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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.
... Specifically, hemp is endowed with secretory structures, namely trichomes, representing the main site to produce secondary metabolites. Among them, cannabinoids and terpenes are contained in a sort of resin released by capitate-stalked glandular hairs localized on flower bracts and, to a minor extent, by capitate sessile and bulbous trichomes occurring also on other vegetative organs [6]. Notably, volatile terpenes of hemp glandular trichomes are recovered as a yellowish and odorous EO [7,8]. ...
... In previous literature contributions, glandular trichomes of various morphotypes have been defined under diverse, controversial terms over time [6,9,[59][60][61]. Therefore, an update of trichome terminology would be highly desirable to redefine the gland morphotypes. ...
... The bulbous hairs occasionally exhibited faintly positive responses to terpenes and polyphenols. Cannabinoid production, however, takes place mainly in the capitate trichomes, especially the stalked ones, as was largely confirmed by gas-liquid chromatographic evidence, by the identification of the candidate biosynthetic genes [6], and by CARS microscopy [10]. Hooked hair-like lithocysts were observed on the bract, bracteole, and inflorescence axis surfaces. ...
New hemp (Cannabis sativa L.) strains developed by crossbreeding selected varieties represent a novel research topic worthy of attention and investigation. This study focused on the phytochemical characterization of nine hemp commercial cultivars. Hydrodistillation was performed in order to collect the essential oils (EO), and also the residual water and deterpenated biomass. The volatile fraction was analyzed by GC-FID, GC-MS, and SPME-GC-MS, revealing three main chemotypes. The polyphenolic profile was studied in the residual water and deterpenated biomass by spectrophotometric assays, and HPLC-DAD-MSn and 1H-NMR analyses. The latter were employed for quali–quantitative determination of cannabinoids in the deterpenated material in comparison with the one not subjected to hydrodistillation. In addition, the glandular and non-glandular indumentum of the nine commercial varieties was studied by means of light microscopy and scanning electron microscopy in the attempt to find a possible correlation with the phytochemical and morphological traits. The EO and residual water were found to be rich in monoterpene and sesquiterpene hydrocarbons, and flavonol glycosides, respectively, while the deterpenated material was found to be a source of neutral cannabinoids. The micromorphological survey allowed us to partly associate the phytochemistry of these varieties with the hair morphotypes. This research sheds light on the valorization of different products from the hydrodistillation of hemp varieties, namely, essential oil, residual water, and deterpenated biomass, which proved to be worthy of exploitation in industrial and health applications.
... For the purpose of phytocannabinoid profiling, NMR spectroscopy is used as (semi)quantitative method alone [20,209] or as an orthogonal technique to LC [216][217][218] or GC [219] for the purpose of qualitative peak assignment of major phytocannabinoids [20], chemical and morphological examination [220], chemotaxonomic classification [20,219,220], metabolomics-based chemovar distinction [51] or quantitative analysis of cannabis plant material without the need of pre-purification step [221], chromatographic separation or use of certified reference standards [219]. Cryogenic NMR spectroscopy combines improved sensitivity and noise reduction with a cryogenic cooling system for the receiver coil and preamplifiers. ...
... For the purpose of phytocannabinoid profiling, NMR spectroscopy is used as (semi)quantitative method alone [20,209] or as an orthogonal technique to LC [216][217][218] or GC [219] for the purpose of qualitative peak assignment of major phytocannabinoids [20], chemical and morphological examination [220], chemotaxonomic classification [20,219,220], metabolomics-based chemovar distinction [51] or quantitative analysis of cannabis plant material without the need of pre-purification step [221], chromatographic separation or use of certified reference standards [219]. Cryogenic NMR spectroscopy combines improved sensitivity and noise reduction with a cryogenic cooling system for the receiver coil and preamplifiers. ...
... Cryogenic NMR spectroscopy combines improved sensitivity and noise reduction with a cryogenic cooling system for the receiver coil and preamplifiers. Its improved spectral quality is employed in compound identification from mass limited samples and as orthogonal analytical technique to HPLC in phytocannabinoid profiling in laser-micro dissected samples of capitate-stalked and capitate-sessile trichomes [220]. ...
Cannabis is gaining increasing attention due to the high pharmacological potential and updated legislation authorizing multiple uses. The development of time- and cost-efficient analytical methods is of crucial importance for phytocannabinoid profiling. This review aims to capture the versatility of analytical methods for phytocannabinoid profiling of cannabis and cannabis-based products in the past four decades (1980–2021). The thorough overview of more than 220 scientific papers reporting different analytical techniques for phytocannabinoid profiling points out their respective advantages and drawbacks in terms of their complexity, duration, selectivity, sensitivity and robustness for their specific application, along with the most widely used sample preparation strategies. In particular, chromatographic and spectroscopic methods, are presented and discussed. Acquired knowledge of phytocannabinoid profile became extremely relevant and further enhanced chemotaxonomic classification, cultivation set-ups examination, association of medical and adverse health effects with potency and/or interplay of certain phytocannabinoids and other active constituents, quality control (QC), and stability studies, as well as development and harmonization of global quality standards. Further improvement in phytocannabinoid profiling should be focused on untargeted analysis using orthogonal analytical methods, which, joined with cheminformatics approaches for compound identification and MSLs, would lead to the identification of a multitude of new phytocannabinoids.
... 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.
... The others include cannabigerol (CBG), cannabichromene (CBC), (−)-Δ 8 -trans-tetrahydrocannabinols (Δ 8 -THC), cannabicyclol (CBLs), cannabielsoin (CBE), cannabinol (CBN), cannabinodiol (CBND), cannabitriol (CBT), and the miscellaneous cannabinoids [5,8]. Cannabinoids biosynthesis is primarily localised on the capitate-stalked trichomes mainly found on the flower and sugar leaf surface of the pistillate plant [9]. Disk-like structures formed on the trichome head synthesizes and secretes the cannabinoids which are then accumulated in the fibrillar matrix, the subcuticular wall, and the cuticle [9,10]. ...
... Cannabinoids biosynthesis is primarily localised on the capitate-stalked trichomes mainly found on the flower and sugar leaf surface of the pistillate plant [9]. Disk-like structures formed on the trichome head synthesizes and secretes the cannabinoids which are then accumulated in the fibrillar matrix, the subcuticular wall, and the cuticle [9,10]. Shortly after the recoveries of the cannabinoids, the cannabinoid receptors (CB1 and CB2) were identified and many endogenous ligands for the cannabinoid receptors were also recognised [11]. ...
Phytocannabinoids are isoprenylated resorcinyl polyketides produced mostly in glandular trichomes of Cannabis sativa L. These discoveries led to the identification of cannabinoid receptors, which modulate psychotropic and pharmacological reactions and are found primarily in the human central nervous system. As a result of the biogenetic process, aliphatic ketide phytocannabinoids are exclusively found in the cannabis species and have a limited natural distribution, whereas phenethyl-type phytocannabinoids are present in higher plants, liverworts, and fungi. The development of cannabinomics has uncovered evidence of new sources containing various phytocannabinoid derivatives. Phytocannabinoids have been isolated as artifacts from their carboxylated forms (pre-cannabinoids or acidic cannabinoids) from plant sources. In this review, the overview of the phytocannabinoid biosynthesis is presented. Different non-cannabis plant sources are described either from those belonging to the angiosperm species and bryophytes, together with their metabolomic structures. Lastly, we discuss the legal framework for the ingestion of these biological materials which currently receive the attention as a legal high.
... The use of aluminum foil is a common Raman trick to avoid the fluorescence produced by the glass [12]. The focus was set on the trichome heads because they are the site of the cannabinoid production [13][14][15][16] and the THC content is expected to be the highest (15 to 25%) and consequently, there is a stronger spectral signal. This was especially important because uninformative spectra were obtained when randomly focusing and irradiating on any bulk marijuana sample. ...
... Since the capitate-stalked and bulbous trichomes of the marijuana contain high cannabinoid accumulation levels [13,14], they were the target structures to be located using a microscope as shown in Figure 2 [15]. Figure 3 shows the average Raman spectrum of marijuana. ...
The Raman analysis of marijuana is challenging because of the sample’s easy photo-degradation caused by the laser intensity. In this study, optimization of collection parameters and laser focusing on marijuana trichome heads allowed collecting Raman spectra without damaging the samples. The Raman spectra of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) standard cannabinoids were compared with Raman spectra of five different types of marijuana: four Sativa varieties (Amnesia Haze, Amnesia Hy-Pro, Original Amnesia, and Y Griega) and one Indica variety (Black Domina). The results verified the presence of several common spectral bands that are useful for marijuana characterization. Results were corroborated by the quantum chemical simulated Raman spectra of their acid-form (tetrahydrocannabinol acid (THCA), cannabidiol acid (CBDA)) and decarboxylated cannabinoids (THC, CBD, and CBN). A chemometrics-assisted method based on Raman microscopy and OPLS-DA offered good classification among the different marijuana varieties allowing identification of the most significant spectral bands.
... Phytocannabinoids and terpenes accumulate in the secretory cavity of the glandular trichomes in C. Sativa [109,110], and are present in the highest quantity on the female flower of the plant. Male plants produce lower levels of phytocannabinoids [111]. ...
... Glandular trichomes consist of a saclike cavity packed with secretory vesicles known as glandular hairs. Glandular trichomes of C. sativa alter morphology and metabolite content during flower maturation, and phytocannabinoids/terpenes are found on the calyx and the underside of anthers of flowers, leaves and bracts [109]. Trichomes rupture due to environmental stress or damage (due to high temperatures and herbivorous Downloaded from http://portlandpress.com/neuronalsignal/article-pdf/doi/10.1042/NS20200080/918663/ns-2020-0080c.pdf by guest on 10 August 2021 consumption), resulting in the release of phytocannabinoids and terpenes as a noxious, sticky liquid on the plant surface. ...
Cannabidiol (CBD), one of the primary non-euphoric components in the Cannabis sativa L. plant, has undergone clinical development over the last number of years as a therapeutic for patients with Lennox-Gastaut syndrome and Dravet syndromes. This phytocannabinoid demonstrates functional and pharmacological diversity, and research data indicate that CBD is a comparable antioxidant to common antioxidants. This review gathers the latest knowledge regarding the impact of CBD on oxidative signalling, with focus on the proclivity of CBD to regulate antioxidants and control the production of reactive oxygen species. CBD is considered an attractive therapeutic agent for neuroimmune disorders, and a body of literature indicates that CBD can regulate redox function at multiple levels, with a range of downstream effects on cells and tissues. However, pro-oxidant capacity of CBD has also been reported, and hence caution must be applied when considering CBD from a therapeutic standpoint. Such pro- and antioxidant functions of CBD may be cell- and model-dependent, and may also be influenced by CBD dose, the duration of CBD treatment and the underlying pathology.
... 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] . ...
... 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
... These include growth conditions such as humidity, light quality and intensity, CO 2 concentration and mineral nutrition (Chandra et al., 2008;Chandra et al., 2017;Bernstein et al., 2019a). The tissue type is also an important factor as within the plant there is a locationand organ-specific distribution of the active secondary metabolites (Happyana et al., 2013;Bernstein et al., 2019a;Bernstein et al., 2019b). Phytocannabinoids are synthesized in glandular trichomes that are located in the highest density on the inflorescences of unfertilized female plants (Lipson Feder et al., 2021), and their accumulation varies in the different aerial parts (flowers, fan leaves, inflorescence leaves, stalk and stem). ...
Medical Cannabis and its major cannabinoids (−)-trans-Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are gaining momentum for various medical purposes as their therapeutic qualities are becoming better established. However, studies regarding their efficacy are oftentimes inconclusive. This is chiefly because Cannabis is a versatile plant rather than a single drug and its effects do not depend only on the amount of THC and CBD. Hundreds of Cannabis cultivars and hybrids exist worldwide, each with a unique and distinct chemical profile. Most studies focus on THC and CBD, but these are just two of over 140 phytocannabinoids found in the plant in addition to a milieu of terpenoids, flavonoids and other compounds with potential therapeutic activities. Different plants contain a very different array of these metabolites in varying relative ratios, and it is the interplay between these molecules from the plant and the endocannabinoid system in the body that determines the ultimate therapeutic response and associated adverse effects. Here, we discuss how phytocannabinoid profiles differ between plants depending on the chemovar types, review the major factors that affect secondary metabolite accumulation in the plant including the genotype, growth conditions, processing, storage and the delivery route; and highlight how these factors make Cannabis treatment highly complex.
... 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 absolutely vital to be able to quantitate the levels of the different acidic and neutral cannabinoids in C. sativa to have a complete cannabinoid profile of the plant. A GC-FID method was developed and validated for the analysis of 20 acidic and neutral cannabinoids, namely cannabidivarinic acid (CBDVA), cannabidiolic acid (CBDA), cannabinolic acid (CBNA), cannabielsoinic acid (CBEA), cannabicyclolic acid (CBLA), cannabichromenic acid (CBCA), trans-Δ9- tetrahydrocannabivarianic acid (Δ9-THCVA),trans-Δ9-tetrahydrocannabinolic acid A (Δ9-THCA), cannabigerolic acid (CBGA), cannabidivarian (CBDV), trans-Δ9-tetrahydrocannabivarian (THCV), cannabidiol (CBD), cannabicyclol (CBL), cannabichromene (CBC), trans-Δ8-tetrahydrocannabinol (Δ8-THC), trans-Δ9-tetrahydrocannabinol (Δ9-THC), cannabigerol (CBG), cannabinol (CBN), cannabitriol (CBT), and cannabielsoin (CBE). The method was applied for the analysis of different cannabis varieties grown at the University of Mississippi, National Center for Natural Products Research (NCNPR). The developed method is simple, sensitive, and reproducible for the quantitation of all 20 acidic and neutral cannabinoids with a limit of detection (LOD) as low as 0.1 ppm while limit of quantitation ranged from 0.25 ppm-0.5 ppm.
... Although CBD is present in both male and female plants, most are found in resin glands on trichomes of the female flower buds (Mahlberg and Kim, 2004). The terpenophenolic compounds are secreted from head cells of trichome glands, specifically from the capitate-stalked glandular hairs (Happyana et al., 2013). Female flowers require mild temperatures and high nitrogen concentrations and light intensity. ...
Forensic laboratories are required to have analytical tools to confidently differentiate illegal substances such as marijuana from legal products (i.e., industrial hemp). The Achilles heel of industrial hemp is its association with marijuana. Industrial hemp from the Cannabis sativa L. plant is reported to be one of the strongest natural multipurpose fibers on earth. The Cannabis plant is a vigorous annual crop broadly separated into two classes: industrial hemp and marijuana. Up until the eighteenth century, hemp was one of the major fibers in the United States. The decline of its cultivation and applications is largely due to burgeoning manufacture of synthetic fibers. Traditional composite materials such as concrete, fiberglass insulation, and lumber are environmentally unfavorable. Industrial hemp exhibits environmental sustainability, low maintenance, and high local and national economic impacts. The 2018 Farm Bill made way for the legalization of hemp by categorizing it as an ordinary agricultural commodity. Unlike marijuana, hemp contains less than 0.3% of the cannabinoid, Δ9-tetrahydrocannabinol, the psychoactive compound which gives users psychotropic effects and confers illegality in some locations. On the other hand, industrial hemp contains cannabidiol found in the resinous flower of Cannabis and is purported to have multiple advantageous uses. There is a paucity of investigations of the identity, microbial diversity, and biochemical characterizations of industrial hemp. This review provides background on important topics regarding hemp and the quantification of total tetrahydrocannabinol in hemp products. It will also serve as an overview of emergent microbiological studies regarding hemp inflorescences. Further, we examine challenges in using forensic analytical methodologies tasked to distinguish legal fiber-type material from illegal drug-types.
... Among these compounds, the most active compounds are Cannabinoids, which are a class of terpenophenolic compounds which are mainly accumulated in the trichome cavity. (11) Cannabinoids are identified and are categorized into 11 subclasses. These different classes of chemicals are of great importance from pharmacological scenarios. ...
Introduction: Cannabis is an annual dioecious plant, which shares its origins with the inception of the first agricultural human societies in Asia. Over the course of time different parts of the plant have been utilized for therapeutic and recreational purposes. Linnaeus was the first person to describe Cannabis as Cannabis sativa (C.sativa). Numerous bioactive phytochemicals are extracted from C. sativa that signal for medicinal development. Methods:The review aims to provide a different perspective of the ethnobotanical, taxonomy and chemical aspects from the ancient times of C. sativa. The study was conducted with the review of scientific papers from Pubmed, Scopus, Wiley Online Library, Springer, Elseveir, Science Direct, Taylor Francis and online textbooks of C. sativa. Results: C. sativa has its origin from Asia. It has traditional spiritual, household and therapeutic uses. Cannabis is a monotypic genera with three different varieties: C sativa var. sativa, C sativa var. indica, C sativa var. ruderalis. A total of 565 chemicals (120 cannabinoids and 445 non cannabinoids) have been recorded in Cannabis. Conclusions: Cannabis is an ethnobotanical rich and phytochemical significant therapeutic plant. Because of lack of scientific research, the taxonomic aspects are still hidden. This study recommends exploratory study on ethnobotanical, taxonomical and phytochemicals of Nepalese Cannabis.
... Entsprechende Oxidoreduktasen ((-)trans-Tetrahydrocannabinolsäure(THCS)-Synthase, Cannabidiolsäure(CBDS)-Synthase, Cannabichromensäure(CBCS)-Synthase) setzen CBGS weiter zu delta9-Tetrahydrocannabinolsäure (delta9-THCS), Cannabidiolsäure (CBDS) oder Cannabichromensäure (CBCS) um [5][6][7]. Die Cannabinoidsäuren werden in geringem Ausmaß bereits in der Pflanze durch Licht oder Wärme zu den neutralen, pharmakologisch wirksamen Cannabinoiden delta9-Tetrahydrocannabinol (delta9-THC), CBD und Cannabichromen (CBC) decarboxyliert [8,9]. Die weitere Decarboxylierung erfolgt durch Erhitzen beim Cannabiskonsum. ...
Zusammenfassung
Hintergrund
Cannabis ist weltweit immer noch die am häufigsten konsumierte illegale Droge, aber auch der Einsatz von Medizinalcannabis oder auch als Lebens‑/Nahrungsergänzungsmittel steigt stetig. Somit sind Kenntnisse über diese verschiedenen Produkte und die Komplexität der rechtlichen Einordnung von Cannabis für die Rechtsmedizin und die forensische Toxikologie von großer Relevanz.
Fragestellung
Ziel der Arbeit ist es, einen Überblick über aktuelle Trends des Cannabiskonsums zu geben und hierbei die verschiedenen Cannabisprodukte darzustellen sowie diese rechtlich einzuordnen.
Material und Methode
Für diese Übersichtarbeit wurde eine Literaturrecherche zu den verschiedenen Cannabisprodukten und ihrer rechtlichen Einordnung durchgeführt.
Ergebnisse
Beim Konsum von Tetrahydrocannabinol(THC)-reichem Cannabis zu Rauschzwecken ist ein Trend hin zu immer höheren THC-Gehalten im Pflanzenmaterial und zusätzlich zu intensiven Konsumformen wie dem „dabbing“ von Butan-Haschisch-Öl zu erkennen. Seit der Betäubungsmittelgesetzesänderung vom 10.03.2017 werden auch Cannabisblüten und -extrakte mit unterschiedlichen THC- bzw. Cannabidiol(CBD)-Gehalten auf Betäubungsmittelrezept stark zunehmend verordnet, einhergehend mit einer Steigerung der THC-Höchstverschreibungsmenge. Ein weiterer Trend besteht in dem Konsum von sogenannten CBD-Lifestyle-Produkten, die als Arzneimittel seit 2016 verschreibungspflichtig sind und als Lebensmittel nach Novel Food-Verordnung jeweils einzeln zu prüfen sind, ob sie als zulassungsbedürftiges neuartiges Lebensmittel einzustufen sind.
Schlussfolgerungen
Insgesamt ergibt sich für die forensisch-chemische sowie forensisch-toxikologische Beurteilung eine zunehmende Komplexität der potenziell konsumierten THC-haltigen Produkte, deren wissenschaftliche Untersuchung sowohl zur möglichen Differenzierbarkeit des Cannabismaterials als auch von biologischen Matrices nach Konsum verschiedener Cannabisprodukte notwendig macht.
... While C. sativa is often cultivated for its phytocannabinoids (i.e., secondary metabolites primarily concentrated in floral tissue that provide various medicinal and intoxicating effects), the plant can also be a source of oilseeds and fibers in its primary stems (Small and Marcus, 2002). The majority of the plant, particularly the upper surface of leaves and flowers, are coated with trichomes (Happyana et al., 2013;Spitzer-Rimon et al., 2019) that produce cannabinoid precursors and ultimately cannabinoids (De Backer et al., 2012;Burgel et al., 2020). Unpollinated pistillate flowers (i.e., sensimilla) are the portion of the plant harvested for recreational and biopharmaceutical use as these inflorescences contain up to 10 times more cannabinoids in them than vegetative tissue . ...
Cannabis sativa L. is an annual, short-day plant, such that long-day lighting promotes vegetative growth while short-day lighting induces flowering. To date, there has been no substantial investigation on how the switch between these photoperiods influences yield of C. sativa despite the tight correlation that plant size and floral biomass have with the timing of photoperiod switches in indoor growing facilities worldwide. Moreover, there are only casual predictions around how the timing of the photoperiodic switch may affect the production of secondary metabolites, like cannabinoids. Here we use a meta-analytic approach to determine when growers should switch photoperiods to optimize C. sativa floral biomass and cannabinoid content. To this end, we searched through ISI Web of Science for peer-reviewed publications of C. sativa that reported experimental photoperiod durations and results containing cannabinoid concentrations and/or floral biomass, then from 26 studies, we estimated the relationship between photoperiod and yield using quantile regression. Floral biomass was maximized when the long daylength photoperiod was minimized (i.e., 14 days), while THC and CBD potency was maximized under long day length photoperiod for ~42 and 49–50 days, respectively. Our work reveals a yield trade-off in C. sativa between cannabinoid concentration and floral biomass where more time spent under long-day lighting maximizes cannabinoid content and less time spent under long-day lighting maximizes floral biomass. Growers should carefully consider the length of long-day lighting exposure as it can be used as a tool to maximize desired yield outcomes.
... THCA-B (6) was discovered four years later by Raphael Mechoulam at the University of Jerusalem (Mechoulam et al. 1969). THCA synthase is expressed primarily in glandular trichomes, where THCA is both formed and stored (Happyana et al. 2013). The most likely explanation for plants producing precannabinoids here lies in their cytotoxicity, as both 4 and THCA cause cell death in cultures of Cannabis as well as insect cells (e.g., Spodoptera frugiperda). ...
Cannabis spp. are some of the most controversial medicinal plants in the world. They contain great amounts of biologically active secondary metabolites, including the typical phenolic compounds called cannabinoids. Because of their low toxicity and complex biological activities, cannabinoids can be useful in the therapy of various diseases, but adverse psychological effects (of Δ9-THC in particular) raise concerns. This review summarizes the current knowledge of selected active C. indica compounds and their therapeutic potential. We summarize the main compounds contained in cannabis, the mechanisms of their effects, and their potential therapeutic applications. Further, we mention some of the clinical tests used to evaluate the efficacy of cannabinoids in therapy.
... Two studies are particularly illustrative: First, as determined by LCMS, the relative trichome concentrations of THC and CBD were found to be *1-2% of that for their precursors THCA-A and CBDA. 66 Also, a second study using quantitative NMR spectroscopy of trichome content (with low-temperature fluid handling) measured the peak concentrations of THC and CBD (during week 7 of the Cannabis flowering period) as about 0.5 mg/g of trichome weight. 65 Therefore, measurable acidic cannabinoid decarboxylation does occur to a limited extent even in the living Cannabis plant during its flowering stage. ...
Introduction: Cannabis is a valuable plant, cultivated by humans for millennia. However, it has only been in the past several decades that biologists have begun to clarify the interesting Cannabis biosynthesis details, especially the production of its fascinating natural products termed acidic cannabinoids. Discussion: Acidic cannabinoids can experience a common organic chemistry reaction known as decarboxylation, transforming them into structural analogues referred to as neutral cannabinoids with far different pharmacology. This review addresses acidic and neutral cannabinoid structural pairs, when and where acidic cannabinoid decarboxylation occurs, the kinetics and mechanism of the decarboxylation reaction as well as possible future directions for this topic. Conclusions: Acidic cannabinoid decarboxylation is a unique transformation that has been increasingly investigated over the past several decades. Understanding how acidic cannabinoid decarboxylation occurs naturally as well as how it can be promoted or prevented during harvesting or storage is important for the various stakeholders in Cannabis cultivation.
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.
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.
Plants of Cannabis sativa L. (Cannabaceae) produce an array of more than 160 isoprenylated resorcinyl polyketides, commonly referred to as phytocannabinoids. These compounds represent molecules of therapeutic importance due to their modulation of the human endocannabinoid system (ECS). While understanding of the biosynthesis of the major phytocannabinoids Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD) has grown rapidly in recent years, the biosynthetic origin and genetic regulation of many potentially therapeutically relevant minor phytocannabinoids remains unknown, which limits the development of chemotypically elite varieties of C. sativa. This review provides an up-to-date inventory of unusual phytocannabinoids which exhibit cannabimimetic-like activities and proposes putative metabolic origins. Metabolic branch points exploitable for combinatorial biosynthesis and engineering of phytocannabinoids with augmented therapeutic activities are also described, as is the role of phytocannabinoid remodelling to accelerate therapeutic portfolio expansion in C. sativa.
Analysis of hemp collection samples based on the content of minor (rare) non-psychotropic cannabinoids, such as cannabichromene (CBC), cannabidivarin (CBDV), and cannabinol (CBN); determination of correlation relationships between them and common compounds; selection of valuable breeding genotypes. Methods. Field, biochemical (gas chromatography of cannabinoid compounds), and statistical (pair, partial, and multiple linear correlations). Results. Quantitative analysis of 210 samples of various ecological-geographical and genetic origin (local and wild forms, self-filing lines, hybrids, varieties, synthetic populations, polyploids) with a tetrahydrocannabinol (THC) content of less than 0.08% in dried plants showed the level of manifestation of the trait from its absence within the sensitivity of the gas chromatograph up to 0.6838% CBC, 0.1719% CBC and 0.3274% CBN. In the studied hemp samples, a medium negative relationship was found between the signs of the CBC and cannabidiol (CBD) contents (r = –0.53), a weak negative relationship between CBC and CBDV contents (r = –0.35), medium positive relationships between the signs of CBC and THC contents (r = 0.57) and CBC and CBN contents (r = 0.59). A medium positive correlation (r = 0.57) was found between the signs of CBDV and CBD contents, while CBN had a strong positive relationship with THC (r = 0.82). There is almost no correlation between cannabigerol (CBG) and the minor cannabinoids under study. The biosynthesis of minor cannabinoid compounds is quite complex. Signs manifestation is affected by many genetic and external factors. Partial correlation coefficients (given that one of the three signs is eliminated) and multiple correlation coefficients (given that the relationship of one sign is determined and two other signs are combined) give grounds to state that the gene for CBCA-synthase affects the production of CBD and, in particular THC. Conclusions. The closeness of the linear relationships between minor cannabinoids and common components allows selecting valuable hemp samples with a high content of one or several compounds under the absence or low content of psychotropic THC.
Cannabis sativa L. has been one of the oldest medicinal plants cultivated since 10,000 years for several agricultural and industrial applications. However, the plant became controversial due to some psychoactive components that have adverse effects on human health. In this review, we analyzed the trends in cannabis research for the past two centuries. We discussed the historical transitions of cannabis from the category of an herbal medicine to an illicit drug and back to a medicinal product post-legalization. In addition, we address the new-age application of immuno-suppressive and anti-inflammatory extracts for the treatment of COVID-19 inflammation. We further address the influence of the legal aspects of cannabis cultivation for medicinal, pharmaceutical, and biotechnological research. We reviewed the up-to-date cannabis genomic resources and advanced technologies for their potential application in genomic-based cannabis improvement. Overall, this review discusses the diverse aspects of cannabis research developments ranging from traditional use as an herbal medicine to latest potential in COVID, legal practices with updated patent status, and current state of art genetic and genomic tools reshaping cannabis biotechnology in modern age agriculture and pharmaceutical industry.
Cembranoids are a class of glandular trichome exudates with a variety of structural variations and biological activities, and are found in high quantities in tobacco (Nicotiana tabacum L.). However, few studies have conducted preparative separation and examined the activities of cembranoids other than (1S,2E,4S,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (α-CBT-diol, 1) and (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (β-CBT-diol, 2); thus, the structure-activity relationship (SAR) of cembranoids remains unclear. Here, to deduce the SAR of cembranoids, raw cembranoid extract was systematically separated through an activity-oriented approach with preparative reversed-phase liquid chromatography. Each fraction was identified, and their antifungal, insecticidal, and cytotoxic bioactivities were evaluated. Composition analysis and activity detection of primary fractions (Fr.1–Fr.10) revealed that cembranoid activity might be influenced by the number of hydroxyl groups and degree of unsaturation (macrocycle and double bonds). Sub-fraction analysis showed that the antifungal activity of cembranoids was related to the number of hydroxyl groups and double bonds, and that their cytotoxic activity was affected by the type and position of substituents. Furthermore, all four factors could influence insecticidal activity. By conducting in-depth studies of single cembranoids, their SARs were summarized. The existence of Δ11,12/Δ7,8 and hydroxylation at C-6/C-4 contributed to the antifungal activity of cembranoids. Variation in the type and position of substituents also significantly affected antifungal activity, of which Δ11,12 was the most influential factor. Additionally, the change in substituent configuration at C-4 and C-12 had no significant effect on antifungal activity. The insecticidal activity of cembranoids benefited from the existence of 4-hydroxyl, which greatly enhanced their binding affinity; however, functional groups substituted at other positions decreased insecticidal activity. Cytotoxic activity was positively correlated with α-configuration at C-4 and hydroxylation at C-6 but inversely correlated with 4-methoxy substitution. Among them, 6-hydroxyl formed the core; the α-/β-configuration at C-12 and 4-/12-substitution of cembranoids did not affect their insecticidal activity. Insecticidal activity might play a key role by restraining acetylcholinesterase activity through hydrophobic interactions between the cembranoid carbon skeleton and residues located at the acetylcholinesterase binding pocket. These findings may provide a foundation for the development of novel cembranoid-based pesticides and medicines.
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.
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.