2.3. Cytotoxicity and Antiviral Activity Assay

1 × 10⁴ Vero cells or SK-N-SH cells were seeded in 96-well plates and incubated at 37 °C, 5% CO₂ overnight. The supernatant was then removed and new medium with amentoflavone or acyclovir was added. After 72 h, the CCK8 reagent (10 μL/well) was added for 2 h to record the OD value by enzyme immunoassay reader at 490 nm. The 50% cytotoxic concentration (CC₅₀) was calculated accordingly [29].

Viral titration was used to determine cytopathic effects (CPEs) in Vero cells and the 50% tissue culture infectious dose (TCID50) was calculated [30]. Subsequently, the TCID50/mL was converted into plaque-forming units (PFU)/mL [31]. The assays were conducted as described in our previous studies [32,33]. Briefly, 1 × 10⁴ or 1.5 × 10⁵ Vero cells were seeded in 96-well or 24-well plates to perform CCK8 assay or plaque assay, respectively. The cells were infected with HSV-1/F, HSV-1/106, HSV-1/153 or HSV-1/Blue (MOI = 0.1) for 2 h, and the culture medium was then replaced with new medium containing amentoflavone at 37 °C, 5% CO₂. After 72 h, the OD value of the cells in 96-well plates was detected by CCK8 assay. The cells in 24-well plates were fixed by paraformaldehyde for 0.5 h, and strained with crystal violet for 0.5 h. Finally, the number of plaques was counted to calculate the inhibition rate of virus infection as described previously [32]. The 50% effective concentration (EC₅₀) was also calculated as described previously [33].