Antimicrobial and Anticancer Potential of Organic Extracts Obtained from the New Ascidian Ecteinascidia litoranea and Associated Microorganisms Collected from the Coast of Ceara.

Introduction - Marine invertebrates and their associated microorganism are a rich source of chemically diverse molecules with pharmacological activities, which have shown important results in combating cancer and infections cause by microbial agents. The present work aims to evaluate the antimicrobial and anticancer potential of extracts obtained from the species Ecteinascidia litoranea and their associated microorganisms. Methods - E. litoranea was collected at Taiba Beach (3°30’23.4”S and 38°53'21.1”W), São Gonçalo do Amarante - CE. A crude extract of the ascidian was obtained by immersing crushed zooids in methyl alcohol (100 mL) for 2 hours then evaporating the solvent under vacuum. For recovery of bacteria, zooids were grounded in sterile Petri dishes then 1 mL of sterile seawater was added. The material was plated fresh (M4) or after heating at 55°C for 10 minutes (M5), by striking 100 uL of the homogenate onto agar filled Petri dishes with the following media: SWA (sea water agar) and SCA (starch casein agar). Distinguishable bacterial colonies were transferred to fresh agar dishes for purification. Pure cultures of each strain recovered were cryopreserved and cataloged by the code BRA-XXX in the microorganism bank (MicroMarin), then grown in both solid and liquid culture medium A1 (starch, peptone and yeast extract) for 7 days. Organic extracts were obtained from the culture of strains in solid medium (S) – by breaking the agar and adding ethyl acetate under sonication – and in liquid medium (L) – by adding ethyl acetate and shaking for 1 hour. Solvent was evaporated under vacuum to yield the respective crude extracts. Anticancer activity of the extracts was evaluated by SRB assay used qualitatively to select potential extracts (5 and 50 μg/mL) on HCT 116 (colon cancer). For antimicrobial activity, the extracts were tested at different concentrations against strains Staphylococcus aureus ATCC 25923 (G+ bacteria), Pseudomonas aeruginosa ATCC 27853 (G- bacteria) and Candida albicans ATCC 90028 (fungus). The minimum inhibitory concentrations (MIC) were obtained by the microdilution test in Mueller Hinton (for bacterial models) or RPMI 1640 media with bicarbonate-free L glutamine (for fungus model), while the minimum bactericidal/fungicidal concentrations (MBC/MFC) was determined by the depleted seed in Mueller Hinton and Sabouraud Dextrose Agar (SDA) with Chloramphenicol for bacteria and fungus, respectively. Results – Of the 29 strains of microorganisms recovered, 55% were isolated from the heated sample (M5). Considering the culture media, SCA (rich medium) yielded the largest portion of the isolates, with 69% and 75% for the M4 and M5 methods, respectively. The crude extract from E. litoranea and the 18 extracts obtained from 10 of the ascidian-associated bacterial strains recovered were not cytotoxic against HCT 116 at the tested concentrations. For the antimicrobial assays, both extracts obtained from BRA-720 (S and L) stood out for their satisfactory antimicrobial activity against S. aureus, with MIC and bactericidal profile addition (MBC) of 5 and 6 µg/mL, respectively. For extracts BRA-677S, BRA-679L and BRA-719S, MICs and CBMs were equivalent (750 µg/ml, 960 µg/ml, 750 µg/ml, respectively). None of the extracts showed activity against P. aeruginosa. BRA-679L showed to be a potent fungicide, showing inhibition and complete elimination of C. albicans, with MIC = 240 µg/ml and CFM = 960 µg/ml. Therefore, associated microorganisms recovered from the ascidian E. litoranea from Ceará coast are promising sources of compounds with antimicrobial activity.
Financial Support: Funcap; CAPES