Summary Of Melipona Quadrifasciata
Preparation of the propolis extracts: the propolis sample of the Brazilian native bee species Melipona quadrifasciata was obtained in May, 2013 in the city of Blumenau, SC, Brazil (26 ° 54'21.3 "S 49 ° 04'49.1 "W). In order to obtain a hydroalcoholic crude extract (HCE), 284.3 grams of propolis were pulverized and macerated in 70% ethanol (m m-1), left in a dark chamber for 7 days at room temperature, filtered in vacuum and taken to complete drying in a rotary evaporator with reduced pressure. In order to increase the yield of the extract, this extraction procedure was repeated 3 times on the same sample. 50 g of the HCE were dissolved in water and submitted to liquid-liquid partition with solvents of different polarities (dichloromethane,
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(2013) [17], with minor modifications. Vero cells were cultured in 24-well plates inoculated with the virus (105 PFU mL-1) and treated simultaneously with varying concentrations of the samples (25, 50, 100 and 200 μg mL-1 for AcF and 50, 100, 200 and 290 μg mL-1 for FM45) determined by the cytotoxicity test. DMEM (2x) containing 25 mM MgCl2 and agarose (1.8%) was added to the cells and then incubated at 37°C with 5% CO2. After 72 h, fixation with 20% formaldehyde in PBS (pH 7.3) was done for 24 hours. To count the plaque forming units (PFU), the staining was done with 0.5% violet crystal in 20% ethanol. The percentage of viral inhibition (%VI) was calculated by the following formula according to Nishimura, Toku and Fukuyasu (1977) [18]: %VI = 1 – [(PFU in treated cells/PFU in virus control)] x 100.
Virucidal assay: A virus suspension (105 PFU mL-1) containing varying concentrations of the samples as above was incubated at 37°C in a water bath for 1 hour. Subsequently, the suspension was added to the cells (0.1 mL per well) and incubated again at 37°C for 1 hour, followed by PRA
(2013) [17], with minor modifications. Vero cells were cultured in 24-well plates inoculated with the virus (105 PFU mL-1) and treated simultaneously with varying concentrations of the samples (25, 50, 100 and 200 μg mL-1 for AcF and 50, 100, 200 and 290 μg mL-1 for FM45) determined by the cytotoxicity test. DMEM (2x) containing 25 mM MgCl2 and agarose (1.8%) was added to the cells and then incubated at 37°C with 5% CO2. After 72 h, fixation with 20% formaldehyde in PBS (pH 7.3) was done for 24 hours. To count the plaque forming units (PFU), the staining was done with 0.5% violet crystal in 20% ethanol. The percentage of viral inhibition (%VI) was calculated by the following formula according to Nishimura, Toku and Fukuyasu (1977) [18]: %VI = 1 – [(PFU in treated cells/PFU in virus control)] x 100.
Virucidal assay: A virus suspension (105 PFU mL-1) containing varying concentrations of the samples as above was incubated at 37°C in a water bath for 1 hour. Subsequently, the suspension was added to the cells (0.1 mL per well) and incubated again at 37°C for 1 hour, followed by PRA