*4.2.4 Phytocannabinoids*

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

such as ethanol, trichloromethane, cyclohexane, n-hexane, petroleum ether, and two in-house oils. Compared with SE, supercritical CO2 extraction, and normal stirring extraction, MASE of artemisinin from *Artemisia annua* considerably reduced the processing time to 12 minutes and resulted in a 92.1% extraction rate (compared to several hours and 60% extraction yield obtained with Soxhlet) [50]. Later, Liu et al. applied the MASE for the isolation and quantification of artemisinin in comparison with the traditional protocols. MASE confirmed shorter extraction time necessity, reduced solvent consumption, and higher recovery of artemisinin than conventional procedures. The best extraction solvent was petroleum ether–acetone (4:1 v/v), because of the high solubility of artemisinin and adequate microwave energy absorption, at 50°C. The highest yield of artemisinin achieved

was 0.55% in 30 minutes among all the extractive methods used [62].

of the content of artemisinin.

*Chemical structures of artemisinin and paclitaxel.*

tional extraction methods was produced [63].

pulmonary cancer.

**Figure 5.**

Recently, Misra et al. developed a rapid and reliable MASE and HPTLC protocol for the analysis of artemisinin. The optimized MASE conditions required 100 mg of dried and grinded drug with a size of 14 mesh dispersed into 10 mL of toluene. The irradiation of the sample at 160 W for 120 seconds led to the extraction of 0.816%

Paclitaxel (**Figure 5**) is a member of the taxane class, and it is one of the most important anticancer drugs approved for human use against ovarian, breast, and

Although the total synthesis of paclitaxel has been reported, its application for the commercial production of this drug is impracticable. Thus, paclitaxel is still produced by extraction from taxol biomass*.* The most commonly used methods for the extraction of paclitaxel require the use of methanol at ambient temperature, although other protocols requiring refluxing methanol, 1:1 methanol-chloroform at ambient temperature, and percolation using ethanol or 95% ethanol-water at ambient temperature have been reported. However, these methods require a long time (12–24 h) for a complete extraction. Incorvia-Mattina et al. reported for the first time in 1997 the use of MASE to optimize the efficiency of the extraction of paclitaxel. The effects of the biomass, solvent ratio and water content on taxane recovery were also determined. Under appropriate MASE conditions an extract equivalent to the one obtained by conven-

Talebi et al. investigated the use of MASE to extract paclitaxel from the needles of *Taxus baccata L.* The extraction parameters were investigated resulting in 90% aq. MeOH as a solvent, a temperature of 95°C, 7 min of extraction time, and a closed-vessel system as the best performing extractive conditions [64].

Recently, another study for the extraction of paclitaxel from biomass through MASE and based on kinetic and thermodynamic analysis has been carried out. The

**46**

*Cannabis sativa* L. has always been considered a controversial plant due to its use as both medicine and illicit drug. Nevertheless, Cannabis is a good source of nutrients, fibers, and natural compounds thus, its industrial and pharmaceutical use is undoubtful. Cannabis produces a peculiar class of natural compounds, namely phytocannabinoids. The two most important and renowned phytocannabinoids are the cannabidiol derivatives (i.e. CBD, CBDV, CBDB, and CBDP) [65, 66] and the tetrahydrocannabinol derivatives (i.e. Δ<sup>9</sup> -THC, Δ<sup>9</sup> -THCV, Δ<sup>9</sup> - THCB, and Δ<sup>9</sup> -THCP) [66, 67] reported in **Figure 6**. Δ<sup>9</sup> -THC is responsible for the recreational use of hemp and therefore its use is banished or tightly regulated by national governments.

CBD-like derivatives are non-psychotropic compounds but with other recognized pharmacological properties such as anti-inflammatory, antioxidant, and anticonvulsant. As an example, Epidiolex, a CBD-based anticonvulsant drug, has been approved in 2018 by Food and Drug Administration for the treatment of seizures associated with Lennox–Gastaut syndrome (LGS), Dravet syndrome, or tuberous sclerosis complex (TSC) in patients 1 year of age and older.

The discovery of a plethora of pharmacological activities ascribed to CBD and other minor phytocannabinoids has increased attention from both scientists and industries for medical, nutraceutical, and cosmetic applications of these cannabinoids.

Several synthetic procedures have been developed and optimized for the industrial preparations of phytocannabinoids and in particular of CBD. However, this process suffers from several drawbacks such as the cost of the starting materials, reagents and solvents, the formation of by-products with consequent cumbersome purification procedures, and the difficulty to control the stereochemistry, the isomerism of the terpenic double bond, and the easy interconversion of CDB into THCs in the synthetic conditions.

Thus, the extraction and purification of phytocannabinoids from *C. sativa* remain the preferred procedure for its cost-effectiveness. Besides, tight monitoring of the chemical consistency of the extracts results therefore mandatory in producing consistent and reliable medical cannabis preparations for human uses. Recently, Nahar et al. reviewed all the procedures adopted at the present for the extraction of naturally occurring phytocannabinoids [68].

**Figure 6.**

*Chemical structures of CBD-like and THC-like major phytocannabinoids present in* C. sativa*.*

Focusing on MASE, Lewis-Bakekr et al. investigated the potential to directly extract and decarboxylate dried Cannabis material with the microwave reactor [69]. Dried plant material, suspended in ethanol, was subjected to heating with stirring in a microwave reactor at 150° C. Extraction yield for the concentrated resin was in the range of 19.6–24.4% and it resulted directly proportional to the heating time and dependent on the cultivar employed in the process. Interestingly, a complete decarboxylation of the phytocannabinoids was achieved in one step following this process and no acid forms of phytocannabinoids such as Δ<sup>9</sup> -THCA and CBDA were detected in the resulting extract. Thus, MASE proves to be a worthy method for extraction and decarboxylation of phytocannabinoids due to the possibility to apply controlled temperatures and shorter extraction times. Moreover, this procedure ensures a more consistent and reproducible *Cannabis* extract with consequent reproducible efficacy of the therapeutic results. Kore et al. investigated and optimized the MASE process applied to *C. sativa* resulting in a patent application where they disclosed an improved method for extracting and decarboxylating cannabinoids from cannabis plant material, before, during, or after extraction [70]. MASE was compared to or used in tandem with other extraction strategies such as ultrasound extraction, SE, and supercritical fluid extraction.

The effect of time and temperature was investigated first. Extraction and decarboxylation of phytocannabinoid resulted in time and temperature dependence. To obtain 100% decarboxylation, the temperature must be sustained over a period without the burning of the cannabis material or the boiling/evaporation of the solvent. Because the solvent of choice is ethanol (b.p. 78°C at 1 atm), to reach a higher boiling temperature (i.e. 100–170°C) the extraction process must be carried out in a sealed vessel and under pressure. 170°C was the highest operative temperature achieved since higher temperatures (>180°C) resulted in the microwave run abortion due to the high pressure reached within the vial.

The extraction of cannabis by MASE at 100°C, 130°C, 150°C, and 170°C for 10 minutes resulted in a 23–25% yield of extract.

Interestingly, it appeared that the addition of a second step, such as SFE, after the MASE did not change the cannabinoid profile in the extract. Thus, MASE alone can perform an almost complete extraction of the cannabinoids from the cannabis plant material. Besides, the extraction and conversion of THCA and CBDA into THC and CBD was better at a temperature above 130°C, than at 100°C.

MASE was compared with the effectiveness of the commonly employed extractive procedure, namely maceration in ethanol, SE, and SFE. The conventional extractive procedures resulted in a low concentration of Δ<sup>9</sup> -THC, THCA, and CBD, whereas the addition of the microwave step resulted in a significant increase in the concentration of CBD and THC. As expected, no THCA was detected.

To sum up, a worth general procedure for the extraction and decarboxylation of CBD and THC from cannabis plant material can be thus resumed: i) the drug is weighed and macerated in a mortar; ii) the grinded drug is charged in a microwave vial along with a stir bar; iii) the drug is submerged with ethanol and the vial is sealed; iv) the vial is irradiated with MW using the following conditions [a) Pre-stirring = 30 sec; b) run time = 10 min; c) temperature = 150°C; d) absorption = Normal]; v) the suspension is filtered, and the filtrate concentrated; iv) residual plant material may be subjected (but not necessarily) to SFE.

Drinic et al. extended these studies over other polyphenols and flavonoids as well as phytocannabinoids [71]. In particular, the effects of different extraction parameters, namely ethanol concentration, extraction time and solid/liquid ratio on extraction yield, total phenol content, total flavonoid content, antioxidant activity, reductive capacity, CBD content, and THC content were investigated. For MASE,

**49**

mandatory.

**5. Conclusion**

*Microwave-Assisted Solid Extraction from Natural Matrices*

the Cannabis extract under the calculated conditions.

of various chemical procedures for the extraction of Δ<sup>9</sup>

by the seeds' bracts. The presence of Δ<sup>9</sup>

contrast, SE provided higher yields of Δ<sup>9</sup>

employed to produce hemp oil. However, the content of Δ<sup>9</sup>

a domestic microwave oven and a round-bottom flask connected with a condenser were used. The solid drug was mixed with the solvent (30, 50, or 70% v/v ethanol) in the selected solid/liquid ratio (S/L = 5, 10, or 15). The extraction was performed irradiating at a potency of 580 W without agitation and for a total extraction time of 10, 20, or 30 min. The results of each extraction were analyzed using response surface methodology. The influence of the three process parameters was investigated on total polyphenols yield, total flavonoids yield, antioxidant activity, and reductive capacity as well. The optimal conditions for the highest CBD content and lowest THC content resulted in 47% ethanol concentration, 10 minutes of extraction time, and an S/L ratio of 5. The model was successfully validated by preparing

Alongside the pharmaceutical uses of Cannabis extracts, hemp seeds are widely

hemp seed oils must be under the limits imposed by the jurisdictions of each State. Indeed, although the hemp seeds produce negligible amounts of THC, their outer surface can be contaminated with the enriched in the phytocannabinoids resin secreted

led to intoxication symptoms in the final consumers. Thus, nowadays the content of THC in hemp products is tightly regulated. Yang et al. investigated the effectiveness

hemp seeds and how the extractive methods could influence their commercialization [72]. Four extraction methods were employed, namely, i) microwave extraction, ii) sonication, iii) SE and iv) SFE. As already investigated by Kore et al., the extraction was performed in ethanol at 150°C with stirring, obtaining a complete conversion of CBDA and THCA into the corresponding neutral form. Hemp seeds were macerated in a mortar, transferred into a microwave vessel, and suspended in ethanol. The suspension was irradiated at 150°C with stirring for 20min in a closed vessel. The yield of the resin (27–38%) achieved was comparable to the other three extraction procedures. In

resulting in a more robust and appropriate extraction methodology for the testing of hemp seed products. Since the same solvent was used in all the compared extractions, the differences in the number of phytocannabinoids can be attributed to the extraction methods themselves. The results suggest that prolonged heating and solvent cycling in extracting phytocannabinoids from lipid-rich materials such as hemp seeds is

MASE has rapidly risen during the latest decades as a method for the extraction of secondary metabolites or compounds of pharmaceutical and nutraceutical interest. The use of microwave can generate peculiar, and otherwise impossible to reach extraction mechanisms. As a result, a reduction of the extraction time, improvement of the extraction efficiency, high reproducibility, and robustness of the procedure can be achieved. An increase of the sample throughput is in addition possible, thus it can be considered as the elective technique when a high number of samples have to be processed specially during the first stage of the NADD process, and for evaluating the quality of the natural matrices [9, 29]. For these reasons, MASE has proven to be effective in all aspects, including economical and practical, compared to traditional extraction techniques, especially over SE. Conversely, in MASE the development of the method must be carefully assessed, and all the variables and factors described above must be thoroughly considered to provide some extraction selectivity. Hence,





*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*

this process and no acid forms of phytocannabinoids such as Δ<sup>9</sup>

ultrasound extraction, SE, and supercritical fluid extraction.

abortion due to the high pressure reached within the vial.

extractive procedures resulted in a low concentration of Δ<sup>9</sup>

10 minutes resulted in a 23–25% yield of extract.

Focusing on MASE, Lewis-Bakekr et al. investigated the potential to directly extract and decarboxylate dried Cannabis material with the microwave reactor [69]. Dried plant material, suspended in ethanol, was subjected to heating with stirring in a microwave reactor at 150° C. Extraction yield for the concentrated resin was in the range of 19.6–24.4% and it resulted directly proportional to the heating time and dependent on the cultivar employed in the process. Interestingly, a complete decarboxylation of the phytocannabinoids was achieved in one step following

were detected in the resulting extract. Thus, MASE proves to be a worthy method for extraction and decarboxylation of phytocannabinoids due to the possibility to apply controlled temperatures and shorter extraction times. Moreover, this procedure ensures a more consistent and reproducible *Cannabis* extract with consequent reproducible efficacy of the therapeutic results. Kore et al. investigated and optimized the MASE process applied to *C. sativa* resulting in a patent application where they disclosed an improved method for extracting and decarboxylating cannabinoids from cannabis plant material, before, during, or after extraction [70]. MASE was compared to or used in tandem with other extraction strategies such as

The effect of time and temperature was investigated first. Extraction and decarboxylation of phytocannabinoid resulted in time and temperature dependence. To obtain 100% decarboxylation, the temperature must be sustained over a period without the burning of the cannabis material or the boiling/evaporation of the solvent. Because the solvent of choice is ethanol (b.p. 78°C at 1 atm), to reach a higher boiling temperature (i.e. 100–170°C) the extraction process must be carried out in a sealed vessel and under pressure. 170°C was the highest operative temperature achieved since higher temperatures (>180°C) resulted in the microwave run

The extraction of cannabis by MASE at 100°C, 130°C, 150°C, and 170°C for

THC and CBD was better at a temperature above 130°C, than at 100°C.

concentration of CBD and THC. As expected, no THCA was detected.

residual plant material may be subjected (but not necessarily) to SFE.

Interestingly, it appeared that the addition of a second step, such as SFE, after the MASE did not change the cannabinoid profile in the extract. Thus, MASE alone can perform an almost complete extraction of the cannabinoids from the cannabis plant material. Besides, the extraction and conversion of THCA and CBDA into

MASE was compared with the effectiveness of the commonly employed extrac-

whereas the addition of the microwave step resulted in a significant increase in the

To sum up, a worth general procedure for the extraction and decarboxylation of CBD and THC from cannabis plant material can be thus resumed: i) the drug is weighed and macerated in a mortar; ii) the grinded drug is charged in a microwave vial along with a stir bar; iii) the drug is submerged with ethanol and the vial is sealed; iv) the vial is irradiated with MW using the following conditions [a) Pre-stirring = 30 sec; b) run time = 10 min; c) temperature = 150°C; d) absorption = Normal]; v) the suspension is filtered, and the filtrate concentrated; iv)

Drinic et al. extended these studies over other polyphenols and flavonoids as well as phytocannabinoids [71]. In particular, the effects of different extraction parameters, namely ethanol concentration, extraction time and solid/liquid ratio on extraction yield, total phenol content, total flavonoid content, antioxidant activity, reductive capacity, CBD content, and THC content were investigated. For MASE,

tive procedure, namely maceration in ethanol, SE, and SFE. The conventional



**48**

a domestic microwave oven and a round-bottom flask connected with a condenser were used. The solid drug was mixed with the solvent (30, 50, or 70% v/v ethanol) in the selected solid/liquid ratio (S/L = 5, 10, or 15). The extraction was performed irradiating at a potency of 580 W without agitation and for a total extraction time of 10, 20, or 30 min. The results of each extraction were analyzed using response surface methodology. The influence of the three process parameters was investigated on total polyphenols yield, total flavonoids yield, antioxidant activity, and reductive capacity as well. The optimal conditions for the highest CBD content and lowest THC content resulted in 47% ethanol concentration, 10 minutes of extraction time, and an S/L ratio of 5. The model was successfully validated by preparing the Cannabis extract under the calculated conditions.

Alongside the pharmaceutical uses of Cannabis extracts, hemp seeds are widely employed to produce hemp oil. However, the content of Δ<sup>9</sup> -THC in the processed hemp seed oils must be under the limits imposed by the jurisdictions of each State. Indeed, although the hemp seeds produce negligible amounts of THC, their outer surface can be contaminated with the enriched in the phytocannabinoids resin secreted by the seeds' bracts. The presence of Δ<sup>9</sup> -THC in the final hemp seed products had led to intoxication symptoms in the final consumers. Thus, nowadays the content of THC in hemp products is tightly regulated. Yang et al. investigated the effectiveness of various chemical procedures for the extraction of Δ<sup>9</sup> -THC from three brands of hemp seeds and how the extractive methods could influence their commercialization [72]. Four extraction methods were employed, namely, i) microwave extraction, ii) sonication, iii) SE and iv) SFE. As already investigated by Kore et al., the extraction was performed in ethanol at 150°C with stirring, obtaining a complete conversion of CBDA and THCA into the corresponding neutral form. Hemp seeds were macerated in a mortar, transferred into a microwave vessel, and suspended in ethanol. The suspension was irradiated at 150°C with stirring for 20min in a closed vessel. The yield of the resin (27–38%) achieved was comparable to the other three extraction procedures. In contrast, SE provided higher yields of Δ<sup>9</sup> -THC and CBD than the other procedures, resulting in a more robust and appropriate extraction methodology for the testing of hemp seed products. Since the same solvent was used in all the compared extractions, the differences in the number of phytocannabinoids can be attributed to the extraction methods themselves. The results suggest that prolonged heating and solvent cycling in extracting phytocannabinoids from lipid-rich materials such as hemp seeds is mandatory.
