PLoS One. 2013 Nov 27;8(11):e79293.

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

Rajasekaran D, Palombo EA, Chia Yeo T, Lim Siok Ley D, Lee Tu C, Malherbe F, Grollo L.

Environment and Biotechnology Centre, Faculty of Life and Social sciences, Swinburne University of Technology, Hawthorn VIC, Australia.

 

Abstract

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.

PMID: 24312177

 

Supplement

Influenza continues to be one of the main causes of global morbidity and mortality due to the inadequacies of available treatment strategies. Based on the antigenicity of the two major surface glycoproteins of the virus, neuraminidase (NA) and haemagglutinin (HA), there are 17 HA (H1-H17) and 9 NA (N1-N9) subtypes [1, 2]. Although annual vaccination is the principal approach for the prevention of infections, antiviral drugs also play a vital role in the control of the disease and transmission of the virus.  Two classes of antiviral drugs, the first generation M2 ion channel inhibitors (Amantadine and Rimantadine), and the second generation neuraminidase inhibitors (NAIs) have been approved for the treatment and prophylaxis of influenza. However, the rapid emergence of drug-resistant variants and the ease of  their transmissibility, and the occurrence of central nervous system side effects emphasize the urgent and continual need to develop new antiviral drugs [3]. The search for plant-based antivirals is promising, as several plants have been shown to possess anti-influenza activity [4, 5]. In this study, fifty medicinal plant extracts from the tropical rainforests of Borneo, selected on the basis of traditional knowledge of medicinal plants, were screened in a range of bioassays to determine whether the extracts possess antiviral potential against Mem-Bel (H3N1) and PR8 (H1N1) influenza viral strains. NAIs, Oseltamivir and Zanamivir were included as positive controls in the assays.

Safety is a major requirement for an antiviral agent, and in the search for new drugs it is important to consider possible secondary effects. Hence, the plant extracts were initially screened for cellular toxicity. Most extracts demonstrated minimal cytotoxicity, similar to the NAIs , suggesting the presence of cytoprotective components as reported elsewhere [6]. In our study, this is an indication that these extracts might serve as potential candidates for the development of safe and less toxic drugs. The extracts were then subjected to a high throughput in vitro micro-inhibition screening assay to assess antiviral activities against H3N1 and H1N1 strains. A number of extracts exhibited inhibitory activity against H3N1; however, only eleven extracts consistently reduced viral infectivity by greater than 50% (Table 1). Some of the extracts were duplicates; extracts 13 and 30 were obtained from the same plant, collected in the same location at different times, while extracts 41 and 42 were obtained from different parts of the same plant. All the eleven extracts were also active against H1N1 strain. The antiviral potentials of the eleven active extracts were tested at different times (-1 h,-2 h, 0 h, +1 h, and +2 h) relative to virus inoculation; the extracts inhibited the viruses by more than 50% at all time points tested, with the exception of Extract 14. Moreover, nine plant extracts (8, 13, 14, 29, 30, 31, 38, 41, 42, and 43) were shown to inhibit the binding of virus onto cells and, with the exception of extracts 29 and 37, all other extracts demonstrated anti-influenza activity against the penetration of virus.

All eleven extracts demonstrated NAI activity following the same mode of action, similar to the commercial drugs Oseltamivir and Zanamivir. The influenza virus HA mediates attachment to the sialic acid residues expressed by the glycoproteins and glycolipids of host cells, which is a critical step in the initiation of infection [7]. Similarly, the viral HA binds to sialic acids on the surface of chicken red blood cells (CRBC) resulting in hemagglutination. Thus, we examined the ability of plant extracts to inhibit virus-induced hemagglutination using a hemagglutination inhibition (HI) assay. As shown in Figure 1, four extracts (8, 41, 42 and 43) demonstrated HI activity, a novel mode of action. Extract controls were included to study the direct effects of the extracts on CRBC, in the absence of Mem-Bel and PR8 viruses. In the absence of viruses, all four HI extracts exhibited hemolysis above 25 µg/mL. Some hemolytic concentrations of extract resulted in HI when the virus was included, suggesting that extract components preferentially target the virus rather than the CRBC. The significant effects exhibited by non-HI extracts in viral binding and penetration could be due to the NAI component, which might play a role in inhibiting viral entry.

FIG1

In general, compounds with HI activity may be considered better than NAIs, since NAIs must be administered during the early phase of infection for pronounced effect. In order to suppress the activities of compounds that might contain sialic acid mimics, competing with the RBC receptors for viral HA, the HI extracts were pre-treated with Receptor Destroying Enzyme (RDE). A significant reduction in the antiviral efficacy was observed and the loss of HI activity was evident, suggesting sialic acid-like components may be the present in the plant extracts (Figure 2). A sialylated molecule that can block attachment of the virus to cellular receptors might act to limit the initial stages of virus infection, compared to NA inhibition that is largely believed to act through preventing release of new virions from virus-infected cells. Since the antiviral efficacy of HI extracts was not affected by temperature or trypsin treatment, it can be concluded that the compound(s) of interest may not be proteinaceous.

Figure 2

Apart from inhibiting HA and NA, the plant extracts could also have affected other proteins in the virus including nucleoprotein, RNA polymerase, matrix protein1, nuclear export protein and non-structural protein 1, which all play vital roles in virus replication. Individual extract components could act on multiple targets, operating in a supportive agonistic, synergistic approach, called ‘‘synergistic multi-target effects’’ [8]. The results presented suggest that plants with reported medicinal properties could be a potential source for novel antiviral drugs. The HI plant extracts investigated could serve as promising candidates for the development of third generation anti-influenza drugs, thereby challenging the NA drug resistant viruses in an attempt to safeguard human health and the global economy.

Table 1: Medicinal plant extracts from Sarawak demonstrating antiviral activity against H3N1 and H1N1 strains

TAB1

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