Antibacterial and Antifungal Activity of Holothuria leucospilota Isolated From Persian Gulf and Oman Sea.

0.1 MB·English

by  Adibpour, Neda

Jundishapur J Microbiol. 2014 January; 7(1): e8708. DOI: 10.5812/jjm.8708
Research Article
Published online 2014 January 1.
Holothuria leucospilota
Antibacterial and Antifungal Activity of Isolated
From Persian Gulf and Oman Sea
1,2 1 3 3
Neda Adibpour ; Farhad Nasr ; Fatemeh Nematpour ; Arash Shakouri ; Abdolghani
1,4,*
Ameri
1Marine Pharmaceutical Sciences Research Center, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran
2Department of Medicinal Chemistry, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran
3School of Marine Sciences, Chabahar Maritime University, Chahbahar, IR Iran
4Department of Food and Drug Control, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran
*Corresponding author
: Abdolghani Ameri, Marine Pharmaceutical Sciences Research Center, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran.
Tel: [phone], Fax: [phone], E-mail: [email protected]
Received: ; Revised: ; Accepted:
October 22, 2012 January 23, 2013 February 23, 2013
Background:
Emergence of antimicrobial resistance toward a number of conventional antibiotics has triggered the search for
antimicrobial agents from a variety of sources including the marine environment.
Objectives: Holothuria leucospilota
The aim of this study was to evaluate the antimicrobial potential of from Qeshm and Kharg Islands
against some selected bacteria and fungi.
Materials and Methods:
In this investigation, sea cucumbers from two coastal cities of Persian Gulf were collected in March and May
2011 and identified by the scale method according to the food and agriculture organization of the United Nations. Antibacterial activity of
hydroalcoholic extracts of the body wall, cuvierian organs and coelomic fluid, methanol, chloroform, and n-hexane extracts of the body
wall were evaluated by the spot test. In addition, their antifungal activity was assessed by the broth dilution method.
Results:
The displayed effect was microbiostatic at concentrations of 1000 and 2000 µg/mL rather than microbicidal. The highest
Escherichia coli Salmonella
activity of hydroalcoholic extracts was exhibited by body wall, cuvierian organs and coelomic fluid against ,
typhi Staphylococcus aureus Pseudomonas aeruginosa;Aspergillus niger A. fumigatus A. flavus A. brasilensis
, and , , and . However, none of the
Shigella dysenteriae Proteus vulgaris Bacillus cereus S.
methanol, chloroform and n-haxane extracts showed appreciable effects against , , ,
epidermidis Candida albicans
and . Moreover, cuvierian organs did not possess any antifungal potential.
Conclusions: H. leucospilota
Our data indicated that water-methanol extracts from the body wall of possess antibacterial and antifungal
activity. However, additional and in-depth studies are required to isolate and identify the active component(s).
Keywords: Holothuria leucospilota
; Anti-Infective Agents; Persian Gulf
1. Background
substances (11) and antimicrobial peptides (12) have been
isolated from the sea cucumbers. Additionally, several ho-
Emergence of antimicrobial resistance toward a num- Holothuria fusco-
lostane-type triterpene glycosides (from
ber of conventional antibiotics has stimulated the search cinerea
) (13) and three new cytotoxic triterpene glycosides
for antimicrobial agents from a variety of sources includ- Mensamaria intercedens
(from Lampert) displaying broad
ing the marine environments. Sea cucumbers are echino-
Holothuroidea range of antibacterial, antifungal and cytotoxic activity
derms from the class . In Vietnamese tradi-
(14), have been isolated.
tional medicine, sea cucumbers had been used as tonics
Although the focus of study on marine organisms such
and delicacies (1). In Malaysia, different species of sea
as echinoderms and holothuroids is increasing, informa-
cucumbers are used to relieve pain and skin irritations
and treat eczema and arthritis (2). Antimicrobial activi- tion regarding exploitation and fishing techniques in
ties of several species of echinoderms from the Gulfs of Iran is scanty and recent. However, due to the expansive
California, Mexico and Caribbean Islands and the Coast coastal area of Iran, most coastal cities have some species
of Norway have been reported (3-5). of Holothurians (15). Since sea cucumbers are not popu-
Various antimicrobial components including steroidal lar in Iran, out of 1400 globally-recorded (16) Holothuri-
glycosides (6), polyhydroxylated sterols (7), naphthoqui- an species, so far only 20 have been recorded in Iran (17).
none pigments (8), lysozymes (9, 10), complement-like The most harvested sea cucumber in Iran is the sandfish,
Implication for health policy/practice/research/medical education:
Holothuria leucospilota
Since little information is available on antimicrobial activity of , this study was designed and conducted to assess the effects of
extracts of different parts of this organism on several bacterial and fungal species.
Copyright © 2014, Ahvaz Jundishapur University of Medical Sciences; Published by Kowsar Corp. This is an open-access article distributed under the terms of the Cre-
ative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Adibpour N et al.
H scabra
. or Khiar Daryaei (as it is called in the local lan- standard ATCC microorganisms were procured from the
guage), harvest of which began in 2004 at Qeshm Island institute for collection and maintenance of pathogenic
(18). and industrial bacteria and fungi of Iranian research or-
ganization for science and technology (IROST), Tehran,
2. Objectives
Iran.
Since the published data about the antimicrobial activ- 3.5. Antibacterial Assay
ity of sea cucumbers in Persian Gulf and Oman Sea is very
scarce, the aim of this study was to evaluate the antimi- Various extracts of different body parts were assayed
H leucospilata
crobial potential of the sea cucumber, . , col- against the test bacteria using the spot test (20). Initially,
lected from Qeshm and Kharg islands in Persian Gulf and various concentrations (2000 - 40000 µg/mL) of differ-
Oman Sea. ent dried extracts were prepared in sterile distilled water,
then 1 mL of each extract was transferred to a sterile pe-
3. Materials and Methods
tri plate, 19 mL of molten Meuller-Hinton agar medium
was added, the contents of plates were gently rotated
3.1. Collection of Samples and allowed to solidify to give the concentration range
of 100 - 2000 µg/mL. Microbial suspensions for each test
Sea cucumbers were harvested freshly from fixed sites
microorganism were prepared and adjusted according
of two coastal cities of Kharg and Qeshm in the Persian to 0.5 McFarland standard. Ten microliters of each tested
Gulf and also from the rocky beach of Tis village in Oman bacteria was deposited on the surface of each plate. Bac-
Sea during March and May of 2011. Samples were rinsed teria were incubated at 37°C for 24 hours. At the end of
with distilled water to remove debris and foreign par- the incubation period, plates were examined for growth
ticles. All samples were identified according to the food and minimal inhibitory concentration of each extract
and agriculture organization of the United Nations (19). was determined. All assays were repeated two times.
Following identification, all samples were maintained at
3.6. Antifungal Assay
-20°C until the usage time.
3.2. Processing of Samples Before starting the antifungal assay, each test strain was
cultured on Sabouraud's dextrose agar and incubated for
C. albicans Aspergillus
Initially, body wall, cuvierian organs, and coelomic 1 - 2 days ( ) or 2 - 3 days ( spp.) at 37°C.
Aspergillus
fluid were separated. Then, each part was cut into several For spp, suspensions of each strain in 3 mL
pieces and dried at room temperature in the dark, then of sterile distilled water containing 500 ppm Tween 80
milled to a fine powder. (Merck, Germany) were prepared by vortex mixing. After
filtration through a sterile celite to remove the hyphal
3.3. Extraction of Samples
fragments and residual agar, the starting inocula were
H. leucospilota adjusted to 104 CFU/mL by sterile distilled water using
Crude methanol extracts of were pre- C. albicans
spectrophotometry at 530 nm. Preparation of
pared by maceration of different body parts in appro-
was done in the same manner without using Tween 80
priate amounts of methanol-water (50:50), mixing, and
(21, 22).
maintenance for 16 hours. Then, the mixture was filtered
Stock solutions of the extracts were prepared in sterile
and the process was repeated for the second time. Fi-
water. The resulting solutions were progressively double
nally, the two portions were pooled together. The filtrate
diluted with the test medium (RPMI 1640 (Sigma, USA)
was concentrated to dryness by rotary evaporation. The
supplemented with L-glutamin without sodium bicar-
obtained powder was subjected to extraction with n-
bonate and buffered to pH 7 with 0.165 M (35.54 g/L) mor-
hexane, chloroform and ethyl acetate and then all the
pholinesulfonic acid (MOPS)) to give the final concentra-
extracts were dried by flash evaporation. tion range of 100 - 2000 µg/mL. Blanks were prepared in
3.4. Test Microorganisms the test medium using the same quantities of water, but
without the test extracts. One mililiter of each strain was
Test bacteria used in the study included standard strains mixed with 1 mL of media-containing test extracts or in
Shigella dysenteriae Proteus vulgaris
of (ATCC 13313), (ATCC 5-mL culture tubes and tubes were incubated at 37°C for
29905), Escherichia coli (ATCC 8739), Salmonella typhimuri- 48 hours. The MIC values were determined as the lowest
um (ATCC 19430), Pseudomonas aeruginosa (ATCC 9027), concentration of the test extract with no visible growth.
Staphylococcus aureus S epidermidis
(ATCC 29737), . (ATCC
4. Results
Bacillus ceureus
12229), and (ATCC 11778). Fungi included
Aspergillus flavus A fumigatus A niger H. leucospilota
clinical isolates of , . , . , Hydroalcoholic extract (50%) of exhibited
A nidulans A restatus A brasil-
. , . and standard strains of . antibacterial and antifungal activity against some of the
iensis Candida albicans
(ATCC 9029) and (ATCC 10231). All test microorganisms (Table 1). Results with antibacterial
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Jundishapur J Microbiol. 2014;7(1):e8708
Adibpour N et al.
H. leucospilota,
effects showed microbiostatic effects rather than micro- cucumber, were screened for antimicro-
cidal. The highest activity was exhibited by the body wall, bial activities against an array of Gram positive and Gram
E. coli S.
Cuvierian organs, and coelomic fluid against , negative bacteria as well as fungi and molds. From all ex-
typhi S. aureus P. aeruginosa
, and . coelomic fluid also in- tracts prepared with different solvents, only the hydroal-
hibited the growth of these bacteria, but at a slower rate. coholic extracts of the body wall, Cuvierian organs, and
Hydoalcoholic extract of the three examined body tissues coelomic fluid exhibited antibacterial and antifungal ac-
S. dysenteriae P. vulgaris B.
did not inhibit the growth of , , tivities in vitro. Previous studies reported antimicrobial
cereus S. epidermidis
and . activities from various species of echinoderms (2, 4, 6).
Table 2 depicts the results of all extracts examined for In most of the studied species, the whole bodies or body
antifungal activity. As presented in this Table, cuvierian walls were tested. Other studies reported antimicrobial
Paracentrotus lividus
extracts did not inhibit the fungal growth even at a high- activities of egg extracts of (23) and
er concentration (2000 µg/mL). However, the body wall Marthasterias glacialis (10). In the latter study, the active
and coelomic fluid inhibited the growth of all fungi at compound was reported to be a lysozyme.
both concentrations; nonetheless, body wall extract at Our study indicates that the component(s) responsible
A. niger
1000 µg/mL did not inhibit the growth of . for antimicrobial activity appear(s) to be concentrated
No significant difference of antibacterial and antifun- mainly in the body wall and coelomic fluid; but little or
H leucospilota
gal activity was observed between the . col- no activity was observed in the cuvierian organs since
lected from Persian Gulf and Oman Sea. there was moderate activity toward some species of bac-
Table 1. H leuco- teria and none against any of the tested fungi and molds.
Antimicrobial Activity of Various Extracts of .
spilata The methanol-water extracts of tissues/organs exhibited
Against Different Species of Bacteria
Test Extracts, µg/mL antimicrobial activities against some selected species of
Organism Body Wall Cuvierian Coelomic Gram positive and Gram negative bacteria as well as fun-
Organs Fluid gi, suggesting that multiple factors are responsible for
2000 1000 2000 1000 2000 1000 these activities. Thus, additional in-depth chemical analy-
B. cereus - a - - - - - ses are required for isolation and purification of the ac-
S.epidermidis tive compound(s) as well as identification of their chemi-
- - - - - -
S. aureus + b + + + + + cal nature and evaluation of their potential strength for
S. dysenteriae novel drugs.
- - - - - - Holo-
S. typhi In conclusion, the body wall and coelomic fluid of
+ + + + + + thuria leucospilota
P. proteus collected from Persian Gulf and Oman
- - - - - -
E.coli Sea showed weak antibacterial and antifungal effects
+ + + + + +
P.aeruginosa against few species of human pathogenic bacteria and
+ + + + + +
a -: no activity. fungi undertaken in this study.
b +: antimicrobial activity. Acknowledgments
Table 2. H leuco- The contents of this paper have been extracted from the
Antimicrobial Activity of Various Extracts of .
spilata Against Different Species of Fungi Pharm D. thesis No. 798.
Test Extracts, µg/mL
Authors’ Contribution
Organism Body Wall Cuvierian Coelomic
Organs Fluid
Neda Adibpour and Abdolghani Ameri developed the
2000 1000 2000 1000 2000 1000
original idea and protocol and contributed to the prep-
C. albicans - a - - - - - aration of the manuscript; Frahad Nasr and Fatemeh
A. niger + b + - - + - Nematpour conducted the antimicrobial tests and prepa-
A. fumigatus
+ + - - + + ration of the samples; collection and identification of Ho-
A. flavus
+ + - - + + lothuria leucospilota was done by Arash Shakouri.
A. brasilensis
+ - - - + +
Financial Disclosure
a -: no activity.
b +: antimicrobial activity.
There is no financial disclosure.
Funding/Support
5. Discussion
This study was supported by grant No. U-90280 from
Hydroalcoholic (50%), n-hexane, chloroform, and meth- Vice Chancellor of Research of Ahvaz Jundishapur Uni-
anol extracts from different tissues and organs of the sea versity of Medical Sciences.
3
Jundishapur J Microbiol. 2014;7(1):e8708
Adibpour N et al.
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