Method for detecting biological effect of Xinshu tablet

Abstract

The invention belongs to the field of drug detection, and particularly discloses a method for detecting a biological effect of a Xinshu tablet, which comprises the following steps of: fishing targeted protein active ingredients of the Xinshu tablet by adopting a surface plasma resonance instrument with a built-in TGF-beta protein chip, and detecting the biological effect of the Xinshu tablet by adopting an SPR (Surface Plasmon Resonance) method based on a fished biological quality marker. According to the method, the quality of the Xinshu tablet is controlled by adopting a surface plasmon resonance method, evaluation of the overall drug effect and composition are combined, the quality of the Xinshu tablet is controlled more comprehensively, and the method has practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of drug detection, and in particular relates to a method for detecting the biological effects of Xinshubao tablets. Background Art

[0002] Xinshubao Tablets, listed in Volume 14 of the Ministry of Health's Standardized Traditional Chinese Medicine Formulas (National Medicine Approval No. Z35020241), are produced by Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd. and are primarily used clinically to treat coronary heart disease, chest tightness and angina pectoris caused by qi deficiency and blood stasis, as well as hypertension, hyperlipidemia, and arteriosclerosis. The formula is based on the theory that "qi is the commander of blood, qi circulates through blood, and unblocked blood leads to relief of pain." It utilizes salvia miltiorrhiza and hawthorn to promote blood circulation, dissipate stasis, and promote menstruation; while white peony root and curcuma root calm the liver, promote qi circulation, and clear the heart to disperse stasis. These four herbs work together to regulate blood circulation, promote qi circulation, disperse stasis, and relieve pain, effectively treating the symptoms. However, a single herb, Acanthopanax senticosus, is used to address the root cause. The ancients said, "It is better to have a handful of Acanthopanax senticosus than a cart full of gold and jade," demonstrating its profound tonic properties. "Compendium of Materia Medica" points out that Acanthopanax can "strengthen muscles and bones, strengthen will", "break up evil wind and blood", and is mainly used to treat "heart, abdomen, hernia and abdominal pain". It is a medicine that can both nourish and dispel evil. The various medicines in this prescription can treat both the symptoms and the root cause of cardiovascular diseases.

[0003] At present, the specific pharmacological substances of Xinshubao tablets are still unclear, and the existing national standard (WS3-B-2667-97) is still imperfect. Therefore, it is urgent to use modern analytical technology to clarify the material basis of Xinshubao tablets and to construct biological and chemical quality evaluation methods to improve its quality standards.

[0004] Surface plasmon resonance analysis technology (SPR) is a refractometer. When a substance binds to a ligand fixed on the surface of a gold film, the refractive index of the gold film surface changes to capture its signal. This technology is the only molecular interaction technology approved by the U.S. and Japanese Pharmacopoeias. It has the advantages of real-time monitoring, high sensitivity, and simple sample pretreatment. At present, it has become an important means to discover the chemical component targets of traditional Chinese medicine, and it is also one of the important methods for biological quality evaluation. However, since SPR cannot characterize the molecular structure information of fishing components, it needs to be combined with UPLC-MS / MS technology to realize the analysis and identification of the structure of fishing components.

[0005] So far, there has been no report on combining SPR technology and UPLC-MS / MS technology to identify the active ingredients of Xinshubao tablets, and there has been no research on establishing a method for detecting the biological effects of Xinshubao tablets by analyzing the identified ingredients. Summary of the Invention

[0006] The purpose of the present invention is to screen the biological quality markers of Xinshubao tablets based on target fishing technology, and to establish a biological effect detection method of Xinshubao tablets using the biological quality markers to accurately control the quality of Xinshubao tablets and ensure the efficacy of Xinshubao tablets.

[0007] The present invention provides a fishing method using the active ingredient of Xinshubao tablets, comprising the following steps:

[0008] A1: Construct protein chip: Dissolve TGF-β protein in water, then dilute with sodium acetate buffer to a 30-50 μg / ml protein solution. Couple the protein solution to the sensor chip.

[0009] A2 Preparation of fishing test solution: Take Xinshubao tablets, add methanol solution to extract, centrifuge the extract, take the supernatant, dry it, add DMSO to dissolve it, filter it, and add PBS solution to the filtrate to mix well.

[0010] A3: inject the sample solution obtained in step A2 into a surface plasmon resonance instrument loaded with the protein chip obtained in step A1 for testing;

[0011] Fishing conditions are as follows: mobile phase: PBS buffer solution containing DMSO; sample flow rate 1-10 μL·min -1 Injection time: 10-30 seconds; Protein binding time: 2-5 minutes; Dissociation reagent: TFA solution, Dissociation time: 15-30 seconds; Recovery reagent: NH4HCO3 solution, Cycle number: 1-100 times;

[0012] A4 uses UPLC-Q-TOF-MS method to perform qualitative detection on the fishing component recovery liquid obtained in step A3.

[0013] Furthermore, the concentration of the sodium acetate buffer in step A1 is 10 mM, and the pH value is 5; the protein solution is 50 μg / ml; the coupling is amino coupling; the parameters of the amino coupling are: sensor chip model: CM5, coupling reagents: EDC and NHS, protein solution flow rate: 5 μl / min, protein binding time: 720 s, temperature: 25°C, blocking reagent: ethanolamine, and blocking time: 7 min;

[0014] Furthermore, the volume ratio of the protein solution, EDC, NHS and ethanolamine is 100:200:140; and the volume ratio of EDC and NHS is 1:1.

[0015] Furthermore, in step A2, the mass volume ratio of the Xinshubao tablet to the methanol solution is 0.1 g: 5-25 ml; the concentration of the methanol solution is 60%;

[0016] The extraction is ultrasonic extraction, and the time is 20 to 60 minutes;

[0017] The centrifugal speed is 10000-20000r, and the time is 5-20min;

[0018] The volume ratio of the supernatant, DMSO, filtrate and PBS buffer is 0.5-1.5 mL: 100 μL: 10 μl: 190 μL; the PBS solution is PBS 1× solution;

[0019] and / or:

[0020] The fishing conditions in step A3 are as follows: mobile phase: PBS buffer solution containing 5% DMSO; sample flow rate 5 μL·min -1 ; Injection time: 20s; Protein binding time 3min; Dissociation reagent: 0.5% TFA solution, dissociation time: 20s; Recovery reagent: 50μM NH4HCO3 solution, Number of cycles: 10 times.

[0021] Furthermore, the qualitative detection in step A4 includes the following steps:

[0022] ① Preparation of reference solution: Take salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid and lithospermic acid reference substances, dissolve and dilute with methanol solution to obtain the reference solution;

[0023] ② The recovered fishing component solution was dried and dissolved in 60% methanol. The dissolved solution and the reference solution obtained in step ① were injected into UPLC-Q-TOF-MS for detection respectively;

[0024] Chromatographic conditions are as follows: chromatographic column: C18 column; mobile phase: acetonitrile-0.1% formic acid water gradient elution program: 0-16.5 min, 7-7% acetonitrile; 16.5-17.50 min, 7-12% acetonitrile; 17.50-22.50 min, 12-16% acetonitrile; 22.5-32.0 min, 16-22% acetonitrile; 32.0-38.0 min, 22-30% acetonitrile; 38.0-41.0 min, 30-52% acetonitrile; 41.0-46.00 min, 52-57% acetonitrile; 46.0-46.1 min, 57-7% acetonitrile; 46.1-48.0 min, 7-7% acetonitrile;

[0025] The mass spectrometry conditions were as follows: ion source: electrospray ion source, positive and negative ion modes; data acquisition mode: full scan mode, range m / z 50-1500.

[0026] Furthermore, the mass concentration of each reference substance in the reference solution of step ① is 0.5-1.5 μg·mL -1 .

[0027] Furthermore, the C18 chromatographic column in the chromatographic conditions of step ② is CORTECS TM UPLC C18 , 2.1mm×100mm, 1.6μm, flow rate 0.25mL·min -1 , column temperature 45°C; injection volume 2 μL;

[0028] The mass spectrometry conditions include a capillary voltage of +3.5 kV in positive ion mode, a capillary voltage of -4.5 kV in negative ion mode, a nebulizer pressure of 2.0 bar, N2 as the drying gas, an N2 volume flow rate of 4.0 L / min, a drying gas temperature of 180°C, a flight migration parameter of 200.0 Vpp, a quadrupole ion energy of 3.0 eV, a collision cell of 150.0 Vpp, an ion transmission time of 80 μs, a pre-pulse storage time of 5 μs, collision gas argon, and a collision energy of 10-50 eV.

[0029] Furthermore, the active ingredients fished out are salvianolic acid A, salvianolic acid C, salvianolic acid D, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, lithospermic acid, salvianolic acid B, chlorogenic acid and rosmarinic acid.

[0030] The present invention also provides a method for detecting the biological effect of Xinshubao tablets, which adopts surface plasmon resonance detection and comprises the following steps:

[0031] 1) Using a surface plasmon resonance instrument to measure the RU value of Xinshubao tablets and the RU value of salvianolic acid B, wherein the protein chip in the surface plasmon resonance instrument is a TGF-β protein chip;

[0032] 2) The ratio is calculated using the following formula. If the ratio is not less than 1, the biological effect test of Xinshubao tablets is qualified;

[0033] Ratio = RU value of Xinshubao tablets / RU value of salvianolic acid B.

[0034] Furthermore, the steps for determining the RU value of Xinshubao tablets and the RU value of salvianolic acid B are as follows:

[0035] A test solution of Xinshubao tablets and a reference solution of salvianolic acid B were placed in a surface plasmon resonance test plate loaded with a TGF-β protein chip. SPR assay conditions included a run time of 60 s, a flow rate of 30 μl / min, a dissociation time of 60 s, a temperature of 25°C, and a running buffer of 5% DMSO in PBS.

[0036] The Xinshubao tablet test solution is prepared by taking Xinshubao tablets according to the method for preparing the fishing test solution described in step A2;

[0037] The salvianolic acid B reference solution is prepared by dissolving salvianolic acid B in DMSO, adding PBS solution and PBS solution containing 5% DMSO to the dissolved solution; the concentration of salvianolic acid B in the dissolved solution is 10 mM; the volume ratio of the dissolved solution, PBS solution and PBS solution containing 5% DMSO is 5 μl:95 μl:900 μl;

[0038] The TGF-β protein chip is constructed by taking TGF-β protein according to the method for constructing a protein chip described in step A1.

[0039] The present invention combines SPR and UPLC-MS / MS technologies to establish a screening system for active components of TGF-β protein. The system has the advantages of simple operation, high sensitivity, and strong specificity. The system was applied to the screening of active components in Xinshubao tablet samples, and a total of 12 small molecule compounds with affinity for TGF-β protein were identified, namely, salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid, and lithospermic acid. Affinity tests were performed to verify their activity. Based on the affinity dissociation equilibrium constant and molecular docking binding energy results, salvianolic acid B was identified as a quality biomarker (Q-biomarker). The quality biomarker was used to construct and verify the biological effect methodology of Xinshubao tablets based on a TGF-β protein chip, providing an experimental basis for the formulation of biological quality standards for Xinshubao tablets.

[0040] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0041] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Pre-enrichment results at different protein concentrations;

[0043] Figure 2 Pre-enrichment results of TGF-β protein in CM5;

[0044] Figure 3 Coupling results of TGF-β protein in CM5;

[0045] Figure 4 Results of TGF-β protein specificity investigation;

[0046] Figure 5 Affinity test of baicalin on TGF-β protein chip;

[0047] Figure 6 (A) MRM graph of baicalin recovered at different cycle numbers; (B) Linear relationship between sample peak area and different recovery cycle numbers;

[0048] Figure 7 (A) MRM graphs of samples recovered at different baicalin concentrations; (B) Fitting curve between the peak area of ​​recovered baicalin and the baicalin concentration;

[0049] Figure 8 SPR biosensor identification and recovery of TGF-β binding components. a. Schematic diagram of the binding and recovery of Xinshubao tablets and TGF-β protein; b. Schematic diagram of the negative control running buffer flow through the system;

[0050] Figure 9 Total ion current of Xinshubao tablets recovered from fishing

[0051] Figure 10 MRM chromatogram of Xinshubao tablets (A). Blank sample (B). Reference substance (C). Fishing sample of Xinshubao tablets;

[0052] Figure 11 Precision test of Xinshubao tablets on TGF-β chip;

[0053] Figure 12 Precision test of salvianolic acid B on TGF-β chip;

[0054] Figure 13 Repeatability test of Xinshubao tablets on TGF-β chip;

[0055] Figure 14 Stability test of Xinshubao tablets on TGF-β chip. DETAILED DESCRIPTION

[0056] The reagents, reagents, and equipment used in the specific embodiments of the present invention are all known products and were obtained by purchasing commercially available products. Among them, recombinant human TGF-β protein (Article No. CA72) was purchased from Suzhou Jinan Protein Technology Co., Ltd.; amino coupling kit (BR-1000-50), CM5 sensor chip (BR-1005-30), HBS-EP (10×) buffer (BR-1006-69), 96-well plate (BR-1005-03), and 96-well plate microporous diaphragm (28-9758-16) were purchased from GE Healthcare; Xinshubao tablets were purchased from Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd.

[0057] Example 1 Establishment of a SPR-based TGF-β protein active ingredient screening system and method investigation

[0058] 1. Solution Preparation

[0059] 1.1 Preparation of test samples

[0060] Take about 0.1g of Xinshubao tablet powder, add 10mL of 60% methanol, and treat it with ultrasound (power 250W, frequency 50kHz) for 30min. Take the extract and centrifuge it at 12000rpm for 10min. Take the supernatant and filter it through a 0.22μm microporous membrane to obtain it.

[0061] Preparation of positive and negative samples: Take an appropriate amount of baicalin reference powder and prepare a 10mM positive sample stock solution with DMSO. Other negative (polygalaside B) and positive samples of different concentrations are prepared by diluting with PBS solution containing 5% DMSO.

[0062] 1.2 Solution configuration required for Biacore experiment

[0063] (1) Preparation of HBS-EP buffer: Use a measuring cylinder to measure 180 mL of ultrapure water and place it in a 200 mL glass bottle. Then add 20 mL of HBS-EP (10× stock solution) and mix well to obtain HBS-EP buffer (1×).

[0064] (2) Preparation of Running buffer: Use a measuring cylinder to measure 380 mL of PBS buffer and place it in a 500 mL glass bottle. Then add 20 mL of DMSO to dilute and mix to obtain PBS buffer containing 5% DMSO.

[0065] (3) Preparation of protein sample: TGF-β protein powder (100 μg) stored in a -80°C refrigerator was centrifuged at 14,000 rpm for 2 min and prepared with sterile water to a concentration of 500 μg mL -1 Take 25 μL of the protein solution and place it in a 1.5 mL centrifuge tube for later use, and store the rest in a sealed container at -80°C.

[0066] 2 Ligand pre-enrichment

[0067] 2.1 Selection of protein concentration

[0068] (1) 10 μg mL -1 Protein solution: 2 μL protein stock solution + 98 μL sodium acetate buffer of different pH values;

[0069] 20 μg mL -1 Protein solution: 4 μL protein stock solution + 96 μL sodium acetate buffer of different pH values;

[0070] 40 μg mL -1 Protein solution: 8 μL protein stock solution + 92 μL sodium acetate buffer of different pH values;

[0071] 50 μg mL -1 Protein solution: 10 μL protein stock solution + 90 μL sodium acetate buffer of different pH values.

[0072] (2) Protein pre-enrichment Flow rate: 10 μL min -1 Flowpath: Flowpath 2, Sample and Reagent Rack 1 mode selected for pre-enrichment; Contact time: 120 s. Pre-enrichment was performed at four protein concentrations.

[0073] (3) Pre-enrichment Figure 1 The results showed that when the concentration was greater than 50 μg·mL -1 When the coupling amount is close to saturation, 50 μg·mL is finally selected. -1 The protein solution was used as the concentration for coupling.

[0074] 2.2 pH selection

[0075] 96 μL of sodium acetate buffer solution with pH values ​​of 4.0, 4.5, 5.0, and 5.5 (10 mM) were added to three 1.5 mL EP tubes, and 4 μL of 500 μg mL -1 ) TGF-β protein solution, mix well, centrifuge to remove bubbles, and take a 50mM NaOH regeneration solution to wash away the protein adsorbed on the chip surface. Observe the signal size generated by TGF-β protein when sodium acetate buffer solutions of different pH values ​​flow through the CM5 sensor chip. The results are shown in Figure 2 The optimal pH is the one with the strongest binding ability signal. In this experiment, the optimal pH for TGF-β protein is 5.0.

[0076] 3. Coupling of TGF-β protein to SPR sensor

[0077] First, the ligand was activated with an amino coupling reagent (EDC / NHS) solution for 14 min, followed by protein coupling. The ligand was adsorbed and covalently attached to the chip surface by pulse injection at a protein flow rate of 5 μL·min -1 , the time is 12min, and finally ethanolamine is used to block the unreacted carboxyl sites on the chip surface for 7min. The results are shown in Figure 3 The coupling amounts of TGF-β protein with the four channels of the CM5 chip were 13463, 12600, 13275, and 12755 RU, respectively. The coupling amounts were large enough and in line with expectations.

[0078] 4. Recovery of compounds bound to TGF-β protein

[0079] (1) The sample flows through the surface of the chip with coupled protein. The active components that can bind to the protein are retained on the chip surface, while other ineffective components flow out with the waste liquid; (2) The connection between the system pipeline and the sample pool is cut off, and the injection needle and pipeline are rinsed with distilled water to prevent the sample solution remaining in them from interfering with the active components bound to the chip; (3) The system flow is reversed, and a 0.5% TFA dissociation solution is injected into the chip surface for 20 seconds to dissociate the active components that can bind to the TGF-β protein; (4) The system flow is reversed, and the dissociation solution containing the active components is recovered and injected into 50mM NH4HCO3. In one cycle, the above steps are repeated 10 times. The specific parameters of the recovery experiment are as follows: the mobile phase is a PBS buffer solution containing 5% DMSO; the sample flow rate is 5μL·min -1 ; The protein binding time is 3 minutes; the dissociation reagent is 0.5% TFA solution; the dissociation time is 20 seconds; the recovery reagent is 50mM NH4HCO3 solution.

[0080] 5UPLC-QqQ-MS analysis conditions

[0081] Using CORTECS UPLC C 18 Chromatographic column (2.1 mm × 100 mm, 1.6 μm); acetonitrile (A)-0.1% formic acid water (B) was used as the mobile phase for gradient elution (0-0.5 min, 8% to 8% A; 0.5-2.5 min, 8% to 15% A; 2.5-4.5 min, 15% to 30% A; 4.5-5.5 min, 30% to 48% A; 5.5-5.8 min, 48 %~48%A; 5.8-6.2min, 48%~71%A; 6.2-6.8min, 71%~71%A; 6.8-7.5min, 71%~97%A; 7. 5-8.8min, 97%~97%A; 8.8-9.0min, 97%~8%A; 9.0-10.0min, 8%~8%A); flow rate is 0.25mL·min -1 Column temperature: 40°C; injection volume: 2 μL. Electrospray ionization was performed in positive-negative switching multiple reaction monitoring mode; capillary voltage: 2.5 kV; desolvation gas flow: nitrogen 800 L·h -1 ; Desolvation temperature: 200℃; Cone gas flow: nitrogen 50L·h -1 ; Ion source temperature: 150℃; Extractor: 3.00V; Collision gas: argon, baicalin detection ion channel: 445.0→269.0, cone voltage: 30V, collision energy: 20eV.

[0082] 6. Chip specificity investigation

[0083] Take a 50μM solution of baicalin, a positive small molecule ligand of TGF-β, a 50μM solution of the negative drug polygalaside B and a running buffer (PBS solution containing 5% DMSO) and inject them into the Biacore T200 system respectively to detect the binding response value of the protein. In addition, the affinity response value between the positive small molecule ligand baicalin and the TGF-β protein was also detected. Specifically: take a 50μM solution of baicalin and use a 5% DMSO PBS buffer solution to prepare a gradient with a concentration of 0 to 50μM, and the flow rate is set to 30μL min -1 The binding time was set to 60s, and the dissociation time was set to 60s. The affinity binding data of baicalin and protein were analyzed by Biacore T200evaluation software using 1:1 steady-state affinity mode.

[0084] Using protein positive small molecule ligands baicalin and polygalaside B as negative controls, after injecting 25μM baicalin and polygalaside B solution into the system, it was found that the binding response value of polygalaside B to protein was about 3RU, which was close to the response value of the mobile phase, while the response value of baicalin to protein was 31.10RU. Figure 4 The preliminary results show that the TGF-β protein sensor chip has specific binding to the ligand. Secondly, to further verify the specificity of the chip, we also tested the affinity between the positive small molecule ligand baicalin and TGF-β protein. The affinity analysis results showed that the equilibrium dissociation constant K of baicalin binding to TGF-β protein is 2.34. D The value is 38.05μM, the results are shown in Figure 5 The results showed that the TGF-β protein chip had binding specificity with the ligand.

[0085] 7 Linear Investigation

[0086] A 25 μM baicalin solution was recovered on a TGF-β protein chip for 1, 2, 5, 10, 20, 50, and 100 cycles. The recovered sample was evaporated to dryness by centrifugation and reconstituted in 100 μL of 50% methanol. The sample was centrifuged at 12,000 rpm for 3 minutes, and the supernatant was injected into a UPLC-QqQ-MS system for analysis. The signal-to-noise ratio (S / N) of the chromatographic peak area was calculated using Masslynx software. The S / N values ​​for the peaks detected by LC-MS were all greater than 300 for the selected cycles, indicating that baicalin could be detected in the recovered sample with a single cycle at a sample concentration of 25 μM. However, traditional Chinese medicine extracts are complex and have a wide range of concentrations. Some components, despite their low concentrations, have good affinity for proteins. Therefore, to ensure that the active components in the recovered traditional Chinese medicine extracts can be effectively detected by LC-MS, subsequent sample recovery experiments were performed for at least 10 cycles.

[0087] To further demonstrate the repeatability of the recovery cycle number method, the sample chromatographic peak area (Y) detected by LC-MS was linearly regressed with the recovery cycle number (X). The results showed that the chromatographic peak and the cycle number showed a good linear relationship (R2 = 0.9911) when the cycle number was between 1 and 100. Figure 6 .

[0088] 8 Detection limit and saturation investigation

[0089] According to existing literature reports, the KD values ​​of active ingredients in traditional Chinese medicine are mostly in the concentration range of 10 to 50 μM. In order to investigate the relationship between sample concentration and the amount of recovered active ingredients, different concentrations of baicalin solution (0.39075, 0.7815, 1.5625, 3.125, 6.25, 12.5, 25, 50 μM) were selected for injection recovery. Each concentration recovery was set up for 10 cycles (n = 3). The recovered sample was evaporated by centrifugation and concentrated, and 100 μL of 50% methanol was added to redissolve it. The sample was centrifuged at 10,000 rpm for 3 minutes, and the supernatant was injected into LC-MS for analysis. The chromatogram is shown in Figure 2. Figure 7 A. The signal-to-noise ratio (S / N) of the chromatographic peak area was calculated using Masslynx software. It was found that when the concentration of baicalin was 0.39075 μM, its recovered sample was not detected on LC-MS. When the concentration was 0.7815 μM, its chromatographic peak signal-to-noise ratio / (S / N) was 5.31, indicating that the sample detection limit concentration was between 0.39075 and 0.7815 μM based on 10 recovery cycles. Therefore, when screening active compounds of traditional Chinese medicine, its concentration should be greater than 0.7815 μM. The curve was fitted with the chromatographic peak area of ​​each concentration as the vertical axis (Y) and the sample concentration as the horizontal axis (X). The results are shown in Figure 3. Figure 7B. The results showed that when the baicalin concentration was between 0.78125 and 25 μM, the amount recovered increased accordingly with the increase in sample concentration. However, when the sample concentration was greater than 25 μM, the amount recovered tended to be flat, demonstrating that SPR-AIRS is saturable and that the appropriate sample concentration should be selected based on the KD value of the component.

[0090] The results of the methodological investigation proved that the SPR-based TGF-β protein active ingredient screening system established in the present invention has good specificity and repeatability, which laid the foundation for the use of SPR to screen the active ingredients of Xinshubao tablets.

[0091] Example 2 Identification and Identification of the Active Ingredients of Xinshubao Tablets

[0092] 1 Establishment of fishing method for active ingredients of Xinshubao tablets

[0093] 1.1 Solution Preparation

[0094] (1) Preparation of fishing test sample: Take about 0.1g of Xinshubao tablet powder, add 10mL of 60% methanol, and treat with ultrasound (power 250W, frequency 50kHz) for 30min, shake well, let it stand, take the supernatant and centrifuge at 12000rpm for 10min, take the supernatant and filter it with 0.22μm microporous filter membrane, take 1mL of the filtrate and place it in a 1.5mL centrifuge tube, centrifuge and concentrate to dryness, add 100μL of DMSO to dissolve it, accurately pipette 10μL, add 190μL of PBS1× solution, mix well, and the product is obtained.

[0095] (2) Preparation of reference solution: Take appropriate amount of reference sample powders of salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid, and lithospermic acid, and prepare them with methanol to a concentration of approximately 1 mg mL. -1 The mother solution of the reference substance was diluted with 50% methanol to a mass concentration of about 1 μg mL -1 For qualitative use.

[0096] 1.2 Solution configuration required for Biacore experiment

[0097] (1) Preparation of HBS-EP buffer: Use a 250 mL graduated cylinder to measure 180 mL of ultrapure water, pour it into a 200 mL glass bottle, add 20 mL of HBS-EP (10× stock solution) using a 10 mL pipette, and shake evenly to obtain HBS-EP buffer (1×).

[0098] (2) Preparation of Running buffer: Use a 250 mL graduated cylinder to measure 190 mL of PBS buffer twice and add it to a 500 mL glass bottle. Then add 20 mL of DMSO to dilute and mix to obtain PBS buffer containing 5% DMSO.

[0099] 1.3 Recovery of TGF-β protein fishing components

[0100] (1) Recovery experiment parameters: The number of recovery test cycles was set to 10 times, the mobile phase was PBS buffer solution containing 5% DMSO; the sample flow rate was 5 μL·min -1 ; The protein binding time is 3 minutes; the dissociation reagent is 0.5% TFA solution; the dissociation time is 20 seconds; the recovery reagent is 50mM NH4HCO3 solution.

[0101] (2) Recovery of the active ingredients of Xinshubao tablets: TGF-β protein was coupled to the four channels of the CM5 chip through amino coupling, and the components of Xinshubao tablets were subjected to SPR analysis. Example 1 investigated the specificity and repeatability of the TGF-β protein chip. It was found that when the sample flowed through the surface of the TGF-β sensor, the signal on the sensor surface was enhanced, indicating that one or more components in the Xinshubao tablet extract bind to the TGF-β protein and induce its signal to increase; Xinshubao tablets are a complex mixture, which contains a variety of inactive ingredients that fail to bind to the TGF-β protein. Regardless of their concentration, they will not cause a response signal. Therefore, the running buffer PBS was used as a negative control to flow through the system, and an entire fishing recovery procedure was run. The results are as follows. Figure 8 As SPR analysis could not provide structural information of the recovered active components, UPLC-Q-TOF-MS / MS was used to further identify the structural information of the fishing components.

[0102] 1.4 Qualitative identification of fishing ingredients in Xinshubao tablets

[0103] Chromatographic column: CORTECS TM UPLC C 18 (2.1×100mm,1.6μm); mobile phase: acetonitrile (A)-containing 0.1% formic acid in water (B), gradient elution (0-16.5min, 7-7%A; 16.5-17.50min, 7-12%A; 17.50-22.50min, 12-16%A; 22.5-32.0min, 16-22%A; 32.0-38.0min, 22-30%A; 38.0-41.0min, 30-52%A; 41.0-46.00min, 52-57%A; 46.0-46.1min, 57-7%A; 46.1-48.0min, 7-7%A), flow rate 0.25mLmin -1 , column temperature 45℃, injection volume 2μL.

[0104] Time-of-flight mass spectrometry was performed using an electrospray ionization source in positive and negative ion modes: capillary voltage +3.5 kV (positive ion mode) and -4.5 kV (negative ion mode), nebulizer pressure 2.0 bar, drying gas (N2) flow rate 4.0 L / min, drying gas temperature 180°C, flight mobility (funnel 1 and 2) parameters 200.0 Vpp, quadrupole ion energy 3.0 eV, collision cell (collision Rf) 150.0 Vpp, ion transfer time 80 μs, prepulse storage time 5 μs, collision gas argon, and collision energy 10-50 eV. Mass spectrometric data were acquired in full-scan mode (calibrated with sodium formate solution) over the m / z 50-1500 range.

[0105] The fishing component recovery liquid collected from the Xinshubao tablets sample was centrifuged, concentrated and evaporated, re-dissolved with 100μL of 60% methanol, and injected into the UPLC-Q-TOF-MS / MS system for analysis. A total of 12 chemical components were identified from the LC-MS total ion current of the fishing recovery liquid of Xinshubao tablets. Finally, after accurate comparison with the reference substance, the 12 identified compounds were: salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid, and lithospermic acid. The total ion current of LC-MS identification of the recovered sample of Xinshubao tablets is shown in Figure 9 The mass spectrometry identification is shown in Table 1.

[0106] Table 1 Identification of fishing ingredients in Xinshubao tablets by UPLC-Q-TOF-MS

[0107]

[0108] 2 Methodological Study on the Fishing Ingredients of Xinshubao Tablets

[0109] 2.1 Solution Preparation

[0110] Take appropriate amount of reference substance powder of salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid and lithospermic acid, and add DMSO to prepare reference substance stock solutions with a concentration of about 10 mM for later use.

[0111] 2.2 UPLC-QqQ-MS quantitative detection conditions

[0112] (1) Chromatographic conditions: CORTECS UPLC C 18Chromatographic column (2.1 mm × 100 mm, 1.6 μm); acetonitrile (A)-0.1% formic acid water (B) was used as the mobile phase for gradient elution (0-0.5 min, 8% to 8% A; 0.5-2.5 min, 8% to 15% A; 2.5-4.5 min, 15% to 30% A; 4.5-5.5 min, 30% to 48% A; 5.5-5.8 min, 48 %~48%A; 5.8-6.2min, 48%~71%A; 6.2-6.8min, 71%~71%A; 6.8-7.5min, 71%~97%A; 7. 5-8.8min, 97%~97%A; 8.8-9.0min, 97%~8%A; 9.0-10.0min, 8%~8%A); flow rate is 0.25mL·min -1 , column temperature 40℃; injection volume 2μL.

[0113] (2) Mass spectrometry conditions: electrospray positive ionization mode; capillary voltage 2.5 kV; desolvation gas flow: nitrogen 800 Lh -1 ; Desolvation temperature: 200℃; Cone gas flow: nitrogen 50L h -1 ; Ion source temperature: 150℃; Extractor: 3.00V; Collision gas: Argon. Specific parameters are shown in Table 2, and the chromatogram is shown in Figure 10 .

[0114] Table 2 MS parameters of 12 components in recovered samples of Xinshubao tablets

[0115]

[0116]

[0117] 2.3 Linearity Study of Active Ingredients in Xinshubao Tablets

[0118] The sample concentration of 12 compounds was diluted to 25 μM, and 1, 2, 5, 10, 20, 50 and 100 recovery cycles were tested on the TGF-β protein chip. The recovered sample solution was centrifuged and concentrated to dryness, and 100 μL of 50% methanol was added for ultrasonic reconstitution. The sample was centrifuged at 12000 rpm for 3 minutes, and the supernatant was injected into UPLC-QqQ-MS for analysis. The sample chromatographic peak area was used as the vertical axis and the recovery cycle number was used as the horizontal axis for linear regression. The linear correlation coefficient (R) of salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid, and lithospermic acid was obtained. 2) were 0.9909, 0.9909, 0.9760, 0.9780, 0.9833, 0.9925, 0.9806, 0.9745, 0.9559, 0.9861, 0.9815 and 0.9909, respectively. The results showed that the cycle times of the 12 components were between 1 and 100, and they showed a good linear relationship with their recovery amounts, indicating that the screening of the 12 components in Xinshubao tablets using TGF-β protein had good repeatability, and it was found that when the concentration of the fishing samples of each component was 25 μM and the cycle times were set to 10, the samples of each recovered component could be effectively detected in LC-MS.

[0119] 2.4 Investigation of the saturation and detection limit of the active ingredients in Xinshubao tablets

[0120] The sample concentrations of the 12 fishing components were prepared into mixed reference solutions with gradient concentrations of 50, 25, 12.50, 6.25, and 3.125 μM, and passed through the Biacore T200 system to collect the recovered liquid bound to the TGF-β protein chip, which was centrifuged and concentrated to dryness, and 100 μL of 50% methanol was added for ultrasonic re-dissolution. The samples were centrifuged at 12000 rpm for 3 minutes, and the supernatant was injected into UPLC-QqQ-MS for analysis. The results showed that when the concentration was 3.125 μM, the chromatographic peak area of ​​each component was small, and S / N>3. 3.125 μM was set as the detection limit of each component. Therefore, in the subsequent recovery test of the above 12 components of TGF-β protein fishing, the compound concentration in the samples of traditional Chinese medicine or Chinese patent medicine should be greater than 3.125 μM after conversion calculation to ensure that the recovered samples can be effectively detected in UPLC-QqQ-MS.

[0121] The correlation analysis between the peak area of ​​the recovered samples and the injection concentration showed that the samples can be divided into two major categories. One category is salvianolic acid, including salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D and lithospermic acid. When the injection concentration of this type of component is greater than 25 μM, the chromatographic peak area of ​​the recovered samples does not increase significantly with the increase of the injection concentration, and basically tends to be stable, that is, this type of component is saturated within the concentration range of the study; the other category is chlorogenic acid (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C) and rosmarinic acid. The peak area of ​​the recovered samples is linearly related to the sample concentration. Linear regression is performed with the peak area of ​​the sample of this type of component as the ordinate and the concentration of the sample as the abscissa. The results show that the correlation coefficient (R 2) were 0.9761, 0.9856, 0.9831, 0.9955, 0.9974, 0.9905 and 0.9982, respectively, indicating that this type of component has a good linear relationship with the peak area when the sample concentration is between 3.125-50 μM, and there is no saturation trend.

[0122] 3. Affinity Verification of the Active Ingredients of Xinshubao Tablets

[0123] 3.1 Sample preparation

[0124] Take 1 μL of the mother solution of each compound under item "2.1", add 19 μL of PBS respectively, and then add 180 μL of PBS buffer solution containing 5% DMSO, mix well to obtain 200 μL sample solution of 50 μM PBS buffer solution containing 5% DMSO, and set aside.

[0125] 3.2 Preparation of calibration fluid

[0126] To eliminate the influence of mobile phase on experimental results, calibration is performed using calibration solutions. Take 0.45 mL and 0.58 mL of DMSO solution, respectively, and add 9.5 mL of PBS solution to prepare 10 mL of calibration solutions containing 4.5% and 5.8% DMSO. Then, according to Table 3, use the calibration solutions to prepare eight calibration solutions with different DMSO concentrations ranging from 4.5% to 5.8%.

[0127] Table 3 Configuration of calibration fluid

[0128]

[0129] 3.3 Affinity Verification

[0130] The sample solution prepared under "3.1" was diluted in half with 5% DMSO in PBS as the dilution solvent to prepare a gradient concentration sample solution. The concentrations were 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.563 μM, 0.781 μM, 0.391 μM, and 0 μM, respectively. The lowest concentration of 0.391 μM was repeatedly injected to test the stability of the SPR system. The samples were injected from low to high concentrations at a flow rate of 30 μM min -1 The data were analyzed using the Biacore T200 evaluation software in a 1:1 steady-state affinity mode. The dissociation equilibrium constants (K D ) values, the results are shown in Table 4.

[0131] Table 4 SPR dissociation equilibrium constants

[0132]

[0133]

[0134] 3.4 Molecular docking

[0135] To evaluate the binding energy and interaction mode between the candidate small molecules screened by SPR and their targets, molecular docking was further performed by computer: first, the small molecule structure was obtained from the PubChem compound database (https: / / pubchem.ncbi.nlm.nih.gov / ), the mechanical structure was optimized using Chem 3D, saved as mol2 format, opened with AutoDock Tools 1.5.6 (The Scripps Research Institute, CA, USA), hydrogenated, and saved as pdbqt format; second, the pdb structure of TGF-β protein (code 5VQP) was downloaded from the Protein Data Bank (http: / / www.rcsb.org / ), water molecules and small molecule ligands were deleted using PYMOL software, hydrogenated using AutoDock Tools 1.5.6, and saved as pdbqt format; third, the AutoDock Vina 1.1.2 Perform molecular docking 50 times to obtain the docking binding energy between the molecule and the protein; finally, based on the docking results, select the model with the optimal conformation and use PYMOL software to visualize the results.

[0136] To ensure the reliability of the docking results, baicalin was used as a positive small molecule and docked together with the candidate small molecules. The lower the binding energy, the better the binding ability of the ligand and protein. The binding energies of the 13 compounds are shown in Table 5. The binding energies of the 13 compounds are all less than -6 kJ·mol -1 , indicating that they can bind well to proteins, among which the docking binding energy of the positive small molecule baicalin is -6.9 kJ·mol -1 Salvianolic acid B has the lowest docking binding energy, which is -8.6 kJ·mol -1 The results were consistent with the affinity constants obtained by SPR analysis.

[0137] Table 5 SPR kinetic constants and molecular docking binding energy

[0138]

[0139] The above affinity verification and molecular docking results showed that the surface plasmon resonance (SPR) method was used to fish out active ingredients that act on the TGF-β protein target from the complex system of Xinshubao tablets. These active ingredients revealed that Xinshubao tablets can exert their drug efficacy through the TGF-β protein target, laying the foundation for the evaluation of the biological effects of Xinshubao tablets.

[0140] The above-mentioned verified method for targeting the active ingredients of Xinshubao tablets based on the TGF-β protein target is:

[0141] A1: Construct protein chip: Dissolve TGF-β protein in water, then dilute with sodium acetate buffer to a 30-50 μg / ml protein solution. Couple the protein solution to the sensor chip.

[0142] A2 Preparation of fishing test solution: Take Xinshubao tablets, extract with methanol solution, centrifuge the extract, take the supernatant, dry it, add DMSO to dissolve it, filter it, and add PBS solution to the filtrate and mix well.

[0143] A3: inject the sample solution obtained in step A2 into a surface plasmon resonance instrument loaded with the protein chip obtained in step A1 for testing;

[0144] Fishing conditions are as follows: mobile phase: PBS buffer solution containing DMSO; sample flow rate 1-10 μL·min -1 Injection time: 10-30 seconds; Protein binding time: 2-5 minutes; Dissociation reagent: TFA solution, Dissociation time: 15-30 seconds; Recovery reagent: NH4HCO3 solution, Cycle number: 1-100 times;

[0145] A4 uses UPLC-Q-TOF-MS method to perform qualitative detection on the fishing component recovery liquid obtained in step A3.

[0146] The specific steps of the above-mentioned Xinshubao tablet active ingredient fishing method are as follows:

[0147] 1) Construction of protein chip:

[0148] Dissolve TGF-β protein in sterile water to obtain a 500 μg / ml TGF-β protein stock solution; take 10 μl of the TGF-β protein stock solution and add 90 μl of 10 mM pH 5.0 sodium acetate buffer and mix well to obtain a 50 μg / ml TGF-β protein solution;

[0149] Start the surface plasmon resonance instrument, set channel 1 as the blank unit and channel 2 as the coupling unit;

[0150] 100 μl of TGF-β protein solution was amino-coupled to a CM5 chip. The coupling parameters were: binding time: 720 s, flow rate: 5 μl / min, temperature: 25°C, coupling reagents: 100 μl each of EDC and NHS (from the amino coupling kit, EDC:NHS volume ratio: 1:1), 140 μl of ethanolamine (from the amino coupling kit) was used for blocking, and the blocking time was 7 min. Running buffer: 1×HBS-EP.

[0151] 2) Preparation of fishing test solution

[0152] Take 0.1g Xinshubao tablets, place them in a 25mL Erlenmeyer flask, add 10mL of 60% methanol, seal, weigh, and extract by ultrasonication for 30min (50kHz, 250W), weigh again, add 60% methanol to make up for the weight loss, centrifuge at 12000r for 10min, accurately aspirate 1mL of the supernatant, concentrate by centrifugation and evaporate to dryness, add 100μL DMSO to redissolve, filter through a 0.22μm pore membrane, accurately aspirate 10μL, add 190μL PBS 1× solution, and mix well.

[0153] 3) Fishing

[0154] The test solution was injected into a surface plasmon resonance instrument loaded with a TGF-β protein chip for fishing. The fishing conditions were as follows: mobile phase: PBS buffer solution containing 5% DMSO; injection time: 20 s; sample flow rate: 5 μL min -1 ; Protein binding time: 3 min; Dissociation reagent: 0.5% TFA solution, dissociation time: 20 s; Recovery reagent: 50 mM NH4HCO3 solution, number of cycles: 10 times;

[0155] 4) UPLC-Q-TOF-MS qualitative detection of components recovered after fishing

[0156] a: Preparation of reference solution: Prepare reference solution powders of salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid, and lithospermic acid, and prepare them into a concentration of 1 mg mL with methanol. -1 The mother solution of the reference substance was diluted with 50% methanol to a mass concentration of 1 μg·mL -1 The solution is used as a reference solution for qualitatively identifying the active ingredient that targets TGF-β protein;

[0157] b: Take the fishing component recovery solution, dry it, add 100 μL of 60% methanol to dissolve it, and inject the dissolved solution and the reference solution obtained in step a into UPLC-Q-TOF-MS for detection:

[0158] Chromatographic conditions: Column: CORTECS TM UPLC C 18 (2.1×100mm,1.6μm); mobile phase: acetonitrile (A)-containing 0.1% formic acid in water (B), gradient elution (0-16.5min, 7-7%A; 16.5-17.50min, 7-12%A; 17.50-22.50min, 12-16%A; 22.5-32.0min, 16-22%A; 32.0-38.0min, 22-30%A; 38.0-41.0min, 30-52%A; 41.0-46.00min, 52-57%A; 46.0-46.1min, 57-7%A; 46.1-48.0min, 7-7%A), flow rate 0.25mLmin -1 , column temperature 45℃, injection volume 2μL.

[0159] Time-of-flight mass spectrometry was performed using an electrospray ionization source in positive and negative ion modes: capillary voltage +3.5 kV (positive ion mode) and -4.5 kV (negative ion mode), nebulizer pressure 2.0 bar, drying gas (N2) flow rate 4.0 L / min, drying gas temperature 180°C, flight mobility (funnel 1 and 2) parameters 200.0 Vpp, quadrupole ion energy 3.0 eV, collision cell (collision Rf) 150.0 Vpp, ion transfer time 80 μs, prepulse storage time 5 μs, collision gas argon, and collision energy 10-50 eV. Mass spectrometric data were acquired in full-scan mode (calibrated with sodium formate solution) over the m / z 50-1500 range.

[0160] Example 3: Bioeffect Detection Method for Screening Bioquality Markers of Xinshubao Tablets Based on Target Fishing Technology

[0161] In Example 2, target fishing screening analysis revealed that 12 phenolic acid components, including salvianolic acid A, salvianolic acid C, salvianolic acid D, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, lithospermic acid, salvianolic acid B, chlorogenic acid, and rosmarinic acid, bound to the TGF-β protein target in a concentration-dependent manner. Among them, salvianolic acid B had the best affinity for TGF-β protein. Salvianolic acid B is an indicator component of Danshen (Salvia miltiorrhiza), the main herb in the Xinshubao tablets formula. Therefore, salvianolic acid B was selected as the positive control for the biological effect detection method of Xinshubao tablets.

[0162] 1 Establishment and validation of Q-biomarker methodology for Xinshubao tablets

[0163] 1.1 Solution Preparation

[0164] (1) Preparation of test solution: Accurately take 0.1 g of Xinshubao tablet powder and place it in a 25 mL conical flask, add 10 mL of 60% methanol, weigh it, ultrasonicate it (50 kHz, 350 W) for 30 min, weigh it again, make up the lost weight by adding 60% methanol, shake it well, centrifuge it at 12000 r for 10 min, accurately aspirate 2 mL of the supernatant, concentrate it by centrifugation and evaporate it to dryness, add 200 μL of DMSO to dissolve it again, filter it through a 0.22 μm microporous membrane, accurately aspirate 10 μL of the filtrate, add 190 μL of PBS1× solution, mix it well, and the solution is ready.

[0165] (2) Preparation of the positive drug, salvianolic acid B: Accurately weigh an appropriate amount of salvianolic acid B powder and prepare a 10 mM stock solution with DMSO. Take 5 μL of the control stock solution, add 95 μL of 1× PBS and 900 μL of 5% DMSO in PBS to prepare a 50 μM salvianolic acid B solution in 5% DMSO in PBS. Mix thoroughly. The negative drug, polygala tenuifolia glycoside B, is prepared in the same manner.

[0166] 1.2 Precision, repeatability and stability tests

[0167] 1.2.1 Precision test

[0168] Take the Xinshubao tablet sample (batch number 2011020), prepare the solution according to the method under "1.1", accurately pipette 6 100 μL reconstituted Xinshubao tablet sample solutions into the Biacore test 96-well plate, and inject them into the SPR system for detection under the conditions of contact time: 60 s, flow rate: 30 μL min -1 , Dissociation time: 60s, Temperature: 25℃; Running Buffer: 5% DMSO in PBS. The RU values ​​obtained by 6 injections were 145.3, 140.4, 144.5, 142.3, 135.9, and 136.9, respectively, with an RSD of 2.8%. Figure 11 The positive drug salvianolic acid B was tested 6 times using the same method, and the RU values ​​were 16.1, 16.5, 16.8, 16.8, 16.6, and 17.4, respectively, with an RSD of 2.6%. Figure 12 , through the detection of samples and positive drugs, the results show that the precision of the instrument is good.

[0169] 1.2.2 Repeatability test

[0170] Six samples of Xinshubao tablets from the same batch (batch number 2011020) were prepared in parallel according to the method under "1.1". 100 μL was accurately pipetted and placed in a 96-well Biacore test plate. The conditions were contact time: 60 s, flow rate: 30 μL / min, dissociation time: 60 s, temperature: 25°C; running buffer: 5% DMSO in PBS solution. The RU values ​​of the six screenings were 134.1, 131.8, 140.5, 137.7, 134.6, and 134.0, respectively, with an RSD of 2.4%, indicating good reproducibility of the method. The results are shown in the table. Figure 13 .

[0171] 1.2.3 Sample stability test

[0172] Take Xinshubao tablets sample (batch number 2105001) and prepare it according to the method under "1.1". Accurately pipette 100 μL and place it in a 96-well Biacore test plate, place it at room temperature, and inject it into SPR detection at 0, 2, 4, 8, 12 and 24 hours respectively. The conditions are contact time: 60 s, flow rate: 30 μL·min-1, dissociation time: 60 s, temperature: 25℃; running buffer: 5% DMSO in PBS solution. The RU values ​​screened at 0, 2, 4, 8, 12 and 24 hours were 139.9, 141.1, 141.5, 138.9, 137.5 and 137.2, respectively, with an RSD of 3.7%, indicating that the test solution was stable at room temperature for 24 hours. The results are shown in the table. Figure 14 .

[0173] 1.3 Selection of positive drug concentration

[0174] 1.3.1 Preparation of Salvianolic Acid B Solution

[0175] A solution of PBS containing 5% DMSO was prepared with a gradient concentration of 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.7813, 0.3906, 0.1953, 0.0977, 0.0488, and 0 μM.

[0176] 1.3.2 Affinity determination of salvianolic acid B

[0177] Affinity analysis parameter settings: Flow path: 2-1, Chip type: CM5, Contact time: 180s. Flow rate: 30μL·min -1, Dissociation time: 300s, Temperature: 25℃. The experimental results show that the K D The value is 6.11×10 -6 M, and finally 50 μM was selected as the concentration of the positive drug.

[0178] 1.4 Optimization of test solution preparation

[0179] Xinshubao tablets were extracted using solvents with different methanol concentrations and injected into UPLC-Q-TOF-MS for analysis. According to the results of total ion current, it was found that the response of Xinshubao tablets extracted with 60% methanol was better than that of other solvents, so 60% methanol was determined to be used for extraction.

[0180] 1.5 Investigation of the amount of DMSO for re-dissolution

[0181] 1.5.1 Preparation of test samples

[0182] Accurately weigh 0.1 g of Xinshubao tablet powder and place it in a 25 mL stoppered bottle. Add 10 mL of 60% methanol, seal, weigh, and extract by ultrasonication for 30 min (50 kHz, 350 W). Then weigh again, make up the weight loss with 60% methanol, centrifuge at 14000 r for 10 min, accurately aspirate 1 mL, concentrate by centrifugation and evaporate to dryness, add different volumes (100, 200, 400, 1000 μL) of DMSO to re-dissolve, and filter through a 0.22 μm pore filter membrane to obtain test samples with different re-dissolution ratios.

[0183] 1.5.2 Screening of Xinshubao Tablets with Different Reconstitution Ratios in TGF-β

[0184] 100 μL of Xinshubao tablet samples with different reconstitution ratios were placed in a 96-well Biacore plate. The sample was screened on the chip with a contact time of 60 s and a flow rate of 30 μL min. -1 Dissociation time: 60s, Temperature: 25°C. Running Buffer: 5% DMSO in PBS. Experimental results with sample solution and TGF-β protein showed that 100μL of DMSO reconstitution gave the best response. Because sample precipitation often occurred when using 50μL, 100μL was selected for reconstitution.

[0185] 1.6 SPR detection of different batches of Xinshubao tablets

[0186] SPR screening of TGF-β protein was performed based on samples of 6 batches of Xinshubao tablets.

[0187] 1.6.1 Preparation of test samples

[0188] Preparation of Xinshubao tablet sample solution: Accurately take 0.1g of Xinshubao tablet powder, place it in a 25mL stoppered bottle, add 10mL of 60% methanol, seal, weigh, ultrasonically treat (50kHz, 350W) for 30min, weigh again, make up the weight loss with 60% methanol, centrifuge at 12000r for 10min, accurately aspirate 2mL of supernatant, centrifuge and concentrate at 4℃ to evaporate, add 200μLDMSO to dissolve it again, filter through 0.22μm microporous membrane, accurately aspirate 10μL, add 190μL of PBS solution, mix well, and obtain.

[0189] Preparation of the positive agent, salvianolic acid B: Accurately weigh an appropriate amount of salvianolic acid B powder and prepare a stock solution with DMSO to a concentration of 10 mM. Add 5 μL of the control stock solution to 95 μL of 1× PBS and 900 μL of 5% DMSO in PBS, and mix thoroughly to a 50 μM solution in 5% DMSO in PBS. The negative agent, protocatechuic aldehyde, is prepared in the same manner.

[0190] 1.6.2 SPR Detection of TGF-β Protein in Xinshubao Tablets

[0191] Take 6 batches of Xinshubao tablets samples, the positive drug salvianolic acid B, and the negative drug polygalaside B test solution, and accurately pipette 100 μL of each into a 96-well Biacore test plate. The sample run time in the chip test is contact time: 60 s; flow rate: 30 μL·min -1 Dissociation time: 60 s, Temperature: 25°C. Running Buffer: 5% DMSO in PBS. The ratios of the RU values ​​of 6 batches of Xinshubao tablets to the RU values ​​of salvianolic acid B are shown in Table 6. The results show that the ratios are at least greater than 1.

[0192] Table 63 Ratios of RU values ​​of batches of Xinshubao tablets samples, intermediates and positive salvianolic acid B

[0193]

[0194] 1.7 Summary

[0195] According to the results of the methodological investigation, the concentration of TGF-β protein solution was selected as 50 μg·mL -1 Protein coupling was performed on a CM5 chip. The pH was determined based on the pre-enrichment results. The test sample was extracted with 60% methanol and reconstituted with DMSO at a volume ratio of 10:1. Salvianolic acid B was selected as the reference substance at a concentration of 50 μM. The final method for detecting the biological effects of Xinshubao tablets was as follows:

[0196] 1) Using a surface plasmon resonance instrument to measure the RU value of Xinshubao tablets and the RU value of salvianolic acid B, wherein the protein chip in the surface plasmon resonance instrument is a TGF-β protein chip;

[0197] 2) The ratio is calculated using the following formula. If the ratio is not less than 1, the biological effect test of Xinshubao tablets is qualified;

[0198] Ratio = RU value of Xinshubao tablets / RU value of salvianolic acid B.

[0199] The specific steps of the above-mentioned biological effect detection method of Xinshubao tablets are as follows:

[0200] 1) Construction of protein chip

[0201] Dissolve TGF-β protein in sterile water to obtain a 500 μg / ml TGF-β protein stock solution; take 10 μl of the TGF-β protein stock solution and add 90 μl of 10 mM pH 5.0 sodium acetate buffer and mix well to obtain a 50 μg / ml TGF-β protein solution;

[0202] Start the surface plasmon resonance instrument, set channel 1 as the blank unit and channel 2 as the coupling unit;

[0203] 100 μl of TGF-β protein solution was amino-coupled to a CM5 chip. The coupling parameters were: binding time: 720 s, flow rate: 5 μl / min, temperature: 25°C, coupling reagents: 100 μl each of EDC and NHS (from the amino coupling kit, EDC:NHS volume ratio: 1:1), 140 μl of ethanolamine (from the amino coupling kit) was used for blocking, and the blocking time was 7 min. Running buffer: 1×HBS-EP.

[0204] 2) Preparation of test solution

[0205] 0.1 g of Xinshubao tablets were placed in a 25 mL conical flask, 10 mL of 60% methanol was added, the flask was sealed, and the weight was weighed. Ultrasonic extraction was performed for 30 min (50 kHz, 250 W), and the weight was reweighed. The weight loss was supplemented with 60% methanol. The tablets were centrifuged at 12000 r for 10 min. 1 mL of the supernatant was accurately aspirated, concentrated by centrifugation, evaporated to dryness, and redissolved in 100 μL of DMSO. The tablets were filtered through a 0.22 μm pore membrane, and 10 μL of the supernatant was accurately aspirated. 190 μL of PBS 1× solution was added and mixed.

[0206] 3) Preparation of Reference Solution: Accurately weigh salvianolic acid B and prepare a stock solution with DMSO to a concentration of 10 mM. Take 5 μL of the reference stock solution, add 95 μL of 1× PBS and 900 μL of 5% DMSO in PBS, and mix thoroughly to prepare a 50 μM solution in 5% DMSO in PBS.

[0207] 4) taking the test solution obtained in step 2) and the reference solution obtained in step 3) and placing them in the surface plasmon resonance test plate containing the protein chip obtained in step 1);

[0208] SPR detection conditions: running time: 60 s; flow rate: 30 μl / min; dissociation time: 60 s; temperature: 25° C.; running buffer: 5% DMSO in PBS.

[0209] 5) Calculate the ratio of the RU values ​​of Xinshubao tablets to that of salvianolic acid B. When the ratio is greater than or equal to 1, the biological effect test of Xinshubao tablets is qualified.

[0210] In summary, the present invention combines SPR and UPLC-MS / MS technology to establish a TGF-β protein active ingredient screening system, which has the advantages of simple operation, high sensitivity and strong specificity; the system is applied to the fishing of active ingredients in Xinshubao tablets samples, and a total of 12 small molecule compounds with affinity to TGF-β protein are fished out, namely, salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid and lithospermic acid, and affinity tests are performed to verify their activity. According to the affinity dissociation equilibrium constant and molecular docking binding energy results, salvianolic acid B is selected as the Q-biomarker to construct and verify the biological effect methodology of Xinshubao tablets, providing an experimental basis for the formulation of biological quality standards for Xinshubao tablets.

Claims

1. A fishing method using the active ingredient of Xinshubao tablets, characterized in that: The steps include: A1: Construct protein chip: Dissolve TGF-β protein in water, then dilute with sodium acetate buffer to a 30-50 μg / ml protein solution. Couple the protein solution to the sensor chip. A2 Preparation of fishing test solution: Take Xinshubao tablets, extract with methanol solution, centrifuge the extract, take the supernatant, dry it, add DMSO to dissolve it, filter it, and add PBS solution to the filtrate and mix well. A3: inject the sample solution obtained in step A2 into a surface plasmon resonance instrument loaded with the protein chip obtained in step A1 for testing; Fishing conditions are as follows: mobile phase: PBS buffer solution containing DMSO; sample flow rate 1-10 μL·min -1 Injection time: 10-30 seconds; Protein binding time: 2-5 minutes; Dissociation reagent: TFA solution, Dissociation time: 15-30 seconds; Recovery reagent: NH4HCO3 solution, Cycle number: 1-100 times; A4 uses UPLC-Q-TOF-MS method to perform qualitative detection on the fishing component recovery liquid obtained in step A3.

2. The fishing method according to claim 1, wherein: The concentration of the sodium acetate buffer in step A1 is 10 mM, and the pH value is 5; the protein solution is 50 μg / ml; the coupling is amino coupling; the parameters of the amino coupling are: sensor chip model: CM5, coupling reagents: EDC and NHS, protein solution flow rate: 5 μl / min, protein binding time: 720 s, temperature: 25°C, blocking reagent: ethanolamine, and blocking time: 7 min.

3. The fishing method according to claim 2, wherein: The volume ratio of the protein solution, EDC, NHS and ethanolamine is 100:200:140; the volume ratio of EDC and NHS is 1:

1.

4. The fishing method according to claim 1, wherein: In step A2, the mass volume ratio of Xinshubao tablets to methanol solution is 0.1 g: 5-25 ml; the concentration of the methanol solution is 60%; The extraction is ultrasonic extraction, and the time is 20 to 60 minutes; The centrifugal speed is 10000-20000r, and the time is 5-20min; The volume ratio of the supernatant, DMSO, filtrate and PBS buffer is 0.5-1.5 mL: 100 μL: 10 μL: 190 μL; the PBS solution is PBS 1× solution; and / or: The fishing conditions in step A3 are as follows: mobile phase: PBS buffer solution containing 5% DMSO; sample flow rate 5 μL·min -1 ;Injection time: 20s; The protein binding time is 3 min; Dissociation reagent: 0.5% TFA solution, dissociation time: 20 s; recovery reagent: 50 μM NH 4 HCO 3 solution, number of cycles: 10 times.

5. The fishing method according to claim 1, wherein: The qualitative detection in step A4 includes the following steps: ① Preparation of reference solution: Take salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid and lithospermic acid reference substances, add methanol solution to dissolve, and obtain; ② The recovered fishing component solution was dried and dissolved in 60% methanol. The dissolved solution and the reference solution obtained in step ① were injected into UPLC-Q-TOF-MS for detection. The chromatographic conditions were: Chromatographic column: C18 column; Mobile phase: Acetonitrile-0.1% formic acid water; Gradient elution program: 0-16.5 min, 7-7% acetonitrile; 16.5-17.50 min, 7-12% acetonitrile; 17.50-22.50 min, 12-16% acetonitrile; 22.5-32.0 min, 16-22% acetonitrile; 32.0-38.0 min, 22-30% acetonitrile; 38.0-41.0 min, 30-52% acetonitrile; 41.0-46.00 min, 52-57% acetonitrile; 46.0-46.1 min, 57-7% acetonitrile; 46.1-48.0 min, 7-7% acetonitrile; The mass spectrometry conditions were as follows: ion source: electrospray ion source, positive and negative ion modes; data acquisition mode: full scan mode, range m / z 50-1500.

6. The fishing method according to claim 5, characterized in that: The mass concentration of each reference substance in the reference solution of step ① is 0.5-1.5 μg·mL -1 .

7. The fishing method according to claim 5, characterized in that: The C18 chromatographic column in step ② is CORTECS TM UPLC C 18 , 2.1mm×100mm, 1.6μm, flow rate 0.25mL·min -1 , column temperature 45°C; injection volume 2 μL; The mass spectrometry conditions include a capillary voltage of +3.5 kV in positive ion mode, a capillary voltage of -4.5 kV in negative ion mode, a nebulizer pressure of 2.0 bar, N2 as the drying gas, an N2 volume flow rate of 4.0 L / min, a drying gas temperature of 180°C, a flight migration parameter of 200.0 Vpp, a quadrupole ion energy of 3.0 eV, a collision cell of 150.0 Vpp, an ion transmission time of 80 μs, a pre-pulse storage time of 5 μs, collision gas argon, and a collision energy of 10-50 eV.

8. The fishing method according to claim 5, wherein: The active ingredients fished out are salvianolic acid A, salvianolic acid C, salvianolic acid D, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, lithospermic acid, salvianolic acid B, chlorogenic acid and rosmarinic acid.

9. A method for detecting the biological effects of Xinshubao tablets, characterized in that: It uses surface plasmon resonance detection, which includes the following steps: 1) Using a surface plasmon resonance instrument to measure the RU value of Xinshubao tablets and the RU value of salvianolic acid B, wherein the protein chip in the surface plasmon resonance instrument is a TGF-β protein chip; 2) The ratio is calculated using the following formula. If the ratio is not less than 1, the biological effect test of Xinshubao tablets is qualified; Ratio = RU value of Xinshubao tablets / RU value of salvianolic acid B.

10. The method according to claim 9, characterized in that: The steps for determining the RU value of Xinshubao tablets and the RU value of salvianolic acid B are as follows: A test solution of Xinshubao tablets and a reference solution of salvianolic acid B were placed in a surface plasmon resonance test plate loaded with a TGF-β protein chip. SPR assay conditions included a run time of 60 s, a flow rate of 30 μl / min, a dissociation time of 60 s, a temperature of 25°C, and a running buffer of 5% DMSO in PBS. The Xinshubao tablet test solution is prepared by taking Xinshubao tablets according to the method for preparing the fishing test solution described in step A2; The salvianolic acid B reference solution is prepared by dissolving salvianolic acid B in DMSO, adding PBS solution and PBS solution containing 5% DMSO to the dissolved solution; the concentration of salvianolic acid B in the dissolved solution is 10 mM; the volume ratio of the dissolved solution, PBS solution and PBS solution containing 5% DMSO is 5 μl:95 μl:900 μl; The TGF-β protein chip is constructed by taking TGF-β protein according to the method for constructing a protein chip described in step A1.

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