[Genome-wide screening and bioinformatics analysis of Cannabis sativa LecRLK gene family].

Lectin receptor-like kinase(LecRLK) is a class of phytokinase with lectin conserved domain, which plays an important role in plant resistance to biological and abiotic stresses, as well as plant growth and development. Cannabis sativa is an important multi-purpose plant, widely used in food, textile, medicine, and other fields. Genome-wide screening and expression analysis of the LecRLK family of C. sativa were performed in this paper, so as to provide scientific reference for functional analysis of the LecRLK family of C. sativa. Based on BLAST and HMM methods, 93 LecRLKs were identified in the whole genome of C. sativa, including 69 G types, 23 L types, and one C types. Subsequently, a series of bioinformatics analyses were performed on the LecRLK family members, and the physicochemical properties of the protein of the LecRLK family members were initially revealed. The prediction of cis-acting elements of promoters in family members showed that family members were regulated by hormones and stress response. The expression analysis showed that some family members were highly expressed in the roots, which may participate in the process of stress resistance. Several members were highly expressed in female flowers and may be involved in female flower development. This study provides a theoretical basis for further study of LecRLK gene function. Meanwhile, the expression analysis screens candidate LecRLK members who may participate in the resistance of C. sativa, which provides a theoretical basis for the subsequent selection of C. sativa varieties against resistance.

Characterization and evaluation of nine Cannabis sativa chloroplast SNP markers for crop type determination and biogeographical origin on European samples.

Cannabis sativa can be classified in two main types, according to psychotropic cannabinoid ∆9-tetrahydrocannabinol (∆9-THC) content: the drug-type and the fiber-type. According to the European Monitoring Center for Drugs and Drug Addiction, most of the European Union countries consider the possession of cannabis, for personal use, a minor offense with possibility of incarceration. Despite of the model of legal supply (i.e., Spanish cannabis clubs, Netherlands coffee shops) or medical use (i.e., Italy), cannabis remains the most used and trafficked illicit plant in the European Union. Differentiating cannabis crops or tracing the biogeographical origin is crucial for law enforcement purposes. Chloroplast DNA (cpDNA) markers may assist to determine biogeographic origin and to differentiate hemp from marijuana. This research aims: to identify and to evaluate nine C. sativa cpDNA polymorphic SNP sites to differentiate crop type and to provide information about its biogeographical origin. Five SNaPshot™ assays for nine chloroplast markers were developed and conducted in marijuana samples seized in Chile, the USA-Mexico border and Spain, and hemp samples grown in Spain and in Italy. The SNapShot™ assays were tested on 122 cannabis samples, which included 16 blind samples, and were able to differentiate marijuana crop type from hemp crop type in all samples. Using phylogenetic analysis, genetic differences were observed between marijuana and hemp samples. Moreover, principal component analysis (PCA) supported the relationship among hemp samples, as well as for USA-Mexico border, Spanish, and Chilean marijuana samples. Genetic differences between groups based on the biogeographical origin and their crop type were observed. Increasing the number of genetic markers, including the most recently studied ones, and expanding the sample database will provide more accurate information about crop differentiation and biogeographical origin.

Utility of a brief measure of cannabis demand: Day-level associations with cannabis use.

BACKGROUND: Cannabis demand (i.e., relative value) is usually assessed as a trait-level risk-factor for cannabis use and consequences. This study examined within-person variability in day-level intensity (i.e., amount consumed at zero cost) and Omax (i.e., maximum cannabis expenditure) and tested hypotheses that demand would be positively associated with day-level cannabis use. METHODS: Young adults (n=85) reporting past-month simultaneous alcohol and cannabis use completed two daily surveys for 30 days. Morning surveys assessed prior-day cannabis use and evening surveys assessed day-level demand (i.e., intensity, Omax). Multilevel models tested day-level effects of intensity and Omax on any cannabis use and flower use frequency and quantity (i.e., grams). RESULTS: Approximately 52 % and 46 % of variability in intensity and Omax, respectively, was due to within-person change. At the day-level, higher intensity and Omax were associated with higher likelihood of any cannabis use, regardless of formulation; Omax was associated with use of flower in particular; and intensity was associated with the highest quantity of use. At the person-level, only Omax was associated with flower use likelihood, and only intensity was associated with flower quantity across days. CONCLUSIONS: Cannabis demand demonstrated day-to-day variability, conceivably in response to various internal states and external factors. Intensity and Omax were related to elevated likelihood of using any cannabis, particularly flower, at the day-level. Overall, these data illustrate the validity and utility of brief cannabis demand measures, which might be used to further understand cannabis' reinforcing value at a fine-grained level.

Diet supplementation with hemp (Cannabis sativa L.) inflorescences: effects on quanti-qualitative milk yield and fatty acid profile on grazing dairy goats.

Hemp (Cannabis sativa L.) is an annual plant belonging to the family of Cannabaceae with several varieties characterized by different fatty acid profile, content in flavonoids, polyphenols, and cannabinoid compounds. Hemp is mostly used in livestock nutrition as oil or as protein cake, but not as inflorescences. The aim of this study was to evaluate the effect of dietary hemp inflorescences on milk yield and composition in grazing dairy goats. Twenty Camosciata delle Alpi goats at their 3rd parity and with a mean body weight of 45.2 ± 2.0 kg, immediately after kidding, were equally allocated into two groups (G: Grazing and GH: grazing and hemp). For three months, all goats were fed on a permanent pasture and received 700/head/day of concentrate; diet of group GH was supplemented with 20 g/head/day of hemp inflorescences. Goats' body weight did not change during the trial. Individual milk yield was daily recorded and samples collected every 20 days for chemical composition and fatty acid profile analysis. No significant differences were found for milk yield and chemical composition. Caproic (C6:0) (1.80 vs. 1.74%; p < 0.01) and lauric acids (C12:0) were significantly higher in milk of group GH (4.83 vs. 4.32%; p < 0.01) as well as linoleic (C18:2) (2.04 vs. 1.93%; p < 0.05), adrenic acid (C22:4) (0.046 vs. 0.031%, p < 0.05), omega-6/omega-3 ratio (3.17 vs. 2.93, p < 0.05) and total conjugated linoleic acids (CLAs) (0.435 vs. 0.417%; p < 0.01). The results of this study suggest that the supplementation of grazing goats' diet with hemp inflorescences may enhance the milk nutritional characteristics by increasing its content of CLAs and other beneficial fatty acids.

Patient out-of-pocket costs for cannabis use during cancer treatment.

BACKGROUND: We assessed patient costs associated with cannabis use during cancer treatment. METHODS: Adults treated for cancer at a large, comprehensive center completed an anonymous survey regarding their thoughts and experiences with cannabis and cancer. Bivariate and weighted multivariable logistic regression assessed clinical and sociodemographic factors associated with patient-reported out-of-pocket costs for cannabis products. RESULTS: Overall, 248 cannabis users provided data on cost and were analyzed. Median monthly out-of-pocket cost for cannabis was $80 (interquartile range = $25-$150). On regression analysis, male gender (odds ratio = 2.5, 95% confidence interval = 1.2 to 5.5, P = .026) and being 45 years of age or older (odds ratio = 7.5, 95% confidence interval = 1.9 to 30.0, P = .0042) were associated with spending $100 a month or more on cannabis. Of the 166 patients who stopped using cannabis early or used less than preferred, 28% attributed it to cost and 26% to lack of insurance coverage. CONCLUSION: Cannabis use during cancer treatment may contribute to significant out-of-pocket costs, with men and younger patients more likely to pay higher costs.

The association of perceived cannabis risks and benefits with cannabis use since cancer diagnosis.

BACKGROUND: Many patients with cancer use cannabis to help alleviate untreated cancer symptoms and side effects. METHODS: We examined associations of perceived benefits and risks and postdiagnosis cannabis use in a weighted sample of adult cancer survivors through a 1-time survey. Fifteen perceived cannabis use benefits and 19 perceived risks were operationalized as both summary scores and report of any benefits or risks. Survey-weighted logistic regression provided covariate-adjusted odds of postdiagnosis cannabis use for each benefit-risk measure. RESULTS: Among the weighted population of 3785 survivors (mean [SD] age = 62.2 [13.5] years), one-third used cannabis after diagnosis. Perceiving any benefits increased the odds of postdiagnosis cannabis use more than 500%, and perceiving any risks lowered the odds by 59%. Each SD increase in endorsed benefits doubled the odds of postdiagnosis cannabis use, while each SD increase in endorsed risks reduced the odds by 36%. CONCLUSION: An accurate understanding of benefits and risks is critical for informed decision making.

An investigation into the sensory properties of luffa (Luffa cylindrica (L.)) seeds and a comparison to other seeds (flax, sunflower, chia, and hemp).

Consumers are interested in new sustainable ingredients but are unwilling to accept undesirable sensory properties in their food products. Luffa (Luffa cylindrica) is mainly harvested and processed for its fibrous network, which is used as an exfoliator, while its seeds are usually discarded. However, the seeds have been found to have various nutritional benefits. As such, this study investigated the sensory properties of luffa seed powder added to yogurt and compared it to other seed powder (flax, sunflower, chia, and hemp). Consumers (n = 107) evaluated their liking of the different seeds added to yogurt using hedonic scales and the sensory properties using check-all-that-apply (CATA). The luffa seeds when mixed with yogurt were associated with off-colour, off-flavour, metallic, strong flavour, bitter, salty, earthy and decreased consumer liking. The flax and sunflower seeds were found to be sweet, nutty, cooked, mild flavour, and to have a smooth texture. The overall liking scores for the flax and sunflower seed samples were significantly higher than the luffa and hemp samples. Future studies should investigate different drying and roasting treatments to improve the sensory properties of the luffa seeds.

Development of Delivery Systems with Prebiotic and Neuroprotective Potential of Industrial-Grade Cannabis sativa L.

This study delves into the transformative effects of supercritical carbon dioxide (scCO2) cannabis extracts and prebiotic substances (dextran, inulin, trehalose) on gut bacteria, coupled with a focus on neuroprotection. Extracts derived from the Bialobrzeska variety of Cannabis sativa, utilising supercritical fluid extraction (SFE), resulted in notable cannabinoid concentrations (cannabidiol (CBD): 6.675 ± 0.166; tetrahydrocannabinol (THC): 0.180 ± 0.006; cannabigerol (CBG): 0.434 ± 0.014; cannabichromene (CBC): 0.490 ± 0.017; cannabinol (CBN): 1.696 ± 0.047 mg/gD). The assessment encompassed antioxidant activity via four in vitro assays and neuroprotective effects against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The extract boasting the highest cannabinoid content exhibited remarkable antioxidant potential and significant inhibitory activity against both enzymes. Further investigation into prebiotic deliveries revealed their proficiency in fostering the growth of beneficial gut bacteria while maintaining antioxidant and neuroprotective functionalities. This study sheds light on the active compounds present in the Bialobrzeska variety, showcasing their therapeutic potential within prebiotic systems. Notably, the antioxidant, neuroprotective, and prebiotic properties observed underscore the promising therapeutic applications of these extracts. The results offer valuable insights for potential interventions in antioxidant, neuroprotective, and prebiotic domains. In addition, subsequent analyses of cannabinoid concentrations post-cultivation revealed nuanced changes, emphasising the need for further exploration into the dynamic interactions between cannabinoids and the gut microbiota.

Cannabis sativa as an Herbal Ingredient: Problems and Prospects.

Cannabis sativa, otherwise known as hemp, is discussed to highlight the various problems and prospects associated with its use as an herbal ingredient. The chemical composition of hemp, with classification based on cannabinoid contents, its biological activities, current global scenarios and legality issues, economic importance, and future prospects, are discussed.

Activity-based detection of synthetic cannabinoid receptor agonists in plant materials.

BACKGROUND: Since late 2019, fortification of 'regular' cannabis plant material with synthetic cannabinoid receptor agonists (SCRAs) has become a notable phenomenon on the drug market. As many SCRAs pose a higher health risk than genuine cannabis, recognizing SCRA-adulterated cannabis is important from a harm reduction perspective. However, this is not always an easy task as adulterated cannabis may only be distinguished from genuine cannabis by dedicated, often expensive and time-consuming analytical techniques. In addition, the dynamic nature of the SCRA market renders identification of fortified samples a challenging task. Therefore, we established and applied an in vitro cannabinoid receptor 1 (CB1) activity-based procedure to screen plant material for the presence of SCRAs. METHODS: The assay principle relies on the functional complementation of a split-nanoluciferase following recruitment of ß-arrestin 2 to activated CB1. A straightforward sample preparation, encompassing methanolic extraction and dilution, was optimized for plant matrices, including cannabis, spiked with 5 µg/mg of the SCRA CP55,940. RESULTS: The bioassay successfully detected all samples of a set (n = 24) of analytically confirmed authentic Spice products, additionally providing relevant information on the 'strength' of a preparation and whether different samples may have originated from separate batches or possibly the same production batch. Finally, the methodology was applied to assess the occurrence of SCRA adulteration in a large set (n = 252) of herbal materials collected at an international dance festival. This did not reveal any positives, i.e. there were no samples that yielded a relevant CB1 activation. CONCLUSION: In summary, we established SCRA screening of herbal materials as a new application for the activity-based CB1 bioassay. The simplicity of the sample preparation, the rapid results and the universal character of the bioassay render it an effective and future-proof tool for evaluating herbal materials for the presence of SCRAs, which is relevant in the context of harm reduction.

Lebanese cannabis oil as a potential treatment for acute myeloid leukemia: In vitro and in vivo evaluations.

ETHNOPHARMACOLOGICAL RELEVANCE: The Cannabis sativa L. ssp. indica (Lam.) plant has been historically utilized as a natural herbal remedy for the treatment of several ailments. In Lebanon, cannabis extracts have long been traditionally used to treat arthritis, diabetes, and cancer. AIM OF THE STUDY: The current study aims to investigate the anti-cancer properties of Lebanese cannabis oil extract (COE) on acute myeloid leukemia using WEHI-3 cells, and a WEHI-3-induced leukemia mouse model. MATERIALS AND METHODS: WEHI-3 cells were treated with increasing concentrations of COE to determine the IC50 after 24, 48 and 72-h post treatment. Flow cytometry was utilized to identify the mode of cell death. Western blot assay was performed to assess apoptotic marker proteins. In vivo model was established by inoculating WEHI-3 cells in BALB/c mice, and treatment commencing 10 days post-inoculation and continued for a duration of 3 weeks. RESULTS: COE exhibited significant cytotoxicity with IC50 of 7.76, 3.82, and 3.34 µg/mL at 24, 48, and 72 h respectively post-treatment. COE treatment caused an induction of apoptosis through an inhibition of the MAPK/ERK pathway and triggering a caspase-dependent apoptosis via the extrinsic and intrinsic modes independent of ROS production. Animals treated with COE exhibited a significantly higher survival rate, reduction in spleen weight as well as white blood cells count. CONCLUSION: COE exhibited a potent anti-cancer activity against AML cells, both in vitro and in vivo. These findings emphasize the potential application of COE as a chemotherapeutic adjuvant in treatment of acute myeloid leukemia.

Potency analysis of twelve cannabinoids in industrial hemp via ultrahigh-performance liquid chromatography-tandem mass spectrometry.

RATIONALE: With an increasing appreciation for the unique pharmacological properties associated with distinct, individual cannabinoids of Cannabis sativa, there is demand for accurate and reliable quantification for a growing number of them. In this study, we developed rapid, sensitive, selective, accurate, and validated liquid chromatography-tandem mass spectrometry for the quantification of cannabinoids. METHODS: Crushed industrial hemp flower and leaf sample was extracted by 95% methanol aqueous, sonicated for 30 min. UPLC-MS/MS analysis using Waters Acquity BEH-C18 column and electrospray ionization(ESI) mass spectrometry detector. RESULTS: The method was validated to demonstrate its reproducibility and precision, linearity, recovery investigation, and investigation of matrix effect. The concentration-response relationship for all analyzed cannabinoids were linear with R2 values >0.99, with intra- and inter-day precision and relative errors below 12%. The recovery and matrix effect were measured as 66.1%-104.1% and 70.42%-110.75%. CONCLUSIONS: This study established a UHPLC-MS/MS method for the simultaneous and rapid quantitative determination of twelve cannabinoids in industrial hemp flowers and leaves in 11 min. The method was used to analyze 43 industrial hemp flower and leaf samples, with the data being statistically analyzed. Based on the statistical analysis of the cannabinoids, hemp from different regions and different varieties were well distinguished by the PLS-DA model, with the main contributing substances being cannabidiol, Δ9-tetrahydrocannabinol, and Δ8-tetrahydrocannabinol.

Oral pre- and early postnatal cannabis exposure disinhibits ventral tegmental area dopamine neuron activity but does not influence cocaine preference in offspring in mice.

Cannabis consumption has increased from 1.5% to 2.5% in Canada between 2012 and 2019. Clinical studies have indicated effects of prenatal cannabis exposure on birth weight, substance use, and neurodevelopmental disorders, but are confounded by several difficult to control variables. Animal models allow for examination of the mechanism of cannabis-induced changes in neurodevelopment and behavior, while controlling dose and timing. Several animal models of prenatal cannabis exposure exist which provide varying levels of construct validity, control of dose, and exposure to maternal stress. Using a voluntary oral consumption model, mouse dams received 5 mg/kg Δ9-tetrahydrocannabinol (THC) whole cannabis oil in peanut butter daily from gestational day 1 (GD1) to postnatal day 10 (PD10). At GD1, GD18, PD1, PD10, and PD15, maternal plasma was collected; pup brains were collected from GD18 onward. Pup brains had higher levels of THC and cannabidiol at each time point, each of which persisted in maternal plasma and pup brains past the end of treatment (PD15). Male and female adolescent offspring were examined for changes to ventral tegmental area (VTA) dopamine neuron activity and cocaine-seeking behavior. Prenatal and early postnatal (GD1-PD10) cannabis-exposed male, but not female mice had decreased gamma-aminobutyric acid (GABAergic) input, depolarized resting membrane potential, and increased spontaneous firing of VTA dopamine neurons. Cannabis-exposed offspring showed faster decay of N-methyl-D-aspartate (NMDA) currents in both sexes. However, no differences in cocaine-seeking behavior were noted. These data characterize a voluntary prenatal cannabis exposure model and demonstrates VTA dopamine neuronal activity is disinhibited in offspring.

Prenatal tobacco and tobacco-cannabis co-exposure: Relationship with attention and memory in middle childhood.

We examined associations between prenatal tobacco exposure (with and without cannabis exposure) and children's performance on laboratory measures of sustained attention, attentional set shifting, and working memory in middle childhood (9-12 years of child age). Participants were recruited in the first trimester of pregnancy and oversampled for prenatal tobacco exposure; with a smaller sample (n = 133; n = 34 non-substance exposed, n = 37 exposed to tobacco only, n = 62 co-exposed) invited (oversampled for co-exposure) to participate in the middle-childhood assessment (M age = 10.6, SD = 0.77; 68% Black, 20% Hispanic). Results for sustained attention indicated lower attention (percent hits) at the first epoch for tobacco only exposed compared to non-exposed and co-exposed; a trend (p = .07) towards increases in impulsive responding across time (a total of 8 epochs) for tobacco exposed (with and without cannabis) compared to non-exposed children; and a significant association between higher number of cigarettes in the first trimester and greater increases in impulsive responding across epochs. However, children prenatally exposed to tobacco (with and without cannabis) demonstrated greater short-term memory compared to children not prenatally exposed, and this difference was driven by higher scores for children prenatally co-exposed to tobacco and cannabis compared to those who were non-exposed. Overall, results suggest that prenatal tobacco exposure, especially in the first trimester, may increase risk for impulsive responding on tasks requiring sustained attention, and that co-use of cannabis did not exacerbate these associations. The higher short-term memory scores among children who were co-exposed compared to non-exposed are perplexing and need replication, particularly in studies with larger sample sizes and samples exposed only to cannabis to examine this more closely.

IgE-Mediated Cannabis Allergy and Cross-Reactivity Syndromes: A Roadmap for Correct Diagnosis and Management.

PURPOSE OF THE REVIEW: With increased access and decriminalization of cannabis use, cases of IgE-dependent cannabis allergy (CA) and cross-reactivity syndromes have been increasingly reported. However, the exact prevalence of cannabis allergy and associated cross-reactive food syndromes (CAFS) remains unknown and is likely to be underestimated due to a lack of awareness and insufficient knowledge of the subject among health care professionals. Therefore, this practical roadmap aims to familiarize the reader with the early recognition and correct management of IgE-dependent cannabis-related allergies. In order to understand the mechanisms underlying these cross-reactivity syndromes and to enable personalized diagnosis and management, special attention is given to the molecular diagnosis of cannabis-related allergies. RECENT FINDINGS: The predominant signs and symptoms of CA are rhinoconjunctivitis and contact urticaria/angioedema. However, CA can also present as a life-threatening condition. In addition, many patients with CA also have distinct cross-reactivity syndromes, mainly involving fruits, vegetables, nuts and cereals. At present, five allergenic components of Cannabis sativa (Can s); Can s 2 (profilin), Can s 3 (a non-specific lipid protein), Can s 4 (oxygen-evolving enhancer protein 2 oxygen), Can s 5 (the Bet v 1 homologue) and Can s 7 (thaumatin-like protein) have been characterized and indexed in the WHO International Union of Immunological Sciences (IUIS) allergen database. However, neither of them is currently readily available for diagnosis, which generally starts by testing crude extracts of native allergens. The road to a clear understanding of CA and the associated cross-reactive food syndromes (CAFS) is still long and winding, but well worth further exploration.

Effects of dietary local hemp seed meal as soybean meal alternative on productive performance, egg quality and yolk fatty acid composition of laying hens.

Supply problems and rising soybean meal prices have an impact on increasing feed costs. Hemp seed meal (HSM) with high protein content has the potential to be used as an alternative to soybean meal. This study evaluated the impact of dietary HSM of Narli Sarayi variety as a substitute for soybean meal on productive performances, egg quality and yolk fatty acid composition. A total of 120 Lohmann Brown laying hens aged 50 weeks were allocated into 4 groups and 10 repetitions. Birds received treatment without HSM (control group), or soybean meal substituted with 4%, 8% and 12% HSM. Dietary 4% significantly increased (p < 0.05) egg production and decreased FCR compared with 8% and 12% HSM group but did not differ from the control group in an overall period of 6 weeks. The inclusion of the 12% HSM group significantly decreased (p < 0.05) egg production. Meanwhile, there was no influence of hemp seed meal (p > 0.05) on feed intake, egg weight, body weight change, egg shape index, albumen index, albumen weight, Haugh unit, yolk weight, yolk index and eggshell thickness. Dietary 8% and 12% HSM significantly increased (p < 0.05) eggshell weight and yolk colour compared with control and 4% HSM groups. There was a significant increase (p < 0.05) in omega-3 fatty acid concentration and a decrease in yolk omega-6 to omega-3 fatty acids ratio with an increase in dietary HSM. It was concluded that dietary up to 12% HSM of the Narli Sarayi variety decreased egg production and increased FCR. Increasing dietary levels of HSM increased eggshell weight, yolk colour and omega-3 fatty acids content and decreased the omega-6 to omega-3 fatty acids ratio.

Modeling the liquid-liquid chromatography separation of cannabinoids from hemp extracts.

The separation of cannabinoids from hemp materials is nowadays one of the most promising industrial applications of liquid-liquid chromatography (LLC). Despite various experimental research efforts to purify cannabinoids, there are currently few works on process modeling. Thus, this study aimed to explore a straightforward approach to model the LLC separation of cannabinoids from two hemp extracts with different compositions. The feed materials were simplified to mixtures of preselected key components (i.e., cannabidiol, tetrahydrocannabinol, cannabigerol, and cannabinol). The elution profiles of cannabinoids were simulated using the equilibrium-cell model with an empirical nonlinear correlation. The model parameters were derived from the elution profiles of single-solute pulse injections. For the validation of the proposed approach, LLC separations with the two hemp extracts were performed in descending mode with the solvent system composed of hexane/methanol/water 10/8/2 (v/v/v). The injected sample concentrations were gradually increased from 5 to 100 mg/mL. The results showed that the approach could describe reasonably well the elution behavior of the cannabinoids, with deviations of only 1-2 min between simulated and experimental elution times. However, to improve the prediction accuracy, the model parameters can be refitted to the elution profiles of 3-4 systematically selected pulse injections with specific hemp extracts.