# Antioxidative and Photop Protective In Vitro Potential of Lavandula Angustifolium
## 2 Materials and Methods
### Determination of Antioxidant Potential of the Lavender Extracts  
The antioxidant activity of the extracts was determined by applying two frequently applied techniques. They are based on the reduction of DPPH and ABTS radicals. These methods were described in detail in our previous papers [39; 42; 43]. Briefly, to determine the antioxidant potential using the DPPH technique, stock DPPH solution in ethanol was prepared as follows: an amount of 0.012 g of DPPH was dissolved in 100 mL of 96% (\(v/v\)) ethanol using a magnetic stirrer. A working solution with an absorbance of 1.00 \(\pm\) 0.02 measured at 517 nm (in 1 cm cuvettes) was prepared from the stock solution by dilution with ethanol. The determination of the antioxidant activity included the addition of 132 \(\upmu\)L of the extract to 2500 \(\upmu\)L of the working solution of DPPH and thorough mixing. After 10 min of incubation in the dark at ambient temperature, the absorbance of the samples at 517 nm was measured (Hitachi U-5100 spectrophotometer) [41; 42]. Three determinations of each extract were performed [20; 40]. The antioxidant activity of the extract was presented as Trolox equivalent antioxidant capacity (TEAC), i.e., in mg Trolox/g raw material (RM) [41]. To calculate the results, a calibration curve of absorbance vs. Trolox concentration (y = \(-\)0.9704x + 0.9093, R\({}^{2}\) = 0.9915) was applied.  
Evaluation of the antioxidant activity with the ABTS method included the addition of 20 \(\upmu\)L of the extract to 2500 \(\upmu\)L of 7 mM ABTS solution and thorough mixing. The samples were incubated for 6 min at room temperature. Measurements of absorbance were conducted at 734 nm. The 7 mM ABTS solution was prepared by dissolving 0.038 g of ABTS in 10 mL of 2.45 mM aqueous potassium persulphate solution. After 24 h, a period necessary to form the ABTS cation radical, the obtained solution was diluted with 50% (\(v/v\)) methanol in such a way that the absorbance of the ABTS solution in 1 cm cuvettes was 1.00 \(\pm\) 0.02 at 734 nm. Three samples of each extract were prepared. The antioxidant potential of the extracts was expressed as TEAC, i.e., in mg Trolox/g RM [41]. For this purpose, the absorbance vs. Trolox concentration calibration curve was prepared (y = \(-\)0.1803x + 1.0185, R\({}^{2}\) = 0.9942). The results are presented as arithmetical mean \(\pm\) standard deviation (SD).  
The in vitro sun protection factor (SPF) was determined using the slightly modified method applied in our previous study [41] according to a methodology described by other authors [44; 45; 46; 47]. To determine the absorbance spectrum, 1 cm quartz cuvettes were used. To obtain samples for SPF determination, 70 \(\mu\)L of extract was added to 2030 \(\mu\)L of the solvent applied for extraction and mixed. UV spectra from 290 nm to 320 nm were measured using Hitachi U-5100 (Hitachi High-Tech Corporation, Tokyo, Japan) and DR6000 Hach (Hach Lange GmbH, Dusseldorf, Germany) spectrophotometers. SPF was calculated using the Mansur equation presented below:  
\[SPF=CF\cdot\sum_{290}^{320}EE(\lambda)\,\cdot\,I(\lambda)\,\cdot\,A(\lambda)\]  
where \(CF\)--correction factor (=10); \(EE(\lambda)\)--erythmogenic effect of radiation with wavelength \(\lambda\); and \(A(\lambda)\)--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. [46]. Table 1 presents the values used in this study to evaluate the SPF value of the studied extracts.