Journal of Ethnopharmacology 79 (2002) 331 334 www.elsevier.

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Effects of propolis from Brazil and Bulgaria on fungicidal activity of macrophages against Paracoccidioides brasiliensis
J.M. Murad a, S.A. Calvi a, A.M.V.C. Soares a, V. Bankova b, J.M. Sforcin a,*
b

Department of Microbiology and Immunology, Biosciences Institute, IB-UNESP, 18618 -000 Botucatu, SP, Brazil Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Soa, Bulgaria Received 1 June 2001; received in revised form 1 November 2001; accepted 14 November 2001

Abstract Paracoccidioidomycosis is the most important systemic mycosis in Latin America. Its etiological agent, Paracoccidoides brasiliensis, affects individuals living in endemic areas through inhalation of airborne conidia or mycelial fragments. The disease may affect different organs and systems, with multiple clinical features, with cell-mediated immunity playing a signicant role in host defence. Peritoneal macrophages from BALB/c mice were stimulated with Brazilian or Bulgarian propolis and subsequently challenged with P. brasiliensis. Data suggest an increase in the fungicidal activity of macrophages by propolis stimulation, independently from its geographic origin. 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Propolis; Macrophage; Yeast; Paracoccidioides brasiliensis; Brazil; Bulgaria

1. Introduction Propolis is a sticky dark-coloured material that honeybees collect from plants, showing a very complex chemical composition (Bankova et al., 1999). It has been used in folk medicine since ancient times, due to its many biological properties, such as antimicrobial, antiinammatory, antioxidant, immunomodulatory activities, among others (Marcucci, 1995). In the temperate zone of the northern hemisphere, bees produce propolis from late spring until early autumn (about 4 months), collecting the material mainly from the bud exudate of poplar trees (Bankova et al., 1998b). In Brazil, propolis production proceeds throughout the entire year and seasonal variations in its chemical composition are not signicant and are predominantly quantitative (Boudourova-Krasteva et al., 1997; Bankova et al., 1998a; Sforcin et al., 2000). Baccharis dracunculifolia DC. was shown to be the main propolis source in Botucatu, Sao Paulo State, followed by Eucalyptus citriodora Hook. and Araucaria angustifolia (Bert.) O. Kuntze (Bankova et al., 1999).
* Corresponding author. E-mail address: sforcin@ibb.unesp.br (J.M. Sforcin).

Macrophages are involved in several processes, such as phagocytosis, enzyme liberation, free radical generation as well as mediators of inammatory processes. Scheller et al. (1988) suggested that the immunostimulant activity of propolis may be associated with macrophage activation and enhancement of macrophage phagocytic capacity. Tatefuji et al. (1996) investigated the effect of six propolis compounds on macrophage mobility and spreading. Paracoccidioidomycosis is a human systemic mycosis caused by the thermally dimorphic fungus Paracoccidioides brasiliensis (Lacaz, 1956). It is one of the most prevalently serious mycoses in Latin America and the great majority of the infected persons develop an asymptomatic pulmonary infection, although some individuals present clinical manifestations, leading to the dissemination of the disease. Clinical and experimental data indicate that cell-mediated immunity plays a signicant role in host defence, whereas high levels of specic antibodies are associated with the most severe form of this disease. Gamma-interferon (IFN-g) has been shown to play a protective role and is one major mediator of resistance against P. brasiliensis infection in mice (Cano et al., 1998). Experimental models have shown the role of macrophages in the mechanisms of resistance against this fungus.

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J.M. Murad et al. / Journal of Ethnopharmacology 79 (2002) 331334

The goal of the present research was to study the effects of various concentrations of propolis on the fungicidal activity of macrophages against P. brasiliensis. We also compared the effects of Brazilian propolis with those produced by a propolis sample from Bulgaria, using the same assay.

removed and macrophage monolayers were reincubated at 37 C for 24 h with Brazilian or Bulgarian propolis (5, 10 and 20 mg per well) or with IFN-g (100 U/ml).

2.4. Fungicidal acti6ity of macrophages against P. brasiliensis

After 24 h of incubation, supernatants were removed and macrophages were challenged with 4 104 fungal cells (at a ratio of 1:50 of fungus:macrophages) and 10% mice fresh serum. After co-culture for 4 h (experimental cultures), cells were harvested with distilled water to lyse the macrophages. Each culture and well washings were contained in 2 ml of distilled water. In order to determine fungicidal activity, the number of colony-forming units (CFU) of P. brasiliensis was determined by plating 0.1 ml of P. brasiliensis culture on agar plates containing Brain Heart Infusion Agar (BHI) supplemented with 4% horse serum and 5% Pb 192 culture ltrate, the latter constituting the source of growth-promoting factor (Singer-Vermes et al., 1992). A control culture containing only 0.1 ml of yeast cells of P. brasiliensis (4 104 viable units per ml) was submitted to the same procedures used for the experimental cultures. Plates were incubated at 37 C for 10 14 days in sealed plastic bags to prevent drying. At the end of this period, the number of CFU in each plate was counted. The percentage of fungicidal activity was determined using the formula: fungicidal activity= 1

2. Methodology

2.1. Propolis samples

Propolis was produced by africanized honeybees (Apis mellifera L.) in the Beekeeping Section of the School of Veterinary Medicine and Animal Husbandry of Botucatu, UNESP. Samples were obtained from plastic nets and were subsequently frozen to promote propolis removal (Toth, 1985). Propolis samples were ground and extracted (30 g of propolis, completing the volume to 100 ml with 70% ethanol) in the absence of bright light, at room temperature, with moderate shaking. After a week, extracts were ltered and diluted in distilled water (Sforcin et al., 1995).

2.2. Paracoccidioides brasiliensis

Yeast cells of P. brasiliensis 18 were maintained by weekly subculturing in a semisolid Fava Nettos culture medium at 35 C and used at the 7th day of culture. The fungal cells were washed in phosphate-buffered saline (PBS; pH 7.2), counted in a hemocytometer and the concentration was adjusted to 4 104 cells per ml. Viability of fungal suspension was determined on a phase contrast microscope. Bright cells were counted as viable, while dark ones as non-viable. Suspensions containing more than 90% viable fungi were used.

CFU experimental cultures CFU control

100

2.5. Statistical analysis

Statistical analysis was performed using Graph Pad Software 1993, San Diego, CA, USA. Analysis of Variance (ANOVA) was performed, followed by the multiple comparison test by TukeyKrammer method (Godfrey, 1985).

2.3. Animals and peritoneal macrophages

Male BALB/c mice weighing approximately 25 30 g and aged between 6 and 8 weeks were used. Peritoneal macrophages were obtained by inoculation of 35 ml of cold PBS in the abdominal cavity. After a soft abdominal massage for 30 s, the peritoneal liquid was collected with a Pasteur pipette and put in sterile plastic tubes (Falcon). This procedure was repeated three or four times for each animal and the tubes were centrifuged at 200 g for 10 min. Cells were pooled and resuspended in cell culture medium (RPMI 1640 supplemented with 5% fetal calf serum (FCS), 2 mM L-glutamine, 40 mg/ml gentamycine, 20 mM HEPES, 2.5 10 5 M 2-mercaptoethanol) and cultured in a 96-welled at-bottomed plate (Corning) at a nal concentration of 2105 cells per well. After 2 h at 37 C, non-adherent cells were

3. Results and discussion Propolis has been used in human and veterinary medicine, because of its therapeutical properties. Its antifungal property has also been established (Marcucci, 1995). Thus, this research was performed to evaluate the possible effect of propolis on the fungicidal activity of macrophages against P. brasiliensis. Macrophages were stimulated with Brazilian propolis and results are shown in Table 1. It can be seen from this table that propolis increased the fungicidal activity

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when compared with control cells, but not signicantly. Although this increase was not statistically signicant, this fact has its biological importance and should be taken into account, since propolis was able to activate macrophage and enhance its fungicidal action, but less efciently than IFN-g-cytokyne used as a positive control. In a recent work in our laboratory using human cells, adequate concentrations of tumour necrosis factor (TNF-a) alone or in a synergistic effect with IFN-g signicantly increased the fungicidal activity of these cells. Dimov et al. (1992) reported that the water-soluble derivative of propolis activated macrophages to produce several mediators, such as IL-1 and TNF. Propolis could exert its function by increasing directly the liberation of fungicidal substances by macrophages, such as oxygen and nitrogen metabolites, as well as inducing production of some cytokynes. The process of phagocytosis is complex and involves the binding of the target to the surface of macrophages

and ingestion, which usually triggers the so-called oxidative burst. Ivanovska et al. (1993), investigating the effects of individual propolis constituents complexed with lysine, found that cinnamic acid tends to inhibit H2O2 release by peritoneal macrophages, while caffeic acid induces its production. Orsi et al. (2000) suggested that propolis acts on the hosts non-specic immunity, inducing a discrete elevation in hydrogen peroxide release and producing a mild inhibition of nitric oxide generation. As to the Bulgarian propolis, it also showed a nonsignicant increase in the fungicidal activity of macrophages (Table 1). Fig. 1 shows a comparison between Brazilian and Bulgarian propolis activities. It may be seen that both samples had a modulatory action on macrophages, without signicant differences between them. In the past few years, propolis has become a subject of increasing interest for both commercial and scientic reasons. Its chemical composition varies according to

Table 1 Fungicidal activity of peritoneal macrophages activated with IFN-g (100 U/ml) or with Brazilian or Bulgarian propolis (P) in different concentrations and challenged with P. brasiliensis 18 yeast Control Brazil Bulgaria 32.3 (2.9) 32.3 (2.9) P5 41.0 (4.9) 35.6 (3.4) P10 40.5 (2.3) 38.3 (3.8) P20 39.0 (4.0) 47.0 (3.5) IFN-g 54.3 (2.0) 54.3 (2.0)

Results are means 9standard deviation (S.D.) of three similar assays. Values are % fungicidal activity of macrophages.

Fig. 1. Comparison between Brazilian and Bulgarian propolis with respect to the fungicidal activity of peritoneal macrophages challenged with P. brasiliensis 18 yeast. *Statistically different from IFN-g (PB 0.05).

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J.M. Murad et al. / Journal of Ethnopharmacology 79 (2002) 331334 Cano, L.E., Kashino, S.S., Arruda, C., Andre, D., Xidieh, C.F., Singer-Vermes, L.M., Vaz, C.A.C., Burger, E., Calich, V.L.G., 1998. Protective role of gamma interferon in experimental pulmonary paracoccidioidomycosis. Infection and Immunity 66, 800 806. Dimov, V., Ivanovska, N., Bankova, V., Popov, S., 1992. Immunomodulatory action of propolis: IV. Prophylactic activity against Gram-negative infections and adjuvant effect of the watersoluble derivative. Vaccine 10, 817 823. Godfrey, K., 1985. Statistics in practice. Comparing the means of several groups. New England Journal of Medicine 313, 1450 1456. Ivanovska, N., Stefanova, Z., Valeva, V., Neychev, H., 1993. Immunomodulatory action of propolis:VII. A comparative study on cinnamic and caffeic acid lysine derivatives, Comptes Rendus de L. Academie Bulgare des Sciences 46, 115 117. Lacaz, C.S., 1956. South American Blastomycosis. Anais da Faculdade de Medicina de Sao Paulo, 29, 7 120. Marcucci, M.C., 1995. Propolis: chemical composition, biological properties and therapeutic activity. Apidologie 26, 83 99. Orsi, R.O., Funari, S.R.C., Soares, A.M.V.C., Calvi, S.A., Oliveira, S.L., Sforcin, J.M., Bankova, V., 2000. Immunomodulatory action of propolis on macrophage activation. The Journal of Venomous Animals and Toxins 6, 205 219. Scheller, S., Gazda, G., Pietsz, G., Gabrys, J., Szumlas, J., Eckert, J., Shani, J., 1988. The ability of ethanolic extract of propolis to stimulate plaque formation in immunized mouse spleen cells. Pharmacological Research Communications 20, 323 328. Sforcin, J.M., Novelli, E.L.B., Funari, S.R.C., 1995. Serum biochemical determinations of propolis-treated rats. The Journal of Venomous Animals and Toxins 1, 31 37. Sforcin, J.M., Fernandes, A. Jr, Lopes, C.A.M., Bankova, V., Funari, S.R.C., 2000. Seasonal effect on Brazilian propolis antibacterial activity. Journal of Ethnopharmacology 73, 243 249. Singer-Vermes, L.M., Ciavaglia, M.C., Kashino, S.S., Burger, E., Calich, V.L.G., 1992. The source of the growth-promoting factor (s) affects the plating efciency of Paracoccidioides brasiliensis. Journal of Medical and Veterinary Mycology 30, 261 264. Tatefuji, T., Izumi, N., Ohta, T., Arai, S., Ikeda, M., Kurimoto, M., 1996. Isolation and identication of compounds from Brazilian propolis which enhance macrophage spreading and mobility. Biological and Pharmacological Bulletin 19, 966 970. Toth, G., 1985. Propolis: medicine or fraud? American Bee Journal 125, 337 338.

its geographical origin, which may inuence its biological properties. Depending on the local ora, one may nd some chemical compounds in lower or higher concentrations in the samples, or even their absence. In this work, Brazilian and Bulgarian propolis samples had similar effects, although they were produced in widely separated geographic regions. Contrary to propolis from the temperate zones, where poplars are its sole source, in Brazil there are many more plants that bees could visit as sources of propolis, and depending on the location, its chemical composition can differ. Since mankind has been using propolis since early times, a better understanding of its action in the immune response will provide a scientic basis for a better therapeutic application in human or veterinary medicine whether it is associated or not with conventional treatments.

Acknowledgements Brazilian authors wish to thank Dr Vassya Bankova, Bulgaria, for providing the Bulgarian propolis sample.

References
Bankova, V., Boudourova-Krasteva, G., Popov, S., Sforcin, J.M., Funari, S.R.C., 1998a. Seasonal variations in essential oil from Brazilian propolis. Journal of Essential Oil Research 10, 693 696. Bankova, V., Boudourova-Krasteva, G., Popov, S., Sforcin, J.M., Funari, S.R.C., 1998b. Seasonal variations of the chemical composition of Brazilian propolis. Apidologie 29, 361 367. Bankova, V., Boudourova-Krasteva, G., Sforcin, J.M., Frete, X., Kujumgiev, A., Maimoni-Rodella, R., Popov, S., 1999. Phytochemical evidence for the plant origin of Brazilian propolis from Sao Paulo State. Zeitschrift fur Naturforschung 54c, 401 405. Boudourova-Krasteva, G., Bankova, V., Sforcin, J.M., Nikolova, N., Popov, S., 1997. Phenolics from Brazilian propolis. Zeitschrift fur Naturforschung 52c, 676 679.