66 SKIN PROTECTION Vehicle control Cumene hydroperoxide 100µM
Cumene hydroperoxide 100µM + T.obliquus caroteinoids0.01%
Figure 6: T.obliquus carotenoids prevent lipid peroxidation. Epifluorescent images of primary human dermal fibroblasts. Cell nuclei are stained in blue. Vehicle treated cells contain only a minor fraction of lipid peroxides and are stained in red (left panel). Addition of 100 μM cumene hydroperoxide lead to a significant increase in lipid peroxidation, as can be seen by the colour change of the dye from red to green (middle panel). T. obliquus carotenoids provide full protection against cumene hydroperoxide induced lipid peroxidation (right panel). The white bar represents 100 μm.
There are two major types of
carotenoids: those that are completely lipophilic are called carotenes and those with polar head groups (i.e. oxygen- containing carotenoids) are called xanthophylls. Lipophilic carotenoids such as β–carotene are incorporated in the lamellar skin barrier lipid system or are integrated between the lipid bilayers of
biomembranes. In contrast, xanthophylls, including lutein, are substances located in the transmembrane region (Fig 2). This means carotenoid accumulation in the skin barrier provides a protective network for lipids in the lamellar lipid system. Carotenoids incorporated in cellular membranes inhibit lipid peroxidation and divert excess energy away from the
vulnerable unsaturated fatty acid tails of the membranes. β–carotene and lutein undergo synergistic interactions with each other and α-tocopherol, which can result in the regeneration of β–carotene.17-21 Tocopherol, in turn, is regenerated by the water soluble vitamin C. It is because of all this that it is important to maintain a fully functional network of antioxidants in our skin.
Methods UV/VIS spectrophotometry: The cosmetic active was diluted 1:10 in 90% acetone and a UV/VIS spectrum over the range 350 - 800 nm was recorded. Oxidative stress with hydrogen
peroxide: Subconfluent, proliferating HaCaT cells were incubated with 500 µM
hydrogen peroxide for 20 minutes with or without 0.005% T. obliquus carotenoids in DMSO (corresponds to a 0.5% concentration of the active). Levels of intracellular reactive oxygen species (ROS) were determined using the Cellular Reactive Oxygen Species Detection Assay Kit (DCFDA). Protection against lipid peroxidation: Primary human dermal fibroblasts were grown over night and incubated with T. obliquus carotenoids at 0.005 % or 0.01 % or correxponding DMSO vehicle controls, respectively. After 30 minutes, fluorescent staining reagent of the Image- iT®
Lipid Peroxidation Kit and 100 μM cumene hydroperoxide were added. Lipid peroxidation was measured by fluorescent imaging and quantification. Reduction of WiFi induced oxidative
stress: For the irradiation experiment, a prototype WiFi emitter system was developed (Fig 7) that very realistically simulates the exposure of skin to the WiFi radiation of smart phone devices. Conventional WiFi emitters were placed beneath a 24-well cell culture dish at a distance of 10 mm. The radiation intensity was set to 0.5 mW or 50 mW, the typical everyday emission ranges of these devices. The experiment was performed under tight temperature control avoiding heating of the culture medium by irradiation and a Faraday cage shielded the surroundings from the WiFi radiation. Subconfluent, proliferating keratinocytes were placed on the WiFi emitters and irradiated for 5 hours directly after application of the T. obliquus carotenoids. Subsequently, levels of intracellular reactive oxygen species (ROS) were determined using the Cellular Reactive Oxygen Species Detection Assay Kit (DCFDA).
Figure 7: Cell culture WiFi emitter system within a Faraday cage. PERSONAL CARE EUROPE Reduction of lipid peroxidation and April 2019
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