Antioxidant activity of Portuguese honey samples: Different contributions of the entire honey and phenolic extract Isabel C.F.R. Ferreira * , Edmur Aires, João C.M. Barreira, Letícia M. Estevinho CIMO/Escola Superior Agrária, Instituto Politécnico de Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-855 Bragança, Portugal The antioxidant activity of Portuguese honeys was evaluated considering the different contribution of entire samples and phenolic extracts. Several chemical and biochemical assays were used to screen the antioxidant properties of entire honeys with different colour intensity and phenolic extracts: reducing power, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging capacity, and inhibition of lipid perox- idation using the b-carotene linoleate model system and the thiobarbituric acid reactive substances (TBARS) assay. The amounts of phenols, flavonoids, ascorbic acid, b-carotene, lycopene and sugars present in the samples were also determined. The highest antioxidant contents and the lowest EC 50 values for antioxidant activity were obtained in the dark honey. An analysis of variance was carried out to evaluate the influence of the colour intensity and extraction method in the antioxidant properties and phenolic contents. A discriminant analysis was also performed, giving satisfactory results once the six samples were clustered in six individual groups obtained through the definition of two discriminant analysis dimensions. 1. Introduction Honey is nectar collected from many plants and processed by honey bees (Apis mellifera). This natural product is widely appreci- ated as the only concentrated form of sugar available worldwide (FAO, 1996) and is also used as a food preservative (Cherbuliez & Domerego, 2003). Honey has been reported to contain about 200 substances (complex mixture of sugars, but also small amounts of other con- stituents such as minerals, proteins, vitamins, organic acids, flavo- noids, phenolic acids, enzymes and other phytochemicals) and is considered to be an important part of traditional medicine (White, 1979). It has been used in ethnomedicine since the early humans, and in more recent times its role in the treatment of burns, gastro- intestinal disorders, asthma, infected and chronic wounds, skin ul- cers, cataracts and other eye ailments has been ‘‘rediscovered” (Castaldo & Capasso, 2002; Marcucci, 1995; Molan, 1992; Orhan et al., 2003). This beneficial role is partially attributed to honeys antibacterial activity. However, since some of these diseases are a consequence of oxidative damage, it seems that part of the ther- apeutic properties of honey is due to its antioxidant capacity. Addi- tionally, the presence of hydrogen peroxide, as well as minerals (particularly copper and iron), in honey, may lead to the generation of highly reactive hydroxyl radicals as part of the antibacterial system (McCarthy, 1995; Molan, 1992); thus, it is evident that mechanisms must be available in honey to control the formation and removal of these reactive oxygen species. Furthermore, honey, as a source of antioxidants, has been proven to be effective against deteriorative oxidation reactions in food, caused by light, heat and some metals (McKibben & Engeseth, 2002), such as enzymatic browning of fruit and vegetables (Chen, Mehta, Berenbaum, Zangerl, & Engeseth, 2000), lipid oxidation in meat (Gheldof & Engeseth, 2002; McKibben & Engeseth, 2002; Nagai, Inoue, Kanamori, Suzuki, & Nagashima, 2006), and inhibit the growth of foodborne pathogens and food spoilage organisms (Mundo, Padil- la-Zakour, & Worobo, 2004; Taomina, Niemira, & Beuchat, 2001). Overall, honey serves as a source of natural antioxidants (Al-Mamary, Al-Meeri, & Al-Habori, 2002; Aljadi & Kamaruddin, 2004; Antony, Han, Rieck, & Dawson, 2000; Beretta, Granata, Ferre- ro, Orioli, & Facino, 2005; Gheldof, Wang, & Engeseth, 2002; Küçük et al., 2007; Nagai, Sakai, Inoue, Inoue, & Suzuki, 2001; Vit, Soler, & Tomas-Barberan, 1997), which play an important role in food preservation and human health by combating damage caused by oxidising agents e.g., oxygen, namely reducing the risk of heart dis- ease, cancer, immune-system decline, cataracts, different inflam- matory processes, etc. (The National Honey Board, 2003). The antioxidants present in honey include both enzymatic: cat- alase (Schepartz, 1966), glucose oxidase, peroxidase (Ioyrish, 1974) and non-enzymatic substances: ascorbic acid, a-tocopherol (Crane, 1975), carotenoids, amino acids, proteins, organic acids, Maillard reaction products (Al-Mamary et al., 2002; Aljadi & Kamaruddin,