Bioactive constituents of propolis

Propolis is a complex mixture of resinous, gummy, and balsamic materials that honeybees collect from living plants and mix with wax and salivary secretions. It has become the subject of intense biological and chemical studies in the last decades. The numerous biological activities of propolis have been confirmed experimentally in both in vivo and in vitro systems, including antibiotic, antitumor, anti-inflammatory, hepatoprotective, and immunomodulatory effects. Although the biological activity of propolis, especially its antimicrobial activity, is always present, in samples from different geographic and climatic zones this activity is due to different chemical constituents. The chemical composition of propolis is very complex and depends on the plants present in the areas where it is collected. It is important to have detailed and reliable comparative data on biological activities combined with chemical data in order to decide whether some specific areas of application of a particular propolis can be formulated as preferable. More than 200 compounds have been identified in propolis from different geographical origins including phenolic acids, flavonoids, terpenes, lignans, amino acids, fatty acids, vitamins, and minerals. In Europe, North America, and the non-tropical regions of Asia the main source of propolis is the resinous exudates of the buds of poplar trees (Populus species). These samples of propolis are characterized by similar chemical composition – the main compounds belong to the phenolic class and include flavonoid aglycones, aromatic acids, and their esters. Brazilian green (Alecrim) propolis, which originates from Baccharis dracunculifolia (Asteraceae) is characterized by the presence of prenylated derivatives of p-coumaric acid and o-hydroxy-acetophenone. These substances are typical chemical constituents of Brazilian propolis, and they are not present in propolis from other regions. Flavonoids different from those in European propolis, as well as diterpenes and lignans, have also been found in samples of Brazilian propolis. Red propolis is typical of Cuba, where its plant source was identified as Clusia nemorosa (Clusiaceae) and of Venezuela, where bees collect it from C. scrobiculata. Typical components of Cuban propolis are polyprenylated benzophenones, and this makes Cuban propolis different from both European and Brazilian propolis. Propolis, independently of its plant source and chemical composition, always possesses antimicrobial and antioxidative activity. The antimicrobial activity of propolis is attributed to the presence of flavonoids such as pinocembrin, galangin, pinobanksin, pinobanksin-3-acetate, and cafeic acid ester. Propolis with strong antioxidant activity contained compounds such as kaempherol, caffeic acid, and phenethyl caffeate. The concentration of bioactive principles in propolis may vary substantially according to the origin of the sample, and such differences may affect its biological activities. Therefore, the chemical constituents of propolis have to be identified and quantified using appropriate methods. These methods involve sample preparation (extraction) and analytical separation and quantification. Ethanol is the most commonly used solvent in propolis extraction, especially at concentrations of 70% and 80%. Some other solvents, such as methanol, hexane and acetone, and chloroform, have also been used for the extraction of propolis. Spectrophotometric methods are assumed to be especially useful for rapid quantification of phenolics in propolis samples. High performance liquid chromatography (HPLC) is currently the most popular and reliable analytical technique for the quantitative and qualitative analysis of phenolics. With the advent of modern chromatographic techniques associated with mass spectrometry or even with nuclear magnetic resonance spectroscopy, many compounds have been isolated and identified in propolis. Capillary electrophoresis (CE) is a new methodological approach in the chemical analysis of propolis, and it is used as an alternative technique to HPLC. Mass spectroscopy (MS) is usually used in conjunction with gas chromatography (GC-MS) for the analysis of volatile and semi-volatile components of propolis. Electrospray ionization (ESI) has revolutionized the way molecules are ionized and transferred to mass spectrometers, and this has greatly expanded the applicability of mass spectrometry for a variety of new classes of molecules with thermal instability, high polarity, and high mass. ESI-MS in the negative ion mode is an effective fingerprinting method for high-throughput screening of propolis.