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Medicinal Cannabis: Ensuring Quality
Medicinal Cannabis: Ensuring Quality
Andrea L. Small-Howard, Ph.D.1
1GBSciences, Inc., Las Vegas, NV.

As therapeutic marijuana enters the medical mainstream, two major challenges face producers and consumers: (1) ensuring a consistent supply of marijuana with minimal day-to-day and batch-to-batch variation in the active secondary metabolites that underlie its therapeutic benefit, and (2) ensuring that this plant-based product is delivered to the consumer in a manner that is free from potentially harmful fungal or bacterial contaminants. Defined growth conditions with controlled nutrient media and environmental parameters offer attractive solutions to these linked problems. However, a potential tension between these two goals arises when we consider that the plant-associated microbiome may be symbiotic determinants of potency, efficacy and metabolite profile. A cultivation approach employing cutting edge botany, chemistry and microbiology will likely hold the solution to these challenges.

Keywords: Cannabis sativa, Cannabinoid, Microbiome, Contaminants
Plant Secondary Metabolite Profiles Are Highly Sensitive To Changes In Environment
By definition, secondary metabolites are complex chemical structures, produced by plants and microorganisms, that aid in growth and development of the organism but are not directly related to survival. Secondary metabolites play key roles in plant defense, and this multitude of carbon based structures are the product of the plant’s impressive abilities in organic synthesis. Humans have exploited the capacity of plants to perform complex syntheses for millennia, recognizing plant secondary metabolism as an important source of medicines, flavors to enhance food, color pigments, fragrances and recreational drugs. The production of secondary metabolites is highly energy intensive and relies on a plethora of enzyme-catalyzed synthetic pathways that are not yet fully defined. These synthetic pathways are sensitive to changes in the physical environment of the whole plant, including light (wavelength and intensity), water, salt, temperature, CO₂ and nutrient availability. These abiotic factors can dramatically influence secondary metabolite production over short timeframes. Stressors are not always detrimental to secondary metabolite production, and the effects can be complex in their strain- and
cultivar-specificity. For example, acute salt stress can actually increase anthocyanin production in roses, but in other species salt stress can diminish anthocyanin yield. All biological processes have a Q10 that links their efficiency to temperature, and the enzymes that catalyze secondary metabolite production are no exception. Small variations of 1-2ºC have been shown to dramatically and specifically compromise pigment production. Light and nutrient/mineral profiles are also critical regulators of secondary metabolite production. Light is a complex and dynamic environmental variable. Intensity, wavelength and circadian exposure rhythms will profoundly influence secondary metabolism. There is considerable subtlety to the relationships between secondary metabolism and light: the distinction between all incident light and photosynthetically active radiation (PAR) does not fully capture this relationship. For example, anthocyanin pigment synthesis is synergistically stimulated by UV in the 280 - 320nm range when combined with red light. Trace elements such as copper, zinc, cadmium and selenium are linked to secondary metabolism both through their electrochemical potential and through action as enzymatic cofactors. As an example, the cancer therapeutic taxol is a Taxus sp secondary metabolite, and its production is extremely sensitive to environmental fluctuations in the rare earth metal lanthanum, and to chromium.
It should be noted that there are very few studies looking at broad representation of the secondary metabolome and the effect of environmental stressors on the thousand of compounds in this metabolome rather than a few marker or bioindicator molecules. This next frontier is critical, in that most extant studies look at one or two metabolites, and compensatory or bystander effects on other metabolites (whether positive or negative) are not captured. In order to develop growth and nutrient conditions that are optimized for the production of commercially important combinations of secondary metabolites, a linkage between the environment and the complete metabolite ‘fingerprint’ needs to be developed.
Figure 1. Drugs, flavors and fragrances all derive from plant secondary metabolism.
The secondary metabolite profile of Cannabis sativa has been exploited by humans for millennia. At the level of plant ecology, cannabinoids provide the plant with antifungal defense, are involved in regulated necrosis that accompanies growth and development, and confer a sticky exudate on leaves that waylays insets involved in fertilization. Several hundred components, comprising the major cannabinoids and numerous ‘entourage’ compounds, exert complex and wide-ranging effects on human physiology. These include the alteration of mood, moderation of pain, impact on memory and fear conditioning, control of appetite and immunomodulation. These physiological effects reflect the cellular and tissue distributions of the cannabinoid receptors (CB1 and CB2, and the ionotropic TRP cation channels), and the actions of the body’s natural endo-cannabinoids. The medicinal potential of plant cannabinoids and associated secondary metabolites derives from these same receptor distributions, and the relationship of the physiological process that are cannabinoiddependent to numerous pathophysiological and disease states.
Figure 2. Plant secondary metabolites of the cannabinoid family from Cannabis spp.
Contemporary approaches to medicinal use of marijuana seek to leverage the full potential of the plant’s chemical synthesis machinery rather than reducing the naturally occurring pharmacology to a single drug molecule. Cannabinoids do not act on the body’s systems in isolation; rather they are co-operative and synergistic with entourage compounds. Thus any therapeutic approach that reduces this complexity risks the loss of efficacy and potency. With this cutting-edge focus on complex mixtures (e.g., cannabis oils), two issues become of paramount importance: (1) ensuring day-to-day and batch-to-batch consistency of the relative proportions of cannabinoids and entourage molecules in the extracted mixtures, and (2) understanding fully the strain- and cultivar-specific nature of these profiles and matching disease targets with effective metabolite fingerprints.  The 
former relies strongly upon control of the plant’s physicochemical environment during growth. The exquisite sensitivity of secondary metabolism to environment makes the achievement of this consistency far more challenging than simply ensuring consistent chemical extraction. Thus a central challenge in the medical marijuana industry is to develop growth conditions that ensure, and predict, efficacy. Overcoming this challenge will represent an inflexion point in the successful application of the naturally occurring pharmacopeia contained within Cannabis sativa to a plethora of human disorders.
Cultivated Marijuana Can Harbor Microbial Contaminants And Other Hazards To Human Health
As an organic material, marijuana that is destined for human ingestion or inhalation, or products derived from
it, are subject to microbial infestation and degradation. Post-harvesting, the lengthy curing process, extraction, storage and distribution may all be opportunities for establishment of new microbial colonies or expansion of those that naturally occur. In addition to being economically detrimental, there are very real possibilities for detrimental effects of these contaminations on human health.

Viral contaminants that have potentially detrimental effects on human health have been detected in commercially supplied marijuana. Likely deriving from water supply or human handling. Hepatitis A and B have been detected in marijuana and the latter was notably linked in one case to cultivation using human feces as a fertilizer, highlighting the potential consequences of an unregulated supply. Bacteria, and the toxins they can produce, contaminate marijuana during growth, after harvesting and during curing. These include Salmonella, Klebsiella, Streptococcus and Enterobactericidae. Moulds (Aspergillus, Penicillium and Fusarium spp have been isolated from marijuana. These, and fungi such as Alternaria alternata and Curvularia lunata, are of particular concern for immunocompromized patients (e.g., AIDS sufferers, the elderly). Histoplasmosis has also been reported in association with marijuana.
Figure 3. Bacteria, viruses and fungi (molds) are classes of pathogen that can be harbored in marijuana products.
These contaminants, as well as pests, pesticides and other chemical factors that can affect human health create a pressing public health problem in both the illicit and licit marijuana production industries. In the latter, the development of controlled, clean growing and curing environments is of paramount importance as numbers of marijuana users and suppliers rise.
Marijuana-Associated Microbiomes May Determine Organism Success And Secondary Metabolite Production.
The abiotic environmental factors discussed above are not the sole influencers of plant secondary metabolism. There is growing evidence that the plant’s associated microbiome may play a role in determining secondary metabolite profile and production. Plants bear a complex leaf and stem microbiome, and specialized compartments such as flowers and nodes may exhibit even more specific microbial associations. All land plants associate with a soil microbiome, with a bidirectional
influence of the host plant upon its microbiosphere, and the soil microbiome upon plant physiology and responses. Root physiology and metabolism affect the microbiome in soil through influence on soil acidity (via root metabolism and oxidative phosphorylation) and O₂ levels. Plants also produce messenger molecules that influence microbial health and diversity (community structure) such as anti-microbial (defense) peptides and molecules that affect quorum sensing by the microbial population. Root-derived carbon, both from decomposition and secreted chemical that contain energy- rich carbon-carbon bonds, provides vital energy that again drives complexity and extent of the associated microbial population. Conversely, the microbiome is a major determinant of plant health and success. As mutualists, these microbes provide nutrients, provide resistivity towards environmental stressors (such as soil acidification, chemical stressors, water limitations) and they synthesize hormones and messengers that regulate growth and productivity.

There is an important distinction, but some likely overlap, between the microbes that ‘contaminate’ medicinal marijuana and have the potential to become pathogenic or problematic for humans, and the microbes that form the normal biota associated with the plant and its growing medium. Microorganisms are essential for plant processes such as growth and defense, and either through a regulatory influence on plant cell metabolism or through their own secondary metabolite profiles, may contribute chemically to the complexity and efficacy of ingested marijuana. The symbiotic and synergistic relationship between the plant and its microbes is a bond that we cannot break lightly: there may be adverse consequences for the success of the plant and for its therapeutically useful signature of secondary metabolites. Conversely, the associated microorganisms may offer their own benefits to the therapeutic goal. A simplistic example of the latter is the 1970’s fad for allowing marijuana to become moldy before recreational use – with fungal metabolites or decomposition associated with marijuana chemistry, desirably increasing potency and psychoactivity. In a more contemporary and rigorous study, Winston presented a seminal description of the microbial communities associated with five distinct cultivars of Cannabis sativa. Three strains were compared in two different soil context. Winston concluded that soil
is the major driver of microbial community composition (biodiversity) but community structure (relative abundance of different microbes) was determined by the plant type. Soil and rhizosphere microbiomes varied less between one another than the endorrhizal microorganisms. Cannabinoid diversity and composition was significantly correlated to the structure of the endorrhizal communities, but the study was unable to dissociate this effect from soil chemistry.

The Winston study raises the intriguing idea that, with further research under conditions that normalize soil chemistry, the effects of associated microbiomes upon a strain’s cannabinoid fingerprint could emerge. More intriguingly, the possibility of manipulating the microbiome to drive desirable properties may join the manipulation of the physical and chemical attributes of the growing medium as an approach to engineering disease-specific strains of cannabis
GrowBLOX™ Defined Growth Systems: An Opportunity For Process Research And Consistent, Safe, Uncontaminated Production Of Medicinal Marijuana
The GrowBLOX™ Controlled Environment Agricultural Chamber (“GrowBLOX™”) is specifically designed to produce optimal environmental growing conditions for medical cannabis cultivation. It is the first chamber of its kind with the ability to monitor and control the growth process in order to produce high-grade medicinal marijuana. The cutting-edge Growblox™ hydroponic technology allows for continuous, healthy cycles of cannabis plants that will reach optimized medicinal potency during the bloom cycle. Each fully enclosed chamber physically isolates each plant to protect from the onset and spread of insect and mite infestations in the grow operation and keep harmful contaminants, such as pesticides, insecticides, and vehicle exhaust, out of the medicinal plants growing inside.

This uniquely controlled growing environment offers the potential to control environmental parameters (light spectrum and intensity, humidity, nutrition, trace element levels, temperature and aeration). The advantage of the GrowBLOX™ chambers is the ability to
experiment and accurately replicate a wide range of environmental conditions and nutrient combinations. By utilizing the GrowBLOX™ chambers, scientists are able to maximize the efficacy and potency of specific cannabis strains. The cultivation parameters can be varied from chamber to chamber, making it possible to test a variety of growing environments more effectively and allows for customization of program settings for each cultivar or strain. All environmental parameters in the chambers can be pre-programmed for each unit and
customized to achieve the desired potency for each strain. This can ensure that the composition and yield from each batch will reflect the genetic makeup of the plants consistently for every harvest. Similarly, CureBLOX™ work in parallel with the GrowBLOX™ technology to ensure chemical consistency and to minimize the potential for microbial contamination during production.
Figure 4. The GrowBLOX™ system for safe, consistent production of medical grade cannabis therapies.
See www.GBSciences.com.
Complex diseases such as Parkinson’s, cancer, diabetes and heart disease require complex solutions. Medicinal marijuana offers these solutions but presents significant challenges in ensuring consistent and uncontaminated production of efficacious multi-compound mixtures for human therapy. Cultivation methodologies such as the GrowBLOX™ system will realize the full potential of medicinal marijuana available to patients in a safe and consistent manner.
Ramakrishna, A., Ravishankar G.A.2011. Influence of abiotic stress signals on secondary metabolites in plants Plant Signal Behav. 6(11): 1720–1731.

Mahajan S, Tuteja N (2005). Cold, salinity and drought . stresses: an overview. Arch Biochem Biophys. 444(2):139-58.

Parida AK, Das AB. 2005. Salt tolerance and salinity . effects on plants: a review. Ecotoxicol Environ Saf. 60(3):324-49

Zhong J, Yoshida M, Fujiyama D, Seki T, Yoshida T. 1993. Enhancement of anthocyanin production by Perilla frutescens cells in a stirred bioreactor with internal light irradiation. J Ferment Bioeng. 75(4):299- 303.

Bossche H.V., et al., 1990. Mycoses in AIDS patients. Plenum Press, NY. 337 pp

Chusid M.J., J.A. Gelfand, C. Nutter & A.S. Fauci. 1975. Pulmonary aspergillosis, inhalation of contaminated marijuana smoke, and chronic granulomatous disease. Ann. Intern. Med. 82: 61-64

McPartland J.M. 1992. The Cannabis pathogen project: report of the second five- year plan. Mycological Society of America Newsletter 43(1): 43.

McPartland J.M. 1991. Common names for diseases of Cannabis sativa L. Plant Disease 75: 226-227.
McPartland J.M. 1983. Fungal pathogens of Cannabis sativa in Illinois. Phytopathology 72: 797.

RamÌrez J. 1990. Acute pulmonary histoplasmosis: newly recognized hazard of marijuana hunters. American Journal Medicine 88 (Supplement 5): 60N-62N.

Schwartz I.R., 1987. Fungal sinusitis and marijuana. JAMA 257: 2914-2915.
Schwartz I.S., 1992. Non-Aspergillus sinusitus and marijuana use. Am. J. Clin. Path. 97: 601.

Schwartz I.S., 1985. Marijuana and fungal infection. Am. J. Clin. Path. 84: 256.
Taylor D.N. et al. 1982. Salmonellosis associated with marijuana. New England J. Med 306: 1249-1253

Taylor D.N. et al. 1982. Salmonellosis associated with marijuana. New England J. Med 306: 1249-1253

Ungerlerder J.T., T. Andrysiak, D.P. Tashkin and R.P. Gale, 1982. Contamination of marijuana cigarettes with pathogenic bacteria. Cancer Treatment Reports 66 (3): 589-590.

Max E. Winston, Jarrad Hampton-Marcell, Iratxe Zarraonaindia, Sarah M. Owens, Corrie S. Moreau, Jack A. Gilbert, Josh Hartsel, Suzanne J. Kennedy, S. M. Gibbons. 2014. Understanding Cultivar- Specificity and Soil Determinants of the Cannabis Microbiome. PLoS One. 9(6): e99641.

Ramakrishna, A., Ravishankar G.A.2011. Influence of abiotic stress signals on secondary metabolites in plants Plant Signal Behav. 6(11): 1720–1731.

Mahajan S, Tuteja N (2005). Cold, salinity and drought stresses: an overview. Arch Biochem Biophys. 444(2):139-58.

Parida AK, Das AB. 2005. Salt tolerance and salinity effects on plants: a review. Ecotoxicol Environ Saf. 60(3):324-49.

Zhong J, Yoshida M, Fujiyama D, Seki T, Yoshida T. 1993. Enhancement of anthocyanin production by Perilla frutescens cells in a stirred bioreactor with internal light irradiation. J Ferment Bioeng. 75(4):299- 303. 

Morimoto S, Tanaka Y, Sasaki K, Tanaka H, Fukamizu T, Shoyama Y, et al. Identification and characterization of cannabinoids that induce cell death through mitochondrial permeability transition in Cannabis leaf cells. J Biol Chem. 2007;282:20739–20751.

Sirikantaramas S, Taura F, Tanaka Y, Ishikawa Y, Morimoto S, Shoyama Y. Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity, is secreted into the storage cavity of the glandular trichomes. Plant Cell Physiol. 2005;46:1578–1582.

ElSohly HN, Turner CE, Clark AM, ElSohly MA. Synthesis and antimicrobial activities of certain cannabichromene and cannabigerol related compounds. J Pharm Sci. 1982;71:1319–1323

Shoyama Y, Sugawa C, Tanaka H, Morimoto S. Cannabinoids act as necrosis- inducing factors in Cannabis sativa. Plant Signal Behav. 2008;3:1111–1112