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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 - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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Antiviral activity of plant extracts against the penetration of H3N1 and H1N1 virus at 60 min.Monolayers of MDCK cells (80% confluent) were chilled at 4°C for an hour and then incubated with 200 TCID50 of H3N1 or H1N1 viruses at 4°C for 3 h. Plant extracts (25 µg/mL in RPMI medium) were then added in triplicate and incubated for 60 minutes at 37°C/5% CO2. Following inactivation and neutralization of unpenetrated virus using acidic and alkaline PBS, respectively, cells were washed with PBS and overlaid with RPMI medium and virus growth medium in equal proportion. Cell viability was evaluated using MTT after three days of incubation at 37°C/5% CO2. Data shown are representative of two independent experiments performed in triplicate. Statistical analysis showed that data were significant with p<0.05 (one way ANOVA).
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pone-0079293-g005: Antiviral activity of plant extracts against the penetration of H3N1 and H1N1 virus at 60 min.Monolayers of MDCK cells (80% confluent) were chilled at 4°C for an hour and then incubated with 200 TCID50 of H3N1 or H1N1 viruses at 4°C for 3 h. Plant extracts (25 µg/mL in RPMI medium) were then added in triplicate and incubated for 60 minutes at 37°C/5% CO2. Following inactivation and neutralization of unpenetrated virus using acidic and alkaline PBS, respectively, cells were washed with PBS and overlaid with RPMI medium and virus growth medium in equal proportion. Cell viability was evaluated using MTT after three days of incubation at 37°C/5% CO2. Data shown are representative of two independent experiments performed in triplicate. Statistical analysis showed that data were significant with p<0.05 (one way ANOVA).

Mentions: As shown in Table 4, all extracts were able to prevent viral penetration, with the exception of extract 29 which was ineffective at all time points and extract 37 which was active only against H3N1 strain. These data support HI results obtained using extracts 8, 41, 42 and 43. Figure 5 shows the effects of 25 µg/mL extracts against H3N1 and H1N1 strains at 60 min. Four plant extracts (8, 30, 31 and 38) demonstrated virus inhibition at all three time points, including the effect of 12.5 and 50 µg/mL of plant extracts at 60 min (data not shown). For three HI extracts (41, 42 and 43), inhibition of virus penetration increased over time as the antiviral activity of the extract at 60 and 120 min was greater than that observed at 30 min against H3N1 strain. It is noteworthy that some extracts lacking HI activity (13/30, 14, 31, 37 and 38) were shown to inhibit virus penetration. Although Zanamivir and Oseltamivir should normally act against virus release and not virus penetration, surprisingly, 50 µg/mL Zanamivir showed 70% viral inhibition of H3N1 after 60 min (data not shown). As expected, Oseltamivir was inactive against both viruses in the assay.


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 - PLoS ONE (2013)

Antiviral activity of plant extracts against the penetration of H3N1 and H1N1 virus at 60 min.Monolayers of MDCK cells (80% confluent) were chilled at 4°C for an hour and then incubated with 200 TCID50 of H3N1 or H1N1 viruses at 4°C for 3 h. Plant extracts (25 µg/mL in RPMI medium) were then added in triplicate and incubated for 60 minutes at 37°C/5% CO2. Following inactivation and neutralization of unpenetrated virus using acidic and alkaline PBS, respectively, cells were washed with PBS and overlaid with RPMI medium and virus growth medium in equal proportion. Cell viability was evaluated using MTT after three days of incubation at 37°C/5% CO2. Data shown are representative of two independent experiments performed in triplicate. Statistical analysis showed that data were significant with p<0.05 (one way ANOVA).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842314&req=5

pone-0079293-g005: Antiviral activity of plant extracts against the penetration of H3N1 and H1N1 virus at 60 min.Monolayers of MDCK cells (80% confluent) were chilled at 4°C for an hour and then incubated with 200 TCID50 of H3N1 or H1N1 viruses at 4°C for 3 h. Plant extracts (25 µg/mL in RPMI medium) were then added in triplicate and incubated for 60 minutes at 37°C/5% CO2. Following inactivation and neutralization of unpenetrated virus using acidic and alkaline PBS, respectively, cells were washed with PBS and overlaid with RPMI medium and virus growth medium in equal proportion. Cell viability was evaluated using MTT after three days of incubation at 37°C/5% CO2. Data shown are representative of two independent experiments performed in triplicate. Statistical analysis showed that data were significant with p<0.05 (one way ANOVA).
Mentions: As shown in Table 4, all extracts were able to prevent viral penetration, with the exception of extract 29 which was ineffective at all time points and extract 37 which was active only against H3N1 strain. These data support HI results obtained using extracts 8, 41, 42 and 43. Figure 5 shows the effects of 25 µg/mL extracts against H3N1 and H1N1 strains at 60 min. Four plant extracts (8, 30, 31 and 38) demonstrated virus inhibition at all three time points, including the effect of 12.5 and 50 µg/mL of plant extracts at 60 min (data not shown). For three HI extracts (41, 42 and 43), inhibition of virus penetration increased over time as the antiviral activity of the extract at 60 and 120 min was greater than that observed at 30 min against H3N1 strain. It is noteworthy that some extracts lacking HI activity (13/30, 14, 31, 37 and 38) were shown to inhibit virus penetration. Although Zanamivir and Oseltamivir should normally act against virus release and not virus penetration, surprisingly, 50 µg/mL Zanamivir showed 70% viral inhibition of H3N1 after 60 min (data not shown). As expected, Oseltamivir was inactive against both viruses in the assay.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

Show MeSH
Related in: MedlinePlus