# Antioxidative and Photoprotective Activity of _Pinus nigra_, _Pinus strobus_ and _Pinus mugo_
## 2 Materials and Methods
For this purpose, calibration curve of absorbance vs. gallic acid concentration was prepared (y = 0.0175x + 0.0102, R\({}^{2}\) = 0.9902). The results are expressed as GAE ( gallic acid equivalent) in mg gallic acid/g raw material. The in vitro _SPF_ (sun protection factor) was evaluated according to the method previously described by others [45; 46; 47]. Absorbance spectrum was taken in 1 cm quartz cuvettes using studied extracts diluted as follows: to 75 uL of extract, 2000 uL of solvent used for extraction was added. UV spectra were taken from 290 nm to 320 nm using Hitachi U-5100 (Tokyo, Japan) and DR6000 Hach (Hach-Lange, Wroclaw, Poland) spectrophotometers. _SPF_ was calculated using Masur equation:  
\[SPF=CF\sum_{290}^{320}EE(\lambda)\cdot I(\lambda)\cdot A(\lambda) \tag{1}\]  
where _CF_ is the correction factor (=10), _EE_(\(\lambda\)) is the erythromycinogenic effect of radiation with wavelength \(\lambda\), and _A_(\(\lambda\)) is the absorbance at wavelength \(\lambda\). The absorbance was determined at 290-320 nm every 5 nm. The values _EE_(\(\lambda\)) \(\cdot I(\lambda)\) are constants, and were determined by [PERSON] et al. [48]. Table 1 presents these values also used in this study to evaluate _SPF_ value of studied extracts. The _UVA_/_UVB_ ratio was calculated using calculated areas under absorption spectrum in _UVA_ region, i.e., 320-400 nm, and _UVB_ region, i.e., 290-320 nm. The following equation was applied:  
\[\textit{UVA}/\textit{UVB} = (\int_{320}^{400}A(\lambda)d\lambda/\int_{320}^{400}d\lambda)/( \int_{290}^{320}A(\lambda)d\lambda/\int_{290}^{320}d\lambda) \tag{2}\]  
These values could be applied to describe protection category according to the Boots Star Rating System [45]. Value of 0-0.2 is described as no protection, 0.21-0.4 as minimal, 0.41-0.6 as moderate, 0.61-0.8 as good, 0.81-0.9 as superior, and values above 0.91 as ultra. The critical wavelength \(\lambda_{cr}\) was calculated according to the following equation:  
\[\int_{290}^{\lambda_{cr}}A(\lambda)\cdot d\lambda=0.9\cdot\int_{290}^{400}A( \lambda)\cdot d\lambda \tag{3}\]  
where \(\lambda_{cr}\) is critical wavelength. According to [47] both parameters, i.e., _UVA_/_UVB_ ratio and the critical wavelength \(\lambda_{cr}\), are very reproducible even if different substrates are applied. \begin{table}
\begin{tabular}{c c} \hline
**Wavelength [nm]** & _EE(\(\lambda\)) \(\cdot I(\lambda)\)_ \\ \hline
290 & 0.0150 \\ \hline
295 & 0.0817 \\ \hline
300 & 0.2874 \\ \hline
305 & 0.3278 \\ \hline
310 & 0.1864 \\ \hline
315 & 0.0839 \\ \hline
320 & 0.0180 \\ \hline \end{tabular}
\end{table} 
Table 1: Values of _EE_(\(\lambda\)) \(\cdot I(\lambda)\)[45; 46]. Statistical analysis. The results are presented as mean \(\pm\) standard deviation (SD). As some groups of results differs from the normal distribution (checked by Lilliefors test), to evaluate the differences between the groups of results, non-parametric tests, i.e., Kruskal-Wallis and Mann-Whitney tests, were applied. Moreover, linear regression between the groups was also evaluated and correlation coefficients and their statistical significance were calculated. All the calculations and figures were carried out using ProStat v. 5.5 and Excel programs.