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Terpenes in Cannabis sativa – From plant genome to humans

Authors:

Abstract

Cannabis sativa (cannabis) produces a resin that is valued for its psychoactive and medicinal properties. Despite being the foundation of a multi-billion dollar global industry, scientific knowledge and research on cannabis is lagging behind compared to other high-value crops. This is largely due to legal restrictions that have prevented many researchers from studying cannabis, its products, and their effects in humans. Cannabis resin contains hundreds of different terpene and cannabinoid metabolites. Many of these metabolites have not been conclusively identified. Our understanding of the genomic and biosynthetic systems of these metabolites in cannabis, and the factors that affect their variability, is rudimentary. As a consequence, there is concern about lack of consistency with regard to the terpene and cannabinoid composition of different cannabis ‘strains’. Likewise, claims of some of the medicinal properties attributed to cannabis metabolites would benefit from thorough scientific validation.
Accepted Manuscript
Title: Terpenes in Cannabis sativa – From plant genome to
humans
Authors: Judith K. Booth, J¨org Bohlmann
PII: S0168-9452(19)30119-0
DOI: https://doi.org/10.1016/j.plantsci.2019.03.022
Reference: PSL 10109
To appear in: Plant Science
Received date: 26 January 2019
Revised date: 27 March 2019
Accepted date: 27 March 2019
Please cite this article as: Booth JK, Bohlmann J, Terpenes in
Cannabis sativa – From plant genome to humans, Plant Science (2019),
https://doi.org/10.1016/j.plantsci.2019.03.022
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1
Review
Terpenes in Cannabis sativa From plant genome to humans
Judith K. Booth1 and Jörg Bohlmann1
1Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, B.C., Canada,
V6T 1Z4
Author for Correspondence: Dr. Jörg Bohlmann
Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, B.C., Canada
Email: bohlmann@msl.ubc.ca
Highlights: In this Review Article we highlight recent progress in research on terpene metabolites in
cannabis. This includes highlighting issues related to claims of medicinal properties of cannabis terpenes.
We also highlight issues related to so-called ‘strains’ and the need to establish properly genotyped
cannabis varieties for research and use in the industry, as well as other areas that require future
research.
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Table of contents
3 Introduction
4 Chemistry, biosynthesis, and genomics of terpene diversity and variation in cannabis
6 Biosynthesis of cannabinoids
7 Effects attributed to terpenes in cannabis
7 Claims of anticancer effects of cannabis and cannabis terpenes may do more harm than good
8 Perspectives and future directions
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Abstract
Cannabis sativa (cannabis) produces a resin that is valued for its psychoactive and medicinal properties.
Despite being the foundation of a multi-billion dollar global industry, scientific knowledge and research
on cannabis is lagging behind compared to other high-value crops. This is largely due to legal restrictions
that have prevented many researchers from studying cannabis, its products, and their effects in humans.
Cannabis resin contains hundreds of different terpene and cannabinoid metabolites. Many of these
metabolites have not been conclusively identified. Our understanding of the genomic and biosynthetic
systems of these metabolites in cannabis, and the factors that affect their variability, is rudimentary. As
a consequence, there is concern about lack of consistency with regard to the terpene and cannabinoid
composition of different cannabis strains. Likewise, claims of some of the medicinal properties
attributed to cannabis metabolites would benefit from thorough scientific validation.
1. Introduction
Cannabis sativa (cannabis) is thought to have originated from central Asia, and has been domesticated
for over 5,000 years [1]. Cannabis varieties that are low in psychoactive cannabinoids are used for the
production of fiber and oilseed. However, the most valuable cannabis product today is the terpene- and
cannabinoid-rich resin with its various psychoactive and medicinal properties. The resin is produced and
accumulates in glandular trichomes that densely cover the surfaces of female (pistillate) inflorescences
and, to a lesser degree, the foliage of male and female plants (Figure 1). In total, more than 150
different terpenes and approximately 100 different cannabinoids [2] (Figure 2) have been identified in
the resin of different cannabis types (Table 1). The predominant cannabinoids in cannabis grown for
medicinal or recreational use are Δ9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA).
While cannabinoids are the primary psychoactive and medicinal components of cannabis resin, volatile
terpenes (monoterpenes and sesquiterpenes) contribute many of the different fragrance attributes that
influence consumer preferences.
Different cannabis types and their derived consumer products are commonly referred to with strain
names. These names often relate to fragrance attributes conferred, at least in part, by terpenes [3].
Different strains may be distinguished by morphological features or differences in the chemical
composition of the resin. However, due to a history of largely illicit cannabis production, cannabis
strains are often poorly defined genetically. Strains may lack reproducibility with regard to profiles of
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terpenes and cannabinoids [4, 5]. The species encompasses large genetic diversity, with most strains
having high levels of heterozygosity and genetic admixture [5, 6]. Cannabis is wind-pollinated, which also
contributes to variability of cannabis metabolites. As a result, many cannabis strains lack the level of
standardization that producers and consumers are accustomed to with other crop plants, such as
genetically and phenotypically well-defined grapevine varieties. In the absence of proper genetic or
genomic characterization, some attempts have been made at chemotaxonomic classification of cannabis
strains based on terpenes, and cannabis plants have also been described as belonging to different
chemotypes (Table 1). However, the complexity of terpene biosynthetic systems, and the many
different sources of terpene variation, renders these efforts often futile; in general, concepts of
chemotaxonomy have been outdated by genome sciences, and chemotypes cannot reliably substitute
for properly genotyped plants.
With the lifting of some of the legal restrictions on cannabis research in Canada, and in some other
jurisdictions, there is now an opportunity to build stronger scientific knowledge of the genomic,
molecular and biochemical properties that define terpene and cannabinoid profiles in different cannabis
strains. This in turn can support the development of a larger number of well-defined cannabis varieties.
Another aspect that requires new research are the various effects that are attributed to cannabis
terpenes in humans. While some of the effects of the cannabinoids have been scientifically explained,
there is a great deal of uncertainty about the effects of cannabis terpenes in humans beyond fragrance
perception.
2. Chemistry, biosynthesis and genomics of terpene diversity and variation in cannabis
Terpene composition is a phenotypic trait that shows much variation across different cannabis strains
(Table 1). The majority of terpenes found in cannabis are hydrocarbons, which are the direct products of
terpene synthase (TPS) enzymes [7, 8], as opposed to more complex terpenes that require modification
by other enzymes such as cytochrome P450s. Therefore, the chemical diversity of cannabis terpenes
reflects the diversity of TPS enzymes encoded in the cannabis (Cs)TPS gene family.
The monoterpene myrcene as well as the sesquiterpenes β-caryophyllene and α-humulene appear to be
present in most cannabis strains. Other common compounds include the monoterpenes α-pinene,
limonene, and linalool as well as the sesquiterpenes bisabolol and (E)-β-farnesene. It is important to
note that some terpenes, in particular sesquiterpenes, remain difficult to identify due to the lack of
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authentic standards for many of these compounds. As a result, reports of terpene profiles in cannabis
may include unknown compounds, rely on tentative identification, or present incomplete profiles of
selected compounds. Stereochemistry is also not consistently described, or is often ignored, in reports
on cannabis terpenes. These issues makes it difficult to fully assess the diversity of terpenes in cannabis
using the available data and make it problematic to compare the results of different studies.
The terpenes found in the cannabis resin, as well as the isoprenoid moiety of the cannabinoid structure,
are produced through the isoprenoid biosynthetic system, which originates in the mevalonic acid (MEV)
pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in plastids. Monoterpenes
and cannabinoids have a common ten-carbon isoprenoid precursor, geranyl diphosphate (GPP, C10),
while sesquiterpenes are produced from the fifteen-carbon isoprenoid farnesyl diphosphate (FPP, C15).
Using GPP or FPP as substrates, monoterpene synthases (mono-TPS) and sesquiterpene synthases
(sesqui-TPS) produce the different structures of mono- and sesquiterpenes found in the cannabis resin
(Figure 2). A recent analysis of the Purple Kush cannabis genome and transcriptome sequences
identified more than 30 different CsTPS genes [8]. Only nine CsTPS have been functionally characterized
and published to date [8, 9]. As with many other plant TPS [7], eight of the nine characterized CsTPS are
multi-product enzymes that generate several different terpene structures from either GPP or FPP [8].
The multi-product nature of CsTPS can explain why some terpenes, such as α-humulene and β-
caryophyllene, typically co-occur in different cannabis samples. The CsTPS responsible for many of the
different terpenes found in cannabis are still unknown.
Variation of the composition of the CsTPS gene family and variation in CsTPS gene expression is likely to
explain observed variations of terpene profiles across the species. However, the level of variation of the
size, composition and expression of the CsTPS gene family, and factors that influence CsTPS gene
expression, are for the most part unknown. For example, variation of terpene biosynthesis at the
genome, transcriptome, proteome and biochemical levels have been shown in other plants to account
for phenotypic intra-specific variation of terpene profiles [eg. 10, 11]. Terpene profiles may also
substantially change as a result of differential CsTPS gene expression over the course of plant
development or in response to environmental factors. In addition, developmental or tissue specific
expression of CsTPS may affect variation of terpene profiles in cannabis products. None of these factors
of terpene variation, which may contribute to poor reproducibility of terpene composition, have been
systematically studied in cannabis.
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The oxygen functionality of simple terpene alcohols found in cannabis such as linalool or bisabolol may
result from the enzymatic activity of CsTPS as has also been shown for TPS in other plants species [8].
Other terpene derivatives detected in cannabis may arise non-enzymatically due to oxidation or due to
thermal- or UV-induced rearrangements during processing or storage, such as caryophyllene oxide, β-
elemene, or derivatives of myrcene [8, 12]. These non-enzymatic modifications may add a level of
variation that is independent of the plant genome and biochemistry. When terpene analysis is
performed with dried plant material, variable quantitative losses of terpenes, especially the more
volatile monoterpenes [13], may be another cause of terpene variation.
To resolve issues of poor reproducibility of terpene profiles in cannabis, it will be essential to perform
rigorous studies with a diversity of cannabis genotypes grown under controlled environmental
conditions and analyze terpene profiles quantitatively and qualitatively over the course of plant
development. This would need to include organ-, tissue- and cell-type specific terpene analysis, and
would have to include controlled experiments to assess effects of environmental conditions such as
light, irrigation, and nutrients. Such experiments should include not only terpene metabolite analysis,
but also a comprehensive transcriptome profiling of CsTPS gene expression. The results of such a study
would enable much needed proper assignment of reproducible terpene profiles to different strains and
support the standardization of cannabis varieties and derived consumer products.
3. Biosynthesis of cannabinoids
Compared to terpene biosynthesis, cannabinoid biosynthesis has been a priority of the limited research
on metabolite biosynthesis in cannabis to date. Much of the core cannabinoid biosynthetic pathway has
been characterized [14, 15, 16, 17]. The primary branch-point intermediate for cannabinoid biosynthesis
is cannabigerolic acid (CBGA). CBGA is produced by the prenylation of the aromatic olivetolic acid with a
geranyl moiety. An aromatic prenyltransferase (aPT) was recently cloned and shown to be active in a
metabolically engineered yeast to produce CBGA [16], and a related cannabis membrane protein with
prenyltransferase activity was previously reported in the patent literature [18]. Similar enzymes were
shown to prenylate acylphloroglucinols to produce bitter acids in hop, a close relative of cannabis [19,
20]. The precise origin of the fatty acid precursors of olivetolic acid is unknown. Genes of three different
cannabinoid synthases THCA synthase (THCAS), CBDA synthase (CBDAS), and cannabichromenic acid
synthase (CBCAS), have been published[21, 22, 23]. However, the genes and enzymes responsible for
the many minor cannabinoids, including propyl sidechain variants, remain unknown.
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4. Effects attributed to terpenes in cannabis
Arguably, the only effect of cannabis terpenes on humans that is unquestionable are the fragrance
attributes of different mono- and sesquiterpene volatiles and their mixtures. Depending on the variable
composition of cannabis terpene profiles, different strains elicit different fragrance impressions, which
may affect consumer preference [24]. However, other attributes assigned to terpenes in cannabis
products, including medicinal properties, remain for now outside of the space of scientific evidence.
The so-called ‘entourage effect’ is a popular idea. It suggests a pharmacological synergy between
cannabinoids and other components of cannabis resin, in particular terpenes [25, 26]. Putative aspects
of the entourage effect include the treatment of depression, anxiety, addiction, epilepsy, cancer, and
infections. The anecdotal notion of a synergistic effect appears to stem from the perception among
cannabis users that different strains have different physiological effects. There is no doubt that the
large chemical space of thousands of plant terpenes and terpenoids includes many biologically active
molecules. Some terpenoids, such as the anticancer drug taxol, are potent and highly valuable
pharmaceuticals, the effects of which are supported by the full range of pharmacological and clinical
studies. In one of the few examples of the entourage effect being tested, terpenes were found not to
contribute to cannabinoid-mediated analgesia in rats [27]. With the possible exception of the
sesquiterpene β-caryophyllene, no molecular mechanism has been demonstrated to explain a potential
synergy of terpenes with cannabinoids. One potential explanation for the effects attributed to terpenes
is revealed in a recent review [28], pointing out that the placebo effect is partially mediated through the
endocannabinoid system, which may explain some of the perceived effects of cannabis products.
The sesquiterpene β-caryophyllene is prominent in many cannabis strains and products. The molecule
binds to the mammalian CB2 cannabinoid receptor, which may provide a plausible mechanism for
interaction with cannabinoids and a starting point for future research [29]. β-caryophyllene is one of the
least variable terpene components of cannabis (Table 1), which would suggest that it cannot explain
strain-specific effects in humans. The proposed synergistic effects of terpenes in the effects of cannabis
in humans is an area that will require careful research, which will now be possible in those jurisdictions
in which some of the legal restrictions have been lifted.
5. Claims of anticancer effects of cannabis and cannabis terpenes may do more harm than good
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Certain monoterpenes have been shown to block tumor formation or inhibit cell cycle progression in
vivo and in rats [30, 31, 32]. However, the amounts of terpenes required to produce anti-proliferative
effects in rats are excessively high with up to 10% of the animals’ diet [30]. Similarly, cannabinoids may
inhibit tumor formation in animal models of cancer [33]. Laboratory studies such as these may have led
to the suggestion that cannabis extracts, with their combination of cannabinoids and terpenes, have
anti-cancer properties [25, 26]. However, to our knowledge, there is no conclusive evidence to support
claims of anticancer activity of terpenes consumed with cannabis products. While the ethanolic extract
of cannabis flowers has higher antitumor activity than pure THC, this effect was not attributed to any of
the five most abundant terpenes [35].
In general, it is important to remember that cannabis is often consumed by smoking or as a vapor. This
includes cannabis consumption by young adults. Consumer habits such as inhaling combusted or
vaporized cannabis products must be considered a health risk, including the potential risk of causing
cancer or other health issues [35, 36], before promoting unsupported claims of anti-cancer effects of
cannabis.
6. Perspective and future directions
Genomics has been slow to reach cannabis, largely due to legal restrictions on funding agencies and
researchers. A first reference quality cannabis genome was published in 2018 [23], enabling the
genome-wide analysis of genes for metabolic pathways systems in cannabis. More genotyping and
sequencing studies are required to encompass the full diversity of the species. A special emphasis is
needed on Eurasian and African landraces, which have been under-sampled. Critical tools for functional
genomics of metabolic systems, and ultimately crop improvement, such as genetic transformation or
genome editing, are not yet established for cannabis research in the public domain. Beyond the genes
that encode enzymes for the biosynthesis of terpenes and cannabinoids in cannabis, research is needed
to elucidate the factors that control expression of these biosynthetic systems. This would include, for
example, the regulation of cell-type specific gene expression in the context of the development of
glandular trichomes, plant architecture, and onset of female flowering.
As restrictions on research with cannabis relax, cannabis is likely to become a more popular research
organism both for the gain of basic and applied knowledge. Cannabis is a useful system for terpene
research as it produces a large volume of a diverse terpene-rich resin on its trichome-covered surfaces.
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The abundance and size of its glandular trichomes make it a useful system for research in cell
specialization and regulation of terpene and cannabinoid metabolism.
At present, of the hundreds of terpene and cannabinoid metabolites that have been identified in
cannabis, the biosynthesis of less than 30 has been characterized. Future biochemical and functional
work on biosynthetic systems in cannabis would benefit from a focused community effort to produce
and archive a complete and reproducible set of metabolite and genomic data for one or a few genotypes
that will serve as a reference framework. In parallel, a larger number of cannabis types need to be
properly genotyped and phenotypically characterized (e.g. with regard to their metabolites) to
overcome current issues with inconsistencies in what is referred to as strains. The goal would be to
establish reproducible cannabis varieties for use in research and in the industry, comparable to the well-
defined grapevine varieties that are used in viticulture. Moving from strains to varieties will require the
cooperation of cannabis researchers, breeders and growers. To our knowledge, so far, no industry
association has taken a lead to set community standards and practices or define community-accessible
varieties. Researchers and industry in Canada, as the first developed nation to have fully legalized
cannabis, are uniquely positioned to lead this effort.
Acknowledgements:
We thank Samuel Livingston for contributions to Figure 1. We thank Sharon Jancsik, Angela Chiang, and
Dr. Sandra Irmisch for comments on the manuscript. Research on cannabis terpenes in the Bohlmann
lab is supported with funds from Genome British Columbia (SIP001 to JB) and the Natural Sciences and
Engineering Research Council of Canada (NSERC). JKB is the recipient of an NSERC graduate student
fellowship.
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cannabinoid versus a botanical drug preparation in preclinical models of breast cancer.,
Biochem. Pharmacol. 157 (2018) pp. 286293.
54. R. Gallily, Z. Yekhtin, L.O. Hanuš, The Anti-Inflammatory Properties of Terpenoids from Cannabis,
3 (2018).
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Figure Legends
Figure 1. Cannabis inflorescence and stalked glandular trichomes. A) Apical inflorescence from the
strain Purple Kush, eight weeks post onset of flowering. B) Floret cluster from the strain Lemon Skunk,
five weeks post onset of flowering. C) Stalked glandular trichomes on the surface of strain Finola
pistillate flowers. Scanning electron microscopy and image credit for C) thanks to Samuel Livingston,
UBC, Department of Botany.
Figure 2. Schematic of terpene and cannabinoid biosynthesis in cannabis. 5-Carbon isoprenoid building
blocks isopententyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are condensed to form
geranyl diphosphate (GPP) (C10) or farnesyl diphosphate (FPP) (C15). Terpene synthases (TPS) convert
GPP or FPP into terpenes. Aromatic prenyltransferases (aPTs) condense GPP with olivetolic acid to form
cannabigerolic acid (CBGA), which is cyclized by cannabinoid synthases to produce cannabinoids.
Cannabinoids: C1: cannabigerolic acid, C2: cannabichromenic acid, C3: cannabidiolic acid, C4:
tetrahydrocannabinolic acid. Monoterpenes: M1: β-pinene, M2: α-pinene, M3: β-thujone, M4: 3-carene,
M5: terpinolene, M6: limonene, M7: terpineol, M8: 1,8-cineole, M9: α-terpinene, M10: linalool, M11:
myrcene, M12: (Z)-β-ocimene. Sesquiterpenes S1: α-elemol, S2: (E)-β-farnesol, S3: (E)-β-farnesene, S4:
bisabolol, S5: (+)-α-bergamotene, S6: δ-cadinene, S7: γ-eudesmol, S8: valencene, S9: eremophilene, S10:
β-himachalene, S11: α-guaiene, S12: germacrene D, S13: alloaromadendrene, S14: β-caryophyllene.
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Table 1. Publications listing cannabis terpene profiles. Purpose refers to the stated objective of the study.
Origin of plant material indicates what the authors stated as the source of their cannabis or extracts. Number
of terpenes identified includes all named or numbered compounds listed by the authors, including those not
identified using authentic standards. Publications are listed in order of date published, from earliest to most
recent.
# of terpenes
identified
Origin of plant
material
Purpose of analysis
Reference
25
Wild-grown in
Kashmir
Plant Biology
57
50
Forensic samples
Classification
38
66
Grown by
researchers
Plant Biology
13
48
Breeders,
researchers, law
enforcement
Classification
39
16
Grown by
researchers
Plant Biology
40
27
Bedrocan BV
Classification
41
49
Grown by
researchers
outdoors
Metabolite survey
42
28
Grown by
researchers
Metabolite survey
43
20
Coffee shops in the
Netherlands and
Bedrocan BV
Classification
44
12
Bedrocan BV
Industrial
45
53
Forensic samples
Metabolite survey
12
13
Grown outdoors
Industrial
46
27
Indoor cultivator in
California
Industrial
47
28
Submissions from
medical patients
Classification
4
28
Grown by
researchers
Plant Biology
48
17
Bedrocan BV
Industrial
49
50
Bedrocan BV
Classification
50
16
Submitted by
dispensary
Classification
3
14
Licensed producers
in Canada
Classification
51
20
Indoor cultivator in
New Mexico,
assorted growers
Classification
52
21
Dispensary in
California
Medical
53
45
Grown outdoors
Medical
54
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... To validate that we observe patterns expected from previous studies, we first looked for correlations between specific terpene pairs. We chose pairs that have been previously observed to display robust positive correlations, likely stemming from constraints on their biochemical synthesis [63][64][65]. Strong positive correlations were seen between α-and β-pinene (Fig 3B; r To systematically understand relationships between all terpene pairs, we performed hierarchical clustering on all pairwise correlations among terpenes (Fig 4A; see Methods). ...
... We also plotted this data in the form of a network diagram configured to display connections between terpenes with the strongest correlations ( Fig 4B). This diagram provides a more compact picture of terpene co-occurrence and likely reflects the underlying biosynthesis pathways that give rise to these correlations [63][64][65]. ...
... constraints of terpene biosynthesis [63,64,69], as has been observed elsewhere [50,65]. The pooled dataset also displays features seen in sample populations from US states not represented here [35]. ...
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