Evidence of synergistic activity of medicinal plant extracts against neuraminidase inhibitor resistant strains of influenza

The frequent emergence of drug resistant influenza viral strains emphasizes the urgent and continual need to develop new antiviral drugs. Given the encouraging findings of previous studies on antiviral compounds from plant sources, this study focused on medicinal plants from Borneo that were traditionally used to treat symptoms of influenza infection. Following the promising results of earlier investigations, four plant extracts that demonstrated multiple modes of viral inhibition were studied against wild-type and neuraminidase (NA) inhibitor-resistant strains of Types A and B influenza viruses. The extracts exhibited more pronounced activities against the wild-type viruses than the NA inhibitor-resistant strains. Variations in the antiviral potential of the extracts collected from different parts of the same plant were also evidenced in the in vitro micro-inhibition assays. Even though all plant extracts affected NA activity of all viruses, only two extracts demonstrated hemagglutination inhibitory (HI) activities against Type A pandemic H1N1 and Type B viruses. Furthermore, Receptor Destroying Enzyme (RDE) treatments of extracts exhibiting HI activities indicated the presence of sialic acid (SA)-like component(s) that may be responsible for HI activity. Since the antiviral potential of extracts was not completely suppressed by RDE, the possibility of non SA-like antiviral components cannot be ruled out. Therefore, synergistic activity between SA-like and non SA-like components contained in the plant extracts may be responsible for the demonstrated antiviral potential. The results also indicated the presence of non SA-like components that may act against other viral proteins apart from hemagglutinin (HA) and NA

Identification of traditional medicinal plant extracts with novel anti-influenza activity

The emergence of drug resistant variants of the influenza virus has led to a need to identify novel and effective antiviral agents. As an alternative to synthetic drugs, the consolidation of empirical knowledge with ethnopharmacological evidence of medicinal plants offers a novel platform for the development of antiviral drugs. The aim of this study was to identify plant extracts with proven activity against the influenza virus. Extracts of fifty medicinal plants, originating from the tropical rainforests of Borneo used as herbal medicines by traditional healers to treat flu-like symptoms, were tested against the H1N1 and H3N1 subtypes of the virus. In the initial phase, in vitro micro-inhibition assays along with cytotoxicity screening were performed on MDCK cells. Most plant extracts were found to be minimally cytotoxic, indicating that the compounds linked to an ethnomedical framework were relatively innocuous, and eleven crude extracts exhibited viral inhibition against both the strains. All extracts inhibited the enzymatic activity of viral neuraminidase and four extracts were also shown to act through the hemagglutination inhibition (HI) pathway. Moreover, the samples that acted through both HI and neuraminidase inhibition (NI) evidenced more than 90% reduction in virus adsorption and penetration, thereby indicating potent action in the early stages of viral replication. Concurrent studies involving Receptor Destroying Enzyme treatments of HI extracts indicated the presence of sialic acid-like component(s) that could be responsible for hemagglutination inhibition. The manifestation of both modes of viral inhibition in a single extract suggests that there may be a synergistic effect implicating more than one active component. Overall, our results provide substantive support for the use of Borneo traditional plants as promising sources of novel anti-influenza drug candidates. Furthermore, the pathways involving inhibition of hemagglutination could be a solution to the global occurrence of viral strains resistant to neuraminidase drugs

Inhibitory effects of plant extracts on H1N1 influenza virus.

<p>Cells at 80% confluency were treated with two-fold serial dilutions of plant extract (0.78–100 µg/mL) and 100 TCID₅₀ of H1N1 simultaneously. All wells were provided with 100 µL of RPMI medium supplemented with 2 µg/mL trypsin (virus growth medium). Cell viability was evaluated using MTT and viral inhibition percentage calculated relative to virus control wells. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with <i>p</i><0.05 (one way ANOVA).</p

Inhibitory effects of plant extracts on the hemagglutination of H3N1 and H1N1 viral strains.

<p>HI activities of four extracts (0.78–100 µg/mL) against 4HAU/25 µL of virus are shown. The following controls were included on each plate; (i) extract controls with extract and chicken red blood cells (CRBC) only, (ii) virus controls containing virus and CRBC and (iii) cell controls containing only CRBC. Monoclonal antibody against the HA of either H3N1 or H1N1 strains were included as a positive control. The antibody titres for monoclonal antibody against H3N1 and H1N1 were 80 and 200, respectively; 1:8 dilution of either of the two antibodies in PBS were employed in the assay. Data are shown from one of three independent experiments, each performed in triplicate.</p

Inhibitory effects of anti-influenza extracts on the penetration of H3N1 and H1N1 strains at 30 and 120 min.

<p>The activity of extracts against the penetration of influenza virus (200 TCID₅₀) at a concentration of 50 and 25 µg/mL is shown along with standard errors. Threshold concentration of active components that prevent virus penetration appears between 25–50 µg/mL of plant extracts. A negative sign indicates lack of anti-influenza activity.</p

Neuraminidase inhibitory activity of anti-influenza extracts.

<p>The plant extracts' neuraminidase inhibitory activity was measured at concentration ranging between 0.3 to 25 µg/mL whereas the controls Zanamivir and Oseltamivir were assayed at 0.01 to 10,000 nM as recommended by the manufacturer. The optimum virus dilution for the neuraminidase inhibition assay was selected by titration of virus stock in an NA activity assay; 1:8 dilution of either of the virus was selected in the NA activity assay to perform NAI assay.</p

Inhibitory effects of anti-influenza extracts on the binding of H3N1 and H1N1 strains.

<p>The activity of extracts against the binding of influenza virus at a concentration of 50, 12.5 and 0.78 µg/mL is shown along with standard errors. Plant extracts were challenged with 200 TCID₅₀ of either of the two virus strains. A negative sign indicates lack of anti-influenza activity.</p

Effect of RDE treatment on the antiviral activity of plant extracts.

<p>A. Inhibitory effect of plant extracts on the hemagglutination of H3N1 viral strain. HI activities of four extracts (25 µg/mL) treated with RDE against 4HAU/25 µL of virus are shown. (i) Virus controls containing virus and CRBC and (ii) cell controls receiving CRBC only are shown. Corresponding RDE treated monoclonal antibody which acts against the HA of H3N1and extracts that mediate HI activity without RDE treatment were included in all plates as positive controls. The experiment was performed in triplicate. B. Loss of efficacy in antiviral inhibition of HI extracts against H3N1 strain. An <i>in vitro</i> micro-inhibition assay was used to assess the ability of plant extracts to inhibit H3N1 (100 TCID₅₀) influenza virus. Extracts were either treated with RDE as per the manufacturer's instructions or left in their native form without RDE treatment. Data shown are representative of two independent experiments performed in triplicate. Statistical analysis showed that data were significant with <i>p</i><0.05 (one way ANOVA).</p

Cytotoxicity effects of plant extracts.

<p>Following overnight incubation of cells seeded at 4×10³ cells per well into 96-well flat-bottomed microtitre plates, the media were aspirated and overlaid with 100 µL of two-fold serial dilutions of plant extract (0.78–100 µg/mL) with an additional 100 µL of growth medium (supplemented RPMI). After three days incubation, cell viability was evaluated using MTT and percentage cell viability calculated relative to cell control wells. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with <i>p</i><0.05 (one way ANOVA).</p

Medicinal plant extracts from Sarawak demonstrating antiviral activity against H3N1 and H1N1 strains.

*<p>Information derived from; Chai 2006 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079293#pone.0079293-Chai1" target="_blank">[50]</a>, Salleh 2002 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079293#pone.0079293-Salleh1" target="_blank">[51]</a>, Yaacob 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079293#pone.0079293-Yaacob1" target="_blank">[52]</a>, Maji 2010 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079293#pone.0079293-Maji1" target="_blank">[53]</a>, Focho 2010 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079293#pone.0079293-Focho1" target="_blank">[54]</a>.</p