Tannins from Hamamelis virginiana bark extract: characterization and improvement of the antiviral efficacy against influenza A virus and human papillomavirus

Abstract

Antiviral activity has been demonstrated for different tannin-rich plant extracts. Since tannins of different classes and molecular weights are often found together in plant extracts and may differ in their antiviral activity, we have compared the effect against influenza A virus (IAV) of Hamamelis virginiana L. bark extract, fractions enriched in tannins of different molecular weights and individual tannins of defined structures, including pseudotannins. We demonstrate antiviral activity of the bark extract against different IAV strains, including the recently emerged H7N9, and show for the first time that a tannin-rich extract inhibits human papillomavirus (HPV) type 16 infection. As the best performing antiviral candidate, we identified a highly potent fraction against both IAV and HPV, enriched in high molecular weight condensed tannins by ultrafiltration, a simple, reproducible and easily upscalable method. This ultrafiltration concentrate and the bark extract inhibited early and, to a minor extent, later steps in the IAV life cycle and tannin-dependently inhibited HPV attachment. We observed interesting mechanistic differences between tannin structures: High molecular weight tannin containing extracts and tannic acid (1702 g/mol) inhibited both IAV receptor binding and neuraminidase activity. In contrast, low molecular weight compounds (<500 g/mol) such as gallic acid, epigallocatechin gallate or hamamelitannin inhibited neuraminidase but not hemagglutination. Average molecular weight of the compounds seemed to positively correlate with receptor binding (but not neuraminidase) inhibition. In general, neuraminidase inhibition seemed to contribute little to the antiviral activity. Importantly, antiviral use of the ultrafiltration fraction enriched in high molecular weight condensed tannins and, to a lesser extent, the unfractionated bark extract was preferable over individual isolated compounds. These results are of interest for developing and improving plant-based antivirals.

Conflict of interest statement

Competing Interests: CAJE is an employee of Dr. Willmar Schwabe GmbH & Co. KG., whose company partly funded this study. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Chemical structures of tannins and pseudotannins in Hamamelis virginiana L.

(A) gallic acid, (B) hamamelitannin, (C) pentagalloylglucose, (D) tannic acid represented with 10 galloylation units, (E) monomeric condensed tannins, (F) polymeric condensed tannins . Pseudotannins having low or no protein precipitating activity (and molecular weights <500 g/mol) are shown in italics.

Figure 2. Antiviral activity of Hamamelis bark extract.

(A–B) Selectivity index determination. Fluorescence of an H1N1 reporter virus A/Puerto Rico/8/34-NS116-GFP (MOI 0.4 on A549 cells) after 24 h of treatment with Hamamelis bark (A) or leaf (B) extract, expressed in relative fluorescent units (RFU, closed circles). Cytotoxicity of bark (A) or leaf (B) extract on A549 cells after 24 h as determined by XTT assay (open triangles). Background absorbance at 650 nm has been subtracted from XTT absorbance at 450 nm. A representative of at least two independent experiments is shown. (C–H) Antiviral activity of the bark extract against wild type strains. A549 cells (or A549Slam for measles) were infected in triplicates with an MOI of 0.1 for H1N1 A/Puerto Rico/8/34 (C), pandemic H1N1 A/Lux/46/2009 (D), seasonal H3N2 A/Lux/01/2005 (E), H7N9 A/Anhui/01/2013 (F), a MOI of 0.01 for measles Schwarz strain/Rimevax (G) or a MOI of 0.05 for adenovirus type 5 ATCC reference strain (H) in presence of Hamamelis bark serial dilutions. TCID50 was determined after 24 h (C–F, H closed circles), 48 h (C, open circles, G), or 72 h (C, closed triangles). (I) Activity of bark extract against 1000 HPV 16 pseudovirions per HaCaT cell in triplicates. Luminescence was read after 24 h and expressed in percent of untreated controls (closed circles). Cytotoxicity of bark extract on HaCaT cells was determined after 24 h by XTT assay (open triangles) as described for panel 2A. OD, optical density.

Figure 3. Antiviral activity of UF-concentrate, a fraction of the bark extract enriched in high molecular weight tannins.

(A–B) A549 cells were infected in triplicates at a MOI of 0.1 with pandemic H1N1 A/Lux/46/2009 (A) or H1N1 A/Puerto Rico/8/34 (B) and serial dilutions of UF-concentrate were added at the same time. TCID50 was determined at 24 h (A, B, closed circles), 48 h (B, open circles) or 72 h (B, closed triangles) post infection. (C) Activity of bark extract against 1000 HPV 16 pseudovirions per HaCaT cell, done in triplicates. Luminescence was read after 24 h and expressed in percent of untreated control (closed circles). (A, C) Cytotoxicity of UF-concentrate on A549 (A, open triangles) or HaCaT cells (C, open triangles) was determined after 24 h by XTT assay. Background absorbance at 650 nm has been subtracted from XTT absorbance at 450 nm. OD, optical density.

Figure 4. Determination of the active antiviral principle in Hamamelis bark extract.

(A) Extent of tannin depletion by precipitation with hide powder. Tannins were depleted from drug solutions by stirring with hide powder for 1 h at room temperature followed by filtration. Phenolics, the main constituting moieties of tannins, were photometrically quantified before (black bars) and after (grey bars) hide powder treatment by Folin-Ciocalteu’s phenol reagent using a pyrogallol standard curve. Pyrogallol equivalents (PGE) of hide powder treated samples were normalized to PGE of untreated samples, set to 100%. (B–C) Antiviral effect of tannins. A549 cells were infected in triplicates with pandemic H1N1 A/Lux/46/2009 (MOI 0.05) and were left untreated or treated for 24 h with bark extract (B), UF-fractions or isolated (pseudo)tannins (C) which had been (grey bars) or had not been (black bars) treated with hide powder. Titers were determined at 24 h post infection by TCID50. n.d., not detectable or TCID50<1.

Figure 5. Effect on different IAV and HPV life cycle steps.

(A–B) Step of the IAV life cycle affected. A549 cells were infected with pandemic H1N1 (MOI 0.1), and treated with 50 µg/ml of bark extract (A) or 10 µg/ml of UF-concentrate (B) starting 2 h before infection or 0, 2, 4 or 6 h after infection. TCID50s were determined 24 h post infection. (C) Effect on HPV attachment. HaCaT cells preincubated for 1 h with the original (black bars) or tannin-free (grey bars) extracts or DMSO and infected for 15 min with 500 HPV pseudovirions per cell were washed five times and collected in SDS sample buffer for Western blotting. Cell-bound HPV16 particles were stained with anti-L1 antibody and relative band intensities to the β-actin band were quantified densitometrically. (D) Effect on HPV capsid disassembly. HaCaT cells were treated with 20 µg/ml of the extracts for 1 h before HPV 16 pseudovirion infection for 7 h followed by fixation and staining with mouse anti-L1-7 antibody. L1-7 recognizes an epitope located inside of the pseudovirion capsid accessible after uncoating. Fluorescence of L1-7-positive pixels was normalized to the cell nucleus signal (Hoechst staining) and expressed as % of untreated. n.d., not detectable or TCID50<1. * significant difference (p<0.05) as compared to “No treatment”.

Figure 6. Effect of virus or cell preincubation with Hamamelis extracts or individual compounds.

(A–B) Preincubation of pandemic H1N1 A/Lux/46/2009 for 2 h with virus growth medium (“no treatment”) or bark extract/UF-fractions (A) or individual compounds (B) before infection of A549 cells (MOI 0.1) and titration 24 h post infection (p.i.). (C–D) Preincubation of A549 cells for 2 h with virus growth medium (“no treatment”) or bark extract/UF-fractions (C) or single compounds (D) before three washes with PBS, infection with pandemic H1N1 (MOI 0.1) and titration 24 h p.i. All experiments were done in at least triplicates. * significant difference (p<0.05) as compared to “No treatment”.

Figure 7. Determination of possible cytotoxicity or unspecific host cell receptor inhibition.

(A) Cell metabolic activity after 24 h of incubation of A549 cells with DMSO (no treatment), 2.5 µM of staurosporine, 10 µg/ml of UF-concentrate or 50 µg/ml of the remaining drugs was determined in triplicates using XTT assay. Optical density (OD) was determined at 450 nm after background (650 nm) subtraction and expressed as % of the untreated samples. (B) Caspase 3/7 activity after 24 h of A549 cell incubation with DMSO (no treatment), 2.5 µM of staurosporine, 10 µg/ml of UF-concentrate or 50 µg/ml of the remaining drugs was assayed in at least triplicates using detection of a luminogenic caspase 3/7 cleavage product. (C) A549 cells were infected in triplicates with adenovirus type 5 (MOI of 0.05) and simultaneously treated with UF-concentrate. TCID50 was determined at 24 h post infection. (D) Interference of the drugs with cellular TNF-α signaling. A549 cells were preincubated for 30 or 0 minutes (“Preinc.”+or −, respectively) with 50 µg/ml of bark extract (“Bark”) or UF-concentrate (“UF-c”). Then, 0 or 30 ng/ml TNF-α were and 15 minutes later, total proteins were extracted. IκB-α and the loading control β-actin were detected on a Western blot using specific primary and Cy-5 and Cy-3 labeled secondary antibodies. * significantly elevated caspase expression (p<0.05) as compared to “No treatment”.