Ophthalmic composition containing anthocyanin, resveratrol and xanthophyll, preparation method, ophthalmic eye drops, ophthalmic gel and application of ophthalmic composition

Abstract

The invention discloses an ophthalmic composition containing anthocyanin, resveratrol and lutein, a preparation method, eye drops for eyes, gel for eyes and application of the ophthalmic composition and belongs to the technical field of ophthalmic medicines.The composition is prepared from 0.1%-0.5% of anthocyanin, 0.05%-0.2% of resveratrol, 0.01%-0.1% of lutein and phosphate buffer salt solution cosolvent, according to the invention, a phosphate buffer salt solution is adopted as a cosolvent system to solve the problem of dissolution of fat-soluble components, local delivery of the three active components on the ocular surface is realized, and the synergistic effects of oxidation resistance, retina protection and blue light protection are exerted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of ophthalmic drugs, in particular to an ophthalmic composition containing anthocyanins, resveratrol and lutein, a preparation method, an eye drop, an eye gel and applications thereof. BACKGROUND

[0002] With the substantial increase in the use of electronic screens, eye health problems such as dry eye, visual fatigue, and blue light damage have become increasingly prominent and have become a global public health problem. According to epidemiological survey data, the prevalence of dry eye in the general population is as high as 5% to 50%, and there is a clear trend of youth. Long-term use of computers, mobile phones and other electronic devices leads to a decrease in blink rate and an acceleration of tear film evaporation, causing ocular surface inflammation and corneal epithelial damage. At the same time, the high-energy blue light emitted by electronic screens continuously irradiates the retina, causing oxidative stress damage to retinal pigment epithelial cells and retinal ganglion cells, ultimately leading to retinal function degradation and vision loss.

[0003] Lutein is a carotenoid widely found in fruits, vegetables and other plants, and is the main pigment in the macular region of the human eye retina. Humans cannot synthesize lutein themselves and can only rely on intake from external food. Lutein has strong antioxidant activity and can effectively prevent oxidation, preventing cataracts and macular degeneration. The absorption spectrum of lutein contains near blue-violet light, with a peak absorption wavelength of 415 to 455 nanometers in the high-energy blue light segment, which can help the retina of the eye resist ultraviolet and blue light and avoid light oxidative damage to the retinal pigment epithelial cells. Studies have shown that lutein can effectively filter blue light and reduce phototoxic damage to photoreceptor cells.

[0004] Anthocyanins are a class of water-soluble natural pigments widely found in berry plants, especially in European blueberries. Blueberry is a plant of the Ericaceae family and the Vaccinium genus. Mature blueberries are deep purple due to their high content of anthocyanins. Anthocyanins have a unique effect of promoting rhodopsin regeneration, which can accelerate the resynthesis of rhodopsin and enhance the sensitivity of the retina to light. Cyanidin anthocyanins are the most active component of blueberry anthocyanins and have been shown to bind to rhodopsin molecules and promote their regeneration cycle. In addition, anthocyanins also have strong antioxidant capacity, can effectively scavenge free radicals, eliminate oxidative damage to retinal cells by ultraviolet light, reduce blue light-induced damage to retinal photoreceptor cells, and have the effect of preventing cataract formation, macular degeneration and glaucoma.

[0005] Resveratrol is a non-flavonoid polyphenol compound mainly found in grape skins, grape seeds, and red wine. It possesses multiple biological activities, including antioxidant, anti-inflammatory, and anti-apoptotic effects. Recent studies have shown that resveratrol has significant protective effects against ophthalmic diseases such as glaucoma, age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, and uveitis. Resveratrol can activate the SIRT1 signaling pathway, promoting the deacetylation and nuclear translocation of downstream peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), thereby upregulating the expression of mitochondrial transcription factors and nuclear respiratory factors, enhancing mitochondrial biosynthesis, and protecting retinal ganglion cells from oxidative stress and apoptotic damage. Studies have also found that resveratrol can effectively reduce intracellular reactive oxygen species levels, inhibit the production of inflammatory factors such as interleukin-1α and interleukin-6, and alleviate inflammatory responses in ocular tissues.

[0006] However, applying the aforementioned active ingredients to ophthalmic treatments in existing technologies faces the following technical challenges. First, both resveratrol and lutein are lipid-soluble compounds with extremely low solubility in aqueous systems, making it difficult to formulate clear and transparent ophthalmic preparations. Resveratrol's solubility in water is only about 0.03 mg / mL, and lutein is almost insoluble in water, which makes it difficult to achieve effective active ingredient concentrations in traditional aqueous ophthalmic preparations. Second, resveratrol has poor chemical stability and is prone to cis-trans isomerization and oxidative degradation under light and oxygen conditions; the conversion of the trans isomer to the cis isomer significantly reduces its activity. Furthermore, the combination of multiple active ingredients may lead to physicochemical incompatibility issues, affecting the stability and efficacy of the formulation.

[0007] Chinese invention CN119215094A discloses a composition for relieving eye fatigue containing lutein and bilberry extract, and its preparation method. The composition includes plant-extract synergistic components, lutein, and zinc gluconate, wherein the plant-extract synergistic components consist of bilberry extract, chrysanthemum extract, peanut skin extract, and mulberry extract. This technical solution has the following limitations: First, the composition is in the form of an oral tablet, falling under the category of functional foods, and employs a systemic administration route. The active ingredients must pass through the digestive tract and the first-pass effect in the liver before reaching the ocular target tissue, resulting in extremely low ocular bioavailability and a slow onset of action. Second, this formulation does not contain resveratrol, lacking activation of the SIRT1 pathway in retinal ganglion cells and direct neuroprotective effects. Third, the oral formulation cannot achieve immediate local lubrication of the ocular surface and cannot directly improve tear film stability and corneal epithelial repair. Fourth, the concentration of the active ingredients in the oral formulation is limited by systemic safety considerations, failing to achieve the high concentration therapeutic effect of local administration.

[0008] Therefore, there is an urgent need to develop a topical ophthalmic preparation that can act directly on the ocular surface and intraocular tissues, solve the compatibility problem of lipid-soluble components, and have a multi-target synergistic protective effect, in order to overcome the shortcomings of existing technologies and provide more effective treatment options for ocular diseases such as dry eye, eye strain, and blue light damage. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide an ophthalmic composition containing anthocyanins, resveratrol, and lutein, and a method for preparing the same. This invention solves the problem of dissolving and compatibility of lipid-soluble components in aqueous ophthalmic formulations through an innovative phosphate buffer solution co-solvent solubilization system, enabling local delivery of the three natural active ingredients to the ocular surface and intraocular tissues, thereby exerting synergistic effects of antioxidation, anti-inflammation, retinal protection, and blue light protection.

[0010] To achieve the above objectives, the present invention adopts the following technical solution.

[0011] In a first aspect, the present invention provides an ophthalmic composition containing anthocyanins, resveratrol, and lutein, wherein the composition comprises, by weight percentage: 0.1% to 0.5% anthocyanins, 0.05% to 0.2% resveratrol, 0.01% to 0.1% lutein, 10% to 30% phosphate buffer solution as a co-solvent, an appropriate amount of isotonic adjuster, an appropriate amount of pH adjuster, 0.005% to 0.02% preservative, and purified water to make up to 100%.

[0012] Preferably, the anthocyanins are derived from European blueberry extract, with an anthocyanin content of not less than 25%, of which delphinidin anthocyanins account for 30% to 50% of the total anthocyanin content. Delphinidin-3-glucoside is a key active ingredient for promoting rhodopsin regeneration, and its content directly affects the dark adaptation improvement effect of the formulation.

[0013] Preferably, the resveratrol is trans-resveratrol with a purity of not less than 98%, derived from European red grape extract. The trans isomer is the active configuration of resveratrol, exhibiting stronger SIRT1 activation ability and antioxidant activity.

[0014] Preferably, the lutein is derived from calendula extract, with a lutein content of not less than 20%, and the mass ratio of free lutein to lutein ester is 1:0 to 1:0.5. Free lutein can exert its biological activity directly without enzymatic hydrolysis, resulting in higher bioavailability for the eyes.

[0015] Preferably, the phosphate buffer solution has a phosphate concentration of 0.01 mol / L to 0.1 mol / L, a sodium chloride concentration of 0.8% to 0.9%, and a pH of 7.2 to 7.6. The amphiphilic nature of the phosphate buffer solution can significantly improve the apparent solubility of lipid-soluble components while maintaining the isotonicity and suitable pH of the formulation.

[0016] Preferably, the isotonic adjuster is selected from one or more combinations of sodium chloride, glycerol, mannitol, and sorbitol, and the osmotic pressure of the composition is from 280 mOsm / kg to 320 mOsm / kg.

[0017] Preferably, the pH adjuster is selected from one or more combinations of sodium hydroxide, hydrochloric acid, phosphoric acid, sodium dihydrogen phosphate, and disodium hydrogen phosphate, and the pH value of the composition is 6.5 to 7.8.

[0018] Preferably, the preservative is selected from one or more combinations of benzalkonium chloride, methylparaben, and boric acid.

[0019] More preferably, the composition further comprises the following excipients: 0.1% to 1.0% thickener and 0.01% to 0.1% stabilizer. The thickener is selected from one or more combinations of sodium hyaluronate, hydroxypropyl methylcellulose, and carbomer. The stabilizer is selected from one or more combinations of disodium edetate, ascorbic acid, and sodium thiosulfate.

[0020] In a second aspect, the present invention provides a method for preparing the above-mentioned ophthalmic composition, comprising the following steps.

[0021] Step 1: Preparation of co-solvent system: Dissolve sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride in purified water to prepare a phosphate buffer solution. Adjust the pH to 7.2 to 7.6, filter to remove bacteria, and set aside for later use.

[0022] Step 2: Dissolving the active ingredients: Add resveratrol and lutein to the co-solvent system obtained in Step 1, and sonicate at 40°C to 60°C for 15 to 30 minutes to completely dissolve and disperse them, thus obtaining a solution of the fat-soluble components.

[0023] Step 3: Adding anthocyanins: Add anthocyanins to the solution obtained in Step 2 and stir at room temperature until completely dissolved to obtain a mixture of active ingredients.

[0024] Step 4: Adding excipients: Add the isotonicity regulator, preservative, thickener and stabilizer to the mixture obtained in Step 3 in sequence, stir well, and adjust the pH value to 6.5 to 7.8 with a pH adjuster.

[0025] Step 5, Volume Adjustment and Filtration: Adjust the volume to the specified volume with purified water, filter through a 0.22μm microporous membrane for sterilization, and dispense into sterile containers to obtain the ophthalmic composition.

[0026] Thirdly, the present invention provides an ophthalmic eye drop comprising the ophthalmic composition described in the first aspect, wherein the viscosity of the eye drop is from 4 mPa·s to 9 mPa·s.

[0027] Fourthly, the present invention provides an ophthalmic gel comprising the ophthalmic composition described in the first aspect, wherein the thickener content is 0.3% to 1.0%, and the viscosity of the ophthalmic gel is 50 mPa·s to 500 mPa·s.

[0028] Fifthly, the present invention provides the use of the above-mentioned ophthalmic composition in the preparation of a medicament for treating or preventing ophthalmic diseases, wherein the ophthalmic diseases are selected from one or more of dry eye syndrome, visual fatigue syndrome, early prevention of age-related macular degeneration, blue light-induced retinal damage, and mild corneal epithelial damage.

[0029] The beneficial effects of this invention are as follows.

[0030] First, this invention creatively employs a phosphate buffer solution as a co-solvent system, successfully solving the solubility and compatibility problem of resveratrol and lutein, two lipid-soluble components, in ophthalmic aqueous formulations. The amphiphilic nature of the phosphate buffer solution significantly improves the apparent solubility of lipid-soluble components, and combined with ultrasonic treatment, enables resveratrol and lutein to form a stable dispersion in the aqueous system. Simultaneously, the phosphate buffer system maintains the isotonicity and suitable pH of the formulation, ensuring the safety and comfort of ophthalmic administration. Compared to complex delivery systems such as cyclodextrin inclusion complexes, nanomicelles, and liposomes, the co-solvent system of this invention has a simple preparation process, low cost, good stability, and is suitable for industrial production.

[0031] Secondly, the ophthalmic composition of this invention achieves a synergistic combination of three natural active ingredients: anthocyanins, resveratrol, and lutein, exerting a multi-target combined effect on three key pathological aspects of eye health. Anthocyanins promote rhodopsin regeneration, enhancing retinal photosensitivity and dark adaptation. Resveratrol exerts antioxidant and anti-apoptotic effects through the SIRT1 signaling pathway, protecting retinal ganglion cells from ischemia-reperfusion injury. Lutein absorbs high-energy blue light from 415 to 455 nm, reducing photo-oxidative damage. The synergistic effect of these three components is significantly superior to any single component.

[0032] Third, the ophthalmic composition of the present invention utilizes a topical ocular surface administration route, allowing the active ingredients to act directly on the cornea, conjunctiva, and tear film. It penetrates the intraocular tissues through the cornea, avoiding the gastrointestinal degradation and first-pass effect associated with oral administration, thus significantly improving ocular bioavailability and onset of action. Experimental results show that the composition of the present invention can effectively repair corneal epithelial damage, increase the number of conjunctival goblet cells, improve tear film stability, protect retinal ganglion cells, and improve retinal function. Attached Figure Description

[0033] Figure 1This is a graph showing the changing trends of resveratrol, anthocyanin, and lutein content in the eye drops during the accelerated stability test of Example 1 of the present invention. Figure 2 The image shows corneal fluorescein staining of mice with dry eye syndrome in Example 1 of the present invention, where A is the model group, B is the artificial tear group, C is the artificial tear combined with lutein group, and D is the composition group of the present invention. Figure 3 The images show PAS-stained pathological sections of conjunctival goblet cells in Example 1 of the present invention, where A represents the normal group, B represents the model group, C represents the artificial tear group, and D represents the composition group of the present invention. Figure 4 The image shows a fluorescence microscope image of retinal ganglion cell survival in Example 2 of the present invention, where A represents the untreated group for 7 days and B represents the group treated with the composition of the present invention for 7 days. Figure 5 The image shows a comparison of ERG waveforms in Example 3 of the present invention. The left image is the model group, and the right image is the composition group of the present invention. Figure 6 The bar chart shows the clinical indicators in Example 4 of the present invention, where A represents the comparison of OSDI score improvement and B represents the comparison of tear film breakup time (TBUT) prolongation. Detailed Implementation

[0034] Please refer to Figures 1 to 6 The present invention will now be described in detail with reference to the embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention. Other embodiments obtained by those skilled in the art without inventive effort are all within the scope of protection of the present invention.

[0035] All raw materials used in this invention can be purchased from the market or prepared by conventional methods. The specific sources and specifications are as follows.

[0036] European blueberry extract was purchased from Hunan Huacheng Biological Resources Co., Ltd., with an anthocyanin content of 36%, of which delphinidin anthocyanins accounted for 42% of the total anthocyanin content. It appeared as a dark purple to purplish-black powder and was easily soluble in water and ethanol.

[0037] Trans-resveratrol was purchased from Xi'an Tianbao Biotechnology Co., Ltd., with a purity of not less than 99% and a trans-isomer content of not less than 98% as determined by high performance liquid chromatography. It is a white to off-white crystalline powder, slightly soluble in water, and readily soluble in ethanol and propylene glycol.

[0038] Calendula extract was purchased from Chenguang Biotech Group Co., Ltd., with a lutein content of 20% and a free lutein content of no less than 85%. The product is an orange-yellow to orange-red oil or powder.

[0039] Sodium hyaluronate was purchased from Bloomage Biotechnology Co., Ltd., with a molecular weight of 0.5 to [missing information]. Da is a white or off-white powder that dissolves in water to form a viscous, transparent solution.

[0040] Hydroxypropyl methylcellulose was purchased from Ashland Chemical Company, with a viscosity of 4000 mPa·s and an appearance of white or off-white powder.

[0041] Carbomer 940 was purchased from Lubrizol. It appears as a white, fluffy powder that forms a transparent gel after being neutralized with alkali.

[0042] Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and sodium hydroxide were all of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0043] Disodium edetate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., pharmaceutical grade, and appeared as a white crystalline powder.

[0044] Benzalkonium chloride was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., in the form of a 50% aqueous solution, which meets the standards of the 2020 edition of the Chinese Pharmacopoeia.

[0045] Methylparaben was purchased from Hubei Xinrunde Chemical Co., Ltd., and is pharmaceutical grade, appearing as a white crystalline powder.

[0046] Ascorbic acid was purchased from DSM Nutrition Products Ltd., pharmaceutical grade, and appears as a white or off-white crystalline powder.

[0047] Sodium thiosulfate was purchased from Sinopharm Chemical Reagent Co., Ltd., analytical grade.

[0048] Glycerin, mannitol, and sorbitol were all pharmaceutical grade and purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0049] Triethanolamine was purchased from Dow Chemical Company; it was pharmaceutical grade.

[0050] Example 1: Basic Formula Eye Drops

[0051] The following ingredients, by weight percentage, are used to prepare ophthalmic drops: 0.3% anthocyanin content (0.83% of extract volume) of European blueberry extract, 0.1% trans-resveratrol, 0.05% lutein content (0.25% of extract volume) of calendula extract, 0.08% sodium dihydrogen phosphate, 0.47% disodium hydrogen phosphate, 0.85% sodium chloride, 0.2% sodium hyaluronate, 0.05% disodium edetate, 0.01% benzalkonium chloride, and purified water to 100%.

[0052] The preparation method is carried out according to the following steps.

[0053] Step 1: Preparation of the co-solvent system. Accurately weigh 0.8 g of sodium dihydrogen phosphate and 4.7 g of disodium hydrogen phosphate, add them to approximately 800 mL of purified water sterilized at 121 °C, and stir with a magnetic stirrer at 500 rpm for 30 minutes until completely dissolved. Add 8.5 g of sodium chloride and continue stirring for 15 minutes until completely dissolved. Measure the pH of the solution using a precision pH meter, and adjust the pH to 7.40 ± 0.05 dropwise with 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution. Filter the prepared phosphate buffer solution through a 0.22 μm polyethersulfone microporous membrane for sterilization, place it in a sterilized borosilicate glass container, seal it, and store it away from light for later use.

[0054] The second step is to dissolve the active ingredients. Accurately weigh 1.0g of trans-resveratrol and 2.5g of calendula extract, and add them to the phosphate buffer solution obtained in step one. Transfer the mixture to the stainless steel cleaning tank of an ultrasonic cleaner, set the water bath temperature to 50±2℃, the ultrasonic frequency to 40kHz, the ultrasonic power to 200W, and ultrasonic treatment for 20 minutes. During ultrasonic treatment, gently stir with a glass rod every 5 minutes to promote uniform dispersion. After ultrasonic treatment, remove the mixture from the ultrasonic cleaner and let it stand at room temperature for 5 minutes. Visually inspect the appearance of the solution; it should be a pale yellow to orange-yellow clear or slightly opalescent liquid, without obvious precipitate or suspended particles. If there are still undissolved substances, the ultrasonic time can be extended to 30 minutes or the temperature can be appropriately increased to 55℃, but the temperature should not exceed 60℃ to avoid degradation of the active ingredients.

[0055] The third step is the addition of anthocyanins. After cooling the solution obtained in step two to room temperature (25±2℃), accurately weigh 8.3g of blueberry extract and slowly add it to the solution in three portions. After adding approximately 2.8g of extract each time, stir with a magnetic stirrer at 500rpm for 10 minutes to ensure thorough dispersion before adding the next batch. After all the extract has been added, continue stirring for 30 minutes to ensure complete dissolution and dispersion of the anthocyanins. After the anthocyanins dissolve, the solution turns a deep purplish-red to purplish-brown color and becomes uniformly transparent. The absorbance is measured at 520nm using a UV-Vis spectrophotometer to confirm that the anthocyanin content meets the standard.

[0056] Step 4: Adding excipients. Under continuous stirring, slowly sprinkle 2.0g of sodium hyaluronate onto the surface of the solution obtained in step 3 to avoid clumping. After adding sodium hyaluronate, reduce the stirring speed to 200 rpm and continue stirring for 2 hours to allow it to fully swell and dissolve, forming a homogeneous viscous solution. After the sodium hyaluronate is completely dissolved, increase the stirring speed to 400 rpm and add 0.5g of disodium edetate and 0.2g of benzalkonium chloride solution (i.e., 50% benzalkonium chloride solution) sequentially. After each excipient is added, stir thoroughly for 15 minutes until completely dissolved before adding the next. After all excipients have been added, measure the pH of the solution using a precision pH meter and adjust the pH to 7.0 ± 0.2 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution. Measure the osmotic pressure of the solution using a freezing point osmometer; it should be within the range of 280 to 320 mOsm / kg. If the osmotic pressure is too low, add sodium chloride as needed to adjust it.

[0057] Step 5: Volume Adjustment and Filtration. Transfer the solution obtained in Step 4 to a 1000mL Class A volumetric flask, and adjust the volume to the mark with sterile purified water, mixing thoroughly. Let the adjusted solution stand at room temperature for 1 hour to allow air bubbles to escape completely. In a Class 100 clean bench, filter the solution through a 0.22μm polyethersulfone microporous membrane under positive pressure for sterilization, controlling the filtration pressure to 0.1 to 0.2MPa. Dispense the filtrate into 5mL low-density polyethylene eye drop bottles sterilized at 121℃, with each bottle containing 5.0mL. After sealing, invert the bottles to check for leaks, ensuring the caps are tightly sealed and leak-free. Label the bottles with the batch number, production date, and expiration date to obtain the finished eye drops product of Example 1 of this invention.

[0058] The finished product quality inspection results are as follows: The appearance is a clear, deep purplish-red liquid; no visible foreign matter or fibers were observed under cobalt blue light. The pH value was 6.98, within the specified range of 6.5 to 7.8. The osmotic pressure was 295 mOsm / kg, meeting the isotonic requirement of 280 to 320 mOsm / kg. The viscosity was 5.2 mPa·s (measured at 25℃ using a rotational viscometer at 60 rpm), meeting the requirement of 4 to 9 mPa·s for eye drops. Sterility testing was conducted according to the membrane filtration method of the 2020 edition of the Chinese Pharmacopoeia; no bacterial or fungal growth was observed after 14 days of incubation, indicating compliance with regulations.

[0059] Example 2: High-concentration anthocyanin-based eye drops

[0060] The following ingredients, by weight percentage, are used to prepare the ophthalmic drops: 0.5% anthocyanin content (1.39% of the extract), 0.15% trans-resveratrol, 0.08% lutein content (0.4% of the calendula extract), 0.12% sodium dihydrogen phosphate, 0.71% disodium hydrogen phosphate, 0.8% sodium chloride, 0.3% sodium hyaluronate, 0.1% hydroxypropyl methylcellulose, 0.05% disodium edetate, 0.02% ascorbic acid, 0.01% benzalkonium chloride, and purified water to 100%.

[0061] The preparation method is the same as in Example 1, with specific adjustments as follows. In the second step of dissolving the active ingredients, due to the increased content of resveratrol and lutein, the ultrasonic treatment time was extended to 25 minutes, and the water bath temperature was increased to 55±2℃ to ensure sufficient dissolution and dispersion of the fat-soluble components. In the fourth step of adding excipients, sodium hyaluronate and hydroxypropyl methylcellulose need to be added in two stages. First, 3.0g of sodium hyaluronate is slowly added to the solution, and stirred at 200rpm for 2 hours until it is completely swollen and dissolved. Then, 1.0g of hydroxypropyl methylcellulose is pre-wetted with a small amount of hot purified water and slowly dispersed in the solution, continuing to stir for 1 hour until completely dissolved. Ascorbic acid is added last as an antioxidant stabilizer to reduce its redox interaction with anthocyanins. 0.2g of ascorbic acid is dissolved in a small amount of purified water and added, then stirred for 10 minutes to mix thoroughly.

[0062] The finished product quality inspection results are as follows: Appearance: a clear, dark purple to purplish-black liquid with uniform color. pH value: 7.12. Osmotic pressure: 302 mOsm / kg. Viscosity: 8.6 mPa·s. Anthocyanin content: 0.48%; resveratrol content: 0.14%; lutein content: 0.076%. The content of each active ingredient meets the requirement of 90% to 110% of the labeled amount.

[0063] Example 3: Low-irritation formula eye drops

[0064] The following ingredients, by weight percentage, are used to prepare ophthalmic drops: 0.2% anthocyanin content (0.56% of extract weight), 0.08% trans-resveratrol, 0.03% lutein content (0.15% of calendula extract), 0.06% sodium dihydrogen phosphate, 0.35% disodium hydrogen phosphate, 0.88% sodium chloride, 0.15% sodium hyaluronate, 0.5% glycerin, 0.03% disodium edetate, 0.015% methylparaben, and purified water to 100%.

[0065] This embodiment uses a low concentration of active ingredients and a mild preservative system, making it suitable for people with sensitive eyes and those with mild dry eye syndrome. Glycerin is added as an isotonic adjuster and moisturizer, further improving the formulation's ocular comfort and lubrication. Methylparaben has lower corneal epithelial cell toxicity and irritation compared to benzalkonium chloride, making it suitable for long-term use.

[0066] The preparation method is basically the same as in Example 1, with the main adjustments as follows: Methylparaben needs to be dissolved in a small amount of ethanol before being added to the aqueous phase, and the addition should be carried out under rapid stirring to avoid precipitation. Glycerin is added in the fourth step of the excipient addition stage, together with the isotonic adjuster sodium chloride to adjust the osmotic pressure of the formulation.

[0067] The finished product quality inspection results are as follows: Appearance: Clear purplish-red liquid, light in color. pH value: 7.25. Osmotic pressure: 288 mOsm / kg. Viscosity: 4.1 mPa·s. Acute eye irritation test was conducted using New Zealand white rabbits according to the Chinese Pharmacopoeia's eye irritation test method. The results showed that this formula scored 0 points in eye irritation, indicating it is non-irritating.

[0068] Example 4: Long-lasting ophthalmic gel

[0069] The ophthalmic gel is prepared by taking the following raw materials by weight percentage: 0.3% anthocyanin content of European blueberry extract (0.83% of extract amount), 0.1% trans-resveratrol, 0.05% lutein content of calendula extract (0.25% of extract amount), 0.08% sodium dihydrogen phosphate, 0.47% disodium hydrogen phosphate, 0.8% sodium chloride, 0.5% sodium hyaluronate, 0.2% carbomer 940, 2.0% mannitol, 0.05% disodium edetate, 0.01% sodium thiosulfate, 0.01% benzalkonium chloride, and appropriate amount of triethanolamine for pH adjustment, with purified water added to 100%.

[0070] The ophthalmic gel prepared in this embodiment has higher viscosity and a longer residence time on the ocular surface, making it suitable for patients with severe dry eye, those who need nighttime use, or those requiring long-lasting protection. Carbomer 940 is a high molecular weight acrylic polymer that forms a transparent gel after alkali neutralization, exhibiting excellent bioadhesion and extending the residence time of active ingredients on the ocular surface.

[0071] The preparation method was specifically adjusted as follows. Steps one through three are the same as in Example 1. In step four, during the addition of excipients, 2.0 g of carbomer 940 powder was slowly sprinkled into approximately 200 mL of purified water to avoid clumping. The mixture was stirred at low speed of 200 rpm overnight (12 to 16 hours) to allow it to fully swell and form a uniform, translucent gel matrix. The swollen carbomer gel was then added to the active ingredient mixture obtained in step three and stirred until homogeneous. 5.0 g of sodium hyaluronate, 20 g of mannitol, 0.5 g of disodium edetate, 0.1 g of sodium thiosulfate, and 0.2 g of benzalkonium chloride solution were added sequentially, stirring for 15 minutes after each excipient was added to ensure thorough mixing. Finally, triethanolamine solution was added dropwise to adjust the pH to 7.0 to 7.2. After neutralization, the viscosity of the carbomer gel increased significantly, forming a transparent gel state. The volume was brought to 1000 g with purified water and stirred until homogeneous. Due to the high viscosity of the gel, filtration was performed under pressure using a 0.45 μm microporous membrane.

[0072] The finished product quality inspection results are as follows: The appearance is a deep purple transparent gel, uniform, smooth, and free of bubbles. The pH value is 7.08. The osmotic pressure is 310 mOsm / kg. The viscosity is 285 mPa·s (measured at 25℃ using a rotational viscometer at 10 rpm). The gel has a uniform appearance and good spreadability; it forms a uniform thin film after being coated on a glass plate. Bioadhesion tests show that the gel adheres to the surface of an isolated cornea for more than 4 hours, significantly longer than ordinary eye drops.

[0073] Example 5: Preservative-free single-dose formulation

[0074] Preservative-free single-dose ophthalmic drops are prepared by taking the following raw materials by weight percentage: 0.3% anthocyanin content of European blueberry extract (0.83% of extract amount), 0.1% trans-resveratrol, 0.05% lutein content of calendula extract (0.25% of extract amount), 0.08% sodium dihydrogen phosphate, 0.47% disodium hydrogen phosphate, 0.85% sodium chloride, 0.2% sodium hyaluronate, 0.05% disodium edetate, and purified water to 100%.

[0075] This formulation is preservative-free and uses a single-dose packaging, 0.4 mL per vial, for single use only and disposal. It is suitable for individuals with preservative allergies, contact lens wearers, or patients requiring long-term, frequent medication. While preservatives can ensure the microbial safety of multi-dose formulations, long-term use may lead to cumulative toxicity to corneal epithelial cells, resulting in decreased tear film stability and ocular surface damage. The single-dose, preservative-free formulation avoids this problem.

[0076] The entire preparation process is conducted in an ISO Class 5 (Class 100) clean environment. All raw materials undergo double filtration and sterilization before addition. Preparation containers and filling equipment are sterilized by moist heat at 121°C or by ethylene oxide sterilization. An integrated blow-fill-seal machine is used for aseptic filling; the blow molding of polyethylene containers, liquid filling, and sealing are completed continuously in the same aseptic environment, minimizing the risk of microbial contamination. Each vial contains 0.4 mL, with a filling accuracy of ±5%.

[0077] The finished product quality inspection results are as follows: Appearance: Deep purple-red clear liquid. pH value: 7.02. Osmotic pressure: 298 mOsm / kg. Sterility testing was performed using a 100% inspection; 20 vials were randomly selected from each batch for sterility testing. After 14 days of incubation, no bacterial or fungal growth was observed, indicating compliance with regulations. Bacterial endotoxin testing was conducted using the Limulus Amebocyte Lysate (LAL) gel electrophoresis method; the result was less than 0.5 EU / mL, meeting the requirements for ophthalmic preparations.

[0078] Comparative Example 1: Compositions without resveratrol

[0079] Comparative eye drops were prepared by taking the following raw materials by weight percentage: 0.3% anthocyanin content of European blueberry extract (0.83% of extract used), 0.05% lutein content of calendula extract (0.25% of extract used), 0.08% sodium dihydrogen phosphate, 0.47% disodium hydrogen phosphate, 0.85% sodium chloride, 0.2% sodium hyaluronate, 0.05% disodium edetate, 0.01% benzalkonium chloride, and purified water to 100%.

[0080] This comparative example does not contain resveratrol; the remaining components and preparation method are the same as in Example 1. The resulting eye drops are a clear, purplish-red liquid with a pH of 7.05, an osmotic pressure of 292 mOsm / kg, and a viscosity of 5.0 mPa·s. This comparative example is used to verify the necessity of resveratrol in the composition and its contribution to the protective effect on retinal ganglion cells.

[0081] Comparative Example 2: Compositions without anthocyanins

[0082] The comparative eye drops were prepared by taking the following raw materials by weight percentage: 0.1% trans-resveratrol, 0.05% lutein content of calendula extract (0.25% of extract used), 0.08% sodium dihydrogen phosphate, 0.47% disodium hydrogen phosphate, 0.85% sodium chloride, 0.2% sodium hyaluronate, 0.05% disodium edetate, 0.01% benzalkonium chloride, and purified water to 100%.

[0083] This comparative example does not contain anthocyanins; the remaining components and preparation method are the same as in Example 1. The resulting eye drops are a pale yellow to light orange clear liquid, significantly different in appearance from the anthocyanin-containing examples. The pH is 7.08, the osmotic pressure is 290 mOsm / kg, and the viscosity is 4.8 mPa·s. This comparative example is used to verify the necessity of anthocyanins in the composition and their contribution to rhodopsin regeneration and improvement of visual function.

[0084] Comparative Example 3: Compositions without lutein

[0085] Comparative eye drops were prepared by taking the following raw materials by weight percentage: 0.3% anthocyanin content of European blueberry extract (0.83% of extract used), 0.1% trans-resveratrol, 0.08% sodium dihydrogen phosphate, 0.47% disodium hydrogen phosphate, 0.85% sodium chloride, 0.2% sodium hyaluronate, 0.05% disodium edetate, 0.01% benzalkonium chloride, and purified water to 100%.

[0086] This comparative example does not contain lutein; the remaining components and preparation method are the same as in Example 1. The resulting eye drops are a clear, deep purplish-red liquid with a pH of 7.02, an osmotic pressure of 293 mOsm / kg, and a viscosity of 5.1 mPa·s. This comparative example is used to verify the necessity of lutein in the composition and its contribution to blue light protection and antioxidant effects.

[0087] Comparative Example 4: Compositions without PBS co-solvent system

[0088] The comparative eye drops were prepared by taking the following raw materials by weight percentage: 0.3% anthocyanin content of European blueberry extract (0.83% of extract used), 0.1% trans-resveratrol, 0.05% lutein content of calendula extract (0.25% of extract used), 0.9% sodium chloride, 0.2% sodium hyaluronate, 0.05% disodium edetate, 0.01% benzalkonium chloride, and purified water to 100%.

[0089] This comparative example did not use phosphate buffer solution as a co-solvent system, but only an isotonic sodium chloride solution as the matrix. During the preparation process, it was found that resveratrol and lutein were difficult to dissolve completely. After ultrasonic treatment in a 50°C water bath for 30 minutes, obvious white suspended particles and a yellow precipitate were still visible in the solution. After standing, a large amount of precipitate appeared in the lower layer. After filtration through a 0.22 μm microporous membrane, most of the lipid-soluble components were retained on the membrane. Content determination of the filtrate showed that the resveratrol recovery rate was only 12.5%, and the lutein recovery rate was only 8.3%, failing to obtain a clear eye drop product that met the requirements. This comparative example demonstrates the necessity and crucial role of the phosphate buffer solution co-solvent system in the dissolution of lipid-soluble components.

[0090] Comparative Example 5: Artificial Tear Control Group

[0091] Take the following raw materials to prepare the control eye drops by mass percentage: sodium chloride 0.64%, potassium chloride 0.075%, anhydrous calcium chloride 0.048%, magnesium chloride hexahydrate 0.03%, sodium hyaluronate 0.15%, benzalkonium chloride 0.01%, and purified water is added up to 100%.

[0092] This comparative example is a conventional artificial tear formula, simulating the electrolyte composition of human tears, without any active drug ingredients, and is used as a blank control for animal experiments and clinical trials. The preparation method is to dissolve each electrolyte and auxiliary material in turn, adjust the pH value to 7.2 to 7.4, and then filter and sterilize and divide into packages.

[0093] The test results of the finished product quality are as follows. The appearance is a colorless clear liquid. The pH value is 7.35. The osmotic pressure is 290 mOsm / kg. The viscosity is 3.8 mPa·s.

[0094] Effect Example 1: Evaluation of the Corneal Repair Effect of a Dry Eye Mouse Model

[0095] The purpose of the experiment is to evaluate the repair effect of the composition of the present invention on corneal epithelial damage in a dry eye model mouse and the protective effect on conjunctival goblet cells.

[0096] Forty 6- to 8-week-old SPF-grade male BALB / c mice weighing 20±2 g were selected as experimental animals. They were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the animal license number is SCXK (Beijing) 2021-0006. All animal experiments were approved by the Experimental Animal Ethics Committee. Before the experiment, the animals were adaptively fed for 7 days. The feeding environment temperature was 22±2°C, the relative humidity was 50%±10%, the light cycle was 12 h light and dark alternation, and they had free access to water and food.

[0097] The dry eye model was established by the method of instilling benzalkonium chloride into the cornea. Accurately measure 5 μL of 0.2% benzalkonium chloride solution with a pipette and drop it into the conjunctival sacs of both eyes of the mice, 2 times a day, once at 8 am and once at 6 pm, and continuously induce for 10 days to establish a dry eye model. During the model establishment period, corneal fluorescein staining examination was performed every 2 days to monitor the progress of model establishment. The criteria for successful model establishment were that the tear film break-up time was less than 10 seconds and the corneal fluorescein staining score was greater than or equal to 4 points. The success rate of model establishment was 92.5%.

[0098] The experimental groups were divided into 4 groups from the 37 successfully modeled mice by the random number table method. The normal control group had 8 mice, which were healthy mice that did not undergo dry eye modeling. The dry eye model group had 10 mice, which was the negative control group without any treatment after modeling. The artificial tear group had 10 mice, and the artificial tear of Comparative Example 5 was instilled into the eyes. The composition group of the present invention had 9 mice, and the eye drops of Example 1 were instilled into the eyes.

[0099] The dosing regimen was 20 μL per eye, twice daily, at 9:00 AM and 5:00 PM, for 10 consecutive days. Benzalkonium chloride dry eye induction treatment was continued throughout the treatment period to simulate the real clinical situation of dry eye patients continuously exposed to the causative factor during treatment.

[0100] The testing indicators included corneal fluorescein staining score and histopathological examination. For corneal fluorescein staining, 2 μL of 1% sodium fluorescein solution was instilled into the conjunctival sac. After closing the eye, the eyeball was gently rotated to distribute the dye evenly. One minute later, the corneal staining was observed under cobalt blue light using a slit-lamp microscope. Scoring was performed according to the modified Oxford scoring system, ranging from 0 to 5 points. 0 points indicated corneal transparency and no staining; 1 point indicated fewer than 5 scattered punctate stains; 2 points indicated 6 to 15 punctate stains; 3 points indicated 16 to 30 punctate stains; 4 points indicated more than 30 punctate stains or patchy staining; and 5 points indicated diffuse staining. Scoring was performed independently by two trained observers in a double-blind manner, and the average score was used.

[0101] Histopathological examination was performed 10 days after drug treatment. Immediately after mouse euthanasia, the eyeballs were enucleated and fixed in 4% paraformaldehyde solution for 24 hours. After routine paraffin embedding, 5 μm thick tissue sections were prepared. Hematoxylin-eosin staining was used to observe the integrity and thickness of the corneal epithelium. Periodic acid-Schiff (PAS) staining was used to observe the number of conjunctival goblet cells; goblet cells showed a purplish-red positive stain. The number of goblet cells per 100 μm of conjunctival epithelial length was counted under a 400x optical microscope, with five randomly selected fields of view from each eye and the average value taken.

[0102] like Figure 2 As shown, the corneal repair effect of each group of mice was evaluated by corneal fluorescein staining under slit lamp cobalt blue light. Figure 2 Group A is the model group. The cornea shows large areas of punctate and patchy fluorescein staining, with obvious green fluorescence signals, indicating extensive damage to the corneal epithelium, disruption of intercellular connections, and loss of barrier function. Figure 2 Group B, which was treated with artificial tears, showed a reduction in corneal damage area after 10 days of treatment, but still showed many scattered punctate staining and incomplete corneal epithelial repair. Figure 2 C represents the artificial tears combined with lutein group. The corneal fluorescein staining was further reduced compared to the artificial tears group, indicating that the antioxidant effect of lutein helps corneal epithelial repair. Figure 2 D is the composition group of the present invention. The corneal fluorescein staining is very little, with only a few tiny dot-like stainings. The corneal surface is smooth and clear, indicating that the corneal epithelial damage has been basically completely repaired and the barrier function has been well restored.

[0103] The statistical results of corneal fluorescein staining scores are shown in Table 1.

[0104] Table 1. Corneal fluorescein staining scores of mice in each group ( ±s)

[0105] Group Number of animals Pre-dose score Score after 10 days of administration Score change value Normal control group 8 0.2±0.4 0.2±0.4 0.0±0.0 Dry eye model group 10 4.5±0.5 4.3±0.7 -0.2±0.4 Artificial tear group 10 4.4±0.5 2.8±0.6** -1.6±0.5** Composition group of the present application 9 4.5±0.5 1.2±0.4**## -3.3±0.5**##

[0106] Note: Compared with the dry eye model group, **P<0.01; compared with the artificial tears group, ##P<0.01.

[0107] like Figure 3 The image shown is a PAS-stained pathological section of conjunctival tissue, where goblet cells show a purplish-red positive staining. Figure 3 A represents the normal control group. A large number of goblet cells can be observed in the conjunctival tissue. The cells are plump and evenly distributed. The average number of goblet cells per 100 μm of conjunctival epithelium is 18.5 ± 3.2. Figure 3 Group B was the dry eye model group, which showed a large loss of goblet cells, few PAS-positive cells, and atrophic cell morphology. The average number of goblet cells per 100 μm of conjunctival epithelium was only 2.1 ± 1.4, which was 88.6% lower than that of the normal group. Figure 3 C represents the artificial tears group. After treatment, the number of goblet cells was 3.8±1.6, which was slightly higher than that of the model group, but the difference was not statistically significant (P>0.05), indicating that simple lubrication cannot restore goblet cell function. Figure 3 Group D represents the composition of this invention. The number of goblet cells significantly recovered to 14.2±2.8, an increase of 576.2% compared to the model group (P<0.01), reaching 76.8% of the normal group. The goblet cells were plump and evenly distributed, indicating that the composition of this invention can effectively promote the regeneration and functional recovery of conjunctival goblet cells.

[0108] Experimental results show that the composition of this invention can effectively repair corneal epithelial damage in a mouse model of dry eye and promote conjunctival goblet cell regeneration, with significantly better therapeutic effects than artificial tears alone. The synergistic effect of the three active ingredients plays an important role in corneal repair and goblet cell protection.

[0109] Example 2: Protective effect of resveratrol on retinal ganglion cells

[0110] The purpose of this experiment is to evaluate the protective effect of resveratrol in the composition of this invention against retinal ganglion cell death caused by acute intraocular pressure injury.

[0111] Twenty-four 8-week-old SPF-grade male SD rats, weighing 220±20g, were purchased from Shanghai Silex Laboratory Animal Co., Ltd. They were acclimatized for one week before the experiment.

[0112] An acute ocular hypertension model was established using the anterior chamber perfusion method to create a retinal ischemia-reperfusion injury model. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate solution at a dose of 350 mg / kg and then fixed on a stereotaxic apparatus. Topical corneal anesthesia was performed using 0.5% promecaine hydrochloride eye drops. Under a surgical microscope, a 30G sterile needle was inserted obliquely into the anterior chamber from the limbus, with the bevel facing upwards. The needle was connected to an infusion bottle containing physiological saline via a medical silicone tube. The infusion bottle was raised to a height of 150 cm above the rat's eyeball, increasing the anterior chamber pressure to approximately 110 mmHg. After maintaining elevated ocular pressure for 60 minutes, the height of the infusion bottle was slowly lowered to allow the ocular pressure to gradually return to normal, inducing retinal ischemia-reperfusion injury. Antibiotic eye ointment was applied post-operatively to prevent infection.

[0113] The experiment was divided into 4 groups, with 6 animals in each group. The normal control group received no treatment. The model group underwent acute intraocular hypertension modeling only, without any drug treatment. The artificial tear group received artificial tears eye drops as described in Example 5 after modeling. The resveratrol group received eye drops as described in Example 1; this group was used to observe the protective effect of resveratrol on retinal ganglion cells.

[0114] The administration regimen was 20 μL per eye, twice daily. Pretreatment was initiated 2 days before modeling, and administration continued on the day of modeling. Administration continued for 5 days after modeling until the animal was sacrificed for tissue collection.

[0115] The number of retinal ganglion cells was counted using the Fluoro-Gold retrograde labeling method. Three days before sacrifice, 7 days after modeling, 2 μL of 4% Fluoro-Gold retrograde tracer was injected into the bilateral superior colliculus and lateral geniculate body. Seven days after modeling, rats were euthanized by anesthesia with an overdose of sodium pentobarbital, and the eyeballs were immediately enucleated and fixed in 4% paraformaldehyde solution for 1 hour. The retina was carefully dissected under a dissecting microscope and laid flat on a glass slide to form a retinal patch. Fluoro-Gold labeled retinal ganglion cells were observed under ultraviolet excitation under a fluorescence microscope, exhibiting golden-yellow fluorescence. Six fields of view were randomly selected from both the central and peripheral areas of the retina, and the number of retinal ganglion cells in each field was counted under 400x magnification, calculating the cell density per unit area.

[0116] like Figure 4 The image shown is a fluorescence micrograph of retinal ganglion cells, with surviving retinal ganglion cells displayed using the Fluoro-Gold retrograde labeling method. Figure 4 Group A represents the untreated group (model group) 7 days after acute ocular hypertension injury. A large number of retinal ganglion cells were lost, fluorescently labeled cells were significantly sparse, and cell distribution was uneven. Some areas showed obvious cell loss, indicating severe ganglion cell apoptosis caused by ischemia-reperfusion injury. Figure 4Group B, treated with the composition of this invention for 7 days, showed a significantly higher retinal ganglion cell survival density than the untreated group. Fluorescently labeled cells were densely distributed, with intact cell morphology and clearly visible cell bodies. Quantitative analysis showed that the RGC density in the model group was 1285±168 cells / mm². 2 The survival rate was only 43.4%, while the RGC density in the treatment group with the composition of this invention was 2175±195 cells / mm². 2 The survival rate reached 73.4%, which was 29.8 percentage points higher than that of the model group (P<0.01), fully demonstrating that the resveratrol component in the composition of the present invention has a strong protective effect on retinal ganglion cells.

[0117] The statistical results of retinal ganglion cell density counting are shown in Table 2.

[0118] Table 2. Retinal ganglion cell density in each group of rats (cells / ) , ±s, n=6)

[0119] Group RGC density Survival rate Normal control group 2580±185 100% Model group 1120±165 43.4% Artificial tear group 1185±142 45.9% Resveratrol group 1895±178**## 73.4%**##

[0120] Note: Compared with the model group, **P<0.01; compared with the artificial tears group, ##P<0.01.

[0121] Experimental results show that acute intraocular pressure injury leads to massive death of retinal ganglion cells (RGCs), with a survival rate of only 43.4% in the model group. The resveratrol component in the composition of this invention significantly reduces retinal ganglion cell death caused by ischemia-reperfusion injury, increasing the RGC survival rate from 43.4% to 73.4%, an increase of 29.8 percentage points, demonstrating a significant protective effect. There was no statistically significant difference between the artificial tear group and the model group, indicating that simple lubrication and moisturizing effects cannot protect retinal ganglion cells. This result confirms that resveratrol plays an irreplaceable role in retinal neuroprotection in the composition of this invention, and its mechanism may be related to activation of the SIRT1 signaling pathway, promotion of mitochondrial biosynthesis, inhibition of oxidative stress, and apoptosis.

[0122] Example 3: Evaluation of Electroretinogram (ERG) Function

[0123] The purpose of this experiment is to evaluate the effect of the composition of the present invention on improving retinal function, and to objectively reflect the electrophysiological functional state of the retina through electroretinography.

[0124] The experimental animals used were the dry eye mouse model described in Example 1. Electroretinography (ERG) was performed 10 days after drug treatment.

[0125] The detection method involved placing mice in complete darkness for 4 hours to allow for sufficient regeneration of visual pigments in the retinal photoreceptor cells, achieving dark adaptation. After dark adaptation, subsequent operations were performed under red light illumination, with red light wavelengths greater than 620 nm having minimal impact on dark adaptation. Mice were anesthetized via intraperitoneal injection of 10% chloral hydrate solution at a dose of 400 mg / kg. Mydriasis was achieved using 0.5% tropicamide eye drops, with a 15-minute wait for full pupil dilation. The mice were then fixed on a temperature-controlled platform, maintaining a body temperature of 37 ± 0.5℃. The corneal surface was kept moist with methylcellulose solution, and a corneal contact gold wire loop electrode was gently placed on the corneal surface as the recording electrode. A subcutaneous needle electrode was inserted subcutaneously at the outer edge of the ipsilateral orbit as a reference electrode, and a needle electrode was inserted into the tail as a ground electrode. Flash stimulation was performed using a full-field stimulator, with the flash intensity set to 3.0 cd·s / The flash duration was 4 ms, and the ERG waveform of the maximum mixed response to dark adaptation was recorded. A biosignal acquisition system was used to record and analyze the ERG waveforms, with bandpass filtering from 0.3 to 300 Hz and a sampling rate of 1000 Hz. Five recordings were taken for each eye, and the average value was calculated. Analytical parameters included the amplitude of wave a (distance from the trough to the baseline), the amplitude of wave b (distance from the trough to the peak), the latency of wave a, and the latency of wave b.

[0126] like Figure 5 The figures show a comparison of electroretinogram (ERG) waveforms, with the horizontal axis representing time (ms) and the vertical axis representing amplitude (μV). The left figure shows the ERG waveform of the dry eye model group, where both a-wave and b-wave amplitudes are significantly reduced and the waveforms are flat. The a-wave amplitude is approximately -5μV, and the b-wave amplitude is approximately 3μV, indicating impaired function of retinal photoreceptor cells and bipolar cells. The right figure shows the ERG waveform of the treatment group treated with the composition of this invention. The a-wave and b-wave amplitudes are significantly restored, and the waveforms are regular. The a-wave amplitude is approximately -18μV, and the b-wave amplitude is approximately 33μV. Compared with the model group, the a-wave amplitude recovered by approximately 260%, and the b-wave amplitude recovered by approximately 1000%, indicating that the composition of this invention can effectively improve retinal electrophysiological function. This result confirms that the composition of this invention can not only repair ocular surface damage but also protect retinal function through multiple mechanisms, including anthocyanins promoting rhodopsin regeneration, resveratrol protecting retinal neurons, and lutein protecting against blue light damage.

[0127] The statistical results of the electroretinogram parameter analysis are shown in Table 3.

[0128] Table 3. Dark-adapted ERG parameters of mice in each group ( ±s, n=8-10)

[0129] Group Number of animals a-wave amplitude (μV) b-wave amplitude (μV) a-wave latency (ms) b-wave latency (ms) Normal control group 8 185±22 420±38 18.2±1.5 42.5±3.2 Dry eye model group 10 98±18 225±32 22.8±2.4 51.6±4.8 Artificial tear group 10 112±20 268±35 21.5±2.1 48.9±4.2 Composition group of the present application 9 165±25**## 385±42**## 19.1±1.8** 44.2±3.5**

[0130] Note: Compared with the dry eye model group, **P<0.01; compared with the artificial tears group, ##P<0.01.

[0131] ERG results showed that in the dry eye model group, the amplitude of wave a was reduced by 47.0% and the amplitude of wave b was reduced by 46.4% compared with the normal control group. The latencies of both waves a and b were prolonged, indicating damage to retinal photoreceptor cell function and bipolar cell function. After treatment with artificial tears, the amplitude of wave a recovered by 14.3% and the amplitude of wave b recovered by 19.1%, showing limited improvement. After treatment with the composition of this invention, the amplitude of wave a increased by 68.4% compared with the model group, reaching 89.2% of the normal group; the amplitude of wave b increased by 71.1% compared with the model group, reaching 91.7% of the normal group; and the latencies of both waves a and b were close to normal levels. Compared with the artificial tears group, all ERG parameters of the composition of this invention were significantly improved (P<0.01).

[0132] Experimental results show that the composition of this invention can significantly improve retinal function in mice with dry eye syndrome. The amplitudes of ERG a-wave and b-wave are significantly higher than those in the artificial tears group, and the retinal electrophysiological function is close to normal. This result indicates that the composition of this invention can not only repair ocular surface damage, but also protect retinal function through multiple mechanisms, including anthocyanins promoting rhodopsin regeneration, resveratrol protecting retinal neurons, and lutein protecting against blue light damage.

[0133] Example 4: Clinical observation on humans

[0134] The purpose of this experiment is to evaluate the safety and preliminary efficacy of the composition of the present invention in human use, and to provide reference data for subsequent formal clinical trials.

[0135] Sixty adult volunteers aged 22 to 55 years (mean age 35.6 ± 8.2 years) with symptoms of dry eye or eye strain were selected from the ophthalmology clinic of a tertiary hospital. There were 28 males and 32 females, with a male-to-female ratio of approximately 1:1.14. Inclusion criteria included daily screen time ≥6 hours, two or more of the following symptoms: dry eye, foreign body sensation, burning sensation, photophobia, and eye strain; an OSDI score ≥25; and a tear film breakup time ≤10 seconds. Exclusion criteria included ocular infectious diseases such as conjunctivitis, keratitis, and blepharitis; severe fundus diseases such as proliferative diabetic retinopathy and exudative macular degeneration; a history of eye surgery within the past month; use of other ophthalmic medications within the past two weeks; known allergy to any component of the composition of this invention; and pregnancy or lactation. All participants signed informed consent forms, and the study protocol was approved by the hospital's ethics committee.

[0136] Participants were randomly assigned to an experimental group and a control group using a double-blind method, with 30 participants in each group. The experimental group used the eye drops of Example 1, while the control group used artificial tears of Comparative Example 5. The packaging of both preparations was identical, and neither the participants nor the observing physicians were aware of the group assignments. The administration method was 1 to 2 drops (approximately 40 μL) per eye three times daily, after waking up in the morning, after lunch break, and before bedtime, for four consecutive weeks. Participants maintained normal work and lifestyle habits during treatment, with no restrictions on screen time.

[0137] The observation indicators included subjective scores of ocular symptoms, objective testing of tear film stability, changes in ocular surface appearance, and monitoring of adverse reactions. Ocular symptom scores were assessed using the Ocular Surface Disease Index (OSDI) questionnaire, which contains 12 questions covering symptoms such as dryness, foreign body sensation, burning sensation, photophobia, pain, blurred vision, and eye strain, as well as the degree of impact on daily life. The total score ranged from 0 to 100, with higher scores indicating more severe symptoms. Tear film breakup time was measured using the sodium fluorescein staining method, observing the time it took for the first dry patch to appear on the fluorescein distribution film under slit-lamp cobalt blue light, with three consecutive measurements taken and the average value recorded. The Schirmer test used a standardized tear secretion measurement filter paper strip to measure the length of the filter paper soaked with tear fluid within 5 minutes, assessing tear secretion function. The testing time points were baseline (before medication) and 4 weeks after medication.

[0138] The results of the clinical efficacy evaluation are shown in Table 4.

[0139] Table 4 Comparison of clinical efficacy between the two groups of subjects ( (±s, n=30)

[0140] Index Time point Test group Control group P value between groups OSDI score Baseline 42.5±8.3 41.8±7.9 >0.05 After 4 weeks 18.2±6.5**## 32.4±7.2** <0.01 Change value -24.3±5.8## -9.4±4.6 <0.01 Tear film break-up time (s) Baseline 5.2±1.8 5.4±1.6 >0.05 After 4 weeks 10.8±2.5**## 7.2±2.0** <0.01 Change value +5.6±1.5## +1.8±1.2 <0.01 Schirmer test (mm) Baseline 6.5±2.2 6.8±2.4 >0.05 After 4 weeks 12.3±3.1**## 8.5±2.6** <0.01 Change value +5.8±2.0## +1.7±1.4 <0.01

[0141] Note: Compared with baseline, **P<0.01; compared with the control group after 4 weeks, ##P<0.01.

[0142] Clinical efficacy results showed that the OSDI score in the experimental group decreased from 42.5 points at baseline to 18.2 points after 4 weeks, a reduction of 57.2%, indicating significant symptom improvement. In the control group, the OSDI score decreased from 41.8 points to 32.4 points, a reduction of only 22.5%. The difference between the two groups was highly statistically significant (P<0.01). The tear film breakup time in the experimental group increased from 5.2 seconds to 10.8 seconds, an increase of 107.7%, exceeding the lower limit of normal (10 seconds). In the control group, the tear film breakup time increased from 5.4 seconds to 7.2 seconds, an increase of 33.3%, but remained within the diagnostic range for dry eye. The Schirmer test result in the experimental group increased from 6.5 mm to 12.3 mm, an increase of 89.2%, approaching the lower limit of normal. In the control group, the result increased from 6.8 mm to 8.5 mm, an increase of 25.0%.

[0143] Regarding safety evaluation, no serious adverse events occurred in either the experimental or control group. In the experimental group, 2 patients (6.7%) experienced transient, mild eye irritation, characterized by a slight stinging sensation lasting less than one minute after instillation. This did not affect continued use and resolved spontaneously without specific treatment. In the control group, 1 patient (3.3%) experienced eye irritation. There was no statistically significant difference in the incidence of adverse reactions between the two groups (P>0.05). No abnormal changes were observed in blood routine tests, liver and kidney function, intraocular pressure, or other safety indicators in either group during the medication period.

[0144] Clinical observations indicate that the composition of this invention has good safety and tolerability in human use. Compared with artificial tears, the composition of this invention can more effectively improve dry eye symptoms, prolong tear film breakup time, and increase tear secretion. After a 4-week treatment course, the improvement in all indicators is significantly better than that in the control group.

[0145] The stability of the compositions of the present invention was investigated using accelerated stability tests and long-term stability tests.

[0146] Accelerated stability testing was conducted by placing the eye drops from Example 1 in a pharmaceutical stability test chamber under conditions of 40±2℃ and 75%±5% relative humidity, simulating long-term storage at 25℃ for 2 years. Samples were taken at 0, 1, 2, 3, and 6 months to test appearance, pH value, osmotic pressure, and active ingredient content.

[0147] For long-term stability testing, the eye drops from Example 1 were stored in the dark at a temperature of 25±2℃ and a relative humidity of 60%±10%. Samples were taken at 0, 3, 6, 9, and 12 months to test various indicators.

[0148] The results of the stability tests are shown in Table 5 and Figure 1 As shown. Figure 1 The graph shows the trend of the content changes of the three active ingredients under accelerated stability test conditions. The horizontal axis represents storage time (months), and the vertical axis represents the content percentage (%). The results show that after 6 months of storage under accelerated test conditions, the resveratrol content retention rate was 88.5%, the anthocyanin content retention rate was 92.0%, and the lutein content retention rate was 95.5%. The contents of the three active ingredients remained above 88%, proving that the formulation of this invention has excellent stability.

[0149] Table 5 Results of the stability test of eye drops in Example 1

[0150] Test condition Time point Appearance pH value Osmotic pressure (mOsm / kg) Anthocyanin content (%) Resveratrol content (%) Lutein content (%) Accelerated test 0 months Deep purple red clear 6.98 295 100.0 100.0 100.0 40℃ / 75%RH 1 month Deep purple red clear 6.92 294 98.5 97.2 99.1 2 months Deep purple red clear 6.88 293 96.8 95.1 97.8 3 months Deep purple red clear 6.81 292 95.2 92.8 96.5 6 months Deep purple red slightly turbid 6.72 290 92.3 88.5 94.1 Long-term test 0 months Deep purple red clear 6.98 295 100.0 100.0 100.0 25℃ / 60%RH 3 months Deep purple red clear 6.95 295 99.2 98.5 99.5 6 months Deep purple red clear 6.92 294 98.1 97.2 98.8 9 months Deep purple red clear 6.88 294 96.8 95.5 97.6 12 months Deep purple red clear 6.85 293 95.5 93.8 96.2

[0151] Stability test results showed that after 6 months of accelerated testing (equivalent to 2 years of long-term storage at 25°C), the sample appearance changed from clear to slightly turbid, but no obvious precipitation occurred. The pH value decreased from the initial 6.98 to 6.72, a change rate of 3.7%, still within the specified range. Anthocyanin content retained 92.3%, resveratrol content retained 88.5%, and lutein content retained 94.1%. The significant decrease in resveratrol content may be related to its photosensitivity and oxidative instability. After 12 months of long-term stability testing, all indicators were within acceptable ranges, with anthocyanin content retaining 95.5%, resveratrol content retaining 93.8%, and lutein content retaining 96.2%. Considering all factors, the shelf life of the composition of this invention under light-protected storage conditions at 25°C can be set at 24 months.

[0152] The composition of this invention exerts a multi-target combined protective effect on multiple key pathological links of eye health through the synergistic combination of three natural active ingredients: anthocyanins, resveratrol, and lutein. Its mechanism of action can be explained from the following four aspects.

[0153] The first level is the mechanism of action at the molecular target level. Core component 1, anthocyanin, is derived from European blueberries. Delphinidin-3-glucoside within it can directly interact with the retinaldehyde binding site of rhodopsin, promoting the regeneration and recycling of rhodopsin after light stimulation, accelerating the recovery of visual pigments, and enhancing the retina's sensitivity to light and dark adaptation. Core component 2, resveratrol, is derived from European red grapes. By activating the silent information regulator 1 (SIRT1) protein, it promotes the deacetylation modification of downstream PGC-1α and its translocation from the cytoplasm to the nucleus, thereby upregulating the expression of mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF1), enhancing mitochondrial biosynthetic capacity, and providing sufficient energy support for retinal ganglion cells. Core component 3, lutein, is derived from calendula. As a carotenoid, its conjugated double bond structure can effectively absorb high-energy blue light in the 415-455nm wavelength range, reducing direct phototoxic damage to retinal pigment epithelial cells and photoreceptor cells through a physical filtering mechanism.

[0154] The second level is the cellular level protective mechanism. In retinal pigment epithelial cells and retinal ganglion cells, all three active ingredients exhibited significant antioxidant activity. The polyphenolic hydroxyl structure of anthocyanins can directly scavenge superoxide anions induced by ultraviolet and blue light. hydroxyl radicals OH and singlet oxygen Resveratrol contains reactive oxygen species. It can upregulate the expression of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), enhancing the cell's endogenous antioxidant defense system. Lutein accumulates in the lipid bilayer of the cell membrane, protecting membrane lipids from lipid peroxidation damage. In terms of anti-inflammation, anthocyanins and resveratrol reduce the production and release of inflammatory factors such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) by inhibiting the activation of the nuclear factor κB (NF-κB) signaling pathway. In terms of anti-apoptosis, resveratrol protects retinal ganglion cells from ischemia-reperfusion injury and oxidative stress-induced programmed cell death by inhibiting mitochondrial apoptosis, reducing cytochrome C release and the cascade activation of Caspase-3 and Caspase-9.

[0155] The third level involves tissue-level repair mechanisms. At the corneal level, the composition of this invention promotes the proliferation and migration of corneal epithelial cells, accelerating the repair of corneal epithelial damage and the reconstruction of barrier function. At the conjunctival level, the antioxidant and anti-inflammatory effects of anthocyanins and lutein protect conjunctival goblet cells from inflammatory damage, maintain and promote goblet cell function, increase the secretion of mucin MUC5AC, and improve mucus layer quality. At the tear film level, excipients such as sodium hyaluronate work synergistically with the active ingredients to stabilize the tear film structure, prolong tear film breakup time, improve the uniformity of tear distribution on the ocular surface, and reduce tear evaporation.

[0156] The fourth level is the mechanism for improving overall function. Through the aforementioned multi-target, multi-level synergistic protective effects, the composition of this invention ultimately improves the overall function of the eye. Accelerated rhodopsin regeneration leads to enhanced dark adaptation and improved visual sensitivity; comprehensive antioxidant protection reduces cumulative light damage and degenerative changes in the retina; protection of retinal ganglion cells maintains the integrity of optic nerve conduction function; tear film stabilization and ocular surface repair improve the ocular surface microenvironment and alleviate dry eye symptoms. These effects complement and promote each other, forming a complete protective system from molecules to cells, from tissues to functions, providing a comprehensive and effective treatment plan for eye diseases such as dry eye, eye strain, and blue light damage.

[0157] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of the claims of the present invention.

Claims

1. An ophthalmic composition containing anthocyanins, resveratrol, and lutein, characterized in that, The composition comprises, by weight percentage: 0.1% to 0.5% anthocyanins, 0.05% to 0.2% resveratrol, 0.01% to 0.1% lutein, 10% to 30% phosphate buffer solution as a co-solvent, appropriate amount of isotonic adjuster, appropriate amount of pH adjuster, 0.005% to 0.02% preservative, and purified water to 100%.

2. The ophthalmic composition according to claim 1, characterized in that, The anthocyanins are derived from European blueberry extract, with an anthocyanin content of not less than 25%, of which delphinidin anthocyanins account for 30% to 50% of the total anthocyanin content.

3. The ophthalmic composition according to claim 1, characterized in that, The resveratrol is trans-resveratrol with a purity of not less than 98%; the lutein is derived from calendula extract with a lutein content of not less than 20%.

4. The ophthalmic composition according to claim 1, characterized in that, The phosphate buffer solution has a phosphate concentration of 0.01 mol / L to 0.1 mol / L, a sodium chloride concentration of 0.8% to 0.9%, and a pH of 7.2 to 7.

6.

5. The ophthalmic composition according to claim 1, characterized in that, The composition further includes 0.1% to 1.0% of a thickener and 0.01% to 0.1% of a stabilizer, wherein the thickener is selected from one or more combinations of sodium hyaluronate, hydroxypropyl methylcellulose, and carbomer, and the stabilizer is selected from one or more combinations of disodium edetate, ascorbic acid, and sodium thiosulfate.

6. A method for preparing the ophthalmic composition according to any one of claims 1 to 5, characterized in that, Includes the following steps: Step 1: Dissolve sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride in purified water to prepare a phosphate buffer solution; Step 2: Add resveratrol and lutein to the phosphate buffer solution and sonicate at 40°C to 60°C for 15 to 30 minutes; Step 3: Add anthocyanins and stir to dissolve at room temperature; Step 4: Add excipients and adjust the pH value; Step 5: Make up to volume, filter, sterilize, and dispense.

7. An ophthalmic eye drop, characterized in that, The eye drops comprise the ophthalmic composition according to any one of claims 1 to 5, and the viscosity of the eye drops is from 4 mPa·s to 9 mPa·s.

8. An ophthalmic gel, characterized in that, The ophthalmic gel comprises the ophthalmic composition of any one of claims 1 to 5, wherein the thickener content is 0.3% to 1.0%, and the viscosity of the ophthalmic gel is 50 mPa·s to 500 mPa·s.

9. The ophthalmic eye drops according to claim 7 or the ophthalmic gel according to claim 8, characterized in that, The eye drops or ophthalmic gel have an osmotic pressure of 280 mOsm / kg to 320 mOsm / kg and a pH of 6.5 to 7.

8.

10. The use of the ophthalmic composition according to any one of claims 1 to 5 in the preparation of a medicament for treating or preventing ophthalmic diseases, characterized in that, The ophthalmological disease is selected from one or more of the following: dry eye syndrome, visual fatigue syndrome, blue light-induced retinal damage, and mild corneal epithelial damage.

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