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ICR State Fiscal Year 2021

ICR – Research Funding

The economic downturn caused by the global COVID-19 pandemic affected the ability of many institutions to continue conducting research and the available funding for research was reduced for FY21. This reduction resulted in a delay of the funding of new research projects through an RFA for FY22, though funding possibilities returned to the ICR in FY22. In spite of this reduction and the inability to fund new projects, the ICR continued to carry out a set of six multi-year projects for PIs Drs.  Sanghyuck Park, Annette Gabaldón, Brian Vanden Heuvel, Barbara Brett-Green, and Eun Soo Kim that had been reviewed and awarded prior to the COVID-19 pandemic.

Research Projects

Understanding Genomic Constituents of Cannabis and Genetic Regulation Underlying Cannabinoid Production

Dr. Sang Hyuck Park, ICR

The objective of the ICR research projects is to provide in-depth scientific evidence for better understanding the biology, chemistry, and physiology of Cannabis sativa L. to improve its agronomic values, as well as to extend the scope of the use of phytocannabinoids as a potential therapeutical agent. In order to achieve this goal, multi-tiered research projects have been initiated.

The main focus has been on the reference-level genome sequencing of commercial hemp varieties and has now reached to the chromosomal level sequencing. Our ongoing genomic comparative analyses will provide clues to unraveling the genomic structure and genetic regulations underlying cannabinoid biosynthesis and other agronomically important traits. 

In addition, the effects of environmental cues (e.g., light spectra, UV radiation) were of great interest and a greenhouse test was set to identify the potential impacts of various wavelengths of light on cannabinoid synthesis and compositional profiles. As a continuation of cannabidiol (CBD) study on the ethanol-intoxicated tobacco hornworm, another insect model species, a fruitfly (Drosophila melanogaster) has been selected to further explore the long-term effects of CBD on their growth, development, and fecundity. More ecological impacts of CBD-enriched hemp are currently being investigated through multiple collaborations.

Anti-Cancer Potential of Fermented Dietary Hempseed (CANNABIS SATIVA L.) Metabolites and the Role of CB Receptors in Human Colon Cancer Cell CACO2 Viability, Proliferation, and Energy Metabolism

Dr. Annette Gabaldón, Biology

The objective of this study is to determine whether metabolites generated through probiotic fermentation of dietary hempseed (Cannabis sativa L.) exert anti-cancer effects. Specifically, we will culture human colorectal carcinoma cells Caco-2 in growth media supplemented with hempseed fermentation supernatant and test for alterations in cell viability, proliferation, and energy metabolism.

Previously, we investigated the ability of two lactic acid-producing probiotics (L. plantarum and L. fermentum) to ferment whole hempseed. Our question of interest was whether hempseed could serve as a prebiotic to support probiotic growth and metabolism. In results that will be reported at the 2019 ICR Conference, we discovered that both probiotic organisms grow very well in media prepared with 5% hempseed powder(HS-5).

From colony forming unit (CFU) assays, we observed the characteristic phases of a bacterial growth curve (i.e., lag, log, stationary). From pH measurements of the fermentation media, we observed significant acidification, indicative of acidic secondary metabolite synthesis. Acidic metabolites yielded from carbohydrate fermentation include lactic acid and short-chain fatty acids (SCFAs), notably acetate, butyrate, and propionate. These SCFAs provide protective effects against colorectal cancer. 

If hempseed fermentation is found to yield high levels of SCFAs, then perhaps this nutrient-rich seed can be developed as a novel dietary anti-cancer therapeutic. We are finalizing SCFA and lactic acid analysis on the HS-5 fermented samples and if this new proposal is funded, we will grow Caco-2 cells in a cell culture system and test for anti-cancer activity using HS-5 fermentation samples that were collected in the study described.

In the event that chemical analyses of the HS-5 fermentation media show low SCFA levels, the Caco-2 study is still worth pursuing because the HS-5 fermented media is expected to be complex. Whole hempseed is a rich nutrient that is high in protein, lipid, digestible and indigestible carbohydrate, vitamins, and phytochemicals.

The HS-5 fermented product that we have generated may also contain protein fermentation metabolites and there is much interest in identifying bioactive fermentation peptides related to human health. In a second study, we are proposing to investigate the role of CB receptors in modulating the viability, proliferation, and cellular energy metabolism of cultured colon cancer cells.

Effects of Dietary Hempseed (Cannabis sativa L.) on Growth Patterns, Body Composition, Bone Mineral Density, and Gut Microbiota Diversity in Memale C57BL/6J Mice

Principal Investigator: Dr. Annette Gabaldon, Biology

The objective of this study is to understand the effects of dietary hempseed (Cannabis sativa L.) ingestion on growth patterns, body composition, bone mineral density, and gut microbiota diversity in female C57BL/6J mice. Hempseed is a nutrient-dense food which contains high amounts of protein, carbohydrate, fiber, lipids, vitamins, minerals, and phytochemicals, but its use as a dietary aide is controversial in the U.S. due to the presence of cannabinoids present in small amounts. The oil from cold-pressed hempseed is mostly polyunsaturated and rich in Omega-3 and Omega-6 fatty acids. The pressed cake is rich in proteins and carbohydrate. Hempseed oil and seeds are beginning to appear in the U.S. market for human consumption, but its use in agricultural animal feed has not gained approval from the FDA. Most of the research on hempseed nutritional properties and health benefits to animals has come from regions where it is a major gran, such as China, Canada, Australia, Italy, and France. Here, we will investigate the influence that dietary hempseed has on growth parameters and intestinal microbiota diversity in young female mice. Forty mice will be randomly divided into four groups: control diet (CON, no hempseed), 5% hempseed in deity (HS5), 10% hempseed in diet (HS10), and 20% hempseed in diet (HS20).

The individual groups of mice will be fed their respective diets daily from ages five to 30 weeks and growth parameters will be evaluated at monthly intervals. Body composition analysis will be performed using dual-energy x-ray absorptiometry (DEXA) scanning, which reports lean mass, fat mass, bone area, bone mineral content, and bone mineral density. Somatometric measurements will include body mass, length, and surface area. At bi-weekly intervals, the mice will be placed singly into metabolic cages for measurement of food intake and collection of feces for microbiota diversity testing by colony forming unit (CFU) assay. Endpoint measurements at 30 weeks of age will include blood plasma total antioxidant capacity, visceral organ component analysis, and DEXA scan on selected individual skeletal bones which will then be tested for mechanical strength properties.

This is a new area of ICR research, but it complements well two ongoing ICR pilot projects. Study 1 is investigating the influence of hempseed supplementation on probiotic growth and synthesis of secondary metabolites in cultured Lactobacillus strains. Study 2 is evaluating new bone formation by cultured human osteoblast (HOB) cells in the presence of estrogen and endocannabinoids (2-AG and anandamide). These studies will help us to gain a better understanding of the functional significance of industrial hempseed and cannabinoids to human nutrition and health. 

Applications of Industrial Hemp

Dr. Brian Vanden Heuvel, Biology

This project enables an inter-departmental, multi-faculty collaborative workflow that builds on 18 months of previous achievements by the principal investigator and co-principal investigators and established infrastructure through previous ICR funding. We believe that it is time to extend our work to applied projects.

Specifically, this proposal asks 1) Can industrial hemp be used as a remediation tool for metals and metalloids from soil and municipal sewage sludge?; 2) Can cannabinoid extraction methods developed in previous ICR projects be scaled up to industrial volumes?; 3) Can waste products from industrial hemp production (stems/leaves) be a reliable source of important biopolymers?; and 4) Can the recently sequenced genomes for industrial hemp be a framework for continued research into what genes are turned on and off during different growth stages, affecting important traits like disease resistance, drought intolerance, yield, specific concentrations of CBCs, THC, or other secondary chemicals?

An Ongoing Investigation Into the Effects of Medicinal Cannabis on Seizures in Adults with Medically Refractory Epilepsy

Dr. Barbara Brett-Green, Psychology

The primary objective for this project is to continue an ongoing study into the effects of medicinal cannabis on seizures in adults with medically refractory epilepsy. The Principal Investigator has established a partnership with Realm of Caring, a nonprofit organization, to provide support services for patients, and iC42, a bioanalytics company, to process participants’ biological samples (blood and urine) for antiepileptic medication and cannabinoid levels. Additional funding awarded by the ICR substantially improved the quality of the original study; however, it also lengthened the original study’s time frame.

Challenging aspects of conducting this research include the six-month length of the study and the expected length of the participant enrollment period. The Principal Investigator and research team are actively engaged in recruiting participants for this study. Recruitment is expected to continue for between six to 12 months until 34 participants are enrolled.

Data analysis and the production of articles for peer-review are expected to continue beyond 2019. Since the ICR initially awarded funding for this study, the Principal Investigator has been engaged in numerous research-related activities, including presenting a progress report at the first annual ICR conference, producing a technical report for the Pueblo County Commissioners, submitting multiple abstracts for presentations at upcoming regional and national conferences, accepting invitations to present at national cannabis conferences and other conferences, and preparing a review article for publication in the upcoming ICR journal.

The primary objective is to keep the Principal Investigator’s lab fully operational so that data collection for this study can be completed. Specific aims are to continue to support the costs of participant stipends, personnel, re-assigned time for the Principal Investigator, travel to present research results at scholarly conferences, and publishing. The Principal Investigator also plans to apply for funding external to the ICR.

Formation of Cannabinoids in Glandular Trichomes of Cannabis (Partnership Project)

Dr. Sang Hyuck Park, Dr. Eun Soo Kim

This study will provide a base for a future thrust relating to a selection of hemp varieties. According to the morphological aspect, three types of glandular trichomes in Cannabis sativa are recognized: bulbous, capitate-sessile, and capitate-stalked trichomes. Capitate-stalked trichomes have garnered research interest, whereas the other two types of trichomes have almost not been investigated during the past twenty years. 

Our goal is to determine the functional activities of the three types of glandular trichomes related to the biosynthesis of cannabinoids. First, we will examine and compare the external features, distribution, and densities of these glandular trichomes using digital microscopy and scanning electron microscopy. Second, we will analyze secretory cavity contents of individual glandular trichomes removed through microcapillary procedures from hundreds trichomes of each type using gas chromatography. Third, we will examine localization and distribution of available antibody probe for CBD in chemically fixed and embedded tissues using transmission electron microscopy.

Registration is Open

Student smiling at camera

Registration is available for Fall B online – please see course details in Course Offerings. 

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