Antimicrobial properties of sea anemone Anthopleura nigrescens from Pacific coast of Costa Rica

Henry Borb′on, Sandra V′aldes, Javier Alvarado-Mes′en, Roy Soto, Ilena VegaLaboratory of Research and Development in Chemical Technology，Department of Chemistry，National University，Heredia，86-000，Costa Rica

2Laboratory of Biochemistry and Biotechnology of Proteins，Department of Biological Science，National University，Heredia，86-3000，Costa Rica

3Laboratory of Natural Products and Biological Essays，Department of Chemistry，National University，Heredia，86-3000，Costa Rica

ARTICLE INFO

Article history：

Received 30 Oct 2015

Receivedinrevisedform16 Nov,2nd

revised form 25 Nov 2015

Accepted 15 Dec 2015

Available online 19 Mar 2016

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Antimicrobial properties of sea anemone Anthopleura nigrescens from Pacific coast of Costa Rica

Henry Borb′on1*, Sandra V′aldes1, Javier Alvarado-Mes′en2, Roy Soto3, Ilena Vega31Laboratory of Research and Development in Chemical Technology，Department of Chemistry，National University，Heredia，86-3000，Costa Rica

2Laboratory of Biochemistry and Biotechnology of Proteins，Department of Biological Science，National University，Heredia，86-3000，Costa Rica

3Laboratory of Natural Products and Biological Essays，Department of Chemistry，National University，Heredia，86-3000，Costa Rica

ARTICLE INFO

Article history：

Received 30 Oct 2015

Receivedinrevisedform16 Nov,2nd

revised form 25 Nov 2015

Accepted 15 Dec 2015

Available online 19 Mar 2016

Keywords：

Biological activity

Antibacterial

Antifungal

Bioactive

Sea anemone

ABSTRACT

Objective：To evaluate the antimicrobial and antifungal activities of the aqueous and partitioned extract of sea anemone Anthopleura nigrescens（A. nigrescens）.

Methods：The sea anemone A. nigrescens was collected, minced, homogenized, lyophilized and then further partitioned with diethyl ether, acetone, ethanol and water. These fractions were evaluated for antimicrobial activity against bacterial and fungal pathogens.

Results：Acetone extract was found to produce a pronounced inhibition of 7.0 mm against Proteus vulgaris and diethyl ether extract inhibited Pseudomonas aeruginosa with an inhibition zone of 6.5 mm. In antifungal activity, ethanol extract showed good activity against Botrytis cinerea, Trichoderma harzianum and Rhizopus oryzae compared with other strains. Acetone and ethanol extract of A. nigrescens showed activity against all of pathogens tested. Slight activity was observed in the water extract with inhibition zone of 1.5 mm.

Conclusions：The present study revealed that sea anemone A. nigrescens may also contain some biologically active agents which have potential activity against pathogenic microorganisms.

Original article http://dx.doi.org/10.1016/j.apjtb.2016.01.014

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Foundation Project: Supported by Institutional Development and Academic Funding（Grant FIDA IIDG01-NX026412）.

Peer review under responsibility of Hainan Medical University. The journal implements double-blind peer review practiced by specially invited international editorial board members.

1. Introduction

Marine ecosystems have a very large diversity of resources, most of them still partially unknown, and a few others exploited for development of new industrial and toxicological products ［1,2］. These natural chemicals have always played an important role in medicine and, in particular, marine metabolites have increasingly become major players in recent drug discovery［3］.

Cnidaria is a phylum containing over 10000 species of animal found exclusively in aquatic（freshwater and marine）environment: they are predominantly marine species, of which 68% belong to the class Anthozoa［4］. Unlike other cnidarians, anthozoans do not have a medusa stage in their development. It means they are sessile marine coelenterates including solitary and colonial polyps, and need to protect themselves against the lethal consequences of environment, including microbial or fungus invasion［5］.

Sea anemones, like other coelenterates, produce many biologically active polypeptides and proteins, including poreforming toxins（cytolysins）, phospholipases, proteinase inhibitors and neurotoxins［6–10］. Cytolytic toxins have attracted a great interest of researchers, because they exhibit antitumor, antimicrobial, antiparasitic and other types of biological activities due to the powerful membranolytic action［11,12］. New trends in drug discovery from natural sources have emphasized investigation of the sea anemones ecosystem to explorenumerous complex and novel chemical entities. These entities are the source of a new lead for treatment of many diseases such as cancer, AIDS, inflammatory condition, arthritis, malaria and a large variety of viral, bacterial and fungal diseases［13,14］. According to Al-Zereini［15］, because of increasing demand for the biodiversity in the screening programs seeking therapeutic drugs from natural products, there is now a greater interest in these kinds of marine metabolites.

Acu?a et al.［16］reported the first record of the sea anemone Anthopleura nigrescens（A. nigrescens）on the Pacific coast of Central America, extending its distribution to the Pacific coast of Costa Rica. Also, A. nigrescens has been reported to produce reducing compounds, saponins, terpenes, steroids, amino acids, and hemolytic sphingomyelin inhibitable cytolytic toxin［17,18］. The present study aimed at determining the antimicrobial activity of tropical sea anemone A. nigrescens from Pacific coast of Costa Rica.

2. Materials and methods

2.1. Material collection and identification

Specimens were collected during low tide in a rocky area on the Pacific coast of Costa Rica, specifically in Mata Lim′on area, province of Puntarenas（latitude 9°55′01.99′′N and longitude 84°42′49.50′′W）. Sea anemones were carefully detached from the rock, washed with the sea water, placed in plastic sample containers, and transported at 4°C to the laboratory. Samples were frozen for transport to prevent decomposition, loss of concentration and their normal activity, as recommended by Ha¨ussermann［19］and S′anchez-Rodríguez et al.［20］. A specimen was deposited at the Actiniaria Collection, Department of Marine Science（College of Natural and Exact Sciences, Mar del Plata National University）with reference C.A. 28.

2.2. Preparation and extraction of material

Crude extract was obtained by mincing and homogenizing the whole animal body in distilled water（1:2, w/v）. The extract was cold steeped overnight at 4°C, decanted and then filtered through Whatman No.1filterpaper.Residueswerecentrifugedat13675r/ min for 20 min at 4°C in an Eppendorf centrifuge 5418R. The supernatant was collected in separate cleaned glass tubes and stored at -22°C. The resulting crude extract was lyophilized and partitioned with diethyl ether, acetone, ethanol and water.

2.3. Antimicrobial activity

The antibacterial and antifungal activity was carried out by disc diffusion assay according to the method of Bauer et al.［21］, and Jorgensen and Ferraro［22］. Antimicrobial activity of sea anemone extract was determined against five bacterial strains viz., Pseudomonas aeruginosa（P. aeruginosa）, Salmonella enterica（S. enterica）, Klebsiella oxytoca（K. oxytoca）, Escherichia coli（E. coli）and Proteus vulgaris（P. vulgaris）and five fungal strains viz., Aspergillus fumigatus （A. fumigatus）, Cladosporium cucumerinum（C. cucumerinum）, Trichoderma harzianum（T. harzianum）, Rhizopus oryzae （R. oryzae）and Botrytis cinerea（B. cinerea）. The bacterial cultures were obtained from Department of Biological Sciences at National University, Costa Rica.

2.3.1. Disc diffusion assay

The strains of microorganisms were inoculated in conical flask containing 100 mL of nutrient broth. These conical flasks were incubated at 37°C for 24 h and were referred to as seeded broth. Media were carried out using Muller Hinton agar, poured on Petri dishes and inoculated with the test microorganisms from the seeded broth using cotton swabs. Sterile discs of 6 mm width were impregnated with 20 μL of test extract and introduced onto the upper layer of the seeded agar plate. The plates were incubated at 37°C for 48 h. The susceptibility of the test strain was recorded by measuring the width of inhibition zone around the discs using vernier caliper. Amoxicillin and ketoconazol were the positive standards. Dimethylsulfoxide was used as negative control［23］.

3. Results

The results of the antibacterial and antifungal activity of the extracts of sea anemone A. nigrescens against the tested pathogens were given in Tables 1 and 2.

3.1. Antibacterial activity

Table 1 Antibacterial activity of A. nigrescens extract against human pathogens.

Table 2 Antifungal activity of A. nigrescens extract against fungal pathogens.

Different degrees of antibacterial inhibition against pathogenic bacteria were obtained with the extracts of A. nigrescens. The growth inhibition zone measured ranged from 1.0 to 7.0 mm for all the sensitive bacteria. The acetone extract exhibited the highest antibacterial activity with inhibition zone of 7.0 mm against P. vulgaris. Similarly, the diethyl ether extract inhibited the growth of P. aeruginosa with an inhibition zone of 6.5 mm.As shown in Table 1, the ethanol and acetone extract showed activity against all the pathogens tested. On the other hand, the water extract was practically zero；it showed the minimum activity or slight activity against K. oxytoca and E. coli（1.0 mm）. No activity was recorded in water extract against S. enterica and P. vulgaris, neither diethyl ether extract against P. vulgaris.

3.2. Antifungal activity

The growth inhibition zone measured ranged from 1.0 to 5.0 mm for all the sensitive fungal pathogens. The ethanol extract showed the highest antifungal activity with inhibition zones of 5.0, 4.5 and 4.5 mm against B. cinerea, R. oryzae and T. harzianum, respectively. Water extract showed the minimum activity［（1.0±0.5）mm］against C. cucumerinum and T. harzianum. As shown in Table 2, the acetone and ethanol extract exhibited activity against all the fungal pathogens tested. No activity was observed in diethyl ether extract against R. oryzae and B. cinerea, neither water fraction against A. fumigatus. Dimethylsulfoxide also had no effect on the growth of any of the 10 microorganisms.

4. Discussion

The antibacterial and antifungal activities were significant in the crude extract of A. nigrescens. Pronounced inhibition was conferred by the acetone extract against the bacterial pathogen P. vulgaris with zone of 7.0 mm, and the same way, diethyl ether extract of A. nigrescens was found to inhibit P. aeruginosa with a maximum zone of 6.5 mm. Thangaraj et al.［24］reported similar activity in acetone and butanol extract of Stichodactyla mertensii against E. coli and Proteus mirabilis in methanolic extract. Rajak ［25］and Balaji［26］found similar properties of sea anemone toxin from Paracondactylis indicus, Paracondactylis sinensis, Heteractis magnifica and Stichodactyla haddoni（S. haddoni）. In the case of antifungal activity, the ethanol extract showed inhibition zones of 5 mm against B. cinerea. The antifungal properties of plant essential oils against B. cinerea were tested by Wang et al.［27］and detailed studies were conducted regarding in vitro activity of eugenol on B. cinerea. Antifungal activity on B. cinerea of flavonoids and diterpenoids isolated from the surface of Pseudognaphalium spp. was reported by Cotoras et al.［28］. Thangaraj et al.［24］reported similar activity of sea anemones Stichodactyla mertensii and Stichodactyla gigantea against B. cinerea in methanolic extract.

John et al.［29］reported the antibacterial activity of sea anemone Anthopleura elegantissima and S. haddoni extracts against human pathogens, which revealed the fact that they have higher potential to produce broad-spectrum antibacterial activity with minimal concentration in non-polar extracts against a wide range of human pathogens. As an earlier report has been made, the crude extract of S. haddoni showed good activity against Gram-negative bacteria by Sureshkumar et al.［30］. Williams et al.［31］reported that the tissue extract of sea anemone showed high inhibition（20 mm）against Klebsiella pneumonia and 24 mm with the hexane tissue extract against fish pathogen.

The results of the inhibitory effect were significant with a broad activity in acetone and ethanol extract in antibacterial and antifungal tests. The evidence indicates the metabolites responsible for such effect are soluble in these solvents, which agrees with Saad et al.［32］, Raja et al.［33］, John et al.［29］, Thangaraj et al.［24］and Madhumitha and Saral［34］, who indicate that the bioassay conducted with different organisms using low polar solvents as extraction solvent showed a better response as antibiotics in general than those performed with ethanol or any other solvent with higher polarity.

Marine organisms collected from Pacific coast of Costa Rica have shown a number of biological activities. This is the first report demonstrating the antibacterial and antifungal activity of A. nigrescens with these pathogens. The inhibitory effect of the extracts in A. nigrescens against pathogenic bacteria strains can introduce the marine organisms like sea anemones as an alternative in future studies for drugs development for the treatment of ailments caused by these pathogens. Furthermore, the recent research studies demonstrate that the Costa Rican coastline is a potential source to identify new cellular targets for further investigation.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

The authors express sincere gratitude to the constant encouragement given by PhD. Felix R. Rom′an, Professor and Director, Center for Education and Training in Agriculture and Related Sciences, Environmental Division, University of Puerto Rico, Mayag¨uez Campus, Puerto Rico. The authors acknowledge the technical guidance provided by MBA. Vanessa Bagnarello, Medical Science University, Costa Rica. We also thank Licda. Rocio Alfaro Avenda?o, Technical Assistant, Department of Chemistry, National University for her assistance and support in theimplementationof this manuscript. This project was supported by Institutional Development and Academic Funding（Government of Costa Rica）grant FIDA-IIDG01-NX026412 under the initiative“Identification of bioactive molecules with biomedical and biotechnological properties from marine invertebrates”and the Joint Research Agreement SIA-0067-13 by the National University of Costa Rica and Medical Science University.

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*Corresponding author:Henry Borb′on, Associate Professor, Department of Chemistry, National University, Heredia, 86-3000, Costa Rica.