FR2845285A1 - Producing an agent for enhancing melanin synthesis, useful for skin tanning, comprises isolating a flavonone fraction from micronized sweet Seville orange - Google Patents

Abstract

Producing an agent (I) for enhancing melanin synthesis by stimulating tyrosinase activity comprising dissolving micronized sweet Seville orange (Citrus aurantium dulcis) in an aqueous medium in an amount of at least 50 g/l, hydrolyzing the solution with at least two enzymes selected from carbohydrases and proteases, separating the soluble and insoluble phases by decantation, filtration or centrifugation, and selecting flavonones by filtration, is new. Independent claims are also included for: (1) (I) produced as above with a solids content of 5-200 g/l, a pH of 3-7, a phenolic compound content of 1.5-70 g/l (including at least 35% flavonones) and a total sugar content of 1.5-70 g/l; (2) a cosmetic composition for promoting skin tanning, comprising 0.1-10% (I) in a dosage form such as a cream, ointment, emulsion or dispersion.

Description

PROCEED OF OBTAINING AN ACTIVE PRINCIPLE FOR STIMULATING TANNING AND

  The present invention relates to a process for obtaining an active principle of

  stimulation of the tan as well as the active ingredient obtained.

  In the western world, in particular, it is a factor of beauty

  particularly sought after, 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 B in particular but a very

  low exposure is more than enough.

  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

basics of social interviews.

  As a result, there is a very high demand from consumers for methods or products which give the skin a more or less

less pronounced.

  The skin, hair and body hairs are colored by a pigment, melanin. And there are two types of melanin, brown eumelanin and

  yellow-orange phaeomelanin.

  This pigment is synthesized by specific cells melanocytes during

of a reaction, melanogenesis.

  The more this reaction is carried out, 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. It is known that these UVs have harmful effects on health because these radiations are more or less penetrating. The majority of UVB, of the order of 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 nonetheless remain dangerous and this permanently, throughout the exposure.

  Faced with these attacks, 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

DNA repair.

  It is necessary to include in suitable cosmetic compositions active agents having a

  action against induced damage such as free radicals.

  be more, if we can accelerate the process of melanogenesis by these same

  active, we can reduce the exposure time for the same degree of tan.

  Apart from the tanning of the skin obtained by stimulation of 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 bHA (dihydroxyacetone) or erytrulose (derivative of DHA), molecules which react with the amino acids of epidermal cells by developing a coloring of color and color. 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 the said cream or emulsion.

  Finally, there are pigmentogenic cosmetic active ingredients which promote

  production of melanin and therefore help to speed up tanning.

  The method and the active principle according to the present invention are positioned in

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 (bOPA) by tyrosinase. This same enzyme also catalyzes the oxidation of DOPA to tbOPAquinone.

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

  phaeomelanin and eumelanin.

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

melanin synthesis.

  Therefore, by stimulating this synthesis, pigmentation is strengthened and plays its

  protective role in the skin by ensuring better photoprotection.

  Melanin absorbs more than 90% of the ultraviolet radiation which has passed through the stratum corneum. Melanin polymers are capable of absorbing UV photons and cellular lipids. Melanin is a trap for

  free radicals 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 appended figures represent: FIG. 1A, a view of a histological section of an untreated, unirradiated control explant, FIG. 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 10 of the present invention and irradiated.

1 / ACTIVE INGREDIENT

  1-1 Process for obtaining: The process for obtaining the active principle of the present invention comprises the following stages: - solubilization of micronized powder of Citrus Aurantium Dulcis (orangette

  sweet, immature fruit) in an aqueous solution at a rate of at least 50 9 / I.

  - 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 flavonone type polyphenols.

1-2 Dry matter rate:

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

  Also, pour 49 of liquid on 49 of sand into an aluminum cup.

  The cup is placed in the 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 / l.

1-3 pH measurement:

  The pH is measured by the potentiometric 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: We know that phenolic compounds form in the presence of ferricyanide

  potassium detectable color complexes.

  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 / I.

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

at 30 9 / I.

  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 Altech Altima 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.

  - flavones (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 bUBOIS method (DUBOIS M & al, (1956) Analytical Chemistry, 28, N 3, p 350-356) In the presence of phenol and acid concentrated sulfuric, the reducing sugars 5 give a yellow-orange compound. After cooling, the) optical unit 1) 0DO is read with 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 p / 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 sugars are obtained

  totals between 1.5 and 70 g / I, more particularly 15 and 30 g / I.

  2 / IN VITRO EVALUATION OF 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 activity

  tyrosinase and the level of melanin synthesized.

  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 produced in parallel in the presence of (x-MSH (Melanocyte Stimuling Hormone),

  melanogenesis stimulating hormone.

  The cells are trypsinized, counted and lysed.

  After centrifugation, the tyrosinase is assayed from the supernatant and the level of

melanin from the pellet.

  2-1 Assay of tyrosinase activity LbOPA is added to this supernatant. After incubation, the optical density DO

  measured at 490 nm makes it possible to determine the activity of tyrosinase.

  The results are as follows:% 0 tyrosinase activity / control Control + 103 + 21 Active ingredient dosed at 0.5% + 59 _ 10 Active ingredient dosed at 1.0% + 64 + 9 Active ingredient dosed at 2.0 % + 74 + 12 The results show a strong tyrosinase activity having values entirely 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 in relation to a standard range of melanin

  synthetic ranging from 0 to 500 pg / l.

  The levels of melanin synthesized by melanocytes are summarized in the following table: Melanin level synthesized in% / control Control ca-MSH + 630 _ 92 Active ingredient dosed at 0.5% + 152 _ 41 Active ingredient dosed at 1.0 % / + 274 + 65 Active ingredient dosed at 2.0% + 309 42

  There is a very large dose-dependent increase in the level of melanin 15 under the action of the active principle.

  3 / EVALUATION ON EXPLANTS bE SKIN bE PIGMENTING ACTIVITY

  One chooses explants of skin of phototype II (clear phototype).

  These samples, kept in a suitable medium, are subjected to UVB irradiations at 0.05 J / cm2, that is to say 1 to 2 times the minimum dose to cause a

erythema this clear phototype.

  Some of the explants are treated topically with 2 mg / cm2 of active ingredient 5 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 10 treated with silver by the so-called FontanaMasson technique.

  It can be seen in the various figures that the explants of the controls treated with the placebo have substantially the same pigmentation as the explants controls

not irradiated.

  On the other hand, the pigmentation of the explants treated with the active principle according to the invention, at 50 °, is important. This pigmentation is all the more visible

  that the explants are subject to UVB.

  4 / EVALUATION OF THE ACCELERATOR EFFECT OF TANNING 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 UV exposure.

  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, an

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 and then 2 days after each irradiation, the

  color of selected and treated areas.

  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 melanic yellow pigmentation

  skin, characteristic of tanning.

  The results obtained are summarized in the following table: After irradiation After 2nd irradiation Active principle at 5% 0.61 1.26 Placebo 0.52 0.94 Pigmenting effect / Placebo + 17% + 34% After irradiation, we have highlighted a significant increase in yellow cutaneous pigmentation of the skin 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

  than an emulsion, cream, lotion, ointment or 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. he

Claims (5)

  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 bulcis in an aqueous solution at a rate of at least 50 g / I, - 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 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 / I - a pH of between 3.0 and 7, 0 - a level of phenolic compounds between 1.5 and 70 g / I   - a level of at least 35% in flavonones of the polyphenols, and - a total sugar content of between 1.5 and 70 g / l.   3. Active ingredient according to claim 2, characterized in that it has: - a dry matter content of between 50 and 85 g / I 20 - a pH between 4.5 and 5.5 - a content of phenolic compounds between 15 and 30 g / I - a level of at least 50% in flavonones of the polyphenols, and   - a total sugar content of between 15 and 30 g / l.   4. A method of tanning the skin by applying the active principle 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, an ointment,   an emulsion or a dispersion.