**2. Microwave-Assisted Solid Extraction (MASE)**

The physical principles underlying MASE are completely different from those of conventional extraction because microwave irradiation can cause a more effective disruption of the cellular structures (cell walls and cellular membranes) thus

**37**

**Figure 1.**

*Microwave heating effect on cells.*

*Microwave-Assisted Solid Extraction from Natural Matrices*

favoring the release of the cellular content and speeding up the extraction process. The interaction of microwave irradiation with the solvents, intracellular water, and ions causes an increase of the dipolar rotation and ionic conductivity of molecules with dipolar moments and ions, which results in a rise of the temperature inside the cell. The vaporization of the intracellular water causes the dehydration of the cellular wall and the reduction of its resistance. This, combined with the abrupt increment of the intracellular pressure, leads to the cell wall and membranes disruption, thus facilitating the passage of the secondary metabolites into the

This is the result of a synergistic combination of heat and mass gradient working in the same direction from the inside to the outside of the cell, as confirmed by

Monomodal and multimodal microwave systems also referred to as single- or multi-mode systems specifically designed for MASE are nowadays available. A single-mode system permits to focus the microwave radiation on a restricted zone where the sample is subjected to a much stronger electric field. Conversely, in a multimode system, the microwave radiation is randomly dispersed within the microwave cavity, where the sample is irradiated [15]. Both mono- and multi-modal microwave devices comprise a magnetron, which generates microwave radiation, a waveguide, which is used to propagate the microwave from the source to the microwave cavity, the applicator, where the sample is placed and a circulator that allows the microwave to move only in the forward direction. The main difference between the two systems relies on the applicator that in the case of a multimodal system is a closed cavity where the microwaves are randomly dispersed, whereas in the monomodal oven the extraction vessel is directly located in-line with the waveguide [15]. Both systems are effective in extracting metabolites from natural sources, and their use is usually related to the amount of natural matrix. Accordingly, the monomodal system is preferred when the amount of natural source to be treated is relatively low (milligram to gram scale), and vice versa. Microwave extraction may be performed using open or closed extraction vessel systems. The open vessel apparatus originated from a modification of domestic MW ovens. The extraction is conducted at ambient pressure and the vessel is directly connected to a condenser to avoid loss of solvent or volatile components. An evolution of the open vessel apparatus is the Focused Microwave-Assisted Soxhlet (FMASE) which combines the classical Soxhlet extraction (SE) technique with MW irradiation. In the closed vessel system the entire extraction vessel is located within the oven, thus allowing better control

Scanning Electron Microscopy (SEM) analysis (**Figure 1**) [14].

of the pressure and temperature during the extraction [16].

The closed-vessel system presents several advantages: i) a higher temperature than open vessel systems can be reached because of the increased pressure inside the vessel raises the boiling point of the solvents used, thus decreasing the time needed for the extraction; ii) the loss of volatile substances is completely avoided because they are confined within the vessel; iii) a low amount of solvent is required because no evaporation occurs and there is little or no risk of airborne contamination thus

*DOI: http://dx.doi.org/10.5772/intechopen.95440*

extractive solvent [12, 13].

*Microwave Heating - Electromagnetic Fields Causing Thermal and Non-Thermal Effects*

inestimable treasure of new potential drugs [5–7].

unique and absent in synthetic NCE, due to the lack of commercially available synthons or cumbersome and prohibitive synthetic procedures [4]. Thus, the screening of libraries of compounds derived from natural sources still remains a worthy procedure for the identification of new and unexplored NCE. Besides, marine sources or lichens are still almost uninvestigated and might therefore represent an

The natural compounds of interest in NADD are secondary metabolites that are not directly involved in the essential functions of the cell cycle and duplication processes and are characterized by high structural variability. From a structural standpoint, they are classified into alkaloids, terpenoids, saponins, lignans, flavonoids, and tannins [8]. Secondary metabolites are produced in different amounts for vexillary functions or defensive responses to biotic or abiotic stress being involved in the system of plant defense [9]. For this reason, specific secondary metabolites may be considered as markers of the plant health and may be used to evaluate the quality of the selected natural matrix and the effects that the environmental factors have on it. The evaluation of their content in natural sources, as well as their recovery, require the exploitation of ad hoc extractive procedures since secondary metabolites are mainly present in the intracellular domain. Thus, appropriate extraction procedures involve the rupture of the cell wall and cellular membranes, thus favoring the passage of the secondary metabolites into the extraction solvent, from which they can be recovered. Over the years different protocols and techniques have been developed for the extraction of metabolites from natural matrices, named Solid–Liquid Extraction (SLEs). SLEs are classified in conventional (or traditional) and non-conventional (or innovative) methodologies [10]. In conventional methodologies, SLE is performed by heating the natural source with conventional heating sources (i.e., flame, heating plate, or mantle) in the presence or not of solvent and with or without stirring. Examples of conventional methodologies are maceration (digestion, infusion, and decoction), percolation, and Soxhlet or steam distillation. Solvents, or generally mixtures of solvents, with wide grades of polarity such as methanol, ethanol, acetone, ethyl acetate, trichloromethane, hexane, *etc*. are employed. Nevertheless, conventional extraction methods usually require a large volume of solvents, long extraction time, and high temperature. Such harsh extraction conditions may lead to thermal or chemical degradation of the metabolites, thus resulting in a low yield of the final extract. Moreover, the upscaling at an industrial level would be impracticable, owing to energy consumption, technological inaccessibility, and environmental considerations [11]. Conversely, non-conventional methodologies exploit innovative chemical–physical principles and cutting-edge technologies to facilitate the extractive procedures and the recovery of the product of interest from the natural source. Varied energy sources and extractive principles may be exploited and, therefore, specific equipments are needed. Microwave-assisted Solid Extraction (MASE), ultrasound-assisted extraction (UAE), pressurized solvent extraction (PLE), and supercritical fluid extraction (SFE) are the most frequently non-conventional methodologies extensively used for NADD [11]. In this chapter, the potential of microwave-assisted irradiation for the extraction of secondary metabolites from plants, and natural resources in general, will be discussed, with a special focus on recent applicative examples of the most

**36**

representative chemical classes.

**2. Microwave-Assisted Solid Extraction (MASE)**

The physical principles underlying MASE are completely different from those of conventional extraction because microwave irradiation can cause a more effective disruption of the cellular structures (cell walls and cellular membranes) thus

favoring the release of the cellular content and speeding up the extraction process. The interaction of microwave irradiation with the solvents, intracellular water, and ions causes an increase of the dipolar rotation and ionic conductivity of molecules with dipolar moments and ions, which results in a rise of the temperature inside the cell. The vaporization of the intracellular water causes the dehydration of the cellular wall and the reduction of its resistance. This, combined with the abrupt increment of the intracellular pressure, leads to the cell wall and membranes disruption, thus facilitating the passage of the secondary metabolites into the extractive solvent [12, 13].

This is the result of a synergistic combination of heat and mass gradient working in the same direction from the inside to the outside of the cell, as confirmed by Scanning Electron Microscopy (SEM) analysis (**Figure 1**) [14].

Monomodal and multimodal microwave systems also referred to as single- or multi-mode systems specifically designed for MASE are nowadays available. A single-mode system permits to focus the microwave radiation on a restricted zone where the sample is subjected to a much stronger electric field. Conversely, in a multimode system, the microwave radiation is randomly dispersed within the microwave cavity, where the sample is irradiated [15]. Both mono- and multi-modal microwave devices comprise a magnetron, which generates microwave radiation, a waveguide, which is used to propagate the microwave from the source to the microwave cavity, the applicator, where the sample is placed and a circulator that allows the microwave to move only in the forward direction. The main difference between the two systems relies on the applicator that in the case of a multimodal system is a closed cavity where the microwaves are randomly dispersed, whereas in the monomodal oven the extraction vessel is directly located in-line with the waveguide [15]. Both systems are effective in extracting metabolites from natural sources, and their use is usually related to the amount of natural matrix. Accordingly, the monomodal system is preferred when the amount of natural source to be treated is relatively low (milligram to gram scale), and vice versa. Microwave extraction may be performed using open or closed extraction vessel systems. The open vessel apparatus originated from a modification of domestic MW ovens. The extraction is conducted at ambient pressure and the vessel is directly connected to a condenser to avoid loss of solvent or volatile components. An evolution of the open vessel apparatus is the Focused Microwave-Assisted Soxhlet (FMASE) which combines the classical Soxhlet extraction (SE) technique with MW irradiation. In the closed vessel system the entire extraction vessel is located within the oven, thus allowing better control of the pressure and temperature during the extraction [16].

The closed-vessel system presents several advantages: i) a higher temperature than open vessel systems can be reached because of the increased pressure inside the vessel raises the boiling point of the solvents used, thus decreasing the time needed for the extraction; ii) the loss of volatile substances is completely avoided because they are confined within the vessel; iii) a low amount of solvent is required because no evaporation occurs and there is little or no risk of airborne contamination thus

**Figure 1.** *Microwave heating effect on cells.*

preventing the oxidation of air-sensitive metabolites; iv) the fumes are contained within the vessel, reducing the hazard of the extractive procedure.

In a closed vessel system high pressure can be reached. The high pressure stimulates various phenomena according to LeChatelier's principle, such as the transition of phase from one form to another, change in reaction dynamics, change in molecular structure, *etc*. hence resulting in the enhancement of extraction efficiency. Accordingly, working under high pressure causes alterations in the structure of some constituents of the cells such as lipids, proteins, enzymes, and outer cell membranes, thus damaging the plant wall and internal structure of the cell and reducing the mass transfer resistance. In this way, the secondary metabolites are released, leaving behind other cellular components. The High-Pressure MASE (HPMAE) is considered a newly emerging technique. It is a time-efficient, convenient, eco-friendly, safe, and energy-efficient extraction method when compared to the traditional or conventional methods of extraction [17].

Nevertheless, the use of closed-vessel systems presents some drawbacks: the amount of samples that can be processed is limited; the addition of reagents or solvents during operation is forbidden and the vessel must be cooled down before it can be opened to avoid loss of volatile constituents [16]. For processing a large amount of material, an open-vessel system is more appropriate. It allows the possibility to add reagents and to refill the solvent during the extraction, and to remove the excess of solvent during the extraction procedure. Moreover, the extraction of thermolabile metabolites is allowed since it usually reaches low temperatures relative to closed-vessel systems. On the other hand, the open-vessel systems ensure less reproducible results compared to the closed-vessel systems and the extraction in open-vessel could require a longer time to obtain results comparable to those achieved in closed-vessel [16].

The microwave energy may be applied also to extraction in solvent-free conditions (Solvent Free Microwave Extraction-SFME) [12]. In this case, the plant material is directly placed into the microwave reactor, without the addition of any solvent. The heating of the water contained by the plant material distends the natural matrices and causes the rupture of the cells releasing their content. This process has been successfully applied for the extraction of the essential oils. After MW heating, the volatile components co-evaporate with the in-situ water contained in the natural matrix; the vapors condense outside the microwave oven into a collector where they separate between essential oils and the aqueous phase. The latter is brought back into the vessel to refresh the amount of water in the sample, whereas the essential oil is collected apart [12].

## **3. Procedure set-up**

The performance of the MASE process is strictly related to the operative conditions employed. Several parameters, such as solvent, solvent-drug ratio, temperature, time, pressure, microwave power, water content, and characteristic of the matrix must be optimized in setting up a MASE procedure. Each of these parameters should not be considered alone, but they are all linked together and the comprehension of the effects and influences of these factors is pivotal for MASE efficiency. A brief discussion about the role of these parameters in the design of a MASE protocol is herein reported.

#### **3.1 Solvent**

The selection of the solvent plays a crucial role in MASE, as well as in other conventional extraction processes, and several solvent parameters (solubility of

**39**

the sample [12].

**3.2 Liquid-Solid (L/S) ratio**

cesses reported in the literature [20].

*Microwave-Assisted Solid Extraction from Natural Matrices*

the analyte, penetration, interaction with the matrix, dielectric constant, and mass transfer kinetic process) should be considered to perform the right choice. *In primis*, the solvent should assure the highest solubility of the analyte of interest while excluding undesired matrix components. However, conversely to the traditional extractive procedures, the chosen solvent must be able to absorb the microwave radiation and to convert it into heat. This depends on the dielectric constant and the dielectric loss of the solvent. Thus, in microwave application, solvents are usually classified in low, medium, or high absorbance whether they absorb at 300 W (i.e. carbon chlorides, 1,4-dioxane, tetrahydrofuran, diethyl ether, ethyl acetate, pyridine, toluene, chlorobenzene, xylenes, and hexane), 200 W (i.e. water, DMF, NMP, butanol, acetonitrile, HMPA, ketones, o-dichlorobenzene, 1,2-dichloroethane, 2-methoxy ethanol, acetic acid, and trifluoroacetic acid) or 100 W (i.e. DMSO, ethanol, methanol, propanol, nitrobenzene, formic acid, ethylene glycol), respectively. For low absorbing solvents, the heating rate can be increased by mixing with solvents with a higher dielectric constant or by adding salts to the mixture [18]. Recently, increasing interest in ionic liquids as MASE solvents or modifiers has been observed. Ionic liquids are organic salts with low melting points, and they are usually liquid at room temperature. They are characterized by extremely low vapor pressure, high stability and can solubilize both polar and non-polar metabolites. Moreover, they have the advantage to be eco-friendly solvents, although their extensive use is limited due to the high commercial costs. Another emerging eco-friendly alternative is the use of deep eutectic solvents. They possess physical and chemical properties similar to the ionic liquids, but they show better biodegradability, toxicity profiles, and solubility properties. Lastly, the content of water in the sample should be taken into account since it influences the heating rate

and facilitates the transport of the analytes into the solvent at higher rates [19]. As stated in the previous paragraph, a Solvent-Free Microwave Extraction (SFME) is also possible. This procedure foresees the direct irradiation with MW of the plant material, fresh or rehydrated. A crucial role is played by the content of water in the sample, because it is the heating of such water to cause the rupture of the cells of the plant material, favoring the release of the content. This process is mainly applied for the extraction of volatile compounds such as essential oil. The oil evaporates by azeotropic distillation with the water contained in the sample. After cooling outside of the microwave reactor the oil separates from the water and can be collected through a modified Clevenger apparatus. Water is refluxed back into the microwave oven to allow the continuous extraction of the oil from

Another important parameter to consider in the set-up of a MASE protocol is the ratio between the amount of sample and the volume of solvent. The latter should be enough to cover the sample during the entire process, especially when the matrix swells during the extraction process. Although in conventional extraction higher is the volume of solvent, higher is the yield of the extract, in MASE larger volume of solvent may result in more energy and time to heat the suspension and in a lower yield due to a non-uniform distribution and exposure to microwave. Usually, an L/S from 10:1 to 20:1 (mL/mg) is found to be the right ratio in many extractive pro-

Strictly correlated to the L/S ratio is the stirring rate since it affects the mass transfer process in MASE. However, the significance of this parameter is rarely explored. By stirring, the mass transfer barrier created by the concentrated compounds in a localized region due to insufficient solvent can also be minimized resulting in better extraction yield. In other words, agitation accelerates the

*DOI: http://dx.doi.org/10.5772/intechopen.95440*

#### *Microwave-Assisted Solid Extraction from Natural Matrices DOI: http://dx.doi.org/10.5772/intechopen.95440*

*Microwave Heating - Electromagnetic Fields Causing Thermal and Non-Thermal Effects*

within the vessel, reducing the hazard of the extractive procedure.

conventional methods of extraction [17].

the essential oil is collected apart [12].

MASE protocol is herein reported.

**3. Procedure set-up**

preventing the oxidation of air-sensitive metabolites; iv) the fumes are contained

Nevertheless, the use of closed-vessel systems presents some drawbacks: the amount of samples that can be processed is limited; the addition of reagents or solvents during operation is forbidden and the vessel must be cooled down before it can be opened to avoid loss of volatile constituents [16]. For processing a large amount of material, an open-vessel system is more appropriate. It allows the possibility to add reagents and to refill the solvent during the extraction, and to remove the excess of solvent during the extraction procedure. Moreover, the extraction of thermolabile metabolites is allowed since it usually reaches low temperatures relative to closed-vessel systems. On the other hand, the open-vessel systems ensure less reproducible results compared to the closed-vessel systems and the extraction in open-vessel could require a longer time to obtain results comparable to those achieved in closed-vessel [16]. The microwave energy may be applied also to extraction in solvent-free condi-

tions (Solvent Free Microwave Extraction-SFME) [12]. In this case, the plant material is directly placed into the microwave reactor, without the addition of any solvent. The heating of the water contained by the plant material distends the natural matrices and causes the rupture of the cells releasing their content. This process has been successfully applied for the extraction of the essential oils. After MW heating, the volatile components co-evaporate with the in-situ water contained in the natural matrix; the vapors condense outside the microwave oven into a collector where they separate between essential oils and the aqueous phase. The latter is brought back into the vessel to refresh the amount of water in the sample, whereas

The performance of the MASE process is strictly related to the operative conditions employed. Several parameters, such as solvent, solvent-drug ratio, temperature, time, pressure, microwave power, water content, and characteristic of the matrix must be optimized in setting up a MASE procedure. Each of these parameters should not be considered alone, but they are all linked together and the comprehension of the effects and influences of these factors is pivotal for MASE efficiency. A brief discussion about the role of these parameters in the design of a

The selection of the solvent plays a crucial role in MASE, as well as in other conventional extraction processes, and several solvent parameters (solubility of

In a closed vessel system high pressure can be reached. The high pressure stimulates various phenomena according to LeChatelier's principle, such as the transition of phase from one form to another, change in reaction dynamics, change in molecular structure, *etc*. hence resulting in the enhancement of extraction efficiency. Accordingly, working under high pressure causes alterations in the structure of some constituents of the cells such as lipids, proteins, enzymes, and outer cell membranes, thus damaging the plant wall and internal structure of the cell and reducing the mass transfer resistance. In this way, the secondary metabolites are released, leaving behind other cellular components. The High-Pressure MASE (HPMAE) is considered a newly emerging technique. It is a time-efficient, convenient, eco-friendly, safe, and energy-efficient extraction method when compared to the traditional or

**38**

**3.1 Solvent**

the analyte, penetration, interaction with the matrix, dielectric constant, and mass transfer kinetic process) should be considered to perform the right choice. *In primis*, the solvent should assure the highest solubility of the analyte of interest while excluding undesired matrix components. However, conversely to the traditional extractive procedures, the chosen solvent must be able to absorb the microwave radiation and to convert it into heat. This depends on the dielectric constant and the dielectric loss of the solvent. Thus, in microwave application, solvents are usually classified in low, medium, or high absorbance whether they absorb at 300 W (i.e. carbon chlorides, 1,4-dioxane, tetrahydrofuran, diethyl ether, ethyl acetate, pyridine, toluene, chlorobenzene, xylenes, and hexane), 200 W (i.e. water, DMF, NMP, butanol, acetonitrile, HMPA, ketones, o-dichlorobenzene, 1,2-dichloroethane, 2-methoxy ethanol, acetic acid, and trifluoroacetic acid) or 100 W (i.e. DMSO, ethanol, methanol, propanol, nitrobenzene, formic acid, ethylene glycol), respectively. For low absorbing solvents, the heating rate can be increased by mixing with solvents with a higher dielectric constant or by adding salts to the mixture [18]. Recently, increasing interest in ionic liquids as MASE solvents or modifiers has been observed. Ionic liquids are organic salts with low melting points, and they are usually liquid at room temperature. They are characterized by extremely low vapor pressure, high stability and can solubilize both polar and non-polar metabolites. Moreover, they have the advantage to be eco-friendly solvents, although their extensive use is limited due to the high commercial costs. Another emerging eco-friendly alternative is the use of deep eutectic solvents. They possess physical and chemical properties similar to the ionic liquids, but they show better biodegradability, toxicity profiles, and solubility properties. Lastly, the content of water in the sample should be taken into account since it influences the heating rate and facilitates the transport of the analytes into the solvent at higher rates [19].

As stated in the previous paragraph, a Solvent-Free Microwave Extraction (SFME) is also possible. This procedure foresees the direct irradiation with MW of the plant material, fresh or rehydrated. A crucial role is played by the content of water in the sample, because it is the heating of such water to cause the rupture of the cells of the plant material, favoring the release of the content. This process is mainly applied for the extraction of volatile compounds such as essential oil. The oil evaporates by azeotropic distillation with the water contained in the sample. After cooling outside of the microwave reactor the oil separates from the water and can be collected through a modified Clevenger apparatus. Water is refluxed back into the microwave oven to allow the continuous extraction of the oil from the sample [12].
