*2.2. Raw Materials and Substrates Characterization*

The tissue substrate was analysed by thermogravimetric analysis using a TA Q-500 (TA Instruments, Waters LLC, New Castle, DE, USA) to evaluate its qualitative composition and thermal degradation profile. This analysis was performed from room temperature to 900 ◦C

under a nitrogen atmosphere at 10 ◦C/min. At 900 ◦C, an isothermal treatment of 30 min under air was performed to evaluate the residual weight under oxidative atmosphere.

Extracted active molecules were analysed by infrared spectroscopy using a Nicolet T380 Thermo Scientific instrument equipped with a Smart ITX ATR accessory with a diamond plate (Thermo Fisher Scientific, Waltham, MA, USA), collecting 256 scans at 4 cm−<sup>1</sup> resolutions.

The polyphenols main composition was determined by high-performance liquid chromatography coupled to mass spectrometry (HPLC-DAD-MS) at 294 nm. An Agilent 1100 HPLC system coupled to a LC/MSD ion trap mass spectrometer with an electrospray ionization (ESI) source (Agilent Technologies, Palo Alto, CA, USA) was used. A HALO C18 column (100 mm × 4.6 mm × 2.7 μm) coupled to a HALO C18 guard column 90 Å (4.6 × 5 mm × 2.7 μm) operating at 25 ◦C was used. The mobile phase was composed of milli-Q water (solvent A) and acetonitrile (solvent B), both added with 0.1% acetic acid. A gradient elution program was used at a flow rate of 0.5 mL/min: starting A at 85% to 60% in 15 min to 30% in 3 min to 10% in 1 min and returning to the initial composition in 3 min. Mass spectra were recorded in the negative ionization mode (m/z 50–900). The electrospray chamber was set at 3.5 kV with a drying gas temperature of 350 ◦C. The N2 pressure and flow rate of the nebulizer were 50 psi and 10 L/min, respectively. Polyphenols were identified by comparing MS experimental data with those of standard compounds prepared in ethanol:water (60%, *v*/*v*). All solutions were filtered through a 0.22 μm nylon membrane prior to injection (6 μL).

The ChNF nature was investigated to verify the effect of the defibrillation process. A drop of diluted ChNF dispersion (1:1000 *v*/*v* in water) was poured on a microscopy slide and dried at room temperature. The slide was mounted on a SEM stub and observed under a field emission scanning electron microscopy (FESEM) using a FEG-Quanta 450 instrument (FEI, Hillsboro, OR, USA).
