Influence of seasonality and production method on the antibacterial activity of propolis

The antibacterial activity of propolis produced throughout the year by different methods of collection (‘intelligent’ collector of propolis ICP; plastic screen PC; conventional scraping CS) on Staphylococcos aureus and Escherichia coli is investigated. Fifteen beehives (five per collector) of Africanized Apis mellifera were used. Monthly produced propolis, with the same collection technique, was mixed for the preparation of the extract. The ethanol extract of propolis (EEP) was prepared at the ratio of 30% (30 g of propolis, completing the volume for 100 mL with ethanol 70%). Two microorganisms, a positive bacterium Gram Staphylococcus aureus and a negative bacterium Gram Escherichia coli, through the methodology of diffusion in agar, were used for the biological activity evaluation of EEP. Results show that propolis presented antibacterial activity, affected by seasonality and by collecting method.

Flavonoids were the principal group of compounds isolated from propolis, but other substances, such as aromatic acids, phenolic compounds, organics acids, minerals, vitamins and amino acids, were also found (SEIDEL et al., 2008;KUROPATNICKI et al., 2013).However, the chemical composition and biological activities of propolis vary and depend on the diversity of plants and geographical locations from which bees collect it (SALATINO et al., 2011).
Propolis production is an inborn trait of honeybees.Several factors, such as seasonality, production method and others, are involved in this process, which must be taken into account when productivity increase is desired (BANKOVA et al., 1998;DAUGSCH et al., 2008;TEIXEIRA et al., 2008).
Propolis has a broad spectrum of biological properties including antifungal, anti-inflammatory, anti-tumoral and antibacterial activities (ARAÚJO et al., 2012;MANNANI et al., 2012;KUROPATNICKI et al., 2013).Several authors have reported that the susceptibility of a range of Gram-positive bacteria to ethanol extracts of propolis may vary according to the site of the propolis collected (GONSALES et al., 2006;MULI et al., 2008).
Current study investigated the microbiological activity of propolis produced by different methods of collection (Intelligent Collector of Propolis, plastic screen and scraping) and in different seasons, on the standard Staphylococcos aureus and Escherichia coli.Africanized A. mellifera were allocated in fifteen Langstroth beehives (five per collector), distributed randomly and managed only for the production of propolis.The collectors comprised plastic screen (PS), 'intelligent' collector of propolis (ICP) and scraping collector (SC).
Extract was prepared by the mixing of the monthly propolis produced from the same technique of collection.The ethanol extract of propolis (EEP) was prepared in the ratio of 30% (30 g of propolis, completing the 100 mL volume, with ethanol 70%).After a week, extracts were filtered and EEP obtained (ORSI et al., 2000).
To determine dry weight (DW; mg mL -1 ) in each sample, 2 mL of EEP were weighed and kept at 105°C during two hours for the evaporation of the volatile phase.Contents were weighed again and DW was obtained by the difference between the initial and final weights.
For the evaluation of the biological activity EEP, two standard microorganisms (American Type Culture Collection), namely, Gram positive (S.aureus -ATCC 25923) and Gram negative (E. coli -ATCC 25922) bacteria were used.The microorganisms had been tested by the methodology of diffusion in agar using paper discs filter.Three antibiotics were used as control: ampicillin, cephalexin and penicillin.A disc containing only alcohol (propolis solvent) was also used as control.
Filter paper discs (8 mm in diameter) saturated with 15 L of each EEP (one extract to each month) were placed on Mueller Hinton agar plates, which were inoculated with test organisms (10 6 UFC mL -1 ), following standard protocol described by the National Committee of Clinical Laboratory Standards (NCCLS, 2005).
The antibacterial activity was determined by reading the formation of the inhibitory halo around the discs after 24 hours incubation, at 37ºC.Each assay was performed in triplicate.
Results were compared by ANOVA, followed by Tukey's test to verify differences between averages, at statistical significance p < 0.05.The results are also evaluated by Correlation of Pearson to verify the existence of relations between the variables analyzed for each extract (ZAR, 1996).

Results
No correlations between climatic variables and propolis production were observed.Seasonality affected the antibacterial activity of EEP against S. aureus.It may be observed that in the winter the ICP and SC methods showed an inhibition halo significantly greater than that in the other seasons.Similarly, the collector also influenced the antibacterial activity against S. aureus, since ICP and SC showed an inhibition halo significantly greater when compared with that of PC (Table 1).
The season affected the antibacterial activity of EEP on E. coli.The propolis produced by ICP in the winter showed an inhibition halo which was significantly greater than that for spring and summer.In the case of SC, the inhibition halo was significantly greater in the winter than in spring, summer and autumn.Collector did not affect the antibacterial activity of EEP on E. coli (Table 2).
There was no statistical significance between the dry weight (DW) of EEP produced in the different seasons and that by collectors, for the two bacteria (Tables 1 and 2).
The antibiotics amoxicillin and cephalexin showed antibacterial activity on S. aureus and E. coli.Penicillin showed antibacterial activity on S. aureus.Ethanol 70% failed to influence the two bacteria (Tables 1 and 2).
Different small letters in the same row and different capital letters in the same column indicate statistical difference between the means (p < 0.05).

Discussion
Several researchers have studied the antibacterial properties of propolis and evidenced its efficient activities against Gram positive and its limited activity against Gram negative bacteria (TOSI et al., 2007;PROBST et al., 2011).Current assay showed that EEP had a greater activity against Gram positive bacteria and less activity against Gram negative bacteria.Above differences might be due to the bacterial wall cell constitution.In fact, gram positive bacteria have a less complex wall cell and lower lipid contents (LOGUERCIO et al., 2005) and thus might have a higher susceptibility to the propolis's chemical constituents.
Other evidences also suggest that propolis composition may be altered as a function of bioactive complex compounds in vegetal sources.Analyzing the possible vegetal sources of the propolis produced in Botucatu, São Paulo State, Brazil, Bankova et al. (1999) observed that the propolis was usually derived from Baccharis dracunculifolia, popularly known as 'vassourinha', Araucaria angustifolia (Paraná pine tree) and Eucalyptus citriodora, even though other secondary vegetal resinous sources may occur around the apiary.Trusheva et al. (2011) reported that propolis produced in different Brazilian states showed activities against S. aureus and verified a positive corelationship between the antibacterial activity and total flavonoid contents.Other researchers underscored a relationship between the flavonoid concentration and the propolis's antibacterial activity (BARBARIC et al., 2011;ISLA et al., 2012).However, Souza et al. ( 2010) researched flavonoid contents in the propolis produced in the same region as current research and failed to observe any difference due to seasonality and production method.They suggested that other substances could have caused the antibacterial activity observed, which may vary according to seasons.
In a study on Brazilian propolis, seasonality affected the antibacterial activity of the propolis produced in the northeastern and southeastern regions of Brazil (CASTRO et al., 2007).The authors suggested that propolis composition may alter as a function of bioactive complex compounds in vegetal sources and may vary over the year (EREMIA;DABIJA, 2007).
Activity mechanism of propolis compounds is complex and may be attributed to the synergy among some of its components.Takaisi-Kikuni and Schilder (1994) verified that the ethanol extract propolis interfered on the growth of Streptococcos agalactie by inhibiting protein synthesis.Koo et al. (2002) suggested that propolis and its components may interfere with the enzymatic activity of some bacteria such as Streptococcos mutans and Streptococcos sangui.A significant synergy may be verified between clinical antibiotics and propolis from two geographical sources against Salmonella typhi (ORSI et al., 2006, 2012a andb).
S. aureus and E. coli were sensitive to the antibiotics amoxicillin, cephalexin and penicillin.Since the alcohol used as solvent for EEP preparation did not present antibacterial activity, the activity against S. aureus was due to compounds present in the propolis, as has been reported by other researchers (FERNANDES JR. et al., 2006;AYALA et al., 2008;MAIA-ARAÚJO et al., 2011).

Conclusion
Propolis showed antibacterial activity against S. aureus and E. coli, influenced by seasonality and the collector method employed.
samples were collected at Nucleus of Education, Sciences and Technology in Rational Beekeeping (NECTAR) in the apiary of Animal Production Department -College of Medicine Veterinary and Animal Sciences -São Paulo State University (UNESP) in Botucatu, São Paulo State, Brazil.

Table 1 .
Antibacterial activity of ethanolic extract of propolis (EEP) and dry weight (DW in mg mL -1 ) produced by 'intelligent' collector of propolis (ICP), scrape (SC) and plastic sreen (PS) in function of the seasonality, Ethanol 70%, Amoxicillin (AM), Cephalexin (CEF) and Penicillin (PEN) against Staphylococcos aureus.The results represent the average and the respective standard deviation.Different small letters in the same row and different capital letters in the same column indicate statistical difference between the means (p < 0.05).

Table 2 .
Antibacterial activity of ethanol extract of propolis (EEP) and dry weight (DW in mg mL -1 ) produced by 'intelligent' collector of propolis (ICP), scrape (SC) and plastic screen (PS) as a function of seasonality, Ethanol 70%, Amoxicillin (AM), Cephalexin (CEF) and Penicillin (PEN) against Escherichia coli.Results represent average and the respective standard deviation.