Application of cytisine-N-methylene-(5,7,4'-trihydroxy) isoflavone in the preparation of drugs for treating cerebral infarction

Abstract

The present invention discloses the use of cytisine-N-methylene-(5,7,4'-trihydroxy) isoflavone in the preparation of a drug for treating cerebral infarction. The invention relates to the fields of medicine, natural products, traditional Chinese medicine, and functional foods. The compound has both alkaloid and isoflavone structures, a novel structure, and good drugability. It also contains both flavonoid and alkaloid structural characteristics and is combined with any excipient or carrier acceptable in pharmaceutical, nutritional, and food science. The compound of the present invention can be used as an active ingredient in the treatment of cerebral infarction, inhibiting ischemic brain damage. It not only has a significant therapeutic effect on cerebral infarction, but also possesses multi-level, multi-target comprehensive pharmacological effects and targeted treatment of various pathological mechanisms.

Description

Technical Field

[0001] The present invention relates to the fields of medicine, natural products, traditional Chinese medicine and functional food, and in particular to the use of cytisine-N-methylene-(5,7,4'-trihydroxy) isoflavone in the preparation of a drug for treating cerebral infarction. Background Art

[0002] As China's aging population worsens, the incidence of ischemic stroke (IS) has also increased significantly, with China experiencing the fastest growth in the number of patients. Statistics show that the global prevalence of stroke is increasing annually, with IS occurring more frequently than hemorrhagic stroke and occurring more frequently in men than in women. The age-standardized prevalence of IS increased from 291.2 per 100,000 in 1990 to 299.1 per 100,000 in 2013. Specifically, the age-standardized prevalence of IS in men increased from 339.2 per 100,000 in 1990 to 346.08 per 100,000 in 2013, and in women from 253.6 per 100,000 in 1990 to 260.4 per 100,000 in 2013.

[0003] Over the past 23 years, the global age-standardized prevalence of ischemic stroke has increased by 2.71%. Stroke is the second leading cause of death among individuals aged 65 and older, characterized by high rates of disability, recurrence, and mortality. Even after surgical treatment, it remains a significant cause of disability and poor health, with many patients experiencing hemiplegia or even paralysis, requiring them to remain bedridden. Individuals with ischemic stroke spend an average of twice as many years or more in the hospital each year as other elderly individuals. Compared to caregivers of other illnesses, caregivers who provide long-term care for stroke patients are more likely to experience severe mental, financial, and physical challenges. Stroke not only severely impacts the patient's quality of life but also places a significant financial burden on their families and society.

[0004] Through the above analysis, the problems and defects of the existing technology are as follows:

[0005] Modern clinical medicine lacks ideal medications for the prevention and treatment of ischemic stroke. While Western medications reported in research and clinical use can improve ischemic stroke symptoms to some extent, their high cost, limited target, and significant side effects significantly limit their clinical application. Currently, rapid reperfusion intravenous thrombolysis and endovascular thrombectomy are the mainstays of stroke treatment. However, due to stringent inclusion criteria and numerous contraindications to thrombolysis, including the risk of hemorrhagic transformation, the clinical application of endovascular therapy remains limited. Therefore, the development of new treatments and medications is crucial.

[0006] In addition, the existing patent documents retrieved mainly discussed the anti-tumor activity experiments of the drug at the cellular level, and did not conduct pharmacological research on cerebral stroke, let alone research on drugs applied to cerebral infarction. Summary of the Invention

[0007] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide the use of cytisine-N-methylene-(5,7,4'-trihydroxy) isoflavone in the preparation of a drug for treating cerebral infarction.

[0008] The technical solution of the present invention is as follows: application of cytisine-N-methylene-(5,7,4'-trihydroxy) isoflavone in the preparation of cerebral infarction medicine.

[0009] Furthermore, the drug comprises cytisine-N-methylene-(5,7,4'-trihydroxy)isoflavone and an excipient acceptable in pharmaceutical science or food science.

[0010] Furthermore, the excipient is at least one of a binder, a filler, a disintegrant, wine, vinegar and starch.

[0011] Furthermore, the synthesis method of cytisine-N-methylene-(5,7,4'-trihydroxy) isoflavone is as follows: firstly, isoflavone is obtained by Suzuki coupling reaction; then, isoflavone and cytisine are subjected to Mannich reaction to synthesize the target product.

[0012] Furthermore, the specific steps of the synthesis method are as follows:

[0013] S1: Using 2,4,6-trihydroxyacetophenone as a raw material, K2CO3 is added to selectively liberate the hydroxyl groups at the 4 and 6 positions, followed by the addition of 0.5-3 equivalents of chloromethyl methyl ether to obtain 2-hydroxy-4,6-dimethyl methyl ether acetophenone, followed by the addition of N,N-dimethyl dimethyl acetal to synthesize aminophenone, and finally, the synthesis of 5,7-dimethyl methyl ether-3-iodochromone is completed under the action of elemental iodine; 5,7-dimethyl methyl ether-3-iodochromone is dissolved in dichloromethane, and the methyl ether at the 5 position is removed to obtain 7-dimethyl methyl ether-3-iodochromone;

[0014] S2: Compound 5 and compound 6 were reacted in a Suzuki coupling reaction to synthesize isoflavones. The methyl ether at position 7 was removed under HCl, and then the methoxy group was removed to obtain gentian isoflavones.

[0015] The compound 5 is: ;

[0016] The compound 6 is: ;

[0017] S3: Cytisine, cytisine isoflavones and formalin are used to catalyze the synthesis of cytisine-N-methylene-(5,7,4 ’ -trihydroxy) isoflavones.

[0018] The beneficial effects of the present invention are:

[0019] (1) The novel structural compounds of the present invention can be used to treat ischemic stroke and inhibit neurological damage caused by cerebral ischemia. The novel compounds are innovative in that they not only have significant anti-ischemic effects, but also possess multi-level, multi-targeted, comprehensive pharmacological effects and targeted effects on various pathological mechanisms. They are ideal monomers for future anti-stroke drugs.

[0020] (2) The present invention provides a preparation and synthesis scheme for a compound having a novel structure, a broom isoflavone alkaloid, and verifies its novel activity in treating cerebral stroke through relevant case studies. The compound is a natural active ingredient and has both alkaloid and isoflavone structures. It has a novel structure, good drugability, and good clinical application prospects.

[0021] (3) The novel structural compound of the present invention has a significant pharmacological effect of resisting cerebral stroke nerve damage, and may become an ideal drug for treating cerebral infarction in the future. It can be used for the prevention and treatment of cerebral infarction in clinical practice, and will generate considerable economic benefits and commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the results of the new compound provided in the examples of the present invention improving oxidative stress damage in cerebral ischemia model mice, wherein A is the SOD content in mouse serum; B is the MDA content in mouse serum (x±SD, n=8), ## indicates P <0.01, ### indicates P <0.001 compared with the Sham group; * indicates P <0.05, ** indicates P <0.01 compared with the Model group;

[0023] Figure 2 This is a schematic diagram showing the effects of the novel compounds provided in the examples of the present invention on improving the motor balance ability of mice modeling cerebral ischemia. A represents the process and steps of the rotarod test; B represents the time it takes for mice to climb the rod before drug administration; C represents the time it takes for mice to climb the rod before drug administration; and D represents the Bederson's behavioral score of each group of mice (x ± SD, n = 8). ## indicates P <0.01 compared with the Sham group; * indicates P <0.05, ** indicates P <0.01 compared with the Model group;

[0024] Figure 3 Schematic diagram of the results of the new compound provided in the examples of the present invention improving the cerebral infarction volume of cerebral ischemia model mice, wherein A is the TTC staining result of mice; B is the statistical diagram of the cerebral infarction volume of each group of mice (x±SD, n=8), * indicates P <0.05, ** indicates P <0.01 compared with the Model group;

[0025] Figure 4 1 is a schematic diagram of the results of studying the effect of immunofluorescence staining on the expression level of GFAP protein in the ischemic brain injury area provided by an embodiment of the present invention;

[0026] Figure 5 : This is a schematic diagram of the results of studying the effects of a new compound on the mRNA levels of inflammation-related factors in mouse brain tissue using the RT-qPCR method provided in an embodiment of the present invention, wherein A is the mRNA level of IL-6 in mouse brain tissue; B is the mRNA level of IL-1β in mouse brain tissue; C is the mRNA level of TNF-α in mouse brain tissue; D is the mRNA level of TNF-β in mouse brain tissue; E is the mRNA level of IL-4 in mouse brain tissue; and F is the mRNA level of IL-10 in mouse brain tissue (x±SD, n=8). # indicates P <0.05, ## indicates P <0.01, ### indicates P <0.001 compared with the Sham group; * indicates P <0.05, ** indicates P <0.01 compared with the Model group;

[0027] Figure 6 Schematic diagram of the effect of the genista isoflavone alkaloids provided in the present invention on the expression of β-catenin and CyclinD1 proteins in mouse brain tissue (x±SD, n=3), wherein A is the immunoblotting result of CyclinD1 and β-actin; B is the statistical graph of β-catenin bands; C is the statistical graph of Cyclin D1 bands; # indicates P <0.05, ## indicates P <0.01 compared with the Sham group; * indicates P <0.05, ** indicates P <0.01, compared with the Model group;

[0028] Figure 7 For identification of neural stem cells (400×).

[0029] Figure 8 GIA improves the viability of C17.2 neural stem cells; A, survival rate of C17.2 cells after treatment with gradient concentrations of GIA; B, quantitative analysis of cell viability; C, growth status of C17.2 cells (200×) (x±SD, n=3), * indicates P <0.05, ** indicates P < 0.01, *** indicates P <0.001, compared with the Control group;

[0030] Figure 9 Increased nuclear expression of Nestin for GIA (400×);

[0031] Figure 10 GIA improves oxidative stress damage in glucose-oxygen deprivation cell models, where A is the SOD content in C17.2 neural stem cells; B is the MDA content in C17.2 neural stem cells; C is the SOD content in primary neural stem cells; D is the MDA content in primary neural stem cells ( x ± SD, n = 3), ## indicates P <0.01, ### indicates P <0.001 compared with the Control group; * indicates P <0.05, ** indicates P <0.01 compared with the OGD group;

[0032] Figure 11 GIA alleviates neuroinflammation in a glucose-oxygen deprivation cell model. A, B, C, D, and C represent the mRNA expression levels of IL-6, IL-1β, TNF-α, and iNOS in 17.2 neural stem cells, respectively; E, F, G, and H represent the mRNA expression levels of IL-6, IL-1β, TNF-α, and iNOS in primary neural stem cells, respectively ( x ± SD, n = 3). ## indicates P < 0.01, and ### indicates P < 0.001 compared with the control group; * indicates P < 0.05, and ** indicates P < 0.01 compared with the OGD group.

[0033] Figure 12 GIA increases the protein levels of β-catenin and CyclinD1 in C17.2 neural stem cells. A is the immunoblotting results of β-catenin, CyclinD1, and β-actin; B is the statistical graph of β-catenin bands; C is the statistical graph of CyclinD1 bands (x±SD, n=3); ## indicates P <0.01 compared with the Control group; * indicates P<0.05, ** indicates P <0.01 compared with the OGD group;

[0034] Figure 13 GIA increases the protein levels of β-catenin and CyclinD1 in primary neural stem cells. A is the immunoblotting results of β-catenin, CyclinD1, and β-actin; B is the statistical graph of β-catenin bands; C is the statistical graph of CyclinD1 bands (x±SD, n=3); ## indicates P <0.01 compared with the Control group; * P <0.05,** P <0.01 compared with the OGD group. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0036] Compounds with therapeutic effects that improve cerebral infarction are combined with any excipient acceptable in pharmaceutical, nutritional, and food science. Generally, a derivative refers to a product derived from a simple compound by replacing hydrogen atoms or atomic groups with other atoms. The derivatives of the present invention retain the same main structure as the isoflavone alkaloid structure, with the effective functional group being an aromatic ketone. Isoflavones are 3-phenyl derivatives and are secondary metabolites of plants. Isoflavones are also effective antioxidants that can inhibit the generation of oxygen free radicals. Therefore, most compounds with an isoflavone structure have antioxidant and certain anti-inflammatory effects.

[0037] The embodiments of the present invention also provide new activities and new applications of the compounds and derivatives having the effect of improving the treatment of cerebral infarction in improving the treatment of cerebral infarction.

[0038] The present invention also provides a pharmaceutical preparation having a therapeutic effect on improving cerebral infarction. The pharmaceutical preparation is composed of cytisine-N-methylene-(5,7,4'-trihydroxy) isoflavone and any excipient acceptable in pharmaceutical and food science (such as binders, fillers, and disintegrants in tablets; wine and vinegar in pills; starch in powders, etc.).

[0039] Another object of the present invention is to provide a compound and derivative having the effect of improving the treatment of cerebral infarction for use in preparing a medicine or functional food having the effect of improving the treatment of cerebral infarction.

[0040] In an embodiment of the present invention, the preparation method of the compound comprises:

[0041] Cytisine is used as a raw material to obtain the isoflavone part through synthesis. The entire compound can be synthesized according to the following steps:

[0042] Step 1: obtaining a series of isoflavones through Suzuki coupling reaction under mild reaction conditions;

[0043] Step 2: The synthesized series of isoflavones and cytisine are subjected to a Mannich reaction to synthesize the target product under the catalysis of DMAP.

[0044] In an embodiment of the present invention, the synthesis method comprises the following steps:

[0045] (1) Using 2,4,6-trihydroxyacetophenone as the raw material, an appropriate amount of K2CO3 is added to selectively free the hydroxyl groups at the 4 and 6 positions, and then 0.5-3 equivalents of chloromethyl methyl ether are added to obtain 2-hydroxy-4,6-dimethyl methyl ether acetophenone. Then, N,N-dimethyl dimethyl acetal is added to synthesize aminophenone. Finally, a simple one-step cyclization reaction is completed under the action of elemental iodine to complete the synthesis of 5,7-dimethyl methyl ether-3-iodochromone. 5,7-dimethyl methyl ether-3-iodochromone (compound 4) is dissolved in dichloromethane, and the methyl ether at the 5 position is removed to obtain 3-iodochromone (compound 5). The synthetic route is:

[0046] ;

[0047] (2) Compound 5 and compound 6 were subjected to Suzuki coupling reaction to synthesize isoflavone (compound 7). The methyl ether at position 7 was removed under 3M HCl, and then the methoxy group was removed to obtain gentian isoflavone (compound 9). The synthetic route is:

[0048] ;

[0049] (3) Synthesis of cytisine-N-methylene-(5,7,4 ’ -trihydroxy) isoflavone, the synthesis route is:

[0050] .

[0051] Example 1

[0052] (1) 2,4,6-trihydroxyacetophenone was used as the raw material. 2.1 times the mass equivalent of K2CO3 was added to selectively free the hydroxyl groups at the 4 and 6 positions. Then, 2.12 times the mass equivalent of chloromethyl methyl ether was added to obtain 2-hydroxy-4,6-dimethyl methyl ether acetophenone. Then, N,N-dimethyl dimethyl acetal was added to synthesize aminophenone at a yield of 88% at a temperature of 74°C. Finally, a simple one-step cyclization reaction was performed under the action of elemental iodine to complete the synthesis of 5,7-dimethyl methyl ether-3-iodochromone in a yield of 75%. 5,7-dimethyl methyl ether-3-iodochromone (compound 4) was dissolved in dichloromethane and placed on a stirrer at medium speed for 4 hours. The methyl ether at the 5 position was removed to obtain 3-iodochromone (compound 5).

[0053] (2) Compounds 5 and 6 were used as raw materials. 3.0 times the mass equivalent of K2CO3 and 0.1 times the mass equivalent of Pd(PPh3)4 were added to dioxane and water, and the mixture was freeze-degassed and replaced three times. The mixture was refluxed at 110°C for 12 hours to synthesize isoflavone 7 with a yield of 75%. Subsequently, compound 7 was demethylated at the 7-position in the presence of 3M HCl, and then demethoxylated in the presence of boron tribromide to obtain genistein (compound 9).

[0054] (3) Using genistein and genistein as raw materials, adding 37 wt% formalin and 0.1 equivalent of DMAP (4-dimethylaminopyridine) in isopropanol, reacting at 80 ° C for 12 hours, genistein-N-methylene-(5,7,4 ’ -trihydroxy) isoflavone compounds.

[0055] The compound obtained in Example 1 was subjected to the following tests:

[0056] 1. Establishment of Cerebral Ischemia Model in Mice

[0057] Rose Bengal is a type II photosensitizer that, upon light activation, directly transfers energy to oxygen molecules, generating large amounts of reactive oxygen species (ROS), which interact with other molecules in the body and cause damage. A saline solution containing Rose Bengal was injected through the tail vein. The mouse skull was then exposed and the left tectum was irradiated with a 560 nm beam for 5 minutes, inducing thrombosis and causing local infarction, thus achieving ischemia.

[0058] Mice were divided into 6 groups, each with 8 C57 mice, including sham-operated control group (normal saline), model group (normal saline), low-dose drug treatment group (0.3 mg / kg), medium-dose drug treatment group (0.6 mg / kg), high-dose drug treatment group (1.2 mg / kg), and positive drug group (ginsenoside 20 mg / kg). The mice were dosed according to body weight for one week. The drug group used the compound monomer GIA (cytisine-N-methylene-(5,7,4 ’ -trihydroxy) isoflavone compounds).

[0059] Determination of superoxide dismutase (SOD) and malondialdehyde (MDA)

[0060] 2.1 Determination of SOD content

[0061] SOD activity was determined using the xanthine oxidase method according to the kit instructions. Reagents were added according to Table 1 below, mixed thoroughly, and incubated at 37°C for 20 minutes. The absorbance of each tube was measured at 450 nm to calculate the SOD activity.

[0062] Table 1 Reagents used in SOD test

[0063]

[0064] 2.2 Determination of MDA content

[0065] MDA content was determined using the thiobarbituric acid (TBA) method. Add the reagents according to Table 2 below, mix thoroughly, then continue adding the reagents listed in Table 2. Incubate at 95°C in a metal bath for 80 minutes, remove from the container, cool under running water, and centrifuge at 3500-4000 rpm for 10 minutes.

[0066] Table 2 Required reagents

[0067]

[0068] Table 3 Required reagents

[0069]

[0070] Tested Figure 1 It can be seen that compared with the sham operation group, the SOD concentration of the model group decreased significantly, and the compound drug treatment can significantly increase this change. Figure 1 As shown in Figure B, the MDA concentration of the model group mice increased significantly, while the drug-treated group and the positive drug could significantly reduce its concentration, and the effect was particularly obvious at medium and high dose concentrations.

[0071] 3. Behavioral Experiments

[0072] 3.1 Rotarod test in mice

[0073] The mice were tested with a rotating rod test before and after the start of the experiment. During the training phase of the rotating rod test, each mouse was tested three times a day for three consecutive days. During training, the rotation speed was kept constant at 10 rpm, and the training was qualified if it remained constant for the rest of the test (90 seconds). During the test phase (the fourth day after injury), the speed of the rod was increased from 4 rpm to 40 rpm for 300 seconds. The climbing time of the mouse was recorded. Each mouse was tested three times, and the average of the three results was taken as the final time. The mouse's falling latency was scored. The test process refers to the test Figure 2 A in.

[0074] 3.2 Bederson's score of mice

[0075] After drug administration, the animals underwent neurobehavioral assessment using the Zea Longa method. The tail of the mouse was lifted approximately 1 foot off the ground, and the condition of the forelimbs was observed. The mouse was placed on a level surface, and the shoulders were pushed to observe any difference in resistance. The mouse was then placed on the ground and its walking was observed. A five-point scoring system (0-4) was used, with higher scores indicating more severe neurobehavioral impairment.

[0076] (1) Those whose behavior is completely normal will be given 0 points;

[0077] (2) When the tail of the rat is lifted off the ground, if the forelimb on the contralateral side of the surgery is internally rotated or adducted, 1 point is scored;

[0078] (3) Place the mouse on the ground and squeeze both sides by hand to check its resistance. If the resistance on the side opposite to the surgery decreases, score 2 points.

[0079] (4) Place the mouse on the ground and observe its walking. If it circles around the side opposite to the surgery, score 3 points;

[0080] (5) If the injury is extremely severe and the patient is unable to move independently, 4 points will be awarded.

[0081] Test results see Figure 2 As shown in Figure 2B, it is the climbing time of the rod in each group of mice before the surgical modeling experiment. It can be clearly seen from the figure that there is no significant difference in the climbing time of each group of mice before the experiment, indicating that the movement balance ability of each group of mice is similar. Figure 2 As shown in Figure C, after drug administration, the climbing time of mice in the model group was significantly reduced compared with the sham operation group; compared with the model, the climbing time of mice in the drug treatment group was significantly increased ( P <0.01), indicating that the treatment with Baimaisan active compounds can significantly improve the motor balance ability of mice. Figure 2As shown in Figure D, the Bederson's score of the sham-operated group was 0. After the surgery, the behavioral scores of the mice increased significantly, indicating that the mice's motor ability was significantly impaired after the photochemical-induced surgery. After drug treatment, the behavioral scores of the mice decreased significantly, indicating that drug treatment can significantly improve the behavioral ability of the mice. This effect was particularly evident in the high-dose treatment group ( P <0.01).

[0082] 4. Get materials

[0083] After the behavioral experiment, the eyeballs were removed and blood was collected. The mice were then placed in a supine position on the operating table. The chest cavity was opened to expose the heart and perfused with normal saline. When the liver turned white, the mouse's limbs became rigid and the brain tissue was removed by decapitation.

[0084] 5. TTC staining

[0085] After the brain tissue was collected, it was quickly placed in a -20℃ refrigerator and frozen for 10 minutes. Then, it was cut into 5 sections using a brain mold, and coronal brain sections (each 2mm). The sections were soaked in TTC dye solution and placed in a 37℃ constant temperature box for 15 minutes. After staining, it was washed with clean water, the staining condition was observed, and photos were taken for record. The test results are shown in Figure 3 , we can see that, if Figure 3 A in the figure shows the TTC staining results of the brain tissue sections of mice in each group. Figure 3 Figure B is a statistical graph of mouse cerebral infarction volume. Compared with the Sham group, the cerebral infarction volume of the Model group mice was significantly larger, indicating that the surgical model was successful and cerebral ischemia occurred in the mice; compared with the Model group, the cerebral infarction volume of the low-dose and high-dose groups mice was significantly reduced ( P <0.05); the cerebral infarction volume of mice in the medium-dose group was more significantly reduced than that in the Model group ( P <0.01); the cerebral infarction volume of mice in the TG group was significantly smaller than that in the Model group ( P <0.05).

[0086] 6. Immunofluorescence Staining

[0087] (1) Slicing: Use a freezing microtome to slice the mouse brain tissue serially, each slice is 30 μm thick, and store in antifreeze at -20°C; (2) Slice selection: Select brain slices and wash them three times with PBS, each time for 5 minutes; (3) Blocking: 1% BSA + 10% sheep serum + 0.3% Triton X-100, 60 min; (4) Apply primary antibody: dilute with Blocker (1% BSA, 0.3% Triton), room temperature for 2 hours, overnight at 4 degrees; (5) Rinse: wash three times with PBS, 5 minutes each time; (6) Apply secondary antibody: dilute with Blocker (1% BSA, 0.3% Triton) 1:400, 120 minutes; (7) Rinse: wash three times with PBS, 5 minutes each time; (8) Nuclear staining: DAPI (1:1000), 15 minutes; (9) Rinse: wash three times with PBS, 5 minutes each time; (10) Seal the slides: seal the slides with anti-fluorescence quencher; (11) Take pictures: observe with fluorescence microscope.

[0088] Test results see Figure 4 Therefore, elevated GFAP levels in ischemic brain tissue are a hallmark of the central nervous system's response to ischemic brain injury. To this end, immunofluorescence was used to examine GFAP protein expression in the brain tissues of each group of mice. The results, shown in Figure 4, show a significant increase in GFAP protein expression after surgical modeling. However, treatment with medium and high doses of the compound significantly decreased GFAP expression, indicating that the active ingredient GIA can effectively alleviate the stress response to ischemic brain injury and provide some protection against neuroinflammation caused by brain injury.

[0089] 7. Western Blot

[0090] (1) Preparation of SDS-PAGE gel

[0091] ① Prepare the separation gel: Wash and dry the glass plate, place it in the clamp, and align it horizontally to ensure a tight seal. Prepare the appropriate separation gel based on the molecular weight of the target protein. See Table 4.

[0092] Table 4 Separation gels

[0093]

[0094] Note: % is the mass concentration percentage.

[0095] Add the prepared separation gel to the glass plate and seal it with water. Leave it at room temperature for 30 minutes, discard the upper layer of water, and absorb the remaining liquid with filter paper.

[0096] ② Prepare the stacking gel: According to the material configuration in Table 5, mix the prepared stacking gel and slowly add it to the glass plate.

[0097] Table 5 Concentrated gel

[0098] Reagent name 5% stacking gel <![CDATA[dd H2O2]]> 2.1 (mL) 30% Arc-bis (29:1) 0.495 (mL) 1.0 M Tris-HCl (pH 6.8) 0.375 (mL) 10% SDS 0.03 (mL) 10% APS 0.03 (mL) TEMED 0.003 (mL)

[0099] Note: % is the mass concentration percentage.

[0100] (2) Loading

[0101] Remove the comb teeth, add 10 µL of protein sample to each well, and add 5 µL of marker at each end.

[0102] (3) Electrophoresis

[0103] Maintain a constant voltage of 80 V. After the sample enters the separation gel, switch to 120 V and electrophorese to the bottom.

[0104] (4) Transfer

[0105] Prepare a "sandwich" in the following order: black plywood - transfer filter paper - glue - PVDF membrane - transfer filter paper - white plywood. Place the transfer cassette into the electrophoresis tank, add transfer buffer, and transfer at 200 mA for 1.5 hours. Surround the electrophoresis tank with ice packs to keep the entire process cool.

[0106] (5) Antibody incubation

[0107] ① Wash the PVDF membrane 3 times for 5 minutes and block it in blocking solution at room temperature for 2 hours;

[0108] ② Wash the PVDF membrane three times for 5 minutes, dilute the primary antibody in blocking buffer, and incubate at 4°C overnight;

[0109] ③ Wash the PVDF membrane 3 times for 10 min, add the secondary antibody, and incubate at room temperature for 1.5 h. Antibody dilution information is shown in Table 6:

[0110] Table 6 Antibody dilution

[0111]

[0112] (6) Gel imaging

[0113] Add ECL chemiluminescence developer, react in the dark for 2 min, then develop and take pictures.

[0114] (7) Image analysis

[0115] Grayscale values ​​were analyzed using Image J software.

[0116] like Figure 5As shown in the results, after modeling, the mRNA expression levels of IL-6, IL-1β, and TNF-α in mice were significantly increased, while the mRNA expression levels of anti-inflammatory factors IL-4, TNF-β, and IL-10 were significantly decreased. After drug treatment, the increase in the level of pro-inflammatory factors was significantly inhibited, and the mRNA expression levels of anti-inflammatory factors such as IL-4 and TNF-β were effectively increased.

[0117] As shown in Figure 6A, the expression levels of β-catenin, CyclinD1, and β-actin proteins in the brain tissues of mice in each experimental group.

[0118] like Figure 6 Figure B shows the quantitative analysis results of β-catenin protein levels. Compared with the Sham group, the β-catenin protein content in the brain tissue of mice in the Model group was reduced ( P <0.001); compared with the Model group, the β-catenin protein content in the brain tissue of mice in the high- and medium-dose groups was significantly increased ( P <0.01). Compared with the Model group, the β-catenin protein content in the brain tissue of mice in the TG treatment group increased ( P <0.01). Figure 6 Figure C shows the quantitative analysis results of Cyclin D1 protein content. Compared with the Sham group, the Cyclin D1 protein content in the brain tissue of the Model group mice was significantly reduced ( P <0.01); compared with the Model group, the Cyclin D1 protein content in the brain tissue of mice in the high-dose and medium-dose groups was significantly increased ( P <0.01), and the effect was particularly significant at low doses. Compared with the Model group, the Cyclin D1 protein content in the brain tissue of mice in the TG group also increased significantly ( P <0.01).

[0119] 8. In vitro cell proliferation study

[0120] (1) Experimental animals: Four pregnant SPF-grade SD rats (3000 ± 20 g), 13 days of gestation, were purchased from Beijing Sibeifu Biotechnology Co., Ltd. with license number SCXK (Beijing) 2019-0010. Embryonic brain tissues of pregnant rats were used for the extraction of primary neural stem cells.

[0121] Experimental cells: The neural stem cells used in the experiment were mouse neural progenitor cells C17.2 cells, which were purchased from Beijing Hancheng Biotechnology Co., Ltd.

[0122] The experimental methods are referenced as follows: Sun T, Chen L, Liu R, Liu QS, Cheng Y. Sophora alopecuroides Alleviates Neuroinflammation and Oxidative Damage of Parkinson's Disease In Vitro and In Vivo. Am J Chin Med. 2023;51(2):309-328.

[0123] Data processing: Data are expressed as x ± SD. Homogeneity of variance and normality tests were performed. If the variances were homogeneous, one-way analysis of variance was performed using SPSS 20.0 software. If the variances were unequal, the Dunnett T3 test was used. If the distribution was not normal, the rank sum test was used. P < 0.05 was considered statistically significant.

[0124] The test results are as follows:

[0125] (1) Identification of neural stem cells

[0126] As shown in Figure 7, in vitro cultured C17.2 cells can be observed using a standard light microscope and fluorescently labeled with Nestin, a hallmark protein of NSCs. In vitro cultured C17.2 cells have good refractive index. Laser confocal microscopy images of C17.2 cells show simultaneous labeling with Nestin and DAPI, with the percentage of Nestin-positive cells reaching 100%.

[0127] (2) Determine the cell administration dose

[0128] The CCK8 kit was used to detect the effect of GIA treatment on the viability of C17.2 cells. 0μg / ml, 5μg / ml, 10μg / ml, 20μg / ml, 40μg / ml, 80μg / ml, and 100μg / ml of GIA were added, and cell viability was detected after 24 hours of culture. The results are shown in Figure 2. Figure 8 A, compared with the control group (0 μg / ml), 5 μg / ml, 10 μg / ml, 20 μg / ml, and 40 μg / ml of GIA treatment had no significant effect on cell survival rate ( P >0.05), 80μg / ml and 100μg / ml GIA treatment significantly decreased cell viability ( P <0.001). Therefore, the concentration of GIA should not exceed 40 μg / ml in subsequent cell experiments.

[0129] Next, the cells were treated with GIA drugs at concentrations of 5μg / ml, 10μg / ml, 20μg / ml, and 40μg / ml, and the OGD model cells were given medium without drugs. Figure 8 As shown in Figure B, GIA treatment at all doses significantly increased cell survival, with the effect being even more pronounced at the two higher concentrations of 20 μg / ml and 40 μg / ml (P < 0.01). This suggests that GIA treatment within a certain concentration range can protect cells from damage caused by glucose and oxygen deprivation and increase cell viability.

[0130] Subsequent cell administration was performed at three concentration gradients of 10 μg / ml, 20 μg / ml, and 40 μg / ml, and cell growth was observed. As shown in Figure 8C, the TG and GIA-treated groups exhibited improved cell growth compared to the model group, with a significant increase in cell number.

[0131] (3) GIA increases the nuclear expression of Nestin

[0132] After cell treatment, Nestin immunocytochemistry experiments were performed to observe the expression and localization of Nestin during cell proliferation. Figure 9 As shown, Nestin is expressed in the cytoplasm of normal C17.2 cells, whereas GIA-treated C17.2 cells show increased expression of Nestin in the nucleus. Based on the location and level of Nestin expression in cells, we performed a localization analysis of Nestin.

[0133] (4) GIA alleviates glucose-oxygen deprivation oxidative stress damage in neural stem cells

[0134] C17.2 neural stem cells and primary neural stem cells were treated with GIA at concentrations of 10μg / ml, 20μg / ml, and 40μg / ml, and the levels of superoxide dismutase and malondialdehyde in the model group and the treatment group were detected 3 hours after the glucose and oxygen deprivation model was established. Figure 10 As shown in Figure 2, compared with the control group, the OGD-treated groups of both cells significantly reduced the superoxide dismutase content in the cells, and the difference was statistically significant ( P <0.001), and the total antioxidant content in cells was significantly increased after treatment with different doses of GIA ( P <0.05, P <0.01). Similarly, the detection of malondialdehyde content also showed a similar trend. The OGD treatment groups of both stem cells significantly increased the malondialdehyde content in the cells, and the difference was statistically significant ( P<0.01). After treatment with different concentrations of GIA, all three concentrations of GIA could significantly reduce the content of malondialdehyde in neural stem cells, and the difference was statistically significant ( P <0.05, P <0.01). The above results indicate that neural stem cells, after being protected by the active ingredient GIA, can alleviate oxidative stress damage caused by glucose deprivation and play a certain role in damage protection.

[0135] (5) GIA alleviates cellular neuroinflammation caused by glucose and oxygen deprivation

[0136] In a glucose-oxygen deprivation cell ischemia model, C17.2 neural stem cells and primary neural stem cells were pre-treated with GIA at concentrations of 10μg / ml, 20μg / ml, and 40μg / ml. The mRNA expression levels of inflammation-related proinflammatory factors IL-6, IL-1β, TNF-α, and iNOS were measured to study the protective effect of GIA on stem cell neuroinflammation. Figure 11 As shown in Figure 2, compared with the blank group, the mRNA expression levels of IL-6, IL-1β, TNF-α, and iNOS in the OGD model group were significantly increased ( P <0.001). GIA treatment at different concentrations significantly reduced the mRNA expression levels of IL-1β, TNF-α, and iNOS. When GIA was administered to C17.2 neural stem cells, low and high doses of GIA reduced the mRNA expression level of IL-6. When GIA was administered to primary neural stem cells, medium and high doses of GIA reduced the mRNA expression level of IL-6 ( P <0.01). The above results indicate that GIA can alleviate neuroinflammation to a certain extent in the glucose-oxygen deprivation cell models of C17.2 neural stem cells and primary neural stem cells.

[0137] (6) GIA increases the levels of β-catenin and CyclinD1 proteins in neural stem cells

[0138] As shown in Figure 12A, compared with the Control group, the β-catenin and CyclinD1 protein levels in C17.2 neural stem cells were significantly reduced ( P <0.01). Specific band statistical analysis is as follows Figure 12 As shown in B and C, compared with the OGD model group, the β-catenin and CyclinD1 protein levels in the GIA treatment groups with different concentrations were significantly increased ( P<0.01). Compared with the OGD group, the Cyclin D1 protein level in the low-dose GIA treatment group increased, but there was no statistical significance ( P >0.05). Figure 13 As shown in A, compared with the Control group, the CyclinD1 protein content in primary neural stem cells was significantly reduced ( P <0.01), but the β-catenin protein level was decreased, but it was not statistically significant ( P >0.05). Specific band statistical analysis is shown in Figure 13B and C. Compared with the OGD model group, the β-catenin and CyclinD1 protein contents in the GIA treatment groups with different concentrations were significantly increased ( P <0.01). Compared with the OGD group, the Cyclin D1 protein level was increased in the low-dose GIA treatment group, and the β-catenin protein level was increased in the high-dose GIA treatment group, but neither was statistically significant ( P >0.05).

[0139] In summary, in vivo animal studies have confirmed that GIA can promote the proliferation of endogenous NSCs and improve the motor balance ability of mice with cerebral ischemia. It was also found that GIA can promote the proliferation of neural stem cells (NSCs).

[0140] In summary, the novel 5'-O-trityl uridine compound can reduce cerebral infarct volume and improve Bederson's motor score in mice with photochemically induced cerebral ischemia, significantly improving the motor function of mice with cerebral ischemia. It can also alleviate oxidative stress damage in mice after ischemia, alleviate neuroinflammation to a certain extent, promote the proliferation of neural stem cells, and significantly improve neurological damage after ischemic stroke.

[0141] The above-described embodiments merely represent preferred implementations of the present invention. While the descriptions thereof are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the scope of protection of the claims of the present invention.

Claims

1. Application of cytisine-N-methylene-(5,7,4'-trihydroxy) isoflavone in the preparation of a drug for treating cerebral infarction, characterized in that: The specific steps of its synthesis method are as follows: S1: Using 2,4,6-trihydroxyacetophenone as a raw material, K2CO3 is added to selectively liberate the hydroxyl groups at the 4 and 6 positions, followed by the addition of 0.5-3 equivalents of chloromethyl methyl ether to obtain 2-hydroxy-4,6-dimethyl methyl ether acetophenone, followed by the addition of N,N-dimethyl dimethyl acetal to synthesize aminophenone, and finally, the synthesis of 5,7-dimethyl methyl ether-3-iodochromone is completed under the action of elemental iodine; 5,7-dimethyl methyl ether-3-iodochromone is dissolved in dichloromethane, and the methyl ether at the 5 position is removed to obtain 7-dimethyl methyl ether-3-iodochromone; S2: Compound 5 and compound 6 are reacted in a coupling reaction to synthesize isoflavones, and the methyl ether at position 7 is removed under HCl, and then the methoxy group is removed to obtain gentian isoflavones; The compound 5 is: ; The compound 6 is: ; S3: Cytisine, cytisine isoflavones and formalin are used to catalyze the synthesis of cytisine-N-methylene-(5,7,4 ’ -trihydroxy) isoflavones.

2. The use according to claim 1, characterized in that The drug comprises cytisine-N-methylene-(5,7,4'-trihydroxy) isoflavone and an excipient acceptable in pharmaceutical or food science.

3. The use according to claim 2, characterized in that The excipient is at least one of a binder, a filler, a disintegrant, wine, vinegar and starch.

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