Cannabidiolic Acid Research Articles

Enantioseparation of chiral phytocannabinoids in medicinal cannabis

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.

Excretion and residue depletion of cannabinoids in beef cattle fed hempseed cake for 111 days

Thirty-two crossbred heifers were fed either a control diet or 20% (dry matter basis) hempseed cake in a complete ration for 111 days; of the cattle fed hempseed cake, four each were harvested with 0, 1, 4, and 8-day withdrawal periods. Urine and plasma were collected during the feeding and withdrawal periods and liver, kidney, skeletal muscle, and adipose tissue were collected at harvest. Total cannabinoid (n = 10) concentration of hempseed cake averaged 11.3 ± 11.7 mg kg−1 across the feeding period with total cannabidiol and tetrahydrocannabinol (CBD/THC) concentrations of 1.3 ± 0.8 mg kg−1. Neutral cannabinoids (cannabinol [CBN], CBD/THC, and cannabidivarin [CBDV]) were not detected in plasma or urine, but CBD/THC was measured in adipose tissue at all withdrawal periods (6.3 ± 2.1 to 10.1 ± 2.5 ng g−1). In contrast, cannabinoid acids (cannabinolic acid [CBNA], cannabidiolic acid [CBDA]/tetrahydrocannabinolic acid [THCA], cannabichromenic acid [CBCA], and cannabidivarinic acid [CBDVA]) were sporadically detected (<15 ng mL−1) in plasma and urine of cattle fed hempseed cake. Cannabinoid acids were depleted from liver by withdrawal day 4, but could still be measured (<1 ng g−1) in kidney of some animals harvested on withdrawal day 8. Assessment of human exposures to CBD/THC residues through the consumption of beef fat from animals fed hempseed cake suggests that the probability of consuming the equivalent of an acute reference dose (ARfD) is remote, even with the use of a conservative reference dose ARfD (1 μg kg−1 body weight).

Developing Prediction Models Using Near-Infrared Spectroscopy to Quantify Cannabinoid Content in Cannabis Sativa.

Cannabis is commercially cultivated for both therapeutic and recreational purposes in a growing number of jurisdictions. The main cannabinoids of interest are cannabidiol (CBD) and delta-9 tetrahydrocannabidiol (THC), which have applications in different therapeutic treatments. The rapid, nondestructive determination of cannabinoid levels has been achieved using near-infrared (NIR) spectroscopy coupled to high-quality compound reference data provided by liquid chromatography. However, most of the literature describes prediction models for the decarboxylated cannabinoids, e.g., THC and CBD, rather than naturally occurring analogues, tetrahydrocannabidiolic acid (THCA) and cannabidiolic acid (CBDA). The accurate prediction of these acidic cannabinoids has important implications for quality control for cultivators, manufacturers and regulatory bodies. Using high-quality liquid chromatography-mass spectroscopy (LCMS) data and NIR spectra data, we developed statistical models including principal component analysis (PCA) for data quality control, partial least squares regression (PLS-R) models to predict cannabinoid concentrations for 14 different cannabinoids and partial least squares discriminant analysis (PLS-DA) models to characterise cannabis samples into high-CBDA, high-THCA and even-ratio classes. This analysis employed two spectrometers, a scientific grade benchtop instrument (Bruker MPA II-Multi-Purpose FT-NIR Analyzer) and a handheld instrument (VIAVI MicroNIR Onsite-W). While the models from the benchtop instrument were generally more robust (99.4-100% accuracy prediction), the handheld device also performed well (83.1-100% accuracy prediction) with the added benefits of portability and speed. In addition, two cannabis inflorescence preparation methods were evaluated: finely ground and coarsely ground. The models generated from coarsely ground cannabis provided comparable predictions to that of the finely ground but represent significant timesaving in terms of sample preparation. This study demonstrates that a portable NIR handheld device paired with LCMS quantitative data can provide accurate cannabinoid predictions and potentially be of use for the rapid, high-throughput, nondestructive screening of cannabis material.

Twice-daily oral administration of a cannabidiol and cannabidiolic acid-rich hemp extract was well tolerated in orange-winged Amazon parrots (Amazona amazonica) and has a favorable pharmacokinetic profile.

To determine the pharmacokinetics of 8 cannabinoids and 5 metabolites after oral administration of single and multiple doses of a cannabidiol (CBD)-cannabidiolic acid (CBDA)-rich hemp extract to orange-winged Amazon parrots (Amazona amazonica) as well as to evaluate the extract's adverse effects. 12 birds. Based on pilot studies, a single-dose study based on 30/32.5 mg/kg of cannabidiol/cannabidiolic acid of a hemp extract was administered orally to 8 fasted parrots, and 10 blood samples were collected over 24 hours after administration. After a 4-week washout period, the hemp extract was administered orally to 7 birds at the previous dose every 12 hours for 7 days, and blood samples were collected at the previous time points. Cannabidiol, Δ9-tetrahydrocannabinol, cannabinol, cannabichromene, cannabigerol, cannabidiolic acid, cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, and 5 specific metabolites were measured by liquid chromatography-tandem/mass-spectrometry, and pharmacokinetic parameters were calculated. Adverse effects and changes in the plasma biochemistry and lipid panels were evaluated. Pharmacokinetic parameters for cannabidiol, cannabidiolic acid, Δ9-tetrahydrocannabinol, Δ9-tetrahydrocannabinolic acid, and the metabolite 11-hydroxy-9-tetrahydrocannabinol were established. For the multiple-dose study, cannabidiol/cannabidiolic acid mean Cmax was 337.4/602.1 ng/mL with a tmax of 30 minutes and a terminal half-life of 8.6/6.29 hours, respectively. No adverse effects were detected during the multidose study. The predominant metabolite was 11-hydroxy-9-tetrahydrocannabinol. Twice daily oral administration of the hemp extract based on 30 mg/kg/32.5 mg/kg of cannabidiol/cannabidiolic acid was well tolerated and maintained plasma concentrations considered to be therapeutic in dogs with osteoarthritis. Findings suggest different cannabinoid metabolism from mammals.

Cannabinoid Receptor 2 Blockade Prevents Anti-Depressive-like Effect of Cannabidiol Acid Methyl Ester in Female WKY Rats.

The pathophysiology of major depressive disorder (MDD) is diverse and multi-factorial, yet treatment strategies remain limited. While women are twice as likely to develop the disorder as men, many animal model studies of antidepressant response rely solely on male subjects. The endocannabinoid system has been linked to depression in clinical and pre-clinical studies. Cannabidiolic Acid-Methyl Ester (CBDA-ME, EPM-301) demonstrated anti-depressive-like effects in male rats. Here, we explored acute effects of CBDA-ME and some possible mediating mechanisms, using a depressive-like genetic animal model, the Wistar-Kyoto (WKY) rat. In Experiment 1, Female WKY rats underwent the Forced swim test (FST) following acute CBDA-ME oral ingestion (1/5/10 mg/kg). In Experiment 2, Male and female WKY rats underwent the FST after injection of CB1 (AM-251) and CB2 (AM-630) receptor antagonists 30 min before acute CBDA-ME ingestion (1 mg/kg, males; 5 mg/kg, females). Serum levels of Brain-Derived Neurotrophic Factor (BDNF), numerous endocannabinoids and hippocampal Fatty Acid Amide Hydrolase (FAAH) levels were assessed. Results indicate that females required higher doses of CBDA-ME (5 and 10 mg/kg) to induce an anti-depressive-like effect in the FST. AM-630 blocked the antidepressant-like effect in females, but not in males. The effect of CBDA-ME in females was accompanied by elevated serum BDNF and some endocannabinoids and low hippocampal expression of FAAH. This study shows a sexually diverse behavioral anti-depressive response to CBDA-ME and possible underlying mechanisms in females, supporting its potential use for treating MDD and related disorders.

Hemp Waste as a Substrate for Hermetia illucens (L.) (Diptera: Stratiomyidae) and Tenebrio molitor L. (Coleoptera: Tenebrionidae) Rearing.

The proper treatment of cannabis agricultural wastes can reduce the environmental impact of its cultivation and generate valuable products. This study aimed to test the potential of cannabis agricultural wastes as a substrate for the rearing of black soldier fly larvae (BSFL) and yellow mealworms (MW). In the case of BSFL, replacing the fibre component (straw) in the substrate with the hemp waste can increase the nutritional value of the substrate and led to bigger larvae. The bigger larvae had lower P and Mg, and higher Fe and Ca. Crude protein also varied based on the size of larvae and/or the content of protein in the initial substrate, which was boosted by replacing straw with hemp material. No other cannabinoids than cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), and cannabidiol (CBD) were found in significant amounts in the larvae. In the case of MW, the larvae grew less on the hemp material in comparison to wheat bran. Replacing wheat bran with the hemp material led to smaller larvae with higher Ca, Fe, K, and crude protein content, but lower Mg and P values. No cannabinoids were detected in the MW fed with the hemp material.

Development and Validation of a GC-FID Method for the Quantitation of 20 Different Acidic and Neutral Cannabinoids.

For decades, Cannabis sativa had been illegal to sell or consume around the world, including in the United States. However, in light of the recent 2018 Farm Bill and the legalization of hemp across the US, various cannabis preparations have flooded the market, making it essential to be able to quantitate the levels of the different acidic and neutral cannabinoids in C. sativa and to have a complete cannabinoid profile of the different chemovars of the cannabis plant. A GC-FID method was developed and validated for the analysis of 20 acidic and neutral cannabinoids as trimethylsilyl (TMS) derivatives. The analyzed cannabinoids include cannabidivarinic acid (CBDVA), cannabidiolic acid (CBDA), cannabinolic acid (CBNA), cannabielsoic acid (CBEA), cannabicyclolic acid (CBLA), cannabichromenic acid (CBCA), trans-Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA), trans-Δ9-tetrahydrocannabinolic acid A (Δ9-THCAA), cannabigerolic acid (CBGA), cannabidiol (CBD), cannabicyclol (CBL), cannabidivarin (CBDV), trans-Δ9-tetrahydrocannabivarin (THCV), cannabichromene (CBC), trans-Δ8-tetrahydrocannabinol (Δ8-THC), trans-Δ9-tetrahydrocannabinol (Δ9-THC), cannabigerol (CBG), cannabinol (CBN), cannabicitran (CBT), and cannabielsoin (CBE). The method limit of detection (LOD) was as low as 0.1 µg/mL, while the limit of quantitation ranged from 0.25 µg/mL to 0.5 µg/mL. The precision (%RSD) was < 10%, while trueness ranged from 90 - 107%. The developed method is simple, accurate, and sensitive for the quantitation of all 20 acidic and neutral cannabinoids. Finally, the proposed method was successfully applied to the quantitation of the cannabinoids in different cannabis chemovars grown at the University of Mississippi.

Comparative Investigation of Antimicrobial and Antioxidant Effects of the Extracts from the Inflorescences and Leaves of the Cannabis sativa L. cv. strawberry

Cannabis sativa products have historically been used for healing purposes; now their biological properties are supported with scientific evidence, but modern research has not yet fully developed its therapeutic potential. This study focuses on the cultivar of C. sativa called strawberry to understand the biological and medical potentials of hydroalcoholic extracts from two different parts of the plant: leaves and inflorescences. Two biological assets were investigated including antioxidant and antimicrobial potential. Additionally, quantitative determination of phenolic and terpenophenol compounds was conducted. The antimicrobial action was highlighted for the hydroalcoholic extract from inflorescences, especially against Escherichia coli and Bacillus subtilis. Among the dermatophytes' strains, the most sensitive was Arthroderma currey. These effects could be related albeit partially to the pattern of the phenolics detected, among which the most prominent one was benzoic acid. On the other hand, antioxidant and antimicrobial effects of the extracts could be also mediated by the main terpenophenolics identified and quantified, namely cannabidiolic acid and cannabidiol. Collectively, the present data point to the potential use of the inflorescences from the C. sativa cultivar strawberry as a valuable plant material for the development of bioactive extracts with antioxidant and antimicrobial effects.

Meta‐analysis and review of cannabinoids extraction and purification techniques

AbstractProjected revenues of cannabis concentrates and extracts in Canada will reach 5 billion dollars, of which infused products will account for half of the total. The pharmacologically active cannabinoids accumulate in the crop's flowers, accounting for as much as 30% of their dry mass, and are absent from the rest of the plant's body. To achieve a cost effective drug formulation requires optimizing cannabis processing techniques. Here, we review the pre‐treatment of Cannabis sativa L., its solvent extraction, and the isolation of its active metabolites. We describe traditional extraction processes such as maceration and percolation with organic solvents, but focus on recent green solvent and methods including supercritical fluid extraction (SCFE) and microwave‐ and ultrasound‐enhanced techniques. Furthermore, we report the decarboxylation kinetics to convert tetrahydrocannabinolic acid and cannabidiolic acid and purification‐isolation techniques to satisfy regulatory and consumer requirements. Cannabinoids decarboxylate in 10–60 min at 100–150°C. Ethanol and petroleum ether recover up to 90% of the neutral cannabinoids from plant inflorescences, but the crude extracts require further refining as the purity is less than 50%. Propane and butane compressed gas extraction facilitate solvent removal but introduce safety hazards related to flammability. SCFE is the safest solvent‐free extraction method with improved terpenoid recovery and purity. Academic and commercial interest in the field is expected to accelerate in the next decade due to recent changes in regulatory schemes across North America, which will reduce legal and stigmatic barriers to research.

Bioassay-Guided Isolation and Identification of Antibacterial Components against Escherichia coli from Industrial Hemp Leaves

Industrial hemp leaves have raised much interest in nutraceuticals and functional foods areas. To expand its application ranges, the antibacterial activities of industrial hemp leaf extract on Escherichia coli, Staphylococcus aureus, and Bacillus cereus were evaluated and the active components were screened. As a result, the industrial hemp leaf extract was found to have strong bacteriostatic effects on E. coli and S. aureus. Bioassay-guided fractionation and isolation from fractions active against E. coli were conducted. Two compounds, cannabidivarinic acid and cannabidiolic acid, were firstly recognized by analytical HPLC by comparing the retention times and UV spectra with standards and later isolated using preparative HPLC. Moreover, the antibacterial mechanisms of cannabidivarinic acid and cannabidiolic acid were investigated by testing the alkaline phosphatase activity, β-galactosidase activity, conductivity, proteins leakage, nucleic acid leakage, and scanning electron microscope observation. The results demonstrated that cannabidivarinic acid and cannabidiolic acid could destroy the cell wall and membrane of E. coli, resulting in the inhibition of enzyme activity and leakage of contents. They could damage the bacteria cell envelope as well. Presented results pointed out cannabidivarinic acid and cannabidiolic acid as promising natural bacteriostatic agents for the food, pharmaceutical, and cosmetic industry.

Evaluation of the efficacy of a cannabidiol and cannabidiolic acid rich hemp extract for pain in dogs following a tibial plateau leveling osteotomy.

To determine the impact of a cannabidiol (CBD) and cannabidiolic acid (CBDA) rich hemp product on acute post-operative pain in dogs following a tibial plateau leveling osteotomy (TPLO), and to evaluate for changes in early bone healing, serum chemistry profiles, and complete blood counts. In this randomized, placebo controlled, blinded clinical trial, 44 client-owned dogs were assigned to receive either a CBD/CBDA product dosed at 2-2.5 mg/kg PO every 12 h or a placebo for 4 weeks following a TPLO. Variables evaluated before (week 0), and at 2 and 4 weeks post-operatively included standardized veterinary assessments for pain score, weight-bearing, and lameness, the Canine Brief Pain Inventory (pain interference score-PIS, pain severity score-PSS), and serum biochemistry. Complete blood counts were performed at weeks 0 and 4. Additionally, orthogonal radiographs evaluating the degree of healing were taken at week 4. A mixed model analysis, analyzing changes of variables of interest from enrollment baseline to all other time points was utilized, with a p-value ≤ 0.05 considered significant. Of the 44 enrolled patients, 3 were lost to follow up and excluded from analysis. No significant differences were noted between placebo (n = 19) and CBD/CBDA (n = 22) groups at any point in pain score, degree of lameness, degree of weight-bearing, PIS, PSS, or radiographic healing of the osteotomy. A significant finding of elevation of ALP above normal reference range in the treatment group was identified (p = 0.02) and eosinophil count was affected by treatment (p = 0.01), increasing from baseline in placebo and decreasing in treatment groups. Finally, a significant difference (p = 0.03) was noted at 2 weeks post-operatively where 4 patients in the placebo group and no treatment patients received trazodone to facilitate activity restrictions. Use of a CBD/CBDA rich hemp product dosed at 2-2.5 mg/kg PO every 12 h did not have a significant impact on pain or delay early bone healing. A statistically significant increase in ALP, decrease in eosinophils, and reduced use of trazodone was identified in the treatment group.

Novel fluorescence spectroscopy method coupled with N-PLS-R and PLS-DA models for the quantification of cannabinoids and the classification of cannabis cultivars.

Cannabis sativa L. inflorescences are rich in secondary metabolites, particularly cannabinoids. The most common techniques for elucidating cannabinoid composition are expensive technologies, such as high-pressure liquid chromatography (HPLC). We aimed to develop and evaluate the performance of a novel fluorescence spectroscopy-based method coupled with N-way partial least squares regression (N-PLS-R) and partial least squares discriminant analysis (PLS-DA) models to replace the expensive chromatographic methods for preharvest cannabinoid quantification. Fresh medicinal cannabis inflorescences were collected and ethanol extracts were prepared. Their excitation-emission spectra were measured using fluorescence spectroscopy and their cannabinoid contents were determined by HPLC-PDA. Subsequently, N-PLS-R and PLS-DA models were applied to the excitation-emission matrices (EEMs) for cannabinoid concentration prediction and cultivar classification, respectively. The N-PLS-R model was based on a set of EEMs (n = 82) and provided good to excellent quantification of (-)-Δ9-trans-tetrahydrocannabinolic acid, cannabidiolic acid, cannabigerolic acid, cannabichromenic acid, and (-)-Δ9-trans-tetrahydrocannabinol (R2 CV and R2 pred >0.75; RPD >2.3 and RPIQ > 3.5; RMSECV/RMSEC ratio < 1.4). The PLS-DA model enabled a clear distinction between the four major classes studied (sensitivity, specificity, and accuracy of the prediction sets were all ≥0.9). The fluorescence spectral region (excitation 220-400 nm, emission 280-550 nm) harbors sufficient information for accurate prediction of cannabinoid contents and accurate classification using a relatively small data set.

DESENVOLVIMENTO DE METODOLOGIA PARA DETERMINAÇÃO DE CANABINOIDES EM PRODUTOS À BASE DE CANNABIS PARA FINS MEDICINAIS

DEVELOPMENT OF A METHODOLOGY FOR THE DETERMINATION OF CANNABINOIDS IN CANNABIS-BASED PRODUCTS FOR MEDICINAL PURPOSES. Medicinal products based on the Cannabis sativa plant are available in Brazil, among them, drug, cannabis products, individual import products, and those provided by patient associations. Despite this, there is no monograph for the evaluation of cannabinoids in Brazilian Pharmacopoeia (BP) or other compendia accepted. Therefore, this work aimed to develop a quick and easy method to quantify cannabinoids in authorized products for medicinal purposes. The method was developed by HPLC-UV, gradient elution composed of formic acid in acetonitrile and water, the flow rate of 0.6 mL min-1 and 220 nm detection. It was possible to resolve the peaks of cannabidiol (CBD), tetrahydrocannabinol (THC), cannabidiolic acid and cannabinol and to quantify CBD (105.35%; 104.07%) and THC (106.62%). The developed method was applied to two products and met the established requirements of precision, accuracy, linearity, and selectivity. This procedure has been tested on four cannabisbased products to attest to its effectiveness. Considering the importance of establishing minimum quality requirements for medicinal products and considering the available products in Brazil, it was found that it is important to evaluate the implementation of other products in the method developed for future validation and later inclusion in the BP.

In silico investigation of the antiemetic effect of cannabidiolic acid (CBDA), the phytocannabinoid of Cannabis sativa, by molecular docking method

In silico investigation of the antiemetic effect of cannabidiolic acid (CBDA), the phytocannabinoid of Cannabis sativa, by molecular docking method

Quality indexes and composition of 13 commercial hemp seed oils

Cold-pressed hemp seed oil can be a valuable source from both nutritional and sustainability points of view. Thirteen hemp seed oils (HSOs) have been characterized, also with useful data about the bioactive compounds present in this product, considering that the hemp seed oil industry lacks of specific standard for the quality control of this oil. Samples were collected from the market and 12 of the 13 HSOs were claimed to be cold-pressed on the label. The hydrolytic and oxidative states were investigated according to the standards provided by the Codex Alimentarius. HSOs showed a wide range of free acidity (from 0.89 ± 0.02 to 4.58 ± 0.03 mg KOH/g) and peroxide values (from 3.97 ± 0.25 to 23.89 ± 0.74 mEqO2/kg). The HSOs had a ratio of ω6:ω3 from 2.60 to 3.67. γ-tocopherol ranged from 594 to 967 mg/kg and a wide range in the content of chlorophylls (from 0.78 to 75.7 mg/kg) and carotenes (from 2.53 to 33.9 mg/kg) was seen. This suggests that different extraction conditions (production plants, extraction temperatures and time) were used. Cannabinoids were also present: cannabidiolic acid (CBDA) (from 4.25 to 91.6 mg/kg), Δ9-tetrahydrocannabinol (Δ9-THC) (up to 5.29 mg/kg) and tetrahydrocannabinolic acid (THCA) (up to 5.00 mg/kg). Lastly, the analysis of the volatile profile highlighted the presence of some specific cannabis terpenes, such as α-pinene and β-pinene.

Exogenous application of stress-related signaling molecules affect growth and cannabinoid accumulation in medical cannabis (Cannabis sativa L.).

Medical cannabis (Cannabis sativa L.) is a source of bioactive phytochemicals with promising pharmacological and therapeutic applications. Enhancing the accumulation of valuable bioactive compounds is potentially a way of increasing the economic importance of this crop. Signaling molecules like salicylic acid (SA), jasmonic acid (JA), and γ-aminobutyric acid (GABA) are involved in the regulation of plant development and responses to biotic and abiotic stresses. Moreover, several phytohormones regulate plant trichome formation and elicit the synthesis of secondary metabolites in many plant species in both in vitro and in vivo systems. Therefore, exogenously delivered plant signaling molecules have the potential to modify the chemical profiles of medical cannabis. In this study, we found that the foliar application of SA, methyl jasmonate (MeJA), and GABA produces changes in the accumulation of the two major cannabinoids, cannabidiolic acid (CBDA) and Δ9- tetrahydrocannabinolic acid (THCA), in leaves and inflorescences of a medical cannabis variety. MeJA at 0.1 mM increased the CBDA content in inflorescences by 15.6%, while SA and MeJA at 0.1 mM increased CBDA and THCA accumulation in leaves by up to 57.3%. Treatments did not change the expression of genes participating in the final steps of the biosynthetic pathway of cannabinoids: olivetolic acid cyclase (CsOAC-1 and CsOAC-2), 2-acylphloroglucinol 4-prenyltransferase (CsPT4), cannabidiolic acid synthase (CsCBDAS), and tetrahydrocannabinolic acid synthase (CsTHCAS). Trichome density was not significantly different from the control plants in any treatment. Besides, we found strong correlations between several plant growth parameters and cannabinoid yields, showing a direct link between plant fitness and the production of cannabinoids.

Robust statistical analysis to predict and estimate the concentration of the cannabidiolic acid in Cannabis sativa L.: A comparative study

Growing awareness of the medicinal and therapeutic benefits of cannabis has resulted in extensive research, increasing numbers of commercial growers, and the provision of commercial testing. For commercial production, the importance of monitoring for consistent cannabinoid composition is essential. Gas chromatography and/or liquid chromatography methods are effective methods. Unfortunately, they are not easily scaled up for frequent or large-scale testing. Precision cultivation for medicinal cannabis requires the industry to test large volumes of plants frequently during different stages of the growth cycle. This cannot be achieved with destructive testing alone. A statistical method with 1H nuclear magnetic resonance (NMR) is proposed and investigated to explore its potential for a cost-effective mass-screening method of plant material samples. In addition, hyperspectral imaging (HSI) is employed for frequent non-destructive measurements. This allows increasing the frequency of observations during the growth of the plant. The cannabidiolic acid (CBDA) concentration is determined by the application of both HSI and NMR spectroscopy and validated against liquid chromatography-mass spectrometry (LCMS). The paper proposes a multivariate statistical regression algorithm that automatically determines the CBDA concentration directly from bucket integration of the NMR spectrum. The algorithm successfully predicted the CBDA concentration of 7 unknown samples from data selected from 4 known samples, with an average R2 value of 0.98. The proposed statistical method was applied to the data collected for HSI. It showed that while the hyperspectral dataset can be correlated with CBDA concentration, it was subject to high variance. However, HSI retains the spatial information of the actual plant structure, allowing the CBDA prediction to be mapped back to the original spatial location in the plant while providing visual information on CBDA concentration within a flower or leaf without requiring the plant component to be destroyed in the process.

Novel fluorescence spectroscopy coupled with PARAFAC modeling for major cannabinoids quantification and identification in cannabis extracts

Cannabinoids are commonly identified and quantified using chromatographic based methods. In the present study, fluorescence spectroscopic method coupled with Parallel Factor Analysis (PARAFAC) modeling was developed and validated as a simple and fast alternative technique for identification and quantification of major cannabinoids. A PARAFAC model was built on a set of excitation-emission matrices, yielding an optimal quantitative and qualitative performance using five components, which were identified as (-)-Δ9-trans-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabichromenic acid (CBCA) and (-)-Δ9-trans-tetrahydrocannabinol/cannabidiol/cannabigerol (THC/CBD/CBG). The identity of the major acidic components, THCA, CBDA and CBGA was verified by the correlation between PARAFAC model scores and their corresponding concentrations measured by HPLC as well as by the similarity between the excitation-emission spectral loadings of each PARAFAC component and the excitation-emission spectra of pure cannabinoids standards. Moreover, the PARAFAC model scores of each component were plotted against the cannabinoids actual concentrations in the extracts to evaluate the performance of the model for predicting the concentration of each compound. All three major acidic cannabinoids revealed good to excellent linear correlations (R2 > 0.7) between the model scores and measured concentrations according to the model calibration, cross-validation and prediction performance criteria. On the other hand, components 4 and 5 identified as CBCA and THC/CBD/CBG, respectively, revealed weaker linear correlation between the PARAFAC scores to the measured concentrations. These findings pave the way for a more comprehensive assessment of cannabis excitation-emission matrices (EEMs) as a cheaper and fast alternative for commonly used chromatographic-based quantification methods.

Thermal decarboxylation of acidic cannabinoids in Cannabis species: identification of transformed cannabinoids by UHPLC-Q/TOF–MS

Decarboxylation of cannabidiolic acid (CBDA) is an important step for efficient production of the active pharmaceutical component cannabidiol (CBD) in Cannabis species. Acidic cannabinoids (ACBs) can be easily transformed into neutral cannabinoids via loss of carbon dioxide when exposed to heat. During the thermal process, several transformed products including psychotropic △9-tetrahydrocannabinol (△9-THC) and its isomers were produced through decarboxylation, hydration, isomerization, and oxidation, as identified by ultra-high-performance liquid chromatography quadrupole/time-of-flight mass spectrometry (UHPLC-Q/TOF MS). Their identification was carried out using authenticated standards and interpreting the MS/MS fragmentations. To investigate thermal decarboxylation, CBDA was extracted and isolated from inflorescence of Cannabis by ultrasonication extraction and two-step column chromatography. To investigate the decarboxylation yield of isolated CBDA and ACBs in Cannabis extract, samples were examined over a range of reaction temperatures (110–130 °C) and times (5–60 min). Time profiles of CBDA degradation and CBD formation were obtained as functions of the reaction temperature. In particular, most of the CBDA was converted into CBD at 130 °C for 20 min; this CBD was partially transformed to psychotropic THC isomers via cyclization. In addition to THC isomers, cannabielsoin acid (CBEA) and cannabielsoin (CBE) were also observed as minor oxidative transformed products. Based on structural identification and profiling data, thermal transformation pathways of CBDA are plausibly suggested. The results of decarboxylation of ACBs will provide important information on production of neutral cannabinoids, especially CBD, in Cannabis plants and quality control of Cannabis-based products in pharmaceutical and cosmetic industries.

Medical Cannabis Use and Inflammatory Cytokines and Chemokines Among Adult Chronic Pain Patients.

Background: Utilizing cannabis as a therapeutic option for chronic pain (CP) has increased significantly. However, data regarding the potential immunomodulatory effects of cannabis in CP patients remain scarce. We aimed at exploring the relationship between cannabis use and inflammatory cytokines and chemokines among a cohort of CP patients. Methods: Adult patients with a CP diagnosis and medical authorization of cannabis were enrolled. Patients completed validated clinical questionnaires and self-reported the effectiveness of cannabis for symptom management. Patients' blood and cannabis samples were analyzed for the presence of four major cannabinoids, two major cannabinoid metabolites, 29 different cytokines/chemokines, and cortisol. The multivariable linear regression model was used to identify cannabis and patient factors associated with immune markers. Results: Fifty-six patients (48±15 years; 64% females) were included, with dried cannabis (53%) being the most common type of cannabis consumed. Seventy percent of products were considered delta-9-tetrahydrocannabinol (Δ9-THC)-dominant. The majority of patients (96%) self-reported effective pain management, and 76% reported a significant decrease in analgesic medication usage (p≤0.001). Compared with males, female patients had higher plasma levels of cannabidiol (CBD), cannabidiolic acid, Δ9-THC, and 11-hydroxy-Δ9-tetrahydrocannabinol but lower concentrations of delta-9-tetrahydrocannabinolic acid and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH). Females had significantly lower eotaxin levels (p=0.04) in comparison to male patients. The regression analysis indicated that high cannabis doses were related to increased levels of interleukin (IL)-12p40 (p=0.02) and IL-6 (p=0.01), whereas female sex was associated with decreased eotaxin (p≤0.01) concentrations. Blood CBD levels were associated with lower vascular endothelial growth factor (p=0.04) concentrations, and THC-COOH was a factor related to decreased tumor necrosis factor alpha (p=0.02) and IL-12p70 (p=0.03). Conclusion: This study provides further support for the patient-perceived effectiveness of cannabis in managing CP symptoms and reducing analgesic medication consumption. The results suggest a potential sex difference in metabolizing cannabinoids, and the varying immune marker concentrations may support a possible immunomodulatory effect associated with patient sex and cannabis product type. These preliminary findings provide grounds for further validation using larger, well-designed studies with longer follow-up periods.