Kenaf seed oil from supercritical carbon dioxide fluid extraction induced G1 phase cell cycle arrest and apoptosis in leukemia cells

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African Journal of Biotechnology Vol. 10(27), pp. 5389-5397, 15 June, 2011
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 ©2011 Academic Journals





Full Length Research Paper

Kenaf seed oil from supercritical carbon dioxide fluid
extraction induced G1 phase cell cycle arrest and
apoptosis in leukemia cells

Jhi Biau Foo1, Latifah Saiful Yazan1,2*, Kim Wei Chan1, Paridah Md. Tahir3 and Maznah
Ismail1,2

1Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor
Darul Ehsan, Malaysia.
2Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor Darul Ehsan, Malaysia.
3Institute of Tropical Forestry and Forest Products (INTROP),
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia.

Accepted 11 April, 2011

This study was designed to determine the cytotoxic effects of kenaf seed oil (Hibiscus cannabinus)
variety V36 extracted using supercritical carbon dioxide fluid extraction (SFE) with different
combinations of pressure (bars) and temperature (° C). Extracted oils were tested on human
promyelocytic HL-60, murine myelomonocytic WEHI-3B and human chronic myelogenous K562
leukemic cell lines. The yield of kenaf seed oil extracted by SFE ranged from 11 to 13% (w/w). Oils were
found to be cytotoxic towards all the leukemia cell lines in a dose-dependent manner with no effects on
normal cells (3T3). Oil from SFE at 600 bar 40° C (V600/40) was more cytotoxic towards HL-60, WEHI-3B
and K562 when compared with other kenaf seed oils (extracted with different parameters) with the IC50
values of 178.78±10.52, 189.43±11.63 and 213.33±15.45 µg/ml, respectively. V600/40-treated leukemia
cells exhibited typical characteristics of apoptosis such as nuclear fragmentation, chromatin
condensation, nuclear margination, membrane blebbing and cellular shrinkage, as viewed under
inverted light microscope and fluorescence microscope. Cell cycle analysis using flow cytometry
revealed that, V600/40 induced G1 phase cell cycle arrest and significantly increased (P < 0.05) the sub-
G1 apoptotic population in the leukemia cells. In conclusion, kenaf seed oil V600/40 induced apoptosis
via G1 phase cell cycle arrest in HL-60, WEHI-3B and K562 leukemia cell lines.

Key words: Kenaf (Hibiscus cannabinus), supercritical carbon dioxide fluid extraction (SFE), leukemia,
cytotoxicity, apoptosis, cell cycle arrest.


INTRODUCTION

Leukemia is a group of heterogeneous neoplastic dis-
order of white blood cells that multiply uncontrollably and
unable to differentiate into mature cells (Lee et al., 2007).
As of 2010, leukemia is diagnosed 10 times more often in
adults than in children and more common in males than
females (American Cancer Society, 2010). People with



*Corresponding author. E-mail: latifah@medic.upm.edu.my. Tel:
+603-89472308. Fax: +603-89436178.
leukemia have many options of treatment such as
chemotherapy (main treatment), antibiotic, blood trans-
fusion, radiation therapy and bone marrow transplantation.
Although, these treatments have prolonged the survival
rate for leukemia patients, the adverse effects of these
treatments are difficult to handle (Chiang et al., 2004).
Thus, there is a need to seek for other remedies in treat-
ing leukemia.
Plants are an excellent source of bioactive components
possessing a wide variety of biological activities and having
great potential therapeutic values (Chiu et al.,2006; Deng

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5390 Afr. J. Biotechnol.



et al., 2006). Crude extracts isolated from these plants
are important source to be developed as anticancer
agents and natural healthy foods for the management of
cancer (Lin et al., 2007). Kenaf (Hibiscus cannabinus) is
a valuable fibre and medicinal plant native to India and
Africa (Mohamed et al., 1995). Recently, it has gained an
important position in Malaysia as a potential plant to
replace tobacco (Mariod et al., 2010). Kenaf is com-
posed of various bioactive components including tannins,
saponins, polyphenolics, alkaloids, fatty acids, phospho-
lipids, tocopherol and phytosterols (Mohamed et al.,
1995). This plant has been reported to be anodyne,
aperitif, aphrodisiac, fattening, purgative, stomachic, and
has long been used in traditional medicine to treat bilious
conditions, bruises and fever (Agbor et al., 2005; Coetzee
et al., 2008; Kobaisy et al., 2001; Mohamed et al., 1995;
Nyam et al., 2009). However, not many studies have
been documented with regards to its anticancer properties,
in particular leukemia (Ghafar et al., 2010; Moujir et al.,
2007; Yazan et al., 2010). Our preliminary study
(unpublished data) showed that, kenaf seed oil was
cytotoxic towards human acute lymphoblastic leukemia
MOLT-4 cells; therefore, raising the possibility that kenaf
seed oil might have some cytotoxic effects towards
myelogenous leukemia cells.
Kenaf seed oil can be extracted conventionally by using
the organic solvents such as n-hexane or petroleum
ether. Nevertheless, the oil is always doubted for the
safety of consumption. Recently, the supercritical carbon
dioxide fluid extraction (SFE) technique for solid materials
was introduced. SFE is the non-toxic, non-explosive,
environmental friendly, cost effective, time saving and
selectivity-adjustable solvent (supercritical carbon dioxide
fluid) extraction (Araújo and Sandi, 2007; Vaquero et al.,
2006). SFE has been extensively studied for the sepa-
ration of bioactive compounds from herbs including kenaf
seeds (Chan and Ismail, 2009; Reverchon and De Marco,
2006). In this study, we investigated the cytotoxicity of
kenaf seed oil extracted by SFE towards myelogenous
leukemia cell lines.


MATERIALS AND METHODS

RPMI-1640 with L-glutamine, fetal bovine serum and penicillin–
streptomycin were purchased from PAA, Austria. Acridine orange
(AO), propidium iodide (PI), RNaseA and MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Sigma-
Aldrich, USA.


Kenaf seed oil extraction

Kenaf seeds variety V36 were purchased from the National
Tobacco Board, Pasir Putih, Kelantan, Malaysia. Kenaf seeds were
cleaned, soaked in water at ambient temperature for 24 h and dried
at constant temperature (50°C) overnight in an oven (FD 115,
Fisher Scientific, Germany). The final moisture content of the dried
seeds was less than 5%. Kenaf seeds were extracted by using
supercritical carbon dioxide extractor (Thar 1000 F, Thar




Technologies, Inc., USA) at different combinations of pressure and
temperature (pressure (bar)/temperature (° C):600/40; 600/60;
600/80) as reported previously (Yazan et al., 2010).


Cell culture

The human promyelocytic leukemia HL-60, murine myelomonocytic
leukemia WEHI-3B, human chronic myelogenous leukemia K562
and normal mouse embryonic fibroblast 3T3 cell lines were
purchased from the American type culture collection (ATCC, USA).
Cells were grown in RPMI 1640, supplemented with 10% of fetal
bovine serum and maintained in a humidified atmosphere of 5%
CO2 at 37° C.


Treatment

All the kenaf seed oils were dissolved in DMSO. Cells at the
concentration of 1 x 105 cells/ml were seeded in a 6-well flat-
bottomed tissue culture plate and treated with different
concentrations of kenaf seed oil ranging from 50 to 800 µg/ml for 72
h. The final concentration of DMSO in the treated wells was kept at
0.5%. DMSO only (0.5%, v/v) was included as the control.


Determination of cytotoxicity (MTT assay)

Following the treatment, cytotoxicity was evaluated using the MTT
assay. Briefly, 10 µl of MTT solution (5 mg/ml) with 50 µl of cell
suspension was added into a 96-well flat-bottomed tissue culture
plate and incubated at 37°C for 4 h. Next, 150 µl of Tris-DMSO
solution was added into each well (Hsu et al., 2005). The
absorbance which was proportional to cell viability was measured at
570 nm and a reference wavelength of 630 nm by using an ELISA
plate reader (Bio-Rad, USA). A graph of percentage of cell viability
versus concentration of kenaf seed oil was plotted and the
concentration that gave 50% inhibition of the cell viability (IC50)
when compared with the control was determined.


Morphological changes of cells treated with kenaf seed oil

The treated and untreated cells were viewed for morphological
changes, characteristic of apoptosis or necrosis under an inverted
light microscope (Olympus, USA).


Determination of mode of cell death

The cells were stained with 1 mg/ml of AO (Sigma-Aldrich, USA)
and 1 mg/ml of PI (Sigma-Aldrich, USA) at the ratio of 1:1 after
treatment with kenaf seed oil. The suspension was placed onto a
clean microscopic slide and viewed under a fluorescence
microscope (Leica, Germany) at 400X magnification.


Cell cycle analysis

The cells were harvested and washed twice with phosphate-
buffered saline (PBS), fixed in ice-cold 70% ethanol and incubated
at -20° C for 2 h. Prior to analysis, the cells were washed once again
with PBS, suspended in 425 µl of PBS, 25 µl of PI (1 mg/ml)
(Sigma-Aldrich, USA) and 50 µl of RNaseA (1 mg/ml) (Sigma-
Aldrich, USA) and incubated at 4°C for 20 min. DNA content was
analyzed by flow cytometer (CyAn ADP, USA) and the population of
cells in each cell-cycle phase was determined by using the submit
v3.4 software (CyAn ADP, USA).

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Foo et al. 5391



Table 1. Yield of kenaf seed oil extracted by SFE and Soxhlet.

Extraction technique
SFE 600/40
SFE 600/60
SFE 600/80
Soxhlet
Yield (%)
11.88 ± 0.63a
12.29 ± 0.52a
12.76 ± 0.46a
19.16 ± 0.72b

Values with the mean of three independent experiments ± SD. a
and b were significantly different (P < 0.05).





Figure 1. Effect of V600/40 on the viability of leukemia (HL-60, WEHI-3B and K562) and 3T3 normal cell lines
after 72 h as determined using the MTT assay. The oil was cytotoxic to the cells in a dose-dependent manner.
Each data point represents the mean of three independent experiments ± SD. *significantly different from the
control (P < 0.05).



Statistical analysis

Statistical analysis was performed using the statistical package for
Social Science (SPSS) version 16.0. Results were analyzed by
one-way analysis of variance (ANOVA). Data were expressed as
mean ± standard deviation (mean ± SD). A difference was conside-
red to be significant at P < 0.05.


RESULTS

Yield of kenaf seed oil

Table 1 shows the difference in the yield of kenaf seed oil
extracted by SFE and n-hexane (Soxhlet). The yield of
kenaf seed oil extracted by SFE ranged from 11 to 13%
(w/w). The yield slightly increased (p > 0.05) in corres-
pondence to the rise in extraction temperature. The yield
from Soxhlet extraction was significantly higher when
compared with SFE (P < 0.05).


Cytotoxicity of kenaf seed oil towards leukemia and
normal 3T3 cell lines

Kenaf seed oil from SFE was more cytotoxic than the one
from Soxhlet towards all the leukemia cell lines in a dose-
dependent manner (Figure 1). Increase in the tempe-
rature of SFE resulted in the increase of the IC50 values
of kenaf seed oil. Oil from SFE at 600 bar 40° C (V600/40)
was more cytotoxic towards HL-60, WEHI-3B and K562
when compared with others with the lowest IC50 values of
178.78±10.52, 189.43±11.63 and 213.33±15.45 µg/ml,
respectively (Table 2). K562 was the least sensitive
towards the extracts (oil). The IC50 value of kenaf seed oil

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5392 Afr. J. Biotechnol.



Table 2. IC50 values of kenaf seed oil variety V36 with different ways of extraction towards leukaemia (HL-
60, WEHI-3B and K562) and 3T3 normal cells after 72 h as determined by using MTT assay.

Cell line
IC50 (µg/ml)
V600/40
178.78 ± 10.52
189.43 ± 11.63
213.33 ± 15.45
>800
V600/60
320.48 ± 11.35
380.32 ± 15.21
472.34 ± 13.12
>800
V600/80
>800
>800
>800
>800
Soxhlet
>800
>800
>800
>800
HL-60
WEHI-3B
K562
3T3

Each data point represents the mean of three independent experiments ± SD.



against 3T3 normal cells was not obtained even at the
treatment of 800 µg/ml of the oil (Table 2). Since V600/40
was the most cytotoxic towards these leukemia cell lines,
further analyses were carried out using this extract.


Morphological changes of HL-60, WEHI-3B and K562
cell lines treated with V600/40

Number of HL-60, WEHI-3B and K562 cells reduced with
increase in the concentration of V600/40. Affected cells
showed some features of apoptosis such as cellular
shrinkage and membrane blebbing (Figure 2).


Fluorescence analysis of mode of cell death

Fluorescence analysis following staining with AO/PI
distinguished viable, apoptotic and necrotic cells. As shown
in Figure 3, the untreated control cells exhibited intact,
round and large green nuclei. The number of viable cells
reduced with increase in the concentration of V600/40.
Nucleus of the cells undergoing apoptosis was green but
fragmented. Other features of apoptosis were also noted
such as chromatin condensation and nuclear margination.
At the highest treatment of the oil (800 µg/ml), majority of
the cells were necrotic (red nucleus) (Figure 3).


Cell cycle analysis

DNA content (Figure 4) and changes in the cell cycle
distribution (Table 3) showed a dose-dependent accumu-
lation of the leukemia cells in the sub-G1 phase after
treatment with V600/40 for 72 h. Cell cycle arrest at the
G1 phase was observed in all the three leukemia cell
lines (Table 3).


DISCUSSION

SFE has been documented as a more favorable extrac-
tion technique due to the advantage over the use of liquid
solvents (Reverchon and De Marco, 2006). Even though
the yield of the oil extracted using the liquid solvent
(Soxhlet) was obviously higher (P < 0.05) (Table 1), the
products from SFE are still of preference because they
are free from any residues of the solvents since CO2 is
easily separated from the oil at the end of extraction
(Pourmortazavi and Hajimirsadeghi, 2007). Besides the
production of non-toxic products, SFE is also claimed to
be non-explosive, environmental friendly, cost effective,
time saving and selectivity-adjustable solvent in the
extraction (Reverchon and De Marco, 2006). SFE at 600
bar was chosen in this study due to higher oil yield when
compared with other pressures (200 and 400 bars) (Chan
and Ismail, 2009; Yazan et al., 2010). Nevertheless, the
yield obtained from our study was lower (by -5%) possibly
due to the differences in the variety, batch of the plant,
cultivation climate, ripening stage and harvesting time of
the seed (Nyam et al., 2009). From Table 1, it seems that
temperature influenced the yield of kenaf seed oil even
though the differences are not significant. For SFE,
pressure and temperature are the two important factors
that contribute to the yield of kenaf seed oil. Elevation in
pressure at certain temperature results in an increase in
the CO2 density, thus, enhancing solubility of the solutes
and increasing the yield. Temperature affects the volatility
of the solute. At constant pressure, the density of CO2
decreases with the increase in temperature, and becomes
more pronounced as the compressibility increases
(Pourmortazavi and Hajimirsadeghi, 2007). On the other
hand, increase in temperature will increase the vapor
pressure of analytes. Therefore, the tendency of
components to be extracted passing through the super-
critical fluid will increase (Reverchon and De Marco,
2006).
In this study, the IC50 was used as an index of
cytotoxicity of kenaf seed oil towards leukemia cells. The
cell viability of the leukemia cells treated with V600/40
reduced with the increase in the concentration of the oil
indicating that the cytotoxic effect was in a dose-
dependent manner (Figure 1). Since the oil was more
cytotoxic towards HL-60 when compared with WEHI-3B
and K562 with IC50 value ranging between 0.125 and 5
mg/ml, it has a good potential for the treatment of leuke-
mia (Manosroi et al., 2006). HL-60 has been reported to
be very sensitive to apoptosis upon anti-cancer agents’
treatment and it is a good in vitro model for testing anti-
leukemic agents (Suh et al., 1995; Yoshida et al., 1996).

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Foo et al. 5393






Figure 2. Morphological changes of (A) HL-60, (B) WEHI-3B and (C) K562 cells treated with V600/40 for 72 h viewed under an inverted light
microscope. Affected cells showed some features characteristic of apoptosis such as cellular shrinkage (CS) and membrane blebbing (BL)
(400X).



Interestingly, it is also of advantage that the oil was least
cytotoxic to the normal 3T3 cell line. Recent study revealed
that, kenaf seed oil was cytotoxic towards the ovarian
cancer cells (CaOV3) (Yazan et al., 2010). In addition,
lignans from kenaf core and bark of acetone extract were
cytotoxic towards cervical cancer (HeLa), epithelial
cancer (Hep-2) and lung cancer (A-549) cell lines (Moujir
et al., 2007). Kenaf seed oil contains various active
compounds such as fatty acid, phenolic acids, phyto-
sterols and tocopherols (Mohamed et al., 1995; Nyam et
al., 2009). Phytosterols showed growth inhibitory effects
on breast (Awad et al., 2007), leukemia (Moon et al.,
2008; Park et al., 2007), lung (Mendilaharsu et al., 1998;
Schabath et al., 2005), ovarian (McCann et al., 2003),
stomach (De Stefani et al., 2000) and prostate cancer
(McCann et al., 2005). Linoleic acid inhibited the prolife-
ration of human skin cancer, breast cancer, colon
cancer, stomach cancer,as well as leukemia in vitro and
in vivo (Hubbard et al., 2000; Kritchevsky, 2000;
MacDonald, 2000; Phoon
phytosterols and linoleic acid are speculated to be
responsible for the cytotoxic effects.
It is obvious that increase in temperature influences the
cytotoxicity of kenaf seed oil. The higher the extraction
temperature of SFE, the higher the IC50 value (Table 2).
Similar condition was observed for Soxhlet (the use of
heat during extraction) where the IC50 value was not
obtained. It is speculated that high extraction temperature
might denature some of the heat sensitive active compo-
nents in the oil (Cossuta et al., 2008), thus, making it less
cytotoxic to the cells.
Kenaf seed oil was also investigated for the mode of
cell death, whether it is apoptosis or necrosis. This is
important as mode of cell death especially apoptosis has
et al., 2001). Thus,

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5394 Afr. J. Biotechnol.





Figure 3. Fluorescence micrograph of AO/PI double-stained of (A) HL-60, (B) WEHI-3B and (C) K562 cells treated with V600/40 for 72 h. Affected cells showed typical characteristics of
apoptosis such as membrane blebbing (BL), chromatin condensation (CC), nuclear margination (NM) and nuclear fragmentation (NF) (400X).

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Foo et al. 5395


























Figure 4. Cell cycle analysis of (A) HL-60, (B) WEHI-3B and (C) K562 cells treated with V600/40 for 72 h. Accumulation of cells at the sub-G1
phase was observed with the increase in concentration of V600/40.



great implication in cancer therapy. Apoptosis is of
advantage and a more favorable one since it plays an
important role in elimination of damaged cells or tumor
cells without causing inflammation (Hou et al., 2005). It is
found that the leukemia cells treated with V600/40 died
primarily via apoptosis as viewed under an inverted light
microscope (Figure 2) and the findings were further sup-
ported by fluorescence analysis following staining with AO

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5396 Afr. J. Biotechnol.



Table 3. Changes in the cell cycle distribution (%) of HL-60, WEHI-3B and K562 cells treated with V600/40 for 72 h.

Apoptotic cell (%)
Sub-G1
Control 3.71 ± 2.22
100 8.01 ± 4.32
178 (IC50) 48.52 ± 2.31*
200 53.36 ± 1.21*
400 92.82 ± 0.56*
800 99.60 ± 0.99*

Control 5.12 ± 2.13
100 3.86 ± 1.24
189 (IC50) 16.22 ± 1.45*
200 19.30 ± 2.26*
400 26.31 ± 0.94*
800 75.29 ± 3.45*

Control 3.99 ± 0.95
100 5.33 ± 1.32
200 29.64 ± 2.34*
213 (IC50) 39.69 ± 1.23*
400 35.95 ± 3.45*
800 43.32 ± 2.11*

Each data point represents the mean of triplicate ± SD. * is significantly different from control (P < 0.05).



and PI (Figure 3). In the analysis, almost all the control
cells (without treatment with V600/40) for the three cell
lines looked healthy, round in shape with similar size and
green color of intact nucleus. In contrast, majority of the
treated cells were apoptotic. They were smaller in size
with green but fragmented nucleus. Other features of
apoptosis such as membrane blebbing, chromatin con-
densation and nuclear margination were also observed
(Kerr et al., 1972) (Figure 3).
The induction of apoptosis was then confirmed by the
accumulation of population of the cells at sub-G1 phase
(P < 0.05) from the cell cycle analysis. Appearance of
sub-G1 cells is the marker of cell death by apoptosis
(Park et al., 2007). Treatment with V600/40 also caused
an arrest at the G1 phase in the three leukemia cell lines.
The mechanisms on how kenaf seed oil caused leukemia
cells to undergo apoptosis at the G1 checkpoint are still
unclear. A study (Hsu et al., 2005) drives a hypothesis
that kenaf seed oil degraded cdc25A which is a
phosphatase associated with CDK4 and CDK2 activities,
following by down-regulation of cyclin D2 and cyclin E
and up-regulate p15INK4b and p27Kip1 genes. These series
of events impose a blockade of mid G1-late G1-S transi-
tion thereby causing G1 phase cell cycle arrest. It will be
followed by a series of typical morphological changes
such as cellular shrinkage, membrane blebbing, chromatin
condensation and nuclear fragmentation, as being obser-
ved in Figures 2 and 3. In conclusion, kenaf seed oil
Cell line
Concentration
(µg/ml)






















Non-apoptotic cell (%)
S
11.24 ± 1.22
11.77 ± 0.23
13.96 ± 1.78
14.09 ± 1.23*
14.69 ± 0.97*
7.5 ± 0.54*

9.39 ± 4.33
13.73 ± 3.76
13.81 ± 1.44
12.02 ± 1.87
11.99 ± 0.96
3.62 ± 0.45*

16.78 ± 2.33
15.84 ± 1.98
18.75 ± 2.11
19.03 ± 3.24
16.43 ± 1.64
13.46 ± 1.23*
G1 G2/M
HL-60
44.84 ± 1.16
46.78 ± 2.31
52.78 ± 2.31*
47.57 ± 0.46
49.66 ± 2.68*
57.52 ± 1.69*

46.77 ± 1.45
50.72 ± 2.34
62.20 ± 1.25*
55.83 ± 2.21*
53.71 ± 0.79*
80.93 ± 0.78*

41.81 ± 1.22
42.15 ± 2.13
48.01 ± 0.97*
49.33 ± 2.78*
52.65 ± 2.89*
48.61 ± 1.56*
43.98 ± 0.95
41.55 ± 2.51
33.26 ± 1.64*
38.35 ± 1.24*
35.65 ± 1.74*
35.14 ± 0.95*

44.84 ± 0.45
34.55 ± 0.78
23.99 ± 1.14*
32.15 ± 1.31*
30.15 ± 2.24*
15.45 ± 3.34*

41.41 ± 3.46
42.15 ± 1.21
38.64 ± 2.13
31.65 ± 0.91*
31.74 ± 1.47*
37.98 ± 0.79
WEHI-3B
K562
V600/40 induced apoptosis via G1 phase cell cycle arrest
in HL-60, WEHI-3B and K562 leukemia cell lines. Interes-
tingly, V600/40 was less cytotoxic towards the normal
mouse embryonic fibroblast 3T3 cell line. Kenaf seed oil
will be a candidate for the development of anti-cancer
agent. A further analysis is needed to investigate the anti-
tumor property of kenaf seed oil in leukemia mice.


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