Iranian Journal of Fisheries Sciences 17(2) 413-426 2018 DOI: 10.22092/IJFS.2018.116076 Antioxidant and cytotoxic activities of metabolites produced by a new marine Streptomyces sp. isolated from the sea cucumber Holothuria leucospilota Gozari M.1,2; Bahador N.2*; Jassbi A.R.3; Mortazavi M.S.4; Eftekhar E.5 Received: January 2017 Accepted: March 2018 Abstract Marine microorganisms are important sources for novel natural products. Hence, the aim of this study was to introduce marine microorganisms with the capability of producing antioxidant and cytotoxic metabolites. For this purpose, ten live sea cucumbers (Holothuria leucospilota) were collected from Larak Island, Persian Gulf. Then, their intestine contents were serially diluted and treated by heating (55°C). 100 µL of treated samples were inoculated on starch casein nitrate agar medium, which is supplemented with nalidixic acid and cycloheximide. The plates were incubated at 28 °C for 4 weeks and the colonies were purified. The antioxidant activity of extracted metabolites from the isolated actinobacteria was evaluated using DPPH (1,1-diphenyl- 2-picrylhydrazyl) radical scavenging activity assay and the cytotoxic activity was screened by Brine-Shrimp micro well cytotoxicity method. In addition, the cytotoxic effect was evaluated against HCT 116 and SW 480 cell lines by MTT cell proliferation assay. A new strain of marine actinobacterium represented maximum antioxidant and cytotoxic activity among 17 actinobacterial isolates. The ethyl acetate culture extract of -1 the isolate scavenged DPPH radicals with IC50 value of 211.2 µg mL . In addition, the extract exhibited high toxicity against HCT 116 and SW 480 tumor cell lines with IC50 values of 26.48 and 18.53 µg mL-1 respectively. The isolate was identified as Streptomyces sp. strain SC 156 and showed 98% similarity with type strains in NCBI database. These results suggested that Streptomyces sp. strain SC 156 could be considered as promising candidate for antioxidant and cytotoxic compound discovery. Keywords: Antioxidant activity, Cytotoxic activity, Holothuria leucospilota, Marine Streptomyces 1-Department of Microbiology, College of Science, Science and Research Branch, Islamic Azad University, Fars, Iran 2-Department of Microbiology, College of Science, Agriculture and Modern Technology, Shiraz Branch, Islamic Azad University, Shiraz, Iran 3-Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. 4-Persian Gulf and Oman Sea Ecological Research Center, Iranian Fisheries Science Research Institute, Agricultural Research, Education and Extension Organization, Bandar Abbas, Tehran, Iran. 5- Molecular Medicine Research Center, Department of Biochemistry, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran Corresponding author’s E-mail: [email protected] 414 Gozari et al., Antioxidant and cytotoxic activities of metabolites produced from a … Introduction position in the microbial biotechnology. Marine organisms are important sources These group of bacteria exhibited high for novel natural products (Blunt et al., physiological diversity and produce 2013). Production of secondary almost half of the discovered bioactive metabolites as a defense strategy is well metabolites which are reported as developed in marine invertebrates like antitumors, antibiotics, antioxidants, sea cucumbers (Bogatyrenko and enzymes and other pharmaceutical Buzoleva, 2016). Among the natural products (Manivasagan et al., metabolites they could produce 2014). Many anticancer drugs including antioxidant compounds to protect anthracyclines, peptides, enediynes, themselves against elevated levels of aureolic acids, antimetabolites, reactive oxygen species (ROS) that are carzinophilin, mitomycins were exposed to in their extreme originated from Actinobacteria environments (Velho-Pereira et al., (Newman and Cragg, 2007; Olano et 2015). They also produce cytotoxic al., 2009). In relation to antioxidant substances to combat against predators compounds, Motohashi et al. (2010) and pathogens in their habitat. It is have isolated two newly modified suggested that some of these secondary Indole-containing peptides, JBIR-34 metabolites are produced by their and JBIR-35, from a sponge-associated symbiotic bacteria however for the Streptomyces species. The confirmation of the above mentioned Streptomyces, Amycolatopsis, hypothesis comprehensive studies need Micromonospora, Saccharopolyspora to be done (Nyholm and Graf, 2012). and Actinoplanes genera are the major Sea cucumbers integrate close bioactive producing Actinobacteria interaction with bacterial communities (Subramani and Aalbersberg, 2012). in marine microenvironments through Among them the members of ingestion and filtration of marine Streptomyces members produce about sediments and seawater respectively 80% of the discovered natural products (Slater et al., 2011). Consequently, this from actinobacteria (Watve et al., complicated interaction facilitate 2001). Although, those organisms are nutrient digestion for sea cucumbers interesting for scientists because of and modulate their immune and their ecological roles and their defensive responses (Amaro et al., taxonomy, but very limited research 2012; Amaro et al., 2009; Hess et al., have focused on the bacterial flora of 2011; Ray et al., 2012; Warnecke et al., the sea cucumbers and their product’s 2007). Many researchers have reported bioactivities. Among the very limited the presence of diverse groups of reports, Ye et al. (Ye et al., 2016) bacteria including: Proteobacteria, isolated a new cytotoxic curvularin Bacteroidetes, Firmicute and glycoside which is synthesized by a Actinobacteria from sea cucumbers marine actinobacterium: (Gao et al., 2014). Among them, Pseudonocardia sp. HS7. In addition to Actinobacteria have outstanding the above report, a novel lineage of Iranian Journal of Fisheries Sciences 17(2) 2018 415 Actinobacteria, Iamia majanohamensis in their culture media for antioxidant was isolated from abdominal epidermis and cytotoxic activities. of a sea cucumber: Holothuria edulis Materials and methods (Kurahashi et al., 2009). Therefore, Sample location considering the importance of the The samples were collected from Larak actinobacterial flora of the sea Island, which is located at 26° 82′ 33''N cucumbers prompted us to isolate and latitude and 56° 33′ 46'' E longitude in identify the marine bacteria from H. the Persian Gulf (Fig. 1). leucospilota and evaluate their exudate Figure 1: Sample location in Larak Island, Persian Gulf (Google Maps, 2014). Sample collection and isolation the treated samples were inoculated on Ten live samples of sea cucumber of starch casein nitrate agar medium the species H. leucospilota were which was prepared with seawater and collected from Larak Island, Persian supplemented with nalidixic acid (20 Gulf, by scuba diving at a depth of 10- mg L-1) and cycloheximide (100 mg L- 15 meters in February 2016. The 1) (Maldonado et al., 2005). The plates animals were dissected and the samples were incubated at 28 °C for 4 weeks were collected from their intestine and the colonies were picked up and contents after rinsing with sterile purified by sub-culturing onto the same seawater, they were dissected and the media. Then, the suspected colonies samples were collected from their were preliminarily characterized using intestine contents. One gram of macroscopic and microscopic intestine content was serially diluted observations and they were purified for with sterile-filtered seawater and then further experiments (Goodfellow et al., treated by heating at 55 °C for 6 min 2012). (Jensen et al., 2005). Then 100 µL of 416 Gozari et al., Antioxidant and cytotoxic activities of metabolites produced from a … Production and extraction of bioactive Brine-shrimp cytotoxicity assay metabolites The cytotoxic activity of all The isolates were inoculated in marine Actinobacterial extracts was screened broth medium (Himedia) and incubated by Brine-shrimp micro well at 28 ˚C under shaking incubator (220 cytotoxicity method (Atta-ur-Rahman, rpm) conditions for 5 days. The filtrated 2001). The ethyl acetate culture extracts fermentation broths were extracted with were diluted to four final concentrations ethyl acetate twice (1:1 v/v) and after in seawater (1000, 500, 250, 125 µg evaporation of the solvent, the culture mL-1). Water insoluble extracts were extracts were kept for the subsequent dissolved in 1mL DMSO prior to serial experiments (Bucar et al., 2013). dilution. 100 µL of Artemia franciscana nauplii suspension (10-15 nauplii 100 Antioxidant bioassay µL-1) was added to 100 µL of each The DPPH (1,1-diphenyl-2- dilution in each 96-well microplate. picrylhydrazyl) radical scavenging After incubation at 25°C for 24 hours, activity was assayed according to the number of live and dead nauplii was microdilution method (Leong and Shui, recorded and LC50 of each crude extract 2002). The ethyl acetate culture extracts was calculated. were diluted in methanol at seven final concentrations (1250, 625, 312, 156, Cytotoxicity assay 78, 39, 19.5 µg mL-1). Five microliters Cytotoxic effect of the potent crude of each concentration were added to extract against human colon cancer cell 195 µL of DPPH solution at 100µM lines (HCT 116) and (SW 480 ) was concentration in methanol. The determined by MTT cell proliferation microplate was incubated at room assay (Peng and Zhao, 2009). Four temperature in the dark place for 30 different final concentrations