基于超高效液相色谱-质谱联用技术对大麻植物中3种成分及化学表型分析

建立了超高效液相色谱-质谱联用(UPLC/PDA-QDa)同时对大麻植物中Δ9-四氢大麻酚(Δ9-THC)、大麻酚(CBN)和大麻二酚(CBD)进行定性与定量分析的方法.缴获的大麻植物用甲醇超声萃取, 采用甲醇(含0.1%甲酸)和超纯水为流动相, 等度洗脱, 流速为0.2 mL/min, 经Waters UPLC BEH C18柱(50 mm×2.1 mm, 1.7 μm)分离, 利用光电二极管阵列检测器(PDA)在220 nm波长下检测, 并通过质谱检测器(QDa)对目标洗脱峰进行追踪确证.在0.5~20 μg/mL浓度范围内, 3种大麻酚类化合物的质量浓度与峰面积呈良好的线性关系, R≥0.999;低、中、高添加水平的平均回收率为82%~102%, 相对标准偏差(RSD)在0.4%~4.1%之间.本方法稳定、简便、灵敏, 能够满足检测需求.根据Δ9-THC、(Δ9-THC+CBN)/CBD、Δ9-THC/CBD或CBN/CBD表型指数, 区分不同产地大麻的化学表型, 为大麻植物的检测分析和质量控制提供了有效手段.     

Direct Quantitation of Phytocannabinoids by One-Dimensional 1H qNMR and Two-Dimensional 1H-1H COSY qNMR in Complex Natural Mixtures

The widespread use of phytocannabinoids or cannabis extracts as ingredients in numerous types of products, in combination with the legal restrictions on THC content, has created a need for the development of new, rapid, and universal analytical methods for their quantitation that ideally could be applied without separation and standards. Based on previously described qNMR studies, we developed an expanded ¹H qNMR method and a novel 2D-COSY qNMR method for the rapid quantitation of ten major phytocannabinoids in cannabis plant extracts and cannabis-based products. The ¹H qNMR method was successfully developed for the quantitation of cannabidiol (CBD), cannabidiolic acid (CBDA), cannabinol (CBN), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabigerol (CBG), cannabigerolic acid (CBGA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabielsoin (CBE), and cannabidivarin (CBDV). Moreover, cannabidivarinic acid (CBDVA) and Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA) can be distinguished from CBDA and Δ9-THCA respectively, while cannabigerovarin (CBGV) and Δ8-tetrahydrocannabivarin (Δ8-THCV) present the same ¹H-spectra as CBG and Δ8-THC, respectively. The COSY qNMR method was applied for the quantitation of CBD, CBDA, CBN, CBG/CBGA, and THC/THCA. The two methods were applied for the analysis of hemp plants; cannabis extracts; edible cannabis medium-chain triglycerides (MCT); and hemp seed oils and cosmetic products with cannabinoids. The ¹H-NMR method does not require the use of reference compounds, and it requires only a short time for analysis. However, complex extracts in ¹H-NMR may have a lot of signals, and quantitation with this method is often hampered by peak overlap, with 2D NMR providing a solution to this obstacle. The most important advantage of the COSY NMR quantitation method was the determination of the legality of cannabis plants, extracts, and edible oils based on their THC/THCA content, particularly in the cases of some samples for which the determination of THC/THCA content by ¹H qNMR was not feasible.     

High-Throughput Quantitation of Cannabinoids by Liquid Chromatography Triple-Quadrupole Mass Spectrometry

The high-throughput quantitation of cannabinoids is important for the cannabis industry. As medicinal products increase, and research into compounds that have pharmacological benefits increase, and the need to quantitate more than just the main cannabinoids becomes more important. This study aims to provide a rapid, high-throughput method for cannabinoid quantitation using a liquid chromatography triple-quadrupole mass spectrometer (LC-QQQ-MS) with an ultraviolet diode array detector (UV-DAD) for 16 cannabinoids: CBDVA, CBDV, CBDA, CBGA, CBG, CBD, THCV, THCVA, CBN, CBNA, THC, Δ8-THC, CBL, CBC, THCA-A and CBCA. Linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, recovery and matrix effect were all evaluated. The validated method was used to determine the cannabinoid concentration of four different Cannabis sativa strains and a low THC strain, all of which have different cannabinoid profiles. All cannabinoids eluted within five minutes with a total analysis time of eight minutes, including column re-equilibration. This was twice as fast as published LC-QQQ-MS methods mentioned in the literature, whilst also covering a wide range of cannabinoid compounds.     

Differential pulsed voltammetry of Δ9-Tetrahydrocannabinol (THC) on disposable screen-printed carbon electrodes: A potential in-field method to detect Δ9-THC in saliva

Due to recent legalization of marijuana across many states in the U.S., there is an increased concern of users driving while impaired/intoxicated with Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the principal psychoactive constituent of cannabis/marijuana. Hence, there is a need for a rapid roadside detection of this drug that can be used to accurately screen drivers. Current field sobriety tests rely on a series of physical and mental exercises administered during DUI investigations to help determine a driver's level of impairment. Due to their portability and effectiveness, screen printed carbon electrodes (SPCEs) are ideal to work with when it comes to devising a low-cost screening device for roadside testing. SPCE's can potentially detect low levels of Δ⁹-THC in an individual's saliva via electrochemical oxidation of Δ⁹-THC. Herein we report a fast, cheap, and accurate approach to electrochemically detect 1–20 μM Δ⁹-THC in a 1 mL sample of artificial oral fluid (AF-OF) diluted to 50 % with a buffer/electrolyte solution using differential pulse voltammetry (DPV) at the surface of a small SPCE. Implications for the use of this method to screen intoxicated drivers are discussed.     

Simultaneous quantification of cannabinoids and metabolites in oral fluid by two-dimensional gas chromatography mass spectrometry

Development and validation of a method for simultaneous identification and quantification of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), and metabolites 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) in oral fluid. Simultaneous analysis was problematic due to different physicochemical characteristics and concentration ranges. Neutral analytes, such as THC and CBD, are present in ng/mL, rather than pg/mL concentrations, as observed for the acidic THCCOOH biomarker in oral fluid. THCCOOH is not present in cannabis smoke, definitively differentiating cannabis use from passive smoke exposure. THC, 11-OH-THC, THCCOOH, CBD, and CBN quantification was achieved in a single oral fluid specimen collected with the Quantisal™ device. One mL oral fluid/buffer solution (0.25 mL oral fluid and 0.75 mL buffer) was applied to conditioned CEREX^® Polycrom™ THC solid-phase extraction (SPE) columns. After washing, THC, 11-OH-THC, CBD, and CBN were eluted with hexane/acetone/ethyl acetate (60:30:20, v/v/v), derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide and quantified by two-dimensional gas chromatography electron ionization mass spectrometry (2D-GCMS) with cold trapping. Acidic THCCOOH was separately eluted with hexane/ethyl acetate/acetic acid (75:25:2.5, v/v/v), derivatized with trifluoroacetic anhydride and hexafluoroisopropanol, and quantified by the more sensitive 2D-GCMS–electron capture negative chemical ionization (NCI-MS). Linearity was 0.5–50 ng/mL for THC, 11-OH-THC, CBD and 1–50 ng/mL for CBN. The linear dynamic range for THCCOOH was 7.5–500 pg/mL. Intra- and inter-assay imprecision as percent RSD at three concentrations across the linear dynamic range were 0.3–6.6%. Analytical recovery was within 13.8% of target. This new SPE 2D-GCMS assay achieved efficient quantification of five cannabinoids in oral fluid, including pg/mL concentrations of THCCOOH by combining differential elution, 2D-GCMS with electron ionization and negative chemical ionization. This method will be applied to quantification of cannabinoids in oral fluid specimens from individuals participating in controlled cannabis and Sativex^® (50% THC and 50% CBD) administration studies, and during cannabis withdrawal.     

Direct quantification of cannabinoids and cannabinoid glucuronides in whole blood by liquid chromatography–tandem mass spectrometry

The first method for quantifying cannabinoids and cannabinoid glucuronides in whole blood by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated. Solid-phase extraction followed protein precipitation with acetonitrile. High-performance liquid chromatography separation was achieved in 16 min via gradient elution. Electrospray ionization was utilized for cannabinoid detection; both positive (Δ⁹-tetrahydrocannabinol [THC] and cannabinol [CBN]) and negative (11-hydroxy-THC [11-OH-THC], 11-nor-9-carboxy-THC [THCCOOH], cannabidiol [CBD], THC-glucuronide, and THCCOOH-glucuronide) polarity were employed with multiple reaction monitoring. Calibration by linear regression analysis utilized deuterium-labeled internal standards and a 1/x ² weighting factor, yielding R ² values >0.997 for all analytes. Linearity ranged from 0.5 to 50 μg/L (THC-glucuronide), 1.0–100 μg/L (THC, 11-OH-THC, THCCOOH, CBD, and CBN), and 5.0–250 μg/L (THCCOOH-glucuronide). Imprecision was <10.5% CV, recovery was >50.5%, and bias within ±13.1% of target for all analytes at three concentrations across the linear range. No carryover and endogenous or exogenous interferences were observed. This new analytical method should be useful for quantifying cannabinoids in whole blood and further investigating cannabinoid glucuronides as markers of recent cannabis intake.     

The influence of cannabis smoke and cannabis vapour on simulated lung surfactant function under physiologically relevant conditions

The use of cannabis for medicinal/recreational purposes is widespread throughout the world. Smoke inhalation is known to cause airway irritation due to noxious substances (ie, benzene) within the mix. Thus, advanced vaporisation platforms (eg, Davinci IQ) have been developed to circumvent negative health implications. Here, we consider the impact that cannabis smoke and cannabis vapour have on simulated lung surfactant performance within a model pulmonary space (ie, 37°C, elevated humidity and related fluid hydrodynamics). In total, 50 mg of herbal material was ignited or placed within a Davinci IQ vaporiser with subsequent activation. The aliquots were collected and then analysed using gas chromatography-mass spectroscopy for composition and cannabinoid (eg, Δ9-tetrahydrocannabinol [Δ9-THC]) concentration. The average content within cannabis smoke was 2.84% (0.07%, SD) Δ9-THC, with the same for cannabis vapour being 0.88% (0.14%, SD). Aerosolised samples were transferred to the lung biosimulator. When compared with the pristine Curosurf system, challenge with cannabis smoke and cannabis vapour reduced the surface pressure term by 26% and 7% and increased film compressibility by 60% and 15% at 80% trough area, respectively. The net effect would be enhanced film elasticity and an increased work of breathing, being more pronounced on cannabis smoke inhalation. The trends noted were ascribed to two factors operating synergistically, namely the amount of Δ9-THC (plus others) within the aerosolised samples and the associated toxicity profile. Further research is required to establish mass-balance effects (ie, titrated outputs) along with detailed chemical profiling of material generated from the unrelated cannabis activation pathways.     

Identification and quantification of cannabinoids in Cannabis sativa L. plants by high performance liquid chromatography-mass spectrometry

High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has been successfully applied to cannabis plant extracts in order to identify cannabinoid compounds after their quantitative isolation by means of supercritical fluid extraction (SFE). MS conditions were optimized by means of a central composite design (CCD) approach, and the analysis method was fully validated. Six major cannabinoids [tetrahydrocannabinolic acid (THCA), tetrahydrocannabinol (THC), cannabidiol (CBD), tetrahydrocannabivarin (THCV), cannabigerol (CBG), and cannabinol (CBN)] were quantified (RSD < 10%), and seven more cannabinoids were identified and verified by means of a liquid chromatograph coupled to a quadrupole-time-of-flight (Q-ToF) detector. Finally, based on the distribution of the analyzed cannabinoids in 30 Cannabis sativa L. plant varieties and the principal component analysis (PCA) of the resulting data, a clear difference was observed between outdoor and indoor grown plants, which was attributed to a higher concentration of THC, CBN, and CBD in outdoor grown plants. Graphical Abstract

Representative figure of the identification and quantification process of cannabinoids     

Determination of cannabinoids in cannabis products using liquid chromatography-ion trap mass spectrometry

A method was developed and validated for the simultaneous determination of five cannabinoids, viz. cannabidiol (CBD), cannabidiol acid (CBD-COOH), cannabinol (CBN), delta9-tetrahydrocannabinol (THC), and 3'-carboxy-delta9-all-trans-tetrahydrocannabinol (THC-COOH) in cannabis products. The cannabinoids were extracted from the grinded cannabis samples with a mixture of methanol-chloroform and analysed using liquid chromatography with ion-trap-mass-spectrometry (LC-IT-MSn). For quantification the two most abundant diagnostic MS-MS ions of the analyte in the sample and external standard were monitored. For confirmation purposes the EU criteria as described in Commission Decision 2002/657/EC were followed. Fully satisfactory results were obtained, that is, unequivocal confirmation according to the most stringent EU criteria was possible. The limits of quantification were 0.1 g/kg for CBD, 0.04 g/kg for CBD-COOH, 0.03 g/kg for CBN, 0.28 g/kg for THC and 9.9 g/kg for THC-COOH. The repeatabilities, defined by R.S.D., were 2% for CBN, THC and THC-COOH at the concentration levels of respectively 0.023, 3.3 and 113 g/kg and 5% for CBD-COOH at the level of 0.34 g/kg (n = 6).     

Determination of cannabinoids from a surrogate hops matrix using multiple reaction monitoring gas chromatography with triple quadrupole mass spectrometry

Cannabinoids are the primary bioactive constituents of Cannabis sativa and Cannabis indica plants. In this work, gas chromatography in conjunction with triple quadrupole mass spectrometry in multiple reaction monitoring mode was explored for determination of cannabinoids from a surrogate hops matrix. Gas chromatography with mass spectrometry is a reasonable choice for the analysis of these compounds; however, such methods are susceptible to false positives for Δ9‐tetrahydrocannabinol, due to decarboxylation of Δ9‐tetrahydrocannabinolic acid, its acid precursor, in the hot injection port. To avoid this transformation, the carboxyl group of Δ9‐tetrahydrocannabinolic acid was protected through a silylation reaction. Multiple reaction monitoring transitions for both unmodified and silylated cannabinoids were developed and the fragmentation pathways for the different species were assigned. Precision and accuracy were evaluated for cannabinoids spiked into hops at different levels. The developed methods provided good linearity (R² > 0.99) for all the cannabinoids with a linear range from 0.15 to 20 mg/L, and with limits of detection in the orders of low‐ to mid‐picogram on column. The recoveries for the cannabinoids were generally between 75 and 120%. Precisions (<6% coefficient of variation) were within acceptable ranges.     

High performance liquid chromatography with electrochemical detection. 1: Separation of Cannabis constituents and quantitation of Δ9 tetrahydrocannabinol

A simple, isocratic high performance liquid chromatography system (HPLC) with electrochemical detection (EC) was used to study Cannabis constituents. Several Cannabis extracts and reference standards were examined. A total of eleven constituents were separated, and three major cannabinoids; Δ⁸, Δ⁹ tetrahydrocannabinol (THC) and cannabidiol (CBD) were identified. A linear relationship was established for the quantitation of the halucinogenic constituent Δ⁹ THC. Minor contaminants in Δ⁹ THC reference standard, which were not detected by gas chromatography (GC) were detected for the first time. The detection limits of Δ⁹ THC and related cannabinoids were in the low nanogram range (2–20 ng).     

Neue cannabinoide—1

Extracts of cannabis contain—as was shown by glass capillary chromatography—a number of so far unknown cannabinoids. They were obtained in pure state by micropreparative gas chromatography followed by thin layer chromatography. The new compounds were characterized by their mass spectra. The structures of four of these compounds were determined by mass spectra, NMR-spectra and microchemical reactions followed by an investigation of the reaction products by gas chromatography-mass spectrometry. The new compounds are:Cannabichromanon = 2,2-Dimethyl-5-hydroxy-3-(3-oxo-butyl)-7-pentyl-4-chromanon (1)Cannabifuran = 1-Hydroxy-9-isopropyl-6-methyl-3-pentyl-dibenzofuran (3)Dehydrocannabifuran = 1-Hydroxy-9-isopropenyl-6-methyl-3-pentyl-dibenzofuran (4)2-Oxo-Δ³-THC = 2-Oxo-Δ³-tetrahydrocannabinol (5)     

Microbial Transformation of (−)-Δ1-3,4-trans-Tetrahydrocannabinol by Cunninghamella blakesleeana LENDER

Incubation of (?)-Δ¹-3, 4-trans-tetrahydrocannabinol (= Δ¹-THC; 3 ) with stationary cultures of Cunninghamella blakesleeana LENDER (Zygomycetales) (ATCC 8688a) yielded a number of metabolic conversion products. Isolation and structure elucidation of 6α-hydroxy-Δ¹-THC ( 4 ), the potential psychoactive 3″-hydroxy-Δ¹-THC ( 2 ) and 4″-hydroxy-Δ¹-THC ( 1 ), and the hitherto unknown metabolites 4″-hydroxy-6-oxo-Δ¹-THC ( 5 ), 4″, 6α-dihydroxy-Δ¹-THC ( 7 ) and 4″, 7 -dihydroxy-Δ¹-THC ( 6 ) is described.     

Chemical characterisation of oxidative degradation products of Δ-THC

The chemical analysis of a sample of Δ⁹-THC, which had been stored in an ethanol/propylene glycol solution for 5 years, resulted in the isolation of several hydroxylated Δ⁹-THC derivatives, the main of which were trans-cannabitriol monoethyl ether (4) and trans-propanediol ethers 7 and 8. cis-Cannabitriol monoethyl ether (5) and the oxidised derivatives 3 and 6 were detected in lesser amounts. The structure elucidation of the unprecedented cannabinoids 3, 5, 7 and 8 was achieved mainly by NMR techniques. Full NMR assignment of compounds 4 and 6 were also made. The detection of cannabitriol (6) and the corresponding solvent-adduct analogues (compounds 4-8) was in agreement with the decomposition mechanisms previously proposed for Δ⁹-THC. The isolation of the endoperoxide 3 represents indirect evidence of the existence of unstable precursors that were suspected to be intermediates in the non-enzymatic oxidation pathway of Δ⁹-THC. Both isomers of cannabitriol monoethyl ether exhibited weak affinity at either CB₁ (K_i=2.25, 6.30 μM) or CB₂ cannabinoid receptors (K_i=1.97, 3.13 μM), the trans isomer always being more potent than the cis isomer.     

Theoretical study of the stereoisomers of tetrahydrocannabinols

A theoretical AM1 semiempirical study of the major active components of marijuana and their stereoisomers is presented. It was found that the calculated partition coefficients, dipole moments, heats of formation, volume, surface area, ovality, and conformation of the pyran ring cannot explain the activity differences between the stereoisomers. The major factor is the orientation of the carbocyclic ring and its C₁ substituent with respect to the phenyl group hydroxyl oxygen. Our study has revealed and supports the involvement of previously described steric features of cannabinoids in determining their biological potency. Based on these conclusions, we predicted the relative activities for the (+)-cis-δ¹-THC, (-)-cis-δ¹-THC, (+)-cis-δ⁶-THC, and (-)-cis-δ⁶-THC stereoisomers, which have not been synthesized. © 1997 John Wiley & Sons, Inc.     

Simultaneous analysis of several synthetic cannabinoids,THC, CBD and CBN,in hair by ultra‐high performance liquid chromatography tandem mass spectrometry. Method validation and application to real samples

A simple procedure for the quantitative detection of JWH‐018, JWH‐073, JWH 200, JWH‐250, HU‐210, Δ⁹‐tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in hair has been developed and fully validated. After digestion with NaOH and liquid–liquid extraction, the separation was performed with an ultra‐high performance liquid chromatography system coupled to a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. The absence of matrix interferents, together with excellent repeatability of both retention times and relative abundances of diagnostic transitions, allowed the correct identification of all analytes tested. The method was linear in two different intervals at low and high concentration, with correlation coefficient values between 0.9933 and 0.9991. Quantitation limits ranged from 0.07 pg/mg for JWH‐200 up to 18 pg/mg for CBD The present method for the determination of several cannabinoids in hair proved to be simple, fast, specific and sensitive. The method was successfully applied to the analysis of 179 real samples collected from proven consumers of Cannabis, among which 14 were found positive to at least one synthetic cannabinoid. Copyright © 2012 John Wiley & Sons, Ltd.     

Valorization of Wild-Type Cannabis indica by Supercritical CO2 Extraction and Insights into the Utilization of Raffinate Biomass

Supercritical CO₂ extraction (SCCO₂) extraction of cannabis oil from Indian cannabis (Cannabis indica) leaves was optimized through a central composite design using CO₂ pressure (150–250 bar), temperature (30–50 °C) and time (1–2 h). From the regression model, the optimal CO₂ pressure, extraction temperature and time were 250 bar, 43 °C and 1.7 h, respectively resulting in the experimental yield of 4.9 wt% of cannabis oil via SCCO₂ extraction. The extract contained cannabidiol, tetrahydrocannabivarin, Δ⁹-tetrahydrocannabinol and Δ⁸-tetrahydrocannabinol as well as two terpenoids such as cis-caryophyllene and α-humulene. Besides SCCO₂ extraction of cannabis oil, the raffinate biomass was utilized to extract polyphenols using water as the extraction medium. Cannabis oil and water extractive were investigated for their half-maximal inhibitory concentration (IC₅₀) values, which were found to be 1.3 and 0.6 mg/mL, respectively. This is comparable to the commercially available antioxidant such as butylated hydroxytoluene with an IC₅₀ value of 0.5 mg/mL. This work on SCCO₂ extraction of cannabinoids and other valuable bioactive compounds provides an environmentally sustainable technique to valorize cannabis leaves.     

Delat9-tetrahydrocannabinol content in cannabis plants of greek origin

The delta9-tetrahydrocannabinol (delta9-THC) content was identified and determined quantitatively using a Gas Chromatography Detector (Gas Chromatography-Electron Ion Detector) instrument in samples of illicit herbal cannabis. Law enforcement authorities sent the samples to the Department of Forensic Medicine and Toxicology, University of Athens, for toxicological analysis. The concentrations of delta9-THC in these samples ranged from 0.08% to 4.41%. Such concentrations suggest that Greece might be at high risk, as an area for the illicit cultivation of "pedigree" cannabis plants. The forensic aspects of cannabis classification are discussed.     

Analysis of Anti-Cancer and Anti-Inflammatory Properties of 25 High-THC Cannabis Extracts

Cannabis sativa is one of the oldest cultivated plants. Many of the medicinal properties of cannabis are known, although very few cannabis-based formulations became prescribed drugs. Previous research demonstrated that cannabis varieties are very different in their medicinal properties, likely due to the entourage effect—the synergistic or antagonistic effect of various cannabinoids and terpenes. In this work, we analyzed 25 cannabis extracts containing high levels of delta-9-tetrahydrocannabinol (THC). We used HCC1806 squamous cell carcinoma and demonstrated various degrees of efficiency of the tested extracts, from 66% to 92% of growth inhibition of cancer cells. Inflammation was tested by induction of inflammation with TNF-α/IFN-γ in WI38 human lung fibroblasts. The efficiency of the extracts was tested by analyzing the expression of COX2 and IL6; while some extracts aggravated inflammation by increasing the expression of COX2/IL6 by 2-fold, other extracts decreased inflammation, reducing expression of cytokines by over 5-fold. We next analyzed the level of THC, CBD, CBG and CBN and twenty major terpenes and performed clustering and association analysis between the chemical composition of the extracts and their efficiency in inhibiting cancer growth and curbing inflammation. A positive correlation was found between the presence of terpinene (pval = 0.002) and anti-cancer property; eucalyptol came second, with pval of 0.094. p-cymene and β-myrcene positively correlated with the inhibition of IL6 expression, while camphor correlated negatively. No significant correlation was found for COX2. We then performed a correlation analysis between cannabinoids and terpenes and found a positive correlation for the following pairs: α-pinene vs. CBD, p-cymene vs. CBGA, terpenolene vs. CBGA and isopulegol vs. CBGA. Our work, thus, showed that most of high-THC extracts demonstrate anti-cancer activity, while only certain selected extracts showed anti-inflammatory activity. Presence of certain terpenes, such as terpinene, eucalyptol, cymene, myrcene and camphor, appear to have modulating effects on the activity of cannabinoids.     

Cannabis-Derived Compounds Cannabichromene and Δ9-Tetrahydrocannabinol Interact and Exhibit Cytotoxic Activity against Urothelial Cell Carcinoma Correlated with Inhibition of Cell Migration and Cytoskeleton Organization

Cannabis sativa contains more than 500 constituents, yet the anticancer properties of the vast majority of cannabis compounds remains unknown. We aimed to identify cannabis compounds and their combinations presenting cytotoxicity against bladder urothelial carcinoma (UC), the most common urinary system cancer. An XTT assay was used to determine cytotoxic activity of C. sativa extracts on T24 and HBT-9 cell lines. Extract chemical content was identified by high-performance liquid chromatography (HPLC). Fluorescence-activated cell sorting (FACS) was used to determine apoptosis and cell cycle, using stained F-actin and nuclei. Scratch and transwell assays were used to determine cell migration and invasion, respectively. Gene expression was determined by quantitative Polymerase chain reaction (PCR). The most active decarboxylated extract fraction (F7) of high-cannabidiol (CBD) C. sativa was found to contain cannabichromene (CBC) and Δ9-tetrahydrocannabinol (THC). Synergistic interaction was demonstrated between CBC + THC whereas cannabinoid receptor (CB) type 1 and type 2 inverse agonists reduced cytotoxic activity. Treatments with CBC + THC or CBD led to cell cycle arrest and cell apoptosis. CBC + THC or CBD treatments inhibited cell migration and affected F-actin integrity. Identification of active plant ingredients (API) from cannabis that induce apoptosis and affect cell migration in UC cell lines forms a basis for pre-clinical trials for UC treatment.