*2.3. Preliminary Analysis*

The pH value of the homogenized system (pH 700, Eutech Instruments, Germany), the activity level (Novasina AW Sprint–TH 500, Switzerland), the Brix degrees (ATAGO, Hand Refractometer N Type Series, Japan) and humidity (Mettler Toledo Moisture Analyzer HB43-S, Switzerland) were measured. The analyses were repeated in triplicate and the results are reported as mean value and standard deviation.

### *2.4. Exhaustive Extraction and Total Phenolics Content*

The fresh cladodes of *Opuntia ficus-indica* both ecotypes were extracted with methanol (48 h × 3 times) at 4 ◦C. The same procedures were followed for a sample of *OFI cult.* and *OFI s.l.* Moreover, the exhaustive analysis was performed on samples dried in the oven for 360 min at 32 ± 1 ◦C; removing the 30% of the weight.

The extraction solutions were filtered in synthetic cloth, concentrated and dried under *vacuum* at 35 ± 1 ◦C for the thermolability of the polyphenols compounds.

The total phenolic content of the cladodes extracts was quantified using *Folin-Ciocalteu* reagen<sup>t</sup> and chlorogenic acid used as standards. The absorbance was measured at 726 nm (Perkin Elmer Lambda 40 UV/VIS spectrophotometer) and the total content was expressed (mean ± S.D. of three determinations) as mg of chlorogenic acid equivalents on 100 g of fresh raw material (Singleton Rossi, 1965).

### *2.5. Extraction of Polyphenols with Supercritical Fluids*

The supercritical extractions by CO2 were performed on a *Spe-ed* SFE 4 extractor (Applied Separation, Allentown, PA, USA) and following the necessary steps: loading in the 50 mL stainless steel vessel, pressurization with CO2 and waiting for a transitory state of temperature, which is kept constant by an oven module. After the reaching of target temperature and pressurization, in order to guarantee the intimate contact between the CO2 and the matrix, the system was kept closed for 20 min (static phase), and only after this time the micrometric valve was opened until a constant flow of 1 was reached. 5 L/min (dynamic phase), measured by a ball float rotameter. A graphical scheme of *Spe-ed* SFE 4 extractor is given in Figure 4.

**Figure 4.** Scheme of Spe-ed SFE 4 extractor unit.

The extract left the vessel through a valve, which was in advance thermostated to avoid CO2 solidification, due to the expansion. Experiments were carried out at 40 ◦C, 110 bar and 250 bar by varying the preventive sample preparation with PSE accessories, as Ottawa Sand and an Spe-edTM PSE Matrix (Hydroscopic Samples Dispersing Agent). All the extracts were stored at −18 ◦C before HPLC analysis.

This extraction method has always attracted because it is safe and eco friendly. In addition, the supercritical fluid, in the specific case CO2, has properties similar to those of gas in the supercritical state so that it can extract in a very short time compared to the classic extraction types, returning an extract already separated from the solvent after depressurization. Supercritical fluids have a high diffusion coefficient and a low viscosity which favours their intimate contact with the matrix. On the other hand, however, this method has limits, especially in relation to the use of the supercritical solvent because CO2 has low polarity. Therefore this type of extraction technique is generally used for the extraction of oils, fats or in any case polar substances. Only the use of other solvents, such as ethanol or water, increases the solvent power of CO2 in the supercritical state.

The different methods to prepare the samples and pressure are listed in Table 1.


**Table 1.** Different preparation of samples before SFE separation.

All samples were cleaned, homogenized and frozen after collection, to be subsequently analysed.

Some samples analysed were centrifuged after defrosting at 3500 rpm for 10 min and only the precipitated part was used to extract the polyphenols while other samples were partially dried at 32 ◦C in the oven to reduce the amount of water.

The extraction time was 1 h. The final extracts were collected in glass tubes covered with an aluminium foil and frozen until analysis. Each extraction was carried out in duplicate.
