Antiviral activity of 7-keto-stigmasterol obtained from green Antarctic algae Prasiola crispa against equine herpesvirus 1 Robson dos Santos Souza Marinho 1 & Carlos José Brito Ramos 2,3,4 & José Paulo Gagliard Leite 5 & Valéria Laneuville Teixeira 2,3,4 & Izabel Christina Nunes de Palmer Paixão 2,3,4 & Cháriston André Dal Belo 6 & Antônio Batista Pereira 6 & Ana Maria Viana Pinto 1 Received: 1 February 2016 /Revised and accepted: 21 August 2016 # Springer Science+Business Media Dordrecht 2016 Abstract The aim of this investigation was to evaluate the in vitro cytotoxic effect and antiviral properties of 7-keto- stigmasterol from the crude extract of the green Antarctic alga Prasiola crispa in dichloromethane against the replication of equine herpesvirus 1 (EHV-1), a worldwide enzootic etiolog- ical agent of clinical signs such as abortion, as well as neuro- logical and respiratory diseases. Extract samples were frac- tionated in silica gel of 70–230 and 230–400 mesh and iden- tified with 1 H and 13 C nuclear magnetic resonance spectros- copy. The cytotoxic effect was assayed with 3-(4,5-dimethyl- thiazol-2yl)-2,5diphenyl tetrazolium bromide (MTT), neutral red, and violet crystal in rabbit kidney (RK-13) cells treated with 12.5, 25, 50, 100, and 200 μM of 7-keto-stigmasterol. The CC 50 of MTT, neutral red, and violet crystal were 1884 ± 7.5, 799 ± 6.7, and 1002 ± 6.3 μM, respectively. To characterize the antiviral action of 7-keto-stigmasterol and acyclovir, RK-13 cells were inoculated with 200 plaque- forming units of EHV-1 and treated with 12.5, 25, and 50 μM of both compounds and the EC 50 value (39 ± 6 μM) of 7-keto-stigmasterol protected 50 % of RK-13 cells, with a selectivity index of 47, 20, and 26 for MTT, neutral red, and violet crystal, respectively. However, acyclovir showed no antiviral activity on the EHV-1 variant studied. EHV-1 was inactivated by mixing a viral suspension with 1 × 10 4 plaque- forming units with different concentrations of 7-keto- stigmasterol and incubated at room temperature (24 °C) for 1 h; a control of untreated infected cells was performed under the same conditions. The samples were then diluted, and the residual infectivity was determined by a plaque assay. The percentages of EHV-1 inactivation with 7-keto-stigmasterol were 12.5 (51 %), 25 (67 %), 50 (91 %), and 100 μM (100 %). The inactivation of EHV-1 by direct interaction with 7-keto-stigmasterol probably interferes with EHV-1 attach- ment to cells with irreversible inactivation of virus infectivity. Keywords Prasiola crispa . Green Antarctic algae . Antiviral activity . Equine herpesvirus 1 Introduction Marine organisms are potentially prolific sources of highly bioactive secondary metabolites applied in countless biologi- cal resources to produce new pharmaceutical agents, including antiviral ones (Carvalho and Roque 2000). The Antarctic Ocean is the primary location of exchanges of the three ocean basins’ energy, mass, and heat, as well as where the evolution of Antarctic biota under extremely rigorous climatic condi- tions result in remarkable biochemical adaptations. As such, * Ana Maria Viana Pinto anapintoj26@gmail.com 1 Instituto Biomédico, Universidade Federal Fluminense, Rua Hernani Melo, 101, São Domingos, Niterói, RJ 24210-130, Brazil 2 Programa de Pós-Graduação em Ciências e Biotecnologia Universidade Federal Fluminense, Centro, Niterói, RJ 24020 141, Brazil 3 Laboratório Algamar, Departamento de Biologia Marinha, Instituto de Biologia, PO Box 100.644, Niterói, RJ 24010-970, Brazil 4 Laboratório de Virologia Molecular e Biotecnologia Marinha, Programa de Pós-graduação em Ciências e Biotecnologia, Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ 24020-141, Brazil 5 Laboratório de Virologia Comparada e Ambiental, Fundação Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ 21040-360, Brazil 6 Centro Interdisciplinar de Pesquisa em Biotecnologia, Universidade Federal do Pampa, Unipampa, Campus São Gabriel, RS 97300-000, Brazil J Appl Phycol DOI 10.1007/s10811-016-0946-9