## Results and Discussion
### Bio-Activity Parameter Values of Portuguese Honey Samples  
Spectrometric assays like DPPH and Folin-Ciocalteu may be routinely used for the in vitro characterization of honey bio-functional properties. The DPPH assay evaluates the antioxidant activity of a sample. The free radical can be neutralized either by direct reduction (via electron transfer) or by radical quenching (via H atom transfer) [21]. It has been reported previously in the literature that dark coloured honeys usually have a higher in vitro antioxidant activity [26; 28; 38]. Among Portuguese honey samples analysed heather honey (darkest honey) showed the higher in vitro antioxidant activity followed by chestnut and eucalyptus honeys (Table 2). The higher in vitro antioxidant activity of heather honey, compared to other types, has been previously reported [14; 21] in agreement with the results of the present study. In general Portuguese honey samples showed a higher in vitro antioxidant activity compared to Polish lime, nectar-honeydew, rape, honeydew, acacia and buckwheat honeys [38]. Greek pine and fir honeys recorded in vitro antioxidant activity values within the range of eucalyptus honeys analysed in the present work [28]. Another important issue to discuss is that the antioxidant activity not only depends on the botanical origin of honey but also on the geographical origin (Table 2). Parameters such as plants' defence against several environmental factors including climatic conditions, ultraviolet radiation, temperature, water stress or mineral nutrient availability may imply significant parameters that affect honey in vitro antioxidant activity [21; 28]. The method of Folin-Ciocalteu is largely used to evaluate total phenolic content despite the arising interferences of this assay since the reagent mixture (phosphotungstic acid and phosphomolybic acid) also reacts with other non-phenolic reducing compounds (ascorbic acid, amino acids, etc.) and affects absorbance measurements, therefore leading to an overestimation of the phenolic content [14]. However, it is a simple and effective procedure to estimate total phenolic content of foodstuffs with bio-functional properties. The lowest TPC content was recorded for eucalyptus honey from Esposende region (Braga) (ca. 422 mg GAE/kg), while the highest phenolic content (ca. 1418 mg GAE/kg) was found in chestnut honey from Vinhais region (Braganca), followed by heather honey (ca. 1380 mg GAE/kg) from Vila Nova de Ceira region (Coimbra) being much higher than the results reported for total phenolic content of heather honey (ca. 500 mg GAE/kg) from Boticas region in Portugal [21]. Some other researchers have also reported a trend for heather honeys to show a higher total phenolic content [14; 20; 39]. Chestnut honey from Croatia [40] recorded a total phenolic content of ca. 430 mg GAE/kg. Such values are much lower compared to the results obtained for Portuguese chestnut honeys. But what is the main reason for such fluctuations? At first, the analytical instrumentation and methodology have definitely a significant effect on the quantification of such compounds. At the same time and in a greater extent, climatic and soil conditions may fully describe such phenomena. Bees collect pollen from different plants grown in different regions. Pollen is a good source of numerous phytochemicals. In that sense, polyphenols and other bio-functional compounds are transferred to honey through pollen, which can justify the observed fluctuations in TPC content among honey samples of different botanical and geographical origins [21]. Finally, the antimicrobial activity of honey against a wide range of microbial contaminants (aerobic mesophilic bacteria, moulds and yeasts, faecal coliforms, sulphite-reducing clostridia, _Salmonella_, etc.) along with its wound healing boosters should be considered in its overall bio-functionality [19; 41].