key: cord-330176-1ugzkf22
authors: Weeratunga, Prasanna; Uddin, Md Bashir; Kim, Myun Soo; Lee, Byeong-Hoon; Kim, Tae-Hwan; Yoon, Ji-Eun; Ma, Jin Yeul; Kim, Hongik; Lee, Jong-Soo
title: RETRACTED ARTICLE: Interferon-mediated antiviral activities of Angelica tenuissima Nakai and its active components
date: 2016-01-05
journal: J Microbiol
DOI: 10.1007/s12275-016-5555-4
sha: 
doc_id: 330176
cord_uid: 1ugzkf22

Angelica tenuissima Nakai is a widely used commodity in traditional medicine. Nevertheless, no study has been conducted on the antiviral and immune-modulatory properties of an aqueous extract of Angelica tenuissima Nakai. In the present study, we evaluated the antiviral activities and the mechanism of action of an aqueous extract of Angelica tenuissima Nakai both in vitro and in vivo. In vitro, an effective dose of Angelica tenuissima Nakai markedly inhibited the replication of Influenza A virus (PR8), Vesicular stomatitis virus (VSV), Herpes simplex virus (HSV), Coxsackie virus, and Enterovirus (EV-71) on epithelial (HEK293T/HeLa) and immune (RAW264.7) cells. Such inhibition can be described by the induction of the antiviral state in cells by antiviral, IFNrelated gene induction and secretion of IFNs and pro-inflammatory cytokines. In vivo, Angelica tenuissima Nakai treated BALB/c mice displayed higher survivability and lower lung viral titers when challenged with lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3, and H9N2). We also found that Angelica tenuissima Nakai can induce the secretion of IL-6, IFN-λ, and local IgA in bronchoalveolar lavage fluid (BALF) of Angelica tenuissima Nakai treated mice, which correlating with the observed prophylactic effects. In HPLC analysis, we found the presence of several compounds in the aqueous fraction and among them; we evaluated antiviral properties of ferulic acid. Therefore, an extract of Angelica tenuissima Nakai and its components, including ferulic acid, play roles as immunomodulators and may be potential candidates for novel anti-viral/anti-influenza agents.

Viral diseases range from trivial infections to plagues that alter the course of history. Because of the enormous variations in viruses and in their epidemiology and pathogenesis, there is no single, magic-bullet approach to control. Each virus presents its own set of problems. As an example, influenza viruses are highly infectious and constitute a major causative agent for recurrent epidemics and pandemics. On average, approximately 10% of the world's population is infected by the virus annually, resulting in an estimated 250,000 deaths, hence posing a serious health threat (Rajasekaran et al., 2013) . Moreover, new and re-emerging infectious viral diseases are a rising global health threat, and the risk of spreading these viruses between continents and countries is even greater. HIV/AIDS, severe acute respiratory syndrome (SARS) and the 2009 H1N1 influenza pandemic and MERS epidemic are only a few of many examples of emerging infectious diseases in the modern world (Morens and Fauci, 2013; WHO, 2015) .

Several preventive and therapeutic measures, including biosecurity, vaccination and antiviral drugs, are routinely used or tried to prevent and treat viral diseases. Vaccines are the basis of prevention of many viral infections; however, there are considerable drawbacks. Because vaccination requires regular monitoring to confirm matching between the vaccines and the circulating virus strains, time-consuming generation processes limit its availability. Failures of influenza vaccines have been widely documented, and in the elderly populationin which most mortality occurs-influenza vaccines are only approximately 50% effective (Reichert et al., 2012) . In the eventuality of a pandemic infection with a new strain, antiviral drugs also represent the first line of defense. Owing to their metabolic properties, viruses are difficult to control, and the limited availability, associated side effects, and rapid development of antiviral resistance have limited the usefulness of these drugs. Therefore, within this evolving environment, groundbreaking strategies and responses are required to reduce the economic and human health risks associated with viral diseases. The development of safe, effective and inexpensive antiviral drugs is among the top global priorities in drug development. Currently, there is a large and ever-expanding global population base that prefers the use of natural products for preventing and treating medical conditions (Gandhiraja et al., 2009 ) and many pharmaceutical companies are attempting to produce new antimicrobial formulations extracted from plants or herbs. Plants are an important sources of lead compounds; up to 40% of modern drugs are derived from plant material (Pan et al., 2013) . For the viral diseases, empirical evidence of the ethno-medical benefits of plants, coupled with bioassay-guided fractionation and isolation, has the potential to identify novel antiviral drugs.

In our study, approximately 400 natural Oriental herbal extracts were screened for antiviral effects. Of these, extracts from Angelica tenuissima Nakai (ATN) showed considerable therapeutic promise because of its broad spectrum of antiviral activities and immune-enhancing properties. Angelica tenuissima Nakai belongs to the family Apiaceae and grows in certain areas in China and rocky slopes in the Korean peninsula (Ka et al., 2005) . It is widely used to treat headache, diarrhea, epilepsy and rheumatic arthralgias in traditional Oriental medicine (Nam et al., 2014) . However, the antiviral activity or immune-modulatory potential of the crude plant extract of Angelica tenuissima Nakai have not been reported in detail. Hence, we evaluated the antiviral activities of total aqueous extracts from this herb against a wide array of viruses in vitro. We also examined the effects of Angelica tenuissima Nakai on innate immune responses. In addition, we used highperformance liquid chromatography (HPLC) analysis to determine the active molecules present in the aqueous fraction. 

Commercial dried bark of the Angelica tenuissima Nakai was obtained and verified by Professor Ki Hwan Bae at the College of Pharmacy, Chungnam National University. The water extract of Angelica tenuissima Nakai was prepared by Vitabio Corporation. Further, the extract quality was assured by Herbal Medicine Improvement Research Center, Korea Institute of Oriental Medicine, Daejeon, Republic of Korea. Hundred gram (100 g) of the dried bark was mixed with 1 L of distilled water and extracted by heating for 2.5 h at 105°C using a medical heating plate (Gyeongseo Extractor Cosmos-600). Then, Angelica tenuissima Nakai was filtered using a filter paper (0.45 μm) (Millex ® ). The extract was next centrifuged at 12,000 rpm for 15 min and the supernatant was collected, and the pH was adjusted to 7.0. The total aqueous extract was then filtered through a syringe filter (0.22 μm) and lyophilized. The final concentration was adjusted to 0.1 mg/ml with phosphate buffered saline (PBS) and kept at 4°C until utilization.

RAW264.7 (ATCC ® TIB-71 TM ), HEK293T (ATCC ® CRL-11268 TM ), HeLa (ATCC ® CCL-2 TM ), MDCK (ATCC ® CCL-34, NBL-2), and A549 (ATCC ® CCL-185 TM ) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotic-antimycotic solution (Gibco) in a humidified incubator at 37°C and 5% CO 2 . Green Fluorescent Protein (GFP)-fused viruses such as PR8-GFP, VSV-GFP, H3-GFP, and EV-71 were kindly provided by Dr. Jae U. Jung, Department of Molecular Microbiology and Immunology, University of Southern California, USA. Challenge viruses were provided by Dr. Y. K. Choi, Chungbuk National University, Cheongju, Republic of Korea. Antiviral assays in Angelica tenuissima Nakai pretreated RAW264.7, HEK293T, and HeLa cells RAW264.7 cells were cultured in 12-well tissue culture plates (8 × 10 5 cells/well) and incubated at 37°C for 12 h. HEK293T and HeLa cells were cultured in six-well tissue culture plates (1 × 10 6 cells/well) under similar conditions. Antiviral assays were performed according to Moon et al. (2012) , with some modifications. DMEM alone (untreated and virus-only groups), DMEM with 1,000 U/ml of recombinant mouse/human interferon (IFN)-β (positive control, Sigma) and DMEM with 1.0 μg/ml (10 μl/ml or 1% v/v) of Angelica tenuissima Nakai were incubated in different wells. At 12 hpt (hour post treatment), all the wells were gently washed with PBS once and RAW264.7 cells were infected with either VSV-GFP (MOI = 1.0) or PR8-GFP (MOI = 1.0), HEK293T cells were infected with VSV-GFP (MOI = 0.2) or HSV-GFP (MOI = 3.0) and HeLa cells were infected with H3-GFP (MOI = 3.0) or EV-71 (MOI = 0.5) viruses using DMEM containing 1% FBS. Two hours post-infection (hpi), the culture medium was renewed, and incubated for an additional 24 h. GFP expression was observed was quantified with the Glomax multi-detection system (Promega). GFP expression, virus titrations and cell viabilities were determined at 12 and/or 24 hpi.

In vitro pro-inflammatory cytokine inducing effect of Angelica tenuissima Nakai was tested using commercial ELISA kits. In RAW264.7 cells, murine interleukin (IL)-6 and IFN-β were measured, as previously described (Wadsworth and Koop, 1999) . RAW264.7 cells were treated with recombinant murine IFN-β (Sigma-Aldrich) and 1.0 μg/ml (10 μl/ml or 1% v/v) Angelica tenuissima Nakai and cultured supernatants were collected at 0, 12, and 24 hpt, clarified by centrifugation at 2,500 × g for 10 min at 4°C and dispensed into murine IFN-β (PBL Interferon Source) ELISA plates or murine IL-6 or murine TNF-α (BD Bioscience) capture antibody-coated ELISA plates. In the case of HEK293T cells, recombinant human IFN-β (Sigma-Aldrich) was used as the positive control and the clarified supernatant was dispensed into human IFNβ (TFB, Inc.) and human IL-6 (Invitrogen) ELISA plates. Murine IFN-β, human IFN-β, and human IL-6 ELISA were performed in duplicate, and other ELISA's was performed in triplicate.

Interferon-mediated antiviral activities of Angelica tenuissima Nakai 59

Determination of the effect of Angelica tenuissima Nakai on type I IFN-related protein phosphorylation in RAW264.7 cells by immunoblot analysis RAW264.7 cells were grown in 6-well tissue culture plates and incubated at 37°C. After 12 h the cells were treated with DMEM containing 10% FBS alone (negative control), DMEM with 100 ng/ml LPS (positive control), or DMEM with 1.0 μg/ml (10 μl/ml or 1% v/v) Angelica tenuissima Nakai, and the cells were harvested at 0, 8, 12, and 24 hpt. The cell pellets were lysed in lysis buffer for immunoblot analysis; loaded on SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (Bio-Rad) for 2 h. The membranes were blocked for 1 h in Tris-buffered saline containing 0.05% Tween-20 (TBST) and 5% bovine serum albumin. Then incubations were performed at 4°C overnight with indicated primary antibody such as anti-IRF3 (Abcam, #ab25950), antiphopho-IRF3 (Ser396), anti-p65, anti-phopho-p65, anti-STAT1, anti-phospho-STAT1, anti-TBK1, or anti-phospho-TBK1, anti-p38, anti-phopho-p38 (Cell Signaling Technology), or anti-β-actin (Santa Cruz SC#47778) antibodies. Horseradish peroxidase-conjugated secondary antibody (Sigma) was used to visualize the respective proteins by means of an enhanced chemiluminescence detection system (ECL-GE Healthcare, UK) using a Las-3000 mini Lumino Image Analyzer.

RAW264.7 cells were grown in 6-well tissue culture plates (1 × 10 6 cells/well) and incubated at 37°C. The cells were treated with DMEM containing 10% FBS alone (negative control), DMEM with 1000 U/ml of recombinant murine/ human IFN-β, DMEM with 1.0 μg/ml (10 μl/ml or 1% v/v) Angelica tenuissima Nakai, and the cells were harvested at 0, 4, 8, 12, and 24 hpt. The total RNA from the cells was isolated using the RNeasy Mini Kit (Qiagen), and cDNA was synthesized using reverse transcriptase (Toyobo). The different levels of cDNA were quantified by real-time polymerase chain reaction (PCR) using a QuantiTect SYBR Green PCR kit (Qiagen) on a Mygenie96 thermal block (Bioneer). The PCR primers are listed in Tables 2 and 3 .

Fifty two female, five-week-old BALB/c mice were separated into four experimental sets, with two groups per set. Of the four sets, one had two groups with 11 mice each (six for lung virus titration at 3 and 5 day's post-infection [dpi]). The other three sets had two groups containing 5 mice each. The mice were orally inoculated 0.1 mg/ml Angelica tenuissima Nakai at a total volume of 200 μl (20 μg/head) 1, 3, and 5 days before infection. The control groups were orally inoculated 200 μl of PBS. All mice were intra-nasally infected (20 μl) with five times the 50% mouse lethal dose (MLD50) of H1N1, H5N2, H7N3 or H9N2. Mice showing a more than 25% body weight loss were regarded to have reached the experimental end point and were humanely killed. The body weight and survival were recorded up to 13 dpi. At 3 and 5 dpi, three mice from each of the two groups from the H1N1infected set were randomly sacrificed to measure the lung virus titers.

Lung tissues were collected from euthanized mice aseptically, and virus titers were measured by 50% tissue culture infectious dose (TCID50) (Quan et al., 2007) . First, lung tissues were mechanically homogenized in 500 ml of PBS solution added with 1% antibiotic/antimycotic solution, centrifuged (15 min, 12,000 × g and 4°C) and stored at -80°C. Madin-Darby Canine Kidney (MDCK) cells cultured in 96-well microtiter plates were infected with 10-fold serial dilutions (in DMEM with 1% FBS) of lung homogenate (50 μl/well) in quadruplicate and incubated at 37°C. At 1 hpi, the media was removed and renewed with medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) trypsin (Thermo Scientific) and incubated for 72 h. Viral cytopathic effects (CPE) were monitored daily and the titers were determined by the hemagglutination assay (HA). The virus titer was calculated by the Reed and Muench method (Zhao et al., 2010) and expressed as Log10 TCID 50 /Lung.

Twenty four, five-week-old BALB/c mice were divided into two groups with 12 mice each. In one group, mice were orally administered 0.1 mg/ml Angelica tenuissima Nakai at a total volume of 200 μl (20 μg/head) 1, 3, and 5 days before the collection of samples. The control group was orally administered 200 μl of PBS. Three mice from each group were randomly selected at 6, 12, 24, and 36 hpt of Angelica tenuissima-Nakai or PBS, and their Bronchoalveolar Lavage Fluid (BALF) and Small-Intestinal Fluids (SIF) were collected as described below. BALF: BALF was collected as previously described with some modifications (Viana et al., 2002) . Briefly, lungs of mice were lavaged four times with 1 ml of Hank's Balanced Salt Solution (HBSS), collected and immediately stored at -20°C and later subjected to ELISA (murine IgA, IL-6, IFN-λ).

SIF: Small intestines of mice were collected as previously described (Lefrançois and Lycke, 2001) . Briefly, mice were euthanized by cervical dislocation; midline incision was performed and retracted the skin. Small intestine was cut ~1 cm above cecum to separate it from others. Then, the intestine was flushed very carefully with 500 μl of HBSS (Sigma-Aldrich), centrifuged and fluids were immediately stored at -20°C. The SIF supernatants were further assayed by the ELISA (murine IgA).

A reversed-phase High Performance Liquid Chromatography (HPLC) system was an Agilent Technologies 1260 Infinity (Agilent Technologies Co.) having G1311C Quaternary Pump, G1329B Auto-sampler, G1316A Column Oven, G1365C Multiple Wavelength Detector a pump L-2130. The column was carried out on a ZORBAX Eclipse XDB-C18 Column (150 × 4.5 mm I.D. S-5 um) and the column oven temperature was kept at 30°C. The mobile phase consisted of 1% Formic acid

(Solvent A) and Acetonitrile (Solvent B) in the gradient mode as: 0-12 min 0-20% B; 12-15 min 20-50% B; 15-20 min 50-58% B; 20-25 min 58-100% B; 25-30 min 100-0% B. The flow rate was 1.0 ml/min, and the elution was monitored at 280 nm. Fraction (FR)-5 (Later identified and confirmed as Ferulic acid) was purified by a semi prep-HPLC Agilent Technologies 1260 Infinity (equipped with a Fraction collector, FC203B, Gilson) using YMC-Pack ODS-A (250 × 10 mm I.D. S-5 μm, 12 nm) (YMC) at a flow rate of 3 ml/min, with constant temperature of 30°C. Purified fractions were further subjected to antiviral assays and cytokine inducing ability in immune (RAW264.7) cells using an effective dose of 5.0 μg/ml. Antiviral activity against PR8-GFP in RAW264.7 cells, virus titration and induction of cytokine secretion were determined according to the protocols described in 'Materials and Methods.'

The viral titers were determined by standard plaque assays using Vero cells (Coil and Miller, 2004) . To assay the viral replication, culture supernatants were collected from VSV-GFP and H3-GFP infected cells both at 12 and 24 hpi and viral titer was determined in Vero cells. In the case of PR8-GFP titration, cells were collected at 24 hpi and subjected to five cycles of freezing at -70°C and thawing at RT and titer was determined in Vero cells.

Viral titers for EV-71, BRV, and HRV were measured by the median tissue culture infectious doses (TCID 50 ) using HeLa cells (Wilden et al., 2009 ) with some modifications. Briefly, supernatants collected from the infected cells were used to infect HeLa cells cultured in 96-well microtiter plates with 10-fold serial dilutions (50 μl/well). After 2 hpi, DMEM (10% FBS) containing TPCK trypsin (Thermo Scientific) was added to the infected wells and incubated for additional 2 days. Viral CPE were observed daily and titers were determined by CPE-TCID50.

RAW264.7, HEK293T and HeLa cells were cultured in 96well plates (2.5 × 10 4 cells/well, 2 × 10 4 cells/well and 2 × 10 4 cells/well, respectively) and incubated at 37°C in a 5% CO 2 atmosphere. After 12 h, two-fold serially diluted Angelica tenuissima Nakai (50 μl/well) was added. At 12 hpt, the cells were infected using DMEM containing 1% FBS. RAW264.7 cells were infected with PR8-GFP (MOI = 1.0) or VSV-GFP (MOI = 1.0), HEK293T cells were infected with VSV-GFP (MOI = 0.2) or HSV-GFP (MOI = 3.0) and HeLa cells were infected with Coxsackie-GFP (MOI = 3.0) or EV-71 (MOI = 0.5) viruses. At 2 hpi, the culture medium was renewed. GFP expression was measured at 24 hpi with the Glomax multi-detection system (Promega). The EC 50 values were then calculated as the Angelica tenuissima Nakai concentration yielding 50% GFP expression. The experiments were performed in triplicate.

The CC 50 was assessed in a cell viability assay through the trypan blue exclusion (Strober, 2001) . The assay was performed using 72-well tissue culture plates. Increasing concentrations (1-160 μl/ml or 0.1-16 μg/ml) of the water extract were added to confluent RAW264.7, HEK293T and HeLa cell monolayers. Clarified cells were stained with 0.4% trypan blue stain (Invitrogen) (ratio: 1:1) at 24 hpt and mounted to a hemocytometer to get the percentages of viable cells. CC50 was calculated as the concentration of the extract resulting in 50% cell viability. The experiment was performed in triplicate.

All the experiments were repeated in triplicate and the data were assessed as the means ± standard deviation (SD). Statistical significance was evaluated using a Student's t-test or one-way analysis of variance (ANOVA), and was considered significant with P<0.05 (*P<0.05 and **P<0.01). Results for percent initial body weight were also compared by Student's t-test. Comparison of survival was done by log-rank test using GraphPad Prism 6 version.

To determine the antiviral effects of Angelica tenuissima Nakai in epithelial cells, we checked the antiviral activity in HEK293T and HeLa cells. In the HEK293T cells, the antiviral activity was determined with GFP-fused VSV or HSV upon pre-treatment with the extract (1.0 μg/ml [10 μl/ml or 1% v/v]). Upon pre-treatment ( Fig. 1A and B), GFP expression was significantly reduced in HEK293T cells in comparison with the untreated groups and showed reduced viral titers by nearly 6-fold or 5-fold against VSV-GFP and HSV-GFP, respectively at 24 hpi. Moreover, a significant reduction in cell death was observed in Angelica tenuissima Nakai extract-treated HEK293T cells compared with the untreated cells. Also, we examined the antiviral activity of Angelica tenuissima Nakai in the HeLa cells against GFP-fused Coxsackie virus (H3-GFP) and Enterovirus-71 (EV-71). Pre-treated cells with Angelica tenuissima Nakai exhibited markedly reduced H3-GFP expression and reduced viral titers by nearly 4.6fold at 24 hpi, together with a significant reduction in cell death (Fig. 1C ). In addition, EV-71-induced CPEs were markedly inhibited upon pre-treatment with Angelica tenuissima Nakai (Fig. 1D ), resulting in a low level of viral replication. Collectively, these results indicate that the total aqueous extract of Angelica tenuissima Nakai reduces the replication of RNA and DNA viruses in epithelial cell lines.

To investigate the antiviral effects in immune cells, we first assessed the replication of divergent GFP-expressing viruses that were treated or untreated with cytotoxic-free (data not shown) Angelica tenuissima Nakai in RAW264.7 cells. A total aqueous extract of Angelica tenuissima Nakai-treated (1.0 μg/ml [10 μl/ml or 1% v/v]) RAW264.7 cells exhibited markedly reduced GFP expression; however, the untreated groups had high levels of GFP expression for VSV ( Fig. 2A) and PR8 (Fig. 2C) . When quantitated, a significant reduction in the GFP expression was observed in the extract-treated cells compared to untreated groups ( Fig. 2B and D middle panel). These findings further correlated with the observed viral titers from VSV-GFP or PR8-GFP infected cells (Fig. 2B and D right panel) . Interestingly, the Angelica tenuissima Nakai-treated cells had a ≥75% cell viability within 24 hpi for all the tested viruses compared with the untreated cells, which were shown to have significantly higher cell death following virus infection ( Fig. 2B and D left panel) . Consequently, these results also suggest that the aqueous extract of Angelica tenuissima Nakai could significantly inhibit the replication of VSV and PR8 viruses in immune cells.

We then developed an improved GFP assay to determine the EC 50 values of Angelica tenuissima Nakai against divergent viruses in vitro using HEK293T and RAW264.7 cells (Magadula and Suleimania, 2010; Lin et al., 2014) . The EC50 is the extract concentration that results in a 50% reduction in virus, and the extract concentration that results in 50% cell viability is the CC50. Because we primarily used GFP-fused viruses, a 50% reduction in GFP expression was considered equivalent to a 50% reduction in virus titer. In the case of EV-71, a 50% reduction in CPE was considered equivalent to a 50% reduction in virus titer. The aqueous extract of Angelica tenuissima Nakai inhibited the replication of VSV-GFP (MOI=0.2) and HSV-GFP (MOI=3.0) by 50% at an EC50 of 0.34±0.04 μg/ml and 0.41±0.12 μg/ml, respectively in HEK293T cells (Table 1 ). In RAW264.7 cells, VSV-GFP (MOI=1.0) and PR8-GFP (MOI =1.0) by 50% at an EC50 of 0.58±0.08 μg/ml and 0.44±0.12 μg/ml, respectively (Table 1 ). In addition, the observed EC 50 values against Coxsackie-GFP (MOI=3.0) and EV-71 (MOI =0.5) were 0.31±0.03 μg/ml and 0.24±0.02 μg/ml, respectively, in HeLa cells. Given its effectiveness and convenience during the experiments, 1.0 μg/ml has been chosen as the optimal dose of Angelica tenuissima Nakai for further in vitro antiviral assays, observed from EC 50 values.

Following treatment with various concentrations, a cell viability test was performed to assess the cytotoxicity of Angelica tenuissima Nakai extract. The extract had a CC50 of 3.47±0.24 μg/ml, 4.34±0.62 μg/ml, and 5.77±0.14 μg/ml in HEK293T, Raw264.7 and HeLa cells, respectively (Table 1) . The selection indexes (SIs) of Angelica tenuissima Nakai for VSV and HSV on HEK293T cells were 10.2 and 8.5, respectively; those for VSV and PR8 on RAW264.7 cells were 7.4 and 9.8, respectively; and those for Coxsackie and EV-71 on HeLa cells were 18.6 and 24.0, respectively. Notably, the SIs of Angelica tenuissima Nakai were several magnitudes higher for the tested viruses in their respective cell lines, a clear indication of the extract's broad prophylactic and therapeutic potential.

Because Angelica tenuissima Nakai possesses antiviral activity, we hypothesized that Angelica tenuissima Nakai might be involved in the Type I IFN signaling pathway. To test this hypothesis, we first evaluated the levels of interferon-β (IFNβ) and the pro-inflammatory cytokines in the extract-treated cell supernatant of HEK293T or RAW264.7 cells (Fig. 3) . Angelica tenuissima Nakai (1.0 μg/ml [10 μl/ml or 1% v/v]) induced high levels of secreted IFN-β and IL-6 in HEK293T cells (Fig. 3A) and TNF-α, IL-6, and IFN-β in RAW264.7 cells at both 12 hpt and 24 hpt (Fig. 3B) . Importantly, the secreted cytokine amounts were significantly higher in HEK-293T cells, although the secreted level was not as high as the levels observed in the RAW264.7 cells in comparison with the non-treated groups. These results suggest that the aqueous extract of Angelica tenuissima Nakai can induce the secretion of IFNs and pro-inflammatory cytokines, which can stimulate the cellular antiviral state for inhibition of viral replication. Further, we checked the phosphorylation of interferon-related signal molecules or p65 molecule related to NF-kB activation to observe correlation of above results with the IFNinducing signaling pathway. For this study, immunoblot analyses were performed using whole-cell lysates of the Angelica tenuissima Nakai-treated (1.0 μg/ml [10 μl/ml or 1% v/v]) RAW264.7 cells. As shown in Fig. 3C , we found that the significant upregulation of type I interferon or NF-kB related signal molecules (IRF3, STAT1, TBK1, p65, and p38) in Angelica tenuissima Nakai-treated cells such as LPS-treated cells. Taken together, these results clearly indicate that the aqueous extract of Angelica tenuissima Nakai can induce the secretion of IFNs and pro-inflammatory cytokines through the activation of signal molecules in the type I IFN and NF-kB signaling pathway.

Given that the Angelica tenuissima Nakai extracts were able to secrete IFN-β and pro-inflammatory cytokines, we further confirmed an interaction between Angelica tenuissima Nakai and the transcriptional levels of antiviral genes or interferonstimulatory genes (ISGs) in vitro. As confirmed by real-time PCR assay, the mRNA expression levels of various antiviral and interferon stimulatory genes in the cells treated with Angelica tenuissima Nakai (1.0 μg/ml [10 μl/ml or 1% v/v]) were up-regulated to levels similar to those found with the IFN-β-treated positive controls (Fig. 4) . Also, to determine the transcription levels of various antiviral genes in the Angelica tenuissima Nakai-treated HEK293T cells (Fig. 4A) , an IFN-β real-time PCR assay was performed from 0 to 24 hpt to monitor the time-dependent mRNA changes. Interestingly, the highest fold inductions of cellular transcriptional levels were observed at 12 hpt for all the tested primers (the primers are listed in Table 2 ). In HEK293T cells, upon treatment of Angelica tenuissima Nakai extract, transcriptional levels for IFN-β, Mx-1, GBP-1, TNF-α, IL-8, and IL-6 were up-regulated by 7.64-fold, 5.6-fold, 4.3-fold, 10.1-fold, 2.6-fold, and 6.9-fold, respectively (Fig. 4A) . Similarly, transcriptional activation patterns were also observed in the Angelica tenuissima Nakai-treated RAW264.7 cells. The results showed that the extract-treated cells displayed a nine-fold increase in the level of IFN-β mRNA at 8 hpt and a thirteen-fold induction at 12 hpt compared with the untreated cells (Fig. 4B) . Hence, we performed a PCR assay for other genes of interest at 0, 8, and 12 hpt using specific primers (the primers are listed in Table 3 ) in RAW264.7 cells. We found that various antiviral gene tran-scriptional levels of were to be up-regulated by Angelica tenuissima Nakai at 8 hpt, including Mx1, OAS-16, PML, PKR, IL-6, ISG-20, and ISG-56, to levels that were 3.1-fold, 3.9fold, 5.4-fold, 6.5-fold, 8.2-fold, 3.2-fold, and 3.3-fold, respectively, higher than those of the control (Fig. 4B) . Thus, overall results suggests that Angelica tenuissima Nakai extracts were able to up-regulate the transcriptional levels of IFN-β, ISGs, and various antiviral related genes in HEK293T and RAW264.7 cells which can induce antiviral state.

To study the Angelica tenuissima Nakai induced prophylactic

Interferon-mediated antiviral activities of Angelica tenuissima Nakai 65 After challenge, the control (PBS) mice groups were suffered severe illnesses and the body weights loss significantly. By approximately 3-4 dpi, most of the infected mice in the con-trol groups displayed severe clinical signs of respiratory disease, including labored respirations and respiratory distress. Moreover, the control groups succumbed to death by 11 dpi for all of the viruses tested. However, the Angelica tenuissima Nakai-inoculated mice showed a ≤20% body weight loss between 4 and 7 dpi and had begun to regain by 8 dpi, returning to their normal state by 12-13 dpi (Fig. 5) . Besides, all the Angelica tenuissima Nakai-inoculated groups had higher survival rates: 100% survival for the groups infected with H5N2 (Fig. 5B) , H7N3 (Fig. 5C) , and H1N1 (Fig. 5A) ; and 80% survival infected with H9N2 (Fig. 5D ). The surviving mice in these groups did not show obvious clinical signs, except for negligible weight loss. Because influenza virus is transmitted primarily by aerosols via the respiratory system of mice and replicated most . 6 . Induction of immune state by Angelica tenuissima Nakai in BALB/c mice. Five-week-old female BALB/c mice were orally administered 0.1 mg/ml Angelica tenuissima Nakai (ATN) at a total volume of 200 μl (20 μg/head) at 1, 3, and 5 days before the collection of samples. Control groups were orally administered with 200 μl of PBS. Three mice from each group were randomly selected at 6, 12, 24, and 36 hpt of Angelica tenuissima Nakai or PBS, and their Bronchoalveolar Lavage Fluid (BALF) and Small-Intestinal Fluid (SIF) were collected and analyzed for the presence of secreted murine (A) IL-6 or IFN-λ, (B) Immunoglobulin A (IgA). The test was performed in duplicate for IgA and IFN-λ and triplicate for IL-6. The data shows representative means ± SD of each cytokine measured over time (*P<0.05 indicates a significant difference between groups compared by Student's t-test). efficiently in lungs (Bouvier and Lowen, 2010) , it is important to examine the ability of Angelica tenuissima Nakai extracts to inhibit viral replication in lungs. To evaluate the ability of Angelica tenuissima Nakai to inhibit viral replication in the H1N1 infected lung tissues, three mice from each group were sacrificed, and their lungs were collected at 3 and 5 days post-infection for viral titration. In comparison of control groups, H1N1 virus replicated efficiently in the lungs with a viral titer of 4.33 log TCID 50 /lung and 5.35 log TCID 50 /lung on 3 and 5 dpi, respectively (Fig. 5E) . Comparatively, the viral loads in the Angelica tenuissima Nakai-treated groups were significantly lower: 2.69 log TCID 50 /lung and 2.05 log TCID50/lung, at 3 dpi and 5 dpi, respectively (Fig. 5E ). Consequently, inoculation of Angelica tenuissima Nakai extracts induce the sufficiently strong inhibition of viral replication and endorsed the survival of mice against diverse influenza A subtypes lethal infections.

To observe the immune status in BALF of Angelica tenuissima Nakai inoculated BALB/c Mice, we measured the level of the cytokines and IgA in the BALF and SIF. After inoculation with Angelica tenuissima Nakai extracts into mice, we detected significant level of IL-6 and IFN-λ which has antiviral function such as Type I IFNs, at each time point starting from 6 to 36 hpt in the BALF. And also, we detected the presence of IgA in BALF and SIF in all the tested samples. However, minor levels of IgA secretion were detected in all the samples from the PBS-treated mice (Fig. 6B) . These results indicate that oral inoculation of Angelica tenuissima Nakai extracts stimulate mucosal immune cells of small intestine and ultimately induce the strong inhibition of influenza virus replication in bronchoalveolar through the mucosal immune response system.

For the main component profile of the water extract of Angelica tenuissima Nakai, we used a HPLC system. A reversed-phase HPLC method was employed with a mobile phase of 1% formic acid (Solvent A) and acetonitrile (Solvent B) in the gradient mode as follows: 0-12 min, 0-20% B; 12-15 min, 20-50% B; 15-20 min, 50-58% B; 20-25 min, 58-100% B; and 25-30 min, 100-0% B. The flow rate was kept constant at 1.0 ml/min for the total run time, and 11 fractions were purified successfully. We then evaluated the antiviral effect of the identified fractions (FR-1 to FR-11) in RAW264.7 cells. Viral replication was monitored with PR8-GFP in response to pre-treatment of various fractions. An effective dose of 5.0 μg/ml was chosen on the basis of our preliminary experiments on the efficacy of purified fractions (data not shown). Interestingly, treatment with FR-5 markedly inhibited the virus replication (Fig. 7C) . Of 11 fractions, FR-5 was detected at a wavelength and retention time of 325 nm and 16.33 min, and the concentration of FR-5 in 6.0 mg/ml (w/v) of Angelica tenuissima Nakai extract was found to be 5.48 μg/ml ( Fig. 7A and B) . Ultimately, FR-5 was confirmed the ferulic acid by LC-Mass. The ferulic acidtreated cells displayed reduced GFP expression and reduced viral titers compared with the untreated cells. Ferulic acid treatment reduced the viral titers by nearly 5.2-fold against PR8-GFP at 24 hpi (Fig. 7C) . Furthermore, treatment with ferulic acid (5.0 μg/ml) markedly increased cytokine secretion in the RAW264.7 cells (Fig. 7D) . Therefore, our results strongly suggest that ferulic acid, a major constituent of Angelica tenuissima Nakai, might be able to induce the antiviral state in cells and subsequent inhibition of virus replication.

Historically, natural products and their derivatives have been invaluable sources of therapeutic agents. Recent technological advances, coupled with unrealized expectations of lead-generation strategies, have led to renewed interest in natural products in drug discovery (Koehn and Carter, 2005) . Plants have a long evolutionary history of developing resistance against viruses and have increasingly drawn attention as potential sources of antiviral drugs. For instance, many plant extracts and compounds of plant origin have been shown to have activity against influenza viruses (Park, 2003; Koehn and Carter, 2005) . At present, plant and herb resources are unlimited, have provided mankind with remedies for many infectious diseases and continue to play a major role in primary health care as therapeutic remedies in developing countries. Here, we evaluated the antiviral activity of Angelica tenuissima Nakai in vitro and in vivo. Despite the known biological properties of the plant, no study has been conducted on the antiviral activity and the underlying mechanism of action. First, we found that the total aqueous extract of Angelica tenuissima Nakai displayed a broad spectrum of antiviral properties in vitro. This herb has been used for a variety of purposes for a long time, and no adverse effects of its use have been reported. Notably, Angelica tenuissima Nakai did not show any significant cytotoxic effect on the tested cell lines. Its cell cytotoxic concentration (CC 50 ) was several magnitudes higher than the effective concentration (EC 50 ) of the tested viruses, and the SIs of the herb for various viruses indicate a higher safety margin of the extract for therapeutic or prophylactic purposes (Table 1 ). In examining the in vitro antiviral activity, we determined that Angelica tenuissima Nakai inhibited the replication of influenza (Fig. 2C) , VSV (Figs. 1A and 2A) , HSV (Fig. 1B) , Coxsackie (Fig. 1C) and EV-71 (Fig. 1D ) viruses in immune (RAW264.7) and epithelial (HEK293T and HeLa) cells. Upon viral infection, host cells initially recognize an infection and quickly evoke antiviral innate immune responses, including secretion of Type I IFNs and pro-inflammatory cytokines (Takeuchi and Akira, 2007) . Secreted IFNs and cytokines induce an antiviral state, which is important to protect the host cells against invading viruses (Tenoever et al., 2007) . Induction of an antiviral state at an early stage of virus infection is critical to control the spread and pathogenesis of viruses (Boasso, 2013) by specific agents could be effective approaches for limiting viral infection (Jackson, 2012; Perlman, 2012) . Likewise, we hypothesized that Angelica tenuissima Nakai induces an antiviral state via the induction of type I interferons and proinflammatory cytokines, and we determined the induction of antiviral, IFN-stimulated genes (Fig. 4 ) and the secretion of IFN-β and IL-6 ( Fig. 3) by Angelica tenuissima Nakai in vitro. To elucidate the features in antiviral signaling, we also evaluated the effect of Angelica tenuissima Nakai on the phosphorylation of IRF-3, p65, TBK1, STAT1, ERK, and p38, which are key signaling molecules in the type I IFN and NF-κB signaling pathways. Upon stimulation of the pattern recognition receptors (PRRs) or unknown receptors of the host cell by foreign materials containing pathogens or diverse ligands, downstream signal transduction is activated, including the activation of adaptor signal molecules or transcriptional factors, and can initiate the induction of Type I IFNs and pro-inflammatory cytokines to up-regulate the antiviral status of the host cell (Mogensen, 2009) . In this study, we found that Angelica tenuissima Nakai extracts treatment can induce the phosphorylation of IRF-3, STAT1, and TBK1 in a time-dependent manner, providing evidence of downstream signal transduction in the type I IFN signaling pathway (Fig.  3C) . Additionally, the activation of NF-κB (p65, p38), which leads to a strong secretion of pro-inflammatory cytokines, was also observed (Fig. 3C ). This phosphorylation could lead to the rapid production of Type I IFNs and various inflammatory cytokines that play a crucial role in stimulating the antiviral state and the subsequent clearance of viruses (Price et al., 2000) .

Oral administration of Angelica tenuissima Nakai extracts not only increased the survival rate of mice subjected to lethal challenges with divergent influenza A subtypes, including H1N1, H5N2, H7N3, and H9N2, but also led to rapid weight recovery (Fig. 5 ). Mice treated with the abstract initially displayed little weight reduction; ultimately, the majority did not lose more than 25% of their body weight. By contrast, all of the mice in the control groups displayed weight loss of more than 25% within 7 dpi and were humanely killed. Influenza virus causes a rapid reduction in the body weight of infected mice; hence, 25% body weight loss is considered the humane end point for sacrificing influenza virus-infected mice . In addition, lung virus titration (Fig.  5E ) correlated with survival, and the extract-treated mice displayed reduced lung viral titers, suggesting that Angelica tenuissima Nakai is sufficiently potent to inhibit viral replication and promote the survival of mice against lethal infections of diverse influenza A viruses. To support the antiviral effects of Angelica tenuissima Nakai in vivo, we observed the immune status in BALF of Angelica tenuissima Nakai inoculated BALB/c Mice. As shown in Fig.  6 , after inoculation with Angelica tenuissima Nakai extracts into mice, we could detect significant level of IL-6 and IFN-λ in BALF. Previous literature has indicated that elevated levels of serum IL-6 correlate with induction of the antiviral state and therefore play an important role in the inhibition of virus replication (Spellberg and Edwards, 2001; Melchjorsen et al., 2003) . And IFN-λ which is an exciting new chapter in the field of IFN research, is also able to activate the same intracellular signaling pathway and many of the same biological activities as those displayed by Type I IFNs, including antiviral activity, in a wide variety of target cells (Lazear et al., 2015) . Moreover, we also found significant level of secreted IgA (SIgA) in BALF and SIF, after inoculation with Angelica tenuissima Nakai extracts. Secretory IgA (SIgA) is the most abundant immunoglobulin in body secretions, including saliva, tears, colostrum and gastrointestinal secretions, and is the main effector of the mucosal immune system, providing an important first line of defense against most pathogens that invade the body at the mucosal surfaces (Woof and Mestecky, 2005; Mantis et al., 2011) . Thus, significant level of IL-6, IFN-λ or SIgA which induced in BALF by Angelica tenuissima Nakai extracts, maybe correlate with the higher survival rates observed in the BALB/c mice against divergent influenza A infection. Macrophages have been widely used in drug development because of their significant role in the immune system, especially in antiviral responses (Wilden et al., 2011; Kim et al., 2013) . Here, we used murine macrophages (RAW264.7) to evaluate the antiviral effect of Angelica tenuissima Nakai in vitro. In addition, we used epithelial cells such as HEK293T and HeLa, which are very susceptible to viral replication. It was recently reported that HEK293T (Graham et al., 2014) and HeLa (Kolli et al., 2013) cells have fewer PRRs than Tolllike receptors (TLRs). This finding means that an aqueous extract of Angelica tenuissima Nakai may contain active components that can bind or penetrate the cell membrane. Those active components may stimulate the cell surface PRRs, cytoplasmic PRRs, or both, and ultimately induce antiviral immune responses in immune or epithelial cells. Surely, we tested Angelica tenuissima Nakai for endotoxin contamination using a limulus amebocyte lysate (LAL) assay and found that it was not contaminated with endotoxin (data not shown). There have been phytochemical reports on various types of compounds from Angelica tenuissima Nakai, including phthalides, coumarins, terpenoids, and phenylpropanoids (Islam et al., 2009; Nam et al., 2014) . Some of these compounds were found to have diverse biological activities, such as antioxidative (Ka et al., 2005) , anticancer (Park et al., 2011) , angiotensin converting enzyme (ACE) inhibitory (Liang et al., 2011) and antifungal effects . To confirm these reports, we conducted HPLC analysis to identify the active compounds present in the total aqueous fraction. We observed 11 major peaks (Fig. 7A) , and 11 fractions (FR-1 to FR-11) were successfully purified. Of the 11 fractions, FR-5 was identified as having significant antiviral properties, like those displayed by Angelica tenuissima Nakai; FR-5 was later confirmed to be ferulic acid (Fig. 7A) . Here, we newly defined the antiviral effects of ferulic acid via Type I IFN stimulation. Thus, the antiviral and immunomodulatory effects of an aqueous extract of Angelica tenuissima Nakai might be due to a cumulative effect of ferulic acid including with other fractions or other unknown active compounds present in the extract. The relationship between the mechanisms of the antiviral effects and active compounds, including ferulic acid, should be studied further.

Interferon-mediated antiviral activities of Angelica tenuissima Nakai 69

In summary, we demonstrated that the aqueous extract of Angelica tenuissima Nakai can be a major alternative antiviral agent. The aqueous extract of Angelica tenuissima Nakai inhibit the diverse viral infection through the induction of Type I IFN signaling and pro-inflammatory cytokines, leading to an antiviral state in epithelial and immune cells. The aqueous extract of Angelica tenuissima Nakai can reduce influenza-induced mortality by disrupting viral replication or preventing viral infection by creating an antiviral state in in vivo (lungs). Given the antiviral activities, Angelica tenuissima Nakai extracts or its components, including ferulic acid may be used as a preventive or therapeutic agent to limit viral replication which have been caused a serious damage to human or livestocks. Furthermore, Angelica tenuissima Nakai may also be used as a target for drug design to prevent the viral infection through the innate immune responses.

Prasanna Weeratunga, Md Bashir Uddin designed and executed all cell biological experiments; Myun-Soo Kim, Byeong-Hoon Lee, Ji-Eun Yoon performed all virus infection experiments, analyzed the data; Jin Yeul Ma, Hongik Kim analyzed the data. Jong-Soo Lee designed the overall study and wrote the paper.

The animal study was conducted under appropriate conditions with the approval of the Institutional Animal Care and Use Committee of Bioleaders Corporation, Daejeon, Korea. Protocol number: BSL-ABLS-13-008.

None of the authors have any financial or personal relationships with other people or organizations that could inappropriately influence or bias this study.

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The authors thank Dr. J. U. Jung of the University of Southern California, USA for providing Green Fluorescence