Pharmaceutical Applications of Chrysine

Abstract

This invention discloses the pharmaceutical application of chelidonine for the treatment of Alzheimer's disease-related diseases. This study, for the first time, demonstrates the downregulation of Aβ42 protein levels by chelidonine, effectively inhibiting the development of Alzheimer's disease and its pre-depressive symptoms at multiple levels, including cells, tissues, and animals. It also significantly improves learning and memory impairment and pre-depressive behavior, demonstrating promising clinical applications in treating related conditions.

Description

Technical Field

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

[0002] Alzheimer's disease (AD), a form of dementia, is characterized by progressive learning and memory impairment, irreversible neuronal loss, and the development of senile plaques in the cerebral cortex and hippocampus, formed by the extracellular accumulation of β-amyloid protein (Aβ42). Imbalances in Aβ42 protein metabolism can cause neuroinflammation, oxidative damage, abnormal tau phosphorylation, and ultimately irreversible neuronal damage, a fundamental driver of AD. In particular, Aβ protein levels begin to rise abnormally long before the onset of overt AD symptoms. Disruption of Aβ protein metabolism in the brain leads to decreased protein clearance and abnormally elevated activity of related secretases, contributing to the development and progression of AD. Therefore, regulating Aβ42 levels in the brain is a key approach to treating AD.

[0003] Major Depressive Disorder (MDD), a common psychiatric disorder, is closely related to Alzheimer's disease (AD). AD patients often experience physiological manifestations of depression in the early stages of their illness. The two conditions share common pathogenesis, such as neurotransmitter imbalances, abnormalities in brain structure and function, inflammation, and neuroplasticity. Because Aβ protein levels are abnormal before overt AD symptoms appear, imbalances in Aβ42 protein metabolism may also contribute to depression in the early stages of AD.

[0004] Currently marketed chemical drugs for the treatment of AD include tacrine, donepezil, revastigmine, galantamine, and menantine. The first four are acetylcholinesterase (AchE) inhibitors, while memantine is an N-methyl-D-aspartic acid (NMDA) receptor antagonist. Both AchE inhibitors and NMDA receptor antagonists are symptomatic treatments, primarily intended to improve cognitive and memory impairments but not to prevent or slow disease progression. Both classes of drugs are associated with significant side effects and may even exacerbate anxiety or depression in some patients. Aducanumab (an Aβ monoclonal antibody) and lecanemab are both antibodies targeting Aβ, designed to reduce Aβ deposition. However, they are expensive, require long-term intravenous infusions, and have significant side effects. In addition, drugs for treating pre-AD depression require special 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, 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 chelidonine hydrate, a pharmaceutically acceptable salt thereof, and a pharmaceutical composition thereof in the treatment of Alzheimer's disease-related diseases.

[0006] Technical solution: The chelidonine of 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 the molecular formula C7H5NO5.

[0008] Chrysine.

[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 the 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 oppressive environments, 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 the preparation of 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 according to the present invention is used in the preparation of a drug 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 a 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 chelidonine 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, which can be an excipient widely used in the field of pharmaceutical production. Excipients are primarily used to provide a safe, stable, and functional pharmaceutical composition, so that the active ingredient dissolves at a desired rate after administration to the subject, or promotes effective absorption of the active ingredient after administration of the composition to the subject. 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 in the composition. The pharmaceutical excipient can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

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

[0031] The pharmaceutical compositions of the present invention can be administered in any form, including transmucosal, oral (solid and liquid formulations), inhalation, ophthalmic, rectal, topical, and parenteral (infusion, injection, implant, subcutaneous, intravenous, intraarterial, and intramuscular) administration. The pharmaceutical compositions of the present invention can also be in controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, hard capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or transmucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, and drops. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other dosage forms include, but are not limited to, eye drops, other ophthalmic formulations; aerosols, such as nasal sprays and inhalers; and 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 early depression in Alzheimer's disease is selected from sertraline, venlafaxine, and trazodone.

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

[0035] More preferably, the usage 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:

[0039] The present invention discovered for the first time the downregulatory effect of leucine on Aβ42 protein levels, which can effectively inhibit the formation of the causes of Alzheimer's disease and its early depressive symptoms at multiple 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 treating related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the effect of chelidonine on the level of Aβ42 in BV2 cells;

[0041] Figure 2 is the effect of chelidonine on the total intrinsic Aβ42 level in BV2 cells;

[0042] Figure 3 Fluorescence images of colocalization of BV2 cells with Aβ42;

[0043] Figure 4 is the sugar water preference rate of each group of mice in the sugar water preference experiment;

[0044] Figure 5 is the immobility time of mice in each group in the tail suspension test;

[0045] Figure 6 is the immobility time of mice in each group in the forced swimming test;

[0046] Figure 7 This is the recognition trajectory of each group of mice in the novel object recognition experiment;

[0047] Figure 8 is the recognition index of each group of mice in the novel object recognition experiment;

[0048] Figure 9 is the escape latency of each group of mice in the water maze experiment;

[0049] Figure 10 is the percentage of each group of mice staying in the target quadrant in the spatial exploration experiment;

[0050] Figure 11 is the percentage of time each group of mice spent in the target quadrant in the spatial exploration experiment;

[0051] Figure 12 is the number of times each group of mice crossed the platform in the spatial exploration experiment;

[0052] Figure 13 This is the swimming trajectory of mice in each group in the space exploration experiment;

[0053] Figure 14 The effect of leucine on Aβ42 levels in the hippocampus and cortex of AD mice. DETAILED DESCRIPTION

[0054] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0055] Example 1: Effect of chelidonine on Aβ42 levels in BV2 cells

[0056] (1) Biomaterials

[0057] BV2 cells were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

[0058] (2) Reagents

[0059] Human Aβ42 peptide (Aβ42) and fluorescein isothiocyanate (FITC)-labeled human Aβ42 peptide (FITC-Aβ42) were purchased from China Qiangyao Biotechnology Co., Ltd. (Suzhou, China). Chrysine was purchased from Selleck Co., Ltd. (S5409, China). DMEM, fetal bovine serum (FBS), and penicillin / streptomycin were purchased from Gibco (New York, NY, 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 Tri-Tian Biotechnology (Wuhan, China, 10904-1-AP). Cy3-labeled goat anti-rabbit IgG (H+L) and DAPI were purchased from Shanghai Beyotime Biotechnology (A0516 and C1002, Shanghai, China).

[0060] (3) Implementation steps

[0061] BV2 cells were treated with RNAi-Axl or RNAi-Control for 24 hours and then co-treated with either 0.1% DMSO and 2 μM soluble Aβ42 or 10 μM chelidonine (Ca) and 2 μM soluble Aβ42 for an additional 24 hours. At the end of treatment, cells were washed with PBS to remove any remaining Aβ42 attached to the cell surface. Cells were then lysed with 1% SDS and sonicated, and intracellular Aβ42 levels were measured using an ELISA kit according to the manufacturer's instructions.

[0062] The specific operations are as follows:

[0063] 1) Move all reagents to room temperature (18-25°C) and equilibrate for 30 minutes.

[0064] 2) Preparation of standard products

[0065] (i) Remove one standard from the kit and centrifuge at 6,000–10,000 rpm for 30 seconds. Dissolve the standard in 1 ml of sample diluent and repeatedly pipette five times toward the bottom of the cryotube to aid dissolution. Mix thoroughly to obtain standard S7 and set aside.

[0066] (ii) Arrange seven 1.5ml microcentrifuge tubes (S0-S6) in sequence and add 250µl of sample diluent to each tube. Pipette 250µl of standard S7 into the first microcentrifuge 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 for serial dilutions of the standard, with S0 being the sample diluent.

[0067] serial number S7 S6 S5 S4 S3 S2 S1 S0 ng / ml 20 10 5 2.5 1.25 0.625 0.312 0

[0068] (iii) Sample addition: Set up separate wells for standards and samples. Add 100µl of standard or sample to each well, gently shake to mix, cover with a plate sticker, and incubate at 37°C for 2 hours.

[0069] (iv) Discard the liquid and spin dry without washing.

[0070] (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.

[0071] (vi) Discard the liquid in the wells, spin dry, and wash the plate three times, soaking for 2 minutes each time, 200µl / well, and spin dry.

[0072] (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.

[0073] (viii) Discard the liquid in the wells, spin dry, and wash the plate five times, soaking for 2 minutes each time, 200µl / well, and spin dry.

[0074] (ix) Add 90 µl of substrate solution to each well and incubate at 37°C in the dark for 15–30 minutes.

[0075] (x) Add 50µl of stop solution to each well to terminate the reaction.

[0076] (xi) Within 5 minutes after the reaction is terminated, measure the optical density (OD) of each well in sequence using a microplate reader at a wavelength of 450 nm.

[0077] The results are as follows Figure 1As shown, treatment of BV2 cells with 10 μM chelidonine (Ca) decreased intracellular Aβ42 protein levels compared to the control (0.1% DMSO-treated cells) (p < 0.01). This effect of chelidonine (Ca) was abolished after Axl knockout. This suggests that chelidonine (Ca) can reduce intracellular Aβ42 levels, suggesting that chelidonine (Ca) can promote microglial degradation of Aβ42, alleviating Alzheimer's disease caused by Aβ42 accumulation, and that this effect is Axl-dependent.

[0078] BV2 cells were first treated with RNAi-Axl and RNAi-Control for 24 hours, and then co-treated with 0.1% DMSO and 1 μM FITC-Aβ42 or 10 μM leucine (Ca) and 1 μM 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.

[0079] The results are as follows Figure 2 As shown in the results, the fluorescence intensity of the 10 μM chelidonine (Ca)-treated group was higher (p < 0.01). After Axl knockout, the increase in fluorescence intensity caused by Ca was eliminated. This indicates that chelidonine (Ca) can increase the total internalized Aβ42 level in BV2 cells, that is, chelidonine (Ca) can promote the phagocytosis of Aβ42 by BV2 cells, and this effect is dependent on Axl.

[0080] BV2 cells were co-treated with 0.1% DMSO and 1 μM FITC-Aβ42 or 10 μM chelidonine (Ca) and 1 μM FITC-Aβ42 and incubated for 6 h before immunofluorescence analysis.

[0081] The specific operations are as follows:

[0082] 1) Wash cells 3 times with PBS.

[0083] 2) Fix the cells with 4% paraformaldehyde for 15 minutes.

[0084] 3) Block cells with 3% BSA for 1 hour.

[0085] 4) Treat cells with Iba1 antibody overnight at 4°C.

[0086] 5) Incubate with Cy3-labeled goat anti-rabbit IgG (H+L) at room temperature for 1 hour.

[0087] 6) DAPI staining for 10 minutes and observation under a confocal microscope. Aβ42 is shown in green, BV2 cells in red, and cell nuclei in blue.

[0088] The results are as follows Figure 3As shown, the colocalization of FITC green-labeled Aβ42 and Cy3 red-fluorescent-labeled microglia increased in the calcineurin (Ca)-treated group, indicating that calcineurin (Ca) can promote the entry of Aβ42 into BV2 cells. Increased phagocytosis of Aβ42 by BV2 cells can effectively prevent the accumulation of Aβ42 in the brain.

[0089] In general, leucine (Ca) not only allows microglia to phagocytize more Aβ42, but also promotes the degradation of Aβ42, thereby 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.

[0090] Example 2: Animal behavior experiment evaluation

[0091] Five-month-old 5XFAD mice and C57 mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.

[0092] (1) AD model mice

[0093] 5XFAD mice show significant AD-like behavioral and pathological features around 5 months of age.

[0094] The 5XFAD transgenic mouse is a classic animal model of Alzheimer's disease. 5XFAD mice express human APP and PSEN1 transgenes, harboring five AD-associated mutations: the Swedish (K670N / M671L), Florida (I716V), and London (V717I) familial Alzheimer's disease (FAD) mutations in APP, and overexpression of the human amyloid β (A4) precursor protein 695 (APP) carrying two FAD mutations (M146L and L286V). 5XFAD mice have high levels of β-amyloid (Aβ) in their brains by 1.5 months of age, and begin to develop neuritic plaques (NP) by 2 months of age. Pathological phenotypes in 5XFAD mice include amyloid plaque accumulation, neuronal loss, and memory impairment.

[0095] (2) Animal grouping and drug administration

[0096] Eight C57 mice served as the control group (WT), and 16 5XFAD mice were randomly divided into a model group (5XFAD) and a model + leucine group (5XFAD+Ca), with 8 mice in each group. Drugs were administered by gavage at 9:00 AM daily for a continuous period, with a gavage volume of 0.01 ml / g (Table 1).

[0097] Table 1 Drug administration of animals in each group

[0098] Group Oral administration Dosage (mg / kg) control group Normal saline / Model Group Normal saline / Model+Ca group Chrysine 30

[0099] After 16 days of administration, sucrose preference, forced swimming, and tail suspension tests were performed to assess the depressive-like behavior of the mice; novel object recognition and water maze tests were used to evaluate the Alzheimer's behavior of the 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.

[0100] (3) Sugar water preference method to detect depression in mice

[0101] For three days prior to testing, mice were acclimated to drinking water and 1% sucrose solution. The positions of the drinking and sugar water tubes were swapped every 12 hours to prevent the mice from developing a preferred position. 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%.

[0102] The results are as follows Figure 4 As shown, the 5XFAD group showed a decreased preference for sugar water (p < 0.01). Compared with the 5XFAD group, administration of leucine significantly increased the sugar water preference of the model mice (p < 0.1). This indicates that 5XFAD mice experience anhedonia, a lack of desire for their favorite sugar water. After administration, the mice's anhedonia symptoms improved and depression was alleviated.

[0103] (4) Tail suspension test to detect depression in mice

[0104] When rodents are suspended by their tails, they experience unavoidable short-term stress and tend to become immobile. The duration of this immobility can reflect their depressive state. The procedure involves suspending a mouse from a hook on top of the test device using approximately 20 cm of medical tape, keeping it suspended upside down, approximately 30 cm from the ground. A camera records the duration of the mouse's immobility for 6 minutes to assess its depressive-like behavior.

[0105] The results are as follows Figure 5 As shown, the immobility time of mice in the 5XFAD group was prolonged in the tail suspension test (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 express despair and remain immobile after being suspended by their tails. After drug administration, this state of despair was significantly improved, and the depression state was alleviated.

[0106] (5) Forced swimming test

[0107] When rodents are trapped in a water-filled cylinder from which they cannot escape, they gradually become immobile, and the duration of this immobility can reflect a state of depression. The specific procedure involves placing a mouse individually in a transparent, open glass cylinder (diameter: 10 cm; water depth: 18 cm; water temperature: 25 ± 1°C). A camera positioned in front of the cylinder records the mouse's swimming for 6 minutes, with the first 2 minutes representing the acclimatization phase. The duration of immobility is then measured during the final 4 minutes of the test to assess depressive-like behavior.

[0108] The results are as follows Figure 6 As shown, the immobility time of mice in the 5XFAD group in the forced swim test was prolonged (p < 0.1), and administration of leucine significantly reduced the immobility time of the model mice compared to the 5XFAD group (p < 0.1). This indicates that the mice's behavioral despair state was alleviated, and depression was improved.

[0109] (6) Novel object recognition method to detect the learning and memory ability of mice

[0110] Testing was conducted in a square, open-air apparatus with a side length of 50 cm. During the acclimation phase, each mouse was placed individually in an open field, facing a wall near the operator, and allowed to explore the field for 6 minutes. The familiarization phase was conducted 24 hours after the acclimation 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 before being returned to its home cage. In this experiment, one cube and one cylinder were used during testing. Testing began 24 hours after the familiarization phase. The two objects were placed in the same locations as before, and the animal was allowed to explore freely for 6 minutes. Exploration time for the familiar and novel objects was recorded for analysis. The discrimination index was calculated as follows: Discrimination index = (percentage time with the novel object - percentage time with the familiar object) / (percentage time with the novel object + percentage time with the familiar object).

[0111] The exploration trajectory of mice is as follows Figure 7 As shown, the WT group showed primarily directional movements, demonstrating purposeful searching; the 5XFAD group exhibited less overall movement and unfocused searching; the drug-treated group improved the mice's exploratory ability, with an increase in directional trajectories. These results suggest that 5XFAD mice are reluctant to explore novel objects and exhibit impaired cognitive abilities; however, after leucine administration, the mice significantly increased their exploration of novel objects, improving their impaired cognitive abilities.

[0112] The discrimination index is an important indicator in the novel object recognition experiment. The value represents the memory ability of mice. Figure 8As shown in the results, the 5XFAD group mice spent significantly less time exploring the novel object than the WT group (p < 0.001). Compared with the 5XFAD group, chelidonine significantly increased the exploration time of the model mice towards the novel object (p < 0.01). Overall, chelidonine can improve the impaired cognitive function of 5XFAD mice.

[0113] (7) Morris water maze method to detect the learning and memory ability of mice

[0114] 1) Device

[0115] 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 made of 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 away from direct light.

[0116] 2) Acquisition training

[0117] At the beginning of training, the platform is placed in the fourth quadrant, and the mouse is gently released into the water at a horizontal plane facing the pool wall at a predetermined position, allowing the mouse to 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 cage with dry bedding. If necessary, it can be exposed to infrared light to accelerate drying. Each mouse is trained 4 times a day (i.e., placed in water from 4 different quadrants), with an interval of 30 minutes each time. Acquisition training is carried out for 5 consecutive days.

[0118] 3) Space exploration training

[0119] Spatial exploration training was conducted two days after the completion of acquisition training (day 6 overall). The platform was removed, and the mice were released from the second quadrant into the water, where they were allowed to explore freely for 90 seconds. The mice's movement paths were recorded, and data such as the time spent in the fourth quadrant, distance traveled, and number of times they crossed the original platform position were analyzed.

[0120] Analysis of navigation results in the Morris water maze. Escape latency is a key indicator of the navigation phase of the Morris water maze. It measures the time it takes for an animal to successfully find the platform after each immersion. Its length reflects the animal's spatial learning ability, with a shorter latency indicating better learning. The Morris water maze behavioral test was used to assess the spatial memory abilities of mice.

[0121] During the acquisition training phase, the time it took for WT mice to successfully find the platform (i.e., latency) 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 abilities of the model mice were impaired. After administration of leucine, the latency of the model mice was significantly reduced (p < 0.01). Figure 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 the 5XFAD group 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 the WT group 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) ( Figure 10 ), the percentage of time spent in the target quadrant (p < 0.01) ( Figure 11 ) and the number of times crossing the platform (p < 0.01) ( Figure 12 ), indicating that the memory ability of 5XFAD mice was impaired, and the impaired memory ability was improved after administration of leucine.

[0122] The swimming trajectory of mice Figure 13 As shown, the normal group showed a predominantly directional pattern, characterized by purposeful searching, while the model group exhibited non-purposeful searching. The drug-treated group showed improved swimming patterns, with an increase in directional trajectories. Overall, leucine intervention can improve the learning and memory abilities of mice with learning and memory impairments.

[0123] Example 3: Elisa detection of Aβ content in mouse brain tissue

[0124] After the behavioral experiments, the mice were harvested and their brain tissue was divided into hippocampal and cortical regions. Cortical and hippocampal samples were lysed in RIPA buffer containing protease inhibitors (Thermo, USA). Aβ42 levels were measured using an ELISA kit and normalized to total protein concentration.

[0125] The specific operations are as follows:

[0126] (1) Move all reagents to room temperature (18-25°C) and equilibrate for 30 minutes.

[0127] (2) Preparation of standard products

[0128] 1) Remove one standard from the kit and centrifuge at 6,000–10,000 rpm for 30 seconds. Dissolve the standard in 1 ml of sample diluent and pipette up and down the bottom of the cryovial five times to aid dissolution. Mix thoroughly to obtain standard S7 and set aside.

[0129] 2) Arrange seven 1.5ml microcentrifuge tubes (S0-S6) in sequence and add 250µl of sample diluent to each. Pipette 250µl of standard S7 into the first microcentrifuge 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 for serial dilutions of the standard, with S0 being the sample diluent.

[0130] serial number S7 S6 S5 S4 S3 S2 S1 S0 ng / ml 20 10 5 2.5 1.25 0.625 0.312 0

[0131] 3) Sample Addition: Set up separate wells for standards and samples. Add 100µl of standard or sample to each well, gently shake to mix, cover with a plate sticker, and incubate at 37°C for 2 hours.

[0132] 4) Discard the liquid and spin dry without washing.

[0133] 5) Add 100µl of biotinylated antibody working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour.

[0134] 6) Discard the liquid in the wells, spin dry, and wash the plate three times, soaking for 2 minutes each time, 200µl / well, and spin dry.

[0135] 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.

[0136] 8) Discard the liquid in the wells, spin dry, and wash the plate five times, soaking for 2 minutes each time, 200µl / well, and spin dry.

[0137] 9) Add 90µl of substrate solution to each well and incubate at 37°C in the dark for 15-30 minutes.

[0138] 10) Add 50µl of stop solution to each well to terminate the reaction.

[0139] 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.

[0140] like Figure 14As shown, the Aβ42 content in the hippocampus and cortex of the 5XFAD group mice was significantly increased (p < 0.0001), while the elevated Aβ42 content in the brain tissue of the 5XFAD mice was reduced in the drug-treated group (p < 0.0001). The results show that leucine intervention can significantly reduce the Aβ content in the brain of 5XFAD mice, reduce Aβ accumulation in the brain, and improve AD symptoms.

Claims

1. Application of chelidonine in the preparation of drugs for treating Alzheimer's disease.

2. The use of leucine in the preparation of drugs for treating early 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 Aβ42 protein metabolic imbalance.

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 medicament for treating Alzheimer's disease.

10. Use of a hydrate of chelidonine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating early depression in Alzheimer's disease.

11. Use of a pharmaceutical composition containing chelidonine, a hydrate thereof or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating Alzheimer's disease.

12. Use of a pharmaceutical composition containing chelidonine, a hydrate thereof or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating early depression in Alzheimer's disease.

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