Hemp is an industrial crop that has been widely investigated due to its diversity of applications in the pharmaceutical, nutraceutical, food, cosmetic, textile, and material industries (Schluttenhofer and Yuan, 2017; Farinon et al., 2020). However, the chemical diversity of the hemp seed metabolome has not been explored in detail and new metabolites with unknown properties are likely to be discovered. In the current study, following an untargeted metabolomics approach by LC-MS and GC-MS, we discovered a previously unreported molecular family in hemp seeds “cinnamic acid glycosyl sulphates” and linked the presence of biologically important molecules to certain accessions. Chemical knowledge of these cultivars and accessions could be used as a base in future to optimize commercial cultivars in the context of nutritional and other functional uses.

Metabolic fingerprinting by LC-MS and data mining tools revealed that the 52 hemp seed accessions can be categorized into five main groups based on the similarities of their metabolic fingerprints. Although it is expected that when growing the same seeds under homogeneous conditions there will be some differences in the metabolic grouping, we believe that we can only have an accurate representation of the true metabolic phenotype of an organism by studying the chemical composition of plants/organs obtained from individuals collected in their natural environment. Germplasm and herbarium collections provide valuable samples for this. In the current study, among all the studied accessions and cultivars, the Spanish accession Kongo Hanf (CAN58) and the French accession CAN37 showed a remarkable structural diversity of bioactive and novel metabolites, some of them accumulated in high amounts. These two varieties showed the highest accumulation of phenylpropionamides (including cinnamic acid amides and their oxidative coupling products lignanamides), as well as new cinnamic acid glycosyl sulphates.

Phenylpropionamides (cinnamic acid amides and lignanamides) represents the main chemical class of bioactive metabolites in hemp seeds (Moccia et al., 2020; Leonard et al., 2021a; Leonard et al., 2021b). These compounds display potent antioxidant, anti-inflammatory, cytotoxic and acetylcholinesterase inhibitory activities, both in vitro and in vivo studies (Yan et al., 2015; Farinon et al., 2020; Moccia et al., 2020; Leonard et al., 2021a). For instance, previous studies have recognized the strong potential of hemp seed phenylpropionamides as protective agents against human chronic diseases (Leonard et al., 2021a), although results from clinical trials or cohort studies are not available to support this. Our results indicate that N-trans-caffeoyltyramine (compound 11) and N-trans-feruloyltyramine (compound 12) constitute the main phenylpropionamides accumulated in hemp seeds, especially in the Spanish accession Kongo Hanf (CAN58) and the French accession CAN37. These two compounds have demonstrated strong antioxidant activities (Chen et al., 2012; Moccia et al., 2020) and in vivo anti-neuroinflammatory action (Maiolo et al., 2018; Zhou et al., 2018). Furthermore, the lignanamides fraction of hemp seeds are also highly active. Like compounds 11 and 12, lignanamides (compounds 16 – 38) are also accumulated in high amounts in the accession CAN58 and CAN37.

Recent studies by Chen et al. (2012) observed stronger DPPH scavenging activity by cannabisin B (compound 17) isolated from hemp seed than common soy (Glycine max (L.) Merr.) isoflavones and secoisolariciresinol diglucoside from flax seed (Linum usitatissimum L.). Cannabisins M, A, C, D, and 3,3’-demethylgrossamide (compounds 21, 23, 27, 32 and 20, respectively) from hemp seed also displayed potent antioxidant activities in vitro (Yan et al., 2015). These compounds are especially accumulated in accessions CAN37, CAN58, CAN68 and CAN42.

The anti-inflammatory properties that these and other hemp seed lignanamides display have also been widely recognized. For example, grossamide, (compound 35) accumulated in higher amounts in accession CAN37 ( Figure 3 ), showed a significant reduction in the production of the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) at a concentration of 10 – 20 µM in a lipopolysaccharide (LPS) induced model (Luo et al., 2017). These cytokines have been linked to the stimulation of inflammatory and auto-immune processes in many diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis as well as different types of cancer (Bradley, 2008; Blauvelt, 2017; Shang et al., 2017). Like grossamide, cannabisin F (compound 34) has also been reported to reduce the production of IL-6 and TNF-a in LPS-induced models, although through a different mechanism (Wang et al., 2019).

Regarding the cytotoxic effects of hemp seed lignanamides, previous studies have reported that it varies in accordance with their chemical structures. For example, cannabisin B has been shown to induce autophagic cell death on HepG2, a human liver cancer cell line (Chen et al., 2013), while cannabisin F is especially active against SH-SY5Y, a human neuroblastoma cell line (Li et al., 2019). Nevertheless, other hemp seed lignanamides, such as grossamide, cannabisin D and cannabisin G, have displayed weak cytotoxic activity against human prostate cancer LNCaP cells (Ma et al., 2002). Considering that CAN37, CAN58, CAN68 and CAN42 have a higher concentration of bioactive cinnamic acid amides and lignanamides, these accessions could be commercially prioritized as potential sources of antioxidant, anti-inflammatory and cytotoxic metabolites or in selective breeding programs to improve the concentration of specific metabolites in other commercialized cultivars. Interestingly, none of the five historic cultivars with deleted status in the EU database ( Table 1 ) showed a particularly high concentration of bioactive metabolites compared to the newer accessions.

In addition to the 29 phenylpropionamide peaks annotated in the current study ( Table 2 ), our results suggest that other lignanamides might also be present. Manual inspection of the FBMN results indicate that there are at least eleven additional nodes representing potentially new phenylpropionamides, some of them showing m/z values previously unreported in hemp seeds. For example, two nodes at m/z 600.270 and m/z 614.286 [M+H]⁺ appear to be structurally related to tricoumaroyl spermidine (compound 14). Manual interpretation of MS data suggests that the node at m/z 600.270 [M+H]⁺ likely represents a hydroxylated analogue, while the node at m/z 614.286 [M+H]⁺ is likely its methoxylated version. Several isomers of m/z 597.33 and m/z 611.23 [M+H]⁺ were also observed. The isolation and complete structural characterization of these potentially new metabolites is still necessary. Recent studies have proved that despite being exhaustively commercialized, hemp seed continues to be a promising source of new lignanamides. For example, Yan et al. (2015) identified the first trimeric feruloyltyramine in hempseeds, of which its coupling resulted in the formation of an 8-O-4 linkage. Considering that hemp hulls are the main site of accumulation of lignanamides, future agricultural waste valorisation programs are highly encouraged. Hemp hulls are a by-product of the hemp seed oil industry (Pojić et al., 2014; Mattila et al., 2018), which represents an opportunity. This is further supported by the fact that the total lignanamides in hemp hull can be up to four times higher than in whole hemp seed (3.36 mg/g vs 0.77 mg/g, respectively) (Mattila et al., 2018).

Aside from phenylpropionamides, hemp seed oil is also a commodity held in high regard for its nutraceutical properties due to a perfectly balanced content of omega 3 and omega 6 polyunsaturated fatty acids (Crescente et al., 2018; Citti et al., 2019). Mean concentration values for the six main fatty acids detected in the current study: palmitic acid oleic acid, linoleic acid, γ-linolenic acid, α-linolenic acid and stearidonic acid ( Table 4 ), are within ranges comparable to previous studies (Galasso et al., 2016; Pavlovic et al., 2019). The Italian cultivar Eletta Campana (CAN48) showed the highest accumulation of fatty acids among the investigated hemp seed accessions. However, CAN32, CAN50 and CAN53 were the accessions with the ratio of linoleic acid: α-linolenic acid closest to 3:1, reported as optimal for human nutrition (Leizer et al., 2000; Simopoulos, 2008; Galasso et al., 2016). These results agree and further expand the studies of Galasso et al. (2016). Similar to previous studies, the cultivar with the lowest proportion of α-linolenic acid was Eletta Campana (CAN48) (Galasso et al., 2016). However, while the mean concentration values for the six fatty acids are also within comparable ranges to previous reports (Galasso et al., 2016; Pavlovic et al., 2019), there are some discrepancies in the concentration of specific fatty acids in certain accessions. For example, we found a higher proportion of oleic acid in CAN48 than in CAN39 and the higher percentage of α-linolenic acid was found in CAN40 instead of CAN24 as reported by Galasso et al. (2016). These discrepancies can be due to different extraction methods and the fact that Galasso et al. (2016) studied the oil composition of seeds coming from individuals grown under the same conditions while our study directly analysed the seeds stored in the IPK genebank.

In addition to the main fatty acids previously mentioned, other fatty acids have also been reported in minor concentrations in hemp seed oil (Galasso et al., 2016; Pavlovic et al., 2019). These metabolites were not reported in the current paper due to identification and quantification discrepancies in these low concentrated metabolites. However, considering that the biological properties of hemp seed oil are related to the presence of the major fatty acids, especially the ratio between linoleic acid and α-linolenic acid, our results are biologically relevant. Complete reports of minor metabolites in hemp seed oil can be found in other studies (Galasso et al., 2016; Pavlovic et al., 2019).

A recent study reported the cannabinoid profile of ten commercially available hemp seed oils (Citti et al., 2019). Besides tetrahydrocannabinol and cannabidiol, another 30 cannabinoids were also identified (Citti et al., 2019). In the current study, we found traces of only three cannabinoids using a non-selective LC-MS method. Our results demonstrated that Eletta Campana (CAN48) was the cultivar with the highest concentration of cannabinoids, although their concentration was around 200 times lower than that of the most abundant metabolite (N-trans-caffeoyltyramine). Previous studies on the same accession (CAN48) obtained from the same genebank (IPK) reported an absolute quantification value of THC of 0.08%, which is well below the 0.3% threshold set by international regulations (Citti et al., 2019). Based on the low THC content of this cultivar, we might assume that all the hemp seed accessions included in the current study meet the international regulatory standards of < 0.3% THC content.

Recent studies have proved that despite being heavily studied, hemp seed continues to be a promising source of new metabolites. In the current study we discovered a previously unknown molecular family in hemp seeds “cinnamic acid glycosyl sulphates” widely distributed across all hemp seed accessions in similar concentrations. The identity of one novel compound from this chemical class was confirmed by isolation and interpretation of spectroscopic data, and four structural analogues, three of them new, is also suggested. The presence of these compounds in all hemp accessions suggest that cinnamic acid glycosyl sulphates might have important implications as quality control markers for the authentication of commercial hemp seed products extracted with highly aqueous solvents. Considering that these highly polar extracts, commonly used in the manufacture of cosmetics, are usually devoid of characteristic hemp seed phenylpropionamides due to insolubility issues, the need of more suitable quality control markers is justified. Further studies are still needed to identify the biological properties of these new metabolites.

In conclusion, our results confirm that some hemp accessions present in the IPK germplasm collection appear to contain more interesting profiles of metabolites than some of the cultivated hemp cultivars, as first suggested by Galasso et al. (2016). We suggest that accessions CAN37 and CAN58, should be scientifically prioritized for the discovery of new metabolites as well as in selective breeding programs for the development of new cultivars with high contents of bioactive phenylpropionamides. However, despite the functional and nutraceutical properties of this crop, the seeds of hemp are not totally free of antinutritional compounds, such as phytic acid (Russo, 2013; Galasso et al., 2016). According to previous reports, including ten of the accessions investigated here, CAN48 presents the highest levels of phytate, although other cultivars not included here like Futura75 and Felina32 have even higher values. The presence of high phytate contents in some cultivars limit their use in human nutrition and other monogastric animals since a high level of phytic acid may lead to mineral deficiencies of macro-and microelements, protein digestibility and poor organoleptic properties (Russo, 2013; Russo and Reggiani, 2015; Galasso et al., 2016). Therefore, an improvement for this trait might be necessary if the high yielding oil cultivar CAN48 is prioritized. According to previous studies (Galasso et al., 2016), the Italian accession CAN40 might be a good candidate to reduce the content of phytate through hybridization and selective breeding. Other accessions such as CAN32, or the Fibrimon cultivars CAN50 and CAN53, could also be prioritized if a 3:1 ratio of linoleic acid/α-linolenic acid is desired.