Drug-type Cannabis Research Articles

Δ9-Tetrahydrocannabinol upregulates fatty acid 2-hydroxylase (FA2H) via PPARα induction: A possible evidence for the cancellation of PPARβ/δ-mediated inhibition of PPARα in MDA-MB-231 cells

Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated nuclear transcription factors, with three characterized subtypes: PPARα, PPARβ/δ, and PPARγ. The biological correlation between the two PPAR subtypes PPARα and γ and carcinogenesis is well-characterized; however, substantially less is known about the biological functions of PPARβ/δ. PPARβ/δ has been reported to repress transcription when PPARβ/δ and PPARα or PPARγ are simultaneously expressed in some cells, and MDA-MB-231 cells express functional levels of PPARβ/δ. We have previously reported that Δ9-tetrahydrocannabinol (Δ9-THC), a major cannabinoid component of the drug-type cannabis plant, can stimulate the expression of fatty acid 2-hydroxylase (FA2H) via upregulation of PPARα expression in human breast cancer MDA-MB-231 cells. Although the possibility of an inhibitory interaction between PPARα and PPARβ/δ has not been demonstrated in MDA-MB-231 cells, we reasoned if this interaction were to exist, Δ9-THC should make PPARα free to achieve FA2H induction. Here, we show that a PPARβ/δ-mediated suppression of PPARα function, but not of PPARγ, exists in MDA-MB-231 cells and Δ9-THC causes FA2H induction via mechanisms underlying the cancellation of PPARβ/δ-mediated inhibition of PPARα, in addition to the upregulation of PPARα.

Terpenoid Chemoprofiles Distinguish Drug-type Cannabis sativa L. Cultivars in Nevada

An unintended consequence of state-mandated cannabis testing regulations has been the resulting database from the analysis of thousands of individual cannabis flower samples from artificially restricted geographical regions. The resulting detailed chemical database can serve as the basis for the development of a chemotaxonomic classification scheme outside of conjectural cultivar naming by strain. Chemotaxonomic classification schemes for cannabis cultivars have previously been reported by others based largely on cannabis strains grown in California under an unregulated testing environment or in Europe from strains grown by a single cultivator. In this study 2,237 individual cannabis flower samples, representing 204 individual strains across 27 cultivators in a tightly regulated Nevada cannabis testing market, were analyzed across 11 cannabinoids and 19 terpenoids. Even though 98.3% of the samples were from Type I cannabis strains by cannabidiolic acid/tetrahydrocannabinolic acid (THCA) ratio of <0.5 CBDA, principal component analysis (PCA) of the combined dataset resulted in three distinct clusters that were distinguishable by terpene profiles alone. Further dissection of individual strains by cultivators within clusters revealed striking fidelity of terpenoid profiles and also revealed a few outliers. We propose that three terpenoid cluster assignments account for the diversity of drug type cannabis strains currently being grown in Nevada.

Bayesian classification criterion for forensic multivariate data

This study presents a classification criteria for two-class Cannabis seedlings. As the cultivation of drug type cannabis is forbidden in Switzerland, law enforcement authorities regularly ask laboratories to determine cannabis plant's chemotype from seized material in order to ascertain that the plantation is legal or not. In this study, the classification analysis is based on data obtained from the relative proportion of three major leaf compounds measured by gas-chromatography interfaced with mass spectrometry (GC–MS). The aim is to discriminate between drug type (illegal) and fiber type (legal) cannabis at an early stage of the growth.A Bayesian procedure is proposed: a Bayes factor is computed and classification is performed on the basis of the decision maker specifications (i.e. prior probability distributions on cannabis type and consequences of classification measured by losses). Classification rates are computed with two statistical models and results are compared. Sensitivity analysis is then performed to analyze the robustness of classification criteria.

A rapid method for the extraction of cannabionoids from cannabis sativa using microwave heating technique

Cannabis sativa and its products (marihuana, hashish and hashish oil) is one of the most used illicit drugs in the world. It is also an agricultural crop so it is important to distinguish between industrial-type and drug-type cannabis by forensic examination of suspected material. Extraction technique is one of the first steps in the laboratory plant material isolation, and therefore the development of new, more efficient method of extraction is desirable. The traditional techniques of extraction of biological active compounds from plant material, such as cannabinoids from cannabis, are based on solvent extraction. These methods are often inefficient in terms of extraction time, amount of solvent, energy consumption and extraction yield. Therefore the importance of continuous development of new, more efficient extraction methods is understandable. In this paper, the short description of cannabis plant and chemical constituents of forensic significance were shown for better understanding. Paper also describes the mechanism of microwave heating and its use in the extraction method through short theoretical background of microwave heating. In addition, examples of application of microwaves for the isolation of some organic compounds from plant material were presented. It is also shown that the microwave extraction can be used in absence of solvent in solvent free microwave technique. The possibility of microwave extraction application in forensic examination of Cannabis sativa is presented by highlighting its simplicity and efficiency.

Evolution of the Content of THC and Other Major Cannabinoids in Drug‐Type Cannabis Cuttings and Seedlings During Growth of Plants*

In Europe, authorities frequently ask forensic laboratories to analyze seized cannabis plants to prove that cultivation was illegal (drug type and not fiber type). This is generally done with mature and flowering plants. However, authorities are often confronted with very young specimens. The aim of our study was to evaluate when the chemotype of cannabis plantlets can be surely determined through analysis of eight major cannabinoids content during growth. Drug-type seedlings and cuttings were cultivated, sampled each week, and analyzed by high-performance liquid chromatography with diode array detection. The chemotype of clones was recognizable at any developmental stage because of high total Δ(9)-tetrahydrocannabinol (THC) concentrations even at the start of the cultivation. Conversely, right after germination seedlings contained a low total THC content, but it increased quickly with plant age up, allowing chemotype determination after 3 weeks. In conclusion, it is not necessary to wait for plants' flowering to identify drug-type cannabis generally cultivated in Europe.

Discrimination of ‘fiber-type’ and ‘drug-type’ Cannabis sativa L. by fluorescent duplex PCR

A fluorescent duplex-PCR test was developed based on polymorphisms of the THCA synthase gene in order to discriminate the fiber- and drug-type Cannabis sativa L. and to indicate the presence of Cannabis trace in suspected materials by the numbers and sizes of PCR-amplified products. DNA analysis of drug-type Cannabis resulted in two different PCR-amplified DNA fragments of 94 and 158 bp, whereas only the 94 bp PCR product was amplified from the fiber-type DNA. DNA test results of another 6 Cannabis sativa L. collected from the field agreed with chemotype determined by GC-MS. However, it was noted that the only intermediate drug-type sample tested gave a drug-type result for DNA testing. Specificity of the duplex PCR was shown by testing with DNA from species that may be related to Cannabis abuse, i.e., common hop ( Humulus lupulus L.), 2 narcotic plants ( Papaver somniferum and Mitragyna speciosa), tobacco ( Nicotiana tabacum) and human. Sensitivity of detection was as low as 100 pg of genomic DNA.

Multi-class differentiation of cannabis seedlings in a forensic context

This article presents an experimental study about the classification ability of several classifiers for multi-class classification of cannabis seedlings. As the cultivation of drug type cannabis is forbidden in Switzerland law enforcement authorities regularly ask forensic laboratories to determinate the chemotype of a seized cannabis plant and then to conclude if the plantation is legal or not. This classification is mainly performed when the plant is mature as required by the EU official protocol and then the classification of cannabis seedlings is a time consuming and costly procedure. A previous study made by the authors has investigated this problematic [1] and showed that it is possible to differentiate between drug type (illegal) and fibre type (legal) cannabis at an early stage of growth using gas chromatography interfaced with mass spectrometry (GC–MS) based on the relative proportions of eight major leaf compounds. The aims of the present work are on one hand to continue former work and to optimize the methodology for the discrimination of drug- and fibre type cannabis developed in the previous study and on the other hand to investigate the possibility to predict illegal cannabis varieties. Seven classifiers for differentiating between cannabis seedlings are evaluated in this paper, namely Linear Discriminant Analysis (LDA), Partial Least Squares Discriminant Analysis (PLS-DA), Nearest Neighbour Classification (NNC), Learning Vector Quantization (LVQ), Radial Basis Function Support Vector Machines (RBF SVMs), Random Forest (RF) and Artificial Neural Networks (ANN). The performance of each method was assessed using the same analytical dataset that consists of 861 samples split into drug- and fibre type cannabis with drug type cannabis being made up of 12 varieties (i.e. 12 classes). The results show that linear classifiers are not able to manage the distribution of classes in which some overlap areas exist for both classification problems. Unlike linear classifiers, NNC and RBF SVMs best differentiate cannabis samples both for 2-class and 12-class classifications with average classification results up to 99% and 98%, respectively. Furthermore, RBF SVMs correctly classified into drug type cannabis the independent validation set, which consists of cannabis plants coming from police seizures. In forensic case work this study shows that the discrimination between cannabis samples at an early stage of growth is possible with fairly high classification performance for discriminating between cannabis chemotypes or between drug type cannabis varieties.

The differentiation of fibre- and drug type Cannabis seedlings by gas chromatography/mass spectrometry and chemometric tools

Cannabis cultivation in order to produce drugs is forbidden in Switzerland. Thus, law enforcement authorities regularly ask forensic laboratories to determinate cannabis plant's chemotype from seized material in order to ascertain that the plantation is legal or not. As required by the EU official analysis protocol the THC rate of cannabis is measured from the flowers at maturity. When laboratories are confronted to seedlings, they have to lead the plant to maturity, meaning a time consuming and costly procedure. This study investigated the discrimination of fibre type from drug type Cannabis seedlings by analysing the compounds found in their leaves and using chemometrics tools. 11 legal varieties allowed by the Swiss Federal Office for Agriculture and 13 illegal ones were greenhouse grown and analysed using a gas chromatograph interfaced with a mass spectrometer. Compounds that show high discrimination capabilities in the seedlings have been identified and a support vector machines (SVMs) analysis was used to classify the cannabis samples. The overall set of samples shows a classification rate above 99% with false positive rates less than 2%. This model allows then discrimination between fibre and drug type Cannabis at an early stage of growth. Therefore it is not necessary to wait plants’ maturity to quantify their amount of THC in order to determine their chemotype. This procedure could be used for the control of legal (fibre type) and illegal (drug type) Cannabis production.

High Frequency Plant Regeneration from Leaf Derived Callus of HighΔ9-Tetrahydrocannabinol YieldingCannabis sativaL.

An efficient in vitro propagation protocol for rapidly producing Cannabis sativa plantlets from young leaf tissue was developed. Using gas chromatography-flame ionization detection (GC-FID), high THC yielding elite female clone of a drug-type CANNABIS variety (MX) was screened and its vegetatively propagated clones were used for micropropagation. Calli were induced from leaf explant on Murashige and Skoog medium supplemented with different concentrations (0.5, 1.0, 1.5, and 2.0 µM) of indole- 3-acetic acid (IAA), indole- 3- butyric acid (IBA), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxy-acetic acid (2,4-D) in combination with 1.0 µM of thidiazuron (TDZ) for the production of callus. The optimum callus growth and maintenance was in 0.5 µM NAA plus 1.0 µM TDZ. The two-month-old calli were subcultured to MS media containing different concentrations of cytokinins (BAP, KN, TDZ). The rate of shoot induction and proliferation was highest in 0.5 µM TDZ. Of the various auxins (IAA, IBA, and NAA) tested, regenerated shoots rooted best on half strength MS medium (1/2 - MS) supplemented with 2.5 µM IBA. The rooted plantlets were successfully established in soil and grown to maturity with no gross variations in morphology and cannabinoids content at a survival rate of 95 % in the indoor growroom.

Effect of Light Intensity on Photosynthetic Characteristics of High Δ9-THC Yielding Varieties of Cannabis sativa L.

Cannabis sativa L. (Cannabaceae), an annual herb is the natural source of cannabinoids that mainly accumulate in glandular trichomes of the plant. Due to the allogamous (cross fertilization) nature of Cannabis sativa it is very difficult to maintain the efficacy of selected high THC yielding elite varieties if grown from seeds under field conditions. Thus, the indoor cultivation, under controlled environmental conditions, using vegetative propagation of selected high yielding female clones can be a better alternative for its mass propagation. In the present study, plants of four drug type Cannabis varieties namely HPM, MX, K2 and W1 were grown indoors, under controlled environmental conditions (25±3°C, 55±5% RH and ˜ 700±24µmol m–2s–1 light at plant canopy level). Gas and water vapor characteristics of these plants were studied at different Photosynthetic Photon Flux Density (PPFD; 000, 500, 1000, 1500 and 2000µmol m–2s–1) for their efficient indoor cultivation. An increasing trend in photosynthesis (PN), transpiration (Tr) and stomatal conductance (gCO2) was observed with increase in PPFD up to 2000µmol m–2s–1 in all the varieties at optimum growth temperature (25±3°C). However, the magnitude of increase and maximum rate of PN (PN max) varied with the varieties. Highest rate of photosynthesis was observed in W1 followed by MX, K2 and HPM. Water Use efficiency (WUE) in W1, MX and HPM increased with light up to highest level tested, whereas, in K2 highest WUE was observed at 1500µmol m–2s–1. Our results show that this species is able to use high level of PPFD for its PN and therefore, may be cultivated in under bright indoor light (˜1500 to 2000µmol m–2s–1) for better growth and biomass. The strict control of other environmental factors however, should be maintained for a higher yield. Acknowledgment: This work was supported with federal funds from the National Institute of Drug Abuse (NIDA), National Institute of Health (NIH), Department of Health and Human Services, USA, under the contract No. N01DA-7–7746.

Inter simple sequence repeats separate efficiently hemp from marijuana (Cannabis sativa L.)

Cannabis sativa L. is a multiple-use plant that provides raw material for the production of seed oil, natural fiber for textiles, automotive and pulp industries. It has also been used in insulating boards, ropes, varnishes, animal feed, and as medicinal agents. Cannabis has potential to be used for phytoremediation: however, its cultivation is strictly controlled due to its psychoactive nature and usage in producing drugs such as marijuana, and hashish. In this study, psychoactive type Cannabis samples, which were seized from 23 different locations of Turkey, and nine hemp type Cannabis accessions, as well as an unknown accession were used. Our interest was to identify the genetic relatedness of the seized samples and to separate drug and hemp type plants. Inter Simple Sequence Repeats (ISSRs) were employed for analysis based on single plant material (SET1) and bulked samples of them (SET2). Data was analysed via cluster analysis and principal coordinate analysis (PCoA). PCoA analyses, by using SET1 and SET2, were able to efficiently discriminate the seized samples from the fiber type accessions. However, separation of the plants was not clear via unweighted pair-group method using arithmetic average (UPGMA) dendogram in SET1, while they were clearly separated in SET2. Hemp type accessions showed high levels of variation compared to drug type Cannabis both in SET1 and SET2.

Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa

Cannabidiolic-acid (CBDA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic-acid into CBDA, the dominant cannabinoid constituent of the fiber-type Cannabis sativa. We cloned a novel cDNA encoding CBDA synthase by reverse transcription and polymerase chain reactions with degenerate and gene-specific primers. Biochemical characterization of the recombinant enzyme demonstrated that CBDA synthase is a covalently flavinylated oxidase. The structural and functional properties of CBDA synthase are quite similar to those of tetrahydrocannabinolic-acid (THCA) synthase, which is responsible for the biosynthesis of THCA, the major cannabinoid in drug-type Cannabis plants.

Hypercholesterolemic Effect of Drug-Type Cannabis sativa L. Seed (Marijuana Seed) in Guinea Pig

Cannabis sativa L. has two drug and nondrug varieties. Nondrug varieties of Cannabis are hemp but drug varieties commonly referred to as marijuana. Marijuana is considered nutritional and narcotic plant. Marijuana has not been studied extensively for its nutritional potential in recent years but whole hempseed typically contains over 30% oil (3%saturated, 28% unsaturated fatty acids) and about 25% protein. The objectives of the present study were to evaluate the effects of whole marijuana seed on lipid profile of guinea pig. This study was experienced on fourteen guinea pigs as case and control groups. In control group, guinea pigs were fed with normal diet while case group had free access to normal diet and marijuana seed for 60 days. At the end of experiment the feeding of animal stopped and after 12hr fasting, the animal anesthetized by ketamine/xylazine combination and 5 ml of blood of heart was taken. The blood parameters were measured by automated biochemical analyzer. Marijuana seed significantly increased total cholesterol and LDL-c levels (p=0.00) while HDL-c and triglyceride levels didn't significantly change. In spite of omega-3, omega-6 and omega-9 fatty acids that highly incorporated in hempseed, short term feeding of marijuana seed has not improved blood lipid profile. It may be due to hidden orexigenic phytocannabinoids that found in drug-type seed. In the light of this research, it is recommended that individuals who are affected to cardiovascular diseases and atherosclerosis, they should not use unclean marijuana seed that cultivated in Isfahan province of Iran in their food preparation on regular basis.

A rapid and simple procedure for the determination of cannabinoids in hemp food products by gas chromatography-mass spectrometry

A rapid and simple procedure using liquid–liquid extraction and subsequent gas chromatographic mass-spectrometric detection has been developed for determination of Δ 9-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in different hemp foods. After addition of Δ 8-tetrahydrocannabinol as internal standard, both solid and liquid specimens were extracted with two volumes of 2 ml of hexane/isopropanol (9:1): Chromatography was performed on a fused silica capillary column and analytes were determined in the selected-ion-monitoring (SIM) mode. The method was validated in the range 1–50 ng/ml liquid samples or 1–50 ng/g solid samples for THC and CBN, and 2–50 ng/ml or ng/g for CBD. Mean recoveries ranged between 78.8 and 90.2% for the different analytes in solid and liquid samples. The quantification limits were 1 ng/ml or ng/g for THC and CBN and 2 ng/ml or ng/g CBD. The method was applied to analysis of various hemp foods. THC content in different products varied 50-fold, whereas CBN and CBD were absent in some samples and achieved hundreds of ng/ml or ng/g in others. The concentration ratio (THC + CBN)/CBD was used to differentiate between the phenotypes of cannabis plants in different specimens. Products possibly originating from drug-type cannabis plants were found in the majority of analyzed specimens.

Biological activity of cannabichromene, its homologs and isomers.

Cannabichromene (CBC) is one of four major cannabinoids in Cannabis sativa L. and is the second most abundant cannabinoid in drug-type cannabis. Cannabichromene and some of its homologs, analogs, and isomers were evaluated for antiinflammatory, antibacterial, and antifungal activity. Antiinflammatory activity was evaluated by the carrageenan-induced rat paw edema and the erythrocyte membrane stabilization method. In both tests, CBC was superior to phenylbutazone. Antibacterial activity of CBC and its isomers and homologs was evaluated using gram-positive, gram-negative, and acid-fast bacteria. Antifungal activity was evaluated using yeast-like and filamentous fungi and a dermatophyte. Antibacterial activity was strong, and the antifungal activity was mild to moderate.