Minor phytocannabinoids, yet of major deeds: from isolation toward unveiling the neurobiological implications

Phytocannabinoids, secondary metabolites derived from Cannabis sativa L., have demonstrated therapeutic potential in several neurodegenerative disorders, including Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease. However, most research has focused primarily on major cannabinoids such as cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (THC), largely overlooking the more than 150 minor cannabinoids present in the plant. Emerging evidence suggests that these lesser-studied compounds may exhibit distinct biological activities and modulate the effects of major cannabinoids, highlighting the need for their systematic investigation. This doctoral thesis aims to (i) develop efficient separation strategies for the isolation of minor phytocannabinoids from plant matrices, (ii) establish targeted and untargeted analytical workflows for cannabis extracts, and (iii) investigate the neurobiological and metabolomic effects of selected minor cannabinoids. In parallel, particular attention was given to the sustainable valorisation of residual plant biomass generated after cannabinoid extraction, in line with green chemistry principles. A centrifugal partition chromatography method was developed and optimised for the isolation of cannabidivarin (CBDV) and cannabidibutol (CBDB). Complementary analytical methods, including HPLC-UV, HPLC-MS/MS, and high-resolution HPLC-TOF-MS, were established for the comprehensive profiling of ethanolic and aqueous extracts. Biological investigations demonstrated that CBDV modulates neuronal activity in murine hippocampal neurons and alters cellular metabolic profiles in human neuronal cultures. Notably, CBDV was found to counteract the effects induced by co-administration of CBD and THC. Building on the concept of full biomass utilisation, the residual cannabis material remaining after cannabinoid extraction was employed as a phytochemical source for the green synthesis of silver nanoparticles. An ultrasound-assisted method was developed, initially optimised using Ruta graveolens extracts and subsequently applied to cannabis residues. Although nanoparticles obtained from different plant sources exhibited similar morphology, they displayed distinct cytotoxic profiles against tumour cell lines, attributable to differences in the composition of the phytochemical corona. The thesis is structured as follows: an introductory chapter reviews the pharmacology of minor cannabinoids and current methodologies for their extraction and analysis. This is followed by four experimental chapters addressing (i) the isolation of minor cannabinoids using liquid–liquid chromatography, (ii) the effects of CBD, THC, and CBDV on neuronal transmission in murine models, (iii) metabolomic alterations in human neurons exposed to CBDV, and (iv) the development of silver nanoparticles from plant extracts and cannabis residues. The thesis concludes with a general discussion and perspectives.