Pharmaceutical application of chelidonamic acid

Abstract

The invention discloses a pharmaceutical application of chelidonine in the aspect of treating Alzheimer's disease related diseases. According to the present invention, the down-regulation effect of chelidonine on the Abeta42 protein level is found for the first time, can effectively inhibit the formation of the inducements of Alzheimer's disease and early-stage depression symptoms on the multi-level levels of cells, tissues and animals, has significant improvement effects on learning and memory disorders and early-stage depression behaviors, and has clinical application prospects in treatment of related diseases.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical application of chelidonine, and in particular to a pharmaceutical application of chelidonine in the treatment of diseases related to Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD), as a type of dementia, is mainly manifested by progressive degeneration of learning and memory abilities and irreversible neuronal loss, as well as the appearance of senile plaques formed by the extracellular accumulation of β-amyloid protein (β-Amyloid, 1-42, Aβ42) in the cerebral cortex and hippocampus. Among them, the imbalance of Aβ42 protein metabolism can cause neuroinflammation, oxidative damage, abnormal phosphorylation of tau protein and ultimately cause irreversible neuronal damage, which is the fundamental link leading to the occurrence of AD. In particular, the level of Aβ protein begins to increase abnormally long before the onset of obvious AD symptoms. As the metabolic balance of Aβ protein in the brain becomes disordered, the protein clearance ability is reduced, and the activity of related secretases is abnormally increased, which promotes the occurrence and development of AD. Therefore, regulating the level of Aβ42 in the brain has become a key link in the treatment of AD.

[0003] Major Depressive Disorder (MDD), as a common mental disorder, is closely related to AD. AD patients often have physiological manifestations of depression in the early stages of the disease. The two have certain common pathogenesis, such as imbalance of neurotransmitters, abnormalities in brain structure and function, inflammation and neuroplasticity. Since Aβ protein levels are abnormal before obvious AD symptoms appear, imbalance in Aβ42 protein metabolism is also a trigger for depression in the early stages of AD.

[0004] Currently, the marketed chemical drugs for the treatment of AD include Tacrine, Donepezil, Revastigmine, Galantamine and Menantine. The first four are acetylcholinesterase (AchE) inhibitors, and Memantine is an N-Methyl-D-aspartic acid (NMDA) receptor antagonist. Both AchE inhibitors and NMDA receptor antagonists are symptomatic drugs, mainly for improving cognitive and memory disorders, and cannot prevent or delay the progression of the disease. Both types of drugs have serious side effects and may even aggravate anxiety or depression symptoms in some patients. At the same time, Aducanumab (Aβ protein monoclonal antibody) and lecanemab are antibodies against Aβ protein, which reduce the deposition of Aβ protein, but they are expensive, require long-term intravenous infusion, and have greater side effects. In addition, drugs for the treatment of pre-AD depression need to be used with extreme caution, as the pathophysiological characteristics and drug tolerance of AD patients are different from those of ordinary depression patients. In addition, therapeutic drugs have limited efficacy, many side effects, slow onset of action, and may even aggravate cognitive dysfunction. Therefore, there is an urgent need to find new, safer and more effective therapeutic drugs. Summary of the invention

[0005] Purpose of the invention: The first purpose of the present invention is to provide a new pharmaceutical application of chelidonine in the treatment of Alzheimer's disease-related diseases. The second purpose is to provide a new pharmaceutical application of a hydrate of chelidonine, a pharmaceutically acceptable salt thereof, and a pharmaceutical composition thereof in the treatment of Alzheimer's disease-related diseases.

[0006] Technical solution: The chelidonine described in the present invention is used in the preparation of a drug for treating Alzheimer's disease or pre-Alzheimer's depression.

[0007] Chelidamic acid is a glutamate decarboxylase inhibitor with a molecular formula of C 7 H 5 NO 5 .

[0008] Chrysene.

[0009] Preferably, the drug is a drug for treating Alzheimer's disease with APP and PSEN1 gene mutations.

[0010] More preferably, the drug is a drug for treating Alzheimer's disease carrying Swedish K670N / M671L, Florida I716V, and London V717I familial mutations in the APP gene.

[0011] More preferably, the drug is a drug for treating Alzheimer's disease carrying familial mutations M146L and L286V in the PSEN1 gene.

[0012] Preferably, the drug is capable of reducing Aβ42 protein levels.

[0013] More preferably, the drug can promote the phagocytosis and degradation of Aβ42 protein.

[0014] More preferably, the drug can inhibit the formation of Aβ42 protein plaques.

[0015] More preferably, the drug can improve irreversible neuronal damage caused by imbalance in Aβ42 protein metabolism.

[0016] More preferably, the drug can improve neuroinflammation, oxidative damage, abnormal tau protein phosphorylation, and glial cell proliferation caused by imbalance in Aβ42 protein metabolism.

[0017] Preferably, the drug can improve learning and memory disorders and depressive behavior.

[0018] Further preferably, the learning and memory disorders include not wanting to contact new things or objects, difficulty remembering new information, inability to learn and acquire new knowledge and skills, longer learning and memory time, easier forgetting of learned content, and difficulty remembering routes or directions; the depressive behaviors include behaviors that are more prone to despair in an oppressive environment, negative and pessimistic behaviors, loss of interest or pleasure in activities that are usually of interest, loss of desire for favorite things, and anhedonia behaviors.

[0019] The hydrate of chelidonine or a pharmaceutically acceptable salt thereof of the present invention is used in preparing a drug for treating Alzheimer's disease or pre-Alzheimer's depression.

[0020] The pharmaceutical composition containing chelidonine, its hydrate or its pharmaceutically acceptable salt described in the present invention is used in preparing a medicine for treating Alzheimer's disease or pre-Alzheimer's depression.

[0021] Preferably, the drug is capable of reducing Aβ42 protein levels.

[0022] More preferably, the drug can promote the phagocytosis and degradation of Aβ42 protein.

[0023] More preferably, the drug can inhibit the formation of Aβ42 protein plaques.

[0024] More preferably, the drug can improve irreversible neuronal damage caused by imbalance in Aβ42 protein metabolism.

[0025] More preferably, the drug can improve neuroinflammation, oxidative damage, abnormal tau protein phosphorylation, and glial cell proliferation caused by imbalance in Aβ42 protein metabolism.

[0026] Preferably, the drug can improve learning and memory disorders and depressive behavior.

[0027] Preferably, the hydrate is chelidonine containing water, wherein the water exists in the form of adsorbed water or crystal water, such as chelidonine monohydrate. When the hydrate is used, its amount needs to be converted into the amount of chelidonine.

[0028] Preferably, the pharmaceutically acceptable salt is a salt formed by chelating acid and any one of the following bases: alkali metal ion base, alkaline earth metal ion base, aluminum ion base, zinc ion base, choline, ammonia water, ammonia gas, ethylenediamine, triethylamine, triethanolamine, piperazine, meglumine.

[0029] Preferably, the pharmaceutical composition further contains a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier can be an excipient widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, so that the active ingredient dissolves at a desired rate after the subject receives the administration, or promotes the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical excipient can be an inert filler, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipient can include one or more of the following excipients: adhesives, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, sweeteners.

[0030] The pharmaceutical composition of the present invention can be prepared by any method known to those skilled in the art, for example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding, and freeze-drying.

[0031] The pharmaceutical composition of the present invention can be administered in any form, including mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical, parenteral (infusion, injection, implant, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or sustained release dosage form (such as liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, hard capsules, caplets, soft capsules, tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, emulsions for injection. Examples of other preparation forms include, but are not limited to, eye drops, other ophthalmic preparations; aerosols, such as nasal sprays, inhalants; suppositories and lozenges suitable for parenteral administration.

[0032] Preferably, the chelidonine, its hydrate, its pharmaceutically acceptable salt, and its pharmaceutical composition are used in combination with a drug for treating Alzheimer's disease or pre-Alzheimer's depression.

[0033] More preferably, the drug for treating Alzheimer's disease is selected from tacrine, donepezil, rivastigmine, galantamine, and memantine; and the drug for treating pre-Alzheimer's depression is selected from sertraline, venlafaxine, and trazodone.

[0034] Further preferably, the drug combination comprises component one: any one or more of chelidonine, its hydrate, its pharmaceutically acceptable salt, and its pharmaceutical composition; component two: drugs for treating Alzheimer's disease, such as cholinesterase inhibitors (donepezil, etc.), NMDA receptor antagonists (memantine), etc.; and optional component three: drugs for treating pre-depression in Alzheimer's disease, such as selective serotonin reuptake inhibitors (sertraline, etc.), serotonin and norepinephrine reuptake inhibitors (venlafaxine, etc.), trazodone, etc.

[0035] More preferably, the dosage ratio of component one, component two and component three is 2:1:(0-2).

[0036] More preferably, the drug combination is administered simultaneously or sequentially.

[0037] More preferably, when sequential administration is adopted, the order of administration is not particularly limited.

[0038] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention discovered for the first time the down-regulatory effect of chelidonine on Aβ42 protein levels, which can effectively inhibit the formation of the causes of Alzheimer's disease and its early depressive symptoms at the multi-level levels of cells, tissues and animals, and has a significant effect on improving learning and memory disorders and early depressive behaviors, and has clinical application prospects for the treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the effect of chelidonine on the level of Aβ42 in BV2 cells; Figure 2 is the effect of chelidonine on the total intrinsic Aβ42 level in BV2 cells; Figure 3 This is the fluorescence image of co-localization of BV2 cells and Aβ42; Figure 4 is the sugar water preference rate of each group of mice in the sugar water preference experiment; Figure 5 is the immobility time of mice in each group in the tail suspension test; Figure 6 is the immobility time of mice in each group in the forced swimming test; Figure 7 This is the recognition trajectory of each group of mice in the novel object recognition experiment; Figure 8 is the recognition index of each group of mice in the novel object recognition experiment; Fig. 9 is the escape latency of each group of mice in the water maze experiment; Fig.10 It is the percentage of the mice in each group staying in the target quadrant in the spatial exploration experiment; Fig.11 is the percentage of time each group of mice spent in the target quadrant in the spatial exploration experiment; Fig.12 is the number of times each group of mice crossed the platform in the spatial exploration experiment; Fig.13 This is the swimming trajectory of each group of mice in the space exploration experiment; Fig.14 The effect of leucine on Aβ42 levels in the hippocampus and cortex of AD mice. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below in conjunction with embodiments.

[0041] Example 1: Effect of chelidonine on Aβ42 levels in BV2 cells (1) Biomaterials BV2 cells were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

[0042] (2) Reagents Human Aβ42 peptide (Aβ42) and FITC-labeled human Aβ42 peptide (FITC-Aβ42) were purchased from China Qiangyao Biotechnology Co., Ltd. (Suzhou, China). Chrysogenycin was purchased from Selleck Co., Ltd. (S5409, China). DMEM, fetal bovine serum (FBS), and penicillin / streptomycin were purchased from Gibco (New York, USA). Human amyloid beta peptide 1-42, Aβ1-42 ELISA Kit was purchased from Huamei Biotechnology (Wuhan, China, CSB-E10684h). Iba1 antibody was purchased from Wuhan Sanying Biotechnology (Wuhan, China, 10904-1-AP). Cy3-labeled goat anti-rabbit IgG (H+L) and DAPI were purchased from Shanghai Bio-Tech Biotechnology (Shanghai, China, A0516 and C1002).

[0043] (3) Implementation steps BV2 cells were treated with RNAi-Axl and RNAi-Control for 24 hours, and then co-treated with 0.1% DMSO and 2μM soluble Aβ42 or 10μM chelidonine (Ca) and 2μM soluble Aβ42 for 24 hours. At the end of the treatment, the cells were washed with PBS to remove the remaining Aβ42 attached to the cell surface. Then, the cells were lysed with 1% SDS plus ultrasonic disruption, and the intracellular Aβ42 level was measured by ELISA kit according to the manufacturer's instructions.

[0044] The specific operations are as follows: 1) Move all reagents to room temperature (18-25°C) and equilibrate for 30 minutes.

[0045] 2) Preparation of standard products (i) Take out a standard from the kit and centrifuge at 6000-10000 rpm for 30 seconds. Dissolve it with 1 ml of sample diluent and repeatedly pipette the tip of the pipette at the bottom of the cryotube for 5 times to help dissolve it. Mix thoroughly to obtain standard S7 and set aside for later use.

[0046] (ii) Arrange 7 1.5ml centrifuge tubes (S0-S6) in sequence and add 250µl of sample diluent to each. Pipette 250µl of standard S7 into the first centrifuge tube (S6) and gently pipette to mix. Pipette 250µl from S6 into the second EP tube (S5) and gently pipette to mix. Repeat this process to dilute the standard sample in multiples, with S0 being the sample diluent.

[0047] serial number S7 S6 S5 S4 S3 S2 S1 S0 ng / ml 20 10 5 2.5 1.25 0.625 0.312 0 (iii) Sample addition: Set up standard wells and test sample wells respectively. Add 100µl of standard or test sample to each well, shake gently to mix, cover with plate sticker, and incubate at 37℃ for 2 hours.

[0048] (iv) Discard the liquid and spin dry without washing.

[0049] (v) Add 100 µl of biotinylated antibody working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour.

[0050] (vi) Discard the liquid in the wells, spin dry, and wash the plate three times. Soak for 2 minutes each time, 200µl / well, spin dry.

[0051] (vii) Add 100 µl of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour.

[0052] (viii) Discard the liquid in the wells, spin dry, and wash the plate 5 times. Soak for 2 minutes each time, 200µl / well, spin dry.

[0053] (ix) Add 90 µl of substrate solution to each well and incubate at 37°C in the dark for 15-30 minutes.

[0054] (x) Add 50 µl of stop solution to each well to terminate the reaction.

[0055] (xi) Within 5 minutes after the reaction is terminated, measure the optical density (OD value) of each well in sequence at a wavelength of 450 nm using a microplate reader.

[0056] The results are as follows Figure 1 As shown in the figure, compared with the control (0.1% DMSO-treated cells), the intracellular Aβ42 protein level of BV2 cells treated with 10μM chelidonine (Ca) decreased (p < 0.01). After Axl knockout, the effect of chelidonine (Ca) disappeared. This shows that chelidonine (Ca) can reduce intracellular Aβ42, that is, chelidonine (Ca) can promote the degradation of Aβ42 by microglia and alleviate Alzheimer's disease caused by Aβ42 accumulation, and this effect depends on Axl.

[0057] BV2 cells were first treated with RNAi-Axl and RNAi-Control for 24 hours, and then co-treated with 0.1% DMSO and 1μM FITC-Aβ42 or 10μM leucine (Ca) and 1μM FITC-Aβ42 for 24 hours. The cells were then collected with PBS in the dark, and the fluorescence intensity, that is, the Aβ42 level, in the cells was measured by flow cytometry.

[0058] The results are as follows Figure 2As shown in the figure, the fluorescence intensity of the group treated with 10 μM chelidonine (Ca) was higher (p < 0.01), and the fluorescence intensity increase effect of Ca disappeared after Axl knockout. This indicates that chelidonine (Ca) can increase the total intrinsic Aβ42 level in BV2, that is, chelidonine (Ca) can promote the phagocytosis of Aβ42 by BV2 cells, and this effect depends on Axl.

[0059] BV2 cells were co-treated with 0.1% DMSO and 1 μM FITC-Aβ42 or 10 μM chelidonine (Ca) and 1 μM FITC-Aβ42 for 6 h, and then immunofluorescence experiments were performed.

[0060] The specific operations are as follows: 1) Wash cells 3 times with PBS.

[0061] 2) Fix the cells with 4% paraformaldehyde for 15 min.

[0062] 3) Block the cells with 3% BSA for 1 hour.

[0063] 4) Treat cells with Iba1 antibody at 4°C overnight.

[0064] 5) Incubate with Cy3-labeled goat anti-rabbit IgG (H+L) at room temperature for 1 hour.

[0065] 6) DAPI staining for 10 minutes, and finally observation under a confocal microscope. Green is Aβ42, red is BV2 cells, and blue is cell nuclei.

[0066] The results are as follows Figure 3 As shown in the figure, the co-localization of FITC green labeled Aβ42 and Cy3 red fluorescent labeled microglia in the Ca-treated group increased, which also shows that Ca-treated group can promote the entry of Aβ42 into BV2 cells. Increasing the phagocytosis of Aβ42 by BV2 cells can effectively avoid the accumulation of Aβ42 in the brain.

[0067] In general, leucine (Ca) can not only enable microglia to phagocytize more Aβ42, but also promote the degradation of Aβ42, thus effectively preventing the accumulation of Aβ42 in the brain, and thus preventing and alleviating a series of neuroinflammation, oxidative damage and other problems caused by Aβ42 accumulation.

[0068] Example 2: Animal behavior experiment evaluation Five-month-old 5XFAD mice and C57 mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.

[0069] (1) AD model mice 5XFAD mice show significant AD-like behavioral and pathological features around 5 months of age.

[0070] 5XFAD transgenic mice are a classic animal model of Alzheimer's disease. 5XFAD mice express human APP and PSEN1 transgenes, with a total of five AD-related mutations: Swedish (K670N / M671L), Florida (I716V), and London (V717I) familial Alzheimer's disease (FAD) mutations in APP, and human PS1 overexpression mutant human amyloid β (A4) precursor protein 695 (APP) carrying two FAD mutations (M146L and L286V). 5XFAD mice have a large amount of β-amyloid (Aβ) in the brain at 1.5 months of age, and neuritic plaques (NP) begin to appear at 2 months of age. The pathological phenotypes of 5XFAD mice include amyloid plaque aggregation, neuronal loss, and memory dysfunction.

[0071] (2) Animal grouping and drug administration Eight C57 mice were used as the control group (WT), and 16 5XFAD mice were randomly divided into a model group (5XFAD) and a model + calycine group (5XFAD+Ca), with 8 mice in each group. Drugs were administered by intragastric gavage at 9 am every day for a continuous period of time, with an intragastric volume of 0.01 ml / g (Table 1).

[0072] Table 1 Drug administration of animals in each group Group Intragastric administration Dosage (mg / kg) Control group Normal saline / Model Group Normal saline / Model+Ca group Chrysine 30 After 16 days of administration, sugar water preference, forced swimming and tail suspension tests were performed to evaluate the depressive-like behavior of mice; novel object recognition and water maze tests were used to evaluate the Alzheimer's behavior of mice. The testing time was from 9 am to 5 pm every day, and the drug was administered by gavage 30 minutes before the experiment.

[0073] (3) Sugar water preference method to detect depression in mice Three days before the test, mice were adapted to drinking water and 1% sucrose solution, and the positions of drinking water and sugar water tubes were exchanged every 12 hours to prevent mice from forming positional preferences. Before the sugar water preference test, mice were deprived of water for 12 hours. At the beginning and end of the sugar water preference test, the bottles were weighed and the sugar water preference rate (%) was calculated. Sugar water preference rate = 1% sucrose solution consumption / (1% sucrose solution consumption + drinking water consumption) × 100%.

[0074] The results are as follows Figure 4 As shown, the 5XFAD group mice showed a decreased preference for sugar water (p < 0.01). Compared with the 5XFAD group, administration of chelidonine significantly increased the sugar water preference rate of the model mice (p < 0.1). This indicates that 5XFAD mice will have anhedonia symptoms and lack desire for their favorite sugar water. After administration, the anhedonia symptoms of the mice were improved and depression was relieved.

[0075] (4) Tail suspension test to detect depression in mice When rodents are suspended by their tails, they are under short-term unavoidable stress and will gradually tend to an immobile posture. The duration of their immobility can reflect their depressive state. The specific operation method is: hang the mouse on the hook at the top of the detection device with a medical tape about 20 cm long, and keep the mouse hanging upside down, about 30 cm from the ground. The camera records the immobility time of the mouse within 6 minutes to evaluate the mouse's depressive-like behavior.

[0076] The results are as follows Figure 5 As shown in the figure, the immobility time of mice in the 5XFAD group in the tail suspension test was prolonged (p < 0.1). Compared with the 5XFAD group, administration of leucine significantly reduced the immobility time of the model mice (p < 0.1). This indicates that 5XFAD mice are more likely to show despair and remain immobile after their tails are suspended. After administration of the drug, this state of despair was significantly improved, and the depression state was alleviated.

[0077] (5) Forced swimming test Rodents in a water-filled cylinder from which they cannot escape will gradually tend to immobile, and their immobility time can reflect the state of depression. The specific operation method is: place the mouse alone in a transparent open glass cylinder (diameter: 10 cm; water depth: 18 cm; water temperature: 25±1℃). The camera in front of the glass cylinder records the mouse's swimming process for 6 minutes, of which the first 2 minutes are the stage for the mouse to adapt to the environment. The mouse's swimming immobility time is calculated in the last 4 minutes of the test to evaluate the mouse's depressive-like behavior.

[0078] The results are as follows Figure 6 As shown in the results, the immobility time of the mice in the 5XFAD group was prolonged in the forced swimming test (p < 0.1), and compared with the 5XFAD group, administration of leucine significantly reduced the immobility time of the model mice (p < 0.1). In other words, the behavioral despair state of the mice was alleviated and depression was improved.

[0079] (6) Novel object recognition method to detect the learning and memory ability of mice The test was conducted in a square open field apparatus with a side length of 50 cm. During the adaptation phase, each mouse was placed individually in the open field, facing the wall near the operator, and the animal was allowed to explore the field for 6 minutes. The familiarization phase was performed 24 hours after the adaptation step. Two identical cylinders were placed in the open field. The mouse was placed in the open field with its head facing the objects. The mouse was allowed to explore freely for 10 minutes and then returned to its home cage. In this experiment, one cube and one cylinder were used during the test. 24 hours after the familiarization phase, the test was started. The two objects were placed in the same position as before, and the animals were allowed to explore freely for 6 minutes. The exploration time of the familiar object and the novel object was recorded for analysis. The discrimination index was calculated as follows: Discrimination index = (percentage of time with the novel object - percentage of time with the familiar object) / (percentage of time with the novel object + percentage of time with the familiar object).

[0080] The exploration trajectory of mice is as follows Figure 7 As shown, the WT group was mainly directional, showing purposeful search; the 5XFAD group had less overall movement and showed purposeless search; the drug-treated group improved the exploration ability of mice, and the number of directional trajectories increased. The results showed that 5XFAD mice did not want to explore new objects and their cognitive ability was impaired; after the administration of leucine, the mice's exploration of new objects increased significantly, and their impaired cognitive ability was improved.

[0081] The discrimination index is an important indicator in the novel object recognition experiment. The value represents the memory ability of mice. Figure 8 As shown in the figure, the exploration time of the 5XFAD mice on the new objects was significantly lower than that of the WT mice (p < 0.001); compared with the 5XFAD group, administration of chelidonine significantly increased the exploration time of the model mice on the new objects (p < 0.01). In general, chelidonine can improve the impaired cognitive function of 5XFAD mice.

[0082] (7) Morris water maze method to detect the learning and memory ability of mice 1) Device The Morris water maze consists of a pool, a platform, a curtain, and a tracking camera system. The pool is virtually divided into four quadrants, and a cylindrical platform with a diameter of 8 cm and a colorless and transparent material is fixed in the center of the fourth quadrant. Water is poured into the pool so that the final water surface is 0.5 cm higher than the platform. Edible titanium dioxide is added to the water and stirred evenly to make the water in the pool opaque and uniform milky white. The water temperature in the pool is raised to 22°C before use. The curtain is white, and four images are hung in the corresponding quadrant positions as clues for mice to find the platform. The water maze system is placed in a separate, quiet room to avoid direct light.

[0083] 2) Acquisition training At the beginning of the training, the platform is placed in the fourth quadrant, and the mouse is gently released into the water at the horizontal plane facing the pool wall at the predetermined position, so that the mouse can explore freely in the pool. Start timing when the mouse enters the water. End timing when the mouse finds and stays on the platform, and record the corresponding time and movement route. If the mouse fails to find the hidden platform within 90 seconds, guide the mouse to the platform and let it stay on the platform for 30 seconds. After one training session, wipe the mouse and place it in a breeding cage covered with dry bedding. If necessary, it can be irradiated under an infrared lamp to accelerate drying. Each mouse is trained 4 times a day (i.e., placed in the water from 4 different quadrants), with an interval of 30 minutes each time. Acquisition training is carried out for 5 consecutive days.

[0084] 3) Space exploration training On the second day after the acquisition training, i.e. the sixth day in total, spatial exploration training was conducted. The platform was removed and the mice were released from the second quadrant into the water, allowing them to freely explore the pool for 90 seconds. The movement routes of the mice were recorded, and the data such as the time they stayed in the fourth quadrant, the distance traveled, and the number of times they crossed the original platform position were analyzed.

[0085] Analysis of the results of the water maze navigation and positioning. The escape latency is an important indicator of the Morris water maze navigation and positioning phase. It is the time required for the animal to successfully find the platform for the first time after entering the water each time. Its length represents the quality of the animal's spatial learning ability. A short latency indicates that the animal has a good learning ability. The spatial memory ability of mice was examined by the Morris water maze behavioral detection method.

[0086] In the acquisition training stage, the time (latency) for WT mice to successfully find the platform decreased rapidly, while the time required for 5XFAD mice to find the hidden platform was longer (p < 0.001), indicating that the learning and memory ability of the model group mice was impaired; after administration of leucine, the latency of the model mice was significantly reduced (p < 0.01) ( Fig. 9 ), which means that leucine can repair the impaired learning ability of 5XFAD mice. In the spatial exploration experiment, the distance traveled in the target quadrant by 5XFAD mice (p < 0.0001), the duration of stay (p < 0.001) and the number of platform crossings (p < 0.001) were significantly lower than those of WT mice. Compared with the 5XFAD group, the administration of leucine can significantly increase the percentage of distance that the model mice stayed in the target quadrant (p < 0.01) ( Fig.10 ), the percentage of time spent in the target quadrant (p < 0.01) ( Fig.11 ) and the number of times of crossing the platform (p<0.01) ( Fig.12 ), indicating that the memory ability of 5XFAD mice was impaired, and the impaired memory ability was improved after administration of leucine.

[0087] The swimming trajectory of mice Fig.13 As shown in the figure, the normal group was mainly directional, showing purposeful search; the model group showed purposeless search; the drug-treated group improved the swimming pattern of mice, and the number of directional trajectories increased. In general, the intervention of leucine can improve the learning and memory ability of mice with learning and memory disorders.

[0088] Example 3: Elisa detection of Aβ content in mouse brain tissue After the behavioral experiment, the mice were sampled and the brain tissues were divided into hippocampus and cortex. The cortex and hippocampus samples were lysed in RIPA buffer containing protease inhibitors (Thermo, USA). The Aβ42 level was detected by ELISA kit and normalized with total protein concentration.

[0089] The specific operations are as follows: (1) Move all reagents to room temperature (18-25°C) and equilibrate for 30 minutes.

[0090] (2) Preparation of standard products 1) Take out a standard from the kit and centrifuge at 6000-10000 rpm for 30 seconds. Dissolve it with 1 ml of sample diluent and repeatedly pipette 5 times at the bottom of the cryotube to help dissolve it. Mix thoroughly to obtain standard S7 and set aside.

[0091] 2) Take 7 1.5ml centrifuge tubes (S0~S6) and arrange them in sequence. Add 250µl of sample diluent to each tube. Pipette 250µl of standard S7 into the first centrifuge tube (S6) and gently pipette to mix. Pipette 250µl from S6 into the second EP tube (S5) and gently pipette to mix. Repeat this process to dilute the standard in multiples. S0 is the sample diluent.

[0092] serial number S7 S6 S5 S4 S3 S2 S1 S0 ng / ml 20 10 5 2.5 1.25 0.625 0.312 0 3) Sample addition: Set up standard wells and sample wells. Add 100µl of standard or sample to be tested to each well, shake gently to mix, cover with a plate sticker, and incubate at 37℃ for 2 hours.

[0093] 4) Discard the liquid and spin dry without washing.

[0094] 5) Add 100 µl of biotin-labeled antibody working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour.

[0095] 6) Discard the liquid in the wells, spin dry, and wash the plate 3 times. Soak for 2 minutes each time, 200µl / well, spin dry.

[0096] 7) Add 100 µl of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour.

[0097] 8) Discard the liquid in the wells, spin dry, and wash the plate 5 times. Soak for 2 minutes each time, 200µl / well, spin dry.

[0098] 9) Add 90 µl of substrate solution to each well and incubate at 37°C in the dark for 15-30 minutes.

[0099] 10) Add 50µl of stop solution to each well to terminate the reaction.

[0100] 11) Within 5 minutes after the reaction is terminated, measure the optical density (OD value) of each well in sequence at a wavelength of 450 nm using a microplate reader.

[0101] like Fig.14 As shown, the Aβ42 content in the hippocampus and cortex of the 5XFAD group mice was significantly increased (p < 0.0001), and the drug group reduced the increased Aβ42 content in the brain tissue of 5XFAD mice (p < 0.0001). The results show that the intervention of chelidonine can significantly reduce the Aβ content in the brain of 5XFAD mice, reduce the accumulation of Aβ in the brain, and improve AD symptoms.

Claims

1. A use of chelidonine in preparing a drug for treating Alzheimer's disease.

2. Use of chelidonine in the preparation of a drug for treating pre-depression in Alzheimer's disease.

3. The use according to claim 1 or 2, characterized in that: The drug can reduce the level of Aβ42 protein.

4. The use according to claim 3, characterized in that: The drug can promote the phagocytosis and degradation of Aβ42 protein.

5. The use according to claim 3, characterized in that: The drug can inhibit the formation of Aβ42 protein plaques.

6. The use according to claim 3, characterized in that: The drug can improve irreversible neuronal damage caused by imbalance in Aβ42 protein metabolism.

7. The use according to claim 3, characterized in that: The drug can improve neuroinflammation, oxidative damage, abnormal tau protein phosphorylation and glial cell proliferation caused by imbalance in Aβ42 protein metabolism.

8. The use according to claim 1 or 2, characterized in that: The medicine can improve learning and memory disorders and depressive behavior.

9. Use of a hydrate of chelidonine or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating Alzheimer's disease or pre-Alzheimer's depression.

10. Use of a pharmaceutical composition containing chelidonine, a hydrate thereof or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating Alzheimer's disease or pre-Alzheimer's depression.

Images