**4. The Antioxidant Capacity of Inositols**

In addition to the modulation of insulin signaling, inositols are polyols that might act as modulators of oxidative metabolism, helping to decrease the burden of oxidative stress. Oxidative stress is the most common factor responsible for metabolic disturbances caused by insulin resistance. Under normal reduction-oxidation conditions, physiological metabolism, especially via aerobic processes, produces a series of sub-products called reactive oxygen species (ROS) that include superoxides (O2 −), hydrogen peroxides (H2O2), and hydroxyl radicals (OH−) as part of the oxidation of metabolites. Normally, antioxidants present in organisms can compensate the generation of ROS, as they accept electrons of negatively charged oxygen molecules, deriving them into H2O.

A common feature of insulin resistance is the elevated production of cytokines such as TNF-α and interleukin-6. A pro-inflammatory state contributes, along with hyperglycemia and decreased insulin signaling, to a deregulated metabolism and excessive generation of ROS. Oxidative stress in adipose tissue, along with an exacerbated release of cytokines, also promotes a pro-inflammatory state that contributes to the development of insulin resistance, diabetes, and concomitantly increases the risk of obesity-associated metabolic syndrome. In presence of nitric oxide (NO), a quick cross-reaction with O2 − produces cytotoxic peroxynitrite (ONOO−) as part of the reactive nitrogen species (RNS). Both ROS and RNS attack biological components of cells, including DNA, RNA, protein, or lipid peroxidation, causing severe damage to plasma and organelle membranes [120]. Oxidative/nitrosative stress during insulin resistance causes endothelial dysfunction and vascular complications or atherosclerosis. Endothelial cell production of NO is promoted by insulin via PI3K/Akt signaling, which leads to the activation of endothelial nitric oxide synthase (eNOS) [121,122]. However, decreased insulin signaling, along with elevated NADPH oxidase activity and increased generation of O2 −, leads to a low bioavailability and bioactivity of NO [123].

In phenolic compounds, hydroxyl groups can transfer their hydrogen to negatively charged free radicals (R−) in order to be stabilized as neutrally charged radicals (RH). As inositols are polyalcohol molecules, it has long been presumed that they possess antioxidant potential due to the presence of hydroxyl groups. The first approaches to inositol derivative molecules focused on the antioxidant potential of the inositol phosphorylated derivative phytic acid (IP6) as an iron (Fe<sup>3</sup>+) chelator. In normal conditions, free radicals are generated in the Fenton reaction by the oxidation of Fe2<sup>+</sup> as follows: Fe2<sup>+</sup> + H2O2 <sup>→</sup> Fe3<sup>+</sup> + HO• + OH−. However, the sequestering of Fe3<sup>+</sup> by IP6 leads to a rapid depletion of Fe2<sup>+</sup>, which is oxidized by molecular oxygen (O2), but not by H2O2, hence blocking free radical formation [124,125]. Antioxidant activity of IP6 is seen to be especially relevant for the xanthine/xanthine oxidase system, which generates H2O2 by consecutive hypoxanthine to xanthine and xanthine to uric acid oxidation reactions [126].

The beneficial effects of free inositols in oxidative stress have also been related to radical scavenging properties. An in vitro study showed that the addition of DPIN exhibited dose-dependent inhibition of superoxide and nitric oxide formation [127]. In endothelial cell cultures under high glucose conditions, the addition of DPIN, DCI, and synthetic 3,4-dibutyryl-DCI (db-DCI) has been shown to dose-dependently scavenge superoxide in an xanthine/xanthine oxidase system [128]. It was shown that db-DCI was most effective at reducing ROS levels and exhibited an Fe3<sup>+</sup>-related mechanism of action, suggesting that db-DCI acts similarly to IP6, although the detailed mechanism of this has not been determined yet [128]. Interestingly, quebrachitol has also been described as an active component displaying a ONOO− scavenging activity [36].

The efficacy of inositols as antioxidants may also be attributed to an enhanced activity of antioxidant enzymes. Recent studies on Jian carp have shown that MI supplementation increased the activities of catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) in copper (Cu)-induced toxicity, but also superoxide dismutase (SOD) and glutathione-S-transferase (GST), both in normal and Cu-induced damage conditions [129,130]. DPIN has also showed an enhancement of endogenous antioxidant activity. DPIN at a dose of 200 mg/kg inhibits oxidative stress caused by 7,12-dimethylbenz(a)anthracene (DMBA) in rats, along with an increased activity of the antioxidant enzymes SOD, CAT, GPx, and GST [127]. In a mouse model of cisplatin-induced oxidative stress, the administration of DPIN increased GSH, SOD, and CAT activities [131]. The administration of an DCI-enriched extract to streptozotocin-induced diabetic mice significantly increased glutathione (GSH) and decreased malondialdehyde (MDA) in the liver, accompanied by decreased pro-inflammatory TNF-α and increased anti-inflammatory IL-6 and interferon gamma (IFN-γ) in the sera [132].

As described previously, endothelial dysfunction is a common pathology derived from insulin resistance. Regarding this, use of inositols yields a synergistic effect for both antioxidant and insulin-sensitizing activities. The administration of MI and DCI in HUVEC cells promotes Akt phosphorylation [106]. The in vitro addition of DPIN, DCI, and db-DCI impaired contraction by the eNOS inhibitor L-NAME and increased NO effectiveness [128]. It has also been shown that db-DCI decreases reduced PKC activation, hexosamine pathway activity, and advanced glycation end products to basal levels in high glucose conditions [128].

Given the increasing problems arising from an unhealthy diet and living conditions, dietary use of inositols should be considered because of their antioxidant and insulin-sensitizing properties (Figure 3). It is still necessary to determine the exact mechanism of free radical scavenging in inositols. Structure differences are likely to contribute to the net antioxidant capacity, as differences have been observed between the sugar alcohols MI and DCI and methyl derivatives DPIN or db-DCI. There are no available research data on the antioxidant activity of IPGs, since PI-derivative structures like IP6 could be active compounds of inositol activity.

**Figure 3.** Proposed antioxidant mechanisms of inositol derivatives. Phytic acid (IP6) chelates and sequesters Fe3<sup>+</sup>, redirecting Fe2<sup>+</sup> via the Fenton reaction to oxidation with O2. Four molecules of Fe2<sup>+</sup> are necessary to oxidize one molecule of O2, which generates O2 <sup>−</sup>, which is later converted in H2O2, and finally inactive H2O. Reactive oxygen species (ROS) are scavenged by the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Inositols upregulate antioxidant enzyme levels and activity. The activation of insulin signaling in the endothelium results in increased NO production. l-quebrachitol and 3,4-dibutyryl-d-chiro-inositol scavenge peroxynitrite (ONOO−) and ROS species, respectively, although the mechanisms of this have not been unveiled yet.
