In this situation, one of the most important processes is lipid peroxidation, which results in the oxidation of polyunsaturated fatty acids (PUFA), such as arachidonic, linoleic, linolenic, eicosapentaenoic, and docosahexaenoic acids . As a result of the ROS reaction with PUFAs, lipid hydroperoxides are formed, and as a result of oxidative fragmentation, unsaturated aldehydes are generated, including 4-hydroxynenenal (4-HNE), malonodialdehyde (MDA) or acrolein . In addition, the propagation of oxidation chain reactions, especially with regard to docosahexaenoic acid, can lead to oxidative cyclization, resulting in production of isoprostanes or neuroprostanes . The formation of lipid peroxidation products directly affects the physical properties and functioning of the cell membranes in which they are formed . Due to their structure (the presence of a carbonyl groups and carbon-carbon double bonds) and electrophilic character, generated unsaturated aldehydes are chemically reactive molecules that can easily form adducts with the majority of the cell’s nucleophilic components, including DNA, lipids, proteins, and GSH . For example, 4-hydroxynonenal (4-HNE) has been identified as a stimulator of the cytoprotective transcription factor Nrf2, an inhibitor of antioxidant enzymes (e.g., catalase and thioredoxin reductase) and a pro-inflammatory factor acting through the NFκB pathway . These reactions reduce the level of reactive lipid peroxidation products, while increasing the formation of adducts with proteins that promote cell signaling disorders, thus stimulating metabolic modifications that can lead to cellular dysfunction and apoptosis [47,48].
CBD is also an agonist of adenosine A2A receptors , which are G-protein coupled receptors. They are expressed in various cell types, participate in numerous physiological and pathological processes and also regulate inflammatory processes . Adenosine and its agonists exhibit anti-inflammatory activity in vivo . Therefore, adenosine release is one of the mechanisms of immunosuppression during inflammation , and adenosine receptor agonists reduce TNF-α levels [108,109]. It has been shown that CBD by activating A2A adenosine receptors can reduce the level of vascular cell adhesion molecule (VCAM-1) in endothelial cells in SJL/J mice, which may provide a new mechanism to control neuroinflammatory diseases such as multiple sclerosis (MS) .
3.4. Cannabinoid Receptors
In addition, the pinene dimethoxy-dimethylheptyl-CBD derivative HU-308 [(3R, 4S, 6S)-2-[2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl]-7,7-dimethyl-4-bicyclo[3.1.1]hept-3-enyl]methanol] and its enantiomer HU-433 [(3S, 4R, 6R)-2-[2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl]-7,7-dimethyl-4-bicyclo[3.1.1]hept-3-enyl]methanol] were shown to have specific agonistic activity for the CB2 receptor ( Table 2 ), and consequently, anti-inflammatory activity in cultured calvarial osteoblasts from C57BL/6J mice . However, it has been found that HU-433 exhibits greater anti-inflammatory activity with poorer CB2 receptor binding affinity ( Table 2 ) . In contrast, HU-308, a CB2 agonist, was found to decrease TNF-α-induced expression of ICAM-1 and VCAM-1 in sinusoidal endothelial cells of human liver tissue . Another CB2 receptor agonist, HU-910 ((1S,4R)-2-[2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl]-7,7-dimethyl-1-bicyclo[2.2.1]hept-2enyl]methanol)), significantly inhibits the effects of LPS that lead to increased inflammation (assessed by increased TNF-α expression) and increased oxidative stress (assessed by increased levels of 4-HNE and protein carbonyl groups) in mouse Kupffer cells . This suggests that these effects are associated with CB2 receptor activation ( Table 2 ).
CBDV has been found to have antagonistic effects on GPR55 ( Table 2 ), which probably leads to anticonvulsant effects . Therefore, this compound is suggested for use when therapeutic antiepileptic activities are needed. On the other hand, CBDA has been suggested to be an effective compound in analgesia and cancer through its agonistic action on TRPA1 and TRPV1 receptors ( Table 2 ) and antagonistic action on TRPM8, similar to CBD [76,116].
4.1. CB1/CB2 Receptors
CBD also reduces reactive oxygen species (ROS) production by chelating transition metal ions involved in the Fenton reaction to form extremely reactive hydroxyl radicals . It was shown that CBD, acting similarly to the classic antioxidant butylated hydroxytoluene (BHT), prevents dihydrorodamine oxidation in the Fenton reaction . In addition, CBD has been found to decrease β-amyloid formation in neurons by reducing the concentration of transition metal ions .
Table 1. Results for the determination of peroxide number, free acidity and resistance to forced oxidation (OSI) of oils with added CBD.
The value of peroxide number increased as both CBD and α-tocopherol concentrations increased, while the most relevant radical scavenger activity was with CBD. This apparent contradiction, i.e. the lack of correlation between these two assays, has also been reported in previous studies [17,18].
The addition of α-tocopherol, on the other hand, increased the forced oxidation time (OSI) in sunflower oil samples. On the contrary, a slight reduction of this time was detected with the addition of α-tocopherol at 0.01% and 0.1% to refined olive oil. In accordance with what has been found in the literature, the activity of α-tocopherol can be antioxidant and, under certain conditions, pro-oxidant ; sometimes, moreover, a positive correlation between the analysis of the induction time and the total tocopherol content has not been found [13,14].
Materials and methods
In addition, it was possible to compare two oils characterized by a different fatty acid profile: olive oil is mainly characterized by oleic acid while sunflower oil that was used had linoleic acid as the predominant component, therefore more unsaturated. In order to be able to make comparisons, all the analyses were also carried out on the oil samples used as a matrix.
Free radicals, which are formed by lipid oxidation, have a negative action both in food and in biological systems . They are responsible for or involved in degenerative processes, first of all cellular aging . For this reason they are introduced with the diet of antioxidants that play an important role in the containment of free radicals .
Results and discussion of the Study
The antioxidant capacity of CBD (purity 99.8 %, in the form of crystals supplied by Enecta) and α-tocopherol (purity 97 %) was evaluated in oily solutions prepared using refined olive oil according to the European Pharmacopoeia (Ph Eur) and refined sunflower oil at three different concentrations: 0.01 %, 0.1 % and 0.5 % (Figure 2).