*3.2.5. Enzyme-assisted extraction (EAE)*

global decreased yield of total phenolic and flavonoid content. Moreover, under these conditions might occur the formation of free radicals and consequently undesirable changes in the

The PLE method, also known as "accelerated solvent extraction (ASE)," is a very recent new technology for phytochemicals extraction including polyphenols, which associates high temperature and pressure [99]. In this method, high level of pressure (normally between 3.3 and 20.3 MPa) is combined with high level of temperatures (between 40 and 200°C) to improve the solubility and desorption of molecules, increasing their movement from matrix into solvents, and thus increasing the yield of polyphenols extracted [54]. According to Nieto et al. [99], the PLE method is an advanced technique that provides a faster extraction processes and requires a small amount of solvents when compared with the classical extraction approach. Moreover, it allows better the usage of water as extraction solvent, which is limited in the other previous methods. The use of water as an extraction solvent in PLE, as so-called subcritical water extraction (SWE), is always possible, particularly when elevated temperatures are used [100]. When temperatures around 200°C are used, a change in the dielectric water properties occurs, and then, the water behaves like a normal organic solvent, increasing their extraction efficiency [101]. The main advantages of PLE often reported by several researchers are cleanness of the extracts that PLE provides in comparison with classical maceration, Soxhlet, MAE and UAE, which results in reduced background noise during the subsequent analytical quantification, is especially important when the LC-MS analysis due to ion-suppression effects [102]. By opposition, the main limitations often reported are the low selectivity towards the analytes during extraction, and many interferents may be extracted during the extraction process, an exaggerated dilution of the analytes, especially when a large number of cycles are used, and the high requirements in instrumentation, which increases their costs [103–105]. However, these limitations in PLE are a well-known extraction technique and have been used for the

extraction of polyphenols from several different matrices [106–111].

 *extraction (SC-CO2*

easily available in high quantity with high grade of purity [112]. SC-CO<sup>2</sup>

extraction is a process in which the CO<sup>2</sup>

*)*

bly is one of the most widely used fluid because it is nontoxic, nonflammable, inert cheap and

sible to use different combinations of temperature and pressure [112], making this method one of the most versatile for creating a multitude of end products. Due to the multitude of combinations, low temperatures (31.6°C, the critical point of carbon dioxide) and pressure (7.386 MPa) are needed, and the SC-CO2 has been considered very popular in a lab-scale laboratorial facilities. Moreover, since low temperatures and pressure are used, there is a good preventing of thermal degradation of phytochemicals. The main advantage s of SC-CO<sup>2</sup>

[112–116] as follows: (i) more extraction capacity due to their higher diffusion coefficient and lower viscosity than the liquids, which increases a higher mass transfer from solid matrix towards solvents; (ii) it allows higher penetration of solvents into the matrices which increase

is used as supercritical fluid and proba-

extraction is pos-

are

drug molecules [97].

*3.2.4. The supercritical CO2*

The SC-CO<sup>2</sup>

*3.2.3. Pressurized liquid extraction (PLE)*

70 Phenolic Compounds - Natural Sources, Importance and Applications

The EAE is a recent method and is based on the capacity of the enzymes to degrade cell wall components into solvents, in general water, with high stability and high bioactivity [117]. In EAE, the enzymes added to food, plant matrices or agro-food wastes are capable to break and weaken the cell walls, increasing the exposure of their cellular components to extraction [71, 118], and thus increasing the capacity to extract polyphenols from the matrices. In fact, some phytochemicals are dispersed in plant cell cytoplasm, and even, some compounds are bound with the polysaccharide-lignin by hydrogen or hydrophobic chain, which are not accessible with a routine organic solvents [119, 120]. Thus, a previous treatment with enzymes can be the only choice, and an enzymatic pretreatment might be the unique and effective way to release bounded compounds from cells [121].

Cellulases, hemicelullases, pectinases and other enzymes may be used to hydrolyze efficiently the cell wall components, enabling the efficiency of extraction of phenolic compounds. Several papers have been published about the positive effect of EAE on increment of polyphenol extraction yield. In 2012, in a study with grape wastes [71], it was found a strong increment in the release of polyphenols when celluclast®, pectinex® and novoferm® enzymes were used. Similar trends were noted in other works [122, 123] which concluded that EAE should be regarded as an alternative method for improved extraction of insoluble-bound phenolics (linked to carbohydrates and proteins of cell wall matrices) from winemaking byproducts. These and many other authors observed that the ability of enzymes to degrade cell walls and membranes enables the extraction efficiency of bioactive compounds, and in several situations, the EAE technology might be the unique way to extract effectively bioactive compounds from foods and agro-industrial by-products. In addition to these advantages, the EAE method has been recognized as one of the most eco-friendly methods, because it uses water as solvent instead of organic chemicals, often toxics [119], and is one of the modern extraction methods that are gaining more attention because of the need for eco-friendly extraction technologies.
