Application of mulberroside A in preparation of medicine for preventing and treating age-related macular degeneration

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a natural product mulberroside A in preparation of a medicine for preventing and treating age-related macular degeneration. The mulberroside A has protection activity on damaged ARPE-19 cells in an in-vitro experiment, can reduce the generation of ROS and MDA, and plays a remarkable anti-oxidation role. In addition, mulberroside A can up-regulate PARP, and Bcl-2 has a remarkable anti-apoptosis effect in an in-vitro dry AMD model. The mulberroside A has a good retina protection effect in an in-vivo dry AMD model, and can resist partial retina injury induced by NaIO3.

Description

Technical field: The invention belongs to the technical field of biomedicine, and specifically relates to the application of the natural product mulberry glycoside A in the preparation of a drug for preventing and treating age-related macular degeneration. Background technology: Age-related macular degeneration (AMD) is a progressive degenerative disease of the macula and the third leading cause of blindness in the world after cataracts and glaucoma. AMD can be divided into two forms: dry AMD and wet AMD, with dry AMD accounting for as much as 85% to 90%. Currently, there is no effective drug for the treatment of dry AMD in clinical practice. Therefore, the development of anti-dry AMD therapeutic drugs with good therapeutic effects is a clinical problem that needs to be solved urgently.

[0003] Natural products have the advantages of safety and high efficiency, and are still an important source for new drug development. However, due to differences in structure, different natural products have very different activities. After long-term and arduous research and practice, the inventors found that mulberry glycoside A has the effect of preventing and treating AMD from a large number of natural products. Summary of the invention: In view of this, the purpose of the present invention is to provide a use of a natural product, mulberry glycoside A, in the preparation of a drug for preventing and treating age-related macular degeneration, in order to address the problem that there is currently no effective drug for treating dry age-related macular degeneration in clinical practice.

[0005] Specifically, the structure of the mulberry glycoside A of the present invention is as follows: Formula I The ineffective compounds used for comparison of the present invention include: proanthocyanidin B2, vitex glycosides, stevioside, dicoumarol, naringin, mulberry flavone G, neoandrographolide, hydroxysafflor yellow A, verbascoside, ginsenoside Rg1, troxerutin, reduced coenzyme I disodium salt, citronelloside, vitexin, myricetin, salvianolic acid B, rubusoside, clematisin I, veratramide, amygdalin, geniposide, sinapinic acid, chicoric acid, atractylodes lactone III, atractylodes lactone II, astragaloside IV, ginsenoside Rb1, peony glycoside, artemisinin, and catalpol.

[0006] Preferably, the natural product mulberry glycoside A of the present invention can be used to prepare a therapeutic drug for age-related macular degeneration, wherein the age-related macular degeneration in the disease is preferably dry age-related macular degeneration.

[0007] Preferably, the drug has the following effects: protecting retinal lesions, reducing the production of reactive oxygen and malondialdehyde, and alleviating retinal cell apoptosis.

[0008] Specifically, MTT results showed that among the 31 natural products, only mulberry glycoside A had protective activity against ARPE-19 cells, and mulberry glycoside A was basically non-toxic to cells when the concentration increased to 50 µM; flow cytometry, ROS immunofluorescence experiment and MDA detection experiment results showed that mulberry glycoside A could reduce the production of ROS and MDA, and exert a significant antioxidant effect; in addition, flow cytometry and Hoechst-PI staining experiments showed that mulberry glycoside A could reduce the total cell apoptosis rate; Western blot experimental results showed that mulberry glycoside A could upregulate PARP and Bcl-2, that is, mulberry glycoside A had a significant anti-apoptotic effect in the in vitro dry AMD model.

[0009] Specifically, fundus photography and OCT results showed that mulberry glycoside A can reduce the number of drusen and high-reflection points caused by NaIO3 damage, and improve the phenomenon of retinal thinning; HE staining results showed that mulberry glycoside A can reduce swollen bundles of RPE cells and improve disordered cells, that is, mulberry glycoside A has a good retinal protective effect in the in vivo dry AMD model and can resist partial retinal damage induced by NaIO3.

[0010] Another object of the present invention is to provide a pharmaceutical composition for treating age-related macular degeneration, which contains a therapeutically effective amount of the compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutical excipient.

[0011] Preferably, the "pharmaceutical excipients" refer to conventional drug carriers in the pharmaceutical field, such as diluents, excipients such as water, fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium / magnesium stearate, polyethylene glycol, etc. In addition, other adjuvants such as flavoring agents, sweeteners, etc. may be added to the composition.

[0012] The present invention provides a pharmaceutical composition for treating age-related macular degeneration, wherein the pharmaceutical composition is in the form of an ophthalmic preparation, an injection, a tablet, a capsule, an aerosol, a suppository, a film, a pill, an ointment, a controlled-release or sustained-release preparation, and a nano preparation.

[0013] Preferably, the drug preparation is an ophthalmic preparation.

[0014] Preferably, the ophthalmic preparations include but are not limited to dry eye drops, eye ointments, eye sprays, eye gels, eye patches, intraocular injections, ophthalmic microspheres, ocular implants, periocular injections and ophthalmic sustained-release preparations. Description of the drawings: In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0016] Attached Figure 1 :Activity screening of natural products and cell protection activity of mulberry glycoside A. (A, B) 20 μM of 31 natural products [1 (proanthocyanidin B2), 2 (vitexin), 3 (stevioside), 4 (dicoumarin), 5 (naringin), 6 (mulberry flavone G), 7 (neoandrographolide), 8 (hydroxysafflor yellow A), 9 (verbascoside), 10 (ginsenoside Rg1), 11 (troxerutin), 12 (reduced coenzyme I disodium salt), 13 (citrus glycoside), 14 (vitexin), 15 (myricetin), 16 (salvia phenol)] were added to the cytoprotective agent. ARPE-19 cells were pretreated with mulberry glycoside A (A) or different concentrations (5-50 µM) of mulberry glycoside A (B) for 18 h, then treated with 12 mM NaIO3 for 24 h, and the cell survival rate was detected by MTT method. (C) Cells were treated with different concentrations (5-50 µM) of mulberry glycoside A for 42 h, and the cytotoxicity was detected by MTT method. ####P<0.0001 relative to the solvent control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 relative to the NaIO3 group.

[0017] Attached Figure 2 :Mulberiside A attenuates NaIO3-induced oxidative stress in ARPE-19 cells. (AC) ARPE-19 cells were pretreated with mulberiside A (20 µM) for 18 h, treated with NaIO3 (12 mM) for 48 h, and incubated with DCFH-DA probe for 30 min. Intracellular ROS levels were detected by flow cytometry (AB) and immunofluorescence (C). (D) Intracellular MDA content was detected according to the instructions of the MDA kit. ####P<0.0001 relative to the solvent control group, **P<0.01, ***P<0.001 relative to the NaIO3 group.

[0018] Attached Figure 3:Mulberiside A inhibits NaIO3-induced apoptosis of ARPE-19 cells. (AC) ARPE-19 cells were pretreated with mulberiside A (20 µM) for 18 h and treated with NaIO3 (16 mM) for 48 h. Cell apoptosis was detected by flow cytometry after Annexin V / PI double staining (AB), or Hoechst and PI staining were performed for 25 min and then photographed using an inverted fluorescence microscope (C). (D) ARPE-19 cells were plated in 96-well plates using phenol red-free 1640 medium containing 10% inactivated FBS. ARPE-19 cells were pretreated with mulberiside A (20 µM) for 18 h and treated with NaIO3 (8 mM) for 24 h. The LDH release level in the supernatant was detected according to the instructions of the LDH kit. ####P<0.0001 relative to the solvent control group, ***P<0.001, ****P<0.0001 relative to the NaIO3 group.

[0019] Attached Figure 4 :Mulberiside A protects retinal tissue in an in vivo dry AMD model. C57BL / 6J mice were intraperitoneally injected with mulberiside A (100 mg / kg) for 7 consecutive days, followed by tail vein injection of NaIO3 (25 mg / kg), and then continued to be intraperitoneally injected with mulberiside A for 5 consecutive days. (AB, D) Fundus photography (A) and OCT (B, D) were used to detect the extent of mouse retinal lesions. The black arrows indicate drusen and the red arrows indicate high reflective points. (C) Five days after the tail vein injection of NaIO3, the mice were killed by cervical dislocation and the eyeballs were removed and HE staining was used to examine the histological changes. The image magnification was 40x. The white arrows indicate the deformation of the outer nuclear layer (ONL), and the yellow arrows indicate the swelling and bundling of RPE cells. Ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), rod and cone layer (R&CL), retinal pigment epithelium (RPE). ####P<0.0001 relative to the solvent control group, ****P<0.0001 relative to the NaIO3 group. Specific implementation method: The present invention also provides a pharmacodynamic evaluation method of the natural product mulberry glycoside A in a cell model and an animal model, as well as specific application details such as their dosage, administration method and administration cycle.

[0021] Example 1 Screening of natural products for anti-dry AMD activity in vitro and ARPE-19 cell protective activity of mulberry glycoside A: MTT experiment: When ARPE-19 cells grow to the logarithmic growth phase, they are inoculated in 96-well plates at 5000 / well, with 100 μL of culture medium per well. After the cells adhere to the wall overnight, different concentrations of drugs are added for incubation for 18 h, and then 12 mM NaIO3 is added for 24 h. 20 μL of MTT solution is added to each well. After 4 h of action, the culture medium and MTT mixture are aspirated with a syringe, and 120 μL of DMSO is added to each well. The cells are shaken on a shaker until the blue-purple crystalline formazan is completely dissolved. The light absorption value (OD value) at 490 nm is detected using an enzyme-linked immunosorbent assay. Cell activity (%) = (mean OD value of the drug group / mean OD value of the solvent group) × 100%, and the experiment is repeated three times.

[0022] The ARPE-19 cell line is a fast-growing immortalized human cell line. NaIO3 is a stress-inducing agent that has been widely used in both in vitro and in vivo models of dry AMD. Therefore, this example uses NaIO3-injured ARPE-19 cells as an in vitro model of dry AMD. These natural products with anti-inflammatory and antioxidant effects were screened for activity in the ARPE-19 cell NaIO3-injured model, as shown in the attached Figure 1 As shown in Figure A, only Mulberiside A among the 31 natural products has a significant protective effect. Compared with the NaIO3-treated group, Mulberiside A increased the activity of ARPE-19 cells by nearly 20%. Therefore, this example further verifies the concentration gradient cell activity and toxicity of Mulberiside A. Figure 1 As shown in Figure 2, mulberry glycoside A has a significant protective effect on ARPE-19 cells at 10 μM. As the concentration gradually increases, the protective effect also gradually increases, but the protective effect slightly decreases at 50 μM. Figure 1 As shown in Figure C, mulberry glycoside A did not show obvious toxicity within a concentration of 50 µM. The above results show that mulberry glycoside A has a significant protective effect in the NaIO3-induced ARPE-19 cell injury model.

[0023] Example 2 Mulberry glycoside A reduces NaIO3-induced oxidative stress damage in ARPE-19 cells: Flow cytometry and immunofluorescence techniques detect reactive oxygen species (ROS) using the fluorescent probe DCFH-DA. DCFH-DA is hydrolyzed by esterase in cells to generate DCFH, which is then oxidized by ROS to fluorescent DCF. The fluorescence intensity of DCF is used to assess the level of intracellular ROS. ARPE-19 cells were treated under specific conditions and labeled with DCFH-DA to analyze the level of ROS. The MDA experiment assesses cellular oxidative stress by measuring the concentration of malondialdehyde (MDA), which involves preparing TBA storage solution and working solution, establishing a standard curve, and calculating the relative content of MDA. All experiments were repeated three times to ensure the accuracy of the results.

[0024] Oxidative stress refers to cell damage caused by reactive oxygen intermediates and is associated with many disease processes, especially age-related diseases. Lipid oxidation leads to the production of malondialdehyde (MDA). Therefore, detecting the levels of intracellular ROS and MDA can determine the occurrence of oxidative stress in cells. In order to evaluate whether mulberry glycoside A has an antioxidant effect in an in vitro dry AMD model, the level of ROS in ARPE-19 cells was first detected. Figure 2 As shown in Figures AC, compared with the solvent control group, the intracellular ROS level increased significantly after NaIO3 injury, while pretreatment with mulberry glycoside A significantly reduced the ROS level, and incubation with mulberry glycoside A alone did not affect the cellular ROS level. Then, the content of intracellular lipid peroxidation product MDA was detected, as shown in the attached Figure 2 As shown in D, the MDA content of cells treated with NaIO3 increased significantly, while pretreatment with mulberry glycoside A greatly reduced the MDA content. In summary, NaIO3 treatment significantly increased the level of oxidative stress in ARPE-19 cells, while pretreatment with mulberry glycoside A significantly reduced the level of oxidative stress in cells.

[0025] Example 3 Mulberoside A inhibits NaIO3-induced apoptosis of ARPE-19 cells: ARPE-19 cells were seeded in 6-well plates during the logarithmic growth phase. After drug treatment, flow cytometry and Hoechst-PI staining experiments were used to detect cell apoptosis. In flow cytometry, cells were trypsinized, washed with PBS, stained with a dye solution containing Annexin V-FITC and PI, and then apoptosis was detected on a flow cytometer. In the Hoechst-PI staining experiment, cells were first treated with Hoechst dye solution, then treated with PI dye solution, and finally observed under a fluorescence microscope. All experiments were repeated three times to ensure the reliability of the results. In the LDH experiment, INT solution (1×) and LDH detection working solution were first prepared, and ARPE-19 cells were cultured in phenol red-free 1640 medium plus 10% inactivated FBS. After the cells were treated with drugs, the supernatant was collected and the LDH detection working solution was added. After incubation, the OD value was measured on an enzyme-linked immunosorbent assay, and the relative content of LDH was calculated. The experiment was repeated three times.

[0026] Increased ROS can induce autophagy and apoptosis by damaging cellular components. Previous studies have reported the presence of apoptosis in oxidant-induced RPE injury. In order to investigate the exact role of mulberry glycoside A in NaIO3-induced RPE injury, we used flow cytometry to detect the type of cell death. Figure 3 As shown in AB, compared with the solvent control group, after NaIO3 treatment, Annexin V-positive and PI-positive cells increased significantly, and about 50% of ARPE-19 cells underwent apoptosis. Morusin A pretreatment significantly reduced the total apoptosis rate of cells. This example also used Hoechst and PI staining to detect the morphology and fluorescence intensity of the cell nucleus after drug incubation. The results are shown in the attached Figure 3 As shown in Figure C, compared with the solvent control group, after ARPE-19 cells were treated with NaIO3, apoptotic cells produced stronger blue and red fluorescence, the cell nuclei shrank and deformed, and appeared shiny and white. The fluorescence intensity of cells pretreated with mulberry glycoside A was significantly weakened, and the number of shrunken, deformed, shiny and white cell nuclei was also significantly reduced. The detection of lactate dehydrogenase LDH level can indirectly detect the toxicity level of cells. The results are shown in the attached figure. Figure 3 As shown in D, NaIO3 treatment significantly increased the level of LDH release, while mulberry glycoside A significantly reduced the release of LDH. In summary, mulberry glycoside A can significantly reduce NaIO3-induced apoptosis of ARPE-19 cells.

[0027] Example 4 Mulberoside A protects retinal tissue in an in vivo dry AMD model: C57BL / 6J male mice were housed under conditions consistent with NIH guidelines and were randomly divided into four groups after acclimatization: solvent control group, NaIO3 model group, NaIO3+mulberry glycoside A group, and mulberry glycoside A group. Mice in the NaIO3 model group and NaIO3+mulberry glycoside A group received tail vein injection of sodium iodate, while the control group and mulberry glycoside A group received PBS. The NaIO3+mulberry glycoside A group and mulberry glycoside A group also received intraperitoneal injection of mulberry glycoside A, while the other groups received the corresponding solvents. For tail vein injection, mice were fixed and vasodilated with alcohol, and injections were performed using a 30G insulin syringe. After injection, cotton swabs were used to press and stop bleeding. For fundus photography, OCT imaging, and mouse retinal HE staining experiments, mice were first anesthetized with intraperitoneal injection of sodium pentobarbital and mydriasis was dilated with compound tropicamide eye drops. For fundus photography and OCT, mice were placed on a tray and adjusted until the fundus was clearly visible, and images were acquired using a small animal fundus camera and an OCT scanner, respectively. In the HE staining experiment, the eyeballs were removed immediately after the mice were killed, fixed, the anterior segment and vitreous body were cut off, and then dehydrated, embedded and sliced. Finally, hematoxylin-eosin staining was performed and the retinal structure was observed and photographed under a microscope. After all operations, the mouse cornea was smeared with eye ointment and placed on a heating pad to keep it moist and warm, waiting for awakening.

[0028] Mice are widely used as in vivo models of dry AMD. NaIO3 can produce a large amount of ROS in retinal tissue, aggravating the oxidative stress response, thereby inducing AMD formation. The degree of damage increases with the increase of NaIO3 dose. Therefore, this experiment used NaIO3 (25 mg / kg) injected into the tail vein of mice to simulate the in vivo dry AMD model. The experimental results are shown in the attached figure. Figure 4 As shown in A, after 5 days of tail vein injection of NaIO3 (25 mg / kg), fundus photographs showed a large number of diffuse punctate lesions in the mouse retina, namely drusen-like deposits (black arrows). Continuous intraperitoneal injection of mulberoside A (100 mg / kg) sharply reduced the area of ​​retinal degeneration in mice, and drusen-like deposits were significantly reduced. Compared with the solvent control group, mice given mulberoside A alone showed no lesions. OCT was used to detect retinal lesions in mice, and the experimental results are shown in the attached figure. Figure 4 As shown in B, compared with the solvent control group, 5 days after tail vein injection of NaIO3, the retina of mice was unclear, disordered, and had more high-reflective lesions (red arrows), and the retinal thickness was also significantly thinner (see Figure 2). Figure 4 D) Continuous intraperitoneal injection of mulberry glycoside A significantly reduced the number of retinal hyperreflective lesions in mice, and the reduction of retinal thickness was improved to a certain extent (Appendix Figure 4 D). After killing the mice, the eyeballs were removed and HE staining was performed to examine the histological changes. The results are shown in the attached Figure 4As shown in C, NaIO3 (25 mg / kg)-induced retinal damage showed significant changes in the RPE layer, rod and cone layer (R&CL) (also known as the photoreceptor (PR) layer), and outer nuclear layer (ONL). Compared with the solvent control group, the cells in all these layers in the NaIO3 model group were disordered, and raised deposits were formed on the retinal pigment epithelium (RPE) (swelling and bundling of RPE cells, yellow arrows). Compared with the NaIO3 model group, continuous intraperitoneal injection of mulberoside A improved the arrangement of cells in all these layers and significantly protected the structure of the RPE layer by reducing the number of deposits. In summary, mulberoside A can resist partial damage to retinal tissue induced by NaIO3 in the in vivo dry AMD model.

[0029] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of mulberry glycoside A in the preparation of a drug for treating age-related macular degeneration, wherein the structural formula of mulberry glycoside A is shown in Formula I: Formula I The mulberry glycoside A is a compound monomer extracted and separated from natural plants and an organic compound synthesized by artificial chemical synthesis.

2. The use according to claim 1, wherein the age-related macular degeneration is preferably dry age-related macular degeneration.

3. The use according to claims 1 and 3, characterized in that: The drug has the following but not limited to the following effects: inhibiting retinal cell apoptosis and protecting retinal lesions by reducing the generation of reactive oxygen and malondialdehyde.

4. A pharmaceutical composition for treating age-related macular degeneration, comprising a therapeutically effective amount of the compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutical excipient.

5. The pharmaceutical composition according to claim 5, wherein the compound according to claim 1 or a pharmaceutically acceptable salt thereof is the sole active ingredient.

6. The pharmaceutical composition according to claim 6, characterized in that: The pharmaceutical composition is in the form of an ophthalmic preparation, an injection, a tablet, a capsule, an aerosol, a suppository, a film, a pellet, an ointment, a controlled-release or sustained-release preparation, and a nano preparation.

7. The use according to claim 7, characterized in that: The ophthalmic preparations include eye drops, eye ointments, eye sprays, eye gels, eye patches, intraocular injections, ophthalmic microspheres, ocular implants, periocular injections and ophthalmic sustained-release preparations.

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