Exhaustion time (s)

*
120 * *
*
*
2000

60
1000

0 0
Control 25 50 100 EEJ CA Control 25 50 100 EEJ CA

WEJ (mg/kg) WEJ (mg/kg)

(a) (b)

Figure 3: Efect of EJ on immobility and exhaustion time of fatigue animal models. (a) Immobility time in the FST and (b) exhaustion time
in TST at 28 days. Values are means ± SEM. n � 5 per group. WEJ, EJ water extract; EEJ, EJ ethanol extract; and CA, chlorogenic acid.
∗
P < 0.05, signifcantly diferent from the control mice.
6 Journal of Food Biochemistry

500 5000
#
#
Lactate in serum (nmole)

400 4000

LDH in serum (U/L)

# #
* *
300 * 3000 *
* * *
* * *
* *
200 2000 * *
*

100 1000

0 0
Normal Control 25 50 100 EEJ CA Normal Control 25 50 100 EEJ CA
WEJ (mg/kg) WEJ (mg/kg)
FST FST
TST TST
(a) (b)

1000 # 15 #
#

Cortisol in serum (μg/ml)

* * * * *
MDA in serum (μM)

#
* * * * * 10
*
*
500
* * *
* * *
5 * *

0 0
Normal Control 25 50 100 EEJ CA Normal Control 25 50 100 EEJ CA
WEJ (mg/kg) WEJ (mg/kg)
FST FST
TST TST
(c) (d)

Figure 4: Efect of EJ on metabolic factors associated with fatigue in the serum after FSTand TST. After the last FSTand TST, serum samples
were obtained from heart. Levels of (a) lactate, (b) LDH, (c) MDA, and (d) cortisol were measured with each kit. Values are the mean ± SEM.
n � 5 per group. WEJ, EJ water extract; EEJ, EJ ethanol extract; and CA, chlorogenic acid. #P < 0.05, signifcantly diferent from the normal
mice. ∗ P < 0.05, signifcantly diferent from the control mice.

Table 1: Efect of EJ on fatigue-causing factors in serum after FST and TST.

Normal Control WEJ (25 mg/kg) WEJ (50 mg/kg) WEJ (100 mg/kg) EEJ (50 mg/kg) CA (1 mg/kg)
BUN (mg/ #
22.2 ± 1.22 68.34 ± 7.91 54.08 ± 7.13∗ 53.93 ± 3.47∗ 46.8 ± 2.45∗ 44.64 ± 7.14∗ 33.24 ± 2.81∗
dL)
FST ALT (U/L) 31.4 ± 4.51 109.6 ± 10.69# 63.4 ± 7.7∗ 56 ± 9.22∗ 50.8 ± 2.38∗ 70.8 ± 10.33∗ 47 ± 7.18∗
# ∗ ∗ ∗
AST (U/L) 104.2 ± 12.19 309 ± 19.65 175.4 ± 16.8 164.2 ± 14.07 135.4 ± 8.26 152.4 ± 12.34∗ 175.8 ± 16.25∗
CK (mg/dL) 0.19 ± 0.02 0.91 ± 0.06# 0.68 ± 0.03∗ 0.6 ± 0.04∗ 0.5 ± 0.03∗ 0.61 ± 0.06∗ 0.61 ± 0.06∗
BUN (mg/
21.44 ± 3.49 86.4 ± 8.72# 53.04 ± 4.18∗ 48.06 ± 1.17∗ 31.96 ± 4.24∗ 33.74 ± 6.77∗ 33.12 ± 3.71∗
dL)
TST ALT (U/L) 31.8 ± 9.04 108 ± 6# 66.8 ± 8.32∗ 60.6 ± 5.13∗ 48.4 ± 6.73∗ 63 ± 11.47∗ 43.6 ± 13.2∗
AST (U/L) 70.6 ± 16.23 409.8 ± 59.58# 210.2 ± 21.25∗ 174 ± 9.3∗ 154 ± 14.54∗ 184.6 ± 33.23∗ 165 ± 30.88∗
CK (mg/dL) 0.13 ± 0.02 0.81 ± 0.08# 0.56 ± 0.03∗ 0.48 ± 0.02∗ 0.43 ± 0.01∗ 0.45 ± 0.02∗ 0.55 ± 0.02∗
After the last FST and TST, serum samples were obtained from the heart. Levels of BUN, ALT, AST, and CK were analyzed with each kit. Values are the
mean ± SEM. n � 5 per group. WEJ, EJ water extract; EEJ, EJ ethanol extract; CA, chlorogenic acid. #P < 0.05, signifcantly diferent from normal mice.
∗
P < 0.05, signifcantly diferent from control mice.

However, the increased glucose levels seen in the control the muscle were remarkably augmented by the adminis-
group were signifcantly lowered by the administration of tration of WEJ (50 and 100 mg/kg) compared to the control
WEJ, EEJ, or CA (P < 0.05). Te free fatty acids contents in group (Figures 5(b)–5(d), P < 0.05). Administration of EEJ
the serum and the glycogen and citrate synthase contents in and CA signifcantly upregulated the free fatty acids and
Journal of Food Biochemistry 7

1.5 100

Free fatty acid in serum (nmole/μl)

Glucose in serum (mg/dL)
#
#
1.0
* * * * * * * *
* 50 *
* * * * *
# *
0.5
#

0.0 0
Normal Control 25 50 100 EEJ CA Normal Control 25 50 100 EEJ CA
WEJ (mg/kg) WEJ (mg/kg)

FST FST
TST TST
(a) (b)
5 6

Citrate synthase in muscle (μg/ml)

Glycogen in muscle (μg/ml)

4 *
*
*
3
3
2
* *
*
1 *
# # *

0 0
Normal Control 25 50 100 EEJ CA Normal Control 25 50 100 EEJ CA
WEJ (mg/kg) WEJ (mg/kg)

FST FST
TST TST
(c) (d)
6
Glycogen in liver (μg/ml)

4 *
* *
* * *
3 * *
* *
2
# #
1

0
Normal Control 25 50 100 EEJ CA
WEJ (mg/kg)
FST
TST
(e)

Figure 5: Efect of EJ on glucose, free fatty acid, glycogen, and citrate synthase in the serum and muscle after the FSTand TST. After the last FST
and TST, serum and muscle samples were obtained from each mouse. Levels of (a) glucose and (b) free fatty acid in serum were analyzed by
a DRI CHEM NX500 analyzer. Levels of (c) glycogen and (d) citrate synthase in muscle were measured with each kit. (e) Levels of glycogen in
liver were measured with glycogen kit. Values are the mean ± SEM. n � 5 per group. WEJ, EJ water extract; EEJ, EJ ethanol extract; and CA,
chlorogenic acid. #P < 0.05, signifcantly diferent from the normal mice. ∗ P < 0.05, signifcantly diferent from the control mice.
8 Journal of Food Biochemistry

citrate synthase contents compared to the control group levels were found to increase signifcantly after excessive
after the FST or TST (Figures 5(b) and 5(d), P < 0.05). Te exercise [24]. Physiological changes which occur during
glycogen content after the FST and TST was increased by EEJ fatigue are closely associated with increases in AST and ALT
and CA, but not signifcantly (Figure 5(c)). Te glycogen levels suggestive of liver failure [25]. Te serum levels of CK
contents in the liver were remarkably augmented by the are increased by exercise-induced muscle damage [26]. An
administration of WEJ, EEJ, or CA compared to the control increase in cortisol is associated with stress. During long-
group (Figure 5(e), P < 0.05). term exercise, the release of cortisol in the adrenal cortex is
induced by an increase in the secretion of the adrenocor-
ticotropic hormone in the anterior pituitary resulting in
3.6. Efect of EJ on Fatigue-Related Infammatory Cytokines in fatigue [27]. Fatigue is induced by a lack of energy due to
Serum after the FST and TST. To investigate whether EJ mitochondrial dysfunction [28]. Terefore, energy storage
played a role in the down regulation of the infammatory and supply are important factors in exercise endurance.
cytokines, the levels of IL-1β, IL-4, IL-6, and TNF-α in serum When exercising without an energy source (glucose, free
were analyzed using the ELISA method after the FST and fatty acids, and glycogen), physical fatigue increases,
TST. As seen in Figure 6, the serum levels of infammatory resulting in a signifcant reduction in endurance [4, 29]. A
cytokines in the control group were remarkably augmented defciency of citrate synthase could cause fatigue due to
compared with the normal group (P < 0.05). Te serum lactate accumulation in the blood, and a signifcant decrease
levels of infammatory cytokines in the mice administered in citrate synthase was shown in chronic fatigue syndrome
WEJ, EEJ, or CA showed a signifcant decrease compared (CFS) patients compared to healthy controls [30]. Many
with the control group (Figure 6, P < 0.05). substances with antifatigue efects have been shown to de-
crease the levels of lactate, LDH, MDA, BUN, ALT, AST,
3.7. Efect of EJ on SOD and Catalase Activities after the FST CK, and cortisol and increase the levels of glycogen, free fatty
and TST. In the in vitro test, EJ augmented the activities of acids, and citrate synthase [1–6, 18, 25]. Previous studies
SOD and catalase (Figure 2). To confrm the antioxidative have shown that EJ improves muscle function and reduces
efect of EJ in the in vivo test, the SOD and catalase activities muscle loss [31, 32]. In the present study, EJ and CA re-
were analyzed after the FST and TST. As shown in markably decreased the levels of lactate, LDH, MDA, BUN,
Figures 7(a)–7(c), the SOD activities in the serum, liver, and ALT, AST, CK, and cortisol, whereas they augmented the
muscle of the control mice were remarkably lower compared glycogen, free fatty acids, and citrate synthase levels.
with the normal group (P < 0.05). However, the SOD ac- Terefore, these results indicate that EJ and CA could have
tivities in the serum, liver, and muscle of the WEJ or CA contributed to exercise performance by ameliorating fatigue.
group were remarkably augmented compared with the Glucose is the predominant energy source during ex-
control group after the FST and TST (Figures 7(a)–7(c), ercise. Decreases in blood glucose levels lead to fatigue and
P < 0.05). Te EEJ only increased the SOD activity after impair endurance exercise [33]. Gibb et al. [34] reported that
TST compared with the control group (Figures 7(a)–7(c)). exercise-induced fatigue results in a decrease of glucose
Furthermore, the administration of WEJ, EEJ, or CA sig- levels, while triglycerides are degraded and stored in the
nifcantly upregulated the levels of catalase in the liver form of free fatty acids in the blood. In contrast, other
compared with the control group after the FST (Figure 7(d), researchers reported that the FST and TST increased the
P < 0.05). WEJ (100 mg/kg) signifcantly increased the serum glucose levels and decreased serum free fatty acids,
catalase activity compared with the control group after the and that many substances with antifatigue efects decreased
TST (Figure 7(d)). the glucose levels and increased the free fatty acids levels
[4, 19, 35]. Also, the previous result indicated that blood
4. Discussion glucose levels continued to rise steadily during exercise and
gradually decreased to levels similar to the baseline during
In this study, we demonstrated that EJ and CA have an the recovery period (1-2 h after exercise cessation) [36]. In
antifatigue efect through their ability to decrease the levels this study, blood was collected immediately after the ces-
of metabolic factors associated with fatigue and increase the sation of FST and TST, and glucose levels increased in the
levels of substances which play a role in improving fatigue in control group, while EJ reduced glucose levels. Terefore,
the C2C12 myotubes in vitro model and the FST and TST in these results indicate that EJ can regulate the increase in
the in vivo models. glucose levels induced by exercise. Moreover, when exercise-
Te metabolic factors involved in the pathophysiology of induced stress increases, cortisol can supply the body with
fatigue include lactate, LDH, MDA, BUN, ALT, AST, CK, glucose, increasing blood glucose levels [37]. Exercise-
and cortisol, whereas recovery from fatigue is induced by induced exhaustion has been reported to decrease the
glucose, free fatty acids, glycogen, and citrate synthase [4]. availability of fatty acids and improve insulin sensitivity in
Accumulation of lactate by LDH reduces the pH of the blood glucose metabolism [38]. Rau et al. [39] reported that stress
and muscle tissue, damages various organs, and is one of the induces hyperglycemia and stress-induced hyperglycemia
causes of fatigue [20]. During exercise, MDA increased by releases stress hormones and cytokines. Terefore, we
oxidative stress causes muscle damage and fatigue [21–23]. speculate that EJ might alleviate stress-induced hypergly-
BUN is another sensitive index of fatigue status, and BUN cemia. However, further investigation is necessary to
Journal of Food Biochemistry 9

15 1.5
#
IL-1β in serum (ng/ml) #

IL-4 in serum (ng/ml)

*
10 1.0
# *
* * *
*
* * * *
5 * 0.5
* * * # *
* * *
* *

0 0.0
Normal Control 25 50 100 EEJ CA Normal Control 25 50 100 EEJ CA
WEJ (mg/kg) WEJ (mg/kg)

FST FST
TST TST
(a) (b)
0.8 30

TNF-α in serum (ng/ml)

# *
IL-6 in serum (ng/ml)

20
*
# * * * *
0.4 * #
*
* * *
* * * 10 *
* * *
* *

0.0 0
Normal Control 25 50 100 EEJ CA Normal Control 25 50 100 EEJ CA
WEJ (mg/kg) WEJ (mg/kg)

FST FST
TST TST
(c) (d)

Figure 6: Efect of EJ on fatigue-related infammatory cytokines in the serum after the FST and TST. After the last FST and TST, serum
samples were obtained from heart. Levels of (a) IL-1β, (b) IL-4, (c) IL-6, and (d) TNF-α were analyzed by the ELISA method. Values are the
mean ± SEM. n � 5 per group. WEJ, EJ water extract; EEJ, EJ ethanol extract; and CA, chlorogenic acid. #P < 0.05, signifcantly diferent
from the normal mice. ∗ P < 0.05, signifcantly diferent from the control mice.

determine whether EJ has an antihyperglycemic property in also preventing cell damage. Moreover, CA reduced expression
hyperglycemia animal models such as hyperglycemic mice of IL-1β, TNF-α, and IL-6 via the inhibition of nuclear factor
or obese diabetic mice. (NF)-κB activation, which in turn reduced rat liver fbrosis and
Oxidative stress causes mitochondrial dysfunction and cell infammation [45].
damage, leading to infammatory disorders, and is a major Te muscles of a mouse include the EDL, TA, soleus, and
cause of fatigue [40]. In the CFS patients, the levels of anti- gastrocnemius muscles [47, 48]. Among them, both EDL and
oxidants (SOD and catalase) were remarkably decreased, while soleus muscles are representative examples of fast-twitch and
there was an increase in the generation of reactive oxygen slow-twitch muscles [48]. Fast-twitch muscles are primarily
species (ROS) [41]. Excessive oxidative stress induces a rise in utilized during short bursts of intense exercise, while slow-
infammatory markers, such as IL-1β, IL-4, IL-6, and TNF-α twitch muscles are predominantly used during prolonged or
[42]. Blundell et al. [43] reported that the levels of infammatory endurance activities [49]. Te rate of glycogen accumulation
cytokines in CFS patients were augmented compared to the varies depending on the muscle type such as fast-twitch and
normal control. Oxidative stress after strenuous exercise slow-twitch muscles [50]. Following exercise, the levels of
resulted in increased TNF-α levels, resulting in muscle fatigue citrate synthase decreased in the EDL muscles; on the contrary,
[29]. Terefore, substances that decrease the levels of in- the levels of citrate synthase increased in the soleus muscles
fammatory cytokines by increasing SOD and catalase activities [51]. During exercise, stanozolol administration induced
could play a role in reducing fatigue [44]. In vitro and in vivo a signifcant increase of SOD activity in both EDL and soleus
data indicate that CA has antioxidant activity and reduces muscles [52]. In the current study, we demonstrated that
oxidative stress in a variety of diseases [45]. According to Jiang citrate synthase content and SOD activity decreased in the
et al. [46], CA reduces infammation and oxidative stress while muscle tissues (EDL, TA, soleus, and gastrocnemius muscles)
10 Journal of Food Biochemistry

1.0 20
SOD activity in serum

SOD activity in liver

* *

(mU/ml)
(U/ml)

0.5 * 10
* * * * * *
* * * * *
# * * #
#
#

0.0 0
Normal Control 25 50 100 EEJ CA Normal Control 25 50 100 EEJ CA
WEJ (mg/kg) WEJ (mg/kg)

FST FST
TST TST
(a) (b)
80 1.2

1.0

Catalase activity in liver

SOD activity in muscle

0.8

(mU/ml)
(mU/ml)

40 * 0.6
* *
* *
* *
* * 0.4 * *
*
# # * *
0.2
#
#
0 0.0
Normal Control 25 50 100 EEJ CA Normal Control 25 50 100 EEJ CA
WEJ (mg/kg) WEJ (mg/kg)

FST FST
TST TST
(c) (d)

Figure 7: Efect of EJ on SOD activities in the serum, liver, and muscle after the FST and TST. After the last FST and TST, samples were
analyzed for SOD and catalase activities. Te activities of SOD in (a) serum, (b) liver, and (c) muscle. (d) Te activity of catalase in liver.
Values are the mean ± SEM. n � 5 per group. WEJ, EJ water extract; EEJ, EJ ethanol extract; and CA, chlorogenic acid. #P < 0.05, sig-
nifcantly diferent from the normal mice. ∗ P < 0.05, signifcantly diferent from the control mice.

of the control group, with no change in glycogen content. In alleviate pathological fatigue caused by COVID-19. How-
contrast, EJ increased glycogen and citrate synthase contents ever, because exercise-induced physiological fatigue can be
and SOD activity within the muscle tissues (EDL, TA, soleus, distinguished from pathological and psychological fatigue,
and gastrocnemius muscles). Terefore, further research based further research on pathological and psychological fatigue
on muscle tissue types should be conducted to elucidate the models should be conducted to confrm the improvement
detailed efects of EJ on muscle biomarkers. In the CFS mice efect of EJ on COVID-19-induced pathological fatigue.
group, compared to the acute-exercise-treated mice group, In the present study, administration of EJ and CA de-
hepatic glycogen levels signifcantly decreased, but muscle creased the levels of IL-1β, IL-4, IL-6, and TNF-α and in-
glycogen levels remained unchanged [53]. An antifatigue creased the activities of SOD and catalase in C2C12
material, Sarcodon imbricatus signifcantly enhanced glycogen myotubes and after FST and TST in animal models.
levels in the liver of acute-exercise-treated mice [53]. Tis Terefore, these data suggest that EJ and CA display anti-
response was similar to our research fndings. fatigue potential through their ability to increase anti-
Published literature indicates that fatigue was one of the oxidative activity. Additionally, we confrm that CA plays an
most common symptoms of COVID-19, and in many pa- important role in fatigue by being an active component of
tients even after recovery. Fatigue caused by COVID-19 is EJ. In conclusion, this study showed that EJ and CA can
pathological fatigue, and as mentioned above, the bio- signifcantly relieve excessive fatigue. Terefore, we suggest
markers for the pathophysiology of fatigue are similar. that EJ may have a potential therapeutic role in relieving
Terefore, we assume that EJ might have the ability to fatigue in people sufering from the fatigue symptom.
Journal of Food Biochemistry 11

Data Availability weight loaded forced swim test,” Pharmacognosy Journal,

vol. 5, no. 2, pp. 66–71, 2013.
Te data used to support the fndings of this study are in- [7] Z. Fang, K. Huang, C. H. Gil, J. W. Jeong, H. R. Yoo, and
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antioxidants for patients with chronic subjective dizziness,”
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efects of infectious diseases such as coronavirus disease 2019 from the seed coat of Euryale ferox Salisb. and identifcation of
(COVID-19), and many people complained of fatigue even three phenolic compounds by LC-ESI-MS/MS,” Molecules,
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the fatigue symptom.
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Conflicts of Interest [12] S. H. Kim and T. Y. Shin, “Anti-infammatory efect of leaves
of Eriobotrya japonicacorrelating with attenuation of p38
Te authors declare that they have no conficts of interest. MAPK, ERK, and NF-κB activation in mast cells,” Toxicology
in Vitro, vol. 23, no. 7, pp. 1215–1219, 2009.
Supplementary Materials [13] A. M. Kuraoka-Oliveira, J. A. S. Radai, M. M. Leitão,
C. A. Lima Cardoso, S. E. Silva-Filho, and C. A. Leite Kassuya,
Supplementary Table 1: Kovats retention index of com- “Anti-infammatory and anti-arthritic activity in extract from
pound in EJ. Supplementary Figure 1: UHPLC-MS chro- the leaves of Eriobotrya japonica,” Journal of Ethno-
matograms of chlorogenic acid (CA) and euscaphic acid pharmacology, vol. 249, Article ID 112418, 2020.
(EA) in EJ (Loquat leaf extract). (a) UHPLC chromatogram [14] Y. H. Kwon, J. Y. Jang, J. H. Lee, Y. W. Choi, Y. H. Choi, and
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of EJ. (b) CA MS spectra of the peak. (c) EA MS spectra of
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the peak. Arrows indicated the CA or EA. Supplementary HO-1 signaling pathways in C2C12 cells,” Applied Sciences,
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EEJ, EJ ethanol extract; and CA, chlorogenic acid. Data “Antioxidant favonoids and chlorogenic acid from the leaves
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