During this past year I’ve not written any blog posts and felt ready to beat myself up about it when I realized that I’ve been creating narrative intensively on multiple fronts. With designing and rolling out two new programs I’ve had to articulate the education mileposts for each, as well as the scope and sequence of competencies learned with each course; articulate the role of newly hired faculty in developing these courses, while overseeing the build out; to communicate with accrediting bodies how our program is different from competitors. I’ve developed the following courses:
Extraction, Analytical Methods and Formulation Strategies
Botanical Pharmacognosy Research and Development of Products
And it’s not just about the writing of text in a document. Rather, to connect with the various tribes or stakeholders requires deep listening; it’s the primitive call to connect even in these most mundane of circumstance.
Also, my work over the past year in creating various formulary, combining cannabis and herbal products for a range of products too is an act of deep listening. Each client is in discovery mode, trying to articulate their own vision for their company and a place for their product in a complex marketplace.
I just needed to step back and appreciate that I’ve been telling stories all along, and an active engagement with my audience refining multiple narrative over time into a cohesive whole. As the poet Peter Klappert, one of my writing mentors, once asked me, “are you writing for yourself or to be part of a community?” There’s no right or wrong answer. There’s just a direction that accompanies your answer. I hope this finds you well and in connection with your tribes.
As the FDA progresses toward a regulatory framework for hemp based cannabinoid products, I’ll touch on some unique research from the production side, focusing on lighting and hydration strategies in greenhouse settings and provide examples of how formulary might help drive the selection. Look for discussions on extraction methods or strain/chemovar choice at another time.
At a 2019 Cannabis Science Conference East talk, typical production methods for cannabis production were described as follows (Kern, 2019):
High light intensity and CO2 concentration
Vegetative growth at 18 hours
Flowering < 12 hours
Use of Blue light during flowering
The blue light treatment is used to optimize several factors:
Increase terpenes with all blue light last three days of flower development
Blue light at end of day can increase leaf expansion increasing photosynthesis and plant mass yield
Blue light at night can slightly increase ∆9-tetrahydrocannabinol (THC)
Research by Mosaleeyanon et al. (2005) showed that light impacts secondary metabolite production, with increased photosynthetic rates leading to increased total hypericin (hypericin + pseudohypericin) concentration in St. Johnswort.
Hawley et al. (2018) experimented with the use of subcanopy lighting (SCL) of red/blue (“Red-Blue”) or red-green-blue (“RGB”) light versus no SCL in a greenhouse production cannabis. They used a plant layout design to limit the amount of additional radiation from the subcanopy and to prevent the treatment light from influencing other plants. They ran the experiment without gyping (removal of the bottom 20 cm of stems). The SCL treatment created differences in the lower canopy metabolite concentrations:
∆9-THC and ∆9-THCA significantly increased using both RGB and RB.
CBD, CBG did not increased by either treatment.
Terpenes alpha-pinene and borneol significantly increased in RGB.
cis-Nerolidol significantly increased in both RGB and RB.
The SCL treatment created differences in upper canopy concentrations:
cis-Nerolidol significantly increased in both RGB and RB.
Alpha-pinene, limonene, myrcene and linalool significantly increased in RGB.
From a QA perspective, the RB SCL treatment also provided the most consistent levels of cannabinoids and terpenes in both upper and lower canopy.
Hawely et al. (2018) suggested previous research (Miller et al., 1995: Zur et al., 2000) had demonstrated light spectra rich in green light was largely absorbed by terpenes, and that the plant had to up regulate the biosynthetic precursors in response to increased green light stimulus. The precursor molecules turn out to be part of both terpene and cannabinoid metabolic pathways, leading to an enriched biosynthetic stream. Greater precursors available led to increased production of both class of metabolites
Two examples of how formulary might take advantage of ecological influences
CBC and CBG concentrations have been shown to be present in leaves at equal or greater levels than in the flowers (Bernstein, Gorelick and Koch, 2019). This suggests that lighting and harvesting strategies may include adding leaf material to an extract and/or optimizing the impact of different light spectra on leaf metabolite profiles.Taking it a step further, cold water extraction of immature leaf of selectively bred cannabis chemotypes yields enriched CBC fraction (Potter, 2009)
Since CBC is the second most prevalent cannabinoid compound found in cannabis (Russo 2011), and research has shown topical anti-inflammatory activity (De Petrocellis et al., 2012: Cascio & Pertwee 2014; Oláh et al., 2016), and sebum reduction in acne, then leaf material might be combined with flowers in an extract. And the impact of under canopy lighting should also be investigated more extensively to determine if the leaf yield of cannabinoids and terpenes can be improved. Applying the RBC-SCL strategy to increase terpenes, the following enriched fractions might improve the results as an anti-acne topical: alpha-pinene as an effective antibacterial (Appendino et al., 2008); limonene, pinene and linalool decreasing sebum/sebocytes (Biro et al., 2009).
CBG displays moderate 5-HT1A antagonist suggesting antidepressant properties (Formukong et al., 1988), and linalool and limonene also show anti-anxiety via 5-HT1A (Russo et al., 2005). Additionally, research on CBD supports the use of CBD as an anxiolytic (Bergamaschi et al., 2011; Shannon et al., 2019; Siloti et al., 2019).
In a second approach to using ecological factors to alter metabolite profiles in cannabis, Caplan et al. (2019) applied drought stress to container-grown cannabis plants, gradually drying substrate under controlled environment for 11 days. The treatment resulted in greater metabolite yield per unit growing area:
THCA 43% higher than the control,
CBDA 47% higher
∆9-tetrahydrocannabinol (THC) 50% higher
cannabidiol (CBD) 67% higher
Experimenting with the yield in various chemovars based on multiple ecological inputs will make for a challenging project. The use of ecological inputs as one strategy for creating treatment specific strains might eventually be combined with chemovar selection and extraction protocols to create a highly specific therapeutic product.
Appendino G, Gibbons S, Giana A, Pagani A, Grassi G, Stavri M, et al. Antibacterial cannabinoids from Cannabis sativa: a structure-activity study. J Nat Prod. 2008;71:1427–1430.
Bergamaschi, M. M., Queiroz, R. H., Chagas, M. H., de Oliveira, D. C., De Martinis, B. S., Kapczinski, F., Quevedo J, Roesler R, Schröder N, Nardi AE, Martín-Santos R, Hallak JE, Zuardi AW, Crippa, J. A. (2011). Cannabidiol reduces the anxiety induced by simulated public speaking in treatment-naïve social phobia patients. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology, 36(6), 1219–1226. doi:10.1038/npp.2011.6.
Biro T, Olah A, Toth BI, Czifra G, Zouboulis CC, Paus R. Proceedings 19th Annual Conference on the Cannabinoids. Pheasant Run, St. Charles, IL: International Cannabinoid Research Society; 2009. Cannabidiol as a novel anti-acne agent? Cannabidiol inhibits lipid synthesis and induces cell death in human sebaceous gland-derived sebocytes; p. 28.
Caplan, D., Dixon, M. and Zheng, Y. (2019) Increasing Inflorescence Dry Weight and Cannabinoid Content in Medical Cannabis Using Controlled Drought Stress. HortScience. 54(5): 964–969. DOI: https://doi.org/10.21273/HORTSCI13510-18
Cascio, M. G., & Pertwee, R. G. (2014). Known pharmacological actions of nine nonpsychotropic phytocannabinoids. In R. G. Pertwee (Ed.), Handbook of cannabis (pp. 137–156). Oxford, UK: Oxford University Press. http://doi.org/10.1093/acprof: oso/9780199662685.003.0007.
De Petrocellis, L., Orlando, P., Moriello, A. S., Aviello, G., Stott, C., Izzo, A. A., et al. (2012). Cannabinoid actions at TRPV channels: Effects on TRPV3 and TRPV4 and their potential relevance to gastrointestinal inflammation. Acta Physiologica, 204(2), 255–266. http://doi.org/10.1111/j.1748-1716.2011.02338.x.
Formukong, E. A., Evans, A. T., & Evans, F. J. (1988). Analgesic and antiinflammatory activity of constituents of Cannabis sativa L. Inflammation, 12(4), 361–371.
Kern, R. (2019) Moving Towards Sustainable Cultivation Practices, Agate Biosciences. 2019 Cannabis Science Conference East. Baltimore, MD.
Marcu, JP. (2016) Chapter 62 – An Overview of Major and Minor Phytocannabinoids. Ed. Victor R. Preedy. Neuropathology of Drug Addictions and Substance Misuse. Volume 1: Foundations of Understanding, Tobacco, Alcohol, Cannabinoids and Opioids (pp. 672-678). King’s College London, London, UK. Academic Press. https://doi.org/10.1016/B978-0-12-800213-1.00062-6
Miller, E.S., G. Mackinney, and F.P. Zscheile. 1935. Absorption spectra of alpha and beta carotenes and lycopene1. Plant Physiol. 10:375–381.
Mosaleeyanon, K., S.M.A. Zobayed, F. Afreen,and T. Kozai. 2005. Relationships between net photosynthetic rate and secondary metabolite contents in St. John’s wort. Plant Sci. 169:523–531.
Oláh A, Markovics A, Szabó-Papp J, Szabó PT, Stott C, Zouboulis CC, Bíró T. (2016) Differential effectiveness of selected non-psychotropic phytocannabinoids on human sebocyte functions implicates their introduction in dry/seborrhoeic skin and acne treatment. Exp Dermatol. 25(9):701-7. doi: 10.1111/exd.13042. https://www.ncbi.nlm.nih.gov/pubmed/27094344.
Potter DJ (2009). The propagation, characterisation and optimisation of Cannabis sativa as a phytopharmaceutical. PhD, King’s College, London, 2009.
Russo EB, Burnett A, Hall B, Parker KK. Agonistic properties of cannabidiol at 5-HT-1a receptors. Neurochem Res. 2005;30:1037–1043.
Russo, EB. (2011) Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects. British J Pharmacology. 163: 1344-1364.
Shannon, S., Lewis, N., Lee, H., & Hughes, S. (2019). Cannabidiol in Anxiety and Sleep: A Large Case Series. The Permanente journal, 23, 18–041. doi:10.7812/TPP/18-041.
Silote GP, Sartim A, Sales A, Eskelund A, Guimarães FS, Wegener G, Joca S. (2019) Emerging evidence for the antidepressant effect of cannabidiol and the underlying molecular mechanisms. J Chem Neuroanat. 98:104-116. doi: 10.1016/j.jchemneu.2019.04.006. Epub 2019 Apr 27.
Zur, Y., A.A. Gitelson, O.B. Chivkunova, and M.N. Merzlyak. 2000. The spectral contribution of carotenoids to light absorption and reflectance in green leaves. Pap. Natl. Resources 2:272.
The theory of evolutionary radiation of flowering plants has been linked to interaction with pollinators and herbivores (Ehrlich and Raven, 1964: Herrera et al., 2002). One class of phytochemicals that researchers investigated to understand the mechanisms for a co-evolutionary impact is pyrrolizidine alkaloids (PAs). Much of the past research has been on the toxicity of these compounds in various structural iterations. When they contain 1,2 double-bond (unsaturated) in their base, necine moiety (Figure 1), PAs can be activated to become hepatotoxic, carcinogenic, genotoxic and teratogenic to humans (IPCS 1988).
This toxicity impacted the dietary supplement market in 2001, when the USA FDA required withdrawal of PA-containing Comfrey (Symphytum officinalis) preparations from the market. Nevertheless, other organisms have benefited from ingesting the compounds.
Biological data revealed how insects interactions with PA containing plants can be protective to the insect (Berenbaum and Aangerl, 1993; Nelson and Kursar, 1999). More recently, Liu, Vrieling and Klinkhamer (2018) explored these synergistic effects with herbivores. Their study evaluated the anti-herbivory effect of PAs on the study insects, Western Flower Trips (WFT). They found that plant secondary metabolite fractions underestimates the effect on herbivory. All fractions contained N-oxides and free bases. Most of the anti-herbivory effect was restored when fractions were recombined.
The insects were more susceptible to the more polar fraction of butanol than the chloroform fraction. Both N-oxides and free bases were present in higher concentration in the butanol as opposed to the chloroform, but total PA amounts were 3x
greater in later. They also noted that when combined with chlorogenic acid (CGA), the N-oxide was more active than when combined with free base, which is the opposite of what was expect based on toxicity profiles.
One of the more important findings from their study was that aside from the N-oxide and freebase forms of the PA, other metabolites can act as antagonists or synergists between CGA and the PA retrorsine. The complexity of the phytochemical background altered the interactions between plant metabolites and their potential bioactivity. What we don’t know is if the use of laboratory spiking studies will work in the field, and will it have population level effects necessary for long term alterations to behavior?
Another piece of interesting research focused on where these compounds get sequestered in the plant. Stegemann et al. (2018) investigated the accumulation and role of PAs’ in comfrey (Symphytum officinale) flowers and fruits. It was originally believed that PA synthesis occurred in the plant roots, which was then distributed to the rest of the structure. They reported secondary sites of synthesis in young leaves subtending developing inflorescences, with transport from leaf to flower to protect reproductive structures.
The authors found variability in accumulation patterns in different tissue, strongly suggesting that the synthesis is developmentally driven. Generally, the level of N-oxides to tertiary PAs was ~95% in all tissue , and the fresh weight PA concentration (ppm) present in different plant parts are listed as follows (from least to most):
complete flower (141)
The patterns of the alkaloids themselves were different in various tissue. Application of methyl jasmonate didn’t appear to alter the levels of expression, thus they appear not to be constitutively produced. The highest levels appeared at peak inflorescence, dropping as the flowers withered. And additional PAs are stored in young leaves and reproductive structures.
In table below, even though only trace amounts of all PAs are located in petals and pollen, almost all is the PA is myosorpine. It’s not clear why that pattern of individual PAs is unique to specific tissue. The presence of specific PAs in different tissues may be “leftover” from the original metabolic pathway, with the plant shuttling those particular metabolites to specific tissue.
We can compare these unique distribution and accumulation patterns to plants that normally don’t produce PAs. Now et al. (2016) tested horizontal transfer of natural products from leachate of rotting plants containing PAs into plants that do not produce those compounds. The accumulation of PA’s occurred in the leaves of guest plant, not flowers, suggesting xylem transfer driven via transpiration. Accumulation in flowers via transport (as opposed to being a production site) is typically source-sink-translocation via phloem. The researchers found that the PAs accumulated as salt like N-oxides. They had expected the presence of free base PAs since they are able to cross biomembranes via simple diffusion. Either the guest plant has a transporter able to translocate N-oxides, or the free base PAs are taken up and oxidized to N-oxide. It made me wonder if this root uptake of exogenous PA’s might happen in all plant species, or was it limited specific plant species, genus or families?
Finally, Wink (2019) discussed how PAs are often stored by specialized insects and that their chemical ecology is intricate. As an example Wink noted how they function as nuptial gift for defense of eggs in the caterpillar larvae of Creatonotus spp. after metamorphosis into adult insects. The presence of the PAs secreted on to the eggs protects them from potential predators. Because of that benefit, it appears that increased PA ingestion by male caterpillars enhances their attractiveness.
This last puzzle in the world of PA chemical ecology had me wondering what examples might exist in the human world that mimic this type of behavior. Although beer consumption has long been thought to enhance the appearance of human male virility, my own experience has disproved that particular absurdity. Do you have any examples?
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Herrera, C.M., Medrano, M., Rey, P.J., Sánchez-Lafuente, A.M., García, M.B., Guitián, J., and Manzaneda, A.J. (2002) Interaction of pollinators and herbivores on plant fitness suggests a pathway for correlated evolution of mutualism- and antagonism-related traits. PNAS. 99 (26): 16823-16828. DOI: 10.1073/pnas.252362799.
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Sahzly, A. and Wink, M. (2014) Diversity of Pyrrolizidine Alkaloids in the Boraginaceae Structures, Distribution, and Biological Properties. Diversity 6(2):188 – 282. https://doi: 10.3390/d6020188.
Stegemann T, Kruse LH, Brütt M, and Ober D. (2018) Specific Distribution of Pyrrolizidine Alkaloids in Floral Parts of Comfrey (Symphytum officinale) and its Implications for Flower Ecology. J Chem Ecol. doi: https://10.1007/s10886-018-0990-9.