Before the problem of propolis standardization, the greatest problem to carry out the immunological assays was to design the experimental protocols, since researchers have used differ-ent concentrations of propolis in vivo and in vitro as well as different extracts, intake period and routes of administration. Table 1 shows some assays dealing with propolis immunomod-ulatory action according to its dose, chemical composition and main components, and assay conditions.

Little was known about the immunomodulatory action of propolis until the 1990s, but in the last decade new and interest-ing articles were published, providing an important contribution to this research field.

In immunosupression models, administration of a water-soluble derivative (WSD) of propolis (50 mg/kg) to mice prevented the cyclophosphamide effects and enhanced the sur-vival rate of the animals (Dimov et al., 1991). These authors also suggested that propolis modulates the non-specific immunity via macrophage activation. Propolis (0.2-1.0 mg/ml) stimulated cytokines production, such as IL-1 and TNF- , by peri-toneal macrophages of mice (Moriyasu et al., 1994). Propolis (0.150 mg/g) was also able to modulate both in vivo and in vitro production of C1q by macrophages as well as the comple-ment receptor function either directly or via cytokines (Dimov et al., 1992). In vitro assays showed that WSD of propolis (63–1000 g/ml) inhibited the classical and alternative path-ways of the complement system (Ivanovska et al., 1995a). C3 was one of the targets of propolis action, and flavonoids and phenolic compounds were pointed out as its major anticomple-mentary compounds (Georgieva et al., 1997).

It was demonstrated that six isolated compounds of propo-lis, identified as caffeoylquinic acid derivatives, enhanced the motility and spreading of macrophages (Tatefuji et al., 1996). Exposure of macrophages to a varied number of stimuli, such as the interaction with microorganisms and their products, antibodies or complement components-opsonized antigens, phorbol miristate acetate (PMA), Con A, immune complexes, leukotrienes, chemiotactic peptide fMLP (n-formyl-methionyl-leucyl-phenylalanine), cytokines, among others, may result in further metabolic changes, such as oxygen intermediates gener-ation. The production of such reactive species appears to be one of the mechanisms by which macrophages become microbicidal.

NADPH oxidase catalyses the reduction of molecular oxygen to superoxide anion (O2−) and the burst respiratory is paralleled by a higher consumption of oxygen (Krol et al., 1995). O2− is the precursor of other reactive oxygen intermediates, including hydroxyl radical (OH•), hypochlorite (OCl−) and hydrogen per-oxide (H2O2). Oxidants produced by phagocytes may destroy important biomolecules as well as phagocyted microorganisms, and are also involved in the tissue injury associated with inflam-matory diseases (Moonis et al., 1992; Brown, 1995; Babior,2000

Antioxidants are classically defined as molecules that, present in lower concentrations than biomolecules, may prevent, protect or reduce the extension of oxidative damage, such as, for example, glutation peroxidase, catalase and superoxide dismu-tase. Other antioxidants, such as ascorbic acid (vitamin C) and tocopherol (vitamin E) are non-enzymatic antioxidants. Thus, there is a delicate balance between the generation and destruc-tion of oxidant agents, which may be beneficial or deleterious to the organism (Novelli, 2005).

In order to evaluate propolis effect on macrophages micro-bicidal action, our group carried out some works, comparing the effects of Brazilian and Bulgarian propolis. The effect of different concentrations of propolis on macrophages fungicidal action against the thermally dimorphic fungus Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, was analysed. This human mycosis 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, lead-ing to the dissemination of the disease. Clinical and experimental data indicate that cell-mediated immunity plays a significant role in host defense, whereas high levels of specific antibod-ies are associated with the most severe form of this disease. Experimental models have shown the role of macrophages in the mechanisms of resistance against this fungus (Borges-Walmsley et al., 2002).