WO2001007005A1

WO2001007005A1 - Use of a protein fraction of the seed of the vigna trilobata-plant in a cosmetic or dermopharmaceutical composition

Info

Publication number: WO2001007005A1
Application number: PCT/EP2000/007007
Authority: WO
Inventors: Gilles Pauly; Philippe Moser; Véronique Gillon
Original assignee: Cognis France, S.A.
Priority date: 1999-07-26
Filing date: 2000-07-21
Publication date: 2001-02-01
Also published as: FR2796839B1; FR2796839A1; AU6276500A

Abstract

The invention relates to the use of at least one soluble protein fraction extracted from the seed of the vigna trilobata plant as an active substance in a cosmetic or dermopharmaceutical composition for local application on the skin, epithelial appendages and/or mucous membranes.

Description

Use of a protein fraction of the grain of the Vigna trilobata plant in a cosmetic or dermopharmaceutical composition

The present invention relates to the field of cosmetics and dermopharmacology and its object is to use at least one protein extract of the grain of the Vigna trilobata plant, and a cosmetic product containing such an extract.

In developing countries, one of the main goals of the public authorities responsible for nutrition and food intake is to find new sources of food and, in particular, cheap and good proteins.

For this reason, they devote their attention to the search for new food sources in the genus of cultivable vegetable plants or wild plants.

In the southern part of India, it is known that ripe Vigna tribolata grains are roasted and consumed by the "tribal sect" Kurumba: they are also eaten by the poorest inhabitants of these regions.

This plant is mentioned in various traditional articles under the name Phaseolus trilobus Ait (see in particular GC Toms: J. Pharmacy & Pharmacol, Volume 30, 79 P, 1978) and its chemical composition and nutritional potential have been described by S. Siddhuraju, K. Vijayakumari and K. Janardanan ("Nutrional and chemical evaluation of raw seeds of the tribal pulse Vigna trilobata" (L.) Verdc. International Journal of food sciences and nutrition (1992), 43, n ° 2, 97-103).

According to this article, the chemical composition (g / 100 g of flour obtained from grains) of Vigna trilobata grains is as follows:

- Moisture: 5.19%

- Proteins (N x 6.25): 20.31% - Fibers (fiber): 8.81% - Fat: 5.54% - Ash: 2.71%

with an energy of 1594 kJ / 100 g dry matter.

The mineral composition of these grains is rich in potassium (1397 mg / 100 g flour), calcium (464 mg / 100 g flour), magnesium (291 mg / 100 g flour).

From a nutritional point of view, the amino acid composition indicates that the sulfur-containing amino acids (cystine and methionine), threonine and isoleucine can be found in limited quantities, whereas valine, leucine, tyrosine, phenylalanine and lysine are present in sufficient quantities.

The globulin fraction of the proteins is said to have a blood-clotting activity for groups A, B, O.

But the inventors have found in an unexpected and astonishing way that the protein extracts of the Vigna trilobata grains, in addition to their nutritious properties, also in topical application on the skin, the epithelial appendages and the mucous membranes, have special biological effects and have a very good tolerance and that the use as an active ingredient, alone or in conjunction with at least one other active ingredient, of at least one soluble protein fraction extracted from Vigna trilobata in a composition or a cosmetic or dermopharmaceutical product enables specific, identifiable and quantitatively measurable properties when used locally on the Skin, the epithelial appendages and / or the mucous membranes.

Thus, softening, biofilmogenic, as well as confirmed, firming effects, conditioning and correcting effects, as well as anti-irritant effects, soothing, and effects against sensitive skin types, were found in particular through biophysical measurements (preventive and curative treatment of sensitive skin types). It has also been found that these extracts facilitate combing dry and moist epithelial appendages and improve the softness and smoothness of undamaged epithelial appendages.

The tests performed by the inventors also highlighted the following additional properties:

- a pronounced cellular nutritional capacity,

- stimulating effects of cell growth and metabolism (energizing, stimulating, anti-aging activity),

- activation and stimulation of the collagen network and the excretion of glucosaminoglucans (scarring, firming, strengthening effect, reduction of the contraction (atrophy) of the dermis),

- a strong anti-apoptosis effect, - a high cytophotos protective activity against oxidative stress, which is caused by UV-A rays,

- a reduction in the inflammation caused by UV-B rays,

- an anti-protease activity, which can lead to anti-elastase activity.

The following text describes various examples of processes for obtaining the aforementioned extracts and then the tests carried out to identify and evaluate the properties of the said extracts, which were discovered by the inventors.

I) PREPARATION OF THE VIGNA TRILOBATA GRAIN PROTEIN EXTRACTS

The preparation of the proteins is carried out by the conventional techniques for extracting vegetable proteins, the preparation of concentrates or protein isolates or by purification (ultrafiltration, ion exchange chromatography, affinity chromatography, precipitation, adsorption), which are known to the person skilled in the art.

However, the extraction with water or an aqueous solution is preferably carried out at a given pH, possibly by an ultrasound generator. As examples, by way of illustration, but not by way of limitation, various processes for obtaining and preparing protein extracts from two different, divided quantities of Vigna trilobata grains of Indian origin are described below.

The protein content (N x 6.25) of the two grains, A and B, is 20.75% and 20.92%.

EXAMPLE 1

In 1.25 liters of distilled water are added 125 g of flour, which was obtained by crushing dry Vigna trilobata grains.

After stirring for 15 minutes, the pH of the solution is adjusted to the pH of 7.5 using sodium hydroxide solution.

The extraction is carried out for 2 hours at room temperature by keeping the extraction pH at 7.5.

After centrifuging at 5000 g for 10 minutes, the beige substance floating on the surface is collected and then filtered to 0.5 μm.

The extract can be dewatered by conventional techniques such as atomization, freeze drying or the like. After atomization, the powder-like product obtained has a protein content (N x 6.25) of 42.5% (extract 1a).

A second extract, prepared under the same conditions, from a different amount of grains makes it possible to obtain an extract that contains a protein content (N x 6.25) of 46.4% (extract 1 b).

EXAMPLE 2

250 g of flour obtained by crushing Vigna trilobata grains are placed in 2.5 liters of distilled water and the solution is processed as in Example 1. 2.15 liters of beige solution are obtained.

The pH of the solution is adjusted to pH 4.5 with sulfuric acid and left for 30 minutes with stirring.

The solution is then centrifuged for 15 minutes at 5000 g: the precipitate and the matter floating on the surface are collected.

The precipitate is placed as a solution in a water volume that corresponds to 20% of the volume before the precipitation. The pH of the solution is adjusted with NaOH until it stabilizes at 7.5.

The solution is centrifuged again to separate out the insoluble substances. 600 ml of a 3.5% dry extract solution are obtained, which is dewatered by atomization.

After atomization, the powder-like product obtained has a protein content (N x 6.25) of 80.6% (extract 2a).

A second extract is prepared under the same conditions from a different amount of grains, it enables an extract to be obtained which has a protein content (N x 6.25) of 80.7% (extract 2b).

Experiments with precipitates that were fractionated at different pH values give the following results:

Type of fraction Protein content (%) Precipitation pH 6.5 65.3

Precipitation pH 6.0 83.1 Precipitation pH 5.5 85.0

Precipitation pH 5.0 84.8

EXAMPLE 3

The substances floating on the surface and obtained after the precipitation of the proteins at pH 4.5 according to Example 2 are reduced to 0.5 μm and then to 0.22 μm filtered: the clear, obtained solutions are dewatered by atomization.

The powder obtained has a protein content of 15% to 20% and an inhibitory activity of trypsin, which was determined by the Kakade technique with 8.4 TUl / mg to 13.3 TUl / mg.

These fractions can therefore serve as the basis for the purification of protease inhibitors (with regard to the use of an extract which consists of a fraction enriched with protease inhibitors).

EXAMPLE 4

200 ml of crude extract (pH 7.5), which was prepared according to Example 1, are placed in an Amicon 8200 ultrafiltering cell which is provided with a 100,000 Da ultrafiltering membrane (Ref. YM100, diameter 6 cm).

The solution is concentrated to 50 ml (compressed air pressure 3 bar). Permeate P1 and retentate R1 are collected.

150 ml of distilled water are added to the retentate and the solution is concentrated again to 50 ml (retentate R2).

Retentate R2 is dehydrated by freeze-drying: a fraction with an 80.7% protein content (N x 6.25) is obtained.

EXAMPLE 5

A protein concentrate is prepared from 350 g of grains according to Example 2, the initial extraction being carried out in a flour / solvent ratio of 1/15.

The precipitate is dissolved at pH 7.5 in 1.5 liters of distilled water.

500 ml of protein concentrate solution containing 57.53 g / l of proteins (N x 6.25) are obtained at the end. The proteins are hydrolyzed at pH between 7.5 and 8.5 with an alkali protease (2.5% in relation to the proteins of the solution).

The hydrolysis is carried out for 2 hours at an optimized temperature and pH of the enzyme used, the optimized values are known to the person skilled in the art.

The enzyme is inactivated by heating to 100 ° C for at least 10 minutes.

After cooling to room temperature, the solution is centrifuged and then filtered to 0.22 μm.

460 ml of dark, pure filtrate and 6.39% dry extract (proteins 45.2 g / l) are obtained.

After atomization, the powder obtained has a protein content of 70.75% (extract 3a).

Analysis by gel permeation on a Superose 12 HR column of the fractions extracted by these different methods enables at least 10 protein fractions to be labeled which, depending on the extracts, are more or less large and visible, as examples in FIG. 1 (distribution of the protein compounds of the extract 1b) and on FIG. 2 (distribution of the protein compounds of the extract obtained in Example 4), that is to say:

a fraction (designated F1) with a very high molecular weight (over 500,000 Da and close to 1,000,000 Da according to the calibration of the column), - a fraction F2 whose molecular weight is between 250,000 Da and 300,000 Da (295,000 Da),

a fraction F3 whose molecular weight is between 100,000 Da and 150,000 Da (138,000 Da),

four fractions designated F4, F5, F6 and F7 whose molecular weights are between 5,000 Da and 70,000 Da,

- three fractions designated F8, F9 and F10 whose molecular weights are between 500 Da and 3,000 Da. The constituents of the hydrolyzate, which was carried out according to the previous example 5, have an average molecular weight of 3,000 Da (see FIG. 3: curve of the same type as that of FIGS. 1 and 2).

The extracts obtained by the process examples described can be used directly in liquid form or after drying using the conventional drainage techniques (atomization, freeze-drying).

The protein fractions obtained can be used either in their original form, without changing the structures or in the form of one or more natural compounds of at least two or all extracted fractions of different visible molecular weights, which correspond to the different chromatogram peaks shown in the accompanying drawings and are found naturally in the grains (total or partial protein extract) or in isolated form.

The protein fractions can be used in compositions in their form modified or functionalized by any of the following procedures:

- the polymerization of the original proteins;

- chemical hydrolysis of the original proteins;

- the enzymatic hydrolysis of the original proteins by proteases of animal, plant, microbial or fungic origin: pepsin, trypsin, chymotrypsin, papain, pronase, bromelaine, endoproteinase, thermitase,

Proteases of Bacillus subtillis, Aspergillus niger, Aspergillus aryzae (subtilisin, alkalase, neutrase);

- microbial change with the use of Vigna trilobata proteins as a fermentation substrate by various microorganisms, such as yeast (Saccharomyces - yeast), mold (Aspergillus), bacteria, (bacillus and the like);

chemical or enzymatic functionalization by methods such as starch extraction, succinilization or phosphorylization;

- quaternization; - Grafting of the saccharidic or lipidic molecules or any other chemical change by grafting. The extracts or releasable protein fractions according to the invention can also be added to or connected to any other applicable cosmetic vector, for example film-forming agents, liposomes, cyclodextrins, micelles, macro, micro and nanoparticles, as well as macro, micro and nanocapsules or they can be absorbed or transplanted onto organic polymers or mineral carriers.

II) EVIDENCE OF PROPERTIES OF VIGNA TRIBOLATA PROTEIN EXTRACTS

The biological properties and activities of the Vigna trilobata protein extracts could be determined and measured by tests known to those skilled in the art, the results of which are shown below:

1) EFFECTS ON CELL GROWTH

Human fibroblasts are vaccinated in an optimum medium (with SVF) and incubated for 24 hours at 37 ° C.

The growth medium is then replaced by a sub-optimal medium (without SVF) containing various concentrations of extracts as described in the invention. After a 3-day incubation, the growth was evaluated by counting the adherent cells by an automatic particle counter and by dosing the intracellular ATP content.

The tests were carried out in triplicate and repeated two or three times.

The results are calculated in relation to a test measurement scale and then specified in a percentage in relation to the untreated control agent and finally created as an average with SEM (error type of the average).

The results of Figure 4 show that the extracts 1a and 2a at doses between 0.003% and 0.03% (w / v - weight-volume), the number of cells and the ATP content in the human Fibroblasts in in vitro growth increase significantly (Figures 4A and 4B each represent the cells counted after three days and the ATP content after three days for different concentrations with 1a and 2a extracts).

2) EFFECTS ON SURVIVAL

The test is done on fibroblasts according to the same protocol as growth, with an incubation period of 72 hours.

Survival was evaluated by dosing the following levels:

- of proteins,

- of adenosine triphosphate (ATP), a high-energy compound, which is mainly produced by mitochondria and is required for the activity of the numerous enzymes in cell construction,

- of glutathione (GSH), a peptide produced directly by the cell, to combat oxidative stress or various contaminants, such as heavy metals. Its synthesis requires ATP as an energy source.

The way of reporting the results is identical to that of the growth test. FIGS. 5A and 5B each represent the protein content and the GSH content, which were measured after three days for different concentrations with 2a, 2b and 3 extracts.

The results of FIGS. 5A and 5B (FIG. 5) indicate that the protein extracts from Vigna trilobata according to Example 2 at 0.05% (w / v) the content of the proteins and of the glutathione in the human fibroblasts in vitro - Increase survival significantly.

The extract according to Example 3 at 0.01% w / v) also slightly increases the protein and glutathione content in human fibroblasts in in vitro survival.

These results indicate that the original or hydrolyzed protein extracts of Vigna trilobata according to the invention have high abilities to

Improvement of growth and metabolism (synthesis of ATP, the

Proteins and glutathione) by human fibroblasts have what clearly indicates an energizing, stimulating and "anti-aging" activity of these extracts.

3) PROOF OF THE ACTIVATION OF THE CONTRAGGEMENT OF THE COLLAGEN NETWORK

a) Principle

The contraction of the collagen network results from a series of biological functions that are triggered by fibroblasts, it enables scarring of wounds in vivo and also the maintenance of a good dermis.

Therefore, contracting the collagen network in vitro is a good model for evaluating the ability of a substance to activate scarring or to reduce the contracting of an old person's dermis.

b) Execution type

Suspension of human fibroblasts by trypsinization,

Mixing the human fibroblasts with a nutrient medium which contains extracts according to the invention and a solution of type I collagen, incubation at 37 ° C. for 14 days, CO ₂ = 5%,

- measurement of the two orthogonal diameters every 2 to 3 days and calculation of the surface in cm ² ,

- GAG staining with alzian blue and quantity determination of the intensity of the coloring of the light envelope (aureole) surrounding the fibroblasts by an image analyst.

Figures 6 and 7 of the drawings in the appendix each show the surface of the rods in cm ² and the separated GAG contents (in pericellular staining intensity) on the 14th day of incubation with different concentrations of extracts 2a and 2b.

In general, the collagen network does not contract in a culture medium without calf fetus serum (SVF).

The speed and intensity of the network contraction vary considerably from one experiment to another, but when comparing the results within the same experiment, one can say that: - SVF has enabled the collagen network to be contracted by the human fibroblasts,

the protein extracts from Vigna trilobata according to Example 2 alone achieve the contracting of the network as well or better than SVF and this effect is found again in the presence of 2% SVF,

- In addition, the GAG content secreted by the fibroblasts is increased in the presence of extract 2b.

These results indicate a firming and refreshing activity of the extracts according to the invention, as well as an ability to reduce the contraction of the dermis in the elderly.

4) EVIDENCE OF ANTI-APOPTOSE ACTIVITY

a) Principle

Apoptosis is a biological, active process used by living organisms to remove certain cells from their tissues by autolysis, especially the destruction of proteins and nuclear ADN into small fragments that are salted out into the cytoplasm.

These small ADN fragments are taken as parameters for the evaluation of the apoptosis content, which was induced in the keratinocytes in the in vitro culture medium.

Apoptosis can also be induced by oxidative stress (UV-R, inflammation), by lack of growth factors or by toxic substances (pollutants, genotoxic substances ...).

The principle of the test is to demonstrate the ability of the extracts according to the invention to reduce the level of apoptosis induced in a culture medium with cells lacking growth factors.

b) Preparation of the human cells

The human keratinocytes are vaccinated in a complete culture medium (with calf fetus serum or SVF), which contains an ADN marker: bromodeoxy uridine or BrdU. The cells then receive a culture medium without serum, which contains different concentrations of the substances to be tested and which are incubated at 37 ° C. for 1 or 2 days. After the incubation, the cells are recovered by trypsinization and then analyzed.

c) Quantity determination of the content of the apoptotic cells

In this method the cells are lysed and the content of the apoptotic cells is quantified by an ELISA test which reveals BrdU which has been incorporated into the cytoplasmic ADN fragments.

The results are given in a percentage in relation to the control agent (Henseleit U, Rosenbach T, Kolde G: "Induction of apoptosis in human HaCaT keratinocytes."; Archiv. Dermatol. Res. (288) 11, 676-683, 1996).

d) conclusions

FIGS. 8A and 8D represent the cell count and the apoptosis content (content of the ADN fragments) for the extracts 1a, 1b, 2a and 2b for different concentrations.

The results of FIG. 8 show that:

- In keratinocytes, which were treated with the 0.01% extracts 1a and 1b% and 0.03% (w / v), the apoptosis content in relation to the amount of control agent, which was treated in a culture medium without SVF, drops sharply , - in the keratinocytes treated with extracts 2a and 2b in doses between 0.01% and 0.02% (w / v), the apoptosis content in relation to the amount of control agent which was treated in a culture medium without SVF , drops sharply.

The extracts according to the invention have shown considerable abilities to reduce the level of apoptosis induced in a culture medium of human cells which lack a growth factor, which explains the high abilities of these extracts the aging of tissue by a "growth factor". like "effect (type growth factor). 5) PROOF OF THE ABILITIES OF CYTAL PROTECTION AGAINST OXYDATIVE STRESS

a) Principle The aim of this test is to evaluate the anti-oxidative stress capabilities of the protein extracts of Vigna trilobata according to the invention by a test on human fibroblasts in an in vitro culture.

This in vitro test evaluates the capabilities of cytophotos protection of human fibroblasts against UV-A. The UV-A are selected as the study model because they penetrate into the dermis and induce an oxidative stress in the skin, which in particular reveals lipoperoxidation of the cytoplasmic membrane and a decrease in the activity of numerous enzymes, including that of catalase. The lipoperoxides formed split into malonaldialdehyde, which is responsible for the reticulation of numerous biological molecules, such as proteins (enzyme inhibition) and nuclear bases (mutagenesis).

In addition, it has been proven that the inhibition of catalase makes the fibroblasts very sensitive to hydrogen peroxide (H ₂ O ₂ ), which is induced by UV-B or UV-A.

Because H ₂ O ₂ must be removed quickly, otherwise it will be present in the presence of

Iron forms very toxic hydroxyl radicals (HO °).

b) Cytophotos protection test Fibroblasts are vaccinated in a nutrient medium which is defined with calf fetus serum (SVF).

The Vigna trilobata extracts according to the invention were added 2 to 3 days after inoculation.

After a 1 to 2 day incubation at 37 ° C and CO ₂ = 5%, the nutrient medium is replaced by a saline solution and the fibroblasts are irradiated with a UV-A dose (3 to 15 J / cm ² ).

After the end of the irradiation, the MDA content (malonaldialdehyde) is dosed in a salt solution floating on the surface and the content of the proteins, Reduced glutathione (GSH) and active catalase are measured in the fibroblasts.

MDA is dosed by the reaction to thiobarbiturate acid and the proteins according to the so-called Bradford method, whereas GSH is dosed by a fluorescent probe. The content of the active catalase is dosed by a spectrophotometric method and the activity of the catalase is based on the protein content measured in the fibroblasts.

c) conclusion

FIGS. 9A and 9B represent the measured contents of MDA, the proteins, of GSH and the catalase, which represents the relative cytophoto protective activity (% in relation to the control means) of the extracts 1a and 2a.

The results of Figure 9 show that the Vigna trilobata extracts according to the invention have remarkable abilities to reduce the levels of the induced lipoperoxides and the catalase which was inhibited by UV-A, which survived on a culture medium of human fibroblasts in vitro have been applied.

They could at least partially work by activating glutathione synthesis in the fibroblasts.

These results indicate that Vigna trilobata extracts have a high ability to reduce the harmful effects of oxidative stress on the skin.

6) EVIDENCE OF ANTI-IGNITION PROPERTIES IN VITRO

a) Principle The aim of these tests is to demonstrate the anti-inflammatory properties of the Vigna trilobata extracts on a culture medium with human keratinocytes in in vitro survival.

UV-B was chosen as an inducer because it causes cutaneous inflammation (erythema, edema) through the activation of enzymes such as phospholipase A2 (PLA2), which releases arachidonic acid (unsaturated fatty acid), which is present in the biological membranes is. On the one hand, this leads to damage to the membrane and the synthesis of the inflammatory transmission agents (mediator), because the arachidonic acid is converted under the action of the so-called "cyclo-oxygenase" enzymes into prostaglandins (PG), such as PGE2. The PGE2 are released outside the cell and they will induce erythema and edema by fixation on specific recipients.

In addition, the UV-B induces lesions of the ADN into the keratinocytes, which can be direct, such as thymidine dimers or indirectly, as in the case of apoptosis (which was initiated by the UV) where the ADN is fragmented and then in the cytoplasm in the form small fragments is salted out.

b) Execution method

All effects of UV-B were evaluated on in vitro cultures of keratinocytes.

^* Preparation of the keratinocytes

- Inoculate into a complete culture medium, which may contain BrdU (bromodeoxyuridine) for dosing the cytoplasmic ADN fragments. - 3-day incubation at 37 ° C,

- inserting the active ingredients in a glucose saline solution,

Irradiation with UV-B at a dose of 50 mJ / cm ² ,

- Daily incubation at 37 ° C

^* Dosages recovery of the medium floating on the surface for:

- Dosage of the active LDH, to evaluate the damage of UV-B on the membranes,

- Dosage of the PGE2 content (ELISA test)

Recovery of the adherent keratinocytes by trypsinization for:

- counting by an automatic particle counter,

- Dosage of the content of the cytoplasmic ADN fragments (ELISA test) to evaluate the harmful effects of UV-B on the ADN.

The results are calculated in relation to a test scale and then based on the number of cells and expressed in% in relation to the untreated and irradiated control agent. c- conclusion

Figures 10A and 10B of the accompanying drawings illustrate the relative activities related to the anti-inflammatory properties of extracts 1a, 1b, 2a and 2b.

The results of Figure 10 indicate that the various extracts of Vigna trilobata according to the invention (0.01% or 0.02%) had the effects of UV-B on the number of keratinocytes, on the content of the exposed LDH and PGE2 and have significantly reduced the content of the fragments of the cytoplasmic ADN.

These Vigna trilobata extracts according to the invention therefore have high abilities to reduce lesions introduced on the biological membranes and on the ADN by UV-B and to reduce the inflammatory process induced by the UV-B on the human keratinocytes.

7) DETECT AN ANTI-SENSITIVE SKIN PROPERTY BY TESTING WITH CAPSAIZINE

a) Principle of the experiment The experiment consists of a sensory evaluation of the effects produced after standardized use of capsaizine to 0.075% in vivo on humans: irritation, burning, pain and erythema. The test is carried out by an expert assistant under standardized conditions with regard to temperature and relative humidity on a test person who is very sensitive to capsaicine on the face; both persons have been trained to describe the effects observed during the test.

b) Evidence of anti-sensitive skin effects

Extract 2a, which was introduced at 1.5% in an H / E emulsion, is applied to a cutaneous zone limited to the cheek. A cream dosed with 0.075% capsaizine was also applied 30 minutes after drying. During the next 30 minutes, the effects produced by capsaizine are evaluated on a semi-quantitative measuring scale with 4 points (from 0 = none to 3 = severe): - by the test person: irritation, burning, pain according to their sensation,

- by the expert assistant: erythema.

The test was conducted against a placebo (H / E emulsion only) and against a control agent (not pre-treated skin to which only capsaicine cream is applied).

Under these conditions, the 2a extract improves the parameters for pain and irritation by 83% (25% for placebo) and the parameters for erythema by 22% (14% for placebo), making its soothing, anti-irritant and anti-sensitive skin -Effect is proven.

8) EVIDENCE OF A TIGHTENING EFFECT BY A QUANTITATIVE IN VIVO MEASUREMENT IN PEOPLE, BY A HORIZONTAL EXPANSION MEASUREMENT TECHNIQUE

a) Principle of the experiment

The experiment consists of measuring the displacement of the skin according to a sinusoidal, constant force that is exerted parallel to its surface. The measuring operations take place according to a device of the type to which the French patent application N ° 98 12125 refers to the name of the applicant.

The processing of the signals of the force and extension by means of a hysteresis ellipse enables the calculation of the dynamic effort or DSR (Dynamic Spring Salary). The application of a firming product on the surface of the skin manifests itself by an increase in DSR, a consequence with constant strength, a decrease in the expansion of the skin during stress. The equipment is completely computer controlled. The tests are carried out under standardized conditions with regard to temperature and relative humidity.

b) Evidence of activity

A patch with super glue is stuck on a cutaneous zone on the back of the test person's hand. Five horizontal extensiometer measurements are then made on the untreated control agent skin. Then the placebo Carrier applied. After drying for 5 minutes, five measuring processes are used to check the effect of the carrier.

The skin is then treated again with the active ingredient, the firming effect of which is measured by five last measuring processes after waiting for δ minutes. The average of the five measurements is determined during each stage. The final results are produced as a percentage of the change in DSR between the one by the placebo carrier (water) and the activity of the active ingredient.

Under these conditions, the 2a extract, which was tested on a healthy, female test person in an aqueous solution at a 1.5% concentration, increased the DSR by 76%, thereby demonstrating its firming cutaneous effect.

9) EVIDENCE OF A SOFTENING EFFECT THROUGH A QUANTITATIVE IN VIVO MEASUREMENT IN PEOPLE WITH FRICTIOMETRY TECHNOLOGY

a) Principle of the experiment

The experiment consists in applying a constant force to the skin using a sliding shoe that is rotated at a controlled speed and pressure. The moment of friction is measured. It enables the calculation of the coefficient of friction of the sliding shoe on the skin. The coefficient of friction depends on the condition of the skin surface, especially its moisture and softness. The more hydrated and softer the skin is to be touched, the more the coefficient of friction increases. The equipment is completely computer controlled. The tests are carried out under standardized conditions with regard to temperature and relative humidity.

b) Evidence of activity

A cutaneous, 9 cm ² zone on the inner surface of the test person's forearm is used to perform a friometric measurement on the skin without treatment (TO). Then the active ingredient is applied. After drying for 15 minutes, a friction measurement is used to check the effectiveness of the active ingredient (T15). At the same time, an adjacent cutaneous control agent zone is measured under the same conditions. The result is in the percentage difference of Change between TO and T15 of the treated zone in relation to the control agent zone indicated.

Under these conditions, the 2a extract tested on a healthy female test subject as an aqueous solution with a 1.5% concentration increases the coefficient of friction by 20.9%, which proves its effectiveness in improving cutaneous softness and moisture.

The object of the present invention is also a cosmetic or dermopharmaceutical composition for topical application to the skin, the epithelial appendages and / or the mucous membranes, which as an active ingredient alone or in combination with at least one other active ingredient, contains at least one protein fraction which extracted from Vigna trilobata grains.

This cosmetic composition, as a single active ingredient or combined with at least one other active ingredient, can contain at least one extract of the aforementioned type which is used to produce at least one of the particular biological effects which have been described above, or even several of these effects as a combination.

The cosmetic or dermopharmaceutical composition according to the invention may advantageously contain between 0.001% and 50% by weight protein fraction (s) extracted from Vigna trilobata grains (the extracts were obtained by one of the aforementioned methods), which may be can be incorporated into suitable cosmetic vectors such as liposomes, macro, micro and nanocapsules, macro, micro and nanoparticles and other analog and known forms.

The aforementioned extracts can not only be used for skin care and hygiene applications (products for the face and body, day or night cosmetics, sunscreens, strengthening, regenerating products, anti-wrinkle cosmetics, slimming products, agents against aging processes), but also in the field of epithelial appendage care and hygiene in the form of various finished products, such as lotions or shampoos, creams, foaming agents, soaps, sticks, gels, hydrogels, sprays, emulsions, Protective agents, repairing, softening, film-forming and photo-protective agents; Perm and hair dye products.

As non-limiting examples, various examples of the practical implementation of cosmetic compositions according to the invention, as well as the main stages of their manufacturing processes, are described below.

EXAMPLE 1

A cosmetic product in the form of a cream for nourishing, firming, refreshing and energizing, which is intended to combat cutaneous aging, the contraction of the dermis and the loss of elasticity of the skin, can consist of the following phases:

Fat phase ceteareth 25 2.00

Ceteareth 6 and stearyl alcohol 1, 00

Cetyl alcohol 4.00

Glycol stearate 4.00

Petrolatum 5.00 Kapische / Kaprylische Triglyzeride 5.00

Aqueous phase

Glycerin 10.00

Vigna proteins according to Example 1 of the method 5.00 distilled water 8.50

Elestab 4112 preservative (Laboratoires Serobiol.) 0.40

Perfume 0.30 distilled water qsp (sufficient amount for) 100.00

The preparation process of the aforementioned cream essentially consists of heating the fat phase to 80 ° C, also heating the aqueous phase to 80 ° C and dissolving Elestab 4112 therein, separately preparing the mother liquor of the Vigna extract, the fat phase in place the aqueous phase under turbine stirring, then add the mother liquor of the Vigna extract at about 50 ° C and finally continue stirring until cooling. EXAMPLE 2

A cosmetic product in the form of a cream for softening, biofilming, relieving, scarring, which is particularly intended to combat aggression by sun exposure and pollution, and to care for sensitive skin types (anti-irritation, to reduce inflammation) can be from the following Phases exist:

Fat phase

Glycol stereate 14.00 octyl dodecanol 6.00

Dibutyl adipate 6.00

Ceteareth 12 1.50

Ceteareth 20 1.50

Aqueous phase

PVP (polyvinylpyrrolidone) 0.50

Glycerin 4.00

Elestab 388 (Laboratoires Serobiologiques) 2.00

Vigna proteins according to Example 2 (2a extract) 5.00 distilled water 9.00

Perfume 0.20 distilled water qsp (sufficient amount for) 100.00

The preparation process of the aforementioned cream consists mainly of heating the fat phase to 80 ° C, also heating the aqueous phase to 80 ° C, dissolving Elestab 388 and PVP therein, the fat phase into the aqueous phase with turbine stirring at 80 ° P pour, then gradually cool while stirring, then add the mother dispersion of the Vigna extract at about 50 ° C and finally continue stirring until it cools down.

EXAMPLE 3

A cosmetic product in the form of a conditioning, non-rinsed and biofilm-forming hair tonic, which is particularly intended to make combing of the epithelial appendages easier and to improve its softness and suppleness, can have the following composition: Vigna proteins according to Example 1 (1a extract) 2.00 distilled water 9.50

Hydroxyethyl cellulose 0.50

Elestab 305 (Laboratoires Serobiologiques) 0.50 perfume 0.10

Cremophor RH 410 0.30 distilled water qsp (sufficient amount for) 100.00

The method of preparation of the non-rinsed lotion consists mainly in dissolving Elestab 305 and hydroxyethyl cellulose in water heated to about 50 ° C, in the distribution of the perfume and Crempphors RH 410 therein, then in the cooling of the mixture to room temperature, then in Dissolution of the Vigna extract in it and finally in the execution of a filtering process.

Of course, the invention is not limited to the described embodiments. Changes remain possible, particularly with regard to the nature of the various elements or by replacing them with technical equivalents, without thereby departing from the scope of the invention.

Claims

1. Use as an active ingredient in a cosmetic or dermopharmaceutical composition for topical application on the skin, the epithelial appendages and / or the mucous membranes of at least one soluble protein fraction extracted from the grain of the Vigna trilobata plant.

2. Use according to claim 1, characterized in that the extract from the Vigna trilobata grains is used as an active ingredient, the activities of stimulating growth and cellular metabolism, stimulating the contraction of the collagen network, stimulating the excretion of glycol aminoglycans Anti-apoptosis activities, which has anti-inflammatory activities and / or the cytophoto-protective activities.

3. Use according to one of claims 1 and 2, characterized in that the protein fraction (s) was extracted by water or a saline solution at a given pH value / n.

4. Use according to any one of claims 1 to 3, characterized in that the protein fraction / s was extracted as an aqueous solution by an ultrasonic generator / n.

5. Use according to one of claims 3 and 4, characterized in that the protein fraction / s is / are purified by a process which is selected in the group formed by precipitation, adsorption, ion exchange chromatography, affinity chromatography and ultrafiltration ,

6. Use according to any one of claims 1 to 5, characterized in that the extracted fraction consists of a fraction enriched with protease inhibitors.

7. Use according to any one of claims 1 to 6, characterized in that the protein fraction (s) forms an active substance / s, which consists of a chemical or enzymatic hydrolyzate which has been prepared from original proteins.

8. Use according to any one of claims 1 to 6, characterized in that the final protein fraction (s) was obtained by polymerizing the original proteins.

9. Use according to any one of claims 1 to 6, characterized in that the extracted protein fraction (s) has / have been chemically modified by grafting.

10. Use according to any one of claims 1 to 9, characterized in that the extracted fractions which form the active ingredient contain at least two protein fractions, the visible molecular weights of which are different.

11. Use according to any one of claims 1 to 5, characterized in that the extract consists of a total extract which is formed by the total extractable protein fractions that are naturally present in the grains.

12. A cosmetic or dermopharmaceutical composition for topical application to the skin, epithelial appendages and / or

Mucous membranes, characterized in that it contains as an active ingredient, alone or in combination with at least one other active ingredient, at least one protein fraction which has been extracted from Vigna trilobata grains.

13. Cosmetic or dermopharmaceutical composition according to claim 12, characterized in that it contains, as an active ingredient which stimulates growth and cellular metabolism, at least one soluble protein fraction which has been extracted from Vigna trilobata grains.

14. Cosmetic or dermopharmaceutical composition according to one of claims 12 and 13, characterized in that it contains as an active ingredient which stimulates the contraction of the collagen network, at least one soluble protein fraction which has been extracted from Vigna trilobata grains.

15. Cosmetic or dermopharmaceutical composition according to any one of claims 12 to 14, characterized in that it is the active ingredient which the Elimination of glycosaminoglycans stimulates at least one soluble protein fraction that has been extracted from Vigna trilobata grains.

16. Cosmetic or dermopharmaceutical composition according to any one of claims 12 to 15, characterized in that it is used as an anti-apoptosis

Active ingredient contains at least one soluble protein fraction, which was extracted from Vigna trilobata grains.

17. Cosmetic or dermopharmaceutical composition according to any one of claims 12 to 16, characterized in that it contains as a cytoprotective protective agent against UV-A effects at least one soluble protein fraction which has been extracted from Vigna trilobata grains.

18. Cosmetic or dermopharmaceutical composition according to any one of claims 12 to 17, characterized in that it is used as an active ingredient for

Protection against inflammation induced by UV-B contains at least one soluble protein fraction extracted from Vigna trilobata grains.

19. Cosmetic or dermopharmaceutical composition according to any one of claims 12 to 18, characterized in that it contains, as an active ingredient for the preventive and curative treatment of sensitive skin types, at least one soluble protein fraction which has been extracted from Vigna trilobata grains.

20. Cosmetic or dermopharmaceutical composition according to any one of claims 12 to 19, characterized in that it contains as a firming agent for the skin and / or the mucous membranes at least one soluble protein fraction extracted from Vigna trilobata grains.

21. Cosmetic or dermopharmaceutical composition according to any one of claims 12 to 20, characterized in that it is used as a softening

Active ingredient for the skin and / or the mucous membranes contains at least one soluble protein fraction, which was extracted from Vigna trilobata grains.

22. Cosmetic or dermopharmaceutical composition according to any one of claims 12 to 19, characterized in that it is an active ingredient for the

Hair that makes combing dry or wet hair easier and Improved softness and smoothness of undamaged hair, contains at least one soluble protein fraction extracted from Vigna trilobata grains.

23. Cosmetic or dermopharmaceutical composition according to any one of claims 12 to 22, characterized in that it contains between 0.001% and 50% by weight of protein fractions extracted from Vigna trilobata grains which may be converted into cosmetically suitable carriers (vectors ) were initiated.