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ICR State Fiscal Year 2020

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 https://www.nature.com/articles/s41598-019-47017-7, 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.

Interactions of Penicillium Spinulosum with Hemo and Nonpsychoactive Hemp Compounds

Dr. Sandra Bonetti, Chemistry

In nature, fungi and plants have developed close ecological relationships due to their co-evolution on earth. The interactions between fungi and plants are diverse and may be beneficial or detrimental depending on environmental conditions and the organisms involved. Mycorrhizal fungi are symbiotically associated plant host root systems, which provide enhanced water and nutrient absorption for the plant, while the fungi benefit from photosynthetic carbohydrate nutrients supplied by the plant.

However, many of the interactions are not positive, as many fungal species are plant pathogens, which have evolved to consume plants. The plant pathogenic fungi, among which are Penicillium species, possess an array (arsenal) of enzymes that allow the fungal mycelia to penetrate and destroy the plant cell walls and metabolize plant compounds.

In addition, many harvested plants and plant products, including those from marijuana and hemp, have been found to be contaminated with fungi. Subsequently, consumption of the fungal-contaminated materials poses a threat to human health due to ingestion of mycotoxins and the potential for fungal infection by Aspergillus species in immunocompromised individuals. In this project, the interaction of Penicillium spinu/osum, a non-human plant pathogen, with hemp will be investigated. The two specific aims of this proposal are to (1) determine how the introduction of hemp seeds affects fungal (P. spinulosum) growth and metabolism and (2) determine whether P.spinulosum metabolizes hemp compounds by identifying the fungal products.

Exploring Factors to Mitigate Customer's Perceived Risk of CBD Oil Usage

Dr. Laee Choi, Marketing

Perceived risk refers to the nature and amount of risk perceived by a consumer in contemplating a particular purchase decision. Cox and Rich (1964) suggest that the amount of risk perceived by the customer is a function of two factors: the amount at stake determined by the importance of the buying goals and the individual's feeling of subjective certainty. Especially, when deciding the usage of innovative and new products such as CBD oil, risks customers perceive may be higher than the usage of general products because of the high amount at stake and low subjective certainty that results from lack of both direct and indirect experiences with CBD oil.

Researchers and marketers are paying attention to CBD oil due to the ground-breaking effectiveness of CBD oil and legalization of its usage, while there is no investigation pertaining to how customers' perceived risk should be handled. Thus, this study first aims to examine what factors-based on product, brand/finn, and information sourcesinfluence perceived risks and how marketing strategies for each factor help to reduce perceived risks.

Second, when customers have high perceived risks, they are less likely to buy the product or brand (Aqueveque, 2006). However, some factors may moderate the negative relationship between perceived risks and the intention to buy. To answer these research questions, the data will be collected from U.S. customers. The survey based and experimental-based study will be developed to test the proposed hypotheses. As statistical techniques, this study will utilize structural equation modeling (SEM), PROCESS macro, and dominance analysis.

Anti-Cancer Potential of Fermented Deitary 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 shows 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.

Sexually Demorphic Effects of Cannabinoids on Cognitive and Reflexive Learning and Memory-Dependent Neuronal Network Activity in Mouse Hippocampus and Amygdala

Dr. Jeff Smith, Biology

The broad objectives of this proposed research are to quantitatively identify the degree to which electrical activity in specific neuronal populations that support fear learning and memory in the mammalian brain is targeted in a sexually dimorphic way by the cannabinoids; Dexanabinol, a synthetic cannabinoid used in human clinical trials for tumors and traumatic brain injury, and Cannabidiol, a widely available cannabinoid that is used to treat inflammation and childhood epilepsy.

The goal of this project is to characterize discreet anatomical and physiological targets of these drugs that will provide insight into the mechanism by which they exert known effects on fear learning and memory, and to show how these effects are divergent with gender.

The specific aims are I, to determine whether the learning-dependent density of excitatory and inhibitory neural activity within the hippocampus and amygdala is sexually dimorphic, whether Dexanabinol or Cannabidiol affect the activity patterns during acquisition of cognitive vs. reflexive memory, and whether gender influences any observed drug effects, and II, to identify gender specific variability in the effects of cannabinoids on markers of intrinsic plasticity within individual neurons that are active in processing different fear memory types which are more or less reliant on cognitive processing.

This work is significant because the information provided will support future informed decision making for safe prescription, or counter indication, of these prominent cannabinoids to individual consumers, male and female.

Data Analytics in Cannabis Research

Dr. Kuang-Yuan Huang, CIS

There are two goals of the proposed research. First, we will continue our ICR-funded research on analyzing the big data collected from a large cannabis online support forum. Specifically, the goal is to analyze the social network data constructed from the social interaction history among cannabis quitters in the online support forum. Since there are several hundreds of thousands of discussion threads on the forum, the resulting data will be large in volume and contains useful social information for analysis.

These findings will have impacts both on research and practice, in that they provide understandings of online social support seeking and provision behaviors of cannabis quitters, and that they provide guidance on the design of online cannabis support forums conducive of social support exchange. The second goal is to create a practical mobile application to assist cannabis rehabilitation based on our research findings from FY18.

The research in FY18 studied the inter-relationships between online support seekers’ emotion states and the social support they received from other members of an online cannabis support forum. The results of FY18-19 can be included into the design of a mobile application to facilitate cannabis rehabilitation. This application will be equipped with expert knowledge regarding different social support types appropriate for a given difficulty experienced by cannabis quitters. We are planning to design, develop, and demonstrate the online/mobile app to be used by cannabis users for further data collection.

Cannabis Use Survey Instrument Development and Implementation

Dr. Karen Yescavage, Psychology

The objective of this project is to engage in survey instrument development, assessing its psychometric properties, in order to build an empirical cannabis knowledge base. Once survey is both reliable and valid, objective is to research the reasons why people use and how they use cannabis, both medicinally and recreationally. Specific aims are two-fold: (1) to assess to what extent, if at all, cannabis is being used in place of other types of drugs or alcohol (and/or reasons why cannabis is not being used as a substitute), and (2) to explore any trends in cannabis use (e.g., strength and strain type) as a function of reason (e.g., insomnia, social anxiety).

Applications of Industrial Hemp (Multi-Year Project)

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?

Rheological characteristics of hemp-based filament composites for 3D printing (Multi-Year Project)

Dr. Neb Jaksic, Engineering 

This research investigates flow characteristics of various hemp-based composites used in 3D printing. It is an expansion to the research already in progress. While the current research is based on the goal of optimizing the hemp-plastic ratio in filament composites, this proposed research emphasizes understanding of underlying principles and it should further help in material optimization. This application seeks funding mainly for an instrument necessary for this research. We are also seeking limited funding for faculty and students compensation. In particular, the funding is requested for one rheometer, two faculty members (one from Engineering and one from Chemistry), two undergraduate students, and one graduate student.

3D printing is a prototyping and manufacturing process where molten plastic filament is extruded from a nozzle creating a layer of a 3D object. A number of these layers are fused together to create 3D objects. The process may create complicated (or personalized) objects and assemblies that cannot be created by any other manufacturing method.

To minimize the cost of filament material and to increase the strength of the created objects, different reinforcing agents can be added to the filament composite. We are studying hemp as a reinforcing agent because hemp is stronger than plastic and is less expensive. However, if the hemp particles in the filament are too large they can clog the 3D printing nozzle and therefore stop the process. This results in wasted material and time (time to clear the nozzle and time to re-print the object). If the hemp particles are too small, then the created objects are not as strong as they could be.

In addition, we are considering recyclability of 3D printed objects. Namely, the process of creating new filament by directly recycling 3D printed parts does not yield useful filament – such filament clogs 3D printers’ nozzles. This research aims to characterize hemp-based material before and after 3D printing from the “viscosity” point of view.

This research will provide the following analyses of polymers and hemp-based composites used in 3D printers: 1) Rheological analysis (viscosity) of molten (varied temperatures) filament before and after 3D printing using HempBioPlastic (HBP@), hemp-PLA, hemp-ABS, hemp-Nylon, and wood-PLA; and 2) Polymer analysis (structure and configuration) of HempBioPlastic, hemp-PLA, hemp-ABS, hemp-Nylon, and wood-PLA liquid solutions using Liquid Chromatography (LC) methods with appropriate detectors. We will verify our results by using instruments from engineering and chemistry department. We plan to submit abstracts for the ICR Conference (2019 and 2020), present our results at the ICR conference, and submit two manuscripts for the Journal of Cannabis Research (2019 and 2020).

An ongoing investigation into the effects of medicinal cannabis on seizures in adults with medically refractory epilepsy (Multi-Year Project)

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.

Identification And Characterization of Hemp Components That Control Abnormal Proliferation of Human Rheumatoid Arthritis Fibroblast-like Synoviocytes And Triple-negative Breast Cancer Cells (Partnership Project) 

Dr. Sang Hyuck Park, Ms.Kyungho Lee 

Both Rheumatoid arthritis (RA) and triple-negative breast cancer (TNBC) are characterized by abnormal proliferation and an association with an affected immune response. Abnormal proliferation and inflammation disrupt endoplasmic reticulum (ER) homeostasis and induce the unfolded protein response (UPR). The UPR is associated with the pathogenesis of various human diseases including autoimmune diseases, neurodegenerative disease, and diverse cancers. Therefore, the UPR could be served as a favorable therapeutic target for the treatment of RA and cancer.

Previously, we have shown that hempseed oil and DHA, one of the polyunsaturated fatty acids, showed the anti-rheumatoid and anti-cancer effects in association with the UPR pathway. Therefore, it is highly possible to find hemp components that control abnormal proliferation of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) and cancer cells under inflammatory conditions.

Therefore, in this research, we propose to identify hemp components that control abnormal proliferation of human RA-FLSs and TNBC cells and investigate underlying mechanisms, focused on the UPR.

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.

How Does CBD Modulate Learning and Memory in Humans? (Partnership Project) 

Dr. Moussa Diawara, Dr. Jeff Smith, Dr. Libby Stuty, Ms. Amy Uhernik

Cannabidiol (CBD) is a form of cannabinoid that lacks the psychoactive (hight) effect associated with Tetrahydrocannabinol (THC), the primary cannabinoid found in cannabis. Research findings point to CBD as a possible memory-enhancing agent that could be used to treat cognitive disorders of L&M whereas treatment of PTSD might not be an appropriate, or neurodegenerative disease of L&M like Alzheimer's.

We hypothesize that CBD is a modulator of human L&M. Our broad objective is to examine the potential effect of CBD on human L&M in relatively large scale surveys and in isolation from other confounding factors (i.e., other drugs being specifically combined with CBD or particular preexisting medical conditions). We specifically aim to determine whether CBD, when administered alone to healthy human subjects is a modulator of human L&M, and aim to determine whether particular demographic factors present in the general population (e.g., starting age of cannabis use, race, gender, smoking, mental health status, SSRI (or other drug use) will affect the modulatory effect of CBD on L&M. 

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