WO2004032890A2

WO2004032890A2 - Method for obtaining an active principle stimulating suntan and resulting active principle

Info

Publication number: WO2004032890A2
Application number: PCT/FR2003/002942
Authority: WO
Inventors: Jean-Jacques Paufique
Original assignee: Societe Industrielle Limousine D'application Biologique (Silab)
Priority date: 2002-10-07
Filing date: 2003-10-07
Publication date: 2004-04-22
Also published as: FR2845285B1; AU2003299762A8; FR2845285A1; AU2003299762A1; WO2004032890A3

Abstract

The invention concerns a method for obtaining an active principle stimulating tyrosinase activity to increase melanin synthesis, characterized in that it comprises the following successive steps: solubilizing micronized powder of Citrus Aurantium bulcis in an aqueous solution at a rate of at least 50 g/l, simultaneous or successive enzymatic hydrolyses with at least two enzymes selected among the carbohydrase and protease families, separating by filtration, centrifuging or decantantion, and selecting by filtration flavonone-type polyphenols.

Description

PROCESS FOR OBTAINING A TANNING STIMULATION PRINCIPLE / ACTIVE, AND ACTIVE INGREDIENT OBTAINED

The present invention relates to a process for obtaining an active principle for stimulating tanning as well as the active principle obtained.

In the Western world, in particular, it is a particularly sought after factor of beauty, tanning or at least a tanned complexion. Indeed, tanning is an outward sign of good health, even if we know that these two parameters are in no way linked. The body needs a slight daily exposure for the synthesis of vitamin D in particular but a very low exposure is largely sufficient.

More specifically, tanning is rather a social sign which corresponds to a certain capacity to take vacations, in the sun and is the subject of one of the bases of social interviews.

As a result, there is a very high demand from consumers for methods or products that give the skin a more or less pronounced coloring. The skin, hair and body hairs are colored by a pigment, melanin. There are two types of melanin, eumelanin, brown in color and phaeomelanin, yellow-orange in color.

This pigment is synthesized by specific cells, melanocytes during a reaction, melanogenesis. The more this reaction is implemented, the more the melanin is synthesized and the more the skin becomes colored. In fact, this reaction is a defense reaction of the skin against the aggressions of the sun and more particularly of the ultraviolet rays of the sun.

We know that these UV rays have harmful effects on health because these radiations are more or less penetrating. The majority of UVB, around 70%, is stopped by the stratum corneum, but 20% penetrate to the mucous body and 10% reach the superficial dermis.

As for UVA, they pass through the stratum corneum and 20 to 30% reach the deep dermis. These UVA are less energetic but are nonetheless dangerous and this permanently, throughout the exposure.

Faced with these aggressions, the skin's defense reactions result in a high production of melanin but also a thickening of the stratum corneum, a release of antioxidants or even by the use of repair systems for the DNA. It is necessary to include in suitable cosmetic compositions active agents having an action against induced damage such as free radicals. In addition, if we can speed up the process of elanogenesis by these same active ingredients, we can reduce the exposure time for the same degree of tan. Apart from the tanning of the skin obtained by stimulating melanogenesis under the effect of ultraviolet radiation, there are other means.

Thus self-tanners are known which are creams or emulsions containing molecules such as DHA (dihydroxyacetone) or erytrulose (derivative of DHA), molecules which react with the amino acids of epidermal cells by developing a coloring of color and of variable intensity which resists water.

However, these self-tanners do not have sufficient durability for certain consumers and the brown coloring only remains thanks to regular applications of said cream or emulsion. Finally, there are cosmetic active pigments which promote the production of melanin and therefore help to speed up tanning.

The method and the active principle according to the present invention fall into this category. In this type of action, we know that the synthesis of melanin, in its different forms, results from a succession of enzymatic reactions which transform an amino acid, tyrosine, into melanin pigments.

The different essential steps include a hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA) by tyrosinase. This same enzyme also catalyzes the oxidation of DOPA to DOPAquinone.

Through successive reactions, this DOPAquinone in the presence of peptides such as glutathione and a specific amino acid cysteine, leads to the synthesis of phaeomelanin and of eumelanin.

The object of the present invention is to provide a process for obtaining an active principle which stimulates this activity of tyrosinase and therefore increases the synthesis of melanin.

Therefore, by stimulating this synthesis, pigmentation is strengthened and plays its protective role in the skin, ensuring better photoprotection.

Melanin absorbs more than 90% of the ultraviolet radiation that has passed through the stratum corneum. Melanin polymers are capable of absorbing UV photons and cellular lipids. Melanin constitutes free radical traps thus protecting the melanocytes.

The process for obtaining and the characteristics of the active ingredient obtained are now described in detail as well as the effects obtained measured. The attached figures represent:

- Figure 1A, a view of a histological section of an untreated, non-irradiated control explant, - Figure 1B, a view of a histological section of a positive control explant treated and irradiated with UVB,

- Figure 2A, a view of a histological section of an explant treated with placebo and not irradiated, - Figure 2B, a view of a histological section of an explant treated with placebo and irradiated with UVB,

FIG. 3A, a view of a histological section of an explant treated with the active principle of the present invention and not irradiated, and

- Figure 3B, a view of a histological section of an explant treated with the active ingredient of the present invention and irradiated.

1 / ACTIVE INGREDIENT 1-1 Method of obtaining:

The process for obtaining the active principle of the present invention comprises the following steps: - solubilization of micronized powder of Citrus Aurantium Dulcis (sweet orange, immature fruit) in an aqueous solution at a rate of at least 50 g / l.

- simultaneous or successive enzymatic hydrolysis with at least two enzymes chosen from the carbohydrase and protease families,

- separation of the soluble and insoluble phases by filtration, centrifugation or decantation, and

- selection by filtration of polyphenols of type f lavonones.

1-2 Dry matter rate •

The measurement consists of starting from a liquid volume and measuring the residual dry matter after drying.

Also, pour 4g of liquid on 4g of sand in an aluminum cup. The cup is placed in an oven at 105 ° C until a constant weight is obtained. The dry matter content obtained is between 5 and 200 g / 1, more particularly between 50 and 85 g / 1.

1-3 pH measurement.

The pH is measured by the potentio etric method, at room temperature. The pH is between 3.0 and 7.0, more particularly between 4.5 and 5.5.

1-4 Determination of phenolic compounds:

It is known that phenolic compounds form detectable colored complexes in the presence of potassium ferricyanide.

As the intensity of the coloring is proportional to the quantity of phenolic compounds, it suffices to compare with a standard range of hesperidin in the range, in this case 40 to 120 mg / l.

The content of phenolic compounds varies from 1.5 to 70 g / l, more particularly from

15 to 30 g / 1.

1-5 Characterization of total phenolic compounds: The characterization of phenolic compounds is carried out by HPLC,

High Performance Liquid Chromatography.

A C18 type column is used for this purpose, in particular that marketed under the name Alltech ltima C18., With a pre-column having the same characteristics. A solution of formic acid and acetonitrile is used as solvents.

A gradient between these two solvents makes it possible to separate the different phenolic compounds.

A detector at 280 nm allows the analysis and provides the following results:

- flavonones (of the hesperidin type) representing at least 35% and up to at least 50% of the total polyphenols.

- f lavones (luteolin-7-glucoside type)

- hydroxycinnamic acids (chlorogenic acid and ferulic acid type). 1-6 Total sugar content '•

The total sugar content is obtained by the DUBOIS method (DUBOIS M

& al, (1956) Analytical Chemistry, 28, N ° 3, p 350-356)

In the presence of phenol and concentrated sulfuric acid, the reducing sugars give a yellow-orange compound. After cooling, the Optical Density

DO is read in a spectrophotometer at 490 nm. A test curve is established with a standard range of a combination of 1/3 of mannose, 1/3 of glucose and 1/3 of galactose, with concentrations of 25 to 100 μg / l.

The values of the sugar level are read directly from the curve and in the case of the active principle according to the present invention, values of total sugars are obtained between 1.5 and 70 g / l, more particularly 15 and 30 g / l .

2 / IN VITRO EVALUATION OF THE PIGMENTING ACTIVITY

The principle consists in studying the pigmenting activity of the active principle according to the present invention on melanocyte cultures by measuring the tyrosinase activity and the level of synthesized melanin.

The operating protocol consists in inoculating B16F1 melanocytes in a complete medium in the presence of 0.5%, 1% and 2% (volume / volume) of the active principle and the mixture is left to incubate under suitable conditions. A positive control is carried out in parallel in the presence of -MSH (Melanocyte Stimuling Hormone), a hormone which stimulates melanogenesis.

The cells are trypsinized, counted and lysed.

After centrifugation, the tyrosinase is measured from the supernatant and the melanin level from the pellet. 2-1 Determination of tyrosinase activity

L-DOPA is added to this supernatant. After incubation, the OD optical density measured at 490 nm makes it possible to determine the activity of tyrosinase.

The results are as follows:

The results show a strong tyrosinase activity having values quite comparable with those of the hormone a-MSH, the effect being dose-dependent.

2-2 Determination of the melanin level;

This melanin level is determined relative to a standard range of synthetic melanin ranging from 0 to 500 μg / l. The levels of melanin synthesized by melanocytes are summarized in the following table:

There is a very large dose-dependent increase in the level of melanin under the action of the active ingredient.

3 / EVALUATION ON SKIN EXPLANTS OF THE PIGMENTING ACTIVITY Skin explants of phototype II (clear phototype) are chosen. These samples, kept in a suitable medium, are subjected to UVB irradiation at 0.05 J / cm ² , that is to say 1 to 2 times the minimum dose to cause erythema, this clear phototype.

Some of the explants are treated topically with 2 mg / cm ² of active principle according to the invention dosed at 5% for some, others are treated with a placebo medium and others, also called controls, remain only in the conservation medium.

The explants are irradiated every day.

The results are deduced from the observations of the histological sections treated with silver by the so-called Fontana-Masson technique.

It can be seen from the various figures that the explants of the controls treated with the placebo have substantially the same pigmentation as the non-irradiated control explants.

On the other hand, the pigmentation of the explants treated with the active principle according to the invention, at 5%, is important. This pigmentation is all the more visible as the explants are subjected to UVB.

4 / EVALUATION OF THE ACCELERATING TANNING EFFECT

The study relates to human volunteers, the aim being to quantify the accelerating effect of tanning of an emulsion containing 5% of active principle according to the present invention, during and after the exposure to UV. To this end, the tests are carried out against placebo on two areas of the forearms, the color of which is measured with a Chromameter, a device of the trade of the Minolta brand. The emulsion containing the active principle and the placebo emulsion are applied twice a day for 15 days then these areas are irradiated twice at two days apart then 2 days after each irradiation, the color of the areas retained is again measured. and processed. The parameters that can be measured:

- L • clarity (from dark to pale)

- a '• the color zone from green to red.

- b: the area from blue to yellow.

b is the parameter studied because it represents the yellow melanin skin pigmentation, characteristic of tanning.

The results obtained are summarized in the following table:

After the irradiations, a significant increase in the yellow skin pigmentation of the skin was demonstrated, which results in a more intense tan.

One of the cosmetic formulas used and therefore capable of being produced industrially comprises:

- Arachidyl alcohol / Behenyl alcohol / Arachidyl glucoside (Montanov 2002): 3%

- Isononyl isononanoate (Lanol 99): 2%

- Cetaryl octanoate (Lanol 1688): 10% - Polyacrylamide / Isoparaffin C13-14 / Laureth-7 (Sepigel 305): 2%

- Active ingredient - 5%

- Water: 77.5%

- Preservative: 0.5%. The active principle can be introduced into any suitable cosmetic support such as an emulsion, a cream, a lotion, an ointment or a dispersion.

Thus, a method of tanning the skin consists in applying a composition containing 0.1% and 10.0% of active principle to cause a large increase in melanin.

Claims

1. Method for obtaining an active principle for stimulating the activity of tyrosinase so as to increase the synthesis of melanin, characterized in that it consists in successively carrying out the following steps:

- solubilization of micronized powder of Citrus Aurantium Dulcis in an aqueous solution at a rate of at least 50 g / 1,

- separation of the soluble and insoluble phases by filtration, centrifugation or decantation, and - selection by filtration of flavonone type polyphenols.

2. Active principle obtained by implementing the method according to claim 1, characterized in that it has:

- a dry matter content of between 5 and 200 g / 1

- a pH between 3.0 and 7.0 - a level of phenolic compounds between 1.5 and 70 g / 1

- a level of at least 35% in flavonones of the polyphenols, and

- a total sugar content between 1.5 and 70 g / 1.

3. Active ingredient according to claim 2, characterized in that it has •

- a dry matter content between 50 and 85 g / 1 - a pH between 4.5 and 5.5

- a level of phenolic compounds between 15 and 30 g / 1

- a level of at least 50% in flavonones of the polyphenols, and

- a total sugar content between 15 and 30 g / 1.

4. A method of tanning the skin by applying the active ingredient according to claim 2 or 3, characterized in that it causes a large increase in melanin when said active ingredient is dosed between 0.1% and 10.0%.

5. Cosmetic composition intended to promote the tanning of the skin, characterized in that it comprises between 0.1% and 10.0% of active principle according to claim 2 or 3 in a galenical form such as a cream, a ointment, an emulsion or a dispersion.