Yield of new hemp varieties for medical purposes under semi-arid Mediterranean environment conditions

Under the effects of climate change new drought tolerant crops are imperative to introduce in irrigated agricultural areas of Mediterranean countries. In this sense, hemp (Cannabis sativa L.) represents an alternative in many semi-arid agricultural areas of Mediterranean basin because of its low water requirements and cost effectiveness when it is developed under non controlled conditions. The aim of this work was to evaluate the potential yield of five new hemp varieties (Sara, Pilar, Aida, Theresa, and Juani) cultivated under high tunnel conditions in a semi-arid Mediterranean area, and also to study the effect of plant density on active biomass production and cannabinoids biosynthesis (cannabidiol, CBD and cannabigerol, CBG) at different plant positions. The trial was conducted under plastic macro-tunnels during two seasons (2014 and 2015), from May to October. The agronomic response and the chemical profiles of the studied varieties were evaluated at the end of each season. Moreover, it was monitored the differentiation in terms of active biomass production and cannabinoids biosynthesis in different plant organ positions (at upper, medium, and lower). Additionally, during the second season, three different plant densities (PD1, 9,777; PD2, 7,333; and PD3, 5,866 plants· ha-1) were tested in order to define the the best of them for maximizing CBD and CBG productions. The findings highlighted significant differences in yield between cultivars within the CBD and CBG. Moreover, plant density was a determinant factor related to active biomass production and cannabinoids contents, PD3 representing a suitable strategy to maximize the cannabinoids production minimizing the requirements of rooted apical cuttings. These results allowed concluding that these new hemp cultivars together with the adopted agronomic practices in this experience would be very appropriate for CBD and CBG productions, being determinant to consider the plant density and the cultivar for both studied chemotypes.Under the effects of climate change new drought tolerant crops are imperative to introduce in irrigated agricultural areas of Mediterranean countries. In this sense, hemp (Cannabis sativa L.) represents an alternative in many semi-arid agricultural areas of Mediterranean basin because of its low water requirements and cost effectiveness when it is developed under non controlled conditions. The aim of this work was to evaluate the potential yield of five new hemp varieties (Sara, Pilar, Aida, Theresa, and Juani) cultivated under high tunnel conditions in a semi-arid Mediterranean area, and also to study the effect of plant density on active biomass production and cannabinoids biosynthesis (cannabidiol, CBD and cannabigerol, CBG) at different plant positions. The trial was conducted under plastic macro-tunnels during two seasons (2014 and 2015), from May to October. The agronomic response and the chemical profiles of the studied varieties were evaluated at the end of each season. Moreover, it was monitored the differentiation in terms of active biomass production and cannabinoids biosynthesis in different plant organ positions (at upper, medium, and lower). Additionally, during the second season, three different plant densities (PD1, 9,777; PD2, 7,333; and PD3, 5,866 plants· ha-1) were tested in order to define the the best of them for maximizing CBD and CBG productions. The findings highlighted significant differences in yield between cultivars within the CBD and CBG. Moreover, plant density was a determinant factor related to active biomass production and cannabinoids contents, PD3 representing a suitable strategy to maximize the cannabinoids production minimizing the requirements of rooted apical cuttings. These results allowed concluding that these new hemp cultivars together with the adopted agronomic practices in this experience would be very appropriate for CBD and CBG productions, being determinant to consider the plant density and the cultivar for both studied chemotypes

Exploring the Mysteries of <em>Cannabis</em> through Gas Chromatography

In the last decades, cannabinoids, the active constituents of Cannabis sativa L., have been attracting a strong interest, regarding the health effects associated with the use of Cannabis and Cannabis-derived products. The progressive legalization of this species in several countries has prompted an increasing concern about the characterization and quantification of cannabinoids in diverse chemotypes of the plant, as well as the obtained final products. Therewith, Process and Product Quality Assurance (PPQA) becomes a mandatory practise to verify the Good Manufacturing Practices (GMP). Gas chromatography is one of the most used techniques in this sense due to its high attainable resolution. However, sample complexity and the thermal lability of cannabinoids hinder the analysis. In this chapter, a fully description of the recent advances in the Cannabis sativa L. analysis by gas chromatography will be presented, including different approaches that have come up to solve the obstacles encountered

Thermal desorption-ion mobility spectrometry: A rapid sensor for the detection of cannabinoids and discrimination of Cannabis Sativa L. chemotypes

Existing analytical techniques used for the determination of cannabinoids in Cannabis sativa L. (Cannabis) plants mostly rely on chromatography-based methods. As a rapid alternative for the direct analysis of them, thermal desorption (TD)-ion mobility spectrometry (IMS) was used for obtaining spectral fingerprints of single cannabinoids from Cannabis plant extracts and from plant residues on hands after their manipulation. The ionization source was 63Ni, with automatic switchable polarity. Although in both ionization modes there were signals in the TD-IMS spectra of the plant extracts and residues that could be assigned to concrete cannabinoids and chemotypes, most of them could not be clearly distinguished. Alternatively, the global spectral data of the plant extracts and residues were pre-processed and then, using principal component analysis (PCA)-linear discriminant analysis (LDA), grouped in function of their chemotype in a more feasible way. Using this approach, the possibility of false positive responses was also studied analyzing other non-Cannabis plants and tobacco, which were clustered in a different group to those of Cannabis. Therefore, TD-IMS, as analytical tool, and PCA-LDA, as a strategy for data reduction and pattern recognition, can be applied for on-site chemotaxonomic discrimination of Cannabis varieties and detection of illegal marijuana since the IMS equipment is portable and the analysis time is highly short

Regulation of Expression of Cannabinoid CB2 and Serotonin 5HT1A Receptor Complexes by Cannabinoids in Animal Models of Hypoxia and in Oxygen/Glucose-Deprived Neurons

Background: Cannabidiol (CBD) is a phytocannabinoid with potential in one of the most prevalent syndromes occurring at birth, the hypoxia of the neonate. CBD targets a variety of proteins, cannabinoid CB2 and serotonin 5HT1A receptors included. These two receptors may interact to form heteromers (CB2-5HT1A-Hets) that are also a target of CBD. Aims: We aimed to assess whether the expression and function of CB2-5HT1A-Hets is affected by CBD in animal models of hypoxia of the neonate and in glucose- and oxygen-deprived neurons. Methods: We developed a quantitation of signal transduction events in a heterologous system and in glucose/oxygen-deprived neurons. The expression of receptors was assessed by immuno-cyto and -histochemistry and, also, by using the only existing technique to visualize CB2-5HT1A-Hets fixed cultured cells and tissue sections (in situ proximity ligation PLA assay). Results: CBD and cannabigerol, which were used for comparative purposes, affected the structure of the heteromer, but in a qualitatively different way; CBD but not CBG increased the affinity of the CB2 and 5HT1A receptor-receptor interaction. Both cannabinoids regulated the effects of CB2 and 5HT1A receptor agonists. CBD was able to revert the upregulation of heteromers occurring when neurons were deprived of oxygen and glucose. CBD significantly reduced the increased expression of the CB2-5HT1A-Het in glucose/oxygen-deprived neurons. Importantly, in brain sections of a hypoxia/ischemia animal model, administration of CBD led to a significant reduction in the expression of CB2-5HT1A-Hets. Conclusions: Benefits of CBD in the hypoxia of the neonate are mediated by acting on CB2-5HT1A-Hets and by reducing the aberrant expression of the receptor-receptor complex in hypoxic-ischemic conditions. These results reinforce the potential of CBD for the therapy of the hypoxia of the neonate

Similarities and differences upon binding of naturally occurring Δ9-tetrahydrocannabinol-derivatives to cannabinoid CB1 and CB2 receptors.

We have here assessed, using Δ9 -tetrahydrocannabinol (Δ9 -THC) for comparison, the effect of Δ9 -tetrahydrocannabinolic acid (Δ9 -THCA) and of Δ9 -tetrahydrocannabivarin (Δ9-THCV) that is mediated by human versions of CB1, CB2, and CB1-CB2 receptor functional units, expressed in a heterologous system. Binding to the CB1 and CB2 receptors was addressed in living cells by means of a homogeneous assay. A biphasic competition curve for the binding to the CB2 receptor, was obtained for Δ9 -THCV in cells expressing the two receptors. Signaling studies included cAMP level determination, activation of the mitogen-activated protein kinase pathway and ß-arrestin recruitment were performed. The signaling triggered by Δ9 -THCA and Δ9 -THCV via individual receptors or receptor heteromers disclosed differential bias, i.e. the bias observed using a given phytocannabinoid depended on the receptor (CB1, CB2 or CB1-CB2) and on the compound used as reference to calculate the bias factor (Δ9 -THC, a selective agonist or a non-selective agonist). These results are consistent with different binding modes leading to differential functional selectivity depending on the agonist structure, and the state (monomeric or heteromeric) of the cannabinoid receptor. In addition, on studying Gi-coupling we showed that Δ9 -THCV and Δ9 -THCA and Δ9 -THCV were able to revert the effect of a selective CB2 receptor agonist, but only Δ9-THCV, and not Δ9-THCA, reverted the effect of arachidonyl-2′ -chloroethylamide (ACEA 100 nM) a selective agonist of the CB1 receptor. Overall, these results indicate that cannabinoids may have a variety of binding modes that results in qualitatively different effects depending on the signaling pathway that is engaged upon cannabinoid receptor activatio

Cannabigerol action at cannabinoid CB1 and CB2 receptors and at CB1-CB2 heteroreceptor complexes

Cannabigerol (CBG) is one of the major phytocannabinoids present in Cannabis sativa L. that is attracting pharmacological interest because it is non-psychotropic and is abundant in some industrial hemp varieties. The aim of this work was to investigate in parallel the binding properties of CBG to cannabinoid CB1 (CB1R) and CB2 (CB2R) receptors and the effects of the compound on agonist activation of those receptors and of CB1-CB2 heteroreceptor complexes. Using [3H]-CP-55940, CBG competed with low micromolar Ki values the binding to CB1R and CB2R. Homogeneous binding in living cells, which is only possible for the CB2R, provided a nanomolar Ki value. In contrast, CBG competed the binding of [3H]-WIN-55,212-2 to CB2R but not to CB1R (2.7 versus >30 µM). The phytocannabinoid modulated signaling mediated by receptors and receptor heteromers even at low concentrations of 0.1-1 µM. cAMP, pERK, ÿ-arrestin recruitment and label-free assays in HEK-293T cells expressing the receptors and treated with endocannabinoids or selective agonists proved that CBG is a partial agonist of CB2R. The action on cells expressing heteromers was similar to that obtained in cells expressing the CB2R. The effect of CBG on CB1R was measurable but the underlying molecular mechanisms remain uncertain. The results indicate that CBG is indeed effective as regulator of endocannabinoid signaling

Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors

The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB2 receptors (CB(2)Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB2R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB2R. Using membrane preparations from CB2R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB2R where the synthetic cannabinoid, [H-3]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB2R-selective compound, CM-157. The effect on binding to CB2R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the K-D. CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB2R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities

Cannabigerol Action at Cannabinoid CB1 and CB2 Receptors and at CB1–CB2 Heteroreceptor Complexes

Cannabigerol (CBG) is one of the major phytocannabinoids present in Cannabis sativa L. that is attracting pharmacological interest because it is non-psychotropic and is abundant in some industrial hemp varieties. The aim of this work was to investigate in parallel the binding properties of CBG to cannabinoid CB1 (CB1R) and CB2 (CB2R) receptors and the effects of the compound on agonist activation of those receptors and of CB1–CB2 heteroreceptor complexes. Using [3H]-CP-55940, CBG competed with low micromolar Ki values the binding to CB1R and CB2R. Homogeneous binding in living cells, which is only technically possible for the CB2R, provided a 152 nM Ki value. Also interesting, CBG competed the binding of [3H]-WIN-55,212-2 to CB2R but not to CB1R (Ki: 2.7 versus &gt;30 μM). The phytocannabinoid modulated signaling mediated by receptors and receptor heteromers even at low concentrations of 0.1–1 μM. cAMP, pERK, β-arrestin recruitment and label-free assays in HEK-293T cells expressing the receptors and treated with endocannabinoids or selective agonists proved that CBG is a partial agonist of CB2R. The action on cells expressing heteromers was similar to that obtained in cells expressing the CB2R. The effect of CBG on CB1R was measurable but the underlying molecular mechanisms remain uncertain. The results indicate that CBG is indeed effective as regulator of endocannabinoid signaling

Correction : Chaparro et al. Incidence, Clinical Characteristics and Management of Inflammatory Bowel Disease in Spain: Large-Scale Epidemiological Study. J. Clin. Med. 2021, 10, 2885

The authors wish to make the following corrections to this paper [...]