Pharmaceutical Biology ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: http://www.tandfonline.com/loi/iphb20 Antiproliferative effect of extract from endophytic fungus Curvularia trifolii isolated from the “Veracruz Reef System” in Mexico Alan Couttolenc, Cesar Espinoza, José J. Fernández, Manuel Norte, Gabriela B. Plata, José M. Padrón, Alla Shnyreva & Ángel Trigos To cite this article: Alan Couttolenc, Cesar Espinoza, José J. Fernández, Manuel Norte, Gabriela B. Plata, José M. Padrón, Alla Shnyreva & Ángel Trigos (2016) Antiproliferative effect of extract from endophytic fungus Curvularia trifolii isolated from the “Veracruz Reef System” in Mexico, Pharmaceutical Biology, 54:8, 1392-1397, DOI: 10.3109/13880209.2015.1081254 To link to this article: http://dx.doi.org/10.3109/13880209.2015.1081254 Published online: 22 Apr 2016. Submit your article to this journal Article views: 91 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iphb20 Download by: [University of Veracruzana] Date: 15 February 2017, At: 09:50 PHARMACEUTICAL BIOLOGY, 2016 VOL. 54, NO. 8, 1392–1397 http://dx.doi.org/10.3109/13880209.2015.1081254 RESEARCH ARTICLE Antiproliferative effect of extract from endophytic fungus Curvularia trifolii isolated from the ‘‘Veracruz Reef System’’ in Mexico Alan Couttolenca, Cesar Espinozab, Jose´ J. Ferna´ndezc, Manuel Nortec, Gabriela B. Platac, Jose´ M. Padro´nc, Alla Shnyrevad and A´ ngel Trigosb aDoctorado en Ciencias Biome´dicas, Universidad Veracruzana, Xalapa-Veracruz, Me´xico; bLaboratorio de Alta Tecnologı´a de Xalapa, Universidad Veracruzana, Xalapa-Veracruz, Me´xico; cInstituto Universitario de Bio-Orga´nica ‘‘Antonio Gonza´lez’’ (IUBO-AG), Universidad de La Laguna, La Laguna, Spain; dDepartment of Mycology and Algology, Faculty of Biology, Moscow Lomonosov State University, Moscow, Russia ABSTRACT ARTICLE HISTORY Context: It is well known that marine fungi are an excellent source of biologically active secondary Received 22 March 2015 metabolites, and by 2011, it was reported that over 400 bioactive metabolites were derived from Accepted 5 August 2015 marine fungi. Published online 21 April Objective: This study establishes the basis for future research on antiproliferative compounds of 2016 marine endophytes inhabited in the Veracruz Reef System. KEYWORDS Materials and methods: Isolation of the 34 fungal strains was carried out by microbiological Antitumor activity; bioactive method from samples of sponges, corals, and other biological material from the Veracruz Reef natural products; biopros- System. The fungal biomass and broth were separated and extracted with a mixture of solvents pecting; marine fungi MeOH:CHCl3. Characterization and molecular identification of the fungal strains were performed through microbiological methods and the analysis of the ITS-rDNA regions. Antiproliferative activity was tested at a dose of 250 mg/mL on human solid tumor cell lines HBL-100, HeLa, SW1573, T-47D, and WiDr by the SRB assay after 48 h-exposure to the fungal extracts. Results: The extracts from five isolates showed an antiproliferative effect against one or more of the tested cell lines (percentage growth 5 50%). The mycelial extract from the isolate LAEE 03 manifested the highest activity against the five cell lines (% PG of 17 HBL-100, 19 HeLa, 23 SW1573, -6 T-47D, and 10 WiDr) and the strain was identified as Curvularia trifolii (Kauffman) Boedijn (Pleosporaceae). Discussion and conclusion: The results obtained indicate that the extract from a marine derived C. trifolii has the antiproliferative effect, thus suggesting that this organism is a good candidate for further analysis of its metabolites. Introduction among other organisms. An example of this was the It is widely known that fungi represent an excellent isolation of sporolids A and B obtained from a micro- source of biologically active secondary metabolites. scopic fungus of genus Cladosporium isolated from an However, it was not until 1981 with the isolation of alga (Shigemori et al., 2004). Both the compounds the antibiotic siccaine from a marine fungus that these manifested a cytotoxic activity against murine lymph- organisms started to draw attention (Kupka et al., 1981). oma cell line L1210. Similarly, varitrol obtained from the As stated in the literature (Gareth, 2011), the number of fungus Emericella variecolor Berk. & Broome showed bioactive metabolites derived from marine fungi has cytotoxic activity against cancer cell lines from kidney increased yearly (from 1 in 1981; 100 in 2002; 272 in and breast (Saleem et al., 2007). 2004; and 4400 in 2011). The marine fungi are a source Coral reefs are considered as one of the ecosystems of bioactive natural products of diverse chemical nature with high biodiversity in coastal oceans, and as a source such as alkaloids, macrolides, terpenes, and peptides for isolation of endophytic microorganisms, including among others (Zhang et al., 2009). fungi (Golubic et al., 2005). The national park Veracruz According to statistics (Bungi & Ireland, 2004), 85% of Reef System (Sistema Arrecifal Veracruzano, SAV) is the the reported compounds derived from marine fungi were second major reef system in Mexico, and it is formed by obtained from endophytic fungi isolated from vegetal 23 reef structures (Ake-Castillo, 2011). It was declared a substrates, algae, mollusks, and marine invertebrates, marine national park in 1992. Although it has been well CONTACT A´ngel Trigos [email protected] Laboratorio de Alta Tecnologı´a de Xalapa, Universidad Veracruzana, Calle Me´dicos No. 5, Col. Unidad del Bosque, 91010 Xalapa-Veracruz, Me´xico ß 2016 Informa UK Limited, trading as Taylor & Francis Group PHARMACEUTICAL BIOLOGY 1393 studied for macroscopic organisms, so far there are only regions by direct PCR technique. PCR products were a few studies on endophytic fungi from this unique first purified by the SiO2-coated magnetic beads (Sileks ecosystem. This work intends to establish the basis for M, Moscow, Russia) and then were sequenced in both future research on bioactive compounds in the Veracruz directions using forward and reverse primers ITS1F and Reef System that based on the antiproliferative activity of ITS4. Sequencing reactions were performed on an ABI marine endophytes. PRISM 3100 Genetic Analyzer (Applied Biosystems, Waltham, MA). Newly obtained sequences were com- pared against nucleotide entries in the NCBI GenBank Materials and methods (http://www.ncbi.nlm.nih.gov/) by using search tool Collection and characterization of biological Blastn to confirm species identity. For phylogenetic materials analysis, sequences were aligned using ClustalW algo- Samples of corals, algae, and sponges were collected from rithm of MegAlign from MEGA6 software (Tamura La Blanca reef (Lat. 19.0865, Long. À95.9984), part of et al., 2013). Phylogenetic tree of nucleotide alignments the (SAV), in two recollections carried out in the period was constructed using Maximum Likelihood algorithm from June to October 2011 by diving in shallow water (Juke-Cantor) of the MEGA6 software. Bootstrap ana- lysis was performed with 1000 replications. (1–3 m) and were identified by Miguel Lozano (‘‘Instituto de Ciencias Marinas y Ecologı´a’’ Universidad Veracruzana, Mexico). The samples were Culture and extraction cut into small pieces, placed in a sterilized tube with sea A culture medium composed of yeast extract (4 g/L), water, and refrigerated for transportation to the labora- soluble starch (10 g/L), peptone (2 g/L), and sea water tory. The coral and algae samples collected were (75%) was employed to culture the fungal strains. For processed as follows: the tissue was rinsed under flowing each strain, a total volume of 500 mL was distributed in water, and subsequently cut and disinfected with 4% 250 mL Erlenmeyer flasks (50 mL of medium in each) sodium hypochlorite, then rinsed with sterile water and and the flasks were inoculated with a small piece of agar after that, placed in Petri plates with potato dextrose agar with the fungal strain (approximately 1 cm2) and were (PDA) supplied with 50% marine water and Petri plates put into an orbital shaker for 14 d at 25 ± 2 C. After that with Marine Agar 2216; both media were supplied with time, the culture broth and the biomass were separated the antibiotic chloramphenicol (0.2 mg/L). Petri plates by vacuum filtration. Both biomass and culture broth were incubated at room temperature (25 ± 2 C) until were frozen and lyophilized. When dry, the biomass and marine samples developed mycelia (approximately for the broth were extracted with a mixture of solvents 14–21 d). MeOH:CHCl3 1:1 for 7 d. The extraction was repeated several times. The extracts were then filtered and Strain identification concentrated in a rotatory evaporator; the lyophilized extracts were employed in the chemosensitivity testing The identification of the strains was carried out by the assay. observation of reproductive structures under a micro- scope and employing taxonomic keys (Barnett & Hunter, 1998; Lima & Furtado, 2007). For the microscopic Cell lines and culture observation, thin layer PDA cultures (microculture The human solid tumor cell lines HBL-100, HeLa, technique) were used. The mycelial cultures were SW1573, T-47D, and WiDr were used in this study. observed after seven days of incubation at 25 C These cell lines were a kind gift from Prof. G. J. Peters employing lactophenol. The microscopic morphology