44 ANTI-POLLUTION UNTREATED CIGARETTE SMOKE EXTRACT
CIGARETTE SMOKE EXTRACT + CITRUS LIMON FRUIT EXTRACT ACTIVE
Keratinocytes
30 25 20 15 10 5 0
UNTREATED CIGARETTE SMOKE EXTRACT
CIGARETTE SMOKE EXTRACT + CITRUS LIMON FRUIT EXTRACT ACTIVE
Fibroblasts 80 60 -55% 40 20 0 Untreated Fibroblasts Cigarette
+ Citrus limon fruit extract active
Figure 5: Quantification ꙋH2AX positive cells labelled by immunofluorescence in keratinocytes or dermal fibroblasts after exposure to cigarette smoke extract and treatment with citrus limon fruit extract active molecule (10 ppm) during 24h. ꙋH2AX in pink and nuclei in blue
citrus limon fruit extract active inhibits the appearance of these damaged cells in the skin by 85% (Figure 4).
In vitro tests: protein quantification in cells exposed to cigarette extract NHEK and normal human dermal fibroblasts (NHDF) were seeded in 96-well plates for 24 hours. The cells were then exposed to a cigarette extract and incubated with the citrus limon fruit extract active molecule at 10ppm. After 24 hours, cells were washed and fixed
with formalin. After permeabilization, cells were incubated with anti- ꙋH2AX antibodies and revealed with a secondary antibody linked to fluorescein. Finally, cells nuclei were marked with DAPI. The labelled protein was observed and quantified by automated fluorescence microscopy (ArrayscanTM
Cellomics). Results show that cigarette smoke extract
increases the amount of DNA damage within skin cells quantified using ꙋH2AX: +28% of keratinocytes and +77% of fibroblasts
SIRT6 gene +66% in fibroblasts
Figure 6: Gene expression quantified by qPCR on skin cells after 24h of citrus limon fruit extract active molecule treatment (3ppm)
containing DNA lesions. Indeed, this histone is phosphorylated within the DNA that has been damaged and broken in the nucleus of the cells. This deleterious effect is counteracted in
the presence of citrus limon fruit extract active molecule which drastically reduces the number of cells with DNA lesions: in keratinocytes we observe a 71% decrease, in fibroblasts a 53% decrease (Figure 5).
In vitro tests: gene expression quantification In the protocol it was used qPCR microfluidic technology according to the protocol of Fluidigm®. Microfluidic technology comes
120 100 80 60 40
Untreated
Citrus limon fruit extract active
+14%
from crossing the world of nanotechnology and gene analysis by q-PCR. System miniaturization has led to the development of a chip that currently allows analyzing 96 conditions versus 96 genes. The chip we designed integrates genes that cover the entire range of skin activity including SIRT6 gene. NHDF and NHEK were seeded at the
concentration of 10,000 cells by well in 96 wells plates. After adhesion during the night, the citrus limon fruit extract active molecule was introduced at 3ppm for 24 hours. Then, the supernatant was eliminated, and
the cells collected in specific lysis solution for mRNA extraction. Lysates were transferred on plate in order to purify mRNA. Afterwards, a reverse transcription system was used. According to the Fluidigm protocol, specific
stages for 96x96 chip preparation were starting. A preamplification step was carried out with the primers used in the chip. Pre- amplified cDNA/PCR mix and primers were deposited on the chip. The mix blending was undertaken by the IFC controllerTM the chip was placed in the BioMarkTM
and then system to Untreated
Citrus limon fruit extract active 10ppm
Figure 7: Sirtuin 6 expression quantified by immunofluorescence in skin cells after 48h of citrus limon fruit extract active molecule treatment (10ppm)
PERSONAL CARE July 2024
carry out real time PCR. The citrus limon fruit extract active
molecule increases the basal expression the Sirtuin 6 gene by 66% (Figure 6). The Sirtuin 6 is a NAD-dependent protein deacetylase tightly bound to chromatin in the nucleus. It acts directly on DNA histones to regulate
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-71%
Fibroblasts
Keratinocytes
Sirtuin 6 production (%)
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