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Analysis of cannabinoids in laser-microdissected trichomes of medicinal Cannabis sativa using LCMS and cryogenic NMR
Abstract
Trichomes, especially the capitate-stalked glandular hairs, are well known as the main sites of cannabinoid and essential oil production of Cannabis sativa. In this study the distribution and density of various types of Cannabis sativa L. trichomes, have been investigated by scanning electron microscopy (SEM). Furthermore, glandular trichomes were isolated over the flowering period (8weeks) by laser microdissection (LMD) and the cannabinoid profile analyzed by LCMS. Cannabinoids were detected in extracts of 25-143 collected cells of capitate-sessile and capitate stalked trichomes and separately in the gland (head) and the stem of the latter. Δ(9)-Tetrahydrocannabinolic acid [THCA (1)], cannabidiolic acid [CBDA (2)], and cannabigerolic acid [CBGA (3)] were identified as most-abundant compounds in all analyzed samples while their decarboxylated derivatives, Δ(9)-tetrahydrocannabinol [THC (4)], cannabidiol [CBD (5)], and cannabigerol [CBG (6)], co-detected in all samples, were present at significantly lower levels. Cannabichromene [CBC (8)] along with cannabinol (CBN (9)) were identified as minor compounds only in the samples of intact capitate-stalked trichomes and their heads harvested from 8-week old plants. Cryogenic nuclear magnetic resonance spectroscopy (NMR) was used to confirm the occurrence of major cannabinoids, THCA (1) and CBDA (2), in capitate-stalked and capitate-sessile trichomes. Cryogenic NMR enabled the additional identification of cannabichromenic acid [CBCA (7)] in the dissected trichomes, which was not possible by LCMS as standard was not available. The hereby documented detection of metabolites in the stems of capitate-stalked trichomes indicates a complex biosynthesis and localization over the trichome cells forming the glandular secretion unit.
... Cannabis sativa L. is an annual, wind-pollinated, herbaceous plant (Happyana et al., 2013). Though generally dieceous (male and female owers are located on separate plants), for ber and oilseed production it has been bred to be monoecious (male and female owers on the same plant). ...
... The former have secretory cells that are well known as the main sites of cannabinoid and essential oil production in cannabis. They are called capitate-stalked trichomes and consist of two parts, the gland (head) and the stem (Happyana et al., 2013). Other types of glandular trichomes are capitate-sessile and bulbous. ...
... Capitate-stalked trichomes are large and globular; they are mostly found in cannabis owers during the owering stage. Non-glandular trichomes are found on stems, leaves, petioles, stipules, and bracts of the plant (Addo et al., 2021;Happyana et al., 2013). ...
This thesis addresses the post-harvest processing of Cannabis sativa L. inflorescences. More specifically, this work focuses on the curing of female cannabis flowers--a technique for preservation and to enhance the aroma. In contrast to the conventional quick-drying after harvesting, curing entails the slow drying of the plant's
flowers. A process which takes more time but in return can lead to a refined product, comparable to an aged wine.
... Hemp also contains a multitude of compounds from various other classes, including flavonoids, terpenes, stilbenoids and lignans, among others, resulting in a highly complex composition (ElSohly et al., 2017;Liu et al., 2022), which remains to be effectively understood and leveraged. The inflorescences, especially the female ones, are considered the most valuable plant parts for the phytochemicals, owing to the abundance in glandular trichomes (Flores-Sanchez and Verpoorte, 2008;Happyana et al., 2013). ...
... CBDA was the highest peak in the pooled QC sample (Fig. S3). Smaller peaks were observed for the other biosynthetically relevant cannabinoids, i.e., CBGA, THCA, and CBCA (Flores-Sanchez and Verpoorte, 2008; Happyana et al., 2013), in addition to CBNDA, which is considered degradation product of CBDA (Berman et al., 2018;Citti et al., 2020). Further, a large number of CBEA and CBTA isomers were found, followed by CBRA isomers. ...
... These structures exhibit significant morphological and chemical diversity, playing a crucial role in the plant's interaction with its environment [1]. The family Cannabaceae, particularly economically important species like Humulus lupulus L. (Hop) and Cannabis sativa L. (hemp), have long captured the attention of researchers due to the ability of their secretory structures to produce chemical substances with therapeutic value [2][3][4][5][6][7]. ...
... Glandular trichomes have undergone extensive study within Cannabaceae, especially in C. sativa [2,4,25,39,86] and H. lupulus [30,[32][33][34][35][36][37][38]87], with some reports for the genera Trema, Celtis and Pteroceltis [88]. The prevalence of glandular trichomes in various parts of the plant body, particularly in wind-pollinated species as Hops and other Urticalean Rosids [88][89][90], suggests their primary role in defense against herbivory [85,91] rather than attracting pollinators. ...
Cannabaceae species garner attention in plant research due to their diverse secretory structures and pharmacological potential associated with the production of secondary metabolites. This study aims to update our understanding of the secretory system in Hops (Humulus lupulus L.), an economically important species especially known for its usage in beer production. For that, stems, leaves, roots, and inflorescences were collected and processed for external morphology, anatomical, histochemical, ultrastructural and cytochemical analyses of the secretory sites. Our findings reveal three types of secretory structures comprising the secretory machinery of Hops: laticifer, phenolic idioblasts and glandular trichomes. The laticifer system is articulated, anastomosing and unbranched, traversing all plant organs, except the roots. Phenolic idioblasts are widely dispersed throughout the leaves, roots and floral parts of the species. Glandular trichomes appear as two distinct morphological types: capitate (spherical head) and peltate (radial head) and are found mainly in foliar and floral parts. The often-mixed chemical composition in the secretory sites serves to shield the plant from excessive UVB radiation, elevated temperatures, and damage inflicted by herbivorous animals or pathogenic microorganisms. Besides the exudate from peltate glandular trichomes (lupulin glands), latex and idioblast content are also likely contributors to the pharmacological properties of different Hop varieties, given their extensive presence in the plant body.
... The highest contents of cannabinoids and terpenoids are found in the glandular trichomes on cannabis bracts. The highest density of glandular hairs is found on the bract surrounding each female cannabis flower and the subtending leaflets of the female inflorescence [80,81]. ...
... Cannabinoids are produced in the sessile and stalked trichomes of C. sativa plants [80,81]. Trichomes are particularly abundant on the inflorescences of the plant, present in a lower number on leaves, petioles and stems, and absent on the roots and seeds. ...
The phytochemistry of fibre hemp (Cannabis sativa L., cv. Futura 75 and Felina 32) cultivated in Lithuania was investigated. The soil characteristics (conductivity, pH and major elements) of the cultivation field were determined. The chemical composition of hemp extracts and essential oils (EOs) from different plant parts was determined by the HPLC/DAD/TOF and GC/MS techniques. Among the major constituents, β-caryophyllene (≤46.64%) and its oxide (≤14.53%), α-pinene (≤20.25%) or α-humulene (≤11.48) were determined in EOs. Cannabidiol (CBD) was a predominant compound (≤64.56%) among the volatile constituents of the methanolic extracts of hemp leaves and inflorescences. Appreciable quantities of 2-monolinolein (11.31%), methyl eicosatetraenoate (9.70%) and γ-sitosterol (8.99%) were detected in hemp seed extracts. The octadecenyl ester of hexadecenoic acid (≤31.27%), friedelan-3-one (≤21.49%), dihydrobenzofuran (≤17.07%) and γ-sitosterol (14.03%) were major constituents of the methanolic extracts of hemp roots, collected during various growth stages. The CBD quantity was the highest in hemp flower extracts in pentane (32.73%). The amounts of cannabidiolic acid (CBDA) were up to 24.21% in hemp leaf extracts. The total content of tetrahydrocannabinol (THC) isomers was the highest in hemp flower pentane extracts (≤22.43%). The total phenolic content (TPC) varied from 187.9 to 924.7 (average means, mg/L of gallic acid equivalent (GAE)) in aqueous unshelled hemp seed and flower extracts, respectively. The TPC was determined to be up to 321.0 (mg/L GAE) in root extracts. The antioxidant activity (AA) of hemp extracts and Eos was tested by the spectrophotometric DPPH● scavenging activity method. The highest AA was recorded for hemp leaf EOs (from 15.034 to 35.036 mmol/L, TROLOX equivalent). In the case of roots, the highest AA (1.556 mmol/L, TROLOX) was found in the extracts of roots collected at the seed maturation stage. The electrochemical (cyclic and square wave voltammetry) assays correlated with the TPC. The hydrogen-peroxide-scavenging activity of extracts was independent of the TPC.
... The medicinal properties of the cannabis plant are attributed to its secondary metabolites, specifically the terpenophenolic compounds classified as phytocannabinoids, which contain a large number of bioactive metabolites [12,13]. Phytocannabinoids (referred to hereinafter as cannabinoids) are synthesised in, secreted by and stored in trichomes, hair-like epidermal structures that can be found across most aerial parts of the cannabis plant but that appear in the highest abundancy over pistillate inflorescences [14][15][16][17][18][19]. ...
... So far, 120 cannabinoids have been scientifically characterised, and these can be found in varying blends and ratios among different genotypes [27,28]. The most studied compounds, Δ 9 -tetrahydrocannabinol (THC) and cannabidiol (CBD), form a substantial proportion of the overall phytocannabinoid content and are considered "major cannabinoids", while most other cannabinoids appear in trace quantities and are classified as "minor cannabinoids" [10,12,29,30]. ...
Maintaining specific and reproducible cannabinoid compositions (type and quantity) is essential for the production of cannabis-based remedies that are therapeutically effective. The current study investigates factors that determine the plant’s cannabinoid profile and examines interrelationships between plant features (growth rate, phenology and biomass), inflorescence morphology (size, shape and distribution) and cannabinoid content. An examination of differences in cannabinoid profile within genotypes revealed that across the cultivation facility, cannabinoids’ qualitative traits (ratios between cannabinoid quantities) remain fairly stable, while quantitative traits (the absolute amount of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), cannabigerol (CBG), Δ9-tetrahydrocannabivarin (THCV) and cannabidivarin (CBDV)) can significantly vary. The calculated broad-sense heritability values imply that cannabinoid composition will have a strong response to selection in comparison to the morphological and phenological traits of the plant and its inflorescences. Moreover, it is proposed that selection in favour of a vigorous growth rate, high-stature plants and wide inflorescences is expected to increase overall cannabinoid production. Finally, a range of physiological and phenological features was utilised for generating a successful model for the prediction of cannabinoid production. The holistic approach presented in the current study provides a better understanding of the interaction between the key features of the cannabis plant and facilitates the production of advanced plant-based medicinal substances.
... Undoubtedly, one of the most common assays is the DPPH approach. The application of this test facilitates an understanding of a variety of chemical processes and offers several obvious advantages, such as affordability, experiment simplicity, reproducibility, applicability at room temperature, and automation possibilities [126]. However, the overlapping spectra of substances that are absorbed in the same wavelength range as DPPH is a significant drawback. ...
... Due to the involvement of non-phenolic reducing agents present in the system when reducing the Folin-Ciocalteu reagent, TPC overestimation is a significant concern for the Folin-Ciocalteu test. Reducing sugars and certain amino acids are some of these pollutants [126]. ...
In recent years, there has been a growing interest in the application of antioxidants in food and pharmaceuticals due to their association with beneficial health effects against numerous oxidative-related human diseases. The antioxidant potential can be measured by various assays with specific mechanisms of action, including hydrogen atom transfer, single electron transfer, and targeted scavenging activities. Understanding the chemistry of mechanisms, advantages, and limitations of the methods is critical for the proper selection of techniques for the valid assessment of antioxidant activity in specific samples or conditions. There are various analytical techniques available for determining the antioxidant activity of biological samples, including food and plant extracts. The different methods are categorized into three main groups, such as spectrometry, chromatography, and electrochemistry techniques. Among these assays, spectrophotometric methods are considered the most common analytical technique for the determination of the antioxidant potential due to their sensitivity, rapidness, low cost, and reproducibility. This review covers the mechanism of actions and color changes that occur in each method. Furthermore, the advantages and limitations of spectrophotometric methods are described and discussed in this review.
... Volatile essential oils as secondary metabolites are mostly produced in peltate glandular trichomes (PGTs), including some monoterpenes, limonene and carvone (Alonso et al., 1992;Champagne and Boutry, 2013;Markus Lange and Turner, 2013;Wang et al., 2016). There are five types of trichomes on Cannabis sativa L., three of them are glandular trichomes, namely capitate-stalked, capitate-sessile, and bulbous trichomes (Dayanandan and Kaufman, 1976;Happyana et al., 2013). The glandular trichomes are as the main production and storage site to terpenes, and cannabinoids which is famous for their psychoactive and therapeutic effects (Kim and Mahlberg, 1991;Mahlberg and Kim, 2004). ...
... While trichomes perpendicular to the laser beam are necessary, which increase the randomness and uncontrollability (Olsson et al., 2009). In C. sativa, capitate-sessile and capitate-stalked trichomes were isolated over the flowering period using LCM with neither fixation nor freeze (Happyana et al., 2013). In Colquhounia coccinea, peltate glandular trichomes was collected directly from unfixed leaves using LCM (Li et al., 2013). ...
Trichomes, which are classified as glandular or non-glandular, are hair-like epidermal structures that are present on aerial parts of most plant species. Glandular secretory trichomes (GSTs) have the capacity to secrete and store specialized metabolites, which are widely used as natural pesticides, food additives, fragrance ingredients or pharmaceuticals. Isolating individual trichomes is an essential way for identifying trichome-specific gene functions and discovering novel metabolites. However, the isolation of trichomes is difficult and time-consuming. Here, we report a method to isolate the GSTs from leaf epidermis dispense with fixation using laser capture microdissection (LCM). In this study, 150 GSTs were captured efficiently from Artemisia annua leaves and enriched for artemisinin measurement. UPLC analysis of microdissected samples indicated specific accumulation of secondary metabolites could be detected from a small number of GSTs. In addition, qRT-PCR revealed that the GST-specific structural genes involved in artemisinin biosynthesis pathway were highly expressed in GSTs. Taken together, we developed an efficient method to collect comparatively pure GSTs from unfixed leaved, so that the metabolites were relatively obtained intact. This method can be implemented in metabolomics research of purely specific plant cell populations and has the potential to discover novel secondary metabolites.
... fibre and oil from it (Happyana et al., 2013). Now, cannabis grows all over the world, including in Pakistan, America, Europe, and Brazil. ...
This work examines the anticancer activity, the anti-inflammatory nature, and the cytotoxicity of the ethanol extract obtained from the female flowers of Cannabis sativa L using molecular methods in vitro, animal testing in vivo, as well as computational methods and simulations in silico. From the GC-MS analysis, the following bioactive compounds were found: cannabidiol (CBD), tetrahydrocannabinol (THC), and humulene. The antiproliferative activities of the extract were determined on HeLa cells by using MTT, Crystal Violet, and Trypan Blue assays with an IC50 value suggesting 51%-77.6% lethality. The bioinformatics analysis of molecular docking proved significant ligand-protein interactions of CBD, THC, and humulene with cancer-associated proteins such as PD-1/PD-L1, TNF-α, and MMP-9. In vivo, breast cancer was first established in female Sprague-Dawley rats with 7,12-dimethylbenz(a)anthracene (DMBA) then treated with cannabinoids either singularly or in combination. Detailed treatment demonstrated that the use of the three cannabinoids simultaneously yielded the best anticancer and anti-inflammatory outcomes together with the best tumor reduction. The concentration of serum biomarkers of inflammation and tumor progression was substantially reduced in treated groups compared to the control group, which proves the synergistic effects of these cannabinoids in breast cancer therapy. This study emphasizes the importance of medical Cannabis sativa derivatives in cancer treatment.
... The capitate-stalked trichomes consist of two parts: the gland (head) and the stem. The head contains disk cells which are presumed to be the site of cannabinoid production, while the stem is not yet functionally characterized (Happyana et al., 2013). ...
... Decarboxylation during the processing of flowers leads to their pharmacologically active forms, with tetrahydrocannabinol (THC) having psychoactive and intoxicating properties, while cannabidiol (CBD) has pharmaceutical uses as an anti-convulsant and antiinflammatory drug (Lu and Mackie, 2016). In living plant tissues, the decarboxylated THC or CBD usually represents less than 2% the total (carboxylated plus decarboxylated) pools (Happyana et al., 2013). ...
Cannabis sativa L. is one of the oldest domesticated crops. Hemp-type cultivars, which predominantly produce non-intoxicating cannabidiol (CBD), have been selected for their fast growth, seed, and fibre production, while drug-type chemovars were bred for high accumulation of tetrahydrocannabinol (THC). We investigated how the generation of CBD-dominant chemovars by introgression of hemp- into drug-type Cannabis impacted plant performance. The THC-dominant chemovar showed superior sink strength, higher flower biomass and demand-driven control of nutrient uptake. By contrast, the CBD-dominant chemovar hyperaccumulated phosphate in sink organs leading to reduced carbon and nitrogen assimilation in leaves, which limited flower biomass and cannabinoid yield. RNA-seq analyses determined organ- and chemovar-specific differences in expression of genes associated with nitrate and phosphate homeostasis as well as growth-regulating transcription factors that were correlated with measured traits. Among these were genes positively selected for during Cannabis domestication encoding an inhibitor of the phosphate starvation response SPX DOMAIN GENE3, nitrate reductase and two nitrate transporters. Altered nutrient sensing, acquisition or distribution are likely a consequence of adaption to growth on marginal, low-nutrient input lands in hemp. Our data provide evidence that such ancestral traits may become detrimental for female flower development and consequently overall CBD yield in protected cropping environments.
... Some researchers reported that the relative trichome concentration of Δ 9 -THC and CBD is 1-2% of that of their precursors THCA and CBDA, as determined by LCMS 34 . As shown in Fig. 4, the secretory cavity contents demonstrated higher proportion of CBDA (86%) compared to the air-dried inflorescences extracts (84%). ...
Studies with secretory cavity contents and air-dried inflorescence extracts of the CBD-rich hemp strain, Cannabis sativa cv. ‘Cherry Wine’, were conducted to compare the decarboxylation rates of acidic cannabinoids between two groups. The secretory cavity contents acquired from the capitate-stalked glandular trichomes by glass microcapillaries, and inflorescence samples air-dried for 15 days of storage in darkness at room temperature were analysed by high-pressure liquid chromatography. The ratio of acidic cannabinoids to the total cannabinoids was ranging from 0.5% to 2.4% lower in the air-dried inflorescence samples compared to the secretory cavity samples as follows. In the secretory cavity content, the percentage of acidic cannabinoids to the total cannabinoids was measured as 86.4% cannabidiolic acid (CBDA), 6.5% tetrahydrocannabinolic acid (THCA), 4.3% cannabichromenic acid (CBCA), 1.4% cannabigerolic acid (CBGA), and 0.6% cannabidivarinic acid (CBDVA), respectively. In the air-dried inflorescence, however, the acidic cannabinoids were detected with 84% CBDA, 4.8% THCA, 3.3% CBCA, 0.8% CBGA, and 0.3% Δ⁹-tetrahydrocannabivarinic acid (Δ⁹-THCVA), respectively. The ratio of cannabidiol (CBD) to cannabidiolic acid (CBDA) was close to 1:99 (w/w) in secretory cavity contents, however, it was roughly 1:20 (w/w) in the air-dried inflorescence. In addition, Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV) and Δ⁹-tetrahydrocannabivarinic acid (Δ⁹-THCVA) were only detected in the air-dried inflorescence sample, and the ratio of Δ⁹-THCV to Δ⁹-THCVA was about 1:20 (w/w). Besides, cannabidivarinic acid (CBDVA) was only observed in the secretory cavity content.
... More research has been done on CBG, a weak partial agonist for CB1 and CB2, and found in higher abundance (0.1-0.9 ng/mL) than CBN in the Cannabis plant (Happyana et al., 2013;Zagzoog et al., 2020). ...
Chronic pain conditions affect nearly 20% of the population in the United States. Current medical interventions, such as opioid drugs, are effective at relieving pain but are accompanied by many undesirable side effects. This is one reason increased numbers of chronic pain patients have been turning to Cannabis for pain management. Cannabis contains many bioactive chemical compounds; however, current research looking into lesser-studied minor cannabinoids in Cannabis lacks uniformity between experimental groups and/or excludes female mice from investigation. This makes it challenging to draw conclusions between experiments done with different minor cannabinoid compounds between labs or parse out potential sex differences that could be present. We chose five minor cannabinoids found in lower quantities within Cannabis: cannabinol (CBN), cannabidivarin (CBDV), cannabigerol (CBG), Δ8-tetrahydrocannabinol (Δ8-THC), and Δ9-tetrahydrocannabivarin (THCV). These compounds were then tested for their cannabimimetic and pain-relieving behaviors in a cannabinoid tetrad assay and a chemotherapy-induced peripheral neuropathy (CIPN) pain model in male and female CD-1 mice. We found that the minor cannabinoids we tested differed in the cannabimimetic behaviors evoked, as well as the extent. We found that CBN, CBG, and high dose Δ8-THC evoked some tetrad behaviors in both sexes, while THCV and low dose Δ8-THC exhibited cannabimimetic tetrad behaviors only in females. Only CBN efficaciously relieved CIPN pain, which contrasts with reports from other researchers. Together these findings provide further clarity to the pharmacology of minor cannabinoids and suggest further investigation into their mechanism and therapeutic potential. Significance Statement Minor cannabinoids are poorly studied ligands present in lower levels in Cannabis than cannabinoids like THC. In this study we evaluated 5 minor cannabinoids (CBN, CBDV, CBG, THCV, and Δ8-THC) for their cannabimimetic and analgesic effects in mice. We found that 4 of the 5 minor cannabinoids showed cannabimimetic activity, while one was efficacious in relieving chronic neuropathic pain. This work is important in further evaluating the activity of these drugs, which are seeing wider public use with marijuana legalization.
... The process of cannabinoid metabolism and synthesis has been clarified, but the molecular regulation mechanism is unknown. Currently, the most studied cannabinoids are THC and CBD, both terpenoids and isomers of each other [34], and both active substances in cannabis with high levels and medicinal properties. In plant tissues, cannabinoids are synthesized in the carboxylated form. ...
HD-Zip (Homeodomain-Leucine Zipper) is a family of transcription factors unique to higher plants and plays a vital role in plant growth and development. Increasing research results show that HD-Zip transcription factors are widely involved in many life processes in plants. However, the HD-Zip transcription factor for cannabis, a valuable crop, has not yet been identified. The sequence characteristics, chromosome localization, system evolution, conservative motif, gene structure, and gene expression of the HD-Zip transcription factor in the cannabis genome were systematically studied. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to verify its function. The results showed that cannabis contained 33 HD-Zip gene members. The number of amino acids is 136–849aa, the isoelectric point is 4.54–9.04, and the molecular weight is 23264.32–93147.87Da. Many cis-acting elements are corresponding to hormone and abiotic stress in the HD-Zip family promoter area of cannabis. Sequencing of the transcriptome at 5 tissue sites of hemp, stems, leaves, bracts, and seeds showed similar levels of expression of 33 members of the HD-Zip gene family at 5 tissue sites. Bioinformatics results show that HD-Zip expression is tissue-specific and may be influenced by hormones and environmental factors. This lays a foundation for further research on the gene function of HD-Zip.
... In C. sativa, the biosynthesis of the cannabinoids occurs in glandular trichomes, which are located on the aerial parts, and especially in the female flowers (Flores-Sanchez and Verpoorte 2008; Happyana et al. 2013). These results agree with those of Elhendawy et al. (2019), who reported that Content courtesy of Springer Nature, terms of use apply. ...
Friedelin and epifriedelanol are pentacyclic triterpenoids that preferentially accumulate in the roots of hemp (Cannabis sativa L.) and are valued for their antidiabetic, hypolipidemic, antioxidant, liver protective, anti-ulcer, anti-inflammatory, antimicrobial, anticancer, and antisenescence properties. The aim of the present study was to investigate the influence of media, carbon sources, and elicitation on the production of C. sativa hairy root biomass and their metabolites. The highest biomass (9.45 ± 0.00 g/100 mL flask fresh weight (FW) from the hairy root culture was obtained in MS liquid medium supplemented with 3% sucrose after 28 days. The highest levels of epifriedelanol (3.79-fold) and friedelin (3.25-fold) were found at the end of the exponential phase. Based on the carbon sources experiment, the presence of 3% sucrose provided the highest accumulation of epifriedelanol (0.930 ± 0.013 mg/g DW) and friedelin (0.574 ± 0.024 mg/g DW) in the hairy root culture after 28th days. The effects of methyl jasmonate (MJ) and salicylic acid (SA) on the enhancement of friedelin and epifriedelanol in C. sativa hairy root cultures were investigated. Between the two elicitors tested, SA at 100 µM showed the highest production of epifriedelanol (5.018 ± 0.35 mg/g DW) and SA at 50 µM showed maximum content of friedelin 1.56 ± 0.34 mg/g DW in hairy root culture. These represented 5.22- and 2.88-fold increase over the control (0.96 ± 0.01 mg/g DW and 0.54 ± 0.03 mg/g DW) after 96 h of treatment, respectively. The maximum accumulations of epifriedelanol (3.59 ± 0.12 mg/g DW) and friedelin (1.31 ± 0.01 mg/g DW) were observed in the treatment with MJ (100 µM) after 24 h of exposure and were 3.73- and 2.44-fold higher than the control, respectively. The result of this study indicates that hairy root culture of C. sativa enhanced friedelin and epifriedelanol contents, which can be used for commercial production in pharmaceutical companies.
... CBGA is synthesized by condensation of the monoterpene precursor geranyl diphosphate (GPP) with aromatic polyketo acetic acid (OA) (Booth et al., 2020). THC and CBD are formed during storage by decarboxylation of their acidic forms or interaction with heat and light (Happyana et al., 2013). The production of plant secondary metabolites (such as essential oils and aromatic compounds) is regulated by many biochemical, physiological, genetic, and metabolic factors. ...
Cannabis sativa is highly cultivated owing to its secondary metabolites, especially cannabinoids, which possess several medicinal effects. This study aims to examine the effect of foliar application of kinetin (KT) on the growth, cannabinoid content, and transcriptome of C. sativa at the flowering stage. A pot experiment was conducted in a greenhouse under a 16h/8h light/dark cycle for growing, and the photoperiod was adjusted to 10h/14h to induce flowering. The plant was sprayed with 0, 20, 40, and 100 mg/L KT every 2 days for a total of six times. Phenotypic parameters (fresh weight, cannabinoid content, and enzyme activity) and metabolome were analyzed. Foliar application of 20 and 40 mg/L of KT increased the growth and cannabinoid content of C. sativa, particularly that of cannabidiol. Similarly, treatment with 20 mg/L of KT increased the Δ9-tetrahydrocannabinolcontent to 0.24%, which is below the legal value of 0.30% in North American countries. Plants treated with 20 and 40 mg/L of KT showed decreased activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase and 1-deoxy-D-xylulose 5-phosphate synthase. KEGG pathway analysis showed that differentially expressed genes (DEGs) in the KT-treated cannabis were mainly enriched in 12 pathways, including sesquiterpenoid and triterpenoid biosynthesis, linoleic acid metabolism, flavonoid and phenylpropanoid biosynthesis, and other secondary metabolic pathways; 14DEGs (including eight upregulated and six downregulated genes) were enriched in sesquiterpene and triterpene biosynthesis pathways. Overall, these findings showed that KT plays a significant role in regulating terpenoid biosynthesis and the cannabinoid content of C. sativa
... These results are consistent with those ofKundu et al (2018), who reported that the highest biomass (5.77 ± 0.06 g per 50 mL FW) was encountered during the log phase (day 28) in Sphagneticola calendulacea. Likewise, Ghimire et al. (2019) reported that maximum hairy root growth and biomass were observed during 7-27 days in A. scaber cultures In C. sativa, the biosynthesis of the cannabinoids occurs in glandular trichomes, which are located on the aerial parts, and especially in the female owers (Flores-Sanchez and Verpoorte, 2008;Happyana et al. 2013). These results agree with those ofElhendawy et al (2018), who reported that glandular trichomes are not found on the surface of the roots, which therefore do not produce cannabinoids. ...
Friedelin and epifriedelanol are pentacyclic triterpenoids that preferentially accumulate in the roots of hemp (Cannabis sativa L.) and are valued for their antidiabetic, hypolipidemic, antioxidant, liver protective, anti-ulcer, anti-inflammatory, antimicrobial, anticancer, and antisenescence properties. The aim of the present study was to investigate the influence of media, carbon sources, and elicitation on the production of C. sativa hairy root biomass and these metabolites. The MS liquid medium promoted the highest fresh weight (9.45 ± 0.00 g/100 mL flask) biomass production in hairy root cultures after 28 days. The highest levels of epifriedelanol (3.79-fold) and friedelin (3.25-fold) were found at the end of the exponential phase. The presence of 3% sucrose provided the highest accumulation of epifriedelanol ( 0.930 ± 0.013 mg/g DW) and friedelin (0.574 ± 0.024 mg/g DW) in the roots. The effects of methyl jasmonate (MJ) and salicylic acid (SA) on the enhancement of friedelin and epifriedelanol in C. sativa hairy root cultures were investigated. Between the two elicitors, SA showed the highest production of epifriedelanol (up to 5.018 ± 0.35 mg/g DW) and friedelin up to 1.56 ± 0.34 mg/g DW in 28-day-old stationary phase hairy roots. These represented 5.22- and 2.88-fold increase over the control (0.96 ± 0.01 mg/g DW and 0.54 ± 0.03 mg/g DW) after 96 h of treatment, respectively. The maximum accumulations of epifriedelanol (3.59 ± 0.12 mg/g DW) and friedelin (1.31 ± 0.01 mg/g DW) were observed in the treatment with MJ (100 µM) after 24 h of exposure and were 3.73- and 2.44-fold higher than the control, respectively. These findings suggest that elicitation is an effective technique for enhancing the yields of these valuable bioactive pentacyclic triterpenoids in C. sativa hairy root cultures in a relatively short period of time.
... This substance is derived from the Cannabis sativa plant, belonging to the Cannabaceae family, native to South Asia and Central Asia [2] and, nowadays, extensively cultivated in Africa, Canada, Europe, and the United States [3]. The cannabis plant is rich in phytochemicals, primarily present in resin, within small crystals known as trichomes, located on the surface of the blossoms of mature unfertilized female specimens [4]. Marijuana (MJ) is obtained by drying the leaves and blossoms of the plant, while hashish is produced by drying the resin that accumulates on the leaves. ...
Cannabis, a plant known for its recreational use, has gained global attention due to its widespread use and addiction potential. Derived from the Cannabis sativa plant, it contains a rich array of phytochemicals concentrated in resin-rich trichomes. The main cannabinoids, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), interact with CB1 and CB2 receptors, influencing various physiological processes. Particularly concerning is its prevalence among adolescents, often driven by the need for social connection and anxiety alleviation. This paper provides a comprehensive overview of cannabis use, its effects, and potential health risks, especially in adolescent consumption. It covers short-term and long-term effects on different body systems and mental health and highlights the need for informed decision making and public health initiatives, particularly regarding adolescent cannabis use.
... In the plant trichrome, THC is stored in its acidic form, ∆ 9 -tetrahydrocannabinol acid (THCA) [50]. THCA is devoid of psychotropic effects [51]. ...
Intestinal inflammation is mediated by a subset of cells populating the intestine, such as enteric glial cells (EGC) and macrophages. Different studies indicate that phytocannabinoids could play a possible role in the treatment of inflammatory bowel disease (IBD) by relieving the symptoms involved in the disease. Phytocannabinoids act through the endocannabinoid system, which is distributed throughout the mammalian body in the cells of the immune system and in the intestinal cells. Our in vitro study analyzed the putative anti-inflammatory effect of nine selected pure cannabinoids in J774A1 macrophage cells and EGCs triggered to undergo inflammation with lipopolysaccharide (LPS). The anti-inflammatory effect of several phytocannabinoids was measured by their ability to reduce TNFα transcription and translation in J774A1 macrophages and to diminish S100B and GFAP secretion and transcription in EGCs. Our results demonstrate that THC at the lower concentrations tested exerted the most effective anti-inflammatory effect in both J774A1 macrophages and EGCs compared to the other phytocannabinoids tested herein. We then performed RNA-seq analysis of EGCs exposed to LPS in the presence or absence of THC or THC-COOH. Transcriptomic analysis of these EGCs revealed 23 differentially expressed genes (DEG) compared to the treatment with only LPS. Pretreatment with THC resulted in 26 DEG, and pretreatment with THC-COOH resulted in 25 DEG. To evaluate which biological pathways were affected by the different phytocannabinoid treatments, we used the Ingenuity platform. We show that THC treatment affects the mTOR and RAR signaling pathway, while THC-COOH mainly affects the IL6 signaling pathway.
... Trichomes correspond to epidermal protuberances present on the surface of the plant and are of two types: glandular or nonglandular (Happyana et al, 2013). ...
The cannabis plant contains the naturally occurring substance cannabidiol, also known as CBD. As opposed to its more widely known relative, tetrahydrocannabinol (THC), cannabidiol (CBD), does not possess any psychoactive or euphoria-inducing properties, and is widely regarded as harmless and non-addictive. Due to its alleged medicinal advantages, which are thought to include pain relief, anxiety reduction, epilepsy management and anti-inflammatory characteristics, CBD has attracted a lot of attention in recent years, in both human and veterinary medicine. The different kinds of CBD products available include oils, tinctures, capsules, lotions, and even edibles in the form of cookies and candy. In the field of veterinary medicine, the use of CBD has become more and more prevalent in recent years, and a formulation of treats for dogs and cats containing varying quantities of cannabidiol have been put on the market. Despite growing in popularity, CBD's legal status is still a little hazy in many nations, and more study is required to fully comprehend both its advantages and disadvantages. This article aims to review CBD's history, mechanisms of action, potential therapeutic roles as well as adverse effects that have been encountered thus far in clinical studies.
... In the plant trichrome THC is stored in its acidic form ∆ 9 -tetrahydrocannabinol acid (THCA) [64]. THCA is devoid of psychotropic effects [65]. ...
Inflammatory bowel diseases (IBD) includes Crohn's disease and ulcerative colitis, are idiopathic chronic relapsing inflammatory disorders of the intestinal tract. Different studies indicate that phytocanna-binoids, could play a possible role in the treatment of IBD by relieving the symptoms involved in the dis-ease. Phytocannabinoids act through the endocannabinoid system, which is distributed throughout the mammalian body in the cells of the immune system and in the intestinal cells. Our in vitro study analyzed the putative-anti-inflammatory effect of nine-selected pure cannabinoids in J774A1 macrophages cells and enteric glial cells (EGC’s) triggered to undergo inflammation with lipopolysaccharide (LPS). The an-ti-inflammatory effect of several phytocannabinoids was measured by their ability to reduce TNF tran-scription and translation in J774A1 macrophages and to diminish S100B and GFAP secretion and tran-scription in EGC’s. Our results demonstrate that THC at the lower concentrations tested exerted the most effective anti- inflammatory effect in both J774A1 macrophages and EGC’s compared to the other phy-tocannabinoids tested herein. We then performed RNA-seq analysis of EGC’s exposed to LPS in the presence or absence of THC or THC-COOH. Transcriptomic analysis of these EGC’s revealed 23 differ-entially expressed genes (DEG) compared to treatment with only LPS. Pretreatment with THC resulted in 26 DEG and pretreatment with THC-COOH resulted in 25 DEG. To evaluate which biological pathways were affected by the different phytocannabinoid treatments we used the Ingenuity platform. We show that THC treatment affected the mTOR and RAR signaling pathway while THC-COOH affected mainly the IL6 signaling pathway.
... For example, wild tomatoes (Solanum habrochaites) are resistant to pests through physical structures or chemical compounds like sesquiterpenes [6] . The anti-malarial drug, artemisinin, could be synthesized, stored and secreted by the annual wormwood glandular trichomes [7] . The psychoactive and therapeutic anesthetic, cannabinoids, is extracted from the glandular trichomes of Cannabis sativa [8] . ...
Trichomes are specialized epidermal appendages, which can be divided into glandular or non-glandular types based on their diverse morphology. The glands of glandular trichomes are responsible for the biosynthesis and storage of many natural metabolites. Recent progress has been made in characterizing the regulatory mechanisms of trichome formation and metabolite biosynthesis in the trichome. In this paper, we describe the structural and morphological features of glandular trichomes in vegetable crops, mainly focusing on tomato and cucumber. We discuss the developmental processes and regulatory mechanisms involved in trichome formation, including the roles of regulatory factors, phytohormones and environmental influences. We also highlight recent advances in the regulatory mechanisms underlying glandular trichome-related metabolites. This review provides a basis for understanding the formation of multicellular trichome and their secondary metabolites.
... Cannabis sativa L. has been used as an essential source of herbal raw materials throughout history with its fiber, seed, oil, and pleasing and therapeutic properties [6]. Plants produce sticky and distinctive-smelling cannabinoids in glandular trichomes, specialized biosynthetic organs on female flowers and leaves [7]. Numerous research studies have utilized the analysis of trichome metabolism to illustrate variations in trichome characteristics such as size, density, and the relative concentration of cannabinoids [8]. ...
Industrial hemp, a versatile and sustainable plant, possesses a broad array of applications. It offers fiber from its stems, food from its seeds, and oil from its flowers and seeds. Its importance lies in its contribution to economic, social, and environmental sustainability, thereby playing a crucial role in fostering a sustainable future. The extensive literature on industrial hemp emphasizes its potential as a sustainable resource. This review aims to underscore hemp's significance globally and highlights various aspects of industrial hemp production, encompassing breeding techniques, challenges, economic projections, and potential utilization.
... Cannabinoids are terpenophenolic compounds with a ring structure derived from a C 10 monoterpene subunit. The production of cannabinoids mainly occurs in the secretory head cells of the glandular trichomes [26] that are particulary concentrated in the bracts and flowers of the female inflorescence [27]. There are over 120 cannabinoids which are classified into 11 general types based upon their structure: Δ 9 -THC, Δ 8 -THC, cannabigerol (CBG), cannabichromene (CBC), cannabidiol (CBD), cannabinodiol (CBND), cannabielsoin (CBE), cannabicyclol (CBL), cannabinol (CBN), cannabitriol (CBT) and miscellaneous types [23]. ...
The lungs, in addition to participating in gas exchange, represent the first line of defense against inhaled pathogens and respiratory toxicants. Cells lining the airways and alveoli include epithelial cells and alveolar macrophages, the latter being resident innate immune cells important in surfactant recycling, protection against bacterial invasion and modulation of lung immune homeostasis. Environmental exposure to toxicants found in cigarette smoke, air pollution and cannabis can alter the number and function of immune cells in the lungs. Cannabis (marijuana) is a plant-derived product that is typically inhaled in the form of smoke from a joint. However, alternative delivery methods such as vaping, which heats the plant without combustion, are becoming more common. Cannabis use has increased in recent years, coinciding with more countries legalizing cannabis for both recreational and medicinal purposes. Cannabis may have numerous health benefits owing to the presence of cannabinoids that dampen immune function and therefore tame inflammation that is associated with chronic diseases such as arthritis. The health effects that could come with cannabis use remain poorly understood, particularly inhaled cannabis products that may directly impact the pulmonary immune system. Herein, we first describe the bioactive phytochemicals present in cannabis, with an emphasis on cannabinoids and their ability to interact with the endocannabinoid system. We also review the current state-of-knowledge as to how inhaled cannabis/cannabinoids can shape immune response in the lungs and discuss the potential consequences of altered pulmonary immunity. Overall, more research is needed to understand how cannabis inhalation shapes the pulmonary immune response to balance physiological and beneficial responses with potential deleterious consequences on the lungs.
... Several findings reveal variability in trichome size, density, and cannabinoid concentration using metabolic profiling of trichomes. However, the genetic mechanisms driving the alterations in trichome formation and subsequent cannabinoid content remain unexplained (Happyana et al., 2013;Hussain et al., 2021;Small & Naraine, 2016;Richard et al., (2007) discovered that incorporating hemp seed into the rat diet boosted plasma linoleic acid and alpha-linolenic acid levels considerably. These fatty acids regulate fluidity, electrolyte transport, and hormone activity because they are a structural component of phospholipids in cell membranes (Vogl et al., 2004). ...
Cannabis sativa L. is a flowering plant in the family Cannabaceae, and has been cultivated since ancient times for its fibres, oils, resins, dried inflorescences, and leaves. It can be used for a variety of industrial purposes. Over the years, the therapeutic and pharmacological efficacy of its phytoconstituents is shown in a variety of human diseases and health. The use and exploitation of the plant have sparked controversy; however, there are recent legalizations of its use for medical and other purposes in many countries within the corresponding legislative framework. In addition to this legalization, C. sativa is encouraging the very rapid growth of the cannabis oriented pharmaceutical industry. This chapter summarized recent developments in the science of C. sativa and its products about their industrial application, while also addressing gaps in the existing knowledge and future research directions for this high-value multi-use, and potential industrial plant with universal benefits.
... In a first step, to form olivetolic acid (OA), consecutive condensations occur between hexanoyl-CoA and malonyl-CoA followed by a cyclization (Gagne et al. 2012). Subsequently, a Friedel-crafts-like alkylation between the synthesized OA and geranyl phosphate takes place to produce cannabigerolic acid (Happyana et al. 2013). Cannabigerolic acid is later converted into cannabidiolic acid (CBDA) (Gagne et al. 2012), the most abundant cannabinoid in this plant (Hanus et al. 2005). ...
In recent decades, the therapeutic potential of cannabinoids and analogous
compounds has been intensively investigated. The endocannabinoid system has already been identified in the skin and, although much remains to be discovered about its contribution and importance for the maintenance of skin homeostasis, it has been increasingly associated as promising for dermatological disorders’ management. Cannabidiol (CBD), the main non-intoxicating phytocannabinoid in cannabis, has been shown to have hydrating, sebostatic, antipruritic, antimicrobial, anti-inflammatory, antioxidant, wound healing, photoprotective, anti-fibrotic and antitumoral, as well as modulating hair growth. Thus, CBD has gained attention concerning its application in cutaneous pathologies such as atopic dermatitis, psoriasis, acne, epidermolysis bullosa,
systemic sclerosis, seborrheic dermatitis, androgenetic alopecia and cutaneous melanoma, although its bioactivities still lack scientific evidence and some of its mechanisms of action remain to be elucidated. Given its physicochemical characteristics, its topical administration becomes challenging, and it is necessary to develop new technological strategies to overcome the skin intact barrier. This review describes the latest evidence that exists on the application of CBD to the skin, the problems inherent to its chemical structure and that compromise its cutaneous administration, and the different strategies and formulations that
have been studied to improve it, also clarifying some CBD-containing cosmetics products that are already available on the market.
... Absolute data shows more variation than the standardized data. This effect is likely a result of trichome density [19]. Part of the plant exposed to more light will have a higher density of trichomes. ...
... It is the glandular trichomes that are known to synthesize, store, and secrete specialized secondary metabolites that gives the leaves of many plants a unique fragrance. These specialised phytochemical metabolites include those with antimicrobial properties such as those found in the leaves of Plectranthus species [20,[23][24][25]. ...
Plectranthus amboinicus is widely recognized as a potential source of antimicrobial compounds due to the presence of bioactive components (essential oils) secreted by the glandular trichomes borne on the leaves. As such, an understanding of the effect of leaf development on the production of these essential oils (EOs) is of crucial importance to its medicinal applications. The current study represents the first comparative investigation of the effect of different stages of leaf development (lag, log, and stationary phase) upon the yield and bioactivity of phytochemicals produced. The effects of leaf extracts on the antimicrobial activity, cell surface hydrophobicity, biofilm formation, and motility of P. aeruginosa and Staphylococcus aureus were evaluated. Cryo-scanning electron microscopy was used to record the abundance and distribution of both glandular and non-glandular trichomes during leaf development. Gas chromatography–mass spectrometry analysis revealed that the potent phytochemical thymol is present primarily in log (30.28%) and stationary phase (20.89%) extracts. Log phase extracts showed the lowest minimum inhibitory concentration (25 mg/ml) when compared to other phases of development. Stationary phase extracts were shown to exhibit the highest biofilm dispersal activity against P. aeruginosa (80%), and log phase extracts against biofilms of S. aureus (59%). Log phase extracts showed the highest biofilm inhibitory activity against P. aeruginosa (66%) and S. aureus (63%). In conclusion, log phase leaf extracts of P. amboinicus exhibited a multimodal mechanism of action by displaying antimicrobial, antibiofilm activities and reducing the motility and hydrophobicity, which are important virulence factors in P. aeruginosa and S. aureus pathogenesis.
... Considerable literature describes the sites of cannabinoid biosynthesis in Cannabis plants [32][33][34][35]. Cannabinoids and terpenes accumulate in the glandular trichomes of the plants; in particular, female flowers show the highest density of glandular trichomes. ...
Cannabis (Cannabis sativa L.) is widely cultivated and studied for its psychoactive and medicinal properties. As the major cannabinoids are present in acidic forms in Cannabis plants, non-enzymatic processes, such as decarboxylation, are crucial for their conversion to neutral active cannabinoid forms. Herein, we detected the levels of cannabidivarin (CBDV), cannabidiol (CBD), cannabichromene (CBC), and Δ9-tetrahydrocannabinol (Δ9-THC) in the leaves and vegetative shoots of five commercial Cannabis cultivars using a combination of relatively simple extraction, decarboxylation, and high-performance liquid chromatography analyses. The CBDV, CBC, and Δ9-THC levels were 6.3–114.9, 34.4–187.2, and 57.6–407.4 μg/g, respectively, and the CBD levels were the highest, ranging between 1.2–8.9 μg/g in leaf and vegetative shoot tissues of Cannabis cultivars. Additionally, correlations were observed between cannabinoid accumulation and transcription levels of genes encoding key enzymes for cannabinoid biosynthesis, including CsCBGAS, CsCBDAS, CsCBCAS, and CsTHCAS. These data suggest that the high accumulation of cannabinoids, such as CBC, Δ9-THC, and CBD, might be derived from the transcriptional regulation of CsCBGAS and CsCBDAS in Cannabis plants.
... When grown for cannabinoid extraction only female plants are cultivated due to the high concentration of glandular trichomes found on the female flowers. These glandular trichomes are the site of cannabinoid biosynthesis and storage [2]. Cannabidiolic acid (CBDA), THCA, and cannabichromeneic acid (CBCA) are the three most abundant cannabinoids found in Cannabis. ...
The objectives of this study were to model the temporal accumulation of cannabidiol (CBD) and tetrahydrocannabinol (THC) in field-grown floral hemp in North Carolina and establish harvest timing recommendations to minimize non-compliant crop production. Field trials were conducted in 2020 and 2021 with BaOx and Cherry Wine cultivars. Harvest events started two weeks after floral initiation and occurred every two weeks for 12 weeks. Per-plant threshed biomass accumulation exhibited a linear plateau trend. The best fit model for temporal accumulation of THC was a beta growth curve. As harvest date was delayed, total THC concentrations increased until concentrations reached their maximum, then decreased as plants approached senescence. Logistic regression was the best fit model for temporal accumulation of CBD. CBD concentrations increased with later harvest dates. Unlike THC concentrations, there was no decline in total CBD concentrations. To minimize risk, growers should test their crop as early as possible within the USDA’s 30-day compliance window. We observed ‘BaOx’ and ‘Cherry Wine’ exceeding the compliance threshold 50 and 41 days after flower initiation, respectively.
... Searching the literature, many analytical methods were used for the chemical profiling of the cannabinoid content in cannabis. Some of these methods donʼt require derivatization before analysis, such as HPLC-UV and LC-MS, but might require more prepurification steps [20][21][22][23][24][25][26][27][28]. LC-MS/MS methods were used for the determination of illicit drugs in biological matrices [29]. ...
For decades, Cannabis sativa had been illegal to sell or consume around the world, including the United States. However, in light of the recent 2018 Farm Bill and the legalization of hemp across the US, various cannabis preparations have flooded the market, making it essential to be able to quantitate the levels of the different acidic and neutral cannabinoids in C. sativa and to have a complete cannabinoid profile of the different chemovars of the cannabis plant. A GC-FID method was developed and validated for the analysis of 20 acidic and neutral cannabinoids as trimethylsilyl (TMS) derivatives. The analyzed cannabinoids include cannabidivarinic acid (CBDVA), cannabidiolic acid (CBDA), cannabinolic acid (CBNA), cannabielsoic acid (CBEA), cannabicyclolic acid (CBLA), cannabichromenic acid (CBCA), trans-Δ9-tetrahydrocannabivarianic acid (Δ9-THCVA), trans-Δ9- tetrahydrocannabinolic acid A (Δ9-THCA), cannabigerolic acid (CBGA), cannabidiol (CBD), cannabicyclol (CBL), cannabidivarin (CBDV), trans-Δ9-tetrahydrocannabivarin (THCV), cannabichromene (CBC), trans-Δ8-tetrahydrocannabinol (Δ8-THC), trans-Δ9-tetrahydrocannabinol (Δ9-THC), cannabigerol (CBG), cannabinol (CBN), cannabicitran (CBT), and cannabielsoin (CBE). The method limit of detection (LOD) was as low as 0.1µg/mL, while the limit of quantitation ranged from 0.25 µg/mL to 0.5 µg/mL. The precision (%RSD) was <10%, while trueness ranged from 90% -107%. The developed method is simple, accurate, and sensitive for the quantitation of all 20 acidic and neutral cannabinoids. Finally, the proposed method was successfully applied to the quantitation of the cannabinoids in different cannabis chemovars grown at the University of Mississippi.
... Hemp EOs are secreted by trichomes present in inflorescences and, to a minor extent, in leaves (Happyana et al., 2013) (Fig. 9.4). The resin of the glandular trichomes is a defensive system against insects and can block other sources of environmental stress, such as attacks by bacteria, fungi or competition with surrounding vegetation (Appendino et al., 2008). ...
Hemp is a crop that has been used since ancient times for its medicinal and textile applications, which is experiencing a resurgence today. This growing interest is due to the fact that hemp is a crop with multipurpose applications: a source of cellulosic and woody fibers, produces oil-rich seeds, is a raw material for phytochemicals and is driven by consumer demand for more natural and sustainable products. Residues recovered during the harvesting and processing of hemp fibers and/or seeds can be utilized to obtain an essential oil rich in phytochemicals with multiple applications. We review the recent progress and developments in hemp essential oil as a complex mixture of bioactive compounds with antiinflammatory, antibacterial, insecticidal and therapeutic properties, and whose exploitation can add value to hemp cultivation. Essential oils are widely used globally, and their use is constantly increasing. This could boost the utilization and market value of hemp essential oil.
... However, botanical extract from cannabis remains the principal source of these compounds 5 . Δ 9 -Tetrahydrocannabinol (THC) and cannabidiol (CBD), the two most widely studied and clinically approved cannabinoids, are normally only found in the small outgrowths in the flowers of female plants, known as trichomes, meaning most of the plant is wasted biomass [5][6][7] . Additionally, purification can be very expensive and often leads to a complex mixture of cannabinoids. ...
Microbial production of cannabinoids promises to provide a consistent, cheaper, and more sustainable supply of these important therapeutic molecules. However, scaling production to compete with traditional plant-based sources is challenging. Our ability to make strain variants greatly exceeds our capacity to screen and identify high producers, creating a bottleneck in metabolic engineering efforts. Here, we present a yeast-based biosensor for detecting microbially produced Δ⁹-tetrahydrocannabinol (THC) to increase throughput and lower the cost of screening. We port five human cannabinoid G protein-coupled receptors (GPCRs) into yeast, showing the cannabinoid type 2 receptor, CB2R, can couple to the yeast pheromone response pathway and report on the concentration of a variety of cannabinoids over a wide dynamic and operational range. We demonstrate that our cannabinoid biosensor can detect THC from microbial cell culture and use this as a tool for measuring relative production yields from a library of Δ⁹-tetrahydrocannabinol acid synthase (THCAS) mutants.
... Cannabis sativa L. is an annual herbaceous flowering plant, that belongs to the Cannabaceae family and originates from Eastern Asia (Happyana et al., 2013;Pourseyed Lazarjani et al., 2020). It has, over the years, been used in many different ways as a source of fiber, as a pain reliever for medical purposes, and as a recreational drug (Radwan et al., 2017). ...
Although still illegal in many countries, food products containing cannabis or marijuana extracts have become very popular in recent years. In the present study, an LC-MS method was developed for the quantitative analysis of seven cannabinoids in various solid and liquid cannabis-based goods. The proposed analytical approach demonstrated satisfactory performance characteristics in terms of linearity (R²≥0.995), accuracy (recovery: 70.0-110%), precision (intraday RSD: 0.950-6.03%, interday RSD: 1.02-6.94%), sensitivity (LOD≤2.19 ng/mL, LOQ≤6.59 ng/mL) and carry-over effect (average carryover signals ≤3.90%). Solid-phase extraction (SPE), and ultrasound-assisted extraction (UAE) were utilized for the extraction of the analytes from liquid cannabis edibles (beer and energy drink), while Soxhlet and ultrasound-assisted extraction (UAE) were used for solid products (chocolates, hemp seeds, hemp tea). Infusion and decoction processes were followed for cannabis hemp tea and roasted coffee, respectively. UAE provided higher extraction efficiencies for cannabis-based edibles in solid form, while infused-cannabis beverages were extracted more efficiently using the SPE procedure. Cannabidiol (CBD) and cannabigerol (CBG) were the most detectable cannabinoids in all examined samples. Significantly high levels of cannabinoids were detected in cannabis tea extract prepared by the UAE procedure (total cannabinoids: 5440 μg/g). According to the suppliers, all examined samples were supposed to be free of Δ⁹-tetrahydrocannabinol (Δ⁹-THC). However, five products were found to contain considerable amounts of this compound (0.600-180 μg/g). Only in the case of cannabis beer, cannabis roasted coffee, and cannabis energy drink, Δ⁹-THC was not detected.
... Hemp produces a variety of secondary compounds that are most highly concentrated in the capitate stalked trichomes found on the apical inflorescences of female plants [4,5]. This region of the plant is the most abundant producer of CBD, the primary legal cannabinoid gaining commercial interest within the US and abundant in chemotype III hemp 1. ...
Hemp (Cannabis sativa) is a multi-use crop garnering newfound attention from researchers and consumers. While interest has emerged, a lack of substantiated research still exists regarding effects of adverse weather events on physiological health and secondary metabolite production of hemp. The aim of this experiment was to assess cold tolerance of hemp using the cultivars ‘FINOLA’ and ‘AutoCBD’. Effects of cultivar, plant age, cold acclimation, frequency of cold treatments, and intensity of cold treatments were all considered in regard to their influence on physiological stress, biomass, and cannabinoid profile. Few effects of sequential cold treatments were noted, and they were not moderated by cold acclimation, which tended to have negative effects across many responses. This detrimental effect of cold acclimation conditions was further observed in decreased total CBD % and total THC % compared to non-acclimated plants. These findings bear consideration when assessing the unpredictability of a changing climate’s effects on the heath and cannabinoid profile of hemp.
... In cannabis, due to the complex metabolome, the combination of several analytical methods usually gives the most comprehensive picture [2]. For instance, LC/QQQ/MS and NMR metabolomics analyses revealed the presence of several cannabinoids detected in extracts of cells of capitate-sessile and capitate-stalked trichomes as well [59]. Extracting and analysing the chemical profile of specific trichome lines holds great potential for use in future multi-omics experiments, as transcriptomic analyses could also be performed on these specific cell types [33]. ...
Cannabis (Cannabis sativa L.), also known as hemp, is one of the oldest cultivated crops, grown for both its use in textile and cordage production, and its unique chemical properties. However, due to the legislation regulating cannabis cultivation, it is not a well characterized crop, especially regarding molecular and genetic pathways. Only recently have regulations begun to ease enough to allow more widespread cannabis research, which, coupled with the availability of cannabis genome sequences, is fuelling the interest of the scientific community. In this review, we provide a summary of cannabis molecular resources focusing on the most recent and relevant genomics, transcriptomics and metabolomics approaches and investigations. Multi-omics methods are discussed, with this combined approach being a powerful tool to identify correlations between biological processes and metabolic pathways across diverse omics layers, and to better elucidate the relationships between cannabis sub-species. The correlations between genotypes and phenotypes, as well as novel metabolites with therapeutic potential are also explored in the context of cannabis breeding programs. However, further studies are needed to fully elucidate the complex metabolomic matrix of this crop. For this reason, some key points for future research activities are discussed, relying on multi-omics approaches.
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Keywords: cannabis; genomics; metabolomics; multi-omics; transcriptomics
Cucumber (Cucumis sativus) trichomes play a critical role in resisting external biological and abiotic stresses. Glandular trichomes are particularly significant as they serve as sites for the synthesis and secretion of secondary metabolites, while non-glandular trichomes are pivotal for determining the appearance quality of cucumbers. However, current methods for separating trichomes encounter challenges such as low efficiency and insufficient accuracy, limiting their applicability in multi-omics sequencing studies. This protocol introduces an efficient system designed for the precise separation of glandular and non-glandular trichomes from cucumber fruit. The process begins with the pre-cooling of sorbitol buffer or ethanol solution and the RNA-free treatment of laboratory supplies, followed by sterilization and pre-cooling. After filling glass bottles with pre-cooling buffer and glass beads, cucumber ovaries are then placed in the glass bottles and the trichome is harvested by bead-beating method. The separation process involves sequential filtration through various steel sieves and centrifugation to separate trichomes. The separated trichomes obtained from this method are well-suited for subsequent multi-omics sequencing analyses. This protocol achieved high precision in separating glandular and non-glandular trichomes, significantly enhancing the efficiency of separation and sample collection processes. This advancement not only addresses existing limitations but also facilitates comprehensive studies aimed at exploring the genetic and biochemical diversity present within cucumber trichomes, thereby opening avenues for broader agricultural and biological research applications.
Key features
• Use cucumber fruits on the day of flowering.
• Pre-cooling and RNA-free treatment ensure supply quality and purity.
• Efficiently separate glandular and non-glandular trichomes.
• Trichome samples are suitable for multi-omics sequencing analysis.
Cannabis sativa L., one of humanity’s oldest cultivated crops, has a complex domestication history due to its diverse uses for fibre, seed, oil and drugs, and its wide geographic distribution. This review explores how human selection has shaped the biology of hemp and drug-type Cannabis, focusing on acquisition and utilisation of nitrogen and phosphorus, and how resulting changes in source-sink relations shape their contrasting phenology. Hemp has been optimized for rapid, slender growth and nutrient efficiency, whereas drug-type cultivars have been selected for compact growth with large phytocannabinoid producing female inflorescences. Understanding these nutrient use and ontogenetic differences will enhance our general understanding of resource allocation in plants. Knowledge gained in comparison with other model species, such as tomato, rice or Arabidopsis thaliana can help inform crop improvement and sustainability in the Cannabis industry.
Drypetes is a plant genus of the family Putranjivaceae. Drypetes have high medicinal importance and the selected species sepiaria is used traditionally to treat various diseases. The present study was undertaken to analyze the chemical composition of Ethanolic crude extract from the leaves of Drypetes sepiaria by using GC-MS and LC-MS. The crude Ethanolic extract of Drypetes sepiaria was subjected to phytochemical screening for the presence of various phytochemicals. The total phenolic content was found to be 212.3 mg/g of Gallic acid equivalent. The IC 50 value is less compared to standard ascorbic acid. The total flavonoid content was found to be 1.76 mg/g of Quercetin. 19 different compounds were identified using LC-MS.
For millennia, various cultures have utilized cannabis for food, textile fiber, ethno-medicines, and pharmacotherapy, owing to its medicinal potential and psychotropic effects. An in-depth exploration of its historical,
chemical, and therapeutic dimensions provides context for its contemporary understanding. The criminalization
of cannabis in many countries was influenced by the presence of psychoactive cannabinoids; however, scientific
advances and growing public awareness have renewed interest in cannabis-related products, especially for
medical use. Described as a ’treasure trove,’ cannabis produces a diverse array of cannabinoids and noncannabinoid compounds. Recent research focuses on cannabinoids for treating conditions such as anxiety,
depression, chronic pain, Alzheimer’s, Parkinson’s, and epilepsy. Additionally, secondary metabolites like
phenolic compounds, terpenes, and terpenoids are increasingly recognized for their therapeutic effects and their
synergistic role with cannabinoids. These compounds show potential in treating neuro and non-neuro disorders,
and studies suggest their promise as antitumoral agents. This comprehensive review integrates historical,
chemical, and therapeutic perspectives on cannabis, highlighting contemporary research and its vast potential in
medicine.
Cannabis sativa has long been used for neurological and psychological healing. Recently, cannabidiol (CBD) extracted from cannabis sativa has gained prominence in the medical field due to its non-psychotropic therapeutic effects on the central and peripheral nervous systems. CBD, also acting as a potent antioxidant, displays diverse clinical properties such as anticancer, antiinflammatory, antidepressant, antioxidant, antiemetic, anxiolytic, antiepileptic, and antipsychotic effects. In this review, we summarized the structural activity relationship of CBD with different receptors by both experimental and computational techniques and investigated the mechanism of interaction between related receptors and CBD. The discovery of structural activity relationship between CBD and target receptors would provide a direction to optimize the scaffold of CBD and its derivatives, which would give potential medical applications on CBD-based therapies in various illnesses.
Graphical Abstract
Is Cannabis a boon or bane? Cannabis sativa has long been a versatile crop for fiber extraction (industrial hemp), traditional Chinese medicine (hemp seeds), and recreational drugs (marijuana). Cannabis faced global prohibition in the 20th century because of the psychoactive properties of ∆9-tetrahydrocannabinol; however, recently, the perspective has changed with the recognition of additional therapeutic values, particularly the pharmacological potential of cannabidiol. A comprehensive understanding of underlying mechanism of cannabinoid biosynthesis is necessary to cultivate and promote globally the medicinal application of Cannabis resources. Here, we comprehensively review the historical usage of Cannabis, biosynthesis of trichome-specific cannabinoids, regulatory network of trichome development, and synthetic biology of cannabinoids. This review provides valuable insights into the efficient biosynthesis and green production of cannabinoids, and the development and utilization of novel Cannabis varieties.
C-Alkylation of aromatic groups, as in Friedel—Crafts chemistry, is an energetically difficult process with significant chemo- and regioselectivity problems, especially if other nucleophiles, such as hydroxy groups or nitrogen atoms, are present in the substrate. Nature provides alkylating enzymes that selectively transfer a methyl, prenyl, or glycosyl group to carbon atoms of aromatic moieties under mild conditions, at room temperature, and mostly with excellent chemo- and regioselectivity. In this review, current enzymatic processes are highlighted and the increasing availability of cosubstrates, cofactors, and suitable enzymes is discussed as a prerequisite for scaling up such processes.
Plant growth-promoting rhizobacteria (PGPR) are a sustainable crop production input; some show positive effects under laboratory conditions but poorly colonize host field-grown plants. Inoculating with PGPR in microbial growth medium (e.g., King's B) could overcome this. We evaluated cannabis plant (cv. CBD Kush) growth promotion by inoculating three PGPR (Bacillus sp., Mucilaginibacter sp., and Pseudomonas sp.) in King's B at vegetative and flower stages. At the vegetative stage, Mucilaginibacter sp. inoculation increased flower dry weight (24%), total CBD (11.1%), and THC (11.6%); Pseudomonas sp. increased stem (28%) dry matter, total CBD (7.2%), and THC (5.9%); and Bacillus sp. increased total THC by 4.8%. Inoculation with Mucilaginibacter sp. and Pseudomonas sp. at the flowering stage led to 23 and 18% increases in total terpene accumulation, respectively. Overall, vegetative inoculation with PGPR enhanced cannabis yield attributes and chemical profiles. Further research into PGPR inoculation onto cannabis and the subsequent level of colonization could provide key insights regarding PGPR-host interactions.
Cannabinoids are bioactive meroterpenoids comprising prenylated polyketide molecules that can modulate a wide range of physiological processes. Cannabinoids have been shown to possess various medical/therapeutic effects, such as anti-convulsive, anti-anxiety, anti-psychotic, antinausea, and anti-microbial properties. The increasing interest in their beneficial effects and application as clinically useful drugs has promoted the development of heterologous biosynthetic platforms for the industrial production of these compounds. This approach can help circumvent the drawbacks associated with extraction from naturally occurring plants or chemical synthesis. In this review, we provide an overview of the fungal platforms developed by genetic engineering for the biosynthetic production of cannabinoids. Different yeast species, such as Komagataella phaffii (formerly P. pastoris) and Saccharomyces cerevisiae, have been genetically modified to include the cannabinoid biosynthetic pathway and to improve metabolic fluxes in order to increase cannabinoid titers. In addition, we engineered the filamentous fungus Penicillium chrysogenum for the first time as a host microorganism for the production of Δ9-tetrahydrocannabinolic acid from intermediates (cannabigerolic acid and olivetolic acid), thereby showing the potential of filamentous fungi as alternative platforms for cannabinoid biosynthesis upon optimization.
Projected revenues of cannabis concentrates and extracts in Canada will reach 5 billion dollars, of which infused products will account for half of the total. The pharmacologically active cannabinoids accumulate in the crop's flowers, accounting for as much as 30% of their dry mass, and are absent from the rest of the plant's body. To achieve a cost effective drug formulation requires optimizing cannabis processing techniques. Here, we review the pretreatment of Cannabis Sativa L., its solvent extraction, and the isolation of its actives metabolites. We describe traditional extraction processes such as maceration and percolation with organic solvents, but focus on recent green solvent and methods including supercritical fluid extraction (SCFE) and microwave‐ and ultrasound‐enhanced techniques. Furthermore, we report the decarboxylation kinetics to convert tetrahydrocannabinolic acid and cannabidiolic acid and purification‐isolation techniques to satisfy regulatory and consumer requirements. Cannabinoids decarboxylate in 10—60 min at 100—150 °C. Ethanol and petroleum ether recover up to 90% of the neutral cannabinoids from plant inflorescences but the crude extracts require further refining as the purity is less than 50%. Propane and butane compressed gas extraction facilitate solvent removal but introduce safety hazards related to flammability. SCFE is the safest solvent‐free extraction method with an improved terpenoid recovery and > 80% purity. Academic and commercial interest in the field is expected to accelerate in the next decade due to recent changes in regulatory schemes across North America, which will reduce legal and stigmatic barriers to research.
Buddleja officinalis is a traditional Chinese medicinal plant covered with glandular and non-glandular trichomes on leaves. Phytochemical investigation of its leaves led to the identification of one undescribed tetranorcycloartane 3-oxo-25,26,27,29-tetranorcycloartan-24-oic acid (1) and one first identified natural product tetranorcycloartane 3-oxo-25,26,27,29-tetranorcycloartan-24-oic methyl ester (2), along with an undescribed megastigmane glucoside (3) and 14 known constituents (4–17). Structures of undescribed chemicals were elucidated by comprehensive 1D and 2D NMR, MS and CD analysis. Further chemical investigation resulted in six triterpenoids (4–9) being localized to the trichomes of B. officinalis. The major trichome components cycloeucalenone (4) and 24-oxo-29-norcycloartan-3-one (5) showed potent antifeedant activity against a generalist insect cotton bollworm (Helicoverpa armigera), but no obvious activity against the specialist herbivore Hyphasis inconstans. Compounds 4 and 7 also displayed inhibitory effects on seed germination of Arabidopsis thaliana. In addition, 1 and 4 exhibited moderate antibacterial activity toward three gram-positive bacteria.
Hamppu (Cannabis sativa L.) on monimuotoinen viljelykasvi, josta on kehitetty lajikkeita eri
käyttötarkoituksiin: öljy-, kuitu-, lääke- ja huumekasviksi. Hyöty- eli teollisuushamppulajikkeisiin luetaan öljy- ja kuituhamppu. Tällä hetkellä Suomessa hampun kannabinoideja sisältävien kasvinosien käyttö
elintarvikkeina ei ole sallittua, vaan edellyttää uuselintarvikelain mukaisen uuselintarvikeluvan hankkimista. Suomessa elintarvikkeet eivät saa sisältää huumausaineeksi luokiteltavaa Δ9-tetrahydrokannabinolia (THC). Koska Δ9-THC:n enimmäismäärälle ole määritelty raja-arvoa, sen tulkitaan tarkoittavan nollatoleranssia. Lainsäädäntö vaihtelee EU-maissa ja on oletettavissa, että Euroopan komissio linjaa kantansa Δ9-THC:n enimmäispitoisuudeksi uuselintarvikkeiden luetteloinnin yhteydessä, jolloin Suomessakin tilanne arvioidaan uudelleen.Hampunsiementuotannon sivuvirtana syntyy kasvimateriaalia, joka sisältää kannabinoideja Tällä hetkellä viljelijät eivät hyödynnä sivuvirtamateriaalia muuten kuin maanparannusaineena.Öljyhamppua tällä hetkellä viljeleville ja aiemmin viljelleille viljelijöille osoitetulla kyselytutkimuksella kartoitettiin heidän näkemyksiään ja kiinnostustaan hampunkorjuusta jäävien
sivuvirtojen hyödyntämiseen. Kyselyyn vastasi 44 viljelijää. Vastaajista noin puolet oli kiinnostuneita hampun sivuvirtamateriaalin hyödyntämisestä ja valmiutta lisäinvestointien tekemiseen löytyi. Vastaavasti noin puolet eivät olisi valmiita tekemään lisäinvestointeja, vaikka sivuvirtamateriaalista tulisi myyntituote. Alustava arvoketjuanalyysi osoittaa, että kustannusten ja riskien hallinta on haastavaa.
In the last decade there has been an increasing demand for hemp derivatives from legal Cannabis sativa L. (THC content < 0.3%) to be used in different industrial applications, because of the spread of its cultivation and preference in sustainable agricultural systems. In the European Union about 25,000 hectares are cultivated, and more than seventy cultivars are allowed to be grown in agricultural systems. During hemp processing a huge amount of biomass, mostly given by leaves and inflorescences, can be generated, and be reused to produce niche products. Among the latter, the essential oil, a liquid, odorous product composed mainly of monoterpenes and sesquiterpenes, represents a promising future candidate in different fields such as pest management science, pharmaceuticals, cosmetics, and others. In this chapter we review scientific literature dealing with the chemical compositions of the essential oil obtained from different cultivars of industrial hemp highlighting the potential use of their constituents as pharmaceutically active drugs, insecticides, acaricides, and antimicrobials.
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.
Cannabinoids and in particular the main psychoactive Δ9-THC are promising substances for the
development of new drugs and are of high importance in biomedicine and pharmacy. This review gives an overview
of the chemical properties of Δ9-THC, its synthesis on industrial scale, and the synthesis of important
metabolites. The biosynthesis of cannabinoids in Cannabis sativa is
extensively described in addition to strategies for optimization of this plant for cannabinoid employment
in medicine. The metabolism of Δ9-THC in humans is shown and, based on this, analytical procedures
for cannabinoids and their metabolites in human forensic samples as well as in C.sativa
will be discussed. Furthermore, some aspects of medicinal indications for Δ9-THC and its ways of administration
are described. Finally, some synthetic cannabinoids and their importance in research and medicine are delineated.
Cannabis sativa is an interesting crop for several industrial uses, but the legislations in Europe and USA require a tight control of cannabinoid
type and content for cultivation and subsidies release. Therefore, cannabinoid survey by gas chromatography of materials under selection is an important step in hemp breeding.
In this paper, a number of Cannabis accessions were examined for their cannabinoid composition. Their absolute and relative content was examined, and results
are discussed in the light of both the current genetic model for cannabinoid’s inheritance, and the legislation’s requirements.
In addition, the effectiveness of two different types of markers associated to the locus determining the chemotype in Cannabis was evaluated and discussed, as possible tools in marker-assisted selection in hemp, but also for possible applications in
the forensic and pharmaceutical fields.
Cannabis sativa has been cultivated throughout human history as a source of fiber, oil and food, and for its medicinal and intoxicating properties. Selective breeding has produced cannabis plants for specific uses, including high-potency marijuana strains and hemp cultivars for fiber and seed production. The molecular biology underlying cannabinoid biosynthesis and other traits of interest is largely unexplored.
We sequenced genomic DNA and RNA from the marijuana strain Purple Kush using shortread approaches. We report a draft haploid genome sequence of 534 Mb and a transcriptome of 30,000 genes. Comparison of the transcriptome of Purple Kush with that of the hemp cultivar 'Finola' revealed that many genes encoding proteins involved in cannabinoid and precursor pathways are more highly expressed in Purple Kush than in 'Finola'. The exclusive occurrence of Δ9-tetrahydrocannabinolic acid synthase in the Purple Kush transcriptome, and its replacement by cannabidiolic acid synthase in 'Finola', may explain why the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) is produced in marijuana but not in hemp. Resequencing the hemp cultivars 'Finola' and 'USO-31' showed little difference in gene copy numbers of cannabinoid pathway enzymes. However, single nucleotide variant analysis uncovered a relatively high level of variation among four cannabis types, and supported a separation of marijuana and hemp.
The availability of the Cannabis sativa genome enables the study of a multifunctional plant that occupies a unique role in human culture. Its availability will aid the development of therapeutic marijuana strains with tailored cannabinoid profiles and provide a basis for the breeding of hemp with improved agronomic characteristics.
We performed a comparative analysis of the genome-wide DNA methylation profiles from three human embryonic stem cell (HESC) lines. It had previously been shown that HESC lines had significantly higher non-CG methylation than differentiated cells, and we therefore asked whether these sites were conserved across cell lines.
We find that heavily methylated non-CG sites are strongly conserved, especially when found within the motif TACAG. They are enriched in splice sites and are more methylated than other non-CG sites in genes. We next studied the relationship between allele-specific expression and allele-specific methylation. By combining bisulfite sequencing and whole transcriptome shotgun sequencing (RNA-seq) data we identified 1,020 genes that show allele-specific expression, and 14% of CG sites genome-wide have allele-specific methylation. Finally, we asked whether the methylation state of transcription factor binding sites affects the binding of transcription factors. We identified variations in methylation levels at binding sites and found that for several transcription factors the correlation between the methylation at binding sites and gene expression is generally stronger than in the neighboring sequences.
These results suggest a possible but as yet unknown functional role for the highly methylated conserved non-CG sites in the regulation of HESCs. We also identified a novel set of genes that are likely transcriptionally regulated by methylation in an allele-specific manner. The analysis of transcription factor binding sites suggests that the methylation state of cis-regulatory elements impacts the ability of factors to bind and regulate transcription.
RNA isolated from the glands of a Delta(9)-tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands.
We identified a unique enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid (CBDA) in Cannabis sativa L. (CBDA strain). The enzyme, named CBDA synthase, was purified to apparent homogeneity by a four-step procedure: ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The active enzyme consists of a single polypeptide with a molecular mass of 74 kDa and a pI of 6.1. The NH2-terminal amino acid sequence of CBDA synthase is similar to that of Delta1-tetrahydrocannabinolic-acid synthase. CBDA synthase does not require coenzymes, molecular oxygen, hydrogen peroxide, and metal ion cofactors for the oxidocyclization reaction. These results indicate that CBDA synthase is neither an oxygenase nor a peroxidase and that the enzymatic cyclization does not proceed via oxygenated intermediates. CBDA synthase catalyzes the formation of CBDA from cannabinerolic acid as well as cannabigerolic acid, although the kcat for the former (0.03 s-1) is lower than that for the latter (0.19 s-1). Therefore, we conclude that CBDA is predominantly biosynthesized from cannabigerolic acid rather than cannabinerolic acid.
The disc cell wall facing the secretory cavity in lipophilic glands of Cannabis was studied for origin and distribution of hyaline areas, secretory vesicles, fibrillar matrix and particulate material. Secretions evident as light areas in the disc cell cytoplasm pass through modified regions in the plasma membrane and appear as hyaline areas in the cell wall. Hyaline areas, surrounded with a filamentous outline, accumulate near the wall surface facing the secretory cavity where they fuse to form enlarged hyaline areas. Fibrillar matrix is related to and may originate from the dense outer layer of the plasma membrane. This matrix becomes distributed throughout the wall material and contributes in part to the composition of the surface feature of secretory vesicles. Thickening of the cell wall is associated with secretions from the disc cells that facilitates movement of hyaline areas, fibrillar matrix and other possible secretions through the wall to form secretory vesicles and intervesicular materials in the secretory cavity. The outer wall of disc cells in aggregate forms the basilar wall surface of the secretory cavity which facilitates the organization of secretory vesicles that fill the secretory cavity.
Δ1-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into
THCA, the precursor of Δ1-tetrahydrocannabinol. We cloned a novel cDNA (GenBank™ accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28
amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide
is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced
THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression
of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme
contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing
His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase
activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent.
This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis.
Tetrahydrocannabinolic acid (THCA) synthase is the enzyme responsible for the production of tetrahydrocannabinol (THC), the psychoactive component of marijuana (Cannabis sativa L.). We suggest herein that THCA is biosynthesized in the storage cavity of the glandular trichomes based on the following observations. (i) The exclusive expression of THCA synthase was confirmed in the secretory cells of glandular trichomes by reverse transcription-PCR (RT-PCR) analysis. (ii) THCA synthase activity was detected in the storage cavity content. (iii) Transgenic tobacco expressing THCA synthase fused to green fluorescent protein showed fluorescence in the trichome head corresponding to the storage cavity. These results also showed that secretory cells of the glandular trichomes secrete not only metabolites but also biosynthetic enzyme.
Laser-assisted microdissection (LAM) is a powerful tool for isolating specific tissues, cell types and even organelles from sectioned biological specimen in a manner conducive to the extraction of RNA, DNA or protein. LAM, which is an established technique in many areas of biology, has now been successfully adapted for use with plant tissues. Here, we provide an overview of the processes involved in conducting a successful LAM study in plants and review recent developments that have made this technique even more desirable. We also discuss how the technology might be exploited to answer some pertinent questions in plant biology.
Plant Methods is a new journal for plant biologists, specialising in the rapid publication of peer-reviewed articles with a focus on technological innovation in the plant sciences. The aim of Plant Methods is to stimulate the development and adoption of new and improved techniques and research tools in plant biology. We hope to promote more consistent standards in the plant sciences, and make readily accessible laboratory and computer-based research tools available to the whole community. This will be achieved by publishing Research articles, Methodology papers and Reviews using the BioMed Central Open Access publishing model. The journal is supported by a prestigious editorial board, whose members all recognise the importance of technological innovation as a driver for basic science.
Laser microdissection is a useful tool for collecting tissue-specific samples or even single cells from animal and plant tissue sections. This technique has been successfully employed to study cell type-specific expression at the RNA, and more recently also at the protein level. However, metabolites were not amenable to analysis after laser microdissection, due to the procedures routinely applied for sample preparation. Using standard tissue fixation and embedding protocols to prepare histological sections, metabolites are either efficiently extracted by dehydrating solvents, or washed out by embedding agents.
In this study, we used cryosectioning as an alternative method that preserves sufficient cellular structure while minimizing metabolite loss by excluding any solute exchange steps. Using this pre-treatment procedure, Arabidopsis thaliana stem sections were prepared for laser microdissection of vascular bundles. Collected samples were subsequently analyzed by gas chromatography-time of flight mass spectrometry (GC-TOF MS) to obtain metabolite profiles. From 100 collected vascular bundles (approximately 5,000 cells), 68 metabolites could be identified. More than half of the identified metabolites could be shown to be enriched or depleted in vascular bundles as compared to the surrounding tissues.
This study uses the example of vascular bundles to demonstrate for the first time that it is possible to analyze a comprehensive set of metabolites from laser microdissected samples at a tissue-specific level, given that a suitable sample preparation procedure is used.
Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy.
Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC) and selective ion trap mass spectrometry. The complete approach involves (i) enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii) gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment), (iii) identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv) identification of phosphorylation sites of these enriched proteins using automatic detection of H3PO4 neutral loss peaks and data-dependent MS3-fragmentation of the corresponding MS2-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures.
A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to proteins extracted from C. reinhardtii. Thus, the method can easily be adapted to suit the sample of interest since it is inexpensive and the components needed are widely available. Reproducibility of the approach was tested by monitoring phosphorylation sites on specific proteins from seeds and C. reinhardtii in duplicate experiments. The whole process is proposed as a strategy adaptable to other plant tissues providing high confidence in the identification of phosphoproteins and their corresponding phosphorylation sites.
Cannabis as a medicine was used before the Christian era in Asia, mainly in India. The introduction of cannabis in the Western medicine occurred in the midst of the 19th century, reaching the climax in the last decade of that century, with the availability and usage of cannabis extracts or tinctures. In the first decades of the 20th century, the Western medical use of cannabis significantly decreased largely due to difficulties to obtain consistent results from batches of plant material of different potencies. The identification of the chemical structure of cannabis components and the possibility of obtaining its pure constituents were related to a significant increase in scientific interest in such plant, since 1965. This interest was renewed in the 1990's with the description of cannabinoid receptors and the identification of an endogenous cannabinoid system in the brain. A new and more consistent cycle of the use of cannabis derivatives as medication begins, since treatment effectiveness and safety started to be scientifically proven.
Development of the secretory cavity and formation of the subcuticular wall of glandular trichomes in Cannabis sativa L. was examined by transmission electron microscopy. The secretory cavity originated at the wall‐cuticle interface in the peripheral wall of the discoid secretory cells. During the presecretory phase in development of the glandular trichome, the peripheral wall of the disc cells became laminated into a dense inner zone adjacent to the plasma membrane and a less dense outer zone subjacent to the cuticle. Loosening of wall matrix in the outer zone initiated a secretory cavity among fibrous wall materials. Membrane‐bound hyaline areas, compressed in shape, arose in the wall matrix. They appeared first in the outer and subsequently in the inner zone of the wall. The membrane of the vesicles, and associated dense particles attached to the membrane, arose from the wall matrix. Hyaline areas, often with a conspicuous electron‐dense content, were released into the secretory cavity where they formed rounded secretory vesicles. Fibrous wall material released from the surface of the disc cells became distributed throughout the secretory cavity among the numerous secretory vesicles. This wall material was incorporated into the developing subcuticular wall that increased five‐fold in thickness during enlargement of the secretory cavity. The presence of a subcuticular wall in the cavity of Cannabis trichomes, as contrasted to the absence of this wall in described trichomes of other plants, supports a polyphyletic interpretation of the evolution of the secretory cavity in glandular trichomes among angiosperms.
Cannabinoid levels of individual mature glandular trichomes from two clones and two strains of Cannabis sativa L., which included both drug and fiber phenotypes, were investigated by gas-liquid chromatographic analyses. Capitate-stalked glands were selectively harvested from vein and nonvein areas of pistillate bracts while capitate-sessile glands were harvested from these areas of leaves. The qualitative cannabinoid profile characteristic of the strain or clone was maintained in the individual capitate-stalked glands while the quantitative cannabinoid profiles varied with each strain or clone and between vein and nonvein areas as well. Capitate-sessile glands were found to contain conspicuously lower levels of cannabinoids than capitate-stalked glands. This study emphasizes that glands of Cannabis represent a dynamic system within the cannabinoid synthesizing activities of this plant.
Trichome density and type and cannabinoid content of leaves and bracts were quantitated during organ ontogeny for three clones of Cannabis sativa L. Trichome initiation and development were found to occur throughout leaf and bract ontogeny. On leaves, bulbous glands were more abundant than capitate-sessile glands for all clones, although differences in density for each gland type were evident between clones. On pistillate bracts, capitate-sessile glands were more abundant than the bulbous form on all clones, and both types decreased in relative density during bract ontogeny for each clone. The capitate-stalked gland, present on bracts but absent from vegetative leaves, increased in density during bract ontogeny. The capitate-stalked gland appeared to be initiated later than bulbous or capitate-sessile glands during bract development and on one clone it was first found midway in bract ontogeny. Nonglandular trichomes decreased in density during organ ontogeny, but the densities differed between leaves and bracts and also between clones. Specific regulatory mechanisms appear to exist to control the development of each trichome type independently. In addition, control of trichome density seems to be related to the plant organ and clone on which the gland type is located. Cannabinoid synthesis occurs throughout organ development and is selectively regulated in each organ. Typically, cannabinoid synthesis occurred at an increasing rate during bract development, whereas in developing leaves synthesis occurred at a decreasing rate. Cannabinoid content on a dry weight basis was generally greater for bracts than leaves. Analyses of leaves indicate that other tissues in addition to glands may contain cannabinoids, while for bracts the gland population can accommodate the cannabinoid content for this organ. The functional significance of trichomes and cannabinoids in relation to evolution is discussed.
Total synthesis of biologically interesting (?)-cannabichromene, (?)-cannabichromenic acid, and (?)-daurichromenic acid is described. The key step in the synthetic strategy involves the formation of benzopyrans by ethylenediamine diacetate-catalyzed reactions of resorcinols with \alpha,\beta-unsaturated aldehydes.
As part of our program to study the phytochemistry of high potency Cannabis sativa L. [1,2], seven new non-cannabinoid constituents were isolated, namely 5-acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone (1), 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene (2), 4-hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3), 4,7-dimethoxy-1,2,5-trihydroxyphenanthrene (4), α-cannabispiranol (5), cannflavin C (6) and β-sitosteryl-3-O-β-D-glucopyranoside-2'-O-palmitate (7). In addition, four known compounds, chrysoeriol (8), 6-prenyl-apigenin (9), cannflavin A (10) and β-acetyl cannabispiranol (11), were identified, with 8 and 9 being reported for the first time form cannabis. The antimicrobial, antileishmanial, antimalarial and anti-oxidant activities of these isolates were evaluated.
Furthermore the analgesic activity of 2, 3 and 4 was evaluated in mice using the hot-plate and tail-flick nociception models.
Acknowledgements: The project described was supported by Grant Number 5P20RR021929–02 from the National Center for Research Resources and by the National Institute on Drug Abuse, contract # N01DA-5–7746. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. We are grateful to Dr. Bharathi Avula for assistance with the HR-ESI-MS, and to Dr. Melissa Jacob, Ms. Marsha Wright and Dr. Babu Tekwani for conducting the antimicrobial and antiprotozoal testing.
References: 1. Radwan, M.M., et al. (2008). Planta Med. 74: 267–272.
2. Ahmed, S.A., et al. (2008)J. Nat. Prod. (In press).
Cannabis sativa L., one of the oldest plants known in medicine, is the most widely used illicit drug in the world today. A total of almost 500 natural constituents have been isolated and/or identified from cannabis [1], with Δ⁹-THC the main biologically active component [2]. The availability of high potency marijuana on the illicit market with unprecedented Δ⁹-THC concentrations (> 20% by dry weight)[3] has renewed our interest in the discovery of new constituents from cannabis. Phytochemical investigation of a high potency variety of C sativa L. resulted in the isolation of six new metabolites, (±)-6,7-trans-epoxycannabigerolic acid (1), (±)-6,7-cis-epoxycannabigerolic acid (2), (±)-6,7-cis-epoxycannabigerol (3), (±)-6,7-trans-epoxycannabigerol (4), 5ʹ-methyl-4-pentylbiphenyl-2,2ʹ,6-triol (5), and 7-methoxycannabispirone (5), along with seven known compounds (cannabigerolic acid, 5ʹ-methoxycannabigerolic acid, cannabispirone, β-cannabispiranol, dehydrocannabifuran, cannaflavin B and cannabigerol). The antimicrobial and antileishmanial activities were investigated. Acknowledgements: This work is supported by the Center of Research Excellence in Natural Products Neuroscience, The University of Mississippi, contract # 1P20RR021929-01, and by the National Institute on Drug Abuse, contract # N01DA-5-7746. We are grateful to Dr. Bharathi Avula for assistance with the HR-ESI-MS, and to Dr. Melissa Jacob and Ms. Marsha Wright for conducting the antimicrobial testing. References: [1] Grotenhermen F, Russo E, In Cannabis and Cannabinoids: Pharmacology, Toxicology, and Therapeutic Potential. Grotenhermen F, Russo E, Ed.; The Haworth Press, Inc.: Binghamton, New York, 2002; Definitions and Explanations, pp. xxvii–xxxi. [2] Clarke RC, Watson DP, In Cannabis and Cannabinoids: Pharmacology, Toxicology, and Therapeutic Potential. Grotenhermen F, Russo E, Ed.; The Haworth Press, Inc.: Binghamton, New York, 2002; Chapter 1 – Botany of Natural Cannabis Medicines, p.3–14.
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Development of the secretory cavity and formation of the subcuticular wall of glandular trichomes in Cannabis sativa L. was examined by transmission electron microscopy. The secretory cavity originated at the wall-cuticle interface in the peripheral wall of the discoid secretory cells. During the presecretory phase in development of the glandular trichome, the peripheral wall of the disc cells became laminated into a dense inner zone adjacent to the plasma membrane and a less dense outer zone subjacent to the cuticle. Loosening of wall matrix in the outer zone initiated a secretory cavity among fibrous wall materials. Membrane-bound hyaline areas, compressed in shape, arose in the wall matrix. They appeared first in the outer and subsequently in the inner zone of the wall. The membrane of the vesicles, and associated dense particles attached to the membrane, arose from the wall matrix. Hyaline areas, often with a conspicuous electron-dense content, were released into the secretory cavity where they formed rounded secretory vesicles. Fibrous wall material released from the surface of the disc cells became distributed throughout the secretory cavity among the numerous secretory vesicles. This wall material was incorporated into the developing subcuticular wall that increased five-fold in thickness during enlargement of the secretory cavity. The presence of a subcuticular wall in the cavity of Cannabis trichomes, as contrasted to the absence of this wall in described trichomes of other plants, supports a polyphyletic interpretation of the evolution of the secretory cavity in glandular trichomes among angiosperms.
Cannabinoid levels of individual mature glandular trichomes from two clones and two strains of Cannabis sativa L., which included both drug and fiber phenotypes, were investigated by gas-liquid chromatographic analyses. Capitate-stalked glands were selectively harvested from vein and nonvein areas of pistillate bracts while capitate-sessile glands were harvested from these areas of leaves. The qualitative cannabinoid profile characteristic of the strain or clone was maintained in the individual capitate-stalked glands while the quantitative cannabinoid profiles varied with each strain or clone and between vein and nonvein areas as well. Capitate-sessile glands were found to contain conspic uously lower levels of cannabinoids than capitate-stalked glands. This study emphasizes that glands of Cannabis represent a dynamic system within the cannabinoid synthesizing activities of this plant. GLANDULAR trichomes are prominent features on the shoot system of Cannabis saliva L. Sev eral studies using histochemical and analytical procedures
Twelve new cannabinoid esters, together with three known cannabinoid acids and Δ9-THC, were isolated from a high potency variety of Cannabis sativa L [1,2]. The structures were determined by extensive spectral analysis to be: β-fenchyl-Δ9-THCA ester (1), α-fenchyl-Δ9-THCA ester (2), bornyl-Δ9-THCA ester (3), epi-bornyl-Δ9-THCA ester (4), α-terpenyl-Δ9-THCA ester (5), 4-terpenyl-Δ9-THCA ester (6), α-cadinyl-Δ9-THCA ester (7), γ-eudesmyl-Δ9-THCA ester (8), inseparable mixture of two sesquiterpenyl-Δ9-THCA esters (9), α-cadinyl-CBGA ester (10), γ-eudesmyl-CBGA ester (11), 4-terpenyl-CBNA ester (12), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid A (Δ9-THCA), cannabinolic acid A (CBNA) and cannabigerolic acid (CBGA). CB-1 receptor assays [3–6] indicated that these esters, as well as the parent Δ9-THCA, are not active compared to Δ9-THC. Acknowledgements: This work is supported by the National Institute on Drug Abuse (contract # N01DA-5-7746) and by the Center of Research Excellence in Natural Products Neuroscience, The University of Mississippi (contract # 1P20RR021929-01). We are grateful to Dr. Bharathi Avula for assistance with the HR-ESI-MS, and to Dr. Melissa Jacob and Ms. Marsha Wright for conducting the antimicrobial testing. References: [1] ElSohly MA, et al. (2000) Journal of Forensic Science 45: 24–30. [2] ElSohly MA, Slade D (2005) Life Sciences 78: 539–548. [3] Devane WA, (1988) Molecular Pharmacology 34: 605–613. [4] Munro S, et al. (1993) Nature 365: 61–65. [5] Barth F (2005) Annual Reports in Medicinal Chemistry 40: 103–118. [6] Ashton JC, Giass M (2007) Current Neuropharmacology 5: 73–80.
Synthetic (−)-Δ9-trans-tetrahydrocannabinol impregnated on placebo Cannabis decomposed only 6.3% after being stored for 1 year at −18°. Storage at 5° and room temperature under various conditions led to severe decomposition. The amount of cannabinol observed when (−)-Δ9-trans-tetrahydrocannabinol decomposed indicates that cannabinol is not the only decomposition product.
Sessile- and capitate-stalked secretory glands are sites of cannabinoid accumulation in Cannabis (Cannabaceae). Analyses show cannabinoids to be abundant in glands isolated from bracts or leaves of pistillate plants. Cannabinoids are concentrated in the secretory cavity formed as an intrawall cavity in the outer wall of the disc cells. Specialized plastids, lipoplasts, in the disc cells synthesize lipophilic substances, such as terpenes, that migrate through the plasma membrane and into the cell wall adjacent to the secretory cavity. These substances enter the cavity as secretory vesicles. An antibody probe for THC shows it to be most abundant along the surface of vesicles, associated with fibrillar material in the cavity, in the cell wall and in the cuticle; little THC was detected in the cytoplasm of disc or other cells. The phenol, phloroglucinol, is abundant in both gland types. A working hypothesis for the site of cannabinoid synthesis is proposed, and must be examined further. Knowledge of the mechanism of cannabinoid synthesis and localization can contribute to efforts to further reduce the THC content in hemp strains for potential agricultural use in the United States and elsewhere.
The biosynthesis of cannabinoids was studied in cut sprouts of Cannabis sativa by incorporation experiments using mixtures of unlabeled glucose and [1-13C]glucose or [U-13C6]glucose. 13C-labeling patterns of cannabichromenic acid and tetrahydrocannabinolic acid were analyzed by quantitative NMR spectroscopy. 13C enrichments and coupling patterns show that the C10-terpenoid moiety is biosynthesized entirely or predominantly (> 98%) via the recently discovered deoxyxylulose phosphate pathway. The phenolic moiety is generated by a polyketide-type reaction sequence. The data support geranyl diphosphate and the polyketide, olivetolic acid, as specific intermediates in the biosynthesis of cannabinoids.
Delta 9-tetrahydrocannabinol (THC) localization in glandular trichomes and bracteal tissues of Cannabis, prepared by high pressure cryofixation-cryosubstitution, was examined with a monoclonal antibody-colloidal gold probe by electron microscopy (EM). The antibody detected THC in the outer wall of disc cells during the presecretory cavity phase of gland development. Upon formation of the secretory cavity, the immunolabel detected THC in the disc cell wall facing the cavity as well as the subcuticular wall and cuticle throughout development of the secretory cavity. THC was detected in the fibrillar matrix associated with the disc cell and with this matrix in the secretory cavity. The antibody identified THC on the surface of secretory vesicles, but not in the secretory vesicles. Gold label also was localized in the anticlinal walls between adjacent disc cells and in the wall of dermal and mesophyll cells of the bract. Grains were absent or detected only occasionally in the cytoplasm of disc or other cells of the bract. No THC was detected in controls. These results indicate THC to be a natural product secreted particularly from disc cells and accumulated in the cell wall, the fibrillar matrix and surface feature of vesicles in the secretory cavity, the subcuticular wall, and the cuticle of glandular trichomes. THC, among other chemicals, accumulated in the cuticle may serve as a plant recognition signal to other organisms in the environment.
Three new cannabichromanone derivatives were isolated from high potency cannabis, along with the known cannabichromanone. Full spectroscopic data, including the use of electronic circular dichroism and Mosher ester analysis to determine the absolute configuration of these compounds, are reported. All isolates were tested for antimicrobial, antimalarial, antileishmanial and anti-oxidant activity.
A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Delta11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1alpha was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes.
Nine new cannabinoids (1-9) were isolated from a high-potency variety of Cannabis sativa. Their structures were identified as (+/-)-4-acetoxycannabichromene (1), (+/-)-3''-hydroxy-Delta((4'',5''))-cannabichromene (2), (-)-7-hydroxycannabichromane (3), (-)-7R-cannabicoumarononic acid A (4), 5-acetyl-4-hydroxycannabigerol (5), 4-acetoxy-2-geranyl-5-hydroxy-3-n-pentylphenol (6), 8-hydroxycannabinol (7), 8-hydroxycannabinolic acid A (8), and 2-geranyl-5-hydroxy-3-n-pentyl-1,4-benzoquinone (9) through 1D and 2D NMR spectroscopy, GC-MS, and HRESIMS. The known sterol beta-sitosterol-3-O-beta-d-glucopyranosyl-6'-acetate was isolated for the first time from cannabis. Compounds 6 and 7 displayed significant antibacterial and antifungal activities, respectively, while 5 displayed strong antileishmanial activity.
Six new non-cannabinoid constituents were isolated from a high potency Cannabis sativa L. variety, namely 5-acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone (1), 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene (2), 4-hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3), 4,7-dimethoxy-1,2,5-trihydroxyphenanthrene (4), cannflavin C (5) and beta-sitosteryl-3-O-beta-d-glucopyranoside-2'-O-palmitate (6). In addition, five known compounds, alpha-cannabispiranol (7), chrysoeriol (8), 6-prenylapigenin (9), cannflavin A (10) and beta-acetyl cannabispiranol (11) were identified, with 8 and 9 being reported for the first time from cannabis. Some isolates displayed weak to strong antimicrobial, antileishmanial, antimalarial and anti-oxidant activities. Compounds 2-4 were inactive as analgesics.
Authors investigated some fiber and some hashish hemp sorts concerning their trichomes on the leaves. Histochemical reactions were developed using the Fast Blue Salt (FBB) reagent applied so far only thin layer chromatography. Glandular hairs were found giving positive reactions due to cannabinoids contained by the cells. The electron microscopic features were studied and the cannabinoid content was measured with GC. A correlation was found between the number of typical glandular hairs and cannabinoid content.
Zusammenfassung Die Synthese von (—)-Δ1(7)-Tetrahydrocannabinol (THC) auf (—)-Δ1(6) wird beschrieben. Das Hauptprodukt der oxidativen Zerstörung von (—)-Δ1-THC ist Cannabinol. Beschleunigte Versuche der Luftoxidation von (—)-Δ1-THC und (—)-Δ1(6)-THC haben gezeigt, dass das letztere beständiger ist. Dieser Unterschied kann mit der Reaktivität des C3-benzylisch-allylischen Wasserstoffes in (—)-Δ1-THC erklärt werden.
Gland number and cannabinoid content for three clones of Cannabis were compared. Cannabinoid levels and mature glands were quantified throughout progressive stages of pistillate bract development in order to define possible interrelationships between glands and cannabinoids. Similar trends occurred among the clones for each gland type while individual cannabinoids displayed a different pattern in each clone. Gland initiation and development as well as cannabinoid synthesis were found to occur during bract ontogeny for all three clones. A positive correlation existed for the total number of capitate glands per bract compared to the total cannabinoid content of the bract. The study also suggested that the glands may contain the majority of the cannabinoids present in the bract.
A new enzyme, geranylpyrophosphate:olivetolate geranyltransferase (GOT), the first enzyme in the biosynthesis of cannabinoids could be detected in extracts of young leaves of Cannabis sativa. The enzyme accepts geranylpyrophosphate (GPP) and to a lesser degree also nerylpyrophosphate (NPP) as a cosubstrate. It is, however, specific for olivetolic acid; its decarboxylation product olivetol is inactive as a prenyl acceptor.
Cannabichromenic acid synthase was purified to apparent homogeneity by sequential column chromatography including DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The enzyme catalysed the oxidocyclization of cannabigerolic acid and cannabinerolic acid to cannabichromenic acid. The K(m) values for both substrates were in the same order of magnitude although the Vmax value for the former was higher than that for the latter. These results suggested that cannabichromenic acid is predominantly formed from cannabigerolic acid rather than cannabinerolic acid. The enzyme required neither molecular oxygen nor hydrogen peroxide, indicating that the cannabichromenic acid synthase reaction proceeds through direct dehydrogenation without hydroxylation.
The biosynthesis of cannabinoids was studied in cut sprouts of Cannabis sativa by incorporation experiments using mixtures of unlabeled glucose and [1-(13)C]glucose or [U-(13)C(6)]glucose. (13)C-labeling patterns of cannabichromenic acid and tetrahydrocannabinolic acid were analyzed by quantitative NMR spectroscopy. (13)C enrichments and coupling patterns show that the C(10)-terpenoid moiety is biosynthesized entirely or predominantly (> 98%) via the recently discovered deoxyxylulose phosphate pathway. The phenolic moiety is generated by a polyketide-type reaction sequence. The data support geranyl diphosphate and the polyketide, olivetolic acid, as specific intermediates in the biosynthesis of cannabinoids.
Laser capture microdissection (LCM) is a powerful system which allows the isolation of selectively targeted cells from a tissue section for the analysis of gene-expression profiles of individual cells. The technique has been successfully used for the isolation of specific mammalian cells, mainly cancer cells. However, LCM has never been reported to be applied to the gene expression analysis of plant cells. We used a modified LCM system and successfully applied it to target and isolate phloem cells of rice leaf tissue whose morphology is apparently different from the surrounding cells. Total RNA was extracted from microdissected (approximately 150) phloem cells and the isolated RNA was used for the construction of a cDNA library following the T7 RNA polymerase amplification. Sequence analysis of 413 randomly chosen clones from the library revealed that there was a high level of redundancy in the population and the clones could be subclassified into 124 different groups that contained related sequences. Approximately 37% of both the redundant population and the non-redundant subgroups had novel components while approximately 63% were either homologues to the known genes reported to be localized in phloem of different plant species, or were homologues to other known genes. In situ hybridization revealed that putative amino acid permease, one of the non-redundant clones, was specifically expressed in the phloem. The results proved the effectiveness of construction of a specialized cDNA library from the specific plant cells.
Laser microdissection (LM) allows the collection of homogeneous tissue- and cell-specific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue- or even cell-specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there is as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryo-sectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two-dimensional gel electrophoresis (2-DE), or by high-efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC-MS/MS than for 2-DE, the combination of LMPC and LC-MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.
The cannabis plant (Cannabis sativa L.) and products thereof (such as marijuana, hashish and hash oil) have a long history of use both as a medicinal agent and intoxicant. Over the last few years there have been an active debate regarding the medicinal aspects of cannabis. Currently cannabis products are classified as Schedule I drugs under the Drug Enforcement Administration (DEA) Controlled Substances act, which means that the drug is only available for human use as an investigational drug. In addition to the social aspects of the use of the drug and its abuse potential, the issue of approving it as a medicine is further complicated by the complexity of the chemical make up of the plant. This manuscript discusses the chemical constituents of the plant with particular emphasis on the cannabinoids as the class of compounds responsible for the drug's psychological properties.
Laser microdissection was used as a tool to harvest secretory cavities (SC) from leaves of Dilatris pillansii Barker (Haemodoraceae) and from leaves and flowers of herbarium specimens of Dilatris corymbosa Berg. and Dilatris viscosa L. Cryogenic (1)H NMR spectroscopy and HPLC analysis of microdissected samples indicated specific accumulation of methoxyphenylphenalenones in the SC. The structures of two novel and a known natural product in the secretory tissue were confirmed by comparison with authentic compounds isolated from rhizomes and roots from which further phenylphenalenones and phenylphenalenone glucosides were isolated and identified by spectroscopic methods. How it will be possible to use the LMD technique to localize natural products in specific plant cell populations is also discussed.