## 2 Materials and Methods
### Samples for Research
#### 2.2.2 Methods for Evaluating the Antioxidant Activity, Total, and Individual Polyphenolic Compounds
Determination of Total Polyphenolic Content  
The determination was performed by the [PERSON] & Rossi's method [68], which is based on the reducing effect of phenolic compounds on a mixture of phosphotungstic (H\({}_{3}\)PW\({}_{12}\)O\({}_{40}\)) and phosphomolybtic (H\({}_{3}\)PMO\({}_{12}\)O\({}_{40}\)) acids (Sigma-Aldrich Chemie GmbH, Darmstadt, Germany), converted into a mix of oxides: wolfram blue (W\({}_{8}\)O\({}_{23}\)) and molybdenum oxide (Mo\({}_{8}\)O\({}_{23}\)) (Sigma-Aldrich Chemie GmbH, Darmstadt, Germany). The resulting blue color had a maximum absorption at \(\lambda\) = 765 nm and it was proportional to the amount of phenolic substances. The results were calculated according to a standard calibration curve constructed with gallic acid solutions and were expressed as mg gallic acid equivalents (GAE) in 100 g of fresh raw material. The determinations were performed on a VIS spectrophotometer Camspec M107 (Spectronic-Camspec, Ltd., Leeds, United Kingdom). Determination of Pigment Content  
For the analysis of chlorophyll \(a\) (C\({}_{\text{a}}\)), and chlorophyll \(b\) (C\({}_{\text{b}}\)), each sample was extracted with 100% acetone (Merck KgaA, Darmstadt, Germany) in a sample to a solvent ratio (1:50 \(w/v\)). The extraction was performed in an ultrasonic bath VWR USC100T (Singapore, Malaysia) with a frequency of 45 kHz, power 30 W at 40 \({}^{\circ}\)C for 20 min. The extraction procedure was repeated twice, and the acetone extracts were filtered through filter paper. The absorbance (A) of the combined final extracts was measured at three wavelengths: 662 nm, 645 nm, and 470 nm. The pigment amount was calculated according to Equations (1) and (2) reported by [PERSON] and [PERSON] [69]. \[C_{a}=11.76\cdot A_{662}-2.35\cdot A_{645} \tag{1}\]  
\[C_{b}=18.61\cdot A_{645}-3.96\cdot A_{662} \tag{2}\]  
HPLC Determination of Phenolic Acids and Flavonoids  
Gallic acid (3,4,5-trihydroxybenzoic acid), neochlorogenic acid, protocatehuic acid (3,4-dihydroxybenzoic acid), chlorogenic acid, catechin, vanillic acid (4-hydroxy-3-methoxy-zoic acid), caffe acid (3,4-dihydroxycinnamic acid), epicatechin, _p_-coumaric acid, ferulic acid, rutin, ellagic acid, naringin, myricetin, quercetin, cinnamic acid, naringenin, and kaempfer were quantified on a Nexera-I LC2040C Plus UHPLC system (Shimadzu Corporation, Kyoto, Japan) with a UV detector and a binary pump. A wavelength of 280 nm was used to determine most of the analytes, whereas chlorogenic acid, caffe acid, ferulic acid, rutin, quercetin, and kaempfer were detected at 360 nm. The separation of phenols and flavonoids was performed on an Agilent TC-C18 column (5 um, 4.6 mm \(\times\) 250 mm) at 25 \({}^{\circ}\)C. The mobile phases consisted of 0.5% acetic acid (A) and 100% acetonitrile (B) at a flow rate of 0.8 mL/min. The gradient condition started with 14% B, between 6 min and 30 min linearly increased to 25% B, then to 50% B at 40 min. The sample injection volume was 20 um. The results were calculated with standard curves for each analytical standard and expressed as mg/100 g sample. Protein Analysis  
The crude protein content was analyzed by the micro-Kjeldahl method (N \(\times\) 6.25). The determination of nitrogen expressed as ammonia content of the digested sample was performed by the acetylacetone-formaldehyde colorimetric method using ammonium sulfate as a standard [70].