Application of alkaloid compound Mallochiphilone in preparation of medicine for inhibiting neuroinflammation

Abstract

The invention discloses an application of an alkaloid compound Mallochiphilone in preparation of a medicine for inhibiting neuroinflammation, and relates to the technical field of biological medicines. The research of the invention finds that the alkaloid compound Mallochiphilone has remarkable anti-neuroinflammation activity, and proves the potential of the alkaloid compound Mallochiphilone in the aspect of inhibiting key pathological characteristics of the Alzheimer disease. Through comprehensive application of network pharmacology, molecular docking, ADMET evaluation and activity evaluation strategies, the action mechanism of the alkaloid compound on AD potential treatment targets is deeply analyzed, the potential anti-AD activity of the alkaloid compound is disclosed, important scientific support is provided for research and development of anti-AD drugs, and the development process of novel anti-AD drugs is expected to be promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of an alkaloid compound Mallochiphilone in preparing a drug for inhibiting neuroinflammation. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease characterized by a gradual decline in cognitive function, memory loss, and other cognitive dysfunctions. The pathological characteristics of AD are mainly manifested in plaques formed by the deposition of beta-amyloid protein (Aβ) in the brain and neurofibrillary tangles caused by hyperphosphorylation of tau protein. In addition, neuroinflammation is also a key factor in the pathogenesis of AD. It not only participates in the early initiation of the disease, but also leads to a persistent inflammatory state, which in turn triggers neuronal degeneration and dysfunction. With the acceleration of the aging of the global population, the prevalence of AD continues to rise, and it is expected to become a serious public health problem in the next few decades. Although there are some drugs on the market to alleviate the symptoms of AD, these drugs cannot cure the disease. Therefore, it is particularly urgent to develop effective treatments.

[0003] The ocean is a huge treasure trove of biodiversity and contains rich natural product resources. Marine natural products show great potential in new drug discovery due to their unique chemical structure and significant biological activity. In recent years, compounds isolated from marine organisms have shown potential application value in many fields, such as anti-tumor, anti-diabetes, and anti-neurodegenerative diseases. In particular, certain marine alkaloid compounds have attracted widespread attention due to their complex structure and diverse biological activities, and are considered to be potential candidate drugs for the treatment of neurodegenerative diseases.

[0004] Network pharmacology is an emerging research method that integrates knowledge and technologies from the fields of systems biology, bioinformatics, and network science to study the complex interaction network of drugs in the body and their effects on diseases from a holistic perspective. This method helps to deeply understand the multi-target mechanism of action of drugs and is of great significance for the development of drugs for complex diseases. Molecular docking technology is a computational method used to predict the binding mode and affinity between small molecule drugs and protein receptors. Through molecular docking, the binding strength of compounds to targets can be evaluated and the interaction mechanism between molecules can be analyzed, thereby improving the efficiency of new drug discovery and reducing experimental costs.

[0005] ADMET (absorption, distribution, metabolism, excretion and toxicity) evaluation is a key step in the drug development process, used to predict the pharmacokinetic properties and safety of compounds in vivo. Through ADMET evaluation, compounds with potential toxicity can be screened in advance and drug design can be optimized. Activity evaluation verifies the effectiveness of compounds through in vitro or in vivo experiments, and further confirms the efficacy and mechanism of action of compounds in specific pathological models.

[0006] The present invention intends to discover new anti-AD compounds by comprehensively using network pharmacology, molecular docking, ADMET evaluation and activity evaluation strategies, thereby promoting the development of new anti-AD drugs. Summary of the invention

[0007] The purpose of the present invention is to provide the use of the alkaloid compound Mallochiphilone in the preparation of a drug for inhibiting neuroinflammation to solve the problems existing in the above-mentioned prior art. By comprehensively using network pharmacology, molecular docking, ADMET evaluation and activity evaluation strategies, the present invention deeply analyzes the mechanism of action of the alkaloid compound Mallochiphilone on potential therapeutic targets for AD and reveals its potential anti-AD activity. These research findings provide important scientific support for the research and development of anti-AD drugs and are expected to promote the development of new anti-AD drugs.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides the use of an alkaloid compound in the preparation of a drug for inhibiting neuroinflammation, wherein the alkaloid compound is a substance described in (1) or (2):

[0010] (1) The compound Mallochiphilone has the structural formula:

[0011]

[0012] (2) A salt or ester of the compound Mallochiphilone.

[0013] Furthermore, the neuroinflammation refers to neuroinflammation of Alzheimer's disease.

[0014] The present invention also provides a drug for inhibiting neuroinflammation, wherein the active ingredient includes an alkaloid compound;

[0015] The alkaloid compound is the substance described in (1) or (2):

[0016] (1) The compound Mallochiphilone has the structural formula:

[0017]

[0018] (2) A salt or ester of the compound Mallochiphilone.

[0019] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0020] Furthermore, the auxiliary materials include solubilizers, binders, disintegrants, fillers, stabilizers, preservatives, coating materials and / or fragrances.

[0021] Furthermore, the dosage form of the drug is granules, tablets, capsules, powders, pills or oral liquids.

[0022] The present invention discloses the following technical effects:

[0023] (1) Based on the network pharmacology strategy, the present invention constructed a "drug-target-gene ontology-pathway-disease" network diagram of the alkaloid compound Mallochiphilone against Alzheimer's disease, revealing the mechanism of action of Mallochiphilone and its potential therapeutic targets. Compared with traditional experimental methods, this method has the characteristics of low cost, high efficiency and comprehensive information, providing an important reference for the subsequent research and development of alkaloid drugs.

[0024] (2) Using molecular docking technology, the present invention reveals the affinity and binding site between Mallochiphilone and its core anti-Alzheimer's disease target, providing a preliminary basis for further application of Mallochiphilone in the prevention and treatment of Alzheimer's disease. This not only provides a reference for the in-depth study of the marine natural product Mallochiphilone, but also has important significance for the development and utilization of rich marine biological resources.

[0025] (3) The present invention uses a deep learning-based ADMET tool to comprehensively evaluate the absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of Mallochiphilone. Through this series of evaluations, it is not only verified that Mallochiphilone has good pharmacokinetic properties, but also ensures its safety and efficacy as a candidate drug.

[0026] (4) Through in vitro experimental verification, the present invention found that Mallochiphilone has significant anti-neuroinflammatory activity, confirming its potential in inhibiting the key pathological features of Alzheimer's disease. The experimental results show that at a certain concentration, Mallochiphilone can effectively inhibit neuroinflammation, laying a solid foundation for subsequent preclinical research and providing important support for the development of anti-Alzheimer's disease drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 The key HMBC (arrow) and 1 H- 1 H COSY (bold) correlation;

[0029] Figure 2 is the HR-ESI-MS spectrum of the compound Mallochiphilone;

[0030] Figure 3 For the compound Mallochiphilone 1 H NMR spectrum (700 MHz, DMSO-d6);

[0031] Figure 4 For the compound Mallochiphilone 13 C NMR spectrum (176 MHz, DMSO-d6);

[0032] Figure 5 For the compound Mallochiphilone 1 H- 1 H COSY spectrum (700 MHz, DMSO-d6);

[0033] Figure 6 It is the HMBC spectrum of the compound Mallochiphilone (176MHz, DMSO-d6);

[0034] Figure 7 HSQC spectrum of the compound Mallochiphilone (176 MHz, DMSO-d6);

[0035] Figure 8The results of the analysis of the anti-neuroinflammatory activity of the compound Mallochiphilone are shown in Figure 1; A is a statistical graph of the cell viability of BV2 cells treated with Mallochiphilone for 24 hours detected by CCK8; B is a statistical graph of the NO content in BV2 microglia cells pretreated with Mallochiphilone for 1 hour and then exposed to 1μg / mL LPS for 24 hours determined by Griess reagent; CG is a statistical graph of the transcription levels of pro-inflammatory factors iNOS (C), 1L-1β (D), TNF-α (E), IL-6 (F) and COX-2 (G) in BV2 microglia cells exposed to a specified concentration of Mallochiphilone for 1 hour and then treated with 1μg / mL LPS for 12 hours determined by qRT-PCR; different lowercase letters indicate significant differences (P<0.05);

[0036] Fig. 9 It is a schematic diagram of the experimental process based on the four-in-one model of "network pharmacology-molecular docking-ADMET-activity evaluation";

[0037] Fig.10 It is a classification diagram of Mallochiphilone-related targets;

[0038] Fig.11 The Venn diagram of the intersection of the compound Mallochiphilone and AD targets;

[0039] Fig.12 The network analysis diagram of the anti-AD mechanism of the compound Mallochiphilone; A is the protein-protein interaction (PPI) network diagram of Mallochiphilone and its anti-AD targets (the top 10 targets selected according to the three ranking methods); B is the neighborhood component centrality (MNC) network diagram; C is the node degree (Degree) network diagram; D is the edge permeation component (EPC) network diagram;

[0040] Fig.13 GO and KEGG enrichment analysis diagrams of the anti-AD target of the compound Mallochiphilone; wherein A is the GO analysis diagram of the anti-AD target of the compound Mallochiphilone; B is the KEGG analysis diagram of the anti-AD target of the compound Mallochiphilone;

[0041] Fig.14 3D view of the docking interaction between the compound Mallochiphilone and EGFR (A), mTOR (B) and CASP3 (C);

[0042] Fig.15This is a diagram showing the physicochemical properties and drug similarity analysis of the compound Mallochiphilone. DETAILED DESCRIPTION

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0046] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0047] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0048] The South China Sea rough sea cucumber symbiotic fungus (Penicillium mallochii) ACD-5 involved in the present invention has been disclosed in the document "The screening for marine fungal strains with high potential in alkaloids production by in situcolony assay and LC-MS / MS based secondary metabolic profiling", and was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 24, 2022, with a deposit number of GDMCC No.62411, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology.

[0049] Example 1 Preparation of Azaphilone Compound Mallochiphilone

[0050] (1) Fermentation

[0051] The ACD-5 strain of the symbiotic fungus of sea cucumbers in the South China Sea was inoculated into a solid culture medium and allowed to ferment for 30 days to obtain a culture; wherein the formula of the solid culture medium is: 1L of pure water contains 20g of artificial sea salt and 1000g of brown rice.

[0052] (2) Rough extraction

[0053] The solid culture obtained in step (1) was extracted three times with methanol ultrasonically and concentrated, each time for 30 minutes, to obtain a crude extract.

[0054] (3) Separation and purification

[0055] The crude extract obtained in step (2) is crudely separated by silica gel column chromatography (mobile phase: n-hexane, n-hexane-ethyl acetate (v / v=1:1), ethyl acetate, ethyl acetate-methanol (v / v=1:1), methanol, three column volumes each), silica gel column chromatography (mobile phase: n-hexane-ethyl acetate (v / v=15:1, 10:1, 5:1, 3:2, 2:3 and 1:5), ethyl acetate, ethyl acetate-methanol (v / v=1:1), methanol), silica gel column chromatography (mobile phase: dichloromethane-methanol (v / v=80:1, 40:1)), gel column chromatography (mobile phase: dichloromethane-methanol, v / v=1:1), reverse phase silica gel column chromatography (mobile phase: methanol-water (v / v=70:30, 80:20)), and the target substance with specific chromatographic characteristics is gradually traced and purified until a pure compound is obtained. The target substance is characterized by a red substance with an Rf value of about 0.6 on a GF254 TLC chromatographic plate of dichloromethane: methanol (v / v = 10:1), and concentrated sulfuric acid-anisaldehyde is colored as brown-green spots. Under the conditions of high-performance liquid chromatography analysis using a Kinetex C18 chromatographic column (4.6mm×100mm, 5μm), 75% methanol-water isocratic elution for 10min, and a flow rate of 0.6mL / min, the retention time is 6.28min. The target substance is a red needle-shaped crystal, which is easily soluble in methanol.

[0056] (4) Identification

[0057] The collected product was subjected to spectral analysis and structural identification. The results showed that the collected product was a pure compound with a molecular formula of C 28 H 38 ClNO6, named Mallochiphilone, has a structural formula as shown in formula (I), and is a new structural compound:

[0058]

[0059] In addition, the compound Mallochiphilone has the following physical and chemical properties and spectral characteristics: Mallochiphilone is a red needle-shaped crystal. 1 H. 13 The C NMR data are shown in Table 1, and the relevant spectral information is shown in Figure 1-Figure 7In the HMBC experiment, correlations can be observed between H-21 and C-22 and C-23, H-22 and C-21, C-23 and C-24, H-23 and C-21, C-22, C-24 and C-25, H-24 and C-22, C-23 and C-25, and H-25 and C-23, C-24, C-26 and C-27. The correlations between H-21 and H-22, H-22 and H-21, H-23, H-23 and H-22, H-24, H-24 and H-23, H-25, H-25 and H-24, H-26 / 27, and H-26 / 27 and H-25 are shown in Figure 2. 1 H- 1 H COSY experiment determined ( Figure 1 ), the compound is a new structure compound, and has a substituent group at the 2-N position that is different from the known Azaphilone compounds. The charge-to-mass ratio in the high-resolution mass spectrometry is 520.2468 ([M+H] + , theoretical value 520.2460) confirmed that its molecular formula is C 28 H 38 ClNO6. Table 1 Compounds of Mallochiphilone 1 H. 13 C NMR data (in DMSO-d6, TMS, 1 HNMR is 700MHz, 13 CNMR is 175MHz, δ is in ppm)

[0060]

[0061] Example 2 Analysis of the Anti-neuroinflammatory Activity of the Compound Mallochiphilone

[0062] 1. Experimental methods

[0063] (1) Cell viability assay

[0064] In order to evaluate the cytotoxicity of Mallochiphilone on BV2 mouse microglia, the present invention used the Cell Counting Kit-8 (CCK-8) method to detect cell viability. BV2 cells were cultured at 5×10 3The cells were seeded at a density of 100 μL / well in a 96-well plate and cultured at 37°C and 5% CO2 for 24 hours to ensure cell adhesion and growth. Subsequently, the cells were treated with 1.92 μM, 19.23 μM and 38.46 μM Mallochiphilone for 24 hours to simulate the cell exposure environment at different concentrations. After treatment, the old culture medium was discarded, and a mixture of 10 μL CCK-8 and 90 μL fresh DMEM was added to each well and cultured for another hour. Finally, the absorbance was measured at a wavelength of 450 nm on a microplate reader to evaluate the cell viability after Pol treatment. The formula for calculating cell viability is: Cell viability (%) = A 450 (Mallochiphilone treated group) / A 450 (Control group)×100%.

[0065] (2) Experiment on inhibition of NO generation

[0066] In order to explore the regulatory effect of Mallochiphilone on inflammatory response, the present invention constructed a BV2 microglial inflammation model induced by lipopolysaccharide (LPS). BV2 cells were cultured at 2×10 4 Cells were seeded at a density of 100 μg / well in a 96-well plate and cultured at 37°C and 5% CO2 for 24 hours. The cells were pretreated with 1.92 μM, 19.23 μM, and 38.46 μM Mallochiphilone for 1 hour and then stimulated with 1 μg / mL LPS for 24 hours to simulate the inflammatory environment. The supernatant was collected and added with Griess reagent, the absorbance was measured at a wavelength of 540 nm by an enzyme reader, and the NO concentration was calculated by the standard curve to evaluate the inhibitory effect of Mallochiphilone on NO production. The setting of blank group and control group is used to eliminate the deviation of experimental operation and ensure the accuracy of the results.

[0067] (3) Inflammatory factor and enzyme gene expression test:

[0068] In order to further understand the effect of Mallochiphilone on the expression of inflammation-related genes, the present invention used real-time fluorescence quantitative PCR (qRT-PCR) to detect the gene expression levels of IL-1β, IL-6, TNF-α, iNOS and COX-2. BV2 cells were cultured at 6×10 5Cells were seeded at a density of 100 μg / well in 6-well plates and cultured at 37°C, 5% CO2 for 24 hours. Cells were pretreated with 1.92 μM, 19.23 μM, and 38.46 μM Mallochiphilone for 1 hour and then stimulated with 1 μg / mL LPS for 12 hours to simulate inflammatory stimulation. Cell RNA was extracted using Trizol reagent and subjected to a series of steps including chloroform lysis, isopropanol precipitation, and ethanol washing to ensure the purity and integrity of RNA. RNA quality and concentration were measured by spectrophotometer and A was calculated. 260 / A 280 Ratio and RNA concentration. Reverse transcription reaction was performed using HiScript III qRT SuperMix to convert RNA into cDNA to provide template for qRT-PCR. In the qRT-PCR reaction, specific primers were used to amplify the target gene, and the fluorescence value of each cycle was measured by a fluorescence quantitative PCR instrument to analyze the changes in gene expression.

[0069] Data analysis was performed using statistical software R4.1.2, and P values ​​were calculated by one-way analysis of variance (ANOVA) and Tukey's multiple comparison test to evaluate the effects of different concentrations of Mallochiphilone on cell viability, NO, and inflammatory factors.

[0070] 2. Experimental results

[0071] Figure 8 The effects of the compound Mallochiphilone on BV2 cell viability and its effects on NO production, COX-2 and pro-inflammatory factor expression in LPS-induced BV2 cells were shown. The CCK8 test results showed that Mallochiphilone had no obvious toxic side effects on BV2 cells in the concentration range of 1.92μM to 38.46μM. This indicates that Mallochiphilone has good cell compatibility within the concentration range tested. In addition, Mallochiphilone can reduce the production of NO in BV2 cells after LPS stimulation, especially at the highest concentration (38.46μM), the inhibitory effect is most significant. Mallochiphilone can also effectively inhibit the expression of IL-1β, IL-6, TNF-α, iNOS and COX-2 genes in BV2 cells after LPS stimulation. Especially at a concentration of 38.46μM, Mallochiphilone showed the best protective effect. These results indicate that Mallochiphilone not only has good cytocompatibility but also can significantly inhibit LPS-induced inflammatory responses at higher concentrations, showing its potential in anti-neuroinflammation.

[0072] Example 3 Analysis of the mechanism of action of Mallochiphilone and its potential therapeutic targets

[0073] The present invention reveals the mechanism of action of Mallochiphilone and its potential therapeutic targets by constructing a "drug-target-gene ontology-pathway-disease" network diagram for Mallochiphilone against Alzheimer's disease. The schematic diagram of the experimental process based on the four-in-one model of "network pharmacology-molecular docking-ADMET-activity evaluation" is shown in the figure. Fig. 9 shown.

[0074] 1. Experimental methods

[0075] 1.1 Acquisition of Mallochiphilone targets and Alzheimer's disease targets and identification of overlapping targets

[0076] S1. Obtain the SMILE code of Mallochiphilone: ​​Based on the structure of Mallochiphilone, the RDkit software package was used to obtain its SMILE (Simplified Molecular-Input Line-Entry System) code to provide accurate molecular representation for subsequent target prediction.

[0077] S2. Prediction of the target of Mallochiphilone: ​​The target of Mallochiphilone was predicted using the SwissTargetPrediction platform (http: / / www.swisstargetprediction.ch / ). The platform can provide target prediction services based on molecular structure.

[0078] S3. Collect Alzheimer's disease-related targets: Use the keyword "Alzheimer's Disease" to collect Alzheimer's disease-related targets from the DisGeNET database (https: / / www.disgenet.org / ) and the DrugBank database (https: / / www.drugbank.ca / ), and perform deduplication and proofreading to ensure the accuracy and uniqueness of the target data.

[0079] S4. Identify overlapping targets: Use the ggvenn package in R language to draw a Venn diagram, compare the targets of Mallochiphilone with those related to Alzheimer's disease, and identify overlapping targets between the two. These overlapping targets may be the key to the anti-Alzheimer's disease effect of Mallochiphilone.

[0080] 1.2 Construction of Mallochiphilone target-Alzheimer's disease target network and screening of core targets

[0081] S1. Upload the intersection targets to the STRING database: Upload the intersection targets of Mallochiphilone and Alzheimer's disease targets obtained in 1.1 to the STRING database (https: / / string-db.org / ), set the species to human (Homo sapiens), and construct a protein interaction network. The STRING database provides information on known and predicted interactions between proteins.

[0082] S2. Export protein interaction network data: Export the raw data of the protein interaction network and import it into Cytoscape software. Use Cytoscape software to visualize the protein interaction network, further analyze the network structure, and identify key nodes in the network.

[0083] S3. Screening of core targets: The MNC, degree, and EPC algorithms of the CytoHubba plug-in in the Cytoscape software were used to analyze and obtain the top 10 core targets. These core targets have high connectivity and centrality in the network and may be the main targets of Mallochiphilone.

[0084] 1.3 Enrichment analysis of Mallochiphilone anti-Alzheimer's disease targets

[0085] Metascape platform (https: / / metascape.org / ) was used to perform GO and KEGG analysis on Mallochiphilone's anti-Alzheimer's disease. GO analysis mainly focuses on three aspects: biological process (BP), cellular component (CC) and molecular function (MF). R language was then used to visualize the core biological processes and pathways, revealing the distribution of the main biological functions and related pathway information involved in Mallochiphilone's anti-Alzheimer's disease.

[0086] 1.4 Docking study of mallochiphilone and its core target molecules against Alzheimer's disease

[0087] S1. Obtaining the structure of Mallochiphilone: ​​The structure of Mallochiphilone was obtained using RDkit.

[0088] S2. Download protein structures of core targets: Download protein structures of core targets (top 3) against Alzheimer's disease from the RCSB PDB database.

[0089] S3. Preprocessing protein structure: Use PyMol software to preprocess the protein (remove water molecules and separate original ligands), etc., in preparation for molecular docking.

[0090] S4. Molecular docking and visualization: AutoDock Vina software was used for molecular docking to study the affinity and action sites of Mallochiphilone with these core targets. After docking, PyMOL software was used to visualize the results to gain a deeper understanding of the interaction mechanism between Mallochiphilone and the targets.

[0091] 1.5 ADMET analysis of Mallochiphilone

[0092] ADMETlab 2.0 (https: / / admetmesh.scbdd.com / service / evaluation / cal) was used to predict the ADMET (absorption, distribution, metabolism, excretion and toxicity) and drug-like properties of Mallochiphilone. The pharmacokinetic properties and potential toxicity of Mallochiphilone were evaluated through the prediction results, providing an important reference for subsequent drug development.

[0093] 2. Experimental results

[0094] 2.1 Screening of anti-Alzheimer's disease targets of Mallochiphilone

[0095] Through the retrieval of literature and databases, we collected targets related to Alzheimer's disease (AD). Then, we used the SwissTarget Prediction platform to predict the potential targets of Mallochiphilone. The prediction results showed that its targets can be divided into 10 categories ( Fig.10 ), among which kinase accounted for the highest proportion, reaching 34%. In order to obtain the relevant targets of Mallochiphilone against AD, a Venn diagram was constructed to map the potential targets of Mallochiphilone with the AD-related targets and obtain the intersection, and finally 68 overlapping targets were obtained ( Fig.11 ).

[0096] 2.2 Interaction network of target proteins of Mallochiphilone against Alzheimer's disease

[0097] In order to further explore the potential therapeutic targets and their interactions for Alzheimer's disease (AD), a protein interaction network (PPI) containing 68 intersection targets was constructed using the STRING database and Cytoscape software. Fig.12As shown in the figure, the nodes in the network represent target proteins, and the edges represent the interactions between them. Through the topological analysis of NetworkAnalyzer, a complex network containing 68 nodes and 241 edges was revealed, among which targets such as EGFR, mTOR, CASP3, PIK3CA, and GSK3B stood out due to their high degree values, showing their key roles in the network ( Fig.12 Further analysis used the maximum neighborhood component (MNC), node degree (Degree), and edge permeation component (EPC) methods to identify key targets in the network. Fig.12 The BD shows the top ten targets in each method. It is worth noting that three hub targets - EGFR (epidermal growth factor receptor), mTOR (mammalian target of rapamycin), and CASP3 (caspase 3) - are consistently ranked high in all three algorithms, highlighting their central position in the network and the important role they may play in AD. The relationship between these three targets and neuroinflammation, a key pathogenesis of AD, is as follows: EGFR: As a cell surface receptor, it is involved in regulating cell proliferation and survival. Its overactivation is associated with the release of proinflammatory factors in neuroinflammation and is a potential target for neuroprotection. mTOR: As an intracellular signaling molecule, it controls cell growth and metabolism. Its abnormal activation promotes the expression of inflammatory mediators and exacerbates the inflammatory response. Regulating the mTOR signaling pathway is essential for controlling neuroinflammation. CASP3: As an executor protease, it is involved in the process of cell apoptosis. Its activation can inhibit NF-κB-mediated inflammatory signals and plays an important role in regulating neuroinflammation and cell apoptosis.

[0098] 2.3 Enrichment analysis of key targets of Mallochiphilon in the treatment of Alzheimer's disease

[0099] In order to further explore the potential pharmacological mechanism of Mallochiphilone in the fight against Alzheimer's disease (AD), the present invention conducted enrichment analysis on the screened targets, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. GO analysis covers three main aspects: biological process, cellular component and molecular function. Fig.13Figure A shows the results of the top five significantly enriched items. In terms of biological processes, it was found that the targets of Mallochiphilone were significantly enriched in the response of cells to organic nitrogen compounds, nitrogen compounds, and organic cyclic compounds. This suggests that Mallochiphilone may affect the pathological process of AD by regulating the response of cells to these compounds. In terms of cellular components, enrichment analysis pointed to structures such as dendrites, dendritic branches, and membrane rafts, which are key cellular components in the nervous system and are closely related to neurotransmission and signal transduction. In terms of molecular function, the targets of Mallochiphilone mainly involve protein kinase activity, phosphotransferase activity with alcohol groups as receptors, and kinase activity. These functions are closely related to cell signaling and neuroprotection, further confirming the role of Mallochiphilone in regulating AD-related biological processes.

[0100] In addition, KEGG pathway analysis was performed on 68 core targets, with a threshold of P < 0.01, and a total of 152 significant pathways were identified, of which the top 15 pathways were as follows Fig.13 As shown in B. These pathways include the Rap1 signaling pathway, the PI3K-Akt signaling pathway, and neuroactive ligand-receptor interactions, revealing the nature of AD as a complex disease regulated by multiple pathways. At the same time, these results also indicate that Mallochiphilone may exert its anti-AD effect by affecting these key signaling pathways. It is worth noting that neuroinflammation-related pathways ranked high in the enrichment analysis, which is consistent with the view that neuroinflammation is one of the key pathogenesis of AD. These findings not only emphasize the importance of neuroinflammation in AD, but also provide strong molecular mechanism support for Mallochiphilone as a potential anti-AD drug.

[0101] 2.4 Docking study of the compound Mallochiphilone with its core target molecules for anti-Alzheimer's disease

[0102] In order to further explore the potential therapeutic effect of Mallochiphilone on Alzheimer's disease, this study focused on its core disease-related targets: EGFR, mTOR and CASP3. The interaction affinity of Mallochiphilone with these targets was evaluated by molecular docking technology, and the results showed that its affinities were -8.3kcal / mol (EGFR), -8.0kcal / mol (mTOR) and -6.3kcal / mol (CASP3), respectively. These affinity values ​​indicate that Mallochiphilone has a high binding affinity with these targets. Further analysis of the interaction forces between Mallochiphilone and the targets showed that they mainly include hydrophobic interactions and salt bridges. For example, the interaction with EGFR involves hydrophobic interactions formed by Mallochiphilone at amino acid residues such as Leu694, Val702, and Ala719; the interaction with mTOR includes hydrophobic interactions at residues such as Ile2228, Gln1937, and Leu1900, as well as a salt bridge formed at Arg2224. These specific amino acid site interactions may be critical for the anti-Alzheimer's disease activity of Mallochiphilone ( Fig.14 ). Therefore, this study not only revealed the high-affinity binding of Mallochiphilone to the core target, but also identified the key sites of its interaction with the target, providing an important structural basis for further drug development and optimization.

[0103] 2.5 ADMET analysis of Mallochiphilone

[0104] ADMETlab 2.0 was used to perform computational predictions of the marine natural product Mallochiphilone in order to evaluate its drug similarity and ADMET properties. Fig.15 As shown in Table 2, most of the physical and chemical properties of Mallochiphilone are within a reasonable range. It also complies with multiple drug-making rules (such as the Lipinski rule and the Pfizer rule) and has certain blood-brain barrier permeability. The above results show that Mallochiphilone is a lead compound with drug-making potential.

[0105] Table 2

[0106]

[0107] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of alkaloid compounds in the preparation of drugs for inhibiting neuroinflammation, characterized in that: The alkaloid compound is the substance described in (1) or (2): (1) The compound Mallochiphilone has the structural formula: (2) A salt or ester of the compound Mallochiphilone.

2. The use according to claim 1, characterized in that: The neuroinflammation refers to neuroinflammation in Alzheimer's disease.

3. A drug for inhibiting neuroinflammation, characterized in that: The active ingredients include alkaloid compounds; The alkaloid compound is the substance described in (1) or (2): (1) The compound Mallochiphilone has the structural formula: (2) A salt or ester of the compound Mallochiphilone.

4. The drug according to claim 3, characterized in that The drug also includes pharmaceutically acceptable excipients.

5. The drug according to claim 4, characterized in that The auxiliary materials include solubilizers, binders, disintegrants, fillers, stabilizers, preservatives, coating materials and / or fragrances.

6. The drug according to claim 4, characterized in that The dosage form of the medicine is granules, tablets, capsules, powders, pills or oral liquids.

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