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Antiviral activity of 7-keto-stigmasterol obtained from green Antarctic algae Prasiola crispa against equine herpesvirus 1

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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 etiological agent of clinical signs such as abortion, as well as neurological and respiratory diseases. Extract samples were fractionated in silica gel of 70–230 and 230–400 mesh and identified with 1H and 13C nuclear magnetic resonance spectroscopy. The cytotoxic effect was assayed with 3-(4,5-dimethylthiazol-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 CC50 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 EC50 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 × 104 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 attachment to cells with irreversible inactivation of virus infectivity.

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Acknowledgments

The authors thank Brazil’s Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Edital do Programa de Fomento à Pesquisa at the Universidade Federal Fluminense (FOPESQ-UFF), and Fundação Oswaldo Cruz (FIOCRUZ) for financial support. All authors would like to express their appreciation to Pronex-FAPERJ (grant no. E-26/110.574/2010). VLT (301420/2010-6) and ICNPP (303368/2013-6) are grateful to CNPq for Productivity Fellowships and to FAPERJ for the Cientista do Nosso Estado Fellowship (E-26/103.176/2011 and E-26/103.024/2011). CSB thanks FAPERJ for the Digital Scholarship Center Fellowship (E-26/100.770/2012), and JPGL, AMVP, and ICNPP are all grateful to FIOCRUZ and FOPESQ-UFF for financial support.

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Authors and Affiliations

  1. Instituto Biomédico, Universidade Federal Fluminense, Rua Hernani Melo, 101, São Domingos, Niterói, RJ, 24210-130, Brazil

    Robson dos Santos Souza Marinho & Ana Maria Viana Pinto

  2. Programa de Pós-Graduação em Ciências e Biotecnologia Universidade Federal Fluminense, Centro, Niterói, RJ, 24020 141, Brazil

    Carlos José Brito Ramos, Valéria Laneuville Teixeira & Izabel Christina Nunes de Palmer Paixão

  3. Laboratório Algamar, Departamento de Biologia Marinha, Instituto de Biologia, PO Box 100.644, Niterói, RJ, 24010-970, Brazil

    Carlos José Brito Ramos, Valéria Laneuville Teixeira & Izabel Christina Nunes de Palmer Paixão

  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

    Carlos José Brito Ramos, Valéria Laneuville Teixeira & Izabel Christina Nunes de Palmer Paixão

  5. Laboratório de Virologia Comparada e Ambiental, Fundação Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ, 21040-360, Brazil

    José Paulo Gagliard Leite

  6. Centro Interdisciplinar de Pesquisa em Biotecnologia, Universidade Federal do Pampa, Unipampa, Campus São Gabriel, RS, 97300-000, Brazil

    Cháriston André Dal Belo & Antônio Batista Pereira

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  1. Robson dos Santos Souza Marinho
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  2. Carlos José Brito Ramos
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  8. Ana Maria Viana Pinto
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Correspondence to Ana Maria Viana Pinto.

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dos Santos Souza Marinho, R., Ramos, C.J.B., Leite, J.P.G. et al. Antiviral activity of 7-keto-stigmasterol obtained from green Antarctic algae Prasiola crispa against equine herpesvirus 1. J Appl Phycol 29, 555–562 (2017). https://doi.org/10.1007/s10811-016-0946-9

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  • DOI: https://doi.org/10.1007/s10811-016-0946-9

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