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.