Uncovering the metabolite complexity and variability of cultivated hemp (Cannabis sativa L.): A first phytochemical diversity mapping in Greece.

The high value of fiber-type Cannabis sativa L. (hemp) due to its phytochemicals has yet to be fully recognized and leveraged. Besides cannabidiol (CBD), which is the most prevalent non-psychoactive cannabinoid, hemp contains numerous other cannabinoids with unexplored bioactivities, in addition to various compound classes. Previous works have aimed to correlate chemical profiles of C. sativa inflorescences with important parameters, mostly based on experiments under controlled conditions. However, mapping studies that explore the phytochemical diversity of hemp in a more realistic context are crucial to guide decisions at multiple levels, especially in areas where hemp cultivation was recently re-authorized, including Mediterranean countries. In this work, a powerful strategy was followed to map the phytochemical diversity of cultivated hemp in Greece, being the first study of its kind for this environment. A panel of 98 inflorescence samples, covering two harvesting years, eleven geographical regions and seven commonly used EU varieties, were studied using a combination of targeted and untargeted approaches. Quantitative results based on UPLC-PDA revealed relatively constant CBD/THC (total) ratios, while profiling by LC-HRMS effectively probed the phytochemical variability of samples, and led to the annotation of 88 metabolites, including a multitude of minor cannabinoids. Multivariate analysis substantiated a strong effect of harvesting year in sample discrimination and related biomarkers were revealed, belonging to fatty acids and flavonoids. The effect of geographical region and, especially, variety on chemical variation patterns was more intricate to interpret. The results of this work are envisioned to enhance our understanding of the real-world phytochemical complexity of C. sativa (hemp), with a view to maximized utilization of hemp for the promotion of human well-being.

Extraction solvent selection for Cannabis sativa L. by efficient exploration of cannabinoid selectivity and phytochemical diversity.

INTRODUCTION: Cannabis sativa L. is attracting worldwide attention due to various health-promoting effects. Extraction solvent type is critical for the recovery of bioactive compounds from the plant, especially cannabinoids. However, the choice of solvent is varied and not adequately warranted elsewhere, causing confusion in involved fields. OBJECTIVE: The present work aimed to investigate the effect of extraction solvent on C. sativa (hemp) with regard to cannabinoid recovery and phytochemical profile of the extracts, considering most of the related solvents. METHODOLOGY: The majority of solvents reported for C. sativa (n = 14) were compared using a representative hemp pool. Quantitative results for major and minor cannabinoids were rapidly and reliably obtained using ultrahigh-performance liquid chromatography coupled with photodiode array detection (UPLC-PDA). In parallel, high-performance thin-layer chromatographic (HPTLC) fingerprinting was employed, involving less toxic mobile phase than in relevant reports. Various derivatisation schemes were applied for more comprehensive comparison of extracts. RESULTS: Differential selectivity towards cannabinoids was observed among solvents. MeOH was found particularly efficient for most cannabinoids, in addition to solvent systems such as n-Hex/EtOH 70:30 and ACN/EtOH 80:20, while EtOH was generally inferior. For tetrahydrocannabinol (THC)-type compounds, EtOAc and n-Hex/EtOAc 60:40 outperformed n-Hex, despite its use in the official EU method. Solvents that tend to extract more lipids or more polar compounds were revealed based on HPTLC results. CONCLUSION: Combining the observations from UPLC quantitation and HPTLC fingerprinting, this work allowed comprehensive evaluation of extraction solvents, in view of robust quality assessment and maximised utilisation of C. sativa.

Effective determination of the principal non-psychoactive cannabinoids in fiber-type Cannabis sativa L. by UPLC-PDA following a comprehensive design and optimization of extraction methodology.

Cannabidiol (CBD) and cannabidiolic acid (CBDA) represent the most abundant non-psychoactive cannabinoids in fiber-type Cannabis sativa L. (hemp) and both have demonstrated high therapeutic potential. Hence, efficient extraction coupled with reliable determination of these compounds is crucial for informed utilization of hemp and is increasingly needed in the present state of harmonization efforts. In this context, a systematic approach for extraction optimization was followed, which initially involved comparison of three widely available extraction techniques, i.e. ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and dynamic maceration (DM). These were applied on samples of different hemp varieties (n = 3) using ethanol as a safe and efficient solvent. UAE showed the most promising results and was further optimized by means of response surface methodology (RSM), based on a circumscribed central composite design. The conditions maximizing CBD, CBDA, and total CBD content as well as extraction yield were determined with high desirability (0.97) and were experimentally confirmed. The optimized UAE method was also compared with a previously reported extraction procedure, demonstrating superior performance. For the quantitation of CBD and CBDA in hemp extracts, a reversed-phase UPLC-PDA method was developed and validated. Chromatographic separation was achieved in less than 10 min, while satisfactory results for linearity (R2 > 0.996), precision (RSD < 2.0%), and accuracy (recovery rates of 93.1-101.0%) were obtained for both analytes. Limits of detection were determined as 0.07 and 0.04 µg mL-1 for CBD and CBDA, respectively, indicating sufficient sensitivity. The good performance of the method was verified by the evaluation of additional parameters (e.g. matrix effect, extraction recovery), which was largely enabled by the use of isolated standards. The whole analytical workflow, involving both optimized UAE extraction and UPLC-PDA determination, entails simplified manipulation and may offer a reliable and cost-effective approach for routine quality control of hemp regarding the principal cannabinoids.

Cannabidiol Modulates the Motor Profile and NMDA Receptor-related Alterations Induced by Ketamine.

Cannabidiol (CBD) is a non-addictive ingredient of cannabis with antipsychotic potential, while ketamine (KET), an uncompetitive NMDA receptor inhibitor, has been extensively used as a psychotomimetic. Only few studies have focused on the role of CBD on the KET-induced motor profile, while no study has investigated the impact of CBD on KET-induced alterations in NMDA receptor subunit expression and ERK phosphorylation state, in brain regions related to the neurobiology and treatment of schizophrenia. Therefore, the aim of the present study is to evaluate the role of CBD on KET-induced motor response and relevant glutamatergic signaling in the prefrontal cortex, the nucleus accumbens, the dorsal and ventral hippocampus. The present study demonstrated that CBD pre-administration did not reverse KET-induced short-lasting hyperactivity, but it prolonged it over time. CBD alone decreased motor activity at the highest dose tested (30 mg/kg) while KET increased motor activity at the higher doses (30, 60 mg/kg). Moreover, KET induced regionally-dependent alterations in NR1 and NR2B expression and ERK phosphorylation that were reversed by CBD pre-administration. Interestingly, in the nucleus accumbens KET per se reduced NR2B and p-ERK levels, while the CBD/KET combination increased NR2B and p-ERK levels, as compared to control. This study is the first to show that CBD prolongs KET-induced motor stimulation and restores KET-induced effects on glutamatergic signaling and neuroplasticity-related markers. These findings contribute to the understanding of CBD effects on the behavioral and neurobiological profiles of psychotogenic KET.