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Parasitology Research
Founded as Zeitschrift für
Parasitenkunde
ISSN 0932-0113
Volume 112
Number 2
Parasitol Res (2013) 112:603-610
DOI 10.1007/s00436-012-3172-7
Anthelmintic activity of carvacryl acetate
against Schistosoma mansoni
Josué de Moraes, Aline A.Leite
Carvalho, Eliana Nakano, Antonia
Amanda Cardoso de Almeida, Thiago
Henrique da Costa Marques, et al.
1 23
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ORIGINAL PAPER
Anthelmintic activity of carvacryl acetate
against Schistosoma mansoni
Josué de Moraes &Aline A. Leite Carvalho &
Eliana Nakano &Antonia Amanda Cardoso de Almeida &
Thiago Henrique da Costa Marques &
Luciana Nalone Andrade &
Rivelilson Mendes de Freitas &
Damião Pergentino de Sousa
Received: 28 August 2012 /Accepted: 8 October 2012 / Published online: 21 October 2012
#Springer-Verlag Berlin Heidelberg 2012
Abstract Blood flukes of the genus Schistosoma are the
causative agents of human schistosomiasis, a debilitating
disease that afflicts over 200 million people worldwide.
Praziquantel is the drug of choice but concerns over prazi-
quantel resistance have renewed interest in the search for
alternative drug therapies. Carvacrol, a naturally occurring
monoterpene phenol and food additive, has been shown
high medicinal importance, including antimicrobials activi-
ties. The aim of this study was to evaluate in vitro effect of
carvacryl acetate, a derivative of carvacrol, on Schistosoma
mansoni adult worms. We demonstrated that carvacryl
acetate at 6.25 μg/mL has antischistosomal activity, affect-
ing parasite motility and viability. Additionally, confocal
laser scanning microscopy pictures revealed morphological
alterations on the tegumental surface of worms, where some
tubercles appeared to be swollen with numerous small blebs
emerging from the tegument around the tubercles.
Furthermore, experiments performed using carvacryl acetate
at sub-lethal concentrations (ranging from 1.562 to 6.25 μg/
mL) showed an inhibitory effect on the daily egg output of
paired adult worms. Thus, carvacryl acetate is toxic at high
doses, while at sub-lethal doses, it significantly interferes
with the reproductive fitness of S.mansoni adult worms.
Due to its safety and wide use in the industry, carvacryl
acetate is a promising natural product-derived compound
and it may represent a step forward in the search for novel
anthelmintic agents, at a time when there is an urgent need
for novel drugs.
Introduction
Schistosomiasis is a major global health problem and
principal among a number of neglected tropical diseases
for the chronic disability inflicted that impacts both
personal and societal productivity (Gryseels et al.
2006; Van der Werf et al. 2003). The causative agents
of schistosomiasis are parasitic flatworms of the genus
Schistosoma. There are five species of schistosomes that
can infect humans, of which Schistosoma mansoni is the
most important one. Pathology associated with schistosomia-
sis is not directly due to the adult worms but rather to the large
numbers of eggs that become trapped in tissues (Pearce and
MacDonald 2002).
J. Moraes (*):E. Nakano
Parasitology Laboratory, Butantan Institute,
Avenida Vital Brasil 1500,
05503-900 São Paulo, SP, Brazil
e-mail: josuem@usp.br
J. Moraes
e-mail: josuemoraes@ig.com.br
J. Moraes :A. A. L. Carvalho
Supervision of Health Surveillance, SUVIS Casa Verde,
Municipal Health Department of São Paulo,
São Paulo, SP, Brazil
A. A. C. de Almeida :R. M. de Freitas
Department of Biochemistry and Pharmacology, Post-graduation
Program in Pharmaceutics Science of Federal University of Piauí,
Teresina, Piauí, Brazil
T. H. C. Marques :R. M. de Freitas
Post-graduation Program in Biotechnology
(RENORBIO) of Federal University of Piauí,
Teresina, Piauí, Brazil
L. N. Andrade :D. P. de Sousa
Department of Pharmacy, Federal University of Sergipe,
São Cristóvão, Sergipe, Brazil
Parasitol Res (2013) 112:603–610
DOI 10.1007/s00436-012-3172-7
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Globally more than 700 million people are at risk and at
least 200 million people are currently infected with
Schistosoma, especially in Africa, Asia and Latin America
(Steinmann et al. 2006). The strategy to control Schistosoma
infections is chemotherapy with praziquantel, which means
large-scale administration of anthelmintic drug to at-risk
populations. However, because reinfection occurs after
treatment, chemotherapy must be repeated regularly and,
hence, drug resistance is a growing concern (Botros and
Bennett 2007; Caffrey 2007; Utzinger and Keiser 2004).
Worries about reliance on one antischistosomal drug and
the possibility of the emergence of drug resistance make the
development of novel anthelmintic drugs an urgent task
(Botros and Bennett 2007; Caffrey 2007). In this regard,
many scientific research centres around the world are ex-
ploring medicinal plants due to global belief of their efficacy
in treatment. In fact, the use of herbal remedies seems to be
the alternative choice of treatment in regions where infec-
tion diseases are endemic (Allegretti et al. 2012; Kayser et
al. 2003; Moraes 2012; Newman and Cragg 2012). Extracts,
oils and compounds with different chemical groups of sev-
eral medicinal plants were tested for potential therapeutic
activity against schistosome infection. For example, anti-
schistosomal activities have been described with steroidal
alkaloids from Solanum lycocarpum fruits (Miranda et al.
2012), epiisopiloturine, an imidazole alkaloid isolated from
the leaves of Pilocarpus microphyllus (Veras et al. 2012),
piplartine, an amide isolated from Piper tuberculatum (de
Moraes et al. 2011; Moraes et al. 2011), some phlorogluci-
nol compounds obtained from the rhizomes of Dryopteris
species (Magalhães et al. 2010), essential oils of Baccharis
trimera (de Oliveira et al. 2012)andPiper cubeba
(Magalhães et al. 2012) as well as several plant extracts
(Ferreira et al. 2011; Mostafa et al. 2011; Koné et al. 2012).
Among natural products, essential oils are promising
therapeutic tools (Anthony et al. 2005). In addition, use of
essential oils is particularly advisable because herbs and
spices are commonly added in food to obtain a specific taste
(Baser 2008; Yu et al. 2012). Carvacrol is a monoterpene
phenol present in essential oils from several species of
medicinal and aromatic plants. It has been widely used both
as a food or food additive in the food industry for long time.
In recent years, its multiple functions were well studied in
different fields (Baser 2008). Carvacrol has shown several
biological activities such as antioxidant (Anthony et al.
2012; Prieto-Garcia et al. 2007), antiviral (Lai et al. 2012),
antibacterial (Sokovićet al. 2010), fungicidal (Ahmad et al.
2011), insecticidal (Cetin and Yanikoglu 2006; Tang et al.
2011) and acaricidal (Coskun et al. 2008) properties.
Moreover, this monoterpene has been shown to have anti-
protozoal activities against Giardia lamblia (Machado et al.
2010), Leishmania chagasi and Trypanosoma cruzi
(Escobar et al. 2010) parasites. Although the carvacrol is
widely used and well known for its medicinal properties,
this natural product has been poorly investigated for their
anthelmintic potential (Baser 2008).
The aim of this study was to evaluate in vitro effect of
carvacryl acetate, a derivative of carvacrol, on S.mansoni
adult worms. We demonstrate that a carvacryl acetate have
antischistosomal activity, affecting parasite motility, viabil-
ity, morphology and egg production. Confocal laser scan-
ning microscopy pictures are presented to underscore our
results.
Materials and methods
Compounds
Carvacryl acetate (98 % purity, Fig. 1) was obtained by
acetylation of carvacrol, using acetic anhydride as acylating
agent and pyridine as catalyst. As described below, first in a
50-mL flask, equipped with magnetic stirrer, coupled to a
Friedrich condenser and an inert atmosphere was added
carvacrol (5 g, 0.033 mol), pyridine (7.5 mL) and acetic
anhydride (12.5 mL). Then subjected to magnetic stirring
and under constant reflux for 24 h. Continuing the proce-
dure for preparing the reaction mixture was poured into ice
water (60 mL) and extracted from reaction product in a
separator funnel using chloroform as the solvent (three times
60 mL). The chloroform phases were combined and washed
with saturated copper sulphate (three times 60 mL). The
chloroform phase was washed with water (three times
60 mL) and dried with anhydrous Na
2
SO
4
. Subsequently,
the solvent was evaporated on a rotary evaporator. The
reaction product was subjected to column chromatography
using silica gel as stationary phase and a mixture of hexane/
ethyl acetate (95:5) as mobile phase. There was obtained
4.779 g (0.025 mol) of carvacryl acetate and 76 % yield
(Vogel et al. 1996). The structural identification of carvacryl
acetate was performed by analysis of
1
H and
13
CNMR, IR
and compared with literature data. Praziquantel was pur-
chased from Merck (Rio de Janeiro, RJ, Brazil).
Fig. 1 Chemical structure of carvacryl acetate
604 Parasitol Res (2013) 112:603–610
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Parasite culture and maintenance
The life cycle of S.mansoni (BH strain) is maintained in
Biomphalaria glabrata snails and Mesocricetus auratus
hamsters at the Butantan Institute (São Paulo, Brazil)
according to standard procedures previously described
(Moraes 2012). After 7 weeks, adult S.mansoni specimens
were recovered from hamster previously infected with 150
cercariae by perfusion in Roswell Park Memorial Institute
(RPMI) 1640 medium (Invitrogen, São Paulo, Brazil) with
heparin (Smithers and Terry 1965).
The maintenance and care of experimental animals com-
plied with the National Institutes of Health guidelines for the
use of laboratory animals and were approved by the
Institutional Review Board of the Butantan Institute (São
Paulo, Brazil).
In vitro antischistosomal assay
For schistosomes preparations and culture, the worms were
washed in RPMI 1640 medium kept at pH 7.5 with HEPES
20 mM, supplemented with 10 % foetal bovine serum
(Gibco BRL) and containing 200 μg/mL streptomycin and
200 IU/mL penicillin (Invitrogen, São Paulo, SP, Brazil).
After washing, one pair of adult worms was transferred to
each well of a 24-well culture plate (TPP, St. Louis, MO)
containing the same medium at 37 °C in a 5 % CO
2
atmo-
sphere as previously described (de Moraes et al. 2011;
Moraes et al. 2011; Veras et al. 2012).
The in vitro assays with S.mansoni were performed as
described in previous publications (de Moraes et al. 2011;
Moraes et al. 2011; Moraes 2012; Veras et al. 2012).
Carvacrol was dissolved in dimethyl sulfoxide (DMSO;
0.5 % final concentration; Sigma-Aldrich, St. Louis, MO)
and used at the concentrations 1.562, 3.125, 6.25, 12.5, 25
and 50 μg/mL. The final volume in each well was 2 mL.
The control worms were assayed in RPMI 1640 medium
and RPMI 1640 with 0.5 % DMSO as negative control
group and 10 μg/mL praziquantel as positive control group.
The parasites were kept for 120 h and monitored every 24 h.
The effect of the drug was assessed under an inverted
microscope (Nikon, Melville, NY) with emphasis on
changes in worm motor activity, morphological/tegumental
changes and mortality rate. All experiments were carried out
in triplicate and were repeated at least three times.
Assessment of the reproductive fitness of adult schistosomes
Schistosome egg output in vitro was determined by counting
the number of eggs, as established in our previous works (de
Moraes et al. 2011; Moraes et al. 2011; Veras et al. 2012).
Briefly, the adult worm pairs were incubated with sub-lethal
concentrations of carvacrol for up to 120 h. Changes in the
pairing and egg production were examined on daily basis
using an inverted microscope. The cumulative numbers of
eggs laid by each worm couple were established only for
those cultures containing alive and coupled male and female
worms.
Confocal laser scanning microscopy study
To observe morphological changes in the tegument of adult
parasites, schistosomes were processed for confocal laser
scanning microscopy as previously described (de Moraes
et al. 2011; Moraes et al. 2011; Moraes 2012). Briefly, at the
end of the drug treatment period (120 h) or in the case of
death, the parasites were fixed in a formalin-acetic acid-
alcohol solution (FAA) and analysed under a confocal mi-
croscope (LSM 510 META, Carl Zeiss, Standorf Göttingen,
Vertrieb, Germany) at 488-nm excitation and 505-nm emis-
sion (Moraes et al. 2009).
Statistical analysis
Statistical tests were performed with GRAPHPAD PRISM
(version 5.0) software. Significant differences were deter-
mined by a one-way analysis of variance and by applying
Tukey’s test for multiple comparisons. A difference in me-
dian was considered to be significant at a level of 5 %.
Results
The effect of carvacryl acetate on the motility and survival
time of S.mansoni
The results of the in vitro studies with 49-day-old adult S.
mansoni exposed to carvacryl acetate at concentrations of
3.125 to 25 μg/mL and control groups are summarised in
Tab l e 1. When paired worms were maintained in RPMI
1640 containing 0.5 % of DMSO for 120 h, their appearance
was similar to those maintained in the same medium without
DMSO. During this period, schistosomes showed normal
motor activity of the worm body and peristalsis of gut. In
contrast, worms exposed to positive control (10 μg/mL
praziquantel) exhibited strong spasmodic contractions im-
mediately post-exposure, followed by a significantly re-
duced motor activity of the worm body. Additionally, all
the parasites died after 24 h and tegumental damage could
be observed along the surface. These observations in the
negative and positive control groups were similar to de-
scribed in the literature (Moraes et al. 2011; Veras et al.
2012).
Furthermore, when adult schistosomes were exposed to
carvacryl acetate at concentrations of 3.125 μg/mL, they
showed normal motility for up to 120 h. On the other hand,
Parasitol Res (2013) 112:603–610 605
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when schistosomes were exposed to carvacrol at 6.25 μg/
mL or higher, a reduction in their motor activity and mor-
tality were observed (Table 1). In fact, all pairs of S.man-
soni adult worms (male and female) were dead after 24 and
120 h of incubation with carvacryl acetate at a concentration
of 6.25 and 25 μg/mL, respectively. Interestingly, similar to
positive control (10 μg/mL praziquantel), when adult
schistosomes were exposed to carvacryl acetate at concen-
trations higher than 6.25 μg/mL, strong spasmodic contrac-
tions, accompanied by very feeble movements of the oral
sucker, were seen in the all worms.
Confocal laser scanning microscopy studies
The tegumental surface of adult S.mansoni has been de-
scribed in a number of confocal laser scanning microscopy
studies (de Moraes et al. 2011,2012; Moraes et al. 2011;
Moraes 2012; Veras et al. 2012). In the absence of the drug,
schistosomes showed normal viability without any tegu-
mental changes for up to 120 h. In the presence of carvacryl
acetate at concentrations of 6.25 to 25 μg/mL, spasmodic
contraction of the worm’s musculature was observed and
some of the tubercles appeared to be swollen, with numer-
ous small blebs emerging from the tegument around the
tubercles. These morphological changes induced by car-
vacryl acetate depended on the concentrations used (Fig. 2).
The effect of carvacryl acetate on egg prodution
S.mansoni is a trematode parasite that is dioecious and
sexually dimorphic. Males have a ventral groove called the
gynecophoral canal in which females reside for maturation,
mating and egg production (Steinauer 2009). Given the
importance of the eggs to disease progression and transmis-
sion, the ability of carvacryl acetate to promote separation of
the coupled worms into individual male and female and/or
to inhibit the oviposition was also investigated. These
experiments were performed using carvacryl acetate at
sub-lethal concentrations (ranging from 1.562 to 6.25 μg/
mL), and the parasites were monitored every 24 h for up to
120 h. The parasites incubated with the carvacryl acetate
and those belonging to the negative control groups remained
coupled until the end of the incubation period. As shown in
Fig. 3, the total number of eggs laid was significantly
reduced (P<0.001). The highest sub-lethal concentration
of carvacryl acetate (6.25 μg/mL) caused approximately a
90 % reduction in egg production when compared with the
untreated worms. Thus, carvacryl acetate is toxic at higher
doses, while at sub-lethal doses, it significantly interferes
with the reproductive fitness of S.mansoni adult worms.
Discussion
Only one drug, praziquantel, is widely available for treating
schistosomiasis, a parasitic disease that affects more than
200 million people worldwide. Currently, concerns over
praziquantel resistance have renewed interest in the search
for alternative drug therapies (Botros and Bennett 2007;
Caffrey 2007). In this context, plants and their natural
Table 1 In vitro effects of carvacryl acetate against 49-day-old adult
Schistosoma mansoni
Group Incubation
period (h)
Dead
worms
(%)
a
Motor activity
reduction (%)
a
Slight Significant
Control
b
24 0 0 0
48 0 0 0
72 0 0 0
96 0 0 0
120 0 0 0
0.5 % DMSO 24 0 0 0
48 0 0 0
72 0 0 0
96 0 0 0
120 0 0 0
Praziquantel
at 10 μg/mL
24 100 0 100
48 100 0 100
72 100 0 100
96 100 0 100
120 100 0 100
Carvacryl acetate
at 3.125 μg/mL
24 0 0 0
48 0 0 0
72 0 0 0
96 0 0 0
120 0 0 0
Carvacryl acetate
at 6.25 μg/mL
24 0 0 0
48 0 70 30
72 0 20 80
96 100 0 100
120 100 0 100
Carvacryl acetate
at 12.5 μg/mL
24 0 0 0
48 40 20 80
72 100 0 100
96 100 0 100
120 100 0 100
Carvacryl acetate
at 25 μg/mL
24 100 0 100
48 100 0 100
72 100 0 100
96 100 0 100
120 100 0 100
a
Percentages relative to the 20 worms investigated
b
RPMI 1640
606 Parasitol Res (2013) 112:603–610
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products constitute good sources of interesting lead com-
pounds that could be potentially active against schistosomes
(Moraes 2012). Carvacrol is the major constituent of some
essential oils from medicinal and aromatic plants. It is a safe
food additive and several studies have indicated that carva-
crol has biocidal activities against bacteria, fungi, protozoa
and arthropods (Baser 2008). In the present study, we
assessed the in vitro anthelmintic activity of carvacryl ace-
tate, a derivative of carvacrol, on S.mansoni. To our knowl-
edge, we have for the first time evaluated the effect of this
natural product-derived compound against schistosomes.
The results showed that both male and female parasites
are susceptible to carvacryl acetate (Table 1). It is well
known that male and female of S.mansoni worms differ in
their sensitivity to therapeutic. For example, male worms are
often more susceptible than female worms in ginger extract
(Zinger officinale) and praziquantel studies (Staudt et al.
1992; Sanderson et al. 2002; Pica-Mattoccia and Cioli
2004). In contrast, Mitsui et al. (2009) showed higher sur-
vival rates for male than for female worms in artenusate
studies. Remarkably, our data demonstrated that carvacryl
acetate exhibited an optimal in vitro activity against the
Fig. 2 Confocal laser scanning
microscopy investigations of
carvacryl acetate in vitro
schistosomicidal effect. In these
experiments, pairs of adult
worms were incubated in 24-
well culture plates containing
RPMI 1640 medium and treated
with different concentrations of
carvacryl acetate. After 120 h of
incubation or in the case of
death, adult male worms were
fixed in FAA solution and the
fluorescent images were
obtained using confocal mi-
croscopy. aControl worms. b
Worms treated with 6.25, c12.5
and d25 μg/mL of carvacryl
acetate. Blebbing (arrows)on
the dorsal mid-body tegument
of a male worm are visible.
Scale bars,50μm
Fig. 3 Effect of carvacryl
acetate on S.mansoni
oviposition. Adult worm
couples were incubated in 24-
well culture plates containing
RPMI 1640 medium and car-
vacryl acetate at the indicated
concentrations. At the indicated
time periods, the cumulative
number of eggs per worm cou-
ple was scored using an
inverted microscope. Values are
the means±SD (bars) of ten
worm couples. *P<0.05; **P<
0.01; ***P<0.001 compared
with untreated groups
Parasitol Res (2013) 112:603–610 607
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adult stage with no differential sensitivity between male and
female worms. Not only carvacryl acetate, but also other plant
constituents such as amide piplartine, alkaloid epiisopiloturine
and essential oils, have shown no differences between male
and female worms (Caixeta et al. 2011;Moraesetal.2011;
Magalhães et al. 2012; Veras et al. 2012).
The tegument of schistosomes is an important target for
antischistosomal drugs (Moraes 2012). Using electron mi-
croscopy, including scanning electron microscopy and trans-
mission electron microscopy, or confocal laser scanning
microscopy, alterations in the tegument of Schistosoma spe-
cies have been documented for various antischistosomal
drugs such as praziquantel and its enantiomers, artemether
and artenusate, mefloquine, miltefosine, epiisopiloturine
and piplartine (Staudt et al. 1992; Jiraungkoorskul et al.
2005,2006; Manneck et al. 2010; Eissa et al. 2011;
Moraes et al. 2011; Veras et al. 2012). In this study, the
effect of carvacryl acetate on the tegumental surface of adult
schistosomes was monitored using a confocal laser scanning
microscope. Because the molecular weight of carvacryl ace-
tate is very small (only 192.26 g/mol) and it has a lipophilic
profile (Yu et al. 2012), it is reasonable to believe that car-
vacryl acetate can easily and rapidly cross the tegument–
helminth barrier. In fact, in the present study, we observed
tegumental damage on the S.mansoni adult worms, which
intensified progressively as the incubation period and the
concentration of carvacryl acetate increased. Blebbing was
visible on the tegument of schistosomes (Fig. 2). Blebbing is
an indicator for stress and has been observed in previous
studies evaluating antischistosomal drugs such as mefloquine,
miltefosine, artesunate and praziquantel (Manneck et al. 2010;
Eissa et al. 2011; Jiraungkoorskul et al. 2005,2006).
The reproductive fitness of schistosomes was also evalu-
ated in this study by pairing and egg production (Veras et al.
2012). It has been reported that other plant constituents,
such as curcumin, piplartine, epiisopiloturine, as well as
essential oils have effects on the egg production (de Melo
et al. 2011; de Oliveira et al. 2012; Magalhães et al. 2009;
Moraes et al. 2011; Veras et al. 2012). In the present study,
we observed that carvacryl acetate reduced egg output with-
out affecting pairing (Fig. 3), but the mechanism remains to
be elucidated. However, evidence for this mechanism has
been described (Araujo et al. 2002; Irie et al. 1983). The
reduction in oviposition seems to be related to degenerative
modifications in the reproductive system, with reduction
and alteration of the viteline follicles and the ovary of the
females as well as modifications in the male’s testicles.
The emergence of parasites resistant to current chemo-
therapies highlights the importance of plant essential oils as
novel antiparasitic agents; however, few oils have been tested
against helminthes (Anthony et al. 2005). The biological
properties of terpenes on different parasitic diseases are still
unclear. The mechanisms of action seem to be related to the
interaction with the parasite redox cycling system leading to
enzyme inhibition and parasite killing (Anthony et al. 2005).
Further studies should be conducted to clarify the anthelmintic
effect of carvacryl acetate on S.mansoni.
Conclusions
In this study, our data demonstrated for the first time that
carvacryl acetate has in vitro anthelmintic properties against
S.mansoni. This natural product-derived compound de-
creased the motor activity and was able to cause the death
of S.mansoni adult worms. In addition, confocal laser
scanning studies also revealed that carvacryl acetate caused
alterations in the tegument of adult schistosomes, including
bleb formation. Furthermore, an inhibitory effect of car-
vacryl acetate on the daily egg output of paired adult worms
during in vitro cultivation was also observed. Because car-
vacrol is widely used as a food additive and its safety is well
studied before (Yu et al. 2012 and references therein), it has
potential as an agent to control Schistosoma infection.
Further studies are necessary, however, to elucidate its mode
of action and actual in vivo application.
Acknowledgements We thank Mr. Alexsander S. Souza (Parasitolo-
gy Laboratory, Butantan Institute, São Paulo, SP, Brazil) for expert
help with the confocal microscopy studies. This work was supported
by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP,
Brazil Grant numbers: 06/56216-8 and 00/11624-5).
Conflict of interest The authors declare that there are no conflicts of
interest.
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