In vitro viral inactivation photosensitized by a vegetal extract

BRENDA KONIGHEIM; MARTA CONTIGIANI; LAURA MUGAS; JAVIER AGUILAR; SUSANA C. NÚÑEZ MONTOYA; JULIANA MARIONI; JOSÉ L. CABRERA

Santiago

Congreso; 25ST I-APS CONFERENCE; 2016

Inter-american photochemical society

Heterophyllaea pustulata Hook. f. (Rubiaceae) is a wild bush that grows in the Andean northwest of Argentina, where is popularly known as "cegadera" (blindness in Spanish). It has phototoxic properties that cause dermatitis and blindness in animals that ingested it and exposed to sunlight. From its chemical study, we found that 9, 10-anthraquinone (AQs) are the predominant metabolites. In complementary studies, we have reported that some of these isolated AQs exhibit photosensitizing properties by generation of superoxide anion radical or singlet molecular oxygen. In addition, we have demonstrated that these AQs show in vitro virucidal effect against Herpes Simplex virus Type 1 (HSV-1), which means an inactivation of viral particles before their entry into the host cells, and this inhibition was increased by light action (photostimulation). The aim of this work was to study the in vitro inactivation of HSV-1 once the virus entered into the host-cells, by the photosensitized action of an enriched-AQs extract obtained from roots of H. pustulata.By HPLC analysis, the chemical composition of the benzene extract (Ben) obtained from roots of this vegetal species were established. Maximum Non-Cytotoxic Concentration (MNCC) on Vero cells (host cells) was estimated from the dose-response curve (%cellular viability vs. concentrations) by Neutral Red uptake assay (NR).The viral inactivation effect of this extract was tested on infected cells with HSV-1 at its MNCC. This effect was evaluated by observation of morphological alterations in cells with optical microscopic (cytopathic effect, CPE) and determination of cellular viability by using NR assay, under two simultaneous conditions: darkness and irradiation (actinic lamp 380-480 nm, Philips TL/03). Active viral particles after both treatments (darkness and irradiation with Ben extract) were detected by extraction of cellular content and its subsequent inoculation in a new Vero cell monolayer to evaluate its viability by using NR.HPLC analysis showed that Ben extract only contains AQs derivatives, being rubiadin 1-methyl ether, rubiadin and damnacanthol the majority compounds. In darkness, the infected cells treated with Ben extract showed the characteristic CPE caused by HSV-1; therefore, the virus was not inactivated by the extract, which was confirmed by the presence of active viral particles. On the other hand, the infected cells incubated with the extract under irradiation exhibited a different CPE. Since no active viral particles were detected in this last experimental condition, we demonstrated that the CPE observed is due to the action of the photosensitized extract on the cells and not caused by virus.In conclusion, under the light action, the extract containing only photosensitizing AQs achieved the inactivation of viral particles inside of infected cells with HSV-1. These results motivate us to continue to deepen studies with purified AQs, with the aim to provide a basis for the development of new treatments for viral etiology lesions.