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

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 H-1 and C-13 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 mu M of 7-keto-stigmasterol. The CC50 of MTT, neutral red, and violet crystal were 1884 +/- 7.5, 799 +/- 6.7, and 1002 +/- 6.3 mu M, 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 mu M of both compounds and the EC50 value (39 +/- 6 mu M) 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 x 10(4) plaque-forming units with different concentrations of 7-keto-stigmasterol and incubated at room temperature (24 A degrees 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 mu M (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.