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Terpenoids In Cannabis: Facts & Research

conradino23

Grow Journal of the Year: 2017 - Grow Journal of the Month: Sept 2017
Arno Hazekamp, Justin T. Fischedick, ... Renee L. Ruhaak, Chemistry of Cannabis, Comprehensive Natural Products II, Volume 3: Development & Modification of Bioactivity, 2010.

3.24.5.1 Terpenoids

Terpenoids make up a large percentage of the essential oil of C. sativa L. To date, more than 120 terpenoids have been found in Cannabis, including 58 monoterpenoids, 38 sesquiterpenoids, 1 diterpenoid, 2 triterpenoids, and 4 other terpenoids. Two excellent reviews have been published summarizing these compounds and how they were identified. Terpenoids display a wide range of biological activities and hence may play a role in some of the pharmacological effects of various Cannabis preparations.

Although cannabinoids are odorless, the volatile monoterpenoids and sesquiterpenoids are the compounds that give Cannabis its distinct smell. The sesquiterpenoid β-caryophyllene-epoxide (Figure 11), for example, is the main compound that search dogs are trained to recognize. Only one unusual terpenoid can be found in Cannabis: the monoterpenoid m-mentha-1,8(9)-dien-5-ol.

All others can be found ubiquitously in nature. For this reason the terpenoids of Cannabis did not receive much scientific interest, until it was found that the terpenoid profile of Cannabis products can help in determining the origin of Cannabis in custom seizures.


Figure 11. Two special terpenoids found in Cannabis.

3.24.5.1.1 Biosynthesis and composition of Cannabis essential oils

The terpenoids in Cannabis are frequently extracted from herbal material by steam distillation or vaporization. Typical yields of the terpene essential oils from fresh plant material range from 0.05 to 0.29% (v/w). The essential oil of Cannabis is mainly composed of monoterpenoids and sesquiterpenoids with monoterpenoids dominating. Since self-administered Cannabis plant material is usually consumed as an (air-)dried product, the change in terpenoid content and concentration in relation to the fresh plant material is important to note.

It has been reported that the essential oil content of a Cannabis plant changed from 0.29% essential oil (v/w) in the fresh product to 0.8% (v/w) after 1 week of drying, as a result of water loss. Following storage at room temperature for up to 3 months in a paper bag, the total essential oil was then reduced to 0.57% (v/w). Furthermore, it was observed in the essential oil that the relative percentage of monoterpenoids decreased whereas the relative percentage of sesquiterpenoids increased.

Environmental conditions such as plant density, harvest time, pollination, and climate conditions may all play a role in composition and yield of Cannabis essential oils. The cultivar of the plant also plays a role in the terpenoid composition.

A study that analyzed the terpenoids of 157 different strains of Cannabis from various known origins found statistically significant differences in terpenoid composition. Even though these differences were not always indicative of what chemotaxonomic type the Cannabis strains belonged to, it may play a role in differential medicinal effects.

3.24.5.1.2 Biological activities of terpenoids

The observation that whole Cannabis extracts may produce effects greater than expected from THC content alone has led researchers to postulate as to what other components in Cannabis could be responsible for enhancing or modulating the effects of THC. The terpenoids present in Cannabis display a wide range of biological activities that may be involved in regulating the effects of THC as well as producing their own unique pharmacological effects.

An overview of some of the known biological activities of terpenoids that have been identified in Cannabis is shown in Table 3.

Table 3. Summary of terpenoid biological activity
TerpenoidKnown properties
β-MyrceneAnalgesic, anti-inflammatory, antibiotic, antimutagenic
β-CaryophylleneAnti-inflammatory, cytoprotective, antimalarial, CB2 agonist
d-LimoneneImmune potentiator, antidepressant, antimutagenic
LinaloolSedative, antidepressant, anxiolytic, immune potentiator
PulegoneAcetylcholinesterase (AChE) inhibitor, sedative, antipyretic
1,8-CineolAChE inhibitor, stimulant, antibiotic, antiviral, anti-inflammatory, antinociceptive
α-PineneAnti-inflammatory, bronchodilator, stimulant, antibiotic, antineoplastic, AChE inhibitor
α-TerpineolSedative, antibiotic, AChE inhibitor, antioxidant, antimalarial
Terpineol-4-olAChE inhibitor, antibiotic
p-CymeneAntibiotic, anticandidal, AChE inhibitor

Some undesired side effects of THC may be decreased or modulated in the presence of terpenoid compounds. For example, THC is known to cause acetylcholine deficits in the hippocampus, which may lead to short-term memory loss. This effect can be alleviated in rats by administering tacrine, an alkaloid that inhibits acetylcholine esterase, the primary enzyme involved in the breakdown of acetylcholine in cholinergic receptors.

Indeed, tacrine has blocked THC-induced memory loss behavior in rats. Interestingly, many of the terpenoids present in Cannabis display similar acetylcholine esterase inhibition, including pulegone, limonene, limonene oxide, α-terpinene, γ-terpinene, terpinen-4-ol, carvacrol, l- and d-carvone, 1,8-cineole, p-cymene, fenchone, and pulegone-1,2-epoxide.224 For this reason, terpenoids are investigated for the treatment of Alzheimer’s disease.

THC has been known to cause negative psychological reactions such as anxiety and depersonalization. Some of these effects may again be alleviated by the terpenoids present in Cannabis, because of their sedative and antidepressive effects. Cannabis terpenoids such as linalool, citronellol, and α-terpinene were shown to have significant sedative effects, as indicated by decreased activity in a mice motility model after the inhalation of these compounds.

Limonene is a common component of Cannabis essential oil, and it was shown to have a strong antidepressant effect by inhibiting the secretion of hypothalamic–pituitary–adrenal (HPA) stress hormones and normalization of CD4:CD8 ratios. Limonene is also under investigation as an antimutagenic compound because of its multiple anticarcinogensis mechanisms. These effects may reduce some of carcinogenic effects of compounds present in Cannabis smoke.

Cannabis and Cannabis extracts are used in pain relief. Although many of the pain-relieving properties of Cannabis have been attributed to cannabinoids, terpenoids present in Cannabis may also exhibit pain-relieving effects. One of the most abundant terpenoids in Cannabis is β-myrcene, which exhibits a potent analgesic effect as well as anti-inflammatory effect. Other terpenoids present in Cannabis, such as carvacrol, exhibit a potent anti-inflammatory effect, even greater than that of THC.

Cannabis extracts are known to effect blood–brain barrier (BBB) permeability, thereby potentially altering the pharmacokinetics of THC and other cannabinoids. Since terpenoids are well known to interact with lipid membranes, they may be responsible for this observed activity. Terpenoids have also been shown to increase cerebral blood flow, which may enhance cognitive brain functions in a way similar to ginkgolides in Ginkgo biloba.

Terpenoids known to be present in Cannabis have a variety of effects, including antibacterial, antifungal, antiviral, and antimalarial activity. Besides the general health-promoting effect of these antimicrobial activities, they may also be important in reducing the dangers of recreational smoking of herbal Cannabis contaminated with microbial organisms. A number of studies have investigated the antimicrobial effects of Cannabis essential oil. One conclusion was that terpenoids from hash oil (obtained from drug cultivars of Cannabis, high in THC content) displayed an antimicrobial effect that was greater than essential oil derived from fiber cultivars.

Finally, terpenoids present in Cannabis may play an important role in the chemical ecology of the plant. For example, they have been shown to be involved in the pesticidal properties of the Cannabis plant. Terpenoids have been detected in the pollen of male Cannabis plants, which may play an important role in either attracting organisms involved in pollination or in repelling harmful organisms.
 

conradino23

Grow Journal of the Year: 2017 - Grow Journal of the Month: Sept 2017
Monika Fellermeier , Wolfgang Eisenreich , Adelbert Bacher and Meinhart H. Zenk, Biosynthesis of cannabinoids Incorporation experiments with 13C-labeled glucoses, Eur. J. Biochem. 268, 1596-1604, 2001

Up until 1990, the precursors of all terpenoids, isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2) were believed to be biosynthesized via the mevalonate pathway. Subsequent studies, however, showed that many plant terpenoids are biosynthesized via the recently discovered deoxyxylulose phosphate pathway which is summarized in Fig. 2.

The first intermediate of this alternative terpenoid pathway, 1-deoxy-d-xylulose 5-phosphate (11), is formed from d-glyceraldehyde 3-phosphate (10) and pyruvate (9) by the catalytic action of 1-deoxyxylulose 5-phosphate synthase (dxs protein) and is converted to 2C-methyl-derythritol 2,4-cyclodiphosphate (12) by the subsequent catalytic action of dxr, ispD, ispE and ispF proteins which have been found in bacteria as well as plants.

In higher plants, the two terpenoid pathways appear to be compartmentally separated. Specifically, the deoxyxylulose phosphate pathway appears to operate in the plastid compartment, and the mevalonate pathway is located in the cytoplasm.

Screenshot_20190706_122334.png
 

conradino23

Grow Journal of the Year: 2017 - Grow Journal of the Month: Sept 2017
Judith K. Booth, Jorg Bohlmann, Terpenes in Cannabis sativa – From plant genome to humans, Plant Science, Mach 2019.

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, 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 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. Only nine CsTPS have been functionally characterized and published to date.

As with many other plant TPS, eight of the nine characterized CsTPS are multi-product enzymes that generate several different terpene structures from either GPP or FPP. The multi-product nature of CsTPS can explain why some terpenes, such as α-humulene and βcaryophyllene, 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.

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.

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. 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, βelemene, or derivatives of myrcene . 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, 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.

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. 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. 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.

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, pointing out that the placebo effect is partially mediated through t 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. 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.
 

conradino23

Grow Journal of the Year: 2017 - Grow Journal of the Month: Sept 2017
Oier Aizpurua-Olaizola, Umut Soydaner, Yilmaz Simsir, Nestor Etxebarria, Aresatz Usobiaga, Ekin Öztürk, Patricia Navarro, Evolution of the Cannabinoid and Terpene Content during the Growth of Cannabis sativa Plants from Different Chemotypes, Journal of Natural Products, February 2016

RESULTS AND DISCUSSION


Complete data regarding the evolutions of all studied cannabinoids and terpenes in each plant are available in Tables 1−36 in the Supporting Information. Figure 3 shows the development of the total monoterpene and sesquiterpene content in the leaves and the flowers of the three different chemotypes.

The total monoterpene and sesquiterpene content values were calculated by summing all analyzed terpenes of each kind: eight monoterpenes and 20 sesquiterpenes. The same evolution patterns found for THCA and CBDA in the leaves were observed for monoterpenes, i.e., a clear decrease during the first weeks of the vegetative phase, a small increase in the last 2 weeks of the vegetative phase, and a slight decrease during the first weeks of the flowering phase followed by a clear increase.

The maximum concentration in the flowers was also chemotype-dependent, and as for THCA and CBDA, this maximum for the total monoterpenes was found in the ninth week of the flowering phase, while for chemotype II and III plants, the concentrations continued to increase until the end of the experiments.

In contrast, sesquiterpenes exhibited a different evolution in both plant matrices. In the leaves, the pattern was similar until the first weeks of the flowering phase, but after that, the content remained stable. In the flowers, the amount of sesquiterpenes did not change significantly during flowering. All terpenes are derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP).

The condensation of one DMAPP and two IPP molecules leads to the formation of farnesyl diphosphate (FPP), i.e., the precursor of sesquiterpenes, whereas the condensation of one DMAPP and one IPP molecule leads to the formation of geranyl diphosphate (GPP), i.e., the precursor of monoterpenes.

Following the formation of FPP and GPP, sesquiterpenes and monoterpenes are generated by the actions of many specialized terpene synthases (TPSs). However, the expression of these TPSs can differ among the plant tissues and different stages of plant development, thereby resulting in differences in terpene content. Thus, monoterpene synthase expressions were more abundant during this phase, leading to an increase in the monoterpene content during the flowering phase.

To obtain a broader view of the formation of cannabinoids and terpenes, principal component analysis (PCA) and partial least squared regression (PLS) were performed, taking account all of the experimental data (x (224 samples × 36 variables), y (224 samples × 1 time)). Although the PCA model requires more than four PCs to explain the original data structure, up to 60% of the variance is explained by the first three PCs. As observed in Figure 4 in the PC1−PC2 projection, there was a clear distinction between chemotype I plants from the rest (two clusters) and between the leaves and the flowers in each cluster (leaves in blue and flowers in red).

The chemotype II plant was closer to the chemotype III plants than the chemotype I plants, most likely because of its higher CBDA content. From the loading projection, the cannabinoids and terpenes of each class of samples were identified as those that are similar to CBDA and THCA. Thus, the higher CBGA and CBC content can be attributed to chemotype I plants.

Moreover, terpenes, such as βeudesmol, γ-eudesmol, guaiol, α-bisabolene, α-bisabolol, or eucalyptol, were much more pronounced in chemotype III plants, whereas γ-selinene, β-selinene, α-gurjunene, γ-elemene, selina-3,7(11)diene, and β-curcumene were characteristic of the chemotype I plants. This chemotype-dependent terpene distribution was also observed in the correlation analysis of the data.

As indicated in Table 1, terpenes that were more pronounced in chemotype III plants had higher correlation coefficients with CBDA than with THCA. In contrast, the characteristic terpenes of chemotype I had high correlation coefficients with THCA and negative coefficients with CBDA
 

conradino23

Grow Journal of the Year: 2017 - Grow Journal of the Month: Sept 2017
An Introduction to Terpenes Science & Cultivation, Steep Hill White Paper, 2018

The Science and Cultivation of Terpenoids


Isoprene is a five-carbon long molecule that is the foundation of all terpenoids. Think of each five-carbon isoprene as a single chain link: terpenes are constructed of these chain links, with most terpenes being comprised of chains of 5, 10, 15—up to 40 carbons long. Limonene and Linalool are both 10 carbons long—or contain 2 isoprene chain links—whereas Phytol, being a larger, oilier molecule, contains 20 carbons or 4 isoprene links, similar to cannabinoids. In most cases, the larger the terpene molecule, the more viscous and less vaporous it is. Limonene evaporates very quickly, with a pungent fragrance, like acetone.

Cannabinoids, on the other hand, are much larger molecules, are nearly solid at room temperature and have only a faint odor, like pine pitch. The size and structure of the terpenoid also determines factors such as how soluble it is in water, ethyl alcohol, oil, or other solvents; as well as how easily it decomposes from heat, light, and air. Research has demonstrated that there are several factors that potentially affect terpenoid production in cannabis cultivation.

Among the most critical are plant genetics, pest presence, cultivation conditions, the ecological history of resources, and overall plant health. While soil nutrient and microbiological diversity and fertilization have an impact on plant chemistry, research on the impact on terpene production is limited and variable.

To our knowledge, terpene enhancers do not boost production or quality of terpenes. These products may have the potential to increase terpenes, although not directly—these products improve overall plant health, which in turn encourages terpene expression.

As some terpenes are produced by the plants for defense, the presence of these compounds will be affected by pest pressure or drought, depending on the terpene. In cases of defensive terpenes, environmental factors such as pest pressure or drought may increase terpene production, as part of the plant’s built-in survival tactic.

Biotic and abiotic conditions affect terpene production. Mechanisms by which light, temperature, or physical damage modify terpene concentrations have been well-studied. Light and plant competition are critical environmental factors affecting defensive terpenoid development at the cultivation stage, especially regarding fertilization, which changes the biotic/abiotic dynamics of plant nutrients.

Nutrient availability, such as nitrogen, promotes terpene development as these components are crucial for photosynthesis and generation of atp, but at present, there is no conclusive evidence pointing to nitrogen alone as a chemical assisting terpene modulation. However, nutrient interactions with plant development are affected by other factors and direct correlations have not yet been conclusively drawn to these factors and terpene emissions.

Additionally, different terpenes react to different soil types—ph levels, nutrient availability, and soil texture are causal influences. Diversity in the soil microbiome and the presence of beneficial, well-balanced microbes enhances rooting ability, resulting in a healthier plant. In general, healthy microbes enhance plant rooting ability, efficiency, and resistance to stressors.

Certain microbes create specific compounds, which, when absorbed by the plant, may selectively increase terpene production. Growth-promoting plant bacteria and fungi boost crop growth and yield by balancing nutrients, hormones, and mineral/water uptake. They also serve to alleviate stress; reducing plant susceptibility to diseases and pests, thereby promoting induced systemic resistance. These beneficial effects result in the accelerated production of secondary metabolites, which include terpenes (Ormeño, et al.)

While research indicates that some pesticides and fungicides have the capacity to act as plant growth regulators (PGRs), increasing terpene production, it’s important to note that this does not necessarily contribute to a healthier plant overall. Lower concentrations of these compounds may increase terpene synthesis, and higher concentrations of specific fungicides may also result in decreased levels of terpene synthesis.

Here, any substance which modulates the geranyl pyrophosphate pathway in plants has the potential to increase terpenes, but not enough is known about the long-term effects on cannabis cultivation, and as such, should be approached with caution.

Terpene Storage


Like any plant material, once harvested, cannabis is perishable. As volatile organic compounds, incorrectly stored, terpenes dissipate quickly from cured cannabis. Post-production, terpene retention depends on environmental conditions and time—for example, light boilers dissolve terpenes faster, and considerable terpene reduction has been observed during handling and storage.

To prevent the structural breakdown of terpenes, the cannabis trichomes where the terpenes are concentrated must be kept intact and unbruised. Optimally, cannabis should be stored in airtight, rigid containers, away from sunlight, at ideal temperatures of 50°f, with long-term and short-term storage solutions varying slightly.

Storage in blue, uv-resistant glass is ideal; with nsf polypropylene, polycarbonate, and polyethylene suitable storage alternatives. Light terpenes will dissolve in soft plastics, like plastic bags. Storage in soft containers or plastics is not recommended, as crushed trichome heads promote oxidation of both terpenoids and cannabinoids, promoting rapid deterioration of cannabis and reducing overall potency.

When suspended in lipid oils, shelf-stability of terpenes changes. Terpenoids reduce the speed at which lipids go rancid; however, the mixture’s concentration would affect stability—with a higher concentration of terpenes resulting in more stable product, dependent on lipid type and storage conditions.

Dilute essential oils, terpenes stabilized in lipids, can be drawn for comparison when discussing degradation of terpenes in cannabis products. When you break a seal on a bottle of lipid oil, the degradation clock starts ticking due to oxygen-exposure—unless repacked with nitrogen or argon gas, which is highly unlikely in most cannabis production operations.

Water vapor expedites the degradation process. Color, texture, and clarity of lipid-suspended cannabis products will change over time. Rancidity occurs when the fats decompose into other compounds, causing the development of an unpleasant smell and/or taste.
 

conradino23

Grow Journal of the Year: 2017 - Grow Journal of the Month: Sept 2017
Dave Hawley, Thomas Graham, Michael Stasiak and Mike Dixon, Improving Cannabis Bud Quality and Yield with Subcanopy Lighting, HortScience, November 2018

Abstract

The influence of light spectral quality on cannabis (Cannabis sativa L.) development is not well defined. It stands to reason that tailoring light quality to the specific needs of cannabis may increase bud quality, consistency, and yield. In this study, C. sativa L. ‘WP:Med (Wappa)’ plants were grown with either no supplemental subcanopy lighting (SCL) (control), or with red/blue (“Red-Blue”) or red-green-blue (“RGB”) supplemental SCL.

Both Red-Blue and RGB SCL significantly increased yield and concentration of total Δ9-tetrahydrocannabinol (Δ9-THC) in bud tissue from the lower plant canopy. In the lower canopy, RGB SCL significantly increased concentrations of α-pinine and borneol, whereas both Red-Blue and RGB SCL increased concentrations of cis-nerolidol compared with the control treatment. In the upper canopy, concentrations of α-pinine, limonene, myrcene, and linalool were significantly greater with RGB SCL than the control, and cis-nerolidol concentration was significantly greater in both Red-Blue and RGB SCL treated plants relative to the control.

Red-Blue SCL yielded a consistently more stable metabolome profile between the upper and lower canopy than RGB or control treated plants, which had significant variation in cannabigerolic acid (CBGA) concentrations between the upper and lower canopies.

Overall, both Red-Blue and RGB SCL treatments significantly increased yield more than the control treatment, RGB SCL had the greatest impact on modifying terpene content, and Red-Blue produced a more homogenous bud cannabinoid and terpene profile throughout the canopy. These findings will help to inform growers in selecting a production light quality to best help them meet their specific production goals.

The production and consumption of drug-type cannabis (C. sativa L.) has seen increased acceptance and legalization in North America in recent years (ArcView Market Research, 2017). “Drug-type” cannabis, as opposed to “hemp” or “fiber-type,” is characterized by high concentrations of Δ9-tetrahydrocannabinol-9-carboxylic acid (∆9-THCA) and relatively low concentrations of cannabidiolic acid (CBDA) (van Bakel et al., 2011; Vollner et al., 1986). “Drug-type” cannabis will henceforth be referred to in this study more simply as cannabis. Like any other cash crop, producers seek to maximize yield, while also optimizing or otherwise standardizing quality.

Floral bud tissue is of primary interest when attempting to maximize cannabis yield. Floral bud has a relatively high density of glandular trichomes rich in cannabinoids and terpenes that are of medicinal and recreational interest (Happyana et al., 2013). There are relatively few peer-reviewed studies on optimizing environmental parameters for bud yield, and commercial cannabis producers are typically guarded with their production strategies.

Nonetheless, one could assume that producers are using the typical production strategies of high light intensities and CO2 concentrations in an effort to achieve higher yields. The specifics of optimal light qualities and CO2 concentrations are known to vary with species, cultivars, and production strategies (Blom et al., 2016; Critten, 1991; Fu et al., 2012; Ilić et al., 2012; Li et al., 2017; Nemali and van Iersel, 2004).

Given the paucity of scientifically peer-reviewed cannabis production data, it is likely that producers have not yet determined the optimal light (quality and quantity) and CO2 inputs for their specific cultivars and production methods (e.g., indoor), but are supplying reasonable levels based on black-market production information for what would be optimal in similar species.

Optimizing and standardizing bud quality is considerably more challenging than just increasing yields in cannabis. This is particularly challenging because it is not yet established what “optimal” bud quality is, medicinally. Furthermore, the definition of “optimal” may vary according to the nature of the medical disorder being treated.

Clinical studies have yet to determine which specific compound or combination of compounds provides any medicinal benefits to users, or the quantities and ratios of these compounds that are optimal in treating various ailments. The currently held theory is that two groups of metabolites together may have medicinal applications: cannabinoids, a class of compounds reserved to only a few plant species; and certain terpenes, common to many plant species (Potter, 2014).

There is some evidence to suggest that different compounds in these families can act together in an “entourage effect,” medicinally of greater benefit than the compounds alone (Russo, 2011). Given the novelty of legal commercial cannabis production, relatively few developments have been made through breeding, genetic modifications, or production strategies aimed at producing consistent cannabinoid and terpene profiles. Without access to consistent metabolite profiles, clinical studies have been unable to thoroughly assess the medical applications of cannabis on a broad scale.

Most commercial cannabis production occurs in greenhouses or growth chambers with supplemental or sole source electric lighting, respectively (Knight et al., 2010; Potter and Duncombe, 2012; Vanhove et al., 2011, 2012). Many horticultural lighting companies looking to capitalize on the cannabis boom are now offering lighting systems that claim to optimize cannabis production.

Some companies offer data supporting their claims, although these data are rarely replicated, reviewed, or published in a peer-reviewed journal. Although the influence of spectral quality on plant development is well documented in the scientific literature (Beaman et al., 2009; Chang et al., 2009; Goins and Yorio, 2000; Lefsrud et al., 2008; Loughrin and Kasperbauer, 2001; Yorio et al., 2001), none yet, to our knowledge, demonstrate the influence of spectral quality on cannabinoid and/or terpene profiles in cannabis.

Notably, many recent studies have demonstrated relationships between light quality, intensity, and secondary metabolism in a variety of species including St. John’s wort (Mosaleeyanon et al., 2005), mint (Kim et al., 2017), perilla (Lu et al., 2017), lettuce (Miyagi et al., 2017; Son et al., 2017), and Cyclocarya paliurus (Liu et al., 2018). The cannabis secondary metabolome may be comparatively more sensitive to its light environment.

To directly investigate the impacts of lighting on cannabis bud yield and quality, supplemental light-emitting diode (LED) bars of two different spectra were deployed below the cannabis canopy in a commercial production environment. Supplemental SCL, as opposed to overhead lighting, was used in this case because it required minimal modifications of infrastructure in the production room, did not add any bulky hardware around plants that would make general plant husbandry cumbersome, and has been proven in the past to be a viable strategy for manipulating plant development (Jiang et al., 2017; Stasiak et al., 1998).

The objectives of this study were to evaluate bud yield, and cannabinoid and terpene contents when plants were grown with no SCL (control), Red-Blue SCL, or RGB SCL. Two crop cycles are presented; the results of the first crop cycle had variability in metabolome that informed changes to data collection and analysis for the second crop cycle.

Table 2.

Cannabinoid and terpene content in dehydrated cannabis bud tissues. In crop cycle 1, 5.0 g of dehydrated bud tissue was randomly sampled from all bud tissue in a given treatment and replication; in crop cycle 2, bud tissue was similarly sampled, but buds were distinctly sampled from the upper and lower plant canopy. Different letters indicate significant differences between treatments in a given sample set using Tukey’s multiple comparisons test, α = 0.05. Asterisks in place of values in crop cycle 1 indicate unmeasured compounds.

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Cannabinoid concentrations from the lower canopy of crop cycle two are presented in Table 2. Lower canopy concentrations of Δ9-THC, total Δ9-THC, and their biosynthetic precursor Δ9-THCA were significantly increased under RGB and Red-Blue SCL treatments compared with the control. Concentrations of CBDA, total CBD, CBG, total CBG, and CBGA were not significantly different between treatments.

In the lower canopy, RGB SCL significantly increased concentrations of alpha-pinine and borneol, and both Red-Blue and RGB SCL significantly increased concentrations of cis-nerolidol compared with control SCL (Table 2). The other measured terpenes did not differ at α = 0.05, although the general patterns suggest similar overall tendencies which may be borne out in further studies with tighter between chamber error control and a greater number of replications.

In the upper canopy of crop cycle two, there were no significant differences in cannabinoid concentrations between treatments (Table 2); however, there were detectable differences in terpene profiles (Table 2). Alpha-pinene, limonene, myrcene, and linalool were present at significantly higher concentrations in the RGB SCL treatment than in the control treatment, whereas cis-nerolidol concentration was significantly higher in both Red-Blue and RGB SCL than in the control (Table 2).

Fig. 6

Terpene concentrations in the upper and lower canopy of plants grown with control, Red-Blue, and Red-Green-Blue (RGB) subcanopy lighting. Filled diamonds indicate lower canopy; empty diamonds indicate upper canopy. Vertical bars indicate standard error. Shaded cells indicate a significant difference between canopy positions using Student’s t test, α = 0.05.

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Cannabinoid and terpene biosynthesis.

Careful consideration of the biosynthetic pathways for cannabinoid and terpene biosynthesis offers a possible explanation for the differences observed between SCL treatments. Figure 7 provides a simple outline of some of the major steps involved in cannabinoid and terpene biosynthesis relevant to this study. Geranyl pyrophosphate (GPP) is condensed from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), and is the basic subunit of all monoterpenes in higher plants (Banthorpe et al., 1972; Croteau and Purkett, 1989).

In the synthesis of cannabinoids, GPP and olivetolic acid (OA) are combined via GPP-OA transferase to produce CBGA (Fellermeier and Zenk, 1998; Fellermeier et al., 2001). Various synthases then convert CBGA to derivatives such as Δ9-THCA and CBDA (Taura et al., 1995, 1996). All terpenes in this study are also downstream products of DMAPP and IPP, including several carotenoids and xanthophylls that mitigate damage from harmful radiation (Croteau and Purkett, 1989; Demmig-Adams and Adams, 1996; Dorothea, 2008; Lichtenthaler, 1987).

Perhaps by supplementing additional light, particularly a spectrum relatively rich in green light that is normally absorbed by some terpenes (Miller et al., 1935; Zur et al., 2000), the plants up-regulated terpene biosynthesis to manage that environmental condition. In so doing, IPP and DMAPP precursors were also up-regulated to supply the demand for these terpenes.

A greater pool of IPP and DMAPP would also theoretically be available for the production of GPP to be condensed with OA to produce CBGA, and Δ9-THCA and CBDA in turn. Conversion from CBGA to Δ9-THCA via THCA synthase may have happened with a high enough efficiency that most extra CBGA produced was converted to Δ9-THCA, accounting for the lack of increase in observed CBGA concentrations.

By contrast, CBDA synthase may have an extremely low activity in this cannabis variety, so even in the presence of an increased CBGA pool, there is no increase in the amount of CBDA produced.

This explanation is conjecture given that, to the best of our knowledge, there are no studies directly measuring IPP or DMAPP concentrations under varying light qualities. The secondary metabolism in cannabis is complex and requires a great deal more study before we will have a satisfactory explanation for this observation.
 

conradino23

Grow Journal of the Year: 2017 - Grow Journal of the Month: Sept 2017
General terpenoid study!

Effect of Soil Nutrient on Production and Diversity of Volatile Terpenoids from Plants, E Ormeño and C Fernandez, Curr Bioact Compd. 2012 Jan; 8(1): 71–79

2.2. What Ecological Theories Anticipate

In the early 1980s, attention began to be focused on the role of nutrient resource availability in terms of the costs and benefits of producing carbon-based metabolites such as terpenoids. This attention resulted in 2 resource allocation theories used for predicting allocation of carbon and nutrient resources for the production of carbon-based defense compounds, especially phenolics and terpenoids.

The carbon-nutrient balance hypothesis (CNBH) presumes that carbon and nutrient availability in the plant environment determines the production of metabolites. When nutrients, especially nitrogen, are highly scarce, a plant will allocate proportionately more of an abundant resource, such as carbon, to the acquisition of the scarce resource and to the synthesis of defensive compounds.

This was based on the observation that limited nutrient resources curtailed plant growth, rather than photosynthesis, resulting in an excess of carbohydrates. Under such conditions, the CNBH asserts that the excess of carbohydrates is not used for growth but provides, instead, an additional substrate to synthesize defense secondary metabolites. This theory considers that carbon-based defense compounds have no cost since they do not directly compete with growth, because their synthesis is achieved through an excess of carbohydrates.

The growth differentiation balance hypothesis (GDBH), also referred to as “excess carbon hypothesis”, assumes that there are 3 types of balance between growth and terpenoid production. Whenever all required resources for growth are available, that is under soils rich in nutrient resources, the theory prescribes that growth (e.g. cell division, biomass production), will be favored over differentiation (e.g. cell maturation and production of defensive compounds).

As nitrogen becomes scarcer and not optimal, differentiation will predominate, and consequently terpenoid accumulation or emission will increase at the expense of growth, since the plant allocates proportionately more of an abundant resource, such as carbon, to the acquisition of the scarce resource and to the synthesis of defensive compounds. Finally, under limiting nutrient conditions, both primary and secondary metabolisms are at their lowest levels.
 
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