3.2.2. Activity: Preclinical Studies

Curcumin inhibits proinflammatory cytokines, TNF–α, ICAM–1, NOX2 and cyclo– oxygenase–2 expressions, and reduces the leucocyte-endothelium interaction in diabetesinduced rat vascular inflammatory models [100,101]. Curcumin has been widely evaluated with respect to the modulation of endothelial function [102]. Curcumin also presents a neuroprotective potential in the pathogenesis of Alzheimer's disease [103–105]. The many described biological activities of curcumin have been linked to its chemical instability that leads to the rapid autoxidation responsible for its antioxidant behavior. The oxidation of curcumin produces intermediate metabolites that conduct to a final bicyclopentadione [106,107]. Moreover, it has been demonstrated that electrophilic metabolites can covalently adduct to cellular protein targets, in particular those of the NF–κB pathway, which explains the anti-inflammatory activity of the natural compound. The methoxy groups of the molecule have also been shown to possess a role in its anti-inflammatory biological activity [108].

Ischemia and reperfusion injury in skeletal muscles, obtained by clamping the femoral artery and vein, is an experimental model that can provide information on the damage occurring in ischemic events of the limbs. Rats subjected to 4 h femoral occlusion followed by 2 h reperfusion, which received curcumin (100 mg/kg i.p., 1 h before reperfusion), presented a preservation of superoxide dismutase and catalase activities, as well as of the glutathione content in muscle tissues, compared to injured but not treated animals [109]. In the same context, the muscle and plasma levels of malondialdehyde and protein oxidation were reduced, as well as the plasma levels of inflammatory cytokines (TNF–α and IL–1β). The data suggest that curcumin has protective activity against the ischemic damage of skeletal muscles [109].

Using a mouse model of hindlimb ischemia obtained by the ligation of the left femoral artery, Liu et al. observed that curcumin (100 mg/kg, i.p. administration 1 h before the ligation surgery) is capable of improving the running capacity on a treadmill of injured mice compared to the controls, evaluated 1 and 2 weeks following injury [110]. A histological examination of skeletal-muscle sections from the curcumin-treated group showed less muscle degeneration and fibrosis than in the control mice; also, curcumin treatment was able to significantly reduce macrophage infiltration and ischemia- induced inflammation. The regulation of the tissue inflammatory response has been associated with a decrease in the proinflammatory cytokines TNF–α, IL–1β and IL–6 as a result of the diminished expression of NF–κB, namely, of the subunit p65 [110]. These data suggest that curcumin could be a promising agent used against diseases of the ischemic limbs. Another in vivo experiment conducted on streptozotocin-induced diabetic mouse that received hindlimb ischemic surgery demonstrated that curcumin (1000 mg/kg in olive oil, applied by gavage for 14 days) can promote the significant recovery of blood flow detected by laser Doppler imaging, 7 and 14 days after ischemic surgery; histology evidenced neogenic capillary formation, as a consequence of improved angiogenesis and the proliferation of endothelial progenitor cells [111]. Zhang et al. studied the role of curcumin in an experimental PAD model of murine hindlimb ischemia obtained with surgical ligation and the excision of the femoral artery. Mice received 1000 mg/kg of curcumin (in 300 μL of olive oil, gavage) once a day for two weeks [112]. In animals treated with curcumin, the laser Doppler perfusion imaging system showed perfusion recovery 14, 21 and 28 days after ischemia induction, significantly better than in the control. Histology of the ischemic gastrocnemius muscle after 28 days revealed a significant increase in capillary density. Furthermore, 7 days after surgery, a 5-fold increase in the expression of microRNA (miR)–93 (a mediator of neovascularization in PAD) was detected in the muscle tissue, suggesting the role of curcumin in angiogenesis [112].

Another possible favorable mechanism of action of curcumin in the damage of large arteries could be the inhibition of hypoxia-inducible factor 1α (HIF–1α), implicated in the progression of atherosclerosis. Ouyang et al. demonstrated that in a culture of human macrophages, the protein level of HIF–1α, induced by hypoxia, is reduced in a concentration-dependent way by treatment with curcumin (20–40 μM), which acts in a proteasome-dependent manner without affecting the mRNA level of the factor [113]. Moreover, it has been shown that curcumin causes the inhibition of the expression of HIF–1α through ERK signaling pathway. Following HIF–1α inhibition, curcumin also affects macrophage apoptosis induced by hypoxia; also, it significantly reduces the hypoxiainduced release of proinflammatory cytokines IL–6 and TNF–α from macrophages [113].

Curcumin has been evaluated under the aspect of AGEs inhibition, a process that is strictly related to arterial senescence and degeneration. A review has recently taken into account this issue [114]. Curcumin has been shown to reduce in vitro the generation of AGEs, as well as to counteract in vivo AGE formation. The possible mechanisms involved include methylglyoxal trapping, but also the capacity to prevent AGE formation, due to the antioxidant activity and clearance of free radicals. Curcumin can also act on the expression of AGE receptors by suppressing RAGE (a receptor that mediates the detrimental effects of AGEs) and enhancing AGE–R1 (a distinct receptor that mediates the detoxification of AGEs) [115]. It should be mentioned that curcumin may act as favorable modulator of PAD also influencing several pathways linked to DM, the disease whose complications often evolve toward vascular manifestations. A recent overview has considered by bioinformatic methodology the many targets that can be affected by curcumin due to its antioxidant and anti-inflammatory properties, which can modulate the disease's progression [116].

#### 3.2.3. Activity: Clinical Studies

From a strict, clinical point of view, the data on the effectiveness of curcumin in PAD are limited. A meta-analysis of 5 randomized clinical trials conducted on a total of 192 healthy subjects with doses of curcumin ranging from 25 to 200 mg, showed that curcumin supplementation is able to improve vascular function, determined as the flow-mediated dilation of the brachial artery [117]. Alidadi et al. reported 7 randomized clinical trials conducted on humans, ranging from healthy subjects to obese people and type 2 diabetic patients, with a sample size ranging from 21 to 88 subjects [118]. Curcumin doses ranged from 150 mg/day to 2 g/day, over periods of 8–16 weeks. In healthy subjects, curcumin appeared to improve endothelial function, determined by a non-invasive ultrasoundbased technique that measured flow-mediated dilation. On the contrary, in pathological conditions, curcumin treatment did not show an appreciable improvement in endothelial function, also due to the limited number of patients evaluated [118].

Despite its interesting biological properties, curcumin has the limitation of being poorly absorbed by the gastrointestinal tract due to its high hydrophobicity [119]. Several formulations were investigated to enhance curcumin bioavailability [120,121], in particular micro- and nano-formulations. An optimized curcumin formulation was developed and investigated [122]. It consisted of poly (propylene sulfide) (PPS) microparticles used for curcumin encapsulation and the delivery of the active principle on demand at the site of oxidative stress. An oil-in-water emulsion was obtained, which permitted, in the presence of ROS, the transition of hydrophobic PPS into more hydrophilic sulfoxide and sulfone, thus releasing curcumin. Following an in vitro evaluation of the system, the authors tested the formulation in vivo (curcumin-loaded PPS microspheres: 5 mg/kg of curcumin with 10.3 mg/kg of PPS, administered i.m. in the gastrocnemius and adductor muscles) in a streptozotocin-induced mouse model of DM in which hindlimb ischemia was induced [122]. The results show that curcumin microspheres were able to selectively reduce ROS levels in the tissue of the ischemic limbs, significantly increasing blood perfusion and the length of the vasculature, providing evidence for a favorable action of curcumin in the treatment of diabetic PAD. Another more recent attempt to produce "on demand" treatment for PAD has been reported [123]. It consists of PVAX (vanillyl alcohol-incorporated copolyoxalate) nanoparticles loaded with curcumin that release the drug at a pathologically elevated level of H2O2, as occurs at an ischemic site. After optimizing in vitro the formulation of curcumin–PVAX nanoparticles and demonstrating their antioxidant, anti-inflammatory and angiogenic activities, the authors evaluated the activity of the product in a mouse model of unilateral hindlimb ischemia. The in vivo results, obtained with the i.m. injection of curcumin–PVAX nanoparticles into the ischemic area on days 0, 3 and 7, showed significant blood-flow recovery and neovascularization, with almost complete blood-flow restoration 12 days after ischemia. The histological examination demonstrated the suppression of tissue damage and inflammatory responses, with an extensive expression of angiogenic factor CD31 [123]. Several other formulations of curcumin, including nano-formulations or encapsulation, have appeared on the market to ensure the optimal intake of the active compound [124,125].

#### 3.2.4. Safety Profile

Regarding curcumin toxicity, the compound is considered safe and has a good tolerability profile in humans [126–128]. The reported side effects mainly include gastrointestinal disturbances. However, in Italy, several cases of acute hepatitis have been reported to be associated with formulations that provide high bioavailability and high doses of curcumin [129]. In fact, the availability of many different formulations of curcumin with an enhanced bioavailability profile has focused on the question of safety. For instance, Fuloria et al. considered the curcumin toxicity profile that emerged during the most recent preclinical and clinical studies; excessive amounts of curcumin may lead to alterations in testosterone levels in men, influence blood clotting and contrast iron absorption [125].

In VigiBase curcumin has 71 potential side effects [95], such as gastrointestinal disorders (i.e., diarrhea and nausea), nervous system disorders (e.g., asthenia and others) and hepatobiliary disorders (e.g., acute liver failure, increase in alanine aminotransferase and aspartate aminotransferase, and others). More trials are needed to understand the safety of curcumin for pharmacological use, also reducing the number of additives, and considering customized microencapsulation [125].
