Stability-Activity of Verbascoside, A Known Antioxidant Compound, At Different pH Conditions

Objectives. The aim of this work was to assess how chemical modification of verbascoside (VB) from olive mill waste water occurring during incubation at pH values mimicking gastric and intestinal conditions may impact antioxidant activity in two human intestinal cells lines. Quali-quantitative approaches were performed in order to evaluate the specific interactions between VB and its derivative. Methodology. VB was incubated at pH 3 and 7,for 24 h and at 37°C as Temperature. HPLC analysis was performed according to Cardinali et al. (2012) protocol, in order to determine possible modifications of VB concentration and/or possible transformation into derivative products. The antioxidant activity of mixture (composed by VB, IsoVB and oxidized products) and pure compounds, such as VB and IsoVB, was assessed on two human intestinal cell lines (HT-29 and Caco-2) using DCFH-DA probe. The characterization study of interactions was performed using a graphical (Tallarida, 1989) and mathematical approaches (Berenbaum, 1989). Results and conclusion. Verbascoside was found to be stable at pH 3 with a recovery of ~100% after 24 hrs. IN contrast, at pH 7 (approximating intestinal conditions), VB was observed to be unstable with a loss of 62.4% observed and isoVB forming as main auto-oxidative product of VB along with other oxidative products. On the two cell lines, the mixture of oxidative VB products resulted less activity (EC50 ranging from 2.7 to 3.4 ppm) compared to VB (EC50 ranging from 0.24 to 0.29 ppm), IsoVB (EC50 ranging from 0.85 to 1.4 ppm) and VB+IsoVB (EC50 ranging from 0.12 to 0.21 ppm). Cellular uptake also differed between cell lines with higher uptake by Caco-2 relative to HT-29. Both graphical and mathematical methods identified different interactions between VB and IsoVB on two cell lines. In conclusion the antioxidant activity of polyphenols such as VB could be modified in relation to the conditions present in intestinal environment.