In this work, Opuntia ficus indica juice was explored as a potential source of natural antioxidant and anti-inflammatory ingredients towards intestinal inflammation. An adsorption separation process was used to produce a natural flavonoid-rich concentrate (FRC) from Opuntia ficus-indica juice. The FRC effect (co- or pre-incubation) on induced-oxidative stress and induced-inflammation was evaluated in human Caco-2 cells. The main constituents identified and present in the extract are flavonoids (namely isorhamnetins and their derivatives such as isorhamnetin 3-O-rhamnose-rutinoside and isorhamnetin 3-O-rutinoside) and phenolic acids (such as ferulic, piscidic and eucomic acids). Our results showed that co-incubation of FRC with the stress-inducer attenuates radicals production in a much more significant manner than pre-incubation. These results suggest that FRC compounds which cannot pass the cell membrane freely (isorhamnetin derivatives) have an ability to inhibit the formation of H₂O₂-induced radicals in the surrounding environment of intestinal epithelial cells. The capacity of FRC (co-incubation) for suppressing (at the extracellular level) free radicals chain initiation or propagation reaction was probably related with a more pronounced reduction in protein oxidation. A similar response was observed in the inflammatory state, where a marked decrease in IL-8 secretion and blocked degradation of IκBα was achieved for FRC co-incubation. Simultaneously, treatment with FRC significantly reduces NO and TNF-α expression and modulates apparent permeability in Caco-2 cells. In these cases, no significant differences were found between pre- and co-incubation treatments suggesting that bioavailable phenolics, such as ferulic, eucomic and piscidic acids and isorhamnetin, act at the intracellular environment.

α-Tocopherol (TOC) has become an important food additive due to its antioxidant effect and capacity to increase shelf-life of products. However, it is a hydrophobically active compound, which compromises its applicability in soft drinks and fruit beverages. The aim of this study was to develop sustained release forms of α-tocopherol using zein and cyclodextrin as carriers to be further applied in fruit beverage products for improved shelf-life. For this purpose, the encapsulation of α-tocopherol into zein protein was first optimized using a spray-drying technology. Different process conditions, namely temperature (110–180 °C) and solids concentration (0.01–0.04 g mL⁻¹), were tested aiming at maximizing the encapsulation of α-tocopherol and antioxidant capacity using response surface methodology (RSM) following a Face Centred Design (FCD). Results showed that lower inlet gas temperature and lower solids concentration enable the production of α-tocopherol:zein forms with a higher antioxidant activity (oxygen radical absorbance capacity >90 μmol Trolox equivalent antioxidant capacity per g), and the highest encapsulation efficiency (42.05%) was obtained using 0.02 g mL⁻¹ of solids concentration. β-Cyclodextrin was further added to the best system to increase the solubility capacity of α-tocopherol in fruit juice beverages. Despite the lower antioxidant activity (46.2 μmol Trolox equivalent antioxidant capacity per g), α-tocopherol:zein:cyclodextrin forms reduced colour losses when compared to the normal juice, and this effect was probably related to the suppression of anthocyanin degradation due to the formation of inclusion complexes with β-cyclodextrin. Importantly, none of these forms induced cytotoxicity in the Caco2 cell model, confirming the safety of these systems.

In this work, pressurized fluid extraction was explored to recover isothiocyanates (ITCs) and phenolic compounds from watercress. Pretreatment of the raw material was studied, using different conditions of temperature (25 °C; 35 °C), pressure (P_atm; 25 MPa), incubation time (0; 30; 60; 120 min) and moisture content (125; 250; 900% of water, dry basis), aiming at promoting the enzymatic hydrolysis of glucosinolates in ITCs. Extractions of ITCs were performed with supercritical CO₂, and different mixtures of CO₂ : ethanol (0–50, % w/w) were applied to obtain phenolic-enriched ITCs extracts. Extractions were performed at 35 °C and 25 MPa for 2 h and extracts analyzed concerning total ITCs, phenethyl isothiocyanate (PEITC) content, phenolic composition, antioxidant capacity (using ORAC, HORAC and HOSC assays) and antiproliferative effect in a human colorectal cancer cell line (HT-29). Results showed that supercritical CO₂ was highly selective in isolating ITCs from watercress (up to 31.7 ± 1.6 μmol ITC per g). When mixtures of CO₂ : ethanol were used, extraction of phenolics (up to 10.1 ± 0.8 mg GAE per g) and antioxidants (up to 204.4 ± 21.5 μmol TE per g concerning ORAC, 70.8 ± 10.7 μmol CAE per g for HORAC and 189.5 ± 22.9 μmol TE per g for HOSC), was promoted. PEITC was the main compound responsible for the inhibition of cancer cell growth of all extracts (EC 50_{24 h} = 27.8 ± 1.9 μM PEITC). However, extracts obtained by supercritical CO₂ extraction after a 30 minute incubation period with CO₂ and with CO₂ : ethanol mixtures (80 : 20 and 60 : 40, % w/w) were revealed to be effective in isolating other bioactive compounds that enhanced the antiproliferative response of extracts (EC 50_{24 h} values of 23.1 ± 0.9; 20.7 ± 1.9 and 19.8 ± 0.7 μM PEITC, respectively).