## 2 Materials and Methods
### Antioxidants in TPW  
#### 2.7.1 Determination of Antioxidant Activity  
Extraction of antioxidants from TPW was conducted by conventional solvent extraction. Conditions of extraction were as follows: 250 mg of the sample was weighed and extracted with 10 mL of 1 M H\({}_{2}\)SO\({}_{4}\) for 90 min at 90 \({}^{\circ}\)C in closed vials, on a magnetic stirrer with continuous stirring. After extraction, reaction mixtures were filtered and after cooling made up to exactly 10 mL with 1 M H\({}_{2}\)SO\({}_{4}\). Clear extracts were used for determination of antioxidant activity and HPLC determination of particular polyphenic compounds using LC-MS-MS (2.6.2). Optimal sample-to-solvent ratio, temperature, and duration of extraction were identified during preliminary experiments and the solvent (0.1 M H\({}_{2}\)SO\({}_{4}\)) was chosen based on the available literature data. Extractions were conducted in duplicates, and each extract was analyzed in triplicate (n = 6). Antioxidant activity of TPW was assessed by three methodological approaches--determination of total reducing capacity/total phenols (TPs) by the Folin-Ciocalteu method [40]; TEAC (Trolox Equivalent Antioxidant Capacity) measuring radical scavenging activity against 2,2\({}^{\prime}\)-azino-bis(3-ethylbenzothazoline-6-sulfonate) radical cation (ABTS-\({}^{\ast}\)) [41] and Oxygen Radical Antioxidant Capacity (ORAC) [42]. For TP determination, an appropriately diluted TPW extract was mixed with Folin-Ciocalteu reagent and 6% Na\({}_{2}\)CO\({}_{3}\) solution in the 96-well plate. Reaction mixtures were incubated for 30 min, and absorbance was recorded at 725 nm. A calibration curve was established using gallic acid standards (ranging from 1 to 150 mgL\({}^{-1}\)), and TP values were reported as gallic acid equivalents (GAEs). For TEAC the reaction mixture contained adequately diluted extract and ABTS-\({}^{\ast}\) solution in the 96-well plate. The microplate was put into the microplate reader immediately after the addition of ABTS-\({}^{\ast}\) to TPW extracts. Plate was shaken for 60 s and an end-point absorbance was read at 732 nm after an additional 90 s of incubation at 30 \({}^{\circ}\)C (in total 3 min of incubation). The percentage of absorbance reduction was calculated according to Equation (2).  
\[\%I=\frac{(Amax-A3 min)}{Amax}\times 100 \tag{2}\]  
A calibration curve was constructed using Trolox standards (0-40 gL\({}^{-1}\)), and antioxidant activity was expressed in Trolox equivalents (TEs). ORAC was assessed by utilizing fluorescein as a fluorescent marker. The assay was conducted in a 96-well black microplate. Briefly, 5 umOL\({}^{-1}\) fluorescein was added to each well, followed by phosphate buffer (75 mmolL\({}^{-1}\), pH 7.0; blank), Trolox standard (6.25-100 umOL\({}^{-1}\)), or diluted sample. The mixture was pre-incubated at 37 \({}^{\circ}\)C for 10 min, after which the oxidation reaction was initiated by adding 25 \(\upmu\)L of 150 mmol\({}^{-1}\) 2,2\({}^{\prime}\)-Azobis(2-methylpropionamidine) dihydro-drochloride solution (AAPH). Fluorescence intensity (excitation: 493 nm; emission: 515 nm) was monitored over 60 min. The area under the curve (AUC) was calculated for each sample, and the net AUC was determined by subtracting the blank value. A standard curve was generated using known Trolox concentrations, and sample antioxidant activity was reported as TE. Absorbance and fluorescence measurements were performed using multimodal microplate reader (SpectraMax i3x and SpectraMax MiniMax 300 Imaging Cytometer, Molecular Devices, San Jose, CA, USA).