Journal:Metabolomic analysis of cannabinoid and essential oil profiles in different hemp (Cannabis sativa L.) phenotypes

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Full article title Metabolomic analysis of cannabinoid and essential oil profiles in different hemp (Cannabis sativa L.) phenotypes
Journal Plants
Author(s) Eržen, Marjeta; Košir, Iztok J.; Ocvirk, Miha; Kreft, Samo; Čerenak, Andreja
Author affiliation(s) Slovenian Institute of Hop Research and Brewing, University of Ljubljana
Primary contact Email: andreja dot cerenak at ihps dot si
Year published 2021
Volume and issue 10(5)
Article # 966
DOI 10.3390/plants10050966
ISSN 2223-7747
Distribution license Creative Commons Attribution 4.0 International
Website https://www.mdpi.com/2223-7747/10/5/966/htm
Download https://www.mdpi.com/2223-7747/10/5/966/pdf (PDF)
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Abstract

Hemp (Cannabis sativa L.) cannabinoids and terpenoids have therapeutic effects on human and animal health. Cannabis plants can often have a relatively high heterogeneity, which leads to different phenotypes that have different chemical profiles despite being from the same variety. Little information exists about cannabinoid and terpenoid profiles in different hemp phenotypes within the same variety. For this study, 11 phenotypes from three different varieties—Carmagnola Selected (CS), Tiborszallasi (TS), and Finola Selection (FS)—were analyzed. The components of essential oil (29) were analyzed using gas chromatography with flame ionization detection (GC-FID), and 10 different cannabinoids of each phenotype were determined using high-performance liquid chromatography (HPLC).

Principal component analysis (PCA) and analysis of variance (ANOVA) showed that according to the components of essential oil, FS and TS plants were more uniform than CS plants, where there were great differences between CI and CII phenotypes. The content of cannabidiolic acid (CBDA) was the highest in all four FS phenotypes. By comparing cannabinoid profiles, FS was clearly separated from TS and CS, while these two varieties were not clearly distinguishable. Phenotypes TV and CI had the highest total content of tetrahydrocannabinol (Δ9-THC), while all phenotypes of FS had the highest total content of cannabidiol (CBD). The highest total content of cannabigerol (CBG) was determined in phenotype CI. Obtained results are useful for the development of new supplementary ingredients, for different pharmacy treatments, and for further breeding purposes.

Keywords: Cannabis sativa L., Cannabaceae, cannabinoids, essential oils, terpenes, GC-FID, HPLC

Introduction

Hemp (Cannabis sativa L.) originated from central Asia and has been used for human and animal food, as a source of fiber for ropes, and in medicine.[1][2] It contains more than 500 phytochemicals with many therapeutic purposes and has been used to treat epilepsy, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, pain and nausea in cancer patients, diabetes, and eating disorders.[3]

The most well-known phytochemicals are secondary metabolites, such as cannabinoids and terpenoids.[4] More than 150 cannabinoids have already been identified in hemp.[5] The most active and studied compounds are Δ9 tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), and their carboxylated forms.[3] Terpenoids in essential oil are divided into monoterpenes and sesquiterpenes, which are responsible for hemp fragrance and flavor and also contribute to therapeutic effects. There are generally fewer sesquiterpenes than monoterpenes detected in hemp flowers. The highest content of cannabinoids and terpenoids is found in the glandular trichomes on bracts.[6]

Precursors for cannabinoids have two biosynthetic pathways. The polyketide pathway leads to olivetolic acid (OLA), and the plastidial 2-C-methyl-D-erytritol 4-phosphate (MEP) pathway leads to geranyl diphosphate (GPP). Precursors OLA and GPP form cannabigerolic acid (CBG-A), which is a precursor for different cannabinoids, as well as tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and cannabichromenic acid (CBCA).[7] Terpenoids are composed of isoprene units. Similar to cannabinoids, terpenoids also have different biosynthetic pathways. Sesquiterpenes and triterpenes are formed from the cytosolic mevalonic acid (MVA) pathway, while monoterpenes, diterpenes, and tetraterpenes are formed via the plastid-localized (MEP) pathway. Subsequently, precursors of sesquiterpene farnesyl diphosphate (FPP) and monoterpene geranyl diphosphate are formed.[7]

Terpenoids and cannabinoids may have a synergistic effect on human and animal health.[8] An example of the positive effects of the combined use of cannabinoids and terpenoids is acne therapy, in which CBD, limonene, linalool, and pinene are involved. Cannabinoids and terpenoids such as CBG and pinene also have a combined effect on MRSA (methicillin-resistant Staphylococcus aureus).[9][10] However, the issue of synergy remains controversial and needs further investigation.

According to the chemical composition, there are five major hemp chemotypes. Small and Beckstead[11] determined three chemotypes: chemotype I, with a THC content higher than 0.3% and CBD content lower than 0.5%; chemotype II (intermediate type), with a THC and CBD ratio that is roughly equal; and chemotype III, with a higher CBD content than 0.5% and THC content lower than 0.3% of the flower dry matter. Later, Fournier et al.[12] determined two other chemotypes: chemotype IV, with a prevalence of CBG higher than 0.3% of the flower dry matter, and chemotype V, with an undetectable content of cannabinoids.

Numerous scientists have studied species and subspecies of Cannabis.[13] In general, it is known that hemp and marijuana differ based on THC content. Hemp is supposed to have THC content below 0.2–1%, which depends on the legislation of different countries, while marijuana could reach THC content up to 20 to 30% in dry inflorescences.[14] In 2015, Sawler et al.[15] determined that hemp and marijuana significantly differ at the genome level, that different marijuana types are often not genetically close, and that THC is not related to the genetic distinction between hemp and marijuana. Hemp has been used for food and fibers, while marijuana was mostly used in traditional medicine.[16] However, marijuana was prohibited and criminalized all around the world due to the psychoactive nature of THC. In 1988, the United Nations Convention against Illicit Traffic in Narcotic Drugs and Psychotropic Substances prohibited the use, production, and cultivation of Cannabis plants, which was recognized as a narcotic drug with psychotropic compounds, still posing a major problem in the legalization of cannabis with higher THC content.[17] Due to many different research studies based on the positive effects of cannabis on numerous diseases, more and more countries are slowly revising their legislation in favor of growing Cannabis plants for medical and scientific purposes in restricted area.

Cannabis varieties (strains) are often not fully inbred; therefore, they have a relatively high level of heterogeneity and instability, compared to other crops. Different phenotypes can be found within one variety. One of the major breeding challenges is that Cannabis plants are mostly dioecious, and they cannot pollinate themselves; hence, outcrossing commonly occurs.[18] This paper aims to clarify the distinction between three different hemp varieties and their 11 phenotypes according to an analysis of cannabinoids and terpenoids. Three varieties—Carmagnola Selected (CS), Tiborszallasi (TS), and Finola Selection (FS)—were chosen because of the expressed interest of our partners from the industry. All of them are registered on the E.U. variety list and are grown as out-growing varieties. Together, we found out that different phenotypes could be detected within them, and we supposed that they could express different chemical profiles as well, with a further different application in pharmacy. That said, the ultimate objective was to establish a connection between the chemical composition and morphological characteristics of hemp plants and to identify phenotypes with an interesting ratio between cannabinoids for further pharmaceutical applications.

Results and discussion

Differentiation of phenotypes according to visual traits

In total, 11 different phenotypes were selected according to visual traits observed within varieties that were compared with each other, and reference types[19][20][21] (certified types from breeders) were added for each variety (Table 1). However, this is a first preliminary comparison between different phenotypes within mentioned varieties. Photos of all 11 phenotypes are presented in Figures S3–S14 (see Supplementary materials). According to the Slovenian Ministry of Agriculture, Forestry, and Food in 2020, there was 6.67 ha of Carmagnola, 0.86 ha of Tiborszallasi, and 19.77 ha of Finola.

Table 1. The 11 different phenotypes (CI, CII, TI, TII, TIII, TIV, TV, FI, FII, FIII, FIV) that are defined by six different visual traits: size, color, leaf size, inflorescences, anthocyanin coloration of leaf petiole, and branching. For each variety, reference types are added. Note that size is the comparison between the height of plants within phenotypes in each variety. Plant color was described as light, medium, or dark green. Branching is scales, with * meaning little branched, up to **** meaning highly branched. - = No data available.
Phenotype Size Color Leaf size Inflorescences Anthocyanin coloration of leaf petiole Branching Remarks
Carmagnola Selected
CI Tall Light Large Small No ***
CII Tall Dark Small Small Yes ****
Reference type Tall Dark Medium - Medium -
Tiborszallasi
TI Tall Medium Medium Small No ****
TII Medium Dark Medium Medium Yes ***
TIII Small Dark Small Medium Yes **
TIV Medium Dark Large Big No *** Compact flowers
TV Small Medium Small Medium Yes * Strong anthocyanin coloration of the whole plant
Reference type Tall Dark - - - ****
Finola Selection
FI Tall Dark Medium Big No ****
FII Medium Medium Medium Big No ***
FIII Medium Light Medium Medium Yes ****
FIV Medium Dark Medium Big Yes ****
Reference type Small Medium Small-medium - No ***

Five plants with the same observed traits within one phenotype were labeled and separately sampled. Hemp is an open-pollinated plant and, therefore, also more prone to non-uniformity. Pollen can disperse a few kilometers in relation to the wind direction[22], which could be one of the reasons for higher heterogeneity in Cannabis plant varieties. Additionally, newly bred populations and marijuana populations are more uniform and can be easily grouped by their desirable traits, such as high THC/CBD level, high limonene, or other terpenoid levels.[15]

Chemical analysis of the essential oil of hemp (Cannabis sativa L.)

Briefly, the 11 phenotypes from CS, TS, and FS contained 0.09–3.38 mL of essential oil per 100 g of air-dried flower (1.34 mL/100 g, on average). FS achieved the highest average content of essential oil (2.81 mL/100 g air-dried flower), compared to CS (0.38 mL/100 g air-dried flower) and TS (0.54 mL/100 g air-dried flower). The greatest relative difference between phenotypes within varieties was between CS phenotypes (p < 0.0001) in relation to essential oil. CI achieved 0.23 mL/100 g of air-dried flower, while CII achieved 0.53 mL/100 g air dry flower of essential oil. When comparing monoecious and dioecious varieties, we noted that Bertoli et al.[6] discovered a higher content of essential oil in dioecious plants. Nissen et al.[23] also reported about low content of essential oil in the CS variety. Significant differences have been recorded between different hemp varieties according to essential oil and cannabinoid content in previous studies as well.[24][25]

As expected, the most abundant terpenoids were myrcene, β-caryophyllene, α-pinene, and α-humulene in all three varieties.[3][6][23] The proportion of the 10 main components of essential oil were compared with analysis of variance (ANOVA) and presented in superscripts in Table 2 (as well as Table S1, Supplementary materials), in which all 29 analyzed components are presented. Notable points about these analyzed components include:

  • According to ANOVA, phenotypes CI and CII showed significant differences based on α-pinene, β-pinene, 3-Carene, terpinolene, β-caryophyllene, α-humulene, caryophyllene oxide, β-eudesmol, and phytol. The CII phenotype had significantly higher contents of all main monoterpenes (α-pinene, β-pinene, 3-carene, and terpinolene) than phenotype CI, while the contents of all main sesquiterpenes (β-caryophyllene, α-humulene, caryophyllene oxide, β-eudesmol) and phytol were significantly higher in the CI phenotype.
  • When comparing phenotypes of TS, differences were shown in caryophyllene oxide.
  • Terpinolene and α-terpinene were found at different concentrations in FS phenotypes. As is evident from Table 2, the phenotypes of FS had very high proportions of limonene (4.1–5.2%), in comparison to CS and TS phenotypes, which is of great value since FS phenotypes also reached the highest content of essential oil. The lowest proportion of limonene was in the TI phenotype (0.6%).
  • Phenotypes of FS had the highest proportions of α-terpineol, β-eudesmol, and α-bisabolol and very low proportions of caryophyllene, oxide, and undetachable proportion of phytol, cis-nerolidol, etc.
  • The highest proportion of α-pinene was in all TS phenotypes, especially in TIV (11.9%), and in phenotype CII (11.9%). A distinctly lower proportion was identified in FIV (0.7%).
  • The highest proportion of myrcene was also observed in the five TS phenotypes; TI had the highest proportion among the TS phenotypes (29.9%).
  • The highest proportion of, β-pinene, 3-carene, γ-terpinene, terpinolene, borneol, and menthol occurred in CII among all phenotypes, while the highest proportion of α-cedrene, β-caryophyllene, α-humulene, cis-nerolidol, geranyl isobutyrate, caryophyllene oxide, β-eudesmol, and phytol occurred in CI (almost all components of sesquiterpenes). However, the CS variety also had the lowest amount of essential oil compared to other investigated varieties.
  • FIII had the highest proportion of α-terpinene (0.6%) and fenchone (0.2%), and TI had the highest proportion of p-cymene (0.1%).
  • The highest proportion of borneol (0.2%) and geranyl acetate (0.3%) was in FIV. FII contained the most α-terpineol (1.0%).
  • The terpenoids p-cymene, camphor, isoborneol, β-citronellol, and neryl acetate had contents lower than 0.1% or were undetectable.


Table 2. Average essential oil (EO) content (mL/100 g air-dried flower) of the main components in the inflorescence and average composition (%) of essential oil. Groups (a, b, c, and d) were determined by analysis of variance (ANOVA) from different phenotypes of Carmagnola Selected, Tiborszallasi, and Finola Selection. The same letters present similarities between phenotypes, while different letters represent differences between phenotypes according to components of essential oil. The mean ± standard deviation (SD) is reported.
Phenotype CI SD CII SD TI SD TII SD TIII SD TIV SD TV SD FI SD TII SD TIII SD TIV SD
Average EO content 0.23 0.10 0.53 0.16 0.58 0.23 0.39 0.04 0.52 0.15 0.64 0.22 0.56 0.17 2.75 0.33 3.11 0.23 2.82 0.41 2.59 0.38
α- Pinene 2.5
a,b 		1.7 11.6
c,d 		4.6 10.3
b,c,d 		3.3 7.4
a,b,c,d 		3.7 8.3
a,b,c,d 		5.2 11.9
d 		6.3 11.5
c,d 		12.2 3.7
a,b,c 		4.1 6.0
a,b,c,d 		3.4 3.5
a,b 		3.4 0.7
a 		0.4

References

  1. ↑ Hillig, K.W. (2005). "Genetic evidence for speciation in Cannabis (Cannabaceae)". Genetic Resources and Crop Evolution 52: 161–80. doi:10.1007/s10722-003-4452-y. 
  2. ↑ Crini, G.; Lichtfouse, E.; Chanet, G. et al. (2020). "Applications of hemp in textiles, paper industry, insulation and building materials, horticulture, animal nutrition, food and beverages, nutraceuticals, cosmetics and hygiene, medicine, agrochemistry, energy production and environment: A review". Environmental Chemistry Letters 18: 1451–76. doi:10.1007/s10311-020-01029-2. 
  3. ↑ 3.0 3.1 3.2 Namdar, D.; Mazuz, M.; Ion, A. et al. (2018). "Variation in the compositions of cannabinoid and terpenoids in Cannabis sativa derived from inflorescence position along the stem and extraction methods". Industrial Crops and Products 113: 376–82. doi:10.1016/j.indcrop.2018.01.060. 
  4. ↑ Turner, J.C.; Hemphill, J.K.; Mahlberg, P.G. (1978). "Quantitative Determination of Cannabinoids in Individual Glandular Trichomes of Cannabis sativa L. (Cannabaceae)". American Journal of Botany 65 (10): 1103–6. doi:10.1002/j.1537-2197.1978.tb06177.x. 
  5. ↑ Hanuš, L.O.; Meyer, S.M.; Muñoz, E. et al. (2016). "Phytocannabinoids: A unified critical inventory". Natural Product Reports 33: 1357–92. doi:10.1039/C6NP00074F. 
  6. ↑ 6.0 6.1 6.2 Bertoli, A.; Tozzi, S.; Pistelli, L. et al. (2010). "Fibre hemp inflorescences: From crop-residues to essential oil production". Industrial Crops and Products 32 (3): 329–37. doi:10.1016/j.indcrop.2010.05.012. 
  7. ↑ 7.0 7.1 Andre, C.M.; Hausman, J.-F.; Guerriero, G. (2016). "Cannabis sativa: The plant of the thousand and one molecules". Frontiers in Plant Medicine 7: 19. doi:10.3389/fpls.2016.00019. PMC PMC4740396. PMID 26870049. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=PMC4740396. 
  8. ↑ Russo, E.B. (2011). "Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects". British Journal of Pharmacology 163 (7): 1344–64. doi:10.1111/j.1476-5381.2011.01238.x. 
  9. ↑ da Silva, A.C.R.; Lopes, P.M.; de Azevedo, M.M.B. et al. (2012). "Biological Activities of a-Pinene and β-Pinene Enantiomers". Molecules 17 (6): 6305-6316. doi:10.3390/molecules17066305. 
  10. ↑ Appendino, G.; Gibbons, S.; Giana, A. et al. (2008). "Antibacterial Cannabinoids from Cannabis sativa: A Structure−Activity Study". Journal of Natural Products 71 (8): 1427–30. doi:10.1021/np8002673. 
  11. ↑ Small, E.; Beckstead, H.D. (1973). "Common cannabinoid phenotypes in 350 stocks of Cannabis". Lloydia 36 (2): 144–65. PMID 4744553. 
  12. ↑ Fournier, G.; Richez-Dumanois, C.; Duvezin, J. et al. (1987). "Identification of a new chemotype in Cannabis sativa: cannabigerol-dominant plants, biogenetic and agronomic prospects". Planta Medica 53 (3): 277–80. doi:10.1055/s-2006-962705. PMID 3628560. 
  13. ↑ Pollio, A. (2016). "The Name of Cannabis: A Short Guide for Nonbotanists". Cannabis and Cannabinoid Research 1 (1): 234-238. doi:10.1089/can.2016.0027. PMC PMC5531363. PMID 28861494. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=PMC5531363. 
  14. ↑ Schilling, S.; Melzer, R.; McCabe, P.F. (2020). "Cannabis sativa". Current Biology 30 (1): R8–R9. doi:10.1016/j.cub.2019.10.039. PMID 31910378. 
  15. ↑ 15.0 15.1 Sawler, J.; Stout, J.M.; Gardner, K.M. et al. (2015). "The Genetic Structure of Marijuana and Hemp". PLoS One 10 (8): e0133292. doi:10.1371/journal.pone.0133292. PMC PMC4550350. PMID 26308334. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=PMC4550350. 
  16. ↑ Janatová, A.; Fraňková, A.; Tlustoš, P. et al. (2018). "Yield and cannabinoids contents in different cannabis (Cannabis sativa L.) genotypes for medical use". Industrial Crops and Products 112: 363–67. doi:10.1016/j.indcrop.2017.12.006. 
  17. ↑ Aguilar, S.; Gutiérrez, V.; Sánchez, L. et al. (23 April 2018). "Medicinal cannabis policies and practices around the world". International Drug Policy Consortium. https://idpc.net/publications/2018/04/medicinal-cannabis-policies-and-practices-around-the-world. Retrieved 19 April 2021. 
  18. ↑ Ranalli, P. (2004). "Current status and future scenarios of hemp breeding". Euphytica 140: 121–31. doi:10.1007/s10681-004-4760-0. 
  19. ↑ "Cerealicoltura e Colture Industriali". Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria. https://www.crea.gov.it/web/cerealicoltura-e-colture-industriali. Retrieved 20 April 2021. 
  20. ↑ "National Food Chain Safety Office (NEBIH)". ERA-LEARN. European Union. https://www.era-learn.eu/network-information/organisations/national-food-chain-safety-office-1. Retrieved 20 April 2021. 
  21. ↑ "Finnish Food Authority". Finnish Food Authority. https://www.ruokavirasto.fi/en/. Retrieved 20 April 2021. 
  22. ↑ Small, E.; Antle, T. (2003). "A Preliminary Study of Pollen Dispersal in Cannabis sativa in Relation to Wind Direction". Journal of Industrial Hemp 8 (2): 37-50. doi:10.1300/J237v08n02_03. 
  23. ↑ 23.0 23.1 Nissen, L.; Zatta, A.; Stefanini, I. et al. (2010). "Characterization and antimicrobial activity of essential oils of industrial hemp varieties (Cannabis sativa L.)". Fitoterapia 81 (5): 413–19. doi:10.1016/j.fitote.2009.11.010. 
  24. ↑ Novak, J.; Zitteril-Eglseer, K.; Deans, S.G. et al. (2001). "Essential oils of different cultivars of Cannabis sativa L. and their antimicrobial activity". Flavour and Fragrance Journal 16 (4): 259-262. doi:10.1002/ffj.993. 
  25. ↑ Novak, J.; Franz, C. (2003). "Composition of the Essential Oils and Extracts of Two Populations of Cannabis sativa L. ssp. spontanea from Austria". Journal of Essential Oil Research 15 (3): 158–60. doi:10.1080/10412905.2003.9712100. 

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.

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