The aim of this work was to evaluate the efficacy of hemp inflorescence ethanolic extract to protect a vegetable oil rich in polyunsaturated fatty acids (PUFAs), namely, linseed oil, against oxidation. Hemp is rich in bioactive compounds, particularly polyphenols and terpenes, potentially capable of counteracting reactive metals and free radicals and, therefore, we hypothesized, of retarding oxidation in vegetable oil. In this study, the bioactive compounds were extracted from inflorescences with ethanol, since it is generally established that this solvent is of less concern to human health than other organic solvents. This is why it is extensively used in the food sector and does not constitute an environmental issue [24]. Moreover, it is reported that ethanol offers the best performance in terms of extraction yields and antioxidant effects [35]. The hemp inflorescence ethanolic extract was investigated in order to profile the terpenoid compounds, estimate the total phenolic content (TPC), and determine its antioxidant activities. The amount of phytocannabinoids classified the hemp as “fiber-type” Cannabis, chemotype III, with a total content of Δ⁹-tetrahydrocannabinol (THC) <0.6% (which represents the legal limit in Italy [36]) and lower than that of CBD (ratio (THC + CBN)/CBD < 0.02 [37]), wherein CBN represents cannabinol content.

The odor and flavor in Cannabis species are determined by terpenes, contributing to a characterization of the different strains [38]. In this study, the total amounts of monoterpenes and sesquiterpenes (96.41%) were in line with the literature, which reports up to 98% of these two families in cannabis essential oil [39]. The profile of the terpene classes was in agreement with that found by Nissen et al. [40] in the essential oil of the Carmagnola variety, while that of the single terpenes was different. In fact, the authors [40] reported β-myrcene (29.22%), α-pinene (15.12%), and β-caryophyllene (13.90%) as the main terpenes. In our study, the three most abundant were β-pinene, β-caryophyllene, and cedrol, whose health benefits (such as anti-inflammatory, antidepressant, and sedative activities, antihypertensive and relaxant effects, and pharmacological actions including cardioprotective, neuroprotective, gastroprotective, hepatoprotective, and immunomodulatory) have been widely described [38,39,41]. This discrepancy with Nissen et al. [40] is likely due to the difference in varieties, as well as growth factors such as soil and climate conditions, abiotic stress, harvesting time, and tissue type [24]. In addition to cannabinoids and terpenes, phenolic compounds are reported in hemp, including flavonoids (such as flavonols and flavones), lignamides (such as cannabisin-like compounds), and stilbenoids (such as cannabistilbene-like compounds) [18,38]. In particular, apigenin, chlorogenic acid, ferulic acid, luteolin, quercetin, N-trans-caffeoyltyramine, p-coumaric acid, cannflavin A, catechin, cannflavin B, epicatechin, and quercetin-3-glucoside (ranging 8.7–126.1 mg/kg) are the most recurrent polyphenols in the inflorescences of Carmagnola variety [23]. Polyphenols can play a fundamental role in human health, due to their antioxidant properties and their ability to modulate or inhibit certain enzymes such as lipoxygenases [42]. The TPC found in the present study was slightly lower compared with that reported by Izzo et al. [23] in the Carmagnola cultivar of hemp inflorescence extracted with methanol (ranged 26.3–41.5 mg GAE/g). However, it was higher than that documented by other researchers (Frassinetti et al. [43] with about 6 mg GAE/g in hemp sprouts, and Drinić et al. [35] with 6.43 and 5.85 mg GAE/g in 90% ethanol extracts of young and mature hemp inflorescences, respectively). Similarly to terpenes, the profile and content of flavonoids in cannabis are also influenced by factors other than the variety. In fact, their distribution varies enormously from plant to plant and according to the tissue considered, and, as the plant ages, their content decreases [18]. The method of extraction parameters (such as the type of solvent, the extraction time and temperature, and the particle size of the sample) are also influential in a solid–liquid extraction [35]. Indeed, Izzo et al. [23], despite having investigated the same variety as this study, obtained a higher TPC value, since they used methanol as a solvent, which is one of the most effective extractants, but much more toxic than ethanol.

In comparison with the literature, it might be pointed out that the inflorescence extract is more able to scavenge DPPH radicals and chelate ferrous ions than hemp leaves and seeds. In fact, as reported by the authors [44], hemp leaves and seeds displayed a relatively higher EC₅₀ for both RSA and chelating activity (a higher EC₅₀ value indicates a lower activity) than that reported in the present study.

However, similar EC₅₀ values were observed for the DPPH^∙ assay in two different varieties of hempseed kernel extracted with 100% ethanol [45]. Nevertheless, the EC₅₀ of DPPH^∙ found in the present study was much higher than that reported by other research on the Carmagnola cultivar cultivated in different areas of the Po valley (Italy) [20]. However, as stated by Lesma et al. [20], the RSA is influenced not only by the variety, but also by the soil where it is grown and by the weather conditions, potentially explaining the discrepancy between RSA values even within the same cultivar.

Having established its potential as an antioxidant agent, the hemp inflorescence extract was then tested in a bulk oil model system, wherein stripped linseed oil was exposed to high temperatures in order to induce its rapid degradation. This process involves a series of chemical and enzymatic reactions that lead to triacylglycerol hydrolysis and fatty acid primary and secondary oxidation, causing a deterioration in the sensory and nutritional properties of the oil due to the formation of undesirable and toxic compounds [9,46,47].

On the basis of the composition of the main lipid classes, the hemp inflorescence extract cannot be regarded as an agent causing great resistance of the oil against lipolysis; no differences between HO and CO were noted until the last day. During the experiment, the hydrolysis in CO was not severe; a minimal cleavage of TAGs and DAGs (and consequent release of fatty acids) was observed, whereas, in HO, there was no such reaction. Usually, the hydrolysis of TAGs is carried out by enzymes, principally lipases and phospholipases. The latter are water-soluble and act at the interfaces with water naturally present in the oil and the lipids [48]. Lipolytic breakdown is determined mostly by the chemical–physical conditions of the oil, which are governed by upstream factors, such as the condition of the plant from which the oil has been extracted (which affects the concentration of lipase enzymes) and the conditions of the oil processing (which affects the moisture content) [48]. Since the stripping process inhibits lipase activity, the lipolysis that occurred in our samples can be mainly ascribed to chemical reactions.

On the other hand, the inflorescence extract turned out to be an effective antioxidant, preventing both primary and secondary oxidation, with HO showing an increased oxidative stability compared to CO and EO. The protective effect of hemp was already evident from the composition of fatty acids, since all the unsaturated fatty acids exhibited a constant trend throughout the entire experiment, while, in CO and EO, a pronounced oxidation of the PUFAs occurred. In addition, the content of ALA in HO on day 7 was higher than in CO, demonstrating that the hemp extract reduced ALA oxidation. Unlike ALA, LA remained stable in both HO and CO. The most probable reason is that linolenate, having a higher degree of unsaturation, is more prone to oxidation than linoleate with 2.4-fold higher reactivity. Thus, linolenate is the first substrate hit by free radicals [9].

In contrast, EO exhibited completely different kinetics compared to CO and HO, starting from lipolysis. A strong hydrolysis of TAGs was followed by an increase in DAG and FFA content. Since the free PUFAs are much more prone to oxidation than their esterified counterparts [48], a severe worsening of the fatty acid composition took place in EO. Almost all FAMEs were affected by oxidation, with a drastic and significant reduction in ALA and even LA, underlining the more marked progress in oxidation. This pro-oxidant activity has been already described by the other researchers [33,49,50]. In fact, it was stated that many factors control the antioxidant or pro-oxidant effect of α-tocopherol, namely, the nature of the substrate and its mobility within it, its concentration, and the applied conditions, such as the temperature and the potential interactions with other substances in the matrix [51]. Moreover, it has been suggested that, in oils, the most effective antioxidants are polar antioxidants and not nonpolar ones, which are more effective in protecting emulsions (the so-called “polar paradox”) [52].

As expected, the protective effects of hemp inflorescences against oxidation, as well as the pro-oxidant effect of α-tocopherol, were established by primary and secondary oxidation products and the protection factor (Pf). According to Frankel [46], peroxide degradation takes place at very high temperatures, and this could be an explanation for the observed linear trend, since a moderate heating was applied in the experiment (40 °C). CO and HO were characterized, as expected, by a progress in hexanal formation that reflected the peroxide content. Thus, an increasing (CO) or constant (HO) trend in peroxide development led to an increasing (CO) or constant (HO) trend in hexanal formation. The trend of hexanal is also a reflection of ALA oxidation, since it is widely reported as a precursor [53]. Consequently, the progressive oxidation of ALA in CO resulted in a progressive increase in hexanal, while the stability of ALA in HO resulted in an unchanged hexanal content. On the other hand, a completely different process was observed in EO. In fact, despite reaching rancidity after only 2 days (50 meq O₂/kg oil as the maximum value indicates that complete rancidity has been reached), the hexanal content remained unusually low. Other authors documented the same situation, stating that a higher concentration of α-tocopherol led to a higher inhibition of hexanal formation [53,54]. The authors suggested that this suppression is due to the action of α-tocopherol on both peroxyl and alkoxyl radicals. On one hand, α-tocopherol, competing with the lipid substrate, might donate its hydrogen atom to the alkoxyl radical, forming a more stable tocopheroxyl radical, which is then also able to trap the alkoxyl radical [54]. On the other hand, α-tocopherol could act on peroxyl radicals by intercepting them and reducing the decomposition of peroxides and, consequently, the formation of hexanal [53].

The conjugated fatty acids were examined through GC–FID and not by measuring the absorbance because, as reported by Tura et al. [16], the cannabinoids can interfere, leading to an overestimation of data. However, the sum of conjugated linoleic acids (CLAs) and conjugated linolenic acids (CLnAs) did not play an important role in the study, as their concentration was scarce and remained unchanged over time in all samples.

The consistency of all oxidation parameters in HO during storage could be due to the ability of hemp bioactive compounds to withdraw the metal ions and free radicals and, therefore, to prevent and delay, respectively, the free-radical chain reaction on fatty acids. These two abilities are governed predominantly by the phenolic compounds thanks to their molecular structure, which is the factor that most influences the RSA and the metal-chelating capacity [49]. The presence of a phenolic pattern and alkyl and hydroxyl groups on the phenol ring, together with a low bond dissociation energy of the latter (as occurs in apigenin, quercetin, luteolin, catechin, epicatechin, and cannflavin A and B), makes an antioxidant more efficient [49]. In addition, the efficacy of chelating metal ions is enhanced if certain functional moieties (i.e., two hydroxy groups, one hydroxy and one carboxyl group, or one hydroxy and one carbonyl group) are positioned on the carbon atoms adjacent to the phenol ring [49]. Furthermore, antioxidant activity was also demonstrated in terpenes, depending again on their molecular structure. In fact, Wojtunik et al. [55] demonstrated that DPPH^∙ inhibition increased in the presence of π-conjugated bonds, which enabled formation of resonance-stabilized structures when reacting with the radical, delocalizing the charge over the whole molecule. None of the terpenes identified in this study presented conjugated double bonds; however, for some of them, such as linalool, limonene, terpinolene, and β-caryophyllene, a strong antioxidant activity was reported by other authors [56,57,58]. In addition, since the CBD did not degrade during the experiment in bulk oils and, as reported by Tura et al. [16], CBD does not protect fatty acids from oxidation, we can exclude the possibility that CBD is directly involved in the aforementioned antioxidant mechanisms. However, there is a possibility that CBD boosted the antioxidant activity of terpenes and/or polyphenols through a synergistic effect, since an enhancement of the antioxidant activity in hemp extracts was reported when cannabinoids were accompanied by a high amount of polyphenols [59].