Sea cucumbers have been known around the world for their medical benefits. In this study, unadulterated doses of crude extracts from body wall and Cuvierian tubules of Pearsonothuria graeffei were investigated for their antibacterial and antifungal potential. Doses of crude body wall methanol extract (MIC, <218.75 /><218.75 /><437.50 />< 0.05) antifungal property against Candida albicans ATCC 10231 compared to Clotrimazole (10 μg/ml ), Fluconazole (25 μg/ml), and Ketoconazole (10 μg/ml).

1. Antibacterial and antifungal property of extracts derived from the body wall and cuvierian tubules of Pearsonothuria graeffei Semper (Black-spotted Sea
cucumber)
FMCDD
Antibacterial and antifungal property of extracts
derived from the body wall and cuvierian tubules of
Pearsonothuria graeffei Semper (Black-spotted Sea
cucumber)
John Raymund D. Torres
College of Arts and Sciences, Don Mariano Marcos Memorial State University, Agoo, La Union, Philippines 2504
Mobile: +639-3343-64187
E-mail: jonramt@gmail.com
Sea cucumbers have been known around the world for their medical benefits. In this study,
unadulterated doses of crude extracts from body wall and Cuvierian tubules of Pearsonothuria
graeffei were investigated for their antibacterial and antifungal potential. Doses of crude body
wall methanol extract (MIC, <218.75 μg/ml against Staphylococcus aureus ATCC 25923) and body
wall ethyl acetate extract (MIC, <218.75 μg/ml against S. aureus ATCC 25923; MIC, <437.50 μg/ml
against Escherichia coli ATCC 25922) both showed weak antibacterial property compared to
Ciprofloxacin (5 μg/ml). Meanwhile, unadulterated crude body wall methanol extract (MIC, 1750
μg/ml) and body wall hexane extract (MIC, 1750 μg/ml) exhibited a significant (p < 0.05) antifungal
property against Candida albicans ATCC 10231 compared to Clotrimazole (10 μg/ml ),
Fluconazole (25 μg/ml), and Ketoconazole (10 μg/ml).
Keywords: Biological properties, Cuvierian tubule, Pearsonothuria graeffei, sea cucumber
INTRODUCTION
The scientific community has been continuously
searching for novel compounds from different species of
plant for their biological and pharmacological activities
(Dellai et al., 2012). On equal stance, bioprospecting
from the marine environment has also yielded discoveries
of several bioactive metabolites with considerable
biological and medical applications (Bordbar et al., 2011;
Mohklesi et al., 2012). In a comparative study, marine
natural products were described to be superior to
terrestrial natural products in terms of chemical
novelty(Kong et al., 2010) and are eco-friendly. In fact,
marine-derived bioactives possess novel chemical
structures exhibiting novel mechanism of action
(Montaser and Leusch, 2011) or taxa specific like in the
case of sea cucumbers (Li et al., 2008). Novel
compounds from plant and marine sources are natural
products and are more eco-friendly and have more
target-specific properties than synthetic ones (Ali et al.,
2011). This makes them good sources of novel
compounds for future nutraceutical, pharmaceutical, and
medical uses. Usually, the biological properties are
evaluated and the chemical composition of the
sequestered extracts is elucidated. The bioactive
compounds serve as blueprints for biochemical
synthesis of new drugs.In an extensive review of the
high-value components from various sea cucumber
species by Bordbar et al. (2011), information on
Pearsonothuria graeffei is very limited. Oftentimes,
traders and the researchers in private and government
funded studies worldwide are concentrated on fewer but
more valuable (usually referring to its direct and indirect
value) species.
Frontiers in Medicinal Chemistry and Drug Discovery
Vol. 1(1), pp. 002-009, June, 2017. © www.premierpublishers.org. ISSN: 2747-3241
Research Article

2. Antibacterial and antifungal property of extracts derived from the body wall and cuvierian tubules of Pearsonothuria graeffei Semper (Black-spotted Sea
cucumber)
Torres JRD 002
Several studies on some species of sea cucumber with
high-commercial value have shown multiple biological
activities such as antinociceptive (Ridzwan et al., 2003),
antibacterial (Abubakar et al., 2012; Mohklesi et al.,
2012), antifungal (Wang et al., 2012), anticoagulant (Ye
et al., 2012), anti-hypertension, anti-inflammatory
property (Sheean et al., 2007), insecticidal, (Thakur et
al., 2004), molluscicidal, and anti-protozoa (Mojica and
Merca, 2005). It was found that some species of sea
cucumber are rich in saponin glycosides that exhibit
anticancer effect (Aminin et al., 2015; Bordbar et al.,
2011). Laboratory studies also claimed that some
species have potential antioxidant (Mamelona and
Pelletier, 2010) and anti-proliferative effects (Althunibat
et al., 2009).
In spite of the worldwide studies showing the medical
uses of sea cucumber species and its potential source of
bioactive compounds, available references or
publications about sea cucumber species composition in
the Philippines are mostly limited to local
description(Gamboa et al., 2007). There is a much need
for researches on species biology and ecology (Choo,
2008). Hence, this study was conducted to help solve
the lack of researches on this nature. This study
intended to search for novel bioactive compounds by
evaluating the biological activities of crude extracts from
a common species of sea cucumber in the Philippines
which is the Pearsonothuria graeffei Semper.
The species is also known as the Black-spotted sea
cucumber (FAO), Orange fish or Shoab (Egypt), Flower
fish (India, Papua New Guinea, and Vietnam) and
Noolattai (India) (Purcell et al., 2012). In the Philippines,
it is known as Mani-mani, Bulaklak (Brown et al., 2010),
Trompa, or Piña, and sometimes identified as
Bohadschia graeffei or B. drachi. Pearsonothuria graeffei
is regarded as 3
rd
class. This means that it is of the
lowest commercial importance. There is also very little
information on its biology (Purcell et al., 2012), and on its
potential bioactivities. In a global standpoint, there is
great concern on P. graeffei for conservationists even
though at present this species is widely distributed
(Conand et al., 2013).
The present study is also essential since conservation
importance of species is not only attributed to its rarity or
abundance, but also to its utility which are often classified
as direct, indirect, and option values (Freeman, 2003).
The latter is the best way to add more value to our
natural resources. Universally, high-value species of sea
cucumbers are the most common target of fishers which
frequently leads to their overexploitation. The utilization
of natural resources for their direct value is the major
cause of overexploitation (Lawrence et al., 2009) which
also results in the destruction of environment and
extinction of the species. Natural resources are meant to
be utilized as they are termed resources but it does not
mean that the consumption or utilization of these is
unlimited or unregulated. However, maximizing the use,
proper utilization, and effective management of this
species is difficult to attain if there is no enough
information about the natural resource. Furthermore, if
there is very little information about the species, it will be
difficult to request for protection within local communities.
It will also be difficult to request for consideration for
listing under the Convention on International Trade in
Endangered Species (CITES). To make this a reality,
there should be enough evidence proving its importance.
Likewise, unraveling sea cucumber species’ biological
properties will help conservationists in their campaign for
its protection and conservation.
At present, there are very few reported biological
properties of the natural products from P. graeffei.
Biological activity studies on the isolated active
compound from P. graeffei include mutagenicity and
teratogenicity of its saponins in fetal rats (Wang et al.,
2013); on the effect of saponins in hematopoiesis in mice
(Li et al., 2011); on the anti-metastatic effect of non-
sulfated triterpene glycosides namely echinoside A and
Ds-echinoside A (Zhao et al., 2012).Bioprospecting by
means of bioactivity evaluation is one of the promising
processes in the discovery of bioactives from natural
sources. In the event that bioactivity testing leads to
favorable results, necessary follow up research works is
usually conducted to determine the best method to
isolate and manufacture the active compound.
Bioprospecting will add value on our natural resources
which in turn will open an opportunity to search for new
source of natural products with potential bioactivities.
Hence, this study will provide useful insights on the sea
cucumber species’ biology, which will be essential in
management efforts and in pharmaceutical or medical
researches for the development of drugs.
METHODOLOGY
Harvesting of Sea Cucumbers
Upon the issuance of Gratuitous Permit No. 01-14 by the
Bureau of Fisheries and Aquatic Resources (BFAR), the
collection of sea cucumbers was immediately
undertaken. Pearsonothuria graeffei Semper specimens
with length ranging from 20 cm to 50 cm and weighing
from 30g to 180g were collected from Poro Point, San
Fernando City, Philippines. The samples were harvested
by hand picking with the help of fishers who dove at
intertidal (shallow water region) and subtidal (deeper
areas) ranging from 15-20 feet. The sea cucumbers
were temporarily stored in coolers with ice, transported,
and upon arrival at the laboratory, were immediately
washed with distilled water.
Extraction of the Active Principle for Antimicrobial
Susceptibility Test (AST)
Dissection was done by separating the body wall and the
Cuvierian tubules from the internal organs, gonads, and

3. Antibacterial and antifungal property of extracts derived from the body wall and cuvierian tubules of Pearsonothuria graeffei Semper (Black-spotted Sea
cucumber)
Frontiers Med. Chem. Drug Disc. 003
tentacles of the organism. Bulk weighing of the fresh
samples and dissected body parts was done using a
digital electronic weighing scale (ACS TCS System,
3208) . Meanwhile, steps in the preparation of crude
extracts were based on the procedures described by
Ismail et al. (2008). However, the researcher modified
some portions in the procedures in which crude extracts
were obtained after vacuo concentration at 40ºC and not
at 45ºC. The study also utilized extracting solvents such
as ethyl acetate and n-hexane aside from methanol.
Furthermore, the present study did not involved
fractionization and purification of semi-purified fractions to
obtain the crude extracts. The following process explains
the methods used in this study.
Freshly separated body wall and Cuvierian tubules of P.
graeffei were cut into smaller pieces and homogenized
using blender (Oster, Model No. 4172-074, PN30598-
074, Mexico) for five minutes. After which, each of the
homogenized body parts were separately soaked in each
of the analytical reagents namely methanol, ethyl acetate,
and n-hexane, for seven days (1:3 w/v). Then, the
soaked samples were filtered using cheesecloth followed
by running through a separator and centrifugation
(Kokusan H-108N, Japan) at 4000 rpm for 10 minutes.
The supernatants were used for vacuo concentration.
To concentrate and remove the solvents, filtrates were
evaporated under vacuum at 40ºC by the rotary
evaporator (Rotavapor Buchi R215, Switzerland).
Samples soaked in methanol were set under pressure
337 psi, those in ethyl acetate were set under pressure
240 psi, and those in n-hexane were set under pressure
of 335 psi. Since the samples were fresh, excess water
component had to be removed. Hence, water was further
separated under pressure of 72 psi. The resulting slimy
and semi-solid substance served as the crude extracts.
Pearsonothuria graeffei’s body wall soaked in the
analytical grades of methanol, ethyl acetate, and n-
hexane and processed in the described procedure above
were labeled as Body Wall Methanol Extract/BWME,
Body Wall Ethyl Acetate Extract/BWEAE, and Body Wall
Hexane Extract/BWHE, respectively. Meanwhile,
Cuvierian tubules of P. graeffei soaked in the analytical
grades of methanol, ethyl acetate, and hexane and
similarly processed in the previously described protocol
were labeled as Cuvierian Tubule Methanol
Extract/CTME, Cuvierian Tubule Ethyl Acetate
Extract/CTEAE, and Cuvierian Tubule Hexane
Extract/CTHE, respectively. The six crude extracts were
kept in the freezer at -4ºC until use.
Strains of Bacteria and Fungus
Pure cultures of the Staphylococcus aureus (ATCC
25923) and Escherichia coli (ATCC 25922) were
obtained from Natural Sciences Research Unit
Laboratory of Saint Louis University, at Baguio City,
Philippines. The microorganisms were cultured in a
Mueller-Hinton Agar (Tritan Biotech LTD, Rajasthan,
India) under 37ºC .Meanwhile, the isolated strain of
Candida albicans (ATCC 10231) was obtained from G.R.
Medical Laboratory, Ilocos Training and Regional Medical
Center (ITRMC) at Pagdaraoan, San Fernando City, La
Union, Philippines. Candida albicans (ATCC 10231) was
cultured in Potato Dextrose Agar (Hi Media Laboratories
Pvt. Ltd., India) inside the incubator (Memmert Air
Incubator IMB 500, Japan).
Antimicrobial Susceptibility Testing
The procedures for preparation and standardization of
inocula, disk diffusion test and determination of Minimum
Inhibitory Concentration (MIC) were based on the criteria
set by the Clinical Laboratory Standards Institute (CLSI)
(Hudzicki, 2013; Cavalieri et al., 2005). Similarly,
antifungal susceptibility testing was based on the
standard method described by CLSI (Clinical and
Laboratory Standards Institute, 2007).
Antibacterial and Antifungal Property Screening
Antibacterial screening was done following the procedure
described by CLSI (Clinical and Laboratory Standards
Institute, 2007) and in the study of Abubakar et al.
(2012).Antibacterial and antifungal activities of methanol,
ethyl acetate, and n-hexane crude extracts of the body
wall and the Cuvierian tubules were assessed by means
of agar disk diffusion method/ Kirby-Bauer assay. A disk
immersed in a distilled water served as negative control
(Treatment 0; T0), a commercial antibiotic
Ciprofloxacin/CFL (5 µg/ml) served as positive control for
antibacterial screening (Treatment 1; T1A), and
Clotrimazole (10 µg/ml) (Treatment 1B; T1B) served as
the reference drug in the antifungal screening. Six
concentrations namely 10, 100, 200, 500 and 1000
µg.mL
-1
each of Body Wall Methanol Extract (BWME),
Body Wall Ethyl Acetate Extract (BWEAE), Body Wall
Hexane Extracts(BWHE),Cuvierian Tubule Methanol
Extract (CTME), Cuvierian Tubule Ethyl Acetate Extract
(CTEAE), and Cuvierian Tubule Hexane Extract (CTHE),
and the unadulterated crude extracts were prepared and
evaluated for their antimicrobial properties.
Susceptibility Test Using Spectrophotometer
Isolated colonies from an 18-24 hour culture of
Staphylococcus aureus (ATCC 25923), Escherichia coli
(ATCC 25922), and Candida albicans (ATCC 10321)
served as the source of cells in preparing the inocula
suspension. A spectrophotometer (Spectro Dual Split
Beam UVS-2800, Japan) was used to check if the
adjusted suspension turbidity equals the 0.5 McFarland
standard solutions. Turbidity was adjusted using sterile
saline (0.85% NaCl). Afterwards, Minimum Inhibitory
Concentrations (MICs) of the bioactive body wall and the

4. Antibacterial and antifungal property of extracts derived from the body wall and cuvierian tubules of Pearsonothuria graeffei Semper (Black-spotted Sea
cucumber)
Torres JRD 004
Table 1. Zone of Inhibition Mean Values (ZOIμ) Exhibited by Crude Extract and Control Drug Against Standard Strain of
Staphylococcus aureus and Escherichia coli.
Controls/
Extract
ZOI (mm)
Mean Values
Staphylococcus aureus ATCC 25923 (Gram +)
Distilled H2O 0 c
Ciprofloxacin 17 a
BWME 8 b
BWEAE 7 b
Escherichia coli ATCC 25922 (Gram -)
Distilled H2O 0 c
Ciprofloxacin 15 a
BWEAE 4 b
NOTE: Any mean value followed by the same letter(s) is not significantly different from each other.
Legend: BWME – Body Wall Methanol Extract
BWEAE – Body Wall Ethyl Acetate Extract
Cuvierian tubule extracts were determined through a
serial dilution method analyzed spectrophotometrically.
RESULTS AND DISCUSSION
Antibacterial Property
Test concentrations which are 10,100, 200, 500, and
1,000 μg.ml
-1
of the six different crude extracts obtained
from the body parts of P. graeffei Semper did not show
antibacterial action against S. aureus ATCC 25923 and
E. coli ATCC 25922. On the other hand, unadulterated
dose of crude BWME and BWEAE showed inhibitory
activity against the growth of S. aureus while only
unadulterated crude BWEAE showed inhibitory activity
against growth of E. coli.
Although not statistically comparable (p< 0.05) to the
antibacterial effect of the control drug used, the results of
experiments confirmed the antibacterial activity of the
extracts. The limited antibacterial effect or weak growth
inhibition of the crude extracts BWME and BWEAE
against Staphylococcus aureus ATCC 25923 and the
crude extract BWEAE against Escherichia coli ATCC
25922, may be attributed to the bioactives that were
previously described and isolated from Pearsonothuria
graeffei and to many other species of sea cucumber.
Sea cucumber extracts derived from other body parts like
body wall, Cuvierian tubules, coelomic fluid, and gonads
of sea cucumber species, has been provento contain
bioactive compounds (Schillaci et al., 2013; Abubakar et
al., 2012). These bioactive compounds were also
accounted for the various biological and pharmacological
properties of the different extracts from sea cucumber
species. These compounds with known bioactivities are
triterpene glycosides or class of saponins commonly
known as holothurins (Mohklesi et al., 2012; Caulier et
al., 2011).
Several studies also ascribed the antimicrobial properties
of sea cucumber extracts to the presence of glycosides
commonly in the form of triterpene glycosides (saponin)
and the aglycones from the class of compounds called
saponins. Saponins from sea cucumbers are commonly
known as holothurins. This secondary metabolite is also
identified as the predominant secondary metabolite of
holothurians with various reported biological properties
(Zhao et al., 2011). Triterpene glycosides were isolated
in several species of sea cucumbers like Pearsonothuria
graeffei with around 3.5 percent of its body wall dry
matter (Zhao et al., 2012; Van Dyck et al., 2010).
In the present study, the methanol and ethyl acetate
extracts from the body wall of P. graeffei Semper,
showed antibacterial effect against S. aureus ATCC
25923 while only one of the tested extracts, that is,
BWEAE showed antibacterial effect against E. coli ATCC
25922. Comparing the resistance capability of Gram
positive and Gram negative alone (i.e. not including
mutant strains), Gram negative bacteria are considered
to be more resistant to most antibiotics. This enhanced
resistance of the Gram negative bacteria can be
accounted to their double layered wall while the Gram
positive bacteria are characterized with single layered
(Cavalieri et al., 2005).Aside from triterpene glycosides,
fucosylated chondroitin sulfates were also isolated from
sea cucumber Pearsonothuria graeffei (Chen et al.,
2011). Fucosylated chondroitin sulfates from sea
cucumbers have much resemblance to the structure of
sulfated polysaccharides in the form of fucoidans with
known several biological properties (Moghadamtousi et
al., 2014; Patel 2012).

5. Antibacterial and antifungal property of extracts derived from the body wall and cuvierian tubules of Pearsonothuria graeffei Semper (Black-spotted Sea
cucumber)
Frontiers Med. Chem. Drug Disc. 005
Table 2. Interpretation of 24-Hour Zone of Inhibition (ZOI) Mean Values Exhibited by Crude Extracts and Control Drug against Candida
albicans ATCC 10231.
Controls/
Extract ZOI (mm)
ZOI Interpretation
D1 D2 D3 D4 D5 D6 Mean
Candida albicans ATCC 10231
Distilled H2O 0 0 0 0 0 0 0.00
b
R
Fluconazole 0 0 0 0 0 0 0.00 R
Ketoconazole 0 4 0 0 6 0 1.67 R
Clotrimazole 12 11 11 8 9 9 10.00
a
R
BWME 10 11 10 11 10 9 10.17
a
R
BWHE 12 11 11 10 12 11 11.17
a
R
Note: Any mean value followed by the same letter(s) is not significantly different from each other.
Legend: D1- disk no.1 BWME – Body Wall Methanol Extract
D2 - disk no.2 BWHE – Body Wall Hexane Extract
D3 - disk no.3 S - Susceptible
D4 - disk no.4 R - Resistant
D5 - disk no.5
D6 - disk no.6
Alcoholic extracts obtained from Holothuria atra showed
inhibition in the growth of Klebsiella pneumonia,
Escherichia coli, Listeria monocytogenes, and
Staphylococcus aureus which are commonly identified as
human pathogens. The methanol extracts from several
sea cucumbers species exhibited antibacterial activities
(Moguel-Salazar et al., 2013). Methanol extracts from
sea cucumber Stichopus badionotus exhibited
antibacterial activity against methicillin-resistant
Staphylococcus aureus (Mariana et al., 2009). In another
study, methanol extracts from another sea cucumber G.
changii, was also successful in inhibiting the growth of
Staphylococcus aureus (Al-Haj et al., 2010).Furthermore,
the study of Shakouri et al. (2017) proved the potential of
aqueous body wall extract of a sea cucumber Stichopus
variegatus, as source of antimicrobial peptides against
the growth of Escherichia coli. Their study revealed the
antimicrobial effect of 8 mg/ml concentration of aqueous
body wall extract of S. variegatus against the growth of E.
coli in the disc diffusion assay. Similarly, Adibpour et al.
(2014) showed that crude methanol extracts separately
derived from the body wall, cuvierian organs, and
coelomic fluids at both concentrations of 2000 μg/ml and
1000 μg/ml had a potential antimicrobial property
specifically microbiostatic effect on the growth of E. coli
ATCC 8739 while found ineffective against fungi Candida
albicans ATCC 10231. In the present study, crude body
wall methanol extracts (BWME) and body wall ethyl
acetate extracts (BWEAE) both showed weak
antibacterial effect against S. aureus ATCC 25922 and
BWEAE showed weak inhibitory effect against E. coli
ATCC 25922 compared to Ciprofloxacin in disc diffusion
assay. However, determination of the MIC revealed the
potential of the crude extracts from P. graeffei as source
of antimicrobial peptides.
Peptides are also one of the many bioactive compounds
from a species of marine organisms which serve as their
innate immunity against microorganisms’ pathogenic
infection. Marine-derived antimicrobial peptides (AMPs)
are structurally different from their counterparts from
terrestrial species and are commonly taxa or species
specific. They were also reported to exhibit a broad
antimicrobial properties. Many hydrophilic antimicrobial
peptides (AMPs) have been identified in the body wall
mucus of sea cucumber species (Chi-Fai Chung et al.,
2015). The isolated novel peptides Holothurin 1 and
Holothurin 2 from 5-HCC, which were classified as
members of Antimicrobial Peptides (AMPs) with known
antibiofilm contributed in the antibacterial activity of the
Holothuria tubulosa isolates against Staphylococcus
aureus ATCC 25923, a strain similar to what is utilized in
the present study, Staphylococcus aureus ATCC 29213,
Staphylococcus aureus ATCC 6538, Enterococcus
faecalis ATCC 29212, Pseudomonas aeruginosa ATCC
15442, and Staphylococcus epidermidis ATCC 35984
(Schillaci et al., 2013). In addition, it appears that novel
AMPs are likely to be characteristic of natural products or
isolates from several other marine organisms (Smith et
al., 2010). This implies that the crude extracts BWME
and BWEAE of Pearsonothuria graeffei Semper may
contain some class of AMPs that may have contributed
to their antimicrobial effects.The AST evaluation in this
study revealed that crude extracts BWME and BWEAE of
Pearsonothuria graeffei Semper statistically resulted in
limited antibacterial activity against the tested Gram
+
and Gram
–
standard bacterial strains. However, result
of MIC by spectrophometric analysis on BWME (MIC,
<218.75 μg/ml against Staphylococcus aureus ATCC
25923) and BWEAE (MIC, <218.75 μg/ml against S.
aureus ATCC 25923; MIC, <437.50 μg/ml against

6. Antibacterial and antifungal property of extracts derived from the body wall and cuvierian tubules of Pearsonothuria graeffei Semper (Black-spotted Sea
cucumber)
Torres JRD 006
Escherichia coli ATCC 25922) proves the potential of the
P. graeffei crude extracts as antibacterial agent.
Antifungal Property
The preliminary evaluation of antifungal activity of the six
different extracts made use of commercial antifungal drug
Fluconazole/FCZ (Diflucan, Pfyzer) as reference drug.
However, this reference drug did not inhibit the growth of
Candida albicans ATCC 10231 after several trials.
Consequently, a substitute commercial antifungal
referenced drug Ketoconazole/KCZ (Pfyzer) was tested,
but did not successfully inhibit the growth of Candida
albicans ATCC 10231. On the third AST, commercial
Clotrimazole was found to exhibit zone of inhibition per
disk against Candida albicans ATCC 10231 during
preliminary testing using Kirby-Bauer Assay (KBA) which
in turn was used as a reference drug.
All test concentrations which are 10,100, 200, 500, and 1,
000 μg ml
-1
of the six different crude extracts obtained
from the body wall and the Cuvierian tubule of P. graeffei
Semper did not show antifungal action against Candida
albicans ATCC 10231. Only unadulterated crude BWME
and BWHE showed a certain degree of inhibitory activity
against Candida albicans ATCC 10231 compared to the
reference commercial antifungal drug Clotrimazole.
Antifungal drugs ketoconazole, clotrimazole, itraconazole,
and miconazole were reported to only exhibit partial
growth inhibition against the standard Candida albicans
ATCC 10231 strain grown in a bYNBG medium.
However, susceptibility of the pathogenic fungal cell
isolates is time-dependent; that is, after 24-hour period of
incubation the inhibitory effect of different antifungal
agents increased (Blanco et al., 1992).
The AST results for both antibacterial and antifungal tests
in this study, may be attributed to the molecular weight
and the inability to exhibit considerable antibacterial or
antifungal activity of the antimicrobial agents. In studies
evaluating antimicrobial effects of certain extracts, the
molecular weight of the evaluated antimicrobial agent
was reported to have an effect in the ZOI that can be
produced within a certain period of time. In the
KBA/DDA, higher molecular weight will be expected to
move slowly than with antimicrobial agent with low
molecular weight. Thus, a limited antimicrobial property
of ZOI might be read. This is often encountered in testing
using natural products on DDA (Klancnik et al., 2010).
Several years ago, a low probability of clinical resistance
is expected from azole drugs against Candida albicans
due to the fact that the microorganisms are diploid and
with no haploid sexual stage(White et al., 1988).
However, succeeding studies verified the existence of
resistant C. albicans from azole antifungal drugs. The
resistances of Candida albicans isolate to fluconazole
and ketoconazole, clotrimazole (Khan et al., 2009),
miconazole and itraconazole (Bitar et al., 2014) had been
previously recognized. In several studies conducted
concerning C. albicans infections, Fluconazole-resistant
C. albicans are commonly isolated from patients who had
previous use of the drug (Dota et al., 2015; Mulu et al.,
2013; Marchaim et al., 2012).
It was described that resistance of Candida albicans
strains can be the result of normal distribution of MICs or
the development of any of the various mechanisms to
counteract antifungal agents. Fungal cells may also
possess or may activate efflux pumps to fight the effect of
an antifungal agent (Lacka et al., 2011). Another
possible way to explain the resistance of fungal strain is
by their ability to form biofilm which is an important factor
in their pathogenicity (Bitar et al., 2014).
To further evaluate and compare the antifungal activity in
terms of exhibited ZOI values, statistical analysis of the
data was also considered.The One-Way Analysis of
Variance suggests that there is no significant ( p<0.05)
difference among the exhibited ZOIμ of BWME, BWHE,
and Clotrimazole against Candida albicans ATCC 10231.
Furthermore, preliminary tests showed that even the
common antifungal drugs used in treating Candida
infection like the Fluconazole and Ketoconazole were
ineffective in inhibiting Candida albicans ATCC 10231
while the use of crude BWME and BWHE from P.
graeffei exhibited ZOI in DDA and showed inhibitory
property revealing MIC at 1750 μg/ml by
spectrophotometric analysis.
Several studies support that extracts derived from various
species of sea cucumbers possess antifungal properties.
Methanol extract and chloroform extract separately from
the body wall, intestine, and gonad of Holothuria
leucospilota exhibited inhibitory activity against the
growth of Aspergillus niger at concentrations of 2.5, 5,
and 10 mg/ml (Farjami et al., 2014). In a recent study,
antifungal activity was observed from methanol extracts
of the body wall (highest) and Cuvierian organs of sea
cucumber Stichopus hermanni against fungus Aspergillus
niger using Fluconazole as positive control
(Sarhadizadeh et al., 2014).
A significant antifungal property of crude body wall
methanol extracts of P. graeffei against Candida
albicanswas also described. The antifungal property is
attributed to the triterpenoids and steroid glycosides
(class of saponin) present in the tested crude
extracts(Lawrence et al., 2009). Similarly, antifungal
activity of extracts from sea cucumber Actinopyga
lecanora was observed against fungal species Candida
albicans, Aspergillus niger, and Aspergillus flavis. The
antifungal property of the extract was attributed to the
bioactive compound triterpene glycosides(Kumar et al.,
2007). It was also reported that the body wall and
Cuvierian tubule extracts from sea cucumber
Pearsonothuria graeffei also contain considerable
amounts of triterpene glycosides (Zhao et al.,2011; Van
Dyck et al., 2010). It can be inferred that the crude
extracts from P. graeffei Semper (BWME and BWHE) in

7. Antibacterial and antifungal property of extracts derived from the body wall and cuvierian tubules of Pearsonothuria graeffei Semper (Black-spotted Sea
cucumber)
Frontiers Med. Chem. Drug Disc. 007
this study, has the ability to induce antifungal activity
which may be caused by the presence of triterpene
glycosides (saponin).
CONCLUSION
The exhibited limited antibacterial property of
unadulterated doses of BWME and BWEAE against S.
aureus and BWEAE against E. coli can be accounted to
the previously reported bioactive compounds from P.
graeffei. It is inferred that the crude extracts from P.
graeffei are still potential source of antibacterial
compounds. Meanwhile, the statistically comparable (p <
0.05) fungicidal property of unadulterated BWME and
BWHE against Candida albicans ATCC 10231 compared
to Clotrimazole, and fungicidal activity better than
Fluconazole and Ketoconazole implies that BWME and
BWHE of P. graeffei have a great potential as a source of
antifungal agents in curing diseases caused by Candida
albicans.
ACKNOWLEDGEMENT
The author would like to thank CENRO-San Fernando
City La Union , DENR, BFAR R.O.1, Victoria N. Malaya
of the Institute of Fisheries, DMMMSU, Philippines, the
BFAR- NIFTDC,headed by Dr.Westly Rosario, Ms.
Jocelyn J. Marcial, Mr. John Carlo G. Gosilatar, Mr.
Romeo R. Visperas , Ms. Evelyn A. Dangla (Microbiology
& Molecular Pathology Laboratories ), Mr. Regino R.
Regpala (Limnology and Oceanography Laboratories.);
Dr. Alessandro Lovatelli, FAO Fisheries and Aquaculture
Department Rome, Italy; and Dr. Anja Klančnik,
Department of Food Science and Technology,
Biotechnical Faculty, University of Ljubljana, Slovenia.
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Accepted 24 May, 2017.
Citation: Torres JRD (2017). Antibacterial and antifungal
property of extracts derived from the body wall and cuvierian
tubules of Pearsonothuria graeffei Semper (Black-spotted Sea
cucumber). Frontiers in Medicinal Chemistry and Drug
Discovery, 1(1): 009-018.
Copyright: © 2017. Torres JRD. This is an open-access article
distributed under the terms of the Creative Commons Attribution
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reproduction in any medium, provided the original author and
source are cited.