Potentials of clays: COSSMA

Clay masks are mainly well-known for their natural purifying properties. One of the markers used to indicate the degree of skin purity is the number of bacterial porphyrins present in the skin.

Skin porphyrins are metabolic products of Propionibacterium acnes (P. acnes) that can become lodged in pores and can induce the oxidation of sebum, leading to comedogenesis and acne¹.
Therefore, a decrease in porphyrins would be linked to a cleaner skin with less undesired bacteria. Porphyrins can also contribute to the inflammatory reaction around the follicles, which aggravates the presence of inflammatory imperfections².
Supported by several in vitro and in vivo tests, a biotechnological ingredient³obtained from a microorganism isolated from a clay close to the North-Eastern Mediterranean coast of Spain, showed to mimic the effects of clays on the skin and to offer a cleaner, smoother and more beautiful skin for different ethnicities.

figure 1: UV photographs of a volunteer before and after 14 and 28 days of the active treatment. The presence of porphyrins can be seen as coloured fluorescent spots. Photo Lubrizol: Life Science Beauty

Tests in vivo and in vitro

The ability of the biotechnological ingredient to inhibit the biofilm formation of P. acnes, whose role is gaining prominence in the pathogenesis of acne vulgaris was evaluated in vitro. P. acnes in culture medium was incubated with 10 μg/mL Bacillus Ferment for 24 hours. Plates were washed to remove the bacteria not crea-ting biofilm and were treated again with the active ingredient for 48 hours. Finally, plates were washed again, and the viability of P. acnes biofilm was evaluated through an XTT assay. P. acnes biofilm formation was reduced by 54.5% at the end of the treatment, which could help prevent acne.

The aim of the next test was to evaluate the ability of the active ingredient to reduce the skin inflammatory response in the presence of P. acnes. Human keratinocytes were treated with 10 μg/mL Bacillus Ferment with the presence of 100 μL of P. acnes suspension for 24 hours and, interleukin-8 (IL-8) production was assessed by enzyme-linked immunosorbent assay (Elisa). At the end of the active treatment, IL-8 was significantly reduced by 14.2%, helping alleviate acne-prone skin.

The skin regenerative capacity was evaluated through a cicatrisation test. Human keratinocytes were treated with 10 μg/mL Bacillus Ferment for 24 hours. After performing a mechanical wound on the cells, keratinocytes were treated again with the active ingredient and migrated for 18 hours. Skin cell regeneration activity was evaluated by measuring the wound area by before and after images. At the end of the active treatment there was a significant 54.2% regeneration of damaged skin cells culture.

Cleaning & antioxidant effects

The ability of the biotechnological ingredient to clean the skin, by simulating the effect of soaps, was evaluated in vitro by testing the emulsifying properties of the ingredient. 10 μg/mL and 100 μg/mL Bacillus Ferment were dissolved in water and mixed in a test tube with the same amount of an artificial sebum mix. The emulgent activity of the ingredient was assessed by measuring the total emulsified phase. At the end of the treatment, the active ingredient presented emulsifying properties to clean the skin, with an increased emulsified phase of 33% and 39.8% at 10 μg/mL and 100 μg/mL respectively.

The antioxidant effect of the ingredientwas evaluated through the study of the accumulation of reactive oxygen species (ROS) after inducing an oxidative stress in skin cells. Human fibroblasts were treated with 10 μg/mL or 100 μg/mL Bacillus Ferment for 24 hours. Then, cells were treated with hydrogen peroxide

(H2O2) for 30 minutes, to induce an oxidative stress, and finally were stained with the ROS orange dye. The levels of ROS were quantified by fluorescence and imaged by confocal microscopy. At the end of the treatment, the levels of ROS were reduced by 39.6% and 94.0% at 10 μg/mL or 100 μg/mL respectively, suggesting an enhanced antioxidant response.

figure 2: Image of a volunteer before and after the

Cellular oxygen consumption

The aim was to evaluate the ability of the active ingredient to stimulate the oxygen consumption rate of cells, an indicator of normal cellular function and cellular metabolism. Unhealthy cells with dysfunctional mitochondria show a lower oxygen consumption rate, compared to healthy cells⁴. Human fibroblasts were treated with 10 μg/mL or 100 μg/mL Bacillus Ferment for four hours. Oxygen consumption rate was assessed by using a phosphorescent oxygen probe. The treatments with the active ingredient showed an increased oxygen consumption rate by 295.1% at 10 μg/mL and by 617.3% at 100 μg/mL compared to the basal control, suggesting a positive effect on the stimulation of cellular functioning and metabolism. The aim of the next clinical study was to evaluate the ability of the active ingredient to reduce the presence of skin imperfections. 21 female volunteers between 25 and 35 years old applied a cream containing 2% of a solution with the biotechnological ingredient on half face and a placebo cream on the other half, twice a day for 28 days. The effect on improving skin homogeneity was assessed by the evaluation of red spots through the Visia complexion analysis system. The biotechnological ingredient was shown to minimise the appearance of red spots by 6.6% after the treatment, and it visibly demonstrated a reduction of imperfections.

The ability to mimic the multiple effects of clays on the skin was assessed by a panel of volunteers of different ethnicities. 61 female subjects with different ethnicities between 25 and 55 years old, who presented imperfections and showed uneven skin tone, were split into two groups. The first applied a cream containing 3% of a solution with the biotechnological ingredient on the face, while the second group applied a placebo cream, twice a day for 28 days. The skin purifying effect and the skin texture were assessed before and after the active and placebo treatments. On the one hand, the cleansing benefits of the active ingredient were evaluated by calculating the area of porphyrins, a marker of skin purity, in a specific region of the face through the Visia complexion analysis system. After 14 days of the treatment with the biotechnological ingredient, the area of porphyrins on the cheek was decreased by 52.2%, with a maximum decrease of a volunteer of 100%.

The skin texture was evaluated by an expert, who scored the skin smoothness and softness parameters through visual and tactile grading, respecdriaively. The biotechnological ingredient showed to improve skin smoothness and softness by 9.5% and 13.5%, respectively.

References

¹ Saint-Leger D, Bague A, Cohen E, Chivot M. A possible role for squalene in the pathogenesis of acne. I. In vitro study of squalene oxidation. Br J Dermatol 1986. 114(5):535-42.

² Schaller M, et al. Induction of a chemoattractive proinflmmatory cytokine response after stimulation of keraitnocytes with Propionibacterium acnes and coproporphyrin III. Br J Dermatol 2005. 153(1):66-71.

³ Uniclay (INCI name: Glycerin, Water (Aqua), Bacillus Ferment)

⁴ Hynes J, Marroquin LD, Ogurtsov VI, et al. Investigation of drug-induced mitochondrial toxicity using fluorescence- based oxygen-sensitive probes. Toxicol. Sci. 92(1): 186-200, 2006.

Júlia Comas,
Product Specialist,
Lipotec Active Ingredients,
Lubrizol Life Science Beauty,
Gavà Spain,
www.lubrizol.com/beauty

Co-authors:
Elena Cañadas, Global Marketing Director;
Albert Soley, R&D - Lipotec Active Ingredients Platform Manager;
Raquel Delgado, Global Technical Director