Eyedrop based on multifunctional peptide composition, preparation method of eyedrop and application of eyedrop in treatment of diabetic retinopathy

Abstract

The invention provides eyedrops based on a multifunctional peptide composition, a preparation method of the eyedrops and application of the eyedrops to treatment of diabetic retinopathy, and belongs to the technical field of eyedrops. The eye drops contain S-acetyl-L-glutathione, fisheye extracted peptide, hyperforin perforatum flower extracted peptide, aesculus hippocastanum leaf extracted peptide and other active ingredients. Wherein the fisheye extracted peptide is a small-molecule extracted peptide obtained by sequentially carrying out graded enzymolysis on snow fisheye through trypsin and serrapeptase. The hyperforin perforatum flower extracted peptide is a small-molecule extracted peptide obtained by sequentially carrying out graded enzymolysis on hyperforin perforatum flowers through bromelain, serrapeptase and nattokinase. The aesculus hippocastanum leaf extracted peptide is a small molecule extracted peptide obtained by performing common enzymolysis on aesculus hippocastanum leaves through papain and ficin. The multifunctional peptide composition disclosed by the invention exerts the portability of eyedrops in treating diabetic retinopathy, and also has safe and efficient treatment effects.

Description

Technical Field

[0001] The invention belongs to the technical field of eye drops, and in particular relates to eye drops based on a multifunctional peptide composition, a preparation method thereof, and application of the eye drops in treating diabetic retinopathy. Background Art

[0002] Diabetic retinopathy is one of the common and serious microvascular complications of diabetes. The continuous increase in the number of diabetic patients worldwide has led to an increasing prevalence of diabetic retinopathy, making it one of the major blinding eye diseases. According to statistics, some diabetic patients will develop varying degrees of retinopathy after several years of illness, which seriously threatens the patient's vision health and quality of life.

[0003] At present, there are various treatments for diabetic retinopathy. Laser photocoagulation therapy controls the progression of the disease by destroying the abnormal vascular tissue of the retina and reducing the production of angiogenic factors, but it may damage the surrounding normal retinal tissue and cause side effects such as visual field defects. Vitrectomy is suitable for more severe proliferative diabetic retinopathy, such as large amounts of vitreous hemorrhage and retinal detachment, but the surgical risk is high, the postoperative recovery period is long, and there is a risk of complications such as infection and re-detachment of the retina. In terms of drug treatment, although anti-vascular endothelial growth factor (VEGF) drugs can effectively inhibit the formation of new blood vessels, they require frequent intraocular injections, which not only increases the risk of intraocular infection, bleeding, retinal detachment, etc., but also imposes a large economic burden and psychological pressure on patients; although glucocorticoids can reduce retinal inflammatory reactions, long-term use can cause eye complications such as cataracts and glaucoma.

[0004] As a topical medication, eye drops have the advantages of being easy to use, having high patient compliance, and being able to act directly on the site of eye lesions. They have a unique position in the treatment of ophthalmic diseases. However, existing eye drops still have many limitations in the treatment of diabetic retinopathy. Most conventional eye drops focus mainly on relieving general symptoms such as dry eyes, fatigue, and inflammation, and lack targeted effects on the complex pathophysiological mechanisms of diabetic retinopathy. For example, some common lubricating eye drops can only improve the lubrication of the ocular surface, but cannot interfere with the progression of retinal vascular lesions and nerve damage; anti-infective eye drops can only prevent or treat eye infections, but are powerless against core problems of diabetic retinopathy such as abnormal vascular proliferation and retinal hypoxia and ischemia.

[0005] In recent years, researchers have been committed to developing eye drops that can effectively treat diabetic retinopathy. Some studies focus on the development of new drug carriers, such as nanoparticles and liposomes, to improve the stability, bioavailability and penetration of drugs into ocular tissues, so that drugs can act more accurately on the site of retinal disease. At the same time, for the key pathological links of diabetic retinopathy, such as oxidative stress damage, inflammatory response, and cell apoptosis, explore drug ingredients with corresponding mechanisms of action and try to formulate them into eye drops. However, to date, there is still a lack of eye drops that can safely, effectively and conveniently treat diabetic retinopathy. Summary of the invention

[0006] In view of the fact that the existing market still lacks eye drops for treating diabetic retinopathy safely, efficiently and conveniently, the present invention provides an eye drop based on a multifunctional peptide composition, a preparation method thereof and an application thereof in treating diabetic retinopathy, with a specific ratio of S-acetyl-L-glutathione, fish eye extract peptide, Hypericum perforatum flower extract peptide and Aesculus hippocastanum leaf extract peptide as active substances, which makes use of the portability of eye drops for treating diabetic retinopathy and has a safe and efficient treatment effect. The specific technical scheme is as follows:

[0007] Disclosed is an eye drop based on a multifunctional peptide composition, comprising the following raw materials: 1 w / v% to 2 w / v% S-acetyl-L-glutathione, 0.5 w / v% to 1 w / v% fish eye extracted peptide, 0.5 w / v% to 1 w / v% Hypericum perforatum flower extracted peptide, 0.3 w / v% to 0.5 w / v% Aesculus hippocastanum leaf extracted peptide, 0.8 w / v% to 0.9 w / v% osmotic pressure regulator, 0.003 w / v% to 0.008 w / v% preservative, a buffer for adjusting pH value to 7.2 to 7.6, and the remainder being water for injection.

[0008] In the above eye drops, the osmotic pressure regulator is sodium chloride.

[0009] In the above eye drops, the buffer is phosphate buffered saline (PBS).

[0010] In the above-mentioned eye drops, the preparation method of the fish eye extracted peptide comprises the following steps: washing fresh snow fish eyes, removing impurities, crushing into a homogenate with a tissue crusher at a low temperature of 4°C to 6°C to obtain fish eye slurry; adding the fish eye slurry to a concentration of 0.04 mol / L to 0.06 mol / L according to the mass ratio of fish eye slurry to phosphate buffer solution = 1: (8-10); l / L phosphate buffer with a pH of 7.0-8.0, then add 0.5%-1.0% of trypsin by weight of the fish eye slurry, perform enzymatic hydrolysis at 35°C-40°C for 60min-90min, and heat up to inactivate the enzyme; after the temperature drops to 35°C-40°C, add 0.5%-1.0% of serrapeptase by weight of the fish eye slurry, perform enzymatic hydrolysis for 60min-90min, and heat up to inactivate the enzyme; after the temperature drops to room temperature, centrifuge at 6000rpm-8000rpm for 20min-30min to obtain the supernatant; filter the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000kDa and 3000kDa, and freeze-dry to obtain fish eye extracted peptides.

[0011] In the above method for preparing the peptide extracted from fish eyes, the snow fish eye includes the cornea, lens, retina and vitreous body.

[0012] In the above-mentioned eye drops, the preparation method of the peptide extracted from the flower of Hypericum perforatum comprises the following steps: crushing the flower of Hypericum perforatum, adding water of 8 to 10 times the mass of the flower of Hypericum perforatum, adjusting the pH to 6.0 to 7.0, adding 0.5% to 1% bromelain by mass of the flower of Hypericum perforatum, enzymolysis at 50°C to 55°C for 60min to 90min, heating to inactivate the enzyme; after cooling to 35°C to 40°C, adjusting the pH to 7.0 to 8.0, adding 0.5% to 1.0% serrapeptase by mass of the flower of Hypericum perforatum, enzymolysis for 60min The method comprises the steps of: heating the mixture to 35°C to 40°C, adjusting the pH value to 7.2 to 7.8, adding 0.5% to 1.0% of the weight of the flower of Hypericum perforatum, performing enzymolysis for 60min to 90min, and heating the mixture to inactivate the enzyme; cooling the mixture to room temperature, centrifuging the mixture at 6000rpm to 8000rpm for 20min to 30min, and taking the supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff of 1000kDa to 3000kDa, and freeze-drying the mixture to obtain a peptide extracted from the flower of Hypericum perforatum.

[0013] In the above-mentioned eye drops, the preparation method of the peptide extracted from the leaves of Aesculus hippocastanum comprises the following steps: taking the leaves of Aesculus hippocastanum and crushing them, first soaking them in a 0.1v / v% to 0.5v / v% hydrochloric acid aqueous solution with a concentration of 5 to 6 times the mass of the leaves of Aesculus hippocastanum for 1h to 2h, filtering them through a 200-mesh to 250-mesh sieve to obtain a filtrate A and a residue; soaking the residue in a 0.1wt% to 0.5wt% sodium hydroxide aqueous solution with a concentration of 5 to 6 times the mass of the leaves of Aesculus hippocastanum for 1h to 2h, filtering them through a 200-mesh to 250-mesh sieve to obtain a filtrate B; combining the filtrate A and the filtrate B, adjusting the pH to 5.0-6.0 to obtain an extract; add 0.05%-0.15% of papain and 0.05%-0.15% of fig protease by weight of the extract to the extract, perform enzymolysis at 60-65°C for 90-120 minutes, and heat to inactivate the enzymes; after cooling to room temperature, centrifuge at 6000-8000 rpm for 20-30 minutes to obtain a supernatant; filter the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000 kDa and 3000 kDa, and freeze-dry to obtain a horse chestnut leaf extract peptide.

[0014] In the above preparation method, the temperature for heating to inactivate the enzyme is 85°C to 90°C, and the time for heating to inactivate the enzyme is 10min to 20min.

[0015] The above-mentioned method for preparing eye drops based on the multifunctional peptide composition comprises the following steps:

[0016] S1: Add 70% to 80% volume percent of water for injection into a liquid preparation container, then add an osmotic pressure regulator and a preservative, stir while adding, mix and dissolve evenly, then add S-acetyl-L-glutathione, fish eye extract peptide, Hypericum perforatum flower extract peptide and Aesculus hippocastanum leaf extract peptide, stir while adding, mix and dissolve evenly, use a pH meter to detect the pH value of the solution, then add a buffer dropwise to adjust the pH to 7.2 to 7.6, and then dilute with water for injection to the final volume of eye drops to obtain an eye drops solution;

[0017] S2: filtering, sterilizing, and sealing the eye drops solution to obtain eye drops.

[0018] Application of the above-mentioned eye drops based on the multifunctional peptide composition in the treatment of diabetic retinopathy.

[0019] The eye drops based on the multifunctional peptide composition of the present invention, the preparation method thereof and the application thereof in treating diabetic retinopathy have the following beneficial effects:

[0020] 1. During diabetic retinopathy, high blood sugar triggers a series of oxidative stress reactions and produces a large amount of reactive oxygen species (ROS). S-acetyl-L-glutathione can directly bind to ROS and reduce them to harmless substances, thereby reducing the damage of oxidative stress to retinal cells. S-acetyl-L-glutathione can remove reactive oxygen species such as superoxide anions and hydroxyl free radicals, and protect the retinal vascular endothelial cells, nerve cells, etc. from oxidative damage. In the diabetic state, the redox state in the cell is destroyed. S-acetyl-L-glutathione can restore the normal redox environment in the cell and ensure the normal metabolism and physiological function of the cell. The oxidative stress and inflammatory response caused by high blood sugar can lead to retinal cell apoptosis. S-acetyl-L-glutathione can reduce the apoptosis of retinal cells by regulating the apoptotic signaling pathway in the cell, inhibiting the activation of caspase enzymes, etc., and maintaining the normal structure and function of the retina. In diabetic retinopathy, oxidative stress and inflammation lead to kidney damage, which in turn affects the level of Cys C. S-acetyl-L-glutathione indirectly protects kidney function by reducing oxidative stress and inflammatory response. S-acetyl-L-glutathione prevents abnormal glomerular filtration function caused by oxidative stress and inflammation, thereby maintaining Cys C within the normal range. Oxidative stress is an important factor in the upregulation of VEGF expression. S-acetyl-L-glutathione can inhibit the activation of hypoxia-inducible factor-1α (HIF-1α) by scavenging ROS and reducing oxidative stress. HIF-1α is a key regulator of VEGF expression. Inhibiting its activation can reduce the overexpression of VEGF and prevent the abnormal proliferation of retinal neovascularization and increased vascular permeability. Inflammatory response plays an important role in the pathogenesis of diabetic retinopathy. TNF-α is a key inflammatory factor. S-acetyl-L-glutathione reduces inflammatory response through antioxidant effects, thereby inhibiting the activation of inflammatory cells and the release of inflammatory factors such as TNF-α. It regulates the intracellular inflammatory signaling pathways, NF-κB pathways, etc., and reduces the production of TNF-α.

[0021] 2. Fish eye extracted peptides are 1000kDa to 3000kDa extracted peptides obtained by graded enzymatic hydrolysis of snow fish eyes with trypsin and serrapeptase. Fish eye extracted peptides treat diabetic retinopathy by regulating the metabolism and function of retinal cells. Under high blood sugar environment, the metabolism and signal transduction pathways of retinal cells will be disordered. Fish eye extracted peptides contain some bioactive peptides that can regulate signal molecules in cells, protect retinal cells by regulating signal pathways such as PI3K-Akt, and promote cell survival. Fish eye extracted peptides have a protective effect on retinal vascular endothelial cells. Under diabetic conditions, vascular endothelial cells are damaged. Fish eye extracted peptides can enhance the barrier function of endothelial cells and reduce vascular leakage, thereby reducing retinal edema and other lesions. Diabetic retinopathy leads to kidney damage and changes in Cys C levels. Fish eye extracted peptides indirectly regulate the level of Cys C by improving retinal blood circulation and reducing renal hemodynamic changes. At the same time, it can also prevent Cys C from increasing due to inflammatory stimulation by inhibiting inflammatory response. Fish eye extracted peptides reduce the expression of VEGF by regulating intracellular signaling pathways and inhibiting the activation of hypoxia-inducible factor-1α (HIF-1α). During the inflammatory process of diabetic retinopathy, the release of TNF-α increases. Fish eye extracted peptides reduce the level of TNF-α by inhibiting related inflammatory signaling pathways, such as NF-κB. Fish eye extracted peptides contain antioxidant peptides, and at the same time, by activating the antioxidant enzyme system in retinal cells, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), they enhance the antioxidant capacity of cells and reduce the generation and accumulation of ROS.

[0022] 3. Hypericum perforatum flower extract peptides are 1000kDa to 3000kDa extract peptides obtained by graded enzymatic hydrolysis of Hypericum perforatum flowers with bromelain, serrapeptase, and nattokinase. Hypericum perforatum flower extract peptides treat diabetic retinopathy through their anti-inflammatory and antioxidant properties. Inflammation and oxidative stress play a key role in the pathogenesis of diabetic retinopathy. Hypericum perforatum flower extract peptides reduce retinal tissue damage by inhibiting inflammatory response and scavenging ROS. Protect retinal nerve cells by regulating the expression of neuroprotective factors. In diabetic retinopathy, nerve cells are damaged. Hypericum perforatum flower extract peptides promote the secretion of neuroprotective factors and maintain the normal function of nerve cells. Hypericum perforatum flower extract peptides indirectly regulate the level of Cys C by reducing inflammatory response and oxidative stress. Inflammation and oxidative stress can cause kidney damage, which in turn affects the level of Cys C. Hypericum perforatum flower extract peptides maintain the normal range of Cys C by improving the microenvironment of the retina and kidney. Hypericum perforatum flower extract peptides affect the expression of VEGF by regulating the release of inflammatory factors. The inflammatory response stimulates the production of VEGF. Hypericum perforatum flower extract peptides inhibit the inflammatory response, reduce the abnormal upregulation of VEGF, and prevent angiogenesis and vascular leakage. Hypericum perforatum flower extract peptides reduce the level of TNF-α by inhibiting the activity of inflammatory cells and inflammatory signaling pathways. Hypericum perforatum flower extract peptides enhance the antioxidant defense capacity of cells and reduce the production and damage of ROS by regulating the expression of antioxidant enzyme genes in cells and increasing the expression of enzymes such as catalase (CAT).

[0023] 4. Aesculus hippocastanum leaf extract peptide is an extract peptide of 1000kDa to 3000kDa obtained by co-enzymatic hydrolysis of Aesculus hippocastanum leaves with papain and ficin. Aesculus hippocastanum leaf extract peptide treats diabetic retinopathy by improving retinal vascular permeability and regulating vascular function. In the diabetic state, retinal vascular permeability increases, and Aesculus hippocastanum leaf extract peptide enhances vascular barrier function by regulating junction proteins between vascular endothelial cells. It reduces retinal tissue damage by inhibiting inflammatory response and oxidative stress. Inflammation and oxidative stress can cause retinal vascular lesions and nerve cell damage, and Aesculus hippocastanum leaf extract peptide reduces these damages through multiple pathways. Aesculus hippocastanum leaf extract peptide regulates the level of Cys C by improving retinal blood circulation and reducing inflammatory response. Improved blood circulation reduces damage to organs such as the kidneys, and reduced inflammatory response also prevents abnormal increase of Cys C due to inflammation. Aesculus hippocastanum leaf extract peptide affects the expression of VEGF by regulating the function and signal transduction of vascular endothelial cells, and reduces the secretion of VEGF by inhibiting the hypoxic response of vascular endothelial cells, preventing excessive formation of new blood vessels and vascular leakage. Aesculus hippocastanum leaf extract peptides can prevent the migration of inflammatory cells by regulating the components of the extracellular matrix, while inhibiting the inflammatory signaling pathways in inflammatory cells and reducing the production of TNF-α. Aesculus hippocastanum leaf extract peptides can enhance the antioxidant capacity of cells and reduce the accumulation and damage of ROS by regulating the redox balance in cells and the ratio of glutathione / oxidized glutathione (GSH / GSSG).

[0024] 5. Hypericum flower extract peptide and Aesculus hippocastanum leaf extract peptide have synergistic antioxidant effects: Hypericum flower extract peptide and Aesculus hippocastanum leaf extract peptide both have antioxidant properties. Hypericum flower extract peptide directly removes reactive oxygen species (ROS) through its antioxidant components, and can regulate the expression of antioxidant enzyme genes in cells, such as increasing the expression of catalase (CAT). Aesculus hippocastanum leaf extract peptide can also remove ROS, regulate the redox balance in cells, and regulate the glutathione / oxidized glutathione (GSH / GSSG) ratio. The combination of the two can more effectively remove ROS in retinal tissue and reduce the damage of oxidative stress to retinal cells and blood vessels. Hypericum flower extract peptide and Aesculus hippocastanum leaf extract peptide have a synergistic anti-inflammatory effect: Inflammation is a key link in diabetic retinopathy. Hypericum flower extract peptide inhibits the activity of inflammatory cells, reduces the secretion of inflammatory factors such as tumor necrosis factor-α (TNF-α) by inhibiting the activation of inflammasomes in inflammatory cells, and can also regulate signal molecules in cells to prevent the activation of inflammation-related signal pathways. Peptides extracted from the leaves of Aesculus hippocastanum can inhibit the infiltration of inflammatory cells, regulate the components of the extracellular matrix to prevent the migration of inflammatory cells, and inhibit the inflammatory signaling pathways in inflammatory cells. The combined use of the two can more comprehensively inhibit the inflammatory response and reduce inflammatory damage to retinal tissue. The combination of peptides extracted from the flowers of Hypericum perforatum and peptides extracted from the leaves of Aesculus hippocastanum has a synergistic effect in vascular protection: the peptides extracted from the flowers of Hypericum perforatum affect the expression of vascular endothelial growth factor (VEGF) by regulating the release of inflammatory factors, preventing angiogenesis and vascular leakage. Peptides extracted from the leaves of Aesculus hippocastanum can improve the permeability of retinal blood vessels, regulate the junction proteins between vascular endothelial cells, and enhance the vascular barrier function. The combination of the two helps to better protect retinal blood vessels, maintain the normal function and structure of blood vessels, and reduce vascular lesions in diabetic retinopathy.

[0025] 6. The combination of fish eye extract peptides, Hypericum perforatum flower extract peptides and Aesculus hippocastanum leaf extract peptides can exert all-round antioxidant defense: Fish eye extract peptides contain antioxidant peptides that can directly remove ROS and activate the antioxidant enzyme system in retinal cells, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), etc. When used together with Hypericum perforatum flower extract peptides and Aesculus hippocastanum leaf extract peptides, a multi-level antioxidant defense system can be constructed to more efficiently remove various ROS in retinal tissue and reduce oxidative stress damage to the retina from multiple angles. The combination of fish eye extract peptides, Hypericum perforatum flower extract peptides and Aesculus hippocastanum leaf extract peptides can achieve comprehensive anti-inflammatory mechanism complementarity: The anti-inflammatory mechanisms of the three can more comprehensively inhibit all aspects of the inflammatory response, including the activation of inflammatory cells, the release of inflammatory factors and the migration of inflammatory cells, thereby more effectively reducing the inflammation of retinal tissue. The combination of fish eye extract peptides, Hypericum perforatum flower extract peptides and Aesculus hippocastanum leaf extract peptides can synergistically improve vascular and cell functions: fish eye extract peptides have a protective effect on retinal vascular endothelial cells, regulate the expression of VEGF, and prevent excessive formation of new blood vessels and vascular leakage. Hypericum perforatum flower extract peptides and Aesculus hippocastanum leaf extract peptides also have their own effects in vascular protection. At the same time, these three extract peptides also jointly regulate the metabolism and function of retinal cells, promote cell survival through different signaling pathways, protect retinal nerve cells, and improve the condition of diabetic retinopathy at both the vascular and cellular levels. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0027] w / v% means: "w / v%" is the unit of mass volume percentage concentration. "w" represents the mass of the solute (weight), and "v" represents the volume of the solution (volume). W / v% represents the mass of the solute in grams per 100 ml of solution.

[0028] Example 1

[0029] An eye drop based on a multifunctional peptide composition, the eye drop comprises the following raw materials: 1.5w / v% S-acetyl-L-glutathione, 0.8w / v% fish eye extract peptide, 0.8w / v% Hypericum perforatum flower extract peptide, 0.4w / v% Aesculus hippocastanum leaf extract peptide, 0.85w / v% osmotic pressure regulator, 0.005w / v% potassium sorbate preservative, buffer to adjust pH 7.4, and the remainder is water for injection. Among them, the osmotic pressure regulator is sodium chloride; the buffer is phosphate buffered saline (PBS).

[0030] The preparation method of fish eye extracted peptides comprises the following steps: selecting fresh snow fish eyes, including cornea, lens, retina and vitreous parts, washing the fresh snow fish eyes, removing impurities, crushing them into homogenate at a low temperature of 5°C with a tissue crusher to obtain fish eye slurry; adding the fish eye slurry to a phosphate buffer with a concentration of 0.05 mol / L and a pH of 7.5 according to a mass ratio of fish eye slurry: phosphate buffer = 1:9, and then adding 0.8% of trypsin by weight of the fish eye slurry, performing enzymolysis at 37°C for 80 minutes, heating to 88°C for 15 minutes to inactivate the enzyme; after cooling to 37°C, adding 0.8% of serrapeptase by weight of the fish eye slurry, performing enzymolysis for 80 minutes, heating to 88°C for 15 minutes to inactivate the enzyme; after cooling to room temperature, centrifuging at 7000 rpm for 25 minutes to obtain a supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000 kDa and 3000 kDa, freeze-drying, and obtaining fish eye extracted peptides.

[0031] The preparation method of the peptide extracted from the flower of Hypericum perforatum comprises the following steps: crushing the flower of Hypericum perforatum, adding water of 9 times the mass of the flower of Hypericum perforatum, adjusting the pH to 6.5, adding 0.8% bromelain by mass of the flower of Hypericum perforatum, enzymolysis at 52°C for 80 minutes, heating to 88°C for 15 minutes to inactivate the enzyme; after the temperature drops to 37°C, adjusting the pH to 7.5, adding 0.8% serrapeptase by mass of the flower of Hypericum perforatum, enzymolysis for 80 minutes, heating to 88°C for 15 minutes to inactivate the enzyme; after the temperature drops to 37°C, adjusting the pH to 7.5, adding 0.8% nattokinase by mass of the flower of Hypericum perforatum, enzymolysis for 80 minutes, heating to 88°C for 15 minutes to inactivate the enzyme; after the temperature drops to room temperature, centrifuging at 7000rpm for 25 minutes to obtain the supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000kDa and 3000kDa, freeze-drying, and obtaining the peptide extracted from the flower of Hypericum perforatum.

[0032] The preparation method of the peptide extracted from the leaves of Aesculus hippocastanum comprises the following steps: crushing the leaves of Aesculus hippocastanum, soaking them in a 0.3v / v% hydrochloric acid aqueous solution with a mass of 5.5 times that of the leaves of Aesculus hippocastanum for 1.5 hours, filtering them through a 200-mesh sieve to obtain a filtrate A and a residue; soaking the residue in a 0.3wt% sodium hydroxide aqueous solution with a mass of 5.5 times that of the leaves of Aesculus hippocastanum for 1.5 hours, filtering them through a 200-mesh sieve to obtain a filtrate B; combining the filtrate A and the filtrate B, and adjusting the pH to 5.5, obtaining an extract; adding 0.1% of papain and 0.1% of ficin by weight of the extract to the extract, performing enzymatic hydrolysis at 62° C. for 100 min, heating to 88° C. for 15 min to inactivate the enzyme; after cooling to room temperature, centrifuging at 7000 rpm for 25 min, and obtaining a supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000 kDa and 3000 kDa, and freeze-drying to obtain a horse chestnut leaf extract peptide.

[0033] The above-mentioned method for preparing eye drops based on the multifunctional peptide composition comprises the following steps:

[0034] S1: Add 75% by volume of water for injection into a liquid preparation container, then add an osmotic pressure regulator and a preservative, stir while adding, mix and dissolve evenly, then add S-acetyl-L-glutathione, fish eye extract peptide, Hypericum perforatum flower extract peptide and Aesculus hippocastanum leaf extract peptide, stir while adding, mix and dissolve evenly, detect the pH value of the solution with a pH meter, then add a buffer dropwise to adjust the pH to 7.4, and then dilute with water for injection to the final volume of eye drops to obtain an eye drops solution;

[0035] S2: filtering, sterilizing, and sealing the eye drops solution to obtain eye drops.

[0036] Application of the above-mentioned eye drops based on the multifunctional peptide composition in the treatment of diabetic retinopathy.

[0037] Example 2

[0038] An eye drop based on a multifunctional peptide composition, the eye drop comprises the following raw materials: 1w / v% S-acetyl-L-glutathione, 0.5w / v% fish eye extract peptide, 0.5w / v% Hypericum perforatum flower extract peptide, 0.3w / v% Aesculus hippocastanum leaf extract peptide, 0.8w / v% osmotic pressure regulator, 0.003w / v% potassium sorbate preservative, buffer to adjust pH 7.4, and the remainder is water for injection. Among them, the osmotic pressure regulator is sodium chloride; the buffer is phosphate buffered saline (PBS).

[0039] The preparation method of fish eye extracted peptides comprises the following steps: selecting fresh snow fish eyes, including cornea, lens, retina and vitreous parts, washing the fresh snow fish eyes, removing impurities, crushing them into homogenate at a low temperature of 4°C with a tissue crusher to obtain fish eye slurry; adding the fish eye slurry to a phosphate buffer with a concentration of 0.04 mol / L and a pH of 7.0 according to a mass ratio of fish eye slurry: phosphate buffer = 1:8, and then adding 0.5% of trypsin by weight of the fish eye slurry, performing enzymolysis at 35°C for 60 minutes, heating to 85°C for 10 minutes to inactivate the enzyme; after cooling to 35°C, adding 0.5% of serrapeptidase by weight of the fish eye slurry, performing enzymolysis for 60 minutes, heating to 85°C for 10 minutes to inactivate the enzyme; after cooling to room temperature, centrifuging at 6000 rpm for 20 minutes to obtain a supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000 kDa and 3000 kDa, freeze-drying, and obtaining fish eye extracted peptides.

[0040] The preparation method of the peptide extracted from the flower of Hypericum perforatum comprises the following steps: crushing the flower of Hypericum perforatum, adding water 8 times the mass of the flower of Hypericum perforatum, adjusting the pH to 6.0, adding 0.5% bromelain by mass of the flower of Hypericum perforatum, enzymolysis at 50°C for 60 minutes, heating to 85°C for 10 minutes to inactivate the enzyme; after the temperature drops to 35°C, adjusting the pH to 7.0, adding 0.5% serrapeptase by mass of the flower of Hypericum perforatum, enzymolysis for 60 minutes, heating to 85°C for 10 minutes to inactivate the enzyme; after the temperature drops to 35°C, adjusting the pH to 7.2, adding 0.5% nattokinase by mass of the flower of Hypericum perforatum, enzymolysis for 60 minutes, heating to 85°C for 10 minutes to inactivate the enzyme; after the temperature drops to room temperature, centrifuging at 6000rpm for 20 minutes to obtain the supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000kDa and 3000kDa, freeze-drying, and obtaining the peptide extracted from the flower of Hypericum perforatum.

[0041] The preparation method of the peptide extracted from the leaves of Aesculus hippocastanum comprises the following steps: taking the leaves of Aesculus hippocastanum for crushing, first soaking them in a 0.1v / v% hydrochloric acid aqueous solution with a concentration of 5 times the mass of the leaves of Aesculus hippocastanum for 1 hour, filtering them through a 200-mesh screen to obtain a filtrate A and a residue; soaking the residue in a 0.1wt% sodium hydroxide aqueous solution with a concentration of 5 times the mass of the leaves of Aesculus hippocastanum for 1 hour, filtering them through a 200-mesh screen to obtain a filtrate B; combining the filtrate A and the filtrate B, adjusting the pH to 5.0, and obtaining an extract; adding 0.05% of papain and 0.05% of fig protease with a mass of the extract to the extract, performing enzymolysis at 60°C for 90 minutes, heating to 85°C for 10 minutes to inactivate the enzyme; cooling to room temperature, centrifuging at 6000rpm for 20 minutes, and obtaining a supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000kDa and 3000kDa, and freeze-drying to obtain the peptide extracted from the leaves of Aesculus hippocastanum.

[0042] The above-mentioned method for preparing eye drops based on the multifunctional peptide composition comprises the following steps:

[0043] S1: Add 70% by volume of water for injection into a liquid preparation container, then add an osmotic pressure regulator and a preservative, stir while adding, mix and dissolve evenly, then add S-acetyl-L-glutathione, fish eye extract peptide, Hypericum perforatum flower extract peptide and Aesculus hippocastanum leaf extract peptide, stir while adding, mix and dissolve evenly, use a pH meter to detect the pH value of the solution, then add a buffer dropwise to adjust the pH to 7.5, and then dilute with water for injection to the final volume of eye drops to obtain an eye drops solution;

[0044] S2: filtering, sterilizing, and sealing the eye drops solution to obtain eye drops.

[0045] Application of the above-mentioned eye drops based on the multifunctional peptide composition in the treatment of diabetic retinopathy.

[0046] Example 3

[0047] An eye drop based on a multifunctional peptide composition, the eye drop comprises the following raw materials: 1w / v% S-acetyl-L-glutathione, 1w / v% fish eye extract peptide, 0.5w / v% Hypericum perforatum flower extract peptide, 0.5w / v% Aesculus hippocastanum leaf extract peptide, 0.8w / v% osmotic pressure regulator, 0.008w / v% potassium sorbate preservative, buffer to adjust pH 7.4, and the remainder is water for injection. Among them, the osmotic pressure regulator is sodium chloride; the buffer is phosphate buffered saline (PBS).

[0048] The preparation method of fish eye extracted peptides comprises the following steps: selecting fresh snow fish eyes, including cornea, lens, retina and vitreous body parts, washing the fresh snow fish eyes, removing impurities, and crushing them into homogenate at a low temperature of 4°C with a tissue crusher to obtain fish eye slurry; adding the fish eye slurry to a phosphate buffer with a concentration of 0.04 mol / L and a pH of 8.0 according to a mass ratio of fish eye slurry: phosphate buffer = 1:10, and then adding 0.5% of trypsin by weight of the fish eye slurry, performing enzymolysis at 40°C for 60 minutes, heating to 90°C for 10 minutes to inactivate the enzyme; after cooling to 40°C, adding 0.5% of serrapeptidase by weight of the fish eye slurry, performing enzymolysis for 90 minutes, and heating to 85°C for 20 minutes to inactivate the enzyme; after cooling to room temperature, centrifuging at 6000 rpm for 30 minutes to obtain a supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000 kDa and 3000 kDa, and freeze-drying to obtain fish eye extracted peptides.

[0049] The preparation method of the peptide extracted from the flower of Hypericum perforatum comprises the following steps: crushing the flower of Hypericum perforatum, adding water 8 times the mass of the flower of Hypericum perforatum, adjusting the pH to 7.0, adding 0.5% bromelain by mass of the flower of Hypericum perforatum, enzymolysis at 55°C for 60 minutes, heating to 90°C for 10 minutes to inactivate the enzyme; after the temperature drops to 40°C, adjusting the pH to 7.0, adding 1.0% serrapeptase by mass of the flower of Hypericum perforatum, enzymolysis for 60 minutes, heating to 90°C for 10 minutes to inactivate the enzyme; after the temperature drops to 40°C, adjusting the pH to 7.2, adding 1.0% nattokinase by mass of the flower of Hypericum perforatum, enzymolysis for 60 minutes, heating to 90°C for 10 minutes to inactivate the enzyme; after the temperature drops to room temperature, centrifuging at 8000rpm for 20 minutes to obtain the supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000kDa and 3000kDa, freeze-drying, and obtaining the peptide extracted from the flower of Hypericum perforatum.

[0050] The preparation method of the peptide extracted from the leaves of Aesculus hippocastanum comprises the following steps: taking the leaves of Aesculus hippocastanum and crushing them, first soaking them in a 0.1v / v% hydrochloric acid aqueous solution with a concentration of 6 times the mass of the leaves of Aesculus hippocastanum for 2 hours, filtering them through a 200-mesh screen to obtain a filtrate A and a residue; soaking the residue in a 0.1wt% sodium hydroxide aqueous solution with a concentration of 6 times the mass of the leaves of Aesculus hippocastanum for 2 hours, filtering them through a 200-mesh screen to obtain a filtrate B; combining the filtrate A and the filtrate B, adjusting the pH to 6.0, and obtaining an extract; adding 0.05% of papain and 0.15% of fig protease with a mass of the extract to the extract, performing enzymolysis at 60°C for 120 minutes, heating to 85°C for 20 minutes to inactivate the enzyme; cooling to room temperature, centrifuging at 6000rpm for 30 minutes, and obtaining a supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000kDa and 3000kDa, and freeze-drying to obtain the peptide extracted from the leaves of Aesculus hippocastanum.

[0051] The above-mentioned method for preparing eye drops based on the multifunctional peptide composition comprises the following steps:

[0052] S1: Add 70% by volume of water for injection into a liquid preparation container, then add an osmotic pressure regulator and a preservative, stir while adding, mix and dissolve evenly, then add S-acetyl-L-glutathione, fish eye extract peptide, Hypericum perforatum flower extract peptide and Aesculus hippocastanum leaf extract peptide, stir while adding, mix and dissolve evenly, detect the pH value of the solution with a pH meter, then add a buffer dropwise to adjust the pH to 7.2, and then dilute with water for injection to the final volume of eye drops to obtain an eye drops solution;

[0053] S2: filtering, sterilizing, and sealing the eye drops solution to obtain eye drops.

[0054] Application of the above-mentioned eye drops based on the multifunctional peptide composition in the treatment of diabetic retinopathy.

[0055] Example 4

[0056] An eye drop based on a multifunctional peptide composition, the eye drop comprises the following raw materials: 2w / v% S-acetyl-L-glutathione, 1w / v% fish eye extract peptide, 1w / v% Hypericum perforatum flower extract peptide, 0.5w / v% Aesculus hippocastanum leaf extract peptide, 0.9w / v% osmotic pressure regulator, 0.008w / v% potassium sorbate preservative, buffer to adjust pH 7.4, and the remainder is water for injection. Among them, the osmotic pressure regulator is sodium chloride; the buffer is phosphate buffered saline (PBS).

[0057] The preparation method of fish eye extracted peptides comprises the following steps: selecting fresh snow fish eyes, including cornea, lens, retina and vitreous parts, washing the fresh snow fish eyes, removing impurities, and crushing them into homogenate at a low temperature of 6°C with a tissue crusher to obtain fish eye slurry; adding the fish eye slurry to a phosphate buffer with a concentration of 0.06 mol / L and a pH of 8.0 according to a mass ratio of fish eye slurry: phosphate buffer = 1:10, and then adding 1.0% of trypsin by weight of the fish eye slurry, performing enzymolysis at 40°C for 90 minutes, heating to 90°C to inactivate the enzyme for 20 minutes; after cooling to 40°C, adding 1.0% of serrapeptidase by weight of the fish eye slurry, performing enzymolysis for 90 minutes, and heating to 90°C to inactivate the enzyme for 20 minutes; after cooling to room temperature, centrifuging at 8000 rpm for 30 minutes to obtain a supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000 kDa and 3000 kDa, and freeze-drying to obtain fish eye extracted peptides.

[0058] The preparation method of the peptide extracted from the flower of Hypericum perforatum comprises the following steps: crushing the flower of Hypericum perforatum, adding water 10 times the mass of the flower of Hypericum perforatum, adjusting the pH to 7.0, adding 1% bromelain by mass of the flower of Hypericum perforatum, enzymolysis at 55°C for 90 minutes, heating to 90°C to inactivate the enzyme for 20 minutes; after the temperature drops to 40°C, adjusting the pH to 8.0, adding 1.0% serrapeptase by mass of the flower of Hypericum perforatum, enzymolysis for 90 minutes, heating to 90°C to inactivate the enzyme for 20 minutes; after the temperature drops to 40°C, adjusting the pH to 7.8, adding 1.0% nattokinase by mass of the flower of Hypericum perforatum, enzymolysis for 90 minutes, heating to 90°C to inactivate the enzyme for 20 minutes; after the temperature drops to room temperature, centrifuging at 8000rpm for 30 minutes, and taking the supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000kDa and 3000kDa, and freeze-drying to obtain the peptide extracted from the flower of Hypericum perforatum.

[0059] The preparation method of the peptide extracted from the leaves of Aesculus hippocastanum comprises the following steps: taking the leaves of Aesculus hippocastanum and crushing them, first soaking them in a 0.5v / v% hydrochloric acid aqueous solution with a concentration of 6 times the mass of the leaves of Aesculus hippocastanum for 2 hours, filtering them through a 250-mesh screen to obtain a filtrate A and a residue; soaking the residue in a 0.5wt% sodium hydroxide aqueous solution with a concentration of 6 times the mass of the leaves of Aesculus hippocastanum for 2 hours, filtering them through a 250-mesh screen to obtain a filtrate B; combining the filtrate A and the filtrate B, adjusting the pH to 6.0, and obtaining an extract; adding 0.15% of papain and 0.15% of fig protease with a mass of the extract to the extract, performing enzymolysis at 65°C for 120 minutes, heating to 90°C and inactivating the enzyme for 20 minutes; cooling to room temperature, centrifuging at 8000rpm for 30 minutes, and obtaining a supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000kDa and 3000kDa, and freeze-drying to obtain the peptide extracted from the leaves of Aesculus hippocastanum.

[0060] The above-mentioned method for preparing eye drops based on the multifunctional peptide composition comprises the following steps:

[0061] S1: Add 80% volume percent of water for injection into a liquid preparation container, then add an osmotic pressure regulator and a preservative, stir while adding, mix and dissolve evenly, then add S-acetyl-L-glutathione, fish eye extract peptide, Hypericum perforatum flower extract peptide and Aesculus hippocastanum leaf extract peptide, stir while adding, mix and dissolve evenly, use a pH meter to detect the pH value of the solution, then add a buffer dropwise to adjust the pH to 7.6, and then dilute with water for injection to the final volume of eye drops to obtain an eye drops solution;

[0062] S2: filtering, sterilizing, and sealing the eye drops solution to obtain eye drops.

[0063] Application of the above-mentioned eye drops based on the multifunctional peptide composition in the treatment of diabetic retinopathy.

[0064] Example 5

[0065] An eye drop based on a multifunctional peptide composition, the eye drop comprises the following raw materials: 2w / v% S-acetyl-L-glutathione, 0.5w / v% fish eye extract peptide, 1w / v% Hypericum perforatum flower extract peptide, 0.3w / v% Aesculus hippocastanum leaf extract peptide, 0.9w / v% osmotic pressure regulator, 0.003w / v% potassium sorbate preservative, buffer to adjust pH 7.4, and the remainder is water for injection. Among them, the osmotic pressure regulator is sodium chloride; the buffer is phosphate buffered saline (PBS).

[0066] The preparation method of fish eye extracted peptides comprises the following steps: selecting fresh snow fish eyes, including cornea, lens, retina and vitreous parts, washing the fresh snow fish eyes, removing impurities, crushing them into homogenate at a low temperature of 6°C with a tissue crusher to obtain fish eye slurry; adding the fish eye slurry to a phosphate buffer with a concentration of 0.06 mol / L and a pH of 7.0 according to a mass ratio of fish eye slurry: phosphate buffer = 1:8, and then adding 1.0% of trypsin by weight of the fish eye slurry, performing enzymolysis at 35°C for 90 minutes, heating to 85°C for 20 minutes to inactivate the enzyme; after cooling to 35°C, adding 1.0% of serrapeptase by weight of the fish eye slurry, performing enzymolysis for 60 minutes, heating to 90°C for 10 minutes to inactivate the enzyme; after cooling to room temperature, centrifuging at 8000 rpm for 20 minutes to obtain a supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000 kDa and 3000 kDa, freeze-drying, and obtaining fish eye extracted peptides.

[0067] The preparation method of the peptide extracted from the flower of Hypericum perforatum comprises the following steps: crushing the flower of Hypericum perforatum, adding water 10 times the mass of the flower of Hypericum perforatum, adjusting the pH to 6.0, adding 1% bromelain by mass of the flower of Hypericum perforatum, enzymolysis at 50°C for 90 minutes, heating to 85°C for 20 minutes to inactivate the enzyme; after the temperature drops to 35°C, adjusting the pH to 8.0, adding 0.5% serrapeptase by mass of the flower of Hypericum perforatum, enzymolysis for 90 minutes, heating to 85°C for 20 minutes to inactivate the enzyme; after the temperature drops to 35°C, adjusting the pH to 7.8, adding 0.5% nattokinase by mass of the flower of Hypericum perforatum, enzymolysis for 90 minutes, heating to 85°C for 20 minutes to inactivate the enzyme; after the temperature drops to room temperature, centrifuging at 6000rpm for 30 minutes to obtain the supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000kDa and 3000kDa, freeze-drying, and obtaining the peptide extracted from the flower of Hypericum perforatum.

[0068] The preparation method of the peptide extracted from the leaves of Aesculus hippocastanum comprises the following steps: taking the leaves of Aesculus hippocastanum for crushing, first soaking them in a 0.5v / v% hydrochloric acid aqueous solution with a concentration of 5 times the mass of the leaves of Aesculus hippocastanum for 1 hour, filtering them through a 250-mesh screen to obtain a filtrate A and a residue; soaking the residue in a 0.5wt% sodium hydroxide aqueous solution with a concentration of 5 times the mass of the leaves of Aesculus hippocastanum for 1 hour, filtering them through a 250-mesh screen to obtain a filtrate B; combining the filtrate A and the filtrate B, adjusting the pH to 5.0, and obtaining an extract; adding 0.15% of papain and 0.05% of fig protease with a mass of the extract to the extract, performing enzymolysis at 65°C for 90 minutes, heating to 90°C for 10 minutes to inactivate the enzyme; cooling to room temperature, centrifuging at 8000rpm for 20 minutes, and obtaining a supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000kDa and 3000kDa, and freeze-drying to obtain the peptide extracted from the leaves of Aesculus hippocastanum.

[0069] The above-mentioned method for preparing eye drops based on the multifunctional peptide composition comprises the following steps:

[0070] S1: Add 80% volume percentage of water for injection into the liquid preparation container, then add osmotic pressure regulator and preservative, stir while adding, mix and dissolve evenly, then add S-acetyl-L-glutathione, fish eye extract peptide, Hypericum perforatum flower extract peptide and Aesculus hippocastanum leaf extract peptide, stir while adding, mix and dissolve evenly, detect the pH value of the solution with a pH meter, then add buffer dropwise to adjust the pH to 7.3, and then dilute with water for injection to the final volume of eye drops to obtain an eye drops solution;

[0071] S2: filtering, sterilizing, and sealing the eye drops solution to obtain eye drops.

[0072] Application of the above-mentioned eye drops based on the multifunctional peptide composition in the treatment of diabetic retinopathy.

[0073] In the above embodiments, S-acetyl-L-glutathione is from Shaanxi Lesent Biotechnology Co., Ltd.; the activity of trypsin is 4000 U / g; the activity of serrapeptase is 200,000 U / g; the activity of bromelain is 200,000 U / g; the activity of nattokinase is 40,000 U / g; the activity of papain is 200,000 U / g; the activity of ficin is 100,000 U / g. Potassium sorbate preservative is from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.

[0074] Comparative Example 1

[0075] No S-acetyl-L-glutathione was added to the eye drops; other parameters and methods were the same as in Example 1.

[0076] Comparative Example 2

[0077] No fish eye extracted peptides were added to the eye drops; other parameters and methods were the same as in Example 1.

[0078] Comparative Example 3

[0079] No peptide extracted from Hypericum perforatum flowers was added to the eye drops; other parameters and methods were the same as in Example 1.

[0080] Comparative Example 4

[0081] No peptide extracted from Aesculus hippocastanum leaves was added to the eye drops; other parameters and methods were the same as in Example 1.

[0082] Comparative Example 5

[0083] No peptides extracted from Hypericum perforatum flowers and no peptides extracted from Aesculus hippocastanum leaves were added to the eye drops; other parameters and methods were the same as those in Example 1.

[0084] Comparative Example 6

[0085] No fish eye extracted peptides, Hypericum perforatum flower extracted peptides and Aesculus hippocastanum leaf extracted peptides were added to the eye drops; other parameters and methods were the same as in Example 1.

[0086] Comparative Example 7

[0087] In the method for preparing the peptide extracted from fish eyes, serrapeptase is not used for enzymatic hydrolysis; other parameters and methods are the same as those in Example 1.

[0088] Comparative Example 8

[0089] In the preparation method of the peptide extracted from the flower of Hypericum perforatum, serrapeptase and nattokinase are not used for enzymatic hydrolysis; other parameters and methods are the same as those in Example 1.

[0090] Comparative Example 9

[0091] In the method for preparing peptides extracted from Aesculus hippocastanum leaves, ficin is not used for enzymatic hydrolysis; other parameters and methods are the same as those in Example 1.

[0092] The eye drops of the above-mentioned embodiments and comparative examples were tested for safety and efficacy.

[0093] 1. Safety Testing

[0094] (1) Cytotoxicity test: Human retinal pigment epithelial cells (ARPE-19 cell line) cultured in vitro were taken and 5×10 3 The cells were inoculated in a 96-well plate, and after culturing for 24 hours with normal cell culture medium, the cell culture medium containing multifunctional peptide composition eye drops (the volume concentration of the eye drops was 1%) was replaced, and a blank control group (cultured with normal cell culture medium only) and a positive control group (the volume concentration of sodium dodecyl sulfate in the cell culture medium was 0.1%) were set up to continue culturing for 48 hours, and the cell viability was detected by MTT method, and the absorbance value at 570nm was measured by microplate reader, and the relative cell survival rate was calculated, and the cell survival rate = (absorbance value of the test group / absorbance value of the blank control group) × 100%. The results are shown in Table 1 below.

[0095] (2) Eye irritation test: 90 healthy adult New Zealand white rabbits were selected and randomly divided into 15 groups (6 rabbits in each group), of which 1 group was a blank control group, where only physiological saline was dripped. The rabbits were fixed, the lower eyelids were gently pulled open, and 50 μL of the corresponding eye drops were dripped into the conjunctival sac of one eye of each rabbit, and the other eye was dripped with physiological saline as a self-control. At 1h, 2h, 4h and 24h after the dripping of the drug, the eye reactions, including conjunctival congestion, edema, corneal opacity, etc., were observed under a slit lamp microscope, and the results showed no abnormal changes.

[0096] (3) Acute toxicity test: 150 healthy adult Kunming mice were selected and randomly divided into 15 groups (10 mice in each group), of which 1 group was a blank control group, which was only dripped with physiological saline. The multifunctional peptide composition eye drops were dripped once with a dose of 0.2mL / 10g body weight by a large-volume administration method. The mice were observed for 14 consecutive days, and the weight changes, diet, mental state, and death of the mice were recorded. On the 14th day after administration, the mice were killed and grossly dissected to observe whether there were any abnormal changes in the main organs (heart, liver, spleen, lung, kidney, etc.). No poisoning or death occurred in any group, and the product was safe and non-toxic.

[0097] 2. Efficacy testing for treating diabetic retinopathy

[0098] A total of 160 male SD rats were selected and randomly divided into 16 groups (10 rats in each group), one of which was a blank control group and the other was a diabetic model group. After fasting for 12 h, citric acid buffer solution containing streptozotocin (STZ) was intraperitoneally injected to establish the diabetic model. The blank control group was only injected with citric acid buffer solution. After the model was successfully established (blood sugar concentration ≥ 16.7mmo l / L was determined to be a successful diabetes model), the rats in each embodiment group and the comparative example group were administered with corresponding eye drops 3 times a day, 50 μL each time, and the control group and the diabetes model group were administered with equal volume of saline drops; before administration and 8 weeks after administration, fundus camera was used to take fundus photos of rats to observe the retinal lesions, including the number and area changes of lesions such as retinal microaneurysms, hemorrhage, and exudation; after 8 weeks of administration, the rats were killed, the eyeballs were taken, the retinal tissues were separated, and the serum cystatin C (Cys C), vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α) and reactive oxygen species (ROS) in the blood of the rats were detected by enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 2 below.

[0099] Table 1 Cytotoxicity test results

[0100] Sample Cell survival rate (%) Sample Cell survival rate (%) Blank control group 99.73 Comparative Example 1 99.41 Positive control group 56.86 Comparative Example 2 99.65 Example 1 99.82 Comparative Example 3 99.49 Example 2 99.75 Comparative Example 4 99.76 Example 3 99.94 Comparative Example 5 99.48 Example 4 99.80 Comparative Example 6 99.37 Example 5 99.77 Comparative Example 7 99.72 Comparative Example 8 99.85 Comparative Example 9 99.70

[0101] Table 2 Efficacy index test results

[0102]

[0103]

[0104] Retinopathy grades:

[0105] Grade 1-no obvious diabetic retinopathy: no obvious retinopathy or only microaneurysms;

[0106] Grade 2 - Mild non-proliferative diabetic retinopathy: Mild intraretinal hemorrhage and / or hard exudates in addition to microaneurysms;

[0107] Grade 3 - Moderate nonproliferative diabetic retinopathy: Increased intraretinal hemorrhages and / or hard exudates, with possible cotton wool spots;

[0108] Grade 4 - severe non-proliferative diabetic retinopathy (NPDR): This grade is defined as any of the following: (1) more than 20 intraretinal hemorrhages in each of the four quadrants; (2) clear venous beading changes in more than two quadrants; (3) significant intraretinal microvascular abnormalities in more than one quadrant;

[0109] Grade 5 - Proliferative diabetic retinopathy: Neovascularization, vitreous hemorrhage, or preretinal hemorrhage occurs.

[0110] It can be seen from the above experimental results that the multifunctional peptide eye drops prepared in Examples 1 to 5 have safe ingredients, are non-toxic and non-irritating to the eyes, and can significantly improve the symptoms of retinopathy, reduce inflammatory responses, and restore retinal damage caused by diabetes compared with the diabetic model group, and can safely, efficiently and conveniently treat diabetic retinopathy; from the results of Comparative Examples 1 to 6, it can be seen that S-acetyl-L-glutathione, fish eye extract peptides, Hypericum perforatum flower extract peptides, and Aesculus hippocastanum leaf extract peptides all have corresponding effects on improving the symptoms of retinopathy; Hypericum perforatum flower extract peptides and Aesculus hippocastanum leaf extract peptides have good synergistic effects when used in combination; fish eye extract peptides, Hypericum perforatum flower extract peptides and Aesculus hippocastanum leaf extract peptides have better synergistic effects on restoring retinal damage caused by diabetes when used in combination. From the results of comparative example 7, it can be seen that in the preparation method of the fish eye extracted peptide, serrapeptase is not used for enzymatic hydrolysis, the composition of the product obtained by extraction is changed, and the effect of restoring retinal damage caused by diabetes is weakened; from the results of comparative example 8, it can be seen that in the preparation method of the Hypericum perforatum flower extracted peptide, serrapeptase and nattokinase are not used for enzymatic hydrolysis, the composition of the product obtained by extraction is changed, and the effect of restoring retinal damage caused by diabetes is weakened; from the results of comparative example 9, it can be seen that in the preparation method of the European horse chestnut leaf extracted peptide, ficin is not used for enzymatic hydrolysis, the composition of the product obtained by extraction is changed, and the effect of restoring retinal damage caused by diabetes is weakened.

Claims

1. An eye drop based on a multifunctional peptide composition, characterized in that: The invention comprises the following raw materials: 1 w / v% to 2 w / v% of S-acetyl-L-glutathione, 0.5 w / v% to 1 w / v% of peptide extracted from fish eyes, 0.5 w / v% to 1 w / v% of peptide extracted from flowers of Hypericum perforatum, 0.3 w / v% to 0.5 w / v% of peptide extracted from leaves of Aesculus hippocastanum, 0.8 w / v% to 0.9 w / v% of osmotic pressure regulator, 0.003 w / v% to 0.008 w / v% of preservative, buffer for adjusting pH value to 7.2 to 7.6, and the remainder being water for injection.

2. The eye drops based on the multifunctional peptide composition according to claim 1, characterized in that: The osmotic pressure regulator is sodium chloride.

3. The eye drops based on the multifunctional peptide composition according to claim 1, characterized in that: The buffer is phosphate buffer.

4. The eye drops based on the multifunctional peptide composition according to claim 1, characterized in that: The preparation method of the fish eye extracted peptide comprises the following steps: washing fresh snow fish eyes, removing impurities, crushing into homogenate at a low temperature of 4°C to 6°C with a tissue crusher to obtain fish eye slurry; adding the fish eye slurry to a phosphate buffer solution at a mass ratio of 1: (8-10) to obtain a phosphate buffer solution at a concentration of 0.04 mol / L to 0.06 mol / L; l / L phosphate buffer with a pH of 7.0-8.0, then add 0.5%-1.0% of trypsin by weight of the fish eye slurry, perform enzymatic hydrolysis at 35°C-40°C for 60min-90min, and heat up to inactivate the enzyme; after the temperature drops to 35°C-40°C, add 0.5%-1.0% of serrapeptase by weight of the fish eye slurry, perform enzymatic hydrolysis for 60min-90min, and heat up to inactivate the enzyme; after the temperature drops to room temperature, centrifuge at 6000rpm-8000rpm for 20min-30min to obtain the supernatant; filter the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000kDa and 3000kDa, and freeze-dry to obtain fish eye extracted peptides.

5. The eye drops based on the multifunctional peptide composition according to claim 4, characterized in that: The snow fish eye includes a cornea, a lens, a retina and a vitreous body.

6. The eye drops based on the multifunctional peptide composition according to claim 1, characterized in that: The preparation method of the peptide extracted from the flower of Hypericum perforatum comprises the following steps: taking the flower of Hypericum perforatum and crushing it, adding water of 8 to 10 times the mass of the flower of Hypericum perforatum, adjusting the pH to 6.0 to 7.0, adding 0.5% to 1% of bromelain by mass of the flower of Hypericum perforatum, performing enzymatic hydrolysis at 50 to 55° C. for 60 to 90 minutes, and heating to inactivate the enzyme; after the temperature drops to 35 to 40° C., adjusting the pH to 7.0 to 8.0, adding 0.5% to 1.0% of serrapeptase by mass of the flower of Hypericum perforatum, and performing enzymatic hydrolysis for 60 to 90 minutes. in, heating to inactivate enzymes; after the temperature drops to 35°C to 40°C, adjusting the pH to 7.2 to 7.8, adding 0.5% to 1.0% of nattokinase by weight of Hypericum perforatum flower, performing enzymolysis for 60min to 90min, and heating to inactivate enzymes; after the temperature drops to room temperature, centrifuging at 6000rpm to 8000rpm for 20min to 30min, and taking the supernatant; filtering the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff between 1000kDa and 3000kDa, and freeze-drying to obtain a Hypericum perforatum flower extracted peptide.

7. The eye drops based on the multifunctional peptide composition according to claim 1, characterized in that: The preparation method of the peptide extracted from the leaves of Aesculus hippocastanum comprises the following steps: taking the leaves of Aesculus hippocastanum and crushing them, first soaking them in a 0.1v / v% to 0.5v / v% hydrochloric acid aqueous solution with a concentration of 5 to 6 times the mass of the leaves of Aesculus hippocastanum for 1 hour to 2 hours, filtering them through a 200-mesh to 250-mesh sieve to obtain a filtrate A and a residue; soaking the residue in a 0.1wt% to 0.5wt% sodium hydroxide aqueous solution with a concentration of 5 to 6 times the mass of the leaves of Aesculus hippocastanum for 1 hour to 2 hours, filtering them through a 200-mesh to 250-mesh sieve to obtain a filtrate B; combining the filtrate A and the filtrate B, adjusting the pH to 5.0 to 6.0, and obtaining an extract; Add 0.05% to 0.15% of papain and 0.05% to 0.15% of fig protease by weight of the extract to the extract, perform enzymolysis at 60°C to 65°C for 90 to 120 minutes, and heat to inactivate the enzymes; after cooling to room temperature, centrifuge at 6000 rpm to 8000 rpm for 20 to 30 minutes to obtain a supernatant; filter the supernatant with an ultrafiltration membrane to obtain a filtrate with a molecular weight cutoff of 1000 kDa to 3000 kDa, and freeze-dry to obtain a horse chestnut leaf extract peptide.

8. An eye drop based on a multifunctional peptide composition according to claim 4, 6 or 7, characterized in that: The temperature of the heating and enzyme inactivation is 85°C to 90°C, and the time of the heating and enzyme inactivation is 10min to 20min.

9. A method for preparing eye drops, for preparing the eye drops based on the multifunctional peptide composition according to claim 1, characterized in that: The preparation method comprises the following steps: S1: Add 70% to 80% volume percent of water for injection into a liquid preparation container, then add an osmotic pressure regulator and a preservative, stir while adding, mix and dissolve evenly, then add S-acetyl-L-glutathione, fish eye extract peptide, Hypericum perforatum flower extract peptide and Aesculus hippocastanum leaf extract peptide, stir while adding, mix and dissolve evenly, use a pH meter to detect the pH value of the solution, then add a buffer dropwise to adjust the pH to 7.2 to 7.6, and then use the remaining water for injection to make up to the final volume of eye drops to obtain an eye drops solution; S2: filtering, sterilizing, and sealing the eye drops solution to obtain eye drops.

10. Use of the eye drops based on the multifunctional peptide composition according to claim 1 in the preparation of a preparation for treating diabetic retinopathy.

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