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What variety of hemp for cbd oil

Industrial, CBD, and Wild Hemp: How Different Are Their Essential Oil Profile and Antimicrobial Activity?

4 Department of Fruit Science, Viticulture and Enology, Faculty of Horticulture and Landscape Engineering, Tr. A. Hlinku 2, Slovak University of Agriculture in Nitra, 949 76 Nitra, Slovakia; [email protected]

5 Department of Bioenergetics and Food Analysis, Institution of Food Technology and Nutrition, University of Rzeszow, Cwiklinskiej 1, 35-601 Rzeszow, Poland

Tess Astatkie

6 Department of Engineering, Faculty of Agriculture, Dalhousie University, Truro, NS B2N 5E3, Canada; [email protected]

Ivanka B. Semerdjieva

7 Department of Botany and Agrometeorology, Faculty of Agronomy, Agricultural University, 4000 Plovdiv, Bulgaria; [email protected]_v

Dragana Latkovic

8 Department of Field and Vegetable Crops, University of Novi Sad, 21000 Novi Sad, Serbia; [email protected]

1 Crop and Soil Science Department, 3050 SW Campus Way, Oregon State University, Corvallis, OR 97331, USA

2 Institute of Field and Vegetable Crops, Alternative Crops and Organic Production Department, Maksima Gorkog 30, 21000 Novi Sad, Serbia; [email protected]

3 Plant Genetic Research Group, Agrobioinstitute, Agricultural Academy, 8 “Dragan Tsankov” Blvd., 1164 Sofia, Bulgaria; [email protected]

4 Department of Fruit Science, Viticulture and Enology, Faculty of Horticulture and Landscape Engineering, Tr. A. Hlinku 2, Slovak University of Agriculture in Nitra, 949 76 Nitra, Slovakia; [email protected]

5 Department of Bioenergetics and Food Analysis, Institution of Food Technology and Nutrition, University of Rzeszow, Cwiklinskiej 1, 35-601 Rzeszow, Poland

6 Department of Engineering, Faculty of Agriculture, Dalhousie University, Truro, NS B2N 5E3, Canada; [email protected]

7 Department of Botany and Agrometeorology, Faculty of Agronomy, Agricultural University, 4000 Plovdiv, Bulgaria; [email protected]_v

8 Department of Field and Vegetable Crops, University of Novi Sad, 21000 Novi Sad, Serbia; [email protected]

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Abstract

Hemp (Cannabis sativa L.) is currently one of the most controversial and promising crops. This study compared nine wild hemp (C. sativa spp. spontanea V.) accessions with 13 registered cultivars, eight breeding lines, and one cannabidiol (CBD) hemp strain belonging to C. sativa L. The first three groups had similar main essential oil (EO) constituents, but in different concentrations; the CBD hemp had a different EO profile. The concentration of the four major constituents in the industrial hemp lines and wild hemp accessions varied as follows: β-caryophyllene 11–22% and 15.4–29.6%; α-humulene 4.4–7.6% and 5.3–11.9%; caryophyllene oxide 8.6–13.7% and 0.2–31.2%; and humulene epoxide 2, 2.3–5.6% and 1.2–9.5%, respectively. The concentration of CBD in the EO of wild hemp varied from 6.9 to 52.4% of the total oil while CBD in the EO of the registered cultivars varied from 7.1 to 25%; CBD in the EO of the breeding lines and in the CBD strain varied from 6.4 to 25% and 7.4 to 8.8%, respectively. The concentrations of δ9-tetrahydrocannabinol (THC) in the EO of the three groups of hemp were significantly different, with the highest concentration being 3.5%. The EO of wild hemp had greater antimicrobial activity compared with the EO of registered cultivars. This is the first report to show that significant amounts of CBD could be accumulated in the EO of wild and registered cultivars of hemp following hydro-distillation. The amount of CBD in the EO can be greater than that in the EO of the USA strain used for commercial production of CBD. Furthermore, this is among the first reports that show greater antimicrobial activity of the EO of wild hemp vs. the EO of registered cultivars. The results suggest that wild hemp may offer an excellent opportunity for future breeding and the selection of cultivars with a desirable composition of the EO and possibly CBD-rich EO production.

Keywords: Cannabis sativa, essential oil, cannabinoids, cannabidiol, δ9-tetrahydrocannabinol, dronabinol, monoterpenes, sesquiterpenes, wild hemp, hemp cultivars

1. Introduction

Hemp (Cannabis sativa L.) is a new-old crop, one of the most controversial and promising crops due to its multiple utilizations, and contains a wide array of biologically active substances synthesized and accumulated in different plant parts [1]. Industrial hemp has been grown for grain and fiber for many decades in Europe, Asia, and North America. In addition, there is wild hemp (C. sativa spp. spontanea Vavilov, also known as spontaneous), which is native to both Central and Eastern Europe as well as parts of Asia, and is found as a weed in agricultural fields.

Hemp essential oil (EO) can be extracted using various extraction methods, the simplest and most natural using either steam or hydro-distillation, as is the case with many other EO crops. Hydro-distilled or steam distilled hemp EO are generally preferred by consumers and can be incorporated into a number of certified organic products; the market for organic food and non-food products reached US$55 billion in 2019 in the USA alone [2]. Currently, cannabidiol (CBD) production and markets in many countries are depressed due to the 2019 overproduction and the COVID-19 situation. For example, hemp production in the USA has increased rapidly and by mid-2019, there were around 500,000 licensed acres to grow hemp [3]. That acreage in 2019 would have generated an estimated US$11.3 billion income, or around 6% of the total value of all cash crops in the USA [3]. However, due to the depressed markets and expensive feminized seed (US$1/seed), there is now significant interest in hemp EO from industrial hemp cultivars and even from wild hemp. Indeed, a number of producers and processors in the USA are developing new products based on naturally extracted hemp EO or cannabinoids. Overall, research suggests that the hemp EO has medical significance and may also be utilized as an ingredient in commercial insect repellents and biopesticides [4,5]. Some cultivars such as Finola have been employed for commercial production of EO, as hemp EO has commanded high prices in recent years.

Hemp terpenes in the EO contribute to the aroma of various cannabis genotypes, and so far, around 140 different terpenes have been reported in this plant [1,6,7,8]. Current thinking is that terpenes have played a key role in the selection of medical/recreational and CBD type cannabis because their concentration is positively correlated to some of the cannabinoids [9].

The hemp EO profile depends on genotype, growth conditions, and extraction method (steam, hydro, CO2, or solvent extraction) [10]. Of the various groups of terpenes, monoterpenes such as limonene, β-myrcene, α-pinene, β-pinene, and linalool (containing 10 carbon units) comprise the major portion of the volatile oil fraction [10]. These EO constituents are widely found in the EO of other plant species such as spices, EO crops, and medicinal herbs; they are not specific to cannabis. Sesquiterpenes (with 15 carbons) such as β-caryophyllene, α-humulene, caryophyllene oxide, and β-phellandrene are also present in higher concentrations in hemp extracts [9,10]. Monoterpene composition can distinguish between monoecious and dioecious hemp cultivars [10]. Furthermore, certain terpenoids are highly correlated to the concentration of CBD and Δ9-tetrahydrocannabinolic acid (THCA); consequently, they have been proposed as a chemotaxonomic classification tool and to distinguish drug-type cannabis in Nevada [11]. Overall, hemp varieties (cultivars) with a higher concentration of monoterpenes have a more pleasant aroma compared with the varieties with higher amounts of sesquiterpenes. The monoterpenes pinene and limonene are the determinants of cannabis aroma in the immediate vicinity of the plant [12]. Hydro-distilled EO from industrial hemp cultivars may contain CBD [10]; the interaction of environment and genetics plays a role in the hemp EO profile.

Hemp EO (distilled from leaves, inflorescences, and thinner stems) has shown biological activity against several targets of pharmaceutical interests S. aureus, H. pylori, Candida, and Malassezia spp., enzymes, and cancer cell lines [13]. The EOs (collected from inflorescences after blooming) of cvs. Carmagnola, Fibranova, and Futura have shown significant antimicrobial activity against G + and G – bacteria and yeast, and the effect depended on the cultivar and seeding date [14].

This study addresses a knowledge gap and current industry interest towards hemp EO with different origins and profile. The hypothesis was that wild hemp would have a different EO content, composition, and antimicrobial activity compared with the EOs of registered industrial hemp cultivars, new hemp breeding lines, and a hemp strain (unregistered cultivar) that is currently used for the commercial production of CBD. The objective of this study was to compare nine wild hemp accessions (C. sativa spp. spontanea) sampled from agricultural fields in northeastern Serbia with 13 EU registered cultivars, eight breeding lines, and one CBD hemp strain (belonging to C. sativa) with respect to their EO profile and antimicrobial activity.

2. Results and Discussion

2.1. Essential Oil (EO) Content (Yield)

The EO yield of the wild hemp accessions varied from 0.085 to 0.262 mL/100 g for air-dried material and the yield of the breeding lines was 0.06 to 0.14, while the EO yield of the registered cultivars was 0.1 to 0.2 mL/100 g of dried material. However, the overall differences in oil yield between the three groups of hemp were not significantly different, with an overall mean of 0.129 mL/100 dried material ( Table 1 ).

Table 1

Analysis of Variance (ANOVA) p-values that show the significance of the effect of cultivar on 16 constituents, square root of mean squares error (Root MSE) that represents the common standard deviation, and the overall mean oil content (v/w, volume oil per dry weight) and concentration (%) of the five constituents with no significant difference between the cultivars and CBD. The p-values that show significant (p < 0.05) and marginally significant (0.05 ≤ p < 0.1) effect and need multiple means comparison are shown in bold.

Constituent ANOVA p-Value Root MSE Overall Mean
Oil content 0.362 0.040 0.129
α-Pinene 0.439 2.083 2.148
β-Pinene 0.380 0.857 0.923
Isocaryophyllene (γ-Caryophyllene) 0.072 0.236
β-Caryophyllene 0.007 2.768
α-(E)-Bergamotene 0.001 0.431
(Z)-β-Farnesene 0.141 0.878 1.669
Caryophyllene oxide 0.052 0.661
Humulene epoxide 2 0.001 0.503
Selina-6-en-4-ol 0.064 0.380
Caryophylla-4(12),8(13)-dien-5α-ol 0.101 0.397 1.412
Caryophylla-4(12),8(13)-dien-5β-ol 0.002 0.303
14-hydroxy-(Z)-Caryophyllene 0.212 0.263 0.349
β-Bisabolol 0.001 0.072
α-Bisabolol 0.003 1.023
CBD 0.487 5.574 12.39
δ9-Tetrahydrocannabinol (Dronabinol) 0.001 0.237

Overall, the EOs of the wild hemps and registered cultivars in this study were similar to those reported previously: 0.23 to 0.31% in fresh inflorescences [14], 0.29 to 0.19% depending on the collection time with higher EO yield from plants sampled earlier (in September than in October) [13], and 0.1% in stems and 0.15% in the leaves of wild hemp from Austria [15], respectively. However, the EO content of the USA hemp strain, utilized for CBD production in the USA and grown near the field trials was 1.15 to 1.2%.

2.2. Essential Oil (EO) Profile of the Three Groups of Hemp

For the wild hemps, two locations (Kovacica and Susara) were randomly selected among the nine locations (Slavka, Kovacica, Buro, Daleka zemlia, Susara, Saykaj, Perez, Titelski breg, and Paluka) to be used as two replications to represent wild hemp in the statistical analyses. As indicated in the Materials and Methods section, one-way ANOVA was completed to determine the significance of differences between the mean constituents obtained from nine hemp cultivars from Northeast Serbia. The constituents were: α-pinene, β-pinene, isocaryophyllene (γ-caryophyllene), β-caryophyllene, α-(E)-bergamotene, (Z)-β-farnesene, caryophyllene oxide, humulene epoxide 2, selina-6-en-4-ol, caryophylla-4(12),8(13)-dien-5α-ol, caryophylla-4(12),8(13)-dien-5β-ol, 14-hydroxy-(Z)-caryophyllene, β-bisabolol, α-bisabolol, CBD, and δ9-tetrahydrocannabinol.

Overall, the EO profile of the wild hemp was different from that of one of the registered cultivars and the new breeding lines (Supplementary Tables S1–S3). The EO constituents whose concentrations were significantly different are shown in bold in Table 1 . The mean concentrations of the five constituents and CBD from the above list that did not have significant differences between the wild and the registered cultivars are shown in Table 1 . The 10 constituents whose means were significantly different are shown in Table 2 . The comparative concentrations of the EO constituents in this and previous reports cited here are summarized in Table 3 .

Table 2

Mean concentration (%) of isocaryophyllene (γ-caryophyllene) [1], β-caryophyllene [2], α-(E)-bergamotene [3], caryophyllene oxide [4], humulene epoxide 2 [5], selina-6-en-4-ol [6], caryophylla-4(12),8(13)-dien-5β-ol [7], β-bisabolol [8], α-bisabolol [9], and δ9-tetrahydrocannabinol (dronabinol) [10] obtained from the 9 cultivars.

Cultivar
Bacalmas Carmagnola CS 1 Dioica Wild Helena Sequieni Simba ŠPIC
[1] 1.1 a 1.0 a 0.96 a 1.1 a 0.87 a 0.98 a 1.4 a 0.93 a 0.32 b
[2] 27 cd 27 cd 33 bc 26 d 28 bcd 28 bcd 33 b 25 d 40 a
[3] 3.1 a 2.1 bc 0.37 d 1.5 c 1.1 cd 3.8 a 1.2 cd 1.7 c 2.9 ab
[4] 6.6 a 4.2 c 5.4 abc 6.2 ab 5.6 abc 6.3 a 6.6 a 4.7 bc 6.0 ab
[5] 2.0 a 1.4 c 1.4 c 1.5 bc 1.7 ab 1.8 ab 1.9 a 1.6 bc 0.96 d
[6] 0.4 c 1.3 ab 0.56 bc 1.6 a 0.39 c 0.89 abc 0.49 bc 0.66 bc 0.19 c
[7] 1.8 bc 1.1 de 1.14 cde 1.5 cd 2.6 a 1.4 cd 2.3 ab 1.3 cd 0.55 e
[8] 1.7 a 1.1 cd 1.1 d 1.3 bc 1.8 a 1.38 b 1.7 a 1.1 cd 0.67 e
[9] 2.7 bcd 4.1 bc 6.7 a 2.1 bcd 1.6 d 1.75 cd 0.50 d 4.1 b 0.46 d
[10] 0.001 d 0.93 b 0.17 d 0.29 cd 2.4 a 0.14 d 0.42 bcd 0.76 bc 0.001 d

Within each row, means sharing the same letter are not significantly different. 1 CS—cv. Carmagnola Selezionata.

Table 3

Comparative concentrations of essential oil (EO) yield (content, % in biomass) and the concentration of various constituents (% of total oil) in hemp EO from this study and the literature reports.

Wild Hemp Reg. cvs New Lines [10] [14] [17] [20] [12] [22] [13] [19] [23] [24] [18]
Extraction type HD HD HD SD HD HD SE SD HD HS-SPME SE SD/HD HD
Oil content/EO yields 0.09–0.26 0.1–0.2 0.06–0.14 0.11–0.25% 0.23–0.31% 0.1–0.3% 0.3% 0.28% 0.04–0.12%
α-Pinene 0–2.5 0–8.4 0.15–2.9 3–20% 10.9–16.99 2–7.8% 23% 17.9% 11% 9.6–40.1% 8.1–18.2% 1.6–2.9%
β-Pinene 0–0.98 0.06–2 0–1.4 1–8% 6.38–9.33 8.6% 2.9–9.3% 2.6–5.2%
Isocaryophyllene (γ-Caryophyllene) 0.2–1.2 0.6–1.4 0.6–1.6
β-Caryophyllene 15.4–29.7 22.4–55 11.4–22 7–28% 10.56–13.90 18.7% 46% 5.2–22.6% 4.83–5.8 mg mL −1
α-(E)-Bergamotene 0.75 to 1.9 3.9–6.8 8.9–17 1.8–2.7 3.6% 4% 1.54–1.91 mg mL −1 1.9–2.7%
(Z)-β-Farnesene 0.13–3.0 0–2.8 0.4–2.1 4.4% 2.37–2.91 mg mL −1 1.4–3.0%
Caryophyllene oxide 0.24–31.2 3.9–6.8 8.7–17 2–6% 3–11 15% 1.2–13% 2.3–4.5%
Humulene epoxide 2 1.3–3.2 0.4–2.3 2.3–5.6
Selina-6-en-4-ol 0.23 to 1.6 0–1.8 1.1–2.8
Caryophylla-4(12),8(13)-dien-5α-ol 1.1–5.4 0–2.3 2.3–6.6 0.7–2.0%
Caryophylla-4(12),8(13)-dien-5β-ol 1.1–5.4 2.3 2.3–6.6
14-hydroxy-(Z)-Caryophyllene 0–3.4 0–1.3 0.72–2.1
β-Bisabolol 0.9–4.5 0–1.8 2.9–4.0
α-Bisabolol 0.4–.9 0–6.9 0.5–3.5
Cannabidiol, CBD 6.9–52.4 7.1–25 6.4–25 0.9–4.4% 10.0–11.1 1.9–8.6mg/g 0.1–0.15% 0.1–7.6% 24.9%
THC, δ9-Tetrahydrocannabinol 0–3.4 0–1.18 0.4–3.6
Monoterpenes 0–8.03 0.3–13 0.19–10 58.06–68.4 5.3 57.2% 54.2% 47–89% 26.7–54% 2–7%
Sesquiterpenes 40.9–88.3 65–89 70–89 26.11–37.97 75 40.4% 46% 67% 45.1–52.6% 65–75%
Cannabinoids 6.0–56.3 4.6–28 6.4–27 10.2 0.1% 0.1–7.9% 11–24%
E-ocimene/trans-Ocimene 1–10% 10% 0.4–7.1% 2.23–3.42 mg mL −1 2.1–5.1%
α- and β Myrcene 8–45% 12.5–29.2 11.3 22.9% 27% 25% 11% 6.9–37% 1.8–3.2 mg mL −1 7.1–14.2%
Terpinolene 0.12–22% 3.4–10.7 12% 18.7% 6% 7.8–10%
α-Humulene 3–12% 7.1–8.9 19% 13% 1.3–8.7% 1.55–1.97 mg mL −1 5–11.2% 9%
(E)-Caryophyllene 21.4–26 45.4% 28% 14–32.7% 24.5–29%
α- and β-Selinene 7% 4.3–7.3%
Limonene 3.11–4.99 12% 1.6–11.5%
Citral 0.09–3.5 mg mL −1

Hydro-distillation (HD); Steam-distillation (SD); Headspace solid-phase microextraction (HS-SPME); Solvent extraction (SE); Microwave-assisted extraction (MAE).

2.2.1. α-Pinene

The concentration range of α-pinene (bicyclic monoterpene) in the EO was from non-detected (n.d.) in three accessions of wild hemp to 2.5% in wild hemp Slavka; from n.d. to 8.4% in the registered cultivars; and 0.15 to 2.95% in the new breeding lines (Supplementary Tables S1–S3). However, when grouped together, the concentration of α-pinene in the EO was not statistically different between the three groups of hemp. The concentration of α-pinene in the EO of the USA hemp strain (grown in the vicinity of the registered cultivars in this study and extracted the same way for the same time duration) was 3.6 to 4.5% of the total EO. The monoterpenes pinene and limonene are the determinants of cannabis aroma in the immediate vicinity of the plant [12]. α-Pinene concentration in the EO in this study was comparable to previous reports from studies that used either steam or hydro-distillation [15,16,17,18], but was lower than that in other reports [14,17,19] ( Table 3 ). The EO of wild hemp from Austria contained 20% of this compound in stems, but a much lower concentration in the leaves [15], while the EO of spontaneous (wild) hemp from Hungary contained 1.6, 2.9, and 0.7% of this compound in the leaves, male, and female flowers, respectively. These differences may be due to the environment (growing conditions including latitude and altitude), the plant parts analyzed, and/or the genetics (cultivar).

2.2.2. γ-Caryophyllene (Bicyclic Sesquiterpene)

The concentration range of γ-caryophyllene in the EO was 0.2 to 1.23% in wild, 0.6 to 1.4% in the registered cultivars, and 0.57 to 1.6% in the EO of the breeding lines (Supplementary Tables S1–S3). γ-Caryophyllene concentration in the EO of the USA strain was 0.12%.

2.2.3. β-Caryophyllene (Bicyclic Sesquiterpene)

The concentration of β-caryophyllene was 15 to 30% in the EO of wild hemp, 22 to 55% in the registered cultivars, and 11 to 22% in the EO of the breeding lines (Supplementary Tables S1–S3). Overall, statistically, the highest concentration of β-caryophyllene was found in the EO of cv. Spic and the lowest in the EOs of cvs. Simba and Dioica ( Table 2 ). β-caryophyllene in the USA strain EO was 6.8 to 7.5%. β-Caryophyllene in the EO of this study was similar to those reported in previous studies [10,12,14,16,20,21] ( Table 3 ). The data from this study and previous reports suggest that the concentration of β-caryophyllene could vary significantly depending on the cultivar. (E)-β–caryophyllene is known as the major EO constituent in hemp [21]. This is one of the C. sativa EO caryophyllane- and humulane-type sesquiterpenes that include sesquiterpenes (E)-β-caryophyllene, (Z)-β-caryophyllene, caryophyllene oxide, and the ring-opened isomer α-humulene (α-caryophyllene) [21]. This EO compound has been shown to function in vivo as a non-psychoactive CB2 receptor ligand in foodstuff [21].

2.2.4. α-(E)-Bergamotene

The concentration of α-(E)-bergamotene (bicyclic sesquiterpene) ranged from 0.75 to 1.9% in the wild hemp, 0.36 to 4.4% in the registered cultivars, and 0.5 to 2.8% in the breeding lines (Supplementary Tables S1–S3). Overall, the concentration of α-(E)-bergamotene was statistically higher in cvs Bacalmas and Helena, and the lowest in cv. CS (Carmagnola Selezionata) ( Table 2 ). The concentration of this compound in the USA strain EO was 0.95%. Overall, the concentration of this EO compound in wild hemp from this study was similar to that in the literature reports on wild hemp from Austria and Hungary [15,18].

2.2.5. Caryophyllene Oxide

The concentration of caryophyllene oxide (bicyclic sesquiterpenoid) was 0.24 to 31% of the total EO in wild hemp, 3.9 to 6.8% in registered cultivars, and 8.9 to 17% in the EO of the breeding lines (Supplementary Tables S1–S3). The concentration of caryophyllene oxide was statistically higher in the EOs of cvs. Bacalmas, Helena, and Sequieni, and lowest in the EO of cv. Carmagnola ( Table 2 ). The concentration of this compound in the USA strain EO was 1.3 to 1.4%. Caryophyllene oxide in the EO of wild hemp from Austria was 4.5% [15], while its concentration in the EO of spontaneous hemp from Hungary was 4.3, 4.5, and 2.3% in the leaves, male, and female flowers, respectively [18].

2.2.6. Humulene Epoxide 2

The concentration of humulene epoxide 2 (bicyclic sesquiterpenoid) was 1.3 to 3.2% in wild hemp, 0.4 to 2.3% in registered hemp cultivars, and 2.3 to 5.6% in the EO of the breeding lines (Supplementary Tables S1–S3). Overall, the concentration of humulene epoxide 2 was statistically higher in the cvs. Bacalmas and Sequieni, and the lowest in cv. Spic ( Table 2 ). The concentration of this compound in the USA hemp strain EO was 0.47%. The concentration of this compound in the EO of wild hemp from Austria was

2.2.7. Selina-6-en-4-ol

Selina-6-en-4-ol (bicyclic sesquiterpenoid) was 0.23 to 1.6% in the EO of wild hemp, n.d. to 1.8% in the EO of the registered cultivars, and 1.1 to 2.8% in the EO of the breeding lines (Supplementary Tables S1–S3). Its concentration was statistically highest in the EO of cv. Dioica and lower in the EOs of cvs. Bacalmas and Spic ( Table 2 ).

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2.2.8. Caryophylla-4(12),8(13)-dien-5β-ol

The concentration of caryophylla-4(12),8(13)-dien-5β-ol (bicyclic sesquiterpenoid) was 1.1 to 5.4% in the EO of wild hemp; from n.d. to 2.3% in the EO of the registered hemp cultivars; and 2.3 to 6.6% in the EO of the breeding lines (Supplementary Tables S1–S3). Its concentration was statistically the highest in the EO of the wild hemp and the lowest in the EO of cv. Spic ( Table 2 ). The concentration of this compound in the USA strain EO was 0.85 to 0.88%, while its concentration in the EO of spontaneous hemp from Hungary was 2.3, 1.0, and 0.8% in the leaves, male, and female flowers, respectively [18].

2.2.9. β-Bisabolol

The concentration of β-bisabolol (monocyclic sesquiterpenoid) was 0.9 to 4.5% in the EO of wild hemp; from n.d. to 1.8% in the EO of the registered cultivars; and 2.9 to 4.0% in the EOs of the breeding lines (Supplementary Tables S1–S3; Table 2 ). Overall, β-bisabolol was higher in the EOs of cvs. Bacalmas, Sequieni, and in the wild hemp, and the lowest in the EO of cv. Spic ( Table 2 ). The concentration of this compound in the USA strain EO was 0.33%.

2.2.10. α-Bisabolol

The concentration of α-bisabolol (monocyclic sesquiterpenoid) was 0.4 to 2.9% in the EO of wild hemp, n.d to 6.9% in the registered cultivars, and 0.5 to 3.5% in the EO of the breeding lines (Supplementary Tables S1–S3). α-Bisabolol was statistically the highest in the EO of cv. CS and lower in wild hemp and cvs. Bacalmas, Sequieni, and Spic ( Table 2 ). α-Bisabolol in the USA strain EO was 3.0 to 4.4%. Epi-α-bisabolol was reported in the EO of spontaneous hemp from Hungary [18].

The major EO constituents of the USA hemp strain that was grown in close vicinity had a different chemical profile, with major constituents of myrcene (9.2 to 12%), β-caryophyllene (6.5 to 7.5%), limonene (3.8 to 4.2%), β-(E)-ocimene (5.3 to 5.6%), and α-bisabolol (3.9 to 4.4%). Some previous reports identified a different number of EO constituents in hemp EO (e.g., 55 EO constituents, with myrcene, α-pinene, and β-pinene as the main monoterpenes, and β-caryophyllene as the main sesquiterpene) [14]; 84 EO constituents were identified in wild (spontaneous) hemp by Nagy et al. [18]. The latter authors named these hemp plants spontaneous forms, with the main EO constituents of the leaves, male and female flowers being E-caryophyllene (28.3%), α-humulene (8.9 to 9.3%), β-selinene (4.3 to 7.3%), and α-selinene (2.9 to 5.1%) [18]. Apparently, the spontaneous hemp plants from Hungary had a different EO chemical profile compared with the EO of the wild hemp of this study that was collected in the northeastern part of Serbia. The hydro-distilled leaf EO of wild hemp in Austria contained mainly β-caryophyllene (26.2%), α-humulene (13.1%), β-selinene (5.0%), caryophyllenen oxide (4.5%), and α-selinene (4.4%) [15]. In the same study, stem EO contained α-pinene (20.2%), β-caryophyllene (8.3%), β-pinene (7.0%), and myrcene (6.1%) [15]. Apparently, the EO of wild hemp in Austria had a similar composition to some, but not to other wild hemp EOs, in this study.

Previous research has suggested that metabolic fingerprinting can be used for chemotaxonomic purposes in C. sativa [9]. However, this and earlier reports on wild hemp [15,18] have demonstrated that the EO profile of wild hemp can vary significantly. Therefore, genetic analyses may be needed to ascertain if the spontaneous or wild hemps in Europe originated from some of the old industrial hemp cultivars that have been grown in Europe over the last few centuries or from medical cannabis, or are products of spontaneous crossings between the two groups.

2.3. Cannabinoids in the EO of the Three Hemp Groups: Cannabidiol (CBD) and δ9-Tetrahydrocannabinol (THC) Content

2.3.1. Cannabidiol (CBD, Cannabinoid)

In this study, the concentration of CBD in the EO of wild hemp varied from 6.9 to 52.4% of the total oil, the CBD in the EO of the registered cultivars was from 7.1 to 25.4%, while the CBD concentration in the oil of the breeding lines was from 6.4 to 25.4% (Supplementary Tables S1–S3). However, when we grouped them together, because of the high variation, there were no significant differences between the wild accessions, registered cultivars, and the breeding lines with overall means of 12.4% ( Table 1 ). The concentration of CBD in the EO of the USA strain (which is commercially grown for CBD production) varied between 7.5 and 7.8% of the total EO, which is an interesting result. The CBD concentrations in the EO of wild hemp Slavka, Kovacica, Susara, Perez and Titelski breg were 14.9, 22.6, 11.3, 17.5, and 15.7%, respectively, while the CBD concentrations in the EO of Buro and Saykaj were 52.4 and 33.7%, respectively. Therefore, this and previous studies [15,18] support the notion that wild hemps can be used as a source for the commercial production of CBD-enriched EO. This is the first report on such a high concentration of CBD in hydro-distilled hemp EO.

2.3.2. δ9-Tetrahydrocannabinol (THC, Cannabinoid) (Dronabinol)

Overall, the concentration of THC in the EO was significantly different between the three groups of hemp ( Table 3 ). The THC concentration was significantly higher in the EO of wild hemp accessions, with an average of 2.4% of the total oil, and a range between n.d. (in Daleka Zemlia) and 3.4% (in Kovacica) (Supplementary Tables S1–S3). Interestingly, it was much higher than the THC (0.16%) in the EO of the USA hemp strain, which was actually developed from marijuana type hemp. The THC concentration in the EO of most of the registered cultivars varied from n.d. (e.g., in cvs. Spic and Bacalmas) to over 1.2% (in the EO of cv. Chameleon) ( Table 2 ). The THC concentration in the EO of the breeding lines was generally low, n.d., or below 0.4% with the exception of line SK8, where it reached 3.6% of the total oil. Previous research has shown that hydro-distilled EO from industrial hemp varieties contained cannabidiol (CBD) [10].

2.4. Chemical Groups

Overall, the content of monoterpenes fluctuated from n.d. to 8.0% in the EO of wild hemp, 0.3 to 13% in the EO of the registered cultivars, and 0.2 to 10% of the EO of the breeding lines (Supplementary Tables S1–S3). Monoterpene concentration in the EO of the USA hemp strain was 33 to 34% of the oil.

Sesquiterpenes were the largest group of chemical constituents. The content of sesquiterpenes was 41 to 79% of the EO of wild hemp, 65 to 89% of the EO of the registered cultivars, and 70 to 89% of the EO of the breeding lines (Supplementary Tables S1–S3). Sesquiterpenes constituted 54 to 55% of the EO in the USA hemp strain.

Cannabinoids also comprised the second largest group of chemical constituents in the wild hemp. The concentration of cannabinoids was 7 to 56% of the EO of wild hemp, 4.6 to 28% of the EO of the registered cultivars, and 6.4 to 26% of the EO of breeding lines (Supplementary Tables S1–S3). Cannabinoid concentration in the EO or USA hemp strain were 8.2 to 8.5%, surprisingly low. Most of the strains for thee commercial production of CBD were selected from marijuana (drug-type hemp with 4 to 12% of Δ 9 -THC %), however, they were selected to have

In a study of spontaneous hemp, Nagy et al. [18] reported that the EO was majorly composed of sesquiterpene hydrocarbons (57.1 to 62.8%), followed by cannabinoids (11.0 to 29.3%) and oxygenated sesquiterpenes (7.8 to 14.8%). Overall, the results from this study suggest that wild/spontaneous hemp in Europe is chemotaxonomically related to the industrial hemp varieties (cultivars) grown in Europe and deviate from the chemical profile of the USA hemp strain that was developed from marijuana-type cannabis for the commercial production of CBD. The USA hemp strain used in this study was started with feminized seed, which guarantees the production of female only plants, which may be one of the reasons for its much higher EO content.

The pharmacological power of hemp is based on the content of Δ9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) [26]. Other major cannabinoids include cannabinolic acid (CBNA), cannabigerolic acid (CBGA), cannabichromenic acid (CBCA), and cannabinodiolic acid (CBNDA) [1,27]. Current hemp EO and cannabionoid production systems in the USA have been scaled up from marijuana production. Like with marijuana production, hemp ‘Christmas tree’ production systems rely on feminized seed, because female plants with non-fertilized flowers (flower bracts) accumulate significantly higher concentrations of secondary metabolites such as cannabinoids and terpenes compared with fertilized flowers [28]. Currently, hemp chemical production is based on non-registered hemp ‘strains’ that were originally developed by marijuana breeders, which must meet government regulations for hemp with less than 0.3% Δ9-tetrahydrocannabinolic acid (THCA) in the dried biomass. Due to the rapidly growing market for non-psychoactive cannabinoids, mainly cannabidiol (CBD), most of production has been focused on cannabidiolic acid (CBDA). Newer hemp strains bred for these characteristics are more likely to be compliant, and breeding companies are working on the registration of a number of hemp strains with high CBD content as commercial cultivars. The results from this study demonstrate that wild hemp may be a good source for further selection and breeding of cultivars with high concentration of CBD and low concentrations of THC.

2.5. Antimicrobial Activity of the Hemp Essential Oils (EO)

We used antibiotics as a positive control: cefoxitin for Gram-negative (G – ) bacteria and gentamicin for Gram-positive (G + ) bacteria, and fluconazole for yeast. From G – we used SE, Salmonella enterica subsp. enterica; PA, Pseudomonas aeruginosa; and YE, Yersinia enterocolitica. From G + , we used SA, Staphylococcus subsp. aureus; EF, Enterococcus faecalis; and SP, Streptococcus pneumoniae. From yeast, CA, Candida albicans; CK, Candida krusei; and CT, Candida tropicalis were used. The method has been described previously [29].

The EOs of different hemp cultivars, accessions, and strains had differential antimicrobial activity that can be explained with differences in the EO profile. Some of the EOs had similar (although lower) activity to the antibiotics gentamycin, cetofoxin, and fluconazole.

The antibiotic gentamycin was used as a positive control in the data presented in Figure 1 . The EO of wild hemp Buro was the most potent against Staphylococcus subsp. aureus (SA), followed by the EO of wild hemp Saykaj ( Figure 1 A). Similarly, the EOs of wild hemps Buro and Saykaj showed the highest antimicrobial activity against Enterococcus faecalis (EF). The EOs of wild hemp Buro and cv. Dioica were the most potent against Streptococcus pneumoniae (SP) ( Figure 1 B). The EOs of wild hemp Buro and the registered cv. Dioica had higher antimicrobial activity against Streptococcus pneumoniae compared with that of other EOs ( Figure 1 C).

Bar graph of (A) Staphylococcus subsp. aureus (SA), (B) Enterococcus faecalis (EF), and (C) Streptococcus pneumoniae (SP) (gentamycin) antimicrobial activities (inhibition zone in mm) from seven registered cultivars and seven wild accessions. The means represented by the bars sharing the same letter are not significantly different.

Furthermore, the EOs of wild hemp Buro and the registered cv. Dioica had higher antimicrobial activity against Pseudomonas aeruginosa compared with that of the EOs of the other hemps ( Figure 2 A). Cetofoxin (antibiotic) was used as a positive control for the data in Figure 2 . The EO of wild hemp Susara had the highest activity against the G − Yersinia enterocolitica ( Figure 2 B). The EO of wild hemp Buro had the highest activity against the G − Salmonella enterica subsp. enterica, lower than the EOs of wild hemps Daleka Zemlia and Saykaj, and the lowest in the EO of other hemps ( Figure 2 C).

Bar graph of (A) Pseudomonas aeruginosa (PA), (B) Yersinia enterocolitica (YE), and (C) Salmonella enterica subsp. enterica (SE) (cefoxitin) antimicrobial activities (inhibition zone in mm) from seven registered cultivars and seven wild accessions. The means represented by the bars sharing the same letter are not significantly different.

The antibiotic fluconazole was the positive control for the data presented in Figure 3 . The EO of wild hemp Saykaj had the highest bioactivity against Candida albicans (CA), the bioactivity of EO of wild hemp Perez was not significantly different, while the bioactivity of the EOs of the other hemp EOs was lower than that of Saykaj ( Figure 3 A). The EO of wild hemp Susara and the EO of registered cv. CS had the highest bioactivity against Candida krusei (CK), while the bioactivity of the EO of Sequieni was not different from the above ( Figure 3 B). The EO of wild hemp Paluka had the highest bioactivity against Candida tropicalis (CT) ( Figure 3 C).

Bar graph of (A) Candida albicans (CA), (B) Candida krusei (CK), and (C) Candida tropicalis (CT) (fluconazole) antimicrobial activities (inhibition zone in mm) from seven registered cultivars and seven wild hemp accessions. The means represented by the bars sharing the same letter are not significantly different.

Previously, hemp EO has shown biological activity against several targets of pharmaceutical interest: S. aureus, H. pylori, Candida and Malassezia spp., enzymes, and cancer cell lines [13]. In another study, the EOs (collected from inflorescences after blooming) of cvs. Carmagnola, Fibranova, and Futura showed significant antimicrobial activity against Gram (+) and Gram (−) bacteria and yeast, and the effect depended on the cultivar and seeding date [14].

Overall, the findings in this study are consistent with the ones in a recent report [29]. This study provides new information on the antimicrobial activity of the EOs of the registered and wild hemps. Good antimicrobial activity against Enterococcus, Listeria, and Staphylococcus growth were found, which were compared to the conventional antibiotics that were used [30,31]. Novak et al. [31] found that the EO of five different cultivars of hemp had modest antibacterial activity. The Gram-positive bacterial strains tested demonstrated high sensitivity toward cannabidiol, with slightly lower effects by cannabidiolic acid [32]. Due to significant antimicrobial potency of CBD against MRSA, the synergy with conventional antibiotics was tested. However, CBD was not able to revert the resistance pattern or demonstrate synergy with any of the conventional antibiotics tested [33,34].

3. Materials and Methods

3.1. Plant Material

Certified industrial hemp (Cannabis sativa L.) seeds were obtained from the Institute for Field and Vegetable Crops in Novi Sad, Serbia. Field experiments were set up at the Alternative Crops and Organic Production Department in Backi Petrovac, Serbia (N502138 E395689) using 13 different cultivars of industrial hemp ( Table 4 ), some of them included in the European List of Approved hemp varieties (cultivars) [35]. The cultivars used in this study included CS (Carmagnola Selezionata), Spic, Dioica, Helena, Carmagnola, Squieni, Bacalmas, Simba, Silesia, Chameleon, Fibrol, Futura, and Lovrin ( Table 4 ). The results from the first eight varieties were used in the statistical analyses and in the tables. In addition, new hemp breeding lines (named SK1 to SK8) were seeded adjacent to the above experiment and subjected to the same growth conditions.

Table 4

List of the industrial hemp cultivars and breeding lines used in this study.

Cultivar/Breeding Lines Origin Type Registered Plant Height, cm
CS EU/Italy dioecious Yes 318
Spic Serbia monoecious No 226
Dioica EU/France dioecious Yes 298
Helena Serbia monoecious Yes 302
Carmagnola EU/Italy dioecious Yes 326
Sequieni Romania monoecious Yes 278
Bacalmas Hungary dioecious Landrace 312
Simba Serbia dioecious No 256
Silesia Poland monoecious Yes 268
Chameleon Netherlands dioecious Yes 312
Fibrol Hungary monoecious Yes
Futura France monoecious Yes 295
Lovrin Romania dioecious Yes 289

EU—European Union List.

Although the botanical classification of hemp is controversial [36], wild populations belong to uncultivated narrow leaf Cannabis sativa ssp. spontanea Vavilov [37], which is considered native to Central and Eastern Europe and parts of Asia. Wild hemp samples in this study were collected from the edges of agricultural fields in the same region that have been used to grow other crops, but not hemp. These were agricultural fields where no hemp has been grown for the last 30 years. However, around 35–40 years ago, at some of the collection sites, there were hemp processing factories or hemp seed storage facilities. Generally, wild hemp is phenotypically different from plants of either old or new commercial hemp cultivars; wild hemp also has a shorter stature, with fewer branches than plants from registered cultivars. The locations of the wild hemp samples were named after the names of the nearby villages: Buro, Daleka Zemlia, Paluka, Perez, Saykaj, Susara, Slavka, Kovacica, and Titelski Breg ( Table 5 ). The wild hemp samples were dried under the same conditions, and the EO was extracted via the same method and during the same time as the hydro-distillations of other hemp samples.

Table 5

Wild hemp (C. sativa L.) sample collection locations.

Location GPS
Buro N5070877 E459221
Daleka Zemlia N5019382 E395825
Paluka N5083581 E344444
Perez N4989836 E526695
Saykaj N5011419 E429982
Susara N4975406 E510258
Slavka N4994987 E478790
Kovacica N4954007 E472403
Titelski Breg N5012250 E436909

3.2. Performing Experiments

Hemp was grown as a rainfed crop without irrigation, as is traditional for the region, and the production technology, which is typical production system for commercial production of hemp in Middle and Southern Europe, was applied. Hemp was seeded with a corn planter on 27 March, 2019 with 50 cm spacing between rows and at a seeding rate of 30 kg/ha. The soil type was alluvial chernozem with pH 7.2., previous crop was millet. The soil preparation prior to seeding included deep plowing on 12 November, 2018 and fine seedbed preparation on 25 March, 2019. The experimental design was completely randomized with three replicates, with the size of the experimental plots being 2 m × 5 m. Fertilizer (NPK 16:16:16) was applied as a broadcast treatment at 300 kg/ha before deep plowing. An additional 50 kg/ha of nitrogen was applied in the spring before sowing. Weed control was conducted using burnout with glyphosate prior to seeding. Mechanized weed control was performed twice during the first four weeks of vegetation stage though the use of sweep-type cultivators. Hemp closes the canopy at 5-6 weeks after emergence and hence, suppresses weeds very well after that. The trial of the breeding lines was at the same field, approximately 60 m from the main trial, and was subjected to the same agricultural conditions. Plants in both trials were cultivated the same way including seeding time, seeding depth, fertilization, and weed control.

Fresh biomass samples (around 1 kg fresh, in three reps) were obtained on 27 June, 2019 at the beginning of flowering of the male plants. Hemp tissue samples were generated by cutting the top 1.5 feet (46 cm) from the top of female plants (male plants were not included in the samples). Fresh weight was measured, then the samples were hung in a shady area (tobacco dryer) until constant air-dried weight and then extracted.

3.3. Essential Oil (EO) Extraction

The EO extraction was conducted via hydro-distillation of hemp air-dried material using 4-L hydro-distillation Clevenger-type units as described previously for another plant material [38]. The sample size was up to 50 g dry weight (DW) biomass in 1.2 L water. All distillations were performed in two replicates, which was sufficient for statistical analyses. Beginning of the distillation was noted when the first drop of EO was deposited in the collection part of the Clevenger apparatus. All samples were distilled non-stop for 180 min. At the end of the distillation, the heat source was removed, the EOs (along with some water) were collected in glass vials, and placed in a freezer. After all distillations were complete, the EO was separated from water, measured on an analytical scale, and kept in a freezer until the gas chromatography (GC) analyses could be performed.

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3.4. GC-FID (Gas Chromatography-Flame Ionization Detector) and GC-MS (Gas Chromatography–Mass Spectrometry) Analyses

Hemp volatile compounds were analyzed by GC-FID and GC-MS as described previously [38], with the following modifications: the column temperature was initially set at 40 °C, then increased to 300 °C at a rate of 5 °C/min, which was held for 10 min. The flow rate of the carrier gas (He) was maintained at 1.0 mL/min. The injection volume was 1.0 µL at a split ratio of 30:1. The temperatures of the ionization source, the transfer line, and the injector were 230 °C, 280 °C, and 250 °C, respectively. The MSD was operated in full scan mode. All mass spectra were acquired in electron impact (EI) mode with 70 eV in the m/z range of 40–400. The injector and detector temperature (FID) was set at 220 °C and 280 °C, respectively.

All constituents present in the EO samples were identified by comparing their linear retention indices (LRI) and MS fragmentation patterns with those from the National Institute of Standards and Technology (NIST′08) and Adams mass spectra library. The estimated LRI were determined using a mixture of a homologous series of aliphatic hydrocarbons from C8 to C40 under the same conditions described above.

3.5. Antimicrobial Assay

3.5.1. Bacteria and Yeasts Culture

The microorganisms used for antimicrobial activity testing included Gram-negative bacteria Pseudomonas aeroginosa (CCM 1959), Salmonella enterica subsp. enterica (CCM 3807), Yersinia enterocolitica (CCM 5671); Gram-positive bacteria Enterococcus faecalis (CCM 4224), Staphylococcus aureus subs. aureus (CCM 4223), Streptococcus pneumoniae (CCM 4501); and yeasts Candida albicans (CCM 8186), C. krusei (CCM 8271), and C. tropicalis (CCM 8223) (Czech Collection of Microorganisms, Brno, Czech Republic). The bacteria cultures were incubated in Mueller Hinton broth (MHB, Oxoid, Basingstoke, UK) at 37 °C, but yeast cultures were in Sabouraud broth (SB, Oxoid, Basingstoke, UK) at 25 °C overnight.

3.5.2. Disc Diffusion Method

For the agar disc diffusion method, a 100 µL of 10 6 CFU/mL bacterial suspension after incubation was spread on the Mueller Hinton Agar (MHA, Oxoid, Basingstoke, UK). Filter paper discs (6 mm in diameter) were infused with 15 µl of the EO, tested, and placed on the inoculated MHA. MHA was kept at 4 °C for 2 h and then at 37 °C for 24 h. For yeasts, 100 µL of the yeast suspension was spread on Sabouraud agar (SA, Oxoid, Basingstoke, UK) and agars were cultivated at 25 °C for 24 h. After the incubation period, the diameter of inhibition zones was measured (mm). Growth inhibition was compared with the standard drugs. The standard drugs cefoxitin for G − bacteria, gentamycin for G + bacteria and fluconazole for yeasts were used as positive controls. Tests were performed in three separate experiments, and the means were calculated.

3.6. Statistical Analyses

One-way analysis of variance (ANOVA) with two replications was completed to determine the significance of differences among the mean oil content and constituents obtained from nine cultivars. The constituents were: α-pinene, β-pinene, isocaryophyllene (γ-caryophyllene), β-caryophyllene, α-(E)-bergamotene, (Z)-β-farnesene, caryophyllene oxide, humulene epoxide 2, selina-6-en-4-ol, caryophylla-4(12),8(13)-dien-5α-ol, caryophylla-4(12),8(13)-dien-5β-ol, 14-hydroxy-(Z)-caryophyllene, β-bisabolol, α-bisabolol, CBD, and δ9-tetrahydrocannabinol (Dronabinol). This completely randomized design has two replications.

The differences among the mean antimicrobial activities (SA, EF, SP, PA, YE, SE, CA, CK, and CT) obtained from the seven registered cultivars (Bacalmas, Carmagnola, CS, Dioica, Helena, Sequieni, and Simba) and seven wild accessions (Buro, Daleka zemlia, Paluka, Perez, Saykaj, Susara, and Titelski breg) were also compared using one-way ANOVA with three replications.

For each response variable, the validity of model assumptions was verified by examining the residuals as described in Montgomery [40].

4. Conclusions

The results from this study demonstrated that the essential oil (EO) of wild hemp from Serbia is different in its chemical profile and bioactivity from the EO of registered industrial hemp cultivars, the breeding lines, and the hemp strain grown for CBD production in North America. Wild hemp EOs were also somewhat different from the EO profile of wild and spontaneous hemps collected in Austria and Hungary, as reported in the literature. However, although having been named differently, the wild hemp in this study, the wild hemp collected in Austria, and the spontaneous hemp from Hungary might have common origins; the collection sites were approximately in the same region (with a dimeter of approximately 500 km) although collected in three different countries. These wild/spontaneous hemps have been exposed to significant environmental and agricultural (pesticide) pressure over the last few decades. These populations may all belong to Cannabis sativa var. spontanea Vavilov, (a synonym of Cannabis sativa L.), considered native to Central and Eastern Europe and parts of Asia. However, the taxonomy of hemp is still debatable and there is no consensus among taxonomists as to whether it is a single species with several subspecies or multiple species.

The concentration of the four major constituents in the industrial hemp lines and wild hemp varied as follows: β-caryophyllene 11 to 22% and 15.4 to 29.6%; α-humulene (α-caryophyllene) 4.4 to 7.6% and 5.3 to 11.9%; caryophyllene oxide, 8.6 to 13.7% and 0.2 to 31.2%; humulene epoxide 2, 2.3 to 5.6% and 1.2 to 9.5%, respectively. The major EO constituents in the USA hemp strain that was grown in the vicinity of the field trials had different chemical profiles, with the major constituents myrcene (9.2 to 12%), β-caryophyllene (6.5 to 7.5%), limonene (3.8 to 4.2%), β-(E)-ocimene (5.3 to 5.6%), and α-bisabolol (3.9 to 4.4%).

Overall, the EO of wild hemp has shown greater antimicrobial activity against Staphylococcus susp. aureus, Enterococcus faecalis, and G−Yersinia enterocolitica, Salmonella enterica subsp. enterica, and yeasts Candida albicans and Candida tropicalis compared with the EO of registered cultivars.

This is the first report to show that a significant amount of CBD can be accumulated in the EO of wild and registered cultivars of hemp following hydro-distillation. One of the wild hemp EOs showed a very high concentration of CBD.

The EO of the wild hemp had a significantly higher concentration of THC relative to the one from the registered EU cultivars of industrial hemp breeding lines. In addition, it was interesting to see that wild hemp had a higher concentration of CBD in the EO relative to the EO from a strain grown for commercial production of CBD in the USA Therefore, wild hemp collected in Serbia could be used for the development of varieties (registered cultivars) with specific desirable chemical composition, and may also provide excellent material for the selection and breeding of hemp cultivars with high CBD for the commercial production of CBD and other high-value chemicals.

Acknowledgments

The authors are grateful to the Institute for Field and Vegetable Crops in Novi Sad, Serbia; Oregon State University, USA; the Global Hemp Innovation Center (GHIC) in Corvallis, OR, USA.; University of Novi Sad, Department of Field and Vegetable Crops; The Plant Genetic Research Group, AgrobioInstitute in Sofia, Bulgaria; Slovak University of Agriculture in Nitra, Slovakia; the University of Rzeszow, Poland; and the Agricultural University, Plovdiv, Bulgaria. These institutions provided significant in-kind support such as access to laboratories and fields, and access to other infrastructure and research instrumentation. Authors also thank Michelle Jeliazkova for critically reading and editing the final version of the manuscript.

Supplementary Materials

The following are available online, Table S1. Essential oil constituents of wild hemp accessions, in % of total oil. Table S2. Essential oil constituents of new hemp breeding lines in % of total oil (area under the curve). Table S3. Essential oil constituents of registered hemp cultivars.

Author Contributions

Conceptualization, V.D.Z. and V.S.; Methodology, V.D.Z. and V.S.; Software, T.A., M.K. and I.D.; Validation, V.D.Z., V.S., M.K., and I.D.; Formal analysis, I.D., T.A.; Investigation, V.D.Z., V.S., I.D., M.K., I.B.S., and D.L; Resources, M.K., V.S., I.D., I.B.S., and D.L.; Data curation, V.D.Z.; Writing—original draft preparation, V.D.Z.; Writing—review and editing, V.S., I.D., M.K., T.A., I.B.S., and D.L.; Visualization, T.A.; Supervision, V.D.Z. and V.S; Project administration, V.D.Z. and V.S; Funding acquisition, V.D.Z. and V.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Sample Availability: Samples of the essential oils are not available from the authors.

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Introduction to Industrial Hemp – Basic Production Agronomy

Hemp was once a common agricultural crop throughout the United States. Prior to the passage of the 1937 Marijuana Act, hemp was grown primarily as a fiber crop in the United States. The passage of the 2014 Farm Bill established a renewed interest in producing industrial hemp. Much of the interest has focused on producing hemp for cannabidiol (CBD). However, it should be recognized that there is still potential to produce hemp as a fiber or grain crop.

If contemplating hemp production, it is important to keep in mind that there have been very few U.S.-based agronomic research studies on industrial hemp since the early 20th century. Information from previous research is important and useful but may not always be completely applicable for modern production systems. Industrial hemp is an untested crop in New Jersey. Research is needed to provide data on planting, management, fertility, harvesting, and processing specific to New Jersey. As a result, production information gaps may be encountered in the short term.

It is important to note there are differences in production systems based on the end use of the hemp. This fact sheet will provide information for producers investigating growing and marketing industrial hemp. Industrial hemp grown for grain or fiber production more closely matches existing grain and forage cropping systems than that of hemp grown for CBD. Because we currently have no New Jersey-specific research results or production experience, the information provided will be based on information from surrounding states and the regions that have participated in industrial hemp pilot programs. The reader should be aware the information is being provided as guidance and will be updated with New Jersey-specific information once available.

Market Research

Although industrial hemp production may provide an opportunity for New Jersey, it is crucial that producers carefully examine the market and accessibility of market channels as part of their overall operation. As is the case with any emerging agricultural product, limited data exists to quantify the economic feasibility of industrial hemp production in New Jersey.

It is extremely important to know how to market hemp and where to sell it. One of the most common reasons for not succeeding with an alternative or niche crop is from lack of research as to where to sell the crop and its potential value. It is recommended to first determine if there are processors or buyers in close proximity. Producers growing industrial hemp should also determine if there is any requirement to contract with a buyer in order to sell the crop. Keep in mind that certain contracts specify varieties to be grown and may also require the crop to be grown using specific production practices.

Legality and Permits

The New Jersey Department of Agriculture is tasked with developing regulations for producing industrial hemp in the state. These regulations will include creating requirements for licensing of growers, prescribing hemp testing procedures to ensure compliance with federal law, creating a fee structure for the administration of the program, and certifying germinating seeds and hemp cultivars if necessary. Information about the application process to grow industrial hemp in New Jersey will only be available once regulations are finalized as required under the 2018 Farm Bill. Until New Jersey State Hemp Regulations are finalized and the permit process is in place, it is illegal to produce industrial hemp in the state.

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THC (tetrahydrocannabinol) Requirement

The 2018 Farm Bill recently signed by President Trump removes industrial hemp and its derivatives containing less than 0.3% THC from the Controlled Substances Act, thus legalizing the cultivation of industrial hemp and the hemp derivative CBD oil. Federal and state law requires that the concentration of THC must be less than 0.3% in industrial hemp. By definition, industrial hemp is low (less than 0.3%) in tetrahydrocannabinol (THC), the cannabis plant’s primary psychoactive chemical. When selecting varieties for production, growers should look for varieties certified as having

Background

Cannabis sativa is a summer annual plant. It is a very photoperiod sensitive crop. As a result, flowering is initiated according to day length (photoperiod) not physiological maturity. Most hemp varieties initiate flower development when day length is less than approximately 12 hours. Hemp is mostly dioecious (male and female flowers occur on separate plants). Therefore, there are both male plants and female plants. Although some monoecious varieties exist, most cultivated hemp is currently dioecious. There are breeding programs to increase the availability of monoecious varieties. Different plant parts are harvested from hemp for specific purposes. Depending on the harvestable plant part of interest, (i.e. fiber, grain, or cannabinoids) male plants and pollen might be required for production, or completely unnecessary, or even excluded from production through management.

The grain (seed) of hemp can be used in numerous ways. As a dietary supplement it is very rich in omega-3 and omega-6 fatty acids compared to other potential sources. It is relatively high in oil content. Hemp grain processors produce a wide array of products including toasted hemp seed, hemp seed oil, hemp flour, and even hemp coffee. It is used as bird feed and livestock feed, much the same as soybean hulls are used today.

Uses of hemp fiber have evolved greatly since the late 19th and early 20th centuries where it was used primarily for rope and cloth. Today hemp fiber can be used in many products ranging from construction materials, concrete additives, and many other materials.

Cannabidiol (CBD) oil is extracted from resins produced largely in female flowers. CBD is used as a health supplement with purported health benefits including pain relief, inflammation, and others. Much of the anticipated growth in the industrial hemp industry is expected to be related to production of CBD and related value-added products. CBD from hemp is thought to have numerous applications as a nutraceutical, pharmaceutical, or dietary supplement.

Types of Hemp

Grain

Grain varieties are selected for food and nutritional applications. Grain varieties have high protein, fatty acid, and seed fiber content and usually have lower CBD content. Grain varieties are often shorter in height, reducing the amount of biomass that passes through the combine and reducing wrapping in the combine. Grain hemp seed is thin-walled and can be fragile. The fragile seeds must be handled with care when harvested and transported to market.

Fiber

Fiber varieties of hemp produce long fibers and increased biomass. Fiber hemp varieties are generally taller and favor vegetative growth over seed production. These types of hemp have a wide range of uses, including textiles, building materials, pulp/paper, and more. Ideally, producers of hemp fiber will have access to processing facilities nearby due to the bulk of the product and cost of transport.

Dual Use (Hybrid)

Dual Use varieties of hemp produce both fiber and seed, but not to the yield or quality of single purpose cultivars.

Cannabinoid (CBD)

CBD varieties are currently the most lucrative for agricultural production and marketing. These varieties can present regulatory challenges when attempting to produce the highest yield of CBD, while keeping the THC within allowable levels. High CBD varieties are generally grown utilizing only female plants, as the combination of male and female plants leads to increased seed production and decreased cannabinoid yields.

Variety selection will be key to successful production of all hemp types for many reasons; one of the most important varietal traits is days to maturity (latitudinal adaptation). For grain growers, this is similar to how soybean varieties are selected according to maturity group. There are several considerations when selecting the correct variety for production. For example, varieties bred primarily for grain production could have significantly different maturity dates relative to each other, and therefore would have very different establishment dates for maximum yields and a crop that is harvestable with standard equipment. Producers growing hemp for CBD production or for dual-use production systems will likely require different varieties to maximize yields and other characteristics.

As we begin to have more experience with hemp production, we are learning that varieties are regionally-specific. Farmers looking to enter the industrial hemp market for the first time will need to understand varietal options that are available and carefully determine which hemp variety is most suitable to their production and marketing strategies.

Field Selection

Hemp grows best on a loose, well-aerated loam soil, with high fertility, abundant organic matter (> 2%), and a pH of 6.0–7.5. Well-drained soils are best, as poorly-drained, heavy textured soils, or poorly structured soils often result in stand establishment failures. Reports indicate seedlings and young plants are prone to damping-off, resulting in poor stands. Sandy soils can produce hemp with adequate irrigation and fertilization, but these additional costs will need to be evaluated with respect to production economics.

Fertility

Fertilizer requirements are best determined by a soil test. Penn State has developed fertilizer recommendations for hemp. There is limited experience with hemp production in our region. Recommendations are based on the most current information available. In a soil with optimum levels of phosphorus (P) and potassium (K), nutrient recommendations for a 1,500 pound/acre grain yield potential would be 150 pounds of nitrogen (N), 30 pounds of phosphate (P2O5), and 20 pounds of potash (K2O). Fertility recommendations for hemp grown for fiber at optimum P and K soil test levels would be 150 pounds of N, 20 pounds of P2O5, and 20 pounds of K2O per acre.

Planting

Hemp can be planted in rows, like corn, or with a grain drill, like a small grain. The seed is fragile and can be damaged during planting. No herbicides or other pesticides are currently labeled for hemp in the US. Potential production strategies to reduce weed competition include narrow row planting, high planting density, and planting tall varieties. If planted in wider rows, mechanical cultivation may be required when the crop is young to reduce weed competition.

There have been reports that hemp seed germination is impacted by a lack of soil moisture content at planting. This could lead to uneven stands and result in increased weed pressure. Hemp should be seeded in soils with sufficient moisture to promote rapid germination. Rapid germination and stand establishment are essential to out-compete weeds. There are many reports that seedlings can be weak and struggle to become established if planted in drier than optimal soils. However, once successfully established, plants are very hardy.

Earlier maturing varieties may be preferred for grain production, and in some instances, they may be desired for both grain and fiber harvest. Shorter varieties are generally chosen for grain production as this reduces the amount of plant material running through the combine and reduces plant fibers wrapping in the combine.

Recommendations for planting rates vary depending on end use of the crop. Planting rate recommendations are most always provided as pounds of pure live seed (PLS) per acre. Pure live seed (PLS) is the seed in a container that will likely produce a viable plant when planted. A review of planting rate recommendations for hemp grain production reveals recommendations from as low as 20 to as high as 40 pounds per acre. The majority of recommendations encountered ranged from 25–35 pounds per acre for grain. Planting rates for fiber production range from 35–60 pounds per acre and are generally planted in higher densities to promote vegetative growth and bast fiber development.

Good seed-to-soil contact is required to achieve the best germination rate of industrial hemp seed. A firm, level, and relatively fine seedbed (similar to seeding forages) should be prepared before planting. The range in recommendations for planting depth is for ½ to 1 inch deep, with the majority of planting recommendations falling into the ½ to ¾ inch depth.

Insects and Diseases

There is potential for disease and insect pest problems, but information and recommendations are lacking for New Jersey and other states. Like most plants, hemp is prone to insects and pathogens, causing damage and diseases. As the acreage of industrial hemp increases, the number of insect pests and pathogens will tend to increase as well.

Insect pests that have been reported to cause damage across North America include the European corn borer, armyworm, and grasshoppers. Plant diseases including gray mold (Botrytis cinerea) and white mold (Sclerotinia sclerotiorum) have been reported to infect and impact industrial hemp production. No pesticide materials are currently registered for use on hemp, therefore more research is needed in this area to minimize these potential challenges.

Harvest

For seed production, hemp is harvested when seeds begin to shatter. The plants will still be green. At this time about 70 percent of the seeds will be ripe and the seed moisture is often about 22–30 percent. If harvesting is delayed, then grain losses can increase from shattering, bird damage, and lower grain quality. There is also a greater problem with the fiber in the stalks wrapping in the combine. Avoiding taller varieties can help reduce the amount of material going through the combine. Grain combines can be used for grain harvest and some recommendations have suggested settings similar to those used for grain sorghum. Hemp grain is thin-walled and fragile, requiring care in harvest, storage, and transport. Grain should be dried immediately after harvest to less than 10% moisture.

Harvest for fiber production in many ways is similar to harvesting forages. Forage harvesting and handling equipment have been reported to perform well without major modifications. One common caution that can be found with regard to fiber production is that any machinery with rotation, pickup heads, or rolling bearings can easily lead to hemp wrapping to the point where machinery can become plugged. Hemp cutting can be accomplished with a disc bine, a disc mower, or a straight sickle mower. Specialized equipment for cutting hemp for fiber is available from some overseas manufacturers. There are reports that swathers and haybines do not work well, especially with very tall crops, as there is a tendency for long stems to wrap on the reel.

Once cut for fiber, hemp must undergo retting. Retting is a process involving the use of moisture and microbes to break down the bonds holding the hemp stem together, enabling easier separation of fibers. Field retting is most commonly used where the hemp is left in the field to partially decompose naturally from dew, molds, and bacteria. This process can take 4–6 weeks depending on the weather and must be closely monitored. After retting, the stalks are dried to a moisture content of less than 15% and baled. A baler may be used to bale the hemp stalks, at which point the stalks are ready for storage.

There is no established technique for CBD harvest on a large-scale acreage. Research is needed to understand the best way to harvest CDB for large-acreage production. Currently most CBD production is on small-acre plots or in greenhouses. Harvest for CBD production can be very labor intensive. Harvesting hemp at the proper stage is critical for CBD production. The presence of molds and mildews will lower the value of hemp floral biomass. Current reports show the vast majority of hemp growers producing for the CBD market rely on manual labor to cut the stalks. This is accomplished most often with a machete. Once hemp is harvested, growers should immediately move the floral biomass into the drying facility. Slow drying with high airflow will cure the hemp flowers and produce a higher quality end product.

Additional Resources

  1. University of Kentucky Industrial Hemp Agronomic Research. hemp.ca.uky.edu
  2. The Ontario Ministry of Agriculture, Food and Rural Affairs’ hemp production fact sheet. www.omafra.gov.on.ca/english/crops/facts/00-067.htm
  3. Cornell Hemp Resources. hemp.cals.cornell.edu/hemp-resources/hemp-growing-info
  4. Penn State Industrial Hemp Production. extension.psu.edu/industrial-hemp-production

References

  1. Williams, D.W. and R. Mundel. 2018. An Introduction to Industrial Hemp and Hemp Agronomy. Cooperative Extension Service Publication ID-250. College of Agriculture, Food and Environment, University of Kentucky. www2.ca.uky.edu/agcomm/pubs/ID/ID250/ID250.pdf
  2. Hemp as an Agricultural Commodity, Congressional Research Service, June 2018. fas.org/sgp/crs/misc/RL32725.pdf
  3. Cherney, J.H.; Small, E. Industrial Hemp in North America: Production, Politics and Potential. Agronomy 2016, 6, 58. DOI

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Hemp Production for CBD – Revised

University of Nebraska-Lincoln Extension information typically is based on the interpretation of research information from Nebraska or elsewhere in the Midwest. However, such information is not available for hemp production due to previous restrictions on research in the U.S. This publication relies heavily on research findings from Europe and Canada and learning from growers’ experiences. See more stories in this series at https://cropwatch.unl.edu/tags/hemp.

CBD Demand

Demand for CBD, a non-psychoactive compound derived from hemp, has soared for un-validated treatment of many conditions and illnesses. However, an approximate 75% plummet in prices for the CBD feedstock during 2019 indicates that the supply exceeded demand.

CBD-containing products marketed in the US range from cosmetics to chocolate bars to bottled water to pet treats, all with no regulation. The Food and Drug Administration warned marketers of CBD products against the use of non-validated health claims to sell their products. In June 2019 the FDA approved the first CBD-based drug, called Epidiolex, to treat seizures caused by extreme types of epilepsy. The efficacy of CBD for treatment of chronic pain, neuro-inflammation, anxiety, addiction, and anti-psychotic effects has not been well-validated by clinical research.

Hemp grown for CBD is a high-value crop grown more as a horticultural than as an agronomic crop. It has a high labor demand, putting US production at a disadvantage with production in China and other countries with relatively inexpensive labor.

Hemp CBD varieties have not been well-validated for Nebraska but possibilities may include ‘Abocus’, ‘Autopilot’, ‘Boax’, Cherry Wine, Cherry Blossom, Cobbler, and Sweetgrass for high pharmaceutical-grade CBD yield but having less than 0.3% THC.. High CBD varieties are generally grown only as female plants, as the combination of male and female plants leads to seed production and decreased CBD yield. Breeders continue to improve the processes for creating stable feminized seed. Farmers need to be wary of the source of their feminized seed stock and to check test results for validation of feminized seed.

Farmers need to know state regulations for testing hemp for CBD and THC. The Nebraska Department of Agriculture (NDA) regulations for industrial hemp production have been approved by USDA. Plant sampling by NDA staff to test for THC needs to be within 15 days before the date of harvest with the grower present during sampling. If the THC level is >0.3% by dry weight, the crop will not meet the legal definition of industrial hemp and need to be destroyed. Again, THC is expected to increase with stressful growing conditions.

CBD varieties have short plants with much branching, growing as squat bushes. The suggested spacing at this time is 2-4 feet x 6 feet. Planting practices may change for higher plant densities when seed supply is sufficient to greatly reduce the cost of seed. Given the high cost of seed, seedlings should be produced in a greenhouse for transplanting. If planting more than five acres, machine transplanting is recommended which may allow transplanting 2 acres per day. Plants can also be produced from cuttings with similar vigor and productivity compared to plants from seedlings. Propagation from cuttings may improve plant uniformity and is a means to all-female plants. The potting mix for greenhouse production of seedlings is important but needs to be well-drained with good available water holding capacity and nutrient supply. The mix probably should include sandy loam soil, perlite, and some organic material.

The CBD levels can be much reduced by cross-pollination with wild or non-CBD hemp. The CBD plants must be well-separated by distance or time of pollination from hemp weeds or another hemp crop. Also, a few rows of corn or forage sorghum can planted around the plots to reduce pollen flow.

The highest concentration of CBD is in the bracts of female flowers but CBD oil may be extracted from the whole plant. Harvest may be by topping plants for the harvest of mostly leaves and flowers, by picking the leaves and flowers from the plant, or by taking most of the plant cut at 8-12” above the ground. The whole plant harvest may be by shredding such as with a silage chopper or by keeping the plant intact.

Drying the plant material is a major operation as the water content is high when harvested. To reduce the quantity to be dried and handled for CBD production, the woody stems may be removed for land application, composting or dried separately for fiber production. Artificial drying at up to 100 o F should be continuous flow but the temperature of the plant material should not exceed 75 o F. Suspending plants or branches upside down by wires indoors out of the sun and with good air movement for air drying at up to 75 o F is a common practice if the harvest is not too large.

The ground-up plant material is soaked in grain alcohol or ethanol to extract the CBD oil. After soaking, the mix is pressed to extract the liquid. The alcohol is then evaporated off leaving the CBD oil.

Drying for smoke able buds is an option. Smoking of CBD is reported to be more effective than oral consumption. The buds are preferred but some upper leaves may be included. Well-dried material can be kept and sold in sealable plastic bags or glass jars.

Market information is too weak for prediction or advice but information is improving such as with a USDA ERS Feb 2020 report.

For information on budgeting for hemp grain, fiber and CBD production, see worksheets from Pennsylvania State University and from the University of Kentucky.