Title of Invention

A PROCESS FOR THE CONVERSION OF LUTEIN OR OF ITS ESTERS INTO ZEAXANTHIN

Abstract

A process for the conversion of lutein or of its esters which occur in nature into zeaxanthin by base-catalyzed isomerization comprises heating an optionally pre-treated natural lutein-containing product or pure lutein in a mixture of an aqueous solution of an alkali hydroxide and either dimethyl sulphoxide or an organic solvent based on saturated aliphatic and/or aromatic hydrocarbons at temperatures above about 50°C, the process being carried out in the presence of a phase transfer catalyst when an organic solvent based on hydrocarbons is used. Being carotenoids lutein and zeaxanthin are used correspondingly, especially as pigments for egg yolk, the integuments and the subcutaneous fat of poultry, the flesh and the integuments offish and crustaceans, as well as foodstuffs. Zeaxanthin is preferably used in many applications, since in comparable dosages it produces a more intensive golden yellow pigmentation than lutein.

Full Text

The present invention relates to a process for the conversion of lutein or of its esters, which occurs in nature into zeaxanthin. As is known, the xanthophylls lutein and zeaxanthin belong to the broad substance class carotenoids and as natural pigments are widely distributed in nature. They are present primarily in higher plants, algae, fish, crustaceans and bacteria. Lutein and zeaxanthin are often found together and in esterified form in the same source, although the ratio of lutein to zeaxanthin varies substantially depending on the source. Thus, lutein normally makes up a substantially larger amount than zeaxanthin when they occur together in plants, e.g. melons and marigolds [Tagetes (T.) erecta, T. patula and other Tagetes species] and in algae. As an exception there is to be mentioned the larger amount of zeaxanthin over lutein in maize, with the former being even the main representative of carotenoids present in maize grain. Moreover, the stereoisomerism of lutein and of zeaxanthin depends on the source. In plants sources (3R,3'R,6'R)-lutein or (3R,3!R)-zeaxanthin is primarily present, while in animal sources, e.g. in fish and crustaceans, lutein is present in the (3R,3'R,6'R)-, (3R,3'R,6'S)- and (SR^'S^'SHorm and zeaxanthin is present in the (3R,3*S)- and (3S,3'S)-form. Being carotenoids, lutein and zeaxanthin are used correspondingly, especially as pigments, e.g. for egg yolk, the integuments (for example, skin, legs and beak) and the subcutaneous fat of poultry, the flesh and the integuments (skin, scales and shell) of fish and crustaceans as well as foodstuffs. Zeaxanthin is preferably used in many applications, since in comparable dosages it produces a more intensive golden yellow pigmentation than lutein. When use is made of a plant raw material containing the two xanthophylls, such as, for example Tagetes erecta flowers, or of an optionally previously saponified extract thereof, as the source of the desired zeaxanthin, it is clearly economically more convenient, where possible, firstly to chemically convert the lutein into the zeaxanthin and then to isolate the latter from the mixture by extraction than to undertake the extraction without prior conversion. It is also of economical significance to chemically convert pure or almost pure lutein into zeaxanthin. As is known, the conversion of lutein into zeaxanthin can be effected under strongly basic conditions, and a variety of pertinent conversion processes have become known from the scientific literature. Already in the year 1946 Karrer and Jucker realised the isomerization of natural lutein (referred to as "xanthophyir) to zeaxanthin in an ethanol-benzene mixture by heating at 110-110°C with sodium ethanolate for 30 hours in an evacuated bomb tube [see Helv. Chim. Acta 30, 266-267 (1947)]. The yield of zeaxanthin was, however, extremely low. In this manner, no doubt for the first time, lutein, of the formula i , with the isolated double bond in the e-ring [(I), right] being brought into conjugation with the remaining double bonds of the molecule [(II), right: p- ring]. Later [1959, see Arch. Biochem. Biophys. 88, 59-63 (I960)], Kargl and Quackenbush accomplished the analogous base-catalyzed conversion of 8-carotene (e,\|/-carotene) into y-carotene (p,\|/-carotene). However, Buchecker et al. were unable to satisfactorily repeat the earlier work of Karrer and Jucker [Chimia 26, No.3, 134-136 (1972)]. Andrewes reported in Acta Chem. Scand. B 28, No. 1,137-138 (1974) the isomerization of lutein to zeaxanthin by heating a solution of lutein and potasssium methanolate in methanolic dimethyl sulphoxide at 118°C for 20 minutes in a closed tube under nitrogen, which gave a yield of only 10-15%. In a subsequent article (ibid, p. 139-140) Andrewes, Borch and Liaaen-Jensen showed that this isomerization was the conversion of OR^'R^'RHutein into (3R,3'S)-zeaxantJiin. Disadvantageous in the method of Andrewes was not only the still unsatisfactory yields of zeaxanthin, but also the finding established as a result of a re-working that numerous (Z)-isomers of not only zeaxanthin but also lutein are produced and that the reaction is therefore non-selective. Moreover, an isomerization which is effected in a closed reaction vessel is a reaction which cannot readily be controlled. Even when the conversion is carried out in an open system - under argon - it turns out that, after using potassium methylate as the base as previously, an uncontrollable (E) -* (Z) isomerization takes place. A further relevant publication, namely PCT Patent Publication WO 96/02594, has recently appeared. This describes a process for the isomerization of lutein to zeaxanthin in a strongly alkaline solution under controlled temperature and pressure conditions. The base is an alkali metal hydroxide, calcium hydroxide, sodium carbonate, ammonium hydroxide or an organic base, especially ethylamine, ethanolamine or morpholine. A solvent other than water is not used. An enrichment of the zeaxanthin from 4.5% (in the educt) to 15.8 to 24.0% has been said to be achieved by this process; it is, however, not mentioned how many decomposition products result in the isomerization. Also, this yield is not adequate for commercial purposes. The aim of the present invention is to enrich the content of zeaxanthin or to manufacture zeaxanthin in the highest possible yields starting from a natural product containing the two xanthophylls lutein and zeaxanthin (in each case optionally in esterified form) or even from pure or almost pure lutein. It has now surprisingly been found that this object can be achieved by a particular choice of reaction conditions, especially the choice of solvent and base. The yields of zeaxanthin can be decisively improved by this choice. The present invention provides a process for the conversion of lutein or of its esters occurring in nature into zeaxanthin by base-catalyzed isomerization, which process comprises heating an optionally pre-treated natural lutein-containing product or pure lutein in a mixture of an aqueous solution of an alkali hydroxide and either dimethyl sulphoxide or an organic solvent based on saturated aliphatic and/or aromatic hydrocarbons at temperatures above about 50°C, with the process being carried out in the presence of a phase transfer catalyst when an organic solvent based on hydrocarbons is used. As the natural lutein-containing product there is preferably used a lutein-containing raw material of plant origin, which optionally has been pre-treated chemically, e.g. by saponification. Such a raw material can be a concentrate or extract and is accordingly present in the form of a milled powder or a liquid or resinous material ("oleoresin"). Powders or extracts of the yellow or orange blossoms of marigolds (Tagetes erecta etc.), which contain not only (mainly) lutein but also zeaxanthin, are especially preferred for this purpose. According to Quackenbush et al. [J.AOAC, 55 (3), 617-621 (1972)] the lutein:zeaxanthin ratio in these raw materials is about 72-88:16-4. The xanthophylls are present there mainly as esters of palmitic, myristic and stearic acid [W. Gau et al., J. Chromatography 262, 277-284 (1983)]. Since it is preferred to convert the lutein in free form, such esters are conveniently previously saponified to the free lutein before the raw material is used in the process in accordance with the invention, with such saponifications as well as enzymatic hydrolyses carried out for the same purpose being known from the state of the art (see, for example, US Patent 3,535,138 and 3,783,099). Powder (flour) and saponified and non-saponified extracts of Tagetes have been available commercially for a long time and can be used as starting materials for the process in accordance with the invention. Examples of these raw materials are saponified Tagetes extracts from the mexican firms ALCOSA and IOSA. The ALCOSA product "pasta saponificada amarillo" is a brownish paste having a total xanthophyll content of, for example, about 2.7%, consisting of about 92.5% lutein and about 6.6% zeaxanthin, while the IOSA product "HI-GOLD 20" is a greenish-yellow powder, which contains, for example, about 0.96% total xanthophylls (about 65.5% lutein and about 27.2% zeaxanthin) (the percentages are area percentages). These details have been established following the analysis of certain production batches and can vary from batch to batch, which also applies to the analytical results given hereinafter. Other raw materials are "FLORA GLO" [Kemin Ind., Iowa, USA; about 739 g/kg total xanthophylls, of which about 681 g/kg consists of (all-E)-lutein and about 58 g/kg consists of (all E)-zeaxanthin], "ORO GLO Layer dry" (esterified, i.e. non-saponified; likewise Kemin Ind.; about 19.4 g/kg total xanthophylls: about 17.6 g/kg lutein and about 1.8 g/kg zeaxanthin), "HI-GOLD 20 Lutexan"(IOSA; about 16.3 g/kg total xanthophylls: about 11.0 g/kg lutein and about 5.3 g/kg zeaxanthin) as well as "XANTOPINA PLUS" (esterified; Bioquimex S.A., Mexico; about 351 g/kg total xanthophylls: about 330 g/kg lutein and about 21 g/kg zeaxanthin). The commercially obtainable raw material can be processed before use, if desired, in order to concentrate these xanthophylls, for example by extraction, followed by chromatography and optionally also subsequent crystallization. A typical concentration method comprises extracting the raw material with acetone, subjecting the extract to flash chromatography on silica gel using a hexane/ethyl acetate mixture and then pure ethyl acetate as the eluting agent, concentrating the fractions obtained and crystallizing the solid resulting therefrom, e.g. from a mixture of methylene chloride and methanol. By using this method it has been possible to obtain, for example, from 100 g of "pasta saponificada amarillo" (ALCOSA; containing about 2.7% total xanthophylls) about 2.4 g of crystalline material consisting of about 93% (all-E)-(3R,3'R,6'R)-lutein and about 7% (all-E)-(3R,3,R)-zeaxanthin, This product is an example of a pre-treated natural lutein-containing product. An organic solvent based on saturated aliphatic and/or aromatic hydrocarbons is used as the (organic) solvent in the process in accordance with the invention as an alternative to dimethyl sulphoxide. This is especially a liquid alkane or an aromatic hydrocarbon, or a mixture of two or more of these hydrocarbons, e.g. a mixture of several liquid alkanes, of several aromatic hydrocarbons or of one or more of such alkanes with one or more aromatic hydrocarbons. The liquid alkane is especially a straight-chain or branched alkane with at least 5 carbon atoms, preferably with 5-10 carbon atoms, such as, for example, pentane, hexane or heptane. Petroleum ether, preferably high-boiling petroleum ether, is an especially suitable alkane mixture and benzene and toluene are especially suitable aromatic hydrocarbons. As is known, dimethyl sulphoxide is soluble in water, and thus an aqueous-organic solution readily forms with the aqueous alkali hydroxide solution which is also used. In contrast thereto, the alkanes and aromatic hydrocarbons are (almost) insoluble in water, and thus when they are used a phase transfer catalyst must be employed. The alkali hydroxide is conveniently sodium hydroxide or potassium hydroxide, preferably the latter, the concentration of which in the aqueous solution being suitably at least 3 molar (M). Even aqueous solutions having concentrations up to saturation can be used. The concentration of the aqueous alkali hydroxide solution preferably lies in the range of about 7M to about 14M. Tricaprylmethylammonium chloride (Aliquat® 336), tetra(n-butyl)-ammonium hydrogen sulphate, various alkylbenzyldimethyl ammonium chlorides (benzalkonium chloride), benzyltri(n-butyl)ammonium bromide and tri(n-butyl)methylammonium iodide are examples of phase transfer catalysts used in accordance with the invention. Tricaprylmethylammonium chloride is preferably used as the phase transfer catalyst. With respect to ratios, there are conveniently used per mol of (calculated) xanthophyll(s) about 10 to about 450 mol of base (alkali hydroxide) and - where required - about 0.5 to about 5 mol of phase transfer catalyst, preferably about 200 to about 250 mol of base and, respectively, about 0.5 to about 1.5 mol of phase transfer catalyst. The dimethyl sulphoxide: aqueous alkali hydroxide solution volume ratio is generally about 4:1 to about 1:2, preferably about 2.5:1 to about 1:1. When an organic solvent based on saturated aliphatic and/or aromatic hydrocarbons is used, the respective organic solvent: aqueous alkali hydroxide solution volume ratio is generally about 8:1 to about 1:2, preferably about 4:1 to about 1:1. It follows that, having regard to these convenient ratios as well as other factors, especially the amount and composition of xanthophylls in the optionally pre-treated natural lutein-containing product or the amount of lutein itself (starting material), there are generally used about 50 to 300 ml of solvent-alkali hydroxide solution mixture per 10 g of xanthophyll-containing starting material, preferably about 60 to 80 ml per 10 g. Having regard to the fact that the formation of undesired byproducts and the decomposition of the desired zeaxanthin are promoted at too high temperatures and/or too long a reaction period, the conversion in accordance with the invention is carried out at temperatures which as far as possible do not exceed 120°C, i.e. the conversion is conveniently carried out at temperatures in the range of about 50°C to about 120°C. Preferably, the conversion is carried out at temperatures in the range of about 80°C to about 100°C. The reaction period depends, inter alia, on the reaction temperature, the amount and concentration of aqueous alkali hydroxide solution and the amount and nature of the organic solvent used. In general, this period is about 5 to about 65 hours. However, the conversion preferably does not take longer than about 24 hours. Under the above basic reaction conditions not only the lutein present in the educt is converted into the desired zeaxanthin in good yield, but esters of lutein and/or zeaxanthin which may be present, e.g. the aforementioned palmitic, myristic and stearic esters, are for the most part converted into free zeaxanthin. The working up of the mixture after completion of the conversion is effected in a simple manner, conveniently by cooling the mixture to room temperature or about 0°C, optionally adding an alcohol, preferably methanol or ethanol, and filtering off the solid, which mainly consists of enriched zeaxanthin. Moreover, conventional purification techniques, such as, for example, extraction, column chromatography and recrystallization, can be used. The last three treatments are required especially when a phase transfer catalyst is used. A recrystallization which may be carried out is effected especially well using a mixture of methylene chloride and methanol. The present invention is illustrated by the following Examples: Example 1 Pre-treatment of the starting material 300 g of "pasta saponificada amarillo'1 [Industrias ALCOSA S.A. DE C.V.; 23.9 g/kg (all-E)-lutein and 3.1 g/kg (all-E)-zeaxanthin] are suspended in 1.5 1 of acetone and the suspension is stirred at room temperature for 30 minutes. Then, 200 g of Dicalite® (filter aid; Dicalite Europe Nord S.A.) are added thereto and the mixture is stirred for a further 5 minutes. The resulting suspension is then filtered through a layer of Dicalite®* the Dicalite® layer is suspended in 780 ml of acetone and the new suspension is stirred at room temperature for 30 minutes. After filtration of the suspension through a Dicalite® layer this is washed six times with 50 ml of acetone each time. The combined filtrates are concentrated under reduced r pressure at 35°C, and in this manner there are obtained 143 g of a dark, red paste. The paste is then subjected to a flash chromatography on 1 kg of silica (mesh 70-230) using 4 1 of hexane/ethyl acetate (2:1), followed by 1.3 1 of ethyl acetate. The ethyl acetate fraction is concentrated under reduced pressure at 35°C, which gives 20.6 g of a red, semi-crystalline oil. This oil is then crystallized from a methylene chloride/methanol mixture and the crystallizate is dried for 6 hours under a high vacuum at 37°C. In this manner there are obtained 6.36 g of a mixture of (all-E)-lutein and (all-E)-zeaxanthin (93:6 area percent) as dark red crystals. Example 2 Conversion of lutein into zeaxanthin General procedure: The solvent dimethyl sulphoxide or liquid alkane or alkane mixture is added to a xanthophyll-containing material [for example, a mixture of lutein and zeaxanthin obtained according to Example 1, a commercially obtainable saponified, zeaxanthin-enriched Tagetes extract "oleoresina amarillo saponificada" (ALCOSA) or "HI GOLD 20" (IOSA), or a likewise commercially obtainable non-saponified Tagetes extract from the US company Kemin Industries, Iowa] in a round flask equipped with a reflux condenser, stirrer and thermometer. When the liquid alkane or alkane mixture is used, the phase transfer catalyst is then added. Subsequently, the aqueous solution of the alkali hydroxide is added thereto and the reaction mixture is heated. In order to follow the course of the reaction, a sample of the reaction mixture (solution or suspension) can be removed periodically, diluted with methylene chloride, the methylene chloride solution washed to neutrality with saturated aqueous ammonium chloride solution and then with water, subsequently dried over anhydrous sodium sulphate and finally the solution subjected to a HPLC analysis. After completion of the reaction has been established (no further conversion lutein —> zeaxanthin) the homogeneous or heterogeneous solution is cooled to room temperature or 0°C while stirring, with an alcohol, e.g. methanol or n-propanol, optionally being added during the stirring and cooling. The resulting suspension is filtered through a paper or glass fibre filter, the solid is washed several times with the chosen alcohol, is dried under a high vacuum at about 37°C for at least one hour and, if desired, recrystallized from methylene chloride/methanol or methylene chloride/hexane. Procedure and results using the starting material "oleoresina amarillo saponificada11: The starting material contains 23.9 g/kg lutein and 3.1 g/kg zeaxanthin. After extraction, chromatography and crystallization there is obtained a mixture which consists of 93.1% lutein, 6.6% zeaxanthin and 0.5% additional material. The results of the conversion carried out according to the above procedure are compiled in Table 1 hereinafter: Additional remarks: In all cases with the exception of experiments (h) and (h') 100 mg of Aliquat ® 336 (Fluka Chemi AG, Buchs, Switzerland) are added to the reaction mixture as the phase transfer catalyst, since hexane, heptane or petroleum ether is used as the organic solvent; in experiments (h) and (h') 300 mg of Aliquat ® 336 are added. In the case of experiment (a) the temperature is that of the heating bath; in all other cases the actual temperature of the reaction mixture is given. Procedure and results with the starting material "oleoresina amarillo saponificada" (direct use) The starting material has the above composition and is not extracted, chromatographed and crystallized, but used immediately. The results of the conversion (without phase transfer catalyst) carried out according to the above procedure are compiled in Table 2 hereinafter: Procedure and results with the starting material FLORA GLO The starting material has a relative (percentage) composition of 91.3% (all-E)-lutein and 6.6% (all-E)-zeaxanthin and a total xanthophyll content of 73.9% (739 g/kg crude material). This material is already saponified, so that the probability of side-reactions is low. According to the above procedure (without phase transfer catalyst) the crude material is stirred in a mixture of dimethyl sulphoxide (DMSO) and concentrated aqueous potassium hydroxide solution (KOH) at about 80°C or above this temperature. It is noticeable that the reaction takes place very cleanly, i.e. practically from educt to product. Also, the working up of the reaction solution is simple: the lutein-zeaxanthin mixture crystallizes from the solution (cooled to 0°C) and is easily filtered off and rinsed with water in order to remove for the most part traces of excess base. The results of the conversion carried out in this manner are compiled in Table 3 hereinafter: 1 HPLC area percent at 450 nm 2 It was not determined which anhydroluteins and other carotenoids made up this fraction 3 Results of quantitative HPLC analysis 4 Addition of the base only after the DMSO-FLORA GLO suspension had reached 80°C. 5 Addition of the base only after the DMSO-FLORA GLO suspension had reached 90°C. 6 The higher value was obtained in each case after 0.5 and 1 hour by the addition of an aliquot of the reaction solution to methanol, subsequent stirring of the suspension, filtration and HPLC analysis. Determination of the lower value as already described. Procedure and results using the starting material XANTOPINA PLUS The starting material has a composition of 90.9% lutein and 5.3% zeaxanthin in esterified form; the remainder consists of (Z)-isomers of lutein esters and a small amount of anhydrolutein esters. The xanthophyll content of XANTOPINA PLUS is 35.1%. The results of the conversion (without phase transfer catalyst) carried out according to above procedure are compiled in Table 4 hereinafter: WE CLAIM: 1. A process for the conversion of lutein or of its esters which occur in nature into zeaxanthin by base-catalyzed isomerization, which process comprises heating an optionally pre-treated natural lutein-contaming product or pure lutein in a mixture of an aqueous solution of an alkali hydroxide and either dimethyl sulphoxide or an organic solvent based on saturated aliphatic and/or aromatic hydrocarbons at temperatures above about 50°C, with the process being carried out in the presence of a phase transfer catalyst when an organic solvent based on hydrocarbons is used. 2. The process according to claim 1, wherein a lutein-containing raw material of plant origin, which has optionally been pre-treated chemically, e.g. by saponification, is used as the natural lutein-containing product. 3. The process according to claim 2, wherein a powder or extract of the yellow or orange blossoms of marigolds is used as the natural lutein-containing product. 4. The process according to anyone of claims 1 to 3, wherein pentane, hexane, heptane, petroleum ether, benzene or toluene, or a mixture of two or im.re of these solvents, is used as the organic solvent based on hydrocarbons. 5. The process according to anyone of claims 1 to 4, wherein sodium hydroxide or potassium hydroxide, preferably potassium hydroxide, is used as the alkali hydroxide. 6. The process according to anyone of claims 1 to 5, wherein the concentration of the aqueous alkali hydroxide solution is at least 3M. 7. The process according to claim 6, wherein the concentration of the aqueous alkali hydroxide solution is about 7M to aboui 14M. 8. The process according to anyone of claims 1 to 7, wherein the phase transfer catalyst is tricaprylmethylammonium chloride. 9. The process according to anyone of claims 1 to 8, wherein the conversion is carried out at temperatures in the range of 50°C to 120°C. 10. The process according to claim 9, wherein the conversion is carried out at temperatures in the range of 80°C to 100°C. 11. The process for the conversion of lutein or of its esters substantially as herein described, and exemplified.

Documents:

1803-mas-1997-abstract.pdf

1803-mas-1997-assignment.pdf

1803-mas-1997-claims.pdf

1803-mas-1997-correspondence others.pdf

1803-mas-1997-correspondence po.pdf

1803-mas-1997-description complete.pdf

1803-mas-1997-form 1.pdf

1803-mas-1997-form 26.pdf

1803-mas-1997-form 3.pdf

1803-mas-1997-form 6.pdf