A fluorescent probe for screening anti-atherosclerosis traditional Chinese medicine and a preparation method and application thereof

Abstract

The present application belongs to the field of drug screening and biological detection, and particularly relates to a fluorescent probe for screening anti-atherosclerotic traditional Chinese medicines and a preparation method and application thereof. The present application aims to construct an anti-atherosclerotic traditional Chinese medicine screening platform based on fluorescent probe technology, and screen 70 kinds of traditional Chinese medicines. First, a probe for recognizing ONOO⁻ is designed and synthesized as a tool for detecting the ROS content in zebrafish, then a zebrafish atherosclerosis model is constructed by using lipopolysaccharide induction, and the improvement of different traditional Chinese medicine crude extracts on the atherosclerosis in zebrafish is dynamically monitored by using confocal imaging technology. The present application not only provides an innovative method for the efficient screening of anti-atherosclerotic components of traditional Chinese medicines, but also provides potential drug candidates for the prevention and treatment of atherosclerosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of drug screening and biological detection, and particularly relates to a fluorescent probe for screening anti-atherosclerosis traditional Chinese medicine and a preparation method and application thereof. BACKGROUND

[0002] Atherosclerosis is a chronic vascular disease characterized by abnormal lipid deposition, endothelial dysfunction, and chronic inflammation, and is widely involved in the occurrence and development of various diseases, and has a profound impact on multiple systems of the body. In the field of cardiovascular disease, it is the pathological basis of atherosclerotic heart disease, myocardial ischemia and other diseases; in the nervous system, it can promote the progression of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease; in the metabolic system, it is closely related to the occurrence and development of metabolic diseases such as diabetes and non-alcoholic fatty liver disease; in addition, its pathological process is also involved in cancer, inflammatory and immune diseases, and lung diseases (acute lung injury, chronic obstructive pulmonary disease), reproductive system diseases (early ovarian dysfunction) and osteoarthritis and other diseases of multiple systems.

[0003] The pathogenesis of atherosclerosis is complex and diverse, involving lipid metabolism disorder, endothelial cell damage, activation of inflammatory signaling pathways and other links. Among them, the chronic inflammation of the vascular wall caused by lipid deposition is the core pathological feature: the deposited lipids can induce endothelial cell activation, recruit monocytes to differentiate into macrophages, form foam cells, and further activate inflammatory signaling pathways such as NF-κB and MAPK, exacerbating local inflammation. This inflammatory state interacts with vascular wall structural damage to form a vicious cycle of "lipid deposition-inflammation-vascular damage", for example, in atherosclerotic cardiovascular disease, the inflammatory response continuously damages the vascular endothelium, promotes plaque formation and rupture, and increases the risk of acute cardiovascular events.

[0004] Oxidative stress is an important accompanying mechanism in the pathological process of atherosclerosis and is closely related to disease progression. In the atherosclerotic state, imbalance between intracellular oxidation and antioxidant systems can lead to excessive accumulation of reactive oxygen species (ROS), which can not only directly damage vascular cell structure through lipid peroxidation, protein modification, etc., but also further activate inflammatory signaling pathways and exacerbate inflammation. At the same time, ROS produced by mitochondrial dysfunction and abnormal NADPH oxidase may also interact with the pathological links of atherosclerosis and affect the disease process.

[0005] Traditional Chinese medicine (TCM) has shown unique advantages in the treatment of atherosclerosis due to its multi-component, multi-pathway and multi-target characteristics. TCM can intervene in the pathological process of atherosclerosis through multiple pathways such as regulating lipid metabolism, inhibiting inflammatory response and protecting vascular endothelial cell function. For example, herbs and food homologous components such as Danshen and Shanzha can improve endothelial function and inhibit lipid deposition, and compound TCM can relieve the inflammatory state of the vascular wall by regulating the expression of inflammation-related genes. Compared with single-component drugs, TCM has the advantage of comprehensive regulation in complex diseases, and shows lower toxic and side effects in animal models. However, the complex composition of TCM, low efficiency of active substance screening and unclear mechanism of action restrict its modern development. How to efficiently screen active ingredients with clear efficacy and systematically analyze their molecular mechanisms is a bottleneck that needs to be broken through in the study of TCM intervention in atherosclerosis. SUMMARY

[0006] In view of the problems existing in the prior art, the present application provides a fluorescent probe for screening anti-atherosclerotic traditional Chinese medicine. The probe is easy to synthesize, has high sensitivity and suitable emission wavelength, and has good application prospect in the screening of anti-atherosclerotic traditional Chinese medicine.

[0007] The present application also provides a preparation method of the above-mentioned fluorescent probe.

[0008] Another object of the present application is to provide the use of the above-mentioned fluorescent probe in the screening of anti-atherosclerotic traditional Chinese medicine.

[0009] In order to achieve the above-mentioned object, the technical scheme of the present application is as follows:

[0010] The present application provides a fluorescent probe for screening anti-atherosclerotic traditional Chinese medicine, and the structural formula of the fluorescent probe is:

[0011] .

[0012] The present application also provides a preparation method of the above-mentioned fluorescent probe for screening anti-atherosclerotic traditional Chinese medicine, comprising the following steps:

[0013] (1) Dissolve isophorone and malonitrile in ethanol, add piperidine and glacial acetic acid, and carry out reaction, then add water to precipitate solid, and obtain compound 1 after filtration and recrystallization;

[0014] (2) Dissolve compound 1 and 3-chloro-4-hydroxybenzaldehyde in acetonitrile, add piperidine, and carry out reaction, then purify by silica gel column chromatography after the reaction is completed, to obtain fluorophore FP-OH; the structural formula is:

[0015] ;

[0016] (3) under the protection of nitrogen, the fluorophore FP-OH is dissolved in dichloromethane, after adding triethylamine, stirring in ice bath, then slowly adding 3,5-bis(trifluoromethyl) benzene-1-sulfonyl chloride, stirring reaction, after the reaction is completed, silica gel column chromatography purification is carried out, and the fluorescent probe FP-ONOO is obtained.

[0017] Preferably, in step (1), the molar ratio of isophorone: malonitrile is 1:0.8-1.5; and the ratio of the isophorone: piperidine: glacial acetic acid is 50 mmol: 0.3-0.6 mL: 0.2-0.5 mL.

[0018] Preferably, in step (1), the reaction is refluxing at 79 DEG C for 6 h under the protection of nitrogen.

[0019] Preferably, in step (2), the molar ratio of compound 1: 3-chloro-4-hydroxybenzaldehyde is 1:0.8-1.5; and the ratio of compound 1 and piperidine is 1 mmol: 80-150 μL.

[0020] Preferably, in step (2), the reaction is refluxing at 82 DEG C for 4 h under the protection of nitrogen; and the silica gel column chromatography purification is carried out by using a mixed solvent of dichloromethane and methanol as an eluent; and the volume ratio of methanol:dichloromethane is 1:150-300.

[0021] Preferably, in step (3), the molar ratio of fluorophore FP-OH: 3,5-bis(trifluoromethyl) benzene-1-sulfonyl chloride is 1:1-3; and the ratio of fluorophore FP-OH and triethylamine is 1 mmol: 0.1-0.3 mL.

[0022] Preferably, in step (3), the stirring reaction is carried out at room temperature for 1 h; and the silica gel column chromatography purification is carried out by using dichloromethane as an eluent.

[0023] The application also provides the use of the fluorescent probe in screening traditional Chinese medicines with anti-atherosclerosis effect.

[0024] The application provides the fluorescent probe, which can be used for high-throughput screening of various traditional Chinese medicine extracts.

[0025] Preferably, the high-throughput screening specifically comprises the following steps.

[0026] Each traditional Chinese medicine extract to be tested is respectively given to zebrafish for incubation, then lipopolysaccharide (LPS) is used to induce the zebrafish to establish an atherosclerosis model, the fluorescence intensity of each group of zebrafish can be visualized on a laser confocal microscope, and the anti-atherosclerosis ability of each traditional Chinese medicine can be evaluated.

[0027] The lower the fluorescence intensity, the better the anti-atherosclerosis effect.

[0028] The reaction route map of the fluorescent probe prepared by the anti-atherosclerosis traditional Chinese medicine screening method is as follows:

[0029] .

[0030] The present application aims to construct an anti-atherosclerosis traditional Chinese medicine screening platform based on fluorescent probe technology, and screen 70 kinds of traditional Chinese medicines. First, a probe for recognizing ONOO- is designed and synthesized as a tool for detecting the content of ROS in zebrafish, then a zebrafish atherosclerosis model is constructed by using lipopolysaccharide induction, and the improvement of different traditional Chinese medicine extracts on atherosclerosis in zebrafish is dynamically monitored by using confocal imaging technology. The present application not only provides an innovative method for efficient screening of anti-atherosclerosis components of traditional Chinese medicine, but also provides potential drug candidates for the prevention and treatment of atherosclerosis.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] (1) The fluorescent probe of the present application has good chemical and light stability, high sensitivity;

[0033] (2) The preparation process of the fluorescent probe of the present application is simple, the materials are easy to obtain, the cost is low, and it is easy to popularize; high-throughput screening is trace, rapid, sensitive and accurate;

[0034] (3) The fluorescent probe of the present application has good anti-interference ability, and its emission spectrum is in the near-infrared region, which can effectively avoid the fluorescence interference of the chemical components of traditional Chinese medicine, improve the detection sensitivity, and has unique advantages in the screening of effective substances of traditional Chinese medicine. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is the nuclear magnetic hydrogen spectrum of the fluorescent group of the present application;

[0036] Figure 2 It is the mass spectrum of the fluorescent group of the present application;

[0037] Figure 3 It is the nuclear magnetic hydrogen spectrum of the fluorescent probe of the present application;

[0038] Figure 4 It is the mass spectrum of the fluorescent probe of the present application;

[0039] Figure 5 It is the ultraviolet-visible absorption spectrum of the fluorescent probe of the present application and ONOO - ;

[0040] Figure 6 It is the fluorescence emission spectrum (A) of the fluorescent probe of the present application and ONOO - ; and the linear relationship diagram of fluorescence intensity and ONOO - concentration (B);

[0041] Figure 7 Fluorescent column chart of the fluorescent probe of the present application and ONOO - and other active substances; wherein, 1-26: control group, Ala, Asp, Cys, Glu, Gly, Lys, Phe, Thr, GSH, Na + , K + , NO3 - , SO4 2- , SO3 2- , HCO3 - , MAO-A, AChE, β-gal, NTR, H2S, CORM-3, H2O2, HClO, O2 - , ONOO - ;

[0042] Figure 8 Fluorescent imaging of zebrafish after incubation with 70 traditional Chinese medicine extracts (a) and color chart of the anti-atherosclerosis ability of 70 different traditional Chinese medicines in the zebrafish model (b), 70 different traditional Chinese medicines (c);

[0043] Figure 9 Zebrafish oil red O staining chart (A) and IOD quantification results chart (B) of lipid accumulation in atherosclerotic plaques in each group;

[0044] Figure 10 The effect of yam extract on the levels of TC (A), TG (B), LDL-C (C) and HDL-C (D) in zebrafish. DETAILED DESCRIPTION

[0045] The technical solutions of the present application will be further explained and described below through specific examples.

[0046] Unless otherwise specified, the raw materials used in the present application are commercially available.

[0047] Example 1 Synthesis of fluorescent probe

[0048] (1) Dissolve isophorone (6.9 g, 50 mmol) in 80.0 mL of ethanol, add malononitrile (3.3 g, 50 mmol), add 0.4 mL of piperidine and 0.3 mL of glacial acetic acid, reflux at 79°C under nitrogen protection for 6 hours, cool to room temperature, add 100 mL of water and stand for the solid to precipitate, filter the mixture, and recrystallize the filter residue twice to obtain a yellow-white solid, which is compound 1 (2-(3,5,5-trimethylcyclohex-2-en-1-ylidene) malononitrile);

[0049] (2) Compound 1 (186 mg, 2 mmol) was dissolved in 15 mL of acetonitrile, 200 μL of piperidine was added, and the reaction was refluxed at 82°C under nitrogen protection for 4 h. After the reaction was completed, the solvent was removed, and the crude product was purified by silica gel column chromatography (methanol: dichloromethane = 1:200, V / V) to obtain orange-red solid fluorophore FP-OH ((E)-2-(3-(3-chloro-4-hydroxy styryl)-5,5-dimethylcyclohex-2-en-1- ylidene)malonitrile), the proton nuclear magnetic resonance spectrum is shown in Figure 1 , and the mass spectrum is shown in Figure 2 .

[0050] (3) The fluorophore FP-OH (324 mg, 1 mmol) was dissolved in 15 mL of dichloromethane solution under nitrogen protection, 0.2 mL of triethylamine was added under ice bath stirring, and the stirring was continued for 30 min. 3,5-bis(trifluoromethyl)benzene-1-sulfonyl chloride (624 mg, 2 mmol) was added and stirred for 30 min, and then the reaction was carried out at room temperature for 1 h. The solvent was removed by reduced pressure distillation, and the crude product was purified by silica gel column chromatography (dichloromethane as eluent) to obtain yellow solid, which was FP-ONOO ((E)-2-chloro-4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl 3,5-bis(trifluoromethyl)benzenesulfonate).

[0051] The chemical structure of the fluorescent probe FP-ONOO is as follows:

[0052] .

[0053] The proton nuclear magnetic resonance spectrum of the fluorescent probe FP-ONOO is shown in Figure 3 , and the mass spectrum is shown in Figure 4 .

[0054] Example 2: Photophysical detection of peroxynitrite ion (ONOO⁻) by the fluorescent probe

[0055] The fluorescent probe FP-ONOO used in the example was prepared in Example 1.

[0056] (1) A PBS buffer solution containing 20% DMSO, PH=7.4, and a concentration of 10 mM was prepared, and a 1 mM fluorescent probe FP-ONOO solution was prepared with DMSO. The blank group: 30 μL of the fluorescent probe FP-ONOO solution (final concentration of 10 μM) was added to 2970 μL of the buffer solution (total volume of 3 mL). The model group: 60 μL of ONOO -(terminal concentration is 100 μM), add 2910 μL of buffer solution, add 30 μL of fluorescent probe FP-ONOO solution (terminal concentration is 10 μM), the total volume of the reaction system is 3 mL. After mixing thoroughly, add constant temperature mixing incubator and incubate for 60 min (incubation temperature is 37℃, shaking speed is 100 r / min). After the reaction is completed, pour the reaction solution into a quartz dish, place it in a sample cell, test the corresponding ultraviolet-visible absorption wavelength, see Figure 5 .

[0057] (2) Prepare PBS buffer solution containing 20% DMSO, PH = 7.4, and the concentration is 10 mM, and prepare 1 mM fluorescent probe FP-ONOO solution with DMSO. Take PBS buffer solution, add different gradient volumes of ONOO - PBS buffer solution (0-100 μM) and 10 μM fluorescent probe FP-ONOO DMSO solution, mix the solution in an EP tube; 37℃ shaking for 40 min, after the reaction, add the solution to a fluorescence cuvette, detect on a fluorescence spectrometer, with the increase of ONOO - concentration, the fluorescence intensity gradually increases, see Figure 6 A and B.

[0058] (3) Prepare PBS buffer solution containing 20% DMSO, PH = 7.4, and the concentration is 10 mM, and prepare 1 mM fluorescent probe FP-ONOO solution with DMSO, add 10 μM fluorescent probe FP-ONOO DMSO solution, and 100 μM various analytes: Ala, Asp, Cys, Glu, Gly, Lys, Phe, Thr, GSH, Na + , K + , NO3 - , SO4 2- , SO3 2- , HCO3 - , H2S, CORM-3, H2O2, HClO, O2 - , ONOO - , and 10 U / mL β-gal, 10 U / mL AChE, 10 U / mL NTR, 30 μg / mL MAO-A, respectively, in an EP tube, 37℃ shaking for 60 min, after the reaction, add the solution to a fluorescence cuvette, detect on a fluorescence spectrometer, ONOO - causes the fluorescence intensity at 640 nm to increase significantly, and other analytes do not cause the fluorescence intensity to change, see Figure 7 .

[0059] Example 3 Screening of fluorescent probe for anti-atherosclerosis traditional Chinese medicine

[0060] In this example, 70 kinds of traditional Chinese medicines such as hawthorn, mint, herba lycopi, rosemary, and water calamus were used to study the anti-atherosclerosis activity of traditional Chinese medicine. Then the screened traditional Chinese medicine was evaluated for its efficacy in improving atherosclerosis. The fluorescent probe FP-ONOO used in this example was prepared in Example 1.

[0061] (1) Preparation of traditional Chinese medicine extract

[0062] Each kind of traditional Chinese medicine (5 g) was accurately weighed, ground into fine particles, and passed through a 40-mesh sieve. Then 80% ethanol (100 mL) was used to extract at room temperature for 60 min, and the extraction was repeated 3 times. After vacuum filtration, the solvent was evaporated. The dried extract was dissolved in DMSO to prepare a 5 mg / mL stock solution for use.

[0063] (2) Anti-atherosclerosis activity determination of 70 kinds of traditional Chinese medicine in zebrafish

[0064] Two-day-old zebrafish embryos were randomly divided into groups, with 10 embryos in each group. The drug administration group was added with 50 μg / mL of different traditional Chinese medicine crude extract in the culture medium (zebrafish E3 culture medium: 0.5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4 dissolved in 600-1000 mL pure water), and the control group and model group were added with the same volume of culture medium, and incubated at 28.5°C for 24 hours. Then the model group and the drug administration group were added with 50 μg / mL of LPS for 48 hours to induce atherosclerosis. The fluorescent probe FP-ONOO 10 μM was used to incubate zebrafish for 30 minutes, and then washed and detected by laser confocal microscope to detect the ONOO - content in zebrafish. The fluorescence intensity was recorded. Image processing software was used to quantitatively analyze the fluorescence intensity of each group of zebrafish, calculate the average fluorescence intensity, and compare with the model group to evaluate the alleviating effect of traditional Chinese medicine extract on atherosclerosis.

[0065] The results showed that many traditional Chinese medicine extracts could significantly reduce the ONOO - content in zebrafish induced by LPS, especially the fluorescence intensity inhibition rate of hawthorn extract was significantly higher than that of other traditional Chinese medicine extracts, indicating that hawthorn extract had potential application value in alleviating oxidative stress-related diseases. Heat map Figure 8b) in FIG. 1, the deeper the red color, the weaker the inhibitory effect, and the closer to white color, the stronger the inhibitory effect. Hawthorn, radix puerariae, and citrus reticulata extract can significantly reduce the fluorescence intensity of zebrafish, indicating that the level of active oxygen in zebrafish is reduced, and atherosclerosis is alleviated. The corresponding column (star mark) of hawthorn is the lightest color (close to white), indicating that its inhibitory effect is the most significant among all tested Chinese medicines. See Figure 8 a, b, and c in FIG. 1.

[0066] Example 4: In vivo pharmacodynamic study of hawthorn extract on improving atherosclerosis

[0067] This example studies the construction of a zebrafish atherosclerosis model to evaluate the improvement of hawthorn extract on zebrafish atherosclerosis. The fluorescent probe FP-ONOO used in the example is prepared in Example 1.

[0068] (1) Inhibitory effect of hawthorn extract on lipid deposition

[0069] In the present application, high-fat diet is used to feed zebrafish to establish a zebrafish AS model. Zebrafish embryos are randomly divided into groups at 5 days of age. The control group (Control) is fed with ordinary zebrafish feed; the model group (HFD), the low, medium, and high dose groups of hawthorn (SZ-L, SZ-M, and SZ-H), and the positive drug group (RVS, rosuvastatin) are fed with feed containing 4% cholesterol, twice a day. The low, medium, and high dose groups of hawthorn are given 2.5, 5, and 10 μg / mL of hawthorn for drug intervention, respectively, and the positive drug group is given 4 μg / mL of rosuvastatin for intervention, for a total of 7 days of feeding.

[0070] 0.3% oil red O staining solution is prepared using 60% isopropanol, filtered, and stored in a refrigerator at 4°C. After the zebrafish are fixed with 4% paraformaldehyde overnight at 4°C, they are soaked and treated with 60% isopropanol on a shaker, then stained with oil red O staining solution for 6 hours. After staining, the zebrafish are rinsed twice with 60% isopropanol and once with PBS. The rinsed zebrafish are transferred to a clean culture dish, and the red-stained area of lipid deposition is observed and recorded under a microscope.

[0071] The results show that after feeding AB wild-type zebrafish larvae with high-cholesterol feed for 7 days, the lipid level of the model group of zebrafish increases significantly. After giving different doses of hawthorn extract, the drug administration group can effectively reduce the lipid level of zebrafish. See Figure 9 A and B in FIG. 2.

[0072] (2) Improvement of hawthorn extract on lipid metabolism

[0073] After the zebrafish larvae are anesthetized with an appropriate amount of tricaine solution, they are transferred to a pre-cooled centrifuge tube, and the excess culture solution is removed as much as possible to keep the sample clean. An appropriate amount of pre-cooled PBS is added at a ratio of 1:10, and the sample is homogenized three times to ensure that the sample is fully lysed. The sample is centrifuged at 12,000 rpm at 4°C for 10 minutes, and the supernatant is collected as the sample to be tested. Commercial biochemical kits for total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) are used for detection, and an appropriate amount of supernatant is added according to the instructions for color development.

[0074] It is found that in the model group, a high-cholesterol diet significantly increases the levels of TC, TG, and LDL-C in the whole body of zebrafish larvae and decreases the level of HDL-C. However, after administration of different doses of hawthorn extract, the levels of TC, TG, and LDL-C are reduced, and the level of HDL-C is restored, indicating that the hawthorn extract has a good lipid-lowering effect on atherosclerotic zebrafish, as shown in A, B, C, and D in Figure 10

[0075] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. For those skilled in the art, other different forms of changes or modifications can be made on the basis of the above description, and it is impossible to exhaust all the embodiments here. Any obvious changes or modifications derived from the technical solutions of the present application are still within the protection scope of the present application.​

Claims

1. A fluorescent probe for screening anti-atherosclerosis traditional Chinese medicine, characterized in that, The structural formula of the fluorescent probe is: 。 2. The method for preparing the fluorescent probe for screening anti-atherosclerosis traditional Chinese medicines according to claim 1, characterized in that, The method comprises the following steps: (1) isophorone and malonitrile are dissolved in ethanol, piperidine and glacial acetic acid are added, and reaction is carried out; after the reaction is completed, water is added to precipitate a solid, and the solid is obtained by filtration and recrystallization to obtain compound 1; The structural formula of the compound 1 is: ； (2) compound 1 and 3-chloro-4-hydroxybenzaldehyde are dissolved in acetonitrile, piperidine is added, and reaction is carried out; after the reaction is completed, silica gel column chromatography is carried out to obtain a fluorescent group FP-OH; The structural formula of the fluorescent group FP-OH is: ； (3) under the protection of nitrogen, the fluorescent group FP-OH is dissolved in dichloromethane, triethylamine is added, and stirring is carried out in an ice bath; then 3,5-bis(trifluoromethyl)benzene-1-sulfonyl chloride is slowly added, and stirring reaction is carried out; after the reaction is completed, silica gel column chromatography is carried out to obtain the fluorescent probe FP-ONOO; The structural formula of the fluorescent probe FP-ONOO is: 。 3. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of isophorone to malonitrile is 1:0.8-1.5; the ratio of the isophorone, piperidine and glacial acetic acid is 50 mmol:0.3-0.6 mL:0.2-0.5 mL.

4. The production method according to claim 3, characterized by, In step (1), the reaction is carried out under the protection of nitrogen, and reflux is carried out at 79 DEG C for 6 h.

5. The preparation method according to claim 2, characterized in that, In step (2), the molar ratio of compound 1 to 3-chloro-4-hydroxybenzaldehyde is 1:0.8-1.5; the ratio of compound 1 and piperidine is 1 mmol:80-150 μL.

6. The production method according to claim 5, wherein In step (2), the reaction is carried out under the protection of nitrogen, and reflux is carried out at 82 DEG C for 4 h; the silica gel column chromatography is carried out by using a mixed solvent of dichloromethane and methanol as an eluent; the volume ratio of methanol to dichloromethane is 1:150-300.

7. The preparation method according to claim 2, characterized in that, In step (3), the molar ratio of the fluorescent group FP-OH to 3,5-bis(trifluoromethyl)benzene-1-sulfonyl chloride is 1:1-3; the ratio of the fluorescent group FP-OH and triethylamine is 1 mmol:0.1-0.3 mL.

8. The preparation method according to claim 7, characterized in that, In step (3), the stirring reaction is carried out at room temperature for 1 h; the silica gel column chromatography is carried out by using dichloromethane as an eluent.

9. The fluorescent probe according to claim 1 is applied to screening traditional Chinese medicines with anti-atherosclerosis.

Images