Eye drops for treating xerophthalmia

Abstract

The eye drops are prepared from the following components in percentage by mass: 0.1 percent to 0.5 percent of chondroitin sulfate, 0.06 percent to 0.3 percent of sericin protein nanoparticles containing astaxanthin, 0.1 percent to 0.3 percent of sodium hyaluronate, 0.05 percent to 0.1 percent of vitamin E, 0.9 percent to 1.0 percent of sodium chloride, 0.05 percent to 0.1 percent of poloxamer, 0.01 percent to 0.1 percent of EDTA (Ethylene Diamine Tetraacetic Acid), a phosphate buffer solution and the balance of water, wherein the particle size of the sericin nano-particles containing the astaxanthin is 50 to 300 nm; the phosphate buffer solution is used for adjusting the pH value of the eye drops to 6.8-7.9. The eye drops can effectively relieve the dry eye symptom, provide long-time wetting and protection, reduce eye inflammation and oxidative stress, remarkably improve the dry eye symptom and are high in use safety.

Description

Technical Field

[0001] The invention relates to the technical field of ophthalmic drugs, and in particular to dry eye eye drops. Background Art

[0002] Dry eye is a disease that causes dry eye symptoms such as discomfort in the eyes and abnormalities in visual function. It is associated with abnormalities in the amount or quality of tears, and can cause obstructions on the surface of the eye (such as the cornea). The abnormality in the amount of tears mainly refers to a state of low tear secretion, which is an indicator of dry eye based on the amount of tears. In terms of abnormalities in tear quality, abnormalities in tear components (such as low lipid or protein components contained in tears) can deteriorate the stability of the tear film, which can lead to dryness of the surface of the eye even if tears are secreted.

[0003] Astaxanthin is known to be a powerful natural antioxidant with multiple biological activities, including antioxidant, anti-inflammatory, immunomodulatory and photoprotective effects. Astaxanthin is used to make eye drops, which can provide potential therapeutic or preventive effects for a variety of eye problems. At present, there are some patent applications that use astaxanthin as an active ingredient to add to eye drops. For example, CN107106627A discloses an eye drops dispenser and an antioxidant eye drops composition, which mainly includes antioxidant ingredients such as green tea extract, resveratrol, astaxanthin. CN117752597A discloses an eye drops for relieving dry eyes, which contains sorbitol, resveratrol, modified chitosan, golden plum extract, dandelion extract, ethylenediaminetetraacetic acid, astaxanthin, vitamin A, dextran and hyaluronic acid. Although astaxanthin has antioxidant, anti-inflammatory, immunomodulatory and photoprotective effects, there are still some challenges in the preparation of eye drops: ① Solubility: Astaxanthin has poor solubility in water, and appropriate solvents or carriers are needed to improve its solubility and bioavailability. ② Stability: Astaxanthin is easily degraded under light, high temperature and oxidative environment. How to ensure the stability of astaxanthin in eye drops, especially during production sterilization, storage and transportation, is a problem that needs to be solved. In addition, the safety of eye drops is also an issue that needs to be improved. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an eye drop for dry eyes, which uses sericin to coat astaxanthin, and utilizes the high hydrophilicity and film-forming properties of sericin to improve the stability and bioavailability of astaxanthin. At the same time, sericin also has moisturizing, antioxidant and antibacterial properties, which helps to improve dry eyes. It solves the technical problems of poor solubility and stability of astaxanthin directly added to eye drops.

[0006] (II) Technical solution

[0007] The present invention provides an eye drop for dry eyes, which comprises, by mass percentage, 0.1%-0.5% of chondroitin sulfate, 0.06%-0.3% of sericin nanoparticles containing astaxanthin, 0.1%-0.3% of sodium hyaluronate, 0.05%-0.1% of vitamin E, 0.9%-1.0% of sodium chloride, 0.05%-0.1% of poloxamer, 0.01%-0.1% of EDTA, phosphate buffer and the balance of water; wherein the particle size of the sericin nanoparticles containing astaxanthin is 50-300nm; and the phosphate buffer is used to adjust the pH value of the eye drop to 6.8-7.9.

[0008] According to a preferred embodiment of the present invention, the composition of the dry eye eye drops is: 0.1% chondroitin sulfate, 0.1% sericin nanoparticles containing astaxanthin, 0.2% sodium hyaluronate, 0.05% vitamin E, 0.9% sodium chloride, 0.075% poloxamer, 0.05% EDTA, phosphate buffer and the remainder water; the pH of the eye drops is 7.4.

[0009] According to a preferred embodiment of the present invention, the dry eye eye drops do not contain any preservatives.

[0010] In a second aspect, the present invention provides a method for preparing dry eye eye drops, comprising:

[0011] S1. Preparation of sericin nanoparticles containing astaxanthin

[0012] Dissolving sericin in water or phosphate buffer to form a first solution, and dissolving astaxanthin in anhydrous ethanol or 95% ethanol to obtain a second solution; adding the second solution to the first solution, and forming a stable emulsion by high-speed stirring or ultrasonic treatment; cooling to 2-8° C., and then centrifuging to separate the centrifugal precipitate, and freeze-drying to obtain sericin nanoparticles containing astaxanthin;

[0013] S2. Add sodium chloride, sodium hyaluronate, chondroitin sulfate, sericin nanoparticles containing astaxanthin, vitamin E, and poloxamer to sterile water, stir evenly, add phosphate buffer and stir evenly, adjust the pH to 6.8-7.9, filter with a 0.22μm filter membrane, and dispense the filtered solution into sterile eye drop bottles.

[0014] According to a preferred embodiment of the present invention, in S1, the concentration of sericin in the first solution is 1-5%. The preparation process of the first solution is: add sericin powder to water or phosphate buffer, stirring while adding until it is completely dissolved, and controlling the temperature to 25-45° C. during the dissolution process (appropriate heating can promote the dissolution of sericin in water). After dissolution, pass through a 0.45 μm filter membrane to obtain the first solution.

[0015] According to a preferred embodiment of the present invention, in S1, the concentration of astaxanthin in the second solution is 0.1-1%; the preparation process of the second solution is: at room temperature, add astaxanthin powder to anhydrous ethanol or 95% ethanol, stirring while adding until it is completely dissolved, and after dissolution, pass through a 0.45μm filter membrane to obtain a second solution.

[0016] According to a preferred embodiment of the present invention, in S1, the first solution and the second solution are prepared in a volume ratio of 5:1-1:1, and the second solution is slowly added to the first solution while stirring at a high speed (such as 1000-3000 rpm), and stirring is continued for 10-30 minutes until a stable emulsion is formed; the temperature does not exceed 25°C during the emulsification process; the emulsion is quickly transferred to an ice bath or a refrigerator, and rapidly cooled to 4-8°C for 0.5-1h, and centrifuged at a centrifugal force of 10000-15000g for 10-20min, and the precipitate is separated after centrifugation, and the precipitate is freeze-dried to obtain sericin nanoparticles containing astaxanthin.

[0017] Preferably, the separated supernatant can be used to prepare the first solution after sterilization.

[0018] Preferably, after being dispensed into sterile eye drop bottles, the eye drops are sterilized by irradiation with 10-20 kGy gamma rays at 4-10°C and then stored. This sterilization condition can achieve sterilization while minimizing the decomposition of antioxidant components in the eye drops.

[0019] (III) Beneficial effects

[0020] The technical effects of the present invention are:

[0021] (1) The present invention uses sericin to coat astaxanthin, and utilizes the high hydrophilicity and film-forming properties of sericin, which has good solubility in water, to improve the stability and bioavailability of astaxanthin. At the same time, sericin itself also has the properties of moisturizing, viscosity adjustment, anti-oxidation, and antibacterial, which helps to improve dry eye syndrome. It solves the technical problem of poor solubility and stability of astaxanthin directly added to eye drops. In addition, the use of sericin to coat astaxanthin can achieve long-term sustained release of astaxanthin, improve the bioavailability of astaxanthin and the anti-inflammatory and immunomodulatory effects. Sericin can also be used as a natural preservative to greatly improve product safety.

[0022] (2) Experiments have shown that the eye drops of the present invention can effectively relieve dry eye symptoms, provide long-term moisturizing and protection, while reducing ocular inflammation and oxidative stress, and significantly improve dry eye syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1This is a comparison of mouse corneal staining before and after modeling with benzalkonium chloride (BAC).

[0024] Figure 2 This is a comparison of tear secretion in mice before and after modeling with benzalkonium chloride (BAC).

[0025] Figure 3 Comparison of the levels of inflammatory factor IL-1β in the cornea of ​​each group of mice 14 days after administration.

[0026] Figure 4 The graph shows the change of astaxanthin retention rate in the eye drops of Example 3 and Comparative Examples 1-2 as a function of storage time. DETAILED DESCRIPTION

[0027] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0028] The eye drops of the present invention contain chondroitin sulfate, sericin nanoparticles containing astaxanthin, sodium hyaluronate, vitamin E, sodium chloride, poloxamer, EDTA and other ingredients, wherein chondroitin sulfate has excellent moisturizing properties, anti-inflammatory, and corneal protection effects, can absorb and retain moisture, help maintain the moisture of the eye surface, and reduce eye inflammation. Chondroitin sulfate helps protect the corneal epithelium and reduce damage to the cornea caused by external stimuli. It works together with sodium hyaluronate to enhance the moisturizing effect. Astaxanthin is a powerful antioxidant that can neutralize free radicals and reduce the damage to the eyes caused by oxidative stress. It also has the effects of anti-inflammatory and reducing eye inflammation, as well as protecting the retina and maintaining healthy vision. Astaxanthin and vitamin E synergistically provide strong antioxidant and anti-inflammatory effects, and synergistically with chondroitin sulfate and sodium hyaluronate to jointly provide moisturizing and anti-inflammatory effects.

[0029] Sericin is used as an astaxanthin carrier to improve the dispersibility and stability of astaxanthin in eye drops, has a sustained release effect, and can improve the bioavailability of astaxanthin. Sericin also has antibacterial properties; among them, sericin (S1, S2) and small molecule sericin peptide (S3) show antibacterial effects on both Gram-negative and Gram-positive bacteria. Sericin is non-toxic to cells, and with the increase of sericin concentration, it also promotes cell growth. In quantitative tests, sericin showed significant antibacterial effects on Staphylococcus aureus and Escherichia coli. Sericin has good antibacterial properties and good biocompatibility. It can replace chemical preservatives and improve the safety of eye drops. The eye drops of the present invention do not need to be added with chemical preservatives. In addition, the use of preservatives can also be avoided through single-dose packaging, which is suitable for patients who are sensitive to preservatives.

[0030] Sodium hyaluronate has extremely strong moisturizing properties, providing long-lasting moisturizing effects, helping to lubricate the surface of the eyeball, reduce friction, relieve dryness, and form a protective film on the cornea to protect the corneal epithelium from damage. It works synergistically with chondroitin sulfate to enhance the moisturizing effect, and works synergistically with astaxanthin and vitamin E to provide antioxidant and anti-inflammatory effects.

[0031] Sodium chloride adjusts the osmotic pressure to make it close to the osmotic pressure of tears, reducing irritation to the eyes. Poloxamer can solubilize and enhance the dispersibility and stability of drugs. EDTA can chelate metal ions in the solution to prevent catalytic oxidation reactions caused by metal particles introduced by water or equipment during the production of eye drops, thereby improving the stability of eye drops. EDTA can enhance the stability of sericin, vitamin E, etc. by chelating metal ions, thereby enhancing the antiseptic effect.

[0032] The eye drop formula of the present invention has various components that work together in the following ways:

[0033] Enhance stability and solubility: Astaxanthin is coated with sericin, and the high hydrophilicity and film-forming properties of sericin are used to reduce the chance of astaxanthin being oxidized, thereby improving the stability of astaxanthin, its solubility in water, improving its bioavailability and providing a long-lasting sustained-release effect; in addition, it can also enable the eye drops to be better preserved at room temperature, reducing the storage cost of the product.

[0034] Moisturizing and Lubricating: Chondroitin sulfate and sodium hyaluronate work together to provide powerful moisturizing and lubricating effects, alleviating dryness.

[0035] Antioxidant and Anti-Inflammatory: Sericin, astaxanthin, vitamin E and chondroitin sulfate work together to provide antioxidant and anti-inflammatory effects, protecting the eyes from damage caused by oxidative stress and inflammation.

[0036] Preservation and Stabilization: Sericin and EDTA together provide preservative and stabilizing effects, ensuring the long-term stability and safety of eye drops.

[0037] Osmolarity and pH adjustment: Sodium chloride and phosphate buffer work together to adjust the osmolarity and pH of eye drops, making them suitable for ocular use.

[0038] Solubilization and dispersion: Poloxamers improve the dispersion and stability of fat-soluble ingredients, ensuring the uniformity and effectiveness of eye drops.

[0039] In order to verify the technical effect of the present invention, the following is a description in conjunction with specific examples. In the following examples, when not specifically stated, % refers to mass concentration.

[0040] Example 1

[0041] This embodiment provides a method for preparing sericin nanoparticles containing astaxanthin:

[0042] (1) Adding sericin powder to phosphate buffer solution, stirring while adding until completely dissolved, maintaining the temperature at 35-38° C. during the dissolution process, and filtering through a 0.45 μm filter membrane after dissolution to obtain a first solution, wherein the sericin concentration is 3%.

[0043] (2) At room temperature, add astaxanthin powder to 95% ethanol while stirring until it is completely dissolved. After dissolution, filter through a 0.45 μm filter membrane to obtain a second solution in which the astaxanthin concentration is 1%.

[0044] (3) The first solution and the second solution are prepared in a volume ratio of 1:1, and the second solution is slowly added to the first solution while stirring at 3000 rpm. After the addition is completed, stirring is continued for 10 minutes until a stable emulsion is formed, and the emulsification temperature is controlled not to exceed 25°C in a water bath; the emulsion is quickly transferred to an ice bath, rapidly cooled to 4-8°C and cooled for 1 hour, and centrifuged at a centrifugal force of 10000g for 10 minutes. After centrifugation, the precipitate is separated and the precipitate is freeze-dried to obtain sericin nanoparticles containing astaxanthin, and the particle size is 50-200 nm.

[0045] Example 2

[0046] This embodiment provides a method for preparing sericin nanoparticles containing astaxanthin:

[0047] (1) Adding sericin powder to water, stirring while adding until completely dissolved, maintaining the temperature at 40-42° C. during the dissolution process, filtering through a 0.45 μm filter membrane after dissolution to obtain a first solution, wherein the sericin concentration is 1%.

[0048] (2) At room temperature, astaxanthin powder is added to anhydrous ethanol while stirring until it is completely dissolved. After dissolution, the astaxanthin powder is filtered through a 0.45 μm filter membrane to obtain a second solution in which the astaxanthin concentration is 1%.

[0049] (3) The first solution and the second solution are prepared in a volume ratio of 3:1, and the second solution is slowly added to the first solution while stirring at 3000 rpm. After the addition is completed, stirring is continued for 15 minutes until a stable emulsion is formed, and the emulsification temperature is controlled not to exceed 25°C in a water bath; the emulsion is quickly transferred to an ice bath, rapidly cooled to 4-8°C and cooled for 1 hour, and centrifuged at a centrifugal force of 10000g for 15 minutes. After centrifugation, the precipitate is separated and the precipitate is freeze-dried to obtain astaxanthin-containing sericin nanoparticles with a particle size of 100-300 nm.

[0050] Example 3

[0051] In this example, the sericin nanoparticles containing astaxanthin prepared in Example 1 are used to prepare eye drops, and the preparation process is as follows:

[0052] Add 0.1% chondroitin sulfate, 0.1% sericin nanoparticles containing astaxanthin, 0.2% sodium hyaluronate, 0.05% vitamin E, 0.9% sodium chloride, 0.075% poloxamer, and 0.05% EDTA to sterile water, and stir and disperse for 30 minutes; finally, add phosphate buffer to adjust the pH of the eye drops to 7.4. After preparation, filter with a 0.22μm filter membrane, dispense the filtered solution into sterile eye drop bottles, and then sterilize with 20kGy gamma rays at 4-8°C and store.

[0053] Example 4

[0054] In this example, the sericin nanoparticles containing astaxanthin prepared in Example 2 were used to prepare eye drops, and the preparation process was as follows:

[0055] Add 0.15% chondroitin sulfate, 0.15% sericin nanoparticles containing astaxanthin, 0.1% sodium hyaluronate, 0.07% vitamin E, 0.95% sodium chloride, 0.070% poloxamer, and 0.04% EDTA to sterile water, and stir and disperse for 30 minutes; finally, add phosphate buffer to adjust the pH of the eye drops to 7.2. After preparation, filter with a 0.22μm filter membrane, dispense the filtered solution into sterile eye drop bottles, and then sterilize with 20kGy gamma rays at 4-8°C and store.

[0056] Comparative Example 1

[0057] In this comparative example, the sericin nanoparticles containing astaxanthin in the eye drops formula of Example 3 were replaced with an equal amount of astaxanthin powder, and the astaxanthin powder was not coated with sericin. The remaining conditions were the same as those of Example 3. The pH was adjusted to 7.4, filtered with a 0.22 μm filter membrane, and sterilized by irradiation with 20 kGy gamma rays at 4-8° C. to prepare eye drops.

[0058] Comparative Example 2

[0059] In this comparative example, the sericin nanoparticles containing astaxanthin in Example 3 were replaced with sericin and astaxanthin in a mass ratio of 3:1, and sericin and astaxanthin were directly added to sterile water in the form of raw materials, respectively, and the other conditions were the same as those in Example 3. The pH was adjusted to 7.4, filtered with a 0.22 μm filter membrane, and sterilized by irradiation with 20 kGy gamma rays at 4-8° C. to prepare eye drops.

[0060] Animal Experimentation:

[0061] (1) Experimental animals and reagents

[0062] Animal selection: The mice used to construct dry eye were BALB / c mice, half female and half male, aged 8 weeks. They were healthy and had no eye abnormalities. They were given appropriate water and food, maintained in good feeding conditions, and controlled at appropriate temperature and humidity.

[0063] (2) Animal grouping and drug administration

[0064] 42 mice were modeled for dry eye disease. The modeling method was as follows: the mice were placed in an experimental environment and raised for a certain period of time to make preliminary preparations for the modeling. Male and female mice of similar weight were selected and separated into cages. A blank control group (6 mice) was set aside, and the remaining mice were used for experimental modeling. After the mice were separated into cages, a 2 mg / ml benzalkonium chloride (BAC) solution was prepared and the mice were eye-dropped. 5 μl of the prepared benzalkonium chloride was given to each eye, 3 times a day, for 10 consecutive days. After the modeling began, attention was paid to maintaining a certain degree of dryness in the mouse environment, and the mice had a normal diet and drinking water. The success of the modeling was confirmed based on the staining area and tear secretion. Figure 1-2 As shown, the mouse eye staining and tear secretion before and after modeling were compared, and the successful modeling was confirmed through comparison.

[0065] The mice were evenly divided into 7 groups, including blank control group, negative control group, positive control group, experimental group 1, experimental group 2, comparison group 1 and comparison group 2, with 6 mice in each group and 12 eyes. Among them, the blank control group did not establish a model, and only physiological saline was dripped. The negative control group was not given any medicine after modeling. The positive control group was dripped with a commercially available eye drop. Experimental group 1, experimental group 2, comparison group 1 and comparison group 2 were respectively given the eye drops of Example 3, Example 4, Comparative Example 1 and Comparative Example 2 after modeling (no additional medicine was given). Dosing time: 2 times a day, 2 drops (about 5 μL) per eye each time, for 14 consecutive days. The dosage method is shown in Table 1:

[0066] Table 1: Grouping and administration method (quantity is calculated based on the number of eyes)

[0067] quantity deal with Blank control group 12 No model was created, only saline was added during the experiment Negative control group 12 After the model was successfully established, only saline was added during the experiment. Positive control group 12 After the model is successfully established, an equal amount of a commercially available eye drop is instilled daily. Experimental Group 1 12 After the model was successfully established, the eye drops of Example 3 were added during the experiment. Experimental Group 2 12 After the model was successfully established, the eye drops of Example 4 were added during the experiment. Comparison group 1 12 After the model was successfully established, the eye drops of Comparative Example 1 were added during the experiment. Comparison of 2 groups 12 After the model was successfully established, the eye drops of Comparative Example 2 were added during the experiment.

[0068] (3) Experimental results

[0069] ① On the 0th day after the dry eye model was successfully established, the tears of mice in each group were collected, and the tear secretion was measured using phenol red cotton thread, and then the tear secretion of mice in each group was measured 14 days after administration. The specific operation of phenol red cotton thread detection of mouse tears is to grab the mouse, clamp the phenol red cotton thread with ophthalmic forceps and place the cotton thread on the conjunctiva of the lower eyelid of the mouse at one-third of the distance from the outer corner of the eye, and then take it out and fix it after 60 seconds. Immediately use a millimeter ruler to measure the length (mm) of the wet stained part of the phenol red cotton thread and record it. The test results are shown in Table 2. The tear secretion in Table 2 is the mean ± standard deviation.

[0070] Table 2: Tear secretion of mice before and after administration

[0071]

[0072] It can be seen from Table 2 that the tear secretion of mice without modeling always remains at a normal level. After modeling, the tear secretion of the mice in the negative control group, the positive control group, and experimental group 1, experimental group 2, comparison group 1, and comparison group 2 was significantly reduced. Among them, the negative control group only dripped with normal saline, and the tear secretion of the mice in the negative control group was further reduced after 14 days of administration. After 14 days of administration, the tear secretion of the mice in the positive control group, experimental group 1, and experimental group 2 basically returned to normal levels, and the tear secretion of the mice in experimental groups 1-experimental groups 2 was slightly better than that in the positive control group. After 14 days of administration, the tear secretion of the mice in comparison groups 1 and 2 also increased, but the degree of increase was not obvious.

[0073] ② Fluorescein sodium staining: On the 0th day after the dry eye model was successfully established and 14 days after administration, 0.25% fluorescein sodium solution was used for staining. After staying on the ocular surface for 10 seconds, 0.9% sodium chloride was used to rinse the excess dye solution, and the ocular surface was wiped dry with a cotton swab; the slit lamp light source was adjusted to cobalt blue light, and the mouse eyeballs were exposed to the slit lamp for observation. After adjusting the magnification, photos were taken to record the corneal staining of fluorescein sodium in different groups of mice. The cornea was divided into four quadrants: superior temporal, inferior temporal, superior nasal, and inferior nasal. Each quadrant was scored according to 0-3 points. The scoring criteria were: no staining was 0 points; 1-30 punctate staining (mild) was 1 point; more than 30 punctate staining but the staining was not fused (moderate) was 2 points; corneal punctate staining fusion, filaments and ulcers (severe) were 3 points. Each corneal score was the sum of the scores of the four quadrants. The test results are shown in Table 3.

[0074] Table 3: Statistics of corneal staining scores of mice before and after drug administration

[0075]

[0076] The lower the corneal score, the higher the degree of corneal epithelial repair in mice and the better the eye recovery. Conversely, the more severe the corneal damage and the worse the recovery state. As shown in Table 3, the tear secretion of mice without modeling always remained at a normal level. After modeling, the corneal epithelial staining area and corneal score of the mice in the negative control group, positive control group, experimental group 1, experimental group 2, comparison group 1 and comparison group 2 increased significantly. Among them, the negative control group only dripped physiological saline, and the corneal epithelial staining area and score of the mice in the negative control group increased further after 14 days of administration; after 14 days of administration, the corneal epithelial staining area and score of the mice in the positive control group, experimental group 1 and experimental group 2 were significantly reduced, and the corneal improvement of the mice in experimental group 1-experimental group 2 was slightly better than that in the positive control group. After 14 days of administration, the corneal epithelial staining area and score of the mice in comparison group 1 and comparison group 2 also decreased slightly, indicating that the degree of corneal epithelial repair of the mice was not obvious.

[0077] ③ Detection of IL-1β level of mouse corneal inflammatory factor, the detection method is as follows:

[0078] Two mice were randomly selected from each group of mice. Their eyeballs were immediately removed after being killed. The eyeballs were separated with ophthalmic forceps under a microscope, rinsed with pre-cooled PBS, and dried with filter paper. Two corneas were placed in an enzyme-free ep tube, frozen with liquid nitrogen, and temporarily stored in a -80°C refrigerator. The corneal tissue was taken out and placed in an ice box. The tissue was cut into pieces with sterilized and clean ophthalmic instruments. 100 μl of pre-cooled PBS containing PMSF was added to each tube of sample, and the samples were sonicated on ice for 3 minutes with an ultrasonic disruptor. The sonicated samples were centrifuged in a refrigerated centrifuge at 4°C with the parameters set to 12,000 rpm for 15 minutes. The supernatant was placed in an enzyme-free ep tube for later use.

[0079] Serially dilute the bovine serum albumin (BSA) standard. Prepare the BCA working solution, usually by mixing BCA reagents A and B in a 50:1 ratio. Add the standard and the sample to be tested to a microplate or test tube, and add the BCA working solution to mix. Incubate at 37°C for 30-60 minutes, or at room temperature for 2 hours. After cooling to room temperature, measure the absorbance of the sample at 562nm.

[0080] Take out the ELISA kit and equilibrate it at room temperature. Before testing, only take out the required number of strips according to the experimental needs, and keep the others sealed and stored at 4°C; add samples to the wells according to the experimental settings, 50μl per well; add HRP-labeled detection antibodies, 100μl per well, seal the plate with sealing film, and incubate at 37°C for 60min; after the reaction is completed, wash the plate with washing solution for 1min each time, and repeat the washing 5 times; add substrate A and substrate B to the wells in turn, 50μl per well, and incubate at 37°C in dark for 15min; after the reaction is completed, add 50μl of stop solution, and use an enzyme reader to detect the OD value of each well at the wavelength specified by the test kit. Note that the test should be completed within 15min after adding the stop solution; finally, draw a standard curve, calculate the sample concentration, and correct the result with the protein concentration of the sample to be tested. The experimental results are as follows Figure 3 shown.

[0081] like Figure 3 As shown, the blank control group is healthy mice (without modeling treatment) at a lower inflammatory level, and the negative control group is only dripped with physiological saline after successful modeling. After 14 days of administration, the level of inflammatory factor IL-1β is high, indicating that the corneal inflammation of the mice in the negative control group is still very obvious. After 14 days of administration, the inflammatory expression levels of the positive control group, experimental group 1, and experimental group 2 decreased significantly, all approaching the blank control group. This shows that the eye drops prepared in Example 3-4 can indeed significantly inhibit the expression of inflammatory factor IL-1β in the cornea of ​​dry eye mice. Compared with the commercially available eye drops, the eye drops prepared in Example 3-4 of the present invention have a better inhibitory effect on the inflammatory factor IL-1β. Although the comparative examples 1-2 can also inhibit the inflammatory factor IL-1β, the overall effect is not as good as the eye drops prepared in Example 3-4. Compared with comparative example 1, comparative example 2 is better in the ability to inhibit inflammatory factors, which shows that sericin also has the effect of inhibiting the expression level of inflammatory factors.

[0082] Eye drops storage stability test

[0083] The eye drop samples prepared in Example 3 and Comparative Examples 1-2 were stored in a sealed container and kept away from light at 25°C for 16 weeks. The astaxanthin content in the samples was measured every 4 weeks, and the retention rate of astaxanthin (reduced state) in the eye drop samples was measured by HPLC. The test results are shown in FIG. Figure 4 As shown in the figure, the astaxanthin eye drops prepared in Example 3 of the present invention can remain stable for a longer time at room temperature, while the astaxanthin eye drops prepared in Comparative Examples 1-2 are difficult to retain for a long time, and astaxanthin is likely to be partially degraded during the preparation, irradiation sterilization, and storage process.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dry eye eye drop, characterized in that: It contains, by mass percentage: 0.1%-0.5% chondroitin sulfate, 0.06%-0.3% sericin nanoparticles containing astaxanthin, 0.1%-0.3% sodium hyaluronate, 0.05%-0.1% vitamin E, 0.9-1.0% sodium chloride, 0.05%-0.1% poloxamer, 0.01%-0.1% EDTA, phosphate buffer and the remainder water; wherein the particle size of the sericin nanoparticles containing astaxanthin is 50-300nm; the phosphate buffer adjusts the pH of the eye drops to 6.8-7.

9.

2. The dry eye eye drops according to claim 1, characterized in that The composition of the dry eye eye drops is: 0.1% chondroitin sulfate, 0.1% sericin nanoparticles containing astaxanthin, 0.2% sodium hyaluronate, 0.05% vitamin E, 0.9% sodium chloride, 0.075% poloxamer, 0.05% EDTA, phosphate buffer and the balance water; the pH of the eye drops is 7.

4.

3. The dry eye eye drops according to claim 1, characterized in that The eye drops do not contain chemical preservatives.

4. A method for preparing the dry eye eye drops according to any one of claims 1 to 3, characterized in that: The steps include: S1. Preparation of sericin nanoparticles containing astaxanthin Dissolving sericin in water or phosphate buffer to form a first solution, and dissolving astaxanthin in anhydrous ethanol or 95% ethanol to obtain a second solution; adding the second solution to the first solution, and forming a stable emulsion by high-speed stirring or ultrasonic treatment; cooling to 2-8° C., and then centrifuging to separate the centrifugal precipitate, and freeze-drying to obtain sericin nanoparticles containing astaxanthin; S2. Add sodium chloride, sodium hyaluronate, chondroitin sulfate, sericin nanoparticles containing astaxanthin, vitamin E, and poloxamer to sterile water, stir evenly, add phosphate buffer and stir evenly, adjust the pH to 6.8-7.9, filter with a 0.22μm filter membrane, and dispense the filtered solution into sterile eye drop bottles.

5. The preparation method according to claim 4, characterized in that: In S1, the concentration of sericin in the first solution is 1-5%. The preparation process of the first solution is: add sericin powder to water or phosphate buffer, stir while adding until completely dissolved, control the temperature to 25-45° C. during the dissolution process, and filter through a 0.45 μm filter membrane after dissolution to obtain the first solution.

6. The preparation method according to claim 5, characterized in that: In S1, the concentration of astaxanthin in the second solution is 0.1-1%; the preparation process of the second solution is: at room temperature, add astaxanthin powder to anhydrous ethanol or 95% ethanol, stirring while adding until it is completely dissolved, and after dissolution, pass through a 0.45μm filter membrane to obtain a second solution.

7. The preparation method according to claim 5 or 6, characterized in that: In S1, the first solution and the second solution are prepared in a volume ratio of 5:1-1:1, and the second solution is slowly added to the first solution while stirring at a high speed of 1000-3000 rpm for 10-30 minutes until a stable emulsion is formed; the temperature does not exceed 25°C during the emulsification process; then, the emulsion is quickly transferred to an ice bath or refrigerator, and rapidly cooled to 4-8°C for 0.5-1h, and centrifuged at 10000-15000g for 10-20min. After centrifugation, the precipitate is separated and the precipitate is freeze-dried to obtain sericin nanoparticles containing astaxanthin.

8. The preparation method according to claim 7, characterized in that: In S1, centrifugation is performed at 10,000-15,000 g for 10-20 min, and the supernatant separated after centrifugation can be used to prepare the first solution after sterilization.

9. The preparation method according to claim 4, characterized in that: In S2, after being divided into sterile eye drop bottles, they are sterilized by irradiation with 10-20 kGy gamma rays at 4-10°C and put into storage.

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