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Antioxidant, Hemolytic, Antimicrobial, and Cytotoxic Activities of the Tropical Atlantic Marine Zoanthid Palythoa Caribaeorum

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Antioxidant, Hemolytic, Antimicrobial, and Cytotoxic Activities of the Tropical Atlantic Marine Zoanthid Palythoa Caribaeorum Anais da Academia Brasileira de Ciências (2015) 87(2): 1113-1123 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 http://dx.doi.org/10.1590/0001-3765201520140370 www.scielo.br/aabc Antioxidant, hemolytic, antimicrobial, and cytotoxic activities of the tropical Atlantic marine zoanthid Palythoa caribaeorum DANIEL B. ALENCAR1, ARTHUR A. MELO1, GISELLE C. SILVA2, REBECA L. LIMA1, KELMA M.S. PIRES-CAVALCANTE1, RÔMULO F. CARNEIRO1, ADRIANA S. RABELO1, OSCARINA V. SOUSA2, REGINE H.S.F. VIEIRA2, FRANCISCO A. VIANA3, ALEXANDRE H. SAMPAIO1 and SILVANA SAKER-SAMPAIO1 1Departamento de Engenharia de Pesca, Universidade Federal do Ceará, Campus do Pici, Av. Mister Hull, s/n, Caixa Postal 6043, 60455-970 Fortaleza, CE, Brasil 2Instituto de Ciências do Mar, Universidade Federal do Ceará, Av. da Abolição, 3207, 60165-081 Fortaleza, CE, Brasil 3Departamento de Química, Universidade do Estado do Rio Grande do Norte, Campus Universitário Central, Setor III, Rua Prof. Antônio Campos, s/n, 59633-010 Mossoró, RN, Brasil Manuscript received on July 16, 2014; accepted for publication on November 25, 2014 ABSTRACT Marine invertebrates are capable of synthesizing bioactive compounds, which may be beneficial to human health. The aim of this study was to evaluate the antioxidant, hemolytic, antimicrobial and cytotoxic activities of crude extract (70% EtOH), and dichloromethane (DCM), ethyl acetate (EtOAc), and aqueous (Aq) fractions of the marine zoanthid Palythoa caribaeorum. The phenolic compound contents of the crude extract, DCM, EtOAc and Aq fractions were 12.33, 18.17, 10.53, and 3.18 mg GAE per gram, respectively. DPPH radical scavenging activity showed slight variation. IC50 of crude extract, DCM, EtOAc and Aq fractions were 11.13, 11.25, 11.74, and 11.28 µg mL-1, respectively. Among the sample, -1 ferrous ion chelating was the highest in crude extract (IC50 302.90 µg mL ), followed by EtOAc, Aq, and DCM fractions with 457.77, 547.91, and 641.82 µg mL-1, respectively. Ferric-reducing antioxidant power showed optical density at about 0.5. The samples tested exhibited low hemolytic activity under 10% up to a concentration of 50 μg mL-1. No antimicrobial activity was observed against any of the tested bacterial strains. For the cytotoxic activity, LC50 of DCM, crude extract, EtOAc, and Aq were 52.10, 83.06, 86.34, and 117.45 μg mL-1, showing high toxicity. Key words: biological activity, marine invertebrate, natural product, Sphenopidae. INTRODUCTION antitumor, anti-inflammatory, antiviral, antioxidant, In recent decades, the discovery of new substances and antimicrobial (Blunt et al. 2014, Radjasa et al. isolated from marine organisms has increased. 2011, Senthilkumar and Kim 2013). These substances may be biologically active and Antioxidants play an important role in may have well-defined biological properties, e.g., biological systems, including cell signaling pathways and defense against oxidative damage. Correspondence to: Daniel Barroso de Alencar E-mail: [email protected] Intracellular antioxidant activity prevents damage An Acad Bras Cienc (2015) 87 (2) 1114 DANIEL B. ALENCAR et al. caused by reactive oxygen species (ROS). Fujji 1982, Shigemori et al. 1999, Uemura et al. While ROS are produced as a product of normal 1979). Besides these classes of compounds, the cellular functioning, excessive amounts can cause vasoconstrictor palytoxin, a very complex molecule deleterious effects on DNA, RNA, and protein that presents both lipophilicity and hydrophilicity, molecules, which, in theory, contribute to the was first isolated from the cnidarians zoanthid physiology of aging and may be involved in many Palythoa toxica by Moore and Scheuer (1971). humans diseases, such as atherosclerosis, cancer, Later, this toxin was also found in other species of diabetes, and cardiovascular and neurological the same genus, such as P. caribaeorum (Ramos diseases, such as Parkinson’s and Alzheimer’s and Vasconcelos 2010). disease (Choudhari et al. 2014, Guetens et al. 2002). This work aimed to evaluate the crude extract Since multidrug resistance is a well-documented (70% EtOH), and dichloromethane (DCM), ethyl health problem, an important aspect of the search for acetate (EtOAc), and aqueous (Aq) fractions of natural compounds isolated from marine organisms is the marine zoanthid P. caribaeorum at different their antimicrobial activity (Al-Haj et al. 2010, Silva concentrations (12.5, 25, 50, and 100 µg mL-1) et al. 2013, Vieira et al. 2010). for total phenolic content (TPC), antioxidant, Marine organisms are natural sources of diverse hemolytic and antimicrobial activities, as well as synthesized biocompounds that have specific cytotoxicity using the brine shrimp (Artemia sp.) protective functions against grazing, epiphytes lethality bioassay. and infections caused by microorganisms. The biological activity of these biocompounds may MATERIALS AND METHODS also have therapeutic applications of consequence ANIMAL COLLECTION AND PREPARATION OF EXTRACT to human health (Blunt et al. 2014, Radjasa et al. 2011, Senthilkumar and Kim 2013). The zoanthid specimens of Palythoa caribaeorum The zoanthid Palythoa caribaeorum were collected in August 2013 at Paracuru Beach, (Duchassing and Michelotti, 1860) (Sphenopidae) São Gonçalo do Amarante, state of Ceará in is found in large quantities along the Ceará northeastern Brazil, during low tide, under the coastline where colonies form thick, encrusted authorization of SISBIO (No. 33913-1) given by mats on rocky substrates under shallow water. The the Instituto Brasileiro do Meio Ambiente e dos skeleton is hard, while the outer surface is covered Recursos Naturais Renováveis. The specimens were with large, round calyces surrounded by a low, transported to the Laboratório de Produtos Naturais rounded ridge, and the tissues are impregnated with Marinhos (PROMAR-LAB) of the Universidade sediment. Their development is vegetative with Federal do Ceará (UFC) and washed with distilled high rates of regeneration. During low tide, when water in order to remove all epiphytes. they are exposed to the air, they produce a jelly-like Their identification was carried out by substance that protects them from dryness (Rabelo Professor Helena Matthews-Cascon (PhD). Voucher et al. 2013, Soares et al. 2006). specimens were deposited at Coleção de Cnidario Most bioactive substances isolated from the do Laboratório de Malacologia e Invertebrados marine zoanthid of the genus Palythoa have been Marinhos do Ceará (LINCE) of the Departamento chemically classified as fatty acids, ceramides, de Biologia of the Universidade Federal do Ceará steroids, or prostaglandins derived from glycerol (UFC) under number 047. and nitrogenous compounds (Almeida et al. Zoanthids in natura were lyophilized, ground 2012, Diop et al. 1986, Han et al. 2006, Pettit and and extracted with 70% EtOH at 1:20 (w/v). An Acad Bras Cienc (2015) 87 (2) BIOLOGICAL ACTIVITIES OF THE Palythoa caribaeorum 1115 Afterwards, two more re-extractions were carried Scavening effect (%) = out using the same solvent (Alencar et al. 2014). (Abssample − Abssample blank) P. caribaeorum 70% EtOH crude extract was 1− ×100% Eq. 1 Abs concentrated by reduced-pressure distillation, control and then liquid-liquid partitioned with sequential FERROUS ION CHELATING (FIC) ASSAY elution with dichloromethane (DMC), ethyl acetate (EtOAc) and water (Aq). FIC of 70% EtOH crude extract and DMC, EtOAc and Aq fractions (12.5, 25, 50, and 100 µg mL-1) TOTAL PHENOLIC CONTENT (TPC) was determined according to Wang et al. (2009). The absorbance of sample, blank sample and control Total phenolic content of 70% EtOH crude extract, was measured at 562 nm after 10 min incubation at as well as DMC, EtOAc and Aq fractions of P. room temperature, using a Biochrom Asys UVM340 -1 caribaeorum at 1 mg mL , was determined using microplate reader (Cambridge, UK). The sample the Folin-Ciocalteu method (Kumar et al. 2008). consisted of distilled water, 2 mM ferrous chloride Distilled water, Folin-Ciocalteu reagent and (FeCl2), 5 Mm ferrozine and fractions at different 20% Na2CO3 were added to 200 µL of samples. concentrations. Distilled water was used for both Following 30 min of incubation in the dark at blank sample and control, instead of ferrozine and room temperature, absorbance was measured at fractions, respectively. Ethylenediaminetetraacetic 760 nm using a microplate reader (Biochrom Asys acid (EDTA) was used as positive control. The UVM 340). The calculation of TPC was based on percentage of the ferrous ion chelating activity was a previously generated gallic acid standard curve, calculated using the expression below: and the results were expressed in mg gallic acid equivalent (GAE) g-1 of extract. Ferrous ion chelating activity (%) = [Abscontrol − DPPH RADICAL SCAVENGING ASSAY (Abssample − Abssample blank)]×100% Eq. 2 The DPPH scavenging activity of 70% EtOH crude Abscontrol extract and DMC, EtOAc and Aq fractions (12.5, FERRIC-REDUCING ANTIOXIDANT POWER (FRAP) 25, 50, and 100 µg mL-1) was measured according to the method described by Duan et al. (2006). The The ferric-reducing antioxidant power of the absorbance of sample, blank sample, and control 70% EtOH crude extract and DMC, EtOAc and was measured at 517 nm, after 30 min incubation Aq fractions (12.5, 25, 50, and 100 µg mL-1) was in the dark at room temperature, using a Biochrom determined using the method described by Ganesan Asys UVM340 microplate reader (Cambridge, UK). et al. (2008). Initially, 0.2 M phosphate buffer (pH The sample consisted of a mixture of 1 mL DPPH 6.6) and 1% potassium ferricyanide were added to methanolic solution (0.16 mM) with 1 mL of crude fractions at different concentrations. The mixture extract or fractions. The blank sample consisted was then incubated at 50°C for 20 min. After of 2 mL of crude extract or fractions, while the cooling, 10% trichloroacetic acid was added to the control contained 2 mL DPPH methanolic solution mixture. Next, an aliquot of the upper layer of the (0.16 mM) only. Butylated hydroxyanisole (BHA) solution was mixed with distilled water and 0.1% was used as positive control.
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