*3.9. Intraperitoneal Administration of Acteoside Inhibits RSV Infection In Vivo*

To further evaluate the therapeutic effect of acteoside against RSV, we investigated its antiviral activity in BALB/c mice. Mice were intranasally infected with RSV-GFP (1 <sup>×</sup> 106 PFU/mouse), and acteoside was administrated intraperitoneally at 6 hpi at a dose of 80 mg/kg, as described previously [28]. Lungs from all mice were collected at 3 dpi and 5 dpi, and RSV-G mRNA was measured by qRT-PCR. As shown in Figure 6C, the level of viral mRNA was significantly lower in the acteoside-treated group than in the PBS control group at both 3 dpi and 5 dpi. When taken together, acteoside exhibited a strong in vivo antiviral effect and protected mice from RSV infection.

**Figure 5.** Identification and antiviral effect of acteoside (AC) *in-vitro.* (**A**,**B**) Chemical compounds in PAE and CTE were analyzed by the reversed phase HPLC. The monolayer of HEp2 cells was infected with RSV-GFP (0.1MOI) for 2 h with DMEM containing 1% FBS. Then, the medium was replaced with DMEM containing 10% FBS and cells were treated with 10, 30, 50 (ng/mL) AC. (**C**) After 48 h, images were obtained (200× magnification). (**D**) GFP absorbance levels were measured by Gloma multi-detection luminometer (Promega). (**E**) Viruses were titrated from the cell supernatant and cells by standard plaque assay. (**F**) Cell viability was determined by trypan blue exclusion assay at 48 hpi. (**A**) 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 AC. 48 hpi GFP expression level was determined. (**G**) HEp2 cells were treated with various concentrations of AC and cell viability was determined at 48 hpi 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. (**I**) The ratio between CC50 and EC50 considered as Selectivity Index (SI). GFP absorbance, cell viability and virus titer expressed as mean ± SD. Error bars indicate the range of values obtained from counting duplicate in three independent experiments. In vivo experiment was performed in duplicate. (\* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 regarded as significant difference).

**Figure 6.** Antiviral effect of acteoside *in-vitro* and *in-vivo*. (**A**) RSV-GFP infected cells were treated with 50 ng/mL of AC at 2 hpi and cells were harvested at indicated time points. RSV-G protein mRNA transcription level was determined by qRT-PCR. GAPDH was used for normalization. (**B**) Infected cells were treated with 50 ng/mL concentration of AC at 2 hpi and cells were harvested at indicated time points. RSV-G protein expression was determined by immunoblotting with anti-RSV-G protein antibody, and the intensity of the RSV-G was quantified. (**C**) Five weeks old (16 g/mice) BALB/c mice (*n* = 5) were intranasally infected with RSV-GFP (1 <sup>×</sup> 10<sup>6</sup> PFU/mice) in the total volume of 28 <sup>μ</sup>L. 6 hpi AC was intraperitoneally administrated at a dose of 80 mg/Kg body weight of mice. At 3 and 5 dpi, lung tissues were collected, and the transcription level of RSV-G protein mRNA was determined by qRT-PCR. mRNA expression and band intensity expressed as mean ± SD. Error bars indicate the range of values obtained from three independent experiments. In vivo experiment was performed in duplicate. (\* *p* < 0.05 and \*\* *p* < 0.01 regarded as significant difference).

#### **4. Discussion**

Traditional medicines have been used as remedies against infectious diseases for thousands of years, due to their significant anti-inflammatory, anti-microbial activity and low rate of adverse effects [29,30]. These medicines have been gaining in popularity, due to concerns related to the side effects, high cost, and lack of efficacy of conventional Western medicines [31]. In the years 2001 and 2002, approximately one-quarter of the bestselling drugs worldwide were natural products or were derived from natural products [32]. Recent publications show that traditional Chinese medicinal herbs account for 10% of the prescription drugs in China. They are perceived as harmless and natural and are widely used in many parts of the world, individually or in combination [33]. In particular, medicinal plants have shown potential therapeutic effects against a wide range of respiratory tract-related viral infections, including Severe Acute Respiratory Syndrome (SARS) [34–36], Influenza [37,38], and RSV [26,27,39]. Among the thousands of promising medicinal herbs, *Plantago asiatica* and *Clerodendrum trichotomum* are well-known and commonly used in traditional medicine in China, Japan, and South Korea [9,14,15].

In the present study, we screened a library of herb extracts to identify novel therapeutic inhibitors of RSV infection. Interestingly, we identified both *Plantago asiatica* extract (PAE) and *Clerodendrum trichotomum* extract (CTE) as hits with potent antiviral effects against RSV infection in HEp2 cells (Supplementary Figures S1 and S2). Next, we confirmed the dose-dependent anti-RSV activity of both herbs in the HEp2 cell line in detail (Figures 1 and 2).

In addition, the CC50 values of PAE and CTE were several magnitudes higher than the EC50 values (Figure 3), which is consistent with a favorable safety profile. Furthermore, both herb extracts reduced intracellular viral gene transcription and protein synthesis in vitro, and oral administration of the herbs to infected mice significantly reduced viral gene transcription in the lungs (Figure 4). Finally, we found that acteoside, a common phenolic glycoside present in both herb extracts, is involved in the antiviral activity of the herbs against RSV infection (Figures 5 and 6).

PAE and CTE significantly reduced RSV replication and RSV-mediated syncytial formation in the HEp2 cell line in a dose-dependent manner (Figure 1C). Moreover, we were curious to evaluate whether PAE and CTE can work synergistically to enhance anti-RSV activity. However, herb extracts did not show synergistic anti-RSV effect compared to the individual treatment at the same dose (Figure 3A,B). Since, both extracts reduce the virus replication at the same level when treated alone, or together, at same dose (Ex: PAE, 50 μg/mL and CTE, 50 μg/mL or PAE, 25 μg/mL +CTE, 25 μg/mL), it's possible that both herb extracts undergo their own mechanism of action for anti-RSV function other than work synergistically.

However, PAE and CTE did not show any cytotoxic effect in the HEp2 cell line (Figure 3F,G). The CC50 values of PAE and CTE were 938.43 μg/mL and 764.17 μg/mL, respectively, which were several magnitudes higher than EC50 values of 39.82 μg/mL and 27.95 μg/mL, respectively. Even though both extracts were used at a concentration of 50 μg/mL for in vitro experiments, their high SI indicates a broad safety margin for therapeutic purposes.

To support the observation mentioned above, we investigated the intracellular RSV gene transcription in infected epithelial cells. Interestingly, PAE and CTE-treated HEp2 cells showed significantly reduced RSV-G mRNA at 36 and 48 hpi (Figure 4A). This reduction in viral mRNA transcription positively correlated with the low level of viral replication observed at the same time points (Figure 2A,B). Furthermore, PAE- and CTE-treated HEp2 cells showed significantly reduced RSV-G protein synthesis at late time points after virus infection (Figure 4B,C). Therefore, it is possible that PAE and CTE may affect the replication of RSV not only at the transcriptional level, but also at the posttranscriptional level. Syncytium formation by RSV is a well-known mechanism of cell-to-cell infection that contributes significantly to virus spread in vivo. PAE and CTE also significantly reduced the RSV-dependent formation of syncytia in HEp2 cells (Figure 4D). Since both herb extracts could inhibit RSV replication in vitro, we went on to test their antiviral potential in vivo. In vivo replication of RSV can also be accurately assessed by qRT-PCR [40].

Interestingly, our in vivo results (Figure 4E,F) revealed that oral administration of PAE and CTE significantly reduced the amount of RSV-G transcription in the lung at 3 and 5 dpi. These in vivo results are consistent with the low virus titers observed in HEp2 cells treated with PAE and CTE in vitro. Even though the exact underling therapeutic anti-RSV mechanisms of PAE and CTE are still under investigation, our results of the in vitro and in vitro antiviral assays show that both herb extracts significantly abolished cell to cell virus infection of RSV, and ultimate inhibition of virus spreading in the infected sites.

Phenylethanoid glycosides are widely found in edible plants and foodstuffs derived from plants [41,42], and these compounds have numerous biological properties, including anti-hepatotoxic [43], anti-inflammatory, anti-nociceptive [44], and anti-oxidant [45] effects. Phenylethanoid glycosides are one of the major bioactive constituents present in both *Plantago asiatica* [46] and *Clerodendrum trichotomum* [47]. HPLC analysis was conducted to confirm this finding (Figure 5A), and acteoside was identified as one of the major constituents of both PAE and CTE. Acteoside, also called kusagin or verbascoside [48], is a phenylethanoid glycoside isolated from many dicotyledons. Reportedly, acteoside has anti-oxidant

and anti-inflammatory properties, and prevents cell apoptosis [39,49]. A recent study demonstrated that acteoside induced ERK activation and subsequent IFN-γ production; thus, showed antiviral activity against influenza and vesicular stomatitis virus (VSV) [28]. Based on these reported findings, we evaluated the antiviral effect of acteoside against RSV. Interestingly, acteoside reduced RSV replication and virus-induced cell death in HEp2 cells (Figure 5E,F) similar to PAE or CTE treatment. In addition, the SI of acteoside against RSV in vitro indicates a high safety margin for its therapeutic effect (Figure 5I). We also confirmed that acteoside reduced the level of RSV-G mRNA and RSV-G protein synthesis at 36 and 48 hpi (Figure 6A). Specifically, intraperitoneal treatment with acteoside showed an anti-RSV effect in a mouse model (Figure 6C). These results are similar to the antiviral activity of acteoside against Influenza or VSV [28]. However, further studies demonstrating a detailed mechanism of how acteoside inhibit RSV replication are needed.

In conclusion, the favorable safety profile and antiviral activity of PAE, CTE, and acteoside suggest that both herb extracts may be good candidates for antiviral therapy for RSV infection. Thus, oral administration of *Plantago asiatica* and *Clerodendrum trichotomum* or administration of acteoside could have potential therapeutic applications in both humans and livestock.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4915/11/7/604/s1, Figure S1. Herb extract library screening results, Figure S2. Selected herb extracts second round screening results.

**Author Contributions:** K.C. designed and executed all virus infection and mouse experiments; M.S.K., H.-C.L., T.-H.K., J.-H.K., W.A.G.C., P.E., H.M.S.M.W., W-.K.C. performed cell biological experiments and mouse experiments, J.Y.M., H.I.K. analyzed the data. J.-S.L. designed the overall study and wrote the paper.

**Acknowledgments:** This work was supported by the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (Grant No. 315044-3, 318039-3), the Small and Medium Business Administration (Grant No. S2130867, S2165234), the Korean Institute of Oriental Medicine by the Ministry of Education, Science and Technology (MEST) (Grant No. K16281), National Research Foundation (Grant No. 2018M3A9H4078703, 2019R1A2C2008283) and the research fund of Chungnam National University.

**Conflicts of Interest:** The authors declare no conflict of interest.
