Analysis of phytocannabinoids in hemp seeds, sprouts and microgreens

Cannabis sativa L. has emerged in the last decade as a valuable crop for its low environmental impact and as an extraordinary source of food, textiles, plastics, paper, paint, construction material, as well as biofuel and animal feed [1]. Moreover, its ability to produce a peculiar class of bioactive compounds called phytocannabinoids, along with a rich fraction of other antioxidant molecules, has made this plant an interesting material for clinical applications and scientific research. In particular, a large inventory of phytocannabinoids has been reported a few years ago by Hanuš et al. [2], to which other new interesting molecules have been recently added [3], [4], [5], [6], [7], [8]. The phytocannabinoid profile has been extensively investigated in mature inflorescences, as well as in other cannabis derived products, of different cannabis chemovars with an outstanding qualitative and quantitative variability [9], [10], [11], [12], [13], [14]. On the other hand, phytocannabinoids seemed to be absent in early stages of plant growth, particularly in the sprouting process since their biosynthesis is thought to be associated with the presence of the glandular trichomes [15].

In a work by Frassinetti et al., phytocannabinoids could be detected in seeds, probably as a carry-over effect of the surrounding bracts, but not in sprouts [16]. Conversely, other important macronutrients could be determined in hemp sprouts, such as the characteristic flavonoids cannflavins A and B with anti-inflammatory properties [17] potentiated by a balanced amount of ω-3 and ω-6 essential fatty acids [15]. More recently, Pannico et al. compared the chemical profile of hemp microgreens (9 days after sowing) of six hemp cultivars in terms of organic acids, amino acids, and polyphenols and disclosed for the first time the presence of various phytocannabinoids [18]. In particular, the concentration of the non-psychotropic cannabidiolic acid (CBDA) and its decarboxylated counterpart cannabidiol (CBD) reached concentrations above 2000 µg/100 g and 50 µg/100 g respectively, being the most abundant species among all phytocannabinoids detected [18]. Other species found were the narcotic Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and its carboxylated non-narcotic precursor tetrahydrocannabinolic acid (THCA) that reached the maximum concentration of 10 µg/100 g and 130 µg/100 g respectively [18]. Lastly, cannabigerolic acid (CBGA), its decarboxylated derivative cannabigerol (CBG) and the Δ⁹-THC oxidation product cannabinol (CBN) were also detected with the latter being the least abundant of all phytocannabinoids [18]. The profiling of phytocannabinoids in the juvenile stages of C. sativa was recently described by Fulvio et al. (2023), highlighting the capacity of different genotypes to produce and accumulate phytocannabinoids, predominantly cannabichromenic acid (CBCA), at very early vegetative growth stages, as previously reported [19].

All these findings support the idea of hemp sprouts as a functional food described by the presence of all essential amino acids, lower amount of oxalate compared to other sprouts species and the remarkable content of cannflavins A and B [18]. Additionally, the consumption of hemp sprouts was reported to be widely safe in terms of the amount of the narcotic tetrahydrocannabinol (THC), which was far below the USA and EU limit of 0.3% [18]. The latter refers to the total THC limit in the plant and not in the finished product, which has been so far established, at least in Italy, only for some type of hemp food products including hemp seed oil (THC limit 5 ppm), hemp seeds and hemp flour, as well as supplements derived from hemp (THC limit 2 ppm) [20]. A more recent regulation was issued by the European Commission on the maximum values of THC in food, imposing limits of 3 ppm in hemp seeds and hemp flour and 7.5 ppm in hemp seed oil [21]. Total THC is to be intended as the sum of the carboxylated (THCA) and decarboxylated (Δ⁹-THC and Δ⁸-THC) species. However, the thresholds have not been updated for other types of hemp derived products or other phytocannabinoids.

As the literature is still scarce on this matter, the present work aims to provide a detailed overview of all phytocannabinoids present in hemp seeds, sprouts, and microgreens, with a particular focus on the differences between the various tissues of the hemp sprouts. Unlike their mature counterparts, there is no specific time of the year for the sprouts and microgreens growth and the only requirement for their production is the availability of good certified seeds with high germinability. Also, they require minimum space and no special care so they are also suitable for domestic cultivation. As an additional advantage, they contain a higher level of beneficial phytonutrients compared to the mature material that are all preserved as long as they are consumed raw [22]. As the hemp-based food market is growing, there is the need to shed light on the chemical composition of these products and, in particular on their phytocannabinoids profile.

A recently developed untargeted metabolomics approach called phytocannabinomics [9] was employed for the qualitative and semi-quantitative analysis of the different phytocannabinoid species accompanied by a multivariate statistical analysis. At the same time, a targeted analysis of the major phytocannabinoids including CBDA, CBD, THCA, trans-Δ⁹-THC, CBGA, CBG, cannabichromenic acid (CBCA) and cannabichromene (CBC) was carried out for quantitative purposes. The determination of phytocannabinoids levels in hemp products is particularly important as these compounds are known to exert several beneficial health effects and can add value to this new “functional food”. Both qualitative and quantitative analyses were carried out by high performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (HPLC-HRMS). The analysis of CBC-type phytocannabinoids is particularly important as this species are produced in higher levels in the early stages of plant growth to decrease at maturation. Moreover, considering the recent interest in their pharmacological activity, the evaluation of the enantiomeric composition of CBCA in these hemp products was carried out by HPLC-UV to shed some light on its stereochemical behavior at these early growth stages.