SKIN PROTECTION
contaminated growth medium and measured the cells ability to survive, with and without the addition of IBR-Pristinizer® The IC50
of contaminated medium was
determined and served as key to measuring cytotoxicity and the IBR-Pristinizer protective activity. In this assay, IBR-Pristinizer (now referred to as ‘Asteriscus graveolens extract’) displayed, a significant protective effect, increasing the IC50
of smoke
contaminated medium from 39% to 48.5% and 56% with 0.001% and 0.002% Asteriscus graveolens extract in the culture medium, respectively.
Study design In the present study, the protective effects of Asteriscus graveolens extract were evaluated against cigarette smoke-induced cytotoxicity on normal human epidermal keratinocytes (NHEK). More specifically, the effects of the compounds were evaluated on NHEK cultured in a cigarette smoke- intoxicated medium for 24 hours and the viability of the cells was evaluated using a standard MTT reduction assay.
Materials and methods Biological model
Normal human epidermal keratinocytes (NHEK) grown at 37C, 5% CO2
in
keratinocytes growth medium. Asteriscus graveolens extract was tested at 0.001% and 0.002%.
Culture and treatment Keratinocytes were cultured in culture medium for 24 hours. The medium was then removed and replaced by assay medium containing either the test compounds or the solvent control (H2
O at 2%). After 24 hours
of pre-incubation, the medium was replaced by cigarette smoke-intoxicated assay medium containing either the test compounds or H2
O. The experiment was
performed using several concentrations of cigarette smoke-intoxicated assay medium
203% THE MECHANISM .
62% 73%
MKK 3/6
p38 USP15 ATF6 69% 66% MK2 203%
Antioxidant
Inflammation
Cell death Figure 2: Protection mechanisms activated by IBR-Pristinizer against pollution.
(8 concentrations ranging from 5% to 90% of intoxicated medium). Cells were then incubated for 24 hours. All experimental conditions were performed in n=5, except for non-intoxicated control in n=10. After treatment, the cells viability was tested using MTT test and calculated according to this formula: viability (%) = (OD sample/OD control) x 100
Results Preliminary remark: The H2 O solvent
control, tested at 2%, had a protective effect against cigarette-smoke intoxication with a shift of the IC50
value from 39.3% under the control conditions to 48.5 and 56.3%, respectively.
the intoxicated control condition to 43%. Asteriscus graveolens extract, tested at 0.001% and 0.002% had a clear protecting effect against cigarette smoke- induced intoxication, with a marked shift of the IC50
value from 39.3% in
Mechanism of action Protection elucidation following gene expression in keratinocytes induced with cigarette smoke contamination A gene expression study (Affymetrix) in a culture of normal keratinocytes, comparing gene expression with vs. without treatment with Asteriscus graveolens extract, provides an indication for potential protective effects of the plant extract (key data summarised in Table 1).
The modulation of the expression observed includes several genes involved in skin protection: l Protection from oxidative damage and stimulation of gene for detoxifying proteins,
l Genes indicating that Asteriscus graveolens extract could have an anti- inflammatory effect,
l Also genes related to increasing cell survival while protected from the mortal effects of environmental pollutants.
Table 1: Effect of Asteriscus graveolens extract (IBR-Pristinizer) on gene expression in cells insulted with cigarette smoke. Values below 1 reflect a down regulation, values above 1 an up regulation Gene
Gene expression (fold-regulation)
USP15 ATF6
MAPKAPK2 ENC1
YWHAE NFIX
IL6ST
ACER3 SCRN1
CYTP4F11
0.27 0.31 0.34 0.38 0.39 0.25 0.3
0.32 I 0.33 2.03
56 PERSONAL CARE September 2015 Possible effect of observed gene expression regulation
Increase NRF2 and the transcription of anti-oxidant genes; wound healing; antioxidant response Reduced cell death mediated by ER stress known to occur following exposure to cigarette smoke Reduced expression of key inflammatory cytokines known to induce inflammation and cell death Increased NRF2 and transcription of anti-oxidant genes
Anti-apoptotic activity following DNA damage known to occur following exposure to cigarette smoke Stem cell survival Anti-inflammatory
mproving skin barrier Anti-allergic
Cytochrome p450-membrane associated protein detoxify and metabolize hazards
ENC1
Keap1
CYP4F11
CYP4F11 CYP4F11
CYP4F11 CYP4F11 CYP4F11
Toxicants
O–
OH–
ER stress
O–
Ub Ub Ub Ub
OH–
NRF2 NRF2 NRF2
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