*2.1. Biochemical Characterization of P. oceanica Leaf Extract (POE)*

POE was found to contain 3.4 ± 0.2 mg/mL of total polyphenols (TP) equivalent to gallic acid (mg GAE/mL). In addition, POE exhibited antioxidant and radical-scavenging activities of 0.9 ± 0.2 mg/mL and 8.9 ± 0.3 mg/mL ascorbic acid equivalents (mg AAE/mL), as evaluated by FRAP and DPPH assays, respectively (Table 1). The data obtained are consistent with those obtained in previous work [6,8], supporting the efficiency and reproducibility of the extraction method.

**Table 1.** Biochemical properties of POE in terms of total polyphenols (TP) antioxidant (FRAP assay) and radical scavenging activities (DPPH assay).


## *2.2. Effect of POE on Cell Viability*

No significant modifications of cell viability were observed in Caco-2 cells treated till to 24 h with increasing levels of POE (corresponding to polyphenol concentration ranging from 5 μg GAE/mL to 40 μg GAE/mL) (Supplementary Figure S1). These results confirm that POE did not exert a cytotoxic effect in our experimental conditions. Based on these results, all the following experiments were conducted using 15 μg GAE/mL POE.

#### *2.3. Effect of POE on Glucose Transport under Sodium-Dependent or Sodium-Free Conditions*

Caco-2 differentiated cells were used as a model of the intestinal barrier. The effect of POE on glucose uptake was investigated both in the presence or in the absence of sodium. In the presence of sodium, glucose transporters SGLT1 and GLUT2 are both active. In sodium-free conditions only GLUT2 is active.

As shown in Figure 2, POE treatment significantly (*p* < 0.05) decreased glucose transport, both in the presence (Figure 2A) and absence of sodium (Figure 2B).

**Figure 2.** Effect of POE on glucose transport in Caco-2 differentiated cells. Glucose transport was evaluated under (**A**) sodium-dependent or (**B**) sodium-free conditions in differentiated Caco-2 cells treated with POE (15 μg GAE/mL) for 24 h. Values are presented as the mean ± SD of three determinations carried out in triplicate. Data are reported in terms of percentage with respect to control cells. \*: *p* < 0.05.

To understand the decrease in glucose transport observed in POE-treated cells, the effect of POE on the expression of glucose transporters (SGLT1 and GLUT2) in Caco-2 differentiated cells was investigated by Western blot analysis (Figure 3A). POE treatment caused a significant (*p* < 0.05) decrease in the GLUT2 levels (68% ± 2.1%) with respect to control cells, as shown in Figure 3B. No modifications of the levels of transporter SGLT1 were observed in POE-treated cells compared with control cells (Figure 3C).

**Figure 3.** Effect of POE on the levels of glucose transporters. (**A**) Representative Western blot images of (**B**) GLUT2 and (**C**) SGLT1 glucose transporters in differentiated Caco-2 cells incubated in the absence or presence of POE (15 μg GAE/mL) for 24 h. Densitometric data are normalized to the Vinculin expression levels. Data are presented as the mean ± SD of three determinations. \*: *p* < 0.05.

#### *2.4. Effect of POE on Caco-2 Monolayer as a Model of Intestinal Barrier*

The evaluation of transepithelial electrical resistance (TEER) across the monolayer of Caco-2 differentiated cells was used to assess the effect of POE on the integrity of the intestinal barrier.

As shown in Figure 4, POE treatment caused an increase in TEER across the Caco-2 cell monolayer. The effect was significant (*p* < 0.05) after 4 h and reached 118% of the initial value after 24 h of incubation compared with control cells.

**Figure 4.** Effect of POE on Caco-2 monolayer cells. Transepithelial electrical resistance (TEER) in differentiated Caco-2 control cells or treated with POE (15 μg GAE/mL) for 24 h. Values are presented as the mean ± SD of three determinations carried out in triplicate. TEER values are reported in terms of percentage with respect the initial value. \* Control vs. POE-treated cells. (\*: *p* < 0.05).

Zonulin-1 (ZO-1) is a protein bound to the cytoskeleton and it has a pivotal role in tight junction (TJ) integrity. Therefore, to investigate the molecular mechanisms involved in the POE-induced increase in TEER, the levels of ZO-1 were determined by Western blot analysis (Figure 5A).

**Figure 5.** Effect of POE on the ZO-1 levels. (**A**) Representative Western blot images of ZO-1 in differentiated Caco-2 incubated in the absence or treated with POE (15 μg GAE/mL) for 24 h. (**B**) Densitometric data are normalized to Vinculin. Data are presented as the mean ± SD of five determinations. \*\*: *p* < 0.01.

As shown in Figure 5B, a two-fold increase in the ZO-1 levels was observed in POEtreated Caco-2 cells compared with the untreated control cells (*p* < 0.01).

#### *2.5. Effect of POE on High-Glucose-Induced Oxidative Stress*

The chronic exposure to high glucose (HG) induces oxidative stress in Caco-2 cells, as previously described [20].

In this study, the potential protective role of POE (15 μg GAE/mL) on chronic HGinduced oxidative stress was investigated in Caco-2 cells. Cells not exposed to HG were used as control cells. A significant increase (1.95 ± 0.7 A.U.) in intracellular ROS production was confirmed in HG cells compared to control cells (1.02 ± 0.18 A.U.), in accordance with our previous study [20]. HG cells treated with POE showed a significant decrease in intracellular ROS production with respect to HG cells (Figure 6A). Figure 6B shows representative images of the fluorescence intensity indicative of ROS formation in POEtreated cells under HG conditions. The dose-dependent effect (0–40 μg GAE/mL) of POE on HG-induced intracellular ROS formation is reported in Supplementary Figure S2.

**Figure 6.** Effect of POE and *N*-acetylcysteine (NAC) on HG-induced intracellular ROS formation. (**A**) Intracellular ROS production was evaluated in Caco-2 cells incubated in the absence (control cells) or in the presence of high-glucose conditions (HG cells) for 1 week and co-incubated with POE (15 μg GAE/mL), and *N*-acetylcysteine (NAC) (50 μM) for the last 24 h. Values are represented as the mean ± SD of five determinations carried out in triplicate. \*\*\* Control vs. HG; \* HG vs. HG + NAC; \*\*\* HG vs. POE; \*\*\* HG vs. HG + POE. (ANOVA: \*: *p* < 0.05; \*\*\*: *p* < 0.001). (**B**) Fluorescent cells observed under a fluorescent microscope in the presence of POE (15 μg GAE/mL (Lionheart™ FX)).

HG cells treated with *N*-acetylcysteine (NAC) were used as the positive antioxidant control. The POE-induced mitigation of intracellular ROS production in HG cells was comparable to that induced by NAC (50 μM) (Figure 6A).

This finding suggests that POE exerts an antioxidant activity against HG-induced intracellular ROS production in Caco-2 cells.

The HG-induced intracellular ROS levels can cause an increase in advanced glycation end products (AGEs), due to the formation of highly reactive intermediates of the Maillard reaction, such as glycolaldehyde and glyoxal. These molecules are involved in cross-linking of proteins and are precursors of AGEs [21].

In this study, the glycolaldehyde (GA)-modified protein levels were also evaluated by Western blot analysis (Figure 7A) to investigate the effect of POE on AGEs formation in HG-treated cells. Higher levels of GA-modified proteins were observed in HG cells compared to control cells (Figure 7B), as previously described [20]. In the presence of POE, a significant (*p* < 0.05) decrease in GA-modified protein levels was observed in HG cells compared with untreated HG cells (Figure 7B). A slight decrease in the GA-modified protein levels was also observed in Caco-2 cells treated with POE compared with the untreated control cells (Figure 7B).

**Figure 7.** Effect of POE on GA-modified proteins levels in Caco-2 cells. (**A**) Representative Western blot images of GA-modified proteins. (**B**) Densitometric analysis of GA-modified proteins in Caco-2 control cells or HG cells in the absence or presence of POE (15 μg GAE/mL) for 24 h. Densitometric data are normalized to Vinculin. Results are presented as the mean ± SD of five determinations. \*\*\* Control vs. HG; \*\*\* Ctrl vs. POE; \*\*\* HG vs. POE; \*\*\* HG vs. HG + POE (ANOVA: \*\*\*: *p* < 0.001).

These results could explain the effect of POE on HG-induced cell toxicity. In fact, a significant decrease (about 40%) in cell viability was observed in HG cells compared with control cells. POE-treated cells exposed to chronic HG conditions had comparable viability to control cells (Supplementary Figure S3)
