WO2002102347A2

WO2002102347A2 - Method for the preparation of an active component rich in di- and tri-peptides, active component and compositions thus obtained

Info

Publication number: WO2002102347A2
Application number: PCT/FR2002/002114
Authority: WO
Inventors: Jean-Jacques Paufique
Original assignee: Societe Industrielle Limousine D'application Biologique (Silab)
Priority date: 2001-06-19
Filing date: 2002-06-19
Publication date: 2002-12-27
Also published as: FR2825925A1; FR2825925B1; WO2002102347A3

Abstract

The invention relates to a method for the extraction of an active component with a nutritional effect on the skin, characterised in comprising the following steps: solubilisation of animal, vegetable or fish proteins in water, with a proportion of at least 5 % by weight; simultaneous or successive hydrolysis of the above suspension in the presence of one or several enzymes chosen from bacterial aminopeptidases applicable in neutral or acid medium, animal stomach proteases applicable in an acid medium, bacterial proteases applicable in a neutral medium, bacterial proteases applicable in an alkaline medium extracted from Bacillus Licheniformis or Bacillus Subtilis, bacterial endoproteases or pancreatic enzymes of animal origin; concentration of the active fraction of small peptides of molecular mass less than 1400 daltons and a sterilising filtration to give a total mesophilic flora of less than 100 germs/g and an absence of yeasts and moulds. The invention also relates to obtained active component and the cosmetic compositions comprising the above.

Description

PROCESS FOR THE PREPARATION OF AN ACTIVE INGREDIENT RICH IN DI- AND TRI-PEPTIDES, ACTIVE INGREDIENT AND COMPOSITIONS OBTAINED

The present invention relates to a process for the preparation of an active principle rich in di- and tri-peptides. The invention also covers the active ingredient obtained as well as compositions incorporating it.

The purpose of the active ingredient thus extracted is in particular to improve cell proliferation, to compensate for stress, to improve the essential balance for cellular dietetics, to increase the defenses of the skin, to increase the oxygenation of the skin and decrease wrinkles.

In the case of the skin, the active principle according to the present invention makes it possible to rapidly supply nutritional elements to the skin by the remarkable assimilation capacities which it exhibits.

This strong assimilation capacity is based on the fact that its peptide structure has a low molecular weight.

These purified peptide units thus provided accelerate cellular assimilation and vitalize the skin. Skin thus nourished and revitalized also helps to fight against tissue aging and in particular to fill in wrinkles.

In the prior art, a process has been described for manufacturing a hydrolyzate of proteins rich in di- and tri-peptides for artificial nutrition and used in dietetics. This process consists in subjecting an egg white protein or casein protein to the simultaneous or successive action of enzymes, in this case a bacterial protease usable in neutral medium, a bacterial protease usable in alkaline medium and an enzyme pancreatic animal. The purpose of the present invention is to obtain a protein hydrolyzate with a low molecular weight to be absorbed by the skin.

In the prior art, the sector concerned is artificial feeding so that the molecular weight is not a constraint. On the other hand, these products obtained by known methods do not allow applications in cosmetology.

The invention is now described in detail, both in its stages of production and in the characterization of the active ingredient obtained and with regard to test results showing the activities of the product obtained. The description is focused on an example from vegetable proteins obtained from rice (Oryza Sativa) but should be extended as indicated in the additional examples for, among others, soybeans (Glycine max), pumpkin seeds ( Cucurbita Pepo) or sesame (Sesamum indicum) or peas (Pisum Sativum) or lupine (Lupinus albus), cashew, sisymbre, or almonds. More generally, for the implementation of the method according to the invention and for the preparation of the products from this method, all vegetable proteins derived from flour, meal, isolate, concentrate and all protein from animal or marine origin. The invention relates to a method of extracting an active principle having a nutritional action from the skin, characterized in that it comprises the succession of the following stages:

- solubilization of vegetable, animal or marine proteins in water, at a rate of at least 5% by weight.

- simultaneous and / or successive hydrolysis (s) of this suspension in the presence of one or more enzymes chosen from:

- bacterial aminopeptidases in neutral or acidic medium,

- stomach proteases of animal origin usable in an acid medium,

- bacterial proteases in neutral medium, - bacterial proteases usable in alkaline medium extracted from Bacillus Licheniformis or Bacillus Subtilis,

- bacterial endoproteases, or

- pancreatic enzymes of animal origin. concentration of the active fraction in small peptides with a molecular mass of less than 1400 daltons, and

- sterilizing filtration to reach a total mesophilic flora of less than 100 germs / g, and an absence of yeasts and molds.

Vegetable proteins are obtained from cereals, oilseeds, legumes and more particularly rice (Orysa Sativa), soybeans (Glycine max), pumpkin seeds, sesame (Sesamum indicum) or peas (Pinu sativum) The active ingredient obtained by implementing the process has:

- a low molecular mass of less than 1,400 daltons - a dry matter content of between 20.0 and 150.0 g / 1

- a pH between 4.0 and 10.0 and

- a protein content of between 10.0 and 100.0 g / 1 More particularly:

- a dry matter content of between 50.0 and 80.0 g / l, - a pH of between 5.0 and 7.0 and

- a protein content is between 40.0 and 60.0 g / 1. The molecular mass is distributed as follows:

- 20% to 60% having a molecular mass of less than 1400,

- 30%> to 50% having a molecular weight of between 75 and 175 daltons, and - 0 to 5% having a molecular weight of less than 75 daltons.

The invention also covers the nutritional cosmetic composition comprising 0.10% to 5.00% of active principle, associated with excipients and in a suitable dosage form such as: cream, gel, ointment, lotion, milk, emulsion or solution. The various attached figures represent:

FIG. 1, a distribution curve of the peptide fractions obtained in the case of rice,

- Figures 2A-2E, table of results on the proliferation of f ibroblasts over time, respectively for rice, soybeans, peas, pumpkin seeds and sesame seeds,

- Figure 3, table of results on the proliferation of f ibroblasts compared to other compositions,

- Figures 4A-4E, table of cell recovery results after UV stress,

- Figure 5, table of the results of the stimulating activity of the active ingredient,

- Figures 6 A and 6B, tables of results of partial pressure of oxygen and carbon dioxide,

FIGS. 7A and 7B, tables of results of analyzes on volunteers concerning tissue oxygenation, and

- Figure 8, table summarizing the improvements vis-à-vis wrinkles.

1 / PROCESS FOR EXTRACTING AN ACTIVE PRINCIPLE RICH IN DI- AND TRI-PEPTIDES 1-1 / The first product according to the invention from which it is desired to obtain a concentrate rich in di and tri peptides constituting the active principle according to the invention, is rice, including the variety Orysa Sativa. The process consists of carrying out the following steps:

- Solubilization of vegetable proteins from a protein concentrate in water, in particular rice proteins, more particularly the variety Orysa Sativa, at a rate of at least 5% by weight.

- bacterial aminopeptidases in neutral or acidic medium, - stomach proteases of animal origin usable in an acid medium,

- bacterial proteases in neutral medium,

- bacterial proteases usable in alkaline medium extracted from Bacillus Lichenifor is or Bacillus Subtilis, - bacterial endoproteases, or

- pancreatic enzymes of animal origin.

concentration of the active fraction in small peptides with a molecular mass of less than 1400 daltons, and

In the case of rice, a curve shown in FIG. 1 is produced on a column sold under the name Superdex Peptide. Using a 0.02 mol phosphate buffer solution added with 0.25 mol NaCl and working at pH 7.2.

The flow rate on the column is 0.25 ml / min and is expressed on paper by 0.25 cm / min.

It can be seen that the fraction less than 1400 daltons is distributed as follows: For the active principle rich in Di- and Tri-rice peptides:

- f 3: approximately 33% (1400 <molecular mass <175), and

- f 4: approximately 42% (175 <molecular mass <75), and

- f 5: approximately 3% (molecular mass <75), that is to say that the peptides of low molecular masses represent at least 60% to 75% of the total fraction less than 1400 daltons.

These peptide structures according to the invention are specific and of low molecular mass, thus allowing applications in cosmetics as shown by the results which will follow. 2 / CHARACTERIZATION OF THE ACTIVE PRINCIPLE OBTAINED

Dry matter rate:

The dry matter content is obtained by passing the active ingredient extracted in an oven at 105 ° C until a constant weight is obtained. The amount of dry matter obtained, for each of them, is between 50 and 150 g / 1.

We can cite several sources of proteins used which give the following results:

Rice dry matter rate • between 50 and 80g / l Pumpkin seed dry matter rate: between 50 and 60g / l

Dry soybean content: between 50 and 60g / l

Rate of sesame dry matter: between 50 and 60g / l

Pea dry matter rate: between 90 and HOg / l

PH measurement:

The pH is measured by the potentiometric method at room temperature. The pH is between 4.0 and 10, more particularly between 5.0 and 7.0. pH of rice: 7.0 pH of pumpkin seeds: 7.0 pH of soybeans: 7.0 pH of sesame: 7.0 pH of peas: 7.0

Determination of the nitrogen content: The determination is carried out by the method of Kjeldhal or Lowry.

The deduced protein content, whatever the source of protein among the selected products, is between 10 and 100 g / 1 and more particularly between 35 and 90 g / 1. Nitrogen content (Di- and Tri-rice peptides): between 35 and 90g / l Nitrogen content (Di- and Tri-pumpkin seed peptides): between 30 and 60g / l Nitrogen content (Di- and Tri - soy peptides): between 30 and 60g / l Nitrogen content (Di- and Tri-sesame peptides): between 30 and 60g / l Nitrogen content (Di- and Tri-pea peptides): between 70 and 100g / l

3 / EFFECTS OF THE ACTIVE INGREDIENT ON THE PROLIFERATION OF FIBROB HUMAN ASTES 3-1 Effects over time:

The study is carried out on human ibroblasts to which the active ingredient rich in Di- and Tri-peptides obtained from the various sources according to the invention is added at 1%, and 2% by volume.

For the active ingredient from rice, the tests are performed after 48 and 72 hours, while the tests are analyzed only after 72 hours for the active ingredient from other basic products, the percentage of proliferation is measured.

The results are recorded in the table of FIGS. 2A to 2E.

The active ingredient rich in di- and tri-peptides, whatever its origin, strongly increases the proliferation of cells, in a dose-dependent manner, more particularly after 72 hours of treatment.

3-2 Influence of the chemical structure

The aim is to study the influence of the biochemical structure of the active principle on the metabolism of human f ibroblasts in culture.

To this end, the effects of different compositions on proliferation are compared after 72 hours of treatment with the active principle according to the present invention obtained from: - rice with 75% peptides with a molecular mass of less than 1,400 daltons.

- model mixture of amino acids, without structuring into small peptides,

- highly structured protein solution, and

- solution of unselected peptides with only a peptide fraction of molecular mass less than 1400 daltons

It can be seen from the results in the table in FIG. 3 that the active principle rich in di- and tri-peptides, according to the present invention, considerably increases the proliferation of fibroblasts. Its optimal peptide structure is adapted to cell proliferation. The non-selected peptides do not make it possible to improve the proliferation activity of the cells as much as the active principle.

4 / EFFECT U ACTIVE PRINCIPLE HIDDEN THE EFFECTS OF A SOLAR STRESS

The purpose of this study is to demonstrate that the active principle according to the invention promotes recovery after stress, for example solar stress.

For this test, the cell viability of human fibroblasts subjected to UVA and UVB irradiation is measured, whether or not the active principle according to the present invention is present after 48 or 72 hours.

The doses are 6 joules / cm ² in UVA and 0.48 joules / cm ² in UVB and the results are expressed in optical density at 540 nm.

It can be seen in the tables of FIGS. 4A to 4E that solar stress induces high toxicity and causes the death of 60% of fibroblasts. The active ingredient at 1% makes it possible for example to increase the number of cells by activating the metabolic functions of the cells, by 35% for the active ingredient derived from rice 5 / EFFECT OF THE ACTIVE INGREDIENT ON THE EXPRESSION OF mRNAs

The active principle, derived from rice proteins, has an action on the messenger RNA coding for collagens VII and III and fibronectin in nutrient deficiency medium. In fact, human fibroblasts are incubated in a complete medium and in a nutrient deficiency medium, in the presence of the active principle.

The results obtained are substantially identical and are expressed as a percentage of expression of the RNA of the protein studied compared to a control. They are shown in the table in Figure 5.

The active ingredient significantly increases the expression of mRNAs encoding collagens III, VII and fibronectin, therefore constitutive proteins of the dermis. The active principle helps maintain the balance essential for cellular dietetics.

6 / EFFECT OF THE ACTIVE INGREDIENT ON METABOLISM BY MEASURING ATP SYNTHESIS This study makes it possible to evaluate the stimulating activity of the active ingredient according to the present invention on the stock of intracellular adenosinetriphosphate. A formulation of each of the active ingredient at 4%> according to the present invention is applied to several epidermis, after 24 hours of contact, the ATP is measured by luminometry using a commercial test.

Note in the table in Figure 6 which combines almost similar results, whatever the source, that intracellular ATP reserves are reduced by 37% to direct energy towards cell synthesis, thus increasing capacities defense of the skin. 7 / EFFECT OF THE ACTIVE INGREDIENT ON TI55ULAR OXYGENATION

The objective of this study is to quantify, in vivo, on humans, the oxygenating effect of the active ingredient, obtained from rice proteins. For this purpose, the partial pressures of oxygen and carbon dioxide transcutaneous on a zone treated with a formulation at 4% of the active principle against placebo are measured.

The measurements are taken before and after 28 days of twice-daily application of the active principle. The partial pressure measurements are carried out in a known manner for oxygen by measuring the variations in electric microcurrents and for carbon dioxide by determining the pH of an electrolyte also by measuring the variations in electric microcurrents. Changes in pH are proportional to the logarithm of changes in partial pressure of carbon dioxide. The measurements are expressed in mm of mercury.

The tables of FIGS. 7A and 7B group together the very similar results for the active principle and show an increase in the partial pressure of oxygen in 54% of the volunteers and in the partial pressure of O2 in 61% of the volunteers. Thus, it is found that the active principle tends to allow better diffusion of oxygen in cells and tissues.

8 / ANTI-WRINKLE EFFECT OF THE ACTIVE INGREDIENT

The aim of the study is to check the anti-wrinkle efficacy of the active ingredient formulated at 5% against placebo on 20 volunteers, applied twice daily for two months.

The anti-wrinkle study consists of analyzing by observing the footprints of crow's feet, using a profilometer equipped with an image analyzer. The four parameters studied are:

- total wrinkled surface,

- length of wrinkles,

- depth of wrinkles, and - number of wrinkles.

The results are grouped in the table in FIG. 8. The analysis shows a sharp reduction of 32% in the total wrinkled surface and in 24% in the length of the wrinkles and the distribution confirms that this reduction interests 70% of the volunteers. The invention also covers nutritional cosmetic compositions including, 0.10% to 5.00% of active principle, associated with excipients, in a suitable dosage form such as: cream, gel, ointment, lotion, milk, emulsion or solution. .

Claims

1. Method for extracting an active principle having a nutritional action from the skin, characterized in that it comprises the succession of the following stages:

- solubilization of vegetable, animal or marine proteins in water, at a rate of at least 5% by weight. - simultaneous and / or successive hydrolysis (s) of this suspension in the presence of one or more enzymes chosen from:

- bacterial aminopeptidases in neutral or acidic medium,

- stomach proteases of animal origin usable in an acid medium,

- bacterial proteases in neutral medium, - bacterial proteases usable in alkaline medium extracted from Bacillus

Licheniformis or Bacillus Subtilis,

- bacterial endoproteases, or

- pancreatic enzymes of animal origin.

2. Method for extracting an active principle having a nutritional action on the skin according to claim 1, characterized in that the vegetable proteins are obtained from cereals, oilseeds and legumes in particular.

3. Method for extracting an active principle having a nutritional action from the skin according to claim 2, characterized in that the vegetable proteins are obtained from rice (Orysa Sativa), soy (Glycine ax), seeds squash, sesame (Sesamu indicum) or peas (Pinum sativum)

4. Active principle obtained by implementing the method according to one of claims 1 to 3, characterized in that it has:

- a low molecular weight of less than 1,400 daltons

- a dry matter content between 20.0 and 150.0 g / 1 - a pH between 4.0 and 10.0 and

- a protein content between 10.0 and 100.0 g / 1

5. Active ingredient according to claim 4, characterized in that it has:

- a dry matter content of between 50.0 and 80.0 g / 1,

- a pH between 5.0 and 7.0 and - a protein content is between 40.0 and 60.0 g / l.

6. Active ingredient according to claim 4, characterized in that the molecular mass is distributed as follows:

- 20% to 60%> having a molecular mass of less than 1400,

- 30% to 50% having a molecular weight of between 75 and 175 daltons, and - 0 to 5% having a molecular weight of less than 75 daltons.

7. Nutritional cosmetic composition, characterized in that it comprises 0.10% to 5.007o of active principle according to one of claims 4 to 6, associated with excipients and in a suitable dosage form such as: cream, gel, ointment, lotion, milk, emulsion or solution.