Traditional Chinese medicine microemulsion eye drop and preparation method and application thereof

Abstract

The application discloses a traditional Chinese medicine microemulsion eye drop as well as a preparation method and application thereof, and comprises the following components in percentage by weight: 0.001% to 2.0% of baicalein, 0.001% to 2.0% of borneol, 0.1% to 20% of oil for injection, 0.1% to 10% of a surfactant, 0.1% to 10% of a phospholipid, 0.1% to 5% of an osmotic pressure regulator, and the balance of water. The baicalein and borneol are combined to prepare an oil-in-water emulsion containing borneol and baicalein, so that the mutual synergistic effect is achieved, the opportunity of the medicine entering the cornea and the inflammatory site of the eye is increased, the medicine is rapidly introduced into the eye tissue, the curative effect is enhanced, the in-eye retention time is obviously prolonged, the corneal permeability of the medicine is increased, the non-invasive drug administration mode treatment for AMD patients and the treatment for various types of eye fatigue are realized.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to a traditional Chinese medicine microemulsion eye drop, its preparation method, and its application. Background Technology

[0002] Age-related macular degeneration (AMD) is a neurodegenerative disease of the retina and a leading cause of blindness in people over 50 years of age. In 2020, there were approximately 196 million AMD patients worldwide, and this number is projected to rise to 288 million by 2040. With the accelerating aging of my country's population, the number of people experiencing visual impairment due to AMD will increase rapidly. Clinically, AMD is divided into two types: dry (atrophic) and wet (neovascular) AMD, with dry AMD accounting for approximately 90% of all cases. Most AMD patients initially present with dry AMD, which later progresses to wet AMD; therefore, dry AMD is considered a risk factor or initiating stage for wet AMD. In recent years, significant progress has been made in the treatment of wet AMD due to the application of anti-vascular endothelial growth factor drugs. However, there is still no specific treatment for dry AMD. Therefore, AMD remains a leading cause of blindness in my country, and the development of novel AMD treatments is crucial for clinical treatment.

[0003] Baicalein, also known as baicalein aglycone, is one of the main active ingredients in the dried root of *Scutellaria baicalensis*, a perennial herbaceous plant belonging to the genus *Scutellaria* in the family Lamiaceae. It belongs to the flavonoid class of compounds. Baicalein is a yellow needle-like crystal, soluble in acetone, alcohols, and ethyl acetate, but almost insoluble in water. Domestic and international studies have shown that it possesses anti-inflammatory, antioxidant, antibacterial, and antiviral effects. It has shown great promise in inhibiting ocular inflammation, combating oxidative stress damage, and preventing and treating eye diseases such as keratitis and retinal degeneration. However, the poor water solubility and low bioavailability of baicalein make it difficult for its conventional formulations to exert their biological activity through traditional ocular administration.

[0004] Currently, baicalin-related preparations available in China include gastrointestinal medications such as baicalin aluminum capsules, compound baicalin tablets, Shuanghuanglian granules / soft capsules, and Qingkailing tablets / soft capsules, as well as traditional Chinese medicine formulas like Scutellaria baicalensis and Pueraria lobata capsules; liquid oral medications such as Shuanghuanglian oral liquid, Shuanghuanglian compound preparation, and Qingkailing oral liquid; and ophthalmic medications such as bear bile baicalin eye drops. It is evident that most preparations containing baicalin are currently compound traditional Chinese medicine formulations. Due to the presence of many unknown components in these compound preparations, their pharmacological effects are often unclear, and they are prone to causing various unpredictable adverse reactions.

[0005] The eyeball is a highly enclosed organ, making it difficult to deliver medications into the eye, especially to retinal lesions. Currently, large-molecule drugs for retinal diseases are generally administered by injection, which requires invasive intravitreal injection to deliver the drug to the posterior segment of the eye. Frequent and prolonged injections not only create psychological stress for both patients and doctors but also cause trauma to the eye, thus affecting treatment effectiveness.

[0006] The drug delivery logic of small-molecule eye drops differs from that of invasive injections. For ophthalmic diseases, eye drops are easily accepted by patients due to their convenience, non-invasiveness, and low cost; therefore, intraconjunctival administration of eye drops remains the most common method of treatment. However, traditional eye drops have extremely low ocular bioavailability (less than 5%).

[0007] Therefore, in order to increase ocular absorption of the drug, prolong its residence time in the eye, improve bioavailability, and obtain better local therapeutic effects, there is an urgent need to provide a novel baicalein formulation that increases corneal permeability and bioavailability, reduces the frequency of drug use, and minimizes ocular irritation. Summary of the Invention

[0008] The purpose of this invention is to overcome the above-mentioned defects in the prior art and provide a traditional Chinese medicine microemulsion eye drop with high corneal permeability and bioavailability, low eye irritation, and improved efficacy, as well as its preparation method and application.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

[0010] A microemulsion eye drop containing traditional Chinese medicine comprises the following components by weight percentage: 0.001%–2.0% baicalein, 0.001%–2.0% borneol, 0.1%–20% oil for injection, 0.1%–10% surfactant, 0.1%–10% phospholipid, 0.1%–5% osmotic pressure regulator, with the balance being water.

[0011] This invention also discloses a method for preparing the above-mentioned traditional Chinese medicine eye drops, comprising the following steps:

[0012] (1) Mix the injection oil, phospholipids, baicalin and borneol to obtain the oil phase;

[0013] (2) Mix the osmotic pressure regulator, surfactant and water to obtain an aqueous phase;

[0014] (3) At room temperature, the oil phase is added to the aqueous phase and mixed to obtain a primary emulsion;

[0015] (4) The colostrum is mixed with water and then subjected to high-pressure homogenization and filtration sterilization to obtain the Chinese medicine microemulsion eye drops.

[0016] The present invention also discloses the application of the above-described traditional Chinese medicine microemulsion eye drops, or the traditional Chinese medicine microemulsion eye drops prepared by the above-described preparation method, in the preparation of drugs for treating eye diseases or for relieving eye fatigue.

[0017] Implementing the embodiments of the present invention will have the following beneficial effects:

[0018] (1) The traditional Chinese medicine microemulsion eye drops provided by the present invention are prepared by using injection oil, phospholipids, baicalein and borneol as oil phase and water phase (osmotic pressure regulator, surfactant and water) to obtain an oil-in-water emulsion containing borneol and baicalein. By appropriately combining borneol and baicalein and controlling the preparation process conditions, a synergistic effect is achieved, which increases the chance of the drug entering the cornea and the site of ocular inflammation, realizes the function of rapid drug entry into ocular tissue and enhances the therapeutic effect, and significantly prolongs the retention time in the eye. It can also increase the corneal permeability of the drug and treat intraocular or posterior segment diseases by eye drops, so as to make up for the shortcomings of existing eye drops.

[0019] (2) This invention loads borneol and baicalein into a microemulsion and utilizes the "guiding upward" effect of borneol to promote drug penetration through the cornea and increase the distribution of the drug in the eye tissue. From the perspective of baicalein's antioxidant stress and anti-inflammatory effects, it can achieve the effects of clearing reactive oxygen species in the posterior ocular tissue and reducing inflammation.

[0020] In summary, this invention, by combining baicalein with borneol, allows baicalein to reach the posterior segment of the eye. Furthermore, by screening the ratio of the two ingredients, it not only reduces the irritation of borneol to the eye but also effectively solves the problem of baicalein's poor water solubility, improving the bioavailability of the active pharmaceutical ingredient and achieving excellent synergistic effects. This enables non-invasive treatment of intraocular or posterior segment eye diseases, especially for early and mid-stage AMD patients and various types of eye fatigue. Attached Figure Description

[0021] Figure 1 These are schematic diagrams of the formulation appearance of Examples 1 and 2.

[0022] Figure 2 This is a schematic diagram of the particle size measurement results for Example 1.

[0023] Figure 3 This is a schematic diagram of the in vitro release in Examples 1 and 2.

[0024] Figure 4 The images show the irritation effects of Examples 1, 2, and saline solution.

[0025] Figure 5 These are images of irritant pathological tissue sections from Examples 1 and 2, and from physiological saline.

[0026] Figure 6 The figures are the drug distribution curves in rabbit eye tissue for Examples 1 and 2.

[0027] Figure 7 The area under the curve of drug distribution in rabbit eye tissue in Examples 1 and 2 are shown.

[0028] Figure 8 These are pharmacodynamic pathological tissue sections of mice from Examples 1 and 2, the commercially available formulation, the normal group, and the model group.

[0029] Figure 9 The graph shows the apoptosis of mice in Example 1, Example 2, commercially available formulation, normal group, and model group.

[0030] Figure 10 This is a quantitative graph showing the apoptosis status of mice in Example 1, Example 2, commercially available formulation, normal group, and model group.

[0031] Figure 11 ROS staining images of mice in Examples 1 and 2, commercially available formulations, normal group, and model group.

[0032] Figure 12 The images show quantitative ROS staining fluorescence intensity of mice in Examples 1 and 2, the commercially available formulation, the normal group, and the model group.

[0033] Figure 13 The graph shows the detection results of the inflammatory factor IL-6 in mice of Example 1, Example 2, commercially available formulation, normal group and model group.

[0034] Figure 14 The graph shows the detection results of the inflammatory factor MCP-1 in mice of Example 1, Example 2, commercially available formulation, normal group and model group.

[0035] Figure 15 The graph shows the detection results of the inflammatory factor TNF-α in mice of Example 1, Example 2, commercially available formulation, normal group and model group. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0037] This invention discloses a traditional Chinese medicine microemulsion eye drop, comprising the following components by weight percentage: 0.001%–2.0% baicalein, 0.001%–2.0% borneol, 0.1%–20% oil for injection, 0.1%–10% surfactant, 0.1%–10% phospholipid, 0.1%–5% osmotic pressure regulator, with the balance being water.

[0038] Specifically, the herbal microemulsion eye drops disclosed in this invention exhibit a synergistic effect after proper formulation, wherein:

[0039] Borneol, a traditional Chinese medicine, is a crystalline substance extracted from the resin and volatile oil of borneol. First recorded in the *Mingyi Bielu* (Records of Famous Physicians), it is frequently used as an adjuvant or guiding drug in formulations and is also extensively documented in ancient Chinese ophthalmological medical texts. Borneol's guiding effect manifests in altering the direction or site of action of other drugs, or in emphasizing or concentrating their effects in specific directions and sites. For example, in preparations such as Compound Danshen Dripping Pills, Ma Yinglong Hemorrhoid Suppositories, and Quick-Acting Heart-Saving Pills, borneol is used as an adjuvant, playing a guiding and permeation-promoting role in the formulation. Furthermore, the inventors have discovered that combining borneol with baicalein enhances the effect of baicalein on improving endothelial dysfunction after cerebral ischemia, indicating borneol's guiding and permeation-promoting effect. Meanwhile, the inventors also discovered that its "drug-guiding upward" function can act on the "blood-eye barrier". For example, borneol can promote the entry of drugs such as fraxin, guanethidine, and puerarin into the eye. This may be related to the fact that borneol makes the movement of the phospholipid bilayer of the corneal epithelial cell membrane more orderly, increasing corneal permeability and providing a method for ocular drug delivery.

[0040] Therefore, based on these insights, the inventors have developed a method to co-load borneol and baicalein into a microemulsion, allowing baicalein to enter the posterior segment of the eye through combined administration. By screening the ratio of the two, the irritation of borneol to the eye is reduced, and the problem of baicalein's poor water solubility is effectively solved, ultimately achieving non-invasive treatment of posterior segment eye diseases.

[0041] Furthermore, to increase ocular absorption, prolong intraocular retention time, improve bioavailability, and achieve better local therapeutic effects, the microemulsion eye drops provided by this invention consist of nano-sized and uniformly distributed droplets. These droplets dissolve the drug and allow it to be absorbed into the body, reducing in vitro and in vivo degradation. Compared to aqueous solutions, the solubility is improved, increasing ocular retention without increasing foreign body sensation, thus enhancing bioavailability. Simultaneously, this invention uses phospholipids to replace some surfactants, increasing bypass transport and promoting drug absorption.

[0042] By further optimizing and limiting the weight ratio of each component in the herbal microemulsion eye drops of the present invention, the dosage and efficacy of each component are specifically adjusted and controlled, thereby improving the synergistic effect between the components.

[0043] In one specific embodiment, the herbal microemulsion eye drops comprise the following components by weight percentage: 0.01%–1.0% baicalein, 0.01%–1.0% borneol, 2%–15% oil for injection, 0.1%–10% surfactant, 0.2%–1.5% phospholipid, 0.5%–3% osmotic pressure regulator, with the balance being water.

[0044] In one specific embodiment, the mass ratio of baicalein to borneol is 10:1 to 1:1. Preferably, the mass ratio is 5:1 to 1:1. More preferably, the mass ratio is 5:3.

[0045] In one specific embodiment, the mass ratio of baicalein to injectable oil is 1:20-1:200; preferably, the mass ratio is 1:50-1:200.

[0046] In one specific embodiment, the mass ratio of injectable oil to phospholipid is 15:1 to 1:3.

[0047] In one specific embodiment, the injectable oil includes one or more of MCT, LCT, and structured triglycerides. Preferably, the injectable oil includes one or two of MCT and LCT.

[0048] In one specific embodiment, the osmotic pressure regulator includes one or more of glycerol, sucrose, trehalose, xylitol, and sodium chloride.

[0049] In one specific embodiment, the surfactant includes one or more of Tween-80, Solutol HS-15, and poloxamer.

[0050] In one specific embodiment, the surfactant includes one or more of the following: 0.3% to 1.2% Tween-80, 0.2% to 0.9% Solutol HS-15, and 0.5% to 1.6% Poloxamer 188.

[0051] In one specific embodiment, the phospholipid includes one or both of glycerophospholipids and sphingomyelins.

[0052] In one specific embodiment, the phospholipid includes one or more of natural phospholipids, semi-synthetic phospholipids, and fully synthetic phospholipids.

[0053] In one specific embodiment, the natural phospholipids include one or more of egg yolk phospholipids, soybean phospholipids, EPG, ESM, phosphatidylinositol, and cardiac phospholipids.

[0054] In one specific embodiment, the semi-synthetic phospholipid includes hydrogenated soybean phospholipid (HSPC).

[0055] In one specific embodiment, the fully synthetic phospholipid includes one or more of phosphatidylcholine, phosphatidylglycerol, myocardial phospholipid, and distearate phosphatidylethanolamine (DSPE).

[0056] In one specific embodiment, phosphatidylcholine includes one or more of DOPC, DSPC, DPPC, DMPC, and DLPC.

[0057] In one specific embodiment, phosphatidylglycerol includes one or more of DOPG, DSPG, DPPG, DMPG, and DLPG.

[0058] In one specific embodiment, the phospholipid preferably includes one or more of egg yolk phospholipid E80 (0.3%–0.7%), soybean phospholipid PC70 (0.6%–1.2%), and soybean phospholipid PC50 (0.6%–1.2%).

[0059] In one specific embodiment, the herbal microemulsion eye drops also include 0.001% to 1% by weight of an antioxidant to improve the stability of the system.

[0060] In one specific embodiment, the antioxidant includes one or more of sodium bisulfite, sodium sulfite, sodium thiosulfate, ascorbic acid, citric acid, and disodium EDTA.

[0061] In one specific embodiment, the average droplet size in the herbal microemulsion eye drops is <200nm.

[0062] In one specific embodiment, the pH value of the herbal microemulsion eye drops is 5-9.

[0063] This invention also discloses a method for preparing the above-mentioned traditional Chinese medicine eye drops, comprising the following steps:

[0064] (1) Mix the injection oil, phospholipids, baicalin and borneol to obtain the oil phase.

[0065] (2) Mix the osmotic pressure regulator, surfactant and water to obtain an aqueous phase.

[0066] (3) At room temperature, the oil phase is added to the aqueous phase and mixed to obtain the primary emulsion.

[0067] (4) After mixing the colostrum with water, the mixture is subjected to high-pressure homogenization and filtration sterilization to obtain the Chinese medicine microemulsion eye drops.

[0068] In one specific embodiment, step (1) specifically includes: dissolving baicalin and borneol in ethanol, adding injectable oil and phospholipids to the resulting mixed solution, and stirring at room temperature for 10 minutes until a clear and transparent liquid is obtained to obtain the oil phase.

[0069] In one specific embodiment, step (2) specifically includes: stirring the osmotic pressure regulator, surfactant and about 10% of the prescribed amount of purified water in a water bath at 40°C to 60°C until homogeneous to obtain an aqueous phase.

[0070] In one specific embodiment, step (3) includes mixing: adding the oil phase to the aqueous phase and shearing for 5 min to 7 min at 15000 rpm to 20000 rpm.

[0071] In one specific embodiment, in step (4), the pressure of high-pressure homogenization is 800 bar to 1000 bar; the number of high-pressure homogenization cycles is 15 to 25.

[0072] The present invention also discloses the application of the above-described traditional Chinese medicine microemulsion eye drops, or the traditional Chinese medicine microemulsion eye drops prepared by the above-described preparation method, in the preparation of drugs for treating eye diseases or for relieving eye fatigue.

[0073] In one specific embodiment, the drug for treating eye diseases is a drug for treating age-related macular degeneration.

[0074] In one specific embodiment, the medication for relieving eye fatigue is a medication for preventing dry age-related macular degeneration.

[0075] In one specific embodiment, the drug for treating eye diseases or relieving eye fatigue is a drug that alleviates oxidative damage to cells or inhibits inflammatory responses in cells.

[0076] In one specific embodiment, the solution can be diluted to a certain concentration to form a hypotonic or isotonic form, and then administered via topical titration to the eye.

[0077] The following are specific embodiments.

[0078] The reagents used in the following examples are all commercially available products. Unless otherwise specified, standard conditions were followed in the examples.

[0079] Example 1:

[0080] A microemulsion eye drop containing traditional Chinese medicine comprises the following components in weight percentage: baicalein 0.05%; borneol 0.03%; vegetable oil MCT 5%; egg yolk lecithin E80 0.6%; surfactant Tween-80 0.9%; osmotic pressure regulator glycerin 2.2%; and antioxidant sodium sulfite 0.01%.

[0081] The preparation method includes the following steps:

[0082] (1) Take MCT, egg yolk lecithin E80, add baicalein and borneol completely dissolved in 10 mL of ethanol, stir at room temperature until a clear and transparent liquid is obtained; remove ethanol by rotary evaporation under reduced pressure at 45 °C to obtain a homogeneous oil phase.

[0083] (2) Take glycerin for injection, add Tween-80, add about 80% purified water, and stir at 60°C until dissolved to form the aqueous phase.

[0084] (3) The organic phase was added dropwise to the aqueous phase under shearing conditions of 3000 rpm, and the rotation speed was adjusted to 16000 rpm. After shearing for 5 min, the pre-emulsion was obtained. The pre-emulsion was transferred to a graduated cylinder and brought to a final volume of 100 mL. The pre-emulsion was then transferred to a high-pressure homogenizer and homogenized at a pressure of 800 bar. After passing through a 0.22 μm filter, the baicalin microemulsion was obtained.

[0085] Example 2:

[0086] A traditional Chinese medicine microemulsion eye drop comprises the following components in weight percentage: baicalein 0.05%; borneol 0.03%; vegetable oil MCT 10%; egg yolk lecithin E80 0.6%; surfactant Tween-80 0.9%; osmotic pressure regulator glycerin 2.2%; and antioxidant sodium sulfite 0.01%.

[0087] The preparation method in this embodiment is the same as that in Example 1.

[0088] Example 3:

[0089] A microemulsion eye drop containing traditional Chinese medicine comprises the following components in weight percentage: baicalein 0.05%; borneol 0.03%; vegetable oil MCT 5%; soybean lecithin S100 0.6%; surfactant Tween-80 0.9%; osmotic pressure regulator glycerin 2.2%; and antioxidant sodium sulfite 0.01%.

[0090] The preparation method in this embodiment is the same as that in Example 1.

[0091] Example 4:

[0092] A microemulsion eye drop containing traditional Chinese medicine comprises the following components in weight percentage: baicalein 0.05%; borneol 0.03%; vegetable oil MCT 5%; soybean lecithin PC50 0.6%; surfactant Tween-80 0.9%; osmotic pressure regulator glycerin 2.2%; and antioxidant sodium sulfite 0.01%.

[0093] The preparation method in this embodiment is the same as that in Example 1.

[0094] Example 5:

[0095] A microemulsion eye drop containing traditional Chinese medicine comprises the following components in weight percentage: baicalein 0.05%; borneol 0.03%; vegetable oil MCT 5%; egg yolk lecithin E80 0.6%; surfactant HS-15 0.9%; osmotic pressure regulator glycerin 2.2%; and antioxidant sodium sulfite 0.01%.

[0096] The preparation method in this embodiment is the same as that in Example 1.

[0097] Example 6:

[0098] A traditional Chinese medicine microemulsion eye drop comprises the following components in weight percentage: baicalein 0.05%; borneol 0.03%; vegetable oil MCT 5%; egg yolk lecithin E80 0.6%; surfactant Tween-80 1.8%; osmotic pressure regulator glycerin 2.2%; and antioxidant sodium sulfite 0.01%.

[0099] The preparation method in this embodiment is the same as that in Example 1.

[0100] Example 7:

[0101] A traditional Chinese medicine microemulsion eye drop comprises the following components in weight percentage: baicalein 0.05%; borneol 0.01%; vegetable oil MCT 5%; egg yolk lecithin E80 0.6%; surfactant Tween-80 0.9%; osmotic pressure regulator glycerin 2.2%; and antioxidant sodium sulfite 0.01%.

[0102] The preparation method in this embodiment is the same as that in Example 1.

[0103] Example 8:

[0104] A traditional Chinese medicine microemulsion eye drop comprises the following components in weight percentage: baicalein 0.05%; borneol 0.05%; vegetable oil MCT 5%; egg yolk lecithin E80 0.6%; surfactant Tween-80 0.9%; osmotic pressure regulator glycerin 2.2%; and antioxidant sodium sulfite 0.01%.

[0105] The preparation method in this embodiment is the same as that in Example 1.

[0106] Comparative Example 1:

[0107] A baicalin microemulsion eye drop comprises the following components in weight percentage: baicalin 0.05%; vegetable oil MCT 5%; egg yolk lecithin E80 0.6%; surfactant Tween-80 0.9%; osmotic pressure regulator glycerin 2.2%; and antioxidant sodium sulfite 0.01%.

[0108] The preparation method in this embodiment is the same as that in Example 1.

[0109] Comparative Example 2:

[0110] The only difference between this comparative example and Example 1 is that the preparation method of the oil phase is different.

[0111] Specifically: Take MCT, egg yolk lecithin E80, add baicalein and borneol, and stir at room temperature for 30 minutes; the aqueous phase preparation method is as follows: take injectable glycerol, add Tween-80, add sodium sulfite, add about 80% purified water, and stir at 60°C until dissolved to obtain the aqueous phase; the primary emulsion: add the organic phase dropwise to the aqueous phase under shearing conditions of 3000 rpm, adjust the speed to 16000 rpm, and shear for 5 minutes to obtain the primary emulsion; the final emulsion: bring the primary emulsion to a final volume of 100 mL, transfer it to a high-pressure homogenizer, and homogenize it under a pressure of 800 bar to obtain the final emulsion; fill the solution to obtain baicalein microemulsion eye drops containing borneol.

[0112] Commercially available formulations:

[0113] The main active ingredients of Aesculin-Digital Glycoside Eye Drops are digitoxin and aesculin. They improve microcirculation in the macular region of the retina, enhance the delivery of nutrients to the retina, restore damaged nerve cells, and clear metabolic waste products. The two ingredients work together to prevent damage and atrophy of the retina. Given that there are currently no drugs with significant efficacy for dry age-related macular degeneration, this product is marketed as an interventional formulation.

[0114] Application Example 1: Detection of average particle size and particle size distribution (PDI) of baicalin microemulsion eye drops in Examples 1-8 and Comparative Examples 1-2

[0115] Table 1: Average particle size of baicalein microemulsion eye drops

[0116]

[0117] Application Example 2: Stability of Baicalein Microemulsion Eye Drops obtained from Examples 1-8 and Comparative Examples 1-2

[0118] The eye drops prepared in Examples 1-8 and Comparative Examples 1-2 were placed in an environment with a temperature of 60℃±2℃ and a relative humidity of 75±5% for 10 days. The appearance of the samples was observed and the average particle size of the emulsion was measured. The test results are shown in Table 2 below:

[0119] Table 2: Stability Test Results

[0120]

[0121] Application Example 3: In vitro release study

[0122] The formulations of Example 1 and Comparative Example 1 were investigated. The release medium used in this experiment was artificial tears containing 0.5% SDS and 0.2% ascorbic acid.

[0123] The in vitro release characteristics of baicalin microemulsion were determined using a transdermal diffusion analyzer.

[0124] The specific experimental steps were as follows: A semi-permeable membrane was fixed between the supply and receiving cells. A magnetic stir bar was placed in the receiving cell. 0.5 mL of baicalin microemulsion eye drops was placed in the supply cell, and 7.5 mL of [unspecified ingredient] was added to the receiving cell. The diffusion cell was placed in a transdermal diffusion apparatus, with the temperature set at 34℃ and the rotation speed at 300 rpm. Samples were taken at 0.5, 1, 2, 3, 4, 6, 8, and 12 hours. The release medium in the receiving cell was removed, and 7.5 mL of fresh release medium was added. The baicalin content in the release medium was determined by HPLC, and the in vitro release of baicalin was calculated and evaluated. Each sample was tested in triplicate.

[0125] Cumulative release (Q) n The percentage of release (F%) and the cumulative release percentage (F%) are obtained from formulas (1) and (2), respectively:

[0126]

[0127]

[0128] In the formula Q n C0 represents the cumulative release at each time point; C0 represents the measured drug concentration at time point 0; V0 represents the sampling volume at time point 0; n represents the number of samplings; C i V represents the measured drug concentration at the i-th time point; i The sampling volume at time point i; F% is the cumulative release percentage at each time point; C 总 This represents the total drug concentration.

[0129] The in vitro release curves of baicalein solution and baicalein microemulsion eye drops containing borneol were obtained by fitting the cumulative drug release percentage (F%) to the sampling time. Figure 3 .

[0130] In vitro drug release curves were plotted based on the cumulative release amount and percentage at each time point, and fitted using zero-order, first-order, Higuchi, and Ritger-Peppas equations, respectively. The in vitro release results of the baicalin microemulsion eye drops containing borneol are shown in Table 1.

[0131] Table 3. In vitro release fitting results of baicalin microemulsion eye drops containing borneol (a traditional Chinese medicine).

[0132]

[0133] The release data from Example 1 and Comparative Example 1 were fitted with zero-order, first-order, and Higuchi diffusion equations and regression analysis was performed. The correlation coefficients of each regression equation were compared. The in vitro release fitting results showed that the in vitro release of both examples conformed well to the first-order equation.

[0134] Application Example 4: In vitro safety study

[0135] The hemolysis test was performed on the samples from Examples 1, 8, 9, and Comparative Example 1. As an alternative to the Draize test, the hemolysis test uses the amount of hemoglobin released after red blood cell rupture as an indicator to evaluate the damage effect on the red blood cell membrane. It has the advantages of high sensitivity and simple operation, and high reliability. It is more sensitive than the Draize test and can be used to predict eye irritation.

[0136] The specific experimental steps are as follows:

[0137] (1) Using a disposable syringe, draw approximately 10 mL of blood from the middle ear artery of a New Zealand white rabbit. Immediately stir with a sterilized glass rod to remove fibrinogen from the blood. Add physiological saline and mix well. Centrifuge at 2000 rpm for 10 min at 4°C. Aspirate the supernatant. Under the same conditions, add 0.9% physiological saline twice, 5 min each time, until the supernatant is no longer red. Measure the red blood cells and prepare a 2% red blood cell suspension with 0.9% physiological saline. Maintain the operation at 4°C.

[0138] (2) Mix the red blood cell suspension with that from Example 1 at a 1:1 ratio in a test tube, incubate at 32°C for 30 min, terminate the reaction on ice, centrifuge at 800 rpm for 10 min at 4°C, collect the supernatant, use 0.9% physiological saline as a negative control and distilled water as a complete hemolysis control, and repeat the above operation. Transfer 200 μL of the supernatant to a 96-well microplate, and measure the absorbance at 540 nm and 575 nm using a microplate reader to calculate the hemolysis percentage (HR).

[0139]

[0140] Examples 8, 9, and Comparative Example 1 were repeated in place of Example 1, and the hemolysis percentage results are shown in Table 4.

[0141] Table 4: Results of Hemolysis Rate Experiment

[0142]

[0143] Experimental results showed that the hemolysis rate was close to 100% when the borneol concentration was 0.5 mg / mL. However, some studies have shown that 1 mg / mL borneol eye drops are almost non-irritating. This may be because the Draize experiment is based on whole animals and is influenced by many factors. Considering that the hemolysis experiment is more sensitive, the concentration of borneol used was controlled below 0.5 mg / mL.

[0144] Application Example 5: In vivo safety study

[0145] Safety studies were conducted on the formulations of Example 1 and Comparative Example 1. The safety of the baicalin microemulsion eye drops containing borneol was investigated using the Draize eye irritation test and pathological examination.

[0146] The specific experimental steps are as follows:

[0147] (1) Draize Eye Irritation Test: 50 μL of the baicalein microemulsion eye drops containing borneol prepared in Example 1 was instilled into the right eye of each rabbit, while physiological saline was instilled into the left eye for comparison. After administration, the rabbits' eyes were passively closed for 8-10 seconds. Administration was repeated four times daily, with a 4-hour interval between each administration, for seven consecutive days. The condition of the eyes was observed before each administration and at 4, 12, and 24 hours after the last administration, focusing on the reactions of the cornea, iris, and conjunctiva. Simultaneously, after the last administration each day, 2% sodium fluorescein was instilled onto the corneal surface, and the rabbits were manually made to blink, ensuring the sodium fluorescein was evenly dispersed on the ocular surface. Ocular damage was detected using a slit-lamp cobalt blue light. If ocular surface damage was present, green fluorescence would appear at the cornea under cobalt blue light. If no damage was present, no green fluorescence would be observed in the eye.

[0148] (2) Pathological examination: After the eye irritation study was completed, all rabbits were euthanized by intravenous injection of air. Then, the rabbit eyeballs were immediately removed and placed in an eye fixation solution. For histopathological analysis, paraffin sections of each eye tissue were prepared, stained with hematoxylin and eosin (H&E), and the cornea, sclera, iris, and conjunctiva were observed under an optical microscope for inflammatory cell infiltration, hemorrhage, necrosis, etc.

[0149] (3) In the Draize eye irritation test, the baicalin microemulsion eye drops containing borneol (a traditional Chinese medicine) showed no irritation to the cornea, iris, or conjunctiva. Observational images of each eyeball and histopathological sections of each tissue are shown below. Figure 4 , Figure 5 As shown, after 7 days of continuous administration, there was no significant difference between the baicalin microemulsion eye drop preparation group containing borneol and the control group, and no obvious inflammatory reaction was observed, indicating that the preparation has low ocular irritation. The results further confirm the safety of the baicalin microemulsion eye drop preparation containing borneol.

[0150] Comparative Example 1 was used to repeat the above experimental steps instead of Example 1, and the irritation results were observed. The results showed that Comparative Example 1 had no effect on corneal structure and integrity.

[0151] Application Example 6: Investigation of Rabbit Eye Tissue Distribution

[0152] The distribution of the formulations from Example 1 and Comparative Example 1 in rabbit eye tissue was investigated. The content of baicalin in each tissue was determined by HPLC-MS / MS.

[0153] The specific implementation steps are as follows:

[0154] (1) Take 0.1 mL of the preparation prepared in Example 1 and drip it into the conjunctival sac of a rabbit to force the rabbit's eyelids to close for 15 seconds. The eyeballs were removed 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after administration. The surface of the eyeball was repeatedly rinsed with physiological saline solution. The eyeball was dissected and the cornea, sclera, vitreous body and lens were separated. The eyeballs were then dried with filter paper and set aside for later use.

[0155] (2) Weigh each of the separated tissues precisely, add 5 mL of acetonitrile, homogenize, centrifuge at 4000 rpm for 10 min, take the supernatant, blow dry under nitrogen flow, dissolve the residue in 500 μL of methanol, vortex for 5 min, centrifuge at 12000 rpm (4℃) for 10 min, and inject 10 μL of the supernatant.

[0156] (3) Create a curve graph of the drug and calculate the area under the curve.

[0157] (4) The baicalein microemulsion eye drop formulation of Comparative Example 1 was used to replace the baicalein microemulsion eye drop formulation containing borneol in this embodiment, and the tissue distribution was investigated.

[0158] The tissue distribution of different formulations in rabbit eyes over time is as follows: Figure 6 The area under the pharmacokinetic curve (AUC) of the two formulations in different tissues is as follows: Figure 7 The drug-time curve results showed that the drug concentration in all tissues was significantly increased in Example 1, with significant differences between groups. Specifically, the AUC of the cornea, conjunctiva, sclera, iris, and vitreous humor was significantly higher. 0-24 The AUCs of the aqueous humor, lens, and retina were 2.35, 2.80, 4.17, 2.9, and 5.65 times that of Comparative Example 1, respectively. 0-24 The concentration increased by more than 10 times, and the difference was statistically significant (P < 0.05). The experimental results demonstrate that the addition of borneol can effectively increase the concentration of baicalin in the eye.

[0159] Application Example 7: Pharmacodynamic Study

[0160] Pharmacodynamic studies were conducted on Example 1, Comparative Example 1, and the commercially available formulation. The efficacy of the formulation was assessed by comparing it with pathological tissue sections, retinal cell apoptosis, retinal reactive oxygen species levels, and related inflammatory factor levels in the normal group and the model group.

[0161] The specific implementation steps are as follows:

[0162] (1) Forty male C57BL / 6 mice were used. After one week of adaptive culture, 32 mice were selected to establish a dry AMD model (retinal damage model was established by tail vein injection of NaIO3). After modeling, they were randomly divided into 4 groups of 8 mice each: the model group, Example 1, Comparative Example 1, and the commercially available formulation group. Mice in the normal group were injected with physiological saline.

[0163] (2) The drug was administered one day after the model was established. The mouse eyeballs were taken 14 and 28 days after drug administration. Paraffin sections of each eye tissue were prepared, and hematoxylin-eosin (H&E) staining was performed for histopathological analysis. DAPI staining and TUNEL staining were performed to analyze the apoptosis of retinal cells. Frozen sections were embedded, and DHE fluorescent probe and DAPI staining were performed to detect the level of reactive oxygen species in the retina. The level of inflammatory factors in the tissue was measured after homogenization of the eyeballs to observe the therapeutic effect.

[0164] (3) Histopathological section results are shown in Figure 8 The results showed that 14 days after administration, the cells in all layers of the retina of the normal group mice had good morphology, intact structure, and neat and uniform cell arrangement and density. In the model group mice, the outer retinal layer (OPL) had collapsed, and the outer nuclear layer (ONL, yellow arrow) showed disorder, thinning, decreased cell density, and wavy changes. Disordered thinning of the photoreceptor (PR) layer and significant thinning of the entire retina were observed, along with RPE structural atrophy and pigmentation (blue arrows). The damage worsened on day 28. Comparative Example 1 showed thinning of all layers of the retina, decreased cell density, and wavy changes, but still differed significantly from the model group. Comparative Example 1 showed similarities to the model group, with decreased cell density, but still showed significant differences, indicating that baicalin can protect the retina. Compared to Comparative Example 1, Example 1 and the commercially available formulation showed relatively mild damage; the RPE layer showed plaques and a decrease in cell count rather than disappearance, and the degree of disorder and cell reduction in the ONL and PR was significantly milder, with relatively neat cell arrangement. These results indicate that the formulation in Example 1 protected the structure and morphology of the retina. After 28 days of treatment, the retinal thickness in Example 1 was greater than that in the commercially available formulation and Comparative Example 1, suggesting that the presence of baicalin and borneol in the formulation enhanced its therapeutic effect.

[0165] The results of TUNEL retinal apoptosis are shown in […]. Figure 9After 14 and 28 days of treatment, the TUNEL assay kit was used to detect apoptosis in the retina of model mice. Apoptotic cells were marked in red, while normal cells were marked in blue. The percentage of TUNEL-stained cells determined the apoptosis level in the tissue. No apoptotic cells were present in the retinal sections of normal mice, resulting in no obvious red fluorescence. However, TUNEL+ (apoptotic) cells were detected to varying degrees in the INL and ONL of other groups. To quantify the amount of apoptosis, apoptotic cells and total cells in each group were counted using ImageJ software, and the apoptosis rate was calculated (see [link to image]). Figure 10 There were statistically significant differences between the normal group and all other groups (p<0.5). The model group had the highest number of labeled cells, suggesting that sodium iodate induces apoptosis in the retinal region of mice, and the number of apoptotic cells increases over time. The apoptosis rate in the retina of mice in Example 1, the commercially available formulation, and Comparative Example 1 was lower than that in the model group, and the number of apoptotic cells in Example 1 and the commercially available formulation was significantly lower than that in Comparative Example 1 and the model group, with statistically significant differences (p<0.01). This indicates that the formulation in Example 1 can successfully enter the fundus and inhibit sodium iodate-induced apoptosis, which is consistent with the results of histopathological sections.

[0166] Results of retinal reactive oxygen species levels are shown in Figure 11 The results of quantifying fluorescence intensity are shown in [the table]. Figure 12 The results showed that the reactive oxygen species (ROS) level in the model group was significantly higher than that in the other groups. It should be noted that ROS was also detected in the normal control group, as the body produces a certain amount of ROS even under healthy conditions. The experimental results showed that the fluorescence intensity of Example 1, the commercially available formulation, and Comparative Example 1 was significantly lower than that of the model group, proving that baicalin, similar to the commercially available formulation, can quench reactive oxygen species in the eye and clear ROS from the retina. Simultaneously, the fluorescence intensity of Example 1 was lower than that of Comparative Example 1, proving that borneol can enhance the anti-ROS effect of the formulation. There was no significant difference between Example 1 and the commercially available formulation, but Example 1 showed a smaller relative deviation and better precision. The ROS results were consistent with the HE staining and TUNEL results.

[0167] The results of the relevant inflammatory factor level test are shown in Figure 13 , Figure 14 , Figure 15Compared with the normal group, the levels of IL-6, MCP-1, and TNF-α inflammatory factors in the eyeballs of mice in the model group were significantly increased (P<0.0001). Compared with the model group, the levels of the three inflammatory factors decreased to varying degrees after treatment with different eye drop formulations, and the differences were statistically significant. After eye drops in Example 1, the levels of the three inflammatory factors were significantly reduced (P<0.001). However, after eye drops in Comparative Example 1, the levels of inflammatory factors IL-6 and TNF-α did not decrease significantly, which was significantly different from that in Example 1 (P<0.05). The results show that the eye drops in Example 1 can effectively reduce the levels of intraocular inflammatory factors IL-6, MCP-1, and TNF-α, demonstrating a good anti-inflammatory effect. In addition, the commercially available formulation did not significantly reduce the levels of inflammatory factors IL-6 and TNF-α, similar to Comparative Example 1, and both were significantly different from those in Example 1 (P<0.05). This is consistent with the above pharmacodynamic experiment results.

[0168] In summary, the baicalein microemulsion eye drops containing borneol prepared in this invention can increase the corneal permeability and bioavailability of baicalein, improve the therapeutic effect, and reduce the frequency of drug use. It is expected to provide a new approach for the treatment and prevention of AMD as a potential therapeutic drug.

[0169] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of a traditional Chinese medicine microemulsion eye drop in the preparation of drugs for treating eye diseases or for relieving eye fatigue, characterized in that, The medication for treating eye diseases is a medication for treating age-related macular degeneration; the medication for relieving eye fatigue is a medication for preventing dry age-related macular degeneration. The herbal microemulsion eye drops comprise the following components by weight percentage: 0.001%~2.0% baicalein, 0.001%~2.0% borneol, 0.1%~20% oil for injection, 0.1%~10% surfactant, 0.1%~10% phospholipid, 0.1%~5% osmotic pressure regulator, balance water; The mass ratio of baicalein to borneol is 10:1 to 1:1; The mass ratio of baicalin to the injection oil is 1:20-1:200; The average droplet size in the herbal microemulsion eye drops is <200nm; The preparation method of the traditional Chinese medicine eye drops includes the following steps: (1) Mix the injection oil, phospholipids, baicalin and borneol to obtain the oil phase; (2) Mix the osmotic pressure regulator, surfactant and water to obtain an aqueous phase; (3) At room temperature, the oil phase is added to the aqueous phase and mixed to obtain a primary emulsion; (4) The colostrum is mixed with water and then subjected to high-pressure homogenization and filtration sterilization to obtain the Chinese medicine microemulsion eye drops; Step (1) specifically includes: dissolving baicalein and borneol in ethanol, adding injection oil and phospholipids to the resulting mixed solution, stirring at room temperature for 10 minutes until a clear and transparent liquid is obtained, thus obtaining the oil phase.

2. The application according to claim 1, characterized in that, The herbal microemulsion eye drops comprise the following components by weight percentage: 0.01%~1.0% baicalein, 0.01%~1.0% borneol, 2%~15% oil for injection, 0.1%~10% surfactant, 0.2%~1.5% phospholipid, 0.5%~3% osmotic pressure regulator, with the balance being water.

3. The application according to claim 1 or 2, characterized in that, The mass ratio of the injectable oil to the phospholipid is 15:1-1:3; The injectable oil includes one or more of MCT, LCT and structured triglycerides; The osmotic pressure regulator includes one or more of glycerol, sucrose, trehalose, xylitol, and sodium chloride; The surfactant includes one or more of Tween-80, Solutol HS-15, and poloxamer; The phospholipids include one or both of glycerophospholipids and sphingomyelins; The phospholipids include one or more of the following: natural phospholipids, semi-synthetic phospholipids, and fully synthetic phospholipids; The natural phospholipids include one or more of egg yolk phospholipids, soybean phospholipids, EPG, ESM, phosphatidylinositol, and myocardial phospholipids; The semi-synthetic phospholipids include hydrogenated soybean phospholipids; The fully synthetic phospholipids include one or more of phosphatidylcholine, phosphatidylglycerol, myocardial phospholipids, and distearate phosphatidylethanolamine.

4. The application according to claim 1, characterized in that, The herbal microemulsion eye drops also include 0.001% to 1% antioxidant by weight; The antioxidants include one or more of sodium bisulfite, sodium sulfite, sodium thiosulfate, ascorbic acid, citric acid, and disodium ethylenediaminetetraacetate.

5. The application according to claim 1, characterized in that, The pH value of the herbal microemulsion eye drops is 5-9.

6. The application according to claim 1, characterized in that, In step (1), the mixing temperature is 20℃~25℃; In step (2), the mixing temperature is 40℃~60℃; In step (3), the mixing includes: adding the oil phase to the aqueous phase and shearing at 15,000 rpm to 20,000 rpm for 5 min to 7 min; In step (4), the pressure of the high-pressure homogenization is 800 bar to 1000 bar; the number of high-pressure homogenizations is 15 to 25 times.

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