*3.1. Preliminary Analysis*

Cladodes by *Opuntia* genus showed a weakly acid pH and this allows easier conservation of the homogenized system. The results are shown in Table 3.



The higher amount of saccharides was found in the sample of *Opuntia ficus-indica* s.l., than that one of *Opuntia ficus indica* cult., and this is possible due to different factors such as the variety and age of the plant, soil, and climate. The pH was measured, because it can influence the viscosity of the mucilage and it could hamper the extraction, and it is similar for the two types of plant. The water activity and humidity values are quite similar, guarantying the same times of exposition to airflow and heat during the dehydration.

#### *3.2. Exhaustive Extraction, Total Phenolics Content and HPLC Analysis*

The high content of total phenolics was observed with the *Folin–Ciocalteu* assay in the samples dried in the oven, Cult\_AD and Sl\_AD; these show as well the better quantitative yield of extraction, calculated using the following equation [45]:

$$\text{Yield } \%= \frac{\left[\mathcal{W}\_{d.c.} \left(\mathcal{g}\right)\right]}{\left[\mathcal{W}\_{\text{m}} \left(\mathcal{g}\right)\right]} \ast 100\tag{1}$$

where *Wd.e.* is the weight of the dry extract obtained and *Wm* the mass of the plant macerated. The data for the yield of extraction at different sample preparation and the total phenolics content are shown in Table 4.

**Table 4.** Data for the yield of extraction at different sample preparation and total phenolic content expressed as mg of phenols on 100 g of fresh raw material.


In Table 4 it is possible to point out that samples dried give a higher yield of extraction and total phenolics content with respect to the fresh macerated ones, showing that the total polyphenol contents vary depending on the type of treatment and extraction.

Comparing the same fresh sample collected in two different months, it is possible to observe that the extraction yield was slightly higher in the sample Cult\_J with respect to the Fresh frozen macerated of August, but the total phenolic content is almost unchanged between samples of Cult\_J and Cult\_AF. This indicates that the different maturation period does not influence the content of secondary metabolites, such as phenols.

Moreover, the total phenolic content is higher in the dried samples Cult\_AD and Sl\_AD, probably because drying process is designed to dehydrate the matrix in order to stop the common enzymatic processes; the aqueous environment of the cytoplasm of plant cells could damage the active compounds [52].

The highest content of total phenols was found in the sample of Sl\_AD, perhaps because being a wild ecotype is less affected by climate change, more adaptable than a cultivated plant.

In Figure 5 are reported the data, obtained by the HPLC analysis, of extractions with solvent.

Rutin is one of the polyphenols presents in greater amount in the analysed species. Moreover, it is possible to observe that by dring the sample it is possible to obtain extracts with a greater amount of some polyphenols (Cult\_AD and Sl\_AD).

As reported in literature rutin is already described to be present in cladode extracts of different Opuntia species [8,20].

Isoquercitrin is another bioactive compound present in our extract in similar quantity in the two species analysed while the quantity of Nicotiflorin and Narcissin are very low in the extracts. The different exposure to the sun or climate change can cause the secondary metabolism of the plant. As a consequence, the antioxidant profile can be influenced.

**Figure 5.** Quantification in mg of standards in 100 g of dry extract.

#### *3.3. Extraction with Supercritical Fluids and HPLC Analysis*

The yields extractions are calculated by using the following equation according to Yieddes et al. [45]:

$$\text{Yield } \%= \frac{\left[\mathcal{W}\_{\text{extracted}} \left(\text{g}\right)\right]}{\left[\mathcal{W}\_{\text{load}} \left(\text{g}\right)\right]} \* 100$$

where *Wextract* is the weight of the extract obtained, which is the difference between the mass of glass trap with extract and the mass of empty glass trap and *Wload* is the mass of sample load in the column before the extraction. The results are reported in Table 5; instead, the results of HPLC quantification on mg of the compound for 100 g of material loaded (mean ± S.D. of two determinations) for *OFI cult.* are shown in Figure 6, instead of *OFI s.l.* in Figure 7.


**Table 5.** Yield % of extraction (mean ± S.D. of two determinations) of different samples by SFE-CO2.

**Figure 6.** Quantification in mg of four different polyphenols for 100 g of loaded material for *OFI cult*.

**Figure 7.** Quantification in mg of four different polyphenols for 100 g of loaded material for *OFI s.l.*

The yields of extraction with SFE-CO2 are lower than that one with solvent as well as the amount of polyphenols, but the resulting extracts do not need to be separated from solvent, they are purer and cleaner.

It is possible to observe that the better results were obtained with the samples of *OFI cult.* despite the *OFI s.l.* In particular, the best yield of extraction was 2.14% ± 0.35, obtained with the sample CULT\_30E\_20O of *OFI cult* dehydrated at 30% and added with 20% (of the total weight loaded for the extraction) of Ottawa Sand at 110 bar. Given the results obtained from the extractions, it is possible to assume that Ottawa sands, hydrophobic natural silica particles, guarantee a better dispersion than diatomaceous earth, which also acts as a drying agent, being hydrophilic. The Ottawa sands, therefore, by favoring a homogeneous dispersion, under the same initial conditions (type of sample and initial drying conditions), guarantee a tighter

contact between the matrix and the supercritical fluid. At the increase of the dehydration to 90% for the sample CULT\_90E\_20O, a decrease in the yields' % follows, but an improvement is obtained concerning the selective extraction of polyphenols. In this sample, it is possible to observe the highest quantities of Rutin, Narcissin and Nicotiflorin, in accordance with the extractions by maceration, where the greatest quantity of polyphenols was obtained for the samples first dried and then macerated in methanol (Table 4).

The treatment of *OFI cult.* with 20% of Diatomee Sand (sample CULT\_20D) implies a reduction of both quantity and selectivity of extraction of polyphenols, perhaps because the Diatomee Sand is hygroscopic. This sand absorbs water from the surrounding environment reducing the function of co-solvent of water naturally present in the matrix.

Being polyphenols polar compounds, the pressure was then raised to 250 bar for the sample CULT\_20P, maintaining the same sand, trying to increase the polarity of CO2, but the effect of the sand was stronger. It was obtained a reduction of both yield % and selectivity of polyphenols; this shows that high pressures result in a loss of bioactive principles sensitive as polyphenols.

When the sample is centrifuged (sample CULT\_C\_20D) to remove water, it was obtained a good increase of the yield % of extraction and a little improvement on the selectivity of polyphenols.

The yields % of extractions of the *OFI s.l*. were very low, ranging between 0.02 and 0.25%; but the selectivity and purity of the extracts derived therefrom are very high, as can be seen in Figure 8 for the sample SL\_30E\_20D.

**Figure 8.** Chromatogram resulting from the analysis of the extract by SFE-CO2 of *OFI s.l.* dried to 30% and added 20% of Diatomee Sand.
