The skin care elixir of life

PCR-June-Ingredients-Savannah-Figure-5Historical references point to shiitake’s powerful anti-ageing benefits. Emperors of China would eat vast amounts of this mushroom to keep themselves young. Scientific studies have shown that shiitake is rich in antioxidant compounds, which help reduce levels of reactive oxygen species and DNA, preventing protein and lipid damage, contributing to reduced cellular apoptosis and increased cell viability.

The mushroom has also gained attention from the cosmetics industry as it’s considered a powerful anti-ageing candidate to introduce into skin care formulations.

Functional collagen is key for young skin

Young skin is mostly characterised by its firmness, elasticity and resilience, mainly provided by functional collagen and elastin. Type I collagen is one of the most abundant proteins in skin, providing tissue structure and strength. The biosynthesis of collagen is a complex process. It includes multiple steps that contribute to the protein’s proper structural and biological functions. One of the most critical is the post-translational modifications (PTMs). These PTMs include the hydroxylation of lysine residues by the enzyme lysyl hydroxylase (LH), encoded by the PLOD gene. These hydroxylysines are precursors of cross-links that help provide tensile strength and mechanical stability, indicating PLOD importance in offering a resistant and younger looking skin. 

Once the collagen molecule is formed, it can endure several deterioration processes, such as carbamylation. This non-enzymatic post-translational modification (NEPTM) is caused by various factors. Carbamylation has been studied for its implication in modifying the collagen structure, inducing destabilisation, and in reducing fibril organisation and cohesion, leading to skin ageing. When compared with other well-known NEPTM of glycation, carbamylation has shown to have a stronger implication in ageing, due to its stronger accumulation of derived products. 

Efficacy tests with positive resultsPCR-June-Ingredients-Savannah-Figure-1

Lipotec has conducted several efficacy tests on Actifcol, a shiitake mushroom-based extract, to confirm its potential activity in increasing type I collagen and PLOD1 levels, as well as in reducing carbamylation. The results corroborate its rejuvenating capacity.

The expression of certain genes contributing to firmness was evaluated using microarray analysis. Multilayered human skin models were treated with culture media with 10 percent Actifcol, or a buffer solution as a control. After incubation, cells were lysed and RNA isolated. The ingredient showed to upregulate the expression of type I collagen (44.2 percent), PLOD3 (128.4 percent), elastin (69.4 percent) and fibronectin (33.6 percent) mRNA, versus the control.

Human dermal fibroblasts (HDFa) were incubated with one percent Actifcol for 48 hours, or only the medium as a control. The ability of the active ingredient to boost the synthesis of type I collagen was assessed by an enzyme-linked immunosorbent assay (ELISA) technique, resulting in an increase of 36.6 percent (p<0.01), compared to non-treated cells.

HDFa were treated with two or three percent Actifcol, or only the medium as a control. The ELISA technique was used to evaluate the ingredient’s ability to boost the levels of PLOD1, which is associated with an improved quality of collagen. Results showed an increase in the protein by 17.5 (p<0.1) and 21.9 percent (p<0.01), when treated at two or three percent respectively, and compared to the control.

Collagen type I at 20µg/mL was incubated with 0.1 M potassium cyanate to induce carbamylation and it was either treated with two percent Actifcol advanced botanical ingredient or with only the medium as a control. Protein carbamylation was evaluated using the ELISA technique. Levels of carbamylated adducts were then determined with a carbamylated bovine serum albumin (CBL-BSA) standard curve. After the active treatment, carbamylation decreased by 75.9 percent (p<0.0001) versus control, indicating a reduction in collagen deterioration.

 PCR-June-Ingredients-Savannah-Figure-2Mollecular structure and fibril organisation

The ability of Actifcol to improve the structure and organisation of collagen fibrils was assessed in human skin explants placed in culture medium, which underwent carbamylation.

The carbamylated explants were either treated with two percent advanced botanical ingredient, a placebo cream or were left with only the medium. As a control, non-carbamylated explants were kept only in the medium throughout the experiment. At the end of treatments, the molecular structure of collagen was evaluated for each explant by X-ray diffraction technique. This provides patterns characterising collagen’s microstructure. Fibril organisation was assessed using atomic force microscopy Technique (AFM), which can deliver microscopic images of collagen fibril bundles.

When fibrils are correctly arranged, the diffraction peaks related to collagen are narrow and intense. When they are disrupted, they are wider and weaker. After treatment, the diffraction signal was like that of the control, suggesting an improvement in the collagen structure (see Figure 1). Large, straight and parallel bundles, typical of well-preserved collagen, were obtained at the end of treatment, achieving results like those of the non-carbamylated samples (see Figure 2). These results indicate the promising effects of the botanical ingredient in reducing collagen’s carbamylation-induced deterioration process.

 Firmness, maximal deformation and anisotropyPCR-June-Ingredients-Savannah-Figure-3

A clinical test was performed on 19 female volunteers, aged between 41 and 55. Twice a day, they applied a cream containing two percent Actifcol advanced botanical ingredient on the neck, décolleté and half face, and a placebo cream on the other half. After 56 days of treatment, different parameters were evaluated.

The maximal deformation (R0) parameter, inversely proportional to firmness, was evaluated using a cutometer. At the end of treatment, R0 decreased by 13.5, 10.1 and 14.4 percent on the skin of the face, neck and décolleté respectively (see Figure 3).

Skin’s linearity, which determines its displacement and returning profile, was assessed using a device that combines mechanical force and imaging. This provides a 3D view of skin’s suction and return to its original state. After 56 days of product application, skin became more isotropic and uniform in all directions with a recovery to the starting point, typical of a young skin behaviour (see Figure 4).

PCR-June-Ingredients-Savannah-Figure-4Proven youthful and rejuvenating efficacy

Enhanced skin firmness can now be achieved with Lipotec’s Actifcol advanced botanical ingredient. This shiitake-based extract has been selected to look after collagen in all stages of its life cycle, contributing to younger-looking skin.

The ingredient showed, in vitro, to increase the expression of genes associated with firmness, and to increase the synthesis of type I collagen protein. It also boosts PLOD1 protein levels, suggesting an improved quality of collagen.

Actifcol also helps decrease the levels of carbamylation, involved in the collagen deterioration process. When evaluated on human skin explants, the ingredient improved the structure and organisation of collagen fibrils, even after carbamylation.

The rejuvenating efficacy of this advanced botanical ingredient was assessed on women presenting skin flaccidity. The treatment resulted in firmer, smoother and more isotropic skin of the face, neck and décolleté of the volunteers.


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