*2.1. Evaluation of the RBC Oxidative Status (Diamide) and Response to DDS-NHOH before Licorice Intake*

Cytosolic compartments of PG RBC showed much higher monomeric CAII isoform (30 kDa) than CG RBC (35 ± 3.7% compared to 12 ± 3%, respectively, *p* < 0.001). Being the activity of CA strictly depending on its monomerization [36], a CA activity assay was performed, with PG RBC exhibiting almost six times higher values compared to CG RBC (Figure 1). When RBCs were treated with diamide, the amount of the 30 kDa band of CA increased to 24% and 49%, in CG and PG RBC, respectively, (*p* < 0.001), with a parallel activity increase (5.2 ± 2 in CG and 32.7 ± 8.0 in PG, *p* < 0.001). In addition, in the presence of DDS-NHOH, if the process of formation of the 30 kDa isoform was more evident (26% in CG, 68% in PG), the correspondent values of CA activity were not so drastically increased as expected (3.3 ± 0.6 and 12.2 ± 3.1 in CG and PG, respectively).

PG, respectively).

**Figure 1.** Fresh blood was collected from CG and PG RBCs (isolated as described in the Methods section) and was incubated with and without 1.5 mM diamide or 0.3 mM DDS-NHOH. (**a**) Diluted cytosol from 1 μL of packed cells, underwent Western blotting in non-reducing conditions. Bands immunostained with anti-CA antibodies were densitometrically analyzed, and the sum of the 30 and 60 kDa bands was arbitrarily calculated as 100%, taking into account that amount of proteolytic 30 kDa bands accounts for half the larger bands. Values were expressed as the means ± SD of *n* = 12 CG and *n* = 18 PG patients. \* *p* < 0.001, comparison of CA 30 kDa isoform before and after treatments within groups, Student's *t*-test for paired data; † *p* < 0.001, comparison of the 30 kDa band between CG and PG groups, in both experimental conditions (diamide and DDS-NHOH treatments), Student's t-test. (**b**) CA activity: 300 μL of diluted cytosol from CG or PG RBCs, previously incubated with and without 1.5 mM diamide or 0.3 mM DDS-NHOH, were assayed for activity as described in the Methods section. The activity was calculated as the ratio to activity observed in untreated CG (chosen as arbitrary comparison unit, experimentally determined as 1 ± 0.23, mean value ± SD). Data show the means ±SD of *n* = 12 CG and *n* = 18 PG patients. \* *p* < 0.001, comparison of CA activity to CG, before and after diamide treatment, Student's *t*-test for paired data. **Figure 1.** Fresh blood was collected from CG and PG RBCs (isolated as described in the Methods section) and was incubated with and without 1.5 mM diamide or 0.3 mM DDS-NHOH. (**a**) Diluted cytosol from 1 µL of packed cells, underwent Western blotting in non-reducing conditions. Bands immunostained with anti-CA antibodies were densitometrically analyzed, and the sum of the 30 and 60 kDa bands was arbitrarily calculated as 100%, taking into account that amount of proteolytic 30 kDa bands accounts for half the larger bands. Values were expressed as the means ± SD of *n* = 12 CG and *n* = 18 PG patients. \* *p* < 0.001, comparison of CA 30 kDa isoform before and after treatments within groups, Student's *t*-test for paired data; † *p* < 0.001, comparison of the 30 kDa band between CG and PG groups, in both experimental conditions (diamide and DDS-NHOH treatments), Student's *t*-test. (**b**) CA activity: 300 µL of diluted cytosol from CG or PG RBCs, previously incubated with and without 1.5 mM diamide or 0.3 mM DDS-NHOH, were assayed for activity as described in the Methods section. The activity was calculated as the ratio to activity observed in untreated CG (chosen as arbitrary comparison unit, experimentally determined as 1 ± 0.23, mean value ± SD). Data show the means ± SD of *n* = 12 CG and *n* = 18 PG patients. \* *p* < 0.001, comparison of CA activity to CG, before and after diamide treatment, Student's *t*-test for paired data.

amount of the 30 kDa band of CA increased to 24% and 49%, in CG and PG RBC, respectively, (*p* < 0.001), with a parallel activity increase (5.2 ± 2 in CG and 32.7 ± 8.0 in PG, *p* < 0.001). In addition, in the presence of DDS-NHOH, if the process of formation of the 30 kDa isoform was more evident (26% in CG, 68% in PG), the correspondent values of CA activity were not so drastically increased as expected (3.3 ±0.6 and 12.2 ± 3.1 in CG and

#### *2.2. Effect of Licorice Intake on the Membrane and Cytosol Oxidative Status 2.2. Effect of Licorice Intake on the Membrane and Cytosol Oxidative Status*

In the presence of diamide, RBCs from PG showed a much higher Tyr-P level in RBC membranes compared to that from CG (221 ± 26 and 100 ± 9 for PG and CG membranes, In both CG and PG, RBC membranes at T0 (before licorice intake) showed a similar pattern of B3 Tyr-P levels (which remained practically undetectable) and HMWA content (Figure 2, panel b).

respectively, *p* < 0.005). Similarly, membrane band 3 HMWA content also increased to double the basal amount in CG but reached almost four times the basal value in PG (196 ± 19 and 349 ± 45, for membranes from CG and PG, respectively, *p* < 0.001) (Figure 2, panel a, lanes T<sup>0</sup> and Figure 3, panels a and b, compare CG and PG at T0, diamide) [4]. When diamide was replaced by DDS-NHOH, the average increment in the Tyr-P level was almost 6 times in RBCs from PG compared to CG (*p* < 0.001) (Figure 3, panel a). Similarly, in PG, DDS-NHOH treatment induced a higher increase in band 3 HMWA content (average PG increase of about 471 ± 37 % compared to CG, *p* < 0.001), much higher than that evidence in the RBC membranes of CG (196 ± 35 and 135 ± 29 for diamide and DDS-NHOH In the presence of diamide, RBCs from PG showed a much higher Tyr-P level in RBC membranes compared to that from CG (221 ± 26 and 100 ± 9 for PG and CG membranes, respectively, *p* < 0.005). Similarly, membrane band 3 HMWA content also increased to double the basal amount in CG but reached almost four times the basal value in PG (196 ± 19 and 349 ± 45, for membranes from CG and PG, respectively, *p* < 0.001) (Figure 2, panel a, lanes T<sup>0</sup> and Figure 3, panels a and b, compare CG and PG at T0, diamide) [4]. When diamide was replaced by DDS-NHOH, the average increment in the Tyr-P level was almost 6 times in RBCs from PG compared to CG (*p* < 0.001) (Figure 3, panel a). Similarly, in PG, DDS-NHOH treatment induced a higher increase in band 3 HMWA content (average PG increase of about 471 ± 37 % compared to CG, *p* < 0.001), much higher than that evidence in the RBC membranes of CG (196 ± 35 and 135 ± 29 for diamide and DDS-NHOH treatment, respectively, *p* < 0.01) (Figure 3, panels a and b, compare CG and PG at T0, DDS-NHOH).

NHOH).

ure 2).


NHOH, respectively, *p* < 0.001) (panel c).

**Figure 2.** The membranes (10 μg) obtained, as described in the Methods section, were analyzed by Western blotting in non-reducing conditions (panel **a**) and immunostained with anti-band 3 P-Tyr antibodies or in non-reducing conditions and immunostained with anti-band 3 (panel **a** and **b**), anti-P-Tyr or anti-GSH antibodies (panel **b**). For each immunostaining, bands corresponding to the relative proteins were densitometrically estimated and statistically analyzed (panel **b**). The Tyr-P value of both CG and PG RBCs were undetectable. The band 3 HMWA or GSH values were calculated as the ratio to band 3 HMWA or GSH obtained in basal (T0) samples of CG (chosen as arbitrary comparison unit, experimentally determined as 100 ± 5% and 102± 7%, respectively). Data shows the means ± SD of *n* = 12 CG and *n* = 18 PG patients. Comparison from respective baseline values: \**p* < *0*.001, Student's *t*-test for paired data. Comparison CG vs PG: ‡ *p* < *0*.001, Student *t*-test for unpaired data. **Figure 2.** The membranes (10 µg) obtained, as described in the Methods section, were analyzed by Western blotting in non-reducing conditions (panel **a**) and immunostained with anti-band 3 P-Tyr antibodies or in non-reducing conditions and immunostained with anti-band 3 (panel **a** and **b**), anti-P-Tyr or anti-GSH antibodies (panel **b**). For each immunostaining, bands corresponding to the relative proteins were densitometrically estimated and statistically analyzed (panel **b**). The Tyr-P value of both CG and PG RBCs were undetectable. The band 3 HMWA or GSH values were calculated as the ratio to band 3 HMWA or GSH obtained in basal (T<sup>0</sup> ) samples of CG (chosen as arbitrary comparison unit, experimentally determined as 100 ± 5% and 102 ± 7%, respectively). Data shows the means ± SD of *n* = 12 CG and *n* = 18 PG patients. Comparison from respective baseline values: \* *p* < *0*.001, Student's *t*-test for paired data. Comparison CG vs PG: ‡ *p* < *0*.001, Student *t*-test for unpaired data.

treatment, respectively, *p* < 0.01) (Figure 3, panels a and b, compare CG and PG at T0, DDS-

The increased oxidative status of the membrane was also evaluated by glutathionylated protein content (average increase of about 145% and 140% for diamide and DDS-

After 1 week of licorice intake (T1), RBCs from both groups were reanalyzed and the HMWA anti-Tyr-P and anti-GSH content from PG were compared to those from CG (Fig-

Results showed that both the HMWA and protein-bound GSH (GS-SG) contents were reduced after licorice intake (T1), both in CG (with an average decrease of about 30% in HMWA and 10% in GS-SP) and PG (average decrease of 25% in HMWA, but almost 40 The increased oxidative status of the membrane was also evaluated by glutathionylated protein content (average increase of about 145% and 140% for diamide and DDS-NHOH, respectively, *p* < 0.001) (panel c).

% in the GS-SP). Either in CG or PG, no alterations were detectable in the Tyr-P level. Interestingly, at T1, the PG RBCs also showed a net reduction in both diamide- and DDS-NHOH-induced alterations compared with their own values at T0, with an average After 1 week of licorice intake (T1), RBCs from both groups were reanalyzed and the HMWA anti-Tyr-P and anti-GSH content from PG were compared to those from CG (Figure 2).

decrease ranging from 35 ± 15% (diamide-induced Tyr-P level) to 61 ± 5% (decrease of DDS-NHOH-induced glutathionylation) (panel f). All the other parameters ranged between these two values, with a reduction higher than 50%. CG RBCs showed a slight decrease only in diamide-induced Tyr-P values (7 ± 7%), but in all the other parameters the Results showed that both the HMWA and protein-bound GSH (GS-SG) contents were reduced after licorice intake (T1), both in CG (with an average decrease of about 30% in HMWA and 10% in GS-SP) and PG (average decrease of 25% in HMWA, but almost 40 % in the GS-SP). Either in CG or PG, no alterations were detectable in the Tyr-P level.

average decrease ranged from a minimum of 17 ± 9% (DDS-NHOH-induced HMWA Interestingly, at T1, the PG RBCs also showed a net reduction in both diamide- and DDS-NHOH-induced alterations compared with their own values at T0, with an average decrease ranging from 35 ± 15% (diamide-induced Tyr-P level) to 61 ± 5% (decrease of DDS-NHOH-induced glutathionylation) (panel f). All the other parameters ranged between these two values, with a reduction higher than 50%. CG RBCs showed a slight decrease only in diamide-induced Tyr-P values (7 ± 7%), but in all the other parameters the average decrease ranged from a minimum of 17 ± 9% (DDS-NHOH-induced HMWA formation) to a maximum of 48 ± 6% (the decrease in membrane glutathionylation was induced by DDS-NHOH). The variations of all parameters observed between T<sup>0</sup> and T<sup>1</sup> were statistically different, both in the CG and PG groups (*p* < 0.005). Also, the average

values were statistically different between the CG and PG groups (*p* < 0.001) in all conditions (panels a–c), except for DDS-NHOH-induced glutathionylation, because, following licorice intake (T1), the higher level of glutathionylated protein in PG RBCs was almost completely lowered, thus resembling the level in the CG RBCs (panel c, T1). values were statistically different between the CG and PG groups (*p* < 0.001) in all conditions (panels a–c), except for DDS-NHOH-induced glutathionylation, because, following licorice intake (T1), the higher level of glutathionylated protein in PG RBCs was almost completely lowered, thus resembling the level in the CG RBCs (panel c, T1).

formation) to a maximum of 48 ± 6% (the decrease in membrane glutathionylation was induced by DDS-NHOH). The variations of all parameters observed between T<sup>0</sup> and T<sup>1</sup> were statistically different, both in the CG and PG groups (*p* < 0.005). Also, the average

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 5 of 13

**Figure 3.** The effect of 1-week licorice intake on the Tyr-P level (**a**), HMWA, (**b**) and membrane GSH contents (**c**), following diamide or DDS-NHOH stimulation, in the CG and PG groups. RBCs from CG and PG were incubated with 1.5 mM diamide or 0.30 mM DDS-NHOH. The membranes (10 µg) obtained, as described in Methods, were analyzed by Western blotting and immunostained with the anti-P-Tyr antibody and then with anti-β actin as a loading control (Panel **a**). The membranes were also analyzed in non-reducing conditions and immunostained with antiband 3 (Panel **b**) or anti-GSH (not shown) antibodies, in order to evidence the band 3 high molecular weight aggregate (HMWA) and bound glutathione, respectively. The figure is representative of the study population. For each immunostaining, bands corresponding to the relative proteins were densitometrically estimated and statistically analyzed GSH values of diamide or DDS-NHOH treated RBCs were calculated as the ratio percentage to GSH levels obtained in the basal samples of CG at T<sup>0</sup> (chosen as an arbitrary comparison unit, experimentally determined as 101 ± 3%,) (Panel **c**). The Tyr-P value of diamide—or DDS-NHOH—treated RBCs before (T0) and after a week of licorice intake (T1) was calculated as: Tyr-P% = (Tyr-P(x)/Tyr-P(CG) diamide T0%, with the Tyr-P value obtained in diamide samples of CG at T<sup>0</sup> chosen as an arbitrary comparison unit (experimentally determined as 100 ± 5%) (Panel **d**). The band 3 HMWA of diamide or DDS-NHOH treated RBCs were calculated as the ratio percentage to band 3 HMWA obtained in the basal samples of CG at T<sup>0</sup> (chosen as an arbitrary comparison unit, experimentally determined as 100 ± 4 and) (Panel **e**). (Panel **f**) average percent decrease for each parameter, referring to the corresponding values at T0, were calculated and reported. Data show the means ± SD of *n* = 12 healthy subjects (CG) and *n* = 18 patients (PG). Comparison from the respective T<sup>0</sup> values: \* *p* < 0.005, Student's *t*-test for paired data. Comparison CG vs PG: † *p* < 0.001, Student *t*-test for unpaired data. *2.3. Licorice Intake and CA Monomerization and Activity in RBC Cytosol* **Figure 3.** The effect of 1-week licorice intake on the Tyr-P level (**a**), HMWA, (**b**) and membrane GSH contents (**c**), following diamide or DDS-NHOH stimulation, in the CG and PG groups. RBCs from CG and PG were incubated with 1.5 mM diamide or 0.30 mM DDS-NHOH. The membranes (10 µg) obtained, as described in Methods, were analyzed by Western blotting and immunostained with the anti-P-Tyr antibody and then with anti-β actin as a loading control (Panel **a**). The membranes were also analyzed in non-reducing conditions and immunostained with anti-band 3 (Panel **b**) or anti-GSH (not shown) antibodies, in order to evidence the band 3 high molecular weight aggregate (HMWA) and bound glutathione, respectively. The figure is representative of the study population. For each immunostaining, bands corresponding to the relative proteins were densitometrically estimated and statistically analyzed GSH values of diamide or DDS-NHOH treated RBCs were calculated as the ratio percentage to GSH levels obtained in the basal samples of CG at T<sup>0</sup> (chosen as an arbitrary comparison unit, experimentally determined as 101 ± 3%) (Panel **c**). The Tyr-P value of diamide—or DDS-NHOH—treated RBCs before (T<sup>0</sup> ) and after a week of licorice intake (T<sup>1</sup> ) was calculated as: Tyr-P% = (Tyr-P(x)/Tyr-P(CG) diamide T0%, with the Tyr-P value obtained in diamide samples of CG at T<sup>0</sup> chosen as an arbitrary comparison unit (experimentally determined as 100 ± 5%) (Panel **d**). The band 3 HMWA of diamide or DDS-NHOH treated RBCs were calculated as the ratio percentage to band 3 HMWA obtained in the basal samples of CG at T<sup>0</sup> (chosen as an arbitrary comparison unit, experimentally determined as 100 ± 4 and) (Panel **e**). (Panel **f**) average percent decrease for each parameter, referring to the corresponding values at T<sup>0</sup> , were calculated and reported. Data show the means ± SD of *n* = 12 healthy subjects (CG) and *n* = 18 patients (PG). Comparison from the respective T<sup>0</sup> values: \* *p* < 0.005, Student's *t*-test for paired data. Comparison CG vs PG: † *p* < 0.001, Student *t*-test for unpaired data.

#### The cytosolic oxidative status was also evaluated, and the monomerization and activity of CA and the variation of GSH contents were compared between the two groups *2.3. Licorice Intake and CA Monomerization and Activity in RBC Cytosol*

The cytosolic oxidative status was also evaluated, and the monomerization and activity of CA and the variation of GSH contents were compared between the two groups in the presence of diamide or DDS-NHOH at T<sup>0</sup> and T1. As expected, in PG, at T<sup>0</sup> the monomeric

form of this enzyme, representative of increased oxidation [26,36], was much higher compared to that of CG (35 ± 5 % of the 30 kDa isoform in PG compared to 12 ± 4 % present in CG, *p* < 0.001), and, consequently, also the CA activity was by far higher (Figure 2, *p* < 0.001).

What is interesting is that, following licorice intake, net decreases of both monomerization and activity were observed. By evaluating the average decrease in both values between T<sup>1</sup> and T0, at T<sup>1</sup> the percentage of CA activity reduction was 8.5 ± 7 and 50.7 ± 4.3, in CG at basal or DDS-NHOH conditions, respectively (*p* < 0.005). In PG, CA activity reduction was 61.7 ± 16 and 79.1 ± 8.7, at basal or DDS-NHOH conditions, respectively (*p* < 0.005) (Table 1).

**Table 1.** The effect of 1-week licorice intake on CA activity and monomerization and ∆GSH in the cytosol of RBCs, in the presence of diamide or DDS-NHOH in in vitro treatment. Panel **a**: CA activity and monomerization values, obtained at T<sup>0</sup> and T<sup>1</sup> , were expressed as the decrease after licorice intake, in the absence (basal), or presence of DDS-NHOH. The activity of CA was assayed in the RBC cytosol as described in the Methods. The decrease following the licorice intake (∆CA activity %) was calculated as: (1-activity T1/ activity T<sup>0</sup> )%. The monomerization of CA was assayed in diluted cytosol from 1 µL of packed RBCs, obtained as described in the Methods. The cytosol underwent Western blotting in non-reducing conditions and was immunostained with an anti-CA antibody. Densitometrical analysis of the CA bands was carried out and the sum of the 30 and 60 kDa bands was arbitrarily calculated as 100%, taking into account that amount of proteolytic 30 kDa bands accounts for half the larger [36]. Panel **b**: Total glutathione was determined according to the Tietze method [4], as described in the Methods. The total decrease of glutathione content after diamide or DDS-NHOH treatment (∆GSH) was expressed as 1-GSH(Diam or DDS-NHOH)/GSH(Basal) [4,36].


Values are expressed as the mean ± SD of *n* = 12 healthy subjects (CG) and *n* = 18 patients (PG). Comparison from respective basal values: \* *p* < 0.005, Student's *t*-test for paired data. Comparison CG vs. PG: † *p* < 0.001, Student's *t*-test for unpaired data. Comparison between T<sup>0</sup> and T<sup>1</sup> within each group: ‡ *p* < 0.001, Student's *t*-test for paired data.

When the cytosol was analyzed for the GSH content, DDS-NHOH treatment induced a drop in the total glutathione content of about a mean value of 0.85 ± 0.08 in PG RBCs (Table 1, panel b, T0). In CG only, a slight decrease in the glutathione content was observed after both diamide and DDS-NHOH treatments, 0.08 ± 0.03 and 0.20 ± 0.01, respectively, thus confirming that the band 3 Tyr-P level and HMWA formation involved cell GSHrelated anti-oxidant defenses, with the depletion of cytosolic pools to implement the membrane protein glutathionylation. Also, in this case, licorice intake induced a net reduction of the GSH lost, as indicated by the lowering of ∆GSH values in both CG and PG (Table 1).
