A microcapsule product for improving age-related macular degeneration, and its preparation method and application

Abstract

The present invention discloses a microcapsule product for improving age-related macular degeneration, its preparation method and application, and belongs to the field of biomedicine. The active ingredients of the microcapsule product include lutein and resveratrol, and the components are calculated by mass fraction: 1.5% sodium alginate, 0.5% chitosan, 3% to 8% mustard extract, 4% to 5.5% beta-cyclodextrin, 0.5% polysorbate 80, 2.5% to 3.2% pure water, 2.5% to 3.2% almond oil, 0.5% to 2.5% peppermint essential oil, 25% to 28% lutein and 3.5% to 4.5% resveratrol. The present invention combines lutein and resveratrol, and the two can synergistically better improve AMD caused by smoke. It is prepared into a microcapsule product, and through in vitro and in vivo experiments and clinical studies, it is confirmed that the product has a good improvement effect on AMD caused by smoke, and has a very high clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a microcapsule product for improving age-related macular degeneration, and a preparation method and application thereof. Background Art

[0002] Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Early AMD is characterized by the presence of drusen, which are lipoprotein-rich deposits located between the retinal pigment epithelium (RPE) and Bruch's membrane (BrM). Late AMD can manifest as an atrophic, dry, or neovascular wet form. The atrophic form of the disease, or geographic atrophy, leads to loss of RPE cells, followed by loss of underlying photoreceptors and overlying choroidal capillaries. The neovascular form, or choroidal neovascularization, is characterized by the growth of new choroidal vessels through the BrM and RPE into the subretinal space. Because new vessels tend to be more leaky than established vessels, fluid leakage into the subretinal or sub-RPE space can lead to impaired retinal function and increased photoreceptor cell death. Therefore, one of the primary target cells of both early and late AMD pathology is the RPE. Compared with dry AMD, wAMD (wet AMD) has a higher incidence of blindness, severely impairs vision, and significantly impacts patients' daily lives and work. Intraocular injection of ranibizumab is an important treatment for wAMD, promoting the disappearance of macular hemorrhage and edema and inhibiting choroidal neovascularization in the macular region. However, it can also cause adverse reactions such as intraocular infection, inflammation, and conjunctival lesions. Therefore, further development of new interventional treatments for AMD is needed.

[0003] Cigarette smoke is an important environmental factor that triggers the development of AMD, and cigarette smoke-induced retinal pigment epithelium (RPE) damage has been widely used in AMD-related research. Tobacco extracts induce endoplasmic reticulum (ER) stress and oxidative stress in retinal pigment epithelial (RPE) cells, exacerbate the unfolded protein response and damage RPE tight junctions, which is considered to be an important mechanism for the development of AMD. At present, no drugs have been found to address the eye protection needs of smokers and passive smokers. Many smokers and passive smokers suffer from AMD. Although they can improve AMD by taking some products, the treatment effect is not ideal and there are side effects. The main reason is that the drugs or products taken cannot specifically intervene in the treatment of AMD symptoms caused by smoking. Summary of the Invention

[0004] The present invention aims to provide a microcapsule product for improving age-related macular degeneration, as well as its preparation method and application, to address the aforementioned problems of the prior art. The present invention combines lutein and resveratrol, which synergistically improve smoke-induced AMD. These two ingredients are combined into a microcapsule product. In vitro and in vivo experiments and clinical studies have demonstrated that this product has a significant improvement effect on smoke-induced AMD and has great clinical application prospects.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a composition for improving age-related macular degeneration. The active ingredients include lutein and resveratrol.

[0007] The present invention also provides use of the composition in preparing a product for improving age-related macular degeneration.

[0008] The present invention also provides a product for improving age-related macular degeneration, wherein the active ingredient comprises the composition.

[0009] Optionally, the product comprises microcapsules.

[0010] Optionally, the microcapsules are prepared with the composition as the core material, sodium alginate and chitosan as the wall materials, and mustard extract, β-cyclodextrin and polysorbate 80 as emulsifiers.

[0011] Optionally, the raw materials for preparing the microcapsules include the following components, calculated by mass fraction: 1.5% sodium alginate, 0.5% chitosan, 3% to 8% mustard extract, 4% to 5.5% β-cyclodextrin, 0.5% polysorbate 80, 2.5% to 3.2% pure water, 2.5% to 3.2% almond oil, 0.5% to 2.5% peppermint essential oil, 25% to 28% lutein and 3.5% to 4.5% resveratrol.

[0012] Optionally, the age-related macular degeneration is caused by smoke.

[0013] The present invention screens core-shell materials and emulsifiers to obtain the optimal microcapsule formulation, and prepares a microcapsule product with ideal encapsulation effect and stability. Experiments verify that the microcapsule product can effectively improve AMD symptoms caused by cigarette smoke.

[0014] Optionally, the age-related macular degeneration is wet age-related macular degeneration.

[0015] The present invention also provides use of the composition or product in preparing a product for improving the efficacy of ranibizumab in treating wet age-related macular degeneration.

[0016] The present invention also provides use of the composition or the product in preparing a product for preventing adverse reactions caused by ranibizumab in treating wet age-related macular degeneration.

[0017] Through clinical studies, the present invention has found that the above-mentioned microcapsule product can enhance the therapeutic effect of ranibizumab on wet AMD caused by smoking. Compared with the use of ranibizumab alone to treat wet AMD, the addition of the microcapsule product with the optimal formula of the present invention to the joint intervention treatment has a more ideal recovery effect on AMD patients, a higher clinical cure rate, and a lower incidence of adverse reactions.

[0018] The present invention discloses the following technical effects:

[0019] This invention combines lutein and resveratrol, which synergistically improve smoke-induced AMD. This approach addresses the pharmacological and efficacy mechanisms of smoking-induced AMD, filling a gap in this field. The proposed microcapsule product also addresses the absorption issues associated with lutein utilization, improving its encapsulation efficiency and stability.

[0020] The present invention demonstrates the mechanism by which the microcapsule product intervenes in AMD through in vivo and in vitro studies. Clinical trials have also confirmed its efficacy in combination with ranibizumab for treating AMD in smokers. Therefore, the microcapsule product prepared by the present invention has a promising clinical application prospect.

[0021] The microcapsule product provided by the present invention has simple ingredients, natural raw materials, and higher safety. It can be further developed into food or medicine, making it more universal. It also provides a feasible reference for improving the quality of life of smokers and reducing the cost of eye disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The stability of each formula emulsion under 3500rpm centrifugation condition;

[0024] Figure 2 The stability of each formula emulsion under 5500rpm centrifugation condition;

[0025] Figure 3 is the particle size distribution diagram of the emulsion of each formula;

[0026] Figure 4 This is the viscosity test chart of each formula emulsion;

[0027] Figure 5 Statistical diagram of microcapsule diameter in each formula;

[0028] Figure 6 This is a statistical chart of the hardness of microcapsules in each formula;

[0029] Figure 7 The statistical diagram of the swelling degree of microcapsules in each formula;

[0030] Figure 8 This is a statistical chart of the microcapsule embedding efficiency of each formula;

[0031] Figure 9 The statistical diagram of microcapsule solubility of each formula;

[0032] Figure 10 This is the effect of temperature on the stability of microcapsules;

[0033] Figure 11 This is the effect of pH on the stability of microcapsules;

[0034] Figure 12 This is the effect of light on the stability of microcapsules;

[0035] in, Figures 10 to 12 In the table, C represents concentrated lutein stock solution;

[0036] Figure 13 The protein expression of ERP in cells under different nicotine concentrations;

[0037] Figure 14 The protein expression of GRP78 in cells under different nicotine concentrations;

[0038] Figure 15 The protein expression of ERP in cells exposed to nicotine for different time periods;

[0039] Figure 16 The protein expression of GRP78 in cells exposed to nicotine for different time periods;

[0040] Figure 17 This is the protein expression of ERP after cells were treated with microcapsules in a nicotine environment;

[0041] Figure 18 The protein expression of GRP78 after microcapsule treatment of cells in a nicotine environment;

[0042] Figure 19 The effect of M4 microcapsule intervention on the expression level of ER stress and lipid disorder genes induced by smoke;

[0043] Figure 20The effect of M4 microcapsule intervention on the level of reactive oxygen species induced by smoke;

[0044] Figure 21 The effect of M4 formula microcapsule intervention on lipid peroxidation MDA induced by smoke;

[0045] Figure 22 The effect of M4 formula microcapsule intervention on C3 complement concentration under smoke stimulation;

[0046] Figure 23 The effect of M4 formula microcapsule intervention on C3a content under smoke stimulation;

[0047] Figure 24 The effect of M4 formula microcapsule intervention on C3 mRNA expression under smoke stimulation;

[0048] Figure 25 The effect of M4 microcapsule intervention on the gene expression of ER stress and lipid homeostasis markers in RPE cells;

[0049] Figure 26 The effect of M4 microcapsule intervention on the protein expression of ER stress and lipid metabolism markers in RPE cells; Figures 19 to 26 In the table, ** indicates a very significant difference compared with the model group (p < 0.01), * indicates a significant difference compared with the model group (p < 0.05); ## indicates a very significant difference compared with the control group (p < 0.01), # indicates a significant difference compared with the control group (p < 0.05);

[0050] Figure 27 To adjust the effect of microcapsule intervention on smoke-induced reactive oxygen species levels after M4 formulation;

[0051] Figure 28 To adjust the effect of microcapsule intervention on smoke-induced lipid peroxidation MDA after M4 formulation;

[0052] Figure 29 To investigate the effect of microcapsule intervention after adjusting the M4 formula on the expression levels of ER stress and lipid disorder genes induced by smoke;

[0053] Figure 30 This is the statistical results of CRT indicators in different treatment groups in clinical studies;

[0054] Figure 31 This is the statistical result of BCVA index of different treatment groups in clinical research;

[0055] Figure 32 This is the statistical result of retinal hemorrhage area in different treatment groups in clinical research;

[0056] Figure 33These are the statistical results of NEI-VFQ scores in different treatment groups in clinical studies. DETAILED DESCRIPTION

[0057] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0058] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0059] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0060] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0061] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0062] Lutein is a xanthophyllic carotenoid found in various foods, such as marigold flowers, dark green leafy vegetables, and egg yolks. Lutein exhibits antioxidant activity and scavenges reactive oxygen species, such as singlet oxygen and lipid peroxyl radicals. Oxidative stress activates inflammatory mediators, leading to the development of metabolic and inflammatory diseases. Recent basic and clinical studies have investigated the anti-inflammatory effects of lutein based on its antioxidant activity and modulation of oxidant-sensitive inflammatory signaling pathways. Therefore, it may protect against various inflammatory diseases, including neurodegenerative diseases, diabetic retinopathy, osteoporosis, cardiovascular disease, skin diseases, liver damage, obesity, and colitis. Furthermore, lutein exhibits tissue-specific effects, such as regulating lipid profiles in cardiovascular disease, adipocyte differentiation in obesity, and skin function. Therefore, consuming foods rich in lutein may help prevent inflammatory diseases caused by oxidative stress.

[0063] Lutein's photothermal instability has largely limited its development and application. Microencapsulation, which utilizes stable materials to microencapsulate core ingredients (such as paprika oleoresin), is an effective method for enhancing the stability of unstable bioactive ingredients. Among the various microencapsulation technologies that have been developed, spray-dried microencapsulation is a simple method that can protect active compounds from adverse reactions, harsh environments, and interactions with other ingredients. It improves the solubility of active substances, increases stability in harsh environments, and masks unpleasant odors.

[0064] Among the various core-shell microencapsulation materials, ionic polymer components are commonly used, such as sodium alginate. Derived from brown seaweed, sodium alginate is a versatile anionic polymer known for its biocompatibility and low toxicity. As a polyelectrolyte, it helps control solution rheology through external stimuli such as pH and ionic strength. β-cyclodextrin (β-CD) can improve emulsion stability and antioxidant properties when combined with traditional emulsifiers. Its ability to bring antioxidant activity to the oil-water interface makes it valuable in emulsions seeking antioxidant benefits. Polysorbate 80 is a synthetic nonionic surfactant widely used as an emulsifier in emulsions. It has multiple functions, including emulsification, dispersing, wetting, and stabilization. White mustard extract plays a crucial role in the formation of traditional oil-in-water emulsions. This extract contains lecithin, the primary emulsifier in egg yolk, which contributes to the emulsion's stability.

[0065] The complement system is an evolutionarily ancient component of the innate and adaptive immune systems, designed to participate in the elimination of pathogens and non-self cells from an organism. To carry out this function, there are three initiating pathways (classical, lectin, and alternative), all of which culminate in a common terminal pathway through the production of the cognate complement C3 convertase. Activation of the complement system leads to the production of three classes of effector molecules: the soluble anaphylatoxins C3a and C5a, which signal through their respective G protein-coupled receptors (C3aR and C5aR) and participate in chemotaxis and mediate inflammatory responses; the cell-bound opsonins C3b, C3d, and iC3b, which, upon cleavage, are derived from C3 and participate in the removal of pathogens and cells; and the terminal membrane attack complex, which forms nonspecific pores in the cell membrane and participates in cell lysis. The expression of membrane-bound complement inhibitory molecules and soluble serum inhibitors prevents complement-mediated damage to self-cells, thereby protecting them from complement activation. However, because the levels and cellular localization of these inhibitors can be affected by environmental factors such as oxidative stress or cigarette smoke, the surface of self-cells can become targets for complement activation. Cigarette smoke has been shown to directly activate C3 by modifying it, thereby reducing its ability to bind to CFH, and serum levels of complement components are elevated in smokers.

[0066] Lipid accumulation, a hallmark of age-related macular degeneration, accumulates as extracellular lesions in BrM. Analysis of the lipid composition in BrM reveals that the lipoprotein particles are distinct from those found in plasma. They contain free and esterified cholesterol, phosphatidylcholine, and apolipoprotein B100. ER stress has been shown to activate transcription factors called sterol regulatory element-binding proteins (SREBPs), which increase the expression of proteins required for cholesterol and triglyceride biosynthesis and uptake.

[0067] The present invention screens core-shell materials and emulsifiers to optimize the microcapsule formulation, and then prepares a microcapsule powder product with ideal encapsulation and stability through high-pressure homogenization and spray drying. Furthermore, the microcapsule powder product of the present invention can effectively improve the symptoms of AMD caused by cigarette smoke, and the mechanism of intervention for AMD has been further investigated through in vivo and in vitro experiments. Experimental studies have confirmed that the microcapsule powder product of the present invention can effectively improve the symptoms of AMD, an eye disease caused by cigarette smoke, through oxidative stress and complement activation pathways. Furthermore, clinical studies have shown that the microcapsule powder product of the present invention can enhance the therapeutic effect of ranibizumab on wet AMD caused by smoking. Compared with treating wet AMD with ranibizumab alone, the co-intervention treatment with the optimal microcapsule powder product of the present invention resulted in more ideal recovery outcomes for AMD patients, a higher clinical cure rate, and a lower incidence of adverse reactions.

[0068] Reagents used in the present invention: β-CD was purchased from Sigma-Aldrich (Hamburg, Germany); Folin-Ciocalteu phenol reagent (Merck, Darmstadt, Germany); sodium alginate was obtained from brown algae from Sigma-Aldrich (Shanghai, China); chitosan (high molecular weight (HMW) 80 / 3000) was purchased from Sigma-Aldrich (Steinheim, Germany); polysorbate 80 (Tween ) was purchased from Sigma-Aldrich (Germany). Resveratrol (CAS No. 501-36-0) was purchased from Shanghai Koraman Reagent Co., Ltd. Acetic acid was purchased from Sigma-Aldrich (Germany). Solvents used for sample extraction and analysis, including hexane, acetonitrile, methanol, and dichloromethane, were HPLC-grade solvents purchased from Sigma-Aldrich. All other chemicals and reagents were of analytical grade and purchased from Sigma-Aldrich. Lutein ester (CAS No. 547-17-1) was purchased from Shaanxi Bowei Biotechnology Co., Ltd.

[0069] The mustard extract of the present invention is an alcohol extract of common white mustard seeds. The preparation method comprises the following steps: preparing the prepared white mustard seeds by rapidly mixing and grinding them at low temperatures in a fully automatic liquid nitrogen cryo-grinder and maintaining them at -20°C until extraction. The ground seeds are then poured into a mixed solvent of ethanol and water (ethanol:water volume ratio of 8.5:2), ultrasonically treated for 25 minutes (21 kHz, 145W), and then maintained at room temperature for 24 hours. The suspension is filtered onto paper using a Buchner funnel, and the filtrate is dried under vacuum to obtain the mustard extract.

[0070] In a specific embodiment of the present invention, white mustard seeds are purchased from Sichuan, and other commercially available common white mustard seed products can achieve corresponding effects.

[0071] The technical solution of the present invention is demonstrated in detail below with reference to specific embodiments.

[0072] Example 1 Preparation of microcapsules

[0073] 1. Reagents and Materials

[0074] Capsule shell: sodium alginate, chitosan (high molecular weight (HMW) 80 / 3000).

[0075] Emulsifiers: Mustard Extract, β-cyclodextrin (β-CD), Polysorbate 80.

[0076] 2. Microcapsule formula

[0077] Table 1 shows the formulas M1 to M7 for preparing microcapsules.

[0078] Table 1 Microcapsule formulation

[0079]

[0080] 3. Preparation of emulsion

[0081] (1) Shell solution preparation: Sodium alginate and / or chitosan as the wall material were dispersed in 152 g of water according to the formulation in Table 1, heated at 67°C and stirred at 350 rpm for 35 min, and then cooled in cold water (4°C) overnight to ensure complete hydration before use.

[0082] (2) Emulsion preparation: 0.35 g / mL sodium alginate solution (26 mL) and the required amount of emulsifier (mustard extract, β-cyclodextrin, polysorbate 80) were stirred at 22°C for 15 minutes. The required amount of peppermint essential oil or almond oil (0.028-0.072 g / mL) and lutein esters and resveratrol were added to the solution. The emulsion was stirred at 23°C for 16 minutes and then pre-emulsified using a handheld homogenizer for 3 minutes (Biospec Products Inc., USA). The crude emulsion was further homogenized five times by a high-pressure TC5 homogenizer (Stansted Fluid Power, Harlow, UK) at 83.86 MPa and 54°C. The emulsion was cooled to room temperature before spray drying.

[0083] 4. Preparation of microcapsule powder by spray drying encapsulation emulsion

[0084] Microcapsule particle powder was prepared using an experimental spray dryer (BILON-6000Y, Bilon Instruments, Shanghai, China). The homogenized emulsion was stirred continuously during the spraying process and fed into the main chamber at a feed flow rate of 9.5 mL / min. The suction volume and spray pressure were set to 0.58 m 3 The inlet temperature was set at 158°C and the outlet temperature was varied between 70 and 90°C. The spray-dried powder was collected from the bottom of the cyclone and transferred to a ziplock bag, which was then stored at -18°C.

[0085] Example 2 Emulsion Stability Study

[0086] 1. Stability determination

[0087] Experimental Procedure: Emulsion stability was assessed using a Sigma 3-18KS centrifuge (Sigma Laborzentrifugen GmbH, Osterodeam Harz, Germany). The test was repeated three times, with centrifugal forces ranging from 3000 to 7000 rpm for 5 minutes each. The test was performed, and the percentage of non-separated emulsion, termed the centrifugation index (CI), was calculated using the following formula:

[0088] CI=Ve / Vi×100%,

[0089] Where Ve is the volume of the emulsion remaining after centrifugation, and Vi is the volume of the initial emulsion. The test was repeated three times, and the average CI was determined. Test parameters included a centrifugal force of 3500 rpm / 5500 rpm, a temperature of 23°C, and a duration of 5 minutes.

[0090] The experimental results are as follows Figure 1 and Figure 2 As shown, from Figure 1 and Figure 2 It can be seen that among the seven lutein emulsion formulas tested at the two rotation speeds, the stability of formulas M3, M4 and M5 is better than that of the other formulas.

[0091] 2. Particle size distribution measurement

[0092] Experimental Procedure: The particle size and distribution of the oil were analyzed using a Mastersizer 3000 with a Hydro EV unit (Malvern Analytical Ltd., Malvern, UK). The emulsion was added dropwise to water until the laser obstruction reached 9.5-10.5%, maintaining a constant pump speed of 2400 rpm. Refractive indices of 1.330 and 1.478 were used. The average particle size distribution was calculated from five runs. The results are shown in Figure 2. Figure 3 shown.

[0093] from Figure 3 As can be seen, the average particle size of the emulsions of formulations M3 and M4 is approximately 0.5 μm, the average particle size of the emulsion of formulation M1 is approximately 0.9 μm, and the average particle size of the emulsions of the remaining formulations is 0.7 μm. The particle size of the lutein formulation emulsion is relatively small, and the microemulsions prepared are relatively small. The average particle size of the emulsions of formulations M3 and M4 is relatively small, which is more ideal.

[0094] 3. Dynamic viscosity

[0095] Emulsion viscosity was assessed using a Fungilab ALPHA series viscometer (Fungilab, Sant Feliu de Llobregat, Barcelona, ​​Spain). For each measurement, 50 mL of emulsion was added to a dedicated container of the viscometer and placed on the surface of the instrument. An L3 head was used and set to 100 rpm. Viscosity (mPa·s) was recorded at room temperature. Each sample was measured until the dynamic viscosity value stabilized. The results are shown in Figure 2. Figure 4 shown.

[0096] from Figure 4 It can be seen that the emulsions prepared by M3 and M4 have higher viscosities.

[0097] Example 3 Study on properties of microcapsules

[0098] 1. Size of microcapsules (diameter)

[0099] The microcapsule size was determined using a digital caliper (BGS Technic, Wermelskirchen, Germany). Thirty microcapsules were measured to calculate the mean and standard deviation of their diameters, including both dried and freshly prepared samples.

[0100] The results are as follows Figure 5 As shown, from Figure 5 It can be seen that the diameters of the microcapsules prepared by the M3 and M4 formulations are smaller than those of the microcapsules prepared by the other formulations. The diameters of the microcapsules prepared by the M3 and M4 formulations are about 1.6 mm, while the diameters of the microcapsules prepared by the other formulations are about 1.8 mm.

[0101] 2. Hardness of microcapsules

[0102] The force required to compress a 2 mm microcapsule was measured on freshly prepared samples using an XT.plus texture analyzer (Texture Technologies, Brewster, NY, USA). Using a P / 100 platen probe, the force required to compress a 2 mm microcapsule was measured. The maximum force was set to 6500 g. Five microcapsules were analyzed for each sample, and the mean and standard deviation were calculated based on five measurements.

[0103] The results are as follows Figure 6 As shown, from Figure 6 It can be seen that the hardness of the microcapsules prepared by the M3 and M4 formulations is better than that of the microcapsules prepared by other formulations.

[0104] 3. Swelling characteristics of microcapsules

[0105] Experimental procedures: Dried microcapsules were swelled in water. After 4 hours, the microcapsules were weighed and then the swollen microcapsules were separated, filtered through a metal mesh, and wiped dry with a paper towel to remove excess liquid. The swelling index (SI) was calculated using the following formula:

[0106] SI(%)=(Ws-Wi) / Wi×100%,

[0107] Where Ws is the weight of the expanded microcapsules and Wi is the weight of the dried microcapsules.

[0108] The results are as follows Figure 7 As shown, from Figure 7 It can be seen that the swelling coefficients of the M3 and M4 formulations are better than those of the other formulations.

[0109] 4. Microcapsule embedding efficiency

[0110] Method for determining the encapsulation efficiency (EE) of lutein microcapsules: The microencapsulated lutein powder composition was dissolved in anhydrous ethanol to prepare solutions of varying concentrations. The absorbance of the solutions was measured at 446 nm using a double-beam UV / visible spectrometer with an ethanol solution (96%, v / v) as a blank. A standard curve was constructed by plotting the relationship between lutein absorbance and lutein concentration, which was obtained by the equation A = 2.145C + 0.0011 (R 2 =1) description. Accurately weighed lutein microcapsule particles (20 mg) were dissolved in 1 mL of deionized water by ultrasound-assisted method and diluted to 20 mL with anhydrous ethanol. Accurately weighed lutein microcapsule particles (30 mg) were diluted to 20 mL with anhydrous ethanol and shaken for 1 min. The two solutions were then centrifuged at 5°C (4500 rpm, 10 minutes). The absorbance of the supernatant was measured to calculate the surface lutein content and total lutein content of the microcapsules. The encapsulation efficiency was quantitatively calculated using the following formula A, and the results are shown in Figure 8 .

[0111] Formula A:

[0112] from Figure 8 The results show that the encapsulation efficiency of lutein microcapsules prepared by the M1, M6, and M7 formulations was low, and none of them reached an encapsulation efficiency of 50%. The encapsulation efficiency of lutein microcapsules prepared by the other formulations ranged from 51% to 81%, achieving a good encapsulation efficiency, indicating that the system has an excellent loading effect. It can be further seen that the encapsulation effect of lutein microcapsules prepared by the M3 and M4 formulations was even better, with an encapsulation efficiency of over 75%. Among them, the encapsulation efficiency of the M4 formulation reached the highest, 81%, indicating that the microcapsules prepared by the M4 formulation had the most ideal loading effect.

[0113] 5. Solubility measurement

[0114] Experimental steps: Dissolve lutein microcapsule powder (dry basis) in deionized water and centrifuge at 4°C (4000 rpm, 10 min). Then pour out the supernatant. After repeating several times, wash the residue with deionized water into an aluminum box of known mass and dry to constant weight. Solubility (S) is calculated using the following formula B. Results are shown in Figure 9 shown.

[0115] Formula B: S (%) = [1-((total weight of aluminum box and insoluble matter - aluminum box weight) / sample weight)] × 100%

[0116] Good solubility is an important factor for the application of microcapsules in food. Figure 9It can be seen that the lutein microcapsule powder has a good solubility ranging from 42% to 85%. Except for the microcapsules of the M1, M2, M6 and M7 formulations whose solubility did not reach 60%, the solubility of the microcapsule powders of the other three formulations reached above 60%. Among them, the solubility of the microcapsules of the M4 formulation was the highest. The solubility of the microencapsulated lutein composition prepared by the M4 formulation can reach 85%.

[0117] In summary, based on Examples 2 and 3, it can be seen that among the seven microcapsule formulations, formulations M3 and M4 have the best microcapsule properties, and they can effectively encapsulate and stabilize lutein. Therefore, microcapsules from formulations M3 and M4 were selected for further research.

[0118] Example 4 Microcapsule Stability Study

[0119] 1. Study on the effect of temperature on the stability of microcapsules

[0120] Experimental steps: Take 0.5g of M3 and M4 microcapsule powder and 5mL of concentrated lutein stock solution (containing about 0.05g of lutein), dissolve the microcapsule powder in 5mL of anhydrous ethanol, and then add distilled water to 100mL for temperature stability test. Heat the two 20mL samples at 20, 40, 60, 80 and 100℃ for 30 minutes. Finally, detect the change in the lutein OD value in the microcapsule to determine its stability. The test results are as follows: Figure 10 shown.

[0121] Depend on Figure 10 It can be seen that increasing the temperature directly leads to a decrease in the OD of lutein, which indicates that high temperature has a certain destructive effect on lutein. However, overall, the absorbance of microencapsulated lutein only decreases slightly compared to the natural lutein in the control group. When heated to 80°C, the absorbance of natural lutein drops to 35.8%, while the absorbance of microencapsulated lutein only drops to 48.5%. At 100°C, the absorbance of lutein in the microcapsules of the two formulations (M3 and M4) only decreases by the same proportion, only about 16.64%, while the absorbance of natural lutein decreases by about 53.97%. Therefore, the microcapsules of the present invention play a relatively important role in protecting lutein from high temperature damage.

[0122] 2. Study on the effect of pH on the stability of microencapsulated lutein

[0123] Experimental steps: Following the same preparation method as in "1. Study on the effect of temperature on the stability of microcapsules", take 20 ml of the two solutions and adjust the pH to 2, 4, 6, 8 and 10 with HCl and NaOH, and detect the changes in the OD value of the microcapsule lutein. The experimental results are as follows: Figure 11 shown.

[0124] according to Figure 11 As shown in the figure, the more the pH value decreases, the more the absorbance shows a certain positive correlation and continues to decline. When the pH value drops to 2 from 10, the absorbance of lutein in the microcapsule decreases by only about 8.24%. However, the absorbance of natural lutein has decreased by about 18.94%. In addition, we can further see from the figure that when the pH drops to 2 from 4, that is, when entering a strongly acidic environment, the absorbance of natural lutein (i.e., C group control group lutein) presents a very obvious downward trend, and the absorbance downward trend of the microencapsulated lutein of two prescriptions of the present invention is still very slow. Of course, compared to the microcapsule of the M3 prescription, when entering a strongly acidic environment, the absorbance of lutein in the M4 prescription microcapsule does not decrease significantly. This shows that natural lutein is very unstable in a highly acidic environment, and the lutein in the microcapsule can be used for a wider range of acidic and alkaline environments, and the M4 prescription microcapsule is more acid- and alkali-resistant than the M3 prescription microcapsule, and is more stable in a strongly acidic environment.

[0125] 3. Effect of light on lutein stability

[0126] Experimental steps: Following the same preparation method as in "1. Study on the effect of temperature on the stability of microcapsules", the microcapsules were exposed to natural light at room temperature (25°C) for 2, 4, 6, 8 and 10 days, with distilled water as the control, and the absorbance was measured at 445nm. Figure 12 shown.

[0127] from Figure 12 It can be seen that natural lutein is more sensitive to external light than microencapsulated lutein. As the illumination time increases, the absorbance decreases more significantly. In particular, from the 6th to the 10th day of illumination, the absorbance value of natural lutein (Group C) decreased by about 6.9%, while the absorbance value of lutein in the M4 microcapsules only decreased by about 1.4% from the 6th to the 10th day. After 10 days of irradiation, the absorbance of the natural lutein group (Group C) decreased by a total of 8.4%. Under the same conditions, the microencapsulated lutein showed relatively stable performance, with absorbance only decreasing by 3.8% (M4 group) and 4.64% (M3 group). Therefore, under illumination conditions, the microencapsulated lutein of the present invention can effectively improve the light-instability property of lutein, making it more suitable for food production and storage.

[0128] Next, the present invention will explore the beneficial effects of the microcapsules prepared by the M3 and M4 formulations on improving age-related macular degeneration (AMD) caused by smoking, an eye disease. Because tobacco extracts can induce endoplasmic reticulum (ER) stress and oxidative stress in retinal pigment epithelial (RPE) cells, aggravate the unfolded protein response and damage RPE tight junctions. Endoplasmic reticulum protein 29 (ERp29) is a new type of chaperone protein, which is expressed at a reduced level in AMD patients and can reduce endoplasmic reticulum stress in RPE cells and reduce tight junction damage. Therefore, the present invention detected the expression of ERp29 in AMD mice before and after taking the microcapsules of the present invention to determine whether the microcapsules of the present invention can regulate the expression of ERp29 and the ER stress mechanism caused by smoke.

[0129] Example 5 In vitro experimental study on the use of microcapsule products to prevent and improve nicotine-induced age-related macular degeneration (AMD)

[0130] 1. Cell culture and cell treatment

[0131] Human ARPE-19 RPE cell line and human THP-1 monocyte cell line were purchased from Procell Life Science & Technology (Wuhan, China). Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection.

[0132] Human retinal pigment epithelial (ARPE-19) cells were cultured in DMEM / F-12 medium (1:1) supplemented with 10% fetal bovine serum (FBS) in a cell culture incubator at 37°C and 5% CO2. The cells were then cultured in a medium containing CSE solution for 24 hours.

[0133] Preparation of Cigarette Smoke Extract (CSE): Dissolve smoke from one burning cigarette in 1 mL of DMEM / F-12 medium. Filter the mixture using a 0.22 μm filter to remove large particles and bacteria, and adjust the pH of the mixture to approximately 7.4. Label the final CSE solution to 100% and store at -80°C until use.

[0134] 2. Western blot analysis

[0135] Total protein was extracted on ice for 20 min using RIPA lysis buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail. Protein concentration was determined using a BCA protein assay kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. Proteins were separated by 10% SDS-PAGE and then transferred to a 0.45 μm polyvinylidene difluoride membrane (Millipore, MA, USA). After blocking each membrane with 5% skim milk for 2 h at room temperature, the membrane was incubated with the primary antibody at 4 °C overnight. After washing three times with Tris-buffered saline-0.1% Tween-20 (TBST), the membrane was incubated with the secondary antibody for 1 h at room temperature. The membrane was washed again with TBST three times and then visualized using Immobilon Western chemiluminescent HRP substrate (Millipore, MA, USA). The experimental results are shown in Figure 5. Figures 13 to 16 shown.

[0136] from Figure 13 and Figure 14 It can be seen that exposure of cells to nicotine environment will increase the expression of their ERP29 and GRP78. Different concentrations of nicotine have slightly different effects on the expression of ERP and GRP78 in ARPE-19 cells. When the cells are in a 25μM nicotine environment, the effect on the expression of ERP and GRP78 is the most significant, which shows a very significant difference compared with the blank control group (P < 0.001). Of course, when the nicotine concentration is 40μM, the effect on the expression of ERP and GRP78 is also very significantly different compared with the blank control group (P < 0.01).

[0137] In addition, from Figure 15 and Figure 16 It can be seen that the time in the nicotine environment also affects the expression of ERP and GRP78. When exposed to the nicotine environment for 24 hours and 48 hours, the effect on the expression of ERP is significantly different from that of the blank control (P < 0.01). The effect of exposure to the nicotine environment for 24 hours on the expression of GRP78 is more significant (compared with 48 hours), which is significantly different from the blank control group (P < 0.01). After exposure for 48 hours, the expression of GRP78 is significantly different from that of the blank control group (P < 0.05).

[0138] Based on the above results, a nicotine concentration of 25 μM and an exposure time of 24 hours were selected as the subsequent research conditions to study the effect of the microcapsule formulation on improving the changes caused by nicotine. Figure 17 and Figure 18 .from Figure 17 and Figure 18It can be seen that the microcapsules of the two formulations can improve the expression of ERp29 and GRP78 in cells induced by nicotine, and the microcapsules of the M4 formulation are more ideal for improving the expression of ERp29 and GRP78 in cells induced by nicotine than those of the M3 formulation.

[0139] According to the above experimental results, it can be seen that the microcapsules prepared by the M4 formula have more excellent properties. Therefore, the intervention mechanism of the optimal formula M4 microcapsules on smoke-induced AMD will be further studied through in vivo and in vitro experiments.

[0140] Example 6: Cell and animal experiments on the optimal formula M4 microcapsule product for preventing and improving smoke-induced AMD and its mechanism of oxidative stress and complement activation cascade reaction

[0141] 1. Animal experiments

[0142] C57BL / 6J mice were purchased, with 9 mice in each group, and divided into 3 groups: blank control group, model group, and M4 microcapsule treatment group (M4 intervention). The animals were housed in an environment with a 12 / 12 hour light and dark cycle, with free access to food and water. At 8 weeks of age, male mice were divided into two groups (n = 12 per group); the control group was maintained in a filtered air environment, and the model group and treatment group were exposed to cigarette smoke. The smoke environment was simulated for 6 months using a smoking machine (Model TE-5; Teague Enterprises), and the average concentration of total suspended particulate matter was 130 mg / m 3 The mice in the treatment group were gavaged once a day with 24 mg / kg body weight (bw) of microcapsule powder. The control and model groups were gavaged with the same dose of normal saline.

[0143] 2. Cell culture system

[0144] Experiments were conducted on ARPE-19 cells, where the microcapsule powder solution was added to culture medium containing CSE solution.

[0145] Preparation of microcapsule powder solution: 1 g of microcapsule powder was dissolved in 98% ethanol aqueous solution, ultrasonicated for 15 minutes, filtered, and the supernatant was stored at -10°C for later use.

[0146] The ARPE-19 cell line is a human RPE cell line that displays the differentiated phenotype of RPEW cells and forms a polarized monolayer on Transwell filters (Costar). To form a monolayer, cells were expanded in DMEM medium containing 10% fetal bovine serum (FBS) and penicillin:streptomycin. After the cells reached confluence, they were grown in serum-containing medium until they reached stability, as measured by transepithelial resistance (TER). TER values ​​reached 40-45 Ωcm within 2-3 weeks after confluence.2 Complete removal of FBS in the last few days before measurement did not alter viability or monolayer formation, allowing the use of complement-replete normal human serum as a complement source.

[0147] 3. ELISA measurement of C3 and C3a

[0148] To measure the production of cellular C3 and C3a, cell culture supernatants were centrifuged at 20,000 rpm for 5 minutes. Microplates were coated with the corresponding C3 and C3a capture antibodies (BD Bioscience), and 100 μL of supernatant was added. The captured proteins were detected with C3- or C3a-specific antibodies conjugated to horseradish peroxidase, followed by development with the chromogenic substrate OPD (Sigma). The absorbance at 492 nm was measured. Aliquots were assayed in duplicate, and the values ​​were compared with C3 and anaphylatoxin dose-response curves.

[0149] 4. Western Blot Analysis

[0150] Cell culture supernatants were separated by electrophoresis on 10% BisTris polyacrylamide gels (Invitrogen) and proteins were transferred to nitrocellulose membranes. The membranes were probed with polyclonal antibodies against GRP78, CHOP, XBP1, and GAPDH (loading control), and antibody binding was visualized using a chemiluminescent detection kit (Amersham Biosciences Life Science). Band density was analyzed and normalized using an Alpha Innotech Fluorchem 9900 imaging system running Alpha Ease FC software 3.3 (Alpha Innotech, San Leandro, CA), and normalized to a GAPDH control.

[0151] 5. Measurement of oxidative stress

[0152] After the cells were treated on Transwell filters, they were collected, stained with 20mDCFDA (2,7-dichlorofluorescein diacetate dye), washed, and analyzed using a spectrophotometer (excitation wavelength, 485 nm; emission wavelength, 535 nm). Lipid peroxidation was measured to determine the production of malondialdehyde (MDA), a natural byproduct of lipid peroxidation. Cells were collected after the corresponding treatment and homogenized in MDA lysis buffer (Abcam). MDA present in the cells was reacted with thiobarbituric acid to form MDA-thiobarbituric acid adducts, and colorimetric quantification was performed using a spectrophotometer (λ = 532 nm). Standard curves for ROS and MDA measurements were prepared as recommended.

[0153] 6. Reverse transcription PCR

[0154] RPE / choroid / sclera fractions (hereafter referred to as RPE / choroid) were isolated from control and smoke-exposed animals and stored at −80°C until use. Cells were collected after treatment and stored at −80°C. Quantitative RT-PCR analysis was performed: Real-time fluorescence quantitative PCR analysis was performed in triplicate using standard cycling conditions in the 5700 Sequence Detection System (Applied Biosystems). Quantitative values ​​were obtained by cycle number, the target gene was normalized to β-actin, and the fold difference between the experimental and control samples was determined. Fold difference values ​​were compared using the Z test, with significance accepted at p < 0.05. Figures 19 to 26 shown.

[0155] 7. Results

[0156] from Figure 19 It can be seen that the gene expression levels of Grp78 and Chop, Er stress markers, on the RPE / choroid of the model group mice exposed to the smoke environment increased by approximately 2.4 times and 8.3 times, respectively, compared with normal mice. The changes in Grp78 were significantly different from those in the control group (P < 0.05), and the changes in Chop were extremely significantly different from those in the control group (P < 0.01). However, after intervention with the microcapsule formula M4, the levels of these two markers in mice did not change significantly compared with the expression of normal mice, indicating that the microcapsules of the present invention can effectively intervene in the gene expression of Er stress markers Grp78 and Chop. In addition, Srebf-1, a marker indicating changes in cholesterol and fatty acid homeostasis, also increased by approximately 4.7 times in the RPE / choroid of smoke-exposed mice, with a significant difference compared with the control group (P < 0.05). After intervention with the microcapsule formula M4, the expression level of Srebf-1 did not change significantly compared with the normal group of mice. This indicates that smoke environment does affect the expression levels of ER stress markers and lipid imbalance markers on the RPE / choroid, and the microcapsule formulation of the present invention can improve the expression levels of these smoke-induced ER stress markers and lipid imbalance markers on the choroid to normal values.

[0157] Smoke exposure can induce oxidative stress in RPE cells, leading to the production of free radicals and the loss of antioxidant systems. The experimental results of the present invention also confirmed that exposure to smoke for 2 hours can lead to a rapid increase in ROS ( Figure 20); ROS levels remained elevated 12 and 24 hours after CSE exposure. However, after intervention with the microcapsule formulation of the present invention, intracellular ROS levels were significantly reduced after exposure to smoke. The intervention effect of the microcapsules of the present invention was particularly pronounced 12 and 24 hours after smoke exposure, indicating that the microcapsule formulation of the present invention can improve AMD by regulating the oxidative stress pathway induced by smoke.

[0158] Reactive oxygen species (ROS) have been shown to degrade polyunsaturated lipids to form malondialdehyde. ROS can trigger lipid peroxidation or oxidative degradation of lipids. The present invention measures MDA formation 12 hours after smoke exposure to record lipid peroxidation. Figure 21 It can be seen that compared with the control group, the MDA content in the cells of the model group exposed to smoke increased by about 8 times, while after intervention with the microencapsulated lutein formula of the present invention, the MDA content decreased significantly.

[0159] from Figure 22 It can be seen that the measurement of the supernatant of ARPE-19 cells stimulated with smoke for 24 hours showed that the complement molecule C3 in the supernatant increased significantly, reaching about 27ng / mL. After intervention with the microcapsules of the M4 formula, the content was significantly reduced, with a very significant difference compared with the model group (P < 0.01). In addition, Figure 24 Quantitative RT-PCR analysis showed that C3 mRNA levels increased 24 hours after smoke exposure, indicating that smoke induced C3 transcription, and after intervention with the M4 microcapsule formula, the transcription level returned to a level close to that of the normal control group. Figure 23 Measurements of C3a in the smoke showed that short-term smoke exposure resulted in approximately 3.15% of C3 being cleaved into C3a. After intervention with the M4 microcapsule formulation, C3a levels were significantly reduced, with a highly significant difference compared to the model group (P < 0.01). These results suggest that the pathogenesis of smoke-induced AMD involves the complement activation cascade mechanism, as the smoke environment stimulates changes in the complement regulatory proteins C3 and C3a and their expression. C3 and its cleaved C3a complement component are molecular components of drusen, which are hallmark extracellular deposits associated with early AMD. Therefore, the experimental results confirm that the microcapsule formulation of the present invention can improve AMD by regulating the complement cascade mechanism regulated by the C3a signaling pathway.

[0160] Figure 25Quantitative RT-PCR results further demonstrated that smoke stimulation affects cellular ER stress and lipid homeostasis. After smoke stimulation, ER stress markers GRP78 and CHOP increased by approximately 2.8-fold and 12.7-fold, respectively, with CHOP changes showing a highly significant difference compared to the control group (P < 0.01). SREBF-1 also increased by approximately 3.6-fold, showing a significant difference compared to the control group (P < 0.05). Figure 25 ). It was also found that these fold differences were similar to those identified in vivo in mouse RPE ( Figure 19 ).

[0161] In addition, it can be found that after smoke stimulation, GRP78, CHOP and XBP1 not only increased in mRNA level, but also significantly increased in protein expression, which was significantly different from the normal control group (P < 0.05, indicated by #) ( Figure 26 ), while after intervention with M4 microcapsules, the protein levels of GRP78, CHOP, XBP1 and phosphorylated eIF2α were significantly reduced (the changes in GRP78, CHOP, and eIF2α were significantly different compared with the model group (P < 0.05, as shown by *); the changes in XBP1 were extremely significantly different compared with the model group (P < 0.01, as shown by **), indicating that the microcapsules of the present invention can effectively improve AMD caused by cigarette smoke through the complement-mediated cellular ER stress pathway.

[0162] Example 7 Synergistic Effect of Lutein and Resveratrol in Microcapsule Formula on Improving Smoke-Induced AMD

[0163] In order to explore whether the combination of lutein and resveratrol in the microcapsule formulation of the present invention can have a synergistic effect on improving smoke-induced AMD, a comparative experiment was conducted based on Example 6.

[0164] The effects of the original M4 formula were compared with those of the original M4 formula by removing resveratrol from the original M4 formula, formula M4-2, and formula M4-3, which were obtained by removing lutein esters from the original M4 formula, using the same experimental procedures as in Example 6. The microcapsule formulations of formulas M4-2 and M4-3 are shown in Table 2.

[0165] Table 2 Microcapsule formulations of formulations M4-2 and M4-3

[0166]

[0167] The experimental results are as follows Figure 27-Figure 29 As shown. Figure 27-Figure 29 It can be seen that after removing resveratrol or lutein ester from the M4 formula, the improvement effect on the oxidative stress and lipid homeostasis indicators of AMD caused by smoke stimulation is not as good as the original M4 formula, indicating that the combination of lutein ester and resveratrol in the original M4 formula has a synergistic effect on improving AMD symptoms under smoke stimulation.

[0168] Example 8 Human Clinical Study

[0169] A total of 56 patients (56 eyes) with smoking-induced wAMD who were treated at the Affiliated Hospital of Shandong University from January 2020 to December 2022 were included in the study. They were randomly divided into two groups of 28 patients (28 eyes) each. The study was approved by the Medical Ethics Committee. The observation group included 20 males and 8 females; their ages ranged from 45 to 85 years, with an average age of (65.85±3.31) years; 18 of them had left eyes and 10 had right eyes. The control group included 17 males and 11 females; their ages ranged from 48 to 84 years, with an average age of (66.07±3.29) years; 15 of them had left eyes and 13 had right eyes. There was no statistically significant difference in the general data between the two groups (P>0.05), indicating that the two groups were comparable.

[0170] 1. Inclusion criteria: ① Signed informed consent; ② Smokers who meet the Western medical diagnostic criteria for wAMD; ③ Smokers who meet the Traditional Chinese Medicine diagnostic criteria for wAMD; ③ Diagnosed by optical coherence tomography (OCT) and fluorescein fundus angiography.

[0171] 2. Exclusion criteria: ①Combined with other eye diseases; ②History of drug allergy; ③Psychiatric and behavioral abnormalities and low compliance; ④Poor blood pressure and blood sugar control; ⑤Previous to treatment such as photodynamic therapy.

[0172] 3. Treatment Intervention: Both groups received intraocular injections of ranibizumab (Novartis Pharma Schweiz AG, approval number S20181010). Levofloxacin eye drops were administered 3 days before surgery, four times daily. The intraocular injections were performed in the operating room. After disinfection and draping, anesthesia was followed by insertion of a needle into the flattened ciliary body, 3.5–4 mm from the limbus. The eye was penetrated perpendicularly to the eyeball wall, and 0.05 mL of ranibizumab was injected. Sterile cotton swabs were applied, and antibiotic ointment and a bandage were applied. Injections were administered once monthly for three consecutive treatments. The observation group also received 5 mg of microcapsules formulated in M4 three times daily for three consecutive months.

[0173] 4. Observation indicators

[0174] CRT (central retinal thickness): CRT was measured by OCT before and 3 months after treatment.

[0175] BCVA (Best Corrected Visual Acuity): The BCVA was measured using an international standard visual acuity chart before and 3 months after treatment, and converted to LogMAR after correction.

[0176] Retinal hemorrhage area: Before treatment and 3 months after treatment, fundus color photography was performed after mydriasis, and the fundus color images were input into Image-Pro Plus software for measurement.

[0177] Vision-related quality of life: The NEI-VFQ-25 scale was used to assess vision-related quality of life before and 3 months after treatment. It includes 12 dimensions and 25 items, with a score range of 0 to 100 points. There was a positive correlation between vision-related quality of life and the score.

[0178] 5. Determination of treatment effect

[0179] Clinical cure: visual acuity improved (the number of lines on the visual acuity chart increased by ≥2 lines), and the foveal thickness thinned (reduced by ≥60 μm).

[0180] Significant improvement in disease symptoms (such as retinal neovascularization and leakage): Effective results are as follows: improved visual acuity (an increase of 1 to 2 lines on the visual acuity chart) and thinning of the fovea (a decrease of 40 to 59 μm compared to the previous thickness);

[0181] Symptoms of the disease have improved;

[0182] Invalid means failing to meet the above standards.

[0183] Total effective rate of treatment = (number of clinically cured cases + number of effective cases) / total number of cases × 100%

[0184] Data were analyzed using SPSS 22.0 software. Count data were expressed as percentages and tested with the chi-square test, and measurement data were expressed as x ± s and tested with the t test. P < 0.05 was considered statistically significant.

[0185] 6. Results Analysis

[0186] The treatment effects, analysis of various indicators, adverse reactions and disease recurrence within 1 year in the two groups are shown in Tables 3 and 4. Figures 30 to 33 shown.

[0187] Table 3 Comparison of treatment effects (n / %)

[0188] Group Number of cases Clinical cure efficient invalid Total efficiency control group 28 9 / 32.14％ 14 / 46.43％ 5 / 17.86％ 82.14％ Observation Group 28 12 / 42.86％ 15 / 53.57％ 1 / 3.57％ 96.43％

[0189] Table 4 Adverse reactions and disease recurrence rate within 1 year (n / %)

[0190]

[0191] from Figure 30 It can be seen that compared with the effect before treatment of the observation group, after the additional addition of the microcapsule product of the present invention to intervene in the treatment of wAMD, the patient's CRT index decreased to a certain extent, and there was a significant difference compared with before treatment (P < 0.05). At the same time, compared with the control group, the CRT of the observation group showed a significant downward trend after treatment, and the two also had a significant difference (P < 0.05).

[0192] from Figure 31 It can be seen that after the combined intervention of microcapsules and ranibizumab, the BCVA index of AMD patients decreased to a certain extent compared with pre-treatment, with a significant difference between the two (P < 0.05). It also decreased to a certain extent compared with the group treated with ranibizumab alone (control group), with a significant difference between the two (P < 0.05). This shows that the microcapsule formulation of the present invention can enhance the therapeutic effect of AMD drugs.

[0193] from Figure 32 It can be seen that after the combined intervention treatment of microcapsules and ranibizumab, the area of ​​retinal hemorrhage in AMD patients was significantly reduced, with a very significant difference compared to before treatment (P < 0.01). Compared with the effect of the single ranibizumab treatment group (control group) after treatment, it was also reduced to a certain extent, and there was a significant difference between the two (P < 0.05), indicating that the microcapsule formulation product of the present invention can synergistically enhance the effect of drug treatment for AMD.

[0194] from Figure 33 It can be seen that after the synergistic intervention treatment of microcapsules and ranibizumab, the NEI-VFQ-25 scores of AMD patients were significantly increased, with a significant difference compared to before treatment (P < 0.05); at the same time, compared with the group treated with only ranibizumab (control group), the NEI-VFQ-25 scores also increased to a certain extent after the addition of the M4 formula microcapsules of the present invention, and the two were significantly different (P < 0.05).

[0195] From the data changes in Table 3, it can be seen that compared with the treatment of wet AMD with ranibizumab alone, the combined intervention of the microcapsules of the present invention and ranibizumab in the treatment of wet AMD can increase the clinical cure rate of wet AMD caused by smoking by 10.72%, increase the total effective rate by 7.14%, reduce the ineffective rate by 14.29%, and increase the total effective rate by 4.29%.

[0196] As can be seen from the data in Table 4, when the microcapsules of the present invention are used together with ranibizumab to intervene in the treatment of wet AMD caused by smoking, compared with treatment with ranibizumab alone, the two adverse reactions caused by ranibizumab alone, namely retinal detachment and persistent increase in intraocular pressure, are eliminated. The incidence of the adverse reaction of endophthalmitis is reduced by 14.29%, and the adverse reaction of one-time increase in intraocular pressure is reduced by 7.15%. More importantly, the overall incidence of adverse reactions is reduced by 28.57%, and the recurrence rate within 1 year is reduced by 8.34%.

[0197] Based on the above analysis, the microcapsule product of the present invention can significantly enhance the therapeutic effect (cure rate and efficacy) of the drug ranibizumab as a monotherapy for wet AMD, and can significantly reduce the incidence of adverse reactions when the drug ranibizumab is used as a monotherapy for AMD. It has great potential for future clinical development and application.

[0198] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a composition for improving age-related macular degeneration in the preparation of a product for improving age-related macular degeneration, characterized in that: The age-related macular degeneration is caused by smoke; The active ingredients of the composition are lutein and resveratrol; The age-related macular degeneration is wet age-related macular degeneration.

2. The use according to claim 1, characterized in that The product comprises microcapsules.

3. The use according to claim 2, characterized in that The microcapsules are prepared by using the composition described in claim 1 as core material, sodium alginate and chitosan as wall materials, and mustard extract, beta-cyclodextrin and polysorbate 80 as emulsifiers.

4. The use according to claim 3, characterized in that The raw materials for preparing the microcapsules include the following components, calculated by mass fraction: 1.5% sodium alginate, 0.5% chitosan, 3%-8% mustard extract, 4%-5.5% beta-cyclodextrin, 0.5% polysorbate 80, 2.5%-3.2% pure water, 2.5%-3.2% almond oil, 0.5%-2.5% peppermint essential oil, 25%-28% lutein, and 3.5%-4.5% resveratrol.

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