Tetrahydrocannabinolic Acid Research Articles

Investigation of Cannabinoid Acid/Cyclodextrin Inclusion Complex for Improving Physicochemical and Biological Performance.

This study aimed to investigate the enhancement of cannabinoid acid solubility and stability through the formation of a cannabinoid acid/cyclodextrin (CD) inclusion complex. Two cannabinoid acids, tetrahydro-cannabinolic acid (THCA) and cannabidiolic acid (CBDA), were selected as a model drug along with five types of CD: α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD), hydroxypropyl-β-cyclodextrin (HP-β-CD), and methylated-β-cyclodextrin (M-β-CD). Phase solubility studies were conducted using various types of CD to determine the complex stoichiometry. The preparation methods of the CD inclusion complex were optimized by adjusting the loading pH solution and the drying processes (spray-drying, freeze-drying, spray-freeze-drying). The drying process of the cannabinoid acid/M-β-CD inclusion complex was further optimized through the spray-freeze-drying method. These CD complexes were characterized using solubility determination, differential scanning calorimetry (DSC), field-emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), and 1H NMR spectroscopy. DSC, XRD, and FE-SEM studies confirmed the non-crystalline state of the cannabinoid acid/CD inclusion complex. The permeation of THCA or CBDA from the M-β-CD spray-freeze-dried inclusion complex was highly improved compared to those of cannabis ethanolic extracts under simulated physiological conditions. The stability of the cannabinoid acid/M-β-CD inclusion complex was maintained for 7 days in a simulated physiological condition. Furthermore, the minimum inhibitory concentration of cannabinoid acid/M-β-CD inclusion complex had superior anti-cancer activity in MCF-7 breast cancer cell lines compared to cannabinoid acid alone. The improved physicochemical and biological performances indicated that these CD inclusion complexes could provide a promising option for loading lipophilic cannabinoids in cannabis-derived drug products.

A forensic application of genetic markers for distinction between drug-type and fiber-type Cannabis sativa L.

Genetic markers can represent a valuable tool for forensic purposes in discriminating between fiber-type and drug-type cannabis. The aim of this research was to evaluate developed genetic markers for tetrahydrocannabinolic acid synthase (THCAS) when applied on certified hemp (14 varieties) and forensic casework samples of four chemotypes (40 seizures). Chemotype-associated PCR-based markers did not enable reliable selective amplification despite the difference in cannabinoid composition. In order to characterize forensic samples of unknown origin, THCAS sequencing was performed. The comparison of THCAS sequences, including additional accessions, indicated high genetic similarity of hemp varieties. Confiscated samples of intermediate, THC, CBD and CBG type were clearly separated from fiber-type accessions and assigned to drug-type cluster. Despite the unknown origin, their position on the tree support the notion that they are more related to drug-type accessions than to the fiber-type. However, no clear distinction between chemotypes was found. Furthermore, 26 amino acid substitutions were revealed in THCAS that clearly separate hemp varieties and neither of them cluster with any other tested sample.

Validation of a Fast LC-PDA Method for the Quantification of Cannabinoids in Commercial Tea Samples

AbstractCannabis and cannabinoids are currently being used to treat a wide variety of medical conditions including, but not limited to, cancer, epilepsy, and pain management. Since the legalization of cannabis-containing (mainly cannabidiol) products, in many countries, and the subsequent upsurge in the number of available commercial products, an increased need exists for a fast and reliable quality control method. Here, we describe the development and validation of a fast (7.5 min) and reliable method for the quantification of cannabidiol and tetrahydrocannabinol, and their respective acid precursors, cannabidiolic acid and tetrahydrocannabinolic acid, in ten commercial tea products using LC-PDA. Due to the lipophilic nature of cannabinoids, all tea samples were also prepared according to the manufacturer’s recommendations and analyzed with the validated method. By extracting the tea samples with acetonitrile, it was found that the actual cannabidiol content in all products tested deviated significantly from their label claims by having as much as 80% less than claimed. By preparing the samples as described on the packaging, no cannabidiol could be detected, indicating that boiling or hot water fails to extract any significant amount of cannabidiol present in these products. It can therefore be concluded that based on the lipophilic nature of cannabidiol, very little to no cannabidiol will be consumed when these products are used as prescribed and therefore may nullify the various health claims made for these popular products. Graphical Abstract

Identification and Quantification of the Main Psychoactive Ingredients of Cannabis in Urine Using Excitation-Eemission Matrix Fluorescence Coupled with Parallel Factor Analysis.

Cannabis is the most prevalent abused substance after alcohol, and its consumption severely harms human health and thus adversely impacts society. The identification and quantification of cannabis in urine play important roles in practical forensics. Excitation-emission matrix (EEM) fluorescence spectroscopy coupled with parallel factor (PARAFAC) analysis was developed to identify and quantify the four main ingredients of cannabis in urine samples. The main ingredients of cannabis including Δ-9-tetrahydrocannabinol (THC), cannabidiol, cannabinol, and tetrahydrocannabinolic acid (THC-COOH) exhibited diverse fluorescence characteristics, and the concentrations of these compounds depicted a positive linear relationship with the fluorescence intensity at the ng/mL level. The EEM/PARAFAC method adequately characterized and discriminated the four ingredients in calibration and prediction samples with a low root-mean-square error of prediction (RMSEP; 0.03-0.07 μg/mL) and limit of quantitation (LOQ; 0.26-0.71 μg/mL). The prediction results of the EEM/PARAFAC method well correlated with that of GC-MS with a low RMSEP range (0.01-0.05 μg/mL) and LOQ range (0.07-0.44 μg/mL) in urine samples. The EEM spectroscopic investigation coupled with the PARAFAC algorithm results in an organic, solvent-less, fast, reliable tool to perform accurate and rapid screening of cannabis abusers.

Development and Validation of a Simple, Fast, and Accessible HPLC-UV Method for Cannabinoids Determination in Cannabis sativa L. Extracts and Medicinal Oils.

Cannabis sativa L. is a well-recognized medicinal plant. Cannabis regulations in Argentina are insufficient to solve the problem of patient access to full-spectrum cannabis-based products. So, the market of artisanal products with unknown quality and dosage of cannabinoids is increasing, and so is the local demand and need for analyzing these products. However, much of the latest validated methodologies for cannabinoid quantification include expensive instrumentation that is not always available in laboratories of health institutions in Argentina. The aim of this work was to develop and validate a simple and rapid HPLC-UV method for the identification and quantification of principal cannabinoids in cannabis resins, inflorescences, and medicinal oils using standard HPLC equipment. The cannabinoids selected for validation were cannabidiol acid (CBDA), cannabigerol (CBG), cannabidiol (CBD), cannabinol (CBN), delta-9-tetrahydrocannabinol (Δ9-THC), cannabichromene (CBC), and tetrahydrocannabinol acid (THCA). A method for the simultaneous identification and quantification of these 7 main cannabinoids was developed and then validated. Some data parameters were comparable to other reports with more sophisticated analytical instruments for the analysis of cannabis. The assessed limits of detection and the limits of quantitation ranged from 0.9 to 3.66 μg/mL and 2.78 to 11.09 μg/mL, respectively. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area with R2 values of > 0.99 for all 7 cannabinoids. The relative standard deviation (RSD%) varied from 2.34 to 4.82 for intraday repeatability and from 1.16 to 3.15 for interday repeatability. The percentage of recovery values was between 94 to 115% (resins) and 80 to 103% (inflorescence extract). The cannabis industry is growing rapidly, and there is a need for reliable testing methods to ensure the safety and efficacy of cannabis products. In addition, current methods for cannabinoid analysis are often time-consuming and expensive, while the HPLC-UV method herein reported is a simple, rapid, accurate, and cost-effective alternative for the analysis of cannabinoids in cannabis resins, inflorescences, and medicinal oils. This method will be proposed to be included in the Cannabis sativa L. monograph of the Argentine Pharmacopoeia.

Bidimensional heart-cut achiral-chiral liquid chromatography coupled to high-resolution mass spectrometry for the separation of the main chiral phytocannabinoids and enantiomerization studies of cannabichromene and cannabichromenic acid

In this work, a heart-cut bidimensional achiral-chiral liquid chromatography method coupled to high-resolution mass spectrometry was developed for the separation of the main carboxylated phytocannabinoids, namely cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), cannabichromenic acid (CBCA), and cannabicyclolic acid (CBLA), and decarboxylated derivatives, namely cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THC), cannabichromene (CBC), and cannabicyclol (CBL), and the evaluation of their enantiomeric composition in extracts of different Cannabis sativa L. varieties. Optimal conditions for the chiral analysis of CBC- and CBL-type compounds were found with methanol and water (95:5, v/v, with 0.1% formic acid, 1.5 mL/min) on an amylose-based chiral stationary phase. These settings also allowed to evaluate the parameters responsible for CBC and CBCA racemization.

Quality and safety of hemp meal as a protein supplement for nonlactating dairy cows

Hemp seed meal may be a suitable protein supplement for dairy cows, but its quality and safety as a dairy cow feed has not yet been fully investigated. As a result, dry matter intake (DMI), rumen fermentation, blood metabolites, total-tract digestibility, and concentrations of cannabinoids in blood plasma, urine, muscle, and adipose tissues were compared among nonlactating Holstein dairy cows receiving a basal partial mixed ration that was supplemented with either 10.2% dry matter (DM) hemp meal (HM treatment), 13.5% DM canola meal (CM treatment), or 6.25% DM hemp meal and 6.16% DM canola meal (HC treatment). Diets were formulated to be isoenergetic and isonitrogenous. Six nonlactating, nonpregnant Holstein cows were used in a repeated 3 × 3 Latin square design trial with three 3-wk experimental periods. The first 2 weeks of each served as adaptation. Sample and data collection occurred during the third week of each period. Neither the partial mixed ration nor canola meal contain cannabidiol (CBD), cannabidiolic acid (CBDA), d9-tetrahydrocannabinol (THC), or tetrahydrocannabinolic acid A (THCA-A). However, the hemp meal contained 3.0, 4.4, 0, and 0.1 μg/g DM of CBD, CBDA, THC, and THCA-A, respectively. Treatment did not affect DMI, pH, concentrations of volatile fatty acids or ammonia in the rumen, total-tract digestibilities of DM and crude protein, or blood plasma concentrations of glucose, urea, β-hydroxybutyrate, and nonesterified fatty acids. Hence, based on these metabolites, treatment did not affect the nutritional status of the cows. However, the total-tract neutral detergent fiber digestibility of the CM treatment (43%) was higher than that of the HM treatment (38%). No cannabinoids were detected in blood plasma, rumen fluid, and urine. Cannabinoids were also not detected in kidney, liver, urine, muscle, or adipose tissues at the end of the experiment when cows had undergone all treatments. Feces from all treatments did not contain detectable concentrations of THC or THCA-A, but feces of cows on the HC treatment contained 0.42 and 0.40 μg/g DM of CBD and CBDA, respectively. Feces of cows on the HM treatment contained 0.68 and 0.67 μg/g DM of CBD and CBDA, respectively. This indicated that most ingested CBD and CBDA were not absorbed but instead were excreted in the feces. Our data show dietary inclusion rates of up to 10.2% of DM. We find that hemp meal is a high-quality and safe protein supplement for nonlactating dairy cows.

Comparison of recirculation and drain-to-waste hydroponic systems in relation to medical cannabis (Cannabis sativa L.) plants

Rising temperatures, higher water consumption for irrigation, and the discharge of agricultural wastewater may soon adversely affect water resources across the entire planet and its people. Agriculture consumes large amounts of water, and there is a pressing need to explore possibilities for recirculation and reuse of nutrient solutions to reduce the associated environmental and economic costs of hydroponics. This study compared two different nutrient systems for hydroponic cultivation (recirculation, RS, and drain-to-waste system, DS), and assessed their effect on the growth and concentration of elements and secondary metabolites in medical cannabis (Cannabis sativa L.) plants. The dry decomposition method with flame atomic absorption spectroscopy (FAAS) and inductively coupled plasma optical emission spectroscopy (ICP-OES) were used for the elemental analysis of plant organs. Nitrogen content was measured using the Kjeldahl method. A high-performance liquid chromatograph equipped with a diode array detector (HPLC-DAD) was used to analyze the phytocannabinoid profile in ethanolic flower extracts. Gas chromatography with flame ionization detection (GC-FID) and gas chromatography with mass spectrometry (GC-MS) were employed to determine the content of terpenic compounds in hexane extracts. Cultivation of plants with the RS system, achieved an 87% higher maximum yield of the main cannabinoid of medical cannabis chemotype I, tetrahydrocannabinolic acid (THCA), with lower total water and nutrient consumption, but with a longer cultivation time and a lower concentration of the sesquiterpene, β-caryophyllene. In contrast, the DS system enabled better control over the nutrient solution with stable delivery of the exact concentrations of nutrients. DS accelerated plant maturation, but at the cost of higher water and fertilizer consumption and a significantly lower total yield of THCA and monoterpenes.

Determination of eight cannabinoids in foods with enhanced matrix removal-lipid adsorbent by ultra performance liquid chromatography-tandem mass spectrometry

A novel method was developed for the simultaneous determination of eight cannabinoids in six types of food matrices, including chocolate, fondant, biscuit, beverage, cookie and baijiu, using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The sample extraction and cleanup steps were optimized, and various purification methods were investigated to remove the oil matrix and glue in chocolate and fudge, respectively. Enhanced matrix removal-lipid adsorbent (EMR-Lipid) provided efficient, selective cleanup of the evaluated matrices. The sample was extracted using acetonitrile, followed by EMR-Lipid cleanup, and then dried using anhydrous sodium sulfate. The acetonitrile layer was concentrated by nitrogen to near-dry after 100 μL 10% glycerol in methanol was added to improve the recovery by reducing loss during concentration under the stream of nitrogen gas. Eight cannabinoids were separated using a Waters ACQUITY UPLC BEH Shield RP18 column (100 mm×3.0 mm, 1.7 μm). The responses of the cannabinoids in the positive and negative ionization modes were investigated and optimized, and the responses were superior in the negative ion mode compared to those in the positive ion mode. MS detection was performed in the multi-reaction monitoring (MRM) mode using an electrospray source in the negative ion mode. The cannabinoids were quantified using an external standard with matrix calibration curves to reduce the influences of the matrix effects on the quantitative results. The developed method was verified, and the conditions of sample pretreatment were also optimized. The calibration curves of tetrahydrocannabinol, cannabidivarin, tetrahydrocannabivarin, and cannabigerol and those of cannabidiol, cannabinol, cannabidiolic acid, and tetrahydrocannabinolic acid exhibited good linearities, with r>0.995, in the ranges of 2-200 and 0.4-40 ng/mL, respectively. The respective limits of detection (LODs, S/N=3) and quantification (LOQs, S/N=10) of tetrahydrocannabinol, cannabidivarin, tetrahydrocannabivarin, and cannabigerol were 4 and 10 μg/kg, and those of cannabidiol, cannabinol, cannabidiolic acid, and tetrahydrocannabinolic acid were 0.8 and 2 μg/kg. The average recoveries of the cannabinoids were 82.0%-114.9% under three spiked levels with corresponding relative standard deviations (RSDs) of <15% (n=6). EMR-Lipid provided efficient, selective cleanups of food matrices with good accuracy. The method is sensitive, rapid, accurate, simple to execute, and it is suitable for the determination of cannabinol compounds in typical food matrices.

The Cannabinoids, CBDA and THCA, Rescue Memory Deficits and Reduce Amyloid-Beta and Tau Pathology in an Alzheimer’s Disease-like Mouse Model

Most studies related to hemp are focused on Cannabidiol (CBD) and Tetrahydrocannabinol (THC); however, up to 120 types of phytocannabinoids are present in hemp. Hemp leaves contain large amounts of Cannabidiolic acid (CBDA) and Tetrahydrocannabinolic acid (THCA), which are acidic variants of CBD and THC and account for the largest proportion of CBDA. In recent studies, CBDA exhibited anti-hyperalgesia and anti-inflammatory effects. THCA also showed anti-inflammatory and neuroprotective effects that may be beneficial for treating neurodegenerative diseases. CBDA and THCA can penetrate the blood-brain barrier (BBB) and affect the central nervous system. The purpose of this study was to determine whether CBDA and THCA ameliorate Alzheimer's disease (AD)-like features in vitro and in vivo. The effect of CBDA and THCA was evaluated in the Aβ1-42-treated mouse model. We observed that Aβ1-42-treated mice had more hippocampal Aβ and p-tau levels, pathological markers of AD, and loss of cognitive function compared with PBS-treated mice. However, CBDA- and THCA-treated mice showed decreased hippocampal Aβ and p-tau and superior cognitive function compared with Aβ1-42-treated mice. In addition, CBDA and THCA lowered Aβ and p-tau levels, alleviated calcium dyshomeostasis, and exhibited neuroprotective effects in primary neurons. Our results suggest that CBDA and THCA have anti-AD effects and mitigate memory loss and resilience to increased hippocampal Ca2+, Aβ, and p-tau levels. Together, CBDA and THCA may be useful therapeutic agents for treating AD.

Determination of 11 Cannabinoids in Cannabis sativa L. by Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-Q-TOF-MS)

Eleven major cannabinoids from each subdivided tissue of drug-type and fiber-type cannabis plants were determined by means of a liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). The cannabinoids analyzed in this study were tetrahydrocannabinol acid (THCA), Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol acid (CBDA), cannabidiol (CBD), Δ8-tetrahydrocannabinol (Δ8-THC), cannabinol (CBN), cannabichromene (CBC), cannabidivarin (CBDV), cannabigerolic acid (CBGA), cannabigerol (CBG) and tetrahydrocannabivarin (THCV). As a result, THCA was detected in the bracts at 28.4 µg/mg, in the buds at 24.8 µg/mg, and in the leaves at 5.1 to 10.5 µg/mg in the drug-type cannabis plant. In addition, Δ9-THC, CBGA, CBN, CBG, CBC, and THCV were mainly detected in bracts, buds, and leaves. On the other hand, as for the fiber-type cannabis plant, CBDA was detected in the bracts at 27.5 µg/mg, in the buds at 10.6 µg/mg, and in the leaves at 1.5-3.3 µg/mg. In addition, Δ9-THCA, CBD, Δ9-THC, CBC, and CBG were mainly detected in bracts, buds, and leaves.

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).

Sparse reproducible machine learning for near infrared hyperspectral imaging: Estimating the tetrahydrocannabinolic acid concentration in Cannabis sativa L.

Sparse reproducible machine learning for near infrared hyperspectral imaging: Estimating the tetrahydrocannabinolic acid concentration in Cannabis sativa L.

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.

Comparative metabolism of THCA and THCV using UHPLC-Q-Exactive Orbitrap-MS

Delta(9)-tetrahydrocannabinolic acid (THCA) and delta(9)-tetrahydrocannabivarin (THCV) are phytocannabinoids with a similar structure derived from Cannabis sativa and possess a variety of biological activities. However, the relationship between the metabolic characterisation and bioactivity of THCA and THCV remains elusive. To explore the relationship between the metabolism of THCA and THCV and their underlying mechanism of activity, human/mouse liver microsomes and mouse primary hepatocytes were used to compare the metabolic maps between THCA and THCV through comparative metabolomics. A total of 29 metabolites were identified containing 7 previously undescribed THCA metabolites and 10 previously undescribed THCV metabolites. Of these metabolites, THCA was transformed into an active metabolite of delta(9)-tetrahydrocannabinol (THC) in these three systems, while THCV was transformed into THC and CBD. Bioactivity assays indicated that all of these phytocannabinoids exhibited anti-inflammatory activity, but the effects of THCA and THCV were slightly different in macrophages RAW264.7. Prediction of ADMET lab demonstrated that THCV and its metabolites were endowed with the advantage of blood–brain barrier (BBB) penetration compared to THCA. In conclusion, this study highlighted that metabolism plays a critical role in the biological activity of phytocannabinoids.

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.

Microwave- and Ultrasound-Assisted Extraction of Cannabinoids and Terpenes from Cannabis Using Response Surface Methodology.

Limited studies have explored different extraction techniques that improve cannabis extraction with scale-up potential. Ultrasound-assisted and microwave-assisted extraction were evaluated to maximize the yield and concentration of cannabinoids and terpenes. A central composite rotatable design was used to optimize independent factors (sample-to-solvent ratio, extraction time, extraction temperature, and duty cycle). The optimal conditions for ultrasound- and microwave-assisted extraction were the sample-to-solvent ratios of 1:15 and 1:14.4, respectively, for 30 min at 60 °C. Ultrasound-assisted extraction yielded 14.4% and 14.2% more oil and terpenes, respectively, compared with microwave-assisted extracts. Ultrasound-assisted extraction increased cannabinoid concentration from 13.2-39.2%. Considering reference ground samples, tetrahydrocannabinolic acid increased from 17.9 (g 100 g dry matter-1) to 28.5 and 20 with extraction efficiencies of 159.2% and 111.4% for ultrasound-assisted and microwave-assisted extraction, respectively. Principal component analyses indicate that the first two principal components accounted for 96.6% of the total variance (PC1 = 93.2% and PC2 = 3.4%) for ultrasound-assisted extraction and 92.4% of the total variance (PC1 = 85.4% and PC2 = 7%) for microwave-assisted extraction. Sample-to-solvent ratios significantly (p &lt; 0.05) influenced the secondary metabolite profiles and yields for ultrasound-assisted extracts, but not microwave-assisted extracts.

Cold Ethanol Extraction of Cannabinoids and Terpenes from Cannabis Using Response Surface Methodology: Optimization and Comparative Study.

Efficient cannabis biomass extraction can increase yield while reducing costs and minimizing waste. Cold ethanol extraction was evaluated to maximize yield and concentrations of cannabinoids and terpenes at different temperatures. Central composite rotatable design was used to optimize two independent factors: sample-to-solvent ratio (1:2.9 to 1:17.1) and extraction time (5.7 min-34.1 min). With response surface methodology, predicted optimal conditions at different extraction temperatures were a cannabis-to-ethanol ratio of 1:15 and a 10 min extraction time. With these conditions, yields (g 100 g dry matter-1) were 18.2, 19.7, and 18.5 for -20 °C, -40 °C and room temperature, respectively. Compared to the reference ground sample, tetrahydrocannabinolic acid changed from 17.9 (g 100 g dry matter-1) to 15, 17.5, and 18.3 with an extraction efficiency of 83.6%, 97.7%, 102.1% for -20 °C, -40 °C, and room temperature, respectively. Terpene content decreased by 54.1% and 32.2% for extraction at -20 °C and room temperature, respectively, compared to extraction at -40 °C. Principal component analysis showed that principal component 1 and principal component 2 account for 88% and 7.31% of total variance, respectively, although no significant differences in cold ethanol extraction at different temperatures were observed.

Selective Cytotoxicity of Medical Cannabis (Cannabis sativa L.) Extracts Across the Whole Vegetation Cycle Under Various Hydroponic and Nutritional Treatments.

Introduction: The use of Cannabis sativa L. in health care requires stringent care for the optimal production of the bioactive compounds. However, plant phenotypes and the content of secondary metabolites, such as phytocannabinoids, are strongly influenced by external factors, such as nutrient availability. It has been shown that phytocannabinoids can exhibit selective cytotoxicity against various cancer cell lines while protecting healthy tissue from apoptosis. Research Aim: This study aimed to clarify the cytotoxic effect of cannabis extracts on colorectal cell lines by identifying the main active compounds and determining their abundance and activity across all developmental stages of medical cannabis plants cultivated under hydroponic conditions. Materials and Methods: Dimethyl sulfoxide extracts of medical cannabis plants bearing the genotype classified as chemotype I were analyzed by high-performance liquid chromatography, and their cytotoxic activity was determined by measuring cell viability by methylthiazolyldiphenyl-tetrazolium bromide assay on the human colon cancer cell lines, Caco-2 and HT-29, and the normal human epithelial cell line, CCD 841 CoN. Results: The most abundant phytocannabinoid in cannabis extracts was tetrahydrocannabinolic acid (THCA). Its maximum concentrations were reached from the 7th to the 13th plant vegetation week, depending on the nutritional cycle and treatment. Almost all extracts were cytotoxic to the human colorectal cancer (CRC) cell line HT-29 at lower concentrations than the other cell lines. The phytocannabinoids that most affected the cytotoxicity of individual extracts on HT-29 were cannabigerol, Δ9-tetrahydrocannabinol, cannabidiol, cannabigerolic acid, and THCA. The tested model showed almost 70% influence of these cannabinoids. However, THCA alone influenced the cytotoxicity of individual extracts by nearly 65%. Conclusions: Phytocannabinoid extracts from plants of the THCA-dominant chemotype interacted synergistically and showed selective cytotoxicity against the CRC cell line, HT-29. This positive extract response indicates possible therapeutic value.