66 ANTI-POLLUTION
supplemented media. Trolox (150 μg/ml) was used as a positive control, while cells treated with media alone were used as an untreated control. After the test materials were added to the cells, 10 μl of 1 mg/ml urban dust was added to each well. One set of cells was not exposed to urban dust (non-urban dust control). A baseline fluorescence measurement
was then made using a Fluoroskan Ascent fluorometer. Measurements were made on the underside of the 96-well plate so as to obtain direct measurements of the cells. The measurements were made using an excitation wavelength of 485 nm and an emission wavelength of 518 nm. After this baseline measurement was made, the cells were incubated for two hours at 37±2°C and 5±1% CO2
60 minute incubation■ 120 minute incubation ■ , with additional fluorescence
measurements made at the one and two-hour time points. A UVLM-26 lamp was used as the source
of UVB light. UV light intensity was measured using a UVX radiometer coupled to a UVB sensor probe (UV Products) to determine the time required to deliver an approximate dose of 100 mJ/cm2
. After exposure, the PBS was
removed and replaced with fresh FDP media and the cells were incubated for 120 minutes, with fluorescence measurements made at 60 and 120 minutes. After loading the dermal papilla cells with
DCF-DA, the cells were treated with the test materials prepared in 100 μl of phenol red free Hanks balanced salt solution (with 1 mM sodium bicarbonate and 5 mM HEPES) and a baseline fluorescence measurement was obtained as described above. The DP cells were exposed to 470 nM
blue light. Changes in fluorescence were measured after 30 and 60 minutes of blue light exposure as described above. For the ROS assays, mean fluorescence values were calculated for each treatment group and compared using the ANOVA method.
Results & discussion Figure 2 shows the data generated from the study in which particulate pollution was employed to generate ROS in the DP cells. Exposure of the DP cells to 100 μg/ml of Urban Dust for 60 and 120 minutes caused a statistically significant upregulation in cellular ROS. The application of 0.015% Trolox was effective
at reducing the expression of ROS. BHT had no impact on reducing ROS at 0.001% or 0.005%. The antioxidant complex was able to reduce the expression of the ROS significantly at both 1% and 3%, indicating profound efficacy in controlling ROS generated from urban particulate pollution. Results from the UVB study can be seen in
Figure 3. Exposure of the DP cells to 100 mJ/cm2 of UVB radiation for 60 minutes and 120 minutes resulted in statistically significant upregulation of cellular ROS. The application of Trolox after the UVB exposure was able to suppress the expression of ROS in the affected cells. BHT was not able to control the ROS expression at the levels examined in the study. It can be noted, again, that the antioxidant complex is able to effectively reduce the expression of ROS after UVB exposure, comparable to the effects seen from Trolox.
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Figure 2: ROS formation due to exposure of DP cells to 100 μg/ml of urban dust Note: Measured in relative fluorescence units (RFUs). Asterisks indicate statistical significance against untreated DP cells
60 minute post UVB■ 120 minute post UVB ■ *
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* * * * * Non-Exposed Untreated
0.015% Trolox 0.005% BHT 0.001% BHT 3% SC-Help Defense
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Figure 3: ROS formation due to exposure of DP cells to 100 mJ/cm2 Note: Measured in RFUs. Asterisks indicate statistical significance against untreated DP cells
Figure 4 shows the results from the HEV
study. Recent publications have suggested that exposure to LED screens has little impact on skin ageing or ROS expression.4-6
However,
even short exposure to sun light can generate a strong HEV ROS response. This study has proven that the exposure of DP cells to 13.5 mJ/ cm2
of blue light for 30 minutes and 27 mJ/cm2
of blue light for 60 minutes results in a strong expression of ROS. The application of Trolox after the HEV
exposure was able to control the expression of ROS as was BHT, at the longest exposure times. The antioxidant complex was able to also control the expression of cellular ROS effectively at both time points, comparable to the influence seen from Trolox.
Conclusion The results found in the studies discussed demonstrate that DP cells are vulnerable to exogenous stresses that can induce the
of UVB radiation
formation of cellular ROS. The ingredients that have been chosen for the new antioxidant complex are able to effectively reduce the expression of ROS in these treated cells, regardless of the stress imposed. Only Trolox showed a somewhat comparable
indication of ROS control. In these studies, BHT was quite ineffective at reducing cellular ROS in any of the stresses at the concentrations examined, except at the longest exposure to HEV. While BHT is a well-known antioxidant, it is evident that its protective benefits might be over-exaggerated. The scientifically-proven botanical blend that has been specifically developed for use in scalp applications has the profound ability to effectively reduce cellular ROS induced by all three oxidative stresses: pollution, UVB and HEV light. This benefit can manifest itself by control of cellular ROS in the follicle resulting from unwanted exogenous threats including particulate pollution, UVB and HEV light). It
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ROS Formulation (RFU)
ROS Formulation (RFU)
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