FR2825925A1 - Production of an active agent having a nutritional effect on the skin comprises enzymatic hydrolysis of vegetable, animal or marine proteins - Google Patents

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

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PROCESS FOR PREPARING AN ACTIVE INGREDIENT FROM RICE,
ACTIVE PRINCIPLE OBTAINED AND ADAPTED COMPOSITIONS
The present invention relates to a process for the preparation of an active principle from rice and the active ingredient obtained.

 The invention also covers compositions using such an active ingredient.

 In the case of the skin, the active principle according to the present invention aims to have strong assimilation capabilities through the skin to quickly provide nutritional elements. These nutritional elements will facilitate the metabolic functions and allow the cells of the skin subjected to stress, to recover quickly.

 For this purpose, the invention provides an active ingredient from peptide structure elements having a low molecular weight.

 The purified peptide units thus added accelerate cell assimilation and vitalize the skin.

 A skin thus nourished, revitalized helps in particular to fight against the tissue aging thus to fill wrinkles.

 There is known a process for producing a protein hydrolyzate rich in di- and tri-peptides for artificial nutrition and used in dietetics.

 This process consists in subjecting an egg white or casein protein to the simultaneous or successive action of enzymes, in this case a bacterial protease that can be used in a neutral medium, a bacterial protease that can be used in an alkaline medium, and an enzyme. pancreatic of animal origin.

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 In the present application, it is necessary to obtain a protein hydrolyzate with a low molecular weight fraction to be absorbed by the skin.

 In the prior art, as it is artificial feeding, the molecular weight is not a constraint and the products obtained by these methods would not allow any application to cosmetology.

 The invention is now described in detail, both in its steps of the method of obtaining that in the characterization of the product obtained and through suitable cosmetic compositions.

 The various figures represent: FIG. 1, a distribution curve of the peptide fractions obtained.

 FIG. 2, table of results on the proliferation of fibroblasts over time, FIG. 3, table of results on the proliferation of fibroblasts compared to other compositions, FIG. 4, table of the results of cellular recovery after a UV stress. FIG. 5, table of the results of the stimulating activity of the active principle, FIGS. 6A and 6B, tables of oxygen partial pressure results and carbon dioxide, FIGS. 7A and 7B, tables of results of analyzes on volunteers concerning tissue oxygenation, and - figure 8, table summarizing the improvements with respect to wrinkles.

1 / PROCESS FOR EXTRACTING AN ACTIVE INGREDIENT FROM RICE
The product according to the invention, of which it is desired to obtain a concentrate constituting the active ingredient according to the invention, is rice, in particular the variety
Orysa Sativa. The method consists in carrying out the following succession of steps: solubilization of a rice protein concentrate in water, in particular the Orysa Sativa variety, in a proportion of at least 5% by weight.

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 successive hydrolyses of this suspension in the presence of a stomach protease of animal origin that can be used in an acid medium; concentration of the active fraction in small peptides with a molecular mass of less than 1400 daltons; sterilizing filtration to reach a total mesophilic flora less than 100 germs / g, and an absence of yeasts and molds.

 A curve is shown in FIG. 1 on a column sold under the name Superdex Peptide. A 0.02 mol phosphate buffer solution containing 0.25 mol of NaCl is used and the pH is adjusted to 7.2.

 The flow rate on the column is 0.25 ml / min and is printed on paper at 0.25 cm / min.

- f3 : 32, 63% (1400 < masse moléculaire < 175), soit environ 33, 00% - f4 : 42, 01 % (175 < masse moléculaire < 75), soit environ 42, 00% et - f5 : 2, 61 % (masse moléculaire < 75), soit environ 3%, c'est-à-dire les peptides de faibles masses moléculaires, représentent 77, 25% ou au moins 3/4 de la fraction totale inférieure à 1400 daltons. It can be seen that the fraction below 1355 daltons is distributed as follows:

- 32: 63% (1400 <molecular weight <175), ie about 33.00% - f4: 42.01% (175 <molecular weight <75), ie about 42.00% and -f5: 2, 61% (molecular weight <75), ie about 3%, i.e. the peptides of low molecular weight, represent 77, 25% or at least 3/4 of the total fraction less than 1400 daltons.

22, 5% are greater than 1400 daltons
This peptide structure of the active ingredient according to the invention is specific and of low molecular weight, thus allowing applications to cosmetics as shown by the results that follow.

extrait dans une étuve à 105 C jusqu'à obtention d'un poids constant. La quantité de matière sèche obtenue est comprise entre 20 et 150 g/l, plus particulièrement comprise entre 50 et 80 g/t. 2 / CHARACTERIZATION OF THE ACTIVE PRINCIPLE OBTAINED
Dry matter content:
The dry matter content is obtained by passage of the active ingredient

extracted in an oven at 105 C until a constant weight. The quantity of dry matter obtained is between 20 and 150 g / l, more particularly between 50 and 80 g / t.

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PH measurement:
The pH is measured by the potentiometric method at room temperature. The pH is between 4.0 and 10, more preferably between 5.0 and 7.0.

Determination of the nitrogen content:
The determination is carried out by Kjeldhal's method.

 The deduced protein content is between 10 and 100 g /! and more particularly between 40 and 60 girl.

3 / EFFECTS OF THE ACTIVE INGREDIENT ON THE PROLIFERATION OF HUMAN FIBROBLASTS
3-1 Effects over time:
The study is conducted on human fibroblasts to which the active ingredient according to the invention is added at 1% and 2% by volume.

 After 48 and 72 additional hours, the percentage of proliferation is measured.

 The results are shown in the table in Figure 2.

 The active ingredient greatly 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 ingredient according to the present invention on the metabolism of human fibroblasts in culture.

 For this purpose, the effects of four compositions on proliferation are compared after 72 hours of treatment: active ingredient according to the present invention with 77% of peptides with a molecular mass <1400 daltons.

 - model amino acid mixture, these being not structured in small peptides, - solution of highly structured proteins, and

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 - solution of unselected rice peptides with only 37% molecular weight less than 1400 daltons.

 It can be seen from the results of the table of FIG. 3 that the active principle according to the present invention considerably increases the proliferation of fibroblasts. Its optimal peptide structure is adapted to cell proliferation.

 The model amino acid mixture does not modify the proliferation capacity, which shows that it is not the amino acid composition of the active ingredient that gives this active ingredient its ability to promote proliferation.

 Highly structured proteins also do not alter the proliferation capacity and they are not assimilated. The cells must consume energy during a prior hydrolysis to use this protein source.

 The unselected rice peptides do not make it possible to improve the proliferative activity of the cells as much as the active principle.

4 / EFFECT OF THE ACTIVE PRINCIPLE TO COMPENSATE THE EFFECTS OF A SOLAR STRESS
This study aims to demonstrate that the active ingredient 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 irradiations, in the presence or absence of active principle according to the present invention after 48 or 72 hours, is measured.

 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 table in FIG. 4 that solar stress induces a high toxicity and causes the death of 60% of the fibroblasts. The active ingredient at 1% makes it possible to increase the number of cells by 35% by activating the metabolic functions of the cells.

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5 / EFFECT OF THE ACTIVE INGREDIENT ON mRNA EXPRESSION
The active ingredient according to the present invention has an action on messenger RNAs encoding collagen VII and t) and fibronectin in a nutrient-deficient medium.

 Indeed, human fibroblasts are incubated in complete medium and in a nutrient deficient medium, in the presence of the active ingredient.

 The results obtained are expressed as a percentage of expression of the mRNA of the studied protein relative to a control.

 They are shown in the table in Figure 5.

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

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

 Note in the table of Figure 6 that the reserves of intracellular ATP are decreased by 37% to direct the energy towards cell synthesis, thus increasing the defense capabilities of the skin.

7 / EFFECT OF THE ACTIVE INGREDIENT ON TISSUE OXYGENATION
The objective of this study is to quantify, in vivo, on the man, the oxygenating effect of the active principle.

 For this purpose, the partial pressures of oxygen and carbon dioxide transcutaneous on a treated area with a formulation containing 4% of active ingredient against placebo.

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 The measurements are carried out before and after 28 days of bi-daily application of the active ingredient.

 The partial pressure measurements are carried out in a known manner for oxygen by measuring the variations of electrical microcurrent and for carbon dioxide by determining the pH of an electrolyte also by measuring the variations of electrical microcurrent. The pH changes are proportional to the logarithm of carbon dioxide partial pressure changes.

 The measurements are expressed in mm of mercury.

 The tables of FIGS. 7A and 7B combine the results and show an increase in the partial pressure of oxygen in 54% of the volunteers and in the partial pressure of CO 2 in 61% of the volunteers.

 Thus, it is found that the active ingredient tends to allow a better diffusion of oxygen in cells and tissues.

81 ANTI-WRINKLE EFFECT OF THE ACTIVE INGREDIENT
The purpose of the study is to check on 20 volunteers the anti-wrinkle efficacy of the active ingredient formulated at 5% against placebo, applied twice daily for two months.

 The anti-wrinkle study consists of analyzing the footprints of the 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 summarized in the table of Figure 8.

 The analysis shows a sharp decrease of 32% of the wrinkled total area and 24% of the length of the wrinkles and the distribution confirms that this decrease concerns 70% of the volunteers.

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 The invention also covers the cosmetic composition obtained.

 This nutritional cosmetic composition with high assimilative capacity comprises 0.10% to 5.00% of active ingredient according to the invention, combined with excipients and in a suitable dosage form such as: cream, gel, ointment, lotion, milk, emulsion or solution.

Claims (5)

1. A method for extracting an active ingredient having a nutritional action of the skin, characterized in that it comprises the following succession of steps: - solubilization of a rice protein concentrate in water, especially the variety Orysa Sativa, at least 5% by weight, - successive hydrolysis of this suspension in the presence of a stomach protease of animal origin, usable in an acidic medium, - concentration of the active fraction in small peptides of lower molecular mass at 1400 daltons, - sterilizing filtration to achieve total mesophilic flora less than 100 germs / g, and an absence of yeasts and molds.  2. Active ingredient obtained by the process according to claim 1, characterized in that it has: - a low molecular weight of less than 1400 daltons - a dry matter content of between 20 and 150 g / t - a pH between 4.0 and 10.0 and - a protein content of between 10 and 100 g / t 3. Active ingredient according to claim 2, characterized in that the molecular weight is distributed as follows: 22.50% having a molar mass greater than 1400 daltons, 33.00% having a molecular weight between 1400 and 175 daltons, 42.00% 175 and 75 daltons and -3.00% having a molecular weight of less than 75 daltons.  4. Active ingredient according to claim 2 or 3, characterized in that it has: - a solids content is between 50.0 and 80.0 g / l, - a pH between 5.0 and 7, And a protein content is between 40.0 and 60.0 g / t. <Desc / Clms Page number 10>  5. Nutritional cosmetic composition with high assimilative capacity, characterized in that it comprises 0.10% to 5.00% of active principle according to one of claims 2, 3 or 4 associated with excipients and in a suitable dosage form such as: cream, gel, ointment, lotion, milk, emulsion or solution.