FR2991166A1 - ACTIVATOR ACTIVE PRINCIPLES OF SKIN CELL MITOPHAGIA AND USE TO IMPROVE SKIN CONDITION - Google Patents

Abstract

The object of the invention is the use in a cosmetic composition of an effective amount of at least one activator of cutaneous cell mitophage as a cosmetic active ingredient. The invention also relates to cosmetic compositions comprising at least one mitophage activator for cutaneous cells as a cosmetic active ingredient, and a cosmetic method for detoxifying the skin and / or for preventing or combating cutaneous aging.

Description

The present invention relates to the use of mitophagy activators for improving the condition of the skin, in particular for detoxifying and / or preventing and treating skin disorders. / or fight against skin aging. The invention also relates to cosmetic compositions intended to improve the state of the skin, comprising at least one active ingredient activating the mitophage of the cells of the skin and a cosmetic process for detoxifying the skin and / or for preventing and / or or fight against skin aging, comprising the topical application to the skin of such compositions. The skin undergoes many stresses daily which lead to the production and accumulation of deleterious molecules in the cutaneous cells. This intoxication of the skin results in unsightly visible manifestations, in particular the premature appearance of the effects of skin aging: loose skin, wrinkled, rough, dry, with pigmented spots and dilated vessels. Many products exist to try to fight against these unsightly skin manifestations, but we are always looking for more efficient products. The object of the present invention is to meet this need by proposing a new effective means for improving the state of the skin and to protect it against stresses and aggression that may impair its proper functioning. For this purpose, the invention is directed to the use of an active ingredient capable of acting at the level of the mitochondria, in particular an activating active ingredient of mitophagy in the cells of the skin.

Mitochondria are essential for the life and death of eukaryotic cells. In addition to their main role in producing energy in the form of ATP, they are involved in many processes such as amino acid and lipid metabolism, calcium homeostasis and assembly of iron / sulfur cores which are co-factors. essential for many enzymes. Mitochondria are highly dynamic organelles that are constantly elongating and dividing. This mitochondrial dynamic is the biological phenomenon that counteracts the deleterious processes that the cell undergoes through two activities: the repair of non-functional organelles by fusion and the elimination of organelles damaged by fission thanks to mitophagy. There are currently cosmetic products presented as stimulating the mitochondria and thus increasing the cellular energy level. In particular, these are products involved in the energy metabolism of mitochondria, capable of activating cytochrome C, increasing the expression of cytochrome oxidase or increasing the synthesis of ATP. However, these products assume the presence of mitochondria in good working order and in sufficient number.

However, the stress and various aggressions of the skin alter the mitochondria and their functioning in particular because of the accumulation of proteins damaged by reactive oxygen species (RO5) and mutations of mitochondrial DNA. In addition, with age and overexposure to the sun, the skin cells are no longer able to evacuate these damaged mitochondria. In addition, since aging is associated with a decrease in autophagosome formation, the number of damaged mitochondria greatly increases in aged skin cells.

Existing products acting on mitochondria are therefore not efficient enough. By acting on mitophagy, autophagy own mitochondria, the present invention overcomes these deficiencies. The activation of mitophagy makes it possible to fight against the accumulation of deleterious molecules in the cutaneous cells produced in case of stress and thus to prevent and / or attenuate the resulting manifestations. According to a first aspect, the invention therefore relates to an activating active ingredient of mitophage of cutaneous cells, in particular keratinocytes and / or fibroblasts, for its use as a cosmetic active ingredient and / or dermopharmaceutical intended to improve the state of the skin. skin, in particular to detoxify skin cells, fight and / or prevent against aging of the skin. The invention also relates to a cosmetic composition for topical application to the skin comprising in a physiologically acceptable medium, an effective amount of at least one activator of the mitophage of cutaneous cells. Preferentially, the mitophagy activator of the cutaneous cells is present in an amount of between 0.1 and 10% by weight relative to the total weight of the composition.

Finally, the subject of the invention is also a cosmetic process for detoxifying the skin and / or for preventing and / or combating cutaneous aging, comprising the topical application to the skin of a composition comprising at least one activator of mitophage skin cells. The invention is now described in detail.

The object of the invention is therefore an activating active ingredient of cutaneous cell mitophage, for its use as a cosmetic active ingredient and / or dermopharmaceutical intended to improve the state of the skin.

By active principle activator of cutaneous cell mitophage means any molecule or mixture of molecules capable of activating the mitophage of skin cells, in particular fibroblasts and / or keratinocytes. It is preferably an active ingredient activator mitophage of cutaneous cells derived from or obtained from a plant, a yeast or an algae. According to a particularly suitable embodiment, it is an activating active ingredient of cutaneous cell mitophage derived or obtained from: at least one plant selected from Helianthus annuus, Ceratonia si ligua, Nigella sativa, Withania somnifera, Secale Cereale, Sesamum indicum, Ophiopogon japonicus, Alisma plantago-aquatica, Oryza sativa, Tropaeolum majus, Cichorium intybus, Prunus persica, Verbena officinalis, Panicum miliaceum, Rosa canina, Camelina sativa or Phaseolus vulgaris; or - at least one alga selected from Pterocladia capillacea, Palmaria palmata or Chlorella vulgaris; or at least one yeast selected from Metschnikowia hawciiensis, Candida magnoliae, Saccharomyces cerevisiae, Torulaspora delbrueckii, Pichia anomala. Mitophagy is a process of selective mitochondrial degradation by autophagy. This mechanism makes it possible to maintain the mitochondrial population by detecting the defective mitochondria so that they are selectively degraded. It plays a decisive role in cell homeostasis by enabling the eradication of potentially harmful mitochondria, such as the so-called giant mitochondria that appear with age, or small mitochondria that have low membrane potential and are considered as non-functional, even dangerous because they are able to produce excess free radicals, or even altered and possibly depolarized mitochondria that can no longer merge with another and thus leave the fusion-fission cycle. With age or premature aging of the skin due to various stresses and aggression, the mechanism of mitophagy decreases and the number of damaged mitochondria in skin cells increases sharply. We then witness an accumulation of deleterious molecules in the cells of the skin which lead to a deterioration of the functioning and the state of the skin. The use of an active ingredient that activates mitophagy on the skin thus makes it possible to eliminate these defective or even dangerous mitochondria, to restore cellular homeostasis and to improve the state of the skin. An activating active ingredient of cutaneous cell mitophage useful according to the invention may be an active ingredient having a stimulating activity for the expression of VbAC-Ub of the skin cells and / or an activity stimulating the expression of LC3 of the skin cells. skin cells and / or stimulating activity of Parkin expression of skin cells and / or stimulating activity of p62 protein expression of skin cells. The VbAC-Ub, Parkin, p62 and LC3 proteins are indeed involved in the mechanism of mitophagy. When the mitochondria are damaged and lose their membrane potential, the PINK1 kinase accumulates and recruits Parkin (E3 ubiquitin ligase) from the cytoplasm to the damaged mitochondria. Parkin ubiquinates the mitochondrial proteins (Mitofuscin 1 and 2, VDAC), resulting in their sequestrations in autophagosomes (via p62 / 5Q5TM1) which then fuse with lysosomes. Furthermore, an active ingredient activator mitophagy cutaneous cells useful according to the invention may be an active ingredient with mitochondrial RO5 inhibitory activity of skin cells.

According to one aspect of the invention, the activator of cutaneous cell mitophage is capable of being selected by a test carried out on cutaneous cell cultures comprising the following steps: - culture of cutaneous cells, keratinocytes or fibroblasts, in a adapted culture medium, - removal of the culture medium and replacement with a culture medium comprising the active ingredient to be tested, - removal of the culture medium and the active ingredient and replacement with a culture medium comprising a stressing agent inducing an aging stress and an active ingredient to be tested; the aging stress can be for example a moderate aggression H2O2, - withdrawal of the aging agent and replacement of the culture medium, and - analysis of the expression of the proteins of LC3, p62, Parkin, VbAC-Ub or the mitochondrial ROS content, and comparison of the results with those obtained on stressed skin cell cultures not treated with the active principle to be tested. If there is an increase in the expression of LC3, p62, Parkin and / or VbAC-Ub and / or a decrease in the mitochondrial ROS content, then the active ingredient tested can be used in a cosmetic composition as 20 activator of the mitophagic cells of the skin. The cellular consequence of an activation of the mechanism of mitopathy by an active ingredient exhibiting a mitochondrial ROS inhibitory activity and / or stimulating the expression of VbAC-Ub, Parkin, p62 and / or LC3, is the reduction of damaged mitochondria. in cutaneous cells, which makes it possible to detoxify the skin and limit cutaneous aging. According to another aspect, the invention therefore also covers a cosmetic composition for topical application to the skin, intended to detoxify and / or prevent and / or fight against skin aging, comprising in a physiologically acceptable medium, an effective amount of less an activating active ingredient of the mitophage of cutaneous cells and at least one cosmetic adjuvant. Preferentially, the mitophagic activator of cutaneous cells is present in an amount of between 0.1 and 10% by weight relative to the total weight of the composition, in particular between 1% and 4%. The compositions according to the invention may be in different galenic forms, adapted for topical administration to the skin. They can be in particular in the form of creams, oil-in-water emulsions, water-in-oil emulsions, multiple emulsions, solutions, suspensions or powders. They can be more or less fluid and have the appearance of a cream, a lotion, a milk, a serum, an ointment, a gel, a paste or a foam, or in solid form. The invention also relates to a cosmetic process for detoxifying the skin and / or preventing and / or combating cutaneous aging, comprising the topical application to the skin of a composition comprising at least one active ingredient activating the mitophage of the cells. skin. The invention is now illustrated by examples and tests. First, it has been verified that the induction of skin cell aging results in an increase in mitochondrial ROS due to altered mitophagy. Then examples of active ingredients were selected by the implementation of a screening test method and then incorporated into examples of composition. 1 / Visualization of cutaneous cell mitophage after aging stress The objective of this study is to evaluate the effect of aging stress on cutaneous cell mitophagy.

This study was performed by flow cytometry on human keratinocytes subjected to a moderate attack of H2O2. The operating protocol is as follows. At 0, normal human keratinocytes are inoculated in suitable culture medium. The cells are incubated at 37 ° C. At D2, the cells are labeled with a MitoSOXTM solution. This solution makes it possible to mark the mitochondrial RO5. The culture medium is replaced by a MitoSOXTM solution, the cells are incubated for 10 minutes. The MitoSOXTM is then removed and replaced by a solution of H2O2, a solution inducing aging stress, for 60 minutes. The keratinocytes are then trypsinized and analyzed by flow cytometry. The mitochondrial RO5 content is analyzed. Mitochondrial RO5 content (%) Normal cells without aggression 0 Aggravated cells + 74% The results show that aging stress increases the mitochondrial RO5 content in cutaneous cells by moderate aggression in H2O2. Mitophagy in the attacked cells is less effective than those of the normal unaffected cells. 2 / Screening of active principles increasing the mitophage of cutaneous cells The screening is carried out according to the aging stress protocol presented in point 1, starting from active principles derived from plants, algae or yeasts. The objective of this study is to evaluate the effect of active principles on the production of mitochondrial RO5, thus on the mitophage of cutaneous cells.

This study was performed by flow cytometry on human keratinocytes subjected to a moderate attack of H2O2. The operating protocol is described following. At 0, normal human keratinocytes are inoculated in suitable culture medium. The cells are incubated at 37 ° C. At D1, the cells are pretreated with the active ingredients: The culture medium is removed and replaced with medium containing or not the active ingredients at 1% or 2%. The cells are incubated for 24 hours. At D2, the cells are labeled with a MitoSOXTM solution, a solution for labeling the mitochondrial RO5: The culture medium is replaced by a solution of MitoSOXTM, the cells are incubated for 10 minutes. MitoSOXTM is then removed and replaced with a solution of H2O2, a solution inducing aging stress for 60 minutes. The active ingredients at 1% or 2% are added to the medium during the aggression. The keratinocytes are then trypsinized and analyzed by flow cytometry. The results are expressed as a percentage of the mitochondrial RO5 content compared with the witnessed attacked cells.

Mitochondrial ROS content / Strained cell indicator (%) Control Strain cells 0 H202 + Helianthus annuus seed extract 1% -79% H202 + Active ingredient from Ceratonia siliqua fruit at 2% -29% H202 + Active ingredient from 2% -29% H202 Nigella sativa seeds + 2% -37% H202 + active ingredient from 2% -terocladia capillacea + 2% active ingredient from Metschnikowia hawa ensis -39% H202 + Active ingredient from Candida magnoliae 1% -12% H202 + Active ingredient from Saccharomyces cerevisiae 1% -11% H2O + extract of Withania somnifera roots at 1% -10% H202 + Secale Cereale seed extract at 1% -14% H202 + Extract sesame hydrolyzed 1% -19% H202 + Ophiopogon japonicus root extract 1% -16% H202 + Alisma plantago-aquatica 1% extract -26% H202 + Active ingredient from Torulaspora delbrueckii at 1% -11% H202 + 1% -20% H202 Hydrolyzed Rice Protein + 1% -17% H202 Tropaeolum Majus Extract + Pichia Extract nomala at 1% -13% H202 + 1% -13% H202 intusbus Cichorium extract + 2% -29% H202 Prunus persica leaf extract + 1% -22% H202 Verbena officinalis extract + Palmaria extract palmata extract 1% -31% H202 + 1% -20% H202 hydrolyzed Millet extract + Active ingredient from Chlorella vulgaris 1% -8% H202 + Active ingredient from Rosa canina 1% -21% H202 + Active ingredient from Camelina sativa at 1% -16% H202 + Active ingredient from Phaseolus vulgaris at 1% -20% The results show that the active ingredients tested allow a reduction in mitochondrial ROS compared to untreated cells. These active ingredients are therefore activators of mitophage of cutaneous cells. 3 / Examples of cosmetic compositions containing an active ingredient acting on the mitophage of cutaneous cells 3.1. Cream expert An example of a cream containing an active ingredient extracted from Palmaria palmata activator of mitophage, having passed the screening test passed in point 2, can have the following composition: 10 A. Water QSP 100% Glycerol (Univar) 3.33% EbTA 0.13% Satialgine (begussa) 0.66% Talc (Luzenac) 1% 15 B. Miglyol 812 (Condea Chemie) 3.33% Ritaphyl IC5 (Rita) 1.33% Ritachol 55 (Rita) 1.33% Ipm (Univar) 3.33% Ritalan C (Rita) 1.33% 20 Montanov 14 (Seppic) 1.33% Lanol 14M (Seppic) 1.33% Brij 72 (Uniquema) 1% Brij 721 (Uniquema) 0.33% C. Phenoxyethanol (Sigma) 0.9% Ethylhexylglycerin (Seppic) 0, 1% b. Active ingredient from Palmaria palmata according to the invention 3% The cream is obtained by carrying out the following steps: - heating A and B at 80 ° C., with mechanical stirring, - emulsifying B in A with stirring, - at 30 ° C. ° C add C in order, and - continue homogenisation until the cream is uniform. 3.2. Anti-wrinkle cream An example of a cream comprising an active ingredient extracted from Prunus persica activator of mitophage, having passed the screening test passed in point 2, may have the following composition: A. Water QSP 100% 3% B. Butylene glycol (Univar) 5% Isohexadecane (Laserson) 2% Rita IPP (Rita) 3% Montanov 202 (Seppic) Arlacel161 (Uniquema) 1% Ritox 59 (Rita) 1% Lanette O (Cognis) 1% C. Phenoxyethanol (Sigma) 0 , 9% Ethylhexylglycerin (Seppic) 0.1% DC 200 (Dow Corning) 0.5% Active ingredient from Prunus persica according to the invention 2% D. Sepigel 305 (Seppic) 4% The cream is obtained by the use of following steps: - mix A, mix B and heat A and B at 80 ° C, with mechanical stirring, - emulsify B in A with stirring, - at 30 ° C add C in order, and - continue homogenization until the cream is uniform. 3.3. Night cream An example of a cream comprising an active ingredient extracted from Metschnikowia hawciiensis activator of mitophage, having passed the screening test passed in point 2, can have the following composition: A. Water QSP 100% Carbopol ETD 2020 (Noveon) 0.4 % B. cethylic alcohol (Stéarinerie Dubois) 3% Stearyl alcohol (Stéarinerie Dubois) 3% DUB MCT 5545 (Stéarinerie Dubois) 4.5% DC 345 (Dow Corning) 7.5% Monomuls 900 18 (Henkel) 3% C. Phenoxyethanol (Sigma) 0.9% Ethylhexylglycerin (Seppic) 0.1% Active ingredient from Metschnikowia hawciiensis according to the invention 3% D. NaOH QSP pH 4.5 The cream is obtained by carrying out the following steps: - mixing A, well dispersing the gel and mix B, - heat A and B at 80 ° C, - emulsify B in A with stirring, - at 40 ° C add C in order, - continue homogenization until the cream is uniform, - adjust the pH with D, and allow to cool with stirring to 30 ° C. 3.4. Mature skin cream An example of a cream comprising an active ingredient extracted from Helianthus annuus activator of mitophage, having passed the screening test passed in point 2, may have the following composition: A. Water QSP 100% 14 1.75% Glycerol (Univar) 6.5% Propylene glycol (Univar) 0.5% Aculyne 33A (ISP) 2.5% Carbopol EDT 2020 (Goodrich) B. Cetiol 5N (Cognis) 3% Eumulgin B1 (Cognis) 1.75% Ritaphyl IC5 (Rita) 3% Patlac IL (Rita) 3% Isopropyl myristate (Univar) 3% Montane 80 (Seppic) 1.5% Montanox 60 (Seppic) 1.5% DC 345 (Dow Corning) 3% C. Phenoxyethanol (Sigma) 0.9% Ethylhexylglycerin (Seppic) 0.1% D. Active ingredient from Helianthus annuus 1% NaOH QSP pH 5.5 The cream is obtained by carrying out the following steps: - mix A, mix B, and heat A and B at 80 ° C, - emulsify B in A under stirring, at 30 ° C add C and D, adjust to pH 5.5 with NaOH, and continue homogenization until the cream is uniform.

Claims (12)

REVENDICATIONS1. Activating active principle of cutaneous cell mitophage, for its use as a cosmetic active ingredient and / or dermopharmaceutical intended to improve the state of the skin. 2. Active ingredient according to claim 1, for use as a cosmetic and / or dermopharmaceutical active principle for detoxifying the skin and / or preventing or combating cutaneous aging. 3. Active ingredient according to one of the preceding claims, characterized in that it is an active ingredient having mitochondrial ROS inhibitory activity of the skin cells and / or stimulating activity of expression of the cells. VDAC-Ub skin cells and / or stimulating activity of LC3 expression of skin cells and / or stimulating activity of Parkin expression of skin cells and / or stimulating activity of skin cells. expression of the p62 protein of skin cells. 4. Active ingredient according to one of the preceding claims, characterized in that it is an active ingredient having mitochondrial ROS inhibitory activity of the skin cells and / or stimulating activity of expression of the skin cells. VDAC-Ub skin cells and / or stimulating activity of Parkin expression of skin cells and / or stimulating activity of p62 protein expression of skin cells, when subjected to a test carried out on skin cell cultures comprising the following steps - culture of cutaneous cells, keratinocytes or fibroblasts, in a suitable culture medium, - withdrawal of the culture medium and replacement with a culture medium comprising the active principle to be tested, - withdrawal of the culture medium and replacement with a culture medium comprising a stressing agent inducing an aging stress and the active ingredient to be tested, - withdrawal of the aging agent and replacing the culture medium with a culture medium without an active ingredient, and - analyzing the expression of LC3, p62, Parkin and / or VbAC-Ub by said cells and / or mitochondrial ROS content, and comparing results with those obtained on cultures of cutaneous cells not treated with the active principle to be tested. 5. Active ingredient according to one of the preceding claims, characterized in that it is derived or obtained from at least one plant, an alga or a yeast. 6. Active ingredient according to one of claims 1 to 5, characterized in that it is derived from at least one plant selected from Helianthus annuus, Ceratonia siliqua, Nigella sativa, Withania somnifera, Secale Cereale, Sesamum indicum, Ophiopogon japonicus , Alisma plantago-aquatica, Oryza sativa, Tropaeolum majus, Cichorium intybus, Prunus persica, Verbena officinalis, Panicum miliaceum, Rosa canina, Camelina sativa or Phaseolus vulgaris. 7. Active ingredient according to one of claims 1 to 5, characterized in that it is derived from at least one alga selected from Pterocladia capillacea, Palmaria palmata or Chlorella vulgaris. 8. Active ingredient according to one of claims 1 to 5, characterized in that it is derived from at least one yeast selected from Metschnikowia hawciiensis, Candida magnoliae, Saccharomyces cerevisiae, Torulaspora delbrueckii, Pichia anomala. 9. Cosmetic composition for topical application to the skin comprising in a physiologically acceptable medium, an effective amount of at least one active ingredient according to one of the preceding claims and at least one cosmetic adjuvant. 10. Cosmetic composition according to claim 9, characterized in that the active ingredient is present in an amount of between 0.1 and 10% by weight relative to the total weight of the composition. 11. Cosmetic process for detoxifying the skin and / or preventing and / or fighting against skin aging, comprising the topical application to the skin of a composition comprising at least one active ingredient activating the mitophage of skin cells. 12. Cosmetic process for detoxifying the skin and / or preventing and / or combating cutaneous aging, comprising the topical application to the skin of a composition comprising at least one active ingredient according to one of Claims 3 to 8.