Compositions comprising carotenoids and / or bilberry for protecting the eyes of a mammalian subject from or reducing or treating damage due to exposure to blue light

Abstract

Described herein are methods and compositions that comprise an amount of at least one active agent, such as at least one carotenoid and an ingredient or extract of bilberry, preferably an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and an additive.

Description

COMPOSITIONS COMPRISING CAROTENOIDS AND / OR BILBERRY FOR PROTECTING THE EYES OF A MAMMALIAN SUBJECT FROM OR REDUCING OR TREATING DAMAGE DUE TO EXPOSURE TO BLUE LIGHT RELATED APPLICATIONS

[0001] The present patent document claims the benefit of the filing date under 35 U.S.C. §119(e) of Provisional U.S. Patent Application Serial No.63 / 557,173, filed February 23, 2024, which is hereby incorporated by reference. REFERENCE TO APPENDIX [CD ROM / SEQUENCE LISTING]

[0002] The instant application contains a Sequence Listing XML which is being submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 29, 2025, is named “503086_5002857_Seq_Listing.xml” and is 14,408 bytes in size. BACKGROUND

[0003] High-energy visible light has a wavelength in the range of 380 to 530 nm and is present in sunlight, fluorescent light, and light-emitting diode (LED) light. Blue light, having a wavelength in the range of 450 to 495 nm, is high-energy visible light and is related to the pathogenesis of age-related macular degeneration (AMD) and retinitis pigmentosa.

[0004] Blue light is part of the visible light spectrum and has shorter waves, with wavelengths between about 400 and 495 nanometers (Zou, L., et al., (2015)). Blue light can reach the retina may cause potential phototoxic retinal damage that can generate reactive oxygen species (ROS) (Behar-Cohen, F., et al., (2011); Godley, B.F., et al., (2005); Ham, W.T., Jr., et al., (1976); Algvere, P.V., et al., (2006)). It has been speculated that blue light radiation may cause retinal damage or contribute to the development of age-related macular degeneration (AMD) (Taylor, H.R., (1992)). Retinal exposure to excessive levels of blue light can also induce photochemical damage to retinal pigment epithelium (RPE).

[0005] AMD is a degenerative disease that causes blindness in people 60–65 years old (Lim, L.S., et al., (2012)). In addition, oxidative stress, apoptosis, and inflammation play important roles in both the onset and development of AMD (Abu-Amero, K.K., et al., Nutrients, 8:200 (2016)).

[0006] AMD is a main cause of central blindness among the working, aged population worldwide. As of 2020, it affects more than 190 million people globally with the prevalence expected to increase to 288 million people by 2040 as the proportion of elderly persons in the population increases (Apte R.S. (2021)). It affects males and females equally, and it is more common in those of European or North American ancestry. In 2013, it was the fourth most common cause of blindness, after cataracts, preterm birth, and glaucoma (Vos, T., et al. (2015)). It most commonly occurs in people over the age of fifty and in the United States is the most common cause of vision loss in this age group. About 0.4% of people between 50 and 60 have the disease, while it occurs in 0.7% of people 60 to 70, 2.3% of those 70 to 80, and nearly 12% of people over 80 years old (Mehta, S. (2015)).

[0007] The retinal pigment epithelium (RPE) is in the outer retina and the RPE is exposed to high oxygen tension and abundant light influx including blue light, rendering it highly susceptible to oxidative stress. This disproportionate burden of oxidative stress contributes to the development of several retinal diseases (Strauss, O., (2005)).

[0008] Carotenoids, a group of pigments found in various fruits and vegetables, have been considered to play a crucial role in promoting eye health due to their antioxidant properties and other beneficial effects (Khachik, F., et al., (2002)). Approximately 40 natural carotenoids are typically consumed in the human diet, but only 15–20 are routinely detectable in human serum and tissues, including lycopene, beta-carotene, lutein, and zeaxanthin.

[0009] Some epidemiological and experimental studies suggest that the increased consumption of dietary antioxidants can prevent and treat many ophthalmic disorders associated with oxidative stress. Clinical studies showed that supplementation with antioxidant nutrients, including β-carotene, and vitamins C and E, are associated with a reduced risk of AMD.

[0010] For example, lutein and zeaxanthin are found in high concentrations in the macula of the eye. Landrum, J.T. et al (1997) conducted a study of the macular pigment using a lutein supplement and found that it may confer macular protection via antioxidant and light-screening properties.

[0011] Bilberries, rich in polyphenols which belong to the flavonoid family (Määttä- Riihinen KR, et al., (2004); Koponen JM, et al. (2007)), are generally recognized as low energy foods, containing little or no saturated fat, cholesterol, and sodium, a good source of dietary fiber, and the antioxidant vitamins E and C. The anthocyanins are the most abundant flavonoids present in bilberries, and have been shown to be potential antioxidants (Mazza G, et al. (2002)). These compounds can penetrate the nervous system even after short-term feeding (Kalt W, et al. (2008)), and they have been reported to be able to reduce age-associated oxidative stress and the related cognitive decline (Barros D, et al. (2006)).

[0012] With improvement in working and living conditions and the changes in people's lifestyles, more and more exposure to blue light occurs. The prevention and control of blue light damage is becoming more and more important, and effective anti- blue light products are needed. SUMMARY

[0013] In one embodiment, described herein is a composition for protecting the eyes of a mammalian subject from or treating damage to the eyes of the subject due to exposure of the subject’s eyes to blue light comprising: (i) an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and (ii) an additive; wherein the composition effectively protects the eyes of a mammalian subject from or treats damage to the eyes of the subject due to exposure of the subject to blue light. In the composition, the ratio of lutein to zeaxanthin to β-carotene to lycopene to bilberry may be 5:1:1:3.2:12.5. In the composition, the amount of an ingredient or extract of bilberry may be in the range from about 0.1 mg to about 50 mg. In the composition, the amount of at least one of lutein, zeaxanthin, β-carotene, and lycopene may be in the range from about 0.1 mg to about 25 mg. The composition may be formed as tablets, gels, capsules, pills, caplets, dragees, granules, powders, or effervescent tablets, liquid, sprays,functional foods, gums, chewing gum, gummi candy, taffy, caramel candy, fudge, degradable thin films, nondegradable thin films, hard candy, liquids, drinks, or beverages. The composition may be an immediate release composition, a mixed-release composition, or an enterically-coated composition. The composition is effective in protecting against blue light-induced DNA damage. The composition is effective in protecting against blue light-induced oxidative stress. The composition is effective in protecting human retinal pigment epithelium against blue light irradiation. The additive comprises at least one of: (i) a sweetener selected from high fructose corn syrup, mannose, maltose, glucose polymers, sucrose, glucose, dextrose, lactose, galactose, fructose, polysaccharides, rice syrup, honey, saccharin, cyclamates, acetosulfam, sorbitol, sucralose, xylitol, erythritol, Stevia extract, L-aspartyl-L-phenyl-alanine ester, L-aspartyl-D-alanine alkyl amides, L- aspartyl-L-1-hydroxymethylalkaneamide, and L-aspartyl-1-hydroxyethylalkaneamide; (ii) a pH-adjusting agent selected from hydrochloric acid, citric acid, sodium hydrogen carbonate, potassium hydroxide, sodium hydroxide, and sodium carbonate; (iii) a preservative selected from sorbic acid, benzoic acid, sodium benzoate, calcium benzoate, potassium benzoate, potassium sorbate, calcium sorbate, and sodium sorbate; and (iv) a flavoring agent selected from almond oil, amaretto oil, anethole, anise oil, benzaldehyde, blackberry, black walnut oil, blueberry, caraway, caraway oil, cardamom oil, cardamom seed, cherry juice, cherry syrup, cinnamon, cinnamon oil, cinnamon water, citric acid, citric acid syrup, clove oil, cocoa, coriander oil, dextrose, eriodictyon, ethyl acetate, ethyl vanillin, fennel oil, ginger, glucose, glycerin, glycyrrhiza, grape, honey, lavender oil, lemon oil, lime, mannitol, methyl salicylate, myristica oil, orange oil, orange peel, orange syrup, peppermint, peppermint oil, peppermint water, phenylethyl alcohol, pineapple, raspberry juice, raspberry syrup, rosemary oil, rose oil, rose water, sarsaparilla syrup, sorbitol, spearmint, spearmint oil, strawberry, sucrose, thyme oil, tolu balsam, tropical, vanilla, vanillin, and wild cherry syrup. The composition may be a food product. The composition may be a dietary supplement. The composition may be for consumption once or more times a day. In certain embodiments, the composition comprises lycopene, lutein, zeaxanthin and β-carotene, wherein the ratio of lycopene to lutein to zeaxanthin to β- carotene in the composition is 5.7:10:2:0.5.

[0014] Another embodiment relates to a composition comprising: about 10 mg lutein; about 2.0 mg zeaxanthin; about 2.0 mg beta carotene; about 6.4 mg lycopene; about 25 mg bilberry; and an additive. The composition effectively protects the eyes of a mammalian subject from or treats damage to the eyes of the subject due to exposure to blue light. The composition is effective in protecting against blue light-induced DNA damage. The composition is effective in protecting against blue light-induced oxidative stress. The composition is effective in protecting human retinal pigment epithelium against blue light irradiation. The composition may be formed as tablets, gels, capsules, pills, caplets, dragees, granules, powders, or effervescent tablets, liquid, sprays, functional foods, gums, chewing gum, gummi candy, taffy, caramel candy, fudge, degradable thin films, nondegradable thin films, hard candy, liquids, drinks, or beverages. The composition may be an immediate release composition, a mixed-release composition, or an enterically-coated composition. In the composition, the additive comprises at least one of: (i) a sweetener selected from high fructose corn syrup, mannose, maltose, glucose polymers, sucrose, glucose, dextrose, lactose, galactose, fructose, polysaccharides, rice syrup, honey, saccharin, cyclamates, acetosulfam, sorbitol, sucralose, xylitol, erythritol, Stevia extract, L-aspartyl-L-phenyl-alanine ester, L-aspartyl-D-alanine alkyl amides, L- aspartyl-L-1-hydroxymethylalkaneamide, and L-aspartyl-1-hydroxyethylalkaneamide; (ii) a pH-adjusting agent selected from hydrochloric acid, citric acid, sodium hydrogen carbonate, potassium hydroxide, sodium hydroxide, and sodium carbonate; (iii) a preservative selected from sorbic acid, benzoic acid, sodium benzoate, calcium benzoate, potassium benzoate, potassium sorbate, calcium sorbate, and sodium sorbate; and (iv) a flavoring agent selected from almond oil, amaretto oil, anethole, anise oil, benzaldehyde, blackberry, black walnut oil, blueberry, caraway, caraway oil, cardamom oil, cardamom seed, cherry juice, cherry syrup, cinnamon, cinnamon oil, cinnamon water, citric acid, citric acid syrup, clove oil, cocoa, coriander oil, dextrose, eriodictyon, ethyl acetate, ethyl vanillin, fennel oil, ginger, glucose, glycerin, glycyrrhiza, grape, honey, lavender oil, lemon oil, lime, mannitol, methyl salicylate, myristica oil, orange oil, orange peel, orange syrup, peppermint, peppermint oil, peppermint water, phenylethyl alcohol, pineapple, raspberry juice, raspberry syrup, rosemary oil, rose oil, rose water, sarsaparilla syrup, sorbitol, spearmint, spearmint oil, strawberry, sucrose, thyme oil, tolu balsam, tropical,vanilla, vanillin, and wild cherry syrup. The composition may be a food product. The composition may be a dietary supplement. The composition may be for consumption once or more times a day.

[0015] Yet another embodiment relates to a composition for preventing, reducing, or treating one or more signs and / or symptoms of an ophthalmic condition in a subject resulting from exposure of the subject’s eyes to blue light comprising: (i) an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and (ii) an additive; wherein the composition effectively prevents, reduces, or treats one or more signs and symptoms of an ophthalmic condition in the subject. The ophthalmic condition is a blinding eye disorder, such as age-related macular degeneration, cataract, glaucoma, or retinitis pigmentosa. In the composition, the ratio of lutein to zeaxanthin to β-carotene to lycopene to bilberry is 5:1:1:3.2:12.5. The composition may be formed as tablets, gels, capsules, pills, caplets, dragees, granules, powders, or effervescent tablets, liquid, sprays, functional foods, gums, chewing gum, gummi candy, taffy, caramel candy, fudge, degradable thin films, nondegradable thin films, hard candy, liquids, drinks, or beverages. In the composition, the additive comprises at least one of: (i) a sweetener selected from high fructose corn syrup, mannose, maltose, glucose polymers, sucrose, glucose, dextrose, lactose, galactose, fructose, polysaccharides, rice syrup, honey, saccharin, cyclamates, acetosulfam, sorbitol, sucralose, xylitol, erythritol, Stevia extract, L-aspartyl-L-phenyl-alanine ester, L-aspartyl- D-alanine alkyl amides, L-aspartyl-L-1-hydroxymethylalkaneamide, and L-aspartyl-1- hydroxyethylalkaneamide; (ii) a pH-adjusting agent selected from hydrochloric acid, citric acid, sodium hydrogen carbonate, potassium hydroxide, sodium hydroxide, and sodium carbonate; (iii) a preservative selected from sorbic acid, benzoic acid, sodium benzoate, calcium benzoate, potassium benzoate, potassium sorbate, calcium sorbate, and sodium sorbate; and (iv) a flavoring agent selected from almond oil, amaretto oil, anethole, anise oil, benzaldehyde, blackberry, black walnut oil, blueberry, caraway, caraway oil, cardamom oil, cardamom seed, cherry juice, cherry syrup, cinnamon, cinnamon oil, cinnamon water, citric acid, citric acid syrup, clove oil, cocoa, coriander oil, dextrose, eriodictyon, ethyl acetate, ethyl vanillin, fennel oil, ginger, glucose, glycerin, glycyrrhiza, grape, honey, lavender oil, lemon oil, lime, mannitol, methylsalicylate, myristica oil, orange oil, orange peel, orange syrup, peppermint, peppermint oil, peppermint water, phenylethyl alcohol, pineapple, raspberry juice, raspberry syrup, rosemary oil, rose oil, rose water, sarsaparilla syrup, sorbitol, spearmint, spearmint oil, strawberry, sucrose, thyme oil, tolu balsam, tropical, vanilla, vanillin, and wild cherry syrup. The composition may be a food product. The composition may be a dietary supplement. The composition may be for consumption once or more times a day. The one or more signs and symptoms of the ophthalmic condition may be selected from the group consisting of: vision loss, blurred or decreased vision in one or both eyes; distortion of vision; blind spots; distorted vision in the form of metamorphopsia; central scotomas; shadows; missing areas of vision; slow recovery of visual function after exposure to bright light; decreased visual acuity; decreased ability to discern colors; decreased or lost contrast sensitivity; visual hallucinations; flashing lights; and accelerated aging of the eyes. In certain embodiments, the composition comprises lycopene, lutein, zeaxanthin and β-carotene, wherein the ratio of lycopene to lutein to zeaxanthin to β-carotene in the composition is 5.7:10:2:0.5.

[0016] Yet another embodiment relates to a method for treating or preventing blue light-induced ophthalmic condition, or one or more sign or symptom thereof in a mammalian subject at risk of or having blue light-induced ophthalmic condition, or one or more sign or symptom thereof, comprising: administering to the subject a therapeutically effective amount of a composition that is provided in the form selected from the group consisting of capsules, tablets, liquids, and powders for oral ingestion, the composition comprising: (i) an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and (ii) a pharmaceutically acceptable additive. The administering may be intraocularly, iontophoretically, orally, topically, systemically, intravenously, subcutaneously, or intramuscularly. The ophthalmic condition may be a blinding eye disorder. The ophthalmic condition may be age-related macular degeneration, cataract, glaucoma, or retinitis pigmentosa. The ratio of lutein to zeaxanthin to β-carotene to lycopene to bilberry in the administered composition may be 5:1:1:3.2:12.5. The administering may be once or more times a day. The one or more signs and symptoms of an ophthalmic condition may be selected from the group consisting of: vision loss, blurred or decreased vision in one or both eyes; distortion ofvision; blind spots; distorted vision in the form of metamorphopsia; central scotomas; shadows; missing areas of vision; slow recovery of visual function after exposure to bright light; decreased visual acuity; decreased ability to discern colors; decreased or lost contrast sensitivity; visual hallucinations; and flashing lights. In certain embodiments, in the method, the composition comprises lycopene, lutein, zeaxanthin and β-carotene, wherein the ratio of lycopene to lutein to zeaxanthin to β-carotene in the composition is 5.7:10:2:0.5.

[0017] Yet further embodiment relates to a method of protecting a user’s eyes from the harmful effect of blue light, comprising: administering to the user a therapeutically effective amount of a composition that is provided in the form selected from the group consisting of capsules, tablets, liquids, and powders for oral ingestion, the composition comprising: (i) an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and (ii) a pharmaceutically acceptable additive. In certain embodiments, in the method, the composition comprises lycopene, lutein, zeaxanthin and β-carotene, wherein the ratio of lycopene to lutein to zeaxanthin to β- carotene in the composition is 5.7:10:2:0.5.

[0018] Yet another embodiment relates to a method for enhancing protective mechanisms against oxidative stress damage induced by blue light exposure and mitigating DNA damage and cellular senescence in a user’s eyes, comprising: administering to the user a therapeutically effective amount of a composition that is provided in the form selected from the group consisting of capsules, tablets, liquids, and powders for oral ingestion, the composition comprising: (i) an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and (ii) a pharmaceutically acceptable additive. In certain embodiments, in the method, the composition comprises lycopene, lutein, zeaxanthin and β-carotene, wherein the ratio of lycopene to lutein to zeaxanthin to β-carotene in the composition is 5.7:10:2:0.5.

[0019] Yet another embodiment relates to a composition for protecting the eyes of a mammalian subject from or treating damage to the eyes of the subject due to exposure of the subject’s eyes to blue light comprising: (i) an amount of an ingredient or extract of bilberry; and (ii) an additive; wherein the composition effectively protects the eyes of amammalian subject from or treats damage to the eyes of the subject due to exposure to blue light.

[0020] Yet a further embodiment relates to a composition for enhancing protective mechanisms against oxidative stress damage induced by blue light exposure and mitigating DNA damage and cellular senescence in a user’s eyes, comprising lycopene, lutein, zeaxanthin and β-carotene; and an additive; wherein the ratio of lycopene to lutein to zeaxanthin to β-carotene in the composition is 5.7:10:2:0.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0022] Figure 1. Carotenoids reduced blue-light-induced ROS generation in ARPE-19 cells. The cells were exposed to blue light for 1.5 and 24 h and microscopic pictures were taken to show the cell morphology. After blue light exposure, the cells were fixed and stained with DAPI. Scale bar: 50 µm (A). p16INK4Aand p21WAF1 / CIP1mRNA expression levels were evaluated by qRT-PCR following exposure to BLUE LIGHT using GAPDH as a housekeeping gene (B). Cells were treated with indicated concentrations of carotenoids (lycopene, lutein / zeaxanthin (Lu / Zea), or β-carotene) for 48 h. Cell viability was measured by MTT assay (C). Cells were pre-treated with indicated conditions for 48 h and ROS production was analyzed. Each dietary carotenoid and blend dose was 400 µg / mL, and the N-acetylcysteine (NAC) dose was 1 mM (D). Blue-light-induced ROS generation was detected by using CellROXTMstaining and immunofluorescence microscopy. The nuclei were stained with DAPI (E). Data are shown as mean±SD (n=3). Lutein / zeaxanthin; Lu / Zea. #p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. vehicle.

[0023] Figure 2. Antioxidant activity of individual carotenoid. FRAP assay was used to monitor the antioxidant activities of the carotenoids at indicated doses. Images are representative of three independent replicates. Data are shown as mean±SD (n=3). A significant difference (*p < 0.05, **p < 0.01) was determined in comparison to control cells.

[0024] Figure 3. Effect of individual carotenoid on Nrf2 activation and mRNA levels of Nrf2 target genes. ARPE-19 cells were incubated with the indicated condition for 48 h, and ARE-responsive luciferase reporter activity was analyzed (A). The mRNA levels of Nrf2 target genes including Ho-1 (B), Nqo1 (C), Gclc (D), and Sod1 (E) were analyzed by qRT-PCR. (F) Heatmap depicts relative mRNA expression level of antioxidant mechanism associated genes from indicated group. All values were normalized to the expression of GAPDH. Data are shown as mean±SD (n=3). A significant difference (*p < 0.05, **p < 0.01) was determined in comparison to control cells. Lutein / zeaxanthin; Lu / Zea

[0025] Figure 4. Carotenoids alleviate blue light-induced DNA damage and cellular senescence in ARPE-19 cells. Cells were pretreated with 400 µg / ml of carotenoids (lycopene, lutein / zeaxanthin (Lu / Zea), or β-carotene) for 48 h and the cells were exposed to blue light. Representative immunofluorescence images of p-H2AX immunofluorescence (green) observed with a fluorescence microscope are shown. The location of the nucleus was indicated by counterstaining with DAPI (blue) (A). SA-β-gal stain images (B) and their activity (C) were analyzed in ARPE-19 cells with indicated conditions. After blue light irradiation, the cells were maintained in a fresh medium for 24 h. Data are shown as mean±SD (n=3). #p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. vehicle. Scale bar: 50 µm, Lutein / zeaxanthin; Lu / Zea

[0026] Figure 5. Effect of dietary carotenoids on the expression of DNA repair mechanism associated genes. The cells were treated with 400 µg / mL of individual carotenoid for 48 h. Heatmap display that relative mRNA expression level of DNA repair mechanism associated genes from indicated group. All values were normalized to the expression of GAPDH (A). Representative image of an ICW plate. The cells were treated with the indicated concentration for 48 h. Signals from anti-OGG1 appear as green fluorophores. Signals from CellTag (cell number normalization) appear as red fluorophores. Quantification of fluorescence signal intensity compared with untreated control and intensity of CellTag was used for normalization (B). Data are shown as mean±SD (n= 3). A significant difference (*p < 0.05, **p < 0.01) was determined in comparison to control cells. Lutein / zeaxanthin; Lu / Zea.

[0027] Figure 6. Effect of carotenoids on the secretion of IL-6 and VEGF. The cells were pretreated with 400 µg / mL of carotenoids for 48 h. After blue light irradiation the cells were incubated in a fresh medium. IL-6 (A) and VEGF (B) secretion levels were measured in 24 h supernatants. Data are shown as mean±SD (n= 3). #p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. vehicle. Lutein / zeaxanthin; Lu / Zea.

[0028] Figure 7. Bilberry reduced blue light-induced ROS formation in ARPE-19 cells. Cells were pre-treated with indicated conditions for 48 h and ROS production was analyzed. Each ingredient dose was 400 µg / mL. After the pre-treatment, the cells were exposed to BL for 1.5 and 24 h. (A) Blue-light-induced ROS generation was detected by using CellROXTMstaining and (B) immunofluorescence microscopy. The nuclei were stained with DAPI. Scale bar: 50 µm. Data are shown as mean±SD (n=3). Lutein / zeaxanthin; Lu / Zea.

[0029] Figure 8. Bilberry reduced blue light-induced DNA damage in ARPE-19 cells. Cells were pretreated with 400 µg / ml of each ingredient (lycopene, lutein / zeaxanthin (lu / Zea), β-carotene, or bilberry) for 48 h and the cells were exposed to blue light. Representative immunofluorescence images of p-H2AX immunofluorescence (green) observed with a fluorescence microscope are shown. The location of the nucleus was indicated by counterstaining with DAPI (blue). Data are shown as mean±SD (n=3). Scale bar: 100 µm, Lutein / zeaxanthin; Lu / Zea.

[0030] Figure 9. The antioxidant activity of Bilberry extract measured by α-TEAC assay.

[0031] Figure 10. The antioxidant activity of Bilberry extract measured by FRAP assay.

[0032] Figure 11. Analysis of antioxidant properties of individual carotenoids. DETAILED DESCRIPTION OF THE DRAWINGS AND THE PRESENTLY PREFERRED EMBODIMENTS

[0033] The present invention is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the invention. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to thoseskilled in the art. Functionally equivalent methods and apparatuses within the scope of the invention, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this invention is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0034] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0035] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0036] Described herein are methods and nutritional and pharmaceutical compositions for protecting the eyes of a mammalian subject from (i.e., protecting the health of the eyes), and / or reducing or treating damage to the eyes of the subject due to exposure to blue light.

[0037] Provided here is evidence of the anti-oxidative effect of carotenoids, lycopene, lutein, zeaxanthin, β-carotene, and an extract of bilberry against oxidative stress induced by blue light irradiation in ARPE-19 cells, a human RPE cell line derived from the normal eyes. The ROS levels, DNA damage, cellular senescence, and underlying mechanisms of antioxidant effect were analyzed. The study elucidated the protective effect of carotenoids and / or bilberry on the blue light-induced RPE damage model.

[0038] General

[0039] In practicing the present technology, many conventional techniques in molecular biology, protein biochemis-try, cell biology, immunology, microbiology andrecombinant DNA are used. These techniques are well-known and are explained in, e.g., Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989); DNA Cloning: A Practical Approach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis, Gait, Ed (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds (1985); Transcription and Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the series, Meth Enzymol., (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor Laboratory, NY, 1987); and Meth. Enzymol., Vols.154 and 155, Wu & Grossman, and Wu, Eds., respectively.

[0040] Unless otherwise defined, all terms technical and scientific used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention.

[0041] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 ingredients refers to groups having 1, 2, or 3 ingredients. Similarly, a group having 1-5 ingredients refers to groups having 1, 2, 3, 4, or 5 ingredients, and so forth.

[0042] All combinations of method or process steps as used herein may be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

[0043] As used herein, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. The term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements.

[0044] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0045] As used herein, “around,” “about” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate; meaning that the terms “around,” “about” or “approximately” can be inferred if not expressly stated. When the term “about” is used in describing a value or an endpoint of a range, the disclosure should be understood to include both the specific value and end-point referred to.

[0046] As used herein, the terms “comprising,” “including,” “having,” “containing,” “involving,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to.

[0047] As used herein, the “administration” of an agent, drug, or composition or any ingredient thereof to a subject includes any route of introducing or delivering to a subject the agent, drug, or composition or any ingredient thereof to perform its intended function. Administration can be carried out by any suitable route, including orally, intraocularly, intranasally, parenterally (intravenously, intramuscularly, intraperitone-ally, or subcutaneously), or topically. Administration includes self-administration and the administration by another.

[0048] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results inthe prevention of, or a decrease in, the symptoms or damage associated with an ophthalmic condition resulting from exposure to blue light, e.g., blinding eye disorder, such as, e.g., age-related macular degeneration (AMD). The amount of a composition administered to the subject will depend on the type and severity of the signs or symptoms or disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. A skilled artisan will be able to determine appropriate dosages depending on these and other factors. In the methods described herein, the described compositions may be administered to a subject having one or more signs or symptoms of a blue light- induced ophthalmic condition. For example, a “therapeutically effective amount” of the described composition is meant levels in which the physiological effects, i.e., one or more of signs or symptoms of the blue-light induced ophthalmic condition are, at a minimum, reduced and, preferably, ameliorated.

[0049] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0050] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0051] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0052] As used herein, the terms “treating” or “treatment” or “amelioration” refers to both therapeutic treatment and prophylactic measures, wherein the object is to slow down (lessen) the targeted pathologic ophthalmic condition or disorder, or at least one sign or symptom thereof, resulting from exposure of a subject to blue light. A subject is successfully “treated” for a blue light-induced ophthalmic condition, if, after receiving atherapeutic amount of the described composition according to the methods described herein, the subject shows observable and / or measurable reduction in or absence of one or more signs and symptoms of the blue light-induced ophthalmic condition, which are described in detail below. It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. As compared with an equivalent untreated control, such amelioration or degree of treatment is at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, as measured by any standard, suitable technique.

[0053] As used herein, “prevention” or “preventing” or “protecting” of a blue light- induced ophthalmic disorder or condition refers to the use of a composition described herein that, in a statistical sample, reduces the occurrence of the disorder or condition (resulting from exposure to blue light) in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0054] The term “composition” as used herein, unless otherwise specified, refers to a formulation that includes an amount of at least one active ingredient: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry, and is suitable for administration to a mammalian subject. Compositions may be in the form of powders, solids, semi-solids, liquids, gels, and semi-liquids. Compositions may further comprise vitamins, minerals, and other ingredients, which are not active ingredients. The compositions described herein are for generally maintaining or improving the eye health of a mammalian subject, and include those mixtures designed for mammalian subjects who have, are susceptible to, or are at risk of a pathological ophthalmic condition or disorder due to exposure to blue light.

[0055] The term “subject” as used herein refers to a mammal, including but not limited to a human (e.g., an infant, toddler, child or adult), a domesticated farm animal (e.g., cow, horse, or pig), or a pet (e.g., dog or cat), who ingests the composition.

[0056] The terms “susceptible to” or “at risk of” as used herein in reference to a subject, unless otherwise specified, are used interchangeably to mean a subject havinglittle resistance to a certain condition or disease, including being genetically predisposed, having a family history of, and / or having symptoms of the condition or disease; or subject having increased exposure to blue light, i.e., being at risk of blue light-induced ophthalmic condition, or any signs or symptoms thereof.

[0057] All percentages, parts and ratios as used herein, are by weight of the total composition, unless otherwise specified. All such weights as they pertain to listed ingredients are based on the active level and, therefore, do not include solvents or by- products that may be included in commercially available materials, unless otherwise specified.

[0058] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0059] An “extract” is the material which dissolves in a solvent after contacting a plant substance with a suitable solvent. Preferably, the plant substance is contacted for a sufficiently long period of time and at a suitable temperature so that substantially all of the soluble material is removed by the solvent. The solvent together with the dissolved soluble plant material is referred to as the “liquid extract.” The term “extract” refers to the soluble plant material remaining after removal of the solvent. The solvent can be removed by any suitable means, including evaporation, lyophilization, spray drying and the like.

[0060] The term “ingredient or extract of bilberry” refers to one or more natural materials, in the form of a single material or ingredient (e.g., inorganic, organic, salt, etc.) or a mixture of natural materials obtained from the bilberry plant, genus Vaccinium (family Ericaceae), e.g., Vaccinium myrtillus L.

[0061] The term “carotenoid” refers to a group of pigments (i.e., yellow, orange, and red organic pigments) found in various fruits and vegetables. Specific examples of naturally occurring carotenoids are provided below. The most common carotenoids include lycopene and the vitamin A precursor β-carotene.

[0062] As used herein, the expression “active combination” refers to the ability of the combination of the described ingredients to exert a protective / preventative / therapeuticeffect in the context of improving eye health and / or protecting, preventing or treating a blue light-induce pathogenic ophthalmic condition, as disclosed herein. Neither of the components is regarded as an additive, such as carrier, diluent, or excipient.

[0063] The term “damage to eyes” refers to any pathological sign or symptom occurring in subject’s eyes due to exposure of the subject to blue light.

[0064] The term “additive” refers to a substance which helps to absorb any of the components of the described composition, stabilizes the components or helps in the preparation of the composition. Thus, additives can have the function of keeping the components bound together, such as for example starches, sugars or celluloses, a sweetening function, a colorant function, the function of protecting the composition from the external medium, such as for example isolating it from the air and / or moisture, a filler function for a tablet, capsule or any other form of formulation, such as for example di basic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of additives not mentioned in this paragraph. Therefore, the term “additive” is defined as the substance which, included in dosage forms, is added to the active substances or their associations in order to enable their preparation and stability, modify their organoleptic properties or determine the physical / chemical properties of the described composition and its bioavailability. The “pharmaceutically acceptable” additive should not interact with the activity of the active ingredients of the described composition. Additives include carriers, diluents, and excipients. Examples of additives include binding agents, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings and colorants. More specific, non-limiting examples of acceptable additives are starches, sugars, xylitol, sorbitol, calcium phosphate, steroid fats, talc, silica or glycerin, among others.

[0065] Compositions

[0066] Described herein are compositions comprise an amount of at least one active agent, such as at least one carotenoid and an ingredient or extract of bilberry, preferably an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and an additive.

[0067] In certain embodiments, the described composition may be for protecting the eyes of a mammalian subject from, or treating damage to the eyes of the subject due to exposure of the subject’s eyes to blue light. The described composition comprises an amount of at least one active agent, such as at least one carotenoid and an ingredient or extract of bilberry, preferably an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and an additive. The composition effectively protects the eyes of a mammalian subject from or treats damage to the eyes of the subject due to exposure of the subject to blue light.

[0068] In certain other embodiments, the described compositions may be for preventing, reducing or treating one or more signs and / or symptoms of an ophthalmic condition in a mammalian subject, the ophthalmic condition resulting from the exposure of the subject’s eyes to blue light. The described composition comprises an amount of at least one active agent, such as at least one carotenoid and an ingredient or extract of bilberry, preferably an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and an additive. The composition effectively reduces or prevents one or more signs and symptoms of an ophthalmic condition in the subject.

[0069] In certain embodiments, the described composition is effective in protecting against blue light-induced DNA damage.

[0070] In certain other embodiments, the described composition is effective in protecting against blue-light-induced oxidative stress.

[0071] In certain other embodiments, the composition is effective in protecting human retinal pigment epithelium against blue light irradiation.

[0072] Specifically, described herein are nutritional (e.g., supplements) and pharmaceutical compositions that are beneficial for eye health, i.e., that can prevent and / or treat damage to eyes, where the damage to the subject’s eyes occurs as a result of exposure of the subject’s eyes to blue light.

[0073] The nutritional and pharmaceutical compositions and corresponding manufacturing methods described herein may comprise, consist of, or consist essentially of the essential elements of the disclosure as described herein, as well as any additional oroptional element described herein or which is otherwise useful in nutritional and pharmaceutical composition applications.

[0074] The composition may be in any useful form. Non-limiting examples of the product forms include: solids, semi-solids, ready-to-drink liquids, concentrated liquids, gels, and powders. In some embodiments, the composition may be in the form of a flowable or substantially flowable particulate composition. In some embodiments, the composition may be easily scooped and measured with a spoon or similar other device, such that the composition may easily be reconstituted by the intended user with a suitable aqueous liquid, such as water, to form a liquid supplement or pharmaceutical composition for immediate oral or enteral use. In this context, “immediate” use generally means within about 48 hours, most typically within about 24 hours, and in some embodiments, immediately after reconstitution.

[0075] In some embodiments, the composition may include spray dried powders, dry blended powders, agglomerated powders, combinations thereof, or powders prepared by other suitable methods.

[0076] In some embodiments, the composition may be contained in a dosage element suitable for oral consumption. Suitable dosage elements include tablets, hard gelatin capsules, starch capsules, cellulose-based capsules, softgel capsules, and elixirs.

[0077] In some embodiments, the powdered composition may be compressed into a tablet. In some embodiments, the powdered composition may be included in a capsule. Capsules comprise a shell surrounding and containing the composition. The capsule shell dissolves or disintegrates in the digestive tract after the capsule is ingested, thereby releasing the composition to be absorbed by the body of the individual ingesting the capsule. Capsule shells are typically made of gelatin, starch, cellulose, or other components that readily dissolve or disintegrate after ingestion, and the composition, manufacture, and use of capsule shells are well known in the art. In some embodiments, the dosage element for the composition is a softgel capsule. Softgel capsules are particularly suitable for containing liquid-based ingredients, such as nutrients dissolved, dispersed or suspended in a carrier oil. The shell of a softgel capsule is typically made of gelatin plasticized with glycerin and water, although vegetarian softgel capsules made from starch or carrageenan are also available. Softgel capsule shells are typically madeand filled with the composition in continuous processes that are known in the art. Softgel capsules are made by manufacturers such as Catalent Pharma Solutions, LLC (Somerset, N.J.) and Captek Softgel International (Cerritos, Calif.).

[0078] Capsules come in a wide range of sizes, and the capsule size should be chosen to contain an appropriate volume or weight of the composition and, hence, an appropriate dosage of the lipophilic compound. The capsule size can be chosen to contain at least about 0.001 grams of the composition, 0.01 grams of the composition, 0.1 grams of the composition, including from about 0.1 grams to about 30 grams, from about 0.2 grams to about 20 grams, from about 0.25 grams to about 15 grams, from about 0.25 grams to about 10 grams, from about 0.25 grams to about 5 grams, from about 0.25 to about 3 grams, from about 0.25 grams to about 1.5 grams, or about 0.25 to about 1.0 grams of the composition. In some embodiments, for use with humans, the capsules contain from about 0.1 grams to about 1.5 grams or from about 0.2 grams to about 1.0 grams of the composition, as these capsule sizes are most convenient for most adults and children to swallow. In some embodiments, for use with large animals, such as domesticated farm animals, the capsules contain from about 0.1 grams to about 30 grams or from about 1.0 grams to about 30 grams of the composition.

[0079] In certain embodiments, the composition comprises at least one active component selected from carotenoids and bilberry.

[0080] For example, in certain embodiments, the composition comprises an amount of at least one carotenoid. In another example, the composition comprises an amount of at least one of: lutein, zeaxanthin, β-carotene, and lycopene.

[0081] In another embodiment, the composition comprises an amount of an ingredient or extract of bilberry.

[0082] In certain other embodiments, the composition comprises an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry.

[0083] I. Active Components of the Composition

[0084] Carotenoids

[0085] Carotenoids are yellow, orange, and red organic pigments that are produced by plants and algae, as well as several bacteria, archaea, and fungi. Over 1,100 identifiedcarotenoids can be further categorized into two classes – xanthophylls (which contain oxygen) and carotenes (which are purely hydrocarbons and contain no oxygen).

[0086] All are derivatives of tetraterpenes, meaning that they are produced from 8 isoprene units and contain 40 carbon atoms. In general, carotenoids absorb wavelengths ranging from 400 to 550 nanometers (violet to green light).

[0087] Carotenoids serve two key roles in plants and algae: they absorb light energy for use in photosynthesis, and they provide photoprotection via non-photochemical quenching. Carotenoids that contain unsubstituted beta-ionone rings (including β- carotene, α-carotene, β-cryptoxanthin, and γ-carotene) have vitamin A activity (meaning that they can be converted to retinol). In the eye, lutein, meso-zeaxanthin, and zeaxanthin are present as macular pigments.

[0088] Examples of naturally occurring carotenoids that may be included in the described compositions include: (i) hydrocarbons: Lycopersene 7,8,11,12,15,7',8',11',12',15'-Decahydro-γ,γ-carotene; Phytofluene; Lycopene; Hexahydrolycopene 15-cis-7,8,11,12,7',8'-Hexahydro-γ,γ-carotene; Torulene 3',4'-Didehydro-β,γ-carotene; α-Zeacarotene 7',8'-Dihydro-ε,γ-carotene; α- Carotene; β-Carotene; γ-Carotene; δ-Carotene; ε-Carotene; ζ-Carotene; (ii) alcohols: Alloxanthin; Bacterioruberin 2,2'-Bis(3-hydroxy-3-methylbutyl)-3,4,3',4'-tetradehydro- 1,2,1',2'-tetrahydro-γ,γ-carotene-1,1'-diol; Cynthiaxanthin; Pectenoxanthin; Cryptomonaxanthin (3R,3'R)-7,8,7',8'-Tetradehydro-β,β-carotene-3,3'-diol; Crustaxanthin β,-Carotene-3,4,3',4'-tetrol; Gazaniaxanthin (3R)-5'-cis-β,γ-Caroten-3-ol; OH-Chlorobactene 1',2'-Dihydro-f,γ-caroten-1'-ol; Loroxanthin β,ε-Carotene-3,19,3'-triol; Lutein (3R,3′R,6′R)-β,ε-carotene-3,3′-diol; Lycoxanthin γ,γ-Caroten-16-ol; Rhodopin 1,2- Dihydro-γ,γ-caroten-l-ol; Rhodopinol a.k.a. Warmingol 13-cis-1,2-Dihydro-γ,γ-carotene- 1,20-diol; Saproxanthin 3',4'-Didehydro-1',2'-dihydro-β,γ-carotene-3,1'-diol; Zeaxanthin; (iii) glycosides: Oscillaxanthin 2,2'-Bis(β-L-rhamnopyranosyloxy)-3,4,3',4'-tetradehydro- 1,2,1',2'-tetrahydro-γ,γ-carotene-1,1'-diol; and Phleixanthophyll 1'-(β-D- Glucopyranosyloxy)-3',4'-didehydro-1',2'-dihydro-β,γ-caroten-2'-ol; (iv) ethers: Rhodovibrin 1'-Methoxy-3',4'-didehydro-1,2,1',2'-tetrahydro-γ,γ-caroten-1-ol; and Spheroidene 1-Methoxy-3,4-didehydro-1,2,7',8'-tetrahydro-γ,γ-carotene; (v) epoxides:Diadinoxanthin 5,6-Epoxy-7',8'-didehydro-5,6-dihydro—carotene-3,3-diol;Luteoxanthin 5,6: 5',8'-Diepoxy-5,6,5',8'-tetrahydro-β,β-carotene-3,3'-diol; Mutatoxanthin; Citroxanthin; Zeaxanthin furanoxide 5,8-Epoxy-5,8-dihydro-β,β- carotene-3,3'-diol; Neochrome 5',8'-Epoxy-6,7-didehydro-5,6,5',8'-tetrahydro-β,β- carotene-3,5,3'-triol; Foliachrome; Trollichrome; and Vaucheriaxanthin 5',6'-Epoxy-6,7- didehydro-5,6,5',6'-tetrahydro-β,β-carotene-3,5,19,3'-tetrol; (vi) aldehydes: Rhodopinal; Warmingone 13-cis-1-Hydroxy-1,2-dihydro-γ,γ-caroten-20-al; and Torularhodinaldehyde 3',4'-Didehydro-β,γ-caroten-16'-al; (vii) acids and acid esters: Torularhodin 3',4'-Didehydro-β,γ-caroten-16'-oic acid; and Torularhodin methyl ester Methyl 3',4'-didehydro-β,γ-caroten-16'-oate; (viii) ketones: Astacene; Astaxanthin; Canthaxanthin a.k.a. Aphanicin, Chlorellaxanthin β,β-Carotene-4,4'-dione; Capsanthin (3R,3'S,5'R)-3,3'-Dihydroxy-β,κ-caroten-6'-one; Capsorubin (3S,5R,3'S,5'R)- 3,3'-Dihydroxy-κ,κ-carotene-6,6'-dione; Cryptocapsin (3'R,5'R)-3'-Hydroxy-β,κ-caroten- 6'-one; 2,2'-Diketospirilloxanthin 1,1'-Dimethoxy-3,4,3',4'-tetradehydro-1,2,1',2'- tetrahydro-γ,γ-carotene-2,2'-dione; Echinenone β,β-Caroten-4-one; 3'- Hydroxyechinenone; Flexixanthin 3,1'-Dihydroxy-3',4'-didehydro-1',2'-dihydro-β,γ- caroten-4-one; 3-OH-Canthaxanthin a.k.a. Adonirubin a.k.a. Phoenicoxanthin 3-Hydroxy- β,β-carotene-4,4'-dione; Hydroxyspheriodenone 1'-Hydroxy-1-methoxy-3,4-didehydro- 1,2,1',2',7',8'-hexahydro-γ,γ-caroten-2-one; Okenone 1'-Methoxy-1',2'-dihydro-c,γ- caroten-4'-one; Pectenolone 3,3'-Dihydroxy-7',8'-didehydro-β,β-caroten-4-one; Phoeniconone a.k.a. Dehydroadonirubin 3-Hydroxy-2,3-didehydro-β,β-carotene-4,4'- dione; Phoenicopterone β,ε-caroten-4-one; Rubixanthone 3-Hydroxy-β,γ-caroten-4'-one; and Siphonaxanthin 3,19,3'-Trihydroxy-7,8-dihydro-β,ε-caroten-8-one; (ix) esters of alcohols: Astacein 3,3'-Bispalmitoyloxy-2,3,2',3'-tetradehydro-β,β-carotene-4,4'-dione or 3,3'-dihydroxy-2,3,2',3'-tetradehydro-β,β-carotene-4,4'-dione dipalmitate; Fucoxanthin 3'- Acetoxy-5,6-epoxy-3,5'-dihydroxy-6',7'-didehydro-5,6,7,8,5',6'-hexahydro-β,β-caroten-8- one; Isofucoxanthin 3'-Acetoxy-3,5,5'-trihydroxy-6',7'-didehydro-5,8,5',6'-tetrahydro-β,β- caroten-8-one; Physalien; Siphonein 3,3'-Dihydroxy-19-lauroyloxy-7,8-dihydro-β,ε- caroten-8-one or 3,19,3'-trihydroxy-7,8-dihydro-β,ε-caroten-8-one 19-laurate; (x) apocarotenoids: β-Apo-2'-carotenal 3',4'-Didehydro-2'-apo-b-caroten-2'-al; Apo-2- lycopenal; Apo-6'-lycopenal 6'-Apo-y-caroten-6'-al; Azafrinaldehyde 5,6-Dihydroxy-5,6- dihydro-10'-apo-β-caroten-10'-al; Bixin 6'-Methyl hydrogen 9'-cis-6,6'-diapocarotene-6,6'-dioate; Citranaxanthin 5',6'-Dihydro-5'-apo-β-caroten-6'-one or 5',6'-dihydro-5'-apo- 18'-nor-β-caroten-6'-one or 6'-methyl-6'-apo-β-caroten-6'-one; Crocetin 8,8'-Diapo-8,8'- carotenedioic acid; Crocetinsemialdehyde 8'-Oxo-8,8'-diapo-8-carotenoic acid; Crocin Digentiobiosyl 8,8'-diapo-8,8'-carotenedioate; Hopkinsiaxanthin 3-Hydroxy-7,8- didehydro-7',8'-dihydro-7'-apo-b-carotene-4,8'-dione or 3-hydroxy-8'-methyl-7,8- didehydro-8'-apo-b-carotene-4,8'-dione; Methyl apo-6'-lycopenoate Methyl 6'-apo-y- caroten-6'-oate; Paracentrone 3,5-Dihydroxy-6,7-didehydro-5,6,7',8'-tetrahydro-7'-apo-b- caroten-8'-one or 3,5-dihydroxy-8'-methyl-6,7-didehydro-5,6-dihydro-8'-apo-b-caroten- 8'-one; Sintaxanthin 7',8'-Dihydro-7'-apo-b-caroten-8'-one or 8'-methyl-8'-apo-b-caroten- 8'-one; (xi) nor- and seco-carotenoids: Actinioerythrin 3,3'-Bisacyloxy-2,2'-dinor-b,b- carotene-4,4'-dione; β-Carotenone 5,6:5',6'-Diseco-b,b-carotene-5,6,5',6'-tetrone; Peridinin 3'-Acetoxy-5,6-epoxy-3,5'-dihydroxy-6',7'-didehydro-5,6,5',6'-tetrahydro- 12',13',20'-trinor-b,b-caroten-19,11-olide; Pyrrhoxanthininol 5,6-epoxy-3,3'-dihydroxy- 7',8'-didehydro-5,6-dihydro-12',13',20'-trinor-b,b-caroten-19,11-olide; Semi-α- carotenone 5,6-Seco-b,e-carotene-5,6-dione; Semi-β-carotenone 5,6-seco-b,b-carotene- 5,6-dione or 5',6'-seco-b,b-carotene-5',6'-dione; Triphasiaxanthin 3-Hydroxysemi-b- carotenone 3'-Hydroxy-5,6-seco-b,b-carotene-5,6-dione or 3-hydroxy-5',6'-seco-b,b- carotene-5',6'-dione; (xii) retro-carotenoids and retro-apo- carotenoids:Eschscholtzxanthin 4',5'-Didehydro-4,5'-retro-b,b-carotene-3,3'-diol; Eschscholtzxanthone 3'-Hydroxy-4',5'-didehydro-4,5'-retro-b,b-caroten-3-one; Rhodoxanthin 4',5'-Didehydro-4,5'-retro-b,b-carotene-3,3'-dione; Tangeraxanthin 3- Hydroxy-5'-methyl-4,5'-retro-5'-apo-b-caroten-5'-one or 3-hydroxy-4,5'-retro-5'-apo-b- caroten-5'-one; and (xiii) higher carotenoids: Nonaprenoxanthin 2-(4-Hydroxy-3-methyl- 2-butenyl)-7',8',11',12'-tetrahydro-e,y-carotene; Decaprenoxanthin 2,2'-Bis(4-hydroxy-3- methyl-2-butenyl)-e,e-carotene; C.p.4502-[4-Hydroxy-3-(hydroxymethyl)-2-butenyl]-2'- (3-methyl-2-butenyl)-b,b-carotene; C.p.4732'-(4-Hydroxy-3-methyl-2-butenyl)-2-(3- methyl-2-butenyl)-3',4'-didehydro-l',2'-dihydro-β,γ-caroten-1'-ol; and Bacterioruberin 2,2'-Bis(3-hydroxy-3-methylbutyl)-3,4,3',4'-tetradehydro-1,2,1',2'- tetrahydro-γ,γ-carotene-1,1'-diol.

[0089] In certain embodiments, the composition described herein includes the at least one carotenoid; alternatively, at least two carotenoids; alternatively, at least threecarotenoids; alternatively, at least four carotenoids or more from the carotenoids listed above.

[0090] In certain embodiments, the described composition comprises at least one carotenoid selected from the group consisting of lutein, zeaxanthin, beta-carotene, and lycopene.

[0091] Lycopene is commercially available from Lycored (Mehoz Hadarom, Israel); lutein and zeaxanthin are commercially available from DSM (Alsace, France); β-carotene can be purchased from BASF Australia (Victoria, Australia). Bilberry can be purchased from Beijing Gingkgo Group in Beijing, China.

[0092] In certain preferred embodiments, the composition described herein includes lutein, zeaxanthin, beta-carotene, and lycopene.

[0093] In the described compositions, the amount of at least one carotenoid is in the range from about 0.1 mg to about 50 mg.

[0094] For example, a preferable composition may include 1-20 mg of lutein, 0.1-10 mg of zeaxanthin, 0.1-10 mg of beta carotene; and / or 1-20 mg lycopene.

[0095] Specifically, in one embodiment, the composition may include, for example, 10 mg of lutein, 2 mg of zeaxanthin, 2 mg of beta carotene; and / or 6.4 mg lycopene.

[0096] Bilberry

[0097] Bilberry (Vaccinium myrtillus L.) is a member of the Ericaceous family that grows in the forests of northern Europe.

[0098] Bilberry contains 15 different anthocyanins, including 5 anthocyanidins (delphinidin, cyanidin, malvidin, petunidin, and peonidin), and 3 sugars (glucose, galactose, and arabinose).

[0099] Any appropriate method can be used to prepare a bilberry extract. For example, bilberry extract can be made by hydrodistillation, steam distillation, leaching, solvent extraction, pressing and extraction with supercritical carbon dioxide. Preferably, the bilberry extract can be prepared using ethanol extraction method.

[0100] In the described compositions, the amount of an ingredient or extract of bilberry is in the range from about 1 mg to about 100 mg.

[0101] In a preferable embodiment, the amount of an ingredient or extract of bilberry is, for example, 25 mg.

[0102] Mixtures of Active Components

[0103] In certain embodiments, the described composition may include a mixture of an amount of at least one carotenoid and an amount of an ingredient or extract of bilberry.

[0104] In certain embodiments, the described composition comprises an amount of at least one active agent, such as at least one carotenoid and an ingredient or extract of bilberry, preferably an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and an additive.

[0105] In certain embodiments, a preferable composition may include at least one carotenoid in an amount, e.g., 1-20 mg of lutein, 0.1-10 mg of zeaxanthin, 0.1-10 mg of beta carotene and / or 1-20 mg lycopene; and 1-100 mg of an ingredient or extract of bilberry.

[0106] In certain preferred embodiments, a composition may include carotenoids in an amount, e.g., 1-20 mg of lutein, 0.1-10 mg of zeaxanthin, 0.1-10 mg of beta carotene and / or 1-20 mg lycopene; and 1-100 mg of an ingredient or extract of bilberry.

[0107] In certain embodiments, the following ratios of lutein to zeaxanthin to β- carotene to lycopene to bilberry may be used: 5: 1: 1: 3.2 :12.5. Alternatively, the ratios of lutein to zeaxanthin to β-carotene to lycopene to bilberry may be 1.66: 0.33: 4 :4: 1; alternatively, 0.83: 1.16: 4 :4: 1; and, alternatively 0.83: 0.16: 1: 1 :1. In certain embodiments, lutein and zeaxanthin are in a ratio of 5:1 as a blend.

[0108] In certain further embodiments, where the described composition comprises lycopene, lutein, zeaxanthin and β-carotene, and an additive, the ratio of lycopene to lutein to zeaxanthin to β-carotene in the composition is 5.7:10:2:0.5.

[0109] II. Non-Active Components / Additives of the Composition

[0110] In certain embodiments, the described compositions may comprise an additive, such as a “vehicle” or “carrier,” which is preferably an inert substance.

[0111] The function of the vehicle is to facilitate the incorporation of other compounds, to allow a better dosing and administration or to give consistency and shape to the composition. Therefore, the vehicle is a substance that is used to dilute any of the components of the described composition to a determined volume or weight, or evenwithout diluting said components it is capable of allowing better dosing and administration or giving consistency and shape to the composition (e.g., nutraceutical composition). Due to all of this, a vehicle would be considered pharmaceutically or nutraceutically acceptable.

[0112] The compositions can also include an additive, such as one more excipients that are non-toxic and non-inflammatory in a subject.

[0113] In some embodiments, the excipient(s) can provide desirable or improved physical and / or chemical properties such as stability, flow, viscosity, rate of disintegration, taste, delivery, etc.

[0114] The excipient can include a disintegrant, a binder, a surfactant, an emulsifier, a viscosity modifier, a lubricant, a sweetener, a pH-adjusting agent, a preservative, a flavoring agent, a coloring agent, or an antioxidant.

[0115] For example, the excipient may be selected from gellan gum, carob bean gum, locust bean gum, carrageenan, alginates, agar, guar gum, xanthan gum, carboxymethyl cellulose, clear starch, pectin, gelatin, cornstarch, katakuri starch, potato starch, and gum arabic.

[0116] The composition described herein may additionally include additives, for example, sweeteners, flavoring and coloring agents, vitamins, minerals, preservatives, and antioxidants.

[0117] For example, a sweetener may be selected from high fructose corn syrup, mannose, maltose, glucose polymers, sucrose, glucose, dextrose, lactose, galactose, fructose, polysaccharides, rice syrup, honey, saccharin, cyclamates, acetosulfam, sorbitol, sucralose, xylitol, erythritol, Stevia extract, L-aspartyl-L-phenyl-alanine ester, L-aspartyl- D-alanine alkyl amides, L-aspartyl-L-1-hydroxymethylalkaneamide, and L-aspartyl-1- hydroxyethylalkaneamide.

[0118] For example, a pH-adjusting agent may be selected from hydrochloric acid, citric acid, sodium hydrogen carbonate, potassium hydroxide, sodium hydroxide, and sodium carbonate.

[0119] For example, a preservative may be selected from sorbic acid, benzoic acid, sodium benzoate, calcium benzoate, potassium benzoate, potassium sorbate, calcium sorbate, and sodium sorbate.

[0120] For example, a flavoring agent may be selected from almond oil, amaretto oil, anethole, anise oil, benzaldehyde, blackberry, black walnut oil, blueberry, caraway, caraway oil, cardamom oil, cardamom seed, cherry juice, cherry syrup, cinnamon, cinnamon oil, cinnamon water, citric acid, citric acid syrup, clove oil, cocoa, coriander oil, dextrose, eriodictyon, ethyl acetate, ethyl vanillin, fennel oil, ginger, glucose, glycerin, glycyrrhiza, grape, honey, lavender oil, lemon oil, lime, mannitol, methyl salicylate, myristica oil, orange oil, orange peel, orange syrup, peppermint, peppermint oil, peppermint water, phenylethyl alcohol, pineapple, raspberry juice, raspberry syrup, rosemary oil, rose oil, rose water, sarsaparilla syrup, sorbitol, spearmint, spearmint oil, strawberry, sucrose, thyme oil, tolu balsam, tropical, vanilla, vanillin, and wild cherry syrup.

[0121] Additionally, the described compositions may include excipients, such as candelilla wax and sunflower lecithin.

[0122] The described composition can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the reduction, treatment or prevention of a disorder described herein.

[0123] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration.

[0124] For ophthalmic applications, the described compositions may be formulated into solutions, suspensions, and ointments appropriate for use in the eye. For ophthalmic formulations generally, see Mitra (ed.), Ophthalmic Drug Delivery Systems, Marcel Dekker, Inc., New York, N.Y. (1993), and also Havener, W. H., Ocular Pharmacology, C.V. Mosby Co., St. Louis (1983). Ophthalmic pharmaceutical compositions may be adapted for topical administration to the eye in the form of solutions, suspensions, ointments, creams or as a solid insert. For a single dose, from between 0.1 ng to 5000 µg, 1 ng to 500 µg, or 10 ng to 100 µg of the described composition can be applied to the human eye.

[0125] The ophthalmic preparation may contain non-toxic auxiliary substances, such as antibacterial components which are non-injurious in use, for example, thimerosal, benzalkonium chloride, methyl and propyl paraben, benzyldodecinium bromide, benzyl alcohol, or phenylethanol; buffering ingredients such as sodium chloride, sodium borate, sodium acetate, sodium citrate, or gluconate buffers; and other conventional ingredients such as sorbitan monolaurate, tri-ethanolamine, polyoxyethylene sorbitan monopalmitylate, ethylenediamine tetraacetic acid, and the like.

[0126] The ophthalmic solution or suspension may be administered as often as necessary to maintain an acceptable level of described composition in the eye. Administration to the mammalian eye may be about once, twice, or trice daily.

[0127] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral administration, the active compound(s) can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.

[0128] Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition.

[0129] The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder (such as microcrystalline cellulose, gum tragacanth or gelatin); an excipient (such as starch or lactose); a disintegrating agent (such as alginic acid, Primogel, or cornstarch); a lubricant (such as magnesium stearate or Sterotes); a glidant (such as colloidal silicon dioxide); a sweetening agent (such as sucrose or saccharin); and / or a flavoring agent (such as peppermint, methyl salicylate, or orange flavoring).

[0130] In some embodiments, the composition described herein can be prepared with carriers that will protect the composition against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.

[0131] Biodegradable, biocompatible polymers can be used, such as, e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylacetic acid. Such formulations can be prepared using known techniques. The materials can also be obtained commercially.

[0132] Typically, an effective amount of the described compositions, sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. In some embodiments, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example, dosages can be 1 mg / kg body weight or 10 mg / kg body weight every day, every two days or every three days or within the range of 1-10 mg / kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of the composition ranges from 0.1-10,000 micrograms per kg body weight.

[0133] III. Supplementary Therapies

[0134] In certain additional embodiments, the described compositions may be used in combination with procedures that may provide additional or synergistic benefit to the patient, including, e.g., the use of extracorporeal rheopheresis (also known as membrane differential filtration), the use of implantable miniature telescopes, laser photocoagulation of drusen, and microstimulation therapy. This may provide support to the light sensitive photoreceptors of the retina and support normal visual focus.

[0135] Modes of Administration and Effective Dosages

[0136] Any method known to those in the art for administering compositions may be employed.

[0137] Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of the described composition to a mammal, such as a human. When used in vivo for therapy, the described composition may be administered to the subject in effective amounts (i.e., amounts that have desired preventative and / or therapeutic effect(s)). The dose and dosage regimen will depend upon many factors, e.g., the amount or length or exposure to blue light, the extent or severity of the ophthalmic disorder, such as AMD in the subject, the extent or severity of any signs or symptoms of the ophthalmic disorder, the characteristics of the composition used, e.g., its therapeutic index, the subject, and the subject's family history, and genetic background.

[0138] The described compositions may be administered to a mammal in need thereof by any of a number of well-known methods for administering compositions. Insome embodiments, the described composition may be administered systemically, topically, or intraocularly.

[0139] An exemplary treatment regime entails administration once per day, twice per day, or trice per day. Alternatively, the described composition may be administered once a week, twice a week, or three times a week, etc. Intervals can also be irregular.

[0140] In certain embodiments, the described composition may be an immediate release composition, a mixed-release composition, or an enterically-coated composition.

[0141] In therapeutic applications, in some embodiments, a relatively high dosage at relatively short intervals may be required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0142] In some embodiments, the dosage of the described composition is provided at a “low,” “mid,” or “high” dose level.

[0143] In one embodiment, the low dose is provided from about 0.0001 to about 0.5 mg / kg / h, suitably from about 0.01 to about 0.1 mg / kg / h. In one embodiment, the mid- dose is provided from about 0.1 to about 1.0 mg / kg / h, suitably from about 0.1 to about 0.5 mg / kg / h. In one embodiment, the high dose is provided from about 0.5 to about 10 mg / kg / h, suitably from about 0.5 to about 2 mg / kg / h.

[0144] A skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the described compositions can include a single treatment or a series of treatments.

[0145] The mammal treated in accordance present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In some embodiments, the mammal is a human.

[0146] The described composition may be in a form of a dietary supplement.

[0147] The described composition may be a food product.

[0148] Eye Conditions, Signs and Symptoms

[0149] In certain embodiments, the described compositions may be used for prevention of, a decrease in, and / or treatment of the symptoms or damage associated with an ophthalmic condition resulting from exposure of a subject’s eyes to blue light, e.g., blinding eye disorder, such as, e.g., age-related macular degeneration (AMD).

[0150] The signs or symptoms include but are not limited to: vision loss, blurred or decreased vision in one or both eyes; distortion of vision and blind spots (especially in and around the central visual field); distorted vision in the form of metamorphopsia (a grid of straight lines appears wavy and parts of the grid may appear blank); central scotomas; shadows; missing areas of vision; slow recovery of visual function after exposure to bright light; decreased visual acuity (two levels or more, e.g.: 20 / 20 to 20 / 80); decreased ability to discern colors, specifically dark ones from dark ones and light ones from light ones; a decrease or loss in contrast sensitivity; formed visual hallucinations and flashing lights; and others.

[0151] In certain other embodiments, the described compositions and methods may be used to prevent or treat eye conditions that may result from exposure of subject’s eyes to blue light.

[0152] For example, in certain embodiments, the described compositions may be effective for treating or preventing the blinding eye disorder in a subject that may result from exposure of subject’s eyes to blue light.

[0153] The blinding eye disorder may be age-related macular degeneration, cataract, diabetic retinopathy and glaucoma. Additionally, the described compositions may be effective for treating or preventing retinitis pigmentosa.

[0154] In certain other embodiments, the described compositions may be effective for treating or preventing other eye diseases or conditions, or any signs or symptoms thereof that may result from exposure of a subject to blue light.

[0155] I. Age-related Macular Degeneration

[0156] In certain embodiments, the described compositions and methods may be used for preventing and / or reducing age-related macular degeneration (AMD) and / or at least one or more signs or symptoms of AMD, which are described in more detail below. As used herein, “age-related macular degeneration (AMD)” refers to a medical conditionwhich may result in blurred or no vision in the center of the visual field. Early on there are often no symptoms. Over time, however, some people experience a gradual worsening of vision that may affect one or both eyes. While it does not result in complete blindness, loss of central vision can make it hard to recognize faces, drive, read, or perform other activities of daily life. Visual hallucinations may also occur with AMD.

[0157] AMD typically occurs in older people, and is caused by damage to the macular of the retina of the eye. Genetic factors and smoking may play a role. The condition is diagnosed through a complete eye exam. Severity is divided into early, intermediate, and late types. The late type is additionally divided into “dry” and “wet” forms, with the dry form making up 90% of cases.

[0158] In the context of the described embodiments, AMD may be due to exposure to blue light.

[0159] The difference between the two forms is categorized by the change in the macula. Those with dry form AMD have drusen, cellular debris in their macula that gradually damages light-sensitive cells and leads to vision loss. In wet form AMD, blood vessels grow under the macula, causing blood and fluid to leak into the retina.

[0160] Early or intermediate AMD may be asymptomatic, or it may present with blurred or decreased vision in one or both eyes. This may manifest initially as difficulty with reading or driving (especially in poorly lit areas). Other symptoms of AMD include distortion of vision and blind spots (especially in and around the central visual field).

[0161] Other signs and symptoms of macular degeneration include:

[0162] (i) Distorted vision in the form of metamorphopsia, in which a grid of straight lines appears wavy and parts of the grid may appear blank: Patients often first notice this when looking at things like miniblinds in their home or telephone poles while driving. There may also be central scotomas, shadows or missing areas of vision.

[0163] (ii) Slow recovery of visual function after exposure to bright light (photostress test).

[0164] (iii) Visual acuity drastically decreasing (two levels or more), e.g.: 20 / 20 to 20 / 80.

[0165] (iv) Blurred vision: Those with nonexudative (dry) macular degeneration may be asymptomatic or notice a gradual loss of central vision, whereas those withexudative (wet) macular degeneration often notice a rapid onset of vision loss (often caused by leakage and bleeding of abnormal blood vessels).

[0166] (v) Trouble discerning colors, specifically dark ones from dark ones and light ones from light ones.

[0167] (vi) A loss in contrast sensitivity.

[0168] (vii) Formed visual hallucinations and flashing lights have also been associated with severe visual loss secondary to wet AMD.

[0169] Macular degeneration by itself will not lead to total blindness. For that matter, only a small number of people with visual impairment are totally blind. In almost all cases, some vision remains, mainly peripheral. Other complicating conditions may lead to such an acute condition (severe stroke or trauma, untreated glaucoma, etc.), but few macular degeneration patients experience total visual loss.

[0170] The area of the macula constitutes only about 2.1% of the retina, and the remaining 97.9% (the peripheral field) remains unaffected by the disease. Even though the macula provides such a small fraction of the visual field, almost half of the visual cortex is devoted to processing macular information.

[0171] In addition, people with dry macular degeneration often do not experience any symptoms but can experience gradual onset of blurry vision in one or both eyes. People with wet macular degeneration may experience acute onset of visual symptoms.

[0172] In AMD there is a progressive accumulation of characteristic yellow deposits, called drusen (buildup of extracellular proteins and lipids), in the macula (a part of the retina), between the retinal pigment epithelium and the underlying choroid. This accumulation is believed to damage the retina over time. Amyloid beta, which builds up in Alzheimer's disease brains, is one of the proteins that accumulate in AMD, which is a reason why AMD is sometimes called “Alzheimer's of the eye” or “Alzheimer's of the retina.” AMD can be divided into 3 stages: early, intermediate, and late, based partially on the extent (size and number) of drusen.

[0173] AMD-like pathology begins with small yellow deposits (drusen) in the macula, between the retinal pigment epithelium and the underlying choroid. Most people with these early changes (referred to as age-related maculopathy) still have good vision.People with drusen may or may not develop AMD. In fact, the majority of people over age 60 have drusen with no adverse effects. The risk of developing symptoms is higher when the drusen are large and numerous, and associated with the disturbance in the pigmented cell layer under the macula. Large and soft drusen are thought to be related to elevated cholesterol deposits.

[0174] Early AMD is diagnosed based on the presence of medium-sized drusen, about the width of an average human hair. Early AMD is usually asymptomatic.

[0175] Intermediate AMD is diagnosed by large drusen and / or any retinal pigment abnormalities. Intermediate AMD may cause some vision loss, but, like early AMD, it is usually asymptomatic.

[0176] Recently, subgroups of intermediate AMD have been identified, which have a very high risk of progression toward late AMD. This subgroup (depending on the precise definitions) is termed nascent GA and / or iRORA (incomplete retinal pigment epithelium and outer retinal atrophy). These ‘high-risk’ subgroups of intermediate AMD can be used to inform patients of their prognosis. In addition, these can be applied in clinical trials as endpoints.

[0177] In late AMD, enough retinal damage occurs that, in addition to drusen, people will also begin to experience symptomatic central vision loss. The damage can either be the development of atrophy or the onset of neovascular disease. Late AMD is further divided into two subtypes based on the types of damage: Geographic atrophy and Wet AMD (also called Neovascular AMD).

[0178] Dry AMD (also called nonexudative AMD) is a broad designation, encompassing all forms of AMD that are not neovascular (wet AMD). This includes early and intermediate forms of AMD, as well as the advanced form of dry AMD known as geographic atrophy. Dry AMD patients tend to have minimal symptoms in the earlier stages; visual function loss occurs more often if the condition advances to geographic atrophy. Dry AMD accounts for 80–90% of cases and tends to progress slowly. In 10– 20% of people, dry AMD progresses to the wet type.

[0179] Neovascular or exudative AMD, the “wet” form of advanced AMD, causes vision loss due to abnormal blood vessel growth (choroidal neovascularization) in the choriocapillaris, through Bruch's membrane. It is usually, but not always, preceded bythe dry form of AMD. The proliferation of abnormal blood vessels in the retina is stimulated by vascular endothelial growth factor (VEGF). Because these blood vessels are abnormal, these are also more fragile than typical blood vessels, which ultimately leads to blood and protein leakage below the macula. Bleeding, leaking, and scarring from these blood vessels eventually cause irreversible damage to the photoreceptors and rapid vision loss if left untreated.

[0180] II. Retinitis Pigmentosa

[0181] In certain embodiments, the described compositions may be used to treat or prevent retinitis pigmentosa, or at least one sign or symptom thereof in a subject that resulted from the subject’s exposure to blue light.

[0182] Retinitis pigmentosa (RP) is a genetic disorder of the eyes that causes loss of vision. Symptoms include trouble seeing at night and decreasing peripheral vision (side and upper or lower visual field). As peripheral vision worsens, people may experience “tunnel vision.” Complete blindness is uncommon. Onset of symptoms is generally gradual.

[0183] The initial retinal degenerative symptoms of retinitis pigmentosa are characterized by decreased night vision (nyctalopia) and the loss of the mid-peripheral visual field. The rod photoreceptor cells, which are responsible for low-light vision and are orientated mainly in the retinal periphery, are the retinal processes affected first during non-syndromic (without other conditions) forms of this disease. Visual decline progresses relatively quickly to the far peripheral field, eventually extending into the central visual field as tunnel vision increases. Visual acuity and color vision can become compromised due to accompanying loss of the cone photoreceptor cells, which are responsible for color vision, visual acuity, and sight in the central visual field. The progression of disease occurs in both eyes in a similar but not identical pattern. A variety of indirect symptoms characterize retinitis pigmentosa along with the direct effects of the initial rod photoreceptor degeneration and later cone photoreceptor decline. Phenomena such as photophobia, which describes the event in which light is perceived as an intense glare, and photopsia, the presence of blinking, swirling or shimmering lights spontaneously occurring within the visual field, often manifest during the later stages of RP.

[0184] The symptoms of RP can include: night blindness; tunnel vision (due to loss of peripheral vision); loss of peripheral vision; loss of depth perception; photopsia (spontaneously occurring flashes / blinking / swirling / shimmering lights); photophobia (aversion to bright lights); development of the appearance of melanin pigment in a bone spicule pattern in the fundus (not bone tissue); slow adjustment from dark to light environments and vice versa; blurring of vision; poor color separation; loss of central vision; and blindness.

[0185] III. Cataracts

[0186] In certain embodiments, the described compositions may be used to treat or prevent cataracts, or any signs or symptoms thereof in a subject that resulted from the subject’s exposure to blue light.

[0187] A cataract is a cloudy area in the lens of the eye that leads to a decrease in vision of the eye. Cataracts often develop slowly and can affect one or both eyes. Symptoms may include faded colors, blurry or double vision, halos around light, trouble with bright lights, and difficulty seeing at night. This may result in trouble driving, reading, or recognizing faces. Poor vision caused by cataracts may also result in an increased risk of falling and depression. Cataracts cause 51% of all cases of blindness and 33% of visual impairment worldwide.

[0188] Cataracts are most commonly due to aging but may also occur due to trauma or radiation exposure or other risk factors, be present from birth, or occur following eye surgery for other problems. The underlying mechanism involves accumulation of clumps of protein or yellow-brown pigment in the lens that reduces transmission of light to the retina at the back of the eye.

[0189] IV. Glaucoma

[0190] In certain embodiments, the described compositions may be used to treat or prevent glaucoma, or any signs or symptoms thereof in a subject that resulted from the subject’s exposure to blue light.

[0191] Glaucoma is a group of eye diseases that lead to damage of the optic nerve, which transmits visual information from the eye to the brain. Glaucoma may cause vision loss if left untreated. The loss of vision usually occurs slowly over a long period of time.A major risk factor for glaucoma is increased pressure within the eye, known as intraocular pressure (IOP).

[0192] V. Other Eye Conditions

[0193] In certain embodiments, the described compositions and methods may be used for preventing diabetic retinopathy.

[0194] Methods

[0195] Certain embodiments relate to a method for treating or preventing blue light-induced ophthalmic condition, or one or more sign or symptom thereof in a mammalian subject at risk of or having blue light-induced ophthalmic condition, or one or more sign or symptom thereof. The method comprises administering to the subject a therapeutically effective amount of a composition that is provided in the form selected from the group consisting of capsules, tablets, liquids, and powders for oral ingestion, the composition comprising: (i) an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and (iii) a pharmaceutically acceptable additive.

[0196] In the described methods, the administering of the composition may be intraocularly, iontophoretically, orally, topically, systemically, intravenously, subcutaneously, or intramuscularly.

[0197] In the described methods, the ophthalmic condition may be a blinding eye disorder. In certain embodiments, the ophthalmic condition may be age-related macular degeneration, cataract, glaucoma, or retinitis pigmentosa.

[0198] In the described methods, in certain embodiments, the ratio of lutein to zeaxanthin to β-carotene to lycopene to bilberry is 5:1:1:3.2:12.5.

[0199] The administering may be once or more times a day, or as described above.

[0200] The one or more signs and symptoms of an ophthalmic condition may be selected from the group consisting of: vision loss, blurred or decreased vision in one or both eyes; distortion of vision; blind spots; distorted vision in the form of metamorphopsia; central scotomas; shadows; missing areas of vision; slow recovery of visual function after exposure to bright light; decreased visual acuity; decreased ability todiscern colors; decreased or lost contrast sensitivity; visual hallucinations; and flashing lights.

[0201] Another embodiment relates to a method of protecting a user’s eyes from the harmful effect of blue light. The method includes administering to the user a therapeutically effective amount of a composition that may be provided, e.g., in the form selected from the group consisting of capsules, tablets, liquids, and powders for oral ingestion, the composition comprising: (i) an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and (ii) a pharmaceutically acceptable additive.

[0202] Yet another embodiment relates to a method for enhancing protective mechanisms against oxidative stress damage induced by blue light exposure and mitigating DNA damage and cellular senescence in a user’s eyes. The method comprises administering to the user a therapeutically effective amount of a composition that is provided in the form selected from the group consisting of capsules, tablets, liquids, and powders for oral ingestion, the composition comprising: (i) an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and (ii) a pharmaceutically acceptable additive.

[0203] EXAMPLES

[0204] Example 1

[0205] Materials

[0206] Dulbecco′s Modified Eagle′s Medium / Nutrient Mixture F-12 medium was obtained from ATCC.

[0207] Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT). The DuoSet ELISA kit for IL-6 was obtained from R&D Systems (Minneapolis, MN).

[0208] Senescence-associated β-galactosidase assay kit (Cat# 9860 and 23833) was purchased from Cell Signaling Technology (Danvers, MA).

[0209] The anti-OGG1 antibody (Cat# PA131402) was purchased from Fisher. CellTagTM700 and IRDye® 800CW Goat anti-Rabbit IgG secondary antibodies were purchased from LI-COR. CellROXTMwas purchased from ThermoFisher.

[0210] The blue LED light was purchased from RubyLuxLights.

[0211] All other reagents and ferric-reducing antioxidant power (FRAP) assay kits (Cat# MAK369) were purchased from Sigma.

[0212] Lycopene was purchased from Lycored (Mehoz Hadarom, Israel); lutein and zeaxanthin were purchased from DSM (Alsace, France); β-carotene was purchased from BASF Australia (Victoria, Australia).

[0213] All extractions underwent verification via vendor certification.

[0214] Cell culture

[0215] The ARPE-19 cells were maintained in DMEM: F12 Medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a humidified atmosphere with 5% CO2. Cells were passaged by trypsinization every 2–3 days.

[0216] MTT Assays

[0217] ARPE-19 cells (5.0 ×104) were plated in 96-well plates and incubated. After 24 hours, the media was removed and replaced with media containing vision health ingredients (lycopene or beta-carotene or lutein / zeaxanthin) at concentrations ranging from 0–1,600 µM. Plates were incubated with treatments for 24 h. Media containing treatments were removed and replaced with media containing MTT reagent at 20 mg / mL. Plates with MTT were wrapped in foil and incubated for 1 h. Following incubation, the media was carefully aspirated and replaced with sterile DMSO; plates were put on a room temperature shaker for 10 min while wrapped in foil. Absorbance readings were taken at 570 nm in a multi-well plate reader.

[0218] Blue LED light exposure

[0219] ARPE-19 cells (5×104cells / 100 μL) were seeded onto a black 96-well plate and cultured at 37°C for 24 h.400 µg / mL of vision health ingredients were administered to the cells and the cells were incubated for 24 hr or 48 hr. After the pretreatment, CellROXTM(ThermoFisher) was added to the cells, and the cells were exposed to 2.5 mW / cm2of blue LED light at wavelength 410 nm for 90 min. Fluorescence readings were taken at 485 / 520 nm in a multi-well plate reader. For immunocytochemistry, after exposure to blue light, the cells were fixed with 4% paraformaldehyde and washed in PBS at RT. Fluorescence images were acquired using a fluorescence microscope.

[0220] Immunofluorescence and confocal microscopy

[0221] ARPE-19 cells in an 8-well chamber were fixed with 4% paraformaldehyde (PFA) for 10 min and then permeabilized in 0.1% Triton X-100 in PBS for 20 min at room temperature. After being blocked with 5% FBS in PBS for 1.5 h, the samples were incubated with the primary antibodies described at 4°C overnight. The cells were washed with PBST 3 times and incubated with Alexa Fluor 488-conjugated (1:500; ThermoFisher) secondary antibodies diluted in 5% FBS in PBS at room temperature in the dark for 1 h. Coverslips were washed 3 times and stained with 4′,6-diamidino-2- phenylindole (DAPI) for 3-5 min and then imaged by fluorescence microscope.

[0222] RNA isolation and quantitative RT-PCR

[0223] Total RNA was purified from the cells using the RNAeasy kit from Qiagen following the manufacturer’s protocol. Total RNAs (2 µg) were reverse transcribed using iscript cDNA synthesis kit (Biorad). Quantitative RT-PCR was performed with Bio-Rad CFX 96 real-time PCR detection system using SsoFast™ EvaGreen® Supermix. PCR specificity was confirmed by melting curve analysis. A housekeeping gene (GAPDH) was used as an internal standard. Gene-specific primers used were: NQO1 Forward 5′-TGGCTAGGTATCATTCAACTC-3′ SEQ ID NO:1 NQO1 Reverse 5′-CCTTAGGGCAGGTAGATTCAG-3′ SEQ ID NO:2 HO-1 Forward 5′-GCCAGCAACAAAGTGCAAGAT-3′ SEQ ID NO:3 HO-1 Reverse 5′-GGTAAGGAAGCCAGCCAAGAG-3′ SEQ ID NO:4 Sod1_Foward 5′-GCCAAAGGATGAAGAG-3′ SEQ ID NO:5 Sod1_Reverse 5′-CCACAAGCCAAACGAC-3′ SEQ ID NO:6 Gclc_Forward 5′-TTGGAGACCAGAGTATGGGAGT-3′ SEQ ID NO:7 Gclc_Reverse 5′-CTGGGAAATGAAG TTATTGTGC-3′ SEQ ID NO:8 Gapdh_Forward 5′-ACCACA GTCCATGCCATCAC-3′ SEQ ID NO:9 Gapdh_Reverse 5′-TCCACCACCCTGTTGCTGTA-3' SEQ ID NO:10 p16INK4A_Forward 5'-CTCGTGCTGATGCTACTGAGGA-3' SEQ ID NO:11 p16INK4A_Reverse 5'-GGTCGGCGCAGTTGGGCTCC-3' SEQ ID NO:12 p21_Forward 5'-AGGTGGACCTGGAGACTCTCAG-3' SEQ ID NO:13 p21_Reverse 5'-TCCTCTTGGAGAAGATCAGCCG-3' SEQ ID NO:14

[0224] For analysis of expression of antioxidant and DNA repair associated genes, TaqMan™ Array Human DNA Repair Mechanisms (Cat# 4418773, Thermo Fisher Scientific) and TaqMan™ Array, Human Antioxidant Mechanisms (Cat# 4418764, Thermo Fisher Scientific) were used.

[0225] Ferric Reducing Antioxidant Power (FRAP) assay

[0226] The FRAP assay (Shamsi, T.N., et al. (2018)) was performed according to the manufacturer’s manual (Sigma, MO, USA).

[0227] Senescence Associated β-galactosidase Assay

[0228] Senescence-associated β-galactosidase activity was determined according to the manufacturer’s manual (Cat# 9860 and 23833, Cell Signaling Technology).

[0229] Antioxidant Response Element (ARE) Luciferase Reporter Assay

[0230] To assess activation of Nrf2 in the ARE luciferase reporter assay, a synthetic oligonucleotide containing four repeats of the ARE DNA sequence (5′- GTGACTCAGCA‑3′ (SEQ ID NO:15)) was hybridized to its complementary oligonucleotide, and the resulting DNA fragment ligated at the SacI / BglII site of a pGL4.27 / luc2P / minP / Hygro plasmid (Promega). A stable cell line was generated by transfecting human hepatocellular carcinoma HepG2 cells (ATCC) with the vector using Fugene 6 (Roche) according to the manufacturer’s instructions. To test the botanical samples, ARE luciferase reporter HepG2 cells were plated (1 × 104cells / well) in white- walled, clear-bottom, 96-well plates and incubated for 48 h in a humidified, 37 °C, 5% CO2 incubator. The cells were treated with samples at specified concentrations for an additional 48 h. Following incubation, luciferase activity was quantified using a luciferase assay kit (Biotium, Inc.) according to the manufacturer’s instructions. Light emission was read on a SpectraMax M5 spectrophotometer (Molecular Devices).

[0231] In-Cell Western (ICW)

[0232] Cells were dispensed in the same way as for the cell viability assay (4×103cells / well) and were treated with dietary carotenoids (lycopene, lutein / zeaxanthin, or β- carotene) in a 96-well cell culture plate. After 48 h treatment, cells were washed in PBS and directly fixed in the plate with 4% paraformaldehyde in PBS for 10 min at RT, then washed three times using PBS for 5 min. Cells were permeabilized with 0.2% Triton X- 100 in PBS for 15 min and washed three times with PBS. Cells were blocked withblocking buffer (Li-COR) for 1.5 h at RT, followed by overnight min incubation with rabbit anti-OGG1 (Fisher, Cat# PA131402, dilution ratio is 1:200) in blocking buffer including 0.2% tween-20. After three times washed with in PBST, secondary detection was carried out using an infrared fluorescent dye conjugated donkey antibodies absorbing at 800 nm (IRDyeTM 800CW, Rockland) (1 / 500) in blocking buffer including 0.2% tween-20. For normalization, 1 / 500 dilution of CellTagTM700 iodide (Li-COR) in blocking buffer including 0.2% tween-20 was used. After 1 h of incubation and three times washing with PBST, OGG1 was simultaneously visualized using an Odyssey Infrared Imaging Scanner (Li-Cor ScienceTec, Les Ulis, France) with the 680 nm fluorophore (red color) and the 800 nm fluorophore (green dye). Relative fluorescence units from the scanning allowed a quantitative analysis. Relative fluorescent units for OGG1 per cell were divided by vehicle controls to determine the percent change in OGG1 levels relative to control. All experiments were carried out in triplicate.

[0233] Statistical Analyses

[0234] Statistical evaluation was performed by one-sided, two-sample with equal variance t-tests using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Data are presented as the mean ± standard deviation. A value of p < 0.05 was considered statistically significant. For ICW, immunofluorescence, and senescence-associated β- galactosidase assay, representative images are shown. Each of these experiments was independently repeated at least three times.

[0235] Results

[0236] Carotenoids Reduced Blue Light-Induced ROS Generation.

[0237] Degeneration of the RPE induced by light is a popular in vitro model for studies of AMD (Boutzen, J., et al. (2020)). Blue light is a well-known aggressor through inducing oxidative stress in human RPE (ARPE-19) cells (Cheng, K.C., et al.2021)). The condition of blue light exposure was set as 2.5 mW / cm2for 1.5 h to further examination of blue light-induced ROS generation in ARPE-19 cells. Blue light exposure with 2.5 mW / cm2for 1.5 h did not affect cell viability (Fig.1 (A)) but induced cellular senescence. After blue light irradiation, the mRNA expression of senescence markers, p16INK4aand p21WAF1 / CIP1, were increased (Fig.1 (B)). Each carotenoid including lycopene, lutein, zeaxanthin, and β-carotene showed no toxicity in ARPE-19 cells up to 400 µg / mL (Fig.1(C)). Blue light exposure increased ROS levels significantly compared to blue light irradiated vehicles (DMSO), but much lower ROS generation was shown in the presence of N-acetylcysteine (NAC), which was used as a positive control to show this assay is reliable (Fig.1 (D) and 1 (E)). Next, we pretreated 400 µg / mL of the individual carotenoid and carotenoid blend in the APRE-19 cells for 48 h and examined ROS generation levels after blue light exposure. Pretreatment of individual carotenoid reduced ROS generation and the carotenoid blend showed the most reduced ROS levels (Fig.1 (D) and 1 (E)). These results demonstrated that the antioxidant effect of individual carotenoid and carotenoid blend maximized the antioxidant effect to reduce ROS generation induced by blue light exposure in ARPE-19 cells.

[0238] Antioxidant Activity of Carotenoids

[0239] The antioxidant capacity of the individual carotenoid was evaluated using FRAP assay (Benzie, I.F., et al. (1996); and Bohm, V., et al. (2002)). Lycopene, lutein / zeaxanthin (Lu / Zhea), and β-carotene showed significant antioxidant activity in a dose-dependent manner (Fig.2). These results support carotenoids’ antioxidant effect on reducing ROS generation induced by blue light irradiation.

[0240] Carotenoids Activated the Nuclear Factor Erythroid 2-Related Factor (NRF2) Pathway and Increased NRF2 Target Genes.

[0241] The induction of the cytoprotective enzymes in response to ROS is mediated by antioxidant response elements (ARE), which activate the NRF2. The transcription factor NRF2 regulates the expression of antioxidative enzymes including NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), glutamate- cysteine ligase catalytic subunit (GCLC) (Nguyen, T., et al. (2003)).

[0242] ARPE-19 cells were incubated with each carotenoid for 48 h and analyzed ARE activity and expression of NRF2 target genes. All the tested individual carotenoid showed dose-dependent luciferase activity in an ARE reporter assay (Fig.3 (A)). For further investigation of the activation of the NRF2-ARE pathway, we analyzed the expression of NRF2 target genes (Ho-1, Sod1, Nqo1, and Gclc). Lycopene and β-carotene significantly increased the expression of Gclc and Sod1 respectively (Fig.3 (D) and 3 (E)). Lutein / zeaxanthin significantly increased all examined NRF2 target genes (Ho-1, Sod1, Nqo1, and Gclc) (Fig.3 (B-E)). The genes associated with antioxidant mechanismwere significantly increased in Lu / Zea treated cells (Fig.3 (F)). These results demonstrated the carotenoids activated the NRF2 pathway and increased the expression of NRF2 target genes as well as the genes associated with antioxidant mechanism, which might explain the antioxidant effect to reduce blue light-induced oxidative stress.

[0243] Carotenoids Reduced DNA Damage and Cellular Senescence Triggered by Blue Light Exposure.

[0244] ROS-mediated oxidative stress leads to DNA damage and serine139- phosphorylated histone H2A.X (p-H2A.X) has been established as evidence of DNA damage (Kurz, E.U., et al. (2004)).

[0245] We evaluated nuclear p-H2A.X immunofluorescence to assess DNA damage triggered by blue light exposure in ARPE-19 cells. Blue light exposure increased p-H2A.X nuclear foci, demonstrating the presence of a DNA damage response. Pretreatment of individual carotenoid resulted in reduced p-H2AX expression, while the combination of these carotenoids further decreased p-H2AX (Fig.4 (A)).

[0246] It has been known that DNA damage causes cellular senescence (Campisi, J., et al. (2014)). If DNA damage is severe or its accumulation exceeds its elimination by DNA repair mechanisms, cellular senescence will occur (Chen, J.H., et al. (2007)).

[0247] Next, we further analyzed the effects of carotenoids on cellular senescence. Blue light exposure increased the senescence marker, senescence-associated β- galactosidase (SA-β-gal) positive cells, and SA-β-gal activity, but pre-treatment of individual carotenoid showed fewer SA-β-gal positive cells and lower SA-β-gal activity (Fig.4 (B)). Lutein / Zeaxanthin and carotenoid blend showed lower the SA-β-gal activity, 1.2 and 1.1-fold respectively compared to 2.2-fold of vehicle (Fig.4 (C)). These results showed carotenoids’ preventive effect against blue light-induced DNA damage and cellular senescence in ARPE-19 cells.

[0248] Lutein / Zeaxanthin Increased the Expression of DNA Repair Associated Genes.

[0249] Oxidatively damaged DNA is primarily repaired by the base excision repair (BER) pathway, which in mammals is initiated by expression of DNA repair associated genes such as 8-oxo guanine DNA glycosylase-1 (Ogg1) (Cheng, J., et al. (2020) and Han, J., et al. (2003)). It has been known that the expression of OGG1 is regulated byNRF2 pathway (Singh, B., et al. (2013)). For further exploring the underlying mechanism involving carotenoids reduced blue light-induced DNA damage, we investigated expression levels of DNA repair associated genes in ARPE-19 cells. Compared with controls, treatment of Lu / Zea or β-carotene significantly increased the expression levels of genes associated with DNA repair mechanism (Fig.5 (A)). Next, we confirmed the increased expression of OGG1 in Lu / Zea treated cells. OGG1 protein expression significantly increased in the cells treated with Lu / Zea in a dose-dependent manner (Fig. 5 (B)). These results showed that Lu / Zea and β-carotene can influence DNA repair by modulation of expression of DNA repair associated genes.

[0250] Carotenoids Reduced Secretion of IL-6 and VEGF After Blue Light Irradiation.

[0251] IL-6 is the major cytokine in the senescence-associated secretory phenotype (SASP), and it has been known that the increased secretion of IL-6 is involved in the pathogenesis and progression of AMD (Seddon, J.M., et al. (2005)). Hence, the release of IL-6 was analyzed after blue light irradiation in ARPE-19 cells. Secretion of IL-6 was increased significantly after blue light irradiation (Fig.6 (A)). Pre-treatment of lycopene or lutein / zeaxanthin showed significantly reduced secretion of IL-6 compared to vehicle and carotenoid blend showed the highest effect of inhibition of IL-6 secretion compared to individual carotenoid (Fig.6 (A)).

[0252] RPE secreted vascular endothelial growth factor (VEGF) (Miller, J.W., et al. (2013)). VEGF signaling regulates normal vascular development, but it is also involved in pathological angiogenesis in neovascular AMD. We examined whether the carotenoids regulated the secretion of VEGF in ARPE-19 cells. Following blue light exposure, the secretion of VEGF protein levels was increased significantly. However, pre-treatment of individual carotenoids maintained lower levels of VEGF (Fig.6 (B)). Carotenoid blend showed higher effect on inhibition of VEGF release compared to individual carotenoid (Fig.6 (B)). Taken together, these findings demonstrate that both individual carotenoid and combination suppressed the secretion of both IL-6 and VEGF, potentially mitigating the pathological features associated with AMD.

[0253] Discussion

[0254] In this study, we examined the potential phototoxic damage triggered by blue light exposure and evaluated the protective effect of carotenoids including lycopene, lutein, zeaxanthin, and β-carotene against RPE damage. We set the blue light irradiation condition to not affect cell viability but induce cellular senescence (Fig.1 (A) and 1 (B)). Blue light irradiation increased ROS formation, but pretreatment of the carotenoids reduced the ROS levels in ARPE-19 cells (Fig.1 (D) and 1 (E)). The carotenoid blend showed a maximized antioxidant effect compared to individual carotenoid. Thus, taking carotenoids gives health benefits to protect human RPE against blue light-induced oxidative stress.

[0255] Chronic and repetitive non-lethal RPE damage (Marin-Castano, M.E., et al. (2006)) with an oxidative environment appear as important factors for the development of AMD condition (Cano, M. et al. (2010) and Suzuki, M., et al. (2012)). ROS are deleterious to DNA and to a variety of cellular organelles. It has been known that ROS causes DNA damage and triggers cellular senescence, which might be involved in AMD. Indeed, blue light exposure increased DNA damage and cellular senescence probed by p- H2A.X and SA-β-gal positive cells respectively (Fig.4). Pre-treatment of individual carotenoid reduced DNA damage and cellular senescence triggered by blue light exposure in ARPE-19 cells (Fig.4 (A-C)). Moreover, carotenoid blend showed higher effect on lowering DNA damage and cellular senescence compared to the effect of individual carotenoid. Thus, these results demonstrated the protective effect of carotenoids to prevent DNA damage and cellular senescence triggered by blue light exposure.

[0256] We investigated the mechanisms underlying the antioxidant properties of carotenoids. Carotenoids showed significant reducing activity evaluated by FRAP assay (Fig.2) and activated the NFR2-ARE pathway with the increase of NRF2 target genes (Fig.3). The activation of NFR2-ARE is a major defense mechanism against oxidative stress (Ben-Dor, A., et al. (2005) and Buendia, I., et al. (2916)) and NRF2 induction is associated with the inhibition of DNA damage via up-regulation of OGG1 (Singh, B., et al. (2013)). OGG1 is the major DNA repair glycosylase and deficiency of OGG1 has been implicated in aging and AMD (Jarrett, S.G., et al. (2012)). Lu / Zea treatment increased the expression levels of genes associated with DNA repair mechanism including Ogg1 (Fig. 5). Taken together, carotenoids showed protective antioxidant properties with multiplemechanisms, which might be an underlying mechanism for the protective effect against blue light-induced cellular damage.

[0257] The senescent cells accelerate the age-related decrease of tissue regeneration, through the depletion of stem and progenitor cells, and moreover, senescent cells produce SASP including pro-inflammatory cytokines and growth factors (Tchkonia, T., et al. (2013)). Therefore, evaluation of SASP induction in senescent RPE cells could help to further understand the course of AMD. Secretion of IL-6 and VEGF was increased after blue light irradiation, but pretreatment of individual carotenoid and the blend reduced the release of this cytokine and growth factor significantly (Fig.6 (A) and 6 (B)).

[0258] The study demonstrated that blue light irradiation triggered DNA damage and cellular senescence in ARPE-19 cells, which enhanced IL-6 and VEGF secretion. Therefore, senescent cells as those described may trigger the hallmarks of AMD pathogenesis: inflammation, and activation of pro-angiogenic cytokines. The central role of ocular anti-VEGF therapy is highly effective for treating a subset of patients with blinding eye disorders such as diabetic retinopathy and neovascular AMD. Thus, taking carotenoids gives health benefits to protect human RPE against blue light irradiation. A comprehensive knowledge of the senescent response in the RPE will help to understand the pathogenesis of this disease and to establish efficient preventive and therapeutic modalities.

[0259] Conclusion

[0260] In summary, carotenoids, as potent antioxidants, have the potential to enhance protective mechanisms against oxidative stress damage induced by blue light exposure and may mitigate DNA damage and cellular senescence, possibly through DNA repair mechanisms. Carotenoids potentially prevent initiation and progression of the pathological features associated with AMD. Consequently, carotenoids emerge as crucial and essential components in preserving the eye health from the deleterious effect of excessive blue light exposure.

[0261] Example 2: Bilberry reduced blue light-induced ROS generation in ARPE- 19 cells.

[0262] Referring to FIG.7, cells were pre-treated with indicated conditions for 48 h and ROS production was analyzed.

[0263] Each ingredient dose was 400 µg / mL.

[0264] After the pre-treatment, the cells were exposed to blue light for 1.5 and 24 h.

[0265] Figure 7 shows: (A) blue-light induced ROS generation was detected by using CellROXTMstaining; and (B) immunofluorescence microscopy. The nuclei were stained with DAPI. Scale bar: 50 µm. Data are shown as mean±SD(n=3). Lutein / zeaxanthin;Lu / Zea.

[0266] Example 3: Bilberry reduced blue light-induced DNA damage in ARPE-19 cells.

[0267] Cells were pretreated with 400 µg / ml of each ingredient (lycopene, lutein / zeaxanthin (lu / Zea), β-carotene, or bilberry) for 48 h and the cells were exposed to blue light. Representative immunofluorescence images of p-H2AX immunofluorescence (green) observed with a fluorescence microscope are shown in Figure 8. The location of the nucleus was indicated by counterstaining with DAPI (blue). Data are shown as mean±SD (n=3). Scale bar: 100 µm, Lutein / zeaxanthin; Lu / Zea.

[0268] Bilberry alleviated blue light-induced DNA damage in ARPE-19 cells.

[0269] Example 4: The antioxidant activity of Bilberry extract measured by α- TEAC and FRAP assays.

[0270] The α-TEAC and FRAP assays were used to study the antioxidant activities of bilberry at indicated doses.

[0271] It is often difficult to determine which assays would work best for measuring the antioxidant characteristics of extracts, such as the bilberry extract. In view of this, two types of assays, alpha-TEAC and FRAP were used in this study.

[0272] Referring to Figure 9, the bilberry extract showed an anti-oxidant effect in a dose dependent manner.

[0273] Referring to Figure 10, the bilberry extract showed an anti-oxidant effect in a dose dependent manner.

[0274] Both results obtained from the two different assays showed the same conclusion - bilberry extract has significant anti-oxidant effect, which is a free radical scavenging activity.

[0275] Example 5 (prophetic)

[0276] The effects of carotenoid blend (including bilberry) at the specific ratios on ROS inhibition and Nrf2-ARE induction are examined.

[0277] The following ratios of Lutein: Zeaxanthin: β-carotene: Lycopene: Bilberry are tested:

[0278] i) 5: 1: 1: 3.2 :12.5;

[0279] ii) 1.66: 0.33: 4 :4: 1;

[0280] iii) 0.83: 1.16: 4 :4: 1; and

[0281] iv) 0.83: 0.16: 1: 1 :1.

[0282] The method used is the same as described in the Example 1 above. Lutein and zeaxanthin are in the same blend (5:1).

[0283] Blue LED Light Exposure and Analysis of ROS Formation

[0284] ARPE-19 cells (5×104 cells / 100 μL) are seeded onto a black 96-well plate and cultured at 37°C for 24 h. The cells are treated with the ingredients at the ratios noted above and then incubated for 24 hr or 48 hr.

[0285] After the pretreatment, CellROXTM(ThermoFisher) is added to the cells, and the cells are exposed to 2.5 mW / cm2 of blue LED light at wavelength 410 nm for 90 min. Fluorescence readings are taken at 485 / 520 nm in a multi-well plate reader.

[0286] For immunocytochemistry, after exposure to blue light, the cells are fixed with 4% paraformaldehyde and washed in PBS at RT. Fluorescence images are acquired using a fluorescence microscope.

[0287] Nrf2-Antioxidant Response Element (ARE) Luciferase Reporter Assay

[0288] To assess activation of Nrf2 in the ARE luciferase reporter assay, a synthetic oligonucleotide containing four repeats of the ARE DNA sequence (5′- GTGACTCAGCA-3′; SEQ ID NO:15) is hybridized to its complementaryoligonucleotide, and the resulting DNA fragment ligated at the SacI / BglII site of a pGL4.27 / luc2P / minP / Hygro plasmid (Promega).

[0289] A stable cell line is generated by transfecting human hepatocellular carcinoma HepG2 cells (ATCC) with the vector using Fugene 6 (Roche) according to the manufacturer’s instructions.

[0290] To test the various samples at the indicated ratios, ARE luciferase reporter HepG2 cells are plated (1 × 104 cells / well) in white-walled, clear-bottom, 96-well plates and incubated for 48 h in a humidified, 37 °C, 5% CO2incubator. The cells are treated with samples at specified ratios for an additional 48 h. Following incubation, luciferase activity is quantified using a luciferase assay kit (Biotium, Inc.) according to the manufacturer’s instructions. Light emission is read on a SpectraMax M5 spectrophotometer (Molecular Devices).

[0291] Example 6: Carotenoids Reduce Cellular Senescence in RPE

[0292] The retinal pigmented epithelium (RPE) is constantly exposed to visible light, including blue light (BL) that creates reactive oxygen species (ROS), which are harmful to DNA and induce cellular senescence.

[0293] Carotenoids are recognized for their antioxidant properties, but their protective effect on DNA repair and cellular senescence under BL induced oxidative stress has not been evaluated.

[0294] After BL irradiation, the positive senescence-associated-β-galactosidase (SA-β-gal) staining, and gene expression of p16INK4a and p21Waf / Cip1 were upregulated in ARPE-19 cells. Pretreatment with carotenoids reduced ROS, p-H2A.X nuclear foci, and SA-β-gal positive cells induced by BL irradiation. Furthermore, pretreatment with carotenoids reduced the secretion of IL-6 and VEGF triggered by BL. Since increased senescent cells and secretion of IL-6 and VEGF are involved in age- related macular degeneration pathogenesis, the described results support that carotenoid supplementation has a potential role in protecting the eyes from the deleterious effects of excessive BL exposure.

[0295] (1) Introduction

[0296] Blue light (BL) is part of the visible light spectrum with a wavelength range between 400 and 495 nanometers (Zou and Dai, 2015). BL can reach the retina and is considered to have the potential to cause phototoxic retinal damage since it has high energy that can generate reactive oxygen species (ROS) (Godley et al., 2005). It has been considered that BL irradiation may induce retinal damage and contribute to the pathogenesis of age-related macular degeneration (AMD) (Alaimo et al., 2019). Oxidative stress, apoptosis, and inflammation of retinal pigment epithelium (RPE) play crucial roles in both the onset and progression of AMD (Abu-Amero et al., 2016).

[0297] Cellular impairment, including organelle and DNA damage, can induce cellular senescence (Herranz and Gil, 2016). Cellular senescence events such as cell-cycle arrest, as well as prolonged senescence may lead to chronic inflammation, cardiovascular disease, and AMD (Kozlowski, 2012).

[0298] The RPE, in the outer retina, is subjected to high levels of oxygen and abundant light influx including BL, rendering it highly vulnerable to oxidative stress. This excessive burden of oxidative stress invokes the onset and progression of several retinal diseases (Strauss, 2005).

[0299] Carotenoids, a group of pigments contained in various fruits and vegetables, play a drastic role in promoting eye health because of their antioxidant properties and other beneficial effects (Khachik et al., 2002). Approximately 40 natural carotenoids are commonly ingested through the human diet; however, only 15 to 20 of these are commonly detectable in human serum and tissues, including lycopene, β- carotene, lutein, and zeaxanthin (Khachik et al., 2002). Lutein and zeaxanthin are found in high concentrations in the macula of the eye and provide macular protection through their antioxidant and light-filtering properties (Kotagiri et al., 2022). An inverse correlation between AMD and macular pigment density has long been established (LaRowe et al., 2008).

[0300] Here using ARPE-19, a human RPE cell line derived from normal eyes, we provide evidence that the carotenoids – lycopene, lutein, zeaxanthin, and β-carotene – protect against the cellular damage induced by BL irradiation. We analyzed ROS levels, p-H2A.X levels as a marker of DNA damage, and senescence-associated β-galactosidase (SA- β -gal) activity as a marker of cellular senescence. We also explored the underlyingmechanisms of antioxidant effects and DNA repair. Our study revealed the protective roles of carotenoids on the BL-induced RPE damage model.

[0301] (2) Materials and Methods

[0302] Materials

[0303] β-carotene, lycopene, lutein, zeaxanthin, tetrahydrofuran, dimethyl sulfoxide, ethyl lactate, and ceramic balls (2.8 mm, Omni international) were purchased from Sigma Aldrich. An LC-MS grade water, Formic acid (FA), Methanol (MeOH), and Methyl-tertbutyl ether (MeMT) were obtained from Fisher Scientific. Dulbecco′s Modified Eagle′s Medium / Nutrient Mixture F-12 medium was obtained from ATCC. Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT). The human IL-6 DuoSet ELISA kit was obtained from R&D Systems (Minneapolis, MN). Senescence- associated β -galactosidase assay kit (Cat# 9860 and 23833), anti-HDAC2 antibody (Cat# 5113), and anti-GAPDH antibody (Cat# 2118) were obtained from Cell Signaling Technology (Danvers, MA). Anti-OGG1 antibody (Cat# PA131402) was purchased from Fisher. Anti-Nrf2 antibody (sc-365949) was obtained from Santa Cruz Biotechnology (Dallas, TX). CellTagTM 700 and IRDye®800CW Goat anti-Rabbit IgG secondary antibody was obtained from LI-COR (Lincoln, NE). CellROXTM was purchased from ThermoFisher. Blue LED light was obtained from RubyLuxLights. All other reagents were obtained from Sigma. Antioxidant assay kit (Cat#709001) was purchased from Cayman Chemical. Lycopene was purchased from Lycored (Mehoz Hadarom, Israel); lutein and zeaxanthin were purchased from DSM (Alsace, France); β-carotene was purchased from BASF Australia (Victoria, Australia). All extractions underwent verification via vendor certification.

[0304] Cell culture

[0305] The ARPE-19 cells were maintained in DMEM: F12 Medium including 10% fetal bovine serum (FBS), penicillin (100 U / mL), and streptomycin (100 µg / ml) at 37°C in a humidified atmosphere with 5% CO2. Cells were passaged by trypsin-EDTA (0.05%) solution every 2–3 d.

[0306] MTT Assays

[0307] ARPE-19 cells (5×104 cells / well) were seeded into 96-well plates. The carotenoids were dissolved in DMSO and tetrahydrofuran (THF) in a ratio of 2:1 (v / v).After incubation for 24 h, the media was replaced with media containing Lutein / zeaxanthin (Lu / Zea), lycopene, or β-carotene at concentrations ranging from 0 to 10 μg / ml. Twenty-four hours later, 5 μL of MTT reagent (20 mg / ml) was treated and the plates were incubated for 1 h. Following this incubation, the media was aspirated 106 and DMSO was added. Absorbance was analyzed at 570 nm using a microplate reader (Spectramax M5, Molecular Devices, CA, USA).

[0308] Blue LED light irradiation

[0309] ARPE-19 cells (5×104 cells / well) were seeded onto a black 96-well plate and maintained at 37°C for 24 h. The cells were treated with carotenoids at a concentration of 400 μg / mL for Lu / Zea or 745 μM for lycopene and β-carotene for 48 h. The N-acetylcysteine (NAC, 1 mM) was used as a positive control. After the pretreatment, CellROXTM (ThermoFisher) was added to the cells, and the cells were irradiated with 2.5 mW / cm2 of blue LED light at wavelength 410 nm for 90 min. Fluorescence readings were taken at 485 / 520 nm in a multi-well plate reader. For Immunocytochemistry, the BL exposed cells were fixed with 4% paraformaldehyde and washed in PBS at RT. Fluorescence images were taken using a fluorescence microscope. Cells were treated with carotenoids at a concentration of 10 μg / ml of Lu / Zea, lycopene, or β-carotene for 48 h.

[0310] Immunofluorescence

[0311] The cells in an 8-chambered slide (Ibidi, Gräfelfing, Germany) were fixed with 4% paraformaldehyde (PFA), and made permeable using 0.1% tritonX100 in PBS, and then blocked with 5% BSA in PBS for 1.5 h. The cells were incubated with p-Histone H2A.X (Ser 139) antibody (Santa Cruz, sc-517348, dilution ratio is 1:400) at 4°C overnight. The cells were washed with PBST, and the cells were incubated with Alexa Fluor 488-conjugated (1:500; ThermoFisher) antibody diluted in 3% BSA in PBS at room temperature in the dark for 1 h.4′,6-diamidino-2-phenylindole (DAPI) was used to counterstain the cell nuclei and then imaged by fluorescence microscope (Leica DMI6000B, Leica Microsystems, Wetzlar, Germany).

[0312] Isolation of RNA, cDNA synthesis, and real-time PCR

[0313] Total RNA was isolated from the cells using the RNAeasy kit from Qiagen following the manufacturer’s manual. cDNA was synthesized with 2 μg of isolated RNAusing iscript cDNA synthesis kit (Biorad). Real-time PCR was performed with Bio-Rad CFX 96 real-time PCR detection system using SsoFast™ EvaGreen® Supermix. PCR specificity was determined by melting curve analysis. A reference gene (GAPDH) was used as an internal standard. Gene specific primers used were as follows: NQO1 Forward 5′-TGGCTAGGTATCATTCAACTC-3′ SEQ ID NO:1 NQO1 Reverse 5′-CCTTAGGGCAGGTAGATTCAG-3′ SEQ ID NO:2 HO-1 Forward 5′-GCCAGCAACAAAGTGCAAGAT-3′ SEQ ID NO:3 HO-1 Reverse 5′-GGTAAGGAAGCCAGCCAAGAG-3′ SEQ ID NO:4 Sod1_Foward 5′-GCCAAAGGATGAAGAG-3′ SEQ ID NO:5 Sod1_Reverse 5′-CCACAAGCCAAACGAC-3′ SEQ ID NO:6 Gclc_Forward 5′-TTGGAGACCAGAGTATGGGAGT-3′ SEQ ID NO:7 Gclc_Reverse 5′-CTGGGAAATGAAG TTATTGTGC-3′ SEQ ID NO:8 Gapdh_Forward 5′-ACCACA GTCCATGCCATCAC-3′ SEQ ID NO:9 Gapdh_Reverse 5′-TCCACCACCCTGTTGCTGTA-3' SEQ ID NO:10 p16INK4A_Forward 5'-CTCGTGCTGATGCTACTGAGGA-3' SEQ ID NO:11 p16INK4A_Reverse 5'-GGTCGGCGCAGTTGGGCTCC-3' SEQ ID NO:12 p21_Forward 5'-AGGTGGACCTGGAGACTCTCAG-3' SEQ ID NO:13 p21_Reverse 5'-TCCTCTTGGAGAAGATCAGCCG-3' SEQ ID NO:14

[0314] For analysis of expression of antioxidant and DNA repair associated genes, TaqMan™ Array Human DNA Repair Mechanisms (Cat# 4418773, Thermo Fisher Scientific) and TaqMan™ Array, Human Antioxidant Mechanisms (Cat# 4418764, Thermo Fisher Scientific) were used.

[0315] Trolox equivalent antioxidant capacity (TEAC) assays

[0316] TEAC assays were conducted as directed by the manufacturer’s manual.

[0317] Senescence associated β-galactosidase assay

[0318] Senescence-associated β-galactosidase activity was assessed according to the manufacturer’s manual (Cat# 9860 and 23833, Cell Signaling Technology).

[0319] Antioxidant response element (ARE) luciferase assay

[0320] To evaluate the NRF2 activation, a stable cell line was generated as previously described (Yang et al., 2019). To evaluate the botanical samples, the cellswere seeded (1×104 cells / well) in 96-well plates. The cells were treated with the ingredients at indicated concentrations in Figure 3 and then incubated for 48 h. A luciferase assay kit (Biotium, Inc.) was used to analyze luciferase activity as directed by the manufacturer's manual. Light emission was measured using a microplate reader (Spectramax M5, Molecular Devices, CA, USA).

[0321] In-Cell Western (ICW)

[0322] Cells (4×103 cells / well) were seeded in a 96-well plate and were treated with carotenoids (lycopene, Lu / Zea, or β-carotene). After 48 h incubation, cells were washed in PBS and fixed in the plate with 4% PFA in PBS for 10 min at RT. After washing with PBS, the cells were permeabilized with 0.2% Triton X-100 in PBS for 15 min and washed three times with PBS. Cells were blocked with blocking buffer (Li-COR) for 90 min at RT, followed by overnight incubation with rabbit anti-OGG1 (Fisher, Cat# PA131402, dilution ratio is 1:200) in blocking buffer including 0.2% tween-20. After three washes with PBST, the cells were incubated with a secondary donkey anti-rabbit antibody (IRDye 800CW) in a blocking buffer including 0.2% tween-20. For normalization, 1 / 500 dilution of CellTagTM 700 iodide (Li-COR) in blocking buffer including 0.2% tween-20 was used. After 1 h of incubation and three washes PBST, OGG1 was detected using an infrared fluorescent scanner (Odyssey DLx, Li-Cor, Les Ulis, France). Relative fluorescent intensity for OGG1 per cell was divided by vehicle to analyze the percent change in OGG1 intensity relative to the vehicle group. All experiments were repeated in triplicate.

[0323] Isolation of nucleus and cytoplasmic fractions and immunoblotting

[0324] The cells were treated with indicated conditions and washed with PBS. Isolation of nucleus and cytoplasmic fractions was conducted as directed by the manufacturer’s manual (ThermoFisher Scientific, Cat.#: 78833). The concentrations of isolated protein were analyzed using a BCA Protein Assay kit (ThermoFisher Scientific, Cat.# 23227) as directed by the manufacturer’s manual. For immunoblotting, the isolated proteins were prepared in β-mercaptoethanol-containing 4×Laemmli sample buffer (Bio- RAD, Cat.#: 1610747) and were run following immunoblotting protocol described previously (Won et al., 2022). Briefly, the prepared samples were resolved on SDS- PAGE and were transferred to low fluorescence PVDF membrane (Bio-RAD, #1620260).Indicated antibody applied to the membrane and the antibodies are listed in the materials section. The images of immunoblotting were taken using the Odyssey® CLx Imaging System with Image Studio™ Software V.5.0 (LI-COR). Uncropped images are attached in supplementary information.

[0325] Standard stock solutions

[0326] Commercial standards were prepared in Ethyl lactate (lycopene), a 50 / 50 (v / v) cocktail of MeOH / MeMT (lutein and zeaxanthin), and in a 50 / 50 (v / v) cocktail of tetrahydrofuran / dimethyl sulfoxide (β-carotene) at concentration 1 mg / ml. Solutions were kept at -70° C.

[0327] Carotenoid quantitation

[0328] The extraction and chromatography methods (Cortes-Herrera et al., 2019) were adapted with slight modifications. Quantification of carotenoids by UV (lutein, zeaxanthin, and β-carotene) and mass spectrometry (lycopene) were used in the study. Due to the low UV absorption of lycopene, mass spectrometry was used for the measurement of this carotenoid. The ionization of carotenoids in positive ESI did not lead to the formation of protonated ions (M+H)1+, but a molecular cation (M+.). This phenomenon was reported previously for lycopene with a poor concomitant fragmentation of such ions (Cortes-Herrera et al., 2019). Moreover, due to the complicated commercial availability of an isotopically labeled standard for lycopene at the time of analysis, the latter was quantified via a standard addition method that enables matrix effect correction (Hasegawa et al., 2021).

[0329] Cell extraction and quantitation

[0330] Three ceramic balls and 250 μl of ice-cold ethyl lactate were added into each tube with a cell pellet followed by incubation at -70° C for 30 min. Cells were disrupted in TissueLyzer II (Qiagen, Milden, Germany) at the frequency of 15 Hz in two cycles of 7 minutes each at ambient temperature. Disrupted cells were vortexed for 30 minutes (Fisher Genie 2) at frequency 7 and centrifuged at 20,000 x g at ambient temperature for 30 min. Supernatants were transferred into clean tubes and stored on ice until further manipulation.

[0331] The calibration curve for UV quantification was prepared in supernatants of cells not exposed to carotenoids by 2-fold serial dilutions on the day of analysis. Forthe quantification of lycopene, the serial dilutions of the lycopene stock in ethyl lactate were prepared at concentrations 10-fold higher than the desired concentrations in samples destined for quantitation. Triplicates of cell extracts exposed to lycopene were combined and then distributed in 90 μl aliquots. The aliquot representing “0” addition received 10 µl of ethyl lactate. Other aliquots received 10 µl of a correspondent standard addition sample to yield the desired concentration. All test samples and aliquots of the calibration curve were analyzed using an Acquity UPLC H213 class chromatograph equipped with a UV-Vis detector (Waters) and connected to Orbitrap Tribrid Fusion (ThermoFisher) mass spectrometer. Samples were loaded (12 μl) onto HALO C-30, 2.1 x 150 mm column packed with 2.7 um particles (Allentown, PA). Separation was executed at 0.25 ml / min and 10°C using water (A), MeOH (B), and MTBE (C) supplemented with 0.05% (v / v) of FA. The initial, 1st minute gradient of 10 % A and 90% B was followed by 0 to 20% gradient of C (90 to 80 % B), and A (10 to 0%)) for 27 minutes. Then, the separation was continued using binary (B and C) solvent combinations: isocratic 20% C between 28 and 33 minutes, followed by an increase of C from 20 to 27 % between 33 and 35 minutes and a further increase to 37.5% between 35 and 43 minutes. Then, the solvent C was held constant (37.5%) for two minutes. The gradient was switched to 100 % B at the 45th minute and held isocratic for 2 minutes. Finally, the system was equilibrated in 10% A and 90% B for 18 min.

[0332] Data for UV analysis was acquired at 450 nm. The mass spectrometry data was acquired using the HESI 2.0 source in a positive mode (3750V, sheath gas - 10, Aux gas – 6, Ion transfer tube - 250° C, and Vaporizer -175° C). Ions were scanned in the range of 500-600 m / z at the resolution 120,000 (FWHM). Mass spectrometry and chromatography data was used for the confirmation of carotenoids in UV peaks and quantification (peak area) of lycopene at 568.4276 (lutein, zeaxanthin) and 536.4375 (β- carotene and lycopene) with 5 ppm accuracy.

[0333] Statistical Analyses

[0334] GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) was used to conduct one-sided, two-sample with equal variance t-test analyses. Data are presented as the mean ± standard deviation. A value of p < 0.05 was considered statistically significant. For ICW, immunofluorescence, and senescence-associated β-galactosidaseassay, representative images are presented. Each of these experiments was independently repeated at least three times.

[0335] (3) Results and discussion

[0336] (i) Carotenoids Reduced BL-induced ROS Generation

[0337] With the emergence and widespread use of electronic devices in our modern lifestyle, we are constantly exposed to BL. It is known that retinal exposure to excessive levels of BL induces photochemical damage to human RPE (Nunez-Alvarez et al., 2019). Therefore, the BL-induced degeneration of the human RPE presents a more relevant in vitro model for our study than the H2O2 exposure models.

[0338] A compelling body of research shows that dietary antioxidants may play a prophylactic and therapeutic role in a range of ophthalmic disorders associated with oxidative stress. Clinical investigations demonstrate that supplementation with antioxidant nutrients, such as β-carotene, and vitamins C and E, is linked to a reduced risk of AMD (Lin et al., 2009). Furthermore, lutein and zeaxanthin exhibit preferential accumulation in the macula of the retina, where they comprise the macular pigment and offer defense mechanisms to execute a protective function against oxidative damage (Mozaffarieh et al., 2003).

[0339] We quantified the carotenoids absorbed in the cells using UV and mass spectrometry (Cortes-Herrera et al., 2019). ARPE-19 cells were incubated for 24 h in the media including vehicle (DMSO: THF) or indicated carotenoids. A 5:1 ratio blend of lutein and zeaxanthin (Lu / Zea) was employed as a singular ingredient. The cells were treated with 10 μg / ml of Lu / Zea, β-carotene (18.6 μM), or lycopene (18.6 μM). After incubation, cells were rinsed 5 times with PBS and analyzed as described in the methods section. The amounts of absorbed carotenoids were 0.73 μg / 1×106 cells for lycopene, 0.37 μg / 1×106 cells for lutein, 0.07 μg / 1×106 cells for zeaxanthin and 0.06 μg / 1×106 cells for β-carotene (Table 1).

[0340] Table 1. Quantification of absorbed carotenoid in ARPE-19 Lycopene Lutein Zeaxanthin β-carotene Amount in cell pellet 6 0.73 0.37 0.07 0.06 (µg / 1x10 cells)Relative standard deviation 5.4 8.3 4.7 6.8 (%)

[0341] Light induced degeneration of the RPE is a more relevant in vitro model for studies of AMD (Boutzen et al., 2020). We set the conditions of BL exposure to 2.5 mW / cm2 for 1.5 h to further examine BL-induced ROS formation in ARPE-19 cells. BL exposure with 2.5 mW / cm2 for 1.5 h did not affect cell viability (Fig.1 (A)) but did induce cellular senescence. After BL irradiation, the mRNA expression of senescence markers, p16INK4a and p21WAF1 / CIP1, were increased (Fig.1 (B)). In natural dietary sources, lutein and zeaxanthin are found as a 5:1 ratio. (Kotagiri et al., 2022). In this research, to evaluate the optimized antioxidant effect specifically targeted at ocular health, we used the ratio (5:1) of lutein and zeaxanthin (Lu / Zea), along with the carotenoid blend containing lycopene, Lu / Zea, and β-carotene. None of the carotenoids including lycopene, Lu / Zea, and β-carotene showed toxicity in ARPE-19 cells. (Fig.1 (C)). BL exposure in the untreated ARPE-19 cells increased ROS levels significantly compared to BL irradiated vehicles, but much lower ROS generation was shown in the cells with N-acetylcysteine (NAC) as a positive control. (Fig.1 (D) and 1 (E)).

[0342] Next, we pretreated the individual carotenoids and carotenoid blend in the APRE-19 cells for 48 h and examined ROS generation levels after BL exposure. For the carotenoid blend treatment, we blended the individual carotenoids in a specific ratio. This ratio was determined as follows: Lycopene: Lutein: Zeaxanthin: β-carotene = 5.7: 10: 2: 0.5. Pretreatment with individual carotenoids reduced ROS generation. Surprisingly, the carotenoid blend showed the most reduced ROS levels (Fig.1 (D) and 1 (E)). These results demonstrated both the antioxidant effect of individual carotenoids as well as the maximizing effect of the carotenoid blend to reduce ROS generation induced by BL exposure in ARPE-19 cells. These findings highlight the potential health benefits of dietary carotenoids in reducing cell damage in human RPE from oxidative stress induced by the exposure to blue light.

[0343] (ii) Antioxidant Activity of Carotenoids

[0344] We investigated the mechanisms by which carotenoids exert their antioxidant properties.

[0345] Lycopene, Lu / Zea, and β-carotene showed significant antioxidant activity in a dose-dependent manner (Fig.11 (A)). These results support carotenoids’ antioxidant potential for scavenging ROS and reducing ROS generation induced by BL irradiation. The antioxidant capacity of the individual carotenoids was investigated using TEAC assay (Bohm et al., 2002). The antioxidant capacity of carotenoids was reflected differently depending on the analysis method and the antioxidant activity of β-carotene is well reflected without degradation to long-chain decomposition products with analysis by TEAC assay (Mueller and Boehm, 2011). In this context, the TEAC assay was adapted to evaluate the antioxidant activity of lycopene and β-carotene, as well as Lu / Zea.

[0346] (iii) Carotenoids Activated the Transcription of Nuclear Erythroid 2- Related Factor (NRF2)

[0347] The transcription factor NRF2 regulates the expression of antioxidative enzymes including glutamate-cysteine ligase catalytic subunit (GCLC), heme oxygenase- 1 (HO-1), and NAD(P)H quinone oxidoreductase 1 (NQO1) (Chen and Kunsch, 2004). The expression of the antioxidative enzymes in response to ROS is induced by antioxidant response elements (ARE), which activate the NRF2 (Chen and Kunsch, 2004).

[0348] ARPE-19 cells were treated with each carotenoid for 48 h and were analyzed for ARE activity. The individual carotenoids each showed a dose-dependent luciferase activity in the ARE reporter assay (Fig.11 (B)). We evaluated the effect of carotenoids on NRF2 levels within the nucleus using western blotting and immunocytochemistry. It was observed that treatment with individual carotenoids at a concentration of 10 μg / ml of Lu / Zea, β-carotene, or lycopene for 24 hours led to increased NRF2 levels in the nucleus (Fig 11 (C) and 11 (D)). Analysis of NRF2 expression in the nuclear and cytoplasmic fractions revealed a significant increase (p < 0.05) in the NRF2 ratio (nucleus / cytoplasm) by 2.2-fold for lycopene, 2.0-fold for β- carotene, and 1.8-fold for Lu / Zea after treatment (Fig.11 (D)). The individual carotenoid treated groups showed a notable enhancement in NRF2 levels within the nucleus, as depicted in Fig.11 (E). These findings demonstrate that carotenoids enhanced NRF2 levels within nucleus compartments, highlighting their significance in NRF2-ARE signaling pathways. Next, we analyzed gene expressions associated with antioxidantmechanisms. Multiple genes associated with antioxidant mechanisms were significantly increased in Lu / Zea treated cells (Fig.11 (F)). These results show that carotenoids activated the NRF2 pathway and increased expression of genes associated with antioxidant mechanisms. These results give insight into the carotenoids antioxidant ability to reduce BL-induced oxidative stress.

[0349] (iv) Carotenoids Reduced DNA Damage and Cellular Senescence Triggered by BL Exposure

[0350] ROS-mediated oxidative stress leads to DNA damage and serine139- phosphorylated histone H2A.X (p-H2A.X) has been established as evidence of DNA damage (Kurz et al., 2004). We evaluated nuclear p-H2A.X immunofluorescence to assess DNA damage triggered by BL exposure in ARPE-19 cells. BL exposure increased expression of p-H2A.X nuclear foci, which is evident for the activation of the response associated with DNA damage. Pretreatment of cells with individual carotenoids resulted in reduced p-H2A.X expression, while the combination of these carotenoids further decreased p-H2A.X expression (Fig.4 (A)).

[0351] It is known that DNA damage causes cellular senescence (Chen et al., 2007). In instances where DNA damage is excessive or accumulates beyond the capacity of DNA repair mechanisms, cellular senescence will occur (Chen et al., 2007). Next, we analyzed the effects of carotenoids on cellular senescence. BL exposure led to an increase in SA-β-gal expression, indicating higher senescence levels, as well as enhanced SA-β- gal activity but pre-treatment with individual carotenoids showed fewer SA-β-gal positive cells (Fig.4 (B) and lower SA-β-gal activity (Fig.4 (C)). Lu / Zea and the carotenoid blend showed significantly lower SA-β-gal activity, 1.2 and 1.1-fold respectively compared to 2.2-fold of vehicle (Fig.4 (C)). These results showed carotenoids’ preventive effect against BL-induced DNA damage and cellular senescence in ARPE-19 cells.

[0352] Repetitive and chronic non-lethal RPE damage with oxidative stress are regarded as significant factors for the pathogenesis of AMD disease (Cano et al., 2010). ROS are harmful to DNA and a variety of cellular organelles (Algvere et al., 2006). It is known that ROS causes DNA damage and triggers cellular senescence, which might be involved in AMD (Kozlowski, 2012). Here pre-treatment with individual carotenoids reduced DNA damage and cellular senescence triggered by BL exposure in ARPE-19cells (Fig.4). Moreover, the carotenoid blend showed a higher effect on lowering DNA damage and cellular senescence as compared to the effect of individual carotenoids. These results demonstrate the protective effect of carotenoids in reduction of DNA damage and cellular senescence triggered by BL exposure.

[0353] (v) Carotenoids Increased the Expression of DNA Repair Associated Genes

[0354] To further explore the underlying mechanisms involved in carotenoids reduction of BL-induced DNA damage, we investigated expression levels of DNA repair associated genes in ARPE-19 cells. Treatment with Lu / Zea or β-carotene significantly increased the expression levels of genes associated with DNA repair mechanisms when compared to the control group (Fig.5 (A)). Next, we confirmed a significant dose dependent increase in expression of OGG1 protein in Lu / Zea treated cells. (Fig.5 (B)). The base excision repair (BER) pathway appears as a primary DNA repair mechanism, and the BER pathway is initiated by expression of DNA repair associated genes such as 8-oxo guanine DNA glycosylase-1 (Ogg1) in mammals (Wang et al., 2018). It is known that the expression of OGG1 is regulated by NRF2 pathway (Singh et al., 2013). Induction of NRF2 is associated with the inhibition of DNA damage via up-regulation of OGG1 (Singh et al., 2013). OGG1 is the major DNA repair glycosylase and deficiency of OGG1 has been implicated in aging and AMD (Jarrett and Boulton, 2012). Taken together, these results showed that Lu / Zea and β-carotene can influence DNA repair by modulation of expression of DNA repair associated genes, that provide a potential protective mechanism against a BL-induced cellular damage.

[0355] (vi) Carotenoids Reduced Secretion of IL-6 and VEGF after BL Irradiation

[0356] One of the major cytokines (IL-6) in the senescence-associated secretory phenotype (SASP), is known to be involved in the development of AMD (Seddon et al., 2005). In our research, the release of IL-6 was analyzed after BL irradiation in the control ARPE-19 cells. Secretion of IL-6 was increased significantly after BL irradiation (Fig.6 (A)). After pre-treatment with lycopene or Lu / Zea there was a significant decrease in IL-6 secretion as compared to the vehicle. The carotenoid blend showed the highest reduction of IL-6 secretion compared to individual ingredients (Fig.6 (A)).

[0357] RPE naturally secret vascular endothelial growth factor (VEGF) (Miller et al., 2013). VEGF signaling regulates normal vascular development, but it is also known that overexpression of VEGF is involved in pathological angiogenesis in neovascular AMD (Miller et al., 2013). We examined whether the carotenoids regulated the synthesis of VEGF in ARPE-19 cells. Following BL exposure, the secretion of VEGF protein levels was increased significantly. However, pre-treatment with individual carotenoids maintained significantly lower levels of VEGF (Fig.6 (B)). The carotenoid blend showed a higher effect on inhibition of VEGF release compared to individual carotenoids (Fig.6 (B)). Taken together, these findings demonstrate that both individual carotenoids and combination suppressed the secretion of both IL-6 and VEGF, potentially mitigating the pathological features associated with AMD.

[0358] (4) Conclusion

[0359] These findings demonstrated that BL irradiation triggered DNA damage and cellular senescence in ARPE-19 cells, which enhanced IL-6 and VEGF secretion. Senescent cells may initiate the signs of AMD pathogenesis: inflammation, and activation of pro-angiogenic cytokines. Ocular anti-VEGF therapy occupies a pivotal position in treating sight-threatening ophthalmic conditions such as diabetic retinopathy and neovascular AMD (Sene et al., 2015). This work supports that supplementation with dietary carotenoids can promote multiple mechanisms for protection of human RPE against BL irradiation.

[0360] In summary, carotenoids, as potent antioxidants, have the potential to enhance protective mechanisms against oxidative stress damage induced by BL exposure. The carotenoids promote the NRF2 translocation levels to the nucleus and activate the ARE pathway. Furthermore, carotenoids also mitigate DNA damage and cellular senescence, possibly through DNA repair mechanisms.

[0361] Carotenoids demonstrated a potential to prevent the onset and progression of AMD by decreasing the secretion of IL-6 and VEGF, pathological features associated with AMD. Consequently, carotenoids emerge as crucial and essential components in preserving eye health from the deleterious effect of excessive BL exposure.

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Claims

CLAIMS 1. A composition for protecting the eyes of a mammalian subject from or treating damage to the eyes of the subject due to exposure of the subject’s eyes to blue light comprising: an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and an additive; wherein the composition effectively protects the eyes of a mammalian subject from or treats damage to the eyes of the subject due to exposure of the subject to blue light.

2. The composition of claim 1, wherein the ratio of lutein to zeaxanthin to β-carotene to lycopene to bilberry is 5:1:1:3.2:12.

5.

3. The composition of claim 1 or claim 2, wherein the amount of an ingredient or extract of bilberry is in the range from about 0.1 mg to about 50 mg.

4. The composition of any of claims 1-3, wherein the amount of at least one of lutein, zeaxanthin, β-carotene, and lycopene is in the range from about 0.1 mg to about 25 mg.

5. The composition of any of claims 1-4, wherein the composition is formed as tablets, gels, capsules, pills, caplets, dragees, granules, powders, or effervescent tablets, liquid, sprays, functional foods, gums, chewing gum, gummi candy, taffy, caramel candy, fudge, degradable thin films, nondegradable thin films, hard candy, liquids, drinks, or beverages.

6. The composition of any of claims 1-5, wherein the composition is an immediate release composition, a mixed-release composition, or an enterically-coated composition.

7. The composition of any of claims 1-6, wherein the composition is effective in protecting against blue light-induced DNA damage.

8. The composition of any of claims 1-6, wherein the composition is effective in protecting against blue light-induced oxidative stress.

9. The composition of any of claims 1-6, wherein the composition is effective in protecting human retinal pigment epithelium against blue light irradiation.

10. The composition of any of claims 1-9, wherein the additive comprises at least one of: a sweetener selected from high fructose corn syrup, mannose, maltose, glucose polymers, sucrose, glucose, dextrose, lactose, galactose, fructose, polysaccharides, rice syrup, honey, saccharin, cyclamates, acetosulfam, sorbitol, sucralose, xylitol, erythritol, Stevia extract, L-aspartyl-L-phenyl-alanine ester, L-aspartyl-D-alanine alkyl amides, L- aspartyl-L-1-hydroxymethylalkaneamide, and L-aspartyl-1-hydroxyethylalkaneamide; a pH-adjusting agent selected from hydrochloric acid, citric acid, sodium hydrogen carbonate, potassium hydroxide, sodium hydroxide, and sodium carbonate; a preservative selected from sorbic acid, benzoic acid, sodium benzoate, calcium benzoate, potassium benzoate, potassium sorbate, calcium sorbate, and sodium sorbate; and a flavoring agent selected from almond oil, amaretto oil, anethole, anise oil, benzaldehyde, blackberry, black walnut oil, blueberry, caraway, caraway oil, cardamom oil, cardamom seed, cherry juice, cherry syrup, cinnamon, cinnamon oil, cinnamon water, citric acid, citric acid syrup, clove oil, cocoa, coriander oil, dextrose, eriodictyon, ethyl acetate, ethyl vanillin, fennel oil, ginger, glucose, glycerin, glycyrrhiza, grape, honey, lavender oil, lemon oil, lime, mannitol, methyl salicylate, myristica oil, orange oil, orange peel, orange syrup, peppermint, peppermint oil, peppermint water, phenylethyl alcohol, pineapple, raspberry juice, raspberry syrup, rosemary oil, rose oil, rose water, sarsaparilla syrup, sorbitol, spearmint, spearmint oil, strawberry, sucrose, thyme oil, tolu balsam, tropical, vanilla, vanillin, and wild cherry syrup.

11. The composition of any of claims 1-10, wherein the composition is a food product.

12. The composition of any of claims 1-10, wherein the composition is a dietary supplement.

13. The composition of any of claims 1-12, wherein the composition is for consumption once or more times a day.

14. The composition of any of claims 1-13, wherein the composition comprises lycopene, lutein, zeaxanthin and β-carotene, and wherein the ratio of lycopene to lutein to zeaxanthin to β-carotene in the composition is 5.7:10:2:0.5 15. A composition comprising: about 10 mg lutein; about 2.0 mg zeaxanthin; about 2.0 mg beta carotene; about 6.4 mg lycopene; about 25 mg bilberry; and an additive.

16. The composition of claim 15, wherein the composition effectively protects the eyes of a mammalian subject from or treats damage to the eyes of the subject due to exposure to blue light.

17. The composition of claim 15, wherein the composition is effective in protecting against blue light-induced DNA damage.

18. The composition of claim 15, wherein the composition is effective in protecting against blue light-induced oxidative stress.

19. The composition of claim 15, wherein the composition is effective in protecting human retinal pigment epithelium against blue light irradiation.

20. The composition of any of claims 15-19, wherein the composition is formed as tablets, gels, capsules, pills, caplets, dragees, granules, powders, or effervescent tablets, liquid, sprays, functional foods, gums, chewing gum, gummi candy, taffy, caramel candy,fudge, degradable thin films, nondegradable thin films, hard candy, liquids, drinks, or beverages.

21. The composition of any of claims 15-20, wherein the composition is an immediate release composition, a mixed-release composition, or an enterically-coated composition.

22. The composition of any of claims 15-21, wherein the additive comprises at least one of: a sweetener selected from high fructose corn syrup, mannose, maltose, glucose polymers, sucrose, glucose, dextrose, lactose, galactose, fructose, polysaccharides, rice syrup, honey, saccharin, cyclamates, acetosulfam, sorbitol, sucralose, xylitol, erythritol, Stevia extract, L-aspartyl-L-phenyl-alanine ester, L-aspartyl-D-alanine alkyl amides, L- aspartyl-L-1-hydroxymethylalkaneamide, and L-aspartyl-1-hydroxyethylalkaneamide; a pH-adjusting agent selected from hydrochloric acid, citric acid, sodium hydrogen carbonate, potassium hydroxide, sodium hydroxide, and sodium carbonate; a preservative selected from sorbic acid, benzoic acid, sodium benzoate, calcium benzoate, potassium benzoate, potassium sorbate, calcium sorbate, and sodium sorbate; and a flavoring agent selected from almond oil, amaretto oil, anethole, anise oil, benzaldehyde, blackberry, black walnut oil, blueberry, caraway, caraway oil, cardamom oil, cardamom seed, cherry juice, cherry syrup, cinnamon, cinnamon oil, cinnamon water, citric acid, citric acid syrup, clove oil, cocoa, coriander oil, dextrose, eriodictyon, ethyl acetate, ethyl vanillin, fennel oil, ginger, glucose, glycerin, glycyrrhiza, grape, honey, lavender oil, lemon oil, lime, mannitol, methyl salicylate, myristica oil, orange oil, orange peel, orange syrup, peppermint, peppermint oil, peppermint water, phenylethyl alcohol, pineapple, raspberry juice, raspberry syrup, rosemary oil, rose oil, rose water, sarsaparilla syrup, sorbitol, spearmint, spearmint oil, strawberry, sucrose, thyme oil, tolu balsam, tropical, vanilla, vanillin, and wild cherry syrup.

23. The composition of any of claims 15-22, wherein the composition is a food product.

24. The composition of any of claims 15-22, wherein the composition is a dietary supplement.

25. The composition of any of claims 15-22, wherein the composition is for consumption once or more times a day.

26. A composition for preventing, reducing, or treating one or more signs and / or symptoms of an ophthalmic condition in a subject resulting from exposure of the subject’s eyes to blue light comprising: an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and an additive; wherein the composition effectively prevents, reduces, or treats one or more signs and symptoms of an ophthalmic condition in the subject.

27. The composition of claim 26, wherein the ophthalmic condition is a blinding eye disorder.

28. The composition of claim 26, wherein the ophthalmic condition is age-related macular degeneration, cataract, glaucoma, or retinitis pigmentosa.

29. The composition of any of claims 26-28, wherein the ratio of lutein to zeaxanthin to β-carotene to lycopene to bilberry is 5:1:1:3.2:12.

5.

30. The composition of any of claims 26-29, wherein the composition is formed as tablets, gels, capsules, pills, caplets, dragees, granules, powders, or effervescent tablets, liquid, sprays, functional foods, gums, chewing gum, gummi candy, taffy, caramel candy, fudge, degradable thin films, nondegradable thin films, hard candy, liquids, drinks, or beverages.

31. The composition of any of claims 26-30, wherein the additive comprises at least one of: a sweetener selected from high fructose corn syrup, mannose, maltose, glucose polymers, sucrose, glucose, dextrose, lactose, galactose, fructose, polysaccharides, rice syrup, honey, saccharin, cyclamates, acetosulfam, sorbitol, sucralose, xylitol, erythritol, Stevia extract, L-aspartyl-L-phenyl-alanine ester, L-aspartyl-D-alanine alkyl amides, L- aspartyl-L-1-hydroxymethylalkaneamide, and L-aspartyl-1-hydroxyethylalkaneamide; a pH-adjusting agent selected from hydrochloric acid, citric acid, sodium hydrogen carbonate, potassium hydroxide, sodium hydroxide, and sodium carbonate; a preservative selected from sorbic acid, benzoic acid, sodium benzoate, calcium benzoate, potassium benzoate, potassium sorbate, calcium sorbate, and sodium sorbate; and a flavoring agent selected from almond oil, amaretto oil, anethole, anise oil, benzaldehyde, blackberry, black walnut oil, blueberry, caraway, caraway oil, cardamom oil, cardamom seed, cherry juice, cherry syrup, cinnamon, cinnamon oil, cinnamon water, citric acid, citric acid syrup, clove oil, cocoa, coriander oil, dextrose, eriodictyon, ethyl acetate, ethyl vanillin, fennel oil, ginger, glucose, glycerin, glycyrrhiza, grape, honey, lavender oil, lemon oil, lime, mannitol, methyl salicylate, myristica oil, orange oil, orange peel, orange syrup, peppermint, peppermint oil, peppermint water, phenylethyl alcohol, pineapple, raspberry juice, raspberry syrup, rosemary oil, rose oil, rose water, sarsaparilla syrup, sorbitol, spearmint, spearmint oil, strawberry, sucrose, thyme oil, tolu balsam, tropical, vanilla, vanillin, and wild cherry syrup.

32. The composition of any of claims 26-31, wherein the composition is a food product.

33. The composition of any of claims 26-31, wherein the composition is a dietary supplement.

34. The composition of any of claims 26-31, wherein the composition is for consumption once or more times a day.

35. The composition of any of claims 26-34, wherein the one or more signs and symptoms of the ophthalmic condition is selected from the group consisting of: vision loss, blurred or decreased vision in one or both eyes; distortion of vision; blind spots; distorted vision in the form of metamorphopsia; central scotomas; shadows; missing areas of vision; slow recovery of visual function after exposure to bright light; decreased visual acuity; decreased ability to discern colors; decreased or lost contrast sensitivity; visual hallucinations; flashing lights; and accelerated aging of the eyes.

36. The composition of any of claims 26-35, wherein the composition comprises lycopene, lutein, zeaxanthin and β-carotene, and wherein the ratio of lycopene to lutein to zeaxanthin to β-carotene in the composition is 5.7:10:2:0.5 37. A method for treating or preventing blue light-induced ophthalmic condition, or one or more sign or symptom thereof in a mammalian subject at risk of or having blue light-induced ophthalmic condition, or one or more sign or symptom thereof, comprising: administering to the subject a therapeutically effective amount of a composition that is provided in the form selected from the group consisting of capsules, tablets, liquids, and powders for oral ingestion, the composition comprising: (i) an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and (ii) a pharmaceutically acceptable additive.

38. The method of claim 37, wherein the administering is intraocularly, iontophoretically, orally, topically, systemically, intravenously, subcutaneously, or intramuscularly.

39. The method of claim 37, wherein the ophthalmic condition is a blinding eye disorder.

40. The method of claim 37, wherein the ophthalmic condition is age-related macular degeneration, cataract, glaucoma, or retinitis pigmentosa.

41. The method of any of claims 37-40, wherein the ratio of lutein to zeaxanthin to β- carotene to lycopene to bilberry is 5:1:1:3.2:12.

5.

42. The method of any of claims 37-41, wherein the administering is once or more times a day.

43. The method of any of claims 37-41, wherein the one or more signs and symptoms of an ophthalmic condition is selected from the group consisting of: vision loss, blurred or decreased vision in one or both eyes; distortion of vision; blind spots; distorted vision in the form of metamorphopsia; central scotomas; shadows; missing areas of vision; slow recovery of visual function after exposure to bright light; decreased visual acuity; decreased ability to discern colors; decreased or lost contrast sensitivity; visual hallucinations; and flashing lights.

44. The method of any of claims 37-43, wherein the composition comprises lycopene, lutein, zeaxanthin and β-carotene, and wherein the ratio of lycopene to lutein to zeaxanthin to β-carotene in the composition is 5.7:10:2:0.5 45. A method of protecting a user’s eyes from the harmful effect of blue light, comprising: administering to the user a therapeutically effective amount of a composition that is provided in the form selected from the group consisting of capsules, tablets, liquids, and powders for oral ingestion, the composition comprising: (i) an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and (ii) a pharmaceutically acceptable additive.

46. A method for enhancing protective mechanisms against oxidative stress damage induced by blue light exposure and mitigating DNA damage and cellular senescence in a user’s eyes, comprising:administering to the user a therapeutically effective amount of a composition that is provided in the form selected from the group consisting of capsules, tablets, liquids, and powders for oral ingestion, the composition comprising: (i) an amount of at least one of: lutein, zeaxanthin, β-carotene, lycopene, and an ingredient or extract of bilberry; and (ii) a pharmaceutically acceptable additive.

47. The method of claim 46, wherein enhancing protective mechanisms against oxidative stress damage induced by blue light exposure and mitigating DNA damage and cellular senescence in a user’s eyes is by: decreasing senescence-associated β-galactosidase (SA-β-gal); decreasing ROS levels, and / or decreasing p-H2A.X levels.

48. The method of any of claims 46-47, wherein the composition comprises lycopene, lutein, zeaxanthin and β-carotene, and wherein the ratio of lycopene to lutein to zeaxanthin to β-carotene in the composition is 5.7:10:2:

0.

49. A composition for protecting the eyes of a mammalian subject from or treating damage to the eyes of the subject due to exposure of the subject’s eyes to blue light comprising: (i) an amount of an ingredient or extract of bilberry; and (ii) an additive; wherein the composition effectively protects the eyes of a mammalian subject from or treats damage to the eyes of the subject due to exposure to blue light.

50. A composition for enhancing protective mechanisms against oxidative stress damage induced by blue light exposure and mitigating DNA damage and cellular senescence in a user’s eyes, comprising:(i) lycopene, lutein, zeaxanthin and β-carotene, and (ii) an additive; wherein the ratio of lycopene to lutein to zeaxanthin to β-carotene in the composition is 5.7:10:2:0.

Images