EP1301171A1

EP1301171A1 - Use of extracted soluble protein fractions

Info

Publication number: EP1301171A1
Application number: EP01967142A
Authority: EP
Inventors: Christine Jeanmaire; Marc Pauly; Louis Danoux
Original assignee: Cognis France SAS
Current assignee: BASF Health and Care Products France SAS
Priority date: 2000-07-19
Filing date: 2001-07-10
Publication date: 2003-04-16
Also published as: AU2001287595A1; FR2811894B1; FR2811894A1; WO2002005774A1; JP2004503579A; US20040013635A1

Abstract

The invention relates to the use of extracted soluble protein fractions which (a) have at least one band in polyacrylamide gel electrophoresis with sodium dodecylsulfate in non-reducing conditions; (b) have an average molecular weight of 500 to 500.000 dalton; (c) contain 0.005 to 0.5 wt. % total nitrogen and 0.0005 to 0.01 wt. % amino nitrogen in relation to the protein part; (d) are soluble in water and in aqueous electrolyte solutions but insoluble in ethanol or acetone; and (f) in aqueous solutions, form precipitates together with trichloroacetic acid or picric acid; for stimulating the synthesis of proteins that are characteristic for differentiating keratinocytes, especially for stimulating filaggrin synthesis. The invention also relates to the use of the protein fractions for combating skin ageing, especially the formation of wrinkles, and to the use thereof for treating dry skin and/or preventing the skin from drying out.

Description

USE OF EXTRACTED LUBLE PROTEIN FRACTIONS

Field of the Invention

The invention is in the field of cosmetics and relates to the use of special extracts for stimulating the synthesis of proteins which are characteristic of the differentiation of keratinocytes, in particular for stimulating Füaggrin synthesis.

State of the art

Profilaggrin forms the main component of the keratohyalin grains in the cells of the granular layer of the tissue epidermis. The profilaggrin synthesized and phosphorylated in the stratum granulosum is a protein with a molecular weight of around 400 kDa and serves as a precursor for the biosynthesis of filaggrin, to which it is converted by dephosphorylation and proteolysis as the keratinocytes mature. Human filaggrin has a molecular weight of approx. 37 kDa, is cationically charged and is usually found in the stratum corneum of the tissue epidermis [cf. Sarret et al., Path. Biol. 37 (4), 297 (1989)].

Filaggrin is known to play an important role in keratin aggregation in the lower stratum corneum. The degradation products of filaggrin, namely free amino acids and their derivatives, prevent water loss from the stratum corneum. Filaggrin thus represents an essential reservoir for natural moisturizing factors (NMF). It could be shown that a decrease in the Filaggrin concentration, which can be due to age in particular, goes hand in hand with the appearance of dry skin [see T. Tezuka et al. Dermatology, 1994, 188, 21-24] and wrinkling [see I. Contet-Audonneau et al. British J. Dermatology, 1999, 140, 1038-1047].

The object of the present invention was therefore to find new active substances with which the synthesis of proteins which are characteristic of the differentiation of keratinocytes, in particular the synthesis of filaggrin, can be stimulated in order to counteract in particular the aging of the skin and the drying out of the skin. Description of the invention

The invention relates to the use of extracted soluble protein fractions

(a) have at least one band under non-reducing conditions in polyacrylamide gel electrophoresis with sodium dodecyl sulfate;

(b) have an average molecular weight in the range of 500 to 500,000, preferably 5,000 to 100,000 daltons;

(c) based on the protein content, have a total nitrogen content of 0.005 to 0.5% by weight and an amino nitrogen content of 0.0005 to 0.01% by weight;

(d) soluble in water and aqueous electrolyte solutions but insoluble in ethanol or acetone; and

(e) form precipitates in aqueous solutions together with trichloroacetic acid or picric acid,

for stimulating the synthesis of proteins which are characteristic of the differentiation of keratinocytes, in particular for stimulating filaggrin synthesis.

Surprisingly, it was found that the administration of protein fractions which meet the above-mentioned conditions and which are obtained specifically from the seeds of Bambaran stimulate the synthesis of filaggrin and thus counteract the effects of dehydration and wrinkling.

Soluble protein extracts

According to the invention, fractions are preferably used which are obtained by extracting leguminous seeds, in particular by extracting the seeds of the Bambaran nut. Bambara nut (Voandzeia subterranea (L) Thouars) is a seed that is enjoyed locally as a vegetable. It is a vegetable of African origin that is mainly cultivated by the farmers as a "starvation culture", the most important of which is its insensitivity to drought and poor soil and its ability to grow under conditions that are not suitable for the peanut can is. The Bambaranus seeds, which are a wholesome food, contain proteins, carbohydrates and lipids and can be enjoyed in different stages of ripening. Their chemical composition is as follows (g / lOOg flour or 100g dry seeds): > Proteins: 16 to 21% by weight,

> Starch: 39 to 49.5% by weight,

> Tannins (expressed in tannic acid): 0.36 to 0.94% by weight,

> Lipids: 5 to 7.3% by weight,

> Ash: 3.65% by weight.

It is known that the seeds of Voandzeia subterranea contain protease inhibitors, and the trypsin-inhibiting activity estimated according to the so-called cockade technique is from 6.7 to 15.4 TlU / mg of flour according to the literature, the functional properties of the protein isolates of this seed were examined for food purposes. In this context, reference is made to the international patent application WO 98/42305, from which the use of Bambaran nut extracts for cosmetics is known. The document also contains further information on the production of the extracts. Furthermore, it has proven to be advantageous to use fractions which have at least one protease inhibitor.

The extracted soluble protein fractions according to the invention are preferably used

• against skin aging, especially against wrinkling.

• for the treatment of dry skin and / or

• against drying out the skin

For the purposes of the invention, the extracted soluble protein fractions according to the invention act against skin aging, in particular against any type of wrinkles and wrinkles. Another name for this type of care product is anti-aging. The uses include slowing skin aging processes. Furthermore, the protein fractions according to the invention act against drying out of the skin, since by stimulating the synthesis of proteins to differentiate keratinocytes, in particular to stimulate filaggrin synthesis, the proportion of these proteins in the keratinocytes of the stratum corneum is increased. Their breakdown products, the free amino acids and their derivatives prevent the skin from drying out. Filaggrin in particular is an essential reservoir for natural moisturizing factors (NMF). The protein fractions according to the invention can, in addition to the preventive use against drying out of the skin, also be used to treat dry skin and at least the natural moisture content return. Examples

Production Example 1. 250 g of ground Voandzeia subterranea seeds were dispersed in 2.5 L of distilled water. After stirring for 15 min, the pH was adjusted to 7.5 by adding sodium hydroxide solution and the extraction was carried out at room temperature over a period of 1 h. After centrifugation (10 min, 5000 g), the upper oily phase was discarded and the yellowish-colored aqueous phase was purified by ultrafiltration (retention at 15,000 Da, concentration factor 2 to 10) or diafiltration with water. The fraction obtained in this way had a dry residue of 1 to 3% by weight and a protein concentration of 0.3 to 15 g / L (biuret determination). The fraction was then dried by lyophilization. The anti-trypsic activity of the dried product was 50 to 200 TUI / mg (determined according to the Kakade method) or based on the dry residue of the solution from 800 to 2500 TUI / ml.

Preparation Example 2. 400 g of ground Voandzeia subterranea seeds were dispersed in 4 L of distilled water and extracted as described in Example III. 3.2 L of an approximately colorless solution with a dry residue of 3% by weight and a protein concentration of 0.3 to 15 g / L were obtained. The pH of the solution was adjusted to 4.5 by adding sulfuric acid, followed by stirring for 30 minutes. After centrifugation (15 min, 5000 g), the upper oily phase was discarded and the yellowish-colored aqueous phase was purified by ultrafiltration (retention at 15,000 Da, concentration factor 2 to 10) or diafiltration with water. The fraction obtained in this way had a dry residue of 1 to 3% by weight and a protein concentration of 0.3 to 15 g / L (biuret determination). The fraction was then dried by lyophilization. The anti-trypsic activity of the dried product was 50 to 200 TUI / mg (determined according to the Kakade method) or based on the dry residue of the solution from 800 to 2500 TUI / ml.

Preparation Example 3. The extract obtained according to Example Hl was subjected to gel permeation on a Superose 12 HR, FPLC column from Pharmacia. 5 fractions were obtained:

Fraction 1: molecular weight> 500,000 Da Fraction 2: molecular weight 100,000 to 500,000 Da Fraction 3: molecular weight 30,000 to 100,000 Da Fraction 4: molecular weight 5,000 to 30,000 Da Fraction 5: molecular weight <5,000 Da Cell growth test. For the subsequent efficacy tests, on the one hand an extract according to preparation example H2 and on the other hand the commercially available formulation Filladyn® from Laboratoires Serobiologiques, SA, which contained 60% of the extract according to example H2 and also polyols, xantham and buffer salts, was used.

The influence of the preparations on human fibroblasts, with which the regenerative capacity of the cells was to be tested, was examined as follows: A standard cell medium (DMEM) with 10% fetal calf serum (FCS) was inoculated with human fibroblasts. After an incubation period of 1 day at 37 ° C. and a CO ₂ concentration of 5%, the growth medium was exchanged for one without FCS, which, however, contained the test preparations in concentrations of 0.03 to 0.6% by volume. After an incubation period of 3 days, cell growth was determined by determining the content of cellular proteins (Bradford method) and the ATP essential for the phosphorylation of Pro-Filaggrin (Vasseur method). The results are summarized in Table 1. The relative percentage based on a standard is given without addition of the test substances (= 100%).

Table 1 Cell growth

Stimulation of filaggrin synthesis (in vitro). A standard cell medium (DMEM) with 10% fetal calf serum (FCS) was inoculated with human keratinocytes. After an incubation period of 4 days at 37 ° C. and a CO ₂ concentration of 5%, the growth medium was exchanged for one without FCS, which however contained the test preparations in concentrations of 0.025 to 2% by volume. After an incubation of 6 to 7 days, the synthesis of pro-filaggrin / filaggrin was determined immunohistochemically, ie using a monoclonal antibody which recognizes pro-filaggrin or filaggrin. The results are summarized in Table 2. Stimulation of Pro-FHaggrin / FHaggrin synthesis (ex vivo). Human biopsies from plastic surgery were disinfected and cultures were set up (DMEM, 37 ° C). The test substances (0.3% by weight Bambaran nut extract or 5% by weight Filladyn®) incorporated in a hydrogel were applied topically (5 treatments within 3 days). After 3 days, narrow biopsies were made and frozen in liquid nitrogen. Then the formation of pro-filaggrin / filaggrin was again examined immunohistochemically. The results are summarized in Table 3.

Pro-Filaggrin / Filaggrin was determined microscopically using a confocal laser scanning microscope. The images were converted into numerical values and analyzed using the Quantimet Q500IW software from Leica. The percentage of the area that is occupied in the histological sections of Pro-Filaggrin / Filaggrin is given.

Table 2

Pro-filaggrin / filaggrin synthesis

Some formulation examples are given in Table 4 below. Table 4

Examples of cosmetic preparations (water, preservative ad 100 wt .-%)

(1) soft cream, (2,3) moisturizing emulsion, (4,5) night cream

The registered trademarks and brands listed in Table 4 are products of the Cognis Group.

Claims

claims

1. Use of extracted soluble protein fractions

(a) under non-reducing conditions in polyacrylamide gel electrophoresis with sodium dodecyl sulfate, one band has at least one band;

(b) have an average molecular weight in the range of 500 to 500,000 daltons;

2. Use according to claim 1, characterized in that fractions are used which have at least one protease inhibitor.

3. Use according to claims 1 and / or 2, characterized in that fractions are used which are obtained by extraction of leguminous seeds.

4. Use according to claim 3, characterized in that fractions are used which are obtained by extracting the seeds of Bambara nut (Voandzeia subterranea).

5. Use of extracted soluble protein fractions according to one of claims 1 to 4 against skin aging, in particular against skin formation.

6. Use of extracted soluble protein fractions according to one of claims 1 to 4 for the treatment of dry skin and / or against drying out of the skin.