Dihydroquercetin for the treatment of ocular surface diseases

Abstract

The present invention discloses the use of dihydroquercetin in the treatment of ocular surface diseases, which belongs to the field of pharmaceutical technology. The use of dihydroquercetin in the treatment of ocular surface diseases described in the present invention specifically refers to the use of dihydroquercetin in the manufacture of a medicament for the prevention, alleviation, and / or treatment of ocular surface diseases. The present invention is the first to report the use of dihydroquercetin in the prevention, alleviation, and / or treatment of ocular surface diseases. As an antioxidant, dihydroquercetin has particularly good protective effects against diseases caused by oxidative damage to corneal epithelial cells (HCE). Furthermore, within a certain concentration range, dihydroquercetin can promote the proliferation of HCE cells and improve cell viability, and further increase the tear secretion volume and tear film stability of HCE cells, thereby significantly improving ocular surface diseases.

Description

cross reference

[0001] This application claims priority based on Chinese patent application No. 202211536112.X, filed with the China Patent Office on December 2, 2022, entitled "Use of dihydroquercetin in treating ocular surface diseases," the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] The present invention relates to the pharmaceutical field, and in particular to the use of dihydroquercetin in the treatment of ocular surface diseases. [Background technology]

[0003] Oxidative damage is widespread in diseases, particularly in aging and inflammation-related diseases. Radicals generated by oxidation directly attack and destroy tissue cell membranes, and then enter cells through membrane pathways, damaging intracellular proteins and DNA. The involvement of oxygen free radicals in the metabolism of arachidonic acid is an important process during inflammatory responses. The resulting lipid peroxides are chemokines, which exacerbate inflammatory responses. Furthermore, oxidation products induce the production of chemokines distinct from arachidonic acid, inactivate protease inhibitors, and increase collagenase activity, thereby destroying connective tissue. Common eye diseases caused by oxidative damage include dry eye and corneal alkali burns.

[0004] Dry eye is a common ophthalmological condition caused by multiple factors, including insufficient tear production or excessive tear evaporation, which can affect tear stability and ocular surface inflammation. In recent years, domestic and international scholars have conducted extensive research into the pathogenesis of dry eye. The onset and development of this disease are related to the immune inflammatory response, apoptosis, and sex hormone levels, among which T cell-mediated immune inflammatory responses are considered to be the most crucial factor in the development of dry eye. Furthermore, the formation of hyperosmolarity in tears due to decreased tear secretion or increased evaporation is also a key factor in the vicious cycle of dry eye. Hyperosmolarity in tears induces morphological changes, such as apoptosis of conjunctival epithelial cells and a decrease in the number of functional goblet cells, which then triggers an inflammatory cascade and ultimately leads to the death of corneal epithelial cells. The loss of these functional cells leads to a decrease in the mucin and lipid content of tears, further exacerbating tear film instability, thus perpetuating the vicious cycle. This disease causes varying degrees of dryness of the eyes, foreign body sensation, photophobia, and pain, and in severe cases can cause corneal damage that threatens the patient's visual function, making research into clinical treatments for dry eye urgently needed.

[0005] Corneal alkali burns are a common ocular injury and are associated with a high rate of blindness. In the early stages of corneal alkali burns, activation of apoptosis and inflammatory responses contributes to corneal damage. In the later stages, corneal neovascularization significantly impacts corneal transparency and visual acuity. However, the formation of choroidal neovascularization (CNV) in the fundus is a complex pathological process regulated by multiple factors and precisely controlled by a growth factor network consisting of numerous cytokines. Vascular endothelial growth factor (VEGF) is the most potent angiogenic factor discovered to date, and its overexpression is closely associated with the development of CNV. Although numerous drugs are available to inhibit the mechanism of neovascularization, it is increasingly becoming clear that many of these drugs cause adverse reactions during treatment. For example, common side effects of glucocorticoids include Cushing's syndrome, infection, gastrointestinal reactions, edema, glucose and electrolyte metabolism disorders, and neurological disorders. Common side effects of anti-VEGF drugs include conjunctival injection, eye pain, foreign body sensation, corneal abrasion, corneal edema, increased intraocular pressure, black spots, intraocular infection, retinal detachment, or vitreous hemorrhage.

[0006] Therefore, research and development of novel antioxidants that have protective effects against diseases associated with ocular oxidative damage has become extremely important. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in consideration of the above circumstances, and aims to provide dihydroquercetin, which is used to treat ocular surface diseases and has a protective effect against oxidative stress damage to corneal epithelial cells (HCE) induced by H2O2. [Means for solving the problem]

[0008] To achieve the above objectives, the present invention adopts the following technical solutions.

[0009] The present invention provides the use of dihydroquercetin in the manufacture of a medicament for the prevention, alleviation and / or treatment of ocular surface disease.

[0010] Dihydroquercetin (DHQ), also known as taxifolin, is found in many plants, with relatively high concentrations in larch (especially Douglas fir). Dihydroquercetin was first isolated and extracted from the leaves of the coniferous plant Chamaecyparis isobtusa by a Japanese scientist in Fukui. In recent years, dihydroquercetin has also been found to be present in many fruits, such as grapes, mandarins, and grapefruit. Research has shown that dihydroquercetin contains many phenolic hydroxyl groups and possesses diverse biological activities, producing different physiological effects by inhibiting or activating various enzymes. The structural formula of dihydroquercetin is as follows:

[0011] [ka]

[0012] The present invention is the first to use dihydroquercetin to prevent, alleviate and / or treat ocular surface diseases. The ocular surface disease is preferably an ocular surface disease caused by corneal epithelial cell damage and / or apoptosis.

[0013] The corneal epithelial damage is preferably oxidative damage.

[0014] The oxidative damage is preferably damage caused by hydrogen peroxide.

[0015] The dihydroquercetin of the present invention has a protective effect against oxidative stress damage in corneal epithelial cells (HCE) induced by H2O2.

[0016] Specifically, the protective effect means that dihydroquercetin within a certain concentration range has a proliferation effect on HCE cells and can improve the relative cell survival rate of HCE cells after oxidative damage.

[0017] In this invention, a model of corneal epithelial cell (HCE) damage induced by H2O2 was constructed to determine the H2O2 modeling concentration, and modeling was performed at this concentration to cause oxidative damage to HCE cells. Finally, by applying different concentrations of dihydroquercetin to HCE cells, it was found that a certain concentration of dihydroquercetin has a proliferation effect on HCE cells, and that a high concentration can cause a certain degree of toxicity to HCE cells.

[0018] The above modeling concentration is specifically 300 μM. In some embodiments of the present invention, the dihydroquercetin concentration that exhibits a growth effect on HCE cells is 25 to 200 μM, and the dihydroquercetin concentration that causes some toxicity to HCE cells is 600 μM.

[0019] In the present invention, the ocular surface disease is preferably dry eye or a disease associated with corneal neovascularization.

[0020] The results showed that dihydroquercetin treatment could moderately increase the tear secretion and tear film stability of HCE cells, significantly improving dry eye symptoms.

[0021] The present invention also provides a dihydroquercetin ophthalmic preparation containing dihydroquercetin and a solvent.

[0022] The mass concentration of the dihydroquercetin is preferably 0.01% to 0.5%, more preferably 0.03% to 0.3%, and even more preferably 0.03% or 0.3%.

[0023] In the present invention, the dosage form of the preparation is preferably eye drops, eye ointment, periocular and intraocular injection, ophthalmic gel, or liposome.

[0024] Compared with the prior art, the present invention provides the use of dihydroquercetin in the manufacture of a medicament for the prevention, alleviation, and / or treatment of ocular surface diseases. The present invention is the first to use dihydroquercetin for the prevention, alleviation, and / or treatment of ocular surface diseases. As an antioxidant, dihydroquercetin has excellent protective effects against diseases associated with oxidative damage to corneal epithelial cells (HCE). Furthermore, within a certain concentration range, dihydroquercetin can promote HCE cell proliferation and improve cell viability, as well as increase HCE cell tear secretion and tear film stability, thereby significantly improving ocular surface diseases. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1 shows the results of MTT assay measurements of corneal epithelial cells exposed to various concentrations of dihydroquercetin (A), various concentrations of H2O2 (B), and a combination of 300 μM hydrogen peroxide and various concentrations of dihydroquercetin (C) for 24 hours. Here, P indicates significance, ** indicates P<0.01, *** indicates P<0.001, ## indicates P<0.01, and ### indicates P<0.001. [Figure 2] FIG. 2 is a graph showing the results of measuring the tear secretion volume of live mice in an animal experiment. [Figure 3] FIG. 3 is a graph showing the results of measuring the tear film rupture time of live mice in an animal experiment. [Figure 4] FIG. 4 shows the morphology of the mouse corneas of each experimental group after staining with sodium fluorescein. [Figure 5] FIG. 5 is a graph showing the score results obtained by measuring damage to the mouse corneal epithelium by sodium fluorescein staining of the cornea. [Figure 6] FIG. 6 shows the morphology of corneal neovascularization in New Zealand rabbits in each experimental group measured using a slit lamp. [Figure 7]7 is a graph showing the results of measuring the area of ​​corneal neovascularization in New Zealand rabbits in each experimental group, where **** indicates P<0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0026] To further illustrate the present invention, the application of dihydroquercetin provided by the present invention to the treatment of ocular surface diseases will be described in detail below in conjunction with examples.

[0027] Human corneal epithelial cells were purchased from the ATCC Cell Bank in the United States. The cell culture medium used was high-sugar DMEM medium supplemented with 10% (V / V) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL gentamicin. The cells were cultured in a 37°C incubator containing 5% carbon dioxide. 1.5 × 10 cells were used in the logarithmic growth phase. 5 Cells were plated in a 96-well plate at a density of 100 μL / well with six replicate wells per group. The cells were divided into normal cells, a hydrogen peroxide model group, and various concentrations of dihydroquercetin treatment groups. Absorbance at 490 nm was measured using a microplate reader with the MTS kit method (Promega). Viability = Viability of each group / Viability of normal cells × 100%

[0028] Experimental Example 1 (1) MTS measurement method Human corneal epithelial cells were purchased from the ATCC Cell Bank in the United States. The cell culture medium used was high-sugar DMEM medium supplemented with 10% (V / V) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL gentamicin. The cells were cultured in a 37°C incubator containing 5% carbon dioxide. Cells in the logarithmic growth phase were used at a density of 1.5 × 10 5The cells were plated in a 96-well plate at a density of 100 μL / well with six replicate wells per group. After 24 hours, the cells were treated with H2O2 modeling or dihydroquercetin depending on the experimental objective. Colorimetry: 20 μL of MTS solution was added to each well and incubated for 2-4 hours. Colorimetry: The absorbance of each well was measured using an enzyme-linked immunosorbent assay (ELISA) at a wavelength of 490 nm. The results were recorded, and a cell growth curve was plotted with time as the abscissa and absorbance as the ordinate.

[0029] (2) Establishment of a human corneal epithelial cell (HCE) injury model induced by H2O2 1.5 × 10 HCE cells 5 The cells were subcultured by adding 100 μL of H2O2 per well at 100 cells / mL. HCE cells were exposed to different concentrations of H2O2 (100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, and 900 μM) for 24 hours. To ensure the stability and accuracy of the experimental data, six replicate wells were used per group to observe the degree of H2O2-induced cell damage. Colorimetric analysis: 20 μL of MTS solution was added to each well and incubated for 2–4 hours. Colorimetric analysis: The absorbance of each well was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA). The results were recorded, and cell growth curves were plotted with different concentrations of H2O2 as the abscissa and absorbance as the ordinate. The H2O2 concentration at which cell viability was approximately 50% as measured by the MTT assay was used as the modeling concentration for the oxidative damage model. Viability = Viability of each group / Viability of normal cells × 100%

[0030] Figure 1 shows the MTT assay results for corneal epithelial cells exposed to various concentrations of dihydroquercetin (A), various concentrations of HO (B), and a combination of 300 μM hydrogen peroxide and various concentrations of dihydroquercetin (C) for 24 hours. Here, P indicates significance, ** indicates P<0.01, *** indicates P<0.001, ## indicates P<0.01, and ### indicates P<0.001. As shown in Figure 1B, cell viability was 49% at 300 μM HO. Therefore, 300 μM HO was selected as the modeling concentration for the oxidative damage model in subsequent experiments.

[0031] (3) Proliferative and toxic effects of dihydroquercetin on HCE cells Dihydroquercetin at different concentrations (0.01 to 600 μM) was applied to HCE cells. To ensure the stability and accuracy of experimental data, six replicate wells per group were used to observe the drug's proliferation effect on the cells.

[0032] As shown in Figure 1A, after 24 hours of treatment of HCE cells with different concentrations of dihydroquercetin, there was a significant difference in cell viability in the experimental groups with concentrations of 50 μM, 100 μM, and 200 μM compared to the control group, with a significant increase observed compared to the control group. Dihydroquercetin at concentrations of 50-200 μM was shown to have a proliferation-promoting effect on HCE cells. After the dihydroquercetin concentration increased to 600 μM, the cell viability of HCE cells in the dihydroquercetin group was significantly decreased compared to the control group. Dihydroquercetin at a concentration of 600 μM was shown to be toxic to HCE cells.

[0033] (4) Antioxidative damage effect of dihydroquercetin on HCE cells The experiment was divided into eight groups: a normal control group, an H2O2 model group, and a dihydroquercetin + H2O2 intervention group with different concentrations (0.1-200μM). Modeling of HCE cells was performed with 300μM H2O2. After modeling, the HCE cells were exposed to various concentrations of drugs within a safe range. The MTS method was used to measure cell viability in each group and to assess the antioxidant damage protection effect of dihydroquercetin on HCE cells.

[0034] As shown in Figure 1C, the cell viability of each experimental group was lower in the H2O2 model group than in the control group (P<0.001), but higher in the 25-200 μM dihydroquercetin group than in the H2O2 model group (P<0.01). H2O2 can induce oxidative stress damage in HCE cells, and dihydroquercetin has a protective effect against H2O2-induced oxidative stress damage in HCE cells.

[0035] 1.1 Dry eyes 1.1.1 Grouping Thirty out of 50 healthy female 8-week-old C57 / BL6 mice were selected and randomly divided into groups of five, resulting in a total of six groups: blank control group (N), model group (M), positive drug group (CsA), low-concentration herbal medicine combination group (SL), medium-concentration herbal medicine combination group (SM), and high-concentration herbal medicine combination group (SH). When grouped, the mice were labeled on their tails and cages. Labeling information included the experiment name, experiment number, group classification, dosage, and administration time.

[0036] 1.1.2 Rearing Experimental animals were housed in the S-class representative PF laboratory at the Animal Center of Yanghe Medical University. During the experiment, a total of six cages (40 cm long x 20 cm wide x 20 cm high) were used, with five mice per cage. Corn cob bedding was used. Sterile complete mixed feed (purchased from Liaoning Changsheng Biological Co., Ltd.) was used for this experiment, and purified water prepared using the Animal Center's HT-RO1000 water purification system was used for drinking water. Dedicated personnel monitored water quality annually. During the experiment, mice had free access to food and water. They were fasted the night before dissection and specimen collection, but were not deprived of water. The temperature and humidity in the animal experiment room were automatically controlled by an air conditioner to maintain a temperature of 20°C–25°C and a humidity of 40%–70%, and temperature and humidity changes were recorded daily. The lighting system automatically controlled the light cycle in the animal experiment room, evenly distributing light and dark over a 24-hour period. The noise level in the animal experiment room was controlled to below 60 decibels. This experiment was approved and supervised by the Institutional Animal Care and Use Committee (IACUC) and strictly adhered to the national animal welfare regulations, "Guiding Opinions on the Welfare and Care of Animals."

[0037] 1.2 Modeling Method In this experiment, a mouse dry eye model was constructed using an intelligent dry environment control system combined with scopolamine injection induction. During the modeling process, a two-stage dehumidification method was used to control the humidity of the model environment. First, the room humidity was reduced to 40% ± 5% using a temperature-controlled industrial dehumidifier. The humidity of the mouse housing environment was then reduced to 15% ± 3% using an intelligent dry box (dehumidification range: 10%-80%). At the same time, a noiseless, speed-adjustable fan (air speed range: 0-5 m / s) was installed inside the intelligent dry box. The fan was positioned 20 cm away from the mouse cage and at the same vertical height as the mice. These operations effectively controlled each experimental environmental parameter within the required range for the experiment. Mice in the model control group (M), active drug group (CsA), dihydroquercetin low-concentration group (E1), dihydroquercetin high-concentration group (E2), and vehicle control groups were housed in this dry environment (15% ± 3% humidity, 2.1 ± 0.2 m / s wind speed, and 21-23°C temperature). Mice in the blank control group were housed in a normal environment (60%-80% humidity, 21-23°C temperature). Except for the blank control group, mice in each group received subcutaneous injections of 0.5 mg / 0.2 mL scopolamine solution three times daily (9:00 AM, 12:00 PM, and 3:00 PM). The model construction period lasted for 2 weeks. A 5 μL instillation was administered intraocularly into the conjunctival sac of each eye, twice daily (9:00 AM and 3:00 PM). In each treatment group, treatment began the day before modeling and continued for 15 days.

[0038] 1.3 Administration method and dosage All mice were administered intraocularly starting the day before modeling. The blank control and model control groups were not administered any drugs. The positive drug group received 0.05% cyclosporine A (5 μL / dose) into the conjunctival sac. The low-concentration group received 0.03% dihydroquercetin (5 μL / dose). The high-concentration group received 0.3% dihydroquercetin (5 μL / dose). Mice in each group received the drugs twice daily (9:00 AM and 3:00 PM).

[0039] 1.4 Model Evaluation On the 14th day of modeling, after the normal modeling and drug administration had finished, the corneal epithelial damage and tear secretion status of the mice in the blank control group and the model control group were measured. If there was a significant difference between the indicators of the two groups in the statistical analysis, it meant that the modeling was successful.

[0040] 1.5 Experimental Metrics 1.5.1 General Condition Observation During the adaptation period (before group allocation), all mice underwent ophthalmologic examinations (including visual impairment, ocular abnormalities, and corneal damage) once to confirm that the experimental mice were normal. During the adaptation period and experimental course, the mice were weighed weekly. During the administration period, the experimental mice were observed daily before and after administration to record their health and any abnormal signs. The mice were observed for appearance (eyes, ears, mouth, nose, vulva, fur, excrement, limb activity, and mental state), vomiting, activity, eating and drinking, ambulatory status, moribund status, and death.

[0041] 1.5.2 Tear secretion measurement The mouse was immobilized without anesthesia, and the lower eyelid was lifted with soft-tipped forceps. One end of the phenol-red cotton thread was bent and placed in the inferior conjunctival sac, allowing the mouse's lower eyelid to relax and close naturally. After fixing the phenol-red cotton thread, the phenol-red cotton thread was removed after 1 minute of timing. The phenol-red cotton thread was placed on a ruler paper, and the length of the reddened area was calculated and recorded. Statistical analysis of the data was performed using GraphPad Prism 8.0 software.

[0042] Figure 2 is a graph showing the results of measuring tear secretion in live mice in an animal experiment. As shown in Figure 2, N represents the blank control group, M represents the model control group, R represents the solvent control group, Y represents the active drug group (cyclosporine A at a mass concentration of 0.05%), E1 represents the low-concentration dihydroquercetin group, and E2 represents the high-concentration dihydroquercetin group. Furthermore, P indicates significant difference, * represents P<0.05, ** represents P<0.01, and *** represents P<0.001.

[0043] The length (mm) of the phenol red cotton thread was statistically analyzed, and it was found that the longer the phenol red cotton thread, the greater the tear production, and conversely, the less tear production. Before modeling, the tear production of each group of animals was normal, with no significant differences between groups (P > 0.05). As shown in Figure 2, after 14 days of modeling, the positive drug group (CsA), the low-concentration dihydroquercetin group (E1), and the high-concentration dihydroquercetin group (E2) showed a significant increase in tear production compared to the model control group (M), with statistical significance (P < 0.001). In the vehicle control group, tear production did not change significantly compared to the model control group. These results demonstrate that dihydroquercetin treatment moderately increased tear production in dry eye model mice.

[0044] 1.5.3 Tear film rupture time measurement Mice were immobilized without anesthesia, and sodium fluorescein solution was instilled into the conjunctival sac of the mice. The mice's eyelids were passively closed several times, then opened. The time when the first black dot appeared under cobalt blue light from a slit lamp was recorded as the tear film rupture time. Data were statistically analyzed using GraphPad Prism 8.0 software.

[0045] Figure 3 shows the results of measuring tear film rupture time in live mice in an animal experiment. As shown in Figure 3, N represents the blank control group, M represents the model control group, R represents the solvent control group, Y represents the active drug group (0.05% cyclosporine A by mass), E1 represents the low-concentration dihydroquercetin group, and E2 represents the high-concentration dihydroquercetin group. P indicates significant difference, * represents P<0.05, ** represents P<0.01, and *** represents P<0.001. Before modeling, the measured BUT values ​​were within the normal range and there was no statistically significant difference (P>0.05). After modeling using the intelligent dry environment system and drug induction, there was no statistically significant difference in the BUT values ​​measured before and after modeling in the blank control group (P>0.05). BUT measurements were performed on mice in each group on day 14 of modeling.

[0046] As a result, as shown in Figure 3, compared with the blank control group, the other five groups of mice had a somewhat shorter tear film rupture time, with statistical significance (P<0.05). Compared with the model control group, the dihydroquercetin-treated group had a significantly longer tear film rupture time, with statistical significance (P<0.05). This indicates that dihydroquercetin treatment improved the tear film stability of the mice.

[0047] 1.5.4 Measurement of corneal epithelial damage by sodium fluorescein staining of the cornea The corneal morphology of each experimental mouse group was observed before and 14 days after modeling. Both corneas were stained with fluorescein sodium solution and observed and photographed using a slit lamp. After immobilization, 5 μL of 2% fluorescein sodium solution in saline was instilled into the conjunctival sac of the mouse. After 1 minute, the eye was gently rinsed with 2 mL of saline, excess solution was removed with a cotton swab, and the fluorescein-stained mouse corneas were illuminated with cobalt blue light and observed, photographed, and recorded using a slit lamp. Residual green fluorescence on the corneal surface indicated damage to the corneal epithelium at that site. The residual fluorescein sodium on the corneal surface was scored and recorded. Statistical analysis of the data was performed using GraphPad Prism 8.0 software.

[0048] Figure 4 shows the morphology of mouse corneas in each experimental group after sodium fluorescein staining. As shown in Figure 4, the corneas in the blank control group were intact and smooth, with no sodium fluorescein deposits, indicating that the corneal epithelium in the blank control group was intact and undamaged. Except for the blank control group, the model group and each treatment group showed varying degrees of sodium fluorescein deposits, indicating that the corneal epithelium in the model group and each treatment group had different degrees of damage. On day 14 of modeling, the positive drug group and dihydroquercetin-treated group showed dense punctate fluorescent deposits compared to the model control group, but these were significantly reduced compared to the model group.

[0049] Figure 5 shows the mouse corneal epithelial damage scores for each experimental group, measured by fluorescein sodium staining of the cornea. Here, N represents the blank control group, M represents the model control group, R represents the solvent control group, Y represents the positive drug group (0.05% cyclosporine A by mass), E1 represents the low-concentration dihydroquercetin group, and E2 represents the high-concentration dihydroquercetin group. * represents P<0.05, ** represents P<0.01, and *** represents P<0.001. The results shown in these figures indicate that, except for the blank control group, the model group, and each treatment group, the mouse corneal epithelium suffered varying degrees of damage. On day 14 of modeling, the positive drug group and the dihydroquercetin-treated group showed corneal epithelial cell damage compared to the model control group, but this was significantly reduced compared to the model group (P<0.001). In the positive drug group and the groups administered dihydroquercetin at various concentrations, the degree of damage to the mouse corneal epithelium was reduced, indicating a certain degree of protective effect on the cornea.

[0050] 2.1 Establishment of a corneal neovascularization model induced by alkali burn of the cornea 2.1.1 Grouping Fifty 3-month-old healthy New Zealand rabbits (half male, half female) were randomly assigned to six groups, six per group, for a total of six groups. These groups were designated as blank control group (N), model control group (M), active drug group (LEV), low-concentration dihydroquercetin group (E1), high-concentration dihydroquercetin group (E2), and solvent control group (R). When assigned to each group, the rabbits were labeled on their ears and cage labels. Labeling information included the experiment name, experiment number, group classification, dose, and administration time.

[0051] 2.1.2 Rearing Breeding conditions: Experimental animals were kept in the standard rabbit laboratory at the Animal Center of Shenyang He Medical University. During the experiment, a total of six cages were kept, each with one rabbit. They were kept in stainless steel hanging cages, and the animals had free access to food and water. Animals of other species were not kept in the same room.

[0052] Environmental conditions: The animal breeding room was automatically controlled to maintain a temperature of 20-25°C and humidity of 40-70%. Artificial lighting was used, and the light cycle was automatically controlled to 12 hours light and 12 hours dark. Noise levels were kept below 60 dB.

[0053] Animal welfare: Animal use complied with the national animal welfare regulations, "Guiding Opinions on Animal Welfare and Management" (2006, Science and Technology Agency). The animal use plan was approved and overseen by the Institutional Animal Care and Use Committee (IACUC). During the testing process, animals were humanely killed to alleviate their suffering and pain. Animals killed for humane reasons were treated in the same way as animals that died during the experiment. Surviving animals were euthanized after the experiment was completed. Disposal of animal carcasses was entrusted to a specialized institution.

[0054] 2.2 Modeling Method In this experiment, a rabbit corneal neovascularization model was constructed using the chemical injury method. General anesthesia was achieved by intravenous injection of 1% pentobarbital sodium, and ocular surface anesthesia was achieved with proparacaine eye drops. A 6-mm diameter filter paper strip was soaked in 1 mol / L NaOH solution for 1 minute and then applied to the center of the cornea. After 30 seconds, the filter paper was removed, and the ocular surface and conjunctival sac were rinsed with saline for 1 minute. After modeling, levofloxacin was instilled into the modeled eye to prevent postoperative infection.

[0055] 2.3 Administration method and dosage All animals in the treatment groups were administered intraocularly starting the day before modeling. The blank control and model control groups were not administered any drugs. The positive drug group received levofloxacin, administered intraconjunctivally at a dose of 50 μL. The low-concentration group received 0.03% dihydroquercetin, administered intraocularly at a dose of 50 μL. The high-concentration group received 0.3% dihydroquercetin, administered intraocularly at a dose of 50 μL. The New Zealand rabbits in each group received levofloxacin twice daily (9:00 AM and 3:00 PM).

[0056] 2.4 Measurement of corneal neovascularization area Animals in each experimental group were examined for ocular surface inflammation and neovascularization area on day 14 after treatment, and photographs were taken to calculate the neovascularization area. Statistical analysis of the data was performed using GraphPad Prism 8.0 software.

[0057] Figure 6 shows the morphology of corneal neovascularization measured by slit lamp in New Zealand rabbits of each experimental group on day 14 after administration. The black dots indicate the area of ​​corneal neovascularization. In the blank control group, the cornea was clear and free of neovascularization. In the model and vehicle control groups, a large number of dendritic neovascularizations appeared in the cornea, extending along the limbus toward the central cornea, showing significant differences compared to the blank control group. In the positive drug control group and the high-concentration dihydroquercetin-treated group, a small number of neovascularizations appeared in the cornea, showing significant improvement compared to the model group. Figure 7 shows the corneal neovascularization area measurements in New Zealand rabbits of each experimental group. Here, N represents the blank control group, M represents the model control group, R represents the vehicle control group, Y represents the positive drug group (levofloxacin), E1 represents the low-concentration dihydroquercetin group, and E2 represents the high-concentration dihydroquercetin group. ** represents P<0.01, and **** represents P<0.0001. In the model group and the solvent control group, there was a significant difference in the area of ​​corneal neovascularization compared to the blank control group (P<0.0001). In the positive drug control group and the high and low concentration dihydroquercetin administration groups, the area of ​​corneal neovascularization was relatively small, and there was a significant improvement compared to the model group (P<0.01). It was shown that dihydroquercetin could inhibit the appearance of neovascularization and reduce the degree of alkali burn on the cornea.

[0058] These results suggest that dihydroquercetin plays an important role in the treatment of ocular surface diseases. Dihydroquercetin at 50-200 μM promoted HCE cell proliferation, and dihydroquercetin at 25-200 μM protected HCE cells against oxidative stress damage induced by H2O2. Treatment with dihydroquercetin moderately increased tear secretion and improved tear film stability in mice with dry eye. Furthermore, dihydroquercetin reduced the severity of corneal epithelial damage in mice, providing some protective effects for the cornea, inhibiting the development of neovascularization and reducing the severity of alkali burns to the cornea.

[0059] The above description of the embodiments is intended to help understand the method and spirit of the present invention, and it should be noted that those skilled in the art may make some improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications will also fall within the scope of the claims of the present invention.

Claims

1. Use of dihydroquercetin in the manufacture of a medicament for the prevention, alleviation and / or treatment of ocular surface disease.

2. The use according to claim 1, characterized in that the ocular surface disease is an ocular surface disease caused by damage and / or apoptosis of corneal epithelial cells.

3. The use according to claim 2, characterized in that the corneal epithelial damage is oxidative damage.

4. The use according to claim 3, characterized in that the oxidative damage is damage caused by hydrogen peroxide.

5. The use according to claim 2, characterized in that the ocular surface disease is dry eye or a corneal neovascularization-related disease.

6. A dihydroquercetin ophthalmic preparation comprising dihydroquercetin and a solvent.

7. The dihydroquercetin ophthalmic preparation according to claim 6, wherein the mass concentration of the dihydroquercetin is 0.01% to 0.5%.

8. 7. The dihydroquercetin ophthalmic preparation according to claim 6, wherein the ophthalmic preparation is in the form of eye drops, eye ointment, periocular and intraocular injection, ophthalmic gel, or liposome.

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