Compound for preventing and treating retinal ganglion cytopathy as well as preparation method and application thereof

Abstract

The invention provides a compound for preventing and treating retinal ganglion cytopathy as well as a preparation method and application thereof, and particularly relates to the field of biological medicines. The invention provides a strain of Umbellopsisin A, the Umbellopsisin A metabolized by the Umbellopsisin A can significantly increase the number of viable retinal ganglion cells and also can improve visual conduction dysfunction, and the Umbellopsisin A can effectively pass through a blood retina barrier to achieve efficient prevention of omentum ganglion cell death, so that the Umbellopsisin A can be used for preparing medicines for treating retinal ganglion cell death. And retina injury can be repaired.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a compound for preventing and treating retinal ganglion cell lesions, a preparation method thereof, and an application thereof. Background Art

[0002] Retinal ganglion cells (RGCs) are central nervous system neurons located in the retina, and their axons extend to the optic nerve. The degeneration of retinal ganglion cells leads to optic disc lesions and ultimately visual impairment, and is associated with various diseases, such as glaucoma, hereditary optic neuropathy, ischemic optic neuropathy, and demyelinating diseases. More than 70 million people are suffering from glaucoma, and approximately 10% of people have bilateral blindness, making glaucoma the leading cause of irreversible blindness worldwide. Some types of glaucoma are usually asymptomatic in the early stage, indicating that the actual number of affected individuals may be significantly higher than the known number. Current treatments mainly focus on controlling intraocular pressure (IOP) to slow down the progression of the disease. However, even with IOP management, studies have shown that RGC death and visual field loss continue.

[0003] The clinical application of neuroprotective drugs for glaucoma is still highly restricted, and there is currently no FDA-approved therapy specifically for retinal ganglion cell protection. Neuroprotective methods, including NMDA receptor antagonists, α2-adrenergic agonists, neurotrophic factors, calcium channel blockers, antioxidants, statins, and nicotinamide, have shown significant potential in preclinical studies. However, these methods face major challenges in clinical translation, including low bioavailability, limited efficacy in clinical trials, and difficulty in effectively crossing the blood-retinal barrier. Therefore, developing effective strategies to prevent retinal ganglion cell death is a key breakthrough point in glaucoma treatment. In this regard, due to the high structural diversity, wide range of biological activities, multi-target mechanisms, and potentially more favorable safety characteristics of natural products, the discovery of natural products with neuroprotective potential has become a hot research direction in the future. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a compound for preventing and treating retinal ganglion cell lesions, a preparation method thereof, and an application thereof.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a fungus Umbelopsis sp. DWS.372, and the preservation number of the fungus Umbelopsis sp. DWS.372 is: CGMCC NO.41875.

[0007] The present invention provides a compound for preventing and treating retinal ganglion cell lesions. The compound is named umbelopsisin A and has a structure as shown in Formula 1:

[0008]

[0009] Formula I.

[0010] The present invention provides a preparation method of the compound, including the following steps:

[0011] 1) Propagate the umbelliferous mold fungus DWS.372 with Sabouraud dextrose medium to obtain a bacterial solution;

[0012] 2) Inoculate the bacterial solution on rice medium and culture for 20 - 40 days to obtain a rice ferment;

[0013] 3) Extract the rice ferment with ethyl acetate to obtain a crude extract;

[0014] 4) Subject the crude extract to macroporous resin chromatography, and elute successively with water, methanol solution with a mass concentration of 15% - 25%, methanol solution with a mass concentration of 70 - 85%, methanol solution with a mass concentration of 90% - 100%, and ethyl acetate to obtain fractions Fr.A - Fr.E respectively;

[0015] 5) Subject fraction Fr.C to silica gel column chromatography, gradient elute with a mixed solution of petroleum ether and ethyl acetate as the eluent, and after separating the obtained eluate by thin layer chromatography, combine similar components. A total of 16 components are obtained, and they are sorted according to the Rf value from large to small to obtain Fr.1 - Fr.16;

[0016] 6) Separate fraction Fr.8 through Sephadex LH - 20 to obtain ten components Fr.8.1 - Fr.8.10, and purify fraction Fr.8.7 by semi - preparative HPLC to obtain the compound;

[0017] The mobile phase of fraction Fr.8 through Sephadex LH - 20 is methanol, and the flow rate is 0.05 - 0.2 mL / min;

[0018] The mobile phase for purifying fraction Fr.8.7 by semi - preparative HPLC is a solution with a mass ratio of methanol: water = 60 - 70:30 - 40, the flow rate is 2.0 - 4.0 mL / min, and the retention time is 23 - 25 min.

[0019] Preferably, in step 1), the culture temperature for propagation is 20 - 26°C, the culture time is 4 - 8 days, and the concentration of the strains in the bacterial solution is 10 6 ~10 8 CFU / mL.

[0020] Preferably, the preparation method of the rice culture medium in step 2) includes mixing rice and water, and sterilizing at 100-130 °C for 20-40 min to obtain the rice culture medium;

[0021] The mass-volume ratio of rice to water is 90-110 g: 70-90 mL.

[0022] Preferably, the number of times of ethyl acetate extraction in step 3) is 8-12 times;

[0023] The volume-mass ratio of ethyl acetate used for each extraction to the rice is 15-25 L: 6-10 kg.

[0024] Preferably, the mass-volume ratio of the crude extract to water in step 4) is 70-90 g: 3-7 L;

[0025] The mass-volume ratio of the crude extraction to the methanol solution with a mass concentration of 15%-25% is 70-90 g: 4-8 L;

[0026] The mass-volume ratio of the crude extraction to the methanol solution with a mass concentration of 70%-85% is 70-90 g: 10-14 L;

[0027] The mass-volume ratio of the crude extraction to the methanol solution with a mass concentration of 90%-100% is 70-90 g: 3-7 L;

[0028] The mass-volume ratio of the crude extraction to ethyl acetate is 70-90 g: 3-7 L.

[0029] Preferably, the mesh number of the silica gel column in step 5) is 200-300.

[0030] The present invention provides the application of the compound in the preparation of a drug for treating glaucoma.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a strain of Umbelopsis fungus DWS.372. The compound umbelopsisin A metabolized by the Umbelopsis fungus DWS.372 can significantly increase the number of surviving retinal ganglion cells, can also improve visual conduction dysfunction, and the compound umbelopsisin A can effectively pass through the blood-retinal barrier to achieve efficient prevention of retinal ganglion cell death and repair retinal damage. Description of the Drawings

[0033] Figure 1 For the key 1 H- 1 H COSY and HMBC signals of the compound umbelopsisin A;

[0034] Figure 2 Experimental and calculated ECD curves of compound umbelopsisin A;

[0035] Figure 3 Screening of the protective activity of compound umbelopsisin A against glutamate-induced R28 cell damage, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns: no significant difference, all compared with the Glu group;

[0036] Figure 4 Effect diagrams of compound umbelopsisin A protecting against glutamate-induced R28 cell damage. Among them, A is the Hoechst / PI staining experiment, and living cells are labeled with Hochest (blue); dead cells are labeled with PI (red), and the scale bar is 200 μm; B is the histogram of the viability of R28 cells treated with 2 μM compound umbelopsisin A; C is the histogram of the fluorescence intensity of DCFH-DA in R28 cells; D is the detection of intracellular reactive oxygen species levels in R28 cells using the DCFH-DA probe, and the green fluorescence intensity represents the intracellular reactive oxygen species level, and the scale bar is 100 μm; E is the detection of the fluorescence density of DCFH-DA in R28 cells by flow cytometry; ***P<0.001 compared with the Glu group, **P<0.01 compared with the Glu group, #P<0.0001 compared with the Control group;

[0037] Figure 5 Effect diagrams of compound umbelopsisin A protecting against NMDA-induced mouse retinal damage. Among them, A is the immunofluorescence staining of the whole mouse retina 5 days after intravitreal injection of NMDA, and the immunofluorescence staining images of the central, middle, and peripheral parts of the retina. RGCs are labeled with Brn3a (green), and the scale bar is 20 μm. B is a schematic diagram of the retinal area, and RGCs are quantified in three regions (central, middle, peripheral) in three of the four quadrants of the retina; C is a schematic diagram of intravitreal injection in the mouse retina; (D) Histogram of the number of remaining ganglion cells in the central, middle, and peripheral regions of the mouse retina 5 days after intravitreal injection of NMDA;

[0038] Figure 6This is the effect diagram of the changes in the retina of mice 5 days after intravitreal injection of NMDA. A is the H&E staining image of the retina section of mice 5 days after intravitreal injection of NMDA, and the scale bar is 50 μm; B is the measurement of the GCC thickness of mice at ±300, ±600, ±900, ±1200, and ±1500 μm from the optic nerve 5 days after intravitreal injection of NMDA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the Glu group; #P<0.0001 compared with the Control group; ns: no significant difference.

[0039] Figure 7 To investigate the visual function of compound umbelopsisin A in protecting NMDA-induced retinal damage. A is the visual evoked potential map of mice 5 days after intravitreal injection of NMDA, B is the electroretinogram of mice 5 days after intravitreal injection of NMDA, C is the quantitative statistical histogram of the latency of the P1 wave in the visual evoked potential map of mice 5 days after intravitreal injection of NMDA, D is the quantitative statistical histogram of the amplitudes of the N1 and P1 waves in the visual evoked potential map of mice 5 days after intravitreal injection of NMDA, E is the quantitative statistical histogram of the amplitudes of the a and b waves in the electroretinogram of mice 5 days after intravitreal injection of NMDA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 are all compared with the Glu group; ##P<0.01, #P<0.0001 compared with the Control group; ns: no significant difference.

[0040] Biological deposit description

[0041] The present invention provides a strain of fungus Umbelopsis sp. DWS.372, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 31, 2025. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.41875. Detailed implementation manners

[0042] The present invention provides a strain of fungus Umbelopsis sp. DWS.372, and the deposit number of the fungus Umbelopsis sp. DWS.372 is: CGMCC NO.41875.

[0043] The present invention provides a compound for preventing and treating retinal ganglion cell lesions, and the compound is named umbelopsisin A, and its structure is shown in Formula 1:

[0044]

[0045] Formula I.

[0046] The present invention provides a method for preparing the compound, comprising the following steps:

[0047] 1) Propagate the umbelliferous mold fungus DWS.372 with Sabouraud dextrose medium to obtain a bacterial solution;

[0048] 2) Inoculate the bacterial solution into rice medium and culture for 20 - 40 days to obtain a rice ferment;

[0049] 3) Extract the rice ferment with ethyl acetate to obtain a crude extract;

[0050] 4) Subject the crude extract to macroporous resin chromatography, and elute successively with water, methanol solution with a mass concentration of 15% - 25%, methanol solution with a mass concentration of 70 - 85%, methanol solution with a mass concentration of 90% - 100%, and ethyl acetate to obtain fractions Fr.A - Fr.E respectively;

[0051] 5) Subject fraction Fr.C to silica gel column chromatography, elute with a mixed solution of petroleum ether and ethyl acetate as an eluent by gradient elution, and after separating the obtained eluate by thin - layer chromatography, combine similar components. A total of 16 components are obtained, and they are sorted according to the Rf value from large to small to obtain Fr.1 - Fr.16;

[0052] 6) Component Fr.8 is separated by Sephadex LH - 20 to obtain ten components Fr.8.1 - Fr.8.10, and the compound is obtained by semi - preparative HPLC purification of component Fr.8.7;

[0053] In the present invention, in step 1), the culture temperature for the propagation is preferably 20 - 26 °C, more preferably 21 - 25 °C, and even more preferably 23 °C; the culture time is preferably 4 - 8 days, more preferably 5 - 7 days, and even more preferably 6 days; the concentration of the strain in the bacterial solution is preferably 10 6 ~10 8 CFU / mL, more preferably 10 7 CFU / mL.

[0054] In the present invention, in step 2), the culture is a dark culture; the culture time is 20 - 40 days, preferably 25 - 35 days, more preferably 30 days; the culture temperature is preferably 20 - 26 °C, more preferably 21 - 25 °C, and even more preferably 23 °C.

[0055] In the present invention, the preparation method of the rice culture medium in step 2) preferably includes mixing rice and water and sterilizing them to obtain the rice culture medium; the sterilization temperature is preferably 100-130 °C, more preferably 110-125 °C, and even more preferably 121 °C; the sterilization time is preferably 20-40 min, more preferably 25-35 min, and even more preferably 30 min; the mass-volume ratio of rice to water is preferably 70-90 g: 90-110 mL, more preferably 75-85 g: 95-105 mL, and even more preferably 80 g: 100 mL.

[0056] In the present invention, the number of times of ethyl acetate extraction in step 3) is preferably 8-12 times, more preferably 9-11 times, and even more preferably 10 times; the volume-mass ratio of ethyl acetate used for each extraction to the rice is preferably 15-25 L: 6-10 kg, more preferably 18-23 L: 7-9 kg, and even more preferably 20 L: 8 kg; the time for each extraction is preferably 8-16 h, more preferably 10-14 h, and even more preferably 12 h; the extraction temperature is preferably 20-26 °C, more preferably 21-25 °C, and even more preferably 23 °C; after extraction, the extract is preferably concentrated, and the concentration method is preferably rotary evaporation under reduced pressure until a solid is obtained. The concentration temperature is preferably 30-40 °C, more preferably 33-38 °C, and even more preferably 35 °C. The rotation speed is preferably 40-80 revolutions per minute, more preferably 50-70 revolutions per minute, and even more preferably 60 revolutions per minute, and the pressure is preferably 0.098 MPa.

[0057] In the present invention, the mass-volume ratio of the crude extract to water in step 4) is preferably 70-90 g: 3-7 L, more preferably 75-85 g: 4-6 L, and even more preferably 80 g: 5 L.

[0058] The mass-volume ratio of the crude extraction to a methanol solution with a mass concentration of 15%-25% is preferably 70-90 g: 4-8 L, more preferably 75-85 g: 5-7 L, and even more preferably 80 g: 6 L; the concentration of the methanol solution is 15%-25%, preferably 18%-23%, and even more preferably 20%.

[0059] The mass-volume ratio of the crude extraction to a methanol solution with a mass concentration of 70%-85% is preferably 70-90 g: 10-14 L, more preferably 75-85 g: 11-13 L, and even more preferably 80 g: 12 L; the concentration of the methanol solution is 70%-85%, preferably 75%-83%, and more preferably 80%.

[0060] The mass-to-volume ratio of the crude extraction to a methanol solution with a mass concentration of 90% to 100% is preferably 70 to 90 g: 3 to 7 L, more preferably 75 to 85 g: 4 to 6 L, and even more preferably 80 g: 5 L; the concentration of the methanol solution is 90% to 100%, preferably 100%.

[0061] In the present invention, the flow rate of the elution in step 4) is independently preferably 80 to 120 mL / min, more preferably 90 to 110 mL / min, and even more preferably 100 mL / min.

[0062] The mass-to-volume ratio of the crude extraction to ethyl acetate is preferably 70 to 90 g: 3 to 7 L, more preferably 70 to 90 g: 3 to 7 L, further more preferably 75 to 85 g: 4 to 6 L, and even more preferably 80 g: 5 L.

[0063] In the present invention, the mesh number of the silica gel column in step 5) is preferably 200 to 300, more preferably 220 to 280, and even more preferably 250.

[0064] In the present invention, the volume ratios of petroleum ether to ethyl acetate in step 5) are 80:1, 50:1, 20:1, 15:1, 10:1, 5:1, 3:1, 2:1, 1:1 in sequence.

[0065] In the present invention, the mobile phase of the component Fr.8 through Sephadex LH-20 is methanol, and the flow rate is preferably 0.05 to 0.2 mL / min, more preferably 0.07 to 0.15 mL / min, and even more preferably 0.1 mL / min;

[0066] In the present invention, the mobile phase for purifying the component Fr.8.7 by semi-preparative HPLC is methanol: aqueous solution, the flow rate is preferably 2.0 to 4.0 mL / min, more preferably 2.5 to 3.5 mL / min, and even more preferably 3.0 mL / min, the retention time is preferably 23 to 25 min, more preferably 24.07 min, the volume ratio of methanol: water is preferably 60 to 70: 30 to 40, more preferably 62 to 68: 32 to 38, and even more preferably 64: 36. The chromatographic column used in the purification process is preferably Supersil ODS-2, and the specifications are preferably 10.0 mm × 250 mm, 5 μm.

[0067] The present invention provides the use of the compound in the preparation of a drug for treating glaucoma. The compound exerts its function in the drug by significantly increasing the number of surviving retinal ganglion cells and inhibiting the thinning of the retinal ganglion cell complex, etc.

[0068] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0069] Material source

[0070] Table 1 Experimental instruments and reagent consumables

[0071]

[0072]

[0073]

[0074] Example 1 Preparation of compound umbelopsisin A

[0075] (1.1) Strain fermentation

[0076] Preparation of rice medium: Take a 500 mL conical flask, add 80 g of rice and 100 mL of tap water to each flask, mix well, cover with a breathable membrane and tie it with a rubber band, then place it in an autoclave for sterilization at 121 °C for 30 min. After sterilization, cool it to room temperature for standby (a total of 8.0 kg of rice is used for this strain and evenly distributed into 100 500 mL conical flasks).

[0077] SDA medium: Glucose (20 g / L), peptone (10 g / L), yeast extract (1 g / L), agar (15 g / L).

[0078] SDB medium: Glucose (20 g / L), peptone (10 g / L), yeast extract (1 g / L).

[0079] Inoculate the mycelium of the umbellate mold fungus DWS.372 on the SDA solid medium and culture it at 25 °C for 5 days. After good growth, under sterile conditions, use a small steel spatula to cut 5 - 10 mycelial blocks, with the size of the mycelial blocks about 5 mm × 5 mm, and inoculate them into the SDA liquid medium (the SDA liquid medium in a 500 mL conical flask does not exceed 300 mL). The inoculated liquid medium is incubated at room temperature and 120 r / min for 5 days to obtain the seed liquid, and then the seed liquid is inoculated onto the sterilized rice medium under sterile conditions, about 5 mL of seed liquid per bottle, and cultured under static conditions at room temperature for 30 days. Observe once every 2 days to prevent contamination.

[0080] (1.2) Extraction and separation of the compound

[0081] After the fermentation of the strain is completed, all the fermented culture media are collected and extracted with 20 L of ethyl acetate each time. The extraction temperature is 20 - 26 °C, the extraction time is 12 h each time, and the extraction is carried out 10 times. After concentration under reduced pressure using a rotary evaporator (temperature: 35 °C, rotation speed: 60 rpm), the pressure is 0.098 MPa to obtain a crude ethyl acetate extract, about 80 g. The crude extract is subjected to column chromatography using D101 macroporous resin and gradually eluted with water (5 L), 20% methanol - water (6 L), 80% methanol - water (12 L), pure methanol 100% (5 L), and ethyl acetate (5 L) in sequence at a flow rate of 100 mL / min to obtain a total of 5 components (Fr.A - Fr.E). Component Fr.C is separated by column chromatography on a 200 - mesh normal - phase silica gel column using petroleum ether:ethyl acetate as the eluent for gradient elution. The volume ratios of petroleum ether:ethyl acetate are 80:1, 50:1, 20:1, 15:1, 10:1, 5:1, 3:1, 2:1, 1:1 in sequence. After using thin - layer chromatography (TLC) and coloring with sulfuric acid - ethanol, similar components are combined to obtain a total of 16 components, which are sorted according to the Rf values from large to small to get Fr.1 - Fr.16. Component Fr.8 is separated by Sephadex LH - 20 (CH3OH) to obtain ten sub - components (Fr.8.1 - Fr.8.10), and one bottle is collected every 20 mL. Component Fr.8.7 is purified by semi - preparative HPLC (the volume ratio of CH3OH / H2O is 64:36, the treatment time is 30 min, the flow rate is 3.0 mL / min, and the chromatographic column is Supersil ODS - 2: 10.0 mm×250 mm, 5 μm) to obtain a compound (8.2 mg, t R = 24.07 min).

[0082] (1.3) Detection of the compound

[0083] Red powder, UV(CH3OH - H2O)λ max : 220 nm; IR(KBr)ν max 3438, 1790, 1600, 1227, 1095 cm -1 ; HRESIMS(positive)m / z 366.1348[M + H] + (calcd forC 21 H 19 O5, 366.1336), and the degree of unsaturation is 13.

[0084] The 1H NMR spectrum of compound umbelopsisin A shows 19 proton signals. The 1 H NMR(600 MHz) and 13The \(^{13}\)C NMR (150 MHz) data (CDCl\(_3\)) are shown in Table 2 and include two exchangeable proton signals: δ H 4.96 (1H, s, 6-OH), 8.23 (1H, s, -NH); five vinylic proton signals: δ\(_H\) 7.14 (1H, d, \(J\) = 7.3 Hz, H-11), 7.03 (1H, t, \(J\) = 7.3 Hz, H-12), 7.17 (1H, d, \(J\) = 7.3 Hz, H-13), 7.35 (1H, d, \(J\) = 7.3 Hz, H-14), 7.22 (1H, s, H-16); four methyl signals: δ\(_H\) 3.81 (3H, s, H\(_3\)-18), 2.02 (3H, s, H\(_3\)-19), 2.13 (3H, s, H\(_3\)-20), 2.29 (3H, s, H\(_3\)-21). The \(^{13}\)C NMR spectrum shows 21 carbon signals (Table 2), including two ester carbonyl carbon signals (δ C 173.0, 168.3), fourteen olefinic carbon signals (δ C 153.4, 151.0, 136.7, 133.0, 125.4, 125.2, 122.7, 120.4, 120.3, 118.4, 117.2, 111.5, 109.1, 105.3), one sp 3 quaternary carbon signal (δ C 58.9), four methyl carbon signals (δ C 53.6, 15.3, 11.6, 9.2). 1 H- 1 The H-H COSY spectrum shows the following spin-coupling segments: H-11 / H-12 / H-13 / H-14, combined with the following signals shown in the HMBC spectrum: H-11 / C-13, C-15; H-12 / C-10, C-14; H-16 / C-10, C-15, and combined with the HSQC spectrum, indicating the presence of an indole structure. The HMBC spectrum shows the following signals: H\(_3\)-19 / C-3, C-4, C-5; H\(_3\)-20 / C-4, C-5, C-6; H\(_3\)-21 / C-6, C-7, C-8; 6-OH / C-5, C-6, C-7, and combined with the HSQC spectrum, indicating the presence of a fully substituted benzene ring structure. There are still two ester groups, one quaternary carbon, one methyl, and the remaining one degree of unsaturation belongs to a ring. Therefore, a benzofuranone fragment is established (for the specific structure, see Figure 1)。Since C-2 is a quaternary carbon, the indole ring fragment is connected through C-9 and C-2. In addition, there is a methyl formate substituent at C-2. Thus, the planar structure of this compound is determined. To determine the absolute configuration of this compound, the ECDs of two configurations 1a(2S) and 1b(2R) were calculated at the B3LYP-D3(BJ) / TZVP / / B3LYP-D3(BJ) / TZVP level Figure 2 ), and the absolute configuration of this compound was determined to be 2S, and it was named umbelopsisin A.

[0085] Table 2 1 H NMR(600MHz) and 13 C NMR(150MHz) data (CDCl3)

[0086]

[0087]

[0088] Example 2 Bioactivity Evaluation of Compound umbelopsisin A

[0089] (2.1) Protective Effect of Compound umbelopsisin A on Glutamate-Induced R28 Cell Damage The R28 cell line is an immortalized rat retinal progenitor cell line obtained from the Department of Anatomy, Central South University. Complete medium was prepared by adding 10% fetal bovine serum and 1% penicillin-streptomycin to DMEM medium. R28 cells were cultured at 37 °C and 5% CO2. R28 cells were seeded in 96-well plates at a density of 0.5×10 4 cells / well. After 24 hours, the original culture medium was aspirated, and the experimental group was treated with glutamate (10 mM) solution containing different concentrations of the compound (0, 0.5, 1, 2, 3, 4 μM). The Control group continued to be cultured with complete medium. After 24 hours, 10% CCK-8 solution was used to replace the original medium in all wells. After the plate was cultured in the incubator for 3 hours, the absorbance at a wavelength of 450 nm was measured using a microplate reader. Cell viability was calculated by Graphpad Prism 9.0.0, and the results are shown in detail in Figure 3 .

[0090] The experimental results showed that compared with the Control group, the cell viability was significantly increased after treatment with compound umbelopsisin A. Among them, compound umbelopsisin A had the most prominent activity and had neuroprotective activity at a concentration of 1 μM. At a concentration of 2 μM, the cell viability recovered to 100.6±6.9% (P<0.001, n = 3).

[0091] The apoptosis and necrosis detection kit is used to visually evaluate the viability of R28 cells. The R28 cells were seeded in a 12-well plate at a density of 10×10 4 cells / well. After 24 hours, the original culture medium was aspirated. The cells were divided into three groups: the cells in the Control group continued to be maintained in complete medium, the Glu group was treated only with 10 mM glutamate solution, and the administration group (Glu+1) was treated with complete culture medium containing 2 μM compound umbelopsisin A and 10 mM glutamate. After 24 hours, the original culture medium was discarded, and the cells were rinsed twice with PBS. Then, 500 μL of staining buffer, 2.5 μL of Hoechst staining solution, and 2.5 μL of PI staining solution were added to each well. After incubation for 30 minutes, the cells were rinsed once with PBS. The cell nuclei were observed and photographed under a fluorescence microscope (see A, B, and C in Figure 4 ).

[0092] The results showed that the Hoechst / PI staining experiment further confirmed the protective effect of compound umbelopsisin A. The fluorescence images showed that after treatment with glutamate for 24 hours in the model group, compared with the Control group, the red fluorescence increased significantly, indicating more R28 cell death. Compared with the model group, the red fluorescence in the administration group decreased significantly, indicating less R28 cell death, suggesting that compound umbelopsisin A could protect R28 cells from glutamate-induced damage.

[0093] (2.2) Compound umbelopsisin A inhibits glutamate-induced oxidative stress in R28 cells

[0094] The intracellular reactive oxygen species (ROS) level of R28 cells was detected using a ROS detection kit: First, the cells were digested into a single-cell suspension, and the DCFH-DA reagent was diluted to a working concentration of 10 μM. Subsequently, the DCFH-DA working solution was added to the cells for incubation, and the cells were gently shaken every 5 minutes during incubation. After incubation for 20 minutes, the cells were washed three times with serum-free medium. The mean fluorescence intensity of ROS was analyzed by flow cytometry. In addition, the cells washed with PBS were incubated again with the DCFH-DA working solution, and after completion, they were washed with serum-free medium, and the fluorescence changes of intracellular ROS were observed and recorded under a fluorescence microscope ( Figure 4 D in). The fluorescence images showed that compared with the Control group, the green fluorescence in the Control group increased significantly, indicating an increase in the intracellular ROS level, while the green fluorescence in the administration group was less, indicating a decrease in the intracellular ROS level.

[0095] In addition, the flow cytometry experiment also showed that the intracellular ROS level was significantly decreased after treatment with compound umbelopsisin A ( Figure 4 E in).

[0096] Example 3 Animal Experiment

[0097] (3.1) NMDA-induced Mouse Retinal Injury Model

[0098] Eight-week-old C57BL / 6 mice were selected, fed with standard laboratory food and water, and acclimated in an environment with a 12-hour light-dark cycle. The mice were divided into three groups: Control group (injected with an equal volume of normal saline), NMDA group (injected only with 20 mM NMDA), and NMDA+1 group (injected with 20 mM NMDA and 20 μM compound umbelopsisin A). After the mice were anesthetized with 1% sodium pentobarbital solution, tropicamide eye drops were used to dilate the pupils, proparacaine hydrochloride was used for topical ocular surface anesthesia, and levofloxacin eye drops were used to prevent infection. Under the microscope, a 34-gauge needle was inserted through the limbus corneae to establish an injection channel, and then 1 μL of 20 mM NMDA solution with or without 20 μM compound umbelopsisin A was injected using a microsyringe. The mice in the Control group were injected with an equal volume of normal saline. After injection, the needle was left in the vitreous cavity for 30 seconds and then withdrawn, and gentamicin-dexamethasone eye ointment was applied to the eye surface. Subsequent experiments were carried out five days after injection.

[0099] (3.2) Retinal Immunofluorescence Staining

[0100] Five days after intravitreal injection, the mice were anesthetized again with 1% sodium pentobarbital solution. After perfusion of precooled normal saline through the ventricle, the eyeballs were removed and fixed in 4% paraformaldehyde for 1 hour. The retinal tissues were carefully dissected under the microscope. A blocking solution containing 5% bovine serum albumin (BSA) and 0.5% Triton X-100 was prepared, and the retina was blocked at room temperature for 2 hours. Subsequently, the retina was incubated overnight at 4°C with Brn-3a antibody (rabbit origin, diluted 1:100). The next day, after washing with PBS containing 0.5% Triton X-100, it was incubated with a fluorescently labeled secondary antibody (goat anti-rabbit Alexa Fluor 488, diluted 1:1000) at room temperature for 1.5 hours. After washing, the retinal tissue was laid flat on a glass slide and sealed with an anti-fading mounting medium. Images were observed and recorded through a fluorescence microscope, and quantitative analysis of Brn-3a-positive retinal ganglion cells (RGCs) was performed using ImageJ software. The results are shown in Figure 5 .

[0101] The results showed that compared with the Control group, the green fluorescence in the NMDA group was significantly reduced, indicating a decrease in the number of surviving retinal ganglion cells. Compared with the NMDA group, the green fluorescence in the NMDA+1 group was significantly increased, indicating that compound umbelopsisin A could significantly increase the number of surviving retinal ganglion cells.

[0102] (3.3) H&E Staining

[0103] Five days after the intravitreal injection in step (3.1), the mice were euthanized and their eyeballs were removed, immersed in an eyeball fixative for 24 hours. After dehydration in gradients of 80%, 95%, and 100% ethanol and clearing in xylene, the eyeballs were embedded in paraffin. Sections with a thickness of 3 μm were cut along the sagittal plane of the eyeballs, stained with H&E, and observed under a microscope. The thickness of the ganglion cell layer was measured at 300, 600, 900, 1200, and 1500 μm from the center of the optic nerve using CaseViewer software. The results are shown in Figure 6 .

[0104] Figure 6 A in Figure 6 showed that compared with the Control group, the retinal ganglion cell complex in the NMDA group was significantly thinner. B in Figure 6 showed that the NMDA+1 group significantly inhibited the thinning of the retinal ganglion cell complex at 300 and 600 μm from the optic nerve center.

[0105] (3.4) Functional visual electrophysiological detection (f-VEP and f-ERG)

[0106] Visual evoked potential (VEP) is a cluster of electrical signals generated by the visual cortex in the occipital lobe of the brain in response to visual stimuli. Changes in latency and amplitude can identify lesions between the retina and the visual cortex. Using this principle, it was detected whether compound umbelopsisin A has a protective effect on the visual function of mice. The experimental steps are as follows:

[0107] Five days after the intravitreal injection in step (3.1), the mice were anesthetized with 1% sodium pentobarbital solution. After 15 minutes of dark adaptation, electrodes were implanted subcutaneously in the back, at the anterior angle, and on the occipital bone of the mice, serving as the ground electrode, cathode, and anode, respectively. The contralateral eye was covered with thick gauze and aluminum foil, and a multifocal visual evoked potential recording system was used to record and evaluate visual function. Before the electroretinogram experiment, the mice needed to undergo overnight dark adaptation. During the experiment, they were anesthetized in the dark, mydriatic eye drops (tropicamide) were used to dilate the pupils, and they were placed on a heating pad to maintain body temperature. A semicircular gold ring was used as the recording electrode and gently placed on the corneal surface. A subcutaneous stainless steel needle was inserted subcutaneously at the root of the tail and under the corner of the eye as the ground electrode and reference electrode, respectively. The Reti-scan system was used to record the flash response at different light intensities. The experimental results show the waveforms measured at 3.0 cd / s / m -2 . The results are shown in Figure 7 A, C, and D in showed that compared with the Control group, the amplitude of the N1 wave in the model group was significantly reduced, and the latency of the P1 wave was significantly prolonged, while the amplitude of the N1 wave in the drug-administered group increased and the latency of the P1 wave shortened.

[0108] Electroretinogram (ERG) is a test that measures the electrical responses of retinal cells and is used to evaluate the function of the retina. Measuring the electrical responses of retinal cells using ERG, the images show ( Figure 7 B and E in []) that, compared with the Control group, the amplitudes of the a-wave and b-wave in the NMDA group were significantly reduced, and the latency was also significantly prolonged, and this change was improved in the NMDA + 1 group. Figure 7 The results of [] indicate that the compound umbelopsisin A can improve NMDA-induced visual conduction dysfunction.

[0109] All data in Example 2 were expressed in the form of mean ± standard error (X-±SEM). Data analysis was performed using GraphPad Prism 9.0.0 software, and one-way or two-way analysis of variance was carried out. All statistical tests were performed using a two-tailed test criterion, where: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 or #P < 0.05, ##P < 0.01, P < 0.001, #P < 0.0001.

[0110] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art of the present technology, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An Umbelopsis sp. fungus DWS.372, characterized in that, The preservation number of the umbelopsis fungus DWS.372 is: CGMCC NO.41875.

2. A compound for preventing and treating retinal ganglion cell lesions, characterized in that, The compound is named umbelopsisin A, and its structure is shown in Formula 1: Formula I.

3. A method for preparing the compound according to claim 2, characterized in that, It includes the following steps: 1) Propagate the umbelopsis fungus DWS.372 described in Claim 1 with Sabouraud dextrose medium to obtain a bacterial solution; 2) Inoculate the bacterial solution on the rice medium and culture for 20 - 40 days to obtain a rice ferment; 3) Extract the rice ferment with ethyl acetate to obtain a crude extract; 4) Subject the crude extract to macroporous resin chromatography, and elute successively with water, a methanol solution with a mass concentration of 15% - 25%, a methanol solution with a mass concentration of 70 - 85%, a methanol solution with a mass concentration of 90% - 100%, and ethyl acetate to obtain fractions Fr.A - Fr.E respectively; 5) Subject fraction Fr.C to silica gel column chromatography, elute with a mixed solution of petroleum ether and ethyl acetate as the eluent by gradient elution, and use thin - layer chromatography to separate the obtained eluate and combine similar components. A total of 16 components are obtained, and they are sorted according to the Rf value from large to small to obtain Fr.1 - Fr.16; 6) Component Fr.8 is separated by Sephadex LH - 20 to obtain ten components Fr.8.1 - Fr.8.10, and the component Fr.8.7 is purified by semi - preparative HPLC to obtain the compound described in Claim 2; The mobile phase of component Fr.8 through Sephadex LH - 20 is methanol, and the flow rate is 0.05 - 0.2 mL / min; The mobile phase for purifying component Fr.8.7 by semi - preparative HPLC is a solution with a volume ratio of methanol: water = 60 - 70:30 - 40, the flow rate is 2.0 - 4.0 mL / min, and the retention time is 23 - 25 min.

4. The preparation method according to claim 3, characterized in that, Step 1) The culture temperature for propagation is 20 - 26 °C, the culture time is 4 - 8 d, and the concentration of the strains in the bacterial liquid is 10 6 - 10 8 CFU / mL.

5. The preparation method according to claim 3, characterized in that, The preparation method of the rice medium in step 2) includes mixing rice and water, sterilizing at 100 - 130 °C for 20 - 40 min to obtain the rice medium; The mass - to - volume ratio of rice to water is 90 - 110 g:70 - 90 mL.

6. The preparation method according to claim 5, characterized in that, The number of times of ethyl acetate extraction in step 3) is 8 - 12 times; The volume - to - mass ratio of ethyl acetate to the rice used for each extraction is 15 - 25 L:6 - 10 kg.

7. The preparation method according to claim 3, characterized in that, The mass - to - volume ratio of the crude extract to water in step 4) is 70 - 90 g:3 - 7 L; The mass - to - volume ratio of the crude extract to a methanol solution with a mass concentration of 15% - 25% is 70 - 90 g:4 - 8 L; The mass - to - volume ratio of the crude extract to a methanol solution with a mass concentration of 70% - 85% is 70 - 90 g:10 - 14 L; The mass - to - volume ratio of the crude extract to a methanol solution with a mass concentration of 90% - 100% is 70 - 90 g:3 - 7 L; The mass - to - volume ratio of the crude extract to ethyl acetate is 70 - 90 g:3 - 7 L.

8. The preparation method according to claim 3, characterized in that, The mesh number of the silica gel column in step 5) is 200 - 300.

9. Use of the compound described in Claim 2 in the preparation of a drug for treating glaucoma.

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