**4. Discussion**

Most of the studies involving natural products are performed with the original compounds found in the plant or food matrix, and very few are carried out with the metabolites obtained after the biotransformation in the body. Since ellagitannins are poorly absorbed in the gastrointestinal tract, urolithins have been proposed as the bioactive metabolites responsible for the beneficial effects of pomegranates and other plants containing ellagitannins. Pomegranate juice and extracts have exhibited neuroprotective properties, so here we investigate whether urolithin A, one of the major intestinal metabolites of ellagic acid, might act as a neurotherapeutic and antioxidant agent.

Neuro-2a is a neuroblastoma cell line derived from mice, which has been used because of its ability to produce microtubular proteins. Few works have been done with urolithins using this cell line, but it has been demonstrated that urolithin A protects against ischemic neuronal injury by activating autophagy [29]. This research supports our results on mitochondrial activity, demonstrating that urolithin A is not cytotoxic at this range of physiological concentrations (0.5–50 μM).

The antioxidant activity of urolithin A has already been established in different cell lines using in vitro procedures [30–32]. However, we here investigate whether urolithin can be considered as a neuroprotective agent due to its role as a direct free radical scavenger, as an indirect antioxidant improving the physiological antioxidant defense system of the cells, and as an inhibitor of oxidases such as monoamine oxidase A and tyrosinase.

Frequently, when the ROS and MDA levels decrease, the activity of the cytosolic enzymes (superoxide dismutase, catalase) increases; previous experiments in HepG2 cells treated with urolithin A and H2O2 have demonstrated that cells significantly increased SOD activity compared with cells subjected to oxidative stress with H2O2 [30]; in fact, SOD activity was ameliorated by urolithin in kidney cells from mice [33]. Computational studies performed with different pomegranate juice constituents revealed that they can act as pro-oxidants or antioxidants. Molecular docking studies seem to be controversial, as they have determined that urolithin A may inhibit cytosolic enzymes such as catalase, superoxide dismutase, and glutathione [34]; however, urolithin A seems to improve the activity of these enzymes in our present study. With the aim of elucidating molecular mechanisms involved in the cytoprotective and antioxidant properties of urolithin A, peroxiredoxins expression (Prx) was quantified in this work. These peroxiredoxins are antioxidant enzymes that catalyze the reduction of hydroperoxide. Prx1 and Prx3 play a great role in response to oxidative stress. In this way, the expression of Prx1 and Prx3 was increased when cells were treated with urolithin A, which translates into an antioxidant effect of urolithin A on Neuro-2a cells. Apart from the role of urolithin A as a free radical scavenger, this phenolic compound seems to act in a more specific way through the modulation of antioxidant enzymes. As observed above, urolithin A increased the expression of peroxiredoxins; this fact may explain the cytoprotective properties of urolithin A improving cell viability, decreasing ROS production, and increasing the activity of other physiological antioxidant defense systems, such as catalase, SOD, or glutathione reductase and peroxidase. Other polyphenols, such as resveratrol, have been described in the literature as antioxidant compounds with the ability to induce these cytoprotective proteins known as peroxiredoxins [35]; nevertheless, this is the first time that urolithin A is reported to increase the expression of peroxiredoxins 1 and 3.

This is also the first time that glutathione reductase and peroxidase activities were quantified in neuronal cells subjected to urolithin A treatments. In order to assess this pro-oxidant or antioxidant relationship of urolithin A, other researchers evaluated the in vitro antioxidant activity of the metabolite in HepG2 cancer cells [36]; unexpectedly, urolithin A exerted a pro-oxidant effect on these hepatoblastoma cells, however greater results were obtained in ORAC assay (6.67 ± 0.11). Regarding oxygen radical absorbance capacity, mice treated with urolithin A exhibited significantly different ORAC values after an hour of oral administration [37]. Nevertheless, urolithins may have different antioxidant potency, as shown in previous studies [38], and the ORAC value of urolithin C was lower than ours; the antioxidant properties derived from its role as a free radical scavenger were also confirmed by the DPPH in other studies [39–41].

ROS generation is directly linked to neurodegenerative diseases, such as Alzheimer's, Parkinson's, or Huntington's diseases. In this way, after demonstrating the potential of this metabolite in Neuro2-a cells, different bioassays were carried out involving CNS enzymatic targets known as acetylcholinesterase (AChE), monoamine oxidase A (MAO-A), and tyrosinase; these enzymes are considered pharmacological targets whose inhibition may lead to neuroprotective effects. Urolithin A acted as an inhibitor of oxidase enzymes such as MAO-A and tyrosinase, thus preventing oxidative damage of certain tissues. A human study in older adults with mild memory complaints suggests that 8 ounces of pomegranate juice taken daily over one-month improves verbal memory and alters neural activity during a visual source memory task [42]. Synthetized urolithins have also demonstrated comparable activity to AChE inhibitors such as rivastigmine, galantamine, and donepezil [43]. In silico computational studies predicted that urolithins can penetrate the blood-brain barrier, preventing β-amyloid fibrillation in a *C. elegans* model [44]. In addition, urolithin A has been identified as a proper anti-inflammatory and anti-ageing compound [38]. Recently, 10 μM urolithin A was demonstrated to possess depigmentation efficacy by suppressing tyrosinase activity, attenuating melanogenesis in B16 melanoma cells [45]. Tyrosinase has been recognised as a potential pharmacological target because an excess of tyrosinase activity or dopamine may lead to neurotoxicity through dopamine quinone formation; in this sense, tyrosinase inhibitors might have protective properties in neuronal cells. MAO-A is responsible for the catalytic oxidative deamination of monoamines generating hydrogen peroxide; for this reason, MAO-A inhibitors have been proposed as neuroprotective agents as well. The inhibitory activity of pomegranate polyphenols on MAO-A has already been tested, which could explain its effects in the CNS [22]; however, to the best of our knowledge this is the first report of urolithin A as an antioxidant agent capable of neutralizing MAO-A oxidative reactions.
