134 ANTI-AGEING


combinations. At the end of the incubation, the cells were washed with PBS and the activity of luciferase was determined by using the SteadyGlo Luciferase assay system (Promega Corporation, Madison, WI, USA) in the Multiwell Plate Reader Victor3 (Perkin Elmer).


ATP content determination 1 x 104


HDF were seeded in 96-well plates


and grown for 20 h. The cells were then incubated with the compounds for 24h. After the treatment, the cells were incubated at room temperature for 30 min in the culture medium and an equal volume of CellTiter-Glo Reagent (Promega Corporation, Madison, WI, USA) was added to each well, mixed for 2 minutes to induce cell lysis. The luminescent signal of the samples was measured after 15 min at 595 nm by the instrument Victor 3 (PerkinElmer).


Statistical analyses


All the cellular analyses were conducted in triplicates and repeated three times. The results were presented as means ± standard deviations (SD) of three independent experiments. All the statistical tests were performed with the aid of the software GraphPad Prism version 8. A one- or two- way analysis of variance (ANOVA) was used followed by a Tukey’s multiple comparisons post-test; a p value lower than 0.05 was considered statistically significant. The number of asterisks in the graphs indicates the level of significance (***p value ≤ 0.001; **≤0.01; * ≤0.05).


Clinical tests The tests were performed on 20 female volunteers, aged from 35 to 55 years old. Skin elasticity was determined on the right and left half of the face by the Cutometer MPA 580 (Courage & Khazaka, Köln, Germany), before any application (D1/T0) and after 28


a


90 80 70 60 50 40 30 20 10 0


160 140 120 100 80 60 40 20 0


untreated *** *** ***


RrHEs 0.002%


RrHEs 0.001% GD11 100ng/ml


Figure 5: ATP synthesis assay in fibroblasts. Cells were treated with the indicated compounds and the amount of newly synthesised ATP was measured. The values were expressed as percentages to the untreated control, arbitrarily set as 100%. The error bars represent standard deviations, and the asterisks indicate statistically significant measures.


consecutive days of product use, either the cream containing the rose ingredient or the placebo. The measurements were based on the resistance of the skin to the negative pressure (firmness) and its ability to return into its original position (elasticity), allowing to get information about the elastic and mechanical properties of skin surface and objectively quantify skin ageing. In this study, parameters R0 (related to skin firmness) and R2 (related to overall elasticity) were considered. In parallel, photographs of the right and left half of the face were taken with Visioface Quick (Courage & Khazaka), which was a high- resolution integrated reflex camera (over 12 Megapixel). The VisioFace was operated with the software Complete Skin Investigation (CSI) that allowed to calculate the depth of the wrinkles of the eye contour (in pixel). All the obtained measurements were subjected to statistical treatment using Student’s t-test for repeated measurements and were considered statistically significant if p-value was < 0.05.


b ** *** * *


350 300 250 200 150 100 50 0


Neonatal HDF


36 yo HDF 36 yo HDF+RrHEs 0.002%


HDF+RrHEs 0.01%


36 yo 10ng/ml TSA *** ** ** **


n nHDF control n 36 yo HDF n 36 yo HDF+RrHEs 0.002% n 36 yo HDF+RrHEs 0.01% n 36 yo HDF+GDF11 100ng/ml


*** *** * **


Results and discussion Preparation of the Rosa rugosa extracts and cytotoxicity assay To evaluate the potential cell toxicity of the rose culture extracts, those derived from both cell cultures and somatic embryo cultures (RrHEc and RrHEs), and to establish the right concentrations to use in the cell assays, cytotoxicity tests (MTT assays) were conducted on human fibroblasts by testing scalar dilutions of the Rosa rugosa extracts, ranging from 0.1% to 0.001% w/v. The extracts’ results proved to be non-cytotoxic at all the tested doses (data not shown), and for convenience the concentrations of 0.002 % and 0.01 % were chosen for all the following analyses.


Activity of the Rosa rugosa extracts on the expression of GDF11, Sirtuins 1 and Sirtuin 6 From a preliminary screening of plant compounds, we found out that the extract derived from Rosa rugosa somatic embryo cultures (RrHEs) induced the gene


Pro-Col I


Pro-Col III


Figure 6: Protein level determination of GDF11 and collagens in neonatal and 36 years old fibroblasts. (A) Cells of different ages were treated with the indicated compounds and the GDF11 protein level was measured by ELISA assay, using a recombinant GDF11 standard curve as reference. (B) After treating the cells with the indicated compounds, the production of pro-collagen I (Pro-Col I) and pro-collagen III (pro-Col III) was measured by ELISA assay. The error bars represent standard deviations, and the asterisks indicate statistically significant measures.


PERSONAL CARE EUROPE April 2020


ng of GDF11


Luciferase (% to untreated cells=100)


ProCollagen typel/III production (% to 36yoHDF=100)


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