Protective Effect of Flavonoids from Ohwia caudata against Influenza a Virus Infection

To identify new potential anti-influenza compounds, we isolated six flavonoids, 2′-hydroxyl yokovanol (1), 2′-hydroxyl neophellamuretin (2), yokovanol (3), swertisin (4), spinosin (5), and 7-methyl-apigenin-6-C-β-glucopyranosyl 2″-O-β-d-xylopyranoside (6) from MeOH extractions of Ohwia caudata. We screened these compounds for antiviral activity using green fluorescent protein (GFP)-expressing H1N1 (A/PR/8/34) influenza A-infected RAW 264.7 cells. Compounds 1 and 3 exhibited significant inhibitory effects against influenza A viral infection in co-treatment conditions. In addition, compounds 1 and 3 reduced viral protein levels, including M1, M2, HA, and neuraminidase (NA), and suppressed neuraminidase (NA) activity in RAW 264.7 cells. These findings demonstrated that 2′-hydroxyl yokovanol and yokovanol, isolated from O. caudate, inhibit influenza A virus by suppressing NA activity. The moderate inhibitory activities of these flavonoids against influenza A virus suggest that they may be developed as novel anti-influenza drugs in the future.


Introduction
According to the World Health Organization, the influenza A virus (IVA) infects about 10% of the population worldwide [1]. Influenza A is an acute, infectious respiratory disease with high mortality and epidemic potential. Currently, vaccines and therapeutic agents, including neuraminidase (NA) inhibitors, proton channel protein (M2) inhibitors, and entry inhibitors, are used to prevent or treat influenza [2].
NA is a viral enzyme that consists of four identical subunits and is localized to the viral membrane. NA plays an important role in the spread of IVA by assisting in the release of virions by cleaving neuraminic acids and glycoprotein linkages. As a consequence, NA inhibition is an attractive target for anti-influenza studies [2].
Amantadine and rimantadine specifically target the influenza A virus through their inhibition of the viral M2 protein, which is a proton channel found only in the influenza A virus [3]. In early 2006, the US Centers for Disease Control and Prevention recommended that the use of amantadine be discontinued, as the number of H3N2 cases with amantadine resistance rapidly increased to 92.3% of all cases in the US [4,5]. Since 2006, amantadine resistance has also been reported throughout the rest of the world. Currently, the M2 inhibitors amantadine and rimantadine are no longer used due to drug resistance [4,5]. In contrast, NA inhibitors have inhibitory effects on both type A and type B influenza viruses and these drugs, including oseltamivir and zanamivir, which are recommended as antiviral agents against influenza infections [3]. However, as with other drugs, NA inhibitors exhibit drug resistance as well as side effects [2]. Therefore, it is essential that anti-influenza A therapeutic agents with minimal side effects and high efficacy be identified. For this purpose, various studies have examined the use of natural materials, including those based on traditional medicines with anti-influenza effects [6].
Ohwia caudata is a shrub belonging to the family Fabaceae, which is formerly placed in the genus Desmodium (as Desmodium caudatum). It has traditionally been used for treating rheumatic backache, diarrhea, icterohepatitis, and colds [7]. In a previous report, the chemical composition of O. caudata was examined and found to include flavonoids, triterpenoids, and alkaloids. Previous studies demonstrated that the flavonoids from O. caudata exhibited free radical scavenging and anti-amyloid beta (Aβ) aggregation activities [8]. However, the potential inhibitory effects of O. caudata on influenza A have not yet been examined.
In this study, we sought to isolate potential antiviral compounds from the leaves and stems of O. caudate. Six flavonoids were isolated and their antiviral activity was investigated using influenza A-infected RAW 264.7 cells. In addition, we identified compounds that affected NA inhibition.
Molecules 2020, 25, x FOR PEER REVIEW 2 of 10 agents with minimal side effects and high efficacy be identified. For this purpose, various studies have examined the use of natural materials, including those based on traditional medicines with antiinfluenza effects [6]. Ohwia caudata is a shrub belonging to the family Fabaceae, which is formerly placed in the genus Desmodium (as Desmodium caudatum). It has traditionally been used for treating rheumatic backache, diarrhea, icterohepatitis, and colds [7]. In a previous report, the chemical composition of O. caudata was examined and found to include flavonoids, triterpenoids, and alkaloids. Previous studies demonstrated that the flavonoids from O. caudata exhibited free radical scavenging and anti-amyloid beta (Aβ) aggregation activities [8]. However, the potential inhibitory effects of O. caudata on influenza A have not yet been examined.
In this study, we sought to isolate potential antiviral compounds from the leaves and stems of O. caudate. Six flavonoids were isolated and their antiviral activity was investigated using influenza A-infected RAW 264.7 cells. In addition, we identified compounds that affected NA inhibition.

Compounds 1 and 3 Inhibit Influenza Vires A (IVA) Infection in RAW 264.7 Cells
We next evaluated flavonoid compounds 1-6 for potential anti-influenza A activity. We first examined the viability of RAW 264.7 cells after treatment with various concentrations of flavonoid compounds 1-6. As shown in Figure 2, flavonoid compounds 1-6 did not show cytotoxicity at 5 to 25 μM. Therefore, we used by concentration at 25 μM on screening. Next, RAW 264.7 cells were cotreated with flavonoid compounds 1-6 and A/PR/8/34-GFP (10 MOI) IVA. Both compounds 1 and 3 reduced green fluorescent protein (GFP) expression in cells in the co-treatment assay (Figures 3 and  S1). However, in pre-treatment and post-treatment assays, flavonoid compounds 1-6 did not show any effects (data not shown). These data indicate that compounds 1 and 3 significantly inhibited influenza A viral activity in a co-treatment assay when compared with that of the vehicle. These results suggest that the effect of compounds 1 and 3 in the co-treatment assay could directly affect the virus or prevent the virus from entering the cells.

Compounds 1 and 3 Inhibit Influenza Vires A (IVA) Infection in RAW 264.7 Cells
We next evaluated flavonoid compounds 1-6 for potential anti-influenza A activity. We first examined the viability of RAW 264.7 cells after treatment with various concentrations of flavonoid compounds 1-6. As shown in Figure 2, flavonoid compounds 1-6 did not show cytotoxicity at 5 to 25 µM. Therefore, we used by concentration at 25 µM on screening. Next, RAW 264.7 cells were co-treated with flavonoid compounds 1-6 and A/PR/8/34-GFP (10 MOI) IVA. Both compounds 1 and 3 reduced green fluorescent protein (GFP) expression in cells in the co-treatment assay ( Figure 3 and Figure S1). However, in pre-treatment and post-treatment assays, flavonoid compounds 1-6 did not show any effects (data not shown). These data indicate that compounds 1 and 3 significantly inhibited influenza A viral activity in a co-treatment assay when compared with that of the vehicle. These results suggest that the effect of compounds 1 and 3 in the co-treatment assay could directly affect the virus or prevent the virus from entering the cells.
We next examined the expression of A/PR/8/34-GFP virus-induced GFP in RAW 264.7 cells co-treated with multiple concentrations of compounds 1 and 3 (12.5 and 25 µM) and IVA (A/PR/8/34-GFP). Compounds 1 and 3 inhibited virus-induced GFP expression in a dose-dependent manner when compared with that of the vehicle ( Figure 4A,B). Consistent with these data, an MTT assay revealed that co-treatment with compounds 1 and 3 suppressed H1N1 virus-induced cell death in RAW 264.7 cells ( Figure 4C). These data demonstrate that compounds 1 and 3 can inhibit A/PR/8/34-GFP virus-induced GFP expression and viral cytopathic effect (CPE) when compared with that of the vehicle.   We next examined the expression of A/PR/8/34-GFP virus-induced GFP in RAW 264.7 cells cotreated with multiple concentrations of compounds 1 and 3 (12.5 and 25 μM) and IVA (A/PR/8/34-GFP). Compounds 1 and 3 inhibited virus-induced GFP expression in a dose-dependent manner when compared with that of the vehicle ( Figure 4A,B). Consistent with these data, an MTT assay revealed that co-treatment with compounds 1 and 3 suppressed H1N1 virus-induced cell death in RAW 264.7 cells ( Figure 4C). These data demonstrate that compounds 1 and 3 can inhibit A/PR/8/34-GFP virus-induced GFP expression and viral cytopathic effect (CPE) when compared with that of the vehicle.

Compounds 1 and 3 Suppress Viral Protein Expression
To determine whether compounds 1 and 3 decreased the expression of viral proteins in IVA-infected RAW 264.7 cells, we performed Western blotting analysis of protein expression. As shown in Figure 5, compounds 1 and 3 suppressed A/PR8/34 viral protein levels (M1, M2, NP, HA, NA, and NS1). In total, 25 µM concentrations of compounds 1 and 3 significantly inhibited NA protein levels by 76.9% and 76.4%, respectively, when compared with the IVA-infected control (vehicle). In addition, we observed a decrease in the expression of NA in a dose-dependent manner in infected RAW 264.7 cells by immunofluorescence analysis (Figure 6). These data suggest that compounds 1 and 3 reduce viral protein levels in IVA-infected RAW 264.7 cells.

Compounds 1 and 3 Suppress Viral Protein Expression
To determine whether compounds 1 and 3 decreased the expression of viral proteins in IVAinfected RAW 264.7 cells, we performed Western blotting analysis of protein expression. As shown in Figure 5, compounds 1 and 3 suppressed A/PR8/34 viral protein levels (M1, M2, NP, HA, NA, and NS1). In total, 25 μM concentrations of compounds 1 and 3 significantly inhibited NA protein levels by 76.9% and 76.4%, respectively, when compared with the IVA-infected control (vehicle). In addition, we observed a decrease in the expression of NA in a dose-dependent manner in infected RAW 264.7 cells by immunofluorescence analysis (Figure 6). These data suggest that compounds 1 and 3 reduce viral protein levels in IVA-infected RAW 264.7 cells.    (B) Western blot band was quantified by using the Image J software. The bar graphs show the mean ± SD of 3 independent experiments (* p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the vehicle (IVA-infected control)).

Compounds 1 and 3 Reduced NA Activity
Next, we investigated the effects of compounds 1 and 3 on NA activity according to previously described materials and methods. Zanamivir, which is an FDA-approved drug for treating influenza, was used as a positive control. The NA activity of H1N1 (P/PR/8/34) was significantly reduced with compounds 1, 3, and zanamivir. The results suggest that compounds 1 and 3 has an inhibitory effect on the influenza A virus by inhibiting the NA of H1N1(P/PR/8/34) in a dose-dependent manner compared with that of the vehicle (Figure 7).

Compounds 1 and 3 Reduced NA Activity
Next, we investigated the effects of compounds 1 and 3 on NA activity according to previously described materials and methods. Zanamivir, which is an FDA-approved drug for treating influenza, was used as a positive control. The NA activity of H1N1 (P/PR/8/34) was significantly reduced with compounds 1, 3, and zanamivir. The results suggest that compounds 1 and 3 has an inhibitory effect on the influenza A virus by inhibiting the NA of H1N1(P/PR/8/34) in a dose-dependent manner compared with that of the vehicle (Figure 7).
Previous reports shown that flavonoids such as kaempferol, quercetin, and naringenin have anti-influenza effect via inhibited NA activity [14]. However, compounds 1 and 3 isolated from O. caudate in this study had not previously been reported to have anti-influenza effects. In this study, we found that compound 1, 2′-hydroxyl yokovanol, and compound 3, yokovanol, inhibited the infection of influenza and reduced the NA activity.
In the structure-activity relationships of six flavonoids (1-6), compounds 1 and 3 showed the inhibitory effect on NA and viral infection. By comparison of other compounds, a 2,2-dimethyl-2H pyran ring located at C-7 and C-8 seems to be a key functional element. Several flavonoids of O. caudate were found to possess anti-bacterial, anti-inflammatory, and anti-pyretic activities. However, to our knowledge, the present study is the first to report anti-influenza activity of the chemical components isolated from O. caudate.

General Information
Optical rotations were determined using a Jasco DIP-370 automatic polarimeter. The nuclear magnetic resonance (NMR) spectra were recorded using a JEOL ECA 600 spectrometer (JEOL Ltd., Tokyo, Japan)( 1 H, 600 MHz, 13 C, 150 MHz), The licence controller qualification (LCQ) advantage trap mass spectrometer (Thermo Finnigan, San Jose, CA, USA) was equipped with an electrospray ionization (ESI) source, and high-resolution electrospray ionization mass spectra (HR-ESI-MS) were Previous reports shown that flavonoids such as kaempferol, quercetin, and naringenin have anti-influenza effect via inhibited NA activity [14]. However, compounds 1 and 3 isolated from O. caudate in this study had not previously been reported to have anti-influenza effects. In this study, Molecules 2020, 25, 4387 7 of 10 we found that compound 1, 2 -hydroxyl yokovanol, and compound 3, yokovanol, inhibited the infection of influenza and reduced the NA activity.
In the structure-activity relationships of six flavonoids (1-6), compounds 1 and 3 showed the inhibitory effect on NA and viral infection. By comparison of other compounds, a 2,2-dimethyl-2H pyran ring located at C-7 and C-8 seems to be a key functional element. Several flavonoids of O. caudate were found to possess anti-bacterial, anti-inflammatory, and anti-pyretic activities. However, to our knowledge, the present study is the first to report anti-influenza activity of the chemical components isolated from O. caudate.

Plant Material
The leaves and stems of O. caudata were collected in Jeju, Korea, in August 2010 and identified by Prof. Young Ho Kim. A voucher specimen (CNU 10107) was deposited at the Herbarium of College of Pharmacy, Chungnam National University, Korea.

Extraction and Isolation
The dried leaves and stems of O. caudata (1.0 kg) were extracted with MeOH under reflux for 9 h (5 L × 3 times) to yield 83.0 g of extract. The extracts were remained for emergency and used for a previous experiment (Li et al. 2014). This extract was suspended in water and partitioned with n-hexane to yield 26.0 g of hexane extract and 55.0 g of water extract. The water extract was partitioned with n-BuOH to yield 10.5 g of n-BuOH extract. Compounds 1 (48.0 mg), 2 (21.0 mg), and 3 (16.0 mg) were isolated from the hexane extract. Compound 4 (55.0 mg), 5 (46.0 mg), and 6 (38.0 mg) were isolated from n-BuOH extract.

Cell Viability MTT Assay
Cells were seeded in 24-well plates for 24 h and then were treated with various concentrations of flavonoid compounds for 24 h. Cell viability was measured using the MTT assay. Cells were treated with 5 mg/mL of MTT solution for 30 min, and then purple formazan was dissolved in DMSO and the absorbance at 540 nm was measured with a microplate reader (Epoch, BioTek, Irvine, CA, USA).

Virus Infection and Antiviral Activity Assay
Raw 264.7 cells were cultured in 24-well plates at a density of 1 × 10 5 cells/well for 18 h. In order to confirm the antiviral effect, three conditions (pre-treatment, co-treatment, and post-treatment) were used for viral infection (10 MOI) and flavonoid compounds 1-6 ( Figure S2). Influenza virus GFP expression was measured under a fluorescence microscope (Nikon, Tokyo, Japan) and the reduction of the viral infection effect was determined by measuring GFP expression using flow cytometry [16].

Immunofluorescence
IVA-infected Raw 264.7 cells were cultured on cover slips and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After washing three times with phosphate-buffered saline (PBS), the fixed cells were permeabilized with 0.1 M glycine for 5 min at room temperature. After three washes with PBS, the cells were incubated with blocking solution (5% BSA in PBS) for 30 min and then with NA antibody (GenoTex, Irvine, CA, USA) overnight at 4 • C for 24 h. Afterward, cells were washed with PBS and then incubated with Alexa568-tagged secondary antibody for 30 min on the rocker. The cells were washed with PBS and Hoechst 33,342 stained for 15 min. After washing, the cover slips were mounted on a slide using mounting media. Cells were visualized with a fluorescence microscope (Lionheart FX automated microscope, BioTek, Irvine, CA, USA).