*2.2. Cytoprotective Activity in H2O2-Mediated HUVECs Injury*

Evaluation of cytoprotective activity was carried out on the identified peptides FTVN and EPTF, as well as their combination in the same proportion, to see if there was any synergy effect between the two peptides. Cell viability was evaluated using the MTT assay after cultured HUVECs were treated with sample peptides and subsequently challenged with 600 μM of H2O2, a concentration which was determined to significantly decrease cell viability in a previous report [22]. Compared with untreated cells that were not exposed to peptides or H2O2 (control), the addition of H2O2 significantly decreased the cell viability of HUVEC by 65.43%. Meanwhile, HUVECs pretreated with 100 μg/mL peptide samples showed remarkably increased cell viability of 85.46%, 83.11%, and 86.58% for FTVN, EPTF, and their combination (1:1), respectively. The results indicate the cytoprotective effect of FTVN, EPTF, and their combination. On the other hand, no significant difference was shown in improving HUVECs cell viability between the peptide samples or the combination of FTVN and EPTF. However, the concentration of the samples had a significant effect on cell viability (Figure 3A). Similar results were found in fluorescence microscopy with Calcein-AM/PI double staining analysis, where H2O2 treatment significantly reduced the green fluorescence of live cells, but pretreatment with FTVN, EPTF, and their combination reversed this effect. Moreover, in H2O2 treatment, more cells are stained with PI, while the peptides and their combination reduce the cells stained with PI, indicating a cytoprotective effect (Figure 3B).

**Figure 3.** (**A**) The cell viability of HUVECs after BAPs pretreatment at different concentrations. (**B**) Calcein-AM/PI staining assay on EPTF, FTVN, and their combination pretreatment at 0.1 mg/mL. HUVECs were incubated with samples for 2 h before being challenged with 600 μM H2O2 for 24 h. The data are provided as means <sup>±</sup> SD (*<sup>n</sup>* = 4). a–c Different letters show significance difference at *p* < 0.05.

### *2.3. Inhibition of Intracellular ROS Generation*

It is hypothesized that ROS in cells is one of the causes of H2O2-induced HUVEC damage. Therefore, we also investigated the presence of ROS in the cells. The DCFH-DA fluorescent probe revealed the amount of intracellular ROS was significantly higher in HUVECs exposed to H2O2 alone compared to the control group. This is indicated by the visibility of the DCF fluorescence signal. The DCF fluorescence signal was decreased in HUVECs with pretreatment of FTVN and EPTF or their combination in H2O2-induced HUVEC injury (Figure 4A). Quantitatively, ROS generation in the cells significantly increased as a result of H2O2 exposure to HUVECs, whereas pretreatment of FTVN, EPTF, and the combination of both significantly reduced the intracellular ROS generation by approximately 40% (Figure 4B).

**Figure 4.** ROS generation in cells and the defense mechanism against it. (**A**) Visualization and (**B**) fluorescence intensity quantification on ROS existence. (**C**) Western blot analysis and (**D**) HO-1 expression value. (**E**) Nrf2 nuclear accumulation immunofluorescence stained HUVECs were incubated with 0.1 mg/mL of peptides for 2 h before being challenged with 600 μM H2O2 for 24 h (ROS generation and HO-1 analysis) or within 2 h for Nrf2 analysis. The data are provided as means <sup>±</sup> SD (*<sup>n</sup>* = 4). a–c (ROS generation) and <sup>±</sup> SD (*<sup>n</sup>* = 3). a–c (HO-1 and Nrf2 analysis). Different letters show significance difference at *p* < 0.05.
