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

Abstract

The invention discloses an euphausia superba oil composition for promoting growth of retinal cells and improving oxidative damage of the retinal cells and application of the euphausia superba oil composition, and belongs to the technical field of functional foods.The euphausia superba oil composition is prepared from, by weight, 0.1-500 parts of euphausia superba oil, 0.1-25 parts of lutein ester and 0.1-5 parts of zeaxanthin; the content of phospholipid in the euphausia superba oil is greater than or equal to 38 wt%, and the content of omega-3 polyunsaturated fatty acid is greater than or equal to 9 wt%. The raw materials of the composition are easy to obtain, the safety is high, retinal cell proliferation can be promoted, the inhibition effect of H2O2 on retinal cells is improved, H2O2-induced high inflammation and high oxidative stress states are relieved, damage of H2O2 to retinal cells is relieved, and the composition has wide application prospects in the aspect of preparing products for optic nerve protection.

Description

Technical Field

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

[0002] The frequent use of electronic devices has led to an increase in the attention to visual health, and the demand for eye-care functional foods is continuously increasing. The main consumer groups of related eye-care functional products include students, office workers, and middle-aged and elderly people. Students need to relieve visual fatigue and prevent myopia due to long-term eye use for learning; office workers are strongly in need of improving dry eyes and reducing eye pressure due to long-term exposure to electronic screens; middle-aged and elderly people are more concerned about preventing ocular degenerative diseases such as macular degeneration. Traditional eye-care functional foods such as lutein esters and zeaxanthin supplements cannot meet the different eye-care needs of different age groups, and it is necessary to promote the development of eye-care functional products towards precision and personalization.

[0003] Antarctic krill is a shrimp species in the genus Euphausia of the family Euphausiidae. It is rich in protein, has a variety of amino acids, and contains various minerals and trace elements such as calcium, iron, zinc, potassium, selenium, and magnesium. Antarctic krill oil is a natural oil extracted from Antarctic krill, rich in various nutrients, with a low triglyceride content and a high polar lipid content. Antarctic krill oil has strong antioxidant properties and multi-dimensional health effects, and has attracted much attention in recent years due to its unique nutritional value and health effects.

[0004] A variety of eye protection functional foods have also been reported in the prior art. For example, a Chinese patent document with publication number CN103316032A discloses a composition containing hydroxytyrosol and its application. The composition is composed of hydroxytyrosol and hyaluronic acid, lutein, proanthocyanidins, and taurine in a certain proportion as active ingredients, and then is made into an oral preparation with an appropriate amount of common excipients. The composition has the effects of improving eyesight and improving eye and visual function. Chinese patent document with publication number CN106491947A discloses a composition for relieving visual fatigue and its preparation. The invention quantitatively combines Chinese medicinal materials such as bilberry powder, marigold powder, chrysanthemum powder, glossy privet powder, wolfberry powder, mulberry leaf powder, cassia seed powder, qingxie powder, cicada shell powder, cornus powder, Cistanche powder, asparagus powder, ophiopogon powder, bitter almond powder, and fructus aurantii powder, so that the effective ingredients of each raw material medicine produce obvious synergistic effect, can effectively remove the damage of free radicals to eyeball tissue, protect eye microvessels, improve fundus microcirculation, and increase ciliary muscle blood flow, thereby eliminating symptoms of visual fatigue and improving vision. A Chinese patent document with publication number CN117298142A discloses an anti-eye fatigue composition, a preparation method and an application thereof. 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. The composition can effectively relieve symptoms such as dry eyes, astringent eyes, and swollen eyes caused by eye fatigue, and can also resist damage caused by blue light exposure, relieve eye fatigue, and protect vision.

[0005] However, the components of the above-mentioned eye protection 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 have high safety, and the composition can be taken as a common food, health product or medicine for a long time.

[0007] The specific technical solutions adopted are as follows: 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; The phospholipid content of the Antarctic krill oil is ≥38 wt% (in the prior art, the highest 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 highest ω-3 polyunsaturated fatty acid content in Antarctic krill oil is 27.4 wt%).

[0008] Preferably, the Antarctic krill oil composition for promoting the growth of retinal cells and improving the oxidative damage of retinal cells comprises, by weight, 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 phospholipid content in the Antarctic krill oil is ≥40 wt%, and the ω-3 polyunsaturated fatty acid content is ≥18 wt%.

[0009] More preferably, the Antarctic krill oil composition for promoting the growth of retinal cells and improving the oxidative damage of retinal cells comprises, by weight, 200 parts by weight of Antarctic krill oil, 10 parts by weight of lutein ester, and 2 parts by weight of zeaxanthin.

[0010] The preparation method of Antarctic krill oil includes: S1 Using Antarctic krill powder as raw material, countercurrent extraction of the raw material with ethanol to retain active ingredients, and then centrifuging to remove solid residues to obtain crude oil; S2 Concentrating and filtering the crude oil; S3 Further rectifying the crude oil treated in step S2 to make the contents of ω-3 polyunsaturated fatty acids and phospholipids meet the requirements, and filling the obtained Antarctic krill oil into a container, filling it with nitrogen and sealing it, and storing it in the dark and cold for standby.

[0011] The sources of lutein ester include but are not limited to marigold flowers, spinach, kale, egg yolks, pumpkins, etc.; the sources of zeaxanthin include but are not limited to marigold flowers, corn, broccoli, oranges, mangoes, goji berries, etc.

[0012] Furthermore, promoting the growth of retinal cells means promoting the proliferation of retinal cells, and improving the oxidative damage of retinal cells means at least one of the following three ways: (1) Improving the inhibitory effect of H2O2 on retinal cells (specifically referring to alleviating the death of retinal cells caused by H2O2); (2) Alleviating the high inflammation and high oxidative stress states induced by H2O2, and reducing the expression of inflammation-related genes and oxidative stress-related genes; (3) Alleviating the damage of H2O2 to retinal cells, and increasing the expression of microtubule-associated protein-2 (MAP-2) and axon length in retinal cells.

[0013] The components in Antarctic krill oil include ω-3 polyunsaturated fatty acids, phospholipids, astaxanthin, etc. The inventor found in the research that the compounding of lutein esters, zeaxanthin, and Antarctic krill oil has a synergistic effect in eye protection. The synergistic protective effect of the three components on retinal cells is mainly reflected in: ① The antioxidant and blue light filtering work synergistically. Lutein esters (absorbing short-wave blue light) and zeaxanthin (absorbing 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 to reduce the risk of age-related macular degeneration (AMD); ② Phospholipids in Antarctic krill oil enhance the bioavailability of lutein esters, ω-3 polyunsaturated fatty acids such as DHA promote pigment deposition in the macular area, and astaxanthin improves microcirculation and enhances nutrient delivery; ③ ω-3 polyunsaturated fatty acids in krill oil and lutein esters, zeaxanthin respectively protect retinal cells from two aspects of antioxidant and anti-inflammatory by inhibiting inflammatory factors and the NF-κB pathway; ④ ω-3 polyunsaturated fatty acids such as DHA in Antarctic krill oil maintain the integrity of the retinal cell membrane, astaxanthin enhances mitochondrial function, and lutein esters and zeaxanthin improve visual function.

[0014] The present invention also provides the application of the Antarctic krill oil composition for promoting the growth of retinal cells and improving the oxidative damage of retinal cells in the preparation of a product for optic nerve protection.

[0015] The present invention also provides a product for optic nerve protection, the components of which include the Antarctic krill oil composition for promoting the growth of retinal cells and improving the oxidative damage of retinal cells.

[0016] Furthermore, the components of the product for optic nerve protection also include pharmaceutically acceptable excipients or food-grade acceptable additives.

[0017] Specifically, the forms of the product for optic nerve protection include but are not limited to powders, tablets, capsules, granules or beverages.

[0018] More preferably, the form of the product for optic nerve protection is soft capsules or gummy candies, and the addition amount of the Antarctic krill oil composition for promoting the growth of retinal cells and improving the oxidative damage of retinal cells is 20.0 - 60.0 wt%.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention obtains a krill oil composition that can promote the growth of retinal cells and improve the oxidative damage of retinal cells by compounding specific proportions of krill oil, lutein esters, and zeaxanthin. The raw materials of this composition are easy to obtain and have high safety. It can promote the proliferation of retinal cells and improve the oxidative damage of retinal cells (improve the inhibitory effect of H2O2 on retinal cells; alleviate the high inflammation and high oxidative stress states induced by H2O2, reduce the expression of inflammation-related genes and oxidative stress-related genes; alleviate the damage caused by H2O2 to retinal cells, increase the expression of microtubule-associated protein-2 and axon length in retinal cells), and can be made into ordinary foods, health products, or drugs for long-term consumption. Description of the Drawings

[0020] Figure 1 It is a statistical chart of the proliferation rate of cells treated with three-component compositions at different concentrations. Among them, when the three-component composition treatment groups at different concentrations are compared with the control group, * indicates P <0.05, ** indicates P <0.01.

[0021] Figure 2 It is a diagram showing the effect of the three-component composition and the two-component composition at the corresponding concentration on the proliferation of 661W retinal cells. Among them, when the three-component composition or two-component composition treatment group is compared with the control group, ** indicates P <0.01.

[0022] Figure 3 It is a diagram showing the effect of the three-component composition and the two-component composition at the corresponding concentration and krill oil on the apoptosis of 661W retinal cells. Among them, when the three-component composition treatment group is compared with the two-component composition / krill oil treatment group, ** indicates P <0.01, when the three-component composition is compared with the H2O2 treatment group, ## indicates P <0.01.

[0023] Figure 4 It is a diagram showing the effect of the three-component composition and the two-component composition at the corresponding concentration and krill oil on the mRNA expression levels of inflammation-related genes ( Tnf-α, Il-1β ) and oxidative stress-related genes ( Cat, Sod ) in 661W retinal cells. Among them, A is the expression level of inflammation-related genes, and B is the expression level of oxidative stress-related genes. When the three-component composition treatment group is compared with the two-component composition / krill oil treatment group, ** indicates P <0.01, when the three-component composition is compared with the H2O2 treatment group, ## indicates P <0.01.

[0024] Figure 5Effect of the three-component composition, the two-component composition at corresponding concentrations, and Antarctic krill oil on MAP-2 in 661W retinal cells. Among them, A is the fluorescence imaging diagram, B is the quantitative diagram of 661W MAP-2 fluorescence intensity, and C is the quantitative diagram of synaptic length in 661W retinal cells. Compared with the two-component composition / Antarctic krill oil treatment group, ** indicates P <0.01. Compared with the H2O2 treatment group, ## indicates P <0.01. Detailed implementation mode

[0025] The present invention will be further clarified below in conjunction with the embodiments and the drawings. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The operation methods without specific conditions indicated in the following embodiments are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. The content not described in detail in this specification belongs to the prior art well known to those skilled in the art. The experimental materials used in the embodiments can be purchased from conventional biochemical reagent companies without special instructions.

[0026] The Antarctic krill oil used in the embodiments is prepared by the following method: (1) Preparation and feeding: Weigh the thawed Antarctic krill powder and put it into the hopper. (2) Countercurrent extraction: Use ethanol to carry out countercurrent extraction on the raw materials to retain the active ingredients. (3) Preliminary separation: Centrifuge to remove solid residues to obtain crude oil. (4) Concentration and filtration: Sequentially pass through scraping concentration and spherical concentration to gradually volatilize ethanol until the water and volatile matter are less than 2%, and filter the liquid through filter paper. (5) Rectification: Adjust the contents of ω-3 polyunsaturated fatty acids and phospholipids to meet the industry standard of krill oil SC / T 3506-2020 by low-temperature molecular distillation method to 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%). (6) Finished product filling: Fill the obtained Antarctic krill oil into a barrel, fill it with nitrogen and seal it, and store it in the dark and refrigerated.

[0027] In addition, the Antarctic krill oil microcapsule powder used in the embodiments can be prepared by the following method: (1) Preparation and weighing: Accurately weigh solid corn syrup, Antarctic krill oil and other processing aids in a certain proportion. (2) Dissolution and emulsification: Add the weighed raw and auxiliary materials to the stirring tank, fully dissolve and emulsify them, and mix them evenly. (3) Spray drying: spray dry the above-mentioned feed liquid; (4) Screening and packaging: screen the coarse powder obtained by spray drying, add an appropriate amount of silicon dioxide to the fine powder after screening and mix evenly, and finally pack the obtained Antarctic krill oil microcapsule powder into an aluminum foil bag and store it in the dark and refrigerated.

[0028] Example 1 (soft capsule) Weigh 200 parts by mass of Antarctic krill oil, 10 parts by mass of lutein esters, 2 parts by mass of zeaxanthin, and linseed oil (an appropriate amount, mainly for filling and dispersing, and can also be replaced with soybean oil, sunflower oil, MCT oil, etc., and the specific addition amount is customized according to market demand). After mixing evenly in proportion, the content is obtained. The content of the Antarctic krill oil composition in the content is 35.3 wt% (the specific addition amount can be other contents within the range of 20.0 - 60.0 wt%). The rubber shell is prepared from gelatin, glycerol and water in a ratio of 1:0.4:1, and the soft capsule is obtained by pill pressing and drying.

[0029] Example 2 (gel candy) Mix 200 parts by mass of Antarctic krill oil, 10 parts by mass of lutein esters, 2 parts by mass of zeaxanthin, linseed oil (an appropriate amount, mainly for filling and dispersing, and can also be replaced with soybean oil, sunflower oil, MCT oil, etc., and the specific addition amount can be customized according to market demand), blueberry fruit powder (an appropriate amount, and the specific addition amount can be customized according to market demand), and glyceryl monostearate (an appropriate amount, and the specific addition amount can be customized according to market demand) evenly in proportion to obtain the content. The content of the Antarctic krill oil composition in the content is 35.3 wt% (the specific addition amount can be other contents within the range of 20.0 - 60.0 wt%). Separately, mix gelatin, glycerol, and xylitol solution in a ratio of 1:0.4:1 to form a glue solution, and fill it with a molding machine to obtain the gel candy.

[0030] Example 3 (tablet) Weigh 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 croscarmellose sodium, 10 mg of lutein esters, 2 mg of zeaxanthin, 10 mg of silicon dioxide, 5 mg of magnesium stearate, 3 mg of stevioside, and 2 mg of strawberry essence respectively. Dry-mix the above-mentioned raw and auxiliary materials and directly press them into tablets to obtain chewable tablets of lutein ester Antarctic krill oil microcapsule powder.

[0031] Example 4 (powder) 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 lutein ester Antarctic krill oil modulated milk powder.

[0032] Sample analysis 1 Materials and methods 1.1 Cell culture The 661W retinal cell line (Fenghui Biotechnology Co., Ltd.) was used for the experiment. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% newborn calf serum, 100 IU / mL penicillin and 100 µg / mL streptomycin at 37°C, 5% CO2 and saturated humidity. The medium was changed and passaged routinely. When the cell density reached 90%, the culture medium was discarded, the cells were washed twice with phosphate buffered saline, and the cells were digested with 0.25% EDTA trypsin. The cell density was adjusted and inoculated in a cell culture plate and cultured until the cells stably adhered to the wall.

[0033] 1.2 Cell proliferation assay 1.2.1 Effects of three-component compositions of different formulations on the proliferation of 661W retinal cells 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.

[0034] Formula 1: 50 mg Antarctic krill oil + 10 mg lutein ester + 2 mg zeaxanthin; Formula 2: 100 mg Antarctic krill oil + 10 mg lutein ester + 2 mg zeaxanthin; Formula 3: 200 mg Antarctic krill oil + 10 mg lutein ester + 2 mg zeaxanthin; Formula 4: 400 mg Antarctic krill oil + 10 mg lutein ester + 2 mg zeaxanthin.

[0035] After treating 661W retinal cells with the above three-component composition for 48 h, the cell viability and proliferation were detected by an MTT kit to determine the appropriate addition amount of Antarctic krill oil and the effective concentration range of the three-component composition that can promote the proliferation of 661W retinal cells. The operation procedure of the cell proliferation experiment is as follows: After culturing the cells for 48 h, they 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 incubating at 37 °C for 4 h, the supernatant was aspirated, rinsed twice with PBS, 200 μL of dimethyl sulfoxide was added to each well to dissolve the formed crystals, shaken at room temperature for 15 min, and then the optical density (OD) value was measured at 490 nm on a spectrophotometer.

[0036] 1.2.2 Effects of the three-component composition and its corresponding two-component compositions at different concentrations on the proliferation of 661W retinal cells According to the experimental results in 1.2.1, a suitable formulation was selected for subsequent experiments. Based on the lowest effective concentration of the three-component composition, a control group (containing 0.1% DMSO), lutein ester + zeaxanthin group, Antarctic krill oil group, and lutein ester + zeaxanthin + Antarctic krill oil group were set up. The 661W retinal cells were treated with the three-component composition and its corresponding two-component compositions at different concentrations for 48 h. After 48 h, the cell viability and proliferation were detected by an MTT kit to determine whether the proliferation effect of the three-component composition on 661W retinal cells was superior to that of the two-component compositions. The operation of the cell proliferation experiment was the same as in step 1.2.1.

[0037] 1.3 Cell apoptosis experiment The 661W retinal cells were seeded in a 96-well plate at a density of 5×10 3 cells per well, and groups of H2O2, lutein ester + zeaxanthin, Antarctic krill oil, and Antarctic krill oil + lutein ester + zeaxanthin were set up. When the cell density reached 30%, the cells were pretreated with 400 μM H2O2 for 12 h, and then the 661W retinal cells were treated with the three-component composition and its corresponding two-component compositions at different concentrations for 48 h. After 48 h, the cell viability and proliferation were detected by an MTT kit to determine whether the anti-apoptotic effect of the three-component composition on 661W retinal cells was superior to that of the two-component compositions. The operation of the cell experiment was the same as in step 1.2.1.

[0038] 1.4 Real-time fluorescence quantitative polymerase chain reaction experiment The 661W retinal cells were seeded in a 96-well plate at a density of 5×10 3Cells were inoculated in 12-well plates at a density of [density value], and groups of H2O2, lutein ester + zeaxanthin, Antarctic krill oil, and Antarctic krill oil + lutein ester + zeaxanthin were set up. When the cell density reached 30%, 400 µM H2O2 was used for pretreatment for 12 h, and then the 661W retinal cells were treated with the three-component composition and the two-component composition at their corresponding concentrations for 48 h. After 48 h, the culture medium was discarded, and the cells were rinsed twice with PBS, and then total cellular RNA was extracted using Trizol reagent (Novizan, Nanjing). The RNA concentration was measured using a Nano-300 spectrophotometer (Aosheng, Hangzhou), and cDNA was synthesized using a cDNA reverse transcriptase kit (Novizan, Nanjing). Quantitative real-time PCR was performed using SYBR Green real-time PCR Master Mix (Novizan, Nanjing) on a CFX Connect real-time PCR system (Bio-rad, USA).

[0039] The transcription of β-Actin was used as a housekeeping gene for data normalization, and the gene primer sequences are shown in the following table: Table 1 RT-qPCR primer sequences

[0040] 1.5 Cell immunofluorescence staining experiment The 661W retinal cells were seeded in 24-well plates containing cell culture inserts (10×10 mm) at a density of 5×10 3 per well, and groups of H2O2, lutein ester + zeaxanthin, Antarctic krill oil, and lutein ester + zeaxanthin + Antarctic krill oil were set up. When the cell density reached 30%, 400 µM H2O2 was used for pretreatment for 12 h, and then the 661W retinal cells were treated with the three-component composition and the two-component composition at their corresponding concentrations for 48 h. After 48 h, the culture medium was discarded, and the cells were rinsed twice with PBS, and then fixed with 4% paraformaldehyde PFA for 15 min. After washing away the excess paraformaldehyde with PBS, the cells were permeabilized with PBS containing 0.1% Triton X-100 for 15 min. After washing with PBS, the cells were blocked at room temperature for 1 h. After the blocking was completed, the blocking solution was removed, and then the primary antibody MAP-2 (ET1602-12, Huaan, Hangzhou, China) was added and incubated overnight at 4°C. After the incubation of the primary antibody, the cells were washed with PBS, and then the secondary antibody (R37116, Thermo Fisher, Massachusetts, USA) was added and incubated at room temperature for 1 h. Finally, the secondary antibody was removed, and DAPI (P36981, Thermo Fisher, Massachusetts, USA) was added for mounting. The stained cell sections were photographed using a fluorescence microscope.

[0041] 1.6 Statistical analysis All experimental data were expressed as "mean ± standard error", and one-way ANOVA was used for statistical analysis, followed by Newman-Keuls test. P A significance difference was indicated when <0.05; P An extremely significant difference was indicated when <0.01.

[0042] 2 Experimental results 2.1 Effects of four formulations on the proliferation of 661W retinal cells To determine the proliferative effect of the combination of Antarctic krill oil, lutein esters, and zeaxanthin on 661W retinal cells, a three-component composition with four different addition amounts of Antarctic krill oil was designed and used to treat the cells for 48 h for the experiment. The results showed that formulation 1 significantly promoted the proliferation of 661W retinal cells at an addition concentration of 200 μg / mL, and the proliferative effect increased with the increase in concentration. When the concentration reached 400 μg / mL, the cell proliferation rate reached 105.8% ( Figure 1 ). At the same time, formulations 2, 3, and 4 significantly promoted the proliferation of 661W retinal cells at a concentration of 100 μg / mL, and the cell proliferation rates were 108.8%, 112.6%, and 113.5% respectively, showing a concentration dependence. When the concentration reached 400 μg / mL, the cell proliferation rates reached 109.9%, 127.4%, and 130.5% respectively. However, although the content of Antarctic krill oil in formulation 4 was twice that of formulation 3, the increase in the proliferation rate of 661W retinal cells was relatively weak. Therefore, formulation 3 at the lowest effective concentration (100 μg / mL) was used for subsequent experiments.

[0043] 2.2 Effects of formulation 3, two-component compositions at corresponding concentrations, and Antarctic krill oil on the proliferation of 661W retinal cells The results showed that the combination of lutein esters and zeaxanthin alone and Antarctic krill oil alone did not significantly promote the proliferation of 661W retinal cells. When Antarctic krill oil was used in combination with lutein esters and zeaxanthin, the three-component composition could significantly promote the proliferation of 661W retinal cells, and the proliferation rate could reach more than 110%, indicating that there may be an interaction between different components, jointly promoting cell proliferation ( Figure 2 ).

[0044] 2.3 Effects of formulation 3, its corresponding two-component combination at the same concentration, and Antarctic krill oil on the apoptosis of 661W retinal cells The results of the cell apoptosis experiment showed that the treatment with H2O2 significantly inhibited the proliferation of 661W retinal cells and increased the mortality of 661W retinal cells. The two-component composition of lutein esters and zeaxanthin and Antarctic krill oil alone could not reverse this effect. However, the three-component composition supplemented with Antarctic krill oil could significantly improve the inhibitory effect caused by H2O2 (Figure 3 ), alleviating retinal cell death induced by H2O2. In summary, Antarctic krill oil is a key component for promoting the proliferation of 661W retinal cells, and in synergistic action with lutein esters and zeaxanthin, further enhances the cell proliferation ability.

[0045] 2.4 Effects of Formula 3, its corresponding two-component composition, and Antarctic krill oil on inflammation and oxidative damage of 661W retinal cells After treatment with H2O2, the mRNA expression levels of inflammation-related genes ( Tnf-α, Il-1β ) and oxidative stress-related genes ( Cat, Sod ) in 661W retinal cells were significantly increased compared with the control group, indicating that the cells were damaged at this time. Treatment with only the two-component composition (lutein esters and zeaxanthin) or Antarctic krill oil alone for 48 h failed to effectively reduce the expression of inflammation-related genes ( Tnf-α, Il-1β ) and oxidative stress-related genes ( Cat, Sod ), and failed to improve the inflammatory and oxidative stress states of the cells. However, after adding Antarctic krill oil, the three-component composition significantly alleviated the high-inflammatory and high-oxidative stress states induced by H2O2. ( Figure 4 A and B in

[0046] 2.5 Immunofluorescence experiment to compare the improvement effects of Formula 3, its corresponding two-component composition, and Antarctic krill oil on the growth and morphology of 661W retinal cells Figure 5 As shown in A in Figure 5 , after treatment with H2O2, the fluorescence intensity of MAP-2 in 661W retinal cells was significantly decreased, and the cell axons were shortened, indicating that the neuronal morphology and structural integrity of the cells were damaged. Treatment with the two-component composition of lutein esters and zeaxanthin and Antarctic krill oil alone failed to reverse this damage. However, when lutein esters, zeaxanthin, and Antarctic krill oil were compounded, both the fluorescence intensity of MAP-2 and the axon length of 661W retinal cells were significantly improved, and the effect of the three-component composition was significantly better than that of the two-component composition ( Figure 5 B and C in

[0047] 3 Experimental conclusions This invention explored the effects of Antarctic krill oil, lutein esters, and zeaxanthin on the proliferation of 661W retinal cells and the protection against cell damage induced by H2O2 through a series of experiments. The results showed that the three-component composition of Antarctic krill oil, lutein esters, and zeaxanthin played a key role in promoting the proliferation of 661W retinal cells and protecting the cell structure.

[0048] First, the combination of Antarctic krill oil with lutein esters and zeaxanthin can significantly promote the proliferation of 661W retinal cells, and the promoting effect increases with the increase of the content of Antarctic krill oil in the formula. In addition, in terms of cell protection, H2O2 treatment significantly inhibited the proliferation of 661W retinal cells and induced inflammatory responses and oxidative stress. The binary composition of lutein esters and zeaxanthin and the treatment with Antarctic krill oil alone failed to effectively reverse these damages. However, when treated with the ternary composition, the proliferation ability of the cells was significantly improved and the cell damage was effectively alleviated. The results of the MAP-2 immunofluorescence staining experiment showed that the ternary composition containing Antarctic krill oil significantly improved the decrease in MAP-2 protein expression and the shortening of axons caused by H2O2 treatment, further supporting the important role of Antarctic krill oil in protecting the structure and neuronal morphology of 661W retinal cells.

[0049] In summary, Antarctic krill oil plays a significant synergistic effect in promoting the growth of 661W retinal cells and protecting cells from H2O2-induced damage in combination with lutein esters and zeaxanthin. The significant effect of the ternary composition indicates that the synergistic effect is crucial for alleviating cell damage. This finding provides important scientific basis for the ternary composition of Antarctic krill oil-lutein ester-zeaxanthin in promoting neurodevelopment and protecting the neuronal cell morphology, and lays a foundation for subsequent clinical applications and mechanism studies.

[0050] The above-described embodiments have elaborated on 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 used to limit the present invention. Any modifications, supplements, or substitutions in a similar manner within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Euphausia superba oil composition for promoting the growth of retinal cells and improving the oxidative damage of retinal cells, characterized in that, By weight, it includes 0.1 to 500 parts by weight of Antarctic krill oil, 0.1 to 25 parts by weight of lutein esters, and 0.1 to 5 parts by weight of zeaxanthin; In the Antarctic krill oil described, the phospholipid content is ≥38 wt%, and the ω-3 polyunsaturated fatty acid content is ≥9 wt%.

2. The Antarctic krill oil composition for promoting the growth of retinal cells and improving the oxidative damage of retinal cells according to claim 1, wherein By weight, it includes 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; In the Antarctic krill oil described, the phospholipid content is ≥40 wt%, and the ω-3 polyunsaturated fatty acid content is ≥18 wt%.

3. The Antarctic krill oil composition for promoting the growth of retinal cells and improving the oxidative damage of retinal cells according to claim 1, wherein By weight, it includes 200 parts by weight of Antarctic krill oil, 10 parts by weight of lutein esters, and 2 parts by weight of zeaxanthin.

4. The Antarctic krill oil composition for promoting the growth of retinal cells and improving the oxidative damage of retinal cells according to claim 1, wherein The preparation method of Antarctic krill oil includes: S1 Using Antarctic krill powder as the raw material, ethanol is used for countercurrent extraction of the raw material to retain the active ingredients, and then centrifuged to remove the solid residue to obtain crude oil; S2 Concentrate and filter the crude oil; S3 Further rectify the crude oil treated in step S2 to make the ω-3 polyunsaturated fatty acid and phospholipid contents meet the requirements, and obtain Antarctic krill oil.

5. The Antarctic krill oil composition for promoting the growth of retinal cells and improving the oxidative damage of retinal cells according to claim 1, wherein Promoting the growth of retinal cells means promoting the proliferation of retinal cells; improving the oxidative damage of retinal cells means at least one of the following three ways: (1) Improving the inhibitory effect of H2O2 on retinal cells; (2) Alleviating the high inflammation and high oxidative stress states induced by H2O2, and reducing the expression of inflammation-related genes and oxidative stress-related genes; (3) Alleviating the damage of H2O2 to retinal cells, and increasing the expression of microtubule-associated protein-2 and the axon length in retinal cells.

6. Use of the Antarctic krill oil composition for promoting the growth of retinal cells and improving the oxidative damage of retinal cells according to any one of claims 1-5 in the preparation of a product for optic nerve protection.

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

8. The product for optic nerve protection according to claim 7, wherein, The components also include pharmaceutically acceptable excipients or food-grade acceptable additives.

9. The product for protecting the optic nerve according to claim 7, characterized in that, The product form includes powder, tablet, capsule, granule or drink.

10. The product for protecting the optic nerve according to claim 7, characterized in that, The product form is soft capsule or gummy candy, and the addition amount of the Antarctic krill oil composition for promoting the growth of retinal cells and improving the oxidative damage of retinal cells is 20.0-60.0 wt%.

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