WO2007060302A1 - Use of a composition containing an ecdysteroid - Google Patents

Abstract

The invention concerns the use in cosmetics, pharmaceutics or as nutritional supplement, of a composition containing an ecodysteroid, an ecodysteroid derivative, or a natural plant or animal extract containing same, to stimulate the natural defences of cells, such as protein p53, against the consequences of their being exposed to ultraviolet rays in particular solar radiation.

Description

 Use of a composition containing an ecdysteroid

The present invention relates to the use of a composition containing an ecdysteroid, an ecdysteroid derivative or a natural plant or animal extract comprising, to stimulate the natural defenses, such as the p53 protein, cells of the skin and mucous membranes against the consequences of exposure to ultraviolet radiation.

Ecdysteroids are polyhydroxylated steroids carrying a 7-ene-6-one group, a 14 alpha-OH, and having a cis junction between the A and B cycles. They are insect-shedding hormones, but also occurs frequently in plants (Lafont R., Chem.Listy 97,280-281, 2003).

Many effects on mammals, including humans, have already been highlighted:

- stimulation of growth and reproduction, cell proliferation and differentiation, protein, carbohydrate and lipid metabolism, nervous and cardiovascular systems, detoxification of liver and kidney;

- anti-inflammatory and immunomodulatory effects;

- antibiotic effects (Lafont R. and Dinan L. Journal of Insect Science 3: 7,30pp, 2003, available online: insectscience.org).

CN-86 01 06791-A relates to a cosmetic skin protection product containing beta-ecdysone, which prevents aging of the skin, preserves hydration, and eliminates acne and brown spots.

The document WO-90/03778-A relates to compositions based on hydrated lamellar phases or liposomes containing for example an ecdysteroid, and which can be used in particular as healing or anti-wrinkle compositions.

The document WO-94/04132-A relates to a cosmetic or dermatological composition containing for example an ecdysteroid, intended in particular to give the skin a smoother and softer appearance, to reinforce the water barrier function of the skin, as well as the cohesion of the cells of the epidermis. The document FR-2801504-A relates to an extract of the Ajuga turkestanica plant containing at least one ecdysteroid and at least one iridoid, as well as cosmetic compositions containing it, for improving the differentiation of keratinocytes, or for regulating the fluid flow and reabsorption water in the epidermis, or to moisturize the epidermis.

US-6,641,848 relates to a composition comprising a combination of tri-terpene saponins and ecdysteroids to stimulate the synthesis of collagen IV in the skin.

The document FR-2863378-A relates to the use of ecdysteroids for the preparation of cosmetic or dermatological anti-hair loss compositions.

Document FR-863886-A relates to the use of an ecdysterone receptor ligand for the preparation of a composition intended to maintain cutaneous homeostasis.

In addition, ecdysterone is widely used in sericulture to control the moult of the silkworm, and is part of the composition of many dietary supplements to increase muscle mass (Lafont R. et al, 2003, already cited).

Furthermore, the repeated exposure of the skin to ultraviolet radiation causes damage to the DNA present in the cells of the skin and the mucous membranes, in particular in the cells called fibroblasts which are responsible for the renewal of the macromolecules constituting the extracellular matrix of the skin. derm (Goukassian D. et al., The FASEB J., 14: 1325-34, 2000).

This damage is naturally repaired. But when this is not the case, it can have serious consequences for the cells, and this can even lead to photocarcinogenesis (Goukassian DA and Gilchrest BA, Rejuvenation Res.7 (3): 175-85,2004). In addition, it has long been known that skin cells exposed to the sun lose their reproductive capacity more quickly (Gilchrest BA, J Gerontol., 35 (4): 537-41, 1980).

The cells that can no longer reproduce are called "senescent", and are characterized by the strong expression of a beta-galactosidase active at pH 6. There are more cells expressing this beta-galactosidase among the keratinocytes or fibroblasts of the skin of older people, than 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 undergone ultrasonic stresses. Repeated purples (Chainiaux F. et al., Int J. Biochem, Cell Biol 34 (11): 1331-9,2002).

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

It thus appears that it would be very interesting to be able to have means to stimulate the natural repair of the damage caused to the cutaneous cells by ultraviolet radiation, so as to delay the appearance of senescent cells which have lost the capacity to reproduce.

Thus, it is an object of the present invention to provide a means for stimulating the natural defenses, such as p53 protein, of cells against damage to skin cells by exposure to ultraviolet radiation.

This aim, as well as others which will appear later, is achieved by the use of a composition containing an ecdysteroid, an ecdysteroid derivative or a natural plant or animal extract comprising for stimulating the natural defenses of the cells of the skin and mucous membranes against the consequences of exposure to ultraviolet radiation.

Advantageously, the ecdysteroid according to the invention is ecdysterone. Preferably, the ecdysteroid is in the form of a derivative such as a mono- or multiacyl derivative and in particular acetylated derivative, an acetonide, an ester of phosphoric acid.

Advantageously, the ecdysteroid used is contained in a plant extract such as an extract of Cyanotis arachnoidea, Cyanotis vaga, Ajuga turkestanica, Ajuga iva, Ajuga decumbens, Serratula coronata, Leuzea (or Rhaponticum) carthamoides, Paris polyphylla, Pfaffia paniculata, Pfaffia Iresinoides, Pfaffia stenophylla, Polypodium vulgare, Polypodium aureum, Polypodium lepidopteris, Silene otitis, Cyatula officinalis, Cyatula capitata, Achyrantes aspera, Achyrantes bidentata, Spinacia oleracea, Chenopodium quinoa, or in an animal extract such as an extract of Bombyx mori.

According to the present invention, the composition may also comprise:

at least one antioxidant such as vitamin E, vitamin C, a carotenoid, a flavonoid, an isofiavonoid, a polyphenol, a procyanidin, resveratrol, epigallocatechin gallate, genistein, daidzein, formononetin, andequol, a green tea extract;

at least one anti-inflammatory agent such as an extract of Glycyrrhiza glabra, glycyrrhetinic acid.

Preferably, the dermatological or cosmetic composition according to the invention intended to stimulate the repair of cellular damage caused to the skin by ultraviolet exposures, contains from 0.001 to 95% and more specifically from 0.01 to 1% by weight. ecdysterone.

Advantageously, this composition is more particularly applied to the skin. It can also be administered orally, for example as a dietary supplement.

Preferably, this composition is used to stimulate the activity of the p53 protein in cells of the skin or mucous membranes.

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 an ecdysteroid, we 006/002407

very significantly decreases the number of cells that pass to the senescent state characterized by the appearance of the beta-galactosidase activity associated with it ("Senescence Associated Beta-Galactosidase" or "SA Beta-gal").

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 it.

The ecdysteroid used, in this case ecdysterone, in the examples and compositions below is contained in a plant extract such as an extract of

Cyanotis arachnoidea, Cyanotis vaga, Ajuga turkestanica, Ajuga iva, Ajuga decumbens, Serratula coronata, Leuzea (or Rhaponticum) carthamoides, Paris polyphylla, Pfaffia paniculata, Pfaffia iresinoides, Pfaffia stenophylla, Polypodium vulgare, Polypodium aureum, Polypodium lepidopteris, Silenitis otitis, Cyatula officinalis, Cyatula capitata, Achyrantes aspera, Achyrantes bidentata, Spinacia oleracea,

Chenopodium quinoa, or in an animal extract such as an extract of Bombyx mori.

Example 1

Ecdysteroid attenuation of damage to cultured fibroblasts by repeated exposure to ultraviolet radiation.

The ecdysteroid used is a sample of ecdysterone of greater than 90% purity extracted from Cyanotis arachnoidea, supplied by the Kunming Institute of Botany (Kunming, Yunnan, China).

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 ecdysterone over a period of 24 hours is verified in the range 0.4 to 250 μg / ml.

For 5 days the fibroblasts are exposed once a day to a dose of ultraviolet B of 500 mJ / cm ² . Following each irradiation, the culture medium is replaced by medium containing (0.4), 2, 10, 50 and 250 μg / ml of ecdysterone.

Before each new irradiation, these media are replaced by a medium containing no ecdysterone.

After the last irradiation, the cells are left for 72 hours in contact with medium supplemented with ecdysterone, 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 concentration of ecdysterone, by colorimetry, as well as by Bioanalyzer fluorescence.

The mean values of the percentages of cells expressing beta-gal SA are as follows:

On constate que :

We observe that :

- the damage caused by repeated exposure to ultraviolet B rays has led to a significant increase in fibroblasts exhibiting the characteristic beta-galatic activity of cells that can no longer reproduce (termed "senescent"),

- the treatment of the cells with ecdysterone after the irradiations, allowed a mitigation of the damages caused by the ultra-violet ones from the concentration of 10 μg / ml and significantly from 50 μg / ml, which is translated by a number of "senescent" fibroblasts which remains equal to or less than that of cultures that have not undergone any irradiation.

It thus appears that the use of an ecdysteroid makes it possible, according to the invention, to attenuate the cellular damage caused by exposure to ultraviolet radiation.

Example 2

The same experiment as in Example 1 is carried out by only incorporating ecdysterone during the 72 hour recovery period after the five irradiations.

The assay of beta-galactosidase activity gives the following results:

Thus we see that ecdysterone does not have a strictly restorative effect after a series of exposure to ultraviolet B radiation. Example 3

The same experiment is carried out as in Example 1, but the ecdysterone is incorporated in the culture medium of the cells 24 hours before each irradiation with ultraviolet B (including before the first irradiation) as well as during the period of 72 hours. hours after the five irradiations.

The determination of the beta-galactosidase activity in the cells gives the following results:

Thus, it can be seen that the treatment of fibroblasts with ecdysterone before UV irradiations makes it possible to attenuate the appearance of senescent cells.

Example 4

A cell, after having undergone a stress that has caused damage to the DNA contained in this cell, activates the p53 protein, called "guardian of the genome", which induces the processes of repairing primary damage (see Magalhaes JP et al. Gerontol Expert 39 (9): 1379-89, 2004).

Stimulation of p53 protein expression in the fibroblasts of Example 1 exposed to repeated ultraviolet irradiation was evaluated as in Example 1: 250 mg / l of ecdysterone was added to the medium. 24h culture before each exposure and after the last exposure. A first "control" culture was subjected to the same treatments with 250 mg / l of ecdystero, but was not irradiated with ultraviolet light.

A second "control" crop experienced the same ultraviolet exposures as the treated cultures, but in the total absence of ecdysterone.

Six hours after the last irradiation, the proteins were extracted from the fibroblasts and their content assessed by means of the "Micro BCA kit" system from Pierce.

Then 5 .mu.g of nuclear proteins were incubated in a microplate coated with an oligonucleotide carrying the specific DNA sequence capable of binding the p53 transcription factor.

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 as follows:

On a ainsi trouvé de façon surprenante que le traitement à l'ecdystérone en l'absence d'UV a multiplié par 6 la teneur des fibroblastes en protéine p53.

It has surprisingly been found that ecdysterone treatment in the absence of UV has increased the fibroblast content to p53 by a factor of 6.

It has also been confirmed that ultraviolet exposures, in the absence of ecdysterone, have increased the fibroblast content to p53 by a factor of 9. Finally, it has been shown that fibroblasts exposed to ultraviolet in the presence of ecdysterone expressed 16 times more p53 protein than the "control" cells, thus neither treated nor exposed.

This example makes it possible to demonstrate, surprisingly, that ecdysterone stimulates the p53 defense protein necessary for preventing damage caused by ultraviolet radiation, such as the appearance of senescent cells having lost the capacity to reproduce.

Composition N ° 1:

Cosmetic cream stimulating natural defenses.

The procedure is as for the production of a conventional oil-in-water emulsion, that is to say that is mixed with stirring at 85 ° C a hydrophobic phase and an aqueous phase each containing raw materials intended to give the product the Usual properties: stability, safety, easy spreading, nice final touch, etc.

It is cooled with stirring and, when the temperature is lowered to 45 ° C., 0.1% by weight of ecdysterone is added.

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 N ° 2: Cosmetic cream stimulating natural defenses and antioxidant protection.

As above, an oil-in-water emulsion containing alpha-tocopherol and alpha-tocopherol acetate are prepared; and during cooling, at 45 ° C., ecdysterone and vitamin C phosphate are added in an amount sufficient to arrive at the concentrations by weight in the final composition of:

- 0.1% ecdysterone

- alpha-tocopherol 0.05%

- alpha-tocopherol acetate 0.2%

- Vitamin C phosphate, 0.2% Mg salt. This formulation is a day cream. Composition N ° 3:

Cosmetic cream stimulating natural defenses and filter protection.

As in the case of Composition No. 1, an oil-in-water emulsion with addition of titanium oxide is used, then a dry alcoholic extract of Leuzea carthamoides is added with stirring at 45 ^° C. at a rate of 2% by weight. The protection factor SPF is equal to 6.

The product is intended to be applied in the morning on uncovered skin areas, and if possible the application must be renewed in the middle of the day.

Composition N ° 4:

Cosmetic gel stimulating natural defenses for sensitive skin.

An aqueous acrylic gel titrated with 0.1% by weight of ecdysterone and containing in addition 2% by weight of hydroglycolic extract of Scutellaria baicalensis is prepared.

Composition N ° 5:

Dermatological gel for stimulating natural defenses.

An aqueous acrylic gel containing 0.2% by weight of ecdysterone is prepared. This gel is applied in the morning and during the day on exposed skin areas.

Composition N ° 6:

Dietary supplement to stimulate the natural defenses of the skin before any exposure to the sun. Tablets containing 15 mg of ecdysterone per tablet and a powder formulation comprising 20% by weight of ecdysterone are prepared by conventional techniques.

These tablets should be taken with a little water during the morning and mid-day meals, at a rate of one to two tablets per dose depending on the intensity and duration of sunshine expected. Composition IM ° 7:

Dietary supplement to stimulate the natural defenses of the skin before any exposure to the sun.

Capsules containing 15 mg of an extract of Cyanotis arachnoidea comprising about 90% by weight of ecdysterone are prepared, and the balance, about 10% by weight, consisting of a gelatin shell.

These capsules are to be taken as the tablets of the composition No. 6.

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

Capsules containing a powder formulation containing 20% by weight of ecdysterone dosed with 10 mg of ecdysterone, 15 mg of green tea extract and 25 mg of vitamin C per capsule are prepared.

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 N ° 9:

Drink to stimulate the natural defenses of the skin before any exposure to the sun. A soy milk drink containing 20% by weight concentrated spinach juice and 0.05% by weight ecdysterone is made. 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

1. Use of a composition containing an ecdysteroid, an ecdysteroid derivative or a plant or animal extract comprising it for stimulating the natural defenses of cells against the consequences of their exposure to ultraviolet radiation. 2. Use according to claim 1, characterized in that the ecdysteroid is ecdysterone. 3. Use according to claim 1, characterized in that the ecdysteroid is in the form of a derivative such as a mono- or multi-acyl derivative and in particular acetylated, an acetonide, an ester of phosphoric acid. 4. Use according to claim 1, characterized in that the ecdysteroid used is contained in a plant extract such as an extract of Cyanotis arachnoidea, Cyanotis vaga, Ajuga turkestanica, Ajuga iva, Ajuga decumbens, Serratula coronata, Leuzea (or Rhaponticum) carthamoides, Paris polyphylla, Pfaffia paniculata, Pfaffia iresinoides, Pfaffia stenophylla, Polypodium vulgare, Polypodium aureum, Polypodium lepidopteris, Silenitis otitis, Cyatula officinalis, Cyatula capitata, Achyrantes aspera, Achyrantes bidentata, Spinacia oleracea, Chenopodium quinoa, or in an extract animal such as an extract of Bombyx mori. 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, an extract of green tea; at least one anti-inflammatory agent such as an extract of Glycyrrhiza glabra, glycyrrhetinic acid. 6. Use according to claim 2, characterized in that it comprises from 0.001 to 95% by weight of ecdysterone. 7. Use according to claim 6, characterized in that it comprises from 0.01 to 1% by weight of ecdysterone. 8. Use according to any one of claims 1 to 7, characterized in that the composition is intended for topical application. 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.