Tag: Cannabinoids

Quantitative Analysis of Cannabinoids from Cannabis sativa Using 1H-NMR

Arno HAZEKAMP, Young Hae CHOI, and Robert VERPOORTE

July 2004 CHEMICAL & PHARMACEUTICAL BULLETIN 52(6):718-21

DOI: 10.1248/cpb.52.718

A 1H-NMR method has been developed for the quantitative analysis of pure cannabinoids and for cannabinoids present in Cannabis sativa plant material without any chromatographic purification. The experiment was performed by the analysis of singlets in the range of d 4.0—7.0 in the 1H-NMR spectrum, in which distinguishable signals of each cannabinoid are shown. Quantitation was performed by calculating the relative ratio of the peak area of selected proton signals of the target compounds to the known amount of the internal standard, anthracene. For this method, no reference compounds are needed. It allows rapid and simple quantitation of cannabinoids with a final analysis time of only 5 min without the need for a pre-purification step.
Abstract

*Qualitative and Quantitative Aspects of the Inheritance of Chemical Phenotype in Cannabis

Giuseppe Mandolino, Manuela Bagatta, Andrea Carboni, Paolo Ranalli, Etienne de Meijer

March 2003 Journal of Industrial Hemp 8(2):51-72

DOI: 10.1300/J237v08n02_04

Four crosses were made between Cannabis plants with “pure” CBD and THC chemotypes. The F1 plants obtained were self-fertilised to produce F2s. Chemotypical distributions were analysed by gas-chromatography. A segregation analysis of the different F2 progenies obtained showed that chemotype, estimated as CBD/THC ratio, behaves as a qualitative character, and a model for a single locus B, with two co-dominant alleles, BD and BT is described. The CBD/THC ratios in the F1 offsprings were found to be significantly different in the heterozygous plants from the different pedigrees. The amount of CBD plus THC in the same pedigrees was also described. Heterosis was found to be a common feature, but not a general one, of cannabinoid accumulation in the F1s. Distribution of the values of cannabinoid content in classes was found to be normal. RAPD markers linked to the segregating chemotypes (“pure” CBD and “pure” THC) were identified by bulk segregant analysis, and the degree of linkage of these markers with the chemotype was described.
Abstract

Randomized, dose-ranging safety trial of cannabidiol in Dravet syndrome

Orrin Devinsky, MD, Anup D. Patel, MD, Elizabeth A. Thiele, MD, Matthew H. Wong, MD, Richard Appleton, MD, Cynthia L. Harden, MD, Sam Greenwood, PhD, Gilmour Morrison, and Kenneth Sommerville, MD

Neurology, 90(14), e1204–e1211. 

doi: 10.1212/wnl.0000000000005254

To evaluate the safety and preliminary pharmacokinetics of a pharmaceutical formulation of purified cannabidiol (CBD) in children with Dravet syndrome.
Objective

Prospects for cannabinoid therapies in basal ganglia disorders

Javier Fernández‐Ruiz , Miguel Moreno‐Martet, Carmen Rodríguez‐Cueto , Cristina Palomo‐Garo , María Gómez‐Cañas, Sara Valdeolivas, Manuel Guzmán, Carmen Guaza,, Julián Romero

British Journal of Pharamcology (2011) 163 1365-1378

doi: 10.1111/j.1476-5381.2011.01365.x

Cannabinoids are promising medicines to slow down disease progression in neurodegenerative disorders including Parkinson’s disease (PD) and Huntington’s disease (HD), two of the most important disorders affecting the basal ganglia. Two pharmacological profiles have been proposed for cannabinoids being effective in these disorders. On the one hand, cannabinoids like Δ9‐tetrahydrocannabinol or cannabidiol protect nigral or striatal neurons in experimental models of both disorders, in which oxidative injury is a prominent cytotoxic mechanism. This effect could be exerted, at least in part, through mechanisms independent of CB1 and CB2 receptors and involving the control of endogenous antioxidant defences. On the other hand, the activation of CB2 receptors leads to a slower progression of neurodegeneration in both disorders. This effect would be exerted by limiting the toxicity of microglial cells for neurons and, in particular, by reducing the generation of proinflammatory factors. It is important to mention that CB2 receptors have been identified in the healthy brain, mainly in glial elements and, to a lesser extent, in certain subpopulations of neurons, and that they are dramatically up‐regulated in response to damaging stimuli, which supports the idea that the cannabinoid system behaves as an endogenous neuroprotective system. This CB2 receptor up‐regulation has been found in many neurodegenerative disorders including HD and PD, which supports the beneficial effects found for CB2 receptor agonists in both disorders. In conclusion, the evidence reported so far supports that those cannabinoids having antioxidant properties and/or capability to activate CB2 receptors may represent promising therapeutic agents in HD and PD, thus deserving a prompt clinical evaluation
Abstract

Production of Δ9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing Δ9-tetrahydrocannabinolic acid synthase from Cannabis sativa l.

Zirpel, B., Stehle, F., & Kayser, O. 

Biotechnology Letters, 37(9), 1869–1875. (2015). 

doi: 10.1007/s10529-015-1853-x

Objective- The D9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed intracellularly in different organisms to investigate the potential of a biotechnological production of D9- tetrahydrocannabinolic acid (THCA) using whole cells.
Results- Functional expression of THCAS was obtained in Saccharomyces cerevisiae and Pichia (Komagataella) pastoris using a signal peptide from the vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest volumetric activities obtained were 98 pkat ml-1 (intracellular) and 44 pkat ml-1 (extracellular) after 192 h of cultivation at 15 C using P. pastoris cells. Low solubility of CBGA prevents the THCAS application in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid (CBGA) to THCA. Finally, 1 mM (0.36 g THCA l-1 ) THCA could be produced by 10.5 gCDW l -1 before enzyme activity was lost.
Abstract

Production of Δ1-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris.

Taura, F., Dono, E., Sirikantaramas, S., Yoshimura, K., Shoyama, Y., & Morimoto, S. 

Biochemical and Biophysical Research Communications, 361(3), 675–680 (2007)

doi: 10.1016/j.bbrc.2007.07.079

D1 -Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of D1 -tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted 1.32 nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure–function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.
Abstract

Prenylation of olivetolate by a hemp transferase yields cannabigerolic acid, the precursor of tetrahydrocannabinol

Fellermeier M, Zenk MH (1998)

FEBS Lett 427: 283–285

DOI: 10.1016/S0014-5793(98)00450-5

A new enzyme, geranylpyrophosphate:olivetolate geranyltransferase (GOT), the first enzyme in the biosynthesis of cannabinoids could be detected in extracts of young leaves of Cannabis sativa. The enzyme accepts geranylpyrophosphate (GPP) and to a lesser degree also nerylpyrophosphate (NPP) as a cosubstrate. It is, however, specific for olivetolic acid; its decarboxylation product olivetol is inactive as a prenyl acceptor.
Abstract

Plant cannabinoids: a neglected pharmacological treasure trove

Raphael Mechoulam

British Journal of Pharmacology (2005) 146, 913–915

doi: 10.1038/sj.bjp.0706415

Most of the cannabinoids in Cannabis sativa L. have not been fully evaluated for their pharmacological activity. A publication in this issue presents evidence that a plant cannabinoid, D9-tetrahydrocannabivarin is a potent antagonist of anandamide, a major endogenous cannabinoid. It seems possible that many of the non-psychoactive constituents of this plant will be of biological interest.
Abstract

PKS Activities and Biosynthesis of Cannabinoids and Flavonoids in Cannabis sativa L. Plants

Isvett Josefina Flores-Sanchez and Robert Verpoorte

Plant Cell Physiol (2008) 49 (12): 1767-1782.

doi: 10.1093/pcp/pcn150

Polyketide synthase (PKS) enzymatic activities were analyzed in crude protein extracts from cannabis plant tissues. Chalcone synthase (CHS, EC 2.3.1.74), stilbene synthase (STS, EC 2.3.1.95), phlorisovalerophenone synthase (VPS, EC 2.3.1.156), isobutyrophenone synthase (BUS) and olivetol synthase activities were detected during the development and growth of glandular trichomes on bracts. Cannabinoid biosynthesis and accumulation take place in these glandular trichomes. In the biosynthesis of the first precursor of cannabinoids, olivetolic acid, a PKS could be involved; however, no activity for an olivetolic acid-forming PKS was detected. Content analyses of cannabinoids and flavonoids, two secondary metabolites present in this plant, from plant tissues revealed differences in their distribution, suggesting a diverse regulatory control for these biosynthetic fluxes in the plant.
Abstract

Phytocannabinoids beyond the Cannabis plant – do they exist?

Gertsch, J., Pertwee, R. G., & Di Marzo, V. 

British Journal of Pharmacology, 160(3), 523–529.(2010).

doi: 10.1111/j.1476-5381.2010.00745.x

It is intriguing that during human cultural evolution man has detected plant natural products that appear to target key protein receptors of important physiological systems rather selectively. Plants containing such secondary metabolites usually belong to unique chemotaxa, induce potent pharmacological effects and have typically been used for recreational and medicinal purposes or as poisons. Cannabis sativa L. has a long history as a medicinal plant and was fundamental in the discovery of the endocannabinoid system. The major psychoactive Cannabis constituent D9 -tetrahydrocannabinol (D9 -THC) potently activates the G-protein-coupled cannabinoid receptor CB1 and also modulates the cannabinoid receptor CB2. In the last few years, several other non-cannabinoid plant constituents have been reported to bind to and functionally interact with CB receptors. Moreover, certain plant natural products, from both Cannabis and other plants, also target other proteins of the endocannabinoid system, such as hydrolytic enzymes that control endocannabinoid levels. In this commentary we summarize and critically discuss recent findings.