70 ANTI-POLLUTION
■ Forming a skin barrier by applying products that form a protective film so as to keep toxic and ROS-generating molecules away from skin and to inhibit, or at least reduce, penetration of pollutants ■ Applying antioxidants to replace those consumed by ROS or to inactivate ROS molecules before they can react with the skin and trigger MMP production ■ Using chelators, such as EDTA, to inhibit heavy metal reactions from being catalysed
ESR studies ex vivo Proderm has carried out a series of ex vivo studies using electron spin resonance (ESR) on pig skin sourced from abattoir waste in order to measure free radical generation directly after the application of different doses of UV light. The samples were first exposed to two model pollutants: cigarette smoke applied in a smoke chamber, as a model indoor pollutant; and urban dust in a gel formulation. The latter is widely used as a NIST standard reference material for PM in the US. A natural hydrophilic phenylethanoid
antioxidant, H1, was tested in some of the samples against a common antioxidant, tocopherol. EDTA was also tested as a chelator. The results showed that H1 clearly reduces the formation of free radicals (Figure 1), whereas tocopherol has a weak effect for low UV doses only (Figure 2). EDTA “was a disaster, at least in this experiment”, Bielfeldt said. It did not reduce free radicals at all and had a slightly pro-oxidative effect (Figure 3). Overall, the tests indicated that H1 has a
stronger effect than tocopherol or EDTA, both with or without cigarette smoke (Figure 4), on the two models. Urban dust generates a comparable level of free radicals, compared to cigarette smoke, Bielfeldt said. In addition, 5% urban dust in a gel vehicle induces approximately the same number of free radicals on skin as fresh cigarette smoke over ten minutes.
In vivo findings Proderm also carried out in vivo assessments on the backs and forearms of volunteers. In these, fresh cigarette smoke as the model pollutant was applied to the skin using a pump to transport the smoke to a chamber that is fixed on the skin. The new developed system administers a number of well standardised
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(smoked) Figure 5: In vivo induction of malondialdehyde by cigarette smoke PERSONAL CARE November 2021 Water (smoked) EDTA p<0.001 p<0.001 p<0.001
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Test sample (no. and short label) 1. Water (negative control)
2. Water + cigarette smoke (vehicle, positive control) 3. Antioxidant H1 (1%), (treatment control) 4. Antioxidant H1 (1%) + cigarette smoke 5. Tocopherol (1%)
6. Tocopherol (1%) + cigarette smoke 7. EDTA (0.1%)
8. EDTA (0.1%) + cigarette smoke 9. Physiogel (control for UrbanDust) 10. Physiogel + cigarette smoke 11. Physiogel + Urban Dust 5% 12. Physiogel + Urban Dust 2.5%
Figure 4: Induced free radicals compared to no UV irradiation
puffs of smoke to the skin. Swabs were then taken to harvest lipids
from the skin and put into a buffer solution. These were assessed by measuring the concentration of malondialdehyde (MDA) and squalene monohydroperoxide (SQQOH) as markers for sebum lipid oxidation (Figure 5 & 6). Both of these are already present on untreated skin that has not been subject to smoke. The results were quite similar to the ESR findings. H1 reduced the level of both MDA and SQQOH on skin exposed to smoke to a greater degree than tocopherol, which itself had almost no effect on SQOOH. It did not, however, bring the level of either marker down to that on the untreated skin that was not subject to smoke. Water as a control and EDTA both had no effect, though EDTA at least did not act as a pro- oxidant as it had in the ex vivo trials.
In vivo human study designs Based on these tests, Bielfeldt continued, Proderm has developed in vivo test designs to support claims of instant and long-term protection. In each MDA and SQOOH levels are quantified by LC/MS or GC/MS on ten participants, for the effects of smoke on the barrier lipids on the upper back and for its effects on sebum lipids in the forearms. The test products can be up to three leave-on
cosmetic products, plus a positive control (an untreated area exposed to smoke) and a negative control (an untreated area not exposed to smoke). For the instant protection tests, products
are applied, left for minutes, exposed to pollution for 15 minutes and tested. In the long-term tests, they are applied daily for four days and tested after 15 minutes of exposure to pollution on the fifth day.
Outlook Proderm’s testing, Bielfeldt claimed, showed that the ex vivo data it generated on the release of free radicals were confirmed in vivo findings. Cigarette smoke and assessment of lipid peroxidation work both ex vivo and in vivo; the results on selected antioxidants were found to be comparable; and the NIST standard and cigarette smoke lead to comparable results “These findings confirm the reliability of our in vivo cigarette smoke model,” he said. Thus, cosmetic claims of ‘anti-pollution efficacy’ can be assessed successfully by the use of in vivo model studies with cigarette smoke. The company also performs studies with rinse off products using harmless PM 2.5 carbon particles and has also established an in vivo anti-pollution method that uses the ozone radical as the model pollutant, which it will soon add to its portfolio of testing methods.
Induced free radicals (%) with ± standard deviation
22.2 62.1 19.1
34.4 20.4 51.7
28.2 79.4 23.4 62.3 54.1 42.6
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PC
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Figure 6: In vivo induction of SQOOH by cigarette smoke
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