ANTIBACTERIAL ACTIVITY OF PROPOLIS COLLECTED IN DIFFERENT REGIONS OF BRAZIL

The aim of this study was to investigate the antibacterial activity of propolis samples from Goiás, Paraná and São Paulo States, Brazil, and their flavonoids content. Ethanolic extracts of propolis (EEP) were prepared (30g of propolis in 70% ethanol), and the microorganisms Staphylococcus aureus and Escherichia coli were tested. The methodology employed was agar diffusion using filter paper discs. Ampicillin and tetracycline were used as controls. Antibacterial activity was determined by the reading of inhibition zone diameters (mm) after 24 hours incubation at 37°C. Results demonstrated that EEP inhibited the growth of Staphylococcus aureus but not that of Escherichia coli. Tetracycline and ampicillin showed an efficient action against both bacteria. Flavonoids content was variable, depending on the propolis sample. According to the results, it may be concluded that EEP showed effective action against Gram-positive bacteria, independently on their geographic origin, and a positive correlation between antibacterial activity and flavonoids content.

However, its biological properties may vary according to different plant sources (2).In Brazil, there are many plants that bees could visit as sources of propolis, and depending on the geographic location, its chemical composition may differ.
Based on these observations, the aim of this work was to investigate the antibacterial activity of ethanolic extracts of propolis (EEP) produced in different regions of Brazil against Staphylococcus aureus and Escherichia coli and to determinate the flavonoids content of propolis samples.

Propolis samples
Propolis (twenty-two samples) was collected by Apis mellifera in different regions of Brazil: Goiás, São Paulo and Paraná States (Table 1).Propolis samples were ground and extracted (30g of propolis, completing the volume to 100ml with 70% ethanol) in absence of bright light, at room temperature, and after a week, extracts were filtered (15).

Microorganisms
Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 35218 strains were obtained from the American Type Culture Collection, Rocksville Md., USA.

Microbiological tests
Agar disc diffusion method was employed for the determination of antimicrobial activities of EEP (13).Suspensions of tested microorganisms (0.5 Mac Farland scale) were spread into solid media plates.Filter paper discs (6mm in diameter) were impregnated with 20µl of each EEP sample and with ethanol (control) and the inoculated plates were incubated at 37 º C for 24 hours.Diameters of the inhibition zones were measured in millimeters.All the tests were performed in triplicate.

Statistical Analysis
Results were analyzed using Analysis of variance.The probability of 0.05 was chosen as the significant level (24).Pearson correlation was used in order to verify a possible correlation between EEP and flavonoids content.

RESULTS AND DISCUSSION
Flavonoids content (%) varied from 0.05 (sample 8 -Anhembi, São Paulo State) to 0.63 (sample 15 -Itaporanga, São Paulo State) (Table 2).They also varied according to the geographic region.Massuda (12) verified that propolis inhibited Staphylococcus aureus growth (10 to 14mm inhibition diameters), and Sato (20) obtained diameters varying from 0 to 11mm, suggesting variability in the biological activity of EEP.Differences in propolis biological activity may be related to its botanical origin, reflecting differences in its chemical composition.
With regard to Escherichia coli, EEP did not show any antibacterial activity.
Tetracycline and ampicillin showed antibacterial activity, with inhibition diameters of 31 and 29mm, respectively (Table 2).As a control of propolis solvent, 70% ethanol did not show antibacterial activity on the studied strains (Table 2).These results suggest that antibacterial action of EEP against Staphylococcus aureus was due to propolis constituents.
A positive correlation (p<0.01) between the flavonoids content and the antibacterial activity of EEP against Staphylococcus aureus was observed (Table 2).Flavonoids are reported to be the most important group of compounds with propolis biological activity (2).
We can conclude from this work that EEP have antibacterial activity mainly on Grampositive bacteria, showing a positive correlation with flavonoids content.

Sato ( 20 )
verified moderate antibacterial action of EEP against Escherichia coli (inhibition diameters from 8.0 to 8.8 mm), and Orsi et al. (17) demonstrated an elevated minimal inhibitory concentration of propolis against Salmonella sp, concluding that propolis shows limited action on Gram-negative bacteria.In this work, EEP was effective only against Gram-positive bacteria.