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Figure 3 – Wound confluence with Koffee ’ Up at 0.1 % & 0.2 % & controls over 48 hours activity , demonstrating the activation of the Nrf2 pathway to be nine times greater than with an untreated control . We also observed an activation of the Nrf2 pathway up to six times greater with Koffee ’ Up at 0.1 %.
Reduction of ROS production
In order to demonstrate that the activation of the Nrf2 pathway is well correlated to an improvement of antioxidant defence , the intracellular reactive oxygen species production has been measured . TBHP has been used to induce oxidative stress , as observed by the drastic increase of intracellular ROS accumulation with THBP .
Figure 4 – Activation of Nrf2 pathway by Koffee ’ Up vs . controls
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The intracellular accumulation of reactive oxygen species ( ROS ) is detected by fluorimetry using a 2 ’, 7- ‘ dihydrofluorescein-diacetate ( DCFH-DA ) probe . Keratinocyte cells were cultured in a complete culture medium of DMEM supplemented with 10 % serum ; the medium was renewed when the cells reached 80 % confluence . For inhibition , the cells were preincubated with the test substances diluted in culture medium for 30 minutes and oxidative stress was induced with TBHP . After three hours , the cells were incubated with 10 μM DCFH-DA in the culture medium at 37 º C for
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30 minutes . The acetate groups on DCFH-DA were removed by an intracellular esterase , trapping the probe inside the keratinocytes . The cells were washed at 37 º C and the production of ROS measured by changes in fluorescence due to the intracellular accumulation of DCF caused by the oxidation of DCFH . Intracellular ROS , as indicated by DCF fluorescence was detected using Incucyte FLR software ; the data were analysed by the total green object integrated intensity ( GCU x μm 2 x well ) of Essen BioScience ’ s IncuCyte HD imaging system . The data were expressed in percentage relative to untreated basal condition . The antioxidant property of Koffee ’ Up is observed by the reduction of intracellular ROS accumulation with a dose-dependent effect . Indeed , relevant 43 % and 54 % reductions in ROS production were observed with 0.1 % and 0.2 % Koffee ’ Up respectively . •
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* Koffee ' Up is a registered trade mark of Givaudan
Melanie Pelican
COMMUNICATION SPECIALIST
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