WO2009080857A1

WO2009080857A1 - Method for obtaining melanins and use thereof

Info

Publication number: WO2009080857A1
Application number: PCT/ES2008/070219
Authority: WO
Inventors: Manuel Jaren Galan; Juan Garrido Fernandez; Juan Jose Negro Balmaseda
Original assignee: Consejo Superior De Investigaciones Cientificas
Priority date: 2007-12-21
Filing date: 2008-11-27
Publication date: 2009-07-02
Also published as: ES2322530A1; ES2322530B1

Abstract

The invention relates to a method for obtaining and subsequently quantifying melanin pigments, characterised in that it includes the following steps: initial alkaline treatment, separation of pheomelanins, production of a dry eumelanin concentrate by means of precipitate washing and subsequent centrifugation, dissolution of the dry eumelanin concentrate by means of time-controlled alkaline oxidation, and neutralisation of the solutions in order to obtain dry concentrates of pheomelanin and pseudopheomelanin. In particular, the invention describes the production of dry concentrates of natural pheomelanin, eumelanin and pseudopheomelanin from bird feathers or coloured mammal hair and to the use thereof as a raw material for industrial use in cosmetics (creams, make-up and dyes) and dermatology (for the treatment of diseases associated with depigmentation).

Description

PROCEDURE FOR OBTAINING MELANINS AND THEIR APPLICATIONS

SECTOR OF THE TECHNIQUE

The present invention is included within the chemical sector. Specifically, it describes the obtaining and subsequent quantification of purified melanins by means of a low cost technique.

Specifically, it describes the obtaining of dry concentrates of natural pheomelanin, eumelanin and pseudofeomelanin from bird feathers or colored mammalian hairs, which can be used as raw material for industrial purposes in cosmetics

(creams, makeup and dyes) and dermatology (for treatment of diseases that occur with depigmentation).

STATE OF THE TECHNIQUE Melanin is a general term that describes a family of quinol-phenolic pigments of diverse origin and chemical nature. Depending on their biological origin, the melanins are divided into three groups: microbial, animal and vegetable. There are also synthetic melanins. Animal melanins are the most common pigments in the vertebrate tegument (McGraw 2006), including the skin of humans (Jablonski 2006). From the chemical point of view, there are two basic types of melanins: pheomelanins and eumelanins, which give rise to the huge palette of melanin colors observed according to their concentration, relative proportions and contrast with depigmented areas. Pheomelanins contain sulfur and are perceived as reddish or blond colorations, both in the hair or skin of mammals and in the feathers of birds. Eumelanins are black or brown, and that is the color they confer to the integumentary structures.

Unlike carotenoids, exclusively vegetable pigments that must be ingested in the diet, and that are responsible for the brightest colors of birds, including yellow and red, the Melanins are synthesized by the body itself from amino acid precursors. They also differ in that while melanins can appear within the same pen as bands or points alternated with depigmented areas or with different degrees of mechanization, carotenoids occupy continuous regions of color. This feature allows infinite variants, and thus, for example, diurnal birds of prey (Order Falcon forms, with about 300 species), have distinctive and specifically melanin plumages and even numerous species among them have great individual variation. Among the feral pigeons (Columba livia) more basic types of coloration have been described than in any species of wild bird, and that phenotypic multiplicity again responds to multiple combinations of melanin tones.

As for the role of melanins in the tegument, it is essentially photoprotective, since it avoids the damage caused by the sun's ultraviolet (UV) radiation. They have, however, other potential functions: due to their structure they can protect cells and tissues from oxidative stress by trapping free radicals (McGraw 2006) and, in the case of bird feathers, it has been suggested that melanized feathers are more resistant to the deterioration by abrasion or mechanical breakage (Burtt 1986), and perhaps also to the attack of bacteria (Burtt and Ichida 1999) although this last aspect has been questioned recently (Grande and Cois 2004).

Despite the enormous prevalence of melanins in the vertebrate tegument, and the enormous interest aroused by color analysis in the context of evolutionary and behavioral studies (eg, HiII and McGraw 2006), there are no simple analytical procedures to identify and quantify them. Melanins have a complex structure and it has historically been very difficult to analyze them biochemically (Ito 2000). At present, chemical methods are used to extract them

(Ito and Fujita 1985) or enzymatic (Liu et al. 2003). Chemical methods, which are based on acid / base extractions and oxidative fragmentations and are considered very aggressive because they alter the molecular structure of the melanin, give rise to colorless metabolites that are subsequently separated by high performance liquid chromatography (HPLC) and quantified by UV detection. visible and electrochemical detector (Liu et al. 2003). The chemical methods of melanin extraction can be divided into two large groups: the first group consists of the extraction of melanins with suitable solvents, mainly alkalis, and subsequent elimination of the accompanying mixture. The second group of methods consists in extracting all other materials, with the exception of melanin, by acid hydrolysis and washing with a suitable solvent until melanin is obtained. On the other hand, enzymatic extraction methods are based on the use of proteases that degrade keratins that envelop melanins in the hair or feathers (Liu 2003).

As regards the plumages of birds, it has been described that in every melanized feather ugly and eumelanin co-exist in different proportions. For the determination and quantification of both types of melanins in feathers, the procedures of Ito and Fujita (1985) are still used with some subsequent modifications (Ito and Wakamatsu 1994, Wakamatsu and Ito 2002). Their methods are based on the HPLC analysis of the degradation products of the eumelanin after being oxidized and of the pheomelanin after being hydrolyzed. To calculate its concentration, however, it is necessary to use correction factors, since the conversion into degradation products is incomplete.

Because laboratory methods are relatively complex, until recently, only the proportion and concentration of eumelanins and pheomelanins in the plumage of 13 bird species has been determined (McGraw 2006). This contrasts with the identification and quantification of carotenoids in 150 bird species, and indicates that Melanization studies in birds are in their infancy. A simplified method with repeatable results is necessary and that has been the objective of the present invention. Based on the fact that the pheomelanin is easily solubilizable in alkaline medium but the eumelanin is insoluble in both acidic and alkaline medium, the procedure presented in this invention describes, for the first time, a method for obtaining the pheromellic fraction on the one hand and of the eumelanic fraction on the other, starting from an alkaline digestion from bird feather or mammalian colored hair. In addition, the possibility of solubilizing the insoluble concentrate of eumelanin in the form of pseudofeomelanin in alkaline medium, allows spectrophotometrically quantifying the relative concentration of pheomelanin and eumelanin without resorting to HPLC. On the other hand, this method allows to obtain dry concentrates of the three fractions, pheomelanins, eumelanins and pseudosfeomelanins, which can be used as a natural raw material for use in cosmetics or dermatology

DESCRIPTION OF THE INVENTION Brief Description The present invention describes, for the first time, a procedure for obtaining, from a raw material of zero utility such as the bird feather or certain colored mammalian hairs, of purified extracts of pheomelanin , eumelanin and pseudofeomelanin. Said procedure is simple and inexpensive, and allows the use of the extracts obtained both for photoprotective creams, as for makeup for cosmetic purposes or dermatological purposes in diseases that occur with depigmentation of the skin. In addition, the present invention presents a simple and reliable method of quantifying melanins. Therefore, one aspect of the present invention constitutes a method of separation, quantification and obtaining of concentrates. solids of pheomelanin, eumelanin and pseudoeumelanin based on the known solubility of the pheomelanins in alkaline medium and comprising the following steps: i) dissolution of a homogenate of free spine feathers in alkaline medium, preferably with 20% NaOH in water, in a bath with sonication, preferably at 6O ⁰ C, for at least 20 minutes, preferably 40 minutes, ii) separation of the soluble pheromellic fraction, preferably by centrifugation, of the precipitated eumelanic fraction, iii) obtaining a precipitate of eumelanin free of Pheomelanins, by washing, preferably with an aqueous solution of 20% NaOH and constant stirring, and subsequent centrifugation, iv) solubilization in alkaline medium, preferably 20% aqueous NaOH solution, of the precipitated eumelanic fraction, free of pheomelanins, by oxidative transformation in pseudofeomelanin, preferably with H2O2 at

30% in water, stopped by the action of an antioxidant, preferably 40% HNaS03 in water, and subsequent centrifugation, v) relative quantification of the pheomellanic and pseudofeomellanic solutions spectrophotometrically at 450 nm, and vi) obtaining solid pheomelanin concentrates, and pseudofeomelanin by neutralization, preferably with HCI, and subsequent centrifugation.

Another aspect of the invention constitutes the use of solid concentrates of pheomelanin, eumelanin and pseudofeomelanin obtained by the process of the invention, as raw material for industrial purposes for the preparation of cosmetic compositions.

(creams, ointments or aerosols) or dermatological (in diseases that They run with depigmentation) or sunscreens against UV radiation.

The cosmetic compositions thus obtained may comprise other products, for example: skin makeup, eyelashes, eyebrows, lips or nails, hair or hair dyes, tattoo dyes, or any other cosmetic product.

Detailed description

The present invention faces the problem of offering a simplified and reproducible procedure for the determination of melanins, and more specifically for the determination of the basic types of melanins, eumelanins and pheomelanins, and a method of extracting and obtaining both types of melanins in solid state.

The present method for determining eumelanins and pheomelanins is based on an initial alkaline treatment to separate pheomelanins, and a time-controlled alkaline oxidation of insoluble eumelanins to transform them, stoichiometrically, into soluble pseudofeomelanins in alkaline medium. Subsequently, the relative concentration of pheomelanins and eumelanins is measured, spectrophotometrically, by analyzing the former directly and the latter from their oxidatively obtained soluble product, without resorting to HPLC. It is important to highlight that there is currently no method in which the feomellanic and eumelanic fractions are obtained separately starting from an alkaline digestion from a bird's feather or colored mammalian hair.

Specifically, the procedure for the initial separation of the pheomelanins begins with an alkaline digestion of the raw material used by the addition of 20% NaOH dissolved in water. Digestion is carried out in a bath with controlled temperature sonication, preferably at 6O ⁰ C, for 40 minutes with periodic agitation of the sample. Once the alkaline digestion is finished, the soluble pheromellic fraction is separated from the precipitated eumelanic fraction, preferably by centrifugation. More specifically, the sample is centrifuged for 15 minutes at 17500 g and 4 ⁰ C, removing the supernatant consisting of the fraction of solubilized pheomelanins and the material solubilized after digestion, formed essentially by free amino acids from the keratin of the pen. The precipitate is reserved for the subsequent study of the eumelanic fraction.

The determination of the colored solution, which constitutes the pheromellic fraction, is performed in a spectrophotometer. More specifically, the determination is made by means of the absorbance measurement at 450nm, subtracting the one obtained at 600nm against a blank of 20% NaOH aqueous solution. The result obtained is divided by the original sample weight to express the concentration of pheomelanins as UA / g of raw material.

For the determination of the concentration of the precipitated eumelanic fraction, obtained after the digestion with NaOH, first a washing is carried out to eliminate possible remains of pheomelanins. Specifically, 1 ml of 20% NaOH in water is added to the precipitated eumelanic fraction, stirred, until a suspension is obtained, and centrifuged at 1750 Og for 15 minutes. The supernatant is neglected and the washing process is repeated a second time. The resulting precipitate, which is dried in an oven at 6O ⁰ C, constitutes the eumelanic fraction, insoluble under alkaline conditions, free of pheomelanins. The determination of the content of eumelanins, once the precipitate is clean of remains of solubilized pheomelanins, requires prior solubilization of the precipitate. This solubilization is carried out through an oxidation process that begins with the addition, to the precipitate of eumelanins, of 1 mL of 20% NaOH in water, remaining under constant agitation conditions until obtaining the complete resuspension of the precipitate. To this suspension are added subsequently 0.02 ml_ of 30% H2O2 in water, the mixture is stirred vigorously and immersed in a bath at 6O ⁰ C with sonication for 10 minutes counted from the moment of the addition of the hydrogen peroxide. The oxidation reaction of the eumelanins is stopped by the addition of an antioxidant, specifically 0.050 ml_ of 40% HNaS03 in water, and stirring. After the oxidation reaction, centrifugation and spectrophotometric measurement of the solution of eumelanins transformed by oxidation in pseudofeomelanins are carried out under identical conditions to those described above for the pheromellic fraction, the results also being expressed as UA / g pen.

An additional feature of the present invention is the obtaining of dry concentrates of pheomelanin and pseudofeomelanin from a process of neutralization of the solutions used for quantifications. Specifically, the solutions of pheomelanin and pseudofeomelanins are neutralized, separately, with two-stage hydrochloric acid (HCI) using first 20% HCI until reaching a pH close to 8-9 units and subsequently, with 0.1 N HCI, The solution is neutralized until reaching a pH between 6-7 units. The neutralized solution is allowed to cool and centrifuged at 1750Og for 20 minutes. The supernatant obtained is neglected and the precipitate, once washed by resuspension with deionized water and subsequent centrifugation under identical conditions, is finally dried in an oven at 6O ⁰ C. The precipitate obtained is constituted by the pheromellanic or pseudofeomellanic fractions respectively.

The precipitated solids (feomelánico, pseudofeomelánico and eumelánico) are intensely colored and interpose easily with diluents by mechanical dispersion. They are not soluble in hydrophilic or lipophilic media, they are simply dispersible in solids or semi-solids of high viscosity, giving different colors depending on their concentration and the original coloration of the excipient. The proposed determination methodology represents a remarkable advance in the field of melanins. With simple and inexpensive treatments, the content of pheomelanins can be directly quantified and indirectly that of eumelanins by correlation with the direct measurement of pseudofeomelanins. The quantification is approximate since the degree of purity that is reached in the ugly and eumelanin precipitates that allow to establish the extinction coefficient for its exact quantification has not yet been established. In any case, obtaining this parameter is not vital since the results obtained by absorbance measurement divided by the sample weight will only be lacking in multiplying by this factor, and for the general purpose of measuring the melanin content that is compare between species, the extinction coefficient would be simplified by appearing in the two members of the equation. As an additional advantage, this methodology allows to obtain dry concentrates of pheomelanin, pseudofeomelanin and eumelanin that can be used as a natural raw material for industrial purposes in cosmetics or dermatology. Melanins are the natural pigments of the skin and one of its functions is photoprotection. By obtaining them in isolation, it is possible to achieve, by combining them with different proportions, all existing skin tones, and could be used in the form of cream, ointments or aerosols as a second skin that will perform the photoprotective function against UV radiation , without this function having to suffer our own skin (for example, for sunscreens). At the same time, the wide range of natural shades that can be achieved would also allow its use as a makeup agent for the natural color of the skin, both for cosmetic purposes (for example, such as skin makeup, eyelashes, eyebrows or nails, hair dyes. ..) as with dermatological purposes in diseases that occur with depigmentation of the skin. DESCRIPTION OF THE FIGURES

Figure 1.- It is shown how the process of solubilization of pheomelanins takes place as a function of time. Figure 2. Verification of the linearity between the content of pheomelanins in the samples and the absorbance measured in three different bird species.

Figure 3.- Detail of electron microscopy of the solid obtained after the separation of the pheromellic fraction and washing with water from the precipitate. Set of melanosomes containing eumelanin. Figure 4.- Evolution of the insoluble matter content and color of an eumelanic nature in samples treated under conditions of alkaline oxidation in the presence and absence of antioxidant.

Figure 5.- FT-IR spectrum of pheomelanin, eumelanin and its alkaline oxidation product, pseudofeomelanin. Figure 6.- Verification of the linearity between the content of eumelanins in the samples and the absorbance measured in three different bird species.

EXAMPLES OF REALIZATION

Example 1.- Methodologies of extraction, quantification and precipitation of pheomelanins.

1.1. -Separation of pheomelanins by solubilization in alkaline medium

The procedure set up is based on the known solubility of the pheomelanins in alkaline medium. Rooster pen of black coloration is used in the base and dark golden coloration in the tips. Cut into small pieces of approximately 1 g of rachis-free feathers and discard and mix to homogenize. Twenty samples of approximately 5 mg are taken and placed in Eppendorf tubes, 1.5 ml_ capacity and hermetically sealed, and 1 ml_ of 20% NaOH are added simultaneously. The tube set is Enter in a thermostated sonication bath at 6O ⁰ C. Two tubes are removed every 5 minutes, to minimize re-extraction after the established time, they are immersed in an ice bath and centrifuged at 4 ⁰ C and 17.50Og for 15 minutes . Next, the absorbance of the supernatant at 450 nm is measured by subtracting the absorbance measured at 600 nm against a blank of 20% NaOH accusing solution, expressing the results in absorbance units per gram of pen of each sample. Figure 1 shows how the process of solubilization of pheomelanins takes place as a function of time. Since t = 0 there is a gradual increase in the solubilized color that stabilizes from t = 20. From this moment there is no increase in the amount of solubilized color, which remains constant throughout the subsequent time. The constancy in time of the amount of color extracted guarantees that the extraction has been completed and that the dissolution of pheomelanins is stable and no degradation of the color occurs under the solubilization conditions.

With the application of this same procedure to structurally different feathers, it has been observed that in thick and hard feathers such as vulture, the alkaline digestion is slower, while in small and low compaction feathers, the digestion of keratins and The solubilization of the pheomelanins occurs rapidly. For all the samples studied, the solubilization time has been in all cases less than 30 minutes, so it has been set as a digestion time 40 minutes, time in which there is a guarantee that the pheomelanins have been completely solubilized in all samples.

The solubilization of pheomelanins is practically completed in the first twenty minutes (Figure 1), however, the reaction time is extended up to 40 minutes to achieve the perfect disintegration of the material of the pen. In pens with eumelanic content, this factor is important for subsequent treatments since the previous digestion of 40 minutes completely releases the melanosomes making them accessible to subsequent oxidative treatment (Figure 2).

The amount of color extracted is a function of the weight used, following the Lambert-Beer law, in a wide weight range as long as the rest of the parameters set for the extraction remain identical. Using feathers of three species with ugly and eumelanic content, in Figure 2 it can be seen that the linearity between featherweight and absorbance obtained has a correlation coefficient (r) in all three cases greater than 0.98.

The reproducibility of the measures carried out has been verified using feathers of three different species, griffon vulture (Gyps fulvus), bearded vulture (Gypaetus barbatus) and elanium (Elanus caeruleus). In each species feathers have been taken and a homogenate of which four samples have been taken. In each sample, the pheomelanin content has been determined, expressing the results in UA / g and the means, standard deviation, confidence interval and percentage of variability in the determination have been calculated. The results are shown in table 1.

Table 1. Reproducibility of the process of extraction and quantification of pheomelanins

The variability between replicates, when using griffon vulture feathers as raw material, becomes 3.35%, which indicates that the methodology used has a high reproducibility. However, in some samples this variability becomes up to 8%. The fact of that the variability of the quantification method is a function of the sample, clearly shows that the homogeneity is a critical factor to obtain a good reproducibility. The variability that the raw material itself has been verified, for this purpose the feomelanin content in the basal, middle and distal part of the pen has been determined in the primary vulture feather pen. The results are shown in Table 2.

Table 2. Variability in the content of pheomelanins in different parts of the griffon vulture feather

In each area of the pen there is a different feomellan content. Therefore, it is not possible to speak of a feomellanic content in a given bird species, or even in a certain bird's feather. The concentration varies depending on the species of bird, the type of feather and even the area of the feather. The applied methodology has, however, an acceptable repeatability and to apply it it is necessary to specify very well what raw material is the one that is going to be analyzed because no comparable results will be obtained if the analysis is performed on a specific area of different feathers or on a Homogenized pens in which the different parts are included.

1.2.- Obtaining solid-state pheomelanins

To obtain the solid state pheomelanin concentrate, the alkaline solution containing the solubilized pheomelanins is brought to neutrality with 20% HCI until the solution reaches a pH alkaline close to neutrality and it is finished accessing to the point of neutrality using HCI 0.1 N to be able to adjust more accurately that the final pH does not fall below 6. Under neutral conditions, the pheomelanins become insoluble forming a fine suspension that separates by centrifugation at 1750Og for 20 minutes and temperature of 4 ⁰ C. The suspension is formed by a fine powder with little tendency to precipitate, so that sometimes the precipitation is not complete and the supernatant retains some color. In this case it may be necessary to repeat the centrifugation process and even in more energetic conditions to achieve complete precipitation. The supernatant is removed and the precipitate is resuspended in deionized water, subsequently centrifuged in identical conditions to the previous ones and the washing process is repeated a second time finally obtaining a precipitate enriched in pheomelanins.

Example 2.- Development of methodologies for extraction and quantification of eumelanins.

2.1.- Obtaining the solid precipitate of eumelanins

In pens with eumelanic content, after the treatment of solubilization of pheomelanins, the precipitate retains an intense black color consisting of melanosomes containing eumelanin (Figure 3). The obtained residue is washed and centrifuged up to three times with 20% aqueous NaOH solution to remove the remains of colored solution from the first extraction. Under these conditions a new re-extraction does not occur and the color that is observed are remains of the first extraction that impregnate the residue and generally, especially in feathers of low feomellanic content, after the first wash, the rest is absolutely colorless, constituting the eumelanic precipitate free of pheomelanins. 2.2.- Solubilization of the solid precipitate of eumelanins in the form of time-controlled alkaline oxidation.

The solubilization of the solid eumelanic residue, free of pheomelanins, is carried out by an oxidative treatment in alkaline medium. For this, the resuspension of the eumelanic precipitate in 1 ml of 20% NaOH solution is carried out first by stirring and subsequent sonication in an ultrasonic bath. After resuspension, the oxidative treatment is carried out by adding 0.02 ml_ of 30% H2O2. The reaction is carried out in an ultrasonic bath at 6O ⁰ C and allowed to run for 10 minutes.

To study the processes that occur during the time after the addition of H ₂ O ₂ , a rooster pen with ugly content and eumelanins is used. In order to be able to track the solid material solubilization process, the previously described methodology is modified promptly by scaling it twenty times. 10 g of cannon-free feather are chopped and undone. 45 0.1 g samples are taken, each deposited in 50 mL centrifuged tubes previously tared and the pheomelanin removal treatment is applied using 20 mL of 20% NaOH in each of the samples. After the extraction of pheomelanins, the eumelanic precipitate is washed repeatedly with 20% NaOH and centrifuged to remove salts and all soluble material. The residue subsequently washed is dried in an oven and the weight of each of the samples is determined to know the content of the eumelanic concentrate from which it is started before starting the oxidative treatment. For consistency with the study of pheomelanins, measures of eumelanin content are also expressed in absorbance units per gram of initial pen.

The dried residue is resuspended in 20 mL of 20% NaOH. At t = 0 0.4 mL of 30% H ₂ O ₂ is added to all samples and the samples are immediately deposited under the reaction conditions.

Three samples are periodically removed to which 1 mL of 40% HNaS03 and stirred vigorously. The sample is centrifuged at 1750Og for 15 minutes at 4 ⁰ C and the supernatant is removed for spectrophotometric measurement. The precipitate is washed repeatedly with deionized water and dried in the oven to determine its weight. Subsequently, t = 10, weight determination is not performed because there is no precipitate in any of the samples. Until reaching point t = 10 minutes, 15 samples have been removed from the reaction conditions. At t = 10 minutes, 1 ml of 40% HNaS03 is added to fifteen of the thirty samples that are maintained under the reaction conditions, stirred vigorously and returned to the bath. With these samples the study of the progress of the reaction will be carried out in the presence of the antioxidant while maintaining the reaction conditions. In the rest of the samples nothing is added and the reaction is allowed to pass to know what happens with the eumelanins formed in the absence of the oxidizing agent.

Above t = 10, six samples are periodically removed from the bath, three of them corresponding to samples to which antioxidant has been added and another three corresponding to the group in which the antioxidant has not been previously added and which are added in the moment in which the sample is taken from the bathroom. Figure 4 summarizes everything that occurs during the reaction time.

After the addition of H2O2 there is an immediate solubilization of the color, as well as a sharp decrease in the amount of solid material. The rate of color solubilization slows down as the amount of solid material in suspension decreases. The maximum solubilized color is reached after 5-7 minutes of reaction. From that moment there appears a slight decrease in the absorbance of the solubilization, indicative of the fact that the oxidizing medium in addition to allowing the solubilization of the originally eumelanic color, also causes its destruction, so it must be taken into account that there is a parallel reaction of destruction of color that coexists while its solubilization occurs and that is done visible once the contribution of new color by solubilization is less than oxidative destruction. In the ten minute the reaction has unfolded between that which occurs in the presence of antioxidant and in the absence of this. After the addition of sulphite, there is no destruction of the color in any sample, while in the samples in which no sulfite has been added, a progressive decrease in the amount of color per pen weight appears, clearly indicative of the existence of a reaction of degradation of the solubilized product that has been previously generated. The degradation reaction that appears clearly follows a kinetics of order 0, which indicates that there is no limitation of the oxidizing substrate to degrade the solubilized color. It can be ensured that the degradation reaction has existed in the reaction medium from the moment the eumelanins begin to oxidize and solubilize, which occurs immediately after t = 0. The parallel degradation reaction is no longer masked by

The melanin solubilization reaction just at the moment when they are depleted, that is, at t = 10, and after this point it appears as the only reaction. The isolation of the degradation reaction above t = 10 allows to extrapolate the incidence of the degradation during the time in which the reaction is masked by the solubilization of oxidized eumelanins.

Extrapolating the values measured during the reaction of destruction of oxidized eumelanins between the time t = 10 and t = 20, with the theorists that would exist at t = 0 allow to know, that if there had been no degradation reaction in the interval between t = 0 and t = 10, the measured absorbance value should have been 152.

On the other hand, in the samples in which the antioxidant has been added, the degradation reaction has been paralyzed, and the color that is measured is stable, at least within the 20 minutes that the reaction has been allowed to pass. The antioxidant stabilization procedure is effective and allows for a reasonable timeframe for the spectrophotometric measurement, knowing that no degradative reaction is occurring. However, the measurement carried out in this way is affected by the error of the under-quantification of oxidized eumelanins by the destruction that has occurred of them during the period necessary for the solubilization of the oxidized eumelanins. In fact, the measured value is 136, when according to the extrapolation carried out of the degradation line it should have been 152. The simultaneous study of both reactions allows to know the real value that is measured and the theoretical value that should have been measured, by Io that you can calculate the conversion factor between one and the other, which is 1, 119. That is, the actual measurement that is made at t = 10 must be multiplied by 1, 119 to obtain the value that theoretically the initial sample would have given if there had not been a parallel degradation reaction.

The quantification of eumelanins cannot be carried out directly because they are insoluble, but by means of oxidative treatment, the original eumelanin is transformed into another compound colored and soluble in alkaline medium that can be quantified as in the case of pheomelanin and correlate the value obtained with the eumelanic content original. The characteristics of the product formed are partly reminiscent of those of the pheomelanin, such as its solubility in alkaline medium and show an apparent coloration with golden brown tones, very different from the intense black color that intact eumelanins have.

The analysis of the Fourier Transformed Infrared (FT-IR) spectrum of oxidized pheomelanins, eumelanins and eumelanins (Figure 5) shows that the eumelanins and pheomelanins differ in the wave number zone between 1000 and 1400 and in Ia which usually absorb functional groups such as sulfonides or sulfones. After the oxidizing treatment of the eumelanins in alkaline medium, a new compound is formed that maintains approximately the original eumelanin spectrum but the differences found in the wave number zone between 1000-1400 and the product spectrum are attenuated obtained, it becomes much more similar to that of pheomelanins, without becoming identical, so that the original eumelanin seems to have been spectrally transformed into a compound similar to pheomelanin, with which it also shares other physical and chemical properties, such as solubility in alkaline medium and apparent color. This new compound formed by oxidation of eumelanins, and similar in certain properties to pheomelanin, has been called pseudofeomelanin.

Due to the transformation that eumelanin undergoes, it is possible to quantify it spectrophotometrically as a pseudofeomelanin at 450 nm by subtracting the absorbance at 600 nm and dividing the absorbance measured by the pen weight used. The sample taken at = 10 and immediately stabilized with HNaSO ₃ is the one suitable for quantifying pseudofeomelanin by measuring its absorbance. The result obtained must be corrected by multiplying the value obtained by 1, 119 to contemplate the losses caused by co-oxidation.

The methodology for measuring the content of eumelanins by quantification of their corresponding pseudofeomelanins formed by alkaline oxidation is adequate within a wide range of concentration, since the amount of product that is formed is directly correlated with the amount of raw material used following the law from Lambert-Beer. Using feathers of three species of birds of very different melanin content in Figure 6, it can be observed that the measurement procedure perfectly correlates the melanin content with the weight of the feather. Likewise, the methodology shows a very acceptable repeatability (Table 3). Feathers of the three same species used for the study of linearity in the measure are used and it is obtained that, as in the case of pheomelanins, depending on the sample, this percentage can range between 3.74% and 7.92%. Obviously, this variability among replicates is the result of a heterogeneity in the raw material used. In a study similar to that carried out with pheomelanins, it is found that, depending on the portion of pen used, the content of eumelanin, quantified as pseudofeomelanin, varies, and within each portion the percentage of variability narrows to below 5% in all cases (Table 4).

Table 3. Reproducibility of the process of extraction and quantification of eumelanins in the form of pseudofeomelanins

Table 4. Variability in the content of quantified eumelanins as pseudofeomelanins in different parts of feather of Griffon Vulture

Example 3.- Obtaining solid state pseudofeomelanins

As in the case of the pheomelanins, it is possible to obtain a concentrate of pseudofeomelanins, by precipitation in neutral medium, following the same procedure of adding concentrated HCI and subsequently diluted until reaching a pH between 6 and 7. Under these conditions the Pseudofeomelanins form a fine suspension that is separated by centrifugation. BIBLIOGRAPHIC REFERENCES

- Burtt, E. H. 1986. An analysis of physical, physiological and optical aspects of avian colouration with emphasis on Word warblers. Ornithol Monogr. 38: 1-126.

- Burtt, E. H. and Ichida, J. M. 1999. Occurrence of feather degrading battery in the plumage of birds. Auk 116: 364-372. - Grande, J. M., Negro, JJ. & Torres, MJ. The evolution of bird colouration. A role for feather-degrading bacteria? Ardeola 51: 375-383.

- HiII, G. E. & KJ. McGraw (Eds.). 2006.Bird Coloration VoI I. Mechanisms and Measurements. Harvard University Press. London, England

- Ito, S. & Fujita, K. 1985. Microanalysis of eumelanin and phaeomelanin in hair and melanomas by chemical degradation and liquid chromatography.

Analyt Biochem. 144: 527-536.

- Ito, S. Wakamatsu, K. 1994. An improved modification of permanganate oxidation that gives constant yield of pyrrole-2,3,5-tricarboxylic acid. Pigment CeII Res. 1: 141-144. - Jablonski, N. 2006. The Skin. A natural history. California University Press.

- Liu, Y. et al. 2003. Comparison of the structural and physical properties of human hair eumelanin following enzymatic or acid / base extraction. Pigment CeII Res. 16: 355-365. - McGraw, KJ. 2006. Mechanics of melanin-based coloration. Pp. 243-294 in Bird Coloration VoI I. Mechanisms and Measurements. G. E. HiII and KJ. McGraw (Eds.). Harvard University Press. London, England

- Wakamatsu, K. & Ito, 2002. Advanced chemical methods in melanin determination. Pigement CeII Res. 15: 174-183.

Claims

1.- Procedure for obtaining melanin pigments characterized in that it comprises the following steps: a) initial alkaline treatment, b) separation of the pheomelanins, c) obtaining the dry concentrate of eumelanin by washing the precipitate and subsequent centrifugation, d) solubilization of the dry concentrate of eumelanin by time-controlled alkaline oxidation, and e) neutralization of the solutions to obtain dry concentrates of pheomelanin and pseudofeomelanin.

2. Method according to claim 1, characterized in that the initial alkaline treatment of a) is carried out with NaOH preferably 20% in water, in a bath with sonication, preferably at 6O ⁰ C, for at least 20 minutes, and preferably during 40 minutes with periodic agitation of the sample.

3. Method according to claim 1, characterized in that the separation of the pheomelanins from b) is carried out by centrifugation, preferably for 15 minutes at 17500 g and 4 ⁰ C, the supernatant containing the fraction of solubilized pheomelanins being removed.

A - Procedure according to claim 1, characterized in that the washing for

The obtaining of eumelanins in solid state of c) is preferably performed by resuspension in 20% NaOH and subsequent centrifugation at 17500 g for 15 minutes repeating the operation three times. 5. Method according to claim 1, characterized in that the alkaline oxidation of the precipitated eumelanic fraction of d) is preferably carried out by adding 30% H ₂ O ₂ in water to the eumelanic precipitate resuspended in 20% NaOH, sonication at 6O ⁰ C for 10 minutes of Ia sample, and stopping the reaction by adding an antioxidant, preferably 40% HNaS03 in water.

6. Method according to claim 1, characterized in that the obtaining of the solid concentrates of pheomelanin and pseudofeomelanin of e) is carried out by means of a neutralization process of the solutions, preferably with 20% HCI followed by 0.1 N HCI, and subsequent centrifugation .

7. Method according to claims 1 to 6, characterized in that the raw material used consists of bird feathers or colored mammalian hairs.

8. Melanin quantification process characterized in that it comprises obtaining melanins according to claims 1 to 7 and its spectrophotometric measurement, preferably by absorbance measurement at 450 nm by subtracting the one obtained at 600 nm from a blank of aqueous NaOH solution at 20%, and divided by the weight of the original sample to express the concentration of eumelanins, in the form of pseudofeomelanins, and of pheomelanins as UA / g of raw material.

9. Use of the melanins obtained according to claims 1 to 7 for the preparation of cosmetic compositions.

10. Use of the melanins according to claim 9 wherein the cosmetic composition comprises any of the following products: skin makeup, eyelashes, eyebrows, lips or nails, hair or hair dyes, tattoo dyes, or any other product of cosmetic use.

11. Use of the melanins obtained according to claims 1 to 7 for the preparation of sunscreens against UV radiation.

12. Use of the melanins obtained according to claims 1 to 7 for the preparation of compositions for dermatological purposes in diseases that occur with skin depigmentation.