Characterization and mechanisms of anti-influenza virus metabolites isolated from the Vietnamese medicinal plant Polygonum chinense

Background Polygonum chinense Linn. is a common medicinal plant in Southeast Asia and has been used in traditional medicine in Vietnam. The plant contains phytochemicals with various biological properties; however, its antiviral effect has not yet been demonstrated. This study was aimed to evaluate the anti-influenza virus activity of crude extracts of P. chinense, to characterize antiviral metabolites therefrom and to investigate their mechanisms of antiviral action. Methods The methanol (MeOH) extract and organic solvent layers of P. chinense were prepared by extraction and partition with relevant solvents. The ethyl acetate (EtOAc) layer showing antiviral activity was chromatographed repeatedly on SiO2 and Sephadex LH-20 columns to give eight pure metabolites. Their chemical structures were determined by NMR and MS spectral data. Anti-influenza virus activity of the eight metabolites against virus strains A/Puerto Rico/8/34 (H1N1, PR8), A/Hong Kong/8/68 (H3N2, HK) and B/Lee/40 (Lee) was evaluated on the basis of cytopathic effect (CPE) and plaque inhibition assays. Time-of-addition, confocal microscopy and neuraminidase inhibition assay were performed for mode-of-action studies of active ingredients. Results The MeOH extract of P. chinense showed anti-influenza virus activity with EC50 values ranging from 38.4 to 55.5 μg/mL in a CPE inhibition assay. Among the eight pure metabolites isolated from P. chinense, ellagic acid (PC5), methyl gallate (PC7) and caffeic acid (PC8) significantly inhibited viral replication in a dose-dependent manner in both plaque inhibition and CPE inhibition assays with EC50 values ranging from 14.7 to 81.1 μg/mL and CC50 values higher than 300 μg/mL. Mode-of-action studies suggested that PC5 and PC7 suppress virus entry into or replication in cells, while PC8 targets influenza viral neuraminidase, even oseltamivir-resistant one. Conclusion These results demonstrated that P. chinense and its metabolites possess effective anti-influenza virus activities. The botanical materials of P. chinense could be a promising multitargeted inhibitor of influenza A and B viruses and applied to development of a novel herbal medicine.


Background
Influenza virus is an enveloped virus with negativestrand, eight-segmented RNA genomes, belonging to the Orthomyxoviridae family. Each viral segment is encapsidated by a virus-encoded nucleoprotein (NP), called viral ribonucleoprotein (vRNP) [1]. Influenza virions are pleomorphic, roughly spheroidal and approximately 100 nm in diameter [2]. The viral envelope is distinguished by a lipid bilayer containing three transmembrane proteins-hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2, ion channel) on the outside and matrix protein 1 (M1) beneath the membrane. The virus causes pandemics and annual influenza epidemics. Influenza outbreaks result in morbidity and mortality in the human population and commonly occur during winter, or the rainy season in tropical countries [3,4].
Pharmaceutical ingredients can be classified into two groups: NA inhibitors, such as oseltamivir and zanamivir, and M2 inhibitors, such as amantadine and rimantadine. These have been approved and used to treat and prevent influenza infections. The NA inhibitors are effective against both influenza A and B viruses, while the M2 inhibitors are effective only against influenza A virus [5]. However, long-term use of these drugs is limited by their toxicity and emergence of resistance [6]. Therefore, the development of new, low-toxic anti-influenza viral drugs is required.
Polygonum chinense (P. chinense) Linn. belonging to the family Polygonaceae is a common medicinal plant found in China, India, Japan and Southeast Asian countries. In Vietnam, the plant is widespread nationwide, especially in open forest and highland [7]. This plant has been used in traditional medicine for skin infectious diseases such as eczema and zona, indigestion, gastroenteritis and hepatitis, as well as for healing inflammatory wounds or insect stings and snakebites [7,8]. Several phytochemistry studies of P. chinense have been revealed that the plant contains terpenoids, alkaloids, flavonoids, tannins, steroids and glycosides [8][9][10]. Aqueous leaf extract of P. chinense possesses gastroprotective activity [11]. Its methanol (MeOH) extract showed antimicrobial, antioxidant and cytotoxic activities in vitro [8,12]. Ethanol extract and isolated bioactive substances exerted antidiarrheal effects [13]. However, its antiviral potential has not been investigated. During screening of plant extracts against influenza viruses, we found that the methanol extract of P. chinense exhibited antiviral activity. Therefore, the objectives of this study were to examine the antiviral activities of its crude extracts against influenza virus strains A/Puerto Rico/8/34 (H1N1, PR8), A/Hong Kong/8/68 (H3N2, HK) and B/Lee/40 (Lee), to isolate and identify effective metabolites and to investigate their mechanisms of action.

Structure determination and characterization of the isolated compounds
The chemical structures were determined by spectroscopic methods, including electrospray ionization (ESI)mass spectroscopy (MS) and nuclear magnetic resonance (NMR) spectroscopy, and by comparison of their spectral data with values reported previously [12,16]. The ESI-MS spectra of the purified compounds were recorded by LC-MS (Agilent 1100 Series LC/MSD Trap XCT Plus, Agilent Technologies, Palo Alto, CA). The 1 H and 13 C NMR, COSY, HMQC and HMBC spectra were recorded in deuterated NMR solvents using a Bruker AMX-500 FT-NMR spectrometer (Bruker Analytische Messtechnik Gmbh, Rheinstetten, Germany).

Cells, viruses and antiviral agents
Madin-Darby canine kidney (MDCK) cells and 293 T cells were purchased from ATCC (Rockville, MD) and maintained in minimum essential medium (MEM; Invitrogen, Carlsbad, CA) and in Dulbecco's minimal essential medium (DMEM; Invitrogen), respectively, supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37°C in 5% CO 2 . Influenza viruses PR8, HK and Lee were obtained from ATCC. Influenza A viral strains (PR8 and HK) were amplified by infection of 10-day-old chicken eggs at 37°C for 3 days and influenza B virus (Lee) by infection of MDCK cells under serum-free conditions. Viruses were partially purified from harvested egg allantoic fluid or cell culture supernatant by centrifugation at 3000 rpm for 5 min. Viral titers were determined by HA assay or plaque assay, and stocks were stored at −70°C before use. Antiviral agents, ribavirin (RBV) (Sigma-Aldrich) and oseltamivir carboxylate (OSV-C; US Biological, Swampscott, MA) were used as control compounds. The test extracts and purified compounds, with the exception of the water layer which was dissolved in water, were dissolved in DMSO to 40 mg/mL and stored at −20°C.

Rescue of PR8 virus with the H275Y mutation in NA
DNAs derived from eight-segmented PR8 genomic RNAs were individually cloned into a vector harboring convergent RNA polymerase I and II promoters using a universal primer and genome-specific primers, generating pVP-PB2, −PB1, −PA, −HA, −NP, −NA, −M and -NS [17][18][19]. Their sequences were compared to those in GenBank (accession numbers AB671295 for PB2, KC866596.1 for PB1, KC 866595.1 for PA, AB671289.1 for HA, KC866598.1 for NP, CY033579.1 for NA, EF467824.1 for M and EF467817 for NS, respectively). Using the plasmid pVP-NA, a histidinto-tyrosin mutation at amino acid 275 (H275Y) was introduced with a primer (5′-GAATGCACCTAATTTTTACT ACGAGGAATGTTC-3′: sequences corresponding to the mutant tyrosin in bold) and its complementary one. The QuickChange II Site-Directed Mutagenesis Kit (Stratagene, LA Jolla, CA) were used to generate pVP-NA(H275Y). For production of PR8 virus with the NA mutation H275Y [rgPR8(H275Y)] by a reverse genetics system, 293 T cells were co-trasfected with pVP-NA(H275Y) and the rest seven pVP plasmids [17]. The rescued virus from the culture supernatants was propagated into embryonated chicken eggs and viral titer was determined as mentioned above.

Cytopathic effect (CPE) inhibition assay
Cell viability was measured according to our previous report [20]. In brief, MDCK cells were seeded in 96-well plates and grown until 100% confluence. Cells were mock-infected or infected with influenza virus at a multiplicity of infection (MOI) of 0.001 for 1 h at 35°C under serum-free conditions. Unabsorbed virus was removed by washing with PBS, and threefold serial dilutions of compounds dissolved in MEM with 2 μg/ mL TPCK-trypsin were added to each well. On day 3 postinfection (p.i.), cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich). After reading the absorbance at 540/690 nm, the half maximal cytotoxic concentration (CC 50 ) and the half maximal effective concentration (EC 50 ) were calculated using GraphPad Prism 6 (Graph-Pad Software, La Jolla, CA). The selectivity index (S.I.) is the ratio of CC 50 to EC 50 .

Plaque inhibition assay
MDCK cells seeded in 48-well plates were infected with the influenza viruses at an MOI of 0.001 for 1 h at 33°C (PR8) or 35°C (HK and Lee). Test compounds and reference antiviral compounds were serially diluted in overlay medium [MEM containing 0.5% carboxymethylcellulose (CMC; Sigma) and 2 μg/mL TPCK-trypsin]. After washing with PBS, the cells were treated with the compounds in the overlay medium for 3 days at the temperature used for virus infection. Viral plaques were counted by staining with crystal violet [20].

Time-of-addition
To determine when the purified materials, PC5, PC7 and PC8, express their anti-influenza viral activity, MDCK cells were treated with increasing concentrations (from 4 to 100 μM) of each compound for 2 h before (pre-treatment), during (co-treatment) or after (posttreatment) PR8 infection [40 plaque forming units (PFU) per well in 48-well plates] at 37°C. After washing with PBS, cells were incubated with overlay medium for 3 days at 33°C and stained with crystal violet to count the number of viral plaques.

Confocal microscopy
Confluently grown MDCK cells in 4-well slides were infected with the influenza viruses at an MOI of 1 for 4 h in the presence either of 1 mM PC5, PC7 or PC8, or of 10 μM epigallocatechin gallate (EGCG), which was used as a control to block viral entry [19]. After fixation and permeabilization, cells were blocked with 10% goat serum and 1% bovine serum albumin (BSA; Sigma-Aldrich) in PBS. Viral NP was detected with mouse anti-NP antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and Alex Fluor 488-conjugated goat anti-mouse IgG (Invitrogen). Nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions.

NA inhibition assay
Twenty-five microliters of PR8 or rgPR8(H275Y) (2 × 10 5 PFU/mL) in PBS were mixed with an equal volume of 3-fold serially diluted PC5, PC7, PC8 (from 6 mM to 80 μM) or OSV-C (from 60 to 0.8 nM) in PBS in 96-well plates. After addition of 50 μL 200 μM NAfluor fluorescent substrate (Applied Biosystems, Foster City, CA), the reaction was incubated at 37°C for 1 h and stopped with 100 μL of stop solution. The fluorescent intensity was measured with excitation at 350 nm and emission at 450 nm.

Statistical analysis
Statistically significant differences were determined using two-tailed Student's t-test. P < 0.05 was considered significant.

Results and discussion
Screening of antiviral activities in extracts from P. chinense The anti-influenza viral activity of MeOH extract and organic solvent layers from P. chinense was evaluated by CPE inhibition assay. Cell viability was measured using MTT to estimate the CC 50 and EC 50 values before and after infection of MDCK cells with PR8, HK and Lee ( Table 1). As expected, OSV-C (an NA inhibitor) and RBV (a viral polymerase inhibitor) efficiently inhibited the replication of all viral strains tested, confirming the reliability of the assay. MeOH extract of P. chinense showed moderate inhibition of influenza viruses: EC 50 values were 55.0 μg/mL for PR8, 38.8 μg/mL for HK and 55.5 μg/mL for Lee. The EtOAc layer derived from the MeOH extract by liquid-liquid partitioning exhibited EC 50 values of 46.9, 23.2 and 50.8 μm/mL, respectively. Moreover, the MeOH extract and EtOAc layer were not cytotoxic to MDCK cells at the highest concentration used, 300.0 μg/mL. The BuOH layer also displayed antiviral efficacy similar to EtOAc layer. However, the aqueous layer showed limited or no activity.

Isolation and identification of substances from P. chinense
Using repeated chromatographic processes, eight substances, PC1 to PC8, were further isolated from the EtOAc layer (Fig. 1). Compound PC1 was purified as yellow crystals from Fr. 2. The ESI-MS (positive) spectrum of PC1 showed a pseudo-molecular ion peak [M + H] + at m/ z 303; this suggested it to have a molecular formula of C 15 H 10 O 7 . The 1D-NMR spectrum of PC1 showed five 1 H-NMR peaks, presenting aromatic proton signals in the down-field region and 15 carbon signals with characteristics of a C6-C3-C6 structure in the 13 C-NMR spectrum. By comparison of the 1D-NMR data of PC1 with those in the literature, PC1 was identified as quercetin [15].
Compounds PC2, PC3, PC5 and PC7 were determined from their 13 C-NMR spectra as derivatives of gallic acid with six aromatic carbon signals and a carbonyl group. The molecular weight and formula of PC3 were determined to be 170 and C 7    . The chemical structure of PC5 was determined by comparison with 1 H-and 13 C-NMR data published previously [12]. Compound PC8 was purified from Fr. 6 as an amorphous solid. Its chemical structure was identical to that of caffeic acid on the basis of 1 H-and 13 C-NMR data analysis and comparison of these data with those in the literature [13]. Compounds PC4 and PC6 were identified by comparing their R f values with those of authentic compounds that were spotted and developed on the same TLC. PC4 was identified as quercitrin (quercetin 3rhamnoside) and contained a minor gallic acid impurity. Therefore, this compound was not tested in our bioassays. The 1 H-and 13 C-NMR spectra of PC6 were compared with those reported previously [14]. Among eight compounds isolated from P. chinense, PC6 was identified as βsitosterol, while the others were related to flavonoid and phenolic metabolites found commonly in the Polygonaceae family [10,13,15]. All PC compounds, except for PC4, have been reported to possess antifungal, antioxidant and other pharmaceutical properties [13,16]. In this study, it was aimed to investigate anti-influenza virus activities of the P. chinense constituents systematically and their mode-of-action.

Anti-influenza virus activity of isolated compounds
To study the in vitro anti-influenza virus effects of the isolated substances, CPE inhibition assay was performed (Table 2 and Fig. 2). As reported in previous   [21,22], PC1 showed anti-influenza A virus activity with an EC 50 of 12.6 μg/mL and 13.1 μg/mL for PR8 and HK, respectively, without cytotoxicity at 300 μg/mL (Table 2). This result was in agreement with the report by Wu et al. [22], in which quercetin revealed broad-spectrum inhibitory activity against influenza A viruses such as PR8, A/FM-1/47/1 (H1N1) and A/Aichi/2/68 (H3N2) by interaction with the viral HA2 subunit. However, in our study, the compound induced overgrowth of uninfected MDCK cells, resulting in 150-160% increases in cell viability at 100 and 300 μg/mL (Fig. 2). This undesirably increased optical density was likely not due to the colorimetric properties of PC1, as it was not detected in MTT-based viability tests of other cells, such as Vero (African green monkey kidney cell line), MT4 (human T-cell line) or HuT 78 (human T-cell lymphoma cell line), in the presence of PC1 (data not shown). It was expected that PC1 non-specifically stimulated cell viability of MDCK cells. PC6 displayed marginal or no anti-influenza activity and cytotoxicity to MDCK cells, with a CC 50 value of 300 μg/mL (Table 2 and Fig. 2). Accordingly, PC1 and PC6 were excluded from further antiviral assays. Compounds PC2, PC3, PC5 and PC7 are gallic acid and its derivatives (Fig. 1), all of which, except for PC2, exhibited activity against influenza A and B strains ( Table 2). In terms of selectivity, PC7 showed the most desirable antiviral profile with EC 50 values of 18.1, 17.1 and 19.2 μg/mL against PR8, HK and Lee, respectively. However, it also contained latent cytotoxicity, reducing cell viability by 49% at the maximum concentration, 300 μg/mL (Fig. 2). The inhibition of PR8 by PC7 was Fig. 2 Dose response curves of PC1 to PC3 and PC5 to PC8 isolated from P. chinense against PR8, HK and Lee and the MDCK host cell line. Cell monolayers were mock-infected or infected with influenza A and B viruses at a MOI of 0.001 for 1 h. After washing with PBS, compounds serially diluted in MEM with 2 μg/mL TPCK-trypsin were added to the wells and incubated for 3 days. Cell viability was measured by MTT assay. RBV was used as a positive control. The values are means ± standard deviation (SD) from three replicates described in a previous report [23]. Nevertheless, it is noteworthy that this may be the first report of the antiinfluenza activity of methyl gallate (PC7) against additional strains A/H3N2 and B. Consistent with other researchers' observation [21,24], we found that gallic acid (PC3) and ellagic acid (PC5) are active against influenza viruses, even though PC3 showed cytotoxicity with CC 50 of 111.1 μg/mL ( Table 2). The anti-influenza viral activity of gallic acid and its derivatives as well as quercetin could be associated with their planar structure and cell membrane permeability, as described previously [21]. The last compound, PC8 showed desirable antiviral activity with EC 50 values ranging from 14.7 to 37.8 μg/mL without cytotoxicity or an abnormally increase in cell viability at the maximum concentration treated (300.0 μg/mL) ( Table 2 and Fig. 2).
The antiviral activity of the three most effective compounds, PC5, PC7 and PC8, was compared by plaque inhibition assay, a standard method for measuring the activity of viral inhibitors. Consistent with results from the CPE assay (Table 2), they inhibited plaque formation of influenza A and B viruses in a dose-dependent manner (Fig. 3). Our findings suggest that among the three compounds, PC8 has the greatest antiviral efficacy and lowest cytotoxicity. Even though PC5 suppressed plaque formation significantly at all concentrations tested (P < 0.05), it was less potent than PC7 and PC8 (Fig. 3a). PC7 showed potent antiviral activity with relatively high S.I. values (Table 2), but it seemed to have marginal cytotoxicity given that crystal violet staining resulted in faint background images at concentrations higher than 33 μg/mL (Fig. 3b). This observation was in agreement with the CPE assay graph (compare Figs. 2  and 3), confirming the reliability of the cell culturebased antiviral screening system. The antiviral activity of caffeic acid (PC8) against influenza A virus, including H1N1 and H3N2 strains, was reported previously [25]. Herein, we evaluated the spectrum of its efficacy also against influenza B virus (Fig. 3c).

Mode-of-action of PC5, PC7 and PC8
Although anti-influenza viral activity of ellagic acid (PC5), methyl gallate (PC7) and caffeic acid (PC8) in vitro or in vivo was presented in several previous reports [23,25,26], their mechanisms of action have not yet been elucidated clearly except for PC8, that targets viral NA protein [27]. To investigate their effects on virus entry and early replication in cells, time-of-addition experiment was performed (Fig. 4). Treatment of PC5 for 2 h before (pretreatment) or after (post-treatment) reduced viral plaque titers, while plaque reduction was more distinct when it was treated during virus infection (co-treatment) (Fig. 4a). It means that PC5 could affect both viral and cellular factors working at early steps of the virus life cycle and potentially interfere with their interaction. PC7 also showed the most significant antiviral activity when it was co-treated, resulting in about 35% reduction (Fig. 4b). It marginally reduced the viral infectivity by 15% at the maximum concentration (100 μM) in the post-treatment experiment, while no inhibition when treated before virus infection. It is expected that PC7 might regulate a host factor essential for influenza virus entry or early replication. PC8 resulted in no significant antiviral activity when treated before or after virus infection, but its co-treatment barely showed the efficacy at the maximum concentration (Fig. 4c). It can be suggested that PC8 is partially involved in suppression of the influenza virus entry step. To ensure that PC5 and PC7 suppress influenza virus entry or early replication, we analyzed viral NP protein production in cells by confocal microscopy (Fig. 5). Here, the 4-h incubation of influenza A virus PR8 was optimized for nuclear localization of NP. The result clearly showed that both PC5 and PC7 inhibit the early steps similar to EGCG, which is a virus entry blocker [20]. As expected, PC8 did not affect NP signal intensity or its localization within 4 h after virus infection.
To further examine whether PC8, known as an NA inhibitor [27], targets not only wild-type NA protein but also its OSV-C-resistant one, fluorescence NA inhibition assay was performed using live influenza viruses, PR8 and rgPR8(H275Y) (Fig. 6). Although NA inhibitory effect of PC8 against PR8 was less potent than that of OSV-C, it showed consistent activity against rgPR8(H275Y), that is a reverse genetically rescued virus resistant to OSV-C (compare panels A and B in Fig. 6). It suggested that PC8 targets influenza virus NA protein as OSV-C does but in a different binding manner. Moreover, previous reports proposed that the NA activity is involved in influenza virus entry as well as release of progeny virus [28]. Thus, it can be explained the reason for marginal inhibition of the initial stage of PR8 infection by the NA inhibitor, PC8 (Fig. 4c).

Conclusion
The present study is the first report on anti-influenza virus activity of P. chinense and identification of the active components. MeOH extract, EtOAc and BuOH layers of the plant were active against influenza viruses of A/H1N1, A/H3N2 and B. Among constituents isolated from the EtOAc layer, ellagic acid, methyl gallate and caffeic acid exhibited considerable inhibitory activity against the pathogens. Based on the mode-of-action studies, ellagic acid and methyl gallate suppress the early steps of the viral life cycle. In contrast caffeic acid targets the NA protein, comparably suppressing both wild-type and OSV-C-resistant influenza viruses. Our study suggests that the multi-functional botanical materials of P. chinense could be promising inhibitors of influenza A and B viruses and applied to development of a novel herbal medicine.