*3.2.2. The ultrasound-assisted extraction (UAE) method*

anthocyanins) may be degraded under MAE extraction conditions [79, 80]. The temperature used for extraction is proportional to the power (watts) and time and inversely proportional to the heat capacity of the solvent and the mass of sample [80]. Higher temperatures and small amounts of sample increase the rate of solvent diffusion and promote faster

Lemon balm (*Melissa officinalis L*.) **EAE** + **PLE** were applied for the extraction

**Table 3.** Application of advanced methods in extraction of polyphenols in different foods and agro-waste matrices

**Method Botanic matrix Results reported by the authors References**

**anthocyanins extracted**.

volume in a shorter time.

**extraction in less time**.

**polyphenols**

at flow rate.

compounds.

**phenolics**.

**antioxidant phenolics** with lower solvent

tool to extract both **anthocyanins** and **total** 

**environmental friendly** and a **good alternative** for the extraction of natural

**concentrated in polyphenols**.

**yield of polyphenols extracted**.

A combined method of **EAE** + **UAE** was used to extract polyphenols. The combined methods enhanced the content of polyphenol and antioxidant activity

allowed to obtain **extracts more** 

maceration **improved the release of bound** 

**Novoferm**® were used to release phenolic compounds from grape wastes. The pretreatment with **enzymes increased the** 

of phytochemicals. The results showed that **EAE + PLE enhanced the total phenolic content and the antioxidant capacity**.

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

MAE Blueberries (*Vaccinium corymbosum* L.) The usage of **MAE increased the yield of** 

MAE Grape seeds (*Vitis vinifera*) The **MAE** was able **to extract maximum** 

UAE *Cassia auriculata* leaves The **UAE** process **enhanced the phenolics** 

UAE Mulberry pulp (*Morus nigra*) The **UAE can be a reliable and economic**

PLE Mango (*Mangifera indica* L.) **High yields** of **polyphenols** were obtained

PLE Asparagus (*Asparagus officinalis* L) **PLE revealed cheaper, faster and** 

EAE Pomegranate peels (*Punica granatum* L.**)** The incorporation of **enzymes** in the

EAE Grape residues (*Vitis vinifera*) **Celluclast**®, **Pectinex**® **Ultra**® and

SC-CO<sup>2</sup> Pitanga leaves (*Eugenia uniflora* L.) **SC-CO2**

68 Phenolic Compounds - Natural Sources, Importance and Applications

Broccoli inflorescences (*Brassica oleracea*

L. var. italica)

extraction kinetics [80].

Combined approaches

Combined approaches

[63–73].

The UAE is a very simple method that relies on the mechanical effect caused by the implosion of micro-sized bubbles, which cause a rapid tissue disruption allowing the release of compounds into the solvent [84]. This is a very simple method with relatively low cost, and it can be used on both small laboratory and large industrial scale [84, 85]. The use of UAE has been widely used in the last years in the extraction of polyphenols from different parts of plants such as leaves, stems, stalks, fruits, seeds [85–93]. In general, the experimental procedure involves the use of ultrasounds with frequencies ranging from 20 to 2000 kHz, which increases the permeability of cell walls and produces cavitation.

Several studies have reported that UAE allows a better and faster extraction of polyphenols with less degradation when compared with other extraction methods. For example, UAE shown to be highly efficient in the extraction of carnosic acid and rosmarinic compared to classical methods of extraction [94]. In a recent study [95], the maximum extraction yield of total polyphenols (13.2 mg/g dry weight) from spruce wood bark was obtained when UAE system was used. Also, an increment in anthocyanin content in purple sweet potato was observed when UAE was used [96]. All these studies have in common the same trend: under UAE, the rate speed dissolution of compounds into extraction solvent was always higher, and thus, the solvent volume used and need to extract phytochemicals was lower compared to the classical extraction methods. Based on these studies and others, it seems that UAE has the advantage of being less expensive due to lower solvent volume used, higher amount of samples tested and lower time needed to perform the extraction process. Also, they agree that the lower temperatures and shorter sonication periods (time) are better to enhance the extraction of polyphenols contributing also to the preservation of the thermolabile and unstable compounds. However, some studies [97, 98] reported that sonication for long periods (>40 min) with higher energy levels (above >20 kHz) could have a deleterious effect on phytochemicals due to the decrease of diffusion area and diffusion rate and increased diffusion distance, leading to a 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 drug molecules [97].

### *3.2.3. Pressurized liquid extraction (PLE)*

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].

#### *3.2.4. The supercritical CO2 extraction (SC-CO2 )*

The SC-CO<sup>2</sup> extraction is a process in which the CO<sup>2</sup> is used as supercritical fluid and probably is one of the most widely used fluid because it is nontoxic, nonflammable, inert cheap and easily available in high quantity with high grade of purity [112]. SC-CO<sup>2</sup> extraction is possible 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> are [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 the effectiveness and polyphenols extraction yield; (iii) it allows different combinations of pressure and temperature and thus allows a better adaptation of the extraction conditions to the different types of food and plant matrices, increasing the solubility of their different components in the supercritical fluids; (iv) it allows the CO<sup>2</sup> recycling at the end of the process, without any disgrace of chemical residue to environment at the end of the extraction and separation process.
