**4.1 Evaluation of plant productivity**

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

bound to the sample matrix [17].

**3.3 Time**

compounds [22].

target metabolite [24].

**3.4 Temperature and microwave power**

extraction speed by accelerating the desorption and dissolution of compounds

Extraction times in MASE lasts from a few minutes up to half an hour, and this represents an advantage for the extraction of thermal or oxygen labile compounds since it avoids the degradation of the compounds of interest [21]. The extraction yield is directly proportional to the extraction time, although it has been observed that this increment is very small for an extremely long time. Moreover, for longer extraction time overheating may occur, especially with high absorbent solvents, thus exposing thermolabile compounds to degradation. Whether longer extraction time is required, consecutive and shorter extraction cycles are preferable. The solvent can be collected after each extraction cycle and a fresh solvent could be added to the residue to guarantee the exhaustion of the matrix. This discontinuous procedure has been applied for the extraction of several secondary metabolites from plant material, allowing an enhanced yield and low decomposition of thermolabile

Temperature and MW power are strictly correlated. Power is the driving force of the process since it provides the energy necessary to excite the dipolar moments and the ionic conduction of the constituent of the sample, resulting in a proportional increase of the temperature and promoting the destruction of the natural matrix. Thus, the power of the microwave irradiation has to be carefully dosed in function of the amount of the sample, the solvent employed, the extraction time required, and the chemical stability of the secondary metabolites of interest [23]. Increasing the power results in an improved extraction yield and shorter extraction time. However, this result is true until the reaching of an optimal temperature beyond which a decrease in yield is observed, mainly due to the thermal stability of the

Accordingly, the temperature is a key parameter to enhance the efficacy of MASE and to avoid at the same time the degradation of the sample. Thus, the choice of the extraction temperature is strictly related to the properties of the solvent, the chemical stability of the metabolites of interest, and the microwave system used. In particular, at high temperatures the viscosity and the surface tension of the solvent diminish; moreover, the capability to solubilize the analytes, and to wet and penetrate the matrix increase, thus resulting in improved extractive efficacy. Also, when operating in a close-vessel, there is the advantage of heating the solvent above

To conclude, the development of a proper MASE methodology must consider at least four variables: solvent, liquid/solid ratio, temperature, and time. To speed up the set-up of the procedure, the Design of Experiment (DoE) approach may be applied. This is a systematic statistic-based tool to assess the best experimental conditions both in the academic and industrial fields [26]. Thanks to this approach, all the variables and their interactions can be evaluated while doing the minimum

Over the years, microwave-assisted extraction has been successfully applied to extract diverse classes of secondary metabolites (i.e. polyphenols, flavonoids, coumarins, terpenoids, cannabinoids, and alkaloids) from natural sources, for evaluating the plant productivity, for extracting bioactive compounds both for drug

its boiling point, thus leading to a more performing extraction [25].

**40**

number of runs.

discovery or for commercial purposes.

Numerous applications report about the use of the MW to assist the extraction of organic and organometallic compounds from various matrices (soils, sediments, water samples, botanicals), with special emphasis on environmental applications [27, 28]. Extraction of natural matrices is essential to compare their productivity under different stress conditions [9], harvesting time [29], and places [30]. MASE offers the possibility of performing multiple extractions and therefore, it is suitable for the rapid screening of a numerous set of samples to evaluate the productivity of organisms.

An example is a work performed by Martino et al., regarding the MASE of *Melilotus officinalis,* harvested in different environmental situations, to compare the amount of coumarin and related compounds, and to find the best condition for its cultivation [22]. The Authors developed a rapid, reliable, and reproducible method of extraction from *M. officinalis* inflorescence of coumarin (**Figure 2**), melilotic acid, and o-coumaric acid, considered as productivity markers of the plants. A comparison of different extraction techniques evidenced that MASE is the best procedure in terms of both yields and extraction time [22]*.* The optimal extraction conditions consisted of two successive irradiations of 5 min each at 50°C, with a cooling step in between, and it resulted suitable for application to large sets of samples [22].

Another example is the setup of a fast and reproducible extraction methodology of vitexin and its isomer isovitexin from *Crataegus monogyna* (**Figure 2**) for evaluating the plant productivity and determining the best ecological conditions for hawthorn cultivation in northern Italy (Lombardy). These metabolites have a high pharmaceutical value due to their anti-hyperalgesic and neuroprotective effects and their activity against oxidative stress, cancer, and inflammation [31].

Within this context, Martino et al. set up a MASE procedure that can be applied for quantitative extraction of both metabolites from *C. monogyna* in just 3 minutes [50% aqueous methanol (v/v), 120°C, 120 W], bringing advantages both in terms of time (3 min *vs* 6 hours) and solvent consumption (0.05 vs 0.10 g/mL) over standard extraction methods [30]. The developed MASE protocol combined with isocratic

**Figure 2.** *Secondary metabolites extracted via MASE approach and considered as markers of the plant productivity.*

HPLC analysis is suitable for the rapid screening of plant materials collected in different environmental conditions, and to determine the best ecological conditions for its cultivation. To extract vitexin and isovitexin from *Crotalaria sessiliflora,* Tang et al. exploited a microwave-assisted cloud-point extraction (MACPE). MACPE combines cloud-point extraction (CPE) with MAE. This has emerged as a technique to extract and separate bioactive compounds from medicinal plants [32]. Of note, using MACPE, hydrophobic compounds present in the aqueous phase can be favorably extracted into the hydrophobic core of micelles [33]. Applying MACPE, vitexin and isovitexin have been obtained in high yields and short times [34].

MASE can also be applied to study the effect of micronutrients or pollutants on secondary metabolites production. Amri et al. investigated the impact of soil copper (II) concentrations on nutrient uptake and the antioxidant system of *Marrubium vulgare*. Owing to waste deposition and agricultural practices, copper (II) tends to accumulate in high and toxic concentrations, leading to an alteration of the vital physiological or biochemical functions of the plants. As it is the case of *M. vulgare,* these effects may have a great impact on human health, since such a plant is used worldwide for its medicinal properties. To perform the study, the Authors selected marrubiin (**Figure 2**) as a reference compound, since it is the main secondary metabolite produced by *M. vulgare* leaves. A MASE protocol was developed for the easy extraction of marrubiin. This procedure allowed to evaluate the quality of a wide range of samples of white horehound. To optimize the process, the Authors used the statistical DoE approach. DoE findings indicated that the highest extraction efficiency of marrubiin with high repeatability was obtained using 100% ethanol at 120°C for 15 min, with significant benefits in terms of extraction times and environmental impact, given that ethanol is completely biodegradable. The MASE methodology may be applied for the characterization of *M. vulgare* herbal drug samples, thus evaluating their exposure to abiotic stress, revealing their phytochemical status, and facilitating the identification of raw materials obtained from a plant grown under stress conditions.

To sum up, MASE procedures is a versatile technique suitable for the evaluation of the plant productivity, and to assess the quality of vegetal matrices, since it is fast, reproducible, suitable for extraction of a large number of samples and requires a low amount of natural matrix.
