Industrial hemp (Cannabis sativa L.) waste products as a source of biopesticides and bioactive compounds for pharmaceutical, nutraceutical and cosmeceutical purposes

In the last decade, there has been a raising demand for hemp products from legal Cannabis sativa L. with tetrahydrocannabinol (THC) content < 0.3%, to be employed for different industrial uses, due to the spread of its cultivation and preference in sustainable agriculture. In the European Union about 25,000 hectares are grown, and more than 70 varieties are allowed to be cultivated in agricultural systems. During hemp processing, a massive amount of biomass, mostly represented by leaves and inflorescences, can be produced, and re-used to manufacture niche products. Among the latter, the essential oil (EO), a liquid, odorous product consisting mainly of monoterpenes and sesquiterpenes, can be a promising candidate to be employed in the next years in different sectors such as pest science, pharmaceuticals, cosmetics, and so on. On this basis, in Chapter 1 of this thesis, I overviewed the scientific literature regarding the chemical compositions of the EOs deriving from different varieties of industrial hemp, evidencing the potential of the use of their compounds as pharmaceutically active drugs, pesticides, and antimicrobial agents. In Chapter 2, I used the microwave-assisted extraction (MAE) to obtain a hemp EO enriched in bioactive constituents, especially cannabidiol (CBD), from the dry inflorescences of the Italian variety Carmagnola Selezionata (CS). For this aim, the operative parameters to enhance the EO yield and CBD content in terms of microwave irradiation power (W/g), extraction time (min), and water added to the plant matrix after moistening (%), were optimized through a central composite design (CCD) approach using a Milestone Ethos X device. The conventional hydrodistillation (HD) was used for comparative purposes. The qualitative compositions of EOs by MAE and HD were analysed by Gas Chromatography-Mass Spectrometry (GC-MS), whereas the quantitative detection of CBD and main terpenes was achieved by Gas Chromatography-Flame Ionization Detection (GC- FID). Moreover, the enantiomeric distribution of the chiral components (α-pinene, β-pinene, limonene, (E)-caryophyllene, and caryophyllene oxide) was evaluated by chiral chromatography. Results showed that the MAE treatment, using high irradiation power and relatively long extraction times, significantly enhanced the amount of CBD in the EO while maintaining high oil yield values with respect to the conventional HD. The enantiomeric excess of the three chiral monoterpenes was determined, with the (+)-enantiomers being prevalent, while (E)-caryophyllene and caryophyllene oxide were enantiomerically pure. So, the MAE was successfully applied to dry hemp inflorescences to recover a CBD-rich EO that may be exploited in diverse industrial applications. In Chapter 3, I investigated the chemical composition of EOs obtained from two hemp varieties, namely Felina 32 and CS, employing monoecious, male, and female inflorescences, and their mosquitocidal properties on larvae and pupae of two main malaria vectors, Anopheles gambiae and An. stephensi, were evaluated. Then, to demonstrate the safe use of hemp EOs for operators, their potential pro- or anti-inflammatory effect along with their toxicological profile, were assessed on dermal fibroblasts and keratinocytes. Given the promising outcomes achieved by insecticidal and anti-inflammatory studies, a preliminary evaluation of EOs encapsulation into nanoemulsions (NEs) has been carried out to improve their poor physicochemical stability by developing an eco-friendly formulation. This work pointed out the potential application of male inflorescences, which are usually discarded during hemp product processing. These EOs could be exploited as potential sustainable botanical insecticides, due to their capability to be active against mosquitoes and the possibility of employing them to develop stable and safe formulations. The LC50 values detected in this study (<80 ppm) are lower, on average, than those of several plant EOs, with the advantage of exploiting an industrial waste product. From MTT assay and gene and protein expression analysis, EOs displayed no cytotoxicity at the appropriate doses and possessed an anti-inflammatory effect on the tested human cell lines. These evidences support further applied research on hemp EOs in order to encourage their industrial exploitation. In Chapter 4, I applied MAE as a green extraction method for boosting the yield and quality profile of hemp EO when compared with other conventional extraction methods. During this process, two by-products are obtained, namely the aqueous residue, including bioactive phenolics, and the residual deterpenated biomass, which can be again extracted for purification of phytocannabinoids. So far, the hemp industry has not exploited these products, although they can be precious for the food, cosmetic, nutraceutical, and pharmaceutical markets. This work determined and optimized the variables influencing MAE efficiency, namely microwave irradiation power, extraction time, and added water, that were studied using a CCD approach, and results were useful to optimize the extraction process for recovering three valuable fractions, namely EO, polyphenols and phytocannabinoids. The products obtained through the optimized conditions were evaluated in terms of yield, chemical profile, and antioxidant potential. In addition, the by-products from the optimized run were further analysed for their biological activity performing both enzymatic and non-enzymatic assays. The aqueous residue showed a powerful α-glucosidase inhibition, a good effect in terms of superoxide radical scavenging activity, a modest efficacy in terms of inhibition of advanced glycation end products formation and no activity in terms of lipase inhibition. The residual deterpenated biomass did not display considerable biological activity. This study demonstrated the valorisation of industrial hemp EO and its by-products, resulting from a sustainable and eco-friendly extraction technique, through an almost waste-free approach. Cannabinoids along with other valuable bioactive constituents such as glycosidic flavones, may be recovered from the residues of the EO extraction, representing interesting compounds in the pharmaceutical, cosmetic, and nutraceutical sectors. In Chapter 5, two extracts of industrial hemp with different polarity (aqueous and hexane) were assessed by determining their antioxidant profile and their neuroprotective potential on pharmacological targets in the central nervous system (CNS). Several assays on in vitro antioxidant capacity (DPPH, superoxide radical, FRAP, ORAC), as well as inhibition of physiological enzymes, namely acetylcholinesterase (AChE) and monoaminooxidase A (MAO-A) were performed to discover how these extracts may be helpful to prevent neurodegenerative diseases. Neuro-2a cell line was selected to evaluate the cytotoxic and neuroprotective potential of these extracts. Both extracts displayed notable antioxidant capacity in the FRAP and ORAC tests, particularly the hexane extract, and interesting results for the DPPH and superoxide radical uptake assays, with the aqueous extract being performant, especially in the latter. In enzyme inhibition assays, the aqueous extract exhibited AChE and MAO-A inhibitory activities, while the hexane extract only achieved IC50 value for AChE inhibitory bioassay. Neuro-2a tests confirmed that polyphenolic extract was not cytotoxic and exerted cytoprotective properties against hydrogen peroxide and antioxidant response, reducing reactive oxygen species (ROS) production. Thus, these extracts could be a source of substances with potential benefit on human health, especially concerning neurodegenerative disorders. In Chapter 6, I investigated a novel research topic represented by new hemp breeding lines developed by crossbreeding of selected cultivars. In this context, the study focused on the phytochemical characterization of 9 hemp commercial varieties. HD was carried out in order to recover the EO, and also the residual water and deterpenated biomass. The terpene fraction was analysed by GC-FID, GC-MS, and Solid Phase Microextraction (SPME-GC-MS), showing diverse chemotypes. The polyphenolic profile was assessed on the residual water and deterpenated biomass by spectrophotometric tests, and by High-Performance Liquid Chromatography-Diode-Array Detection (HPLC-DAD-MSn ) and Proton Nuclear Magnetic Resonance (1H-NMR) analyses. The latter were used for quali-quantitative evaluation of cannabinoids in the deterpenated material compared with the untreated one. Moreover, the glandular and non-glandular indumentum of the 9 commercial cultivars was investigated by means of Light Microscopy (LM) and Scanning Electron Microscopy (SEM) in an attempt to find a potential correlation with the phytochemical and morphological traits. The EO and residual water were rich in monoterpene, sesquiterpene hydrocarbons, and flavonol glycosides, respectively, while the deterpenated biomass was revealed to be a source of neutral cannabinoids. The micromorphological survey allowed us to partly link the phytochemistry of these varieties with the hair morphotypes. This work led to the valorisation of several products from HD of new hemp cultivars, namely EO, residual water, and deterpenated biomass, that were found to be worthy of exploitation in both industrial and health applications. In Chapter 7, I reported the research work carried out abroad, specifically at Teagasc- Agriculture and Food Development Authority and University College (UCD) in Dublin, Ireland. During this period, I focused on Ultrasound Assisted Extraction (UAE) and MAE of Finola hemp inflorescences by using two advanced and novel equipments (an UAE probe system and an UAE-MAE pilot scale extractor), and employing only water as extraction solvent, for the recovery of phenolics, cannabinoids and proteins. This variety possessed limited amounts of cannabinoids, especially represented by cannabidiolic acid (CBDA), and only traces of phenolic compounds. Regarding proteins, the obtained results were more satisfactory. In fact, the applied techniques allowed to recover hemp extracts with higher protein content than that of raw (untreated) hemp and, in some cases, with respect also to conventional extracts. The percentage amount of proteins was especially enhanced by adding ethanol to the aqueous extracts resulting from probe UAE, to facilitate their precipitation. The most promising extracts were characterized in terms of free amino acid (FAA) profile, revealing the presence of all the essential AA, with an evident improvement in their content with respect to raw hemp and partly to conventional extracts as well. The data were statistically processed through Principal Component Analysis (PCA) and cluster analysis. The extracts were also evaluated through LC-MS protein analysis, which is still in progress. The hemp residues after probe UAE were subjected to Scanning Electron Microscopy (SEM) for the observation of the treatment effect on the samples surface, highlighting a certain micro-structures modification by cavitation induced by these radiations. Overall, the results of my PhD thesis provided new insights into the potential of hemp- derived compounds as valuable sources for the pharmaceutical, nutraceutical and cosmeceutical markets, and for the development of innovative botanical insecticides.