Anti-Influenza Activity of an Ethyl Acetate Fraction of a Rhus verniciflua Ethanol Extract by Neuraminidase Inhibition

Antigenic mismatch can cause influenza vaccines to be ineffective, and influenza viruses resistant to antiviral drugs are rising. Thus, development of antiviral agents against these viruses is an immediate need. Rhus verniciflua (RVS) has long been used in herbal medicine and as a nutritional supplement. The effect of RVS and its components on influenza virus has not, however, been reported. We found that RVS treatment significantly reduced viral replication when evaluated with green fluorescent protein- (GFP-) tagged virus (influenza A virus, A/PR/8/34-GFP) in Madin–Darby canine kidney (MDCK) cells. RVS showed significant inhibition of neuraminidase from A/PR/8/34. Subsequently, three fractions were prepared from an ethanolic crude extract of RVS. In vitro assays indicated that an ethyl acetate fraction (RVSE) was more potent than H2O and CHCl3 fractions. RVSE significantly suppressed influenza virus infection in MDCK cells via neuraminidase inhibition. Additionally, RVSE treatment inhibited expression of several virus proteins and decreased mortality of mice exposed to influenza A/PR/8/34 by 50% and reduced weight loss by 11.5%. Active components in RVSE were isolated, and 5-deoxyluteolin (5) and sulfuretin (7) demonstrate the highest neuraminidase inhibitory activity against influenza A virus. RVS, RVSE, and their constituents may be useful for the development of anti-influenza agents.

NA is a glycoprotein present on the surface of influenza viruses and is required for release of progeny virions from infected cells. NA acts by cleaving sialic acid groups on cell surfaces that bind to viral hemagglutinin. Thus, NA inhibitors prevent progeny virions from budding from infected cells. The active site of NA is highly conserved in both influenza A and B [14][15][16]. However, H274Y and E119G/D/A mutations in the NA gene decrease susceptibility to NA inhibitors oseltamivir and zanamivir, respectively. Such resistance leads to the current demand for the development of new NA inhibitors [17,18].
Phytochemicals from medicinal plants provide valuable building blocks for new drug development [19,20]. Rhus verniciflua Stokes (RVS), which produces various bioactive constituents, has been used as a traditional herbal medicinal plant for various diseases, such as gastroenteritis, diabetes, arthritis, hypertension, stroke, and cancer. Aromatic compounds from RVS significantly block PD-1/PD-L1 and CTLA-4/CD80 interactions [21]. Antiviral efficacy of RVS has been investigated for human immunodeficiency virus type 1 and fish pathogenic viruses, but not for influenza [22,23].
Thus, we examined the effects of an RVS ethyl acetate fraction (RVSE) on inhibiting the replication of influenza virus in vitro and in vivo. We initially assessed the potential of RVSE to inhibit influenza virus replication and underlying mechanisms of action in vitro, focusing on the inhibition of NA activity. Subsequently, we investigated RVSE for protection of mice from a lethal challenge with influenza virus. RVSE significantly averted influenza virus infection in Madin-Darby canine kidney (MDCK) cells via inhibition of NA. RVSE treatment also decreased mortality and prevented weight loss in mice exposed to influenza A/PR/8/34 virus. We also isolated and identified 10 major components in the RVSE and found that 5-deoxyluteolin (5) and sulfuretin (7) demonstrated the highest NA inhibitory activity. RVS, RVSE, and their components were effective in inhibiting the NA activity of both influenza virus A and B, suggesting that RVSE and its components may be good candidates and building block for novel anti-influenza drugs.

Preparation of RVS and RVSE.
Dried bark of RVS (8.0 kg) was exhaustively extracted under reflux with 70% ethanol three times, each time with 50 L solvent. The total extract (330.0 g) was suspended in deionized water and partitioned with CHCl 3 (80.0 g). The water fraction was then partitioned sequentially with ethyl acetate (EA) (125.0 g).
2.5. MTS Assay. Cell viability was determined using the Cell-Titer 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), following the manufacturer's instructions. MDCK cells (1 × 10 4 cells/well) were seeded into 96-well plates, and RVS and RVSE were added to wells at concentrations of 0-400 μg/mL. After 48 h, MTS solutions were added to each well, and the cells were incubated for additional 2 h. Subsequently, absorbance at 490 nm was recorded using a GloMax® Explorer Multimode Microplate Reader (Promega, Madison, WI, USA). The values of MTS assay were represented by the mean ± SEM of four independent experiments.

Docking Simulation and Interaction
Analysis. Two RVSE components 5 and 7 were docked onto the predefined binding pocket of the H1N1 NA crystal structure (PDB code: 3TI6) retrieved from the Protein Data Bank (www.rcsb.org) using SwissDock [30]. After docking simulation, the lowest energy scoring binding mode for each component was selected. The hydrogen bonding and hydrophobic interactions between H1N1 NA and each component were investigated with LigPlot+ v1.4.5 [31]. Amino acid residues involved in interactions were indicated with green (H-bonds) and red (hydrophobic interactions).

Immunofluorescence Staining.
For the immunofluorescence analysis, we used a slightly modified version of a previously used immunofluorescence analysis method [24]. Briefly, MDCK cells were cultured in 4-well tissue culture slides (1 × 10 5 cells/well) for 18 h. Subsequently, A/PR/8/34-GFP (MOI = 5) were mixed with different concentrations of RVSE (25 and 100 μg/mL), and the mixtures were incubated at 37°C for 1 h. MDCK cells were infected with these mixtures at 37°C for 2 h. Thereafter, the virus was removed, and the cells were washed three times with PBS and were cultured in a CO 2 incubator at 37°C for 24 h. Cells were then washed three times with cold PBS and fixed with 4% paraformaldehyde in PBS and 1% Triton X-100 for 10 min each at room temperature. After blocking, the fixed cells were incubated overnight at 4°C with M2-specific antibodies, washed three times (5 min per wash) with TBS, and incubated with Alexa Fluor 568 goat anti-rabbit IgG antibody (1 : 1,000; Life Technologies, Eugene, OR, USA) and washed three times (5 min per wash) with TBS. Next, the cells were incubated with DAPI for 10 min and measured using fluorescence microscopy.
2.14. Western Blot Analysis. MDCK cells were cultured in 6-well plates (1 × 10 6 cells/well) for 18 h. H1N1 was mixed with different concentrations of RVSE (12.5, 25, 50, and 100 μg/mL), and the mixtures were incubated at 37°C for 3 Oxidative Medicine and Cellular Longevity 1 h. MDCK cells were infected with these mixtures at 37°C for 2 h. Afterwards, the virus was removed, cells were washed three times with PBS, and the medium was replaced by complete DMEM. After 24 h, cells were harvested and subjected to western blotting using whole cell extracts [24]. The PVDF membrane was then blocked with 5% BSA in TBS-T buffer for 1 h and incubated overnight at 4°C with primary anti-PA, anti-NA, anti-NP, anti-PB1, anti-PB2, anti-M1, and anti-NS-1 and anti-β-actin antibodies (1 : 1,000 dilution). Primary antibodies were washed three times (5 min per wash) with TBS-T buffer and incubated with HRP-conjugated secondary antibodies (1 : 5,000 dilution) at room temperature for 1 h. Relative intensities of protein bands were measured using ImageJ program [24]. The experiment was repeated independently three times, and similar results were obtained in each replicate.

Inhibition of NA Activity by RVS and Its
Fractions. NI is recognized as a quality anti-influenza drug target that prevents progeny virions from being released from infected cells. We investigated the ability of RVS and its fractions for inhibition of NA activity. NI assay showed that RVS effectively inhibited NA activity of A/PR/8/34 at concentrations above 12.5 μg/mL (Figure 1(a)). Additionally, we con-firmed that the EA fraction of RVS inhibits NA activity of H1N1 (A/PR/8/34) by 98.3%. This inhibition is superior to other fractions (chloroform, 8.0%; water, 24.5%) (Figure 1(b)). We also assessed the inhibition of NA activity by RVSE Figure 5: RVSE reduced the expression of influenza A virus proteins in infected MDCK cells. The reduction of M2 proteins in MDCK cells was observed with fluorescence microscopy using the influenza A virus protein M2-specific antibodies (a). MDCK cells were also stained with DAPI (blue), and the merged images represent M2 (red). Viruses were titrated from the supernatant via the hemagglutination inhibition assay. The supernatant titer of H1N1-infected cells treated with RVSE (12.5-100 μg/mL) was significantly decreased compared with that without RVSE treatment (b, c). MDCK cells were cultured in 6-well plates (1 × 10 6 cells/well) for 18 h. Then, H1N1 was mixed with different concentrations of RVSE (12.5, 25, 50, and 100 μg/mL), and the mixtures were incubated at 37°C for 1 h. MDCK cells were infected with these mixtures at 37°C for 2 h. Afterwards, the virus was removed, the cells were washed three times with PBS, and the medium was replaced by complete DMEM. After 8 h, the cells were harvested, and western blotting was performed using the whole cell extracts. Influenza H1N1 virus protein levels (PA, NA, NP, PB1, PB2, M1, and NS-1) in MDCK cell lysates were detected using western blotting, and β-actin was analyzed as a loading control (d, e). The blots of NA and NS-1 were stripped and reprobed using β-actin antibody. The data are representative of three independent experiments that gave similar results. Bar graph (mean ± SEM) statistics were determined by three experiments' data using one-way ANOVA with Tukey's post hoc test, * * * p < 0:001; * * p < 0:01. n.s.: not significant, compared with the (RVSE untreated) samples. 8 Oxidative Medicine and Cellular Longevity ( Figure 2 (Figure 3(a)). The following experiments were conducted at an RVS concentration below 100 μg/mL.

RVS Inhibited the Infection of Influenza Virus in MDCK
Cells. MDCK cells treated with RVS concentrations of 0, 12.5, 25, 50, or 100 μg/mL were infected with A/PR/8/34-GFP (Figure 3(b)). RVS-treated MDCK cells showed significantly reduced GFP expression levels compared with untreated cells 24 h after infection (Figure 3(b)). Additionally, flow cytometry analysis using fluorescence detection indicated that RVS effectively inhibits viral replication in MDCK cells (Figure 3(c)). RVS-treated cells showed a significantly reduced viral load following infection with influenza virus compared with untreated cells.

RVSE Inhibited Infection of MDCK Cells by Influenza
Virus. Viral replication was investigated at the concentration of RVSE up to 100 μg/mL, which did not show the cytotoxicity to MDCK cells (Figure 4(a)). Viral replication in MDCK cells treated with varying concentrations of RVSE and infected with A/PR/8/34-GFP was inhibited as measured by decreasing levels of GFP expression compared with those of untreated cells 24 h after infection (Figure 4(b)). Flow cytometry analysis using fluorescence detection showed that RVSE effectively inhibits viral replication (Figures 4(c) and 4(d)). Further, we investigated viral replication in RVSE-treated

Caucasian human lung carcinoma A549 cells infected with A/PR/8/34-GFP (Figures 4(e) and 4(f)). RVSE-treated MDCK and A549 cells exhibited significantly reduced viral loads following infection with influenza virus.
We also evaluated the effect of RVSE on expression of influenza A virus proteins, such as M2, using immunofluorescence analysis in RVSE-treated MDCK cells 24 h after infection with A/PR/8/34-GFP ( Figure 5(a)). The expression of influenza A virus protein M2 was inhibited by RVSE concentrations of 25 and 100 μg/mL following infection with A/PR/8/34-GFP at 24 h ( Figure 5(a)).

Inhibitory Effect of the RVSE on the Influenza Virus In
Vivo. We initially examine the impact of RVSE on influenza A virus infection in mice. Mice, which were treated once daily with RVSE (10 mg/kg), maintained a relatively stable body weight, and no significant clinical symptoms were observed throughout the study (data not shown). Untreated A/PR/8/34-infected mice displayed significant body weight loss by 3 dpi before dying within 3 dpi (Figure 6). By contrast, RVSE-treated mice exhibited significantly increased survival after A/PR/8/34 infection (Figure 6(a)). Survival rate in the RVSE-treated group 10 dpi was 50%, higher than that in the 9 Oxidative Medicine and Cellular Longevity viral control group (10%). Further, RVSE treatment did protect against body weight loss following viral infection (by approximately 11.5%) compared with the findings in untreated mice (Figure 6(b)).

3.7.
Anti-Influenza Efficacy of the Components Identified from RVSE. We further assessed NI efficacy of 10 components isolated from RVSE (Figures 7(a) and 7(b)). Components 5 and 7 ( Figure 8). HPLC analysis also showed that RVS contained components 5 and 7 at 3.4 and 3.2 mg/g, and RVSE showed concentrations of 44 and 20 mg/g, respectively.
3.9. Protein-Ligand Docking Simulation and Pharmacophore Analysis of the Components in RVSE. Oseltamivir carboxylate inhibits the release of replicated viruses from infected host cells by interacting with NA, and we thus investigated the molecular interactions between H1N1 NA (PDB code: 3TI6) and two components in RVSE with a protein-ligand docking simulation and pharmacophore analysis using SwissDock and LigPlot+ software. The pharmacophore analysis showed that 5-deoxyluteolin formed seven hydrophobic interactions and two hydrogen bonds and sulfuretin formed five hydrophobic bonds and four hydrogen bonds with NA ( Figure 9). Specifically, 5-deoxyluteolin and sulfuretin were stably bound to NA by common molecular interactions: the hydrophobic interactions of AC-rings in 5 and 7 with S370, W403, and K432 and hydrogen bonds with R371; and hydrophobic interaction of B-ring in 5 and 7 with R118 and D151.

Discussion
An ongoing urgent medical need currently exists to develop new strategies to combat influenza virus infection [39][40][41][42]. Antiviral drugs are the only way to treat the disease when a vaccine is not available. Currently, COVID-19 is causing deaths worldwide, and the number of cases of simultaneous COVID-19 and influenza virus infection is increasing. Such coinfection is common during periods of increased novel COVID-19 transmission [2][3][4]. Patients with simultaneous infection are at high risk of poor outcomes [2][3][4].
The NA is a glycoprotein present on the surface of the influenza virus. The enzyme is required for release of progeny virions from infected cells by cleaving sialic acid groups on   Figure 9: Protein docking simulation between NA and RVSE components. Binding affinity of components 5 and 7 with NA (09H1N1, PDB ID: 3TI6) was predicted by protein docking simulation using SwissDock. LigPlot+ software was applied to analyze their key hydrophobic and hydrogen bonds. 11 Oxidative Medicine and Cellular Longevity the cell surface that bind to viral hemagglutinin. NA inhibitors prevent the release of progeny virions by interacting with the highly conserved active site of NA [14][15][16]. Unfortunately, H274Y and E119G/D/A mutations of NA decrease susceptibility to NA inhibitors oseltamivir and zanamivir, respectively, resulting in continued demand for the development of new agents [17,18]. NA remains an attractive target for developing anti-influenza drugs.
New and effective preventive and therapeutic agents might be found among natural products [39][40][41]. We investigated the antiviral activity of plant extracts against influenza virus infection [19,20]. We selected an ethanolic extract from the bark of RVS [43] for this study due to the beneficial effects of this plant on human health, which have already been reported in many literature. RVS has been used as a traditional herbal medicine for various symptoms, such as gastroenteritis, diabetes, arthritis, hypertension, stroke, and cancer [36,43]. Previously, we demonstrated that RVS and its active constituents blocked immune checkpoint PD-1/PD-L1 CTLA-4/CD80 [21]. The antiviral efficacy of RVS has been investigated, but not for influenza virus [22,23].
We examined the antiviral activity of RVS against influenza virus and found that treatment with RVS markedly reduced viral replication in MDCK cells, evaluated using a GFP-tagged virus. Also, RVS displayed significant inhibitory activity against NA from A/PR/8/34. Subsequently, we isolated an RVS fraction with substantial antiviral activity via NI. Three fractions were prepared from the ethanolic crude extract of RVS. In vitro assays indicate that RVSE is more potent than H 2 O and CHCl 3 fractions. We confirm that RVSE significantly suppresses influenza virus infection in MDCK and A549 cells through concentration-dependent NI. RVSE treatment also inhibited expression of virus proteins, PA, NA, NP, PB1, PB2, M1, and NS-1, and decreased mortality in mice exposed to the influenza A/PR/8/34 virus by 50% and prevented weight loss by approximately 11.5% (Figures 6(a) and 6(b)).
Active compounds in RVSE were isolated, and 10 components were identified. 5-Deoxyluteolin (5) and sulfuretin (7) have the highest inhibitory activity against NA. The strong inhibition of NA activity of RVSE likely results from high concentrations of components 5 and 7.
Structure-activity relationships among isolated flavonoids indicate that components 5-8 with a double bond between C-2 and C-3 show greater inhibition of NA activity than components 1-4. This finding suggests the double bond at C-2/3 is a key functional element. Components 5 and 7 are not substituted at C-3, compared with components 6 and 8  Figure 10: Identification of structure-activity relationship for NA inhibitory activity in compounds from RVSE.
that display a hydroxyl group at C-3. Thus, lack of substitution at C-3 might be an important functional feature ( Figure 10). These findings may be useful in evaluating the structure-activity relationships of other flavonoids for antiinfluenza activity. We identified constituents of RVSE using HPLC and confirmed that components 5 and 7 inhibit NA activity using an NA-Fluor™ influenza NA assay. We also examined amounts of identified phytochemicals and found that active components 5 and 7 were relatively abundant in RVSE, at 44 and 20 mg/g, respectively. We show some evidence that the antiviral effects of RVSE and its components are due to these components. NA sequences among viral strains may provide a more interesting interpretation for anti-influenza activity of components 5 and 7.
In the early 2000s, NA inhibitor-resistant influenza viruses emerged by the mutations in E119, H274, R292, and N294 of NA [44,45]; thus, the research that investigates antiviral efficacy of natural products, including RVSE and components 5 and 7, against these resistant viruses will be of interest.
In summary, we demonstrate that RVSE significantly averted influenza virus infection in MDCK and A549 cells by NI. Further, RVSE treatment decreased mortality in mice exposed to influenza A/PR/8/34 virus and prevents weight loss compared with that in untreated mice. Further, we confirmed that 5-deoxyluteolin and sulfuretin (components 5 and 7) inhibited NA activity notably among the ten components isolated and identified from RVSE. RVS, RVSE, and its components are effective in inhibiting the NA activity of both influenza virus A and B. RVSE and its components may provide good candidates and building blocks for novel anti-influenza drugs. However, additional mechanistic and in vivo studies are required to elucidate in detail the mode of action of active components 5 and 7.

Data Availability
The datasets generated and/or analyzed during the present study are available from the corresponding author on reasonable request. Figure S1: 1 H and 13 C NMR spectrum of compound 1 in methanol-d 4 (600 and 150 MHz). Figure S2: 1 H and 13 C NMR spectrum of compound 2 in methanol-d 4 (600 and 150 MHz). Figure S3: 1 H and 13 C NMR spectrum of compound 3 in methanol-d 4 (600 and 150 MHz). Figure S4: 1 H and 13 C NMR spectrum of compound 4 in methanol-d 4 (600 and 150 MHz). Figure S5: 1 H and 13 C NMR spectrum of compound 5 in methanol-d 4 (600 and 150 MHz). Figure  S6: 1 H and 13 C NMR spectrum of compound 6 in methanol-d 4 (600 and 150 MHz). Figure S7: 1 H and 13 C NMR spectrum of compound 7 in methanol-d 4 (600 and 150 MHz). Figure S8: 1 H and 13 C NMR spectrum of compound 8 in methanol-d 4 (600 and 150 MHz). Figure S9: 1 H and 13 C NMR spectrum of compound 9 in methanol-d 4 (600 and 150 MHz). Figure S10