ISSUE FOCUS 54 FEED & ADDITIVE MAGAZINE May 2024 The University of Antwerp in Belgium, for instance, has established its own validated ICOS model via fluorescence-based measurements in animal gut epithelial cells (i.e. the IPEC-J2 porcine type). Such an assay creates the following benefits for an antioxidant compound or -product compared to other evaluation methods: 1. assessing its bio-availability or uptake by gut epithelial cells (its systemic, whole-body effect) 2. simulating its protective effects on gut integrity (vs. ROS-induced inflammation) and thus also disease susceptibility 3. measuring its antioxidant effect inside the cell (where most oxidative stress is typically generated) 4. allowing a head-to-head comparison with Vitamin E (its water-soluble analogue Trolox®) INTRA-CELLULAR OXIDATIVE STRESS TEST The aim of this ex vivo test is to measure the integrity of IPEC-J2 gut epithelial cells, after an induced oxidative challenge by either hydrogen peroxide (H2O2) or menadione, which are strong ROS generators. This by measuring the oxidation across the cell membrane via fluorescence, with the use of an indicator for ROS radicals. This indicator diffuses into the animal cell and if ROS are present therein, it becomes oxidised. When this occurs, the molecule turns fluorescent and thus can be measured (Figure 4). Therefore, the higher the oxidation, the higher the fluorescence. ICOS & VITAMIN E EQUIVALENCE In a peer-reviewed, independent study conducted by the University of Antwerp1, the above ICOS fluorescence test on animal cells was used to evaluate the biological efficacy of a dietary antioxidant (ELIFE®). The latter is a concentrated blend of different, EUsourced botanical extracts rich in primarily short monomeric and oligomeric polyphenols (covering different subclasses of flavonoids and phenolic acids). Within the setup of this ICOS test, the phytobiotic antioxidant blend was added to the IPEC-J2 cell culture at increasing dosages from 0 (control) up to 1,000 ppm. This was done both with and without an initial triggering of oxidation (generating of ROS). The fluorescence results of the ELIFE® treatments were compared to a treatment of Trolox® (VE analogue), which was added in an amount equating to 1,100 ppm of synthetic VE50 ADS. The intra-cellular ROS content by fluorescence measurement was expressed in arbitrary units. The outcome of the test (Figure 5) proved that ELIFE®’s polyphenols, which are primarily of the short and water-soluble type, penetrated the gut epithelial cells and exerted a significant intra-cellular antioxidant activity. Such a biological action is key, as most ROS radicals are produced inside animal cells due to the high mitochondrial activity. Figure 4. Fluorescence principle of measuring the impact of an added antioxidant (AOX) on intra-cellular ROS presence, after an oxidative challenge to animal gut epithelial cells.
RkJQdWJsaXNoZXIy MTUxNjkxNQ==