Application of cryptochlorogenic acid in preparation of medicine for preventing and treating RSV (Respiratory Syndrome Virus) infection

Abstract

The invention discloses application of cryptochlorogenic acid in preparation of a medicine for preventing and treating RSV (Respiratory Syndrome Virus) infection, and belongs to the technical field of biology. The invention focuses on a multi-component synergistic mechanism of the anti-virus effect of the Yinqiao drug pair, takes a targeted ACSL4 protein as an entry point, researches the pharmacodynamic material basis of the Yinqiao drug pair by combining SPR affinity fishing with computational chemistry, and finds that the Yinqiao drug pair contains nine active components capable of being combined with the ACSL4 protein. Subsequently, a molecular docking and dynamic simulation technology is adopted to verify the combination mode of the active ingredients and proteins, the combination of the key ingredients of chlorogenic acid and forsythiaside is more stable, cryptochlorogenic acid becomes a key pharmacodynamic substance for regulating lipid peroxidation and playing an antiviral role, and cryptochlorogenic acid and forsythiaside become a key pharmacodynamic substance for regulating lipid peroxidation and playing an antiviral role. Research results provide theoretical basis and data support for traditional Chinese medicine prevention and treatment of RSV virus infectious diseases and research and development of traditional Chinese medicine targeted drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the use of cryptochlorogenic acid in preparing medicines for preventing and treating RSV infection. Background Art

[0002] Respiratory syncytial virus (RSV) is a non-segmented, single-stranded, negative-sense RNA virus belonging to the Paramyxoviridae family and the Pneumovirus genus. It is highly contagious and prone to outbreaks in infants, children, and immunocompromised elderly individuals. It is a leading cause of acute lower respiratory tract infections (ALRTIs) in infants and young children worldwide. As a common respiratory virus, RSV has a complex and diverse infection mechanism, capable of causing a variety of pathological responses. Targeted metabolomics results indicate that abnormal increases in AA and its derived metabolites after RSV infection may be closely related to ferroptosis.

[0003] The ACSL4 / LPCAT3 / ALOX15 signaling axis is one of the core regulatory pathways for ferroptosis. It promotes lipid peroxide accumulation by driving PUFA activation and esterification, as well as iron-dependent oxidation reactions, leading to cell membrane rupture and ferroptosis. Acyl-CoA synthetase long chain family member 4 (ACSL4), which mediates the production of the AA metabolites 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE), is crucial for ferroptosis. Studies have demonstrated that ACSL4 expression levels directly determine cell sensitivity to ferroptosis, and its absence significantly inhibits the formation of oxidized AA-PE-containing substances.

[0004] Currently, safe and universally applicable specific prevention and treatment measures for RSV infection have yet to be established in clinical practice. Therefore, the exploration of innovative drugs that are tailored to my country's medical realities is urgently needed. As the culmination of thousands of years of clinical practice and theoretical heritage, medicine, with its holistic regulatory and multi-target intervention approach, offers unique advantages in epidemic prevention and control. RSV infection falls under the Traditional Chinese Medicine (TCM) syndrome of "wind-heat invading the lungs" and "lung heat congestion," consistent with the pathological characteristics of "epidemic toxins invading the lungs with wind-heat." The heat-clearing combination of honeysuckle and forsythia (the Yinqiao herbal pair) can reduce inflammatory cytokines and, by intervening in imbalanced AA peroxidation metabolism, inhibit RSV-induced ferroptosis, thereby reducing viral load in lung tissue and improving RSV infection. The complex composition of traditional Chinese medicines and natural remedies, such as the Yinqiao herbal pair, requires further clarification and analysis of their anti-RSV activity. SPR allows for the direct screening and further identification of bioactive compounds that bind to relevant targets, laying the foundation for the development of effective compounds. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide the use of cryptochlorogenic acid in the preparation of drugs for the prevention and treatment of RSV infection. Taking the regulation of ACSL4 by the Yinqiao drug pair as the starting point, the bioactive components of the Yinqiao drug pair that have potential binding to the ACSL4 protein are identified through surface plasmon resonance (SPR) affinity fishing combined with computational chemistry methods, in order to provide a theoretical reference for the research and development and application of targeted drugs for the prevention and treatment of RSV viral infectious diseases in traditional Chinese medicine.

[0006] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides the use of cryptochlorogenic acid in the preparation of an effective substance targeting ACSL4 protein.

[0007] The screening process for the efficacy substance targeting ACSL4 protein comprises the following steps: (1) Prepare the extract of Yinqiao herbal medicine, perform protein coupling, take the Yinqiao extract and add PBS to dilute it in multiple ratios, flow the dilution through the ACSL4 immobilized chip, dissociate, and collect the eluate; (2) The eluate was taken for mass spectrometry detection, and molecular docking, molecular dynamics simulation, SPR point screening and affinity detection were used to screen the active ingredients that stably bind to the ACSL4 protein, and the effective substances of Yinqiao medicine targeting the ACSL4 protein were obtained.

[0008] In step (1), the protein coupling method is to activate the chip channel using a mixed solution of EDC and NHS at a flow rate of 10 μL / min, and the ACSL4 protein is diluted to 50 μg / mL using a pH 4.0 sodium acetate solution and fixed to the chip at a flow rate of 10 μL / min, and blocked with ethanolamine.

[0009] In step (2), the mass spectrometry detection mode is Full MS-ddMS 2 , using positive and negative ion modes for scanning.

[0010] In step (2), the molecular dynamics simulation step size is 2 fs, the non-bonded interaction cutoff value is 1 nm, the pressure is set to 1.0 bar, and the coordinate trajectory is recorded every 5000 steps.

[0011] In step (2), the SPR spot screening flow rate is set to 10 μL·min -1 , injection time 150 s, dissociation time 300 s.

[0012] In step (2), the affinity detection screening flow rate is set to 30 μL·min -1, duration 150 s, dissociation time 300 s.

[0013] The efficacy substance forms a high-frequency binding interaction through THR278, SER281, THR469, VAL324 and LYS690 residues, and targets and binds to the ACSL4 protein.

[0014] In a second aspect, the present invention provides the use of an effective substance targeting ACSL4 protein in the preparation of a drug for preventing and treating RSV infection.

[0015] The effective substance is cryptochlorogenic acid.

[0016] Beneficial effects: This study focuses on the multi-component synergistic mechanism of Yinqiao herbal medicine's antiviral effects. By targeting the ACSL4 protein, the authors investigated the pharmacological basis of the Yinqiao herbal medicine pair through surface plasmon resonance (SPR) affinity analysis combined with computational chemistry. The results revealed that the Yinqiao herbal medicine pair contains nine active ingredients that bind to the ACSL4 protein. Subsequently, molecular docking and kinetic simulation techniques were used to further verify the binding patterns of these active ingredients with the protein, revealing that the key ingredients, chlorogenic acid and forsythiaside, bind more stably. Among them, cryptochlorogenic acid is the key pharmacological agent in the Yinqiao herbal medicine pair that regulates lipid peroxidation and exerts antiviral effects. This study identified cryptochlorogenic acid, an active monomer component, through affinity screening from a combinatorial chemical mixture library, providing a theoretical basis and data support for the prevention and treatment of RSV viral infections with Traditional Chinese Medicine (TCM) and the development of targeted TCM drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the sensor diagram of ACSL4 protein coupled with CM5 sensor chip.

[0018] Figure 2 These are the interaction signals between different concentrations of Yinqiao water extract and ACSL4 protein.

[0019] Figure 3 This is the TIC diagram of Yinqiao eluate detected by UHPLC-MS / MS; A: positive ion mode; B: negative ion mode.

[0020] Figure 4 This is the molecular docking of the active ingredients of Yinqiao with ACSL4 protein; among them, A: isofuscin A; B: forsythiaside E; C: isochlorogenic acid B; D: cleotidylcholine; E: cryptochlorogenic acid; F: scutellarin; G: sucrose; H: uridine; I: dextrorotatory quinic acid.

[0021] Figure 5The RMSD of the simulated trajectory of the active ingredient of Yinqiao medicine and ACSL4 protein; where A: isofuspensoside A; B: forsythiaside E; C: isochlorogenic acid B; D: clerophyllin; E: cryptochlorogenic acid; F: styrocoside; G: sucrose; H: uridine; I: dextrorotatory quinic acid.

[0022] Figure 6 This is the RMSF of the simulated trajectory of the active ingredient of Yinqiao herb and ACSL4 protein; where A: isofusin A; B: forsythiaside E; C: isochlorogenic acid B; D: clerophyllin; E: cryptochlorogenic acid; F: styrocoside; G: sucrose; H: uridine; I: dextrorotatory quinic acid.

[0023] Figure 7 is the Rg of the simulated trajectory of the active ingredient of Yinqiao medicine and ACSL4 protein; among them, A: isofuscin A; B: forsythiaside E; C: isochlorogenic acid B; D: cleotidylcholine; E: cryptochlorogenic acid; F: styrocoside; G: sucrose; H: uridine; I: dextrorotatory quinic acid.

[0024] Figure 8 This is the SASA of the active ingredient of Yinqiao medicine and the simulated trajectory of ACSL4 protein; among them, A: isofuscin A; B: forsythiaside E; C: isochlorogenic acid B; D: cleotidylcholine; E: cryptochlorogenic acid; F: styrocoside; G: sucrose; H: uridine; I: dextrorotatory quinic acid.

[0025] Figure 9 This is a single-concentration SPR screening of the active ingredients of Yinqiao medicine and ACSL4 protein.

[0026] Figure 10 This is the SPR affinity analysis of cryptochlorogenic acid and ACSL4 protein.

[0027] Figure 11 For the detection of mouse organ index.

[0028] Figure 12 Detection of inflammatory factor levels in mouse lung tissue; Figure A is the detection of IL-1β content, and Figure B is the detection of IL-6 content.

[0029] Figure 13 This is a slice showing the effect of cryptochlorogenic acid on lung tissue pathology in RSV-infected mice; Figure 13 A in the middle is the pathological section of lung tissue in the normal group. Figure 13 Middle B is the pathological section of lung tissue in the model group. Figure 13 Middle C is the lung tissue pathological section of the ribavirin positive control group. Figure 13 Middle D is the pathological section of lung tissue in the cryptochlorogenic acid group. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0031] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present invention, rather than to limit the scope of the present invention.

[0032] The drugs and reagents used in the present invention are as follows: Acyl-CoA synthetase 4 (ACSL4) protein, isofusin A, cryptochlorogenic acid, isochlorogenic acid B, dextrorotatory quinic acid, serotonin, forsythiaside E, sucrose, uridine, and schizonepetaside (Shanghai Taoshu Biotechnology Co., Ltd., batch numbers: 241943, 243778, 155119, 154914, 114105, 132999, 156366, 130953, 163779, 227707); CM5 sensor chip, PBS-P+ (Cytiva Biotechnology Co., Ltd., Sweden, batch numbers: 2024010372, 2024060123); Surfactant P20, NaOH 50 mM, pH 4.0 sodium acetate buffer solution (GE Healthcare, batch numbers: 2024024364, 2023120344, 2024021211); DMSO, 1-ethyl-3-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), and ethanolamine (Sigma, batch numbers: 2024042335, 2024025326, 2024031292, 2023124993).

[0033] The experimental instruments used in the present invention are as follows: Q Exactive Plus Orbitrap high-resolution liquid spectrometer, U3000 ultra-high performance liquid chromatography (Thermo Fisher Scientific, USA); ACQUITY UPLC HSS T3 (2.1×100 mm, 1.8 μm) (Waters, USA); Biacore T200 (GE Healthcare, USA); 5810R low-temperature centrifuge, MiniSpin small centrifuge (Eppendorf, Germany); Vortex-2 Genie vortex mixer (Ika); WD-9415C ultrasonic cleaner (Beijing Liuyi Instrument Factory).

[0034] The complete culture medium used in the present invention is: DMEM + 10% FBS + 1% double antibody, which is mixed and stored at 4°C. The maintenance culture medium used is: DMEM + 2% FBS, which is mixed and stored at 4°C.

[0035] Example 1: Fishing with Yinqiao Extract 1. Experimental Methods (1) Protein coupling: A mixed solution of EDC and NHS (1:1) was used to activate channel 4 of the chip at a flow rate of 10 μL / min. ACSL4 protein was diluted to 50 μg / mL with sodium acetate solution at pH 4.0. The protein was fixed to the chip at a flow rate of 10 μL / min and blocked with ethanolamine. At the same time, channel 3 was activated in the same way as the reference channel. A protein-free acetate buffer was selected and flowed through the chip, and then blocked with ethanolamine. The system buffer was protein coupling buffer solution: 1.0×PBS-P+ (pH 7.4).

[0036] (2) Preparation and determination of the extract of Yinqiao herbal medicine: Weigh 50 g each of honeysuckle and forsythia slices, add 10 times the amount of water to soak for 1 hour, heat and reflux to extract for 45 minutes, filter, add 10 times the amount of water to extract for 45 minutes again, filter through gauze, combine the two filtrates and concentrate the extract to make a 1 g / mL concentration of crude drug. Replace the system buffer: 1.0×PBS-P+ (pH7.4), 5% (v / v) DMSO. Before the instrument is tested, take the Yinqiao extract and add PBS to dilute it to 1000 mg / mL, 500 mg / mL, and 250 mg / mL. Pass it through the ACSL4 immobilized chip for 60 seconds, dissociate for 60 seconds, repeat each concentration gradient 6 times, and collect the eluate.

[0037] (3) Mass spectrometry detection and identification: The eluate obtained in step (3) was diluted 50 times with a methanol-water mixed solvent (1:1, v / v), and ultrasonicated for 30 min. The supernatant was separated and purified by centrifugation at 14,000 rpm for 5 min. The supernatant was transferred through a 0.22 μm filter membrane (pre-rinsed 3 times) to collect the filtrate, which was placed in an injection bottle for UHPLC-MS / MS analysis.

[0038] Mass spectrometry data were collected by Q Exactive Orbitrap high-resolution mass spectrometry with detection mode of Full MS-ddMS. 2, positive and negative ion modes were scanned separately, and the detailed parameter settings were as follows: Scanning range: m / z 100-1200, Ion source voltage: 3.2kV, Capillary temperature: 320℃, Full ms resolution: 70000, MS / MS resolution: 17500, Aux gas heater temperature: 350℃, Sheath gasflow rate: 40 Arb, Aux gas flow rate: 15 Arb, AGC Target: 1e6, TopN: 5, Collisionenergy: 30 / 40 / 50 in NCE mode.

[0039] Raw mass spectrometry data were analyzed using Compound Discover 3.2 software. The software performed characteristic peak extraction and element matching, elemental composition analysis, and molecular formula deduction using isotope pattern matching. Mass deviation was maintained within 5 ppm. Characteristic peaks of each substance were identified using the mzcloud online database and the in-house mzVault Traditional Chinese Medicine Natural Products Database. Positive results were considered to meet the following criteria: mass deviation <5 ppm, consistent isotope distribution, and an mzVault best match score ≥90.

[0040] 2. Experimental Results ACSL4 protein was best enriched in pH 4.0 sodium acetate solution and could be covalently coupled to the CM5 chip with a response signal of 9025 RU, indicating specific binding between the protein and the chip. This protein could be used in subsequent fishing experiments with Yinqiao extract ( Figure 1 Different concentrations of Yinqiao extract were passed through the chip surface to detect its binding activity to ACSL4 protein. The results showed that Yinqiao extract specifically bound to ACSL4 protein in a concentration-dependent manner ( Figure 2 ).

[0041] The total ion currents of the eluate samples recovered from Yinqiao extract in positive and negative ion modes are shown in the following figure: Figure 3 The results show that the peak shapes and positions are well distributed under these detection conditions. After database comparison and mass spectrometry analysis, nine active components that bind to the ACSL4 protein were identified: isofusin A, cryptochlorogenic acid, isochlorogenic acid B, dextrorotatory quinic acid, serotonin, forsythiaside E, sucrose, uridine, and schizonepetaside. Mass spectrometry identification information for each component is shown in Table 1.

[0042] Table 1: Mass spectrometry identification results of fishing ingredients Example 2: Molecular docking In order to verify the binding of the active ingredients of Yinqiao extract with ACSL4 protein at the microscopic level, the interaction with ACSL4 protein was preliminarily explored and molecular docking of each ingredient with it was performed.

[0043] 1. Experimental Methods (1) Active site prediction: The pdb structure of the ACSL4 protein was downloaded from the UniProt database. The protein crystal water was removed using Pymol software. The protein file was imported into Proteins Plus (https: / / proteins.plus / ). The DoGSiteScorer algorithm was used to perform protein surface topology analysis using a multi-scale Gaussian difference filter. Volume, hydrophobicity, and closure were used as descriptors and incorporated into the support vector machine to predict the druggability score of the active binding site. The site with the highest score was selected as the docking coordinate file.

[0044] (2) Molecular docking: The 3D structures of each fishing component were downloaded from PubChem in sdf format, and energy minimization was performed using Chem 3D and saved in mol2 format. The optimized proteins and small molecules were imported into AutoDock Tools 1.5.6 for hydrogenation and charge calculation, and saved in pdbqt format. AutoDock Vina was used for molecular docking to find the optimal conformation and calculate its binding affinity, and the results were visualized using Pymol software.

[0045] 2. Experimental Results As shown in Table 2, each component showed good binding ability with ACSL4 protein, and the binding patterns between the systems were shown in Table 2. Figure 4 It can be seen that most components form high-frequency binding interactions with five residues in the binding pocket of ACSL4. Among them, hydrogen bonds with THR278, SER281, and THR469 enhance binding affinity and specificity, while hydrophobic interactions with VAL324 and LYS690 minimize the hydrophobic surface to enhance the overall stability of the complex. Both contribute to the binding of each component to the target protein.

[0046] Table 2: Molecular docking results Molecular docking results showed that the binding energy between each fishing component and ACSL4 was lower than -5 kcal / mol, suggesting that there may be strong interactions, mainly driven by hydrogen bonding and hydrophobic interactions.

[0047] Example 3: Molecular dynamics simulation (1) Determine the initial conformation: Based on the docking binding energy, the optimal conformation of each molecule bound to ACSL4 was selected as the starting position. Dynamic simulations were performed using AMBER22 (San Francisco, CA, USA) to study the key interactions. The composite file obtained by molecular docking was uploaded and the protein and ligand files were saved separately. System preprocessing was performed, including protein structure analysis, dehydration and residue hydrogenation, ligand hydrogenation, and charge calculation.

[0048] (2) Constructing the simulation system: Loading the system simulation force field, in which the protein uses the ff14SB force field and the small molecule ligand uses the gaff force field, selecting the TIP3P water model to place the composite system for solvation treatment, adding a 1.2 nm water molecule layer, and randomly adding counter ions Na + / Cl - The system is neutralized to make it electrically neutral. The final result is saved as a topology file containing molecular force field parameter information, molecular bonding information, and atomic coordinate files.

[0049] (3) Energy minimization: After the simulation system is constructed, the steepest descent method is used to quickly remove the stress in the system and optimize the structure for unconstrained minimization. After obtaining the lowest energy conformation, the conjugate gradient method is used to perform constrained main chain minimization.

[0050] (4) System heating and conformational equilibrium: The optimized system is heated and pressurized to critical values, and then the actual dynamics simulation is performed after it reaches equilibrium. First, the system is heated from 0 K to 300 K within 300 ps, ​​the system is confined in the canonical ensemble, namely the NVT ensemble, and the system is pre-equilibrated at 300 K for 10 ns.

[0051] (5) Dynamic simulation: After pre-equilibration, a 200 ns molecular simulation was performed in an isothermal and isobaric ensemble, maintaining periodic boundary conditions. All covalent bonds involving hydrogen were constrained using the SHAKE method. During the simulation, the simulation step size was 2 fs, the non-bonded interaction cutoff was 1 nm, the pressure was set to 1.0 bar, and the coordinate trajectory was recorded every 5000 steps.

[0052] (6) Analysis of simulation results: The MMGBSA algorithm was used to calculate the binding free energy of each system. The binding free energy can be expressed as Gibbs free energy: Among them, -TΔS represents the entropy term, which can be ignored; enthalpy change ( ΔH ) is meteorological energy ( ΔE MM ) and solvation energy ( ΔG sol ) and meteorological energy ( ΔE MM) can be decomposed into the van der Waals interaction energy ( ΔE VDWAALS ) and electrostatic energy ( ΔE EEL ); and the solvation energy can be decomposed into polar solvation energy ( ΔE EGB ) and nonpolar solvation energy ( ΔE ESURF ).

[0053] Right now : The final decomposition is: 2. Experimental Results In order to fully consider factors such as the solvation effect of the system binding and further illustrate the binding of the complex system, the MM-GBSA method was used to calculate the average binding free energy to evaluate the binding stability and binding affinity of different small molecules with ACSL4 protein, and the average energy of each complex system was obtained. The results are shown in Table 3. It can be seen that among the various energy contributions, the interaction between the ACSL4 protein complex system is mainly dominated by the van der Waals energy (ΔE VDWAALS ) and electrostatic energy (ΔE EEL ), while solvation primarily inhibits binding. The binding free energy values ​​are all negative; lower values ​​indicate stronger binding stability, thermodynamically stable complexes, and spontaneous binding.

[0054] Table 3: Binding free energy of small molecule compounds to ACSL4 protein Example 4: Trajectory Feature Analysis 1. Experimental Methods Kinetic analysis was performed using AmberTools 23. The Cpptraj module was used to further analyze the binding of each component to the ACSL4 protein using parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and solvent accessible surface area (SASA).

[0055] Note: The calculation formulas for RMSD, RMSF, Rg and SASA are as follows: in, is the atomic number; are atomic coordinates; is the trajectory coordinate of the atom; is the total simulation time; is the atomic position; The average position of atoms during the entire simulation; is the center of mass coordinate of the molecule; is the solvent accessible surface area of ​​the atom.

[0056] 2. Experimental Results RMSD is used to evaluate the overall conformational changes of each composite system relative to the initial structure during the simulation process, indicating the differences in structural superposition between different structures. In addition, the ligand RMSD reflects the change in ligand configuration rather than the change in position. The smaller the RMSD value, the more stable the structural change. By calculating the RMSD values ​​of the above composite systems during the simulation time, it can be seen that during the entire simulation process, the RMSD-ligand of each composite system can basically remain stable after a certain period of time, indicating that each molecule has not undergone major conformational changes during the simulation process and is relatively stable; while the RMSD-protein of each composite system fluctuates within a certain range, but remains relatively stable. In addition, there are several groups of systems whose RMSD changes significantly after relative equilibrium, which may be due to changes in the protein structure conformation during the simulation process. The simulation time can be further extended to observe the equilibrium state ( Figure 5 ).

[0057] RMSF can characterize the flexibility and movement intensity of each residue in the ACSL4 protein during the simulation process, indicating the degree of fluctuation relative to the average position. The higher the RMSF value, the greater the protein flexibility in this region, which may be located in the ligand-protein binding functional area. The RMSF values ​​of the above composite system were calculated during the simulation time, and the results are shown as follows: Figure 6 shown.

[0058] Rg is usually used to characterize the compactness of protein structure, that is, the change in the looseness of protein peptide chains during the simulation process. The smaller the Rg value, the more compact the protein conformation and the more rigid the structure. The Rg values ​​of the above composite system were calculated during the simulation time. The results are as follows Figure 7 shown.

[0059] SASA can characterize the change in the surface area of ​​the solution that the ligand can directly contact over time, reflecting the relative exposure of the ligand in the solvent, and thus indirectly reflecting the tightness of the protein wrapping the ligand. The smaller the SASA value, the tighter the protein wraps the molecule. By calculating the SASA value of the above composite system during the simulation time, the difference in SASA values ​​of each molecule can be compared, thereby better predicting the interaction and stability of each molecule with the ACSL4 protein. The results are as follows Figure 8 shown.

[0060] The RMSD, RMSF, Rg and SAS binding to ACSL4 protein showed that the RMSD value fluctuation range of each complex system was less than 2 Å, indicating that each system was stable during the simulation; compared with other complex systems, the ACSL4 protein bound to isoflavone A and chlorogenic acid showed a lower RMSF value at the amino acid residue level, suggesting that it has lower volatility in protein backbone and side chain movement and a more stable conformation; compared with isoflavone A, the Rg and SASA values ​​of chlorogenic acid completed the dynamic adjustment of the system structure and tended to be stable when the simulation reached 100ns, suggesting that it may play a role by catalyzing the conformation of key residues.

[0061] Example 5: Single-point screening and affinity analysis 1. Experimental Methods Take a 96-well plate and dilute the compound obtained by fishing with 5% DMSO in Running Buffer to an initial concentration of 100 μM for single-point screening. The flow rate is set to 10 μL min -1 The injection time was 150 s, and the dissociation time was 300 s. Based on the SPR spot screening and MD calculation results, compounds were selected for affinity analysis. Compounds were serially diluted with Running Buffer containing 5% DMSO and allowed to interact with F protein from low to high concentrations through the chip. The flow rate was set at 30 μL / min. -1 , duration 150 s, dissociation time 300 s.

[0062] Data for samples F4-F3 were collected using Biacore T200 Control software (v.2.0, GE Healthcare). The data were globally fitted to a 1:1 Langmuir binding model using Biacore T200 evaluation software to obtain the association and dissociation constants.

[0063] 2. Experimental Results The monomeric substances of each component were diluted to 100 μmol / L, and the binding potential was screened by SPR single concentration detection. The results showed that each component could specifically bind to ACSL4 protein, among which cryptochlorogenic acid and isofelianaside A had higher RU values ​​( Figure 9 ).

[0064] Combining molecular dynamics simulation and its trajectory characteristics, cryptochlorogenic acid was selected for affinity detection analysis and diluted to different concentrations (0.3125, 0.625, 1.25, 2.5, 5 and 10 μmol / L) to bind to the target protein. The binding curve is shown in Figure 10 It can be seen that cryptochlorogenic acid binds well to the target protein and shows a concentration-dependent trend.

[0065] The binding curve of chlorogenic acid was further analyzed by using the Kinetics / Affinity module in BIAevaluation software, and the 1:1 Langmuir interaction model was fitted to calculate the dissociation constant (K D ), the binding rate constant (K a ) and the dissociation rate constant (K d The result was chlorogenic acid K. D The value is 1.42×10 -6 mol / L, Ka value is 2.65×10 5 The Kd value was 0.376 1 / s. These results indicate that the binding mode between chlorogenic acid and ACSL4 protein is fast on and off, and the binding affinity is significant.

[0066] Example 6: Verification of the effect of cryptochlorogenic acid on RSV infection After 3 days of adaptive feeding, BALB / c mice were randomly divided into 4 groups (n=6), namely normal group, model group (RSV infection), ribavirin positive control group (RSV+ribavirin), and cryptochlorogenic acid group (RSV+cryptochlorogenic acid). The ribavirin positive control group was given 50 mg·kg·d -1 The dose of chlorogenic acid group was 100 mg·kg·d -1 .

[0067] After the mice in each group were anesthetized with isoflurane inhalation, 100 TCID 50 RSV virus was infected by intranasal drip, 50 μL per mouse, once a day for 3 consecutive days. The normal group was infected by intranasal drip with the same volume of maintenance medium. The drug-treated group was infected with 10 mL·kg -1 The mice in the normal group and the model group were given the same volume of normal saline once a day for 4 consecutive days, and their body weights were recorded every day.

[0068] (1) Mouse organ index detection Mice were sacrificed under sterile conditions by cervical dislocation. The thymus, spleen, and lungs were removed, washed with PBS, and dried. Organ weights were measured and recorded. A final weighing was performed before sacrifice, and organ indices were calculated using the formula: Organ index = (organ weight / final body weight) × 100%.

[0069] Compared with the normal group, the lung index of RSV-infected mice was significantly increased, while the thymus index and spleen index were significantly decreased, indicating that the RSV-infected mouse model was successfully established. After administration of the drug to RSV-infected mice, the lung index was restored. The chlorogenic acid group and the ribavirin group showed varying degrees of recovery compared to the model group. The thymus index and spleen index are important indicators of immune function. They can be restored after the intervention of chlorogenic acid, indicating that chlorogenic acid may have a positive effect on immune function ( Figure 11 ).

[0070] (2) Comparison of inflammatory factor levels in lung tissue The right lung lobule of the mouse was weighed, infiltrated with PBS (1:9), placed in grinding beads, ground at 50 Hz for 90 seconds, and then centrifuged at 3000 r / min and 4°C for 15 minutes. The supernatant was divided into centrifuge tubes and stored in a -80°C refrigerator. The levels of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the lung tissue were determined according to the instructions of the ELISA kit. The test results showed that compared with the normal group, the levels of inflammatory factors IL-1β and IL-6 in the lung tissue of the model group mice were significantly increased. Compared with the model group, the drug group can significantly adjust the levels of IL-1β and IL-6 in the lung tissue of the mice, among which the ribavirin and cryptochlorogenic acid groups had similar effects ( Figure 12 ).

[0071] (3) Histopathological analysis One lobe of the left lung was fixed in 4% paraformaldehyde for 48 hours, then dehydrated and paraffin-infused with gradient alcohol, then embedded in paraffin, cooled in a -20°C freezer, and placed in a microtome for tissue sectioning, and dried in a 60°C oven; the paraffin sections were dewaxed to water in sequence, treated with high-definition constant stain pretreatment solution for 1 minute, stained with hematoxylin and eosin, dehydrated in gradient ethanol-n-butanol-xylene until transparent, and mounted with neutral gum. Pathological images of the mouse lung tissue were collected under a microscope. The results showed that the lung tissue of the normal group mice had abundant alveoli, clear structure, uniform alveolar septa, and no pathological changes were found; the lung tissue structure of the model group mice was severely abnormal, with alveolar atrophy and collapse, widened septa, and thickening of the alveolar capillary wall. Inflammatory cell infiltration, mainly neutrophils, and red blood cell aggregation appeared in the alveolar cavity, and the inflammatory reaction was obvious; compared with the model group, the lung tissue structure and lesion degree were improved after the intervention of cryptochlorogenic acid, indicating that cryptochlorogenic acid can effectively improve the lung tissue damage of RSV-infected mice ( Figure 13 ).

[0072] In summary, the present invention conducted affinity fishing screening for active ingredients in the Yinqiao herbal combination that target the ACSL4 protein. The recovered eluate was then identified using UHPLC-MS / MS, ultimately identifying nine components with well-defined chemical structures. To focus on the ligand binding properties of the single target ACSL4 and elucidate the ligand-receptor binding mechanism, molecular docking techniques were employed, and molecular dynamics simulations were used to assess the conformational stability and kinetic properties of the complex system. These findings revealed that chlorogenic acids (cryptochlorogenic acid) and forsythiasides (isoforsythiaside A) exhibited higher binding potential to the ACSL4 protein. Furthermore, animal experiments confirmed that cryptochlorogenic acid is the key pharmacological agent in the Yinqiao herbal combination that regulates lipid peroxidation and exerts antiviral effects. This invention elucidates the pharmacological basis for the Yinqiao herbal combination's targeting of the ACSL4 protein, providing new insights into the synergistic mechanisms of multi-component interactions in Traditional Chinese Medicine.

[0073] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Application of cryptochlorogenic acid in the preparation of effective substances targeting ACSL4 protein.

2. The use according to claim 1, characterized in that The efficacy substance forms a high-frequency binding interaction through THR278, SER281, THR469, VAL324 and LYS690 residues, and targets and binds to the ACSL4 protein.

3. Application of cryptochlorogenic acid in the preparation of drugs for preventing and treating RSV infection.

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