Beneficial Effect of Cordyceps militaris on Exercise Performance via Promoting Cellular Energy Production

Abstract

Cordyceps militaris has been reported to the diverse pharmaceutical effects including cancer, inflammatory diseases, and bacteria or virus infection. However, the effect of C. militaris on exercise performance has not yet been elucidated. In this study, we investigated the beneficial effect of C. militaris on exercise performance. To evaluate exercise performance, we prepared C. militaris ethyl acetate extract (CMEE) and conducted grip strength tests every week after administration. Additionally, blood samples were collected at the end of the experiment for biochemical analysis. The administration of CMEE slightly increased grip strength, and this result was similar to the red ginseng treated group. According to the result of biochemical analysis, CMEE had an effect on the biomarkers related to ATP generation pathway but had little influence on the muscle fatigue related biomarkers. Therefore, C. militaris has the possibility of improving exercise performance, which could be associated with the increase in ATP production rather than the decrease in muscle fatigue during exercise.

Full text

RESEARCH ARTICLE
Beneficial Effect of Cordyceps militaris on Exercise Performance via
Promoting Cellular Energy Production
Eunhyun Choi
a
, Junsang Oh
a
and Gi-Ho Sung
a,b
a
Translational Research Division, Biomedical Institute of Mycological Resource, International St. Mary’s Hospital and College of
Medicine, Catholic Kwandong University, Incheon, Republic of Korea;
b
Department of Microbiology, College of Medicine,
Catholic Kwandong University, Incheon, Republic of Korea
ABSTRACT
Cordyceps militaris has been reported to the diverse pharmaceutical effects including cancer,
inflammatory diseases, and bacteria or virus infection. However, the effect of C. militaris on
exercise performance has not yet been elucidated. In this study, we investigated the benefi-
cial effect of C. militaris on exercise performance. To evaluate exercise performance, we pre-
pared C. militaris ethyl acetate extract (CMEE) and conducted grip strength tests every week
after administration. Additionally, blood samples were collected at the end of the experi-
ment for biochemical analysis. The administration of CMEE slightly increased grip strength,
and this result was similar to the red ginseng treated group. According to the result of bio-
chemical analysis, CMEE had an effect on the biomarkers related to ATP generation pathway
but had little influence on the muscle fatigue related biomarkers. Therefore, C. militaris has
the possibility of improving exercise performance, which could be associated with the
increase in ATP production rather than the decrease in muscle fatigue during exercise.
ARTICLE HISTORY
Received 5 June 2020
Revised 8 September 2020
Accepted 28 September 2020
KEYWORDS
Cordyceps militaris; exercise
performance; ATP; AMPK
1. Introduction
Health promotion is a positively related to reducing
the risk factors for cardiovascular diseases, metabolic
disorders, and bone diseases; whereas an inability to
maintain exercise is well known to be associated with
increase in these diseases [1]. Recently, the emergence
of the concept of well-being has resulted in the
increasing interest in the maintenance or enhancement
of exercise performance [2,3]. For improving exercise
performance, there are many dietary supplements,
including amino acids, vitamins, minerals, and botani-
cals or mushrooms [4]. Among numerous medicinal
mushrooms, Cordyceps militaris is considered as the
valuable mushroom because of their various health
benefits, including anti-cancer [5], immune modulat-
ing [6], anti-aging [7], anti-viral and anti-bacterial [8],
and anti-fatigue effects [9]. However, the influence of
C. militaris on enhancing exercise performance as well
as its underlying mechanism has yet to be proven in
animal models.
The exercise performance is correlated with
recovery of muscle fatigue, endurance, and activat-
ing the neuromuscular system [10,11]. To evaluating
of muscle fatigue or endurance in animals, there is
usually the measurement of the specific enzymatic
activity and/or biochemical analysis in blood
samples. These biomarkers are related to muscle
damage or fatigue and energy metabolism, such as
creatine kinase (CK), lactate dehydrogenase (LDH),
blood urea nitrogen (BUN), insulin-responsive glu-
cose transporter 4 (GLUT4), pyruvate dehydrogen-
ase (PDH), AMP-activated protein kinase (AMPK),
and TCA cycle, lipid metabolism, and electron
transport chain-involved oxidative enzymes [12–14].
The purpose of present study is to evaluate the
effect on improving exercise performance of C. mili-
taris ethanol extract (CMEE) in mice during the
grip strength test. In addition, we have analyzed the
several biochemical biomarkers, such as blood con-
centrations of LDH, aspartate aminotransferase
(AST), alanine aminotransferase (ALT), BUN, creat-
ine, phosphocreatine, adenosine-50-thriphosphate
(ATP), GLUT4, PDH, AMPK, and peroxisome pro-
liferator-activated receptor-c(PPAR-c), involved in
the muscle fatigue and the energy production or
metabolism pathway during exercise.
2. Materials and methods
2.1. Reagents
The Korean Red Ginseng Powder Special was pur-
chased from Geumsan Red Ginseng Land
CONTACT Gi-Ho Sung sung97330@gmail.com
These authors contributed equally to this work.
ß2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Korean Society of Mycology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/),
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
MYCOBIOLOGY
2020, VOL. 48, NO. 6, 512–517
https://doi.org/10.1080/12298093.2020.1831135

(Geumsan-gun, Chungcheongnam-do, Republic
of Korea).
2.2. Preparation of C. militaris ethanol
extract (CMEE)
Artificial cultured C. militaris was provided from
Mushtech Co., Ltd. (Hoengseong, Korea). Cultivated
whole fruiting bodies of C. militaris, containing
2.33 mg/g of cordycepin, were dried at 50 C and
crushed in a blender and then the crude powder
was extracted with ethanol at 85 C for 6 hr. The
ethanol extract was vacuum filtered using a filter
paper (Whatman No. 2) and then was evaporated at
65 C by an evaporator (N-1000; Eyela, Tokyo,
Japan) under reduced pressure. The concentrated
extract was frozen at 80 C and then lyophilized
using a freeze-dryer.
2.3. Animals and treatment
Male CrljOri:CD1 (ICR) mouse (6 weeks) were pur-
chased from Orientbio, Inc. (Gapyeong, Korea). The
animals were housed in groups of 5 per cage under
standard laboratory conditions (temperature
23 ± 3 C, relative humidity 53 ± 15%, and 12 hr
light/dark cycle of 150–300 Lux). Food and sterilized
water were available ad libitum. Animals were div-
ided equally into 5 groups: G1, vehicle-treated nor-
mal control group; G2, red ginseng 100 mg/kg
treated comparative group (orally once daily for
12 weeks); G3–G5, C. militaris treated groups (50,
150, and 300 mg/kg, orally once daily for 12 weeks).
All animals were closely monitored, and there were
no clinical symptoms observed during the entire
experimental period. All experiment protocols were
approved by the Institutional Animal Care and Use
Committee at the KNOTUS Co. Ltd. (Guri, Korea;
Certificate No: IACUC 17-KE-296).
2.4. Grip strength test
Grip strength was measured with a computerized
grip strength meter (47200; Ugo-Basile, Varese,
Italy). When the animals (n¼10 per group per test)
grasped the transducer metal bar with their fore-
paws, the experimenter pulled the animals back-
wards by the tail until grip was lost. Basal grip
strength values were recorded for each animal as
once a week for 12 weeks.
2.5. Biochemical analysis
Animals were anesthetized with isoflurane at
12 weeks and blood was collected from the postcaval
vein of the subjects into a vacutainer tube
containing a clot activator. After the serum was sol-
idified at room temperature, then allowed to stand
for about 15 min, the serum was separated by centri-
fuged for 10 min at 3,000 rpm. The quantitative
determination of contents of lactate dehydrogenase
(LDH), aspartate aminotransferase (AST), alanine
aminotransferase (ALT), blood urea nitrogen
(BUN), and creatine were analyzed by Hitachi
Chemistry Analyzer (7180; Hitachi, Tokyo, Japan),
and ELISA assay was allowed for the quantitative
determination of phosphocreatine (Cat. No.
MBS2700694; Mybiosource, San Diego, CA, USA),
adenosine triphosphate (ATP) (Cat. No. Ab83355;
Abcam, Cambridge, UK), insulin induces glucose
transporter 4 (GLUT4) (Cat. No. MBS727326;
Mybiosource), pyruvate dehydrogenase (PDH) (Cat.
No. MAK183; Sigma-Aldrich, St. Louis, MO, USA),
AMP-activated protein kinase (AMPK) (Cat. No.
MBS2505028; Mybiosource), and peroxisome prolif-
erator-activated receptor-c(PPAR-c) (Cat. No.
MBS2501353; Mybiosource).
2.6. Statistical analysis
All data are expressed as the mean ± standard error
(SE). Statistical analysis was performed using Prism
5.04 program (GraphPad Software Inc., San Diego,
CA, USA). The statistical comparisons were per-
formed using one-way analysis of variance
(ANOVA) followed by Dunnett’s test. A significant
difference was defined as p<0.05.
3. Results
3.1. Effect of CMEE on grip strength
To evaluate the influence of the CMEE on exercise,
we employed the grip strength test model and used
the red ginseng as positive control. The red ginseng
is one of the most famous medicinal herb and has
been reported to the various health beneficial effects
including the protection of muscle damage, the
relief of fatigue, and the improvement of exercise
endurance [15]. There were no significant changes
in body weight during the experiment (Figure 1).
Despite the exercise improving effect of CMEE was
not in dose-dependent manner, CMEE (G3–G5)
and red ginseng (G2) increased grip strength by
approximately 10 gf compared to the control group
(G1) at 11 and 12 weeks after administration
(Table 1).
3.2. Effect of CMEE on muscle fatigue and
ATP production
To elucidate the molecular mechanism of improving
exercise performance of CMEE, we performed
MYCOBIOLOGY 513

biochemical and ELISA analysis on blood samples
collected at the end of the experiment. The results of
biochemical analysis showed that there were no sig-
nificant changes in the concentration of AST, ALT,
LDH, creatinine, and creatine in all groups (Figure
2). The level of BUN was decreased in CMEE and
red ginseng treated groups. Especially, the levels of
BUN both G3 and G5 groups were significantly
reduced compared G1 and G2 groups (Figure 3(A)).
In addition, the results of ELISA analysis demon-
strated red ginseng showed dramatically increased the
levels of ATP, AMPK, PPAR-c, GLUT4, PDH, and
phosphocreatine, which were related to cellular
energy generation (Figure 3). In the case of CMEE-
treated groups, the level of AMPK were significantly
elevated all groups (G3–G5), and the levels of ATP,
GLUT4, PDH, PPAR-c, and phosphocreatine were
also increased compared to G1 control group but
there were no statistical significance, except for G4
group (Figure 3).
4. Discussion
Although it has been well known the various
pharmaceutical benefits of C. militaris on the health,
the precision mechanism of enhancing exercise per-
formance remains poorly understood. In the present
study, we evaluated whether the administration of
C. militaris has an influence on increasing exercise
performance in an animal model. To explain the
effect of exercise performance by C. militaris,we
analyzed peripheral blood biomarkers, related to
muscle fatigue and ATP generation.
To measurement of muscle strength in rodent
models, grip strength test is general used due to
convenient and noninvasive method [16]. During
the experiment, we found no significant difference
in body weight between normal control group (G1)
and experimental groups (G2–G5) (Figure 1). As a
result of grip strength test, all CMEE-treated groups
(G3–G5) were slightly higher than that of normal
control group (G1) at 11 and 12 weeks. These results
were similar to red ginseng-treated group (G2), a
positive control (Table 1). These observations sug-
gest that CMEE could contribute to improve exer-
cise performance.
The alleviation of exercise-induced muscle fatigue
and the increase on energy metabolism can be asso-
ciated with enhancing exercise performance [17–19].
We investigated whether the beneficial effect CMEE
on exercise performance is in consequence of the
reduction of muscle fatigue and the revitalization of
energy production. The results of biochemical ana-
lysis showed that the levels of fatigue-related bio-
markers such as ALT, AST, LDH, creatinine, and
creatine were no change except for BUN (Figure 2).
In contrast, the results of ELISA analysis showed
that the concentrations of AMPK, GLUT4, PDH,
PPAR-c, and phosphocreatine involved in energy
production as well as ATP increased (Figure 3).
AMPK is a sensor of intracellular ATP level and is
activated by ATP depletion. During the exercise,
ATP-consuming process is accelerated, as a result
AMP/ATP or ADP/ATP ratio increased. AMPK
activation leads to decrease ATP-consuming process
and increase ATP generation, which maintains the
Figure 1. Body weight change during the experiment. Mice
were fed with vehicle (G1), 100 mg/kg of red ginseng (G2),
or 50, 150, and 300 mg/kg of CMEE (C3–C5) for 12 weeks.
Data were expressed as mean ± S.D. (n¼10 mice in each
group). p<0.05, compared with normal control (G1).
Table 1. Effect of CMEE on grip strength (gf, grams of force).
Week
Grip strength (gf)
G1 G2 G3 G4 G5
0 110.9 ± 14.8 114.4 ± 13.7 125.2 ± 16.0 125.3 ± 14.5 119.1 ± 14.2
1 120.8 ± 14.9 125.8 ± 11.2 117.0 ± 16.0 127.1 ± 16.8 123.5 ± 13.5
2 121.7 ± 16.0 124.2 ± 12.3 124.3 ± 12.1 124.0 ± 17.6 117.0 ± 8.4
3 121.2 ± 9.7 131.9 ± 10.8 127.8 ± 11.4 129.7 ± 9.0 123.2 ± 6.9
4 124.8 ± 4.1 132.9 ± 10.1 126.8 ± 15.4 128.3 ± 9.3 123.4 ± 8.0
5 126.2 ± 7.3 133.8 ± 9.5 130.7 ± 13.9 130.9 ± 10.5 129.8 ± 8.4
6 129.5 ± 4.3 134.6 ± 7.4 131.6 ± 13.6 134.1 ± 9.9 136.5 ± 12.8
7 132.1 ± 6.4 136.9 ± 8.5 135.5 ± 13.3 135.9 ± 9.2 140.0 ± 13.5
8 136.1 ± 5.6 142.6 ± 8.8 140.1 ± 11.3 140.7 ± 8.6 142.3 ± 11.4
9 137.8 ± 7.3 145.0 ± 5.4 143.7 ± 9.5 143.8 ± 7.4 144.3 ± 6.3
10 138.2 ± 5.9 145.8 ± 6.9 144.4 ± 8.1 145.6 ± 5.9 145.7 ± 6.7
11 136.3 ± 4.5 146.3 ± 4.6 143.0 ± 5.1 148.9 ± 3.5 145.9 ± 3.2
12 136.4 ± 1.5 147.2 ± 3.6 142.6 ± 3.3 148.8 ± 2.8 146.7 ± 3.4
Mice were fed with vehicle (G1), 100 mg/kg of red ginseng (G2), or 50, 150, and 300 mg/kg of CMEE (C3–C5) for
12 weeks. Data were expressed as mean ± S.D. (n¼10 mice in each group). p<0.001, p<0.01, compared with nor-
mal control (G1).
514 E. CHOI ET AL.

energy homeostasis. Interestingly, AMPK promotes
the transcription of GLUT4 gene and regulates lipo-
genesis homeostasis [20]. GLUT4 plays an important
role in glucose homeostasis in skeletal muscle, and
transports glucose from blood to skeletal muscle.
Glucose in muscle cells is phosphorylated by hexoki-
nase and then enters glycolysis process or stored as
glycogen [21]. Glycolysis is the metabolic process,
which produces pyruvate from glucose. Pyruvate is
converted to acetyl-CoA, an important metabolic
Figure 2. Serum biochemical analysis results of the muscle fatigue related biomarkers. Mice were fed with vehicle (G1),
100 mg/kg of red ginseng (G2), or 50, 150, and 300 mg/kg of CMEE (C3–C5) for 12 weeks. Data are expressed as mean ± S.D.
(n¼10 mice in each group). p<0.001, p<0.01, p<0.05, compared with normal control (G1).
Figure 3. ELISA analysis results of the energy production related biomarkers. Mice were fed with vehicle (G1), 100 mg/kg of
red ginseng (G2), or 50, 150, and 300 mg/kg of CMEE (C3–C5) for 12 weeks. Data are expressed as mean ± S.D. (n¼10 mice in
each group). p<0.001, p<0.01, p<0.05, compared with normal control (G1).
MYCOBIOLOGY 515

intermediate of TCA cycle by PDH [22]. In add-
ition, the muscle contractile activity is dependent on
phosphocreatine activity that is involved in ATP
regeneration [23], and PPAR-cis a metabolic regu-
lator and is related to glucose and lipid metabolism
[24]. PPAR-cactivation upregulates PPAR-c-con-
trolled genes including mitochondrial biogenesis
and aerobic respiration, consequently, has beneficial
effects in skeletal muscle. Because these factors are
involved in ATP generation and exercise endurance,
the increased expression and activity of them is
associated with enhancing exercise performance.
Therefore, the administration of CMEE is influenced
on ATP production pathway, and consequently to
assist the improvement of exercise performance.
5. Conclusion
We investigated the effect of the administration of
CMEE on exercise performance in grip strength test
and analyzed biochemical biomarkers for elucidating
the mechanism of improving exercise performance.
Our results demonstrated that the administration of
CMEE enhances exercise performance by upregulat-
ing the ATP generation pathway rather than allevi-
ating muscle fatigue. Taken together, our finding
suggests that C. militaris could be useful material
for an adjuvant and a functional food improving
exercise performance.
Disclosure statement
No potential conflict of interest was reported by
the author(s).
Funding
This research was supported by Bio-industry
Technology Development Program [316025-05] of
IPET (Korea Institute of Planning and Evaluation
for Technology in Food, Agriculture, Forestry, and
Fisheries), and the National Research Foundation
(NRF) grant funded by the Korea government
(MSIT) [No. 2019R1A2C2005157].
ORCID
Eunhyun Choi http://orcid.org/0000-0002-8464-7156
Junsang Oh http://orcid.org/0000-0002-0811-2491
Gi-Ho Sung http://orcid.org/0000-0002-1861-5543
References
[1] Warburton DE, Nicol CW, Bredin SS. Health ben-
efits of physical activity: the evidence. CMAJ.
2006;174:801–809.
[2] Loureiro A, Veloso S. Green exercise, health and
well-being. In: Fleury-Bahi G, Pol E, Navarro O,
editors. Handbook of environmental psychology
and quality of life research. Cham (Switzerland):
Springer; 2017. p. 149–169.
[3] Penedo FJ, Dahn JR. Exercise and well-being: a
review of mental and physical health benefits asso-
ciated with physical activity. Curr Opin Psychiatry.
2005;18:189–193.
[4] Jagim AR, Harty PS, Camic CL. Common ingredi-
ent profiles of multi-ingredient pre-workout sup-
plements. Nutrients. 2019;11:254.
[5] Park JG, Son YJ, Lee TH, et al. Anticancer efficacy
of Cordyceps militaris ethanol extract in a xeno-
grafted leukemia model. Evid Based Complement
Alternat Med. 2017;2017:8474703.
[6] ShinS,KwonJ,LeeS,etal.Immunostimulatory
effects of Cordyceps militaris on macrophages through
the enhanced production of cytokines via the activa-
tion of NF-kappaB. Immune Netw. 2010;10:55–63.
[7] Li XT, Li HC, Li CB, et al. Protective effects on mito-
chondria and anti-aging activity of polysaccharides
from cultivated fruiting bodies of Cordyceps militaris.
Am J Chin Med. 2010;38:1093–1106.
[8] Dong CH, Yang T, Lian T. A comparative study
of the antimicrobial, antioxidant, and cytotoxic
activities of methanol extracts from fruit bodies
and fermented mycelia of caterpillar medicinal
mushroom Cordyceps militaris (Ascomycetes). Int
J Med Mushrooms. 2014;16:485–495.
[9] Song J, Wang Y, Teng M, et al. Studies on the
antifatigue activities of Cordyceps militaris fruit
body extract in mouse model. Evid Based
Complement Alternat Med. 2015;2015:174616.
[10] Wan JJ, Qin Z, Wang PY, et al. Muscle fatigue:
general understanding and treatment. Exp Mol
Med. 2017;49:e384.
[11] Huang WC, Chiu WC, Chuang HL, et al. Effect of
curcumin supplementation on physiological fatigue
and physical performance in mice. Nutrients. 2015;
7:905–921.
[12] Kim H, Park S, Han DS, et al. Octacosanol supple-
mentation increases running endurance time and
improves biochemical parameters after exhaustion
in trained rats. J Med Food. 2003;6:345–351.
[13] Dalla Corte CL, de Carvalho NR, Amaral GP,
et al. Antioxidant effect of organic purple grape
juice on exhaustive exercise. Appl Physiol Nutr
Metab. 2013;38:558–565.
[14] Ping FW, Keong CC, Bandyopadhyay A. Effects of
acute supplementation of Panax ginseng on endur-
ance running in a hot & humid environment.
Indian J Med Res. 2011;133:96–102.
[15] Shin EJ, Jo S, Choi S, et al. Red ginseng improves
exercise endurance by promoting mitochondrial
biogenesis and myoblast differentiation. Molecules.
2020;25:865.
[16] Takeshita H, Yamamoto K, Nozato S, et al.
Modified forelimb grip strength test detects aging-
associated physiological decline in skeletal muscle
function in male mice. Sci Rep. 2017;7:42323.
[17] Vollestad NK, Sejersted OM. Biochemical corre-
lates of fatigue. A brief review. Eur J Appl Physiol
Occup Physiol. 1988;57:336–347.
[18] Tung YT, Hsu YJ, Liao CC, et al. Physiological
and biochemical effects of intrinsically high and
low exercise capacities through multiomics
approaches. Front Physiol. 2019;10:1201.
516 E. CHOI ET AL.

[19] Layzer RB. Muscle metabolism during fatigue and
work. Baillieres Clin Endocrinol Metab. 1990;4:
441–459.
[20] Ke R, Xu Q, Li C, et al. Mechanisms of AMPK in
the maintenance of ATP balance during energy
metabolism. Cell Biol Int. 2018;42:384–392.
[21] Richter EA, Hargreaves M. Exercise, GLUT4, and
skeletal muscle glucose uptake. Physiol Rev. 2013;
93:993–1017.
[22] Morales-Alamo D, Guerra B, Santana A, et al.
Skeletal muscle pyruvate dehydrogenase
phosphorylation and lactate accumulation during
sprint exercise in normoxia and severe acute hypoxia:
effects of antioxidants. Front Physiol. 2018;9:188.
[23] Guimaraes-Ferreira L. Role of the phosphocreatine
system on energetic homeostasis in skeletal and car-
diac muscles. Einstein (Sao Paulo). 2014;12:126–131.
[24] Thomas AW, Davies NA, Moir H, et al. Exercise-
associated generation of PPARgamma ligands acti-
vates PPARgamma signaling events and upregu-
lates genes related to lipid metabolism. J Appl
Physiol. 2012;112:806–815.
MYCOBIOLOGY 517