Figure 4.

Induction of ARE/Nrf2 reporter activity in keratinocytes by combinations of TNC with carnosic acid or rosemary extract. KERTr keratinocytes were transfected, and ARE/Nrf2 reporter activity was measured as described in Materials and Methods. Concentrations of lycopene from the TNC were 10 µM. (a) Combination of TNC with carnosic acid. Concentrations of carnosic acid were as indicated in the figure. (b) Combination of TNC with rosemary extract. The extract was added to give the indicated concentrations of carnosic acid. Lycoderm^TM was added in a concentration that provided 10 µM of lycopene and 5 µM of carnosic acid. The results were normalized to cell number, which was estimated by the crystal violet method. Values are the means ± SEM of 2–4 experiments, each done in triplicate. The sum of the effects of each compound alone was calculated () and compared to the effects of the combinations of polyphenols and carotenoids (). * p < 0.05 indicates that the combination is significantly different from the sum of each compound alone. The vertical line marked “C” indicates ARE/Nrf2 induction by the combination that is above the calculated sum of the induction by the separate compounds. (c–g) Combination index calculations of the synergy between TNC and carnosic acid. Different concentrations of TNC, carnosic acid, or their combinations at the ratios indicated in the figure were added 6 h after transfection. (c) Dose-effect curve at concentrations of 1.25–20 µM and at TNC:carnosic acid ratios of 1:1, 2:1, and 4:1. The µM concentrations shown on the x-axis are of each of the compounds alone or of carnosic acid in the combinations. Results are from two experiments, each done in triplicate. (d) Fraction affected-combination index plots of the data presented in (c). (e–g) Average CI values for the TNC–carnosic acid combinations. (h,i) Different concentrations of lycopene in TNC, carnosic acid in rosemary extract, or their combinations at a ratio of 4:1 were added 6 h after transfection. (h) Dose-effect curve at concentrations of 1.25–20 µM and at TNC:carnosic acid ratios of 4:1. The µM concentrations shown on the x-axis are of each of the compounds alone or of carnosic acid (from rosemary extract) in the combinations. Lycoderm^TM containing lycopene:carnosic acid at a ratio of 1.8:1 was added at the indicated concentrations of carnosic acid. (i) Fraction affected-combination index plot of the data presented in (h).