Cannabidiol-enriched hemp essential oil obtained by an optimized microwave-assisted extraction using a central composite design

https://doi.org/10.1016/j.indcrop.2020.112688Get rights and content

Highlights

  • Hemp essential oil was obtained by microwave-assisted extraction.

  • The operative conditions were optimized using a central composite design.

  • CBD-enriched essential oil can be obtained by MAE optimization.

  • Hydrodistillation was carried out for comparative purposes.

  • The enantiomeric ratio of chiral terpenes was determined.

Abstract

The increase of cultivation of industrial hemp (Cannabis sativa L.) all over the world offers new opportunities for the industry to manufacture innovative products from this multipurpose crop. In this regard, the hemp essential oil represents a niche product with potential interest for the pharmaceutical, nutraceutical, cosmeceutical and agrochemical companies. On this basis, in the present work we used the microwave-assisted extraction (MAE) to get an essential oil enriched in bioactive compounds, especially cannabidiol (CBD), from the dry inflorescences of the Italian variety CS (Carmagnola Selezionata). For this purpose, the operative conditions to increase the essential oil yield and CBD concentration in terms of microwave irradiation power (W/g), extraction time (min) and water added to the plant matrix after moistening (%), were optimized using a central composite design (CCD) approach using a Milestone ETHOS X device. The conventional hydrodistillation (HD) performed for 240 min was used for comparative purposes. The qualitative compositions of essential oils obtained by MAE and HD were analysed by GC-MS, whereas the quantitative detection of CBD and main terpenoids (α-pinene, β-pinene, myrcene, limonene, terpinolene, (E)-caryophyllene, α-humulene and caryophyllene oxide) was achieved by GC-FID. Furthermore, the enantiomeric distribution of the chiral constituents (α-pinene, β-pinene, limonene, (E)-caryophyllene and caryophyllene oxide) was determined using chiral chromatography. Results showed that the MAE treatment, using high irradiation power and relatively long extraction times, increased significantly the content of CBD in the essential oil while maintaining high oil yield values when compared with conventional HD. The enantiomeric excess of three chiral monoterpenes (α-pinene, β-pinene and limonene) was determined, with the (+)-enantiomers being predominant, whereas (E)-caryophyllene and caryophyllene oxide were enantiomerically pure. In conclusion, the MAE was successfully applied to hemp dry inflorescences in order to obtain a CBD-rich essential oil which may be exploited in several industrial applications.

Introduction

Hemp (Cannabis sativa L.), also known as the ‘fibre-type’ cannabis, is a legal crop cultivated from ages all around the world and its different parts, e.g. fibre, seeds, leaves and flowers are exploited in several sectors such as automotive industry, construction, paper, innovative materials, bioenergy, textile, varnishes and inks, as well as in medicine, foods, nutraceuticals and cosmetics (Ranalli and Venturi, 2004).

Hemp is an eco-friendly and sustainable crop since it enriches the soil in organic matter (> 10 t/ha), requires no agrochemical input and moderate fertilizer requirement, and owns adsorption properties toward pesticides, that is helpful in sustainable agricultural systems (Amaducci et al., 2008; Finnan and Styles, 2013; Vukčević et al., 2015). Indeed, hemp can be used in crop rotation with wheat, barley, corn and sunflower (Finnan and Styles, 2013). Different varieties are cultivated in the EU (EC Regulation, 2004), with most of them coming from France and Italy (Cappelletto et al., 2001). At present, the EU is the third producer of hemp in the world after China and Canada, with 25,000 ha of cultivations (Di Candilo, 2006). In Italy the hemp cultivation area is estimated at around 4000 ha with about 2000 farmers, most of them using organic agriculture (EC Regulation, 2007), and an overall income of ∼ 40 mln € per year.

Nowadays, C. sativa provides the bulk material for medical preparations, namely Bedrocan®, Epidiolex®, Sativex®, and others, that are used for the treatment of chronic diseases, multiple sclerosis, neuropathic pain and epilepsy (Barnes, 2006; Devinsky et al., 2018; Palmieri et al., 2019).

The availability of hemp biomass produced during manufacturing and processing of fibre and seeds represents a valuable resource to exploit and valorize on an industrial level. Thus, the increase of hemp cultivation in the years to come may represent an important occasion to valorize the potential of this multipurpose crop by developing innovative products from the huge amount of biomass produced during plant processing (Calzolari et al., 2017). In this regard, the hemp essential oil could be a product of interest for the pharmaceutical, nutraceutical and cosmeceutical industries and useful in integrated pest management (IPM) programs (Fiorini et al., 2019; Benelli et al., 2018a, b). In addition, its production may satisfy the increasing demand for oily extracts from cannabis (Fiorini et al., 2019). The essential oil may act as a good pesticide, notably against aphids, houseflies and ticks (Benelli et al., 2018a, b; Tabari et al., 2020), giving an added value to the whole production chain.

The hemp essential oil is produced in the capitate trichomes that are particularly abundant in inflorescences and, to a minor extent, in leaves (Happyana et al., 2013). The main volatile components can be divided into three groups depending on the cultivar, plant organ, storage, processing and extraction technique: i.e. monoterpenes including α-pinene, myrcene and terpinolene, sesquiterpenes such as (E)-caryophyllene, α-humulene and caryophyllene, and cannabinoids with cannabidiol (CBD) as the predominant compound, whereas δ-9-tetrahydrocannabinol (THC) is missing or occurs at trace levels (Mead, 2017; Benelli et al., 2018a, b; Bertoli et al., 2010). Thus, the hemp essential oil is an interesting non-psychotropic product showing a complex mixture, made up of terpenes and cannabinoids, able to produce the so-called ‘entourage-effect’ (Nahler et al., 2019).

CBD is a non-psychotropic cannabinoid endowed with notable immunomodulatory, anticonvulsant, anti-inflammatory, neuroprotective and anticancer effects (Appendino et al., 2011; Morelli et al., 2014; Russo, 2016; Nabissi et al., 2016; Watt and Karl, 2017; Gertsch, 2018). It acts on CB2 receptors and modulates the psychotropic effects of THC. Noteworthy, it was shown that CBD may defend the plant against herbivore attacks due to its antifeedant properties (Park et al., 2019). (E)-caryophyllene is an FDA-approved additive, recently recognized as a ligand of CB receptors with a non-cannabinoid structure. Notably, it is a selective agonist of CB-2 receptors, modulating the inflammatory processes and may also synergize the CBD action (Gertsch, 2008; Chicca et al., 2014; Sut et al., 2018). This sesquiterpene has also been found as an effective mosquitocidal and acaricidal agent (Pavela et al., 2020; Tabari et al., 2020). α-Humulene is reported as an anti-inflammatory and anticancer agent (Legault and Pichette, 2007), and owns insecticidal and acaricidal potential (Benelli et al., 2018c; Tabari et al., 2020). Caryophyllene oxide, the degradation product of (E)-caryophyllene, is an FDA-approved food additive owning anticancer activity and synergistic effects with chemoterapics (Fidyt et al., 2016; Hanušová et al., 2017). α-Pinene interacts with the cholinergic system improving memory and learning and counterbalances the toxicity of THC (Lewis et al., 2018). Myrcene has sedative and relaxant effects (Do Vale et al., 2002).

The most common extraction techniques to get essential oil from hemp both at laboratory and industrial scale are steam- (SD) and hydro-distillation (HD). However, they show some disadvantages, e.g., they are time-consuming, request high energy and water input, and sometimes cause thermal degradation of thermosensitive molecules (Filly et al., 2014). In the last years, solvent-free approaches have been designed for the extraction of volatile organic compounds (VOCs) from medicinal and aromatic plants (MAPs). Among them, microwave-assisted extraction (MAE) appears to be an effective, reliable, green technology to improve the extraction of VOCs from different kinds of matrices without the use of organic solvents (Lucchesi et al., 2004). This novel and efficient method works through microwave radiation causing vibration of water and other polar molecules with an increase of temperature and evaporation of water that disrupts cells and matrices with the release of VOCs from the matrix by azeotropic distillation (Filly et al., 2014). In this way, the diffusion of target compounds is easier and faster, saving time and energy. When applied to the extraction of essential oils, MAE revealed to have higher yields and lower costs, compared with conventional techniques such as SD and HD (Filly et al., 2014; Petigny et al., 2014). MAE efficacy is related to the selection of suitable operative conditions. Specifically, for every plant matrix and solvent composition, the effectiveness of the extraction process is dependent on the solvent-to-feed ratio, extraction temperature and time, and microwave irradiation power. Usually, an increase of microwave power and extraction time is associated with an increase of the yield even if this effect tends to level off after certain values. However, excessive heating of the matrices has to be avoided since it could damage some thermosensitive compounds. Concerning the solvent-to-feed ratio, a general trend cannot be defined since the results are strictly related to the matrix and solvent type (Veggi et al., 2012).

Recently, we showed that pretreatment with microwaves or oven heating has a significant effect on modulating the chemical profile of the hemp essential oil, for instance increasing the content of bioactive CBD and (E)-caryophyllene (Fiorini et al., 2019). On this basis, we decided to optimize for the first time the MAE process to get a bioactive-enriched essential oil from hemp using the Milestone ETHOS X for the microwave green extraction of natural products (Turk et al., 2018).

For this purpose, a statistical approach, the response surface methodology (RSM) - central composite design (CCD), was applied in order to understand the relationship between the hemp volatile constituents, essential oil yield and extraction parameters. The determined mathematical models were validated and then used to maximize the oil yield and recovery of bioactive compounds such as phytocannabinoids. The RSM methodology proved to be an effective tool in the MAE optimization of the extraction recovery of some essential oils (Petigny et al., 2014; Abedi et al., 2017; Mollaei et al., 2019), although it has been rarely applied for the evaluation of the extraction efficiency on single bioactive compounds. To the best of our knowledge, the optimization of the marker hemp volatile compounds as a function of extraction conditions has never been performed. To complete the work, we also determined the enantiomeric distribution for the main hemp optically active compounds, namely α-pinene, β-pinene, limonene, (E)-caryophyllene and caryophyllene oxide, by using chiral chromatography.

Section snippets

Plant material

Dry inflorescences of hemp were provided by Coop Canapa – Società Cooperativa Agricola, San Severino Marche, Italy (https://www.coopcanapa.it). They were obtained from female individuals of C. sativa cv CS (Carmagnola Selezionata) cultivated in Castelbellino (N 43°30′07.80″; E 13°11′16.33″, 200 m a.s.l.) and harvested in October 2018. Hemp inflorescences were dried under darkness at 20 °C and 50 % R.H. until constant weight, afterward they were crushed into small pieces and stored into jute

Results and discussion

In the present study, we optimized for the first time the extraction of the hemp essential oil using the ETHOS X, developed and patented by Milestone. This device showed to maximize the volatile terpenes and terpenoids extraction from the Cannabis plant, maintaining THC and CBD’s content in the fresh plant matrix (Milestone srl, 2019). Indeed, this procedure works well when the fresh inflorescences are used. In this case, the essential oil is devoid or contain trace levels of cannabinoids (

Conclusions

In the last years, the global market of CBD and hemp derivatives is continuously growing, acquiring importance from multiple standpoints, namely economics, medicine, industry and agriculture. This has been reflected by a significant increase in hemp cultivation area worldwide. In the US, the market of CBD accounted for 200  mln $ in 2017 and is expected to increase further, with a prediction of 450 mln $ in 2020. Noteworthy, the European Union is destined to become the largest world market for

CRediT authorship contribution statement

Dennis Fiorini: Formal analysis, Funding acquisition, Investigation, Writing - original draft, Writing - review & editing. Serena Scortichini: Formal analysis, Investigation, Methodology. Giulia Bonacucina: Writing - review & editing. Nicolas G. Greco: Formal analysis, Investigation, Methodology. Eugenia Mazzara: Formal analysis, Investigation, Methodology. Riccardo Petrelli: Funding acquisition, Writing - review & editing. Jacopo Torresi: Formal analysis, Investigation, Methodology. Filippo

Declaration of Competing Interest

Authors declare no competing interest.

Acknowledgements

The authors thank the Coop Canapa for providing the plant material, FKV – Milestone Srl for its assistance during the optimization procedure of ETHOS X, and Regione Marche (Programma di Sviluppo Rurale 2014/2020 – M16.1.A.2 Finanziamento dei Gruppi Operativi PSRMarche20142020_M16.1.A.2_ID_SIAR29012) and the University of Camerino (Fondo di Ateneo per la Ricerca) for financial support.

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