3-O-galloylated procyanidins from Rumex acetosa L. inhibit the attachment of influenza A virus

Abstract

Infections by influenza A viruses (IAV) are a major health burden to mankind. The current antiviral arsenal against IAV is limited and novel drugs are urgently required. Medicinal plants are known as an abundant source for bioactive compounds, including antiviral agents. The aim of the present study was to characterize the anti-IAV potential of a proanthocyanidin-enriched extract derived from the aerial parts of Rumex acetosa (RA), and to identify active compounds of RA, their mode of action, and structural features conferring anti-IAV activity. In a modified MTT (MTTIAV) assay, RA was shown to inhibit growth of the IAV strain PR8 (H1N1) and a clinical isolate of IAV(H1N1)pdm09 with a half-maximal inhibitory concentration (IC50) of 2.5 µg/mL and 2.2 µg/mL, and a selectivity index (SI) (half-maximal cytotoxic concentration (CC50)/IC50)) of 32 and 36, respectively. At RA concentrations>1 µg/mL plaque formation of IAV(H1N1)pdm09 was abrogated. RA was also active against an oseltamivir-resistant isolate of IAV(H1N1)pdm09. TNF-α and EGF-induced signal transduction in A549 cells was not affected by RA. The dimeric proanthocyanidin epicatechin-3-O-gallate-(4β→8)-epicatechin-3'-O-gallate (procyanidin B2-di-gallate) was identified as the main active principle of RA (IC50 approx. 15 µM, SI≥13). RA and procyanidin B2-di-gallate blocked attachment of IAV and interfered with viral penetration at higher concentrations. Galloylation of the procyanidin core structure was shown to be a prerequisite for anti-IAV activity; o-trihydroxylation in the B-ring increased the anti-IAV activity. In silico docking studies indicated that procyanidin B2-di-gallate is able to interact with the receptor binding site of IAV(H1N1)pdm09 hemagglutinin (HA). In conclusion, the proanthocyanidin-enriched extract RA and its main active constituent procyanidin B2-di-gallate protect cells from IAV infection by inhibiting viral entry into the host cell. RA and procyanidin B2-di-gallate appear to be a promising expansion of the currently available anti-influenza agents.

Figure 1. Structural features of flavan-3-ols, oligomeric proanthocyanidins, hydrolyzable tannins, depsides and building blocks of tannins tested for antiviral activity; compounds isolated from Rumex acetosa extract RA are marked by asterisk.

Figure 2. Antiviral and cytotoxic activity of RA on MDCK II cells.

1×10⁴ pfu IAV/well in serum-free medium (antiviral activity, black bars) or serum-free medium (cytotoxic activity, white bars) were incubated with RA at different concentrations indicated for 1 h at 37°C. 48 h after adding the reaction mixtures to 96-well plates, the antiviral activity and cell vitality were determined by MTT_IAV assay and cytotoxicity assay, respectively. The following IAV laboratory strains and isolates were used: (A) laboratory strain PR8 [A/Puerto Rico/8/34], (B) clinical isolate I1 [A(H1N1)pdm09], (C) clinical isolate NRW172 [A(H1N1)pdm09], (D) clinical isolate NRW173 [A(H1N1)pdm09]. Values represent mean ±SD of ≥3 independent experiments. * p<0.05, ** p<0.01 (two-tailed, unpaired Student's t-test). Statistical significance of antiviral activity was calculated for nontoxic concentrations only (A: 1 to 10 µg/mL, B: 1 to 7.5 µg/mL, C: 1 to 25 µg/mL, D: 1 to 10 µg/mL).

Figure 3. Reduction of IAV plaque formation by the Rumex acetosa extract RA (A), epigallocatechin-3-O-gallate (6) (B) and procyanidin B2-digallate (8) (C).

IAV and test compounds were co-incubated for 1 h at 37°C prior to the addition to MDCK II cells. Heparin served as positive control (D). Values (% of plaque reduction) ±SD relate to the respective mock-treated controls ( = 100%). * p<0.05, ** p<0.01 (two-tailed, unpaired Student's t-test).

Figure 4. Effect of Rumex acetosa extract RA (A), epigallocatechin-3-O-gallate (6) (B) and procyanidin B2-digallate (8) (C) on the penetration of IAV.

Effects on the penetration of IAV were determined by a modified plaque reduction assay. Test compounds were added for 30 min. after attachment of IAV to MDCK II cells at 4°C. Values (% of plaque reduction) ±SD relate to the respective mock-treated controls ( = 100%) and represent ≥3 independent experiments. * p<0.05, ** p<0.01 (two-tailed, unpaired Student's t-test).

Figure 5. Effect of RA on the electrophoretic mobility of recombinant soluble HA.

Mock-treated HA (lane 1), RA (10 mg/mL) (lane 2), and HA treated with RA (0.1 to 10 mg/mL) as indicated for 1 h (lanes 3 to 9) were loaded onto 10% bis-tris SDS-PAGE gels and analyzed by Coomassie-staining. The positions of molecular weight marker (mwm) and HA are indicated. HA conglomerates in the gel pockets are marked by arrowhead.

Figure 6. Effect of EGCG (6) (A, B) and procyanidin B2-di-gallate (8) (C, D) on electrophoretic mobility and detection of HA by immunoblotting.

Recombinant soluble HA was either mock-treated (lanes 1), incubated with EGCG (6) (2.18 mM) or procyanidin B2-di-gallate (8) (1.13 mM) dissolved in PBS for the times indicated (lanes 3 to 9) or incubated with PBS only (lanes 10 to 13); EGCG (6) (2.18 mM) and procyanidin B2-di-gallate (8) (1.13 mM) incubated in the absence of HA served as control (lanes 2). Figure 6A, C: Coomassie-stained SDS-PAGE. Figure 6B, D: Detection of HA by immunoblot using a penta-His-specific monoclonal antibody. The expected position of monomeric (approx. 75 kDa) and dimeric HA (approx. 150 kDa) is indicated. Required parameters are missing or incorrect.

Figure 7. Protein-ligand docking of epicatechin-3-O-gallate-(4β→8)-epicatechin-3′-O-gallate (8) into the sialic acid binding cavity of hemagglutinin.

(A) 3D model; protein: green: hydrophobic, purple: polar, red: exposed; ligand: yellow: carbon, light grey: hydrogen, red: oxygen, blue: nitrogen; (B) Interactions of Tyr98 and Trp153; (C) 2D.

Figure 8. Influence of extract RA on TNF-α (A) and EGF (B) induced signal transduction in A549 cells.

Lanes 1 and 2 represent cells preincubated for 1 h with medium, lanes 3 and 4 with RA (100 µg/mL). (A) Stimulation of cells with TNF-α (20 ng/mL, 30 min.) (lane 2, 4), and detection of phosphorylated NF-κB; loading control β-actin; (B) stimulation of cells with EGF (30 ng/mL, 10 min.) (lane 2, 4), and detection of phosphorylated ERK1/2; loading control α-tubulin.