*3.4. Determination of the E*ff*ective Concentration (EC50) and Cytotoxic Concentration (CC50) of PAE and CTE*

EC50 values of the herb extracts were determined against RSV on HEp2 cells. For this experiment, a GFP assay was performed with some modifications, as described previously [25,26]. Briefly, RSV-GFP virus was used, and a 50% reduction in GFP expression was considered equivalent to a 50% reduction in virus titer. As shown in Figure 3D,F, PAE and CTE inhibited RSV-GFP infection (MOI, 0.1) by 50% at concentrations of 39.82 μg/mL and 27.95 μg/mL, respectively. Next, we determined the CC50 values of the two extracts based on a cell cytotoxicity assay using HEp2 cells. The assay showed CC50 values of 938.43 μg/mL and 764.17 μg/mL for PAE and CTE, respectively (Figure 3F,G). Interestingly, the cell viability at the effective concentrations of both extracts was greater than 80%. The selectivity index (SI) indicates the safety of a crude extract against RSV infection [19]. The SIs of PAE and CTE were 23.5 and 27.3, respectively (Figure 3H). This data suggests that both PAE and CTE could be used safely as therapeutic agents against RSV infection.

**Figure 3.** Synergistic effect and effective concentration (EC50), cytotoxic concentration (CC50) of PAE and CTE in HEp2 cells. (**A**) HEp2 cells were infected with RSV-GFP (0.1 MOI) for 2 h with DMEM containing 1% FBS. Cells were treated with PAE, CTE or combination of both at different concentrations with DMEM containing 10% FBS. At 48 hpi GFP expression level was determined. (**B**) Virus titer was measured from both supernatant and cells by standard plaque assay at 48 hpi and expressed as PFU. (**C**) Dose information of PAE and CTE single or combination treatment. (**D**,**E**) HEp2 cells were infected with RSV-GFP (O.1MOI) for 2 h with DMEM containing 1% FBS. Then, the medium was changed to DMEM containing 10% FBS and cells were treated with various concentrations of PAE (**D**) or CTE (**E**). 48 hpi GFP expression level was determined. (**F**,**G**) HEp2 cells were treated with various concentrations of PAE (**F**) or CTE (**G**), and cell viability was determined at 48 h post treatment (hpt) by cell cytotoxicity assay kit. (**H**) To calculate EC50 value, 50% reduction of GFP expression was considered as equivalent to the 50% reduction in virus titer. The ratio between CC50 and EC50 was considered as Selectivity Index (SI). GFP absorbance and cell viability expressed as mean ± SD. Error bars indicate the range of values obtained from counting duplicate in three independent experiments. (\*\* *p* < 0.01 and \*\*\* *p* < 0.001 regarded as significant difference).
