2.5. Cytopathic effect (CPE) inhibition assay

MDCK cells were inoculated with influenza virus A/California/7/2009 (H1N1). The 50% tissue culture infective dose (TCID₅₀) determined according to the Reed-Muench method (^{Reed, 1938}) was 10⁻⁵. Assay of antiviral activity of epiprogoitrin, progoitrin, epigoitrin and goitrin in vitro were performed using the CPE and CCK8 methods. MDCK cells were grown in a 96-well culture plate (1 × 10⁴ cells/well) for 24 h. To identify the probably affected viral life cycle, cells were treated with 100 μL of virus at 100 TCID₅₀ and 100 μL of sample solutions at various concentrations (10⁻³ mol/L-10⁻⁷ mol/L) using three different protocols including prevention, treatment and virus neutralization. For protocol 1 (prevention), cells were pre-incubated at 37 °C with sample solutions for 2 h before viral adsorption. Then the cells were washed with PBS and inoculated with virus for 2 h without the compounds and further grown for 72 h. For protocol 2 (treatment), cells were first inoculated at 37 °C with the virus for 2 h, then washed with PBS and cultured with the compounds for 72 h. For protocol 3 (virus neutralization), sample solutions were mixed with the virus and incubated at room temperature for 30 min, and the mixtures were added to cells and cultured for 72 h. After 3 days’ incubation, the solution was discarded and the cells were washed with PBS, then 190 μL of DMEM and 10 μL of CCK8 were added to each well. The plates were incubated in the dark for 3 h at 37 °C. The absorbance was read at 450/630 nm by a microplate spectrophotometer (Gemini EM, Molecular Device, USA). Six duplicates were used for each dilution ratio. Cell controls with/without the sample solutions and virus controls were included. The inhibition ratio was calculated using the following formula: Inhibition activity (%) = (ODsample – ODvirus)/(ODcellular control – ODvirus) × 100%, where ODsample is the optical density of the tested sample at a certain concentration, ODvirus is the optical density of the influenza virus control, and ODcellular control is the optical density of normal cells. The 50% inhibition concentration (IC₅₀) was determined by linear extrapolation of the results from serious doses tested.