Fluorescent compound for detecting ursolic acid, and preparation method and application thereof

Abstract

The present application relates to the technical field of fluorescent chemical sensor, in particular to a fluorescent compound for detecting ursolic acid and a preparation method and application thereof. The fluorescent compound for detecting ursolic acid has the structural formula shown in the following formula I: The fluorescent compound of the present application has the property of aggregation-induced emission, can realize rapid and sensitive 'lighting type' response to ursolic acid, and solves the problems of high cost, complex process and long time consumption in the prior art for detecting natural plant compounds.

Description

Technical Field

[0001] This invention relates to the field of fluorescent chemical sensor technology, and in particular to a fluorescent compound for detecting ursolic acid, its preparation method, and its application. Background Technology

[0002] Plant-derived natural compounds have wide applications in medicine, food, and cosmetics. Ursolic acid, a triterpenoid compound, is one such compound and possesses various biological effects, including sedation, anti-inflammation, antibacterial, antidiabetic, anti-ulcer, and hypoglycemic effects. Clinically, it has shown significant and rapid reduction of alanine aminotransferase (ALT) and serum transaminase levels, jaundice relief, appetite enhancement, anti-fibrosis, and liver function restoration. In particular, recent studies have discovered that ursolic acid can significantly inhibit the proliferation of HL-60 cells and induce apoptosis, making it a potentially low-toxicity and effective novel anticancer drug.

[0003] Ursolic acid exists in its free form or as a glycoside bound to sugars in approximately 62 species of plants belonging to 46 genera and 7 families. It is primarily extracted from leaves of privet, bearberry, loquat, and paulownia. The extraction method generally involves refluxing pulverized plant leaves with ethanol to obtain a fluid extract, followed by washing, drying, dissolving, decolorizing, and pH adjustment to obtain a crude ursolic acid extract. This traditional plant extraction method suffers from resource scarcity, reliance on arable land, and long extraction cycles, making it difficult to meet the growing health needs of the population. In recent years, the synthesis of natural plant compounds using microbial cell factories has become a key competitive area of ​​investment for many countries. Due to the superior properties of pentacyclic triterpenoids, including ursolic acid, they have become the primary target for cell factory synthesis of natural plant compounds. However, rapid, sensitive, and specific detection of these compounds remains a challenge and difficulty in cell factory synthesis.

[0004] Existing methods for detecting natural plant compounds, such as liquid chromatography-mass spectrometry (LC-MS), require large, expensive, complex, and time-consuming equipment, and cannot achieve real-time tracking and monitoring in cell factories. Currently, fluorescent probes are attracting increasing attention due to their various advantages. Designing and synthesizing fluorescent probes that specifically illuminate natural plant compounds has become the most promising method for detection. Constructing technologies for real-time monitoring of key compounds and dynamic metabolic regulation based on induced aggregation-luminescence (AIE), as well as high-throughput screening techniques for high-yield strains, are the fastest and most sensitive methods.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] One object of the present invention is to provide a fluorescent compound for the detection of ursolic acid, which can specifically detect ursolic acid.

[0007] Another object of the present invention is to provide a method for preparing a fluorescent compound for detecting ursolic acid.

[0008] Another object of the present invention is to provide an application for the detection of fluorescent compounds of ursolic acid.

[0009] To achieve the above-mentioned objectives of the present invention, one aspect of the present invention provides a fluorescent compound for detecting ursolic acid, having the structural formula shown in Formula I:

[0010]

[0011] Another aspect of the present invention provides a method for preparing the above-mentioned fluorescent compound for detecting ursolic acid, comprising the following steps:

[0012] (a) 4-Ethylaniline, 4-pyridinecarboxaldehyde and catalyst were mixed in a solvent and heated. Then, 2,3-butanedione was added and reacted to obtain the first intermediate.

[0013] (b) The first intermediate is reacted with iodomethane in a solvent in the dark to obtain the second intermediate;

[0014] (c) The second intermediate and methyl trifluoromethanesulfonate were reacted in a solvent and stirred in the dark. The reaction solution was then added to ethyl acetate, and the precipitate was collected to obtain the fluorescent compound used for the detection of ursolic acid.

[0015] In a specific embodiment of the present invention, in step (a), the molar ratio of 4-ethylaniline to 4-pyridinecarboxaldehyde is 1:(0.8 to 1.2); the molar ratio of 4-ethylaniline to 2,3-butanedione is 1:(0.4 to 0.6).

[0016] In a specific embodiment of the present invention, in step (a), the temperature is raised to 80–100°C during the mixing and heating process. Further, after adding 2,3-butanedione, the reaction is carried out at 80–100°C for 2–4 hours.

[0017] In a specific embodiment of the present invention, in step (a), the catalyst is p-toluenesulfonic acid and the solvent is glacial acetic acid.

[0018] In a specific embodiment of the present invention, in step (b), the molar ratio of the amount of iodomethane to the amount of 4-ethylaniline is (1.5~2):1.

[0019] In a specific embodiment of the present invention, in step (b), the light-protected reaction is carried out at room temperature. Further, the light-protected reaction time is 20–30 hours.

[0020] In a specific embodiment of the present invention, in step (b), the solvent is dichloromethane.

[0021] In a specific embodiment of the present invention, the molar ratio of the second intermediate to the methyl trifluoromethanesulfonate is 1:(0.4 to 0.5).

[0022] In a specific embodiment of the present invention, in step (c), the light-protected stirring reaction is carried out at room temperature. Further, the light-protected stirring reaction time is 10–15 hours.

[0023] In a specific embodiment of the present invention, in step (c), the solvent is methanol.

[0024] In another aspect, this invention provides the application of any of the fluorescent compounds described above for detecting ursolic acid in the detection of ursolic acid, wherein the detection method includes:

[0025] (A) Add the fluorescent probe and the solution containing the natural compound of the plant to be tested to a good solvent of the fluorescent compound, and mix them evenly to obtain the test solution;

[0026] (B) Perform fluorescence detection on the test solution and analyze the obtained fluorescence spectrum;

[0027] The fluorescent probe comprises the fluorescent compound and a good solvent for the fluorescent compound.

[0028] In a specific embodiment of the present invention, the detection method further includes: performing fluorescence detection on the control solution under the same detection conditions; the preparation of the control solution includes: adding the fluorescent probe to the good solvent and mixing evenly.

[0029] In a specific embodiment of the present invention, the analysis includes: obtaining the emission wavelength λ of the test solution and the control solution at the maximum emission wavelength λ. m Given the fluorescence emission intensities I and I0, calculate the fluorescence intensity change value I / I0 or (I-I0) / I0.

[0030] In a specific embodiment of the present invention, in step (A), the concentration of the fluorescent compound in the test solution is 10. -4 ~10 -6 The concentration of the plant natural compound is 10 mol / L. -4 ~10 -6 mol / L. Further, the volume percentage of the good solvent in the test solution is 95%–99.99%.

[0031] In a specific embodiment of the present invention, the good solvent for the fluorescent compound includes at least one of water, PBS buffer, and SD culture medium.

[0032] In a specific embodiment of the present invention, the concentration of the natural plant compound to be tested in the solution is 10. -3 ~10 -6 mol / L. Further, the solvent in the solution containing the natural plant compound to be tested is dimethyl sulfoxide.

[0033] In a specific embodiment of the present invention, the concentration of the fluorescent compound in the fluorescent probe is 10. -3 ~10 -6 mol / L.

[0034] In a specific embodiment of the present invention, the natural plant compound to be tested includes at least one of ursolic acid, glycyrrhizin, arbutin, mogroside, echinacoside, gallic acid, 5-hydroxytryptophan, icariin, 4-hydroxycoumarin, glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizin and tocopherol.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] (1) The fluorescent compound of the present invention has aggregation-induced emission properties and can achieve a rapid and sensitive "light-up" response to ursolic acid, which solves the problems of high cost, complex process and long time consumption in the detection of natural plant compounds by existing methods;

[0037] (2) The detection method of the present invention can specifically and in real time detect ursolic acid in glycyrrhizin, arbutin, mogroside, echinacoside, gallic acid, 5-hydroxytryptophan, icariin, ursolic acid, 4-hydroxycoumarin, glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizin, and tocopherol, which is of great significance to the development of the synthesis of natural plant compounds. Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 The second intermediate provided in the embodiments of the present invention in CD3OD 1 H NMR spectrum;

[0040] Figure 2 The second intermediate provided in the embodiments of the present invention in CD3OD 13 C NMR spectrum;

[0041] Figure 3The ESI-Ms spectrum (cation) of the second intermediate provided in the embodiments of the present invention;

[0042] Figure 4 The ESI-Ms spectrum (anion) of the second intermediate provided in the embodiments of the present invention;

[0043] Figure 5 ESI-Ms spectra (cations) of fluorescent compounds provided in embodiments of the present invention;

[0044] Figure 6 ESI-Ms spectra (anions) of fluorescent compounds provided in embodiments of the present invention;

[0045] Figure 7 The image shows the ultraviolet absorption spectrum of the fluorescent compound in aqueous solution provided in Example 1 of this invention. The concentration of the fluorescent compound is 1 × 10⁻⁶. -5 mol / L;

[0046] Figure 8 The fluorescence spectrum of the fluorescent compound provided in Example 1 of this invention in aqueous solution, wherein the concentration of the fluorescent compound is 1 × 10⁻⁶. -5 mol / L, excitation wavelength 460 nm;

[0047] Figure 9 The fluorescence spectra of the fluorescent compound provided in Example 1 of this invention in Glycerin / PBS mixed solvents with different glycerol contents are shown. The concentration of the fluorescent compound is 1×10⁻⁶. -5 mol / L, excitation wavelength 460 nm;

[0048] Figure 10 for Figure 9 The fluorescence intensity of the fluorescent compound changes with the Glycerin content (vol.%), where I0 is the corresponding fluorescence intensity in pure PBS solvent;

[0049] Figure 11 The fluorescence spectrum of the test solution obtained by mixing the fluorescent compound provided in Example 2 of this invention with different natural plant compounds;

[0050] Figure 12 for Figure 11 A bar chart showing the fluorescence intensity ratio (I / I0) of different natural plant compounds.

[0051] Figure 13 The image shows the fluorescence response spectra of the fluorescent compound provided in Example 3 of this invention to different concentrations of ursolic acid in PBS. The concentration of the fluorescent compound is 1 × 10⁻⁶. -5 The concentration of ursolic acid is 1 × 10 mol / L. -6 ~1×10 -5mol / L, excitation wavelength 460 nm;

[0052] Figure 14 for Figure 13 Graph showing the relationship between fluorescence intensity enhancement ratio (I-I0) / I0 and ursolic acid concentration;

[0053] Figure 15 The image shows the fluorescence response spectra of the fluorescent compound provided in Example 5 of this invention to different concentrations of ursolic acid in SD medium. The concentration of the fluorescent compound is 1×10⁻⁶. -5 The concentration of ursolic acid is 1 × 10 mol / L. -6 ~1×10 -5 mol / L, excitation wavelength 460 nm;

[0054] Figure 16 for Figure 15 Graph showing the relationship between fluorescence intensity enhancement ratio (I-I0) / I0 and ursolic acid concentration;

[0055] Figure 17 Fluorescence spectra of ursolic acid added to a fluorescent probe at different times, as provided in an embodiment of the present invention. Detailed Implementation

[0056] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0057] This invention provides a fluorescent compound for detecting ursolic acid, having the structural formula shown in Formula I:

[0058]

[0059] The fluorescent compound of this invention has aggregation-induced emission properties, which can achieve a rapid and sensitive "light-up" response to ursolic acid, solving the problems of high cost, complex process and long time consumption in the detection of natural plant compounds by existing methods.

[0060] Another aspect of the present invention provides a method for preparing the above-mentioned fluorescent compound for detecting ursolic acid, comprising the following steps:

[0061] (a) 4-Ethylaniline, 4-pyridinecarboxaldehyde and catalyst were mixed in a solvent and heated. Then, 2,3-butanedione was added and reacted to obtain the first intermediate.

[0062] (b) The first intermediate was reacted with iodomethane in a solvent in the dark to obtain the second intermediate;

[0063] (c) The second intermediate and methyl trifluoromethanesulfonate were reacted in a solvent and stirred in the dark. The reaction solution was then added to ethyl acetate, and the precipitate was collected to obtain a fluorescent compound for the detection of ursolic acid.

[0064] The first intermediate and the second intermediate have the structural formulas shown in equations II and III, respectively:

[0065]

[0066] The synthetic route for the fluorescent compound of this invention can be found as follows:

[0067]

[0068] In practice, step (a) may include: mixing 4-ethylaniline, 4-pyridinecarboxaldehyde, and the catalyst in a solvent, heating the mixture to 80–100°C, and stirring for 30–35 minutes. Then, slowly adding 2,3-butanedione into the reaction system using a syringe, and continuing to stir the reaction for 2–4 hours after the addition is complete. After the reaction is complete, the solvent is removed by rotary evaporation to obtain a black oily substance, which includes the first intermediate. As an intermediate, it can be used directly for subsequent reactions without purification.

[0069] In practice, step (b) may include: dissolving the black oily substance in a solvent, adding iodomethane, stirring at room temperature in the dark for 20-30 hours, removing the solvent by rotary evaporation to obtain the crude product; and then recrystallizing the product at least once with a mixed solvent of methanol and ethyl acetate to obtain the golden yellow second intermediate.

[0070] In practice, step (c) may include: mixing the second intermediate, methyl trifluoromethanesulfonate and solvent evenly, stirring and reacting at room temperature in the dark for 10-15 hours, then adding the reaction liquid dropwise to ethyl acetate to generate a yellow precipitate, collecting the filter cake by suction filtration, and drying to obtain the fluorescent compound used for detecting ursolic acid.

[0071] In a specific embodiment of the present invention, in step (a), the molar ratio of 4-ethylaniline to 4-pyridinecarboxaldehyde is 1:(0.8 to 1.2); the molar ratio of 4-ethylaniline to 2,3-butanedione is 1:(0.4 to 0.6).

[0072] In different embodiments, in step (a), the molar ratio of 4-ethylaniline to 4-pyridinecarboxaldehyde can be a range of 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, or any two of these; and the molar ratio of 4-ethylaniline to 2,3-butanedione can be a range of 1:0.4, 1:0.45, 1:0.5, 1:0.55, 1:0.6, or any two of these.

[0073] In a specific embodiment of the present invention, in step (a), the temperature is raised to 80–100°C during the mixing and heating process. Further, after adding 2,3-butanedione, the reaction is carried out at 80–100°C for 2–4 hours.

[0074] In different embodiments, the temperature can be raised to 80°C, 85°C, 90°C, 95°C, 100°C, or any combination thereof. After adding 2,3-butanedione, the reaction time at 80–100°C can be 2 hours, 3 hours, 4 hours, etc., and the specific reaction time can be adjusted according to the reaction progress monitored by TIL.

[0075] In a specific embodiment of the present invention, in step (a), the catalyst is p-toluenesulfonic acid and the solvent is glacial acetic acid. The amounts of both the catalyst and the solvent are conventional; for example, the amount of catalyst can be 10 mol% of the raw material 4-ethylaniline.

[0076] In a specific embodiment of the present invention, in step (b), the molar ratio of iodomethane to 4-ethylaniline is (1.5-2):1.

[0077] In different embodiments, the molar ratio of iodomethane to 4-ethylaniline can be 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, or any combination thereof.

[0078] In a specific embodiment of the present invention, in step (b), the light-protected reaction is carried out at room temperature. Further, the light-protected reaction time is 20–30 hours.

[0079] In a specific embodiment of the present invention, in step (b), the solvent is dichloromethane.

[0080] In a specific embodiment of the present invention, the molar ratio of the second intermediate to methyl trifluoromethanesulfonate is 1:(0.4 to 0.5).

[0081] In different embodiments, the molar ratio of the second intermediate to methyl trifluoromethanesulfonate can be 1:0.4, 1:0.42, 1:0.45, 1:0.48, 1:0.5, or any combination thereof.

[0082] In a specific embodiment of the present invention, in step (c), the reaction is carried out at room temperature with stirring in the dark. Further, the reaction time with stirring in the dark is 10–15 h.

[0083] In a specific embodiment of the present invention, in step (c), the solvent is methanol.

[0084] Another aspect of this invention provides the application of any of the above-mentioned fluorescent compounds for detecting ursolic acid in the detection of ursolic acid, wherein the detection method includes:

[0085] (A) Add the fluorescent probe and the solution containing the natural compound of the plant to be tested to a good solvent of the fluorescent compound, and mix them evenly to obtain the test solution;

[0086] (B) Perform fluorescence detection on the test solution and analyze the obtained fluorescence spectrum;

[0087] Fluorescent probes consist of fluorescent compounds and good solvents for those compounds.

[0088] In the detection method of the present invention, ursolic acid forms aggregated particles in a good solvent of fluorescent compounds. The fluorescent compounds are adsorbed on the surface of the ursolic acid aggregated particles based on hydrophobic interactions, which restricts the intramolecular movement of the fluorescent compounds, resulting in a significant increase in the fluorescence intensity of the system.

[0089] In a specific embodiment of the present invention, the detection method further includes: performing fluorescence detection on the control solution under the same detection conditions; the preparation of the control solution includes: adding the fluorescent probe to a good solvent and mixing them evenly.

[0090] The control solution was prepared in the same manner as the test solution, except that the solution containing the natural compound of the plant to be tested was replaced with an equal volume of the solvent used in the solution containing the natural compound of the plant to be tested, i.e., a blank solvent without the natural compound of the plant to be tested. Subsequent preparations followed the same method.

[0091] In a specific embodiment of the present invention, the analysis includes: obtaining the emission wavelengths of the test solution and the control solution at the maximum emission wavelength λ. m Given the fluorescence emission intensities I and I0, calculate the fluorescence intensity change value I / I0 or (I-I0) / I0.

[0092] Among them, the maximum emission wavelength λ m It can be any value in the range of 520 to 560 nm.

[0093] In a specific embodiment of the present invention, in step (A), the concentration of the fluorescent compound in the test solution is 10. -4 ~10 -6 mol / L, the concentration of plant natural compounds is 10 -4 ~10-6 mol / L. Furthermore, the volume percentage of good solvent in the test solution is 95%–99.99%.

[0094] In different embodiments, in step (A), the concentration of the fluorescent compound in the test solution can be 10. -4 mol / L, 10 -5 mol / L, 2×10 -5 mol / L, 5×10 -5 mol / L, 8×10 -5 mol / L, 10 -6 mol / L, 2×10 -6 mol / L, 5×10 - 6 mol / L, 8×10 -6 The concentration of plant natural compounds can be in the range of mol / L or any combination thereof; the concentration can be 10 mol / L. -4 mol / L, 10 -5 mol / L, 2×10 -5 mol / L, 5×10 -5 mol / L, 8×10 -5 mol / L, 10 -6 mol / L, 2×10 -6 mol / L, 5×10 - 6 mol / L, 8×10 -6 The range of mol / L or any combination thereof.

[0095] In different implementations, the volume percentage of good solvent in the test solution can be 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or any combination thereof.

[0096] In a specific embodiment of the present invention, a good solvent for the fluorescent compound includes at least one of water, PBS buffer, and SD culture medium.

[0097] The fluorescent compound of this invention can be used to detect ursolic acid not only in water and PBS buffer, but also in SD medium system, enabling the tracking and real-time monitoring of ursolic acid synthesis in cell factories, especially the detection of ursolic acid content, which can be used for screening high-yield strains.

[0098] In a specific embodiment of the present invention, the concentration of the natural plant compound to be tested in the solution is 10. -3 ~10 -6mol / L. Furthermore, in the solution containing the natural compound of the plant to be tested, the solvent is dimethyl sulfoxide.

[0099] In different implementation methods, the concentration of the target plant natural compound in the solution can be 10. -3 mol / L, 10 -4 mol / L, 2×10 -4 mol / L, 5×10 -4 mol / L, 8×10 -4 mol / L, 10 -5 mol / L, 2×10 -5 mol / L, 5×10 -5 mol / L, 8×10 -5 mol / L, 10 -6 The range of mol / L or any combination thereof.

[0100] In a specific embodiment of the present invention, the concentration of the fluorescent compound in the fluorescent probe is 10. -3 ~10 -6 mol / L.

[0101] In different embodiments, the concentration of the fluorescent compound in the fluorescent probe can be 10. -3 mol / L, 10 -4 mol / L, 2×10 -4 mol / L, 5×10 -4 mol / L, 8×10 -4 mol / L, 10 -5 mol / L, 2×10 -5 mol / L, 5×10 -5 mol / L, 8×10 -5 mol / L, 10 -6 mol / L, 2×10 -6 mol / L, 5×10 -6 mol / L, 8×10 -6 The range of mol / L or any combination thereof.

[0102] In a specific embodiment of the present invention, the natural plant compound to be tested includes at least one of ursolic acid, glycyrrhizin, arbutin, mogroside, echinacoside, gallic acid, 5-hydroxytryptophan, icariin, 4-hydroxycoumarin, glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizin and tocopherol.

[0103] The detection method of the present invention can specifically detect ursolic acid in ursolic acid, glycyrrhizin, arbutin, mogroside, echinacoside, gallic acid, 5-hydroxytryptophan, icariin, 4-hydroxycoumarin, glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizin and tocopherol using a fluorescent probe.

[0104] In a specific embodiment of the present invention, the detection method further includes: detecting the content of ursolic acid.

[0105] In a specific embodiment of the present invention, the detection of ursolic acid content includes: preparing test solutions containing different concentrations of ursolic acid, performing fluorescence detection under the same conditions, and recording the results at the maximum emission wavelength λ. m The fluorescence emission intensity was measured; a standard curve was plotted with the concentration of ursolic acid in the test solution as the abscissa and (I-I0) / I0 as the ordinate; the maximum emission wavelength λ was obtained based on the fluorescence spectrum of the target test solution. m The concentration of ursolic acid in the target test solution was calculated by substituting the fluorescence emission intensity into the standard curve.

[0106] Example 1

[0107] This embodiment provides a fluorescent compound for detecting ursolic acid, having the structural formula shown in Formula I below:

[0108]

[0109] The method for preparing the fluorescent compound for detecting ursolic acid in this embodiment includes the following steps:

[0110] (1) 4-Ethylaniline (0.02 mol, 2.42 g), 4-pyridinecarboxaldehyde (0.02 mol, 2.14 g), p-toluenesulfonic acid (0.002 mol, 0.34 g), and glacial acetic acid (100 mL) were added sequentially to a 250 mL round-bottom flask equipped with a magnetic stirrer. The mixture was heated to 90 °C and stirred for 30 min. Then, 2,3-butanedione (0.01 mol, 0.86 g) was slowly added to the reaction solution using a syringe. After the addition was complete, stirring was continued for 3 h. No precipitate was formed in the reaction solution. A black oily substance was obtained after removing the solvent by rotary evaporation.

[0111] (2) The black oily substance obtained in step (1) was dissolved in 50 mL of CH2Cl2, and then excess iodomethane (0.035 mol, 5 g) was added. The mixture was stirred at room temperature in the dark for 24 h. After the reaction was completed, the solvent was evaporated to dryness, and the crude product was recrystallized from a mixed solvent of methanol and ethyl acetate to obtain a golden product. The golden product was further recrystallized from the mixed solvent of methanol and ethyl acetate to finally obtain 0.39 g of the pure second intermediate product.

[0112] (3) Add the second intermediate product (0.08 mmol, 50 mg), methyl trifluoromethanesulfonate (0.32 mmol, 52.51 mg) and 7 mL of methanol to a test tube equipped with a magnetic stirrer. Stir the reaction solution at room temperature in the dark for 12 h. Then add the reaction solution dropwise to ethyl acetate to obtain a yellow precipitate. Filter under reduced pressure and collect the filter cake. After drying, the final product of 55.46 mg is obtained.

[0113] Figures 1-4 Characterization results for the second intermediate: 1 H NMR (400MHz, CD3OD, ppm): δ8.52-8.50(m,4H),7.72-7.70(m,4H),7.48-7.46(m,4H),7.40- 7.38(m,4H),7.20(s,2H),4.22(s,6H),2.81-2.75(q,J=8Hz,4H),1.34-1.30(t,J=8Hz,6H); 13 C NMR(101MHz,CD3OD)δ144.06,129.60,125.78,123.02,100.40,46.15,28.07,14.62; ESI-Positive(m / z)calcd.forC 34 H 34 N4 2+ [M] 2+ :249.14,found:249.14,ESI-Negative(m / z)calcd.for I - [M] - :126.90,found:126.86.

[0114] Figures 5-6 Characterization results for the final product: ESI-Positive(m / z)calcd.for C 34 H 34 N4 2+ [M] 2+ :249.14,found:249.14,ESI-Negative(m / z)calcd.for CF3SO3 - [M] - :148.95,found:148.91.

[0115] according to Figures 1-6 Characterization results of the second intermediate and the final product show that the target fluorescent compound I was synthesized in this embodiment.

[0116] Figure 7The ultraviolet absorption spectrum of the fluorescent compound in aqueous solution provided in the embodiments of the present invention Figure 8 The fluorescence spectrum of the fluorescent compound in aqueous solution provided in the embodiments of the present invention.

[0117] The aggregation-induced emission properties of fluorescent compound I in a water and glycerol system were verified. The test results are shown in [Figure number missing]. Figure 9 and Figure 10 This demonstrates that the fluorescent compounds of the present invention possess significant AIE properties.

[0118] Example 2

[0119] This embodiment provides a detection method for ursolic acid, comprising the following steps:

[0120] (1) Weigh 3.98 mg of fluorescent compound I and add 5 mL of PBS buffer to prepare a solution with a concentration of 1 × 10⁻⁶. -3 A solution of mol / L is named a fluorescent probe.

[0121] (2) Weigh out the corresponding mass of each of the plant natural compounds to be tested (ursolic acid, glycyrrhizin, arbutin, mogroside, echinacoside, gallic acid, 5-hydroxytryptophan, icariin, 4-hydroxycoumarin, glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizin, and tocopherol) and dissolve them in DMSO solution to prepare a solution with a concentration of 1×10⁻⁶. -3 A DMSO solution of a plant natural compound at a concentration of mol / L; taking ursolic acid as an example, weigh 2.28 mg of ursolic acid, add 5 mL of DMSO, and prepare a solution with a concentration of 1×10⁻⁶ mol / L. -3 A solution with a concentration of mol / L is named ursolic acid solution.

[0122] (3) Take 14 sample bottles and label them 1 to 14. Add 0.99 mL of PBS buffer to sample bottle 1 and add 0.98 mL of PBS buffer to the other sample bottles.

[0123] Add 10 μL of fluorescent probe to each of the 14 sample vials sequentially. Add 10 μL of DMSO solution of the 13 plant natural compounds from step (2) to each of the 14 sample vials. The concentration of fluorescent compound I in each sample vial is 1 × 10⁻⁶. -5 mol / L, except for sample vial #1, the concentration of plant natural compounds in all other sample vials was 1×10 mol / L. -5 mol / L.

[0124] (4) Perform fluorescence emission spectroscopy tests on the solutions in sample bottles 1-14. The excitation wavelength is 460 nm. The fluorescence intensity of sample bottle 1 is taken as I0, and the fluorescence intensity of the other sample bottles is taken as I. The I / I0 ratio reflects the fluorescence response of fluorescent compound I to different plant natural compounds, thus obtaining the response spectra of fluorescent compound I to different plant natural compounds. Figures 11-12 As shown in the figure, the fluorescence intensity of the mixed solution of fluorescent compound I and ursolic acid is significantly enhanced compared to sample bottle 1, while the fluorescence intensity of the solution containing other plant natural compounds does not change significantly compared to sample bottle 1. Therefore, the fluorescent probe of this invention can be used to specifically detect ursolic acid in a variety of plant natural compounds.

[0125] Example 3

[0126] This embodiment provides a method for calculating the detection limit of a fluorescent compound for ursolic acid, including the following steps:

[0127] Weigh 3.98 mg of fluorescent compound I and dissolve it in 5 mL of PBS buffer to prepare a concentration of 1 × 10⁻⁶. -3 A 1 mol / L solution of fluorescent compound I was prepared in PBS and then diluted with PBS buffer to a concentration of 1 × 10⁻⁶. -5 A PBS solution of fluorescent compound I at a concentration of mol / L; 2.28 mg of ursolic acid was dissolved in 50 mL of DMSO to prepare a solution with a concentration of 1×10⁻⁶. -4 A 1 mol / L ursolic acid solution. Take 1 mL of a 1×10 mol / L solution. -5 Fluorescence emission spectroscopy was performed on a PBS solution of fluorescent compound I at a concentration of mol / L, followed by the addition of 1×10⁻⁶ mol / L solution dropwise using a pipette. -4 A mol / L ursolic acid solution was prepared and fluorescence emission spectroscopy was performed (e.g., Figure 13 As shown in the figure, the volume of ursolic acid solution added each time was fixed at 10 μL. The above operation was repeated three times, and then the average fluorescence intensity was used as the ordinate, and the concentration of ursolic acid in the tested solution was used as the abscissa (left graph) to determine the linear relationship between ursolic acid concentration and fluorescence intensity.

[0128] The detection limit was then calculated using the formula 3σ / k, where σ is the standard deviation of the lowest response value during testing, and k is the slope of the linear relationship between ursolic acid concentration and fluorescence intensity. The relationship between the fluorescence intensity enhancement ratio of fluorescent compound I in response to ursolic acid and its concentration is shown in the figure. Figure 14 As shown, the detection limit of fluorescent compound I for ursolic acid in PBS buffer is 1.9 × 10⁻⁶. -7 mol / L (86.77 μg / L).

[0129] Example 4

[0130] This embodiment provides a detection method for ursolic acid, comprising the following steps:

[0131] (1) Weigh 3.98 mg of fluorescent compound I and add it to 5 mL of SD culture medium to prepare a solution with a concentration of 1×10⁻⁶. -3 A solution of mol / L is named a fluorescent probe.

[0132] (2) Weigh out the corresponding mass of each of the plant natural compounds to be tested (ursolic acid, glycyrrhizin, arbutin, mogroside, echinacoside, gallic acid, 5-hydroxytryptophan, icariin, 4-hydroxycoumarin, glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizin, and tocopherol) and dissolve them in DMSO solution to prepare a solution with a concentration of 1×10⁻⁶. -3 A DMSO solution of a plant natural compound at a concentration of mol / L; taking ursolic acid as an example, weigh 2.28 mg of ursolic acid, add 5 mL of DMSO, and prepare a solution with a concentration of 1×10⁻⁶ mol / L. -3 A solution with a concentration of mol / L is named ursolic acid solution.

[0133] (3) Take 14 sample bottles and label them 1 to 14. Add 0.99 mL of SD culture medium solution to sample bottle 1 and add 0.98 mL of SD culture medium solution to the other sample bottles.

[0134] Add 10 μL of fluorescent probe to each of the 14 sample vials sequentially. Add 10 μL of DMSO solution of the 13 plant natural compounds from step (2) to each of the 14 sample vials. The concentration of fluorescent compound I in each sample vial is 1 × 10⁻⁶. -5 mol / L, except for sample vial #1, the concentration of plant natural compounds in all other sample vials was 1×10 mol / L. -5 mol / L.

[0135] (4) Fluorescence emission spectroscopy was performed on the solutions in sample vials 1-14, with an excitation wavelength of 460 nm. The fluorescence intensity of sample vial 1 was taken as I0, and the fluorescence intensity of the other sample vials was taken as I. The fluorescence response of fluorescent compound I to different plant natural compounds was represented by (I-I0) / I0, thus obtaining the response spectra of fluorescent compound I to different plant natural compounds. The fluorescent probe of this invention can specifically detect ursolic acid in a variety of plant natural compounds in an SD culture medium system.

[0136] Example 5

[0137] This embodiment provides a method for calculating the detection limit of a fluorescent compound for ursolic acid, including the following steps:

[0138] Weigh 3.98 mg of fluorescent compound I and dissolve it in 5 mL of SD medium to prepare a concentration of 1 × 10⁻⁶.-3 A solution of fluorescent compound I at mol / L in SD medium was prepared and then diluted with SD medium solution to a concentration of 1×10⁻⁶. -5 A solution of fluorescent compound I at mol / L in SD medium; 2.28 mg of ursolic acid was dissolved in 50 mL of DMSO to prepare a solution with a concentration of 1×10⁻⁶. -4 A 1 mol / L ursolic acid solution. Take 1 mL of a 1×10 mol / L solution. -5 Fluorescence emission spectroscopy was performed on a solution of fluorescent compound I in SD medium at a concentration of mol / L. Subsequently, 1×10⁻⁶ mol / L was added dropwise to the solution using a pipette. -4 A mol / L ursolic acid solution was prepared and fluorescence emission spectroscopy was performed (e.g., Figure 15 As shown in the figure, the volume of ursolic acid solution added each time was fixed at 10 μL. The above operation was repeated three times, and then the average fluorescence intensity was used as the ordinate, and the concentration of ursolic acid in the tested solution was used as the abscissa (left graph) to determine the linear relationship between ursolic acid concentration and fluorescence intensity.

[0139] The detection limit was then calculated using the formula 3σ / k, where σ is the standard deviation of the lowest response value during testing, and k is the slope of the linear relationship between ursolic acid concentration and fluorescence intensity. The relationship between the fluorescence intensity enhancement ratio of fluorescent compound I in response to ursolic acid and its concentration is shown in the figure. Figure 16 As shown, the detection limit for ursolic acid by fluorescent compound I in SD culture medium was 6.2 × 10⁻⁶. -7 mol / L (283.15 μg / L).

[0140] Example 6

[0141] This embodiment provides a method for detecting the response rate of ursolic acid, including the following steps:

[0142] Weigh 3.98 mg of fluorescent compound I and dissolve it in 5 mL of PBS buffer to prepare a concentration of 1 × 10⁻⁶. -3 A 1 mol / L solution of fluorescent compound I was prepared in PBS and then diluted with PBS buffer to a concentration of 1 × 10⁻⁶. -5 A PBS solution of fluorescent compound I at a concentration of mol / L; 2.28 mg of ursolic acid was dissolved in 5 mL of DMSO to prepare a solution with a concentration of 1×10⁻⁶ mol / L. -3 A 1 mol / L ursolic acid solution. Take 1 mL of a 1×10 mol / L solution. -5 Fluorescence emission spectroscopy was performed on a PBS solution containing 1 mol / L of fluorescent compound I. Subsequently, 10 μL of ursolic acid solution was added dropwise using a pipette, and fluorescence emission spectra were measured at 5 min, 10 min, 15 min, 20 min, and 30 min after the addition. The results are as follows: Figure 17 As shown. From Figure 17It can be seen that the fluorescence emission spectra tested at various time points after the addition did not change, indicating that the fluorescence response of fluorescent compound I to ursolic acid is instantaneous.

[0143] As can be seen from the above, the fluorescent compound I of the present invention can specifically and in real time detect ursolic acid in glycyrrhizin, arbutin, mogroside, echinacoside, gallic acid, 5-hydroxytryptophan, icariin, ursolic acid, 4-hydroxycoumarin, glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizin, and tocopherol, and can also achieve quantitative detection of ursolic acid, which is of great significance to the development of the synthesis of natural plant compounds.

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fluorescent compound for detecting ursolic acid, characterized in that, It has the following structural formula as shown in Equation I: 。 2. The method for preparing the fluorescent compound for detecting ursolic acid according to claim 1, characterized in that, Includes the following steps: (a) 4-Ethylaniline, 4-pyridinecarboxaldehyde and catalyst were mixed in a solvent and heated. Then, 2,3-butanedione was added and reacted to obtain the first intermediate. (b) The first intermediate is reacted with iodomethane in a solvent in the dark to obtain the second intermediate; (c) The second intermediate and methyl trifluoromethanesulfonate were reacted in a solvent and stirred in the dark. The reaction solution was then added to ethyl acetate, and the precipitate was collected to obtain the fluorescent compound used for the detection of ursolic acid.

3. The preparation method according to claim 2, characterized in that, In step (a), the molar ratio of 4-ethylaniline to 4-pyridinecarboxaldehyde is 1:(0.8 to 1.2); the molar ratio of 4-ethylaniline to 2,3-butanedione is 1:(0.4 to 0.6).

4. The preparation method according to claim 3, characterized in that, In step (a), the temperature is raised to 80-100°C during the mixing and heating process.

5. The preparation method according to claim 3, characterized in that, In step (a), after adding 2,3-butanedione, the reaction is carried out at 80-100°C for 2-4 hours; In step (a), the catalyst is p-toluenesulfonic acid and the solvent is glacial acetic acid.

6. The preparation method according to claim 2, characterized in that, In step (b), the molar ratio of the amount of iodomethane to the amount of 4-ethylaniline is (1.5~2):

1.

7. The preparation method according to claim 6, characterized in that, In step (b), the light-protected reaction is carried out at room temperature.

8. The preparation method according to claim 6, characterized in that, The light-protected reaction takes 20–30 hours.

9. The preparation method according to claim 6, characterized in that, In step (b), the solvent is dichloromethane.

10. The preparation method according to claim 2, characterized in that, The molar ratio of the second intermediate to the methyl trifluoromethanesulfonate is 1:(0.4 to 0.5).

11. The preparation method according to claim 10, characterized in that, In step (c), the light-protected stirring reaction is carried out at room temperature.

12. The preparation method according to claim 10, characterized in that, The reaction time, involving stirring in the dark, is 10–15 hours.

13. The preparation method according to claim 10, characterized in that, In step (c), the solvent is methanol.

14. The application of the fluorescent compound for detecting ursolic acid according to claim 1 in the detection of ursolic acid, characterized in that, The detection method includes: (A) Add the fluorescent probe and the solution containing the natural compound of the plant to be tested to a good solvent of the fluorescent compound, and mix them evenly to obtain the test solution; (B) Perform fluorescence detection on the test solution and analyze the obtained fluorescence spectrum; The fluorescent probe comprises the fluorescent compound and a good solvent for the fluorescent compound.

15. The application according to claim 14, characterized in that, The detection method further includes: performing fluorescence detection on the control solution under the same detection conditions; the preparation of the control solution includes: adding the fluorescent probe to the good solvent and mixing evenly.

16. The application according to claim 15, characterized in that, The analysis includes: obtaining the emission wavelength λ of the test solution and the control solution at the maximum emission wavelength λ. m Given the fluorescence emission intensities I and I0, calculate the fluorescence intensity change value I / I0 or (I-I0) / I0.

17. The application according to claim 14, characterized in that, In step (A), the concentration of the fluorescent compound in the test solution is 10. -4 ~10 -6 The concentration of the plant natural compound is 10 mol / L. -4 ~10 -6 mol / L.

18. The application according to claim 17, characterized in that, In the test solution, the volume percentage of the good solvent is 95% to 99.99%.

19. The application according to claim 14, characterized in that, Good solvents for the fluorescent compound include at least one of water, PBS buffer, and SD medium.

20. The application according to claim 19, characterized in that, The concentration of the natural plant compound to be tested in the solution is 10. -3 ~10 -6 mol / L.

21. The application according to claim 19, characterized in that, The solvent in the solution containing the natural compound of the plant to be tested is dimethyl sulfoxide.

22. The application according to claim 19, characterized in that, In the fluorescent probe, the concentration of the fluorescent compound is 10. -3 ~10 -6 mol / L.

23. The application according to claim 14, characterized in that, The natural plant compounds to be tested include ursolic acid.

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