Synergistic herbal compositions for eye health

Abstract

The present invention discloses a synergistic herbal compositions comprising: (i) a zinc complex or chelate of enriched punicalagins, wherein enriched punicalagins are derived from Punica granatum fruit or fruit peel and (ii) a second ingredient selected from Tagetes erecta flower extract; process for their preparation, methods of treatment, and use of the compositions for eye health benefits.

Description

[0001] SYNERGISTIC HERBAL COMPOSITIONS FOR EYE HEALTH

[0002] TECHNICAL FIELD OF INVENTION:

[0003] The present invention relates to a synergistic herbal composition for promoting eye health. Specifically, the compositions comprise: (i) a zinc complex or chelated form of enriched punicalagins derived from Punica granatum (pomegranate) fruit or fruit peel as a first component; and (ii) a second component selected from one or more phytochemicals derived from an extract of Tagetes erecta (marigold) flowers. The disclosure also provides methods for preparing the compositions, and methods of use, and applications thereof in the prevention and management of ocular conditions.

[0004] BACKGROUND OF THE INVENTION:

[0005] The human eye is a critical sensory organ responsible for vision. It functions by capturing light from the external environment and transmitting visual information to the brain for interpretation. Multiple anatomical components, including the cornea, iris, pupil, lens, retina, and optic nerve, operate in a highly coordinated manner to enable this process. Proper functioning of these structures is essential for maintaining visual acuity and overall ocular health.

[0006] However, the eyes are susceptible to a variety of internal and external stressors that can compromise their function. Oxidative stress, exposure to blue light, aging, and lifestyle-associated conditions such as obesity and diabetes, as well as environmental aggressors like cigarette smoke and ultraviolet (UV) radiation, are known to have a detrimental effect on ocular tissues. These risk factors contribute to the development of various ocular pathologies, including dry eye disease (DED), age-related macular degeneration (AMD), myopia, and diabetic retinopathy, among others.

[0007] Of particular concern is the retina, which is one of the most metabolically active tissues in the body and a major consumer of oxygen. This high metabolic demand, coupled with constant exposure to light, predisposes retinal cells to elevated levels of reactive oxygen species (ROS). The accumulation of ROS leads to oxidative damage, resulting in photoreceptor degeneration, dysfunction of retinal pigment epithelial (RPE) cells, and eventual cellular apoptosis. These processes are now recognized as key contributors to the pathogenesis of DED, AMD, and related degenerative ocular conditions.

[0008] Current strategies for managing eye health include both pharmacological and non- pharmacological approaches. These range from dietary and lifestyle interventions to artificial tear substitutes, punctal plugs, antioxidant or anti-inflammatory medications (administered topically or systemically), and surgical procedures. Among these, nutritional supplementation has garnered significant interest due to its non-invasive nature and growing body of clinical evidence supporting its efficacy and safety in preserving ocular health and mitigating disease progression. Despite the availability of various treatment options, there remains a need for compositions that offer synergistic, targeted, and well-tolerated support for eye health, particularly those that address oxidative stress at the retinal level. The present invention addresses this need by developing novel synergistic herbal compositions with enhanced antioxidative and protective properties.

[0009] OBJECTIVE OF THE INVENTION:

[0010] The primary objective of the present invention is to provide a synergistic and safe herbal composition that promotes and supports eye health.

[0011] The other objective is to provide a composition comprising

[0012] (i) a zinc complex or chelated form of enriched punicalagins derived from the fruit or fruit peel of Punica granatum (pomegranate); and (ii) a second ingredient selected from one or more phytochemicals derived from an extract of Tagetes erecta (marigold) flowers; to support the prevention and management of various eye conditions. SUMMARY OF THE INVENTION:

[0013] In an aspect, the present invention provides a synergistic herbal compositions comprising (i) an zinc complex or chelate of punicalagins enriched in the range of 20-70% from Punica granatum fruit or fruit peel, wherein zinc is present in a concentration of 0.05-2.0%; and (ii) a second ingredient selected from one or more phytochemicals derived from Tagetes erecta flower extract standardized to lutein or zeaxanthin or its esters in the range of 1-40%; or optionally at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents; for eye health benefits.

[0014] In an aspect, the present invention provides a process for the preparation of synergistic herbal compositions comprising (i) a zinc complex or chelate of enriched punicalagins derived from Punica granatum fruit or fruit peel as first ingredient and (ii) a second ingredient selected from Tagetes erecta flower extract standardized to contain lutein or zeaxanthin or its esters or mixtures thereof; or optionally at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents.

[0015] In another aspect, the present invention provides methods of obtaining at least one eye health benefit selected from but not limited to preventing or alleviating symptoms of dry eye disease (DED), age-related macular degeneration (AMD), cataract, vision loss, glaucoma, and diabetic retinopathy; reducing the harmful effects of blue light exposure, including digital eye strain, visual fatigue, burning eyes, blurred vision, and headaches; and enhancing visual performance, including improvements in visual acuity, light adaptation, and dark adaptation in a human; wherein the method comprises supplementing the human in a need thereof with an effective dose of a composition comprising; (i) a zinc complex or chelate of enriched punicalagins in the range of 20-70% derived from Punica granatum fruit or fruit peel, wherein, zinc is present in a concentration of 0.05-2.0%; as first ingredient and (ii) a second ingredient selected from Tagetes erecta flower extract standardized to contain one or more phytochemicals selected from lutein or zeaxanthin or its esters in the range of 1-40%; (iii) optionally further containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers, and diluents.

[0016] In yet another aspect, the present invention provides the use of the synergistic herbal compositions comprising; (i) a zinc complex or chelate of enriched punicalagins in the range of 20-70% derived from Punica granatum fruit or fruit peel, wherein, zinc is present in a concentration of 0.05-2.0%; and (ii) a second ingredient selected from Tagetes erecta flower extract standardized to contain one or more phytochemicals selected from lutein or zeaxanthin or its esters in the range of 1-40%; (iii) optionally further containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers, and diluents; for improving at least one eye health benefit in a human in a need thereof, selected from but not limited to preventing or alleviating symptoms of dry eye disease (DED), age-related macular degeneration (AMD), cataract, vision loss, glaucoma, and diabetic retinopathy; reducing the harmful effects of blue light exposure, including digital eye strain, visual fatigue, burning eyes, blurred vision, and headaches; and enhancing visual performance, including improvements in visual acuity, light adaptation, and dark adaptation.

[0017] DETAILED DESCRIPTION OF THE INVENTION:

[0018] The invention will now be described in detail in connection with certain preferred and optional embodiments so that various aspects thereof may be more fully understood and appreciated.

[0019] The terms “metal complex,” “metal chelate” and “metal salt” convey the same meaning and are used interchangeably in the specification. Thus, Punica granatum fruit rind extract containing a metal complex of punicalagins; Punica granatum fruit rind extract containing a metal chelate of punicalagins; Punica granatum fruit rind extract containing metal salt of punicalagins; convey the same meaning and are interchangeable. The terms “fruit peel,” “fruit rind” as used herein, conveys the same meaning and is interchangeable.

[0020] The term “zinc complex or chelate of enriched punicalagins” means that the zinc complex contains punicalagins in the range of 20-70% derived from Punica granatum fruit or fruit peel, along with zinc which is present in a concentration of 0.05-2.0%.

[0021] The term “enrichment process of punicalagins zinc complex” according to the invention includes the process steps, viz., extraction of Punica granatum fruit or fruit peel, complexation with zinc ion and enrichment of the complex over an adsorbent resin column.

[0022] Geographical Origin

[0023] The source of the herb used in the invention is as follows:

[0024] 1. Punica granatum was collected from Potulanage Palli village, Dharmavaram Mandal, Ananthapur district, Andhra Pradesh.

[0025] 2. Tagetes erecta was collected from Rayapadu village, Tallur Mandal, Guntur district, Andhra Pradesh.

[0026] The inventors of the present invention randomly screened numerous plant extracts and their phytochemicals in in vitro assays related to eye health. Punicalagins and marigold extracts containing carotenoids exhibited good in vitro efficacies. Surprisingly, the zinc complex of punicalagins showed higher efficacy than punicalagins.

[0027] A summary of each plant material used in the preferred embodiment of the present invention is provided below.

[0028] Punica granatum fruit: Punica granatum, commonly known as pomegranate, is native to the Mediterranean region and has a long history in folk and ayurvedic medicine. Punica granatum is of great significance with rich phenolic compounds. Punicalagin (2,3-hexahydroxydiphenoyl-4,6-gallagylglucose) is the major phytochemical in Punica granatum fruit peel and is a mixture of punicalagin A (punicalagin a) and punicalagin B (punicalagin P), as shown in the figure- 1 below. Thus, the term “Punicalagins” in the entire specification, has the same meaning.

[0029] Figure-1 : Chemical structures of punicalagins. Tagetes erecta L: Tagetes erecta L (Marigold) is a large flowered annual herbaceous plant and is used in folk medicine to cure various diseases. Marigold flowers are rich in carotenoids such as lutein, zeaxanthin or its esters. The chemical structures of carotenoid pigments are shown in Figure-2.

[0030] (a) R1=R2= H(Lutein); Rl= Palmityl or myrsityl or stearyl, R2= H; R1=H, R2= palmityl or myrsityl or stearyl; Rl= R2= palmityl or myrsityl or stearyl

[0031] (b) R1=R2= H (Zeaxanthin); Rl= Palmityl or myrsityl or stearyl, R2= H; R1=H, R2= palmityl or myrsityl or stearyl; Rl= R2= palmityl or myrsityl or stearyl Figure-2: Chemical structure of (a) Lutein and its esters (b) Zeaxanthin and its esters

[0032] Zinc: Zinc is an important element in the body and is found in modest amounts in a variety of foods. Since the human body does not store excess zinc, it must be consumed regularly as part of the diet. Zinc helps maintain the physiological functions of the retina, cell membranes, and protein structure of the eye.

[0033] The inventor imagined that an enriched punicalagin-zinc complex / chelate would markedly enhance the eye health benefits of Punica granatum extract.

[0034] Punicalagin, being hydrolysable tannin, is susceptible to degradation during the process, and needs special measures to keep it intact. The stability and bioavailability of polyphenols can be enhanced by complex / chelate formation with metals, including zinc. The inventors recognised that enrichment of punicalagin-zinc salt is advantageous over enrichment of punicalagin followed by punicalagin-zinc salt formation.

[0035] Hence, the main objective of the present invention is to prepare for the first time zinc complex or chelate of enriched punicalagins in the range of 20-70% from Punica granatum fruit or fruit peel.

[0036] The inventors surprisingly found that the Punica granatum peel extract containing zinc complex of enriched punicalagins exhibited higher efficacy in reactive oxygen species (ROS) and superoxide scavenging, and more protection from superoxide-induced retinal epithelial cell apoptosis, thus suggesting a greater potential of eye health benefits.

[0037] Enrichment through macroporous resins: Macroporous resin is a type of polymer material that can adsorb or capture selected substances onto its surface. It is commonly used in various industries for purification, separation, and removal of specific molecules or ions from a mixture. Adsorption of target compounds onto macroporous (adsorbent) resins from the crude extracts, followed by their elution using organic solvent, has proved to be an effective and efficient strategy for the purification of natural products. Microporous resins are selected from but not limited to AB, ADS, SDA, XAD, LSA, HPD, PAD, DS, PA, and HP.

[0038] The inventors chose to perform enrichment of punicalagins after complexation with zinc, owing to the degradative nature of free punicalagins in the presence of water and heat. As per inventor’s knowledge, enrichment of Punica granatum fruit rind extract containing zinc complex of punicalagins was not reported in the prior art.

[0039] Punica sranatum fruit or fruit peel extract containing zinc complex of enriched punicalagins: The pomegranate fruit or fruit peel 70% aqueous ethanol extract was prepared and treated with zinc oxide to form a zinc complex of punicalagins. The extract is then enriched using macroporous resin such as PA-800 to obtain a zinc complex of enriched punicalagins. The enriched extract was analysed by HPLC and it was found to contain 49% of punicalagins and 0.69% zinc by ICP- MS (Example 1). Similarly, other solvent extracts of pomegranate fruit or fruit peel were also converted into zinc complex and enriched through PA-800 resin (examples 2-4). For comparison, pomegranate fruit or fruit peel extract containing enriched punicalagins without zinc and pomegranate fruit or fruit peel extract without enrichment were also prepared. (Comparative examples 1 A & IB). Complex formation: The aqueous alcoholic extract of P. granatum fruit or fruit peel contains punicalagins as the major phytochemical, which contain several hydroxyl groups (Figure-1) capable of forming complexes with metals. The zinc complex of P. granatum fruit or fruit peel aqueous alcohol extract containing punicalagins was prepared using zinc oxide. The process was typically performed in water, followed by fine filtration to remove any unreacted zinc oxide. ICP-MS analysis of the resulting product showed the presence of zinc (0.69%). As zinc oxide is practically insoluble in water, the existence of zinc in the product confirms the formation of zinc complex with punicalagins. Furthermore, purification on adsorbent resin column separates inorganic metal salts present in the extracts from the organic compounds; however, the enriched punicalagin extract of the current invention still showed 0.69% zinc. If zinc did not exist as a complex with punicalagins, the enriched product obtained from the resin column should not contain any zinc. This confirms that the zinc present in the product is indeed in complex formation with punicalagins.

[0040] Reactive Oxygen Species (ROS) in ocular health and vision protection:

[0041] Reactive oxygen species (ROS) are highly active molecules, which include free radicals like superoxide (O2 •), hydroxyl (*OH), and peroxyl, as well as nonradical oxidants such as hydrogen peroxide (H2O2), hypochlorous acid, and ozone (O3). Their origin can be from endogenous source (e.g., mitochondrial respiration, NADPH oxidases) or from the exogenous factors (e.g., UV light, pollutants, high- fat diets, and radiation). While ROS serves important physiological roles, excessive levels lead to oxidative stress, and damaging to lipids, proteins, and DNA.

[0042] The eye, particularly the retina, is highly susceptible to oxidative damage due to its high metabolic activity, significant oxygen demand, and continuous exposure to light. This makes ROS a major contributor to the development and progression of dry eye disease (DED), age-related macular degeneration (AMD), cataracts, glaucoma, vision loss, and diabetic retinopathy. Retinal photoreceptors are rich in polyunsaturated fatty acids such as DHA, which are highly susceptible to lipid peroxidation. Although the retina is equipped with ROS-scavenging systems and continuously renews damaged photoreceptor segments, age-related decline and persistent oxidative stress can overwhelm these defense mechanisms.

[0043] Increased screen time and exposure to blue light from devices such as phones, monitors, and TVs further elevate ROS levels, contributing to digital eye strain, visual fatigue, blurred vision, burning eyes, headaches, and disrupted circadian rhythms. Blue light penetrates deeply into ocular tissues and accelerates oxidative damage to the eyes.

[0044] Hyperglycemia, a hallmark of diabetes, plays a central role in the development of several ocular pathologies by inducing oxidative stress. Chronic high blood glucose levels lead to the excessive production of reactive oxygen species (ROS) in retinal and lens cells. This oxidative burden leads to: (i) Cellular apoptosis and inflammation in retinal tissues, contributing to diabetic retinopathy and finally loss of vision; (ii) Oxidative modification of lens proteins and lipids, accelerating cataract formation, (iii) Damage to the trabecular meshwork and optic nerve, increasing intraocular pressure and the risk of glaucoma. Furthermore, oxidative stress disrupts mitochondrial function and impairs vascular integrity, resulting in retinal hypoxia and neurodegeneration. Thus, mitigating oxidative stress in hyperglycemia is critical for preserving visual function and preventing diabetes- related ocular complications.

[0045] The Punica granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins was evaluated in comparison with punicalagins enriched extract without zinc and regular extract with without zinc for ROS inhibition in the presence of hydrogen peroxide (H2O2). The results are summarized in Table 1. Notably, the zinc-complexed extract (Example 1) demonstrated superior antioxidant efficacy compared to the corresponding extracts without zinc (Comparative Examples 1 A and IB), highlighting the enhanced activity conferred by the metal complexation. Table 1 : Inhibition or scavenging of ROS in H2O2-induced ARPE-19 human retinal pigment epithelial cells by the P. granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins and, and regular extracts without zinc.

[0046] For example, in the H2O2-induced ARPE-19 cells, the Punica granatum fruit or fruit peel 70% aqueous ethanol extract containing zinc complex of enriched punicalagins (PG-1) exhibited 50.60% ROS inhibition at 10 pg / mL. In contrast, the extract containing enriched punicalagins without zinc (PGC-1) showed only 36.54% inhibition, and the regular fruit or fruit peel extract (PGC-2) demonstrated just 29.12% inhibition under the similar experimental conditions. This marked difference highlights a surprising and unexpected enhancement in H2O2-induced ROS inhibition, which can be attributed to the presence of the zinc-punicalagin complex (Table 1).

[0047] Compositions

[0048] Encouraged by the observed eye health benefits of the Punica granatum fruit or fruit peel 70% aqueous ethanol extract containing a zinc complex of enriched punicalagins, the inventors planned to develop a series of compositions combining this extract with marigold (Tagetes erecta) flower extracts. Accordingly, various Punica granatum fruit or fruit peel extracts complexed with zinc and different standardized marigold flower extracts were prepared, as outlined in Examples 1-5 and Examples 6-18 respectively.

[0049] The Punica granatum extracts were standardized to punicalagins using HPLC and to zinc content using ICP-MS. The marigold flower extracts were standardized to lutein esters or lutein using validated HPLC methods.

[0050] Subsequently, multiple compositions (C-l-C-28) incorporating Punica granatum zinc complexes and marigold flower extracts were prepared, as described in Examples 19-26.

[0051] Efficacy screening of extracts and their compositions: The individual extracts and compositions (C-l to C-28) were evaluated for their biological activity using well-established cell-based assay models. These assays were designed to assess the potential of each test sample for ocular health and vision protection. Specifically, the following parameters were measured in the ARPE-19 human retinal epithelial cells:

[0052] (i) reactive oxygen species (ROS) inhibition or scavenging in hydrogen peroxide (H2O2)-induced cells

[0053] (ii) protection against mitochondrial superoxide-induced stress in blue light-exposed cells

[0054] (iii) ROS scavenging in high-glucose induced cells

[0055] (iv) protection from blue light-induced apoptosis

[0056] This in vitro screening strategy constituted a key component of the overall evaluation to identify effective compositions with potential application in nutraceutical and functional food formulations aimed at promoting and preserving eye health. Inhibition of ROS by Punica sranatum-Zinc Complex and Marigold Extracts in the presence of H2O2: The potential of Punica granatum fruit or fruit peel extract containing Zinc complex of enriched with punicalagins, in combination with Tagetes erecta (marigold) flower extracts, was evaluated for ROS scavenging in the presence of hydrogen peroxide (H2O2) using a cellular assay model. Compositions C-l through C-28 were tested and compared their ROS inhibitions against those of their respective individual components to assess antioxidant efficacy. Remarkably, the results revealed that the compositions produced significantly greater ROS scavenging than the expected additive effects of their individual ingredients, indicating a synergistic interaction between the two botanical sources.

[0057] For example, the P. granatum extract containing zinc complex of enriched punicalagins (PG-1) at 7.5 pg / mL achieved 37.95% inhibition of ROS production, while the T. erecta flower extract containing lutein esters (TE-1) at 2.5 pg / mL showed only 7.20% inhibition. However, when combined as in Composition C-l (PG-ETE-l in a 3: 1 ratio at a total concentration of 10 pg / mL), the inhibition rose dramatically to 71.70%. This result far exceeded the expected additive effect of 45.15% (i.e., 37.95% + 7.20%), clearly demonstrating synergism. Similar synergistic effects were observed with compositions C-2 through C-5, all of which featured PG-1 and TE-1 in varying ratios and concentrations, as outlined in Table 5.

[0058] Furthermore, compositions C-6 through C-l 6 comprising PG-1 in combination with T. erecta flower extracts containing either lutein or lutein esters consistently showed higher-than-expected ROS inhibition compared to their individual components. The enhanced efficacy of these combinations is detailed in Tables 6 and -7, which underscore the synergistic antioxidant potential of these dual-extract compositions. Protection against mitochondrial superoxide-induced damage from blue light exposure: With increasing reliance on digital devices and prolonged exposure to artificial light sources, the human eye is subjected to elevated levels of blue light, which can trigger excessive production of reactive oxygen species (ROS), particularly superoxide radicals. These oxidative stressors play a pivotal role in the pathogenesis of several vision-related disorders. Chronic blue light exposure is strongly linked with the enhanced progression of dry eye disease (DED), age- related macular degeneration (AMD), cataracts, glaucoma, diabetic retinopathy, and overall vision loss.

[0059] Moreover, enhanced mitochondrial superoxide generation produces stress in ocular tissues that contribute to the symptoms of digital eye strain, including visual fatigue, burning sensations, blurred vision, and headaches, which are increasingly prevalent across populations of all ages. The retina, rich in metabolically active photoreceptors and highly susceptible to oxidative insult, is especially vulnerable to such damage. Beyond disease prevention, mitigating superoxide-induced damage also supports enhanced visual performance including improved visual acuity, light adaptation, and dark adaptation.

[0060] Protective strategies that reduce mitochondrial superoxide accumulation and overall cellular oxidative burden offer promising avenues for preserving longterm eye health and function. Nutritional interventions, particularly those involving targeted antioxidants and synergistic phytonutrient formulations, are emerging as effective, population-wide solutions to combat blue light-induced oxidative stress and its visual consequences.

[0061] Protection against mitochondrial superoxide stress by Punica ^ranalum-zmc complex and Marigold extracts under blue light exposure: The antioxidant potential of Punica granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins, in combination with Tagetes erecta (marigold) flower extracts, was evaluated for its ability to protect against blue light-induced superoxide generation in mitochondria using a cellular assay model. Compositions C-l through C-10 were assessed and compared to their efficacy with those of respective individual components to determine their potential in mitigating superoxide-related oxidative stress in ARPE-19 cells. Surprisingly, the compositions demonstrated significantly higher protective effects than the corresponding additive effect calculated based on values of their individual constituents, indicating a synergistic interaction between the two botanical extracts.

[0062] For instance, P. granatum extract (PG-1) at 2.48 pg / mL conferred 36.11% protection against superoxide formation, while T. erecta flower hexane extract containing lutein esters (TE-1) at 0.83 pg / mL achieved 14.37% protection. In contrast, their combination as in Composition C-l (PG-1 :TE-1 in a 3: 1 ratio; at 3.3 pg / mL) resulted in 81.71% inhibition of superoxide, far surpassing the expected additive effect of 50.48%. This substantial enhancement clearly supports the existence of a synergistic effect.

[0063] Similarly, compositions C-2 through C-5 containing varying ratios of PG-1 and TE-1 also demonstrated greater-than-expected efficacy, as summarized in Table 9. Additional compositions (C-6 through C-10), which combined PG-1 with TE-4, also showed superior superoxide protection at the mitochondrial level compared to their individual components, confirming consistent synergistic behaviour across multiple extract pairings (Table 10).

[0064] Protection against high glucose-induced ROS production- implications for eye health and visual performance: Elevated glucose levels, as seen in diabetic and prediabetic states, significantly contribute to the overproduction of overall cellular reactive oxygen species (ROS), particularly superoxide radicals. Increased oxidative stress disrupts cellular homeostasis and damages ocular tissues, playing a central role in the development and progression of multiple vision-related conditions. High glucose-induced ROS has been strongly implicated in dry eye disease (DED), age-related macular degeneration (AMD), cataract formation, glaucoma, diabetic retinopathy, and eventual vision loss. Ocular tissues, especially the retina, are highly metabolically active and vulnerable to oxidative damage under hyperglycaemic stress. Excess ROS impairs photoreceptor function, disrupts the blood-retinal barrier, and accelerates retinal cell apoptosis. These effects not only contribute to chronic eye diseases but also exacerbate visual fatigue, blurred vision, and other symptoms associated with poor glycaemic control.

[0065] Protective interventions that reduce or inhibit high glucose-induced ROS offer promising avenues for preserving visual health, particularly in populations at risk of or living with diabetes. Such strategies can help alleviate symptoms, prevent disease progression, and support enhanced visual performance, including improved visual acuity, light sensitivity, and dark adaptation.

[0066] Protection against high glucose-induced ROS production by Punica granatum- zinc complex and Marigold extracts: The antioxidant potential of Punica granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins, in combination with Tagetes erecta (marigold) flower extracts, was evaluated for its protective efficacy against high glucose-induced ROS generation using a cellular assay model. Compositions C-l through C-10 were assessed and compared with their individual components to determine their ability to the scavenging of ROS in the ARPE-19 cells. Notably, the compositions demonstrated significantly higher efficacy than the expected additive effects of the individual extracts, indicating a synergistic interaction.

[0067] For example, P. granatum extract (PG-1) at 7.5 pg / mL exhibited 15.38% inhibition of ROS, while T. erecta flower hexane extract containing lutein esters (TE-1) at 2.5 pg / mL showed 6.94% inhibition. However, when combined as in Composition C-l (PG-ETE-l at a 3: 1 ratio, totaling 10 pg / mL), the ROS inhibition increased markedly to 55.67%, significantly exceeding the expected additive effect of 22.32%. This substantial increase reflects synergistic efficacy between the two extracts. Similarly, compositions C-2 through C-5, formulated with different ratios of PG-1 and TE-1, consistently demonstrated higher-than-expected ROS inhibition (scavenging), as presented in Table 11. In addition, compositions C-6 through C- 10, which are a combination of PG-1 with a different marigold extract (TE-4), also showed superior protective effects compared to the corresponding standalone constituents, confirming the robust and reproducible synergistic behaviour across various extract combinations (Table 12).

[0068] Protection from blue light-induced apoptosis-impact on eye health and vision: Blue light from digital screens and artificial lighting penetrates deep into ocular tissues, generating ROS and triggering mitochondrial dysfunction, which leads to apoptosis, especially in photoreceptors and retinal ganglion cells. This process contributes to the development of DED, AMD, cataracts, glaucoma, diabetic retinopathy, vision loss, and symptoms of digital eye strain such as visual fatigue, burning eyes, and headaches.

[0069] As screen exposure continues to rise across all age groups, it is crucial to implement interventions that mitigate blue light-induced retinal cell damage or cell death in the eyes to protect public eye health. These strategies not only help prevent or manage ocular diseases but also enhance visual performance, including visual acuity, light adaptation, and dark adaptation.

[0070] Protection from blue light-induced apoptosis by Punica granatum-zinc complex and Marigold extracts: The combination of Punica granatum fruit or fruit peel extract (zinc complex of enriched punicalagins) and Tagetes erecta (marigold) flower extracts was evaluated for their ability to prevent apoptosis in blue light- exposed human retinal epithelial cells (ARPE-19). The efficacies of the Compositions C-l to C-10 were compared to those of their corresponding individual components, revealing significantly greater protection in the combined formulations, indicating synergistic efficacy. For instance, PG-1 (7.5 pg / mL) and TE-1 (2.5 pg / mL) individually provided 30.86% and 15.82% protection, respectively. In contrast, their combination in Composition C-l (10 pg / mL, 3: 1 ratio) achieved 82.68% protection, well above the expected additive value of 46.67%. Similar synergistic effects were observed in compositions C-2 to C-5 (PG-1 + TE-1) and C-6 to C-10 (PG-1 + TE-4), as detailed in Tables 13 and 14.

[0071] Formulations: The synergistic herbal compositions are formulated with at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents; wherein the excipients, carriers and diluents are selected from monosaccharide’s such as glucose, dextrose, fructose, galactose etc.; disaccharides such as but not limited to sucrose, maltose, lactose, lactulose, trehalose cellobiose, chitobiose etc.; polycarbohydrates such as starch and modified starch such as sodium starch glycolate, pre-gelatinized starch, soluble starch, and other modified starches; dextrins that are produced by hydrolysis of starch or glycogen such as yellow dextrin, white dextrin, maltodextrin etc.; polyhydric alcohols or sugar alcohols such as but not limited to sorbitol, mannitol, inositol, xylitol, isomalt etc.; cellulose based derivatives such as but not limited to microcrystalline cellulose, hydroxy propyl methyl cellulose, hydroxy ethyl cellulose etc.; silicates such as but not limited to neusilin, veegum, talc, colloidal silicon dioxide etc.; metallic stearates such as but not limited to calcium stearate, magnesium stearate, zinc stearate etc.; organic acids such as citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid etc.; fatty acid esters and esters of poly sorbate, natural gums such as but not limited to acacia, carrageenan, guar gum, xanthan gum etc.; vitamin B group, nicotinamide, calcium pantothenate, amino acids, proteins such as but not limited to casein, gelatin, pectin, agar; organic metal salts such as but not limited to sodium chloride, calcium chloride, dicalcium phosphate, zinc sulphate, zinc chloride, zinc gluconate, etc.; natural pigments, flavors, class I & class II preservatives and aqueous, alcoholic, hydro-alcoholic, organic solutions of above listed ingredients alone or in combination. Preparation of compositions with excipients (C-29 and C-30): As an example, Composition C-29 was prepared by blending 45 g of Punica granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins (PG-1), 15 g of Tagetes erecta flower aqueous ethanol-ethyl acetate extract containing lutein esters (TE-4), 30.5 g of acacia gum, and 2 g of silicon dioxide. Similarly, Composition C-30 was formulated by mixing 45 g of PG-1, 15 g of T. erecta flower hexane extract containing lutein esters (TE-1), along with 30.5 g of acacia gum and 2 g of silicon dioxide.

[0072] Process: The process for the preparation of synergistic compositions comprising;

[0073] (i) a zinc complex or chelate of enriched punicalagins in the range of 20-70% derived from Punica granatum fruit or fruit peel, wherein, zinc is present in a concentration of 0.05-2.0%; and (ii) a second ingredient selected from Tagetes erecta flower extract standardized to lutein or zeaxanthin or its esters in the range of 1-40%; optionally at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents; wherein the process comprises the following steps of;

[0074] (i) Extraction: Extracting dried Punica granatum fruit or fruit peel with a suitable solvent selected from C1-C5 alcohols, including but not limited to ethanol, methanol, n-butanol, water, and mixtures thereof, or organic solvents such as acetone and ethyl acetate;

[0075] (ii) Zinc complexation: Concentrating the extract via solvent evaporation and treating the concentrated solution with a zinc compound selected from zinc oxide, zinc hydroxide, or zinc carbonate to form a zinc-punicalagin complex or chelate;

[0076] (iii) Column purification: Filtering the mixture and subjecting it to column chromatography using a resin selected from PA-800, SP-700, or HP-20. The elution is carried out using aqueous ethanol;

[0077] (iv) Drying of zinc chelate: The eluted fraction is concentrated and dried, preferably under reduced pressure, to yield the enriched zinc complex or chelate of punicalagins; (v) Blending with second active: The obtained enriched zinc-punicalagin complex is blended with a Tagetes erecta flower extract standardized to lutein, zeaxanthin, or their esters. This step may include the addition of at least one acceptable carrier, excipient, or diluent to enhance the formulation's stability;

[0078] (vi) Final drying: The final composition is subjected to drying under vacuum conditions to obtain the finished synergistic composition in powder or granulate form suitable for further formulation.

[0079] Evaluation of compositions containing Punica granatum fruit or fruit peel extract containing zinc and Tagetes erecta flower extract in a scopolamine- induced dry eye disease in Sprague Dawley rats: This study investigates the therapeutic potential of novel compositions containing Punica granatum fruit or fruit peel 70% aqueous ethanol extract with a zinc complex of enriched punicalagins (PG-1) and Tagetes erecta flower extract (TE-4) containing lutein esters. The efficacy of composition 29 was evaluated in an in vivo model of scopolamine-induced dry eye disease (DED) in Sprague Dawley rats (Example 1). The results demonstrated a significant and synergistic improvement in multiple ocular health parameters when compared to individual extracts.

[0080] Efficacy in ocular health: The in vivo study confirmed a notable enhancement in ocular health outcomes with composition 29. When compared to the individual effects of PG-1 or TE-4, the combination demonstrated superior improvements, indicating a synergistic interaction between the components.

[0081] Tear secretion: PG-1 and TE-4, each administered at a dose of 20 mg / kg body weight (BW), showed improvements in tear secretion by 71.52% and 86.81%, respectively, over DED control animals. In contrast, Composition-29, comprising PG-1 and TE-4 in a 3: 1 ratio, exhibited a 121% improvement. This surprising enhancement provides compelling evidence of a synergistic effect when the two extracts are combined, data summarized in Table 15. Tear Film Break-Up Time (TBUT): Composition-29 significantly improved TBUT, a 382% increase relative to DED controls. This improvement was surprisingly higher than the results obtained from either extract administered alone, further reinforcing the presence of synergistic activity, as detailed in Table 16.

[0082] Reduction in corneal staining score: Administration of composition-29 led to a 91.32% reduction in corneal staining scores compared to the DED control group. This was a significantly greater reduction than that observed with individual PG-1 or TE-4 administration, as summarized in Table 17.

[0083] In-vivo efficacy of the composition in protecting against blue light-induced retinal damage in rats: The widespread adoption of digital devices such as smartphones, tablets, and computer screens has markedly increased human exposure to blue light, a component of high-energy visible (HEV) light in the 450-495 nm wavelength range. Unlike ultraviolet (UV) radiation, blue light penetrates ocular tissues more deeply and reaches the retina with greater intensity. This has raised growing concern over its potential to induce cumulative photochemical damage to the retina, particularly under conditions of prolonged or repeated exposure. Emerging evidence suggests that prolonged exposure to blue light can compromise retinal integrity and may contribute to the pathogenesis of degenerative ocular conditions. Epidemiological studies have linked sustained exposure to blue light to an increased risk of age-related macular degeneration (AMD), a leading cause of reduced visual acuity in the young and aging populations. At the cellular level, exposure to blue light has been shown to generate excessive reactive oxygen species (ROS), impair mitochondrial function, damage nuclear and mitochondrial DNA, and promote premature senescence in retinal cells.

[0084] Among retinal structures, the photoreceptor layer and the outer nuclear layer (ONL), which contains the nuclei of photoreceptor cells, are particularly vulnerable to oxidative stress and energy imbalance. These layers serve as sensitive indicators of retinal health and are critical in maintaining visual function. In this context, the present study aimed to investigate the morphological changes upon blue light exposure on the retina, with particular emphasis on changes in photoreceptor layer thickness and ONL thickness. These parameters offer quantifiable and reliable endpoints for evaluating structural damage and assessing potential interventions for blue light-induced retinal injury.

[0085] This study evaluated the therapeutic potential of novel compositions containing Punica granatum fruit or fruit peel 70% aqueous ethanol extract containing a zinc complex of enriched punicalagins (PG-1) and Tagetes erecta flower extract (TE- 4) containing lutein esters. The efficacy of these extracts, particularly their combination in Composition-29, was assessed in a rat model of blue light-induced retinal damage (Example 33). The results demonstrated a significant and synergistic protective effect when the extracts were combined, compared to their individual administration.

[0086] Improvement in photoreceptor layer thickness: When administered individually at a dose of 20 mg / kg body weight, PG-1 and TE-4 increased photoreceptor layer thickness by 32.92% and 49.20%, respectively, relative to the blue light control group. Surprisingly, the composition-29 containing PG-1 and TE-4 in a 3: 1 ratio produced a markedly enhanced improvement of 74.91%. This substantial increase supports the presence of a synergistic interaction between the two extracts. Results are presented in Table 18.

[0087] Improvement in ONL thickness: Similarly, PG-1 and TE-4 at 20 mg / kg improved ONL thickness by 26.25% and 32.30%, respectively, compared to the blue light control group. In comparison, composition-29 achieved a 46.20% improvement in ONL thickness. This enhanced response further reinforces the synergistic potential of the PG-1 and TE-4 combination. The data are summarized in Table 19. The foregoing findings from this study clearly demonstrate that Compositions confer synergistically enhanced therapeutic benefits in the management of eye disease. The combination of Punica granatum fruit or fruit peel extract with a zinc complex of enriched punicalagins and Tagetes erecta flower extract containing lutein esters significantly outperformed the individual extracts in improving key measures of eye health; including tear secretion, tear film stability, corneal integrity, Photoreceptor Layer Thickness, and ONL thickness. These results support the development of such synergistic formulations for the effective treatment of eye diseases.

[0088] Therefore, in an important embodiment, the present invention provides synergistic herbal compositions comprising the combination of a first ingredient containing a zinc complex or a chelate of enriched punicalagins, wherein the said punicalagins are in the range of 20% to 70% by weight, said complex or chelate being derived from the fruit or fruit peel of Punica granatum, and wherein zinc is present in the range of 0.05% to 2.0% by weight; and a second ingredient comprising an extract of Tagetes erecta flowers, standardized to contain one or more phytochemicals selected from the group consisting of lutein esters, zeaxanthin esters, lutein, zeaxanthin, and mixtures thereof, wherein said phytochemicals are present in the range of 1% to 40% by weight; for eye health benefits.

[0089] In one preferred embodiment, the present invention provides synergistic herbal composition as described above, wherein the eye health benefits include, but are not limited to, preventing or alleviating symptoms of dry eye disease (DED), age- related macular degeneration (AMD), cataract, vision loss, glaucoma, and diabetic retinopathy; reducing the harmful effects of blue light exposure, including digital eye strain, visual fatigue, burning eyes, blurred vision, and headaches; and enhancing visual performance, including improvements in visual acuity, light adaptation, and dark adaptation. In one preferred embodiment, the present invention provides synergistic herbal compositions as disclosed above; wherein the concentration of Punica granatum fruit or fruit peel extract containing zinc in the composition varies in the range of 10%-90% by weight, and Tagetes erecta flower extract in the composition varies in the range of 90%-10% by weight.

[0090] In another embodiment, the process of extraction of Punica granatum and Tagetes erecta involves the use of techniques known in the art, such as maceration, digestion, decoction, infusion, percolation, Soxhlet extraction, refluxing, continuous, ultrasound-assisted, microwave-assisted, pressurized liquid / fluid extraction, and supercritical carbon dioxide extraction. Further, the extracts or fractions may be further purified using column chromatography or resin chromatography using a microporous resin.

[0091] In another embodiment, the present invention discloses synergistic herbal compositions as described above, wherein, the Lutein esters are selected from any lutein geometrical isomers, lutein 3 -O-palmitate, lutein 3-O-palmitate, lutein dipalmitate, lutein 3 -O-myristate, lutein 3-0- myristate, lutein dimyristate, lutein 3 -O-palmitate-3-O-myri state, lutein 3 -O-stearate, lutein 3-0- stearate, lutein distearate, lutein 3 -O-palmitate-3-O- stearate, lutein 3 -O-laurate, lutein 3-0- laurate, lutein di laurate, lutein 3 -O-palmitate-3 -O-laurate, lutein 3 -O-oleate, lutein 3-0- oleate, lutein di oleate, lutein 3 -O-palmitate-3-O-oleate, lutein 3 -0- myristate-3-O-laurate etc.

[0092] In another embodiment, the compositions disclosed above, wherein the phytochemical reference marker compound, pharmacologically active marker or group of markers or its zinc salt, may be estimated by several analytical techniques known in the art, such as but not limited to HPLC, gravimetry, UV, GC, and ICP-mass. In another embodiment, the present invention provides synergistic herbal compositions as disclosed above, wherein the extracts are produced using at least one solvent selected from C1-C5 alcohols such as but not limited to ethanol, methanol, n-propanol, isopropyl alcohol; ketones selected from acetone, methyl isobutyl ketone, chlorinated solvents selected from methylene dichloride and chloroform; water and mixtures thereof; C1-C7 hydrocarbons such as but not limited to hexane, pentane; esters such as but not limited to ethyl acetate and mixtures thereof.

[0093] In one preferred embodiment, the present invention provides synergistic herbal compositions comprising the combination of a first ingredient selected from a zinc complex or chelate of enriched punicalagins, where the punicalagins are in the range of 20-70% derived from Punica granatum fruit or fruit peel, wherein, zinc is present in a concentration of 0.05-2.0%; and a second ingredient selected from Tagetes erecta flower extract standardized to lutein or zeaxanthin or their esters in the range of 1-40%; further containing optionally at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents.

[0094] In another embodiment, the present invention provides a process for the preparation synergistic herbal compositions comprising a step of combining a zinc complex or chelate of enriched punicalagins as a first ingredient, wherein the punicalagins are present in the range of 20-70% derived from Punica granatum fruit or fruit peel, wherein, zinc is present in a concentration of 0.05-2.0%; from Tagetes erecta flower extract as a second ingredient which is standardized to contain one or more phytochemicals selected from lutein or zeaxanthin or its esters in the range of 1-40%; and optionally at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents. In another embodiment of the invention, the composition as disclosed above is formulated into a dosage form selected from a dry powder form, a liquid form, a beverage, a food product, a dietary supplement, or any suitable form such as a tablet, a capsule, a soft chewable tablet, gummies or gummy bar.

[0095] In another embodiment of the invention, the composition as disclosed above is formulated into a nutritional / dietary supplement that can be contemplated / made into the dosage form of healthy foods or food for specified health uses, such as solid food like chocolate or nutritional bars, semisolid food like cream, jam, or gel or beverage such as refreshing beverage, lactic acid bacteria beverage, drop, candy, chewing gum, gummy candy, yogurt, ice cream, pudding, soft adzuki bean jelly, jelly, cookie, tea, soft drink, juice, milk, coffee, cereal, snack bar.

[0096] In another embodiment of the invention, the composition disclosed above is formulated into a controlled-release tablet, using controlled-release polymer-based coatings, such as those employing techniques including nanotechnology, microencapsulation, colloidal carrier systems, and other drug delivery systems to obtain the desired therapeutic benefits.

[0097] In yet another embodiment, the present invention provides methods of obtaining at least one ocular health benefit selected from but not limited to preventing or alleviating symptoms of dry eye disease (DED), age-related macular degeneration (AMD), cataract, vision loss, glaucoma, and diabetic retinopathy; reducing the harmful effects of blue light exposure, including digital eye strain, visual fatigue, burning eyes, blurred vision, and headaches; and enhancing visual performance, including improvements in visual acuity, light adaptation, and dark adaptation in a human; wherein the method comprises supplementing the human in a need thereof with an effective dose of a composition comprising the combination of a first ingredient selected from a zinc complex or chelate of enriched punicalagins in the range of 20-70% derived from Punica granatum fruit or fruit peel, wherein, zinc is present in a concentration of 0.05-2.0%; Tagetes erecta flower extract as a second ingredient standardized to contain one or more phytochemicals selected from lutein or zeaxanthin or its esters in the range of 1-40%; optionally further containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers, and diluents.

[0098] In yet another embodiment, the present invention provides synergistic herbal compositions comprising the combination of an enriched punicalagins in the range of 20-70% derived from Punica granatum fruit or fruit peel as a first ingredient and a second ingredient selected from Tagetes erecta flower extract standardized to contain lutein or zeaxanthin or its esters in the range of 1 40% by weight; for eye health benefits.

[0099] In yet another embodiment, the present invention provides methods of obtaining at least one ocular health benefit selected from but not limited to preventing or alleviating symptoms of dry eye disease (DED), age-related macular degeneration (AMD), cataract, vision loss, glaucoma, and diabetic retinopathy; reducing the harmful effects of blue light exposure, including digital eye strain, visual fatigue, burning eyes, blurred vision, and headaches; and enhancing visual performance, including improvements in visual acuity, light adaptation, and dark adaptation; wherein the method comprises supplementing human with an effective dose of a composition comprising the combination of an enriched punicalagins in the range of 20-70% derived from Punica granatum fruit or fruit peel as a first ingredient; and a second ingredient selected from Tagetes erecta flower extract standardized to contain lutein or zeaxanthin or its esters in the range of 1-40%; optionally further containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers, and diluents.

[0100] In yet another embodiment, the present invention provides use of synergistic herbal compositions comprising the combination of a first ingredient selected from a zinc complex or chelate of enriched punicalagins, wherein the punicalagins are in the range of 20-70% derived from Punica granatum fruit or fruit peel, wherein, zinc is present in a concentration of 0.05-2.0%; and a second ingredient selected from Tagetes erecta flower extract standardized to lutein or zeaxanthin or its esters in the range of 1-40%; optionally further containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers, and diluents; for improving at least one ocular health benefit in a human selected from but not limited to preventing or alleviating symptoms of dry eye disease (DED), age-related macular degeneration (AMD), cataract, vision loss, glaucoma, and diabetic retinopathy; reducing the harmful effects of blue light exposure, including digital eye strain, visual fatigue, burning eyes, blurred vision, and headaches; and enhancing visual performance, including improvements in visual acuity, light adaptation, and dark adaptation.

[0101] Those of ordinary skill in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the embodiments or examples disclosed herein but is intended to cover modifications within the objectives and scope of the present invention as defined in the specification. The examples are given solely for illustration and are not to be construed as limitations of the present disclosure, as many variations thereof are possible without departing from the spirit of the disclosure.

[0102] EXAMPLES:

[0103] Example 1: Punica granatum fruit or fruit peel 70% aqueous ethanol extract containing zinc complex of enriched punicalagins (PG-1).

[0104] Punica granatum fruit or fruit peel powder (50 g) was extracted with 70% aqueous ethanol(— ml) under heating for 3 h. The resulting 70% aqueous ethanol extract was evaporated under reduced pressure to 100 mL of volume, and water (400 mL) was added, followed by zinc oxide (430 mg). The mixture was stirred at ambient temperature for 3 h. The mixture was filtered and washed with water (50 mL). The filtrate was passed through a column packed with PA-800 resin (150 mL) for 5 h. The column was sequentially eluted with water (450 mL) and 10% aqueous ethanol (900 m ). The combined 10% aqueous ethanol eluent was evaporated to give the Punica granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins (PG-1) as a pale brown color solid (4.7 g).

[0105] Comparative Example 1A: Punica granatum fruit or fruit peel 70% aqueous ethanol extract containing enriched punicalagins without zinc (PGC-1).

[0106] P. granatum fruit or fruit peel 70% aqueous ethanol extract containing enriched punicalagins without zinc was prepared as described in Example 1 without the step of adding zinc oxide, but with purification on resin column to give the product.

[0107] Comparative Example IB: Punica granatum fruit or fruit peel 70% aqueous ethanol extract containing punicalagins (PGC-2).

[0108] Punica granatum fruit or fruit peel 70% aqueous ethanol extract containing punicalagins was prepared as described in example 1 without the steps of adding zinc oxide and resin column purification to give the product (PGC-2) as a pale brown color solid (18.0 g).

[0109] Example 2: Punica granatum fruit or fruit peel 95% aqueous acetone extract containing zinc complex of enriched punicalagins (PG-2).

[0110] Punica granatum fruit or fruit peel 95% aqueous acetone extract containing zinc complex of enriched punicalagins was prepared as described in Example 1 using 95% aqueous acetone as extraction medium in place of 70% aqueous ethanol.

[0111] Example 3: Punica granatum fruit peel 0.4% aqueous HC1 extract containing zinc complex of enriched punicalagins (PG-3).

[0112] P. granatum fruit peel 0.4% aqueous HC1 extract containing zinc complex of enriched punicalagins was prepared as described in Example 1 using 0.4% aqueous HC1 extraction medium in place of 70% aqueous ethanol. The pH of the 0.4% aqueous HC1 extract was adjusted from 1.3 to 4.65 using 10% aqueous sodium hydroxide solution (41 m ) before the addition of zinc oxide to give the product (PG-3) as a pale brown-colored solid (6.0 g).

[0113] Example 4: Punica granatum fruit or fruit peel water extract containing zinc complex of enriched punicalagins (PG-4).

[0114] Punica granatum fruit or fruit peel water extract containing zinc complex of enriched punicalagins was prepared as described in Example 1 using water as the extraction medium in place of 70% aqueous ethanol. Example 5: Standardization of Punica granatum fruit or fruit peel extracts.

[0115] Punica granatum fruit or fruit peel extracts disclosed in examples 1 to 4 and comparative examples 1A and IB were analysed for punicalagin by analytical HPLC and zinc by ICP-MS, and the results are summarized in Table 2.

[0116] Table 2: Analysis data of Punica granatum fruit or fruit peel extracts containing punicalagin and zinc

[0117] ND - Not detected

[0118] Example 6: Tagetes erecta flowers hexane extract containing lutein esters (TE-1). Dry Tagetes erecta flowers (50 g) were extracted 3 times with hexane for 3 h. The combined hexane extract was evaporated under reduced pressure to give the oleoresin (TE-1) as a dark orange color semi-solid (3.2 g).

[0119] Example 7; Isolation of lutein dipalmitate from dry Tagetes erecta flowers.

[0120] Tagetes erecta oleoresin prepared as described in Example 6, was adsorbed over silica gel and chromatographed over a silica gel column using hexane: ethyl acetate (99: 1) as eluents to give the product as an orange color semi-solid (600 mg). The product was triturated with ethanol (20 mL), and the residue was dried under vacuum to yield lutein dipalmitate as an orange color semi-solid (500 mg). The product was well characterized by NMR.

[0121] 'H NMR: 6.61 (4 H, m), 6.35 (2 H, dd, J= 15.2 Hz, 3.2 Hz), 6.25 (2 H, d, J=8.0 Hz), 6.11 (5 H, m), 5.44 (3 H, m), 5.06 (1 H, m), 2.42 (2 H, m), 2.28 (4 H, m), 2.11 (2 H, m), 1.94 (12 H, d), 1.83 (3 H, m), 1.76 (3 H, s), 1.60 (9 H, m), 1.55 (3 H, s), 1.43 (2 H, m), 1.09 (6 H, d), 1.002 (3 H, s), 0.881 (10 H, m).

[0122] Example 8: Tagetes erecta flowers ethyl acetate extract containing lutein esters (TE-2).

[0123] Dry Tagetes erecta flowers ethyl acetate extract containing lutein esters was prepared as described in example 6, by employing ethyl acetate in place of hexane to give the oleoresin (TE-2) as a dark orange colored semi-solid (4.6 g).

[0124] Example 9: Heptane extract of Tagetes erecta flowers containing lutein esters (TE-3).

[0125] Heptane extract of dry Tagetes erecta flowers containing lutein esters, was prepared as described in example 6, by employing heptane instead of hexane to give the oleoresin (TE-3) a dark orange colored semi-solid (3.3 g).

[0126] Example 10: Tagetes erecta flowers aq. ethanol followed by ethyl acetate extract containing lutein esters (TE-4). Dry Tagetes erecta flowers (50 g) were extracted with aq. ethanol followed by ethyl acetate for 3 h. The combined ethyl acetate extract was evaporated under reduced pressure to give the oleoresin (TE-4) as a dark orange colored semi solid (3-2 g).

[0127] Example 11: Tagetes erecta flowers acetone extract containing lutein esters (TE-

[0128] 5).

[0129] Acetone extract of dry Tagetes erecta flowers containing lutein esters, was prepared as described in example 6, by employing acetone instead of hexane to give the oleoresin (TE-5) as a dark orange colored semi-solid (5.1 g).

[0130] Example 12: Tagetes erecta flowers ethanol extract containing lutein esters (TE-

[0131] 6).

[0132] Dry Tagetes erecta flowers ethanol extract containing lutein esters was prepared as described in example 6, by employing ethanol instead of hexane to give the oleoresin (TE-6) as dark orange color semi-solid (14.5 g).

[0133] Example 13: Standardization of Tagetes erecta flower extracts.

[0134] Tagetes erecta flower extracts disclosed in examples 6 and 8-12 were analysed for lutein esters by analytical HPLC in two different methods.

[0135] Method-1: Lutein esters were hydrolysed to free lutein, which was then quantified and the result was then used to estimate the original lutein ester content.

[0136] Method-2: Lutein esters were quantified based on their relative retention times, using lutein dipalmitate as the reference standard.

[0137] Table 3: HPLC analysis data of Tagetes erecta flower extracts containing lutein esters (Method- 1).

[0138] Example 14: Tagetes erecta flowers hexane extract containing lutein and zeaxanthin (TE-7).

[0139] Tagetes erecta oleoresin prepared as described in example 6, was dissolved in isopropyl alcohol at 60°C and treated with 50% aqueous potassium hydroxide solution. The mixture was stirred at 60-70°C for 1 h, cooled to RT, and water was added. The mixture was stirred for 10 more min and was extracted repeatedly with ethyl acetate. The combined ethyl acetate extract was washed with water (100 m ) and then evaporated at 50°C to yield the product as a dark orange colored semi-sold (2.2 g).

[0140] Example 15: Isolation of lutein from Tagetes erecta flowers.

[0141] Tagetes erecta extract prepared as described in example 14 was adsorbed over silica gel. The adsorbed silica gel was chromatographed over a silica gel column using hexane: EtOAc (70:30) as eluents to give lutein as an orange color solid (70 mg), mp: 174-178°C (Lit. mp: 190°C). The product was well characterized by NMR.

[0142] 'H NMR: 6.75 (4 H, m), 6.43 (2 H, m), 6.27 (5 H, m), 6.11 (5 H, m), 5.53 (2 H, m), 4.58 (2 H, m), 4.13 (1 H, m), 3.83 (1 H, m), 2.33 (3 H, m), 2.00 (12 H, d), 1.75 (5 H, m), 1.62 (3H, s), 1.36 (14 H, m), 1.11 (6 H, d), 0.94 (2 H, m), 0.86 (3 H, s).

[0143] Example 16: Tagetes erecta flowers ethyl acetate extract containing lutein & zeaxanthin (TE-8). Dry Tagetes erecta flowers ethyl acetate extract containing lutein and zeaxanthin was prepared as described in example 14 by employing Tagetes erecta oleoresin prepared as described in example 8 to give the product as a dark orange colour semi-sold (2.6 g).

[0144] Example 17: Tagetes erecta flowers heptane extract containing lutein and zeaxanthin (TE-9).

[0145] Dry Tagetes erecta flowers heptane extract containing lutein and zeaxanthin was prepared as described in example 14 by employing Tagetes erecta oleoresin prepared as described in example 9 to give the product as a dark orange color semi-sold (2.3 g).

[0146] Example 18: Standardization of Tagetes erecta flower extracts.

[0147] Tagetes erecta flower extracts disclosed in the above examples 14 and 16-17 were analysed for lutein and zeaxanthin by analytical HPLC, and the results are summarized in Table 5.

[0148] Table 4: HPLC analysis data of Tagetes erecta flower extracts containing lutein and zeaxanthin.

[0149] Example 19: Preparation of various compositions containing Punica granatum fruit or fruit peel 70% aqueous ethanol extract containing zinc complex of enriched punicalagins (PG-1) and Marigold flowers hexane extract containing lutein esters (TE-1) in the following ratios.

[0150] Comp-1 (C-l): C-l was prepared by combining PG-1 and TE-1 in the ratio of 3:1. Comp-2 (C-2): C-2 was prepared by combining PG-1 and TE-1 in the ratio of 2: 1.

[0151] Comp-3 (C-3): C-3 was prepared by combining PG-1 and TE-1 in the ratio of 1 :1.

[0152] Comp-4 (C-4): C-4 was prepared by combining PG-1 and TE-1 in the ratio of 1 :2.

[0153] Comp-5 (C-5): C-5 was prepared by combining PG-1 and TE-1 in the ratio of 1 :3.

[0154] Example 20: Preparation of various compositions containing Punica granatum fruit or fruit peel 70% aqueous ethanol extract containing zinc complex of enriched punicalagins (PG-1) and Tagetes erecta flowers aq. ethanol followed by ethyl acetate extract containing lutein esters (TE-4) in the following ratios.

[0155] Comp-6 (C-6): C-6 was prepared by combining PG-1 and TE-4 in the ratio of 3 : 1.

[0156] Comp-7 (C-7): C-7 was prepared by combining PG-1 and TE-4 in the ratio of 2: 1.

[0157] Comp-8 (C-8): C-8 was prepared by combining PG-1 and TE-4 in the ratio of 1 : 1.

[0158] Comp-9 (C-9): C-9 was prepared by combining PG-1 and TE-4 in the ratio of 1 :2.

[0159] Comp- 10 (C-10): C-10 was prepared by combining PG-1 and TE-4 in the ratio of 1 :3.

[0160] Example 21: Preparation of various compositions containing Punica granatum fruit or fruit peel 70% aqueous ethanol extract containing zinc complex of enriched punicalagins (PG-1) and Tagetes erecta flowers ethyl acetate extract containing lutein esters (TE-2) in the following ratios.

[0161] Comp-11 (C-l l): C-l l was prepared by combining PG-1 and TE-2 in the ratio of 2: 1.

[0162] Comp-12 (C-12): C-12 was prepared by combining PG-1 and TE-2 in the ratio of 1 : 1.

[0163] Comp-13 (C-13): C-13 was prepared by combining PG-1 and TE-2 in the ratio of 1 :2.

[0164] Example 22: Preparation of various compositions containing Punica granatum fruit or fruit peel 70% aqueous ethanol extract containing zinc complex of enriched punicalagins (PG-1) and Tagetes erecta flowers heptane extract containing lutein esters (TE-3) in the following ratios. Comp-14 (C-14): C-14 was prepared by combining PG-1 and TE-3 in the ratio of 2: 1.

[0165] Comp- 15 (C-15): C-15 was prepared by combining PG-1 and TE-3 in the ratio of 1 : 1.

[0166] Comp- 16 (C-16): C-16 was prepared by combining PG-1 and TE-3 in the ratio of 1 :2.

[0167] Example 23: Preparation of various compositions containing Punica granatum fruit or fruit peel 95% aqueous acetone extract containing zinc complex of enriched punicalagins (PG-2) and Tagetes erecta flowers ethanol extract containing lutein esters (TE-6) in the following ratios.

[0168] Comp- 17 (C-17): C-17 was prepared by combining PG-2 and TE-6 in the ratio of 2: 1.

[0169] Comp- 18 (C-18): C-18 was prepared by combining PG-2 and TE-6 in the ratio of 1 : 1.

[0170] Comp- 19 (C-19): C-19 was prepared by combining PG-2 and TE-6 in the ratio of 1 :2.

[0171] Example 24: Preparation of various compositions containing Punica granatum fruit or fruit peel water extract containing zinc complex of enriched punicalagins (PG-4) and Tagetes erecta flowers acetone extract containing lutein esters (TE-5) in the following ratios.

[0172] Comp-20 (C-20): C-20 was prepared by combining PG-4 and TE-5 in the ratio of 2: 1.

[0173] Comp-21 (C-21): C-21 was prepared by combining PG-4 and TE-5 in the ratio of 1 : 1.

[0174] Comp-22 (C-22): C-22 was prepared by combining PG-4 and TE-5 in the ratio of 1 :2.

[0175] Example 25: Preparation of various compositions containing Punica granatum fruit or fruit peel 70% aqueous ethanol extract containing zinc complex of enriched punicalagins (PG-1) and Tagetes erecta flowers ethyl acetate extract containing lutein and zeaxanthin (TE-8) in the following ratios.

[0176] Comp-23 (C-23): C-23 was prepared by combining PG-1 and TE-8 in the ratio of 2: 1.

[0177] Comp-24 (C-24): C-24 was prepared by combining PG-1 and TE-8 in the ratio of 1 : 1.

[0178] Comp-25 (C-25): C-25 was prepared by combining PG-1 and TE-8 in the ratio of 1 :2.

[0179] Example 26: Preparation of various compositions containing Punica granatum fruit or fruit peel 95% aqueous acetone extract containing zinc complex of enriched punicalagins (PG-2) and Tagetes erecta flowers heptane extract containing lutein and zeaxanthin (TE-9) in the following ratios.

[0180] Comp-26 (C-26): C-26 was prepared by combining PG-2 and TE-9 in the ratio of 2: 1.

[0181] Comp-27 (C-27): C-27 was prepared by combining PG-2 and TE-9 in the ratio of 1 : 1.

[0182] Comp-28 (C-28): C-28 was prepared by combining PG-2 and TE-9 in the ratio of 1 :2.

[0183] Example 27: Formulation of the compositions

[0184] Comp-29: Transferred PG-1 (45 g), TE-4 (15 g), acacia gum (7.5 g), micro crystalline cellulose (30.5 g), and silicon dioxide (2 g) into a double polybag. The mixture was uniformly blended, then subjected to pulverization and sieving through a #40 mesh to give the composition as a fine powder (Comp-29).

[0185] Comp-30: Transferred PG-1 (45 g), TE-1 (15 g), acacia gum (7.5 g), micro crystalline cellulose (30.5 g), and silicon dioxide (2 g) into a double polybag. The mixture was uniformly blended, then subjected to pulverization and sieving through a #40 mesh to obtain the composition as a fine powder (Comp-30). Example 28: General procedure of ROS assay in H2O2-induced human retinal epithelial cells (ARPE-19)

[0186] ROS assay was performed in H2O2-induced ARPE-19 human retinal pigment epithelial cells. Briefly, an equal number of ARPE-19 cells (50,000 cells / well) 5 was seeded in a 96-well black clear bottom plate in 200pL of DMEM + 10% FBS medium and the plate was incubated at 37°C in a CO2 incubator overnight. The next day, the media was removed and HBSS containing DCF -DA (working concentration, lOpM) was added to all the wells and further incubated for 1 hour at 37°C in a CO2 incubator. After the incubation period, the media containing 0 DCF-DA was removed and the cells were replenished with 150pl of DMEM + 10% FBS medium. Then, cells were pretreated with 50pl of different concentrations of test samples for 2 hours, incubated at 37°C in a CO2 incubator. After 2 hours, the cells were treated with 2.5 mM H2O2, except for vehicle (cells + 0.2% DMSO), and incubated further for 4 hours in a CO2 incubator. After 4 hours, 5 Relative Fluorescence Units (RFU) were measured in a Spectramax M5e spectrophotometer at a wavelength of Ex / Em: 490 / 535 nm, and the percentage inhibition of ROS was calculated using the following formula:

[0187] % scavenging (Inhibition) of ROS= [(Normalized ROS (RFU) in Induction) - (Normalized ROS (RFU) in Test compounds)] / Normalized ROS (RFU) in 0 Induction) x 100

[0188] The results were presented in tables 5-8.

[0189] Table 5: ROS inhibition (scavenging) by the compositions containing P. granatum fruit or fruit peel extract containing zinc complex of enriched 5 punicalagins (PG-1); and Tagetes erecta flowers hexane extract containing lutein esters (TE-1) in H2O2-induced ARPE-19 cells

[0190] Table 6: ROS inhibition by the compositions containing P. granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins (PG-1); and Tagetes erecta flowers aq. ethanol followed by ethyl acetate extract containing

[0191] 5 lutein esters (TE-4) in H2O2-induced ARPE-19 cells.

[0192] Table 7: ROS inhibition by the compositions containing P. granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins (PG-1 or PG-2); and Tagetes erecta flowers extract containing lutein esters (TE-2 or TE-3 or TE-6) 0 in H2O2-induced ARPE-19 cells

[0193] Table 8: ROS inhibition by the compositions containing P. granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins (PG-4 or PG-1 or PG-2); and Tagetes erecta flowers extract (TE-5 or TE-8 or TE-9) in H2O2-

[0194] 5 induced ARPE-19 cells.

[0195] Example 29: General procedure for mitochondrial Superoxide scavenging assay in in the Blue light induced ARPE-19 cells

[0196] Superoxide assay was performed using ARPE-19 human retinal pigment epithelial cells, using MitoSOX™ Mitochondrial Superoxide Indicators (Thermo Fisher Scientific, Waltham, MA). Briefly, an equal number of ARPE-19 cells (30,000 cells / well) was seeded in a 96-well cell culture plate in DMEM + 10% FBS medium, incubated at 37°C in a CO2 incubator overnight. The next day, cells were replenished with DMEM+10% FBS media and pretreated with different concentrations of test compounds for 2 hours, followed by incubation at 37°C in a CO2 incubator. After 2 hours, cells were exposed to 8 Volts (0.53mW or 38.4J7cm2) of blue light for 6 hrs except for the vehicle control (cells+0.2% DMSO) which was kept in a separate plate and incubated further for 6 hrs at 37°C, in the presence of 5% CO2. After 6 hrs of treatment, the culture media was removed and HBSS containing 2.5pM MitoSOX reagent was added to the wells and incubated for 30 min. at 37°C, 5% CO2 incubator. Then, the cells were transferred to FACS tubes and centrifuged at 180g for 5 min. The supernatant was discarded, and the cells were resuspended in 0.5 mL of FACS buffer (2% FBS in lx PBS) and washed. Finally, the cells were resuspended in 250 pL of FACS buffer and acquired on a BD FACSVerse flow cytometer. The data were then analyzed. The percentage of the cell population protected from the mitochondrial superoxide stress was calculated using the following formula.

[0197] 5 % Cell Population Protected from Mitochondrial Superoxide stress =

[0198] [(Normalized % cell population stressed in Induction) - (Normalized % Superoxide +ve cell Population)] / Normalized % cell population stressed in Induction x 100

[0199] The results are presented in Tables 9-10. 0

[0200] Table 9: Percent population of blue light-exposed ARPE-19 cells protected from mitochondrial superoxide stress by the compositions containing P. granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins (PG-1); and Tagetes erecta flowers hexane extract containing lutein esters (TE-1) 5

[0201] Table 10: Percent population of blue light-exposed ARPE-19 cells protected from mitochondrial superoxide stress by the compositions containing P. granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins (PG-1); and Tagetes erecta flowers aq. ethanol followed by ethyl acetate extract containing lutein esters (TE-4).

[0202] 5 Example 30: General procedure for ROS scavenging assay in high glucose- induced ARPE-19 cells

[0203] An equal number of ARPE-19 cells (30000 cells / well) suspended in DMEM medium supplemented with 10%FBS and 30 mM glucose (high glucose) was seeded into a 96-well black plate and incubated overnight at 37°C in a CO2 0 incubator. Cells grown in DMEM medium containing 17 mM glucose (normal glucose) served as the vehicle control. The next day, the media was removed, and HBSS containing DCF-DA (working concentration of 10 pM) was added to all wells, and incubated for 1 hour at 37 °C in a CO2 incubator. After the incubation period, the DCF-DA media was removed, and the cells were replenished with 5 DMEM containing 10% FBS and high glucose (30 mM) for the experimental wells and with DMEM plus 10% FBS (normal glucose, 17 mM) for the vehicle control wells. The cells were treated with 50 pL of various concentrations of test samples and incubated further for 4 hours at 37 °C in a CO2 incubator. Thereafter, the relative fluorescence was measured using a spectrophotometer at Ex / Em wavelengths of 490nm / 535nm. The percent inhibition of ROS was calculated using the following formula.

[0204] 5 % ROS scavenging = [(Normalized ROS (RFU) in Induction) - (Normalized ROS

[0205] (RFU) in Test wells)] / Normalized ROS (RFU) in Induction) x 100 The results are presented in Tables 11-12.

[0206] Table 11: ROS scavenging in high glucose-induced ARPE-19 cells by the 0 compositions containing P. granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins (PG-1); and Tagetes erecta flowers hexane extract containing lutein esters (TE-1).

[0207] Table 12: ROS scavenging in high glucose-induced ARPE-19 cells by the 5 compositions containing P. granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins (PG-1); and Tagetes erecta flowers aq. ethanol followed by ethyl acetate extract containing lutein esters (TE-4).

[0208] Example 31: General procedure for apoptosis assay in blue light-exposed ARPE- 19 cells

[0209] An apoptosis assay was performed in ARPE-19 human retinal pigment epithelial 5 cells exposed to blue light. Briefly, an equal number of ARPE-19 cells (30,000 cells / well) was seeded in a 96-well cell culture plate in DMEM + 10% FBS medium, incubated overnight at 37°C in a 5% CO2 incubator.. The next day, cells were replenished with DMEM+10% FBS media and pretreated with different concentrations of test compounds for 2 hours, followed by incubation at 37°C in a 0 CO2 incubator. After 2 hours, cells were exposed to 8 Volts (0.53mW or 38.4J7cm2) of blue light for 4 hrs, except for the vehicle (cells + 0.2% DMSO) control which was kept in a separate plate, incubated further for 6 hrs at 37°C, 5% CO2 incubator. After 4 hours, the media was removed and washed with HBSS. The cells were then transferred into FACs tubes and centrifuged at 180g for 5 5 min. The cells were resuspended in 0.2 mL of Annexin-V binding buffer. Five microliters of Annexin-V FITC were added to each tube and incubated for 20 min at room temperature in the dark. After 20 min, the cells were centrifuged at 180g for 5 min. The cells were resuspended in 250pl of FACS buffer (2% FBS in IX PBS) and acquired in BD FACS Verse flow cytometer. The percentage of the cell population protected from apoptosis was calculated using the following formula:

[0210] % Cell Population Protected from Apoptosis = [(Normalized % cell population

[0211] 5 Annexin-V FITC+ve exposed to blue light) - (Normalized % Annexin-V FITC+ve cell Population in test)] / Normalized % cell population Annexin-V FITC+ exposed to blue light x 100.

[0212] The results are presented in Tables 13-14. 0 Table 13: Percentage of blue light-exposed ARPE-19 cell population protected from apoptosis by the compositions containing P. granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins (PG-1); and Tagetes erecta flowers hexane extract containing lutein esters (TE-1). 5 Table 14: Percentage of blue light-exposed ARPE-19 cell population protected from apoptosis by the compositions containing P. granatum fruit or fruit peel extract containing zinc complex of enriched punicalagins (PG-1); and Tagetes erecta flowers aq. ethanol followed by ethyl acetate extract containing lutein esters (TE-4).

[0213] 5 Example 32: In vivo study of eye health compositions on scopolamine-induced dry eye disease in Sprague Dawley rats

[0214] Following a 7-day acclimatization period, twenty male and twenty female Sprague Dawley rats (age: 8-10 weeks) were randomly assigned to five groups based on their body weights. Each group consisted of 8 animals: four males and 0 four females. The first group, vehicle control (Gl), and the second group, DED control (G2), were supplemented with vehicle. The other groups were supplemented with PG-1 (G3; 20 mg / kg BW), TE-4 (G4; 20 mg / kg BW), and comp-29 (G5; 20 mg / kg BW) from day 3 to day 15 of the study. Rats (G2 to G5) were given 0.1 mL of scopolamine (15 mg / mL) subcutaneously (s.c.) thrice daily 5 for five days (day 1 - day 5) to induce dry eye disease (DED). Rats in the vehicle control and DED control groups were given 0.5% w / v CMC-Na as a vehicle, and groups 3 to 5 rats received oral supplementation of the respective test items from day 1 to day 15 of the study. On day 15, the DED parameters, such as tear secretion, tear film breakup time, and corneal staining score, were assessed.

[0215] Tear secretion was measured using the Schirmer’s tear test strip method. The rats were mildly anesthetized, and a test strip was placed in the inferior conjunctival fornix and left in plac for 5 minutes. The length of the wet strip was measured in millimeters (mm).

[0216] Tear film break-up time (TBUT) was evaluated under cobalt blue light after applying 1% sodium fluorescein into both eyes of anesthetized rats. The average of three observations of the time taken for appearing dry spots (in seconds) on the corneal surface after blinking was recorded.

[0217] Corneal fluorescein staining was used to assess corneal epithelial damage. A drop of 1% fluorescein (Fluoro touch strips) was applied to the conjunctivae, and eyes were examined for fluorescein staining using a slit-lamp biomicroscope under a cobalt blue light. Scores ranging from 0 to 4 were given, depending on the number of corneal staining spots.

[0218] The results of the compositions are summarized in Tables 15-17.

[0219] Table 15: In vivo efficacy of compositions in improving tear secretion in scopolamine-induced Sprague Dawley rats.

[0220] Table 16: In vivo efficacy of compositions in improving tear film break-up time (TBUT) in scopolamine-induced Sprague Dawley rats.

[0221] Table 17: In vivo efficacy of compositions in reducing Corneal staining scored in scopolamine-induced DED in Sprague Dawley rats. Example 33: In-vivo efficacy of the composition in protecting against blue light- induced retinal damage in Sprague Dawley rats

[0222] Following acclimatization, thirty male Sprague Dawley rats (aged 6-8 weeks) were randomly assigned to five experimental groups (n = 6) based on their body weights. The designated treatment groups were as follows: G1 - Vehicle Control; G2 - Blue Light (BL) Control; G3 - BL + PG-1 (20 mg / kg), G4 - BL + TE-4 (20 mg / kg) and G5 - BL + Comp-29 (20 mg / kg). Following randomization, all animals were administered their respective vehicle or test item orally (p.o.) via gavage once daily for 21 consecutive days. The blue light exposure regimen was conducted from days 8 to 21. During this period, all groups, excluding the vehicle control (Gl), were exposed to blue light. Specifically, one hour after the oral administration of the respective test items, animals were exposed to blue light at a wavelength of 460 nm and an intensity of 10,000 lux for one hour daily. To optimize ocular response to blue light, animals in groups G2 to G5 underwent a 24-hour dark adaptation period, preceding the initial (day 8) and following the final (day 21) blue light exposure sessions. This dark adaptation was performed to enhance ocular sensitivity to the subsequent light stimulus.

[0223] Histopathological Evaluation: Following blood collection, all animals were euthanized by CO2 asphyxiation, followed by exsanguination and necropsy. Eyes were collected and fixed in Davidson’s fixative for a minimum of 48 hours, then transferred to 10% neutral buffered formalin (NBF), dehydrated, and embedded in paraffin. Paraffin-embedded tissues were sectioned (3-6 pm thick) using a microtome (Leica Biosystems, Nussloch, Germany) and stained with hematoxylin and eosin (H&E). Microscopic evaluation was conducted using an Axio Al microscope (Carl Zeiss AG, Germany) at 20* magnification. The following histological parameters were assessed: thickness of the outer nuclear layer (ONL) and photoreceptor layer (PL).

[0224] Data Analysis: Data are presented as mean ± standard deviation (SD). Mean differences between the groups were assessed using one-way ANOVA, followed by suitable post hoc analyses to determine specific pairwise comparisons. Differences were considered statistically significant at p < 0.05. Significant differences were indicated by * (vs. Gl) or # (vs. G2). All analyses were conducted using GraphPad Prism version 5.01 (GraphPad Software, Inc., CA, USA). The results are summarized in Tables 18-19.

[0225] Table 18: In vivo efficacy of compositions in improving photoreceptor layer thickness in blue light-exposed Sprague Dawley rats

[0226] Values are presented as mean ± SD. One-way ANOVA followed by Dunnet’s test.

[0227] #p<0.05 vs. Gl- Vehicle control. *p<0.05 vs. G2- Blue light control.

[0228] Table 19: In vivo efficacy of compositions in improving ONL thickness in blue light-exposed Sprague Dawley rats

[0229] Values are presented as mean ± SD. One-way ANOVA followed by Dunnet’s test. #p<0.05 vs. Gl- Vehicle control. *p<0.05 vs. G2- Blue light control.

[0230] Although the invention has been described in detail in the foregoing for the purpose of illustration, it is to be understood that such detail is solely for that purpose and that variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention except as it may be limited by the claims.

Claims

We Claim,1. A synergistic herbal composition for eye health benefits comprising; a) Punica granatum fruit or fruit peel extract containing a zinc complex or a chelate of enriched punicalagins as a first ingredient, wherein the zinc complex or a chelate of punicalagins in the range of 20% to 70% by weight, and the zinc in the range of 0.05% to 2.0% by weight; and b) Tagetes erecta flower extract, standardized to contain one or more phytochemicals selected from the group consisting of lutein esters, zeaxanthin esters, lutein, zeaxanthin, and mixtures thereof as a second ingredient, wherein said phytochemicals are present in the range of 1% to 40% by weight.

2. The synergistic herbal composition as claimed in claim 1, wherein the eye health benefits include, but not limited to, preventing or alleviating symptoms of dry eye disease (DED), age-related macular degeneration (AMD), cataract, vision loss, glaucoma, and diabetic retinopathy; reducing the harmful effects of blue light exposure, including digital eye strain, visual fatigue, burning eyes, blurred vision, and headaches; and enhancing visual performance, including improvements in visual acuity, light adaptation, and dark adaptation.

3. The synergistic herbal composition as claimed in claim 1, wherein the concentration of the Punica granatum fruit or fruit peel extract containing zinc, varies in the range of 10%-90% by weight, and Tagetes erecta flower extract in the composition varies in the range of 90%-10% by weight.

4. The synergistic herbal compositions as claimed in claim 1, wherein the synergistic herbal composition optionally contains, atleast one componentselected from pharmaceutically nutraceutically, or dietically acceptable excipients, carriers, and diluents.

5. The synergistic herbal compositions as claimed in claim 4, wherein the excipients, carriers and diluents are selected from monosaccharides such as glucose, dextrose, fructose, galactose etc.; disaccharides such as but not limited to sucrose, maltose, lactose, lactulose, trehalose cellobiose, chitobiose etc.; polycarbohydrates such as starch and modified starch such as sodium starch glycolate, pre-gelatinized starch, soluble starch, and other modified starches; dextrins that are produced by hydrolysis of starch or glycogen such as yellow dextrin, white dextrin, maltodextrin etc.; polyhydric alcohols or sugar alcohols such as but not limited to sorbitol, mannitol, inositol, xylitol, isomalt etc.; cellulose based derivatives such as but not limited to microcrystalline cellulose, hydroxy propyl methyl cellulose, hydroxy ethyl cellulose etc.; silicates such as but not limited to neusilin, veegum, talc, colloidal silicon dioxide etc.; metallic stearates such as but not limited to calcium stearate, magnesium stearate, zinc stearate etc.; organic acids such as citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid etc.; fatty acid esters and esters of poly sorbate, natural gums such as but not limited to acacia, carrageenan, guar gum, xanthan gum etc.; vitamin B group, nicotinamide, calcium pantothenate, amino acids, proteins such as but not limited to casein, gelatin, pectin, agar; organic metal salts such as but not limited to sodium chloride, calcium chloride, dicalcium phosphate, zinc sulphate, zinc chloride, zinc gluconate, etc.; natural pigments, flavors, class I & class II preservatives and aqueous, alcoholic, hydro-alcoholic, organic solutions of above listed ingredients alone or in combination.

6. The synergistic herbal composition as claimed in claim 4, wherein the composition is formulated into a dosage form selected from dry powder form, liquid form, beverage, food product, dietary supplement, or anysuitable form such as a tablet, a capsule, a soft chewable tablet, gummies, or gummy bar.

7. The synergistic herbal composition as claimed in claim 4, wherein the composition is formulated into a nutritional / dietary supplement that can be contemplated / made into the dosage form of healthy foods or food for specified health uses, such as solid food like chocolate or nutritional bars, semisolid food like cream, jam, or gel or beverage such as refreshing beverage, lactic acid bacteria beverage, drop, candy, chewing gum, gummy candy, yogurt, ice cream, pudding, soft adzuki bean jelly, jelly, cookie, tea, soft drink, juice, milk, coffee, cereal, snack bar.

8. The synergistic herbal composition as claimed in claim 4, wherein the composition is formulated into a controlled-release tablet, using controlled-release polymer-based coatings by techniques including nanotechnology, microencapsulation, colloidal carrier systems, and other drug delivery systems for obtaining the desired therapeutic benefit.

9. A process for preparing synergistic compositions comprising; (i) a zinc complex or chelate of enriched punicalagins in the range of 20-70% derived from Punica granatum fruit or fruit peel, wherein, zinc is present in a concentration of 0.05-2.0%; and (ii) Tagetes erecta flower extract standardized to contain lutein or zeaxanthin or its esters in the range of 1- 40%; optionally at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents; wherein the process comprises the following steps of;(i) Extraction: Extracting dried Punica granatum fruit or fruit peel with a suitable solvent selected from C1-C5 alcohols, including but not limited to ethanol, methanol, n-butanol, isopropyl alcohol; ketones such as acetone, methyl isobutyl ketone, chlorinated solvents such as methylene dichloride and chloroform; C1-C7hydrocarbons such as but not limited to hexane, heptane; esters such as but not limited to ethyl acetate and mixtures thereof; water and mixtures thereof;(ii) Zinc complexation: Concentrating the extract via solvent evaporation and treating the concentrated solution with a zinc compound selected from zinc oxide, zinc hydroxide, or zinc carbonate to form a zinc-punicalagin complex or chelate;(iii) Column purification: Filtering the mixture and subjecting it to column chromatography using a resin selected from PA-800, SP- 700, or HP -20. and aqueous ethanol as an eluent;(iv) Drying of zinc chelate: Concentrating and drying the eluted fraction preferably under reduced pressure, to yield the enriched zinc complex or chelate of punicalagins;(v) Blending with second active: Blending the said enriched zinc- punicalagin complex with a Tagetes erecta flower extract standardized to contain lutein, zeaxanthin, or their estersand optionally with the addition of at least one acceptable carrier, excipient, or diluent to obtain final formulation with enhanced stability.(vi) Final drying: Subjecting the final composition to drying under vacuum conditions to obtain the finished synergistic composition in powder or granulate form, suitable for further formulation.

10. The synergistic composition as claimed in claim 9, wherein the solvent used for the preparation of extracts or fractions or mixtures thereof; is selected from C1-C5 alcohols such as ethanol, methanol, n-propanol, isopropyl alcohol; ketones such as acetone, methyl isobutyl ketone, chlorinated solvents such as methylene di chloride and chloroform; C1-C7 hydrocarbons such as but not limited to hexane, heptane; esters such as but not limited to ethyl acetate and mixtures thereof; water and mixtures thereof.

11. A method of obtaining at least one eye health benefit selected from group comprising of preventing or alleviating symptoms of dry eye disease (DED), age-related macular degeneration (AMD), cataract, vision loss, glaucoma, and diabetic retinopathy; reducing the harmful effects of blue light exposure, including digital eye strain, visual fatigue, burning eyes, blurred vision, and headaches; and enhancing visual performance, including improvements in visual acuity, light adaptation, and dark adaptation in a human; wherein the method comprises supplementing the human in need thereof with an effective dose of a composition comprising Punica granatum fruit or fruit peel extract containing a zinc complex or a chelate of enriched punicalagins, wherein said punicalagins are in the range of 20% to 70% by weight as a first ingredient, and wherein zinc is present in the range of 0.05% to 2.0% by weight; and an extract of Tagetes erecta flowers, standardized to contain one or more phytochemicals selected from the group consisting of lutein esters, zeaxanthin esters, lutein, zeaxanthin, and mixtures thereof as a second ingredient, wherein said phytochemicals are present in the range of 1% to 40% optionally further containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers, and diluents.

12. A method of obtaining at least one eye health benefit selected from group comprising of preventing or alleviating symptoms of dry eye disease (DED), age-related macular degeneration (AMD), cataract, vision loss, glaucoma, and diabetic retinopathy; reducing the harmful effects of blue light exposure, including digital eye strain, visual fatigue, burning eyes, blurred vision, and headaches; and enhancing visual performance, including improvements in visual acuity, light adaptation, and dark adaptation in a human; wherein the method comprises supplementing the human in need thereof with an effective dose of a composition comprising a combination of a first ingredient selected from an enriched punicalagins, wherein the said punicalagins are in the range of 20-70% derived fromPunica granatum fruit or fruit peel,; and a second ingredient selected from Tagetes erecta flower extract standardized to contain lutein or zeaxanthin or its esters in the range of 1-40%; optionally further containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers, and diluents.

13. Use of a synergistic herbal composition comprising the combination of a first ingredient selected from a zinc complex or chelate of enriched punicalagins, wherein the said punicalagins are in the range of 20-70% derived from Punica granatum fruit or fruit peel, wherein, zinc is present in a concentration of 0.05-2.0%; and a second ingredient selected from Tagetes erecta flower extract standardized to contain lutein or zeaxanthin or its esters in the range of 1-40%; optionally further containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers, and diluents; for improving at least one eye health benefit selected from but not limited to preventing or alleviating symptoms of dry eye disease (DED), age-related macular degeneration (AMD), cataract, vision loss, glaucoma, and diabetic retinopathy; reducing the harmful effects of blue light exposure, including digital eye strain, visual fatigue, burning eyes, blurred vision, and headaches; and enhancing visual performance, including improvements in visual acuity, light adaptation, and dark adaptation.

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