Bioactivity of aqueous and organic extracts of sea cucumber Holothuria leucospilota (Brandt 1835) on pathogenic Candida and Streptococci
Bioactivity of aqueous and organic extracts of sea cucumber Holothuria leucospilota (Brandt 1835) on pathogenic Candida and Streptococci
0 B. Heidari Department of Marine Sciences, Caspian Sea Basin Research Center, University of Guilan , Rasht , Iran
1 M. Ghadiri S. Kazemi B. Heidari (&) M. Rassa Department of Biology, Faculty of Science, University of Guilan , Namjoo St., Rasht 4193833697 , Iran
Holothurians (sea cucumbers) exist almost in all benthic marine environments and they are an important species for the commercial fisheries. In this study, the antimicrobial activity of different tissues of the sea cucumber, as a marine invertebrate, was investigated. The bacteria, Streptococcus mutans and Streptococcus sobrinus, the fungi, Candida parapsilosis, Candida albicans and Candida glabrata, were chosen for antimicrobial test, because they are opportunistic pathogens. The body wall, respiratory tree, and gastrointestinal tract of the sea cucumbers were utilized to make the extracts. Phosphate-buffered saline (PBS), ethanol (Et), and acetonitrile (ACN) were used as solvents for aqueous and organic extractions. The results showed that there were antimicrobial active compounds against infective streptococci and candid fungi. Streptococcus mutans was generally more tolerant than Streptococcus sobrinus to the sea cucumber extracts. The extracts of the respiratory tree with ACN and PBS, as solvents, showed the highest effect against Candida albicans, Candida parapsilosis and Candida glabrata. The extracts of gastrointestinal tract and the body wall with Et and PBS, respectively, had not any antifungal activity. The antimicrobial activities had been found in different extracts of the sea cucumber tissues, which could be related to the protein and non-protein compounds.
During the last decades, the marine natural products have been attracted much attentions of biologists and
chemists of all around the world. Marine organisms produce natural products which can be pharmaceutically
useful or being a toxic compound. During the last decade, researches on marine crustaceans, sponges, jellyfish,
sea anemones, corals, bryozoans, mollusks, tunicates and echinoderms have been increased, and these
investigations have been specially focused on desirable antimicrobial properties
(Bhakuni and Rawat 2005;
Casas et al. 2011; Haug et al. 2002)
. Echinoderms are a group of benthic organisms, which are exposed to
relatively high concentrations of pathogens which can be harmful to the animal
(Haug et al. 2002)
cucumbers are a class of Holothuroidea with body shape of a worm and without any spines.
Many sea cucumbers have novel defensive mechanisms which compensates their lack of spines
. The sea cucumbers have multiple biological activities such as wound healing
(Fredalina et al. 1999)
(Gowda et al. 2008; Haug et al. 2002; Kiani et al. 2014; Ridzwan et al. 1995)
et al. 2012)
, agglutinating activities
(De Melo et al. 2014; Gowda et al. 2008)
and antioxidant properties
(Osama et al. 2009)
Different habitats of human oral cavity such as teeth, gingival sulcus, tongue, cheeks, hard and soft palates,
and tonsils can be colonized easily by bacteria
(Dewhirst et al. 2010)
. The oral diseases including dental caries
created by aerobic and anaerobic bacteria are of the most major health problems in the world
(Aas et al. 2008;
Anusavice 2002; Becker et al. 2002; Beighton 2005; Chhour et al. 2005)
. The streptococci are the major dental
caries aetiologic agents and they are the most common pathogens isolated from the human dental plaque and
play an important role in the development and progression of dental caries
(Nishikawara et al. 2006; Okada
et al. 2005)
. Streptococcus mutans (Clark 1924) and Streptococcus sobrinus (Coykendall 1974) are the two
species which are responsible for the dental caries
(Ghasempour et al. 2013; Nascimento et al. 2004; Okada
et al. 2005)
. These groups of bacterial species are characterized by their ability of producing extracellular
glucans from sucrose and via their acidic productions in animal and human studies
(Ghasempour et al. 2013;
Nishikawara et al. 2006)
The Candida strains are the main factor for serious fungal infections such as vulvovaginal candidiasis
(VVC) which can infect 75% of adult women during their life
(Sharanappa and Vidyasagar 2013)
, and it also
increases the chance of cutaneous infections, vaginitis, intestinal and systemic infections
(Molero et al. 1998)
The Candida strains are considered to be the fourth effective factor in hematological infections for patients
hospitalized and this kind of infection is responsible for about 40% of death
(Anaissie et al. 2003)
opportunistic fungal strains such as C. albicans are more frequent than other species and they can be isolated
from oral infections
(Kantarcioglu and Yucel 2002)
. The other Candida species with direct effects can cause
systemic infections in immunosuppressant patients (such as AIDS, cancer patients and solid-organ transplant
recipients). Because of the pathogenic microbe resistances to the conventional antibiotics, the calls for the
investigations on new antimicrobials metabolites have been increased. The marine biological reserves,
especially 17 species of Holothurians
(Dabbagh et al. 2011)
of the Persian Gulf, have high potential for
antimicrobial investigations, but their biological benefits are unknown due to their unfamiliarity. Therefore, in
this work, the antimicrobial effects of various extracts of different tissues of the Persian Gulf sea cucumber on
the infectious oral streptococci and Candida strains were investigated.
Materials and methods
Sampling and preparation of specimens
Live specimens of the sea cucumbers (N = 10) were collected via scuba diving off (4–5 m depth) from
Qeshm Island of the Persian Gulf in October (Fig. 1). The samples were transferred on ice to the Marine
Biology Laboratory of the University of Guilan. After dissection, all tissues of animals including body wall,
respiratory tree and gastrointestinal tract were separately frozen in liquid nitrogen for 5 min and stored at
- 70 C until the extraction process. The specimens were identified using ossicles obtained from the
epidermal tissues of dorsal and ventral surfaces of the body wall
(Kiani et al. 2014)
Preparation of extracts
The tissues (body wall, respiratory tree and gastrointestinal tract) were separately homogenized (Misonix
Sonicator 3000, USA) with PBS (NaH2PO4 2H2O, 0.1 M; NaOH, 0.2 M) (1:5 w/v) for 5 min. The obtained
samples were shaken (Infros, RFI-150) on ice for 2 h at 120 rpm and then they were centrifuged at 4000 rpm
for 10 min at 4 C. The supernatants were collected and stored at 4 C, and the residues were centrifuged in
two volumes of PBS. Then, the combined supernatants were centrifuged and lyophilized by freeze–dryer
(Christ Alpha 1–2/LD, Osterode am Harz, Germany) for 9 h (- 55 C and 0.02 mbar) and they were stored at
- 70 C until antibacterial test.
Et and ACN extracts
The organic extracts have been prepared with Et 96% and ACN (Merck Schuchardt, Germany). As above, all
tissues were separately homogenized in Et and ACN with combination of (1:5 w/v) for 5 min. Subsequently,
the solutions were shaken at 40 C for 24 h (120 rpm) and centrifuged at 4000 rpm for 10 min at 4 C. The
supernatants were sampled and the residues were centrifuged in two volumes of the solvents. Finally, the
collected supernatants were lyophilized by freeze-dryer for 9 h (- 55 C and 0.02 mbar) and stored at
- 70 C until antibacterial test.
Bacterial and yeast strains
The Gram-positive bacteria Streptococcus mutans (ATCC 35668) and Streptococcus sobrinus (ATCC 27607)
were purchased from the Persian Type Culture Collection (PTCC, Tehran, Iran) and were used as test
microorganisms. The strains were grown on blood agar (Biolife, Italy) containing 7% fresh horse blood and
maintained at 37 C for 48 h. The candida strains Candida albicans (5027 ATCC 10231), C. parapsilosis
(5297 DSM 11226) were obtained from the Persian Type Culture Collection (PTCC, Tehran, Iran) as well as
C. glabrata (30005) was purchased from Iranian Biological Resource Center (IBRC-M, Tehran, Iran).
The antibacterial activities of the aqueous (PBS) and organic extractions were evaluated by the well diffusion
(Kiani et al. 2014)
. S. mutans and S. sobrinus were cultured in blood agar for 24 h. The crude aqueous
and organic extracts were tested separately with two concentrations of 600 and 1500 lg were dissolved in 40
lL of double distilled water (d.d.H2O) and dimethyl sulfoxide (DMSO) (Merck, Germany) as solvents,
respectively. After that, the dissolved extracts were transferred into the wells (diameter 6 mm), punched out
on plates, containing the bacteria and incubated at 37 C for 24 h. The antibacterial activities were determined
by measuring the diameter of zone of inhibition (mm).
Vancomycin (30 lg disc-1), Tetracycline (30 lg disc-1), Chloramphenicol (30 lg disc-1), Nalidixic acid
(30 lg disc-1), Sulfadiazine (1.0 lg disc-1), Oxacillin (1 lg disc-1), Bacitracin (15 lg disc-1), and
Cefepime (30 lg disc-1) were utilized as positive controls. DMSO (40 lL well-1) and dd H2O (40 lL well-1)
were also tested as negative controls to ensure that they did not interfere with the tests.
The antifungal activities of the sea cucumber extracts were evaluated using Sabouraud Dextrose Agar (SDA)
well diffusion method
(Periyasamy et al. 2012; Stepanovic et al. 2003)
against Candida albicans (Persian
Type Culture Collection (PTCC): 5027 ATCC 10231), C. parapsilosis (PTCC: 5297 DSM 11226) and C.
glabrata (Iranian Biological Resource Center (IBRC-M): 30005). All Candida strains were unloaded from
lyophilized ampoules to SDA media after dissolving in 0.5 mL of the distilled water and cultivated by linear
method. The organic and aqueous extracts with two concentrations of 600 and 1500 lg were dissolved in 30
lL of DMSO and 30 lL of distilled water, respectively. All extractions were added to the labeled wells with
diameter 6 mm on the plates containing the fungi, and then, the loaded plates were incubated at 37 C for
Three antibiotics drugs including Ketoconazole, Fluconazole and Nystatin (with concentration 30 lg mL-1)
were prepared to be used as positive controls, DMSO (40 lL well-1) and distilled water (40 lL well-1) were
also utilized as negative controls.
To test whether the sea cucumber contain factors that display toxic effect on eukaryotic cells, the hemolytic
activity of aqueous and organic extracts obtained from body wall, respiratory tree and gastrointestinal tract
was determined. The assay was performed using the method described by
Haug et al. (2002)
heparinized horse red blood cells (RBCs) washed three times in PBS (pH 7) and they were suspended in PBS
to reach to a hematocrit value of 10%. The samples were diluted to a protein concentration of 500 lg mL-1.
The test mixture consisted of 40 lL PBS, 50 lL extract, and 10 lL of the RBCs suspension which was
incubated at 37 C for 1 h and then it was centrifuged at 3000 rpm for 10 min. Finally, the absorbance of the
supernatants (60 lL) was measured at wavelength of 550 nm. Baseline hemolysis and 100% hemolysis were
defined as the amount of hemoglobin released in the presence of PBS and 0.1% Triton X-100 (Sigma),
The specimens were identified using the ossicle of the sea cucumber. After taking digital photographs of
ossicle, the identification of species was performed via the diagnostic keys
The raw data were initially tested versus the assumptions of normality and homogeneity of variance.
Levene’s test and Kolmogorov–Smirnov test were used to assess the homogeneity and normality of variance,
respectively. The variances of the populations from which different samples were drawn were equal. The
oneway ANOVA followed by Duncan test with a confidence level of 95% were used to determine the differences
between various extracts using IBM SPSS Statistics 19. The significant differences between the two
concentrations of 600 and 1500 lg well-1 were assessed by independent t test. All experiments (treatments
and controls) were carried out for three times and the obtained data were reported as the mean ± SD.
Due to the dorsal and ventral ossicles obtained from epidermal tissues of the body wall, it was concluded that
the studied species was Holothuria leucospilota (Brandt 1835) (Fig. 2).
Due to the results, all tissues of the echinoderm species showed antimicrobial activities against oral
streptococci and Candida sp.
Extract activities against S. Sobrinus and S. mutans
At concentration of 1500 lg well-1 of the various extracts of different tissues, antibacterial effects against S.
sobrinus and S. mutans were observed (Fig. 3). The maximum diameter of inhibition zones (statistically
significant, at p \ 0.05 level) was belonged to the ACN extract of the respiratory tree and Et extract of the
body wall, while the other extracts showed similar inhibitory effects on the growth of S. sobrinus (statistically
insignificant, at p [ 0.05 level) (Fig. 3).
At concentration of 1500 lg.well-1, the maximum diameter of inhibition zone against S. mutans was
obtained by the PBS extract of gastrointestinal tract (9.67 mm) while the ACN extracts of gastrointestinal tract
and respiratory tree had less significant inhibitory effects, respectively (p \ 0.05) (Fig. 3). The other extracts
had no effects on S. mutans.
The ACN and Et extracts of gastrointestinal tract, at concentration of 600 lg well-1, had no antibacterial
effects against S. sobrinus. The highest antibacterial effect on S. sobrinus was observed for the ACN extract of
the respiratory tree (p \ 0.05) (Fig. 4). There was no significant difference (p [ 0.05) between the
antibacterial effects of the various extracts of the body and the PBS extract of gastrointestinal tract (Fig. 4). The Et
extract of the respiratory tree had no effect on S. sobrinus at both concentrations.
In concentration of 600 lg well-1, only the PBS extract of gastrointestinal tract had inhibitory effect on the
growth of S. mutans (Fig. 4).
The comparison of antibacterial effects of two concentrations using statistical independent t test showed
that the extracts with concentration of 1500 lg well-1 had overall more significant effect (p \ 0.05) against S.
sobrinus and S. mutans (Fig. 5a, b).
Activity of extracts with a concentration of 600 lg.well-1 against Candida sp.
The ACN extract of the respiratory tree and the body wall had significantly the maximum and minimum
inhibitory effects on the growth of C. parapsilosis, respectively (p \ 0.05) (Fig. 6). The other extracts had no
significant antifungal activity (p [ 0.05).
The ACN extracts of the body wall, respiratory tree and gastrointestinal tract showed the highest antifungal
activity on C. albicans and followed by the Et extracts of the respiratory tree and body wall (p \ 0.05)
The maximum inhibitory activity against C. glabrata belonged to the PBS extract of the respiratory tree and
followed by the ACN extract of the body wall (p \ 0.05). In addition, there were no significant differences
between antifungal effects of the ACN extract of the respiratory tree and the PBS extract of gastrointestinal
tract (p [ 0.05) (Fig. 6).
Activity of extracts with a concentration of 1500 lg.well-1 against Candida sp.
The Et. extracts of all tissues of the sea cucumber had significantly higher antifungal activities than the other
extracts against C. parapsilosis (p \ 0.05) (Fig. 6). The ACN extracts of all tissues had the same antifungal
effects (p [ 0.05).
The maximum inhibitory activity against C. albicans remarkably belonged to the ACN extract of the
respiratory tree (p \ 0.05) (Fig. 7). The ACN and Et extracts of the gastrointestinal tract as well as the Et
extract of the body wall had the same inhibitory effects (p [ 0.05).
The extracts of the different tissues of the sea cucumber showed the diverse significant antifungal effects on
C. glabrata (Fig. 7). The ACN extract of the respiratory tree had significantly the maximum inhibitory effect
(p \ 0.05). The Et and PBS extracts like ACN extracts of the respiratory tree and gastrointestinal tract had the
minimum antifungal effects (p \ 0.05). In addition, the ACN extract of the body wall and PBS extract of the
gastrointestinal tract had higher effect than Et extract of the body wall (p \ 0.05) (Fig. 7).
Hemolytic activity of the sea cucumber extracts
The hemolytic activity of the PBS, Et and ACN extracts from the body wall, respiratory tree and
gastrointestinal tract of H. leucospilota was evaluated using horse red blood cells. Several extracts showed high
hemolytic activity (Table 1). In general, the highest and lowest hemolytic activities were obtained from the
ACN and PBS extracts of all tissues, respectively. In addition, the antimicrobial activity of the positive and
negative controls against the bacteria and fungi was summarized in Figs. 8 and 9.
Marine organisms have high positions in traditional medical sciences and they also utilize as the main sources
for pharmacological investigations
. It has been discovered that echinoderms have stronger
antibacterial effects than Porifera, Mollusca, Bryozoa or Annelida
(Ridzwan et al. 1995)
. The sea cucumbers
are benthic animals with no shell and they do not have the ability to escape from predators but they have some
effective chemical responses. In this study, it had been tried to evaluate the antimicrobial effects of the various
extracts of the different parts of the sea cucumber Holothuria leucospilota on oral streptococci and Candida
strains. These extracts with different solvents (Et, ACN and PBS) allowed us to investigate the presence of
antimicrobial activity of the substances potentially presented in the lipid- or water-soluble fraction. Since the
antimicrobial effects in the different tissues of H. leucospilota with various solvents were found, therefore it
was concluded that the protein and non–protein factors could be responsible for such effects.
Antibacterial effects of the extracts
The antibacterial activity has previously been described in some species of echinoderms
(Gowda et al. 2008;
Haug et al. 2002; Kazemi et al. 2016; Kiani et al. 2014; Ridzwan et al. 1995)
. Haug et al. (2002) studied the
antibacterial activity of different parts of the green sea urchin Strongylocentrotus droebachiensis (Mu¨ ller,
1776), the common starfish Asterias rubens (Linnaeus, 1758) and the sea cucumber Cucumaria frondosa
aThe data is presented as % hemolysis compared to control (Triton X-100)
Hemolytic activity (%)a
(Gunnerus, 1767) against Gram-positive and -negative bacteria. They showed antibacterial activities in the
extracts of several tissues including the body wall, coelomocyte and especially the gastrointestinal organ and
the eggs from A. rubens and C. frondosa. Ridzwan et al. 1995 tested the extracts of the sea cucumbers
Holothuria atra (Jaeger, 1933), H. scabra (Jaeger, 1833) and Bohadschia argus (Jaeger, 1833) against seven
species of bacteria and they found that the lipid and methanol extracts had no inhibitory activities, whereas the
PBS extract had severe inhibitory activity. The methanol–acetone extract of the body wall of the sea cucumber
Parastichopus parvimensis (Clark, 1913) had antibacterial properties against Bacillus subtilis (Ehrenberg
1835) and Escherichia coli (Migula, 1895)
(Villasin and Pomory 2000)
. The present study demonstrates the
presence of antibacterial substances in the body wall, respiratory tree and gastrointestinal tract of the sea
cucumber, H. leucospilota. As the most tropical sea cucumbers feed on live microorganisms and the organic
contents found in sand or slime
, there is the possibility that some pathogenic microorganisms are
taken together with the food substances. Therefore, the presence of some active antibacterial substance(s) is
predictable for the body defense in different parts of the sea cucumber.
In the present study, among the tissues of sea cucumber, almost all extracts of the gastrointestinal tract had
effect on both species of Streptococcus, whereas the effects of the extracts of body wall and respiratory tree
were different. All of the extracts prepared from the body wall did not show any inhibitory effect on S. mutans,
whereas its antibacterial activity against S. sobrinus indicated the presence of the effective active compounds
in this tissue. The concentrated extracts of all tissues (1500 lg well-1) had more significant inhibitory effects
than other ones (600 lg well-1). The different effects of the sea cucumber extracts can be related to the
animal’s habitat and its feeding on microorganisms presented in the sea bed. In addition, the antibacterial
activity might be both due to the factors of the innate immune system or bacterial symbionts
(Strahl et al.
living on the organisms.
According to the results, as some extracts had shown antibacterial activities against these two oral
streptococci, they consist of compounds which can be used for further and more in depth studies due to inhibition
of oral infections caused by these bacteria.
Antifungal effects of the extracts The antifungal activity has previously been described in some species of echinoderms (Batrakov et al. 1980; Kumar et al. 2007; Mohammadizadeh et al. 2013; Mokhlesi et al. 2011). The antifungal activity of the extracts of the respiratory tree was more than the extracts of the body wall and intestine of the sea cucumber,
Holothuria scabra, against Candida albicans and Aspergillus niger
(Mohammadizadeh et al. 2013)
different organs of the sea cucumber, Bohadschia marmorata, including Cuvier organ, body wall and
coelomic fluid showed antifungal activities in which the Et extract of the body wall and water–alcohol extract of
the Cuvier organ had the highest effects
(Mokhlesi et al. 2011)
. In the present study, the organic extracts (ACN
and Et) of the different tissues of the sea cucumber, Holothuria leucospilota, had generally more antifungal
effects than the PBS extracts. The observed differences between the results of this study and the other ones
could be attributed to the animal habitat and the sea bed that the cucumber feed on it. In addition, the extracts
of the respiratory tree had generally more antifungal activity against Candida strains which was followed by
the body wall and gastrointestinal tract extracts, respectively. The antifungal activity of the respiratory tree in
the sea cucumber could be referred to the defensive role of it which was demonstrated by
Purcell et al. (2012)
The antimicrobial activity of the body wall is evident as the first defensive line.
The studied species possesses hemolytic activity in their tissues. Several hemolytic factors had been
isolated from echinoderms, including proteins
(Canicatti and D’Ancona 1990)
(Hatakeyama et al.
, complement-like factors
, and the variety of saponins
(Iorizzi et al. 2001)
. In the
present study, it seems that the hemolytic activities of the extracts generally are higher than their antimicrobial
activities. Some extracts had highly hemolytic activity without any effects on streptococci (e.g., Et extract of
respiratory tree) while some extracts showed more antibacterial activity than the hemolytic one (e.g., the PBS
extract of respiratory tree). From the pharmaceutical point of view, it is a requirement that the antibacterial
drugs have no side effects such as hemolytic activity
(Haug et al. 2002)
The present study shows that the sea cucumber, Holothuria leucospilota, as an echinoderm, can be a source of
antibiotics. Streptococcus sobrinus had generally been less tolerant than S. mutans against the sea cucumber
extracts, therefore almost all extracts except Et extract of respiratory tree can inhibit the growth of S. sobrinus.
The growth inhibition of Streptococcus sobrinus and S. mutans was generally controlled better by PBS and
organic extracts, respectively. The extract of the respiratory tree had the maximum antifungal effect against
Candida in comparison to the other tissues. In addition, it seemed that the sea cucumber extracts controlled the
growth of Candida parapsilosis better than the other candida fungi. Finally, the extracts of the sea cucumber
should be further analyzed to isolate, purify and determine the chemical structure of the antimicrobial
compounds for their applications in the medicine and dentistry industries.
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