ReviewPropolis and the immune system: a review
Introduction
Propolis has attracted researchers’ interest in the last decades because of several biological and pharmacological properties, such as immunomodulatory, antitumor, antimicrobial, anti-inflammatory, antioxidant, among others (Bankova et al., 2000). Besides, propolis-containing products have been intensely marketed by the pharmaceutical industry and health-food stores (Banskota et al., 2001). The ethnopharmacological approach, combined with chemical and biological methods, may provide useful pharmacological leads. Thus, this review aimed to discuss its chemical composition and plant sources as well as to discuss some mechanisms of action of this bee product on the immune system and against tumor cells.
Propolis is in no way a new discovery. The use of propolis goes back to ancient times, at least to 300 BC, and it has been used as a medicine in local and popular medicine in many parts of the world, both internally and externally. Egyptians, Greeks and Romans reported the use of propolis for its general healing qualities and for the cure of some lesions of the skin. Propolis has always been reputed as an anti-inflammatory agent and to heal sores and ulcers. Ancient Egyptians used it to embalm their dead, and more recently it was used during the Boer War for healing wounds and tissue regeneration (Ghisalberti, 1979). However, its use continues today in remedies and personal products, and the list of preparations and uses is endless. It is still one of the most frequently used remedies in the Balkan States (Bankova, 2005a), and it has only been in the last decades that scientists have investigated its constituents and biological properties.
Propolis is a resinous material collected by bees from bud and exudates of the plants, which is transformed in the presence of bee enzymes. Its color varies from green, red to dark brown. Propolis has a characteristic smell and shows adhesive properties because it strongly interacts with oils and proteins of the skin. In general, propolis in natura is composed of 30% wax, 50% resin and vegetable balsam, 10% essential and aromatic oils, 5% pollen, and other substances (Burdock, 1998).
Etymologically, the Greek word propolis means pro, for or in defence, and polis, the city, that is “defence of the hive”. Bees use it to seal holes in their honeycombs, smooth out internal walls as well as to cover carcasses of intruders who died inside the hive in order to avoid their decomposition. Propolis also protects the colony from diseases because of its antiseptic efficacy and antimicrobial properties (Salatino et al., 2005).
After its administration to mice or to humans propolis does not seem to have side effects (Kaneeda and Nishina, 1994, Sforcin et al., 1995, Sforcin et al., 2002b, Jasprica et al., 2007). According to Burdock (1998) propolis is non-toxic, and its DL50 ranges from 2 to 7.3 g/kg in mice. This author suggested that the safe concentration for humans could be 1.4 mg/kg and day, or approximately 70 mg/day. After treatment of rats with different concentrations of propolis (1, 3 and 6 mg/kg/day), different extracts (water or ethanol) and varying the time of administration (30, 90 and 150 days) no significant alterations in total lipids, triglycerides, cholesterol, HDL-cholesterol concentrations, nor in AST and LDH specific activities were observed (Mani et al., 2006). The body weight of rats was measured in all these protocols, and propolis administration did not induce alterations in their weight. Cuesta et al. (2005) have not observed either mortality or growth rate alteration after daily intake of propolis in the diet during 6 weeks.
Although few in number, some cases of propolis allergy and contact dermatitis have been reported (Hausen et al., 1987, Hegyi et al., 1990, Silvani et al., 1997, Callejo et al., 2001), differently from the common allergy to honey, which contains allergens derived from flowers. Beekeepers usually show sensitivity to propolis (Rudeschko et al., 2004, Gulbahar et al., 2005). Ethanol and water extracts of propolis possess anti-allergic action, inhibiting histamine release in rat peritoneal mast cells (Miyataka et al., 1998). However, in higher concentrations (300 μg/ml), propolis directly activated mast cells, promoting inflammatory mediators release, what could be linked to allergic processes in propolis-sensitive individuals (Orsi et al., 2005b).
Recently, the presence of radioactive particles in propolis samples was investigated, since these particles may be concentrated in the soil, contaminating the plants, insects and its products, and, consequently, humans as well. Cesium (Cs137) was not found in the samples, and only natural radioactive particles such as potassium (K40) and beryllium (Be7) were found. These data suggested that propolis may be studied as an environmental contamination indicator in order to understand the soil–plant–bee–propolis chain (Orsi et al., 2006a).
Propolis antimicrobial property has been widely investigated, and several authors have demonstrated its antibacterial action (Grange and Davey, 1990, Kujumgiev et al., 1999, Sforcin et al., 2000, Orsi et al., 2005c, Orsi et al., 2006b, Scazzocchio et al., 2006). Fernandes et al. (2001) investigated the antibacterial action of propolis produced by Africanized honeybees, comparing with that produced by the stingless bees (subfamily Meliponinae). Propolis produced by Partamona sp. and Melipona sp. had a similar activity to that produced by Apis mellifera.
Propolis also shows antiviral (Amoros et al., 1992, Serkedjieva et al., 1992, Vynograd et al., 2000, Ito et al., 2001, Huleihel and Isanu, 2002, Gekker et al., 2005), antifungal (Dobrowolski et al., 1991, Sforcin et al., 2001) and antiparasite activities (Higashi and De Castro, 1994, De Castro and Higashi, 1995, Salomão et al., 2004, Freitas et al., 2006).
Propolis extraction methods may influence its activity, since different solvents solubilize and extract different compounds. The most common extracts used in biological assays are ethanol, in different concentrations, methanol and water (Cunha et al., 2004). Its chemical composition is very complex: more than 300 components have already been identified, and its composition is dependent upon the source plant and local flora. Moreover, propolis composition is completely variable creating a problem for the medical use and standardization (Marcucci, 1995, De Castro, 2001).
The term “propolis” is not characterizing with respect to the chemical composition, unlike the term “bee venom” for example (Bankova, 2005a), so that the biological studies with propolis must be carried out identifying its botanical sources and chemical composition as well.
Propolis samples, collected in the Beekeeping Section of the University, UNESP, Campus of Botucatu, SP, Brazil, were analysed by gas-chromatography (GC), gas chromatography–mass spectrometry (GC–MS) and thin layer chromatography (TLC), revealing that its main components are phenolic compounds (flavonoids, aromatic acids and benzopyranes), di- and triterpenes, essential oils, among others. Flavonoids are present in small quantities in Brazilian propolis (kaempferid, 5,6,7-trihydroxy-3,4′-dimethoxyflavone, aromadendrine-4′-methyl ether); a prenylated p-coumaric acid and two benzopyranes: E and Z 2,2-dimethyl-6-carboxyethenyl-8-prenyl-2H-benzopyranes; essential oils (spathulenol, (2Z,6E)-farnesol, benzyl benzoate and prenylated acetophenones); aromatic acids (dihydrocinnamic acid, p-coumaric acid, ferulic acid, caffeic acid, which are common for poplar propolis, 3,5-diprenyl-p-coumaric acid, 2,2-dimethyl-6-carboxy-ethenyl-8-prenyl-2H-1-benzo-pyran); di- and triterpenes were identified, among others.
In the temperate zone of the Northern Hemisphere bees collect propolis only in summer, including late spring and early autumn. In Brazil, propolis collection proceeds throughout the entire year and seasonal variations could be expected. This aspect has a practical application: propolis could be collected during the seasons with higher concentrations of biologically active compounds. Thus, propolis produced by Africanized (Apis mellifera L.) and Italian (Apis mellifera ligustica) bees all over a year was investigated in order to analyse its constitution as well as to compare its activities in different biological assays. Data showed that seasonal variations in propolis composition are not significant and are predominantly quantitative. This fact indicated that bees collect propolis from the same plant group, with a predominant vegetal source. Also, no differences were seen between Africanized and Italian bees, since propolis composition was qualitatively identical (Boudourova-Krasteva et al., 1997, Bankova et al., 1998a, Bankova et al., 1998b).
Bud exudates of different poplar species are the main sources of propolis in temperate zone, including Europe, Asia and North America. Samples originating from these regions are characterized by similar chemical composition; the most important constituents appeared to be phenolics: flavonoids, aromatic acids and their esters. In Russia, the main plant source of propolis is Betula verrucosa Ehrh., and its main biologically active substances are flavones and flavonols, whereas in Cuba and Venezuela, Clusia spp. are its main vegetal sources and polyprenylated benzophenones are the main active components (Bankova, 2005a).
The main vegetal source of propolis samples in Botucatu, SP, Brazil, is Baccharis dracunculifolia DC., followed by Eucalyptus citriodora Hook and Araucaria angustifolia (Bert.) O. Kuntze. Plants visited by bees in our apiary (UNESP, Campus of Botucatu) were collected, identified in the Department of Botany of our Institute. Leaves from Araucaria and Baccharis and trunk from Eucalyptus (parts of the plants preferably visited by bees) were investigated using GC–MS. The main components identified in Baccharis dracunculifolia and in propolis were almost the same: dihydrocinnamic acid, p-coumaric acid, prenyl- and diprenyl-p-coumaric acids and flavonoids in similar concentrations. On the other hand, some components were entirely absent in Baccharis exudates. Overall, the main components of Eucalyptus citriodora are aromatic acids, a class of compounds usually found in propolis, and sugars. Araucaria angustifolia exudates contained only traces of aromatic acids, consisting mainly of diterpenic acids (Bankova et al., 1999).
It is important to mention that the identification of these three plants does not exclude the possibility that other plants could also contribute as vegetal sources of propolis, however it has been reported that bees do not change its chemical composition in a specific geographic region because they visit essentially the same vegetal sources. Africanized bees have a preference for Baccharis dracunculifolia as sources of propolis in Brazil (Teixeira et al., 2005). Volatile substances, in the resiniferous ducts or gland trichomes, trigger bee attraction.
Bankova (2005b) reported that the distinct chemistry of propolis from different origins leads to the expectation that the biological properties of different propolis types will be dissimilar. Propolis is the defence of bees against infections, and the antibacterial and antifungal activities are mainly due to flavonones, flavones, phenolic acids and their esters for European propolis, while such activities are due to prenylated p-coumaric acids and diterpenes for Brazilian propolis. The fact that different chemistry leads to the same type of activity and in some cases even to activity of the same magnitude is amazing. A universal chemical standardization would be impossible, and for this reason, a detailed investigation of propolis composition, its botanical origin and biological properties are meaningful (Bankova, 2005a). The use of chemically characterized propolis samples for biological assays is the way to study its properties, and to do comparative studies. This author discussed this aspect very well, mentioning that the composition of the plant source determines propolis composition. Combined with the knowledge of active principles, it gives clues to standardization and quality control. Measurement of the concentrations of groups of active compounds instead of that of individual components would be the right approach in the case of propolis. There is still a lot of work to be done in order to achieve a reliable standardization on propolis types and formulate recommendations for practitioners as well as to connect a particular propolis sample to a specific biological activity (Bankova, 2005a, Bankova, 2005b).
Section snippets
Propolis action on macrophages
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 different 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 immunomodulatory action according to its dose, chemical composition and main components, and assay conditions.
Little was known about the
Conclusions
Propolis’ chemical composition as well as the identification of its vegetal sources enables us to carry out the assays with chemically characterized samples. The fact that no seasonal effect was seen on Brazilian propolis composition and variations were predominantly quantitative suggests to use samples collected in the same place all over the year, although in some regions, such as the temperate zone of the Northern Hemisphere, bees collect propolis mainly in summer. Biochemical,
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