**1. Introduction**

The endocannabinoid system is an important molecular system responsible for controlling homeostasis and is becoming an increasingly popular target of pharmacotherapy. Endocannabinoids are ester, ether, and amide derivatives of long chain polyunsaturated fatty acids (PUFAs), such as arachidonic acid, and they act mainly as cannabinoid receptor ligands [1]. Endocannabinoids belong to a large group of compounds with a similar structure and biological activity called cannabinoids. Cannabinoids are chemical derivatives of dibenzopyrene or monoterpenoid, and to date over four hundred have been identified. The most important of these are Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-tetrahydrocannabinol (Δ8-THC), cannabinol (CBN), and cannabidiol (CBD), and they are members of a large group of biologically active compounds found in *Cannabis sativa* L. [2]. The medical use of cannabinoids, in particular phytocannabinoids, has been one of the most interesting approaches to pharmacotherapy in recent years.

CBD is one of the main pharmacologically active phytocannabinoids [3]. It is non-psychoactive, but has many beneficial pharmacological effects, including anti-inflammatory and antioxidant effects [4]. In addition, it belongs to a group of compounds with anxiolytic, antidepressant, antipsychotic, and anticonvulsant properties, among others [5]. The biological effects of cannabidiol, including the various molecular targets, such as cannabinoid receptors and other components of the endocannabinoid system, with which it interacts, have been extensively studied. The therapeutic potential of CBD has been evaluated in cardiovascular, neurodegenerative, cancer, and metabolic diseases, which are usually accompanied by oxidative stress and inflammation [6]. One of the best studied uses of CBD is for therapeutic effect in diabetes and its complications in animal and human studies [7]. CBD, by activating the cannabinoid receptor, CB2, has been shown to induce vasodilatation in type 2 diabetic rats [8,9], and by activating 5-HT1A receptors, CBD showed a therapeutic effect in diabetic neuropathy [10]. Moreover, this phytocannabinoid accelerated wound healing in a diabetic rat model by protecting the endothelial growth factor (VEGF) [11]. In addition, by preventing the formation of oxidative stress in the retina neurons of diabetic animals, CBD counteracted tyrosine nitration, which can lead to glutamate accumulation and neuronal cell death [12].

This review summarizes the chemical and biological e ffects of CBD and its natural and synthetic derivatives. Particular attention was paid to the antioxidant and anti-inflammatory e ffects of CBD and its derivatives, bearing in mind the possibilities of using this phytocannabinoid to protect against oxidative stress and the consequences associated with oxidative modifications of proteins and lipids. Although CBD demonstrates safety and a good side e ffect profile in many clinical trials [4], all of the therapeutic options for CBD discussed in this review are limited in a concentration-dependent manner.

#### **2. Molecular Structure of CBD**

CBD is a terpenophenol compound containing twenty-one carbon atoms, with the formula C21 H30 O2 and a molecular weight of 314.464 g/mol (Figure 1). The chemical structure of cannabidiol, 2-[1R-3-methyl-6R-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol, was determined in 1963 [13]. The current IUPAC preferred terminology is 2-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol. Naturally occurring CBD has a (−)-CBD structure [14]. The CBD molecule contains a cyclohexene ring (A), a phenolic ring (B) and a pentyl side chain. In addition, the terpenic ring (A) and the aromatic ring (B) are located in planes that are almost perpendicular to each other [15]. There are four known CBD side chain homologs, which are methyl, *n*-propyl, *n*-butyl, and *n*-pentyl [16]. All known CBD forms (Table 1) have absolute *trans* configuration in positions 1R and 6R [16].

**Figure 1.** Chemical structure of cannabidiol (CBD) [16].

The CBD chemical activity is mainly due to the location and surroundings of the hydroxyl groups in the phenolic ring at the C-1- and C-5- positions (B), as well as the methyl group at the C-1 position of the cyclohexene ring (A) and the pentyl chain at the C-3- of the phenolic ring (B). However, the open CBD ring in the C-4 position is inactive. Due to the hydroxyl groups (C-1- and C-5- in the B ring), CBD can also bind to amino acids such as threonine, tyrosine, glutamic acid, or glutamine by means of a hydrogen bond [17].



CBD has potential antioxidant properties because its free cationic radicals exhibit several resonance structures in which unpaired electrons are distributed mainly on ether and alkyl moieties, as well as on the benzene ring [18].

#### **3. Biological Activity of CBD**

CBD has a wide spectrum of biological activity, including antioxidant and anti-inflammatory activity, which is why its activity in the prevention and treatment of diseases whose development is associated with redox imbalance and inflammation has been tested [4,19,20]. Based on the current research results, the possibility of using CBD for the treatment of diabetes, diabetes-related cardiomyopathy, cardiovascular diseases (including stroke, arrhythmia, atherosclerosis, and hypertension), cancer, arthritis, anxiety, psychosis, epilepsy, neurodegenerative disease (i.e., Alzheimer's) and skin disease is being considered [20–22]. Analysis of CBD antioxidant activity showed that it can regulate the state of redox directly by a ffecting the components of the redox system and indirectly by interacting with other molecular targets associated with redox system components.

#### *3.1. Direct Antioxidant E*ff*ects of CBD*

CBD has been shown to a ffect redox balance by modifying the level and activity of both oxidants and antioxidants (Figures 2 and 3). CBD, like other antioxidants, interrupts free radical chain reactions, capturing free radicals or transforming them into less active forms. The free radicals produced in these reactions are characterized by many resonance structures in which unpaired electrons are mainly found on the phenolic structure, suggesting that the hydroxyl groups of the phenol ring are mainly responsible for CBD antioxidant activity [18].

CBD reduces oxidative conditions by preventing the formation of superoxide radicals, which are mainly generated by xanthine oxidase (XO) and NADPH oxidase (NOX1 and NOX4). This activity was shown in the renal nephropathy model using cisplatin-treated mice (C57BL/6J) [23] and in human coronary endothelial cells (HCAEC) [24]. In addition, CBD promoted a reduction in NO levels in the liver of doxorubicin-treated mice [25] and in the paw tissue of Wistar rats in a chronic inflammation model [26].

**Figure 2.** Direct antioxidant e ffects of CBD (closed arrows indicate reducing e ffects; opened arrows indicate inducing action).

CBD also reduces reactive oxygen species (ROS) production by chelating transition metal ions involved in the Fenton reaction to form extremely reactive hydroxyl radicals [27]. It was shown that CBD, acting similarly to the classic antioxidant butylated hydroxytoluene (BHT), prevents dihydrorodamine

oxidation in the Fenton reaction [28]. In addition, CBD has been found to decrease β-amyloid formation in neurons by reducing the concentration of transition metal ions [29].

In addition to the direct reduction of oxidant levels, CBD also modifies the redox balance by changing the level and activity of antioxidants [19,26]. CBD antioxidant activity begins at the level of protein transcription by activating the redox-sensitive transcription factor referred to as the nuclear erythroid 2-related factor (Nrf2) [30], which is responsible for the transcription of cytoprotective genes, including antioxidant genes [31]. CBD was found to increase the mRNA level of superoxide dismutase (SOD) and the enzymatic activity of Cu, Zn- and Mn-SOD, which are responsible for the metabolism of superoxide radicals in the mouse model of diabetic cardiomyopathy type I and in human cardiomyocytes treated with 3-nitropropionic acid or streptozotocin [32]. Repeated doses of CBD in inflammatory conditions were found to increase the activity of glutathione peroxidase and reductase, resulting in a decrease in malonaldehyde (MDA) levels, which were six times higher in untreated controls [26]. Glutathione peroxidase activity (GSHPx) and glutathione level (GSH) were similarly changed after using CBD to treat UVB irradiated human keratinocytes. The high affinity of CBD for the cysteine and selenocysteine residues of these proteins is a possible explanation for this observation [33]. It is known that under oxidative conditions, alterations in enzymatic activity may be caused by oxidative modifications of proteins, mainly aromatic and sulfur amino acids [34]. It has also been suggested that the reactive CBD metabolite cannabidiol hydroxyquinone reacts covalently with cysteine, forming adducts with, for example, glutathione and cytochrome P450 3A11, and thereby inhibiting their biological activity [35]. In addition, CBD has been found to inhibit tryptophan degradation by reducing indoleamine-2,3-dioxygenase activity [36]. CBD also supports the action of antioxidant enzymes by preventing a reduction in the levels of microelements (e.g., Zn or Sn), which are usually lowered in pathological conditions. These elements are necessary for the biological activity of some proteins, especially enzymes such as superoxide dismutase or glutathione peroxidase [25].

By lowering ROS levels, CBD also protects non-enzymatic antioxidants, preventing their oxidation, as in the case of GSH in the myocardial tissue of C57BL/6J mice with diabetic cardiomyopathy [32] and doxorubicin-treated rats [25]. An increase in GSH levels after CBD treatment was also observed in mouse microglia cells [37] and in the liver of cadmium poisoned mice [25]. This is of grea<sup>t</sup> practical importance because GSH cooperates with other low molecular weight compounds in antioxidant action, mainly with vitamins such as A, E, and C [38]. CBD exhibits much more antioxidant activity (30–50%) than α-tocopherol or vitamin C [4].

#### *3.2. The Consequences of Direct Antioxidant Action of CBD*

The result of an imbalance between oxidants and antioxidants is oxidative stress, the consequences of which are oxidative modifications of lipids, nucleic acids, and proteins. This results in changes in the structure of the above molecules and, as a result, disrupts their molecular interactions and signal transduction pathways [39]. Oxidative modifications play an important role in the functioning of redox-sensitive transcription factors (including Nrf2 and the nuclear factor kappa B (NFκB). As a consequence, oxidative modifications play a role in the regulation of pathological conditions characterized by redox imbalances and inflammation, such as cancer, inflammatory diseases, and neurodegenerative diseases [40,41].

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 [42]. 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 [43]. 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 [44]. The formation of lipid peroxidation products directly affects the physical properties and functioning of the cell membranes in which they are formed [42]. 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 [45]. 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 [46]. 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].

In addition to lipid peroxidation, oxidative conditions also favor the oxidative modification of proteins by ROS. The aromatic and sulfhydryl amino acid residues are particularly susceptible to modifications, and can result in production of levodopa (l-DOPA) from tyrosine, ortho-tyrosine from phenylalanine, sulfoxides and disulfides from cysteine, and kynurenine from tryptophan, among others [49]. The resulting changes in the protein structures cause disruption of their biological properties and, as in the case of lipid modification, affect cell metabolism, including signal transduction [46,50].

One of the most noticeable CBD antioxidant effects is the reduction in lipid and protein modifications [25,51]. CBD supplementation has been found to reduce lipid peroxidation, as measured by MDA levels, in mouse hippocampal (HT22) neuronal cells depleted of oxygen and glucose under reperfusion conditions [51]. A reduction in lipid peroxidation following CBD supplementation has also been shown in C57BL/6J mouse liver homogenates, assessed by 4-HNE levels [52]. CBD also protected the brain against oxidative protein damage caused by D-amphetamine in a rat model of mania [53]. On the other hand, CBD induced ubiquitination of the amyloid precursor protein (APP), an indicator of cellular changes in the brain of people with Alzheimer's disease, when evaluated in human neuroblastoma cells (SHSY5YAPP+) [54]. In addition, CBD treatment has recently been shown to exhibit an unusual protective effect by transporting proteins including multidrug-1 resistance protein and cytosol transferases, such as S-glutathione-M1 transferase, prior to modification by lipid peroxidation products. This prevents elevation of 4-HNE and MDA adduct levels in fibroblast cell culture [55]. It was also shown that this phytocannabinoid reduced the level of small molecular αβ-unsaturated aldehydes in the myocardial tissue of Sprague-Dawley rats and mice with diabetic cardiomyopathy, and in the liver of mice from the acute alcohol intoxication model [21,25,32]. Additionally, CBD caused a reduction in the level of PUFA cyclization products, such as isoprostanes, in the cortex of transgenic mice (APPswe/PS1ΔE9) with Alzheimer's disease [56]. Thus, CBD protects lipids and proteins against oxidative damage by modulating the level of oxidative stress, which participates in cell signaling pathways.

#### *3.3. Indirect Antioxidant E*ff*ects of CBD*

Various cell metabolic systems, including the endocannabinoid system, are involved in the regulation of redox balance. Thus, the action of CBD as a phytocannabinoid may support the biological activity of the endocannabinoid system. CBD has recently been shown to modulate the endocannabinoid system activity by increasing anandamide (AEA) levels [5], which can affect cannabinoids signaling, including their interaction on cannabinoid receptors [57]. However, it is known that the peroxisome proliferator-activated receptor alpha (PPAR-α), for example, activated by endocannabinoids, directly regulates the expression of antioxidant enzymes such as superoxide dismutase by interacting with their promoter regions [58]. Therefore, it is believed that the most important antioxidant activity of CBD, like endocannabinoids, is associated with its effect on receptors. CBD, depending on the concentration, can activate, antagonize or inhibit cannabinoid receptors (CB1 and CB2), as well as ionotropic (TRP) and nuclear (PPAR) receptors (Figure 4) [52,59,60].
