Evaluation of the Cytotoxic Effect and PRRS Antiviral Activity of Glycyrrhizinic Acid in Aqueous Solution and with the Presence of Solid Lipid Nanoparticles

Aims: To study the effect of glycyrrhizinic acid (GA) solutions on uninfected and PRRS (Porcine Reproductive and Respiratory Syndrome) virus-infected cells in culture. An attempt of testing loaded nanoparticles with GA on cells was also explored.

Study Design: Different concentrations of GA in solution were tested on MARC cells as well as on virus-infected cells and observed during 144 h. Solid lipid nanoparticles with GA were tested on cell culture previously infected. In the end, cytotoxicity (CC50), inhibition of the cytopathic effect (EC50), virus titer and selectivity index were determined. Trypan blue staining (TB) and MTT assay were also performed and statistically analysed.

Place and Duration of Study: Laboratorio de Posgrado e Investigación en Tecnología Farmacéutica y Laboratorio de Microbiología y Virología de las Enfermedades Respiratorias del Cerdo, both at Facultad de Estudios Superiores Cuautitlán, UNAM; between March 2015 and August 2017.

Methodology: For CC50 determination solutions of GA were added and TB and MTT assay was performed. EC50 was evaluated on virus-infected cells that were treated with GA solutions. Viability and selectivity index were calculated. Virus titer was calculated by the Reed & Muench method. Nanoparticles containing GA (0.54 mg/ml) were obtained by the microemulsion method and tested on cells previously infected with the virus. TB staining and MTT assay were performed at the end of the study.

Results: Cell viability was verified by MTT assay and TB dye exclusion test. Only the 0.7, 0.8 and 0.9 mg/ml solutions were statistically different from controls P < .05. The CC50 of GA was above 4.2 mg/ml. The EC50 was calculated at 0.48 mg/ml. The viral titer decreased two logarithms compared to the control. The selectivity index was 8.7. Viability decreased significantly P < .05 compared to controls only at 0.9 mg/ml. An SLN assay on PRRS-infected cells showed that cell viability was comparable to that exerted by the virus-infected control cells P > .05. So cells treated with SLN interfere with MTT assay.

Conclusion: GA showed a reduction of PRRS in vitro replication. SLN interferes with the MTT assay and it is necessary to perform more assays to conclude on the antiviral activity of GA when administered in nanoparticles.