## 2 Materials and Methods
### Samples for Research
#### 2.2.2 Methods for Evaluating the Antioxidant Activity, Total, and Individual Polyphenolic Compounds  
Samples Preparation  
A sample of 1 g of lyophilized algae was extracted twice with 80% acetone (Merck KGaA, Darmstadt, Germany) containing 0.5% of formic acid (Merck KGaA, Darmstadt, Germany) [65] in solid to the solvent ratio (1:10 \(w/v\)) for 60 min with a constant stirring on a magnetic stirrer Ika RCT Basic (IKA\({}^{\text{\textregistered}}\)-Werke GmbH & Co. KG, Staufen (Staufen im Breisgau) Germany). The mixture was centrifuged for 20 min at 6000\(\times\)_g_ by centrifuge Nahita, model 2640/12 (Nahita, Beriain, Spain). The supernatants were collected and used for subsequent analysis of the antioxidant activity and total and individual polyphenolic compounds content. Oxygen Radical Absorbance Capacity (ORAC) Method  
An ORAC method developed by [PERSON] et al. [66] was used in this study with slight modifications. The capacity of particular antioxidants to neutralize peroxyl radicals was measured. AAPH (2,2\({}^{\circ}\)-azo-bis (2-amidinopropane) dihydrochloride) (Sigma-Aldrich Chemie GmbH, Darmstadt, Germany) was used as a free radical generator. A sample of 10 \(\upmu\)L of extract and 170 \(\upmu\)L of fluorescein (70 nmol/L) were tempered for 20 min at 37 \({}^{\circ}\)C in the apparatus. Twenty micrololters of AAPH (51.5 mM final concentration) was added to the reaction mixture. The reaction volume was 200 \(\upmu\)L, and all solutions were prepared in phosphate buffer (75 mM, pH 7.4). The mixture was shaken, and the fluorescence was evaluated every minute until a zero value was reached. To express the antioxidant activity, the results obtained for standard solutions of Trolox (6.25, 12.5, 25, 50, and 100 \(\upmu\)M, Sigma-Aldrich Chemie GmbH, Darmstadt, Germany) were used, based on which a standard curve was generated. Antioxidant concentration was directly proportional to the area under the decaying fluorescence curve, and the obtained data were presented in \(\upmu\)mol Trolox equivalents. The measurements were performed on a Fluostar OPTIMA fluorometer (BMG LABTECH, Offenburg, Germany). The excitation wavelength was 485 nm, and the emission wavelength was 520 nm. Hydroxyl Radical Averting Capacity (HORAC) Method  
The HORAC method developed by [PERSON] et al. [67], was used to measure the complexability of an antioxidant under Fenton reaction conditions caused by an interaction between Co (II) and H\({}_{2}\)O\({}_{2}\). The Co (II) solution was prepared in the following way: 15.7 mg CoF\({}_{2}\)\(\times\) 4H\({}_{2}\)O and 20 mg picolinic acid (Sigma-Aldrich Chemie GmbH, Darmstadt, Germany) were dissolved in distilled water. A sample of 10 \(\upmu\)L of extract and 170 \(\upmu\)L of fluorescein (final concentration 60 nM) prepared in phosphate buffer (75 mM, pH = 7.4) (Merck KGaA, Darmstadt, Germany) was heated at 37 \({}^{\circ}\)C for 20 min directly in the apparatus. Then, 10 \(\upmu\)L of H\({}_{2}\)O\({}_{2}\) (27.5 mM, final concentration) and 10 \(\upmu\)L of cobalt solution (Co (II), 230 \(\upmu\)M final concentration, (Merck KgaA, Darmstadt, Germany) were added to the reaction mixture. After shaking, the initial fluorescence was measured every minute until zero fluorescence was reached. Gallic acid solutions (100, 200, 400, 500, and 600 \(\upmu\)M, Sigma-Aldrich Chemie GmbH, Darmstadt, Germany) were prepared in phosphate buffer (75 mM, pH = 7.4, Merck KgaA, Darmstadt, Germany) and were used to construct the standard curve. The area under the decaying fluorescence curve of a gallic acid solution with a concentration of 1 umol is taken as one HORAC unit. The results were expressed in umol equivalents of gallic acid. The measurements were performed on a FLUOstar OPTIMA fluorimeter (BMG LABTECH, Offenburg, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.