Hypochlorous acid response type near-infrared fluorescent probe for screening Alzheimer's disease antioxidants as well as preparation method and application of hypochlorous acid response type near-infrared fluorescent probe

Abstract

The invention belongs to the technical field of fluorescent probes and biological detection, and particularly relates to a hypochlorous acid response type near-infrared fluorescent probe for screening Alzheimer's disease antioxidants as well as a preparation method and application of the hypochlorous acid response type near-infrared fluorescent probe. The near-infrared fluorescent probe FP-MT-HClO provided by the invention is subjected to a specific reaction under the action of HClO, generates obvious fluorescence signal enhancement, outputs near-infrared fluorescence signals at about 665 nm, and has the characteristics of quick response, high sensitivity, good selectivity, strong anti-interference capability and the like. An in-vitro screening model used for simulating an inflammatory oxidative stress link related to the Alzheimer's disease is constructed based on the probe, and rapid screening and activity evaluation of candidate antioxidants capable of reducing the HClO level in cells are achieved. The method disclosed by the invention is good in repeatability, is suitable for in-vitro screening of candidates related to inflammatory oxidative stress, and can be used for early evaluation and mechanism research of an oxidative stress process related to Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe and biological detection technology, specifically relating to a hypochlorous acid-responsive near-infrared fluorescent probe for screening Alzheimer's disease antioxidants, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a group of central nervous system degenerative diseases characterized by progressive cognitive decline and memory impairment. Its pathogenesis is complex and significantly heterogeneous. Current research generally agrees that pathological changes such as abnormal deposition of β-amyloid (Aβ), hyperphosphorylation of Tau protein, and neurofibrillary tangles are closely related to disease progression, and are accompanied by multiple factors including neuroinflammatory responses, oxidative stress damage, mitochondrial dysfunction, and neurotransmitter homeostasis imbalance. Due to the insidious onset, long course, and lack of effective etiological treatments for AD, early evaluation and screening of intervention candidates targeting key pathological processes have significant research and application value.

[0003] In the pathological process of Alzheimer's disease (AD), oxidative stress is considered one of the important factors driving neuronal damage and inflammation amplification. HClO, as a typical highly reactive chlorine species, can be generated by the peroxidase system in the inflammatory microenvironment. It is characterized by high reactivity and a broad range of targets, and can induce a series of oxidative stress-related changes, such as protein oxidation / chlorination modification, lipid peroxidation, and nucleic acid damage, thereby aggravating cellular dysfunction and promoting the sustained amplification of inflammatory signals. Therefore, establishing reproducible and quantifiable detection and evaluation methods for HClO-mediated oxidative stress processes, and using them for the screening and validation of candidate antioxidants, is an important need in AD research.

[0004] Currently, the screening and evaluation of antioxidant candidates often employ chemical free radical scavenging assays (such as DPPH, ABTS, etc.) or cellular-level endpoint indicators (such as cell viability, malondialdehyde / superoxide dismutase, etc.). However, these methods are mostly in vitro or endpoint detection, which cannot reflect the dynamic changes of specific bioactive species within cells. Furthermore, intracellular oxidative stress involves the interconversion of multiple reactive oxygen species (ROS), nitrogen species, and chloride species. Using general-purpose ROS probes or non-specific indicators often suffers from insufficient selectivity, unclear signal sources, and difficulty in quantitative comparison, thus limiting the reliability and interpretability of screening results in inflammatory oxidative stress scenarios.

[0005] For the detection of HClO, various technical approaches exist, including colorimetric methods, electrochemical methods, indirect analysis by chromatography / mass spectrometry, and fluorescence detection. Traditional physicochemical analysis methods typically rely on complex instruments or sample pretreatment, making it difficult to achieve real-time imaging and high-throughput evaluation of intracellular HClO. While fluorescence detection offers advantages such as sensitivity, speed, and visualization, some existing HClO fluorescent probes may suffer from problems such as short emission wavelengths leading to strong interference from biological autofluorescence, limited tissue penetration, insufficient photostability or water solubility, and high intracellular background signals. Furthermore, in natural product screening scenarios, they are more susceptible to interference from sample-borne fluorescence or color, affecting screening accuracy and repeatability. Therefore, there is an urgent need to develop a detection and evaluation method for HClO at the cellular level with higher selectivity, lower background interference, and compatibility with screening procedures to support the rapid screening and activity verification of candidate antioxidants related to inflammatory oxidative stress. Summary of the Invention

[0006] To address the problems of insufficient selectivity, large background interference, difficulty in quantitative or visual comparison of specific intracellular active species, and difficulty in adapting to high-throughput screening processes in existing detection methods for evaluating inflammatory oxidative stress and screening antioxidant candidates, this invention provides a hypochlorous acid-responsive near-infrared fluorescent probe for screening antioxidants for Alzheimer's disease.

[0007] The present invention further provides a method for preparing the above-mentioned hypochlorous acid-responsive near-infrared fluorescent probe.

[0008] Another objective of this invention is to provide the application of the aforementioned hypochlorous acid-responsive near-infrared fluorescent probe in the preparation of drugs for screening Alzheimer's disease antioxidants; this invention also provides a method for screening and evaluating cellular-level candidate antioxidants based on the fluorescent probe, in order to achieve rapid screening and activity verification of candidates that can reduce intracellular HClO levels.

[0009] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides a hypochlorous acid-responsive near-infrared fluorescent probe for screening antioxidants for Alzheimer's disease, the structure of which is shown in formula (I): Formula (I).

[0010] This invention also provides a method for preparing the above-mentioned hypochlorous acid-responsive near-infrared fluorescent probe, comprising the following steps: (1) Dissolve Basic Blue 3 and Na2CO3 in a mixed solvent, slowly add sodium dithionite aqueous solution to the reaction system, heat and reflux under inert gas protection to obtain intermediate reaction solution; (2) After mixing Na2CO3 and intermediate reaction solution, triphosgene dichloromethane solution was slowly added dropwise, and the reaction was heated under inert gas protection; after the reaction was completed, the solution was extracted, dried and purified to obtain the fluorophore intermediate FP-Cl-HClO; (3) Dissolve FP-Cl-HClO in dichloromethane, add 5-methoxytryptamine and organic base for substitution reaction, remove solvent and purify after reaction to obtain hypochlorous acid responsive near-infrared fluorescent probe.

[0011] Preferably, in step (1), the molar ratio of Basic Blue 3, Na2CO3 and sodium dithionite is 1:4:4; the mixed solvent is composed of water and dichloromethane in a volume ratio of 2:1; and the concentration of the sodium dithionite aqueous solution is 0.28 mmol / mL.

[0012] Preferably, in step (1), the heating reflux reaction is carried out under nitrogen protection at 40 °C for 1 h.

[0013] Preferably, in step (2), the molar ratio of Na2CO3 to triphosgene is 6.67:1; the concentration of the triphosgene dichloromethane solution is 125-130 mg / mL; the heating reaction is a reflux reaction at 40 °C for 3 h under nitrogen protection; the purification is carried out by column chromatography; the eluent for column chromatography is composed of ethyl acetate and petroleum ether in a volume ratio of 1:40.

[0014] Preferably, in step (2), the structural formula of the fluorophore intermediate FP-Cl-HClO is shown in formula (II): Equation (II).

[0015] Preferably, in step (3), the molar ratio of FP-Cl-HClO, 5-methoxytryptamine and organic base is 1:2:2; the organic base is triethylamine; the substitution reaction is a stirred reaction at room temperature for 4 h; the purification is carried out by silica gel column chromatography; the eluent for silica gel column chromatography is composed of dichloromethane and methanol in a volume ratio of 25:1.

[0016] This invention also provides the application of the above-mentioned hypochlorous acid-responsive near-infrared fluorescent probe in the preparation of drugs for screening Alzheimer's disease antioxidants.

[0017] Another object of the present invention is to provide a kit containing the above-mentioned hypochlorous acid-responsive near-infrared fluorescent probe for screening Alzheimer's disease antioxidants.

[0018] This invention also provides a method for screening candidate antioxidants based on the above-mentioned hypochlorous acid-responsive near-infrared fluorescent probes, used to screen candidates that can reduce intracellular HClO levels. The method includes the following steps: (1) Provide test cells and establish an inflammatory oxidative stress model; (2) Pretreatment by co-incubating the candidate samples with the test cells; (3) Treatment with an inducer to induce an increase in intracellular HClO levels; (4) Add the fluorescent probe FP-MT-HClO to the treated cell system and incubate; (5) The fluorescence signals of each group of cells were obtained by fluorescence imaging or fluorescence intensity detection, and the degree of reduction of fluorescence signal of the candidate treatment group relative to the model group was used as the evaluation index to screen out candidate antioxidants that can reduce the level of HClO in cells.

[0019] Preferably, the test cells are RAW264.7 cells; and the inducer is LPS.

[0020] Preferably, the candidate pretreatment concentration is 10–50 μM and the pretreatment time is 0.5–4 h; the LPS induction concentration is 0.1–5 μg / mL and the induction time is 6–36 h.

[0021] Preferably, the fluorescent probe is incubated at a concentration of 1–20 μM for 10–60 min; the detection method includes one or more of laser confocal imaging, ELISA reader detection, or flow cytometry detection.

[0022] Preferably, the evaluation index is the fluorescence inhibition rate, calculated using the following formula: Inhibition rate (%) = (F model -F sample ) / F model ×100%.

[0023] Among them, F model F represents the fluorescence intensity of the model group. sample The fluorescence intensity represents the fluorescence intensity of the candidate treatment group; a higher inhibition rate indicates a stronger ability of the candidate to reduce intracellular HClO levels.

[0024] The reaction route diagram of the hypochlorous acid-responsive near-infrared fluorescent probe prepared in this invention is as follows: .

[0025] The near-infrared fluorescent probe FP-MT-HClO prepared in this invention exhibits a specific reaction and significant fluorescence signal enhancement under the action of HClO, emitting a near-infrared fluorescence signal at approximately 665 nm. It features rapid response, high sensitivity, good selectivity, and strong anti-interference ability. Based on this fluorescent probe, an in vitro screening model for simulating Alzheimer's disease-related inflammatory oxidative stress was constructed: using RAW264.7 cells as test cells, candidate natural products or compounds were pretreated to induce an increase in intracellular HClO levels using lipopolysaccharide (LPS). Subsequently, the near-infrared fluorescent probe was added, and fluorescence imaging or fluorescence intensity detection was performed. The degree of reduction in intracellular fluorescence intensity of the candidate relative to the model group was used as an evaluation index, enabling rapid screening and activity evaluation of candidate antioxidants that can reduce intracellular HClO levels. The method of this invention has good reproducibility, is suitable for in vitro screening of candidates related to inflammatory oxidative stress, and can be used for early evaluation and mechanistic research of Alzheimer's disease-related oxidative stress processes.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The hypochlorous acid-responsive near-infrared fluorescent probe provided by the present invention has high selectivity for HClO and can output near-infrared fluorescent signals, which is beneficial to reduce the background interference of cell autofluorescence and some natural products and improve the reliability of screening results. (2) The preparation route of the hypochlorous acid-responsive near-infrared fluorescent probe provided by the present invention is clear, the operation steps are simple, the raw materials are readily available, and it is convenient for large-scale preparation and promotion and application. (3) The cell-level screening method based on hypochlorous acid-responsive near-infrared fluorescent probe of the present invention has a clear process and controllable parameters, which can realize the rapid evaluation of the ability of candidates to reduce the intracellular HClO level and meet the requirements of high-throughput screening of trace amounts, speed, sensitivity and accuracy. Attached Figure Description

[0027] Figure 1 This is the electron cloud density map of the HClO-responsive near-infrared fluorescent probe FP-MT-HClO of this invention; Figure 2 The near-infrared fluorescent probe FP-MT-HClO of this invention 1 H NMR spectrum; Figure 3 The near-infrared fluorescent probe FP-MT-HClO of this invention 13 C NMR spectrum; Figure 4 This is a high-resolution mass spectrum (HRMS) image of the near-infrared fluorescent probe FP-MT-HClO of this invention; Figure 5This is the UV-Vis absorption spectrum of the near-infrared fluorescent probe FP-MT-HClO before and after the reaction with HClO in this invention; Figure 6 The fluorescence emission spectra (A) of the near-infrared fluorescent probe FP-MT-HClO of the present invention when reacted with different concentrations of HClO, and the linear relationship between fluorescence intensity at 665 nm and HClO concentration (B). Figure 7 This is a kinetic curve showing the change in fluorescence intensity at 665 nm over time when the near-infrared fluorescent probe FP-MT-HClO (10 μM) of this invention reacts with different concentrations of HClO. Figure 8 This is a bar chart showing the fluorescence response of the near-infrared fluorescent probe FP-MT-HClO of this invention when it interacts with HClO and other potential interfering substances; where 1–17 represent, in order: control group, Gly, Lys, Phe, Thr, GSH, Na + K + SO4 2- HCO3 - ,AChE,β-gal,NTR,H2O2,O2 - ONOO - HClO; Figure 9 The graph shows the effect of different pH buffer systems on the fluorescence response of the near-infrared fluorescent probe FP-MT-HClO in this invention in the reaction with HClO. Figure 10 A cytotoxicity evaluation diagram showing the effect of different concentrations of the near-infrared fluorescent probe FP-MT-HClO of this invention on macrophage survival. Figure 11 The figure shows the experimental results of LPS-induced fluorescence signals in cells; where A is the blank control group (A1 is fluorescence imaging, A2 is bright field imaging), B is the low concentration group of 1 μg / mL LPS (B1 is fluorescence imaging, B2 is bright field imaging), C is the high concentration group of LPS (C1 is fluorescence imaging, C2 is bright field imaging), D is the LPS+4-ABAH inhibitor control group (D1 is fluorescence imaging, D2 is bright field imaging), and E is the relative fluorescence intensity of different groups. Figure 12 Figure 1 shows the experimental results of exogenous HClO inducing fluorescence signals in cells; where A is the blank control group (A1 is fluorescence imaging, A2 is bright field imaging), B is the 20 μM HClO treatment group (B1 is fluorescence imaging, B2 is bright field imaging), C is the 50 μM HClO treatment group (C1 is fluorescence imaging, C2 is bright field imaging), D is the antioxidant intervention group with 50 μM HClO + NAC (D1 is fluorescence imaging, D2 is bright field imaging), and E is the relative fluorescence intensity of different groups. Figure 13 This is a fluorescence imaging diagram of cells after incubation with different natural products based on near-infrared fluorescent probes according to the present invention; in the figure, 1-28 are, in order, control group, model group, methyl vanillate, eugenol, guaiacol, sesamol, juniperol, menthone, oxymatrine, ethyl ferulic acid, naringenin, polygalactoside, hesperidin, proanthocyanidins, glycyrrhizic acid, shikonin, emodin methyl ether, berberine, eugenol, malic acid, rosin, hesperidin, iridoside, ginsenosides, tanshinone, arbutin, hyperoside, and rhein; Figure 14 This is a chromatogram of the inhibitory activity of different natural products against HClO based on near-infrared fluorescent probes according to the present invention. In the figure, the components are, in order, control group, model group, methyl vanillate, eugenol, guaiacol, sesamol, juniperol, menthone, oxymatrine, ethyl ferulic acid, naringenin, polygalactoside, hesperidin, proanthocyanidins, glycyrrhizic acid, shikonin, emodin methyl ether, berberine, eugenol, malic acid, rosin, hesperidin, iridoside, ginsenosides, tanshinone, arbutin, hyperoside, and rhein. Figure 15 To develop a bar chart of relative fluorescence intensity for screening different natural products based on near-infrared fluorescent probes; in the figure, the horizontal axis from left to right represents the control group, model group, methyl vanillate, eugenol, guaiacol, sesamol, juniperol, menthone, oxymatrine, ethyl ferulic acid, naringenin, polygalactoside, hesperidin, proanthocyanidins, glycyrrhizic acid, shikonin, emodin methyl ether, berberine, eugenol, malic acid, rosin, hesperidin, iridoside, ginsenosides, tanshinone, arbutin, hyperoside, and rhein. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise stated, all reagents used in the present invention are commercially available reagents; all solvents used are of analytical grade or higher purity.

[0029] Example 1

[0030] (1) Preparation of the fluorophore FP-Cl-HClO Weigh out 1.0 g (0.7 mmol) of Basic Blue 3 and 297 mg (2.8 mmol) of Na2CO3, and dissolve / disperse them in a mixed solvent of water (8.0 mL) and dichloromethane (4.0 mL). Dissolve 487 mg (2.8 mmol) of sodium dithionite in water (10.0 mL) and slowly add it dropwise to the above reaction system. React at 40 °C for 1 h under nitrogen protection to obtain an intermediate reaction solution.

[0031] Na₂CO₃ (297 mg, 2.8 mmol) was added to a dry three-necked flask. The intermediate reaction solution was transferred to the flask, and a solution of triphosgene (125 mg, 0.42 mmol) in dichloromethane (10 mL) was slowly added dropwise at low temperature. The reaction was then carried out at 40 °C for 3 h under a nitrogen atmosphere. After the reaction was completed, the mixture was extracted with a dichloromethane / water system, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:40, v / v) to give the fluorophore intermediate FP-Cl-HClO (white solid, yield 66.8%).

[0032] (2) Preparation of near-infrared fluorescent probe FP-MT-HClO 5-Methoxytryptamine (190 mg, 1.0 mmol) and triethylamine (TEA, 140 μL, 1.0 mmol) were added to a dichloromethane solution (5 mL) containing the fluorophore FP-Cl-HClO (173 mg, 0.5 mmol), and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the solvent was removed to obtain the crude product, which was purified by silica gel column chromatography (CH2Cl2 / MeOH = 25:1, v / v) to obtain the near-infrared fluorescent probe FP-MT-HClO (pale blue solid, 50 mg, yield 20%).

[0033] The chemical structure of the near-infrared fluorescent probe FP-MT-HClO is as follows: .

[0034] The electron cloud density diagram of the near-infrared fluorescent probe FP-MT-HClO is as follows: Figure 1 As shown; its 1 The H NMR spectrum is shown in [reference]. Figure 2 , 13 The C NMR spectrum is shown below. Figure 3 High-resolution mass spectra can be found in [the image]. Figure 4 .

[0035] Example 2: Spectral response performance of FP-MT-HClO to HClO The near-infrared fluorescent probe FP-MT-HClO used in this embodiment was prepared in Example 1.

[0036] (1) Ultraviolet-Visible Absorption Spectroscopy Test Prepare PBS buffer solution (pH 7.4, 10 mM, containing 20% ​​DMF, v / v), and prepare probe FP-MT-HClO stock solution (1 mM) with DMF.

[0037] Blank system: Take 2970 μL of PBS buffer solution and add 30 μL of probe stock solution to make the final probe concentration 10 μM, with a total volume of 3.0 mL.

[0038] Reaction system: 60 μL of HClO stock solution (5 mM) was mixed with 29-10 μL of PBS buffer solution, and then 30 μL of probe stock solution was added to bring the final concentration of HClO to 100 μM and the final concentration of the probe to 10 μM, for a total volume of 3.0 mL. After incubation at 37 ℃ and 100 r / min for 1 min with shaking, the UV-Vis absorption spectrum was measured. The probe itself had weak absorption, but a significantly enhanced absorption peak appeared after reacting with HClO, indicating that the probe specifically reacted with HClO. The results are shown in the figure below. Figure 5 .

[0039] (2) Relationship between fluorescence titration and linearity Prepare reaction systems with different concentrations of HClO (0–100 μM) according to the above-described PBS buffer system (pH 7.4, 10 mM, containing 20% ​​DMF), ensuring the final concentration of the fluorescent probe is maintained at 10 μM (the total volume can be set to 3.0 mL or scaled proportionally according to the instrument's cuvette specifications). After incubating the reaction system with shaking at 37 ℃ (e.g., 40 min), the fluorescence emission spectrum was measured. As the HClO concentration increased, the fluorescence intensity at approximately 665 nm gradually increased, showing a good linear relationship between fluorescence intensity and HClO concentration. The results are shown in [Figure number missing]. Figure 6 A and Figure 6 In section B, the kinetic curves of fluorescence intensity at 665 nm over time when the near-infrared fluorescent probe FP-MT-HClO (10 μM) prepared in this invention reacts with different concentrations of HClO are shown in the figure. Figure 7 As shown.

[0040] (3) Selectivity and anti-interference ability In the same buffer system, HClO and other potential interfering substances (such as amino acids, GSH, inorganic ions, related enzymes, and typical active species) were added to bring the final probe concentration to 10 μM and the final concentration of each analyte to 100 μM. After incubation at 37 ℃ with shaking for 60 min, the fluorescence signal was measured. The results showed that HClO significantly induced fluorescence enhancement at 665 nm, while the changes caused by other analytes were not significant, indicating that the probe has high selectivity for HClO. (See [link to relevant documentation]). Figure 8 .

[0041] (4) pH range The probe was added to 20% DMF / PBS buffer solutions at different pH values ​​to achieve a final concentration of 10 μM, and HClO was added to achieve a final concentration of 35 μM. After mixing, the solutions were incubated in a constant-temperature mixer for 1 h, and the fluorescence intensity was measured. The results showed that the probe maintained a good response to HClO across different pH ranges, especially at the physiological pH of 7.4, where the response was optimal. (See attached figures). Figure 9 .

[0042] (1) Cell culture and probe cytotoxicity evaluation RAW264.7 macrophages were used as a model cell for evaluation. Cells were digested / collected, centrifuged, resuspended, counted, and their density adjusted to 1×10⁻⁶ cells / year. 5 Cells / mL; 100 μL of cell suspension was seeded into each well of a 96-well plate and incubated overnight at 37 ℃ in a 5% CO2 incubator. The next day, different concentrations (0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM) of near-infrared fluorescent probe were added and co-incubated with cells for 24 h. After discarding the culture medium, 100 μL of 10% CCK-8 working solution was added to each well and incubated at 37 ℃ for 2 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. The results showed no significant cytotoxicity within the tested concentration range, indicating that the probe has good biocompatibility at conventional concentrations and is suitable for cell-level imaging and screening. The results are shown in the figure. Figure 10 .

[0043] (2) Imaging verification of LPS-induced endogenous HClO generation RAW264.7 cells were divided into the following groups: Figure 11 In the diagram, A represents the blank control group (A1 is fluorescence imaging, A2 is bright-field imaging), B represents the low-concentration LPS group (1 μg / mL) (B1 is fluorescence imaging, B2 is bright-field imaging), C represents the high-concentration LPS group (3 μg / mL) (C1 is fluorescence imaging, C2 is bright-field imaging), and D represents the LPS+4-ABAH inhibitor control group (D1 is fluorescence imaging, D2 is bright-field imaging). E represents the relative fluorescence intensity of different groups. After 24 h of treatment, each group was incubated with 10 μM probe FP-MT-HClO for 30 min, washed with PBS, and then fluorescence images were acquired using laser confocal microscopy for quantitative analysis. The results showed that LPS dose-dependently enhanced intracellular fluorescence signals, and this effect could be inhibited by 4-ABAH.

[0044] (3) Validation of exogenous HClO and antioxidant intervention Settings such as Figure 12In the diagram, A represents the blank control group (A1 is fluorescence imaging, A2 is bright-field imaging), B represents the 20 μM HClO treatment group (B1 is fluorescence imaging, B2 is bright-field imaging), C represents the 50 μM HClO treatment group (C1 is fluorescence imaging, C2 is bright-field imaging), and D represents the 50 μM HClO+NAC antioxidant intervention group (D1 is fluorescence imaging, D2 is bright-field imaging); E represents the relative fluorescence intensity of different groups. After treatment under the set conditions, 10 μM probe FP-MT-HClO was added and incubated for 30 min. After washing with PBS, confocal imaging was performed and fluorescence intensity was quantitatively analyzed. The results showed that exogenous HClO could enhance the cell fluorescence signal, while NAC could inhibit this enhancement effect (see [reference]). Figure 12 .

[0045] The fluorescent probe FP-MT-HClO used in this embodiment was prepared in Example 1.

[0046] (1) Model building and candidate preprocessing RAW264.7 cells were seeded in 96-well plates and cultured until stable. Twenty-six test monomeric natural products were prepared as 10 mM stock solutions using DMSO. Groups included: a normal control group (using the same volume of DMSO instead of the monomeric natural products), a model group (LPS-induced), and candidate product treatment groups. Candidate product treatment groups were pretreated with complete culture medium containing the corresponding monomer to a final concentration of 20 μM for 2 h. Subsequently, except for the normal control group, all other groups were incubated with LPS (final concentration 1 μg / mL) for another 24 h to establish an inflammatory oxidative stress model; the normal control group was cultured in complete culture medium.

[0047] (2) Probe loading and detection After induction, discard the culture medium and wash once with PBS. Add phenol red-free medium (containing 1% FBS) with 10 μM probe FP-MT-HClO to each well and incubate at 37 ℃ in 5% CO2 in the dark for 30 min. After incubation, wash three times with PBS to remove probes that have not entered the cells.

[0048] (3) Imaging acquisition and quantification Fluorescence images were acquired using a laser confocal microscope in the excitation / emission channels corresponding to the probes; at least three non-overlapping fields of view were selected for each well. The average fluorescence intensity of cells in each group was calculated using software such as ImageJ.

[0049] (4) Screening and Judgment The average fluorescence intensity of the model group is F model The average fluorescence intensity of the candidate treatment group was F. sample Calculate the fluorescence inhibition rate: Inhibition rate (%) = (F model -Fsample ) / F model ×100%.

[0050] A higher inhibition rate indicates a stronger ability of the candidate to reduce intracellular HClO levels.

[0051] Figure 13 The images show fluorescence imaging of cells after incubation with different natural products, revealing changes in intracellular HClO after treatment with different natural products. The numbers in the images correspond to the following groups in order: control group, model group, methyl vanillate, eugenol, guaiacol, sesamol, juniperol, menthone, oxymatrine, ethyl ferulic acid, naringenin, polygalactoside, hesperidin, proanthocyanidins, glycyrrhizic acid, shikonin, emodin methyl ether, berberine, eugenol, malic acid, rosin, hesperidin, iridoside, ginsenosides, tanshinone, arbutin, hyperoside, and rhein. Figure 14 To screen the chromaticity diagrams of different natural products' inhibitory activity against HClO, the corresponding order in the diagram is the same as... Figure 13 In the color scale diagram, from top to bottom, the lower the value, the better the inhibition of activity. Figure 15 This is a bar chart showing the relative fluorescence intensity. Figure 15 From left to right, the groups are: control group, model group, methyl vanillate, eugenol, guaiacol, sesamol, juniperol, menthone, oxymatrine, ethyl ferulic acid, naringenin, polygalactoside, hesperidin, proanthocyanidins, glycyrrhizic acid, shikonin, emodin methyl ether, berberine, eugenol, malic acid, rosin, hesperidin, iridoside, ginsenosides, tanshinone, arbutin, hyperoside, and rhein. Screening results showed that proanthocyanidins (No. 14) exhibited the best HClO inhibitory activity, with reduced fluorescence intensity in its treatment group, suggesting that proanthocyanidins have significant anti-inflammatory and antioxidant potential and can be further studied as candidate compounds for intervention in Alzheimer's disease-related inflammatory oxidative stress.

[0052] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A hypochlorous acid-responsive near-infrared fluorescent probe for screening of Alzheimer's disease antioxidants, characterized by, The structure of the near-infrared fluorescent probe is shown as formula (I): Formula (I).

2. A method for preparing the hypochlorous acid-responsive near-infrared fluorescent probe according to claim 1, characterized by, The method comprises the following steps: (1) dissolving basic blue 3 and Na2CO3 in a mixed solvent, slowly adding an aqueous sodium hydrosulfite solution into the reaction system, and heating and refluxing the reaction under inert gas protection to obtain an intermediate reaction liquid; (2) mixing Na2CO3 and the intermediate reaction liquid, slowly adding a triphosgene dichloromethane solution, and heating and reacting under inert gas protection; after the reaction is completed, extraction, drying, and purification are performed to obtain a fluorophore intermediate FP-Cl-HClO; (3) dissolving FP-Cl-HClO in dichloromethane, adding 5-methoxy tryptamine and an organic base for a substitution reaction, removing the solvent after the reaction is completed, and purifying to obtain a hypochlorous acid responsive near-infrared fluorescent probe.

3. The production method according to claim 2, characterized by, In step (1), the molar ratio of basic blue 3, Na2CO3, and sodium hydrosulfite is 1:4:4; the mixed solvent is composed of water and dichloromethane in a volume ratio of 2:1; and the concentration of the aqueous sodium hydrosulfite solution is 0.28 mmol / mL.

4. The production method according to claim 2 or 3, characterized by, In step (1), the heating and refluxing reaction is carried out under nitrogen protection at 40 ℃ for 1 h.

5. The preparation method according to claim 2, characterized in that, In step (2), the molar ratio of Na2CO3 and triphosgene is 6.67:1; the concentration of the triphosgene dichloromethane solution is 125-130 mg / mL; the heating reaction is carried out under nitrogen protection at 40 ℃ for 3 h; and the purification is performed by column chromatography; the eluent for column chromatography is composed of ethyl acetate and petroleum ether in a volume ratio of 1:

40.

6. The production method according to claim 2 or 5, characterized by, In step (2), the structure of the fluorophore intermediate FP-Cl-HClO is shown as formula (II): Formula (II).

7. The preparation method according to claim 2, characterized in that, In step (3), the molar ratio of FP-Cl-HClO, 5-methoxy tryptamine, and the organic base is 1:2:2; the organic base is triethylamine; the substitution reaction is carried out at room temperature for 4 h; and the purification is performed by silica gel column chromatography; the eluent for silica gel column chromatography is composed of dichloromethane and methanol in a volume ratio of 25:

1.

8. Use of the hypochlorous acid responsive near-infrared fluorescent probe according to claim 1 in the preparation of a drug for screening an antioxidant for Alzheimer's disease.

9. A kit for screening antioxidants for Alzheimer's disease, characterized by, The kit contains the hypochlorous acid responsive near-infrared fluorescent probe according to claim 1.

Images