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586
doi: 10.4103/2221-1691.248095 ©2018 by the Asian Pacific Journal of Tropical Biomedicine.
Anti-cancer and anti-inflammatory activities of aronia (Aronia melanocarpa) leaves
Nhuan Do Thi, Eun-Sun Hwang
Department of Nutrition and Culinary Science, Hankyong National University, 327 Chungang-ro, Anseong-si, Gyeonggi-do, 456-749, Korea
ART ICL E I NFO ABS TRA CT
Article history:
Received 6 October 2018
Revision 23 October 2018
Accepted 12 December 2018
Available online 26 December 2018
Keywords:
Aronia leaf
Cancer
Metatasis
Lipopolysaccharides-induced
Inflammation
Corresponding author: Eun-Sun Hwang, Department of Nutrition and Culinary
Science, Hankyong National University, 327 Chungang-ro, Anseong-si, Gyeonggi-do,
456-749, Korea.
Tel: +82-31-670-5182
Fax: +82-31-670-5187
E-mail: ehwang@hknu.ac.kr
Foundation project: This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF-2016R1A2B4014977).
1. Introduction
Aronia (Aronia melanocarpa) was grown in Poland, North
America, and Europe, but now it is cultivated worldwide, including
Korea[1]. Aronia contains many health-promoting substances such
as anthocyanins, proanthocyanidins, vitamin C, pectins, tannins,
flavonoids, and phenolic acids[2,3]. Aronia is also known as ‘black
chokeberry’ and is considered by many to be the healthiest fruit in
the world, offering more health benefits than any other superfood[4].
Aronia fruits have been reported to have antioxidant, anticancer,
hypoglycemic, hypolipidemic, hypotensive, anti-inflammatory and
antibacterial properties[3-7].
Objective: To determine the anti-cancer effect of aronia leaf extract on SK-Hep1 cells
using migration, metallo metrix proteinase-2/-9 (MMP-2/-9) and MT-1 MMP expression
and to evaluate the anti-inflammatory activities of the leaf extract. Methods: The effect of
aronia leaf extract on cancer prevention was investigated. SK-Hep1 human liver cancer cell
line was treated with aronia leaf extract at various concentractions. MTT assay was used to
measure cancer cell growth inhibition, and wound migration assay was used for metastasis
determination. The expression of MMP-2/-9 was measured at the protein level using
zymography and the expression of MMP-2/-9 and MT-1 MMP was examined at the gene level
by RT-PCR. Raw 264.7 macrophage cells were stimulated with lipopolysaccharides to induce
inflammation, and then the inhibition of inflammation was evaluated by treatment of aronia
leaf extract. Expressions of interleukin-6, tumor factor-毩, and nitric oxide (NO) were also
determined. Results: SK-Hep1 cell growth was inhibited in proportion to the concentration of
aronia leaf extract. In migration assay, aronia leaf extract showed 61.3%-96.3% wound size
inhibtion after treating 50-200 µg/mL of aronia leaf extract for 24 h. At the protein level, the
expression of MMP-2 and MMP-9 decreased as the concentration of aronia leaf extract treated
with SK-Hep1 cells increased. In addition, the same pattern as in the protein was also observed
in the mRNA levels. The expressions of MMP-2 and MMP-9 protein were inhibited by 92.2%
and 53.8%, respectively after treatment with 200 µg/mL aronia leaf extract. In addition,
Raw 264.7 cells treated with aronia leaf extract did not affect cell survival. There was dose
dependent inhibition of interleukine-6, tumor necrosis factor-毩 and nitric oxide after treating
aronia leaf extract in lipopolysaccharides-treated Raw 264.7 cell. Conclusions: The results
show that aronia leaf has anticancer and and antimetastatic properties in SK-Hep1 and Raw
264.7 cells.
Asian Pacific Journal of Tropical Biomedicine 2018; 8(12): 586-592
Asian Pacific Journal of Tropical Biomedicine
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How to cite this article: Thi ND, Hwang ES. Anti-cancer and anti-inflammatory
activities of aronia (Aronia melanocarpa) leaves. Asian Pac J Trop Biomed 2018;
8(12): 586-592.
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Nhuan Do Thi et al./ Asian Pacific Journal of Tropical Biomedicine 2018; 8(12): 586-592
Aronia leaves have been reported to contain a variety of
physiologically active substances such as chlorogenic acid, its
isomers, caffeic acid, quercetin 3-O-glucopyranoside and rutin[4].
In particular, aronia young leaves contain a variety of phenolic
compounds, such as caffeic acid, neochlorogenic acid and
chlorogenic acid, compared to old leaves[8]. In some published
studies, there is evidence that leaves of plants have physiological
potency over fruit[9]. Denev et al.[10] reported aronia leaf extract
contains a combination of antioxidant and strongly inhibited peroxyl
radical formation in vitro with the same order of that for 毩-tocophenol
and significantly reduced CCl4-induced hepatic lipid peroxidation in
vivo. In addition, streptozotocin was administered to rats to induce
diabetes mellitus, and then it was confirmed that blood glucose was
decreased at a significant level when the exract of aronia leaf was
consumed[11]. Aronia leaves have been studied and reported to be
effective in the prevention and treatment of cancer, leukemia and
other chronic diseases[9]. Aronia leaf extract is known to be involved
in the death of human leukemia cell line, HL60 cell and human
promyelocytic leukemia cells, which is thought to be due to the
action of polyphenol compounds contained in the extract of aronia
leaf[9].
Inflammation, which occurs as a tissue damage or as an early
symptom of infection, initially occurs locally, but spreads to the
surroundings over time[12]. The immune response is promoted
by bacterial products and substances such as endogenous toxins
or lipopolysaccharide. Previously published studies have shown
that MMPs act as regulators in both increasing or decreasing
inflammation[13]. In general, MMPs are known to increase in
diseases where tissue damage or inflammation occurs, and to break
down proteins and regulate cytokine/chemokine activity in the
body. There is increasing evidence that it works in inflammation to
regulate the inflow of white blood cells[13-15]. Epidemiologic studies
show that many cancers such as lung, stomach, colon, bladder
cancers are associated with microbial infection and these cancers
and inflammation are related by epidemiology, histopathology,
inflammatory profiles[16-18]. To study the inflammatory response,
Raw 264.7 cells are generally treated with lipopolysaccharides (LPS)
known to cause inflammation, which is widely used and evaluated
as the most reliable method to date. LPS, called endotoxin, is a
macromolecule composed of lipids and polysaccharides. Raw 264.7
cells are able to predict the extent of inflammation when stimulated
with nitric oxide (NO) synthase secretion by measuring the amount
of NO produced at that time[19,20].
In this study, the effect of aronia leaf extract on cancer cell
metastasis was investigated by measuring the degree of inhibition
of cancer cell migration and MMP-2/-9 and MT-1 MMP expression
after treatment of aronia leaf extract in SK-Hep1 cancer cells. Raw
264.7 cells were treated with LPS to induce inflammation, and
then treated with different concentrations of aronia leaf extract.
The amount of inflammation was measured to evaluate the anti-
inflammatory effect of aronia leaf.
2. Materials and methods
2.1. Chemicals
Cell culture supplies and chemicals were purchased from Sigma
Chemical (St. Louis, MO, USA) and Gibco Life Technologies, Inc.
(Paisley, UK). Organic solvents were purchased from Burdick &
Jackson (Batavia, IL, USA).
2.2. Cell culture
SK-Hep1 human hepatocellular carcinoma cells and Raw 264.7
murine cells were obtained from the Korean Cell Line Bank (Seoul,
South Korea). Cells were cultured in a 37 曟 incubator containing
5% CO2. The cell culture medium, Dulbeco’s Modified Eagle’s
Media, was supplemeted with 10% fetal bovine serum and penicillin/
streptomycin.
2.3. Aronia leaf extraction
Aronia leaves were used in the experiment by receiving leaves of
‘Nero’ varieties harvested in June at aroina farm in Korea. The leaves
were washed in flowing water, frozen at -80 曟 for 24 h, then placed
in a freeze dryer and dried for 2 d. The dried sample was put into a
food grinder and made into a fine powder and stored at -80 曟. The
samples were extracted as follows; The powder sample (5 g) was
mixed with 125 mL of 80% ethanol and shaken for 2 h in a 85 曟
water bath to extract the bioactive substances. This procedure was
repeated three times. The supernatant was collected separately from
the mixture and filtered through Whatman #2 filter paper under the
vaccum. The extracted and filtered samples were evaporated under
the vacuum at 40 曟 using a rotary evaporator. After evaporation,
50 mL of water was added to the sample to completely dissolve the
sample. The sample was then lyophilized to prepare powder, which
was then stored at -20 曟 for subsequent experiments.
2.4. MTT assay
MTT assay was used to determine proliferation of SK-Hep1 and
Raw 264.7 cells. SK-Hep1 cells were grown in 96-well plate at a
concentration of 1 伊 105 and Raw 264.7 cells at a density of 5 伊 103
cells per 200 µL of medium. Cells were cultured for 2 h, then the
medium in the 96-well plate was removed and replaced with 100 µL
of fresh medium containing aronia leaf extract diluted to different
concentrations (0-400 µg/mL). The cell viability was determined
after 24 or 48 hours of incubation by the method of Hwang and
Lee[21].
2.5. Wound migration assay
SK-Hep1 cells were treated with 50-200 µg/mL of aronia leaf
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588 Nhuan Do Thi et al./ Asian Pacific Journal of Tropical Biomedicine 2018; 8(12): 586-592
extract and cultured for 12-48 h to observe the migration rate of
cancer cells. The cells were incubated in a 6-well plate, and then a
line was drawn in the middle of the well with a thin plastic pipette
tip to induce the wound, and the migration of the attached cells was
observed under a microscope. The wound migration distance by the
effect of aronia leaf extract was measured by the method of Thi and
Hwang[7].
2.6. Determination of MMP-2/-9 expression with
zymography
SK-Hep1 cells were treated with aronia leaf extract at various
concentrations of 50-400 µg/mL and cultured for 48 h in serum-free
DMEM. After 48 hours of culture, the medium was collected and the
protein content contained in the medium was measured. The MMP-2
and MMP-9 proteins were separated using sodium dodecyl sulfate
(SDS) gel electrophoresis in a 10% polyacrylamide gel. The MMP-2
and MMP-9 levels were determined by the method of Hwang and
Lee[21].
2.7. Determination of MMP-2/-9 and MT-1 MMP with
RT-PCR
SK-Hep1 cells were treated with aronia leaf extract at various
concentrations of 50-200 µg/mL and cultured for 24 h in DMEM.
Total RNA was isolated using TRIzol reagent, RNA purity and
concentration were measured, and cDNA was synthesized after
mixing with oligo-dT primer. After denaturation at 95 曟 for 2 min,
PCR was carried out for 30 cycles of 3 steps at 92 曟 for 1 min,
55 曟 for 60 s, and 73 曟 for 1 min, and the final extension was
carried out at 73 曟 for 10 min. The amplified PCR product was
electrophoresed on 1.5% agarose gel and the amount of expressed
MT1-MMP and MMP-2/-9 messenger RNA (mRNA) was
quantitated by the method of Hwang and Lee[21].
2.8. NO determination
In a 96-well plate, Raw 264.7 cells (at a concentration of 5 伊 105 cell/
well) were plated and stabilized. After the medium was removed and
LPS was dissolved in fresh medium at a concentration of 2 mg/mL,
100 µL was added to each well. After 1 hour of incubation, different
concentrations of aronia leaf extract (50-200 µg/mL) were added.
Then, the media were collected and used for NO determination after
12 hours of incubation. Since NO production is directly correlated
with the amount of nitrite concentration in the medium, the amount
of nitrite was calculated by Griess reaction in this experiment. After
adding 50 µL of the Griess reagent to 50 µL of the collected medium
and incubating at room temperature for 15 min, the absorbance was
measured at 540 nm. Sodium nitrite (NaNO2) standard solution was
diluted by different concentrations to make a standard curve, and the
NO content in each sample was calculated.
2.9. Interleukin 6 (IL-6) and tumor necrosis factor (TNF-
毩
)
measurement
IL-6 and TNF-毩 were measured after LPS treated Raw 264.7 cells
were cultured and incubated for a certain period of time with different
concentrations of aronia leaf exteact. Cells treated with different
concentrations of aronia leaf extract were mixed with 100 µL of capture
antibody solution, reacted at 4 曟 for 18 h, and then 100 µL of mixed
samples were added to a 96-well plate. The samples were incubated
for 2 h at room temperature with 100 rpm shaing device, 50 µL of
detection antibody solution was added and incubated another 1 h at the
same speed of shaker. To the diluted antibody solution, 50 µL of diluted
Avidin-HRP solution was added and reacted at room temperature
for 30 min. Then, 50 µL of TMB substrate solution was added and
incubated for 20-30 min while shaking at 100 rpm in a dark place.
The stop soluation of 50 µL was added to terminate the reaction. IL-6
and TNF-毩 conents were determined using ELISA reader at 570 nm
and 450 nm, and the the amount of IL-6 and TNF-毩 contained in the
samples was calculated based on the standard curve.
2.10. Statistical analysis
All measurements were expressed as mean 依 SD and statistical
analysis was performed using the SPSS software package (Ver. 17).
All results were compared using one-way ANOVA analysis and
significance was shown at the P < 0.05 level.
3. Results
3.1. Effect of aronia leaf extract on SK-Hep1 cell proliferative
activity
To investigate the effect of aronia leaf on SK-Hep1 cell proliferation,
the exract was diluted to various concentrations (0-400 µg/mL) and
cultured for 24 or 48 h. Aronia leaf extract significantly inhibited SK-
Hep1 human hepatoma cancer cell growth in both dose- and time-
dependent manner (Figure 1). SK-Hep1 cancer cell growth was
significantly reduced by 4.4%-27.6% after treating 12.5-400 µg/mL
of aronia leaf extract for 24 h compared with the control. Similar to the
results of 24 h, SK-Hep1 cell proliferation decreased with increasing
concentration of aronia leaf extract even in the samples cultured for 48
h. As the concentration of aroina leaf extract increased from 12.5 to
400 µg/mL, the proliferation of cancer cells decreased by 8.9%-50.4%.
The growth of SK-Hep1 cells was reduced by 13.4% and 18.7%,
respectively, when 100 and 200 µg/mL of aronia leaf extract were
added for 24 h. In addition, when SK-Hep1 cells were cultured for 48
h under the same conditions, the numbers of cells were decreased by
29.7% and 39.8%, respectively, compared to the control.
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Figure 1. Effect of aronia leaf extract on cell viability in SK-Hep1 human
hepatoma cells.
3.2. Effect of aronia leaf extract on migration
As shown in Figure 2, the migration distance of cancer cells was
shorter as the concentration of aronia leaf extract was higher and the
incubation time was longer. There was dose dependent inhibition
of migration distance with 70.6%-98.4% wound size inhibtion after
treating 50-200 µg/mL of aronia leaf extract for 12 h. In the case of
treatement with 100 µg/mL aronia leaf extract, the mobility of SK-
Hep1 cells was controlled in proportion to the treated time, and the
wound size in cells cultured for 12, 24 and 48 h were 91.5%, 89.6%
and 76.3%. As the treated time of aronia leaf extreact increased, the
cells recovered mobility.
3.3. Effect of aronia leaf extract on MMP
As shown in Figure 3, cell culture media were collected and
separated the MMP-2 and MMP-9 protein with gelatin zymography.
The quantification of MMP-2 and MMP-9 by gelatin gel staining
and de-staining revealed that SK-Hep1 cells secreted the largest
amount of MMP-2 and MMP-9 in the control group without aronia
leaf extract. After 48 h, aronia leaf extract treatment at 50 and
400 µg/mL decreased MMP-9 expression by 15.5% and 75.8%,
respectively. Aronia leaf extract at 50 and 400 µg/mL decreased
MMP-2 expression by 37.7% and 100.0%, respectively and aronia
leaf extract at 400 µg/mL clearly suppressed MMP-2 activity. These
results suggested that the expression of MMP-2 and MMP-9 protein
in SK-Hep1 cells was inhibited by higher concentration of aronia
leaf extract.
Figure 3. Effect of aronia leaf extract on MMP-2/-9 expression in SK-Hep1
human hepatoma cells.
0 h
12 h
24 h
48 h
0 50 100 200
Aronia leaf extract (µg/mL)
Control
50 µg/mL
100 µg/mL
200 µg/mL
0 12 24 48
Time (h)
120
100
80
60
40
20
0
Wound size (% of control)
Figure 2. Effect of aronia leaf extract on wound healing migration of SK-Hep1 human hepatoma cells.
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590 Nhuan Do Thi et al./ Asian Pacific Journal of Tropical Biomedicine 2018; 8(12): 586-592
3.4. Effect of aronia leaf extract on mRNA MMP and MT1-
MMP
The mRNA expression of MT1-MMP, MMP-2, and MMP-9
decreased as the concentration of aronia leaf extract increased
(Figure 4). The MMP-9 mRNA expression was reduced by 34.7%
and 63.3%, respectively, in samples treated with 50 and 100 µg/mL
of aronia leaf extracts compared to the control. The MMP-2 mRNA
expression was inhibited by 32.4% and 50.0%, respectively, in the
same concentration of aronia leaf treatment. When 200 µg/mL of
aronia leaf extract was treated, the MMP-2 mRNA expression was
completely inhibited. The expression of MMP-9 mRNA in leaf
extracts was much higher than that of MMP-2 mRNA. The MT-1
MMP expression was reduced by 5.3%, 15.8% and 31.6% compared
to the control group after SK-Hep1 cells were treated with 50, 100,
and 200 µg/mL of aronia leaf extracts for 24 h.
3.5. Effect of aronia leaf extract on Raw 264.7 cell
proliferation
The degree of inhibition of Raw 264.7 cell proliferation was
measured (Figure 5). The concentration of aronia leaf extract up to
400 µg/mL used in this experiment did not increase or inhibit the
survial of Raw 264.7 cells.
3.6. Effects of aronia leaf extract on NO production
Raw 264.7 cells with LPS (1 µg/mL) stimulation were treated with
aronia leaf extract (50-200 µg/mL) and cultured for 12 h to determine
NO content. NO production that was in control and LPS treated
without aronia leaf extract was 30.3 and 96.6 µM, respectively. It
appeared that aronia leaf extract had potential to inhibit LPS-induced
production of NO compared to the LPS-treated control in Raw 264.7
cells. Aronia leaf extract at 50, 100, and 200 µg/mL suppressed the
production of NO to 85.2, 70.5, and 58.2 µM, respectively in a dose
dependent manner.
3.7. TNF-
毩
and IL-6 measurement
The Raw 264.7 cells were inoculated with aronia leaf extract of
0-200 µg/mL and cultured for 12 h. As the concentration of aronia
leaf extract increased, TNF-毩and IL-6 expression decreased,
especially IL-6. These expressions showed aronia leaf extract
inhibited the proinflammatory mediators in a dose dependent manner
(Figure 6).
TNF-毩 production that was in control and LPS treated without
aronia leaf extract was 260.4 and 1 300.0 µg/mL, respectively. The
results also demonstrated that aronia leaf extract had potential to
inhibit LPS-induced IL-6 production compared to the LPS-treated
control in Raw 264.7 cells. Aronia leaf extract at 50, 100 and 200
µg/mL suppressed dose dependently the production of TNF-毩 by
10.0%, 20.6%, and 28.4%, respectively compared to LPS-treated
control without aronia leaf extract.
IL-6 production that was in control and LPS treated without aronia
leaf extract was 296.3 and 9 810.3 µg/mL, respectively. As shown
in Figure 6, aronia leaf extract had potential to inhibit LPS-induced
IL-6 production compared to the LPS-treated control in Raw 264.7
cells. Aronia leaf extract at 50, 100 and 200 µg/mL suppressed dose
dependently the production of IL-6 by 20.1%, 32.9%, and 87.3%,
respectively compared to LPS-treated control without aronia leaf
extract.
0 50 100 200
Aronia leaf extract (µg/mL)
GAPDH
MMP-9
MMP-2
MT-1 MMP
0 50 100 200
0 50 100 200
0 50 100 200
Concentration (µg/mL)
Concentration (µg/mL)
Concentration (µg/mL)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.4
0.3
0.2
0.1
0.0
0.24
0.20
0.16
0.12
0.08
0.04
0.00
MMP-9/GAPDH
MMP-2/GAPDH
MT-1 MMP/GAPDH
Figure 4. Effect of aronia leaf extract on MMP-2/-9 and MT-1 MMP gene
expression in SK-Hep1 human hepatoma cells.
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Nhuan Do Thi et al./ Asian Pacific Journal of Tropical Biomedicine 2018; 8(12): 586-592
Figure 5. Effect of aronia leaf extract on the cytotoxicity of Raw 264.7
macrophage cells.
Figure 6. Effect of aronia leaf extract on the level of TNF-毩 and IL-6 in
LPS-stimulated Raw 264.7 macrophage cells.
a-eValues with different superscript letters within the same column are
significantly different at P <0.05.
4. Discussion
The anticancer activity of aronia leaf extract was investigated by
the multiple cellular mechanism assays. The extract of aronia leaf
inhibited SK-Hep1 human hepatoma cell growth and metastasis of
cancer cells in a dose dependent manner. It has been reported that
the growth and migration of cancer cells are very important in the
metastasis of cancer, and thus, the effect of aronia leaf extract on
SK-Hep1 cancer cell migration was examined. The mechanism of
antitumor effect was investigated by measuring the effect of aronia
leaf extract on the expression of MMP-2 and MMP-9, which are
one of the matrix malloproteinases and are reported to be directly
involved in invasion and metastasis of cancer cells[22]. Among the
proteolytic enzymes, MMP-2/-9 are the most important enzymes
involved in the degradation of basic membranes, and thus they are
the enzymes directly related to the invasion and metastasis of cancer
cells[20,23]. When SK-Hep1 cells were treated with 200 µg/mL of
aronia leaf extract for 24 h, it was confirmed that up to 96.3% of
the cells inhibited migration to the wound site. The results of this
study demonstrated that aronia leaf extract inhibited extremely cell
migration.
The potential anti-inflammatory properties of aronia leaf extract
were also investigated. Aronia leaf extract inhibited NO production
in a dose-dependent manner in Raw 264.7 cells induced by LPS. In
addition, in the Raw 264.7 cell line stimulated with LPS, the aronia
leaf extract decreased the expression of TNF–毩and IL-6, which
are secreted before inflammation, in proportion to the treatment
concentration of aronia leaf extract. TNF-毩 and IL-6 are among the
most important cytokines released by activated macrophages. During
inflammatory response, TNF-毩induces the expression of IL-6
together. Similar to the physiological function of TNF-毩, IL-6 is one
of the major cytokines and is induced by a variety of stimuli including
LPS. Also, IL-6 is one of the major initiators of acute response and
plays an important role in the immune response to inhibit chronic
inflammation. Studies have shown that IL-6 level is increased in a
variety of inflammatory diseases such as arthritis, Crohn’s disease,
and systemic lupus erythematosus[24,25]. LPS activiates a wide
variety of transcription factors and induces a number of genes that
are expressed during inflammation[26]. IL-6 induces inflammation
and is actually found in serum and tumors of cancer cells and cancer
patients[18]. IL-6 has the ability to promote tumors and, in fact, it is
known that the increase in IL-6 expression is related to the growth
of aggressive tumor cells in most cancers[27,28]. Therefore, inhibition
of cancer cell growth and progression is necessary to discover and
develop a substance that inhibits IL-6 or IL-6 receptor[18]. Our results
indicate that aronia leaf extract inhibits growth and inflammation
of cancer cells. These suggest that aronia leaf can be used as a
candidate for the development of cancer and inflammation inhibitory
food supplement or drug.
Conflict of interest statement
All authors declare that there is no conflict of interest.
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592 Nhuan Do Thi et al./ Asian Pacific Journal of Tropical Biomedicine 2018; 8(12): 586-592
Funding
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF-
2016R1A2B4014977).
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