FR2930446A1 - USE OF A COMPOSITION CONTAINING ERGOSTEROL OR A NATURAL EXTRACT OF MICROORGANISM OR PLANT OR ANIMAL. - Google Patents

Abstract

The present invention relates to the use of a cosmetic composition or as a dietary supplement, containing ergosterol, an ergosterol derivative, or a natural extract of microorganisms, plant or animal containing it, to stimulate the natural defenses of cells, such as the p53 protein, against the consequences of oxidative stress such as exposure to ultraviolet radiation, especially solar radiation, and to prevent the appearance of "senescent cells" that have lost the ability to reproduce and exert adverse effects on the surrounding tissues.

Description

The present invention relates to the use of a composition containing ergosterol or a natural extract of microorganism, or plant, or animal, comprising, to protect the cells of the skin and mucous membranes against the consequences of a oxidative stress such as exposure to ultraviolet radiation.

It is known from document WO-99/13858-A to use ergosterol to stimulate the proliferation of cutaneous cells, document FR-2829390-A to use shiitake mushroom extracts to improve the cutaneous barrier, and JP-2003267873-A to use an ergosterol derivative for lightening the skin. In addition, ergosterol is used in human nutrition as a precursor of Vitamin D. In addition, the repeated exposure of the skin to ultraviolet radiation causes damage to the cells of the skin and mucous membranes, particularly in the skin. cells called fibroblasts which are responsible for the renewal of the macromolecules constituting the cell-free extellular matrix of the dermis (Goukassian D. et al., The FASEB J., 14: 1325-34,2000). This damage is naturally repaired; but when this is not the case, this may have consequences for the proper functioning of the cells (Goukassian D.A. and Gilchrest B.A., Rejuvenation Res.7; 3): 175-85,2004). In addition, it has long been known that skin cells exposed to the sun lose their ability to reproduce more quickly when introduced into in vitro culture (Gilchrest, BA, J Gerontol., 35 (4): 537-41, 1980). Cells that can no longer reproduce are called senescent cells by the specialists in Cell Biology, and are characterized by the strong expression of an active beta-galactosidase at pH 6. More cells expressing this beta-galactosidase are found among the keratinocytes or fibroblasts of the skin of the elderly, as in the cultures of cells aged by successive subcultures (Dimri GP et al, Proc Nat Acad Sci USA 92: 9363-67,1995), or in cultures having under repeated ultraviolet stress (Chainiaux F. et al., Int.J. Biochem Cell Cell Biol 34 (11): 1331-9,2002).

Moreover, it has recently been shown (Debacq-Chainiaux F. et al., J. Cell Sci 118 (Pt4): 743-58, 2005) that fibroblasts lose with age a part of their ability to repair damage caused by ultraviolet (UV) radiation (Goukassian D. et al, 2000, cited above).

Finally, it appears that senescent cells in vivo have adverse effects on the surrounding tissue (Funk W.D. et al., Exp Oeil Res.258: 270-8, 2000) and that it seems important to have means to prevent their appearance. It thus appears that it would be very interesting to have means available to prevent the appearance of senescent cells which have lost the capacity to reproduce and which have adverse effects on the surrounding tissues. Also, an object of the present invention is to provide a means for protecting cutaneous cells against the appearance of senescent cells that are the consequence of oxidative stress such as exposure to ultraviolet radiation.

This purpose, as well as others which will appear later, is achieved by the use of a composition containing ergosterol, an ergosterol derivative or a natural extract of microorganisms, plant or animal including, to protect skin cells and mucous membranes against the consequences of oxidative stress such as exposure to ultraviolet radiation.

Advantageously, the ergosterol is in the form of a water-soluble derivative such as an ester of phosphoric acid or of succinic acid. Preferably, the ergosterol used is contained in a yeast extract such as Saccharomyces cerevisiae. Advantageously, the ergosterol used is contained in a fungus extract. Preferably, the composition according to the present invention also comprises at least one antioxidant such as vitamin E, vitamin C, a carotenoid, a flavonoid, an isoflavonoid, a polyphenol, a procyanidin, resveratrol, epigallocatechin gallate, genistein, daidzein, formononetin, equol, a green tea extract. Advantageously, it comprises from 0.00001 to 95%, and preferably from 0.001 to 1% by weight, by weight of ergosterol. According to the present invention, this composition is intended for application to the skin or the mucous membranes or to be ingested.

Preferably, the composition stimulates the activity of the p53 protein in cells of the skin or mucous membranes. The following description, which is in no way limiting, relates in particular to embodiments of the present invention which will enable those skilled in the art to better understand the latter.

Now, it has now been found quite unexpectedly and surprisingly, that if the fibroblast culture medium subjected to repeated ultraviolet exposures, is supplemented, before the irradiations, by the addition of ergosterol, one very significantly decreases the number of cells that pass to the senescent state characterized by the appearance of beta-galactosidase activity associated with it (Senescence Associated beta-galactosidase or SA beta-gal). The ergosterol used is advantageously contained in a yeast extract such as Saccharomyces cerevisiae.

EXAMPLE 1 Ergosterol Attenuation of Damage to Fibroblasts in Culture by Repeated Exposure to Ultraviolet Radiation The ergosterol used is a product marketed by LGC Promochem (France). Dermal fibroblasts of the same BJ strain as used by Chainiaux F. et al (2002, already cited) are cultured by conventional methods.

The absence of cytotoxicity of ergosterol over a period of 24 hours is verified in the range 0.1 to 0.5 μg / ml. For 5 days the fibroblasts are exposed once a day to a dose of ultraviolet B of 200 mJ / cm 2. 24 hours before the first UV treatment, and following each irradiation, the culture medium is replaced by medium containing (0.1), (0.25), and (0.5) pg / ml ergosterol. Before each new irradiation, these media are replaced by a medium containing no ergosterol. After the last irradiation, the cells are left for 72 hours in contact with medium supplemented with ergosterol, renewed every 24 hours. At the end of this last period of contact, the beta-galactosidase activity at pH6 (beta-galactosidase associated with senescence or SA beta-gal) is evaluated according to the method of Dimri GP et al (already cited), on at least 400 cells for each ergosterol concentration.

The experiment was performed twice. The mean values of the percentages of cells expressing beta-gal SA are summarized in Table 1 below: Table I Condition SA beta-gal SA beta-gal lere experiment 2nd experiment Control without UV 31.04 41.19 UV and 0 pg / ml of ergosterol 38.65 49.19 UV and 0.1 μg / ml of ergosterol 25.13 34.32 UV and 0.25 μg / ml of ergosterol 24.82 35.78 UV and 0, 5 μg / ml of ergosterol 27.80 31.78 It is found that: the damage caused by the repeated exposures to ultraviolet B rays caused a significant increase of fibroblasts presenting the beta-gal SA activity characteristic of the cells that do not can no longer reproduce (described as senescent), the treatment of cells by ergosterol before and after irradiations, has allowed a mitigation of damage caused by ultraviolet at a concentration of 0.1 pg / mI, which is translated by a number of senescent fibroblasts which remains lower than that of cultures without i no irradiation. It thus appears that the use of ergosterol makes it possible, according to the invention, to attenuate the cellular damage caused by exposure to ultraviolet radiation.

Example 2: To protect the cells against oxidative stress, it is advantageous to activate the p53 protein (see Magalhaes J.P. et al., Expert Gerontol 39 (9): 1379-89, 2004).

Stimulation of p53 protein expression in the fibroblasts of Example 1 was evaluated under the influence of 0.5 μg / ml of ergosterol in the culture medium for 24 hours. A positive control was achieved by irradiating a culture of the same cells by a dose of 150 mJ / cm 2 of UVB.

After these treatments, the nuclear proteins were extracted from the fibroblasts. 5 g of nuclear proteins were then incubated in a microplate coated with an oligonucleotide carrying the specific DNA sequence capable of binding the p53 protein.

Finally, the content of bound p53 protein was evaluated by colorimetry using an antibody specific for this protein and a peroxidase-conjugated antibody (TransAM test of Active Motif, Rixensart, Belgium). The results obtained are summarized in Table II below: Table 1 It was thus surprisingly found that ergosterol treatment in the absence of UV increased by 30% the p53 protein content in the nucleus of the cells. fibro-blasts. It has also been confirmed that ultraviolet exposures, in the absence of ergosterol, increased the p53 protein content in the fibroblast nucleus by a factor of 4. This example surprisingly demonstrates that ergosterol stimulates the p53 defense protein necessary to prevent damage caused by ultraviolet radiation such as the appearance of senescent cells that have lost the ability to reproduce. and which have adverse effects on the surrounding tissues. Various compositions are given hereinafter by way of example to illustrate the different modes of action or use of ergosterol.

Composition N ° 1: Cosmetic cream of! protection against oxidative stress. The procedure is as for the production of a conventional oil-in-water emulsion, that is to say that a hydrophobic phase containing 0.05% ergosterol and an aqueous phase is mixed with stirring at 85 ° C. containing each of the raw materials intended to give the product the usual properties of use: stability, safety, easy spreading, pleasant final touch, etc. It is then cooled with stirring. p53 Test conditions Opg / ml control ergosterol 1 0.5 μg / ml ergosterol 1,3 Opg / ml ergosterol + UV 45 The proportion of hydrophobic phase relative to the aqueous phase is such that the final proportion of ergosterol is 0.04% by weight. This cream is applied to the face, neck and hands, preferably in the morning so that the principle stimulating the natural defenses has time to penetrate to the living cells, to be in contact in the middle of the day when the intensities solar irradiation are at their maximum.

Composition No. 2: Cosmetic cream for protection against oxidative stress. As above, an oil-in-water emulsion containing ergosterol, alpha-tocopherol and alpha-tocopheryl acetate is prepared; when cooling to 45 ° C, vitamin C phosphate is added in an amount sufficient to reach the concentrations by weight in the final composition of: - ergosterol 0.02% - alpha-tocopherol 0.05% - alpha acetate tocopherol 0.2% - Vitamin C phosphate, Mg 0.2 salt This formulation is a day cream.

Composition No. 3: Cosmetic cream for protection against oxidative stress and filter protection. As in Composition No. 1, an oil-in-water emulsion with an addition of titanium oxide sufficient to obtain an SPF protection factor equal to 6 is carried out, then a dry alcoholic extract of Saccharomyces cerevisiae is added with stirring at 45 °. C in sufficient quantity to arrive at 2% of extract by weight in the final preparation. The product is intended to be applied in the morning on uncovered skin areas, and if possible, the application should be renewed mid-day.

Composition No. 4: Cosmetic gel for protection against oxidative stress, for sensitive skin. An aqueous-alcoholic acrylic gel titrated with 0.01% by weight of ergosterol and containing, in addition, 2% by weight of glycolic acid extract of Glycyrrhiza glabra.35 Composition No. 5: Dietary supplement to protect the skin before exposure to the sun or pollution.

Tablets containing 10 μg of ergosterol per tablet and a powder formulation comprising 0.1% by weight of ergosterol are prepared by conventional techniques.

These tablets should be taken with a little water during the morning and mid-day meals, one to two tablets per dose depending on the intensity and duration of exposure expected.

Composition No. 6: Dietary supplement to protect the skin before exposure to the sun or pollution.

Gelatin capsules containing 100 mg of a dry extract of

Saccharomyces cerevisiae comprising about 2% by weight of ergosterol.

These capsules are to be taken as the tablets of the composition No. 5.

Composition No. 7: Dietary supplement to prepare the skin for sun exposure.

Capsules containing 60 mg of a 2% by weight ergosterol powder formulation, 15 mg of green tea extract and 25 mg of vitamin C per capsule are made.

The recommended daily dose is 2 to 4 capsules a day in two doses in the morning and in the middle of the day with a little water at the time of the meal. Composition No. 8: Drink to protect the skin during sun exposure. A soy milk drink containing 0.01% by weight of ergosterol is produced.

This drink is presented in doses of 100 ml.

It is advisable to drink a dose in the morning and a dose in the middle of the day at mealtimes.

Claims (10)

REVENDICATIONS1. Use of a composition containing an ergosterol, an ergosterol derivative or an extract of a microorganism, plant or animal comprising it, for protecting cells against the consequences of oxidative stress such as exposure to ultraviolet radiation purples. 2. Use according to claim 1, characterized in that the ergosterol is in the form of a water-soluble derivative such as an ester of phosphoric acid or succinic acid. 3. Use according to claim 1, characterized in that the ergosterol used is contained in a yeast extract such as Saccharomyces cerevisiae. 4. Use according to claim 1, characterized in that the ergosterol used is contained in a mushroom extract. 5. Use according to any one of claims 1 to 4, characterized in that it also comprises at least one antioxidant such as vitamin E, vitamin C, a carotenoid, a flavonoid, an isoflavonoid, a polyphenol, a procyanidin, resveratrol, epigallocatechin gallate, genistein, daidzein, formononetin, equol, green tea extract. 6. Use according to claim 1, characterized in that it comprises from 0.00001 to 95% by weight of ergosterol. 7. Use according to claim 6, characterized in that it comprises from 0.001 to 1% by weight of ergosterol. 8. Use according to any one of claims 1 to 7, characterized in that the composition is intended for application to the skin or mucous membranes. 9. Use according to any one of claims 1 to 7, characterized in that the composition is intended to be ingested. 10. Use according to any one of claims 1 to 7, characterized in that the composition stimulates the activity of the p53 protein in the cells of the skin or mucous membranes.