The cold-pressed technique is the most commonly used technique to extract commercial HSO, leaving almost half of the total biomass (HSM) as a byproduct. This waste, which represents a huge cost for companies, is a rich source of compounds with a high nutritional value as well as of secondary metabolites that remain in the squeezing residue. In particular, the unsaponifiable fraction of HSO and HSM, which until now received very little attention by researchers, represents the true inherent potential of industrial hemp products for their possible use in the nutraceutical field [20,21].

Although HSOs do not have to meet specific quality parameters to be commercialized, often these parameters are compared with those of other vegetable oils, in particular extra virgin olive oil, which is also obtained exclusively by cold pressing. However, Pharmacopoeia establishes a maximum value of 6% and 10 mEq O₂/kg for HSO acidity and peroxide value, respectively [22]. Acidity in edible oils is indicative of its conservation status and is strictly dependent on several variables including harvesting type, drying process, and storage [23]. Hand-harvested seeds had lower acidity than machine-harvested ones (~ 2% vs. 3%), as well as clean ventilated seeds with respect to the not-cleaned ventilated seeds (~ 3% vs. 6%). Furthermore, it has been reported that drying and storage increases this index by 0.1% and 0.05% per month. In a recent study, the authors observed that Federcanapa samples’ acidity values (1.9–16%) were higher with respect to certified Futura 75 (1.1%) and even more than the Monoica variety (0.81%) [23], which is the one closest to the USO 31 and Futura 75 HSOs (≤0.65%) analyzed in this study. Similar results were also obtained by Izzo and co-workers [24], who analyzed thirteen commercial HSOs belonging to different varieties cultivated in Italy and produced as a monovarietal or blended oil, finding acidity and peroxide index values ranging from 1.3–9.9% and 1.75–7.62 meq O₂/kg, respectively. Another study reported acidity and peroxide values for HSOs of 1.76% and 1.94 meq O₂/kg, respectively [25]. Results comparable to those obtained in the present study were instead observed by Al Jourdi et al. [26], who analyzed three HSOs harvested in ecological crops from Romania, finding acidity and peroxide values ranging from 0.5 to 0.75% and 0.6–1.2 meq O₂/kg, respectively. Furthermore, since the oxidation of PUFAs may generate conjugated dienes (K₂₃₂) and trienes (K₂₇₀), spectrophotometric analyses were carried out to calculate the ΔK value, which was ≤0.008, significantly lower with respect to that previously reported for HSOs (≤0.047) [24].

Other than quality parameters, it has been demonstrated that cultivar, variety, geographical origin, harvesting, drying, and storage conditions also affect the expression of minor constituents in HSOs such as total phenols, and the values have been found ranging from 32.50 to 160.80 mg GAE/g [24], which are well below those found in this study (≥ 290 mg GAE/g). On the contrary, a superimposable value was observed for Finola HSO total phenols and flavonoids content [6]. Regarding the total phenol content in HSMs, it has been recently reported that it may range from 0.39 to 0.91 mg/g [27,28]. However, Chen et al. [29] showed higher results in defatted HSM (3.9–15.6 mg GAE/g), with values superimposable to those obtained in the present study (2.88–16.32 mg GAE/g). According to the literature, specimens of the same plant species growing under different environmental conditions show significant differences in the production and accumulation of specific secondary metabolites such as polyphenols [30]. Furthermore, these differences increase based on the growing year and plant genotype. At this purpose, Irakli et al. [4] analyzed seven industrial hemp varieties harvested in three successive growing years, finding lignans and phenolic acids such as protocatechuic, p-hydroxybenzoic, and cinnamic acid as the main polyphenols. On the contrary, Faugno et al. [31], investigating if the polyphenol profile of USO 31 hempseed is affected by different cropping techniques, observed that flavonol glycosides were the main and characterizing USO 31 HSO polyphenols, according to our results.

The same trend was also observed in our previous study on the Finola variety, in which flavonoids were the most abundant polyphenols class HSO [6].

However, the geographical provenience of hemp should be strictly considered in order to select the most suitable variety for a specific nutraceutical purpose, without forgetting that, in any case, the final expression in secondary metabolites is strongly influenced by a whole series of parameters involving growing, harvesting, storage, and oil pressing conditions [31,32].

Together with polyphenols, other secondary metabolites that play a pivotal role in counteracting oxidation events in vegetable oils by stopping radical chain reactions against PUFAs and positively affecting their storage are tocopherols [33,34]. This is also the reason why extra virgin oils have a higher oxidative stability than refined oils. Among tocopherols, the γ-isomer is found to be the most abundant, followed by α-, δ-, and β-isomers, according to previous results [6,35,36]. This is of great importance because, although α-tocopherol is the best antioxidant among the tocopherol isomers, γ-tocopherol was reported to decrease low-density lipoprotein oxidation and to increase superoxide dismutase and nitric oxide synthase activity with a higher efficiency than α-tocopherol [37]. The USO 31 and Futura 75 HSOs investigated in the present study showed a higher total tocopherols content with respect to that previously reported, ranging from 3.47 to 13.25 mg/100 g [6,33]. The closest tocopherols content (63.03–85 mg/100 g) was observed instead by Anwar et al. [38], who analyzed HSOs from three agro-ecological zones of Pakistan. Finally, similar results were obtained by The et al. [25], who found a total tocopherols value of 59.16 mg/100 g. The greatest difference in terms of tocopherols was found in the content of γ-tocopherol, which, in the varieties investigated in this study, was approximately three times greater than that reported in the literature (77.43–92.45 vs. 28.23 mg/100 g) [33]. On the contrary, the α-tocopherol content was very close to the ranges previously reported (1.83–3.53 mg/100 g) [33]. However, it should not be forgotten that even substantial differences in terms of tocopherols can be recorded for the same reasons mentioned above for polyphenols, such as storage time, oxygen exposure, and temperature [39].

The endocannabinoid system (ECS) plays a critical homeostatic role in modulating PUFA signaling to maintain a balanced inflammatory and redox status. Dietary interventions based on animal or plant origin foods rich in PUFAs are increasingly used to support the ECS tone, promoting a healthy metabolism, brain, and emotional well-being and improving the risk factors associated with cardiovascular diseases. For this specific nutraceutical application, HSOs and HSMs represent matrices of primary choice because of their unique ω6:ω3 ratio (3:1), which is recommended for human nutrition and considered to be very important to reduce the risk of arteriosclerosis and coronary heart disease [40,41]. Moreover, the European Commission has authorized the use of hempseed and co-products in animal nutrition as well. Indeed, as demonstrated by a recent in vivo study, hempseed cake is useful in dogs’ nutrition as it leads, after 30 days, to a reduction in liver and renal markers and cholesterol, due to the healthier fatty acid profile [42].

PUFAs indeed are able to exert several health properties such as hypocholesterolemic, anti-hypertensive, anti-inflammatory, immunostimulant, anti-diabetic, cardioprotective, dermo-protective, and anti-obesity effects [36,43,44].

According to our results, the main fatty acids in the HSM were linoleic (54.09−55.42%), α-linolenic (17.31−18.42%), and oleic (12.96−13.93%) acid, followed by palmitic (6.48−7.90%), stearic (3.18−3.86%), and γ-linolenic (2.61−2.76%) acid [18]. Similar results were obtained for several commercial HSOs from different industrial hemp varieties, with the main fatty acids (α-linoleic, α- and γ-linolenic, and oleic acid) alone representing 80–90% of the total fatty acids [24,25,33,42,45,46]. Longer chain fatty acids, i.e., C:20 or higher, were also detected, but their concentration was significantly lower according to our results [46]. Furthermore, according to the British Department of Health and WHO/FAO experts, the PUFA/SFA ratio detected in our study (4.34–6.11) falls perfectly, also according to previous results (6.02–7.14) [18], within the recommendations, which suggests values above 0.40 [47,48]. Even in this case, several factors may affect fatty acid content and composition such as cultivars, pedo-climatic conditions, and farming, in particular during seed development [24].

All these compounds contribute to determining the antioxidant properties of HSOs and HSMs. Antioxidants play a pivotal role in protecting cell constituents against oxidative damage, which is well known to be the first step for the onset of various chronic diseases [49]. For this reason, antioxidant capacity is widely used as a screening parameter to characterize foods or medicinal plants as well as their bioactive phytochemicals. Currently, few works that evaluate antioxidant and free-radical scavenging activity through an in vitro test set, which are useful to characterize the behavior of extracts in different environments and reaction mechanisms, are available. Comparing the results of the present study with our previous work about the Finola HSO variety, what is immediately evident is that, in accordance with the richer phytochemical profile of the USO 31 and Futura 75 HSOs, the antioxidant activity appears significantly higher (1.1–2.9 times) than that observed for the Finola variety in all the tests carried out. The best antioxidant activity was recorded by the DPPH and ORAC test according to previous results [6,23] because they are generally considered more sensitive than other common antioxidant methods, such as TEAC, although highly variable values ranging from 0.2 to 22.3 mM TE/g were recorded [50]. Since the antioxidant activity in addition to fatty acids is mainly attributable to the polyphenols and tocopherols content, it is evident how the parameters that influence the expression and accumulation of these bioactive compounds, such as harvesting and storage, also consequently influence antioxidant activity. Recently, the antioxidant potential of HSOs was also demonstrated in vivo by Vitorović et al. [51], who analyzed the effects of HSOs on oxidative stress markers and on the life cycle of D. melanogaster under non-stress and hydrogen peroxide-induced stress conditions, demonstrating how the antioxidant effect is closely related to the HSO dose. On the contrary, regarding the antioxidant properties of HSMs, it has been recently demonstrated that, in the circular economy perspective of the re-use of hempseed byproducts for nutraceutical purposes, the antioxidant activity of HSM, already reduced with respect to the HSO, generally decreases after extrusion [28]. This result might have the same explanation as the reduction in the total phenols and flavonoids content, mainly due to the high screw speed and barrel moisture. Indeed, the friction generated during extrusion increases the internal product’s temperature, therefore leading to phenolics loss. Furthermore, it seems that this effect is amplified with ingredients rich in proteins and carbohydrates [28].

Regarding the nutritional profile of HSMs, according to our results, the moisture content ranged from 6.98% to 7.88% [18]. Proteins were the most abundant nutrient (10.62–44.36%), followed by lipids (8.26–18.60%) and sugars (0–4.96%). However, the greatest variability was recorded in the crude fiber content with values that could reach 29.54% [18].

Interestingly, the anti-nutrients factors in the USO 31 and Futura 75 HSMs analyzed in this study were well below those determined in a previous work on HSMs of the Helena variety [18] and those obtained for watermelon, pumpkin, and paprika seed flour [52]. Finally, the phytic acid content was lower than that of Canola meal, as reported by Bell et al. [53], and of the HSM of Italian and French varieties [54]. This is very important because the presence of certain anti-nutrients may limit the recovery of HSM as edible-grade products in human nutrition, as they influence protein digestibility, organoleptic properties, and the bioavailability of macro- and microelements. However, when evaluating the consumption of protein-rich meals, the bioavailability of amino acids must always be evaluated. Indeed, a recent study, which investigated and compared the postprandial events related to satiety and protein metabolism following the acute consumption of high-protein meals of animal and plant origins, demonstrated important differences in the bioavailability of amino acids, suggesting an important and favorable impact of the food matrix in plant-based meals compared to meat-based ones [55]. Amino acids are emerging as new biomarkers for metabolic disorders; just think that phenylalanine and tyrosine have been associated with insulin resistance in men [56] and that alanine, glutamine, glycine, as well as other amino acids such as histidine, arginine, and tryptophan did not show any association with insulin resistance. High dietary intakes of branch-chain amino acids (BCAAs) have been associated with type two diabetes (T2D), suggesting that nutritional strategies that help to maintain a lower concentration of these amino acids could be used in T2D prevention. Even in this case, however, it has been shown that BCAA intake from plant sources has a lower impact on health. From this point of view, the HSMs analyzed in the present study had a very interesting amino acid profile, being rich, in particular the USO 31 variety, in essential amino acids and poor, in general, in BCAAs. Furthermore, from the tested meals in the above study, hemp had the highest fiber and total fat content, which exert a pivotal role in the up-regulation of glucagon-like peptide one (GLP-1) gene expression [57] and satiety [55]. Therefore, in the light of the results obtained, USO 31 and Futura 75 HSMs prove to be very promising matrices from a nutritional point of view.