3.5.6. Peptide Absorption Analysis

In order to evaluate the intestinal crossing capacity of the four peptides, Transwell ® insert model peptide absorption, was performed. For this purpose **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E** (75 μg) were added to the di fferentiated Caco-2 cells grown (21 days) on the Transwell ® insert. After 10 min and 120 min, apical and basolateral compartments were collected and analyzed by RP-HPLC and mass spectrometry. As shown in Figure 8, all peptides, although with di fferent extent and fragmentation pattern, can cross the intestinal barrier model.

In the apical compartment (AP), the measured amount of **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E** after 120 min was 60.74%, 88.28%, 87.59% and 68.4%, respectively of the total amount of peptide added (Table 5). These values were due both to the absorption and to a slight fragmentation depending on the action of the brush border peptidases present in the AP. In particular, **N-15-M** gave rise to **V-13-M** and **A-11-M** and **Q-14-R** originated **N-8-R**, **V-10-R**, **I-11-R** and **G-13-R,** and, finally, **A-16-S**, **S-15-S** and **F-14-E** were produced from **A-17-E**. The peptide **E-11-F** gave rise to one fragment, **D-10-F** detectable only by mass spectrometry analysis. The increasing amount of **V-13-M** and **A-11-M** (**N-15-M)** and **A-16-S**, **S-15-E** and **F-14-E** (**A-17-E**) was visible at 120 min (Figure 8A,D, respectively). On the other hand, after 120 min a grea<sup>t</sup> amount of the four peptides was found in the basolateral compartment both with HPLC and with mass spectrometry analysis (Figure 8). In particular, **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E** were estimated to be 0.13%, 0.21%, 0.02% and 0.05% of the initial amount of the peptides, added in the apical compartment (Table 5). All the details about peptides and their fragments absorption analysis were reported in Table 5.

**Figure 8.** Uptake of the peptides by Caco-2 cells monolayer and their detection in AP and BL compartments. Each peptide (75 μg) was administered to the monolayer cells and samples of AP and BL were collected at the indicated time. (**A–D**) RP-HPLC chromatograms of the four peptides in the apical compartment at 10 and 120 min. (**A'–D'**) MS analysis of the peptides present in the basolateral side after 120 min.


**Table 5.** Features of the studied peptides (bold) and their produced fragments, using RP-HPLC and MS analysis.

> a n. d.: not detected with RP-HPLC analysis.
