Journal:Essential oil of Cannabis sativa L: Comparison of yield and chemical composition of 11 hemp genotypes

Full article title	Essential oil of Cannabis sativa L: Comparison of yield and chemical composition of 11 hemp genotypes
Journal	Molecules
Author(s)	Pieracci, Yienia; Ascrizzi, R.; Terreni, Valentina; Pistelli, Luisa; Flamini, Guido; Bassolino, Laura; Fulvio, Flavia; Montanari, Massimo; Paris, Roberta
Author affiliation(s)	University of Pisa, CREA – Cereal and Industrial Crop Research Centre, University of Foggia
Primary contact	Email: roberta dot ascrizzi at gmail dot com
Year published	2021
Volume and issue	26(13)
Article #	4080
DOI	10.3390/molecules26134080
ISSN	1420-3049
Distribution license	Creative Commons Attribution 4.0 International
Website	https://www.mdpi.com/1420-3049/26/13/4080/htm
Download	https://www.mdpi.com/1420-3049/26/13/4080/pdf (PDF)

Abstract

Cannabis sativa L. is an annual species cultivated since antiquity for different purposes. While in the past hemp inflorescences were considered crop residues, at present they are regarded as valuable raw materials with different applications, among which extraction of the essential oil (EO) has gained increasing interest in many fields. The aim of the present study is the evaluation of the yield and the chemical composition of the EO obtained by hydrodistillation from 11 hemp genotypes, cultivated in the same location for two consecutive growing seasons. The composition of the EOs was analyzed by gas chromatography–mass spectrometry (GC–MS) and then subjected to multivariate statistical analysis. Sesquiterpenes represented the main class of compounds in all the EOs, both in their hydrocarbon and oxygenated forms, with relative abundances ranging from 47.1 to 78.5%; the only exception was the Felina 32 sample collected in 2019, in which cannabinoids predominated. Cannabinoids were the second most abundant class of compounds, of which cannabidiol was the main one, with relative abundances between 11.8 and 51.5%. The statistical distribution of the samples, performed on the complete chemical composition of the EOs, evidenced a partition based on the year of cultivation, rather than on the genotype, with the exception of Uso-31. Regarding the extraction yield, a significant variation was evidenced among both the genotypes and the years of cultivation.

Keywords: monoecious, dioecious, by-products, monoterpenes, sesquiterpenes, cannabinoids, flowering behavior, cannabidiol

Introduction

Cannabis sativa L. is an annual herb belonging to the Cannabaceae family, which has been cultivated since antiquity as a source of fiber, seed oil, food, and medicine, as well as for recreational and religious purposes. It has evolved as a dioecious species, with female and male flowers on different individuals, but selection processes have led to the development of monoecious genotypes that bare male and female flowers on the same individual. Thus, depending on the intended use, the morphology of the plants varies significantly between genotypes in terms of height, biomass, and seed yield.

The female inflorescences and leaves of the Cannabis plant are covered in glandular trichomes, which are considered biofactories of phytochemicals due to their ability to synthesize and store different secondary metabolites, of which phytocannabinoids are the best known and studied. On the basis of their cannabinoid content, in particular of their cannabidiol (CBD)/tetrahydrocannabinol (THC) ratio, Cannabis sativa L. genotypes are divided into five distinguished chemical phenotypes: (i) chemotype I, or drug-type (with the predominant cannabinoid being THC); (ii) chemotype II, or intermediate-type (with the predominant cannabinoids being CBD and THC); (iii) chemotype III or fiber-type (with the predominant cannabinoid being CBD); chemotype IV (with a prevalence of [[cannabigerol] [CBG]); and chemotype V, classifying materials with undetectable amounts of any cannabinoid.

Since 2001, when the regulatory European Commission (E.C.) No. 2860/2000 entered into force, the European Union (E.U.) authorized the cultivation of hemp complying with the 0.2% w/w Δ⁹-THC threshold. As a consequence, hemp cultivation for fiber and seed production was resumed, and more attention was paid to agro-industrial waste of the hemp chain, among which inflorescences, as valuable sources of bioactive molecules to feed the pharmaceutical, cosmeceutical, and manufacturing industries, in the perspective of the sustainable circular economy. In recent years, the extraction of hemp-based essential oil (EO) has gained increasing interest as a value-added product, thanks to its various fields of application. Hemp EO showed its best outcomes as an environmentally friendly insecticide against aphids, housefly populations, and mosquitoes, as a noteworthy toxic effect against Aedes albopictus is reported. Moreover, it exerts positive toxic activity towards the snail Physella acuta, an intermediate host of nematode and trematode human parasites, as well as being a common disease for rice fields. In the agricultural field, hemp EO exhibits strong allelopathic activity against invasive weed germination, as well as seedling growth. Interestingly, EOs were reported to be effective against dermatophyte species, thus exerting a role in preventing skin disorders. Moreover, its use as a beverage flavoring agent has been reported.

Essential oil from Cannabis is a complex mixture of volatile compounds made up of more than 100 terpenes and terpenoids (the oxygen-containing terpenes), known as the major contributors of the peculiar aromatic profile of different Cannabis strains. Monoterpenes (10 C) and sesquiterpenes (15 C) constitute the largest content of the hemp essential oil, in both their hydrocarbon and oxygenated forms, followed by diterpenes (20 C). Almost every compound identified in the EO has its own characteristic fragrance, and their combination is responsible for the unique aromatic bouquet of different strains, which can influence consumers’ preferences; generally, hemp varieties with high percentages of monoterpenes are considered more pleasant than those with high percentages of sesquiterpenes. Several factors, such as genotype, flowering behavior (dioecy or monoecy), cultivation technique, plant density, stage of development at harvest, material processing, and storage conditions can influence the composition of hemp EO (chemical profile) and its extraction yield. Monoterpenes could be present in higher quantities in the fresh material, while drying and storage could determine their loss, leading to the increment of the relative portion of sesquiterpenes. Environmental and weather conditions also seem to be an important factor for both the EO composition and yield; indeed, dry conditions between flowering stage and seed maturity can prevent trichome damage and EO yield losses.

All these factors affecting the yield and chemical composition of hemp essential oils—including the lack of standardization of growing and operating conditions—make it hard to compare the chemical composition of the essential oils extracted from different Cannabis sativa L. genotypes in diverse studies. Given that, this study aimed to evaluate the EO yield and the volatile profile of 11 different hemp genotypes, both monoecious and dioecious, belonging to three chemotypes (III–V), and the differences that occurred during two years of cultivation, in order to promote their employment as value-added by-products based on their peculiar characteristics.

Results and discussions

Plant material features

Five monoecious (Uso-31, Carmaleonte, Codimono, Futura 75, and Felina 32) and six dioecious (Bernabeo, Carmagnola, CS, Fibranova, Fibrante, and Eletta Campana) European industrial hemp genotypes were cultivated in an open field for two consecutive years. The main characteristics of the cultivated plant material—including chemotypes, flower type, and flowering time—are reported in Table 1.

Table 1. Agronomic trial data from the years 2019–2020. Height is expressed in cm, the diameter at the collar in mm, the density as number of plants/m2, and the dried inflorescences yield as g/m2. Genotype Chemotype Flower type Flowering time Sowing date Harvest date Height Collar diameter Plant density Dried inflorescences yield 2019 2020 2019 2020 2019 2020 2019 2020 2019 2020 2019 2020 Carmaleonte III Monoecious Medium 19 April 23 April 16 September 16 September 177 ± 42.5 223.6 ± 35.7 7.9 ± 2.8 13.8 ± 3.3 62 ± 24 2.2 ± 1.1 460 20 Codimono III Monoecious Medium 19 April 23 April 25 September 16 September 183 ± 39.9 255.2 ± 32.8 6.6 ± 2.2 13.0 ± 3.6 77 ± 27.2 5.7 ± 0.7 440 51 Futura 75 III Monoecious Medium 19 April 23 April 18 September 16 September 77.3 ± 17.6 238.6 ± 58.2 255.6 ± 4.9 13.1 ± 4.4 8.6 ± 0.3 7.4 ± 1.3 71 67 Felina 32 III Monoecious Medium 4 June 23 April 20 September 16 September 151 ± 33 222.3 ± 43.3 5.9 ± 1.9 13.3 ± 4.5 61.3 ± 23.8 4.2 ± 1.5 160 40 Uso-31 V Monoecious Early 4 June 23 April 20 September 31 August 108 ± 31 184.4 ± 26.0 5.2 ± 1.9 11.6 ± 2.3 60.6 ± 11.3 3.2 ± 0.3 200 29 Bernabeo IV Dioecious Late 19 April 23 April 28 September 8 October 211.6 ± 50.1 277.3 ± 38.9 8.6 ± 2.8 19.5 ± 6.7 64.3 ± 15.3 4.3 ± 3.2 430 39 Carmagnola III Dioecious Late 19 April 23 April 9 October 8 October 228.3 ± 44.8 272.4 ± 34.5 8.2 ± 2.5 19.2 ± 5.1 61 ± 1 2.7 ± 2.0 410 24 Fibranova III Dioecious Late 19 April 23 April 30 September 8 October 273.7 ± 45.6 314.5 ± 34.8 9.4 ± 2.8 17.1 ± 5.0 78.3 ± 14.4 6.3 ± 3.2 440 57 Fibrante III Dioecious Late 19 April 23 April 11 October 8 October 269.1 ± 47.6 202.8 ± 5.2 10.1 ± 2.7 15.0 ± 1.3 85.3 ± 11 0.7 ± 0.4 420 6 Eletta Campana III Dioecious Late 19 April 23 April 16 October 8 October 260.3 ± 44.3 304.5 ± 40.4 9.1 ± 2.6 17.5 ± 3.6 94.7 ± 29.1 5.7 ± 2.1 420 51 CS III Dioecious Late 19 April 23 April 1 October 8 October 246 ± 51.8 333.7 ± 37.5 9.2 ± 3 18.1 ± 3.8 60.3 ± 22.3 6.3 ± 1.7 440 57

Sowing was performed on April 19, 2019 and April 23, 2020, with a 25 cm distance between rows on randomized blocks with three repetitions, and a plot size of 20 m².

The harvest date differed according to the flower behavior and flowering time of each genotype. Generally, monoecious and early-flowering strains were harvested earlier than dioecious and late-flowering genotypes in both years (Table 1).

The comparison between plant heights showed differences between the two years, with higher plants obtained in 2020. The same trend was observed when comparing plant collar diameters. Plant density, defined as number of plants/m², and dried inflorescences yield, measured as g/m², were significantly lower in 2020, while dried inflorescences yield calculated for single plant was by far higher in 2020 than in 2019. These differences in plant growth in the two years of cultivation could be due to the lack of rainfall in the pre- and post-emergence period of 2020, compared to that of 2019, which could have caused a reduced germination rate and, therefore, a lower plant density. Conversely, 2019 was very rainy in May and June, favoring seed germination and following plant density. This rainfall trend was inverted in June; indeed, the rainy period (July and August) in 2020 could have allowed for a biomass production greater than that observed in 2019, as is clearly shown in Table 1. Finally, September 2019 was rainier than 2020, and this could have influenced the EOs’ yield.

Essential oil compositions and yields

The complete compositions and yields of the essential oils obtained from the dried inflorescences of 11 genotypes of Cannabis sativa L., cultivated by CREA-CI (Italy) in an experimental farm located in Rovigo and harvested during the years 2019 and 2020, are reported in Table 2 and Table 3, respectively. In total, 116 compounds were identified, representing 90.6–99.4% of the total composition.

TABLE 2

TABLE 3

All the EOs, with the exception of Felina 32-2019, were characterized by a predominance of sesquiterpenes, in both their hydrocarbon and oxygenated forms, ranging from 47.1% in Carmagnola-2020 (Table 3) to 78.5% in Fibrante-2019 (Table 2). As reported in Table 4, both the hydrocarbon and oxygenated sesquiterpenes presented significant differences in all the EO compositions as a function of the genotype, the year of cultivation, and their interaction.

Table 4. Two-way ANOVA performed on: (i) SIMPER-selected compounds; (ii) all detected chemical classes; (iii) EO extraction yield.   LSR, ** p < 0.01; *** p < 0.0001. n.s. = not significative. SIMPER-selected compounds (≥1% dissimilarity contribution) Genotype (G) Year (Y) Genotype × Year (G × Y) cannabidiol *** n.s. *** β-caryophyllene *** *** ** 14-hydroxy-9-epi-(E)-caryophyllene *** *** *** caryophyllene oxide *** ** *** caryophylla-4(14),8(15)-dien-5-ol (unidentified isomer 1) *** *** *** α-humulene *** *** *** caryophylla-4(14),8(15)-dien-5-ol (unidentified isomer 2) *** *** *** humulene oxide II *** ** *** selina-3,7(11)-diene *** *** *** α-bisabolol *** *** *** phytol *** *** *** α-pinene *** *** ** selin-6-en-4-ol *** *** ** myrcene *** *** *** hexahydrofarnesylacetone *** *** *** β-selinene *** *** *** All detected chemical classes Genotype (G) Year (Y) Genotype × Year (G × Y) Monoterpene hydrocarbons *** *** *** Oxygenated monoterpenes *** *** *** Sesquiterpene hydrocarbons *** *** *** Oxygenated sesquiterpenes *** *** *** Diterpene hydrocarbons *** *** *** Oxygenated diterpenes *** *** *** Phenylpropanoids *** ** ** Cannabinoids *** n.s. *** Apocarotenoids *** *** *** Other non-terpene derivatives *** ** ** Extraction yield (% w/w) Genotype (G) Year (Y) Genotype × Year (G × Y) EO hydrodistillation yield (% w/w) *** *** ***

In 2019 EOs, sesquiterpene hydrocarbons exhibited a significantly higher relative concentration in Futura 75, followed by Fibrante and Carmagnola. Lower relative abundances were, instead, found in Bernabeo, Eletta Campana, and Felina 32 (Table 2). In 2020 samples, instead, this class of compounds was significantly more abundant in Carmaleonte, Codimono, Eletta Campana, Fibranova, and Futura 75, while their lowest presence was detected in Uso-31 (Table 3). Furthermore, sesquiterpene hydrocarbons were significantly higher in 2020 than in 2019 for each analyzed hemp genotype, except for Carmagnola and Uso-31. Among this chemical class, the main volatile compounds were β-caryophyllene and α-humulene, in accordance with Ascrizzi et al., Vuerich et al., and Menghini et al., who reported these constituents as being typical of hemp varieties.

Regarding the oxygenated sesquiterpenes, among the 2019 EOs, Carmaleonte and Uso-31 presented higher relative abundances (59.6 and 60.9%, respectively), whilst the lowest was exhibited by Felina 32 (30.9%). Conversely to the hydrocarbon forms, oxygenated sesquiterpenes were significantly more abundant in all the 2019 samples, compared to those of 2020. These secondary metabolites are degradation products deriving from the oxidation of the corresponding terpenes due to air exposure (i.e., during prolonged storage), and are considered responsible for the antioxidant activity of many EOs. Moreover, higher oxygenated compounds’ relative abundances reflect more favorable growth conditions, as was also confirmed by the higher plant density detected in the 2019 samples. The analyzed samples presented caryophyllene oxide and humulene oxide II as the main components of this chemical class, which were also reported as the main epoxides found in the hemp varieties analyzed by Micalizzi et al. Furthermore, 14-hydroxy-9-epi-(E)-caryophyllene, reported by Ascrizzi at al., was present in low relative abundances.

Notes

This presentation is faithful to the original, with only a few minor changes to presentation. Some grammar and punctuation was cleaned up to improve readability. In some cases important information was missing from the references, and that information was added.