Phenolic Characterization of Northeast Portuguese Propolis: Usual and Unusual Compounds

Anal Bioanal Chem (2010) 396:887–897 DOI 10.1007/s00216-009-3232-8 ORIGINAL PAPER Phenolic characterization of Northeast Portuguese propolis: usual and unusual compounds Soraia I. Falcão & Miguel Vilas-Boas & Letícia M. Estevinho & Cristina Barros & Maria R. M. Domingues & Susana M. Cardoso Received: 8 June 2009 /Revised: 8 October 2009 /Accepted: 12 October 2009 /Published online: 10 November 2009 # Springer-Verlag 2009 Abstract In this study, an ethanolic extract from Portu- Introduction guese propolis was prepared, fractionated by high- performance liquid chromatography, and the identification Propolis or bee glue is a sticky dark-colored substance of the phenolic compounds was done by electrospray mass produced from the collected buds or exudates of plants spectrometry in the negative mode. This technical approach (resin) by bees (Apis melifera L.). The resin is masticated, allowed the identification of 37 phenolic compounds, salivary enzymes are added, and the partially digested which included not only the typical phenolic acids and material is mixed with beeswax and used in the hive to seal flavonoids found in propolis from temperate zones but also the walls, strengthen the borders of combs, and embalm several compounds in which its occurrence have never been dead invaders. Propolis is a building material, but it is also referred to in the literature. Four of the novel phenolic the most important “chemical weapon” of bees against compounds were methylated and/or esterified or hydroxyl- pathogen microorganisms [1]. ated derivatives of common poplar flavonoids, although six Propolis has been used as remedy by humans since peculiar derivatives of pinocembrin/pinobanksin, contain- ancient times [2]. In the last years, this product has been the ing a phenylpropanoic acid derivative moiety in their subject of intensive studies, highlighting its biological and structure, were also identified. Furthermore, the Portuguese pharmacological properties, such as the antimicrobial [3], propolis sample was shown to contain a p-coumaric ester antioxidative, antihepatotoxic [4], antitumoral, antiinflam- derivative dimer. matory [5], anti-HIV-1 [6], antineurodegenerative [7], and antituberculosis [8]. Propolis was also tested as food Keywords Phenolic compounds . Flavonoids . Phenolic preserver due to its bactericidal and bacteriostatic properties acids . Mass spectrometry. Electrospray ionization [9]. Furthermore, most of its components are natural constituents of food and recognized as safe substances [9]. S. I. Falcão : M. Vilas-Boas : L. M. Estevinho : The successful medical applications of propolis led to an S. M. Cardoso (*) increased interest in its chemical composition [10]. In general, CIMO—Escola Superior Agrária, resin comprising flavonoids and related phenolic acids Instituto Politécnico de Bragança, represent approximately half of the propolis constituents, Campus de Sta. Apolónia, Apartado 1172, 5301-855 Bragança, Portugal while beeswax, volatiles, and pollen represent approximately e-mail: [email protected] 30%, 10%, and 5%, respectively [11]. Still, the chemical : composition of the bee glue is extremely dependent on the C. Barros M. R. M. Domingues plants found around the hive, as well on the geographic and Centro de Espectrometria de Massa, Departamento de Química, Populus Universidade de Aveiro, climatic characteristics of the site. Buds from 3810-193 Aveiro, Portugal species are the main source of resins in Europe and North America propolis (“poplar type” propolis) [2]. Alternatively, S. M. Cardoso in regions where these plants are not native, other species Instituto Politécnico de Coimbra, Clusia Baccharis CERNAS—Escola Superior Agrária, such as in Cuba and in Brazil are used as Bencanta 3040-316 Coimbra, Portugal resin sources, increasing its diversity and complexity [12]. 888 S.I. Falcão et al. Less commonly, species such as Betula, Ulmus, Pinus, cinnamic acid, and p-coumaric acid were purchased from Quercus, Salix,andAcacia are also used [13]. Sigma Chemical Co (St Louis, MO, USA). Genkwanin More than 300 constituents have been identified in and luteolin were obtained from Lancaster Synthesis different types of propolis [11], with the phenolics being the (Morecambe, England). The analytical grade reagents most abundant compounds. In propolis from temperate formic acid and ethanol were obtained from Panreac zones, the most frequently reported phenolic components (Barcelona, Spain), and methanol and acetonitrile with include the flavonoids pinocembrin, galangin, and chrysin HPLC purity were purchased from Lab-Scan (Lisbon, and the phenolic acids caffeic acid, ferulic acid, and the Portugal). Water was treated in a Mili-Q water purification cinnamic acid [2]. Instead, the propolis from tropical zones, system (TGI Pure Water Systems, USA). in particular those from the Southeastern region of Brazil, were shown to be rich in prenylated phenylpropanoids [11], Samples although non-typical flavonoids from “poplar type” propolis, such as kaempferide and isosakuranetin, have been found Three distinct propolis samples were collected in the fall of [14, 15]. Moreover, Cuban propolis has recently caught the 2007 from Apis mellifera hives located in different apiaries attention of scientists because its peculiar enrichment in in the Northeast of Portugal. They were obtained after the polyisoprenylated benzophenones turns it chemically distinct honey extraction, by scratching the hive walls and frames, from both the European and the Brazilian bee glue [16]. followed by the removal of debris of wood and bees. These A large number of analytical methods have been used for propolis samples were then stored at −20 °C until analysis. the analysis of phenolic compounds in propolis, including spectrophotometry [17], thin-layer chromatography [18], gas Extraction of phenolic compounds chromatography–mass spectrometry (GC–MS) [19], high- performance liquid chromatography (HPLC) [20], liquid Prior to the extraction, the propolis was grounded and chromatography–mass spectrometry (LC–MS) [21–23], elec- homogenized. The samples were extracted with 80% of trospray ionization–mass spectrometry (ESI–MS) and elec- ethanol/water (1/10, v/v) at 70 °C for 1 h, the resulting trospray ionization-tandem mass spectrometry (ESI–MSn) mixtures were filtered, and the residues were re-extracted in [24], capillary electrophoresis [25], and nuclear magnetic the same conditions. After the second extraction, the resonance (NMR) [26]. Among these methods, MS with ESI filtrated solutions were combined, concentrated, frozen at is one of the most-used techniques in the research of natural −20 °C, and freeze-dried. products, as it is able to analyze complex mixtures because of its high selectivity. This method also provides adequate HPLC analysis structural information, and thus, it facilitates the structural identification of unknown compounds. The phenolic profile of the propolis extracts was analyzed The chemical composition of European propolis was by reversed-phase HPLC, based on the method of Gardana determined by GC–MS [11, 19], LC–MS, and ESI–MS [21– et al. [23] with some modifications, as described below. 24]. Also Moreira et al. [27] have recently described the The HPLC analysis was performed on a Knauer Smartline phenolic content and the antioxidant potential of Portuguese separation module equipped with a Knauer smartline propolis from two different regions, including the Northeast, autosampler 3800, a cooling system set to 4 °C, and a but still, their phenolic components were not elucidated. In this Knauer UV detector 2500. Data acquisition and remote context, this paper aims to characterize the phenolic compo- control of the HPLC system were done by ClarityChrom® sition of Northeast Portuguese propolis by ESI–MS. For that, software (Knauer, Berlin, Germany). The column was a the ethanolic extract was fractionated by reversed-phase 250 mm × 4 mm id, 5 µm particle diameter, end-capped HPLC, and the major phenolic compounds of each fraction Nucleosil C18 (Macherey-Nagel), and its temperature was were identified by detailed analysis of their fragmentation maintained at 30 °C. The mobile phase comprised (A) 0.1% pathways. New compounds were found in propolis for the first formic acid in water and (B) 0.1% formic acid in time, and their structure will be assigned in the present study. acetonitrile, which were previously degassed and filtrated. The solvent gradient started with 80% A and 20% B, reaching 30% B at 10 min, 40% B at 40 min, 60% B at Materials and methods 60 min, 90% B at 80 min, followed by the return to the initial conditions. For the HPLC analysis, the ethanolic Chemicals extract (10 mg) was dissolved in 1 mL of 80% of ethanol. All samples were filtered through a 0.2 μm Nylon The phenolic standards naringenin, quercetin, kaemp- membrane (Whatman) and 10 μL of each solution was ferol, pinocembrin, chrysin, caffeic acid, ferulic acid, injected. Chromatographic data were acquired at 280 nm, Phenolic characterization of Northeast Portuguese propolis 889 and the eluted fractions were manually collected according electrospray ionization–mass spectrometry (ESI–MS and to the visualization of the UV profile. MSn), even when its retention time matched with that of an available reference compound. Mass spectrometry analysis by ESI–MS and ESI–MSn ESI–MS and MSn The freeze-dried HPLC fractions were dissolved in methanol and directly injected into the ESI source by means of a syringe These analyses were carried out in the negative ion mode pump, at flow

Load more