Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage and application thereof

Abstract

The present invention discloses an Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage, and its application. This composition belongs to the field of functional food technology. The composition comprises, by weight, 0.1 to 500 parts of Antarctic krill oil, 0.1 to 25 parts of lutein ester, and 0.1 to 5 parts of zeaxanthin. The Antarctic krill oil has a phospholipid content of 38% or greater and an ω-3 polyunsaturated fatty acid content of 9% or greater. The composition, made from readily available raw materials and highly safe, can promote retinal cell proliferation, improve the inhibitory effect of H2O2 on retinal cells, alleviate H2O2-induced hyperinflammation and oxidative stress, and mitigate H2O2-induced damage to retinal cells. The composition has broad application prospects in preparing products for optic nerve protection.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional foods, and specifically relates to an Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage and an application thereof. Background Art

[0002] The frequent use of electronic devices has led to increased attention to visual health, and demand for functional eye-protecting foods is growing. The main consumer groups for these products include students, office workers, and the elderly. Students spend long hours studying and using their eyes, so their eye protection needs focus on relieving visual fatigue and preventing myopia. Office workers, who spend long periods in front of electronic screens, have a strong need to improve dry eyes and reduce eye pressure. The elderly are more concerned about preventing degenerative eye diseases, such as macular degeneration. Traditional functional eye-protecting foods, such as lutein esters and zeaxanthin supplements, cannot meet the diverse eye protection needs of different age groups, necessitating the development of functional eye-protecting products towards precision and personalization.

[0003] Antarctic krill, a member of the genus Euphrasia, belongs to the family Euphrasiatiidae, order Euphrasiatiformes. They are rich in protein, a diverse range of amino acids, and contain numerous minerals and trace elements, including calcium, iron, zinc, potassium, selenium, and magnesium. Antarctic krill oil, a natural oil extracted from Antarctic krill, is rich in various nutrients, low in triglycerides and high in polar lipids. Its strong antioxidant properties and multifaceted health benefits have garnered significant attention in recent years for its unique nutritional value and health benefits.

[0004] A variety of functional eye-protecting foods have also been reported in the prior art. For example, Chinese patent document CN103316032A discloses a composition containing hydroxytyrosol and its application. The composition comprises hydroxytyrosol and hyaluronic acid, lutein, proanthocyanidins, and taurine in a certain proportion as active ingredients, which are then prepared into an oral preparation with an appropriate amount of commonly used excipients. The composition has the effect of improving eyesight and improving eye and visual function. Chinese patent document CN106491947A discloses a composition and preparation for relieving visual fatigue. The invention quantitatively combines traditional Chinese medicines such as bilberry powder, marigold powder, chrysanthemum powder, glossy privet fruit powder, wolfberry powder, mulberry leaf powder, cassia seed powder, qingxiezi powder, cicada slough powder, cornus officinalis powder, cistanche powder, asparagus powder, ophiopogon powder, bitter almond powder, and fructus aurantii powder to produce a significant synergistic effect between the active ingredients of the raw materials. The composition can effectively scavenge free radical damage to eye tissue, protect eye microvessels, improve fundus microcirculation, and increase ciliary muscle blood flow, thereby eliminating symptoms of visual fatigue and improving vision. Chinese patent document with publication number CN117298142A discloses an anti-asthenopia composition, its preparation method and application. The composition includes the following active ingredients: wolfberry polysaccharide, catechin, myricetin, luteolin, chlorogenic acid, ophiopogon polysaccharide, ophiopogon saponin D, ophiopogon methylflavanone A, paeoniflorin, methyl gallate, paeoniflorin and zinc gluconate. It can effectively relieve symptoms such as dry eyes, astringent eyes, and swollen eyes caused by asthenopia, and can also resist damage from blue light exposure, relieve visual fatigue and protect eyesight.

[0005] However, the components of the above-mentioned eye-protecting functional foods are complex and difficult to adapt to the needs of large-scale production. Summary of the Invention

[0006] The present invention provides an Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage. The raw materials of the composition are easy to obtain and are highly safe. The composition can be consumed for a long time as a common food, health product or medicine.

[0007] The specific technical solutions adopted are as follows:

[0008] An Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage, comprising, by weight, 0.1 to 500 parts of Antarctic krill oil, 0.1 to 25 parts of lutein ester, and 0.1 to 5 parts of zeaxanthin;

[0009] The phospholipid content in the Antarctic krill oil is ≥38 wt% (in the prior art, the maximum phospholipid content in Antarctic krill oil is 73.2 wt%), and the ω-3 polyunsaturated fatty acid content is ≥9 wt% (in the prior art, the maximum ω-3 polyunsaturated fatty acid content in Antarctic krill oil is 27.4 wt%).

[0010] Preferably, the Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage comprises, by weight, 100 to 400 parts by weight of Antarctic krill oil, 1 to 20 parts by weight of lutein esters, and 1 to 4 parts by weight of zeaxanthin; the phospholipid content in the Antarctic krill oil is ≥40 wt%, and the ω-3 polyunsaturated fatty acid content is ≥18 wt%.

[0011] Further preferably, the Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage comprises, in parts by weight, 200 parts by weight of Antarctic krill oil, 10 parts by weight of lutein esters and 2 parts by weight of zeaxanthin.

[0012] The preparation method of Antarctic krill oil includes:

[0013] S1 uses Antarctic krill powder as raw material, uses ethanol to perform countercurrent extraction to retain the active ingredients, and then centrifuges to remove solid residues to obtain crude oil;

[0014] S2 concentrates and filters the crude oil;

[0015] S3 further distills the crude oil treated in step S2 to ensure that the ω-3 polyunsaturated fatty acid and phospholipid contents meet the requirements. The obtained Antarctic krill oil is placed in a nitrogen-filled, sealed container, and refrigerated away from light for later use.

[0016] Sources of lutein esters include but are not limited to marigold flowers, spinach, kale, egg yolk, pumpkin, etc.; sources of zeaxanthin include but are not limited to marigold flowers, corn, broccoli, orange, mango, wolfberry, etc.

[0017] Furthermore, promoting retinal cell growth refers to promoting retinal cell proliferation, and improving retinal cell oxidative damage refers to at least one of the following three ways:

[0018] (1) Improve the inhibitory effect of H2O2 on retinal cells (specifically, alleviate the death of retinal cells caused by H2O2);

[0019] (2) Alleviate the high inflammation and high oxidative stress state induced by H2O2, and reduce the expression of inflammation-related genes and oxidative stress-related genes;

[0020] (3) Alleviate the damage caused by H2O2 to retinal cells and increase the expression of microtubule-associated protein-2 (MAP-2) and axon length in retinal cells.

[0021] The ingredients in Antarctic krill oil include ω-3 polyunsaturated fatty acids, phospholipids, astaxanthin, etc. The inventors found in their research that the combination of lutein esters, zeaxanthin and Antarctic krill oil has a synergistic effect in protecting the eyes. The synergistic protective effect of the three ingredients on retinal cells is mainly reflected in: ① Antioxidation and blue light filtering synergistically enhance the effect. Lutein esters (absorb short-wave blue light) and zeaxanthin (absorb long-wave blue light) form a macular pigment protection layer, and astaxanthin in Antarctic krill oil scavenges free radicals. The three build a multi-level antioxidant network, reducing the risk of age-related macular degeneration (AMD); ② The phospholipids in Antarctic krill oil Improve the bioavailability of lutein esters. DHA and other ω-3 polyunsaturated fatty acids promote pigment deposition in the macular area. Astaxanthin improves microcirculation and enhances nutrient delivery. ③ The ω-3 polyunsaturated fatty acids, lutein esters, and zeaxanthin in krill oil protect retinal cells from both antioxidant and anti-inflammatory perspectives by inhibiting inflammatory factors and the NF-κB pathway, respectively. ④ DHA and other ω-3 polyunsaturated fatty acids in Antarctic krill oil maintain the integrity of retinal cell membranes. Astaxanthin enhances mitochondrial function. Lutein esters and zeaxanthin improve visual function.

[0022] The present invention also provides the use of the Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage in the preparation of products for optic nerve protection.

[0023] The present invention also provides a product for protecting optic nerves, the components of which include the Antarctic krill oil composition that promotes retinal cell growth and improves retinal cell oxidative damage.

[0024] Furthermore, the product components for optic nerve protection also include pharmaceutically acceptable excipients or food-acceptable additives.

[0025] Specifically, the product forms for optic nerve protection include but are not limited to powders, tablets, capsules, granules or drinks.

[0026] Further preferably, the product for optic nerve protection is in the form of soft capsules or gel candies, and the addition amount of the Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage is 20.0-60.0wt%.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention obtains an Antarctic krill oil composition capable of promoting retinal cell growth and improving oxidative damage to retinal cells by compounding Antarctic krill oil, lutein esters and zeaxanthin in specific proportions. The raw materials of the composition are easily available and highly safe. The composition can promote retinal cell proliferation and improve oxidative damage to retinal cells (improve the inhibitory effect of H2O2 on retinal cells; alleviate the high inflammation and high oxidative stress state induced by H2O2, reduce the expression of inflammation-related genes and oxidative stress-related genes; alleviate the damage to retinal cells caused by H2O2, and increase the expression of microtubule-associated protein-2 and axon length in retinal cells). The composition can be made into ordinary food, health products or medicines for long-term consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The cell proliferation rate statistics after treatment with the three-component composition at different concentrations, where * indicates that the three-component composition treatment groups at different concentrations are significantly different from the control group. P <0.05, ** indicates P <0.01.

[0030] Figure 2 The effect of the three-component composition and the two-component composition at corresponding concentrations on the proliferation of 661W retinal cells is shown in FIG. , wherein the three-component composition or two-component composition treatment group is compared with the control group, and ** indicates P <0.01.

[0031] Figure 3 This is a graph showing the effects of the three-component composition, the two-component composition at corresponding concentrations, and Antarctic krill oil on 661W retinal cell apoptosis. The three-component composition treatment group was compared with the two-component composition / Antarctic krill oil treatment group. ** indicates P <0.01, the three-component composition compared with the H2O2 treatment group, ## indicates P <0.01.

[0032] Figure 4 The effects of the three-component composition, the two-component composition at corresponding concentrations, and Antarctic krill oil on the expression of inflammation-related genes in 661W retinal cells ( Tnf-α, Il-1β ) and oxidative stress-related genes ( Cat, Sod ) mRNA expression levels, where A represents the expression level of inflammation-related genes and B represents the expression level of oxidative stress-related genes. ** indicates that the three-ingredient composition treatment group and the two-ingredient composition / Antarctic krill oil treatment group P <0.01, the three-component composition compared with the H2O2 treatment group, ## indicates P <0.01.

[0033] Figure 5The effects of the three-ingredient combination, the two-ingredient combination at corresponding concentrations, and Antarctic krill oil on MAP-2 in 661W retinal cells. A is a fluorescence imaging image, B is a quantitative image of 661W MAP-2 fluorescence intensity, and C is a quantitative image of synaptic length in 661W retinal cells. Compared with the two-ingredient combination / Antarctic krill oil treatment group, ** indicates P <0.01, the three-component composition compared with the H2O2 treatment group, ## indicates P <0.01. DETAILED DESCRIPTION

[0034] The present invention will be further illustrated below in conjunction with the examples and accompanying drawings. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The operating methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or under the conditions recommended by the manufacturer. Contents not described in detail in this specification sheet belong to the prior art known to those skilled in the art. The experimental materials used in the examples, unless otherwise specified, can be purchased from conventional biochemical reagent companies.

[0035] The Antarctic krill oil used in the examples was prepared by the following method:

[0036] (1) Material preparation and feeding: weigh the thawed Antarctic krill meal and put it into the hopper;

[0037] (2) Countercurrent extraction: Use ethanol to perform countercurrent extraction on the raw materials to retain the active ingredients;

[0038] (3) Preliminary separation: centrifugation to remove solid residue and obtain crude oil;

[0039] (4) Concentration and filtration: Use scraper concentration and spherical concentration in sequence to gradually evaporate the ethanol until the water and volatile matter are less than 2%, and filter the liquid through filter paper;

[0040] (5) Distillation: Adjust the contents of ω-3 polyunsaturated fatty acids and phospholipids by low-temperature molecular distillation to meet the krill oil industry standard SC / T 3506-2020, and obtain Antarctic krill oil (the phospholipid content in Antarctic krill oil is 40 wt%~62 wt%, and the ω-3 polyunsaturated fatty acid content is 18 wt%~24 wt%);

[0041] (6) Finished product filling: The Antarctic krill oil is placed in barrels, sealed with nitrogen, and stored in a refrigerated place away from light.

[0042] In addition, the Antarctic krill oil microcapsule powder used in the examples can be prepared by the following method:

[0043] (1) Material preparation and weighing: Accurately weigh solid corn syrup, Antarctic krill oil and other processing aids according to a certain ratio;

[0044] (2) Dissolution and emulsification: Add the weighed raw materials and auxiliary materials into the mixing tank and fully dissolve and emulsify them, and mix them evenly;

[0045] (3) Spray drying: spray drying the above liquid;

[0046] (4) Screening and packaging: The coarse powder obtained by spray drying is screened, and an appropriate amount of silicon dioxide is added to the sieved fine powder and mixed evenly. Finally, the obtained Antarctic krill oil microcapsule powder is packed into aluminum foil bags and stored in a refrigerator away from light.

[0047] Example 1 (soft capsule)

[0048] Weigh 200 parts by weight of Antarctic krill oil, 10 parts by weight of lutein esters, 2 parts by weight of zeaxanthin, and flaxseed oil (an appropriate amount, primarily for filling and dispersing purposes; soybean oil, sunflower oil, MCT oil, etc. can also be substituted; the specific amount added is customized based on market demand) and mix them uniformly in proportion to obtain a mixture containing 35.3% by weight of the Antarctic krill oil composition (the specific amount added can be selected from a range of 20.0-60.0% by weight). The capsule is prepared from gelatin, glycerin, and water in a ratio of 1:0.4:1, and the soft capsules are pelletized and dried.

[0049] Example 2 (jelly candy)

[0050] 200 parts by weight of Antarctic krill oil, 10 parts by weight of lutein ester, 2 parts by weight of zeaxanthin, flaxseed oil (an appropriate amount, primarily for filling and dispersing purposes, which can also be substituted with soybean oil, sunflower oil, MCT oil, etc., and the specific amount added can be customized according to market demand), blueberry powder (an appropriate amount, the specific amount added can be customized according to market demand), and glyceryl monostearate (an appropriate amount, the specific amount added can be customized according to market demand) are uniformly mixed in proportion to obtain a filling. The content of the Antarctic krill oil composition in the filling is 35.3 wt% (the specific amount added can be selected from a range of 20.0-60.0 wt%). Separately, gelatin, glycerin, and xylitol solution are mixed in a ratio of 1:0.4:1 to prepare a glue solution, which is then filled using a molding machine to obtain a gel candy.

[0051] Example 3 (Tablets)

[0052] 418 mg of microcrystalline cellulose, 1000 mg of Antarctic krill oil microcapsule powder (containing 200 mg of Antarctic krill oil), 100 mg of hydroxypropyl methylcellulose, 50 mg of cross-linked carboxymethyl cellulose sodium, 10 mg of lutein ester, 2 mg of zeaxanthin, 10 mg of silicon dioxide, 5 mg of magnesium stearate, 3 mg of steviol glycosides, and 2 mg of strawberry flavor were weighed separately. The above raw materials were dry-mixed and directly tableted to obtain lutein ester Antarctic krill oil microcapsule powder chewable tablets.

[0053] Example 4 (Powder)

[0054] Calculated by weight, every 20g of the modulated milk powder includes 8.7g of whole milk powder, 7.85g of skim milk powder, 1.52g of galacto-oligosaccharide, 0.80g of lactose, 78mg of vitamin premix, 40mg of mineral premix, 1g of Antarctic krill oil microcapsule powder (containing 200mg of Antarctic krill oil), 10mg of lutein ester, and 2mg of zeaxanthin. The above raw and auxiliary materials are batched, dry-mixed, and nitrogen-filled and packaged to obtain the lutein ester Antarctic krill oil modulated milk powder.

[0055] Sample analysis

[0056] 1 Materials and Methods

[0057] 1.1 Cell culture

[0058] Experiments were conducted using the 661W retinal cell line (Fenghui Biotechnology Co., Ltd.) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% newborn calf serum, 100 IU / mL penicillin, and 100 µg / mL streptomycin. The cells were cultured at 37°C, 5% CO2, and saturated humidity, with routine medium changes and passages. When the cell density reached 90%, the medium was discarded, and the cells were washed twice with phosphate-buffered saline. The cells were trypsinized with 0.25% EDTA, plated at an adjusted cell density, and cultured until they were stably attached.

[0059] 1.2 Cell proliferation assay

[0060] 1.2.1 Effects of three-component compositions of different formulations on the proliferation of 661W retinal cells

[0061] This experiment designed four three-component compositions (hereinafter referred to as three components) with different addition amounts of Antarctic krill oil. The specific formulas are as follows.

[0062] Formula 1: 50 mg Antarctic krill oil + 10 mg lutein ester + 2 mg zeaxanthin;

[0063] Formula 2: 100 mg Antarctic krill oil + 10 mg lutein ester + 2 mg zeaxanthin;

[0064] Formula 3: 200 mg Antarctic krill oil + 10 mg lutein ester + 2 mg zeaxanthin;

[0065] Formula 4: 400 mg Antarctic krill oil + 10 mg lutein ester + 2 mg zeaxanthin.

[0066] After treating 661W retinal cells with the three-component combination for 48 hours, cell viability and proliferation were assessed using an MTT assay to determine the appropriate dosage of Antarctic krill oil and the effective concentration range of the three-component combination for promoting 661W retinal cell proliferation. The cell proliferation assay was performed as follows: After 48 hours of culture, cells were rinsed once with PBS, and 20 µL of MTT (5 g / L) and 200 µL of serum-free DMEM medium were added to each well. After incubation at 37°C for 4 hours, the supernatant was discarded, and the cells were rinsed twice with PBS. The resulting crystals were dissolved by adding 200 µL of dimethyl sulfoxide to each well. The cells were shaken at room temperature for 15 minutes, and the optical density (OD) was measured at 490 nm on a spectrophotometer.

[0067] 1.2.2 Effects of the three-component composition and its corresponding two-component composition on the proliferation of 661W retinal cells

[0068] Based on the experimental results in 1.2.1, an appropriate formulation was selected for subsequent experiments. Using the lowest effective concentration of the three-component combination as the starting point, a control group (containing 0.1% DMSO), a lutein ester + zeaxanthin group, an Antarctic krill oil group, and a lutein ester + zeaxanthin + Antarctic krill oil group were set up. 661W retinal cells were treated with the three-component combination and corresponding concentrations of the two-component combination for 48 hours. After 48 hours, cell viability and proliferation were assessed using an MTT assay to determine whether the three-component combination was more effective than the two-component combination in promoting 661W retinal cell proliferation. The cell proliferation assay was performed in the same manner as in step 1.2.1.

[0069] 1.3 Cell apoptosis assay

[0070] 661W retinal cells were plated at 5 × 10 3661W retinal cells were seeded at a density of 100 cells / well in a 96-well plate. The H2O2, lutein ester + zeaxanthin, Antarctic krill oil, and Antarctic krill oil + lutein ester + zeaxanthin groups were set up. When the cell density reached 30%, 661W retinal cells were pretreated with 400 µM H2O2 for 12 hours. The three-component combination and its corresponding concentrations of the two-component combination were then treated for 48 hours. After 48 hours, cell viability and proliferation were assessed using an MTT assay to determine whether the three-component combination had a superior anti-apoptotic effect on 661W retinal cells compared to the two-component combination. The cell-based experimental procedures were the same as in step 1.2.1.

[0071] 1.4 Real-time fluorescence quantitative polymerase chain reaction experiment

[0072] 661W retinal cells were plated at 5 × 10 3 661W retinal cells were seeded at a density of 100 μl in 12-well plates and treated with H2O2, lutein esters + zeaxanthin, Antarctic krill oil, and Antarctic krill oil + lutein esters + zeaxanthin. When the cell density reached 30%, they were pretreated with 400 μM H2O2 for 12 hours. The cells were then treated with the three-component combination and the corresponding concentrations of the two-component combination for 48 hours. After 48 hours, the culture medium was discarded, the cells were rinsed twice with PBS, and total RNA was extracted using Trizol reagent (Novagen, Nanjing). RNA concentration was measured using a Nano-300 spectrophotometer (Aosheng, Hangzhou), and cDNA was synthesized using a cDNA reverse transcriptase kit (Novagen, Nanjing). Quantitative real-time PCR was performed using the CFX Connect real-time PCR system (Bio-rad, USA) using SYBR Green real-time PCR Master Mix (Novagen, Nanjing).

[0073] β-Actin transcripts were used as housekeeping genes for data normalization. The gene primer sequences are shown in the following table:

[0074] Table 1 RT-qPCR primer sequences

[0075]

[0076] 1.5 Cell immunofluorescence staining experiment

[0077] 661W retinal cells were plated at 5 × 10 3661W retinal cells were plated at varying densities in 24-well plates containing cell slides (10 × 10 mm) and treated with H2O2, lutein esters + zeaxanthin, Antarctic krill oil, and lutein esters + zeaxanthin + Antarctic krill oil. When the cell density reached 30%, the cells were pretreated with 400 µM H2O2 for 12 hours. The cells were then treated with the three-component combination and the corresponding concentrations of the two-component combination for 48 hours. After 48 hours, the culture medium was discarded, the cells were rinsed twice with PBS, and fixed with 4% paraformaldehyde (PFA) for 15 minutes. Excess paraformaldehyde was washed with PBS, and the membrane was permeabilized by adding 0.1% TrionX-100 in PBS for 15 minutes. After washing with PBS, the cells were blocked for 1 hour at room temperature. The blocking solution was removed, and the primary antibody MAP-2 (ET1602-12, Huaan, Hangzhou, China) was added and incubated overnight at 4°C. After incubation with the primary antibody, the sections were washed with PBS and blocked with a secondary antibody (R37116, Thermo Fisher Scientific, Massachusetts, USA) for 1 h at room temperature. Finally, the secondary antibody was removed, and DAPI (P36981, Thermo Fisher Scientific, Massachusetts, USA) was added for mounting. The stained cell sections were then photographed using a fluorescence microscope.

[0078] 1.6 Statistical analysis

[0079] All experimental data were expressed as “mean ± standard error”, and statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test. P When <0.05, it indicates a significant difference; P When the value was <0.01, the difference was considered extremely significant.

[0080] 2 Experimental Results

[0081] 2.1 Effects of the four formulations on 661W retinal cell proliferation

[0082] To determine the proliferative effect of Antarctic krill oil combined with lutein esters and zeaxanthin on 661W retinal cells, four three-component compositions with different Antarctic krill oil addition levels were designed and the cells were treated for 48 hours for experiments. The results showed that formula 1 significantly promoted the proliferation of 661W retinal cells at a concentration of 200 μg / mL, and the proliferation effect increased with increasing concentration. When the concentration reached 400 μg / mL, the cell proliferation rate reached 105.8% ( Figure 1Meanwhile, Formulas 2, 3, and 4 all significantly promoted the proliferation of 661W retinal cells at a concentration of 100 µg / mL, with cell proliferation rates of 108.8%, 112.6%, and 113.5%, respectively. This concentration-dependent effect was observed, with cell proliferation rates reaching 109.9%, 127.4%, and 130.5%, respectively, at 400 µg / mL. However, despite containing twice the amount of Antarctic krill oil as Formula 3, Formula 4 exhibited a relatively weaker increase in 661W retinal cell proliferation. Therefore, subsequent experiments were conducted using Formula 3 at its lowest effective concentration (100 µg / mL).

[0083] 2.2 Effects of Formula 3, the two-component combination at corresponding concentrations, and Antarctic krill oil on 661W retinal cell proliferation

[0084] The results showed that the combination of lutein esters and zeaxanthin, as well as Antarctic krill oil, alone failed to significantly promote the proliferation of 661W retinal cells. However, when Antarctic krill oil, lutein esters, and zeaxanthin were used together, the three-ingredient combination significantly promoted the proliferation of 661W retinal cells, with a proliferation rate of over 110%. This suggests that there may be an interaction between the different components, which together promotes cell proliferation. Figure 2 ).

[0085] 2.3 Effects of Formula 3 and its two-component combination at corresponding concentrations and Antarctic krill oil on apoptosis in 661W retinal cells

[0086] The results of the cell apoptosis experiment showed that H2O2 treatment significantly inhibited the proliferation of 661W retinal cells and increased the mortality rate of 661W retinal cells. Neither the two-component combination of lutein esters and zeaxanthin nor Antarctic krill oil could reverse this effect. However, the three-component combination with Antarctic krill oil significantly ameliorated the inhibitory effect caused by H2O2 ( Figure 3 ), alleviating H2O2-induced retinal cell death. In summary, Antarctic krill oil is a key component in promoting the proliferation of 661W retinal cells, and it works synergistically with lutein esters and zeaxanthin to further enhance cell proliferation.

[0087] 2.4 Effects of Formula 3, its corresponding concentrations of two-component combination, and Antarctic krill oil on inflammation and oxidative damage in 661W retinal cells

[0088] After H2O2 treatment, inflammation-related genes ( Tnf-α, Il-1β ) and oxidative stress-related genes ( Cat, Sod ) mRNA expression levels were significantly increased compared to the control group, indicating that the cells were damaged. Treatment with only the two-component combination (lutein esters and zeaxanthin) or Antarctic krill oil alone for 48 h did not effectively reduce the expression of inflammation-related genes ( Tnf-α, Il-1β ) and oxidative stress-related genes ( Cat, Sod ) expression, but failed to improve the inflammatory and oxidative stress status of cells. However, after adding Antarctic krill oil, the three-ingredient combination significantly alleviated the high inflammation and high oxidative stress status induced by H2O2. ( Figure 4 A and B in ).

[0089] 2.5 Immunofluorescence experiment comparing the effects of Formula 3, its corresponding concentration of two-component combination, and Antarctic krill oil on the growth and morphology of 661W retinal cells

[0090] Figure 5 Middle A shows that after H2O2 treatment, the MAP-2 fluorescence intensity of 661W retinal cells decreased significantly, and the cell axons shortened, indicating that the neural morphology and structural integrity of the cells were damaged. The two-component combination of lutein ester and zeaxanthin and Antarctic krill oil alone failed to reverse this damage. However, when lutein ester, zeaxanthin and Antarctic krill oil were combined, the MAP-2 fluorescence intensity and axon length of 661W retinal cells were significantly improved, and the effect of the three-component combination was significantly better than that of the two-component combination ( Figure 5 This suggests that Antarctic krill oil plays a key role in protecting cell structure and neural morphology, and the synergistic effect of the three-ingredient combination may have a stronger protective effect on repairing H2O2-induced cell damage.

[0091] 3 Experimental Conclusions

[0092] This study investigated the effects of Antarctic krill oil, lutein esters, and zeaxanthin on the proliferation of 661W retinal cells and their protection against H2O2-induced cell damage. The results demonstrated that the three-ingredient combination of Antarctic krill oil, lutein esters, and zeaxanthin played a key role in promoting 661W retinal cell proliferation and protecting cell structure.

[0093] First, the combination of Antarctic krill oil with lutein esters and zeaxanthin can significantly promote the proliferation of 661W retinal cells, and the pro-proliferation effect increases with the increase of Antarctic krill oil content in the formula. In addition, in terms of cell protection, H2O2 treatment significantly inhibited the proliferation of 661W retinal cells and induced inflammatory response and oxidative stress. The two-component combination of lutein esters and zeaxanthin and Antarctic krill oil treatment alone failed to effectively reverse these damages, but when treated with the three-component combination, it significantly improved the cell proliferation ability and effectively improved cell damage. The results of the MAP-2 immunofluorescence staining experiment showed that the three-component combination containing Antarctic krill oil significantly improved the phenomenon of reduced MAP-2 protein expression and axonal shortening caused by H2O2 treatment, further supporting the important role of Antarctic krill oil in protecting the structure and neural morphology of 661W retinal cells.

[0094] In summary, Antarctic krill oil, in combination with lutein esters and zeaxanthin, exhibited significant synergistic effects in promoting 661W retinal cell growth and protecting cells from H2O2-induced damage. The significant effects of the three-ingredient combination suggest that synergy is crucial for alleviating cell damage. This finding provides important scientific evidence for the role of the Antarctic krill oil-lutein ester-zeaxanthin combination in promoting neurodevelopment and protecting retinal cell morphology, and lays the foundation for subsequent clinical applications and mechanistic research.

[0095] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage, characterized in that: In parts by weight, it consists of 100-400 parts by weight of Antarctic krill oil, 1-20 parts by weight of lutein ester and 1-4 parts by weight of zeaxanthin; The preparation method of Antarctic krill oil includes: S1 uses Antarctic krill powder as raw material, uses ethanol to perform countercurrent extraction to retain the active ingredients, and then centrifuges to remove solid residues to obtain crude oil; S2 concentrates and filters the crude oil; S3 further distilling the crude oil treated in step S2 to ensure that the ω-3 polyunsaturated fatty acid and phospholipid contents meet the requirements, thereby obtaining Antarctic krill oil; The Antarctic krill oil has a phospholipid content of ≥38 wt%, and an ω-3 polyunsaturated fatty acid content of ≥9 wt%. Promoting retinal cell growth refers to promoting retinal cell proliferation; Improving oxidative damage to retinal cells means at least one of the following three ways: (1) Improve the inhibitory effect of H2O2 on retinal cells; (2) Alleviate the high inflammation and high oxidative stress state induced by H2O2, and reduce the expression of inflammation-related genes and oxidative stress-related genes; (3) Alleviate the damage caused by H2O2 to retinal cells and increase the expression of microtubule-associated protein-2 and axon length in retinal cells.

2. The Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage according to claim 1, characterized in that: The Antarctic krill oil has a phospholipid content of ≥40 wt% and an ω-3 polyunsaturated fatty acid content of ≥18 wt%.

3. The Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage according to claim 1, characterized in that: In parts by weight, it consists of 200 parts by weight of Antarctic krill oil, 10 parts by weight of lutein ester and 2 parts by weight of zeaxanthin.

4. Use of the Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage according to any one of claims 1 to 3 in the preparation of a product for optic nerve protection.

5. A product for optic nerve protection, characterized in that: The components include the Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage according to any one of claims 1 to 3.

6. The product for optic nerve protection according to claim 5, characterized in that: The components also include pharmaceutically acceptable excipients or food-acceptable additives.

7. The product for optic nerve protection according to claim 5, characterized in that: Product forms include powder, tablets, capsules or granules.

8. The product for optic nerve protection according to claim 5, characterized in that The product is in the form of soft capsules or gel candies, and the addition amount of the Antarctic krill oil composition for promoting retinal cell growth and improving retinal cell oxidative damage is 20.0-60.0 wt%.

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