Orally stable lipophilic nutrients composition for ophthalmic care

Abstract

The present invention relates to a stable, bioavailable, oral lipophilic nutrient composition comprising RR-zeaxanthin, curcuminoids, and / or lutein, along with nutraceutically acceptable excipients. In particular, the present invention provides a stable, bioavailable liquid oral composition comprising zeaxanthin isomers, curcuminoids, and / or lutein. The stable, bioavailable lipophilic nutrient composition of R, R-zeaxanthin, curcuminoids, and / or lutein is used for ophthalmic care, preferably for age-related eye diseases (AREDS), Age-related Macular degeneration (AMD), Visual dysfunction, Retinopathy, and other Eye diseases and disorders. The present invention further provides a process for preparing a nutraceutically stable and bioavailable lipophilic nutrient composition comprising zeaxanthin isomers, curcuminoids, and / or lutein.

Description

[0001] ORALLY STABLE LIPOPHILIC NUTRIENTS COMPOSITION FOR OPHTHALMIC CARE

[0002] FIELD OF THE INVENTION

[0003]

[0001] The present invention relates to a stable, bioavailable, oral lipophilic nutrient composition comprising carotenoids and polyphenols. The lipophilic nutrient composition of the present invention comprises zeaxanthin, curcuminoids, and / or lutein along with nutraceutically acceptable excipients. In particular, the present invention relates to an oral liquid oil suspension composition of zeaxanthin isomers, curcuminoids, and / or lutein.

[0004]

[0002] The present invention further relates to the use of a stable and bioavailable oral lipophilic nutrient composition of zeaxanthin isomers, curcuminoids, and / or lutein for ophthalmic care, preferably age-related eye diseases (AREDS), Age-related Macular degeneration (AMD), Visual dysfunction, Retinopathy, and other Eye diseases and disorders.

[0005]

[0003] The present invention also relates to a process for the preparation of a stable, bioavailable oral lipophilic nutrient composition of zeaxanthin isomers, curcuminoids, and / or lutein.

[0006] BACKGROUND OF THE INVENTION

[0007]

[0004] Zeaxanthin is a xanthophyll, which is a type of oxygenated carotenoid, which has no provitamin A activity. It is a structural isomer of lutein. Chemically, zeaxanthin is known as (all-E)-l,l'-(3,7,12,16-tetramethyl-l,3,5,7,9,ll,13,15,17- octadecanonaene-l,18-diyl) bis[2,6,6-trimethylcyclohexene-3-ol] and synonyms are 3R,3'R-beta, beta-carotene-3, 3'-diol; all-trans-p-carotene-3,3’-diol; (3R,3’R)-dihydroxy-p-carotene; zeaxanthol; anchovyxanthin. Zeaxanthin has 11 conjugated double bonds, which are distributed between the ionone rings and the polyene chain and two hydroxyl groups, which are more polar and hydrophobic than other carotenoids. In principle, the polyene chain double bonds present in zeaxanthin can exist in a cis or trans conformation.

[0008]

[0005] Zeaxanthin can be produced synthetically by the Wittig reaction or by extraction and subsequent purification by saponification and crystallization from Marigold (Tagetes erecta) red flowers / Paprika (Capsicum annuum L). Products obtained by these two processes are of fundamentally different compositions, concerning the content of zeaxanthin, by-products and impurities. Intended use of zeaxanthin is as a colour and a nutritional supplement in a wide range of foods such as baked goods, beverages, breakfast cereals, chewing gum, egg products, fats and oils, gravies and sauces, hard and soft candy, infant and toddler foods (other than infant formula), milk products, processed fruits and fruit juices, soups and soup mixes in levels range from 0.50 to 70 mg / kg. Zeaxanthin is an orange-red crystalline powder and is practically insoluble in water, sparingly soluble in ethanol and soluble in chloroform.

[0009]

[0006] The chemical formula of zeaxanthin is C40H56O2, and its molecular weight is 568.87g / mol. The chemical structure is of R, R-Zeaxanthin is as follows -

[0010]

[0007] Curcumin is a yellow-orange compound extracted from curcuma rhizomes, especially Curcuma longa L, which belongs to Zingiberaceae family obtained through extraction with solvent and extract purification through crystallization. Curcumin is also known as turmeric yellow, kurkum, diferuloylmethane, kachahaldi, haldi, natural yellow 3 etc, haridara and INS No. 100(i).

[0011]

[0008] Chemically, curcumin is known as (lE,6E)-l,7-bis(4-hydroxy-3-methoxyphenyl) hepta- l,6-diene-3, 5-dione. Curcumin consist of curcuminoids like is Desmethoxycurcumin and bis- desmethoxycurcumin. It has antioxidant, anti-inflammatory, antitumor, anti-hyperglycemic, antimalarial, antineoplastic, antibacterial, antiviral, flavouring agent, a nutraceutical, an antifungal agent, a dye and anti -Alzheimer’s properties. Curcumin provides a protective effect against hydrogen peroxide (H2O2) exposure in retinal pigment epithelial (RPE) cells by suppressing apoptosis and oxidative stress as well as by acting as an ROS scavenger.

[0012]

[0009] The average concentration of the three major curcuminoids of different samples of Curcuma longa L. are 5.69%-2.86% curcumin, 1.47% desmethoxycurcumin and 1.36% bidesmethoxycurcumin. It is a natural antioxidant practically water-insoluble and has low bioavailability. Curcumin is an oil-soluble pigment, practically insoluble in water at acidic and neutral pH, and soluble in alkali. Preparations of water-soluble curcumin by incorporation into various surfactants.

[0013]

[0010] The chemical formula of curcumin is C2IH20O6, and its molecular weight is 368.4 g / mol.

[0014] The chemical structure is depicted as follows -

[0015] [Oil] Lutein is a xanthophyll and naturally occurring carotenoids or antioxidant. Lutein is an unsaturated polyene hydrocarbon with a carbon chain of 40 carbon atoms and two -OH groups in p-ionone rings. It has two cyclic end groups, one beta- and one alpha-ionone ring. The principal natural stereoisomer of lutein is (3R,3'R,6'R)-beta, epsilon-carotene-3, 3'-diol.

[0016]

[0012] Lutein is a lipophilic molecule that is generally insoluble in water. It has a long chromophore of conjugated double bonds (polyene chain) that gives it distinctive lightabsorbing properties. Chemically known as (lR)-4-[(lE,3E,5E,7E,9E,llE,13E,15E,17E)-18- [(lR,4R)-4-hydroxy-2, 6, 6-trimethylcy cl ohex-2-en-l-yl]-3, 7, 12, 16-tetr am ethyloctadeca.

[0017] Lutein is a fat-soluble carotenoid pigment and has low water solubility. Lutein is also known as luteol and xanthophyll and is known to improve or even prevent age-related macular disease which is the leading cause of blindness and vision impairment.

[0018]

[0013] Chemical formula of lutein is C40H56O2, and its molecular weight is 568.87 g / mol. The chemical structure is as depicted below -

[0019]

[0014] The eye is a highly specialized and complex organ in the human body. Eye can be broadly divided into the anterior (front of the eye) and posterior (back of the eye) segments. An anterior segment of the eyes consists of sclera and conjunctiva, cornea, iris and pupil, trabecular meshwork, and lens while a posterior segment of the eye consists of vitreous humour, retina, optic nerve, and choroid. The working principle of eye is similar to a camera with film. Incoming light is first focused by the cornea and lens onto the retina (film), which is then converted into electrical signals to be sent to the brain for further processing via the optic nerve.

[0020]

[0015] Age-related macular degeneration (hereinafter referred to as AMD) is the most common cause of irreversible blindness in people over the age of 50 in the developed world. Although the pathogenesis of AMD is poorly understood, oxidative stress has been implicated as a major contributing factor. As lutein and zeaxanthin are powerful antioxidants selectively absorbed and maintained in the retina, their role in AMD has been studied extensively. Similarly, curcumin could play a significant role in AMD by reducing inflammation and oxidative stress. It has also been shown decrease apoptosis of retinal pigmented epithelial cells and a reduction of inflammatory markers.

[0021]

[0016] Age-related eye disorder includes, but are not limited to, age-related macular degeneration, visual dysfunction, and retinopathy which are eye diseases / disorders. AMD happens when ageing causes damage to the macula. The macula is responsible for sharp, central vision, which is needed for activities such as reading, driving, recognizing faces etc. Macular degeneration is a medical condition when the cells in the macula start to break down, leading to vision loss. There are 2 types of AMD - Dry AMD and Wet AMD. Dry AMD is also called as atrophic AMD. In dry AMD, the macula gets thinner with age and it happens in 3 stages i.e., early, intermediate, and late.

[0022]

[0017] Wet AMD also known as advanced neovascular AMD and is less common type of late AMD that usually causes faster vision loss. Any stage of dry AMD can turn into wet AMD whereas wet AMD is always a late stage. It happens when abnormal blood vessels grow in the back of the eye and damage the macula.

[0023]

[0018] Both types of AMD pathology start with the formation of insoluble aggregates, drusen, which form in the extracellular matrix between Bruch’s membrane and retinal pigment epithelial (RPE) cells.

[0024]

[0019] Drusen are focal yellow-white deposits of extracellular debris, which consist of complement proteins, esterified and non-esterified cholesterol, apolipoproteins, carbohydrates, and trace elements, above the retinal pigment epithelium (RPE) or between the RPE and Bruch’s membrane.

[0025]

[0020] Based on the extracellular deposits, drusens can be categorised as soft drusen, hard drusen, calcified drusen and pseudo drusen. Hard drusen is considered to be part of the normal ageing process while a large number of soft drusen and retinal pigment abnormalities are well- recognized as precursors for advanced AMD. Calcified drusen are defined as calcium deposition on the surface.

[0026]

[0021] AREDS (Age-related Eye Disease Study) was a prospective, multicentric, randomised clinical trial conducted between 1992 and 2006, mainly sponsored by the National Eye Institute (NEI) of the National Institutes of Health (NIH) and thereafter they are categorised as follows: AREDS category 1 - no or a few small drusen (<63 microns in diameter).

[0027] AREDS category 2 (Early AMD) - combination of multiple small drusen, a few intermediate drusen (63 to 124 microns in diameter) or RPE abnormalities.

[0028] AREDS category 3 (Intermediate AMD) - characterised by extensive intermediate drusen, at least one large drusen (>125 microns in diameter) or geographic atrophy not involving the centre of the fovea.

[0029] AREDS category 4 (Advanced / Late AMD) - characterised by breakdown of light-sensitive cells and / or supporting tissue in the central retinal area (advanced dry form) or abnormal and fragile blood vessels under the retina (wet form)

[0030]

[0022] The naturally occurring lipophilic nutrients of the present invention are derived from various plant and animal sources. Fat-soluble nutrients such as vitamins, carotenoids, terpenoids, glycerides, fatty acids such as saturated and monounsaturated acids, and curcumin are well known and generally used to maintain good health. The oral bioavailability of these nutrients is low. In order to increase the solubility of nutrients and to enhance the therapeutic efficacy, different techniques are employed by person skilled in the art including particle size reduction, use of solubilizers, salt formation, complexation with excipients such as Beta cyclodextrin, saccharin, surfactant, nano-milling, solid dispersion, nanosuspension, melt granulation and the like. The antioxidant properties of these nutraceuticals beneficial in various issues with eye health such as xerophthalmia (dry eyes), age-related macular degeneration, eye disease / disorder, age-related cataracts, dry eye syndrome, glaucoma, asthenopia, diabetic retinopathy, myopia, inflammation and other central nervous system degenerative diseases, and the like are known to improve with the administration of lipophilic nutrients orally.

[0031]

[0023] Epidemiological studies suggest that the levels of lutein and zeaxanthin in the retina and circulating system are inversely associated with the risk of age-related macular degeneration (AMD). Lutein and zeaxanthin are carotenoids specifically accumulated in the retina, which is believed to protect the retina from light and other environmental and / or pathological stimuli- induced damage.

[0032]

[0024] Macular pigments have a unique distribution within the retina. The concentration of lutein and zeaxanthin are highest in the macula, especially in the centre of the macula (the fovea) while zeaxanthin has a peak concentration in the central fovea, lutein predominates in the periphery.

[0025] Macular pigments enhance visual function in a variety of ways. The filtration of blue light reduces chromatic aberration which can enhance visual acuity and contrast sensitivity. Lutein and zeaxanthin also reduce discomfort associated with glare and improve visual acuity, photo stress recovery time, macular function and neural processing speed.

[0033]

[0026] Formulating dietary supplements with antioxidant, anti-inflammatory and cytoprotective properties to reduce injuries caused due to oxidative stress and inflammation is beneficial for the early management of dry AMD and for delaying progression to the neovascular stage.

[0034]

[0027] United States Publication No. US 20100291053 discloses a method of treating an inflammatory condition or disease with long-chain polyunsaturated fatty acid and at least one carotenoid, wherein the carotenoids are selected from astaxanthin, zeaxanthin, lycopene, lutein etc and the long chain fatty acid is a triglyceride, diglyceride etc.

[0035]

[0028] United State Publication No. US 20070231371 discloses dietary formulation having at least three ingredients - antioxidants such as lutein, zeaxanthin, retinal etc; reducers of body weight or body fat for e.g. medium chain triglycerides & anti-inflammatory agents such as curcumin and its use for increasing longevity in an animal.

[0036]

[0029] United State Publication No. US 20080175957 discloses composition having sensitive ingredients within a first protective coating; method of stabilizing sensitive ingredients and method of producing a composition having the same, wherein the sensitive ingredient is at least one carotenoid, polyphenol catechin, vitamin, mineral, unsaturated fatty acid etc)

[0037]

[0030] United State Publication No. US 20080305096 discloses method of providing controlled release composition of biologically active substance combined with one or more soluble fibres for the subject’s digestive system.

[0038]

[0031] United State Publication No. US 20100062040 discloses method of forming water- soluble microparticles and water-soluble microparticles having at least one food supplement such as lutein, zeaxanthin, fucoxanthin, flaxseed oil, borage oil, black currant oil, pine nut oil and fish oils etc.

[0039]

[0032] Chinese Publication No. CN 107823150 A discloses a rapidly dispersible tablet of waterinsoluble active ingredient, excipient and lubricant.

[0040]

[0033] Indian Patent No. IN 394537 discloses dispersible composition for enhancing the bioaccessibility of lipophilic compounds having lipophilic activities such as bisdemethoxycurcumin, bis-o-demethylcurcumin, tetrahydro curcumin, lutein, zeaxanthin etc; hydrophilic active; micelle forming agent and acidifier; The patent also covers its preparation process.

[0041]

[0034] US 11141386 discloses eye health supplement softgel dosage form having zeaxanthin, lutein, Vitamin E, zinc, curcumin and omega-3 oils.

[0042]

[0035] International Publication No. WO 2024086307 discloses extended-release preparation with carrier component and payload component selected from polyphenol, an antioxidant, a metabolic intermediate, or a combination thereof.

[0043]

[0036] International Publication No. WO 2024086306 discloses an extended-release preparation having carrier component, payload component and nutrient component, wherein nutrient is carotenoid and payload component are polyphenol and an antioxidant.

[0044]

[0037] Indian Publication no. IN 201941024774 discloses composition for enhancing the bioavailability of phytochemicals having phytochemicals are carotenoids, quinones, fatty acids and their derivatives either alone or mixtures thereof and bio-enhancing agent derived from Curcuma longa.

[0045]

[0038] Indian Publication No.7274 / DELNP / 2013 discloses population of particles, i.e., micelles, reverse micelles or clusters wherein each particle comprises carotenoid compound and cargo molecules, wherein the carotenoid compound is lutein, zeaxanthin, canthaxanthin, phytoene, phytofluene and cargo molecule is selected from the group consisting of food fermentation products; lecithin, curcuminoids.

[0046]

[0039] As seen above, there are numerous lipophilic nutrient compositions of zeaxanthin, curcumin contains curcuminoids, lutein with many active ingredients available, but there is no specific disclosure of zeaxanthin isomers such as RR Zeaxanthin, lutein, and Curcuminoids as directed in the specification. Also prior arts directed few formulations for enhancing the bioavailability and / or absorption and / or solubility of lipophilic agents. Owing to the lipophilic nature, these nutrients exhibit highly low aqueous solubility and permeability through body membranes, thus contributing to the low oral bioavailability of these nutrients. Due to poor bioavailability or solubility of lipophilic nutrients, the delivery of many potentially important therapeutic agents to the eye is difficult. Overcoming the difficulty of delivering such a potentially important composition of lipophilic nutrients is a major challenge during the treatment, prevention, and / or improvement of most eye disorders. The present invention provides the formulation to enhance the solubility and / or bioavailability of lipophilic nutrients during oral administration. The invention also provides the oil suspension of therapeutically effective lipophilic nutrients prepared using the micronization technique.

[0047]

[0040] Multiple solid oral dosage forms comprising zeaxanthin, curcumin, and / or lutein are also available in the market, but there exist limited options when it comes to the liquid oral dosage of nutrient composition. Liquid dosage forms are more effective as compared with solid dosage forms, as they eliminate the need for drug / supplement dissolution in gastrointestinal tract before the release of an active. The liquid dosage is also advantageous for people who find it difficult to swallow the solid dosage form, preferably in the geriatric and pediatric population.

[0048]

[0041] Thus, there exists a long-felt need for an effective liquid composition of lipophilic nutrients to overcome the problems as stated above and also remain stable for a longer time in order to administer in an aqueous solution. Another problem encountered in the art is large- scale production of lipophilic nutrient composition containing zeaxanthin isomers, curcuminoids, and / or lutein.

[0049] BRIEF DESCRIPTION OF THE INVENTION

[0050]

[0042] The present invention relates to stable and bioavailable oral lipophilic nutrients composition of an carotenoids and polyphenol. The present invention more particularly relates to stable and bioavailable oral lipophilic nutrient composition of zeaxanthin isomers, curcuminoids and / or lutein.

[0051]

[0043] The present invention also provides a composition of R, R-zeaxanthin, curcuminoids, and / or lutein to increase the bioavailability, absorption, solubility, and permeability of lipophilic agents. Particularly, the present invention relates to a stable and bioavailable oral liquid composition of R, R-zeaxanthin, curcuminoids, and / or lutein.

[0052]

[0044] The present invention further relates to using zeaxanthin isomers, curcuminoids, and / or lutein, a stable and bioavailable oral lipophilic nutrient composition, for ophthalmic care, preferably age-related eye diseases (AREDS), Age-related Macular degeneration (AMD), Visual dysfunction, Retinopathy, and other Eye diseases and disorders.

[0053]

[0045] The present invention also discloses the process for preparing a stable and bioavailable liquid composition of zeaxanthin isomers, Curcuminoids, and / or lutein.

[0054] BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

[0055] Figure 1. Bioavailability of test products in plasma of Sprague Dawley rats Figure 2. Bioavailability of test products in retina of Sprague Dawley rats

[0056] Figure 3. Bioavailability of test products in brain tissue of Sprague Dawley rats

[0057] Figure 4. Histology of rat retina: Outer nuclear layer (ONL) thickness from LED induced retinal damage model

[0058] Figure 5. Effects of supplements on serum oxidative stress markers in retinal damage models

[0059] Figure 6. Relative expression of protein Nrf2 and HO-1 in retinal tissues

[0060] Figure 7. Relative expression of protein ATG5 and ATG7 in retinal tissues

[0061] Figure 8. Relative expression of protein ATF4 & 6 in retinal tissues

[0062] Figure 9. Relative expression of protein mTOR in retinal tissues

[0063] Figure 10. Relative expression of apoptotic markers in retinal tissues

[0064] Figure 11. Relative expression of NF-KB in retinal tissues

[0065] Figure 12. Relative expression of NLRP Inflammasome in retinal tissues

[0066] Figure 13. Relative expression of serum inflammatory markers

[0067] Figure 14. Relative expression of MCP1 in retinal tissues

[0068] Figure 15. Relative expression of NCAM in retinal tissues

[0069] Figure 16. Relative expression of ICAM1 in retinal tissues

[0070] Figure 17. Relative expression of VEGF in retinal tissues

[0071] Figure 18. Relative expression of TGFp in retinal tissues

[0072] Figure 19. Relative expression of GAP43 and GFAP in retinal tissues

[0073] DETAILED DESCRIPTION OF THE INVENTION

[0074]

[0046] Before explaining any one embodiment of the present disclosure by way of experimentation, results, and pertinent procedures, it is to be understood that the disclosure is not limited in its application to the details as explained in below embodiments set forth in the following description or illustrated in the experimentation and / or results. The disclosure is further capable of other embodiments which can be practiced or carried out in various ways. However, it is not intended that the invention be limited to the specific terms so selected and it is to be understood that such specific terms include all technical equivalents that operate in a similar manner to accomplish a similar purpose.

[0075]

[0047] Definitions: The features and advantages of the invention may be more readily understood by those of ordinary skill in the art upon reading the following detailed description. It is to be appreciated that certain features of the invention that are, for clarity reasons, described above and below in the context of separate embodiments, may also be combined to form a single embodiment. Embodiments identified herein as exemplary or preferred are intended to be illustrative and not limiting.

[0076]

[0048] Source of Phytochemicals used in the Invention:

[0077] Lutein: Marigold flowers, Green leafy vegetables, egg yolk, peppers, grapes etc.

[0078] Zeaxanthin Isomers: Paprika, Marigold flowers, Leafy greens, Egg yolks, Corns, Oranges, red grapes, mango, honeydew melon, and orange peppers, Goji berries, Pumpkin etc.

[0079] Curcumin / Curcuminoids: Turmeric rhizome

[0080]

[0049] As used herein, reference to an element by the indefinite article “a”, “an” and “the”, does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0081]

[0050] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”

[0082]

[0051] The terms “approximately” and “about” shall mean to be nearly the same as a referenced number or value. As used herein, the terms “approximately” and “about” should be generally understood to encompass ±10% of a specified amount, frequency or value.

[0083]

[0052] The term “bioavailability” shall mean the extent and rate at which the active moiety (nutrient) enters systemic circulation, thereby accessing the site of action.

[0084]

[0053] The term “nutraceutically acceptable” means biologically or pharmacologically compatible for in use in animals or humans, and preferably shall mean approved by drug regulatory agencies or listed in the Pharmacopeia or official compendia for use in animals, and more particularly in humans.

[0085]

[0054] The term “phytochemical” refers herein is as any of the various biologically active compounds found in plants.

[0055] The term “stable and bioavailable oral lipophilic nutrient composition” or “composition of zeaxanthin, curcuminoids and / or lutein”, as used throughout the specification, refers to the oral liquid dosage form.

[0086]

[0056] As used within the scope of the invention, the term “lipophilic” though refers to lipid- like, it generally covers all compounds that are poorly water-soluble. Non-limiting examples are carotenoids, fat-soluble vitamins, fatty acids, glycerides, capsaicin, curcumin and mixtures thereof.

[0087]

[0057] Preferably the lipophilic nutrient is selected from the group consisting of, but not limited to, carotenoids, such as alpha-carotene, beta- carotene, canthaxanthin, 8'-apo-beta-carotenal, 8'-apo-beta-carotenoic acid esters such as the ethyl ester, ethanolic extracts of Terminalia, astaxanthin, mesozeaxanthin, astaxanthin ester, betacryptoxanthin, lycopene, lutein, lutein (di) ester, zeaxanthin or crocetin, alpha or beta-zeacarotene or mixtures thereof; vitamin A, vitamin D, vitamin E, vitamin K, Coenzyme Q10 or derivatives thereof such as their acetates, e.g. vitamin A acetate or tocopherol acetate, or their longer chain fatty acid esters, e.g. vitamin A palmitate or tocopherol palmitate, capsaicin, dihydrocapsaicin, derivatives thereof, polyunsaturated fatty acids (PUFAs) or derivatives thereof, and triglycerides rich in polyunsaturated fatty acids such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), Omega 6 oils or derivatives thereof, gamma-linolenic acid (GLA), Omega 3, Salacia extract and or mixtures thereof.

[0088]

[0058] Preferably the lipophilic nutrients of the instant invention are selected from the carotenoids such as, but not limited to, lutein, beta-carotene, lycopene, zeaxanthin, astaxanthin, astaxanthin ester, a-cryptoxanthin, neoxanthin and canthaxanthin. More preferably the carotenoids used herein is the combination of lutein with zeaxanthin. The person skilled in the art would understand that this list of potential lipophilic active substances is not exhaustive, and there are many other lipophilic actives that offer nutritional, medicinal, and pharmaceutical benefits, for eye health which may also be utilized in the current invention.

[0089]

[0059] In one embodiment, the present invention provides a stable and bioavailable oral lipophilic nutrient composition comprising zeaxanthin, curcuminoids and lutein. Preferably, the present invention provides the stable and bioavailable nutraceutical composition comprising R, R-zeaxanthin, curcuminoids and lutein.

[0090]

[0060] In one more embodiment, curcumin extract in the range 90-99 %, which further comprises curcuminoids, per se., curcumin 70-80%, desmethoxycurcumin 10-20 %, and bisdemethoxycurcumin 2-6 %. In another embodiment, the present invention provides the stable and bioavailable oral lipophilic nutrient composition comprising zeaxanthin, curcuminoids and lutein along with the nutraceutically acceptable excipients selected from carrier, solubility enhancer, antioxidant, flavouring agent, solubilizer and the like. Preferably, the present invention provides the stable and bioavailable nutraceutical composition comprising R, R-zeaxanthin, curcuminoids and lutein along with the nutraceutically acceptable excipients selected from carriers, solubility enhancers, antioxidants, flavouring agents, solubilizers and the like.

[0091]

[0061] In another embodiment, the present invention provides the stable and bioavailable oral lipophilic nutrients composition comprising zeaxanthin and curcuminoids. Preferably, the present invention provides the stable and bioavailable nutraceutical composition comprising R, R-zeaxanthin and curcuminoids.

[0092]

[0062] In another embodiment, the present invention provides the stable and bioavailable oral lipophilic nutrient composition comprising zeaxanthin and curcuminoids lutein along with the nutraceutically acceptable excipients selected from carriers, solubility enhancers, antioxidants, flavouring agents, solubilizers and the like. Preferably, the present invention provides the stable and bioavailable nutraceutical composition comprising R, R-zeaxanthin and curcuminoids along with the nutraceutically acceptable excipients selected from carriers, solubility enhancers, antioxidants, flavouring agents, solubilizers and the like.

[0093]

[0063] It is well known that excipients such as antioxidants, solubilizers, solubility enhancers, flavouring agents etc are routinely incorporated into nutraceutical compositions, human drug products, dietary supplements, food products, veterinary drug products, botanical compositions and the products which are derived using actives from synthetic or natural sources and are meant for administration to mammals.

[0094]

[0064] As used within the scope of the invention, the “carrier”, as used throughout the specification, is selected from the group consisting of polyethylene glycol 200, polyethylene glycol 400, medium chain triglyceride (also referred as MCT) oil, 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 their mixtures thereof. In a preferred embodiment, the present invention provides the lipophilic nutrient composition comprising the medium chain triglyceride (also referred as MCT) oil as a carrier. According to further embodiment, the amount of MCT oil used in the present invention ranges from about 30% to about 60% by weight of composition, preferably it ranges from about 40% to about 55% by weight of composition and more preferably about 45% to about 50% by weight of composition of the MCT oil is used for the present invention.

[0095]

[0065] In another embodiment, the MCT oil is present in an amount ranging from about 120mg to about 350mg; preferably from about 145 mg to about 320 mg.

[0096]

[0066] As used within the scope of the invention, the “antioxidant” is selected from the group consisting of commonly used excipients including, but not limited to tocopherol, mixed tocopherol, glutathione, citric acid, alfa-tocopherol, rosemary extract, lipoic acid, selenium, sodium ascorbate, flavonoid, ascorbyl palmitate, resveratrol or the like and their combinations thereof. In one preferred embodiment, the present invention uses mixed Tocopherol 70%-SF in oil as an antioxidant. According to a further embodiment, the mixed tocopherol 70%-SF in oil is present in an amount ranging from about 0.5% to about 10% by weight of the composition, preferably the mixed tocopherol 70%-SF in oil present in the composition of the invention is about 1% to about 5% by weight of the composition.

[0097]

[0067] In another embodiment, antioxidants are present in amounts ranging from about 4 mg to about 18 mg; preferably from about 6 mg to about 14 mg.

[0098]

[0068] As used within the scope of the invention, the term "oil", as used herein, means any neutral, nonpolar chemical substance that is a viscous liquid at ambient temperatures and is both hydrophobic (immiscible with water) and lipophilic (miscible with other oils), and is selected from plant or synthetic source or their mixtures thereof. The present invention provides the oil that can be used for suspension or solubilization of lipophilic nutrients or as a flavouring agent in the composition.

[0099]

[0069] The term “solubility enhancer” means a functional excipient included in the composition to increase the solubility of a substance or to improve the absorption of nutrients or pharmacologically active drug. It can also be used as a carrier for lipophilic active or may have additional function such as solubilization, flavouring agent, retinal targeting or additional synergistic benefit such as eye lubrication, eye protection from intense light, improvement of eye function such as blinking, improving co-ordination between eye and brain, improvement in functioning of optic nerve, and the like. It can be used as a single oil or a combination of oils varying in amounts based on total weight of the composition.

[0070] The oil as used herein includes but is not limited to vegetable oils such as soya oil, palmkernel oil, olive oil, cotton oil, medium chain triglyceride oil (MCT oil), maize oil, coconut oil, palm oil, d- limonene, sesame oil, linseed oil (flaxseed oil), sunflower oil, walnut oils, cedar leave oil, hazelnut oil, corn oil, fish oil, hydrogenated castor oil, safflower oil, peanut oil, corn oil, cotton seed oil, canola oil, Wintergreen oil, tea tree oil, thyme oil, castor oil, bay oil, aj wain oil, clove oil, anise oil, eucalyptus oil, cassia oil, nutmeg oil, oil of bitter almonds, and oil of sage. In one preferred embodiment of the present invention, olive oil is used as a solubility enhancer for lipophilic nutrients wherein the olive oil is present in amount ranging from about 1% to about 15% by weight of the composition. Preferably the solubility enhancer used in the present invention is olive oil present in an amount ranging from about 3% to about 12% by weight of the composition, more preferably it presents in an amount ranging from about 5% to about 10% by weight of the composition.

[0100]

[0071] In another embodiment, the olive oil is present in an amount ranging from about 10 mg to about 50mg; preferably from about 18 mg to about 42 mg.

[0101]

[0072] In one preferred embodiment, linseed oil (also known as flaxseed oil) is also used in the composition of the present invention. According to a further embodiment, linseed oil is present in an amount ranging from about 0.5% to about 10% by weight of the composition, preferably the linseed oil present in the composition is about 1% to about 5% by weight of composition. The linseed oil used in the present invention helps in treating dry eye syndrome.

[0102]

[0073] In another embodiment, the linseed oil is present in an amount ranges from about 4 mg to about 18 mg; preferably from about 6mg to about 14 mg.

[0103]

[0074] As used within the scope of the invention, the term “flavouring agent” means a natural or artificial compound. Flavors incorporated in the composition may be chosen from natural and synthetic flavour oils or extracts from fruits, flowers, leaves, plants, and combinations thereof. Such compounds include, by way of example and without limitation, cinnamon oil, d- limonene, menthol, orange, vanillin, lime, cocoa, fruit essences, including berry, apple, pineapple, pear, peach, blueberry, kiwi, raspberry, cherry, plum, strawberry, and apricot, peppermint oil, citrus oils such as lemon, lime and grapefruit oils; and all of these flavourings are commercially available. In one preferred embodiment, the present invention uses d- limonene as a flavouring agent for lipophilic nutrient compositions. According to a further embodiment, d-limonene used in the present invention is present in an amount ranging from about 0.5% to about 10% by weight of the composition, preferably amount of d-limonene present in the invention is about 1% to about 5% by weight of the composition.

[0104]

[0075] In another embodiment, the flavouring agent is present in an amount ranging from about 4. mg to about 18 mg; preferably from about 6 mg to about 14 mg.

[0105]

[0076] As used within the scope of the invention, the “solubilizer” is selected from the group consisting of egg yolk lecithin, hydroxy propyl betacyclodextrin, vitamin E TPGS, polysorbate 80, poloxamer 188, polysorbate 20, linoleic acid, PEG 4000, oleic acid, sorbitan trioleate, polyethylene glycol 7-stearate, sodium ascorbate, phosphatidylcholine or lysophosphatidylcholine and their combination thereof. In one preferred embodiment, the phosphatidylcholine is used as a solubilizer for the lipophilic nutrient compositions. According to further embodiment, phosphatidylcholine present in the composition ranges from about 0.5% to about 10% by weight of the composition, preferably the amount of phosphatidylcholine present in the invention is about 1% to about 5% by weight of the composition.

[0106]

[0077] In another embodiment, the solubilizer is present in amount ranging from about 4mg to about 18 mg; preferably from about 6mg to about 13mg.

[0107]

[0078] In one embodiment, the present invention provides the stable and bioavailable oral lipophilic nutrient composition comprising R, R-zeaxanthin, curcuminoids and lutein along with the nutraceutically acceptable excipients selected from medium chain triglyceride oil, olive oil, mixed tocopherol, flax seed or linseed oil, d-limonene, phosphatidylcholine and their combination thereof.

[0108]

[0079] In one embodiment, the present invention provides the stable and oral lipophilic nutrient composition comprising R, R-zeaxanthin and curcuminoids along with the nutraceutically acceptable excipients selected from medium chain triglyceride oil, olive oil, mixed tocopherol, flax seed or linseed oil, d-limonene, phosphatidylcholine and their combination thereof.

[0109]

[0080] In one embodiment, the stable and oral lipophilic nutrient composition of R, R- zeaxanthin, curcuminoids and lutein exhibits improved bioavailability and is preferably used for treating, preventing, improving or reducing the risk of progression of age-related eye disorder (AREDS) including, but not limited to, age-related macular degeneration (AMD), visual disfunction, retinopathy, eye disorder / disease, asthenopia, diabetic retinopathy, myopia, inflammation, and other central nervous system degenerative diseases.

[0081] In another embodiment, the stable and bioavailable oral lipophilic nutrient composition for treating, preventing, improving or reducing the AREDS, age age-related macular degeneration, eye disorder / disease, visual dysfunctionl and retinopathy, comprising

[0110] • R, R-zeaxanthin;

[0111] • curcuminoids;

[0112] • lutein; and

[0113] • nutraceutically acceptable excipients selected from medium chain triglyceride oil, olive oil, mixed tocopherol, flax seed or linseed oil, d-limonene, phosphatidyl and their combination thereof.

[0114]

[0082] In one embodiment, the stable and oral lipophilic nutrient composition of R, R- zeaxanthin and curcuminoids exhibits improved bioavailability and is preferably used for treating, preventing, improving or reducing the risk of progression of age-related eye disorders including, but not limited to, age-related macular degeneration, visual dysfunction, retinopathy, asthenopia, diabetic retinopathy, myopia, inflammation and other central nervous system degenerative diseases.

[0115]

[0083] In another embodiment, the stable and bioavailable oral lipophilic nutrient composition for treating, preventing, improving or reducing the AREDS, age age-related macular degeneration, eye disorder / disease, visual dysfunction, and retinopathy, comprising:

[0116] • R, R-zeaxanthin;

[0117] • curcuminoids; and

[0118] • nutraceutically acceptable excipients selected from medium chain triglyceride oil, olive oil, mixed tocopherol, flax seed or linseed oil, d-limonene, phosphatidyl and their combination thereof.

[0119]

[0084] In another embodiment, the stable and bioavailable oral lipophilic nutrient composition for treating, preventing, improving or reducing the AREDS, age age-related macular degeneration, eye disorder / disease, visual dysfunction, and retinopathy, comprising:

[0120] • R, S-zeaxanthin;

[0121] • curcuminoids; and

[0122] • nutraceutically acceptable excipients.

[0085] In another embodiment, the stable and bioavailable oral lipophilic nutrient composition for treating, preventing, improving or reducing the AREDS, age age-related macular degeneration, eye disorder / disease, visual dysfunction, and retinopathy, comprising:

[0123] • Lutein

[0124] • R, R & R, S-zeaxanthin in equal and varied ratios

[0125] • curcuminoids; and

[0126] • nutraceutically acceptable excipients.

[0127]

[0086] In one embodiment, the present invention provides stable and oral lipophilic nutrient composition comprising carotenoids present in an amount ranging from about 0.5% to about 20% by weight of the composition, preferably present in an amount ranging from about 1% to about 15% by weight of the composition. In another embodiment, the carotenoids used in the composition of the present invention are lutein and zeaxanthin, preferably, lutein and R, R- zeaxanthin.

[0128]

[0087] In one aspect, the present invention provides stable and oral lipophilic nutrient composition comprising lutein present in an amount ranging from about 1 mg to about 35 mg, preferably from about 5 mg to about 30 mg and more preferably from about 8 mg to about 25 mg.

[0129]

[0088] In another aspect, the present invention provides stable and oral lipophilic nutrient composition comprising R, R-zeaxanthin present in an amount ranging from about 0.2 mg to about 10 mg, preferably from about 1 mg to about 8 mg and more preferably from about 1.5 mg to about 5 mg.

[0130]

[0089] In another aspect, the present invention provides stable and oral lipophilic nutrient composition comprising R, S-zeaxanthin present in an amount ranging from about 0.2 mg to about 10 mg, preferably from about 1 mg to about 8 mg.

[0131]

[0090] In another embodiment, the present invention provides stable and oral lipophilic nutrient composition comprising curcuminoids present in an amount ranging from about 10% to about 50% by weight of the composition, preferably in an amount ranging from about 20% to about 45% by weight of the composition and more preferably the curcuminoids is present in an amount ranging from about 25% to about 40% by weight of the composition.

[0132]

[0091] In one aspect, the present invention provides stable and oral lipophilic nutrient composition comprising curcuminoids present in an amount ranging from about 40 mg to about 350 mg, preferably from about 60 mg to about 300 mg and more preferably from about 80 mg to about 250 mg.

[0133]

[0092] In another aspect of the invention, Lutein and Zeaxanthin are derived from marigold flowers or from green leafy vegetables. Marigold flowers are botanically known as Tagetes which belongs to the Asteraceae family. Lutein and zeaxanthin are also categorised as potent antioxidants.

[0134]

[0093] In one embodiment, a stable and bioavailable oral lipophilic nutrient composition of the present invention is available in oral liquid dosage forms but not limited to dosages such as oil suspensions, dispersions, flocculated suspension, deflocculated suspension, emulsions, solutions, elixir, suspensions, and the like. Preferably, the oral lipophilic nutrient composition is prepared in the form of oil suspension for oral administration.

[0135]

[0094] In another embodiment, the present invention provides a process for preparing a stable and bioavailable oral liquid composition of R, R-zeaxanthin, curcuminoids, and / or lutein. In another embodiment, the present invention provides the process to prepare the liquid oral dosage form comprising R, R-zeaxanthin, curcuminoids and lutein, wherein the process comprises -

[0136] (a) Adding the mixture of lutein and R, R-zeaxanthin along with curcumin as a curcuminoids in the air jet mill;

[0137] (b) micronizing the mixture of step (a);

[0138] (c) adding the inactive excipients selected from carrier, solubility enhancer, antioxidant, flavouring agent, solubilizer and the like

[0139] (d) preparing the oil suspension using ball mill / homogenizer;

[0140] (e) obtaining the final product.

[0141] Block representation of manufacturing process is depicted as follows:

[0142] Note - Micronization ofLuten / RR Zeaxanthin and Curcuminoids is done separately, not after combination, and particle size is ensured from 0.1 to 20 microns.

[0143]

[0095] In another embodiment, the present invention provides the process to prepare the liquid oral dosage form comprising R, R-zeaxanthin, RS Zeaxanthin, curcuminoids, and lutein, wherein the process comprises the same method as above without any changes.

[0144]

[0096] In another embodiment, the present invention also provides the process to prepare the liquid oral dosage form comprising R, R-zeaxanthin and curcuminoids wherein the process comprises- (a) Adding R, R-zeaxanthin along with curcuminoids in the air jet mill; Adding Lutein, R,

[0145] R-zeaxanthin and curcuminoids micronized separately using Air jet mill

[0146] (b) micronizing the mixture of step (a);

[0147] (c) adding the inactive excipients selected from carrier, solubility enhancer, antioxidant, flavouring agent, solubilizer and the like (d) preparing the oil suspension using ball mill / homogenizer;

[0148] (e) obtaining the final product.

[0097] In another embodiment, the present invention also provides the process to prepare the liquid oral dosage form comprising R, S-zeaxanthin and curcuminoids wherein the process comprises the same as like above without any changes.

[0149]

[0098] The invention is further illustrated by the following examples which are provided to be exemplary of the invention and do not limit the scope of the invention. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

[0150]

[0099] Examples:

[0151]

[0100] Example A: Process for Preparation of Oil Suspension

[0152]

[0101] Example 1 - Composition of Lutein (10 mg), R,R-Zeaxanthin (2 mg), and Curcuminoids (100 mg) (Dose 335 mg).

[0153]

[0102] A weighed quantity of micronized marigold paprika extract concentrate (26.12 gm) containing 84.85% total xanthophyll content, 67.90 % (trans, R,R)-lutein and 14.47% (trans, R,R)-zeaxanthin content, and Curcumin micronized concentrate (172.54 gm) containing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxycurcumin 15.94% and bisdem ethoxy curcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. Both concentrates were used to formulate an oil suspension of the desired dose. Concentrates were mixed by adding oil (231.34 gm) medium Chain triglycerides (MCT) and 14% w / w (70.0 gm) other ingredients such as 6.0% olive oil, 2.0% linseed oil from flaxseed oil, 2.0% D- Limonene from orange peel oil, 2.0% Lecithin (Phosphatidylcholine Sunflower source), 2.0% mixed tocopherol 70% Sunflower source by using ball mill at room temperature. The resulting oil suspension was sieved and unloaded (488gm). In the formulated oil suspension, the particle size was (DV90) 3.40pm, measured using a Malvern particle size analyser 3000 (wet method).

[0154]

[0103] The content of (trans, R,R)- lutein, (trans, R,R)- Zeaxanthin, and curcuminoid were 3.36%, 0.66% and 32.69%, respectively, determined by UV / Vis spectrometer and HPLC.

[0155]

[0104] Example 2: Lutein (10 mg), R,R-Zeaxanthin (2 mg) and Curcuminoids (200 mg), (Dose 650 mg).

[0156]

[0105] A weighed quantity of micronized marigold paprika extract concentrate (13.46 gm) containing 84.85% total xanthophyll content, 67.90 % (trans, R,R)-lutein and 14.47% (trans, R,R)-zeaxanthin content, and Curcumin micronized concentrate (178.46 gm) containing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxycurcumin 15.94% and bisdem ethoxy curcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. Both concentrates were used to formulate an oil suspension of the desired dose. Concentrates were mixed by adding oil (238.08 gm) medium Chain triglycerides (MCT) and 14% w / w (70.0 gm) other ingredients such as 6.0% olive oil, 2.0% linseed oil from flaxseed oil, 2.0% D- Limonene from orange peel oil, 2.0% Lecithin (Phosphatidylcholine Sunflower source), 2.0% mixed tocopherol 70% Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (490gm). In the formulated oil suspension, the particle size was (DV90) 3.63pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,R)- lutein, (trans, R,R)- Zeaxanthin, and curcuminoid were 1.86%, 0.36% and 34.54%, respectively, determined by UV / Vis spectrometer and HPLC.

[0157]

[0106] Example 3: Composition of Lutein (10 mg), R,R-Zeaxanthin (2 mg) and Curcuminoids (200 mg), (Dose 580 mg). Batch size 100.0 gm

[0158]

[0107] A weighed quantity of micronized marigold paprika extract concentrate (2.76 gm) containing 84.85% total xanthophyll content, 67.90 % (trans, R,R)-lutein and 14.47% (trans, R,R)-zeaxanthin content and Curcumin micronized concentrate (40.00 gm) containing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxycurcumin 15.94% and bisdem ethoxy curcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. All concentrates were used to formulate oil suspensions of the desired dose. Concentrates were mixed by adding oil (43.24 gm) medium Chain triglycerides (MCT) and 14% w / w (14.0 gm) other ingredients such as 6.0% olive oil, 2.0% linseed oil from flaxseed oil, 2.0% D-Limonene from orange peel oil, 2.0% Lecithin (Phosphatidylcholine Sunflower source), 2.0% mixed tocopherol 70% Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (96.5 gm). In the formulated oil suspension, the particle size was (DV90) 2.52pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,R)- lutein, (trans, R,R)- Zeaxanthin, and curcuminoid were 1.74%, 0.38% and 38.42%, respectively, determined by UV / Vis spectrometer and HPLC.

[0159]

[0108] Example 4: Composition of Lutein (10 mg), R,R-Zeaxanthin (10 mg) and Curcuminoids (200 mg), (Dose 700 mg). (Batch size 100.0 gm)

[0160]

[0109] A weighed quantity of micronized marigold paprika extract concentrate (2.31gm) containing 84.85% total xanthophyll content, 67.90 % (trans, R,R)-lutein and 14.47% (trans, R,R)-Zeaxanthin content; Paprika extract R,R-Zeaxanthin micronized concentrate (2.29gm) containing RR Zeaxanthin content of 60.00% and Curcumin micronized concentrate (33.14gm) containing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxycurcumin 15.94% and bisdemethoxycurcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. All concentrates were used to formulate oil suspensions of the desired dose. Concentrates were mixed by adding oil (48.26 gm) medium Chain triglycerides (MCT) and 14% w / w (14.0 gm) other ingredients such as 6.0% olive oil, 2.0% linseed oil from flaxseed oil, 2.0% D-Limonene from orange peel oil, 2.0% Lecithin (Phosphatidylcholine Sunflower source), 2.0% mixed tocopherol 70% Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (96.0 gm). In the formulated oil suspension, the particle size was (DV90) 2.51pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,R)- lutein, (trans, R,R)- Zeaxanthin, and curcuminoid were 1.62%, 1.65% and 32.26%, respectively, determined by UV / Vis spectrometer and HPLC.

[0161]

[0110] Example 5: Composition of Lutein (10 mg), R,R-Zeaxanthin (10 mg), and Curcuminoids (200 mg), (Dose 630 mg).

[0162]

[0111] A weighed quantity of micronized marigold paprika extract concentrate (2.57gm) containing 84.85% total xanthophyll content, 67.90 % (trans, R,R)-lutein and 14.47% (trans, R,R)-Zeaxanthin content; Paprika extract R,R-Zeaxanthin micronized concentrate (2.54gm) containing RR Zeaxanthin content of 60.00% and Curcumin micronized concentrate (36.83 gm) containing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxycurcumin 15.94% and bisdemethoxycurcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. All concentrates were used to formulate oil suspensions of the desired dose. Concentrates were mixed by adding oil (44.06 gm) medium Chain triglycerides (MCT) and 14% w / w (14.0 gm) other ingredients such as 6.0% olive oil, 2.0% linseed oil from flaxseed oil, 2.0% D-Limonene from orange peel oil, 2.0% Lecithin (Phosphatidylcholine Sunflower source), 2.0% mixed tocopherol 70% Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (97.0 gm). In the formulated oil suspension, the particle size was (DV90) 2.57pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,R)- lutein, (trans, R,R)- Zeaxanthin, and curcuminoid were 1.73%, 1.80% and 34.86%, respectively, determined by UV / Vis spectrometer and HPLC.

[0163]

[0112] Example 6: R,R-Zeaxanthin (8 mg) and Curcuminoids (200 mg), (Dose 650 mg). Batch Size 500 gm

[0164]

[0113] A weighed quantity of Paprika extract R,R Zeaxanthin micronized concentrate (11.54 gm) containing R,R Zeaxanthin content was 73.21% and Curcumin micronized concentrate (178.46 gm) containing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxy curcumin 15.94% and bisdemethoxycurcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. Both concentrates were used to formulate an oil suspension of the desired dose. Concentrates were mixed by adding oil (240.0 gm) medium Chain triglycerides (MCT) and 14% w / w (70.0 gm) other ingredients such as 6.0% olive oil, 2.0% linseed oil from flaxseed oil, 2.0% D-Limonene from orange peel oil, 2.0% Lecithin (Phosphatidylcholine Sunflower source), 2.0% mixed tocopherol 70% Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (494gm). In the formulated oil suspension, the particle size was (DV90) 3.96pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,R)- Zeaxanthin, and curcuminoid were 1.47 and 32.21%, respectively determined by UV / Vis spectrometer and HPLC.

[0165]

[0114] Example 7: R,R-Zeaxanthin (8 mg) and Curcuminoids (100 mg), (Dose 335 mg). Batch

[0166] Size 500 gm

[0115] A weighed quantity of Paprika extract R,R Zeaxanthin micronized concentrate (22.39 gm) containing R,R-Zeaxanthin content of 73.21% and Curcumin micronized concentrate (173.13 gm) containing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxy curcumin 15.94% and bisdem ethoxy curcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. Both concentrates were used to formulate an oil suspension of the desired dose. Concentrates were mixed by adding oil (234.48 gm) medium Chain triglycerides (MCT) and 14% w / w (70.0 gm) other ingredients such as 6.0% olive oil, 2.0% linseed oil from flaxseed oil, 2.0% D-Limonene from orange peel oil, 2.0% Lecithin (Phosphatidylcholine Sunflower source), 2.0% mixed tocopherol 70% Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (491gm). In the formulated oil suspension, the particle size was (DV90) 5.06pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,R)- zeaxanthin and curcuminoid were 2.83 and 32.80%, respectively, determined by UV / Vis spectrometer and HPLC.

[0167]

[0116] Example 8: R,R-Zeaxanthin (8 mg) and Curcuminoids (200 mg), (Dose 580 mg). Batch Size 100 gm

[0168]

[0117] A weighed quantity of Paprika extract R,R Zeaxanthin micronized concentrate (2.59 gm) containing R,R-Zeaxanthin content of 60.0% and Curcumin micronized concentrate (40.0 gm) containing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxycurcumin 15.94% and bisdemethoxycurcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. Both concentrates were used to formulate an oil suspension of the desired dose. Concentrates were mixed by adding oil (43.41 gm) medium Chain triglycerides (MCT) and 14% w / w (14.0 gm) other ingredients such as 6.0% olive oil, 2.0% linseed oil from flaxseed oil, 2.0% D-Limonene from orange peel oil, 2.0% Lecithin (Phosphatidylcholine Sunflower source), 2.0% mixed tocopherol 70% Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (96 gm). In the formulated oil suspension, the particle size was (DV90) 2.59pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,R)- Zeaxanthin and curcuminoid was 1.56% and 38.25%, respectively, as determined by UV / Vis spectrometer and HPLC.

[0169]

[0118] Example 9: R,R-Zeaxanthin (4 mg) and Curcuminoids (200 mg), (Dose 580 mg).

[0170]

[0119] A weighed quantity of Paprika extract R,R-Zeaxanthin micronized concentrate (1.29gm) containing R,R-Zeaxanthin content of 60.0% and Curcumin micronized concentrate (40.0 gm) containing total curcuminoid contain total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxy curcumin 15.94% and bisdem ethoxy curcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. Both concentrates were used to formulate an oil suspension of the desired dose. Concentrates were mixed by adding oil (44.71 gm) medium Chain triglycerides (MCT) and 14% w / w (14.0 gm) other ingredients such as 6.0% olive oil, 2.0% linseed oil from flaxseed oil, 2.0% D-Limonene from orange peel oil, 2.0% Lecithin (Phosphatidylcholine Sunflower source), 2.0% mixed tocopherol 70% Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (95.0 gm). In the formulated oil suspension, the particle size was (DV90) 2.33pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,R)-Zeaxanthin and curcuminoid was 0.86% and 38.64%, respectively, as determined by UV / Vis spectrometer and HPLC.

[0171]

[0120] Example 10: Zeaxanthin isomers 8 mg (5 mg R,S (meso) Zeaxanthin + 3 mg R,R Zeaxanthin) + 200 mg Curcuminoids, (Dose 580mg).

[0172]

[0121] A weighed quantity extract of R,S Zeaxanthin concentrate-micronized (1.38 gm) containing 71.31 % (trans, R, S) and 6.60 % (trans, R,R) zeaxanthin content, 0.69gm R,R zeaxanthin concentrate micronized containing 70.0 % (trans, R,R) Zeaxanthin content, Curcumin micronized concentrate (39.83 gm) containing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxycurcumin 15.94% and bisdemethoxy curcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. All concentrates were used to formulate oil suspensions of the desired dose. Concentrates were mixed by adding oil (44.10 gm) medium Chain triglycerides (MCT) and 14% w / w (14.0 gm) other ingredients such as 6.0% olive oil, 2.0% linseed oil from flaxseed oil, 2.0% D-Limonene from orange peel oil, 2.0% Lecithin (Phosphatidylcholine Sunflower source), 2.0% mixed tocopherol 70% Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (97.50 gm). In the formulated oil suspension, the particle size was (DV90) 6.36pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,S)- zeaxanthin, (trans, R,R)- zeaxanthin and curcuminoid was 0.98%, 0.58 and 38.64%, respectively, as determined by UV / Vis spectrometer and HPLC.

[0173]

[0122] Example 11: Zeaxanthin isomers 10 mg (5 mg R,S (meso) Zeaxanthin + 5 mg R,R Zeaxanthin) + 200 mg Curcuminoids, (Dose 580mg).

[0174]

[0123] A weighed quantity extract of R,S Zeaxanthin concentrate-micronized (1.38 gm) contain ing 71.31 % (trans, R, S) and 6.60 % (trans, R,R) zeaxanthin content, 1.21gm R,R zeaxanthin concentrate micronized contain ing 70.0 % (trans, R,R) Zeaxanthin content, Curcumin micronized concentrate (39.83 gm) contain ing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxy curcumin 15.94% and bisdem ethoxy curcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. All concentrates were used to formulate oil suspensions of the desired dose. Concentrates were mixed by adding oil (43.60 gm) medium Chain triglycerides (MCT) and 14% w / w (14.0 gm) other ingredients such as 6.0% olive oil, 2.0% linseed oil from flaxseed oil, 2.0% D-Limonene from orange peel oil, 2.0% Lecithin (Phosphatidylcholine Sunflower source), 2.0% mixed tocopherol 70% Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (97.50 gm). In the formulated oil suspension, the particle size was (DV90) 6.36pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,S)- zeaxanthin, (trans, R,R)- zeaxanthin and curcuminoid was 0.99%, 0.93%, and 38.57%, respectively, as determined by UV / Vis spectrometer and HPLC.

[0175]

[0124] Example 12: Zeaxanthin isomers 8 mg (5 mg R,S (meso) Zeaxanthin + 3 mg R,R Zeaxanthin) + 200 mg Curcuminoids, (Dose 580mg).

[0176]

[0125] A weighed quantity extract of R,S Zeaxanthin concentrate-micronized (1.38 gm) contain ing 71.31 % (trans, R, S) and 6.60 % (trans, R,R) zeaxanthin content, 0.69gm R,R Zeaxanthin concentrate micronized contain ing 70.0 % (trans, R,R)-zeaxanthin content, Curcumin micronized concentrate (39.83 gm) contain ing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxy curcumin 15.94% and bisdemethoxy curcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. All concentrates were used to formulate oil suspensions of the desired dose. Concentrates were mixed by adding sunflower oil (57.80 gm) and mixed tocopherol 70% (0.30gm) Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (97.50 gm). In the formulated oil suspension, the particle size was (DV90) 7.23pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,S)- zeaxanthin, (trans, R,R)- zeaxanthin and curcuminoid was 0.99%, 0.57 and 37.93%, respectively, as determined by UV / Vis spectrometer and HPLC.

[0177]

[0126] Example 13: R,S Zeaxanthin 8mg and Curcuminoids 200mg, (Dose 580mg),

[0178]

[0127] A weighed quantity extract of R,S Zeaxanthin concentrate-micronized (2.76 gm) contain ing 71.31 % (trans, R, S) and 6.60 % (trans, R, R) zeaxanthin content, and Curcumin micronized concentrate (39.83 gm) contain ing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxy cur cumin 15.94% and bisdemethoxycurcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. Both concentrates were used to formulate an oil suspension of the desired dose. Concentrates were mixed by adding oil (43.41 gm) medium Chain triglycerides (MCT) and 14% w / w (14.0 gm) other ingredients such as 6.0% olive oil, 2.0% linseed oil from flaxseed oil, 2.0% D-Limonene from orange peel oil, 2.0% Lecithin (Phosphatidylcholine Sunflower source), 2.0% mixed tocopherol 70% Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (97.60 gm). In the formulated oil suspension, the particle size was (DV90) 10.2pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,S)- zeaxanthin, (trans, R, R)-zeaxanthin and curcuminoid was 2.09%, 0.20, and 38.30%, respectively, as determined by UV / Vis spectrometer and HPLC.

[0179]

[0128] Example 14: R,S Zeaxanthin 4mg and Curcuminoids 200mg, (Dose 580mg),

[0180]

[0129] A weighed quantity extract of RS Zeaxanthin concentrate-micronized (1.38 gm) contain ing 71.31 % (trans, R, S) and 6.60 % (trans, R, R) zeaxanthin content, and Curcumin micronized concentrate (39.83 gm) contain ing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxy curcumin 15.94% and bisdemethoxycurcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. Both concentrates were used to formulate an oil suspension of the desired dose. Concentrates were mixed by adding oil (44.79 gm) medium Chain triglycerides (MCT) and 14% w / w (14.0 gm) other ingredients such as 6.0% olive oil, 2.0% linseed oil from flaxseed oil, 2.0% D-Limonene from orange peel oil, 2.0% Lecithin (Phosphatidylcholine Sunflower source), 2.0% mixed tocopherol 70% Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (97.60 gm). In the formulated oil suspension, the particle size was (DV90) 3.74pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,S)- zeaxanthin, (trans, R, R)-zeaxanthin and curcuminoid was 0.99%, 0.11 and 38.28%, respectively, as determined by UV / Vis spectrometer and HPLC.

[0181]

[0130] Example 15: R,S Zeaxanthin 8mg and Curcuminoids 200mg, (Dose 580mg),

[0182]

[0131] A weighed quantity extract of RS Zeaxanthin concentrate-micronized (2.76 gm) contain ing 71.31 % (trans, R, S) and 6.60 % (trans, R, R) zeaxanthin content, and Curcumin micronized concentrate (39.83 gm) contain ing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxy curcumin 15.94% and bisdemethoxy curcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. Both concentrates were used to formulate an oil suspension of the desired dose. Concentrates were mixed by adding sunflower oil (57.11 gm) and mixed tocopherol 70% (0.30gm) Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (96.80 gm). In the formulated oil suspension, the particle size was (DV90) 3.57pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,S)- zeaxanthin, (trans, R, R)-zeaxanthin and curcuminoid was 1.97%, 0.18 and 38.27%, respectively, as determined by UV / Vis spectrometer and HPLC.

[0183]

[0132] Example 16: R,S Zeaxanthin 4mg and Curcuminoids 200mg, (Dose 580mg)

[0184]

[0133] A weighed quantity extract of RS Zeaxanthin concentrate-micronized (1.39 gm) contain ing 71.31 % (trans, R, S) and 6.60 % (trans, R, R) zeaxanthin content, and Curcumin micronized concentrate (39.83 gm) contain ing total curcuminoid content of 96.08% (curcumin 76.04%, desmethoxy curcumin 15.94% and bisdemethoxy curcumin 4.10%) were determined by UV / Vis-spectrophotometer and HPLC. Both concentrates were used to formulate an oil suspension of the desired dose. Concentrates were mixed by adding sunflower oil (58.48 gm) and mixed tocopherol 70% (0.30gm) Sunflower source by using ball mill at room temperature. The resulted oil suspension was sieved and unloaded (96.80 gm). In the formulated oil suspension, the particle size was (DV90) 6.92pm, measured using a Malvern particle size analyser 3000 (wet method). The content of (trans, R,S)- zeaxanthin, (trans, R, R)-zeaxanthin and curcuminoid was 0.98%, 0.10 and 38.28%, respectively, as determined by UV / Vis spectrometer and HPLC.

[0185] A clinical study is being carried out in vivo and is illustrated below; further additional studies are underway.

[0186]

[0134] Example 17. Bioavailability of test products in plasma of Sprague Dawley rats

[0187]

[0135] This study was conducted to evaluate the bioavailability of the test products in rat plasma after oral administration done using Sprague Dawley rats.

[0188] Sixty animals were used in the study investigation, divided into six group as shown below;

[0189] Gl- Vehicle control (MCT oil): lOml / kg

[0190] G2- Ocusorb (lOmg Lutein & 2 mg RR Zeaxanthin) + Curcumin (200 mg): 67 mg / kg

[0191] G3- RR-Zeaxanthin (8 mg) + Curcumin (200 mg): 67 mg / kg

[0192] G4- AREDS2 (Marketed sample as per patent no US 8,603,522 B2): 128 mg / kg

[0193] (AREDS 2 Eye Supplement comprising Lutein & Zeaxanthin, Vitamin C, Bilberry Extract, Vitamin E, Astaxanthin, and Vitamin A).

[0194] G5- Ocusorb (lOmg Lutein & 2 mg RR Zeaxanthin) + Curcumin (100 mg): 35 mg / kg

[0195] G6- RR-Zeaxanthin (8 mg) + Curcumin (100 mg): 35 mg / kg

[0196]

[0136] At the end of treatment period, blood was collected under anaesthesia from all rats. Plasma was separated from blood samples, and Lutein, Zeaxanthin & Curcumin levels were estimated. Plasma was prepared for LC-MS / MS analysis by using solid Phase Extraction (SPE) method. At the time of analysis, the samples were removed from the deep freezer, kept at room temperature, and allowed to thaw. Plasma samples were prepared by adding 50 pL plasma to 2.0 ml centrifuge tube, and 150 pL of precipitating agent (0.1% formic acid in acetonitrile) was added and then vortexed for 2 min. The resulting solution was centrifuged at 4000 rpm for 7 min, and the supernatant layer was separated and injected into the LC-MS / -MS system for analysis.

[0197]

[0137] Results (Figure 1):

[0198] • There was a significant increase in plasma Lutein level in treated Group 2 & 4 as compared to the Normal Control group Gl. • Plasma Zeaxanthin level was significantly increased in all the groups except for G1 with higher concentration achieved in case of G2, 3 & 4 compared to Normal Control group Gl.

[0199] • It was surprisingly found that a significant increase in plasma Curcumin level in G2, & G3 and moderate increase in G5 compared to Normal Control group Gl.

[0200]

[0138] Conclusion: We observed significantly increased levels of lutein, R,R zeaxanthin, and curcumin levels in rat plasma samples after oral administration of the product.

[0201]

[0139] Example 18. Bioavailability of test products in retina of Sprague Dawley rats

[0202]

[0140] This study was conducted to evaluate the bioavailability of the test products in rat retina after oral administration done using Sprague Dawley rats.

[0203]

[0141] Sixty animals were used in the study investigation, divided into six group as shown below;

[0204] Gl- Vehicle control (MCT oil): lOml / kg.

[0205] G2- Ocusorb (lOmg Lutein & 2 mg RR Zeaxanthin) + Curcumin (200 mg): 67 mg / kg.

[0206] G3- RR-Zeaxanthin (8 mg) + Curcumin (200 mg): 67 mg / kg.

[0207] G4- AREDS2 (Marketed sample as per patent no US 8,603,522 B2): 128 mg / kg.

[0208] (AREDS 2 Eye Supplement comprising Lutein & Zeaxanthin, Vitamin C, Bilberry Extract, Vitamin E, Astaxanthin, and Vitamin A).

[0209] G5- Ocusorb (lOmg Lutein & 2 mg RR Zeaxanthin) + Curcumin (100 mg): 35 mg / kg.

[0210] G6- RR-Zeaxanthin (8 mg) + Curcumin (100 mg): 35 mg / kg.

[0211]

[0142] All animals were sacrificed using CO2 gas. The eye was removed for further evaluation, and the retina portion of each rat's eye was separated and homogenized. The retina homogenate were processed to estimate the levels of Lutein, Zeaxanthin & Curcumin by LC-MS / MS method.

[0212]

[0143] Results (Figure 2):

[0213] • There was significant increase in Lutein level in retina in Group 2 and moderate increase in group 4, 5 & 6 as compared to the Normal Control group Gl.

[0214] • Zeaxanthin level in retinal tissue was significantly increased in all the groups compared to Normal Control group Gl, with highest increase in case of G2.

[0215] • There was significant increase in retinal Curcumin level in G2 and moderate increase in G3, 5 & 6 compared to Normal Control group Gl.

[0144] Conclusion: We observed significantly increased levels of lutein, zeaxanthin and curcumin levels in rat retinal tissue after oral administration of the product.

[0216]

[0145] Example 19. Bioavailability of test products in brain tissue of Sprague Dawley rats

[0217]

[0146] This study was conducted to evaluate the bioavailability of the test products in rat brain after oral administration done using Sprague Dawley rats.

[0218]

[0147] Sixty animals were used in the study investigation, divided into six group as shown below;

[0219] Gl- Vehicle control (MCT oil): lOml / kg

[0220] G2- Ocusorb (lOmg Lutein & 2 mg RR Zeaxanthin) + Curcumin (200 mg): 67 mg / kg

[0221] G3- RR-Zeaxanthin (8 mg) + Curcumin (curcuminoids) (200 mg): 67 mg / kg

[0222] G4- AREDS2 (Marketed sample as per patent no US 8,603,522 B2): 128 mg / kg

[0223] G5- Ocusorb (lOmg Lutein & 2 mg RR Zeaxanthin) + Curcumin (100 mg): 35 mg / kg

[0224] G6- RR-Zeaxanthin (8 mg) + Curcumin (100 mg): 35 mg / kg

[0225]

[0148] All animals were sacrificed using CO2 gas. Brain tissue was removed for further evaluation and homogenized. The brain tissues were processed to estimate the levels of Lutein, Zeaxanthin & Curcumin by LC-MS / MS method.

[0226]

[0149] Results (Figure 3):

[0227] • Both lutein and zeaxanthin levels were not detected in the brain for any of the groups.

[0228] • There was significant increase in brain Curcumin level in G2 compared to Normal Control group Gl.

[0229]

[0150] Conclusion: We observed significantly increased levels of curcumin levels in rat brain tissue after oral administration of the product.

[0230]

[0151] Example 20. Evaluation of protective effect of formulations on rat retina by histological analysis

[0231]

[0152] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0232]

[0153] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0233] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) was injected intraperitoneally using a 4% stock solution to induce retinal damage 1. Experimental groups with dose of the product administered in each group Normal control (C)

[0234] 2. Disease control (I)

[0235] 3. Ocusorb (O): 5.5 mg / kg

[0236] 4. Curcumin (Cl): 11 mg / kg

[0237] 5. Curcumin (C2): 22 mg / kg

[0238] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0239] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0240] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0241] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0242] 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0243] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0244] 12. AREDS2 formula (AR): 128 mg / kg

[0245] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0246] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0247]

[0154] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0248]

[0155] Histological analysis: The fixed eye tissues were dehydrated and embedded in paraffin. Sections containing the optic nerve head was cut at 4 pm, deparaffinised, and stained with haematoxylin and eosin (HE). Pathological pictures of retinas were taken under a microscope. Histological alterations were evaluated by changes of the thickness of retinal outer nuclear layer (ONL).

[0249]

[0156] Results: There was significant induction of retinal damage in both LED and NaIO3 induced retinal damage as measured by ONL retinal thickness (pm). However, supplementation with our novel formulations OC1, OC2, ZCl,and ZC2 induced significant protection against the retinal damage with maximum protection in case of AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg and AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg. Of various formulations used in the study protective effect was provided in the following order: AROC2>ARZC2>AR> ZC2>OC2> ZC1>OC1

[0250]

[0157] Conclusions: The novel oral formulations OC1, OC2, ZC1 and ZC2 used in the study provided significant protection against retinal damage induced by LED or NaIO3 with maximum protection observed by AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg and AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0251]

[0158] Histology of rat retina: Outer nuclear layer (ONE) thickness from LED induced retinal damage model (Figure 4)

[0252]

[0159] Histology of rat retina: Outer nuclear layer (ONL) thickness: Comparative analysis of the retinal damage models

[0253]

[0160] Example 21. Effects of supplements on serum oxidative stress markers in retinal damage models

[0254]

[0161] Introduction: Oxidative stress and cumulative oxidative damage with age may cause structural degeneration of the choriocapillaris, resulting in decreased blood flow to the RPE and photoreceptors. Impaired circulation reduces the clearance of lipids and cellular by products, which accumulate as drusen which in turn stimulate an inflammatory response that eventually lead to initiation of AMD pathology. Role of oxidative stress in AMD pathogenesis has been further confirmed by use of anti-oxidant vitamins when supplemented as in AREDS study helped to reduce the risk of disease. Similarly cigarette smoking which increases oxidant load and impairs antioxidant defense mechanisms is a known risk factor for AMD.

[0255]

[0162] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0256]

[0163] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0257]

[0164] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) was injected intraperitoneally using a 4% stock solution to induce retinal damage

[0258] Experimental groups with dose of the product administered in each group

[0259] 1. Normal control (C)

[0260] 2. Disease control (I)

[0261] 3. Ocusorb (O): 5.5 mg / kg

[0262] 4. Curcumin (Cl): 11 mg / kg

[0263] 5. Curcumin (C2): 22 mg / kg

[0264] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0265] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0266] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0267] 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0268] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0269] 12. AREDS2 formula (AR): 128 mg / kg

[0270] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0271] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0272]

[0165] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0273]

[0166] Lipid peroxidation marker, MDA, was analysed by HPLC. Antioxidant Enzymes were measured using commercial kits by ELISA

[0274]

[0167] Results: LED / NaIO3 exposure significantly increased oxidative stress with increased levels of MDA while decreased anti-oxidant capacity in form of decreased Superoxide dismutase (SOD), Catalase and Glutathione peroxidase (GPx) activity. However, supplementation with our novel formulation OC2 and ZC2 resulted in significant reduction in MDA levels while antioxidants, SOD, CAT and GPx activity was significantly increased with maximum effect in case of AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg and AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg. Of various formulations used in the study protective effect was provided in the following order: AROC2>ARZC2>AR>OC2>ZC2

[0275]

[0168] Conclusions: Supplementation with OC2 and ZC2 reduced oxidative stress while improving the anti-oxidant response, with maximal effect by AROC2 followed by ARZC2 (Figure 5)

[0276]

[0169] Example 22. Effect of supplements on markers of oxidative stress regulators (Nrf2 / HO-l signalling)

[0277]

[0170] Introduction: The Nrf2 (Nuclear factor erythroid 2-related factor 2) / HO-1 signalling pathway plays an important role in protecting retinal cells from oxidative stress, inflammation, and damage, which are central factors in the development of macular degeneration, particularly age-related macular degeneration (AMD). Nrf2 is a transcription factor that plays a crucial role in cellular defense mechanisms against oxidative stress. Under normal conditions, Nrf2 is bound to Keapl (Kelch-like ECH-associated protein 1), which keeps it in the cytoplasm. When cells experience oxidative stress, Nrf2 dissociates from Keapl and trans locates to the nucleus, where it activates the expression of various genes involved in antioxidant defense, detoxification, and cell survival. HO-1 (Heme oxygenase-1) is one of the key target genes activated by Nrf2. It is an enzyme that breaks down heme into biliverdin, free iron, and carbon monoxide, all of which have potent antioxidant and anti-inflammatory effects. HO-1 has been shown to protect against retinal cell damage in various eye diseases, including macular degeneration. Nrf2 / HO-l Activation: The activation of the Nrf2 / HO-l pathway can help mitigate this oxidative damage. By enhancing the antioxidant and cytoprotective response, this pathway can reduce the cellular damage that underlies the progression of AMD.

[0278]

[0171] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams, were used for the study.

[0279]

[0172] LED-induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0280]

[0173] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) was injected intraperitoneally using a 4% stock solution to induce retinal damage

[0281] Experimental groups with dose of the product administered in each group

[0282] 1. Normal control (C)

[0283] 2. Disease control (I)

[0284] 3. Ocusorb (O): 5.5 mg / kg

[0285] 4. Curcumin (Cl): 11 mg / kg

[0286] 5. Curcumin (C2): 22 mg / kg

[0287] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0288] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0289] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0290] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0291] 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0292] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0293] 12. AREDS2 formula (AR): 128 mg / kg

[0294] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0295] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0296]

[0174] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0175] Western blot analysis for protein markers: Tissues were lysed in a protein lysis buffer, lysates centrifuged at 12,000 rpm at 4°C for 10 min and supernatants were collected. Protein concentrations is determined and protein lysates (20 pg of each sample) are separated by SDS / polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with primary antibodies in 5% non-fat dry milk for overnight at 4°C. Membranes were washed three times with PBST and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody at room temperature for 2 h. The density of each band were measured using Image! software. The protein expression level of each molecular were normalized to p-actin protein expression level.

[0297]

[0176] Results: LED / NaIO3 exposure significantly decreased the level of both Nrf2 and HO-1 which was restored significantly by supplementation with all groups and with maximum effect in case of AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg and AREDS2 + RR- Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg.

[0298]

[0177] Conclusions: LED / NaIO3 exposure reduced the levels of both Nrf2 and HO-1 in retinal tissues and supplementation with OC2 and ZC2 restored the levels with maximal effect by combination of AROC2 or ARZC2.

[0299]

[0178] Relative expression of protein Nrf2 and HO-1 in retinal tissues (Figure 6)

[0300]

[0179] Example 23. Effect of supplements on Autophagy-related proteins 5 and 7 (Atg5 and Atg7)

[0301]

[0180] Introduction: Autophagy is a crucial cellular process for maintaining cellular homeostasis, especially in the retina, where cells are subjected to high levels of oxidative stress and metabolic activity. In age-related macular degeneration (AMD), a degenerative disease of the retina, autophagy dysfunction has been implicated in disease progression. Specifically, autophagy-related proteins ATG5 and ATG7 are vital in the formation of autophagosomes, which are essential for the degradation and recycling of cellular components. ATG5 is involved in the formation of the autophagosome, a critical structure in the autophagic process. ATG5 is a key part of the ATG12-ATG5 conjugate, which plays a central role in autophagosome membrane elongation and maturation. ATG7 functions similarly to a El-like enzyme in autophagy and is responsible for the conjugation of ATG12 to ATG5, as well as other key reactions in the autophagy pathway. ATG7 is essential for the vesicle nucleation step of autophagy.

[0302]

[0181] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0303]

[0182] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0304]

[0183] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) was injected intraperitoneally using a 4% stock solution to induce retinal damage

[0305] Experimental groups with dose of the product administered in each group

[0306] 1. Normal control (C)

[0307] 2. Disease control (I)

[0308] 3. Ocusorb (O): 5.5 mg / kg

[0309] 4. Curcumin (Cl): 11 mg / kg

[0310] 5. Curcumin (C2): 22 mg / kg

[0311] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0312] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0313] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0314] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0315] 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0316] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0317] 12. AREDS2 formula (AR): 128 mg / kg

[0318] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0319] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0320]

[0184] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0321]

[0185] Western blot analysis for protein markers: Tissues were lysed in a protein lysis buffer, lysates centrifuged at 12,000 rpm at 4°C for 10 min and supernatants were collected. Protein concentrations is determined and protein lysates (20 pg of each sample) are separated by SDS / polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with primary antibodies in 5% non-fat dry milk for overnight at 4°C. Membranes were washed three times with PBST and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody at room temperature for 2 h. The density of each band were measured using Image! software. The protein expression level of each molecular were normalized to p-actin protein expression level.

[0322]

[0186] Results: LED / NaIO3 exposure significantly increased the levels of ATG5 & 7 which was restored significantly by supplementation with all groups and with maximal effect by AROC2 followed by ARZC2

[0323]

[0187] Conclusions: LED / NaIO3 exposure significantly increased the levels of ATG5 & 7 which was restored by supplementation with OC2 and ZC2 with maximal effect by AROC2 followed by ARZC2.

[0324]

[0188] Relative expression of protein ATG5 and ATG7 in retinal tissues (Figure 7)

[0325]

[0189] Example 24. Effect of supplements on ER stress related proteins ATF6 and ATF4

[0326]

[0190] Introduction: Endoplasmic reticulum (ER) stress plays a crucial role in the pathophysiology of age-related macular degeneration (AMD), as it impacts the function of retinal cells, particularly retinal pigment epithelium (RPE) cells. Two critical ER stress-related proteins — ATF6 (Activating Transcription Factor 6) and ATF4 (Activating Transcription Factor 4) — are central players in the unfolded protein response (UPR), a cellular response mechanism activated during ER stress to restore homeostasis. Dysfunction in the UPR and the failure to resolve ER stress can lead to cellular damage and contribute to AMD.

[0327]

[0191] ATF6 is an ER membrane-bound protein that, when activated by ER stress, translocates to the nucleus to activate the transcription of genes involved in restoring ER homeostasis, such as those encoding chaperones (e.g., BiP / GRP78) and ER-associated degradation (ERAD) proteins. ATF6 activation is part of the UPR's adaptive response to mitigate ER stress and prevent cellular damage. However, if the stress is too severe or prolonged, ATF6 can also contribute to apoptotic signalling. ATF4 is another key protein involved in the UPR, primarily activated by PERK (Protein kinase RNA-like endoplasmic reticulum kinase), one of the main sensors of ER stress. ATF4 upregulates the expression of genes involved in amino acid metabolism, redox regulation, and apoptosis under stress conditions. When ER stress is not resolved, ATF4 can promote apoptosis by activating pro-apoptotic factors such as CHOP (C / EBP homologous protein), which leads to cell death.

[0192] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0328]

[0193] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0329]

[0194] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) is injected intraperitoneally using a 4% stock solution to induce retinal damage

[0330] Experimental groups with dose of the product administered in each group

[0331] 1. Normal control (C)

[0332] 2. Disease control (I)

[0333] 3. Ocusorb (O): 5.5 mg / kg

[0334] 4. Curcumin (Cl): 11 mg / kg

[0335] 5. Curcumin (C2): 22 mg / kg

[0336] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0337] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0338] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0339] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0340] 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0341] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0342] 12. AREDS2 formula (AR): 128 mg / kg

[0343] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0344] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0345]

[0195] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0346]

[0196] Western blot analysis for protein markers: Tissues were lysed in a protein lysis buffer, lysates centrifuged at 12,000 rpm at 4°C for 10 min and supernatants were collected. Protein concentrations is determined and protein lysates (20 pg of each sample) are separated by SDS / polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with primary antibodies in 5% non-fat dry milk for overnight at 4°C. Membranes were washed three times with PBST and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody at room temperature for 2 h.. The density of each band were measured using Image! software. The protein expression level of each molecular were normalized to p-actin protein expression level.

[0347]

[0197] Results: LED / NaIO3 exposure significantly increased the levels of ATF4 & 6 which was restored significantly by supplementation with all groups, and maximal effect by AROC2 followed by ARZC2

[0348]

[0198] Conclusions: LED / NaIO3 exposure significantly increased the levels of ATF4 & 6 which was restored by supplementation with OC2 and ZC2 with maximal effect by AROC2 followed by ARZC2.

[0349]

[0199] Relative expression of protein ATF4 & 6 in retinal tissues (Figure 8)

[0350]

[0200] Example 25. Effect of supplements on Mammalian target of the rapamycin (mTOR)

[0351]

[0201] Introduction: The mammalian target of rapamycin (mTOR) is a central regulator of cell growth, metabolism, and survival. It is involved in numerous cellular processes, including protein synthesis, autophagy, and cellular responses to nutrients, energy, and stress. Given its critical role in cellular homeostasis, mTOR has been implicated in the pathophysiology of several diseases, including age-related macular degeneration (AMD), a leading cause of blindness in older adults. In the context of AMD, mTOR plays a complex role, with its activity influencing several disease pathways, including oxidative stress, inflammation, and autophagy. Both dry AMD (non-exudative) and wet AMD (exudative) are influenced by dysregulated mTOR signalling, leading to retinal cell damage, particularly in the retinal pigment epithelium (RPE) and photoreceptors.

[0352]

[0202] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0353]

[0203] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0354]

[0204] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) is injected intraperitoneally using a 4% stock solution to induce retinal damage

[0355] Experimental groups with dose of the product administered in each group

[0356] 1. Normal control (C)

[0357] 2. Disease control (I)

[0358] 3. Ocusorb (O): 5.5 mg / kg 4. Curcumin (Cl): 11 mg / kg

[0359] 5. Curcumin (C2): 22 mg / kg

[0360] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0361] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0362] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0363] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0364] 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0365] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0366] 12. AREDS2 formula (AR): 128 mg / kg

[0367] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0368] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0369]

[0205] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0370]

[0206] Western blot analysis for protein markers: Tissues were lysed in a protein lysis buffer, lysates centrifuged at 12,000 rpm at 4°C for 10 min and supernatants were collected. Protein concentrations is determined and protein lysates (20 pg of each sample) are separated by SDS / polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with primary antibodies in 5% non-fat dry milk for overnight at 4°C. Membranes were washed three times with PBST and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody at room temperature for 2 h. The density of each band were measured using Image! software. The protein expression level of each molecular were normalized to [3-actin protein expression level.

[0371]

[0207] Results: LED / NaIO3 exposure significantly increased the levels of mTOR which was restored significantly by supplementation with all groups and maximal effect by AROC2 followed by ARZC2

[0372]

[0208] Conclusions: LED / NaIO3 exposure significantly increased the levels of mTOR which was restored by supplementation with OC2 and ZC2 with maximal effect by AROC2 followed by ARZC2

[0373]

[0209] Relative expression of protein mTOR in retinal tissues (Figure 9)

[0374]

[0210] Example 26. Effect of supplements on Apoptotic markers in retinal damage models

[0211] Introduction: Apoptosis, or programmed cell death, plays a significant role in the pathogenesis of Age-related Macular Degeneration (AMD), a leading cause of vision loss, particularly in older adults. In AMD, the retinal cells, particularly those in the macula (the central part of the retina responsible for sharp vision), undergo various stressors that trigger apoptosis. This can contribute to both the dry and wet forms of AMD, which have different underlying mechanisms but both involve retinal cell death.

[0375]

[0212] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0376]

[0213] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0377]

[0214] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) is injected intraperitoneally using a 4% stock solution to induce retinal damage

[0378] Experimental groups with dose of the product administered in each group

[0379] 1. Normal control (C)

[0380] 2. Disease control (I)

[0381] 3. Ocusorb (O): 5.5 mg / kg

[0382] 4. Curcumin (Cl): 11 mg / kg

[0383] 5. Curcumin (C2): 22 mg / kg

[0384] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0385] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0386] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0387] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0388] 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0389] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0390] 12. AREDS2 formula (AR): 128 mg / kg

[0391] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0392] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0393]

[0215] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0394]

[0216] Western blot analysis for protein markers: Tissues were lysed in a protein lysis buffer, lysates centrifuged at 12,000 rpm at 4°C for 10 min and supernatants were collected. Protein concentrations is determined and protein lysates (20 pg of each sample) are separated by SDS / polyacrylamide gel and transferred to a nitrocellulose membrane.

[0395]

[0217] The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with primary antibodies in 5% non-fat dry milk for overnight at 4°C. Membranes were washed three times with PBST and incubated with horseradish peroxidase conjugated secondary anti -mouse antibody at room temperature for 2 h. The density of each band were measured using Image! software. The protein expression level of each molecular were normalized to p-actin protein expression level.

[0396]

[0218] Results: LED / NaIO3 exposure significantly decreased pro-apoptotic Bax and caspase-3 levels which was restored significantly by supplementation with all groups and with maximal effect by AROC2 followed by ARZC2

[0397]

[0219] Conclusions: LED / NaIO3 exposure significantly decreased pro-apoptotic Bax and caspase-3 levels which was restored by supplementation with OC2 and ZC2 with maximal effect by AROC2 followed by ARZC2.

[0398]

[0220] Relative expression of apoptotic markers in retinal tissues (Figure 10)

[0399]

[0221] Example 27. Effect of supplements on NF-KB that drives inflammation in AMD

[0400]

[0222] Introduction: NF-KB (Nuclear Factor kappa B) is a key regulator of immune and inflammatory responses, and it plays a significant role in the pathogenesis of Age-related Macular Degeneration (AMD). Its activation has been implicated in the chronic inflammation and tissue damage that are characteristic of both the dry and wet forms of AMD. NF-KB is a family of transcription factors that regulate the expression of various genes involved in immune response, inflammation, and cell survival. In its inactive state, NF-KB is typically bound to inhibitory proteins (such as IKBS) in the cytoplasm. Upon activation, these inhibitory proteins are degraded, allowing NF-KB dimers (most commonly p65 / p50) to translocate to the nucleus, where they initiate the transcription of genes involved in inflammation and cell survival. .

[0401]

[0223] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0402]

[0224] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0225] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) is injected intraperitoneally using a 4% stock solution to induce retinal damage

[0403] Experimental groups with dose of the product administered in each group

[0404] 1. Normal control (C)

[0405] 2. Disease control (I)

[0406] 3. Ocusorb (O): 5.5 mg / kg

[0407] 4. Curcumin (Cl): 11 mg / kg

[0408] 5. Curcumin (C2): 22 mg / kg

[0409] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0410] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0411] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0412] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0413] 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0414] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0415] 12. AREDS2 formula (AR): 128 mg / kg

[0416] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0417] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0418]

[0226] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0419]

[0227] Western blot analysis for protein markers: Tissues were lysed in a protein lysis buffer, lysates centrifuged at 12,000 rpm at 4°C for 10 min and supernatants were collected. Protein concentrations is determined and protein lysates (20 pg of each sample) are separated by SDS / polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with primary antibodies in 5% non-fat dry milk for overnight at 4°C. Membranes were washed three times with PBST and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody at room temperature for 2 h. The density of each band were measured using Image! software. The protein expression level of each molecular were normalized to [3-actin protein expression level.

[0420]

[0228] Results: LED / NaIO3 exposure significantly increased the levels of NF-KB which was restored significantly by supplementation with all groups and with maximal effect by AROC2 followed by ARZC2

[0229] Conclusions: LED / NaIO3 exposure significantly increased the levels of NF-KB which was restored by supplementation with OC2 and ZC2 with maximal effect by ARZC2 followed by AROC2.

[0421]

[0230] Relative expression of NF-KB in retinal tissues (Figure 11)

[0422]

[0231] Example 28. Effect of supplements on NLRP Inflammasome: mediator of inflammation in AMD

[0423]

[0232] Introduction: The NLRP inflammasome is a key component of the innate immune system and plays a critical role in inflammatory responses. It has been implicated in the pathogenesis of Age-related Macular Degeneration (AMD), particularly in the dry form of the disease, where inflammation and immune activation are major contributors to retinal damage. The NLRP inflammasome is a multiprotein complex that is formed in response to cellular stress, infection, or damage. The inflammasome is composed of NLRP (NOD-like receptor family pyrin domain containing) proteins, such as NLRP1, NLRP3, and NLRC4, along with ASC (apoptosis- associated speck-like protein containing a CARD) and caspase-1. The assembly of this inflammasome complex leads to the activation of caspase-1, which subsequently triggers the production of pro-inflammatory cytokines, mainly IL-ip and IL-18. These cytokines are central to inflammation and immune responses. In the context of AMD, NLRP3 is the most studied member of the NLRP inflammasome.

[0424]

[0233] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0425]

[0234] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0426]

[0235] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) is injected intraperitoneally using a 4% stock solution to induce retinal damage

[0427] Experimental groups with dose of the product administered in each group

[0428] 1. Normal control (C)

[0429] 2. Disease control (I)

[0430] 3. Ocusorb (O): 5.5 mg / kg

[0431] 4. Curcumin (Cl): 11 mg / kg

[0432] 5. Curcumin (C2): 22 mg / kg

[0433] 6. Ocusorb + Curcumin (OC1): 35 mg / kg 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0434] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0435] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0436] 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0437] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0438] 12. AREDS2 formula (AR): 128 mg / kg

[0439] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0440] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0441]

[0236] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0442]

[0237] Western blot analysis for protein markers: Tissues were lysed in a protein lysis buffer, lysates centrifuged at 12,000 rpm at 4°C for 10 min and supernatants were collected. Protein concentrations is determined and protein lysates (20 pg of each sample) are separated by SDS / polyacrylamide gel and transferred to a nitrocellulose membrane.

[0443]

[0238] The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with primary antibodies in 5% non-fat dry milk for overnight at 4°C. Membranes were washed three times with PBST and incubated with horseradish peroxidase conjugated secondary anti -mouse antibody at room temperature for 2 h. The density of each band were measured using Image! software. The protein expression level of each molecular were normalized to [3-actin protein expression level.

[0444]

[0239] Results: LED / NaIO3 exposure significantly increased the levels of NLRP which was restored significantly by supplementation with all groups and with maximal effect by AROC2 followed by ARZC2

[0445]

[0240] Conclusions: LED / NaIO3 exposure significantly increased the levels of NLRP which was restored by supplementation with OC2 and ZC2 with maximal effect by AROC2 followed by ARZC2.

[0446]

[0241] Relative expression of NLRP Inflammasome in retinal tissues (Figure 12)

[0447]

[0242] Example 29. Effect of supplements on serum inflammatory markers during retinal damage in rats

[0243] Introduction: Inflammation plays a central role in the pathogenesis of Age-related Macular Degeneration (AMD), both in its dry (atrophic) and wet (neovascular) forms. In AMD, the inflammatory response is triggered by various factors, including oxidative stress, drusen accumulation, and retinal damage, leading to the activation of immune cells and the release of pro-inflammatory cytokines and other markers. These inflammatory markers are key contributors to the progressive damage of retinal cells and the breakdown of the blood-retina barrier. Tumor Necrosis Factor-alpha (TNF-cx) is a potent pro-inflammatory cytokine that promotes inflammation, cell death, and the breakdown of extracellular matrix components. It is involved in the pathogenesis of both dry and wet AMD by inducing oxidative stress, apoptosis of retinal cells, and activating other inflammatory pathways, including the NF-KB pathway. Interleukin-1 beta (IL-ip) is a key cytokine produced by activated microglia, retinal pigment epithelium (RPE) cells, and other retinal cells. It is involved in the regulation of the immune response in AMD and is a product of NLRP3 inflammasome activation. IL-ip promotes inflammation and induces damage in retinal cells, especially in the dry form of AMD, where it contributes to RPE dysfunction and photoreceptor degeneration. Interleukin-6 (IL -6) is another important cytokine that is upregulated in AMD and is associated with inflammation and cellular damage. It is produced by various retinal cells, including RPE cells, and can exacerbate inflammation in the retina. IL-6 is involved in the activation of other inflammatory pathways and contributes to retinal cell apoptosis and degeneration.

[0448]

[0244] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0449]

[0245] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0450]

[0246] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) is injected intraperitoneally using a 4% stock solution to induce retinal damage

[0451] Experimental groups with dose of the product administered in each group

[0452] 1. Normal control (C)

[0453] 2. Disease control (I)

[0454] 3. Ocusorb (O): 5.5 mg / kg

[0455] 4. Curcumin (Cl): 11 mg / kg

[0456] 5. Curcumin (C2): 22 mg / kg

[0457] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0458] 7. Ocusorb + Curcumin (OC2): 67 mg / kg 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0459] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0460] 10. RR_Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0461] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0462] 12. AREDS2 formula (AR): 128 mg / kg

[0463] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0464] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0465]

[0247] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0466]

[0248] ELISA assay: Blood was collected from rats at the end of the experiment and serum was separated and ELISA was carried out using established commercial kits for various inflammatory markers.

[0467]

[0249] Results: LED / NaIO3 exposure significantly increased pro-inflammatory response upon LED / NaIO3 exposure which is significantly restored by supplementation with all groups and maximum response in the case of combination of supplements; AROC2 and ARZC2.

[0468]

[0250] Conclusions: LED / NaIO3 exposure significantly increased pro-inflammatory response upon LED / NaIO3 exposure which is restored by supplementation with OC2 and ZC2 with maximum response in case of combination of supplements; AROC2 and ARZC2.

[0469]

[0251] Relative expression of serum inflammatory markers. (Figure 13)

[0470]

[0252] Example 30. Effect of supplements on Monocyte chemoattractant protein-1 (MCP1)

[0471]

[0253] Introduction: Monocyte Chemoattractant Protein-1 (MCP-1), also known as CCL2 (C-C Motif Ligand 2), is a chemokine that plays a critical role in the recruitment and activation of monocytes, macrophages, and other immune cells to sites of inflammation. In Age-related Macular Degeneration (AMD), MCP-1 is implicated in driving the chronic inflammation that underlies both the dry (atrophic) and wet (neovascular) forms of the disease. MCP-1 is upregulated in the retina and choroid in AMD, and its production is primarily induced by inflammatory stimuli, such as oxidative stress, drusen accumulation, and the activation of the NLRP3 inflammasome. Here’s how MCP-1 contributes to AMD pathogenesis. MCP-1 is a potent chemokine that attracts monocytes, which are precursors of macrophages, to sites of injury or inflammation. In AMD, the presence of drusen (debris accumulating between the retinal pigment epithelium and Bruch's membrane) and oxidative stress triggers MCP-1 release from retinal pigment epithelium (RPE) cells, microglia, and endothelial cells. Once monocytes are recruited to the retina, they differentiate into macrophages, which then release additional pro-inflammatory cytokines (e.g., TNF-cx, IL-ip) and contribute to retinal inflammation, oxidative stress, and cell death.

[0472]

[0254] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0473]

[0255] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0474]

[0256] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) is injected intraperitoneally using a 4% stock solution to induce retinal damage

[0475] Experimental groups with dose of the product administered in each group

[0476] 1. Normal control (C)

[0477] 2. Disease control (I)

[0478] 3. Ocusorb (O): 5.5 mg / kg

[0479] 4. Curcumin (Cl): 11 mg / kg

[0480] 5. Curcumin (C2): 22 mg / kg

[0481] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0482] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0483] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0484] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0485] 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0486] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0487] 12. AREDS2 formula (AR): 128 mg / kg

[0488] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0489] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0490]

[0257] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0491]

[0258] Western blot analysis for protein markers: Tissues were lysed in a protein lysis buffer, lysates centrifuged at 12,000 rpm at 4°C for 10 min and supernatants were collected. Protein concentrations are determined and protein lysates (20 pg of each sample) are separated by SDS / polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with primary antibodies in 5% non-fat dry milk for overnight at 4°C. Membranes were washed three times with PBST and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody at room temperature for 2 h. The density of each band were measured using Image! software. The protein expression level of each molecular were normalized to p-actin protein expression level.

[0492]

[0259] Results: LED / NaIO3 exposure significantly increased the levels of MCP1 which was restored significantly by supplementation with all groups and maximal effect by AROC2 followed by ARZC2.

[0493]

[0260] Conclusions: LED / NaIO3 exposure significantly increased the levels of MCP1 which was restored by supplementation with OC2 and ZC2 with maximal effect by AROC2 followed by ARZC2.

[0494]

[0261] Relative expression of MCP1 in retinal tissues (Figure 14)

[0495]

[0262] Example 31. Effect of supplements on NCAM protein

[0496]

[0263] Introduction: Neural Cell Adhesion Molecule (NCAM) is a glycoprotein involved in cellcell adhesion and plays an important role in neuronal development, synaptic plasticity, and tissue regeneration. NCAM is part of the immunoglobulin superfamily and is expressed in various tissues, including the retina, where it is crucial for maintaining the integrity and function of retinal cells. In the context of Age-related Macular Degeneration (AMD), NCAM has been implicated in several ways, particularly in the inflammatory response, cell adhesion, tissue repair, and neuroprotection within the retina. Although the role of NCAM in AMD is not as widely studied as other inflammatory markers or pathways, there are several aspects where it may influence AMD pathogenesis.

[0497]

[0264] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0498]

[0265] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0499]

[0266] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) is injected intraperitoneally using a 4% stock solution to induce retinal damage

[0500] Experimental groups with dose of the product administered in each group 1. Normal control (C)

[0501] 2. Disease control (I)

[0502] 3. Ocusorb (O): 5.5 mg / kg

[0503] 4. Curcumin (Cl): 11 mg / kg

[0504] 5. Curcumin (C2): 22 mg / kg

[0505] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0506] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0507] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0508] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0509] 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0510] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0511] 12. AREDS2 formula (AR): 128 mg / kg

[0512] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0513] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0514]

[0267] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0515]

[0268] Western blot analysis for protein markers: Tissues were lysed in a protein lysis buffer, lysates centrifuged at 12,000 rpm at 4°C for 10 min and supernatants were collected. Protein concentrations is determined and protein lysates (20 pg of each sample) are separated by SDS / polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with primary antibodies in 5% non-fat dry milk for overnight at 4°C. Membranes were washed three times with PBST and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody at room temperature for 2 h. The density of each band were measured using Image! software. The protein expression level of each molecular were normalized to -actin protein expression level.

[0516]

[0269] Results: LED / NaIO3 exposure significantly decreased the levels of NCAM which was restored significantly by supplementation with all groups and maximal effect by AROC2 followed by ARZC2

[0517]

[0270] Conclusions: LED / NaIO3 exposure significantly decreased the levels of NCAM which was restored by supplementation with OC2 and ZC2 with maximal effect by AROC2 followed by ARZC2.

[0518]

[0271] Relative expression of NCAM in retinal tissues (Figure 15)

[0272] Example 32. Effect of supplements on Intercellular adhesion molecule-1 (ICAM-1) protein

[0519]

[0273] Introduction: Intercellular Adhesion Molecule-1 (ICAM-1) is an important cell adhesion molecule that plays a crucial role in immune cell trafficking and the inflammatory response. It is a member of the immunoglobulin superfamily and is primarily expressed on the surface of endothelial cells, epithelial cells, and immune cells such as macrophages and neutrophils. ICAM-1 is involved in the adhesion and transmigration of leukocytes (e.g., T-cells, monocytes) from the bloodstream into tissues during inflammation. In the context of Age-related Macular Degeneration (AMD), ICAM-1 plays a significant role in the chronic inflammation observed in both the dry (atrophic) and wet (neovascular) forms of the disease. Here's a detailed look at how ICAM-1 contributes to the pathogenesis of AMD. Chronic inflammation is a central feature of AMD, and ICAM-1 is a key mediator in recruiting immune cells to sites of retinal injury and inflammation. The accumulation of drusen (debris between the retinal pigment epithelium (RPE) and the Bruch’s membrane) and oxidative stress are two primary factors that drive inflammatory responses in AMD. ICAM-1 is upregulated in the retina during these conditions, contributing to immune cell infiltration and exacerbating the disease.

[0520]

[0274] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0521]

[0275] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0522]

[0276] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) is injected intraperitoneally using a 4% stock solution to induce retinal damage

[0523] Experimental groups with dose of the product administered in each group

[0524] 1. Normal control (C)

[0525] 2. Disease control (I)

[0526] 3. Ocusorb (O): 5.5 mg / kg

[0527] 4. Curcumin (Cl): 11 mg / kg

[0528] 5. Curcumin (C2): 22 mg / kg

[0529] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0530] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0531] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0532] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0533] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0534] 12. AREDS2 formula (AR): 128 mg / kg

[0535] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0536] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0537]

[0277] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0538]

[0278] Western blot analysis for protein markers: Tissues were lysed in a protein lysis buffer, lysates centrifuged at 12,000 rpm at 4°C for 10 min and supernatants were collected. Protein concentrations is determined and protein lysates (20 pg of each sample) are separated by SDS / polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with primary antibodies in 5% non-fat dry milk for overnight at 4°C. Membranes were washed three times with PBST and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody at room temperature for 2 h. The density of each band were measured using Image! software. The protein expression level of each molecular were normalized to [3-actin protein expression level.

[0539]

[0279] Results: LED / NaIO3 exposure significantly increased the levels of ICAM1 which was restored significantly by supplementation with all groups and maximal effect by AROC2 followed by ARZC2

[0540]

[0280] Conclusions: LED / NaIO3 exposure significantly increased the levels of ICAM1 which was restored by supplementation with OC2 and ZC2 with maximal effect by AROC2 followed by ARZC2.

[0541]

[0281] Relative expression of ICAM1 in retinal tissues (Figure 16)

[0542]

[0282] Example 33. Effect of supplements on markers of angiogenesis (VEGF)

[0543]

[0283] Introduction: Vascular Endothelial Growth Factor (VEGF) is a key signaling protein involved in the regulation of angiogenesis (the formation of new blood vessels), vascular permeability, and inflammation. In the context of Age-related Macular Degeneration (AMD), VEGF plays a crucial role, particularly in the wet (neovascular) form of the disease. It is central to the pathological changes that lead to vision impairment in AMD patients. In wet AMD, VEGF is responsible for the abnormal growth of new, leaky blood vessels beneath the retina in a process known as choroidal neovascularization (CNV). The development of these new blood vessels is one of the hallmarks of wet AMD and leads to significant vision loss.

[0544]

[0284] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0545]

[0285] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0546]

[0286] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) is injected intraperitoneally using a 4% stock solution to induce retinal damage

[0547] Experimental groups with dose of the product administered in each group

[0548] 1. Normal control (C)

[0549] 2. Disease control (I)

[0550] 3. Ocusorb (O): 5.5 mg / kg

[0551] 4. Curcumin (Cl): 11 mg / kg

[0552] 5. Curcumin (C2): 22 mg / kg

[0553] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0554] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0555] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0556] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0557] 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0558] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0559] 12. AREDS2 formula (AR): 128 mg / kg

[0560] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0561] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0562]

[0287] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0563]

[0288] Western blot analysis for protein markers: Tissues were lysed in a protein lysis buffer, lysates centrifuged at 12,000 rpm at 4°C for 10 min and supernatants were collected. Protein concentrations is determined and protein lysates (20 pg of each sample) are separated by SDS / polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with primary antibodies in 5% non-fat dry milk for overnight at 4°C. Membranes were washed three times with PBST and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody at room temperature for 2 h. The density of each band were measured using Image! software. The protein expression level of each molecular were normalized to p-actin protein expression level.

[0564]

[0289] Results: LED / NaIO3 exposure reduced the levels of both VEGF in retinal tissues and supplementation with all groups significantly restored the levels with maximal effect by combination of AROC2 or ARZC2

[0565]

[0290] Conclusions: LED / NaIO3 exposure reduced the levels of both VEGF in retinal tissues and supplementation with OC2 and ZC2 restored the levels with maximal effect by combination of AROC2 or ARZC2.

[0566]

[0291] Relative expression of VEGF in retinal tissues (Figure 17)

[0567]

[0292] Example 34. Effect of supplements on TGF

[0568]

[0293] Introduction: Transforming Growth Factor Beta (TGF- ) is a multifunctional cytokine that regulates a wide range of cellular processes, including cell growth, differentiation, immune response, and extracellular matrix (ECM) remodeling. It plays a critical role in maintaining tissue homeostasis and promoting wound healing. In the context of Age-related Macular Degeneration (AMD), TGF- has a complex role, influencing both inflammation and fibrosis, as well as contributing to the pathophysiology of the disease. TGF-P is involved in several key aspects of AMD, particularly in the inflammatory response, vascular changes, and fibrosis that occur in both the dry (atrophic) and wet (neovascular) forms of the disease. The following outlines the major roles of TGF-P in AMD

[0569]

[0294] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0570]

[0295] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0571]

[0296] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) is injected intraperitoneally using a 4% stock solution to induce retinal damage

[0572] Experimental groups with dose of the product administered in each group

[0573] 1. Normal control (C)

[0574] 2. Disease control (I)

[0575] 3. Ocusorb (O): 5.5 mg / kg 4. Curcumin (Cl): 11 mg / kg

[0576] 5. Curcumin (C2): 22 mg / kg

[0577] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0578] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0579] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0580] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg

[0581] 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0582] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0583] 12. AREDS2 formula (AR): 128 mg / kg

[0584] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0585] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0586]

[0297] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0587]

[0298] Western blot analysis for protein markers: Tissues were lysed in a protein lysis buffer, lysates centrifuged at 12,000 rpm at 4°C for 10 min and supernatants were collected. Protein concentrations is determined and protein lysates (20 pg of each sample) are separated by SDS / polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with primary antibodies in 5% non-fat dry milk for overnight at 4°C. Membranes were washed three times with PBST and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody at room temperature for 2 h. The density of each band were measured using Image! software. The protein expression level of each molecular were normalized to p-actin protein expression level.

[0588]

[0299] Results: LED / NaIO3 exposure increased TGFp levels in retinal tissues which was restored significantly by supplementation with all groups and maximal effect by combination of AROC2 or ARZC2

[0589]

[0300] Conclusions: LED / NaIO3 exposure increased TGFp levels in retinal tissues which was restored by supplementation with OC2 and ZC2 with maximal effect by combination of AROC2 or ARZC2.

[0590]

[0301] Relative expression of TGFp in retinal tissues (Figure 18)

[0591]

[0302] Example 35. Effect of supplements on neuronal proteins (GAP43 & GFAP)

[0303] Introduction: GAP-43 (Growth-Associated Protein 43) and GFAP (Glial Fibrillary Acidic Protein) are proteins that play significant roles in the central nervous system (CNS) and are involved in neuronal plasticity, regeneration, and response to injury. In the context of Age- Related Macular Degeneration (AMD), these proteins are implicated in the retinal injury response, neurodegeneration, and glial activation. Both GAP-43 and GFAP are used as biomarkers of retinal changes associated with AMD and contribute to understanding the disease mechanisms. GAP-43 is a protein found in the nervous system, particularly in neuronal growth cones and synaptic terminals, where it is involved in axonal growth, neuroplasticity, and neuronal regeneration. GAP-43 is often used as a marker for neuronal injury and plasticity, and its expression is generally upregulated in response to neurodegeneration and injury. GFAP is a protein expressed primarily in astrocytes and Muller cells (the retinal glial cells), and it is used as a marker of gliosis (the activation of glial cells in response to injury). Muller cells in the retina play a critical role in maintaining retinal integrity and supporting neuronal health. In response to retinal injury or degeneration, GFAP expression in Muller cells is upregulated, indicating glial activation and neuroinflammation. In AMD, GFAP expression in Muller cells is often used as a marker for retinal gliosis, which is a reaction to retinal damage and neurodegeneration.

[0592]

[0304] Animal models used: Albino Sprague-Dawley rats, aged 8-10 weeks, with an average weight of 180-200 grams were used for the study.

[0593]

[0305] LED induced retinal damage model: Rats were anesthetized and their pupils dilated and exposed to 750 lux of diffuse white LED light for 48 hours to induce retinal damage.

[0594]

[0306] NaIO3-induced retinal degeneration model: Rats were anesthetized, and NaIO3 (50 mg / kg) is injected intraperitoneally using a 4% stock solution to induce retinal damage

[0595] Experimental groups with dose of the product administered in each group

[0596] 1. Normal control (C)

[0597] 2. Disease control (I)

[0598] 3. Ocusorb (O): 5.5 mg / kg

[0599] 4. Curcumin (Cl): 11 mg / kg

[0600] 5. Curcumin (C2): 22 mg / kg

[0601] 6. Ocusorb + Curcumin (OC1): 35 mg / kg

[0602] 7. Ocusorb + Curcumin (OC2): 67 mg / kg

[0603] 8. RR-Zeaxanthin (Zl): 0.8 mg / kg

[0604] 9. RR-Zeaxanthin (Z2): 1.6 mg / kg 10. RR-Zeaxanthin + Curcumin (ZC1): 35 mg / kg

[0605] 11. RR-Zeaxanthin + Curcumin (ZC2): 67 mg / kg

[0606] 12. AREDS2 formula (AR): 128 mg / kg

[0607] 13. AREDS2 + Ocusorb + Curcumin (AROC2): 128 + 67 mg / kg

[0608] 14. AREDS2 + RR-Zeaxanthin + Curcumin (ARZC2): 128 + 67 mg / kg

[0609]

[0307] Supplementation: Rats were administered supplements in MCT oil via oral gavage. Supplementation began three days prior to the induction of retinal damage and continued for 14 days following the injury.

[0610]

[0308] Western blot analysis for protein markers: Tissues were lysed in a protein lysis buffer, lysates centrifuged at 12,000 rpm at 4°C for 10 min and supernatants were collected. Protein concentrations is determined and protein lysates (20 pg of each sample) are separated by SDS / polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, followed by incubation with primary antibodies in 5% non-fat dry milk for overnight at 4°C. Membranes were washed three times with PBST and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody at room temperature for 2 h. The density of each band were measured using Image! software. The protein expression level of each molecular were normalized to [3-actin protein expression level.

[0611]

[0309] Results: LED / NaIO3 exposure significantly decreased the levels of GAP43 which was restored significantly by supplementation with all groups and maximal effect by AROC2 followed by ARZC2. LED / NaIO3 exposure significantly increased the levels of GFAP which was restored significantly by supplementation with all groups and maximal effect by AROC2 followed by ARZC2.

[0612]

[0310] Conclusions: LED / NaIO3 exposure significantly decreased the levels of GAP43 which was restored by supplementation with OC2 and ZC2 with maximal effect by AROC2 followed by ARZC2. LED / NaIO3 exposure significantly increased the levels of GFAP which was restored by supplementation with maximal effect by AROC2 followed by ARZC2.

[0613]

[0311] Relative expression of GAP43 and GFAP in retinal tissues (Figure 19)

[0614]

[0312] A mechanistic study for AREDS / Retinal damage is planned and is underway, which will be captured in the complete filing. A provisional outline of studies is brief as follows:

[0615]

[0313] These studies will help to understand further the detailed molecular pathways involved in retinal damage and subsequent protection by the invented formulations' dosages.

[0314] Damage to RPE cells caused by oxidative stress is closely related to the pathogenesis of AMD. Oxidative stress induced by H2O2 or Sodium iodate often results in classical oxidative damage in RPE cells and studies have shown that antioxidants can efficiently maintain the viability of RPE cells in a state of oxidative stress. A large number of studies suggest that naturally occurring antioxidant agents, such as blueberry anthocyanins and Lutein and zeaxanthin, can ameliorate age-related changes associated with oxidative damage in RPE cells.

[0616]

[0315] We propose to treat the RPE cell line with H2O2 or Sodium iodate, and then cell viability will be assessed, followed by assessment of cell death (apoptosis), reactive oxygen species, and anti-oxidant levels. This will be followed by focused protein and transcript profiling to explore specific mechanistic pathways associated with pathogenesis of AMD. Similarly detailed mechanistic studies also will be initiated using rat retinal tissue exposed to LED- induced retinal damage and subsequently protection by our novel formulations.

Claims

CLAIMS1. The stable and bioavail able oral lipophilic nutrient composition comprising zeaxanthin isomers, preferably RR Zeaxanthin, curcuminoids, and lutein.

2. The stable and bioavailable oral lipophilic nutrient composition as claimed in claim 1 comprises from about 1% w / w to about 10% w / w zeaxanthin isomers and lutein together.

3. The stable and bioavailable oral lipophilic nutrient composition, as claimed in any of the preceding claims, comprises about 5 mg to about 30 mg lutein.

4. The stable and bioavailable oral lipophilic nutrient composition comprising zeaxanthin isomers, preferably RR Zeaxanthin and curcuminoids.

5. The stable and bioavailable oral lipophilic nutrient composition claimed in any of the preceding claims comprises about 20% w / w to about 50% w / w curcuminoids.

6. The stable and bioavailable oral lipophilic nutrient composition, as claimed in any of the preceding claims, comprises from about 50 mg to about 300 mg curcuminoids.

7. The stable and bioavailable oral lipophilic nutrient composition as claimed in any of the preceding claims zeaxanthin isomers comprises R, R-zeaxanthin.

8. The stable and bioavailable oral lipophilic nutrient composition as claimed in any of the preceding claims comprises from about 1 mg to about 15 mg R, R-zeaxanthin.

9. The stable and bioavailable oral lipophilic nutrient composition as claimed in any of the preceding claims further comprises at least one nutraceutically acceptable excipient.

10. The nutraceutically acceptable excipients as claimed in claim 9 are selected from the group consisting of carrier, solubility enhancer, antioxidant, flavoring agent, solubilizer, and the like.

11. The carrier as claimed in claim 10 is selected from the group consisting of polyethylene glycol 200, polyethylene glycol 400, medium chain triglyceride oil, 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, hydrolysed starch and their mixtures thereof.

12. The carrier, as claimed in claim 11, is present in an amount ranging from about 40% to about 60% by weight of the composition.

13. The carrier as claimed in claim 11 is medium-chain triglyceride oil.

14. The antioxidant as claimed in claim 10 is selected from the group consisting of tocopherol, mixed tocopherol, glutathione, citric acid, alfa-tocopherol, rosemary extract, lipoic acid, selenium, sodium ascorbate, flavonoid, ascorbyl palmitate, resveratrol, and their combinations thereof.

15. The antioxidant as claimed in claim 14 is present in an amount ranging from about 0.5% to about 5% by weight of the composition.

16. The antioxidant, as claimed in claim 14, is mixed tocopherol 70%-SF in oil.

17. The solubility enhancer, as claimed in claim 10, is selected from the group consisting of soya oil, palm-kernel oil, olive oil, cotton oil, medium chain triglyceride oil (MCT oil), maize oil, coconut oil, palm oil, d- limonene, sesame oil, linseed oil (flaxseed oil), sunflower oil, walnut oils, cedar leave oil, hazelnut oil, corn oil, fish oil, hydrogenated castor oil, safflower oil, peanut oil, corn oil, cotton seed oil, canola oil, Wintergreen oil, tea tree oil, thyme oil, castor oil, bay oil, ajwain oil, clove oil, anise oil, eucalyptus oil, cassia oil, nutmeg oil, oil of bitter almonds, oil of sage and their combinations thereof.

18. The flavouring agent, as claimed in claim 10, is selected from the group consisting of cinnamon oil, d-limonene, menthol, orange, vanillin, lime, cocoa, fruit essences, including berry, apple, pineapple, pear, peach, blueberry, kiwi, raspberry, cherry, plum, strawberry, and apricot, peppermint oil, citrus oils such as lemon, lime and grapefruit oils and their combinations thereof.

19. The flavouring agent, as claimed in claim 18, is present in an amount ranging from about 0.5% to about 5% by weight of the composition.

20. The flavouring agent, as claimed in claim 18, is d-limonene.

21. The solubilizer as claimed in claim 10 is selected from the group consisting of egg yolk lecithin, hydroxy propyl beta-cyclodextrin, vitamin E TPGS, polysorbate 80, poloxamer 188, polysorbate 20, linoleic acid, PEG 4000, oleic acid, sorbitan trioleate, polyethylene glycol 7-stearate, sodium ascorbate, phosphatidylcholine or Lys phosphatidylcholine, and their combinations thereof.

22. The solubilizer, as claimed in claim 21, is present in an amount ranging from about 0.5% to about 5% by weight of the composition.

23. The solubilizer, as claimed in claim 21, is phosphatidylcholine.

24. The stable and bioavailable oral lipophilic nutrient composition comprising R, R- zeaxanthin, curcuminoids, and lutein, along with the nutraceutically acceptable excipients selected from medium chain triglyceride oil, olive oil, mixed tocopherol, flaxseed or linseed oil, d-limonene, phosphatidyl, and their combination thereof.

25. The stable and oral lipophilic nutrient composition comprising R, R-zeaxanthin, and curcuminoids, along with the nutraceutically acceptable excipients selected from medium chain triglyceride oil, olive oil, mixed tocopherol, flaxseed or linseed oil, d- limonene, phosphatidyl, and their combination thereof.

26. The stable and bioavailable oral lipophilic nutrient composition for preventing and improving or reducing the risk of progression of age-related eye disorders (AREDS), comprising R, R-zeaxanthin, curcuminoids, and lutein.

27. The stable and bioavailable oral lipophilic nutrient composition for preventing and improving or reducing the AREDS, age-related macular degeneration (AMD), eye disorder / disease, visual dysfunction, and retinopathy, comprising R, R-zeaxanthin, curcuminoids, and lutein.

28. The stable and bioavailable oral lipophilic nutrient composition comprising R, R- zeaxanthin, curcuminoids, and lutein is used for improving Outer nuclear layer (ONL) thickness, oxidative stress markers selected from MDA, SOD, CAT, and GSH-Px, oxidative stress regulators (Nrf2 / HO-l signaling, autophagy-related proteins 5 and 7 (Atg5 and Atg7), ATF4 & ATF 6, mammalian target of the rapamycin (mTOR), pro- apoptotic Bax and caspase-3 levels, NF-KB, NLRP inflammasome, serum inflammatory markers selected from IL-1B, TNF-alpha, and IL -6, Monocyte chemoattractant protein-1 (MCP1), Neural Cell Adhesion Molecule (NCAM), Intercellular Adhesion Molecule 1 (ICAM-1), angiogenesis marker VEGF, TGFp, neuronal proteins like GAP43 & GFAP .

29. The stable and bioavailable oral lipophilic nutrient composition for preventing and improving or reducing the AREDS, age related macular degeneration, eye disorder / disease, visual dysfunction and retinopathy, comprising:R, R- zeaxanthin;Curcuminoids;• lutein; and• nutraceutically acceptable excipients selected from medium chain triglyceride oil, olive oil, mixed tocopherol, flaxseed or linseed oil, d-limonene, phosphatidyl, and their combination thereof.

30. The stable and bioavailable oral lipophilic nutrient composition for preventing and improving or reducing the risk of progression of age-related eye disorder, comprising R, R-zeaxanthin, and curcuminoids.

31. The stable and bioavailable oral lipophilic nutrient composition for preventing and improving or reducing the AREDS, age-related macular degeneration, eye disorder / disease, visual dysfunction, and retinopathy, comprising R, R-zeaxanthin, and curcuminoids.

32. The stable and bioavailable oral lipophilic nutrient composition for preventing and improving or reducing the AREDS, age-related macular degeneration, eye disorder / disease, visual dysfunction, and retinopathy, comprising• R, R-zeaxanthin;• curcuminoids; and• nutraceutically acceptable excipients selected from medium chain triglyceride oil, olive oil, mixed tocopherol, flaxseed or linseed oil, d-limonene, phosphatidyl, and their combination thereof.

33. The process to prepare the oral dosage form comprising R, R-zeaxanthin, curcuminoids, and lutein, wherein the process comprises:(a) adding the mixture of lutein and R, R-zeaxanthin, along with curcuminoids, in the air jet mill;(b) micronizing the mixture of step (a);(c) adding the inactive excipients selected from carrier, solubility enhancer, antioxidant, flavoring agent, solubilizer, and the like;(d) preparing the oil suspension using a ball mill / homogenizer; and(e) obtaining the final product.

34. The process to prepare the oral dosage form comprising R, R-zeaxanthin, and curcuminoids, wherein the process comprises:(a) adding the mixture of R, R-zeaxanthin, and curcuminoids in the air jet mill; (b) micronizing the mixture of step (a);(c) adding the inactive excipients selected from carriers, solubility enhancers, antioxidants, flavoring agents, solubilizers, and the like:(d) preparing the oil suspension using a ball mill / homogenizer; and(e) obtaining the final product.

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