FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
The Patent Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
EFECT OF LIPOPHILIC NUTRIENTS ON DIABETIC EYE DISEASES
OMNIACTIVE HEALTH TECHNOLOGIES LTD., an Indian Company,
registered under the Indian Companies Act, 1956 having its registered office
located at Rajan House, Appasaheb Marathe Marg, Prabhadevi, Maharashtra,
India 400025
The following specification particularly describes the invention and the manner in
which it is to be performed

Field of the invention:
The present invention relates to a method of delaying the development and maturation of eye related complications of diabetes by administering a composition containing lipophilic nutrients. More particularly, the present invention relates to a method of delaying the development and maturation of eye related complications of diabetes by administering a composition containing lutein and its isomers, lutein ester, zeaxanthin isomers, turmeric extract, curcumin or curcuminoids, derived from plant extract/oleoresin containing xanthophylls/ xanthophylls esters which are safe for human consumption and are particularly useful as dietary supplements for nutrition and health promoting benefits.
Background of the invention
Eye is the most important and complex organ in the human body that is protected by the bony orbit of the eye. It is divided into anterior segment which consists of the cornea, iris, lens, ciliary body, and the anterior portion of the sclera. The posterior segment is bounded anteriorly by the lens and extends to the back of the eye. The retina optic disc is also included in the posterior segment. The light passes through anterior portion called cornea, aqueous humor, crystalline lens, pupil, vitreous humor to reach the retina of the eye, this path way is called visual axis of the eye. The lens refracts light rays and helps focus the image of an object on the retina (fovea) through accommodation.
Diabetes and diabetic complications: Diabetes is one of the most occurring non-communicable, heterogeneous, metabolic disorder characterized by hyperglycemia resulting from defective insulin production, resistance to insulin action or both, there are two forms of diabetes, type 1 and type 2. Type 1 diabetes mellitus is the

consequence of an autoimmune-mediated destruction of pancreatic p-cells, leading to insulin deficiency.
Type 2 diabetes mellitus is characterized by insulin resistance and relative, rather than absolute, insulin deficiency. According to the latest WHO estimation currently there are about 366 million diabetic people in the world, it is expected to increase to 552 million by 2030 and India has about 62 million diabetics. Prolonged exposure to chronic hyperglycemia can lead to various complications including vascular and non-vascular complications. Vascular complications are further divided into macrovascular and microvascular complications. The insulin independent tissues like retina, kidney, peripheral nerves and lens are most effective tissues in long term complications of the diabetes, which results in the development of diabetic retinopathy, nephropathy, neuropathy and cataract respectively.
Diabetic cataract: Cataract is characterized by opacity of the eye lens, and the leading cause of blindness worldwide. The development of cataract in diabetes is 2-5 times more when compared with the non-diabetic counterparts. Furthermore, patients with diabetes mellitus have higher complication rates from cataract surgery. Both diabetes and cataract pose an enormous health and economic burden, particularly in developing countries, where diabetes treatment is insufficient and cataract surgery often inaccessible.
Many clinical interventions have been reported to countering cataract including diabetic cataract but are not completely successful at clinical practice.
Diabetic Retinopathy: Diabetic retinopathy (DR) is one of the most common micro vascular complications of diabetes. DR occurs in 70% of all persons having diabetes for more than 15 years and is the most common cause of blindness. DR is a disease of retina, resulting in loss of vision, macular edema, recurrent vitreous hemorrhages,

fractional or secondary rhegmatogenous retinal detachment, and so forth. Since the last two decades there have been significant developments in the emerging field of pharmacotherapy of DR. The advent of laser photocoagulation three decades back, was really useful in limiting vision loss in most of the cases and is still considered gold standard therapy for the treatment of DR. However, corticosteroids and anti-VEGF agents have shown promising results with regard to prevention of neo-vascularisation, but remained limited in use due to their short-duration effects. Therefore, pharmacotherapy of DR is still an adjunct to pan retinal photocoagulation.
In recent years, a great deal of attention has been focused on biological activities of carotenoids. Carotenoids are naturally occurring xanthophylls in plants that are involved in light harvesting reactions and protection of plant organelles against singlet oxygen induced damage. Dietary carotenoids serve as antioxidants in the tissues (Thurnham DL. Carotenoids: function and fallacies. Proc Nutr Soc 1994; 53: 77-87) and protect the body from oxidative damage. Mammalian species do not synthesize carotenoids and therefore these have to be obtained from dietary sources such as fruits and vegetables and or dietary supplements. Numerous epidemiological studies support a strong inverse relationship between consumption of carotenoid rich fruits and vegetables and incidence of degenerative diseases (Coleman H, Chew E. Nutritional supplementation in age-related macular degeneration. Curr Opin Ophthalmol 2007; 18(3): 220-223)
Lutein is one of the major xanthophylls present in green leafy vegetables and egg yolk. Lutein and zeaxanthin are known to selectively accumulate in the macula of the human retina. They have been thought to work as antioxidants and as blue light filters to protect the eyes from such oxidative stresses as cigarette smoking and sunlight exposure, which can lead to age-related macular degeneration and cataracts.

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 the 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 31 positions in the two end rings are both chiral atoms.
Thus, trans-zeaxanthin has two chiral centers at the carbon atoms C3 and C31, 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 twofold opposite effects (R,S; optically inactive) which is called meso-formofzeaxanthin.
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 the extra conjugated double bond, zeaxanthin is believed to be a stronger anti-oxidant compared to lutein.

Regarding the location of xanthophylls at a cellular level, they are reported to be bound to specific proteins referred to as 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 et al., Photophysical Properties of Xanthophylls in Caroteno proteins from Human Retina, Photochemistry and Photobiology. 78, 138-145, 2003)
Epidemiological studies suggested that higher dietary intake of lutein and zeaxanthin reduce the risk of cataracts and age-related macular degeneration. Previous studies showed that rats treated with combination of insulin and lutein exhibited delayed development and maturation of cataract than treated with lutein or insulin alone. Serum lutein and zeaxanthin concentrations in DR patients was found to be significantly lower than those in the normal subjects, and their intake was proved to improve the visual acuity, contrast sensitivity and macular edema, suggesting that lutein and zeaxanthin supplementation might be targeted as potential therapeutic agents in treating DR. .
Curcumin has been identified as the active principle of turmeric and has been shown to exhibit antioxidant, anti-inflammatory, antimicrobial, and anticarcinogenic activities. Curcumin is a natural extract from the spice Turmeric. Turmeric is derived from the plant Curcuma Longa, a member of the ginger family. Curcumin is a known

antioxidant which inherently has many health benefits. Curcumin was shown to induce apoptosis in human retinal endothelial cells and decrease VEGF release into media in vitro and it also inhibits diabetes-induced elevation of serum VEGF levels in rat. Vascular endothelial growth factor (VEGF) expression, induced by high glucose levels and hypoxia, is a main feature in retinopathy. Several studies have also shown that VEGF may also play a role in the development of the earliest stages of retinopathy.
Though dietary supplements such as curcumin, lutein, zeaxanthin, etc have offered some benefits in preclinical studies, the translation has been very poor and the doses used in clinical trials are unfeasible to practice in reality. One of the major reasons for the lack of clinical success with curcumin is linked to its extensive intestinal and hepatic metabolic biotransformation resulting in poor bioavailability. Recently, the focus is to address bioavailability concerns of the supplements with a view to improve the therapeutic efficacy.
The lipophilic nutrients such as curcumin, lutein, zeaxanthin, ginger, etc are poorly absorbed if administered either as oil suspensions or as beadlets, which are the currently used forms. The main reason for poor absorption is their poor solubility in water. Due to their insolubility their bioavailability is very poor. Lipophilic nutrients have limited absorption in the body due to limited solubility in the gastrointestinal tract. Generally, the bioavailability of such nutrients is below 40%. The bioavailability can be enhanced by reducing the particle size, which in turn will enhance their efficiency of micellization. Dispersion of nutritional products at molecular level is generally regarded as a technique of reducing the particle size. Such molecular dispersions provide higher efficiency for micellization of nutrients in water and thereby increase the bioavailability.

The molecular dispersions of lipophilic nutrients can be obtained by dispersing the solution of lipophilic nutrient in a polar or non polar organic solvent certain water soluble hydrophilic solid or liquid carrier systems. Upon removal of solvent under vacuum, the resultant dispersion remains as a homogenous liquid or solid dispersions which is suitable for filling in to soft gel capsules or in to Heaps, tablets, capsules and other oral solid or liquid preparations. Because of such dispersions, the absorption of lipophilic nutrients can be enhanced several folds. The said technology is protected by the Applicant under granted patent number IN253078.
Hence, it is interesting to search the effects of a composition containing soluble lipophilic nutrients in diabetic rats with respect to its beneficial effect on retina by nutrigenomics approach and the effect was compared with regular lipophilic nutrients.
Nutrigenomics is the science of interaction between nutrients and genes. It is the study of how genetic expression affect your need for certain nutrients and help maintain optimal health throughout your life. Nutrigenomics promotes an increased understanding of how nutrition influences metabolic pathways and homeostatic control, how this regulation is disturbed in the early phases of diet-related disease, and the extent to which individual sensitizing genotypes contribute to such diseases. Our goal is to better understand how phytonutrients affect gene expression.
Prior Art
Numerous prior art references are available that provide compositions containing carotenoids used for the prevention/treatment of diabetic eye diseases.
Brown et al. (Am J ClinNutr. 1999) Dietary antioxidants, including carotenoids, are hypothesized to decrease the risk of age-related cataracts by preventing oxidation of

proteins or lipids within the lens. However, prospective epidemiologic data concerning this phenomenon are limited. The authors examined prospectively the association between carotenoid and vitamin A intakes and cataract extraction in men. US male health professionals (n = 36644) who were 45-75 y of age in 1986 were included in this prospective cohort study. Others were subsequently included as they became 45 y of age. During 8 y of follow-up, 840 cases of senile cataract extraction were documented. They observed a modestly lower risk of cataract extraction in men with higher intakes of lutein and zeaxanthin but not of other carotenoids (alpha-carotene, beta-carotene, lycopene, and beta-cryptoxanthin) or vitamin A after other potential risk factors, including age and smoking, were controlled for. Men in the highest fifth of lutein and zeaxanthin intake had a 19% lower risk of cataract relative to men in the lowest fifth (relative risk: 0.81; 95% CI: 0.65, 1.01; P for trend = 0.03). Among specific foods high in carotenoids, broccoli and spinach were most consistently associated with a lower risk of cataract. Lutein and zeaxanthin may decrease the risk of cataracts severe enough to require extraction, although this relation appears modest in magnitude. This study is cohort study done with US male population. This study establishes a relation between nutrition deficiency and cataract.
EP 2618832 A2 provides a composition comprising an enzyme selected from the group comprising superoxide dismutase (SOD) and SOD mimics and the like, in association with lutein and at least one stereoisomer of zeaxanthin; the invention also includes a kit of parts comprising such composition, wherein the kit comprises a first part comprising the enzyme, and a second part comprising lutein and at least one zeaxanthin isomer; according to the invention, the composition or the kit of part may be included in a functional food, a nutraceutical composition or a food or dietary supplement, a medicament or a pharmaceutical composition, or a veterinarian product: the invention also relates to a composition for use in treating,

preventing or stabilizing a disease, condition or disorder of the eye associated to oxidative stress, comprising administering to a subject in need thereof a medicament or a pharmaceutical composition according to the invention. However, the said invention does not make use of Zeaxanthin isomers. And it is a proven fact that zeaxanthin isomers present better antioxidant profile than zeaxanthin alone.
WO2010032267A2 provides a herbal formulation for prevention and treatment of Diabetes and associated complications comprising extracts from selected Indian medicinal herbs. The invention has associated formulations for different diabetes related complications, which are individually useful in clinical requirements such as improving renal health and preventing renal diseases, preventing diabetic retinopathy and prevention and treatment for oxidative damage to heart and btood vessels. The invention is versatile and can be processed into extracts/ concentrates and further pharmacologically modified to tablets or capsules or granules or syrups or herbal health drink or inhalable herbal medicinal preparations or ocular preparations or transdermal absorbable preparations such as ointments / gels or injectable medicine. This is a poly herbal formulation and there is no synergistic data to support the claim.
CN 102178925A discloses a lutein ophthalmic preparation for protecting eyesight, which is prepared from the following raw materials: 5 to 13 parts of water-soluble lutein (based on C4oH5602), 50 to 80 parts of taurine, 0.1 to 0.5 parts of selenium (based on Se), 10 to 25 parts of zinc (based on Zn), 0.5 to 1.0 part of water-soluble vitamin A, and 0.8 to 2.0 parts of glutathione; an auxiliary material consists of a diluent, a wetting agent, an isoosmotic adjusting agent, a preservative, an antioxygen and water for injection; formulations comprise eye drops, eye lotion and the like; and the lutein ophthalmic preparation is suitable for eye diseases such as myopia, long sight, cataract, glaucoma, retinal pigment degeneration,

macular degeneration and the like. Various nutritious factors are reasonably compatible, a blank of a lutein external preparation is filled, and the bioavailability and health-care effect are obviously improved; and through actual application by 300,000 people, the total effective rate for the myopia, cataract and diabetic eye disease is over 90 percent and the lutein ophthalmic preparation has positive promotion valve. This ophthalmic preparation contains only one macular carotenoid i.e Lutein and does not mention use of zeaxanthin isomers.
Sasaki et al. (IOVS, March 2009, Vol. 50, No. 3) The aim of this study was to investigate, with the use of a mouse endotoxin-induced uveitis (EIU) model, the neuroprotective effects of lutein against retinal neural damage caused by inflammation. EIU was induced by intraperitoneal injection of lipopolysaccharide (LPS). Each animal was given a subcutaneous injection of lutein or vehicle three times: concurrently with and 3 hours before and after the LPS injection. Analysis was carried out 24 hours after EIU induction. Levels of rhodopsi'n protein and STAT3 activation were analyzed by immunoblotting. Lengths of the outer segments of the photoreceptor cells were measured. Dark-adapted full-field electroretinograms were recorded. Oxidative stress in the retina was analyzed by dihydroethidium and fluorescent probe. Expression of glial fibrillary acidic protein (GFAP) was shown immunohistochemically. The ElU-induced decrease in rhodopsin expression followed by shortening of the outer segments and reduction in a-wave amplitude were prevented by lutein treatment. Levels of STAT3 activation, downstream of inflammatory cytokine signals, and reactive oxygen species (ROS), which are both upregulated during EIU. were reduced by lutein. Pathologic change of Mi'iller glial cells, represented by GFAP expression, was also prevented by lutein. The present data revealed that the antioxidant lutein was neuroprotective during EIU, suggesting a potential approach for suppressing retinal neural damage during inflammation. Lutein is a nutritional supplement and subjects have to take daily dose for prevention

or treatment of any disease. In this study, Lutein was administered through injection. Daily supplementation of lutein via injection is painful causing discomfort to the
subject.
CA 2760932 Al describes ophthalmic formulations that deliver a variety of therapeutic agents, including but not limited to rapamycin (sirolimus). analogs thereof (rapalogs) or other mTOR inhibitors, to a subject for an extended period of time. The ophthalmic formulations may be placed in an aqueous medium of a subject, including but not limited to intraocular or periocular administration, or placement proximate to a site of a disease or condition to be treated in a subject. A method may be used to administer a therapeutic agent to treat or prevent age-related macular degeneration, macular edema, diabetic retinopathy, uveitis, dry eye, or a hyperpermeability disease in a subject.
Overcoming the difficulty of delivering therapeutic/ preventive agents to specific regions of the eye presents a major challenge to treatment of most eye disorders. Due to poor bioavailability of lipophilic nutrients the delivery of many potentially important therapeutic/ preventive agents to the eye is hindered.
From above it is clear that there is a need to provide a technology which can overcome the difficulty of delivering the therapeutic/ preventive agents for diabetic eye complications even at reduced dose levels.
Objectives of the present invention
The main objective of the present invention is to provide molecular dispersions of lipophilic nutrients which are useful for delaying the development and maturation of eye related complications of diabetes and which are safe for human consumption and

are particularly useful as dietary supplements for nutrition and health promoting benefits.
Another objective of the present invention is to provide molecular dispersions of lipophilic nutrients such as curcumin or trans-lutein and zeaxanthin isomers namely (R,R)-zeaxanthin and (R,S)-zeaxanthin or trans-lutein and (R,R)-zeaxanthin in a solid or liquid hydrophilic carrier, derived from plant extract/oleoresin containing xanthophylls/ xanthophylls esters which are useful for delaying the development and maturation of eye related complications of diabetes.
Yet another objective of the present invention is to provide molecular dispersions of composition containing at least 80% by weight of total xanthophylls, out of which the trans-lutein content is 80-95% w/w; (R,R)-zeaxanthin is 14-20% w/w; (R,S)-zeaxanthin is 0.01-1% w/w or trans-lutein content is 80-95% w/w; (R,R)-zeaxanthin is 14-20% w/w and traces of other carotenoids derived from the plant extracts/oleoresin containing xanthophylls/xanthophylls esters or curcumin which contains 5-95% of curcuminoids.
Still another objective of the present invention is to provide molecular dispersions of xanthophyll composition containing trans-lutein and zeaxanthin isomers namely (R,R)-zeaxanthin and (R,S)-zeaxanthin or trans-lutein and (R.R)-zeaxanthin in a solid or liquid hydrophilic carrier, wherein the complex has higher antioxidant potential than the free xanthophylls and which are useful for delaying the development and maturation of eye related complications of diabetes.
Still another objective of the present invention is to provide molecular dispersions of curcumin composition containing curcuminoids in a solid or liquid hydrophilic

carrier, which is useful for delaying the development and maturation of eye related complications of diabetes.
Yet another objective of the present invention is to provide molecular dispersions of lipophilic nutrients which have higher efficiency for micellization which enhances the bioavailability resulting in increased levels of lipophilic nutrients in tissues due to which these molecular dispersions are effective even at the lower concentrations and are useful for delaying the development and maturation of eye related complications of diabetes.
Still another objective of the present invention is to provide molecular dispersions of lipophilic nutrients in solid or liquid hydrophilic carriers which have higher bioavailability.
Yet another objective is to provide the molecular dispersions of lipophilic nutrients which are prepared by using safe solvents (GRAS) and are suitable for human consumption, with minimum solvent residues.
Further objects and advantages of the invention will become apparent from a consideration of the ensuing description.
Summary of the invention
The usefulness of the product is described herein below which is illustrative as shown in the examples and should not be construed to limit the scope of the present invention in any manner whatsoever.

The present invention relates to a method of delaying the development and maturation of eye related complications of diabetes by administering a composition containing lipophilic nutrients. More particularly, the present invention relates to a method of delaying the development and maturation of eye related complications of diabetes by administering a composition containing lutein and its isomers, lutein ester, zeaxanthin isomers, turmeric extract, curcumin or curcuminoids, derived from plant extract/oleoresin containing xanthophylls/ xanthophylls esters which are safe for human. consumption and are particularly useful as dietary supplements for nutrition and health promoting benefits.
The said dispersions are in the form of powders, tablets, capsules, sachets, beadlets, microencapsulated powders, oil suspensions, liquid dispersions, pellets, soft gel capsules, chewable tablets or liquid preparations.
It may be noted that a novel feature of the present invention is the use of molecular dispersions of trans-lutein and zeaxanthin isomers namely (R.R)-zeaxanthin and (R.S)-zeaxanthin or trans-lutein and (R.R)-zeaxanthin or curcumin containing curcuminoids in a solid or liquid hydrophilic carrier with enhanced water solubility and bioavailability which helps in effectively delivering the molecules and shows potential in delaying the development and maturation of eye related complications of diabetes. The use of this combination of carotenoids namely, trans-lutein and zeaxanthin isomers having higher antioxidant potential in highly water soluble form with enhanced bioavailability for delaying the development and maturation of eye related complications of diabetes has not been reported in the prior art.
Description of the invention
Diabetes mellitus can cause a variety of eye problems, the most common being diabetic retinopathy (DR) and diabetic cataract which are the most common causes

of blindness. Antioxidant compounds are considered to have high antioxidant potential in the prevention of many human ailments such as age related macular degeneration, cataract, diabetic eye complications and various other diseases.
Lutein is a naturally occurring antioxidant found in green leafy vegetables like spinach. Lutein is also found in eye mainly present in macula lutea. It is well known that lutein is a carotenoid and powerful antioxidant. It has been used in cataracts and macular degeneration which is an age related degenerative disorder. Lutein has also shown protective antioxidant activity in human HepG2 cell lines.
Zeaxanthin is one of the most common carotenoid alcohols found in nature. Lutein and zeaxanthin have identical chemical formulas and are isomers, but they are not stereoisomers. The only difference between them is in the location of the double bond in one of the end rings. This difference gives lutein three Chiral centers whereas zeaxanthin has two. Because of symmetry, the (3R,3'S) and (3S.3'R) stereoisomers of zeaxanthin are identical. Therefore, zeaxanthin has only three stereoisomeric forms. The (3R,3'S) stereoisomer is called meso-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 the extra conjugated double bond, zeaxanthin is believed to be a stronger anti-oxidant compared to lutein. It has been demonstrated that the complex of lutein and zeaxanthin isomers act as a better antioxidant than the free xanthophylls, facilitating improved protection from oxidative damages.
Curcumin. a yellow pigment from Curcuma longa, is a major component of turmeric and is commonly used as a spice and food-coloring agent. It is also used as a cosmetic and in some medical preparations. The desirable preventive or putative

therapeutic properties of curcumin have also been considered to be associated with its antioxidant and anti-inflammatory properties. Curcumin is thought to play a vital role against a variety of chronic pathological complications such as cancer, atherosclerosis, and neurodegenerative diseases.
The lipophilic nutrients are poorly absorbed if administered either as oil suspensions or as beadlets, which are the currently used forms. The main reason for poor absorption is their poor solubility in water. Due to their insolubility their bioavailability is very poor. Dispersion of nutritional products at molecular level provides higher efficiency for micellization of nutrients in water and thereby increases the bioavailability.
The present invention therefore provides use of lipophilic nutrients composition containing at least 80% by weight of total xanthophylls, out of which the trans-lutein content is 80-95% w/w; (R,R)-zeaxanthin is 14-20% w/w; (R,S)-zeaxanthin is 0.01-1% w/w or trans-lutein content is 80-95% w/w; (R,R)-zeaxanthin is 14-20% w/w and traces of other carotenoids derived from the plant extracts/oleoresin containing xanthophylls/xanthophylls esters or curcumin which contains 5-95% of curcuminoids in highly water soluble form with enhanced bioavailability in delaying the development and maturation of eye related complications of diabetes.
The said composition comprises lipophilic nutrients; stabilizer; water soluble hydrophilic carrier and optionally a surfactant.
The said composition contains at least 80% by weight of total xanthophylls. out of which the trans-lutein content is 80-95% w/w; (R,R)-zeaxanthin is 14-20% w/w; (R;S)-zeaxanthin is 0.01-1% w/w or trans-lutein content is 80-95% w/w; (R,R)-zeaxanthin is 14-20% w/w and traces of other carotenoids derived from the plant

extracts/oleoresin containing xanthophylls/xanthophylls esters or curcumin which contains 5-95% of curcuminoids.
The stabilizer used is selected from Ascorbic acid, BHA, BHT, ascorbyl palmitate, rosemary extract, mixed natural tocopherols, alpha tocopheryl acetate, sodium ascorbate. castor oil derivatives, sodium lauryl sulfate and mixtures thereof.
The carrier used is selected from polyethylene glycol 200, polyethylene glycol 400, ethylene glycol, propylene glycol, glycerol, sorbitol, glucose syrup, corn steep liquor, mannitol, polyethylene glycol 6000, polyethylene glycol 10000, Polyethylene glycol 20000, polyvinyl pyrrolidone, hydroxy! propyl methyl cellulose, sucrose, glucose, sodium chloride, hydroxyl propyl cellulose, polyvinyl alcohol, soluble starch, hydrolyzed starch and mixtures thereof.
The said surfactant is selected from a group comprising of polysorbate 20, polysorbate 60, polysorbate 80, lecithin, sucrose fatty acid esters, glyceryl fatty acid esters, sodium lauryl sulfate and mixtures thereof.
Studies with rats were carried out to test the activity of lipophilic nutrients in diabetic eye complications with four samples viz water soluble composition of trans-lutein and zeaxanthin isomers (sold under the brand name UltraSol Lutemax2020™); concentrate containing trans-lutein and zeaxanthin isomers (sold under the brand name Lutemax2020®; water soluble composition containing curcumin (sold under the brand name UltraSol CurcuWin™) and curcumin powder.
Earlier studies demonstrated that Lutemax2020 delayed diabetic cataract in rats at 1% in the diet but not at (0.1%). Further Lutemax2020® (1%) only delayed but not completely prevented diabetic cataract and hence the water soluble composition of

trans-lutein and zeaxanthin isomers (UltraSol Lutemax2020™) was used to test further the effect of the said composition in prevention/treatment of diabetic eye complications such as diabetic cataract and diabetic retinopathy.
The following examples are given by the way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
To determine the effect of water soluble composition containing trans-lutein and zeaxanthin isomers and water soluble composition containing curcumin in comparison to regular composition containing trans-lutein and zeaxanthin isomers and regular composition containing curcumin in preventing or delaying diabetic cataract and. diabetic retinopathy the rats were induced diabetes by using streptozotocin (STZ).
Example 1
Effect of UltraSol Lutemax2020™ and UltraSol CurcuWin™ in diabetic
cataract
Experimental design
Male Wistar strain (WNIN) rats (2 months old; Average BW of 213±14 g) obtained from the National Center for Laboratory Animal Sciences, National Institute of Nutrition, Hyderabad, India (NCLAS, NIN). Animals were maintained at NCLAS. NIN and kept for acclimatization in experimental room for two weeks. Diabetes was induced in overnight fasted animals by a single intraperitoneal injection of STZ (30 mg/ kg) in 0.1 M citrate buffer, pH 4.5. Another set of rats, which received only vehicle, served as the control (Group I; n=12). Fasting blood glucose levels were measured 72 h after STZ injection. Animals having blood glucose levels >150 mg/dL were considered diabetic and those were only divided into five groups (Group II-

VI). A group of control rats (n=6) were fed with 0.01% soluble curcumin (Group VII) and soluble % lutein alone (Group VIII).
All the animals were housed in individual cages maintained on their respective diets for 12 weeks and drinking water was provided ad libitum throughout the study
period.
Table 1: Experimental groups and diets

Group Number of animals Diet
1 Control 12 AIN93
11 Diabetic 14 AIN93
III Diabetic +SC 12 A IN 93 with soluble curcumin (SC) 0.01 %
IV Diabetic +RC 12 A1N 93 with regular curcumin (RC) 0.01 %
V Diabetic+SL 12 AIN 93 with soluble lutein (SL) 0.5 %
VI Diabetic +RL 12 AIN 93 with regular lutein (RL) 0.5 %
VII Control +SC 6 AIN 93 with soluble curcumin (SC) 0.01 %
VIII Control + SL 6 AIN 93 with soluble lutein (SL) 0.5 %
Animal Care: Institutional and national guidelines for the care and use of animals were followed and all experimental procedures involving animals were approved by the IAEC (institutional animal ethical committee) of National Institute of Nutrition.
Animals were housed in individual cages in a temperature (22°C) and humidity
controlled room with a 12-h light/dark cycle. All the animals had free access to
water.
Food intake (daily) and body weights (weekly) were monitored.

Slit lamp examination and Cataract grading: Eyes were examined every week using a slit lamp bio microscope on dilated pupils. Initiation and progression of lens opacity was graded into five categories (0-4).
Mortality: During the course of the study, 3 animals in Group II and 2 animals each from Groups III-VI have died to hyperglycemia as expected.
Blood/ Lens collection and processing: Blood was drawn once in every week from retro orbital plexus for glucose and insulin estimation. At the end of 12 weeks, animals were sacrificed by C02 asphyxiation and lenses were dissected by posterior approach and stored at -70°C until further analysis. A 10% homogenate was prepared from 3-5 pooled lenses in 50 mM phosphate buffer, pH 7.4. All the biochemical parameters were analyzed in the soluble fraction of the lens homogenate (15,000x g at 4°C) except for lens malondialdehyde (MDA). which was determined in the total homogenate.
Biochemical estimations: Lens MDA, as thiobarbituric acid reacting substances (TBARS), protein carbonyl content were determined according to the methods described previously (3). Total, soluble and insoluble protein was assayed by Lowry method using BSA as standard.
Plasma lutein levels: Plasma lutein levels were measured by HPLC using 4.6 * 150 mm, 5 urn, spherisorb waters CI8 column connected to Dionex UltiMate 3000 Rapid Separation Liquid Chromatography (RSLC). The column was equilibrated with mobile phase, isocratic solvent mixture of acetonitrile: dichloroethane: methanol in ration of 70:20:10 (v/v) at flow rate of 0.5 ml/min at 25 °C. 2 u.1 of Plasma samples (extracted with hexane) were loaded on to column and lutein was detected at 300-600 nm.

SDS-PAGE and size exclusion chromatography of lens proteins: Subunit profile and cross-linking of soluble proteins were analyzed on 10% polyacrylamide in the presence of SDS under reducing conditions. Crystallin distribution in the soluble protein fraction was performed by size exclusion chromatography on a 600x7.5 mm TSK-G4000 SW column (TOSOH Co., Japan) using a HPLC system. The column was equilibrated with 0.1 M sodium phosphate buffer pH 6.7 containing 0.1 M sodium chloride at a flow rate of 1 ml/min.
Statistical analysis: One-way ANOVA was used for testing statistical significance between groups of data and individual pair difference was tested by means of Duncan's multiple-range test. Heterogeneity of variance was tested by the nonparametric Mann Whitney test where p < 0.05 was considered as significant.
Results
Fasting blood glucose: Figure 1 summarizes the results of fasting plasma glucose in different groups of animals throughout the treatment period. The plasma glucose concentrations of the diabetic control rats were significantly higher than those of the non-diabetic control rats throughout the experiment. Though there was lower mean fasting plasma glucose levels observed in groups treated with SC and SL but there is no significant effect of treatment on plasma glucose in diabetic rats was observed.
Figure 1: Effects of treatment on fasting plasma glucose in STZ-induced diabetic
rats. (Figure 1 is shown in the drawings accompanied with the specification). The
data is expressed as mean ± SEM. Control (Non-diabetic control); D (Diabetic
control); D+RL (diabetic + regular lutein): D+SL (Diabetic + soluble lutein);
D+RC
(Diabetic + regular curcumin); D+SC (Diabetic + soluble curcumin) ***=p<0.001.

Cataract development and progression: Onset and progression of cataract is monitoring by slit lamp biomicroscope examination as described below; Eyes were, examined every week using a slit lamp biomicroscope (Kowa SL15, Portable. Japan) on dilated pupils. Initiation and progression of lenticular opacity was graded into five categories as follows: clear, clear lenses and no vacuoles present; stage 1, vacuoles cover approximately one half of the surface of the anterior pole, forming a subcapsular cataract; stage 2. some vacuoles have disappeared and the cortex exhibits a hazy opacity; stage 3, a hazy cortex remained and dense nuclear opacity is present; and stage 4, a mature cataract is observed as a dense opacity in both cortex and nucleus (Figure 2).
Figure 2: Delay of diabetic cataract in rats by treatment. (Figure 2 is shown in the drawings accompanied with the specification). The data is expressed as mean ± SEM. Control (Non-diabetic control); D (Diabetic control); D+RL (diabetic + regular lutein); D+SL (Diabetic + soluble lutein); D+RC (Diabetic + regular curcumin); D+SC (Diabetic + soluble curcumin)
The onset of cataract due to hyperglycemia was observed in diabetic animals after three weeks of STZ injection. The average incidence of cataract was calculated and presented in Figure 2. Though there was no delay in the onset there is a clear delay in the progression and maturation of cataract in all the treatment groups when compared to Group-D. Group-D animals showed lens opacification
(stage-IV) by the end of 10th week while the treatment groups showed around stage-2.5 to 3. Our data clearly indicates there is a significant delay in the progression and maturation of cataract intervention groups from sixth week onwards when compared to group-D. At the end of ten weeks; the severity of cataracts was significantly lower in groups D+RL (stage 3.1), D+SL (stage 2.7), D+RC (stage 3.0) and D+SC (stage 3.2) than in Group-D (Stage 4), indicating that

intervention with any agent delayed the maturation of diabetic cataract due to slow progression. Further SL seems to be more effective than RL but SC has not shown any superiority in efficacy over RC in progression of cataract. All the lenses in Group-C during the entire experimental period appeared to be normal, clear and free of opacities.
LENS BIOCHEMICAL ANALYSIS:
Individual lenses were weighed and pooled in to 4 lenses for a pool and 4-5 pools per group. A 10 % homogenate was prepared in 50 mM sodium phosphate buffer pH 7.4, with tissue homogenizer with intermittent time gaps to avoid excess heat generation. Separate aliquots of total homogenate (TH) were made 250 ul for TBARS assay, 150 ul for sorbitol estimation and 20 ul for protein estimation. Remaining homogenate was centrifuged at 10,000 RPM for 30 min at 4 °C. Supernatant was separated in to labelled vials, as total soluble protein (TSP).
Determining soluble percentage of protein in lens homogenate: Protein estimation was done in lens homogenate and soluble fraction by Lowry's method. Amount of protein present per gram weight of lens was calculated. Percentage of soluble protein was calculated by the multiplying the fraction of soluble protein
with 100.
We analyzed the total and soluble protein content in the lenses of all the experimental groups. There was a significant decrease in both total and soluble protein in Group-D compared with the control group. This could be due to a partial leakage of proteins into the aqueous humor or aggregation of proteins and ^solubilization. Among the treatment groups SL and RC significantly prevented of loss of soluble protein compared to group- D. whereas, SL alone had shown significant difference against group D in percentage of soluble protein. Though SC and RL had shown partial beneficial effect in preventing insolubilization of lens

proteins but were not significant statistically.
Table 2: Protein content in total and soluble fraction of lens homogenate. The data is expressed as mean ± SEM. n=6; Control (Non-diabetic control); D (Diabetic control); D+RL (diabetic + regular lutein); D+SL (Diabetic + soluble lutein); D+RC (Diabetic + regular curcumin); D+SC (Diabetic + soluble curcumin); ***=p<0.00.1,**=P<0.01 and *=P<0.05 Vs C; U= PO.01 and #= P<0.05 Vs D
Table: 2

Group Total protein (mg/gm lens) Soluble protein (mg/gm lens) Percentage soluble protein
Control (C) 516.54+9.3 388.77+8.2 75.19+2.2
D 281.34+31.0*** 128.87+11.0*** 46.87+2.5***
D+RL 311.79+7.8 156.55+4.7 50.35+1.9
D+SL 376.53+20.9 218.78+14.3 58.04+1.8 #
D+RC 368.50+19.2 202.63+23.6 # 54.43+3.8
D+SC 351.83+41.5 178.99+25.9 50.16+1.3
SDS-PAGE protein profiling: Differences in protein distribution pattern were observed by running the lens protein samples on 12% polyacrylamide gel. 30 u,g of protein was loaded per well along with molecular weight marker for SDS-PAGE (Broad range SDS-PAGE marker, BioRad). The SDS-electrophoretic pattern of the soluble protein fraction showed a band corresponding to aggregated proteins at~50 kDa in group-D in relation to the group-C and with reduced band intensity

in treatment groups RL, SL, RC and SC. Figure 3 represents the SDS-PAGE pattern of soluble fraction of lens proteins, as shown in the drawings accompanied with the specification.
SIZE EXCLUSION HIGH PERFORMANCE LIQUID CHROMATOGRAPHY:
Size exclusion chromatography on TSK-3000 HPLC column resulted in resolution of crystalline proteins of lens. HPLC profile demonstrated the reduced peak area in low molecular weight region and whereas increased peak area in the high molecular weight protein region in Group-D (red line) TSP compared to group-C (black line). This suggests there was a phenomenon of protein aggregation in diabetic conditions. Intervention with all except RL i.e. SL, RC and SC normalized the profile of TSP. Figure 4 represents the size exclusion chromatography of lens protein soluble fraction, as shown in the drawings accompanied with the specification.
SORBITOL LEVELS: We assessed accumulation of sorbitol in the lens of all experimental animals and the data were presented in the figure 8. Group-D showed significantly elevated levels of sorbitol (5.877 ± 0.27) when compared with group-C (0.301 ± 0.04 u moles/gm lens of sorbitol). Among the intervention groups, except SC, remaining treatments has not lowered sorbitol accumulation compared to group-D. Group-SC showed significantly lower sorbitol levels when compared to group-D and significantly higher when compared with group-C. This might be attributed to the additional pharmacological action of curcumin as aldose reductase inhibition.
Figure 5: Spectrofluoremetric measurement of lens sorbitol (Figure 5 is shown in the drawings accompanied with the specification).The data is expressed as mean ± SEM. n=6; Control (Non-diabetic control); D (Diabetic control); D+RL (diabetic

+ regular lutein); D+SL (Diabetic + soluble lutein); D+RC (Diabetic + regular curcumin): D+SC (Diabetic + soluble curcumin); *=P<0.05 Vs C; # = PO.05 Vs D.
Plasma lutein levels: Plasma lutein levels were measured by HPLC. Since rodent chow diet does not contain carotenoids, lutein was not detected in control and diabetic rats. However, lutein could be detected in lutein supplemented groups. Feeding of diabetic rats with regular lutein results in 0.01 micromoles/L of plasma lutein (Figure 6). Very interestingly, feeding of soluble lutein led to seven fold increase in plasma lutein 0.07 micromoles (Figure 6) suggesting that soluble lutein increases bioavailability of lutein significantly and that might be the reason for improved beneficial effects with soluble lutein compared to regular lutein.
Figure 6: HPLC measurement of plasma lutein levels (Figure 6 is shown in the drawings accompanied with the specification). The data were expressed as mean ± SEM. n=6; Control (Non-diabetic control); D (Diabetic control); D+RL (diabetic + regular lutein); D+SL (Diabetic + soluble lutein).
Conclusion
Supplementation of curcumin rescued photoreceptor degeneration in transgenic rats with P23H rhodopsin mutation. Feeding of dietary antioxidant curcumin was effective in delaying streptozotocin (STZ)-induced diabetic cataract in rats mainly through its antioxidant property. In addition, curcumin inhibited diabetes-induced expression of vascular endothelial growth factor (VEGF) in rat retina and lens aldose reductase (AR).
Soluble lutein is more effective in delaying diabetic cataract compared to regular lutein at dose of 0.5% in the diet which is reflected in molecular analysis related to

cataract genesis. Increased bioavailability of soluble lutein might explain the observed biological effects of soluble lutein compared to regular lutein. However, the efficacy of soluble curcumin was almost comparable with regular curcumin.
Example 2
Effect of UltraSol Lutemax2020 in diabetic retinopathy by Nutrigenomics
approach
The effect of soluble lutein (UltraSol Lutemax2020 TM) was investigated in diabetic rats with respect to its beneficial effect on retina by nutrigenomics approach and the effect was compared with regular lutein (Lutemax2020lj.
Methodology
Animal model: The streprtozotocin (STZ) rat model of diabetes has been one of the most commonly used models of human disease with respect to diabetes. It is known to mimic many of the acute and some of the chronic complications observed in human diabetes. This model has the advantage of being highly reproducible and the time lines for various complications to develop are well recognized and reproducible. Given the established similarities of some of the structural, functional and biochemical abnormalities to human disease, it is considered an appropriate model to assess mechanisms of diabetes and evaluate potential therapies.
Experimental design: Male Wistar-NIN rats with an average body weight of 120 gms are obtained from the National center for laboratory animal sciences, National Institute of Nutrition. Hyderabad (NCLAS, NIN). Animals were maintained at NCLAS, NIN and kept for acclimatization in experimental room for two weeks. Diabetes was induced in overnight fasted animals by a single intraperitoneal injection of STZ (30 mg/ kg) in 0.1 M citrate buffer, pH 4.5. Another set of rats, which received only vehicle, served as the control (Group-C; n=6). Fasting blood

glucose levels were measured 72 h after STZ injection. Animals having blood glucose levels >150 mg/dL were considered diabetic and all the animals are divided in to four groups as shown in the table 3 below.
Table 3

Group No. of animals Diet
I Control 6 AIN 93
II Diabetic 9 A1N93
III Diabetic + Soluble lutein (SL) 8 AIN 93 with soluble lutein 0.5 %
IV Diabetic + Regular lutein (RL) 6 AIN 93 with regular lutein 0.5 %
All the animals were housed in individual cages maintained on their respective diets for 12 weeks and drinking water was provided ad libitum throughout the study period. Daily food intake and weekly body weights, fasting glucose levels were noted. Before sacrifice electro retinogram was performed and glycosylated hemoglobin (HbAlC) was estimated. At the end of 12 weeks rats were euthanized and retinas harvested for histological and molecular analysis (gene and protein expression).
Electeroretinogram (ERG) Analysis: Diabetic retinoapthy is characterized by-disturbances in retinal function. The function of retina can be assessed by electroretinogram. Diabetes results in ischemia and apoptosis in different retinal cell layers this result in changes in the functions of the retina. It is well reported that OPs are more affected in diabetes than a- or b- waves. OPs represent the functional aspects of inner retinal layers, ganglion cell layer and inner plexiform layer.

Animals were dark-adapted for overnight and prepared for the ERG procedure under dim red illumination. The pupils of the rats were dilated with atropine eye drops. The ground electrode was a subcutaneous needle in the tail, and the reference electrode was an ear clip electrode. The active contact lens electrodes were placed on the cornea. The recordings were performed with a UTAS Visual Diagnostic System. The responses were differentially amplified with a gain of 1,000 using alternating current-coupled UBA-4204 Amplifier. A flash stimuli of -2 to 8 dB were delivered via a with BigShot™ Ganzfeld System (LKC Technologies; Gaithersburg, MD, USA). The oscillatory potentials were extracted from the wave form and the sum of all OPs was calculated.
Quantitative real-time PCR: Total RNA was extracted from the retina of rats using Tri reagent. Isolated RNA was further purified by RNeasy Mini Kit (Qiagen) and quantified by measuring the absorbance at 260 and 280 nm on ND1000 spectrophotometer (NanoDrop technologies, Delaware, USA). The quality of RNA preparation was assessed by electrophoresis on a denaturing agarose gel. Two \xg of total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription kit. Reverse transcription reaction was carried out with thermocylcer (ABI 9700). Real-time PCR (ABI-7500) was performed in triplicates with 25ng cDNA templates using SYBR green master mix with gene specific primers. Normalization and validation of data were carried using p-actin as an internal control and data were compared, between samples according to comparative threshold cycle (2-∆∆ct) method.
SDS-PAGE and Immunobtotting:Retma. were homogenized in a buffer containing 20 mMTris, 100 mMNaCl, 1 mM EDTA (TNE buffer; pH7.5) containing 1 mM DTT, 1 mM PMSF, 1 μg/ml of each aprotinin. leupeptin, and pepstatin. The homogenate was centrifuged at 12,000xg for 20 min. The supernatant was collected and used for immunoblot analysis. Equal amount of protein from was subjected to

12% SDS-PAGE and proteins were transferred onto PVDF membrane. Nonspecific binding was blocked with 5% BLOT-QuickBlocker reagent (WB57, Calbiochem) in PBST and incubated overnight at 4°C with primary antibodies diluted in PBS. After washing with PBST, membranes were then incubated with anti-rabbit IgG (1:3500) or anti-mouse IgG (1:3500) (which one is appropriate) secondary antibodies conjugated to HRP. The immunoblots were developed with enhanced chemiluminescence detection reagents (RPN2232, GE Health Care, Buckinghamshire, UK) and digital images were recorded by Image analyzer (Syngene, G-box). Quantification of band intensity was performed with Image J software.
Histopathology: The eye balls from selected animals were collected in fixative 4 % parafarmaldehyde solution in separately labeled vials. The tissues were given some nicks so as to facilitate penetration of fixative into deep tissue. They were kept at room temperature for 24-48 hrs fallowed replacing the fixative with 20 mM sodium phosphate buffer. Buffer was scheduled to replace with fresh buffer weekly once till histopathological processing. Tissues were embedded in paraffin and sections were taken in microtome. Coated slides are used for immunihistochemistry and immuofluorescence whereas uncoated slides for H & E staining.
Statistical Analysis: The data were expressed as mean ± SEM. n=6; C (Non-diabetic control); D (Diabetic control); D+RL (diabetic + regular lutein); D+SL (Diabetic + soluble lutein); *** =p<0.001, ** =P<0.01 and *=P<0.05 Vs C; ## = PO.01 and # = PO.05 Vs D.
Results were analysed for statistical significance by one way ANOVA followed by Dunnett's multiple comparison test for comparing all the groups with control group. Between group significance was checked by two tailed unpaired t-test.

Results
Electroretinograph: In diabetic rats (D) amplitude of OPs were reduced (334.2 uV) compared to normal control (C) animals (498.4 uV). It is also noted that implicit time for OPs is increased in group-D. Ingestion of antioxidant lutein resulted in lowering the reduction in OP amplitudes suggested by sum of OPs; RL (442.6) and SL (561.9). Group-SL had shown significant difference in sum of OPs compared to group-D, in fact it is better than normal control rats.
Figure 7: Representative wave forms of OPs from different groups and total amplitudes (Figure 7 is shown in the drawings accompanied with the specification). C, Non diabetic control; D, Diabetic control; D+RL, Diabetes+Regular Lutein; D+SL, Diabetes+Soluble Lutein.
Morphology of retina: In control rats C, all the layers of the retina are intact and with maximum thickness of retina and also noted with dense INL, and distinguishable separation (OPL) between INL and ONL. In contrast diabetic retina (D), is with significantly reduced total retinal thickness and is also marked by less dense ONL and almost merged INL and ONLs. Treatment with lutein prevented gross morphological changes to a significant extent in diabetic retina. However, soluble formulation of lutein was shown to be better than the regular lutein, indicated by dense ONLs (Fig 8). Figure 8 represents histology of retina, as shown in the drawings accompanied with the specification.

Table 4: Thickness of layers of retina (u.) of different groups.

Retinal layers Total retinal thickness GCL+1PL INL OPL ONL PRL
C 121.74 46.813 10.767 5.024 25.697 19.426
D 58.848 12.508 9.257 0.791 23.052 12.184
RL 89.409 31.518 12.117 1.41 23.181 12.600
SL 112.714 40.877 16.074 4.718 31.196 23.072
A representative table of retinal layers thickness (in μm) measured by the use of Lieca application suit, Leica, Switzerland. C, Non diabetic control; D, Diabetic control; D+RL, Diabetes+Regular Lutein; D+SL, Diabetes+Soluble Lutein; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photo receptor layer.
Expression of retinal markers (genes) by real-time PCR, imunohistochemistochemistry and immonoblotting:
Rhodopsin (Rho): Rhodopsin (Rho) is a biological pigment in photoreceptor cells of the retina that is responsible for the first events in the perception of light. The mRNA levels of Rho gene as quantified by real time PCR showed decreased levels in the retina of diabetic animals. Treatment with RL and SL prevented its decline and however feeding of SL had significant effect when compared to RC and even better than normal rats (Fig.9A, as shown in the drawings accompanied with the specification). Further, we quantified protein level of Rho by immunofluorescence, which is in coincidence with mRNA level. Immunofluorescence imaging of Rho protein showed decreased expression in diabetic rat retina in comparison to normal control rat retina. Treatment with RL and SL prevented loss of Rho protein expression in diabetic retina indicated by intensed Rho positive fluorescence. Furthermore, SL is found to be distinctly more effective than RL in preventing loss

of Rho protein expression in rat retina in diabetic animals respectively (Fig. 9B. as shown in the drawings accompanied with the specification).
Nerve Growth Factor (NGF): NGF is the best-characterized neurotrophin, known to play a key role in the survival and differentiation of select neurons in the peripheral and central nervous system. Since its discovery in the 1950s, NGF has shown promise in the treatment of progressive neurodegenerative disorders. In animals, NGF is known to promote nerve terminal outgrowth and neuron recovery after ischemic, traumatic, and toxic injuries. We checked the status of Ngf gene expression by real time PCR and found down regulation in the retinas of diabetic animals. Treatment with RL could not prevent its decline whereas SL prevented it's down regulation (Fig.lOA, as shown in the drawings accompanied with the specification). The protein levels of NGF as estimated by immunofluorescence yielded same results. Immunofluorescence imaging of NGF protein showed its decreased expression in diabetic rat retina in comparison to normal control rat retina (Fig.lOB. as shown in the drawings accompanied with the specification). Treatment with SL prevented loss of NGF protein in diabetic retina indicated by intense NGF positive fluorescence. Whereas RL unable to prevent the loss of NGF protein effectively in diabetic rat retina.
Vascular endothelial growth factor (VEGF): Vascular endothelial growth factor (VEGF) is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. It is part of the system that restores the oxygen supply to tissues when blood circulation is inadequate. When VEGF is over expressed, it can contribute to disease. VEGF causes widespread retinal vascular dilation, produces breakdown of the blood-retinal barrier, and is implicated in ocular neovascularization. Western blotting for VEGF indicated the up regulation of VEGF expression in diabetic retina (Fig. 11, as shown in the drawings accompanied with the

specification). Treatment with both RL and SL inhibited diabetes induced VEGF over expression.
Platelet-derived growth factor (PDGF): PDGF is a growth factor that plays a significant role in blood vessel formation (angiogenesis), the growth of blood vessels from already-existing blood vessel tissue. Several studies have shown elevated PDGF concentrations in vitreous samples from patients with diabetic retinopathy. Like VEGF, PDGF is a proangiogenic growth factor that may promote aberrant neovascularization in diabetic retinopathy. Furthermore, PDGF may stimulate the formation and traction of epiretinal membranes in patients with diabetic retinopathy, leading to tractional retinal detachment. Indeed, the development of inhibitors that antagonize PDGF signaling in pathologic retinal neovascularization remains an active area of ophthalmic drug development. Immunohistochemistry for PDGF indicated the up regulation of protein in diabetic retina (Fig. 12, as shown in the drawings accompanied with the specification). Treatment with both RL and SL inhibited diabetes induced PDGF over expression.
Plasma lutein; To understand the effect of soluble lutein administration on its bioavailability we measured plasma levels of carotenoids by HPLC method. The data showed undetectable lutein levels in plasma of rats fed with normal diet (AIN-93). The plasma lutein levels were in detectable range in rats fed with AIN-93 diets mixed with regular lutein and soluble lutein. More interestingly, rats fed with SL diet were found to contain seven folds higher lutein levels compared with rats fed with RL diet. This in particular confirmed the success of this formulation in improving the bioavailability of lutein.
Figure 13 represents Serum lutein levels measured by RP-HPLC, as shown in the drawings accompanied with the specification. Values are expressed as mean ± SEM (n=6)., $$$<0.001 D+SL Vs. D+RL.

Conclusion
Lutein administration to rats prevented diabetes induced abnormalities in the retina. Lutein retained the functionality of retina of rats which is lost in diabetic rats as checked by ERG. It is also evident by the morphological study of retina as done by H & E staining. Lutein prevented decline in the expression of rhodopsin and nerve growth factor which have vital role in maintaining health of the retina. Lutein prevented over expression of VEGF and PDGF that are involved in stress and angiogenesis. Interestingly rats treated with soluble lutein showed profound benefit when compared with regular lutein and the reason can be.easily answered as increased bioavailability as shown by increased plasma levels. Further, the antioxidant and anti-inflammatory potential of lutein may contribute for its beneficial effect. Hence soluble lutein can be used to treat and or prevent diabetic retinopathy.
Example 3 Effect of UltraSol CurcuWin ' in diabetic retinopathy by Nutrigenomics
approach
The effect of soluble curcumin (UltraSol CurcuWin™) was investigated in diabetic rats with respect to its beneficial effect on retina by nutrigenomics approach and the effect was compared with regular curcumin.
Methodology
Same as mentioned in example 2. All the animals were divided into four groups as
shown below

Table 5

Group No. of animals Diet
1 Control 6 AIN93
II Diabetic 9 AIN93
III Diabetic + Soluble lutein (SC) 8 AIN 93 with soluble curcumin 0.01 %
IV Diabetic + Regular lutein (RC) 6 AIN 93 with regular curcumin 0.01 %
Ekctroretinograph: In diabetic rats (D) amplitude of OPs were reduced (334.2 uV) compared to normal control (C) animals. It is also noted that implicit time for OPs is increased in group-D. Ingestion of antioxidant curcumin resulted in lowering the reduction in OP amplitudes suggested by sum of OPs; RC (445.7), SC (455.3). SC not only lowered the reduction in sum of OPs but also normalized the implicit times.
Figure 14 represents wave forms of OPs from individual animals of different groups and total amplitudes, as shown in the drawings accompanied with the specification. C, Non diabetic control; D, Diabetic control; D+RC, Diabetes+Regular Curcumin; D+SC, Diabetes+Soluble Curcumin.
Morphology of Retina: In control rats C, all the layers of the retina are intact and with maximum thickness of retina and also noted with dense INL, and distinguishable separation (OPL) between INL and ONL. In contrast diabetic rats retina (D), is with significantly reduced total retinal thickness and is also marked by less dense ONL and almost merged INL and ONLs. Treatment with curcumin prevented gross morphological changes to a significant extent in diabetic retina. However, soluble formulation of this active principle was shown to be better than the regular active principle, indicated by dense ONLs.

Figure 15, Representative histology of retina, as shown in the drawings accompanied with the specification.
Table 6: Thickness of layers of retina (μ) of different groups.

Retinal layers Total retinal thickness GCL+IPL INL OPL ONL PRL
C 121.74 46.813 10.767 5.024 25.697 19.426
D 58.848 12.508 9.257 0.791 23.052 12.184
RC 106.014 35.692 13.648 5.685 30.108 19.520
SC 124.430 54.004 14.926 6.597 29.320 21.857
A representative table of retinal layers thickness (in μm) measured by the use of Lieca application suit, Leica, Switzerland. C, Non diabetic control; D, Diabetic control; D+RC, Diabetes+Regular Curcumin; D+SC, Diabetes+Soluble Curcumin. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photo receptor layer.
Expression of retinal markers (genes) by real-time PCR, immunohistochemistry and immunoblotting:
R/todopsin (Rlw): Rhodopsin. is a biological pigment in photoreceptor cells of the retina that is responsible for the first events in the perception of light. The mRNA levels of Rho gene as quantified by real time PCR showed decreased levels in the retina of diabetic animals. Treatment with RC and SC prevented its decline and however SC treatment has more beneficiary effect when compared to RC (Fig. 16.A, as shown in the drawings accompanied with the specification). We even quantified protein level of Rho by immunofluorescence, which is in coincidence with mRNA level. Immunofluorescence imaging of Rho protein showed its decreased expression in diabetic rat retina in comparison to normal control rat retina. Treatment with RC and SC prevented loss of Rho protein expression in diabetic retina indicated by intensed Rho positive fluorescence. Furthermore, SC is found to be more effective

than RC in preventing loss of Rho protein expression in rat retina in diabetic animals respectively (Fig. 16.B, as shown in the drawings accompanied with the
specification).
Nerve Growth Factor (NGF): NGF is the best-characterized neurotrophin, known to play a key role in the survival and differentiation of select neurons in the peripheral and central nervous system. Since its discovery in the 1950s. NGF has shown promise in the treatment of progressive neurodegenerative disorders. In animals, NGF is known to promote nerve terminal outgrowth and neuron recovery after ischemic, traumatic, and toxic injuries. We checked the status of Ngf gene expression by real time PCR and found down regulation in the retinas of diabetic animals. Treatment with both regular as well as soluble curcumin showed equal beneficiary effect in preventing down regulation of Ngf gene under diabetic conditions (Fig. 17.A. as shown in the drawings accompanied with the specification). The protein levels of NGF as estimated by immunofluorescence yielded same results. Immunofluorescence imaging of NGF protein showed its decreased expression in diabetic rat retina in comparison to normal control rat retina (Fig.H.B, as shown in the drawings accompanied with the specification). Treatment with RC and SC prevented loss of NGF protein in diabetic retina indicated by intense NGF positive fluorescence.
Vascular endothelial growth factor (VEGF):
Vascular endothelial growth factor (VEGF) is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. It is part of the system that restores the oxygen supply to tissues when blood circulation is inadequate. When VEGF is over expressed, it can contribute to disease. VEGF causes widespread retinal vascular dilation, produces breakdown of the blood-ret*nal barrier, and is implicated in ocular neovascularization.

Western blotting for VEGF indicated the up regulation of VEGF expression in diabetic retina (Fig. 18, as shown in the drawings accompanied with the specification). Treatment with both RL and SL inhibited diabetes induced VEGF over expression.
Platelet-derived growth factor (PDGF): PDGF is a growth factor that regulates cell growth and division. In particular, it plays a significant role in blood vessel formation (angiogenesis), the growth of blood vessels from already-existing blood vessel tissue. Several studies have shown elevated PDGF concentrations in vitreous samples from patients with diabetic retinopathy. Like VEGF, PDGF is a proangiogenic growth factor that may promote aberrant neovascularization in diabetic retinopathy. Furthermore, PDGF may stimulate the formation and traction of epiretinal membranes in patients with diabetic retinopathy, leading to tractional retinal detachment. Indeed, the development of inhibitors that antagonize PDGF signaling in pathologic retinal neovascularization remains an active area of ophthalmic.drug development. Immunohistochemistry for PDGF indicated the up regulation of protein in diabetic retina (Fig. 19, as shown in the drawings accompanied with the specification). Treatment with both RC and SC inhibited diabetes induced PDGF over expression. Soluble curcumin was more effective than regular curcumin (Fig. 19, as shown in the drawings accompanied with the specification).
Conclusion
Curcumin administration to rats prevented diabetes induced abnormalities in the retina. Curcumin retained the functionality of retina of rats which is lost in diabetic rats as checked by ERG. It is also evident by the morphological study of retina as evaluated by H & E staining where thickness of various layers of retina was measured. Curcumin prevented decline in the expression of rhodopsin and nerve growth factor which have vital role. Curcumin prevented over expression of

VEGF and PDGF that are involved in stress and angiogenesis. Interestingly rats treated with soluble curcumin showed profound benefit when compared with regular curcumin and this might be due to increased bioavailability. Hence soluble curcumin can be used to treat and or prevent diabetic retinopathy.
Advantages of the invention
1. Provides dry free flowing, water soluble/miscible form of lipophilic nutrient.
2. Provides a suitable method of delivering poorly water soluble, oily, lipophilic nutrient in the form of granule, powder, tablets, ointment, paste, mouth wash, gargle, sachet, capsules or in to beverages.

4. Provides a dosage form of poorly water soluble, oily, lipophlic nutrient with high bioavailability.
5. The said composition is effective to regulate blood glucose levels.

6. The said composition containing lipophilic nutrients is effective to regulate amacrine cells dysfunction in diabetic retinopathy.
7. The said composition is effective to prevent loss of retinal layers, Rhodopsin levels and NGF protein levels in diabetic retinopathy.
8. The said composition is effective to inhibit diabetes induced PDGF over expression in diabetic retinopathy.
9 The said composition is effective to prevent accumulation of in-soluble lens proteins in diabetic cataract.

10. The said composition is effective to prevent accumulation of sorbital levels in lens in diabetic cataract.
11. The said composition is effective to reduce protein aggregation and normalize the profile of total soluble protein in diabetic cataract.

We claim:
1. A method of delaying the development and maturation of eye related complications of diabetes by administering a composition containing lipophilic nutrients which is safe for human consumption and is particularly useful as a dietary supplement for nutrition and health promoting benefits.
2. The method of Claim 1, wherein the eye related complications of diabetes is cataract and retinopathy.
3. The method as claimed in any of the presiding claims, wherein the lipophilic nutrients are selected from a group comprising lutein and its isomers, lutein ester, zeaxanthin isomers, turmeric extract, curcumin, ginger, and the like and the mixtures thereof.
4. The method as claimed in any of the presiding claims, wherein the said composition is effective to regulate blood glucose levels.
5. The method as claimed in any of the presiding claims, wherein said composition is effective to regulate glycated hemoglobin (HbAlc) levels.
6. The method as claimed in any of the presiding claims, wherein said composition is effective to regulate amacrine cells dysfunction in diabetic retinopathy.
7. The method as claimed in any of the presiding claims, wherein the said composition is effective to prevent loss of retinal layers, Rhodopsin levels and NGF protein levels in diabetic retinopathy.

8. The method as claimed in any of the presiding claims, wherein the said
composition is effective to inhibit diabetes induced PDGF over expression in
diabetic retinopathy.
9. The method as claimed in any of the presiding claims, wherein the said composition is effective to prevent accumulation of in-soluble lens proteins in diabetic cataract.
10. The method as claimed in any of the presiding claims, wherein the said composition is effective to prevent accumulation of sorbitol levels in lens in diabetic cataract.
11. The method as claimed in any of the presiding claims, wherein the said composition is effective to reduce protein aggregation and normalize the profile of total soluble protein in diabetic cataract.
12. The method as claimed in any of the presiding claims, wherein the composition
comprises lipophilic nutrients; stabilizer; water soluble hydrophilic carrier and optionally a surfactant.
13. The method as claimed in any of the presiding claims, wherein the composition
contains at least 80% by weight of total xanthophylls, out of which the trans-
lutein content is 80-95% w/w; (R,R)-zeaxanthin is 14-20% w/w; (R,S)-
zeaxanthin is 0.01-1% w/w or trans-lutein content is 80-95% w/w; (R,R)-
zeaxanthin is 14-20%) w/w and traces of other carotenoids derived from the plant
extracts/oleoresin containing xanthophylls/xanthophylls esters or curcumin
which contains 5-95%o of curcuminoids.

14. The method as claimed in any of the presiding claims, wherein the stabilizer is selected from Ascorbic acid, BHA, BHT, ascorbyl palmitate, rosemary extract, mixed natural tocopherols, alpha tocopheryl acetate, sodium ascorbate, castor oil derivatives, sodium lauryl sulfate and mixtures thereof.
15. The method as claimed in any of the presiding claims, wherein the carrier used is selected from polyethylene glycol 200, polyethylene glycol 400, ethylene glycol, propylene glycol, glycerol, sorbitol, glucose syrup, corn steep liquor, mannitol, polyethylene glycol 6000, polyethylene glycol 10000, Polyethylene glycol 20000, polyvinyl pyrrolidone, hydroxyl propyl methyl cellulose. sucrose, glucose, sodium chloride, hydroxyl propyl cellulose, polyvinyl alcohol, soluble starch, hydrolyzed starch and mixtures thereof.
16. The method as claimed in any of the presiding claims, wherein the said surfactant is selected from a group comprising of polysorbate 20, polysorbate 60, polysorbate 80, lecithin, sucrose fatty acid esters, glyceryl fatty acid esters, sodium lauryl sulfate and mixtures thereof.
17. The method as claimed in any of the presiding claims, wherein the said composition containing lipophilic nutrients are in the form of powders, tablets, capsules, sachets, beadlets, microencapsulated powders, oil suspensions, liquid dispersions, pellets, soft gel capsules, chewable tablets or liquid preparations.