Microcapsule product for improving age-related macular degeneration and preparation method and application thereof

Abstract

The invention discloses a microcapsule product for improving age-related macular degeneration as well as a preparation method and application thereof, and belongs to the field of biological medicines. The effective components of the microcapsule product comprise lutein and resveratrol, and the microcapsule product comprises the following components in percentage by mass: 1.5% of sodium alginate, 0.5% of chitosan, 3%-8% of mustard extract, 4%-5.5% of beta-cyclodextrin, 0.5% of polysorbate 80, 2.5%-3.2% of pure water, 2.5%-3.2% of almond oil, 0.5%-2.5% of peppermint essential oil, 25%-28% of lutein and 3.5%-4.5% of resveratrol. The xanthophyll and the resveratrol are combined for use, and the xanthophyll and the resveratrol cooperate with each other, so that AMD caused by smoke can be better improved. In-vivo and in-vitro experiments and clinical research prove that the product has a good improvement effect on AMD caused by smoke and has an extremely high clinical application prospect.

Description

Technical Field

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

[0002] Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly. Early AMD is characterized by the presence of drusen, lipoprotein-rich deposits located between the retinal pigment epithelium (RPE) and Bruch's membrane (BrM). Late-stage AMD can present as atrophic, dry, or neovascularized wet AMD. The atrophic form, or geographic atrophy, leads to the loss of RPE cells, followed by the loss of underlying photoreceptors and the covering 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 leak more easily than established vessels, fluid leakage into the subretinal or subRPE space leads to impaired retinal function and increased photoreceptor cell death. Therefore, one of the primary target cells in the pathology of both early and late AMD is the RPE. Compared to dry AMD, wet AMD has a higher rate of blindness, severely impairs vision, and significantly impacts patients' daily lives and work. Intraocular injection of ranibizumab is an important method for treating wAMD, which helps promote the resolution of macular hemorrhage and edema and inhibits choroidal neovascularization in the macular region. However, adverse reactions such as intraocular infection, inflammation, and conjunctival lesions may occur. Therefore, further development of new interventional treatments for AMD is needed.

[0003] Cigarette smoke is a significant environmental factor contributing to 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 RPE cells, exacerbating unfolded protein responses and damaging RPE tight junctions, which is considered an important mechanism in AMD development. Currently, no drugs have been found to address the eye health needs of smokers and passive smokers. Many smokers and passive smokers suffer from AMD, and although some products can improve AMD, the treatment effects are not ideal and side effects are present. The main reason is that the medications or products cannot specifically target and treat the AMD symptoms induced by smoking. Summary of the Invention

[0004] The purpose of this invention is to provide a microcapsule product for improving age-related macular degeneration (AMD), its preparation method, and its application, thereby addressing the problems existing in the prior art. This invention combines lutein and resveratrol, whose synergistic effect can better improve AMD caused by smoke inhalation. The two are prepared into a microcapsule product, and in vitro and in vivo experiments, as well as clinical studies, have confirmed that this product has a good effect on improving AMD caused by smoke inhalation, demonstrating extremely high clinical application potential.

[0005] To achieve the above objectives, the present invention provides the following solution:

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

[0007] The present invention also provides the use of the composition in the preparation of products that improve age-related macular degeneration.

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

[0009] Optionally, the product may include microcapsules.

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

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

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

[0013] This invention, through screening of core-shell materials and emulsifiers, optimizes the microcapsule formulation and prepares a microcapsule product with ideal encapsulation effect and stability. Experiments have verified 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 the use of the described composition or product in the preparation of products that improve the efficacy of ranibizumab in the treatment of wet age-related macular degeneration.

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

[0017] Through clinical research, this invention has found that the above-mentioned microcapsule products can enhance the therapeutic effect of ranibizumab on smoking-induced wet AMD. Compared with the treatment of wet AMD with ranibizumab alone, the combined intervention with the microcapsule products of the optimal formulation of this invention results in a more ideal recovery effect, a higher clinical cure rate, and a lower incidence of adverse reactions in AMD patients.

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

[0019] This invention combines lutein and resveratrol, whose synergistic effect can better improve AMD caused by smoke. Simultaneously, it addresses the problem of AMD caused by smoking at the pharmacological and pharmacodynamic level, filling a gap in this field. The proposed microcapsule product also solves the absorption problem in lutein utilization, improving the encapsulation efficiency and stability of lutein.

[0020] This invention has demonstrated the mechanism by which the microcapsule product intervenes in AMD through in vivo and in vitro experimental studies, and clinical trials have also confirmed its good therapeutic effect in AMD in smokers when used in combination with ranibizumab. Therefore, the microcapsule product prepared by this invention has extremely high clinical application prospects.

[0021] The microcapsule product provided by this invention has a simple composition, uses natural raw materials, and is safer. It can be further developed into food or medicine, and has a wider applicability. At the same time, it provides a feasible reference for improving the quality of life of smokers and reducing the cost of eye disease treatment. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The stability of each emulsion formulation under centrifugation at 3500 rpm was determined.

[0024] Figure 2 The stability of each emulsion formulation under centrifugation at 5500 rpm was determined.

[0025] Figure 3 Particle size distribution diagrams for each emulsion formulation;

[0026] Figure 4 Viscosity test charts for each emulsion formulation;

[0027] Figure 5 A statistical chart showing the diameter of microcapsules in each formulation;

[0028] Figure 6 A statistical chart of the hardness of microcapsules in each formulation;

[0029] Figure 7 A statistical chart of the swelling degree of microcapsules in each formulation;

[0030] Figure 8 Statistical chart of microcapsule encapsulation efficiency for each formulation;

[0031] Figure 9 A statistical chart showing the solubility of microcapsules in each formulation;

[0032] Figure 10 The results show the effect of temperature on the stability of microcapsules;

[0033] Figure 11 Results showing the effect of pH on microcapsule stability;

[0034] Figure 12 The results show the effect of light on the stability of microcapsules;

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

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

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

[0038] Figure 15 The protein expression of ERP in cells at different time points under nicotine exposure;

[0039] Figure 16 The expression of GRP78 protein in cells at different time points under nicotine exposure;

[0040] Figure 17 Protein expression of ERP after microcapsule treatment of cells in a nicotine environment;

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

[0042] Figure 19 The effects of M4 microcapsule intervention on ER stress and lipid-dysregulated gene expression levels induced by smoke.

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

[0044] Figure 21 The effect of M4 microcapsule intervention on smoke-induced lipid peroxidation (MDA);

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

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

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

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

[0049] Figure 26 The effect of M4-formula microcapsule intervention on the protein expression of ER stress and lipid metabolism markers in RPE cells; among which, Figures 19-26 In the figure, ** indicates a highly significant difference compared to the model group (p < 0.01), * indicates a significant difference compared to the model group (p < 0.05); ## indicates a highly significant difference compared to the control group (p < 0.01), # indicates a significant difference compared to 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 adjust the effect of microcapsule intervention after M4 formulation on the expression levels of genes dysregulated by smoke-induced ER stress and lipid disorders;

[0053] Figure 30 This is a statistical result of CRT indicators in different treatment groups during a clinical study;

[0054] Figure 31 This is a statistical result of BCVA index in different treatment groups during a clinical study;

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

[0056] Figure 33This is a statistical result of the NEI-VFQ score for different treatment groups in the clinical study. Detailed Implementation

[0057] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0059] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0060] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0062] Lutein is a carotenoid obtained from various foods, such as marigold flowers, dark green leafy vegetables, and egg yolks. Lutein possesses antioxidant activity, scavenging reactive oxygen species, such as singlet oxygen and lipid peroxide free 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 regulation of oxidant-sensitive inflammatory signaling pathways. Therefore, it may help prevent various inflammatory diseases, including neurodegenerative diseases, diabetic retinopathy, osteoporosis, cardiovascular diseases, skin diseases, liver damage, obesity, and colitis. Furthermore, lutein has tissue-specific effects, such as regulating lipid profiles in cardiovascular diseases, adipocyte differentiation in obesity, and skin function. Therefore, consuming foods rich in lutein may help prevent inflammatory diseases caused by oxidative stress.

[0063] The photothermal instability of lutein has largely limited its development and application, while microencapsulation is an effective method to enhance the stability of unstable bioactive ingredients. It utilizes stable materials to microencapsulate core components (such as capsicum oleoresin). Among the various microencapsulation technologies developed, spray-dried microcapsules are a simple method that protects active compounds from adverse reactions, harsh environments, and interactions with other components. It improves the solubility of active substances, increases stability in harsh environments, and masks unpleasant odors.

[0064] Among various microencapsulated core-shell materials, ionic polymers are commonly used, such as sodium alginate. Sodium alginate, derived from brown algae, is a multifunctional 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), when used in combination with traditional emulsifiers, can improve emulsion stability and antioxidant properties. It can bring antioxidant actives to the oil-water interface, making it valuable in emulsions seeking antioxidant effects. Polysorbate 80 is a synthetic nonionic surfactant widely used as an emulsifier in emulsions. It has multiple functions, including emulsification, dispersion, wetting, and stabilization. White mustard extract plays a crucial role in the formation of traditional oil-in-water emulsions. This extract contains lecithin, a major emulsifier in egg yolk, which contributes to emulsion stability.

[0065] The complement system is an evolutionarily ancient part of the innate and adaptive immune system, designed to participate in the clearance of pathogens and non-self cells from the organism. To perform this function, there are three initiation pathways (classical, lectin, and alternative), all leading to a common terminal pathway through the production of homologous complement C3 convertases. Activation of the complement system results in the production of three classes of effector molecules: soluble anaphylatoxins C3a and C5a, which signal via their respective G protein-coupled receptors (C3aR and C5aR) and participate in chemotaxis and mediate inflammatory responses; cell-bound opsonins C3b, C3d, and iC3b, which, upon cleavage, originate 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. Expression of membrane-bound complement inhibitory molecules and soluble serum inhibitors can protect self-cells from complement-mediated damage, thus protecting them from complement activation. However, because the levels and cellular localization of these inhibitors can be influenced by environmental factors such as oxidative stress or cigarette smoke, the surface of self-cells can become a target for complement activation. Cigarette smoke has been shown to directly activate C3 by modifying C3, thereby reducing its ability to bind to CFH, and serum levels of complement components are elevated in smokers.

[0066] Lipid accumulation is a hallmark of age-related macular degeneration, and in the BrM (Breast Microvascular Membrane), it manifests as extracellular lesions. Analysis of the lipid composition in the BrM revealed that the lipoprotein particles differ from those found in plasma. They contain free and esterified cholesterol, phosphatidylcholine, and apolipoprotein B100. ER stress has been shown to activate a transcription factor called sterol regulatory element-binding protein (SREBP), thereby increasing the expression of proteins required for cholesterol and triglyceride biosynthesis and uptake.

[0067] This invention, through screening of core-shell materials and emulsifiers, optimizes the microcapsule formulation to obtain the best possible microcapsule composition. A microcapsule powder product with ideal encapsulation effect and stability is then prepared using high-pressure homogenization and spray drying methods. Furthermore, the microcapsule powder product of this invention effectively improves symptoms of AMD caused by cigarette smoke. In vivo and in vitro experiments further investigated the mechanism of intervention in AMD. Experimental studies confirmed that the microcapsule powder product of this invention can effectively improve the symptoms of this ocular disease caused by cigarette smoke through oxidative stress and complement activation pathways. In addition, clinical studies have found that the microcapsule powder product of this invention can enhance the therapeutic effect of ranibizumab on smoking-induced wet AMD. Compared with ranibizumab alone in the treatment of wet AMD, the combined intervention with the microcapsule powder product of this invention resulted in more ideal recovery effects, a higher clinical cure rate, and a lower incidence of adverse reactions in AMD patients.

[0068] The reagents and materials used in this invention are as follows: β-CD was purchased from Sigma-Aldrich (Hamburg, Germany); Folin-Ciocalteu phenol reagent (Merck, Darmstadt, Germany); sodium alginate was sodium alginate salt from brown algae obtained from Sigma-Aldrich (Shanghai, China). Chitosan (high molecular weight (HMW) 80 / 3000) was purchased from Sigma-Aldrich (Steinheim, Germany). Polysorbate 80 (Tween) The following reagents were purchased from Sigma-Aldrich (Germany): Resveratrol (CAS No.: 501-36-0) and Acetic acid (Shanghai Koraman Reagent Co., Ltd.). Solvents used for extraction and analysis of samples containing hexane, acetonitrile, methanol, and dichloromethane were HPLC-grade solvents purchased from Sigma-Aldrich. All other chemicals and reagents were analytical grade and purchased from Sigma-Aldrich. Lutein ester (CAS No.: 547-17-1) was purchased from Shaanxi Baiwei Biotechnology Co., Ltd.

[0069] The mustard extract in this invention is an ethanol extract of common white mustard seeds. The preparation method is as follows: Prepared white mustard seeds are placed in a fully automated liquid nitrogen cryogenic grinder for rapid, low-temperature mixing and grinding, maintained at -20°C until extraction. The ground seeds are then poured into a mixed solvent of ethanol and water (ethanol:water volume ratio 8.5:2), ultrasonically treated for 25 minutes (21kHz, 145W), and kept at room temperature for 24 hours. The suspension is filtered through a Buchner funnel onto paper, and the filtrate is dried under vacuum to obtain the mustard extract.

[0070] In a specific embodiment of the present invention, the white mustard seeds were purchased from Sichuan, and other commercially available ordinary white mustard seed products can achieve the same effect.

[0071] The technical solution of the present invention will be shown 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 formulation

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

[0078] Table 1 Microcapsule Formulation

[0079]

[0080] 3. Preparation of emulsion

[0081] (1) Preparation of shell solution: According to the formula in Table 1, sodium alginate and / or chitosan are dispersed in 152g of water as wall material, heated at 67°C and stirred at 350rpm for 35min, and then cooled in cold water (4°C) overnight to ensure complete hydration, and set aside for use.

[0082] (2) Emulsion Preparation: 26 mL of 0.35 g / mL sodium alginate solution and the required amounts of emulsifier (mustard extract, β-cyclodextrin, polysorbate 80) were stirred at 22°C for 15 minutes. The required amounts of peppermint oil or almond oil (0.028-0.072 g / mL), lutein ester, and resveratrol were added to the solution. The emulsion was stirred at 23°C for 16 minutes, followed by pre-emulsification for 3 minutes using a handheld homogenizer (Biospec Products Inc., USA). The crude emulsion was further homogenized five times using 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 and encapsulation of emulsion

[0084] Microcapsule powder was prepared using an experimental spray dryer (BILON-6000Y, Bilon Instruments, Shanghai, China). The homogenized emulsion was continuously stirred during spraying and fed into the main chamber at a feed rate of 9.5 mL / min. The suction volume and spray pressure were set to 0.58 m³ / min. 3 / min and 0.48MPa. The inlet temperature was set to 158℃, and the outlet temperature varied between 70 and 90℃. The spray-dried powder was collected from the bottom of the cyclone separator and transferred to a self-sealing bag, then stored at -18℃.

[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 centrifugation at 3000 to 7000 rpm for 5 minutes each time. The percentage of non-stratified emulsion was calculated using the following formula, known as the centrifugation index (CI):

[0088] CI = Ve / Vi × 100%

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

[0090] 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 formulations tested at two different rotation speeds, formulations M3, M4, and M5 exhibited better stability than the other formulations.

[0091] 2. Particle size distribution measurement

[0092] Experimental Procedure: The particle size and distribution of the oil were analyzed using a Mastersizer 3000 equipped with a Hydro EV device (Malvern Analytical Ltd., Malvern, UK). The emulsion was added dropwise to water until the laser obscuration 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 after five runs. The results are as follows: Figure 3 As shown.

[0093] from Figure 3 It can be seen that the average particle size of emulsions M3 and M4 is approximately 0.5 μm, the average particle size of emulsion M1 is approximately 0.9 μm, and the average particle size of the remaining emulsions is 0.7 μm. The overall lutein emulsion formulations have relatively small particle sizes, resulting in the preparation of microemulsions. Among these, emulsions M3 and M4 have even smaller and more ideal average particle sizes.

[0094] 3. Dynamic viscosity

[0095] Emulsion viscosity was evaluated using a Fungilab ALPHA series viscometer (Fungilab, Sant Feliu de Llobregat, Barcelona, ​​Spain). For each measurement, 50 mL of emulsion was added to the viscometer's dedicated container and placed on the instrument surface. An L3 head was used and set to 100 rpm. Viscosity (mPa·s) was recorded at room temperature. Measurements were taken for each sample until the dynamic viscosity value stabilized. Results are as follows: Figure 4 As shown.

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

[0097] Example 3: Study on the properties of microcapsules

[0098] 1. Size (diameter) of microcapsules

[0099] Microcapsule sizes were determined using digital calipers (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 microcapsules prepared by the M3 and M4 formulations have smaller diameters than those prepared by the other formulations. The microcapsules prepared by the M3 and M4 formulations have a diameter of about 1.6 mm, while the microcapsules prepared by the other formulations have a diameter of about 1.8 mm.

[0101] 2. Hardness of microcapsules

[0102] XT.plus texture analyzer (Texture Technologies, Brewster, NY, USA). Using a P / 100 pressure plate probe, the force required to compress 2 mm microcapsules on freshly prepared samples 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 the five measurements.

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

[0104] 3. Swelling properties of microcapsules

[0105] Experimental Procedure: The dried microcapsules were swelled in water. After 4 hours, the microcapsules were weighed, separated, filtered through a metal mesh, and dried 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 microcapsule and Wi is the weight of the dried microcapsule.

[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 superior to those of the other formulations.

[0109] 4. Encapsulation efficiency of microcapsules

[0110] Method for determining the encapsulation efficiency (EE) of lutein microcapsules: Microencapsulated lutein powder composition was dissolved in anhydrous ethanol to prepare solutions of different concentrations. The absorbance of the solutions was measured at 446 nm using a double-beam UV / Vis spectrometer with an ethanol solution (96%, v / v) as a blank. A standard curve was constructed by establishing the relationship between lutein absorbance and lutein concentration, which is given by the equation A = 2.145C + 0.0011(R²). 2 =1) Description. Accurately weighed lutein microcapsule particles (20 mg) were dissolved in 1 mL of deionized water using an ultrasonic-assisted method, and then diluted to 20 mL with anhydrous ethanol. Accurately weighed lutein microcapsule particles (30 mg) were diluted to 20 mL with anhydrous ethanol, while shaking for 1 min. Both solutions were then centrifuged at 5 °C (4500 rpm, 10 min). The absorbance of the supernatant was measured, and the surface lutein content and total lutein content of the microcapsules were calculated. The encapsulation efficiency was quantitatively calculated using Formula A below, and the results are shown in [Figure 1]. Figure 8 .

[0111] Formula A:

[0112] from Figure 8 The results show that the encapsulation efficiency of lutein microcapsules prepared by formulations M1, M6, and M7 was relatively low, all failing to reach 50%. In contrast, the encapsulation efficiencies of the remaining formulations ranged from 51% to 81%, achieving better encapsulation efficiency and indicating that the system possesses excellent loading capacity. Furthermore, formulations M3 and M4 showed even better encapsulation effects, achieving encapsulation efficiencies exceeding 75%, with formulation M4 reaching the highest encapsulation rate of 81%, indicating that the microcapsules prepared by formulation M4 had the most ideal loading capacity.

[0113] 5. Measurement of solubility

[0114] Experimental Procedure: Dissolve lutein microcapsule powder (dry basis) in deionized water and centrifuge at 4°C (4000 rpm, 10 min). Then discard the supernatant. Repeat several times, wash the residue with deionized water into a pre-determined mass aluminum container, and dry to constant weight. Solubility (S) is calculated using Formula B below. Results are shown in [Figure Number]. Figure 9 As shown.

[0115] Formula B: S(%) = [1 - ((total weight of aluminum box and insoluble matter - weight of aluminum box) / weight of sample)] × 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 good solubility, ranging from 42% to 85%. Except for the M1, M2, M6 and M7 formulations, whose solubility did not reach 60%, the solubility of the other three formulations of microcapsule powder all reached more than 60%. Among them, the M4 formulation of microcapsules had the highest solubility, and the solubility of the microencapsulated lutein composition prepared by the M4 formulation could reach 85%.

[0117] In summary, based on Examples 2 and 3, among the seven microcapsule formulations, formulations M3 and M4 exhibit the best microcapsule properties, demonstrating superior encapsulation and stabilization effects on lutein. Therefore, microcapsules from formulations M3 and M4 will be selected for further research.

[0118] Example 4: Stability Study of Microcapsules

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

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

[0121] Depend on Figure 10 It is evident that increasing temperature directly leads to a decrease in the OD of lutein, indicating that high temperatures have a certain destructive effect on lutein. However, overall, the absorbance of microencapsulated lutein only decreased slightly compared to the control group, natural lutein. When heated to 80°C, the absorbance of natural lutein decreased to 35.8%, while the absorbance of microencapsulated lutein only decreased to 48.5%. At 100°C, the absorbance of lutein in the microcapsules (M3, M4) of both formulations decreased by only the same proportion, approximately 16.64%, while the absorbance of natural lutein decreased by approximately 53.97%. Therefore, the microcapsules of this invention play a significant role in protecting lutein from high-temperature damage.

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

[0123] Experimental Procedure: Following the same preparation method as in "1. Study on the Stability of Microcapsules by Temperature", 20 ml of each solution was taken, and the pH was adjusted to 2, 4, 6, 8, and 10 using HCl and NaOH. The changes in the OD value of lutein in the microcapsules were then detected. The experimental results are as follows: Figure 11 As shown.

[0124] according to Figure 11 As shown, the greater the decrease in pH, the more positively correlated and continuously decreasing the absorbance value. When the pH value decreased from 10 to 2, the absorbance of lutein in the microcapsules decreased by only about 8.24%. However, the absorbance value of natural lutein decreased by about 18.94%. Furthermore, we can see from the figure that when the pH value decreased from 4 to 2, i.e., entering a strongly acidic environment, the absorbance value of natural lutein (i.e., the lutein in the control group C) showed a very obvious decreasing trend, while the absorbance value of the microencapsulated lutein in the two formulations of this invention still showed a very slow decreasing trend. Of course, compared with the microcapsules of formulation M3, the absorbance value of lutein in the microcapsules of formulation M4 did not decrease significantly when entering a strongly acidic environment. This indicates that natural lutein is very unstable in a highly acidic environment, while the lutein in the microcapsules can be used in a wider range of acidic and alkaline environments. Moreover, the microcapsules of formulation M4 are more resistant to acids and alkalis than the microcapsules of formulation M3, and are more stable in strongly acidic environments.

[0125] 3. Effects of light on lutein stability

[0126] Experimental procedure: Following the same preparation method as in "1. Study on the stability of microcapsules by temperature", the microcapsules were exposed to natural light at room temperature (25℃) for 2, 4, 6, 8, and 10 days, with distilled water as a control. The absorbance was measured at 445 nm. The experimental results are shown below. Figure 12 As shown.

[0127] from Figure 12 It can be seen that natural lutein is more sensitive to external light than microencapsulated lutein. The absorbance decreases more significantly with prolonged illumination, especially from day 6 to day 10 of illumination, when the absorbance of natural lutein (group C) decreased by approximately 6.9%, while the absorbance of lutein in the M4 microcapsules decreased by only approximately 1.4% during the same period. After 10 days of irradiation, the absorbance of the natural lutein group (group C) decreased by a total of 8.4%, while under the same conditions, the microencapsulated lutein exhibited relatively stable performance, with absorbance decreasing by only 3.8% (M4 formula) and 4.64% (M3 formula). Therefore, under light conditions, the microencapsulated lutein of this invention can effectively improve the light-instability of lutein, making it more suitable for food production and storage.

[0128] Next, this invention will investigate the beneficial effects of microcapsules prepared from formulations M3 and M4 on improving smoking-induced age-related macular degeneration (AMD), an eye disease. This is because tobacco extracts induce endoplasmic reticulum (ER) stress and oxidative stress in retinal pigment epithelial (RPE) cells, exacerbating unfolded protein responses and impairing RPE tight junctions. Endoplasmic reticulum protein 29 (ERp29), a novel chaperone protein, is reduced in expression in AMD patients and can alleviate RPE cell ER stress and tight junction damage. Therefore, this invention examined the expression of ERp29 in AMD mice before and after administration of the microcapsules of this invention to determine whether the microcapsules can regulate ERp29 expression and the mechanism of smoke-induced ER stress.

[0129] Example 5: In vitro experimental study of microcapsule products for the prevention and improvement of nicotine-induced age-related macular degeneration (AMD).

[0130] 1. Cell culture and cell treatment

[0131] Human ARPE-19RPE 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 epithelium (ARPE-19) cells were cultured in DMEM / F-12 1:1 medium containing 10% fetal bovine serum (FBS) at 37°C in a cell culture incubator with 5% CO2. The cells were then cultured for another 24 hours in medium containing CSE solution.

[0133] Preparation of Cigarette Smoke Extract Solution (CSE Solution Preparation): The smoke from one burning cigarette was dissolved in 1 mL of LDM / F-12 medium. The mixture was filtered using a 0.22 μm microporous filter to remove large particulate impurities and bacteria, and the pH of the mixture was adjusted to approximately 7.4. The final CSE solution was labeled 100% and stored at -80°C for later use.

[0134] 2. Western imprint analysis

[0135] Total protein was extracted on ice for 20 min using RIPA lysis buffer (Beyotime, Shanghai, China) containing a mixture of protease inhibitors. 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 0.45 μm polyvinylidene fluoride membranes (Millipore, MA, USA). Each membrane was blocked with 5% skim milk for 2 hours at room temperature, and then incubated with primary antibody overnight at 4°C. After washing three times with Tris-buffered saline-0.1% Tween-20 (TBST), the membrane was incubated with secondary antibody for 1 hour at room temperature. The membrane was washed three times again with TBST and then visualized using Immobilon Western chemiluminescence HRP substrate (Millipore, MA, USA). Experimental results are as follows. Figures 13 to 16 As shown.

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

[0137] In addition, from Figure 15 and Figure 16 It can be seen that the duration of exposure to nicotine also affects the expression of ERP and GRP78. When exposed to nicotine for 24 hours and 48 hours, the effect on ERP expression was significantly different from the blank control (P < 0.01). The effect of nicotine exposure for 24 hours on GRP78 expression was more significant (compared to 48 hours), and it was significantly different from the blank control (P < 0.01). However, the expression of GRP78 after 48 hours of exposure was significantly different from the blank control (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 conditions for subsequent research to investigate the effect of the microcapsule formulation on mitigating nicotine-induced changes. The results are as follows: Figure 17 and Figure 18 .from Figure 17 and Figure 18It can be seen that the microcapsules of both formulations can improve the expression of ERp29 and GRP78 in nicotine-induced cells, and the microcapsules of formulation M4 are more ideal in improving the expression of ERp29 and GRP78 in nicotine-induced cells than those of formulation M3.

[0139] Based on the aforementioned experimental results, it can be seen that the microcapsules prepared by the M4 formulation have superior properties. Therefore, the next step is to further investigate the intervention mechanism of the optimal M4 microcapsules on smoke-induced AMD through in vivo and in vitro experiments.

[0140] Example 6: Cell and animal experiments on the optimal formulation of M4 microcapsules for the prevention and improvement of smoke-induced AMD, and a study on the mechanisms of oxidative stress and complement activation cascades involved.

[0141] 1. Animal experiments

[0142] Nine C57BL / 6J mice were purchased and divided into three groups: a blank control group, a model group, and an M4 microcapsule treatment group (M4 intervention). Animals were housed in a 12 / 12-hour light / dark cycle environment 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 kept in a filtered air environment, while the model and treatment groups were exposed to cigarette smoke. After 6 months of simulating a smoke environment using a smoking machine (model TE-5; Teague Enterprises), the average concentration of total suspended particulate matter was 130 mg / m³. 3 Mice in the treatment group were monitored twice daily. Each mouse in the treatment group was administered the microcapsule powder via gavage once daily at a dose of 24 mg / kg body weight (bw). The control and model groups were administered the same dose of physiological saline via gavage.

[0143] 2. Cell Culture System

[0144] Experiments were conducted on ARPE-19 cells by adding microcapsule powder solution to a culture medium containing CSE solution.

[0145] Preparation of microcapsule powder solution: Dissolve 1g of microcapsule powder in 98% ethanol aqueous solution, sonicate for 15 minutes, filter, and store the supernatant at -10℃ for later use.

[0146] The ARPE-19 cell line is a human RPE cell line exhibiting the differentiation phenotype of RPEW cells and forming 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 reaching confluence, cells were grown in serum medium until stable, based on transepithelial resistance (TER) measurements. TER values ​​reached 40-45 Ωcm within 2-3 weeks after confluence.2 A stable platform. Complete removal of FBS in the final days before measurement does not alter survival or monolayer formation, thus allowing the use of complement-sufficient normal human serum as a complement source.

[0147] 3. ELISA measures C3 and C3a

[0148] To measure the production of C3 and C3a by cells, cell culture supernatant was 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 using C3 or C3a-specific antibodies conjugated with horseradish peroxidase, followed by development with OPD (Sigma) chromogenic substrate. Absorbance was measured at 492 nm. Two aliquots were measured, and the results were compared with dose-response curves for C3 and anaphylactic toxins.

[0149] 4. Western blot analysis

[0150] Cell culture supernatant was separated by electrophoresis on a 10% BisTris polyacrylamide gel (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 observed using a chemiluminescence assay kit (Amersham Biosciences Life Science). Band density was analyzed and normalized using an Alpha Innotech Fluorchem 9900 imaging system while running Alpha Ease FC software 3.3 (Alpha Innotech, San Leandro, CA), and normalization was performed for the GAPDH control.

[0151] 5. Measurement of oxidative stress

[0152] Cells were collected after treatment on a Transwell filter, stained with 20 mCDCFDA (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 appropriate treatment and homogenized in MDA lysis buffer (Abcam). The MDA present in the cells was reacted with thiobarbituric acid to form an MDA-thiobarbituric acid adduct, and quantified colorimetrically 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 components (hereinafter 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 quantitative PCR analysis in triplicate was performed using the 5700 sequence detection system (Applied Biosystems) under standard cycling conditions. Quantitative values ​​were obtained by cycle number, the target gene was normalized to β-actin, and the fold change between experimental and control samples was determined. Z-tests were used to compare fold changes, with p < 0.05 considered significant. Experimental results are shown below. Figures 19 to 26 As shown.

[0155] 7. Results

[0156] from Figure 19 It can be seen that the gene expression levels of the Er stress markers Grp78 and Chop on the RPE / choroid of mice affected by the smoke environment were increased by approximately 2.4 times and 8.3 times, respectively, compared with normal mice. The change in Grp78 was significantly different from the control group (P < 0.05), and the change in Chop was extremely significantly different from the control group (P < 0.01). However, after intervention with the microcapsule formulation M4, the expression levels of these two markers in mice did not change significantly compared with normal mice, indicating that the microcapsules of this invention can effectively intervene in the gene expression of the Er stress markers Grp78 and Chop. Furthermore, Srebf-1, a marker indicating altered cholesterol and fatty acid homeostasis, also increased by approximately 4.7 times in the RPE / choroid of smoke-affected mice, which was significantly different from the control group (P < 0.05). However, after intervention with the microcapsule formulation M4, the expression level of Srebf-1 did not change significantly compared with normal mice. This indicates that smoky environments do indeed affect the expression levels of ER stress markers and lipid dysregulation markers on the RPE / choroidal membrane, and the microcapsule formulation of this invention can improve the expression levels of these smog-induced ER stress markers and lipid dysregulation markers on the choroidal membrane to normal values.

[0157] Smoke exposure induces oxidative stress in RPE cells, leading to free radical production and depletion of the antioxidant system. The experimental results of this invention also confirm that exposure to smoke for 2 hours results in a rapid increase in ROS levels. Figure 20ROS levels remained elevated 12 and 24 hours after CSE exposure. However, after intervention with the microcapsule formulation of this invention, intracellular ROS levels decreased significantly after exposure to a smoke environment, especially after 12 and 24 hours of exposure. The microcapsule intervention effect of this invention was more pronounced, indicating that the microcapsule formulation of this invention can improve AMD by regulating the oxidative stress pathway induced by the smoke environment.

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

[0159] from Figure 22 It can be seen that measurements of the ARPE-19 cell supernatant after 24 hours of smoke stimulation showed a significant increase in complement molecule C3, reaching approximately 27 ng / mL. However, after microcapsule intervention with the M4 formulation, the C3 level significantly decreased, showing a highly significant difference compared to the model group (P < 0.01). Furthermore, Figure 24 Quantitative RT-PCR analysis showed an increase in C3 mRNA levels 24 hours after smoke exposure, indicating that smoke induces C3 transcription. After intervention with the M4 microcapsule formulation, transcription levels recovered to near the normal control group level. Furthermore, from... Figure 23 The results of C3a measurement showed that short-term smoke exposure led to the cleavage of approximately 3.15% of C3 into C3a. After intervention with the M4 microcapsule formulation, the C3a content also decreased significantly, showing a highly significant difference compared to the model group (P < 0.01). These results indicate that the pathogenesis of smoke-induced AMD involves a complement activation cascade mechanism. The smoke environment stimulates changes in the expression of complement regulatory proteins C3 and C3a, and C3 and its cleaved C3a are complement components of drusen, a hallmark extracellular deposit associated with early AMD. Therefore, the experimental results confirm that the microcapsule formulation of this invention can improve AMD by regulating the complement cascade mechanism regulated by the C3a signaling pathway.

[0160] Figure 25Quantitative RT-PCR results further indicated that smoke stimulation affects cellular ER stress and lipid homeostasis. After smoke stimulation, the ER stress markers GRP78 and CHOP increased by approximately 2.8-fold and 12.7-fold, respectively, with CHOP 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 Furthermore, these fold differences were found to be similar to the differences identified in mouse RPE in vivo (). Figure 19 ).

[0161] Furthermore, it was found that after smoke stimulation, GRP78, CHOP, and XBP1 not only showed increased mRNA levels but also significantly increased protein expression, with significant differences compared to the normal control group (P < 0.05, # shown). Figure 26 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 from those in the model group (P < 0.05, *); the changes in XBP1 were extremely significant compared with those in the model group (P < 0.01, **), indicating that the microcapsules of the present invention can effectively improve AMD induced by cigarette smoke through the complement-mediated cellular ER stress pathway.

[0162] Example 7: Synergistic effect of lutein and resveratrol in microcapsule formulation on improving smoke-induced AMD

[0163] To investigate whether the combined use of lutein and resveratrol in the microcapsule formulation of this invention can have a synergistic effect on improving AMD caused by smoke, a comparative experiment was conducted based on Example 6.

[0164] Formula M4-2, obtained by removing resveratrol from the original M4 formula, and formula M4-3, obtained by removing lutein esters from the original M4 formula, were compared with the original M4 formula in terms of their effects. The experimental procedure was the same 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] Experimental results are as follows Figures 27-29 As shown. By Figures 27-29 It can be seen that after removing resveratrol or lutein ester from the M4 formulation, the improvement effect on oxidative stress and lipid homeostasis indicators of AMD caused by smoke stimulation was not as good as the original M4 formulation. This indicates that the combination of lutein ester and resveratrol in the original M4 formulation has a synergistic effect on improving the symptoms of AMD under smoke stimulation.

[0168] Example 8: Human Clinical Study

[0169] A total of 56 patients (56 eyes) with smoking-induced wAMD treated at the Affiliated Hospital of Shandong University from January 2020 to December 2022 were included in the clinical data. They were randomly divided into two groups of 28 patients (28 eyes) each. The study was approved by the Medical Ethics Committee. In the observation group, there were 20 males and 8 females, aged 45–85 years, with a mean age of (65.85±3.31) years; 18 cases involved the left eye and 10 involved the right eye. In the control group, there were 17 males and 11 females, aged 48–84 years, with a mean age of (66.07±3.29) years; 15 cases involved the left eye and 13 involved the right eye. There were no statistically significant differences in general characteristics between the two groups (P>0.05), making them comparable.

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

[0171] 2. Exclusion criteria: ① Comorbid eye diseases; ② History of drug allergy; ③ Abnormal mental and behavioral patterns and low compliance; ④ Poor blood pressure and blood sugar control; ⑤ Previous treatment such as photodynamic therapy.

[0172] 3. Treatment Intervention: Both groups received intraocular injection of ranibizumab (Novartis Pharma Schweiz AG, approval number S20181010). Levofloxacin eye drops were administered four times daily for three days prior to surgery. The intraocular injection was performed in the operating room. After disinfection and draping, and anesthesia, the needle was inserted at the ciliary body flattening point 3.5–4 mm from the limbus, perpendicular to the eyeball wall, and 0.05 mL of ranibizumab was injected. Sterile cotton swabs were applied, antibiotic eye ointment was applied, and the eye was bandaged. Injections were given once a month for three consecutive months. The observation group additionally received M4 microcapsules, 5 mg three times daily, for three months.

[0173] 4. Observation Indicators

[0174] CRT (Central Retinal Thickness): CRT was measured using OCT before treatment and 3 months after treatment.

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

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

[0177] Visual-related quality of life: The NEI-VFQ-25 scale was used to assess visual-related quality of life before treatment and 3 months after treatment. It includes 12 dimensions and 25 items, with a score range of 0 to 100. Visual-related quality of life was positively correlated with the score.

[0178] 5. Evaluation of treatment effectiveness

[0179] Clinical cure: Visual acuity improved (≥2 more lines on the visual acuity chart), and the thickness of the fovea of ​​the macula decreased (≥60μm less than before);

[0180] Significant improvement in disease symptoms (such as retinal neovascularization leakage): Effective results include improved visual acuity (1-2 more lines on the visual acuity chart) and thinning of the fovea (40-59 μm less than before);

[0181] The symptoms of the disease have improved;

[0182] Invalid means that the above standards have not been met.

[0183] Overall effective rate = (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 analyzed using the χ² test, while continuous data were expressed as mean ± standard deviation (x±s) and analyzed using the t-test. P < 0.05 was considered statistically significant.

[0185] 6. Results Analysis

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

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

[0188] Group Number of examples Clinical cure efficient invalid Overall 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. Occurrence of adverse reactions and recurrence rate within 1 year (n / %)

[0190]

[0191] from Figure 30 As can be seen, compared with the pre-treatment effect of the observation group, after the additional intervention and treatment of wAMD with the microcapsule product of this invention, the CRT index of the patients was reduced to a certain extent, which was significantly different from that before treatment (P<0.05). At the same time, compared with the control group, the CRT of the observation group showed a significant decreasing trend after treatment, and there was also a significant difference between the two (P<0.05).

[0192] from Figure 31 It can be seen that when microcapsules and ranibizumab were used in combination for treatment, the BCVA index of AMD patients decreased to a certain extent compared with that before treatment, and the difference between the two was statistically significant (p<0.05). Furthermore, it also decreased to a certain extent compared with the group treated with ranibizumab alone (control group), and the difference between the two was statistically significant (P<0.05). This indicates 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 when microcapsules and ranibizumab were used together for treatment, the area of ​​retinal hemorrhage in AMD patients was significantly reduced, with a highly significant difference compared to before treatment (P<0.01). Compared with the ranibizumab-only treatment group (control group), the effect was also reduced to a certain extent after treatment, with a significant difference between the two (P<0.05). This indicates that the microcapsule formulation 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 of microcapsules and ranibizumab, the NEI-VFQ-25 score of AMD patients increased significantly, showing a significant difference compared with that before treatment (P<0.05); at the same time, compared with the group treated with ranibizumab alone (control group), the NEI-VFQ-25 score also increased to a certain extent after the addition of the M4 group microcapsules of this invention, and the two showed a significant difference (P<0.05).

[0195] As can be seen from the changes in the data in Table 3, compared with ranibizumab alone for the treatment of wet AMD, the combined intervention of the microcapsules of this invention and ranibizumab for the treatment of wet AMD can increase the clinical cure rate of smoking-induced wet AMD 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 this invention are used in combination with ranibizumab to treat smoking-induced wet AMD, compared with ranibizumab alone, the two adverse reactions caused by ranibizumab alone are eliminated: retinal detachment and persistent intraocular pressure elevation. The incidence of endophthalmitis is reduced by 14.29%, and the incidence of one-time intraocular pressure elevation is reduced by 7.15%. More importantly, the overall incidence of adverse reactions is reduced by 28.57%, and the recurrence rate within one 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 ranibizumab monotherapy in wet AMD, and can significantly reduce the incidence of adverse reactions when ranibizumab monotherapy is used to treat AMD. It has great potential in future clinical development and application.

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

Claims

1. A composition for improving age-related macular degeneration, characterized in that: Active ingredients include lutein and resveratrol.

2. Use of the composition according to claim 1 in preparing a product for improving age-related macular degeneration.

3. A product for improving age-related macular degeneration, characterized in that: The active ingredient comprises the composition according to claim 1.

4. The product according to claim 3, characterized in that The product comprises microcapsules.

5. The product according to claim 4, characterized in that The microcapsule is prepared by using the composition of claim 1 as a core material, sodium alginate and chitosan as wall materials, and mustard extract, β-cyclodextrin and polysorbate 80 as emulsifiers.

6. The product according to claim 5, characterized in that The raw materials for preparing the microcapsules include the following components 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.

7. The product according to claim 3, characterized in that The age-related macular degeneration is triggered by smoke.

8. The product according to claim 3, characterized in that The age-related macular degeneration is wet age-related macular degeneration.

9. Use of the composition of claim 1 or the product of any one of claims 3 to 8 in the preparation of a product for improving the efficacy of ranibizumab in treating wet age-related macular degeneration.

10. Use of the composition of claim 1 or the product of any one of claims 3 to 8 in the preparation of a product for preventing adverse reactions caused by ranibizumab in the treatment of wet age-related macular degeneration.

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