A fluorescence probe based on AIE, a preparation method thereof and application thereof in preparation of a COX-2 inhibitor screening kit

Abstract

The application provides a fluorescent probe based on AIE, a preparation method of the fluorescent probe and application of the fluorescent probe in preparation of a COX-2 inhibitor screening kit. The probe can be used for in vitro determination of COX-2 activity, the probe has good response relationship and selectivity for COX-2, the detection limit is as low as 0.007 mu g / mL, the probe has high sensitivity, and the fluorescent probe can also be used for screening anti-inflammatory active ingredients from lotus seed pods.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a fluorescence probe based on AIE and a preparation method thereof and application thereof in preparation of a COX-2 inhibitor screening kit. BACKGROUND

[0002] Inflammation is a defensive response of the body to harmful stimuli, but excessive inflammatory response can cause serious damage to the body, and even lead to loss of body function and death. Inflammation is closely related to many common diseases, including arthritis, colitis, diabetic nephropathy and cardiovascular system diseases. Cyclooxygenase-2 (COX-2) is rapidly expressed under the stimulation of a series of pro-inflammatory cytokines, and regulates local and systemic inflammatory response by catalyzing the conversion of arachidonic acid into prostaglandin, and has been considered as one of the important targets for playing an anti-inflammatory and analgesic role. At present, a variety of small molecule fluorescent probes have been developed to realize the fluorescence detection of COX-2. However, traditional fluorophores may encounter aggregation quenching (ACQ) problem under high concentration or solid state, which will reduce the detection sensitivity and limit the application of the probe. In recent years, fluorescence probes based on aggregation-induced emission effect (AIE) have gradually emerged, and such aggregation-induced emission materials have excellent light stability and high sensitivity, and have been widely used in the fields of biological sensing, biological imaging and photodynamic therapy.

[0003] Traditional Chinese medicine theory believes that the core pathogenesis of inflammation is mostly the aggregation of evil qi and the accumulation of masses, mostly heat-toxicity and blood stasis. Lotus house, as one of traditional Chinese medicines in China, is often used to treat abortion, carbuncle and sore, incomplete lochia, postpartum stasis and the like due to its functions of removing blood stasis, stopping bleeding, clearing heat and detoxifying, and has a good relieving effect on pain and swelling pain caused by blood stasis. Modern pharmacological studies have shown that lotus house extract can reduce the production of pro-inflammatory cytokines TNF-α and interleukin-6 in LPS-stimulated HepG2 cells, inhibit the expression level of inflammatory mediators COX-2 and iNOS protein, and also can improve the excessive acetaminophen-induced liver toxicity inflammation. SUMMARY

[0004] Therefore, the present application aims to overcome the defects in the prior art, and provides a fluorescence probe based on AIE and a preparation method thereof and application thereof in preparation of a COX-2 inhibitor screening kit, for specifically detecting COX-2 activity, so as to solve the problems of insufficient sensitivity and poor specificity of the existing COX-2 activity detection tools.

[0005] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0006] In a first aspect, the present application provides a fluorescence probe based on AIE, the structural formula of which is shown as formula III:

[0007] Formula III.

[0008] In a second aspect, the present application also provides a preparation method of the AIE-based fluorescent probe.

[0009] The method comprises the following steps:

[0010] Step one, coupling 2,3-bis(4-bromophenyl)-2-butenedinitrile with 4-boronic acid triphenylamine to generate a compound shown in Formula I; the reaction formula is shown as follows:

[0011]

[0012] Formula I

[0013] Step two, substituting the compound shown in Formula I with 6-amino-1-hexanol to generate a compound shown in Formula II; the reaction formula is shown as follows:

[0014]

[0015] Formula I Formula II

[0016] Step three, esterifying the compound shown in Formula II with indometacin to generate a compound shown in Formula III, i.e. the AIE-based fluorescent probe; the reaction formula is shown as follows:

[0017]

[0018] Formula II Formula III.

[0019] Preferably, the substitution reaction of step one further comprises performing in the presence of a base, a palladium catalyst and an organic solvent under an inert atmosphere, wherein the base is selected from one or more of potassium carbonate, potassium phosphate, cesium carbonate, sodium carbonate; the palladium catalyst is selected from one or more of tetrakis(triphenylphosphine)palladium, dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium, dichloro(tri(dimesityl)phosphine)palladium, 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene palladium dichloride; and the organic solvent is selected from one or more of tetrahydrofuran, toluene, 1,4-dioxane.

[0020] Preferably, the molar ratio of 2,3-bis(4-bromophenyl)-2-butenedinitrile, 4-boronic acid triphenylamine, base and palladium catalyst is 100: (50-60): (500-800): (3-5).

[0021] Preferably, the reaction condition of the substitution reaction of step one is that the temperature is 60-80°C and the reaction time is 12-16h.

[0022] Preferably, the coupling reaction of step two further comprises performing in an inert atmosphere in the presence of a base, a palladium catalyst, a ligand and an organic solvent, wherein the base is selected from one or more of cesium carbonate, potassium phosphate, sodium tert-butoxide, potassium tert-butoxide; the palladium catalyst is selected from one or more of tris(dibenzylideneacetone)dipalladium, tetrakis(triphenylphosphine)palladium, dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium, palladium acetate; the ligand is selected from one or more of 1,1'-binaphthalene-2,2'-bisdiphenylphosphine, 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl, 2-dicyclohexylphosphino-2'-methylbiphenyl, 1,1'-bis(diphenylphosphino)ferrocene; and the organic solvent is selected from one or more of toluene, 1,4-dioxane, tetrahydrofuran, N,N-dimethylformamide.

[0023] Preferably, the molar ratio of the compound of formula I, 6-amino-1-hexanol, the palladium catalyst, the ligand and the base is 365: (400-480): (15-22): (30-40): (650-800).

[0024] Preferably, the reaction conditions of the coupling reaction of step two are: the temperature is 80-120℃, and the reaction time is 8-16h.

[0025] Preferably, the esterification reaction of step three further comprises performing in an inert atmosphere in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 4-dimethylaminopyridine and an organic solvent, wherein the organic solvent is selected from one or more of dichloromethane, anhydrous diethyl ether, tetrahydrofuran, N,N-dimethylformamide.

[0026] Preferably, the molar ratio of the compound of formula II, indomethacin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 4-dimethylaminopyridine is 165: (220-280): (400-550): (25-42).

[0027] Preferably, the reaction conditions of the esterification reaction of step three are: the temperature is room temperature, and the reaction time is 8-14h.

[0028] In a third aspect, the present application further provides an application of the above-mentioned AIE-based fluorescent probe in COX-2 activity detection or COX-2 inhibitor screening.

[0029] In a fourth aspect, the present application further provides an application of the above-mentioned AIE-based fluorescent probe in the preparation of a COX-2 activity detection kit or a COX-2 inhibitor screening kit.

[0030] In a fifth aspect, the present application provides a COX-2 activity detection method based on an AIE-based fluorescent probe, which comprises the following steps:

[0031] (1) COX-2 stock solution is diluted to obtain COX-2 detection solutions with different concentrations; the probe solution prepared by weighing the AIE-based fluorescent probe powder is sub-packed and stored in a refrigerator at -80°C for standby;

[0032] (2) The probe solution, COX-2 solution with different concentrations and PBS buffer solution are sequentially added to a 96-well plate to obtain a mixed solution, the mixed solution is shaken and placed in an incubator for incubation;

[0033] (3) The emission spectrum of the mixed solution after incubation is measured at an excitation wavelength of 460 nm, and the fluorescence intensity at 600 nm is recorded, so that the concentration data of COX-2 of the mixed solution are obtained, that is, the COX-2 activity detection is realized;

[0034] The detection range of the detection method for COX-2 is 0-0.5 μg / mL, the linear equation is F-F0=769.26C+9.5256 (R 2 =0.9972), wherein F is the fluorescence intensity of COX-2+probe; F0 is the fluorescence intensity without COX-2, and the detection limit is 0.007 μg / mL.

[0035] Preferably, the final concentration of the probe solution in the mixed solution of step (2) is 5 μM, the PBS buffer solution is a PBS buffer solution with pH=6.8, the incubation temperature is 37°C, and the incubation time is 20 minutes.

[0036] Preferably, the AIE-based fluorescent probe powder in step (1) is dissolved in a PBS buffer solution containing 10% THF.

[0037] Preferably, in step (2), 20 μL of 50 μM probe solution, 20 μL of COX-2 solution with different concentrations and 160 μL of PBS buffer solution are sequentially added to a 96-well plate.

[0038] In a sixth aspect, the application further provides a COX-2 activity inhibition test method using an AIE-based fluorescent probe, which comprises the following steps:

[0039] (1) COX-2 is diluted with a PBS buffer solution to obtain COX-2 detection solutions with different concentrations; an AIE-based fluorescent probe TPADFN-IMC powder is weighed to prepare a probe solution, which is sub-packed and stored in a refrigerator at -80°C for standby;

[0040] (2) The sample solution to be tested, PBS and COX-2 solution are sequentially added to a 96-well plate, incubated, and then the probe solution is added, and the fluorescence intensity at an excitation wavelength of 460 nm and an emission wavelength of 600 nm is determined after incubation again;

[0041] The inhibition rate I is calculated by the formula: I (%)=(F-Fs) / (F-F0)X100%, wherein F is the fluorescence intensity of COX-2 and + probe; Fs is the fluorescence intensity of the sample to be tested, COX-2 and + probe; F0 is the fluorescence intensity without COX-2.

[0042] Preferably, in step (2), 20 μL of the sample to be tested solution, 40 μL of PBS, 20 μL of 0.4 μg / mL COX-2 solution are added to the 96-well plate, and after incubation, 20 μL of 50 μM probe solution is added.

[0043] Compared with the prior art, the present application has the following advantages:

[0044] The present application constructs a fluorescence probe based on the principle of aggregation-induced emission (AIE) effect, which is used for detecting COX-2 and screening COX-2 natural inhibitors. The probe has the advantages of simple operation, low cost, high sensitivity, etc. and is composed of FRET effect between R-CDs and CoOOH NSs. It has been verified that the probe can determine COX-2 activity in vitro, and the probe has good response relationship and selectivity to COX-2, with a detection limit as low as 0.007 μg / mL, and has high sensitivity. The determination method can also be used to screen anti-inflammatory active ingredients from lotus seed pods. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 The FT-IR diagram of the AIE-based fluorescence probe TPADFN-IMC prepared in Example 1;

[0046] Figure 2 The influence diagram of the AIE-based fluorescence probe TPADFN-IMC prepared in Example 1; probe concentration: 5 μM; wherein (a) fluorescence emission spectrum of TPADFN-IMC in different solvents; (b) fluorescence spectrum of TPADFN-IMC in different solvents; (c) fluorescence of TPADFN-IMC in different proportion of water content mixed solvents; detection solvent: THF / PBS buffer; (d) change of fluorescence spectrum of TPADFN-IMC in different proportion of water content; detection solvent: THF / PBS buffer; (e) change of fluorescence intensity of TPADFN-IMC in different proportion of water content; detection solvent: THF / PBS buffer; (f) fluorescence lifetime decay curve of TPADFN-IMC;

[0047] Figure 3Stability verification chart of the probe; detection solvent: 10% THF, probe concentration: 5 μM; wherein chart (a) is the effect of ultraviolet irradiation time on the fluorescence intensity of TPADFN-IMC; (b) is the effect of storage time on the fluorescence intensity of TPADFN-IMC; (c) is the optimization of the probe concentration condition in the detection system; (d) is the optimization of the pH condition of the buffer in the detection system; (e) is the optimization of the incubation temperature condition in the detection system; (f) is the optimization of the incubation time condition in the detection system;

[0048] Figure 4 COX-2 activity detection chart of the AIE probe (10% THF, probe concentration: 5 μM); wherein chart (a) is the change of the fluorescence intensity of the AIE probe with the COX-2 concentration; (b) is the response relationship of the AIE probe to COX-2; (c) and (d) are the anti-interference test of the AIE probe;

[0049] Figure 5 Fluorescence enhancement mechanism chart of the TPADFN-IMC probe; wherein chart (a) is the particle size before the AIE probe is combined with COX-2; (b) is the particle size after the AIE probe is combined with COX-2; (c) is the visualization of the molecular docking of the AIE probe and COX-2; (d) is the interaction residue of the AIE probe and COX-2;

[0050] Figure 6 Inhibition rate of celecoxib with different concentrations on COX-2;

[0051] Figure 7 Lotus seedpod liquid chromatogram, component preparation chart and component COX-2 activity evaluation chart; wherein chart (a) is the lotus seedpod liquid chromatogram and component preparation; (b) is the component COX-2 activity evaluation;

[0052] Figure 8 Potential anti-inflammatory active ingredients in lotus seedpod; wherein chart (a) is the chemical structure chart of the anti-inflammatory active ingredient, (b) is the IC 50 value of seven COX-2 inhibitory active ingredients;

[0053] Figure 9 Molecular docking schematic diagram of the seven active ingredients and COX-2;

[0054] Figure 10 COX-2 enzyme inhibition rate of the combination of lotus seedpod extract and active ingredients. DETAILED DESCRIPTION

[0055] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application.

[0056] In the present text, unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0057] In the present text, where values are described as ranges, it is to be understood that the disclosure includes disclosure of all possible sub-ranges within the range and specific numerical values falling within the range, regardless of whether the specific numerical values or the specific sub-ranges are explicitly stated.

[0058] In the present text, reference to "a plurality of" or the like, unless otherwise specified, means greater than or equal to two or equal to two. For example, "one or more" means one or greater than or equal to two.

[0059] In the present text, reference to "preferred", "more preferred" is only to describe the better effect of the embodiment or example, and it should be understood that it does not constitute a limitation on the scope of protection of the present application.

[0060] In the present text, reference to "further" or the like is used for the purpose of description, indicating a difference in content, but should not be understood as a limitation on the scope of protection of the present application.

[0061] In the present text, the term "and / or" is a description of the relationship between the objects, indicating that there can be three relationships. For example, A and / or B, indicating: A or B, or A and B, the three relationships.

[0062] In the present text, the term "about" means + / - 10% of the specified value, preferably + / - 5%, more preferably + / - 1%.

[0063] In the present text, "include", "including", "have", "containing", and the like are all open terms, that is, they mean including but not limited to.

[0064] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. Although only preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application.

[0065] The following is the synthesis route of the AIE fluorescent probe in Example 1:

[0066]

[0067] The present application will be described in detail below with reference to examples.

[0068] Example 1 Preparation of AIE-based fluorescent probe TPADFN-IMC

[0069] Preparation of fluorescent group: under nitrogen protection, 2,3-bis(4-bromophenyl)-2-butenedinitrile 3.1 g, 4-boronic acid triphenylamine 1.1 g, anhydrous potassium carbonate 5.5 g and tetrakis(triphenylphosphine)palladium 0.28 g were added into a 250 mL reaction bottle, then 120 mL of tetrahydrofuran and 20 mL of water were added, the reaction bottle was placed on a constant temperature heating magnetic stirrer, and the reaction was stirred at 60°C for 15 h; after the reaction was completed, the tetrahydrofuran was removed by evaporation under reduced pressure (EYELA-1300, Tokyo Rikakikai Co., Ltd., Japan), and the remaining system was extracted with water / dichloromethane (1:1 by volume) for 3 times, and the organic phase was collected and filtered through a Buchner funnel; after the filtrate was dried under reduced pressure, the crude product was separated by silica gel column chromatography, using petroleum ether / dichloromethane (V:V=4:1) as the eluent, and the corresponding eluent was collected and dried to obtain compound 1 (1.36 g, yield 63%).

[0070] Preparation of linker coupling product: under nitrogen protection, 810 mg of compound 1, 206 mg of 6-amino-1-hexanol, 66.8 mg of tris(dibenzylideneacetone)dipalladium, 90 mg of 1,1'-binaphthalene-2,2'-biphenylphosphine and 950 mg of cesium carbonate were added into a 100 mL reaction bottle, then 30 mL of toluene was added for dissolution, and the reaction was stirred and heated to 100°C for 12 h; after the reaction was completed, the organic phase was collected and filtered by suction; after the filtrate was dried by evaporation under reduced pressure, the crude product was separated by silica gel column chromatography, using petroleum ether / ethyl acetate (V:V=8:1) as the eluent, and the eluent was collected and dried to obtain compound 2 (240.3 mg, yield 28%).

[0071] Final preparation of AIE-based fluorescent probe TPADFN-IMC: under nitrogen protection, 200 mg of compound 2, 121 mg of indomethacin, 189 mg of 1-(3-dimethylamino propyl)-3-ethyl carbodiimide and 8.1 mg of 4-dimethylamino pyridine were added into a 100 mL reaction bottle, then 40 mL of dry dichloromethane was added for dissolution, and the reaction was stirred at room temperature for 10 h; after the reaction was completed, the organic phase was collected and dried with anhydrous magnesium sulfate, and the filtrate was dried by suction and under reduced pressure; the crude product was separated by silica gel column chromatography, using petroleum ether / dichloromethane (V:V=10:1) as the eluent, and the corresponding eluent was collected and dried to obtain AIE-based fluorescent probe TPADFN-IMC (205.1 mg, yield 67%), which was abbreviated as TPADFN-IMC and stored in a 4°C refrigerator.

[0072] Structure characterization of intermediates and probes: the structure of compounds 1-3 was identified by nuclear magnetic resonance instrument (model Avance III 400, Burker Company, Germany) and mass spectrometer (model Q-TOF6250, Agilent Company, USA); the sample was dissolved in deuterated chloroform, and the nuclear magnetic resonance hydrogen spectrum (HNMR) and carbon spectrum (CNMR) were determined: 1 13

[0073] HNMR of compound 1 1 (400 MHz, Chloroform-d) δ 7.92 (2H, d, J = 8.5 Hz), 7.72 (6H, m), 7.52 (2H, d, J = 8.7 Hz), 7.30 (4H, dd, J = 8.5, 7.3 Hz), 7.5 (m, 6H), 7.08 (2H, t, J = 7.3 Hz), 13 (101 MHz, Chloroform-d) δ 147.43, 144.41, 132.71, 132.47, 131.24, 130.33, 129.87, 129.55, 129.36, 127.98, 127.19, 126.45, 125.87, 125.04, 123.63, 123.21, 122.82, 116.83, mass spectrum (m / z) [M+H] + = 553.1072 (molecular formula C 34 H 22 BrN3);

[0074] HNMR of compound 2 1 (400 MHz, Chloroform-d) δ 7.84 (3H, m), 7.69 (1H, m), 7.54 (1H, d, J = 8.8 Hz), 7.32 (5H, d, J = 8.0 Hz), 7.17 (7H, d, J = 8.4 Hz), 7.08 (2H, t, J = 7.7 Hz), 6.66 (1H, d, J = 9.3 Hz), 3.72 (1H, m), 3.24 (1H, dd, J = 14.0, 6.9 Hz), 1.72 (6H, d, J = 7.7 Hz), 1.47 (4H, m), 13 ​​CNMR (101 MHz, Chloroform-d) δ 147.45, 142.90, 133.05, 132.35, 131.30, 130.67, 130.25, 129.40, 129.20, 127.82, 126.93, 124.91, 124.79, 123.34, 120.21, 118.30, 117.36, 112.17, 62.81, 43.26, 32.62, 29.25, 26.87, 25.57, Mass (m / z) [M+H] + = 589.2956 (Molecular Formula C 40 H 36 N4O);

[0075] Compound 3 (TPADFN-IMC) was prepared according to the following scheme: 1 HNMR (600 MHz, Chloroform-d) δ 7.81 (4H, phenyl, dd, J = 19.3, 8.7 Hz), 7.67 (4H, phenyl, dd, J = 16.1, 8.5 Hz), 7.51 (2H, phenyl, d, J = 8.6 Hz), 7.46 (2H, phenyl, d, J = 8.5 Hz), 7.29 (4H, phenyl, m), 7.15 (6H, phenyl, m), 7.06 (2H, phenyl, t, J = 7.4 Hz), 6.98 (1H, phenyl, d, J = 2.5 Hz), 6.87 (1H, phenyl, d, J = 9.0 Hz), 6.68 (1H, phenyl, dd, J = 9.0, 2.5 Hz), 6.63 (2H, phenyl, d, J = 8.9 Hz), 4.12 (2H, -CH2-O-, t, J = 6.5 Hz), 3.84 (3H, -OCH3, s), 3.67 (2H, -CH2-C=0, s), 2.40 (3H, -CH3, s), 1.62 (5H, m), 1.32 (5H, m), 13 CNMR (101 MHz, Chloroform-d) δ 170.96, 168.35, 156.01, 147.45, 135.93, 133.85, 132.34, 131.20, 130.67, 130.24, 129.39, 129.17, 127.81, 126.93, 124.78, 123.32, 120.25, 114.96, 112.72, 112.15, 111.58, 101.48, 64.83, 55.76, 43.13, 30.51, 28.54, 26.54, 25.65, Mass (m / z) [M+H] += 929.3581 (Molecular formula C 59 H 50 ClN5O4), which confirmed the successful synthesis of the probe.

[0076] In addition, the characteristic functional groups of TPADFN-IMC molecule were further confirmed by Fourier infrared spectrum. The wide absorption peak at 3391.85 cm -1 belongs to the stretching vibration of N-H, 2922.32, 2856.17 cm -1 are the stretching vibration of C-H of methyl and methylene on the linking group. 2204.96 cm -1 is the stretching vibration absorption peak of cyano group. 1088.48, 1069.16 cm -1 are the C-Cl rocking vibration absorption peaks on the aromatic ring, 1605.97, 1518.15, 1488.93 cm -1 nearby appear multiple absorption peaks, which belong to the stretching vibration of C=C, reflecting the conjugated structure of benzene ring. 1324.70 cm -1 is the stretching vibration absorption peak of C-N of -CON-. The above results show that TPADFN-IMC is formed by coupling the indometacin with the fluorescent molecule through the coupling group. Figure 1 .

[0077] Characterization of the optical properties of the probe: After obtaining TPADFN-IMC, different polar solvents were used to explore its optical properties, including dimethyl sulfoxide (DMSO), acetonitrile (CH3CN), N,N-dimethylformamide (DMF), methanol (MeOH), ethanol (EtOH), tetrahydrofuran (THF), dichloromethane (CH2Cl2). The UV-Vis absorption spectrum of the probe in different solvents was determined by using a UV-visible spectrophotometer (model Carry 90, Agilent, USA), and the results showed that the maximum absorption wavelength of TPADFN-IMC in CH2Cl2 and acetonitrile was 450 nm. However, the maximum absorption wavelength of TPADFN-IMC red shifted to 460 nm in DMF, MeOH, EtOH, THF, and the maximum absorption peak red shifted to 475 nm in DMSO, which may be due to the solvent effect (a). Figure 2 The fluorescence spectrum was determined by using a fluorescence spectrophotometer (model FL7000, Hitachi, Japan), and with the increase of the solvent polarity, the emission spectrum of TPADFN-IMC changed with the change of the solvent polarity. In EtOH, the emission wavelength was 560 nm, and in DMSO, the emission wavelength red shifted to 580 nm and the fluorescence intensity decreased. In addition, in 10% THF PBS buffer, the fluorescence intensity of the probe increased significantly due to the AIE effect (b). Figure 2b). Further explore the aggregation-induced emission properties of TPADFN-IMC, select THF and PBS buffer as good and non-good solvents for the probe, by adjusting the proportion of poor solvent in the mixed solvent system to control the state of TPADFN-IMC in the mixed solvent. As shown in Figure 2 c, the probe has almost no fluorescence in THF solution, and as the water content in THF solution increases from 10% to 90%, the fluorescence of TPADFN-IMC under 360 nm ultraviolet light gradually brightens. Figure 2 d is the fluorescence emission spectrum of TPADFN-IMC at 10%-90% water content. With the increase of the content of poor solvent, the fluorescence intensity of TPADFN-IMC is continuously enhanced. As shown in Figure 2 e, when the water content is more than 70%, the fluorescence emission intensity of TPADFN-IMC is significantly enhanced, and in 90% aqueous solution, it is close to the highest level (λex=460 nm). These results show that TPADFN-IMC exists in the form of dispersion in the mixed solvent system with high water content, and increasing the water content can make the fluorescence probe molecules gradually aggregate, resulting in fluorescence enhancement. Therefore, in the following study, 10% THF PBS buffer is selected as the probe dissolution environment. As shown in Figure 2 f, the average lifetime is 4.95 ns, indicating that the probe molecules stay on the excited state level for a short time and have good stability.

[0078] Example 2 Fluorescent detection of COX-2 activity

[0079] Solution preparation before detection: accurately weigh a certain amount of COX-2, and prepare a 0.5 μg / mL stock solution with PBS buffer (0.1 M, pH=6.8). According to the experimental requirements, dilute to obtain 0.001-0.8 μg / mL COX-2 detection solution; weigh TPADFN-IMC powder, and prepare a 50 μM probe solution with THF / PBS buffer (10:90 v / v, pH=6.8). After aliquot, store in a-80℃ refrigerator for standby.

[0080] COX-2 activity detection steps: add 20 μL TPADFN-IMC probe solution (50 μM), 20 μL COX-2 solution with different concentrations and 160 μL PBS buffer (pH=6.8) into a 96-well plate in turn, shake for 2 minutes, and then put it into a 37℃ incubator for incubation for 20 minutes; use a multifunctional enzyme marker to measure the fluorescence intensity of the mixture at an excitation wavelength of 460 nm and an emission wavelength of 600 nm, and the excitation and emission slit width are set to 5 nm.

[0081] COX-2 inhibition activity test: First, add 20 μL of the test sample solution, 40 μL of PBS buffer (pH=6.8) and 20 μL of COX-2 solution to a 96-well plate and incubate at 37℃ for 10 minutes; then add 20 μL of LTPADFN-IMC probe solution and continue incubation at 37℃ for 20 minutes. Measure the fluorescence intensity at an excitation wavelength of 460 nm and an emission wavelength of 600 nm. The inhibition rate is calculated using the following formula: Inhibition rate (%) = (F-Fs) / (F-F0)×100%, where F is the fluorescence intensity containing COX-2 and the probe, Fs is the fluorescence intensity containing the test sample, COX-2 and the probe, and F0 is the fluorescence intensity of the probe without COX-2. Celecoxib was selected as the positive control drug, and 0~0.5 μM celecoxib solutions were prepared for parallel experiments.

[0082] Example 3

[0083] (1) Sample preparation

[0084] Preparation of lotus seedpod extract: Weigh 1 g of lotus seedpod powder and place it in a 50 mL stoppered conical flask. Add 20 mL of 80% methanol, seal tightly, and weigh. Place the conical flask in an ultrasonic cleaner and extract ultrasonically for 30 minutes at an ultrasonic power of 400 W. After standing and cooling, make up the lost mass. Centrifuge the resulting solution at 13400 rpm for 6 minutes. Filter the supernatant through a 0.22 μm filter membrane and store the filtrate at 4℃ for later use.

[0085] Preparation of reference solution: Accurately weigh (+)-catechin, piperidin, proanthocyanidin B1, and myricetin-3- O Appropriate amounts of glucoside, hyperoside, isoquercitrin, and quercetin reference standard powders were prepared into reference standard stock solutions with methanol concentrations of 2.0 mg / mL or 1.0 mg / mL, respectively. These stock solutions were stored in a refrigerator at 4°C and diluted with methanol to the appropriate concentration as needed before use.

[0086] Chromatographic conditions for fraction collection

[0087] The lotus seedpod component preparation was performed by high performance liquid chromatograph coupled with automatic fraction collector; the chromatographic column was ACQUITY UPLC BEH C18 (50 mm x 2.1 mm, 1.7 μm); the mobile phase was composed of 0.1% formic acid aqueous solution (A phase) and methanol (B phase), the gradient elution program was as follows: the B phase content was 5% to 27% within 0-28 min; the B phase content was 27% to 33% within 28-35 min; the B phase content was kept at 33% within 35-53 min; the B phase content was 33% to 70% within 53-80 min; the B phase content was kept at 70% within 80-81 min; the flow rate was 1.0 mL / min, the column temperature was 35 °C, the detection wavelength was 254 nm (photodiode array detector DAD), and the injection amount was 10 μL; the fractions were collected according to the time mode, and a total of 17 fractions were collected, and the specific collection time was as follows: Fr.1 (5.2-5.9 min), Fr.2 (6.0-6.6 min), Fr.3 (9.3-9.9 min), Fr.4 (11.8-12.6 min), Fr.5 (15-15.7 min), Fr.6 (18.5-19.5 min), Fr.7 (21.6-22.4 min), Fr.8 (25.1-25.9 min), Fr.9 (42.5-43.3 min), Fr.10 (43.4-44.5 min), Fr.11 (44.6-45.7 min), Fr.12 (52.5-54.2 min), Fr.13 (54.3-56.6 min), Fr.14 (62-63 min), Fr.15 (64.2-65.1 min), Fr.16 (65.9-66.8 min), Fr.17 (69.7-70.7 min); the fractions collected after enrichment for 4 times were blown dry with nitrogen, redissolved with methanol to an appropriate concentration, and used for subsequent activity test.

[0088] (2) Characterization of lotus seedpod extract

[0089] The active component of lotus seedpod was qualitatively analyzed by ultra-high performance liquid chromatograph coupled with time-of-flight mass spectrometer; the analysis column was Phenomenex Kinetex C18 (100 mm x 2.1 mm, 1.7 μm), and the guard column was Agilent Eclipse XDB-C18 (1.8 μm, 2.1 x 5 mm); the mobile phase was 0.1% formic acid aqueous solution (A phase) and acetonitrile (B phase), and the gradient elution program was as follows: the B phase content was kept at 5% within 0-2 min; the B phase content was 5% to 30% within 2-15 min; the B phase content was 30% to 64% within 15-26 min; the B phase content was 64% to 90% within 26-35 min; the flow rate was 0.3 mL / min, the column temperature was 30 °C, and the injection amount was 2 μL.

[0090] Mass spectrometry parameters: Ion source is electrospray ionization source (ESI), while recording positive and negative ion mode; dry gas flow 9.0 L / min, dry gas temperature 350°C, nebulizer gas pressure 40 psig, fragment voltage 120 V, capillary voltage 3500 V, quadrupole RF 750 V, skimmer voltage 65 V; collision energy settings in MS / MS analysis are 10 V and 30 V, mass range 50-1000; MS and MS / MS data are processed on SCIEX OS software 2 Ion fragment information, determine the molecular formula, relative molecular mass error and characteristic ion fragments, combined with published literature, PubMed, PubChem and ChemSpider database comparison, determine the chemical composition of the lotus seed pod extract.

[0091] (3) Molecular docking

[0092] The crystal structure of COX-2 (PDB ID: 3NL1) was downloaded from the Protein Data Bank (PDB) for molecular docking experiments; AutodockVina was used as the docking engine, and Gasteiger charge was used to calculate the electrostatic interaction. The docking parameters were set as gridbox center X = 44.7, Y = -35.9, Z = 25.3, and dimensions X = 107, Y = 91, Z = 115. The three-dimensional structure of the screened active compound was downloaded from the PubChem database and optimized for minimum energy in Chem3D software. AutoDockTool was used to add hydrogen, charge, and adjust the chemical bond for the ligand. All ligands were docked 100 times for sufficient sampling, and the best conformation was determined by calculating the binding energy. The binding site and hydrogen bond interaction of the active compound with the COX-2 active pocket were analyzed.

[0093] Example 4 Stability of TPADFN-IMC

[0094] To study the light stability of TPADFN-IMC, the effects of UV irradiation time and storage time on its fluorescence intensity were investigated. After the probe was irradiated by a 365 nm UV lamp for a certain period of time, its fluorescence intensity was recorded at the optimal excitation wavelength. As shown in Figure 3 a and Figure 3 b, TPADFN-IMC has good stability within 10 h of irradiation time. Even when the irradiation time reaches 16 h, no significant change in the fluorescence intensity of the probe is observed, indicating that UV light has little effect on the fluorescence intensity of the probe, and the synthesized probe has good anti-photobleaching ability. In addition, the fluorescence intensity of TPADFN-IMC solution is very stable when stored at 4°C for 8 days. These experimental results indicate that the probe has good stability.

[0095] Example 5 Optimization of the conditions for TPADFN-IMC detecting COX-2

[0096] Probe concentration optimization: The concentration of COX-2 was fixed at 0.4 μg / mL, and the concentration of TPADFN-IMC was changed (1-10 μM). The fluorescence intensity difference (F-F0, F is the fluorescence intensity after adding COX-2, and F0 is the fluorescence intensity without adding COX-2) was measured. The results showed that when the probe concentration was 5 μM, F-F0 was the largest. Therefore, the optimal probe concentration was determined to be 5 μM. Figure 3 c).

[0097] Buffer pH optimization: Considering that the TPADFN-IMC molecule has a cyano functional group structure, the pH of the buffer used in the detection system may affect the fluorescence emission of the probe itself and the enzyme activity of COX-2. The effect of the buffer on the fluorescence intensity in the pH range of 5.5-8.0 was investigated. At pH = 6.8, F-F0 was the largest, and the COX-2 enzyme activity remained at a high level. Therefore, PBS buffer with pH = 6.8 was selected as the detection system. Figure 3 d).

[0098] Incubation condition optimization: Incubation temperature has a significant effect on the activity of COX-2, so the temperature during the detection process was optimized. The effects of incubation temperature (25-45 °C) and time (5-30 minutes) on fluorescence intensity were investigated. The results showed that when incubated at 37 °C for 20 minutes, the fluorescence intensity reached a stable and maximum level. Therefore, the optimal incubation temperature was determined to be 37 °C, and the incubation time was 20 minutes. Figure 3 e and Figure 3 f).

[0099] Example 6 Fluorescent quantitative detection of COX-2

[0100] Under the optimal detection conditions, the relationship between the fluorescence intensity of TPADFN-IMC and the activity of COX-2 was studied using different concentrations of COX-2. As shown in Figure 4 a, with the increase of the concentration of COX-2, the fluorescence intensity of TPADFN-IMC gradually increased with the increase of the concentration of COX-2, and there was no obvious shift in the emission wavelength. Figure 4 b shows the relationship between the change in fluorescence intensity at 600 nm of the probe and the concentration of COX-2. In the range of 0-0.5 μg / mL, there was a good linear relationship between the change in fluorescence intensity and the concentration of COX-2, and the linear equation was F-F0 = 769.26C + 9.5256 (R 2= 0.9972). Wherein, F represents the fluorescence intensity of the probe at 600 nm in the presence of COX-2, F0 represents the fluorescence intensity of the probe at 600 nm in the absence of COX-2, C is the concentration of COX-2, unit is pg / mL, and the detection limit is 0.007 pg / mL. In summary, the probe has excellent sensitivity for COX-2 detection.

[0101] Specificity test: specific recognition of the detected substance is one of the important characteristics of the fluorescent probe. In order to evaluate the selectivity of TPADFN-IMC, various potential interferents were introduced in the presence and absence of COX-2, including metal cations, anions, biological small molecules and proteases. The interference of the detection system, the results show that when the above-mentioned interference substances exist, the fluorescence intensity of the system has no significant change, and only COX-2 can make the fluorescence intensity of the probe significantly enhanced, indicating that the probe has good specificity for COX-2 Figure 4 c and Figure 4 d).

[0102] Example 7 Mechanism of fluorescence enhancement of TPADFN-IMC

[0103] In view of the fact that the fluorescence intensity of TPADFN-IMC solution is enhanced after adding COX-2, and the degree is constantly enhanced with the increase of COX-2 concentration, it is speculated that the fluorescence enhancement process occurs in the combination of the probe and the active pocket of COX-2, forming aggregates, limiting intramolecular motion, resulting in enhanced fluorescence intensity. In order to clarify the fluorescence enhancement mechanism of COX-2 to TPADFN-IMC, first of all, DLS was used to study the self-assembly behavior of TPADFN-IMC in the presence and absence of COX-2. The results show that the average diameter of TPADFN-IMC (5 mM) aggregates is 18.2 nm (see Figure 5 a). When exposed to COX-2 (0.4 pg / mL), the average diameter of TPADFN-IMC becomes 168.3 nm (see Figure 5 b). DLS results provide evidence for the self-assembly of TPADFN-IMC and COX-2 to enhance the AIE process. Molecular docking was used to explore the spatial conformation and binding mode of the probe TPADFN-IMC into the active cavity of COX-2. COX-2 (PDB: 3NL1) was selected as the research model. As Figure 5As shown in Fig. c, the docking results show that the recognition group indomethacin in TPADFN-IMC is more inclined to insert into the hydrophobic cavity inside COX-2. In addition, the methoxy group, carbonyl group and cyano group on TPADFN-IMC can form non-covalent bonds with amino acid residues such as CYS-21, TYR-116, GLN-42, CYS-26, ABG-22 in the hydrophobic pocket of COX-2, enabling TPADFN-IMC to be firmly anchored in the specific cavity of COX-2 Figure 5 d), thereby limiting the intramolecular motion of the probe and producing an AIE effect to enhance fluorescence emission.

[0104] Example 8 Evaluation of COX-2 inhibitory activity

[0105] To investigate the applicability of the established AIE fluorescence detection system for evaluating the COX-2 inhibitory activity of samples, the classic COX-2 inhibitor celecoxib was used as a positive drug. During the interaction between the probe and COX-2, when the inhibitor is introduced into the system, the inhibitor will compete with TPADFN-IMC for the hydrophobic active pocket in COX-2. The active site is occupied by the inhibitor, resulting in no binding site for the probe TPADFN-IMC and COX-2, and showing fluorescence intensity inhibition. As shown in Fig. 8, Figure 6 with the increase of celecoxib concentration, the COX-2 activity is inhibited, which limits the binding process of the enzyme and the probe, resulting in weaker fluorescence enhancement effect than when no corresponding inhibitor is added. The calculated IC50 of celecoxib is 0.12 μM, which is consistent with the related reported results. The above results show that the method established in this study is sensitive and reliable, and can be used for evaluating the COX-2 inhibitory activity of samples.

[0106] Example 9 Screening and identification of anti-inflammatory active ingredients of lotus seed pods

[0107] Active component screening: The 17 lotus seed pod components collected by HPLC were tested for COX-2 inhibitory activity, and the inhibition rates of each component were calculated. The active components with an inhibition rate greater than 60% were screened, including Fr. 4, Fr. 6, Fr. 7, Fr. 10, Fr. 12, Fr. 13, Fr. 15, Fr. 17 Figure 7 a). It is shown that these components have potential COX-2 inhibitors, which are speculated to be the material basis for the anti-inflammatory activity of lotus seed pods.

[0108] Active ingredient identification: UPLC-Q-TOF-MS / MS was used to identify the active components, combined with comparison with reference substances, secondary fragment information and literature data, and 19 chemical components were identified. Taking component 12 as an example to explain the process of compound identification, as shown in Fig. 9, Figure 7As shown in b, a quasi-molecular ion peak with m / z 609.1389 [MH] was detected at 13.264 min in negative ion mode. - Based on secondary mass spectrometry fragments, it is inferred that rhamnose (C6H) has been removed. 12 O6) produces fragment ions with m / z 463.0873, which further lose glucose to generate (C) with m / z 300.0247. 15 H8O7 - The compound was compared with rutin standard, and the molecular ion peak, chromatographic retention time, and secondary mass spectrometry fragments were consistent, confirming that the compound was rutin. A quasi-molecular ion peak (m / z 463.0851 [MH]) was detected at both 14.731 min and 15.895 min. - The two are isomers. Based on secondary mass spectrometry fragmentation, it is inferred that the sugar group was removed to generate a fragment at m / z 300.0253. After comparison with the standard, the compounds were identified as hyperoside and isoquercitrin, respectively.

[0109] Selection and Validation of Active Compounds: Based on literature reports, availability of standards, and sample measurability, (+)-catechins, piperidin, proanthocyanidin B1, and myricetin-3- were ultimately selected. O - Seven potential active ingredients, including glucoside, hyperoside, isoquercitrin, and quercetin, with structures as follows: Figure 8 As shown in a. The COX-2 inhibitory activity of the seven active ingredients was verified, and the results showed that all had varying degrees of inhibitory effect, among which (+)-catechin, taxanein, and quercetin had stronger inhibitory effects. Figure 8 b);

[0110] Molecular docking was used to analyze the interaction between seven active compounds screened from lotus seedpods and COX-2, and the lowest binding energy conformations of each chemical component were visualized. Figure 9 The binding energies of all seven components to COX-2 are less than -5.0 kcal / mol, with quercetin having the lowest binding energy (-9.7 kcal / mol). Quercetin can form seven hydrogen bonds with the GLY-121, ALA-142, GLU-451, GLY-30, and CYS-32 residues of COX-2 through its phenolic hydroxyl groups.

[0111] Quantitative analysis: The results showed that the contents of (+)-catechin ranged from 0.910 to 2.469 mg / g, hyperoside from 0.817 to 2.275 mg / g, and isoquercitrin from 0.942 to 2.688 mg / g. The total contribution of the seven components to the COX-2 inhibitory activity of lotus seedpod extract was 54%.

[0112] To determine the contribution rate of the screened anti-inflammatory active ingredients in lotus seedpods, the total inhibition rate of the mixed standard of active compounds was calculated, such as...Figure 10 As shown, the total contribution rate of the screened active compounds to the COX-2 inhibitory activity of the lotus seed pod extract is 54% calculated by area under curve. With the increase of dilution times, both have the same change trend, which further indicates that the 7 screened compounds are the main material basis for the anti-inflammatory activity of the lotus seed pod.

[0113] The present application establishes a fluorescence detection strategy based on the aggregation-induced emission (AIE) effect, realizes high-sensitivity and high-specificity detection of COX-2 activity by designing and synthesizing a TPADFN-IMC probe, and combines chromatographic separation and mass spectrometry identification technology to screen anti-inflammatory active ingredients in traditional Chinese medicine lotus seed pod; the probe solves the aggregation quenching (ACQ) problem of traditional fluorescence probes, has good light stability and anti-interference ability, and the detection limit is as low as 0.007 μg / mL; at the same time, the 7 screened active ingredients clarify the material basis of the anti-inflammatory activity of the lotus seed pod, and provide a powerful tool for the research and development of COX-2 inhibitors and the research on the anti-inflammatory mechanism of traditional Chinese medicine.

[0114] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A fluorescent probe based on AIE, characterized in that: Its structural formula is shown in Formula III: Formula III.

2. The method for preparing the AIE-based fluorescent probe according to claim 1, characterized in that: Includes the following steps: Step 1: 2,3-bis(4-bromophenyl)-2-butenidonitrile is coupled with triphenylamine 4-borate to generate the compound shown in Formula I; the reaction formula is shown below: Formula I Step 2: The compound shown in Formula I undergoes a substitution reaction with 6-amino-1-hexanol to generate the compound shown in Formula II; the reaction formula is shown below: Formula I Formula II Step 3: The compound shown in Formula II undergoes an esterification reaction with indomethacin to generate the compound shown in Formula III, which is the AIE-based fluorescent probe; the reaction formula is shown below: Formula II and Formula III.

3. The method for preparing an AIE-based fluorescent probe according to claim 2, characterized in that: The coupling reaction in step one further includes being carried out under an inert atmosphere in the presence of a base, a palladium catalyst, and an organic solvent. The base is selected from one or more of potassium carbonate, potassium phosphate, cesium carbonate, and sodium carbonate. The palladium catalyst is selected from one or more of tetra(triphenylphosphine)palladium, dichloro[1,1'-bis(diphenylphosphine)ferrocene]palladium, dichloro(triphenylphosphine)palladium, and 1,3-bis(2,6-diisopropylphenyl)imidazolium-2-ylpyridinium dichloride. The organic solvent is selected from one or more of tetrahydrofuran, toluene, and 1,4-dioxane. The molar ratio of bis(4-bromophenyl)-2-butenidonitrile, triphenylamine 4-boronic acid, base, and palladium catalyst is 100:(50~60):(500~800):(3~5); The reaction conditions for the coupling reaction in step one are: temperature of 60~80℃ and reaction time of 12~16h.

4. The method for preparing an AIE-based fluorescent probe according to claim 2, characterized in that: The substitution reaction in step two further includes being carried out under an inert atmosphere in the presence of a base, a palladium catalyst, a ligand, and an organic solvent. The base is selected from one or more of cesium carbonate, potassium phosphate, sodium tert-butoxide, and potassium tert-butoxide; the palladium catalyst is selected from one or more of tris(dibenzylacetone)palladium, tetra(triphenylphosphine)palladium, dichloro[1,1'-bis(diphenylphosphine)ferrocene]palladium, and palladium acetate; the ligand is selected from one or more of 1,1'-binaphthyl-2,2'-bisdiphenylphosphine, 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl, 2-dicyclohexylphosphine-2'-methylbiphenyl, and 1,1'-bis(diphenylphosphine)ferrocene; and the organic solvent is selected from one or more of toluene, 1,4-dioxane, tetrahydrofuran, and N,N-dimethylformamide. The molar ratio of the compound represented by Formula I, 6-amino-1-hexanol, palladium catalyst, ligand, and base is 365:(400~480):(15~22):(30~40):(650~800). The reaction conditions for the substitution reaction in step two are: temperature of 80~120℃ and reaction time of 8~16h.

5. The method for preparing an AIE-based fluorescent probe according to claim 2, characterized in that: Step three of the esterification reaction also includes the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 4-dimethylaminopyridine, and an organic solvent in an inert atmosphere, wherein the organic solvent is selected from one or more of dichloromethane, anhydrous diethyl ether, tetrahydrofuran, and N,N-dimethylformamide. The molar ratio of the compound represented by Formula II, indomethacin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine is 165:(220~280):(400~550):(25~42). The reaction conditions for the esterification reaction in step three are: room temperature and reaction time of 8-14 hours.

6. The application of the AIE-based fluorescent probe of claim 1 in the detection of COX-2 activity for non-disease diagnosis or treatment purposes or in the screening of COX-2 inhibitors for non-disease diagnosis or treatment purposes.

7. The use of the AIE-based fluorescent probe as described in claim 1 in the preparation of a COX-2 activity detection kit or a COX-2 inhibitor screening kit.

8. A method for detecting COX-2 activity using the AIE-based fluorescent probe of claim 1 for non-disease diagnosis or treatment purposes, characterized in that: The detection method includes the following steps: (1) Dilute the COX-2 stock solution to obtain COX-2 detection solutions of different concentrations; weigh the probe solution prepared by the AIE-based fluorescent probe powder as described in claim 1, dispense it and store it in a -80℃ refrigerator for later use; (2) Add probe solution, COX-2 solution of different concentrations and PBS buffer to a 96-well plate in sequence to obtain a mixed solution. Shake the mixed solution and place it in an incubator for incubation. (3) Measure the emission spectrum of the incubated mixture at an excitation wavelength of 460 nm and record the fluorescence intensity at 600 nm to obtain the concentration data of COX-2 in the mixture, thus realizing the detection of COX-2 activity; The detection method described above has a detection range of 0–0.5 μg / mL for COX-2, and the linear equation is F - F0 = 769.26C + 9.5256 (R0). 2 =0.9972), where F is the fluorescence intensity with COX-2 and probe; F0 is the fluorescence intensity without COX-2; C is the concentration of COX-2 in μg / mL; and the detection limit is 0.007 μg / mL.

9. The detection method according to claim 8, characterized in that: In step (2), the final concentration of the probe solution in the mixed solution is 5 μM, the PBS buffer is pH 6.8, the incubation temperature is 37°C, and the incubation time is 20 minutes; in step (1), the AIE-based fluorescent probe powder is dissolved in 10% THF PBS buffer.

10. A method for testing COX-2 activity inhibition using the AIE-based fluorescent probe of claim 1 for non-disease diagnosis or treatment purposes, characterized in that: The testing method includes the following steps: (1) COX-2 was diluted with PBS buffer to obtain COX-2 detection solutions of different concentrations; AIE fluorescent probe TPADFN-IMC powder was weighed and prepared into probe solutions, which were then aliquoted and stored at -80℃ for later use. (2) First, add the sample solution to be tested, PBS, and COX-2 solution to the 96-well plate, incubate, then add the probe solution, incubate again, and measure the fluorescence intensity at an excitation wavelength of 460 nm and an emission wavelength of 600 nm. The inhibition rate I was calculated using the formula: I(%)=(F-Fs) / (F-F0)×100%, where F is the fluorescence intensity of the sample containing COX-2 and the probe; Fs is the fluorescence intensity of the sample, COX-2 and the probe; and F0 is the fluorescence intensity without COX-2.

Images