*3.4. New Identified Peptides*

From the reported analysis, 23 peptides were chosen and synthetized. Of them, 15 were not retrieved on the BIOPEP database, indicating that they were completely new in the scientific investigation. The remaining 8 were already registered in the sequence database, although with activities different from the antioxidant one. These peptides were tested for their antioxidant properties in vitro and in the Caco-2 cells model. In particular, 4 of them were selected for their antioxidant effects exerted on the cells and further analyzed to understand their mechanism of action.

#### 3.4.1. Antioxidant Properties In Vitro and In Caco-2 Cells

The 23 peptides were evaluated for their antioxidant capacity using ABTS and DPPH scavenging tests. As reported in Table 3, many peptides displayed a grea<sup>t</sup> antioxidant capacity with different extent, while some of them were ineffective. All the synthetic peptides were also tested in the cellular model for their potential cytotoxicity. In addition, they were also checked for their capability to protect against oxidative stress induced by 200 μM TbOOH. As reported in Table 3, column c, all peptides were not cytotoxic. Moreover, most of the synthetized peptides protected the viability of the cells once treated with the oxidative agent, in particular **V-12-Q**, **N-15-M**, **E-11-F**, **K-15-L** and **I-14-L**. These peptides showed a recovery of viability of about 10% (Table 3, column d).

**Table 3.** Evaluation of antioxidant properties of the identified peptides in vitro using 2,2-azinobis(3-ethylbenzo-thiazoline 6-sulfonate) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging tests and in Caco-2 cells pretreated with the 23 peptides. Oxidative stress was induced by 200 μM TbOOH.



**Table 3.** *Cont*.

a n. d.: not detected with the used assay.

#### 3.4.2. Inhibition of ROS Production by Bioactive Peptides

The identified peptides were further examined for their capacity to inhibit ROS production in Caco-2 cells. To this purpose, Caco-2 cells were treated with the peptides for 24 h and subsequently incubated with CM-DCFDA as described in the Material and methods. ROS production in Caco-2 cells pretreated with the peptides was similar to the untreated control, while, when the oxidative stress was induced by 250 μM TbOOH we observed a marked decrease in fluorescence after pretreatment with **N-15-M**, **E-11-F**, **Q-14-R**, **E-18-H**, **H-18-Q**, **A-17-E**, **D-17-T**, **S-17-Q**, **V-9-E**, **P-9-E** and **F-12-F** (Figure 2).

**Figure 2.** Estimation of reactive oxygen species (ROS) production in Caco-2 cells treated with the indicated peptides (0.05 mg/mL) in the absence (red) or presence (green) of 250 μM TbOOH. The values at 5000 s were reported and the means of at least three experiments (four replicates for each experiment) were compared with the treated control. (\*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05).

#### *3.5. Analysis of the Mechanism of Action of the Antioxidant Peptides*

Considering both the protection of the viability and the inhibition of ROS production in Caco-2 cells, **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E** were selected for their powerful antioxidant effects. Therefore, these four peptides were further analyzed to define their mechanism of action.

## 3.5.1. Nrf2 Translocation to the Nucleus

An antioxidant action inside the cell could be due to activation of the Keap1/Nrf2 pathway. For this reason, the translocation of Nrf2 from the cytosol to the nucleus in Caco-2 cells treated with the four peptides **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E** (0.05 mg/mL) was evaluated. Cells (1 × 106) were treated for 24 h with the peptides and then processed to obtain the nuclear fraction, as described in Materials and methods. Nrf2 present in the nuclear fraction was detected by Western blot analysis. Peptides **N-15-M**, **Q-14-R** and **A-17-E** increased significantly the levels of Nrf2 in the nucleus as reported in Figure 3, while **E-11-F** was completely ineffective.

**Figure 3.** Nrf2 translocation from cytosol to nucleus in Caco-2 cells in the presence of **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E**. (**A**) Cells were treated with 0.05 mg/mL of each peptide for 24 h. Nuclear fractions were isolated and proteins were subjected to WB detection as indicated in paragraph 2.15. (**B**) Densitometric analysis of four experiments compared with the control were reported, using PCNA as loading control. (\*\*\* *p* < 0.001, \* *p* < 0.05).

## 3.5.2. Antioxidant Enzymes Gene Expression Analysis

After the observation that **N-15-M**, **Q-14-R** and **A-17-E** showed a large increase of Nrf2 in the nucleus, the levels of gene expression of antioxidant enzymes were analyzed by RT-PCR. Cells (5 × 105) were treated for 24 h with the four peptides and processed as described in Section 2.16. The transcription of these enzymes is regulated by the translocation of Nrf2 to the nucleus where it can bind ARE.

As shown in Figure 4, the peptides **N-15-M**, **Q-14-R** and **A-17-E** were able to induce an increase of the *GSR, TXNRD1, NQO1* and *SOD1* mRNA levels. On the other hand, **E-11-F**, that did not show any effect on the translocation of Nrf2 to the nucleus, also in this case did not exert any effect on the antioxidant enzymes gene expression.

**Figure 4.** Antioxidant enzymes gene expression analysis. The gene expression of glutathione reductase (GRS, **A**), thioredoxin reductase (TXNRD1, **B**), NADPH quinone oxidoreductase (NQO1, **C**) and superoxide dismutase (SOD1, **D**) was evaluated in cDNA obtained from Caco-2 cells treated with **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E** (0.05 mg/mL) for 24 h. β-actin was used as reference. Means of at least four experiments were compared with the control. (\*\* *p* < 0.01, \* *p* < 0.05).

3.5.3. Antioxidant Enzymes Detection in the Presence of the Four Peptides

In order to confirm the observation regarding the increase of gene expression induced by **N-15-M**, **Q-14-R** and **A-17-E**, western blot analysis of lysates of cells treated with the four peptides was performed. Cells (5 × 105) were incubated in the presence of the four peptides (0.05 mg/mL) for 24 h. Aliquots of the samples (30 μg) were subjected to WB to detect glutathione reductase (GR), NADPH quinone oxidoreductase (NQO1), superoxide dismutase (SOD1) and thioredoxin reductase 1 (TrxR1). As shown in Figure 5, the most active peptides enhanced also the protein levels of these enzymes. In particular, cells treated with **Q-14-R** and **A-17-E** showed a large increase of GR, TrxR1 and NQO1 protein levels (Figure 5).

#### 3.5.4. TrxR1 and GR Activities in Cell Lysates

The activities of TrxR1 and GR in cells treated with **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E** were also analyzed. Cells were incubated with the indicated peptides in the same conditions as described above and 50 μg of protein cell lysates were used to determine TrxR1 activity by following DTNB reduction at 412 nm and NADPH oxidation at 340 nm for GR. As showed in Figure 6, a slight increase of the activities of the two antioxidant enzymes was observed in cells treated with **N-15-M** and **Q-14-R.**

**Figure 5.** Antioxidant enzymes detection by WB analysis. (**A**) Protein levels of glutathione reductase (GR), thioredoxin reductase (TrxR1), NADPH quinone oxidoreductase (NQO1) and superoxide dismutase (SOD1) in Caco-2 cell lysates treated with the four peptides (0.05 mg/mL) for 24 h. (**B–E**) Densitometric analysis of four experiments were compared with the control and normalized using GAPDH as loading control. (\*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05).

**Figure 6.** GR (**A**) and TrxR1 (**B**) activities in Caco-2 cells treated with **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E** (0.05 mg/mL). The activity of the two antioxidant enzymes was analyzed in Caco-2 cells treated with the four peptides for 24 h. Means of at least four experiments were compared with the control. (\* *p* < 0.05).
