**3. Discussion**

In this study, we investigated the effect of licorice intake on RBC improvements towards oxidative stress in endometriosis.

In this study, we investigated the effect of licorice intake on RBC improvements towards oxidative stress in endometriosis. Dapsone-induced hemolytic anemia is closely related to erythrocyte membrane alterations, leading to premature cell removal, which can occur both extra-vascularly (witness hyperbilirubinemia), or intravascularly by dapsone-induced cell fragility. All hema-Dapsone-induced hemolytic anemia is closely related to erythrocyte membrane alterations, leading to premature cell removal, which can occur both extra-vascularly (witness hyperbilirubinemia), or intravascularly by dapsone-induced cell fragility. All hematological side effects reported for dapsone therapy are due to the N-hydroxy metabolite of the drug, dapsone hydroxylamine (DDS-NHOH) [20,25,37].

tological side effects reported for dapsone therapy are due to the N-hydroxy metabolite of the drug, dapsone hydroxylamine (DDS-NHOH) [20,25,37]. These alterations should be taken into account in choosing therapy for endometriosis patients. Endometriosis is a chronic inflammatory disease with a genetic, epigenetic, and These alterations should be taken into account in choosing therapy for endometriosis patients. Endometriosis is a chronic inflammatory disease with a genetic, epigenetic, and environmental background [38]. It has been recently shown that the presence of endometriosis susceptibility genes whose wide variations of penetrance would be seriously influenced by phenotypic alterations [39]. Environmental changes, such as iron overload during menstruation, can induce a Fenton-mediated oxidative assault, which would affect

DNA hypermethylation and chromatin remodeling, thus stressing gene instability by introducing point mutations and/or DNA single- and double-strand breaks, all leading to a significant increase in cancer risk [39]. For these reasons, redox and inflammatory modifications, which can accumulate in endometriosis patients, may be not only at the origin of endometriosis but also responsible for the further development/worsening of the disease [39].

In this study, we addressed the potential effect of licorice in ameliorating PG RBC tolerance to dapsone treatments. We have previously demonstrated that GA, one of the licorice intestinal metabolism products, was able to prevent diamide-induced band 3 Tyr-P levels and HMWA formation, as well as band 3 proteolytic degradation in in vitro experiments performed with normal RBCs [35]. To investigate if this important GA shielding effect could be efficacious also in endometriosis to lower potential oxidant injuries, we analyzed the same parameters in RBCs after volunteers were given one week of licorice intake. Interestingly, all parameters resulted in positively affected PG RBCs, with net reductions in DDS-NHOH-induced alterations ranging from 35 to 61%, compared to T0. That this effect was due to the GA licorice component was confirmed by the correlation between the plasma GA content and diamide, used as a reference, with its effects amply studied and described in previous studies [35,40], or DDS-NHOH effects. Only with diamide, for bound GSH and HMWA formation parameters, it seemed that weak a correlation was present in PG, but not in CG. This could be explained by the fact that diamide-induced alterations are different from those by DDS-NHOH. Diamide is known to induce disulfide bond formation, thus clustering membrane band 3 and leading to HMWA formation. Band 3 is normally distributed between detergent soluble (66%) and detergent-insoluble (33%) fractions of RBC membranes, and following diamide treatment, band 3 aggregated in HMWA increased only in the detergent soluble fraction [41]. On the contrary, DDS-NHOH induces a complete rearrangement of HMWA, which starts at the soluble fraction but slowly migrates to increase the insoluble counterpart [26].

Among the cytosolic enzymes, an important role is played by CA, a metallo-enzyme, converting CO<sup>2</sup> to HCO3- and H +, which regulates many physiological processes such as acid–base balance homeostasis, respiration, carbon dioxide, ion transport, and bone resorption [42]. To date, their biological functioning has not been clarified, but recent evidence has pointed out how abnormal levels or activities [34] of many CA isoforms were associated with different diseases such as cancer (overexpression of CA IX/XII due to the hypoxia cascade activation), epilepsy (abnormal levels/activities of brain CA isoforms), and obesity (dysregulation of the mitochondrial isoforms CA VA/B) [42].

In human RBCs, the upregulation or high activity level of CA 2, the main isoform [36], has been related to glaucoma [42], and for this its functioning could represent an important parameter to be evaluated, mainly due to the recent finding showing an oxidative-related net increase of CA 2 activity in endometriosis patients. In fact, by increasing oxidative conditions, CA 2, normally present as an inactive dimer, can be activated following a monomerization process [36]. In the present study, the mean CA activity from PG was about 30 times higher than that from CG with a mean monomerization three times superior compared with CG, and 5 times lower GSH (∆GSH). These data identify CA as an important parameter in the evaluation of the oxidative status in endometriosis, as well as a novel paradigm in the prevention of potential clinical complications.

In PG, one week of GA intake succeeded in considerably reducing both untreated and, much more interestingly, DDS-NHOH treated RBC effects on CA, with 5 times activity reduction and 3 times less monomerization, whereas in CG, both CA activity and monomerization, and GSH drop, returned to the level of the DDS-NHOH untreated RBC.

GA, a licorice metabolite, mitigates DDS-NHOH-induced side effects by lowering membrane sensitivity to oxidative stress and preserving cell GSH content.

For a long time, licorice has been considered in many natural medical resources, and these findings emphasize how GA can protect RBCs from strong oxidant-induced

denaturation, thus preventing risks from extensive and prolonged exposure to oxidative stress in impaired anti-oxidant conditions.
