132 ANTI-AGEING
used as ingredients in skin care, thanks to their high variety of secondary metabolites with beneficial activity on the skin.15 Moreover, they represent a safer and more sustainable alternative to the more popular plant extracts.16
In the specific case of Rosa
rugosa, a white rose commonly known as Japanese Rose, the authors first prepared stem cell cultures, starting from the plant leaves, and then induced the cells to form special plant “embryo-like” structures, called “somatic embryos”, with the main goal of inducing cell differentiation and enhancing the accumulation of even more bioactive metabolites. Indeed, it is known that, similarly to the development of seed embryos, during the somatic embryogenesis plant cells activate a broad repertoire of genes and biosynthetic pathways, leading to the production of specific molecules, which are normally not expressed by undifferentiated cell cultures.17
In the present
article, we have reported that the extract of Rosa rugosa somatic embryo-enriched cultures, differently from an analogous extract obtained from non-induced cell cultures, was able to promote youth phenotypes and mitochondria activity in skin cells by acting through GDF11 pathway. In particular, the extract derived from Rosa rugosa somatic embryo cultures produced a broad activation of anti-ageing and protection mechanisms in skin cells, thus it was particularly suitable to be developed as a new active ingredient for skin care, more specifically to fight ageing associated dysfunctions.
Materials and methods Plant tissue cultures Rosa rugosa (white Japanese rose) stem cells were produced by proliferation of meristematic leaf cells on solid Agar plates. When the calli reached 2 cm-size, they were transferred to liquid growth medium (Gamborg B5 medium, supplemented with 500 mg/L myo-inositol, 30g/L sucrose and phytohormones) and grown as suspension cultures at 27°C in the dark under constant orbital stirring. After the cells reached a density of about 0.3 Kg per L, they were collected and lysed in a phosphate buffer at pH 7.4 to prepare a water soluble (hydrosoluble) cell culture extract. In parallel, some liquid cultures were induced to differentiate into somatic embryo structures, by repeated treatment with phytohormones and monitoring the developing of differentiated tissues. After 4 weeks, the cultures were collected and the hydrosoluble extract was prepared similarly to that of cell cultures. The resulting extracts were collected and lyophilised in order to obtain a fine powder that was easy to dissolve in water or cell culture media for testing. As abbreviations, we will use RrHEs to refer to Rosa rugosa Hydrosoluble Extract from
PERSONAL CARE EUROPE n GDF11 n SIRT1 n SIRT6
300 250 200 150 100 50 0
** ** ** *** ** ** ** ** **
Untreated RrHEs 0.002% RrHEs 0.01% RrHEc 0.002% RrHEc 0.01% TSA 10ng/ml
Figure 1: Gene expression analysis of GDF11, SIRT1 and SIRT6 in fibroblasts. Cells were treated with the indicated compounds, and the expression level of the genes was analysed by RT-PCR. The values obtained from the PCR band quantification 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.
200 180 160 140 120 100 80 60 40 20 0
* *** * * ** **
Untreated RrHEs 0.002%
RrHEs 0.01%
0.002%+ inhibitor
RrHEs
RrHEs 0.01%+ inhibitor
GDF11 100ng/ml
GDF11+ inhibitor
Figure 2: Smad2 reporter activity in fibroblasts. Cells were transfected with the Smad2 vector, containing the luciferase reporter gene, and the promoter activity was monitored after the treatment with the indicated compounds. The error bars represent standard deviations, and the asterisks indicate statistically significant measures between the sample and its respective control.
Somatic embryo cultures and RrHEc to mean that obtained from Rosa rugosa cell cultures.
Chemical analyses Lyophilised samples of RrHEs were solubilised in water, centrifuged for 15 minutes at 14000 rpm and then diluted 1:1 (v/v) in TriFluoroacetic Acid (TFA) at 2%. An aliquot of each sample (12 μg) was analysed by Liquid chromatography–mass spectrometry (LC-MS/MS) using a high resolution mass spectrometer based on Orbitrap technology (Q-Exactive, ThermoFisher), equipped with EASY-Spray source and interfaced with a system of liquid nanoflow UHPLC liquid chromatography (Ultra High Performance Liquid Cromatography) (UltiMate 3000 UHPLC, ThermoFisher Dionex). The samples were first loaded onto a C18 PepMap 100 precolumn and desalted for 3 minutes with a 0.1% formic acid solution, then they were
eluted and loaded onto the EASY-Spray analytical column (15cm x 75μm ID PepMap RSLC C18, 3μm, 100 Angstrom) using 0.1% formic acid in water (solvent A) at a flow of 300 nL/min. The elution from the column was carried out by increasing the concentration of solvent B (acetonitrile/0.1% formic acid). Mass data (MS) and MS/MS were acquired from the mass spectrometer operating in positive ion mode with a vaporisation temperature of 350 °C, a capillary temperature of 280 °C and a capillary voltage of 1.9kV. The spectrometer was set in data- dependent acquisition (DDA) mode by selecting the 5 most intense ions (isolation window 1.0 m/z, stepped collision energy 20, 40) with a dynamic exclusion range of 15s, selecting the ions with single and double charge. The MS/MS data from the tandem fragmentation of the samples, analysed in triplicate, and the control blank (injection of 5 μL solvent A) were processed using the
April 2020
Luciferase activity (% to untreated cells=100)
Gene expression (% to untreated cells=100)
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