Application of cyanidin derivative

Abstract

The invention relates to the technical field of biological medicines, in particular to application of five cyanidin derivatives in preparation of medicines for treating and / or preventing neurodegenerative diseases. The cyanidin derivative provided by the invention can inhibit the activity of acetylcholin esterase, relieve oxidative stress, reduce AD related gene expression and protect neuron integrity; due to the multi-target action mechanism, the symptoms of AD patients can be more comprehensively improved, and the disease progression is delayed.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of cyanidin derivatives. Background Art

[0002] Alzheimer's Disease (AD) is a neurodegenerative disease characterized by progressive cognitive impairment and behavioral abnormalities. It is the most common type of dementia globally, severely affecting the quality of life and lifespan of patients. With the increasing aging of the global population, the incidence of AD shows an upward trend year by year, imposing a heavy burden on society and families. Currently, the pathogenesis of AD is not fully understood, and the mainstream hypotheses include the cholinergic hypothesis, the β-amyloid (Aβ) hypothesis, and the tau protein hypothesis. Among them, the cholinergic hypothesis holds that a significant decrease in the level of acetylcholine (ACh) in the brains of AD patients is one of the key factors leading to cognitive impairment.

[0003] Currently, the treatment of AD mainly relies on cholinesterase inhibitors (such as donepezil and rivastigmine) and NMDA receptor antagonists (such as memantine). Although these drugs can improve the cognitive function of patients to a certain extent, their efficacy is limited, and obvious side effects exist. For example, donepezil may cause gastrointestinal reactions such as nausea, vomiting, and loss of appetite, while memantine may cause central nervous system adverse reactions such as dizziness and hallucinations. In addition, these drugs can only delay the progression of the disease and cannot cure AD fundamentally. Therefore, the development of new, highly effective, and low-toxic AD treatment drugs has become a major demand in the global medical and pharmaceutical fields.

[0004] Cyanidin is a type of anthocyanin compound widely present in nature, with various biological activities such as antioxidant, anti-inflammatory, and anti-apoptotic effects. In recent years, with the in-depth study of the medicinal value of natural products, the potential application of cyanidin and its derivatives in the field of neuroprotection has gradually attracted attention. Research shows that cyanidin can exert neuroprotective effects through multiple mechanisms, including inhibiting oxidative stress, regulating the apoptosis pathway, and improving neuroinflammation. However, the current research on the application of cyanidin and its derivatives in the treatment of Alzheimer's disease is still in its initial stage, and there is no clear evidence indicating that it can be directly used in the clinical treatment of AD. Cyanidin derivatives are a class of cyanidin analogs obtained through chemical modification or natural extraction, with diverse structures and functions. On the basis of retaining the core structure of cyanidin, by introducing different substituents (such as glycosyl, methoxy, etc.), their biological activities and pharmacokinetic properties can be significantly enhanced. For example, certain cyanidin derivatives have been proven to have stronger antioxidant capabilities and higher cell uptake rates. Summary of the Invention

[0005] Cyanidin is a type of anthocyanin compound widely present in nature, with various biological activities such as antioxidant, anti-inflammatory, and anti-apoptotic effects. Cyanidin derivatives are a class of cyanidin analogs obtained through chemical modification or natural extraction, having diverse structures and functions. Based on retaining the core structure of cyanidin, by introducing different substituents (such as glycosyl, methoxy, etc.), the biological activities and pharmacokinetic properties of these derivatives can be significantly enhanced. For example, certain cyanidin derivatives have been proven to have stronger antioxidant capacity and higher cellular uptake rates.

[0006] The inventors found in their research that some cyanidin derivatives have strong inhibitory activity against acetylcholinesterase (AChE). An important pathological feature of AD is the loss of cholinergic neurons in the brain, leading to a significant decrease in the level of acetylcholine (ACh) at the synaptic site; acetylcholine is an important neurotransmitter, closely related to memory and cognitive functions; in addition, the increased activity of AChE will cause the rapid decomposition of ACh, further exacerbating cognitive dysfunction. The present invention prevents and / or treats Alzheimer's disease by reducing the activity of acetylcholinesterase.

[0007] The present invention provides the use of a cyanidin derivative in the preparation of a medicament for treating and / or preventing neurodegenerative diseases, and the cyanidin derivative is selected from the following compounds:

[0008]

[0009] In some specific embodiments of the present invention, the cyanidin derivative is:

[0010]

[0011] In the present invention, the neurodegenerative diseases are selected from Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, multiple system atrophy, fragile X syndrome, muscular dystrophy, cerebral small vessel disease, neuronal ceroid lipofuscinosis, Wilson's disease, primary lateral sclerosis, progressive supranuclear palsy, and ataxia.

[0012] In some specific embodiments of the present invention, the neurodegenerative disease is Alzheimer's disease.

[0013] In some specific embodiments of the present invention, the Alzheimer's disease is induced by AlCl3.

[0014] In the present invention, the drug regulates the activity of at least one enzyme among acetylcholinesterase, superoxide dismutase, choline acetyltransferase, monoamine oxidase, catalase, glutathione peroxidase, acetylcholine receptor, tyrosine hydroxylase, glutamate decarboxylase, and phosphodiesterase.

[0015] In some specific embodiments of the present invention, the drug is a drug that inhibits acetylcholinesterase.

[0016] In some specific embodiments of the present invention, the drug is an AchE inhibitor.

[0017] In some specific embodiments of the present invention, the drug is a drug that regulates the expression of at least one gene among ache, tau, appb, and aβ.

[0018] In some specific embodiments of the present invention, the drug is a drug that inhibits the expression of at least one gene among ache, tau, appb, and aβ.

[0019] In some specific embodiments of the present invention, the dosage form of the drug is any one of solid preparations, liquid preparations, gel preparations, semi-fluid preparations, and aerosol preparations.

[0020] The present invention also provides a pharmaceutical composition, and the drug contains an effective dose of cyanidin derivatives.

[0021] In some specific embodiments of the present invention, the drug further contains a pharmaceutically acceptable carrier and / or excipient.

[0022] The term "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable (i.e., does not cause an unacceptable level of undesirable biological effects or interact in a harmful manner).

[0023] "Pharmaceutically acceptable carrier" refers to sterile water or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders that are immediately used for reconstitution into sterile injectable solutions or dispersions before use. Examples of suitable water and non-aqueous carriers, diluents, solvents, or vehicles can be listed but are not limited to water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and its suitable mixtures, vegetable oils (such as olive oil, sesame oil), and injectable organic esters, such as ethyl oleate.

[0024] The carrier can be selected to have appropriate fluidity, and can be maintained by, for example, using coating materials such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants.

[0025] In addition, when preparing pharmaceutical preparations, adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents can also be included. Preservatives can prevent the action of microorganisms, for example, the action can be achieved through various antibacterial agents and antifungal agents. Specific examples include: nipagin, chlorobutanol, phenol, sorbic acid, potassium sorbate, etc.

[0026] The term "treatment" refers to prophylactic (e.g., prophylactic medication), curative, or palliative treatment. Specifically, treatment herein refers to the administration or delivery of the cyanidin derivatives and / or pharmaceutical compositions described in the present invention to an individual who may have a medical disorder, symptoms related to the disorder, secondary diseases or abnormalities of the disorder, or is prone to suffering from the above-mentioned disorders, in order to partially or completely alleviate, improve, or relieve one or more symptoms or characteristics of a specific abnormality and / or disease condition, or delay its occurrence, hinder its progression, reduce its severity and / or reduce the incidence rate. Treatment can also be carried out on individuals who have not shown signs of disease, abnormality, and / or disease condition and / or individuals who have shown early signs, in order to reduce the risk of developing pathological changes related to the disease, abnormality, and / or disease condition.

[0027] In the present invention, the medicinal use of the cyanidin derivative: administering / delivering an effective dose of the cyanidin derivative and / or pharmaceutical composition to an individual suffering from a neurodegenerative disease.

[0028] In the present invention, an "individual" refers to a mammal, including a human, who can receive the cyanidin derivatives and / or pharmaceutical compositions described in the present invention. Unless otherwise specified, the terms "administer" and "deliver" can be used interchangeably herein, and they refer to providing the cyanidin derivatives and / or pharmaceutical compositions of the present invention to an individual in need of treatment.

[0029] In the present invention, an "effective dose" is the amount of the cyanidin derivative and / or pharmaceutical composition sufficient to elicit the desired therapeutic response. The effective dose of the medicament does not necessarily have to be able to cure the disease or disorder, but can delay, hinder, or prevent the occurrence of the disease or disorder, or can alleviate the symptoms related to the disease or disorder. The therapeutically effective dose can be divided into one, two, or more doses and administered once, twice, or more times in a suitable dosage form within a specified period. The specific therapeutically effective dose or sufficient dose depends on various factors, such as the specific condition to be treated, the physiological condition of the individual (e.g., the weight, age, or gender of the individual), the type of mammal or animal being treated, the duration of treatment, the nature of concurrent treatments (if any), and the specific formulation and structure of the active ingredient used. The effective amount can be expressed in any suitable manner, for example, it can be expressed as the total weight of the cyanidin derivative and / or pharmaceutical composition (e.g., expressed in grams, milligrams, or micrograms) or as the ratio of the cyanidin derivative and / or pharmaceutical composition to body weight (e.g., expressed as milligrams per kilogram of body weight (mg / kg)).

[0030] The beneficial effects of the present invention are as follows:

[0031] (1) The present invention provides the use of five cyanidin derivatives in the preparation of drugs for treating and / or preventing neurodegenerative diseases; the cyanidin derivatives can inhibit acetylcholinesterase activity, alleviate oxidative stress, reduce the expression of AD-related genes, and protect neuronal integrity; this multi-target mechanism of action enables it to more comprehensively improve the symptoms of AD patients and delay the progression of the disease;

[0032] (2) In the present invention, experiments show that: cyanidin derivatives can significantly reduce neuronal damage and pathological changes in the zebrafish model induced by AlCl3 and improve the survival rate of neurons; this indicates that it has effective neuroprotective ability and is expected to provide a more effective neuroprotective treatment plan for AD patients. Brief Description of the Drawings

[0033] Figure 1 Effects of cyanidin derivatives on the movement disorder of AD zebrafish (A is the total distance; B is the average speed; C is the representative swimming trajectory diagram of larvae in each group);

[0034] Figure 2 Effects of cyanidin derivatives on the cholinergic system dysfunction and oxidative stress of AD zebrafish;

[0035] Figure 3 Effects of cyanidin derivatives on the expression of AD-related genes;

[0036] Figure 4 HE staining diagram;

[0037] Figure 5 Nissl staining diagram;

[0038] Figure 6 Binding model and affinity of cyanidin derivatives with the crystal structure of AchE protein;

[0039] Figure 7 Molecular dynamics simulation results of the binding of AchE protein with cyanidin derivatives;

[0040] Figure 8 Relative free energy distribution and comparison of the complex structure of the binding of AchE protein with cyanidin derivatives;

[0041] Figure 9 ADMET and drug similarity analysis of five cyanidin derivatives by the online prediction tool ADMET lab 2.0. Detailed Embodiments

[0042] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] Example 1

[0044] 1 Materials and Methods

[0045] 1.1 Chemicals and Reagents

[0046] AlCl3·6H2O was purchased from Sigma-Aldrich (St. Louis, MO, USA). Donepezil and eugenol were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. Seven cyanidin derivatives were all purchased from Shanghai Yuanye Biotechnology Co., Ltd., and the detailed information is shown in Table 1. Phosphate-buffered saline (PBS) was purchased from Wuhan Punosai Life Science Co., Ltd. The zebrafish AChE kit was provided by Shanghai Enzyme-linked Biotechnology Co., Ltd. The superoxide dismutase (SOD) and glutathione (GSH) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing). The lipid peroxidation (MDA) detection kit was purchased from Beyotime Biotechnology Co., Ltd. Trizol and the reverse transcription kit were purchased from Takara Biotechnology (Beijing) Co., Ltd. ChamQ Universal SYBR qPCR Master Mix was purchased from Nanjing Novozymes Biotech Co., Ltd. The primers were synthesized by Shanghai Bioengineering Co., Ltd.

[0047] Table 1 Cyanidin Derivatives

[0048]

[0049]

[0050] 1.2 Animals

[0051] Before the experiment, adult wild-type zebrafish (AB strain) at 6-8 months old used in the present invention were acclimated in the laboratory aquaculture system (Beijing ESEN) for at least 1 week. The temperature was 25±2°C, the pH value was 7.4±0.2, the photoperiod was 14 h light / 10 h dark cycle, and the brine shrimp were fed regularly twice a day. The larvae were obtained by natural mating. Zebrafish larvae at 3 days post-fertilization (dpf) were used in the present invention. All experiments followed the standard ethical guidelines and were carried out under the supervision of the Animal Ethics Committee of the Northwest Institute of Plateau Biology.

[0052] 1.3 Establishment and Grouping of Zebrafish AD Model

[0053] Zebrafish larvae at 3 - 6 dpf were treated with 80 μM AlCl3 to establish an AD model. The experimental method for zebrafish larvae was as follows: Healthy and normally developed zebrafish larvae at 3 dpf were selected and placed in 6 - well plates, with 40 larvae per well and 3 replicate wells. Subsequently, the larvae were divided into several experimental groups: control group (standard system water), model group (treated with 80 μM AlCl3), model + Dpz group (treated with 80 μM AlCl3 + 4 μM donepezil), and model + cyanidin derivative group (treated with 80 μM AlCl + 4 μM cyanidin derivative).

[0054] 1.4 Behavioral analysis

[0055] Three days after drug administration, 12 larvae were randomly selected from each group and rinsed with E3 culture medium. Zebrafish larvae cultured to 6 dpf were placed individually in 96 - well culture plates containing 300 μL of E3 medium per well. The culture temperature was maintained at 28.5°C. A 10 - minute acclimation period was carried out for the zebrafish larvae in the 96 - well plates in the instrument. All experiments were carried out within 30 minutes, including 3 alternating light - dark cycles (each cycle lasting 5 minutes). The movement activities of each larva were recorded using VisuTrack artificial intelligence software (Shanghai Xinxin Information Technology Co., Ltd., Shanghai, China), and their movement distance and average speed were analyzed.

[0056] 1.5 Determination of biochemical indexes

[0057] After co - treatment with AlCl3 and each drug, at 6 dpf, the zebrafish were sacrificed using eugenol. Ninety zebrafish from each group were taken and placed in 1.5 - mL EP tubes, and pre - cooled normal saline was added at a ratio of 1:9 (mass: volume). Mechanical homogenization was carried out using an ultrasonic homogenizer under ice - bath conditions to completely break the tissues. The prepared homogenate of each group was centrifuged at 12,000 rpm for 15 minutes, and the supernatant was taken for the detection of biochemical indexes AChE, SOD, MDA, and GSH. All determinations were repeated 3 times and the average values were taken.

[0058] 1.6 Detection of gene expression

[0059] Real - time fluorescence quantitative PCR technology was used to detect the expression levels of 4 AD - related genes, ache, tau, appb, and aβ, in zebrafish larvae. After the behavioral tests were completed, the zebrafish larvae were euthanized using eugenol solution and then rinsed 3 times with 1×PBS to remove residual water. Twenty larvae from each group were stored in 2 - mL EP tubes for subsequent experiments. Total RNA of each sample was extracted using Trizol, and OD was measured 260 / 280The value is greater than 1.8. The extracted RNA samples were reverse transcribed into cDNA according to the manufacturer's instructions provided by the cDNA synthesis kit. Quantitative PCR was performed using the SYBR Green I method, and the reaction was carried out three times. The average value of the 3 reactions was taken for analysis. The expression of β-actin was selected as the internal reference gene for normalization, and the ΔΔCt method was used for quantitative analysis. The primer sequences are shown in Table 2.

[0060] Table 2 Primer Sequences

[0061]

[0062] 1.7 HE staining and Nissl staining

[0063] Histopathological studies were performed on the brain tissues of zebrafish larvae. Zebrafish larvae at 6 dpf were collected and fixed in 4% (w / v) paraformaldehyde overnight. Subsequently, the tissues were dehydrated through a series of ethanol steps, embedded in paraffin, and sectioned into longitudinal sections with a thickness of 5 μm. The tissue sections were then subjected to HE and Nissl staining. Subsequently, they were dewaxed in xylene, dehydrated with alcohol, and covered with neutral balsam. Changes in telencephalic neurons were observed under a magnifying glass at 400 times magnification. The positive rate of Nissl bodies in the Nissl-stained sections of each group was quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

[0064] 1.8 Molecular docking

[0065] The three-dimensional structure of the receptor protein AChE (PDB ID: 4moe) was obtained from the RCSB Protein Data Bank (PDB) (https: / / www1.rcsb.org / ). The three-dimensional structure file of the small molecule compound cyanidin derivative was downloaded from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), and the molecular force field was optimized using Chem3D to obtain the optimal molecular structure. The protein was hydrogenated using the AutoDock tool 1.5.6, the grid plate parameters were configured to define the docking range, and the semi-flexible docking method was adopted. Molecular docking was performed using AutoDockVina 1.2.0 software, the Lamarckian genetic algorithm was selected, and the binding free energy and docking result files were obtained. The docking results were visualized using a combination of PyMOL 2.3.0 software and Discovery Studio 2019.

[0066] 1.9 Molecular dynamics simulation

[0067] Molecular dynamics simulations were performed on the protein-ligand complexes obtained by molecular docking using Gromacs 2022.4 software. Amber14sb was selected as the protein force field, Gaff2 as the ligand force field, and the TIP4P water model was chosen to add solvent to the protein-ligand system. Before the formal kinetic simulation, the conjugate gradient algorithm was used to perform 50,000 steps of energy minimization on the complex, and then the isothermal (310K) system (NVT) and isobaric (1 atmosphere) system (NPT) were used to further equilibrate the system at a time step of 2 fs for 100 ps. Finally, an unconstrained molecular dynamics simulation of 100 ns was performed at ambient temperature and pressure, and the structural coordinates were saved every 10 ps. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and number of hydrogen bonds between the protein and the ligand were analyzed. The average binding free energy between the protein and the ligand was calculated using the MM / GBSA method.

[0068] 1.10 In silico prediction of ADMET and drug-like properties of cyanidin derivatives

[0069] ADMET evaluation models have been recognized as essential tools in the drug development process, significantly improving the efficiency of drug design and optimization of lead compounds. In the present invention, the pharmacokinetic characteristics and toxicity of cyanidin derivatives were comprehensively evaluated using ADMETlab 2.0 (https: / / admetmesh.scbdd.com / pub / ). This online platform provides reliable predictions of the ADMET properties (absorption, distribution, metabolism, elimination, and toxicity) of compounds and their drug-like properties.

[0070] 1.11 Statistical analysis

[0071] Data were represented as the mean ± standard error of the mean (SEM) obtained from at least 3 independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey's test. All statistical analyses were carried out using GraphPad Prism 9.4 software (GraphPad Software, Inc., La Jolla, CA, USA). A P < 0.05 was considered statistically significant.

[0072] 2 Experimental results

[0073] 2.1 Effects of cyanidin derivatives on AlCl3-induced locomotor defects in zebrafish larvae

[0074] To investigate AlCl3-induced locomotor impairment in zebrafish and the protective effects of cyanidin derivatives, behavioral experiments were performed on zebrafish larvae at 6 dpf. The swimming trajectories of zebrafish are shown as Figure 1 in C. AsFigure 1 As shown, compared with the control group, exposure to AlCl3 led to a significant decrease in the total distance Figure 1 (A) and average speed Figure 1 (B) of zebrafish (P<0.01). The results indicate that AlCl3 has a damaging effect on the locomotor ability of zebrafish. Therefore, the establishment of the zebrafish AD model was successful.

[0075] After treatment with 4 μM donepezil and cyanidin derivatives, compared with the AD group, the swimming distance and average speed of zebrafish both increased Figure 1 (A, B). The results show that donepezil improved the locomotor dysfunction of zebrafish induced by AlCl3, indicating its potential therapeutic effect. Interestingly, a similar trend of behavioral changes was also observed in the cyanidin derivative treatment group. When zebrafish were co-treated with AlCl3 and different types of cyanidin derivatives, their locomotor disorders were significantly reduced, and C3R had the best effect. These results suggest that the application of cyanidin derivatives can effectively alleviate the locomotor behavioral disorders of zebrafish larvae caused by AlCl3, indicating that it may act as a protective agent against the AD-like symptoms of zebrafish caused by AlCl3.

[0076] 2.2 Effects of cyanidin derivatives on AlCl3-induced cholinergic system dysfunction and oxidative stress

[0077] To investigate the alleviating effect of cyanidin derivative treatment on AlCl3-induced cholinergic dysfunction and oxidative stress in the AD model, the activities of AChE, SOD, GSH, and MDA in zebrafish larvae were measured in this invention. The AChE activities of each group are as Figure 2 shown in A. Compared with the control group, AlCl3 significantly increased the AChE activity. However, cyanidin derivatives and Dpz showed a partial recovery of the AlCl3-induced AChE alteration (P<0.01), which was consistent with the results observed in the behavioral assessment. Compared with the normal control group, zebrafish exposed to AlCl3 showed a significant increase in MDA levels and a significant decrease in GSH and SOD levels, as Figure 2 shown in B-D (P<0.01). Co-treatment with cyanidin derivatives and Dpz at a concentration of 4 μM could significantly increase the SOD enzyme activity, increase the GSH level, and decrease the MDA concentration to a certain extent (Dpz, C3R, C3S, and C3X; P<0.05). Among them, C3R had the best effect, and C3Ga and C3G had poor or almost no effect on SOD, GSH, and MDA. The results indicate that some cyanidin derivatives can effectively alleviate AlCl3-induced cholinergic system dysfunction and oxidative stress in zebrafish by enhancing the activities of nerve conduction markers and antioxidant enzymes.

[0078] 2.3 Effects of cyanidin derivatives on the expression of AD-related genes

[0079] As Figure 3 shown, in zebrafish larvae exposed to AlCl3, the expression levels of ache, tau, appb, and aβ were significantly upregulated (P<0.01). However, treatment with cyanidin derivatives C3GG, C3R, C3A, C3S, and C3X reversed this phenomenon (P<0.01, except for C3A). These findings suggest that cyanidin derivatives have the potential to alleviate AD-like symptoms in zebrafish. PCR results further indicate that AlCl3 causes severe cholinergic damage to zebrafish, while treatment with C3GG, C3R, C3A, C3S, and C3X cyanidin derivatives can significantly alleviate its adverse reactions.

[0080] 2.4 Protective effects of cyanidin derivatives on the pathological damage of the brain tissue of AlCl3-induced larvae

[0081] The results of HE staining showed that the brain tissue of zebrafish in the normal group presented a normal and complete overall structure ( Figure 4 A). In addition, the midbrain showed a large number of neatly arranged neurons, characterized by spherical or slightly oval nuclei and stable morphology. In contrast, zebrafish brain sections treated with AlCl3 showed severe balloon degeneration and contracted neurons, showing black staining, without visible nuclei or nucleoli ( Figure 4 B). In addition, tissue defects, as well as some atrophied and sickle-shaped cells, were observed in the midbrain region. When treated with cyanidin derivatives (C3GG, C3R, C3A, C3S, and C3X) and Dpz, the presence of abnormal neurons in the zebrafish brain could be effectively reduced, the number of healthy neurons increased, and cell damage and neuronal edema necrosis were alleviated ( Figure 4 C-H). Notably, C3R treatment significantly restored the abnormal changes caused by AlCl3, and no abnormal brain morphology was observed, indicating its significant ability to resist AlCl3-induced neuronal degeneration ( Figure 4 E).

[0082] In addition, the present invention detected the loss of neurons using Nissl staining. It was found that the nerve cells in the control group were clear and complete, and the proportion of Nissl bodies was high (34.36%). In contrast, the AlCl3 treatment group showed obvious loss of Nissl bodies (13.28%), with damaged and disordered cells, smaller cell size, and cytoplasmic condensation or non-staining. Notably, treatment with cyanidin derivatives (C3GG, C3R, C3A, C3S, and C3X) and Dpz significantly alleviated cell damage ( Figure 5), resulting in an increased proportion of Nissl bodies. The Nissl body ratio of Dpz was 30.91%, C3GG was 9.72%, C3R was 36.94%, C3A was 14.22%, C3S was 17.97%, and C3X was 20.46%. The results of these neuroprotective effects were consistent with those of behavioral and biochemical experiments, further confirming the results of HE staining. These research results demonstrate that cyanidin derivatives can significantly alleviate the pathological damage of neurons in zebrafish induced by AlCl3, and among them, C3R exhibits the most obvious protective effect.

[0083] 2.5 Molecular docking results

[0084] The binding models and affinities of cyanidin derivatives with the crystal structure of AchE protein are as Figure 6 shown. The interaction forces between cyanidin derivatives and the potential target AchE mainly include polar interactions (hydrogen bonds) and non-polar interactions ( Figure 6 A-E). Among them, the binding affinity of C3R is the highest, at -9.5 kcal / mol, and the binding affinities of C3A, C3S, and C3X are comparable, at -9.1 kcal / mol. The docking score of C3GG is -9.0 kcal / mol. It is worth noting that the docking score of the positive control drug Dpz is -9.2 kcal / mol. The results show that cyanidin derivatives have a strong interaction with AchE, and their binding affinity is comparable to that of the positive control drug (Dpz), indicating their potential pharmacological effects. This finding may potentially clarify the molecular mechanism of cyanidin derivatives as effective AchE inhibitors.

[0085] 2.6 Molecular dynamics simulation results

[0086] To verify the molecular docking results, the present invention analyzed the molecular dynamics simulation trajectories of the binding of AchE protein to cyanidin derivatives. The present invention detected the RMSD, RMSF, Rg, and the number of hydrogen bonds, SASA, relative free energy distribution, and structural comparison of the complex at 0, 25, 50, 75, and 100 ns. In addition, the present invention used the MM / GBSA method to calculate the average binding free energy between the protein and the ligand. As Figure 7 shown in A-C, the fluctuations of the RMSD, RMSF, and Rg values of the AchE-cyanidin derivative complex are small and remain stable throughout the simulation, indicating that the formation of the complex has good stability. The number of hydrogen bonds formed between the AchE protein and cyanidin derivatives remains stable, indicating the effective stability of the complex ( Figure 7 D). In addition, throughout the simulation, the SASA curve of the AchE-cyanidin derivative complex shows stable fluctuations, indicating that cyanidin derivatives have little effect on the overall structure of the protein ( Figure 7 E).

[0087] The present invention further analyzed the relative free energy distribution and comparison of the complex structure, as Figure 8 shown in A, the complex formed by the AChE protein and the cyanidin derivative is very stable in terms of free energy distribution. As Figure 8 shown in B, the results show that the cyanidin derivative forms a more stable complex with the AChE protein, while the complexes formed by C3GG and C3S with the AChE protein are less stable. Finally, the average binding free energy between these proteins and the cyanidin derivative was calculated using the MM / GBSA method. The average binding free energies of C3GG, C3R, C3A, C3S, and C3X are -32.29, -46.37, -32.79, -41.50, and -46.13 kcal / mol, respectively, indicating that these compounds have strong binding affinities with AChE. It is worth noting that C3R has the highest binding affinity with AChE ( Figure 8 C).

[0088] 2.7 Prediction of ADMET and drug-like properties

[0089] The results of the ADMET and drug-like properties of the cyanidin derivative are shown in Figure 9 . The results show that the cyanidin derivative generally has good ADMET and drug-like properties. In terms of MDCK permeability and blood-brain barrier (BBB) permeability, the cyanidin derivative shows good activity. In addition, the cyanidin derivative shows satisfactory safety, performing well in multiple indicators, such as low hERG blocker (indicating cardiotoxicity) and human liver toxicity (H-HT). In addition, the cyanidin derivative shows a low risk of inhibiting CYP2C19, CYP2C9, CYP2D6, and CYP3A4 enzymes, while showing a high metabolic rate and low liver toxicity. These compounds meet one or more drug-like criteria, such as MCE-18 and Pfizer rules, indicating that they have good pharmacological properties.

[0090] In summary, the cyanidin derivative exerts a neuroprotective effect by inhibiting AchE; the cyanidin derivative can effectively alleviate the AD-like behavioral disorder induced by AlCl3, reduce the activity of AchE, and alleviate oxidative stress. In addition, the cyanidin derivative can reduce the expression of AD-related genes and enhance cholinergic neurotransmission. The cyanidin derivative can promote neuronal integrity, regulate neuron count, and improve brain tissue pathology. In vitro computer simulations provide further evidence to support the development of the cyanidin derivative as an AD therapeutic agent by inhibiting AchE activity. These findings highlight the preventive potential of the cyanidin derivative, especially C3R, C3S, and C3X, in the treatment of neurodegenerative diseases.

[0091] Although embodiments of the present invention have been shown and described, those of ordinary skill in the art will appreciate that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of cyanidin derivatives in the preparation of drugs for treating and / or preventing neurodegenerative diseases, characterized in that: The cyanidin derivatives are selected from the following compounds:

2. The use according to claim 1, characterized in that: The cyanidin derivative is:

3. The use according to claim 1, characterized in that: The neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, multiple system atrophy, fragile X syndrome, muscular dystrophy, cerebral small vessel disease, neuronal ceroid lipofuscinosis, hepatolenticular degeneration, primary lateral sclerosis, progressive supranuclear palsy and ataxia.

4. The use according to claim 1, characterized in that: The neurodegenerative disease is Alzheimer's disease; further, the Alzheimer's disease is induced by AlCl3.

5. The use according to claim 1, characterized in that: The drug is a drug that regulates at least one enzyme activity of acetylcholinesterase, superoxide dismutase, choline acetyltransferase, monoamine oxidase, catalase, glutathione peroxidase, acetylcholine receptor, tyrosine hydroxylase, glutamate decarboxylase, and phosphodiesterase; further, the drug is a drug that inhibits acetylcholinesterase.

6. The use according to claim 1, characterized in that: The drug is an AchE inhibitor.

7. The use according to claim 1, characterized in that: The drug is a drug that regulates the expression of at least one gene among ACHE, TA, APPB and Aβ.

8. The use according to claim 7, characterized in that: The drug is a drug that inhibits the expression of at least one gene among ACHE, TAU, APPB and Aβ.

9. The use according to claim 1, characterized in that: The dosage form of the drug is any one of a solid preparation, a liquid preparation, a gel preparation, a semi-fluid preparation, and an aerosol preparation.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition contains an effective dose of cyanidin derivatives.

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