On-line version ISSN 1678-4936
J. Venom. Anim. Toxins vol.8 no.2 Botucatu 2002
ANTI-INFLAMMATORY ACTIVITY OF PROPOLIS EXTRACTS: A REVIEW
1 Departamento de Ciências Médicas e da Saúde, Câmpus de Dourados, Universidade Federal de Mato Grosso do Sul, Rodovia Dourados-Itahum, km 12, Caixa Postal 533, 79804-970, Dourados, MS, Brasil; 2 Departamento de Bioquímica e Microbiologia, Instituto de Biociências, UNESP, Caixa Postal 199, 13506-900, Rio Claro, SP, Brasil.
ABSTRACT: Since primeval times, the inflammatory process has been described in many different ways. Several anti-inflammatory therapies have been used in different biological models. However, in a recent "back to nature move", modern man is searching for natural products with medicinal properties, particularly those obtained from plants and bees. Propolis has been used in folk medicine for a very long time. The many compounds present in propolis require investigation.
Physical-chemical analysis studies have not sufficiently established quality standards of propolis containing products. These standards should depend especially on their different pharmacological activities. There are few studies reporting on the in vitro anti-inflammatory activity of propolis containing products. It is necessary to evaluate the anti-inflammatory potential of commercial products containing propolis.
KEYWORDS: propolis, anti-inflammatory activity, bees, Apis mellifera, natural products.
More than two thousand years ago, the ancient Greeks used the term phlogosis and the Romans inflammation to designate the same phenomenon nowadays called inflammation. Since the first description of this phenomenon by Aulus Cornelius Celsus, the inflammation process has been described in many different ways. In the Middle Ages, inflammation was thought to be the heat accumulation originating in the heart, followed by blood flow, mucous, and bile; this was the humoral inflammation theory. This was then overtaken by the vascular theory in the 18th century.
The concept of inflammation has evolved since the discovery of cells in the 19th century. By this time, inflammation was seen to be preceded by cell and tissue injuries, and that vascular changes including leukocyte emigration were secondary events (12,85).
During the 1920s, the idea that the vascular system facilitated quick accumulation of great quantities of phagocytes and antibodies was reviewed. The first physical-chemical analysis of inflammation, cell stress and local tissue changes, promoted by an increasing concentration of oxidants and osmotic pressure, were also made at this time (21). Therefore, modern investigators have considered inflammation a primary event of the host defense system.
Inflammation can be basically defined as a change of the morphological equilibrium in a specific area of the tissue caused by different kinds of agents: physical, chemical, or biological (141). It can be represented by capillary dilatation with fluid accumulation (oedema) and by phagocyte emigration and accumulation (neutrophils, monocytes, macrophages), which also contribute to hyperalgesia generation and loss of tissue function (42). Other characteristics, such as erithema and fever, can also be observed during inflammatory events. The last feature occurs after cytokine release (IL-1, TNF-a ) by activated macrophages, leading to a vessel dilatation resulting from smooth muscular relaxation and followed by an increase in local blood flow (hypothermia) (118,127). The increased hematocrit leads to an erythrocyte aggregation, and leukocytes move from the central axial of the vessel to the periphery ("Roleux") (19).
Similarly, many intra- and extracellular phospholipases are activated from the cytoplasmic membrane phospholipids and activate other enzymes, such as cyclooxygenase (COX) and lipoxygenase (LOX), which act on arachidonic acid (AA) and eicosanoid metabolism (105,106,138). The fibrinolytic system, kinins, complement, vaseactive amines (histamine and serotonin), and nitric oxide may lead to inflammation when physiologically altered (117,138).
Inflammatory events involve micro-vascular changes with increased vascular permeability, flow exudation, including plasmatic protein and amplification of endogenous chemical mediators (20).
Neuropeptides of the skin nerves may interact with target-cells in the skin, releasing more skin neuropeptides, such as P substance, vase intestinal peptides (VIP), and peptides regulator of calcitonin gene, which modulates not only the function of inflammatory and immunocompetent cells but also endothelial and epithelial cells (70).
Excessive quantities of free radicals (FR) trigger neutrophil NADPH oxidase and dissociate a variety of redox systems, including xanthine dehydrogenase of endothelial cell in inflamed areas (136). Low-density lipoprotein oxidative changes, the restraint inactivation of a -1-protease, DNA damage, and heat-shock protein synthesis are also affected by FR excess (67). Collagen and hyaluronic acid changes may also occur, interfering with synovial liquid viscosity, forming carbon radicals that react against themselves, decreasing collagen molecule flexibility (40).
Reactive oxygen intermediates may participate in inflammation events, such as: (a) polymorphonuclear leukocyte (PMN) and monocyte/macrophage chemotaxis; (b) specific stimulus related to respiratory burst, especially in inflammatory cells with greater FR production; (c) low concentration of scavenger enzymes in interstitial spaces; and (d) formation of kelant metal immune complexes which can also produce OH° (24).
Endothelial-leukocyte cellular adhesion occurs in a sequence of events, and specific molecules are expressed in different stages. Selectins (E, P, and L), integrins (VLA-4 and LFA-1), and members of immunoglobulin super-families (ICAM-1 and ICAM-2) move leukocytes from the vascular lumen to the tissues (34,93,135). In response to several mediators, the vascular endothelium expresses specific glycoproteins on the cell surface, which intermediate blood leukocyte connection and extravasation, important for tissue repair (10,31,124). In general, the irritating phlogogen agent, and the frequency and magnitude of body response have allowed a classification of inflammation into immunogenic (self) and non-immunogenic (non-self) (119); the latter is subdivided in acute and chronic phases (127).
The acute-phase response involves serous, fibrinous, supurative or exudative events as well as micro-vascular and cell events; this response to phlogogen occurs within 72 hours. The chronic-phase response includes proliferative events and histological alterations, different from those in the acute phase, characterized by cell emigration and intensive mitosis. Formation of giant multinuclear cells takes place, and all these events are induced by phlogogen (119). In addition, inflammation may be physiological or pathological (39), depending mainly on histological aspects. Specific-immune events, such as hypersensitive reactions (types I, II, III, and IV) can also lead to inflammation (103,122,123).
In spite of the above classifications, inflammation comprises a great variety of reactions occurring in the body, for instance arthritis. Several etiological and metabolic pathways are involved in the inflammatory response (96). All distinct inflammation events, culminating in oedema and pain in bone articulations are generally denominated arthritis. Inflammation studies require different experimental models, so that different metabolic pathways may be elucidated. Inflammation is an important causative agent of human morbidity and mortality, such as systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), and multiple organ failure (MOF) (8). In this way, inflammation events permit molecule identification and allow the development of drugs capable of acting on a variety of related metabolic pathways.
The action of several corticosteroids on asthma, rhinitis, and dermatitis is well known (6,7,88). These drugs have been used in anti-inflammatory therapy in clinical and pre-clinical assays (35,55,86). Like other hormones, corticosteroids act on many different tissues and body systems. At physiological concentrations, they maintain normal blood pressure, heart function, respond to inflammatory prostaglandin (PG) action, and maintain blood volume, diminishing vascular endothelium permeability (48,102). However, these effects are accentuated at high pharmacological concentrations, leading to target-cell dysfunction (mast cells, macrophages, vascular smooth muscles, and mucous glands) (38,48).
Glucocorticoids are used due to their general anti-inflammatory activity, which ranges from clinical suppression of rheumatoid arthritis to palliative treatment of some allergic manifestations, such as bronchitis, asthma, and anaphylatic reactions (6,7,44). They also act upon cytokines involved in eosinophil, basophil, and lymphocyte recruitment (112).
The activity of these compounds depends on the presence of a hydroxyl group in carbon-11 (102). This group acts in a similar manner to the anti-inflammatory activity of macrocortins, rinocortins, or lipocortins (101). Simultaneous findings have been reported on calcium anexin family members and protein phospholipids of several intrinsic groups in the phosphorilation control (25,45,104).
Glucocorticoid anti-inflammatory effects with the LC-1 protein act on arachidonic acid (AA) metabolites, phospholipase A2 (PLA2) inactivation, and COX and LOX (88). These events are present in triggering of apoptotic processes in various cell types, protection against splenic artery occlusion, and reperfusion of peritoneal macrophages (22,37) and neutrophils. However, the exact biological functions are not yet totally clear (59).
Induction of anti-inflammatory mechanisms is also present in synovial arthritis, though the ability to regulate articulation inflammation is still unclear (139,140). The inhibitory action of glucocorticoids on endothelium-leukocyte interaction affects the expression of cellular adhesion molecules (CAM).
Glucocorticoids inhibit the expression of ELAM-1 and ICAM-1 in the endothelium and suppress the expression of LFA-1 in stimulated lymphocytes (28,34,100). Toxicity is high during long-term anti-inflammatory therapy, which limits its unrestricted use (44).
The effects of non-steroid anti-inflammatory drugs (NSAID) on the synthesis of inflammatory prostaglandins, especially the PGE2, are widely known (105,106). The molecular mechanisms of these inflammation compounds have only recently been elucidated (99,128).
The pharmacological aim of most NSAID is the COX-enzyme (PGHS or PGH2) (66,99). Two isoforms of these enzymes are described, COX-1 and COX-2, differing in tissue expression and distribution (137). The mechanism of all NSAID studied until now is based on their involvement with the hydrophobic region of both isoforms; the manner in which the molecule structure of the drug relates to this region may vary (138).
Many other classes of COX-2 selective inhibitors have been developed: 1) sulfonamides (including NS-398, flosulide and L-745, 3337); 2) tricyclic methylsulfone derivatives (including DuP-697, SC-58125, and SC-57666) (131). In relation to LOX inhibitors, the monomethylamine analogs, such as LY269415 and LY-221068, are not only antioxidants but also strong inhibitors of iron-dependent lipidic peroxidation (91).
All the NSAID tested in animal models and man prevent pain, oedema, and erithema leading to reduced inflammatory reactions (138). Administration of therapeutic doses in rats is effective in reducing degenerative diseases associated with adjuvant-induced arthritis. In humans, NSAID have been considered very effective in acute and chronic inflammation, such as arthritis, tendinitis, and pericarditis (137). COX-1 inhibition by NSAID may cause side effects, such as gastrointestinal and renal disorders (129,131,134).
After the advent of glycobiology, clinical studies with CAM have allowed the development of anti-inflammatory strategies (34). Inflammatory reactions as glomerulonephritis, ulcerative colitis, syndrome of breath stress, rheumatoid arthritis, autoimmunity, and atherosclerosis partly result from the pathological activation of endothelial-leukocyte adhesion (97).
Recent studies have shown that mimetic CAM may block inflammation induction, though the differences of adhesion are not clear (10). In these cases, the following components are used: quimerics-immunoglobulins-selectins-proteins, carbohydrate, and peptide carbohydrates anti-sense oligonucleotides, and low-molecular weight inhibitors (13,32).
On the other hand, inhibition of FR production has been stimulated by endogenous antioxidant synthesis and administration of some factors and co-factors for exogenous antioxidant formation (98).
Physiological production of FR is related to: 1) activation and migration of phagocytic cells; 2) COX activation during AA metabolism; 3) catecholamine oxidation; 4) uric acid formation through xanthine oxidase; and 5) microsomal enzyme P-450 action on mitochondrial internal membrane through cytochrome oxidase complex by oxygen reduction. Oxygen is transported by hemoglobins (67); however, excessive quantities of FR are closely related to several inflammatory events, resulting in the production of oxygen toxic reactive species (OTRS) (136).
Natural antioxidant mechanisms (enzymatic or non-enzymatic) have been stimulated. Small molecules and kelants of metallic ion (selenium, zinc, copper, manganese, vitamins A, C, and E, cysteine, and reduced glutathione, and some plasma compounds) participate in inflammatory events and act on ORTS (11,24). Similarly, in vitro inhibition of PMN adhesion by SOD and micro-vascular extravasation by SOD catalysis, DMSO, and L-metionin have been reported (121).
Kelant groups such as oxin, D-penicillin, desferrioxamin-B, EDTA, dimercaprol, estibofen, and melarsoprol as chemotherapy agents act on metallic ion excess in the body (30).
Based on these data, in a recent back to nature move, modern man is searching for natural products with medicinal properties, particularly those from plants and bees (26, 81,126).
Several plants produce resinous exudates with strong anti-microbial and anti-necrotic properties (83), in addition to impermeability provided by populus - a substance from Populus sp (16). Bees collect resin exudates from certain plants and add their secretion, wood fragments, pollen, and wax; this product from bees and plants is called propolis. The word propolis comes from the Greek pro meaning in defense of and polis city, representing defense of bee cities (or beehives). Propolis has been used in folk medicine since primeval times (43). It was used in Egyptian rituals to embalm their dead (74); as violin varnish in Italy in the 17th century (56,83); and as a local antiseptic for umbilical cords in the Middle Ages (76). Nowadays, propolis is still used in homemade remedies and cosmetics (16). Two characteristics of propolis are its smell and its various colors from dark green to brown.
Propolis chemical composition has been correlated with plant diversity around the beehive (5,61). In general, raw propolis is composed of 50% resin and balsam, 30% wax, 10% essential and aromatic oils, 5% pollen, and 5% other substances, including wood fragments (83). More than 210 different compounds have been identified so far, such as aliphatic acids, esters, aromatic acids, fatty acids, carbohydrates, aldehydes, amino acids, ketones, chalkones, dihydrochalcones, terpenoids (73,74), vitamins (84), and inorganic substances.
These compounds have been determined by high-performance liquid and gas chromatography (HPLC and GC) (4,5,93), high-performance thin layer chromatography (HPTLC) (62), and colorimetric (89) and spectrophotometric methods (92). Biological properties have also been determined by flow injection analysis (FIA) (74) and x-ray influorescense for element quantification (15).
Ethanol, the most commonly used solvent for propolis preparations, and other solvents such as ethylic ether, water, methanol, petroleum ether, and chloroform are used for extracting and identifying many propolis compounds (73,74). Moreover, glycerin, propylene glycol and some solutions have been used in propolis preparations by the pharmaceutical and cosmetic industry (125).
Propolis compounds have recently become the subject of investigation in order to determine its therapeutic application (60); flavonoids are the most biologically active (9,16,53).
However, some hypersensitive reactions induced by propolis and its isolated constituents, especially those derived from cinnamic acids have been reported (49,50-52). Oedema and erithema in the face and hands of violin polishers in Cremona, Italy, are related to contact dermatitis with propolis (83). Allergic reactions caused by propolis have also been reported (52).
Propolis has low acute oral toxicity, as shown by the LD50 tested in mice (2.000 to 7.300 mg/kg) and flavonoids evaluated in rats (8.000 to 4.000 mg/kg) (54). No side effects have been seen in oral administration to mice higher than 4.000 mg/kg/day for two weeks (23) and in drinking water at 1.400 mg/kg/day and for 90 days, and to rats at 2.740 mg/kg/day for 60 days (57). On the other hand, intraperitoneal administration of ethanolic propolis extracts has slight effects on animals under narcotic-induced hypothermia. Propolis oral administration does not show any significant alteration in some important enzyme levels in rats (116).
Considering that propolis is a complex mixture, synergistic interactions between its compounds must also be considered as an important factor in its anti-inflammatory activities (18,64,131,132).
In the last 20 years, there has been increased commercial interest (14) in propolis use due to its therapeutic properties (17,71,87). Propolis is commercially found in sprays, ointments, capsules, capillary lotions, and toothpastes (16) because of its bacteriostatic activity and pharmacological properties.
Propolis can be used in decubitus scabs, (2) dermatitis, psoriasis, itching, burning, (17), leg ulcers, (78) simplex and genital herpes (33), and in oral hygienic odontology (71,87). Propolis extracts show: anti-bacterial (27,46,130,132), anti-viral (1,113), anti-fungal (29), anti-protozoan (23,120) antioxidant (95,110,111), and anti-tumor properties (47,77,109). Propolis is also effective in antibody formation in immunized animals (108) as a hepatoprotective (69) and for its hypertensive effects (3). It increases natural killer activity against tumor cells and shows anti-microbial action against GRAM-positive bacteria (114). Propolis minimal inhibitory concentration (MIC) has been performed in order to establish the anti-microbial activity profiles of its extracts (36,115). Nowadays, many in vitro and in vivo experiments are performed with propolis ethanolic extracts (PEE) and propolis aqueous extracts (PAE) to confirm its anti-inflammatory activity. PAE anti-inflammatory effects was observed in platelet aggregation inhibition, in vitro PG biosynthesis, and adjuvant-induced paw oedema in vivo, when orally administered and in a dose-dependent-manner (58). PAE inhibits acute inflammation model (PGE2-induced paw oedema) and chronic inflammation (formaldehyde-induced arthritis). However, PAE has no effect against carrageenin-induced paw oedema and Freunds adjuvant-induced arthritis (29).
The anti-inflammatory effects of orally administered PEE were observed in the inhibition of carrageenin-induced oedema, Freunds adjuvant-induced arthritis, cotton-pellet-induced granuloma, and vascular permeability and analgesia in a dose-dependent manner (94).
Propolis suppresses the generation of LOX and COX during acute peritonitis induced by in vivo zimosan and in vitro murine peritoneal macrophages, inhibiting in vivo high production of LTB4 and LTC4. However, its oral supplements do not affect PGE2 generation in zimosan-treated mice ex vivo or in vitro, increasing LT and PG production by peritoneal macrophages (81). Propolis extracts act on host non-specific immunity activating macrophages, inducing H2O2 release, and inhibiting nitric oxide generation in a dose-dependent manner (90).
Propolis has inhibitory effects on mieloperoxidase activity, NADPH-oxidase (41,133), ornithine decarboxilase, tirosine-protein-kinase, and hyaluronidase from guinea pig mast cells (82). This anti-inflammatory activity can be explained by the presence of active flavonoids and cinnamic acid derivatives (9,65,107,133). The former includes acacetin, (43) quercetin, and naringenin (65,81) (terpenoid constituents may exert an addictive effect (58)); the latter includes caffeic acid phenyl ester (CAPE) and caffeic acid (CA) (41,81).
CAPE action was observed on the generation of several cell oxidative processes: a) myeloperoxidase activity (MPO) by PMN infiltration in mouse ear induced by tumor promoter; b) respiratory burst of human PMN; and c) formation of oxide-base in epidermal DNA isolated in mice treated in vivo (41). It was also observed that CAPE and CA are strong LOX inhibitors, suppressing leukotriene production by peritoneal macrophages. Their action on LTC4 was smaller in vivo (81). Quercetin inhibits LOX, and at high concentrations blocks COX. Naringenin only inhibited LTC4 causing weakness. Propolis constituents have the ability to produce free radicals in inflammatory events (53) including neutrophil chemiluminescence (63). However, some models of inflammation induction (where phlogogen is attenuated in vitro) do not demonstrate propolis extract effects on already established inflammation (68,119).
All these data have demonstrated the strong and different inhibitory action of several propolis preparations or its isolated constituents on inflammation events. However, its anti-inflammatory effects depend mainly on the administration route and its potency (81).
In an attempt to establish quality standards for propolis containing products, physical-chemical analysis studies have not been sufficient (72,131,132,133) mainly for the great variety of compounds detected in propolis from tropical regions (75). These standards should depend specifically on their different pharmacological activities (79).
There are few studies reporting on in vivo anti-inflammatory activity of propolis containing products (80). Based on these data, an evaluation of the anti-inflammatory potential of commercial propolis containing products from several phyto-geographic origins is of major importance for its indication in inflammatory processes.
The authors are grateful to Ms. Lívia Simão de Carvalho, a medical student at Federal University of Mato Grosso do Sul, Campus of Dourados.
1 AMOROS M., SIMÕES CMO., GIRRE L., SAUVAGER F., CORMIER M. Synergistic effect of flavonoids against herpes simplex virus type 1 in cell culture comparison with the antiviral activity of propolis. J. Nat. Prod., 1992, 55, 1732-40. [ Links ]
2 AZEVEDO IBS., SAMPAIO RF., MONTES JC., CONTRERAS RLL. Tratamentos de escaras de decúbito com própolis. Rev. Bras. Enf., 1986,39, 33-7. [ Links ]
3 BANKOVA V., POPOV SS., MAREKOV NL. A study on flavonoids of propolis. J. Nat. Prod., 1983, 46, 478-4. [ Links ]
4 BANKOVA V., DYULGEROV AL., POPOV S., MAREKOV N. A GC/MS study of the propolis phenolic constituents. Z. Naturforsch. C, 1987, 42, 147-51. [ Links ]
5 BANKOVA V., CHRISTOV R., STOEV G., POPOV S. Determination of phenolics from propolis by capillary gas chromatography. J. Chromatogr., 1992, 607, 150-3. [ Links ]
6 BARNES PJ. Molecular mechanisms of antiasthma therapy. Ann. Med., 1995, 27, 531-5. [ Links ]
7 BARNES PJ. Mechanisms of action of glucocorticoids in asthma. Am. J. Respir. Crit. Care Med., 1996, 154, S21-7. [ Links ]
8 BAUE AE., DURHAM R., FAIST E. Systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), multiple organ failure (MOF) are we winning the battle? Shock, 1998, 10, 79-89. [ Links ]
9 BAUMANN J., BRUCHHAUSEN FV., WURM G. Flavonoids and related compounds as inhibitors of arachidonic acid peroxidation. Prostaglandins, 1980, 20, 627-39. [ Links ]
10 BEVILACQUA MP., NELSON RM., MANNORI G., CECCONI O. Endothelial-leukocyte adhesion molecules in human disease. Ann. Rev. Med., 1994, 45, 361-78. [ Links ]
11 BISBY RH. Interactions of vitamin E with free radicals and membranes. Free Radic. Res. Commun., 1990, 8, 266-306. [ Links ]
12 BOGLIOLO, L. Patologia. 2.ed. São Paulo: Guanabara, 1976. 1097p. [ Links ]
13 BOSCHELLI DH. Inhibitors of leukocyte-endothelial cell-adhesion: a new generation of antiinflammatory therapeutics? Drugs Future, 1995, 20, 805-16. [ Links ]
14 BREYER HFE. Própolis: produção com Apis mellifera L. In: CONGRESSO BRASILEIRO DE APICULTURA, 11, Teresina, 1996. Anais... Teresina: Confederação Brasileira de Apicultura, 1996, 193-7. [ Links ]
15 BUENO MIMS., CUNHA IB., MARCUCCI MC. Quantificação elementar em própolis por fluorescência de raios-x. In: CONGRESSO BRASILEIRO DE APICULTURA, 11, Teresina, 1996. Anais... Teresina: Confederação Brasileira de Apicultura, 1996, 341. [ Links ]
16 BURDOCK GA. Review of the biological properties and toxicity of bee propolis (propolis). Food Chem. Toxicol., 1998, 36, 347-63. [ Links ]
17 CARVALHO PSR., TAGLIAVINI DG., TAGLIAVINI RL. Cicatrização cutânea após aplicação tópica de creme de calêndula e da associação de confrei, própolis e mel em feridas infectadas: estudo clínico e histológico em ratos. Rev. Ciênc. Bioméd. São Paulo, 1991, 12, 39-50. [ Links ]
18 CHENG PC., WONG G. Honey bee products: prospects in medicine. Bee World, 1992, 77, 8-15. [ Links ]
19 CHIEN S. Rheology in the microcirculation in normal and low flow states. Adv. Shock Res., 1982, 8, 71-80. [ Links ]
20 CIRINO G. Multiple controls in inflammation: extracellular and intracellular phospholipase A2 inducible and constitutive cyclooxygenase and inducible nitric oxide synthase. Biochem. Pharmacol., 1998, 55, 111-20. [ Links ]
21 COTRAN, R., KUMAR, V., ROBBINS, S.L. Robbins: patologia estrutural e funcional. 4.ed. Rio de Janeiro: Guanabara, 1991. 1231p. [ Links ]
22 CUZZOCREA S., TAILOR A., ZINGARELLI B., SALZMAN AL., FLOWER RJ., SZABO C., PERRETI M. Lipocortin 1 protects against splenic artery occlusion and reperfusion injury by affecting neutrophil migration. J. Immunol., 1997, 159, 5089-97. [ Links ]
23 DECASTRO SL., HIGASHI KO. Effect of different formulations of propolis on mice infected with Trypanossoma cruzi. J. Ethnopharmacol., 1995, 46, 55-8. [ Links ]
24 DELMAESTRO RF. An approach to free radicals in medicine and biology. Acta Physiol. Scand., 1980, 492, 153-68. [ Links ]
25 DELMER DP., POTIKHA TS. Structures and functions of annexins in plants. Cell. Med. Life Sci., 1997, 53, 546-53. [ Links ]
26 DI STASI LC. Plantas medicinais: arte e ciência: um guia de estudo interdisciplinar. São Paulo: UNESP, 1996. 230p. [ Links ]
27 DIMOV V., IVANOVSKA N., BANKOVA V., POPOV S. Immunomodulatory action of propolis: IV. prophylactic activity against gram-negative infections and adjuvant effect of the water-soluble derivative. Vaccine, 1992, 10, 817-23. [ Links ]
28 DINARELLO CA., ENDRES S. Role for interleukin-1 in the pathogenesis of hypersensitivity diseases. J. Cell. Biochem., 1989, 39, 229-38. [ Links ]
29 DOBROWOLSKI JW., VOHORA SB., SHARMA K., SHAS SA., NAQUI SAH., DANDYIA PC. Antiinflammatory, antibacterial, antifungal, antiamoebic, and antipyretic studies on propolis bee products. J. Ethnopharmacol., 1991, 35, 77-82. [ Links ]
30 DORMANDY TL. Free radicals pathology and medicine: a review. J. R. Coll. Physicians Lond., 1989, 23, 221-7. [ Links ]
31 DURAN WW., MILAZZO VJ., SABIDO F., HOBSON RW. Platelet-activating factors modulates leukocyte adhesion to endothelium in ischemia-reperfusion. Microvasc. Res., 1996, 51, 108-15. [ Links ]
32 DWEK RA. Glicobiology more functions for oligosaccharides. Science, 1995, 269, 1234-5. [ Links ]
33 ESANU V. Research in the field of antiviral chemotherapy performed in the Stefan S. Nicolau Instituto of Virology. Rev. Roum. Med., 1984, 35, 281-293. [ Links ]
34 FARSKY SP., MELLO SBV. Participação de moléculas de adesão no desenvolvimento da resposta inflamatória. Rev. Hosp. Clin. Fac. Med. São Paulo, 1995, 50, 80-9. [ Links ]
35 FAUCHERON JL., PARC R. Non-steroidal anti-inflammatory drug-induced colitis. Int. J. Colorectal Dis., 1996, 11, 99-101. [ Links ]
36 FERNANDES JR A., LOPES CAM., SFORCIN JM., FUNARI SRC. Population analysis of susceptibility to propolis in reference strains of Staphylococcus aureus and Escherichia coli. J. Venom. Anim. Toxins, 1997, 3, 287-94. [ Links ](SciELO)
37 FILEP JP., DELALANDRE A., PAYETTE Y., FÖLDES-FILEP E. Glucocorticoid receptor regulates expression of L-selectin and CD11/CD18 on human neutrophils. Circulation, 1997, 96, 295-301. [ Links ]
38 FINOTTO S., MEKORI YA., METCALFE DD. Glucocorticoids decrease tissue mast cell number by reducing the production of the c-kit ligant stem cell factor by resident cells: in vitro and in vivo evidence in murine systems. J. Clin. Invest., 1997, 99, 1721-8. [ Links ]
39 FIOCCHI C. Immunity and inflammation: separated or unificated In: MAC DERMOTT RP. Clinical immunology in gastroenterology and hepathology: from bench to bedside. Cleveland: American Gastroenterology Association, 1994, 182-8. [ Links ]
40 FREEMAN BA., CRAPO JD. Biology of disease: free radicals and tissue injury. Lab. Invest., 1982, 47, 412-26. [ Links ]
41 FRENKEL K., WEI H., BHIMANI R., YE J., ZADUNAISKY JA., HUANG MT., FERRARO T., CONNEY AH., GRUNBERGER D. Inhibition of tumor promoter mediated process in mouse skin and bovine lens by caffeic acid phenethyl ester. Cancer Res., 1993, 53, 1255-61. [ Links ]
42 GALLIN JI. Inflammation. In: PAUL WE. Fundamental immunology. 2.ed. New York: Raven Press, 1989, 721-33. [ Links ]
43 GHISALBERTI EL. Propolis: a review. Bee World, 1979, 60, 59-84. [ Links ]
44 GOODWIN JS. Anti-inflammatory drugs. In: STITES DP., TERR AI., PARSLOW TG. Basic and clinical immunology. 8.ed. Stanford: Appleton & Lange, 1994, 786-94. [ Links ]
45 GOPPELT-STEVEBE M. Molecular mechanisms involved in the regulation of prostaglandin biosynthesis by glucocorticoids. Biochem. Pharmacol., 1997, 53, 1389-95. [ Links ]
46 GRANGE JM., DAVEY RW. Antibacterial properties of propolis (bee glue). J. Res. Soc. Med., 1990, 83, 159-60. [ Links ]
47 GRUNBERGER D., BANERJEE R., EISINGER K., OLTZ EM., EFROS L., CALDWELL M., ESTEVEZ V., NAKANISHI K. Preferential cytotoxicity on tumor cells by caffeic phenetyl ester isolated from propolis. Experientia, 1988, 44, 230-2. [ Links ]
48 GUYTON AC., HALL JE. Textbook of medical physiology. 9.ed. Philadelphia: W.B. Saunders, 1996. 1148p. [ Links ]
49 HASHIMOTO T., TORI M., ASAKAWA Y., WOLLENWEBER E. Synthesis of two allergenic constituents of propolis and poplar bud excretion. Z. Naturforsch., 1988, 43, 470-2. [ Links ]
50 HAUSEN BM., WOLLENWEBER E. Propolis allergy III. sensitization studies with minor constituents. Contact Dermatitis, 1988, 19, 296-303. [ Links ]
51 HAUSEN BM., WOLLENWEBER E., SENFF H., POST B. Propolis allergy I. origin properties usage and literature review. Contact Dermatitis, 1987, 17, 163-70. [ Links ]
52 HAUSEN BM., WOLLENWEBER E., SENFF H., POST B. Propolis allergy II. the sensitizing properties of 1, 1-dimethylallyl caffeic acid ester. Contact Dermatitis, 1987, 17, 171-7. [ Links ]
53 HAVSTEEN B. Flavonoids, a class of natural products of high pharmacological potency. Biochem. Pharmacol., 1983, 32, 1141-8. [ Links ]
54 HOLLANDS I., VIDAL A., GRA B., SOTOLONGO M. Evaluation of the subchronic toxicity of Cuban propolis. Rev. Cubana Cienc. Vet., 1991, 22, 91-100. [ Links ]
55 JOHNSON KS., EISCHEN FA., GIANNASI DE. Chemical composition of North American bee propolis and biological activity towards larvae of greater waxe moth (Lepidoptera: Pyiralidae). J. Chem. Ecol., 1994, 20, 1783-92. [ Links ]
56 JOLLY VG. Propolis varnish for violins. Bee World, 1978, 59, 158-62. [ Links ]
57 KANEEDA J., NISHINA T. Safety of propolis: acute toxicity. Honney Bee Sci., 1994, 15, 29-33. [ Links ]
58 KHAYYAL MT., EL-GHAZALY MA., EL-KHATIB AS. Mechanisms involved in the antiinflammatory effect of propolis extract. Drugs Exp. Clin. Res., 1993, 19, 197-203. [ Links ]
59 KIM GY., LEE HB., LEE SO., RLEE HJ., NA DB. Chaperone-like function of lipocortin 1. Biochem. Mol. Biol. Int., 1997, 43, 521-3. [ Links ]
60 KLEINROK Z., BORZECK Z., SCHELLER S., MATRA W. Biological properties and clinical application of propolis: X. Preliminary pharmacological evaluation of ethanol extract of propolis (EEP). Arzneim. Forsch., 1978, 28, 291-2. [ Links ]
61 KÖNIG B. Plant sources of propolis. Bee World, 1985, 66, 136-39. [ Links ]
62 KOO MH., PARK YK. Investigation of flavonoid aglycones in propolis collected by two different varieties of bees in the same region. Biosci. Biotechnol. Biochem., 1997, 61, 367-9. [ Links ]
63 KROL W., CZUBA Z., SCHELLER S., GABRYS J., GRABIEC S., SHANI J. Anti-oxidant property of ethanolic extract of propolis (EEP) as evaluated by inhibiting the chemiluminescence oxidation of luminol. Biochem. Int., 1990, 21, 593-7. [ Links ]
64 KROL W., SCHELLER S., SHANI J., PIETZ G., CZUBA Z. Synergistic effect of ethanolic extract of propolis and antibiotics on the growth of Staphylococcus aureus. Arzneim. Forsch., 1993, 43, 607-9. [ Links ]
65 KROL W., SCHELLER S., CZUBA Z., MATSUMO T., ZYDOWICZ G., SHANI J., MOS M. Inhibition of neutrophils' chemiluminescence by ethanol extract of propolis (EEP) and its phenolic components. J. Ethnopharmacol., 1996, 55, 19-25. [ Links ]
66 KURUMBAIL RG., STEVENS AM., GIERSE JK., MCDONALD JJ., STEGEMAN RA., PAK JY., GILDEHAUS D., MIYASHIRO JM., PENNING TD., SEIBERT K., ISAKSON PC., STALINGS WC. Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents. Nature, 1996, 384, 644-8. [ Links ]
67 LARA PF. Radicais livres. São Paulo: Departamento de Farmacologia do ICB, USP, 1991. 10p. [ Links ]
68 LEDÓN N., CASACÓ A., GONZÁLEZ R., MERINO N., GONZÁLEZ A., TOLÓN Z. Antipsoriatic, anti-inflammatory, and analgesic effects of an extract of red propolis. Acta Pharmacol. Sin., 1997, 18, 274-6. [ Links ]
69 LIN S-C., LIN Y-H, CHEN C-FA., CHUNG C-YU, HSU S-H. The hepatoprotective and therapeutic effects of propolis ethanol extract on chronic alcohol: induced liver injuries. Am. J. Chin. Med., 1997, 25, 325-32. [ Links ]
70 LUGER TA., LOTTI T. Neuropeptides: role in inflammatory skin diseases. J. Eur. Acad. Dermatol. Venereol., 1998, 10, 207-11. [ Links ]
71 MAGRO-FILHO O., CARVALHO ACP. Topical effect of propolis in the repair of sulcoplasties by the modified Kazanjian technique. J. Nihon Univ. Sch. Dent., 1994, 36, 102-11. [ Links ]
72 MALASPINA O., PALMA MS. Própolis: qualidade e legislação. In: CONGRESSO BRASILEIRO DE APICULTURA, 11, Teresina, 1996. Anais... Teresina: Confederação Brasileira de Apicultura, 1996, 199-202. [ Links ]
73 MARCUCCI MC. Propolis: chemical composition, biological properties and therapeutics activity. Apidologie, 1995, 26, 83-99. [ Links ]
74 MARCUCCI MC. Propriedades biológicas e terapêuticas dos constituintes químicos da própolis. Quím. Nova, 1996, 19, 529-36. [ Links ]
75 MARCUCCI MC., RODRIGUEZ J., FERREREZ F., BANKOVA V., GROTO R., POPOV S. Chemical composition of Brazilian propolis São Paulo State. Z. Naturforsch. C., 1998, 53, 117-9. [ Links ]
76 MASSON, B. Própolis: um antibiótico natural. São Paulo: Ediouro, 1981. 80p. (Saúde e curas naturais). [ Links ]
77 MATSUNO T., CHEN C., BANET P. A tumoricidal and antioxidant compound isolated from an aqueous extract of propolis. Med. Sci. Res., 1997, 25, 583-4. [ Links ]
78 MENDOZA OH., PEREZ JA., RAMOS CL., ARIOZA MC. Propoleo y ulceras fleboblásticas. Rev. Cubana Circ., 1991, 30, 28-33. [ Links ]
79 MENEZES H., BACCI JR M., OLIVEIRA SD., PAGNOCCA FC. Antibacterial properties of propolis and products containing propolis from Brazil. Apidologie, 1997, 28, 71-6. [ Links ]
80 MENEZES H., ALVAREZ JM., ALMEIDA E. Mouse ear edema modulation by different propolis ethanol extracts. Arzneim. Forsch., 1999, 49, 705-7. [ Links ]
81 MIRZOEVA OK., CALDER PC. The effect of propolis and its components on eicosanoid production during the inflammatory response. Prostaglandins Leukot. Essent. Fatty Acids, 1996, 55, 441-9. [ Links ]
82 MIYATAKA H., NISHIKI M., MATSUMOTO H., FUJIMOTO T., MATSUKA M., SATOH T. Evaluation of propolis. I. Evaluation of Brazilian and Chinese propolis by enzymatic and physico-chemical methods. Biol. Pharm. Bull., 1997, 20, 496-501. [ Links ]
83 MONTI M., BERTI E., CARMINATI G., CUSINI M. Occupational and cosmetic dermatitis from propolis. Contact Dermatitis, 1983, 9, 163. [ Links ]
84 MOREIRA TF. Composição química da própolis: vitaminas e aminoácidos. Rev. Inst. Adolfo Lutz, 1990, 7, 12-9. [ Links ]
85 MOVAT, H.Z. The acute inflammatory reaction. In: MOVAT HZ. Inflammation, immunity and hipersensitivity. New York: Harper & Row, 1971: 430-98. [ Links ]
86 MURPHY PJ., MYERS BL., BADIA P. Non-steroidal anti-inflammatory melatonin in humans. Physiol. Behav., 1996, 59, 133-9. [ Links ]
87 MURRAY MC., WORTHINGTON HV., BLINKHORN AS. A study to investigate the effect of a propolis - containing mouthrinse on the inhibition of de novo plaque formation. J. Clin. Periodontol., 1997, 24, 796-8. [ Links ]
88 MYGIND N. Alergia. Rio de Janeiro: Revinter, 1993. 483p. [ Links ]
89 NAGY M., GRANCAI D. Colorimetric determination of flavonoids in propolis. Pharmazie, 1996, 51, 100-1. [ Links ]
90 ORSI RO., FUNARI SRC., SOARES AMVC., CALVI SA., OLIVEIRA SL., SFORCIN JM., BANKOVA V. Immunomodulatory action of propolis on macrophage activation. J. Venom. Anim. Toxins, 2000, 6, 205-19. [ Links ](SciELO)
91 PANETTA JA., BENSLAY DN., SHADLE JK., TOWNER RD., HO PPK. Anti-inflammatory effects of LY221068 and LY269415. Agents Actions, 1991, 34, 100-2. [ Links ]
92 PARK YK., KOO MH., SATO HH., CONTADO JL. Estudo de alguns componentes da própolis coletada por Apis mellifera no Brasil. Arq. Biol. Tecnol., 1995, 38, 1253-9. [ Links ]
93 PARK YK., KOO MH, IKEGARI M., CONTADO JL. Comparison of the flavonoid aglycone contents of Apis mellifera propolis from various regions of Brazil. Arq. Biol. Tecnol., 1997, 40, 97-106. [ Links ]
94 PARK E-K., KIM S-H, PARK S-S. Anti-inflammatory activity of propolis. Arch. Pharm. Res., 1996, 19, 337-41. [ Links ]
95 PASCULAL C., GONZALEZ RG., TORRICELLA RG. Scavenging action of propolis extract against oxygen radicals. J. Ethnopharmacol., 1994, 41, 9-13. [ Links ]
96 PEARSON CM. Experimental models in rheumatoid disease. Arthritis Rheum., 1964, 7, 80-6. [ Links ]
97 PENBERTHY TW., JIANG Y., GRAVES DT. Leukocyte adhesion molecules. Crit. Rev. Oral Biol. Med., 1997, 8, 380-8. [ Links ]
98 PERCÁRIO S., BAPTISTA SF. Radicais livres, antioxidantes e oligoelementos. J. Biomol. Med. Free Radic., 1996, 2, 10-3. [ Links ]
99 PICOT D., LOLL PJ., GAVARITO M. The x-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature, 1996, 367, 243-9. [ Links ]
100 PITIZALIS C., KINGSLEY G., PANAYL GS. Adhesion molecules rheumatoid arthritis: role in the pathogenesis and prospects for therapy. Ann. Rheum. Dis., 1994, 53, 287-92. [ Links ]
101 POMPEO A., LUINI A., HIRATA F., BALDASSRRE M., BUCCIONE R. Neutrophil extracted lipocortin inhibitis corticotropin secretion in the AtT-20 D 16:16 clonal mouse pituitary cell live: lipocortin inhibition of ACTH release in vitro. Regul. Pept., 1997, 72, 169-77. [ Links ]
102 ROBBERS JE., SPEEDIE MK., TYLER VE. Pharmagmnosy and pahrmacobiotechnology. Baltimore: Williams & Wilkins, 1996. 337p. [ Links ]
103 ROITT I., BROSTOFF J., MALE D. Imunologia. 4.ed. São Paulo: Manole, 1997. 476p. [ Links ]
104 ROTHHUT B. Participation of annexins in protein phosphorylation. Cell. Med. Life Sci., 1997, 53, 522-6. [ Links ]
105 SAMUELSSON B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science, 1983, 220, 568-75. [ Links ]
106 SAMUELSSON B., DAHLÉN S-E., LINDGREN JA., ROUZER CA., SERHAN, CN. Leukotrienes and lipoxins: structures, biosynthesis and biological effects. Science, 1987, 237, 1171-5. [ Links ]
107 SCHELLER S., SZAFLARSKI J., TUSTANOWSKI J., NOLEWAJKA E., STOJKO A. Biological properties and clinical application of propolis I. some physico-chemical properties of propolis. Arzneim. Forsch., 1977, 27, 889-90. [ Links ]
108 SCHELLER S., GAZDA G,. PIETZ G., GABRYS J. The ability of ethanol extract of propolis to stimulate plaque formation in immunized mouse spleen cells. Pharmacol. Res. Commun., 1988, 20, 323-8. [ Links ]
109 SCHELLER S., KROL W., SWIACK J., OWCZAREK S., GABRYS J., SAANI J. Antitumoral property of ethanolic extract of propolis in mice-bearing Ehrlich carcinoma, as compared to bleomycin. Z. Naturforsch. C., 1989,44, 1063-5. [ Links ]
110 SCHELLER S., WILCZOKS T., IMIELSKI S., KROL W., GABRYS J., SHANI J. Free radical scavenging by ethanol extract of propolis. Int. J. Radiat. Biol., 1990, 57, 461-5. [ Links ]
111 SCHELLER S., KROL W., SEDLACZEK R., ZYDOWICZ G., WOJCIK L., SHANI J. Ethanolic extract of propolis (EEP), a natural antioxidant, prolongs life span of male and female mice. Pharmacology, 1994, 13, 123-5. [ Links ]
112 SCHWIEBERT LA., BECK LA., STELLATO C., BICKER CA., BOCHNER BS., SCHLEIMER RP. Glucocorticosteroid inhibition of cytokine production: relevance to antiallergic actions. J. Allergy, 1996, 97, 145-52. [ Links ]
113 SERKEDJIEVA J., MANOLOVA N., BANKOVA V. Anti-influenza virus effect of some propolis constituents and their analogues (esters of substituted cinnamic acids). J. Nat. Prod., 1992, 55, 294-7. [ Links ]
114 SFORCIN JM. Seasonal effect on the immunomodulatory and antibacterial properties of propolis and on the biochemical profile of rats. J. Venom. Anim. Toxins, 1997, 3, 49. [ Links ](SciELO)
115 SFORCIN JM., FERNANDES JÚNIOR A., LOPES CAM., FUNARI SRC., BANKOVA V. Seasonal effect of Brazilian propolis on Candida albicans and Candida tropicalis. J. Venom. Anim. Toxins, 2001, 7, 139-44. [ Links ](SciELO)
116 SFORCIN JM., FUNARI JM., NOVELLI ELB. Serum biochemical determination of propolis-treated rats. J. Venom. Anim. Toxins, 1995, 1, 31-7. [ Links ](SciELO)
117 SIES H., ELOHÉ L., ZIMMER G. Molecular aspects of inflammation. Berlin: Springer-Verlag, 1991. 290p [ Links ]
118 SIGAL LH., RON Y. Immunology and inflammation: basic mechanisms and clinical consequences. New York: Mcgraw-Hill, 1994. 760p [ Links ]
119 SOSA S., BARICEVIC D., CINCO M., PADOVAN D., TUBARO A., DELLA LOGGIA R. Preliminary investigation on the anti-inflammatory and anti-microbial activities of propolis. Pharm. Pharmacol. Lett., 1997, 4, 168-71. [ Links ]
120 STARZYK J., SCHELLER S., SZAFLARSKI J., MOSKWA M., STOJKO A. Biological properties and clinical application of propolis II. Studies on the antiprotozoan activity of ethanol extract of propolis. Arzneim. Forsch., 1997, 27, 1198-9. [ Links ]
121 SUZUKI M., INAVEN W., KUIETYS PR., GRISHAM MB., MEININGER CJ., SHELLING ME., GRANGER HJ., GRANGER DN. Superoxide mediants reperfusion induced leukocyte endothelial cell interactions. Am. J. Physiol., 1989, 257, H1740-50. [ Links ]
122 TERR AI. Mecanismos da inflamação. In: STITES DP., TERR AI. Imunologia básica. 7.ed. Rio de Janeiro: Prentice Hall, 1992. 187p. [ Links ]
123 TERR AI. Inflammation. In: STITES DP., TERR AI., PARSLOW TG. Basic and clinical immunology. 8.ed. Stanford: Appleton & Lange, 1994. 870p. [ Links ]
124 TERVAERT JW., KALLENBERG CG. Cell adhesion molecules in vasculitis. Curr. Opin. Rheumatol., 1997, 9, 16-25. [ Links ]
125 TOSI B., DONINI A., ROMAGNOLI C., BRUNI A. Antimicrobial activity of some commercial extracts of propolis prepared with different solvents. Phytother. Res., 1996, 1014, 335-6. [ Links ]
126 TREVISAN MDP. Própolis. Inf. Agropec., 1983, 9, 50-2. [ Links ]
127 TROWBRIDGE HO., EMLING RC. Inflammation: a review of the process. 4.ed. Chicago: Quintessence, 1993. 172p. [ Links ]
128 VANE JP., BOTTING RM. Mechanisms of anti-inflammatory drugs. Scand. J. Rheumatol., 1996, 25, 9-21. [ Links ]
129 VENTURINI C.M., ISAKSON P., NEEDLEMAN P. Non-steroidal anti-inflammatory drug-induced renal failure: a brief review of the role of cyclooxygenase isoforms. Curr. Opin. Nephrol. Hypertens., 1998, 7, 79-82. [ Links ]
130 VICENTE E., HIROOKA EY. Estudos preliminares da atividade antimicrobiana de própolis. Semina, 1987, 8, 76-9. [ Links ]
131 VOLPERT R., ELSTNER EF. Biochemical activities of propolis extracts I. standardization and antioxidative properties of ethanolic and aqueous derivatives. Z. Naturforsch. C., 1993, 48, 851-7. [ Links ]
132 VOLPERT R., ELSTNER EF. Biochemical activities of propolis extracts II. photodynamic activities. Z. Naturforsch. C., 1993, 48, 858-62. [ Links ]
133 VOLPERT R., ELSTNER EF. Interactions of different extracts of propolis with leukocytes and leukocytic enzymes. Arzneimitt. Forsch., 1996, 46, 47-51. [ Links ]
134 WALLACE JL. Non-steroidal anti-inflammatory drugs and gastroenteropathy: the second hundred years. Gastroenterology, 1997, 112, 1000-16. [ Links ]
135 WINN RK., VEDDER NB., MIHELIC D., FLAHERTY LC., LANGDALE L., HARLAN JM. The role of adhesion molecules in reperfusion injury. Agents Actions, 1993, 41, 113-26. [ Links ]
136 WINROW VR., WINYARD PG., MORRIS CJ., BLASE DR. Radicais livres em inflamação: segundos mensageiros secundários e mediadores da destruição tecidual. In: CHEESEMAN KH., SLATER TF. Radicais livres em Medicina. London: British Medical Bulletin, Interlivros, 1992, 27-43. [ Links ]
137 WINZELER S., ROSENSTEIN BD. Non-steroidal anti-inflammatory drugs: a review. AAOHN J., 1998, 46, 253-9. [ Links ]
138 WU KK-Y. Biochemical pharmacology of nonsteroidal anti-inflammatory drugs. Biochem. Pharmacol., 1998, 55, 543-7. [ Links ]
139 YANG Y., LEENCH M., HUTCHINSON P., HOLDWORTH SP., MORAND EF. Anti-inflammatory effect of lipocotin 1 in experimental arthritis. Inflammation, 1997, 21, 583-96. [ Links ]
140 YANG Y., HUTCHINSON P., SANTOS LL., MORAND EF. Glucocorticoid inhibition of adjuvant arthritis synovial macrophage nitric oxide production: role of lipocortin 1. Clin. Exp. Immunol., 1998, 111, 117-22. [ Links ]
141 ZANINI AC., OGA S. Farmacologia aplicada. São Paulo: Atheneu, 1979. 625p. [ Links ]
Received June 4, 2001
Accepted July 16, 2001
E. C. de Almeida - Departamento de Ciências Médicas e da Saúde - Câmpus de Dourados, Universidade Federal de Mato Grosso do Sul, Rodovia Dourados-Itahum, km 12, Caixa Postal 533, 79804-970, Dourados, MS, Brasil.
Phone: 55 67 422-7118