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Antioxidant and anti-inflammatory properties of C-phycocyanin
from blue-green algae
C. Romay1, J. Armesto1, D. Remirez1, R. Gonza
´lez1, N. Ledon1and I. Garcı
´a2
1Pharmacology Department, National Center for Scientific Research, CNIC, P.O. Box 6990, Havana, Cuba, Fax 53 7 330497,
e-mail: ricardo@quimica.cneuro.cu
2Pharmacy and Foods Faculty, University of Havana, Ave. 23, No. 21425, La Coronela, Havana, Cuba
Received 21 April 1997; returned for revision 14 May 1997; accepted by M. J. Parnham 28 October 1997
Abstract. Objective: Phycocyanin is a pigment found in
blue-green algae which contains open chain tetrapyrroles
with possible scavenging properties. We have studied its
antioxidant properties.
Materials and methods: Phycocyanin was evaluated as a
putative antioxidant in vitro by using: a) luminol-enhanced
chemiluminescence (LCL) generated by three different
radical species (O¹
2,OH
•
,RO
•
) and by zymosan activated
human polymorphonuclear leukocytes (PMNLs), b) deoxy-
ribose assay and c) inhibition of liver microsomal lipid
peroxidation induced by Feþ2-ascorbic acid. The antioxi-
dant activity was also assayed in vivo in glucose oxidase
(GO)-induced inflammation in mouse paw.
Results: The results indicated that phycocyanin is able to scav-
enge OH•(IC50 ¼0:91mg/mL) and RO•(IC50 ¼76mg/mL)
radicals, with activity equivalent to 0.125mg/mL of
dimethyl sulphoxide (DMSO) and 0.038mg/mL of trolox,
specific scavengers of those radicals respectively. In the
deoxyribose assay the second-order rate constant was 3:56×
1011 M¹1S¹1, similar to that obtained for some non-steroidal
anti-inflammatory drugs. Phycocyanin also inhibits liver
microsomal lipid peroxidation (IC50 ¼12 mg/mL), the CL
response of PMNLs (p< 0.05) as well as the edema index in
GO-induced inflammation in mouse paw (p< 0.05).
Conclusions: To our knowledge this is the first report
of the antioxidant and anti-inflammatory properties of
c-phycocyanin.
Key words: Antioxidant – Chemiluminescence – C-
Phycocyanin – Blue-green algae – Free radical scavenger
Introduction
C-phycocyanin is a protein-bound pigment found in blue-
green algae. Phycocyanin monomers are themselves made
up of two distinguishable protein subunits designated aand
b, which contain at least three covalently attached bilin
chromophores, open chain tetrapyrroles with no metal
complexes [1]. These prosthetic groups account for about
4% of the algae mass, indicating the presence of about
sixteen chromophoric groups per unit molecular weight [2].
It occurs in four different structural forms, monomeric,
trimeric, hexameric and decameric [3], and is the most
abundant pigment in blue-green algae, accounting for more
than 20% of algal dry weight [4].
The chemical structure of the bilin chromophores in
c-phycocyanin, (open chain tetrapyrroles) are very close to
that of bilirubin. Stocker et al. [5] reported that bilirubin is an
antioxidant of possible physiological importance because it
could scavenge peroxy radicals by donating a hydrogen atom
attached to the C-10 bridge of the tetrapyrrole molecule to
form a carbon-centered radical with resonance stabilization
extending over the entire bilirubin molecule.
It is well known that reactive oxygen species (ROS) are
involved in a diversity of important processes in medicine
including, among others: inflammation, atherosclerosis,
cancer, reperfusion injury [6]. One way by which a substance
can interfere with these processes is by acting as antioxidant
or free radical scavenger.
Taking these data into account, we postulated that
c-phycocyanin may be a putative antioxidant and decided to
evaluate it in some in vitro and in vivo experimental models.
Materials and methods
Reagents
Luminol and xanthine oxidase (XO 20U/mL) were obtained from
Boehringer Mannheim GmbH (Germany). Trolox and p-iodophenol
were from Aldrich Chemical Co. (Milwaukee, WI, USA). Superoxide
dismutase (SOD) (3333U/mL) from bovine erythrocytes and Hypox-
anthine (HX) were from Serva Feinbiochimica (Heidelberg, Germany).
Tert-butylhydroperoxide (t-BOOH) and butyl hydroxytoluene (BHT)
were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
Sodium dodecylsulphate (SDS), hydrogen peroxide (H2O2), ascorbic
Inflamm. res. 47 (1998) 36–41
qBirkha
¨user Verlag, Basel, 1998
1023-3830/98/010036-06 $ 1.50+0.20/0 Inflammation Research
Correspondence to: C. Romay
m
acid, 2-deoxyribose and thiobarbituric acid (TBA), were from BDH Ltd
(Poole, UK). FeCl3and glucose oxidase (GO) (1.4U/mg) were from
Merck (Darmstadt, Germany). All other reagents were of analytical
grade.
Phycocyanin was obtained from Arthospira maxima species and
purified by the method of Neufeld and Riggs [7].
Phosphate-buffered saline solution (PBS) consisted of NaCl
0.14M, KCl 2.7 mM, Na2HPO412 mM, KH2PO41.5mM, CaCl
0.9mM and MgCl20.49 mM.
Animals
Male OF1mice weighing 22–25 g and male Sprague Dawley rats (220–
250 g) were purchased from the National Center for Laboratory
Animals Production (CENPALAB, Havana, Cuba). The animals were
housed under controlled temperature (t ¼25 8C) and air humidity
(60%) with a 12 h light-dark cycle, and kept on a standard laboratory
diet and drinking water ad libitum.
Chemiluminescence measurements
The scavenging action of c-phycocyanin was determined against
different types of oxygen radicals, which were generated by specific
chemical reactions and detected by LCL.
A well known scavenger for each radical was used as control
for the paradigm and to compare its effect with that produced by
c-phycocyanin.
Chemiluminescence was measured in millivolts in an LKB Wallac
1250 luminometer coupled to an LKB 2210 two channel recorder.
Superoxide radical scavenging activity was determined as
described by Pascual et al. [8]. The reaction consisted of 800 mLofa
mixture containing 68 mM glycine buffer pH 8.6, 10 mM luminol and
5mM p-iodophenol. Fifty mL of distilled water or c-phycocyanin
aqueous solutions were added. Then 2 mL of xanthine oxidase (2 U/mL)
were added and the reaction was initiated with 10 mL of hypoxanthine
1mM. The intensity was registered immediately. SOD was used in the
system as a control.
Hydroxyl radicals were generated from the Fenton reaction. The
method used was described by Pascual and Romay [9]. In brief, to
800 mL of a mixture containing: 50 mM K2HPO4-KH2PO4buffer
pH7.8, 2 mM EDTA and 0.1 mM luminol, 0.1 mL of distilled water,
sample or specific scavenger (DMSO) was added. Then 5 mLof6mM
H
2
O
2was also added and mixed. The reaction was started with 10 mLof
20mM FeSO4and after rapid mixing, the CL signal was immediately
registered.
Determination of alkoxyl radical scavenging activity was per-
formed by measuring the inhibition of the CL produced by the reaction
of t-BOOH with ferrous ions in the presence of luminol, as previously
described [9]. The reaction mixture consisted of: 800 mLof50mM
glycine buffer pH 8.6, 50 mM SDS, 0.025 mM luminol. Ten mLof
distilled water was added and mixed prior to the addition andmixing of
5mL of 7.3 mM t-BOOH.
The reaction was started with 10 mL of 0.4 mM FeSO4and
immediately after rapid mixing the chemiluminescence signal (mV)
was recorded. Trolox, a water soluble analogue of vitamin E, was used
as specific scavenger of these radicals.
Double quenching experiments were done in each CL system in
order to determine whether the effect of the phycocyanin was due to the
scavenging of the desired oxygen free radical or the trapping of other
free radical species. These experiments were done by measuring the
luminous signal before (Io) and after (I) adding increasing concentra-
tions of c-phycocyanin in the absence and presence (sufficient to cause
50% inhibition) of the specific scavenger (SOD, DMSO or trolox). The
slope of the plot is equal to kt where tis the life time of the radical in
the absence of phycocyanin. When both phycocyanin and the specific
scavenger compete for the same radical, a decrease in the slope must be
expected.
Effect of phycocyanin on the CL response of isolated PMNLs
PMNL preparation
Human leukocytes were obtained as described previously [8] from
10 mL heparinized (20 U/mL) venous blood from healthy volunteers,
who had not taken any drug during the week before blood sampling.
The blood was mixed with an equal volume of an ACD-dextran-glucose
mixture consisting of: 1.5 mL acid citrate dextrose (ACD) in 0.9%
NaCl (24.5 g glucose/L, 22g sodium citrate dihydrate/L and 7.3 g citric
acid/L), 5 mL of 6% (w/v) Dextran T-500 in 0.9% of NaCl and 3.5 mL
of 5% glucose in 0.9% NaCl.
After mixing well and allowing to stand for 45–60 min at room
temperature, the upper layer containing the leukocytes was removed by
aspiration and three times its volume of ammonium chloride 0.8%
added in order to hemolyze the remaining red cells. The cells were
centrifuged for 10 min at 500 g at 48C. Then, 2 mL of 0.9% NaCl were
added, followed by 3mL of cold distilled water which were mixed and
allowed to stand for 2 min, then mixed with 3.6% NaCl and centrifuged
for 10 min at 500g. Finally the cells were resuspended in approximately
5×106cells per mL PBS.
Opsonisation of particles
Cells from a fresh culture of Saccharomyces cerevisiae were washed
and then put into a boiling water bath for 30min. After washing in
saline they were resuspended at a concentration of 2 ×108particles/mL
PBS. The opsonisation procedure was carried out by incubating a
mixture of 200 mL human serum with 1.8 mL of yeast suspension for
30 min at 30 8C immediately prior to the experiment. A luminol stock
solution of 10¹2M in DMSO was prepared and was diluted to 10¹4M
in PBS prior to use.
Chemiluminescence assay
Chemiluminescence was performed as described previously [8, 10] with
minor modifications. Briefly, 200 mL of opsonised yeast cells, 450 mL
of phosphate-buffered saline (PBS), 200 mL luminol 10¹4M and 50 mL
of water or different phycocyanin concentrations, were incubated for
10 min in the measuring cuvette. Just before the assay, 100 mLofan
isolated leukocyte suspension were added and the light intensity was
measured every 3min at 37 8C. In a system without cells there was
no interaction between luminol and phycocyanin. The viability of
PMNLs after being exposed to the higher c-phycocyanin concentration
(incubated at 37 8C water bath for 40 min) was assessed by the Trypan
blue exclusion test. The viability obtained was 98%.
Inhibition of damage to 2-deoxyribose
Evaluation of the inhibition of damage to 2-deoxyribose, measured as
formation of thiobarbituric acid-reactive material [11], was carried out
as an alternative measure of the hydroxyl radical scavenger capacity of
c-phycocyanin. Mixtures contained, in a final volume of 1.2 mL:
deoxyribose 2.8 mM, KH2PO4/KOH buffer 15 mM pH 7.4, FeCl3
20 mM, EDTA 100 mM, H2O22.8 mM and ascorbic acid 100 mM.
FeCl3and ascorbate solutions were made up in bidistilled water just
before use. FeCl3and EDTA were premixed prior to addition to the
reaction mixture. Ascorbic acid was added in order to start the reaction.
Reaction mixtures were incubated at 37 8C for 1 h. After addition of
1 mL of TBA 1% (w/v) in 0.05 mM NaOH and 1 mL of TCA 2.8%
in water, the mixture was heated at 100 8C for 20min. The pink
chromogen that progressively developed was then measured at 532 nm
after cooling, against appropriate blanks. The second-order rate
constants kswere calculated using the data obtained in the presence
of EDTA from the slope of a plot of 1/A532nm against the test compound
concentration.
37Vol. 47, 1998 C-Phycocyanin as antioxidant
m
Inhibition of liver microsomal lipid peroxidation induced by
Feþ2-ascorbic acid
Rat liver microsomes were prepared by differential ultracentrifugation
as previously described [12] and stored at ¹80 8C until use. Protein was
assayed by the Lowry method [13].
The procedure was carried out as described [14]. The microsomes
(final concentration 1.3 mg protein/ml) were incubated at 37 8C in Tris
buffer 50 mM pH7.4 before induction of lipid peroxidation with 10 mM
FeSO4and freshly prepared 0.2mM ascorbic acid. The reaction was
stopped by adding 0.3 mL of the incubation mixture to 2 mL of ice-cold
TBA-TCA-HCl-BHT.Afterheatingfor15 minat 80 8C andcentrifugation
for15 minat 2000g,the absorbanceat 535nm wasdetermined.The TBA-
TCA-HCl solution was prepared by dissolving 41.6mg TBA/10 mL
TCA (16.8% w/v in 0.125N HCl). To 10mL TBA-TCA-HCL, 1mL of
BHT (1.5mg/mL ethanol) was added.
All the spectrophotometric measurements were done in a Spekol
220 from Carl Zeiss (Jena, Germany).
GO-induced inflammation in mouse paw
The animal model used was described by Spillert et al. [15].
Phycocyanin (50, 100 or 200mg/Kg in saline) or DMSO (1 g/Kg), as
positive control, were administered orally and i.p, respectively, to male
OF1 mice. One hour later, the mice were injected in the right hind foot
with 50mL of physiological saline and in the left foot with 50 mLof
100 U/mL GO. The animals were killed at 1.5h post injection, both hind
feet amputated at the tibiotarsal joint and each paw weighed. The
38 C. Romay et al. Inflamm. res.
Fig. 1. Effect of c-phycocyanin on chemiluminescence intensity
produced by alkoxy radicals generated by the reaction of tert-
butylhydroperoxide with ferrous ion. (A) Decrease in Chemilumines-
cence signal (mV) with increasing c-phycocyanin (X) or trolox (W)
concentration. Each point represents the mean of three determinations.
Vertical bars represent 61 SD. (B) Double quenching experiment
carried out to determine the chemiluminescence signal before (Io) and
after (I) adding increasing concentrations of c-phycocyanin in the
absence (X) and presence (W) of trolox (46 ng/mL). Each point
represents the mean Io/I value obtained from three determinations.
Fig. 2. Effect of c-phycocyanin as scavenger of hydroxyl radicals
produced by the Fenton reaction. (A) Decrease in chemiluminescence
signal (mV) with increasing c-phycocyanin (X) or DMSO (W)
concentrations. Each point represents the mean of three determinations.
Vertical bars represent 61 SD. (B) Io/I vs c-phycocyanin concentration
plot obtained as in Figure 2 in the absence (X) and presence (W)of
DMSO (149 mg/mL). Each point represents the mean Io/I value
obtained from three determinations.
m
difference in weight of hind paws of each animal was called the edema
index (EI).
Statistical analysis
One-way ANOVA followed by Duncan’s multiple comparison test
were used to calculate the significance of the differences between the
means. The IC50 was calculated using a GraphPad InPlot software
(GraphPad Software Inc., version 4.03, 1992).
Results
Chemiluminescent measurements
The CL produced by the reaction of t-BOOH with ferrous
ion was used for evaluation of alkoxyl radical scavenging
capacity of c-phycocyanin. The results show that c-phycocya-
nin inhibited the CL in this system (Fig. 1A). A comparison
with trolox, a water soluble analogue of vitamin E, indicates
that 0.038 mg/mL of trolox causes approximately the same
effect as 76 mg/mL of c-phycocyanin in terms of 50%
inhibition of the CL produced in this system.
C-phycocyanin was evaluated as a scavenger of hydroxyl
radicals by determining the inhibition of CL produced by the
Fenton reaction with luminol. As shown in Figure 2A, the
chemiluminescence signal (mV) was inhibited by increas-
ing phycocyanin concentrations. In this system, 0.91 mg/mL
of phycocyanin caused the same inhibition (50%) as
0.125 mg/mL of DMSO, which was used as control.
In the HX-XO chemiluminescence system (Fig. 3A),
phycocyanin inhibited the signal in a dose-dependent
manner. However, when the HX-XO reaction was used to
reduce nitroblue tetrazolium (NBT) dye, no inhibitory effect
was observed (data not shown). In the double quenching
experiments (Fig. 1B and 2B), the slope decreased when both
phycocyanin and the specific scavenger were added together,
indicating competition for the radical generated. This
behaviour was not observed in the O¹
2-generator system
(Fig. 3B) in which the slope is independent of the previous
addition of the specific scavenger (SOD) and is even
increased.
Effect of phycocyanin on the CL response of PMNLs
Human leukocytes stimulated with opsonized yeast cells
produced a typical time-dependent CL response with a
maximal intensity at approximately 16 min followed by a
decline. Table 1 shows the means of the areas under the
curve (AUC) obtained for each phycocyanin concentration
39Vol. 47, 1998 C-Phycocyanin as antioxidant
Fig. 3. Effect of various c-phycocyanin concentrations on chemilumi-
nescence produced in the luminol-XO-HX system. (A) Decrease in
chemiluminescence signal (mV) with increasing c-phycocyanin (X)or
SOD (W) concentrations. Each point represents the mean of three
determinations. Vertical bars represent 61 SD. (B) Io/I vs c-
phycocyanin concentration plot obtained as in Figure 2 in the absence
(X) and presence (W) of SOD (23ng/mL). Each point represents the
mean Io/I value from three determinations.
Table 1. Effect of phycocyanin on the chemiluminescence response of
human leukocytes.
AUC Inhibition %
X
¯6SD
Control 6662 6221:3a–
Phycocyanin 1 mg/mL 5800 6246:5b12.9
2 mg/mL 4647 6227:0c30.2
3 mg/mL 3841 6296:5d42.3
Different letters: p < 0.05 vs. each other, n ¼5.
Table 2. Effect of phycocyanin on GO-induced inflammation in the
mouse paw.
Treatment Edema index (g) Inhibition %
DMSO (1g/Kg) 0:045 60:007a64.05
Phycocyanin 50mg/Kg 0:110 60:010b4.43
100 mg/Kg 0:080 60:007c35.46
200 mg/Kg 0:061 60:009d51.05
Glucose oxidase 100 U/mL 0:125 60:008b–
Different letters: p < 0.05 vs. each other, n ¼7 mice per group.
Phycocyanin was administered orally 1h before GO. The oedema was
measured 1.5 h after injection of GO into the paw. The oedema index
indicates the difference in weight between the hind paws.
m
and control. The CL signal was significantly reduced with
respect to control by increasing phycocyanin concentrations.
Significant statistical differences were also obtained among
phycocyanin concentrations.
Deoxyribose assay
In the deoxyribose assay, a more conventional method,
phycocyanin inhibited the deoxyribose damage in a
concentration-dependent fashion. The IC50 calculated for
c-phycocyanin was 0.86 mg/mL. In this system, a typical
competition profile was obtained (Fig. 4). This was suitable
for the calculation of a second-order rate constant of
3:56 ×1011 M¹1S¹1, considering a molecular weight of
36 700 g/mol for c-phycocyanin monomer [16].
Microsomal lipid peroxidation inhibition
Addition of c-phycocyanin (8, 12, 20 mg/mL) to isolated
microsomes in the presence of Feþ2-ascorbate, resulted in a
concentration dependent decrease in lipid peroxidation
(IC50 ¼12 mg/ml). Both the rate and the final extent of
lipid peroxidation were reduced by adding c-phycocyanin
(Fig. 5).
In the presence of iron alone, c-phycocyanin did not
cause lipid peroxidation, indicating that it does not have pro-
oxidant effect in this system. Furthermore, its inhibitory
effect was exerted during the incubation time, since it had
not effect when it was added with the TBA reagent.
GO-induced inflammation in mouse paw
Differences in weight between the hind paws of animals in
the various groups are shown in Table 2. Phycocyanin at
doses of 100 and 200 mg/Kg orally was able to inhibit
significantly peroxide-induced inflammation in a dose
dependent fashion with a ED50 of 170:361:62 mg/Kg.
DMSO was used as a positive control and it also inhibited the
inflammatory response induced by GO in mouse paw.
Discussion
In this study, we have applied established in vitro and
in vivo assays in order to evaluate the antioxidant action of
c-phycocyanin. This natural product was able to scavenge
alkoxy and hydroxyl radicals. Two methods were used to
evaluate hydroxyl radical scavenging by c-phycocyanin
because hydroxyl radical is one of the most potent oxidizing
species and its extreme reactivity naturally poses problems
with regard to its detection [17]. In both methods, inhibition
was observed at relatively high concentrations of the
product.
Phycocyanin also quenched the CL signal generated in
the HX-XO system, but this effect can not be ascribed to O¹
2
scavenging, as demonstrated by double-quenching and NBT
reduction assays. One possible explanation for this behaviour
is that phycocyanin quenches CL by binding to an inter-
mediate or co-oxidising species that may be involved in the
CL reaction [18].
The inhibitory effect observed on microsomal lipid
peroxidation most probably is due to a metal-binding
capacity of c-phycocyanin, since chain-breaking antioxi-
dants often introduce a lag period into the peroxidation
process, corresponding to the time taken for the antioxi-
dant to be consumed, whereas metal-binding antioxidants
will give a constant inhibition throughout the reaction
[19]. Another indication of such an action, is the ability of
c-phycocyanin to inhibit deoxyribose damage in a site-
specific manner (in the absence of EDTA) [19]. Further
experiments must be performed to obtain more evidence for
the ability of phycocyanin to chelate metal ions.
In the deoxyribose assay, a second-order rate constant
calculated for phycocyanin was similar to that obtained,
by the same method, for some non-steroidal anti-inflamma-
tory drugs, such as indomethacin and ibuprofen (1:8×1010
M¹1S¹1) [20].
Chemiluminescence of PMNLs is the final result of
luminol oxidation by strong oxidants, such as oxygen
radicals and peroxides, emanating from enzymatic
reactions. For addition to the myeloperoxidase-H2O2-
halide system, the release of arachidonic acid by phospho-
lipase A2and of diacylglycerol and inositol trisphosphate by
phospholipase C, the metabolism of arachidonic acid by the
40 C. Romay et al. Inflamm. res.
Fig. 4. Plots of 1/A532nm against concentration of c-phycocyanin using
data obtained from deoxyribose oxidation in the presence (X)or
absence (W) of EDTA. Values for each point are means of three
experiments. Vertical bars represent 61 SD.
Fig. 5. Time course of microsomal non-enzymatic lipid peroxidation as
affected by various concentrations of c-phycocyanin: (X) no addition,
(W) 8 mg/mL, (O) 12 mg/mL, (K) 20mg/mL. Values for each point are
means of three determinations. Vertical bars represent 61 SD.
m
cyclooxygenase and lipooxygenase pathways, the activation
of membrane NADPH oxidase by diacylglycerol and
calcium mobilization by inositol trisphosphate are all able
to induce the CL reaction. Inhibition of any of these
mechanisms suppresses the CL response [21].
Phycocyanin was able to inhibit the LCL in a dose-
dependent fashion, most probably through its capacity to
scavengefreeradicals (OH•,H2O2,RO
•)and peroxidesarising
during the respiratory burst of phagocytic cells. However, it is
also possible that phycocyanin could diminish CL signals in
other ways, e.g. by affecting enzymes involved in the
production of reactive oxygen species by activated phago-
cytes, NADPH oxidase and myeloperoxidase, or by interfer-
ing either with the binding of the stimulant or the arachidonic
acid metabolism pathway. In this regard, we have recent
evidence for inhibition by phycocyanin of LTB4release in an
animal model of inflammation (manuscript in preparation).
The peroxide-induced inflammatory response is a valu-
able in vivo model in order to test agents with potential
scavenging effect against H2O2and OH•. GO injected into
the mouse paw reacts with endogenous glucose and generates
H2O2which subsequently produces OH•radicals; both
together are responsible for tissue damage and for the
accompanying inflammatory changes [15]. Phycocyanin
reduced the edema produced by glucose oxidase in the
mouse paw. This anti-inflammatory effect must be due, at
least in part, to the scavenging of hydroxyl radicals, taking
into account the fact that DMSO, a well known scavenger of
OH•radicals, also inhibited the inflammatory response
induced by GO.
Currently, there is a consensus that much of the damage
induced by H2O2in vivo is due to its conversion to highly-
reactive oxidants, mainly OH•[19]. Therefore, the scaveng-
ing action of phycocyanin against OH is probably relevant to
its anti-inflammatory effects.
Very recently, research carried out in our laboratory has
confirmed the anti-inflammatory effects of phycocyanin in
other experimental models of inflammation such as cotton
pellet granuloma in the rat and TPA-induced inflammatory
response in the mouse ear (manuscript in preparation).
Finally, to our knowledge, this is the first time that both
antioxidant and anti-inflammatory properties have been
described for c-phycocyanin.
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