A Simple Chiral 1H NMR Method for the Discrimination of (R)- and (S)-Cannabichromene in Complex Natural Mixtures and Their Effects on TRPA1 Activity.

In recent years, the enantiomeric ratio of cannabichromene (CBC) within the cannabis plant has attracted significant attention. Cannabichromene is one of the well-known cannabinoids found in cannabis, along with THC (tetrahydrocannabinol) and CBD (cannabidiol). Cannabichromene exists as a scalemic mixture, meaning it has two enantiomers, (S)-cannabichromene and (R)-cannabichromene, with the ratio between these enantiomers varying among different cannabis strains and even within individual plants. This study presents an accurate and robust chiral NMR method for analyzing cannabichromene's enantiomeric ratio, a well-investigated cannabinoid with numerous pharmacological targets. The use of Pirkle's alcohol as the chiral solvating agent (CSA) or, alternatively, the use of (S)-ibuprofen as a chiral derivatizing agent (CDA) facilitated this analysis. Moreover, the chiral NMR method proves to be a user-friendly tool, easily applicable within any NMR facility, and an expanded investigation of cannabichromene chirality may provide insights into the origin, cultivation, treatment, and processing of Cannabis sativa plants. This study also undertakes a pharmacological examination of the (R)- and (S)-cannabichromenes concerning their most extensively studied pharmacological target, the TRPA1 channels, with the two enantiomers showing the same strong agonistic effect as the racemic mixture.

Genetic Evaluation of In Vitro Micropropagated and Regenerated Plants of Cannabis sativa L. Using SSR Molecular Markers.

Simple sequence repeat (SSR) markers were used to evaluate the genetic stability of the acclimatized micropropagated and regenerated plants of a high cannabidiol (H-CBD) and a high cannabigerol (H-CBG) variety of Cannabis sativa L. Shoot regeneration and proliferation were achieved by culturing calli in Murashige and Skoog basal medium (MS) supplemented with several concentrations of 6-benzyladenine (BA) or thidiazuron (TDZ). Calli derived mostly from stem explants, rather than leaves, cultured on MS supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) or combination of kinetin (KIN) with 1-Naphthaleneacetic acid (NAA) or 2,4-D. Rooting of the regenerated plantlets accomplished on half-strength MS medium supplemented with indole-3-butyric acid (IBA). Previous studies performed have developed an efficient in vitro micropropagation protocol for mass production. Both in vitro methodologies can be employed in genetic breeding via molecular techniques. The genetic stability of micropropagated and regenerated plants was accomplished using twelve SSR primer pairs that produced reproducible and clear bands, ranging from 90 to 330 bp in size, and resulted in amplification of one or two alleles, corresponding to homozygous or heterozygous individuals. The SSR amplification products were monomorphic across all the micropropagated and regenerated plants and comparable to mother plants. The monomorphic banding pattern confirmed the genetic homogeneity of the in vitro cultured acclimatized and mother plants as no somaclonal variation was detected in clones for these specific SSRs. Our results evidently suggest that the developed culture protocols for in vitro multiplication is appropriate and applicable for clonal mass propagation of the C. sativa varieties and demonstrate the reliability of this in vitro propagation system.

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.

An Alternative In Vitro Propagation Protocol of Cannabis sativa L. (Cannabaceae) Presenting Efficient Rooting, for Commercial Production.

An alternative in vitro propagation protocol for medical Cannabis sativa L. cultivars for pharmaceutical industrial use was established. The aim of the protocol was to reduce the culture time, offering healthy and aseptic propagating material, while making the whole process more economic for industrial use. The propagation procedure was performed using plastic autoclavable vented and non-vented vessels, containing porous rooting fine-milled sphagnum peat moss-based sponges, impregnated in ½ Murashige and Skoog liquid growth medium, supplemented with indole-3-butyric acid (IBA) at various concentrations (0, 2.46, 4.92, and 9.84 µM) or by dipping nodal cuttings into 15 mM IBA aqueous solution. The highest average root numbers per cutting, 9.47 and 7.79 for high cannabidiol (H_CBD) and high cannabigerol (H_CBG) varieties, respectively, were achieved by dipping the cuttings into IBA aqueous solution for 4 min and then placing them in non-vented vessels. The maximum average root length in H_CBD (1.54 cm) and H_CBG (0.88 cm) was ascertained using 2.46 µM filter sterilized IBA in non-vented vessels. Filter-sterilized IBA at concentrations of 2.46 µM in vented and 4.92 µM in non-vented vessels displayed the maximum average rooting percentages in H_CBD (100%) and H_CBG (95.83%), respectively. In both varieties, maximum growth was obtained in non-vented vessels, when the medium was supplemented with 4.92 µM filter-sterilized IBA. Significant interactions between variety and vessel type and variety and IBA treatments were observed in relation to rooting traits. Approximately 95% of plantlets were successfully established and acclimatized in field. This culture system can be used not only for propagating plant material at an industrial scale but also to enhance the preservation and conservation of Cannabis genetic material.

Biotechnological Approaches on Two High CBD and CBG Cannabis sativa L. (Cannabaceae) Varieties: In Vitro Regeneration and Phytochemical Consistency Evaluation of Micropropagated Plants Using Quantitative 1H-NMR.

High cannabidiol (CBD) and cannabigerol (CBG) varieties of Cannabis sativa L., a species with medicinal properties, were regenerated in vitro. Explants of nodal segments including healthy axillary bud, after sterilization, were placed in Murashige-Skoog (MS) culture medium. The shoots formed after 30 days were subcultured in full- or half-strength MS medium supplemented with several concentrations of 6-benzyl-amino-purine (BA) or thidiazuron (TDZ). The highest average number and length of shoots was achieved when both full and half-strength MS media were supplemented with 4.0 µM BA. The presence of 4.0 µM TDZ showed also comparable results. BA and TDZ at concentrations of 4.0, 8.0 µM and 2.0, 4.0 µM respectively, displayed the maximum shooting frequency. The new shoots were transferred on the same media and were either self-rooted or after being enhanced with different concentrations of indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Presence of 2.0 or 4.0 µM IBA or 4.0 µM NAA resulted to the optimum rooting rates. The maximum average number and length of roots per shoot was observed when the culture media was supplemented with 4.0 µM IBA or NAA. Approximately 92% of the plantlets were successfully established and acclimatized in field. The consistency of the chemical profile of the acclimatized in vitro propagated clones was assessed using quantitative 1H-NMR high throughput screening. In each variety, analysis of the micropropagated plant in comparison with the mother plant showed no statistically significant differences (p ≤ 0.05) in CBD+ cannabidiolic acid (CBDA) and CBG+ cannabigerolic acid (CBGA) content respectively, thus indicating stability of their chemical profile.