**4. Discussion**

The trend in the human diet is to consume foods with a high nutritional value, replacing the more conventional foods, as well as using them as supplements to provide one's diet the energy and essential nutrients required for proper mental and physical development [26]. In terms of human nutrition, bee pollen is considered a natural substance that constitutes a potential source of compounds with diverse nutritional and antioxidant relevance. Since pollen comes from different plant species, the evaluation of quality, safety and its characterization depend on the botanical and geographical origins. Hence the importance of their characterization and differentiation [1,15,27,28]. According to Campos et al. [29] the correct control of the processing procedures declares a consistent composition and could be considered as an indicator of outstanding quality and properties of food products. Some countries are establishing internal regulations with the intention of favoring the quality control of this product. In addition, local growth and the potential for the sale of bee pollen through exports has motivated the creation of international regulations to standardize the analytical methods for the physical-chemical and nutritional analysis of bee pollen [1].

The beneficial functions of phenolic compounds for human health have been demonstrated by reducing oxidative stress and inhibiting macromolecular oxidation; they positively collaborate in reducing the risk of degenerative diseases [30]. Within the phenolic compounds, the flavonoids in the bee pollen matrix have been recognized as quality factors in terms of antioxidant capacity [3,29]. However, phenolic composition of bee products is conditioned by their botanical origin, hence the need to evaluate the particular botanical characteristics of bee pollen based on geographical origin. In the present study, the bee pollen samples with higher presence of pollen types from *Erica*, *Castanea*, *Trifolium repens* type and *Echium* had the highest TPC and RSA expressed in DPPH. The bee pollen with the highest TFC had the highest antioxidant capacity expressed as ABTS, coinciding with the pollen types with the highest presence of *Campanula* type and *Lythrum*.

The Galician territory is characterized by a transition zone composed of different types of climate, resulting an environmental diversity that favors the abundance of plant resources for bees. The plant species of greatest beekeeping interest in Northwest Spain are from the families Fagaceae, Rosaceae, Fabaceae and Ericaceae. *Castanea* and *Rubus* taxa (Fagaceae and Rosaceae, respectively) produce a high quantity of nectar and pollen during the flowering stage (between May and July in the lowest lands and the mountains, respectively), with important productions of unifloral honeys of this botanical origin [31,32]. Bramble and blackberry plants are the most common of the *Rubus* genus (Rosaceae), with almost 300 species growing in Central European. *Rubus ulmifolius*, *R. caesius*, *R. sampaianus*, *R. praecox* or *R. henriquesii*, among others, are the most widespread in Galicia [33,34]. *Castanea* trees produce a great amount of nectar and pollen, and are considered one of the best beekeeping resources in this geographical area, given the important production of honeys of this botanical origin [32]. Species of the family Ericaceae, and other taxa of Fabaceae, mainly *Trifolium repens* and *Genista* type, constitute the typical scrub of the area, with important beekeeping interest [35]. Undoubtedly, the botanical richness that characterizes the Galician territory confers the chemical particularities of bee products.

The TFC quantified in bee pollen from Northwest Spain was similar to that found in samples from locations in southern Spain [17] and Portugal [7,30,36]. Pascoal et al. [15] reported a higher TPC in bee pollen produced in Portugal (Northeast area), with a dominant abundance of pollen types *Erica*, *Echium* and *Castanea*. The abundance of these pollen types probably increased the phenol content in the set of bee pollen samples, coinciding with the significant relationship found in the PCA of this research. *Erica*, *Castanea* and *Echium* turned out to be the plant variables with weight in PCA, coinciding with the variables close to TPC (Figure 3). Mârghitas et al. [37] reported similar TPC in honeybee-collected pollen pellets from Romania separated by colorimetry (*Crataegus monogyna*, *Centaurea cyanus*, *Salix*), but TFC was higher than the bee pollen analyzed in this study.

On the other hand, bee pollen produced in the Sonoran Desert (North of Tucson, USA) with a pollen spectra characterized by the presence of *Prosopis*, *Yucca*, *Washingtonia*, *Larrea*, *Mimosa* and Chenopodiaceae had higher TPC [2] compared to the pollen samples of the present study. Other researchers also reported higher TPC in bee pollen from Brazil, with a dominant pollen representation of *Cecropia*, *Eucalyptus*, *Mimosa pudica*, *Elaeis*, *Eupatorium* and *Scoparia* [10], and with dominant pollen *Cocos nucifera*, *Miconia*, *Spondias* and *Eucalyptus* [13]. Kostic et al. [28] characterized sunflower bee pollen (*Helianthus annuus*) with *Taraxacum officinale* as accompanying pollen from Serbia by palynological analysis, with lower TPC and TFC than bee pollen from Galicia. *Taraxacum officinale* was also found in the pollen spectrum of studied bee pollen in our study, coinciding with the samples of lower TPC and TFC (Figure 3).

There are several antioxidant compounds involved in the oxidation of the bee products, and the total antioxidant activity is the most accurate measurement [38]. The objective of the determination of the antioxidant activity of bee pollen is to analyze the generation of free radicals due to the disappearance of antioxidants. The DPPH and ABTS methods are the most used and most stable to evaluate the antioxidant capacity of hydrogen-donating antioxidants (aqueous radical scavengers) and chain-breaking antioxidants (lipid peroxyl radical scavengers), although they also show differences [38]. The positive relationship between TFC and antioxidant activity of the bee pollen samples was referenced [10]. The abundance of specific species identified in the extract of pollen samples with high radical scavenging activity and TPC was related, such as *Sinapis alba*, *Robinia pseudoacacia* [12], *Salix alba* [37] or *Mimosa* [2]. In our study, the abundance of *Castanea*, *Erica*, *Rubus*, *Campanula* type and *Genista* type differentiated the bee pollen samples with higher radical scavenging activity, highly related to TPC and TFC (Figure 4 and Table 4). Bee pollen of *Taraxacum officinale* from Romania [37] and Poland [12] despite high TPC showed low antioxidant capacity by DPPH assay. Several researchers have concluded that the antioxidant capacities are not clearly associated with its total phenolic content [12,30,37]. It is possible that the presence of particular phenolic compounds determines the increase in antioxidant activity [13]. The polyphenolic profile is variable in bee pollen, and the antioxidant activity of polyphenols depends on the number and location of the hydroxyl groups it contains in its chemical structure [38]. Its chemical structure is conducive to scavenging free radicals, because the hydrogen atom from the aromatic hydroxyl group readily donates to the radical species and the stability of the quinone structure it turned out to support an unpaired electron [39]. This strong association is mainly attributed to flavonoids and cinnamic acid derivatives [3,12]. Hence, the importance of relating the botanical origin with the individual phenolic compounds, because they can contribute to the discrimination of the antioxidant capacity of some pollen samples based on their floral origin.

It has also been documented that differences in environmental conditions, soil or plant physiology may interfere in the free radical reactions and the ability to remove reactive oxygen species in this bee product [3]. Some authors supported the close relationship between the antioxidant capacity and the collection period of bee pollen, highlighting a higher antioxidant activity in bee pollen produced in a period of more UV-intense, specifically from the beginning to the end of summer [2]. Therefore, in addition to correct management practices, the time of collection of the bee product will influence the chemical and functional characteristics (closely linked to the flowering period) and must be taken into account by the beekeeper.
