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ETAS, an Enzyme-treated Asparagus Extract, Attenuates Amyloid
-Induced Cellular Disorder in PC12 Cells
Junetsu Ogasawaraa,*, Tomohiro Itob, Koji Wakameb, Kentaro Kitadateb, Takuya Sakuraia, Shogo Satoa,
Yoshinaga Ishibashia, Tetsuya Izawac, Kazuto Takahashid, Hitoshi Ishidad, Ichiro Takabatakea,e,
Takako Kizakia and Hideki Ohnoa
aDepartment of Molecular Predictive Medicine and Sport Science, Kyorin University, School of Medicine,
Tokyo 181-8611, Japan
bAmino Up Chemical Co., Ltd., Hokkaido 004-0839, Japan
cGraduate School of Health and Sports Sciences, Doshisha University, Kyoto 610-0394, Japan
dThird Department of Internal Medicine, Kyorin University, School of Medicine, Tokyo 181-8611, Japan
eCelelign Orthodontic Clinic, Tokyo 102-0083, Japan
junetsu@ks.kyorin-u.ac.jp.
Received: October 31st, 2013; Accepted: January 8th, 2014
One of the pathological characterizations of Alzheimer’s disease (AD) is the deposition of amyloid beta peptide (A) in cerebral cortical cells. The deposition
of Ain neuronal cells leads to an increase in the production of free radicals that are typified by reactive oxygen species (ROS), thereby inducing cell death.
A growing body of evidence now suggests that several plant-derived food ingredients are capable of scavenging ROS in mammalian cells. The purpose
of the present study was to investigate whether enzyme-treated asparagus extract (ETAS), which is rich in antioxidants, is one of these ingredients. The
pre-incubation of differentiated PC 12 cells with ETAS significantly recovered A-induced reduction of cell viability, which was accompanied by reduced
levels of ROS. These results suggest that ETAS may be one of the functional food ingredients with anti-oxidative capacity to help prevent AD.
Keywords: Alzheimer’s disease, Amyloid beta, ETAS, ROS, PC 12 cell.
Alzheimer’s disease (AD) is a chronic neurodegenerative disease,
which gives rise to dementia through impairments in learning,
memory, and thought in the elderly. The number of people who
suffer from AD worldwide is predicted to reach 100 million by the
year 2050 [1]. The establishment of new medical treatments for AD
will undoubtedly have a critical role in the improvement of quality
of life (QOL) for human beings. It is widely accepted that the
pathological characteristics of AD feature two states: abnormal
accumulation of amyloid beta (A) peptide and intracellular
phosphorylated tau-protein. In particular, A-induced neuronal cell
toxicity is known to be accompanied by an accumulation of
intracellular reactive oxygen species (ROS) [2-5], which leads to
apoptotic cell death [6,7]. Thus, finding materials that will
contribute to a reduction in A-mediated ROS accumulation in
neuronal cells would be beneficial in the prevention of AD-induced
cellular disorder. It is assumed that diet therapy would have fewer
side effects than pharmaceutical therapy. Therefore, a survey of
food ingredients that could inhibit AD-induced cell toxicity could
be valuable in establishing a new treatment directed at the
prevention of AD.
Growing evidence now suggests that plant-derived polyphenols
have a direct effect on the suppression of ROS production in
neuronal cells [8-10]. Indeed, epigallocatechin gallate (EGCG), a
major compound of green tea polyphenol, has demonstrated a
protective effect against nitric oxide-induced apoptosis in neuronal
PC12 cells [11]. Also, 5-hydroxymethyl-2-furfural, which is widely
available in plant foods, is now known as a natural antioxidant in
mammalian cells [12, 13]. These results suggest that plant-derived
food ingredients might be natural neuroprotective substances via
activation of the ROS scavenging system. Enzyme-treated asparagus
Figure 1: Effect of ETAS on A-induced cell death in differentiated PC 12 cells. Cell
viability was assayed by MTT assay, as described in the Experimental section. Each
value represents the mean ± SD (n = 8).* P < 0.05.
extract (ETAS), which can be isolated from the stem segment of
green asparagus, comprises 5-hydroxymethyl-2-furfural and/or its
derivative [14]. ETAS is thus expected to be an agent that has an
inhibitory effect on A-induced neurotoxicity through its anti-
oxidant capacity.
In previous studies, PC12 pheochromocytoma cells have been
widely used as in vitro research models on AD because of a
differential ability to act as neurocytes. Indeed, several
phytochemical constituents and food ingredients now are known to
attenuate cell cytotoxicity with or without Ain PC12 cells [15-20].
Thus, an investigation using PC12 cells as a neurocyte model would
be useful for the functional evaluation of ETAS on A-mediated
neuronal cell cytotoxicity. In addition, AD has been tightly linked to
the cortical deposition of fibrillar A plaques, which are mainly
composed of A1-42 peptide [21,22], which suggests that the
NPC Natural Product Communications 2014
Vol. 9
No. 4
561 - 564
562 Natural Product Communications Vol. 9 (4) 2014 Ogasawara et al.
Figure 2: Effect of ETAS on A-induced cellular disorder in differentiated PC 12 cells.
Cellular disorder was assayed according to the LDH released from cells to the
incubation medium, as described in the Experimental section. Each value represents the
mean ± SD (n = 8).* P < 0.05.
Figure 3: Effect of ETAS on the A-induced production of ROS in differentiated PC
12 cells. Levels of ROS were assayed by DCFH-DA assay, as described in the
Experimental section. Each value represents the mean ± SD (n = 8).* P < 0.05.
addition of A1-42 peptide would be a suitable AD model for
cultured cells. The present study was focused on evaluating the
preventive effect of ETAS on A1-42-induced cellular disorder in
differentiated PC12 cells.
As shown in Figure 1, the addition of A1-42 to differentiated
PC12 cells revealed a significant decrease in cell viability compared
with non-treated control cells. In contrast, no significant change in
cell viability was observed by the addition of ETAS alone to
differentiated PC12 cells, suggesting that ETAS is safe as a food
ingredient-derived pharmaceutical additive [14]. It is worth noting
that Apeptide-induced cell death in these cells was significantly
inhibited by pre-treatment with ETAS (Figure 1). On the basis of
these results, the rate of lactate dehydrogenase (LDH) release in the
cell culture medium, another cell viability marker, was also
measured to confirm the current observations. As expected, the
A1-42-induced increase in LDH release was significantly
attenuated by the pre-addition of ETAS in differentiated neuronal
PC12 cells (Figure 2). These results indicate that ETAS has the
ability to protect against A-mediated cell death and/or membrane
damage in neuronal PC12 cells. Moreover, as a relevant
mechanism(s), A1-42 peptide-mediated cellular toxicity
reportedly activates the intracellular ROS production system [23].
Indeed, in Figure 3, the pre-treatment of ETAS significantly
inhibited the A-induced production of intracellular ROS levels,
although the addition of A1-42 alone to neuronal PC12 cells
provoked a significant increase in intracellular ROS levels
compared with non-treated control cells. These results suggest that a
reduction in intracellular ROS levels by ETAS also might be
associated with the ETAS-induced inhibition of cellular disorder in
neuronal PC12 cells.
It is noteworthy that A1-42-induced cellular toxicity in neuronal
PC12 cells was prevented by pre-treatment with ETAS. In recent
studies of A-mediated dysfunctional cells, plant foods such as
yuzu, i.e., Citrus junos Tanaka [24], Allium [25], and Panax
notoginseng [26] have inhibited hippocampal A accumulation,
DNA fragmentation, and caspase-3 activity, respectively. It is
noteworthy that the application of plant food-derived ingredients
such as limonene isolated from yuzu peel [24], aqueous extract of
Allium sativum [27], and water and ethanol extract of Panax
notoginseng [28] has been used to attenuate significantly
intracellular ROS levels, thereby improving the scavenging action
of oxidative stress in several cytotoxic cells. Moreover, 5-hydroxy-
methyl-2-furfural, which is a common Maillard reaction product
that is comprised of ETAS [14], has shown an anti-oxidant effect on
several types of cells [13, 29]. Consequently, in the present study,
plant food and its ingredient-mediated suppression of oxidative
stress would play a key role in the attenuation of cell viability by
the addition of A peptide, because cell death triggered by Ais
known to be accompanied by intracellular ROS production in PC12
cells [30, 31]. This concept has been supported by our results
whereby alterations in LDH releases are synchronized with the
generation of ROS in neuronal PC12 cells (Figures 2 and 3).
On the other hand, it is difficult to elucidate the mechanism
underlying the ETAS-induced suppression of ROS. However, it
may be logical to speculate that the attenuation of ROS by ETAS
occurred via the inhibitory action of a ROS production enzyme,
because 5-hydroxymethyl-2-furfural is known to act as an
inhibitor of xanthine oxidase, an oxidative enzyme [32]. Moreover,
5-hydroxymethyl-2-furfural is also known to increase the
expression of anti-oxidative enzymes glutathione peroxidase and
superoxide dismutase on the gene level [27]. Thus, the coordinated
effect of ETAS on a decrease in the excessive production of ROS,
and/or an increase in the removal of defective ROS, could be
associated with a reduction in ROS, which is upregulated by the
addition of A-peptide in neuronal PC12 cells.
In conclusion, ETAS has the capacity to prevent A-induced
cellular disorder via the suppression of intracellular ROS
production. Thus, ETAS is expected to prevent AD through its anti-
cytotoxic capacity.
Experimental
ETAS, an enzyme-treated asparagus extract: The safety of ETAS
as a pharmacological additive has been confirmed [14]. ETAS is
composed of a mixture of 5-hydroxymethyl-2-furfural and its
derivative, which is named asfural [14]. ETAS was dissolved in
DMSO, and freshly prepared solutions were used for each
experiment. ETAS is commercially available (Amino Up Chemical
Co., Ltd., Sapporo Japan).
Materials: PC12 cells were purchased from RIKEN BRC CELL
BANK (RIKEN, Tsukuba, Japan), A 1-42 from Anaspec
(Fremont, CA), DMEM medium and 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) from Sigma (St.Louis,
MO), dichlorofluorescin diacetate (DCFHDA) from Invitrogen
(Carlsbad, CA), horse serum from Gibco (BRL, Palo Alto, CA),
fetal bovine serum from MP Biomedicals, LLC (Solon, OH), and
the assay kit for LDH from Promega (Madison, WI). All other
chemicals and reagents were from Sigma.
Cell culture and assay procedures: PC12 cells were cultured with
growth medium that consisted of DMEM with 10% horse serum,
5% calf serum, 50 unit/mL penicillin and 100 mg/mL streptomycin.
Inhibitory effect of ETAS on amyloid beta-induced cellular disorder Natural Product Communications Vol. 9 (4) 2014 563
The cells (5×104 cells) were seeded in a 96-well plate and
incubated at 37°C for 24 h. Thereafter, the induction of
differentiation in the PC 12 cells was maintained at 37°C for 72 h
by adding 50 ng/mL of nerve growth factor. The differentiated
neuronal PC 12 cells were divided into 4 groups: Control, ETAS,
A and ETAS/A. For control, the neuronal PC 12 cells were
incubated with DMSO (0.1%, v/v) at 37°C for 48 h. For ETAS or
A, the neuronal PC 12 cells were incubated with DMSO (0.1%,
v/v) at 37°C for 24 h following incubation with ETAS (2 mg/ mL)
or A (50 M) for another 24 h, respectively. For ETAS/Athe
neuronal PC 12 cells were pre-incubated with ETAS (2 mg/ mL) for
24 h following incubation with A (50 M) for another 24 h. After
that, samples were prepared and used for each assay, as described
below.
Cell viability assay: Cell viability was measured by quantitative
colorimetric assay with MTT [33]. The MTT solution, at a final
concentration of 500 μg/mL /well, was added and cells were
incubated at 37°C for 4 h. Supernatants were then removed by
aspiration. The reaction was stopped by adding DMSO. The optical
density of each well was determined at a wavelength of 570 nm
using a microplate reader.
LDH release assay: To further determine cell viability, the levels of
extracellular LDH in the cell culture medium were measured using
an LDH assay kit according to the manufacture’s protocol. The
extracellular LDH released to the medium was represented as a
percentage of total intracellular LDH contents. The total amount of
intracellular LDH was determined by solubilizing the cells with
0.2% triton X-100.
Measurement of free radicals: The levels of intracellular ROS
were measured by the alteration in DCF fluorescent intensity. After
the medium was removed, the cells were incubated with 10 M
DCFH-DA at 37°C for 30 min. The cells were washed 3 times with
PBS to remove the extracellular DCFH-DA following re-suspension
in PBS. The optical density of each well was determined at a
wavelength of 490 nm and an emission wavelength of 540 nm using
a microplate reader.
Statistical analysis: Values are expressed as the mean ± S.D. An
analysis of variance was performed to establish that there were
significant differences between the groups, and then the
significance of the differences between the mean values was
assessed using Scheffé’s test. A P-value of < 0.05 was regarded as
significant.
Acknowledgments - This study was supported in part by a Grant-
in-Aid for Scientific Research from the Japan Ministry of
Education, Culture, Sports, Science and Technology. All authors
declare that there are no conflicts of interest between themselves
and Amino Up Chemical. Co., Ltd.
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