EVALUATION OF CELL ANTIOXIDANT ACTIVITY OF OXI-P® ON HUMAN HEPG2 CELLS
Antioxidant Activity by using the LUCS Technology
Oxi-P® is a phytocomplex of some selected plant extracts possessing a high antioxidant power.
Traditional chemical tests such as ORAC do not inform about the physiological functions of antioxidants. These assays have been discredited for years and strongly discouraged by the main health monitoring authorities (see for example EFSA’s opinion and USDA’s opinion). The study of antioxidant effects on live cell models currently represents the most advanced approach for demonstrating antioxidant physiological effects.
We analyzed the Antioxidant Activity of Oxi-P® by using the ultimate technology based on LUCS (Light-Up Cell System) a patented (EP2235505 and US20110008783) approach developed by the Anti Oxidant Power Company, Toulouse, France. The LUCS test is based on the production of cellular radical species following the addition in the culture medium of a photo-inducible fluorescent nucleic acid biosensor. The effect of light application in presence of the cellular biosensor triggers the production of singlet oxygen which in turn causes the production of ROSs (Reactive Oxygen Species) in a biochemical cascade linked to an increase of emitted fluorescence.
LUCS assay measures the ability of a plant extract to neutralize oxidative stress (see Figure 1).
Figure 1. LUCS technology. The Thiazole Orange (TO) biosensor is added to the cell culture medium and becomes fluorescent after association with the human HepG2 cell nucleic acids. When TO is photoactivated by LED illumination at 480 nm, it’s relaxation is accompanied by an energy transfer to the intracellular dioxygen molecule (3O2) resulting in the production of singlet oxygen (1O2) and a cascade of Reactive Oxygen Species (ROS) production including superoxide anion (O2●–) and hydroxyl radical (OH●). The antioxidant effect of a plant extract can then be measured by its ability to counteract this intracellular ROS production.
The effect is measured by a delay in the kinetic evolution of fluorescence emission. The approach has been standardized on high throughput 96-well plates to allow for reliable statistical analysis.
METHODOLOGY USED WITH Oxi-P®: To evaluate Oxi-P® Antioxidant Activity, HepG2 cells were first seeded in 96-well plates at a density of 75 000 cells/well in Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with Fetal Calf Serum (FCS) and kept in the incubator for 24h at 37°C with 5% CO2. Cells were then incubated in the presence of Oxi-P® (8 concentrations obtained by serial log2 dilutions) during 4 h at 37°C/5% CO2. Experiments were realized in DMEM medium without FCS. At least two independent experiments were realized each on triplicate wells. Cells were treated after the 4 h incubation with the fluorescent biosensor during 1 h and fluorescence was measured (RFU at 535 nm) according to a recurrent 480 nm LED application procedure (20 iterations) of the whole 96-well plate. Kinetic profiles were recorded. Dose-response curves are also presented. Antioxidant cell index (AOP index) is calculated from normalized kinetic profiles according to the formula:
AOP index (%) = 100 – 100 (0∫20 RFUOxi-P® / 0∫20 RFUcontrol)
Dose-response curves, obtained by compiling AOP indices according to logarithm(10) of the sample concentration, are submitted to a sigmoid fit according to the formula:
AOP index = AOPindexmin + (AOPindexmax – AOPindexmin)/(1 + 10(Log(EC50-SC)*HS))
OXI-P® SHOWS A STRONG ANTIOXIDANT ACTIVITY ON HUMAN HEPG2 CELLS
Oxi-P® shows a strong and direct antioxidant power on human HepG2 cells, with an Antioxidant Index (AOP) of 925 (theoretical maximum = 1000) at 5 mg/mL concentration.Oxi-P® also shows a significant antioxidant effect at 2.50 mg/ML, whereas a 90% effect I reached at 4.71 mg/mL.
Kinetic data are shown in Figure 2, whereas Dose-Response data are shown in Figure 3.
Figure 2. Intracellular antioxidant effect is revealed by a time delay in the fluorescence increase triggered by the LED-dependent photo-activation of TO. The figure shows the fluorescence profiles of human HepG2 cells treated 4 h at different Oxi-P® concentrations. Control (non-treated, Ctrl DMEM medium) curve is illustrated in black. Differences between control and Oxi-P® Area Under Curves (AUC) allow to establishing an index (AOP index) that qualifies the intracellular antioxidant effect of Oxi-P®. The data also show that Oxi-P® is not cytotoxic.
Figure 3. Calculation of LUCS index at different Oxi-P® concentrations allows to establish dose-effect profiles that fits well with sigmoid regression, allowing the evaluation of the EC10 (Efficacy Concentration 10%, threshold of cellular effect), the standard EC50 (Efficacy Concentration 50%) and the EC90 (Efficacy Concentration 90%, weakest dose at which the compound acts with a maximum effect). The figure illustrates the dose-effect curve obtained after 4 hours treatment with Oxi-P® on human HepG2 cells.
These quantitative results show that OxiP-® is effective on human HepG2 cells by exerting a strong antioxidant effect. These data may be used as human cell-based references to improve preparation processes of products based on Oxi-P®.
For more information on LUCS technology please refer to the following links: