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

Identification of Anti-Neuroinflammatory Bioactive Compounds in Essential Oils and Aqueous Distillation Residues Obtained from Commercial Varieties of Cannabis sativa L

Neuroinflammation, which is mainly triggered by microglia, is a key contributor to multiple neurodegenerative diseases. Natural products, and in particular Cannabis sativa L., due to its richness in phytochemical components, represent ideal candidates to counteract neuroinflammation. We previously characterized different C. sativa commercial varieties which showed significantly different chemical profiles. On these bases, the aim of this study was to evaluate essential oils and aqueous distillation residues from the inflorescences of three different hemp varieties for their anti-neuroinflammatory activity in BV-2 microglial cells. Cells were pretreated with aqueous residues or essential oils and then activated with LPS. Unlike essential oils, aqueous residues showed negligible effects in terms of anti-inflammatory activity. Among the essential oils, the one obtained from 'Gorilla Glue' was the most effective in inhibiting pro-inflammatory mediators and in upregulating anti-inflammatory ones through the modulation of the p38 MAPK/NF-kappa B pathway. Moreover, the sesquiterpenes (E)-caryophyllene, alpha-humulene, and caryophyllene oxide were identified as the main contributors to the essential oils' anti-inflammatory activity. To our knowledge, the anti-neuroinflammatory activity of alpha-humulene has not been previously described. In conclusion, our work shows that C. sativa essential oils characterized by high levels of sesquiterpenes can be promising candidates in the prevention/counteraction of neuroinflammation

Optimization of Solvent-Free Microwave-Assisted Hydrodiffusion and Gravity Extraction of Morus nigra L. Fruits Maximizing Polyphenols, Sugar Content, and Biological Activities Using Central Composite Design

Black mulberry, Morus nigra L. (family: Moraceae), is a healthy food and medicinal plant. Microwave hydrodiffusion and gravity (MHG) is one of the most innovative applications of solvent-free microwave extraction. The aim of this study was to optimize for the first time the MHG solvent-free extraction of polyphenols and sugars from M. nigra fruits. Optimization was carried out using a central composite design (CCD) with selected responses such as extraction yield, total polyphenol (TPC), flavonoid (TFC), anthocyanin (TAC), and sugar (TSC) contents, in addition to DPPH radical scavenging, and a-glucosidase (AGHi), lipase (Li), and xanthine oxidase (XOi) inhibition as tools to evaluate the best parameters for efficient and rapid extraction of black mulberry. The optimized extract was characterized in terms of the aforementioned parameters to validate the models, and was further analyzed for 36 individual polyphenols using HPLC-MS/MS. The optimized MHG extract was finally compared with traditional extracts, and demonstrated much better performance in terms of TPC, TAC, and Li, while the traditional extracts showed better XOi and AGHi. In conclusion, MHG is a valuable green technique for the production of non-degraded black mulberry polyphenol-rich extract and we suggest its larger use in the pharmaceutical and food industries

Aniseed, Pimpinella anisum, as a source of new agrochemicals: phytochemistry and insights on insecticide and acaricide development

Pimpinella anisum L. (Apiaceae), known around the world as aniseed, is a widely cultivated crop, native of the sub-Mediterranean area. Its essential oil (EO) is exploitable in different fields such as food and beverages, pharmaceutics, cosmetics, and nutraceuticals. Regardless of the geographic origin, the EO exhibited consistent transanethole predominancy. Among the numerous biological properties exerted by aniseed EO, its antimicrobial, antifungal, insecticidal, and acaricidal effects have been extensively investigated for the formulation of biopesticides against larvae and adults of various pests and vectors. Hereafter, the published data on the insecticidal and acaricidal activity of aniseed EO and its major compounds on agricultural pests, stored-product pests, and arthropods of medical and veterinary interest is reviewed. For each study, the arthropod and the developmental stage on which the aniseed EO or the aniseed EO-based formulation were tested, the mode of action, the main constituents, and the exerted mortality, as well as the toxicity to non-target organisms and the possible sub-lethal effects are reported. The advantages of the possible use of aniseed EO as a biopesticide are analysed, as well as the current weaknesses and the critical points to be overcome to open the doors to the industrial utilization of Apiaceae EOs by the agrochemical industry

A Design of Experiment (DoE) Approach to Model the Yield and Chemical Composition of Ajowan (Trachyspermum ammi L.) Essential Oil Obtained by Microwave-Assisted Extraction

Ajowan (Trachyspermum ammi L.) is a spice traditionally used in Middle Eastern medicine and contains a valuable essential oil (EO) exploited in different fields, such as pharmaceutics, agrochemicals and food additives. This EO is mostly characterized by the thymol to which most of its biological properties are related. Given the economic value of ajowan and its increasing demand across the globe, the extraction method used for its EO is of paramount importance in terms of quality and quantity of the final product. In the present study, we used the design of experiment (DoE) approach to study and optimize the extraction of the ajowan EO using the microwave-assisted extraction (MAE), a novel extraction technique with high efficiency, low energy consumption, short process length and low environmental impact. A two-step DoE (screening followed by surface response methodology) was used to reduce the number of experiments and to improve the cost/benefit ratio. Reliable mathematical models, relating the more relevant EO features with the extraction conditions, were obtained and used to identify the best experimental conditions able to maximize the yield and thymol concentration. The optimized MAE procedure assures an EO with a higher yield and thymol amount compared with the standard hydrodistillation procedure

Prolonged sublethal effects of essential oils from non-wood parts of nine conifers on key insect pests and vectors

Conifers are of high economic importance due to their exploitation for timber, gardening and landscaping, construction and paper, varnishes, adhesives and waterproof coatings, perfumes and pharmaceuticals, bioplastics and biodiesel. They are endowed in all parts with secretory channels producing a semi-solid resin and a liquid fraction composed of a mixture of monoterpenes and sesquiterpenes, which are obtainable as a steam distillable essential oil. In most of industrial processes involving conifers, a huge biomass, made up mostly of green twigs, is produced, causing a waste disposal concern. The reuse of this material to produce essential oils may be important in a circular economy scenario. On this basis, in the present study we selected nine conifer species grown in central Italy (Abruzzo), namely Cupressus arizonica, x Cupressocyparis leylandii, Cupressus lusitanica, Juniperus chinensis cv. ‘stricta’, Juniperus communis, Juniperus × pfitzeriana, Picea abies, Pinus halepensis, and Pinus sylvestris, and evaluated the insecticidal potential of their essential oils from the non-wood renewable parts (twigs). For comparative purposes, in the case of J. communis two essential oils obtained from both male and female individuals were considered. The essential oils were obtained by hydrodistillation (yield in the range 0.3–2.0 %), and analyzed for chemical composition by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC–MS), and for the enantiomeric distribution of the main optically active isomers by chiral chromatography. The essential oils were assessed for their insecticidal activity against three important insect species, i.e. the larvae of a moth pest, Spodoptera littoralis, the larvae of the mosquito vector Culex quinquefasciatus, and the adults of the housefly, Musca domestica. Pyrethrum extract was the positive control. The essential oils with the highest potential as insecticide were those obtained from twigs of x C. leylandii, P. abies and P. sylvestris. These oils were made up mainly of monoterpenes, with δ-3-carene (20.5 %), α-pinene (16.7 %), limonene (15.4 %) and sabinene (10.4 %); camphene (14.5 %) and limonene (13.4 %), bornyl acetate (15.6 %) and borneol (11.2 %); α-pinene (25.7 %), limonene (15.6 %) and δ-cadinene (11.4 %), as the main constituents, respectively. α-Pinene occurred in these oils mostly with the (+) enantiomeric form; limonene was present mainly as (-)-enantiomer in C. leylandii whereas in P. abies and P. sylvestris the (+)-enantiomer prevailed. Furthermore, being exposed to LD20-30/LC30 of these three essential oils significantly reduced the longevity, development and vitality of the target insects. Overall, our findings showed that the x C. leylandii, P. abies and P. sylvestris essential oil bioactivity fairly overpassed that of the large majority of essential oils considered in earlier studies, allowing us to successfully candidate these botanical byproducts for further insecticide formulations

Encapsulation of Hemp (<i>Cannabis sativa</i> L.) Essential Oils into Nanoemulsions for Potential Therapeutic Applications: Assessment of Cytotoxicological Profiles

Industrial hemp (Cannabis sativa L.), due to its bioactive compounds (terpenes and cannabinoids), has gained increasing interest in different fields, including for medical purposes. The evaluation of the safety profile of hemp essential oil (EO) and its encapsulated form (nanoemulsion, NE) is a relevant aspect for potential therapeutic applications. This study aimed to evaluate the toxicological effect of hemp EOs and NEs from cultivars Carmagnola CS and Uso 31 on three cell lines selected as models for topical and inhalant administration, by evaluating the cytotoxicity and the cytokine expression profiles. Results show that EOs and their NEs have comparable cytotoxicity, if considering the quantity of EO present in the NE. Moreover, cells treated with EOs and NEs showed, in most of the cases, lower levels of proinflammatory cytokines compared to Etoposide used as a positive control, and the basal level of inflammatory cytokines was not altered, suggesting a safety profile of hemp EOs and their NEs to support their use for medical applications

Modulation of neuroinflammation by essential oils obtained from different hemp varieties

Nowadays, the progressive aging of the population due to the increase in longevity is a tangible event for the worldwide population. It represents a success for the scientific community thanks to the progress of science, but at the same time, it will mean a great challenge for the immediate future. Indeed, with the increase in life expectancy, we have witnessed a parallel increase in chronic-degenerative diseases, especially neurodegenerative diseases which mainly affect the elderly population. The etiology of neurodegenerative diseases is multifactorial, but they share key pathological features like oxidative stress and neuroinflammation. During neuroinflammation, a crucial role is played by microglia which constitute resident immune cells in the central nervous system. Nevertheless, pathological conditions like Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, stroke, and other neurological disorders, are associated with the hyperactivation of microglial cells. This phenomenon leads to the release of several pro-inflammatory mediators like cytokines and reactive oxygen species (ROS) leading to neuronal damage. Recently, the regulation of microglial immune-related functions through the endocannabinoid system has emerged as a potential neuroprotective strategy. Essential oils (EOs) obtained from hemp inflorescences are rich in interesting bioactive molecules like terpenes and terpenoids able to prevent the progression and help in the management of several chronic-degenerative diseases including neurodegenerative diseases. In this regard, improving the knowledge of different hemp varieties or cultivars to obtain EOs enriched in specific bioactive phytochemicals may be advantageous. This study aimed to investigate the anti-inflammatory properties of EOs obtained from inflorescences of different hemp varieties in BV-2 microglial cells. Cells were treated with increasing concentrations of EOs for 2 h and then activated with LPS 100 ng/mL for 24 h. One of the tested EOs, named Gorilla Glue (GG), demonstrated promising anti-inflammatory potential. Nitric oxide production, measured by Griess assay, was significantly reduced by 5x10-3 μL/mL EO treatment with respect to LPS. The treatment with EO decreased the gene expression of inflammatory mediators such as IL-1β, IL-6, TNF-α, COX-2, iNOS, and NLRP3 and increased the gene expression of anti-inflammatory mediators such as IL-4 and Arg1 compared to LPS. The anti-inflammatory effect could be ascribed to the significant reduction of the NFkB-nuclear translocation, probably mediated by the inhibition of the phosphorylation of p38 MAPK, as evidenced by immunofluorescence and Western Blot analyses, respectively. GG EO was able to counteract LPS-induced oxidative stress by significantly reducing ROS levels and increasing GSH levels. The main compounds found in GG EO were (E)-caryophyllene (17.6%), selina-3,7(11)-diene (12.5%), selina-4(15),7(11)-diene (7.1%) and α-humulene (4.7%). We tested (E)-caryophyllene and α-humulene for their anti-inflammatory activity. α-humulene was able to counteract the increase of all the pro-inflammatory mediators tested, while (E)-caryophyllene only upregulated the anti-inflammatory cytokine IL-4. These results suggest a specific contribution of different bioactive compounds to the positive effects shown, thus suggesting this EO is a promising protective agent in neurodegenerative diseases due to its anti-inflammatory properties

Antitrypanosomal Activity of Anthriscus Nemorosa Essential Oils and Combinations of Their Main Constituents

This study aimed to investigate the susceptibility of Trypanosoma brucei to the Anthriscus nemorosa essential oils (EOs), isolated compounds from these oils, and artificial mixtures of the isolated compounds in their conventional and nanoencapsulated forms. The chemical composition of the essential oils from the aerial parts and roots of Anthriscus nemorosa, obtained from a wild population growing in central Italy, were analyzed by gas chromatography/mass spectrometry (GC/MS). In both cases, the predominant class of compounds was monoterpene hydrocarbons, which were more abundant in the EOs from the roots (81.5%) than the aerial parts (74.0%). The overall results of this work have shed light on the biological properties of A. nemorosa EO from aerial parts (EC50 = 1.17 μg/mL), farnesene (EC50 = 0.84 μg/mL), and artificial mixtures (Mix 3–5, EC50 in the range of 1.27 to 1.58 μg/mL) as relevant sources of antiprotozoal substances. Furthermore, the pool measurements of ADP (adenosine diphosphate) and NTPs (nucleoside triphosphates) in the cultivated bloodstream form of trypanosomes exposed to different concentrations of EOs showed a disturbed energy metabolism, as indicated by increased pools of ADP in comparison to ATP (adenosine triphosphate) and other NTPs. Ultimately, this study highlights the significant efficacy of A. nemorosa EO to develop long-lasting and effective antiprotozoal formulations, including nanoemulsions

Microwave-assisted hydrodiffusion and gravity extraction: an efficient method to produce pomegranate juice

Green technologies based on microwaves have been developed by the food industry to produce organoleptically acceptable fruit juices without preliminary processing. Microwave irradiation coupled with hydrodiffusion and gravity (MHG) combines microwave heating with the earth's gravity, allowing the collection of hydrophilic substances released from the irradiated matrix. To the best of our knowledge, MHG extraction has never been experimented to produce pomegranate juice. In this work, we have evaluated it as a potential alternative to the conventional squeezing. A central composite design study (CCD) allowed the selection of the best extractive conditions (irradiation power and extraction time) to obtain a pomegranate juice with higher yield, polyphenol (e.g., catechin and delphinidin-3,5-glucoside) content, and related bioactivities (antioxidant and antidiabetic) than the one obtained by squeezing while maintaining the chemical-physical properties. Thus, this technique appears to be a functional alternative to producing high value pomegranate juice