## 3 Results
### The Plant Extracts Increase the Selected Intestinal Epithelial Differentiation Markers
Con—cells treated with the appropriate amount of solvent. Phalloidin–Texas Red staining for actin indicates the presence of a brush border in the differentiated Caco-2 cells (arrows). Nuclei are counterstained with Hoechst 3342. Differential interference contrast images (DIC) show the spheroid structure. **(b)** The projection areas of the spheroids from all the experimental groups do not differ significantly during the culture (mean \(\pm\) SD, n = 5); ns—differences not significant. and a similar trend was observed for the sea buckthorn leaf extract (RL), although the differences did not reach statistical significance (Figure 2b-d). Figure 2: Mucin (alcian blue) and intestinal alkaline phosphatase (ALPI) staining of Caco-2/HT-29-MTX spheroid cultures (**a**) and 2D cultures on glass slides (**b**). Quantitative analysis of microscopic images from mucin (**c**) and ALPI staining (**d**). The plots are expressed in arbitrary values (mean \(\pm\) SD) of image analysis software (ImageJ Fiji), the lower the value, the darker staining colour (the same letters represent lack of statistically significant differences at \(p<0.05\)). (**e**) Trans-epithelial electric resistance (TEER, mean \(\pm\) SD) measured in Caco-2/HT-29-MTX monolayer cultures on inserts (Transwell chambers, BD Biosciences); ns—differences not significant. (**f**) ALPI enzymatic activity (mean \(\pm\) SD) in lysates from the differentiated Caco-2/HT-29-MTX cultures, expressed as the initial rate of p-nitrophenol phosphate (p-NPP) hydrolysis (\(\mu\)mols of p-NP/(min \(\times\) mg protein)); ns—differences not significant. (**g**) Immunoblotting detection of ALPI and villim-1 in the lysates from the differentiated Caco-2/HT-29-MTX cultures. Probing for \(\beta\)-actin serves as a loading control. The numbers below the protein bands express the band intensity normalised to \(\beta\)-actin in relation to the control (Con = 100%). (**h**) In-gel detection of ALPI enzymatic activity using NBT/BCIP colour reaction. RL—sea buckthorn extract (50 \(\mu\)g/mL); RO—sea buckthorn fruit extract (50 \(\mu\)g/mL); DL—Cornelian cherry leaf extract (50 \(\mu\)g/mL); DO—Cornelian cherry fruit extract (50 \(\mu\)g/mL); Con—cells treated with the appropriate amount of solvent; NT—non-treated cells. Panels (**c,d**) created with [[https://www.biorender.com/](https://www.biorender.com/)]([https://www.biorender.com/](https://www.biorender.com/)). Formation of the impermeant cell barrier with functional tight junctions is a hallmark of well-developed intestinal mucosa. 2D Caco-2/HT-29-MTX cultures on inserts are a widely established model for studying the intestinal epithelial barrier, and its integrity can be assessed by measuring the trans-epithelial electric resistance (TEER). In our experimental setting, the presence of the plant extracts during the 2-week culture did not affect the TEER values (Figure 2e). Nevertheless, all the tested plant extracts increased the protein levels of ALPI and villin 1, a structural protein that binds actin filament bundles, stabilising the brush border structure (Figure 2g). The differences in the ALPI activity in the lysates (Figure 2f,h) were not statistically significant.