Title of Invention

A PROCESS FOR THE PREPARATION OF XANTHOPHYLL COMPOSITION CONTAINING ESSENTIAL MACULAR PIGMENTS

Abstract The present invention provides a xanthophyll composition containing at least 80% by weight of total xanthophylls ,( out) of which the trans-lutein content is at least 80% by weight and the remaining being zeaxanthin isomers, namely, : R,R-zeaxanthin and R,S -zeaxanthin and traces of other carotenoids derived from the plant extract. oleoresin containing xanthophylls esters which is safe for human consumption and useful for nutrition and health care. The invention also provides a process for the preparation of the composition .
Full Text FORM 2 THE PATENTS ACT 70
(Act 39 of 70)
PROVISIONAL SPECIFICATION (See SectionlO)


XANTHOPHYLL COMPOSITION CONTAINING ESSENTIAL MACULAR PIGMENTS AND A PROCESS FOR ITS PREPARATION.
OMNIACTTVES HEALTH TECHNOLOGIES LTD., having its registered office located at Rajan House, Appasaheb Marathe Marg, Prabhadevi, Mumbai 400025, Maharashtra, India
The following description describes the nature of the invention


Field of the Invention :
The present invention relates to xanthophyll composition containing essential macular pigmnets and a process for its preperation. The present invention particularly relates to a xanthophyll composition containing trans-lutein and zeaxanthin isomers and a process for its preparation . The present invention more particularly relates to xanthophylls composition containing high concentration of trans -lutein and low concentration of R,R'-zeaxanthin and R,S'-zeaxanthin and a process for its preparation .
The xanthophyll composition of the present invention is useful as dietary supplements for nutrition and health care and colorants for food and feeds.
Back ground of the Invention :The macula is in the center of the retina , directly behind the lens in the eye . It is a tiny area with an yellow color consisting of xanthophylls like lutein , R,R-zeaxanthin and R,S-zeaxanthin and hence called macular xanthophylls. These act as anti-oxidants protecting the retina from oxidative degradation and help in sharp vision needed to read , write , drive and see objects clearly. A life time slow and steady damage of the macula can lead to age related macular degeneration(AMD)and cataract. The macular xanthophylls in the diet or supplementation can help in maintaining healthy eyes. Lutein and R,R-zeaxanthin can be derived form fruits and vegetables while R,S-zeaxanthin from sea foods or dietary supplements or from bio conversion of lutein within the body. Of the various classes of the pigments , the carotenoids are among the most widely distributed in nature with red , yellow and orange color having varied functions like light harvesting and protection against destructive photo oxidation in terrestrial plants.
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 entry into the human blood stream to exert their effect. Each carotenoid shows an individual pattern of absorption, plasma transport and metabolism.
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Carotenoids absorb light in the 400-500 pm 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 reported to have several health benefits, which include prevention and or protection against serious health disorders.
Carotenoids are non-polar compounds classified into two sub-classes, namely, polar compounds called xanthophylls or oxy-carotenoids and non-polar hydrocarbon carotenes like beta-carotene, lycopene, etc. Both the sub-classes have at least nine conjugated double bonds responsible for the characteristic 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,beta.-cryptoxanthin, astaxanthin, etc. As natural colorants and also for their role in human health, xanthophylls like lutein R,R-zeaxanthin AND R,S-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 colors respectively. Lutein and zeaxanthin can be present in plant material in the 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 corn, contain lutein in the form of its esters. It is also present in the blood stream and various tissues in human body and particularly in the macula, lens andretina of the eye.
Lutein is chemically designated as beta,epsilon-carotene 3,3'-diol. Zeaxanthin is formed by the addition of two hydroxy groups to beta-carotene. Since the hydroxy positions are
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in 3 and 3'-, the chemical name for zeaxanthin is beta,beta-carotene-3,3'-diol. The common name of zeaxanthin is derived from zea mays because this carotenoid was first identified in corn (zea mays).
It can be seen that lutein is not symmetrical as the position of double bond in the left ring is not identical with the double bond position in the right ring. Zeaxanthin is completely symmetrical with regards to left and right rings due to an extra conjugated double bond compared to lutein.
Xanthophylls can show both optical (R and S-stereo isomers) and geometrical isomers (trans,E- and cis,Z-). The conformation of R- and S-stereo isomers is based on CD spectral and chiral column HPLC studies while the conformation of cis- and trans-isomers is based on electronic, infrared, NMR, HPLC-MS and HPLC-NMR on-line spectroscopy studies. It is well known that when an organic molecule has a carbon atom with four different types of atoms or groups attached to it, that carbon atom is designated as chiral carbon atom. The chiral carbon atom is responsible for two different spatial arrangements leading to formation of optical isomers while the number of double bonds of the polyene chain and the presence of a methyl group and the absence of steric hindrance decide the number of trans- and cis-isomers. In the case of trans-zeaxanthin, the carbon atoms at 3 and 3' positions in the two end rings are both chiral atoms. Thus, trans-zeaxanthin has two chiral centers at the carbon atoms C3 and C3\ based on the positions of the secondary hydroxy groups attached to them. Therefore, there are four possible stereo isomers of trans-zeaxanthin namely, (3R-3'R)-isomer, (3S-3'S)-isomer and (3R-3'S)- or (3S-3'R)-isomer. In these isomers (3R-3'S)- & (3S-3'R) are identical. Thus, there are three chiral isomers of trans-zeaxanthin. The isomer causing rotation of polarized light in a right handed manner is called R-stereo isomer, the isomer causing left handed rotation S-stereo isomer and the third isomer possessing a two fold opposite effects (R,S; optically inactive) which is called meso-form of zeaxanthin.
The conjugated double bonds of lutein and zeaxanthin contribute to the distinctive colors of each pigment, and also influence the ability of these to quench singlet oxygen. Due to
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the extra conjugated double bond, zeaxanthin is believed to be a stronger anti-oxidant compared to lutein.
The macular pigment of the eye is composed primarily of three xanthophylls pigments, namely (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin and (3R,3'S)-zeaxanthin the order 36, 18 and 18% of the total carotenoid content of the retina along with the remaining 20% consisting of minor carotenoids like oxo-lutein, epi-lutein and epsilon,epsilon,-carotene 3,3'-dione (J. T. Landrum and R. A. Bone, Lutein, zeaxanthin and the macular pigment, Arch. Biochem . Biophys., 385, 28(2001)). Althoug these xanthophylls pigments are found throughout the tissues of the eye, the highest concentration is seen in the macula lutea region of the retina, including a central depression in the retina called the fovea. The concentration of xanthophylls pigments increases progressively towards the center of the macula and in the fovea, the concentration of these xanthophylls pigments are approximately thousand fold higher than in other human tissues. (Landrum et al., Analysis of zeaxanthin distribution within individual human retinas, Methods in Enzymology, L. Packer (editor) 213A, 457-467, Academic Press 1992). The fovea is a relatively small area within the macula, in which the cone photoreceptors reach their maximal concentration. About 50% of the total amounts of the xanthophylls are concentrated in the macula where zeaxanthin dominates over lutein by ratio of 2:1 (Handel man et al., Measurements of carotenoids in human and monkey retinas, in Methods in Enzymology, L. Packer (editor) 213A, 220-230, Academic press, NY, 1992; Billsten et al., photophysical properties of xanthophylls in carotene proteins from human retina,Photochemistry and photobiology, 78, 138-145, 2003). At the center of the retinal fovea, zeaxanthin is 50:50 mixture of (trans-3R,3'R)-zeaxanthin and (trans-3R,3'S)-zeaxanthin along with small quantity of (3S,3'S)-zeaxanthin (J. T. Landrum and R. A. Bone, Lutein, zeaxanthin and the macular pigment, Arch.Biochem.,Biophy., 385, 28-40, 2001).
The fovea is particularly important for proper visual function (eg, acuity) and disease and damage to this area is known to result in legal blindness. For example, age-related macular degeneration (AMD) is characterized by pathological changes in the retina, retinal pigment epithelium (RPE) and/or the choroids and preferentially affects the
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macular region of the retina. This is the leading cause of irreversible vision loss in the United States among that more than 65 y old and there is no established treatment available for most patients. The loss of central vision results in the possible inability to recognize faces, to read or write or drive a car and therefore has a significant effect on an individual's ability to live independently. There is ample epidemiologic evidence which supports a role for dietary intake of lutein and zeaxanthin in different isomeric forms in protection against age-related cataract and macular degeneration. The detection of oxidation products of lutein and zeaxanthin in the human retina supports the hypothesis that dietary lutein and zeaxanthin may act as antioxidants in the macular region. (Khachik et al., Identification of lutein and zeaxanthin oxidation products in human and monkey retinas,Invest.Opthalmol.and Vis.Sci.,38,1802-18M,1997)
Of the 40 to 50 carotenoids typically consumed in the human diet, lutein and zeaxanthin, are deposited at an up to 5 fold higher content in the macular region of the retina as compared to the peripheral retina. Zeaxanthin is preferentially accumulated in the foveal region, whereas lutein is abundant in the perifoveal region.
Regarding the location of xanthophylls at a cellular level, they are reported to be bound to specific proteins, xanthophylls binding protein (XBP). The XBP is suggested to be involved in the uptake of lutein and zeaxanthin from the blood stream and stabilization of the same in the retina. The study of xanthophylls and XBP by femto-second transient absorption spectroscopy showed better stability for (3R,3'S)-zeaxanthin enriched XBP compared to (3R,3'R)-zeaxanthin while the photo physical properties of the xanthophylls(3R,3'R)-zeaxanthin and (3R, 3'S,meso)-zeaxanthin are generally identical. It is likely that the meso-zeaxanthin is better accommodated with XBP wherein the protein protects the xanthophylls from degradation by free radicals. Thus, the complex may be a better antioxidant than the free xanthophylls, facilitating improved protection of ocular tissue from oxidative damages. (Billsten etal., photophysical properties of xanthophylls in carotene proteins from human retina, Photochemistry and Photobiology, 78,138-145,2003)
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Several functions have been attributed to macular pigments including the reduction of the damaging effects of photo-oxidation from blue light absorbed by the eye, reduction of the effects of light scatter and chromatic aberration on visual performance, and protection against the adverse effects of photochemical reactions because of the antioxidant properties of the carotenoids.
The ability to increase the amount of macular pigment by dietary supplementation with lutein has been demonstrated (Landrum et al., Dietary Lutein supplementation increases macular pigment, FASEB. J, 10, A242, 1996). The reduced vision function due to cataract and the adult blindness due to AMD can be substantially controlled by consuming fruits and vegetables and dietary supplements containing lutein and zeaxanthin and meso-zeaxanthin available from sea foods denying the vegetarian population. Although meso-zeaxanthin present in eye is considered a metabolic product originating from lutein, the need for dietary supplementation of meso-zeaxanthin is now recognized to improve the macular pigment density.
(Landrum and Bone, Functional Foods and Nutraceuticals, 1 Sep. 2001). Similarly, the study has shown that R,R-zeaxanthin gains entry to blood and finally to macula. (Breithaupt et al., Comparison of plasma responses in human subjects after the ingestion of (3R,3'R)-zeaxanthin dipalmitate from Wolfberry(Lyium barbarum) and non-esterified (3R,3'R)-zeaxanthin using chiral HPLC ,Brit J. Nutr. 91, 707-713, 2004). Lutein and zeaxanthin dietary supplements in human trials have been shown to raise the macular pigment density and serum concentrations of these carotenoids (Bone et al.,Lutein and zeaxanthin dietary supplements raise macular pigment density and serum concentrations of these carotenoids humans,J.Nutr.,133,992-998,2003).
Dietary Sources of Lutein and Zeaxanthin
Lutein is a common carotenoid found in most fruits and vegetables, while zeaxanthin in the (R,R)-isomer form is present only in minute quantities in most fruits and vegetables. Dietary sources of zeaxanthin are limited to greens, certain yellow/orange fruits and vegetables such as corn, nectarines, oranges, papaya, persimmons and squash. Capsicum
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annum is another most common spice widely used which is a good source of zeaxanthin. Wolfberry (Lycium barbarum, fructus lycii or Gou Qi Zi) plant has small red berries which are commonly used in Chinese home cooking and has been shown to have a high content of zeaxanthin (mainly as zeaxanthin dipalmitate) but negligible amounts of lutein. The dried fruit of wolfberry is prescribed by Chinese herbalists as a therapeutic agent for a number of eye diseases. In France, lutein dipalmitate (Helenien) isolated from the blossom leafs of Helenium autumnale is reported to be used for the treatment of the visual disorders.
As already mentioned earlier, the dietary source of meso-zeaxanthin is mainly from seafoods like shrimps, fish, turtle, etc, thereby the vegetarian population is deprived of meso-zeaxanthin. However, there are patents available for pharmaceuticals composition containing meso-zeaxanthin for treatment of retinal disorders like increasing the deposition of macular pigments in the human eye and therapeutic treatment or prophylaxis of AMD (Howard et al.,Mesozeaxanthin formulations for treatment of retinal disorders , U.S. Pat. No. 6,329,432,2001).
Lutein and zeaxanthin occur naturally in trans-isomeric form in fruits, vegetables and flowers (marigold). Because of processing conditions due to heat and light, a small percentage of trans- is converted into cis-isomeric form. Therefore, the preferred bio-available form is trans-isomeric as evidenced from the data of geometric isomers compositional analysis of human plasma. (Khachik et al., Isolation and structure elucidation of geometric isomers of lutein, zeaxanthin in extracts of human plasma, J. Chrom. 582, 153-156, 1992). In view of this, it is desirable to use the trans-isomeric form of meso-zeaxanthin in dietary supplements.
To date little is known about the mechanism of formation, uptake and deposition of meso-zeaxanthin in the retina of the eye. Khachik et al. have reported the presence of 2-3% of (3R,3'S, meso)-zeaxanthin in twenty normal human plasma samples and proposed the metabolic pathways of its formation from dietary lutein and zeaxanthin. It is not clear whether the deposition of meso-zeaxanthin in the retina routes through serum or are
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produced from lutein/zeaxanthin within the retina. (Khachik. et al., in a chapter on Dietary carotenoids and their metabolites as potentially useful chemo protective agents against cancer, in "Antioxidant food supplement in human health, Eds. Packer et al., Academic Press London, page pp 203-29, 1999). However, Breithaupt et al. did not find the presence of meso-zeaxanthin in human plasma obtained 24 hrs after ingestion of (3R,3'R)-zeaxanthin (ester or free form) in a single blind cross over study using two groups each consisting of six volunteers. The chiral LC-ApcI-MS was used for detection in the pooled plasma sample. (Breithaupt et al., Comparison of plasma responses in human subjects after the ingestion of (3R,3'R)-zeaxanthin dipalmitate from Wolfberry(Lyium barbarum) and non-esterified (3R,3'R)-zeaxanthin using chiral HPLC ,Brit. J. Nutr. 91, 707-713,2004)
There is evidence and reasons supporting the hypothesis that the carotenoids lutein,
zeaxanthin and meso-zeaxanthin are readily bio-available and consequently increase
macular pigment levels (Landrum and Bone, Meso-zeaxanthin-A cutting edge carotenoid,
Functional Foods and Nutraceuticals, 10 Sep. 2001,Bone et al ., Macular pigment
response to a supplement containing meso zeaxanthin ,Nutr.Metabol.ll.l-8(2007),Bone
et al., Macular pigment response to a xanthophyll supplement of lutein ,zeaxanthin and
meso zeaxanthin.Proc.Nutr.soc.,105A,(2006),Thurnham et al., Macular zeaxanthin and
lutein -a review of dietary sources and bio availability and some relation ship with
macular pigment optical density and are-related macular
disease,Nutn.Res.Reviews,20,163-179(2007),Thurnham et al.,A supplementation study in human subjects with a combination of meso-zeaxanthin,(3R,3'R)-zeaxanthin and (3R,3,R,6,R)-LUTEIN,Brit.J.Nutr.99,l-8(2008)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 ultra-violet light from sun rays and quencher of photo-induced free radical and reactive oxygen species, etc. A number of commercial products from natural source are now available to facilitate the formulation of industrial and commercial products with lutein or (R,R)-zeaxanthin.
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Evidence of the protective role of trans-lutein ,(R,R)-zeaxanthin and (R,S) -zeaxanthin in maintaining eye health has been found based on correlation between dietary supplements vs serum levels and the macular pigment density (Bone et al., Macular pigment response to a supplement containing meso zeaxanthin ,Nutr.Metabol.ll.l-8(2007),Bone et al., Macular pigment response to a xanthophyll supplement of lutein ,zeaxanthin and meso zeaxanthin.Proc.Nutr.soc.,105A,(2006), Thumham et al.,A supplementation study in human subjects with a combination of mesozeaxanthin, (3R,3'R) zeaxanthin and (3R,3'R,6'R) lutein,Brit.J.Nutr.99,l-8(2008).Therefore, it is desirable and useful for industry and nutritional product formulators to have a xanthophylls concentrate consisting of all the macular xanthophylls from a commercial scale process, made from natural source material same as that which is already accepted by the market for lutein, (R)R)-zeaxanthin and (R,S) -zeaxanthin. The product so prepared should be made form safe solvents for producing dietary supplements suitable for human consumption, with min solvent residues and specifications of lutein and zeaxanthin isomers keeping in mind visual function and market requirements.
Prior art :
The macular of the eye is composed chiefly of lutein , (R,R)-zeaxanthin and (R,S) -zeaxanthin in the app ratio of 2: l:lin the retina. These pigments represent 72% of the total carotenoids in the eye and the balance being other carotenoids ( Landrum and R.A.Bone , Lutein zeaxanthin and the macular pigments , Arch.Biochem..Biophys. 38,528-40 ,2001 ). The predominant xanthophylls pigment in the diet is lutein and comes form fruits and vegetables. The average intake of lutein in the USA is between 1-3 mg per day which includes about 10-20% (R,R)-zeaxanthin ( E.Y .Chew and J.P.Xan Gio vanni , Lutein in encyclopedea of dietary supplements ,page 409-420,2005 published by Marcel Dekker ). (R,S)-zeaxanthin is not found in normal diet but present in certain sea foods like shrimp, fish and turtle. ( Moeka et.al .,The first isolation of enantiomeric and meso-zeaxanthin in nature . Bio Chem.PhysioL, 83B,121-124 , 1986).In the recent years , substantial amounts of zeaxanthins namely (R,R) and (R,S) -zeaxanthins are reported to be present in the chicken egg yolks in Mexico originating from chicken feeds containing both RR, and RS zeaxanthin. ( Thurnam et.al , A supplementation study in human
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subjects with a combination of meso-zeaxanthin,(3R,3'R)-zeaxanthin and (3R,3'R,6'R)-lutein,Brit.J.Nutr.99,l-8(2008)).During the last 5 years , dietary supplements containing R,S-zeaxanthin are available is US markets sold under the brand names Lutein Plus and LMZ3 and in Europe as Lutein Plus and Mac shield . In addition , there are pharmaceutical formulations containing R,S-zeaxanthins for increasing macular pigment density in the human eye and for therapeutic treatment or prophylaxis of diseases and disorders of macula. ( Howard et.al.,Meso-zeaxanthin formulations for treatment of retinal disorders, US Patent No. 6,379,432 , 2001 ). The dietary lutein supplements chiefly contain lutein 8-90% and low amounts of zeaxanthin and total absence of (R,S)-zeaxanthin . The xanthophylls composition of blended samples : namely lutein plus ; lutein 50% ,(R,R)-zeaxanthin 37% and (R,S)-zeaxanthin 13 %; lutein 25% , zeaxanthin 6% and (R,S)-zeaxanthin 68% ; luein 54% ,zeaxanthin 6% and (R,S)-zexanthin 40% ; meso zeaxanthin concentrate , lutein 5% ,zeaxanthin 5% and (R,S)-zeaxanthin 85%; yolk lyophilised from chicken eggs lutein 34%, zeaxanthin 12.80% and (R,S)-zeaxanthin 7.20% and other carotenoids.Pure (R,S)-zeaxanthin more than 99% purity , (R,S)-zeaxanthin (synthetic ) 99%; xanhophyll composition lutein 3.53% (R,R)-zeaxanthin 5.77% and (R,S)-zaxanthin 90.57%.( LANG , Composition and methods for inhibiting the progression of macular degeneration and promoting healthy vision, US patent No.7,267,830, 2007).Recently a combination of dietary supplements cobeadlet formulation consisting of lutein 2 mg zeaxanthin 0.5 mg and (R,S)-zeaxanthin 0.5mg is reported for inhibiting the process of macular degeneration and promoting healthy vision.
As early as in 1946, Karrer and Jucker reported the sodium ethoxide catalyzed summarization reaction of lutein to zeaxanthin (P. Karrer and E. Jucker, Helv. Chim. Acta, 30, 266, 1947). Later in 1971-72 Buchecker et al. assigned R-chirality to lutein based on PMR analysis and attempts to isomerize lute in to R,R-zeaxanthin failed (Chimia, 25,192,1971; ibid, 26,134, 1972).
Andrewes et al. (1974)reported the stereo chemical aspects of isomerization reaction of (3R,3'R, 6R)-lutein (optically active) which resulted in (3R,3'S)-zeaxanthin which was trans-isomeric and optically inactive based on CD spectral studies (Isomerization of epsilon-carotene to beta-carotene and lutein to zeaxanthin in the Journal Acta Chem.
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Scand , B28, 139). The above process results in low yield of 10 to 15% optically inactive (3R,3'S, meso)-zeaxanthin and uses benzene and DMSO which are objectionable for use in food and health supplements.
Rodriguez has described a method of isomerising lutein to yield a mixture of zeaxanthin epimers by employing non aqueous media and heating a mixture of alkali and propylene glycol. Though the circular dichroism spectrum indicated the formation of meso-isomer of zeaxanthin, no attempts were made to quantify the meso-zeaxanthin content and also provide the composition of the isomerised products. (U.S. Pat. No. 5,973,211, 1999).
However, to the best of our knowledge xanthophylls composition containing high concentration of trans -lutein and low concentration xanthophylls composition containing of R,R'-zeaxanthin and R, S'-zeaxanthin is not known . More particularly xanthophylls composition containing at least 85% of trans-lutein and balance comprising of (R,R)-zeaxanthin and (R,S) -zeaxanthin derived from the same natural source such as marigold flower petals is not known
Xanthophylls composition containing high concentration of trans -lutein and low concentration of R,R'-zeaxanthin and R,S-zeaxanthin will form an ideal and essential dietary supplements which would help in maintaining similar healthy vision . Further the composition may be deemed to be safe based on the factors supporting reagents employed in the process and the literature data available regarding the toxicological safety for the individual xanthophylls. (Ref).Objectives of the present Invention:Therefore, the main objective of the present invention is to provide a xanthophyll composition containing trans-lutein, R,R-zeaxanthin and R,S-zeaxanthin and trace amounts of other carotenoids which is safe for human consumption and useful for nutrition and health care.Another objective of the present invention is to provide a xanthopyll compositon containing at least 85% by weight of total xanthophylls out of which the trans-lutein content is at least 85% and the remaining 10% being zeaxanthin isomers and trace amounts of other carotenoids.According to a feature of the present invention there is provided a xanthophylls composition wherein the composition contains at least 85% by weight of total xanthophylls, out of which at least 85% being trans-lutein,
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4% being R,R-zeaxanthin, 5% being R,S-zeaxanthin and trace amounts of other carotenoids. According to another features of the present invention there is provided a xanthophylls composition containing at least 85% by weight of total xanthophylls, out of which at least 80% being trans-Iutein, the remaining 15% being zeaxanthin isomers and trace amounts of other carotenoids. Yet another objective of the present invention is to provide a process
For the preparation of xanthophyll composition containing trans-lutein, R,R-zeaxanthin and R,S-zeaxanthin and trace amounts of other carotenoids which is safe for human consumption and useful for nutrition and health care.
Yet another objective of the present invention is to provide a process for the reparation of xanthophylls composition containing at least 82% by weight of total xanthophylls, out of which the trans-lutein content is atleast 83% , the remaining being R,R -zeaxanthin , R,S -zeaxanthin and trace amounts of other carotenoids which is safe for human consumption and useful for nutrition and health care.The above objectives of the present invention have been achieved by the present invention based on our following findings that:
a) The saponification step to convert xanthophylls esters present in the source into the de esterified form can be combined with limited isomerisation of lutein to produce xanthophylls composition containing trans-lutein R,R-zeaxanthin and R,S -zeaxanthin and trace amounts of other carotenoids which is safe for human consumption and useful for nutrition and health care.b) in the saponification step pothudroxide can be dissolved in 1-proponaol with out the addition water.c) the temperature of the saponification /isomerisation can be around 95 degree's and performed for a period in the range of 1-2 hrs.d) the composition of xanthophylls during saponification/ isomerisation can be analyzed and monitored using HPLC to obtain a specific composition of xanthophylls.
e) in the extraction of xanthophylls , organic acid ester can be used and the final product further purified as per the procedure disclosed by Kumar et.al . in their US patent no 6,743,953 of 2004 titled "Process for the preparation of xanthophyll crystals".
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f) the GRAS reagent employed in the process can be recovered and used if required, there by making the process economical. Summary of the Invention:Accordingly , the present invention provides a xanthophyll composition containing trans-lutein , R,R-zeaxanthin and R,S-zeaxanthin and trace amounts of other carotenoids which is useful for nutrition and health care According to another embodiment of the present invention there is provided a xanthophyll composition containing high concentration of trans-lutein , and low concentration of R,R-zeaxanthin and R,S-zeaxanthin along with trace amounts of other car Accordingly , the present invention provides a xanthophyll composition containing trans-lutein , R,R-zeaxanthin and R,S-zeaxanthin and trace amounts of other carotenoids which is useful for nutrition and health care
According to another embodiment of the invention there is provided a xanthophyll composition containing at least 85% by weight of total xanthophylls ,out of which at least 85% being trans-lutein content,the remaining 10% being R,R-zeaxanthin and RS-zeaxanthin and trace amounts of other carotenoids which is useful for nutrition and healthcare.
According to another embodiment of the invention there is provided a xanthophylls composition containing at least 85%by weight of total xanthophylls, out of which 85% being trans-lute in ,4% being R,R-zeaxanthin and 5%being R,S- zeaxanthin and trace amounts of other carotenoids which is useful for nutrition and healthcare. According to yet another embodiment of the present invention there is provided a xanthophyll composition containing at least 85%by weight of total xanthophylIs,out of which 80% being trans-lutein ,the remaining 15% being zeaxanthin isomers and trace amounts of other carotenoids which is useful for nutrition and healthcare.
According to another embodiment of the present invention provides a process for the preparation of xanthophyll composition containing trans-lutein , R,R-zeaxanthin and R,S-zeaxanthin and trace amounts of other carotenoids which is useful for nutrition and health i care which comprises (a)Saponifying and partially isomerising the xanthophyll esters present in the extract/oleoresin of suitable plant material by admixing the
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extract/oleoresin with alcoholic alkali solution without addition of water and heating the resultant mass at a temperature in the range of 90-100 degree C
(b)admixing the resultant saponified/partially isomerised crude concentrate obtained in step(a) with water to form a diluted oily mixture
( c) Extracting the diluted oily mixture obtained in step(b) with ethylactate to get extract containing xanthophylls and
(d) Purifying the concentrate obtained in step ( c) by washing with non polar and polar solvents and filtering and drying the residue under vacuum
By varying the reaction temperature , period and the amount of alkali a composition containing the desired concentration of rans-lutein and that of R,S-zeaxanthin can be obtained,whereas the concentration of R,R-zeaxanthin remains practically without any change
While selecting the suitable solvent for use for extracting saponified and partially isomerised xanthophylls and diluting with water before extraction step ,of the process of the present invention ,the criteria used are the same as earlier reported by Kumar et al.in their US patent No.6,743,953,2001 titled "Process for the preparation for xanthophyll crystals".
Accordingly, the criteria considered in the selection of the solvent are l)it should be water miscible (ii)the solvent should be non halogenated (iii)the solvent should have GRAS status (iv)the solubility of xanthophylls in the solvent should behigh (v) the boiling point of the solvent should be in the range of 50-80 degree C(vi)stability of xanthophylls in the solvent used should be high.
Ethylacetate satisfies all the above requirements and in addition eliminating the impurities at low temperature in the process conditions.Accordingly, the present invention provides a process for the preparation of xanthophyll crystals containing atleast 82%by weight of total xanthophylls,out of which the trans-lutein is atleast 83%,the remaining being R,R-zeaxanthin and R,S-zeaxanthin and trace amounts of other
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carotenoids which is safe and useful as dietary supplements The extract/oleoresin used in the process of the present invention can be obtained from any plant source,preferabIy the oleoresin containing xanthophyll esters 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 1-propanol. The ratio of extract/oleoresin containing xanthophyll esters and the a The alkali used for preparing the alcoholic alkali solution may be selected from sodium ' hydroxide or potassium hydroxide, preferably potassium hydroxide.
The saponification and partial isomerisation 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 80.degree.C, preferably at 7O.degree.C. The unused alcohol is recovered by distillation under vacuum, and reused, if required, in step (a).
The amount of water added in the step (b) of the process for dilution of the reaction mixture may be in the range of 1:4 to 1:6, preferably 4, weight by volume. The ratio of ethyl acetate employed for mixing
with the diluted reaction mixture may range from 1:1 to 1:3 preferably 1:1 by volume/volume.
The non-polar solvent used may be selected from the hydrocarbon solvents like pentane, hexane and heptane, and the like preferably hexane.
The alcohol which may be used for further purification may be selected from methanol, ethanol, isopropanol, etc, preferably ethanol.
The details of the invention is given in the example given whic is given to illustrate the invention only and should not be construed to limit the scope of the present invention.
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Example. 1 :
Marigold oleoresin (50 g) containing 124.10 g/kg xanthophyll content (by spectrophotometric method) was mixed with 25 g potassium hydroxide in 25 ml 1-Propanol .The reaction mixture was heated and maintained at 95.degree.C. for a period of 1 hr. The degree of hydrolysis , trans-lutein and zeaxanthin content were monitored by HPLC during the reaction stage. The reaction mass obtained was stirred with 250 ml distilled water 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 (30 g).
This resultant crude extract (30 g) was subjected to purification by stirring with 150 ml of hexane at room temperature for Ihr, followed by filtration. The precipitate (9 g) obtained was washed with 90 ml ethanol at room temp for 1 hr followed by filtration . The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs.
The yield of the xanthophyll concentrate was 4.30 g having xanthophyll content 82.59 % by weight (as determined by UV/Vis spectrophotometry). The composition of the product was 83.32% trans-lutein 15.32% , zeaxanthins and 0.10% other carotenoids determined by HPLC analysis.
Example. 2:
Marigold oleoresin (50 g) containing 160.07 g/kg xanthophyll content (by spectrophotometric method) was mixed with 25 g potassium hydroxide in 25 ml 1-
17

Propanol .The reaction mixture was heated and maintained at 95.degree.C. for a period of 2 hr. The degree of hydrolysis , trans-lutein and zeaxanthin content were monitored by HPLC during the reaction stage. The reaction mass obtained was stirred with 250 ml distilled water 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 (36.90 g).
This resultant crude extract (36.90 g) was subjected to purification by stirring with 185 ml of hexane at room temperature for lhrs followed by filtration. The precipitate 10.33 g) obtained was washed with 103 ml ethanol at room temp for 1 hr followed by filtration . The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs.
The yield of the xanthophyll concentrate was 7.02 g having xanthophyll content 85.59 % by weight (as determined by UV/Vis spectrophotometry).The composition of the product was 88.45% trans-lutein and 10.36 %, zeaxanthin determined by .HPLC analysis.
Example. 3 :
Marigold oleoresin (52 g) containing 132.2 g/kg xanthophyll content (by spectrophotometric method) was mixed with 26 g potassium hydroxide in 26 ml 1-Propanol .The reaction mixture was heated and maintained at 95.degree.C. for a period of 1 hr. The degree of hydrolysis , trans-lutein and zeaxanthin content were monitored by HPLC during the reaction stage. The reaction mass obtained was stirred with 250 ml distilled water 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 (30.30 g).
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This resultant crude extract (30.30 g) was subjected to purification by stirring with 150 ml of hexane at room temperature for lhr, followed by filtration. The precipitate (8.00 g) obtained was washed with 80 ml ethanol at room temp for 1 hr followed by filtration . The resulting orange crystals were vacuum dried at atnbient temperature for 72 hrs. The yield ofthexanthophyll concentrate was 4.40 g having xanthophyll content 82.59 % by weight (as determined by UV/Vis spectrophotometry). The composition of the product was 86.64% trans-lutein and 11.49 %, zeaxanthins determined by .HPLC.
Dated this 23rd day of June, 2008
ABHIJIT BHATTACHARYA CHIEF OPERATING OFFICER OMNI ACTIVE HEALTH TECHNOLOGIES LTD
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Documents:

1322-MUM-2008-ABSTRACT(15-7-2013).pdf

1322-MUM-2008-ABSTRACT(26-6-2009).pdf

1322-MUM-2008-CLAIMS(26-6-2009).pdf

1322-MUM-2008-CLAIMS(AMENDED)-(15-7-2013).pdf

1322-MUM-2008-CLAIMS(AMENDED)-(23-11-2012).pdf

1322-MUM-2008-CORRESPONDENCE(2-9-2009).pdf

1322-MUM-2008-CORRESPONDENCE(26-6-2009).pdf

1322-MUM-2008-CORRESPONDENCE(30-3-2012).pdf

1322-mum-2008-correspondence(ipo)-(22-10-2009).pdf

1322-mum-2008-correspondence.pdf

1322-MUM-2008-DESCRIPTION(COMPLETE)-(26-6-2009).pdf

1322-mum-2008-description(provisional).doc

1322-mum-2008-description(provisional).pdf

1322-MUM-2008-FORM 1(15-7-2013).pdf

1322-mum-2008-form 1.pdf

1322-MUM-2008-FORM 18(26-6-2009).pdf

1322-mum-2008-form 2(26-6-2009).pdf

1322-MUM-2008-FORM 2(TITLE PAGE)-(15-7-2013).pdf

1322-MUM-2008-FORM 2(TITLE PAGE)-(23-11-2012).pdf

1322-MUM-2008-FORM 2(TITLE PAGE)-(26-6-2009).pdf

1322-mum-2008-form 2(title page).pdf

1322-mum-2008-form 2.doc

1322-mum-2008-form 2.pdf

1322-MUM-2008-FORM 3(15-7-2013).pdf

1322-MUM-2008-FORM 3(23-11-2012).pdf

1322-MUM-2008-FORM 5(26-6-2009).pdf

1322-MUM-2008-MARKED COPY(15-7-2013).pdf

1322-MUM-2008-MARKED COPY(23-11-2012).pdf

1322-MUM-2008-PETITION UNDER RULE-137(15-7-2013).pdf

1322-MUM-2008-RECEIPT(2-9-2009).pdf

1322-MUM-2008-REPLY TO EXAMINATION REPORT(23-11-2012).pdf

1322-MUM-2008-REPLY TO HEARING(15-7-2013).pdf

1322-MUM-2008-SPECIFICATION(AMENDED)-(15-7-2013).pdf

1322-MUM-2008-SPECIFICATION(AMENDED)-(23-11-2012).pdf


Patent Number 257071
Indian Patent Application Number 1322/MUM/2008
PG Journal Number 36/2013
Publication Date 06-Sep-2013
Grant Date 30-Aug-2013
Date of Filing 24-Jun-2008
Name of Patentee OMNIACTIVE HEALTH TECHNOLOGIES LTD.
Applicant Address RAJAN HOUSE, APPASAHEB MARATHE MARG, PRABHADEVI, MUMBAI
Inventors:
# Inventor's Name Inventor's Address
1 SUNIL KUMAR T.K. OMNIACTIVE HEALTH TECHNOLOGIES LTD. RAJAN HOUSE, APPASAHEB MARATHE MARG, PRABHADEVI, MUMBAI-400025.
2 SHERENA P.A. OMNIACTIVE HEALTH TECHNOLOGIES LTD. RAJAN HOUSE, APPASAHEB MARATHE MARG, PRABHADEVI, MUMBAI-400025.
PCT International Classification Number A61K31/045
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA