*3.1. Antiviral E*ff*ects of PAE and CTE*

A library of herb extracts was screened to detect antiviral activity against RSV. Among them, PAE and CTE were selected. The ability of the two herb extracts to inhibit the replication of GFP-tagged RSV (RSV-GFP) was further confirmed in a dose-dependent experiment. The expression of RSV-GFP, the virus titer, and the recovery of RSV-induced cell death were evaluated in HEp2 cells upon herb treatment. As shown in Figure 1A,B, HEp2 cells and A549 cells treated with PAE and CTE (10, 30, or 50 μg/mL) exhibited a marked reduction in GFP expression compared to untreated HEp2 cells and A549 cells. Moreover, all doses of the two-herb extract significantly reduced the RSV titer compared to the untreated group (Figure 1C). Interestingly, treatment with PAE and CTE significantly reduced RSV-induced HEp2 cell and A549 cell death at 48 hpi (Figure 1D). Therefore, both herb extracts could significantly reduce RSV replication in HEp2 cells and A549 cells. Since the 50 μg/mL concentration was the most effective at inhibiting viral replication and virus-induced cytotoxicity, this concentration was used for further in vitro experiments.

#### *3.2. Therapeutic E*ff*ect of PAE and CTE against RSV Infection*

The ability of PAE and CTE to inhibit virus replication after the infection was determined. HEp2 cells were infected with RSV-GFP 0.1 MOI, and herb extracts were added at the indicated time points. GFP expression was measured at 48 hpi. As expected, increased GFP expression at 48 hpi was observed as the amount of time between viral infection and herb treatment increased (Figure 2A,B). Similarly, RSV titers increased with increasing time between viral infection and herb extract treatment (Figure 2C,D). Next, to assess the effect of PAE and CTE on virus replication, an assay was performed, and GFP expression was measured at different times after virus infection. As shown in Figure 2E,F, a 50 μg/mL concentration of each herb extract significantly reduced the GFP expression at 36 hpi and 48 hpi but, interestingly, not at 12 hpi or 24 hpi.

**Figure 1.** Antiviral activity of *Plantago asiatica* extract (PAE) and *Clerodendrum trichotomum* extract (CTE) in HEp2 cells and A549 cells. HEp2 cells and A549 cells were seeded into 12 well cell culture plates with the cell number of 2.5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well. Twelve hours later, the medium was changed to 1% fetal bovine serum (FBS) containing Dulbecco's Modified Eagle's Medium (DMEM) and cells were infected with Green Fluorescent Protein fused Respiratory syncytial virus (RSV-GFP) 0.1multiplicity of infection (MOI) or kept uninfected. Two hours later, the medium was replaced with 10% FBS containing DMEM and cells were treated with 10, 30, 50 (μg/mL) PAE or CTE. Cells without any treatment regard as virus only. (**A**) After 48 h, images were obtained (200× magnification). (**B**) GFP absorbance levels were measured by Gloma multi-detection luminometer (Promega). (**C**) Virus titration was done from the cell supernatant and cells by standard plaque assay and expressed as plaque forming unit (PFU). (**D**) Cell viability was determined by trypan blue exclusion assay at 48hour post infection (hpi). GFP absorbance, cell viability and virus titer expressed as mean ± standard deviations (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).
