## 2 Materials and Methods
### Antioxidant Activity  
#### 2.8.1 Reducing Power  
The reducing power was assayed according to the procedure described [28], with some modifications, and adapted to the 96-well microplate scale. Trolox was considered as the antioxidant reference. The stock solution of Trolox was prepared in methanol, whilst three bark polar extracts of each _Pinus_ spp. were dissolved in methanol/water (50:50, \(v/v\)) at 1 mg mL\({}^{-1}\). Different volumes of extract and standard stock solutions were added to deionised water up to 1000 \(\upmu\)L and then mixed with 1000 \(\upmu\)L of 0.2 M phosphate buffer solution (pH 6.6) and 1000 \(\upmu\)L of 1% (\(w/v\)) potassium ferrocyanide solution, obtaining the concentration ranges of 3.4-49.5 \(\upmu\)g mL\({}^{-1}\) and 3.6-71.1 \(\upmu\)g mL\({}^{-1}\) for the extracts and Trolox, respectively. The reactional mixture was kept at 50 \({}^{\circ}\)C for 20 min. Thereafter, 1000 \(\upmu\)L of 10% (\(w/v\)) trichloroacetic acid solution was added to stop the redox reaction, and the mixture was left to cool to room temperature for 10 min. An aliquot of 125 \(\upmu\)L of  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Standard** & \(\lambda\) **(nm) 1** & **Concentration** & **Linear Regression** & **LOD 3** & **LOQ 4** \\  & **Range (\(\upmu\)g mL\({}^{-1}\))** & **Equation 2** & & **(\(\upmu\)g mL\({}^{-1}\))** & **(\(\upmu\)g mL\({}^{-1}\))** \\ \hline Catechin & 280 & 0.50–30.29 & \(y=30528x-19621\) & 0.9973 & 2.08 & 6.93 \\ Gallic acid & 280 & 0.52–31.20 & \(y=104717x-38562\) & 0.9986 & 1.56 & 5.19 \\ Naringenin & 280 & 0.51–41.04 & \(y=394428x+172880\) & 0.9999 & 0.44 & 1.48 \\ Protacatechic acid & 280 & 0.50–30.26 & \(y=112312x-18937\) & 0.9997 & 0.66 & 2.20 \\ Quercetin & 370 & 0.45–27.05 & \(y=364286x-66014\) & 0.9949 & 2.55 & 8.51 \\ \hline \hline \end{tabular}  
* Wavelength used in the HPLC-UV quantification. 2 \(y\) and \(x\) represent peak area and concentration in \(\upmu\)g mL\({}^{-1}\), respectively. 3 LOD, limit of detection. 4 LOQ, limit of quantification.  
\end{table}
Table 1: UHPLC-UV standard data used for the quantitative analysis of the identified compounds in _Pinus_ spp. bark polar extracts.  
the mixture was transferred to the microplate well, and 125 \(\mu\)L of deionised water and 25 \(\mu\)L of 0.1% (\(w/v\)) ferric (III) chloride solution were added. Then, the absorbance was recorded against the blank at 690 nm in a Thermo Scientific Multiskan(tm) FC microplate reader. An increased absorbance means a higher reducing power of sample. The half-maximal inhibitory concentration (IC\({}_{50}\)) was considered as the sample concentration at which the absorbance was measured as 0.500, being calculated through the respective linear regression equations from the graph plotting absorbance at 690 nm against the sample concentration. Each concentration was tested in triplicate.  
The reducing power represents a ferric-ion-based total antioxidant capacity assay, in which the potassium ferricyanide (K\({}_{3}\)[Fe(CN)\({}_{6}\)]) is the oxidant species and reacts with the antioxidant, forming potassium ferrocyanide (K\({}_{4}\)[Fe(CN)\({}_{6}\)]) and the oxidized antioxidant (Equation (1)). Then, potassium ferrocyanide combines with ferric chloride (FeCl\({}_{3}\)), yielding the Prussian blue chromophore (KFe[Fe(CN)\({}_{6}\)]) (Equation (2)). The reducing power of the tested extract or compound increases as much as the absorbance of Prussian blue [29].  
\[K_{3}[Fe(CN)_{6}]+ antioxidant\stackrel{{\rightarrow}}{{\leftarrow}}K_{4} [Fe(CN)_{6}]+ oxidized antioxidant \tag{1}\]  
\[K_{4}[Fe(CN)_{6}]+FeCl_{3}\stackrel{{\rightarrow}}{{\leftarrow}}KFe [Fe(CN)_{6}] \tag{2}\]