Seasonal effect of brazilian propolis on Candida albicans and Candida tropicalis

SEASONAL EFFECT OF BRAZILIAN PROPOLIS ON Candida albicans AND Candida tropicalis

J. M. SFORCIN¹, A. FERNANDES JÚNIOR¹, C. A. M. LOPES¹, S. R. C. FUNARI¹, V. BANKOVA²

1 Departamento de Microbiologia e Imunologia, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, São Paulo, Brasil; 2 Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113, Sofia, Bulgaria.

ABSTRACT: Recently, propolis has been attracting the attention of researchers due to various biological activities and therapeutic properties. In Brazil, propolis is produced all year long, so there may be some seasonal variations. This work was carried out in order to compare propolis collected during the four seasons by its in vitro antimicrobial activity on yeast pathogens isolated from human infections. Propolis was produced by africanized honeybees in Botucatu, São Paulo State, collected throughout a year and pooled by season. Hydroalcoholic solutions of propolis were prepared with each pool and diluted in agar, using serial concentrations of propolis from each pool. A determination of minimal inhibitory concentration (MIC) was performed. The results show that Candida tropicalis and Candida albicans were susceptible to low concentrations of propolis, the latter showing a higher susceptibility. No differences were seen in relation to seasonal effects in the minimal inhibitory concentration of propolis.

KEY WORDS: propolis, season, Candida, antimicrobial activity.

INTRODUCTION

Propolis, a natural resinous product of honeybees, is known to possess antimicrobial properties (3).

In Brazil, propolis is produced all year long, so there may be some seasonal variations. No data has been found in literature concerning this. The goal of this work was to compare propolis collected during the four seasons by its in vitro antimicrobial activity on yeast pathogens isolated from human infections.

Propolis was collected from the Beekeeping Section of Botucatu School of Veterinary Medicine and Animal Husbandry from 10 colonies of africanized honeybees (Apis mellifera) throughout the year between December 21 1994 and December 21 1995 from plastic nets. At the end of each month, the nets were taken and frozen for propolis removal. Samples were pooled according to the four seasons.

Propolis obtained from each season was stirred and 30% ethanolic propolis extracts (EPE) were prepared (30 g of propolis completing the volume to 100 ml of 96o ethanol). EPE were prepared aseptically and left with moderate shaking at room temperature protected from bright light. A week later, the extracts were filtered and stored in the dark (5).

Strains of yeast pathogens from clinical specimens of patients treated at the University Hospital of the School of Medicine of Botucatu, UNESP, were used as follows: Candida albicans (n=15) and Candida tropicalis (n=15). The microorganisms were identified by current standard microbiological method, as per Kreger-Van Rij (2).

The determination of minimal inhibitory concentration (MIC) by an agar dilution method was performed, following the National Committee of Clinical Laboratory Standards guidelines (4).

Strains of yeasts were grown in Sabouraud Dextrose Agar (Difco) at 35°C/24 h. After incubation, five colonies of each strain were suspended in 5 ml of sterile phosphate buffer solution (PBS) and diluted 1/100 in PBS to yield a final inoculum of approximately 5 x 10⁴ cells/ml.

Serial concentrations (% V/V) of propolis from each season were made on plates containing Sabouraud Dextrose Agar at the following percentages: 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, 9.0, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, and 14.0.

Each antimicrobial test also included a duplicate set of plates containing culture medium plus ethanol in order to obtain 5.0, 10.0, and 15.0% concentrations of solvent as a control.

After the inoculation procedures using a multiloop replicator, the plates were incubated at 35oC for 24 h and MIC endpoints were read as the lowest propolis concentration that resulted in no visible growth or haze on the surface of the culture medium. Populational analyses of data were carried out by calculating the MIC for 50% and 90% of the strains of each group of microorganisms tested (1).

We observed that C. tropicalis and C. albicans are susceptible to low concentration of propolis (Table 1). Of the tested yeasts, C. albicans showed a higher susceptibility than C. tropicalis. Fernandes Jr. et al. also confirmed propolis action on these strains, with values of 0.40-1.80 (% V/V) for C. tropicalis and 0.60-3.00 (% V/V) for C. albicans. These results were slightly lower than ours. Their solutions were prepared to yield a final concentration of 50% (w/v), whereas ours 30% (w/v).

Table 1.
Susceptibility of Candida albicans and Candida tropicalis to ethanolic extracts of propolis obtained during the 4 seasons. Minimal inhibitory concentration (MIC) of propolis at 50% and 90% of the strains.

An inhibitory ethanol effect was seen only in the concentration of 10.0%, it being very distant from that obtained from propolis. It may be concluded that the observed antifungal activity was exclusively due to propolis components. Souza Neto et al. also observed the fungistatic and fungicidal action of propolis on C. albicans, reporting that ethanol 96o GL used as solvent showed no antifungal effect on this microorganism (6).

No differences were seen in relation to the seasonal effect in the minimal inhibitory concentration of propolis. A possible explanation for this new and relevant result may be the fact that it was obtained from the same geographical region, and differences in its chemical composition and biological activities could be found between samples collected in different regions, mainly due to the local flora and perhaps to phenological factors.

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• 01 FERNANDES JR A., SUGIZAKI MF., FOGO ML., FUNARI SRC., LOPES CAM. In vitro activity of propolis against bacterial and yeast pathogens isolated from human infections. J. Venom. Anim. Toxins, 1995, 1, 63-9. [SciELO]
• 02 KREGER-VAN RIJ NJW. The yeasts: a taxonomic study. In KREGER-VAN RIJ NJW. Ed. The yeasts 3.ed. Amsterdam: Elsevier, 1987: 963.
• 03 MARCUCCI MC. Propolis: chemical composition, biological properties and therapeutic activity. Apidologie, 1995, 26, 83-99.
• 04 NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Villanova: 1993 (Approved standard M7-A3. NCCLS, 771).
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• 06 SOUZA NETO BA., SOUZA ET., LILENBAUM W., SANTOS CS., SILVA LCS. Ação da própolis perante Candida albicans Ciênc. Méd., 1989, 8, 39-42.

CORRESPONDENCE TO:

J. M. SFORCIN - Departamento de Microbiologia e Imunologia, Instituto de Biociências, Universidade Estadual Paulista/UNESP, Distrito de Rubião Junior, s/n, CEP 18618-000, Botucatu, São Paulo, Brasil.

Publication Dates

• Publication in this collection
  02 Apr 2001
• Date of issue
  2001