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DPPH Method


Cell damages are induced by Reactive Oxygen Species (ROS). ROS are free radicals, reactive anions containing oxygen atoms or oxygen containing molecules able to generate free radicals. Some examples are hydroxyl radical, superoxide and hydrogen peroxide.

Main source of ROS in vivo is aerobic respiration, but ROS are also produced during beta-oxidation of fatty acids, in the xenobiotic compounds metabolism trough cytochrome P450, in phagocytosis stimulation of pathogens or lipopolysaccharides, etc. ROS and oxidative stress in general are involved in some chronic conditions such as Alzheimer and Parkinson disease, cancer and aging.

Figure: Main oxigen radical species


Starting from an O2 molecule and adding one electron to the external orbital the reduction product of molecular oxygen: the superoxide anion (O2 .- ). It is produced during the oxidative phosphorylation, by enzymes (i.e. xanthine oxidase) and leukocytes. Due to its toxicity all aerobic organisms developed different isoforms of the antagonist enzyme: the superoxide dismutase (SOD). SOD is a very efficient enzyme able to combine the superoxide anion with two H+ catalyzing the dismutation reaction through a metal based co-factor yielding H2O2 and O2 as final products. If not properly and promptly inactivated the superoxide anion can create damages to membranes lipids, proteins and DNA.

Figure: Superoxide radical


In normal conditions, in our body, ROS are inactivated through enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). SOD is a key enzyme able to inactivate the superoxide radical, one of the most reactive and therefore the most dangerous radical species.

Figure: In vivo generation of superoxide anion and its enzymatic inactivation paths


To reduces the harmful effects of ROS, cells have developed different defensive strategies including enzymatic and non-enzymatic systems. Considering the antioxidant enzymes, some of these are playing a preventative role eliminating directly ROS. Among these enzymes superoxide dismutase is the first line of defense removing the superoxide anion, the first and most reactive radical derived by molecular oxygen. SOD is therefore one of the main antioxidant defensive system present in almost all the cells exposed to oxygen. The SOD catalyzed reaction is a dismutation with a second-order kinetic based on the following half reactions:

DPPH Method

The antiradical capacity has been assessed using the DPPH method. The sample is placed in a concentrated solution of a standard free radical (1,1-diphenyl-2-picryl-hydrazyl) and its concentration is measured via spectrophotometry to assess the ability of the phytocomplex to quench the radicals. Superox-D has an high antiradical capacity due to quenching mechanisms.

16 folds more antiradical compared to melon

37 folds more antiradical compared to SOD from melon

Figure: Structure of the radical DPPH


Antiradical capacity (DPPH method) of carot, melon and commercial SOD from melon





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Superox-D: effects on intestine epithelium model

Superox-D has been tested for its ability of being up taken and able to provide protection at intestinal level. A human intestine model of enterocyte-like cell Caco-2 was selected been widely used as a model of the intestinal epithelial barrier. It has been well documented that Caco-2 monolayers represent a reliable correlate for studies on the absorption of drugs and other compounds after oral intake in humans.

Up take studies


To assess the ability of being efficiently absorbed in the intestine, the Caco-2 cell model were incubated for 2 hours with media containing different concentration of Superox-D. The ability of Superox-D of being up taken was assessed measuring the Total Antioxidant Activity (TAA) of cell cytosol. The data reported in Figure show that Superox-D is able to effectively penetrate the cell membrane and to induce an increased antioxidant capacity in cell cytosol. This effect is due to the ability of Superox-D of being promptly up taken by the intestine proving the high bioavailability of the product.

Antioxidant protection studies

The protective effect of Superox-D was assessed by measuring the ability of some enterocyte-like cell Caco-2 model culture of better resisting to intense antioxidant stress. The cell lines were pre treated for 2 hours with media containing different concentration of Superox-D. After the incubation with Superox-D a fresh control medium was used in all samples and the effect of an oxidative stress caused by tert-butyl hydroperoxide (t-BOOH) was assessed with a fluorometric assessment. As shown in Figure the cell lines pre treated with Superox-D proved to be far more resistant to the radical stress induced during the assay with a dose dependent behavior. Superox-D is therefore able of keeping the intestine protected from the harmful radical species.

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