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On-line version ISSN 1678-4936
J. Venom. Anim. Toxins vol.8 no.1 Botucatu 2002
ABSENCE OF SEASONAL EFFECT ON THE IMMUNOMODULATORY ACTION OF BRAZILIAN PROPOLIS ON NATURAL KILLER ACTIVITY.
1 Department of Microbiology and Immunology, Institute of Biosciences, UNESP, 18618-000, Botucatu, SP, Brazil.
ABSTRACT: Propolis, a beehive product widely used in folk medicine as an antiinflammatory agent, has been attracting researchers' attention to scientifically elucidate its biological properties and therapeutic activities. The aim of this paper was to study the possible effect of propolis on natural killer activity, since propolis immunomodulatory action has been suggested, especially on non-specific immunity. Propolis was produced by africanized honeybees (Apis mellifera L.), collected throughout a whole year, and pooled by season. Hydroalcoholic solutions of propolis were prepared with each pool and administered to rats by gavage over three days. Natural killer activity of non-adherent spleen cells was evaluated by the 51Cr-release cytotoxicity assay against Yac-1 target cells. Our results indicated that the natural killer activity was increased in spleen cells from propolis-treated animals. There were no significant differences related to the seasonal effect on the immunomodulatory action of propolis.
KEY WORDS: propolis, season, immunomodulation, natural killer activity, cytotoxic assay.
Propolis is a natural waxy mixture produced by honeybees with several biological properties, such as antibacterial, antiviral, fungicidal, antiprotozoan, antitumoral, antioxidant, and free radical scavenging (13).
The effect of propolis on the immune system has also been investigated by some authors, who showed its ability to activate macrophages (5,16) and stimulate antibody production by SRBC-immunized mice (21). Frankiewicz and Scheller (7), investigating propolis effects on elderly patients, and Scheller et al. (23), evaluating its activity in alveolitis fibroticans cases, observed the immunorestorative property of propolis via granulocyte activation and increasing T cell percentages and immunoglobulin levels, suggesting the therapeutic role this product. Several authors have reported the antitumoral property of propolis. Scheller et al. (22) observed the cytotoxic effect of ethanolic extract of propolis in mice-bearing Ehrlich carcinoma in vivo. Rao et al. (18) showed that caffeic acid ester, an active propolis component, possesses antitumoral activity against human colon carcinogenesis. Guarini et al. (9) reported that caffeic acid causes growth inhibition of human melanoma and glioblastoma multiforme cells. Cytotoxic chemotherapy has significantly improved the treatment of many human malignancies. These, however, are very resistant human tumors, and in the most advanced stages, are virtually incurable. Thus, propolis carcinostatic property against these tumors is extremely relevant. Frenkel et al. (8) observed that caffeic acid phenethyl ester is a potent chemopreventive agent, which may be useful in treating diseases with strong inflammatory and/or oxidative stress components, such as various types of cancer. Basic et al. (1) observed that aqueous extract of propolis, administered to mice 7 days before tumor cells inoculation, significantly diminished lung metastases. Matsuno (14) isolated a new clerodane-type diterpenoid from Brazilian propolis, which showed cytotoxicity on human hepatocellular and lung carcinoma.
Although the direct carcinostatic effects of propolis or its isolated components have been demonstrated, an important question is whether propolis acts on the immunocompetent cells to help tumor cell destruction. Resistance to spontaneous tumor development has been associated with the cytotoxic activity of natural killer cells (NK), found both in man and experimental animals. NK cells are characterized as a lymphocyte subpopulation different from T and B cells, and non-adherent and non-phagocytic cells, showing lytic activity mainly towards several types of tumor and virus-infected cells (26). Similar to macrophages, NK cells are considered the host primary defense mechanism.
The aim of this work was to evaluate the cytotoxic activity of rat NK-like cells after propolis treatment, using a murine lymphoma (Yac-1) as target cells. The seasonal effect on the immunomodulatory action of Brazilian propolis was also analyzed, as in Brazil, propolis collection is made throughout a whole year and some seasonal variations are possible, but have not been reported in literature.
MATERIALS AND METHODS
Propolis was collected in the Beekeeping Section of Botucatu School of Veterinary Medicine and Animal Husbandry, UNESP. Propolis samples were obtained from colonies of africanized honeybees (Apis mellifera L.) and collected throughout a whole year from plastic nets. At the end of each month, nets were taken and frozen to facilitate propolis removal (25). Samples were pooled by season.
Hydroalcoholic solutions of propolis
Ethanolic extracts of propolis (EEP) were obtained by mixing each seasons pooled propolis with 95° ethyl alcohol (30 g propolis to 100 ml of 95º ethyl alcohol). This was protected from bright light and moderately shaken at room temperature. After a week, the extracts were filtered and used to prepare 10% propolis hydroalcoholic solutions as described previously (24).
Animal groups and treatment
Forty-two male rats (Rattus norvegicus) weighing 200 g were divided into 6 groups (G1, G2, G3, G4, G5, and G6) of 7 rats each. G1, G2, G3, and G4 received 10% propolis hydroalcoholic solutions from spring, summer, autumn, and winter samples, respectively. G5 received 10% hydroalcoholic solution (Labsynth) to observe a possible ethanol effect as propolis solvent. G6, control, received physiologic salt solution (0.9% NaCl). Animals received 0.4 ml by gavage twice a day for 3 days just before sacrifice, according to Hollands et al. (10).
Natural killer activity
The analysis of NK activity of non-adherent spleen cells was performed according to Sato et al. (20) with few alterations. Excised rat spleens were pressed on a nylon mesh and converted into a single-cell suspension in RPMI 1640 medium (Cultilab). Cells were layered on a Ficoll-Hypaque gradient (d=1.089 g/ml) and centrifuged. Mononuclear cells thus obtained were washed twice with RPMI 1640 medium supplemented with gentamicin (40 mg/ml), 20 mM HEPES (Sigma Chemical Co.), 2 x 10-5 M 2-mercaptoethanol (Sigma Chemical Co.), 0.2% sodium bicarbonate, and 10% heat-inactivated fetal calf serum (Cultilab, Campinas, Brazil). Mononuclear cells ressuspended in this complete medium were incubated in a glass Petri dish for 1 h at 37°C to remove adherent cells. Non-adherent cells were collected, washed twice with complete medium, and used as effector cells. These cells were assayed for natural killer activity against murine lymphoma (Yac-1 cells), previously labeled with 51Cr (100 mCi) as sodium chromate (IPEN/CNEN, São Paulo, Brazil). Using U-bottomed 96-well microplates, the cytotoxic activity was analyzed, yielding effector:target (E:T) ratios of 50:1, 25:1, 12.5:1, and 6.25:1 in triplicate. Plates were incubated for 4 h at 37°C under 5% CO2 tension and 100 ml of supernatant of each well was collected for 51Cr-release measurement. Maximum and spontaneous 51Cr-release were determined by incubating tumor cells with detergent (Triton X-100, diluted 1:2) or medium alone, respectively.
Radioactivity was determined in a gamma counter (Gamma Nuclear, Hungary) and cytotoxicity was calculated as follows: % specific lysis = [(cpm of experimental release - cpm of spontaneous release)/(cpm of maximum release - cpm spontaneous release)] x 100.
Analysis of Variance was used to examine the treatment effect at 50:1 (E:T) ratio, and comparison between means was performed by Tukey test, with 0.05 significance level (27).
We observed that propolis treatment for 3 days induced an increase in the cytotoxic activity of non-adherent spleen cells. As shown in Table 1, the results detected at 50:1 (E:T) ratio demonstrated that treatment with propolis obtained from each season was significantly different from the control group (p<0.05).
|Table 1. Seasonal effect on the immunomodulatory action of propolis: natural killer activity.|
Hydroalcoholic solution-treated animals showed NK activity (38.44%) between those of the control group and groups treated with propolis obtained in the 4 seasons (Table 1; Figure 1). Statistical analysis showed no seasonal effect on the immunomodulatory action of propolis.
|Figure 1. Natural killer activity of non-adherent spleen cells of the control groups and groups treated with propolis from each season (x + s). Effector: target ratio = 50:1.|
Propolis (bee glue) is a resinous hive product collected by bees endowed with versatile biological activities and successfully used in medicine and cosmetics (13). Because of the speculation on the immune response to honeybee products, propolis has been attracting researchers' attention lately.
As shown in Table 1 and Figure 1, propolis administration to rats for 3 days led to increased NK activity (mean of the 4 seasons = 43.36%) when compared to the control group (36.35%). This finding confirms a previous observation that propolis action on the immune system has a short-term effect (21).
Propolis stimulates cytokine production, such as IL-1 and TNF by mice peritoneal macrophages, which as well as indicating the activation state of these cells, may control the synthesis of complement system components, modulating both in vivo and in vitro C1q production by macrophages and complement receptor function (5,6).
NK cells are under cytokine action, such as interferon (a, b, g), TNF-a, IL-1, IL-2, IL-4, IL-10, IL-12, IL-13, IL-15, TGF-b (4,11,12,15,17,19), but the activation mechanism of these cells by propolis still remains obscure. We suggest that propolis-activated macrophages could produce cytokines, such as TNF-a and IL-12, which act on NK cells, increasing its cytotoxic activity.
NK activity observed in hydroalcoholic solution-treated animals was intermediate between the control group and groups treated with propolis obtained in the four seasons. Blank et al. (2) observed that chronic ethanol consumption may suppress NK activity in mice, once the cytotoxic activity of these cells decayed after a week with ethanol treatment. In 1993, Blank et al. (3) suggested that this suppression may be due to ethanol metabolites, affecting NK cell calcium-dependent programming and signal transduction, or that NK cells are negatively inhibited via paracrine regulation by other cells of the immune system. In this work, however, impairment of lytic activity of alcohol-treated rats was not observed; this may be due to factors, such as short time interval of treatment or alcohol concentration.
Several authors have been studying the in vivo antitumoral property of propolis (1,22). Our results suggest an important role of propolis as a stimulant agent of NK-like cells, increasing its cytotoxic activity in vitro against tumor cells. Further investigations will permit a better understanding of cells and cytokines involved in NK activity.
Another new result obtained in this work was the lack of seasonal effect on propolis immunostimulatory action. This may be due to the regional source of this product, once it was produced during the four seasons in the same geographical region. One could suggest that possible differences in its chemical composition and biological activities might be found between samples produced in different areas, mainly due to local flora.
The authors are grateful to Dr. Paulo Roberto Curi for statistical analysis, Mrs. Sônia Maria Faraldo for computer assistance, and Mrs. Célia Sforcin Guimarães for English editing. This study was performed with technical assistance of Luiz Henrique Alquati, Maria Conceição Tenore do Carmo, and Luiz Carlos Fioravante.
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Received October 25, 2000
Accepted March 5, 2001
J. M. SFORCIN - Departamento de Microbiologia e Imunologia, Instituto de Biociências, UNESP, Campus de Botucatu, 18618-000, Botucatu, SP, Brasil.