**1. Introduction**

Nowadays, the consumption of natural extracts from plants and seeds present in nature and the discovery of the many benefits connected to their intake, have favoured and promoted research in the field of extraction and use.

For this purpose, in recent years, extract of plant parts (seeds, cladodes, stem, fruits, etc.) of the family *Opuntiaceae* have been investigated, as for instance: *Opuntia joconostle* seeds for cholesterol-lowering properties [1]; *Opuntia humifusa* cladodes, for cytotoxic activity against the human breast cancer cell lines MCF-7 and human colon SW-480 cells, and stem and fruits able to inhibit the growth of U87MG glioblastoma cells [2]; *Opuntia*

**Citation:** Iacopetta, D.; Baldino, N.; Caruso, A.; Perri, V.; Lupi, F.R.; de Cindio, B.; Gabriele, D.; Sinicropi, M.S. Nutraceuticals Obtained by SFE-CO2 from Cladodes of Two *Opuntia ficus-indica* (L.) Mill Wild in Calabria. *Appl. Sci.* **2021**, *11*, 477. https://doi.org/10.3390/app11020477

Received: 10 December 2020 Accepted: 2 January 2021 Published: 6 January 2021

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*stricta* for antioxidant, anti-inflammatory and cytotoxic activities [3,4]. Instead, our attention was focused on the cladodes of the cactus pear (*Opuntia ficus-indica* (L.)) Mill., a tropical or subtropical invasive plant, widespread and well adapted to Mediterranean area due to the climate.

*Opuntia* was an important source of the agricultural economy and diet of the ancient Mexicans of the Aztec empire [5] and it was also traditionally used to differentiate the properties of the peasants and to curb fires; the cladodes have high water content, 95% in mass [6].

In some countries, like Mexico, Italy and South Africa, the most commercial variety *Opuntia ficus-indica* (L.) Mill. is cultivated on the considerable surface for industrial purposes; in Mexico, its culture stretches on a surface of 300,000 ha [7]. The immature cladodes of Opuntia ficus indica are a part of the edible plant and also used in Mexican cuisine for many traditional dishes and in folk medicine for certain diseases, such as obesity, gastrointestinal disorders [8], ulcers and healing of wounds [8].

*Opuntia ficus indica* fruits are used as a laxative in Turkey, to reduce kidney stones, rheumatism pains, and as a sedative [9]. It was also been reported that the extracts of fruits and stems exhibit hypoglycemic, anti-ulcer [10], anti-allergic, analgesic and antioxidant activity [11]. From the fruits and the stem extracts was isolated the *β*-sitosterol, an active anti-inflammatory principle [12], whereas the fruit and flower infusions significantly increase diuresis [13].

Several researches show that the prickly pear is a plant very rich in vitamins, minerals, amino acids and sugars [14]. In particular, its fruit, with a pleasant flavor, has a high content of potassium, phosphorus, magnesium, sodium, calcium, vitamins C and E, dietary fibers, pigments betalains (betacyanins and betaxanthins), polyphenols and their glycosides, glutamine, proline and taurine; the cladodes, consumed as vegetables and used also as animal feed too [15], are rich of mucilages, pectins, sterols, vitamins and polyphenols.

The blades of the prickly pear are however very perishable and can be stored for about 5 days at room temperature or up to 10 days in refrigerated environments [16]. They contain high quantities of pectins and fibers that are capable of increasing fecal mass and intestinal motility, which therefore improve plasma levels of cholesterol and glucose. [17].

The young cladodes of OFI, also known as nopalitos, contain functional polyphenols (Figure 1), like Ruthin, Iso-quercitrin, Narcissin and Nicotiflorin [18] and a series of polysaccharides with high molecular weight and important functional properties, as rheological [19], medicinal and nutritional [20].

Rutin is found in many fruits and vegetables and has been used in over 130 therapeutic medicinal preparations that have been registered as drugs worldwide [21]. In literature, it has been found that Rutin improves the memory of mice in Alzheimer's as it reduces the levels of Aβ-oligomer and attenuates oxidative stress and neuroinflammation. [22].

Isoquercitrin, as glycosylated flavonoid, is rapidly absorbed and transformed, in the gastro-intestinal tract, in glucuronidated Quercetin [23,24] that was found to be the major form in plasma after oral administration of Isoquercitrin in rats [25]. Nicotiflorin, as well, shows protective effects on memory dysfunction in multi-infarct dementia model rats [26]. Narcissin is known for its biological proprieties, such as hepatoprotective, antioxidant, anti *α*-glucosidase, and cytotoxicity against human myelogenous erythroleukemia cells [27].

In general, flavonoids can be found in many varieties of fruit, vegetables and cereals [28–31]. They play a very important role in a series of pathologies, both in their control and in their prevention, such as in Alzheimer's, cancer, etc., perhaps because they act eliminating free radicals. [32–36].

**Figure 1.** Polyphenols present in cladodes of *Opuntia ficus-indica*.

Conventionally the used techniques to obtain polyphenols extracts, such as Soxhlet, maceration [37], organic solvent extraction [38–40], autoclave treatment and microwave [37] or ultrasound-assisted extraction [39–44], usually require several hours or days and spending a large volume of solvents, frequently toxics, and with problematic garbage disposal. The correct separation between solute and solvent is important, as a possible degradation of the thermolabile components would compromise the benefits of the extract. Furthermore, any residual solvent could lower the quality and quantitative yield of the extraction.

Instead, for the extraction of polyphenols from cladodes, in this work it was used a green extraction technique: the supercritical extraction (SFE) with CO2 [37], generally used to obtain oils from natural products [45–48].

This method has been used for the purpose of investigating the possibility of phytochemicals extraction without any solvent and to optimize the selectivity between phenolics and other substances in the cladodes of two ecotypes of *Opuntia ficus-indica* wild in Calabria, *O. ficus-indica cult.* (*OFI cult*.) and *O. ficus-indica s.l.* (*OFI s.l*.).

Supercritical fluid technology is the most innovative method for preparing bioactive products from plants used as supplements for functional foods [49] and it results as promising technology both in food farming and pharmaceutical industry. Supercritical fluids have high solvation capabilities, which are similar to those of liquids, as well as diffusion properties similar to those of gases. For these reasons, the extraction using supercritical fluids is particularly suitable for the extraction of biocompounds from plant matrices [50].

Although there are several usable supercritical fluids, supercritical CO2 is the most commonly used. This application success is due to its low critical constants (Tc = 31.1 ◦C, Pc = 7.38 MPa), to the fact that it is non-toxic and non-flammable and because it is available at high purity at low cost. Moreover, other advantages are a high diffusion coefficient and low viscosity; being gas at atmospheric condition, the CO2 immediately seeps out when brought to the environment, so the products obtained are free from the "extraction solvent" and thermal degradation compounds.

The extracts obtained by SFE-CO2 are also considered as *Generally Recognized As Safe* (GRAS) for the *American Food and Drug Administration*, being possible to add them to all food without undesirable effects for health. Some data have also been reported on the application of SFE-CO2 from vegetable by-products [51].

Today, new chemistry knowledge at the molecular level regarding the functional and structural properties of active principles may allow a better selection of the products and extracts that can satisfy the request of the market, also according to the specific needs of both food and pharmaceutical industries [52]. Thus, with this innovative and advantageous technique, we extracted the Rutin, Isoquercitrin, Nicotiflorin and Narcissin from hard to manipulate plant matrices, namely the cladodes, with the aim to use them as safe supplements in the food and pharmaceuticals companies.

### **2. Materials and Methods**
