PEPTIDES
180 160 140 120 100 80 60 40 20 0
+72% +46% MM Juvenile HDF MM +81 µg/ml MM +161 µg/ml MM Centella Reversa Senescent HDF
Figure 5: Graph of recovering detoxification capacity in senescent fibroblasts treated with the Centella asiatica biotechnology active compared to senescent (100%)
than growth medium at 10% (growth medium composition contained benchmark growth factors, used as positive control), showing a growth factor-like effect. Non-treated HDF were propagated and grown in growth medium.
In vitro senescence reversal in HDF cultures In order to analyze the effect of the product, on the cellular ageing of HDF, the expression variation of different senescence markers was evaluated in aged fibroblasts. To obtain a culture of ‘senescent HDF’, the
primary culture was subcultivated repeatedly in maintenance medium at each time it becomes a confluence of 80-90%, for more than 20 times, by means of routine subcultivation method. These cells were characterized and showed
clearly senescent phenotype: morphological and structural changes (increased cell size, change of shape from thin, long, and spindle-like to flattened and irregular, increased membrane rigidity and irregularity, increased number of vacuoles), decreased proliferative index (doubling time > 60 hours) and Beta-Galactosidase (β-Gal) positive staining. These in vitro results demonstrated that
Juvenile HDF +322 µg/ml MM
200 180 160 140 120 100 80 60 40 20 0
+83% +48%
81
MM
MM
+81 µg/ml MM
+161 µg/ml MM Centella Reversa Senescent HDF
Figure 6: Graph of genomic integrity protection in senescent fibroblasts treated with the Centella asiatica biotechnology active compared to senescent (100%)
specific oligopeptide fractions arising from the plant totipotent cell cultures of Centella asiatica have the potential to revert the senescence phenotype of senescent fibroblasts. It has been demonstrated that these oligopeptide fractions have a strong effect reverting the cell senescence processes (such as cell detoxifying or DNA protection) and promoting very efficiently cell proliferation in senescent fibroblasts.
Cell proliferation: β-Gal β-Gal is a hydrolase specifically over- expressed in senescent cells.9
The Centella
asiatica biotechnology active reduced β-Gal levels up to +41% (Fig. 3). Staining with reagent from commercial kit (X-gal): the blue colour indicates the presence of β-Gal.
Cell detoxification: Peroxiredoxin Peroxiredoxin-4 (PrxIV) is a peroxidase exerting protection against oxidative stress. Antioxidant enzyme which inactivates ROS.10,11
DNA protection: Poly(ADP-Ribose) Polymerase-1 Poly(ADP-Ribose) Polymerase-1 (PARP-1) is a nuclear enzyme responsible for the stability of telomeres. Potentiates DNA repair, protecting telomeres and avoiding their shortening.12-14 Increased levels of PARP-1 were obtained in
senescent fibroblasts treated with the Centella asiatica biotechnology active compared to senescent (100%) (Figure 6). A dose response effect based on total peptide content is observed.
Cell morphology: Fibroblast re-shaping After eight days incubation with maintenance medium (MM) and + 10 µg/ml Centella asiatica biotechnology active, reversed morphology of aged fibroblasts to a characteristically younger one was observed (Figure 7).
Increased levels
of PrxIV in senescent fibroblasts treated with the Centella asiatica biotechnology active up to 72% were obtained compared to senescent (100%). A dose response effect based on total peptide content is observed (Figure 5).
Clinical evaluation In vivo tests measure biomechanical properties and wrinkles (firmness and elasticity and clinical evaluation of wrinkle intensity), and facial complexion analysis using VISIA, showing a holistic rejuvenating effect (wrinkles, texture, pores, visible spots, red areas, porphyrins). These studies demonstrate that the asset
+322 µg/ml MM
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November 2023 PERSONAL CARE
% PRX-4 expression levels
% PARP-1 expression levels
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