ANTI-POLLUTION
ingredient. The product was used twice a day (morning and night), after facial cleansing. At day 0 and day 28 of the study,
corneocytes were collected from the volunteers’ skin. Carbonylated proteins of these cells were stained on D-Squames with the anti-carbonylated proteins probe (fluorescein-5-thiosemicarbazid (FTZ) (Lifetechnologies, Ref. F-121) and incubated for 30 minutes in the dark and at room temperature. The FTZ reacts with carbonyl groups in
the corneocytes, then carbonylated proteins are visualized by the green fluorescence. The staining was observed in epifluorescence and assessed by image analysis. Results show that after 28 days of twice-daily use, the citrus limon fruit extract active induced a significant decrease of carbonylated proteins by 61% on 90% of the volunteers (Figure 2).
In vitro tests: quantification of IL8 production in cells The citrus limon fruit extract active ingredient controls the expression of the main inflammation mediators & decreases skin redness. The anti-inflammatory properties of the test product were evaluated following the application of the cytokine IL1β to NHEK. IL1β was added to the NHEK culture medium at 10ng/ml for 24 hours. After induction of the inflammatory
phenotype, the culture media were removed and replaced with culture media containing the citrus limon fruit extract active molecule at 3ppm and Dexamethasone at 0.4ppm as positive control for 24 hours. After a 24-hour incubation period, the supernatants were collected and used in an ELISA test to quantify IL8. Results show that the citrus limon
fruit extract active molecule significantly decreases the amount of IL-8 produced by 30% compared to the untreated control. Dexamethasone is more effective in this study since it decreases the IL8 produced by 43%, but this compound is considered a drug and not a cosmetic active ingredient (Figure 3).
In vivo tests: reduction of redness The complexion was evaluated by the CLBT® clinical method. This evaluation, conducted by trained assessors, is based on a visual analysis in vivo of the following descriptors, clearly defined by consensus: a beige, olive,
Skin explants 40 *** 30 20 10 0 Untreated -85% UNTREATED UVA
Figure 2: The citrus limon fruit extract active ingredient decreases carbonylated proteins, which are a marker of oxidative stress in cells
100% 75% *** 50% 25% 0% Untreated IL 1-β IL 1-β
Dexamethasone 0.4ppm
IL 1-β
Citrus limon fruit extract active 3ppm
Figure 3: IL8 production quantified by ELISA in keratinocytes exposed to a pro-inflammatory treatment using IL1ß and Dexamethasone or citrus limon fruit extract active molecule during 24h. ** 0.001<p<0.01 *** p<0.001
red-pink and light pink skin colouring of each volunteer’s facial skin. The scoring of perceived skin descriptor
intensity (from 10 to 100%) is made possible with structured and analogical scales. Results show that after 28 days of application, a significant decrease of the volunteers’ skin redness is observed (-30%).
Ex vivo tests: protein quantification in skin explants exposed to UVA Skin explants were exposed to UVA 50J/ cm2
for three days and treated with 2% of citrus limon fruit extract active (100ppm of active molecule) 24 hours before, during and 24 hours after exposure. Explants were embedded in paraffin and cut into sections.
The skin sections were deparaffined and
labelled with anti-Lamin B1, Elastin, MMP1 or ꙋH2AX antibodies (Lamin B1, Elastin and MMP1 are discussed in the next paragraphs.). Then skin sections were rinsed in PBS solution and revealed with secondary antibody coupled with an Alexa Fluor . Finally, the cell nuclei were labelled with
DAPI. The fluorescent protein was observed and quantified by fluorescence microscopy (Leica, DM5000).
The citrus limon fruit extract significantly
reduces the number of cells with DNA damage after irradiation of skin explants with UVA. Indeed, exposure to UVA increases by 35% in the quantity of cells presenting DNA damage detected by the staining of ꙋH2AX. The
UVA + CITRUS LIMON FRUIT EXTRACT ACTIVE
** -30% DAY 0 DAY 28
43
UVA
UVA + Citrus limon fruit extract
Figure 4: Quantification of ꙋH2AX positive cells labelled by immunofluorescence in skin explants after exposure UVA and treatment with citrus limon fruit extract active (2%), ꙋH2AX in green and nucleus in blue
www.personalcaremagazine.com July 2024 PERSONAL CARE
IL8 production
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