FR2854069A1 - A method of obtaining a soya active principle for use in anti-aging and anti-wrinkle compositions by solubilizing soya cake at acid pH and soya fiber at basic pH, hydrolyzing each with proteases, concentrating and combining - Google Patents

Abstract

A method of obtaining an active principle with anti-aging, anti-wrinkle and firming properties by preparing and then combining two products from soya by (a) solubilizing soya cake at 50 g/l in water at acid pH and conducting simultaneous or successive enzymatic hydrolyses in the presence of proteases and (b) solubilizing soya fibers at basic pH and conducting one or more enzymatic hydrolyses with the aid of one or more proteases. In both cases the hydrolyzate is filtered, decanted or centrifuged to separate the solubilized phase and the insoluble products and the active phase is subjected to successive concentration by filtration, ultrafiltration, osmosis or nanofiltration. At least 10% of product (b) is combined with product (a). Independent claims are included for an active principle with anti-wrinkle properties obtained by the method and with an amount of dry matter 30-150 g/l, a pH of 4.0-8.0, a protein content 15-90 dl/g and an amber color and for a composition including 0.1-10 % of the claimed active principle in an appropriate galenic composition such as an emulsified gel, a milk, an emulsion, a lotion, and ointment or a powder.

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 is made up of three essential layers - the epidermis, a stratified surface epithelium, mainly made up 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 Npropeptides and Cpropeptides 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 decrease in fibroblasts.

  bes studies have shown that the aging process is the cause not only of a decrease in the proliferation capacity of fibroblasts but also of a reduction in metabolic activity.

  It should also be noted that, between the dermis and the epidermis, there is the dermal-epidermal junction JbE which ensures the connection of the two layers, this JbE contributing strongly to the modulation of the surface skin.

  Thus, when fibroblasts exhibit reduced metabolic activity, they lose their ability to adhere to collagen fibers, making JbE 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 results from a disturbance of the interactions between the collagens and the proteoglycans, versican, decorin, biglycan and hyaluronan.

  - the hypodermis, consisting essentially 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 method, 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. 20 1 / PROCEb b OBTAINING THE ACTIVE INGREDIENT ACCORDING TO THE PRESENT INVENTION The process consists in combining two products A and B, both obtained from soya. 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 / l by weight, in water at acid pH, - simultaneous or successive enzymatic hydrolysis in 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 nanof i Itration.

  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 INGREDIENT OBTAINED 2-1 / Dry matter: The dry matter content is obtained by passing 10 9 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 / I, more particularly 60 to 90 g / I. 25 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 Lowry method. The results give a protein content of 15 to 90 g / l, more particularly 35 to 55 g / l.

  2-4 / Product color: The color of the active ingredient obtained is amber.

  3 / EFFECT OF THE ACTIVE INGREDIENT ON COLLAGENS I AND III: 3-1 / Protocol: The collagens concerned are more particularly the collagens of type I 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 concerning 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 principle or 20 in the presence of 0.05 and 0.10 g / ml 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: Synthesis of collagen I (ng / ml) Change in% Collagen I / control Control 101 + 35 Active ingredient 0.5% 102 + 38 + 1 Principle active 1.0% 164 + 48 + 62 Active ingredient 2.0% 278 + 64 + 175 Vitamin C 0.05 pg / ml 116 + 41 + 15 Vitamin C 0.10 // ml 261 + 85 + 158 Note that the synthesis of collagen I from 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 pc / ml. 3-2-2 / Human fibroblasts at the bottom of wrinkles / Collagen I: Synthesis of collagen I (ng / ml) Change in% Collagen I / control Witness 34 + 10 Active ingredient 0.5% 34 + 12 Active ingredient 1.0% 66 + 15 + 94 Active ingredient 2.0% 94 + 19 + 176 Vitamin C 0.05 pg / ml 36 + 12 + 6 Vitamin C 0.10 pg / ml 72 + 16 + 112 The previous results are even better in the case of fibroblasts from the bottom of wrinkles. We arrive at a 176% increase in the collagen I synthesis of fibroblasts from the background 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: Collagen III synthesis (ng / ml) Change in% Collagen III / control Control 5.6 + 2.1 Active ingredient 0.5% 11.9 + 3.8 + 113 Active ingredient 1.0% 13.1 + 5.1 + 134 Active ingredient 2.0% 17.3 + 4.7 + 209 Vitamin C 0.05 p / ml 5.9 +, 9 + 5 Vitamin C 0 , 10 p // ml 10.25 + 3.3 + 83 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 fibroblasts at the bottom of wrinkles / Collagen III: Synthesis of collagen III (ng / ml) Variation in% Collagen h III / control Control 0.4 + 0.21 Active ingredient 0.5% 0.4 + 0.25 Active ingredient 1.0% 0.52 + 0.31 + 130 Active ingredient 2.0% 1.29 + 0.26 + 273 Vitamin C 0.05 pg / ml 0.40 + 0.24 Vitamin C 0.10 d / ml 0.48 + 0.25 + 120 The results on the background fibroblasts 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: On can see that the active principle according to the present invention promotes the synthesis of the two collagen fibers I and III, predominant in the dermis, and tends to restore the metabolic activity of fibroblasts at the bottom of wrinkles.

  4 / EFFECT bU ACTIVE PRINCIPLE ON SYNTHESIS bE CARBOXYPEPTIbASE: 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 the 10 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 of 5 p // ml of vitamin C. The cells are recovered and the total RNA are extracted. The carboxypeptidase mRNAs and the internal control mRNA, 13-actin, are determined. 15 The results are found in the table: Carboxypeptidase mRNA level (%) Control 100 Active ingredient 1% 115 + 6 Active ingredient 2% 121 + 5 Vitamin C 5 d / ml 113 + 5 Thus the active ingredient according to the present invention promotes the expression of carboxypeptidase mRNAs by 21%, which makes it possible to increase the transformation of procollagens into collagens and to improve the structure of the dermis.

  5 / EFFECT OF THE ACTIVE INGREDIENT ON SYNTHESIS bE DECORINE: 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: Rate of decorin mRNA (%) Control 100 Active ingredient 1% 110 + 3 Active ingredient 2% 131 + 9 Vitamin C 5 p / ml 116 + 7 The active ingredient according to the present The 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 capacity of fibroblasts to visibly contract a collagen gel.

  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 gel I. 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: Capacity of contraction (%) Human fibroblasts Normal human fibroblasts against the background of wrinkles Control 44 + 2 15 + 1

WITHOUT

Vitamin C 45 + 6.5 22 + 0.5

UVA

  Active ingredient 2% 47 + 4.5 25 + 7 Control 17 + 3.5 0

WITH

Vitamin C 25 + 4.5 2 + 2

UVA

  Active ingredient 2% 37 + 7.5 13 + 3 It is found that the active ingredient makes it possible to improve the contraction of normal human fibroblasts, to restore the ability of background fibroblasts 5 to wrinkle and to limit the UVA alteration 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 irradiations, 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 / cm2 of active principle formulated at 3% in an emulsion or with 5 mg / cm2 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 by 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. he

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

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

  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.

  Active principle according to the invention Elasticity 3% Tonicity - 8% 9 / EFFECT OF THE ACTIVE PRINCIPLE ON WRINKLES: The analysis relates to the observation of fingerprints by a profilometer equipped with an image analyzer.

  These imprints are made of silicone polymer and produced at the level of crow's feet on volunteers divided into 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 Active ingredient according to the invention Vitamin C Group A | Group B Group C Total wrinkled surface - 26% - 30% - 26% Number of wrinkles - 21% - 22% - 13% Length of wrinkles - 25% - 21% - 20% These results are significant according to the Student test on matched data.

  Figures 7 to 12 show the different results.

  In FIGS. 7 and 8, the images of the skin of volunteers from 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 20 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 test on paired data.

  Wrinkle of the lion Total wrinkled surface - 29% Number of wrinkles - 35% Length of wrinkles - 27%

Claims (9)

  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 solubilization of soybean meal at a rate of at least 50 g / I by weight, in water at acid pH, - simultaneous or successive enzymatic hydrolysis in the presence of proteases, - filtration, decantation or centrifugation 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 soybean fibers in water at basic pH, - at least one enzymatic hydrolysis at 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 ac phase tive by filtration, ultrafiltration, osmosis or nanofiltration, and to combine these two products A and B. 2. Method for obtaining an active ingredient having anti-wrinkle properties according to claim 1, characterized in that at least 10% of product B is combined with product A. 3. Active ingredient 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 / I - pH between 4.0 and 8 , 0 ,.   - protein level between 15 to 90 g / I, and - amber color of the active ingredient obtained.   4. Active principle according to claim 3, characterized in that it has the following parameters: - dry matter content between 35 to 55 g / I - pH between 6.3 and 7.3 - protein content between 60 to 90 g / I, and 10 - 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 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, a ointment or powder.