Light Spectrum Effects on Metabolite Profile of Cannabis

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
  •  Photoperiod
    • 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:

Neon Cannabis Leaf
  • 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.

References:

  1. 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.
  2. 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 Neuropsychopharmacology36(6), 1219–1226. doi:10.1038/npp.2011.6.
  3. Bernstein, N, Gorelick, J, and Koch, S. (2019) Interplay between chemistry and morphology in medical cannabis (Cannabis sativa L.). Industrial Crops and Products. 129: 185-194. https://doi.org/10.1016/j.indcrop.2018.11.039
  4. 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.
  5. 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
  6. 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.
  7. 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.
  8. 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.
  9. Hawley, D., Graham, T., Stasiak, M., & Dixon, M. (2018). Improving Cannabis Bud Quality and Yield with Subcanopy Lighting, HortScience horts, 53(11), 1593-1599. Retrieved Oct 11, 2019, from https://journals.ashs.org/hortsci/view/journals/hortsci/53/11/article-p1593.xml.
  10. Kern, R. (2019) Moving Towards Sustainable Cultivation Practices, Agate Biosciences. 2019 Cannabis Science Conference East. Baltimore, MD.
  11. 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
  12. Miller, E.S., G. Mackinney, and F.P. Zscheile. 1935. Absorption spectra of alpha and beta carotenes and lycopene1. Plant Physiol. 10:375–381.
  13. 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.
  14. 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.
  15. Potter DJ (2009). The propagation, characterisation and optimisation of Cannabis sativa as a phytopharmaceutical. PhD, King’s College, London, 2009.
  16. Russo EB, Burnett A, Hall B, Parker KK. Agonistic properties of cannabidiol at 5-HT-1a receptors. Neurochem Res. 2005;30:1037–1043.
  17. Russo, EB. (2011) Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects. British J Pharmacology. 163: 1344-1364.
  18. Shannon, S., Lewis, N., Lee, H., & Hughes, S. (2019). Cannabidiol in Anxiety and Sleep: A Large Case Series. The Permanente journal23, 18–041. doi:10.7812/TPP/18-041.
  19. 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.
  20. 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.

Pyrrolizidine Alkaloids as a Fulcrum for Co-Evolution

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).

Symphytum officinale

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

Necine structure of Pyrrolizidine Alkaloids
Figure 1: Necine structure of Pyrrolizidine Alkaloids (Sahzly and Wink, 2014) 

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):

  • sepals (tr)
  • petals (6)
  • pollen (14)
  • ovaries (98)
  • complete flower (141)
  • fruits (183)

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.

7-acetylintermedine ~65%

  • ovaries
  • fruits
  • whole flower

~45%

  • leaves
  • roots
7-acetyllycopsamine ~30%

  • leaves
  • roots
intermedine ~15% all
lycopsamine ~15%

  • ovaries
  • fruits
  • whole flowers

5%

  • leaves

12%

  • roots
myosorpine 99%

  • petals
  • pollen
3-acetylmyoscorpine largely trace

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?

References:

  1. Berenbaum MR, Zangerl AR (1993) Furanocoumarin metabolism in Papilio polyxenes: biochemistry, genetic variability, and ecological significance. Oecologia 95:370–37. https://doi.org/10.1007/BF00320991.
  2. Ehrlich P & Raven P (1964Butterflies and plants: a study in coevolutionEvolution 18586– 608. DOI: 10.2307/2406212
  3. El 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.
  4. International Programme on Chemical Safety (IPCS) (1988) Pyrrolizidine alkaloids. Environmental health criteria 80. WHO, Geneva.
  5. 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.
  6. Liu, X., Vrieling, K., and Klinkhamer, PGL. (2018) Phytochemical background medicates effects of pyrrolizidine alkaloid in Western Flower Trips. J. Chem. Eco. 45(2): 116–127.
  7. https://doi.org/10.1007/s10886-018-1009-2.
  8. Nelson AC, Kursar TA (1999) Interactions among plant defense compounds: a method for analysis. Chemoecology 9:81–92. https://doi.org/10.1007/s000490050037.
  9. Now, M., Wittke, C., Lederer, I., Klier, B., Kleinwächter, M., and Selma, D. (2016) Interspecific transfer of pyrrolizidine alkaloids: An unconsidered source of contaminations of phytopharmaceuticals and plant derived commodities. Food Chem. 213: 163–168. https://doi.org/10.1016/j.foodchem.2016.06.069.
  10. 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.
  11. 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.
  12. Wink, M. (2019) Quinolizidine and Pyrrolizidine Alkaloid Chemical Ecology – a Mini-Review on Their Similarities and Differences. J Chem Ecol. 45(2):109-115. https://doi.org/10.1007/s10886-018-1005-6.

Case Study to Develop NDIs for Cannabis Oil

In their final trimester, Masters students in the Herbal Product Design and Manufacture Program at Maryland University of Integrative Health (MUIH) take a case study course where companies and various stakeholders present real world problems for our students to solve. The current iteration will investigate the significant public interest in cannabis (hemp)-based and CBD-containing food and herbal products. Image of Marijuana leaf with CBD molecule superimposed over it.

Many questions were generated about the science, safety, and quality of these products at the May 31, 2019 public hearing with the Food and Drug Administration.  Key take-away messages from this meeting are the following:

  • Data are needed to determine safety thresholds for CBD.
  • Datasets/information should be objective, of adequate quality and available for transparent review.
  • Lab testing and data analyses need to be replicable.
  • Consumers need consistent information and labeling.
  • State/government entities need support in knowing what to do.
  • We need to understand the implications for children when they take CBD-containing products at different dosage ranges.

Because public has expressed considerable interest in marijuana products (see state legal status map: https://disa.com/map-of-marijuana-legality-by-state,)the FDA is considering how to issue a regulation creating an exception, such as allowing the marketing of CBD in conventional foods or as a dietary supplement.  Regardless, the former FDA commissioner noted that such foods must safe based standards for food additives and related food safety assessment procedures, such as those presented in the NDI guidance (https://www.fda.gov/news-events/press-announcements/statement-fda-commissioner-scott-gottlieb-md-signing-agriculture-improvement-act-and-agencys).

This has its own challenges.  The FDA has identified several safety based-questions about CBD products that need answers as noted above.  For example:

  • How much CBD is safe to consume in a day?
  • How does it vary depending on what form it’s taken?
  • Are there drug interactions that need to be monitored?
  • What are the impacts to special populations, like children, the elderly, and pregnant or lactating women?
  • What are the risks of long-term exposure?

Many industry insiders believe that hemp-based cannabis products, such as cannabis oil or tinctures will be addressed using the DSHEA statutory framework, but that these products will require New Dietary Ingredient (NDI) filings. A description of the 2016 NDI guidance document can be found at https://www.fda.gov/media/99538/download.

To that end, our case study this trimester students will collaborate with stakeholder Dr. Roger Clemens.  He is internationally known scientist, and Professor of Pharmacology and Pharmaceutical Sciences and Assistant Professor of Regulatory Science within USC’s School of Pharmacy, International Center for Regulatory Science. Dr. Clemens is a past president of the Institute of Food Technologists (IFT) and is the current presiding officer of the International Academy of Food Science and Technology.  He co-founded, established and contributes to a Food, Medicine and Health column published monthly in Food Technology for the past 15 years.  He continues to serve on several editorial boards (e.g., Food Chemical Toxicology, Journal of Food Science, Toxicology Research and Applications, Journal of Dietary Supplements) and continues to serve as a reviewer for many high-impact journals.  Dr. Clemens has published > 50 original manuscripts and commentaries. In addition, he has presented more than 250 invited lectures at domestic and international scientific conferences.  He is an elected Fellow in four scientific organizations.

They will be tasked with designing an effective model for a hemp oil NDI (New Dietary Ingredient), which will serve them in two important ways: 1) acquire a deeper understanding of hemp constituents and their safety (classic ADME and toxicology), public health implications, and potential medicinal application; and 2) learn to write an NDI, which is vital in the herbal supplement industry.

Wish them the best of luck.

Plant Defense Signaling: How is this related to good medicine?

Botrytis cinerea growing on a PDA
Botrytis cinerea growing on a PDA (Wikipedia)

Plants make numerous small molecules (metabolites) that are either directly toxic to insects, grazing animals, fungi and bacteria, or that stimulate the production of other toxic metabolites. The production of various types of metabolites such as alkaloids, terpenes, and phenolics can be turned on (induced) after damage to the plant occurs. The control mechanisms involve  jasmonate, salicylate, phytohormone (plant hormone) ethylene, the volatile (gaseous) methyljasmonate and methylsalicylate signaling pathways.

The signal process is complex. It works on a local level or systemically by traveling throughout the entire plant. It behaves like a network, allowing chemical communication between parts of a plant or within a population of plants in a given locale. With recent developments in metabolic profiling, highly sensitive separation and detection systems are used to create metabolite profiles, high-throughput gene expression analysis is used to detect genes transcripts and rigorous statistical mining resulted in some interesting data that reveals more about how plant defense signaling is controlled.

Researchers used metabolic profiling of overall patterns rather than relying on targeted metabolites. This is important, since previous work focused on a few major metabolites and provided conflicting data. They found differences in profiles from plants exposed to generalist insect feeders versus plants treated with phytohormones. Although both jasmonate and salicylate pathways were activated in each treatment (co-induced), the metabolite patterns were distinct; both treatments lead to a stronger localized rather than systemic response; and there appeared to be a great deal of cross-talk between both pathways influencing pools of precursor metabolites.

Botrytis cinerea growing on tomato leaf
Botrytis cinerea growing on tomato leaf

Another study followed the spatial accumulation of hydroxycinnamates (phenylpropanoids) and lignins in cell walls of Arabidopsis (a model organism) in response to changes in the ethylene signaling induced by the necrotrophic fungal pathogen, Botrytis cinerea. They correlated metabolic profiles with cytological (cell based) changes to provide biological validation of the analytical data.

When a fungus like Botrytis attacks a plant, it generally destroys cells and eventually the entire plant. In the presence of the fungus, plant genes for the biosynthesis of phenylpropanoids and lignins are expressed (turned on) to modify and reinforce the plant cell wall against fungal penetration. Botrytis induces over 30 ethylene regulated transcription factors – cellular molecules that target and induce underlying genes to become active. So these researchers used a metabolite profile of 3 ethylene mutants, plants that had gene mutations at different DNA sequence points of the ethylene signaling pathway.

It turned out that the mutant plants were less resistant to the fungus and that ethylene resistance, when present, appeared after the fungus had made contact with the plant cell wall and had begun to build the structures necessary to penetrate the cell wall barrier. One phenylpropanoid metabolite in particular, ferulate, seemed to be highly influenced by ethylene signaling. Ferulate cross-links the polymer strands of cell wall polysaccharides, enhancing their structural integrity as a barrier.

So what does this have to do with good medicine? Don’t focus on one or two metabolites to make an efficacious extract. Despite what you hear, we are not trying to mimic “magic bullet” medicine. Expose plants to a full set of ecological challenges to produce a metabolite profile of greater diversity. Mono-crop farming doesn’t cut it. Damn if those hippies had it right after all.

Using ePortfolios to Guide Student Learning, Part I

This post also focuses on complex ecologies, found in education, not the wild. It reviews the adoption of an ePortfolio in our Therapeutic Herbalism Masters program at Maryland University of Integrative Health, which was designed to accomplish three main tasks:

  1. Encourage and provide opportunities for our students to experience meta-cognitive learning about the competencies they have acquired/developed..
  2. Create a professional web presence for the student to market their expertise.
  3. Assess whether our program learning outcomes are being met in the classroom.

The Student Learning Portfolio allowed student to collect artifacts of their acquired competencies. These were often based on assignments from each of their courses.  Ideally, they reflected skills developed, competencies, and career readiness resulting from each course and the integration of those experiences. The process was meant to enhance the ability of students to be self-sufficient reviewing their ability to succeed in a chosen profession.

Image of learning Zones

Their final product was creation of a Professional ePortfolio, where the competency artifacts selected by a student showcased the knowledge, skills, and abilities that are in demand in the professional marketplace.

During our review of the tool it became apparent two important lynch pins were missing. Firstly was the articulation of appropriate professional competencies as measurable program learning outcomes. Secondly, our faculty had limited experience applying learning outcomes and reviewing meta-cognitive tools such as the ePortfolio. They were not prepared to highlight the connection between chosen artifact from their course, as well as the underlying learning process and how it was linked to an overriding professional competency.

A series of faculty retreats refined more effective and measurable program learning outcomes. In addition, the institutional assessment process that emerged out of a regular Middle States Higher Education Commission review of the university helped create more specific measurement goals. The combined effect enabled both faculty and students to identify appropriate artifacts and learning processes.

We articulated specific competencies that were missing or ill-defined in previous versions, including:

  • Improved research literacy skills – finding and assessing the validity of scientific research – in support of their analytical work on assignments.
  • Succinct summarization of primary, peer-reviewed resources and the synthesizing of new ideas from those summaries that contribute unique ideas to the field
  • For clinical students, populated their portfolio with case study write-ups.
  • Created effective narratives of how they worked with incomplete data (medicine making, research or diagnosis) in finding a solution using iterative problem solving.

Other challenges that appeared included the need to help students learn how to select learning artifacts that reflect project-based learning. Since the feedback loop in assessment provides data about how program outcomes are being met in the classroom, the process of student metacognitive review of both object and learning processes revealed difficulty in effectively linking the two.

Apparently this is not unusual in ePortfolio development (Land & Greene, 2000). At the core of this issue is the requirement for more engaged and knowledgeable faculty to embed assignments and course long assessment arcs focused on strengthening the linkage between an object (paper, case study, etc.…) and the underlying programmatic learning objective.

Faculty training in applying ePortfolio to their own professional development would improve their the ability to guide students into choosing suitable learning artifact and how to articulate those to employment marketplace.

Reference

Land, S.M. & Greene, B.A. (2000) Project-based learning with the world wide web: A qualitative study of resource integration. Educational Technology Research and Development. 48: 45. https://doi.org/10.1007/BF02313485.

Fermentation as an Extraction Method

Although most cultures have fermented food as a staple in their dietary patterns, little has been reported on the use of fermentation as an herbal extraction method. The process may contribute more than just modifying solvent pH. Rizzello et al. (2013) reported using a lactic acid fermentation with specific yeast strains that improved the antioxidant activity of Echinacea purpurea. They compared fermentation extraction to either a methanol or water extract without fermentation. The greatest antimicrobial activity was associated with low molecular mass compounds negated in the presence of digestive enzymes, suggesting small peptides as the active agent.  The authors cited other experiments with grapes, soy and cereal grains where the fermentation process increased bio-availability of  certain compounds and produced novel chemical species.

This process is worth exploring by both herbal supplement companies and herbalist as a new medicine making method. Traditional texts provide some guidance.  Enzymatic processes can optimize extraction of plant cellular content at lower temperatures. And research (Mishra et al., 2010; Mulay and Khale, 2011) applying traditional Ayurvedic methods of  fermented extraction found reduced toxicity in the final product. This opens up a little explored market around functional foods as well.

Reference:

  1. Mishra AKGupta AGupta VSand RBansal P2010Asava and arishta: an Ayrvedic medicine – an overview. Int J Pharm Biol Arch. 1(1):2430.
  2. Mulay SKhale A2011Asavarishtas through improved fermentation technology. Int J Pharma Sci Res. 2(6):14211425.
  3. Rizzello, CG et al. (2013) Lactic acid fermentation as a tool to enhance the functional features of Echinacea spp. Microbial Cell Factories. 12:44-59.

 

Not Blueberry Pie, but Close

Vaccinium myrtillus
Image via Wikipedia

Bilberry (Vaccinium myrtillus) contains varying levels of phenolic compounds – anthocyanins, chlorogenic acid derivatives, hydroxycinnamic acids, flavonol glycosides, catechins, and proanthocyanidins. Research by Martz et al (2010) elucidated how levels of bilberry leaf phenolics differed along an ecological gradient in boreal forests running north to south in Finland. These regions differ in latitude, altitude, over story cover, levels of continuous light, temperature and associated frost spells.

An analysis of bilberry leaves showed that major phenolic changes in bilberry leaves appeared in the first stages of leaf development. As important, synthesis and accumulation of flavonoids was delayed in the forest compared to the high light sites. Two-fold higher flavonoid levels appeared in leaf tissue growing in high-light intensity sites, higher latitudes, and/or higher altitudes compared to in lower altitudes and low-light intensity sites.

Close and Mcarther (2002) previously theorized that the presence of greater phenolic levels in leaf tissue found in northern regions was a response to colder temperatures, which would limit essential enzyme function, during periods of maximal photo-oxidative stress (Close and Mcarther, 2002).  However, Martz et al (2010) also showed that leaf flavanoid genes were highly expressed in shade, but that the timing of expression appeared to alter the relative metabolite levels in shade compared to sun exposed bilberry leaf.

Mudge et al., (2016), researched phenolic profiles of wild elderberry fruits (Sambucus nigra subsp. canadensis) over two years in eastern US, noting that flavanols (quercitin, isoquercitin, rutin) and chlorogenic acid metabolite concentrations were higher in the southeast, particularly interior. They suggested the variation of phytochemical profiles of the berries were impacted by genetic or environmental factors without understanding on which was more important.

What’s missing from the data picture includes a more complex measurement of ecological influences, such as response to herbivory and rhizosphere fungal associations? This type of whole community data would help to build a more complete picture of  plant response.

This requires sampling, sampling, sampling.

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  1. Martz, F., Jaakola, L., Julkunen-Tiitto, R. and Stark, S. (2010). Phenolic Composition and Antioxidant Capacity of Bilberry (Vaccinium myrtillus) Leaves in Northern Europe Following Foliar Development and Along Environmental Gradients. J Chem Ecol, published online, 19 August 2010
  2. Close, D.C., and Mcarther, C. (2002). Rethinking the role of many plant phenolics—protection from photodamage not herbivores? Oikos. 99:166–172
  3. Mudge, E., Applequist, W. L., Finley, J., Lister, P., Townesmith, A. K., Walker, K. M., & Brown, P. N. (2016). Variation of Select Flavonols and Chlorogenic Acid Content of Elderberry Collected Throughout the Eastern United States. Journal of food composition and analysis : an official publication of the United Nations University, International Network of Food Data Systems, 47, 52–59.