An eye drop for treating dry eye and a method of preparing the same

Abstract

The present application relates to a kind of eye drops for treating dry eye and its preparation method, eye drops include by mass percentage: 0.05-0.5% of the extract of Monarda, 0.1-0.4% of silk fibroin 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 silk fibroin nanoparticles coated with lutein is 50-300nm, water is pharmaceutical sterile water, and the pH of eye drops is adjusted to 7.0-7.8 by phosphate buffer solution.The eye drops of the present application can effectively relieve dry eye symptoms, provide long-term wetting and protection, while reducing ocular inflammation and oxidative stress, can promote the repair of corneal injury, and effectively reduce the expression level of inflammatory factors, and high use safety.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an eye drop for treating dry eye syndrome and its preparation method. Background Technology

[0002] Dry eye syndrome is a common eye disease characterized by insufficient tear production or poor tear quality, leading to inadequate lubrication of the eyes and causing a range of uncomfortable symptoms. In severe cases, it can affect vision. The exact causes of dry eye syndrome are varied, but can be broadly categorized into two types: insufficient tear secretion (lack of the aqueous layer) and excessive evaporation (abnormal lipid layer). The main causes of dry eye syndrome are: 1. Insufficient tear secretion; 2. Excessive tear evaporation. Insufficient tear secretion can be further caused by: ① Aging: The function of the lacrimal glands gradually declines with age. ② Autoimmune diseases: such as... Sjögren's syndrome. ③ Medication side effects: Some medications, such as antihistamines, antidepressants, and contraceptives, may reduce tear secretion. ④ Environmental factors: Dry or windy climates, prolonged exposure to air-conditioned rooms. ⑤ Laser correction surgery: Some ophthalmic surgeries may temporarily or permanently alter tear production or distribution. Causes of excessive tear evaporation include: ① Meibomian gland dysfunction; ② Prolonged use of electronic devices can lead to reduced blinking frequency, decreasing the opportunity for tear film renewal. ③ Long-term contact lens wear increases friction on the eye surface and may also interfere with normal tear distribution. Symptoms of dry eye include foreign body sensation, burning sensation, redness, increased discharge, photosensitivity, fluctuating vision, and difficulty driving at night, severely interfering with daily life.

[0003] Lutein is a naturally occurring carotenoid that plays an important role in eye health. It possesses strong antioxidant properties, helping to reduce free radical damage to eye tissues and thus protecting the eyes from oxidative stress. Lutein also absorbs harmful blue light, reducing its potential damage to the retina, which is especially important for people who frequently use electronic screens. For example, CN118384216A discloses an An Tong Cao tea eye wash, whose composition includes: 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 being pure water. CN117771294A also discloses a borneol compound eye protection preparation, comprising at least borneol fresh branch extract, lutein, vitamin A, and zeaxanthin, formed by processing the above components. However, there are some challenges or unresolved issues in the development of lutein into eye drops: (1) Solubility: Lutein has very limited solubility in water, usually less than 1 mg / L, which means it is difficult to dissolve directly in water and difficult to increase the concentration of lutein in eye drops and maintain its stability. (2) Bioavailability: Since lutein is lipid-soluble, its ability to cross 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 eye irritation is a major challenge in the research and development process. (3) Stability: Lutein is easily degraded under the influence of light, temperature and other factors. How to ensure the stability of lutein in eye drops, especially in the production sterilization, storage and transportation process, 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] (a) Technical problems to be solved

[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 syndrome and its preparation method. It mainly uses sericin to encapsulate lutein, and utilizes the high hydrophilicity and film-forming properties of sericin to improve dispersibility, ability to cross the corneal barrier, and stability in the eye drop, thereby improving the bioavailability of lutein. At the same time, sericin also has moisturizing, antioxidant, and antibacterial properties, which help improve dry eye syndrome. It solves the technical problems of poor solubility and stability that exist when lutein is directly added to eye drops.

[0006] (II) Technical Solution

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

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

[0009] According to a preferred embodiment of the present invention, the peppermint extract is a freeze-dried powder of an ethanol extract of peppermint. The preparation method is as follows: the peppermint (Eriodictyon californicum) raw material is washed, dried in the shade or blow-dried in the dark, mixed with water, pulverized by ultra-micro pulverization, and extracted with ethanol by ultrasonic extraction to obtain an extract. The extract is concentrated by rotary evaporation under reduced pressure and the ethanol is recovered. The concentrate is freeze-dried to obtain the peppermint extract.

[0010] According to a preferred embodiment of the present invention, the eye drops are composed of: 0.2% peppermint extract, 0.15% lutein-coated sericin nanoparticles, 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 being 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 eye syndrome, comprising:

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

[0014] Sericin was dissolved in water or phosphate buffer to form a first dispersion, and lutein was dissolved in ethyl acetate or 95% ethanol to obtain a second dispersion. The second dispersion was added to the first dispersion, and the mixture was subjected to high-speed stirring or ultrasonic treatment to form a stable emulsion. The mixture was cooled to 2-8℃, centrifuged, the centrifuged precipitate was separated, and freeze-dried to obtain lutein-coated sericin nanoparticles.

[0015] S2, Preparation of Peppermint Extract

[0016] Add water to peppermint and pulverize it with an ultra-micro pulverizer to obtain a slurry. Extract it with anhydrous ethanol using ultrasonic assistance to obtain an ethanol extract. Centrifuge at 2000-4000 rpm to remove the precipitate and keep the supernatant. Recover the ethanol by rotary evaporation under reduced pressure at ≤45℃ to produce a concentrated liquid. Freeze-dry the concentrated liquid to obtain peppermint extract powder.

[0017] S3. Add all raw materials except phosphate buffer to sterile water, stir well, add phosphate buffer and stir well, adjust pH to 7.0-7.8, filter with a 0.22μm 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 as follows: sericin powder is added to water or phosphate buffer while stirring until completely dissolved, and the temperature is controlled at 25-45℃ during the dissolution process (appropriate heating can promote the dissolution of sericin in water). After dissolution, it is filtered through a 0.45μm filter membrane to obtain the first dispersion; the concentration of lutein in the second dispersion is 1-2%; the preparation process of the second dispersion is as follows: at room temperature, lutein is added to ethyl acetate or 95% ethanol while stirring until completely dissolved, and after dissolution, it is filtered through a 0.45μm filter membrane to obtain the second dispersion.

[0019] According to a preferred embodiment of the present invention, in step S1, the first dispersion and the second dispersion are prepared at a volume ratio of 4:1 to 1:1. The second dispersion is slowly added to the first dispersion while simultaneously stirring at high speed (e.g., 1000-3000 rpm) for 10-30 minutes until a stable emulsion is formed. The temperature during emulsification does not exceed 25°C. The emulsion is then rapidly transferred to an ice bath or refrigerator and rapidly cooled to 4-8°C for 0.5-1 hour. It is then centrifuged at 10000-15000 g for 10-20 minutes. After centrifugation, the precipitate is separated and freeze-dried to obtain lutein-coated sericin nanoparticles. Preferably, the supernatant obtained after sterilization can be used to prepare the first dispersion.

[0020] According to a preferred embodiment of the present invention, in S2, peppermint is mixed with water and pulverized using an ultrafine pulverizer at a speed of 40-50 krpm for 10-30 min to obtain an ultrafine powder slurry. 3-5 times the volume of ethanol is added, and ultrasonic-assisted extraction is performed at an ultrasonic frequency of 40-60 kHz for 0.5-1 h to obtain an ethanol extract. The extract is then centrifuged at 2000-4000 rpm for 15-25 min to remove the precipitate, resulting in a supernatant. The supernatant is evaporated under reduced pressure at ≤45℃ to recover the ethanol and remove some water to obtain a concentrated solution. The concentrated solution is freeze-dried at -40℃ to obtain peppermint extract powder.

[0021] Preferably, after being dispensed into sterile eye drop bottles, the solution is sterilized by irradiation with 10-20 kGy gamma rays at 4-10°C before being stored. This sterilization condition achieves sterilization while minimizing the decomposition of antioxidant components in the eye drops.

[0022] (III) Beneficial Effects

[0023] (1) This invention uses sericin to coat lutein. Utilizing sericin's high hydrophilicity, film-forming properties, and good solubility in water, it improves the solubility, stability, and ability to cross the corneal barrier of lutein in eye drops, thereby enhancing its bioavailability. Simultaneously, sericin itself possesses moisturizing, viscosity-regulating, antioxidant, and antibacterial properties, which help improve dry eye syndrome. This solves the technical problems of poor solubility and susceptibility to sterilization, light, and temperature affecting the stability of lutein in eye drops. Furthermore, coating lutein with sericin provides a long-lasting, sustained-release effect, significantly improving its bioavailability and fully leveraging its antioxidant properties to protect the eyes from oxidative stress. Sericin also has excellent antibacterial properties, serving as a natural preservative and greatly improving the safety of eye drops.

[0024] (2) This invention also uses peppermint extract in the eye drops. Peppermint, also known as North American holy herb or California holy herb (Eriodictyon californicum), is rich in mucilage and active ingredients. Specifically, peppermint contains various flavonoids and polyphenols, which have anti-inflammatory effects and help reduce eye inflammation; it also has strong antioxidant properties, which can neutralize free radicals, reduce oxidative stress damage to the eyes, and protect the composition of the eye drops. The mucilage can regulate the viscosity of the eye drops and form a protective film on the surface of the eye, helping to keep the eyes moist. In the eye drops, it can replace auxiliary antioxidants such as sodium hyaluronate and vitamin E.

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

[0026] Figure 1 This is a comparison image of red ochre staining of the cornea of ​​mice before and after modeling with benzalkonium chloride (BAC).

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

[0028] Figure 3 The levels of corneal inflammatory factor IL-6 in mice were compared 14 days after administration to each group of mice.

[0029] Figure 4 The curves show the changes in lutein retention rate in eye drops of Example 1 and Comparative Examples 1-2 over storage time. Detailed Implementation

[0030] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] The eye drops of this invention contain chondroitin sulfate, lutein-encapsulated sericin nanoparticles, peppermint extract, sodium chloride, poloxamer, EDTA, and epidermal growth factor (EGF). Chondroitin sulfate, a natural polysaccharide, increases tear viscosity and stability, helping to form a stable tear film and thus relieving dry eye symptoms. Lutein is a potent antioxidant that absorbs harmful blue light and protects the retina from light damage. Sericin nanoparticles enhance the water solubility, stability, corneal barrier crossing ability, and bioavailability of lutein, making it easier for the eyes to absorb. Peppermint extract has anti-inflammatory and antioxidant properties. It can reduce eye inflammation and oxidative stress, thus helping to relieve dry eye symptoms. The mucilage in peppermint extract has strong moisturizing properties, providing long-lasting hydration, helping to lubricate the surface of the eyeball, reducing friction, relieving dryness, and forming a protective film on the cornea to protect the corneal epithelium from damage. When chondroitin sulfate is used in combination with lutein and peppermint extract, it provides better tear film stability and protection. The mucin synergistically enhances the moisturizing effect of chondroitin sulfate, while peppermint extract and lutein synergistically provide antioxidant and anti-inflammatory effects. In summary, the combined use of lutein with chondroitin sulfate and peppermint extract provides stronger antioxidant protection, while also enhancing the stability and effectiveness of lutein, thus protecting ocular tissues.

[0032] In addition, sodium chloride is used to adjust the osmotic pressure of the eye drops to match that of tears, reducing eye irritation. Poloxamer acts as a solubilizer and enhances the dispersibility and stability of the drug. EDTA chelates metal ions in the solution, preventing catalytic oxidation reactions caused by metal particles introduced into the eye drops during production by water or equipment, thus improving the stability of the eye drops. EDTA, by chelating metal ions, can further enhance the stability of sericin, peppermint extract, lutein, etc. EGF is a polypeptide that promotes cell proliferation and wound healing. EGF can help repair damaged ocular surface cells and promote corneal epithelial regeneration, thereby improving dry eye symptoms, mainly playing a role in repair and regeneration.

[0033] The eye drop formulation of this invention comprises components that work synergistically through the following mechanisms:

[0034] Anti-inflammatory and antioxidant: 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, enhancing the repair capabilities of eye tissues.

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

[0038] Sericin, as a lutein carrier, enhances the dispersibility and stability of lutein in eye drops, providing sustained-release effects and improving lutein bioavailability. Sericin also possesses antibacterial properties; specifically, sericin (S1, S2) and small-molecule sericin peptide (S3) exhibit antibacterial activity against both Gram-negative and Gram-positive bacteria. Sericin is non-toxic to cells and promotes cell growth with increasing concentration. Quantitative tests showed significant antibacterial activity against Staphylococcus aureus and Escherichia coli. Sericin exhibits good antibacterial properties and biocompatibility, making it a viable alternative to chemical preservatives and improving the safety of eye drops. The eye drops of this invention can be used without chemical preservatives. Furthermore, single-dose packaging avoids the use of preservatives, making it suitable for patients sensitive to preservatives.

[0039] To verify the technical effects of the present invention, specific embodiments are described below. Unless otherwise specified in the following embodiments, % refers to mass concentration.

[0040] Example 1

[0041] This embodiment provides an eye drop for treating dry eye syndrome, and its preparation method is as follows:

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

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

[0044] (3) Prepare the first and second dispersions at a volume ratio of 1:1. Slowly add the second dispersion to the first dispersion while stirring at 3000 rpm. After the addition is complete, continue stirring for 30 minutes until a stable emulsion is formed. The temperature during emulsification should not exceed 25°C. Quickly transfer the emulsion to an ice bath and rapidly cool it to 4-8°C for 1 hour. Centrifuge at 12000g for 15 minutes. After centrifugation, separate the precipitate and freeze-dry it to obtain lutein-coated sericin nanoparticles. The supernatant can be sterilized and used to prepare the first dispersion. The particle size of the sericin nanoparticles is 50-200 nm.

[0045] (4) Clean the peppermint thoroughly, dry it under light-proof conditions, add water at a mass ratio of 1:1 and mix, then pulverize it in an ultra-micro pulverizer at a speed of 40k rpm for 20min to obtain an ultra-micro powder slurry. Add 4 times the volume of ethanol and treat it at an ultrasonic frequency of 40kHz for 45min to obtain an ethanol extract. Continue to centrifuge at 3000rpm for 20min to remove the precipitate and obtain the supernatant. Evaporate the supernatant under reduced pressure at ≤45℃ to recover the ethanol and remove some water to obtain a concentrated solution. Freeze-dry the concentrated solution at -40℃ to obtain peppermint extract powder.

[0046] (5) Add the following to sterile water: 0.2% peppermint extract, 0.15% lutein-coated sericin nanoparticles, 0.1% chondroitin sulfate, 0.9% sodium chloride, 0.065% poloxamer, and 0.05% EDTA. After adding, 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. Dispense the filtered solution into sterile eye drop bottles and sterilize by irradiation with 15 kGy gamma rays at 4-8℃. Store in a warehouse.

[0047] Example 2

[0048] The only difference between this embodiment and Embodiment 1 is that 0.05% epidermal growth factor (EGF) was added in step (5). Similarly, the pH was adjusted to 7.4, filtered with a 0.22 μm filter membrane, and the filtered solution was dispensed into sterile eye drop bottles. Then, it was 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 syndrome, and its preparation method is as follows:

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

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

[0053] (3) Prepare the first dispersion and the second dispersion at a volume ratio of 2:1. Slowly add the second dispersion to the first dispersion while stirring at 3000 rpm. After the addition is complete, continue stirring for 30 minutes until a stable emulsion is formed. The temperature during emulsification should not exceed 25°C. Quickly transfer the emulsion to an ice bath and rapidly cool it to 4-8°C for 1 hour. Centrifuge at 10000g for 20 minutes. After centrifugation, separate the precipitate and freeze-dry it to obtain lutein-coated sericin nanoparticles. The supernatant can be sterilized and used to prepare the first dispersion. The particle size of the sericin nanoparticles is 100-300 nm.

[0054] (4) Clean the peppermint thoroughly, dry it under light-proof conditions, add water at a mass ratio of 1:1 and mix, then pulverize it in an ultra-micro pulverizer at a speed of 50k rpm for 20min to obtain an ultra-micro powder slurry. Add 5 times the volume of ethanol and treat it at an ultrasonic frequency of 40kHz for 50min to obtain an ethanol extract. Continue to centrifuge at 3000rpm for 20min to remove the precipitate and obtain the supernatant. Evaporate the supernatant under reduced pressure at ≤45℃ to recover the ethanol and remove some water to obtain a concentrated solution. Freeze-dry the concentrated solution at -40℃ to obtain peppermint extract powder.

[0055] (5) Add the following to sterile water: 0.15% peppermint extract, 0.2% lutein-coated sericin nanoparticles, 0.15% chondroitin sulfate, 0.9% sodium chloride, 0.075% poloxamer, 0.05% EDTA, and 0.05% epidermal growth factor (EGF). After adding all the above components, stir thoroughly for 30 minutes. Finally, add phosphate buffer and stir evenly. Adjust the pH to 7.4 and filter with a 0.22 μm filter membrane. Dispense the filtered solution into sterile eye drop bottles and sterilize by irradiation with 15 kGy gamma rays at 4-8℃. Store in a warehouse.

[0056] Comparative Example 1

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

[0058] Comparative Example 2

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

[0060] Comparative Example 3

[0061] This comparative example is based on Example 1, except that the peppermint extract was replaced with an equal amount of sodium hyaluronate, and the peppermint extract was removed; all other conditions were the same as in Example 1. The pH was adjusted to 7.4, filtered through a 0.22 μm filter membrane, and sterilized by irradiation with 15 kGy gamma rays at 4-8°C to obtain eye drops.

[0062] Animal experiments:

[0063] (1) Laboratory animals and reagents

[0064] Animal selection: BALB / c mice, half female and half male, aged 8 weeks, were used to construct the dry eye syndrome. They were healthy and showed no observed ocular abnormalities. They were provided with adequate water and food, and kept in good husbandry conditions with controlled temperature and humidity.

[0065] (2) Animal grouping and administration

[0066] Forty-five mice were used to establish a dry eye model. The modeling method was as follows: Mice were housed in an experimental environment for a certain period of time, and pre-modeling preparations were made. Male and female mice of similar weight were selected and separated into different cages, with a blank control group of 5 mice. The remaining mice were used for the experimental model. After separation, a 2 mg / ml benzalkonium chloride (BAC) solution was prepared and applied to the eyes of each mouse, 5 μl of the prepared benzalkonium chloride solution, three times a day for 10 consecutive days. After the modeling began, the environment of the mice was kept relatively dry, and the mice were allowed normal food and water. Figure 1-2 As shown, the comparison of red ochre staining and tear secretion in the mouse eyes before and after modeling confirms successful modeling.

[0067] Mice were randomly divided into nine groups: a blank control group, a negative control group, a positive control group, experimental group 1, experimental group 2, experimental group 3, and control group 1, control group 2, and control group 3, with five mice in each group and ten eyes in each group. The blank control group received no modeling and only instilled physiological saline. The negative control group received no medication after modeling. The positive control group received 0.3% sodium hyaluronate eye drops (trade name: Aili). Experimental groups 1, 2, 3, and control groups 1, 2, and 3 received the eye drops from Examples 1, 2, 3, 1, 2, and 3, respectively, after modeling (without additional medication). Administration was twice daily, 2 drops (approximately 5 μL) per eye each time, for 14 consecutive days. The administration method is shown in Table 1.

[0068] Table 1: Grouping and Administration Methods (Quantity based on the number of eyes)

[0069]

[0070] (3) Experimental Results

[0071] ① On day 0 of successful dry eye model establishment, tear fluid was collected from mice in each group. Tear secretion was measured using phenol red cotton thread. Tear secretion was then measured again 14 days after drug administration. The specific procedure for phenol red cotton thread tear detection was as follows: the mouse was grasped, and the phenol red cotton thread was placed on the conjunctiva of the lower eyelid, one-third of the way from the outer corner of the eye, for 60 seconds. The thread was then removed and fixed, and the length (mm) of the moistened stained portion of the cotton thread was immediately measured and recorded. The results are shown in Table 2, where tear secretion is expressed as mean ± standard deviation.

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

[0073]

[0074] Table 2 shows that the tear secretion of mice without modeling remained at a normal level. After modeling treatment, the tear secretion of mice in the negative control group, positive control group, and experimental groups 1, 2, 3, control groups 1, 2, and 3 significantly decreased. The negative control group, which only received saline, showed a further decrease in tear secretion after 14 days of treatment, indicating that saline could not alleviate dry eye in mice. After 14 days of treatment, the tear secretion of mice in the positive control group, experimental groups 1, 2, and 3 basically returned to normal levels. Experimental groups 2 and 3 showed better recovery than experimental group 1 and the positive control group, mainly because experimental groups 2 and 3 contain EGF, which can help repair damaged ocular surface cells. After 14 days of treatment, the tear secretion of mice in control groups 1, 2, and 3 also increased, but the increase was not significant. Control group 2 showed better results than control group 1, while control group 3 showed a poorer effect on increasing tear secretion.

[0075] ② Rose Bengal Staining: On day 0 of successful dry eye model establishment and 14 days after drug administration, mice were stained with 1% rose benzene solution. After 10 seconds on the ocular surface, excess stain was rinsed off with 0.9% sodium chloride solution, and the ocular surface was dried with cotton swabs. The slit lamp light source was adjusted to natural light, and the mice's eyes were exposed under the slit lamp for observation. After adjusting the magnification, the staining of the cornea of ​​mice in different groups with sodium fluorescein was recorded. The cornea was divided into four quadrants: superior temporal, inferior temporal, superior nasal, and inferior nasal. Each quadrant was scored from 0 to 3 points. The scoring criteria were: no staining 0 points; 1-30 punctate stains (mild) 1 point; more than 30 punctate stains but no fusion (moderate) 2 points; corneal punctate staining fusion, filamentous structures, and ulcers (severe) 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: Statistical table of corneal staining scores in mice before and after drug administration

[0077]

[0078] A lower corneal score indicates a higher degree of corneal epithelial repair and better eye recovery in mice, while a higher score indicates more severe corneal damage.

[0079] Table 3 shows that the tear secretion of mice without modeling remained at a normal level. After modeling treatment, the corneal epithelial staining area and corneal score of mice in the negative control group, positive control group, experimental group 1, experimental group 2, experimental group 3, control group 1, control group 2, and control group 3 significantly increased. Specifically, the negative control group received only saline drops; after 14 days of treatment, the corneal epithelial staining area and score of mice in the negative control group further increased. In the positive control group, experimental group 1, experimental group 2, and experimental group 3, after 14 days of treatment, the corneal epithelial staining area and score of mice significantly decreased. Furthermore, the corneal improvement 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. In control groups 1, 2, and 3, after 14 days of treatment, the corneal epithelial staining area and score of mice also decreased slightly, indicating that the degree of corneal epithelial repair in mice was not significant. Among the comparison groups, group 2 showed a greater reduction in staining score than group 1, while group 3 showed the least reduction. This indicates that peppermint extract in eye drops plays a crucial role in repairing corneal damage and promoting eye recovery.

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

[0081] Two mice were randomly selected from each group and sacrificed. Immediately afterward, the eyeballs were removed. Under a microscope, the eyeballs were separated using ophthalmic forceps, rinsed with pre-cooled PBS, and dried with filter paper. The two corneas were assembled into an enzyme-free EP tube and frozen in liquid nitrogen for temporary storage at -80°C. The corneal tissue was removed and placed in an ice box. The tissue was minced using sterilized ophthalmic instruments. 100 μl of pre-cooled PBS containing PMSF was added to each sample tube, and the samples were sonicated on ice for 3 min using an ultrasonic homogenizer. The sonicated samples were centrifuged in a frozen centrifuge at 4°C and 12,000 rpm for 15 min. The supernatant was collected and placed in an enzyme-free EP tube for later use.

[0082] Serial dilution of bovine serum albumin (BSA) standard. Prepare BCA working solution, typically 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, then add the BCA working solution and mix well. 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 562 nm.

[0083] After equilibrating the ELISA kit to room temperature, remove only the required number of strips according to the experimental needs before detection, and keep the rest sealed and stored at 4℃. Add 50 μl of sample to each well according to the experimental settings. Add 100 μl of HRP-labeled detection antibody to each well, seal with a sealing film, and incubate at 37℃ for 60 min. After the reaction, wash the plate with washing buffer for 1 min each time, repeating 5 times. Add substrate A and substrate B sequentially to each well, 50 μl each, and incubate at 37℃ in the dark for 15 min. After the reaction, add 50 μl of stop solution. Measure the OD value of each well at the wavelength specified in the kit using a microplate reader. Note that the detection should be completed within 15 min of adding the stop solution. Finally, plot a standard curve, calculate the sample concentration, and correct the results using the protein concentration of the sample. Experimental results are as follows: Figure 3 As shown.

[0084] like Figure 3 As shown, the blank control group consisted of healthy mice (without modeling treatment) with low levels of inflammation. The negative control group, after successful modeling, only received saline solution. After 14 days of treatment, their levels of the inflammatory factor IL-6 were high, indicating that corneal inflammation in the negative control group mice remained significant. After 14 days of treatment, the inflammatory expression levels in the positive control group, experimental group 1, experimental group 2, and experimental group 3 decreased significantly, all approaching those of the blank control group, indicating that the eye drops of this invention can effectively help the corneas of mice recover health. Compared with the positive control group, the eye drops of Examples 1-3 were more effective in relieving dry eye syndrome. Although Comparative Examples 1-3 also inhibited the inflammatory factor IL-6, their overall effect was not as good as that of Examples 1-3. Furthermore, a comparison between Comparative Example 3 and Comparative Examples 1-2 showed that the absence of peppermint extract in Comparative Example 3 significantly weakened its effect in relieving dry eye syndrome, indicating that peppermint plays an important role in relieving dry eye syndrome.

[0085] Eye Drops Storage Stability Test

[0086] The eye drop samples prepared in Example 1 and Comparative Examples 1-2 were stored in sealed containers and kept in the dark at 25°C for 16 weeks. Samples were taken every 4 weeks to measure the lutein content, and the retention rate of lutein (reduced form) in the eye drop samples was determined by HPLC. The test results are as follows: 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. Lutein is likely to undergo partial degradation during preparation, irradiation sterilization, and storage.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 eye, characterized by, It comprises by mass percentage: 0.05-0.5% of the extract of the mountain mint, 0.1-0.4% of the silk fibroin 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 silk fibroin nanoparticles coated with lutein is 50-300nm, the water is pharmaceutical sterile water, and the phosphate buffer adjusts the pH of the eye drops to 7.0-7.8; The mountain mint extract is a freeze-dried powder of the ethanol extract of the mountain mint, and the preparation method is as follows: washing the mountain mint raw material, drying in the dark, adding water, using the ultrafine grinding method to grind, using ethanol ultrasonic extraction to obtain the extract, reducing pressure rotary evaporation to concentrate and recover ethanol, freezing and drying the concentrated solution to obtain the mountain mint extract.

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

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

8.

4. The eye drop according to claim 1, characterized by, The eye drops do not contain chemical preservatives.

5. A method for preparing the eye drops for treating dry eye according to any one of claims 1 to 4, characterized by, The steps include: S1, preparing the silk fibroin nanoparticles coated with lutein Dissolving the silk fibroin 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, forming a stable emulsion by high-speed stirring or ultrasonic treatment; cooling to 2-8℃ for cooling treatment, centrifuging, separating the centrifugal precipitate, and freeze-drying to obtain the silk fibroin nanoparticles coated with lutein; S2, preparing the extract of the mountain mint Adding water to the mountain mint, using an ultrafine grinder to grind to obtain a slurry, using anhydrous ethanol for ultrasonic-assisted extraction to obtain an ethanol extract, centrifuging at 2000-4000rpm to remove the precipitate, leaving the supernatant, recovering ethanol under reduced pressure rotary evaporation at ≤45℃, producing a concentrated solution, freeze-drying the concentrated solution to obtain the powder of the mountain mint extract; S3, adding the remaining raw materials except the phosphate buffer to sterile water, stirring uniformly, adding the phosphate buffer after stirring uniformly, adjusting the pH to 7.0-7.8, filtering with a 0.22μm filter membrane, and dispensing the filtered solution into sterile eye drop bottles.

6. The production method according to claim 5, wherein In S1, the concentration of sericin in the first dispersion is 1-5%; the preparation process of the first dispersion is as follows: sericin powder is added to water or phosphate buffer while stirring until completely dissolved, the temperature is controlled at 25-45℃ during the dissolving process, and then the solution is filtered through a 0.45 μm filter to obtain the first dispersion; the concentration of lutein in the second dispersion is 1-2%; the preparation process of the second dispersion is as follows: lutein is added to ethyl acetate or 95% ethanol at room temperature while stirring until completely dissolved, and then the solution is filtered through a 0.45 μm filter to obtain the second dispersion.

7. The production method according to claim 5 or 6, characterized by, In S1, the first dispersion and the second dispersion are prepared at a volume ratio of 4:1-1:1, the second dispersion is slowly added to the first dispersion while high-speed stirring, and the stirring is continued for 10-30 min until a stable emulsion is formed; the temperature during the emulsification process does not exceed 25℃; the emulsion is quickly transferred to an ice bath or refrigerator for rapid cooling to 4-8℃ for 0.5-1 h, and then centrifuged at a centrifugal force of 10000-15000 g for 10-20 min; after centrifugation, the precipitate is separated and freeze-dried to obtain sericin nanoparticles coated with lutein.

8. The preparation method according to claim 5, characterized in that, In S2, the water is mixed with the mountain mint, and then an ultrafine pulverizer is used to pulverize the mixture at a speed of 40-50 k rpm for 10-30 min to obtain an ultrafine powder slurry; 3-5 times the volume of ethanol is added for ultrasonic-assisted extraction, the ultrasonic frequency is 40-60 kHz, and the treatment time is 0.5-1 h to obtain an ethanol extract; the extract is centrifuged at 2000-4000 rpm for 15-25 min to remove the precipitate, and the supernatant is obtained; the supernatant is evaporated under reduced pressure at ≤45℃ to recover ethanol and remove part of the water to obtain a concentrated solution; the concentrated solution is freeze-dried at -40℃ to obtain a mountain mint extract powder.

9. The preparation method according to claim 5, characterized in that, In S3, the mountain mint extract is dispensed into sterile eye drop bottles, and then sterilized by 10-20 kGy gamma rays at 4-10℃, and then stored.

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