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Mississippi-Grown Cannabis sativa L.: Preliminary Observation on Chemical Definition of Phenotype and Variations in Tetrahydrocannabinol Content versus Age, Sex, and Plant Part
Abstract
Nine strains of Cannabis sativa L. (marijuana) were grown for research by the University of Mississippi. The seeds for these strains were obtained from Iowa, Minnesota, Mexico, Turkey, Italy, France, and Sweden. The cannabinoid content was determined using GLC, and the material was divided into two chemical phenotypes according to cannabinoid content. These phenotype categories are used to differentiate between drug-type and fiber-type Cannabis sativa. In addition, the ( - )-δ9−trans-tetrahydrocannabinol content was determined for both male and female plants, various plant parts, and a Turkish variety during various stages in its growth.
... Although genotype has a significant influence on phytocannabinoid production [26,[51][52][53], its concentration is highly dependent on biotic and abiotic factors [54]. The majority of agronomic research conducted under field conditions has focused mostly on fiber production and yield [55,56]. Information that has been published on hemp production allows some parallels to be drawn. ...
... In 1971, two research projects were developed to evaluate the concentrations of phytocannabinoids in different plant organs [25,26]. According Fetterman and collaborators, all the hemp plant parts contained phytocannabinoids, and they showed that the decrease in THC content in different parts of the plants followed the order of bracts, flowers, leaves, smaller stems, larger stems, roots, and seeds [26], with similar results in other works [27,113]. ...
... In 1971, two research projects were developed to evaluate the concentrations of phytocannabinoids in different plant organs [25,26]. According Fetterman and collaborators, all the hemp plant parts contained phytocannabinoids, and they showed that the decrease in THC content in different parts of the plants followed the order of bracts, flowers, leaves, smaller stems, larger stems, roots, and seeds [26], with similar results in other works [27,113]. Another study confirmed that bracts were the organ with the highest concentration of phytocannabinoids [52]. ...
The main characteristic of Cannabis sativa L. is the production of compounds of medicinal interest known as phytocannabinoids. Environmental factors and crop management practices are directly related to the yield of these compounds. Knowing how these factors influence the production of phytocannabinoids is essential to promote greater metabolite yield and stability. In this review, we aim to examine current cannabis agronomic research topics to identify the available information and the main gaps that need to be filled in future research. This paper introduces the importance of C. sativa L., approaching state-of-the-art research and evaluating the influence of crop management and environment conditions on yield and phytocannabinoid production, including (i) pruning; (ii) light and plant density; (iii) ontogeny; (iv) temperature, altitude, and CO2 concentration; (v) fertilization and substrate; and (vi) water availability, and presents concluding remarks to shed light on future directions.
... Cannabis contains over 400 compounds, including more than 60 cannabinoids, the main active molecules being tetrahydrocannabinol (delta-9-THC) and cannabidiol (CBD) (Pollastro et al. 2018). These two cannabinoids were often considered the only chemicals involved in the medicinal properties and psychoactive effects associated with cannabis, and remain the only ones screened when evaluating cultivar chemotypes (De Meijer et al. 2009;Fetterman et al. 1971;Hazekamp et al. 2016;Nie et al. 2019;UNODC 1968). However, increasing evidence supports the relevance of terpenes and terpenoids, molecules responsible for the flavour and scent of the plants, both as synergetic to cannabinoids and as active compounds by themselves (Henry 2017;Hillig 2004;Nuutinen 2018;Russo 2011). ...
... Terpenes are widely used as biochemical markers in chemosystematics studies to characterize plant samples due to the fact that they are under strong genetic control and relatively unaffected by environmental factors (Aizpurua-Olaizola et al. 2016;Casano et al. 2011;Hillig 2004). Cannabinoid content, on the other hand, can vary greatly among generations of the same strain, and also due to the sex, age and part of the plant (Fetterman et al. 1971;Hazekamp et al. 2016). ...
... In the first place, this approach requires technology for quantitative chemical analysis that is beyond the reach of many dispensaries and growers. Furthermore, variations in the concentration of cannabinoids are high even within the same "strain", depending on factors such as age, environmental conditions, generation, and geographical location (Casano et al. 2011;Fetterman et al. 1971;Nuutinen 2018;Russo 2011). Finally, the predictive value of chemotypes has been questioned in markets where consumers increasingly demand higher THC content (Freeman et al. 2019, Freeman et al. 2018Jikomes and Zoorob 2018;Smart et al. 2017). ...
Abstract Background Widespread commercialization of cannabis has led to the introduction of brand names based on users’ subjective experience of psychological effects and flavors, but this process has occurred in the absence of agreed standards. The objective of this work was to leverage information extracted from large databases to evaluate the consistency and validity of these subjective reports, and to determine their correlation with the reported cultivars and with estimates of their chemical composition (delta-9-THC, CBD, terpenes). Methods We analyzed a large publicly available dataset extracted from Leafly.com where users freely reported their experiences with cannabis cultivars, including different subjective effects and flavour associations. This analysis was complemented with information on the chemical composition of a subset of the cultivars extracted from Psilabs.org . The structure of this dataset was investigated using network analysis applied to the pairwise similarities between reported subjective effects and/or chemical compositions. Random forest classifiers were used to evaluate whether reports of flavours and subjective effects could identify the labelled species cultivar. We applied Natural Language Processing (NLP) tools to free narratives written by the users to validate the subjective effect and flavour tags. Finally, we explored the relationship between terpenoid content, cannabinoid composition and subjective reports in a subset of the cultivars. Results Machine learning classifiers distinguished between species tags given by “Cannabis sativa” and “Cannabis indica” based on the reported flavours: = 0.828 ± 0.002 (p
... The phytocannabinoid content and composition is different in female and male plants, and varies during plant development and among the different organs [2,[69][70][71]. For instance, the Δ 9 -THC content of drug-type cannabis leaves is more or less constant during development, while that of the bracts increases considerably during flowering [72]. ...
... Chemotaxonomic evaluation has led to the establishment of three major chemotypes: fiber-type chemovariants contain predominantly CBD, intermediate chemovariants have equal amounts of CBD and Δ 9 -THC, while the latter is the major compound in drug-type plants [2,71,72,[77][78][79]. In spite of quantitative variations observed in phytocannabinoid contents (e.g., [75]), it has been demonstrated that the chemotype is not influenced by sex or developmental age [70]. ...
... Furthermore, phytocannabinoids provide a unique chemical fingerprint for cannabis identification and can be unequivocally identified by several analytical methods [16,93]. These include high performance liquid chromatography (HPLC) [10,75,89,[94][95][96], gas-liquid chromatography (GLC) [72,75,[97][98][99][100] or gas chromatography (GC) [5,6,28,30,71,72,84] coupled with flame ionization or mass spectrometric (MS) detection. In addition to discrimination between the different cannabis species, cultivars, chemovariants and samples collected by forensic scientists on the basis of their phytocannabinoid composition, these data may even provide information about plant cultivation (indoor or outdoor) and about the country of origin [30]. ...
Background & objective:
Cannabis is one of the earliest cultivated plants. Cannabis of industrial utility and culinary value is generally termed as hemp. Conversely, cannabis that is bred for medical, spiritual and recreational purposes is called marijuana. The female marijuana plant produces a significant quantity of bio- and psychoactive phytocannabinoids, which regained the spotlight with the discovery of the endocannabinoid system of the animals in the early 90's. Nevertheless, marijuana is surrounded by controversies, debates and misconceptions related to its taxonomic classification, forensic identification, medical potential, legalization and its long-term health consequences.
Method:
In the first part, we provide an in-depth review of the botany and taxonomy of Cannabis. We then overview the biosynthesis of phytocannabinoids within the glandular trichomes with emphasis on the role of peculiar plastids in the production of the secreted material. We also compile the analytical methods used to determine the phytocannabinoid composition of glandular trichomes. In the second part, we revisit the psychobiology and molecular medicine of marijuana.
Results & conclusion:
We summarize our current knowledge on the recreational use of cannabis with respect to the modes of consumption, short-term effects, chronic health consequences and cannabis use disorder. Next, we overview the molecular targets of a dozen major and minor bioactive cannabinoids in the body. This helps us introduce the endocannabinoid system in an unprecedented detail: its up-todate molecular biology, pharmacology, physiology and medical significance, and beyond. In conclusion, we offer an unbiased survey about cannabis to help better weigh its medical value versus the associated risks.
... According to recent studies, genetics, growing conditions, manner of drying and storage, and methods of processing and extraction may affect the concentration and profile of pharmaceutically active ingredients derived from cannabis [12]. Within a specific cultivar, the ratio of THC and CBD remains consistent in both male and female plants [13] [14] as well as in leaves and flowers throughout vegetative growth and flowering stages [15] [16] [17]. ...
... However, the density of floral bracts and bracteoles that carry glandular trichomes, where cannabinoids and terpenes are biosynthesized and stored, is higher in female plants than in males [2]. Concentration also varies in different plant parts, decreasing in the order of inflorescences, leaves, stem, seeds, and roots [13] [18]. The ratio of THC to CBD is a qualitative trait and the total yield of THC plus CBD is a quantitative trait [19]. ...
Cannabis has attracted a new wave of research attention as an herbal medicine. To deliver compliant, uniform, and safe cannabis medicine, growers should optimize growing environments on a site-specific basis. Considering that environmental factors are interconnected, changes in a factor prompts adjustment of other factors. This paper reviews existing work that considers indoor growing conditions (light, temperature, CO2 concentration, humidity, growing media, and nutrient supply), management practices (irrigation, fertilization, pruning & training, and harvest timing), and post-harvest treatment (drying and storage) for cannabis indoor production.
... However there is no method to identify these phenotypes other than measuring the concentration of (-)-trans-∆ 9 -tetrahydrocannabinol. 16 According to the local law of the legal growth of industrial C. sativa the concentration of tetrahydrocannabinol in the dry matter has to be less than 0.2% in order to be considered as industrial C. sativa. 17 The study was carried out to establish the criteria of classification of chemical phenotype by measuring the ratio of cannabinols over the cannabidiols using gas-liquid chromatography. ...
... 17 The study was carried out to establish the criteria of classification of chemical phenotype by measuring the ratio of cannabinols over the cannabidiols using gas-liquid chromatography. 16,18 Earlier study used combined spectrophotometric method in UV-VIS-IR range for classification of chemical phenotype. 19 Another plant from Cannabaceae family is Humulus lupulus L. It is grown widely for beer brewing industry. ...
In this work, a comparative research of biologically active organic molecules in its natural environment using the terahertz (THz) time domain spectroscopy (TDS) and Fourier transform spectroscopy (FTS) systems is carried out. Absorption coefficient and refractive index of Nicotiana tabacum L. leaves containing nicotine, Cannabis sativa L. leaves containing tetrahydrocannabinol, and Humulus lupulus L. leaves containing α-acids, active organic molecules that obtain in natural environment, were measured in broad frequency range from 0.1 to 13.5 THz at room temperature. In the spectra of absorption coefficient the features were found to be unique for N. tabacum, D. melanoxylon, C. sativa and H. lupulus. Moreover, those features can be exploited for identification of C. sativa plant sex. The refractive index can be also used to characterize different species.
... From sequential harvesting studies on C. sativa chemotypes, it is evident that the concentration of cannabinoids in the flowers increases as the plant enters the full flowering phase (Barnicomparini et al., 1984;Vogelmann et al., 1988;Pacifico et al., 2008;Muntendam et al., 2012). The potency of representatives of the genus Cannabis is significantly influenced by the pollination (Fetterma et al., 1971;Fairbairn and Rowan, 1977;Mandolino et al., 2003), phytogeographic region (Hillig and Mahlberg, 2004), illumnation conditions (Mahlberg and Hemphill, 1983), UV-B radiation (Zhang and Bjorn, 2009), temepature and humidity, fertilization and the applied (in-vs. outdoor) breeding method (Latta and Easton, 1975;Staquet et al., 1978;Vanhove et al., 2011). ...
... In durg type strains, the most prominent and unique terpenoids are β-caryophyllene-epoxide (the compound sensed by the drug searching dogs) and m-mentha -1,8(9)-dien-5-ol (Stahl and Kunde, 1973;Russo, 2011), depicted in Figure 9. The level of terpenoid production in planta depends on the applied cultivation and breeding methods as well as the harvest time and the mode of processing (Brenneisen, 2007;Fischedick et al., 2010b). ...
Cannabis sativa L. (Cannabaceae) is the oldest known medicinal plant. For millennia, the plant has also been used for fibre and oil production.The most prominent feature of C. sativa is the psychoactive effect ascribed to its secondary metabolites, cannabinoids (mainly to tetrahydrocannabinol, THC). However, many other pharmacological properties of the aforementioned specialized compounds have been described. Currently, the demand for THC for various medical applications is substantial, while cultivation and breeding of Cannabis in most countries is strictly regulated and limited to serving research purposes and meeting therapeutic needs. Therefore, the hereby proposed and discussed production of THC using in vitro cultures could be a viable alternative. In the work presented here, in vitro organogenesis from callus cultures (undifferentiated plant cell masses grown on solid media) was successfully established, ultimately resulting in regeneration of the complete C. sativa plant. Further, production of THC as well as other important cannabinoids was achieved in cell suspension, hairy root and trichome cultures of Cannabis. The optimal combination of phytohormones, as applied to the B5 growth medium, fostering the development of meristemoids from callus cultures was: 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BA) and adenine hemisulfate salt (AS) in respective concentrations of 0.5, 5 and 40 mg/l. Concurrently, the most favourable augmentation protocols of the B5 medium for the induction and differentiation of shootlets (small plants with leaves but without roots) were: 0.5 mg/l of gibberelic acid (GA3) or 0.25 mg/l of thidiazouron (TDZ) and 3 mg/l of GA3 (8.5 ± 1.73 and 7.25 ±1.03 shootlets/callus, respectively). The subsequent root formation of shootlets was most prominent after supplementation with 1.5 mg/l of indole-3-acetic acid (IAA). In vitro acclimatized plants growing in Erlenmeyer flasks formed tetrahydrocannabinolic acid (THCA), cannabigerolic acid (CBGA) and cannabidiolic acid (CBDA), retrieved at respective concentrations of about 0.33, 0.45 and 157.1 mg/g fresh weight. In contrast, ex vitro acclimatized plants (grown hydroponically for 8 weeks) synthesized THCA, THC, CBGA, cannabigerol (CBG) and CBDA at corresponding concentrations of 1.54, 28.30, 6.0, 0.125 and 1121.4 mg/g fresh weight. The obtained results confirmed the generation of pharmacologically important cannabinoids; however, the biosynthetic abilities of the investigated cell and hairy root cultures did not provide sufficient levels of the valuable metabolites to warrant scaling-up of the proposed in vitro production platform.
... The plants were propagated from seed grown, and phenotypic observations for sex were collected as previously described (Faux et al. 2014). Subsequently, 8200 leaf samples were used for this analysis which includes the main 3 types of Cannabis (types 1, 2, and 3) from various related and unrelated lineages (Gray et al. 2016;Fetterman et al. 1971;Clarke and Merlin 2013;Small and Beckstead 1973a;Small and Beckstead 1973b Table 4) and collaborative research projects (Supplemental Table 2). The Y chromosome containing genome assemblies used within our analyses ...
Background
Cannabis sativa is a primarily dioecious angiosperm that exhibits sexual developmental plasticity. Developmental genes for staminate male flowers have yet to be elucidated; however, there are regions of male-associated DNA from Cannabis (MADC) that correlate with the formation of pollen producing staminate flowers. MADC2 is an example of a PCR-based genetic marker that has been shown to produce a 390-bp amplicon that correlates with the expression of male phenotypes. We demonstrate applications of a cost-effective high-throughput male genotyping assay and other genotyping applications of male identification in Cannabis sativa .
Methods
In this study, we assessed data from 8200 leaf samples analyzed for real-time quantitative polymerase chain reaction (qPCR) detection of MADC2 in a commercial testing application offered through Steep Hill Laboratories. Through validation, collaborative research projects, and follow-up retest analysis, we observed a > 98.5% accuracy of detection of MADC2 by qPCR. We also carried out assay development for high-resolution melting analysis (HRM), loop-mediated isothermal amplification (LAMP), and TwistDx recombinase amplification (RPA) assays using MADC2 for male identification.
Results
We demonstrate a robust high-throughput duplex TaqMan qPCR assay for identification of male-specific genomic signatures using a novel MADC2 qPCR probe. The qPCR cycle quotient (Cq) value representative of MADC2 detection in 3156 males and the detection of tissue control cannabinoid synthesis for 8200 samples and the absence of MADC2 detection in 5047 non-males demonstrate a robust high-throughput real-time genotyping assay for Cannabis . Furthermore, we also demonstrated the viability of using nearby regions to MADC2 with novel primers as alternative assays. Finally, we also show proof of concept of several additional commercially viable sex determination methodologies for Cannabis sativa .
Discussion
In industrial applications, males are desirable for their more rapid growth and higher quality fiber quality, as well as their ability to pollinate female plants and produce grain. In medicinal applications, female cultivars are more desirable for their ability to produce large amounts of secondary metabolites, specifically the cannabinoids, terpenes, and flavonoids that have various medicinal and recreational properties. In previous studies, traditional PCR and non-high-throughput methods have been reported for the detection of male cannabis, and in our study, we present multiple methodologies that can be carried out in high-throughput commercial cannabis testing.
Conclusion
With these markers developed for high-throughput testing assays, the Cannabis industry will be able to easily screen and select for the desired sex of a given cultivar depending on the application.
... Cannabis spp can be categorized into psychoactive species (drug phenotype) or nonpsychoactive species (fiber phenotype) depending on the (%delta-9-THC + %CBN)/ (%CBD) ratio; if the ratio exceeds one then the Cannabis is regarded as psychoactive species (drug phenotype), else it is considered as fiber phenotype. [1] The nonpsychoactive species of C. sativa L, known as industrial hemp (THC content <1%) is extensively cultivated and utilized in the textile, paper, and rope industries after processing. The outer layer of the hemp crop called bast fiber has industrial applications. ...
Cannabis was extensively utilized for its medicinal properties till the 19th century. A steep decline in its medicinal usage was observed later due to its emergence as an illegal recreational drug. Advances in technology and scientific findings led to the discovery of delta-9-tetrahydrocannabinol (THC), the primary psychoactive compound of cannabis, that further led to the discovery of endogenous cannabinoids system consisting of G-protein-coupled receptors - cannabinoid receptor 1 and cannabinoid receptor 2 along with their ligands, mainly anandamide and 2-arachidonoylglycerol. Endocannabinoid (EC) is shown to be a modulator not only for physiological functions but also for the immune system, endocrine network, and central nervous system. Medicinal research and meta-data analysis over the last few decades have shown a significant potential for both THC and cannabidiol (CBD) to exert palliative effects. People suffering from many forms of advanced stages of cancers undergo chemotherapy-induced nausea and vomiting followed by severe and chronic neuropathic pain and weight loss. THC and CBD exhibit effective analgesic, anxiolytic, and appetite-stimulating effect on patients suffering from cancer. Drugs currently available in the market to treat such chemotherapy-induced cancer-related ailments are Sativex (GW Pharmaceutical), Dronabinol (Unimed Pharmaceuticals), and Nabilone (Valeant Pharmaceuticals). Apart from exerting palliative effects, THC also shows promising role in the treatment of cancer growth, neurodegenerative diseases (multiple sclerosis and Alzheimer's disease), and alcohol addiction and hence should be exploited for potential benefits. The current review discusses the nature and role of CB receptors, specific applications of cannabinoids, and major studies that have assessed the role of cannabinoids in cancer management.
... The fiber-type Cannabis, or "hemp", has a THC dry weight in the flowering heads of <0.3% or <0.2% depending on the jurisdiction [9,11,18]. Hemp can often be accompanied by a higher cannabidiol (CBD) content (THC:CBD < 1), while the elite drug-type cultivars typically report a THC:CBD ratio >1, or >0.3% THC in the flower heads [9,37]. ...
The recent legalization of Cannabis sativa L. in many regions has revealed a need for effective propagation and biotechnologies for the species. Micropropagation affords researchers and producers methods to rapidly propagate insect-/disease-/virus-free clonal plants and store germplasm and forms the basis for other biotechnologies. Despite this need, research in the area is limited due to the long history of prohibitions and restrictions. Existing literature has multiple limitations: many publications use hemp as a proxy for drug-type Cannabis when it is well established that there is significant genotype specificity; studies using drug-type cultivars are predominantly optimized using a single cultivar; most protocols have not been replicated by independent groups, and some attempts demonstrate a lack of reproducibility across genotypes. Due to culture decline and other problems, the multiplication phase of micropropagation (Stage 2) has not been fully developed in many reports. This review will provide a brief background on the history and botany of Cannabis as well as a comprehensive and critical summary of Cannabis tissue culture. Special attention will be paid to current challenges faced by researchers, the limitations of existing Cannabis micropropagation studies, and recent developments and future directions of Cannabis tissue culture technologies.
... The fiber-type Cannabis, or "hemp", has a THC dry weight in the flowering heads of <0.3% or <0.2% depending on the jurisdiction [9,11,18]. Hemp can often be accompanied by a higher cannabidiol (CBD) content (THC:CBD < 1), while the elite drug-type cultivars typically report a THC:CBD ratio >1, or >0.3% THC in the flower heads [9,37]. ...
The recent legalization of Cannabis sativa L. in many regions has revealed a need for effective propagation and biotechnologies for the species. Micropropagation affords researchers and producers methods to rapidly propagate insect/disease/virus free clonal plants, store germplasm, and forms the basis for other biotechnologies. Despite this need, research in the area is limited due to the long history or prohibitions and restrictions. Existing literature has multiple limitation: many publications use hemp as a proxy for drug-type Cannabis when it is well established that there is significant genotype specificity, studies using drug-type cultivars are predominantly optimized using a single cultivar, most protocols have not been replicated by independent groups, and some attempts demonstrate a lack of reproducibility across genotypes. Due to culture decline and other problems the multiplication phase of micropropagation (stage 2) has not been fully developed in many reports. This review will provide a brief background on the history and botany of Cannabis as well as a comprehensive and critical summary of Cannabis tissue culture. Special attention will be paid to current challenges faced by researchers, the limitations of existing Cannabis micropropaga-tion studies, and recent developments and future directions of Cannabis tissue culture technologies.
... Consistent with Asian provenance, 'Carmagnola' contained more THC than other European landraces and cultivars. Fetterman et al. (1971) first noted this: they measured 0.32% THC in 'Carmagnola'. Small and Marcus (2003) measured 0.38% THC. ...
This paper presents a scoping review of recent literature regarding the phylogenetics of Cannabis, descending the taxonomic ranks of family, genus, species, subspecies, and varieties. The family Cannabaceæ now consists of ten genera. Cannabis is a monotypic genus consisting of one species, Cannabis sativa L. The monotypic model is supported by DNA “barcode” studies. Two subspecies are recognized, and the number of varieties has increased to six. When and where Cannabis evolved has been explored, using molecular clock analyses and fossil pollen studies, respectively. The ubiquitous vernacular taxonomy of drug-type plants— “Sativa” and “Indica”—is deconstructed, and placed in a formal botanical framework. Italy has been a crossroads of Cannabis fiber-type plant genotypes, between Europe and Asia.
... For the differentiation between 'fibre-type' and 'drug-type' plants the phenotypic ratio is calculated as (E % THC + E % CBN)/ % CBD. Ratios >1 indicate the 'drug-type' (Fetterman et al. 1971, Gambaro et al. 2002. Also the single ratios THC/CBD and CBN/CBD may be determined and 1 represents the border between both chemotypes (Stefanidou et al. 1998). ...
Cannabis is used as a co-medication by patients with cancer or chronic inflammatory diseases. Anti-inflammatory effects of Δ9-tetrahydrocannabinol (THC) and other cannabinoids are frequently linked to the modulation of the Nuclear Factor kappaB (NF-KB). Advantages of using whole plant preparations have also been reported. The composition of preparations such as traditional hydroethanolic cannabis extracts (CE) varies due to the type of plant and preparation. This dissertation aimed to contribute insights into chemical standardisation and pharmacological profiling as part of a European Project developing CE medicines. The chemical profile of CE from different starting materials was determined using HPLC and 1H-NMR. Their pharmacological properties were measured as the ability to modulate the activation of NF-KB in IL-6 reporter gene stably transfected HeLa cells, to induce in vitro cytotoxicity in cancer cell lines (MTT-assay) and to activate caspase 3/7. The effect of pure cannabinoids and their combinations with plant phenolics and classical anti-inflammatory/cytotoxic drugs was also investigated. The HPLC/NMR profiles showed cannabinoid dominance even in polar extracts and a substantial portion of cannabinoid acids depending on CE age and storage. Markers for standardisation indicating plant type, solvent and stability -such as the ratio between neutral and carboxylated cannabinoids- are proposed. CE toxicity correlated with the total cannabinoid but not necessarily the THC content. Also all main pure phytocannabinoids proved to be equally toxic. Some CE were more toxic than pure phytocannabinoids, other CE reduced the effects of the compounds alone. In most cases toxicity correlated with the effect on NF-KB activation and also with the caspase 3/7 activation indicating apoptotic signalling. It appears also that the NF-KB activity of cannabinoids/CE is neither CB1 nor CB2 receptor dependent. The results show that there is a strong link between NF-KB and the toxic effect of cannabis in cancer cell lines. The in vitro effect of CE can differ from that of pure cannabinoids and is more influenced by factors other than the chemotype. Thus standardised CE of plants with predominantly non-psychotropic cannabinoids such as cannabidiol or cannabigerol may be as useful as traditional THC-type derived CE for the co-treatment in cancer and inflammatory diseases.
... The ratio of THC and CBD in the leaves and the flowers of the plant is generally used as a marker to classify cannabis varieties. According to Fetterman et al. (1971), varieties having high THC and low CBD (THC/CBD > 1) were characterized as drug type otherwise (THC/CBD < 1) fiber type variety. Whereas, Small and Beckstead (1973a;1973b) distinguished C. sativa in three phenotypes with an additional class containing THC~CBD. ...
Cannabis has been reported to contain over 560 different compounds, out of which 120 are cannabinoids. Among the cannabinoids, Δ9-tetrahydrocannabinol and cannabidiol are the two major compounds with very different pharmacological profile and a tremendous therapeutic potential. However, there are many challenges in bringing cannabis from grow-farms to pharmaceuticals. Among many, one important challenge is to maintain the supply chain of biomass, which is consistent in its cannabinoids profile. To maintain this process, male plants are removed from growing fields as they appear. Even with that practice, still there are fair chances of cross fertilization. Therefore, controlled indoor cultivation for screening, selection of high yielding female plants based on their cannabinoids profile, and their conservation and multiplication using vegetative propagation and/or micropropagation is a suitable path to ensure consistency in biomass material. In this chapter, the botany and propagation of elite cannabis varieties will be discussed.
... The phenotypic system of hemp classification [9] defines 2 chemotypes based on the combined quotient of Δ 9 -THC, cannabinol (CBN), and cannabidiol (CBD) contents, which are the most abundant cannabinoids in the majority of samples reported in the literature. Any sample with a value of this ratio greater than 1 is classified as drug-type marijuana and has potent psychotropic effects while quotients lower than 1 indicate a fiber-type. ...
Phytochemical investigation of the lipids extracted from seeds of Cannabis sativa by GC-MS showed 43 cannabinoids, 16 of which are new. The extract is dominated by Δ9-tetrahydrocannabinolic acid (A) and its neutral derivative trans-Δ9-tetrahydrocannabinol-C5 (THC) Cis and trans-Δ9-tetrahydrocannabinol-C7 isomers with an ethyl-pentyl branched chain together with minor amounts of trans-Δ9-tetrahydrocannabinol with a methyl-pentyl C6 branched side chain were identified as new natural compounds. Four cannabichromene isomers with a C5 side chain are postulated to be derived from the double bond migration at the terminal isoprenyl unit. C7 cannabichromene together with the neutral and acidic forms of cannabinol-C7 were also detected. The mass spectrum of these homologues as trimethylsilyl (TMS) derivatives are presented, and the fragmentation patterns are discussed.
... Abhängig vom THC-Gehalt kann zwischen Drogenhanf und Faserhanf unterschieden werden. Die Phänotypen von Cannabis sativa werden durch das Verhältnis (THC+CBN)/CBD charakterisiert (Drogenhanf oder Marihuana > 1; Faserhanf < 1) [15][16][17][18]. Die größten Drüsenhaare werden an weiblichen Hanfpflanzen in den Blütenregionen und hier besonders auf den Blättern und Samenhüllblättern vorgefunden. ...
Zusammenfassung
Seit dem 1996 aufgehobenen Anbauverbot für Pflanzen der Spezies Cannabis sativa L. (sog. Faserhanf) mit geringem Gehalt des psychoaktiven Inhaltsstoffs Δ9-Tetrahydrocannabinol (THC) wird eine Vielzahl daraus hergestellter Lebensmittel angeboten. Als Beurteilungshilfe für die amtliche Lebensmittelüberwachung werden in dieser Übersichtsarbeit alle Aspekte von Hanf als Lebensmittel diskutiert, eine Einführung in die Botanik der Hanfpflanze gegeben und die aktuelle Gesetzeslage in Deutschland und der Europäischen Union dargestellt. Forensisch-toxikologische Aspekte insbesondere hinsichtlich des Einflusses von Hanflebensmitteln auf Drogentests werden beschrieben und eine Übersicht über die analytischen Möglichkeiten zur Absicherung der THC-Richtwerte gegeben. Abschließend werden Vorschläge für die lebensmittelchemische und rechtliche Beurteilung von Hanflebensmitteln gemacht. Neue Aspekte in diesem Update betreffen insbesondere sog. Cannabidiol (CBD)-Öle und deren Novel-Food-Status. Seit 1998 wurde ein Rückgang der THC-Konzentrationen für mehrere Produktgruppen beobachtet. Die von der EU vorgeschriebene Verwendung von zertifiziertem Hanfsamen und die verstärkte Kontrolle der Hersteller haben offensichtlich zu einem deutlichen Rückgang der THC-Konzentrationen in Hanflebensmitteln geführt. Der maximale THC-Gehalt in derzeit verfügbaren traditionellen Hanflebensmitteln ist zehn- bis hundertfach niedriger als in den Studien der 1990er-Jahre. Es ist zu beachten, dass frühere GC-Studien immer die Summe von THC und THC-Säuren bestimmten. In den letzten Jahren liefern LC-MS-Methoden Informationen über den spezifischen Gehalt an THC in Hanfprodukten. Dennoch wurden seitdem immer noch Lebensmittel mit inakzeptablen THC-Gehalten vorgefunden, was zu einer ganzen Reihe von öffentlichen Warnungen im EU-Schnellwarnsystem für Lebens- und Futtermittel (RASFF) führte. Daher ist eine kontinuierliche Qualitätskontrolle erforderlich, um den THC-Wert niedrig zu halten. Dazu gehört sowohl die Verwendung von Sorten mit niedrigem THC-Gehalt als auch die richtige Saatgutreinigung. Jüngstes Interesse gilt dem CBD, das wegen seiner vermeintlich günstigen gesundheitlichen Eigenschaften vermarktet wird. Während natürliche Gehalte in den oben genannten Lebensmitteln toleriert werden, werden reine CBD-Extraktprodukte entweder als Arzneimittel oder als sogenanntes Novel Food behandelt, die beide vor dem Inverkehrbringen zugelassen werden müssen. Nicht-traditionelle Hanf-Extraktprodukte weisen zudem häufig so hohe THC-Konzentrationen auf, dass die Produkte als gesundheitsschädlich beurteilt werden müssen.
Summary
In 1996, the prohibition of the cultivation of plants of the species Cannabis sativa L. (so-called fibre hemp) with minor content of the psychoactive Δ9-tetrahydrocannabinol (THC) was lifted. Nowadays, a wide variety of hemp food products is offered on the market. As a help for evaluation of such products, this review article provides the official food control with information on all aspects of hemp as foodstuff. An introduction to the botany of the hemp plant and the current law situation in Germany and the European Union is presented. In particular, the forensic-toxicological aspects regarding the influence of hemp food on drug tests are described. Furthermore, an overview of the analytic techniques used to verify compliance with the guidance values is given. Finally, suggestions for the food regulatory and food chemical evaluation of hemp food products are made. New aspects in this update concern so-called cannabidiol (CBD) oils and their novel food status. Since 1998, a decrease in the THC concentrations for several product groups has been observed. The prescribed use of certified hemp seed by the EU and the increase of controls on manufactur-ers have obviously led to a significant decline of THC concentrations in hemp food products. The maximum THC content in currently purchasable traditional hemp food products is ten- to a hundred-fold lower than those found in the studies of the 1990s. It is of note that earlier GC studies always consider the sum of THC and THC-acids. In the last years, LC-MS methods provide information about the isolated content of THC in hemp products. Nevertheless, food products with inacceptable THC contents were still observed since then, which led to a series of public warnings in the EU Rapid Alert System for Food and Feed (RASFF). Therefore, ongoing quality control is needed to maintain low THC levels. This includes both the use of low THC varieties and proper seed cleaning. Recent interest surrounds CBD, which is purported for various health properties. While natural contents in the above-mentioned food products are tolerated, pure CBD extract products are either treated as medicines or as so-called novel food, which both need to be approved before being placed on the market. In addition, nontraditional hemp extract products often exhibited extreme THC concentrations, which must be evaluated as hazardous to health.
... It is possible that high-elevation populations of a naturally higher THC-producing variety were recognized and targeted by people in the Pamir region, possibly even explaining the prominence of ritual sites in the high mountains. Moreover, the content of THC also varies across plant parts, decreas-ing from the bract, flower, leave, stem, root, and seed in turn (10,23). The lack of seeds in the burners may suggest that nonfloral plant parts were burned, or it may suggest that seeds were removed from the floral structures because they do not contain the desired secondary compounds. ...
Cannabis is one of the oldest cultivated plants in East Asia, grown for grain and fiber as well as for recreational, medical, and ritual purposes. It is one of the most widely used psychoactive drugs in the world today, but little is known about its early psychoactive use or when plants under cultivation evolved the phenotypical trait of increased specialized compound production. The archaeological evidence for ritualized consumption of cannabis is limited and contentious. Here, we present some of the earliest directly dated and scientifically verified evidence for ritual cannabis smoking. This phytochemical analysis indicates that cannabis plants were burned in wooden braziers during mortuary ceremonies at the Jirzankal Cemetery (ca. 500 BCE) in the eastern Pamirs region. This suggests cannabis was smoked as part of ritual and/or religious activities in western China by at least 2500 years ago and that the cannabis plants produced high levels of psychoactive compounds.
... Cannabis spp can be categorized into psychoactive species (drug phenotype) or nonpsychoactive species (fiber phenotype) depending on the (%delta-9-THC + %CBN)/ (%CBD) ratio; if the ratio exceeds one then the Cannabis is regarded as psychoactive species (drug phenotype), else it is considered as fiber phenotype. [1] The nonpsychoactive species of C. sativa L, known as industrial hemp (THC content <1%) is extensively cultivated and utilized in the textile, paper, and rope industries after processing. The outer layer of the hemp crop called bast fiber has industrial applications. ...
ABSTRACT
Cannabis was extensively utilized for its medicinal properties till the 19th century. A steep decline in its medicinal usage as observed later due to its emergence as an illegal recreational drug. Advances in technology and scientific findings led to the discovery of delta-9-tetrahydrocannabinol (THC), the primary psychoactive compound of cannabis, that further led to the discovery of endogenous cannabinoids system consisting of G-protein-coupled receptors – cannabinoid receptor 1 and cannabinoid receptor 2 along with their ligands, mainly anandamide and 2-arachidonoylglycerol. EC is shown to be a modulator not only for physiological functions but also for the immune system, endocrine network, and central nervous system. Medicinal research and meta-data analysis over the last few decades have shown a significant potential for both THC and cannabidiol (CBD) to exert palliative effects. People suffering from many forms of advanced stages of cancers undergo chemotherapy-induced nausea and vomiting followed by severe and chronic neuropathic pain and weight loss. THC and CBD exhibit effective analgesic, anxiolytic, and appetite-stimulating effect on patients suffering from cancer. Drugs currently available in the market to treat such chemotherapy-induced cancer-related ailments are Sativex (GW Pharmaceutical), Dronabinol (Unimed Pharmaceuticals), and Nabilone (Valeant Pharmaceuticals). Apart from exerting palliative effects, THC also shows promising role in the treatment of cancer growth, neurodegenerative diseases (multiple sclerosis and Alzheimer’s disease), and alcohol addiction and hence should be exploited for potential benefits. The current review discusses the nature and role of CB receptors, specific
applications of cannabinoids, and major studies that have assessed the role of cannabinoids in cancer management.
KEY WORDS: 2-Arachidonoylglycerol, analgesic, anandamide, cannabidiol, cannabinoid receptor 1, cannabinoid receptor 2,
cannabinol, delta-9-tetrahydrocannabinol, endocannabinoid system
... In 1970s, three chemotypes of Cannabis plants have been recognized based on Δ9-THC/CBD ratio: a Δ9-THC/CBD ratio > > 1.0 is characteristic of "drug-type" plants (chemotype I), a Δ9-THC/CBD ratio close to 1.0 is for "intermediate-type" plants (chemotype II), and a Δ9-THC/CBD ratio < < 1.0 is characteristic of "fiber-type" plants (chemotype III) [14][15][16]. ...
Cannabis has been one of the oldest source of food, textile fiber and psychotropic substances. Cannabinoids are the main biologically active constituents of the Cannabis genus, with a demonstrated medicinal value. Its production is becoming legalized and regulated in many countries, thus increasing the need for a rapid analysis method to assess the content of cannabinoids. Gas chromatography (GC) is the preferred analytical method for the determination of these compounds, although is a slow and costly technique. Near infrared spectroscopy (NIR) has the potential for the quantitative prediction of quality parameters, and also of pharmacologically active compounds, but no references about cannabinoids prediction has been previously reported. The aim of the present research was to develop a fast, economical, robust and environmentally friendly method based on NIR technology that allow the quantification of the main cannabinoids present in Cannabis sativa L.
Samples:
A total of 189 grinded and dried samples from different genotypes and registered varieties were used. The content of the cannabinoids CBDV, Δ9-THCV, CBD, CBC, Δ8-THC, Δ9-THC, CBG and CBN were determined by gas chromatography. Spectra were collected in a dispersive NIR Systems 6500 instrument, and in a Fourier transform near Infrared (FT-NIR) equipment. The sample group was divided into calibration and validation sets, to develop modified partial lest squares (PLS) regression models with WINISI IV software with the dispersive data, and PLS models using OPUS 7.2 with the FT-NIR ones. Excellent coefficient of determination of cross validation (R2CV from 0.91 to 0.99), were obtained for the prediction of CBD, CBC, Δ8-THC, Δ9-THC, CBG and CBN, with standard error of prediction (SEP) values among 1.5-3 times the standard error of laboratory (SEL); and good for CBDV and Δ9-THCV cannabinoids (R2 values of 0.89 and 0.83, respectively) with the dispersive instrument. Similar calibration and validation statistics have been obtained with the FT-NIR instrument with the same sample sets, using its specific OPUS software. In conclusion, a methodology of quantitative determination of cannabinoids in Cannabis raw materials has been developed for the first time using NIR and FT-NIR instruments, with similar good predictive results. This new analytical method would allow a simpler, more robust and precise estimation than the current standard GC.
... Simpson, 2013 has highlighted the importance of gender in phytochemical research and its impact on pharmacological properties of a species. For example in the dioecious species Cannabis sativa L., the female plants are used for marijuana, whereas the male plants are preferred for fiber (Fetterman et al., 1971). In Dodonaea polyandra Merr. ...
Ethnopharmacological relevance:
More than 15,000 angiosperm species are dioecious, i.e., having distinct male and female individual plants. The allocation of resources between male and female plants is different, and also variation in secondary metabolites and sex-biased herbivory is reported among dioecious plants. However, little is known about the ethnobotany of dioecious species and whether preferences exist for a specific gender, e.g., in food, medicine or timber.
Aim of the study:
The aim of this study was: 1) to study whether Indian folk healers have preference for plant genders, and to document their knowledge and use of dioecious species; 2) to understand the concept of plant gender in Indian systems of medicine and folk medicine, and whether Ayurvedic literature includes any references to gender preference.
Materials and methods:
Lists of dioecious plants used in Indian systems of medicine and folk medicine were compiled. Ethnobotanical data was collected on perceptions and awareness of dioecious plants, and preferences of use of specific genders of dioecious species using semi-structured interviews with folk healers in Tamil Nadu, India. In addition, twenty Ayurvedic doctors were interviewed to gain insight into the concept of plant gender in Ayurveda.
Results:
Indian systems of medicine contain 5-7% dioecious species, and this estimate is congruent the number of dioecious species in flowering plants in general. Informants recognized the phenomenon of dioecy in 31 out of 40 species, and reported gender preferences for 13 species with respect to uses as timber, food and medicine. Among informants different plant traits such as plant size, fruit size, and visibility of fruits determines the perception of a plant being a male or female. Ayurvedic classical literature provides no straightforward evidence on gender preferences in preparation of medicines or treatment illness, however it contains details about reproductive morphology and sexual differentiation of plants.
Conclusions:
A knowledge gap exists in ethnobotanical and ethnopharmacological literature on traditional knowledge of dioecious plants. From this explorative study it is evident that people have traditional knowledge on plant gender and preferential usages towards one gender. Based on this, we propose that researchers conducting ethnobotanical and ethnopharmacological studies should consider documenting traditional knowledge on sexual systems of plants, and test the existence of gender specific usages in their conceptual framework and hypothesis testing. Incorporating such concepts could provide new dimensions of scientific knowledge with potential implications to conservation biology, chemical ecology, ethnoecology and drug discovery.
... Two early CGEs that lacked Afghani plants were nevertheless instructive, because they analyzed plants of Indian heritage prior to the era of widespread hybridization. Fetterman et al. (1971) Small and Beckstead (1973) analyzed 350 accessions from around the world. Many accessions came from botanical gardens, of questionable provenance (e.g., three indica accessions with no measurable THC). ...
The formal botanical taxonomy of Cannabis sativa Linnaeus and C. indica Lamarck has become entangled and subsumed by a new vernacular taxonomy of “Sativa” and “Indica.” The original protologues (descriptions, synonymies, and herbarium specimens) by Linnaeus and Lamarck are reviewed. The roots of the vernacular taxonomy are traced back to Vavilov and Schultes, who departed from the original concepts of Linnaeus and Lamarck. The modified concepts by Vavilov and Schultes were further remodeled by underground Cannabis breeders in the 1980s and 1990s. “Sativa” refers to plants of Indian heritage, in addition to their descendants carried in a diaspora to Southeast Asia, South- and East Africa, and even the Americas. “Indica” refers to Afghani landraces, together with their descendants in parts of Pakistan (the northwest, bordering Afghanistan). Phytochemical and genetic research supports the separation of “Sativa” and “Indica.” But their nomenclature does not align with formal botanical C. sativa and C. indica based on the protologues of Linnaeus and Lamarck. Furthermore, distinguishing between “Sativa” and “Indica” has become nearly impossible because of extensive cross-breeding in the past 40 years. Traditional landraces of “Sativa” and “Indica” are becoming extinct through introgressive hybridization. Solutions for reconciling the formal and vernacular taxonomies are proposed.
... As a dimentionless ratio, THC/CBD cancels two quantities (THC%, CBD%), and therefore provides a more valid comparison of many studies that grew plants under many different conditions. Fetterman et al. (1971) presented data as a quotient of THC+CBN/CBD, and assigned plants to two populations: "drug-types" with a quotient >1.0, and "fiber-types" with a quotient <1.0. Unlike individual cannabinoid quantities, the ratio remained fairly stable in plants. ...
This chapter has two parts. The first part details five characters that contribute to phenotypic diversity in Cannabis. Cannabinoids can be assayed by quantity (dry weight percentage of cannabinoids in harvested material) or by quality (the THC/CBD ratio, or chemotype). Cannabinoid quality is largely genetic, possibly monogenic. We dissect the monogenic inheritance model (two alleles at a single gene locus). Essential oil is composed of volatile, aromatic terpenoids. Terpenoid content varies between different varieties. Hemp seed oil consists of polyunsaturated fatty acids, including omega-6 and omega-3 fatty acids, which are under genetic control. Protein has received less attention than oil, despite hemp’s value as a protein supplement. Bast fibers are phloem (sap-conducting) cells in stalks. The second part presents the current breeding status of phenotypes for various uses. Breeding for fiber production includes monoecious cultivars, dioecious cultivars, high percentage of primary fiber, fast-retting phenotypes, and unique morphological markers in low-THC plants. Selective cross-breeding for cannabinoids includes prevalent-THC, prevalent-CBD, and cannabinoid-free plants. Relatively few cultivars have been bred specifically for seed production.
... Early studies noted that cannabis used for fiber tended to have higher levels of CBD, whereas cannabis used for drug purposes had higher levels of THC. 22 Small and Beckstead identified three chemical types (chemotypes) based on ratios of THC and CBD: type I, which contained high THC (>0.3%) and low CBD (<0.5%), type II high THC (>0.3%) and high CBD (>0.5%), and type III high CBD (>0.5%) and low THC (<0.3%). 23 The three chemotype concepts were confirmed by Hillig and Mahlberg among cultivars originating from different geographic locations in addition to noting other minor cannabinoids that were characteristic of certain cultivars. ...
Introduction: With laws changing around the world regarding the legal status of Cannabis sativa (cannabis) it is important to develop objective classification systems that help explain the chemical variation found among various cultivars. Currently cannabis cultivars are named using obscure and inconsistent nomenclature. Terpenoids, responsible for the aroma of cannabis, are a useful group of compounds for distinguishing cannabis cultivars with similar cannabinoid content.
Methods: In this study we analyzed terpenoid content of cannabis samples obtained from a single medical cannabis dispensary in California over the course of a year. Terpenoids were quantified by gas chromatography with flame ionization detection and peak identification was confirmed with gas chromatography mass spectrometry. Quantitative data from 16 major terpenoids were analyzed using hierarchical clustering analysis (HCA), principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA).
Results: A total of 233 samples representing 30 cultivars were used to develop a classification scheme based on quantitative data, HCA, PCA, and OPLS-DA. Initially cultivars were divided into five major groups, which were subdivided into 13 classes based on differences in terpenoid profile. Different classification models were compared with PLS-DA and found to perform best when many representative samples of a particular class were included.
Conclusion: A hierarchy of terpenoid chemotypes was observed in the data set. Some cultivars fit into distinct chemotypes, whereas others seemed to represent a continuum of chemotypes. This study has demonstrated an approach to classifying cannabis cultivars based on terpenoid profile.
... (Raman, 1998;Sytsma et al., 2002;Hillig, 2005 The viability and functioning secretion is correlated with senescence of epidermal cells (Fairbairn, 1972;Hammond & Mahlberg, 1977;Turner et al., 1977Turner et al., , 1980bTurner et al., , 1981Croteau, 1988;Werker, 2000;Guy & Stott, 2005;Happyana et al., 2013) ( [CBD]. If the ratio exceeded 1, plants are classified as "chemo-type," otherwise as "fiber-type," and this was the first study to differentiate between the drugand fiber-type, by Fetterman et al. (1971). Therefore, this ratio was subsequently used to discriminate chemotype, intermediate type, and fiber-type (Turner, Cheng, Lewis, Russell, & Sharma, 1979). ...
Cannabis sativa L. (Cannabaceae) is one of the oldest medicinal plants used by humans. For millennia, the plant has also been used for fiber, oil production, and simply as additive for food products. This chapter gives an overview of botanical aspects of the genus Cannabis, such as, macroscopical and microscopical features, taxonomic classification, the current varieties, genomics. Furthermore, geographical distribution, agricultural status, and germplasm conservation are provided. This chapter presents the current state of knowledge of different cultivation forms, including outdoor, indoor, and micropropagation.
... A phenotype ratio greater than 1.0 was classified as drug-type. Less than 1.0 was classified as fiber-type (Grlic 1968;Fetterman et al. 1971). ...
Cannabis, both herbal and resin, has been the most popular illicit drug in Israel in recent years. Until 2005, the main sources of cannabis resin (known also as hashish) to the Israeli drug market were Lebanon and India. Hashish from these sources can be distinguished by its external appearance. The aim of this study was to determine if there is any difference in the quality of the hashish from each source. For this purpose, we quantified the main cannabinoids, cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THC), and cannabinol (CBN) of hashish from different police seizures of known origins, Lebanon, India and Morocco, that had been submitted to the Analytical Chemistry Laboratory of the Division of Identification and Forensic Science at the Israeli National Police Headquarters and subsequently to the Hebrew University for quantitative analysis. The results, based on many different seizures, showed that the average CBD content of hashish from Lebanon varied from 5.69% to 12.79% (an average 8.98 ± 0.59%), THC of hashish from Lebanon varied from 0.93% to 4.20% (an average of 2.38 ± 0.27 %), CBD of hashish from Morocco varied from 1.52% to 5.14% (an average of 3.72 ± 0.19%), THC of hashish from Morocco varied from 5.08% to 13.41% (an average of 9.21 ± 0.40%), CBD of hashish from India varied from 0.78% to 13.13% (an average of 4.59 ± 1.07%), and THC of hashish from India varied from 0.53% to 16.45% (an average of 6.35 ± 1.50%), At the same time, several other cannabinoids present in the samples in lower amounts were identified (cannabidivarol, CBDV; cannabicitran; Δ9-tetrahydrocannabivarol, Δ9-THCV; cannabivarol, CBV; cannabicyclol, CBL; cannabichromene, CBC; cannabielsoin, CBE; Δ8-THC; and cannabigerol, CBG). The samples, predominantly from Lebanon, Morocco, and India, were evaluated for chemical phenotype (drug type and fiber type) to determine the geographical origin of these samples.Indian hashish, seized by Czech Republic authorities, was analyzed qualitatively for many cannabinoids and terpenes. In this sample three new cannabinoids were identified: cannabiorcochromene, cis-Δ9-tetrahydrocannabivarol, and cannabinerol.
... As of 2007, most taxonomists have listed cannabis as a single species; Cannabis sativa L. (Clarke &Watson, 2007). By THC content itself, THC/CBD ratio or [THC+CBN]/CBD ratio defined as phenotypic index (Hillig & Mahlberg, 2004;Fetterman et al., 1971;Small & Beckstead, 1973) has been used for classifying drug-, intermediate-and fiber phenotypes in cannabis plants. The chemotypes do not change in plants at different ages or in different sexes, as demonstrated by the consistency of major cannabinoids ratios; CBD/THC or CBG/CBD throughout the entire life cycle until flowering (Pacifico et al., 2008). ...
... A first study was done by Grlic [21] who recognized different ripening stages. Later, Fettermann [22] described different phenotypes based on quantitative differences in the content of main cannabinoids and he was the first to distinguish the drug-and fibre-type. Further extension of this approach was done by Small and Beckstead [23], Turner [24] and Brenneisen [25]. ...
Recently, the exponential growth of the legal cannabinoid market has revitalized the interest in this traditional in Cannabis sativa L. or industrial hemp in various industries around the world. Today, strict regulatory mechanisms over this crop exist to control the expression of federally banned cannabinoids produced by the plant, hindering its industrial potential. As the interest in cannabis grows, and its production expands to new soils and climates, research is needed to assess how different cultivars will fare in these new regions. Here, were determining the changes in cannabinoid concentration for three CBD varieties of industrial hemp (Cannabis sativa L): Bubba Kush (BK), Emerald Flower (EF), and Golden Sunset (GS). Crops were cultivated under open-field conditions in a randomized block design. Plants were sampled weekly until senescence to assess the changes in Cannabidiol (CBD) and Tetrahydrocannabinol (THC) concentrations across time. A cannabinoid extraction and quantification was developed to quantify cannabinoids (post-anthesis) via HPLC-DAD. Total CBD and THC reached their peak
concentration at 5-8 weeks within the study. After seven weeks, the decline of secondary metabolite concentration was observed, causing a decrease in cannabinoid concentration. Although the fluctuation of cannabinoids was dynamic within each variety, the study provides information and insights on the proper management and harvesting
of Cannabis sativa L or industrial hemp in South Florida.
The fibrous hemp Cannabis sativa L. called also: industrial hemp and cannabis, is a plant widespread all over the world, grown mainly for its fiber, used for various purposes. Hemp fruits (also known as seeds or nuts) are mainly used to produce of oil for food, cosmetic and as bird feed. The nuts contain about 30–35% of oil with 80 to 90% of essential fatty acids (EFA), of which 55–56% is linoleic acid (LA) from the omega-6 group and 22–25% α-linolenic acid (ALA) from the omega-3 group, as well as amino acids, participating in the formation of albumin and globulin (edestin), vitamins, and minerals. The article describes in detail: the nutritional value of hemp nuts, the possibilities for the use of hemp nuts, discussion about the cannabinoid content of fibrous hemp nuts, and legislative issues concerning the cultivation of industrial hemp. The paper reviews the literature on the most important nutritional properties of the seeds and products made from the processing of hemp seeds, making it a very versatile raw material used among others in the food industry.
Coincident with the cannabis legalization and the increased interest in the medicinal use of the plant, the cannabis marketplace and farming have seen tremendous growth. It is reported that there are more than 2000 cannabis varieties available to customers. However, the data that is available to the growers and breeders regarding the cannabinoid contents of various varieties remains low. Here, a high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous separation and determination of 11 cannabinoids. A total of 104 hemp bud materials belonging to 20 varieties were collected from farms in the state of Maryland and analyzed with the HPLC method. The contents of the 11 cannabinoids in various varieties were compared and discussed, highlighting the varieties that showed a high yield of cannabinoids and good consistency that are more appropriate for cannabinoid production.
Cannabis sativa has a long history of domestication both for its bioactive compounds and its fibers. This has produced hundreds of varieties, usually characterized in the literature by chemotypes, with Δ9-THC and CBD content as the main markers. However, chemotyping could also be done based on minor compounds (phytocannabinoids and others). In this work, a workflow, which we propose to name cannabinomics, combines mass spectrometry of the whole metabolome and statistical analysis to help differentiate C. sativa varieties and deciphering their characteristic markers. By applying this cannabinomics approach to the data obtained from 20 varieties of C. sativa (classically classified as chemotype I, II, or III), we compared the results with those obtained by a targeted quantification of 11 phytocannabinoids. Cannabinomics can be considered as a complementary tool for phenotyping and genotyping, allowing the identification of minor compounds playing a key role as markers of differentiation.
Background
Cannabis has been used worldwide for centuries for industrial, recreational and medicinal use, however, to date no successful attempts at editing genes involved in cannabinoid biosynthesis have been reported. This study proposes and develops an in silico best practices approach for the design and implementation of genome editing technologies in cannabis to target all genes involved in cannabinoid biosynthesis.
Results
A large dataset of reference genomes was accessed and mined to determine copy number variation and associated SNP variants for optimum target edit sites for genotype independent editing. Copy number variance and highly polymorphic gene sequences exist in the genome making genome editing using CRISPR, Zinc Fingers and TALENs technically difficult. Evaluation of allele or additional gene copies was determined through nucleotide and amino acid alignments with comparative sequence analysis performed. From determined gene copy number and presence of SNPs, multiple online CRISPR design tools were used to design sgRNA targeting every gene, accompanying allele and homologs throughout all involved pathways to create knockouts for further investigation. Universal sgRNA were designed for highly homologous sequences using MultiTargeter and visualised using Sequencher, creating unique sgRNA avoiding SNP and shared nucleotide locations targeting optimal edit sites.
Conclusions
Using this framework, the approach has wider applications to all plant species regardless of ploidy number or highly homologous gene sequences.
Significance statement
Using this framework, a best-practice approach to genome editing is possible in all plant species, including cannabis, delivering a comprehensive in silico evaluation of the cannabinoid pathway diversity from a large set of whole genome sequences. Identification of SNP variants across all genes could improve genome editing potentially leading to novel applications across multiple disciplines, including agriculture and medicine.
Despite being a controversial crop, Cannabis sativa L. has a long history of cultivation throughout the world. Following recent legalisation in Canada, it is emerging as an important plant for both medicinal and recreational purposes. Recent progress in genome sequencing of both cannabis and hemp varieties allows for systematic analysis of genes coding for enzymes involved in the cannabinoid biosynthesis pathway. Single nucleotide polymorphisms in the coding regions of cannabinoid synthases play important role in determining plant chemotype. Deep understanding of how these variants affect enzymes activity and accumulation of cannabinoids will allow breeding of novel cultivars with desirable cannabinoid profile. Here we present a short overview of the major cannabinoid synthases and present the data on the analysis of their genetic variants and their effect on cannabinoid content using several in-house sequenced Cannabis cultivars.
Cannabis (Cannabis sativa L.), commonly known as hemp, is an annual herb belongs to the family Cannabacea. The seeds of this plant have considerable content of oil and unsaturated fatty acids, and its fiber is used in the paper and textile industries. Tetrahydrocannabinol and cannabidiol are main cannabinoid compounds of this plant, which have high importance for their well-known pharmaceutical properties. Therapeutic effects of secondary metabolites of hemp on different diseases, such as cancer, Multiple Sclerosis (M.S.), and AIDS and their anxiety and palliative characteristics have been reported in several studies. Considering oil content, and therapeutic and industrial properties of the hemp as well as, its high diversity in Iran, more studies are needed to better recognize this plant and the economic production of its therapeutic compounds. In the present paper, a comprehensive review of agronomic, therapeutic and phytochemical characteristics of hemp is presented.
The diversity of non-glandular and glandular hairs of Cannabis sativa L. (marihuana) are described by scanning electron microscopy. The non-glandular hairs are of two major types, as distinguished by size differences and locations, and all of them are highly silicified. The presence of silica as well as cystoliths of calcium carbonate help in the identification of marihuana even in its ash residues. X-ray microanalyses of Cannabis hairs are compared with those of Humulus lupulus and Lantana camera, whose hairs have been considered to resemble those of marihuana. Glandular hairs are found to be of two major categories. One group consists of glands whose heads are generally made up of eight cells and the other group whose heads are generally made up of two cells but never more than four cells. All glands of both categories are stalked. Some glands of the first category are massively stalked and these are restricted solely to anthers and bracts of staminate and pistillate plants. The massive stalk is considered to be made up of epidermal and hypodermal cells that have grown in response to some stimulation during anthesis. Fine details of the shoot system of Cannabis, such as cuticular ridges on epidermal cells, warty protuberances on non-glandular hairs, and surface views of glands in developing stages are also reported. Glandular hairs on the bracts of Humulus lupulus resemble those of Cannabis.
Three distinct types of glandular hairs of increasing morphological complexity which occur on flowering tops of Cannabis sativa L. (marihuana) are described from scanning electron microscopy. These gland types—termed bulbous, capitate-sessile, and capitate-stalked, described from pistillate plants—occur in greatest abundance on the outer surface of bracts ensheathing the ovary. Bulbous and capitate-sessile glands, which arise at an early stage in bract development, are scattered over the bract surface. Mature bulbous glands have a small swollen head on a short stalk, whereas capitate-sessile glands have a large globular head attached directly to the bract surface. Because of their numbers and large size, capitate-sessile glands are the most conspicuous gland type during the early phase of bract development. Capitate-stalked glands, which have a large globular head on a tall, multicellular stalk, differentiate during subsequent bract development. These stalked glands arise first along the bracteal veins and then over the entire bract surface. A voluminous, fluid secretory product accumulates in the glandular head of all three types. These glands are believed to be a primary site of localization of the marihuana hallucinogen, tetrahydrocannabinol.
The relationship between glandular trichomes and cannabinoid content in Cannabis sativa L. was investigated. Three strains of Cannabis, which are annuals, were selected for either a drug, a non-drug, or a fiber trait and then cloned to provide genetically uniform material for analyses over several years. The distribution of the number and type of glands was determined for several organs of different ages including the bract and its subtending monoleaflet leaf and the compound leaf on pistillate plants. Quantitation of glands on these structures was integrated with gas chromatographic analyses of organ cannabinoid profiles. A negative correlation was found between cannabinoid content and gland number for each of the three clones. Isolated heads of the capitate-stalked glands also were analyzed for cannabinoid content and found to vary in relation to clone and gland age. These studies indicate that cannabinoids may occur in plant cells other than glandular trichomes. The results of these studies emphasize the need for stringent sampling procedures in micromorphological studies on trichome distribution and analytical determinations of cannabinoid content in Cannabis.
Cannabis has gained a lot of popularity in last few years not only because of its use as illicit drug but due to its use as food, fiber and medicine. It is a complex mixture of constituents which contain a unique class of secondary metabolites called phytocannabinoids. In general, so far a total of 565 constituents including 120 phytocannabinoids have been reported in Cannabis sativa. This chapter discusses the chemistry of phytocannabinoids in the plant with particular emphasis on the Δ9-THC type of cannabinoids and different analytical methods available for cannabinoids analysis in cannabis plant and cannabis products.
Cannabis sativa has been utilized for millennia, primarily as a source of a stem fiber (both the plant and the fiber termed “hemp”) and a resinous intoxicant (the plant and its drug preparations commonly termed “marijuana”), and secondarily as a source of edible seeds. In domesticating the species for these divergent purposes, humans have altered the morphology, chemistry, distribution and ecology of cultivated forms by comparison with related wild plants. Wild-growing plants appear to be either escapes from domesticated forms or the results of thousands of years of widespread genetic exchange with domesticated plants, making it impossible to determine if unaltered primeval or ancestral populations still exist. There are conflicting botanical classifications of Cannabis, including splitting it into several alleged species. The different approaches to classifying and naming plants such as Cannabis, with interbreeding domesticated and wild forms, are examined. It is recommended that Cannabis sativa be recognized as a single species, within which there is a high-THC subspecies with both domesticated and ruderal varieties, and similarly a low-THC subspecies with both domesticated and ruderal varieties. Alternative approaches to the classification of Cannabis that do not utilize scientific nomenclature are noted.
Cannabis preparations, derived from the hemp plant, Cannabis sativaL., have been used by man for their euphoric effects for over four thousand years (Nahas, 1973) and represent the most widely used of all illicit drugs. The active constituents, which are found mainly in the resin associated with the flowering tops of female plants, belong to a group of compounds classified as cannabinoids and which are unique to Cannabis.To date, over sixty of these compounds have been identified (Turner et al.,1980a) with the structure of the major active constituent, Δ1-tetrahydrocannabinol (Δ1-THC, Figure 10.1a), being elucidated as recently as 1964 (Gaoni and Mechoulam, 1964). Two numbering systems are in use for this compound, the monoterpene system (Figure 10.5a) used in this chapter, and the benzopyran system (Figure 10.5b) under which THC is named Δ9-THC. Other major cannabinoids are shown in Figures 10.2 to 10.4. Of these, cannabidiol (CBD, Figure 10.2a), a biochemical precursor of Δ1 -THC, and cannabinol (CBN, Figure 10.3a), a chemical degradation product, are generally the most abundant. Both of these compounds are devoid of psychotomimetic properties although they do show other pharmacological activities.
Mass spectrometry occupies a prominent role in the historical evolution of the instrumental analysis of forensic evidence. Most forensic evidence submissions are scheduled drugs, and mass spectrometry, combined with gas chromatography, has provided the gold standard in drug identification for decades. In the earliest ‘forensic’ applications in the 1950s and 1960s, mass spectrometry was used to help identify the elemental compositions and structures of natural products/drugs such as the cannabinoids and tropane alkaloids. In the 1970s, mass spectrometry went out of favor for years for the identification of cocaine because mass spectrometry could not provide information about the enantiomeric form(s) of cocaine that were necessary for prosecution around that time. Once the rules for prosecution were clarified, MS, especially in the form of GC-MS, became the gold standard for almost all organic forensic analyses. Reviews of crime labs in the US and abroad in the 1970s showed several interesting results that are still just as true almost 40 years later: 1) that mass spectrometers are among the most desired new pieces of equipment in existing crime labs, 2) that most forensic analyses involve the confirmation of drugs of abuse, and 3) that mass spectrometry is very valuable for forensic toxicology and is an area destined for more growth in the future. Recent advances in high resolution mass spectrometers, in multi collector isotope ratio mass spectrometers, and in ambient pressure sampling provide a hopeful outlook for continued growth and impact of mass spectrometry in legal applications.
Cannabis sativa L. is among the oldest known cultivated plants, with a long history of medical use. Cannabis produces a unique class of terpenophenolic compounds called cannabinoids, 104 of which have been isolated, the major biologically active one being Δ9-tetrahydrocannabinol. Cannabidiol, an antiepileptic, is also important. Cannabis is an annual, normally dioecious and occasionally monoecious, wind-pollinated species and is highly allogamous (cross-fertilization) in nature. Therefore, maintaining the efficacy of selected high Δ9-tetrahydrocannabinol-yielding elite varieties grown from seeds under field or greenhouse conditions is very difficult. Thus, a careful screening of elite mother clones using GC-FID and their propagation using vegetative cuttings or advanced biotechnological approaches, including micropropagation, is the most suitable way to maintain quality. This chapter describes the botany, species debate, phenotype, screening of high-yielding clones using GC-FID, indoor and outdoor cultivation, micropropagation, quality assurance of propagated plants, harvesting, processing, and storage.
Cannabis is known to produce more immediate and stronger effects when smoked than when taken orally in similar amounts. However, insufficient data are available on the pharmacology and toxicology of cannabis smoke. It has been reported that the psychoactive effects of smoked marihuana are strong er than can be expected on the basis of the tetrahydrocannabinol (THC) content [3]. It is therefore important to investigate the pharmacology of the products obtained after smoking, rather than to concentrate only on the pharmacology of the natural products. Some factors influencing the relative ratios are the variety of seed, the region of cultivation, the type of soil, and the method of preparation and storage of the sample. Consequently, results reported for pharmacological and clinical research vary considerably. The complexity of marihuana or hashish, as indicated above, hardly allows a proper characterization of changes occurring during smoking (pyrolysis); the known pyrolytic products are given in Annex IV of a recent United Nations report [11].
There are several methods of analysis available for detection of delta-9-tetrahydrocannabinol (delta-9-THC), a biologically active component of marihuana derived from Cannabis sativa L. These range from simple colorimetric tests (Beam 1911) to the Duquenois-Levine reaction which replaced the Beam test (Hahas, 1975). Analytical laboratories having available to them gas chromatography instrumentation utilize the procedures of Lerner (1986), Fetterman et al. (1971), Turner and Hadley (1973), or the DEA (1976) method when small quantities of material are evaluated.
For the past decade no subject, with the exception of the Vietnam War, has aroused such intense, continuous discussion and controversy in the Western World as has marihuana. This is particularly true in the United States. Some insight into the reasons for this may be obtained by perusal of the brilliant treatise by Bonnie and Whitebread (1974) which traces the development of legal measures to control the use of marihuana in the United States. Additional insights may also be gained by a consideration of how other cultures view the use of this “deceptive weed” (Rubin, 1975).
This article focuses on Cannabis, primarily, the development of biomarkers to classify and uniquely identify marijuana. DNA methods such as short tandem repeat, amplified fragment length polymorphism, and single-nucleotide polymorphism are discussed with an emphasis on forensic data-basing applications and the benefits and limitations of each method. It also provides an overview of marijuana regarding the history of cultivation in the United States, associative evidence at crime scenes and current law enforcement concerns in the US, and microscopic and chemical methods for classification, geo-sourcing and high-throughput screening.
1.The psychopharmacological activity of ?1-tetrahydrocannabinol, (I); ?1(6)-tetrahydrocannabinol (4' hexyl), (II); ?1(6)-tetrahydrocannabinol, (III); 1-ethoxyhexahydrocannabinol, (IV); 8-ethoxy-iso-hexahydrocannabinol, (V); ?1(6)-tetrahydrocannabinolic acid Me ester, Isomer I, (VI); ?1(6)-tetrahydrocannabinolic acid Me ester, Isomer II, (VII); cannabigerol, (VIII); ?1(6)-tetrahydrocannabinol (3' hexyl), (IX); cannabichromene, (X); has been examined in a variety of animal species.2.Compounds (I) and (III) caused severe motor disturbances and a stuporous state in dogs and ptosis, “tameness” and peculiar postural changes in monkeys. In the latter animal, compound (II) elicited similar effects.3.Compounds (I) and (III) after intraperitoneal but not subcutaneous administration, suppressed the gerbil digging activity; reduced the rat conditioned avoidance response and induced a cataleptoid reaction in mice, rats and gerbils. In addition, compound (I) reduced the performance of mice on the rotating-rod. Both compounds, administered subcutaneously, induced a measurable ataxic gait in rats.4.Amphetamine reversed the behavioural changes elicited by compounds (I) and (III) in monkeys, as well as the cataleptoid reaction in rats.5.None of the other compounds provoked observable changes in any of the species studied.6.It is suggested that Rhesus monkeys might serve as a suitable model for assessing the psychopharmacological activity of active cannabinoids.
The United States Pharmacopoeia, " 11th rev from the Department of Pharmacog-nosy, School of Pharmacy This research was supported by NIMH Contract No. Ph-43-68-1307. The authors thank NIMH for supplying the seed for the Mississippi-grown marijuana
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lishing Co., Easton, Pa., 1936, p. 104. (11) " The United States Pharmacopoeia, " 11th rev., Mack Pub-ACKNOWLEDGMENTS AND ADDRESSES Received August 7, 1970, from the Department of Pharmacog-nosy, School of Pharmacy, University of Mississippi, University, MS 38677 Accepted for publication March 15, 1971. Presented to the Pharmacognosy and Natural Products Section, APHA Academy of Pharmaceutical Sciences, Washington, D. C. meeting, April 1970. This research was supported by NIMH Contract No. Ph-43-68-1307. The authors thank NIMH for supplying the seed for the Mississippi-grown marijuana. Vol. 60, No. 8, Augusi 1971 lt49