Application of asarone in preparation of drug for treating and / or preventing ocular diseases, and composition and application thereof

Abstract

The present invention relates to the technical field of biomedicine, and provides an application of asarone in the preparation of a drug for treating and / or preventing ocular diseases, and a composition and an application thereof. For the purposes of the present invention, ocular diseases include diseases characterized by retinal ganglion cell damage, macular degeneration, cataracts, or dry eye syndrome; and asarone comprises at least one of alpha-asarone, beta-asarone, or gamma-asarone. The present invention also provides an asarone composition. The asarone or the asarone composition of the present invention is administered by eye drops, orally, or by injection, and can effectively reduce intraocular pressure, protect retinal ganglion cells, improve retinal function, reduce vascular leakage in neovascular age-related macular degeneration, reduce MDA content, reduce retinal cell apoptosis, increase tear secretion volume in dry eye syndrome, and inhibit visual dysfunction caused by dry age-related macular degeneration and macular degeneration. The present invention provides an effective prevention and treatment basis for asarone in the treatment and / or prevention of the described ocular diseases.

Description

Application of asarone in preparation of medicine for treating and / or preventing eye diseases and a composition and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to application of asarone in preparation of medicine for treating and / or preventing eye diseases and a composition and application thereof. BACKGROUND

[0002] Optic nerve, as an important part of central nervous system, is composed of axons of retinal ganglion cells (RGC). Optic nerve is vulnerable to injury, including primary injury from mechanical direct trauma, axonal disruption and shear stress, and secondary injury from inflammation, neurotoxic factors and vascular dysfunction. Optic nerve injury is a serious disease that causes patients to have difficulty in life and clinicians to have difficulty in treatment, and brings heavy burden to patients' families and society. Various ophthalmic diseases are closely related to optic nerve injury, such as glaucoma, optic neuritis, traumatic optic nerve injury and diabetic retinopathy.

[0003] Glaucoma is a disease characterized by intermittent or persistent elevated intraocular pressure and characteristic optic atrophy and visual field defects. Pathological elevation of intraocular pressure is the main risk factor. Persistent high intraocular pressure can lead to irreversible and non-selective death of retinal ganglion cells. The pathogenesis of glaucoma is complex, and there is still a huge unmet clinical need for the treatment of glaucoma.

[0004] Optic neuritis (ON) refers to all inflammatory diseases related to optic nerve, with visual loss or vision loss as the main clinical manifestation, with or without eye movement pain, and is usually unilateral. Current treatments for ON include hormone therapy, immunosuppression, and immune adsorption. The above therapies have the problems of large adverse reactions and poor treatment compliance, so it is of great significance to develop new drugs for treating ON.

[0005] Diabetic retinopathy (DR) is one of the most common eye complications in diabetic patients. If not treated in time, DR can cause retinal edema, hemorrhage, and neovascularization, which can lead to retinal detachment and even threaten vision. The causes of DR are not clear at this stage, and the main therapies include retinal laser photocoagulation, anti-VEGF drugs, corticosteroid drugs, and combination of multiple drugs.

[0006] Age-related macular degeneration (AMD) is a group of ocular fundus diseases with degenerative changes in the macular region of retina and choroid closely related to age. AMD is clinically divided into atrophic (dry) and exudative (wet) types, wherein the pathogenesis of dry AMD is affected by multiple factors, including genetic factors, RPE cell aging, oxidative stress, etc. Dry AMD has complex pathogenesis, a large number of patients, and few effective treatment drugs. Therefore, it is of great clinical value to develop new drugs for treating and / or preventing dry AMD.

[0007] Cataract is caused by damage to the lens capsule, which causes protein denaturation and opacity. The pathogenesis of cataract is related to oxidative stress, apoptosis, autophagy, etc. At present, there is no effective drug therapy for cataract. Early cataract is mostly treated with eye drops, and middle and late stage patients are mostly treated with surgery. Therefore, it is of great significance to develop drugs that can treat or delay the progression of cataract.

[0008] Dry eye syndrome (Dry eye syndrome, Xerophthalmia) is one of the most common ophthalmic diseases caused by low secretion of lacrimal gland due to various causes in the eye and / or whole body, which leads to abnormal tear film and dry epithelium of the eye. It is an ocular surface disease caused by various obstacles to the natural function and protection mechanism of the external eye, which causes the instability of the tear film during blinking. The existing treatment methods include artificial tears, immunomodulators, and surgical treatment, but the effect is poor.

[0009] Optic nerve injury, macular degeneration, cataract and dry eye are the main blinding eye diseases that cause visual impairment. Current treatment strategies for these diseases are mostly targeted at a single target (such as anti-VEGF treatment for wet macular degeneration, etc.), and such therapies have limitations such as high risk of invasive operation (such as vitreous injection) and inability to reverse neurodegenerative diseases. Therefore, it is of great clinical significance to develop multi-target drugs with neuroprotective effect (such as inhibition of RGC apoptosis), anti-inflammatory (regulation of complement or immune pathways) and repair function (promotion of blood-retinal barrier reconstruction), and to use non-invasive drug delivery routes to improve patient compliance. SUMMARY

[0010] In view of the deficiencies of the prior art described above, the application provides the use of asarone in the preparation of a drug for treating and / or preventing eye diseases and a composition and its use.

[0011] In order to achieve the above-mentioned purposes of the application, the following technical solutions are provided:

[0012] The application provides the use of a-sesamin, β-sesamin or γ-sesamin in the preparation of a drug for treating and / or preventing an eye disease, wherein the eye disease includes a retinal ganglion cell injury disease and macular degeneration, cataract or dry eye.

[0013] Preferably, the retinal ganglion cell injury disease includes glaucoma, optic neuritis or diabetic retinopathy.

[0014] Preferably, the eye disease is macular degeneration, and the a-sesamin is used.

[0015] Preferably, the macular degeneration includes dry macular degeneration, vascular leakage caused by neovascular age-related macular degeneration or visual dysfunction caused by macular degeneration.

[0016] The application provides a composition containing an effective concentration of a-sesamin, β-sesamin or γ-sesamin, wherein the final concentration of the a-sesamin, β-sesamin or γ-sesamin is 0.5-30 g / L.

[0017] Preferably, the a-sesamin is the only active ingredient in the composition.

[0018] Preferably, the composition further contains an oil solvent, an emulsifier, an osmotic pressure regulator, a pH regulator and water.

[0019] Preferably, the oil solvent is soybean oil, the emulsifier is egg yolk lecithin, the osmotic pressure regulator is glycerol and the pH regulator is sodium hydroxide.

[0020] Preferably, the composition is in the form of eye drops, oral preparations or injections.

[0021] Preferably, the eye drops contain the following components: 0.5-5 g / L of a-sesamin, 4-50 g / L of soybean oil, 1-15 g / L of egg yolk lecithin, 2-25 g / L of glycerol and 0.001-0.005 g / L of sodium hydroxide.

[0022] Preferably, the oral preparations or injections contain the following components: 4-30 g / L of a-sesamin, 80-300 g / L of soybean oil, 10-20 g / L of egg yolk lecithin, 20-30 g / L of glycerol and 0.02-0.1 g / L of sodium hydroxide.

[0023] The application provides the use of the composition in the preparation of a drug for treating and / or preventing an eye disease, wherein the eye disease includes glaucoma, optic neuritis, diabetic retinopathy, macular degeneration, cataract or dry eye.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] 1、The composition of the asarain in the present application comprises the following components: at least one of alpha-asarain, beta-asarain and gamma-asarain, preferably alpha-asarain, soybean oil, egg yolk lecithin, glycerol, sodium hydroxide and water. The asarain or asarain composition in the present application can be administered by eye drops, oral administration and injection, and can effectively reduce the intraocular pressure, protect the retinal ganglion cells, improve the retinal function, reduce the vascular leakage caused by neovascular age-related macular degeneration, reduce the MDA content in the retinal tissue of optic neuritis and cataract, reduce the retinal cell apoptosis in diabetic retinopathy and increase the tear secretion of dry eye rats. The present application provides an effective prevention and treatment basis for alpha-asarain in the treatment and / or prevention of the above eye diseases.

[0026] 2、The alpha-asarain composition provided by the present application can effectively alleviate the retinal damage caused by single tail vein injection of sodium iodate, improve the oxidative damage of retinal cells, increase the ERG waveform amplitude, increase the retinal thickness, improve the cell arrangement of the ONL layer of the retina, increase the expression of Rhodopsin, RPE65 and ZO-1 in the retina, reduce the number of apoptotic cells in the retina, and down-regulate the expression of inflammation-related proteins P2X7, NLRP3 and Caspase-1 in the retina. At the same time, alpha-asarain can effectively enhance the activity of ARPE-19 cells after sodium iodate injury and reduce the apoptosis of ARPE-19 cells; and has a significant protective effect on the damaged retina. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a graph of the intraocular pressure of the pre-administration for 1 week and post-administration for 1-4 weeks of experimental example 1 (n=10).

[0028] Figure 2 is a graph of the amplitude of the dark adaptation 3.0 ERG a wave of each group of pre-administration for 1 week and post-administration for 1-4 weeks of experimental example 1 (n=10).

[0029] Figure 3 is a graph of the amplitude of the dark adaptation 3.0 ERG b wave of each group of pre-administration for 1 week and post-administration for 1-4 weeks of experimental example 1 (n=10).

[0030] Figure 4 is a graph of the amplitude change of the visual evoked potential F-VEP of each group of pre-administration for 1 week and post-administration for 2 weeks of experimental example 2 (n=10).

[0031] Figure 5 is a graph of the retinal thickness of each group of pre-administration for 1 week and post-administration for 2 weeks of experimental example 2 (n=10), compared with the Control group, #### P<0.0001, compared with the model control group, *** P<0.001.

[0032] Figure 6 is a chart of the amplitude of the dark adaptation ERG a wave of each group in Example 3, pre-administration for 1 week and administration after modeling for 2 weeks (n=10).

[0033] Figure 7 is a chart of the amplitude of the dark adaptation ERG b wave of each group in Example 3, pre-administration for 1 week and administration after modeling for 2 weeks (n=10).

[0034] Figure 8 is a chart of the amplitude of the visual evoked potential F-VEP of each group in Example 3, pre-administration for 1 week and administration after modeling for 2 weeks (n=10).

[0035] Figure 9 is a chart of the leakage grade score of the fundus angiography image of each group in Example 4, pre-administration for 3 days and administration after modeling for 7 days, compared with the model control group, * P<0.05.

[0036] Figure 10 is a chart of the maximum central thickness of the choroid CNV of each group in Example 4, pre-administration for 3 days and administration after modeling for 7 days (n=12).

[0037] Figure 11 is a column chart of the effect of α-asarone on glutamate-induced apoptosis of R28 cells in Example 5 (n=6).

[0038] Figure 12 is a chart of the retinal MDA content of each group of rats in Example 6 (n=10).

[0039] Figure 13 is a chart of the number of retinal apoptotic cells of each group in Example 7 (n=10).

[0040] Figure 14 is a chart of the retinal MDA content of each group of rats in Example 8.

[0041] Figure 15 is a chart of the tear secretion of each group of rats in Example 9.

[0042] Figure 16 is a chart of the ERG amplitude of each group in Example 10, pre-administration for 1 week and administration after modeling for 3 weeks (n=12), with the vertical coordinate being amplitude (μV).

[0043] Figure 17 is an OCT image of the retinal thickness of each group in Example 10, pre-administration for 1 week and administration after modeling for 3 weeks (n=14), with the range indicated by the arrow being the retina.

[0044] Figure 18 is a chart of the retinal thickness of each group in Example 10, pre-administration for 1 week and administration after modeling for 3 weeks, compared with the Control group, ### P<0.001; compared with the model control group, *** P<0.001.

[0045] Figure 19 is a graph showing the results of retinal HE staining of each group in Example 10 (n=10), the ONL layer is indicated by an arrow, scale bar = 50 μm.

[0046] Figure 20 is a graph showing the retinal ONL layer thickness of each group in Example 10 (n=10), compared with the Control group, #### P<0.0001; compared with the model control group, **** P<0.0001.

[0047] Figure 21 is a graph showing the results of retinal TUNEL staining and Rhodopsin, RPE65, ZO-1 immunofluorescence of each group in Example 10 (n=5).

[0048] Figure 22 is a graph showing the results of counting the number of apoptotic cells in retinal TUNEL staining of each group in Example 10 (n=7), compared with the Control group, #### P<0.0001; compared with the model control group, **** P<0.0001.

[0049] Figure 23 is a graph showing the protein expression of RPE65, P2X7, NLRP3, Caspase-1 in the retina of each group in Example 10 (n=6).

[0050] Figure 24 is a graph showing the statistical results of protein expression of RPE65, P2X7, NLRP3, Caspase-1 in the retina of each group in Example 10 (n=6), compared with the Control group, # P<0.05, #P<0.01, #### P<0.0001; compared with the model control group, * P<0.05; *** P<0.001; **** P<0.0001.

[0051] Figure 25 is a graph showing the pharmacodynamic effect of α-asarone on sodium iodate damaged ARPE-19 cells (n=6), compared with the Control group, #### P<0.0001; compared with the model control group, **** P<0.0001.

[0052] Figure 26 is a graph showing the effect of α-asarone on sodium iodate-induced apoptosis of ARPE-19 cells (n=6).

[0053] Figure 27 is a column chart of the effect of experimental example 11 α-asarone on sodium iodate-induced apoptosis of ARPE-19 cells (n = 6), compared with the Control group, #### P < 0.0001, compared with the model control group, **** P < 0.0001.

[0054] Figure 28 is a statistical chart of the amplitude of ERG in experimental example 12 (n = 18), in which the vertical axis is the amplitude (μV). DETAILED DESCRIPTION

[0055] The application and a composition and the use thereof in the preparation of a medicament for treating and / or preventing an ocular disease are described in detail below.

[0056] The first aspect of the present application provides the use of asarone in the preparation of a medicament for treating and / or preventing an ocular disease, including retinal ganglion cell damage diseases and macular degeneration, cataract or dry eye; the asarone includes α-asarone, β-asarone or γ-asarone.

[0057] The structural formula of α-asarone or β-asarone or γ-asarone is as follows:

[0058] In the present application, the retinal ganglion cell damage diseases include glaucoma, optic neuritis or diabetic retinopathy.

[0059] In some embodiments of the present application, the ocular disease is macular degeneration, and the asarone is α-asarone.

[0060] In some embodiments of the present application, the macular degeneration includes dry macular degeneration, vascular leakage caused by neovascular age-related macular degeneration or visual dysfunction caused by macular degeneration.

[0061] The second aspect of the present application provides a composition comprising an effective concentration of at least one of α-asarone, β-asarone and γ-asarone, preferably α-asarone, the final concentration of the α-asarone being 0.5-30 g / L, further preferably 10-20 g / L, and more preferably 15 g / L.

[0062] The present application has unexpectedly found through experimental research that the order of the pharmacological effects of α-asarone, β-asarone and γ-asarone in the preparation of a medicament for treating and / or preventing the ocular disease described in the present application is: α-asarone > β-asarone > γ-asarone.

[0063] In some embodiments of the present application, the α-asarone in the composition is the only effective active ingredient.

[0064] In some embodiments of the present application, the composition further comprises an oil solvent, an emulsifier, an osmotic pressure regulator, a pH regulator, and water. In the present application, the oil solvent comprises soybean oil, the emulsifier comprises egg yolk lecithin, the osmotic pressure regulator comprises glycerol, and the pH regulator comprises sodium hydroxide.

[0065] The composition provided by the present application is a preparation prepared by adding the therapeutically effective amount of the compound to pharmaceutically acceptable adjuvants. The preparation can be prepared into eye drops, solutions, suspensions, emulsions, injections, gels, tablets, capsules, aerosols, transdermal preparations, eye ointments, etc. The dosage forms can be prepared according to the conventional production method in the pharmaceutical field, and are administered by eye, injection, oral administration, pulmonary inhalation, transdermal or intravitreal injection, as long as the therapeutically effective amount of the compound reaches the effective drug concentration in the blood or at the site of action.

[0066] The composition provided by the present application can be administered by eye, orally or by injection.

[0067] In some embodiments of the present application, the dosage form of the composition is eye drops, oral preparations or injections.

[0068] In the present application, the eye drops use water as a solvent and contain the following components at the following concentrations: α-asarone 0.5-5 g / L, soybean oil 4-100 g / L, egg yolk lecithin 1-15 g / L, glycerol 2-25 g / L, and sodium hydroxide 0.001-0.005 g / L.

[0069] The concentration of α-asarone in the eye drops is 0.5-5 g / L, further preferably 1-4 g / L, and more preferably 2 g / L;

[0070] The concentration of soybean oil is 4-100 g / L, further preferably 20-80 g / L, and more preferably 50 g / L;

[0071] The concentration of egg yolk lecithin is 1-15 g / L, further preferably 5-10 g / L, and more preferably 8 g / L;

[0072] The concentration of glycerol is 2-25 g / L, further preferably 10-20 g / L, and more preferably 15 g / L;

[0073] The concentration of sodium hydroxide is 0.001-0.005 g / L, further preferably 0.002-0.004 g / L, and more preferably 0.003 g / L.

[0074] In the present application, the oral agent or injection agent uses water as solvent and comprises the following components in the following concentrations: alpha-sinacen 4-30 g / L, soybean oil 80-300 g / L, egg yolk lecithin 10-20 g / L, glycerol 20-30 g / L, and sodium hydroxide 0.02-0.1 g / L.

[0075] The concentration of alpha-sinacen in the oral agent or injection agent is 4-30 g / L, further preferably 10-20 g / L, and more preferably 15 g / L.

[0076] The concentration of soybean oil is 80-300 g / L, further preferably 100-200 g / L, and more preferably 150 g / L.

[0077] The concentration of egg yolk lecithin is 10-20 g / L, further preferably 12-18 g / L, and more preferably 15 g / L.

[0078] The concentration of glycerol is 20-30 g / L, further preferably 22-28 g / L, and more preferably 25 g / L.

[0079] The concentration of sodium hydroxide is 0.02-0.1 g / L, further preferably 0.04-0.08 g / L, and more preferably 0.06 g / L.

[0080] The soybean oil, egg yolk lecithin, and glycerol in the present application are preferably injection-grade soybean oil, injection-grade egg yolk lecithin, and injection-grade glycerol. The water in the present application is preferably injection-grade water.

[0081] If necessary, the composition of the present application can further comprise a pharmaceutically acceptable bacteriostatic agent, such as acids, alcohols, quaternary ammonium salts, p-hydroxybenzoic acid esters, benzoic acid, sorbic acid, and the like.

[0082] The third aspect of the present application provides use of the composition in the preparation of a medicament for treating and / or preventing an eye disease, including glaucoma, optic neuritis, diabetic retinopathy, macular degeneration, cataract, or dry eye.

[0083] The technical solutions provided by the present application are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present application.

[0084] The high-pressure homogenizer used in the examples of the present application is a Dongni Bio-Experimental High-Pressure Nanometer Homogenizer, Model AH-NANO-TKE061. SD rats and C57BL / 6 mice are purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0085] Example 1 Preparation of alpha-sinacen oral emulsion and alpha-sinacen emulsion injection

[0086] 1. Preparation process

[0087] Table 1 Raw materials and dosages

[0088] 1) Pour soybean oil for injection into the oil tank, start the stirrer and begin heating, then add α-asarone, egg yolk lecithin for injection at 70°C, keep the oil temperature at 70°C, stir at 3000 rpm / min, disperse the lecithin evenly in the oil phase, stir for 20 min to completely dissolve the lecithin.

[0089] 2) Add 45 L of water for injection to the preparation tank. Then add the weighed glycerin for injection and sodium hydroxide, start the stirrer to stir evenly, pass through the filter (0.22 μm, 5 inches pp, Hangzhou Kebaituo Filter Co., Ltd.) into the water phase tank. Control the water phase temperature to be 70°C. Protect with nitrogen, adjust the water phase to be sufficient.

[0090] 3) Slowly press the oil phase into the water phase with nitrogen at a stirring speed of 3000 rpm / min, control the flow rate, about 5 min to transfer, get the initial emulsion.

[0091] 4) The initial emulsion is passed through a high-pressure homogenizer for 2 times of homogenization, each time for 30 min, the drug solution temperature should be controlled at 60°C. The homogenization conditions are as follows:

[0092] Table 2 Pressure and temperature of high-pressure homogenizer

[0093] After the homogenization starts, adjust the homogenization pressure, and only when the specified pressure is reached can the drug solution be transferred to the next storage tank.

[0094] 2. Filling, sterilization, storage

[0095] 1) After homogenization, cool the drug solution to 30°C, transfer the drug solution to the stationary tank for storage, and wait for filling. The drug solution is filtered with nitrogen and sent to the filling machine;

[0096] 2) Filling, seal

[0097] The prepared emulsion is filtered through a 5 μm PP filter (20 inches, Hangzhou Kebaituo Filter Co., Ltd.), filled into cleaned ampoules by the filling machine, and immediately protected with nitrogen and sealed.

[0098] 3) Sterilization

[0099] In the rotary sterilization cabinet, the product is sterilized (121°C x 10 min, F0 value ≥ 12).

[0100] 4) Packaging, storage

[0101] After the sterilized product is checked by light, it is packed into cartons to obtain α-asarone emulsion injection with a concentration of 10 mg / mL.

[0102] Example 2 Preparation of α-asarone emulsion eye drops

[0103] 1. Preparation process

[0104] Table 3 Raw materials and amounts

[0105] The raw materials and amounts of the α-asarone emulsion eye drops in this example are shown in Table 3, and the preparation method is the same as in Example 1.

[0106] 2. Sterilization, filling, and storage

[0107] 1) Sterilization

[0108] A non-final sterilization process was used to sterilize the polyester (PET) packaging material using a vaporized hydrogen peroxide sterilization box, and the filling and sealing process for the preparation of the α-asarone emulsion eye drops must be performed under corresponding clean environment conditions.

[0109] 2) Filling

[0110] After homogenization, the drug solution was cooled to 30°C, transferred to a holding tank for storage, and waited for filling. The drug solution was filtered with nitrogen gas and sent to the filling machine; the prepared emulsion was filtered through a 5 μm PP filter (20 inches, Hangzhou Kebaiter Filter Material Co., Ltd.), and then filled into pre-cleaned, dried, and sterilized packaging containers made of polyester (PET).

[0111] 3) Packaging and storage

[0112] After lamp inspection, the product was packed into cartons to obtain the α-asarone emulsion eye drops with a concentration of 2 mg / mL.

[0113] Comparative Example 1

[0114] Unlike Example 2, the emulsion eye drops prepared in Comparative Example 1 did not contain α-asarone. Except for the absence of α-asarone, the other reagents and preparation methods were the same as in Example 2, and the following experiment is referred to as "blank emulsion eye drops".

[0115] Experimental Example 1 Protective effect of α-asarone emulsion eye drops on chronic high intraocular pressure glaucoma rats induced by magnetic microspheres

[0116] 1. Reagents and materials

[0117] 30 SPF level SD rats, 6-8 weeks old, male, weighing 180-220 g; magnetic polystyrene microspheres (Magnetic polystyrene microspheres, abbreviated as PS); α-asarone emulsion eye drops (prepared according to the method in Example 2, with a concentration of 2 mg / mL).

[0118] 2. Animal grouping and administration method

[0119] The experimental rats were randomly divided into three groups, namely the normal control group (Control group, n=10), the model control group (PS group, n=10), and the α-asarone emulsion eye drop group (α-asarone group, n=10).

[0120] One week in advance, the α-asarone emulsion eye drop group was given 20 μL of α-asarone emulsion eye drops (2 mg / mL) in the form of eye drop administration every 3.5 h during 8:30-18:00; on the 8th day, the model control group and the α-asarone group injected 10 μL of magnetic polystyrene microspheres with a diameter of 5 μm and a concentration of 50 mg / mL into the right anterior chamber of the rats under a surgical microscope to induce unilateral intraocular pressure elevation, and the normal control group was injected with the same amount of sterile normal saline at the same site. After modeling, the α-asarone group was given 20 μL of α-asarone emulsion eye drops every 3.5 h during 8:30-18:00 for 4 weeks. The PS group was given the same volume of blank emulsion eye drops in the form of eye drop administration.

[0121] 3. Evaluation method and results

[0122] The intraocular pressure of rats was measured before modeling and 1, 2, 3, and 4 weeks after modeling, and the results are shown in Figure 1 and Table 4; the waveform amplitude of ERG dark adaptation 3.0 was detected at 1, 2, 3, and 4 weeks, and the results are shown in Figures 2-3 and Tables 5-6. The data results are presented in the form of mean ± standard deviation, and after normality test and variance homogeneity test of experimental data, single factor analysis of variance (One-way ANOVA) is used for statistics; post hoc comparison uses LSD test (homogeneous variance) or Dunnett-t test (inhomogeneous variance); P<0.05 is considered to have significant difference, all data are processed and analyzed using IBM SPSS Statistics 27, and all statistical graphs are plotted using GraphPad Prism 9.3.0. The specific results are as follows:

[0123] Table 4 Comparison of intraocular pressure of rats in each group before modeling and after administration (mmHg) (n=10)

[0124] Table 5 Change of ERG a-wave amplitude in dark adaptation 3.0 (μV) (n=10)

[0125] Table 6 Change of ERG b-wave amplitude in dark adaptation 3.0 (μV) (n=10)

[0126] Note: compared with Control group, ## P<0.01; ### P<0.001; #### P<0.0001; compared with PS group, * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001.

[0127] a wave is generated by the outer layer cells of the retina (mainly rod cells and cone cells), reflecting the function of photoreceptors; b wave is generated by the inner layer cells of the retina (mainly bipolar cells and Muller cells), reflecting the function of the intermediate layer of the retina.

[0128] As shown in Tables 4-6 and Figures 1-3, the intraocular pressure of rats slowly decreased after continuous administration for 4 weeks. Under dark adaptation flash stimulation, the amplitude size at each time point was ranked as: Control group > α-asarone group > PS group. The difference between α-asarone group and PS group was significant (P<0.001 or P<0.0001), indicating that α-asarone prophylactic administration can effectively reduce intraocular pressure, increase the amplitude of flash stimulation, and improve the retinal function of chronic high intraocular pressure rats.

[0129] The inventors found through experimental research that the pharmacodynamics of α-asarone emulsion eye drops is basically consistent with that of α-asarone emulsion injection administered at a dose of 30 mg / kg by intraperitoneal injection (i.p.) and α-asarone oral emulsion administered at a dose of 60 mg / kg by gavage (i.g.). To simplify the description, the following experiments only show the pharmacodynamic results of α-asarone emulsion eye drops.

[0130] Example 2 Protective effect of α-asarone emulsion eye drops on N-methyl-D-aspartate (NMDA)-induced retinal ganglion cell injury

[0131] 1. Reagents and materials

[0132] A total of 28 SD rats, 6-8 weeks old, SPF grade, male, weighing 180-220 g; NMDA; α-asarone emulsion eye drops (prepared according to the method in Example 2, concentration of 2 mg / mL).

[0133] 2. Animal grouping and administration method

[0134] The experimental rats were randomly divided into three groups, namely normal control group (Control group, n=10), model control group (NMDA group, n=10), and α-asarone emulsion eye drop group (α-asarone group, n=10).

[0135] One week in advance, every 3.5 hours during 8:30-18:00, the rats in the α-asarone emulsion eye drop group were given 20 μL of α-asarone emulsion eye drops (2 mg / mL) by eye drop administration; on the 8th day, the model control group and the α-asarone group slowly injected 3 μL of NMDA (50 mM) solution into the vitreous cavity of the right eye of the rats under an optical microscope using a 33G needle to induce neuroexcitatory toxicity, and the normal control group was injected with an equal amount of sterile normal saline. After the modeling was completed, every 3.5 hours during 8:30-18:00, the rats in the α-asarone group were given 20 μL of α-asarone emulsion eye drops for 2 weeks. The NMDA group was given the same volume of blank emulsion eye drops by eye drop administration at the same time.

[0136] 3. Evaluation method and results

[0137] At 2 weeks after modeling, the amplitude changes of flash visual evoked potential (F-VEP) of rats in each group were detected, and the results are shown in Figure 4 and Table 7. At 2 weeks after modeling, the eyeballs of rats in each group were removed to make paraffin sections, which were stained with HE, and the retinal thickness of rats in each group was counted, and the results are shown in Figure 5 and Table 8. The data results are presented in the form of mean ± standard deviation. After normality test and homogeneity test of experimental data, single factor analysis of variance (One-way ANOVA) was used for statistics; post hoc comparison used LSD test (homogeneity of variance) or Dunnett-t test (heterogeneity of variance); P<0.05 was considered to have significant difference. All data were processed and analyzed using IBM SPSS Statistics 27, and all statistical graphs were plotted using GraphPad Prism 9.3.0. The specific results are as follows:

[0138] Table 7 Change of amplitude of visual evoked potential F-VEP (n=10)

[0139] Table 8 Change of retinal thickness of rats in each group (n=10)

[0140] Note: Compared with the Control group, #### P<0.0001; compared with the NMDA group, *** P<0.001.

[0141] As shown in Tables 7-8 and Figures 4-5, after 2 weeks of continuous administration, the thickness of the rat retina increased, and part of the electrophysiological function recovered. There were significant differences between the administration group and the model group (P<0.001), indicating that α-asarone prophylactic administration had a protective effect on NMDA-induced RGC death, retinal morphology, and optic nerve function damage in rats.

[0142] Experimental Example 3: The therapeutic effect of α-Asarum cerebroside emulsion eye drops on traumatic optic neuropathy in rats.

[0143] 1. Reagents and Materials

[0144] Thirty male SD rats aged 6–8 weeks, SPF grade, weighing 180–220 g; α-Asarum lactone emulsion eye drops (prepared according to the method in Example 2, concentration 2 mg / mL).

[0145] 2. Animal grouping and administration method

[0146] The experimental rats were randomly divided into three groups: normal control group (Control group, n=10), optic nerve crush model control group (ONC group, n=10), and α-asarone emulsion eye drops group (α-asarone group, n=10).

[0147] One week prior to the event, rats in the α-asarone emulsion eye drop group were administered 20 μL of α-asarone emulsion eye drops (2 mg / mL) every 3.5 hours between 8:30 AM and 6:00 PM daily. On day 8, optic nerve clipping surgery was performed on the model control group and the α-asarone emulsion eye drop group, while the normal control group received no treatment. After model establishment, rats in the α-asarone emulsion group were administered 20 μL of α-asarone emulsion eye drops every 3.5 hours between 8:30 AM and 6:00 PM daily for two weeks. The ONC group was simultaneously administered an equal volume of blank emulsion eye drops.

[0148] 3. Evaluation Methods and Results

[0149] ERG waveforms were observed in the second week after modeling, and the results are shown in Figures 6-7 and Tables 9-10. Simultaneously, the amplitude changes of flash visual evoked potentials (F-VEP) in each group of rats were measured in the second week, and the results are shown in Figure 8 and Table 11. Data are presented as mean ± standard deviation. After testing for normality and homogeneity of variance, one-way ANOVA was used for statistical analysis. Post-hoc comparisons were performed using the LSD test (homogeneous variance) or the Dunnett-t test (unequal variance). A p-value < 0.05 was considered statistically significant. All data were processed and analyzed using IBM SPSS Statistics 27, and all statistical graphs were created using GraphPad Prism 9.3.0. Specific results are as follows:

[0150] Table 9. Variation of ERG a-wave amplitude in dark adaptation (μV)(n=10)

[0151] Table 10 Changes in amplitude of dark-adapted ERG b-wave (μV) (n=10)

[0152] Table 11 Changes in amplitude of visual evoked potential F-VEP (n=10)

[0153] Note: compared with the Control group, # P<0.05; #### P<0.0001; compared with the ONC group, * P<0.05; ** P<0.01; **** P<0.0001.

[0154] As shown in Tables 9-11 and Figures 6-8, the amplitude of each flash frequency measured when pre-administered for 1 week and administered for 2 weeks after modeling was ranked as: Control group > α-asarone group > ONC group, indicating that α-asarone can effectively increase the waveform amplitude of each flash stimulation and alleviate the rat optic nerve damage caused by external pressure.

[0155] Example 4 Protective effect of α-asarone emulsion eye drops on laser-induced choroidal neovascularization mouse model

[0156] 1. Reagents and materials

[0157] 30 C57BL / 6 mice, 7-8 weeks old, SPF level, male, weighing 19-21 g; α-asarone emulsion eye drops (prepared according to the method in Example 2, concentration of 2 mg / mL).

[0158] 2. Animal grouping and administration method

[0159] The experimental mice were randomly divided into two groups, namely a normal control group (Control group, n=6), a choroidal neovascularization (Choroidal Neovascularization) model control group (CNV group, n=12), and an α-asarone emulsion eye drop group (α-asarone group, n=12).

[0160] The α-asarone emulsion eye drops group rats were given 20 μL of α-asarone emulsion eye drops (2 mg / mL) by eye drop administration every 3.5 h during 8:30-18:00 for 3 days; on the 4th day, the CNV group and the α-asarone group were modeled by 532 nm laser, and the normal control group was not treated. After the modeling was completed, the α-asarone emulsion group rats were given 20 μL of α-asarone emulsion eye drops every 3.5 h during 8:30-18:00 for 2 weeks. The CNV group was given the same volume of blank emulsion eye drops by eye drop administration at the same time.

[0161] 3. Evaluation method and result

[0162] On the 7th day after laser photocoagulation, the mice in each group were subjected to fluorescein fundus angiography (FFA) examination, and the results are shown in Figure 9 and Table 12; at the same time, the eyeballs of mice in each group were removed to make paraffin sections, which were subjected to HE staining, and the maximum central thickness of CNV of mice in each group was counted. The results are shown in Figure 10 and Table 13. The data results are presented in the form of mean ± standard deviation, after normality test and variance homogeneity test of experimental data, single factor analysis of variance (One-way ANOVA) is used for statistics; post hoc comparison uses LSD test (variance equal) or Dunnett-t test (variance not equal); P<0.05 is considered to have significant difference, all data are processed and analyzed using IBM SPSS Statistics 27, and all statistical graphs are plotted using GraphPad Prism 9.3.0.

[0163] Table 12 Leakage grade score of fundus angiography of mice in each group (n=12)

[0164] Table 13 Change of maximum central thickness of CNV of mice in each group (μm)(n=12)

[0165] Note: compared with the Control group, #### P<0.0001; compared with the CNV group, * P<0.05; ** P<0.01.

[0166] As shown in Tables 12-13 and Figures 9-10, pre-administration of α-asarone can reduce laser spot fluorescein leakage in laser-induced mice, reduce the generation of CNV, and the results of ex vivo eyeball HE staining and fundus fluorescence angiography are basically consistent. There is a significant difference (P<0.05) between the administration group and the model group, which indicates that α-asarone prophylactic administration can effectively inhibit the formation of laser-induced CNV and has a certain therapeutic effect on CNV mice.

[0167] Protective effect of α-asarone on glutamate-induced injury of R28 cells

[0168] 1. Reagents and materials

[0169] R28 cells were obtained from Wuhan Ponsen Life Science Co., Ltd. L-glutamic acid (Macron, L810369); CCK-8 (Biosharp, BS350C); DMEM / F12 medium (containing double antibodies) (Keygen Biotech, KGM12500-500); fetal bovine serum (ZETA, 120214018-4S); PBS (Keygen Biotech, KGB5001); DMSO (Solarbio, D8371).

[0170] 2. Experimental procedures

[0171] (1) Screening of glutamate modeling concentration: R28 cells were cultured with different concentrations of glutamate for 24 h. It was found that the cell viability was about 60% in the presence of 10 mM glutamate. Therefore, 10 mM glutamate was selected for modeling.

[0172] (2) Screening of α-asarone efficacy: R28 cells in the logarithmic growth phase cultured in complete medium were inoculated into 96-well plates at a density of 1 × 10 4 cells / 100 μL / well, and the edge wells were filled with sterile PBS. The cells were incubated at 37 °C and 5% CO2 for 24 h until they were completely adherent. The supernatant was discarded, and 50 μL of α-asarone solution with a final concentration of 0.5 μM, 1 μM, or 2 μM was added. The cells were incubated at 37 °C and 5% CO2 for 2 h. Then, 50 μL of glutamate solution with a final concentration of 10 mM diluted with serum-free medium was added to each well, and the cells were incubated at 37 °C and 5% CO2 for 24 h. The control group was added with the same volume of serum-free medium. The model group was added with 50 μL of serum-free medium and 50 μL of glutamate solution with a final concentration of 10 mM diluted with serum-free medium. The rest of the operations and drug administration of the control group and the model group were the same as those of the drug administration group. Then, 110 μL of 10% CCK-8 solution was added to each well, and the cells were incubated at 37 °C and 5% CO2 for 2 h. The OD value at 450 nm was detected by a microplate reader, and the cell viability of R28 was calculated according to the following formula.

[0173] Cell viability = (average absorbance value of the experimental group - average absorbance value of the zero adjustment well) / (average absorbance value of the normal control group - average absorbance value of the zero adjustment well).

[0174] Figure 11 is a cell drug effect result measured after 2 h of pre-administration of a- asarone at a final concentration of 0.5, 1, 2 μM, and then 24 h of administration of glutamate at a final concentration of 10 mM. As can be seen from the figure, compared with the Glu group, the absorbance of the different pre-administration groups increased significantly, and the cell viability was the largest at 2 μM. This shows that the pre-administration of a-asarone has a protective effect on R28 cells.

[0175] Experimental Example 6 Protective Effect of a-Asarone Emulsion Eye Drops on Inflammation-induced Optic Neuritis Rat Model

[0176] 1. Reagents and Materials

[0177] 6-8 week old SD rats, a total of 34, SPF level, female, weighing 180-220 g; guinea pig spinal cord homogenate; complete Freund's adjuvant (CFA); pertussis vaccine; a-asarone emulsion eye drops (prepared according to Example 2, concentration 2 mg / mL)

[0178] 2. Animal grouping and administration method

[0179] The SD rats were adaptively fed for one week, and 10 of the SD rats were randomly selected as a normal control group (Control group, n = 10). The remaining 24 SD rats were injected subcutaneously with guinea pig spinal cord homogenate and complete Freund's adjuvant (CFA) as an antigen, and pertussis vaccine was injected subcutaneously into the hind limbs to establish a rat experimental autoimmune encephalomyelitis (EAE) model. The Control group was injected with the same amount of normal saline in the same way. Twelve of the successfully modeled rats were randomly selected as an a-asarone group, and the a-asarone emulsion eye drops (2 mg / mL) with a volume of 20 μL were administered to the a-asarone group of rats by eye drop administration every 3.5 h during 8:30-18:00 for three consecutive days. The remaining 12 rats were an EAE group, and the same volume of blank emulsion eye drops was administered by eye drop administration every 3.5 h during 8:30-18:00 for three consecutive days.

[0180] 3. Evaluation method and results

[0181] After 3 weeks of administration, the retinas of rats were removed to detect the MDA content, and the results are shown in Table 14, Figure 12. The data results are presented in the form of mean ± standard deviation, and after normality test and homogeneity test of experimental data, one-way ANOVA is used for statistics; post-hoc comparison uses LSD test (homogeneity of variance) or Dunnett-t test (inhomogeneity of variance); P<0.05 is considered to have significant difference, and all data are processed and analyzed using IBM SPSS Statistics 27, and all statistical graphs are plotted using GraphPad Prism 9.3.0.

[0182] Table 14 MDA content of retinas of rats in each group (n=10)

[0183] Note: compared with the Control group, #### P<0.0001; compared with the EAE group, **** P<0.0001.

[0184] As shown in Table 14, Figure 12, the MDA content results show that the EAE group is significantly higher than the α-asarone group and the Control group, and the difference is significant (P<0.0001); while there is no significant difference between the Control group and the α-asarone group, indicating that the EAE group has severe oxidative damage, while the α-asarone group has no obvious damage. Therefore, α-asarone can alleviate inflammation-induced retinal damage.

[0185] Example 7 Protective effect of α-asarone emulsion eye drops on streptozotocin-induced diabetic retinopathy rat model

[0186] 1. Reagents and materials

[0187] A total of 34 SD rats, 6-8 weeks old, SPF level, male, weighing 180-220 g; streptozotocin (STZ); α-asarone emulsion eye drops (prepared in Example 2, concentration of 2 mg / mL).

[0188] 2. Animal grouping and administration method

[0189] The SD rats were adaptively fed for one week, and 10 SD rats were randomly selected as the normal control group (Control group, n=10). The remaining 24 SD rats were given STZ solution (60 mg / kg) by intraperitoneal injection (i.p.), and 72 h after injection of STZ, the blood glucose value was measured 3 times, and the value ≥16.7 mmol / L was considered to be a successful model. The Control group was injected with the same amount of normal saline in the same way.

[0190] The 12 rats with successful modeling were randomly selected as the a-asarone group, and the a-asarone emulsion eye drops (2 mg / mL) with a volume of 20 μL were given to the a-asarone group of rats in the form of eye drop administration every 3.5 h during 8:30-18:00 for 12 consecutive weeks; the remaining 12 rats were STZ group, and the same volume of blank emulsion eye drops were given in the form of eye drop administration every 3.5 h during 8:30-18:00 for 12 consecutive weeks.

[0191] 3. Evaluation method and result

[0192] After 12 weeks of administration, the eyeballs of the rats were removed for TUNEL staining to observe the retinal cell apoptosis, and the results are shown in Table 15 and FIG. 13. The data results are presented in the form of mean ± standard deviation, and after normality test and homogeneity test of experimental data, one-way ANOVA is used for statistics; post-hoc comparison uses LSD test (homogeneous variance) or Dunnett-t test (inhomogeneous variance); P<0.05 is considered to have significant difference, all data are processed and analyzed using IBM SPSS Statistics 27, and all statistical graphs are plotted using GraphPad Prism 9.3.0.

[0193] Table 15 Retinal Apoptotic Cell Number (n=10)

[0194] Note: Compared with the Control group, ### P<0.001; compared with the STZ group, *** P<0.001.

[0195] As shown in Table 15 and FIG. 13, the TUNEL staining results show that the number of apoptotic cells in the STZ group is significantly more than that in the a-asarone group and the Control group, and there is a significant difference (P<0.001); while there is no significant difference between the Control group and the a-asarone group, indicating that the retinal damage in the STZ group is more serious due to high blood sugar, while the a-asarone group has no obvious damage. Therefore, a-asarone can reduce the retinal damage of STZ-induced diabetic rats.

[0196] Example 8 Protective effect of a-asarone emulsion eye drops on D-galactose-induced cataract rat model

[0197] 1. Reagents and materials

[0198] 6-8 weeks old SD rats, 34 in total, SPF level, male, body weight 180-220 g; D-galactose; a-asarone emulsion eye drops (prepared in Example 2, concentration 2 mg / mL)

[0199] 2. Animal grouping and administration method

[0200] The SD rats were adaptively fed for one week, and 10 SD rats were randomly selected as the normal control group (Control group, n = 10). The remaining 24 SD rats were given D-galactose (200 mg / kg) by subcutaneous injection, once a day, for 4 consecutive weeks. The degree of lens turbidity of the rats was observed under slit lamp illumination. If the lens appeared turbid, the model was determined to be successfully constructed. The Control group was injected with the same amount of normal saline in the same way.

[0201] Twelve successfully modeled rats were randomly selected as the a-asarone group, and the a-asarone emulsion eye drops (2 mg / mL) with a volume of 20 μL were given to the a-asarone group of rats by eye drop administration every 3.5 h during 8:30-18:00 for 4 consecutive weeks. The remaining 12 rats were used as the D-galactose group (Veh group), and the same volume of blank emulsion eye drops was given by eye drop administration every 3.5 h during 8:30-18:00 for 12 consecutive weeks.

[0202] 3. Evaluation method and results

[0203] After 3 weeks of administration, the rat retinas were removed to detect the MDA content, and the results are shown in Table 16 and FIG. 14. The data results are presented in the form of mean ± standard deviation. After normality test and variance homogeneity test on the experimental data, single factor analysis of variance (One-way ANOVA) was used for statistics; post-hoc comparison used LSD test (variance equal) or Dunnett-t test (variance not equal); P < 0.05 was considered to have significant difference. All data were processed and analyzed using IBM SPSS Statistics 27, and all statistical graphs were plotted using GraphPad Prism 9.3.0.

[0204] Table 16 MDA content of retinas of rats in each group (n = 10)

[0205] Note: compared with the Control group, #### P < 0.0001; compared with the Veh group, **** P < 0.0001.

[0206] The results are shown in Table 16 and Fig. 14. The MDA results show that the MDA content of the Veh group was significantly more than that of the Control group and the a-cubebin group, and there was a significant difference (P < 0.0001); while there was no significant difference between the Control group and the a-cubebin group, indicating that the Veh group had severe oxidative damage due to hyperglycemia, while the a-cubebin group had no obvious damage. Therefore, a-cubebin can alleviate the oxidative damage to the retina caused by D-galactose induction.

[0207] Experimental Example 9 Protective Effect of a-Cubebin Emulsion Eye Drops on a Scopolamine Hydrobromide-Induced Dry Eye Syndrome Rat Model

[0208] 1. Reagents and materials

[0209] 34 SD rats, 6-8 weeks old, SPF level, female, weighing 180-220 g; scopolamine hydrobromide (SCOP); a-cubebin emulsion eye drops (prepared according to the method in Example 2, concentration 2 mg / mL).

[0210] 2. Animal grouping and administration method

[0211] The SD rats were adaptively fed for one week, and 10 of the SD rats were randomly selected as the normal control group (Control group, n = 10). The remaining 24 SD rats were given SCOP (12.5 mg / kg) by subcutaneous injection, 3 h / time (9:00, 12:00, 15:00, 18:00).

[0212] Twelve of the model rats were randomly selected as the a-cubebin group, and the a-cubebin emulsion eye drops (2 mg / mL) with a volume of 20 μL were given to the a-cubebin group rats by eye drop administration every 3.5 h during 8:30-18:00 for one week. The remaining 12 rats were randomly selected as the D-galactose group (Veh group), and the same volume of blank emulsion eye drops were given to the rats by eye drop administration every 3.5 h during 8:30-18:00 for one week.

[0213] 3. Evaluation method and results

[0214] The tear secretion of rats in each group was detected on the 7th day after modeling, and the results are shown in Table 17 and FIG. 15. The data results are presented in the form of mean ± standard deviation. After normality test and homogeneity test of experimental data, one-way ANOVA was used for statistics; post-hoc comparison used LSD test (homogeneous variance) or Dunnett-t test (inhomogeneous variance); P<0.05 was considered to have significant difference. All data were processed and analyzed using IBM SPSS Statistics 27, and all statistical graphs were plotted using GraphPad Prism 9.3.0.

[0215] Table 17 Tear secretion of rats in each group (n=10)

[0216] Note: compared with the Control group, #### P<0.0001; compared with the SCOP group, * P<0.05.

[0217] As shown in Table 17 and FIG. 15, the results of tear secretion detection of rats in each group showed that the tear secretion of the SCOP group was significantly less than that of the Control group and the alpha-asarone group, and had a significant difference, indicating that SCOP caused a significant decrease in tear secretion and led to the occurrence of dry eye, while alpha-asarone could alleviate the decrease in tear secretion induced by SCOP and play a role in protecting the ocular surface.

[0218] From the experimental results of experimental examples 1-9, it can be known that the alpha-asarone and the composition thereof of the present application can effectively treat and / or prevent retinal ganglion cell damage related diseases (such as glaucoma, optic neuritis, diabetic retinopathy), neovascular age-related macular degeneration, cataract, dry eye disease.

[0219] Experimental Example 10 Protective effect of alpha-asarone oral emulsion / alpha-asarone emulsion injection on sodium iodate-induced dry age-related macular degeneration rat model

[0220] The inventors found through experimental research that when the model rats were given alpha-asarone oral emulsion at a dose of 60 mg / kg by gavage (i.g.), the comprehensive pharmacodynamic performance was basically consistent with that of alpha-asarone emulsion injection at a dose of 30 mg / kg by intraperitoneal injection (i.p.). To avoid repetition, only the pharmacodynamic results of alpha-asarone emulsion injection are listed below.

[0221] 1. Reagents and materials

[0222] 6-8 weeks old SD rats, a total of 30 (purchased from Chengdu Dashuo Experimental Animal Co., Ltd.), SPF level, male, body weight 180-220 g; sodium iodate; a-sinacen oral emulsion / a-sinacen emulsion injection (prepared according to the method in Example 1, the dose is 10 mg / mL)

[0223] 2. Animal grouping and administration method

[0224] The experimental rats were randomly divided into three groups, namely the normal control group (Control group, n = 6), the model control group (SI group, n = 12), and the a-sinacen emulsion injection group (a-sinacen group, n = 12).

[0225] One week in advance, the a-sinacen group of rats was given a-sinacen at a dose of 30 mg / kg by intraperitoneal injection (i.p.), and the SI group was given the same volume of blank emulsion injection (preparation method same as Example 1, where the a-sinacen prescription amount is 0 kg) by the same method; on the 8th day, the SI group and the a-sinacen group were given sodium iodate at a dose of 30 mg / kg for modeling, and the Control group was given the same volume of normal saline; all rats were modeled by single tail vein injection.

[0226] After modeling, the rats in the a-sinacen group were given a-sinacen at a dose of 30 mg / kg, and the SI group was given the same volume of blank emulsion injection by the same method, once a day, for 3 weeks of continuous intraperitoneal administration. The data results are presented in the form of mean ± standard deviation. After normality test and homogeneity test of experimental data, single factor analysis of variance (One-way ANOVA) was used for statistics; post-hoc comparison used LSD test (homogeneous variance) or Dunnett-t test (heterogeneous variance); P < 0.05 is considered to be significantly different. All data were processed and analyzed using IBM SPSS Statistics 27, and all statistical graphs were plotted using GraphPad Prism 9.3.0.

[0227] 3. Evaluation method and results

[0228] On the 7th, 14th, and 21st days after modeling, ERG was detected to observe the waveform changes, and the results are shown in Figure 16 and Tables 18-21; on the 22nd day, OCT was detected to observe the changes in retinal thickness, and the results are shown in Table 22, Figure 17, and Figure 18. After the 22nd day, the eyeballs of rats in each group were removed to make paraffin sections, which were subjected to HE staining and immunofluorescence staining, and the results are shown in Table 23, Figure 19, and Figure 20, as well as Table 24, Figure 21, and Figure 22; the retinas of rats in each group were removed to extract proteins for detecting the contents of P2X7, NLRP3, and Caspase-1 proteins, and the results are shown in Figure 23 and Figure 24.

[0229] The specific results are as follows:

[0230] (1)ERG detection results

[0231] Table 18 Change of amplitude of b-wave in dark adaptation 0.01 ERG (μV) (n=12)

[0232] Table 19 Change of amplitude of a-wave in dark adaptation 3.0 ERG (μV) (n=12)

[0233] Table 20 Change of amplitude of b-wave in dark adaptation 3.0 ERG (μV) (n=12)

[0234] Table 21 Change of total amplitude of oscillatory potential in dark adaptation 3.0 ERG (μV) (n=12)

[0235] Note: The detection time refers to the 7th, 14th, 21st day after modeling; compared with the Control group, #### P<0.0001; compared with the SI group, * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001.

[0236] As shown in Tables 18-21 and Figure 16, under the flash stimulation of dark adaptation 0.01 ERG, dark adaptation 3.0 ERG, and dark adaptation 3.0 oscillatory potential, the amplitude at each time point was ranked as: Control group > α-asarone group > SI group. After 14 days and 21 days of continuous administration, the α-asarone group had significant or extremely significant difference compared with the SI group (P<0.05 or P<0.0001), indicating that α-asarone can effectively increase the amplitude of each flash stimulation and reduce the damage of sodium iodate to the retinal photoreceptor cells.

[0237] (2) OCT detection results

[0238] Table 22 Retinal thickness of each group before pre-administration for 1 week and after modeling for 3 weeks (μm) (n=14)

[0239] Note: compared with the Control group, #### P<0.0001; compared with the model control group, **** P<0.0001.

[0240] As shown in Table 22, FIG. 17 and FIG. 18, the retinal thickness of the SI group and the a-cubebine group was significantly smaller than that of the Control group (P<0.001), and the retinal thickness of the a-cubebine group was significantly higher than that of the SI group (P<0.001), indicating that a-cubebine can significantly improve the damage of sodium iodate to the cells of each layer of the retina and maintain the normal thickness of the retina.

[0241] (3) HE staining results

[0242] Table 23: ONL thickness of each group in the pre-administration of 1 week and post-administration of 3 weeks (μm) (n=10)

[0243] Note: compared with the Control group, #### P<0.0001; compared with the model control group, **** P<0.0001.

[0244] FIG. 19 shows the retinal structure of each group detected at the time point of pre-administration of 1 week and post-administration of 3 weeks. As shown by HE staining, the ONL cells of the Control group were arranged in an orderly and compact manner, and the thickness was uniform; the ONL cells of the SI group were arranged in a disordered manner and the thickness was uneven, and there was obvious damage; the ONL cells of the a-cubebine group were arranged in an orderly and compact manner and the thickness was uniform, and there was no obvious damage.

[0245] FIG. 20 shows the average value of the ONL thickness of each group and the statistical results thereof. As shown in the figure, the ONL thickness of the SI group and the a-cubebine group was significantly smaller than that of the Control group (P<0.0001), and the retinal thickness of the a-cubebine group was significantly higher than that of the SI group (P<0.0001). Therefore, a-cubebine can effectively reduce the damage of sodium iodate to the ONL of the retina, including the change in the thickness of the ONL and the arrangement of the cells.

[0246] (4) Immunofluorescence

[0247] Table 24: Statistical results of the number of apoptotic cells in the retinal TUNEL staining of each group in the pre-administration of 1 week and post-administration of 3 weeks (number) (n=7)

[0248] Note: compared with the Control group, #### P<0.0001; compared with the model control group, **** P<0.0001.

[0249] From Fig. 21 A1-A3, Table 24, Fig. 22, it can be seen that the number of apoptotic cells in the SI group was significantly more than that in the a-corydine group and the control group, and there was a significant difference (P < 0.0001); while there was no significant difference between the control group and the a-corydine group, indicating that the SI group was severely damaged, while the a-corydine group had no obvious damage.

[0250] From Fig. 21 B1-B3, C1-C3, it can be seen that Rhodopsin and RPE65 expression decreased in the SI group, while normal expression in the a-corydine group, indicating that the function of the rod cells and RPE layer in the SI group was impaired, while a-corydine could effectively protect the functional integrity of the rod cells and RPE layer in the retina.

[0251] From Fig. 21 D1-D3, it can be seen that ZO-1 expression decreased in the SI group while normal expression in the a-corydine group, indicating that the RPE structure in the SI group was destroyed, while a-corydine could effectively protect the structural integrity of the RPE layer in the retina.

[0252] (5) Western Blot experiment

[0253] After 1 week of preventive administration and 3 weeks of modeling and re-administration, the rats were sacrificed, the rat retinas were removed and stored in a -80°C refrigerator for Western Blot experiment to detect the expression of RPE65, P2X7, NLRP3, Caspase-1 proteins in the retina. The results are shown in Fig. 23 and Fig. 24.

[0254] From Fig. 23, 24, it can be seen that RPE65 expression decreased in the SI group while normal expression in the a-corydine group, indicating that a-corydine could effectively protect the function and structure of RPE in the retina after SI induction; P2X7, NLRP3, Caspase-1 may be involved in the promotion of inflammation, photoreceptor degeneration and other links in the pathogenesis of AMD, promoting the development of dry macular degeneration, P2X7, NLRP3, Caspase-1 expression increased in the SI group while decreased in the a-corydine group, indicating that a-corydine could reduce the inflammatory response induced by SI.

[0255] In summary, a-corydine can effectively alleviate the retinal damage caused by SI, increase the amplitude of ERG waveform, increase the thickness of the retina, improve the arrangement of ONL cells, reduce the number of apoptotic cells, increase the expression of Rhodopsin, RPE65, ZO-1 in the retina, and down-regulate the expression of P2X7, NLRP3, Caspase-1 protein, and has a significant protective effect on damaged retina.

[0256] The results show that a-cubebin and its various preparation products can prevent or treat dry age-related macular degeneration, and are expected to provide a new candidate drug for the clinical prevention and treatment of dry age-related macular degeneration.

[0257] Experimental Example 11 Protective effect of a-cubebin bulk drug on sodium iodate damaged ARPE-19 cells

[0258] 1. Reagents and materials

[0259] Human retinal pigment epithelial cells (ARPE-19) were from Wuhan Punsai Life Science and Technology Co., Ltd.; sodium iodate (Macklin, S817792); a-cubebin was from the School of Pharmacy, West China University of Sichuan; CCK-8 (Biosharp, BS350C); DMEM / F12 medium (containing double antibodies) (Kaiji Biological, KGM12500-500); fetal bovine serum (ZETA, 120214018-4S); PBS (Kaiji Biological, KGB5001); DMSO (Solebao, D8371).

[0260] 2. Experimental steps

[0261] DMEM / F12 complete medium: mix DMEM / F12 medium (containing double antibodies) and fetal bovine serum at a volume ratio of 9:1, and store in a 4°C refrigerator.

[0262] a-cubebin stock solution: weigh 104.3 mg of a-cubebin powder into a 2 mL EP tube, add 1 mL of cell-grade DMSO, filter with a 0.22 μm filter, and obtain a 500 mM a-cubebin stock solution.

[0263] Sodium iodate stock solution: weigh 198.0 mg of sodium iodate powder into a 15 mL centrifuge tube, add 10 mL of serum-free medium, sonicate to dissolve, and filter with a 0.22 μm filter to obtain a 100 mM sodium iodate stock solution.

[0264] (1) Sodium iodate modeling concentration screening: take ARPE-19 cells in logarithmic growth phase cultured in complete medium, and dilute them to 1×10 4The cells were seeded at a density of 100 μL / well in 96-well plates, with the edge wells filled with sterile PBS. The plates were incubated at 37°C and 5% CO2 for 24 h until the cells were fully adherent. The supernatant was discarded, and sodium iodate solution with final concentrations of 0 mM, 5 mM, 7.5 mM, 10 mM, 12.5 mM, and 15 mM was added. The plates were then incubated at 37°C and 5% CO2 for 24 h. Subsequently, 110 μL of the solution was added to each well. ARPE-19 cells were cultured in 10% CCK-8 solution at 37°C and 5% CO2 for 2 hours. The OD value at 450 nm was measured using a microplate reader, and ARPE-19 cell viability was calculated. Using the formula "Cell viability = [(Average absorbance of experimental group - Average absorbance of zeroing well) / (Average absorbance of normal control group - Average absorbance of zeroing well]", a sodium iodate concentration of 12.5 mM was selected as the concentration for damage in subsequent cell experiments, specifically for the following cellular pharmacodynamic experiments.

[0265] (2) Screening of α-Asarum pharmacodynamics: ARPE-19 cells in the logarithmic growth phase cultured in complete culture medium were subjected to 1×10⁻⁶ HCl. 4 Cells were seeded at a density of 100 μL / well in 96-well plates, with the edge wells filled with sterile PBS. Cells were incubated at 37°C and 5% CO2 for 24 h until complete adhesion. The supernatant was discarded, and 50 μL of α-asarone solution with final concentrations of 0.25 μM, 0.5 μM, 1 μM, 2 μM, and 4 μM were added to each well. Cells were incubated at 37°C and 5% CO2 for 2 h. Then, 50 μL of sodium iodate solution diluted with serum-free medium to a final concentration of 12.5 mM was added to each well of the above-mentioned drug-treated groups, and the cells were incubated at 37°C and 5% CO2 for another 24 h. The control group received only the same volume of serum-free medium. The model group received 50 μL of serum-free medium followed by 50 μL of sodium iodate solution diluted with serum-free medium to a final concentration of 12.5 mM. The remaining procedures for the control and model groups were the same as those for the drug-treated groups. Subsequently, 110 μL of 10% CCK-8 solution was added to each well, and the cells were cultured at 37°C and 5% CO2 for 2 h. The OD value at 450 nm was detected using a microplate reader, and the ARPE-19 cell viability was calculated using the formula "cell viability = [(average absorbance value of experimental group - average absorbance value of zeroing well) / (average absorbance value of normal control group - average absorbance value of zeroing well]". The results are shown in Figure 25.

[0266] Figure 25 is a cell pharmacodynamic result measured after pre-administration of a- asarone at a final concentration of 0.25, 0.5, 1, 2, 4 μM for 2 h, and then administration of sodium iodate at a final concentration of 12.5 mM for 24 h. As can be seen from the figure, compared with the SI group, the absorbance of each concentration of the prophylactic administration group increased and had a significant difference, and the cell viability was the largest at 2 μM. This shows that a- asarone prophylactic administration can effectively reduce the damage to ARPE-19 cells caused by sodium iodate through oxidative stress, and the subsequent experiment selects 2 μM a- asarone.

[0267] (3) Annexin V-FITC / PI apoptosis detection: detection of the effect of a- asarone on ARPE-19 cell apoptosis: ARPE-19 cells in the logarithmic growth phase cultured in complete medium were inoculated into a 12-well plate at a density of 2 x 10 5 cells / 1000 μL / well, and cultured at 37 °C, 5% CO2 for 24 h until the cells were completely adherent; the cells were divided into a blank control group, a biological control group, a single positive control group 1 (Annexin V-FITC Reagent), a single positive control group 2 (PI Reagent), and an experimental group: a Control group, an SI group, and an a- asarone group; at the end of the culture, the supernatant was discarded, 1 mL of a- asarone solution at a concentration of 2 μM was added to the wells of the a- asarone group, and the cells were cultured at 37 °C, 5% CO2 for 2 h; then 500 μL of sodium iodate solution at a final concentration of 12.5 mM diluted with serum-free medium was added to each well of the above SI group and a- asarone group, and the cells were cultured at 37 °C, 5% CO2 for 24 h. The blank control group, the biological control group, and the Control group were treated in the same way; the single positive control groups 1 and 2 and the SI group were treated in the same way.

[0268] After 24 h of drug administration, the drug solution was sucked into a 2 mL EP tube, 1 mL PBS was added to wash 3 times, 200 μL of trypsin without EDTA was added for 2 min of room temperature digestion, 500 μL of DMEM / F12 complete medium was added to terminate the digestion, the cells were collected in a 2 mL EP tube, centrifuged at 300 x g for 5 min, the supernatant was discarded, washed once with PBS, centrifuged at 300 x g for 5 min, 100 μL of diluted 1 x Annexin V Binding Buffer was added to gently resuspend the cells. 2.5 μL of Annexin V-FITC Reagent and 2.5 μL of PI Reagent were added to the cell suspension of the experimental group. Meanwhile, 2.5 μL of Annexin V-FITC Reagent and 2.5 μL of PI Reagent were added to the single positive control group 1 and 2, respectively, 2.5 μL of Annexin V-FITC Reagent and 2.5 μL of PI Reagent were added to the biological control group, and no dye was added to the blank control group. After adding all the dyes, gently vortex to mix, incubate at room temperature for 20 min in the dark, add 200 μL of diluted 1 x Annexin V Binding Buffer, and mix the sample. When detected by flow cytometry, select the FITC and PerCP / Cy5.5 channels for detection. The results are shown in Figures 26 and 27.

[0269] Figures 26 and 27 are results of detecting cell apoptosis by the Annexin V-FITC / PI method after pre-administration of α-asarone at a final concentration of 2 μM for 2 h, and then administration of sodium iodate at a final concentration of 12.5 mM for 24 h.

[0270] As can be seen from Figures 26 and 27, compared with the Control group, the total apoptosis rate of the SI group and the α-asarone group was significantly increased, and the difference was significant (P < 0.0001); compared with the SI group, the total apoptosis rate of the α-asarone group was significantly reduced, and the difference was significant (P < 0.0001). It can be seen that α-asarone can significantly reduce the apoptosis of ARPE-19 cells induced by SI.

[0271] Experimental Example 12 Protective Effect of α-Asarone Emulsion Eye Drops on Sodium Iodate-induced Dry Age-related Macular Degeneration Rat Model

[0272] 1. Reagents and materials

[0273] 22 SPF level, male, 6-8 weeks old SD rats (purchased from Chengdu Dashuo Experimental Animal Co., Ltd.), weighing 180-220 g; sodium iodate; α-asarone emulsion eye drops (prepared according to the method in Example 2, concentration of 2 mg / mL).

[0274] 2. Animal grouping and administration method

[0275] The experimental rats were randomly divided into three groups, namely the normal control group (Control group, n=6), the model control group (SI group, n=8), and the α-asarone emulsion eye drop group (α-asarone group, n=8).

[0276] The α-asarone emulsion eye drop group was given 20 μL of α-asarone emulsion eye drops (2 mg / mL) by eye drop administration every 3.5 h from 8:30 to 18:00 every day one week in advance; on the 8th day, the model control group and the α-asarone emulsion eye drop group were given 30 mg / kg of sodium iodate, and the normal control group was given the same dose of normal saline, and all rats were modeled by single tail vein injection. After modeling, the α-asarone emulsion eye drop group was given 20 μL of α-asarone emulsion eye drops every 3.5 h from 8:30 to 18:00 every day for 2 weeks.

[0277] 3. Evaluation method and results

[0278] On the 14th day after modeling, the changes in ERG observation waveform were detected, and the data results were presented in the form of mean ± standard deviation. After normality test and variance homogeneity test on the experimental data, one-way ANOVA was used for statistics; post-hoc comparison used LSD test (homogeneous variance) or Dunnett-t test (inhomogeneous variance), P<0.05 was considered to have significant difference, all data were processed and analyzed using IBM SPSS Statistics 27, all statistical graphs were plotted using GraphPad Prism 9.3.0, and the results are shown in FIG. 28 and Tables 25-28.

[0279] Table 25 Change in 0.01 ERG b-wave amplitude in dark adaptation (μV)(n=18)

[0280] Table 26 Change in 3.0 ERG a-wave amplitude in dark adaptation (μV)(n=18)

[0281] Table 27 Change in 3.0 ERG b-wave amplitude in dark adaptation (μV)(n=18)

[0282] Table 28 Change in 3.0 oscillatory potential total amplitude in dark adaptation (μV)(n=18)

[0283] Note: The detection time refers to the 14th day after modeling; compared with the Control group, # P<0.05; ## P<0.01; compared with the model control group, * P<0.05; ** P<0.01

[0284] As shown in Tables 25-28 and Figure 28, under the dark adaptation 0.01 ERG, the dark adaptation 3.0 ERG, and the dark adaptation 3.0 oscillatory potential flash stimulation, the amplitude size ranking measured when pre-administered for 1 week and administered for 2 weeks after modeling was: the normal control group > the α-asarone emulsion eye drop group > the model control group, indicating that α-asarone can effectively increase the amplitude of each flash stimulation and reduce the retinal damage caused by SI.

[0285] As can be seen from the experimental results of Experimental Examples 10-12, α-asarone and various preparation products thereof provide a more optimal administration route and drug selection for treating and / or preventing age-related macular degeneration.

[0286] The above examples only exemplarily illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.

Claims

1. Use of asarone in the preparation of a medicament for treating and / or preventing an ocular disease, the ocular disease including a retinal ganglion cell damage disease and macular degeneration, cataract or dry eye; the asarone including α-asarone, β-asarone or γ-asarone.

2. Use according to claim 1, characterized in that, The retinal ganglion cell damage disease includes glaucoma, optic neuritis or diabetic retinopathy.

3. Use according to claim 1, characterized in that, The ocular disease is macular degeneration, and the asarone is α-asarone.

4. Use according to claim 3, characterized in that, The macular degeneration includes dry macular degeneration, vascular leakage caused by neovascular age-related macular degeneration or visual dysfunction caused by macular degeneration.

5. A composition characterized in that, The composition includes an effective concentration of asarone, the asarone including at least one of α-asarone, β-asarone and γ-asarone, and the final concentration of the asarone is 0.5-30 g / L.

6. The composition of claim 5, wherein, The α-asarone is the only effective active ingredient in the composition.

7. The composition of claim 6, wherein, The composition further includes an oil solvent, an emulsifier, an osmotic pressure regulator, a pH regulator and water.

8. The composition of claim 7, wherein, The oil solvent includes soybean oil, the emulsifier includes egg yolk lecithin, the osmotic pressure regulator includes glycerol and the pH regulator includes sodium hydroxide.

9. The composition of claim 8, wherein, The dosage form of the composition is eye drops, oral preparation or injection.

10. The composition of claim 9, wherein, The eye drops include water as a solvent and the following components: α-asarone 0.5-5 g / L, soybean oil 4-50 g / L, egg yolk lecithin 1-15 g / L, glycerol 2-25 g / L and sodium hydroxide 0.001-0.005 g / L.

11. The composition of claim 9, wherein, The oral preparation or injection includes water as a solvent and the following components: α-asarone 4-30 g / L, soybean oil 80-300 g / L, egg yolk lecithin 10-20 g / L, glycerol 20-30 g / L and sodium hydroxide 0.02-0.1 g / L.

12. Use of the composition of any one of claims 5-11 in the preparation of a medicament for treating and / or preventing an ocular disease, the ocular disease including glaucoma, optic neuritis, diabetic retinopathy, macular degeneration, cataract or dry eye.

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