3.7.1. NRU Assay

The effect of BGT and PLOMW on 3T3 fibroblasts was measured by *in vitro* acute toxicity assay through the neutral red uptake (NRU, Figure 4). The treatment with increased concentrations of BGT and PLOMW non-altered cell viability compared to the control, in both treatments.

**Figure 4.** 3T3 cells viability (%) measured through NRU cytotoxicity assay upon treatment with increased concentrations of BGT and PLOMW (mg/mL). Each column represents the mean + SD of 3 wells/group.

### 3.7.2. Cell Viability by MTT Assay

To evaluate the cytotoxic and adverse cellular effects of BGT and PLOMW, was used the MTT assay. This test assesses the activity of mitochondrial enzymes in healthy cells by measuring the absorbance of purple formazan, formed through an NADP-dependent reaction catalyzed by succinate dehydrogenase in metabolically active cells [50].

**BGT PLOMW** 

As shown in Figure 5, the treatments with increased concentrations of BTG and PLOMW for 24 h, not altered Caco-2 cells viability.

**Figure 5.** Effect of BTG and PLOMW on Caco-2 cells viability.

3.7.3. Bio-Adhesive Ability Assay

In this study, to evaluate the bio-adhesive ability of active substances was used a technique described by Rizza [30]. Our results showed a similar muco-adhesivity of BTG and PLOMW (Figure 6) at 12.5 mg mL−<sup>1</sup> concentration. This concentration represents the final dose of dietary use.
