EP0799049A1

EP0799049A1 - COSMETIC OR PHARMACEUTICAL AND PARTICULARLY DERMATOLOGICAL COMPOSITION, AND CULTURE MEDIUM CONTAINING A $i(SMELOPHYLLUM CAPENSE) EXTRACT

Info

Publication number: EP0799049A1
Application number: EP95943268A
Authority: EP
Inventors: Frédéric Bonte; Marc Dumas; Catherine Lavaud; Georges Massiot
Original assignee: LVMH Recherche GIE
Current assignee: LVMH Recherche GIE
Priority date: 1994-12-23
Filing date: 1995-12-22
Publication date: 1997-10-08
Also published as: WO1996020000A1; FR2728465A1; US5807555A; JPH10511380A; FR2728465B1

Abstract

A Smelophyllum capense extract useful for preparing a cosmetic or pharmaceutical and particularly dermatological composition, or as an active principle or additive for a culture medium. Said Smelophyllum capense extract promotes collagen synthesis, in particular for controlling skin ageing or preparing artificial skin or dermis, preventing wrinkling or reducing wrinkle depth, or promoting skin firming.

Description

Cosmetic or pharmaceutical composition, in particular dermatological and culture medium, containing an extract of Smelophyllum capense.

The present invention essentially relates to a cosmetic or pharmaceutical composition, in particular dermatological and culture medium, containing an extract of Smelophyllum capense.

More specifically, the present invention relates to the use of an extract of Smelophyllum capense, for the preparation of a cosmetic or pharmaceutical composition, in particular a dermatological composition, in particular stimulating the synthesis of collagen, and intended in particular for combating the effects skin aging, to obtain a skin tightening, to improve healing or to treat various pathologies accompanied by a collagen deficiency; or as one of the active principles or adjuvants of a cell or tissue culture medium, especially for the mass culture of skin cells, in particular fibroblasts, as well as cosmetic or pharmaceutical compositions or culture media with application.

The Smelophyllum capense plant belongs to the Sapindaceae family which includes around 1,700 plant species. The plants of this family contain saponins which have long been used as detergent and fishing poison.

The Smelophyllum capense plant is a tree of relatively modest height, measuring 3 to 4 m, which is found endemic in particular in the forests of the southern part of South Africa (see RB Drumont, " Trees of Southern Africa, C. Struik Publisher, Capetown, 1984, page 531) It has now been discovered that extracts of Smelophyllum capense are of great interest in cosmetics, especially for skin care.

In particular, a surprising activity has been discovered from the extracts of Smelophyllum capense on the synthesis of collagen, in particular collagen type I, hereinafter abbreviated as "collagen I". However, the skin essentially contains collagen I, a protein synthesized by fibroblasts, the major cells of the dermis. This protein plays a supporting role and is responsible for the rheological qualities of the dermis, in particular for its firmness and for maintaining its architecture (EU KUCHARZ, "The collagens Biochemistry and pathophysiology", Springer Verlag, Berlin 1992). Furthermore, it has been shown that fibroblasts in the dermis of the elderly secrete less collagen than those of young subjects (M. DUMAS et al, Mech, Aging Dev. (1994) 73, 179-187). Thus, with age, there is a decrease in the rheological qualities of the skin and its response to the stresses to which it is subjected daily. The skin distends, reacts less well to tension, loses its tone and wrinkles deepen.

Therefore, the action of the extracts of Smelophyllum capense on the synthesis of collagen makes these particularly useful to fight against the effects of skin aging, such as wrinkles or sagging of the tissues of the skin support, or to improve healing . The main object of the present invention is therefore to solve the new technical problem consisting in providing a new formulation of a cosmetic or pharmaceutical composition, in particular a dermatological composition, having good efficacy in preventing or treating the effects of skin aging, as well as on skin tightening, or to improve healing.

The main object of the present invention is also to solve this new technical problem in a particularly simple, satisfactory and usable manner on an industrial scale, especially in the cosmetic or pharmaceutical industry. Thus, according to a first aspect, the present invention relates to the use of an extract of Smelophyllum capense as a cosmetic agent, in particular for the care of the skin.

According to an alternative embodiment of the invention, an extract of Smelophyllum capense is used for the preparation of a cosmetic or pharmaceutical composition, in particular dermatological, stimulating in particular the synthesis of collagen, in particular that of collagen I, and intended in particular for improve skin healing, improve biomechanical properties and surface appearance of the skin, obtain skin firming, combat the effects of skin aging or treat various pathologies accompanied by collagen deficiency.

According to an advantageous alternative embodiment, the extract of Smelophyllum capense is used for the preparation of a composition, with anti-aging skin effect, in particular anti-wrinkle, intended to prevent the appearance of wrinkles or attenuate their depth. According to a particular embodiment, the extract of

Smelophyllum capense above is obtained by extraction with a polar solvent, such than a lower alcohol, advantageously chosen from methanol or a hydro-ethanolic mixture, preferably from the bark of the plant, more preferably from the root bark of this plant.

In particular, the extract of Smelophyllum capense can be obtained according to the method described below by way of indication, but in no way limiting.

A first extraction of the bark, preferably from the root bark, from the plant, is carried out with a polar solvent, advantageously chosen from the group consisting of: water, alcohols preferably containing from 1 to 4 atoms carbon, chlorinated solvents preferably having 1 to 2 carbon atoms, organic esters preferably having 3 to 6 carbon atoms; or a mixed solvent based on any mixture of the aforementioned solvents.

More preferably, the solvent of first extraction is chosen from the group consisting of water, methanol, ethanol, a methanol-water mixture or an ethanol-water mixture, chloroform, dichloromethane. More preferably, it is water, methanol, ethanol or their mixtures.

The ratio of the bark to the extractant is not critical and will generally be between 1: 5 and 1:20 parts by weight.

The extraction is generally carried out at temperatures between room temperature and the boiling point of the solvent used for the extraction.

Preferably, this first extraction is carried out at reflux under atmospheric pressure for a period of 2 to 4 h. In addition, it is advantageously preceded by cold maceration for 2 to 4 hours in the extraction solvent.

At the end of extraction, the solvent phase containing the extract is filtered and then concentrated and / or evaporated to dryness under reduced pressure. A first crude extract of Smelophyllum capense according to the invention is thus obtained. This crude extract can be purified by various methods well known to those skilled in the art. According to a second aspect, the present invention also relates to a cosmetic composition characterized in that it comprises, as active ingredient, a cosmetically effective amount of an extract of Smelophyllum capense preferably dispersed in a cosmetically acceptable excipient.

According to a particular variant embodiment, this cosmetic composition stimulates the synthesis of collagen, in particular that of collagen I, and is intended in particular to combat the effects of skin aging, or to get skin firming. Such a composition, for example, can advantageously be used as a composition to prevent the appearance of wrinkles or attenuate their depth.

According to a third aspect, the invention also relates to a pharmaceutical composition, in particular a dermatological composition, stimulating the synthesis of collagen, in particular that of collagen I, characterized in that it comprises, as active ingredient, a pharmaceutically effective amount of an extract of Smelophyllum capense dispersed in a pharmaceutically acceptable excipient. According to a particular embodiment, said composition is intended to improve skin healing or to treat various pathologies accompanied by a deficiency in collagen.

According to a fourth aspect, the invention also covers a cosmetic or pharmaceutical treatment process, in particular dermatological treatment, characterized in that it comprises the application of a cosmetically or pharmaceu¬ tically effective amount for a cosmetic or pharmaceutical treatment of a Smelophyllum capense extract, in particular dispersed in a cosmetic or pharmaceutically acceptable excipient. The particularly advantageous cosmetic or pharmaceutical applications currently result from the preceding description as well as from the following description in relation to the examples, and from the claims. The same applies to the concen¬ tration in extract.

In one or other of the preceding aspects, the extract of Smelophyllum capense will preferably be used at a concentration of between 0.0001% and 1% by weight relative to the total weight of the final composition. Preferably, this concentration is between 0.01% and 0.25% by weight relative to the total weight of the final composition.

Also in any one of the preceding aspects, the composition according to the invention preferably also contains an active substance chosen from the group consisting of ascorbic acid, madecassic acid, asian acid, madecassoside, asiaticoside, alpha-1-proteinase inhibitor, collagenase inhibitors, such as retinoic acid, elastase inhibitors, lysine, proline, 2-oxoglutarate, and ginsenoside Rrj-

According to a fifth aspect, the present invention also relates to the use of an extract of Smelophyllum capense as one of the active principles or one of the adjuvants of a cell or tissue culture medium, in particular for the mass culture of skin cells, in particular fibroblasts. Advantageously, this use relates to the preparation of skin or an artificial dermis.

The invention also relates in this aspect to a method of treating fibroblasts in culture by an effective concentration of an extract of Smelophyllum capense to obtain stimulation of the synthesis of collagen, in particular collagen I.

Thus, in the context of this culture for example for the preparation of artificial skin, in particular of artificial dermis, by cell culture, according to techniques well known to those skilled in the art, the process is obtained using the invention, a very good quality artificial skin or dermis, particularly as regards the biomechanical properties.

According to a preferred embodiment of the use or the culture method, the fibroblasts are treated with an extract of Smelophyllum capense at a concentration at the time of culture of between 0.3 / g / ml and 30 / g / ml of culture medium.

According to another advantageous variant, ascorbic acid or one of its derivatives can be added to the culture medium at a non-cytotoxic concentration, in particular between 0.001 mM and 0.5 mM. According to yet another advantageous variant, one or more of the following substances can be added to the culture medium: L-glutamine, L-lysine, 5-hydroxy-L-lysine, L-proline and 4-hydroxy-L-proline, said substances may each be present at a concentration of between 2 and 10 mM. Other objects, characteristics and advantages of the invention will become apparent to those skilled in the art from the following description made with reference to several currently preferred embodiments of the invention, as well as to the examples which follow . In the examples, all the percentages are given by weight, unless otherwise indicated.

Example 1

Preparation of a methanolic extract of the root bark of Smelophyllum capense

Smelophyllum capense root bark powder is prepared by grinding dried root bark of this plant from South Africa. Then 200 g of this powder are macerated for 2 h in 2 l of methanol. The whole is brought to reflux for 3 h, then it is allowed to cool and filtered. on sintered glass. The filtrate is evaporated to dryness. The residue, weighing 44.6 g, is taken up in 350 ml of methanol. Precipitation is carried out by adding 1.75 l of ethyl ether to this solution, then filtering. The precipitate is then dried over a solid dehydrating agent such as P2O5. An amount of approximately 27 g of this precipitate is then dialyzed for 4 days against 270 ml of demineralized water, then lyophilized. 13.6 g of extract according to the invention, called extract 1, are thus obtained.

Example 2

Demonstration of the activity of a methanolic extract of the root bark of Smelophyllum capense prepared according to example 1. extract Ii. on the synthesis of collagen by human fibroblasts in culture

Fibroblast culture

Cultures of healthy adult dermis fibroblasts are carried out using the explant method using a facial skin sample obtained from a woman aged 60 years during a facelift.

Fibroblasts are grown to confluence in 199 E medium (Gibco) supplemented with 2 mM L-glutamine (Gibco) and 10% v / v fetal calf serum (Gibco) at 37 ^° C in an atmosphere of 5% of humidified CO2. To assess the collagen content, the confluent primary cultures are harvested with a 0.1% solution of trypsin and 0.02% EDTA in phosphate buffered saline (PBS) at pH 7.2 and the cells are then seeded at the density of 10 ^ fibroblasts per well, in microculture plates (Falcon) with 96 wells, in the presence of the same culture medium as that described above. 24 h after seeding, the medium is removed and replaced by a medium of the same composition as the medium described above, except that it does not contain serum and that 25 μM of acid have been added L-ascorbic in the form of sodium salt. In addition, this new medium contains or not the product to be tested (extract 1 ^) depending on whether it is a treated culture or a control culture. Then incubated for an additional 48 h at 37 ^* C. The test product (extract I _j) was dissolved in DMSO prior to incorporation into the culture medium (final DMSO concentration in the medium is 0.1% v / v).

Cell viability At the end of the incubation period, the medium is removed and an MTT cell viability test was carried out in accordance with the publication by Denizot F. et al. J. Immunol. Methods, (1986) 89, 271-277. The cells are incubated with 100 μl of a tetrazolium salt solution at 0.5 mg / ml (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium or MTT bromide) in medium E 199 without phenol red (Gibco) for 3 h at 37 ^° C. Then 100 .mu.l of isopropanol are added to each well to solubilize the formazan blue dark derivative formed by living cells. The absorbance rate is measured at 540 nm.

Collagen measurement

The amount of type I collagen secreted by the cells in the serum-free medium after 48 h of incubation with the mixture or the individual component is determined by an ELISA test as previously described in the publication by Dumas M. et al. , Mech. Aging Dev. (1994) 73, 179-187, and that of Grimaud JP et al., In: Methods of Enzymatic Analysis (Bermeyer, HU, ed.), VCH Publishers, einheim, (1986), 186-201. The serum-free incubation mediums and the remaining cells homogenized by sonication in ice is collected and transferred to wells of a plastic immunoplate (NUNC) for an incubation of 24 hours at 4 ^° C to allow the adhesion of secreted collagen. Protease inhibitors (ethylmaleimide, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetate, each in a final concentration of 1 mM) are added during this period. The plates are then reinserted with PBS. A similar washing step is carried out after each plaque treatment.

After 24 h of incubation at 4 ^° C with the serum albumin to prevent nonspecific binding, antibodies of anti-human type I collagen rabbit (Institut Pasteur, Lyon, France) was added for 1.5 h at 22 ^* C and the bound antibodies are reacted with anti-rabbit goat IgG conjugated to alkaline phosphatase (Interchim, Montluçon, France). The absorbance of para-nitrophenol formed from para-nitrophenylphosphate (Sigma) by alkaline phosphatase is measured at 405 nm. The optical densities are converted into a nanogram of collagen using a standard curve established with purified human type I collagen (Jacques Boy Institute, France).

Statistics

Collagen levels (mean + SD, standard deviation, n = 6) are compared with those determined on untreated cells (medium + L-ascorbate + DMSO) by Student's t test with p <0.05 as the level of significance. Results

The percentage of stimulation A of the synthesis of collagen gene I is calculated by comparing the amounts of collagen I secreted in the control cultures (receiving no product to be tested) C ₀ and in the treated cultures, from the following formula:

Ct - C _r

A = x lOO

-o

All the results are shown in Table I below.

TABLE I

* S = significant NS = not significant

Examination of Table I first shows that the rate of cell viability in the treated cultures did not vary significantly compared to that of the control cultures. The extract 1 ^ therefore does not exhibit cytotoxicity.

In this table, the amounts of collagen I secreted by the fibroblasts are expressed in ng / 10,000 cells / 48 h. It will be observed, from Table I, that the methanolic extract of

Smelophyllum capense produced a significant activity of stimulation of the synthesis of collagen I by fibroblasts. However, it is known in particular by the publication of Dumas M. et al., in: Mech. Aging Dev. (1994) 7_3_, 179-187, that the amount of collagen I decreases during skin aging.

Thus, the extract of Smelophyllum capense can be used for products intended to combat skin aging or requiring an increase in local synthesis of collagen as is the case during a treatment of wrinkles or to help improve the skin healing.

Thus, the extracts of Smelophyllum capense can advantageously be used, thanks to their property of stimulating the synthesis of collagen, as active agent, in cosmetic or pharmaceutical compositions, in particular dermatological, as defined above.

Various formulations of cosmetic compositions are given below:

EXAMPLE 3 Skin Firming Massage Gel

- Smelophyllum capense 1 ^ root bark extract from Example 1 ... 0.1 g

- Ethanol 25 g

- Glycerin 2 g

- Propylene glycol 2 g - Carbopol 940® 1.25 g

- Aqueous excipient with possibly flavored preservative .... q.s.p. 100.00 g

Part of the aqueous excipient is used with Carbopol to separately prepare a gel, the other part of the aqueous excipient is used to be mixed with the other components and the gel is added to the solution obtained in order to obtain a gelled composition forming the massage gel.

This massage gel composition can be used three times a week for two months at the bust.

Example 4

Body care lotion for skin firming

- Smelophyllum capense root bark extract I _j from Example 1 0.5 g

- Solubilizing agent (Cremophor RH40®) 2 g

- Hyaluronic acid 1 g - Aqueous excipient containing a possibly flavored preservative qs 100.00 g The extract is first dissolved in the solubilizing agent, then is added to the aqueous excipient to which hyaluronic acid is added.

The lotion obtained can be used for a three-week treatment on areas sensitive to sagging, such as the belly, thighs.

EXAMPLE 5 Anti-Wrinkle Emulsion

- Smelophyllum capense Ij root bark extract from Example 1 0.10 g

- Scented emulsified excipient q.s.p. 100.00 g

EXAMPLE 6 Healing composition

- Root bark extract of Smelophyllum capense l \ according to Example 1 0.5 g

- Emulsified excipient, type water in oil q.s.p. 100.00 g

Example 7

Firming and beautifying cream

- Smelophyllum capense Ij root bark extract from Example 1 ... 0.05 g

- Asiaticoside 0.1 g - Ascorbyl phosphate (magnesium salt) 0.05 g

- Vegetable glycoceramides 0.05 g

- Vitamin A palmitate 0.01 g

- Preservatives 0.05 g

- Emulsified excipients q.s.p. 100.00 g

This cream, applied twice a day, restores the qualities of firmness, elasticity as well as the skin barrier properties and the radiance of young skin.

Claims

1. Use of an extract of Smelophyllum capense as a cosmetic agent, in particular for the care of the skin.

2. Use of an extract of Smelophyllum capense for the preparation of a cosmetic or pharmaceutical composition, in particular dermatological, stimulating the synthesis of collagen, in particular that of collagen I, and intended in particular to improve cutaneous healing, to improve the biomechanical properties and the surface appearance of the skin, to obtain a firming of the skin, to fight against the effects of skin aging, or to treat various pathologies accompanied by a deficiency in collagen.

3. Use according to claim 1 or 2, characterized in that the aforementioned composition is a composition with anti-aging skin effect, in particular anti-wrinkle, intended to prevent the appearance of wrinkles or attenuate their depth.

4. Use according to one of claims 1 to 3, characterized in that the aforementioned extract is a bark extract, in particular an extract of the root bark of Smelophyllum capense.

5. Use according to one of claims 1 to 4, characterized in that the above-mentioned extract of Smelophyllum capense is obtained by extraction with a polar solvent, such as a lower alcohol advantageously chosen from methanol or a hydro-ethanolic mixture , preferably from the bark of the plant, more preferably from the root bark of this plant.

6. Cosmetic composition, characterized in that it comprises, as active ingredient, a cosmetically effective amount of an extract of Smelophyllum capense dispersed in a cosmetically acceptable excipient.

7. Pharmaceutical composition, in particular dermatological composition, stimulating the synthesis of collagen, in particular that of collagen I, characterized in that it comprises, as active ingredient, a pharmaceutically effective amount of an extract of Smelophyllum capense dispersed in a pharmaceutically acceptable excipient.

8. Composition according to one of claims 6 or 7, characterized in that the concentration of extract of Smelophyllum capense is between 0.0001% and 1% by weight, preferably between 0.01% and 0.25% by weight, relative to the total weight of said composition.

9. Composition according to one of claims 6 to 8, characterized in that the abovementioned extract is obtained from the bark of the plant, in particular from the bark of the root of the plant.

10. Composition according to one of claims 6 to 9, characterized in that the above-mentioned extract of Smelophyllum capense is obtained by extraction with a polar solvent, such as a lower alcohol, advantageously chosen from methanol or a hydro- ethanolic.

11. Composition according to one of claims 6 to 10, characterized in that it also contains an active substance chosen from the group consisting of ascorbic acid, madecassic acid, asian acid, madecassoside, l asiaticoside, alpha-1-proteinase inhibitor, collagenase inhibitors, such as retinoic acid, elastase inhibitors, lysine, proline, 2-oxoglutarate, and ginsenoside RQ.

12. Cosmetic or pharmaceutical treatment method, in particular dermatological treatment, characterized in that it comprises the application of a cosmetically or pharmaceutically effective amount for a cosmetic or pharmaceutical treatment of an extract of Smelophyllum capense, in particular dispersed in an excipient cosmetically or pharmaceutically acceptable.

13. Method according to claim 12, characterized in that the extract of Smelophyllum capense is present in a composition comprising a concentration of extract of Smelophyllum capense of between 0.0001% and 1%, preferably between 0.01% and 0 , 25%, by weight relative to the total weight of said composition.

14. Method according to claim 12 or 13, characterized in that it is a cosmetic treatment to stimulate the synthesis of collagen, in particular that of collagen I, to improve skin healing, to combat the effects of skin aging, to prevent the appearance of wrinkles or reduce their depth, or to obtain skin firming.

15. Use of an extract of Smelophyllum capense as one of the active principles or adjuvants of a cell or tissue culture medium, in particular for the mass culture of skin cells, in particular fibroblasts, for example for the preparation of artificial skin or dermis.

16. Method for treating fibroblasts in culture by an effective concentration of an extract of Smelophyllum capense to obtain stimulation of the synthesis of collagen, in particular collagen I.

17. The method of claim 16, characterized in that the fibro¬ blasts are treated with an extract of Smelophyllum capense at a concentration between 0.3 μg / ml and 30 μg / ml of culture medium.

18. The method of claim 16 or 17, characterized in that a culture medium of ascorbic acid or one of its derivatives is added at a non-cytotoxic concentration, in particular between 0.001 mM and 0.5 mM.

19. Method according to one of claims 16 to 18, characterized in that one or more of the following substances are added to the culture medium: L-glutamine, L-lysine, 5-hydroxy-L-Iysine, L- proline and 4-hydroxy-L-proline, said substances each being able to be present each at a concentration of between 2 and 10 mM.