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![A crude plant ethanol extract (namely, Futura 75) was analyzed by applying the ICCA protocol. The chromatogram of (−)-Δ 9 -cis-THC [(−)-3] spiked with CBD (2) (marked with an asterisk) has been added for peak identification. The dashed lines indicate the retention time of the (+)-Δ 9 -cis-THC (if present) on the column with inverted chirality. In the inset (on the right) it is possible to identify and integrate (on the (R,R)-Whelk-O1 column) the peak relative to (+)-Δ 9 -cis-THC [(+)-3] (pointed with a red arrow).](publication/353448538/figure/fig1/AS:1049618666254339@1627259999979/A-crude-plant-ethanol-extract-namely-Futura-75-was-analyzed-by-applying-the-ICCA.png)
A crude plant ethanol extract (namely, Futura 75) was analyzed by applying the ICCA protocol. The chromatogram of (−)-Δ 9 -cis-THC [(−)-3] spiked with CBD (2) (marked with an asterisk) has been added for peak identification. The dashed lines indicate the retention time of the (+)-Δ 9 -cis-THC (if present) on the column with inverted chirality. In the inset (on the right) it is possible to identify and integrate (on the (R,R)-Whelk-O1 column) the peak relative to (+)-Δ 9 -cis-THC [(+)-3] (pointed with a red arrow).
Source publication
The cis-stereoisomers of Δ⁹-THC [(−)-3 and (+)-3] were identified and quantified in a series of low-THC-containing varieties of Cannabis sativa registered in Europe as fiber hemp and in research accessions of cannabis. While Δ⁹-cis-THC (3) occurs in cannabis fiber hemp in the concentration range of (−)-Δ⁹-trans-THC [(−)-1], it was undetectable in a...
Contexts in source publication
Context 1
... Thus, a column switch will result in inverted retention times for a pair of enantiomers, making it possible to identify enantiomers and evaluate enantiomeric excesses even when only one enantiomer of a chiral compound is available. To implement this strategy, samples of synthetic (−)-Δ 9 -cis -THC [(−)-3] 16 as well as (−)-CBD [(−)-2] were analyzed in the popular hemp strain Futura 75 on two columns (R,R)-Whelk-O1 and (S, S)-Whelk-O1, which met the ICCA requirements ( Figure 3). ...
Context 2
... the (S,S)-Whelk-O1 column, (−)-Δ 9 -cis-THC [(−)-3] eluted at 4.75 min, which is well before CBD (2), the main phytocannabinoid constituent of the extract (Figure 3, blue trace = standards; green trace = extract). In accordance with the ICCA protocol, 18 (+)-Δ 9 -cis-THC [(+)-3] (red trace) eluted at the same retention time on the (R,R)-Whelk-O1 column. ...
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Citations
... This justifies why THC (and other molecules with partial or full agonistic activity on CB1, including the weak psychoactive D9-cis-THC enantiomer, recently demonstrated to occur naturally in Hemp-derived nutraceuticals, both phytocannabinoids and non-cannabinoids, with potential effects on the gastrointestinal tract and the brain-gut axis. (Schafroth et al., 2021), should be absent (or at low, non-psychotropic levels) in hemp-derived food products, as a minimum health safety requirement. With antagonistic or negative allosteric activity on CB1 receptors as an outstanding feature, the pharmacology of CBD is very complex (summarized in Table 5) and underlies its broad biological activities, including neuroprotection, analgesia, antiinflammatory and immune-modulating effects, anxiolytic, spasmolytic, anticonvulsant and antipsychotic effects, mood stabilization, and normalization of sleep disorders, just to name a few (Kicman and Toczek, 2020;Mlost et al., 2020;Britch et al., 2021;Franco et al., 2021;Vitale et al., 2021). ...
Nutrition security is a challenge of the XXI century for achieving a sustainable health. Hemp cultivation contributes to the European Green Deal objectives and is a potential solution for producing a more sustainable food chain and contributing to the nutrition security of the global population. Hemp, Cannabis sativa cultivars containing less than 0.2% of Δ9-tetrahydrocannabinol (THC), is a multipurpose crop which can be used to produce feed, food and supplements among other products (biodegradable plastics, paper, paint). Hemp seeds are the hemp component most used in the food context, and the products derived from them (oil, cake, flour and proteins) are gaining popularity in human nutrition. In the European Union (EU), only marketing of hemp seeds and their derivatives, such as hemp seed oil, hemp seed flour, defatted hemp seed, and germinated hemp seed is authorized. Other parts of the plant are considered as novel foods. Nutrition claims “high dietary fiber, high protein, low saturated fat, high omega-3 fatty acids, high polyunsaturated fat, high unsaturated fat” can be attributed to those hemp products. In addition, hemp is a source of bioactive compounds, cannabinoids and others, with great impact in health including that of the brain-gut axis which is essential for achieving optimal physical and emotional conditions. The present chapter represents an updated revision of the state of the art on the potential of hemp in nutrition security.
... Thus, ∆ 9 -THCA-A (5b) retains significant activity in displacement-based assays, while ∆ 9 -THCA-A (5b) is even more powerful than ∆ 9 -THC (1) in these assays, but both compounds are almost inactive in functional assays of β-arresting 2 recruitment, and additional studies identified ∆ 9 -THCV (5a) as a CB1 antagonist or very weak agonist and ∆ 9 -THCA-A (5b) as an allosteric activator of CB1 [15]. (−)-cis-∆ 9 -THC (6, Figure 2) duplicated, albeit with less potency, the activity of (trans)-∆ 9 -THC at CB1 [16], while CBN (7) is a weak partial agonist of CB1 in both radioligand-based and in functional assays [17]. Conversely, anhydrocannabimovone (8, Figure 2) shows significant binding activity at CB1 [18]. ...
... antagonist or very weak agonist and Δ 9 -THCA-A (5b) as an allosteric activator of CB1 [15]. (-)-cis-Δ 9 -THC (6, Figure 2) duplicated, albeit with less potency, the activity of (trans)-Δ 9 -THC at CB1 [16], while CBN (7) is a weak partial agonist of CB1 in both radioligand-based and in functional assays [17]. Conversely, anhydrocannabimovone (8, Figure 2) shows significant binding activity at CB1 [18]. ...
... Significant activity in binding and functional assays has been reported for the acidic phytocannabinoids Δ 9 -THCA-A (5b) and CBDA (9b), with CBDV (9a) showing minor potency [17]. (-)-cis-Δ 9 -THC (6) [16], CBDV (9a), CBN (7) and anhydrocannabimovone (8) [18] showed significant activity in binding assays. Marginal activity was, conversely, reported for CBGA (10b), as well as for other minor phytocannabinoids [17]. ...
Despite the very large number of phytocannabinoids isolated from Cannabis (Cannabis sativa L.), bioactivity studies have long remained focused on the so called “Big Four” [Δ9-THC (1), CBD (2), CBG (3) and CBC (4)] because of their earlier characterization and relatively easy availability via isolation and/or synthesis. Bioactivity information on the chemical space associated with the remaining part of the cannabinome, a set of ca 150 compounds traditionally referred to as “minor phytocannabinoids”, is scarce and patchy, yet promising in terms of pharmacological potential. According to their advancement stage, we sorted the bioactivity data available on these compounds, better referred to as the “dark cannabinome”, into categories: discovery (in vitro phenotypical and biochemical assays), preclinical (animal models), and clinical. Strategies to overcome the availability issues associated with minor phytocannabinoids are discussed, as well as the still unmet challenges facing their development as mainstream drugs.
... Cannabinoids content in different hemp cultivars and hemp seed oil, hemp distillate, hemp shatter and oils were performed by LC-PDA [65,72] and LC-DAD [86] detectors, GC-tandem mass spectrometry (GC-MS/MS) [81], LC-tandem mass spectrometry (LC-MS/MS) [76], and HRMS [70]. ...
... A GC-MS/MS method separated and quantified Δ9-THC stereoisomers (Δ9-cis-THC and Δ9-trans-THC) in hemp strains [81]. An enantioselective analytical method established the absolute configuration and enantiomeric excess of naturally occurring Δ9-cis-THC using the inverted chirality column approach in normal-phase enantioselective UHPLC. ...
Δ9-tetrahydrocannabinol (Δ9-THC) isomers, especially Δ8-tetrahydrocannabinol (Δ8-THC), are increasing in foods, beverages, and e-cigarettes liquids. A major factor is passage of the Agriculture Improvement Act (AIA) that removed hemp containing less than 0.3% Δ9-THC from the definition of “marijuana” or cannabis. CBD-rich hemp flooded the market resulting in excess product that could be subjected to CBD cyclization to produce Δ8-THC. This process utilizes strong acid and yields toxic byproducts that frequently are not removed prior to sale and are currently inadequately studied.
Pharmacological activity is qualitatively similar for Δ8-THC and Δ9-THC, but most preclinical studies in mice, rats, and monkeys documented greater ∆9-THC potency. Both isomers caused graded dose-response effects on euphoria, blurred vision, mental confusion and lethargy, although Δ8-THC was at least 25% less potent. The most common analytical methodologies providing baseline resolution of ∆8-THC and ∆9-THC in non-biological matrices are liquid-chromatography coupled to diode-array detection (LC-DAD or LC-PDA), while liquid chromatography coupled to mass spectrometry is preferred for biological matrices. Other available analytical methods are gas-chromatography-mass spectrometry (GC-MS) and quantitative nuclear magnetic resonance (QNMR). Current knowledge on the pharmacology of ∆8-THC and other ∆9-THC isomers are reviewed to raise awareness of the activity of these isomers in cannabis products, as well as analytical methods to discriminate ∆9-THC qualitatively, and quantitatively and ∆8-THC in biological and non-biological matrices.
... As reported in the literature, the (-)-trans isomer is the most abundant form found in cannabis extracts, while the others are present only in trace [8]. Although Smith reported the isolation of cis-Δ 9 -THC by HPLC-UV in seized marijuana samples for the first time in 1977 [9], the real existence of cis-Δ 9 -THC has been dismissed until a work by Schafroth et al. appeared in 2021 [10]. The latter described the quantification of cis-Δ 9 -THC in low THC-containing industrial hemp, thus confirming the natural occurrence of this stereoisomer of Δ 9 -THC. ...
... The latter described the quantification of cis-Δ 9 -THC in low THC-containing industrial hemp, thus confirming the natural occurrence of this stereoisomer of Δ 9 -THC. However, the authors declared they were unable to isolate the natural compound despite the apparently high amount, in most cases comparable to that of trans-Δ 9 -THC [10]. Their experiments led to the conclusion that cis-Δ 9 -THC might originate either from the same process that generates CBD and trans-Δ 9 -THC through a specific oxidocyclase or the one that leads to cannabichromene (CBC) from cannabigerolic acid (CBGA) through a pericyclic cyclase [10]. ...
... However, the authors declared they were unable to isolate the natural compound despite the apparently high amount, in most cases comparable to that of trans-Δ 9 -THC [10]. Their experiments led to the conclusion that cis-Δ 9 -THC might originate either from the same process that generates CBD and trans-Δ 9 -THC through a specific oxidocyclase or the one that leads to cannabichromene (CBC) from cannabigerolic acid (CBGA) through a pericyclic cyclase [10]. Nonetheless, the origin of this molecule is far from being elucidated as all analyses were carried out on heated cannabis extracts, which unavoidably are characterized by the predominant presence of the decarboxylated species of all phytocannabinoids [9,10]. ...
Cannabidiolic acid (CBDA) and trans-Δ⁹-tetrahydrocannabinolic acid (trans-Δ⁹-THCA) are known to be the major phytocannabinoids in Cannabis sativa L., along with their decarboxylated derivatives cannabidiol (CBD) and trans-Δ⁹-tetrahydrocannabinol (trans-Δ⁹-THC). The cis isomer of Δ⁹-THC has been recently identified, characterized and quantified in several Cannabis sativa varieties, which had been heated (decarboxylated) before the analysis. Since decarboxylation alters the original phytocannabinoids composition of the plant, this work reports the identification and characterization of the carboxylated precursor cis-Δ⁹-THCA. The compound was also synthesized and used as analytical standard for the development and validation of a liquid chromatography coupled to high resolution mass spectrometry-based method for its quantification in ten Cannabis sativa L. samples from different chemotypes. The highest concentrations of cis-Δ⁹-THCA were found in CBD-rich varieties, lower levels were observed in cannabigerol (CBG)-rich varieties (chemotype IV) and in those varieties with a balanced level of both CBD and THC (chemotype III), while its levels were not detectable in cannabichromene (CBC)-rich varieties (chemotype VI). The presence of the cis isomer of THC and THCA raises the question on whether to include or not this species in the calculation of the total amount of THC to classify a cannabis variety as a drug-type or a fiber-type (hemp).
... Only recently, due to the increasing popularity of cannabis products and recent legalization, there has been a growing interest towards the characterization of cannabinoids also in terms of enantiomeric purity and stereostability. A relevant example is the discovery of (+)-9 -THC in trace amounts in medicinal cannabis extracts [5,6] , where only the ( −)-9 -THC was believed to be present. It is well known that the biological activity of cannabinoids is affected by the stereochemistry since interactions between these molecules and endocannabinoid receptors are mediated by specific stereochemical requirements [7] . ...
The growing popularity of cannabis products and recent legalization of cannabis for recreational purposes have contributed to the increase of the demand for analytical methods able to give a detailed characterization of cannabis samples and derivatives. In this context, one of the aspects that is strongly emerging is about the hazardous potential of uncharacterised minor cannabinoids, including chiral ones, for which achiral potency testing methods currently employed do not give any information. For this reason, there is a growing interest towards the development of liquid chromatographic methods for the enantioseparation of cannabinoids. Much work is needed in this field where one of the major limitations is the lack of optically pure standards. This manuscript reports about the chromatographic behavior of five popular cannabinoids (including the cannabichromene racemate, CBC) on nine immobilised polysaccharide-based chiral stationary phases (CSPs) differently substituted, under reversed phase conditions. Results showed that chemo-selectivity of CSPs is not affected by changes in mobile phase composition, in the range of mobile phase investigated. In addition, the presence of electron withdrawing groups on the CSPs systematically leads to shorter retention times compared to when electron donating groups are present. An application of separation of cannabinoids from a real hemp extract on two of the chiral columns employed in this work revealed the presence of both CBC enantiomers in the sample.
... As reported in the literature, the (-)-trans isomer is the most abundant form found in cannabis extracts, while the others are present only in trace [8]. Although Smith reported the isolation of cis-Δ 9 -THC by HPLC-UV in seized marijuana samples for the first time in 1977 [9], the real existence of cis-Δ 9 -THC has been dismissed until a work by Schafroth et al. appeared in 2021 [10]. The latter described the quantification of cis-Δ 9 -THC in low THC-containing industrial hemp, thus confirming the natural occurrence of this stereoisomer of Δ 9 -THC. ...
... The latter described the quantification of cis-Δ 9 -THC in low THC-containing industrial hemp, thus confirming the natural occurrence of this stereoisomer of Δ 9 -THC. However, the authors declared they were unable to isolate the natural compound despite the apparently high amount, in most cases comparable to that of trans-Δ 9 -THC [10]. Their experiments led to the conclusion that cis-Δ 9 -THC might originate either from the same process that generates CBD and trans-Δ 9 -THC through a specific oxidocyclase or the one that leads to cannabichromene (CBC) from cannabigerolic acid (CBGA) through a pericyclic cyclase [10]. ...
... However, the authors declared they were unable to isolate the natural compound despite the apparently high amount, in most cases comparable to that of trans-Δ 9 -THC [10]. Their experiments led to the conclusion that cis-Δ 9 -THC might originate either from the same process that generates CBD and trans-Δ 9 -THC through a specific oxidocyclase or the one that leads to cannabichromene (CBC) from cannabigerolic acid (CBGA) through a pericyclic cyclase [10]. Nonetheless, the origin of this molecule is far from being elucidated as all analyses were carried out on heated cannabis extracts, which unavoidably are characterized by the predominant presence of the decarboxylated species of all phytocannabinoids [9,10]. ...
The evaluation of the chiral composition of phytocannabinoids in the cannabis plant is particularly important as the pharmacological effects of the (+) and (-) enantiomers of these compounds are completely different. Chromatographic
attempts to assess the presence of the minor (+) enantiomers of the main phytocannabinoids, cannabidiolic acid (CBDA) and trans-Δ9-tetrahydrocannabinolic acid (trans-Δ9-THCA), were carried out on heated plant extracts for the determination of the corresponding decarboxylated species, cannabidiol (CBD) and
trans-Δ9-tetrahydrocannabinol (trans-Δ9-THC), respectively. This process produces an altered phytocannabinoid composition with several new and unknown decomposition products. The present work reports for the first time the stereoselective synthesis of the pure (+) enantiomers of the main phytocannabinoids, trans-CBDA, trans-Δ9-THCA, trans-CBD and trans-Δ9-THC, and the development and optimization of an achiral-chiral liquid chromatography method coupled to UV and high-resolution mass spectrometry detection in reversed phase conditions (RP-HPLC-UV-HRMS) for the isolation of the single compounds and evaluation of their actual enantiomeric composition in plant. The isolation of the peaks with the achiral stationary phase ensured the absence of interferences that could potentially co-elute with the analytes of interest in the chiral analysis. The method applied
to the Italian medicinal cannabis variety FM2 revealed no trace of the (+) enantiomers for all phytocannabinoids under investigation before and after decarboxylation, thus suggesting that the extraction procedure does not lead to an inversion of configuration.
Until recently, chirality has not been a major focus in the study of cannabinoids, as most cannabinoids of interest, such as cannabidiol and tetrahydrocannabinol, exist as a single isomer from natural sources. However, this is changing as more cannabinoids are identified, and compounds such as cannabichromene and cannabicyclol are emerging as potential investigatory candidates for varying indications. Because these molecules are chiral, the separation and study of the individual enantiomers’ biological and physiological effects should therefore be of interest. The purpose of this study was to identify analytical separation conditions and then adapt those conditions to preparative separation. This was accomplished with a column-screening approach on Daicel’s immobilized polysaccharide chiral stationary phases using non-traditional mobile phases, which included dichloromethane, ethyl acetate, and methyl tert-butyl ether under high-performance liquid chromatography conditions. CHIRALPAK® IK was found to separate all four compounds well with mobile phases containing hexane-dichloromethane (with or without an acidic additive). From these methods, the separation productivities were calculated to better visualize the separation scalability, which shows that the kilogram-scale separations of each are feasible.
Synthetic cannabinoid receptor agonists (SCRAs) are distributed on the drug market to produce THC‐like effects while evading routine drug testing and legislation. The cyclobutylmethyl (CBM) and norbornylmethyl (NBM) side chain specifically circumvented the German legislation and led to exploratory SCRAs being seen in 2019–2021. The NBM SCRAs were detected post‐amendment of the new psychoactive substances act in 2020, which scheduled all CBM SCRAs. All six SCRAs are full agonists at the cannabinoid receptor 1 compared to Δ9‐tetrahydrocannabinol and CP‐55940. The CBM SCRAs showed binding affinities of Ki: 29.4–0.65 nm and potencies of EC50: 483–40.1 nm (CBMICA << CBMINACA < CBMeGaClone). The norbornyl derivatives exhibited high affinities (Ki: 1.87–0.25 nm), with indazole the most affine. Functional activity data confirmed that the indazole derivative is the most potent of all three NBM SCRAs (EC50: 169–1.78 nm). The sterically demanding NBM side chain increased the affinity and activity of almost all core structures. Future studies should be conducted on similarly voluminous side chain moieties. The life cycle of all SCRAs was less than a year. Notably, Cumyl‐CBMICA was the most prevalent while also having the worst cannabimimetic properties. Quantification of Cumyl‐CBMICA during peak consumption in late 2019 and early 2020 revealed an increase in the concentration on the herbal material, which, together with forum entries and blog posts, corroborate the low in vitro cannabimimetic properties. Seizure prevalence data indicate that almost all SCRAs continue to be identified in 2022, potentially due to left‐over samples.
Cannabinoid production is one of the key attributes of the plant Cannabis sativa and the characterization of the genes involved is an essential first step to develop tools for their optimization. We used bioinformatic approaches to annotate and explore variation in the coding genes for critical enzymes comprising the cannabinoid pathway: Olivetol Synthase (OLS), Olivetolic Acid Cyclase (OAC), and Cannabigerolic Acid Synthase (CBGAS), in multiple C. sativa genomes. These upstream genes of the Cannabinoid Oxidocyclase Genes THCAS, CBDAS, and CBCAS generate the necessary precursor molecules to produce the cannabinoids THC and CBD. We found that these genes vary in copy number and confirm that OLS, OAC, CBGAS, and the Cannabinoid Oxidocyclases are on separate chromosomes, while homologs are found in proximity. CBGAS, located on Chromosome X, suggests potential dosage effects in female plants. Except for the Cannabinoid Oxidocyclase genes, the other genes have multiple exons, up to 10 in CBGAS. Through differential exon usage explorations in CBGAS we found evidence for potential regulatory differences. This study provides valuable insight on the genomic identity and variation of cannabinoid biosynthesis genes that will benefit future research on the origin and evolution of this pathway, driver of economic, social, and medicinal value.
