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Effects of Rhizome Extract of Dioscorea batatas and Its Active Compound, Allantoin, on the Regulation of Myoblast Differentiation and Mitochondrial Biogenesis in C2C12 Myotubes

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With the aging process, a loss of skeletal muscle mass and dysfunction related to metabolic syndrome is observed in older people. Yams are commonly use in functional foods and medications with various effects. The present study was conducted to investigate the effects of rhizome extract of Dioscorea batatas (Dioscoreae Rhizoma, Chinese yam) and its bioactive compound, allantoin, on myoblast differentiation and mitochondrial biogenesis in skeletal muscle cells. Yams were extracted in water and allantoin was analyzed by high performance liquid chromatography (HPLC). The expression of myosin heavy chain (MyHC) and mitochondrial biogenesis-regulating factors, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), sirtuin-1 (Sirt-1), nuclear respiratory factor-1 (NRF-1) and transcription factor A, mitochondrial (TFAM), and the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) were determined in C2C12 myotubes by reverse transcriptase (RT)-polymerase chain reaction (RT-PCR) or western blot. The glucose levels and total ATP contents were measured by glucose consumption, glucose uptake and ATP assays, respectively. Treatment with yam extract (1 mg/mL) and allantoin (0.2 and 0.5 mM) significantly increased MyHC expression compared with non-treated myotubes. Yam extract and allantoin significantly increased the expression of PGC-1α, Sirt-1, NRF-1 and TFAM, as well as the phosphorylation of AMPK and ACC in C2C12 myotubes. Furthermore, yam extract and allantoin significantly increased glucose uptake levels and ATP contents. Finally, HPLC analysis revealed that the yam water extract contained 1.53% of allantoin. Yam extract and allantoin stimulated myoblast differentiation into myotubes and increased energy production through the upregulation of mitochondrial biogenesis regulators. These findings indicate that yam extract and allantoin can help to prevent skeletal muscle dysfunction through the stimulation of the energy metabolism.
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Molecules 2018, 23, 2023; doi:10.3390/molecules23082023 www.mdpi.com/journal/molecules
Article
Effects of Rhizome Extract of Dioscorea batatas and
Its Active Compound, Allantoin, on the Regulation of
Myoblast Differentiation and Mitochondrial
Biogenesis in C2C12 Myotubes
Junnan Ma 1, Seok Yong Kang 1, Xianglong Meng 1, An Na Kang 1, Jong Hun Park 1,2 ,
Yong-Ki Park 1,2 and Hyo Won Jung 1,2,*
1 Department of Herbology, College of Korean Medicine, Dongguk University, Gyeongju 38066, Korea;
nnzhgn@gmail.com (J.M.); seokppo2@hanmail.net (S.Y.K.); sszywzh@126.com (X.M.);
ank4200@gmail.com (A.N.K.); hasangpaul@hanmail.net (J.H.P.); yongki@dongguk.ac.kr (Y.-K.P.)
2 Korean Medicine R&D Center, Dongguk University, Gyeongju 38066, Korea
* Correspondence: tenzing2@hanmail.net; Tel.: +82-54-770-2367
Received: 24 July 2018; Accepted: 10 August 2018; Published: 13 August 2018
Abstract: With the aging process, a loss of skeletal muscle mass and dysfunction related to metabolic
syndrome is observed in older people. Yams are commonly use in functional foods and medications
with various effects. The present study was conducted to investigate the effects of rhizome extract
of Dioscorea batatas (Dioscoreae Rhizoma, Chinese yam) and its bioactive compound, allantoin, on
myoblast differentiation and mitochondrial biogenesis in skeletal muscle cells. Yams were extracted
in water and allantoin was analyzed by high performance liquid chromatography (HPLC). The
expression of myosin heavy chain (MyHC) and mitochondrial biogenesis-regulating factors,
peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), sirtuin-1 (Sirt-1),
nuclear respiratory factor-1 (NRF-1) and transcription factor A, mitochondrial (TFAM), and the
phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) were
determined in C2C12 myotubes by reverse transcriptase (RT)-polymerase chain reaction (RT-PCR)
or western blot. The glucose levels and total ATP contents were measured by glucose consumption,
glucose uptake and ATP assays, respectively. Treatment with yam extract (1 mg/mL) and allantoin
(0.2 and 0.5 mM) significantly increased MyHC expression compared with non-treated myotubes.
Yam extract and allantoin significantly increased the expression of PGC-1α, Sirt-1, NRF-1 and
TFAM, as well as the phosphorylation of AMPK and ACC in C2C12 myotubes. Furthermore, yam
extract and allantoin significantly increased glucose uptake levels and ATP contents. Finally, HPLC
analysis revealed that the yam water extract contained 1.53% of allantoin. Yam extract and allantoin
stimulated myoblast differentiation into myotubes and increased energy production through the
upregulation of mitochondrial biogenesis regulators. These findings indicate that yam extract and
allantoin can help to prevent skeletal muscle dysfunction through the stimulation of the energy
metabolism.
Keywords: allantoin; Chinese yam; C2C12 cells; Dioscorea batatas; Dioscoreae Rhizoma; myoblast
differentiation; mitochondrial biogenesis
1. Introduction
Dietary glucose is supplied by meals, and glucose is stored as glycogen in the liver, kidney, and
muscle to enable metabolic energy function [1]. Skeletal muscle is largely responsible for regulating
carbohydrate metabolism and achieving energy balance in normal feeding [2]. Accordingly, skeletal
muscle function deficit, in particular, age-related and disease-related muscle loss, is associated with
Molecules 2018, 23, 2023 2 of 14
many chronic diseases including sarcopenia, diabetes and obesity [3]. Such conditions are still
difficult to control because causes of muscle loss are multifactorial and influenced by genetics.
Recently, there has been increased interest in various functional foods and medicines for promotion
of muscle function and maintenance of muscle balance in condition between protein synthesis and
degradation [4].
The root (Dioscoreae Rhizoma, Chinese yam) of Dioscorea batatas Decaisne (=D. oposita Thunberg)
is a perennial trailing plant of the Dioscoreaceae family. The yam, which is one of the most important
herbs in traditional medicine, has long been used as a food and medication with various
pharmaceutical functions. In herbology, the yam is neutral in nature, sweet in flavor, and mainly
manifests its therapeutic actions in the spleen, lung, and kidney meridians [5,6]. Therefore, yams are
utilized to cure yin deficiency in metabolic disorders such as diabetes and hyperthyroidism by
tonifying and replenishing qi in meridian organs. Yam is also known to have digestive functions in
the stomach and intestines, as well as immune-regulatory and antiaging effects. In modern
pharmacology, the yam has been studied for its effects on asthma [7], cancer [810], diabetes [11,12],
and liver damage [13], as well as its anti-oxidation, anti-inflammation, and anti-aging effects [14,15].
The yam contains various compounds such as dioscin [16], steroidal saponins [16,17], saponins, gallic
acid, vanillic acid [14], allatoin [12], and protodioscin [18]. In recent years, natural dietary compounds
have gained increasing attention as adjuvant therapy due to their relative low toxicity and synergistic
effects with current chemotherapeutic agents [19].
Allantoin, a diureide of glyoxylic acid, is an active and abundant component of yam [10,12,20].
In vivo studies have shown allantoin to have anti-asthmatic [20], antidiabetic [12,21] and
antihypertensive [22] activities, as well as memory-enhancing effects in Alzheimer’s disease [23].
We recently conducted a study that provided scientific evidence of the abilities of various herbs
to improve obesity, diabetes and sarcopenia based on clinical practice and found that some herbs
have good effects that occur via regulation of the differentiation and mitochondria biogenesis in
skeletal muscle [24,25]. Therefore, we investigated the effects of yam water extract and its bioactive
compound, allantoin, on myoblast differentiation and mitochondrial biogenesis in C2C12 myotubes.
2. Results
2.1. Effects of Yam Extract and Allantoin on Myoblast Differentiation into Myotubes
To investigate the effects of yam extract and allantoin on myoblast differentiation into myotubes,
we determined the expression of Myosin heavy chain (MyHC) mRNA and protein as differentiation
markers in C2C12 myotubes using reverse transcriptase (RT)-polymerase chain reaction (RT-PCR)
and western blot analysis. Treatment with yam extract (p < 0.05 for 1 mg/mL) and allantoin (p < 0.001
for 0.2 and 0.5 mM) significantly increased the expression of MyHC mRNA (Figure 1A) and protein
(Figure 1B) in C2C12 myotubes compared with non-treated cells as a negative control. We also observed
MyHC expression in C2C12 myotubes using immunocytochemistry (Figure 1C), which revealed that
MyHC-positive myotubes with an elongated and widened cylinder-shape and multiple nuclei were
present in greater numbers in yam extract and allantoin-treated cells than non-treated cells.
Metformin-treated cells as a positive control group were also shown to exhibit an increase of MyHC
expression, but this was lower than for treatment with yam extract or allantoin. These results indicate
that yam extract and allantoin can induce myoblast differentiation into myotubes in skeletal muscle
cells. In MTT viability assay, we confirmed that the concentrations of yam extract and allantoin for
treatment in C2C12 myoblasts did not affect the viability (data not shown).
Molecules 2018, 23, 2023 3 of 14
Figure 1. Effects of yam extract and allantoin on the expression of MyHC protein and mRNA in C2C12
myotubes. C2C12 myoblasts were differentiated with DMEM containing 2% HS for 5 days, then
treated with or without yam extract (0.5 and 1.0 mg/mL) or allantoin (0.2 and 0.5 mM) for 24 h.
Metformin (2.5 mM) was used as a positive control. The expression of MyHC mRNA (A) and protein
(B) was determined by RT-PCR and western blot, respectively. GAPDH and β-actin were used as
internal controls. All data were presented as the means ± SEM of three independent experiments. Y,
yam extract; A, allantoin; and M, metformin. * p < 0.05 and *** p < 0.001 vs. non-treated cells; (C) the
myotubes were stained with anti-MyHC antibody and DAPI, then observed by fluorescence
microscopy (original magnification = 200×). Green, MyHC-positive cells; and blue, DAPI-positive
nuclei.
2.2. Effects of Yam Extract and Allantoin on the Expression of Mitochondria Biogenesis-Regulating Factors
in Myotubes
To investigate the effects of yam extract and allantoin on mitochondrial biogenesis in myotubes,
we measured the expression of the biogenesis regulating factors, peroxisome proliferator-activated
receptor gamma coactivator 1-alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1), transcription
factor A, mitochondrial (TFAM) and sirtuin 1 (Sirt-1) mRNA and protein in C2C12 myotubes by RT-
PCR and western blot, respectively. Treatment of yam extract (0.5 and 1 mg/mL) and allantoin (0.2
and 0.5 mM) in the myotubes for 24 h increased the expression of PGC1α (Figure 2A,B), NRF-1
(Figure 2C,D), TFAM (Figure 2E,F) and Sirt-1 (Figure 2G,H) mRNA (Figure 2A,C,E,G) and protein
(Figure 2B,D,F,H) as compared with non-treated cells. In particular, allantoin in a high concentration
(0.5 mM) significantly increased the expression of all regulators of mRNA (p < 0.01 for PGC1α, p <
0.05 for NRF-1, TFAM and Sirt-1) and protein (p < 0.01 for PGC1α, NRF1, and TFAM, p < 0.05 for
Sirrt1) compared with non-treated cells. Metformin significantly increased the expression of PGC1α
(p < 0.05 for mRNA and protein) and NRF-1 (p < 0.05 for protein) compared with non-treated cells.
Molecules 2018, 23, 2023 4 of 14
These results indicate that yam extract and allantoin can enhance mitochondrial biogenesis through
upregulation of the expression of the transcription factors.
Figure 2. Effects of yam extract and allantoin on the expression of mitochondrial biogenesis-
regulating factors in C2C12 myotubes. Differentiated myotubes were treated with or without yam
extract (0.5 and 1.0 mg/mL) or allantoin (0.2 and 0.5 mM) for 24 h, after which the expression of PGC1α
(A,B), NRF-1 (C,D), TFAM (E,F) and Sirt-1 (G,H) mRNA (A,C,E,G) and protein (B,D,F,H) was
analyzed by RT-PCR (A,C,E) and western blot (B,D,F), respectively. Metformin (2.5 mM) was used as
a positive control. GAPDH and β-actin were used as internal controls. Each band was presented as a
representative figure and the histogram was calculated from the band density value of each
experiment. All data were presented as the means ± SEM of three independent experiments. Y, yam
extract; A, allantoin; and M, metformin. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. non-treated negative
control.
Molecules 2018, 23, 2023 5 of 14
2.3. Effects of Yam Extract and Allantoin on the AMPK and ACC Pathways in Myotubes
Next, we investigated the effects of yam extract and allantoin on the signaling pathway activated
with mitochondrial biogenesis based on an evaluation of the phosphorylation of AMP-activated
protein kinase (AMPK) and Acetyl-CoA carboxylase (ACC) in C2C12 myotubes by western blot.
Treatment with yam extract (0.5 and 1 mg/mL) and allantoin (0.2 and 0.5 mM) resulted in the
increased phosphorylation of AMPK (Figure 3A) and ACC (Figure 3B) in the myotubes. Moreover,
treatment with allantoin at a high concentration (0.5 mM) significantly increased the phosphorylation
of AMPK (p < 0.001) and ACC (p < 0.01) compared with non-treated cells. Metformin as an AMPK
activator also significantly increased the phosphorylation of AMPK (p < 0.001) and ACC (p < 0.05) in
myotubes. Metformin significantly increased the phosphorylation of AMPK as AMPK activator (p <
0.001) and ACC (p < 0.05) compared with non-treated cells. These results indicate that yam extract
and allantoin can increase the mitochondrial biogenesis in myotubes through activation of the
AMPK/ACC signaling pathway.
Figure 3. Effects of yam extract and allantoin on the phosphorylation of AMPK and ACC protein
inC2C12 myotubes. Differentiated C2C12 myotubes were treated with or without yam extract (0.5
and 1.0 mg/mL) or allantoin (0.2 and 0.5 mM) for 24 h and the phosphorylation of AMPK (A) and
ACC (B) protein was investigated by western blot. Metformin (2.5 mM) was used as a positive control.
Each band was presented as a representative figure and a histogram was calculated from the band
density value of each experiment. β-actin were used as an internal control. All data were presented
as the means ± SEM of three independent experiments. Y, yam extract; A, allantoin; and M, metformin.
* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. non-treated negative control.
2.4. Effects of Yam Extract and Allantoin on Glucose Uptake in Myotubes
To investigate the effects of yam extract and allantoin on glucose uptake into myotubes, we
evaluated the expression of Glucose transporter type 4 (GLUT-4) protein and measured the glucose
levels in culture medium and in cells using western blot, glucose consumption assay, and glucose
uptake assay, respectively. The results revealed that the expression of GLUT-4 in the myotubes was
significantly increased by treatment with yam extract (0.5 and 1 mg/mL) and allantoin (0.2 and 0.5
mM) in a dose-dependent manner. Moreover, allantoin treatment induced a significant increase in
GLUT-4 expression (p < 0.05 for 0.2 and 0.5 mM), which was more effective than yam extract treatment
(Figure 4A). Additionally, glucose was significantly decreased in culture medium of allantoin-treated
myotubes (p < 0.05 for 0.2 and 0.5 mM, Figure 4B), while the cellular levels were significantly
increased (p < 0.05 for 0.5 mM, Figure 4C). Metformin treatment also significantly decreased glucose
levels in culture medium (p < 0.01) and significantly increased cellular glucose levels (p < 0.001)
compared with non-treated cells. Metformin significantly increased GLUT-4 expression (p < 0.05) and
glucose uptake (p < 0.05) in myotubes compared with non-treated cells. These results indicate that
Molecules 2018, 23, 2023 6 of 14
yam extract and allantoin can stimulate glucose uptake in myotubes by increasing the GLUT-4
expression.
2.5. Effects of Yam Extract and Allantoin on ATP Production in Myotubes
To investigate the effects of yam extract and allantoin on energy production in myotubes, we
measured the ATP contents in myotubes. As shown in Figure 4D, the treatment of myotubes with
yam extract and allantoin led to dose-dependent increases in ATP production, with significantly
increased ATP levels being observed in response to allantoin (p < 0.05 for 0.5 mM). Metformin
significantly increased ATP production (p < 0.05) in myotubes compared with non-treated cells. These
results indicate that yam extract and allantoin can enhance the energy production in myotubes, which
might be related to the upregulation of the mitochondrial biogenesis-regulating factors, as shown in
Figure 2.
Figure 4. Effects of yam extract and allantoin on the expression of GLUT-4 and the levels of glucose
in C2C12 myotubes. Differentiated myotubes were treated with or without yam extract (0.5 and 1
mg/mL) or allantoin (0.2 and 0.5 mM) for 24 h. (A) The expression of GLUT-4 protein was determined
by western blot. Metformin (2.5 mM) was used as a positive control and β-actin was used as an
internal control. Each band was presented as a representative figure and a histogram was calculated
from the band density value of each experiment. The levels of glucose in culture medium (B) and in
the cells (C) were measured by a glucose consumption assay and glucose uptake assay, respectively.
The contents of ATP in the myotubes were measured using an ATP assay kit (D). All data were
presented as the means ± SEM of three independent experiments. Y, yam extract; A, allantoin; and M,
metformin. * p < 0.05 and ** p < 0.01 vs. non-treated negative control.
2.6. HPLC Analysis
To analyze the content of allantoin in yam extract, we conducted HPLC analysis and then
compared the retention time of samples with that of authentic standard (Figure 5A). The content of
allantoin in yam water extract was subsequently calculated by comparison of peak areas (Figure 5B).
The equation of the calibration curves for allantoin was y = 16039x16.79. In addition, the correlation
coefficient of the calibration curve was higher than 0.9995, and the concentration of allantoin in the
Molecules 2018, 23, 2023 7 of 14
extract was 1.53%. The relative standard deviations of precision and repeatability were 0.67% and
1.85%, respectively.
Figure 5. HPLC analysis of allantoin in the yam extract: (A) allantoin as a standard compound, and
(B) allantoin in the water extract. a, allantoin (retention time: 3.253 min).
3. Discussion
Social, health, and technological developments have resulted in increases in the proportion of
older people increasing worldwide along with increasing life expectancy [25]. The aging process is
responsible for many changes in body composition, particularly a loss of skeletal muscle mass.
Muscle mass loss and dysfunction in older people are associated with various types of disease, injury
or aging, which significantly increases the cost of health care [26]. Age-related reductions in muscle
mass known as sarcopenia induce negative effects on muscle strength and muscle quality, as well as
decreased physical function, all of which lead to mobility impairment, disability, fatigue, risk of
metabolic disorders, falls, and mortality in older adults [2]. Recent research strategies have focused
on factors associated with muscle mass and strength, as well as nutritional interventions; specifically,
diets rich in proteins and antioxidant supplements and various exercise-related interventions are
thought to increase muscle strength and physical function [25,27]. Although potent pharmaceutical
treatments such as hormone therapies, angiotensin converting enzyme inhibitors and ghrelin
agonists have been studied, there has been little convincing evidence of their effects or they have
induced adverse side effects [27]. Nevertheless, it is necessary to find and implement interventions
to prevent and treat sarcopenia in the ageing population.
Dietary supplemental herbs with many beneficial effects have long been considered to enhance
health status and physical strength as well as to improve abnormal status among the elderly [28].
Yams are commonly used in medications because of their various pharmaceutical functions, which
include the enhancement of the digestive process in the stomach and intestines, immune regulation,
and antiaging, antiinflammation and antioxidation effects. In traditional medicine, yams are known
as a nourishing herb that alleviates yin deficiency in the spleen, lung, and kidney by providing a
supplementary energy (qi), therefore, it is used to treat metabolic syndromes such as obesity,
diabetes, and hypothyroidism [29]. In addition, yams have been used to prevent the aging process
(e.g., muscle weakness) because they control muscle function by spleen control [30]. However, there
is little known about the medicinal effects of yams on muscle function. In the present study, we
investigated whether yam extract and its active compound, allantoin, could help enhance the muscle
function in myotubes. The results revealed that yam extract and allantoin significantly increased
myoblast differentiation into myotubes in C2C12 cells and mitochondrial biogenesis through the
Molecules 2018, 23, 2023 8 of 14
upregulation of the mitochondrial transcription factors, PGC1α, TFAM, NRF-1, and Sirt-1 via
activation of the AMPK/ACC signaling pathway.
To overcome muscle wasting in sarcopenia, it is necessary to stimulate the myogenesis pathway
or inhibit the muscle wasting process. Satellite cells such as C2C12 myoblasts undergo expansion and
migration and differentiate into multinucleated fibers, myotubes via myoblasts fusion [31]. Myoblast
differentiation is orchestrated by myogenic regulatory factors (MRFs) such as myoblast
determination protein (MyoD), MRF4, myogenic factor 5 (Myf5) and myogenin [31]. Mature
myotubes also express structural muscle proteins such as tropomyosin and MyHC, which is the
motor protein of muscle thick filaments and a specific mature marker protein [32]. In the present
study, the treatment of yam extract and allantoin significantly increased the expression of MyHC
mRNA and protein in C2C12 myotubes, which was higher than Metformin-treatment, suggesting
that yams and allantoin can facilitate myoblast differentiation in muscle cells; however, future
investigations of the regulation of MRFs and their signals are still needed to better understand the
effects of yams and allantoin on myogenesis. Meanwhile, in MTT assay, allantoin did not decrease
the viability up to 1 mM in C2C12 myotubes, but its treatment showed a lower expression of MyHC
than treatment with 0.5 mM (data not shown). Therefore, we used concentrations of allantoin at 0.2
and 0.5 mM for this study.
Skeletal muscle, which is a key tissue involved in the control of the energy metabolism, processes
up to 75% of insulin-stimulated glucose disposal by the translocation of GLUT4 to the plasma
membrane in response to the activation, or resulting in the activation, of the AMP-activated protein
kinase (AMPK) pathway [33]. AMPK is a key energy sensor controlling metabolic homeostasis at
both the cellular and whole-body levels and is involved in many other cellular processes including
cell cycle regulation and endothelial and vascular relaxation [34]. Therefore, it has been considered a
subject in recent studies of metabolic syndrome such as obesity, insulin resistance, dyslipidemia, and
diabetes mellitus [34,35]. In myoblast differentiation, cellular ATP consumption elevates the cellular
AMP/ATP ratio which stimulates ATP generation through AMPK activation [35,36]. AMPK
activation mediates the increased expression of GLUT-4 and mitochondrial biogenesis and regulates
fatty acid oxidation via the phosphorylation of ACC and the expression of Sirt-1 [37]. Sirt-1 is another
downstream regulator of the glucose and lipid metabolism that is known to improve insulin
sensitivity and to stimulate mitochondrial biogenesis in skeletal muscle via interaction with
AMPK/PGC1α [38]. PGC1α is a major regulator of mitochondrial biogenesis that activates the
expression of its downstream transcription factors, NRF-1and TFAM [39]. In the present study, yam
extract (0.5 and 1 mg/mL) and allantoin (0.2 and 0.5 mM) increased the expression of PGC1α, Sirt-1,
NRF-1, and TFAM in C2C12 myotubes. These results indicate that the increasing levels of glucose
uptake and ATP in the myotubes are connected with the upregulation of the mitochondrial
biogenesis regulating factors PGC1α, Sirt-1, NRF-1, and TFAM, as well as with activation of the
AMPK/ACC pathway. Therefore, yam extract and allantoin could help to elevate energy production
by increasing mitochondrial biogenesis in skeletal muscle. Recently, it was reported that metformin
increases mitochondrial energy formation in L6 muscle cells [40]. We investigated the effects of yam
extract and allantoin on the expression of mitochondrial biogenesis regulating factors, PGC1α, Sirt-
1, NRF-1, and TFAM, at one-time treatment point; however, these should be considered at multiple
time points to observe any changes in mitochondrial biogenesis.
In Dioscorea species, the rhizomes of D. batatas, D. opposite, and D. japonica are commonly used
as cultivated edible yams, but many wild varieties have rhizomes with different tastes and are not
generally edible. However, it was reported that batatasin IV, raspberry ketone, 2-methoxy-4′-
hydroxyacetophenone, (3R,5S)-3,5-dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl) heptane, β-
sitosterol, blumenol A, dihydropinosylvin, stilbostemin N, butyl-β-D-fructofuranoside, allantoin,
dioscin, and coreajaponins A(1) and B(2) were found in 50% EtOH extract of D. japonica [41], and
steroidal saponins such as protodioscin, dioscin, and gracilin were found in CH3CN extract of in D.
tokoro (wild yam) [18], and batatasin I and 6,7-dihydroxy-2,4-dimethoxy phenanthrene were found
in MeOH extract of D. batatas aerial bulbil [42]. Allantoin was identified in the water extract of flesh
and peel of D. opposite by HPLC-PAD [9], and of the tuber and bulbil of D. batatas by HPLC [43]. In
Molecules 2018, 23, 2023 9 of 14
our analysis, allantoin (0.36 mg/mL, 1.53%) was found in the water extract of D. batatas rhizome.
Meanwhile, we detected additional small peak beside to allantoin in HPLC analysis. Recently, it was
reported that the adenosine is detected at similar retention time with allantoin because of similar
polar and structure [44], however, it will be necessary to analysis.
We used metformin as a reference drug to compare the efficacy of yam extract and allantoin on
mitochondrial biogenesis. Metformin treatment to C2C12 myotubes significantly increased the
glucose uptake and ATP levels with upregulation of PGC1α and NRF-1 expression, and
phosphorylation of AMPK, but these effects were seen to be lower than in allantoin. Thus, the
antidiabetic effects of allantoin have been reported in streptozotocin-induced diabetic rats through
modulating lipid profiling and increasing GLP-1 release [12] and glucose uptake with GLUT-4
expression in skeletal muscle [20,45]. Our results also suggest that allantoin has an improvement
potential for muscle dysfunction in disease conditions through increasing energy formation in
muscle. Under diabetic conditions including insulin resistance, hyperglycemia is a risk factor for age-
related loss of muscle mass in sarcopenia, which induces muscle synthesis reduction, chronic
inflammation, and mitochondrial dysfunction [46]. Therefore, in our further study, the effects of yam
extract and allantoin will be investigated in skeletal muscle dysfunction in type 2 diabetes mouse
models which show insulin resistance and impaired mitochondrial function in muscle.
4. Materials and Methods
4.1. Materials
Allantoin and metformin were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMEM
and penicillin/streptomycin solution were acquired from Corning (Corning, NY, USA). Fetal bovine
serum (FBS), horse serum (HS) and penicillin/streptomycin (P/S) solution were obtained from Merck
Millipore (Temecula, CA, USA). An ATP colorimetric assay kit was procured from BioVision Inc.
(Milpitas, CA, USA). Anti-Sirt-1, TFAM, NRF-1, AMPK, phospho-AMPK, total-AMPK, phospho-
ACC, and total-ACC antibodies were purchased from Cell Signaling Technology (Danvers, MA,
USA). Anti-MyHC and GLUT4 antibodies were acquired from Santa Cruz Biotechnology (Dallas, TX,
USA). Anti-PGC1α antibody and radioimmunoprecipitation assay (RIPA) buffer were obtained from
Thermo Fisher Scientific (Waltham, MA, USA).
4.2. Preparation of Yam Extract
Dried rhizome of D. batatas was purchased from an herbal company (Kwangmyungdang, Ulsan,
Korea) and identified by Professor Y.-K. Park, a medical botanist in herbology at College of Korean
Medicine, Dongguk University (DUCOM). A voucher specimen was deposited at the herbarium of
DUCOM (2017DR). Yams (200 g) were extracted by boiling in2 L of water for 3 h, filtered through
Whatman Grade 1 filter paper (Sigma-Aldrich, St Louis, MO, USA), concentrated under a vacuum
rotary evaporator (Eyela. Co., Ltd., Tokyo, Japan) at 60 °C, and then lyophilized in a freeze-dryer
(IlShinBioBase Co., Yangju, Korea) at −80 °C under 5 mTorr. Yam extract (yield = 11.4%) was stored
at 4 °C, dissolved in Phosphate Buffer solution (PBS), and filtered through a syringe filter (0.45
μm, Corning, Wiesbaden, Germany) before being used in in vitro experiments.
4.3. Cell Culture and Drug Treatments
C2C12 myoblasts, a mouse skeletal muscle line, were purchased from the American Type
Culture Collection (ATCC; Manassas, VA, USA) and grown in DMEM supplemented with 10% (v/v)
FBS and1% P/S solution in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. At 85–95%
confluence, myoblasts were induced to differentiate in DMEM with 2% HS once every day for an
additional 5 days. The C2C12 myotubes were then treated with or without different concentrations
of yam extract or allantoin. Metformin (2.5 mM) was used as a positive control drug. Allantoin and
metformin were dissolved in 1× PBS (pH 7.4).
Molecules 2018, 23, 2023 10 of 14
4.4. Western Blot
After cells were lysed in ice-cold RIPA buffer containing a phosphatase inhibitor cocktail
(Thermo Fisher Scientific), lysates were centrifuged at 12,000× g for 20 min at 4 °C. Protein
concentrations of the lysates were then quantified using the protein assay solution (BioRad, Hercules,
CA, USA). Next, 50 μg of protein was separated by SDS-polyacrylamide gel electrophoresis (PAGE)
and transferred onto nitrocellulose membrane. The membrane was then blocked with 5% skim milk
for 1 h at room temperature, after which it was immunoblotted with primary antibodies against
MyHC, PGC1α, NRF-1, TFAM, Sirt-1, AMPK (total or phosphor-forms), and ACC (total or phosphor
forms), as well as β-actin as an internal control overnight at 4 °C. Following immunoblotting, the
membranes were washed three times with 1× tris-buffered saline (pH 7.4) containing 0.1% tween-20
(TBST) buffer, then reacted with horseradish peroxidase (HRP)-labeled anti-mouse or anti-rabbit IgG.
All immunoblots were subsequently washed with 1× TBST three times, then developed using ECLTM
Western blotting detection reagent (GE Healthcare, Pittsburgh, PA, USA). Finally, bands were
detected using a ChemiDoc MP Imaging System (BioRad, Hercules, CA, USA) and quantified by
densitometry using the Image J programing software (1.51p 22 for Windows, NIH, Bethesda, MD,
USA).
4.5. Reverse Transcriptase (RT)-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from cells by TRIzol reagent (GibcoBRL Life Technologies Inc., Grand
Island, NY, USA) according to the manufacturer’s instructions. The RNA concentration was then
quantified using a Nano Drop ND-1000 spectrophotometer (NanoDrop Technologies, Inc.,
Wilmington, DE, USA). Next, cDNA was generated from 1 μg of total RNA using a Reverse
Transcription System kit (Promega, Fitchburg, WI, USA), after which RT-PCR was conducted using
a Blend Taq PCR kit (Toyobo, Osaka, Japan) and primers specific to the target genes (Table 1). For
PCR, the samples were subjected to pre-denaturation at 94 °C for 2 min, followed by 30 cycles of
denaturation for 30 s at 94 °C, annealing for 30 s at 56–60 °C, and extension for 1 min at 72 °C. Finally,
the bands were detected with the BioRad ChemiDoc MP imaging system and quantified by
densitometry using the Image J programming software.
Table 1. Primer sequences of the target genes for PCR.
Primers
Accession No.
Sequence (53)
MyHC
Forward
NM 001039545.2
TGA ACT GGA GGG TGA GGT AG
Reverse
NM 001039545.2
TTC GGT CTT CTT CTG TCT GG
PGC1α
Forward
XM 006503779.3
CAC CAA ACC CAC AGA AAA CAG
Reverse
XM 006503779.3
GGG TCA GAG GAA GAG ATA AAG TTG
NRF-1
Forward
XM 017321445.1
ACC CTC AGT CTC ACG ACT AT
Reverse
XM 017321445.1
GAA CAC TCC TCA GAC CCT TAA C
TFAM
Forward
XM 017313918.1
CAC CCA GAT GCA AAA CTT TCA G
Reverse
XM 017313918.1
CTG CTC TTT ATA CTT GCT CAC AG
Sirt-1
Forward
NM 001159589.2
GAT CCT TCA GTG TCA TGG TT
Reverse
NM 001159589.2
GAA GAC AAT CTC TGG CTT CA
Gapdh
Forward
XM_017321385.1
CAG CCT CGT CCC GTA GAC A
Reverse
XM_017321385.1
CGC TCC TGG AAG ATG GTG AT
4.6. Immunocytochemistry
Differentiated myotubes were seeded on Thermanox plastic cover slips (NuncTM, Thermo Fisher
Scientific) and differentiated using a common culture method for 5 days. Samples on cover slips were
then fixed with 4% paraformaldehyde for 10 min, after which they were permeabilized with 0.1%
Triton X-100 (Sigma-Aldrich) for 20 min. After washing with PBS, cover slips were blocked with
5% bovine serum albumin (BSA) for 30 min at room temperature, then incubated with anti-MyHC
antibody overnight at 4 °C. Cover slips were subsequently labelled with AlexaFluor 488-conjugated
Molecules 2018, 23, 2023 11 of 14
goat anti-rabbit antibody for 1 h at room temperature, then counterstained with DAPI for 5 min.
Finally, the expression of MyHC was observed using a fluorescence microscope (Leica DM2500, Leica
microsystems, Wetzlar, Germany).
4.7. Glucose Assay
Glucose consumption was determined in culture media using a glucose assay kit (Sigma-
Aldrich, St. Louis, MO, USA). Briefly, cell culture supernatants were harvested and diluted with
deionized water, after which 50 μL of the diluted sample was mixed with an equal volume of assay
buffer including o-dianisidine in a 96-well plate. The mixture was then incubated at 37 °C for 30 min,
at which time the reaction was stopped by adding 50 μL of H2SO4 and the absorbance (O.D.) at 540
nm was measured in a microtiter reader (UVM340, Asys Hitech Gmbh, Eugendorf, Austria). The
glucose consumption in each sample was calculated using a calculation formula from a standard
curve.
Next, the cellular levels of glucose were measured in C2C12 myotubes using a glucose uptake
cell-based assay kit (Cayman Chemical Co., Ann Arbor, MI, USA). Briefly, C2C12 myotubes were
treated with or without yam extract and allantoin at different concentrations in glucose-free medium
containing 100 μg/mL of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (NBDG)
for 4 h. After harvesting the cells, cell-based assay buffer (200 μL) was added to each well. The amount
of 2-NBDG taken up by the myotubes was then measured with fluorescent filters that detected
fluorescein (excitation/emission = 485/650) using a Glomax multi detection system (Promega
Biosystems, Sunnyvale, CA, USA)
4.8. ATP Assay
Total ATP contents were determined using an ATP colorimetric assay kit (BioVision) according
to the manufacturer’s instructions. Briefly, C2C12 myotubes were harvested and homogenized in 100
μL ATP assay buffer, after which 50 μL of deproteinized cell lysate was mixed with 50 μL of reaction
mix containing ATP probe, ATP converter, and developer in a 96-well plate. The mixture was
subsequently incubated at room temperature for 30 min, at which time the absorbance (O.D.) at 570
nm was measured using a microtiter reader. Finally, the concentration of ATP (μM) in each sample
was calculated using a calculation formula generated from a standard curve.
4.9. HPLC Analysis
To identify allantoin in yam extract, HPLC was conducted using an Agilent 1260 infinity II
quaternary system equipped with a G7129A vial sampler and a WR G7115 Adiode array detector
(Agilent, Waldbronn, Germany) and a ZORBAXNH2 column (4.6 × 150 mm, 5-micron).
Chromatographic separation was performed using a gradient solvent system consisting of
acetonitrile (HPLC grade, Merck, Darmstadt, Germany) (B) and water (HPLC grade, Merck) (A). The
gradient program was as follows: 0 min, 25% B; 5 min, 17% B; 10 min, 17% B. The injection volume
was 10 μL and the column eluent was monitored at UV 200 nm while chromatography was
performed at 30 °C with a flowrate of 1.0 mL/min. The HPLC pattern of allantoin in yam water extract
has been reported in the literature [9,26].
4.10. Statistical Analysis
The data are presented as means ± standard errors of means (SEMs) of three independent
experiments. Differences between groups were identified by the Student’s t-test using the GraphPad
Prism program (ver. 5.0, GraphPad Software, La Jalla, CA, USA) and p-values < 0.05 were considered
statistically significant.
5. Conclusions
Yam water extract and its active compound, allantoin, significantly improved C2C12 myoblast
differentiation into myotubes by increasing the MyHC expression. In addition, these compounds
Molecules 2018, 23, 2023 12 of 14
significantly increased the glucose uptake and ATP production in myotubes through the
upregulation of the mitochondrial biogenesis-regulating factors PGC1α, NRF-1, TFAM, and Sirt-1
and activation of the AMPK/ACC signaling pathway. In particular, the effects of allantoin on
biogenesis were shown to be more pronounced than metformin. Our results suggest that yam and
allantoin can help prevent energy loss in muscle dysfunction and are applicable for use as natural
sources for food materials and medication for the prevention of sarcopenia and treatment.
Author Contributions: Y.-K.P. and H.W.J. designed the study. J.M., S.Y.K., J.H.P. and H.W.J. performed the
experiment and conducted statistical analysis. J.M. and H.W.J. wrote the manuscript. All authors revised the
manuscript and approved the final version.
Funding: This research was supported by the Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education (No. 2016R1D1A1B04935601).
Conflicts of Interest: The authors no conflict of interest.
Abbreviations
ACC
Acetyl-CoA carboxylase
AMPK
AMP-activated protein kinase
BSA
Bovine serum albumin
DMEM
Dulbecco’s modified Eagle’s medium
FBS
Fetal bovine serum
HRP
Horseradish peroxidase
HS
Horse serum
MRFs
Myogenic regulatory factors
Myf5
Myogenic factor 5
MyoD
Myoblast determination protein
MyHC
Myosin heavy chain
NRF-1
Nuclear respiratory factor-1
RT-PCR
Reverse transcriptase-polymerase chain reaction
PAGE
Polyacrylamide gel electrophoresis
PBS
Phosphate buffered saline
PGC1α
Peroxisome proliferator-activated receptor gamma coactivator
RIPA
Radioimmunoprecipitation assay
Sirt-1
Sirtuin 1
TBST
Tris-buffered saline containing 0.1% tween-20
TFAM
Transcription factor A, mitochondrial
HPLC
High Performance Liquid Chromatography
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Ethnopharmacological relevance TADIOS is a n herbal formulation prepared from a mixture of Taraxacum officinale (L.) Weber ex F.H.Wigg, Dioscorea batatas Decaisne and Schizonepeta tenuifolia (Benth.) Briquet. These plants have traditionally been used in Asia to treat a variety of respiratory diseases. A bulk of literature on traditional Korean medicine describe their activities and functions for respiratory problems. Therefore, we hypothesized that the combination of these plants might be effective in alleviating respiratory symptoms. Aim of the study In this study, we investigated whether TADIOS ameliorates LPS-induced acute lung injury via regulation of the Nrf2-HO-1 signaling pathway. Materials and Methods The LPS-induced acute lung injury mouse model was used to determine the anti-inflammatory and anti-oxidative stress effects of TADIOS. The amount of marker compounds contained in TADIOS was quantified using high-performance liquid chromatography (HPLC) analysis. The protein level of pro-inflammatory cytokines in culture supernatant was measured by ELISA. Changes in the RNA level of pro-inflammatory cytokines in mice lungs and RAW264.7 cells were measured by quantitative RT-PCR. The relative amounts of reactive oxygen species (ROS) were measured by DCF-DA assay. Western blot analysis was used to evaluate expression of cellular proteins. Effects of TADIOS on antioxidant responsive elements (AREs) were determined by luciferase assay. The severity of acute lung injury was evaluated by Hematoxylin & Eosin (H&E) staining. To test the effects of TADIOS on LPS-induced oxidative stress, myeloperoxidase (MPO) activity and the total antioxidant capacity were measured. Results TADIOS was prepared by extraction of a blend of these three plants by ethanol, and quality control was performed through quantification of marker compounds by HPLC and measurement of bioactivities using cell-based bioassays. In the murine macrophage cell line RAW264.7, TADIOS effectively suppressed the production of pro-inflammatory cytokines such as IL-6 and IL-1b, and also ROS induced by LPS. When RAW264.7 cells were transfected with a luciferase reporter plasmid containing nucleotide sequences for ARE, TADIOS treatment increased the level of relative luciferase units in a dose-dependent manner. In the LPS-induced acute lung injury mouse model, orally administered TADIOS alleviated lung damage and neutrophil infiltration induced by LPS. Consistent with the in vitro data, treatment with TADIOS inhibited the LPS-mediated expression of pro-inflammatory cytokines and oxidative stress, and activated the Nrf2-HO-1 axis. Conclusion Our data suggest the potential for TADIOS to be developed as a safe and effective therapeutics for the treatment of acute respiratory distress syndrome.
... Dioscorea Rhizome, the root of D. batatas Decaisne, is widely used as a traditional herbal medicine for the treatment of diabetes, hyperthyroidism, liver damage, neuropathy, and asthma [6][7][8]. Moreover, it has anti-tumor, anti-oxidant, antiinflammatory, and anti-osteoporotic immunomodulatory activities [9][10][11][12]. ...
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