Bioassimilable protein-melanin complex, preparation and uses

Abstract

The present invention relates to a bioassimilable protein-melanin complex, advantageously soluble in water, comprising a protein extract, advantageously rich in S-sulfonated residues and melanin. The claimed complex further exhibits subsequent good assimilation in the human body. The invention also relates to a method for preparing the claimed bioassimilable protein-melanin complex, use of said complex and compositions containing at least said complex.

Claims

1. Bioassimilable protein-melanin complex, comprising at least one protein extract, soluble or partially soluble, and melanin, said protein-melanin complex being bioactive and said protein extract comprising at least one cysteine residue or at least one tyrosine residue or at least one cysteine residue and one tyrosine residue, characterized in that the proteins and/or the protein fragments of said protein extract have been rendered soluble by grafting at least one polar group on the proteins and/or the protein fragments of said protein extract.

2. Protein-melanin complex according to claim 1, characterized in that said proteins and/or said protein fragments of said protein extract have been rendered soluble by grafting at least one polar group on at least one cysteine, preferentially on the sulphur atom of said cysteines, advantageously a polar group selected from the phosphate, sulphate or sulphite groups, preferentially sulphite.

3. Protein-melanin complex according to any one of claim 1 or 2, characterized in that said cysteine bears a sulphite group on its sulphur atom.

4. Protein-melanin complex according to any one of claims 1 to 3, characterized in that said protein extract is soluble in water from 0.1 to 99%, preferentially from 0.1 to 75%, very preferentially from 0.1 to 50%.

5. Protein-melanin complex according to any one of claims 1 to 4, characterized in that it comprises between 0.01% and 99.99% of protein extract, preferentially between 1% and 95%, very preferentially between 30% and 95%, yet more preferentially between 50% and 95%.

6. Protein-melanin complex according to any one of claims 1 to 5, characterized in that it comprises between 0.01% and 99.99% of melanin, preferentially between 0.5% and 20%, very preferentially between 1% and 15%.

7. Protein-melanin complex according to any one of claims 1 to 6, characterized in that the protein extract comprises peptides with a length comprised between 2 and 1,000 amino acids, preferentially between 2 and 500 amino acids, very preferentially between 2 and 100 amino acids.

8. Protein-melanin complex according to any one of claims 1 to 7, characterized in that said protein extract and/or said melanin originate from different or identical protein and melanin sources, taken separately or originate from one and the same melanoprotein source, advantageously used alone.

9. Protein-melanin complex according to any one of claims 1 to 8, characterized in that said protein extract and/or said melanin originate from wool, bristles, hair, claws, horns or also feathers, plants, fruits, the ink from cephalopods, bacteria or synthetic sources preferentially originating from wool.

10. Protein-melanin complex according to any one of claims 1 to 9, characterized in that said protein extract and/or said melanin originate from the wool of sheep, mouflons, goats, chamois, takin, ibexes, Siberian ibexes, thars, Himalayan thars, serows, goral, musk ox, urial, bharal, isard, rabbits, hares, pikas, llama, alpaca, guanaco, vicuna, camel, dromedary, yak or feathers such as those from magpie, crow or blackbird, preferentially from the wool of sheep, very preferentially from black sheep (Ouessant, Noire du Velay, Valais Blacknose, Noir de Thibar, Black Welsh Mountain, Balwen, Zwartbles or also Hebridean sheep).

11. Process for the preparation of a bioassimilable protein-melanin complex as described in any one of claims 1 to 10, in which in a first step the protein part of said melanoprotein source is rendered solubilizable, advantageously water-solubilizable, in a second step the protein part of the mixture obtained in the first step is fractionated to peptides, in order to obtain a mixture comprising the sought protein-melanin complex.

12. Process for the preparation of a bioassimilable protein-melanin complex according to claim 11, characterized in that it comprises moreover a third step in which melanin is added to the mixture obtained in the second step in order to obtain a mixture comprising the sought protein-melanin complex.

13. Process according to any one of claim 11 or 12, characterized in that the first step is carried out by grafting groups, advantageously polar, on said proteins and/or protein fragments (peptides), particularly on the cysteines, very particularly on the sulphur atom of said cysteines, preferentially a polar group selected from the phosphate, sulphate or sulphite groups, preferentially sulphite.

14. Process according to claim 13, characterized in that the grafting of groups is the grafting of a sulphite on the sulphur atom of the cysteines by S-sulphonation, advantageously by oxidative sulphitolysis.

15. Process according to any one of claims 11 to 14, characterized in that the second step of hydrolysis is carried out by acid hydrolysis, basic hydrolysis, enzymatic hydrolysis, or also by partial oxidation using peracetic acid, hydrogen peroxide or an equivalent, preferentially by enzymatic hydrolysis.

16. Bioassimilable protein-melanin complex as described in claims 1 to 10 for stimulating melanogenesis.

17. Bioassimilable protein-melanin complex according to claim 16, for stimulating melanogenesis in the skin, the hair, the bristles, the wool, the keratinized appendages, the brain, the inner ear canal, and/or the eyes.

18. Bioassimilable protein-melanin complex as described in claims 1 to 10, for protecting living organisms from ionising or non-ionising radiation.

19. Bioassimilable protein-melanin complex as described in claims 1 to 10, for protecting living organisms from far or near ultraviolet radiation (A, B or C), visible light, infrared radiation (A, B and/or C) or also X-rays.

20. Bioassimilable protein-melanin complex as described in claims 1 to 10, for trapping free radicals.

21. Bioassimilable protein-melanin complex as described in claims 1 to 10, for combating tissue aging.

22. Bioassimilable protein-melanin complex as described in claims 1 to 10, for increasing cell regeneration, the elasticity and/or the hydration of the skin, for reinforcing cell cohesion, particularly in the skin and/or the keratinized appendages.

23. Bioassimilable protein-melanin complex as described in claims 1 to 10, for combating wrinkles and fine lines in the skin.

24. Bioassimilable protein-melanin complex as described in claims 1 to 10, for combating oxidative stress and treating or preventing its consequences.

25. Bioassimilable protein-melanin complex as described in claims 1 to 10, for trapping heavy metals, particularly in the human organism.

26. Bioassimilable protein-melanin complex as described in claims 1 to 10, for preventing and/or treating age-related maculopathy (ARM) and/or age-related macular degeneration (ARMD).

27. Bioassimilable protein-melanin complex as described in claims 1 to 10, characterized in that it is in any known galenic form, particularly in the form of a powder, a liquid, a cream, a lotion, a patch or also a spray.

28. Use of at least one bioassimilable protein-melanin complex as described in claims 1 to 10 or 16 to 28, for the preparation of cosmetic, dermatological, pharmaceutical or also food compositions.

29. Composition, advantageously cosmetic, dermatological, pharmaceutical or also food, comprising at least one bioassimilable protein-melanin complex as described in claims 1 to 10 or 16 to 28.

Description

[0111] FIG. 1: Diagram showing the experimentation areas used during the trials.

[0112] FIG. 2: Illustration of the variation in the parameters L* (2A) and ITA (2B) in the study of the effect of the food supplement comprising the bioassimilable protein-melanin complex (product), on the immediate cutaneous pigmentation (UVA exposure).

[0113] FIG. 3: Illustration of the variation in the melanic index (MI) in the study of the effect of the food supplement comprising the bioassimilable protein-melanin complex (product), on the immediate cutaneous pigmentation (UVA exposure).

[0114] FIG. 4: Illustration of the variations in the parameters L* (4A) and ITA (4B) in the study of the effect of the food supplement comprising the bioassimilable protein-melanin complex (product), on the delayed pigmentation (UVB/UVA exposure).

[0115] FIG. 5: Illustration of the variations in the melanic index (MI) in the study of the effect of the food supplement comprising the bioassimilable protein-melanin complex (product), on the delayed pigmentation (UVB/UVA exposure).

[0116] FIG. 6: Illustration of the variation in the parameters L* (6A) and ITA (6B) in the study of the effect of the food supplement comprising the bioassimilable protein-melanin complex (product), on an unexposed area.

[0117] FIG. 7: Illustration of the variation in the melanic index (MI) in the study of the effect of the food supplement comprising the bioassimilable protein-melanin complex (product), on an unexposed area.

[0118] FIG. 8: Illustration of the variation in the parameters L* in the study of the effect of the food supplement comprising the bioassimilable protein-melanin complex (product), on hair colour.

[0119] FIG. 9: Illustration of the results of the bioassimilability study of the elements of the protein-melanin complex according to the invention.

[0120] FIG. 10: Calibration curve established in order to determine the quantity of melanin present in the samples obtained at the end of the bioassimilability study.

[0121] FIG. 11: Illustration of the results of the study of the modification in the protein content of the skin for the protein-melanin complexes. [Control, .square-solid.Active Group].

[0122] Other elements, characteristics and advantages of the invention will become apparent on reading the following examples given by way of illustration and with reference to the attached figures.

[0123] FIG. 1 illustrates the location of the areas of skin subjected to UV radiation during the trials presented in the examples. The presence should be noted of a measurement area of the Minimal Erythema Dose (MED) defined as the lowest dose of UltraViolet (UV) radiation causing the first perceptible erythema appearing on the major part of the UV exposure site, in the 16 to 24 hours after the exposure, and a measurement area of the minimal dose of persistent pigment darkening (MPPD) defined as the Minimal Persistent Pigment Darkening Dose (MPPD).

[0124] The different control areas 1A and 1B receive defined doses of UV (MED and MPPD) and make it possible to measure the epidermal response before taking the protein-melanin product.

[0125] The areas 2A, 3A and 4A make it possible to measure the epidermal response after taking the protein-melanin product under UVA every 10 days. This corresponds to the measurement of the immediate pigmentation due to the melanin which migrates to the surface of the skin in response to the UVA radiation.

[0126] The area 4B makes it possible to measure the epidermal response after taking the protein-melanin product and under UV A/B. This corresponds to the measurement of the delayed pigmentation due to the increased production of melanin measured after 30 days of treatment and which results in a more intense and persistent tan in response, which corresponds to the activation of the synthesis of the melanin (melanogenesis).

[0127] The area NE is the measurement area before and after taking the complex, without UV irradiation. This corresponds to research into the activity of the product on melanogenesis in the absence of irradiation.

[0128] FIG. 2 illustrates the results obtained in the study of the variation in the parameters L* (2A) and ITA (2B) after administration of the food supplement comprising the bioassimilable protein-melanin complex (product), on the immediate cutaneous pigmentation (UVA exposure). It should be noted that on DO two hours after one exposure to the UVA, the skin was significantly darker (reduction in the parameter L*; FIG. 2A) and more pigmented (reduction in the parameter ITA; FIG. 2B) after the use of the food supplement comprising the bioassimilable protein-melanin complex (product) according to the invention.

[0129] This effect was observed from the 10th day of use of the product and the efficacy also increases with time (maximum effect observed after 30 days of use of the product: reduction of 225% in the parameter ITA compared to before the use of the product). These results are statistically significant (p<0.001%).

[0130] FIG. 3 illustrates the results obtained in the study of the variation in the melanic index (MI) after administration of the food supplement comprising the bioassimilable protein-melanin complex (product), on the immediate cutaneous pigmentation (UVA exposure). It should be noted that after 10 days of use of the product, the measurements with the Mexameter showed the skin to be slightly more pigmented, two hours after the UVA exposure (+14%, variation at the limit of significance). After 20 and 30 days of use of the product, the increase in the melanic index is much greater and becomes statistically significant (on D30: +106%, p<0.001).

[0131] FIG. 4 illustrates the results obtained in the study of the variation in the parameters L* (4A) and ITA (4B) after administration of the food supplement comprising the bioassimilable protein-melanin complex (product), on the delayed pigmentation (UVB/UVA exposure). It should be noted that after 30 days of use of the food supplement comprising the bioassimilable protein-melanin complex (product), the measurements with the Spectrocolorimeter showed the skin to be significantly darker (reduction in the parameter L*) and more pigmented (reduction in the parameter ITA) after one, two or three UVB/UVA exposures (after three exposures: significant reduction in the parameter ITA of 206% compared to before the use of the product).

[0132] These results are statistically significant (p<0.001%).

[0133] FIG. 5 illustrates the results obtained in the study of the variation in the melanic index (MI) after administration of the food supplement comprising the bioassimilable protein-melanin complex (product), on the delayed pigmentation (UVB/UVA exposure). It should be noted that after 30 days of use of the food supplement comprising the bioassimilable protein-melanin complex (product), the measurements with the Mexameter showed a skin that is slightly more pigmented (increase in the melanic index) after one, two or three UVB/UVA exposures (after three exposures, increase in the melanic index of 217% compared to before use). These results are statistically significant (p<0.001%).

[0134] FIG. 6 illustrates the results obtained in the study of the variation in the parameters L* (6A) and ITA (6B) after administration of the food supplement comprising the bioassimilable protein-melanin complex (product), on an unexposed area. It should be noted that during the entire period of taking the food supplement comprising the bioassimilable protein-melanin complex (product), the measurements with the Spectrocolorimeter have not shown a relevant variation in the colour of the skin on an area not exposed to UV: variations of 0% to 2%, which are not significant for the most part and not relevant from a biological point of view (not visible to the naked eye).

[0135] FIG. 7 illustrates the results obtained in the study of the variation in the melanic index (MI) after administration of the food supplement comprising the bioassimilable protein-melanin complex (product), on an unexposed area. It should be noted that between 10 and 39 days of use of the food supplement comprising the bioassimilable protein-melanin complex (product), the measurements with the Mexameter have shown a very slight increase in the melanic index (significant variation or at the limit of significance between 4% and 6%).

[0136] FIG. 8 illustrates the results obtained in the study of the variation in the parameters L* in the study of the effect of the food supplement comprising the bioassimilable protein-melanin complex (product), on hair colour. It should be noted that during 120 days of use of the food supplement comprising the bioassimilable protein-melanin complex (product), the measurements with the Spectrocolorimeter have shown a significantly darker hair colour (13% after 120 days of use, p<0.001). The effect increases with the treatment time. A variation in the shade of the hair was also observed with a colour that is less yellow and less red.

[0137] FIG. 9 illustrates the results of the study of bioassimilability of the protein-melanin complex according to the invention. A bioassimilability of the proteins in the simulated gastro-intestinal liquids of 40.50.8%, a bioassimilability of the tyrosine equal to 1.850.2%, and a bioassimilability of melanin in the simulated gastro-intestinal fluids of 9.430.7% is noted.

[0138] The total bioassimilability of the protein-melanin complex was mathematically determined by adding the contents of proteins, tyrosine and melanin; the result is a value for the bioassimilability of the protein-melanin complex of 49.93% (FIG. 9).

[0139] FIG. 10 shows the curve used for the quantitative determination of melanin by spectrophotometric methods (see Example 5).

[0140] FIG. 11 illustrates the results of the change in the protein content of the skin collected from subjects having received the protein-melanin complex according to the invention [.square-solid.Active Group] compared to a group of subjects not having received said protein-melanin complex [ontrol].

[0141] The protein content (cohesion) is measured by using the Corneofix foil method (Courage+Khazaka electronic GmbH).

[0142] The Corneofix F 20 foils are applied onto the stratum corneum of a clean face on T0 and T90 days. Determination of the content of the proteins captured in the foils of the Corneofix F 20 allows evaluation of skin cohesion.

[0143] An improvement in skin cohesion implies a reduction in the quantity of proteins captured in the foils between the samplings on days T0 and on T90.

[0144] Taking the protein-melanin complex significantly improved skin cohesion with respect to the control on day 90 with a reduction of 19.7% in the protein content with respect to the control (P<0.001).

EXAMPLE 1

Preparation of a Bioassimilable Protein-Melanin Complex According to the Invention

First Step: Oxidative Sulfitolysis Applied to Wool

Procedure:

[0145] 100 kg of washed degreased sheep's wool from the Noire du Velay breed is introduced into a tank.

Preparation of the Copper-Ammonium Solution and the Sulphites Solution:

[0146] Preparation of the Soluble Copper-Ammonium Hydroxide Solution in a Basic Medium:

[0147] In another tank, approximately 20 litres of an aqueous solution of ammonium hydroxide at 25% is mixed with 8 kg of pure copper, in the form of copper sulphate. Then approximately 750 litres of water is added, in which 50 kg of sodium sulphites has previously been dissolved.

[0148] Progress of the Reaction, Controlled Parameters:

[0149] The reaction mixture is added to the tank containing the wool then set in motion. The temperature is maintained between 20 and 40 C., and air is injected in order to provide oxygen. When the reaction medium has become sufficiently fluid the mixture is stirred. This operation is conducted for a duration of approximately 1 to 2 days, until the fibres have lost all mutual cohesion.

Second Step: Hydrolysis and Enzymatic Inactivation

[0150] Then 350 g of the enzyme Protex 6L from Genencor or Multifect PR 6L from Brenntag (alkaline serine protease) is added the enzymatic activity of which is 580,000 U/g in the medium at a pH maintained between 9 and 10, and at a temperature comprised between 40 and 50 C.;

[0151] It is left to hydrolyze for 24 hours under stirring.

[0152] As hydrolysis proceeds, with the pH increasing, sodium hydroxide is added in order to maintain the pH constant between 9 and 10.

[0153] When the pH no longer varies, therefore it is no longer necessary to add sodium hydroxide, or when it is considered that the hydrolysis is sufficient, the hydrolysis step is then terminated.

[0154] The reaction medium then contains, among others, the protein-melanin complex that is the subject of the invention, which can be used as it is or purified and concentrated by any techniques known to a person skilled in the art.

EXAMPLE 2

Composition Comprising the Protein-Melanin Complex Obtained in Example 1

[0155] 2A) lotion for slowing hair loss and restoring the colour to grey hair by topical application topic on the scalp and the hair of the head.

TABLE-US-00001 Protein-melanin complex 7.0 g Urea 1.0 g Sodium lactate 0.5 g Phenoxyethanol 0.1 g Fragrance qs Water qsf 100.0 g [0156] 2B) preparatory cream for accelerating tanning and regenerating the epidermis

TABLE-US-00002 Protein-melanin complex 5.0 g Glyceryl behenate 2.0 g Isononyl isononanoate 3.0 g Shea butter 1.5 g Glycerine 4.0 g Tocopherol 0.1 g Vaseline oil 8.0 g Polyglycerol ester 0.5 g Fragrance qs Glycerol undecylanate 0.1 g Water qsf 100.0 g [0157] 2C) Food supplement, preparatory to and accelerator of tanning, in capsule form

TABLE-US-00003 Protein-melanin complex in 325.0 mg microgranulated powder form Vitamin C 90.0 mg Copper gluconate 1.0 mg Magnesium stearate 2.0 mg Silicon dioxide 0.2 mg 1 Hard gelatin capsule 94.0 mg

EXAMPLE 3

Evaluation of the Effect of a Bioassimilable Protein-Melanin Complex According to the Invention on Skin Pigmentation

[0158] The objective of this study is to evaluate the effect of the bioassimilable protein-melanin complex according to the invention, prepared in Example 1, on skin pigmentation under UV stimulation, after use of the product studied for 10, 20 and 30 days.

[0159] The following were studied during this evaluation: [0160] the effect of the product on skin pigmentation after exposure to UVA and measurement of the immediate pigmentation (2 hours after exposure) with a Spectrocolorimeter and a Mexameter after use of the product for 10, 20 and 30 days. This pigmentation mainly involves the activation of the melanin already present in the skin. [0161] the effect of the product on skin pigmentation after repeated exposure to UV(B+A) and measurement of the delayed pigmentation with a Spectrocolorimeter and a Mexameter after 30 days' use of the product. This pigmentation mainly involves the synthesis of new melanin.

3.1) Tools and Protocols

UV Exposures

[0162] In order to measure the Minimal Erythema Dose (MED: the lowest dose of UltraViolet (UV) radiation causing the first perceptible erythema appearing on the major part of the UV exposure site), in the 16 to 24 hours after the exposure and the Minimal Persistent Pigment Darkening Dose (MPPD), a Xenon lamp of the Solar Light Multiport 601-300W type, equipped with a WG320 (1.25 mm) filter for the UV(A+B) and a WG335 (3 mm) filter (UVA) for eliminating the UVB with a power rating of 300 W was used.

[0163] The spectra emitted are from 290 to 400 nm for the UV (A+B) and from 320 to 400 nm for the UVA.

[0164] A UG11 (1 mm) filter and dichroic mirror were used to eliminate infra-red radiation and visible light.

[0165] The skin surface exposed is delimited using a mask comprising six holes (diameter 8 mm) 0.5 cm.sup.2.

[0166] In order to determine the MED the UV flux of each optical fibre is adjusted by the technician in order to obtain a geometrical progression of 15%. The system is used with a constant flux, all the fibres are open at the same time.

[0167] In order to determine the MPPD the UVA flux of each optical fibre is determined by the technician in order to obtain a geometric progression of 25%. The system is used with a constant flux, all the fibres are open at the same time.

[0168] For the exposures to UV (A+B) and UVA, a Xenon lamp of the Solar Light Monoport 1000W High Power Solar SimulatorModel LS1000 type, having a UVA (A+B) spectrum from 290 to 400 nm and UVA spectrum from 320 to 400 nm was used. The skin surface exposed is 44 cm at maximum.

Measurements With the Spectrophotometer at the Level of the Skin

[0169] The colorimetric measurement of the skin is carried out using a Spectrocolorimeter MINOLTA CM700-d, equipped with an 8 mm diameter head.

[0170] The Spectrocolorimeter converted the colours situated within the range of human perception into a numerical code comprising three parameters: [0171] L*: represents lightness (from dark to pale), [0172] a*: represents the range from the greens to the reds, [0173] b*: represents the range from the blues to the yellows.

[0174] a* and b* are chrominance parameters and L* is a luminance parameter.

[0175] It then becomes possible to express, in the smallest detail, the differences between two skin areas which appear to be of the same colour. After calibration, the measurements are carried out directly on the skin using a pulsed Xenon light source and a double beam system for measuring the light emitted and correcting any slight deviation.

[0176] This instrument is commonly used in cosmetics and in medicine for measuring the colour of the skin.

[0177] The parameters L* (characteristic of lightness) and b* (characteristic of cutaneous melanin yellow pigmentation) are studied during an investigation of cutaneous pigmentation.

[0178] These two parameters are utilized through calculation of the Individual Typological Angle (ITA), which defines the degree of pigmentation of the skin of a person by incorporating the lightness (L*) and the melanization parameter (b*), according to the following formula:

ITA=[Arc tan((L*50)/b*)]180/

[0179] The higher the ITA, the lighter the skin.

[0180] Each measurement is the average of three acquisitions.

Measurements With the Mexameter at the Level of the Skin

[0181] The Mexameter is an instrument from Courage and Khazaka, equipped with a 5 mm diameter head, which particularly measures the melanin and haemoglobin content of skin. These two components are mainly responsible for the colour of the skin.

[0182] The measurement is based on the absorption principle. The special probe of the Mexameter MX18 emits light at three predefined wavelengths (568 nm (green), 660 nm (red) and 880 nm (infrared)). A photo-detector measures the light reflected by the skin. This measurement is based on the same optical principle which consists of measuring the light absorbed and reflected at the red and infrared wavelengths for melanin.

[0183] A Melanic Index (Mx) is calculated from the intensity of the light absorbed and reflected at 600 and 880 nm respectively.

[0184] An Erythema Index (Ex) is calculated from the intensity of the light absorbed and reflected at 568 and 660 nm respectively.

[0185] Only the Melanic Index, which represents the melanin content of the skin, was analyzed. An increase in this parameter characterizes an increase in skin pigmentation.

Progress of the Test

[0186] The test was carried out on 35 subjects

On D-10

[0187] The subjects come to the laboratory without having applied any product on their backs since the previous evening and the areas of exposure on their backs are defined as shown in FIG. 1 [0188] UVB/UVA exposure for determining the MED. [0189] UVA exposure for determining the MPPD. [0190] 2 hours after UVA exposure: reading the MPPD

On D-9

[0191] The subjects come to the laboratory without having applied any product on their backs since the previous evening. [0192] Between 16 and 24 hours after UVB/UVA exposure: reading the MED. [0193] Measurements with the Spectrocolorimeter on areas 1A, 1B and NE. [0194] Measurements with the Mexameter on areas 1A, 1B and NE. [0195] UVB/UVA exposure on area 1B at 0.8 MED. [0196] UVA exposure on area 1A at 1.25 MPPD. [0197] 2 hours after UVA exposure: measurements with the Spectrocolorimeter and Mexameter on area 1A.

On D-6

[0198] The subjects come to the laboratory without having applied any product on their backs since the previous evening. [0199] Measurements with the Spectrocolorimeter and Mexameter on areas 1B and NE. [0200] UVB/UVA exposure on area 1B at 0.8 MED.

On D-3

[0201] The subjects come to the laboratory without having applied any product on their backs since the previous evening. [0202] Measurements with the Spectrocolorimeter and Mexameter on areas 1B and NE. [0203] UVB/UVA exposure on area 1B at 0.8 MED.

On D0

[0204] The subjects come to the laboratory without having applied any product on their backs since the previous evening. [0205] Measurements with the Spectrocolorimeter and Mexameter on areas 1B and NE.
The product is taken at a rate of 2 capsules per day, in the morning with breakfast, until the end of the trial.

On D10

[0206] The subjects come to the laboratory without having applied any product on their backs since the previous evening. [0207] Measurements with the Spectrocolorimeter and Mexameter on areas 2A and NE. [0208] UVA exposure on area 2A at 1.25 MPPD. [0209] 2 hours after UVA exposure: measurements with the Spectrocolorimeter and Mexameter on area 2A.

On D20

[0210] The subjects come to the laboratory without having applied any product on their backs since the previous evening. [0211] Measurements with the Spectrocolorimeter and Mexameter on areas 3A and NE. [0212] UVA exposure on area 3A at 1.25 MPPD. [0213] 2 hours after UVA exposure: measurements with the Spectrocolorimeter and Mexameter on area 3A.

On D30

[0214] The subjects come to the laboratory without having applied any product on their backs since the previous evening. [0215] Measurements with the Spectrocolorimeter on areas 4A, 4B and NE. [0216] Measurements with the Mexameter on areas 4A, 4B and NE. [0217] UVB/UVA exposure on area 4B at 0.8 MED. [0218] UVA exposure on area 4A at 1.25 MPPD. [0219] 2 hours after UVA exposure: measurements with the Spectrocolorimeter and Mexameter on area 4A.

On D33

[0220] The subjects come to the laboratory without having applied any product on their backs since the previous evening. [0221] Measurements with the Spectrocolorimeter and Mexameter on areas 4B and NE. [0222] UVB/UVA exposure on area 4B at 0.8 MED.

On D36

[0223] The subjects come to the laboratory without having applied any product on their backs since the previous evening. [0224] Measurements with the Spectrocolorimeter and Mexameter on areas 4B and NE. [0225] UVB/UVA exposure on area 4B at 0.8 MED.

On D39

[0226] The subjects come to the laboratory without having applied any product on their backs since the previous evening. [0227] Measurements with the Spectrocolorimeter and Mexameter on areas 4B and NE.

Analysis of the Data

[0228] Statistical analysis of the data makes it possible to determine the significance of the variations under the effect of the tested product.

[0229] The comparison is based on the values obtained at the different evaluation times.

[0230] The test used is the Student's t-test on paired data. The application conditions are the random and simple nature of the samples and the normality of the population of differences.

[0231] The principle of the test is to pose a null hypothesis (H0) of an absence of difference between the average effect at different evaluation times (d=0) and an alternative hypothesis H1 (our research hypothesis) of a difference between the evaluation times (d<>0).

[0232] The probability p of observing a difference between the times which is at least as great as that observed if the null hypothesis is true is then determined. [0233] If p5%, the null hypothesis is rejected. The alternative hypothesis H1 of a significant difference between the evaluation times is therefore accepted. [0234] If p>5%, the null hypothesis is accepted. The data have not revealed a significant difference between the evaluation times.

3.2) Results

[0235] In the tables shown below, the results for L* are dark to light data, the results for ITA are data ranging from more pigmented to less pigmented.

3.2.1) Effect of the Food Supplement Comprising the Bioassimilable Protein-Melanin Complex (Product) on Immediate Skin Pigmentation (UVA Exposure)

Spectrocolorimeter

[0236] A summary of the results is shown in Table 1 below.

TABLE-US-00004 TABLE 1 D 10 D 20 D 30 Variation Variation Variation AU % p AU % p AU % p L* 2.4 +/ 0.3 118 <0.001 3.9 +/ 0.4 189 <0.001 6.1 +/ 0.4 248 <0.001 ITA 2.6 +/ 0.4 82 <0.001 6.2 +/ 0.4 161 <0.001 7.2 +/ 0.5 225 <0.001 AU: average +/ SEM

[0237] These results are also illustrated in FIGS. 2A and 2B. Two hours after a UVA exposure, the skin was significantly darker (decrease in the parameter L*) and more pigmented (decrease in the parameter ITA) after the use of the food supplement comprising the bioassimilable protein-melanin complex (product) according to the invention.

[0238] This effect was observed from use of the product for 10 days and the effectiveness also increases over time (maximum effect observed after use of the product for 30 days: decrease of 225% in the parameter ITA compared to before use of the product.)

Mexameter

[0239] A summary of the results is shown in Table 2 below.

TABLE-US-00005 TABLE 2 D 10 D 20 D 30 Variation Variation Variation AU % p AU % p AU % p MI 3.3 +/ 1.8 14 0.076 20 +/ 1.9 84 <0.001 25.3 +/ 3.6 106 <0.001 MI = Melanic Index; AU: average +/ SEM

[0240] These results are also illustrated in FIG. 3.

[0241] After use of the product for 10 days, the measurements with the Mexameter showed a slightly more pigmented skin two hours after the UVA exposure (+14%, variation at the limit of significance). After use of the product for 20 and 30 days, the increase in the Melanic Index is much greater and becomes statistically significant (at D30: +106%, p<0.001).

3.2.2) Effect of the Food Supplement Comprising the Bioassimilable Protein-Melanin Complex (Product) on Delayed Pigmentation (UVB/UVA Exposure)

Spectrocolorimeter

[0242] A summary of the results is shown in Table 3 below.

TABLE-US-00006 TABLE 3 D 30 Variations AU % p After 1 exposure to UV L* 2.2 +/ 0.0 170 <0.001 ITA 4.2 +/ 0.3 190 <0.001 After 2 exposures to UV L* 4.1 + 0.3 180 <0.001 ITA 6.7 +/ 0.6 166 <0.001 After 3 exposures to UV L* 7.0 +/ 0.3 249 <0.001 ITA 11.0 +/ 0.5 206 <0.001

[0243] These results are also illustrated in FIGS. 4A and 4B.

[0244] After use of the food supplement comprising the bioassimilable protein-melanin complex (product) for 30 days, the measurements with the Spectrocolorimeter showed a significantly darker (decrease in the parameter L*) and more pigmented (decrease in the parameter ITA) skin after one, two or three UVB/UVA exposures (after three exposures: significant decrease in the parameter ITA of 206% compared to before the use of the product).

Mexameter

[0245] A summary of the results is shown in Table 4 below.

TABLE-US-00007 TABLE 4 D 30 Variations AU % p MI After 1 exposure to UV 15.1 +/ 0.0 193 <0.001 After 2 exposures to UV 26.5 + 1.9 167 <0.001 After 3 exposures to UV 43.1 +/ 2.7 217 <0.001 MI = Melanic Index; AU: average +/ SEM

[0246] These results are also illustrated in FIG. 5.

[0247] After use of the food supplement comprising the bioassimilable protein-melanin complex (product) for 30 days, the measurements with the Mexameter showed a significantly more pigmented skin (increase in the melanic Index) after one, two or three UVB/UVA exposures (after three exposures, increase in the melanic Index of 217% compared to before use).

3.2.3) Effect of the Food Supplement Comprising the Bioassimilable Protein-Melanin Complex (Product), on an Unexposed Area

Spectrocolorimeter

[0248] A summary of the results is shown in Table 5 below.

TABLE-US-00008 TABLE 5 L* b* ITA Variation Variation Variation AU % p AU % p AU % p D 10 0.6 +/ 0.2 1 0.004 0.5 +/ 0.2 2 0.010 0.3 +/ 0.2 1 0.139 D 20 0.1 +/ 0.2 0 0.604 0.0 +/ 0.2 0 0.804 0.0 +/ 0.0 0 0.000 D 30 0.1 +/ 0.2 0 0.722 0.1 +/ 0.2 1 0.552 0.0 +/ 0.0 0 0.000 D 33 0.1 +/ 0.2 0 0.549 0.1 +/ 0.2 0 0.689 0.0 +/ 0.0 0 0.000 D 36 0.0 +/ 0.2 0 0.975 0.2 +/ 0.2 1 0.358 0.0 +/ 0.0 0 0.000 D 39 0.1 +/ 0.2 0 0.613 0.1 +/ 0.2 0 0.714 0.0 +/ 0.0 0 0.000 AU: average +/ SEM

[0249] These results are also illustrated in FIGS. 6A and 6B.

[0250] At no time while the food supplement comprising the bioassimilable protein-melanin complex (product) was being taken did the measurements with the Spectrocolorimeter show a relevant variation in the colour of the skin on an area not exposed to UV: variations of 0% to 2%, not significant for the majority and not relevant from the biological point of view (not visible to the naked eye).

Mexameter

[0251] A summary of the results is shown in Table 6 below.

TABLE-US-00009 TABLE 6 Variation AU % p D 10 3.0 +/ 1.3 5 0.027 D 20 2.7 +/ 1.4 5 0.064 D 30 3.4 +/ 1.4 6 0.020 D 33 3.3 +/ 1.2 6 0.011 D 36 2.1 +/ 1.2 4 0.088 D 39 3.1 +/ 1.2 5 0.014 AU: average +/ SEM

[0252] These results are also illustrated in FIG. 7.

[0253] After use of the food supplement comprising the bioassimilable protein-melanin complex (product) for 10 to 39 days, the measurements with the Mexameter showed a very slight increase in the melanic index (significant variation or at the limit of significance between 4% and 6%).

3.3) Conclusion

[0254] The main objective of this test was to assess the effect of the food supplement comprising the bioassimilable protein-melanin complex (product) on skin pigmentation under UV stimulation, after use of the tested product for 0, 20 and 30 days.

[0255] Under the conditions of this test, the food supplement comprising the bioassimilable protein-melanin complex (product) tested: [0256] allowed a significant improvement in the immediate pigmentation 2 hours after a UVA exposure. This effect was observed both with the Spectrocolorimeter and with the Mexameter, from use for 10 days. The effectiveness of the product also increased after use for 30 days.

[0257] An increase in the pigmentation was observed of 225% with the Spectrocolorimeter and of 106% with the Mexameter. [0258] allowed a significant improvement in the delayed pigmentation after one, two or three UVB/UVA exposures. This effect was observed both with the Spectrocolorimeter and with the Mexameter, from use of the product for 30 days. After 3 UVB/UVA exposures, an increase in the pigmentation was observed of 206% with the Spectrocolorimeter and of 217% with the Mexameter. [0259] caused a very slight increase in the Melanic Index on an area not exposed to UV (variation of 4% to 6%). This effect was observed from use of the product for 10 days.

[0260] It is noteworthy that the product was greatly liked by the majority of the subjects. 66% of the subjects found their skin to have more colour and 76% wished to continue to use the product. The product has moreover been well tolerated by the sample group of testers. No sensation of discomfort or intolerance has been reported by the subjects.

EXAMPLE 4

Evaluation of the Effect of a Bioassimilable Protein-Melanin Complex According to the Invention on the Pigmentation of the Hair

[0261] The objective of this study is to evaluate the effect of the bioassimilable protein-melanin complex according to the invention, prepared in Example 1, on the pigmentation of the hair.

[0262] The study was conducted with 32 volunteers with grey and/or salt-and-pepper hair and applying no dye for the duration of the study.

[0263] Measurements of the hair colour are carried out using a Chromametre on D0, D60, D90 and D120 in order to evaluate the effect of the product on the pigmentation of the hair.

4.1) Measurements with the Chromametre on the Hair

[0264] On D0, the hair was shaved over a mini-area of 1 cm.sup.2 in order to perfectly locate the measurements during the entire study. This mini-area was identified in the CRF using centimetric measurements.

[0265] Four colorimetric measurements were carried out on the root of the hair surrounding the shaved mini-area (up, down, to the left and to the right) using a MINOLTA CR-400 Chromametre MINOLTA CR-400, equipped with an 8 mm diameter head. Analysis of the data was carried out on the average of four measurements.

[0266] At the following kinetic time, the mini-area was identified and shaved again if necessary and the measurements were carried out with the Chromametre as explained above.

[0267] The Chromametre converts the colours situated in the range of human perception into a numerical code comprising three parameters:

[0268] L*: represents the lightness (from dark to pale),

[0269] a*: represents the range from the greens to the reds,

[0270] b*: represents the range from the blues to the yellows.

[0271] a* and b* are the parameters of chrominance and L* a parameter of luminance.

[0272] It then becomes possible to express, in the smallest details, the differences between two areas which appear to be the same colour. After calibration, the measurements are carried out directly on the hair using a pulsed Xenon light source and a double beam system for measuring the light emitted and correcting any slight deviation.

[0273] This instrument is commonly used in cosmetics and in medicine for measuring the colour of the skin or the hair.

4.2) Progress of the Trial

[0274] On D0: [0275] The subjects come to the laboratory without having applied any product on the hair since the previous evening. [0276] Definition and shaving of a mini-area of 1 cm.sup.2 on the scalp. [0277] Measurements with the Chromametre on the root of the hair around this mini-area. Four measurements are carried out up, down, to the left and to the right of the area.

[0278] On D60, D90, D120: [0279] The subjects come to the laboratory without having applied any product on the hair since the previous evening. [0280] Location of the mini-area defined on D0. This area is shaved again. [0281] Measurements with the Chromametre on the root of the hair around this mini-area. Four measurements are carried out up, down, to the left and to the right of the area.

4.3) Analysis of the Data

[0282] Statistical analysis of the data makes it possible to determine the significance of the variations under the effect of the tested product.

[0283] The comparison is based on the values obtained at the different evaluation times.

[0284] The test used is the Student's t-test on paired data. The application conditions are the random and simple nature of the samples and the normality of the population of differences.

[0285] The principle of the test is to pose a null hypothesis (H0) of absence of difference between the average effect at different evaluation times (d=0) and an alternative hypothesis H1 (our research hypothesis) of a difference between the evaluation times (d<>0).

[0286] The probability p of observing a difference between the times which is at least as great as that observed if the null hypothesis is true is then determined. [0287] If p5%, the null hypothesis is rejected. The alternative hypothesis H1 of a significant difference between the evaluation times is therefore accepted. [0288] If p>5%, the null hypothesis is accepted. The data have not made it possible to demonstrate a significant difference between the evaluation times.

4.4) Results

Chromametre

[0289] A summary of the results is shown in Table 7 below.

TABLE-US-00010 TABLE 7 J60 J90 J120 Variation Variation Variation A.U. % p A.U. % p A.U. % p L* 2.2 +/ 0.3 4 <0.001 4.9 +/ 0.4 9 <0.001 7.5 +/ 0.5 13 <0.001 a* 0.5 +/ 0.2 8 0.012 1.4 +/ 0.2 22 <0.001 2.4 +/ 0.2 37 <0.001 b* 0.6 +/ 0.1 6 <0.001 1.3 +/ 0.2 14 <0.001 2.0 +/ 0.2 22 <0.001 AU: average +/ SEM

[0290] These results are also illustrated in FIG. 8.

[0291] After use of the food supplement comprising the bioassimilable protein-melanin complex (product) for 30, 90 and 120 days, the measurements with the Chromametre showed a significantly darker hair colour (13% after use for 120 days, p<0.001). The effect increases with the treatment time. A variation of the shade of the hair was also observed with a colour that is less yellow and less red.

EXAMPLE 5

Study of the Bioassimilability of the Protein-Melanin Complex According to the Invention

Experimental System

[0292] Evaluation of the bioassimilability of the protein-melanin complex according to the invention and of the different components of said complex in the simulated gastric and intestinal fluids was studied following the dialysis tube procedure [D. W. BOLLINGER et al.; J. Agric. Food Chem.; 2005. 53: 3287-3294].

Pepsin Digestion.

[0293] A quantity of 100 mg of melanoprotein complex according to the invention was mixed with 1.0 mL of a 0.85 N solution of hydrochloric acid (HCl).

[0294] 24,000 U of porcine pepsin per mL were then added and the sample was incubated in a water bath at 39 C. for 120 min.

Pancreatin Digestion.

[0295] At the end of the pepsin digestion, the samples were transferred into dialysis tubes 18 cm long and 1.3 mL of a 0.8 M solution of NaHCO.sub.3 containing 22.60 mg of porcine pancreatin/mL (8USP) were added to pepsin digesta. The dialysis tubes were sealed at each end with clips. The dialysis tubes with a porosity greater than 12,000 and with a diameter of 1.6 cm (Sigma Chemical Co., reference D6191) were placed in a 250 mL flask containing 100 mL of 0.05 M succinate buffer. The samples were incubated at 39 C. under stirring at 120 cycles per minute for 4 h.

[0296] After incubation of the pancreatin, the components available in the dialysis medium were determined by different experimental procedures.

[0297] All the experiments were carried out in triplicate and the results are expressed as the average of the results of the 3 experiments.

Quantitative Determination of the Proteins by the Lowry Method

[0298] 1 mL of each sample was mixed with 4.5 mL of Lowry reagent (9.8 mL of Na.sub.2CO.sub.3 (2% W/V), 0.1 mL of CuSO.sub.4-5H.sub.2O (1% W/V) and 0.1 mL of Na.sup.+K.sup.+ tartrate (0.5% W/V). The solution was mixed and incubated for 10 min. Then 0.5 mL of Folin-Ciocalteau reagent 1N was added and left to react for 30 min. Finally, the absorbance was measured at 660 nm [O. H. LOWRY et al.; J. Biol. Chem.; 1951. 193: 265-275].

[0299] The data are expressed in percentage of available proteins.

Quantitative Determination of the Tyrosine by HPLC

[0300] The HPLC system consisted of a Jasco BIP-je pump, a Rheodyne 7725 injector (230 L loop), and a Jasco UVDEC-100 V UV detector.

[0301] A C8 type column (0.415 mm) packed with particles 5 m in size was used for the determination. The separation was monitored at 210 nm. A mobile phase constituted by an acetonitrile-water mixture (5:95 v/v) was used. The flow rate was maintained at 1.5 mL min.sub.1

[0302] Identification of the peaks was carried out by comparing the retention time of the samples with a standard solution of tyrosine.

[0303] The data are expressed in percentage of available tyrosine.

Quantitative Determination of Melanin by Spectrophotometric Methods

[0304] After the incubations of pepsin and pancreatin, the quantity of available melanin was determined by spectrophotometry using a Jasco V-530 UV/V is spectrometer, according to the method developed by Ozeki and his collaborators [Ozeki, H., et al.; 1996. Spectrophotometric characterization of eumelanin and pheomelanin in hair. Pigment Cell Research 9:265-270 or Ozeki, H. et al.; 1995. Chemical characterization of hair melanins in various coat-color mutants of mice. Journal of Investigative Dermatology 105:361-366], method slightly modified by the inventors.

[0305] The samples obtained after the pepsin and pancreatin digestions were dried under vacuum and the residue was redissolved in Soluene-350 (Perkin Elmer), a strong organic base formulated with toluene. The samples were analysed in order to determine the absorbances at 500 nm (A500). The A500 values correspond to the total melanin contained in the sample.

[0306] The total concentrations of melanin were calculated with reference to a calibration curve established from five different standard solutions of melanin (0.02, 0.04, 0.06, 0.08 and 0.10 mg/ml) in Soluene-350 (see FIG. 10). The absorbance of the standard solutions was measured at 500 nm in order to establish a calibration curve and the correlation coefficient (R2), slope and the y-axis intercept of the regression equation obtained were calculated by the least squares method.

[0307] The absorbance values of the melanic samples obtained by the bioavailability studies in vitro carried out in triplicate, and the corresponding bioavailability percentages, are shown in Table 8 below.

TABLE-US-00011 TABLE 8 Abs Bioavailability (%) 0.7535 8.92 0.7832 9.31 0.8526 10.06

[0308] The final in vitro value of the bioavailability (9.430.68%) was calculated as the average of the data obtained by the three experiments carried out (Table 1)

[0309] The data are expressed as a percentage of available melanin.

Results

[0310] The results are presented in FIG. 9

[0311] The method using dialysis tubes is a rapid and low cost method for evaluating the bioassimilability of different types of compounds.

[0312] As explained previously, the components of the protein-melanin complex are determined by using different experimental procedures.

[0313] More precisely, for the quantification of the content of proteins in the gastro-intestinal digesta, the Lowry assay was used.

[0314] A bioassimilability of the proteins in the simulated gastro-intestinal liquids of 40.50.8% was measured.

[0315] The quantification of tyrosine was carried out by HPLC analysis by comparing the chromatograms of the samples to that of a standard solution of tyrosine.

[0316] A bioassimilability equal to 1.850.2% was thus determined.

[0317] Finally, the bioassimilability of melanin in the simulated gastro-intestinal fluids was determined by spectrophotometric analysis.

[0318] A bioassimilability equal to 9.430.7% was thus determined.

[0319] The total bioassimilability of the protein-melanin complex was mathematically determined by adding the content of proteins, tyrosine and melanin; the result is a bioassimilability value of the protein-melanin complex of 49.93 (FIG. 9).

EXAMPLE 5

Evaluation of the Effect of a Bioassimilable Protein-Melanin Complex According to the Cell Cohesion in the Human Skin

[0320] Measurement of the protein content of the skin makes it possible to evaluate the cohesion of the cells.

[0321] Evaluation of skin cohesion as a function of its protein content is useful for evaluating the effectiveness of the cell cohesion due to the treatment received by the subjects of the study. A reduction in the quantity of proteins exuded on the surface of the skin reflects an increase in the cell cohesion.

[0322] The protein content (cohesion) is measured by using the Corneofix foil method with the Corneofix F 20 kit (Courage+Khazaka electronic GmbH), according to the supplier's protocol. The non-invasive samples of 10 layers of the stratum corneum of a clean face were thus obtained in order to determine the protein content.

[0323] The Lowry method (Oliver H. Lowry, Nira J. Rosebrough, A Lewis Farr and Rose J. Randall, Protein measurement with the Folin phenol reagent, J. biol. Chem., vol. 193, n 1, 1951, p. 265-275) is used for measuring the protein content. It is based on the ability of copper to bind to proteins under alkaline conditions, and when the Folin reagent is added, a complex is formed with the protein which is visible at 550 nm.

[0324] The subjects are divided into 2 groups, a control group and a so-called active group.

[0325] The active group [.square-solid.] received 500 mg/day in 1 dose of melanoprotein complex according to the invention for 90 days.

[0326] The control group [] received 500 mg/day in 1 dose of maltodextrin for 90 days.

[0327] The subjects of the control group (not having received this protein-melanin complex according to the invention), showed no improvement in the protein content of the skin from the base line on day 90. The subjects having received the protein-melanin complex according to the invention, improved the protein content of their skin significantly on day 90 compared to the base line. Up to less than 15.9% of proteins captured in the Corneofix F 20 foils (P<0.001 was noted.

[0328] Taking the protein-melanin complex also significantly improved the protein content of the skin compared to the control on day 90 with a difference of 19.7% compared to the control (P<0.001).

[0329] 95.8% of the subjects of the active group showed an improvement in skin cohesion.

[0330] The results of this study are shown in FIG. 11.