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To cite this article: Mohammed H. Mona, Nahla E. E. Omran, Merveet A. Mansoor & Zainab
M. El-Fakharany (2012) Antischistosomal effect of holothurin extracted from some Egyptian sea
cucumbers, Pharmaceutical Biology, 50:9, 1144-1150, DOI: 10.3109/13880209.2012.661741
research ARTICLE
Abstract
Context: Holothuria polii (H. polii) Linnaeus (Holothuriidae), Actinopyga mauritiana (A. mauritiana) Quoy & Gaimard
(Holothuriidae) and Bohadschia vitiensis(B. vitiensis) Semper (Holothuriidae) are sea cucumbers inhabiting the coasts
of Egypt. Their tegument and the cuvierian gland contained a substance called holothurin that was used in traditional
medicine. These three species are abundant in the Egyptian coast, however there are no reports about their efficacy
as antiparasitic agent.
Objective: The antischistosomal effect of the holothurin extracted from the three species of sea cucumber is
investigated.
Materials and methods: The ethanol extract was made from the tegument of both H. polii and A. mauritiana while
it was made from the cuvierian gland of B. vitiensis. The body wall (or cuvierian gland) of the sea cucumber was
blended with 95% ethanol in a volume = 4 tissue weight. Extraction was done at room temperature for one day
then filtered. The ethanol was removed by evaporation using Rotavapour (BCHI 461 water bath REIII) at 40C. Later
the aqueous residue was placed in a vacuum oven at 20C for about 48 h to remove water. The resulting dried mass
was then stored at 4C until use. The percentage yield and the LD50 were calculated for each extract. Each extract
was administered orally to Shistosoma mansoni infected mice in acute and chronic phases of infection. The dose of
one-tenth of LD50 of each extract was administrated to mice (5.4, 62.2, and 10 mg/kg body weight/mouse for H. polii
extract (HPE), A. mauritiana extract (AME), and cuvierian gland of B. vitiensis, respectively) for 24 h. The effects of each
extract on the worm burden and total egg count was studied. The effects of each extract on the worm tegument
using scanning electron microscope (SEM) were investigated in vivo and in vitro.
Results: The percentage yield of cuvierian gland extract (CGE) was higher (70%) than the tegument AME (33.4%) and HPE
(9.3%). The 24 h LD50 of investigated sea cucumber ethanol extracts were 54.46, 627, and 100 mg/kg body weight/mouse
for HPE, AME, and CGE. Oral administration of HPE caused decrease in male and female worm burden of 30-day infected
mice to reach 60 and 90%, respectively. HPE decreased the egg count significantly in those mice with 30-day (1.75 egg
counts/g tissue, p < 0.05) and 45-day (3.25 egg counts/g tissue, p < 0.05) infections. SEM studies of recovered worms
from treated mice with all extracts showed different tegumental changes like formation of blebs, wrinkling, formation
of numerous pores, and rupturing of some tubercles. These effects were more pronounced in those worms treated in
vitro represented by severe shrinkage of the tegument, deformation of spines, rupturing, and collapsing of tubercles.
Discussion and conclusion: Results support the hypothesis that holothurin is a promising antischistosomal agent.
Keywords: Antiparasitic, glycoside, tegument
Introduction
in Malaysia (Hassan et al., 1996). Modern research has
Sea cucumber is a worldwide echinoderm of the class shown that sea cucumber extracts have wound heal-
Holothuroidea, with an elongated body and leathery ing effects (Hassan et al., 1996), besides, they contain
skin, which is found on the sea floor. Sea cucumbers anticoagulant, antithrombotic compounds, cholesterol,
have long been used as a source of traditional medicines lipid reducing compounds (Zancan & Mourao, 2004),
Address for Correspondence: Nahla E. E. Omran, Medical Campus, El-Bahr Street, Faculty of Science, Tanta University, Tanta, Egypt.
E-mail: nahlaayo@hotmail.com
(Received 25 August 2011; revised 08 November 2011; accepted 10 January 2012)
1144
Antischistosomal effect of holothurin 1145
anticancer compounds (Hatakeyama etal., 2002; Kariya glycone (Figure 1B). This type of glycoside was named
et al., 2004), antiinflammatory (Yamonouchi, 1955; bohadschioside (Stonik, 1986).
Nigrelli, 1972; Idid et al., 2001) and an antifungal com- Schistosomiasis is a disease caused by an infection
pounds (Sedov etal., 1990; Omran, 2006). Yoshida etal. with parasitic blood flukes of the genus Schistosoma. This
(2007) found that a lectin from sea cucumber Cucumaria fluke is distributed across Africa, the Arabian Peninsula,
echinata Von Marenzeller (Cucumariidae) impaired the South America, and some locations in the Caribbean.
development of the malaria parasite when produced by More than 2 billion people, about one third of the worlds
transgenic mosquitoes. population, are infected with these parasitic helmint-
Yamonouchi (1955) was the first to coin the term hes (WHO, 2005). Several schistosomocide drugs were
holothurin to signify the biologically active compound administered to treat schistosomiasis, but praziquantel
produced by the body wall of holothuroid cucumbers. (PZQ) showed the successful results in the treatment
This compound is found also in the cuvierian tubules because of its effectiveness and insignificant side effects
and the coelomic fluid. Holothurin was isolated from (Adam etal., 2005). However, after 30 years of PZQ usage,
Pacific, Indo Pacific, and Atlantic species belonging to a decreased susceptibility to the drug and the emergence
the Holothuriidae family and particularly from species of drug-resistant strains was reported (Ismail etal., 1999;
of Holothuria and Actinopyga genera (Levin etal., 1984). King etal., 2000; Botros etal., 2005; Riad etal., 2009).
The structure of the holothurin extracted from the two The present study investigated the antiparasitic activity
latter species has been established by Kitagawa et al. of sea cucumber extract against Schistosoma mansoni (S.
(1978, 1982). Holothurin formed from triterpene glyco- mansoni) as a model of endemic parasite in Egypt. Three
sides possess a 12--hydroxy-9(11)-ene fragment in the species of sea cucumber were used Holothuria polii (H.
glycone and disaccharide or tetrasaccharide part with an polii), Bohadschia vitiensis (B. vitiensis), and Actinopyga
O-sulphate group (Figure 1A). This structure is restricted mauritiana (A. mauritiana). The efficacy of the holothu-
to species of Holothuria and Actinopyga genera (Stonik, rin was tested at the acute and the chronic phases of the
1986), while holothurin extracted from Bohadschia spe- infection and estimated by monitoring the worm burden,
cies contains glycosides without an O-sulphate group egg count, and the worm surface topography in vivo and
and with holost-9-(11)-en-12--3-ol as a genuine in vitro using scanning electron microscope (SEM).
Mice infection
Collection of S. mansoni cercariae and the infection of
mice were carried out according to Christensen et al.
(1984). The mice were individually exposed to about 100
10 cercariae by the tail immersion technique.
Praziquantel
This drug is a chemotherapeutic treatment of schis-
tosome infection. It was purchased from Alexandria
Company for drugs and chemicals (Alexandria, Egypt).
PZQ was administered at days 30 and 45 in one oral dose
of 685 g/kg (b.w.) (El-Bolkiny & Al-Sharkawi, 1997).
Figure 1.Structure of some glycosides from Holothurin family Collection of sea cucumbers
(Stonik 1986): A, Holothurin structure from Holothuria and The sea cucumber H. polii was collected from the inter-
Actinopyga genera. B, Bohadschiosides from genus Bohadschia. tidal region at Abu-Kir coast (Mediterranean Sea) during
Pharmaceutical Biology
Antischistosomal effect of holothurin 1147
Table 1. Lethal dose values after 24 h exposure time to Sea Figure 7. The effect of different sea cucumber extracts on the worm
Cucumber extracts. burden of 45-days infected mice. HPE, Holothuria polii extract;
Species LD50 AME, Actinopyga mauritiana extract; CGE, cuvierian gland extract;
Holothuria polii 54.46 mg/mouse PZQ, praziquantel; cont 30. Control infected mice for 30 days.
Bohadschia vitiensis 100 mg/mouse
actinopyga Mauritiana 627.1 mg/mouse
Discussion
The emergence of drug-resistant schistosome strains has
brought great attention toward natural bioactive com-
pounds. Several studies have been done to evaluate natu-
ral substances of plant origin as antischistosomal agents
such as Curcuma longa (Rizk etal., 2000), myrrh (Massoud,
1998; Sheir etal., 2001), garlic (Riad etal., 2009), and ginger
(Al-Sharkawi etal., 2007; Mostafa etal., 2011). To the best
of our knowledge the current study is the first trial to use
this substance (holothurin) as an antischistosomal agent.
Among the investigated extracts, HPE showed
decreased male and female worm burden to 60 and 90%,
respectively, in 30-day infected mice. This reduction was
accompanied with liver egg count reduction that reached
56%. Comparing these results with those obtained from
PZQ treated mice revealed that HPE may be a promising
antischistosomal agent. Another point that should be
taken into consideration is that the effect of HPE is signif-
icant when administered at an early stage of infection (30
days of infection; the acute phase). Comparable findings
were obtained by Riad etal. (2007) who reported a sig-
nificant reduction in the egg load after treating infected
mice with aqueous garlic extract at acute phase.
Despite the significant effect of HPE during the early
stage of infection, it did not reveal any significant change in
the worm burden of 45 day-infected mice. It caused, how-
ever, 56% reduction in the liver egg count. Accordingly, it
can be suggested that the reduction in the egg count is not
due to the decrease in the worm burden but it might be
due to its ability to decrease worm fecundity.
Recovered worms from treated mice with all extracts
showed different tegumental changes like formation of
blebs, wrinkling, formation of numerous pores, and rup-
turing of some tubercles. These tegumental alterations
would impair the functioning of the defence system of
the worm, so that it could easily be attacked by the hosts
immune system (Xiao etal., 2000). Moreover, it results in
the inability of the worm to adhere to the walls of the host
blood vessels, causing the schistosome to be dislodged
and moved by blood stream from mesenteric veins to
the portal vein and intravenous hepatic capillaries and
become lodged in the liver which is followed by the death
and disintegration of the parasites (Mehlhorn etal., 1981;
Figure 9. Photomicrographs of control and treated Schistosoma Riad etal., 2009; Gnanasekar etal., 2009).
mansoni tegument. A, Control male worm showing os, vs and gpc. There is no available information about the effect of
B, The tegument of the middle part of the control worm covered
with Tb armed with s and sponge like Tg. C, In vivo treated worms holothurin on the tegument. But there are other related
with HPE showing a few blebs (B) and transverse folds (F). D, studies about the effect of antischistosome drugs that
In vivo treated worm with HPE showing the RTg and deformed reported tubercular disruption and loss of tubercular
Tb. E, In vivo treated male worms with AME showing transverse spines as a result of treatment (Amin & Mikhail, 1989;
folds (F) and (RTb). F, In vivo treated worms with CGE showing Mostafa & Soliman 2002; Shaohong etal., 2006; Taha &
the tegument with numerous blebs (B) and rough tegument with
transverse folds (F). G, In vitro treated worm with AME showing Soliman 2007; Riad etal., 2009).
severe shrinkage of both Tg and vs. H, In vitro treated worm with Although the in vivo administration of AME and
AME showing CTb with deformed s. I, In vitro treated worm with CGE showed no effect on the worm burden or on the
CGE showing CTb and RTb. J, In vitro treated worm with HPE egg count, the SEM revealed severe destruction of the
showing RTb and CTb. os, oral sucker; vs, ventral sucker; gpc, worm tegument as manifested by severe shrinkage of the
gynechophoric canal; Tb, tubercles; s, spines; Tg, tegument; HPE,
Holothuria polii extract; RTg, rough tegument; AME, Actinopyga tegument, collapsing and rupturing of tubercles beside
mauritiana extract; RTb, ruptured tubercles; CGE, cuvierian deformation of spines, suggesting that these two extracts
gland extract; CTb, collapsed tubercles. express strong schistosomocide. These defects could
Pharmaceutical Biology
Antischistosomal effect of holothurin 1149
be attributed to the act of their metabolic pathways in Ismail M, Botros S, Metwally A, William S, Farghally A, Tao LF, Day
infected mice that suppressed the activity of the extracts TA, Bennett JL. (1999). Resistance to praziquantel: direct evidence
from Schistosoma mansoni isolated from Egyptian villagers. Am J
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regimen or to the period of treatment that resulted in Kariya Y, Mulloy B, Imai K, Tominaga A, Kaneko T, Asari A, Suzuki
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reach mesenteric and portal vessels. partial characterization of fucan sulfates from the body wall of
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Conclusion King CH, Muchiri EM, Ouma JH. (2000). Evidence against
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antiparasitic drug with conventional therapy such as PZQ. Kitagawa I, Yamanaka H, Kobayashi M, Nishino T, Yosioka I, Sugawara
Further study must be conducted to improve the effect of T. (1978). Saponins and sapogenols XXVII. Revised structures of
holotoxin A and holotoxin B, two antifungal oligoglycosides from
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Declaration of interest taxonomic revision of holothurian Bohadschia graeffei (Semper) as
The authors report no conflicts of interest. refer to elucidation of a new genus. Biol Morya, 3, 3338.
Massoud A, Salama O, Bennett JL. (1998). Therapeutic efficacy of new
antischistosomal drug, derived from myrrh, in active intestinal
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