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.

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.

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.