WO2008023283A2

WO2008023283A2 - Stabilized esters of lutein

Info

Publication number: WO2008023283A2
Application number: PCT/IB2007/004104
Authority: WO
Inventors: Thomas Eidenberger
Original assignee: Omnica Gmbh
Priority date: 2006-08-25
Filing date: 2007-08-24
Publication date: 2008-02-28
Also published as: WO2008023283A3

Abstract

The invention describes lipoic acid or dihydrolipoic acid esters of xanthophylls, such as lutein, that have improved stability and bioavailablity versus simple mixtures of lipoic or dihydrolipoic acid and the xanthophyll.

Description

STABILIZED ESTERS OF LUTEIN

FIELD OF THE INVENTION

[001] The invention relates generally to compositions, methods to prepare, isolate and purify xanthophylls such as that of lutein, zeaxanthin and related compounds that are esterified with specific nutritionally beneficial carboxylic acids or esters thereof that provide a stabilizing effect from environmental factors, such as degradation, as well as advantageously providing value as a nutritional ingredient upon hydrolysis with digestion.

BACKGROUND OF THE INVENTION [002] Carotenoids are yellow, red and orange pigments that are widely distributed in nature. Although specific carotenoids have been identified in various fruits and vegetables, bird feathers, egg-yolk, poultry skin, crustaceans and macular eye region, they are especially abundant in marigold petals, corn and leafy vegetables. The correlation between dietary carotenoids and carotenoids found in human serum and plasma indicate that only selected groups of carotenoids make their way into the human blood stream to exert their effect.

[003] Carotenoids absorb light in the 400-500 nm region of the visible spectrum. This physical characteristic imparts the yellow/red color to the pigments. Carotenoids contain a conjugated backbone composed of isoprene units, which are usually inverted at the center of the molecule, imparting symmetry. Changes in geometrical configuration about the double bonds result in the existence of many cis- and trans-isomers. Mammalian species do not synthesize carotenoids and therefore these have to be obtained from dietary sources such as fruits, vegetables and egg yolks. In the recent years, carotenoids have been attributed several health benefits, which include prevention and or protection against serious health disorders.

[004] Carotenoids are non-polar compounds classified into two sub-classes, namely more polar compounds called xanthophylls or oxy-carotenoids and non-polar hydrocarbon carotenes like [beta] -carotene, lycopene, etc. Both the sub-classes have at least nine conjugated double bonds responsible for the characteristic color of the carotenoids. Xanthophylls have ring structures at the end of the conjugated double bond chain with polar functionalities, such as hydroxyl or keto groups. Examples of xanthophylls include lutein, zeaxanthin, capsanthin, canthaxanthin, β-cryptoxanthin, astaxanthin, etc. As natural colorants and also for their role in human health, xanthophylls containing lutein and zeaxanthin have attracted the renewed attention of scientists and researchers in the biomedical, chemical and nutritional field in recent years.

[005] Lutein and zeaxanthin contribute to yellow and orange-yellow color respectively. Lutein and zeaxanthin can be present in plant material in free form (non- esterified) and also as esters. Lutein is present in green leafy vegetables like spinach, kale and broccoli in the free form while fruits like mango, orange, papaya, red paprika, algae and yellow corn. These sources generally contain lutein in the form of its fatty esters. Lutein is also present in the blood stream and various tissues in human body and particularly the macula, lens and retina of the eye. [006] Marigold (Tagetes erecta) flower petals are a rich source of lutein in its esterified form. The ester portion(s) are fatty acids. Dried marigold flowers contain approximately 1-1.6% carotenoids by weight and lutein esters content accounts for 90% of the total carotenoids. The xanthophyll fatty acid esters composition in marigold oleoresin chiefly consists of lutein in its ester form as di-palmitate, myristate-palmitate, palmitate-stearate, dimyristate and monoesters.

[007] Lutein obtained by the hydrolysis of lutein esters from marigold has been found to be identical to the lutein found in fruits, vegetables and in human plasma and the macular region. After absorption, the human body cannot distinguish the source of lutein. Therefore, a widely cultivated and commercially processed raw material like marigold, which is already used by the food and feed industry, is an attractive source for lutein in view of abundant availability and cost considerations.

[008] Essentially, lutein esters and lutein in the free form are commercially important nutraceuticals obtained from marigold flowers. Dried flowers are used for obtaining marigold extract or oleoresin. By subjecting the extract/oleoresin to saponification, xanthophylls in the free form are obtained. The resultant alkali salts of fatty acids obtained from the saponification are removed and the xanthophyll containing mixture of lutein and zeaxanthin purified further.

[009] In the fresh marigold flowers, lutein esters exist in trans-isomeric form, whereas exposure to heat, light, oxygen, acid, etc. catalyses isomerization from trans- to cis-lutein geometric isomeric forms. As a nutraceutical and food additive, the trans- isomeric form of lutein is preferred because of better bioavailability and deeper yellow color compared to the corresponding cis-isomeric form.

[010] The free form of lutein (de-esterified) is unstable against the effects of heat and light. In its natural state, lutein is found esterified with fatty acids. Therefore, lutein is generally utilized and isolated in a form that includes fatty acids to provide esters of lutein, which are generally stable against environmental conditions. The saturated fatty acids have no value as a nutritional ingredient. Only the lutein has value as a nutritional ingredient. [011] Therefore, a need exists for a process to prepare esters of lutein that provide stability against environmental conditions such as light, oxygen, acid, etc. and further provide a beneficial nutritional value to the recipient.

BRIEF SUMMARY OF THE INVENTION

[012] The present invention surprisingly provides esterified xanthophyll materials, such as lutein and zeaxanthin, wherein a nutritionally beneficial carboxylic acid derivative is used to esterify the hydroxyl containing xanthophyll. The mono, di, or nutritionally beneficial triesterified xanthophyll provides a doubly advantageous nutritional effect upon digestion. That is, upon uptake by the recipient, hydrolysis can occur such that a free xanthophyll, for example lutein, is liberated from the ester as well as the nutritionally beneficial carboxylic acid from the ester. The carboxylic acid is selected such that the carboxylic acid also has nutritional value to the recipient. Such carboxylic acids include lipoic acid (LA) and dihydrolipoic acid (DHLA). Up until the present invention, esterified xanthophylls were composed of the free xanthophyll and a fatty acid. Fatty acids typically do not provide a nutritional benefit to the recipient. [013] The yields of the xanthophyll esters produced by the processes of the invention are extremely high, generally being at least 85% or better in terms of overall yield from starting material.

[014] In one embodiment, the present invention provides an esterified xanthophyll that is a condensation product (esterified) of a free xanthophyll having at least one hydroxyl moiety and at least one nutritionally beneficial activated carboxylic acid derivative. The resultant product of the condensation reaction is an ester.

[015] In another embodiment, the present invention provides an esterified xanthophyll that is a condensation product of a partially or fully hydrolyzed xanthophyll that has at least one hydroxyl moiety and at least one nutritionally beneficial activated carboxylic acid derivative. The resultant product of the condensation reaction is an ester.

[016] hi still another embodiment, the present invention provides a xanthophyll ester comprising the formula:

-5-

or

[017] wherein each R¹, R², R³, individually if present, is a hydrogen, an acyl residue of a fatty acid, or an acyl residue of a nutritionally beneficial carboxylic derivative, provided at least one of R¹, R², R³, when present, is an acyl residue of a nutritionally beneficial carboxylic derivative or mixtures thereof.

[018] Dependent upon the hydroxyl content of the starting xanthophyll, the product can be a mono, di, or tri (or more) ester.

[019] Therefore, suitable examples of hydroxyl containing xanthophylls include, but are not limited to, lutein, zeaxanthin, capsanthin, β-cryptoxanthin, astaxanthin, antheraxanthin, diatoxanthin, 7,8-didehydroastaxanthin, fucoxanthin, fucoxanthinol, lactucaxanthin, neoxanthin, peridinin, siphonaxanthin, violaxanthin, etc.

[020] Additionally, the some xanthophylls contain enolizable ketone, such as for example, canthaxanthin, alpha-carotene, etc., that can be reacted in the enol form with a carboxylic acid derivative such that the enol is captured by the carboxylic acid derivative to form an enolate. A suitable example of such xanthophyll containing ketones include canthaxanthin.

[021] The present invention also provides a method to prepare an esterified xanthophyll by treating a free xanthophyll having at least one hydroxyl moiety, with at least one nutritionally beneficial activated carboxylic acid derivative under conditions such that esterification occurs.

[022] The present invention further provides a method to prepare an esterified xanthophyll by treating a partially or fully hydrolyzed xanthophyll having at least one hydroxyl moiety with at least one nutritionally beneficial activated carboxylic acid derivative under conditions such that esterification occurs.

[023] The present invention also provides a method to prepare an esterified xanthophyll comprising the step of treating one or more of initial compounds having a formula

-8-

-9- or

[024] wherein each R¹, R², R³, individually if present, is a hydrogen or an acyl residue of a fatty acid provided at least one of R , R , R , when present, is a hydrogen, with a nutritionally beneficial carboxylic acid derivative under conditions suitable for esterification to occur, such that esterification provides a product wherein R¹, R², R³, individually if present, is a hydrogen, an acyl residue of a fatty acid, or an acyl residue of a nutritionally beneficial carboxylic derivative, provided at least one of R¹, R², R³, when present, is an acyl residue of a nutritionally beneficial carboxylic derivative or mixtures thereof. [025] hi particular embodiments, the nutritionally beneficial carboxylic acid derivative is lipoic acid or dihydrolipoic acid.

[026] hi still yet another embodiment, the present invention provides a method of preventing or inhibiting free radical oxidation in a mammal, the method comprising administering an antioxidative amount of an esterified compound as described herein. [027] While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. As will be apparent, the invention is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present invention. Accordingly, the detailed descriptions are to be regarded as illustrative in nature and not restrictive. DETAILED DESCRIPTION

[028] The present invention relates to the preparation, isolation and purification of certain esterified oxygenated carotenoids from various plant sources as described herein. That is, the carotenoids need to have at least one free hydroxyl that can be esterified with at least one nutritionally beneficial carboxylic acid derivative. [029] In the specification and in the claims, the terms "including" and

"comprising" are open-ended terms and should be interpreted to mean "including, but not limited to. . . . " These terms encompass the more restrictive terms "consisting essentially of and "consisting of." [030] Carotenoids are a class of hydrocarbons (carotenes) and the corresponding oxygenated derivatives are xanthophylls. They consist of eight isoprenoid units joined in such a manner that the arrangement of isoprenoid units is reversed at the center of the molecule so that the two central methyl groups are in a 1,6- position relationship and the remaining nonterminal methyl groups are in a 1,5-position relationship. All carotenoids may be formally derived from the acyclic C₄₀H₅₆ structure (I) (Compound I), having a long central chain of conjugated double bonds, by (I) hydrogenation, (2) dehydrogenation, (3) cyclization, or (4) oxidation, or any combination of these processes. The class also includes compounds that arise from certain rearrangements or degradations of the carbon skeleton (I) (lycopene), provided hat the two central methyl groups are retained.

[031] About 600 carotenoids have been isolated from natural sources. These carotenoids have been listed with their trivial and semisystematic names in Key to Carotenoids (Pfander, 1987) and in the Appendix of Carotenoids, Volume IA (KuIl & Pfander 1995) which also includes literature references for their spectroscopic and other properties. The structure is still uncertain for many of the carotenoids, including stereochemical assignments, hi the cases where the structure is uncertain, resolution, followed by structural elucidation with modern spectroscopic methods (including high resolution nuclear magnetic resonance (NMR) spectroscopy) is necessary. About 370 of the naturally occurring carotenoids are chiral, bearing from one to five asymmetric carbon atoms, and in most cases one carotenoid occurs only in one configuration in

Nature.

[032] AU specific names of cartenoids are based on the stem name carotene, which corresponds to the structure and numbering as in Compound II (carotene).

[033] The name of a specific compound is constructed by adding two Greek letters as prefixes (Compound fragments 3) to the stem name carotene. The Greek letter prefixes are cited in alphabetical order noted in compounds Ha.

Ha [034] The oxygenated carotenoids (xanthophylls) most frequently include hydroxy, methoxy, carboxy, oxo, and epoxy functionality. Important and characteristic carotenoids (Compounds III through X) are lycopene (gamma, gamma-carotene) (I), beta-carotene (beta, beta-carotene) (III), alpha-carotene ((6'R)-beta, epsilon-carotene) (IV), beta-cryptoxanthin ((3R)-beta,beta-caroten-3-ol) (V), zeaxanthin ((3R,3'R)-beta, beta carotene-3,3'-diol) (VI), lutein ("xanthophyll", (3R,3'R,6'R)-beta, epsilon-carotene- 3,3'-diol) (VII), neoxanthin ((3S,5R,6R,3^tS,5¹R,6'S)-5',6'-epoxy-6,7-didehydro-5,6,S',6^I- tetrahydro-beta,beta-carotene-3,5,3'-triol) (VIII), violaxanthin ((3S,5R,6R,3'S,5'R,6'S)- 5,6,5',6'-diepoxy-5,6,5',6'-tetrahydro-beta,beta-carotene-3,3'-diol) (IX), fucoxanthin ((3S,5R,6S,3'S,5'R,6'R)-5,6-epoxy-3,3^5'-trihydroxy-6^7'-didehydro-5,6,7,8,5^6'- hexahydro-beta,beta-caroten-8-one 3'-acetate) (X), canthaxanthin (beta,beta-carotene- 4,4'-dione) (XI), and astaxanthin ((3S,3'S)-3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) (XII).

SIV

[035] Normally carotenoids occur in Nature as the (all-E)-isomer. Some carotenoids undergo isomerization very easily during processing. For processing, it must be kept in mind that (E/Z)-isomerization can occur when a carotenoid is kept in solution. Normally the percentage of the (Z)-isomers is rather low, but it is enhanced at higher temperatures. Furthermore, the formation of (Z)-isomers is increased by exposure to light.

[036] In commercial practice, xanthophylls of food grade quality and free of Z- lutein isomers are seldom achieved because of lack of selectivity in the raw material and improper processing conditions including high temperature drying. This results in the formation of xanthophylls of food grade quality but having higher levels of Z-lutein. The present invention avoids such increased levels of undesired Z-lutein, in part because of the relatively low temperatures used in the processes. [037] Humans and animals cannot synthesize xanthophylls like lutein and zeaxanthin, and the source of this has to be from diet. The occurrence of lutein and zeaxanthin in the macula has specific functions, viz., protection of the cells and tissues from ultra-violet light and reduced cataract risk. Lutein and zeaxanthin are known to comprise the macular pigment and lutein isomerizes into zeaxanthin in the macula. [038] There is evidence suggesting that lutein may have a protective effect against cancers of the breast, colon, lung, skin, cervix and ovaries and could bear promise in treatment of cardiovascular disease. Therefore, providing lutein to an individual for use in their diet or as nutritional supplements supports better human health and healthy vision. [039] Therefore, there is a high demand for xanthophyll materials for use as antioxidants, prevention of cataract and macular degeneration, as lung cancer- preventive agents, as agents for the absorption of harmful ultra-violet light from the rays of the sun and quencher of photo-induced free radical and reactive oxygen species, etc. [040] The present invention relates to esterification processes for producing esterified xanthophylls having enhanced nutritional benefits, and in particular, esters of lutein. Up until the time of this invention, lutein was generally provided as an extract in the form of fatty acid esters. Fatty acids, except for omega type fatty acids, are generally not nutritionally beneficial to the recipient.

[041] The term "xanthophyll ester" is intended to include the mono-, di-, or tri-

(or more) esters of "free" xanthophyll. Typically the plant source contains the xanthophyll in the esterified form as a mono- or di-C12 -C 18 long chain, fatty acid such as lauric, myristic, oleic, linolenic and/or palmitic acids. Lutein in marigold flowers, zeaxanthin in wolfberries and capsanthin and capsorubin in pepper plants are present as xanthophyll diesters. The free or non-esterified xanthophyll can be found in other plants such as spinach, broccoli, kale and corn. In the present invention, at least one nutritionally beneficial carboxylic acid residue such as lipoic acid (LA) or dihydrolipoic acid (DHLA) has replaced at least one of the fatty acid moieties.

[042] The term "free xanthophyll" (or free lutein, etc.) is intended to mean a carotenoid having a hydroxyl portion that remains after hydrolysis of the xanthophyll ester. [043] The phrase "partially hydro lyzed xanthophyll " is intended to mean a xanthophyll material originally exists as in fatty acid ester form (having as many as 3 or more fatty acid residues) that has been treated such that one or more (preferably all) of the fatty acid esters of the xanthophyll have (has) been hydrolyzed from the xanthophyll to provide a xanthophyll material that has at least one free hydroxyl group, and in particular, all fatty acid residues hydrolyzed from the xanthophyll, thereby providing a xanthophyll that is partially or fully hydrolyzed. Therefore, there is at least one hydroxyl group that is available for esterification with a carboxylic acid derivative. [044] The phrase "nutritionally beneficial carboxylic acid derivative" or residue is intended to mean that the carboxylic acid (in whatever form) has a nutritional value in and of itself. That is, the carboxylic acid, its ester, amide, etc. form when ingested by a recipient would receive a nutritional benefit due to the ingestion and, optionally, hydrolysis of the carboxylic acid derivative.

[045] The phrase "carboxylic acid derivative" is intended to mean those materials known in the art that provide a carboxylic acid or an equivalent species that can react with a hydroxyl group. Such moieties include, for example, carboxylic acids per se, carboxylic esters, carboxylic amides, carboxyl halides (acyl halides), anhydrides, activated carboxylic acid intermediates such as carboxyl imidazoles, tosyl carboxylates, etc. that are known in the art to provide a carboxylic species that can react with a hydroxyl group to afford an ester upon condensation (esterification). [046] The term alpha lipoic acid is intended to mean the compound represented by the formula:

[047] as one of its enantiomers or racemic form. [048] Alpha-lipoic acid was first isolated as an acetate replacing factor. It is slightly soluble in water and soluble in certain organic solvents. Alpha-lipoic acid was initially identified as a vitamin after its isolation, but it was later found to be synthesized by mammals, including humans, as well as by plants. The complete enzyme pathway that is responsible for the de novo synthesis has not yet been definitively elucidated. Several studies have indicated that octanoate serves as the immediate precursor for the 8-carbon fatty acid chain, and cysteine appears to be the source of sulfur. As an amide (lipoamide), it functions as a cofactor in the multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids such as pyruvate, alpha-keto glutarate, and branched chain alpha-keto acids.

[049] Alpha-lipoic acid is one of the strongest naturally occurring antioxidants.

Alpha-lipoic acid (LA) is also known as thioctic acid, l,2-dithiolane-3-pentanoic acid, l,2-dithiolane-3-valeric acid and 6,8-thioctic acid. Alpha-lipoic acid has a chiral carbon atom and occurs in two enantiomeric forms (R- and S-). The form of alpha-lipoic acid sold in stores is a synthetic mixture of the natural isomer (R-) and the unnatural isomer (S-). The natural form of R-LA is not as stable as the synthetic mixture. One manufacturer, Asta Medica, sells R-LA for diabetes and has made a stable form of R- LA by crystallizing it with Tris buffer, a commonly used synthetic, but unnatural, buffer. [050] Various enantiomeric forms of alpha-LA, and combinations and derivatives thereof (including its reduced form), have been used to treat numerous conditions. For example, LA's have been used in the treatment of circulatory disorders. LAs and vitamins have been found useful for producing analgesic, anti-inflammatory, antinecrotic, anti-diabetic and other therapeutic effects. Certain alkylated derivatives of LA have been used in treatment of retroviral diseases.

[051] Alpha-lipoic acid, and its reduced form, dihydrolipoic acid (DHLA) have antioxidant properties. Lipoate (a term for carboxylic acid esters and salts), or its reduced form, DHLA, reacts with reactive oxygen species such as superoxide radicals, hydroxyl radicals, hypochlorous acid, peroxyl radicals, and singlet oxygen. It also protects membranes by interacting with vitamin C and glutathione, which may in turn recycle vitamin E. In addition to its antioxidant activities, DHLA may exert prooxidant actions to reduction of iron. Alpha-lipoic acid administration has been shown to be beneficial in a number of oxidative stress models such as ischemia-reperfusion injury (IRI), diabetes (both alpha-lipoic acid and DHLA exhibit hydrophobic binding to proteins such as albumin, which can prevent glycation reactions), cataract formation, HIV activation, neurodegeneration, and radiation injury. Furthermore, lipoate can function as a redox regulator of proteins such as myoglobin, prolactin, thioredoxin, and NF-kappa-B transcription factor.

[052] Lipoates may also have other activities. For example, DHLA has been found in vitro to be an anti-inflammatory agent, which at the same time interferes with nitric oxide release from inflammatory macrophages and protects target cells from oxygen radical attack.

[053] Lipoic acid is also a coenzyme for several enzymes. Lipoic acid is a coenzyme for both alpha-keto acid dehydrogenase complex enzymes (i.e. pyruvate dehydrogenase complex and alpha-keto glutarate dehydrogenase complex), branched chain alpha-keto acid dehydrogenase complex, and the glycine cleavage system. In the enzyme system, the body forms a multi-enzyme complex involving lipoic acid, that breaks down molecules of pyruvate produced in earlier metabolism, to form slightly smaller, high energy molecules, called acetyl-coenzyme A. This results in molecules that can enter into a series of reactions called the citric acid cycle, or Krebs cycle, which finishes the conversion of food into energy. Essentially, lipoic acid stimulates basal glucose transport and has a positive effect on insulin stimulated glucose uptake. [054] Under physiological conditions, LA exists as lipoamide in at least five proteins where it is covalently linked to a lysyl residue. Four of these proteins are alpha-ketoacid dehydrogenase complexes, the pyruvate dehydrogenase complex, the branched chain keto-acid dehydrogenase complex and the alpha-ketoglutarate dehydrogenase complex. Three lipoamide-containing proteins are present in the E2 enzyme dihydrolipoyl acyltransferase, which is different in each of the complexes and specific for the substrate of the complex. One lipoyl residue is found in protein X, which is the same in each complex. The fifth lipoamide residue is present in the glycine cleavage system.

[055] Recently LA has been detected in the form of lipoyllysine in various natural sources. In the plant material studied, lipoyllysine content was highest in spinach. When expressed as weight per dry weight of lyophilized vegetables, the abundance of naturally existing lipoate in spinach was over three- and five-fold higher than that in broccoli and tomatoes, respectively. Lower concentrations of lipoyllysine were also detected in garden pea, Brussels sprouts and rice bran. [056] In animal tissues, the abundance of lipoyllysine in bovine acetone powders can be represented in the following order of 1) kidney, 2) heart, 3) liver, 4) spleen, 5) brain, 6) pancreas and 7) lung.

[057] α-lipoic acid is known as an active pharmaceutical agent for treating various diseases, such as liver diseases or diabetic and alcoholic polyneuropathy. DE 198 18 563 discloses the use of α-lipoic acid or its salts for reducing appetite and/or reducing body weight. Therefore, lipoic acid (in combination with the xanthophyll) provides a nutritional benefit to provide treatment to such diseases. [058] LA suffers from certain disadvantages, however. In particular, the natural form R-LA is unstable above 40°C, so it can degrade under some warehousing conditions. Also LA is hygroscopic. The present invention surprisingly provides an ability to provide stabilization of this natural form of LA with a coganer such as a xanthophyll, while providing the benefit of the xanthophyll. [059] Dihydrolipoic acid (DHLA) is a constituent of cellular metabolism. DHLA has two thiol residues that make is susceptible to radical species, thus provides antioxidant functionality to the biomolecule. Oxidation reduction (redox reactions) involves the transfer of an electron from a donor to an acceptor. When the donor loses an electron, it is transformed from its reduced form to its oxidized form. When an acceptor gains an electron, it changes from its oxidized form to its reduced form. Together, the oxidized and reduced forms of a redox component, such as lipoic acid and DHLA or CoQ-10 (ubiquinone) and reduced CoQ-10 (ubiquinol) are called "redox couples."

[060] Dihydrolipoic acid is the reduced (has electrons added) form of lipoic acid (thioctic acid). When DHLA is oxidized (has electrons removed) lipoic acid is produced. It should be understood that DHLA can be either the R or S enantiomer or it can be racemic. Likewise, lipoic acid can also be enantiomerically pure or racemic.

[061] Dihydrolipoic acid (dihydrothioctic acid)

[062] The term "aprotic solvent" is intended to include those solvents that do not include an acidic proton, a hydroxyl proton or easily hydrolysable hydrogen atom or a solvent that does not yield or accept a proton. Suitable aprotic solvents include, for example, methylene chloride, C5 to ClO alkanes (branched and unbranched), aromatic hydrogens, etc.

[063] The phrase "addition of a sufficient quantity of transesterification carboxylic acid derivative" is intended to mean that amount of a carboxylic acid derivative, such as LA or DHLA, used with the free xanthophyll or partially hydrolyzed xanthophyll that results in the disappearance of the free xanthophyll or partially hydrolyzed xanthophyll and results in the esterified form of the xanthophyll, wherein at least one of the hydroxyl groups of the xanthophyll is condensed with a nutritionally beneficial carboxylic acid derivative. This amount can be determined by monitoring the reaction process progress by an analytical technique used in the art, such as thin layer chromatography, gas chromatography, high performance (pressure) liquid chromatography, mass spectral analysis, etc., using known standards of the desired product(s). The product of the reaction can be a mono ester, a di-ester, tri-ester, etc. or mixtures thereof. Selection of one or more esterifying agents can result in mixed diesters. For example, a mixture of LA and DHLA would produce a mixture of mono esterified lutein with an LA and/or DHLA residue, diesters of LA and DHLA as well as a mixed diester of LA/DHLA. Depending on the amounts of xanthophyll and carboxylic acid derivative(s) utilized, free xanthophyll or xanthophyll with non- esterified hydroxyl group(s) can remain. [064] The carboxylic acid derivative that can be added to the reaction mixture can be a branched or unbranched, substituted or unsubstituted Cl through ClO carboxylic acid, or mixtures thereof that provide a nutritional benefit to the recipient upon hydrolysis during the digestive process. Exemplary carboxylic acids include lipoic acid and dihydrolipoic acid. [065] The ratio of carboxylic acid derivative in the step where it is added to the xanthophyll or plant material is from between about 1 :5 to about 5:1 (w/w), preferably from about 1 :2 to about 2:1. [066] Preferably, the weight ratio of lipoic acid and/or dihydrolipoic acid to lutein is from 1.01:1 to 1.2:1, preferably 1.1:1 per hydroxyl group. It has been found that the reaction rate reaches an optimum around a molar ratio of around 1 weight part lutein to 1.1 weight parts of lipoic acid/DHLA per hydroxyl group. A higher excess of lipoic acid/DHLA does not increase the reaction rate significantly.

[067] hi order to carry out the process of the present invention, the xanthophyll, especially lutein, can be provided in a suitable solvent, such as dichloromethane, and a solution of the carboxylic acid derivative (lipoic acid and/or DHLA) in the same solvent may be added to the solution of lutein. [068] In one example, a catalytic amount of acid such as hydrochloric acid, can be added and water can be removed via azeotropic distillation.

[069] hi another embodiment of the process according to the invention, the reaction is carried out in the presence of a dehydrating agent. [070] The dehydrating agent is a diimide, as known in the art, and is preferably dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine or mixtures thereof.

[071] The optimal amount of the dehydrating agent to be used can depend on the environmental conditions: hi one example, where DCC is used as the dehydrating agent, with a high room humidity of around 70-80%, a preferred weight ratio of DCC to xanthophyll, such as lipoic acid, can be around 1.3:1, in cases of low humidity, the weight ratio of DCC to lipoic acid may be around 1.2: 1 per hydroxyl group.

[072] The rate of the conversion of the xanthophyll to the mono, di or triesters of the present invention increases with the reaction time. Preferably, the reaction is carried out for a time period of from 1 hour to 12 hours. [073] Dehydration type reactions can be carried out at around room temperature or slightly below. Preferably, the reaction temperature is about 20⁰C.

[074] After the reaction is complete, the product is concentrated under reduced pressure. Reclaimed solvent may be recycled. The product can be precipitated by e.g. 95% ethanol and washed with the same solvent. [075] The compounds according to the present invention are especially useful as food ingredients, in the cosmetic industry and/or in a pharmaceutical product. Especially the antioxidative properties of lutein and its positive effects on the skin and the eyes are fully preserved in the compound according to the present invention. [076] The term "xanthophyll solid" is intended to mean a material that generally precipitates out of the reaction mixture in solid form. Ideally, and in one embodiment, it has been found that the present processes produce solids that are actually crystals. This helps provide highly pure xanthophyll product that requires little if any further purification.

[077] The transesterification can be conducted at room temperature although elevated reaction temperatures are possible. Suitable reaction temperatures for the heating the mixture containing the xanthophyll(s) or suitable plant material with the nutritionally beneficial carboxylic acid derivative range from about 35°C to about 100°C, more particularly from about 40⁰C to about 80°C and more particularly from about 40⁰C to about 5O⁰C. Advantageously, it is desirable to use lower reaction temperatures, such as about 40⁰C to about 50⁰C to prevent isomerization of the double bonds of the xanthophylls.

[078] If the process of the invention is conducted at room temperature, the xanthophyll solid may precipitate from solution. Otherwise, if the transesterification reactions were conducted at elevated temperatures, it is generally advantageous to cool the reaction temperature to about room temperature to allow the solidification process of the xanthophyll product. Ideally, the solid product results in a crystalline in form. [079] Generally, after the transesterifciation is complete, the lower boiling aprotic solvent(s) are removed by distillation, preferably under vacuum, or other suitable methods known in the art to remove solvent(s). [080] Suitable sources for the xanthophylls include marigold, Chinese wolf- berry, mango, peach, prune, acorn squash, orange, broccoli, green beans, peas, brussel sprouts, cabbage, kale, spinach, kiwi, honeydew, or mixtures thereof. [081] hi one embodiment of the invention, commercially available dried and ground marigold flowers (Tagetes erecta) are used as a source of lutein. [082] hi another embodiment, wolfberry fruits (Lycium barbarum) are used as a source of zeaxanthin, whereas red peppers {Capsicum annum) are a source of capsanthin and capsorubin. [083] For example, the processes of the invention provide that the isolated xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue is lutein, zeaxanthin, capsorubin, capsanthin, astaxanthin, canthaxanthin or mixtures thereof. In one aspect when marigold oleoresin is used as the starting material, the lutein is present at about 80%, zeaxanthin is present below about 20% and capsorubin, capsanthin, astaxanthin, canthaxanthin are all present below about 5%, all based on total weight of the isolated xanthophyll solids.

[084] In another aspect, the isolated xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue solids can be further treated with one or more solvents to remove any residual impurities. Typically the solvent is a protic solvent, such as an alcohol, e.g. , ethanol, a hydritic hydrocarbon, e.g., a polyol, such as glycerin, or glycol, or polar solvents such as acetone, ethyl acetate, or methyl ethyl ketone, . [085] hi another aspect, the process of the invention provides that the isolated xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue solids yield is at least 85%, more particularly 90% and even more particularly 95% on a weight basis based the original amount of xanthophyll in the oleoresin. [086] The purity of the xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue content prepared by the present process is generally at least 90%, more particularly 95%, and even more particularly 99% or better, e.g., 99.5%.

[087] The purified xanthophyll esters derivatized with at least one nutritionally beneficial carboxylic acid residue of the present invention can be utilized in the treatment of a diseases or conditions noted throughout this specification. They can also be used generally as nutritional supplements.

[088] Therefore, the xanthophyll esters derivatized with at least one nutritionally beneficial carboxylic acid derivative can be used to treat cancers of the breast, colon, lung, skin, cervix and ovaries, treatment of cardiovascular disease as well as for use as antioxidants, prevention of cataract and macular degeneration, as lung cancer-preventive agents, as agents for the absorption of harmful ultra-violet light from the rays of the sun and quencher of photo-induced free radical and reactive oxygen species, liver diseases or diabetic and alcoholic polyneuropathy as well as reducing appetite and/or reducing body weight.

[089] Typically the xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue can be purified. For example, ultrafiltration can be used to remove unwanted components by molecular weight cut offs. The retentate from the filtration can be stored as a liquid or, for example, can then be further concentrated into a powder by spray drying, freeze drying, flash drying, fluidized bed drying, ring drying, tray drying, vacuum drying, radio frequency drying or microwave drying. Ultimately, the product should contain at least 95% by weight xanthophyll content, in particular about 99%, more particularly 99.5% or better.

[090] The xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue can be further purified by one or more methods known in the art, such as chromatography, gel chromatography, high performance liquid chromatography, crystallization, affinity chromatography, partition chromatography and the like. Identification of the particular xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue can be accomplished by methods know to those skilled in the art and include ¹H NMR, chemical degradation, chromatography and spectroscopy, especially homo- and heteronuclear two-dimensional NMR techniques for the characterization of the isolated isoprenoid compounds.

[091 ] The term "purified" or "isolated" is used in reference to the purification and/or isolation of one or more xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue as described above. Again using conventional methods known in the art, various xanthophylls can be separated into purified materials. In one aspect of the invention, the xanthophyll are substantially purified and isolated by techniques known in the art. The purity of the purified compounds is generally at least about 90%, preferably at least about 95%, and most preferably at least about 99% and even more preferably at least about 99.9% (e.g. about 100%) by weight. [092] The xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue product(s) do not include residual solvent(s) as determined by analytical methods, such as gas chromatography. Therefore, the phrase "no measurable amount of residual solvent" is intended to mean that the xanthophyll isolate, when tested by an analytical method such as gas chromatography, does not show a measurable quantity of any solvent. [093] In certain aspects, therefore, the xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue product contains less than 1 part per million, ideally less than 1 part per billion, more ideally less than 1 part per trillion of any detectable solvent. In the most ideal situation, there is no residual solvent left in the isolated xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue product.

[094] Therefore, the present invention further provides bioavailable isolated xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue described herein that are useful to treat various afflictions noted herein. The xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue can be administered by a number of methods, as discussed infra.

[095] The compositions of the invention can be incorporated into various foods, drinks, snacks, etc. hi one aspect, the composition can be sprinkled onto a food product, prior to consumption. If sprinkled onto a food product, a suitable carrier such as starch, sucrose or lactose, can be used to help distribute the concentration of the xanthophyll(s) making it easier to apply to the food product.

[096] The compositions of the present invention can also be provided as supplements in various prepared food products. For the purposes of this application, prepared food product means any natural, processed, diet or non-diet food product to which a composition of the invention has been added. The compositions of the present invention can be directly incorporated into many prepared diet food products, including, but not limited to diet drinks, diet bars and prepared frozen meals. Furthermore, the compositions of the inventions can be incorporated into many prepared non-diet products, including, but not limited to candy, snack products such as chips, prepared meat products, milk, cheese, yogurt, sport bars, sport drinks, mayonnaise, salad dressing, bread and any other fat or oil containing foods. As used herein, the term "food product" refers to any substance fit for human or animal consumption. [097] The compositions of the invention can be added to various drinks, such as fruit juices, milkshakes, milk, etc.

[098] The preferred method of administration is oral. The compositions of the invention can be formulated with suitable carriers such as starch, sucrose or lactose in tablets, capsules, solutions, syrups and emulsions. The tablet or capsule of the present invention can be coated with an enteric coating that dissolves at a pH of about 6.0 to 7.0. A suitable enteric coating, which dissolves in the small intestine but not in the stomach, is cellulose acetate phthalate. [099] Formulation of the compositions of the invention into a soft gel capsule can be accomplished by many methods known in the art. Often the formulation will include an acceptable carrier, such as an oil, or other suspending or emulsifying agent. [0100] Suitable optional carriers include but are not limited to, for example, fatty acids, esters and salts thereof, that can be derived from any source, including, without limitation, natural or synthetic oils, fats, waxes or combinations thereof. Moreover, the fatty acids can be derived, without limitation, from non-hydrogenated oils, partially hydrogenated oils, fully hydrogenated oils or combinations thereof. Non- limiting exemplary sources of fatty acids (their esters and salts) include seed oil, fish or marine oil, canola oil, vegetable oil, safflower oil, sunflower oil, nasturtium seed oil, mustard seed oil, olive oil, sesame oil, soybean oil, corn oil, peanut oil, cottonseed oil, rice bran oil, babassu nut oil, palm oil, low erucic rapeseed oil, palm kernel oil, lupin oil, coconut oil, flaxseed oil, evening primrose oil, jojoba, wheat germ oil, tallow, beef tallow, butter, chicken fat, lard, dairy butterfat, shea butter or combinations thereof. [0101] Specific non-limiting exemplary fish or marine oil sources include shellfish oil, tuna oil, mackerel oil, salmon oil, menhaden, anchovy, herring, trout, sardines or combinations thereof, m particular, the source of the fatty acids is fish or marine oil (DHA or EPA), soybean oil or flaxseed oil. Alternatively or in combination with one of the above identified carrier, beeswax can be used as a suitable carrier, as well as suspending agents such as silica (silicon dioxide). [0102] The formulations of the invention are also considered to be nutraceuticals. The term "nutraceutical" is recognized in the art and is intended to describe specific chemical compounds found in foods that can prevent disease or ameliorate an undesirable condition.

[0103] The formulations of the invention can further include various ingredients to help stabilize, or help promote the bioavailability of the components of the beneficial compositions of the invention or serve as additional nutrients to an individual's diet. Suitable additives can include vitamins and biologically-acceptable minerals. Non- limiting examples of vitamins include vitamin A, B vitamins, vitamin C, vitamin D, vitamin E, vitamin K and folic acid. Non-limiting examples of minerals include iron, calcium, magnesium, potassium, copper, chromium, zinc, molybdenum, iodine, boron, selenium, manganese, derivatives thereof or combinations thereof. These vitamins and minerals can be from any source or combination of sources, without limitation. Non- limiting exemplary B vitamins include, without limitation, thiamine, niacinamide, pyridoxine, riboflavin, cyanocobalamin, biotin, pantothenic acid or combinations thereof. [0104] Various additives can be incorporated into the present compositions.

Optional additives of the present composition include, without limitation, hyaluronic acid, phospholipids, starches, sugars, fats, antioxidants, amino acids, proteins, flavorings, coloring agents, hydro lyzed starch(es) and derivatives thereof or combinations thereof. [0105] As used herein, the term "antioxidant" is recognized in the art and refers to synthetic or natural substances that prevent or delay the oxidative deterioration of a compound. Exemplary antioxidants include tocopherols, flavonoids, catechins, superoxide dismutase, lecithin, gamma oryzanol; vitamins, such as vitamins A, C (ascorbic acid) and E and beta-carotene; natural components such as camosol, camosic acid and rosmanol found in rosemary and hawthorn extract, proanthocyanidins such as those found in grapeseed or pine bark extract, and green tea extract. [0106] Compositions comprising the xanthophyll of the invention can be manufactured by methods of conventional mixing, dissolving, granulating, dragee- making levigating, emulsifying, encapsulating, entrapping or lyophilization processes. The compositions can be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate processing of the xanthophyll compositions into preparations that can be used. [0107] The compositions of the invention can take a form suitable for virtually any mode of administration, including, for example, oral, buccal, systemic, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation.

[0108] Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.

[0109] Useful injectable preparations include sterile suspensions, solutions or emulsions of the xanthophyll extract compositions in aqueous or oily vehicles. The compositions can also contain formulating agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers, and can contain added preservatives. [0110] Alternatively, the injectable formulation can be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use. To this end, the xanthophyll compositions can be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.

[0111] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art. [0112] For oral administration, the compositions of the invention can take the form of, for example, lozenges, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets can be coated by methods well known in the art with, for example, sugars, films or enteric coatings. [0113] Liquid preparations for oral administration can take the form of, for example, elixirs, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl p hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, preservatives, flavoring, coloring and sweetening agents as appropriate.

[0114] Preparations for oral administration can be suitably formulated to give controlled release of the xanthophyll composition as is well known. [0115] For buccal administration, the compositions can take the form of tablets or lozenges formulated in conventional manner. [0116] For rectal and vaginal routes of administration, the xanthophyll compositions can be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.

[0117] For nasal administration or administration by inhalation or insufflation, the xanthophyll compositions can be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. hi the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator (for example capsules and cartridges comprised of gelatin) can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. [0118] For prolonged delivery, the xanthophyll compositions can be formulated as a depot preparation for administration by implantation or intramuscular injection. The xanthophyll compositions can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt. Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch, which slowly releases the xanthophyll compositions for percutaneous absorption, can be used. To this end, permeation enhancers can be used to facilitate transdermal penetration of the compositions. Suitable transdermal patches are described in for example, U.S. Pat. No. 5,407,713; U.S. Pat. No. 5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat. No. 5,336,168; U.S. Pat. No. 5,290,561; U.S. Pat. No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S. Pat. No. 5,088,977; U.S. Pat. No. 5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No. 4,921,475. [0119] Alternatively, other delivery systems can be employed. Liposomes and emulsions are well-known examples of delivery vehicles that can be used to deliver xanthophyll compositions. Certain organic solvents such as dimethylsulfoxide (DMSO) can also be employed, although usually at the cost of greater toxicity. [0120] The compositions can, if desired, be presented in a pack or dispenser device, which can contain one or more unit dosage forms containing the xanthophyll compositions. The pack can, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration. [0121] Soft gel or soft gelatin capsules can be prepared, for example, without limitation, by dispersing the formulation in an appropriate vehicle (e.g., rice bran oil, and/or beeswax) to form a high viscosity mixture. This mixture is then encapsulated with a gelatin based film using technology and machinery known to those in the soft gel industry. The capsules so formed are then dried to constant weight. Typically, the weight of the capsule is between about 100 to about 2500 milligrams and in particular weigh between about 1500 and about 1900 milligrams, and more specifically can weigh between about 1500 and about 2000 milligrams.

[0122] For example, when preparing soft gelatin shells, the shell can include between about 20 to 70 percent gelatin, generally a plasticizer and about 5 to about 60% by weight sorbitol. The filling of the soft gelatin capsule is liquid (principally a carrier such as rice bran oil or wheat germ oil and/or beeswax if desired) and can include, apart from the xanthophyll, a hydrophilic matrix. The hydrophilic matrix, if present, is a polyethylene glycol having an average molecular weight of from about 200 to 1000. Further ingredients are optionally thickening agents and/or emulsifying agent(s). hi one embodiment, the hydrophilic matrix includes polyethylene glycol having an average molecular weight of from about 200 to 1000, 5 to 15% glycerol, and 5 to 15% by weight of water. The polyethylene glycol can also be mixed with propylene glycol and/or propylene carbonate.

[0123] In another embodiment, the soft gel capsule is prepared from gelatin, glycerine, water and various additives. Typically, the percentage (by weight) of the gelatin is between about 30 and about 50 weight percent, in particular between about 35 and about weight percent and more specifically about 42 weight percent. The formulation includes between about 15 and about 25 weight percent glycerine, more particularly between about 17 and about 23 weight percent and more specifically about 20 weight percent glycerine. [0124] The remaining portion of the capsule is typically water. The amount varies from between about 25 weigh percent and about 40 weight percent, more particularly between about 30 and about 35 weight percent, and more specifically about 35 weight percent. The remainder of the capsule can vary, generally, between about 2 and about 10 weight percent composed of a flavoring agent(s), sugar, coloring agent(s), etc. or combination thereof. After the capsule is processed, the water content of the final capsule is often between about 5 and about 10 weight percent, more particularly 7 and about 12 weight percent, and more specifically between about 9 and about 10 weight percent.

[0125] As for the manufacturing, it is contemplated that standard soft shell gelatin capsule manufacturing techniques can be used to prepare the soft-shell product. Examples of useful manufacturing techniques are the plate process, the rotary die process pioneered by R. P. Scherer, the process using the Norton capsule machine, and the Accogel machine and process developed by Lederle. Each of these processes is mature technologies and is all widely available to any one wishing to prepare soft gelatin capsules. [0126] Emulsifying agents can be used to help solubilize the ingredients within the soft gelatin capsule. Specific examples of the surfactant, emulsifier, or effervescent agent include D-sorbitol, ethanol, carrageenan, carboxyvinyl polymer, carmellose sodium, guar gum, glycerol, glycerol fatty acid ester, cholesterol, white beeswax, dioctyl sodium sulfosuccinate, sucrose fatty acid ester, stearyl alcohol, stearic acid, polyoxyl 40 stearate, sorbitan sesquioleate, cetanol, gelatin, sorbitan fatty acid ester, talc, sorbitan trioleate, paraffin, potato starch, hydroxypropyl cellulose, propylene glycol, propylene glycol fatty acid ester, pectin, polyoxyethylene (105) polyoxypropylene (5) glycol, polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene hydrogenated castor oil, polyoxyethylene hydrogenated castor oil 40, polyoxyethylene hydrogenated castor oil 60, polyoxyl 35 castor oil, polysorbate 20, polysorbate 60, polysorbate 80, macrogol 400, octyldodecyl myristate, methyl cellulose, sorbitan monooleate, glycerol monostearate, sorbitan monopalmitate, sorbitan monolaurate, lauryl dimethylamine oxide solution, sodium lauryl sulfate, lauromacrogol, dry sodium carbonate, tartaric acid, sodium hydroxide, purified soybean lecithin, soybean lecithin, potassium carbonate, sodium hydrogen carbonate, medium- chain triglyceride, citric anhydride, cotton seed oil-soybean oil mixture, and liquid paraffin.

[0127] The present invention also provides packaged formulations of the compositions of the invention and instructions for use of the product for appropriate condition(s). Typically, the packaged formulation, in whatever form, is administered to an individual in need thereof. Typically, the dosage requirement is between about 1 to about 4 dosages a day.

[0128] Although the present invention describes the preparation, use, manufacture and packaging of the compositions of the invention in soft gelatin capsules for treatment of various conditions, it should not be considered limited to only soft gelatin capsules. Ingestible compositions of the invention can be delivered in traditional tablets, pills, lozenges, elixirs, emulsions, hard capsules, liquids, suspensions, etc. as described above.

[0129] The xanthophyll compositions of the invention, or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular related condition being treated. The composition can be administered therapeutically to achieve therapeutic benefit or prophylactically to achieve prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and/or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient can still be afflicted with the underlying disorder. For example, administration of a composition of the invention to a patient suffering from pain provides therapeutic benefit not only when the underlying condition is eradicated or ameliorated, but also when the patient reports a decrease in the severity or duration of the physical discomfort associated with the pain. [0130] For prophylactic administration, the composition can be administered to a patient at risk of developing one of the previously described conditions. [0131] The amount of composition administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, etc. Determination of an effective dosage is well within the capabilities of those skilled in the art. [0132] Total dosage amounts of a xanthophyll ester derivatized with at least one nutritionally beneficial carboxylic acid residue composition will typically be in the range of from about 0.0001 or 0.001 or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the components, its bioavailability, the mode of administration and various factors discussed above. Dosage amount and interval can be adjusted individually to provide plasma levels of the compound(s) which are sufficient to maintain therapeutic or prophylactic effect. For example, the compounds can be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. Skilled artisans will be able to optimize effective local dosages without undue experimentation. [0133] The following paragraphs enumerated consecutively from 1 through 44 provide for various aspects of the present invention, hi one embodiment, in a first paragraph (1), the present invention provides an esterified xanthophyll comprising the condensation product of a free xanthophyll comprising at least one hydroxyl moiety and at least one nutritionally beneficial activated carboxylic acid derivative. [0134] 2. The esterified xanthophyll of paragraph 1, wherein the xanthophyll comprises at least 2 hydroxyl groups for esterification. [0135] 3. The esterified xanthophyll of paragraph 2, wherein the nutritionally beneficial carboxylic acid derivative is a mixture of two of more nutritionally beneficial carboxylic acid derivatives.

[0136] 4. The esterified xanthophyll of any of paragraphs 1 through 3, wherein the nutritionally beneficial carboxylic acid derivative is a carboxylic acid, carboxylic ester or carboxylic halide.

[0137] 5. The esterified xanthophyll of any of paragraphs 1 through 3, wherein the nutritionally beneficial carboxylic acid derivative is lipoic acid or dihydrolipoic acid. [0138] 6. The esterified xanthophyll of any of paragraphs 1 through 3, wherein the nutritionally beneficial carboxylic acid derivative is a lipoic ester or a dihyrolipoic ester.

[0139] 7. The esterified xanthophyll of any of paragraphs 1 through 6, wherein the free xanthophyll is lutein, zeaxanthin, a cryptoxanthin or neoxanthin, violaxanthin, fucoxanthin, astaxanthin, or peridinin. [0140] 8. An esterified xanthophyll comprising the condensation product of a partially or fully hydrolyzed xanthophyll comprising at least one hydroxyl moiety and at least one nutritionally beneficial activated carboxylic acid derivative. [0141 ] 9. The esterified xanthophyll of paragraph 8, wherein the partially or fully hydrolyzed xanthophyll comprises at least 2 hydroxyl groups for esterification. [0142] 10. The esterified xanthophyll of paragraph 9, wherein the nutritionally beneficial carboxylic acid derivative is a mixture of two of more nutritionally beneficial carboxylic acid derivatives.

[0143] 11. The esterified xanthophyll of any of paragraphs 8 through 10, wherein the nutritionally beneficial carboxylic acid derivative is a carboxylic acid, carboxylic ester or carboxylic halide. [0144] 12. The esterified xanthophyll of any of paragraphs 8 through 10, wherein the nutritionally beneficial carboxylic acid derivative is lipoic acid or dihydrolipoic acid.

[0145] 13. The esterified xanthophyll of any of paragraphs 8 through 10, wherein the nutritionally beneficial carboxylic acid derivative is a lipoic ester or a dihyrolipoic ester.

[0146] 14. The esterified xanthophyll of any of paragraphs 8 through 13, wherein the partially or fully hydrolyzed xanthophyll is lutein, zeaxanthin, a cryptoxanthin or neoxanthin, violaxanthin, fucoxanthin, astaxanthin, or peridinin.

[0147] 15. A xanthophyll ester comprising the formula:

-37-

or

[0148] wherein each R¹, R², R³, individually if present, is a hydrogen, an acyl residue of a fatty acid, or an acyl residue of a nutritionally beneficial carboxylic derivative, provided at least one of R¹, R², R³, when present, is an acyl residue of a nutritionally beneficial carboxylic derivative or mixtures thereof.

[0149] 16. The xanthophyll ester of paragraph 15, wherein the acyl fatty acid residue is from formic, acetic, propionic, butyric, valeric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, oleic acid or mixtures thereof.

[0150] 17. The xanthophyll ester of either paragraph 15 or 16, wherein the acyl residue of a nutritionally beneficial carboxylic acid is from lipoic acid or dihydrolipoic acid.

[0151] 18. The xanthophyll ester of paragraph 15, wherein each R¹, R², R³, individually if present, is a hydrogen or an acyl residue of a nutritionally beneficial carboxylic derivative, provided at least of one of R¹, R², R³, when present, is an acyl residue of a nutritionally beneficial carboxylic derivative.

[0152] 19. The xanthophyll ester of paragraph 18, wherein the acyl residue of a nutritionally beneficial carboxylic acid is from lipoic acid or dihydrolipoic acid. [0153] 20. The xanthophyll ester of paragraph 18, wherein each R¹ , R², R³, individually if present, is an acyl residue of a nutritionally beneficial carboxylic derivative.

[0154] 21. The xanthophyll ester of paragraph 20, wherein the acyl residue of a nutritionally beneficial carboxylic acid is from lipoic acid or dihydrolipoic acid. [0155] 22. A method of preventing or inhibiting free radical oxidation in a mammal, the method comprising administering an antioxidative amount of a compound as in any of paragraphs 1 through 21.

[0156] 23. A method to prepare an esterified xanthophyll comprising the step of: treating a free xanthophyll comprising at least one hydroxyl moiety with at least one nutritionally beneficial activated carboxylic acid derivative under conditions such that esterification occurs.

[0157] 24. The method of paragraph 23, wherein the xanthophyll comprises at least 2 hydroxyl groups for esterification.

[0158] 25. The method of paragraph 24, wherein the nutritionally beneficial carboxylic acid derivative is a mixture of two of more nutritionally beneficial carboxylic acid derivatives.

[0159] 26. The method of any of paragraphs 23 through 25, wherein the nutritionally beneficial carboxylic acid derivative is a carboxylic acid, carboxylic ester or carboxylic halide. [0160] 27. The method of any of paragraphs 23 through 25, wherein the nutritionally beneficial carboxylic acid derivative is lipoic acid or dihydrolipoic acid.

[0161 ] 28. The method of any of paragraphs 23 through 25, wherein the nutritionally beneficial carboxylic acid derivative is a lipoic ester or a dihyrolipoic ester.

[0162] 29. The method of any of paragraphs 23 through 28, wherein the free xanthophyll is lutein, zeaxanthin, a cryptoxanthin or neoxanthin, violaxanthin, fucoxanthin, astaxanthin, or peridinin. [0163] 30. A method to prepare an esterified xanthophyll comprising the step of: treating a partially or fully hydrolyzed xanthophyll comprising at least one hydroxyl moiety with at least one nutritionally beneficial activated carboxylic acid derivative under conditions such that esterification occurs. [0164] 31. The method of paragraph 30, wherein the partially or fully hydrolyzed xanthophyll comprises at least 2 hydroxyl groups for esterification.

[0165] 32. The method of paragraph 31 , wherein the nutritionally beneficial carboxylic acid derivative is a mixture of two of more nutritionally beneficial carboxylic acid derivatives. [0166] 33. The method of any of paragraphs 30 through 32, wherein the nutritionally beneficial carboxylic acid derivative is a carboxylic acid, carboxylic ester or carboxylic halide.

[0167] 34. The method of any of paragraphs 30 through 32, wherein the nutritionally beneficial carboxylic acid derivative is lipoic acid or dihydrolipoic acid. [0168] 35. The method of any of paragraphs 30 through 32, wherein the nutritionally beneficial carboxylic acid derivative is a lipoic ester or a dihyrolipoic ester.

[0169] 36. The method of any of paragraphs 30 through 35, wherein the partially or fully hydrolyzed xanthophyll is lutein, zeaxanthin, a cryptoxanthin or neoxanthin, violaxanthin, fucoxanthin, astaxanthin, or peridinin. [0170] 37. A method to prepare an esterified xanthophyll comprising the step of: treating one or more of initial compounds having a formula

-41- [0171] wherein each R¹, R², R³, individually if present, is a hydrogen or an acyl residue of a fatty acid provided at least one of R¹, R², R³, when present, is a hydrogen, with a nutritionally beneficial carboxylic acid derivative under conditions suitable for esterification to occur, such that esterification provides a product wherein R , R , R , individually if present, is a hydrogen, an acyl residue of a fatty acid, or an acyl residue of a nutritionally beneficial carboxylic derivative, provided at least one of R , R , R , when present, is an acyl residue of a nutritionally beneficial carboxylic derivative or mixtures thereof.

[0172] 38. The method of paragraph 37, wherein the acyl fatty acid residue of the initial compound is from formic, acetic, propionic, butyric, valeric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, oleic acid or mixtures thereof.

[0173] 39. The method of either paragraph 37 or 38, wherein the acyl residue of a nutritionally beneficial carboxylic acid in the product is from lipoic acid or dihydrolipoic acid. [0174] 40. The method ofparagraph 37, wherein each R¹, R², R³ of the product, individually and if present, is a hydrogen or an acyl residue of a nutritionally beneficial carboxylic derivative, provided at least of one of R , R , R , where present, is an acyl residue of a nutritionally beneficial carboxylic derivative.

[0175] 41. The method ofparagraph 40, wherein the acyl residue of a nutritionally beneficial carboxylic acid is from lipoic acid or dihydrolipoic acid. [0176] 42. The method ofparagraph 40, wherein each R¹, R², R³ of the product, individually and if present, is an acyl residue of a nutritionally beneficial carboxylic derivative. [0177] 43. The method of paragraph 42, wherein the acyl residue of a nutritionally beneficial carboxylic acid is from lipoic acid or dihydrolipoic acid.

[0178] 44. A method to treat one or more of to cancers of the breast, colon, lung, skin, cervix and ovaries, treatment of cardiovascular disease as well as for use as antioxidants, prevention of cataract and macular degeneration, as lung cancer- preventive agents, as agents for the absorption of harmful ultra-violet light from the rays of the sun and quencher of photo-induced free radical and reactive oxygen species, liver diseases or diabetic and alcoholic polyneuropathy, reducing appetite and/or reducing body weight comprising the step of administering an effective amount of a compound described in any of paragraphs 1 through 21.

[0179] The following examples are not to be meant as limiting but are presented to provide additional information and support for the invention.

[0180] Examples

[0181] Example 1 (lutein ester)

[0182] 8.5 g lutein were added to a 250 ml flask with 150 ml dichloromethane.

The mixture was stirred until homogeneous.

[0183] 6.8 g lipoic acid dissolved in 50 ml dichloromethane were added into another beaker and stirred until dissolution was complete. [0184] 8.8 g dicyclohexylcarbodiimide, 0.5g 4-dimethylaminopyridine and 50 ml dichloromethane were added to a third beaker and stirred until dissolution was complete.

[0185] Under continuous stirring, the lipoic acid solution was added to the lutein solution over a period of about 15 minutes. The mixture was stirred until homogeneous. To that solution was added the solution containing DCC and dimethylaminopyridine over a period of about 15 minutes. The temperature was maintained below about

20°C,and the reaction mixture was stirred for 12 hours.

[0186] After the reaction was complete, the reaction liquid was filtered, and the filtrate was vacuum-concentrated to about 100 ml. [0187] In order to precipitate the product, 700 ml 95% ethanol were added to a beaker with stirring and the concentrated reaction liquid was slowly added thereto over a period of about 60 minutes while maintaining the temperature below 25°C. Stirring was continued for about 5 minutes after addition was complete, then the precipitate was filtered off and washed three times with 150 ml 95% ethanol. [0188] The washed reaction product was vacuum dried below 45°C about 6 hours. The vacuum was maintained below about 0.095 MPa. [0189] The reaction product appeared as a red powder, with a melting point of

76 to 80°C.

[0190] The product was analyzed by NMR-analysis. The results of the NMR- analysis are provided below:

1. Lutein portion

2. Lipoic acid portion (2 mol lipoic acid correspond to 1 mol of lutein)

*: may be effected by the esterification R = H

[0191] From the data above, based on chemical shifts (δ) and peak integration, there is a peak integration differential between the lutein and the ester product of approximately 2. The product, therefore, is a diester of lutein with two lipoic acid residues.

[0192] Example 2 (zeaxanthin ester)

[0193] 8.0 g zeaxanthin were added with stirring to 150 ml dichloromethane in a

250 ml flask until the solution was homogeneous. [0194] 7.0 g lipoic acid were added with stirring to 50 ml dichloromethane.

[0195] 9.0 g dicyclohexylcarbodiimide, 0.5 g 4-dimethylaminopyridine and 50 ml CH₂Cl₂ were mixed in another beaker with stirring.

[0196] The lipoic acid solution was added to the zeaxanthin solution over a period of 15 minutes with stirring until a homogeneous solution was obtained. To the homogeneous solution was added the solution containing DCC and dimethylaminopyridine over a period of about 15 minutes. The temperature was maintained below about 2O⁰C and the reaction mixture was stirred for 12 hours. [0197] After the reaction was complete, the reaction liquid was filtered, and the filtrate was condensed to 100 ml under vacuum.

[0198] The concentrated filtrated was added to a solution of 700 ml 95% ethanol with stirring over a period of about 60 minutes while maintaining the temperature of the mixture below about 25°C.

[0199] Stirring was continued for about 5 minutes after the addition and the resulting precipitate was filtered and washed three times with 150 ml 95% ethanol. [0200] The washed reaction product was vacuum dried below about 45⁰C for about 6 hours. The resulting product was a red powder. The product was analyzed by 1H NMR. The results of the ¹H NMR are provided below:

[0201] Stability test

[0202] Materials: [0203] Ig lutein ester of lipoic acid, from example 1;

[0204] Ig simp Ie mixture of lutein and lipoic acid, prepared as follows: Ig lutein powder was mixed with 0.72 g lipoic acid.

[0205] Method: [0206] A I g sample of each of the above materials was heated at 5O⁰C for 24 hrs. A lO mg portion of each sample was then dissolved in 10 mL THF and diluted with ethanol to 25 mL.

[0207] A 0.1 mL sample was then diluted with ethanol to 25 mL and lutein content was determined by UV at 446nm.

[0208] Sample UV

; Decomposing ratio = 1 -UV

255 x m

[0209] A : sample absorbency

[0210] m : sample weight

[0211] 255 : absorbance coefficient

[0212] 6250 : dilution ratio

[0213] Results:

[0214] Conclusion:

[0215] The lutein ester of lipoic acid is more stable than just simply combining a mixture of lutein and lipoic acid. [0216] Bioavailability Testing

[0217] Material and treatment : [0218] Five male Sprague-Dawley rats (weighing 220-25Og, 7-8weeks of age) can be used in each treatment group. Treatment groups would include two groups. Group A would be dosed with the lutein ester of lipoic acid (from example 1); Group B would be dosed with a simple mixture of lipoic acid and lutein (Mixture of Ig lutein powder with 0.72g lipoic acid, as described above). [0219] The rats would be dosed orally with the samples at 20 mg/Kg.

[0220] Method and detection:

[0221] Blood sampling (0.5 mL) would be taken after dosing (t=0) and 10, 20,

30, 40, 50, 60, 90, 120, 150, 180, 240 minutes after dosing. [0222] An HPLC assay would be used to detect for the quantity of lutein in blood samples. A wavelength of 446nm would be used to analyze for lutein.

[0223] Analysis method:

[0224] The mean lutein plasma concentration of samples versus time after a single oral dose would be compared.

[0225] Expected results:

[0226] The results should be that the lutein plasma concentration of Group A is several times higher than that of Group B.

[0227] Conclusion:

[0228] Data obtained from the rats given oral administration of lutein can reveal that the lutein ester of lipoic acid was better absorbed physiologically while also providing stability to the sample versus the simple mixture of lutein and lipoic acid. [0229] These results would account for the better bioavailability of the lutein ester of lipoic acid product of the present invention. [0230] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. An esterified xanthophyll comprising: the condensation product of a free xanthophyll comprising at least one hydroxyl moiety and at least one nutritionally beneficial activated carboxylic acid derivative.

2. The esterified xanthophyll of claim 1, wherein the xanthophyll comprises at least 2 hydroxyl groups for esterification.

3. The esterified xanthophyll of claim 2, wherein the nutritionally beneficial carboxylic acid derivative is a mixture of two of more nutritionally beneficial carboxylic acid derivatives.

4. The esterified xanthophyll of claim 3, wherein the nutritionally beneficial carboxylic acid derivative is a carboxylic acid, carboxylic ester or carboxylic halide.

5. The esterified xanthophyll of claim 1, wherein the nutritionally beneficial carboxylic acid derivative is lipoic acid or dihydrolipoic acid.

6. The esterified xanthophyll of claim 1, wherein the nutritionally beneficial carboxylic acid derivative is a lipoic ester or a dihyrolipoic ester.

7. The esterified xanthophyll of claim 1, wherein the free xanthophyll is lutein, zeaxanthin, a cryptoxanthin or neoxanthin, violaxanthin, fucoxanthin, astaxanthin, or peridinin.

8. An esterified xanthophyll comprising: the condensation product of a partially or fully hydro lyzed xanthophyll comprising at least one hydroxyl moiety and at least one nutritionally beneficial activated carboxylic acid derivative.

9. A xanthophyll ester comprising the formula:

-53-

or

wherein each R¹, R², R³, individually if present, is a hydrogen, an acyl residue of a fatty acid, or an acyl residue of a nutritionally beneficial carboxylic derivative, provided at least one of R¹, R², R³, when present, is an acyl residue of a nutritionally beneficial carboxylic derivative or mixtures thereof.

10. The xanthophyll ester of claim 9, wherein the acyl fatty acid residue is from formic, acetic, propionic, butyric, valeric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, oleic acid or mixtures thereof.

11. The xanthophyll ester of claim 9, wherein the acyl residue of a nutritionally beneficial carboxylic acid is from lipoic acid or dihydrolipoic acid.

12. The xanthophyll ester of claim 9, wherein each R¹, R², R³, individually if present, is a hydrogen or an acyl residue of a nutritionally beneficial carboxylic derivative, provided at least of one of R¹, R², R³, when present, is an acyl residue of a nutritionally beneficial carboxylic derivative.

13. The xanthophyll ester of claim 12, wherein the acyl residue of a nutritionally beneficial carboxylic acid is from lipoic acid or dihydrolipoic acid.

1 0 ¹X

14. The xanthophyll ester of claim 12, wherein each R , R , R , individually if present, is an acyl residue of a nutritionally beneficial carboxylic derivative.

15. The xanthophyll ester of claim 14, wherein the acyl residue of a nutritionally beneficial carboxylic acid is from lipoic acid or dihydrolipoic acid.

16. A method of preventing or inhibiting free radical oxidation in a mammal, the method comprising administering an antioxidative amount of a compound as claimed in claim 1.

17. A method of preventing or inhibiting free radical oxidation in a mammal, the method comprising administering an antioxidative amount of a compound as claimed in claim 8.

18. A method of preventing or inhibiting free radical oxidation in a mammal, the method comprising administering an antioxidative amount of a compound as claimed in claim 9.

19. A method to prepare an esterified xanthophyll comprising the step of: treating a free xanthophyll comprising at least one hydroxyl moiety with at least one nutritionally beneficial activated carboxylic acid derivative under conditions such that esterification occurs.

20. A method to prepare an esterified xanthophyll comprising the step of: treating a partially or fully hydrolyzed xanthophyll comprising at least one hydroxyl moiety with at least one nutritionally beneficial activated carboxylic acid derivative under conditions such that esterification occurs.

21. A method to prepare an esterified xanthophyll comprising the step of: treating one or more of initial compounds having a formula

wherein each R¹, R², R³, individually if present, is a hydrogen or an acyl residue of a fatty acid provided at least one of R¹, R², R³, when present, is a hydrogen, with a nutritionally beneficial carboxylic acid derivative under conditions suitable for esterification to occur, such that esterification provides a product wherein R¹, R², R³, individually if present, is a hydrogen, an acyl residue of a fatty acid, or an acyl residue of a nutritionally beneficial carboxylic derivative, provided at least one of R¹, R², R³, when present, is an acyl residue of a nutritionally beneficial carboxylic derivative or mixtures thereof.

22. A method to treat one or more of cancers of the breast, colon, lung, skin, cervix and ovaries, treatment of cardiovascular disease as well as for use as antioxidants, prevention of cataract and macular degeneration, as lung cancer- preventive agents, as agents for the absorption of harmful ultra-violet light from the rays of the sun and quencher of photo-induced free radical and reactive oxygen species, liver diseases or diabetic and alcoholic polyneuropathy, reducing appetite and/or reducing body weight comprising the step of administering an effective amount of a compound described in claim 1.

23. The method of claim 22, further comprising a pharmaceutically acceptable carrier.

24. A method to treat one or more of cancers of the breast, colon, lung, skin, cervix and ovaries, treatment of cardiovascular disease as well as for use as antioxidants, prevention of cataract and macular degeneration, as lung cancer- preventive agents, as agents for the absorption of harmful ultra-violet light from the rays of the sun and quencher of photo-induced free radical and reactive oxygen species, liver diseases or diabetic and alcoholic polyneuropathy, reducing appetite and/or reducing body weight comprising the step of administering an effective amount of a compound described in claim 8.

25. The method of claim 24, further comprising a pharmaceutically acceptable carrier.

26. A method to treat one or more of cancers of the breast, colon, lung, skin, cervix and ovaries, treatment of cardiovascular disease as well as for use as antioxidants, prevention of cataract and macular degeneration, as lung cancer- preventive agents, as agents for the absorption of harmful ultra-violet light from the rays of the sun and quencher of photo-induced free radical and reactive oxygen species, liver diseases or diabetic and alcoholic polyneuropathy, reducing appetite and/or reducing body weight comprising the step of administering an effective amount of a compound described in claim 9.

27. The method of claim 26, further comprising a pharmaceutically acceptable carrier.