## 2 Results and Discussion
### Total Phenolic and Flavonoid  
The colourimetric Folin-Ciocalteu and aluminium chloride assays, which are repeatable, easy, and reliable tests, are widely performed in accordance with phenolic and flavonoid standards, assuming equal responses from all phenolic and flavonoid compounds, especially when estimating the total amount of that bioactive compound's classes in plant-based materials. The total phenolic and flavonoid content of the bee pollen samples that were studied are given in Table 2. \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Sample**} & **TPC** & **TFC** & **DPPH\({}^{\bullet}\)** & **ABTS\({}^{\bullet\star}\)** & **Reducing** \\  & **(mg QE/g)** & **(mcg QE/g)** & **(EC\({}_{\text{50:}}\) mg/mL)** & **(mM)** & **Power** \\  & **GAE/g)** & & & **Trolov/mg)** & **(mg GAE/g)** \\ \hline BP1 & 2.9 \(\pm\) 0.8 \({}^{\text{f}}\) & 0.7 \(\pm\) 0.1 \({}^{\text{e}}\) & 0.38 \(\pm\) 0.00 \({}^{\text{b}}\) & 0.98 \(\pm\) 0.02 \({}^{\text{e}}\) & 0.02 \(\pm\) 0.00 \({}^{\text{e}}\) \\ BP2 & 16.0 \(\pm\) 0.8 \({}^{\text{c}}\) & 4.8 \(\pm\) 0.8 \({}^{\text{a}}\) & 0.15 \(\pm\) 0.00 \({}^{\text{d}}\) & 1.33 \(\pm\) 0.02 \({}^{\text{b}}\) & 0.03 \(\pm\) 0.00 \({}^{\text{d}}\) \\ BP3 & 6.8 \(\pm\) 1.0 \({}^{\text{e}}\) & 2.5 \(\pm\) 0.3 \({}^{\text{c}}\) & 0.25 \(\pm\) 0.01 \({}^{\text{b}}\) & 1.22 \(\pm\) 0.15 \({}^{\text{c}}\) & 0.09 \(\pm\) 0.01 \({}^{\text{a}}\) \\ BP4 & 14.8 \(\pm\) 0.3 \({}^{\text{d}}\) & 1.2 \(\pm\) 0.2 \({}^{\text{d}}\) & 0.37 \(\pm\) 0.00 \({}^{\text{b}}\) & 1.59 \(\pm\) 0.15 \({}^{\text{a}}\) & 0.02 \(\pm\) 0.01 \({}^{\text{e}}\) \\ BP5 & 6.8 \(\pm\) 1.1 \({}^{\text{e}}\) & 2.3 \(\pm\) 0.3 \({}^{\text{c}}\) & 0.75 \(\pm\) 0.04 \({}^{\text{a}}\) & 1.11 \(\pm\) 0.05 \({}^{\text{d}}\) & 0.03 \(\pm\) 0.00 \({}^{\text{d}}\) \\ BP6 & 19.3 \(\pm\) 0.4 \({}^{\text{b}}\) & 0.7 \(\pm\) 0.0 \({}^{\text{e}}\) & 0.20 \(\pm\) 0.02 \({}^{\text{d}}\) & 1.35 \(\pm\) 0.05 \({}^{\text{b}}\) & 0.04 \(\pm\) 0.01 \({}^{\text{c}}\) \\ BP7 & 35.8 \(\pm\) 1.5 \({}^{\text{a}}\) & 3.1 \(\pm\) 0.1 \({}^{\text{b}}\) & 0.07 \(\pm\) 0.00 \({}^{\text{e}}\) & 1.34 \(\pm\) 0.16 \({}^{\text{b}}\) & 0.05 \(\pm\) 0.02 \({}^{\text{b}}\) \\ \hline \hline \end{tabular}  
* BP: bee pollen. GAE: gallic acid equivalent, QE: quercetin equivalent. EC\({}_{\text{50:}}\) 50% efficient concentration. Different letters (\({}^{\text{e}-\text{f}}\)) in each column indicate significant (\(p<0.05\)) differences between samples.  
\end{table} 
Table 2: Total phenolic (TPC), flavonoid compounds (TFC), and antioxidant activity of bee pollen. Figure 1: Microscopic view of the major pollen species seen in bee pollen samples. _Cardius_ sp. (**A**); _Olea_ sp. (**B**); _Cistaceae_ sp. (**C**); _Rubus_ sp. (**D**); _Echium_ sp. (**E**); and _Castana_ sp. (**F**). The botanical origin, as well as the geographical location from which the samples were collected, resulted in some significant differences in total phenolic content. For the TPC, the lowest value was recorded for BP1 samples, _Carduus_ sp., with 2.9 \(\pm\) 0.8 mg GAE/g, whereas the highest value was observed in the BP7 sample, corresponding with _Castanea_ sp., which had a value of 35.8 \(\pm\) 1.5 mg GAE/g. A high total phenolics value was also observed in monoflorl _Castanea_ bee pollen from Italy [24]. The BP6 and BP7 samples collected in Braganca had significantly (\(p\) < 0.05) higher values compared with samples from Nisa. BP4 and BP6, which presented the same botanical origin, _Rubus_ sp., from different geographical areas, showed a total phenolic amount of 14.8 \(\pm\) 0.3 and 19.3 \(\pm\) 0.4 mg GAE/g, respectively, which are in accordance with the values previously described for these pollens [24]. The differences observed can be related to the fact that they are different sub-individuals of the same species and pollen origin.