WO2004096168A1

WO2004096168A1 - Method for obtaining an active ingredient from soya having anti-aging properties, compositions and use of said compositions for cosmetic purposes

Info

Publication number: WO2004096168A1
Application number: PCT/FR2004/050168
Authority: WO
Inventors: Jean Paufique
Original assignee: Societe Industrielle Limousine D'application Biologique, Dite Silab
Priority date: 2003-04-23
Filing date: 2004-04-22
Publication date: 2004-11-11
Also published as: FR2854069B1; FR2854069A1

Abstract

The invention relates to a method for obtaining an active ingredient with anti-wrinkle and firming properties, characterised in comprising the production of two products A and B from soya, said two products being obtained by the series of the following steps: Product A - solubilisation of a soya filter cake at a rate of at least 50 g/l by weight, in water at an acidic pH, simultaneous or successive enzymatic hydrolyses in the presence of proteases, filtration, decantation or centrifugation to separate the solubilised phase from the insoluble products and successive concentrations of the active phase by filtration, ultrafiltration, osmosis or nanofiltration, Product B - solubilisation of soya fibres in water at a basic pH, at least one enzymatic hydrolysis by means of at least one protease, filtration, decantation or centrifugation to separate the solubilised phase and the insoluble products and successive concentrations of the active phase by filtration, ultrafiltration, osmosis or nanofiltration and mixing of said two products A and B. The invention further relates to the uses thereof and the compositions including said active ingredient.

Description

PROCESS FOR OBTAINING AN ACTIVE INGREDIENT FROM SOYBEANS,

HAVING ANTI-AGING PROPERTIES, COMPOSITIONS AND

USES OF THESE COSMETIC COMPOSITIONS

The present invention relates to a process for obtaining an active principle from soy which has anti-aging properties.

The invention covers the compositions using this active principle as well as the uses of these compositions for cosmetic purposes. Life expectancy increases and aging leads to thinner, drier and above all less elastic skin.

This is manifested in a very visible manner by the appearance on the face of wrinkles which correspond inter alia to changes in the structure of the dermis.

Studies have shown that wrinkles can be linked to stiffening of the skin and sagging of the deep dermis.

Indeed, the skin is a complex organ which consists of three essential layers:

- the epidermis, a stratified surface epithelium, consisting mainly of keratinocytes. - the dermis, connective tissue, consists of fibroblasts. The dermis comprises an extra cellular matrix, ECM, synthesized from proteins, the main ones of which are essentially type I and III collagens, glycoproteins other than collagens, elastin and proteoglycans. The specific interactions between these different classes of proteins give rise to this functional tissue.

MEC owes its mechanical properties essentially to the fibrous network of collagens. These fibrillar collagens are synthesized from collagens in the form of long precursors, procollagens I and II, which are then transformed into collagen molecules by cleavage of N-propeptides and C-propeptides by specific proteases such as lysyl oxidases or carboxypeptidases.

The presence of wrinkles is therefore essentially induced by a reduced level of precursors of collagen fibrils, which decreases the amount of collagen, by a reduced level of elastin and finally a reduction in f ibroblasts.

Studies have shown that the aging process is the cause not only of a decrease in the proliferation capacity of f ibroblasts but also of a reduction in metabolic activity. It should also be noted that, between the dermis and the epidermis, there is the JDE dermo-epidermal junction which ensures the connection of the two layers, this JDE contributing greatly to the modulation of the surface skin.

Thus, when fibroblasts exhibit reduced metabolic activity, they lose their ability to adhere to collagen fibers, making JDE less effective.

We also know that age and / or repeated exposure to the sun lead to damage and alterations of the dermis by modifying the connections between fibroblasts and CEM, essentially made up of collagen.

Finally, it should be noted that another degradation of the dermal network comes from a disturbance of the interactions between collagens and proteoglycans, versican, decorin, biglycan and hyaluronan.

- the hypoder e, essentially consisting of fat cells. We know, and many studies have shown, that the external appearance of the skin is a reflection of the organization of these different layers.

The process for obtaining according to the present invention makes it possible to obtain an active principle which, formulated in a suitable galenical composition, makes it possible to act on: - the synthesis of collagens I and III,

- the synthesis of carboxypeptidase,

- the synthesis of decorin,

- the contractile power of fibroblasts,

- the alteration of the dermal matrix, - the biomechanical properties of the skin.

Tests on the studies of anti-aging properties, in particular anti-wrinkle tests and the study of the biomechanical properties of the skin on volunteers which make it possible to verify the tangible results obtained by application of the compositions including the active principle obtained by the process. according to the invention. The invention is now described in detail according to a particular, nonlimiting embodiment, through a process, the characterization of the active principle obtained and the compositions allowing, by application, to obtain the desired cosmetic results. Figures are appended to show the visible results obtained.

1 / PROCESS FOR OBTAINING THE ACTIVE INGREDIENT ACCORDING TO THE PRESENT INVENTION: The process consists in combining two products A and B, both obtained from soybeans. These two products are each obtained by the succession of the following stages: Product A

- solubilization of soybean meal at a rate of at least 50 g / 1 by weight, in water at acid pH,

- simultaneous or successive enzymatic hydrolysis in the presence of proteases, - filtration, decantation or centrifugation in order to separate the solubilized phase and the insoluble products, and

- successive co ncentrations of the active phase by filtration, ultrafiltration, osmosis or nanofiltration.

Product B

- solubilization of soybean fibers in water at basic pH,

- at least one enzymatic hydrolysis using at least one protease,

- filtration, decantation or centrifugation in order to separate the solubilized phase and the insoluble products, and - successive concentrations of the active phase by filtration, ultrafiltration, osmosis or nanofiltration.

We then proceed to the association of the two products A and B. More particularly, we associate with product A at least 10% of product B.

2 / CHARACTERIZATION OF THE ACTIVE PRINCIPLE OBTAINED:

2-1 / Dry matter:

The dry matter content is obtained by passing 10 g of product placed in an oven at 105 ° C, until a constant weight is obtained.

The results give a weight of 30 to 150 g / 1, more particularly 60 to 90 g / 1. 2-2 / pH:

The pH is measured by the potentiometric method, at room temperature and the results give a value between 4.0 and 8.0, more particularly between 6.3 and 7.3. 2-3 / Protein level:

The protein level is obtained by the Lo ry method. The results give a protein content of 15 to 90 g / 1, more particularly 35 to 55 g / 1. 2-4 / Product color:

The color of the active ingredient obtained is amber.

3 / EFFECT OF THE ACTIVE INGREDIENT ON COLLA6ENES I AND III:

3-1 / Protocol: The collagens concerned are more particularly type I collagens and

III which represent the major components of the dermis as indicated in the preamble.

The study concerns normal human fibroblasts and specific fibroblasts taken by biopsy at the bottom of a frontal wrinkle. The tests relating to the active principle according to the invention are carried out in comparison with vitamin C which is known to have a favorable effect on the synthesis of the collagens concerned.

After seeding and incubation of fibroblasts in a suitable medium, the fibroblasts are treated in the presence of 0.5%; 1.0%; 2.0% of active ingredient or in the presence of 0.05 and 0.10 μg / l of vitamin C diluted in the culture medium.

Witnesses are made.

After incubation, the cell supernatants are recovered for an ELISA assay of the collagens. 3-2 / Results:

3-2-1 / Normal human fibroblasts / Collagen I

It is noted that the collagen I synthesis of normal human fibroblasts is increased more significantly with the active principle according to the invention than with vitamin C at the higher of the two dosages.

The synthesis of collagen I is increased by 175% with the active ingredient to 2% and 158% with vitamin C at 0.10 μg / ml. 3-2-2 / Human background fibroblasts of wrinkles / Collagen I:

The previous results are even better in the case of fibroblasts originating from the background of wrinkles. We arrive at an increase of 176% in the synthesis of collagen I of fibroblasts at the bottom of wrinkles with the active principle according to the present invention, whereas for vitamin C, at the highest dosage, the increase is limited to 112%.

3-2-3 / Normal human fibroblasts / Collagen III:

The synthesis of collagen III is increased by 209% with the active ingredient to 2% according to the present invention while vitamin C only leads to an increase of 83%.

3-2-4 / Human background fibroblasts / Collagen III

The results on fibroblasts from the background of wrinkles show an increase in the synthesis of collagen III by 273% compared to the 120% obtained in the presence of vitamin C.

Conclusion:

It can be seen that the active principle according to the present invention promotes the synthesis of the two collagen fibers I and III, which are predominant in the dermis, and tends to restore the metabolic activity of fibroblasts at the bottom of wrinkles.

4 / EFFECT OF THE ACTIVE INGREDIENT ON THE SYNTHESIS OF CARBOXYPEPTIDASE: The carboxypeptidase is a protease allowing the transformation of procollagen into collagen by cutting chains.

The study relates to the evaluation of the effect of the active ingredient on the expression of

Messenger RNAs of the carboxypeptidase.

For this, human fibroblasts are incubated in the presence of 1%; 2% of active principle according to the invention or 5 μg / ml of vitamin C.

The cells are recovered and the total RNAs are extracted. We determine the

Carboxypeptidase mRNA and internal control mRNAs, β-actin.

The results are grouped in the table:

Thus the active principle according to the present invention promotes the expression of the mRNA of the carboxypeptidase by 21%, which makes it possible to increase the transformation of procollagens into collagens and improving the structure of the dermis.

5 / EFFECT OF THE ACTIVE INGREDIENT ON THE SYNTHESIS OF DECORIN:

Decorin is a proteoglycan essential for the organization of the extracellular matrix.

The tests are also carried out in comparison with the vitamin as before. The results are grouped in the following table:

The active ingredient according to the present invention, at a concentration of 2%, leads to an increase in the expression of decorin mRNAs by 31%, which promotes the improvement of the structural organization of the dermis.

6 / EFFECT OF THE ACTIVE INGREDIENT ON THE CONTRACTILE POWER OF FIBROBLASTS: 6-1 / Protocol:

To determine the capacity of fibroblasts to adhere to the collagen network and to maintain a three-dimensional network of the dermis, the ability of fibroblasts to visibly contract a collagen gel is analyzed in vitro.

Normal fibroblasts, fibroblasts of the background of wrinkles are tested in parallel, these two types being or not subjected to UVA irradiations. The fibroblasts of the two types subjected and not subjected to UVA are added to a collagen I gel.

The diameter of the gels thus formed is determined after incubation in the presence of the active principle or of vitamin C.

6-2 / Results:

It is noted that the active principle makes it possible to improve the contraction of normal human fibroblasts, to restore the capacity of fibroblasts at the bottom of wrinkles and to limit the alteration by UVA of this contractile power of the two types of fibroblasts.

7 / CAPACITY OF THE ACTIVE INGREDIENT TO LIMIT FIBROSIS ON THE DERMAL MATRIX '- This study is carried out on explants which are exposed to UVA irradiation, treated or not with the active principle according to the invention.

The explants are irradiated at 8 joules / cm and then treated with 5 mg / cm ² of active principle formulated at 3% in an emulsion or with 5 mg / cm ² of vitamin C at 1% in the same emulsion. This treatment is repeated once a day for five consecutive days.

4 μm cuts are made using known techniques for cutting frozen samples.

These samples are colored and the images are analyzed with suitable software. Figures 1 to 6 show the results obtained.

There is a limitation in the alteration of collagen fibers of explants treated with the active ingredient subjected to repetitive UVA irradiation.

8 / EFFECT OF THE ACTIVE INGREDIENT ON THE BIOMECHANICAL PROPERTIES5 OF THE SKIN: The efficacy of the active ingredient against placebo on the biomechanical properties of the skin is being studied.

The test consists in measuring the deformation of the skin treated with the active ingredient at 4% and against placebo for 28 days by twice daily application.

A probe makes it possible to measure the deformation of the skin obtained during a measurement involving several successive aspirations of a given duration.

One can then determine from the curves obtained: instantaneous deformation, immediate retraction, total elongation and residual deformation.

Elasticity is the ratio of the residual strain to the total elongation. If this ratio increases, the elasticity increases.

The tone is determined by the difference in total elongation minus immediate retraction.

The following results are significant according to the Student test on paired data.

9 / EFFECT OF THE ACTIVE INGREDIENT ON WRINKLES:

The analysis relates to the observation of fingerprints by a profiler equipped with an image analyzer. These imprints are made of silicone polymer and produced at the level of crow's feet on volunteers distributed by groups A, light wrinkles treated with the active principle according to the invention, B, pronounced wrinkles treated with the active principle according to the present invention and C , pronounced wrinkles treated with vitamin C dosed at 0.5%. The dosage formulation is an emulsion.

The total wrinkled surface, the total length of the wrinkles and the number of wrinkles are then measured.

The results are summarized in the following table:

These results are significant according to the Student test on paired data.

Figures 7 to 12 show the different results.

In FIGS. 7 and 8, the images of the skin of the volunteers of group A, before and after treatment with the active principle according to the invention, dosed at 4%.

In FIGS. 9 and 10, the images of the skin of the volunteers of group B before and after treatment with the active ingredient dosed at 4%.

In FIGS. 11 and 12, the images of the skin of volunteers from group C before and after treatment with vitamin C formulated at 0.5%. There is an improvement in the various parameters, this whether the wrinkles are light or pronounced.

The active principle is introduced into a galenical composition adapted to allow application to the skin under optimized conditions. Mention may be made, as dosage form, of a gel, an emulsified gel, a milk, an emulsion, a lotion, an ointment or a powder.

These results were confirmed by the anti-wrinkle study of the active ingredient on the lion's wrinkle (frontal wrinkle). The study involved two groups of volunteers, one testing a placebo emulsion, the other the emulsion containing the active ingredient formulated at 4%. The technique of taking impressions and analysis is identical to that described above. These results are significant according to the

Student on paired data.

Claims

1. Method for obtaining an active principle having anti-aging, anti-wrinkle and firming properties, characterized in that it consists in preparing two products A and B from soybeans, these two products being obtained by the succession of next steps: Product A

- simultaneous or successive enzymatic hydrolysis in the presence of proteases,

- filtration, decantation or centrifugation in order to separate the solubilized phase and the insoluble products, and

- successive concentrations of the active phase by filtration, ultrafiltration, osmosis or nanofiltration,

Product B

- solubilization of soy fibers in water at basic pH, - at least one enzymatic hydrolysis using at least one protease,

- successive concentrations of the active phase by filtration, ultrafiltration, osmosis or nanofiltration, and to combine these two products A and B.

2. Method for obtaining an active principle having anti-wrinkle properties according to claim 1, characterized in that one associates with product A at least 10% of product B.

3. Active principle obtained from the process according to claim 1 or 2, characterized in that it has the following parameters: - dry matter content between 30 to 150 g / 1

- pH between 4.0 and 8.0 ,.

- protein level between 15 to 90 g / 1, and

- amber color of the active ingredient obtained.

4. Active ingredient according to claim 3, characterized in that it has the following parameters:

- dry matter content between 35 to 55 g / 1

- pH between 6.3 and 7.3

- protein level between 60 to 90 g / l, and - amber color of the active ingredient obtained.

5. Use of the active principle according to claim 3 or 4, characterized in that it makes it possible to promote the synthesis of the two collagen fibers I and III, predominant in the dermis.

6. Use of the active principle according to claim 3 or 4, characterized in that it allows an increase in the expression of mRNAs of decorin, which promotes the improvement of the structural organization of the dermis.

7. Use of the active principle according to claim 3 or 4, characterized in that it promotes the expression of the mRNA of the carboxypeptidase, which makes it possible to increase the transformation of procollagens into collagens and to improve the structure of the dermis.

8. Use of the active principle according to claim 3 or 4, characterized in that it makes it possible to improve the contraction of fibroblasts and to limit the alteration by UVA of the contractile power of fibroblasts.

9. Composition including from 0.1 to 10% of active principle according to one of claims 3 or 4, in a suitable galenical composition such as a gel, an emulsified gel, a milk, an emulsion, a lotion, an ointment or a powder.