Non-psychoactive Cannabinoid Research Articles

Antioxidants help favorably regulate the kinetics of lipid peroxidation, polyunsaturated fatty acids degradation and acidic cannabinoids decarboxylation in hempseed oil

The seed of the hemp plant (Cannabis sativa L.) has been revered as a nutritional resource in Old World Cultures. This has been confirmed by contemporary science wherein hempseed oil (HSO) was found to exhibit a desirable ratio of omega-6 and omega-3 polyunsaturated fatty acids (PUFAs) considered optimal for human nutrition. HSO also contains gamma-linoleic acid (GLA) and non-psychoactive cannabinoids, which further contribute to its’ potential bioactive properties. Herein, we present the kinetics of the thermal stability of these nutraceutical compounds in HSO, in the presence of various antioxidants (e.g. butylated hydroxytoluene, alpha-tocopherol, and ascorbyl palmitate). We focussed on oxidative changes in fatty acid profile and acidic cannabinoid stability when HSO was heated at different temperatures (25 °C to 85 °C) for upto 24 h. The fatty acid composition was evaluated using both GC/MS and 1H-NMR, and the cannabinoids profile of HSO was obtained using both HPLC-UV and HPLC/MS methods. The predicted half-life (DT50) for omega-6 and omega-3 PUFAs in HSO at 25 °C was about 3 and 5 days, respectively; while that at 85 °C was about 7 and 5 hours respectively, with respective activation energies (Ea) being 54.78 ± 2.36 and 45.02 ± 2.87 kJ/mol. Analysis of the conjugated diene hydroperoxides (CDH) and p-Anisidine value (p-AV) revealed that the addition of antioxidants significantly (p < 0.05) limited lipid peroxidation of HSO in samples incubated at 25–85 °C for 24 h. Antioxidants reduced the degradation constant (k) of PUFAs in HSO by upto 79%. This corresponded to a significant (p < 0.05) increase in color stability and pigment retention (chlorophyll a, chlorophyll b and carotenoids) of heated HSO. Regarding the decarboxylation kinetics of cannabidiolic acid (CBDA) in HSO, at both 70 °C and 85 °C, CBDA decarboxylation led to predominantly cannabidiol (CBD) production. The half-life of CBDA decarboxylation (originally 4 days) could be increased to about 17 days using tocopherol as an antioxidant. We propose that determining acidic cannabinoids decarboxylation kinetics is a useful marker to measure the shelf-life of HSO. The results from the study will be useful for researchers looking into the thermal treatment of hempseed oil as a functional food product, and those interested in the decarboxylation kinetics of the acidic cannabinoids.

Combination of UV and MS/MS detection for the LC analysis of cannabidiol-rich products

Cannabidiol (CBD), a major non-psychoactive cannabinoid, has received a lot of attention due to its potential anti-inflammatory, pain-relieving, and anti-anxiety properties. This has led to a recent boom in CBD-rich commercial products, which are sold without prescription in the form of oils, candies, and cosmetics. Since these products are derived from cannabis, the presence of the psychoactive tetrahydrocannabinol (THC) has to be tested before they enter the market. Here, we present a high-throughput approach based on liquid chromatography coupled to UV and tandem mass spectrometric detection for the determination of CBD, THC, and six other minor cannabinoids (cannabigerolic acid, cannabidivarin, cannabinol, cannabigerol, cannabidiolic acid, and tetrahydrocannabinolic acid) in a wide range of concentrations and in a variety of matrices, including oils, hydrophobic ointments, water-soluble liquids, plant material and gelatinous gummies. Each product was dissolved in a suitable solvent and further diluted to avoid matrix interference. The diluted samples were analyzed by reversed-phase chromatography, coupled to a UV detector followed by a triple quadrupole mass spectrometer, used in the multiple reaction monitoring mode. The UV signal was utilized for the quantification of samples containing high levels of CBD, while the mass spectrometer was used for low levels of THC and other minor cannabinoids. This allowed us to meet the required sensitivity for THC while significantly expanding the range of analyzed CBD, all within an 8-min long chromatographic run. The samples were further quantified using calibration in solvent, the approach was validated, and the validation criteria were met for all matrices except for two (i.e., emulsions and gels). The lower limit of quantification for THC was 0.5 μg/g in gummies, 1.0 μg/g in oils, ointments and liquids, and 5.0 μg/g in plant material. CBD was analyzed in the range of 0.5–60,000 μg/g in gummies, 1–120,000 μg/g in oils, ointments and liquids, and 5–300,000 μg/g in plant material. The developed method was used for the analysis of thirteen real products with a wide range of CBD content, with positive THC findings in twelve of them.

T177. CANNABIDIOL AS A POTENTIAL PREVENTATIVE TREATMENT IN A NEUREGULIN-1 MOUSE MODEL OF SCHIZOPHRENIA

BackgroundSchizophrenia is caused by interactions between genes of predisposition and environmental insults. The main pharmacological treatments for schizophrenia are antipsychotic drugs; however, these are associated with a range of side effects (weight gain, metabolic disease, diabetes), have limited treatment compliance, and do not work for approximately 30% of patients. Cannabidiol (CBD) is a non-psychoactive cannabinoid that has shown promise as an antipsychotic-like drug in both human and rodent studies, however its potential as a preventative drug via anti-inflammatory pathways has not yet been investigated. Brain maturation during adolescent development creates a window where CBD could potentially reverse later behavioural deficits via pathways such as anti-inflammation, and reverse the deficits caused by Δ⁹-tetrahydrocannabinol (THC), a psychoactive compound of the cannabis plant linked to the development of schizophrenia.Here we investigated the therapeutic potential of CBD administered in adolescence to protect against the development of schizophrenia-like behaviours, as well as whether CBD could protect against later sensitivity to THC. For this study we used a well-established genetic mouse model of schizophrenia (Nrg1 TM HET) that shows face, construct, and predictive validity for schizophrenia, and is also more susceptible to the effects of cannabinoids.MethodsIn this study, male Nrg1 TM HET mice and wild type (WT) mice were treated with 30 mg/kg of CBD or vehicle intraperitoneally for three weeks during adolescence (PND 35–60). Mice were tested CBD-free in adulthood (5–6 mo) in the open field task, which measures locomotion and anxiety (related to positive symptoms in schizophrenia); social interaction, which measures mouse interactions (relevant to negative symptoms); pre-pulse inhibition (PPI), which measures sensorimotor gating (deficits also found in schizophrenia patients); and fear conditioning, which measures associative learning and memory. Mice were then treated after one week washout with either 3 mg/kg of THC or vehicle, and run in a battery of open field, social interaction, and PPI tests. Brains and bloods were collected to investigate neuroinflammation after another week of washout. Western blots were used to investigate Iba1 levels, while ELISA was used to investigate levels of cytokines IL-10, IL-1B, and TNF-a.ResultsCBD treatment significantly reduced anxiety in Nrg1 TM HET mice but not WT mice, however it had no effect on locomotion in either group in the open field task. CBD treatment lowered the frequency of total social interaction in both genotypes, and reduced the frequency of social interactions of Nrg1 TM HET to WT levels. CBD reduced PPI in Nrg1 TM HET mice but not WT mice, and Nrg1 TM HET mice had lower overall PPI than WT mice. There was no effect of CBD on fear-associated memory. CBD effects on THC sensitivity in these mice are ongoing, as are brain cytokine and protein analyses.DiscussionThese results suggest that Nrg1 TM HET mice are more susceptible to the effects of chronic CBD in adolescence, despite a significant washout. This confirms previous data from our laboratory demonstrating increased susceptibility of Nrg1 TM HET male mice to cannabinoid constituents, and suggests persistent brain changes following adolescent exposure to CBD. Potential reasons for these persistent changes include an altered neuroinflammatory profile in Nrg1 TM HET mice. CBD has previously been shown to be anxiolytic in other studies and our results confirm its potential as an anxiolytic, particularly in our model of schizophrenia.

Cannabidiol and Other Non-Psychoactive Cannabinoids for Prevention and Treatment of Gastrointestinal Disorders: Useful Nutraceuticals?

Cannabis sativa is an aromatic annual flowering plant with several botanical varieties, used for different purposes, like the production of fibers, the production of oil from the seeds, and especially for recreational or medical purposes. Phytocannabinoids (terpenophenolic compounds derived from the plant), include the well-known psychoactive cannabinoid Δ9-tetrahydrocannabinol, and many non-psychoactive cannabinoids, like cannabidiol. The endocannabinoid system (ECS) comprises of endocannabinoid ligands, enzymes for synthesis and degradation of such ligands, and receptors. This system is widely distributed in the gastrointestinal tract, where phytocannabinoids exert potent effects, particularly under pathological (i.e., inflammatory) conditions. Herein, we will first look at the hemp plant as a possible source of new functional food ingredients and nutraceuticals that might be eventually useful to treat or even prevent gastrointestinal conditions. Subsequently, we will briefly describe the ECS and the general pharmacology of phytocannabinoids. Finally, we will revise the available data showing that non-psychoactive phytocannabinoids, particularly cannabidiol, may be useful to treat different disorders and diseases of the gastrointestinal tract. With the increasing interest in the development of functional foods for a healthy life, the non-psychoactive phytocannabinoids are hoped to find a place as nutraceuticals and food ingredients also for a healthy gastrointestinal tract function.

The cannabidiol conundrum: potential benefits and risks of cannabidiol products for children.

Since the federal ban on hemp products was lifted in December 2018, cannabidiol (CBD), a nonpsychoactive cannabinoid derived from hemp, has become increasingly popular and accessible. CBD is sold in the form of oils, tablets, and foods in locations including gas stations, cafés, and drug stores. Despite a lack of reliable evidence, many parents praise its purported therapeutic effects on a variety of childhood ailments. Epidiolex was the first CBD-based medication to be approved by the US Food and Drug Administration in 2018 for the treatment of two rare and severe forms of epilepsy, known as Lennox-Gastaut and Dravet syndrome, in patients of at least 2 years of age. Its efficacy was assessed through three randomized, double blind, and placebo-controlled trials in a sample of 516 patients with either Lennox-Gastaut syndrome or Dravet syndrome. Despite this development, there are few or no large-scale, rigorous studies concerning the effects of CBD on any other pediatric conditions that parents have tried to alleviate with CBD. The purpose of this review is to explore recent literature regarding the efficacy and safety of CBD in treating various health conditions in children; the risks of consuming CBD products, and the role of pediatricians in helping parents navigate often-confusing information about CBD. Although CBD use has dramatically increased in recent years, both its potential to treat conditions and its risks have not yet been subjected to rigorous study. Pediatricians should be aware of the risks posed by poor-quality standards and labeling practices for cannabinoid products. Due to the confusing nature of the numerous sources of information about CBD, pediatricians are in a position to provide and clarify information about CBD to parents and understand the risks it poses to children.

Development of extraction technique and GC/FID method for the analysis of cannabinoids in Cannabis sativa L. spp. santicha (hemp).

Nowadays, the interest in industrial Cannabis sativa L. herb has been increasing in the world. As a result, it is becoming one of the most studied plants due to its multifunctional benefits. To the best of our knowledge, no study has been conducted so far to determine the impact of extraction methods and conditions on the extraction yields of CBD and CBG from the Cannabis sativa L. ssp. Santhica. Therefore, we aimed to investigate a simple and sensitive GC-FID method to determine CBD and CBG in hemp extract. As regards sample preparation, three extraction techniques were compared, including maceration (ME), ultrasound-assisted extraction (UAE) and reflux-heat extraction (RHE), in order to obtain a high recovery of the CBD of interest from the plant material. The GC-FID method developed in this study represents a powerful tool for the extraction and analysis of non-psychoactive cannabinoids from hemp varieties to be used for the preparations of extracts with a high content of bioactive compounds for both pharmaceutical and nutraceutical applications. A simple extraction procedure for CBD and CBG from hemp was also optimized in this work, by using ultrasound assisted extraction method with 96% ethanol, material/solvent ratio 1:10 and extraction time 10 min at room temperature. The overall analytical method was fully validated in agreement with international guidelines. Therefore, proving a powerful and reliable tool for both the selection of hemp varieties with a high content of bioactive compounds and the quality control of its derivatives.

The synthetic cannabinoid dehydroxylcannabidiol restores the function of a major GABAA receptor isoform in a cell model of hyperekplexia

The functions of the glycine receptor (GlyR) and GABAA receptor (GABAAR) are both impaired in hyperekplexia, a neurological disorder usually caused by GlyR mutations. Although emerging evidence indicates that cannabinoids can directly restore normal GlyR function, whether they affect GABAAR in hyperekplexia remains unknown. Here we show that dehydroxylcannabidiol (DH-CBD), a synthetic nonpsychoactive cannabinoid, restores the GABA- and glycine-activated currents (IGABA and IGly , respectively) in HEK293 cells coexpressing a major GABAAR isoform (α1β2γ2) and GlyRα1 carrying a human hyperekplexia-associated mutation (GlyRα1R271Q). Using coimmunoprecipitation and FRET assays, we found that DH-CBD disrupts the protein interaction between GABAAR and GlyRα1R271Q Furthermore, a point mutation of GlyRα1, changing Ser-296 to Ala-296, which is critical for cannabinoid binding on GlyR, significantly blocked DH-CBD-induced restoration of IGABA and IGly currents. This S296A substitution also considerably attenuated DH-CBD-induced disruption of the interaction between GlyRα1R271Q and GABAAR. These findings suggest that, because it restores the functions of both GlyRα1 and GABAAR, DH-CBD may represent a potentially valuable candidate drug to manage hyperekplexia.

A Warning against the Negligent Use of Cannabidiol in Professional and Amateur Athletes.

Cannabidiol (CBD) is a non-psychoactive cannabinoid, widely marketed to athletes for claimed effects such as decreased anxiety, fear memory extinction, anti-inflammatory properties, relief of pain and for post-exercise recovery. The World Anti-Doping Agency (WADA) has excluded CBD from its list of prohibited substances. Nevertheless, caution is currently advised for athletes intending to use the compound—except CBD, all other cannabinoids are still on the prohibited list. CBD products, specifically non-medicinal, so-called full-spectrum cannabis extracts, may contain significant levels of these substances, but also contaminations of tetrahydrocannabinol (THC) (>2.5 mg/day in >30% of products on the German market) potentially leading to positive doping tests. Labelled claims about CBD content and absence of THC are often false and misleading. Contaminations with the psychoactive THC can result in adverse effects on cognition and, in general, the safety profile of CBD with respect to its toxicity is a controversial topic of discussion. For these reasons, we would currently advise against the use of over-the-counter CBD products, especially those from dubious internet sources without quality control.

Could the Combination of Two Non-Psychotropic Cannabinoids Counteract Neuroinflammation? Effectiveness of Cannabidiol Associated with Cannabigerol.

Background and Objectives: Neuroinflammation is associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). In this study, we investigate the anti-inflammatory, anti-oxidant, and anti-apoptotic properties of two non-psychoactive phytocannabinoids, cannabigerol (CBG) and cannabidiol (CBD). Materials and Methods: The motoneuron-like cell line NSC-34 differentiated by serum deprivation and with the additional treatment of all-trans retinoic acid (RA) is a valid model to investigate molecular events linked to neurodegeneration in ALS. Results: Pre-treatment with CBG (at 2.5 and 5 µM doses) alone and in combination with CBD (at 2.5 and 5 µM doses) was able to reduce neuroinflammation induced by a culture medium of LPS-stimulated macrophages. In particular, the pre-treatment with CBD at a 5 µM dose decreased TNF-α levels and increased IL10 and IL-37 expression. CBG–CBD association at a 5 µM dose also reduced NF-kB nuclear factor activation with low degradation of the inhibitor of kappaB alpha (IkBα). CBG and CBD co-administered at a 5 µM dose decreased iNOS expression and increased Nrf2 levels. Furthermore, the pre-treatment with the association of two non-psychoactive cannabinoids downregulated Bax protein expression and upregulated Bcl-2 expression. Our data show the anti-inflammatory, anti-oxidant, and anti-apoptotic effects PPARγ-mediated. Conclusions: Our results provide preliminary support on the potential therapeutic application of a CBG–CBD combination for further preclinical studies.

A new Cannabis germplasm classification system and research strategies of non-psychoactive medicinal cannabis

At present,China manages cannabis into industrial and medical types. Industrial cannabis( THC<0. 3%) is grown for fiber and seed,while medical cannabis( THC>0. 3%) is prohibited from being planted and used. In recent years,breakthroughs have been made in the applications for a variety of fields of CBD,a non-psychoactive cannabinoid of Cannabis. However,China made a minimal contribution to this international research hotspot,mainly due to the outdated way of classification on cannabis. Here we reviewed the medicinal use history of cannabis,achievements in the study of Cannabis germplasm,and first proposed the three-level classification of management: psychoactive medicinal cannabis( THC>0. 3%),non-psychoactive medicinal cannabis( THC<0. 3%,high CBD)and industrial cannabis( THC<0. 3%,for fiber and seed,low CBD). Based on summarizing the research progress of the non-psychoactive medicinal cannabis,four key research strategies of breeding,sex control,plant factory,and synthetic biology are proposed.

In Silico Repositioning of Cannabigerol as a Novel Inhibitor of the Enoyl Acyl Carrier Protein (ACP) Reductase (InhA).

Cannabigerol (CBG) and cannabichromene (CBC) are non-psychoactive cannabinoids that have raised increasing interest in recent years. These compounds exhibit good tolerability and low toxicity, representing promising candidates for drug repositioning. To identify novel potential therapeutic targets for CBG and CBC, an integrated ligand-based and structure-based study was performed. The results of the analysis led to the identification of CBG as a low micromolar inhibitor of the Enoyl acyl carrier protein (ACP) reductase (InhA) enzyme.

Evaluating the efficacy of cannabidiol to manage surgically induced neuropathic pain in a preclinical rat model: Are T cells a sexually dimorphic target?

ABSTRACTBackground: Considering the poorly understood etiology and complex symptoms of chronic neuropathic pain (NP), the lack of effective treatments, and sex-dependent differences in the neuroimmune system as well as in antinociceptive responses to existing pharmacological agents, the potential to therapeutically target the endocannabinoid system as a means of treating this type of intractable pain is clinically relevant and timely. Chronic NP may involve the utilization of distinct immune cell populations in males and females that differentially affect supraspinal and spinal neuromodulation. It is therefore important to investigate the effects of cannabidiol (CBD) on chronic NP-induced nociceptive responses in both sexes.Aims: Evaluating whether the expression of markers associated with CD4+ T cells are affected by CBD in a sexually dimorphic manner will provide key insights into the contribution of these adaptive immune cells to the onset and progression of NP.Methods: Future research will be directed toward examining the potential sex-dependent effects of this nonpsychotropic cannabinoid relative to vehicle in a preclinical model of chronic postsurgical NP. Specifically, (1) differences in nociceptive behavior, (2) chronic changes in neural firing patterns, and (3) up- or downregulation of markers associated with CD4+ T cells in relevant tissues will be evaluated to better understand CBD-mediated neuroimmune modulatory effects in males and females.Conclusions: Chronic postsurgical pain is a growing clinical problem. Current treatment strategies rely on opioid-based therapeutics, which affect patient quality of life and are associated with addiction and withdrawal. Treatment of nerve injuries with CBD could provide an effective alternative to manage NP. Understanding its mechanisms of action will provide important insights into the sex-dependent application of this nonpsychoactive cannabinoid, setting the groundwork for large-scale Canadian clinical trials in women and men presenting with chronic pain.

Cannabidiol, cannabinol and their combinations act as peripheral analgesics in a rat model of myofascial pain

ObjectiveThis study investigated whether local intramuscular injection of non-psychoactive cannabinoids, cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC) and their combinations can decrease nerve growth factor (NGF)-induced masticatory muscle sensitization in female rats. DesignIn awake rats, changes in mechanical sensitivity induced by intramuscular injection of NGF and cannabinoids were measured by applying an electronic von Frey hair over the masseter muscle to measure the withdrawal response. The effect of CBD (5 mg/ml) and CBN (1 mg/ml) or their combinations CBD/CBN (1:1 mg/ml or 5:1 mg/ml) were assessed. To confirm a peripheral action, electrophysiological experiments were undertaken in anesthetized rats to examine whether intramuscular injections of CBD (5 mg/ml) and CBN (1 mg/ml) altered the mechanical threshold of masticatory muscle mechanoreceptors. ResultsIn behavioral experiments, CBD (5 mg/ml) or CBN (1 mg/ml) decreased NGF-induced mechanical sensitization. Combinations of CBD/CBN induced a longer-lasting reduction of mechanical sensitization than either compound alone. No significant change in mechanical withdrawal threshold was observed in the contralateral masseter muscles and no impairment of motor function was found with the inverted screen test after any of the treatments. Consistent with behavioral results, CBD (5 mg/ml), CBN (1 mg/ml) and the combination of CBD/CBN (1:1 mg/ml) increased the mechanical threshold of masseter muscle mechanoreceptors. However, combining CBD/CBN (5:1 mg/ml) at a higher ratio reduced the duration of this effect. This may indicate an inhibitory effect of higher concentrations of CBD on CBN. ConclusionsThese results suggest that peripheral application of these non-psychoactive cannabinoids may provide analgesic relief for chronic muscle pain disorders such as temporomandibular disorders and fibromyalgia without central side effects.

Efficacy of Cannabinoids in a Pre-Clinical Drug-Screening Platform for Alzheimer's Disease.

Finding a therapy for Alzheimer's disease (AD) is perhaps the greatest challenge for modern medicine. The chemical scaffolds of many drugs in the clinic today are based upon natural products from plants, yet Cannabis has not been extensively examined as a source of potential AD drug candidates. Here, we determine if a number of non-psychoactive cannabinoids are neuroprotective in a novel pre-clinical AD and neurodegeneration drug-screening platform that is based upon toxicities associated with the aging brain. This drug discovery paradigm has yielded several compounds in or approaching clinical trials for AD. Eleven cannabinoids were assayed for neuroprotection in assays that recapitulate proteotoxicity, loss of trophic support, oxidative stress, energy loss, and inflammation. These compounds were also assayed for their ability to remove intraneuronal amyloid and subjected to a structure-activity relationship analysis. Pairwise combinations were assayed for their ability to synergize to produce neuroprotective effects that were greater than additive. Nine of the 11 cannabinoids have the ability to protect cells in four distinct phenotypic neurodegeneration screening assays, including those using neurons that lack CB1 and CB2 receptors. They are able to remove intraneuronal Aβ, reduce oxidative damage, and protect from the loss of energy or trophic support. Structure-activity relationship (SAR) data show that functional antioxidant groups such as aromatic hydroxyls are necessary but not sufficient for neuroprotection. Therefore, there is a need to focus upon CB1 agonists that have these functionalities if neuroprotection is the goal. Pairwise combinations of THC and CBN lead to a synergistic neuroprotective interaction. Together, these results significantly extend the published data by showing that non-psychoactive cannabinoids are potential lead drug candidates for AD and other neurodegenerative diseases.

Development of a new method for the analysis of cannabinoids in honey by means of high-performance liquid chromatography coupled with electrospray ionisation-tandem mass spectrometry detection

Fibre-type Cannabis sativa L. (hemp) represents a valuable resource in many different fields, including both the pharmaceutical and food ones. This plant contains non-psychoactive cannabinoids, a class of bioactive compounds biosynthesized in both female and male inflorescences. Among them, cannabidiol (CBD) is the most interesting compound from a medicinal point of view. Indeed, several scientific studies have proved its therapeutic potential in a large number of pathologies, in addition to its biological effects attributable to its antioxidant, neuroprotective and anti-inflammatory properties.The analysis of the amount of cannabinoids in food and food supplements represents a critical issue in the ambit of both the quality assurance and the dietary intake control of these biologically active compounds. In this ambit, a particular attention is necessary for apiary products, since they are widely consumed and they can be produced by bees starting from different floral sources.In the light of all the above, the aim of this study was to develop for the first time a new analytical method based on RP-HPLC with ESI-MS/MS detection for the determination of CBD and related cannabinoids in honey. A quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction procedure with an un-buffered method was selected and optimised as the more suitable protocol. As regards detection, it was carried out by using a linear ion trap quadrupole (QTRAP) mass analyser, operated in the multiple reaction monitoring (MRM) mode. Hemp male inflorescences and pollen were analysed in parallel by means of HPLC-UV/DAD, since bees can transfer pollen into their hives and, consequently, into beehive products.The method developed and validated for the first time in this work was finally applied to the analysis of cannabinoids in honey samples, thus demonstrating to be a useful tool for both quality control and safety assurance.

Use of 13C-qNMR Spectroscopy for the Analysis of Non-Psychoactive Cannabinoids in Fibre-Type Cannabis sativa L. (Hemp).

Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The discovery of the presence of many biologically-active metabolites (cannabinoids) in fibre-type Cannabis (hemp) has recently given rise to the valorisation of this variety. In this context, the present study was aimed at the multi-component analysis and determination of the main non-psychoactive cannabinoids (cannabidiol, cannabidiolic acid, cannabigerol and cannabigerolic acid) in female inflorescences of different hemp varieties by means of 13C quantitative nuclear magnetic resonance spectroscopy (qNMR). The method proposed here for the first time for the determination of cannabinoids provided reliable results in a competitive time with respect to the more consolidated HPLC technique. In fact, it gave sufficiently precise and sensitive results, with LOQ values lower than 750 μg/mL, which is easily achievable with concentrated extracts, without affecting the quality of 13C-qNMR spectra. In conclusion, this method can be considered as a promising and appropriate tool for the comprehensive chemical analysis of bioactive cannabinoids in hemp and other derived products in order to ensure their quality, efficacy and safety.

Decarbonylation: A metabolic pathway of cannabidiol in humans.

Cannabidiol (CBD) is a non-psychoactive cannabinoid, which is of growing medical interest. Previous studies on the metabolism of CBD showed mainly the formation of hydroxylated or oxidized derivatives, the formation of carboxylic acids or modifications of the aliphatic side chain. Using incubation of CBD with hepatic microsomes of mice, the formation of carbon monoxide was reported. We investigated the phase I metabolism of CBD and cannabidivarin (CBDV) using in vitro experiments with human liver microsomes in order to discover so far not considered metabolites. Identification of metabolites was done by liquid chromatography coupled with quadrupole time of flight mass spectrometry (LC-QToF-MS). Within these experiments, we came across decarbonylation of CBD and CBDV. Further investigations were focused on observed decarbonylated CBD (DCBD). To confirm this metabolite in humans in vivo, plasma samples containing large amounts of cannabinoids as well as serum and urine samples, collected after a voluntary intake of a CBD-containing food supplement, were analyzed by LC coupled to triple quadrupole mass spectrometry (LC-QQQ-MS). DCBD was detected in in vitro incubation mixtures, serum samples, and urine samples (after alkaline or enzymatic hydrolysis) collected after the voluntary intake, as well as in plasma samples of cannabis users. DCBD appears to be an important supplementary human metabolite that might be helpful for the analytical confirmation of a CBD uptake and might improve the interpretation of the consumption of CBD-containing products. Results of this study indicate a prolonged detectability of DCBD (in serum) in comparison to CBD after oral CBD ingestion.

Chemical Composition and In Vitro Neuroprotective Activity of Fibre-Type Cannabis sativa L. (Hemp)

Background: Fibre-type Cannabis sativa L. (hemp) usually contains cannabidiolic acid and cannabidiol as the main non-psychoactive cannabinoids. Even though there is evidence of the neuroprotective activity of pure cannabidiol, no in vitro studies have reported so far the role of hemp extracts on neuroprotection. The objective of this study was to evaluate the neuroprotective effect of hemp extracts in in vitro cellular models of neurotoxicity. Methods: One extract was obtained from raw hemp inflorescences, while the other was prepared from the same plant material submitted to a decarboxylation process. The composition of both these extracts was evaluated by HPLC-UV/DAD. Human neuroblastoma SH-SY5Y and microglial BV-2 cell lines treated with rotenone were selected as the model of neurodegeneration. The neuroprotection of hemp extracts was assessed also in serum-free conditions both in the presence and in the absence of rotenone as the toxic agent by using the same cell lines. The neuroprotective potential of cannabidiol was tested in parallel. Results: The decarboxylated hemp extract possesses a mild neuroprotective activity on BV-2 cells treated with rotenone, higher than that of pure cannabidiol. As regards serum-free experiments, the nondecarboxylated hemp extract was the most effective neuroprotective agent toward SH-SY5Y cells, while BV-2 cells were better protected from the toxic insult by the decarboxylated extract and cannabidiol. Conclusion: Both hemp extracts and pure cannabidiol displayed a moderate neuroprotective activity in the neurotoxicity models considered in this study; in addition, they showed a trophic effect on SHSY5Y cells.

Cannabidiol modulates the expression of neuroinflammatory factors in stress- and drug-induced reinstatement of methamphetamine in extinguished rats.

Methamphetamine (METH) is a highly potent and addictive psychostimulant that is frequently abused worldwide. Although the biggest challenge to the efficient treatment of drug dependence is relapse, its mechanism is completely unclear. Plenty of evidence suggests that inflammation contributes to drug-induced reward especially in brain regions that are involved in the reward system, but there is no document about relapse. Cannabidiol (CBD) is a nonpsychoactive cannabinoid that has powerful anti-inflammatory and immunosuppressive properties. A previous research in our laboratory has demonstrated that CBD prevents reinstatement of METH even in 24-hour rapid eye movement (REM) sleep-deprived (RSD) rats. The aim of this study was to assess whether CBD prevents reinstatement of METH through change of gene expression of cytokines such as interleukin-1β, interleukin-6, interleukin-10, and tumor necrosis factor α (TNF-α) in extinguished rats. Real-time polymerase chain reaction (PCR) was used in this research to assay gene expression of cytokines. We found that stress- and drug-induced reinstatement of METH enhanced mRNA expression of cytokines in the prefrontal cortex (PFC) and hippocampus. Furthermore, CBD treatment significantly reduced the mRNA expression of cytokines in the PFC and hippocampus, but CBD treatment in RSD rats increased expression of cytokines in the hippocampus. It seems that enhancement of cytokines leads to change in neurotransmission and so triggers reinstatement of METH.

Role of CB2 Receptor in the Recovery of Mice after Traumatic Brain Injury.

Cannabis is one of the most widely used plant drugs in the world today. In spite of the large number of scientific reports on medical marijuana, there still exists much controversy surrounding its use and the potential for abuse due to the undesirable psychotropic effects. However, recent developments in medicinal chemistry of novel non-psychoactive synthetic cannabinoids have indicated that it is possible to separate some of the therapeutic effects from the psychoactivity. We have previously shown that treatment with the endocannabinoid 2-AG, which binds to both CB1 and CB2 receptors 1 h after traumatic brain injury in mice, attenuates neurological deficits, edema formation, infarct volume, blood-brain barrier permeability, neuronal cell loss at the CA3 hippocampal region, and neuroinflammation. Recently, we synthesized a set of camphor-resorcinol derivatives, which represent a novel series of CB2 receptor selective ligands. Most of the novel compounds exhibited potent binding and agonistic properties at the CB2 receptors with very low affinity for the CB1 receptor, and some were highly anti-inflammatory. This selective binding correlated with their intrinsic activities. HU-910 and HU-914 were selected in the present study to evaluate their potential effect in the pathophysiology of traumatic brain injury (TBI). In mice and rats subjected to closed-head injury and treated with these novel compounds, we showed enhanced neurobehavioral recovery, inhibition of tumor necrosis factor α production, increased synaptogenesis, and partial recovery of the cortical spinal tract. We propose these CB2 agonists as potential drugs for development of novel therapeutic modality to TBI.