Figure 1.
Cytotoxicity effects of plant extracts.
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 p<0.05 (one way ANOVA).
Table 1.
Medicinal plant extracts from Sarawak demonstrating antiviral activity against H3N1 and H1N1 strains.
Table 2.
Cellular toxicity and inhibitory concentration of anti-influenza extracts against H3N1 and H1N1 strains.
Figure 2.
Inhibitory effects of plant extracts on H3N1 influenza virus.
Cells at 80% confluency were treated with two-fold serial dilutions of plant extract (0.78–100 µg/mL) and 100 TCID50 of H3N1 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 p<0.05 (one way ANOVA).
Figure 3.
Inhibitory effects of plant extracts on H1N1 influenza virus.
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 p<0.05 (one way ANOVA).
Figure 4.
Inhibitory effects of plant extracts on the binding of H3N1 and H1N1 virus to MDCK cells.
Cells at 80% confluency and pre-chilled at 4°C for an hour were infected with 200 TCID50 of H1N1 or H3N1 followed by supplementation with plant extract at 25 µg/mL concentration. After 3 h incubation at 4°C, cells were washed twice with ice-cold PBS and overlaid with RPMI and virus growth medium. Cell viability was evaluated using MTT and viral inhibition percentage calculated relative to virus control wells. Effect of plant extracts on virus binding at a concentration of 25 µg/mL is shown. Representatives of two independent experiments performed in triplicate are shown. Statistical analysis showed that data were significant with p<0.05 (one way ANOVA).
Table 3.
Inhibitory effects of anti-influenza extracts on the binding of H3N1 and H1N1 strains.
Figure 5.
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).
Table 4.
Inhibitory effects of anti-influenza extracts on the penetration of H3N1 and H1N1 strains at 30 and 120 min.
Table 5.
Neuraminidase inhibitory activity of anti-influenza extracts.
Figure 6.
Inhibitory effects of plant extracts on the hemagglutination of H3N1 and H1N1 viral strains.
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.
Figure 7.
Effect of RDE treatment on the antiviral activity of plant extracts.
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 in vitro 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 p<0.05 (one way ANOVA).
Figure 8.
Effect of trypsin treatment on the antiviral activity of plant extracts.
A. Inhibitory effect of plant extracts on the hemagglutination of H3N1 viral strain. HI activities of three extracts (50 µg/mL) treated with trypsin against 4HAU/25 µL of virus are shown. (i) Virus controls containing virus and CRBC and (ii) cell controls receiving CRBC only are shown. Extracts that mediate HI activity without trypsin treatment were included in all plates as positive controls. The experiment was performed in triplicate. B. Antiviral inhibition of HI extracts against H3N1 strain. An in vitro micro-inhibition assay was used to assess the ability of plant extracts to inhibit H3N1 (100 TCID50) influenza virus. Extracts (3.13–100 µg/mL) were either treated with trypsin for 24 hours at 37°C, followed by incubation at 56°C for 60 minutes or subjected to temperature without trypsin. Activity of extracts at 50 µg/mL concentration is shown in the figure. 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).