58 ANTI-AGEING
slow down the effects of time and prevent the appearance of wrinkles.
Materials and methods SCBE obtention Commonly known as magnolia berries or five- flavour berries, Schisandra chinensis is grown by producers according to Good Agricultural Practices in the mountainous province of Jilin. The fruit is then dried naturally in the sun. The cultivation of Schisandra by our partner growers enables local people to increase their income and improve their living conditions. Schisandra chinensis berries extracts (SCBE) were obtained by a hydroalcoholic extraction.
In vitro experiments Transcriptional expression NHEK (normal human epidermal keratinocytes) were treated with SCBE 0.01% for 24 hours. Total mRNA was extracted, purified and assayed. Complementary DNA (cDNA) was obtained by reverse transcription (RT) before amplification by quantitative polymerase chain reaction (qPCR). Relative gene expression was determined using the DDCt method after normalizing the data with the expression of the housekeeping gene and then with the untreated control condition. Each experimental condition was performed in biological triplicates (n=3). Variations in gene expression are expressed as % ± standard deviation relative to the untreated control condition.
Protein expression NHEK were treated with SCBE 0.1% or schisandrin alone at 0.0004576% (concentration equivalent to the concentration of schisandrin in SCBE 0.1%) for 24 hours. After fixation and washing, they were incubated with monoclonal primary antibodies specific for LC3 or LAMP2 and then with a secondary antibody coupled to a green fluorochrome. An incubation was also carried out with
DAPI to mark the cell nuclei. Images were taken with an epifluorescence microscope (Leica®, 20x objective) using, for each primary antibody, the same acquisition parameters for the different conditions. Quantification of the green fluorescent signal was performed on at least two separate images per replicate
Figure 1: SIRT6 and FOXO3 signaling pathways, adapted from Li et al., 2021
by normalizing the average green fluorescent signal in relation to the number of cells (DAPI counterstaining). Results are presented as mean protein expression ± standard deviation versus control (untreated NHEK).
Mitochondrial activity NHEK were seeded and then treated with SCBE 0.01% or with schisandrin in equivalent active concentration (0.000045%) for 4h (n=3). A fluorescent probe specific for mitochondrial mass (MitoTracker®) was then added for 30 minutes. After washing, the nuclei were labelled with Hoechst for five minutes before washing and then read using a fluorescence microscope (Leica®) with FITC and DAPI filters. Mitochondrial activity was measured by
quantifying the specific accumulation of the fluorescent probe in active mitochondria compared with the untreated control. Results are presented as mean mitochondrial activity ± standard deviation versus control (untreated NHEK).
Statistical analysis Statistical significances were assessed by Student’s t test. Significant differences from NHEK controls are indicated as follows: *p<0.05, **p<0.01, ***p<0,001.
Clinical study The double-blind study was realized on 40
SIRT6 expression
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
Control
+181% **
3.0 2.5 2.0 1.5 1.0 0.5 0.0
SCBE 0.01% Figure 2: Relative expression of SIRT6 and FOXO3 in SCBE-treated NHEK cells versus control (2-ΔΔCt PERSONAL CARE November 2024 Control ); *p<0.05, **p<0.01
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women, from 36 to 55 years old. Mean age was 49 years old. Subject phototypes were II to IV, presented a dull complexion, a lack of elasticity and wrinkles with a Bazin score of 2 to 3. Bazin score is an ageing scale from 1 to 5, which allows to determine the skin level of ageing according with number, depth and aspect of wrinkles.22
SCBE 1% and placebo
were applied daily in a split-face manner during 28 days. Number of wrinkles and skin roughness
(Ra) were measured on crow’s feet area with an AEVA-HE 3D at D0, D14 and D28. In order to evaluate dynamic elasticity, an air blow was applied on the lower part of the face, and recorded with high-speed rate camera. The elastic deformation of the face was measured at D0 and D28, and the associated recovery time of the skin was measured in milliseconds. The faster the skin returns to its original states, the more elastic it is. Finally, a specific dynamic method was
used in order to reveal future wrinkles that will appear or become deeper in a few years. Skin features density, meaning the ratio of detected features divided by the total surface, was measured on crow’s feet area with an AEVA- HE 3D at D0 and D28. Each subject smiled in the most tension facial position (the subject stays in a fixed position for a few seconds) and then an image was acquired by the AEVA camera. Tension and visible wrinkles on the
FOXO3 expression
+104% *
SCBE 0.01%
Expression relative SIRT6 vs Control (2ΔΔCT
)
Protein relative FOXO3 vs control (%)
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