ANTI-AGEING A ■ Control ■ Reference (FK866) ■ Sunflower sprout extract
120 100 80 60 40 20 0
1 µM Figure 2: In vitro ageing model: Sunflower sprouts boost NAD+
the germination process, seeds undergo a transformation, leading to the conversion, and breakdown of various nutrients.6
This process
significantly enhances the nutritional value of the sprouts, turning them into a potent source of beneficial compounds.
Methods In vitro ageing model Keratinocytes were seeded in 24-well plates, either directly or on glass coverslips, and incubated for 24 hours. The cells were then pretreated with 1% sunflower sprout extract for an additional 24 hours. Ageing stress was induced with BrdU, and cells were further incubated for either 24 or 48 hours, depending on the subsequent analysis. For the analysis of NAD+
with BrdU in the absence or presence of 1% sunflower sprout extract for 24 hours were used. The compound FK866 was included as a control. FK866 is an inhibitor of the enzyme NAMPT, known to be the rate-limiting enzyme for replenishing NAD+ NAD+
levels. levels were assessed in cell lysates
using the NAD/NADHGlo™ assay in accordance with the manufacturer’s protocol. NAD+
levels
were recorded using the GloMax® Discover Microplate Reader. For the analysis of DNA damage, cells grown
on glass coverslips and treated with BrdU for 48 hours in the presence or absence of 1% sunflower sprout extract were used. Cells were fixed, and immunofluorescence staining was performed with a Phospho-Histone H2A.X (Ser139) antibody. Images were captured with a 20x objective, and a quantitative evaluation of the staining was conducted.
Rejuvenation study on skin explants The skin explant, sourced from a 35-year-old Caucasian female donor, was cultured at 37°C with 5% CO2
every 24 hours. To explore potential skin rejuvenation, the skin explants were initially exposed to 6 J/cm2
UV-A radiation.
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Clinical rejuvenation study To evaluate the clinical efficacy of sunflower sprout active, a double-blind, randomized, and placebo-controlled study was conducted. In this study, 22 volunteers aged between 52 and 65 years (average age: 60 years) applied either a cream containing 2% sunflower sprout active or a corresponding placebo cream on each side of their faces twice daily for 42 days. Various skin ageing parameters were
measured after 28 and 42 days of application. These parameters included skin smoothness (data not shown), the lifting of the jawline region, and the depth of crow’s feet wrinkles. For the lifting analysis, 3D images of the face
. The culture medium was refreshed
were captured using the Visia®-CR after 42 days of treatment. The lifting effect was analyzed by measuring the length of three vertical lines between the eyes and the jawline region. The depth of crow’s feet wrinkles was measured on days 0, 28, and 42 using Primos 3D analysis. In order to evaluate whether the volunteers
1 % levels and enhance DNA repair Subsequently, the skin explants were treated
topically either with a placebo cream or a cream containing 2% sunflower sprout active. After applying the cream, the skin explants were incubated for 24 hours and then harvested for further evaluation. To evaluate mitochondrial function and protein
recycling in the skin explants, carbonylation of mitochondrial proteins as a marker of mitochondrial protein damage was analyzed. For this, the mitochondrial proteins were first extracted and then analyzed by immunoblotting using a specific fluorescent probe for detection. Additionally, the collagen density of the skin
levels, cells treated
explants was examined after the treatment. Sections of the skin explants were assessed using XPolar® technology for imaging. The collagen density analysis was conducted on the reticular dermis, the deeper layer of the dermis. This layer is characterized by its densely arranged collagen fibres, embedded within a supportive matrix, which is crucial for the maintenance of skin structure.
appeared younger, the average values of wrinkle depth were compared with a reference dataset comprising over 300 women aged between 30 and 65 years. This dataset includes the recorded age and wrinkle depth of the volunteers.
Results and discussion In vitro ageing model The skin is subjected to a wide array of stress factors. Both external and internal stressors, including chronological ageing, can cause DNA damage. However, the skin’s capacity to repair DNA damage declines with age. As a result, DNA damage becomes a
significant contributor to skin ageing. In the repair process of damaged DNA, NAD+
serves
as a crucial cofactor, which is consumed and consequently depleted. Previous work has shown that sunflower
sprout extract can increase the expression of the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT) that is responsible for NAD+
To simulate skin ageing, an in vitro skin
ageing model in human epidermal keratinocytes was established using DNA damage. In this model, ageing stress is induced both in the absence and presence of 1% sunflower sprout extract by incorporating a thymidine analog, 5-bromodeoxyuridine (BrdU), into DNA, which triggers DNA damage. To demonstrate the dependency of NAD+
levels on the enzyme NAMPT, the cells were additionally treated with the NAMPT inhibitor FK866. The inhibition of NAMPT resulted in a significant reduction in NAD+
levels within the
cells, highlighting the importance of this enzyme in NAD+
recycling and synthesis. depletion of NAD+
Ageing stress by BrdU treatment caused a within the cells. However, co-
treatment of the stressed cells with sunflower sprout extract elevated the NAD+
levels even
beyond those of the control (Figure 2A). These results suggest that sunflower sprout extract enhances the longevity molecule NAD+ cells, even under ageing conditions.
in skin May 2024 PERSONAL CARE synthesis and recycling. B ■ Control ■ Sunflower sprout extract
1200 1000 800 600 400 200 0
1 µM *
33
*p<0.0001 versus untreated control **P<0.0001 versus stressed control
**
1 %
NAD+
levels compared to control (=100) in %
Ageing stress
γH2AX levels compared to control (=100) in %
Ageing stress
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