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

dos Santos Souza Marinho, Robson, Ramos, Carlos José Brito, Leite, José Paulo Gagliard, Teixeira, Valéria Laneuville, de Palmer Paixão, Izabel Christina Nunes, Belo, Cháriston André Dal, Batista Pereira, Antônio, Pinto, Ana Maria Viana
Journal of applied phycology 2017 v.29 no.1 pp. 555-562
rabbits, algae, respiratory tract diseases, median effective concentration, stable isotopes, tetrazolium, etiological agents, methylene chloride, ambient temperature, viruses, cytotoxicity, enzootic diseases, Prasiola, pathogenicity, silica gel, kidneys, Equid alphaherpesvirus 1, nuclear magnetic resonance spectroscopy, antiviral properties, mixing, Antarctic region
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.