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The total carbohydrate contents of Fractions GQP-1, GQP-2 and GQP-3 were determined by HLPC to be 93.2%, 95.4% and 92.7% respectively. GQP2 was the fraction selected for the study of antiviral activity.
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The cytotoxicity of GQP, in contrast to that of ribavirin, increased linearly in a dose-dependent manner as its concentration increased from 1.95 μg/mL to 1000 μg/mL (Fig. 1). For GQP, the concentration required to achieve 50% cytotoxicity (CC50), was greater than the highest concentration tested (> 1000 μg/mL), whereas the corresponding value for ribavirin was considerably lower -below 31.2 μg/mL. These results demonstrated that GQP was less cytotoxic than ribavirin. The highest concentration of GQP used in this experiment was 31.2 μg/mL, whereas the concentration of ribavirin used was 15.6 μg/mL.
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GQP showed an appreciable direct inactivation effect on EV71, as indicated by the cell survival rate determined over a range of GQP concentrations. With concentrations of GQP of 3.9, 7.8, 15.6 and 31.2 μg/mL, cell survival rates of 43.4%, 51.0%, 58.1% and 65.8%, respectively, were observed; by comparison, with 15.6 μg/mL ribavirin, the cell survival rate was 63.9% and this was not significantly different from the value with 31.2 μg/mL GQP (P > 0.01). These values were significantly greater than the value obtained for cells treated with EV71 alone (28.6%) (P < 0.01).
The values for the inhibitory rate calculated for GQP at these concentrations were, respectively, 20.8%, 31.3%, 41.3% and 52.1%. The three lowest concentrations of GQP gave values for the inhibitory rate which were lower than that (49.5%) obtained with 15.6 μg/mL ribavirin (P < 0.05 for 15.6 μg/mL GQP, P < 0.01 for 3.9 and 7.8 μg/mL GQP; see Table 1 and Fig. 2C).
Groups Concentration (μg/mL) Optical density Cell survival rate Inhibitory rate GQP 31.2 0.518±0.040 65.8★★ 52.1 15.6 0.457±0.041 58.1★★▲ 41.3▲ 7.8 0.401±0.028 51.0★★▲▲ 31.3▲▲ 3.9 0.341±0.029 43.4★★▲▲ 20.8▲▲ Ribavirin 15.6 0.503±0.035 63.9★★ 49.5 Normal cell - 0.787±0.041 100★★ - Virus control - 0.225±0.012 28.6 0 GQP or ribavirin was added to Vero cells at the time of EV71 infection (t = 0) except for “Normal cell” and “Virus control” and then incubated at 37 °C in 5% CO2 incubator. When the cytopathic effect (CPE) in the ‘Virus control’ well had reached +++ (when CPE was evident in 50%-75% of the Vero cells) and the cells of the ‘Normal cell’ group were normal, the MTT assay was used to determine.A490 nm . Inhibitory rate (%) = (Aagent - AVirus control ) / (Acell control - AVirus control) ×100%. x±SD, n=3. ★★P < 0.01, compared to “Virus control”; ▲▲P < 0.01, compared to “Ribavirin”; ▲P < 0.05, compared to “Ribavirin”. Table 1. Direct inactivation effect of GQP upon EV71 (n = 3x±SD)
Figure 2. Effects of GQP and ribavirin upon CPE in EV71-infected Vero cells (magnified 100×). A: Normal Vero cells; B: EV71 control; C: GQP added at the point of infection (t =0); D: Prevention of viral absorption by GQP (GQP added 2 h prior to infection; t = -2); E: Inhibition of viral proliferation by GQP (GQP added 2 h post infection; t = +2); F: Inhibition of viral proliferation by ribavirin (ribavirin added 2 h post infection; t = +2). The concentration of GQP and ribavirin were 15.6 μg/mL.
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When cells were pre-incubated with GQP prior to exposure to EV71, cell survival rates were substantially increased, compared to survival rates for controls without GQP. Thus, cell survival rates following pre-incubation with GQP at concentrations of 3.9, 7.8, 15.6 and 31.2 μg/mL were 63.8%, 75.3%, 85.8%, 94.2%, respectively -significantly higher than the cell survival rate (30.6%) for cells exposed to EV71 without pre-incubation with GQP (P < 0.01). The survival rate for each concentration of GQP was also significantly higher than that observed with 15.6 μg/mL ribavirin (P < 0.01). The virus inhibition rates with all concentrations of GQP were also significantly greater than the virus inhibition rate (26.9%) observed with 15.6 μg/mL ribavirin (P < 0.01; see Table 2 and Fig. 2D).
Groups Concentration (μg/mL) Optical density Cell survival rate(%) Inhibitory rate(%) GQP 31.2 0.747±0.082 94.2★★▲▲ 91.6▲▲ 15.6 0.681±0.058 85.8★★▲▲ 79.6▲▲ 7.8 0.597±0.043 75.3★★▲▲ 64.4▲▲ 3.9 0.506±0.045 63.8★★▲▲ 47.8▲▲ Ribavirin 15.6 0.391±0.035 49.3★★ 26.9 Normal cell - 0.793±0.041 100★★ - Virus control - 0.243±0.012 30.6 0 GQP or ribavirin was added to Vero cells 2 h prior to viral infection (t = -2) except for “Normal cell” and “Virus control”. Incubation was at 37 ℃ in 5% CO2 incubator. x±SD, n=3. ★★P < 0.01, compared to “Virus control”; ▲▲P < 0.01, compared to “Ribavirin”. Table 2. The preventative effect of GQP upon EV71 adsorption to Vero cells (n=3 x ± SD)
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GQP was also effective in inhibiting the proliferation of EV71 in Vero cells. Thus, cell survival rates with GQP at concentrations of 3.9, 7.8, 15.6 and 31.2 μg/mL were 57.5%, 65.6%, 75.4% and 82.1%, lespectively -significantly higher than the cell survival rate (30.6%) for cells exposed to EV71 without treatment with GQP (P < 0.01). For the three highest concentrations tested, the cell survival rates with GQP were also higher than the value (60.9%) obtained with ribavirin at 15.6 μg/mL (P < 0.05 for 7.8 μg/mL GQP, P < 0.01 for 15.6 and 31.2 μg/mL GQP; see Table 3 and Fig. 2E-F).
Groups Concentration (μg/mL) Optical density Cell survival rate(%) Inhibitory rate(%) GQP 31.2 0.651±0.056 82.1★★▲▲ 74.2▲▲ 15.6 0.598±0.040 75.4★★▲▲ 64.5▲▲ 7.8 0.521±0.046 65.6★★▲ 50.5▲▲ 3.9 0.456±0.029 57.5★★ 38.7▲ Ribavirin 15.6 0.483±0.035 60.9★★ 43.6 Normal cell - 0.793±0.041 100★★ - Virus control - 0.243±0.012 30.6 0 GQP or ribavirin was added to Vero cells 2 h after EV71 infection (t = +2) except for “Normal cell” and “Virus control” and incubated at 37 °C in 5% CO2 incubator. x±SD, n=3. ★★P < 0.01, compared to “Virus control”; ▲▲P < 0.01, compared to “Ribavirin”; ▲P < 0.05, compared to “Ribavirin”. Table 3. The inhibitory effect of GQP on EV71 proliferation in Vero cells (n=3 x ± SD)
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The time of addition assay revealed that GQP at 31.2 mg/mL consistently inhibited virus-induced cell death by more than 90% if it was added 2 h before infection (i.e. t = -2). The degree of inhibition of cell death that was observed when GQP was added at this time was greater than the degree of inhibition that was observed when it was added at the other times tested (i.e. t = 0; t = +2), and this effect was statistically significant (P < 0.01); and at t = -2 the effect with GQP was also greater than that observed with ribavirin (Tables 1, 2 and 3). GQP added 2 h after viral infection (t = +2) was more effective than GQP added at the same time as infection (t = 0); addition of GQP at the point of infection with EV71 was therefore the least effective of the three time-points for GQP addition that were tested. In contrast to the three lowest concentrations of GQP, ribavirin displayed significantly stronger (P < 0.05 for 15.6 μg/mL GQP, P < 0.01 for 3.9 and 7.8 μg/mL GQP) virus-inhibition activity when it was added either simultaneously with EV71 (t = 0). The inhibition rate for ribavirin under this condition was 63.9% -higher than the value (49.3%) observed for ribavirin addition 2 h before infection (t = -2).