# Antioxidative and Photoprotective Activity of _Pinus nigra_, _Pinus strobus_ and _Pinus mugo_
## 2 Materials and Methods  
Reagents. DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2\({}^{\prime}\)-azino-bis(3-ehylbenzothiazazine-6-sulphonic acid) diammonium salt were purchased from Sigma-Aldrich (St. Louis, MO, USA); Folin-Ciocalteu reagent was purchased from Merck, Darmstadt, Germany). Methanol, ethanol, propan-1-ol, propan-2-ol, sodium persulphate, and sodium carbonate anhydrous were from Chempur, Piekary Slaskie (Poland). Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) and gallic acid were from Sigma-Aldrich. All the reagents were of analytical grade. Raw material. The plant material applied in this study was collected from a natural state in July 2024. It consisted of needles of _Pinus strobus_ (53\({}^{\circ}\)58\({}^{\prime}\)58\({}^{\prime\prime}\) N, 15\({}^{\circ}\)56\({}^{\prime}\)46\({}^{\prime\prime}\) E), _Pinus mugo_ (54\({}^{\circ}\)00\({}^{\prime}\)28\({}^{\prime\prime}\) N, 15\({}^{\circ}\)59\({}^{\prime}\)50\({}^{\prime\prime}\) E), and _Pinus nigra_ (54\({}^{\circ}\)00\({}^{\prime}\)19\({}^{\prime\prime}\) N, 15\({}^{\circ}\)58\({}^{\prime}\)54\({}^{\prime\prime}\) E), growing near Bialogard (West Pomeranian Voivodeship, Poland). All raw material was dried at room temperature in a dark, ventilated place. Samples of raw material marked as PS-01/2024, PM-01/2024, and PN-01/2024, respectively, were deposited in Department of Cosmetic and Pharmaceutical Chemistry, Pomeranian Medical University in Szczecin. Evaluation of antioxidant activity of the extracts  
To determine antioxidant activity of the extracts, two frequently applied methods based on DPPH and ABTS radical reduction were used, as described in our previous papers [2; 40; 41]. Shortly, determination of antioxidant activity using DPPH method include preparation of DPPH solution in 96% (\(v/v\)) ethanol. For this purpose, 0.012 g of DPPH was dissolved in 100 mL of ethanol using magnetic stirrer. To obtain working solution of absorbance, 1.00 \(\pm\) 0.02 at 517 nm in 1 cm cuvettes, stock solution was diluted with 70% (\(v/v\)) ethanol. To 2500 \(\mu\)L of working solution of DPPH, 132 \(\mu\)L of the sample was added and thoroughly mixed. After 10 min incubation in dark at room temperature, absorbance at 517 nm was measured using Hitachi U-5100 spectrophotometer (Tokyo, Japan) [38; 40]. Each extract was evaluated in 3 replicates. The antioxidant activity of the samples was expressed as trolox equivalent antioxidant capacity (TEAC) in mg trolox/g raw material [41]. For this purpose, calibration curve of absorbance vs. trolox concentration was prepared (\(y=-1.1018\)x + 0.9712, R\({}^{2}\) = 0.9926). To evaluate antioxidant activity using ABTS method, to 2500 \(\mu\)L of 7 mM ABTS solution of absorbance 1.00 \(\pm\) 0.02 at 734 nm in 1 cm cuvettes, an aliquot of 25 \(\mu\)L of the extract was added and thoroughly mixed. The samples were then incubated at room temperature for 6 min. Absorbance was measured at 734 nm. Three samples were prepared for each extract [2; 42]. The antioxidant activity of the samples was expressed as trolox equivalent antioxidant capacity in mg trolox/g raw material [41]. For this purpose, calibration curve of absorbance vs. trolox concentration was prepared (\(y=-0.2395\)x + 0.9783, R\({}^{2}\) = 0.9936). The results are expressed as mean \(\pm\) standard deviation (SD). The polyphenol content in examined extracts was evaluated using Folin-Ciocalteu method [43] as described in our previous papers [42; 44]. In short, to 150 \(\mu\)L of examined extract, 150 \(\mu\)L of tenfold diluted commercial Folin-Ciocalteu reagent (Merck) was addedfollowed by adding 1350 uL of sodium carbonate aqueous solution and the same volume of water. The tubes were thoroughly mixed and incubated for 15 min at room temperature. After this time, the spectrophotometric measurement at 765 nm was performed. The polyphenol content in the samples was expressed as gallic acid equivalents in mg gallic acid/g raw material [42; 44].