WO2010058235A1

WO2010058235A1 - Method for the preparation of oxycarotenoids

Info

Publication number: WO2010058235A1
Application number: PCT/IB2008/003141
Authority: WO
Inventors: Danilo Vizcarra Gonzales; Mario David Torres Cardona
Original assignee: Innova Andina S.A.
Priority date: 2008-11-19
Filing date: 2008-11-19
Publication date: 2010-05-27
Also published as: MX2011005264A; CN102272098A; JP2012509307A; PE20100745A1; WO2010058235A8; CL2009002105A1

Abstract

A high-yield process for preparing astaxanthin (3,3'-dihydroxy-β, β- carotene-4, 4'- dione) from silylated derivatives of zeaxanthin (3,3'-dihydroxy-β, β- carotene-3, 3'-diol), whether it be of synthetic or natural origin, is described.

Description

METHOD FOR THE PREPARATION OF OXYCAROTENOIDS

Background of the Invention

Astaxanthin occurs naturally in different seaweed, bacteria, fungi and some animals, frequently forming some type of complex with proteins; also in plants such as the Adonis annua L. and also found in some birds, such as flamingoes, quails, and other species. In addition it can be found in its free form or as a mono- or di-ester derivative.

Normally the amount of this carotenoid found in its natural sources is very limited, for which reason other additional natural sources have been resorted for commercial purposes, such as krill, crustaceans by-products, genetically engineered marigolds, and others. In addition, the use of astaxanthin isolated from leavened flour extracts of Phaffia rhodozyma and the Haematococcus pluvialis seaweed has also been increased. The microalgae Chlorococcum sp. seems to be a promising source of astaxanthin as well as other carotenoides such as canthaxanthin and adonixanthin. This carotenoid has generally been used for the pigmentation of some salmonids that do not synthesize it de novo, which confers the pink tonalities that the market prefers. It has also found applications in the pigmentation of crustaceans, although in these its immunostimulant and antioxidant activities have been most appreciated; this affects their health remarkably and therefore lead to better survival rates when they are cultivated intensively. Other oxicarotenoids, like canthaxanthin, echinenone and adonixanthin, are actually used for the same purposes. It has been demonstrated that astaxanthin exhibits anti-oxidant activity superior to that of most carotenoids (J. Agric. Food Chem. 48, 1150) and thus it is also currently being used successfully for human consumption. The potent antioxidant property of astaxanthin has been implicated in its various biological activities demonstrated in both experimental animals and clinical studies. Astaxanthin has considerable potential and promising applications in human health and nutrition. The most significant activities of astaxanthin, includes its antioxidative and anti-inflammatory properties, its effects on cancer, diabetes, the immune system and ocular health, the promotion of human health, including the antihypertensive and neuroprotective potentials and the effects of dietary astaxanthin on blood pressure, stroke, and vascular dementia (J Nat Prod., 69,443). For decades astaxanthin was obtained synthetically in the form of a racemic mixture of isomers (3R, 3'R), (3R, 3'S) and (3S, 3'S) by companies such as DSM (Roche) and BASF using long and expensive procedures, which has resulted in this carotenoid becoming very expensive. Synthetic astaxanthin. is an identical molecule to that produced in living organisms and it consists of a mixture 1:2:1 of isomers (3S,3S^'), (3R,3R') and (3R.3R) respectively.

Nevertheless, in 1967 the obtaining of astaxanthin from astacene (Chem Comm. 49) was announced, and astaxanthin di-methyl ether had also been obtained by oxidating zeaxanthin di-methyl ether (J. Org. Chem., 32.180) in addition to the synthesis of astaxanthin from cantaxanthin using silylating agents (J. Org. Chem. 43, 1599). Published more recently was news on the obtaining of astaxanthin from zeaxanthin, also in a single step, using oxidants such as sodium bromate or hydrogen peroxide with relative success, althougth with very poor yields (US 6,372,946, US 6,376,717, MX PA03009685), which were reported as between 20 % and 30 %. Later the synthesis of astaxanthin as food coloring agent was reported (ES 2223270) producing the carotenoid aryl esters or of oxymethyl ethers through controlled reaction. The resulting compounds are oxidized by salts or complexes of chromium Vl, manganese and mixtures of these in aqueous medium. Similar procedures were reported early by Torres-Cardona using esterified zeaxanthin to produce esterified astaxanthin (US 7291749), mainly zeaxanthin diacetate as raw material.

Thus it is that today the synthesis of astaxanthin from another carotenoid finds examples extremely limited, as was mentioned above. In the case of zeaxanthin as a substratum, mainly because its hydroxyl groups are sensitive to practically any oxidant resulting in highly degraded synthetic procedures and therefore of very low yield. Therefore the process described herein includes an additional step, which consists of protecting the zeaxanthin's hydroxyl groups first, and then proceeding with a step of simultaneous de-protection and oxidation that, under the technique mentioned, produces a high yield of astaxanthin including the usage of a catalyst and a phase transfer agent for the oxidation step improvement. If the protecting groups step is omitted then echinenone, adonixanthin and other carotenoids are obtained.

Some of the major natural sources (Sajilata et al.) for obtaining zeaxanthin as a raw material for the process that is described herein are yellow corn, orange pepper, orange juice, honeydew, mango, genetically altered marigolds and chicken egg yolk. Zeaxanthin comprise about 90% of the total carotenoids in the anthers of Delonix regia (GuI Mohr) flowers. Zeaxanthin is also the major carotenoid in cold-pressed marionberry, boysenberry, red raspberry, and blueberry seed oils. Zeaxanthin has also been identified in extracts from apricots, peaches, cantaloupe, and a variety of pink grapefruit (Ruby seedless). The stereochemical correlation between capsanthin and zeaxanthin and the cooccurrence of the 2 pigments in Capsicum sp. have suggested a close biogenetic relationship between the two. Of the total pigment content, zeaxanthin contributes to about 6.5%, 7.3%, and 15.9%, respectively, in the red, orange, and yellow varieties of Capsicum annuum In com, xanthophylls are mostly found in the horny endosperm.The total xanthophyll content is estimated to be 11 to 30 mg/kg. Wolfberry (Lycium Chinese), a small fruit used to improve vision in traditional Chinese medicine, contains concentrations of zeaxanthin dipalmitate that can approach 1 g/kg wet weight. Among the sources of microbial xanthophylls, Flavobacterium sp. is reported to produce zeaxanthin as essentially its only carotenoid. The pigment formed by Flavobacterium consists of 95% to 99% zeaxanthin. Flavobacterium produced zeaxanthin is identical to zeaxanthin from Zea mays.. Erwinia herbicola, a nonphotosynthetic bacterium, is yellow-colored due to accumulation of polar carotenoids, primarily mono- and diglucosides of zeaxanthin. The green alga Neospongiococcum excentricum is shown to produce up to 0.65% xanthophylls (dry mass ,basis). A zeaxanthin-overproducing mutant strain zea 7 generated from Dunaliella salina may be considered for commercial exploitation. Zeaxanthin is a natural constituet of the outer membrane of Synechocystis sp. PCC6714 . The microalgae Microcystis aeruginosa is reported to produce the bioactive carotenoid zeaxanthin. Also the blue-green alga Spirulina has zeaxanthin as one of its carotenoids. Zeaxanthinibacter enoshimensis is a zeaxanthin-producing marine bacterium of the family Flavobacteriaceae, isolated from the seawater off Enoshima island in Japan. Mesoflavibacter zeaxanthinifaciens is another novel zeaxanthin-producing marine bacterium of the family Flavobacteriaceae. The carotenoids of the red algae Corallina officinalis, C. elongate, and Jania sp. are reportedly composed of /3-carotene, zeaxanthin, fucoxanthin, 9_-c/sfucoxanthin, fucoxanthinol, 9_-c/s-fucoxanthinol, and 2 epimeric mutatoxanthins. The symbiotic bluegreen algae Cyanophora paradoxa and Glaucocystis nostochinearum synthesize only /3-carotene and zeaxanthin. The thylakoid of the cyanobacteria Anacystis nidulans is reported to have zeaxanthin as one of its major carotenoids. Dunaliella parva , Erythrotrichia carnea,Dunaliella bardawil ,

Prochloron sp. , and Pleurochloris commutata are some of the other microbial sources of zeaxanthin.

In addition there is also the zeaxanthin prepared synthetically. It is composed of trans- zeaxanthin and minor quantities of c/s-zeaxanthin, 12^'-apo-zeaxanthinal, diatoxanthin, and parasiloxanthin.

The zeaxanthin prepared by synthetic processes suffer from several disadvantages; they typically require numerous reaction steps, and each step has a less than 100% yield, so that the final yield of zeaxanthin at the end of the multistep processing tends to be relatively poor. In addition, chemical synthesis tends to yield undesirable S-S and S-

R stereoisomers of zeaxanthin, as well as various conversion products such as oxidized zeaxanthin, and zeaxanthin molecules that have lost one or more of the double bonds in the straight portion or end rings.

And the semi-synthetic zeaxanthin obtained from the isomerization of lutein, mainly from the marigold extracts must be considered. As a general principle in these cases, lutein is heated for a relatively long time in the presence of a strong base. This isomerization reaction does not proceed to completion, however. Due to the elimination of water under the strongly basic reaction conditions, anhydrous secondary products are formed

(Pure Appl. Chem., 74,1369). Lutein from natural sources is usually accompanied by (3R,3'R)-zeaxanthin and always has the (3R, 3'R, 6'R) chirality and zeaxanthin prepared from lutein necessarily has the

(3R, 3^"S) chirality, this is a main disadvantage of the epsilon-beta-arrangement (US

6420614), but can be used as the oxidation step substratum.

Summary of the Invention

The present invention describes the utilization of silyl ethers as protective groups for zeaxanthin, such as -O-Si(CH₃)₃, -O-Si(CH₂-CH₃)3, -O-Si(isopropyl)₃, -O- Si(CH₂CH₃)₂(isopropyl), -O-Si(CH₃)₂(tert-butyl), -O-Si(CH₃)₂(n-hexyl), etc. The protective groups can be converted by hydrolysis into a hydroxyl group again.

Such protection is necessary in order to avoid the extensive oxidation of the said hydroxyl groups. Subsequently silyl derivatives are put under a process of de-protection and oxidation by which, in a single step procedure, the astaxanthin is produced in high yield if a catalyst and a phase transfer agent are used.

Some of the preferred oxidation systems are iodobenzoic acid, nickel peroxide, the Jones reagent, the Collins reagent, pyridinium chlorochromate (PCC), bipyridinium chlorochromate, benzyl-trimethyl-ammonium chlorochromate, pyridinium fluorochromate, pyridinium dichromate, trimethylsilyl chlorochromate, chromic acid, HOBr, HOCI, N-bromosuccinimide, hypochlorites like tert-butyl hypochlorite, sodium or calcium hypochlorite, tetrabutylammonium hypochlorite, N-chlorosuccinimide, perchlorates, bromates, chlorates, iodates and periodates, manganese dioxide, potassium permanganate, 2,3-dichloro-5,6-di-cyanoquinone, p-chloranil, silver oxide, silver carbonate, lead tetraacetate, aluminum isopropoxide, perbenzoic acid and its halogenated derivatives, etc. in the presence of a prefered catalyst and phase transfer agent . The trimethylsiloxy (TMS-O) groups, triethyl -, terf-butyldiphenyl -, tert- butyldimethylsiloxy and commercial silylating equivalent mixtures have been preferred for protecting the hydroxyls in zeaxanthin.

The process is carried out under very soft reaction conditions with temperatures that range from -30 ⁰C to room temperature, and in the presence of a halogenated catalyst such as salts of iodine and bromine; selenium dioxide, vanadium pentoxide and osmium tetraoxide, eerie ammonium nitrate, eerie sulphate, ruthenium tetroxide, ruthenium trichloride or mixtures thereof, which are also appropriate.

A fundamental objective of this invention is to provide a procedure for obtaining astaxanthin with yields of 70 % or greater from zeaxanthin, so that it is possible to have a product economically more accessible and of equal quality compared to those that are now circulating in the world market. The astaxanthin thus prepared is useful in aquaculture, poultry farming and as a nutraceutic in human consumption; mainly in the pigmentation of salmonids, crustaceans and other aquatic species. Another objective of this invention is to provide a procedure to obtain several oxicarotenoids like adonixanthin, β-cryptoxanthin and canthaxanthin when isozeaxanthin is used.

Scheme 1 shows how the transformation of zeaxanthin to astaxanthin takes place when its hydroxyl groups are protected.

Zβaxanthin

Me₃Si-CI Pyridine 1 W10*C

Zβaxanthin irimrthyfcilyfether

Scheme 1. Oxidating of Silylates Zeaxanthin Derivatives

Detailed Description of the Invention

The zeaxanthin used may be 100 % pure or in the form of a product that contains more than 400 g of carotenoid for each kilogram of concentrate containing more of 90 % of zeaxanthin. These normally come from natural sources, so frequently the pigment comes in a greasy matrix that does not interfere in the overall process. In a first stage the zeaxanthin is dissolved in a solvent, which preferably would be pyridine, although halogenated solvents such as methylene chloride, carbon tetrachloride and chloroform are also suitable.

Once dissolved, the carotenoid is made to react with trimethyl-chlorosilane or any commercial silylating mixture at room temperature. Upon conclusion of the reaction the mixture that contains the zeaxanthin trimethyl-silylether will be kept under the same conditions as initially in order to continue with the process of deprotection and oxidation. A minimum amount of the catalyst is added to the mixture from the reaction previously described and then the pyridinium chloro-chromate, which is the oxidant that is preferred, is added.

The advance of the reaction is monitored by TLC, and once it has concluded, evidenced by the total disappearance of zeaxanthin trimethylsilylether, the organic phase is decanted, then proceeding to recover the solvent by vacuum evaporation. The solid residue that contains the astaxanthin is again dissolved in ethanol and brought to evaporation with the double objective of eliminating other volatile residue and carrying out the thermal isomerization cis-trans of carotenoid with the object of minimizing the cis isomers while recovering the alcohol. In this way a constant proportion between the various geometric isomers of astaxanthin is also secured. The zeaxanthin is dissolved in solvent in a proportion of one part of carotenoid to one hundred parts of solvent by weight, although it is preferable to use 20 to 50 parts of solvent to each part of xantophyll.

The mixture of pigment with solvent is shaken vigorously in a sealed reactor so that the inner atmosphere can be controlled, whether in atmospheric conditions or in the absence of oxygen. The temperature of the process is kept between -30 ⁰C and room temperature or between -15 ⁰C and 15 ⁰C, although it is preferable to carry it out between 0⁰C and 10⁰C, both in its group protection stage and in its oxidation stage. The catalyst is used in amounts that range from 0.1 to 2 % of the weight of pigment, although it is preferable to use from 0.5 to 1.0 %, previously dissolved in a portion of the solvent. In the case of oxidant, up to 10 parts of this have been used to each part of zeaxanthin, although it is preferable to use between 3 and 5 parts of oxidant to each part of carotenoid.

The phase transfer agent is used in amounts that range from 0.01 % to 1 % of the weight of xanthophylls. Both the group protection and the oxidation stage are carried out with between 15 min and 5 hours of reaction, although under favorable conditions it is possible to carry it out in between 1 and 3 hours. The protective or silylant agent is used in the amount of 10 parts to each part of zeaxanthin, although it is preferable to use 3 to 6 parts. Below are provided different examples in order to illustrate the process described without this meaning a limitation in its scope:

Example 1

18 g of zeaxanthin were dispersed in 750 ml of chloroform and 1 g of tetrabutylammonium bromide were added successively to a solution prepared with 13 g of sodium bromate and 0.5 g of ruthenium trichloride in 250 ml of deionized water, adjusting the pH to 2.5. The mixture was stirred at 20 ⁰C and the reaction finished 120 min after. The dark red reaction mixture was treated with saturated aqueous sodium carbonate solution and then with 20 % aqueous sulphite.

The organic phase is decanted and washing twice with aqueous bicarbonate 2 % first then with deionized water. 100 mg of alpha-tocopherol, 50 mg of BHT and 100 ml of soy oil are added to the organic phase, then it is evaporated to recover the solvent and an oil suspension of astaxanthin containing 50 g of the pigment per kilogram. The suspension is homogenizated using an Ultra Turrax (IKA T-25) homogenizer obtaining a xanthophylls crystals distribution size from 0.2 to 2 microns, excellent to be formulated in salmonids and crustaceans diets.

Example 2.

500 milliliters of pyridine at room temperature is loaded into a one-liter glass reactor provided with mechanical agitation, and then 10 g of zeaxanthin is added. The mixture is shaken vigorously until completing homogeneity. The temperature of the mixture is adjusted to 20 ⁰C and then 15 ml of a solution of fe/t-butyl dimethyl silyl chloride is added. After 5 hours of reaction, 250 mg of metallic iodine dissolved in pyridine is added, while continuing shaking. Eight g of pyridinium chlorochromate is slowly added and the reaction is continued for a further two hours. Once zeaxanthin is no longer detected by TLC, the reaction is deemed as finished, and the organic phase is decanted. The solid residue is washed with 50 milliliters of additional pyridine and the washing is joined with the decanting, proceeding to discard the residual solid. The organic phase is evaporated to recover the solvent and then 100 milliliters of ethanol is added to the residue even with less than 10 % of pyridine and evaporation is continued until almost dryness. Then the residue is washed with 300 milliliters of water at 40 °C acidulated with acetic acid to a pH of 4.5, discarding the wash. An additional washing takes place and again the wash is discarded.

The solid obtained contains around 6.1 g of astaxanthin and is mixed with 200 ml of red olein added with 150 mg of BHT, then milling (IKA colloid mill, MK 2000) until obtain a crystal size distribution from 0.2 to 2.0 microns . The obtained suspension is homogenizated using an Ultra Turrax homogenizer (IKA Ultra Turrax T-25).

Example 3.

The product obtained from marigold lutein published isomerization methods is purified using our patented methods (US 7,150,890) until having a concentrate with 600 g of carotenoid in all, of which 98 % is zeaxanthin.

The material thus obtained is put under the process described in Example 2, in the end obtaining 7.3 g of astaxanthin, which represents a yield of around 66 % on the basis of the initial carotenoid totals. In addition, lesser amounts of adonixanthin and β- cryptoxanthin are obtained.

Example 4.

5.8 g of synthetic zeaxanthin is dissolved in 1.0 liter of methylene chloride at room temperature and then 25 milliliters of trimethylchlorosilane is added. After 2 hours of reaction, 50 mg of iodine dissolved in the same solvent is added and the vigorous agitation is continued. Four grams of pyridinium dichromate is dissolved in 100 milliliters of water and is added to the previous mixture. Then it is slowly dosed with a 5 % acetic acid solution until complete disappearance of the zeaxanthin, which is monitored by TLC. Once having finished the reaction, the organic phase is separated and is then washed with 200 milliliters of a watery solution of 10 % sodium thiosulfate first and then 200 milliliters of water. The solvent is recovered and the residue is again dissolved in ethanol and is put under thermal isomerization while the alcohol is recovered. The final residue contains around 4.1 g of astaxanthin, which represents a yield of nearly 70 %.

Example 5.

15 g of a purified concentrate of yellow corn xanthophyll is dissolved in 1.0 liter of chloroform that contains 700 g of carotenoid/ kilogram of concentrate, of which 95 % is zeaxanthiπ. The temperature of the mixture adjusts to ⁰C and then 50 milliliters of tert- butyldiphenylsilyl chloride are added. The group-protection reaction is extended for 5 hours and then 100 mg of ruthenium trichloride is added while it continues to be shaken. Then 25 g of bipyridinium chlorochromate is added so that the same reaction temperature is maintained until the complete disappearance of the zeaxanthin. The reactor has been maintained in an inert atmosphere throughout the whole process. Washing with 200 ml of soft water and discard the watery fase, then the organic phase is treated with 200 milliliters of a watery solution of 15 % sodium thiosulfate in order to carry out a first washing; after discarding the wash, there is one more washing with 200 milliliters of a watery 2 % sodium bicarbonate solution, followed by discarding the wash in order to finally carry out one more wash under the same conditions but only with water. All the washing is done at room temperature. The solvent is recovered and the residue is put under the thermal isomerization described in the previous examples in order to finally obtain 7 g of astaxanthin, which corresponds to a yield of approximately 70 %.

Example 6

500 milliliters of pyridine at room temperature is loaded into a one-liter glass reactor provided with mechanical agitation, and then 10 g of isozeaxanthin is added. The mixture is shaken vigorously until completing homogeneity then 250 mg of metallic iodine dissolved in pyridine is added, while continuing shaking. 20 g of pyridinium chlorochromate is slowly added and the reaction is continued for a further two hours. Once isozeaxanthin is no longer detected by TLC, the reaction is deemed as finished, and the organic phase is decanted. The solid residue is washed with 50 mililiters of additional pyridine and the washing is joined with the decanting, proceeding to discard the residual solid. The organic phase is evaporated to recover the solvent and then 100 mililiters of ethanol is added to the residue even with less than 10 % of pyridine and evaporation is continued until almost dryness. Then the residue is washed with 300 milliliters of water at 40 ⁰C, discarding the wash. An additional washing takes place and again the wash is discarded. The solid obtained contains around 6.1 g of canthaxanthin.

Example 7

800 milliliters of dichlorometane at room temperature are loaded into a two-liter glass reactor provided with mechanical agitation, and then 20 g of zeaxanthin is added. The mixture is shaken vigorously until completing dispertion then 50 mg of osmium tetroxide dissolved in the same solvent is added, while continuing shaking. 14.5 g of potassium bromate are dissolved in 300 ml of water and added into the reactor, then the mixing temperature is adjusted at 10 ⁰C. The phase-transfer agent cetyltrimethylammoniumbromide was used to achieve contact of the hydrophilic oxidant with the lipophilic carotenoid zeaxanthin dissolved in methylene chloride. A solution of sulfuric acid 25 % was added dropwise via syringe until zeaxanthin is no longer detected by TLC. The reddish reaction mixture was treated with saturated aqueous sodium carbonate solution and then with 20 % aqueous sulphite. The reaction is deemed as finished, and the organic phase is decanted. The solid residue is washed with 50 mililiters of additional dichlorometane and the washing is joined with the decanting, proceeding to discard the residual solid. The organic phase is evaporated to recover the solvent and then 100 mililiters of ethanol is added to the residue even with less than 10 % of dichlorometane and evaporation is continued until almost dryness. Then the residue is washed with 300 milliliters of water at 40 ⁰C, discarding the wash. An additional washing takes place and again the wash is discarded. The solid obtained contains around 12 g of astaxanthin.

Claims

1. A method for the preparation of astaxanthin from zeaxanthin in which a protector of the zeaxanthin hydroxyl groups is used and later an oxidation agent, catalyst and phase transfer are added, which acts simultaneously by de-protecting it and oxidizing it in a single step procedure in order to obtain astaxanthin.

2. A method in accordance with claim 1 in which the zeaxanthin may be from synthetic, natural or semi-synthetic origin.

3. A method in accordance with claim 1 in which the protective groups for zeaxanthin hydroxyls are silyl ethers.

4. A method in accordance with claim 3 in which the protective groups are silyl derivatives such as triethyl-diphenylsiloxy, tert-butyldiphenylsiloxy, tert- butyldimethylsiloxy, trimethylsiloxy or similar or mixtures thereof.

5. A method in accordance with claim 1 in which the prefered oxidation agent may be selected from iodobenzoic acid, nickel peroxide, the Jones reagent, the Collins reagent, pyridinium chlorochromate (PCC), bipyridinium chlorochromate, benzyl-trimethyl-ammonium chlorochromate, pyridinium fluorochromate, pyridinium dichromate, trimethylsilyl chlorochromate, chromic acid, hypohalogenous acids, N-bromosuccinimide, hypochlorites like tert-butyl hypochlorite, sodium or calcium hypochlorite, tetrabutylammonium hypochlorite, N-chlorosuccinimide, perchlorates, bromates, chlorates, iodates and periodates, manganese dioxide, potassium permanganate, 2,3-dichloro-5,6-di-cyanoquinone, p-chloranil, silver oxide, silver carbonate, lead tetraacetate, aluminum isopropoxide, perbenzoic acid and its halogenated derivatives.

6. A method in accordance with claim 1 in which the catalyst used may be halogenated catalyst such as salts of iodine and bromine, ferric chloride, selenium dioxide, vanadium pentoxide and osmium tetraoxide, eerie ammonium nitrate, eerie sulphate, ruthenium tetroxide, ruthenium trichloride or mixtures thereof.

7. A method in accordance with claim 1 in which the phase transfer agent including quaternary ammonium salts like cetyltrimethylammoniumbrornide and similar, quaternary phosphonium salts, sulfonium salts or mixturfes thereof.

8. A method in accordance with claim 1 in which the organic solvent is pyridine, although halogenated hydrocarbons such as carbon tetrachloride, methylene chloride and chloroform may be used.

9. A method in accordance with claims 1 and 8 in which a co-solvent such as acetic acid, water or acetonitrile can be used, including benzene and its halogenated derivatives or mixtures thereof.

10. A method in accordance with claims 1 , 3 and 4 in which the silylant agent is used in the proportion of 10 parts to each part of zeaxanthin, preferably between 4 and 6 parts to each part of zeaxanthin.

11. A method in accordance with claims 1 and 5 in which the oxidation agent is used in the proportion of 3 to 5 parts to each part of the substratum that contains the zeaxanthin, although preferably 0.5 to 2 parts for each part of zeaxanthin would be used.

12. A method in accordance with claim 1 in which the temperature of reaction for protecting the zeaxanthin's hydroxyl groups is set between -30 ⁰C and 30 ⁰C, although it can be done between -15⁰C and 15 ⁰C, but preferably between O⁰C and 10 ⁰C.

13. A method in accordance with claims 1 and 12 in which the reaction time is set between 15 min and 24 hours, although it can be done between 8 and 18 hours, but preferably between 2 and 5 h.

14. A method in accordance with claim 1 in which the temperature of reaction for the de-protection and oxidation of the zeaxanthin is set between -30 ⁰C and 30 ⁰C, although it can be done between -15⁰C and 15 °C, but preferably between 0°C and 10 °C.

15. A method in accordance with claims 1 and 12 , 13 and 14 in which the time of de-protection and oxidation is between 15 min and 24 hours, although it can be done between 8 and 18 hours, but preferably between 2 and 5 hours.

16. A method in accordance with claim 1 in which the astaxanthin obtained is put under a process of thermal isomerization in the presence of a polar solvent.

17. A method in accordance with claim 1 in which canthaxanthin is obtained when isozeaxanthin is used as the oxidation substratum.

18. A method in accordance with claims 1 and 16 in which the temperature of isomerization may be between 40 ⁰C and 70 ⁰C.

19. A method in accordance with claims 1 and 16 in which the polar solution may be a C2 - C5 alcohol or a ketone, preferably acetone.

20. A method in accordance with claim 1 that can be carried out in atmospheric conditions or inert atmospheres.

21. A method in accordance with claim 1 in which the astaxanthin obtained may be utilized for the pigmentation of salmonids, crustaceans and birds.

22. A method in accordance with claim 1 in which the astaxanthin obtained may be used as antioxidant and inmunostimulant in salmonids, crustaceans and birds.

23.A method in accordance with claim 1 in which the astaxanthin obtained may be used as a nutraceutical for human consumption.

24.A method in accordance with claim 1 which the astaxanthin obtained may be used for the feed pet formulation.