## 3 Materials and Methods
### Biological Activity  
#### 3.6.1 Antioxidant Activity  
Three different assays were used to assess the in vitro antioxidant potential of bee pollen, as follows: the DPPH\({}^{\bullet}\) and ABTS\({}^{\bullet+}\) radical scavenging assays, and the reducing power assay. The DPPH\({}^{\bullet}\) free radical scavenging activity of bee pollen was performed in accordance with the method described by [PERSON] et al. [15], with some modifications. An aliquot comprising 0.15 mL of the pollen extract solutions, with concentrations ranging from 0.034 to 0.5 mg/mL, was mixed with 0.15 mL of DPPH\({}^{\bullet}\) (0.024 mg/mL), and the absorbance was read at 515 nm using an ELX800 Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The percentage of radical inhibition was calculated using the following equation:  
\[\%\ \text{Inhibition}=[(\text{A}_{\text{DPPH}}-\text{A}_{\text{Sample}})( \text{A}_{\text{DPPH}})\times 100] \tag{1}\]  
The amount of antioxidant needed to reduce the initial DPPH\({}^{\bullet}\) concentration by 50\(\%\) (EC\({}_{50}\)) was determined by plotting the inhibition percentage against the extract concentration.  
Reducing the power analysis, which measures the reduction of the ferric ion (Fe\({}^{3+}\))-ligand complex to the ferrous (Fe\({}^{2+}\)) complex, was performed in accordance with the method described in [PERSON] et al. [43]. The bee pollen extract (5 mg/mL) was mixed with a sodium phosphate buffer (pH = 6.6, 1.25 mL) and 1.25 mL of potassium ferricamide (1%), and the mixture was incubated at 50 \({}^{\circ}\)C for 20 min. Then, trichloroacetic acid (10%, 1.25 mL) was added. After centrifuging at 3000\(\times\) g for 10 min, a volume of 1.25 mL was removed from the top solution to a new tube, and it was combined with 1.25 mL of water and 0.25 mL of 0.1% ferric chloride. The absorbance of the final solution was then read as 700 nm. The results were expressed in milligrams of gallic acid equivalent per gram of sample (mg GAE/g).  
The ABTS\({}^{\bullet+}\) [2,2\({}^{\prime}\)-azinobis-(3-ethyl-benzothazoline-6-sulfonic acid)] assay was conducted to determine whether the bee pollen samples could scavenge the ABTS radical cation, using Trolox as the standard; the assay was performed in accordance with the previously described method, with modifications [49]. A stock ABTS\({}^{\bullet+}\) solution was prepared from 7 mM ABTS\({}^{\bullet+}\) and 2.45 mM sodium persulfate in deionized water. The working ABTS\({}^{\bullet+}\) solution was diluted with distilled water to obtain an absorbance of 0.70 (\(\pm\) 0.02)at 734 nm. Bee pollen extract (5 mg/mL, 40 \(\upmu\)L) was added to the diluted ABTS\({}^{\bullet+}\) solution (960 \(\upmu\)L) and mixed immediately. The mixture was incubated for 10 min in the dark, and the absorbance was determined at 734 nm. The percentage of inhibition was calculated using the following formula:  
\[\%\ \text{Inhibition}=((1-\text{A}_{\text{Sample}}))/\text{A}_{\text{Control}}) \times 100 \tag{2}\]  
\(\text{A}_{\text{Sample}}\) was the absorbance of the ABTS\({}^{\bullet+}\) solution containing the sample, and \(\text{A}_{\text{control}}\) was the absorbance of ABTS\({}^{\bullet+}\) solution without the sample. The Trolox equivalent antioxidant capacity (TEAC) of the bee pollen samples (mM Trolox/mg bee pollen extract) was calculated using the calibration curve, as follows:  
\[\text{TEAC (mM Trolox/mg BP extract)}=((\%\ \text{Inhibition Sample-b)}/\text{a}\ \times(\text{aliquot}\ \text{volume (mL)})/(\text{bee pollen weight (mg)}) \tag{3}\]  
where a and b are the slope and intercept of the calibration curve.