Application of daidzin to preparation of medicine and effectiveness verification method of daidzin

Abstract

The invention discloses an application of daidzin to preparation of a medicine and a potency verification method of daidzin, the medicine is applied to improvement of age-related macular degeneration, and the potency verification method comprises in-vitro verification and in-vivo verification; the protection effect of daidzin on oxidative damage caused by SIN-1 is evaluated through an in-vitro experiment, and the protection effect is specifically shown as increase of cell activity and reduction of MDA content; in-vivo verification is completed by means of a rat model, a visual placement reaction experiment is formed, a rat visual function system is evaluated, the MDA content of rat eye peripheral blood is detected, and it is clear that the daidzin can obviously relieve AMD damage, that is, the daidzin has an obvious improvement effect on AMD; and daidzin is good in safety and low in improvement and treatment cost on age-related macular degeneration, and meets the current clinical requirements.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of daidzin in the preparation of drugs, and also relates to a method for verifying the efficacy of this application. Background Art

[0002] Age-related Macular Degeneration (AMD) is a blinding fundus disease closely related to aging, mainly manifested as pathological aging changes in the macular area (the core area of the retina responsible for fine vision and color vision), which can lead to irreversible central vision impairment or loss. This disease is highly prevalent in people over 50 years old.

[0003] At present, the exact pathogenesis of AMD is still unclear. Among them, oxidative stress-induced damage to retinal pigment epithelial cells (RPE) is considered to be one of the key factors in the pathogenesis of AMD. The traditional ways to intervene in age-related macular degeneration mainly include: I. Drug treatment with anti-VEGF drugs and adjuvant drugs. By intravitreal injection of drugs such as ranibizumab, bevacizumab, and conbercept, vascular endothelial growth factor (VEGF) is inhibited, the formation and leakage of new blood vessels are reduced, thereby controlling macular edema and hemorrhage. Regular injections (such as once a month) are required, and antioxidants such as oral vitamin C, vitamin E, and lutein, or hormonal drugs such as triamcinolone acetonide are used to delay the progression of the disease.

[0004] II. Physical therapies using lasers or photodynamics. Among them, laser photocoagulation therapy closes abnormal blood vessels through lasers to reduce bleeding and edema, but it is only applicable to specific lesion types. Photodynamic therapy is to inject a photosensitizer and then activate the drug with a laser of a specific wavelength to selectively destroy abnormal new blood vessels, with less damage to surrounding tissues, but the treatment cost is relatively high.

[0005] III. Surgical treatment, mainly including conventional procedures and innovative procedures. Conventional procedures can be used for submacular hemorrhage removal, choroidal neovascular membrane peeling, etc., which have the effect of directly and quickly removing the lesion. Innovative procedures such as stem cell therapy (combined with choroidal neovascular membrane peeling + stem cell-derived retinal cell transplantation) can repair damaged retinal pigment epithelial cells, and the retinal structure is significantly improved after surgery, but the treatment cost is quite high and the generality is greatly limited.

[0006] IV. Traditional Chinese medicine adjuvant therapy. It mainly relieves the phenomena of visual fatigue and insufficient blood supply to the macular area by means of traditional Chinese medicine, etc. Acupuncture and massage can be used to stimulate eye acupoints to improve local blood circulation. At the same time, diet and living habits can be improved to achieve the purpose of delaying the progression of the disease, but the onset is slow and the effect is not obvious.

[0007] Based on this, through the study of the isoflavone chemical component in Pueraria lobata - daidzin, the prevention and treatment of AMD have been achieved. Summary of the Invention

[0008] The present invention aims at the deficiencies in the prior art and provides an application of daidzin in improving age - related macular degeneration by inhibiting oxidative stress.

[0009] To solve the above - mentioned technical problems, the technical solution of the present invention is: the application of daidzin in preparing a drug, and the drug is applied to improve age - related macular degeneration.

[0010] As a preferred technical solution, the drug improves age - related macular degeneration by inhibiting oxidative stress.

[0011] As a preferred technical solution, the drug is an injection.

[0012] As a preferred technical solution, the administration method of the drug is intraperitoneal injection.

[0013] As a preferred technical solution, the drug includes daidzin and preparation excipients.

[0014] As a preferred technical solution, the preparation excipients include one or a combination of two or more of a suspending agent, a bacteriostatic agent, an antioxidant, a preservative, an isotonicity regulator, and a pH regulator in any ratio.

[0015] The present invention also experimentally verified that daidzin can improve age - related macular degeneration by inhibiting oxidative stress, and daidzin can be applied in the preparation of drugs for improving age - related macular degeneration. The efficacy verification method includes in vitro verification and in vivo verification. The protective effect of daidzin on oxidative damage caused by SIN - 1 is evaluated through the in vitro experiment, which is specifically manifested as an increase in cell viability and a decrease in MDA content; the in vivo verification is completed with the help of a rat model, forming a visual placement reaction experiment to evaluate the visual function system of rats, detecting the MDA content in the peripheral blood of the rat eyes, and clarifying that the application of daidzin can significantly reduce AMD damage, that is, daidzin has an improving effect on AMD.

[0016] The in vitro experiment of the present invention includes the following steps: SP A1 、Mother liquor preparation SP A1-1 Weigh 1 mg of daidzin and dissolve it in 24 μL of DMSO, and mix well at room temperature to prepare a 100 mM mother liquor; SP A1-2 Take 98 μL of DMSO as the dilution solvent, add 2 μL of the mother liquor obtained by mixing daidzin and DMSO, and mix and dilute it into a 2 mM drug - containing solvent; SPA1-3 , take DMSO as the dilution solvent, and use DMSO to perform gradient dilution on the 2 mM drug-containing solvent. By adjusting the amount of DMSO, four drug-containing solvents with concentrations of 2 mM, 1 mM, 0.5 mM, and 0.2 mM are finally prepared in sequence; SP A1-3 , take 2 μL of each of the above drug-containing solvents with different concentrations and place them into the wells of a 96-well plate correspondingly. And 200 μL of DMSO solvent should be pre-placed in each well containing the drug-containing solvent. Use the pre-placed DMSO solvent to adjust the 2 μL of each concentration of the drug-containing solvent added to form drug-containing solvents with concentrations of 20 μM, 10 μM, 5 μM, and 2 μM respectively; SP A1-5 , weigh 1 mg of SIN-1 and dissolve it in 96.8 μL of DMSO, and mix well at room temperature to prepare a 50 mM stock solution for simulating oxidative stress damage of RPE cells; SP A2 Cell culture SP A2-1 , take DMEM as the culture medium, add 10% fetal bovine serum and 1% penicillin-streptomycin to DMEM to form a culture solution. Add retinal pigment epithelial cells (RPE) to the above culture solution for culture. When the cell density reaches 80%, digest the cells and resuspend them, and culture the cells in several wells on a 96-well plate. The standard of the cells in each well is 3000 cells / well; SP A2-2 , group the wells on the 96-well plate with cultured cells to obtain a control group, a model group, and a drug administration group; SP A2-3 , add 2 μL of DMSO to the wells in the control group, and add 2 μL of the 50 mM SIN-1 stock solution to the wells in the model group, so that the cells are cultured in a medium with a concentration of 0.5 mM. After 24 h, perform CCK-8 detection; SP A2-4 , the concentrations of the daidzin-containing drug solvents used in the drug administration group are 2 mM, 1 mM, 0.5 mM, and 0.2 mM respectively, and each well is added separately. The addition amount of each well is 2 μL. After culturing for 4 h, add the same dose and 50 mM SIN-1 stock solution as the model group, and continue to incubate and culture for 24 h. After the incubation and culture are completed, add 20 μL of CCK-8 detection solution for detection.

[0017] As a preferred technical solution, the in vivo experiment includes the following steps, SP B1 Daidzin-containing drug solvent preparation SP B1-1, Weigh two portions of daidzin, 6 mg and 12 mg respectively, and set aside for later use; SP B1-2 , First dissolve 6 mg of daidzin in 0.4 mL of DMSO, then successively add 4 mL of polyethylene glycol 12-hydroxydiol and 35.6 mL of normal saline, and mix the above components at room temperature to obtain a low-dose daidzin drug-containing solvent, and set aside for later use; SP B1-3 , First dissolve 12 mg of daidzin in 0.4 mL of DMSO, then successively add 4 mL of polyethylene glycol 12-hydroxydiol and 35.6 mL of normal saline, and mix the above components at room temperature to obtain a high-dose daidzin drug-containing solvent, and set aside for later use; SP B2 , Rat model SP B2-1 , Take 36 male SD rats of SPF grade, and the body weight of the SD rats is 200 - 220 g. Randomly divide the SD rats into a control group, a sodium iodate group, a daidzin 3 mg / kg group, and a daidzin 6 mg / kg group; SP B2-2 , Inject 50 mg / kg of sodium iodate into the sodium iodate group, the daidzin 3 mg / kg group, and the daidzin 6 mg / kg group respectively through the tail vein to obtain an AMD model, and the control group is injected with the corresponding volume of normal saline to complete the model establishment; SP B2-3 , In the daidzin 3 mg / kg group, according to the dosage of 20 mL / kg of injection volume, inject the low-dose daidzin drug-containing solvent dissolved with 6 mg of daidzin into each SD rat in the intraperitoneal state in the group, inject once a day and continuously inject for 21 days at regular intervals. The low-dose daidzin drug-containing solvent needs to be prepared and used immediately; In the daidzin 6 mg / kg group, according to the dosage of 20 mL / kg of injection volume, inject the high-dose daidzin drug-containing solvent dissolved with 12 mg of daidzin into each SD rat in the intraperitoneal state in the group, inject once a day and continuously inject for 21 days at regular intervals. The high-dose daidzin drug-containing solvent needs to be prepared and used immediately; , In the control group, according to the dosage of 20 mL / kg of injection volume, continuously inject normal saline into the SD rats in the intraperitoneal state in the group for 21 days, inject once a day, and inject at regular intervals; , In the sodium iodate group, according to the dosage of 20 mL / kg of injection volume, continuously inject sodium iodate into the SD rats in the intraperitoneal state in the group for 21 days, inject once a day, and inject at regular intervals; SP B2-3After 21 days of injection in each group of SD rats, blood was collected from the abdominal aorta, and the blood samples were allowed to stand for at least 30 min. The serum was separated by centrifugation at 12,000 rpm for at least 10 min in a temperature environment of 4°C for detection.

[0018] Due to the adoption of the above technical solution, the present invention has the following beneficial effects: The protective effect of daidzin on oxidative damage caused by SIN-1 was evaluated through in vitro experiments, which was specifically manifested as an increase in cell viability and a decrease in MDA content; in vivo verification was completed with a rat model, a visual placement reaction experiment was formed to evaluate the visual function system of rats, the MDA content in the peripheral blood of the eyes of rats was detected, and it was clarified that the application of daidzin could significantly reduce AMD damage, that is, daidzin has an obvious improvement effect on AMD, and daidzin has good safety, low improvement and treatment costs for age-related macular degeneration, meeting the current clinical needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following drawings are only intended to illustrate and explain the present invention, and do not limit the scope of the present invention. Among them: Figure 1 is the experimental result of daidzin reducing RPE cell damage caused by oxidative stress in the embodiment of the present invention; Figure 2 is the result display of the MDA content in RPE cells after daidzin reduces oxidative damage in the embodiment of the present invention; Figure 3 is the influence of daidzin on the MDA content in the blood of AMD rats in the embodiment of the present invention; Figure 4 is the influence of daidzin on the visual function score of AMD rats in the embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention will be further described below in conjunction with the drawings and embodiments. In the following detailed description, only some exemplary embodiments of the present invention are described by way of illustration. Undoubtedly, those of ordinary skill in the art can recognize that various different ways can be used to modify the described embodiments without departing from the spirit and scope of the present invention. Therefore, the drawings and the description are illustrative in nature and are not used to limit the protection scope of the claims.

[0021] The application of daidzin in preparing a drug for improving age-related macular degeneration. Specifically, the drug improves age-related macular degeneration by inhibiting oxidative stress. The drug prepared with daidzin can be an injection, and the administration method of the drug is intraperitoneal injection.

[0022] The drug includes daidzin and preparation excipients. The preparation excipients include one or a combination of two or more of a suspending agent, a bacteriostatic agent, an antioxidant, a preservative, an isotonic regulator, and a pH regulator, which are common excipients in the technical field for treating eye diseases or abnormalities and will not be described in detail herein.

[0023] The present invention also experimentally verified that daidzin can improve age-related macular degeneration by inhibiting oxidative stress, and daidzin can be applied in the preparation of drugs for improving age-related macular degeneration. The potency verification method includes in vitro verification and in vivo verification. The protective effect of daidzin on oxidative damage caused by SIN-1 is evaluated through the in vitro experiment, which is specifically manifested as an increase in cell viability and a decrease in the content of MDA; the in vivo verification is completed with the help of a rat model to form a visual placement reaction experiment, evaluate the visual function system of the rat, detect the content of MDA in the peripheral blood of the rat's eye, and clarify that the application of daidzin can significantly reduce AMD damage, that is, daidzin has an improvement effect on AMD.

[0024] The in vitro experiment in this embodiment includes the following steps. SP A1 、Mother liquor preparation SP A1-1 Weigh 1 mg of daidzin (molecular weight is 416.38) and dissolve it in 24 μL of DMSO, and mix well at room temperature to prepare a 100 mM mother liquor. Among them, daidzin was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., and DMSO was purchased from Beyotime Biotechnology Co., Ltd.

[0025] SP A1-2 Take 98 μL of DMSO as the dilution solvent, add 2 μL of the mother liquor obtained by mixing daidzin and DMSO, and mix and dilute it into a 2 mM drug-containing solvent.

[0026] SP A1-3 Take DMSO as the dilution solvent, and perform concentration gradient dilution of the 2 mM drug-containing solvent with DMSO. Finally, four concentrations of drug-containing solvents of 2 mM, 1 mM, 0.5 mM, and 0.2 mM are prepared in sequence by adjusting the amount of DMSO used.

[0027] SP A1-3 Take 2 μL of each of the above drug-containing solvents and place them into the wells of a 96-well plate correspondingly. And 200 μL of DMSO solvent should be pre-placed in each well containing the drug-containing solvent, and the 2 μL of drug-containing solvent added is adjusted to form drug-containing solvents of 20 μM, 10 μM, 5 μM, and 2 μM respectively with the pre-placed DMSO solvent.

[0028] SP A1-5, 1 mg of SIN-1 (molecular weight 206.63) was weighed and dissolved in 96.8 μL of DMSO, and mixed well at room temperature to prepare a 50 mM stock solution for simulating oxidative stress injury of RPE cells. SIN-1 was purchased from MedChemexpress Biotechnology Company, USA.

[0029] SP A2 Cell culture SP A2-1 , DMEM was used as the culture medium, 10% fetal bovine serum and 1% penicillin-streptomycin were added to DMEM to form the culture solution. Retinal pigment epithelial cells (RPE) were added to the above culture solution for culture. When the cell density reached 80%, they were digested, and the resuspended cells were cultured in several wells of a 96-well plate. The standard of cells in each well was 3000 cells / well. Materials and reagents: Retinal pigment epithelial cells (RPE): Cell Bank of the Chinese Academy of Sciences DMEM medium: Gibco Company, USA Fetal bovine serum: Gibco Company, USA Penicillin streptomycin: Beyotime Biotechnology Co., Ltd. SP A2-2 , The wells on the 96-well plate with cultured cells were grouped to obtain a control group, a model group, and a drug administration group.

[0030] SP A2-3 , 2 μL of DMSO was added to the wells in the control group, and 2 μL of 50 mM SIN-1 stock solution was added to the wells in the model group, so that the cells were cultured in a medium with a concentration of 0.5 mM. After 24 h, CCK-8 detection was performed. The CCK-8 detection reagent was purchased from Beyotime Biotechnology Co., Ltd.

[0031] SP A2-4 , The concentrations of the daidzin-containing drug solvents used in the drug administration group were 2 mM, 1 mM, 0.5 mM, and 0.2 mM, and they were added to each well separately. The addition amount for each well was 2 μL. After culturing for 4 h, a 50 mM SIN-1 stock solution with the same dose as the model group was added, and incubation was continued for 24 h. After the incubation was completed, 20 μL of CCK-8 detection solution was added for detection.

[0032] Specifically, after adding 20 μL of CCK-8 detection working solution into each well, continue to incubate at a temperature of 37 °C for at least 2 h, and perform detection at a wavelength of 450 nm. The specific detection equipment and means are well-known to those of ordinary skill in the art and will not be described in detail here. Normalize the detection data, that is, regard the control group as 100%, and count the cell survival percentage of the model group and the drug administration group. The experimental results are as Figure 1 shown. Soyasaponin at 10 μM and 20 μM can improve cell activity ( ** P < 0.01, * P < 0.05), showing an antioxidant damage effect. The experimental results are as Figure 2 shown. In the cell experiment, after stimulation with SIN-1, the content of MDA increased significantly ( ### P < 0.001). Compared with the model group, the content of MDA decreased after drug administration ( *** P < 0.001), which has proved the antioxidant effect of soyasaponin.

[0033] To verify that the antioxidant effect still exists in the AMD model, the in vivo experiment was also carried out in this example, including the following steps, SP B1 、Preparation of soyasaponin-containing drug solvent SP B1-1 、Weigh two portions of soyasaponin, 6 mg and 12 mg, for standby.

[0034] SP B1-2 、First dissolve 6 mg of soyasaponin in 0.4 mL of DMSO, then sequentially add 4 mL of polyethylene glycol 12-hydroxydiol and 35.6 mL of normal saline, and mix the above components at room temperature to obtain a low-dose soyasaponin-containing drug solvent for standby. In this step, 6 mg of soyasaponin is actually dissolved in 40 ml of solvent. Polyethylene glycol 12-hydroxydiol is purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., and normal saline is purchased from Sinopharm Chemical Reagent Co., Ltd.

[0035] SP B1-3 、First dissolve 12 mg of soyasaponin in 0.4 mL of DMSO, then sequentially add 4 mL of polyethylene glycol 12-hydroxydiol and 35.6 mL of normal saline, and mix the above components at room temperature to obtain a high-dose soyasaponin-containing drug solvent for standby. In this step, 12 mg of soyasaponin is actually dissolved in 40 ml of solvent.

[0036] SP B2 、Rat model SP B2-1, 36 SPF-grade male SD rats were selected, with a body weight of 200 - 220 g. The SD rats were randomly divided into a control group, a sodium iodate group, a daidzin 3 mg / kg group, and a daidzin 6 mg / kg group. The SPF-grade male SD rats were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd.

[0037] SP B2-2 , The sodium iodate group, the daidzin 3 mg / kg group, and the daidzin 6 mg / kg group were respectively injected with 50 mg / kg of sodium iodate via the tail vein to obtain an AMD model, while the control group was injected with the corresponding volume of normal saline to complete the modeling. Sodium iodate was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0038] SP B2-3 , In the daidzin 3 mg / kg group, according to the dosage of 20 mL / kg of injection volume, a low-dose daidzin-containing solvent dissolved with 6 mg of daidzin was injected into each SD rat in the intraperitoneal state in the group, once a day for 21 consecutive days at a fixed time. The low-dose daidzin-containing solvent needed to be prepared and used immediately.

[0039] In the daidzin 6 mg / kg group, according to the dosage of 20 mL / kg of injection volume, a high-dose daidzin-containing solvent dissolved with 12 mg of daidzin was injected into each SD rat in the intraperitoneal state in the group, once a day for 21 consecutive days at a fixed time. The high-dose daidzin-containing solvent needed to be prepared and used immediately.

[0040] In the control group, according to the dosage of 20 mL / kg of injection volume, normal saline was injected into the SD rats in the intraperitoneal state in the group for 21 consecutive days, once a day at a fixed time.

[0041] In the sodium iodate group, according to the dosage of 20 mL / kg of injection volume, sodium iodate was injected into the SD rats in the intraperitoneal state in the group for 21 consecutive days, once a day at a fixed time.

[0042] SP B2-3 , After the 21-day injection of each group of SD rats, blood was taken from the abdominal aorta respectively, and the blood samples were left standing for at least 30 min. In a temperature environment of 4°C, the samples were centrifuged at a speed of 12,000 rpm for at least 10 min using a centrifuge to separate and obtain serum for detection. The content of MDA in the serum of SD rats was detected using an MDA detection kit purchased from Beyotime Biotechnology Co., Ltd. As Figure 3 shown, after modeling, the content of MDA in the serum of SD rats with AMD increased ( ### P < 0.001), and daidzin could significantly reverse this oxidative damage ( *** P < 0.001).

[0043] In this example, in the implementation step SPB2-3 Prior to this, behavioral scoring can be performed on SD rats first, and the specific steps are as follows: Invert and suspend the SD rats, and lower them onto a solid object without touching their whiskers to test whether the SD rats can perceive the position based on vision and thus produce a conditional limb grasping response. By observing whether the SD rats will raise their heads, extend their forelimbs towards the object and grasp it when approaching the object, and score according to the response sensitivity of the SD rats: 0 points indicates that no placement response is observed, 1 point indicates a relatively slow placement response, and 2 points indicates a sensitive placement response. As Figure 4 shown, after modeling, the visual score of the SD rats decreased significantly ( ### P < 0.001), and it improved significantly after administration ( *** P < 0.001), which has proven that daidzin can significantly improve the visual function damage of AMD rats and is related to its antioxidant capacity.

[0044] In the statistical method of this embodiment, all data are expressed as mean ± standard deviation, and statistical analysis is performed using GraphPad Prism 7.0. The differences between multiple groups are evaluated by one-way analysis of variance (One-way ANOVAs), followed by Tukey's test. Behavioral tests are detected by the Krystal-Wallis test method. P < 0.05 indicates that the data is statistically significant.

[0045] In the in vitro experiment of the present invention, it is verified that daidzin can significantly increase the cell viability of retinal pigment epithelial cells (RPE) after SIN-1 injury, and at the same time inhibit the content of MDA in the cells; in the in vivo experiment, it is also verified that daidzin can significantly improve the visual ability of rats, manifested as an increase in the visual score, and is consistent with the results of the in vitro experiment. Daidzin also reduces the oxidative damage of SD rats with AMD. In summary, daidzin can inhibit AMD damage by antioxidant, can be used as a drug for the treatment of AMD, and has good safety, low cost for the improvement and treatment of age-related macular degeneration, meeting the current clinical needs.

[0046] The description of the present invention is given for purposes of illustration and description, and is not intended to be exhaustive or to limit the invention to the disclosed form. Many modifications and variations are obvious to those of ordinary skill in the art. The embodiments are chosen and described in order to best explain the principles of the invention and its practical application, and to enable those of ordinary skill in the art to understand the invention and design various embodiments with various modifications suitable for specific purposes.

Claims

1. The application of soybean glycoside in preparing medicines, characterized in that: The drug is used to improve age-related macular degeneration.

2. The use of soybean glycoside for preparing medicines as claimed in claim 1, characterized in that: The drug improves age-related macular degeneration by inhibiting oxidative stress.

3. The use of soybean glycoside for preparing medicines as claimed in claim 1, characterized in that: The medicine is an injection.

4. The use of soybean glycoside for preparing medicines as claimed in claim 1, characterized in that: The drug is administered by intraperitoneal injection.

5. The use of soybean glycoside as claimed in any one of claims 1 to 4 for preparing medicines, characterized in that: The medicine comprises daidzein and preparation auxiliary materials.

6. The use of soybean glycoside for preparing medicines as claimed in claim 5, characterized in that: The preparation auxiliary materials include one or a combination of any ratio of suspending agents, antibacterial agents, antioxidants, preservatives, isotonicity regulators, and pH regulators.

7. A method for verifying the efficacy of a drug prepared by soybean glycoside, comprising preparing a drug by using the soybean glycoside according to any one of claims 1 to 4 to improve age-related macular degeneration by inhibiting oxidative stress, characterized in that: The efficacy verification method includes in vitro verification and in vivo verification. The in vitro experiment is used to evaluate the protective effect of daidzin on the oxidative damage caused by SIN-1, which is specifically manifested as an increase in cell viability and a decrease in MDA content. The in vivo verification is completed with the help of a rat model, forming a visual placement reaction experiment, evaluating the visual function system of rats, and detecting the MDA content in the peripheral blood of the rat eyes. It is clear that the application of daidzin can significantly reduce AMD damage, that is, daidzin has an improvement effect on AMD.

8. The effectiveness verification method according to claim 7, characterized in that: The in vitro experiment comprises the following steps, SP A1 , mother liquor configuration SP A1-1 1. Weigh 1 mg of daidzein and dissolve it in 24 μL of DMSO. Mix well at room temperature to prepare a 100 mM stock solution. SP A1-2 , take 98 μL of DMSO as a diluent, add 2 μL of the mother solution obtained by mixing soybean glycoside and DMSO, and mix and dilute to a 2 mM drug-containing solvent; SP A1-3 2. Take DMSO as the diluent, dilute the 2mM drug-containing solvent with DMSO in a concentration gradient, and finally obtain four concentrations of 2mM, 1mM, 0.5mM, and 0.2mM drug-containing solvents in sequence by adjusting the amount of DMSO; SP A1-3 2 μL of each concentration of the drug-containing solvent is taken and placed into the wells of the 96-well plate accordingly, and each well to which the drug-containing solvent is added must be pre-placed with 200 μL of DMSO solvent, and the pre-placed DMSO solvent is used to adjust the added 2 μL of drug-containing solvent of each concentration to form 20 μM, 10 μM, 5 μM, and 2 μM drug-containing solvents respectively; SP A1-5 1 mg of SIN-1 was weighed and dissolved in 96.8 μL of DMSO, and mixed at room temperature to form a 50 mM stock solution to simulate oxidative stress damage of RPE cells; SP A2 , Cell culture SP A2-1 1. Take DMEM as the culture medium, add 10% fetal bovine serum and 1% penicillin-streptomycin into DMEM to form a culture solution, add retinal pigment epithelial cells (RPE) into the above culture solution for culture, digest when the cell density reaches 80%, and culture the resuspended cells in several wells of a 96-well plate, and the standard number of cells in each well is 3000 / well; SP A2-2 , grouping the wells in which cells were cultured on the 96-well plate to obtain a control group, a model group, and a drug-treated group; SP A2-3 2 μL of DMSO was added to the wells of the control group, and 2 μL of 50 mM SIN-1 stock solution was added to the wells of the model group. The cells were cultured in a medium with a concentration of 0.5 mM. CCK-8 detection was performed after 24 h. SP A2-4 The concentrations of daidzein-containing solvents used in the drug-treated groups were 2mM, 1mM, 0.5mM and 0.2mM, respectively, and were added to each well. The amount added to each well was 2μL. After 4 h of culture, the same dose of SIN-1 mother solution as that in the model group and the concentration of 50mM was added, and the incubation was continued for 24 h. After completion of the incubation, 20μL of CCK-8 detection solution was added for detection.

9. The effectiveness verification method according to claim 7, characterized in that: The in vivo experiment comprises the following steps, SP B1 , Preparation of daidzein-containing solvent SP B1-1 2. Weigh 6 mg and 12 mg of daidzein in two portions and set aside; SP B1-2 , dissolve 6 mg of daidzein in 0.4 mL of DMSO, then add 4 mL of polyethylene glycol 12-hydroxydiol and 35.6 mL of normal saline in sequence, and mix the above components at room temperature to obtain a low-dose daidzein-containing solvent for use; SP B1-3 1. Dissolve 12 mg of daidzein in 0.4 mL of DMSO, then add 4 mL of polyethylene glycol 12-hydroxydiol and 35.6 mL of normal saline in sequence, and mix the above components at room temperature to obtain a high-dose daidzein-containing solvent for later use; SP B2 , rat model SP B2-1 36 SPF male SD rats with a body weight of 200-220 g were randomly divided into a control group, a sodium iodate group, a daidzin 3 mg / kg group, and a daidzin 6 mg / kg group; SP B2-2 1. The sodium iodate group, daidzein 3 mg / kg group and daidzein 6 mg / kg group were injected with 50 mg / kg sodium iodate through the tail vein to obtain AMD models, and the control group was injected with the corresponding volume of normal saline to complete the modeling; SP B2-3 2. The daidzein 3 mg / kg group was injected with a low-dose daidzein solvent containing 6 mg daidzein at an injection volume of 20 mL / kg into each SD rat in the group in the peritoneal state. The injection was given once a day and regularly for 21 consecutive days. The low-dose daidzein solvent must be prepared and used immediately. In the daidzin 6 mg / kg group, a high-dose daidzin-containing solvent containing 12 mg daidzin was injected into the peritoneal cavity of each SD rat in the group at an injection volume of 20 mL / kg. The injection was given once a day and regularly for 21 consecutive days. The high-dose daidzin-containing solvent had to be prepared and used immediately. The control group was injected with normal saline at a volume of 20 mL / kg into the peritoneal cavity of SD rats in the group for 21 consecutive days, once a day and at regular intervals; In the sodium iodate group, sodium iodate was injected intraperitoneally into SD rats at an injection volume of 20 mL / kg for 21 consecutive days, once a day, and at regular intervals. SP B2-3 After 21 days of injection, each group of SD rats had blood drawn from the abdominal aorta and the blood samples were left to stand for at least 30 minutes. The blood samples were centrifuged at 12,000 rpm for at least 10 minutes at 4°C to separate the serum for testing.

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