Antioxidant activity of Portuguese honey samples: Different contributions of the entire honey and phenolic extract

Abstract

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 peroxidation using the β-carotene linoleate model system and the thiobarbituric acid reactive substances (TBARS) assay. The amounts of phenols, flavonoids, ascorbic acid, β-carotene, lycopene and sugars present in the samples were also determined. The highest antioxidant contents and the lowest EC₅₀ 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.

Introduction

Honey is nectar collected from many plants and processed by honey bees (Apis mellifera). This natural product is widely appreciated 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 constituents such as minerals, proteins, vitamins, organic acids, flavonoids, 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, gastrointestinal disorders, asthma, infected and chronic wounds, skin ulcers, cataracts and other eye ailments has been “rediscovered” (Castaldo and 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 therapeutic properties of honey is due to its antioxidant capacity. Additionally, 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 and Engeseth, 2002, McKibben and Engeseth, 2002, Nagai et al., 2006), and inhibit the growth of foodborne pathogens and food spoilage organisms (Mundo et al., 2004, Taomina et al., 2001). Overall, honey serves as a source of natural antioxidants (Al-Mamary et al., 2002, Aljadi and Kamaruddin, 2004, Antony et al., 2000, Beretta et al., 2005, Gheldof et al., 2002, Küçük et al., 2007, Nagai et al., 2001, Vit et al., 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 disease, cancer, immune-system decline, cataracts, different inflammatory processes, etc. (The National Honey Board, 2003).

The antioxidants present in honey include both enzymatic: catalase (Schepartz, 1966), glucose oxidase, peroxidase (Ioyrish, 1974) and non-enzymatic substances: ascorbic acid, α-tocopherol (Crane, 1975), carotenoids, amino acids, proteins, organic acids, Maillard reaction products (Aljadi and Kamaruddin, 2004, Al-Mamary et al., 2002, Baltrušaityte et al., 2007, Bertoncelj et al., 2007, Gheldof et al., 2001, Gheldof et al., 2002, Schramm et al., 2003, The National Honey Board, 2003), and more than 150 polyphenolic compounds, including flavonoids, flavonols, phenolic acids, catechins, and cinnamic acid derivatives. In the literature, several studies for the identification and quantification of antioxidant components of honeybee products have been reported (Buratti et al., 2007, Ferreres et al., 1994, Gheldof et al., 2002).

Many methods for determining the antioxidative activity in honey have been used, e.g., determination of total phenolic content (Beretta et al., 2005), radical formation and following scavenging as in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical-scavenging activity measurements (Aljadi and Kamaruddin, 2004, Chen et al., 2000, Gheldof and Engeseth, 2002, Gheldof et al., 2002, Gülçin et al., 2003, Kefalas et al., 2001, Meda et al., 2005, Nagai et al., 2001, Taomina et al., 2001), the ferric-reducing/antioxidant power (FRAP) assay (Aljadi & Kamaruddin, 2004) and enzymatic or non-enzymatic measurements of lipid peroxidation inhibition (Chen et al., 2000, McKibben and Engeseth, 2002, Nagai et al., 2001).

Although it has already been demonstrated that honey has antioxidant activity and different antioxidant compounds, nothing is reported about the different contributions of the entire honeys and their phenolic extracts to those properties. Accordingly, in this work, the antioxidant properties of the entire samples, and phenolics extracts were evaluated through several chemical and biochemical assays: DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity, reducing power, inhibition of β-carotene bleaching and inhibition of lipid peroxidation in pig brain tissue through formation of thiobarbituric acid reactive substances (TBARS). This is also the first study reporting antioxidant activity of Portuguese honey, particularly from a region with high amounts of this natural product (Northeast Portugal).