article information

Jundishapur Journal of Natural Pharmaceutical Products: Vol.12, issue 1; e32422
published online: September 18, 2016
article type: Research Article
received: August 21, 2015
revised: May 17, 2016
accepted: July 5, 2016
DOI: https://doi.org/10.5812/jjnpp.32422
Crossmark

Antimicrobial Activity of Sea Cucumber (Stichopus variegatus) Body Wall Extract in Chabahar Bay, Oman Sea

authors:

avatar Arash Shakouri 1 , 2 , * , avatar Mohammad Reza Shoushizadeh 2 , avatar Fatemeh Nematpour 3

Marine Biology Department, Chabahar Maritime University, Chabahar, IR Iran
Marine Pharmaceutical Science Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Graduated in Marine Biology, Chabahar Maritime University, Chabahar City, IR Iran

how to cite: Shakouri A, Shoushizadeh M R, Nematpour F. Antimicrobial Activity of Sea Cucumber (Stichopus variegatus) Body Wall Extract in Chabahar Bay, Oman Sea. Jundishapur J Nat Pharm Prod. 2017;12(1):e32422. https://doi.org/10.5812/jjnpp.32422.

Abstract

Background:

Seas and oceans have been recognized as a rich source of metabolites and bioactive compounds with biodiversity and drug activity. These compounds have accumulated in various organisms. Bioactive compounds can be extracted from various animal groups, such as coral, crabs, moss animals, Echinodermata, case-bearers, fishes, and sponges.

Objectives:

Antimicrobial extracts of the sea cucumber body wall (Stichopus variegatus), collected from Chabahar Bay, were tested for four bacterial, fungal, and yeast species.

Methods:

The disk diffusion method was used in this study. The McFarland 0.5 standard was used in the preparation of suspensions of the mentioned microorganisms under a hood. Next, the species were cultured with a swab on a Mueller-Hinton agar for bacteria and on a Sabouraud dextrose agar for fungi. Subsequently, the disks, with appropriate concentrations, were placed on microbiological growth media.

Results:

The results of the study showed that the best effect belonged to the aqueous methanol extract of the sea cucumber body wall, with an 8 mg/mL concentration on E. coli, with an inhibition zone diameter of 12.26 mm. The lowest inhibition zone diameter belonged to the methanol extract of white strands, with a 2 mg/mL concentration on C. albicans and an inhibition zone diameter of 1.16 mm. The n-hexane and chloroform extracts had no effect.

Conclusions:

Comparison of these tests indicates that sea cucumbers have an innate immunity system, which can be considered a potential source for discovering antimicrobial peptides.

1. Background

Seas and oceans have been recognized as a rich source of metabolites and bioactive compounds with biodiversity and drug activity. Compounds extracted from marine organisms, because of their antifungal and antibacterial activities, have been isolated (1). Sea cucumbers are rich in glycosides (2), especially triterpene glycosides, whose antifungal and antitumor activities have been demonstrated (3). These organisms also have considerable amounts of lectin (4, 5), cerebroside (6), glycosaminoglycan (7, 8), sterol and omega 6, fatty acids and omega 3. The natural compounds in marine organisms can be used as a source of compounds with nutritional, pharmaceutical, and medical applications (9). Compounds with biological activities can be extracted from various animal groups, such as coral, crabs, moss animals, Echinodermata, case-bearers, fishes, and sponges (10).

Echinodermata is an independent and special phylum of animals whose body structure cannot be compared to any other animal. These organisms constitute more than 6,500 species of marine organisms. Sea cucumbers (Holothuroidea) are among the most unusual members of Echinodermata, in terms of both structure and physiology (11). Studies on sea cucumbers have been restricted to its physiology and ecological features for many years (12). Today, sea cucumbers are being tested in terms of their antibacterial, antifungal, anticoagulant, antiviral, cytotoxic, hemolytic, and even anti-HIV activities (13, 14).

Recent studies have shown that sea cucumber extracts have a strong antimicrobial activity. This antimicrobial activity is attributed to triterpene glycosides (15), disulphate glycosides (16), steroid glycosides (17), polyhydroxy sterols (18), naphthoquinone pigments (19), lysozyme, and various precursors (20). Stichopus variegatus (also known as Stichopus hermanni) is a species that is most valued not only as a food source, but it also offers a variety of remedies which have been proven in an animal model system (21). Stichopus variegatus water extract (SVWE) has been reported to be used for rheumatoid arthritis (22), abdominal pain, and liver damage (23), as well as heart ailments (24). Numerous experiments have revealed various in vitro effects, such as antioxidant (25), anti-angiogenesis (26), antimicrobacterial (27), and antileshmanial (28). Experiments have also revealed its potential as a cyctoxic agent on T-lymphoblastic cell lines (29) and its proliferation effect on neurospheres (30).

2. Objectives

In this study, the antimicrobial activity of aqueous methanol, methanol, chloroform, and n-hexane extracts of the sea cucumber (Stichopus variegatus) body wall, collected from Chabahar Bay, in 1 - 8 mg/mL concentrations, were tested in four bacterial species, namely Staphylococcus epidermidis, Proteus vulgaris, Escherichia coli, Staphylococcus aureus, the fungus Aspergillus niger, and a yeast, Candida albicans.

3. Methods

Stichopus variegatus species were collected by divers in the subtidal zone of Chabahar Bay. Samples were placed in a container with sea water and were transferred to the microbiology lab of Chabahar Maritime University, where they were frozen at -20°C. Samples were washed with distilled water after being melted, and their internal organs were removed through an abdominal incision. The dried pieces of the body wall were extracted two times (15). The extraction process was implemented with the aim of separating the solved compounds in a solution or removing compounds from a solid mixture.

There are different techniques, including maceration, digestion, and infusion, that can be employed for extracting the organic compounds from plant and animal tissues. The method for extraction is usually selected in regard to the type of tissue and the extractable material (31). In this study, the maceration technique was used for extracting the body wall of the sea cucumber. The small pieces of the body wall were kept in 300 mL each of aqueous methanol, methanol, chloroform, and n-hexane solutions for 72 hours. After filtering with filter paper, the resulting extracts were condensed under the condition of vacuum with a rotary machine at 40°C and were dried with a freeze drier (15, 31).

Both the antibacterial and antifungal activities of Stichopus variegatus body wall extract, in four concentrations, were analyzed using the disk diffusion method. Initially, several solutions with different concentrations were prepared from the test extracts. Next, they were used for preparing four disks with 1, 2, 4, and 8 mg/mL concentrations. The prepared disks were tested with antibacterial and antifungal experiments. Regarding the studies on other species of sea cucumber, bacterial species Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Proteus volgaris, the yeast Candida albicans, and the fungus Aspergillus niger were used. The McFarland 0.5 standard was used in the preparation of the suspensions of the mentioned microorganisms under a hood. The species were then cultured with a swab on a Mueller-Hinton agar for bacteria and on a Sabouraud dextrose agar for fungi. Subsequently, the disks with the different concentrations were placed on microbiological growth media. Plates containing bacteria were kept in a 37°C incubator for 24 hours, and plates containing fungi were kept at 25°C for 24 - 48 hours. The bacterial and fungal growth inhibitory zones were measured in terms of mm with a caliper (32). In this test, the antibiotics gentamicin and ketoconazole were used as a positive control for bacteria and fungi, respectively.

4. Results

The results gained from testing aqueous methanol, methanol, chloroform, and n-hexane extracts of the body wall of S. variegatus were studied. For the antibacterial and antifungal tests, E. coli, S. epidermidis, and S. aureus were used as bacterial species. C. albicans and A. niger were used as yeast and fungal species. Most chloroform and n-hexane extracts did not show an inhibition zone. The best effect was shown by the aqueous methanol extract of the body wall. It had the highest inhibitory effect on the mentioned strains. The best effect of the aqueous methanol extract, at 8 mg/mL concentration, was related to the E. coli strains, with an inhibition zone of 12.26 mm.

The minimum inhibitory of the methanol extract of the sea cucumber body wall was also on strains of E. coli, at a concentration of 1 mg/mL, with an inhibition zone of 1/36 mm. The maximum inhibitory effect was significant, at a concentration of 8mg/ml of the strain of P. vulgaris, with an inhibition zone of 11 mm. The minimum inhibitory concentration was 1 mg/mL of the E. coli strain, with a 1.36 mm inhibition zone.

The chloroform extract of the sea cucumber body wall affected only the S. aureus strain. Its highest inhibition zone was 9.30 mm, at a concentration of 8 mg/mL, and its lowest inhibition zone was 2.16 mm at a concentration of 1 mg/mL. The n-hexane extract had no effect on the mentioned concentration. The results of the antimicrobial test of white string extracts, at the four concentrations of 1, 2, 4, and 8 mg/mL, are shown in Figures 1 - 4.

Diameter of the Inhibition Zones of Bacterial and Fungal Species Developed on S. variegatus Body Wall Extracts in 1 mg/mL
Diameter of the Inhibition Zones of Bacterial and Fungal Species Developed on S. variegatus Body Wall Extracts in 1 mg/mL
Figure 1.

Diameter of the Inhibition Zones of Bacterial and Fungal Species Developed on S. variegatus Body Wall Extracts in 1 mg/mL

Diameter of the Inhibition Zones of Bacterial and Fungal Species Developed on S. variegatus Body Wall Extracts in 2 mg/mL
Diameter of the Inhibition Zones of Bacterial and Fungal Species Developed on S. variegatus Body Wall Extracts in 2 mg/mL
Figure 2.

Diameter of the Inhibition Zones of Bacterial and Fungal Species Developed on S. variegatus Body Wall Extracts in 2 mg/mL

Diameter of the Inhibition Zones of Bacterial and Fungal Species Developed on S. variegatus Body Wall Extracts in 4 mg/mL
Diameter of the Inhibition Zones of Bacterial and Fungal Species Developed on S. variegatus Body Wall Extracts in 4 mg/mL
Figure 3.

Diameter of the Inhibition Zones of Bacterial and Fungal Species Developed on S. variegatus Body Wall Extracts in 4 mg/mL

Diameter of the Inhibition Zones of Bacterial and Fungal Species Developed on S. variegatus Body Wall Extracts in 8 mg/mL
Diameter of the Inhibition Zones of Bacterial and Fungal Species Developed on S. variegatus Body Wall Extracts in 8 mg/mL
Figure 4.

Diameter of the Inhibition Zones of Bacterial and Fungal Species Developed on S. variegatus Body Wall Extracts in 8 mg/mL

5. Discussion

Various countries have organized many studies on the antimicrobial activities of a number of sea organisms, including Echinodermata, during recent years (33). These studies have made it clear that Echinodermata, in contrast to other sea organisms, such as Porifera, Bryozoa, Mollusca, coral, and annelida (i.e., ringed worms), have the highest antimicrobial activity (21). Most experiments carried out to analyze the antibacterial and antifungal activity of sea cucumbers have focused on methanol, aqueous methanol, ethanol, and chloroform extracts, as well as triterpene compounds (34).

Omran and Allam used the disk diffusion method to analyze the antifungal activity of H. polii (a special variety of sea cucumber) in the Mediterranean sea (35). The experiments indicated that the ethanol extract of the body wall of this sea cucumber, in a concentration of 2.5 mg/mL, has a strong antifungal activity on A. Flavus, A. niger, and C. albicans. The antifungal activity of the raw extract (i.e., aqueous methanol) of H. polii, using the disk diffusion method, in the concentration of 150 to 300 μgr/mL, has a strong inhibitory effect on A. funmigatus and a weaker inhibitory effect on Trichophytonrubram, although it did not show any considerable effect on C. albicans.

Comparing this experiment with studies on similar species of C. albicans and A. niger revealed that the aqueous methanol and methanol extracts of the S. variegatus body wall, in the mentioned concentrations, inhibits the growth of C. albicans. By comparing the test with the study on the same strains of A. niger and C. albicans, it can be stated that the methanol and aqueous methanol extracts of the body wall of the sea cucumber S. variegatus, in concentrations of 1 – 8 mg/mL, have an inhibitory effect on A. niger. Only the methanol water extract of S. variegatus, at these concentrations, had an inhibitory effect on the C. albicans strain. Therefore, it can be said that various extracts of different species of sea cucumbers have different effects, even on similar species.

Using the disk diffusion method, Mokhlesi et al. tested the antibacterial and antifungal activities of three extracts (i.e., ethyl-acetate, aqueous methanol, and methanol) of species of H. leucospilota and B. marmorata on S. aureus, E. coli, P. aeruginosa (as bacterial species), A. niger (as a fungal species) and the yeast C. albicans (31). These tests showed that the extracts had no inhibitory effect on the bacterial species (i.e., no inhibition zone was formed). However, A. niger and C. albicans did not grow in the methanol extract of the white strands of H. leucospilota, the methanol extract of the body wall of B. marmorata, or in the aqueous methanol extract of the white strands of B. marmorata. These effects were stronger for A. niger, as the methanol extract of the body wall of B. marmorata, in a concentration of 8 mg/mL, inhibited the growth of A. niger through forming an inhibition zone with a diameter of 17 mm.

However, the results of this experiment showed that the aqueous methanol and methanol extracts of the body wall of S. variegatus, in concentrations of 1 to 8 mg/mL, inhibited the growth of E. coli and A. niger, and the aqueous methanol and chloroform extracts of the body wall of the sea cucumber had an inhibition effect on the growth of S. aureus. The biggest inhibition zone, developed in a concentration of 8 mg/mL for the aqueous methanol extract of E. coli, was 12.26 mm. For the aqueous methanol extract of S. aureus, it was 11.13 mm, and for the aqueous methanol extract of A. niger, it was 11.16, which was attributed to the body wall extract of sea cucumber. As this test indicates, various species of sea cucumbers have different antimicrobial activities. The difference between the molecular mass and amino acid sequence in the extracted peptides from these organisms is what makes their effect on various bacterial and fungal species different.

Another study dealt with the antibacterial and antifungal activities of alcoholic extracts of various species of Holothurian, including A. miliaria, H. scabra, and H. atra on the Tamil Nadu coast, in concentrations varying from 10 to 200 µg/mL. The bacterial and fungal species tested in the study were E. coli, A. hydrophila, Enterococcus Sp., K. pneumonia, P. aeroginosa, S. Typhi, S. aureus, V. harveyi, and Aspergillus sp., which were inhibited by extracts of sea cucumbers including H. atra, H. scarb, and A. miliaris. For example, the raw extract (aqueous methanol) of Holothuria atra, in the concentration of 130 μgr/mL, inhibited the growth of Aeromonas hydrophila by forming a 2 mm inhibition zone, but it did not affect the growth of E. coli. Likewise, the raw extract of Actinopyga miliaris, in the concentration of 200 μg/mL, inhibited the growth of Aspergillus sp. though forming a 4.5 mm inhibition zone. This test convinces us to conclude that, although all of the tested species were limited to a certain geographical location, different inhibition zones were formed by various concentrations. However, our study indicated that the raw extract (aqueous methanol) of the body wall of S. variegatus, in concentrations of 1, 2, 4, and 8 mg/mL, had an inhibitory effect on E. coli and A. niger. Our results make it clear that there are materials with different compounds in the sea cucumber body which contain antimicrobial activity (36).

Comparison of these tests indicates that sea cucumbers have an innate immunity system which can be considered a potential source for discovering antimicrobial peptides. As a result, sea cucumbers can be introduced as a source rich in compounds with antimicrobial features, which means they can be good candidates for synthesizing pharmaceutical and medical compounds and antibiotics.

Patar et al. (2012) studied the effect of the water extract of sea cucumber Stichopus variegatus on rat spinal astrocytes cell lines. The extracts were prepared in four different concentrations of 0.1, 1.0, 5.0, and 10.0 μg/mL. Their results suggest that treatment with S. variegatus water extract induces proliferation and differentiation of spinal astrocytes, in a dose-dependent manner. The potential of S. variegatus water extract as a growth promoter in vitro demands a further analysis of the effects of S. variegatus water extract on spinal astrocytes proliferative in vitro. Some reports show proliferative effects of S. variegatus extract on fibroblast cell lines, neurite growth, blood vessels in vitro, and neural stem/progenitor cells (37).

Acknowledgements

References

  • 1.

    James DB. Twenty sea cucumbers from seas around India. Naga, The ICLARM Quarterly. 2001;24(1&2):4-8.

  • 2.

    Silchenko AS, Avilov SA, Kalinin VI, Kalinovsky AI, Dmitrenok PS, Fedorov SN, et al. Constituents of the sea cucumber Cucumaria okhotensis. Structures of okhotosides B1-B3 and cytotoxic activities of some glycosides from this species. J Nat Prod. 2008;71(3):351-6. [PubMed ID: 18288810]. https://doi.org/10.1021/np0705413.

  • 3.

    Kitagawa I, Nishino T, Kyogoku Y. Structure of holothurin A a biologically active triterpene-oligoglycoside from the sea cucumber Holothuria leucospilota Brandt. Tetrahedron Letters. 1979;20(16):1419-22.

  • 4.

    Himeshima T, Hatakeyama T, Yamasaki N. Amino acid sequence of a lectin from the sea cucumber, Stichopus japonicus, and its structural relationship to the C-type animal lectin family. J Biochem. 1994;115(4):689-92. [PubMed ID: 8089084].

  • 5.

    Gowda NM, Goswami U, Khan MI. Purification and characterization of a T-antigen specific lectin from the coelomic fluid of a marine invertebrate, sea cucumber (Holothuria scabra). Fish Shellfish Immunol. 2008;24(4):450-8. [PubMed ID: 18282768]. https://doi.org/10.1016/j.fsi.2008.01.002.

  • 6.

    Ikeda Y, Inagaki M, Yamada K, Miyamoto T, Higuchi R, Shibata O. Langmur monolayers of cerebroside with different head groups originated from sea cucumber: Binary systems with dipalmitoylphosphatidylcholine (DPPC). Colloids Surfaces B: Biointerfaces. 2009;72(2):272-83.

  • 7.

    Kariya Y, Watabe S, Kyogashima M, Ishihara M, Ishii T. Structure of fucose branches in the glycosaminoglycan from the body wall of the sea cucumber Stichopus japonicus. Carbohydr Res. 1997;297(3):273-9. [PubMed ID: 9060189].

  • 8.

    Wu M, Xu S, Zhao J, Kang H, Ding H. Free-radical depolymerization of glycosaminoglycan from sea cucumber Thelenata ananas by hydrogen peroxide and copper ions. Carbohydrate Polymers. 2010;80(4):1116-24.

  • 9.

    Jha R, Zi-Rong X. Biomedical compounds from marine organisms. Marine Drugs. 2004;2(3):123-46.

  • 10.

    Yasoda H, Chi Z, Zhu K. Probiotics and sea cucumber farming. SPC Bechedemer Information Bulletin. 2006;24:45.

  • 11.

    Flammang P, Ribesse J, Jangoux M. Biomechanics of adhesion in sea cucumber Cuvierian tubules (Echinodermata, Holothuroidea). Integrative comparat Biol. 2002;42(6):1107-15.

  • 12.

    Du H, Bao Z, Hou R, Wang S, Su H, Yan J, et al. Transcriptome sequencing and characterization for the sea cucumber Apostichopus japonicus (Selenka, 1867). PLoS One. 2012;7(3). e33311. [PubMed ID: 22428017]. https://doi.org/10.1371/journal.pone.0033311.

  • 13.

    Ding XZ, Witt R, Tong WG, Li X, Betts H, Collin P, et al. Antipancreatic cancer effects of ristoleic acid. Pancreatol. 2003;3:209-69.

  • 14.

    Gowda NM, Goswami U, Khan MI. T-antigen binding lectin with antibacterial activity from marine invertebrate, sea cucumber (Holothuria scabra): possible involvement in differential recognition of bacteria. J Invertebr Pathol. 2008;99(2):141-5. [PubMed ID: 18501924]. https://doi.org/10.1016/j.jip.2008.04.003.

  • 15.

    Ismail H, Lemriss S, Ben Aoun Z, Mhadhebi L, Dellai A, Boiron P, et al. Antifungal activity of aqueous and methanolic extracts from the Mediterranean sea cucumber, Holothuria polii. J Mycol Med. 2008;18:23-6.

  • 16.

    Muniain C, Centurión R, Careaga VP, Maier MS. Chemical ecology and bioactivity of triterpene glycosides from the sea cucumber Psolus patagonicus (Dendrochirotida: Psolidae). J Mar Assoc. 2008;88(4):817-23.

  • 17.

    Bryan PJ, McClintock JB, Watts SA, Marion B, Gauthier JJ, Hopkins TS. Bioactive properties of echinoderm body wall extracts: feeding deterrence, antifouling and antimicrobial activity. Alabama Academy Sci J. 1993;64:87.

  • 18.

    Jensen PR, Harvell CD, Wirtz K, Fenical W. Antimicrobial activity of extracts of Caribbean gorgonian corals. Marine Biol. 1996;125(2):411-9.

  • 19.

    Chattopadhyay T, Guha AK, Chatterjee BP. Novel antimicrobial activity of scyllin, a haemolymph lectin of the edible crab Scylla serrata. Biomedical Letters. 1996;53(209):29-40.

  • 20.

    Findlay JA, Daljeet A, Moharir YE. Some constituents of the sea cucumber Cucumaria frondosa. Marine Chem. 1983;12(2-3):228.

  • 21.

    Ridzwan BH. Sea cucumbers, a Malaysian heritage. Research Centre of International Islamic University Malaysia (IIUM), Kuala Lumpur Wilayah Persekutuan: Kuala Lumpur, Malaysia; 2007.

  • 22.

    Ridzwan BH, Zarina MZ, Kaswandi MA, Nadirah M, Shamsuddin AF. The antinociceptive effects of extracts from Stichopus chloronotus Brandt. Pak J Biol Sci. 2001;4:244-6.

  • 23.

    Bordbar S, Anwar F, Saari N. High-value components and bioactives from sea cucumbers for functional foods--a review. Mar Drugs. 2011;9(10):1761-805. [PubMed ID: 22072996]. https://doi.org/10.3390/md9101761.

  • 24.

    Tan WT, Fong Y, Ridzwan BH. The effect of coelomic fluid Stichopus hermanii on isolated perfused rat hearts and the involvement of prostaglandin in its mechanism of action. Pakistan J Biol Sci. 2005;8(1):78-84.

  • 25.

    Hawa I, Zulaikah M, Jamaludin M, Zainal Abidin AA, Kaswandi MA, Ridzwan BH. The potential of the coelomic fluid in sea cucumber as an antioxidant. Malaysian J Nutrition. 1999;5(1&2):55-9.

  • 26.

    Tong Y, Zhang X, Tian F, Yi Y, Xu Q, Li L, et al. Philinopside A, a novel marine-derived compound possessing dual anti-angiogenic and anti-tumor effects. Int J Cancer. 2005;114(6):843-53. [PubMed ID: 15645493]. https://doi.org/10.1002/ijc.20804.

  • 27.

    Ridzwan BH, Kaswandi MA, Azman Y, Fuad M. Screening for antibacterial agents in three species of sea cucumbers from coastal areas of Sabah. Gen Pharmacol. 1995;26(7):1539-43. [PubMed ID: 8690242].

  • 28.

    Singh N, Kumar R, Gupta S, Dube A, Lakshmi V. Antileishmanial activity in vitro and in vivo of constituents of sea cucumber Actinopyga lecanora. Parasitol Res. 2008;103(2):351-4. [PubMed ID: 18452039]. https://doi.org/10.1007/s00436-008-0979-3.

  • 29.

    Kaswandi MA, Hing HL, Sahalan AZ, Farah F, Ridzwan BH, Samsudin MW, et al. Saponin from sea cucumber Stichopus badionotus sluiter as potential cytotoxic agent on CEM-SS T-lymphoblastic cell. J Microsc Soc Thailand. 2004;18:79-84.

  • 30.

    Zhang Y, Song S, Liang H, Wang Y, Wang W, Ji A. Enhancing effect of a sea cucumber Stichopus japonicus sulfated polysaccharide on neurosphere formation in vitro. J Biosci Bioeng. 2010;110(4):479-86. [PubMed ID: 20547343]. https://doi.org/10.1016/j.jbiosc.2010.05.009.

  • 31.

    Mokhlesi A, Saeidnia S, Gohari A, Shahverdi A, Nasrolahi A, Farahani F, et al. Biological activities of the sea cucumber Holothuria leucospilota. Asian J Animal Vet Adv. 2012;7(3):243-9.

  • 32.

    Haug T, Kjuul AK, Styrvold OB, Sandsdalen E, Olsen OM, Stensvag K. Antibacterial activity in Strongylocentrotus droebachiensis (Echinoidea), Cucumaria frondosa (Holothuroidea), and Asterias rubens (Asteroidea). J Inverteb Pathol. 2002;81(2):94-102.

  • 33.

    Shakouri A, Aminrad T, Nabavi MB, Kochanian P, Savari A, Safahiye A. New observation of three species of sea cucumbers from Chabahar bay (Southeast coasts of Iran). J Biological Sci. 2009;9(2):1847.

  • 34.

    Se- kwon K. Advance in food and nutrition research: Implication and application animal and microbs. J Marine Medicinal Food. 2012;65:314-7.

  • 35.

    Omran N, Allam NG. Screening of microbial contamination and antimicrobial activity of sea cucumber Holothuria polii. Toxicol Indust Health. 2013;29(10):944-54.

  • 36.

    Abraham TJ, Nagarajan J, Shanmugam SA. Antimicrobial substances of potential biomedical importance from holothurian species. Indian J Marine Sci. 2002;31(2):161-4.

  • 37.

    Patar A, Jamalullail S, Jaafar H, Abdullah J. The effect of water extract of sea cucumber Stichopus variegatus on rat spinal astrocytes cell lines. Current Neurobiol. 2015;2012.