Nutritional composition for visual function

Abstract

A nutritional composition for preventing, improving, and maintaining visual function. A nutritional composition comprising nutrients and at least one pharmaceutically and / or nutraceutical acceptable excipient. A nutritional composition for preventing, improving, and maintaining dry eye, wherein the nutrients are selected from one or more of lutein, curcumin, and vitamin D3. A process for preparing a nutritional composition comprising lutein, curcumin, and vitamin D3 for use in visual function, more specifically dry eye. The present invention further encompasses nutritional compositions comprising selective proportions of lutein, curcumin, and vitamin D3 formulated in different forms, such as powders, granules, pellets, beadlets, caplets, tablets, capsules, softgel capsules, solutions, emulsions, suspensions, oil suspensions, and dispersions.

Description

Technical Field

[0001] The present invention relates to a nutritional composition for use in the prevention, improvement, and maintenance of visual function in subjects in need thereof. More specifically, the present invention describes a nutritional composition comprising nutrients and at least one pharmaceutically and / or nutraceutically acceptable excipient. More specifically, the present invention relates to a nutritional composition for use in dry eye syndrome for the prevention, improvement, and maintenance of visual function, wherein the nutrient is selected from one or more of lutein, zeaxanthin, curcumin, and vitamin D3. In addition, the present invention relates to a process for preparing a nutritional composition comprising lutein, zeaxanthin, curcumin, and vitamin D3 for use in visual function, more specifically dry eye syndrome.

Background Art

[0002] Dry eye syndrome (DES) is a multifactorial disease of the tear and ocular surface that results in symptoms of decreased tear production, discomfort, and visual disturbances with potential damage to the ocular surface. This is mainly associated with instability of the tear film, increased osmolarity of the tears, and inflammation of the ocular surface. Oxidative stress plays an important role in the induction of ocular surface damage in DES, accompanied by an increase in the levels of malondialdehyde (MDA), a lipid peroxidation marker, in the tear film and ocular surface of dry eye patients, as well as a decrease in antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase.

[0003] Lutein and zeaxanthin are present in high concentrations in the eyes. Generally, the retina and lens, especially the macula at the center of the retina, are very rich in these two xanthophylls. Lutein and zeaxanthin absorb high-energy blue light and protect the retina from phototoxicity. Oxidative stress results from the disruption of the balance between the antioxidant system and the oxidation-promoting system found within cells. It is accepted that overexpression of ROS can be induced on the ocular surface as a result of many acute and chronic diseases, and even normal aging. Recent research has demonstrated that oxidative stress damages the ocular surface and plays an important role in the mechanism of dry eye disease. Curcumin, a yellow polyphenol from the plant turmeric (Curcuma Longa) and known for its anti-inflammatory and antioxidant properties, was used as another component in this preparation. Research has demonstrated the beneficial effects of curcumin in multiple anterior eye diseases such as corneal diseases, dry eye conditions, conjunctivitis, anterior uveitis, cataracts, and glaucoma. Vitamin D is now a multifunctional vitamin known to play important roles in many life activities.

[0004] More importantly, low serum vitamin D levels have been found to be associated with DES, and tear secretion and tear break-up time (TBUT) were positively correlated with serum vitamin D concentration. Furthermore, vitamin D receptors are found in corneal epithelium, endothelium, and retinal pigment epithelium, and also enhance the barrier function of corneal epithelium. Vitamin D can regulate fluid and ion transport in the salivary gland and tear secretion in the lacrimal gland.

[0005] EP 338 478 0 A1 relates to a composition containing linseed oil and hempseed oil having a balanced ratio of omega-3 and omega-6, and / or a volume / volume ratio of linseed oil to hempseed oil contained between 60:40 and 95:5, and a process for its preparation. The composition further contains vitamins, polyphenols, flavonoids, isoflavonoids, bioflavonoids, phytoestrogens, carotenoids, vegetable extracts, lactate dehydrogenase, melatonin, coenzyme Q10, lipophilic substances, molecules having specific rapid metabolism, and combinations thereof.

[0006] US20060020046 A1 describes the use of lycopene in the manufacture of compositions for the primary and secondary prevention of angiogenesis-related pathologies and their coadjuvant treatment, optionally in combination with vitamin E and / or C or other bioactive ingredients disclosed in the specification, as well as certain novel formulations containing lycopene.

[0007] WO2018235939A1 provides an ophthalmic composition containing an inclusion antioxidant.

[0008] WO2008113177A1 provides various compounds and compositions containing polyunsaturated fatty acid monoglycerides and their derivatives. These compounds and compositions may be useful as cancer preventive agents. They may also be useful for enhancing the solubility of various active agents and their bioavailability.

[0009] None of the above-mentioned references speak at all about compositions containing lutein, zeaxanthin, curcumin, and vitamin D3, which are used for visual function and related complications such as dry eye syndrome. The literature states that the first choice for the treatment of DES consists of using topical corticosteroids, which usually improve the symptoms of the disease but are associated with harmful effects with long-term use, after artificial tears. Therefore, there is a high interest in exploring the effectiveness of natural ingredients that can be administered orally to avoid the harmful events associated with the frequent and long-term use of eye drops. There has long been a need to provide a nutritional composition containing selective nutrients for use in the prevention, improvement, and maintenance of visual function, more specifically dry eye syndrome, in subjects in need thereof. SUMMARY OF THE INVENTION

[0010] The main object of the present invention is to develop a nutritional composition comprising a nutrient selected from one or more of lutein, zeaxanthin, curcumin, and vitamin D3, and at least one pharmaceutically and / or nutraceutically acceptable excipient for the prevention, improvement, and maintenance of visual function.

[0011] Another object of the present invention is to develop a nutritional composition comprising lutein, zeaxanthin, curcumin, and vitamin D3 for use in the prevention, improvement, and maintenance of dry eye syndrome.

[0012] A further object of the present invention is to provide a nutritional composition comprising lutein, zeaxanthin, curcumin, and vitamin D3 in optional proportions and / or ratios, which can be formulated in different forms such as solid, semi-solid, and liquid forms for oral administration, selected from, but not limited to, dosage forms such as powders, granules, pellets, beadlets, caplets, tablets, capsules, soft gel capsules, solutions, emulsions, suspensions, oil suspensions, dispersions, etc.

[0013] Another object of the present invention is to reduce inflammatory cytokines such as nuclear factor kappa-light-chain enhancer of activated B cells (NFkB), matrix metallopeptidase 9 (MMP-9), tumor necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1b), interleukin 6 (IL-6), and interleukin 8 (IL-8).

[0014] Another object of the present invention is to reduce oxidative stress markers such as malondialdehyde (MDA) and increase antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx).

[0015] A further object of the present invention is to increase tear volume, tear break-up time, tear stability, and mucin production.

[0016] Another object of the present invention is to reduce corneal surface damage, conjunctival surface damage, the use and frequency of artificial tears.

[0017] Another object of the present invention is to significantly increase tear production, relieve dry eye, reduce eye discomfort, improve tear stability, reduce irritation, burning, and grittiness associated with dry eye, reduce tear loss, reduce ocular surface damage, reduce inflammation in tears, and reduce dependence on artificial tears by developing a composition.

[0018] A further object of the present invention is to provide a preparation process for a nutritional composition containing lutein, zeaxanthin, curcumin, and vitamin D3.

Brief Description of the Drawings

[0019]

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Mode for Carrying Out the Invention

[0020] The invention described herein relates to a nutritional composition and its use for the prevention, improvement, and maintenance of visual function, more specifically dry eye syndrome.

[0021] In the context of the present invention, the term "nutritional composition" is generally used herein to refer to a composition having selectively added nutrients such as lutein, zeaxanthin, curcumin, and vitamin D3, together with one or more pharmaceutically and / or nutraceutically acceptable excipients that promote an improvement in results.

[0022] The inventors of the present invention have arrived at this selective composition of active ingredients through a rigorous experimental method, and there is no prior art that describes the use of this composition for dry eye syndrome. The inventors have found the following literature that describes, with insufficient experiments and no motivation for comparison, that a single ingredient may be effective for dry eye syndrome.

[0023] The published paper at https: / / www.eyeworld.org / article-vitamin-d-and-dry-eye--is-there -an-association states that vitamin D3 induces the production of IL-10, which inhibits the production of other pro-inflammatory cytokines, including IL-1, IL-6, and TNF-α. Considering this, it is physiologically reasonable that vitamin D3 is also involved in supporting inflammatory mediators in the tear film of dry eye patients.

[0024] The paper published on nature.com under the title "Vitamin D Supplementation for Patients with Dry Eye Syndrome Refractory to Conventional Treatment" in Scientific Reports | 6:33083 DOI: 10.1038 / srep33083 describes a study investigating the effect of vitamin D supplementation in patients with dry eye syndrome (DES) refractory to conventional treatment and with vitamin D deficiency. A total of 105 patients with DES refractory to conventional treatment and with vitamin D deficiency were treated with an intramuscular injection of colecalciferol (200,000 IU). Serum 25-hydroxyvitamin D (25(OH)D) levels were measured. Eye discomfort was evaluated using the Ocular Surface Disease Index (OSDI) and the Visual Analogue Pain Score (VAS). The tear break-up time (TBUT), fluorescein staining score (FSS), eyelid margin hyperemia, and tear secretion test were measured before treatment and at 2, 6, and 10 weeks after vitamin D supplementation. The mean serum 25(OH)D level was 10.52 ± 4.61 ng / mL. TBUT and the tear secretion test showed improvement at 2 and 6 weeks after vitamin D supplementation compared to pre-treatment values (all p < 0.05, paired t-test). Eyelid margin hyperemia and symptom severity showed improvement at 2, 6, and 10 weeks after vitamin D supplementation (all p < 0.05). FSS, OSDI, and VAS decreased at 2 weeks compared to pre-treatment values (all p < 0.05). In conclusion, vitamin D supplementation is effective and useful for the treatment of patients with DES refractory to conventional treatment and with vitamin D deficiency.

[0025] However, both published papers only describe vitamin D3 and do not mention at all the composition of lutein, zeaxanthin, curcumin, and vitamin D3. Therefore, considering the comparison of the present invention with the papers, it reflects a significant improvement with the formulated selective composition.

[0026] In the published paper "Curcumin: Therapeutically Potential Planta Medicine 2014; 80: 249-254" by Pescosolido N et al., it has been demonstrated that curcumin has beneficial effects on several eye diseases such as chronic anterior uveitis, diabetic retinopathy, glaucoma, age-related macular degeneration, and dry eye syndrome. The purpose of this review is to report what has been elucidated so far about the properties of curcumin and its potential use in ophthalmology.

[0027] A careful examination of the prior art available in the public domain and the prior art referred to above reveals that none of the prior art mentions anything about the claimed compositions of lutein, zeaxanthin, curcumin, and vitamin D3. However, some of the prior art includes only single components and, with insufficient experimental support, mentions dry eye syndrome. However, there is no prior art teaching any suggestion or motivation regarding the compositions of the present invention.

[0028] Embodiments of the present invention are described in detail as follows.

[0029] According to an embodiment of the present invention, the nutrient is selected from one or more of lutein, zeaxanthin, curcumin, vitamin D3, ginger, ashwagandha, and β-cryptoxanthin, and / or mixtures thereof.

[0030] As in another embodiment of the present invention, a nutritional composition comprising a nutrient and one or more pharmaceutically and / or nutraceutically acceptable excipients is used for the prevention, improvement, and maintenance of dry eye syndrome.

[0031] According to an important embodiment of the present invention, a nutritional composition comprising a nutrient selected from lutein, zeaxanthin, curcumin, and vitamin D3 together with one or more pharmaceutically and / or nutraceutically acceptable excipients is used for the prevention, improvement, and maintenance of dry eye syndrome.

[0032] According to another embodiment of the present invention, the nutritional composition of the present invention is composed of nutrients such as curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione], a polyphenol derivative derived from the spice turmeric. Curcumin is the main active ingredient of turmeric. Turmeric is a well-known indigenous herbal medicine. It should be understood that the term "curcumin" can be interpreted as being within the scope of the term curcuminoid, which generally may include constituents such as curcumin, methoxycurcumin, demethoxycurcumin, bisdemethoxycurcumin, and tetrahydrocurcumin.

[0033] According to a further embodiment, curcumin is present in the range of 20 to 50% by weight of the composition.

[0034] According to an embodiment of the present invention, the nutritional composition of the present invention is composed of nutrients such as lutein, which is an extract from plant materials selected for the preparation of the composition. The plant material is the flower of marigold (Tagetes eracta). Specifically, an extract rich in lutein esters is obtained from the flower of marigold. The marigold extract was self-prepared by saponification and thermal isomerization reactions of the oleoresin of the marigold flower. The final product contains at least 80% total carotenoids, including 60 to 85% lutein and 10 to 20% zeaxanthin isomers. In an embodiment, the extract of the marigold flower is composed of lutein and zeaxanthin isomers in a ratio of 4:1 to 6:1.

[0035] According to a further embodiment, lutein and zeaxanthin are present in the range of 2 to 10% by weight of the composition.

[0036] According to a further embodiment, vitamin D3 is present in the range of 0.01 to 2% by weight of the composition.

[0037] The nutritional composition described in this specification is composed of lutein, zeaxanthin, curcumin, and vitamin D3, either alone or in combination with at least one or more pharmaceutically and / or nutraceutically acceptable excipients that enhance solubility and thus absorption of the composition. More specifically, the nutritional composition is formulated using excipients selected from the group consisting of, but not limited to, antioxidants, stabilizers, carriers, fats, solubilizers, and bioavailability enhancers, or combinations thereof.

[0038] In another embodiment, the carrier used in the preparation of the nutritional composition is selected from the group consisting of, but not limited to, oils such as milk fat, medium-chain triglycerides (MCT), long-chain triglycerides, flaxseed oil, olive oil, thyme oil, fish oil, algal DHA oil, krill oil, safflower oil, sunflower oil, soybean oil, coconut oil, and other vegetable oils, fatty acid esters, hydrocarbons such as terpenes, monoglycerides, and derivatives thereof.

[0039] According to a preferred embodiment, the carrier used is medium-chain triglyceride (MCT) oil. According to a further embodiment, the MCT oil carrier is present in the range of 10 - 60% by weight of the composition. According to a further embodiment, the MCT oil carrier is present in the range of 20 - 50% by weight of the composition.

[0040] In another embodiment, the antioxidant used in the preparation of the nutritional composition is selected from the group consisting of, but not limited to, natural mixed tocopherols, ascorbyl palmitate, rosemary extract, epigallocatechin gallate, catechins, ascorbic acid, and derivatives thereof.

[0041] According to a preferred embodiment, the antioxidant is natural mixed tocopherols.

[0042] According to a further embodiment, the antioxidant is present in the range of 1 - 10% by weight of the composition. According to a further embodiment, the antioxidant is present in the range of 1 - 5% by weight of the composition. According to a further embodiment, the antioxidant is present in the range of 1 - 3% by weight of the composition.

[0043] In another embodiment, the bioavailability enhancer used in the preparation of the nutritional composition is selected from the group consisting of, but not limited to, olive oil, thyme oil, flaxseed oil, d-limonene, monoglycerides, phospholipids such as lecithin / phosphatidylcholine, plant extracts, and derivatives thereof. The bioavailability enhancer also acts as a solubilizing agent.

[0044] According to a further embodiment, the bioavailability enhancer is present in the range of 1 to 20% by weight of the composition. According to a further embodiment, the bioavailability enhancer is present in the range of 1 to 10% by weight of the composition. According to a further embodiment, the bioavailability enhancer is present in the range of 1 to 5% by weight of the composition.

[0045] In another embodiment, the stabilizer used in the preparation of the nutritional composition is selected from the group consisting of, but not limited to, sugar alcohols, triglycerides, antioxidants, and derivatives thereof.

[0046] In another embodiment, the fat used in the preparation of the nutritional composition is selected from the group consisting of, but not limited to, milk fat, fatty acid esters, hydrocarbons such as terpenes, monoglycerides, and derivatives thereof.

[0047] In another embodiment, the solubilizing agent in the preparation of the nutritional composition is selected from the group consisting of, but not limited to, propylene glycol alginate, sugar alcohols, sugar esters, phospholipids, vitamin E TPGS (d-α-tocopherol polyethylene glycol 1000 succinate), β-cyclodextrin, and / or combinations thereof.

[0048] In another embodiment, the pH adjuster in the preparation of the nutritional composition is selected from the group consisting of, but not limited to, citric acid, trisodium citrate, lactic acid, L-arginine, calcium carbonate, magnesium carbonate, and / or combinations thereof.

[0049] The nutritional compositions described herein exhibit improved bioavailability and can be made available in solid, semi-solid, or liquid forms that are orally administrable and are selected from, but not limited to, dosage forms such as oil suspensions, powders, granules, pellets, beadlets, caplets, tablets, capsules, soft gel capsules, solutions, emulsions, suspensions, dispersions, etc.

[0050] As in the preferred embodiment, the nutritional composition is in the form of an oil suspension containing nutrients and pharmaceutically and / or nutraceutically acceptable excipients.

[0051] According to a further embodiment of the present invention, the resulting oil suspension composition has a particle size in the range of 0.1 to 10 microns. The particle sizes of the active herb components before and after micronization were observed and are provided in Table 01. It was observed that micronization had a significant effect on the composition (oil suspension) of lutein, zeaxanthin, curcumin, and vitamin D3, and the composition particle size was reduced to about one-tenth of the particle size for all components measured in the inventors' experiments.

[0052] Particle Size Determination of Particles Particle size analysis is performed using Malvern equipment, details of which are provided below.

[0053] [Table 1]

[0054] [Table 2]

[0055] According to a further embodiment of the present invention, in a method for the prevention, improvement, and maintenance of dry eye syndrome comprising a nutritional composition, a subject is evaluated for oxidative stress markers and inflammatory markers associated with dry eye syndrome.

[0056] According to a further embodiment of the present invention, in a method for the prevention, improvement, and maintenance of dry eye syndrome, the nutritional composition reduces inflammatory cytokines such as nuclear factor kappa-light-chain enhancer of activated B cells (NFkB), matrix metallopeptidase 9 (MMP-9), tumor necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1b), interleukin 6 (IL-6), and interleukin 8 (IL-8).

[0057] According to a further embodiment of the present invention, in a method for the prevention, improvement, and maintenance of dry eye syndrome, the nutritional composition reduces oxidative stress markers such as malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx).

[0058] According to a further embodiment of the present invention, in a method for the prevention, improvement, and maintenance of dry eye syndrome, the nutritional composition increases tear volume, tear break-up time, tear stability, and mucin production.

[0059] According to a further embodiment of the present invention, a method for the prevention, improvement, and maintenance of dry eye syndrome - corneal surface damage, conjunctival surface damage, use and frequency of artificial tears.

[0060] In some embodiments, the process for preparing the nutritional composition includes the following steps: (i) Add one or more carriers while continuing stirring and heat the solution at 65 - 95 °C. (ii) Add an antioxidant to the solution obtained in step (i) and homogenize until completely dissolved. Maintain the temperature of 65 - 95 °C for 20 - 30 minutes, and then cool the suspension to below 30 - 40 °C for further processing. (iii) Add a curcumin extract to the solution obtained in step (ii) while stirring and maintain the temperature in the range of 65 - 95 °C. (iv) Add a bioavailability enhancer to the solution obtained in step (iii). (v) Add marigold extract and vitamin D3 oil to the dispersion in step (iv), and stir at 1200 - 2000 RPM for 20 - 30 minutes. (vi) Add one or more excipients while continuing to stir at 1200 - 2000 RPM. (vii) Filter the dispersion in step 4 through a mesh.

[0061] At the beginning of the following description, it should be understood that the subsequent descriptions are only for explaining specific forms of the present invention. However, such specific forms are merely exemplary embodiments and are not intended to be construed restrictively as implying limitations on the scope of the present invention.

[0062] Example 01: According to a preferred embodiment, the inventors executed batches of various sizes from small to large to support the industrial feasibility of the process, and one batch on the higher side is provided as an example below, but is not limited to this batch size. Since the ratio of excipients to the composition may vary depending on the batch size, it should not limit the scope of the invention. Preparation of an oil suspension composition; batch size 9000 g.

[0063] [Table 3]

[0064] Process details First stage i) Weigh all components, where lutein:zeaxanthin is present in a ratio of 4:1 - 6:1. ii) Set aside 10% of the batch amount of MCT oil for the second stage. iii) Add 90% of the batch amount of MCT oil to a container / reactor and heat to 65 - 95°C. (iv) Add the batch amount of lecithin to the solution obtained in step (ii) and homogenize at 1000 - 3000 RPM for 20 - 30 minutes or until the lecithin is completely dissolved. Maintain a temperature of 65 - 95 °C for 20 - 30 minutes, then cool the suspension to below 30 - 40 °C for further processing. (v) Add the batch amount of curcumin extract and homogenize at 1000 - 3000 RPM for 20 - 30 minutes. (vi) Add the batch amounts of d - limonene, mixed tocopherols 70% - SF, thyme oil, olive oil, and linseed oil to the container / reactor. (vii) Add the batch amount of marigold extract and homogenize at 1000 - 3000 RPM for 25 - 40 minutes or until a homogeneous suspension is obtained.

[0065] Second stage (i) Add the batch amount of vitamin D3 (oil) to the container. (ii) Add approximately 50% of the remaining 10% amount of MCT oil to the container / reactor for mixing vitamin D3 (oil) at 300 - 700 RPM for 2 - 5 minutes. Use the remaining amount of MCT oil for rinsing during the transfer to the suspension preparation container / reactor in the third stage.

[0066] Third stage (i) Add the suspension from the second stage to the container / reactor of the suspension from the first stage and homogenize at 1000 - 3000 RPM for 3 - 5 minutes. (ii) Sieve through a 20# ASTM mesh and then package.

[0067] Clinical study: Objective: To evaluate the effectiveness of a nutritional composition in subjects with dry eye syndrome (DES). Test product (active): Nutritional composition capsules (lutein / zeaxanthin - 20 / 4 mg + curcumin - 200 mg + vitamin D3 - 600 IU) manufactured by OmniActive Health Technologies Limited, India. Placebo: Soybean oil capsules manufactured by OmniActive Health Technologies Limited, India. Study Design: Prospective, randomized, double - blind, repeated - dose, parallel, placebo - controlled, clinical intervention study. Treatment Period: The total study period for the clinical part included a screening period of 8 days, followed by a treatment period of 56 days (8 weeks), and then a study visit at 56 ± 3 days, for a maximum of 67 days. Study Visits: Visit 1: Screening / Baseline visit (- 7 days to 0 days), Visit 2: Randomization visit (0 days), Visit 3: First follow - up visit (14 days ± 3 days), Visit 4: Second follow - up visit (28 days ± 3 days), Visit 5: End of treatment visit (56 days ± 3 days). Dosage and Administration: Subjects were instructed to consume one capsule every morning, after breakfast, at the same time each day, for 56 days (8 weeks). Number of Volunteers: Approximately 60 adult subjects aged 18 to 65 years clinically diagnosed with dry eye syndrome (DES) were planned for randomization. Approximately 30 subjects from the active group completed and were analyzed, and 29 subjects from the placebo group completed and were analyzed. Primary Endpoints: Tear volume, Ocular Surface Disease Index score (OSDI). Secondary Endpoints: Tear - breakup time (TBUT), Standard Patient Evaluation of Eye Dryness (SPEED) score, tear osmolarity, corneal and conjunctival staining, MMP - 9, use of artificial tears.

[0068] Schirmer test The Schirmer test was used to measure tear production in the eye. The test paper was placed at the lateral canthal margin of the lower eyelid for 5 minutes. The wetting length was measured in mm and interpreted for the severity of dry eye syndrome. In this study, the Schirmer test was evaluated at baseline, 14 days, 28 days, and 56 days during consumption of the investigational test product (active).

[0069]

Table 4

[0070] In the between-group analysis using ANOVA, the active group showed a statistically significant increase in the mean Schirmer test strip wetting length (tear volume) on days 28 and 56 compared to the placebo group.

[0071] Ocular Surface Disease Index (OSDI) score OSDI is a symptom-based questionnaire used for the diagnosis of dry eye syndrome. It is a 12-item scale for evaluating the ocular surface symptoms of dry eye, and the score ranges from 0 to 100.

[0072] [Table 5]

[0073] In the between-group analysis using ANOVA, the active group showed a statistically significant decrease in the mean OSDI score on days 14, 28, and 56 compared to the placebo group.

[0074] Tear Break-Up Time (TBUT) TBUT is defined as the time interval (seconds) between a complete blink and the first appearance of a dry spot in the tear film after fluorescein administration. TBUT is used to evaluate the stability of the tear film.

[0075] [Table 6]

[0076] In the between-group analysis using ANOVA, the active group showed a statistically significant increase in the mean TBUT score on days 14, 28, and 56 compared to the placebo group.

[0077] Tear Osmolarity (Tear Stability) Tear film stability is diagnosed in subjects with dry eye syndrome by measuring tear osmolarity. Tear osmolarity is measured in units of mOsms / L. A normal eye has less than 290 mOsms / L or is 290 - 310 mOsmol / L, and the difference between the left and right eyes is ≤ 7 mOsmol / L.

[0078]

Table 7

[0079] In the between-group analysis using ANOVA test, the active group showed a statistically significant increase in tear osmolarity (p < 0.05) on day 56 compared to the placebo group.

[0080] Standardized Patient Evaluation of Eye Dryness (SPEED) Questionnaire SPEED is an 8-item questionnaire that evaluates the frequency and severity of dry eye syndrome. The questionnaire includes scores from 0 - 28. In addition, the questionnaire further analyzes the symptoms according to their severity from no problem to intolerable.

[0081]

Table 8

[0082] In the between-group analysis using ANOVA test, the active group showed a statistically significant decrease in the mean SPEED score on days 14, 28, and 56 compared to the placebo group.

[0083] Matrix metalloproteinase 9 (MMP-9) biomarker for tear inflammation Matrix metalloproteinase 9 (MMP-9) is a biomarker released into the tears due to the inflammatory response in subjects with dry eye syndrome. The presence of MMP-9 was tested at baseline and at the end of the study (day 56). The presence of an MMP-9 level < 40 ng / ml is normal and considered negative.

[0084]

Table 9

[0085] In the between-group analysis using the Z-test, the test product (active) group showed a significant decrease in the number of subjects with positive tear inflammation measured by the MMP-9 marker on the 56th day compared to the placebo group.

[0086]

Table 10

[0087] In the between-group analysis using the Z-test, the test product (active) group showed a significant decrease in the number of subjects with positive tear inflammation measured by the MMP-9 marker on the 56th day compared to the placebo group.

[0088] Use and frequency of artificial tears Artificial tears are used to relieve the symptoms of dry eye by providing lubrication, reducing tear osmolality, and increasing viscosity. The use of artificial tears is symptomatic. During the study, the subjects were prescribed hydroxypropyl methylcellulose 0.7% w / v from randomization at the first visit, on the 14th day (visit 3) until the end of the study (56th day).

[0089]

Table 11

[0090] Table 9: Summary. From day 0 to day 14, 15 subjects (50%) in the active group and 20 subjects (68.97%) in the placebo group used artificial tears. After the 15th day and until the 56th day, 7 subjects (23.33%) in the active group and 20 subjects (68.97%) in the placebo group continued to use artificial tears. There was a -53.33% change in the proportion from baseline (day 0 - day 14) to day 28 and it remained the same until day 56.

[0091] Active group The active group showed a significant decrease in the number of subjects using artificial tears on days 15 - 28 and days 29 - 56 compared to the placebo group.

[0092]

Table 12

[0093] From day 0 to day 14, artificial tears were used 2.47 times / day (100%) in the active group and 2.65 times / day (100%) in the placebo group. A gradual decrease in the frequency of use was observed in the active group on days 15 - 28 and days 29 - 56, which were 1.07 times / day (43.24%), i.e., a 64.4 ± 40.76% decrease from the baseline, and 0.73 times / day (29.73%), i.e., a 75.6 ± 34.43% decrease from the baseline, respectively. In the placebo group, artificial tears were used 2.45 times / day (92.45%) and 2.60 times / day (98.11%) on days 15 - 28 and days 29 - 56, respectively (Table 30), with a 9.17 ± 19.10% and 0.83 ± 32.21% decrease from the baseline.

[0094] In the between - group analysis using the Kruskal - Wallis test, the active group showed a significant decrease in the frequency of use of artificial tears on days 15 - 28 and days 29 - 56 compared to the placebo group.

[0095] Corneal staining score Fluorescein staining is used to examine the state of ocular surface damage, i.e., the surface damage of the cornea and conjunctiva, in subjects with dry eye syndrome. Fluorescein is a synthetic organic compound, an orange - red, immediate - effect, temporary dye. Fluorescein stains damage on the ocular surface as bright green under blue light (slit lamp). Each eye is scored separately based on the damage observed by the researchers of the study.

[0096]

Table 13

[0097] Conjunctival staining score For the conjunctival staining score, the same method as for corneal staining is followed.

[0098]

Table 14

[0099] Results from preclinical studies using a dry eye animal model Seven female Wistar rats (age: 8 weeks, weight: 180 ± 20 g) per treatment group were placed in a controlled environment at 22°C with a 12:12 hour light-dark cycle, and were given solid rat feed and water as appropriate. Dry eye disease was established by topical administration of a benzalkonium chloride (BAC) solution (0.2%, Sigma-Aldrich) to the eyes of the rats twice a day for 14 days. The rats were randomly divided into four groups: I. Normal control group (n = 7), II. Solvent control group (n = 7), III. Blend formulation group 1 - LCD I (100 mg / kg body weight) treatment group (n = 7), and IV. Blend formulation group 2 - LCD II (200 mg / kg body weight) treatment group (n = 7). The blend formulation was administered by forced oral gavage for 4 weeks.

[0100]

Table 15

[0101] The data indicate that the LCD formulation significantly reduced the oxidative stress induced by BAC-induced DES by restoring the level of MDA and increasing the levels of antioxidant SOD and GSH-Px at both doses. Effects of BAC and LCD on the enzyme activities of SOD and GSH-Px in the cornea, and the levels of MDA in serum and cornea. BAC induced oxidative stress on the ocular surface, increased the levels of MDA in serum and cornea, and decreased the levels of SOD and GSH-Px in the cornea, while LCD partially alleviated the effect of oxidative stress induced by BAC (p<0.05). The improvement was more evident in the LCD 2 group compared to the LCD 1 group (p<0.05).

[0102] Histological analysis The eyes were fixed (4% paraformaldehyde, then paraffin) and sliced into 5-μm sections using a microtome. Corneal and conjunctival tissues were stained with hematoxylin and eosin (H&E) and examined using an optical microscope.

[0103]

Table 16

[0104] The inventors observed that the BAC-induced dry eye condition led to an increase in the thickness of the corneal epithelium, and inflammatory cell infiltration and edema were observed under the epithelium. LCD dose-dependently brought about an improvement in the histopathological findings.

[0105] Western blot analysis Muc1, Muc4, Muc5, GAP43, GFAP, NF-κB, TNF-α, IL-1β, IL-6, and IL-8 in corneal tissue were measured using Western blot analysis. A monoclonal mouse antibody against β-actin (A5316; Sigma) was used as a loading control. The blots were performed at least three times to confirm the reproducibility of the results. Densitometric analysis of the bands was detected using the image analysis system Image J (National Institutes of Health, Bethesda, MD, USA).

[0106]

Table 17

[0107] BAC-induced eye symptoms were accompanied by significant increases in the levels of NF-κB, TNF-α, IL-1β, IL-6, and IL-8, and decreases in Muc1, Muc4, Muc5, and GAP43 proteins (p < 0.001). However, LCD restored the expression of the above proteins, and LCD2 was more effective than LCD1 (p < 0.05).

Claims

A composition for use in a method for treating, preventing, and / or ameliorating dry eye syndrome in a subject, said composition comprising: a) a marigold extract containing lutein and zeaxanthin; b) curcumin; c) vitamin D3; and d) at least one excipient wherein the subject is a human and the composition is administered once a day. **Claim 2** The composition according to claim 1, wherein the at least one excipient is selected from the group consisting of antioxidants, bioavailability enhancers, carriers, fats, solubilizers, stabilizers, and combinations thereof. **Claim 3** The composition according to claim 2, wherein the antioxidant is selected from the group consisting of tocopherol, ascorbyl palmitate, rosemary extract, epigallocatechin gallate, catechin, ascorbic acid, and combinations thereof. **Claim 4** The composition according to claim 2, wherein the bioavailability enhancer is selected from the group consisting of olive oil, thyme oil, linseed oil, d-limonene, lecithin, monoglyceride, phosphatidylcholine, plant extracts, and combinations thereof. **Claim 5** The composition according to claim 2, wherein the carrier is selected from the group consisting of milk fat, medium-chain triglycerides (MCT), long-chain triglycerides, linseed oil, olive oil, thyme oil, fish oil, algal DHA oil, oyster oil, safflower oil, sunflower oil, soybean oil, coconut oil, vegetable oil, fatty acid esters, hydrocarbons, terpenes, monoglycerides, oils, and combinations thereof. **Claim 6** The composition according to claim 1, wherein the extract is present in a particle size range of 0.1 micron to 10 microns. **Claim 7** The composition according to claim 1, wherein curcumin is present in a particle size range of 0.1 micron to 10 microns. **Claim 8** The composition according to claim 1, wherein the composition is in a form selected from the group consisting of beadlets, powders, oil suspension forms, granules, capsules, tablets, and films. **Claim 9** Administering the composition reduces the concentration of one or more inflammatory cytokines selected from the group consisting of nuclear factor kappa-light-chain enhancer of activated B cells (NFkB), matrix metallopeptidase 9 (MMP-9), tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8), and combinations thereof in the subject, the composition according to claim 1.

10. Administering the composition reduces the concentration of at least one oxidative stress marker in the subject, the composition according to claim 1.

11. Administering the composition provides to the subject one or more beneficial effects selected from the group consisting of an increase in tear volume, an increase in tear break-up time, an increase in tear stability, an increase in mucin production, a reduction in corneal surface damage, a reduction in conjunctival surface damage, a reduction in the use and frequency of artificial tears, an improvement in tear production, a relief of dry eye, a reduction in eye discomfort, an improvement in tear stability, a reduction in irritation, burning, and grittiness associated with dry eye, a reduction in tear reduction, a reduction in ocular surface damage, a reduction in inflammation in tears, a reduction in dependence on artificial tears, and combinations thereof, the composition according to claim 1.

12. The composition is administered in an amount in the range of 10 mg / kg to 500 mg / kg, the composition according to claim 1.

13. The composition is administered orally, the composition according to claim 12.

14. Administering the composition increases the concentration of one or more antioxidant enzymes in the subject, the composition according to claim 1.

15. The one or more antioxidant enzymes are selected from the group consisting of superoxide dismutase (SOD), glutathione peroxidase (GPx), and combinations thereof, the composition according to claim 14.

16. Administering the composition reduces the concentration of at least one oxidative stress marker in the subject, and the at least one oxidative stress marker is malondialdehyde (MDA), the composition according to claim 1.

17. A composition for use in a method for treating, preventing, and / or ameliorating dry eye syndrome in a subject, the composition comprising a) a marigold extract containing lutein and zeaxanthin, b) curcumin, and c) vitamin D3 A composition, comprising the same, wherein the subject is a human and the composition is administered once a day.

18. A process for preparing the composition according to claim 1, wherein the composition is in the form of an oil suspension, and the process comprises: a) preparing a solution of one or more carriers and a bioavailability enhancer; b) adding a curcumin extract to the solution, followed by adding an antioxidant and the extract containing lutein and zeaxanthin; c) preparing a separate solution of vitamin D3 and a carrier; d) homogenizing the mixture obtained by adding steps (b) and (c); e) filtering the dispersion of step (d) through a mesh A process comprising the same.

19. A composition for use in a method for treating, preventing, and / or ameliorating dry eye syndrome in a subject, the composition comprising: a) an extract of lutein and zeaxanthin consisting of lutein and zeaxanthin in a ratio of 4:1 to 6:1; b) curcumin, and c) vitamin D3 wherein the subject is a human and the composition is administered once a day.

20. A composition for use in a method for treating, preventing, and / or ameliorating dry eye syndrome in a subject, the composition comprising: a) an extract of lutein and zeaxanthin consisting of lutein and zeaxanthin in a ratio of 4:1 to 6:1; b) 200 mg of curcumin, and c) 600 IU of vitamin D3 wherein the subject is a human and the composition is administered once a day.

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