ANTI-AGEING 135 a


18 16 14 12 10 8 6 4 2 0


Placebo


RrHEs 0.002% d


b


9 8 7 6 5 4 3 2 1 0


Placebo RrHEs 0.002% c 0


-2 -4 -6 -8


-10 -12 -14 -16


Placebo RrHEs 0.002%


Figure 7: Clinical tests on human skin. 20 female volunteers were treated for 28 days either with a cream containing 0.002% of RrHEs or with a cream without the extract (placebo). The effects on skin firmness (A), elasticity (B) and on wrinkle depth (C) were measured and expressed in the graphs as percentages of variation compared to day 0. The effects on wrinkle depth were also visualised by a 3D camera and photographed (D).


expression of GDF11 and Sirtuins in human dermal fibroblasts. As shown in Figure 1 the treatment with 0.002% and 0.01% of RrHEs increased GDF11 expression by around 18 and 28%, respectively. Analogously, RrHEs significantly induced the expression of the genes of Sirtuin 1 (Sirt1) and Sirtuin 6 (Sirt6) compared to the untreated control. Differently, the extract obtained from Rosa cell cultures, RrHEc, did not produce any effect on the expression of the genes, suggesting that the effect produced by RrHEs was specific. As positive control of the experiment, the compound Trichostatin A (TSA) was used, as it is an activator of GDF11 synthesis.18 To verify whether the increase of GDF11 synthesis determined by RrHEs was associated with a true activation of GDF11 signalling, we measured the induction of the transcription factor Smad2. The fibroblasts were transfected with the Smad2-luciferase reported plasmid and the increase of luciferase activity in response to RrHEs treatment was evaluated. The results, reported in Figure 2, indicated that the treatments with the extract, similarly to GDF11 itself, led to an increase of about 54 % of the reporter activity. The complete inhibition of this increase caused by the treatment with the compound SB-431542 (at concentration of 2 µg/mL), a potent inhibitor of the GDF11 receptor activity,19


indicated that the activation of Smad2 was specifically mediated by the GDF11 production.


Chemical analysis of the Rosa rugosa somatic embryo extract To further investigate on the nature of the


April 2020


extract derived from Rosa rugosa somatic embryo cultures, we performed a chemical analysis in order to find potential interesting compounds and thus proceed with further biological characterisation. The chemical analysis conducted by Mass Spectrometry revealed the presence of important classes of compounds, such as phenolic acids, carboxylic acids, flavonoids and glucosidic derivatives, chalcones and lignans (Table 1). In particular, isoflavones, such as daidzein, have been reported to have a positive effect on mitochondrial biogenesis and functions,20


which have also been recently related to GDF11 activity.


Activities of the Rosa rugosa somatic embryo extract on mitochondria On the basis of the chemical data, we studied the expression of PGC1α and other related genes in cells treated with RrHEs. As it is shown in Figure 3, we found that the extract, used at 0.002% and 0.01%, increased PGC1α expression by 57% and 28%. GDF11 used as positive control increased PGC1α gene expression by 216%. Moreover, we found that RrHEs induced the expression of NRF1 and TFAM in a way similar to GDF11. Additionally, we analysed the expression of the Sirtuin genes related to mitochondrial functions, and we found that RrHEs increased the expression of both Sirt3 and Sirt4 (Fig 4). Since the increase of mitochondrial biogenesis is related to an upregulation of ATP synthesis, we measured ATP content in cells after the treatment with RrHEs. As shown in Figure 5, the ATP production was enhanced in the


cells by the extract, indicating that the treatment with the extract had a positive effect on cell mitochondrial functions and energy production.


Activities of the Rosa rugosa somatic embryo extract on collagen and GDF11 production To investigate on the potential role of RrHEs as anti-ageing ingredient, we performed a series of analyses of GDF11, Collagen I and Collagen III production in cells deriving from 36-year old donors, treated with the extract derived from rose somatic embryo cultures. As shown in Figure 6A and 6B, both GDF11 synthesis and collagen production were compromised by the cell age: the reduction of GDF11 synthesis was around 25 % (Fig 6A), while that of Col I and Col III of around 60 and 40%, respectively (Fig 6B). However, these reductions were completely (or almost completely) abolished by the treatment with the rose extract, at both the used concentrations. A similar effect was obtained on GDF11 production by TSA, and on collagens by GDF11 itself, indicating that the effect produced by the extract was most likely mediated by the activation of GDF11 signalling.


Clinical tests with the Rosa rugosa somatic embryo extract To validate the in vitro results, the activity of RrHEs was clinically evaluated by a series of in vivo tests on human volunteers, using RrHEs at the concentration of 0.002% in a skin care formula and comparing it with a


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% of firmness increase (R0)


% of elasticity increase (R0)


% of wrinkle depth decrease


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