Eye drops for treating xerophthalmia and preparation method thereof

Abstract

The invention relates to eye drops for treating xerophthalmia and a preparation method of the eye drops. The eye drops are prepared from the following components in percentage by mass: 0.05 to 0.5 percent of a wild mint extract, 0.1 to 0.4 percent of lutein-coated sericin nano-particles, 0.1 to 0.5 percent of chondroitin sulfate, 0.9 to 1.0 percent of sodium chloride, 0.05 to 0.1 percent of poloxamer, 0.01 to 0.1 percent of EDTA (Ethylene Diamine Tetraacetic Acid), a phosphate buffer solution and water. The particle size of the lutein-coated sericin nanoparticles is 50-300nm, the water is medicinal sterile water, and the pH value of the eye drops is adjusted to 7.0-7.8 by the phosphate buffer solution. The eye drops can effectively relieve dry eye symptoms, provide long-time wetting and protection, reduce eye inflammation and oxidative stress, promote repair of corneal injury and effectively reduce the expression level of inflammatory factors, and are high in use safety.

Description

Technical Field

[0001] The invention relates to the technical field of pharmacy, and in particular to an eye drop for treating dry eye and a preparation method thereof. Background Art

[0002] Dry eyes are a common eye disease characterized by insufficient quantity or poor quality of tears, which results in insufficient lubrication of the eyes and causes a series of uncomfortable symptoms, which may affect vision in severe cases. The exact causes of dry eyes vary, but can be roughly divided into two categories: insufficient tear secretion (lack of aqueous layer) and excessive evaporation (abnormal lipid layer). The main causes of dry eyes are: 1. Insufficient tear secretion; 2. Excessive tear evaporation. Among them, the causes of insufficient tear secretion include: ① Age: The function of the lacrimal glands gradually declines with age. ② Autoimmune diseases: such as syndrome. ③ Side effects of drugs: Certain drugs, such as antihistamines, antidepressants, and birth control pills, may reduce tear secretion. ④ Environmental factors: Dry or windy climate conditions, staying in air-conditioned rooms for a long time. ⑤ Laser corrective surgery: Some eye surgeries may temporarily or permanently change the production or distribution of tears. The causes of rapid tear evaporation include: ① Meibomian gland dysfunction; ② Long-term use of electronic devices will lead to fewer blinks and reduce the chance of tear film renewal. ③ Long-term wearing of contact lenses will increase friction on the surface of the eye and may also interfere with normal tear distribution. The uncomfortable symptoms of dry eye include foreign body sensation, burning sensation, red eyes, increased secretions, light sensitivity, vision fluctuations, difficulty driving at night, etc., which seriously interfere with people's daily lives.

[0003] Lutein is a naturally occurring carotenoid that plays an important role in eye health. Lutein has a strong antioxidant property and can help reduce the damage of free radicals to eye tissues, thereby protecting the eyes from the effects of oxidative stress. Lutein can also absorb harmful blue light and reduce the potential damage of blue light to the retina, which is particularly important for people who often face electronic screens. For example, CN118384216A discloses an Antong grass tea eyewash, which is composed of: sodium hyaluronate 0.05-0.5ml, tea polyphenols 0.05-0.5ml, cassia seed 0.3-1ml, vitamin B6 0.1-0.3ml, lutein 0.1-0.5ml, trehalose 0.2-0.5ml, and the remainder is pure water. CN117771294A also discloses a borneol compound eye protection preparation, which at least includes borneol fresh branch extract, lutein, vitamin A, and zeaxanthin, and the materials of the above components are processed to form a borneol compound eye protection preparation. However, there are also some challenges or problems to be solved in the process of developing lutein into eye drops: (1) Solubility: The solubility of lutein in water is very limited, usually less than 1 mg / L, which means that it is difficult to dissolve directly in water, and it is difficult to increase the concentration of lutein in eye drops and keep it stable. (2) Bioavailability: Since lutein is fat-soluble, its ability to pass through the corneal barrier is weak when it enters the eye directly through eye drops. How to improve the bioavailability of lutein is a key technical problem. Developing a suitable carrier system to promote the effective release and absorption of lutein while reducing irritation to the eyes is a major challenge in the research and development process. (3) Stability: Lutein is easily degraded under the influence of factors such as light and temperature. How to ensure the stability of lutein 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 treating dry eye and a preparation method thereof, wherein sericin is mainly used to coat lutein, and the high hydrophilicity and film-forming properties of sericin are utilized to improve the dispersibility and ability to pass through the corneal barrier, as well as the stability in the drops, thereby improving the bioavailability of lutein. At the same time, sericin also has moisturizing, antioxidant and antibacterial properties, which are helpful to improve dry eye. The eye drops solve the technical problems of poor solubility and stability of lutein directly added to eye drops.

[0006] (II) Technical solution

[0007] In a first aspect, the present invention provides an eye drop for treating dry eyes, comprising, by mass percentage: 0.05-0.5% of mountain mint extract, 0.1-0.4% of sericin nanoparticles coated with lutein, 0.1%-0.5% of chondroitin sulfate, 0.9-1.0% of sodium chloride, 0.05%-0.1% of poloxamer, 0.01%-0.1% of EDTA, phosphate buffer and water; the particle size of the sericin nanoparticles coated with lutein is 50-300nm, the water is medicinal sterile water, and the phosphate buffer is used to adjust the pH of the eye drops to 7.0-7.8.

[0008] According to a preferred embodiment of the present invention, the eye drops further contain 0.01%-0.1% of epidermal growth factor (EGF).

[0009] According to a preferred embodiment of the present invention, the mountain mint extract is a freeze-dried powder of an ethanol extract of mountain mint, and its preparation method is: washing the mountain mint (Eriodictyon californicum) raw material, drying it in the shade or blowing it dry away from light, adding water to mix, crushing it by ultrafine grinding method, using ethanol ultrasonic extraction to obtain an extract, concentrating the extract by reduced pressure rotary evaporation and recovering ethanol, and freeze-drying the concentrate to obtain the mountain mint extract.

[0010] According to a preferred embodiment of the present invention, the eye drops are composed of: 0.2% mountain mint extract, 0.15% sericin nanoparticles coated with lutein, 0.1% chondroitin sulfate, 0.05% epidermal growth factor EGF, 0.9% sodium chloride, 0.065% poloxamer, 0.05% EDTA, phosphate buffer and the remainder water; the pH of the eye drops is 7.0-7.8.

[0011] According to a preferred embodiment of the present invention, the eye drops do not contain chemical preservatives.

[0012] In a second aspect, the present invention provides a method for preparing eye drops for treating dry eyes, comprising:

[0013] S1. Preparation of sericin nanoparticles coated with lutein

[0014] Dissolving sericin in water or phosphate buffer to form a first dispersion, dissolving lutein in ethyl acetate or 95% ethanol to obtain a second dispersion; adding the second dispersion to the first dispersion, stirring at high speed or treating with ultrasound to form a stable emulsion; cooling to 2-8°C, centrifuging, separating centrifugal precipitates, and freeze-drying to obtain sericin nanoparticles coated with lutein;

[0015] S2. Preparation of peppermint extract

[0016] Add water to mountain mint, use an ultrafine grinder to grind to obtain a slurry, use anhydrous ethanol to ultrasonically assist extraction to obtain an ethanol extract, centrifuge at 2000-4000 rpm to remove precipitation, retain the supernatant, and recover ethanol by vacuum rotary evaporation at ≤45°C to produce a concentrated solution, and freeze-dry the concentrated solution to obtain a mountain mint extract powder;

[0017] S3. Add the remaining raw materials except phosphate buffer to sterile water, stir evenly, add phosphate buffer and stir evenly, adjust the pH to 7.0-7.8, filter with a 0.22 μm filter membrane, and dispense the filtered solution into sterile eye drop bottles.

[0018] According to a preferred embodiment of the present invention, in S1, the concentration of sericin in the first dispersion is 1-5%; the preparation process of the first dispersion 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 (appropriate heating can promote the dissolution of sericin in water), and filter through a 0.45μm filter membrane after dissolution to obtain the first dispersion; the concentration of lutein in the second dispersion is 1-2%; the preparation process of the second dispersion is: at room temperature, add lutein to ethyl acetate or 95% ethanol, stir while adding until completely dissolved, and filter through a 0.45μm filter membrane after dissolution to obtain the second dispersion.

[0019] According to a preferred embodiment of the present invention, in S1, the first dispersion and the second dispersion are prepared in a volume ratio of 4:1-1:1, the second dispersion is slowly added to the first dispersion, and high-speed stirring (such as 1000-3000rpm) is performed at the same time, and stirring is continued for 10-30min 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 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 coated with lutein. Preferably, the separated supernatant can be used to prepare the first dispersion after sterilization.

[0020] According to a preferred embodiment of the present invention, in S2, the mountain mint is mixed with water, and an ultrafine grinder is used to grind the mixture at a rotation speed of 40-50krpm for 10-30min to obtain an ultrafine powder slurry, and 3-5 times the volume of ethanol is added for ultrasonic-assisted extraction, the ultrasonic frequency is 40-60kHz, and the processing time is 0.5-1h to obtain an ethanol extract, and the mixture is further centrifuged at 2000-4000rpm for 15-25min to remove the precipitate to obtain a supernatant, the supernatant is evaporated under reduced pressure at ≤45°C, the ethanol is recovered and part of the water is removed to obtain a concentrated solution, and the concentrated solution is freeze-dried at -40°C to obtain a mountain mint extract powder.

[0021] 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.

[0022] (III) Beneficial effects

[0023] (1) The present invention uses sericin to coat lutein. The high hydrophilicity, film-forming properties, and good solubility of sericin in water can improve the solubility, stability, and ability of lutein to pass through the corneal barrier in eye drops, thereby improving the bioavailability of lutein. At the same time, sericin itself has the properties of moisturizing, viscosity adjustment, anti-oxidation, and antibacterial, which helps to improve dry eye syndrome. It solves the technical problems of poor solubility of lutein in eye drops and susceptibility to the effects of sterilization, light, and temperature on stability. In addition, the use of sericin to coat lutein can make lutein have long-lasting sustained release, greatly improve the bioavailability of lutein, give full play to the antioxidant properties of lutein, and protect the eyes from oxidative stress. Sericin has good antibacterial properties and, as a natural preservative, can greatly improve the safety of eye drops.

[0024] (2) The present invention also uses mountain mint extract in the eye drops. Mountain mint is also known as North American holy grass or California holy grass (Eriodictyon californicum), which is rich in mucus and active ingredients. Specifically, mountain mint contains a variety of flavonoids and polyphenols, which have anti-inflammatory effects and help alleviate eye inflammation; it also has strong antioxidant properties, can neutralize free radicals, reduce oxidative stress damage to the eyes, and protect the composition of eye drops. Mucus can adjust the viscosity of eye drops and form a protective film on the surface of the eyes, which helps keep the eyes moist. It can replace auxiliary antioxidants such as sodium hyaluronate and vitamin E in eye drops.

[0025] (3) Experiments have shown that the eye drops of the present invention can effectively relieve dry eye symptoms, provide long-term moisturizing and protection, reduce eye inflammation and oxidative stress, promote the repair of corneal damage, and effectively reduce the expression level of inflammatory factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a comparison of the corneal bengalensis staining of mice before and after modeling with benzalkonium chloride (BAC).

[0027] Figure 2 This is a comparison of tear secretion in mice before and after modeling with benzalkonium chloride (BAC).

[0028] Figure 3 Comparison of the levels of corneal inflammatory factor IL-6 in each group of mice after 14 days of drug administration.

[0029] Figure 4 The graphs show the change of lutein retention rate in the eye drops of Example 1 and Comparative Examples 1-2 as a function of storage time. DETAILED DESCRIPTION

[0030] 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.

[0031] The eye drops of the present invention contain ingredients such as chondroitin sulfate, sericin nanoparticles coated with lutein, mountain mint extract, sodium chloride, poloxamer, EDTA, epidermal growth factor EGF, etc., wherein chondroitin sulfate is a natural polysaccharide that can increase the viscosity and stability of tears, help form a stable tear film, and thus relieve dry eye symptoms. Lutein is a powerful antioxidant that can absorb harmful blue light and protect the retina from light damage. Sericin nanoparticles can improve the water solubility, stability, ability to pass through the corneal barrier and bioavailability of lutein, making it easier to be absorbed by the eye. Mountain mint extract has anti-inflammatory and antioxidant properties. It can reduce eye inflammation and reduce oxidative stress, thereby helping to relieve dry eye symptoms. The mucus in the mountain mint extract has extremely strong moisturizing properties, provides a long-term moisturizing effect, helps lubricate the surface of the eyeball, reduces friction, relieves dryness, forms a protective film on the cornea, and protects the corneal epithelium from damage. Chondroitin sulfate, when combined with lutein and mountain mint extract, can provide better tear film stability and protection, mucus and chondroitin sulfate work together to enhance moisturizing effects, and mountain mint extract and lutein work together to provide antioxidant and anti-inflammatory effects. In summary, lutein, when combined with chondroitin sulfate and mountain mint extract, can provide stronger antioxidant protection, while enhancing the stability and effectiveness of lutein and protecting eye tissues.

[0032] In addition, sodium chloride is used to adjust the osmotic pressure of eye drops to match the osmotic pressure of tears and reduce irritation to the eyes. Poloxamer solubilizes and enhances 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. By chelating metal ions, EDTA can further enhance the stability of sericin, mountain mint extract, lutein, etc. EGF is a polypeptide that can promote cell proliferation and wound healing. EGF can help repair damaged ocular surface cells and promote the regeneration of corneal epithelium, thereby improving dry eye symptoms, and mainly plays a repair and regeneration effect.

[0033] The eye drop formula of the present invention has various components that work together in the following ways:

[0034] ANTI-INFLAMMATORY & ANTIOXIDANT: Mountain peppermint extract and lutein work together to reduce inflammation and oxidative stress.

[0035] Tear film stability and protection: Chondroitin sulfate and lutein work together to improve the stability and protection of the tear film.

[0036] Repair and regeneration: EGF promotes cell proliferation and wound healing, and enhances the repair capacity of eye tissues.

[0037] Stability and dispersibility: Poloxamer and EDTA work together to improve the stability and uniform dispersion of eye drops.

[0038] Sericin is used as a lutein carrier to improve the dispersibility and stability of lutein in eye drops, has a sustained release effect, and can improve the bioavailability of lutein. 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.

[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 an eye drop for treating dry eye, and the preparation method thereof is as follows:

[0042] (1) Add sericin powder to phosphate buffer and stir until it is completely dissolved. During the dissolution process, the temperature is controlled at 40-42° C. to promote the dissolution of the sericin. After the dissolution, the sericin is filtered through a 0.45 μm filter membrane to obtain a first dispersion in which the sericin concentration is 2%.

[0043] (2) At room temperature, lutein was added to 95% ethanol while stirring until it was completely dissolved. After dissolution, the lutein was filtered through a 0.45 μm filter membrane to obtain a second dispersion. The concentration of lutein in the second dispersion was 1.5%.

[0044] (3) The first dispersion and the second dispersion are prepared in a volume ratio of 1:1, and the second dispersion is slowly added to the first dispersion while stirring at 3000 rpm. After the addition is completed, stirring is continued for 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, and the temperature is rapidly cooled to 4-8°C for 1 hour, and centrifuged at a centrifugal force of 12000g for 15 minutes. After centrifugation, the precipitate is separated, and the precipitate is freeze-dried to obtain sericin nanoparticles coated with lutein. The separated supernatant can be used to prepare the first dispersion after sterilization. The particle size of the sericin nanoparticles is 50-200nm.

[0045] (4) The mountain mint is cleaned, blown dry under light-proof conditions, mixed with water in a mass ratio of 1:1, and then pulverized in an ultrafine grinder at a speed of 40k rpm for 20 minutes to obtain an ultrafine powder slurry, 4 times the volume of ethanol is added, and the mixture is treated at an ultrasonic frequency of 40kHz for 45 minutes to obtain an ethanol extract, and the mixture is centrifuged at 3000rpm for 20 minutes to remove the precipitate to obtain a supernatant, and the supernatant is evaporated under reduced pressure at ≤45°C, ethanol is recovered and part of the water is removed to obtain a concentrated solution, and the concentrated solution is freeze-dried at -40°C to obtain a mountain mint extract powder.

[0046] (5) Add 0.2% mountain peppermint extract, 0.15% lutein-coated sericin nanoparticles, 0.1% chondroitin sulfate, 0.9% sodium chloride, 0.065% poloxamer, and 0.05% EDTA to sterile water. After addition, stir thoroughly for 30 minutes. Finally, add phosphate buffer and stir evenly. Adjust the pH to 7.4, filter with a 0.22 μm filter membrane, and dispense the filtered solution into sterile eye drop bottles. Stir and sterilize with 15 kGy gamma rays at 4-8°C and put into storage.

[0047] Example 2

[0048] The present embodiment is different from the embodiment 1 only in that 0.05% epidermal growth factor (EGF) is further added in step (5), and the pH is similarly adjusted to 7.4, filtered with a 0.22 μm filter membrane, and the filtered solution is dispensed into sterile eye drop bottles, sterilized by irradiation with 15 kGy gamma rays at 4-8°C, and stored.

[0049] Example 3

[0050] This embodiment provides an eye drop for treating dry eye, and the preparation method thereof is as follows:

[0051] (1) Add sericin powder to water and stir until it is completely dissolved. During the dissolution process, the temperature is controlled at 40-42° C. to promote the dissolution of the sericin. After the dissolution, the sericin powder is filtered through a 0.45 μm filter membrane to obtain a first dispersion in which the sericin concentration is 3%.

[0052] (2) At room temperature, lutein is added to ethyl acetate while stirring until it is completely dissolved. After dissolution, the lutein is filtered through a 0.45 μm filter membrane to obtain a second dispersion. The concentration of lutein in the second dispersion is 2%.

[0053] (3) The first dispersion and the second dispersion are prepared in a volume ratio of 2:1, and the second dispersion is slowly added to the first dispersion while stirring at 3000 rpm. After the addition is completed, stirring is continued for 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, and rapidly cooled to 4-8°C for 1 hour, and centrifuged at a centrifugal force of 10000g for 20 minutes. After centrifugation, the precipitate is separated, and the precipitate is freeze-dried to obtain sericin nanoparticles coated with lutein. The separated supernatant can be used to prepare the first dispersion after sterilization. The particle size of the sericin nanoparticles is 100-300nm.

[0054] (4) The mountain mint is cleaned, dried under light-proof conditions, and water is added in a mass ratio of 1:1, and then the mixture is pulverized in an ultrafine grinder at a speed of 50k rpm for 20 minutes to obtain an ultrafine powder slurry, 5 times the volume of ethanol is added, and the mixture is treated at an ultrasonic frequency of 40kHz for 50 minutes to obtain an ethanol extract, and the mixture is centrifuged at 3000rpm for 20 minutes to remove the precipitate to obtain a supernatant, and the supernatant is evaporated under reduced pressure at ≤45°C, ethanol is recovered and part of the water is removed to obtain a concentrated solution, and the concentrated solution is freeze-dried at -40°C to obtain a mountain mint extract powder.

[0055] (5) Add 0.15% of mountain mint extract, 0.2% of lutein-coated sericin nanoparticles, 0.15% of chondroitin sulfate, 0.9% of sodium chloride, 0.075% of poloxamer, 0.05% of EDTA, and 0.05% of epidermal growth factor (EGF) to sterile water. After the above ingredients are added, stir thoroughly for 30 minutes. Finally, add phosphate buffer and stir evenly. Adjust the pH to 7.4, filter with a 0.22 μm filter membrane, and dispense the filtered solution into sterile eye drop bottles. Stir and sterilize with 15 kGy gamma rays at 4-8°C and put into storage.

[0056] Comparative Example 1

[0057] In this comparative example, the sericin nanoparticles coated with lutein in the eye drops formula of Example 1 were replaced with an equal amount of lutein powder, and the lutein powder was not coated with sericin. The other conditions were the same as those of Example 1. The pH was adjusted to 7.4, filtered with a 0.22 μm filter membrane, and sterilized by irradiation with 15 kGy gamma rays at 4-8°C to prepare eye drops.

[0058] Comparative Example 2

[0059] In this comparative example, the sericin nanoparticles coated with lutein in the eye drops formula of Example 1 were replaced with sericin and lutein in a mass ratio of 2:1.5, and the sericin and lutein powders were directly added to sterile water as raw materials to prepare eye drops, and the other conditions were the same as those of Example 1. The pH was adjusted to 7.4, filtered with a 0.22 μm filter membrane, and sterilized by irradiation with 15 kGy gamma rays at 4-8°C to prepare eye drops.

[0060] Comparative Example 3

[0061] In this comparative example, the mountain mint extract was replaced with an equal amount of sodium hyaluronate on the basis of Example 1, and the mountain mint extract was removed; the other conditions were the same as those of Example 1. The pH was adjusted to 7.4, filtered with a 0.22 μm filter membrane, and sterilized by irradiation with 15 kGy gamma rays at 4-8° C. to prepare eye drops.

[0062] Animal Experimentation:

[0063] (1) Experimental animals and reagents

[0064] 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.

[0065] (2) Animal grouping and drug administration

[0066] 45 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 modeling. Male and female mice of similar weight were selected and separated into cages. A blank control group (5 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 used to drop into the mice's eyes. 5 μl of the prepared benzalkonium chloride was given to each eye, and the eye drops were given 3 times a day for 10 consecutive days. After the modeling begins, pay attention to keeping the mouse environment dry to a certain extent, and the mice eat and drink normally. Figure 1-2 As shown, the comparison of rose bengal staining and tear secretion of mouse eyes before and after modeling was performed. The successful modeling was confirmed by comparison.

[0067] The mice were evenly divided into 9 groups, including blank control group, negative control group, positive control group, experimental group 1, experimental group 2, experimental group 3, comparison group 1, comparison group 2 and comparison group 3, with 5 mice in each group and 10 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 medication after modeling. The positive control group was dripped with 0.3% sodium hyaluronate eye drops (trade name: Aili). Experimental group 1, experimental group 2, experimental group 3, comparison group 1, comparison group 2 and comparison group 3 were respectively given the eye drops of Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3 after modeling (no additional drugs were 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:

[0068] Table 1: Grouping and administration method (quantity is calculated based on the number of eyes)

[0069]

[0070] (3) Experimental results

[0071] 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.

[0072] Table 2: Tear secretion of mice before and after administration

[0073]

[0074] As shown in Table 2, the tear secretion of mice without modeling is always maintained at a normal level. After modeling, the tear secretion of the negative control group, the positive control group, and the mice in Experimental Group 1, Experimental Group 2, Experimental Group 3, Comparison Group 1, Comparison Group 2 and Comparative Example 3 groups were significantly reduced. Among them, the negative control group only dripped physiological saline, and after 14 days of administration, the tear secretion of the mice in the negative control group was further reduced, which shows that physiological saline cannot relieve the dry eye of mice. After 14 days of administration, the tear secretion of the mice in the positive control group, Experimental Group 1, Experimental Group 2 and Experimental Group 3 were basically restored to normal levels, and the recovery of tear secretion in Experimental Group 2 and Experimental Group 3 was better than that in Experimental Group 1 and the positive control group, which is mainly because Experimental Group 2 and Group 3 contain EGF, which can help repair damaged ocular surface cells. After 14 days of administration, the tear secretion of mice in Comparison Group 1, Comparison Group 2 and Comparison Group 3 also increased, but the degree of increase was not obvious. The effect of Comparison Group 2 was better than that of Comparison Group 1, while the effect of Comparison Group 3 on the increase of tear secretion was poor.

[0075] ② Red bengal staining: On the 0th day after the successful establishment of the dry eye model and 14 days after administration, 1% red bengal solution was used for staining. After staying on the ocular surface for 10 seconds, the excess dye was rinsed with 0.9% sodium chloride, and the ocular surface was wiped dry with a cotton swab; the slit lamp light source was adjusted to natural light, and the mouse eyeballs were exposed for observation under the slit lamp. 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 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.

[0076] Table 3: Statistics of corneal staining scores of mice before and after drug administration

[0077]

[0078] The lower the corneal score, the higher the degree of corneal epithelial repair in the mouse and the better the eye recovery. Conversely, the higher the score, the more severe the corneal damage.

[0079] 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, experimental group 3, comparison group 1, comparison group 2 and comparison group 3 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, experimental group 2 and experimental group 3 were significantly reduced, and the corneal improvement of the mice in experimental group 3 and experimental group 2 was slightly better than that in experimental group 1 and the positive control group. This is mainly because experimental groups 2 and 3 contain EGF, which can help repair damaged ocular surface cells. After 14 days of administration, the corneal epithelial staining area and score of the mice in comparison groups 1, comparison groups 2 and comparison groups 3 also decreased slightly, indicating that the degree of corneal epithelial repair in mice was not obvious. The reduction of staining score in comparison group 2 was greater than that in comparison group 1, and the reduction of staining score in comparison group 3 was the lowest. This shows that the mint extract in eye drops plays a key role in repairing corneal damage and promoting eye recovery.

[0080] ③ Detection of mouse corneal inflammatory factor IL-6 level, the detection method is as follows:

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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 only drips physiological saline after successful modeling. After 14 days of administration, the inflammatory factor IL-6 level is high, indicating that the corneal inflammation of the negative control group mice is still very obvious. After 14 days of administration, the inflammation expression levels of the positive control group, experimental group 1, experimental group 2, and experimental group 3 decreased significantly, all moving closer to the blank control group, indicating that the eye drops of the present invention can effectively help the cornea of ​​mice to recover health; compared with the positive control group, the eye drops of Example 1-3 are more effective in relieving dry eye. Although Comparative Example 1-3 can also inhibit the inflammatory factor IL-6, the overall effect is not as good as Example 1-3; in addition, it can be seen from the comparison of Comparative Example 3 with Comparative Example 1-2 that after the lack of mountain mint extract in Comparative Example 3, its relieving effect on dry eye is greatly weakened, which shows that mountain mint plays an important role in relieving dry eye.

[0085] Eye drops storage stability test

[0086] The eye drop samples prepared in Example 1 and Comparative Examples 1-2 were stored in a sealed container and kept away from light at 25°C for 16 weeks. The lutein content in the samples was measured every 4 weeks, and the retention rate of lutein (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 lutein-containing eye drops prepared in Example 1 can remain stable for a longer period of time at room temperature, while the lutein in the lutein eye drops prepared in Comparative Examples 1-2 is difficult to retain for a long time, and the lutein is likely to be partially degraded during the preparation, irradiation sterilization, and storage process.

[0087] 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. An eye drop for treating dry eyes, characterized in that: The invention comprises, by mass percentage, 0.05-0.5% of mountain mint extract, 0.1-0.4% of sericin nanoparticles coated with lutein, 0.1%-0.5% of chondroitin sulfate, 0.9-1.0% of sodium chloride, 0.05%-0.1% of poloxamer, 0.01%-0.1% of EDTA, phosphate buffer and water; the particle size of the sericin nanoparticles coated with lutein is 50-300nm, the water is medicinal sterile water, and the phosphate buffer is used to adjust the pH value of the eye drops to 7.0-7.

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2. The eye drops according to claim 1, characterized in that The eye drops also contain 0.01%-0.1% of epidermal growth factor EGF.

3. The eye drops according to claim 1, characterized in that The mountain mint extract is a freeze-dried powder of an ethanol extract of mountain mint, and the preparation method thereof is as follows: washing the mountain mint raw material, drying it in the shade or blowing it dry away from light, adding water to mix, crushing it by ultrafine grinding method, using ethanol ultrasonic extraction to obtain an extract, concentrating the extract by vacuum rotary evaporation and recovering ethanol, and freeze-drying the concentrate to obtain the mountain mint extract.

4. The eye drops according to claim 2, characterized in that The eye drops are composed of: 0.2% mountain mint extract, 0.15% sericin nanoparticles coated with lutein, 0.1% chondroitin sulfate, 0.05% epidermal growth factor EGF, 0.9% sodium chloride, 0.065% poloxamer, 0.05% EDTA, phosphate buffer and the balance water; the pH of the eye drops is 7.0-7.

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

6. A method for preparing the eye drops for treating dry eyes according to any one of claims 1 to 5, characterized in that: The steps include: S1. Preparation of sericin nanoparticles coated with lutein Dissolving sericin in water or phosphate buffer to form a first dispersion, dissolving lutein in ethyl acetate or 95% ethanol to obtain a second dispersion; adding the second dispersion to the first dispersion, stirring at high speed or treating with ultrasound to form a stable emulsion; cooling to 2-8°C, centrifuging, separating centrifugal precipitates, and freeze-drying to obtain sericin nanoparticles coated with lutein; S2. Preparation of peppermint extract Add water to mountain mint, use an ultrafine grinder to grind to obtain a slurry, use anhydrous ethanol to ultrasonically assist extraction to obtain an ethanol extract, centrifuge at 2000-4000 rpm to remove precipitation, retain the supernatant, and recover ethanol by vacuum rotary evaporation at ≤45°C to produce a concentrated solution, and freeze-dry the concentrated solution to obtain a mountain mint extract powder; S3. Add the remaining raw materials except phosphate buffer to sterile water, stir evenly, add phosphate buffer and stir evenly, adjust the pH to 7.0-7.8, filter with a 0.22 μm filter membrane, and dispense the filtered solution into sterile eye drop bottles.

7. The preparation method according to claim 6, characterized in that: In S1, the concentration of sericin in the first dispersion is 1-5%; the preparation process of the first dispersion 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 0.45μm filter membrane after dissolution to obtain the first dispersion; the concentration of lutein in the second dispersion is 1-2%; the preparation process of the second dispersion is: at room temperature, add lutein to ethyl acetate or 95% ethanol, stir while adding until completely dissolved, and filter through a 0.45μm filter membrane after dissolution to obtain the second dispersion.

8. The preparation method according to claim 6 or 7, characterized in that: In S1, a first dispersion and a second dispersion are prepared in a volume ratio of 4:1-1:1, and the second dispersion is slowly added to the first dispersion while stirring at a high speed 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 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 coated with lutein.

9. The preparation method according to claim 6, characterized in that: In S2, the mountain mint is mixed with water, and an ultrafine grinder is used to grind the mixture at a rotation speed of 40-50k rpm for 10-30 minutes to obtain an ultrafine powder slurry, and 3-5 times the volume of ethanol is added for ultrasonic-assisted extraction, the ultrasonic frequency is 40-60kHz, and the processing time is 0.5-1h to obtain an ethanol extract, and the mixture is further centrifuged at 2000-4000rpm for 15-25min to remove the precipitate to obtain a supernatant, and the supernatant is evaporated under reduced pressure at ≤45°C, the ethanol is recovered and part of the water is removed to obtain a concentrated solution, and the concentrated solution is freeze-dried at -40°C to obtain a mountain mint extract powder.

10. The preparation method according to claim 6, characterized in that: In S3, 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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