Antioxidant properties, total phenols and pollen analysis of propolis samples from Portugal Leandro Moreira, Luís G. Dias, José Alberto Pereira, Leticia Estevinho * CIMO, Escola Superior Agrária, Instituto Politécnico de Bragança, Campus Santa Apolónia, Apartado 1172, 5301-855 Bragança, Portugal article info Article history: Received 5 July 2008 Accepted 26 August 2008 Keywords: Portugal Propolis Polinic analysis Total phenols Antioxidant activity abstract Pollen analysis, total phenols content and antioxidant activity were studied for the first time in Portuguese propolis samples from Bornes and Fundão regions. Total phenols content was determined by colorimetric assay and their amount was of 329 mg/g of GAE in Bornes sample and 151 mg/g of GAE in Fundão propolis. The antioxidant capacity of propolis extracts was assessed through the scavenging effects on DPPH (2,2- diphenyl-1-picrylhydrazyl) and reducing power of iron (III)/ ferricyanide complex assays. A concentra- tion-dependent antioxidative capacity was verified in DPPH and reducing power assays. Low values of EC 50 on DPPH scavenging assay were obtained for Bornes and Fundão propolis (of 6.22 lg/mL and 52.00 lg/mL, respectively). For reducing power the values were 9.00 lg/mL, for Bornes propolis, and 55.00 lg/mL, for Fundão propolis. The high activity of propolis from Bornes could be related with their dif- ferent pollen composition. The results obtained indicate that Portuguese propolis is an important source of total phenols showing antioxidant properties that could be beneficial for human health. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Propolis is a product based on resins collected from resinous sprouts and exudates of some plants by bees of Apis mellifera specie. When the bees reap the propolis, they mix the resinous substance collected from plants with the 13-glicosidase enzyme of their sali- va, causing the hydrolysis of the glucosyl flavonoids, originating flavonoids aglycones (Pereira et al., 2002). In the beehive, the prop- olis is used by the bees to defend them from the invaders (causing death by asphyxia) and promotes conservation of their bodies, pro- tecting the beehive from the resultant plagues of putrefaction. An- other propolis function is the thermal isolation of the beehive, being used to fill eventual cracks or apertures (Bankova et al., 2002). In the past few years, the suspected toxicity of some synthetic compounds used in food has raised the interest in natural products (Stone et al., 2003). Some industries, such as those related to food additive production, cosmetics, and pharmaceuticals, have increased their efforts in obtaining bioactive compounds from nat- ural products by extraction and purification. Antioxidant com- pounds can increase shelf life by retarding the process of lipid peroxidation, which is one of the major reasons for deterioration of food products during processing and storage (Halliwell, 1997; Halliwell and Gutteridge, 1999). Propolis has been used in the traditional medicine since the pri- mordial times of humanity, having acquired popularity between Egyptians Arabs, Greeks, and many other civilizations (Abd El Hady and Hegazi, 2002). Those biological and therapeutic actions were attributed to their phenolic composition (Lahouel et al., 2004). In fact, different works attribute important properties to propolis, namely antibacterial action against different pathogenic bacteria (Kujumgiev et al., 1999), antifungal and anti-inflammatory (Wang et al., 1993), anti-viral (Amoros et al., 1994), curative, anesthetical and anti-tumoural properties (Kimoto et al., 2001; Matsuno, 1995). Recently, Kim et al. (2005) showed that propolis is able to inhibit the action of the enzyme hyaluronidase, allowing slow aging of cells. For all these reasons, this natural product awakened interest in the pharmaceutical industry, mainly in Asian countries, being propolis introduced in different products for human consumption like drinks, foods and cosmetics (Pereira et al., 2002). The studies reported above describe propolis characterization, their biological properties and the action of their composition. However, no previous studies were reported about this Portuguese hive product. In this work, and for the first time, the Portuguese propolis, from two different regions, were studied regarding their total phenols content, pollen characterization and antioxidant activities. Antioxidant potential was accessed by the reducing power assay and the scavenging effect on DPPH (2,2-diphenyl-1- picrylhydrazyl) radicals. 2. Materials and methods 2.1. Samples Two different samples of propolis were analysed. Bornes sample from Serra de Bornes in the Northeast of Portugal and Fundão sample proceed from the Beira Inte- rior Region in the Centre of Portugal (Fig. 1). Between regions, marked differences were registered in terms of climatic conditions and vegetation. 0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.08.025 * Corresponding author. Tel.: +351 273 303342; fax: +351 273 325405. E-mail address: leticia@ipb.pt (L. Estevinho). Food and Chemical Toxicology 46 (2008) 3482–3485 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox