## 2 Materials and Methods
### Analyses of the Extracts  
The obtained extracts were cooled at 20 \(\pm\) 2 \({}^{\circ}\)C and centrifuged for 10 min at 10,000 rpm (Thermo Scientific IEC CL31R Multispeed Laboratory centrifuge, Spinea, Italy). The supernatants were recovered, filtered through PVDF (polyvinylidene difluoride) membrane filters (45 \(\upmu\)m) to eliminate any solid particles that could affect the results of the subsequent spectrophotometric and chromatographic analyses. The filtered supernatants were stored at temperatures below \(-\)18 \({}^{\circ}\)C until analysis.  
The TPC was determined using the Folin-Ciocalteu method [26]. Briefly, 125 \(\upmu\)L of previously diluted extract (1:5) was added to 500 \(\upmu\)L of distilled water and 125 \(\upmu\)L of FC reagent. After 5 min, 1250 \(\upmu\)L of Na\({}_{2}\)CO\({}_{3}\) (70 g/L) was added and the final volume was adjusted to 3000 \(\upmu\)L with distilled water. The absorbance was measured at 760 nm, and results were expressed as mg of gallic acid/g of dry matter.  
The DPPH radical scavenging activity was measured according to the method of [PERSON] et al. [27] with some modifications. Briefly, 100 \(\upmu\)L of each extract was added to 3900 \(\upmu\)L of DPPH solution (40 mg/L) in methanol, and placed in the dark. A blank was prepared by adding 100 \(\upmu\)L of distilled water to 3900 \(\upmu\)L of DPPH. The absorbance of extracts and blank was measured at 515 nm after 90 min. The results of antioxidant activity were expressed as mg of Trolox per g of dry matter. Quantification was performed using a calibration curve prepared with increasing concentrations of Trolox.  
The phenolic profiles of the extracts obtained applying for each technique the conditions able to maximize the TPC were analysed by high-pressure liquid chromatography according to [PERSON] et al. [28]. The HPLC system was equipped with a diode array detector (Agilent 1100 Liquid Chromatograph, Santa Clara, CA, USA) and a 100 \(\times\) 4.6 mm \(\times\) 3 \(\upmu\)m RP-C18 Gemini column (Phenomenex, Aschaffenburg, Germany). Separation was achieved using the following linear gradient of two solvents, Solvent A (1.0% acetic acid in water \(v/v\)) and Solvent B (50% methanol, 50% acetonitrile, \(v/v\)) at 30\({}^{\circ}\) C with a flow rate of 1 mL/min: from 5% to 25% B in 20 min; from 25% to 30% B in 5 min; from 30% to 40% B in 10 min; from 40% to 48% B in 5 min; from 48% to 60% B in 10 min, followed by returning to the initial conditions in 5 min and column equilibration (5 min). The injection volume was 100 \(\upmu\)L. The compounds were identified on the basis of their retention times and comparing their spectra with those of standard compounds. The phenolic compounds were quantified at two wavelengths: 280 nm (gallic acid, 4-hydroxybenzoic acid, vanillic acid, caffe acid, syrigenic acid, chlorogenic acid, ferulic acid, rosmarinic acid, epicatechin gallate, rutin, resveratrol, quercetin and kaempfer) and 320 nm (catechin, epicatechin, epigallocatechin, p-coumaric acid sinapic acid). Quantification was obtained by comparing the sample peak areas with those of standard curves. Data were expressed as mg/g dry matter.