2.9. Plaque Reduction Assay

The Vero cells were seeded on 24-well plates and infected with the virus inoculum for 1 h at 37 °C with gentle shaking. The virus stock was serially diluted to yield 60 plaques/1000 µL. Both the virus and cells were pretreated for 1 h at 37 °C with 25, 50 and 100 µg/mL of PPE and PLE, separately. Similarly, the Vero cells and viral dilution were incubated with punicalagin (20 µg/mL, 10 µg/mL, 5 µg/mL and 2.5 µg/mL), ellagic acid and gallic acid (10 µg/mL, 5 µg/mL and 2.5 µg/mL) for 1h. The infection was then performed at 1 MOI with gentle shaking. Thus, the pretreated virus inoculum was added to pretreated Vero cells to promote viral adsorption. Thus, 1 h later, the monolayer was covered with a medium containing 0.8% methylcellulose in the presence of PPE and PLE crude extracts and compounds. DMSO was included as a control and used at the highest concentration (50 µM). After three days, the cells were fixed, stained with crystal violet, and visualized with an inverted microscope (Leica DMIL, Nuloch, Germany) for plaque detection. The data were analyzed as triplicates ± standard deviation (SD) for each dilution.