Cannabis sativa research trends, challenges, and new-age perspectives

Tajammul Hussain, Ganga Jeena, Thanet Pitakbut, Nikolay Vasilev, and Oliver Kayser

SUMMARY

Cannabis sativa L. has been one of the oldest medicinal plants cultivated for 10,000 years for several agricultural and industrial applications. However, the plant became controversial owing to some psychoactive components that have adverse effects on human health. In this review, we analyzed the trends in cannabis research for the past two centuries. We discussed the historical transitions of cannabis from the category of herbal medicine to an illicit drug and back to a medicinal product post-legalization. In addition, we address the new- age application of immuno-suppressive and anti-inflammatory extracts for the treatment of COVID-19 inflammation. We further address the influence of the legal aspects of cannabis cultivation for medicinal, pharmaceutical, and biotech- nological research. We reviewed the up-to-date cannabis genomic resources and advanced technologies for their potential application in genomic-based cannabis improvement. Overall, this review discusses the diverse aspects of cannabis research developments ranging from traditional use as herbal medicine to the latest potential in COVID-19, legal practices with updated patent status, and current state of art genetic and genomic tools reshaping cannabis biotech-nology in modern age agriculture and pharmaceutical industry.

INTRODUCTION

Cannabis sativa L. is one of the earliest known cultivated plants since agricultural farming started around 10,000 years ago (Schultes et al., 1974). It is a multi-purpose crop plant with diverse agricultural and industrial applications ranging from the production of paper, wood, and fiber, to potential use in the medicinal and pharmaceutical industries. The first-ever report to reveal the prospects of C. sativa L. as a medicinal plant was already published in 1843 and described the use of plant extracts to treat patients suffering from tetanus, hydrophobia, and cholera (O’Shaughnessy, 1843). However, the first chemical constituent identified was oxy-cannabis (1869) (Bolas and Francis, 1869), isolated cannabinoid (1896), and fully identi- fied in 1940 was cannabidiol (CBD) (Jacob and Todd, 1940) followed by tetrahydrocannabinol (THC) (Gaoni and Mechoulam, 1964; Santavy ́ , 1964) and cannabigerol (CBG) in 1964, and cannabichromene (CBC) in 1966 (Gaoni and Mechoulam, 1966). Identification of THC later led to an understanding of the endocannabinoid system followed by the discovery of the first cannabinoid receptor (CB1) in 1988 (Devane et al., 1988; Russo, 2016). CB1 receptor acts as a homeostatic regulator of neurotransmitters for pain relief mechanisms, but the same mode of action was responsible for intoxicating effects from cannabinoids’ excessive use. Thus, the understanding of mode of action of CB1 receptor raised concerns about the adverse effects of cannabis use. Consequently, the plant was removed from the medicinal category and recategorized exclu- sively to the category of drug-type plants.

Cultivation and use of cannabis plants for recreational, medical, and industrial use were strictly banned and severely limited the scientific research in the field. Owing to strict legal regulations, the plant remained unexplored for its incredible potential in drug discovery for an extended period until it was legalized for medical use first in California and later in many countries around the globe. Extensive research followed legalization to explore the chemodiversity of cannabinoids for potential clinical value. In total, more than one thousand compounds—278 cannabinoids, 174 terpenes, 221 terpenoids, 19 flavonoids, 63 flavo- noid glycosides, 46 polyphenols, 92 steroids—have been identified (ElSohly and Slade, 2005; Gould, 2015; Radwan et al., 2017). Nearly 278 of these compounds are cannabinoids and classified as phytocannabinoids (plant-based) to distinguish them from endocannabinoids (non-plant). Cannabimimetic drugs binding to CB1-receptors in the endocannabinoid system can also be found in algae, bryophytes, and monilophytes (Carvalho, 2017; Kumar et al., 2019). The major cannabinoids in cannabis include THC, CBD, and CBC, their precursor CBG and cannabinol (CBN) (Flores-Sanchez and Verpoorte, 2008). To date, 10 CBN-type, 17 CBG-type, 8 CBD-type, and 18 THC-type cannabinoids have been isolated (Gaoni and Mechoulam, 1964). Cannabigerolic acid (CBGA), a CBG-type cannabinoid, is the central precursor for the biosynthesis of psychoactive THC, non-psychoactive CBD, and CBC (ElSohly and Slade, 2005; Gould, 2015; Radwan et al., 2017).

Cannabinoid biosynthesis in plants occurs in specialized biosynthetic organs called glandular trichomes (Happyana et al., 2013) on female flowers and leaves. Several studies use metabolic profiling of trichomes to demonstrate variation in trichome size, density, and relative concentration of cannabinoids (Happyana et al., 2013; Small and Naraine, 2016). However, the genetic mechanisms underlying the developmental changes in trichomes and consecutive cannabinoid content are still unknown. Apart from natural and chemical biosynthesis methods (Bovens et al., 2009), heterologous biosynthesis of cannabinoids has also been reported (Luo et al., 2019). However, the considerable amount of side products is still one of the major bottlenecks (Luo et al., 2019; Thomas et al., 2020) in cannabinoid production.

This review highlights the latest research developments and challenges in cannabis plant sciences, the role of trichomes as biosynthetic sites, with a special focus on plant biology. In addition, we discuss the existing legal practices with patent information for the C. sativa L. We also discuss the new potential use of canna- binoids for COVID-19 treatment. Finally, we address the available genomic and transcriptomic resources and discuss their potential toward the genetic improvement of cannabis. Overall, we provide the first in- depth review of diverse aspects of C. sativa L. from traditional medicinal use to genomics insights and research perspective to broad industrial applications.

CANNABIS RECORDS IN BIBLIOGRAPHIC DATABASES

Cannabis-related publications were searched in four major scientific literature and citation databases of biomed- ical and life-sciences journals: the EuropePMC (https://europepmc.org/) by EMBL-EBI (Data S1), Elsevier’s Sco- pus (https://www.scopus.com/) (Data S2), PubMed Central at NCBI (NCBI PMC: https://www.ncbi.nlm.nih.gov/ pmc/) (Data S3), and Web of Science (WoS: https://www.webofscience.com/wos/) of Clarivate Analytics. The search criteria—‘‘cannabis OR marijuana OR hemp OR cannabinoids OR cannabidiol OR cannabinol’’ were used to examine available research articles. Nearly 80,979, 64,637, 43,182, 28,759 cannabis-related research arti- cles were found in EuropePMC, Scopus, WoS, and NCBI PMC, respectively. The sheer difference in the number of articles could be attributed to the years for which the Cannabis records are present in the databases. Europe PMC currently holds cannabis records for 239 years since the oldest publication in 1783. Whereas, Scopus has data for 194 years (since 1828), NCBI PMC 182 years (since 1840), and WoS only 77 years (since 1945) (Figure 1A). Despite cannabis records for only 77 years WoS records exceed NCBI PMC, owing to the data acquisition policy similar to Scopus, wherein all the cited references for a publication are pulled and listed in the database. Another major reason for the different records in the archives could be owing to the source repositories and partner journals. Although NCBI PMC has only 6.9 million articles from over 10,656 journals by April 2021, Scopus has more than 77.8 million records from nearly 23,500 journals, and WoS comprises over 171 million records including jour- nals, books, and proceedings. However, EuropePMC acquires data from multiple bibliographic repositories such as PubMed, MEDLINE (MED), PMC, AGRICOLA (AGR: AGRICultural OnLine Access), and Chinese Biological Abstracts (CBA) (Figure 1D). It includes more than 45,6 million documents including articles, books, preprints, pat- ents, conference papers, and microPublications. Cannabis citation metadata was publicly available for bulk down- load from EuropePMC, Scopus, and NCBI PMC from 6586, 8647, 3864 journals, respectively (Figure 1B). Among the article identifiers such as DOI, PMCID, and PMID, DOI was found for 85.62% records of EuropePMC, 85.44% of Scopus, and 91.9% of NCBI PMC. Since DOI was the only common identifier, it was used for the comparison of three datasets (Figure 1C). Cannabis records in EuropePMC comprised nearly 76.73% of NCBI PMC and 49.75% of Scopus data (Figure 1C). Hence, metadata from EuropePMC was selected for downstream bibliometric anal- ysis. Majority of Cannabis records in EuropePMC were from MEDLINE (94.94%), followed by 4.29% from PMC, only 0.75% from Agricola (AGR), and 0.02% from CBA (Figure 1D). The distribution of source databases indicates the most explored field in Cannabis research for the last 239 years.

TRENDS OF CANNABIS RESEARCH FROM 1783 TO 2021

C. sativa L. originated in central Asia and later spread to Europe during its cultivation for diverse applications. Archaeological evidence of early medical use was found in fossil records dating back to 315–392AD (Zias et al., 1993). There is a consensus that the plant has been used as traditional medicine (Bridgeman and Abazia, 2017). Based on the research during the past two centuries, we divide the scientific era into four periods (Figure 1E). The period zero (1783–1840) marked the first-ever mention of Cannabis in the category of medicinal plants in the years 1783 (Laurentius Crellius and Huntero, 1783) and 1787 (Wright et al., 1787). There were only 52 articles and 38 reviews in the next five decades (source: EuropePMC). Most reports mentioned the botanical aspects of hemp, the quality of fiber, and few observations about its use in traditional medicines. The first period (1840–1937) started with the detailed evidence-based report of chemical properties and medicinal potential of Cannabis indica (hemp) by William O’Shaughnessy (O’Shaughnessy, 1843) followed by an array of medicinal reports in 1923 articles and 183 reviews in the next 96 years.

Scientific endeavors to experiment, observe, and understand the diverse medicinal applications of cannabis were still in the early stages. However, 1900s witnessed a series of legal regulation in the direction of the criminalization of cannabis. Cannabis was starting to be categorized into the list of narcotic drugs and Poisons Rules including the Pure Food and Drug Act (1906) pushed for stricter measures for cannabis distribution. Later International opium Convention (1925) called for measures to regulate Indian hemp. Ex- ports unless exclusively for medical or scientific purposes or European hemp (for fiber) were banned. Uni- form State Narcotic Drug Act (1925), Geneva Trafficking Conventions (1936) resulted in criminalizing the cultivation, possession, manufacture, and distribution of cannabis derivatives. Marihuana Tax Act (1937) levied heavy taxes on the possession and selling of cannabis, excluding medical, and industrial use. As a consequence, the cultivation and procurement of cannabis for research purpose became increasingly diffi- cult and severely limited the research of medicinal cannabis during this era (Figure 1E: Period I). During the second period (1937–1996), cannabis research suffered major restrictions owing to legal regulations in the first two decades until the identification of the first cannabinoid—cannabidiolic acid in 1954 (Hanus et al., 1975; Krejcı ́ and Santavy ́ , 1955; Krejcı ́ et al., 1958; Santavy ́ , 1964), isolation of the most psychoactive compo- nent of cannabis, the THC in 1964 (Mechoulam et al., 1964). Isolation of the THC, discovery of CB1 (Devane et al., 1988), and CB2 (Munro et al., 1993) receptors, followed by the Compassionate Investigational New Drug program (1978) paved the way for decriminalization laws. The discovery of endocannabinoid and the role of cannabis in the medicinal field have been reviewed in (Hanus, 2009; Kabelik and Santavy, 1955) As a consequence a steep surge was observed in the number of cannabis-related articles from 445 articles and 25 reviews during 1937–1964 to nearly 8,888 articles and 773 reviews during 1964–1996 (Figure 1E: Period II), although with a short period of decline between 1973 and 1982. Finally, the third period (1996-till date) began with the historical Compassionate Use Act of 1996 in California approving medical cannabis. Post- legalization (1996 onwards), cannabis has been extensively explored for its diverse potential in the pharma- ceutical and medicinal industries. During the third period, cannabis research witnessed an unprecedented growth with nearly 67,777 articles, 13,202 reviews, and 493 preprints (source: EuropePMC), of which 97.01% articles were published in the last two decades since 2000 (Figure 1E:period III. Approval of the first cannabis-based inhaler spray in 2005 (Perras, 2005; Pain, 2015) and the first draft of the cannabis genome in 2011 (van Bakel et al., 2011) in this era were the two major accomplishments that exponentially acceler- ated the research development.

The trends of cannabis study in the diverse array of research articles and journals indicate the core interests of the scientific community. To further investigate the most researched field, the journals of cannabis arti- cles were categorized into scientific and social areas. The journals related to social, law, and policy-based studies were merged into the subject category of social research. Although the majority of broad scientific subjects were grouped into the following seven major categories: (i) medicinal (all medical and medicinal subjects), (ii) pharmaceutical-comprised of pharmacology, pharmaceuticals, drug, toxicology, and chemi- cal studies, (iii) neurosciences-comprised of neurological, brain-related, psychiatry, psychology, and cognitive studies, (iv) biochemistry-included biotechnology, microbiology, immunology, virology, and biochemistry, (v) genomics-grouped genetical and genomic studies, (vi) plant biology-included plant sciences, agricultural, botanical aspects, plant-pathogen and environment studies, and lastly, (vii) bioinfor- matics (includes data analytics). Journals that could not be classified into either of the aforementioned cat- egories or social research categories were excluded from downstream evaluation. The Scientific subject areas (74.47% of journals) were further compared for the corresponding number of articles and journals (Figure 1F). A distinct pattern was observed for the Clinical aspects of cannabis which remained a major focus since the very beginning with nearly 94.76% published articles including 64.51% articles in medicinal subject areas, 19.55% in pharmaceutical sciences, and 10.70% in neurosciences. In contrast, plant biology and agricultural sciences comprised only 2.62% of articles, followed by 0.71% genomics, and 0.07% bioinformatics-based cannabis research. Genomics and bioinformatics are relatively new sub- jects growing at a fast pace since the release of the first Cannabis draft genome in 2011 together. Recent advances in sequencing technologies have further propelled genomic and transcriptomic studies with the purpose of dissecting the regulatory networks. The growth of genomic data in public space has met with the fast-paced development of bioinformatics tools for data analysis. In addition, ongoing developments of machine-learning (ML) and artificial intelligence (AI)-based genomic tools will facilitate genetic-level un- derstanding of cannabis metabolism for the selective breeding of genetically modified cannabis with improved metabolic traits.

CANNABIS SATIVA L. PHYSIOLOGY AND LEGAL STATUS

Physiological, morphological, and developmental aspects of Cannabis are key in understanding the plant growth patterns and chemical profiles. However, plant growth and function are substantially influenced by abiotic factors and nutrient availability. Although botanical aspects (Farag and Kayser, 2017), plant archi- tecture, and florogenesis of female C. sativa plants (Spitzer-Rimon et al., 2019) with detailed trichome morphogenesis (Hammond and Mahlberg, 1977) provided crucial insight into plant biology. However, it also became increasingly important to determine the effect of abiotic factors on Cannabis growth and chemical yield, especially for large-scale commercial breeding programs. Hence, in-depth analysis of the effect of soil fertilization, salinity, temperature, and light conditions, as well as nutrient and water- use efficiency is key in establishing industrial-scale systems for the cultivation of hemp and marijuana vari- eties. The first available records about the mineral nutrition of hemp plants were published by Tibeau et al., in 1936 (Tibeau, 1936). Later in 1944, Clarence H Nelson published the effect of varying soil temperature on hemp growth (Nelson, 1944). The first publication with a detailed response of greenhouse cultivated cannabis to nitrogen (N), phosphorus (P), and potassium (K) was published in 1977 (Coffman and Gentner, 1977). Furthermore, two parallel reports by HMG et al., in 1995 discussed the impact of nitrogen fertilization on sex expression in hemp (van der Werf and van den Berg, 1995), and the effect of temperature on leaf and canopy formation (van der Werf et al., 1995). Importantly, most physiological studies in the second and third period (Figure 1A) were published for hemp with a focus on photosynthetic response and biomass yield with varying conditions such as temperature, water availability, nitrogen, and mineral nutrition (Ama- ducci et al., 2002; Aubin et al., 2015; Finnan and Burke, 2013; Papastylianou et al., 2018; Tang et al., 2017, 2018). However, the first study to assess the chemical response of hemp plants was published in 1997 (Bo ́ csa et al., 1997).

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