ES2322530B1 - PROCEDURE FOR OBTAINING MELANINS AND ITS APPLICATIONS. - Google Patents

Abstract

Procedure for obtaining melanins and their Applications.

The present invention relates to a procedure for obtaining and subsequent quantification of pigments of melanins, characterized in that it comprises the stages of: initial alkaline treatment, separation of the pheomelanins, obtaining the dry concentrate of eumelanin by washing the precipitate and subsequent centrifugation, solubilization of dry concentrate of eumelanin by alkaline oxidation controlled over time, and neutralization of solutions for Get dried concentrates of pheomelanin and pseudofeomelanin. Specifically, obtaining dry concentrates of natural pheomelanin, eumelanin and pseudofeomelanin from bird feathers or colored mammalian hairs, and their use as raw material for industrial purposes in cosmetics (creams, makeup and dyes) and dermatology (for treatment of diseases that occur with depigmentation).

Description

Procedure for obtaining melanins and their Applications.

Technical sector

The present invention is encompassed within the chemistry sector Specifically describes obtaining and subsequent quantification of purified melanins by means of a low cost technique Specifically describes obtaining dry concentrates of pheomelanin, eumelanin and pseudofeomelanin natural from bird feathers or colored hairs of mammal, which can be used as raw material for purposes industrial cosmetics (creams, makeups and dyes) and dermatology (for the treatment of diseases that occur with depigmentation).

State of the art

Melanin is a general term that describes a family of quinol-phenolic pigments of diverse Origin and chemical nature. Depending on its origin biological, melanins are divided into three groups: microbial, Animals and vegetables There are also synthetic melanins. The Animal melanins are the most common pigments in the tegument of vertebrates (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 place to the huge palette of melanic colors observed according at its concentration, relative proportions and contrast with zones depigmented Pheomelanins contain sulfur and are perceived like reddish or blond colorations, both in the hair or skin of Mammals like bird feathers. Eumelanins are black or brown, and that is the color they confer on structures integumentary

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, 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 Falconiformes , 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 ultraviolet (W) radiation from the sun. They have however, other potential functions: due to its structure can protect cells and tissues from oxidative stress by trapping free radicals (McGraw 2006) and, in the case of the feathers of the birds, it has been suggested that melanized feathers are more resistant  to deterioration due to 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 (Large and Cols 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, Hill 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 (Ito and Fujita 1985) or enzymatic methods are used to extract them (Liu et al . 2003). The chemical methods, which are based on acid / base extractions and oxidative fragmentation and are considered very aggressive because they alter the molecular structure of melanin, give rise to colorless metabolites that are subsequently separated by high performance liquid chromatography (HPLC) and quantified by UV-visible detection 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 pen co-exist ugly- and eumelanin in different proportions For the determination and quantification of both types of melanins in feathers, currently they are still used Ito and Fujita procedures (1985) with some modifications later (Ito and Wakamatsu 1994, Wakamatsu and Ito 2002). Their methods are based on HPLC analysis of the products of degradation of eumelanin after being oxidized and of pheomelanin After being hydrolyzed. To calculate its concentration it is necessary use, however, correction factors, since the Conversion into degradation products is incomplete.

Because the laboratory methods are relatively complex, until very recently it has only determined the proportion and concentration of eumelanins and Pheomelanins in the plumage of 13 bird species (McGraw 2006). This contrasts with the identification and quantification of carotenoids in 150 bird species, and indicates that studies of Melanization in birds are in their infancy. A simplified method with repeatable results is necessary and that has been the goal of The present invention. Based on the fact that pheomelanin It is easily solubilizable in alkaline medium but eumelanin It 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 the Eumelanic fraction on the other, starting from an alkaline digestion to from bird feather or colored mammalian hair. Besides, the possibility of solubilizing the insoluble concentrate of eumelanin in the form of pseudofeomelanin in alkaline medium, it allows quantifying spectrophotometrically the relative concentration of pheomelanin and Eumelanin without the need for HPLC. On the other hand, this method allows to obtain dry concentrates of the three fractions, pheomelanins, eumelanins and pseudosfeomelanines, which can be used as natural raw material for use in cosmetics or dermatology.

Description of the invention brief description

In the present invention it is described, by for the first time, a procedure for obtaining, from a null utility raw material such as bird feather or certain Colored mammalian hairs, from purified extracts of pheomelanin, eumelanin and pseudofeomelanin. This procedure is simple and inexpensive, and allows the use of extracts obtained for both photoprotective and makeup creams for cosmetic or dermatological purposes in diseases that They go with depigmentation of the skin. In addition, this invention presents a simple and reliable procedure of melanin quantification.

Therefore, one aspect of the present invention it is a procedure of separation, quantification and obtaining solid concentrates of pheomelanin, eumelanin and pseudoeumelanin based on the known solubility of Pheomelanins in alkaline medium and comprising the following stages:

i)

dissolution of a free-feather homogenate of the spine in alkaline medium, preferably with 20% NaOH in water, in a bath with sonication, preferably at 60 ° C, for at least 20 minutes, preferably 40 minutes,

ii)

separation of the soluble pheromellic fraction, preferably by centrifugation, of the eumelanic fraction hasty,

iii)

obtaining an eumelanin precipitate free of pheomelanins, by washing, preferably with a solution 20% aqueous NaOH and constant stirring, and subsequent centrifugation,

iv)

solubilization in alkaline medium, preferably 20% aqueous NaOH solution, of the eumelanic fraction precipitated, free of pheomelanins, by transformation oxidative in pseudofeomelanin, preferably with H2O2 30% in water, stopped by the action of an antioxidant, preferably 40% HNaSO 3 in water, and later centrifugation,

v)

relative quantification of solutions pheromellic and pseudofeomellan spectrophotometrically at 450 nm Y

saw)

obtaining solid pheomelanin concentrates, and pseudofeomelanin by neutralization, preferably with HCl, and subsequent centrifugation.

Another aspect of the invention is 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 elaboration of cosmetic compositions (creams, ointments or aerosols) or dermatological (in diseases that occur with depigmentation) or sunscreens against UV radiation.

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

Detailed description

The present invention faces the problem of  offer a simplified and reproducible procedure for determination of melanins, and more specifically for the determination of the basic types of melanins, eumelanins and pheomelanins, and a procedure to extract and obtain 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 note 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 hair of
mammal.

Specifically, the procedure for Initial separation of the pheomelanins begins with a digestion Alkaline raw material used by adding NaOH 20% dissolved in water. Digestion is performed in a bath with sonication at controlled temperature, preferably at 60 ° C, for 40 minutes with periodic agitation of the sample.

Once the alkaline digestion is finished, it proceeds to the separation of the soluble pheromellic fraction from the precipitated eumelanic fraction, preferably by centrifugation More specifically, the sample is centrifuged for 15 minutes at 17500 g and 4 ° C, removing the constituted supernatant by the fraction of solubilized pheomelanins and by the material solubilized after digestion, mainly formed by Free amino acids from the keratin of the pen. He precipitate is reserved for the subsequent study of the fraction Eumelanic

The determination of the colored solution, which constitutes the pheromellic fraction, it is done in a spectrophotometer More specifically, the determination is made by absorbance measurement at 450 nm by subtracting the obtained from 600 nm against a blank of 20% aqueous NaOH solution. He result obtained is divided by the original sample weight for express the concentration of pheomelanins as UA / g of matter cousin.

For the determination of the concentration of the precipitated eumelanic fraction, obtained after digestion with NaOH, first a wash is done to eliminate possible remains of pheomelanins. Specifically, to the eumelanic fraction precipitate is added 1 mL of 20% NaOH in water, stirred, until a suspension is obtained, and centrifuged at 17500 g for 15 minutes The supernatant is despised and the process of washed a second time. The resulting precipitate, which is dried in an oven at 60ºC, constitutes the eumelanic fraction, insoluble in alkaline conditions, free of pheomelanins.

The determination of the content of eumelanins, once the precipitate is clean of remains of pheomelanins solubilized, requires prior solubilization of the precipitate. This solubilization is carried out by means of a oxidation process that begins with the addition, to the precipitate of eumelanins, 1 mL of 20% NaOH in water, remaining in constant stirring conditions until complete resuspension of the precipitate. To this suspension are added subsequently 0.02 mL of 30% H 2 O 2 in water, the mixture Stir vigorously and immerse in bath at 60 ° C with sonication for 10 minutes counted from the moment of water addition oxygenated. The oxidation reaction of eumelanins stops by the addition of an antioxidant, specifically 0.050 mL of 40% HNaSO 3 in water, and stirring. After the reaction of oxidation proceeds to centrifugation and measurement spectrophotometric solution of eumelanins transformed by oxidation in pseudofeomelanins under identical conditions to described above for the pheromellic fraction, expressing also the results as UA / g pen.

An additional feature of this invention is the obtaining of dry pheomelanin concentrates and pseudofeomelanin from a process of neutralization of solutions used for quantifications. Specifically, the solutions of pheomelanin and pseudofeomelanins are neutralized, separately, with two-stage hydrochloric acid (HCl) using Firstly, 20% HCl until a pH close to 8-9 units and subsequently, with 0.1 N HCl, finish neutralizing the solution until reaching a pH between 6-7 units. The solution neutralized, allowed to cool and centrifuged at 17500 g 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 60 ° C. The precipitate obtained is constituted by the feomelánica or pseudofeomelánica fractions respectively.

The precipitated solids (pheromellic, pseudofeomelánico and eumelánico) are intensely colored and are easily interpose with diluents by mechanical dispersion. Do not they are soluble in hydrophilic or lipophilic medium, they are simply dispersible in solids or semi-solids of high viscosity, giving different colors depending on their concentration and the original coloring of the excipient.

The proposed determination methodology assumes a remarkable advance in the field of melanins. With treatments simple and inexpensive can be quantified directly the Pheomelanin content and indirectly that of eumelanins by correlation with the direct measurement of pseudofeomelanins. The quantification is approximate since the degree of purity achieved in ugly precipitates and eumelanin that allows to establish the extinction coefficient for Its exact quantification. In any case obtaining this parameter is not vital since the results obtained by absorbance measure divided by sample weight only they will be short of multiplying by this factor, and for the general purpose of measuring melanin content which is compare between species, the extinction coefficient is It would simplify by appearing in the two members of the equation.

As an additional advantage, this methodology allows  get dry concentrates of pheomelanin, pseudofeomelanin and eumelanin that can be used as a natural raw material for industrial purposes in cosmetics or dermatology. The melanins are the natural pigments of the skin and one of its functions is the photoprotection By obtaining them in isolation it is possible achieve, by combination between them at different proportions, all shades of existing skin, and could be used in cream, ointment or aerosol form as a second skin that perform the photoprotective function against UV radiation, without that this function had to suffer our own skin (for example, for sunscreens). At the same time, the wide range of natural tones that can be achieved would also allow its use as a natural skin color makeup agent, both for cosmetic purposes (for example as skin makeup, eyelashes, eyebrows or nails, hair dyes ...) as for purposes dermatological diseases that occur with depigmentation of the skin.

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Description of the figures

Figure 1.- It shows how the process takes place of solubilization of pheomelanins as a function of time.

Figure 2. Verification of 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 separation of the pheromellic fraction and  wash with precipitate water. Melanosome set containing eumelanin.

Figure 4.- Evolution of the content in matter insoluble and eumelanic color in samples treated in alkaline oxidation conditions 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 linearity between the content of eumelanins in the samples and the absorbance measured in three different bird species.

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Examples of realization

Example one

Methodologies of extraction, quantification and precipitation of pheomelanins 1.1.- Pheomelanin separation by solubilization in alkaline medium

The procedure set up is based on the known solubility of pheomelanins in alkaline medium. Be uses black colored rooster feather at the base and coloration Dark gold at the tips. It is cut into small pieces of approximately 1 g of rachis-free feathers and fall apart and Mix to homogenize. Twenty samples are taken from approximately 5 mg introduced into Eppendorf tubes, 1.5 mL capacity and with airtight seal, and they are added simultaneously 1 mL of 20% NaOH. The tube set is Enter in a thermostated sonication bath at 60ºC. Every 5 minutes two tubes are removed which, to minimize the re-extraction after the set time, it immersed in an ice bath and centrifuged at 4 ° C and 17,500 g for 15 minutes. Next, the absorbance of the supernatant is measured at 450 nm by subtracting the absorbance measured at 600 nm from a solution blank accuses 20% NaOH, expressing results in absorbance units per gram of pen of each sample. In Figure 1 is shown as the solubilization process proceeds of pheomelanins as a function of time. Since t = 0 there is a gradual increase in solubilized color that stabilizes at from t = 20. From this moment there is no increase in the amount of solubilized color, which remains constant for all the time later. The constancy over time of the quantity  extracted color ensures that the extraction is complete and that the dissolution of pheomelanins is stable and does not occur color degradation under solubilization conditions.

With the application of this same procedure to structurally distinct feathers, it has been observed that in feathers thick and strong as are vulture, alkaline digestion is slower, while in small and low compaction pens, the digestion of keratins and the solubilization of Pheomelanins are produced quickly. For all samples studied the solubilization time has been in all cases less than 30 minutes, so it has been set as the time of digestion 40 minutes, time in which there is a guarantee that the Pheomelanins have been completely solubilized in all samples.

The solubilization of pheomelanins is complete practically in the first twenty minutes (Figure 1), without However, the reaction time is extended to 40 minutes for get the perfect breakdown of the pen material. In pens with eumelanic content, this factor is important for subsequent treatments since the previous digestion of 40 minutes completely releases melanosomes making them accessible to subsequent oxidative treatment (figure 2).

The amount of color extracted is a function of weight used, following the Lambert-Beer law, in a wide range of weight as long as the rest remain identical  of the parameters set for extraction. Using feathers of three species with ugly and eumelanic content, in figure 2 it You can see that the linearity between featherweight and absorbance obtained has a correlation coefficient (r) in the 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 was determined by expressing the results in UA / g and the means, standard deviation, confidence interval and percentage of variability in the determination were calculated. The results are shown in the
Table 1.

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TABLE 1 Reproducibility of the extraction process and quantification of pheomelanins

one

Variability between replicates, when they use griffon vulture feathers as raw material, it 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 that the variability of the quantification method is a function of the sample, clearly shows that homogeneity is a Critical factor to obtain a good reproducibility. It has been proven the variability that the raw material itself has, for this, the primary feather of the griffon vulture has been determined Pheomelanin content in the basal, middle and distal part of the pen. Results are shown in table 2.

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

2

In each area of the pen there is a different Pheromellic content. You cannot therefore talk about a content  Feomelánico in a certain species of bird, not even in a certain feather of the bird. The concentration varies depending on the bird species, the type of feather and even the feather area. The methodology applied has, however, an acceptable repeatability and to apply it you have to specify very well what raw material is the one to be analyzed because they will not be obtained comparable results if the analysis is performed on an area concrete of different pens or on a homogenate of feathers in which includes the different parts.

1.2.- Obtaining solid-state pheomelanins

To obtain the concentrate of solid-state pheomelanins, the alkaline solution containing the  solubilized pheomelanins are brought to neutrality with 20% HCl until solution reaches an alkaline pH close to neutrality and it is finished accessing to the point of neutrality using HCl 0.1 N to be able to adjust more accurately that the final pH does not lower of 6. Under neutral conditions the pheomelanins are made insoluble forming a fine suspension that is separated by centrifugation at 17500 g for 20 minutes and temperature of 4 ° C. The suspension is formed by a fine powder with little tendency to precipitate, so sometimes the precipitation is not complete and the Supernatant retains some color. In this case it can be done it is 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 water deionized, then centrifuged under identical conditions to the previous ones and the washing process is repeated a second time finally obtaining a precipitate enriched in Pheomelanins

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Example 2

Development of extraction and quantification methodologies of eumelanins 2.1.- Obtaining the solid precipitate of eumelanins

In pens with eumelanic content, after solubilization treatment of pheomelanins, the precipitate retains an intense black color consisting of melanosomes that they contain eumelanin (figure 3). The residue obtained is washed and centrifuge up to three times with 20t aqueous NaOH solution to remove the remains of colored solution from the First extraction Under these conditions a new one does not occur re-extraction and the color that is observed are remains of the first extraction that impregnate the residue and generally, especially in feathers of low feomelánico content, after the first washed, the rest is absolutely colorless, constituting the Pheomelanin-free eumelanic precipitate.

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2.2.- Solubilization of the solid precipitate of Eumelanins in the form of alkaline oxidation pseudofeomelanins controlled in time

The solubilization of the solid eumelanic residue, free of pheomelanins, it is carried out through a treatment oxidative in alkaline medium. To do this, it is carried out first place the resuspension of the eumelanic precipitate in 1 mL of 20% NaOH solution by stirring and subsequent sonication in ultrasonic bath. After resuspension, the treatment Oxidative is performed by adding 0.02 mL of 30% H2O2. The reaction is carried out in an ultrasonic bath at 60 ° C and left pass for 10 minutes.

To study the processes that occur during the time after the addition of H 2 O 2, pen is used of rooster with ugly content and eumelanins. In order to be able track the material solubilization process solid, the previously described methodology is modified promptly scaling it twenty times. They chop and undo 10 g cannon-free pen. 45 samples of 0.1 g are taken and deposit each in 50 mL centrifuge tubes previously tare and the elimination treatment of Pheomelanins using 20 mL of 20% NaOH in each of the samples. Finished the extraction of pheomelanins, the precipitate Eumelanic is washed repeatedly with 20% NaOH and centrifuged to remove salts and all soluble material. The residue washing is then dried in an oven and the weight of each of the samples to know the content in concentrate Eumelanic from which it is started before starting treatment oxidative For consistency with the study of pheomelanins, the 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% HNaSO3 is added and stirred vigorously. The sample is centrifuged at 17500 g for 15 minutes at 4 ° 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% HNaSO3 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 agent
oxidizing

Above t = 10 are removed from the bathroom six samples periodically, three of them corresponding to samples to which antioxidant has been added and three others corresponding to the group in which the antioxidant has not been previously added and added at the time The sample is taken from the bathroom. Figure 4 summarizes everything It happens during the reaction time.

After the addition of H2O2, an immediate solubilization of the color occurs, as well as a sharp decrease in the amount of solid material. The color solubilization rate 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 a slight decrease in the absorbance of the solubilization appears, indicative 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 becomes 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 color destruction in any sample, while in the samples in which no sulphite 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, indicating 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 the incidence of degradation to be extrapolated during the time when the reaction is masked by the solubilization of eumelanins
Rusty.

Extrapolating the measured values during destruction reaction of oxidized eumelanins between time t = 10 and t = 20, with theorists that would exist at t = 0 allow know that if there was 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 it has been added the antioxidant, the degradation reaction has paralyzed, and the color that is measured is stable, at least in the within 20 minutes that the reaction has been allowed to pass. The antioxidant stabilization procedure is cash and allows a reasonable timeframe for spectrophotometric measurement being certain that it is not No degradative reaction occurs. However, the measure done in this way is affected by the error of the under-quantification of oxidized eumelanins by the destruction that has occurred during the period necessary for the solubilization of oxidized eumelanins. In fact the value measured is 136, when according to the extrapolation of the line of degradation should have been 152. The simultaneous study of both reactions allow to know the real value that is measured and the theoretical value that should have been measured, so you can calculate the conversion factor between one and the other, which is 1,119. That is, the actual measurement made at t = 10 must multiply by 1,119 to get the value that theoretically I would have given the initial sample if there had not been a reaction Parallel degradation.

Quantification of eumelanins cannot be done directly by being insoluble, but by oxidative treatment the original eumelanin is transformed into another  colored compound and soluble in alkaline medium that can be quantified as in the case of pheomelanin and correlate the value obtained with the original eumelanic content. The characteristics of the product formed in part remind those It has the pheomelanin, as are its solubility in alkaline medium and show an apparent coloration with golden brown tones, very distinct from the intense black color that eumelanins have intact

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

Because of the transformation that the eumelanin, it is possible to quantify it spectrophotometrically as pseudofeomelanin at 450 nm subtracting absorbance at 600 nm and dividing the absorbance measured by the pen weight used. The sample taken at t = 10 and immediately stabilized with HNaSO3 It is suitable for quantifying pseudofeomelanin measuring its absorbance. The result obtained must be corrected multiplying the value obtained by 1,119 to contemplate the losses caused by co-oxidation.

The content measurement methodology in Eumelanins by quantification of their corresponding Pseudofeomelanins formed by alkaline oxidation is suitable 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 of Lambert-Beer Using feathers of three species of birds of very different melanin content in figure 6 you can note that the measurement procedure correlates perfectly Melanin content with pen weight. Likewise, the methodology shows a very acceptable repeatability (Table 3). Be they use feathers of the same three species used for the study linearity in the measurement and it is obtained that, as in the case of pheomelanins, depending on the sample, this percentage It can range between 3.74W and 7.92%. Obviously this variability among replicates is the result of heterogeneity in the raw material used In a study similar to that done with Pheomelanins are found to be, depending on the pen portion used, the eumelanin content, quantified as pseudofeomelanin, varies, and within each portion the percentage of variability narrows to below 5% in all the cases (Table 4).

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

3

TABLE 4 Variability in the content of eumelanins quantified as pseudofeomelanins in different pen parts of Griffon Vulture

4

Example 3

Obtaining solid state pseudofeomelanins

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

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

Claims (12)

1. Procedure for obtaining melanin pigments characterized in that it comprises the following stages:

to)

initial alkaline treatment,

b)

separation of 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, Y

and)

neutralization of solutions for get dried pheomelanin concentrates and Pseudofeomelanin

2. Method according to claim 1, characterized in that the initial alkaline treatment of a) is carried out with NaOH preferably at 20t in water, in a bath with sonication, preferably at 60 ° C, for at least 20 minutes, and preferably for 40 minutes with stirring Periodic 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 ° C, the supernatant containing the fraction of solubilized pheomelanins being removed. 4. Method according to claim 1, characterized in that the washing to obtain solid state eumelanins of c) is preferably carried out by resuspension in 20 ° NaOH and subsequent centrifugation at 17500 g for 15 minutes repeating the operation three times. 5. The 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 60 ° C for 10 minutes of the sample, and stopping the reaction by adding an antioxidant, preferably 40% HNaSO 3 in water. 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% HCl followed by 0.1 N HCl, 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, subtracting that obtained at 600 nm from a blank of aqueous NaOH solution at 20 %, and divided by the original sample weight to express the concentration of eumelanins, in the form of pseudofeomelanins, and of pheomelanins as UA / g of raw material. 9. Use of melanins obtained according to claims 1 to 7 for the preparation of compositions Cosmetics 10. Use of melanins according to claim 9 where the cosmetic composition comprises one of the following products: skin makeup, eyelashes, eyebrows, lips or nails, hair or hair dyes, tattoo dyes, or Any other product for cosmetic use. 11. Use of melanins obtained according to claims 1 to 7 for the preparation of sunscreens against UV radiation. 12. Use of melanins obtained according to claims 1 to 7 for the preparation of compositions with dermatological purposes in diseases that occur with depigmentation of the skin.