EP1802265A2

EP1802265A2 - Method of obtaining a firming active principle and active principle thus obtained

Info

Publication number: EP1802265A2
Application number: EP05797625A
Authority: EP
Inventors: Jean Paufique
Original assignee: Societe Industrielle Limousine dApplication Biologique SA SILAB
Current assignee: Societe Industrielle Limousine dApplication Biologique SA SILAB
Priority date: 2004-09-01
Filing date: 2005-08-25
Publication date: 2007-07-04
Also published as: US20080038388A1; FR2874503B1; WO2006027521A3; US7815948B2; WO2006027521A2; FR2874503A1

Abstract

The invention relates to a method of obtaining a firming active principle resulting from the solubilisation of Cucurbita pepo seed meal treated by means of enzymatic hydrolysis. The invention also relates to the active principle thus obtained and to the uses thereof in cosmetics.

Description

PROCESS FOR OBTAINING A ACTIVE FIRMING, ACTIVE OBTAINED PRINCIPLE AND FUNCTIONALITIES

The present invention relates to a process for obtaining a firming active ingredient from Cucurbita pepo seed cake, able to fight against deformations of the dermis such as stretch marks or wrinkles.

The invention also covers the active ingredient obtained and the functionalities. The simple chronological aging causes alterations of the characteristics of the extracellular matrix which consists of collagen fibers and elastic fibers. However, these fibers have a very low turnover rate in adults.

Collagen fibers account for most of the total mass of the dermis. This network of collagen makes it possible to give birth to a functional tissue that ensures the maintenance of the superficial layers of the skin. Collagens type I, III and V are mainly concerned.

It is therefore necessary to find substances able to maintain the elasticity of the skin thus able to preserve the network of elastic fibers and collagen. We also know that to preserve this capital, we must also fight against the consequences of other phenomena likely to cause profound alterations such as puberty, pregnancy, hormonal changes or skin treatments.

In fact, the purpose of the present invention is to select a natural active ingredient by a suitable method that promotes the synthesis of certain molecules and limit the action of certain enzymes.

Thus, we know that in the family of metalloproteinases:

MMP-I or collagenase-1 degrade type I, II and III collagens, - MMP-2 or gelatin A ₁ play an important role in the final degradation of collagen fibers but also in the hydrolysis of elastin.

Another enzyme, cathepsin L, causes a significant alteration of the elastic network and also degrades collagens. The invention is now described in detail, first of all through its method of obtaining the firming active ingredient, aimed at anti-stretch marks and anti-wrinkles, then through the specific characteristics of the active ingredient obtained and finally through the mechanisms involved, this through results obtained by in vitro and in vivo tests. 1 / Method of obtaining:

The method according to the invention comprises the following steps:

solubilization of Cucurbita pepo seed cake in an aqueous solution at a rate of at least 50 g / l,

simultaneous or successive enzymatic hydrolysis (s), separation of the soluble and insoluble phases by any suitable means, in particular by centrifugation,

inactivation of enzymatic activities,

- mechanical filtration, and

- successive concentrations of the active fraction followed by sterilizing filtration.

Cucurbita pepo is a plant of the cucurbitaceae family.

2 / Characterization of the active ingredient obtained from Cucurbita pepo

2-1 / Dry matter content: The solids content is obtained by passing a given amount of active ingredient obtained by the implementation of the process, placed in an oven at 105 ⁰ C until a constant weight. The solids content for the active ingredient according to the invention is between 20 and 200 g / l, more particularly between 50 and 70 g / l.

2-2 / pH measurement: The pH is measured by the potentiometric method, at room temperature. The pH values of the active ingredient according to the invention are between 5 and 9, more particularly between 6.5 and 7.5.

2-3 / Determination of the nitrogen content: The determination is carried out by the LOWRY method.

The deduced protein content, irrespective of the protein source, is between 15 and 145 g / l, more particularly between 38 and 50 g / l.

2-4 / Characterization of the protein fraction: The distribution of the molecular masses of the different molecular species present in the active principle obtained by the implementation of the method according to the invention is determined by FPLC (Fast Protein Liquid Chromatography).

Markers serve as standards and it is found that the protein fraction has a molecular weight of less than 40,000 Daltons.

2-5 / Characterization of carbohydrates:

2-5-1 / Dosage of simple sugars:

The level of simple sugars is divided into 54.5% glucose and 45.5% fructose. 2-5-2 / Degree of polymerization

The table below shows that the glucidic fraction of the active ingredient according to the present invention comprises glucose and fructose in the form of oligosαcchαrides and polysαcchαrides with a degree of polymerization greater than or equal to 7.

2-6 / Identification of the active fraction:

To carry out this identification, the active principle is studied in its complete composition called PA and the active ingredient of which a molecular fraction has been precipitated by a lowering of the pH, called PA ₅ .

2-6-1 / Comparative analysis:

The efficiencies of the two products are compared in relation to their activities with respect to MMP-I synthesis on normal human fibroblast cultures subjected to UVA irradiation.

The study is carried out by western blot.

The active ingredient makes it possible to limit the synthesis of MMP-I whereas the active ingredient cut by a fraction does not make it possible to obtain this limitation.

This fraction is therefore the active fraction.

2-6-2 / Determination of the active fraction:

The LOWRY method is again used and the protein content for PA and PA ₅ is compared.

It is found that 19% of the proteins disappeared during the lowering of the pH to

5.0.

Molecular analysis by gel filtration liquid chromatography indicates that the protein fraction has a molecular weight of 29,000 daltons.

2-7 / Stability - Solubility:

The active ingredient PA in aqueous solution is stable: for pH values between 2.0 and 10.0,

at a temperature of between 40 and 80 ^° C.,

- with ethanol, in mixtures containing at most 40% pure ethanol. The active ingredient is stable in different cosmetic formulations. Thus the active ingredient is introduced at 5% in the following formulas: - clear gel based on Carbopol, Phenonip and water,

- opaque gel based on Sepigel 305, Phenonip and water,

emulsion gel based on Montanov 202, isopropyl palmitate, Phenonip, Sepigel 305 and water, anionic emulsion based on stearic acid, tethanolamine, isopropyl palmitate, phenonip and water,

cationic emulsion based on quaternium-82, cetyl alcohol, isopropyl palmitate, phenonip and water, or

nonionic emulsion based on Montanov 202, simulsol 165, isopropyl palmitate, phenonip and water.

The active ingredient according to the invention is also stable with respect to raw materials used in cosmetics such as thickeners, emulsifiers or alcoholic solvents up to 40%.

3 / In vitro efficacy tests:

3-1 / Studies of the ability of the active ingredient to limit the degradation of cutaneous fibers. Studies are conducted on human fibroblasts from normal human stretch marks and fibroblasts.

The active ingredient is used at a concentration of 2%.

3-1-1 / Effect of the active ingredient on the synthesis of cathepsin-L.

Since this enzyme exhibits high elastolytic activity in fibroblasts, it is found that the active ingredient according to the invention makes it possible to reduce the synthesis of this enzyme and to return to a level comparable to that of normal human fibroblasts.

After incubation of the two types of fibroblasts in culture medium in the presence or absence of the active ingredient and analysis by western blot, the following results are obtained:

3-1-2 / Effect of the active ingredient on the synthesis of MMP-I.

The MMP-I metalloprotease, which is responsible for the degradation of collagen I and III fibers, must be reduced in synthesis in the presence of the active ingredient according to the present invention and this is what is observed in the results which follow, obtained on cultures of normal human fibroblasts and fibroblasts from stretch marks, analyzed in western blot.

3-1-3 / Effect of the active ingredient on the activity of MMP-2.

MMP-2 metalloproteinase is involved in the degradation of collagen fibers and in the hydrolysis of elastin. It is necessary to fight against its activity and the present analysis shows the anti-MMP-2 activity of the active principle according to the invention. This analysis is conducted by zymography on normal human fibroblast cultures and fibroblasts derived from stretch marks. The results confirm this anti-MMP-2 activity with a 69% reduction.

3-2 / Study on the ability of the active ingredient to reorganize the fibrillar network:

3-2-1 / Effect of the active ingredient on cell renewal:

The aim is to compare the proliferation and replication capacity of normal human fibroblasts and that of stretch mark fibroblasts, in the absence and in the presence of the active ingredient according to the invention.

The results are obtained by spectrophotometric measurement of

MTT.

It is found that the active ingredient according to the invention makes it possible to restore a capacity for proliferation of fibroblasts resulting from stretch marks comparable to that of normal fibroblasts.

3-2-2 / Effect of the active principle on the synthesis of collagen I: The study is carried out by ELISA assay on normal human fibroblasts and on fibroblasts derived from stretch marks. It is found that the active ingredient, back, eJL to i. o 2o% / significantly increases, 62%, collagen I synthesis.

3-2-3 / Effect of the active ingredient on fibrilin synthesis:

Since fibrilin is a protein involved in the organization of the elastic fiber network, the study aims to compare the fibrinin synthesis rate in normal human and stretch mark fibroblast cultures.

The ability of the active ingredient to restore this synthesis capacity for fibroblasts derived from stretch marks and to increase this synthesis for normal fibroblasts is noted.

4 / In vivo efficacy tests:

4-1 / Effect on the microrelief of a skin verqeturée:

Fingerprints are performed on volunteers on specific stretched areas and on non-stretched areas, before and after 42 and 84 days of twice-daily placebo-controlled care.

These fingerprints are analyzed by means of a profilometer equipped with an image analyzer.

The gray levels of the resulting digital images can be determined.

An adapted software makes it possible to obtain the average roughness.

The active ingredient is used in formulation, in this case, with the following galenic formula:

Isopropyl palmitate: 10%

Isononyl isononanoate (Lanol 99) 8% Arachidyl alcohol / Behenyl alcohol / Arachidyl glucoside 5%

(Montanov 202)

Glycerol 3%

Myreth-3 Myristate (Lanol 14M) 2% glycol palmitate (Lanol P) 1% Shea butter 1%

Phenonip 0.7%

Viscolam AT 64 0.5%

Active principle according to the invention 4%

Water (Quantity Enough For) 100% It can be seen that the active ingredient according to the invention makes it possible to significantly reduce the roughness differences of 14% between the stretched zones and the non-stretched zones.

The active ingredient makes it possible to smooth the differences between the stretched areas and the non-stretched zones, the stretch marks are then less visible.

PPI: roughness with active ingredient according to the invention.

PPL: roughness with placebo according to the invention.

4-2 / Effect on the biomechanical properties of vertebral skin: The objective of this study is to quantify in vivo, on volunteers, the firming effect of the active ingredient formulated at 4% in emulsion by evaluation of the biomechanical properties of the skin. skin at the level of stretch marks using a device marketed under the name "Cutometer".

One seeks to measure among the mechanical properties: - cutaneous elasticity, (the more the value of the coefficient tends towards 1 and the more the skin is elastic)

- tonicity (the lower the value of the coefficient and the more toned the skin)

the biological deformation (the value of the parameter tends to 1 and the more the skin is deformed), and - the ability of the skin to return to its original state (the more the value tends towards O and the more the skin returns quickly to its normal state). The galenic formulation is identical to that of the previous test.

Skin elasticity

Pe: cutaneous elasticity with active principle.

Pi: cutaneous elasticity with placebo.

It can be seen that the elasticity variation between the stretched and non-stretched areas remains stable over the zone receiving the placebo and decreases over the area treated with the active ingredient. This result was observed on 65% of the volunteers.

Tone

Pe: skin tonicity with active ingredient.

Cutaneous tonicity with placebo.

The difference in tone is 25% between the two areas of stretch and non-stretched. This result is obtained on 70% of the volunteers. Biological deformation

Pe: cutaneous deformation with active principle.

Pi: cutaneous deformation with placebo.

There is a 15% decrease in biological deformity at 84 days. This result is obtained on 65% of the volunteers.

Ability of the skin to return to its normal state

Pe: ability to return to the normal state of an area treated with the active ingredient.

Pi: ability to return to the normal state of a placebo treated area.

There is a decrease of 30% on the difference of return to the initial state.

4-3 / Effect on firming properties:

One seeks to measure among the firming properties of the active ingredient: - the raw cutaneous elasticity, (the more the value of the coefficient tends towards 1 and the more the skin is elastic) - the cutaneous elasticity net (the more the value of the coefficient tends towards 1 and the more the skin is elastic).

The galenic formulation tested is as follows:

Arachidyl alcohol / Behenyl Alcohol / Arachidyl glucoside (Montanov 202) 5%

Glycol palmitate (Lanol P) 1% / O

Myreth-3 Myristate (Lanol 14 M) 2%

Isopropyl palmitate 10%

Isononyl isononanoate (Lanol 99) 8%

Shea butter 1% Sodium acrylate / sodium acryloyldimethyl taurate copolymer C13-14 Isoparaff in

/ Laureth -7 / sorbitan oleate (Viscolam AT 64) 0.5%

Glycerol 3%

Phenonip 0.7%

Active ingredient 4%

Water (Quantity Enough For) 100%

Raw cut elasticity

Pe: raw elasticity with active principle.

Pi: raw elasticity with placebo.

There is an increase in gross elasticity in the areas treated with the active ingredient compared to the areas receiving the placebo.

Net cut elasticity

Pe: net elasticity with active principle.

Pi: net elasticity with placebo.

There is an increase in net elasticity in areas treated with the active ingredient compared to areas receiving placebo. This result was observed on 68% of the volunteers.

4-4 / Effect on the microrelief of a normal skin:

Fingerprints are performed on the forearms of volunteers, before and after 28 and 56 days of twice-daily placebo-controlled care.

The gray levels of the resulting digital images can be determined.

An adapted software makes it possible to obtain the average roughness.

The active ingredient is used in the same galenic formula as the previous test. It is found that the active ingredient according to the invention significantly reduces the roughness of 6% after 56 days of twice-daily treatment. 4-5 / Anti-wrinkle effect:

The objective of this study is to quantify in vivo the anti-wrinkle efficacy of the active ingredient formulated at 4% emulsion versus placebo.

The test is performed on the crow's feet of 20 healthy female volunteers, mean age 57 years.

The anti-wrinkle effect is analyzed by observation of fingerprints using a profilometer provided with an image analyzer which makes it possible to obtain the following parameters:

- the number of wrinkles,

- the total wrinkled surface, and

- the total length of the wrinkles.

The active principle is applied twice daily in formulation, in this case, with the following galenic formula:

Isononyl isononanoate (Lanol 99) 7%

Arachidyl alcohol / Behenyl alcohol / Arachidyl glucoside 4% (Montanov 202)

Cetearyl ethylhexanoate (Lanol 1688) 3% Isopropyl palmitate 3%

PE6-100 stearate / glyceryl stearate (Simulsol 165) 1%

Phenonip 0.7% Stearyl Stearate δRictachol SS 0.5%

Cetyl alcohol 0.5% Active ingredient according to the invention 4%

Water (Quantity Enough For) 100% The results obtained after 28 and 56 days of twice-daily application are shown in the table below, as a percentage of variation compared to the results obtained with the placebo:

It is found that the active ingredient according to the invention formulated at 4% emulsion significantly reduces the number of wrinkles by 19%, the total wrinkled surface of 39% and the total length of wrinkles by 30% after 56 days of wrinkling. twice-daily application.

Both in vitro and in vivo tests show the activities of the principle extracted from a plant of the cucurbitaceae family, Cucurbita pepo and in particular its ability to fight against stretch marks and wrinkles and more widely against deformations of the dermis.

The active ingredient may be introduced into cosmetic compositions with different galenic forms, some examples of which have been given above in an illustrative and nonlimiting manner.

The galenic formulations may include creams, ointments, aqueous or alcoholic lotions, aqueous or alcoholic emulsions or gel, the concentration of active ingredient ranging from 0.1% by weight to 10%, preferably from 2 to 4%.

Claims

1. A method for obtaining a firming active ingredient and able to fight against deformations of the dermis such as stretch marks or wrinkles, characterized in that it comprises the following steps:

- simultaneous or successive enzymatic hydrolysis (s),

separation of the soluble and insoluble phases by any suitable means, in particular by centrifugation,

- inactivation of enzymatic activities, - mechanical filtration, and

concentration of the active fraction followed by sterilizing filtration.

2. Active ingredient obtained by the implementation of the process according to claim 1, characterized by the following parameters:

dry matter content between 20 and 200 g / l, pH measurement between 5.0 and 9.0,

- nitrogen content between 15 and 145 g / l,

- protein fraction with a molecular mass of less than 40 000 daltons,

- Sugars composed of 54.5% glucose and 45.5% fructose with a degree of polymerization greater than 7.

Active ingredient according to claim 2, characterized by the following parameters:

dry matter content between 50 and 70 g / l,

- pH measurement between 6.5 and 7.5,

- nitrogen content between 35 and 50 g / l; - protein fraction with a molecular mass of less than 40 000 daltons.

4. Active ingredient according to claim 2 or 3, characterized in that it comprises an active fraction, protein fraction of 29,000 daltons.

5. Use in cosmetics of the active principle according to one of claims 2, 3 or 4, characterized in that, applied to the skin in a suitable dosage formulation, it makes it possible to limit the degradation of the cutaneous fibers by limiting the synthesis of cathepsin-L, by reducing the synthesis of MMP-I and MMP-2.

6. Use in cosmetics of the active principle according to one of claims 2, 3 or 4, characterized in that, applied to the skin in a suitable dosage formulation, it restores the proliferation capacity of fibroblasts from stretch marks.

7. Use in cosmetics of the active principle according to one of claims 2, 3 or 4, characterized in that, applied to the skin in a suitable dosage formulation, it allows to increase the synthesis of collagen I.

8. Use in cosmetics of the active ingredient according to one of the claims

2, 3 or 4, characterized in that, applied to the skin in a suitable galenic formulation, it makes it possible to restore the synthesis capacity of fibrilin for fibroblasts derived from stretch marks.

9. Use in cosmetics of the active principle according to one of claims 2, 3 or 4, characterized in that, applied to the skin in a suitable dosage formulation, it reduces the roughness of the skin stretch marks.

10. Use in cosmetics of the active principle according to one of claims 2, 3 or 4, characterized in that, applied to the skin in a suitable dosage formulation, it improves the elasticity, tone and ability to the skin to return to its normal state and limit biological deformation.

11. Use in cosmetics of the active principle according to one of claims 2, 3 or 4, characterized in that, applied to the skin in a formulation adapted gαlénique, it allows to reduce the number, the total surface, and the total length of the wrinkles of the skin.