Title of Invention	NOVEL XANTHOPHYLL CRYSTALS
Abstract	The invention disclosed in this application relates to novel xanthophyll crystals containing high amounts of trans-lutein and / or zeaxanthin. The xanthophyll crystals of the present invention are useful as antioxidant, prevention of cataract and macular degeneration, lung cancer preventive agent, as agents for the absorption of harmful ultra-violet light from sun rays and quencher of photo-induced free radical and reactive oxygen species.

Title of Invention

NOVEL XANTHOPHYLL CRYSTALS

Abstract

The invention disclosed in this application relates to novel xanthophyll crystals containing high amounts of trans-lutein and / or zeaxanthin. The xanthophyll crystals of the present invention are useful as antioxidant, prevention of cataract and macular degeneration, lung cancer preventive agent, as agents for the absorption of harmful ultra-violet light from sun rays and quencher of photo-induced free radical and reactive oxygen species.

Full Text	Introduction This invention relates to novel xanthophyll crystals. This invention particularly relates to xanthophyll crystals containing high content of trans lutein and / or zeaxanthin This invention more particularly relates to novel xanthophyll crystals containing at least 85% total xanthophylls out of which the trans-lutein content is at least 90 %, the remaining being zeaxanthin and other carotenoids This application is divided out of the application no 622 MAS 2002 . In the application no 622 MAS 021 we have described and claimed an improved process for the preparation of xanthophyll crystals containing high content of trans lutein and / or zeaxanthin . More particularly in that application we have described and claimed an improved process for the preparation of xanthophyll crystals containing at least 85% total xanthophylls out of which the trans-lutein content is at least 90 %, the remaining being zeaxanthin and other carotenoids . The xanthophyll crystals of the present invention are useful as antioxidant, prevention of cataract and macular degeneration, lung cancer preventive agent, as agents for the absorption of harmful ultra-violet light from sun rays and quencher of photo-induced free radical and reactive oxygen species. The crystals can also be used for the colouration of food and animal/poultry feed. The xanthophyll crystals prepared by the process of the current invention can also be used for the colouration of food and animal/poultry feed. It is known from various studies that a higher dietary intake of carotenoids is associated with a lower risk for AMD ( Age - related Macular Degeneration). The specific carotenoids, lutein and zeaxanthin, which are primarily obtained from dark green leafy vegetables, were most strongly associated with a reduced risk for AMD. Individuals consuming the highest levels of carotenoids had a statistically significant 43% lower risk for AMD. A significant trend was seen for a lower risk for AMD with increasing amounts of carotenoids in the diet. (JM Seddon et. al .Dietary Carotenoids, Vitamins A, Q and E, and Advanced Age-Related Macular Degeneration, Journal of the American Medical Association, November, 1994, Vol. 272, No. 18, pages 1413-1420) There is a strong inverse association between higher consumption of dark green vegetables, which contain xanthophylls which are rich in lutein and zeaxanthin, and a decreased risk for oxidative stress related diseases such as cataract and cancer as well. Both lutein and zeaxanthin are reported to possess strong anti-tumour promoting properties. (L Packer, M Hiramatsu, T Oshikawa,(Editors), Antioxidant Food Supplements in Human Health, Academic Press, NY, 1999, Pp 223 and Pp 226) Recent studies also reveal that lutein and zeaxanthin can play a useful role in combating conditions which lead to cardio-vascular disease, atherosclerosis, skin cancer, ovarian cancer, etc. Although dark green vegetables are excellent dietary sources of lutein , the isolation and purification of this compound in large quantities from green vegetables is time-consuming and costly due to the high level of chlorophyll pigments. 25g of a fresh, dark green vegetable such as kale would theoretically provide 10 mg of lutein.( F. Khachik etal, 'Lutein, Lycopene, and Their Oxidative Metabolites in Chemoprevention of Cancer/' Journal of Cellular Biochemistry, 1995, Supplement 22:236, pp. 236-246. )Consistent bioavailability and absorption of carotenoids from foods is not as high as supplementation with pure carotenoids. Extracts from the petals of marigold flowers are an excellent source of lutein esters in large quanhties and contain no significant levels of other carotenoids. Lutein and zeaxanthin being fat-soluble nutrients, can be delivered in consistent doses through soft gelatin capsule based supplements, or in stabilized tablets, and other nutritional preparations to overcome deficiencies of diet and improve levels of serum lutein/zeaxanthin, and thereby, the levels of lutein/zeaxanthin in the macula. Background Of The Invention Carotenoids are yellow, red and orange pigments which 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. Carotenoids absorb light in the 400-500 nm region of the visible spectrum. This physical property imparts the characteristic 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. Carotenoids are non-polar compounds classified into two sub-classes namely more polar compounds called xanthophylls or oxycarotenoids and non-polar hydrocarbon carotenes like p-carotene, lycopene, etc.. Both the sub-classes have at least nine conjugated double bonds responsible for the characteristic colors of the carotenoids. Xanthophylls have ring structures at the end of the conjugated double bond chain with polar functions like hydroxyl or keto group. The examples for xanthophylls include lutein, zeaxanthin, capsanthin, canthaxanthin, p-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. Lutein and zeaxanthin contribute to yellow and orange- yellow colour respectively. Lutein and zeaxanthin can be present in plant material in free form and also in ester form. Lutein is present in green leafy vegetables like spinach, kale and broccoli in the free form; fruits like mango, orange and papaya; red paprika, algae, yellow com, contain lutein in the form of its esters etc. It is also present in the blood stream and various tissues in human body and particularly the macula, lens and retina of the eye. Marigold (Tagetes erecta) flower petals are a rich source of lutein in its ester form containing fatty acids. Dried marigold flowers contain approximately 1-1.6 % carotenoids by weight and lutein esters content accounts for 90% of the total carotenoids Q.LX .Antony & M.L. Shankaranarayana, Lutein - A Natural Colorant and a Phytonutrient For Eye Health Protection , The World of Food Ingredients, April/May 64-67, 2001). 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. (W Gau, H.j. Ploschke and C. Wtinsche Mass Spectrometric Identification of Xanthophyll Fatty Acid Esters from Marigold Flowers (Tagetes erecta) Obtained by High Performance Liquid Chromatography and Craig Counter-current Distribution, ]. Chromatogr., 262,277-284,1983 ). Lutein obtained by the hydrolysis of lutein esters from marigold have 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 (F. Khachik, A. Steck and H. Pfander, Isolation and Structural Elucidation of (13Z,13'Z,3R,3'R,6'R)-lutein From Marigold Flowers, Kale, and Human Plasma, J.Agric.Food.Chem ,47,455-461,1999). 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 availability and cost considerations. Essentially, lutein esters and lutein in the free form are commercially important nutraceuticals obtained from marigold flowers. Dried flowers are used for obtaining marigold oleoresin . By subjecting the 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 xanthophylls containing mixture of lutein & zeaxanthin purified further . 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 colour compared to the corresponding cis-isomeric form. The chemical structures of lutein [ (3R,3'R,6'R )-p,e-carotene-3,3'-diol ], zeaxanthin [ ( 3R,3'R )-p,P carotene-3,3'-diol ] are given below in Fig 1 and Fig 2 respectively. 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.(J.M. Moeller, P.F. Jacques and J.B. Blumberg, The Potential Role of Dietary Xanthophylls in Cataract and Age-related Macular Degeneration, J. Am. Coll. Nutr. 19, 522s -527s (2000)] . Lutein and zeaxanthin are known to comprise the macular pigment and lutein isomerizes into zeaxanthin in the macula. (R. A. Bone, J.T. Landrum and L.M. Friedes Distribution of Lutein and Zeaxanthin Stereo-Isomers in the Human Retina, Exp. Eye. Res. 64, 211-218(1997)]. There is evidence suggesting that lutein may have a protective effect against cancers of the breast,colon, lung, skin,cervix and ovaries ( D. A. Cooper, A. L. Eldridge & J. C. Peters, Dietary Carotenoids and Certain Cancers, Heart Disease, and Age Related Macular Degeneration: A Review of Recent Research, Nutr. Rev.,57,201-214,1999; M. L. Slattery, J. Benson, K. Curtin ,M. N. Ma ,D. Schaeffer ,J. D. Potter, Carotenoids and Colon Cancer. Am J Clin Nutr. 71: 335-339, 2000 ) and could bear promise in treatment of cardiovascular disease(A. N. Howard, N. R. Williams, C.R. Palmer, et al, Do Hydroxy-carotenoids Prevent Coronary Heart Disease: A Comparison Between Belfast and Toulouse, Int. J. Vita. Nutr. Res., 66,113-118,1996) Therefore, providing lutein in diet or as nutritional supplements supports better human health and healthy vision. In commercial practice, xanthophylls of food grade quality and free of cis- 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 cis-lutein. In the marigold flower extract, apart from the dominant trans-lutein, the presence of isomers such as 9- , 13-,15-cis-lutein are reported (W. L. Haddon, R. H. Watkins, L. W. Levy, E. Regalado, D. M. Rivaldeneira, R. B. Van Bremen & S. J. Schwartz, Carotenoid Composition of Marigold (Tagetes erecta) Flower Extract Used as Nutritional Supplement., J. Agri Food Chem., 47, 4189-4194,1999). These cis-isomers were earlier wrongly considered as epoxides . As mentioned earlier, lutein in the free form is obtained from lutein fatty acid esters by saponification followed by purification and isolation. Analytically pure (HPLC grade for use in quantification purposes and supported by mass spectral data) samples of lutein and zeaxanthin are not available from chemical suppliers causing difficulty and confusion among analysts, manufacturers and traders. There is also a need for food grade xanthophylls for use as additives in foods and as nutritional and health care supplements. Tyczkowski and Hamilton (J- K. Tyczkowski & P. B. Hamilton, in their article tilted "Preparation of Purified Lutein and Its Di-esters from Extracts of Marigold (Tagetes erecta)" , in Poultry Science 70:651-654,1991) have reported a process for the preparation of free lutein with 99% purity from saponified marigold extract. In the process described, 1 g saponified oleoresin containing 14.7 mg lutein/g was extracted overnight in the dark with 10 ml of hexane: acetone: toluene: absolute alcohol(10:7:7:6 v/v). 10 ml of hexane were then added to the resultant extract followed by another 7 ml of 10% aqueous sodium sulphate . After keeping the solution standing for one hour the clear top layer was separated, condensed under nitrogen to one-third its initial volume, and placed at 4 .degree.C until crystals are formed. The crystals were filtered washed with cold hexane, and dissolved in a minimal amount of warm hexane: acetone (4:1 v/v ) for recrystallization. The final crystals were stored under nitrogen in the dark. The drawback of this method is a multi step process and also time consuming one and also employs harmful organic solvent like toluene. Khachik has described ( F. Khachik, Process for Isolation, Purification and Recrystallisation of Lutein from Saponified Marigold Oleoresin and Uses Thereof, US Patent No. 5,382,714,1995) a process for obtaining lutein, the purity of which is usually greater than 90%, determined by UV/visible spectrophotometry. The purity of the lutein was found to be around 94.79%, its isomers around 3.03% based on HPLC analysis and others consisting of zeaxanthin, etc. The method consists of saponifying marigold extract by admixing with water / alcohol mixture to remove soluble impurities, precipitating lutein crystals by lowering the temperature of the mixture; washing the lutein crystals with water and organic solvent mixture followed by recrystallization. The main drawback of the above mentioned process is in the use of a halogenated solvent. The halogenated solvents are banned for use in human food applications in most of the countries because of apprehensions about their potential carcinogenic effects. Ausich and Sanders ((R. L. Ausich and J. D. Sanders, Process for the Formation, isolation and purification of comestible xanthophyll crystals from plants, US Patent No. 5,648,564,1997) have developed a process for obtaining xanthophyll crystals containing approximately 70-85% total carotenoids, deemed to contain substantially pure xanthophylls. The HPLC analysis of the xanthophylls showed 85-95% trans-lutein, 0.2-1.5% of its geometrical isomers, 2.5-8% zeaxanthin. The process consisted of saponification of marigold oleoresin employing propylene glycol and aqueous alkalL The oleoresin was mixed with propylene glycol and the mixture heated to 55.degree.C, followed by the addition of aqueous potassium hydroxide . Later the temperature of the mixture was raised to 70.degree.C and maintained for 10 hours. When crystals of the lutein separated, the saponified oleoresin was diluted with water and again heated to 70.degree.C under agitation. The lutein crystals were separated by centrifugation and washed with hot water (85.degree.C)., The poor solubility of xanthophyll esters in propylene glycol and the subsequent heating to temperatures around 70.degree.C for 10 hours are the main disadvantages of the above mentioned process, since the lutein undergoes isomerization and decomposition under the above conditions Further, propylene glycol is not a cost- affordable solvent from commercial considerations. Khachik in his US Patent No 6,262,284, 2001 has developed a process for obtaining lutein and zeaxanthin crystals (97% pure) starting from marigold meal instead of marigold extract(F Khachik, Process for extraction and purification of Lutein, zeaxanthin & rare carotenoids from marigold flowers and plants , US Patent No 6,262,284, 2001). This process involved simultaneous extraction and saponification of xanthophyll esters. In this process the marigold meal was mixed with tetrahydrofuran(THF) and 10% ethanolic potassium hydroxide at room temperature, followed by the addition of ethanol and potassium hydroxide to maintain pH around 12. After filtration the solids were washed with tetrahydrofuran and dried under vacuum. The dried material was further washed using a mixture of ethanol and water to bring down the pH to 7 and again washed with ethanol to obtain crude lutein ( 70% purity). This material was further purified by dissolving in THF and using silica gel column chromatography. THF/ water mixture was used as eluant to obtain 97% trans-lutein and 4% zeaxanthin. The main limitations in the above process are saponification of the extract without concentration leading to consumption of large volumes of solvents that are difficult to manage in commercial production. Further formation of peroxides from solvents like THF may cause degradation of the xanthophylls. The use of silica-gel column chromatography is a cumbersome and less economic process for commercial scale production of pure lutein crystals. In a recent patent of Sas and Adams ( B J Sas and C Adams, " Conversion of Xanthophylls in Plant Material for use as food colourant" ,US Patent No 6221417 ) have employed potassium methyl alcoholate for saponifying the biological raw materials such as marigold and paprika in methanol medium. The claims include the use of isopropanol. The process does not use aqueous solution in the saponification stage . The advantages claimed are direct saponification of the xanthophyll esters in the plant material instead of the xanthophyll extracts obtained by solvent extraction which will reduce the step as well as the cost. The process is applicable only for plant materials for direct feeding to poultry etc, and cannot be used for yielding high purity material in the form of xanthophyll crystals. Recently Rodriguez et al. (G Rodriguez, M.D Torres-Cardona, A Diaz,, Purification of Xanthophylls from Marigold Extract that Contain High Levels of Chlorophylls, US Patent No 6329557 , 2001) have disclosed an industrial scale process for obtaining xanthophyll crystals from marigold extract containing high levels of chlorophylls. The method consisted of dissolving the saponified extract in water at room temperature and adjusting the pH to 6 using 25% aqueous phosphoric acid, followed by heating to 70.degree.C under stirring. After cooling and removal of the lower layer containing chlorophylls, the oily material was washed with aqueous acid solution followed by the addition of hexane to obtain xanthophyll precipitate. The precipitate was further purified washing with hexane and drying under vacuum to obtain xanthophyll crystals containing 66 % total xanthophylls in which 92.2% is trans lutein & 6.1.% is zeaxanthin as measured by HPLC . The method is useful for marigold oleoresins containing high levels of chlorophylls. The main disadvantage of this process is in the steps involving the use of acid and heating, as these are likely to degrade xanthophylls and enhance the possibilities of isomerization. More recently , Madhavi and Kagan ( D. L. Madhavi, D I Kagan; Process for the Isolation of Mixed Carotenoids from Plants, US Patent No 6380442 , 2002) have reported a process for the isolation of mixed carotenoids from plants and illustrated the same with examples of marigold oleoresin. The process involved hydrolysis of the carotenoid esters using isopropanol, water and potassium hydroxide for a period of 60-90 minutes at temperatures of about 60.degree.C. The hydrolyzed carotenoids were precipitated from the saponification mixture using large quantity of water. The precipitate was recovered by centrifugation followed by repeated water washing and drying to obtain crystalline material The method is not attractive for commercial applications since the water required is more than 30 times per kg of the input material. In virtually all the processes described in the above mentioned prior art literature, invariably the first step is the saponification of oleoresin (specifically marigold) using an alcoholic and / or aqueous alkali preferably KOH. The saponification steps in these processes (with the exception of US Patent No 6221417J employ external water . The processes involve extracting , re extracting with solvents such as THF, halogenated solvents .Therefore these processes are inappropriate for industrial scale-up operations due to high cost & toxicological considerations In present days ,there is high demand for xanthophyll crystals containing high amounts of trans-lutein and /or zeaxanthin for its use as antioxidants, prevention of cataract and macular degeneration, as lung cancer-preventive agent, as agents for the absorption of harmful ultraviolet light from sun rays and quencher of photo-induced free radical and reactive oxygen species, etc.. It is therefore felt that there is a need for providing an economical and simple process for the production of xanthophyll crystals containing high amounts of trans - lutein for using in food and nutraceutical supplements, employing toxicologically safe solvents for extraction such as GRAS [Generally Recognized As Safe] status . Objectives Of The Invention : Accordingly , the main objective of the present invention is to provide novel xanthophyll crystals containing high amounts of trans-lutein and/or zeaxanthin. Another objective of the present invention is to provide novelxanthophyll crystals containing at least 85% total xanthophylls out of which the trans-lutein content is at least 90 %, the remaining being zeaxanthin and other carotenoids . Yet another objective of the present invention is to provide novel xanthophyll crystals containing at least 85% total xanthophylls out of which the trans-lutein content is at least 90 % , the remaining being zeaxanthin and other carotenoids from oleoresin and extracts of plant materials such as marigold (Tagetes erecta). We have in our application no 622 MAS 02 described and claimed an improved process for the preparation of xanthophyll crystals containing at least 85% total xanthophylls out of which the trans-lutein content is at least 90 %, the remaining being zeaxanthin and other carotenoids wherein the starting material is xanthophyll esters concentrate obtained through solvent or supercritical fluid extraction. That process employs ethyl acetate which is toxicologically safe ( GRAS or Generally Recognized As Safe solvent) The process described therein is simple, convenient, economical and commercially feasible. In addition in the process the recovery of alcohol is effected cost-effectively by recovering the unused alcohol and reusing it for the process thereby making the process more economical The process has been developed based on our main finding that: a. the saponification step can be managed by using alkali ^preferably potassium hydroxide, in excess alcohol without adding water to the reaction mixture b. recovering the unused alcohol and reusing it, if required, in the process c. employing a solvent having the under mentioned characteristics for extracting xanthophylls at the stage after removing the unreacted alcohol and diluting with water and before crystallization step, and if required , dissolving the resulting xanthophyll concentrate using a polar / non- polar solvent mixture to obtain crude crystals, further recrystallizing with alcohol. While selecting the suitable solvent for use in the above mentioned stage of the process of the present invention , a variety of criteria are to be taken into consideration very carefully . Accordingly the major criteria to be considered in solvent selection are (i) it should be water immiscible (ii) the solvent should he non-halogenated (iii) the solvent should have GRAS status (iv) the solubility of xanthophylls in the solvent should be high (v) boiling point of the solvent which should be in the range of 50 to SO.degree.C and (v) stability of xanthophylls in the solvent used should be high. It should be noted though there are many solvents in which lutein is soluble , due to other considerations such solvents cannot be used in this stage of the process of the present invention . For example the solvents in which lutein is highly soluble such as -chloroform , benzene , etc. cannot be used because such solvents are prohibited for human consumption . Hence the solvent used should fall under GRAS status . It is also to be noted that the stage where the solvent is to be used contains large amounts of water . Therefore the solvent to be used in this stage of the process should be water immiscible so as to facilitate phase separation . Accordingly many of the solvents which fall under GRAS status and in which lutein is highly soluble cannot be used . For illustrating the above points we have in the Table 1 given below provided the characteristics of the solvents which were considered for use in the stage of the present invention as explained earlier . From the solvents listed in the Table I , it can be observed that the solvent falling under the category of organic acid esters are only eligible to use in the particular stage of the process of the invention explained above . Out of the aliphatic acid esters which can be employed in the stage of the process of the present invention it is observed that methyl acetate is soluble in water and propyl acetate has a boiling point of 102.degree. C. These characteristics fall outside the requirements explained above making methyl and propyl acetates ineligible for consideration in the process of the present invention . Therefore the only solvent which satisfies the above special characteristics and which can be used according to the present invention is ethyl acetate During our sustained R&D efforts to develop the improved process for the preparation of xanthophyll crystals we observed that the use of the above said special category of solvent in the particular stage of the process explained above helps in effectively and efficiently separating the xanthophyll concentrate from most impurities present in the mixture compared to other processes disclosed in the art. The use of ethyl acetate in the process of the present invention also helps in removing the impurities at a low temperature ( say at room temperature). It should be specifically noted , in this context, that the selection of the particular solvent at a particular stage of the process of the invention is due to the inventive ingenuity of the applicant and is not a mere application of a known and obvious solvent To the best of our knowledge the novel xanthophyll crystals containing at least 90% total xanthophylls with at least 90% trans-lutein content is not known . Accordingly , the present invention provides novel xanthophyll crystals containing at least 85% total xanthophylls out of which the trans-lutein content is at least 90 %, the remaining being zeaxanthin and other carotenoids The process which has been described in the co pending application no 622 MAS 02 to prepared the above said novel Xanthophyll crystals comprises (a)admixing a xanthophyll ester concentrate containing esters of lutein and zeaxanthin with an alcoholic alkali solution and heating the resultant solution at a temperature in the range of 65.degree.C to about SCdegree.C preferably at 70.degree.C (b) maintaining the solution at the same temperature for a period sufficient to saponify the xanthophyll esters present in the concentrate; ( c ) removing alcohol from the reaction mixture under reduced pressure to get a saponified crude concentrate; (d) reusing the recovered alcohol if required in the step (a) above (e) admixing the resultant saponified crude concentrate obtained in step (c) with water to form a diluted oily mixture ; (f) extracting the diluted oily mixture obtained in step (e) with ethyl acetate to get xanthophyll extract (g)washing the resultant extract with water and concentrating the extract under reduced pressure (h) admixing the concentrated extract obtained in step(g) with polar/non-polar solvent mixture and alcohol to remove undesirable materials and to get a precipitate (i) admixing the precipitate obtained in step (h) with alcohol to get xanthophyll crystals and (j) filtering and drying. The concentrate used in the process of the present invention can be obtained from any plant source. Preferably the xanthophyll ester concentrate used in step (a) may be those obtained from naturally occurring plant materials more particularly from marigold flowers , fruits etc. The alcohol used may be selected from a lower aliphatic alcohol such as methanol ethanol, isopropanol, preferably isopropanol. The ratio of xanthophyll ester concentrate and the aliphatic alcohol used may range from 1:2 to 1:5 preferably 1: 3 by weight by volume. The alkali used for preparing the alcoholic alkali solution may be selected from sodium hydroxide or potassium hydroxide, preferably potassium hydroxide. The ratio of xanthophyll ester concentrate to the alkali may be in the range of 1:0.25 to 1: 0.4 weight by weight preferably 1:0.25. The amount of alcoholic alkali solution used in the step (a) may consist of 8-12 parts of the alcohol such as methanol, ethanol, isopropanol, preferably ,isopropanol and 1 part of alkali by volume by weight. The saponification reaction mixture is maintained for a period of 3 to 5 hours, preferably for 3 hours at the temperature in the range of 65.degree.C to about SO.degree.C, preferably at 70.degree.C. The unused alcohol is recovered by distillation under vacuum, and reused, if required , in step (a). The amount of water used in the step (e) of the process for dilution of the saponification mixture may be in the range of 1:4 to 1:6, preferably 1:4, weight by volume . The ratio of ethyl acetate employed for mixing with the diluted saponification reaction mixture to the solvent may range from 1:1 to 1:3 preferably 1:1 by volume / volume The non-polar solvent used in step (h) may be selected from the hydrocarbon solvents like pentane, hexane and heptane, and the like ,preferably hexane. The polar solvent used may be selected from 2-propanone, 2-pentanone, 2-butanone, and the like preferably 2-propanone. The alcohol which may be used in step(i) for further purification may be selected from methanol, ethanol isopropanol etc , preferably methanol Detailed Description Of The Invention: In the present invention, the plant material containing xanthophyll esters is saponified using alkali and an aliphatic alcohol and the resulting xanthophyll solution is subjected to purification by extracting with ethyl acetate and washing with water followed by purification with a mixture of polar/ non- polar solvent mixtures and alcohol to remove the solvent soluble materials, thereby resulting in xanthophyll crystals with high levels of lutein and/or zeaxanthin. Marigold flowers or Chinese wolfberry are considered to be the best possible commercial source for obtaining xanthophyll esters. It is well known that the xanthopyll extract or oleoresin is typically obtained by solvent extraction of dehydrated plant material in milled or pelletized form prepared by drying of fresh or fermented flowers. Generally 15-30 % xanthophyll esters are found in the marigold oleoresins depending on the cultivars , meal quality and extraction processes. The current commercial and industrial practices in the production, handling and supply of marigold flowers / meal and extraction may be summarized as follows: Fresh marigold plant material containing high moisture (80 - 90%) and xanthophyll content of around 0.1-0.2% by weight is used . Silaging of the harvested marigold plant materials is carried out in the fields for extended periods of time, followed by drying using mechanical dryers to reduce the moisture to about 10%. Reducing the size of the dehydrated materials to obtain marigold meal with xanthophyll content of around 1-1.6 % by weight. Preparing the pellets of the meal thus obtained and supplying to the extractors. According to commercial considerations and practices, hexane is the choice for extraction of xanthophyll esters in view of specificity and ease of removal of the solvent residues conforming to standards and specifications. Standardization of the particle size of the meal, extraction condiHons like volumes of solvent and contact time required, removal of solvent, product yield and analysis are routinely performed and validated. This is achieved by monitoring the analytes at various stages employing both spectrophotometric and HPLC analysis. The spectrophotometric analysis (AOAC -16 ^ edition, Method 970.64 ) provides data on the total xanthophyll content by weight while the HPLC analysis (W.L.Hadden, R.H.Watkins, L.W.Levy, E.Regalado, D.M.Rivadeneira, R.B.Van Breemaen, Carotenoid Composition of Marigold {Tagetes erecta ) Flower Extract Used as Nutritional Supplement, J.Agric.Food Chem,47,4189-4194,1999) of the saponified mixture provides percentage composition of the individual xanthophylls in the total xanthophylls. The xanthophyll extract is mixed with alcohol such as isopropanol in which alkali is dissolved forming alkali alcoholate. The ratio of the three constituents of the saponification reaction mixture are about 1 part oleoresin and 3 parts alcohol, preferably isopropanol and 0.25 to 0.4 parts of alkali. Although 2 volumes of alcohol solvent - containing alkali is sufficient for dissolution of the oleoresin , 3 volumes of the solvent mixture is desirable for homogeneity , free flow and better saponification reaction. The saponification is performed by heating the reaction mixture preferably to a temperature of 70.degree.C for a period of 3 hours. The amount of alkali required is related to xanthophyll esters content present in the oleoresin. The exact quantity of alkali required has to be worked out experimentally. Generally, for each 100 g oleoresin, about 25 g alkali is adequate for completion of hydrolysis of the esters. The saponification of the oleoresin results in liberation of xanthophylls in free form along with alkali salts of fatty acids such as palmitic, myristic, stearic acid, etc. The progress of the saponification reaction is monitored by HPLC analysis for the presence of xanthophyll esters till their absence. This analysis is performed by taking an aliquot of the saponification reaction mixture and extraction with hexane: acetone: toluene; absolute alcohol (10:7:7:6 v/v) followed by addition of hexane and 10% sodium sulphate solution and analyzing the upper layer by HPLC. After the complete hydrolysis of the xanthophyll esters, the reaction mixture is subjected to vacuum distillation to recover alcohol. The saponified product obtained is treated with water resulting in a brownish yellow oily layer containing xanthophylls in free form, fatty acid soaps and other impurities. The recovered alcohol may be reused for further saponification of other batch . The solution obtained is subjected to extraction with ethyl acetate The amount of ethyl acetate used may be in the ratio of 1:1 volume . More than 90% xanthophylls are extracted into the ethyl acetate layer . This resultant product is washed with deionised water (2 times) preferably using equal volumes . By this way, most of the fatty acid soapy material is removed into water layer, which is then discarded. The ethyl acetate extract is distilled off under reduced pressure to recover ethyl acetate and the saponified xanthophyll concentrate (xanthophyll content 15-20% by weight by spectrophotometry ) The xanthophyll concentrate obtained is admixed with a solvent or a mixture of solvents , preferably acetone and hexane mixture at room temperature with stirring. Preferably one part of xanthophyll concentrate obtained is admixed with four parts of the solvent or a mixture of solvents. The ratio of polar/non-polar solvent mixture used ranges from 2:8 to 3:7 volume /volume, preferably 2:8 . The xanthophylls separate out as crude crystals and the impurities are dissolved in the solvent / solvent mixture which are removed by filtration. These crude crystals show xanthophyll content of around 50 to 60 % by weight analyzed by spectrophotometry . Finally ,these crude crystals are further purified by washing with aliphatic alcohol such as methanol, ethanol or isopropanol, preferably methanol and vacuum-dried at room temperature to obtain xanthophyll crystals with at least 85 % xanthophylls by weight (analyzed by spectrophotometry) and trans-lutein content of the xanthophylls over 90 % (area % by HPLC method). The purified xanthophyll crystals obtained contained approximately 85-92% total xanthophylls by weight and the composition of the carotenoids of the xanthophylls, as determined by HPLC analysis(peak area %) consist of 90-95% trans-lutein, 4-8% trans-zeaxanthin, 0.5-1.0% cis-lutein isomers and others. It is possible that changes could be effected in the embodiment described above without deviating from the broad spectrum of the concept of the invention . Therefore, it is to be noted that this invention is not limited to the particular embodiments disclosed herein but it is intended to cover all modifications which fall within the spirit and scope of the present invention . The details of the process of preparing the novel xanthophylls crystals are given in the examples provided below which are provided only by way of illustration and therefore should not be construed to limit the scope of the process Example-1 Commercial grade marigold oleoresin (57.98 g) containing 11.54 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 15 g potassium hydroxide in 175 ml isopropanol.) The .. saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 230 ml of water at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(3 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (25.01g) This resultant crude extract (25.01 g) was subjected to purification by stirring with 100 ml of hexane/acetone mixture(80:20) at room temperature for 30 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs. The yield of the xanthophyll crystals was 3.41%(1.98g ). Xanthophyll content was 86.23 % by weight ( as determined by UV/Vis spectrophotometry ) out of which the contents of trans-lutein , zeaxanthin, and other carotenoids were 91.43% , 6.40% and 2.17 % respectively as determined by .HPLC analysis. Example -2 Commercial grade marigold oleoresin (56.3 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 14 g potassium hydroxide in 170 ml isopropanol.) The saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 225 ml of water at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(3 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (22.21 g) This crude extract (22.21 g) was subjected to purification by stirring with 90 ml of hexane/acetone mixture(80:20) at room temperature for 30 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol .The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs. The yield of the xanthophyll crystals was 3.43%(1.93 g). Xanthophyll content was 88.69 % by weight ( as determined by UV/Vis spectrophotometry ) out of which the contents of trans- lutein , zeaxanthin and other carotenoids were 90.78% , 6.27% and 2.95 % respectively as determined by HPLC analysis. Example-3 Commercial grade marigold oleoresin (51.60 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 12.90 g potassium hydroxide in 155 ml isopropanol). The saponification mixture was heated maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 206 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory furmel and extracted with equal volume of ethyl acetate(3 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (21.80g) This crude extract (21.80 g) was subjected to purification by stirring with 87 ml of hexane/acetone mixture(80:20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs. The yield of the xanthophyll crystals was 2.11 g.(4.09%) Xanthophyll content was 90.07 % by weight ( as determined by UV/Vis spectrophotometry ) out of which the contents of trans-lutein,zeaxanthin and other carotenoids were 90.10% , 7.08% and 2.82% respectively as determined by HPLC analysis. Example-4 Commercial grade marigold oleoresin (50.0 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 12.50 g potassium hydroxide in 150 ml isopropanol). The „ saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 200 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(4 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (21.70g) This crude extract (21.70 g) was subjected to purification by stirring with 87 ml of hexane/acetone mixture(80:20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol .The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs. The yield of the xanthophyll crystals was 2.11 g. Xanthophyll content was 90.21 % by weight ( as determined by UV/Vis spectrophotometry ). out of which the contents of trans-lutein ,zeaxanthin and other carotenoids were 90.99% , 7.07% and 1.94% respectively as determined by HPLC analysis. Example-5 Commercial grade marigold oleoresin (47.30 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate prepared by dissolving 11.90 g potassium hydroxide in 142 ml isopropanol. The _ saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 237 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(5 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (19.90g) This crude extract (19.90 g) was subjected to purification by stirring with 80 ml of heptane/ethyl-methyl ketone mixture(80;20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol .The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs. The yield of the xanthophyll crystals was 1.52 g(3.04%). Xanthophyll content was 91.34 % by weight ( as determined by UV/Vis spectrophotometry ). out of which the contents of trans-lutein .zeaxanthin and other carotenoids were 90.20% , 7.25% 2.55 % respectively as determined by HPLC analysis. Example-6 Commercial grade marigold oleoresin (50.40 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 12.85 g potassium hydroxide in 154 ml isopropanol). The „ saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 252 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(5 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali/ soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (21.40g) This crude extract (21.40 g) was subjected to purification by stirring with 80 ml of hexane/acetone mixture(80:20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with ethanol .The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs. The yield of the xanthophyll crystals was 2.299 g (4.54%). Xanthophyll content was 89.05 % by weight ( as determined by UV/Vis spectrophotometry ). out of which the contents of trans-lutein ,zeaxanthin and other carotenoids were 91.97% , 6.83% and 1.20% respectively as determined by HPLC analysis. Example-7 Commercial grade marigold oleoresin (51.60 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 12.90 g potassium hydroxide in 155 ml isopropanol). The _ saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 258 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(5 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (25.60g) This crude extract (25.60 g) was subjected to purification by stirring with 102 ml of hexane/acetone mixture(80:20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with isopropanol. The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs. The yield of the xanthophyll crystals was 1.996 g(3.87%). Xanthophyll content was 85.73 % by weight ( as determined by UV/Vis spectrophotometry ). out of which the contents of trans-lutein, zeaxanthin and other carotenoids were 90.53% , 7.72% and 1.75% respectively as determined by HPLC analysis. Example 8 Commercial grade marigold oleoresin (47,30 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 11.90 g potassium hydroxide in 142 ml isopropanol). The ^ saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 237 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(5 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (23.0g) This crude extract (23.0 g) was subjected to purification by stirring with 92 ml of hexane/ethyl-methyl ketone mixture(80;20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol .The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs. The yield of the xanthophyll crystals was 1.73 g (3.66%). Xanthophyll content was 81.41 % by weight ( as determined by UV/vis spectrophotometry ). out of which the contents of trans-lutein ,zeaxanthin and other carotenoids were 90.04% , 6.95% and 3.01% respectively as determined by HPLC analysis. Example-9 Commercial grade marigold oleoresin (50 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 12.5 g potassium hydroxide in 150 ml isopropanol). The, saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 240 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(5 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extiact (22.20g) This crude extract (22.20 g) was subjected to purification by stirring with 90 ml of hexane/acetone mixture(80:20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol. The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs. The yield of the xanthophyll crystals was 2.78 g (5.56%). Xanthophyll content was 90.58 % by weight ( as determined by UV/Vis spectrophotometry ).). out of which the contents of trans-lutein,zeaxanthin and other carotenoids were 91.26% , 5.68% and 3.06% respectively as determined by HPLC analysis. Advantages of the invention • The commercial grade xanthophyll crystals conatins high content lutein and/or zeaxanthin which make it ideal and suitable for use as human nutritional supplements, as an anti-oxidant, for applications in prevention of cancer and age-related macular degeneration of eye., as anti-oxidant, and as a food/feed colourant. We Claim 1 .A novel xanthophyll crystals having at least 85% total xanthophylls out of which the trans-lutein content is at least 90 % , the remaining being zeaxanthin and other carotenoids 2. A novel xanthophyll crystals as claimed in claim 1 wherein it contains at least 85% total xanthophylls out of which the trans-lutein content is at lest 92 % , zeaxanthin content is around 4 to 8% and the remaining being other carotenoids . 3 .A novel xanthophyll crystals containing high amounts of trans-lutein and / or zeaxanthin substantially as herein described .

Full Text

Introduction
This invention relates to novel xanthophyll crystals. This invention particularly relates to xanthophyll crystals containing high content of trans lutein and / or zeaxanthin This invention more particularly relates to novel xanthophyll crystals containing at least 85% total xanthophylls out of which the trans-lutein content is at least 90 %, the remaining being zeaxanthin and other carotenoids
This application is divided out of the application no 622 MAS 2002 . In the application no 622 MAS 021 we have described and claimed an improved process for the preparation of xanthophyll crystals containing high content of trans lutein and / or zeaxanthin . More particularly in that application we have described and claimed an improved process for the preparation of xanthophyll crystals containing at least 85% total xanthophylls out of which the trans-lutein content is at least 90 %, the remaining being zeaxanthin and other carotenoids .
The xanthophyll crystals of the present invention are useful as antioxidant, prevention of cataract and macular degeneration, lung cancer preventive agent, as agents for the absorption of harmful ultra-violet light from sun rays and quencher of photo-induced free radical and reactive oxygen species. The crystals can also be used for the colouration of food and animal/poultry feed. The xanthophyll crystals prepared by the process of the current invention can also be used for the colouration of food and animal/poultry feed.
It is known from various studies that a higher dietary intake of carotenoids is associated with a lower risk for AMD ( Age - related Macular Degeneration). The specific carotenoids, lutein and zeaxanthin, which are primarily obtained from dark green leafy vegetables, were most strongly associated with a reduced risk for AMD. Individuals consuming the highest levels of carotenoids had a statistically significant 43% lower risk for AMD. A significant trend was seen for a lower risk for AMD with increasing amounts of carotenoids in the diet. (JM Seddon et. al .Dietary Carotenoids, Vitamins A, Q and E, and Advanced Age-Related Macular Degeneration, Journal of the American Medical Association, November, 1994, Vol. 272, No. 18, pages 1413-1420)

There is a strong inverse association between higher consumption of dark green vegetables, which contain xanthophylls which are rich in lutein and zeaxanthin, and a decreased risk for oxidative stress related diseases such as cataract and cancer as well. Both lutein and zeaxanthin are reported to possess strong anti-tumour promoting properties. (L Packer, M Hiramatsu, T Oshikawa,(Editors), Antioxidant Food Supplements in Human Health, Academic Press, NY, 1999, Pp 223 and Pp 226) Recent studies also reveal that lutein and zeaxanthin can play a useful role in combating conditions which lead to cardio-vascular disease, atherosclerosis, skin cancer, ovarian cancer, etc.
Although dark green vegetables are excellent dietary sources of lutein , the isolation and purification of this compound in large quantities from green vegetables is time-consuming and costly due to the high level of chlorophyll pigments. 25g of a fresh, dark green vegetable such as kale would theoretically provide 10 mg of lutein.( F. Khachik etal, 'Lutein, Lycopene, and Their Oxidative Metabolites in Chemoprevention of Cancer/' Journal of Cellular Biochemistry, 1995, Supplement 22:236, pp. 236-246. )Consistent bioavailability and absorption of carotenoids from foods is not as high as supplementation with pure carotenoids.
Extracts from the petals of marigold flowers are an excellent source of lutein esters in large quanhties and contain no significant levels of other carotenoids. Lutein and zeaxanthin being fat-soluble nutrients, can be delivered in consistent doses through soft gelatin capsule based supplements, or in stabilized tablets, and other nutritional preparations to overcome deficiencies of diet and improve levels of serum lutein/zeaxanthin, and thereby, the levels of lutein/zeaxanthin in the macula.
Background Of The Invention
Carotenoids are yellow, red and orange pigments which 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.

Carotenoids absorb light in the 400-500 nm region of the visible spectrum. This physical property imparts the characteristic 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.
Carotenoids are non-polar compounds classified into two sub-classes namely more polar compounds called xanthophylls or oxycarotenoids and non-polar hydrocarbon carotenes like p-carotene, lycopene, etc.. Both the sub-classes have at least nine conjugated double bonds responsible for the characteristic colors of the carotenoids. Xanthophylls have ring structures at the end of the conjugated double bond chain with polar functions like hydroxyl or keto group. The examples for xanthophylls include lutein, zeaxanthin, capsanthin, canthaxanthin, p-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.
Lutein and zeaxanthin contribute to yellow and orange- yellow colour respectively. Lutein and zeaxanthin can be present in plant material in free form and also in ester form. Lutein is present in green leafy vegetables like spinach, kale and broccoli in the free form; fruits like mango, orange and papaya; red paprika, algae, yellow com, contain lutein in the form of its esters etc. It is also present in the blood stream and various tissues in human body and particularly the macula, lens and retina of the eye.
Marigold (Tagetes erecta) flower petals are a rich source of lutein in its ester form containing fatty acids. Dried marigold flowers contain approximately 1-1.6 % carotenoids by weight and lutein esters content accounts for 90% of the total carotenoids Q.LX .Antony & M.L. Shankaranarayana, Lutein - A Natural Colorant and a Phytonutrient For Eye Health Protection , The World of Food Ingredients, April/May 64-67, 2001). 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. (W Gau, H.j. Ploschke and C. Wtinsche Mass Spectrometric Identification of Xanthophyll Fatty Acid Esters from Marigold Flowers (Tagetes erecta) Obtained by High Performance Liquid Chromatography and Craig Counter-current Distribution, ]. Chromatogr., 262,277-284,1983 ).
Lutein obtained by the hydrolysis of lutein esters from marigold have 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 (F. Khachik, A. Steck and H. Pfander, Isolation and Structural Elucidation of (13Z,13'Z,3R,3'R,6'R)-lutein From Marigold Flowers, Kale, and Human Plasma, J.Agric.Food.Chem ,47,455-461,1999). 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 availability and cost considerations.
Essentially, lutein esters and lutein in the free form are commercially important nutraceuticals obtained from marigold flowers. Dried flowers are used for obtaining marigold oleoresin . By subjecting the 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 xanthophylls containing mixture of lutein & zeaxanthin purified further .
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 colour compared to the corresponding cis-isomeric form.
The chemical structures of lutein [ (3R,3'R,6'R )-p,e-carotene-3,3'-diol ], zeaxanthin [ ( 3R,3'R )-p,P carotene-3,3'-diol ] are given below in Fig 1 and Fig 2 respectively.

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.(J.M. Moeller, P.F. Jacques and J.B. Blumberg, The Potential Role of Dietary Xanthophylls in Cataract and Age-related Macular Degeneration, J. Am. Coll. Nutr. 19, 522s -527s (2000)] . Lutein and zeaxanthin are known to comprise the macular pigment and lutein isomerizes into zeaxanthin in the macula. (R. A. Bone, J.T. Landrum and L.M. Friedes Distribution of Lutein and Zeaxanthin Stereo-Isomers in the Human Retina, Exp. Eye. Res. 64, 211-218(1997)].
There is evidence suggesting that lutein may have a protective effect against cancers of the breast,colon, lung, skin,cervix and ovaries ( D. A. Cooper, A. L. Eldridge & J. C. Peters, Dietary Carotenoids and Certain Cancers, Heart Disease, and Age Related Macular Degeneration: A Review of Recent Research, Nutr. Rev.,57,201-214,1999; M. L. Slattery, J. Benson, K. Curtin ,M. N. Ma ,D. Schaeffer ,J. D. Potter, Carotenoids and Colon Cancer. Am J Clin Nutr. 71: 335-339, 2000 ) and could bear promise in treatment of cardiovascular disease(A. N. Howard, N. R. Williams, C.R. Palmer, et al, Do Hydroxy-carotenoids Prevent Coronary Heart Disease: A Comparison Between Belfast and Toulouse, Int. J. Vita. Nutr. Res., 66,113-118,1996) Therefore, providing lutein in diet or as nutritional supplements supports better human health and healthy vision.

In commercial practice, xanthophylls of food grade quality and free of cis- 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 cis-lutein. In the marigold flower extract, apart from the dominant trans-lutein, the presence of isomers such as 9- , 13-,15-cis-lutein are reported (W. L. Haddon, R. H. Watkins, L. W. Levy, E. Regalado, D. M. Rivaldeneira, R. B. Van Bremen & S. J. Schwartz, Carotenoid Composition of Marigold (Tagetes erecta) Flower Extract Used as Nutritional Supplement., J. Agri Food Chem., 47, 4189-4194,1999). These cis-isomers were earlier wrongly considered as epoxides .
As mentioned earlier, lutein in the free form is obtained from lutein fatty acid esters by saponification followed by purification and isolation. Analytically pure (HPLC grade for use in quantification purposes and supported by mass spectral data) samples of lutein and zeaxanthin are not available from chemical suppliers causing difficulty and confusion among analysts, manufacturers and traders. There is also a need for food grade xanthophylls for use as additives in foods and as nutritional and health care supplements.
Tyczkowski and Hamilton (J- K. Tyczkowski & P. B. Hamilton, in their article tilted "Preparation of Purified Lutein and Its Di-esters from Extracts of Marigold (Tagetes erecta)" , in Poultry Science 70:651-654,1991) have reported a process for the preparation of free lutein with 99% purity from saponified marigold extract. In the process described, 1 g saponified oleoresin containing 14.7 mg lutein/g was extracted overnight in the dark with 10 ml of hexane: acetone: toluene: absolute alcohol(10:7:7:6 v/v). 10 ml of hexane were then added to the resultant extract followed by another 7 ml of 10% aqueous sodium sulphate . After keeping the solution standing for one hour the clear top layer was separated, condensed under nitrogen to one-third its initial volume, and placed at 4 .degree.C until crystals are formed. The crystals were filtered washed with cold hexane, and dissolved in a minimal amount of warm hexane: acetone (4:1 v/v ) for recrystallization. The final crystals were stored under nitrogen in the dark. The drawback of this method is a multi step process and also time consuming one and also employs harmful organic solvent like toluene.

Khachik has described ( F. Khachik, Process for Isolation, Purification and Recrystallisation of Lutein from Saponified Marigold Oleoresin and Uses Thereof, US Patent No. 5,382,714,1995) a process for obtaining lutein, the purity of which is usually greater than 90%, determined by UV/visible spectrophotometry. The purity of the lutein was found to be around 94.79%, its isomers around 3.03% based on HPLC analysis and others consisting of zeaxanthin, etc. The method consists of saponifying marigold extract by admixing with water / alcohol mixture to remove soluble impurities, precipitating lutein crystals by lowering the temperature of the mixture; washing the lutein crystals with water and organic solvent mixture followed by recrystallization.
The main drawback of the above mentioned process is in the use of a halogenated solvent. The halogenated solvents are banned for use in human food applications in most of the countries because of apprehensions about their potential carcinogenic effects.
Ausich and Sanders ((R. L. Ausich and J. D. Sanders, Process for the Formation, isolation and purification of comestible xanthophyll crystals from plants, US Patent No. 5,648,564,1997) have developed a process for obtaining xanthophyll crystals containing approximately 70-85% total carotenoids, deemed to contain substantially pure xanthophylls. The HPLC analysis of the xanthophylls showed 85-95% trans-lutein, 0.2-1.5% of its geometrical isomers, 2.5-8% zeaxanthin. The process consisted of saponification of marigold oleoresin employing propylene glycol and aqueous alkalL The oleoresin was mixed with propylene glycol and the mixture heated to 55.degree.C, followed by the addition of aqueous potassium hydroxide . Later the temperature of the mixture was raised to 70.degree.C and maintained for 10 hours. When crystals of the lutein separated, the saponified oleoresin was diluted with water and again heated to 70.degree.C under agitation. The lutein crystals were separated by centrifugation and washed with hot water (85.degree.C).,
The poor solubility of xanthophyll esters in propylene glycol and the subsequent heating to temperatures around 70.degree.C for 10 hours are the main disadvantages of the above mentioned process, since the lutein undergoes isomerization and decomposition under the

above conditions Further, propylene glycol is not a cost- affordable solvent from commercial considerations.
Khachik in his US Patent No 6,262,284, 2001 has developed a process for obtaining lutein and zeaxanthin crystals (97% pure) starting from marigold meal instead of marigold extract(F Khachik, Process for extraction and purification of Lutein, zeaxanthin & rare carotenoids from marigold flowers and plants , US Patent No 6,262,284, 2001). This process involved simultaneous extraction and saponification of xanthophyll esters. In this process the marigold meal was mixed with tetrahydrofuran(THF) and 10% ethanolic potassium hydroxide at room temperature, followed by the addition of ethanol and potassium hydroxide to maintain pH around 12. After filtration the solids were washed with tetrahydrofuran and dried under vacuum. The dried material was further washed using a mixture of ethanol and water to bring down the pH to 7 and again washed with ethanol to obtain crude lutein ( 70% purity). This material was further purified by dissolving in THF and using silica gel column chromatography. THF/ water mixture was used as eluant to obtain 97% trans-lutein and 4% zeaxanthin.
The main limitations in the above process are saponification of the extract without concentration leading to consumption of large volumes of solvents that are difficult to manage in commercial production. Further formation of peroxides from solvents like THF may cause degradation of the xanthophylls. The use of silica-gel column chromatography is a cumbersome and less economic process for commercial scale production of pure lutein crystals.
In a recent patent of Sas and Adams ( B J Sas and C Adams, " Conversion of Xanthophylls in Plant Material for use as food colourant" ,US Patent No 6221417 ) have employed potassium methyl alcoholate for saponifying the biological raw materials such as marigold and paprika in methanol medium. The claims include the use of isopropanol. The process does not use aqueous solution in the saponification stage . The advantages claimed are direct saponification of the xanthophyll esters in the plant material instead of the xanthophyll extracts obtained by solvent extraction which will reduce the step as well as the cost.

The process is applicable only for plant materials for direct feeding to poultry etc, and cannot be used for yielding high purity material in the form of xanthophyll crystals.
Recently Rodriguez et al. (G Rodriguez, M.D Torres-Cardona, A Diaz,, Purification of Xanthophylls from Marigold Extract that Contain High Levels of Chlorophylls, US Patent No 6329557 , 2001) have disclosed an industrial scale process for obtaining xanthophyll crystals from marigold extract containing high levels of chlorophylls. The method consisted of dissolving the saponified extract in water at room temperature and adjusting the pH to 6 using 25% aqueous phosphoric acid, followed by heating to 70.degree.C under stirring. After cooling and removal of the lower layer containing chlorophylls, the oily material was washed with aqueous acid solution followed by the addition of hexane to obtain xanthophyll precipitate. The precipitate was further purified washing with hexane and drying under vacuum to obtain xanthophyll crystals containing 66 % total xanthophylls in which 92.2% is trans lutein & 6.1.% is zeaxanthin as measured by HPLC .
The method is useful for marigold oleoresins containing high levels of chlorophylls. The main disadvantage of this process is in the steps involving the use of acid and heating, as these are likely to degrade xanthophylls and enhance the possibilities of isomerization.
More recently , Madhavi and Kagan ( D. L. Madhavi, D I Kagan; Process for the Isolation of Mixed Carotenoids from Plants, US Patent No 6380442 , 2002) have reported a process for the isolation of mixed carotenoids from plants and illustrated the same with examples of marigold oleoresin. The process involved hydrolysis of the carotenoid esters using isopropanol, water and potassium hydroxide for a period of 60-90 minutes at temperatures of about 60.degree.C. The hydrolyzed carotenoids were precipitated from the saponification mixture using large quantity of water. The precipitate was recovered by centrifugation followed by repeated water washing and drying to obtain crystalline material The method is not attractive for commercial applications since the water required is more than 30 times per kg of the input material.
In virtually all the processes described in the above mentioned prior art literature, invariably the first step is the saponification of oleoresin (specifically marigold) using an alcoholic and /

or aqueous alkali preferably KOH. The saponification steps in these processes (with the exception of US Patent No 6221417J employ external water . The processes involve extracting , re extracting with solvents such as THF, halogenated solvents .Therefore these processes are inappropriate for industrial scale-up operations due to high cost & toxicological considerations
In present days ,there is high demand for xanthophyll crystals containing high amounts of trans-lutein and /or zeaxanthin for its use as antioxidants, prevention of cataract and macular degeneration, as lung cancer-preventive agent, as agents for the absorption of harmful ultraviolet light from sun rays and quencher of photo-induced free radical and reactive oxygen species, etc.. It is therefore felt that there is a need for providing an economical and simple process for the production of xanthophyll crystals containing high amounts of trans - lutein for using in food and nutraceutical supplements, employing toxicologically safe solvents for extraction such as GRAS [Generally Recognized As Safe] status .
Objectives Of The Invention :
Accordingly , the main objective of the present invention is to provide novel xanthophyll crystals containing high amounts of trans-lutein and/or zeaxanthin.
Another objective of the present invention is to provide novelxanthophyll crystals containing at least 85% total xanthophylls out of which the trans-lutein content is at least 90 %, the remaining being zeaxanthin and other carotenoids .
Yet another objective of the present invention is to provide novel xanthophyll crystals containing at least 85% total xanthophylls out of which the trans-lutein content is at least 90 % , the remaining being zeaxanthin and other carotenoids from oleoresin and extracts of plant materials such as marigold (Tagetes erecta).
We have in our application no 622 MAS 02 described and claimed an improved process for the preparation of xanthophyll crystals containing at least 85% total xanthophylls out of which the trans-lutein content is at least 90 %, the remaining being zeaxanthin and other carotenoids

wherein the starting material is xanthophyll esters concentrate obtained through solvent or supercritical fluid extraction.
That process employs ethyl acetate which is toxicologically safe ( GRAS or Generally Recognized As Safe solvent) The process described therein is simple, convenient, economical and commercially feasible. In addition in the process the recovery of alcohol is effected cost-effectively by recovering the unused alcohol and reusing it for the process thereby making the process more economical
The process has been developed based on our main finding that:
a. the saponification step can be managed by using alkali ^preferably potassium hydroxide,
in excess alcohol without adding water to the reaction mixture
b. recovering the unused alcohol and reusing it, if required, in the process
c. employing a solvent having the under mentioned characteristics for extracting
xanthophylls at the stage after removing the unreacted alcohol and diluting with water
and before crystallization step, and if required , dissolving the resulting xanthophyll
concentrate using a polar / non- polar solvent mixture to obtain crude crystals,
further recrystallizing with alcohol.
While selecting the suitable solvent for use in the above mentioned stage of the process of the present invention , a variety of criteria are to be taken into consideration very carefully . Accordingly the major criteria to be considered in solvent selection are (i) it should be water immiscible (ii) the solvent should he non-halogenated (iii) the solvent should have GRAS status (iv) the solubility of xanthophylls in the solvent should be high (v) boiling point of the solvent which should be in the range of 50 to SO.degree.C and (v) stability of xanthophylls in the solvent used should be high.
It should be noted though there are many solvents in which lutein is soluble , due to other considerations such solvents cannot be used in this stage of the process of the present

invention . For example the solvents in which lutein is highly soluble such as -chloroform , benzene , etc. cannot be used because such solvents are prohibited for human consumption . Hence the solvent used should fall under GRAS status . It is also to be noted that the stage where the solvent is to be used contains large amounts of water . Therefore the solvent to be used in this stage of the process should be water immiscible so as to facilitate phase separation . Accordingly many of the solvents which fall under GRAS status and in which lutein is highly soluble cannot be used .
For illustrating the above points we have in the Table 1 given below provided the characteristics of the solvents which were considered for use in the stage of the present invention as explained earlier .

From the solvents listed in the Table I , it can be observed that the solvent falling under the category of organic acid esters are only eligible to use in the particular stage of the process of the invention explained above . Out of the aliphatic acid esters which can be employed in the stage of the process of the present invention it is observed that methyl acetate is soluble in water and propyl acetate has a boiling point of 102.degree. C. These characteristics fall outside the requirements explained above making methyl and propyl acetates ineligible for consideration in the process of the present invention . Therefore the only solvent which satisfies the above special characteristics and which can be used according to the present invention is ethyl acetate
During our sustained R&D efforts to develop the improved process for the preparation of xanthophyll crystals we observed that the use of the above said special category of solvent in the particular stage of the process explained above helps in effectively and efficiently separating the xanthophyll concentrate from most impurities present in the mixture compared to other processes disclosed in the art. The use of ethyl acetate in the process of the present invention also helps in removing the impurities at a low temperature ( say at room temperature).
It should be specifically noted , in this context, that the selection of the particular solvent at a particular stage of the process of the invention is due to the inventive ingenuity of the applicant and is not a mere application of a known and obvious solvent
To the best of our knowledge the novel xanthophyll crystals containing at least 90% total xanthophylls with at least 90% trans-lutein content is not known .

Accordingly , the present invention provides novel xanthophyll crystals containing at least 85% total xanthophylls out of which the trans-lutein content is at least 90 %, the remaining being zeaxanthin and other carotenoids
The process which has been described in the co pending application no 622 MAS 02 to prepared the above said novel Xanthophyll crystals comprises
(a)admixing a xanthophyll ester concentrate containing esters of lutein and zeaxanthin with an alcoholic alkali solution and heating the resultant solution at a temperature in the range of 65.degree.C to about SCdegree.C preferably at 70.degree.C
(b) maintaining the solution at the same temperature for a period sufficient to saponify the xanthophyll esters present in the concentrate;
( c ) removing alcohol from the reaction mixture under reduced pressure to get a saponified crude concentrate;
(d) reusing the recovered alcohol if required in the step (a) above
(e) admixing the resultant saponified crude concentrate obtained in step (c) with water to form a diluted oily mixture ;
(f) extracting the diluted oily mixture obtained in step (e) with ethyl acetate to get xanthophyll
extract
(g)washing the resultant extract with water and concentrating the extract under reduced pressure
(h) admixing the concentrated extract obtained in step(g) with polar/non-polar solvent mixture and alcohol to remove undesirable materials and to get a precipitate

(i) admixing the precipitate obtained in step (h) with alcohol to get xanthophyll crystals and
(j) filtering and drying.
The concentrate used in the process of the present invention can be obtained from any plant source. Preferably the xanthophyll ester concentrate used in step (a) may be those obtained from naturally occurring plant materials more particularly from marigold flowers , fruits etc. The alcohol used may be selected from a lower aliphatic alcohol such as methanol ethanol, isopropanol, preferably isopropanol. The ratio of xanthophyll ester concentrate and the aliphatic alcohol used may range from 1:2 to 1:5 preferably 1: 3 by weight by volume.
The alkali used for preparing the alcoholic alkali solution may be selected from sodium hydroxide or potassium hydroxide, preferably potassium hydroxide. The ratio of xanthophyll ester concentrate to the alkali may be in the range of 1:0.25 to 1: 0.4 weight by weight preferably 1:0.25.
The amount of alcoholic alkali solution used in the step (a) may consist of 8-12 parts of the alcohol such as methanol, ethanol, isopropanol, preferably ,isopropanol and 1 part of alkali by volume by weight.
The saponification reaction mixture is maintained for a period of 3 to 5 hours, preferably for 3 hours at the temperature in the range of 65.degree.C to about SO.degree.C, preferably at 70.degree.C. The unused alcohol is recovered by distillation under vacuum, and reused, if required , in step (a).
The amount of water used in the step (e) of the process for dilution of the saponification mixture may be in the range of 1:4 to 1:6, preferably 1:4, weight by volume .
The ratio of ethyl acetate employed for mixing with the diluted saponification reaction mixture to the solvent may range from 1:1 to 1:3 preferably 1:1 by volume / volume

The non-polar solvent used in step (h) may be selected from the hydrocarbon solvents like pentane, hexane and heptane, and the like ,preferably hexane. The polar solvent used may be selected from 2-propanone, 2-pentanone, 2-butanone, and the like preferably 2-propanone.
The alcohol which may be used in step(i) for further purification may be selected from methanol, ethanol isopropanol etc , preferably methanol
Detailed Description Of The Invention:
In the present invention, the plant material containing xanthophyll esters is saponified using alkali and an aliphatic alcohol and the resulting xanthophyll solution is subjected to purification by extracting with ethyl acetate and washing with water followed by purification with a mixture of polar/ non- polar solvent mixtures and alcohol to remove the solvent soluble materials, thereby resulting in xanthophyll crystals with high levels of lutein and/or zeaxanthin.
Marigold flowers or Chinese wolfberry are considered to be the best possible commercial source for obtaining xanthophyll esters. It is well known that the xanthopyll extract or oleoresin is typically obtained by solvent extraction of dehydrated plant material in milled or pelletized form prepared by drying of fresh or fermented flowers. Generally 15-30 % xanthophyll esters are found in the marigold oleoresins depending on the cultivars , meal quality and extraction processes.
The current commercial and industrial practices in the production, handling and supply of marigold flowers / meal and extraction may be summarized as follows:
Fresh marigold plant material containing high moisture (80 - 90%) and xanthophyll content of around 0.1-0.2% by weight is used .
Silaging of the harvested marigold plant materials is carried out in the fields for extended periods of time, followed by drying using mechanical dryers to reduce the moisture to about 10%.

Reducing the size of the dehydrated materials to obtain marigold meal with
xanthophyll content of around 1-1.6 % by weight.
Preparing the pellets of the meal thus obtained and supplying to the extractors.
According to commercial considerations and practices, hexane is the choice for extraction of xanthophyll esters in view of specificity and ease of removal of the solvent residues conforming to standards and specifications. Standardization of the particle size of the meal, extraction condiHons like volumes of solvent and contact time required, removal of solvent, product yield and analysis are routinely performed and validated.
This is achieved by monitoring the analytes at various stages employing both spectrophotometric and HPLC analysis. The spectrophotometric analysis (AOAC -16 ^ edition, Method 970.64 ) provides data on the total xanthophyll content by weight while the HPLC analysis (W.L.Hadden, R.H.Watkins, L.W.Levy, E.Regalado, D.M.Rivadeneira, R.B.Van Breemaen, Carotenoid Composition of Marigold {Tagetes erecta ) Flower Extract Used as Nutritional Supplement, J.Agric.Food Chem,47,4189-4194,1999) of the saponified mixture provides percentage composition of the individual xanthophylls in the total xanthophylls.
The xanthophyll extract is mixed with alcohol such as isopropanol in which alkali is dissolved forming alkali alcoholate. The ratio of the three constituents of the saponification reaction mixture are about 1 part oleoresin and 3 parts alcohol, preferably isopropanol and 0.25 to 0.4 parts of alkali. Although 2 volumes of alcohol solvent - containing alkali is sufficient for dissolution of the oleoresin , 3 volumes of the solvent mixture is desirable for homogeneity , free flow and better saponification reaction. The saponification is performed by heating the reaction mixture preferably to a temperature of 70.degree.C for a period of 3 hours. The amount of alkali required is related to xanthophyll esters content present in the oleoresin. The exact quantity of alkali required has to be worked out experimentally. Generally, for each 100 g oleoresin, about 25 g alkali is adequate for completion of hydrolysis of the esters.
The saponification of the oleoresin results in liberation of xanthophylls in free form along with alkali salts of fatty acids such as palmitic, myristic, stearic acid, etc. The progress of the

saponification reaction is monitored by HPLC analysis for the presence of xanthophyll esters till their absence. This analysis is performed by taking an aliquot of the saponification reaction mixture and extraction with hexane: acetone: toluene; absolute alcohol (10:7:7:6 v/v) followed by addition of hexane and 10% sodium sulphate solution and analyzing the upper layer by HPLC.
After the complete hydrolysis of the xanthophyll esters, the reaction mixture is subjected to vacuum distillation to recover alcohol. The saponified product obtained is treated with water resulting in a brownish yellow oily layer containing xanthophylls in free form, fatty acid soaps and other impurities. The recovered alcohol may be reused for further saponification of other batch . The solution obtained is subjected to extraction with ethyl acetate The amount of ethyl acetate used may be in the ratio of 1:1 volume . More than 90% xanthophylls are extracted into the ethyl acetate layer . This resultant product is washed with deionised water (2 times) preferably using equal volumes . By this way, most of the fatty acid soapy material is removed into water layer, which is then discarded. The ethyl acetate extract is distilled off under reduced pressure to recover ethyl acetate and the saponified xanthophyll concentrate (xanthophyll content 15-20% by weight by spectrophotometry )
The xanthophyll concentrate obtained is admixed with a solvent or a mixture of solvents , preferably acetone and hexane mixture at room temperature with stirring. Preferably one part of xanthophyll concentrate obtained is admixed with four parts of the solvent or a mixture of solvents. The ratio of polar/non-polar solvent mixture used ranges from 2:8 to 3:7 volume /volume, preferably 2:8 .
The xanthophylls separate out as crude crystals and the impurities are dissolved in the solvent
/ solvent mixture which are removed by filtration. These crude crystals show xanthophyll
content of around 50 to 60 % by weight analyzed by spectrophotometry . Finally ,these crude
crystals are further purified by washing with aliphatic alcohol such as methanol, ethanol or
isopropanol, preferably methanol and vacuum-dried at room temperature to obtain
xanthophyll crystals with at least 85 % xanthophylls by weight (analyzed by

spectrophotometry) and trans-lutein content of the xanthophylls over 90 % (area % by HPLC method).
The purified xanthophyll crystals obtained contained approximately 85-92% total xanthophylls by weight and the composition of the carotenoids of the xanthophylls, as determined by HPLC analysis(peak area %) consist of 90-95% trans-lutein, 4-8% trans-zeaxanthin, 0.5-1.0% cis-lutein isomers and others.
It is possible that changes could be effected in the embodiment described above without deviating from the broad spectrum of the concept of the invention . Therefore, it is to be noted that this invention is not limited to the particular embodiments disclosed herein but it is intended to cover all modifications which fall within the spirit and scope of the present invention .
The details of the process of preparing the novel xanthophylls crystals are given in the examples provided below which are provided only by way of illustration and therefore should not be construed to limit the scope of the process
Example-1
Commercial grade marigold oleoresin (57.98 g) containing 11.54 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 15 g potassium hydroxide in 175 ml isopropanol.) The .. saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 230 ml of water at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(3 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (25.01g)

This resultant crude extract (25.01 g) was subjected to purification by stirring with 100 ml of hexane/acetone mixture(80:20) at room temperature for 30 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs.
The yield of the xanthophyll crystals was 3.41%(1.98g ). Xanthophyll content was 86.23 % by weight ( as determined by UV/Vis spectrophotometry ) out of which the contents of trans-lutein , zeaxanthin, and other carotenoids were 91.43% , 6.40% and 2.17 % respectively as determined by .HPLC analysis.
Example -2
Commercial grade marigold oleoresin (56.3 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 14 g potassium hydroxide in 170 ml isopropanol.) The saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 225 ml of water at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(3 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (22.21 g)
This crude extract (22.21 g) was subjected to purification by stirring with 90 ml of hexane/acetone mixture(80:20) at room temperature for 30 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol .The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs.
The yield of the xanthophyll crystals was 3.43%(1.93 g). Xanthophyll content was 88.69 % by weight ( as determined by UV/Vis spectrophotometry ) out of which the contents of trans-

lutein , zeaxanthin and other carotenoids were 90.78% , 6.27% and 2.95 % respectively as determined by HPLC analysis.
Example-3
Commercial grade marigold oleoresin (51.60 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 12.90 g potassium hydroxide in 155 ml isopropanol). The saponification mixture was heated maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 206 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory furmel and extracted with equal volume of ethyl acetate(3 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (21.80g)
This crude extract (21.80 g) was subjected to purification by stirring with 87 ml of hexane/acetone mixture(80:20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs.
The yield of the xanthophyll crystals was 2.11 g.(4.09%) Xanthophyll content was 90.07 % by weight ( as determined by UV/Vis spectrophotometry ) out of which the contents of trans-lutein,zeaxanthin and other carotenoids were 90.10% , 7.08% and 2.82% respectively as determined by HPLC analysis.
Example-4
Commercial grade marigold oleoresin (50.0 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 12.50 g potassium hydroxide in 150 ml isopropanol). The „ saponification mixture

was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 200 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(4 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (21.70g)
This crude extract (21.70 g) was subjected to purification by stirring with 87 ml of hexane/acetone mixture(80:20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol .The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs.
The yield of the xanthophyll crystals was 2.11 g. Xanthophyll content was 90.21 % by weight (
as determined by UV/Vis spectrophotometry ). out of which the contents of trans-lutein
,zeaxanthin and other carotenoids were 90.99% , 7.07% and 1.94% respectively as
determined by HPLC analysis.
Example-5
Commercial grade marigold oleoresin (47.30 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate prepared by dissolving 11.90 g potassium hydroxide in 142 ml isopropanol. The _ saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 237 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(5 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble

impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (19.90g)
This crude extract (19.90 g) was subjected to purification by stirring with 80 ml of heptane/ethyl-methyl ketone mixture(80;20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol .The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs.
The yield of the xanthophyll crystals was 1.52 g(3.04%). Xanthophyll content was 91.34 % by weight ( as determined by UV/Vis spectrophotometry ). out of which the contents of trans-lutein .zeaxanthin and other carotenoids were 90.20% , 7.25% 2.55 % respectively as determined by HPLC analysis.
Example-6
Commercial grade marigold oleoresin (50.40 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 12.85 g potassium hydroxide in 154 ml isopropanol). The „ saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 252 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(5 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali/ soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (21.40g)
This crude extract (21.40 g) was subjected to purification by stirring with 80 ml of hexane/acetone mixture(80:20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with ethanol .The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs.

The yield of the xanthophyll crystals was 2.299 g (4.54%). Xanthophyll content was 89.05 % by weight ( as determined by UV/Vis spectrophotometry ). out of which the contents of trans-lutein ,zeaxanthin and other carotenoids were 91.97% , 6.83% and 1.20% respectively as determined by HPLC analysis.
Example-7
Commercial grade marigold oleoresin (51.60 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 12.90 g potassium hydroxide in 155 ml isopropanol). The _ saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 258 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(5 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (25.60g)
This crude extract (25.60 g) was subjected to purification by stirring with 102 ml of hexane/acetone mixture(80:20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with isopropanol. The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs.
The yield of the xanthophyll crystals was 1.996 g(3.87%). Xanthophyll content was 85.73 % by weight ( as determined by UV/Vis spectrophotometry ). out of which the contents of trans-lutein, zeaxanthin and other carotenoids were 90.53% , 7.72% and 1.75% respectively as determined by HPLC analysis.

Example 8
Commercial grade marigold oleoresin (47,30 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 11.90 g potassium hydroxide in 142 ml isopropanol). The ^ saponification mixture was heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 237 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(5 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (23.0g)
This crude extract (23.0 g) was subjected to purification by stirring with 92 ml of hexane/ethyl-methyl ketone mixture(80;20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol .The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs.
The yield of the xanthophyll crystals was 1.73 g (3.66%). Xanthophyll content was 81.41 % by weight ( as determined by UV/vis spectrophotometry ). out of which the contents of trans-lutein ,zeaxanthin and other carotenoids were 90.04% , 6.95% and 3.01% respectively as determined by HPLC analysis.
Example-9
Commercial grade marigold oleoresin (50 g) containing 11.82 % xanthophyll content ( by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 12.5 g potassium hydroxide in 150 ml isopropanol). The, saponification mixture was

heated and maintained at 70.degree.C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 240 ml of water(with 5% sodium sulphate) at room temperature. The mixture was taken into a separatory funnel and extracted with equal volume of ethyl acetate(5 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extiact (22.20g)
This crude extract (22.20 g) was subjected to purification by stirring with 90 ml of hexane/acetone mixture(80:20) at room temperature for 60 minutes, followed by filtration. The precipitate of xanthophyll crystals obtained was washed with methanol. The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs.
The yield of the xanthophyll crystals was 2.78 g (5.56%). Xanthophyll content was 90.58 % by weight ( as determined by UV/Vis spectrophotometry ).). out of which the contents of trans-lutein,zeaxanthin and other carotenoids were 91.26% , 5.68% and 3.06% respectively as determined by HPLC analysis.
Advantages of the invention
• The commercial grade xanthophyll crystals conatins high content lutein and/or zeaxanthin which make it ideal and suitable for use as human nutritional supplements, as an anti-oxidant, for applications in prevention of cancer and age-related macular degeneration of eye., as anti-oxidant, and as a food/feed colourant.

We Claim
1 .A novel xanthophyll crystals having at least 85% total xanthophylls out of which the trans-lutein content is at least 90 % , the remaining being zeaxanthin and other carotenoids
2. A novel xanthophyll crystals as claimed in claim 1 wherein it contains at least 85% total xanthophylls out of which the trans-lutein content is at lest 92 % , zeaxanthin content is around 4 to 8% and the remaining being other carotenoids .
3 .A novel xanthophyll crystals containing high amounts of trans-lutein and / or zeaxanthin substantially as herein described .

Documents:

282-che-2004-assignement.pdf

282-che-2004-claims duplicate.pdf

282-che-2004-claims original.pdf

282-che-2004-correspondnece-others.pdf

282-che-2004-correspondnece-po.pdf

282-che-2004-description(complete) duplicate.pdf

282-che-2004-description(complete) original.pdf

282-che-2004-form 1.pdf

282-che-2004-form 26.pdf

282-che-2004-form 3.pdf

282-che-2004-form 6.pdf

282-che-2004-pct.pdf

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Patent Number

209783

Indian Patent Application Number

282/CHE/2004

PG Journal Number

50/2007

Publication Date

14-Dec-2007

Grant Date

06-Sep-2007

Date of Filing

29-Mar-2004

Name of Patentee

M/S. OMNIACTIVE HEALTH TECHNOLOGIES PVT LTD

Applicant Address

RAJAN HOUSE, APPASAHEB MARATHE MARG, PRABHADEVI, MUMBAI 400 025,

Inventors:

#	Inventor's Name	Inventor's Address
1	SHERENA PADINTARAVATTOM ABDULKADIR	KANCOR KANCOR FLAVOURS AND EXTRACTS LTD.XVII/138, KANCOR ROAD, ANGAMALLY (SOUTH), KERALA 683 573.
2	SAJOY SEBASTIAN	KANCOR KANCOR FLAVOURS AND EXTRACTS LTD.XVII/138, KANCOR ROAD, ANGAMALLY (SOUTH), KERALA 683 573.
3	THATTARUPARAMBIL KRISHNADAS SUNIL KUMAR	KANCOR KANCOR FLAVOURS AND EXTRACTS LTD.XVII/138, KANCOR ROAD, ANGAMALLY (SOUTH), KERALA 683 573,

PCT International Classification Number

C07 C 035/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA