NITPA-S sensor for screening anticoagulant active ingredients in wind-transforming pill mother drug and application of NITPA-S sensor

Abstract

The invention belongs to the technical field of fluorescence sensors and traditional Chinese medicine active ingredient screening, and particularly relates to an NITPA-S sensor for screening an anticoagulant active ingredient in a wind-transforming pill mother drug and application of the NITPA-S sensor. The NITPA-S sensor is prepared from NITPA-Py-603 and a thrombin substrate S-2238, the sensor can specifically recognize thrombin, the thrombin and the external substrate S-2238 of the sensor are subjected to a specific hydrolysis reaction, the substrate is hydrolyzed, an NITPA probe is released, and the RIM effect disappears, so that the fluorescence intensity is reduced. According to the invention, the NITPA-S sensor is applied to successfully screen out the anticoagulant active components in the wind-transforming pill mother drug, wherein the anticoagulant active components comprise carracoline, talasamine, nioline, chalmannin and schaftoside. The invention provides a material basis and a theoretical basis for further development of efficient anticoagulant drugs in wind-transforming pills.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence sensors and screening of active ingredients of traditional Chinese medicines, and in particular to a NITPA-S sensor for screening anticoagulant active ingredients in Huafengdan mother medicine and applications thereof. Background Art

[0002] Huafengdan (Huafengdan) is composed of Radix Gastrodiae, Rhizoma Coptidis, Scorpio, Bombyx Batryticatus, Arisaema (processed), Perilla Leaf, Herba Schizonepetae, Atractylodes Rhizome, Realgar, Borax, Artificial Musk, Borneol, Sandalwood, and Cinnabar. It has the effects of calming wind and relieving spasms, clearing phlegm and invigorating the mind, and is clinically used to treat stroke. The Radix Gastrodiae is the main ingredient in Huafengdan. It is fermented in a cellar with ox bile water as the fermentation medium, under suitable temperature, humidity, and moisture conditions. The Radix Gastrodiae contains a variety of toxic Chinese medicinal ingredients, and only some of these compounds (such as neoaconitine, mesaconitine, aconitine, uracil, hypoxanthine, and xanthine) have been determined. Studies have shown that the components that enter the bloodstream of Huafengdan are derived from the Radix Gastrodiae, but the anticoagulant active ingredients in the Radix Gastrodiae are still unclear, limiting its further development and utilization.

[0003] Based on the above problems, the present invention constructed a NITPA-S sensor and combined it with UPLC-Q-TOF / MS technology to screen the highly active anticoagulant components in Huafengdan, aiming to provide technical support for explaining the mechanism of action of Huafengdan mother drug and further developing highly effective anticoagulant drugs in Huafengdan. Summary of the Invention

[0004] The object of the present invention is to provide a NITPA-S sensor for screening anticoagulant active components in Huafengdan mother medicine.

[0005] Another object of the present invention is to provide the application of the above-mentioned NITPA-S sensor in screening anticoagulant active components in Huafengdan mother drug.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The NITPA-S sensor for screening anticoagulant active components in the Huafengdan mother drug of the present invention comprises nanoparticles obtained by cross-linking NITPA-Py-603 and thrombin substrate S-2238.

[0008] The preparation method of the NITPA-S sensor is as follows:

[0009] Prepared in a brown vial with a total reaction volume of 2.0 mL. First, NITPA-Py-603 (50 μM) was dissolved in 90% H2O-THF solution. Subsequently, S-2238 (0.5 mg / mL) and the cross-linker glutaraldehyde (0.75%, v / v) were added and mixed at 200 rpm in the dark for 30 minutes. Finally, the reaction system was uncovered for 12 hours to evaporate the excess tetrahydrofuran. The prepared NITPA-S sensor was stored in a dark refrigerator at 4°C until use.

[0010] Application of the NITPA-S sensor of the present invention in screening anticoagulant active components in Huafengdan mother drug.

[0011] Preferably, the anticoagulant active ingredients in the Huafengdan mother drug are caracoline, talasamine, neolamine, chasmanine and shafotoside.

[0012] Preferably, the method for screening the anticoagulant active ingredient in the Huafengdan mother drug comprises the following steps:

[0013] S1 uses HPLC to separate the components in the mother extract of Huafengdan, collects the fractions at different time periods, blows them dry with nitrogen, and redissolves them with methanol solution to obtain the fractions at different time periods;

[0014] S2 uses NITPA-S sensor to detect the anticoagulant activity of the distillate solution at each time period and calculates the thrombin inhibition rate;

[0015] S3 screened fractions with higher thrombin inhibition rates, and used UPLC-Q-TOF / MS to qualitatively characterize the components, identifying the anticoagulant active components in the Huafengdan mother drug.

[0016] The preparation method of the mother herbal extract is as follows:

[0017] After grinding the stock, pass it through a No. 5 sieve. Accurately weigh 5.0 g and place it in a conical flask. Add 50.0 mL of 70% methanol solution, shake well, and sonicate for 30 minutes (power 500W). Subsequently, transfer the solution to a centrifuge tube and centrifuge at 14,000 rpm for 10 minutes. Pass the supernatant through a 0.22 μm microporous filter membrane, discard the first three drops of filtrate, collect the remaining filtrate, and place it in a liquid phase vial. Store in a refrigerator at 4°C until use.

[0018] Further preferably, in the method for screening the anticoagulant active ingredient in the Huafengdan mother drug, the collecting of fractions at different time periods in step S1 is specifically collecting the fractions every 60 seconds, for a total of 2 collections; the methanol solution is a 70% methanol solution; and the HPLC chromatographic conditions are specifically:

[0019] The chromatographic column was filled with octadecylsilane bonded silica gel; the column temperature was 40°C; the flow rate was 1.0 mL / min; the injection volume was 80 μL; 0.1% formic acid water was used as mobile phase A, and acetonitrile was used as mobile phase B, and gradient elution was performed; the elution procedure of the gradient elution was as follows:

[0020]

[0021] Further preferably, in the method for screening the anticoagulant active ingredient in the Huafengdan mother drug, step S2 is specifically implemented by the following steps:

[0022] S2-1: Mix the buffer solution and thrombin, shake well, and then perform the first incubation;

[0023] S2-2 added the distilled drug solution, shook well, and then incubated for the second time;

[0024] S2-3 Add NITPA-S sensor solution, shake well, and incubate for the third time;

[0025] After the S2-4 reaction was completed, the fluorescence intensity was measured using a microplate reader, and the thrombin inhibition rate was calculated using the standard curve method.

[0026] Still more preferably, in step S2 of the method for screening the anticoagulant active ingredient in the Huafengdan mother drug:

[0027] The buffer solution in step S2-1 is PBS buffer solution or Tris-HCl buffer solution, and the first incubation condition is: incubation at 37° C. for 5 minutes;

[0028] The second incubation conditions in step S2-2 are: incubation at 37°C for 10 min;

[0029] The third incubation condition in step S2-3 is: incubation at 25°C to 47°C for 10 to 30 minutes.

[0030] Still more preferably, in step S2 of the method for screening the anticoagulant active ingredient in the Huafengdan mother drug:

[0031] The buffer solution in step S2-1 is a PBS buffer solution, and its concentration in the incubation system is 10-20 mM;

[0032] The third incubation condition in step S2-3 is: incubation at 25°C to 37°C for 15 to 30 minutes.

[0033] Still more preferably, in step S2 of the method for screening the anticoagulant active ingredient in the Huafengdan mother drug:

[0034] The buffer solution in step S2-1 is a PBS buffer solution, and its concentration in the incubation system is 10 mM;

[0035] The third incubation condition in step S2-3 is: incubation at 37° C. for 20 min.

[0036] Further preferably, in the method for screening anticoagulant active ingredients in Huafengdan mother drug, the UPLC-Q-TOF / MS liquid chromatography conditions in step S3 are: using an electrospray ion source and full scan mode, sampling in positive ion and negative ion modes respectively; the ion spray voltage is 5500V and -4500V; the ion source temperature is 550°C; the gas pressure of the nebulizing gas and the drying gas is 55psi; the scanning range in TOF-MS and TOF-MS / MS mode is m / z 50-1500Da, and the declustering voltage DP is 80V / -80V; the collision energy CE in TOF-MS mode is 10V / -10V, and the collision energy CE in TOF-MS / MS mode is 35V / -35V.

[0037] Beneficial effects of the present invention:

[0038] 1. The present invention successfully constructed a NITPA-S sensor for screening anticoagulant active ingredients in Huafengdan mother medicine. The sensor can specifically identify thrombin. Through the NITPA-S sensor system parameter optimization experiment, it was found that the optimal incubation conditions for thrombin activity detection using the above sensor are: buffer solution is 10mM PBS; incubation temperature is 37℃; incubation time is 20min.

[0039] 2. The present invention adopts NITPA-S sensor and combines UPLC-Q-TOF / MS technology to screen out five highly active anticoagulant components from the Huafengdan mother drug for the first time: caracolline, talasamine, neolamine, chasmanine and shafotoside, which provides technical support for revealing the mechanism of action of the Huafengdan mother drug and further developing high-efficiency anticoagulant drugs in Huafengdan. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The effect of different buffer types and concentrations on thrombin-substrate reaction;

[0041] Figure 2 The effect of different incubation times on thrombin-substrate reaction;

[0042] Figure 3 The effect of different incubation temperatures on thrombin-substrate reaction;

[0043] Figure 4 is the linear regression curve of the difference between thrombin concentration and sensor fluorescence intensity;

[0044] Figure 5 The specificity analysis results of the sensor under the action of different proteases;

[0045] Figure 6 The experimental results of drug interference with NITPA-S sensor system are shown;

[0046] Figure 7 The anticoagulant activity verification test results of the parent drug;

[0047] Figure 8 The effect of 70% methanol solution on the sensor system (in the figure: A is H2O+thrombin+sensor; B is 70% methanol+thrombin+sensor; C is sensor; D is 70% methanol+sensor);

[0048] Figure 9 The inhibition rate of the extract fractions of the motherwort on thrombin at different time periods:

[0049] Figure 10 Schematic diagram of the screening process for anticoagulant active ingredients in Huafengdan mother drug;

[0050] Figure 11 This is the chemical structure of the anticoagulant active component screened out from the mother drug of Huafengdan. DETAILED DESCRIPTION

[0051] The following is a detailed description of the technical solution of the present invention in conjunction with specific embodiments. The following embodiments are only for explanation and illustration, and do not constitute a limitation of the technical solution of the present invention.

[0052] Example 1

[0053] The NITPA-S sensor, used to screen for anticoagulant active ingredients in the Huafengdan herbal formula, comprises nanoparticles cross-linked with NITPA-Py-603 and the thrombin substrate S-2238. The sensor is prepared as follows:

[0054] Prepared in a brown vial with a total reaction volume of 2.0 mL. First, NITPA-Py-603 (50 μM) was dissolved in 90% H2O-THF solution. Subsequently, S-2238 (0.5 mg / mL) and the cross-linker glutaraldehyde (0.75%, v / v) were added and mixed at 200 rpm in the dark for 30 minutes. Finally, the reaction system was uncovered for 12 hours to evaporate the excess tetrahydrofuran. The prepared NITPA-S sensor was stored in a dark refrigerator at 4°C until use.

[0055] Example 2

[0056] The NITPA-S sensor was used to screen the anticoagulant active components in the Huafengdan mother drug. The method is as follows:

[0057] (1) Prepare the mother drug solution of Huafengdan (crush the mother drug and pass it through a No. 5 sieve, accurately weigh 5.0 g and place it in a conical flask, add 50.0 mL of 70% methanol solution, shake well and ultrasonically treat for 30 minutes (power 500W). Then, transfer the solution to a centrifuge tube, centrifuge at 14000 r / min for 10 minutes, take the supernatant and pass it through a 0.22 μm microporous filter membrane, discard the first 3 drops of filtrate, collect the remaining filtrate and put it into a liquid phase vial, and store it in a 4°C refrigerator for later use). Use HPLC to separate the components in the mother drug extract, collect the fractions every 60 seconds, and collect twice in total; after blowing dry with nitrogen, redissolve it with 70% methanol solution to obtain the fraction solutions of different time periods.

[0058] The chromatographic conditions of HPLC are:

[0059] The chromatographic column was filled with octadecylsilane bonded silica gel; the column temperature was 40°C; the flow rate was 1.0 mL / min; 0.1% formic acid water was used as mobile phase A, and acetonitrile was used as mobile phase B, and gradient elution was performed; the elution procedure of the gradient elution was as follows:

[0060]

[0061] (2) Using the NITPA-S sensor, the anticoagulant activity of the fractions at each time period was detected, and the thrombin inhibition rate was calculated. The specific steps are as follows:

[0062] Accurately measure 40 μL of 10 mM PBS buffer solution and 10 μL of thrombin solution (500 U / mL), add them to a 96-well plate, shake well for 1 min, and incubate in a 37°C oven for 5 min.

[0063] Accurately measure 10 μL of the fraction solution prepared in the above steps and add it to the above 96-well plate, shake well for 1 minute, and incubate in a 37°C oven for 10 minutes.

[0064] 40 μL of NITPA-S sensor solution was added to each well in the dark, shaken for 1 min, and incubated in an oven at 37°C for 20 min.

[0065] After the reaction, the fluorescence intensity was measured (Exλ / Emλ=448 / 595 nm), and the thrombin inhibition rate was calculated using the standard curve method.

[0066] (3) The fractions with higher thrombin inhibition rates were screened and the components were characterized by UPLC-Q-TOF / MS to identify the anticoagulant active components in the Huafengdan mother drug.

[0067] The LC-MS conditions of UPLC-Q-TOF / MS are as follows:

[0068] An electrospray ion source and full scan mode were used for sampling in positive and negative ion modes, respectively; the ion spray voltage was 5500 V and -4500 V; the ion source temperature was 550°C; the nebulizer and drying gas pressures were 55 psi; the scan range in TOF-MS and TOF-MS / MS modes was m / z 50-1500 Da, the declustering voltage DP was 80 V / -80 V; the collision energy CE in TOF-MS mode was 10 V / -10 V, and the collision energy CE in TOF-MS mode was 35 V / -35 V.

[0069] Example 3

[0070] The method of Example 2 was used to screen out the following five highly active anticoagulant ingredients in the Huafengdan mother drug: caracoline, talasamine, neolamine, chasmanine and shafotoside.

[0071] In order to further verify the reliability of the present invention and screen out the best solution, the inventors conducted a series of experiments, as follows:

[0072] 1. Instruments and Materials

[0073] The main instruments are shown in Table 1, and the materials and reagents are shown in Table 2.

[0074] Table 1 Main instruments

[0075]

[0076] Table 2 Materials and reagents

[0077]

[0078]

[0079] 2. Experimental methods

[0080] 2.1 Detection principles of thrombin and antithrombin substances

[0081] Thrombin undergoes a specific hydrolysis reaction with the sensor's external substrate, S-2238, resulting in substrate hydrolysis and the release of the NITPA probe, which eliminates the RIM effect and reduces fluorescence intensity. As thrombin concentration increases, fluorescence intensity decreases linearly, as shown by a standard curve (the vertical axis represents the decrease in fluorescence intensity at different thrombin concentrations, and the horizontal axis represents thrombin concentration).

[0082] When screening for thrombin inhibitors, the difference in fluorescence intensity directly reflects the amount of thrombin consumed, allowing the compound's thrombin inhibition rate to be calculated. Based on the compound's thrombin inhibition rate, potential thrombin inhibitors can be screened.

[0083] 2.2 Preparation of NITPA-S sensor

[0084] The NITPA-S sensor was synthesized in a brown vial with a total reaction volume of 2.0 mL. First, NITPA-Py-603 (50 μM) was dissolved in a 90% H₂O-THF solution. Subsequently, S-2238 (0.5 mg / mL) and the crosslinker glutaraldehyde (0.75%, v / v) were added and mixed at 200 rpm in the dark for 30 minutes. Finally, the reaction system was uncovered for 12 hours to evaporate excess tetrahydrofuran. The prepared NITPA-S sensor was stored in a dark refrigerator at 4°C until ready for use.

[0085] 2.3 NITPA-S sensor system parameter optimization

[0086] The hydrolysis of substrate by thrombin is greatly affected by environmental factors. The research team investigated the effects of different buffers, incubation temperatures and incubation times on the NITPA-S sensor system to determine the optimal hydrolysis conditions for the NITPA-S sensor system.

[0087] 2.3.1 Effect of buffer on NITPA-S sensor system

[0088] The research team prepared different concentrations of PBS buffer and Tris-HCl buffer to investigate their effects on the NITPA-S sensor system, as follows:

[0089] (1) Preparation of thrombin solution: Accurately weigh 2000 U / mg of thrombin and dissolve it in an appropriate amount of ultrapure water to prepare a 500 U / mL thrombin solution.

[0090] (2) Preparation of buffer solution: Accurately measure an appropriate amount of 1M Tris-HCl, place it in a volumetric flask, add ultrapure water, and prepare Tris-HCl buffer solutions with concentrations of 25mM and 50mM; accurately weigh an appropriate amount of PBS powder, add ultrapure water to dissolve it, and prepare PBS buffer solutions with concentrations of 25mM and 50mM.

[0091] (3) Thrombin activity detection:

[0092] Accurately measure 40 μL of 50 mM PBS, 25 mM PBS, 50 mM Tris-HCl, and 25 mM Tris-HCl buffer solutions into a 96-well plate, add 10 μL of 500 U / mL thrombin solution, shake well for 1 min, and incubate in a 37°C oven for 5 min.

[0093] Accurately measure 10 μL of ultrapure water and add it to the above 96-well plate, shake it for 1 minute, and incubate it in a 37°C oven for 10 minutes.

[0094] 40 μL of NITPA-S sensor solution was added under light-protected conditions, shaken for 1 min, and incubated in an oven at 37°C for 20 min.

[0095] After the reaction was completed, the fluorescence intensity was measured (Exλ / Emλ=448 / 595 nm).

[0096] 2.3.2 Effect of incubation time on the NITPA-S sensor system

[0097] The research team investigated the effects of different incubation times on the NITPA-S sensor system, as follows:

[0098] Accurately measure 5 portions of 40 μL PBS buffer solution into 96-well plates, add 10 μL of 500 U / mL thrombin solution to each well, shake for 1 min, and incubate in a 37°C oven for 5 min.

[0099] Accurately measure 10 μL of ultrapure water and add it to the above 96-well plate, shake it for 1 minute, and incubate it in a 37°C oven for 10 minutes.

[0100] 40 μL of NITPA-S sensor solution was added to each sample in the dark, shaken for 1 min, and incubated in a 37°C oven for 10 min, 15 min, 20 min, 25 min, and 30 min, respectively.

[0101] After the reaction was completed, the fluorescence intensity was measured (Exλ / Emλ=448 / 595 nm).

[0102] 2.3.3 Effect of incubation temperature on the NITPA-S sensor system

[0103] Three 40 μL portions of PBS buffer solution were accurately measured and placed in 96-well plates. 10 μL of 500 U / mL thrombin solution was added to each well. The plates were shaken for 1 min and incubated in a 37°C oven for 5 min.

[0104] Accurately measure 10 μL of ultrapure water and add it to the above 96-well plate, shake it for 1 minute, and incubate it in a 37°C oven for 10 minutes.

[0105] 40 μL of NITPA-S sensor solution was added to each sample in the dark, shaken for 1 min, and incubated in an oven at 25°C, 37°C, and 47°C for 20 min.

[0106] After the reaction was completed, the fluorescence intensity was measured (Exλ / Emλ=448 / 595 nm).

[0107] 2.4 Maximum Thrombin Activity Detection

[0108] The NITPA-S sensor can be used not only to screen for thrombin inhibitors, but also for thrombin. Using fluorescence differences, the concentration of thrombin can be detected. When screening for thrombin inhibitors, an appropriate amount of thrombin needs to be added to react with the sensor. Determining the maximum thrombin concentration in a given sensor solution is more conducive to screening for thrombin inhibitors and prevents excessive thrombin from causing the sensor to fail to emit fluorescence. Therefore, the research team conducted a test for the maximum thrombin activity, as follows:

[0109] (1) Preparation of thrombin stock solution: Accurately weigh 2000 U / mg of thrombin and dissolve it in an appropriate amount of ultrapure water to prepare a 2000 U / mL thrombin stock solution.

[0110] (2) Preparation of gradient dilutions of thrombin: Use PBS buffer to dilute the thrombin stock solution to 1000U / mL, 800U / mL, 500U / mL, 400U / mL, 300U / mL, 250U / mL, 125U / mL, 62.5U / mL and 31.25U / mL, respectively. After adding to a 96-well plate, the final corresponding concentrations are gradient dilutions of 100U / mL, 80U / mL, 50U / mL, 40U / mL, 30U / mL, 25U / mL, 12.5U / mL, 6.25U / mL and 3.125U / mL.

[0111] (3) Thrombin activity detection:

[0112] Accurately measure 40 μL of PBS buffer solution into a 96-well plate, add 10 μL of thrombin gradient dilution solution, shake well for 1 minute, and incubate in a 37°C oven for 5 minutes.

[0113] Accurately measure 10 μL of ultrapure water and add it to the above 96-well plate, shake it for 1 minute, and incubate it in a 37°C oven for 10 minutes.

[0114] 40 μL of NITPA-S sensor solution was added to each well in the dark, shaken for 1 min, and incubated in an oven at 37°C for 20 min.

[0115] After the reaction was completed, the fluorescence intensity was measured (Exλ / Emλ=448 / 595 nm).

[0116] 2.5 Verification of the specificity of thrombin and sensor

[0117] To verify the specificity between the NITPA-S sensor and thrombin, this study used α-glucosidase (α-glu), albumin, creatine kinase (CK), superoxide dismutase (SOD), lactate dehydrogenase (LDH) and thrombin to incubate the sensor under the same conditions as follows:

[0118] (1) Preparation of different enzyme solutions: Accurately weigh appropriate amounts of various proteases and dissolve them in ultrapure water to prepare various protease solutions with a concentration of 0.25 mg / mL.

[0119] (2) Activity detection of different types of enzyme solutions:

[0120] Accurately measure 40 μL of PBS buffer solution and 10 μL of various protease solutions, add them to the 96-well plate respectively, shake for 1 min, and incubate in a 37°C oven for 5 min.

[0121] Accurately measure 10 μL of ultrapure water and add it to the above 96-well plate, shake it for 1 minute, and incubate it in a 37°C oven for 10 minutes.

[0122] 40 μL of NITPA-S sensor solution was added to each well in the dark, shaken for 1 min, and incubated in an oven at 37°C for 20 min.

[0123] After the reaction was completed, the fluorescence intensity was measured (Exλ / Emλ=448 / 595 nm).

[0124] 2.6 Eliminating the interference of the parent drug on the NITPA-S sensor system

[0125] To verify whether the drug precursor affects the fluorescence intensity of the NITPA-S sensor system, the research team measured the fluorescence intensity of the drug precursor alone and the fluorescence intensity after the drug precursor and NITPA-S sensor were incubated together, as follows:

[0126] (1) Determination of fluorescence intensity of single drug parent: Prepare drug parent solution (crush the drug parent and pass it through a No. 5 sieve. Accurately weigh 5.0 g and place it in a conical flask. Add 50.0 mL of 70% methanol solution, shake well, and ultrasonicate for 30 min (power 500 W). Then, transfer the solution to a centrifuge tube, centrifuge at 14,000 r / min for 10 min, take the supernatant and filter it through a 0.22 μm microporous membrane. Discard the first 3 drops of filtrate, collect the remaining filtrate and place it in a liquid phase vial, and store it in a refrigerator at 4°C for later use). Then, dilute the drug parent solution 10 times. Accurately measure 10 μL of the diluted drug parent solution and 90 μL of ultrapure water and add them to a 96-well plate. Shake well for 1 min, incubate in a 37°C oven for 20 min, and then measure the fluorescence intensity (Exλ / Emλ=448 / 595 nm).

[0127] (2) Incubation of the drug precursor with the NITPA-S sensor: 40 μL of PBS buffer solution was added to a 96-well plate. 10 μL of the drug precursor extract and 40 μL of the NITPA sensor solution were accurately measured and mixed. The plates were shaken for 1 min and incubated in an oven at 37°C for 20 min. The fluorescence intensity (Exλ / Emλ=448 / 595 nm) was measured.

[0128] 2.7 Verification of the anticoagulant activity of the stock solution

[0129] To screen for anticoagulant active components in the stock, it is first necessary to confirm that the stock extract, after incubation with thrombin and the NITPA-S sensor, exhibits fluorescence activity at the excitation wavelength of this study. Only then can each component be used to screen for thrombin, as follows:

[0130] Accurately measure 40 μL of PBS buffer solution and 10 μL of thrombin solution (500 U / mL), add them to a 96-well plate, shake for 1 min, and incubate in a 37°C oven for 5 min.

[0131] The mother extract extracted with 70% methanol solution was diluted 100 times, and 10 μL of the dilution was accurately measured and added to a 96-well plate, shaken for 1 minute, and incubated in an oven at 37°C for 10 minutes.

[0132] 40 μL of NITPA-S sensor solution was added to each well in the dark, shaken for 1 min, and incubated in an oven at 37°C for 20 min.

[0133] After the reaction was completed, the fluorescence intensity was measured (Exλ / Emλ=448 / 595 nm).

[0134] 2.8 Determination of anticoagulant activity of different fractions

[0135] (1) Preparation of distillate solutions at different time periods

[0136] The drug stock solution was separated by HPLC using an XBridge HPLC column (C 18 , 4.6×250mm, 5μm); gradient elution was performed with 0.1% formic acid in water as mobile phase A and acetonitrile as mobile phase B (elution program shown in Table 3); column temperature was set to 40°C; flow rate was set to 1.0mL / min; injection volume was 80μL. HPLC and an automatic collector were used to separate and collect fractions, with fractions collected every 60 seconds for a total of two collections. The fractions were then blown dry with nitrogen and reconstituted with 70% methanol to obtain fractions at different time intervals.

[0137] Table 3 HPLC gradient elution program

[0138]

[0139] (2) Determination of anticoagulant activity of different fractions

[0140] The components have an inhibitory effect on thrombin, resulting in thrombin being unable to completely hydrolyze the substrate, which will produce partial fluorescence. The control group does not add any thrombin inhibitors, and its fluorescence intensity will be greatly reduced. The anticoagulant activity of different fractions can be determined from the fluorescence difference between them. The specific experiment is as follows:

[0141] Fluorescence measurement of the control group: Accurately measure 40 μL of PBS buffer solution and 10 μL of thrombin stock solution (500 U / mL), add them to a 96-well plate, shake for 1 minute, and incubate in a 37°C oven for 5 minutes; accurately measure 10 μL of ultrapure water and add it to the above 96-well plate, shake for 1 minute, and incubate in a 37°C oven for 10 minutes; add 40 μL of NITPA-S sensor solution respectively under light-proof conditions, shake for 1 minute, and incubate in a 37°C oven for 20 minutes; after the reaction, measure the fluorescence intensity (Exλ / Emλ=448 / 595nm) and record it as A1.

[0142] Fluorescence determination of the experimental group: replace 10 μL of ultrapure water in the control group with the sample to be tested. The other steps are the same as those in “Fluorescence determination of the control group”. After the reaction is completed, measure the fluorescence intensity and record it as A2.

[0143] Calculation of inhibition rate: Substitute the fluorescence difference (A2-A1) between the experimental group and the control group into the standard curve, calculate the amount of thrombin consumed by different fractions, and then divide it by 50 U / mL to obtain the inhibition rate.

[0144] 2.9 Eliminating the interference of the solvent of the distillate solution on thrombin

[0145] To verify whether the 70% methanol solution has an impact on the sensor system, the research team conducted the following experiments:

[0146] Accurately measure 40 μL of PBS buffer solution and add it to a 96-well plate, labeled A, B, C, and D. Add 10 μL of thrombin stock solution (500 U / mL) to groups A and B, respectively. Groups C and D serve as blank controls without thrombin. Shake well for 1 min and incubate in a 37°C oven for 5 min.

[0147] Take 10 μL of 70% methanol solution from two portions and add it to the 96-well plates B and D. Take 10 μL of water from two portions and add it to the 96-well plates A and C. Shake well for 1 minute and incubate in a 37°C oven for 10 minutes.

[0148] 40 μL of NITPA-S sensor solution was added to each well under light-protected conditions, shaken for 1 min, and incubated in an oven at 37 °C for 20 min;

[0149] After the reaction was completed, the fluorescence intensity was measured (Exλ / Emλ=448 / 595 nm).

[0150] 2.10 Identification of Anticoagulant Active Components

[0151] The fractions with the highest thrombin inhibition rate were subjected to UPLC-Q-TOF / MS to determine the anticoagulant active components in the fractions. The compounds were identified according to the principle of "high ion responsiveness, strong single active component and easy availability of compound reference substances".

[0152] The mass spectrometry conditions for UPLC-Q-TOF / MS were as follows: an electrospray ion source and full scan mode were used, with sampling performed in positive and negative ion modes, respectively; the ion spray voltage was 5500 V and -4500 V; the ion source temperature was 550°C; the nebulizer and drying gas pressures were 55 psi; the scan range in TOF-MS and TOF-MS / MS modes was m / z 50–1500 Da, and the declustering voltage (DP) was 80 V / -80 V; the collision energy (CE) in TOF-MS mode was 10 V / -10 V, and that in TOF-MS / MS mode was 35 V / -35 V.

[0153] 3. Experimental results

[0154] 3.1 Results of NITPA-S sensor system parameter optimization

[0155] Thrombin is a protease, and its activity is affected by the type, concentration, temperature, and reaction time of the buffer solution. Therefore, the hydrolysis reaction between thrombin and the sensor's external substrate requires optimized incubation conditions. As the substrate is hydrolyzed, the NITPA probe is released, the RIM effect disappears, and the fluorescence intensity decreases. Therefore, the fluorescence intensity difference (F0-F, where F0 is the fluorescence value of the sensor before the reaction and F is the fluorescence value after the reaction) can be used as an indicator of thrombin activity; a larger difference indicates stronger thrombin activity.

[0156] 3.1.1 Effect of buffer on NITPA-S sensor system

[0157] Effects of different buffer types and concentrations on thrombin-substrate reaction Figure 1 As shown in the results, thrombin is most active in 10mM PBS. Furthermore, in the same type of buffer solution, thrombin activity in 10mM PBS is higher than in 20mM PBS, indicating that thrombin requires an appropriate buffer concentration, rather than a higher concentration resulting in greater activity. Therefore, 10mM PBS was selected as the buffer for thrombin.

[0158] 3.1.2 Effect of incubation time on the NITPA-S sensor system

[0159] Effects of different incubation times on thrombin-substrate reaction Figure 2 As shown in the results, the fluorescence intensity of the sensor gradually decreased with increasing reaction time between 10 and 30 minutes. After 20 minutes of incubation, the change in fluorescence intensity leveled off, indicating that thrombin was completely hydrolyzed. Therefore, 20 minutes was selected as the optimal reaction time.

[0160] 3.1.3 Effect of incubation temperature on the NITPA-S sensor system

[0161] Thrombin is a biological protease, and its activity is affected by temperature. The research team investigated the effects of 25°C, 37°C, and 47°C on the thrombin-substrate reaction. The results are as follows: Figure 3 As shown in the figure, thrombin is most active at 37°C, capable of hydrolyzing the sensor's external substrate to the greatest extent, significantly reducing the sensor's fluorescence intensity. At 47°C, thrombin activity is inhibited, failing to effectively reduce the sensor's fluorescence intensity. Therefore, the optimal incubation temperature was determined to be 37°C.

[0162] In summary, the optimal thrombin incubation conditions are: adding thrombin to 10 mM PBS buffer solution, followed by adding 40 μL NITPA-S sensor, and incubating at 37°C for 20 min.

[0163] 3.2 Thrombin maximum activity test results

[0164] The concentration of thrombin directly affects the fluorescence of the NITPA-S sensor. The linear regression curve of the difference between thrombin concentration and sensor fluorescence intensity is shown in Figure 4 As shown. Figure 4 As can be seen, the sensor's fluorescence intensity difference (F0-F1, where F0 is the initial sensor fluorescence value and F1 is the value after the sensor reacts with thrombin) gradually decreases with increasing thrombin activity. When thrombin activity ranges from 3.15 U / mL to 100 U / mL, the fluorescence intensity at 595 nm decreases with increasing thrombin activity. The nonlinear fitting equation is Y = 11.721ln(X)-12.529, where Y is the fluorescence intensity difference before and after the reaction (F0-F1) and X is the thrombin concentration. When the thrombin activity reaches 50 U / mL, the fluorescence intensity drops to its lowest point, indicating that 50 U / mL of thrombin can completely hydrolyze the sensor. Different concentrations of thrombin can reduce the fluorescence difference by a certain amount. Conversely, the amount of thrombin consumed by the active ingredient to restore the sensor's fluorescence difference is proportional to the amount of thrombin it consumes, allowing for the screening of thrombin inhibitors. In subsequent screening of thrombin inhibitors in drug stock, the thrombin concentration was set at 50 U / mL.

[0165] 3.3 Experimental results of thrombin and sensor specificity

[0166] Thrombin hydrolyzes the external substrate of NITPA-S sensor with specificity, which is a characteristic of bioluminescent sensor, namely biological specific recognition. The specificity analysis results of the sensor under the action of different proteases are shown in Figure 2. Figure 5 As shown in the results, with the exception of thrombin, no other proteases reacted significantly with the sensor. Upon addition of thrombin, the fluorescent sensor was hydrolyzed, resulting in the greatest decrease in fluorescence intensity. Therefore, the NITPA-S sensor demonstrates specific recognition of thrombin.

[0167] 3.4 Experimental results of drug interference with NITPA-S sensor system

[0168] In order to screen the anticoagulant component in the drug precursor, it is first necessary to confirm that it does not interfere with the fluorescence of the sensor, so that the change in the sensor fluorescence intensity can be used to screen the best inhibitor in the drug precursor. The results of the drug precursor interference experiment on the NITPA-S sensor system are shown in the figure. Figure 6 The results show that the drug stock solution has no fluorescence response at an excitation wavelength of 448 nm, and after incubation with the NITPA-S sensor, the fluorescence intensity of the NITPA-S sensor is almost unaffected, indicating that the drug stock solution is suitable for screening its antithrombin component using this sensor.

[0169] 3.5 Verification of the Anticoagulant Activity Experimental Results of the Drug Parent

[0170] The mother drug has antithrombotic effects and is often used in combination with other drugs to treat ischemic stroke, with significant clinical efficacy. The research team verified the anticoagulant activity of the mother drug, and the results were as follows: Figure 7 The results show that after adding the drug mother, the fluorescence intensity of the solution increased compared with the group without drug mother (p < 0.05), indicating that the drug mother contains thrombin inhibitors.

[0171] 3.6 Results of the Interference Experiment on Thrombin by the Solvents of the Fractionated Drug Solution

[0172] The effect of 70% methanol solution on the sensor system is as follows Figure 8 As shown in the figure: A is H2O+thrombin+sensor; B is 70% methanol+thrombin+sensor; C is sensor; D is 70% methanol+sensor. Figure 8 It can be seen that the 70% methanol solution has no interference with the sensor system. The fraction can be subsequently re-dissolved with the 70% methanol solution for subsequent determination of the antithrombin activity of the fraction.

[0173] 3.7 Anticoagulant activity assay results of fractions at different time periods

[0174] The inhibition rate of thrombin by the extracts of the mother herb at different time periods is as follows Figure 9In this study, the inhibition rates of 60 fractions were obtained, and groups 22, 35, 36, 37, and 38 were ultimately selected as highly active fractions for subsequent component identification.

[0175] 3.8 Results of the Identification Experiment of Anticoagulant Active Components

[0176] The obtained components 22, 35, 36, 37 and 38 were analyzed by UPLC-Q-TOF / MS. Combined with the qualitative data from mass spectrometry and relevant literature, five main active ingredients were finally identified: caracoline, neorin, shafotoside, chasmanine and talasamine. Figure 10 As shown, the chemical structure of the above-mentioned anticoagulant active ingredient is shown in Figure 11.

[0177] Although the present invention has been described in detail above using general descriptions, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. The NITPA-S sensor used to screen the anticoagulant active ingredients in the Huafengdan mother drug is characterized by: The sensor includes nanoparticles obtained by cross-linking NITPA-Py-603 with the thrombin substrate S-2238.

2. Use of the NITPA-S sensor as claimed in claim 1 in screening anticoagulant active ingredients in Huafengdan medicinal stock.

3. The application according to claim 2, characterized in that: The anticoagulant active ingredients in the Huafeng Pill mother drug are caracoline, talasamine, neolamine, chasmanine and shafotoside.

4. The use according to claim 2, characterized in that The method for screening the anticoagulant active component in the Huafengdan mother drug comprises the following steps: S1 uses HPLC to separate the components in the mother extract of Huafengdan, collects the fractions at different time periods, blows them dry with nitrogen, and redissolves them with methanol solution to obtain the fractions at different time periods; S2 uses NITPA-S sensor to detect the anticoagulant activity of the distillate solution at each time period and calculates the thrombin inhibition rate; S3 screened fractions with higher thrombin inhibition rates, and used UPLC-Q-TOF / MS to qualitatively characterize the components, identifying the anticoagulant active components in the Huafengdan mother drug.

5. The use according to claim 4, characterized in that In the method for screening the anticoagulant active ingredient in the Huafengdan mother drug, the collection of fractions at different time periods in step S1 is specifically collecting fractions every 60 seconds, for a total of 2 collections; the methanol solution is a 70% methanol solution; and the HPLC chromatographic conditions are specifically as follows: The chromatographic column was filled with octadecylsilane bonded silica gel; the column temperature was 40°C; the flow rate was 1.0 mL / min, and the injection volume was 80 μL; 0.1% formic acid water was used as mobile phase A, and acetonitrile was used as mobile phase B, and gradient elution was performed; the elution program of the gradient elution was as follows:

6. The use according to claim 4, characterized in that In the method for screening the anticoagulant active ingredient in the Huafengdan mother drug, step S2 is specifically implemented by the following steps: S2-1: Mix the buffer solution and thrombin, shake well, and perform the first incubation; S2-2: Add the distilled drug solution, shake well, and incubate for the second time; S2-3: Add NITPA-S sensor solution, shake well, and incubate for the third time; S2-4: After the reaction is completed, the fluorescence intensity is measured using a microplate reader, and the thrombin inhibition rate is calculated using the standard curve method.

7. The use according to claim 6, characterized in that In step S2 of the method for screening the anticoagulant active ingredient in the Huafengdan mother drug: The buffer solution in step S2-1 is PBS buffer solution or Tris-HCl buffer solution, and the first incubation condition is: incubation at 37° C. for 5 minutes; The second incubation conditions in step S2-2 are: incubation at 37°C for 10 min; The third incubation condition in step S2-3 is: incubation at 25°C to 47°C for 10 to 30 minutes.

8. The use according to claim 7, characterized in that In step S2 of the method for screening the anticoagulant active ingredient in the Huafengdan mother drug: The buffer solution in step S2-1 is a PBS buffer solution, and its concentration in the incubation system is 10-20 mM; The third incubation condition in step S2-3 is: incubation at 25°C to 37°C for 15 to 30 minutes.

9. The use according to claim 8, characterized in that In step S2 of the method for screening the anticoagulant active ingredient in the Huafengdan mother drug: The buffer solution in step S2-1 is a PBS buffer solution, and its concentration in the incubation system is 10 mM; The third incubation condition in step S2-3 is: incubation at 37° C. for 20 min.

10. The use according to claim 4, characterized in that In the method for screening anticoagulant active ingredients in the Huafengdan herbal formula, the UPLC-Q-TOF / MS conditions in step S3 are as follows: using an electrospray ion source and full scan mode, sampling in positive ion and negative ion modes, respectively; the ion spray voltage is 5500V and -4500V; and the ion source temperature is 550°C. The gas pressure of the nebulizer gas and the drying gas was 55 psi; the scanning range in TOF-MS and TOF-MS / MS modes was m / z 50-1500 Da, and the declustering voltage DP was 80 V / -80 V; the collision energy CE in TOF-MS mode was 10 V / -10 V, and the collision energy CE in TOF-MS / MS mode was 35 V / -35 V.

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